CN118369104A - IRNA compositions and methods for targeting ANGPTL7 - Google Patents

Abstract

The present disclosure relates to a double-stranded ribonucleic acid (dsRNA) composition targeting ANGPTL 7. The invention also relates to methods of using such dsRNA compositions to inhibit ANGPTL7 expression and methods of using such dsRNA compositions to treat ANGPTL 7-related disorders, such as glaucoma.

Description

IRNA compositions and methods for targeting ANGPTL7

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application Ser. No.63/251,203, filed on 1-10-2021, and U.S. provisional application Ser. No. 63/287,414, filed on 8-12-2021. The entire contents of each of the foregoing applications are incorporated herein by reference.

Sequence listing

The present application comprises a sequence listing submitted electronically in XML form and is hereby incorporated by reference in its entirety. The XML copy was created at 9.30 of 2022, named A108868_1510WO_SL.xml, and was 1,323,084 bytes in size.

FIELD OF THE DISCLOSURE

The present disclosure relates to specific inhibition of ANGPTL7 expression.

Background

Glaucoma is a major cause of vision loss. Glaucoma is caused by optic nerve damage and loss of nerve fibers, and is usually associated with elevated intraocular pressure. Lowering intraocular pressure can reduce the progression and progression of glaucoma and the associated vision loss.

Human angiopoietin-like 7 (ANGPTL 7), encoding angiopoietin-like 7 protein (ANGPTL 6), chromosomal region 1p36.22 on chromosome 1, consisting of 6 exons. ANGPTL7 belongs to the ANGPTL family of proteins and is expressed in the corneal stroma and other sites. ANGPTL7 protein levels in aqueous humor of glaucoma patients are reported to be elevated. Human genomic analysis showed that missense and nonsense variants in ANGPTL7 are associated with lower intraocular pressure and lower risk of glaucoma. These findings indicate that interfering with ANGPTL7 is a therapeutic strategy for glaucoma.

Thus, there is a need for agents that selectively and effectively inhibit ANGPTL7 gene expression, thereby effectively treating subjects suffering from ANGPTL 7-related disorders (e.g., glaucoma).

Disclosure of Invention

The present disclosure describes methods and iRNA compositions for modulating ANGPTL7 expression. In certain embodiments, ANGPTL 7-specific iRNA is used to reduce or inhibit ANGPTL7 expression. Such inhibition may be used to treat a disorder associated with ANGPTL7 expression, such as an ocular disorder (e.g., glaucoma or a disorder associated with glaucoma).

Thus, the compositions and methods described herein affect RNA-induced silencing complex (RISC) -mediated cleavage of an RNA transcript of ANGPTL7, such as in a cell or subject (e.g., in a mammal, such as a human subject). Also described are compositions and methods for treating disorders associated with the expression of ANGPTL7, such as glaucoma or disorders associated with glaucoma.

The compositions described herein comprise an iRNA (e.g., dsRNA) comprising an RNA strand (antisense strand) having a region, e.g., a region of 30 nucleotides or less, typically 19-24 nucleotides in length, that is substantially complementary to at least a portion of an mRNA transcript of ANGPTL7 (e.g., human ANGPTL 7) (also referred to herein as an "ANGPTL 7-specific iRNA"). In some embodiments, the ANGPTL7 mRNA transcript is a human ANGPTL7 mRNA transcript, e.g., SEQ ID NO:3 herein. In some embodiments, the ANGPTL7 mRNA transcript is a mouse ANGPTL7 mRNA transcript, e.g., SEQ ID No. 1 herein.

In some embodiments, an iRNA (e.g., dsRNA) described herein comprises a region antisense strand having a region that is substantially complementary to a region of human ANGPTL7 mRNA. In some embodiments, the human ANGPTL7 mRNA has the sequence NM-021146.4 (SEQ ID NO: 3). The sequence of NM_021146.4 is incorporated herein by reference in its entirety. The reverse complement of SEQ ID NO. 3 is provided herein as SEQ ID NO. 4.

In some embodiments, the ANGPTL7mRNA transcript is a mouse ANGPTL7mRNA transcript, e.g., SEQ ID No. 1 herein.

In some embodiments, an iRNA (e.g., dsRNA) described herein comprises an antisense strand having a region that is substantially complementary to a region of mouse ANGPTL7 mRNA. In some embodiments, the mouse ANGPTL7 mRNA has the sequence NM-001039554.3 (SEQ ID NO: 1). The sequence of NM_001039554.3 is incorporated herein by reference in its entirety. The reverse complement of SEQ ID NO. 1 is provided herein as SEQ ID NO. 2.

In some embodiments, the iRNA is substantially complementary to a region of mouse ANGPTL7mRNA that cross-reacts with human ANGPTL7 mRNA. In some embodiments, the iRNA is substantially complementary to a region of mouse ANGPTL7mRNA that is cross-reactive with monkey and rat ANGPTL7 mRNA.

In some aspects, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ANGPTL7, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides having 0,1, 2, or 3 mismatches with a portion of the nucleotide sequence of SEQ ID NO:2 or 4, such that the sense strand is complementary to at least 15 contiguous nucleotides in the antisense strand.

In some aspects, the disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ANGPTL7, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides having 0, 1,2, or 3 mismatches with a portion of the nucleotide sequence of SEQ ID NO:2 or 4, such that the sense strand is complementary to at least 15 contiguous nucleotides in the antisense strand.

In some aspects, the disclosure provides a human cell or tissue having a reduced level of ANGPTL7 mRNA or ANGPTL7 protein compared to an otherwise similar untreated cell or tissue, wherein optionally the cell or tissue is not genetically engineered (e.g., the cell or tissue comprises one or more naturally occurring mutations, e.g., ANGPTL 7), wherein optionally the level is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the human cells or tissue are optic nerve cells, trabecular meshwork cells, schlemm's canal cells (e.g., including endothelial cells), proximal tubular tissue cells, ciliary muscle cells, retinal cells, astrocytes, pericytes, muller cells, ganglion cells (e.g., including retinal ganglion cells), endothelial cells, photoreceptor cells, retinal blood vessels (e.g., including endothelial cells and vascular smooth muscle cells), suprascleral vein or choroidal tissue, e.g., choroidal blood vessels.

In some aspects, the disclosure also provides a cell comprising a dsRNA agent described herein.

In another aspect, provided herein are human eye cells (e.g., optic nerve cells, trabecular meshwork cells), schlemm's canal cells (e.g., including endothelial cells), proximal tubular tissue cells, ciliary muscle cells, retinal cells, astrocytes, pericytes, muller cells, ganglion cells (e.g., including retinal ganglion cells), endothelial cells, photoreceptor cells, retinal blood vessels (e.g., including endothelial cells and vascular smooth muscle cells), suprascleral veins, or choroidal tissues, e.g., choroidal blood vessels, that contain reduced levels of ANGPTL7 mRNA or ANGPTL7 protein as compared to other similar untreated cells. In some embodiments, the level is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

In some aspects, the disclosure also provides a pharmaceutical composition for inhibiting expression of a gene encoding ANGPTL7 comprising a dsRNA agent described herein.

In some aspects, the disclosure also provides a method of inhibiting ANGPTL7 expression in a cell, the method comprising:

(a) Contacting the cell with a dsRNA agent described herein or a pharmaceutical composition described herein; and

(B) Maintaining the cell produced in step (a) for a time sufficient to obtain degradation of mRNA transcripts of ANGPTL7, thereby inhibiting ANGPTL7 expression in the cell.

In some aspects, the disclosure also provides a method of inhibiting ANGPTL7 expression in a cell, the method comprising:

(a) Contacting the cell with a dsRNA agent described herein or a pharmaceutical composition described herein; and

(B) Maintaining the cells produced in step (a) for a time sufficient to reduce the level of ANGPTL7mRNA, ANGPTL7 protein, or both ANGPTL7mRNA and protein, thereby inhibiting expression of ANGPTL7 in the cells.

In some aspects, the present disclosure also provides a method of inhibiting ANGPTL7 expression in an eye cell or tissue, the method comprising:

(a) Contacting the cell or tissue with a dsRNA agent that binds ANGPTL 7; and

(B) Maintaining the cells or tissues produced in step (a) for a time sufficient to reduce the level of ANGPTL7mRNA, ANGPTL7 protein, or both ANGPTL7mRNA and protein, thereby inhibiting expression of ANGPTL7 in the cells or tissues.

In some aspects, the disclosure also provides a method of treating a subject diagnosed with an ANGPTL 7-related disorder (e.g., glaucoma) comprising administering to the subject a therapeutically effective amount of a dsRNA agent described herein or a pharmaceutical composition described herein, thereby treating the disorder.

In any aspect herein, e.g., in the compositions and methods described above, any embodiment herein (e.g., below) may be applied.

In some embodiments, the coding strand of mouse ANGPTL7 has the sequence of SEQ ID No. 1. In some embodiments, the non-coding strand of mouse ANGPTL7 has the sequence of SEQ ID No. 2. In some embodiments, the coding strand of human ANGPTL7 has the sequence of SEQ ID No. 3. In some embodiments, the non-coding strand of human ANGPTL7 has the sequence of SEQ ID No. 4.

In some embodiments, the dsRNA agent comprises a sense strand comprising at least 15 contiguous nucleotides differing by NO more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID nos. 1 or 3 and an antisense strand comprising at least 15 contiguous nucleotides differing by NO more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID nos. 2 or 4.

In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0 or 1, 2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 1 or SEQ ID NO. 3. In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides having 0 or 1, 2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 2 or SEQ ID NO. 4.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 consecutive nucleotides having 0,1, 2, or 3 mismatches with the nucleotide portion of SEQ ID No. 2 or SEQ ID No. 4 such that the sense strand is complementary to at least 17 consecutive nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising 17 consecutive nucleotides having 0 or 1, 2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 1 or SEQ ID NO. 3.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides having 0,1, 2, or 3 mismatches with the nucleotide portion of SEQ ID No. 2 or SEQ ID No. 4 such that the sense strand is complementary to at least 19 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising 19 consecutive nucleotides having 0 or 1, 2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 1 or SEQ ID NO. 3.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides having 0,1, 2, or 3 mismatches with the nucleotide portion of SEQ ID No. 2 or SEQ ID No. 4 such that the sense strand is complementary to at least 21 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising 21 consecutive nucleotides having 0 or 1, 2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 1 or SEQ ID NO. 3.

In some embodiments, the portion of the sense strand is a portion within the sense strand in any of tables 2-7.

In some embodiments, the portion of the antisense strand is a portion within the antisense strand in any of tables 2-7.

In some embodiments, a dsRNA agent for inhibiting expression of ANGPTL7 comprises a sense strand and an antisense strand forming a duplex region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1, 2, or 3 mismatches with one of the antisense sequences listed in any of tables 2-7, and wherein the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1, 2, or 3 mismatches with a sense sequence listed in any of tables 2-7 corresponding to the antisense sequence.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2, or 3 mismatches with one of the antisense sequences set forth in any of tables 2-7. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the sense sequence listed in any one of tables 2-7 corresponding to the antisense sequence.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 17 consecutive nucleotides with 0, 1, 2, or 3 mismatches with one of the antisense sequences set forth in any of tables 2-7. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 17 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the sense sequence listed in any one of tables 2-7 corresponding to the antisense sequence.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 19 consecutive nucleotides having 0, 1, 2, or 3 mismatches with one of the antisense sequences set forth in any of tables 2-7. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 19 consecutive nucleotides having 0, 1, 2, or 3 mismatches with a sense sequence listed in any one of tables 2-7 corresponding to the antisense sequence.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 21 consecutive nucleotides with 0, 1, 2, or 3 mismatches with one of the antisense sequences set forth in any of tables 2-7. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 21 consecutive nucleotides with 0, 1, 2, or 3 mismatches with the sense sequence listed in any one of tables 2-7 corresponding to the antisense sequence.

In some embodiments, the sense strand of the dsRNA agent is at least 23 nucleotides in length, e.g., 23-30 nucleotides in length.

In some embodiments, at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties. In some embodiments, the lipophilic moiety is conjugated to one or more internal positions on at least one strand of the RNA agent. In some embodiments, the lipophilic moiety is conjugated through a linker or carrier. In some embodiments, the lipophilic portion has a lipophilicity of greater than 0 as measured by logKow. In some embodiments, the double stranded RNAi agent has a hydrophobicity of greater than 0.2 as measured by unbound portions of the plasma protein binding assay of the double stranded RNAi agent. In some embodiments, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin.

In various embodiments of the foregoing dsRNA agents, the dsRNA agent targets a hot spot region of an mRNA encoding ANGPTL7, such as a mouse mRNA encoding ANGPTL7 or a human mRNA encoding ANGPTL 7. In one embodiment, the hot spot region comprises nucleotides 1562-1584, 546-568, 709-731, 862-884 and/or 232-256 of SEQ ID NO. 1. In another embodiment, the hot spot region comprises nucleotide 1993-2146、1910-1932、1726-1823、1628-1685、1591-1613、1551-1573、1420-1442、1380-1402、1243-1265、1195-1217、1096-1118、940-962 and/or 299-321 of SEQ ID NO. 3. The dsRNA agent may be selected from ：AD-1094991、AD-1093984、AD-1094129、AD-1094262、AD-1093670、AD-1093672、AD-1565389、AD-1565368、AD-1565357、AD-1565345、AD-1565324、AD-1565303、AD-1565288、AD-1565212、AD-1565141、AD-1565126、AD-1565113、AD-1565091、AD-1565034、AD-1565015、AD-1565004、AD-1564969、AD-1094381、AD-1564428、AD-1564936、AD-1564823、AD-1564802、AD-1564666、AD-1564618 and AD-1563396 below.

In another aspect, the invention provides dsRNA agents that target an angiopoietin-like 7 (ANGPTL 7) mRNA hotspot region.

In some embodiments, the dsRNA agent comprises at least one modified nucleotide. In some embodiments, no more than five of the sense strand nucleotides and no more than five of the antisense strand nucleotides are unmodified nucleotides. In some embodiments, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

In some embodiments, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 3' -terminal deoxythymine (dT) nucleotides, 2' -O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxymodified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic nucleotides, 2' -amino modified nucleotides, 2' -O-allyl modified nucleotides, 2' -C-alkyl modified nucleotides, 2' -C-methoxyethyl modified nucleotides, 2' -O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising unnatural bases, tetrahydrofuran modified nucleotides, 1, 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5' -phosphate esters, nucleotides comprising 5' -phosphate ester mimetics, diol modified nucleotides and 2-O- (N-methylacetamide) modified nucleotides, and combinations thereof. In some embodiments, no more than five of the sense strand nucleotides and no more than five of the antisense strand nucleotides comprise modifications other than: 2' -O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxy modified nucleotides, non-locked nucleic acids (UNA) or diol nucleic acids (GNA).

In some embodiments, the dsRNA comprises a non-nucleotide spacer between two consecutive nucleotides of the sense strand or between two consecutive nucleotides of the antisense strand (wherein optionally the non-nucleotide spacer comprises a C3-C6 alkyl group).

In some embodiments, each strand is no more than 30 nucleotides in length. In some embodiments, the sense strand, the antisense strand, or both the sense and antisense strands comprise a 3' overhang of at least 1 nucleotide. In some embodiments, the sense strand, the antisense strand, or both the sense and antisense strands comprise a 3' overhang of at least 2 nucleotides. In some embodiments, the sense strand, the antisense strand, or both the sense and antisense strands comprise a 2 nucleotide 3' overhang.

In some embodiments, the double stranded region is 15-30 nucleotides in length. In some embodiments, the double stranded region is 17-23 nucleotides in length. In some embodiments, the double stranded region is 17-25 nucleotides in length. In some embodiments, the double stranded region is 23-27 nucleotides in length. In some embodiments, the double stranded region is 19-21 nucleotides in length. In some embodiments, the double stranded region is 21-23 nucleotides in length. In some embodiments, each strand has 19-30 nucleotides. In some embodiments, each strand has 19-23 nucleotides. In some embodiments, each strand has 21-23 nucleotides.

In some embodiments, the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand. In some embodiments, the strand is an antisense strand. In some embodiments, the strand is the sense strand.

In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand. In some embodiments, the strand is an antisense strand. In some embodiments, the strand is the sense strand.

In some embodiments, each of the 5 'and 3' ends of one strand comprises phosphorothioate or methylphosphonate internucleotide linkages. In some embodiments, the strand is an antisense strand.

In some embodiments, the base pair at position 1 at the 5' end of the duplex antisense strand is an AU base pair.

In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions on at least one chain. In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions on at least one chain via a linker or carrier.

In some embodiments, the internal positions include all positions except for the terminal two positions of each terminus of at least one strand. In some embodiments, the internal positions include all but the terminal three positions of each terminus of at least one strand. In some embodiments, the internal position does not include a cleavage site region of the sense strand. In some embodiments, the internal positions include all positions except positions 9-12 from the 5' end of the sense strand. In some embodiments, the internal positions include all positions except positions 11-13 from the 3' end of the sense strand. In some embodiments, the internal position does not include a cleavage site region of the antisense strand. In some embodiments, the internal positions include all positions except positions 12-14 from the 5' end of the antisense strand. In some embodiments, the internal positions include all positions except positions 11-13 from the 3 'end of the sense strand and positions 12-14 from the 5' end of the antisense strand.

In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of: positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counted from the 5' end of each strand. In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of: positions 5, 6, 7, 15 and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counted from the 5' end of each strand.

In some embodiments, the position in the double-stranded region does not include a cleavage site for the sense strand.

In some embodiments, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand, or position 16 of the antisense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1 or position 7 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, position 20, or position 15 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 16 of the antisense strand. In some embodiments, the lipophilic moiety is conjugated to position 6 of the sense strand, counting from the 5' end.

In some embodiments, the lipophilic moiety is an aliphatic, alicyclic, or multi-alicyclic compound. In some embodiments, the lipophilic moiety is selected from the following: lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxyhexanethiol, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) bile acid, dimethoxytrityl or phenoxazine. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and optionally a functional group selected from the group consisting of: hydroxyl, amine, carboxylic acid, sulfonate, phosphate, sulfhydryl, azide, and alkyne. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

In some embodiments, the lipophilic moiety is conjugated through a carrier that replaces one or more nucleotides within the internal position or double-stranded region. In some embodiments, the carrier is a cyclic group selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, tetrahydrothiazolyl, isothiazolyl, quinoxalinyl, pyridazinyl, tetrahydrofuran and decalin; or an acyclic moiety based on a serine backbone or a diethanolamine backbone.

In some embodiments, the lipophilic moiety is conjugated to the double stranded iRNA agent by a linker comprising: ethers, thioethers, ureas, carbonates, amines, amides, maleimide-thioethers, disulfides, phosphodiesters, sulfonamide linkages, click reaction products, or carbamates.

In some embodiments, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

In some embodiments, the lipophilic moiety or targeting ligand is conjugated through a bio-cleavable linker selected from the group consisting of: DNA, RNA, disulfides, amides, functionalized mono-or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

In some embodiments, the 3' end of the sense strand is protected by an end cap, which is a cyclic group with an amine, selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, tetrahydrothiazolyl, isothiazolyl, quinoxalinyl, pyridazinyl, tetrahydrofuran and decalin.

In some embodiments, the dsRNA agent further comprises a targeting ligand. In some embodiments, the targeting ligand targets ocular tissue. In some embodiments, the ocular tissue is the optic nerve, trabecular meshwork, proximal tubular tissue, ganglion (e.g., including retinal ganglion), suprascleral vein, or schlemm's canal (e.g., including endothelial cells).

In some embodiments, the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3 'end or the 5' end of the sense strand. In some embodiments, the ligand is conjugated to the 3' end of the sense strand.

In some embodiments, the ligand comprises N-acetylgalactosamine (GalNAc). In some embodiments, the targeting ligand comprises one or more GalNAc conjugates or one or more GalNAc derivatives. In some embodiments, the ligand is one or more GalNAc conjugates or one or more GalNAc derivatives attached by a monovalent linker or a divalent, trivalent, or tetravalent branching linker. In some embodiments, the ligand is

In some embodiments, the dsRNA agent is conjugated to the ligand, as shown in the following schematic

Wherein X is O or S. In some embodiments, X is O.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification at a first internucleotide linkage present at the 3' terminus of the antisense strand having a linking phosphorus atom of Sp configuration, a terminal, chiral modification at a first internucleotide linkage present at the 5' terminus of the antisense strand having a linking phosphorus atom of Rp configuration, and a terminal, chiral modification at a first internucleotide linkage present at the 5' terminus of the sense strand having a linking phosphorus atom of Rp configuration or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification at the first and second internucleotide linkages present at the 3' terminus of the antisense strand having a linking phosphorus atom of Sp configuration, a terminal, chiral modification at the first internucleotide linkage present at the 5' terminus of the antisense strand having a linking phosphorus atom of Rp configuration, and a terminal, chiral modification at the first internucleotide linkage present at the 5' terminus of the sense strand having a linking phosphorus atom of Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification at the first, second, and third internucleotide linkages present at the 3' terminus of the antisense strand having a connecting phosphorus atom of Sp configuration, a terminal, chiral modification at the first internucleotide linkage present at the 5' terminus of the antisense strand having a connecting phosphorus atom of Rp configuration, and a terminal, chiral modification at the first internucleotide linkage present at the 5' terminus of the sense strand having a connecting phosphorus atom of Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification at the first and second internucleotide linkages present at the 3 'end of the antisense strand having a linking phosphorus atom of Sp configuration, a terminal, chiral modification at the third internucleotide linkage present at the 3' end of the antisense strand having a linking phosphorus atom of Rp configuration, a terminal, chiral modification at the first internucleotide linkage present at the 5 'end of the antisense strand having a linking phosphorus atom of Rp configuration, and a terminal, chiral modification at the first internucleotide linkage present at the 5' end of the sense strand having a linking phosphorus atom of Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification at the first and second internucleotide linkages present at the 3' terminus of the antisense strand having a connecting phosphorus atom of Sp configuration, a terminal, chiral modification at the first and second internucleotide linkages present at the 5' terminus of the antisense strand having a connecting phosphorus atom of Rp configuration, and a terminal, chiral modification at the first internucleotide linkage present at the 5' terminus of the sense strand having a connecting phosphorus atom of Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a phosphate or phosphate mimic at the 5' end of the antisense strand. In some embodiments, the phosphate ester mimic is 5' -Vinyl Phosphonate (VP).

In some embodiments, a cell described herein (e.g., a human cell) is produced by a method comprising contacting the human cell with a dsRNA agent described herein.

In some embodiments, the pharmaceutical compositions described herein comprise a dsRNA agent and a lipid agent.

In some embodiments (e.g., embodiments of the methods described herein), the cell is within a subject. In some embodiments, the subject is a human. In some embodiments, the level of ANGPTL7 mRNA is inhibited by at least 50%. In some embodiments, the level of ANGPTL7 protein is inhibited by at least 50%. In some embodiments, expression of ANGPTL7 is inhibited by at least 50%. In some embodiments, inhibition of expression of ANGPTL7 reduces ANGPTL7 protein levels by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in a biological sample (e.g., an optic nerve sample) from a subject. In some embodiments, inhibition of expression of ANGPTL7 reduces ANGPTL7 mRNA levels in a biological sample (e.g., an optic nerve sample) from a subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

In some embodiments, the subject has been diagnosed with an ANGPTL 7-related disorder. In some embodiments, the subject meets at least one diagnostic criteria for an ANGPTL7 related disorder. In some embodiments, the ANGPTL 7-related disorder is glaucoma or a condition related to glaucoma. In some embodiments, the glaucoma is primary open angle glaucoma.

In some embodiments, the ocular cells or tissue are optic nerve cells, trabecular meshwork cells, schlemm's canal cells (e.g., including endothelial cells), proximal tubular tissue cells, ciliary muscle cells, retinal cells, astrocytes, pericytes, muller cells, ganglion cells (e.g., including retinal ganglion cells), endothelial cells, photoreceptor cells, retinal blood vessels (e.g., including endothelial cells and vascular smooth muscle cells), suprascleral vein or choroidal tissue, e.g., choroidal blood vessels.

In some embodiments, the treatment comprises ameliorating at least one sign or symptom of the disorder. In some embodiments, the at least one sign or symptom comprises a measurement of one or more of intraocular pressure, vision loss, optic nerve injury, ocular inflammation, presence, level, or activity of vision or ANGPTL7 (e.g., ANGPTL7 gene, ANGPTL7mRNA, or ANGPTT protein).

In some embodiments, an ANGPTL7 level above a reference level indicates that the subject has glaucoma or a glaucoma-related condition.

In some embodiments, treating comprises preventing progression of the disorder. In some embodiments, the treatment comprises one or more of the following: (a) inhibiting or reducing intraocular pressure; (b) inhibiting or reducing expression or activity of ANGPTL 7; (c) increasing drainage of aqueous humor; (d) inhibiting or reducing optic nerve damage; or (e) inhibit or reduce retinal ganglion cell death.

In some embodiments, the treatment results in an average decrease of ANGPTL7 mRNA from baseline in the cell or tissue of at least 30%. In some embodiments, the treatment results in an average decrease of ANGPTL7 mRNA from baseline in the cell or tissue of at least 60%. In some embodiments, the treatment results in an average decrease of ANGPTL7 mRNA from baseline in the cell or tissue of at least 90%.

In some embodiments, following treatment, the subject experiences a knockdown duration of at least 8 weeks following a single dose of dsRNA, as assessed by, for example, ANGPTL7 protein in the optic nerve. In some embodiments, the treatment results in a knockdown duration of at least 12 weeks after a single dose of dsRNA, as assessed by, for example, ANGPTL7 protein in the optic nerve. In some embodiments, the treatment results in a knockdown duration of at least 16 weeks after a single dose of dsRNA, as assessed by, for example, ANGPTL7 protein in the optic nerve.

In some embodiments, the subject is a human.

In some embodiments, the dsRNA agent is administered at a dose of about 0.01mg/kg to about 50 mg/kg.

In some embodiments, the dsRNA agent is administered to the subject by intraocular. In some embodiments, the intraocular administration comprises intravitreal administration (e.g., intravitreal injection), transscleral administration (e.g., transscleral injection), subconjunctival administration (e.g., subconjunctival injection), postglobal administration (e.g., postglobus injection), intracameral administration (e.g., intracameral injection), or subretinal administration (e.g., subretinal injection).

In some embodiments, the dsRNA agent is administered to the subject intravenously. In some embodiments, the dsRNA agent is administered to the subject by topical administration.

In some embodiments, the methods described herein further comprise measuring the level of ANGPTL7 (e.g., an ANGPTL7 gene, an ANGPTL7 mRNA, or an ANGPTL7 protein) in the subject. In some embodiments, measuring the level of ANGPTL7 in the subject comprises measuring the level of ANGPTL7 protein in a biological sample (e.g., an optic nerve sample) from the subject. In some embodiments, the methods described herein further comprise performing a blood test, an imaging test, an intraocular pressure measurement test, or an optical nerve biopsy.

In some embodiments, the methods described herein for further measuring the level of ANGPTL7 (e.g., ANGPTL7 gene, ANGPTL7mRNA, or ANGPTL7 protein) in a subject are performed prior to treatment with a dsRNA agent or pharmaceutical composition. In some embodiments, the dsRNA agent or pharmaceutical composition is administered to the subject when the subject is determined to have an ANGPTL7 level greater than a reference level. In some embodiments, the measurement of ANGPTL7 levels in the subject is performed after treatment with the dsRNA agent or pharmaceutical composition.

In some embodiments, the methods described herein further comprise treating the subject with a therapy suitable for treating or preventing an ANGPTL 7-related disorder (e.g., glaucoma), wherein the therapy comprises a drug that reduces intraocular pressure, laser treatment, surgery, or trabeculectomy. In some embodiments, the methods described herein further comprise administering to the subject an additional agent suitable for treating or preventing an ANGPTL 7-related disorder. In some embodiments, the additional agent comprises a prostaglandin analog, a beta blocker, an alpha adrenergic agonist, a carbonic anhydrase inhibitor, a ROCK iRNA agent, an inhibitor of Rho GTPase, an anti-Rho GTPase agent, or an anti-ANGPTL 7 agent.

In some embodiments, the anti-Rho GTPase agent comprises an anti-Rho GTPase antibody or antigen binding fragment thereof.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

The details of various embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several features of the disclosure.

FIG. 1 shows dexamethasone-21-acetate (DEX-Ac) induced inhibition of high intraocular pressure in an ANGPTL7 Knockout (KO) mouse relative to a Wild Type (WT) mouse.

Figure 2 depicts the effect of ANGPTL7 siRNA on intraocular pressure (IOP) in wild type mice. From week 2 to the end of the study, intravitreal injection of 15 μg ANGPTL7 siRNA significantly reduced IOP compared to PBS treated group (n=6) and natural group (no injection, n=5) for two of the six tested sirnas (n=6-8/group). siRNAs #3 and #5 represent AD-1094129 and AD-1094991, respectively. Error bars represent Standard Error of Mean (SEM).

FIG. 3 depicts the effect of ANGPTL7 siRNA on ANGPTL7 expression in the limbal ring of wild-type mice in vivo. qPCR results from microdissection limbal rings showed that siRNA #3 and #5 knockdown levels of ANGPTL7mRNA were highest (> 50%) compared to PBS treated or naive (non-injected) mice, consistent with the IOP reduction observed in mice injected with one of the two sirnas (as shown in fig. 2). Error bars represent SEM.

FIG. 4 depicts the effect of ANGPTL7 siRNA in reducing dexamethasone-21-acetate (DEX-Ac) induced high intraocular pressure in wild type mice.

Detailed Description

IRNA directs sequence-specific degradation of mRNA through a process called RNA interference (RNAi). Described herein are iRNA for modulating (e.g., inhibiting) ANGPTL7 expression and methods of using the same. Also provided are compositions and methods for treating disorders associated with ANGPTL7 expression, such as glaucoma or conditions associated with glaucoma.

Human ANGPTL7, also known as angiopoietin-like 7, ANGPTL7, angiopoietin-related protein 7, angiopoietin-like protein 7, angX, CDT6, cornea-derived transcript 6 protein, angiopoietin-like factor (CDT 6), or dj647m16.1, is a protein encoded by an ANGPTL7 gene. ANGPTL7 is typically expressed in a variety of tissues, including the optic nerve, trabecular meshwork, schlemm's canal (e.g., including endothelial cells), perivascular tissue, ciliary muscle, retina, astrocytes, pericytes, muller cells, ganglion cells (e.g., including retinal ganglion cells), endothelial cells, photoreceptor cells, retinal blood vessels (e.g., including endothelial cells and vascular smooth muscle cells), episcleral veins, or choroidal tissue, e.g., choroidal blood vessels.

Without wishing to be bound by theory, ANGPTL7 may exacerbate the pathogenesis of glaucoma, e.g., by increasing intraocular pressure. ANGPTL7 protein levels were reported to be elevated in aqueous humor in glaucoma patients compared to control patients. Glaucoma stimulation induces secretion of ANGPTL7 protein in primary human trabecular meshwork cells and keratoscleral explants. Overexpression of ANGPTL7 in immortalized human trabecular meshwork cells increases the expression of type I collagen, a potential mechanism for glaucoma progression (Kuchtey et al, 2008Invest.Ophthalmol.Vis Sci.49:3438). Overexpression of ANGPTL7 in primary human trabecular meshwork cells alters the expression of extracellular matrix proteins, including type I, type IV and type V collagens, fibronectin, myofibrins, proteoglycans and MMP1. Silencing of ANGPTL7 during glucocorticoid injury affects the expression of other steroid response proteins (Comes et al, 2011Genes to Cells 16:243-259). Human genomic analysis showed that missense and nonsense variants of ANGPTL7, including p.gln175his and p.arg220cys, were associated with lower intraocular pressure and lower risk of glaucoma (Tanigawa is).

The following description discloses how to make and use iRNA-containing compositions to inhibit the expression of ANGPTL7, as well as compositions and methods for treating conditions associated with increased expression of ANGPTL 7.

In some aspects, provided herein are pharmaceutical compositions comprising ANGPTL7 iRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of ANGPTL7, and methods of using the pharmaceutical compositions to treat disorders associated with expression of ANGPTL7 (e.g., glaucoma or disorders associated with glaucoma).

I. Definition of the definition

For convenience, the following meanings of certain terms and phrases used in this specification, examples, and appended claims are provided. If there is a significant difference between the use of terms in other parts of this specification and their definitions provided in this part, the definitions in this part shall control.

When referring to a number or range of values, the term "about" means that the number or range of values referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or range of values may vary, for example, between 1% and 15% of the number or range of values.

The terms "or more" and "at least" preceding a number or series of numbers should be understood to include the number adjacent to the term "at least" as well as all subsequent numbers or integers that may be logically included, as will be clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 17 nucleotides in a 20 nucleotide nucleic acid molecule" means that 17, 18, 19 or 20 nucleotides have the indicated properties. When "at least" occurs before a series of numbers or ranges, it is to be understood that "at least" can modify each of the numbers in the series or ranges.

As used herein, "no more than" or "less than" is understood to include values adjacent to the phrase and logically lower values or integers, from logical to zero in the context. For example, a duplex with a mismatch to a target site of "no more than 2 nucleotides" has 2, 1, or 0 mismatches. When "no more than" occurs before a series of numbers or ranges, it is to be understood that "no more than" can modify each of the numbers in the series or ranges.

As used herein, "up to" as in "up to 10" is understood to mean and include up to 10, i.e., 0, 1,2, 3, 4, 5, 6,7, 8, 9, or 10.

The ranges provided herein are to be understood to encompass all individual integer values and all subranges within the range.

The terms "activate", "enhance", "up-regulate" expression "," increase "expression of the ANGPTL7 gene, etc., as long as it refers to at least partial activation of ANGPTL7 gene expression, as expressed herein as an increase in the amount of ANGPTL7 mRNA that can be isolated or detected from a first cell or group of cells that transcribes the ANGPTL7 gene and that has been or has been treated such that expression of the ANGPTL7 gene is increased as compared to a second cell or group of cells that is substantially the same as the first cell or group of cells but that has been so treated or has not been so treated (control cells).

In some embodiments, expression of an ANGPTL7 gene is activated by administration of an iRNA as described herein by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%. In some embodiments, the ANGPTL7 gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA as set forth in the present disclosure. In some embodiments, expression of an ANGPTL7 gene is activated by administration of an iRNA as described herein by at least about 85%, 90%, or 95% or more. In some embodiments, ANGPTL7 gene expression in a cell treated with an iRNA described herein is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more as compared to expression in an untreated cell. Activation of expression by small dsrnas is described, for example, in the following: li et al, 2006Proc. Natl. Acad. Sci. U.S. A.103:17337-42 and US 2007/011963 and US2005/226848, each of which is incorporated herein by reference.

The terms "silence," "inhibit expression (inhibit expression of)", "down-regulate expression," "inhibit expression (suppress expression of)", and the like, with respect to ANGPTL7 to which it refers, refer herein to at least partial inhibition of ANGPTL7 expression, as assessed, for example, based on ANGPTL7 mRNA expression, ANGPTL7 protein expression, or another parameter related to the function of ANGPTL7 expression. For example, inhibition of ANGPTL7 expression may be manifested by a decrease in the amount of ANGPTL7 mRNA compared to a control, which ANGPTL7 mRNA may be isolated or detected from a first cell or group of cells that transcribe ANGPTL7, and which have been or have been treated such that expression of ANGPTL7 is inhibited. The control can be a second cell or group of cells that is substantially identical to the first cell or group of cells, except that the second cell or group of cells is not so treated (control cells). The extent of inhibition is typically expressed as a percentage of the control level, e.g.,

Or the extent of inhibition may be given by a decrease in a parameter associated with the function of ANGPTL7 expression, e.g., the amount of protein encoded by the ANGPTL7 gene. The decrease in parameters associated with ANGPTL7 expression function may be similarly expressed as a percentage of control levels. In principle, ANGPTL7 silencing may be determined in any ANGPTL 7-expressing cell, whether constitutive or by genome engineering, and by any suitable assay.

For example, in certain instances, expression of ANGPTL7 is inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA disclosed herein. In some embodiments, ANGPTL7 is inhibited by at least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA disclosed herein. In some embodiments, MYOCs are inhibited by at least about 85%, 90%, 95%, 98%, 99% or more by administration of an iRNA described herein.

The term "antisense strand" or "guide strand" refers to a strand of an iRNA, e.g., a dsRNA, that comprises a region that is substantially complementary to a target sequence.

As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence) as defined herein. In the case where the complementary region is not perfectly complementary to the target sequence, the mismatch may be located in an internal or terminal region of the molecule. In some embodiments, the complementary region comprises 0,1, or 2 mismatches.

As used herein, the term "sense strand" or "follower strand" refers to an iRNA strand comprising a region that is substantially complementary to a region of an antisense strand of a term as defined herein.

As used herein, the term "blunt end" or "blunt end" with respect to a dsRNA refers to the absence of unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhangs, at a given end of the dsRNA. One or both ends of the dsRNA may be blunt ended. When both ends of a dsRNA are blunt, the dsRNA is referred to as "blunt-ended". For clarity, "blunt-ended" dsRNA is a dsRNA that is blunt-ended at both ends, i.e., no nucleotide overhangs at either end of the molecule. The most common such molecules will be double stranded throughout their length.

As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence relative to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize under certain conditions to an oligonucleotide or polynucleotide comprising the second nucleotide sequence and form a duplex structure, as will be understood by the skilled artisan. For example, such conditions may be stringent conditions, where stringent conditions may include the following: 400mM NaCl,40mM PIPES,pH 6.4,1mM EDTA,50 ℃ or 70 ℃ for 12 to 16 hours, and then washing. Other conditions may be applied, such as physiologically relevant conditions that may be encountered inside an organism. The skilled person will be able to determine the set of conditions most suitable for the complementarity test of the two sequences depending on the end use of the hybridizing nucleotides.

Complementary sequences within an iRNA (e.g., within a dsRNA as described herein) comprise base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but typically no more than 5, 4, 3 or 2 mismatched base pairs after hybridization for up to 30 base pairs of the duplex, while retaining the ability to hybridize under conditions most relevant to its end use, such as inhibition of gene expression by the RISC pathway. However, where two oligonucleotides are designed to form one or more single stranded overhangs upon hybridization, such overhangs should not be considered as a defined mismatch with respect to complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for purposes described herein.

As used herein, a "complementary" sequence may also comprise or be formed entirely of non-Watson-Crick base pairs (non-Watson-Crick base pairs) and/or base pairs formed from non-natural and modified nucleotides, so long as the above requirements regarding its hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G: U wobble base pairing or Holstein base pairing (Hoogstein base pairing).

The terms "complementary," "fully complementary," and "substantially complementary" herein may be used with respect to base matching between the sense strand and the antisense strand of a dsRNA or between the antisense strand of an iRNA agent and a target sequence, as understood in the context of its use.

As used herein, a polynucleotide that is "at least partially substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding an ANGPTL7 protein). For example, if the sequence is substantially complementary to an uninterrupted portion of an mRNA encoding ANGPTL7, the polynucleotide is complementary to at least a portion of the ANGPTL7 mRNA. The term "complementary" refers to the ability to pair between nucleobases of a first nucleic acid and a second nucleic acid.

As used herein, the term "complementary region" refers to a region of one nucleotide sequence agent that is substantially complementary to another sequence, e.g., a region of the sense sequence and corresponding antisense sequence of a dsRNA, or the antisense strand and target sequence of an iRNA, e.g., an ANGPTL7 nucleotide sequence, as defined herein. In cases where the complementary region is not perfectly complementary to the target sequence, the mismatch may be located in an internal or terminal region of the antisense strand of the iRNA. Typically, the most tolerable mismatches are in the terminal region, e.g., within 5, 4, 3, or 2 nucleotides of the 5 'or 3' end of the iRNA agent.

As used herein, "contacting" includes directly contacting a cell and indirectly contacting a cell. For example, when a composition comprising iRNA is administered (e.g., intraocular, topical, or intravenous) to a subject, cells in the subject can be contacted.

When referring to iRNA, "introduced into a cell" means promoting or affecting uptake or uptake into the cell. The uptake or uptake of iRNA can occur by unassisted diffusion or active cellular processes, or by adjuvants or devices. The meaning of this term is not limited to cells in vitro; iRNA may also be "introduced into a cell," where the cell is part of a living organism. In this case, the introduction into the cell will comprise delivery to the organism. For example, for in vivo delivery, the iRNA may be injected into a tissue site or administered systemically. In vivo delivery may also be performed by beta-glucan delivery systems, such as those described in U.S. patent nos. 5,032,401 and 5,607,677 and U.S. publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into cells comprises methods known in the art, such as electroporation and lipofection. Additional methods are described below or are known in the art. As used herein, "disorder related to ANGPTL7 expression," "disease related to ANGPTL7 expression," "pathological process related to ANGPTL7 expression," "disorder related to ANGPTL7," "ANGPTL 7-related disease," and the like include any condition, disorder, or disease in which ANGPTL7 expression is altered (e.g., decreased or increased in a level signature relative to a reference level, e.g., a non-diseased subject). In some embodiments, ANGPTL7 expression is reduced. In some embodiments, ANGPTL7 expression is increased. In some embodiments, a decrease or increase in ANGPTL7 expression is detectable in a tissue sample from the subject (e.g., in an optic nerve sample). The decrease or increase may be assessed relative to the level observed in the same individual prior to the development of the disorder or relative to other individuals not suffering from the disorder. The decrease or increase may be limited to a particular organ, tissue or region of the body (e.g., the eye). ANGPTL 7-related disorders include, but are not limited to, glaucoma or conditions related to glaucoma.

As used herein, the term "glaucoma-associated disorder" means any disease or condition associated with elevated intraocular pressure. Non-limiting examples of glaucoma-related conditions that can be treated using the methods provided herein include ocular inflammation, systemic inflammation, anterior uveitis, acute retinal necrosis, sturge-Werber syndrome, axenfeld-Rieger syndrome, ma Fanzeng syndrome, homocystinuria, weill-MARCHESANI syndrome, and autoimmune diseases, such as juvenile rheumatoid arthritis and Marie Strumpell ankylosing spondylitis.

As used herein, the term "double stranded RNA," "dsRNA," or "siRNA" refers to an iRNA comprising an RNA molecule or molecular complex having a hybridization duplex region comprising two anti-parallel and substantially complementary nucleic acid strands that will be referred to as having "sense" and "antisense" orientations relative to a target RNA. The duplex region may have any length that allows for specific degradation of the desired target RNA, e.g., by RISC pathway, but will typically range in length from 9 to 36 base pairs, e.g., from 15 to 30 base pairs in length. It is contemplated that the duplex can have any length between 9 and 36 base pairs within this range, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36, and any subrange therebetween, including but not limited to 15 to 30 base pairs, 15 to 26 base pairs, 15 to 23 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 18 base pairs, 15 to 17 base pairs, 18 to 30 base pairs, 18 to 26 base pairs, 18 to 23 base pairs, 18 to 22 base pairs, 18 to 21 base pairs, 18 to 20 base pairs, 19 to 30 base pairs, 19 to 26 base pairs, 19 to 23 base pairs, 19 to 22 base pairs, 19 to 21 base pairs, 19 to 20 base pairs, 20 to 30 base pairs, 20 to 26 base pairs, 20 to 25 base pairs, 20 to 24 base pairs, 20 to 23 base pairs, 20 to 22 base pairs, 20 to 21 base pairs, 21 to 30 base pairs, 21 to 26 base pairs, 21 to 25 base pairs, 21 to 24 base pairs, 21 to 23 base pairs, and 21 to 22 base pairs. the dsRNA produced in the cell by treatment with Dicer and similar enzymes is typically in the range of 19 to 22 base pairs in length. One strand of the duplex region of dsDNA comprises a sequence that is substantially complementary to a region of the target RNA. The two strands forming the duplex structure may be from a single RNA molecule having at least one self-complementary region, or may be formed from two or more separate RNA molecules. When a duplex region is formed from two strands of a single molecule, the molecule may have duplex regions separated by a single strand nucleotide chain (referred to herein as a "hairpin loop") between the 3 "end of one strand and the 5" end of the corresponding other strand forming the duplex structure. The hairpin loop may include at least one unpaired nucleotide; in some embodiments, the hairpin loop may include at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides. Where the two substantially complementary strands of the dsRNA comprise separate RNA molecules, those molecules need not be, but can be, covalently linked. In some embodiments, when the two strands are covalently linked by means other than a hairpin loop, and the linking structure is a linker.

In some embodiments, the iRNA agent may be a "single stranded siRNA" that is introduced into a cell or organism to inhibit a target mRNA. In some embodiments, the single stranded RNAi agent can bind to RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single stranded sirnas are typically 15 to 30 nucleotides and are optionally chemically modified. The design and testing of single stranded siRNA is described in U.S. Pat. No. 8,101,348 and Lima et al, 2012cell 150:883-894, the entire contents of each of which are hereby incorporated by reference. Any of the antisense nucleotide sequences described herein (e.g., the sequences provided in tables 2-7) can be used as single stranded siRNA described herein, and optionally as chemically modified, e.g., as described herein, e.g., by the methods described in Lima et al, 2012cell 150:883-894.

In some embodiments, the RNA interference agent comprises single-stranded RNA that interacts with the target RNA sequence to direct cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNAs introduced into cells are known as Dicer type III endonucleases break down into siRNA (Sharp et al 2001Genes Dev.15:485). Dicer, a ribonuclease-III like enzyme, uses a characteristic two base 3' overhang to process dsRNA into 19 to 23 base pair short interfering RNA (Bernstein et al 2001Nature 409:363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), wherein one or more helices cleave the siRNA duplex, thereby enabling the complementary antisense strand to direct target recognition (Nykanen et al, 2001cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al 2001Genes Dev.15:188). Thus, in some embodiments, the disclosure relates to single stranded RNAs that promote the formation of RISC complexes to affect silencing of a target gene.

"G", "C", "A", "T" and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil, respectively, as bases. However, it is understood that the terms "deoxyribonucleotide", "ribonucleotide" or "nucleotide" may also refer to modified nucleotides, as described in further detail below, or alternative substitute portions. It will be apparent to those skilled in the art that guanine, cytosine, adenine and uracil can be substituted with other moieties without substantially altering the base pairing properties of oligonucleotides including nucleotides containing such substituted moieties. For example, but not limited to, a nucleotide that includes inosine as its base may be base paired with a nucleotide containing adenine, cytosine, or uracil. Thus, in the nucleotide sequence of the dsRNA proposed in the present disclosure, the nucleotide containing uracil, guanine or adenine may be substituted with a nucleotide containing, for example, inosine. In another example, adenine and cytosine at any positions in the oligonucleotide may be substituted with guanine and uracil, respectively, to form a G-U wobble base pairing with the target mRNA. Sequences containing such substituted moieties are suitable for use in the compositions and methods set forth in the disclosure.

As used herein, the terms "iRNA," "RNAi," "iRNA agent," or "RNAi agent" or "RNAi molecule" refer to agents that contain RNA as that term is defined herein and mediate targeted cleavage of RNA transcripts, for example, through the RNA-induced silencing complex (RISC) pathway. In some embodiments, an iRNA as described herein affects inhibition of ANGPTL7 expression, e.g., in a cell or mammal. Inhibition of ANGPTL7 expression may be assessed based on a decrease in the level of ANGPTL7 mRNA or a decrease in the level of ANGPTL7 protein.

The term "linker" or "linking group" means an organic moiety that connects two moieties of a compound, e.g., covalently connects two moieties of a compound.

The term "lipophilic" or "lipophilic moiety" refers broadly to any compound or chemical moiety having affinity for lipids. One method for characterizing the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient log K _ow, where K _ow is the ratio of the concentration of chemical in the octanol phase to the concentration in the water phase of a two-phase system in equilibrium. Octanol-water partition coefficient is a laboratory measured substance property. However, it can also be predicted by using coefficients attributed to the structural components of the chemical, which coefficients are calculated using the first principle or empirical method (see, e.g., tetko et al, J.chem. Inf. Comput. Sci.41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measurement that a substance tends to be in a non-aqueous or oily environment rather than water (i.e., its hydrophilicity/lipophilicity balance). In principle, the chemical is lipophilic when log k _ow exceeds 0. Typically, the lipophilic moiety has a log k _ow of more than 1, more than 1.5, more than 2, more than 3, more than 4, more than 5, or more than 10. For example, log K _ow of 6-amino hexanol was predicted to be, for example, about 0.7. Using the same method, the log K _ow of cholesterol N- (hex-6-ol) carbamate was predicted to be 10.7.

The lipophilicity of a molecule may vary depending on the functional group it carries. For example, adding a hydroxyl group or an amine group to the end of the lipophilic moiety can increase or decrease the partition coefficient (e.g., log k _ow) value of the lipophilic moiety.

Alternatively, the hydrophobicity of double stranded RNAi agents conjugated to one or more lipophilic moieties can be measured by their protein binding characteristics. For example, in certain embodiments, unbound fraction in a plasma protein binding assay of a double stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of the double stranded RNAi agent, which can then be positively correlated with the silencing activity of the double stranded RNAi agent.

In some embodiments, the determined plasma protein binding assay is an Electrophoretic Mobility Shift Assay (EMSA) using human serum albumin. Exemplary protocols for this binding assay are described in detail in, for example, PCT/US 2019/031170. Hydrophobicity of double stranded RNAi agent measured by fraction of unbound siRNA in the binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 for enhanced in vivo delivery of siRNA.

Thus, conjugation of the lipophilic moiety to the internal location of the double stranded RNAi agent provides optimal hydrophobicity for enhanced in vivo delivery of siRNA.

The term "lipid nanoparticle" or "LNP" is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g. an RNAi agent or a plasmid from which the RNAi agent is transcribed. LNP is described, for example, in U.S. patent nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated by reference.

As used herein, the term "modulate" expression refers to at least partial "inhibition" or partial "activation" of expression of a gene (e.g., an ANGPTL7 gene) in a cell treated with an iRNA composition described herein, as compared to expression of the corresponding gene in a control cell. Control cells comprise untreated cells or cells treated with non-targeted control iRNA.

The skilled artisan will recognize that the term "RNA molecule" or "ribonucleic acid molecule" encompasses not only RNA molecules expressed or found in nature, but also analogs and derivatives of RNA that include one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or known in the art. Strictly speaking, "ribonucleoside" encompasses nucleobases and ribose, and "ribonucleotides" are ribonucleosides having one, two, or three phosphate moieties or analogs thereof (e.g., phosphorothioates). However, the terms "ribonucleoside" and "ribonucleotide" may be considered equivalent as used herein. The RNA can be modified in the nucleobase structure, in the ribose structure, or in the ribose-phosphate backbone structure, for example, as described in more detail herein below. However, molecules comprising ribonucleoside analogues or derivatives must retain the ability to form a duplex. As non-limiting examples, the RNA molecule may also comprise at least one modified ribonucleoside, including but not limited to a 2 '-O-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or an n-dodecanoate didecarboxamide group, a locked nucleoside, an abasic nucleoside, an acyclic nucleoside, a diol nucleotide, a 2 '-deoxy-2' -fluoro modified nucleoside, a 2 '-amino modified nucleoside, a 2' -alkyl modified nucleoside, a morpholino nucleoside, an phosphoramidate, or a non-natural base comprising a nucleoside, or any combination thereof. Alternatively or in combination, the RNA molecule may comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. For each of such multiple modified ribonucleosides in an RNA molecule, the modification need not be the same. In some embodiments, the modified RNAs contemplated for use in the methods and compositions described herein are Peptide Nucleic Acids (PNAs) that have the ability to form a desired duplex structure and allow or mediate specific degradation of the target RNA, e.g., through RISC pathways. For clarity, it should be understood that the term "iRNA" does not encompass naturally occurring double stranded DNA molecules or 100% deoxynucleoside containing DNA molecules.

In some aspects, the modified ribonucleoside comprises a deoxyribonucleoside. In such examples, the iRNA agent can include one or more deoxynucleosides, including, for example, a deoxynucleoside overhang or one or more deoxynucleosides within the double stranded portion of the dsRNA. In certain embodiments, the RNA molecule comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% deoxyribonucleoside or a higher percentage (but not 100%) deoxyribonucleoside, e.g., in one or both strands.

As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA (e.g., dsRNA). For example, when the 3 'end of one strand of a dsRNA extends beyond the 5' end of the other strand, and vice versa, a nucleotide overhang is present. The dsRNA may include an overhang of at least one nucleotide; alternatively, the overhang may include at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five nucleotides or more. Nucleotide overhangs may include or consist of: nucleotide/nucleoside analogs comprising deoxynucleotides/nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may be present on the 5 'end, 3' end or both ends of the antisense strand or sense strand of the dsRNA.

In some embodiments, the antisense strand of the dsRNA has a1 to 10 nucleotide overhang at the 3 'end and/or the 5' end. In some embodiments, the sense strand of the dsRNA has a1 to 10 nucleotide overhang at the 3 'end and/or the 5' end. In some embodiments, one or more nucleotides in the overhang are replaced with a nucleoside phosphorothioate.

In certain embodiments, the antisense strand of the dsRNA has a 1-15 nucleotide overhang at the 3' end. In other embodiments, one or more nucleotides in the overhang are replaced with a phosphorothioate nucleoside.

As used herein, a "pharmaceutical composition" includes a pharmacologically effective amount of a therapeutic agent (e.g., iRNA) and a pharmaceutically acceptable carrier. As used herein, "pharmacologically effective amount," "therapeutically effective amount," or, in short, "effective amount" refers to an amount of an agent (e.g., iRNA) that is effective to produce a desired pharmacological, therapeutic, or prophylactic result. For example, in a method of treating a disorder associated with ANGPTL7 expression (e.g., glaucoma or a condition associated with glaucoma), an effective amount comprises an amount effective to reduce one or more symptoms associated with the disorder, e.g., an amount effective to: (a) inhibiting or reducing intraocular pressure; (b) inhibiting or reducing expression or activity of ANGPTL 7; (c) increasing drainage of aqueous humor; (d) inhibiting or reducing optic nerve damage; or (e) inhibiting or reducing retinal ganglion cell death, or an amount effective to reduce the risk of developing a condition associated with the disorder. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or disorder is reduced by at least 10%, then a therapeutically effective amount of the drug for treatment of the disease or disorder is that amount necessary to reduce the parameter by at least 10%. For example, a therapeutically effective amount of an iRNA that targets ANGPTL7 may reduce the level of ANGPTL7 mRNA or the level of ANGPTL7 protein by any measurable amount, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. The binding agent may comprise starch and gelatin, while the lubricant (if present) is typically magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. The agents contained in the pharmaceutical formulation are described further herein below.

As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle. SNALP represents lipid vesicles that include nucleic acids, such as iRNA or plasmids that transcribe iRNA, that coat reduced aqueous interiors. SNALP is described, for example, in U.S. patent application publication nos. 2006/0243093, 2007/0135572, and international application No. WO 2009/082817. These applications are incorporated herein by reference in their entirety. In some embodiments, SNALP is SPLP. As used herein, the term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within lipid vesicles.

As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referred to using standard nucleotide nomenclature.

As used herein, a "subject" treated according to the methods described herein comprises a human or non-human animal, e.g., a mammal. The mammal may be, for example, a rodent (e.g., a rat or mouse) or primate (e.g., a monkey). In some embodiments, the subject is a human.

A "subject in need thereof" comprises a subject suffering from, suspected of suffering from, or at risk of developing a disorder associated with ANGPTL7 expression (e.g., overexpression (e.g., glaucoma or a condition associated with glaucoma)). In some embodiments, the subject has or is suspected of having a disorder associated with ANGPTL7 expression or overexpression. In some embodiments, the subject is at risk of developing a disorder associated with ANGPTL7 expression or overexpression.

As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a gene (e.g., ANGPTL 7), comprising mRNA that is the RNA processing product of the primary transcript. The target portion of the sequence will be at least long enough to act as a substrate for iRNA-directed cleavage at or near the portion. For example, the target sequence will typically be 9 to 36 nucleotides in length, e.g., 15 to 30 nucleotides in length, including all subranges therebetween. As a non-limiting example of this, the target sequence may be 15 to 30 nucleotides, 15 to 26 nucleotides, 15 to 23 nucleotides, 15 to 22 nucleotides, 15 to 21 nucleotides, 15 to 20 nucleotides, 15 to 19 nucleotides, 15 to 18 nucleotides, 15 to 17 nucleotides, 18 to 30 nucleotides, 18 to 26 nucleotides, 18 to 23 nucleotides, 18 to 22 nucleotides, 18 to 21 nucleotides, 18 to 20 nucleotides, 19 to 30 nucleotides, 19 to 26 nucleotides 19 to 23 nucleotides, 19 to 22 nucleotides, 19 to 21 nucleotides, 19 to 20 nucleotides, 20 to 30 nucleotides, 20 to 26 nucleotides, 20 to 25 nucleotides, 20 to 24 nucleotides, 20 to 23 nucleotides, 20 to 22 nucleotides, 20 to 21 nucleotides, 21 to 30 nucleotides, 21 to 26 nucleotides, 21 to 25 nucleotides, 21 to 24 nucleotides, 21 to 23 nucleotides or 21 to 22 nucleotides.

As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" and the like refer to an amount that provides a therapeutic benefit in treating, preventing, or managing any disorder or pathological process associated with ANGPTL7 expression (e.g., glaucoma or a condition associated with glaucoma). The specific amount that is therapeutically effective may vary depending on factors known in the art, such as the type of disorder or pathological process, the patient's medical history and age, the stage of the disorder or pathological process, and the administration of other therapies.

In the context of the present disclosure, the term "treatment" or the like means preventing, delaying, alleviating or alleviating at least one symptom associated with a disorder associated with ANGPTL7 expression, or slowing or reversing the progression or expected progression of such disorder. For example, when used to treat glaucoma or a condition associated with glaucoma, the methods presented herein can be used to reduce or prevent one or more symptoms of glaucoma or a condition associated with glaucoma, as described herein, or to reduce the risk or severity of the associated condition. Thus, unless the context clearly indicates otherwise, the term "treatment" or the like is intended to encompass prophylaxis, e.g., prophylaxis of a disorder and/or symptoms of a disorder associated with ANGPTL7 expression. Treatment may also mean an extension of survival compared to the expected survival without treatment.

In the context of disease markers or symptoms, "lower" means any decrease, e.g., a statistically or clinically significant decrease in such levels. The reduction may be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The reduction may be to a level acceptable in the normal range of individuals without such disorders.

As used herein, "ANGPTL7" refers to "angiopoietin-like 7", the corresponding mRNA ("ANGPTL 7 mRNA"), or the corresponding protein ("ANGPTL 7 protein"). The sequence of the human ANGPTL7mRNA transcript can be seen in SEQ ID NO. 3. The sequence of the mouse ANGPTL7mRNA transcript can be seen in SEQ ID NO. 1.

Irna agents

Described herein are iRNA agents that inhibit expression of ANGPTL 7.

In some embodiments, the iRNA agent activates expression of ANGPTL7 in a cell or in a mammal.

In some embodiments, an iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of ANGPTL7 in a cell or in a subject (e.g., in a mammalian body, e.g., in a human body), wherein the dsRNA comprises an antisense strand having a region of complementarity which is complementary to at least a portion of an mRNA formed in expression of ANGPTL7, and wherein the region of complementarity is 30 nucleotides or less in length, typically 19 to 24 nucleotides in length, and wherein the dsRNA inhibits expression of ANGPTL7 upon contact with a cell expressing ANGPTL7, e.g., inhibits at least 10%, 20%, 30%, 40% or 50%.

Modulation (e.g., inhibition) of ANGPTL7 expression may be determined, for example, by PCR-based or branched DNA (bDNA) -based methods, or by protein-based methods (e.g., by Western blotting). Expression of ANGPTL7 in cell culture, such as in COS cells, ARPE-19 cells, hTERT RPE-1 cells, heLa cells, primary hepatocytes, hepG2 cells, primary cultured cells, or biological samples from subjects, can be determined by measuring ANGPTL7 mRNA levels, such as by bDNA or TaqMan, or by measuring protein levels, such as by immunofluorescence analysis using, for example, western blot or flow cytometry techniques.

DsRNA typically comprises two RNA strands that are sufficiently complementary and hybridize under conditions that will use the dsRNA to form a duplex structure. One strand of dsRNA (the antisense strand) typically comprises a region of complementarity that is substantially and typically fully complementary to a target sequence derived from the sequence of mRNA formed during expression of ANGPTL 7. The other strand (the sense strand) typically comprises a region complementary to the antisense strand such that the two strands hybridize and form a duplex structure when combined under appropriate conditions. Typically, the duplex structure is between 15 and 30 base pairs in length (inclusive), more typically between 18 and 25 base pairs (inclusive), still more typically between 19 and 24 base pairs (inclusive), and most typically between 19 and 21 base pairs (inclusive). Similarly, the region complementary to the target sequence is between 15 and 30 nucleotides in length (inclusive), more typically between 18 and 25 nucleotides (inclusive), still more typically between 19 and 24 nucleotides (inclusive), and most typically between 19 and 21 nucleotides (inclusive).

In some embodiments, the dsRNA is between 15 and 20 nucleotides in length (inclusive), and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length (inclusive). As one of ordinary skill will recognize, the targeting region that targets the cleaved RNA will typically be part of a larger RNA molecule, typically an mRNA molecule. In related cases, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for targeted cleavage by RNAi (i.e., cleavage via the RISC pathway). In some cases, dsRNA with duplex as short as 9 base pairs can mediate RNAi-directed RNA cleavage. In most general, the target is at least 15 nucleotides in length, e.g., 15 to 30 nucleotides in length.

Those skilled in the art will also recognize that duplex regions are the primary functional portion of dsRNA, e.g., duplex regions of 9 to 36, e.g., 15 to 30 base pairs. Thus, in some embodiments, to the extent that it is processed into a functional duplex, e.g., 15 to 30 base pairs, that targets the desired RNA for cleavage, the RNA molecule or RNA molecule complex having a duplex region of greater than 30 base pairs is a dsRNA. Thus, one of ordinary skill will then recognize that in some embodiments, the miRNA is dsRNA. In some embodiments, the dsRNA is not a naturally occurring miRNA. In some embodiments, an iRNA agent useful for targeting ANGPTL7 expression is not produced in a target cell by cleavage of a larger dsRNA.

The dsRNA as described herein may further comprise one or more single-stranded nucleotide overhangs. dsRNA can be synthesized by standard methods known in the art as discussed further below, for example, by using an automated DNA synthesizer, such as commercially available from, for example, biosearch, applied Biosystems, inc.

In some embodiments, ANGPTL7 is a human ANGPTL7.

In particular embodiments, the dsRNA comprises or consists of a sense strand comprising or consisting of a sense sequence selected from the sense sequences provided in tables 2-7, and an antisense strand comprising or consisting of an antisense sequence selected from the antisense sequences provided in tables 2-7.

In some aspects, the dsRNA will comprise at least sense and antisense nucleotide sequences, whereby the sense strand is selected from the sequences provided in tables 2-7, and the corresponding antisense strand is selected from the sequences provided in tables 2-7.

In these aspects, one of the two sequences is complementary to the other of the two sequences, wherein one of the sequences is substantially complementary to an mRNA sequence produced by expression of ANGPTL 7. Thus, a dsRNA will comprise two oligonucleotides, one of which is described as the sense strand and the second oligonucleotide is described as the corresponding antisense strand. As described elsewhere herein and as known in the art, the complementary sequence of a dsRNA can also be included as a self-complementary region of a single nucleic acid molecule relative to being located on a separate oligonucleotide.

It is well known to those skilled in the art that dsRNAs having duplex structures of 20 to 23 base pairs (but specifically, 21 base pairs) have been known to be particularly effective in inducing RNA interference (Elbashir et al, 2001EMBO 20:6877-6888). However, others have found that shorter or longer RNA duplex structures may also be effective.

In the above embodiments, due to the nature of the oligonucleotide sequences provided in tables 2-7, the dsRNA described herein may comprise at least one strand of at least 19 nucleotides in length. It is reasonably expected that shorter duplexes with one of the sequences in tables 2-7, minus only few nucleotides at one or both ends, will be similarly effective compared to the dsRNA described above.

In some embodiments, the dsRNA has a partial sequence of at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides from one of the sequences of tables 2-7.

In some embodiments, the dsRNA has an antisense sequence comprising at least 15, 16, 17, 18, or 19 consecutive nucleotides of the antisense sequence provided in tables 2-7 and a sense sequence comprising at least 15, 16, 17, 18, or 19 consecutive nucleotides of the corresponding sense sequence provided in tables 2-7.

In some embodiments, the dsRNA comprises an antisense sequence comprising at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of an antisense sequence provided in tables 2-7 and a sense sequence comprising at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of a corresponding sense sequence provided in tables 2-7.

In some such embodiments, although the dsRNA comprises only a portion of the sequences provided in tables 2-7, it is as effective at inhibiting the level of ANGPTL7 expression as a dsRNA comprising the full length sequences provided in tables 2-7. In some embodiments, the dsRNA differs by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% in its inhibition of the expression level of ANGPTL7 compared to a dsRNA comprising the complete sequence disclosed herein.

The iRNA of tables 2, 3, 4 and 5 were designed based on the mouse ANGPTL7 sequence. The iRNA of tables 6-7 were designed based on the human ANGPTL7 sequence. Without wishing to be bound by theory, ANGPTL7 sequences are sufficiently conserved among species that certain iRNA designed based on mouse sequences have activity against ANGPTL7 of primates and other species (including, e.g., humans, monkeys, and rats), and certain iRNA designed based on human sequences have activity against ANGPTL7 of primates or other species. In some embodiments, the iRNA of tables 2-5 has cross-reactivity with human ANGPTL 7. In some embodiments, the iRNA of tables 6 and 7 are cross-reactive with ANGPTL7 from monkeys, mice, rats, and other species.

Thus, in some embodiments, the iRNA of tables 2-7 reduces the level of ANGPTL7 protein or ANGPTL7 mRNA in a cell. In some embodiments, the cell is a rodent cell (e.g., a rat cell) or a primate cell (e.g., a monkey cell or a human cell). In some embodiments, the ANGPTL7 protein or ANGPTL7 MRNA level is reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In some embodiments, an iRNA of tables 2-7 that inhibits ANGPTL7 in a human cell has fewer than 5, 4, 3,2, or 1 mismatches with the corresponding portion of human ANGPTL 7. In some embodiments, the iRNA of tables 2-7 that inhibits ANGPTL7 in a human cell has no mismatch with the corresponding portion of human ANGPTL 7.

The iRNA designed based on rodent sequences may have utility, for example, for inhibiting ANGPTL7 in human cells, for example, for therapeutic purposes, or for inhibiting ANGPTL7 in rodent cells (e.g., for studies characterizing ANGPTL7 in rodent models).

In some embodiments, an iRNA described herein comprises an antisense strand containing at least 15 contiguous nucleotides that has 0, 1,2, or 3 mismatches with a portion of the nucleotide sequence of SEQ ID NO. 4. In some embodiments, an iRNA described herein comprises a sense strand comprising at least 15 contiguous nucleotides that has 0 or 1,2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 3. Human ANGPTL7 mRNA can have the sequence of SEQ ID NO:3 provided herein.

In some embodiments, an iRNA described herein comprises an antisense strand containing at least 15 contiguous nucleotides that has 0, 1,2, or 3 mismatches with a portion of the nucleotide sequence of SEQ ID NO. 2. In some embodiments, an iRNA described herein comprises a sense strand comprising at least 15 contiguous nucleotides that has 0 or 1,2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 1. The mouse ANGPTL7 mRNA may have the sequence of SEQ ID NO:1 as provided herein.

In some embodiments, an iRNA described herein comprises an antisense strand containing at least 15 contiguous nucleotides that has 0, 1, 2, or 3 mismatches with a portion of the nucleotide sequence of SEQ ID NO. 6. In some embodiments, an iRNA described herein comprises a sense strand comprising at least 15 contiguous nucleotides that has 0 or 1, 2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 5. Cynomolgus monkey ANGPTL7 mRNA may have the sequence of SEQ ID No. 5 provided herein.

In some embodiments, an iRNA described herein comprises an antisense strand containing at least 15 contiguous nucleotides that has 0, 1,2, or 3 mismatches with a portion of the nucleotide sequence of SEQ ID NO. 8. In some embodiments, an iRNA described herein comprises a sense strand comprising at least 15 contiguous nucleotides that has 0 or 1,2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 7. The rat ANGPTL7 mRNA may have the sequence of SEQ ID NO. 7 provided herein.

In some embodiments, an iRNA described herein comprises at least 15 consecutive nucleotides of one of the sequences provided in tables 2-7, and may optionally be conjugated to other nucleotide sequences taken from the region in ANGPTL7 adjacent to the selected sequence.

Although the target sequence is typically 15-30 nucleotides in length, there are large differences in the applicability of a particular sequence within this range to directing cleavage of any given target RNA. The various software packages and guidelines shown herein provide guidance for the identification of the optimal target sequence for any given gene target, but empirical methods may also be employed in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed literally or symbolically (including, for example, computer simulations) over the target RNA sequence to identify sequences within a range of sizes that can be used as target sequences. By moving the sequence "window" stepwise to one nucleotide upstream or downstream of the initial target sequence position, the next potential target sequence can be identified until a complete set of possible sequences for any given target size selected is identified. This process, combined with systematic synthesis and testing of the identified sequences (using assays described herein or as known in the art) to identify those that perform best, can identify those RNA sequences that mediate inhibition of optimal target gene expression when targeted with iRNA agents. Thus, further optimization of the expected inhibition efficiency can be achieved by progressively "windowing" one nucleotide upstream or downstream of the given sequence to identify sequences with identical or better inhibition properties.

Furthermore, it is contemplated that for any of the sequences identified, e.g., in tables 2-7, further optimization can be achieved by systematically adding or removing nucleotides to produce longer or shorter sequences and testing these sequences and sequences produced by windowing the target RNA up or down longer or shorter sizes from the point. Likewise, combining this method of generating new candidate targets with testing the effectiveness of iRNA based on these target sequences in inhibition assays as known in the art or as described herein may provide further improvements in inhibition efficiency. Still further, such optimized sequences can be adjusted to further optimize the molecule (e.g., increase serum stability or circulatory half-life, increase thermostability, enhance transmembrane delivery, target specific locations or cell types, increase interaction with silencing pathway enzymes, increase release from endosomes, etc.) as expression inhibitors by, for example, introducing modified nucleotides as described herein or known in the art, adding or altering overhangs or other modifications known in the art and/or discussed herein.

In some embodiments, the disclosure provides an iRNA of any of tables 2-7 that is unmodified or unconjugated. In some embodiments, RNAi agents of the present disclosure have a nucleotide sequence as provided in any one of tables 2-7, but lack one or more ligands or moieties shown in idle current wattmeter. The ligand or moiety (e.g., lipophilic ligand or moiety) may be included in any of the positions provided in the present application.

An iRNA as described herein may contain one or more mismatches with the target sequence. In some embodiments, an iRNA as described herein contains no more than 3 mismatches. In some embodiments, when the antisense strand of the iRNA contains a mismatch to the target sequence, the mismatched region is not centered in the complementary region. In some embodiments, when the antisense strand of the iRNA contains a mismatch to the target sequence, the mismatch is limited to the last 5 nucleotides from the 5 'or 3' end of the complementary region. For example, for a 23 nucleotide iRNA agent RNA strand that is complementary to a region of ANGPTL7, the RNA strand typically does not contain any mismatches within the center 13 nucleotides. The methods described herein or known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of ANGPTL 7. Considering the efficacy of iRNA with mismatches in inhibiting the expression of ANGPTL7 is important, especially if specific complementary regions in the ANGPTL7 gene are known to have polymorphic sequence variations within the population.

The RNA target may have regions or spans of the nucleotide sequence of the target RNA that are relatively easier or more prone to mediate cleavage of the RNA target by RNA interference induced by binding of the RNAi agent to the region than other regions of the RNA target. Increased susceptibility to RNA interference within such a "hot spot region" (or simply "hot spot") means that an iRNA agent targeting that region may have a higher efficacy in inducing iRNA interference than an iRNA agent targeting other regions of the target RNA. For example, without wishing to be bound by theory, the accessibility of a target region of a target RNA may affect the effectiveness of an iRNA agent targeting that region, with some hot spot regions having increased accessibility. For example, secondary structures formed in an RNA target (e.g., within or near a hot spot region) may affect the ability of an iRNA agent to bind to the target region and induce RNA interference.

According to certain aspects of the invention, an iRNA agent can be designed to target a hot spot region of any of the target RNAs described herein, including any identified portion of the target RNA (e.g., a particular exon). As used herein, a hot spot region may refer to a region of about 19-200、19-150、19-100、19-75、19-50、21-200、21-150、21-100、21-75、21-50、50-200、50-150、50-100、50-75、75-200、75-150、75-100、100-200 or 100-150 nucleotides of a target RNA sequence, wherein targeting with an RNAi agent provides a significantly higher effective silencing probability relative to other regions targeting the same target RNA. According to certain aspects of the invention, the hot spot region may comprise a limited region of the target RNA, and in some cases, a substantially limited region of the target, including, for example, less than half the length of the target RNA, e.g., about 5%, 10%, 15%, 20%, 25% or 30% of the length of the target RNA. Conversely, other regions compared to the hot spot may cumulatively comprise at least a majority of the length of the target RNA. For example, the additional regions may cumulatively comprise at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% of the length of the target RNA.

The comparison region of the target RNA can be empirically evaluated to identify hot spots using the effectiveness data obtained from in vitro or in vivo screening assays. For example, RNAi agents targeting individual regions across a target RNA can compare the frequency of effective iRNA agents binding to each region (e.g., the amount of target gene expression inhibited, as measured by mRNA expression or protein expression). In general, hot spots can be identified by observing clusters of multiple effective RNAi agents that bind to limited regions of RNA targets. Hotspots can be well characterized by observing the effectiveness of an iRNA agent that accumulates across at least about 60% of the target region identified as a hotspot, such as about 70%, about 80%, about 90% or about 95% or more of the length of the region, including both ends of the region (i.e., at least about 60%, 70%, 80%, 90% or 95% or more of the nucleotides within the region are targeted by the iRNA agent, including the nucleotides at each end of the region). According to some aspects of the invention, iRNA agents that exhibit at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% inhibition (e.g., no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the remaining mRNA) in that region may be identified as effective.

The suitability of a targeted RNA region can also be assessed by quantitative comparison of inhibition measurements for different regions of defined size (e.g., 25, 30, 40, 50, 60, 70, 80, 90, or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nt). For example, an average inhibition level may be determined for each region, and the average value for each region may be compared. The average inhibition level in the hot spot area may be significantly higher than the average of the average levels in all evaluation areas. According to some aspects, the average level of inhibition in the hot spot region may be at least about 10%, 20%, 30%, 40% or 50% higher than the average of the average levels. According to some aspects, the average level of inhibition in the hotspot zone may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8.1.9 or 2.0 standard deviation. The average level of inhibition may be higher than a statistically significant amount (e.g., p < 0.05). According to some aspects, each inhibition measurement within a hotspot region may be above a threshold amount (e.g., at or below a threshold amount of remaining mRNA). According to some aspects, each inhibition measurement within the region may be significantly higher than the average of all inhibition measurements over all measurement regions. For example, each inhibition measurement in a hot spot region may be at least about 10%, 20%, 30%, 40%, or 50% higher than the average of all inhibition measurements. According to some aspects, each inhibition measurement may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 standard deviation higher than the average of all inhibition measurements. Each inhibition measurement may be higher than the average of all inhibition measurements by a statistically significant amount (e.g., p < 0.05). The criteria for evaluating the hotspot may include various combinations of the above criteria in a compatible case (e.g., at least about an average level of inhibition of the first amount, and no inhibition measure below a threshold level of the second amount that is less than the first amount).

Thus, it is expressly contemplated that any iRNA agent that targets a hot spot region of a target RNA, including the specific exemplary iRNA agents described herein, may be preferred to induce RNA interference of the target mRNA, as targeting such a hot spot region may exhibit a robust inhibitory response relative to targeting a region that is not a hot spot region. RNAi agents targeting a target sequence that substantially overlap (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95% of the length of the target sequence), or preferably lie entirely within a hotspot region, can be considered to target the hotspot region. The hot spot regions of RNA targets of the invention may include any region where the data disclosed herein demonstrates that an effective RNAi agent (including any of the criteria described elsewhere herein) targets more frequently, whether or not the scope of such hot spot regions is explicitly specified.

In various embodiments, the dsRNA agents of the invention target a hot spot region of an mRNA encoding ANGPTL 7. In some embodiments, the dsRNA agents of the invention target a hot spot region of mRNA encoding mouse ANGPTL 7. In one embodiment, the hot spot region comprises nucleotides 1562-1584, 546-568, 709-731, 862-884 and/or 232-256 of SEQ ID NO. 1. In other embodiments, the dsRNA agents of the invention target a hot spot region of mRNA encoding human ANGPTL 7. In one embodiment, the hot spot region comprises nucleotide 1993-2146、1910-1932、1726-1823、1628-1685、1591-1613、1551-1573、1420-1442、1380-1402、1243-1265、1195-1217、1096-1118、940-962 and/or 299-321 of SEQ ID NO. 3. The dsRNA agent may be selected from ：AD-1094991、AD-1093984、AD-1094129、AD-1094262、AD-1093670、AD-1093672、AD-1565389、AD-1565368、AD-1565357、AD-1565345、AD-1565324、AD-1565303、AD-1565288、AD-1565212、AD-1565141、AD-1565126、AD-1565113、AD-1565091、AD-1565034、AD-1565015、AD-1565004、AD-1564969、AD-1094381、AD-1564428、AD-1564936、AD-1564823、AD-1564802、AD-1564666、AD-1564618 and AD-1563396 below.

In some embodiments, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, typically 1 or 2 nucleotides. In some embodiments, dsRNA with at least one nucleotide overhang has better inhibitory properties relative to its blunt-ended counterpart. In some embodiments, the RNA (e.g., dsRNA) of the iRNA is chemically modified to enhance stability or other beneficial properties. The nucleic acids set forth in the present disclosure may be synthesized and/or modified by art-recognized methods, such as those described in "current protocols in nucleic acid chemistry (Current protocols in nucleic ACID CHEMISTRY)", beaucage, s.l., et al, john Wiley & Sons, inc., new York, NY, USA, which is hereby incorporated by reference. Modifications include, for example, (a) terminal modifications, e.g., 5 'terminal modifications (phosphorylations, conjugation, reverse linkages, etc.), 3' terminal modifications (conjugation, DNA nucleotide reverse linkages, etc.), and (b) base modifications, e.g., substitutions with stable bases, labile bases, or bases that base pair with amplified pools of partners, removal of bases (abasic nucleotides) or conjugated bases, (c) sugar modifications (e.g., at the 2 'position or 4' position, or with acyclic sugar) or sugar substitutions, and (d) backbone modifications, modifications or substitutions comprising phosphodiester bonds. Specific examples of RNA compounds useful in the present disclosure include, but are not limited to, RNAs that contain modified backbones or do not contain natural internucleoside linkages. In addition, RNAs having modified backbones include those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have phosphorus atoms in their internucleoside backbones can also be considered oligonucleotides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates (including 3 '-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3' -phosphoramidate and aminoalkyl amine phosphates), thiocarbonylphosphoramidates, thiocarbonylalkyl phosphonates, thiocarbonylalkyl phosphotriesters, and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these esters, and those with reversed polarity, wherein adjacent pairs of nucleoside units are linked in 3'-5' to 5'-3' or 2'-5' to 5 '-2'. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above-described phosphorus-containing bonds include, but are not limited to, U.S. patent No. 3,687,808;4,469,863;4,476,301;5,023,243;5,177,195;5,188,897;5,264,423;5,276,019;5,278,302;5,286,717;5,321,131;5,399,676;5,405,939;5,453,496;5,455,233;5,466,677;5,476,925;5,519,126;5,536,821;5,541,316;5,550,111;5,563,253;5,571,799;5,587,361;5,625,050;6,028,188;6,124,445;6,160,109;6,169,170;6,172,20;6,239,265;6,277,603;6,326,199;6,346,614;6,444,423;6,531,590;6,534,639;6,608,035;6,683,167;6,858,715;6,867,294;6,878,805;7,015,315;7,041,816;7,273,933;7,321,029 and U.S. Pat. RE39464, each of which is incorporated herein by reference.

Wherein the modified RNA backbone that does not contain a phosphorus atom has a backbone formed from: short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatoms or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar moiety of the nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thioacetylacetyl backbones; methylene formylacetyl and thioformylacetyl backbones; an olefin-containing backbone; a sulfamate backbone; methylene imino and methylene hydrazino backbones; sulfonate and sulfonamide backbones; an amide backbone; and other backbones with mixed N, O, S and CH ₂ component moieties.

Representative U.S. patents that teach the preparation of the above-described oligonucleotides include, but are not limited to, U.S. patent nos. 5,034,506;5,166,315;5,185,444;5,214,134;5,216,141;5,235,033;5,64,562;5,264,564;5,405,938;5,434,257;5,466,677;5,470,967;5,489,677;5,541,307;5,561,225;5,596,086;5,602,240;5,608,046;5,610,289;5,618,704;5,623,070;5,663,312;5,633,360;5,677,437 and 5,677,439, each of which is incorporated herein by reference.

In other RNA mimics suitable or contemplated for use in iRNA, the sugar and internucleoside linkages of the nucleotide units, i.e., the backbone, are replaced with new groups. The base unit is maintained to hybridize to the appropriate nucleic acid target compound. One such oligomeric compound, i.e., an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a Peptide Nucleic Acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide containing backbone, especially an aminoethylglycine backbone. The nucleobases are retained and bound directly or indirectly to the aza nitrogen atoms of the amide moiety of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. patent nos. 5,539,082;5,714,331 and 5,719,262, each of which is incorporated herein by reference. Additional teachings of PNA compounds can be found, for example, in ielsen et al, science,1991,254,1497-1500.

Some embodiments presented in this disclosure include RNAs with phosphorothioate backbones and oligonucleotides with heteroatom backbones, particularly-CH ₂--NH--CH₂--、--CH₂--N(CH₃)--O--CH₂ - [ known as methylene (methylimino) or MMI backbones ]、--CH₂--O--N(CH₃)--CH₂--、--CH₂--N(CH₃)--N(CH₃)--CH₂- and-N (CH ₃)--CH₂ - - [ wherein the natural phosphodiester backbone is denoted-O-P-O-CH ₂ - ], and amide backbones of U.S. Pat. No. 5,602,240 cited above.

The modified RNA may also contain one or more substituted sugar moieties. The iRNA, e.g., dsRNA, presented herein may comprise one of the following at the 2' position: OH; f, performing the process; o-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; Or O-alkyl-O-alkyl, wherein alkyl is, Alkenyl and alkynyl groups may be substituted or unsubstituted C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl and alkynyl groups. Exemplary suitable modifications include O[(CH₂)_nO]_mCH₃、O(CH₂)._nOCH₃、O(CH₂)_nNH₂、O(CH₂)_nCH₃、O(CH₂)_nONH₂ and O (CH ₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2' position: c ₁ -C ₁₀ lower alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl 、SH、SCH₃、OCN、Cl、Br、CN、CF₃、OCF₃、SOCH₃、SO₂CH₃、ONO₂、NO₂、N₃、NH₂、 heterocycloalkyl, heterocycloalkylaryl, Aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of iRNA, or groups for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification comprises a 2 '-methoxyethoxy (2' -O-CH ₂CH₂OCH₃), also known as 2'-O- (2-methoxyethyl) or 2' -MOE (Martin et al, helv. Chim. Acta,1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2 '-dimethylaminooxyethoxy, i.e., O (CH ₂)₂ON(CH₃)₂ group, also known as 2' -DMAOE), and 2 '-dimethylaminoethoxyethoxy (also known in the art as 2' -O-dimethylaminoethoxyethyl or 2 '-DMAEOE), i.e., 2' -O-CH ₂-O-CH₂-N(CH2)₂.

In other embodiments, the iRNA agent comprises one or more (e.g., about 1,2, 3, 4, 5,6,7,8, 9, 10, or more) acyclic nucleotides (or nucleosides). In certain embodiments, the sense strand or the antisense strand, or both the sense and antisense strands, each comprise less than five acyclic nucleotides (e.g., four, three, two, or one acyclic nucleotide per strand). The one or more acyclic nucleotides can be found in, for example, the double-stranded region of the sense or antisense strand or both strands; at the 5 'end, 3' end, both 5 'and 3' ends, or both strands of the sense or antisense strand of an iRNA agent. In some embodiments, one or more acyclic nucleotides are present at positions 1-8 of the sense or antisense strand, or both. In some embodiments, one or more acyclic nucleotides are found in the antisense strand at positions 4 to 10 (e.g., positions 6 to 8) from the 5' end of the antisense strand. In some embodiments, one or more acyclic nucleotides are found at one or both 3' -terminal overhangs of the iRNA agent.

As used herein, the term "acyclic nucleotide" or "acyclic nucleoside" refers to any nucleotide or nucleoside having an acyclic sugar (e.g., an acyclic ribose). Exemplary acyclic nucleotides or nucleosides can comprise nucleobases, such as naturally occurring or modified nucleobases (e.g., nucleobases as described herein). In certain embodiments, the bond between any ribose carbon (C1, C2, C3, C4, or C5) is absent from the nucleotide, either independently or in combination. In some embodiments, the bond between the C2-C3 carbons of the ribose ring is absent, e.g., an acyclic 2'-3' -break nucleotide monomer. In other embodiments, the bond between C1-C2, C3-C4, or C4-C5 is absent (e.g., 1'-2', 3'-4', or 4'-5' -nucleotidic monomers). Exemplary acyclic nucleotides are disclosed in US 8,314,227, which is incorporated herein by reference in its entirety. For example, an acyclic nucleotide may comprise any of monomers D-J in fig. 1 to 2 of US 8,314,227. In some embodiments, the acyclic nucleotide comprises the following monomers:

wherein the base is a nucleobase, e.g., a naturally occurring or modified nucleobase (e.g., a nucleobase as described herein).

In certain embodiments, the acyclic nucleotide can be modified or derivatized, such as by coupling the acyclic nucleotide to another moiety, such as a ligand (e.g., galNAc, cholesterol ligand), alkyl, polyamine, sugar, polypeptide, and the like.

In other embodiments, the iRNA agent comprises one or more loop-free nucleotides and one or more LNAs (e.g., LNAs as described herein). For example, one or more loop-free nucleotides and/or one or more LNAs may be present in the sense strand, the antisense strand, or both. The number of acyclic nucleotides in one strand may be the same as or different from the number of LNAs in the opposite strand. In certain embodiments, the sense strand and/or the antisense strand comprises less than five LNAs (e.g., four, three, two, or one LNA) located in a double-stranded region or 3' overhang. In other embodiments, one or both LNAs are located in the double-stranded region or 3' overhang of the sense strand. Alternatively or in combination, the sense strand and/or the antisense strand includes less than five acyclic nucleotides (e.g., four, three, two, or one acyclic nucleotide) in the double-stranded region or 3' overhang. In some embodiments, the sense strand of the iRNA agent includes one or two LNAs in the 3 'overhang of the sense strand, and one or two acyclic nucleotides in the double-stranded region of the antisense strand of the iRNA agent (e.g., at positions 4 to 10 (e.g., positions 6 to 8) of the 5' end of the antisense strand).

In other embodiments, the inclusion of one or more acyclic nucleotides (alone or in addition to one or more LNAs) in the iRNA agent results in one or more (or all) of the following: (i) reduced off-target effects; (ii) a reduction in the accompanying strand involved in RNAi; (iii) increased specificity of the guide strand for its target mRNA; (iv) reduced microrna off-target effects; (v) increased stability; or (vi) increased resistance to degradation of the iRNA molecule.

Other modifications include 2 '-methoxy (2' -OCH ₃), 2 '-aminopropoxy (2' -OCH ₂CH₂CH₂NH₂) and 2 '-fluoro (2' -F). Similar modifications can also be made at other positions on the RNA of the iRNA, particularly at the 3 'position of the sugar on the 3' terminal nucleotide or at the 5 'position of the 2' -5 'linked dsRNA and 5' terminal nucleotide. The iRNA may also have a glycomimetic, such as a cyclobutyl moiety in place of the pentose sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. patent nos. 4,981,957;5,118,800;5,319,080;5,359,044;5,393,878;5,446,137;5,466,786;5,514,785;5,519,134;5,567,811;5,576,427;5,591,722;5,597,909;5,610,300;5,627,053;5,639,873;5,646,265;5,658,873;5,670,633; and 5,700,920, some of which are commonly owned with the present application, and each of which is incorporated herein by reference.

IRNA may also include nucleobase (commonly referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (a) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deaza and 3-deaza and deaza adenine.

Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808, in Modified Nucleosides in Biochemistry, biotechnology AND MEDICINE, herdewijn, p. Editions Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia of Polymer SCIENCE AND ENGINEERING, pages 858-859 Kroschwitz, J.L. journal of John Wiley & Sons,1990, englisch et al, ANGEWANDTE CHEMIE, international edition, 1991,30,613, and those disclosed in Chapter Sanghvi, Y.S., chapter 15, DSRNA RESEARCH AND Applications, pages 289-302, crooke, S.T. and Lebleu, B.edition, CRC Press, 1993. Certain of these nucleobase pairs are particularly useful for increasing the binding affinity of the oligomeric compounds proposed in the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ℃ (Sanghvi, y.s., crooke, s.t. and Lebleu, b. Editions, DSRNA RESEARCH AND Applications, CRC Press, boca Raton,1993, pp.276-278), and are exemplary base substitutions, even more particularly when combined with 2' -O-methoxyethyl sugar modifications.

Representative U.S. patents teaching the preparation of certain modified nucleobases and other modified nucleobases described above include, but are not limited to, U.S. patent nos. 3,687,808; U.S. patent nos. 4,845,205;5,130,30;5,134,066;5,175,273;5,367,066;5,432,272;5,457,187;5,459,255;5,484,908;5,502,177;5,525,711;5,552,540;5,587,469;5,594,121;5,596,091;5,614,617;5,681,941;6,015,886;6,147,200;6,166,197;6,222,025;6,235,887;6,380,368;6,528,640;6,639,062;6,617,438;7,045,610;7,427,672; and 7,495,088; each of the U.S. patents is incorporated herein by reference; and U.S. patent number 5,750,692, also incorporated herein by reference.

The RNA of the iRNA can also be modified to include one or more (e.g., about 1,2, 3,4, 5, 6, 7, 8, 9,10, or more) bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by bridging of two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic system. In certain embodiments, the bridge connects the 4 'carbon and the 2' carbon of the sugar ring. Thus, in some embodiments, an agent of the present disclosure may comprise one or more Locked Nucleic Acids (LNAs) (also referred to herein as "locked nucleotides"). In some embodiments, the locked nucleic acid is a nucleotide having a modified ribose moiety, wherein the ribose moiety includes an additional bridge linking, for example, 2 'and 4' carbons. This structure effectively "locks" the ribose in the 3' -internal structure conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum, increase thermostability, and reduce off-target effects (Elmen, J. Et al 2005Nucleic Acids Research 33 (1): 439-447; mook, OR. Et al, 2007mol. Canc. Ther.6 (3): 833-843; grunwiller, A. Et al, 2003Nucleic Acids Research31 (12): 3185-3193).

Examples of bicyclic nucleosides for polynucleotides of the present disclosure include, but are not limited to nucleosides that include a bridge between 4 'and 2' ribosyl ring atoms. In certain embodiments, antisense polynucleotide agents of the present disclosure comprise one or more bicyclic nucleosides comprising a 4 'to 2' bridge. Examples of such 4 'to 2' bridged bicyclic nucleosides include, but are not limited to 4'-(CH₂)-O-2'(LNA);4'-(CH₂)-S-2';4'-(CH₂)₂-O-2'(ENA);4'-CH(CH₃)-O-2'( also known as "constrained ethyl" or "cEt") and 4'-CH (CH ₂OCH₃) -O-2' (and analogs thereof; See, for example, U.S. patent No. 7,399,845); 4'-C (CH ₃)(CH₃) -O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH ₂-N(OCH₃) -2' (and analogs thereof; see, for example, U.S. patent No. 8,278,425); 4'-CH ₂-O-N(CH₃) -2' (see, e.g., U.S. patent publication No. 2004/0171570); 4'-CH ₂ -N (R) -O-2', wherein R is H, C ₁-C₁₂ alkyl or a protecting group (see, e.g., U.S. patent No. 7,427,672); 4'-CH ₂-C(H)(CH₃) -2' (see, e.g., chattopadhyaya, et al, j. Org. Chem.,2009,74,118-134); and 4'-CH ₂-C(═CH₂) -2' (and analogs thereof; see, for example, U.S. patent No. 8,278,426). The contents of each of the foregoing are incorporated herein by reference for the methods provided therein. Representative U.S. patents teaching the preparation of locked nucleic acids include, but are not limited to, the following: U.S. Pat. nos. 6,268,490;6,670,461;6,794,499;6,998,484;7,053,207;7,084,125; 7,399,845, and 8,314,227, each of which is incorporated by reference in its entirety. Exemplary LNAs include, but are not limited to, 2',4' -C methylene bicyclic nucleotides (see, e.g., wengel et al, international PCT publication Nos. WO 00/66604 and WO 99/14226).

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations, including, for example, α -L-ribofuranose and β -D-ribofuranose (see WO 99/14226).

RNAi agents of the present disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4'-CH (CH ₃) -0-2' bridge. In some embodiments, the constrained ethyl nucleotide is in an S conformation referred to herein as "S-cEt".

RNAi agents of the present disclosure can also include one or more "conformational restriction nucleotides" ("CRNs"). CRNs are nucleotide analogs having a linker linking the C2' and C4' carbons of ribose or the C3 and-C5 ' carbons of ribose. CRN locks the ribose ring in a stable conformation and increases the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in the optimal position for stability and affinity, resulting in less ribose ring wrinkling.

Representative publications teaching the preparation of certain CRNs described above include, but are not limited to, US 2013/0190383; and WO 2013/036868, the contents of each of which are hereby incorporated by reference for the methods provided therein.

In some embodiments, RNAi agents of the present disclosure include one or more monomers that are UNA (unlocking nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which any bonds of the sugar have been removed, forming an unlocked "sugar" residue. In one example, UNA also encompasses monomers where the bond between C1'-C4' has been removed (i.e., covalent carbon-oxygen-carbon bonds between C1 'and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2 'and C3' carbons) has been removed (see 2008Nuc.Acids Symp.Series52:133-134 and Fluiter et al, 2009mol. Biosyst 10:1039).

Representative U.S. disclosures teaching the preparation of UNA include, but are not limited to, US8,314,227; U.S. patent publication No. 2013/0096289;2013/0011922; and 2011/0313020, the contents of each of which are hereby incorporated by reference for the methods provided therein.

In other embodiments, the iRNA agent comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. G-clamp nucleotides are modified cytosine analogs in which the modification confers the ability to hydrogen bond to both Watson-Crick (Watson-Crick) and Hu Gesi-butyl (Hoogsteen) faces of complementary guanine within the duplex, see, e.g., lin and Matteucci,1998J.am.chem.Soc.120:8531-8532. When hybridized to a complementary oligonucleotide, a single G-clamp analog substitution within the oligonucleotide can result in significantly enhanced helix thermostability and mismatch discrimination. inclusion of such nucleotides in iRNA molecules can result in enhanced affinity and specificity for a nucleic acid target, complementary sequence, or template strand.

Potentially stable modifications to the ends of the RNA molecule may include N- (acetamidohexanoyl) -4-hydroxyproline (Hyp-C6-NHAc), N- (hexanoyl-4-hydroxyproline) (Hyp-C6), N- (acetyl-4-hydroxyproline) (Hyp-NHAc), thymidine-2 '-O-deoxythymidine (ether), N- (aminohexanoyl) -4-hydroxyproline (Hyp-C6-amino), 2-behenoyl-uridine-3' -phosphate, inverted base dT (idT), and the like. The disclosure of this modification can be found in PCT publication No. WO 2011/005861.

Other modifications of the RNAi agents of the present disclosure include 5' phosphates or 5' phosphate mimics, e.g., 5' terminal phosphates or phosphate mimics on the antisense strand of the RNAi agent. Suitable phosphate mimics are disclosed, for example, in US2012/0157511, the contents of which are incorporated herein by reference for the methods provided therein.

Irna motif

In certain aspects of the present disclosure, double stranded RNAi agents of the present disclosure comprise agents having chemical modifications, as disclosed, for example, in WO 2013/075035, the contents of which are incorporated herein by reference for the methods provided therein. As shown herein and in WO 2013/075035, excellent results can be obtained by introducing one or more motifs with three identical modifications on three consecutive nucleotides into the sense or antisense strand of the RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent can be fully modified in other ways. The introduction of these motifs interrupts the modification pattern of the sense or antisense strand, if present. The RNAi agent can optionally be conjugated to a lipophilic moiety or ligand, e.g., a C16 moiety or ligand, e.g., on the sense strand. RNAi agents can optionally be modified with (S) -diol nucleic acid (GNA) modifications, e.g., on one or more residues of the antisense strand. The produced RNAi agent exhibits excellent gene silencing activity.

In some embodiments, the sense strand sequence may be represented by formula (I):

5'n_p-N_a-(X)_i-N_b-Y Y-N_b-(Z Z)_j-N_a-n_q 3'(I)

Wherein:

i and j are each independently 0 or 1;

p and q are each independently 0 to 6;

Each N _a independently represents an oligonucleotide sequence comprising from 0 to 25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

Each N _b independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

Each of n _p and n _q independently represents an overhang nucleotide;

Wherein N _b and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represents a motif of three identical modifications on three consecutive nucleotides. In some embodiments, YYY is all 2' -F modified nucleotides.

In some embodiments, N _a and/or N _b include an alternating pattern of modifications.

In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17 to 23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., can occur at positions 6,7, 8;7, 8, 9;8, 9, 10;9, 10, 11;10, 11, 12 or 11, 12, 13), counting from nucleotide 1, starting from the 5' end; or optionally counting from the 5' end, starting from the 1 st paired nucleotide within the duplex region.

In some embodiments, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand can be represented by the formula:

5’n_p-N_a-YYY-N_b-ZZZ-N_a-n_q 3’(Ib)；

5'n _p-N_a-XXX-N_b-YYY-N_a-n_q 3' (Ic); or (b)

5’n_p-N_a-XXX-N_b-YYY-N_b-ZZZ-N_a-n_q 3’(Id)。

When the sense strand is represented by formula (Ib), N _b represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N _a can independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the sense strand is represented by formula (Ic), N _b represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N _a can independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

Each N _b independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides when the sense strand is represented by formula (Id). In some embodiments, N _b is 0, 1, 2, 3, 4, 5, or 6. Each N _a can independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

X, Y and Z may each be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand can be represented by the formula:

5’n_p-N_a-YYY-N_a-n_q 3’(Ia)。

when the sense strand is represented by formula (Ia), each N _a may independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

In some embodiments, the antisense strand sequence of RNAi can be represented by formula (Ie):

5'n_q'-N_a'-(Z'Z'Z')_k-N_b'-Y'Y'Y'-N_b'-(X'X'X')_l-N'_a-n_p'3'(Ie)

Wherein:

k and l are each independently 0 or 1;

p 'and q' are each independently 0 to 6;

Each Na' independently represents an oligonucleotide sequence comprising from 0 to 25 modified nucleotides,

Each sequence comprising at least two differently modified nucleotides;

Each N _b' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

Each of n _p 'and n _q' independently represents an overhang nucleotide;

Wherein N _b 'and Y' do not have the same modification;

And

X ' X ' X ', Y ' Y ' Y ' and Z ' Z ' Z ' each independently represent one of three identical modifications on three consecutive nucleotides.

In some embodiments, N _a 'and/or N _b' include an alternating pattern of modifications.

The Y ' Y ' Y ' motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17 to 23 nucleotides in length, the Y' motif can occur at positions 9, 10, 11 of the antisense strand; 10. 11, 12; 11. 12, 13; 12. 13, 14; or 13, 14, 15, counting from nucleotide 1, from the 5' end; or optionally counting from the 5' end, starting from the 1 st paired nucleotide within the duplex region. In some embodiments, the Y ' Y ' Y ' motif occurs at positions 11, 12, 13.

In some embodiments, the Y 'Y' Y 'motifs are all 2' -Ome modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both 5k and l are 1.

Thus, the antisense strand can be represented by the formula:

5’n_q'-N_a'-Z'Z'Z'-N_b'-Y'Y'Y'-N_a'-n_p’3’ (If)；

5'n _q'-N_a'-Y'Y'Y'-N_b'-X'X'X'-n_p '3' (Ig); or (b)

5'n_q'-N_a'-Z'Z'Z'-N_b'-Y'Y'Y'-N_b'-X'X'X'-N_a'-n_p'3' (Ih).

When the antisense strand is represented by formula (If), nb' represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N _a' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the antisense strand is represented by formula (Ig), each N _b' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N _a' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. In some embodiments, N _b is 0, 1,2, 3,4, 5, or 6.

When the antisense strand is represented by formula (Ih), each N _b' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N _a' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Preferably, N _b is 0, 1, 2,3, 4, 5, or 6.

In other embodiments, k is 0 and l is 0, and the antisense strand can be represented by the formula:

5’n_p'-N_a'-Y'Y'Y'-N_a'-n_q’3’ (Ia)。

When the antisense strand is represented by formula (Ie), each N _a' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

Each of X ', Y ', and Z ' may be the same or different from each other.

Each nucleotide of the sense and antisense strands may be independently modified with LNA, HNA, ceNA, GNA, 2 '-methoxyethyl, 2' -O-methyl, 2 '-O-allyl, 2' -C-allyl, 2 '-hydroxy, or 2' -fluoro. For example, each nucleotide of the sense strand and the antisense strand is independently modified with 2 '-O-methyl or 2' -fluoro. In particular, each X, Y, Z, X ', Y ', and Z ' may represent a 2' -O-methyl modification or a 2' -fluoro modification.

In some embodiments, when the duplex region is 21nt, the sense strand of the RNAi agent can contain YYY motifs present at positions 9, 10, and 11 of the strand, counting from nucleotide 1 of the 5 'end, or optionally, counting from nucleotide 1 of pairing within the duplex region of the 5' end; and Y represents a 2' -F modification. The sense strand may additionally contain an XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2' -F modification.

In some embodiments, the antisense strand may contain a Y ' motif present at positions 11,12,13 of the strand, counting from nucleotide 1 of the 5' end, or optionally, counting from nucleotide 1 of the pairing within the duplex region of the 5' end; and Y 'represents a 2' -O-methyl modification. The antisense strand may additionally contain an X 'motif or a Z' motif as a wing modification at opposite ends of the duplex region; and X 'X' X 'and Z' Z 'Z' each independently represent a 2'-OMe modification or a 2' -F modification.

The sense strand represented by any of the above formulas (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any of the formulas (Ie), (If), (Ig) and (Ih), respectively.

Thus, certain RNAi agents useful in the methods of the present disclosure can include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex being represented by formula (Ii):

Sense of meaning ：5'n_p-N_a-(XXX)_i-N_b-YYY-N_b-(ZZZ)_j-N_a-n_q 3'

Antisense sense ：3'n_p'-N_a'-(X'X'X')_k-N_b'-Y'Y'Y'-N_b'-(Z'Z'Z')_l-N_a'-n_q'5(Ii)

Wherein,

I. j, k and l are each independently 0 or 1;

p, p ', q and q' are each independently 0-6;

Each N _a and N _a' independently represents an oligonucleotide sequence comprising from 0 to 25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

Each N _b and N _b' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

Wherein the method comprises the steps of

Each of n _p'、n_p、n_q' and n _q, which may or may not be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X ' X ' X ', Y ' Y ' Y ' and Z ' Z ' Z ' each independently represent one motif in three identical modifications on three consecutive nucleotides.

In some embodiments, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or i and j are both 0; or i and j are both 1. In some embodiments, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or k and l are both 0; or k and l are both 1.

An exemplary combination of sense and antisense strands forming an RNAi duplex comprises the formula:

5’n_p-N_a-Y Y Y-N_a-n_q 3’

3’n_p'-N_a'-Y'Y'Y'-N_a'n_q’5’(Ij)

5’n_p-N_a-Y-N_b-Z-N_a-n_q 3’

3’n_p-N_a'-Y'Y'Y'-N_b'-Z'Z'Z'-N_a'-n_q’5’(Ik)

5’n_p-N_a-X-N_b-Y-N_a-n_q 3’

3’n_p-N_a'-X'X'X'-N_b'-Y'Y'Y'-N_a'-n_q’5’(Il)

5’n_p-N_a-X-N_b-Y-N_b-Z Z-N_a-n_q 3’

3’n_p-N_a'-X'X'X'-N_b'-Y'Y'Y'-N_b'-Z'Z'Z'-N_a'-n_q’5’(Im)

When the RNAi agent is represented by formula (Ij), each N _a independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the RNAi agent is represented by formula (Ik), each N _b independently represents an oligonucleotide sequence comprising 1 to 10,1 to 7, 1 to 5, or 1 to 4 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the RNAi agent is represented by formula (Il), each N _b、N_b' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N _a independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the RNAi agent is represented by formula (Im), each N _b、N_b' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N _a、N_a' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of N _a、N_a'、N_b and N _b' independently includes an alternating pattern of modifications.

Each of X, Y and Z in formulas (Ii), (Ij), (Ik), (Il), and (Im) may be the same or different from each other.

When the RNAi agent is represented by formulas (Ii), (j), (Ik), (Il) and (Im), at least one Y nucleotide can form a base pair with one Y' nucleotide. Alternatively, at least two Y nucleotides form base pairs with corresponding Y' nucleotides; or all three Y nucleotides form base pairs with the corresponding Y' nucleotide.

When the RNAi agent is represented by formula (Ik) or (Im), at least one Z nucleotide can form a base pair with one Z' nucleotide. Alternatively, at least two Z nucleotides form base pairs with corresponding Z' nucleotides; or all three Z nucleotides form base pairs with the corresponding Z' nucleotide.

When the RNAi agent is represented by formula (Il) or (Im), at least one X nucleotide can form a base pair with one X' nucleotide. Alternatively, at least two X nucleotides form base pairs with corresponding X' nucleotides; or all three X nucleotides form base pairs with the corresponding X' nucleotide.

In some embodiments, the modification on the Y nucleotide is different from the modification on the Y ' nucleotide, the modification on the Z nucleotide is different from the modification on the Z ' nucleotide, and/or the modification on the X nucleotide is different from the modification on the X ' nucleotide.

In some embodiments, when the RNAi agent is represented by formula (Im), the N _a modification is a2 '-O-methyl or 2' -fluoro modification. In some embodiments, when the RNAi agent is represented by formula (Im), the N _a modification is a2 '-O-methyl or 2' -fluoro modification, and N _p '>0, and at least one N _p' is linked to the adjacent nucleotide a through a phosphorothioate linkage. In some embodiments, when the RNAi agent is represented by formula (In), the N _a modification is a2 '-O-methyl or 2' -fluoro modification, N _p '>0, and at least one N _p' is linked to an adjacent nucleotide through a phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties or one or more GalNAc moieties) linked by a divalent or trivalent branched linker. In some embodiments, when the RNAi agent is represented by formula (Im), the N _a modification is a2 '-O-methyl or 2' -fluoro modification, N _p '>0, and at least one N _p' is linked to an adjacent nucleotide through a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties or one or more GalNAc moieties) linked by a divalent or trivalent branched linker.

In some embodiments, when the RNAi agent is represented by formula (Ij), the N _a modification is a2 '-O-methyl or 2' -fluoro modification, N _p '>0, and at least one N _p' is linked to an adjacent nucleotide through a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties or one or more GalNAc moieties) linked through a divalent or trivalent branched linker.

In some embodiments, the RNAi agent is a multimer comprising at least two duplex represented by formulas (Ii), (Ij), (Ik), (Il), and (Im), wherein the duplex is connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes; or each duplex may target the same gene at two different target sites.

In some embodiments, the RNAi agent is a multimer comprising three, four, five, six, or more duplex represented by formulas (Ii), (Ij), (Ik), (Il), and (Im), wherein the duplex is connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes; or each duplex may target the same gene at two different target sites.

In some embodiments, the two RNAi agents represented by formulas (Ii), (k), (Ii), and (Im) are linked to each other at the 5 'end and one or both 3' ends, and optionally conjugated to a ligand. Each agent may target the same gene or two different genes; or each agent may target the same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used in the methods of the present disclosure. Such publications include WO2007/091269, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520; and US 7858769, the contents of each of which are hereby incorporated by reference for the methods provided therein. In certain embodiments, RNAi agents of the present disclosure can comprise GalNAc ligands.

As described in more detail below, RNAi agents containing conjugation of one or more carbohydrate moieties to the RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be linked to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably, cyclic) carrier linked to a carbohydrate ligand. Ribonucleotide subunits in which the ribose sugar of the subunit has been so replaced are referred to herein as Ribose Replacement Modified Subunits (RRMS). The cyclic carrier may be a carbocyclic ring system, i.e. all ring atoms are carbon atoms, or a heterocyclic ring system, i.e. one or more ring atoms may be heteroatoms, such as nitrogen, oxygen, sulfur. The cyclic carrier may be a single ring system or may contain two or more rings, such as fused rings. The cyclic carrier may be a fully saturated ring system or it may contain one or more double bonds.

The ligand may be linked to the polynucleotide by a carrier. The carrier comprises (i) at least one "backbone attachment point", preferably two "backbone attachment points", and (ii) at least one "tether attachment point". As used herein, "backbone attachment point" refers to a functional group, such as a hydroxyl group, or a bond that is generally useful and suitable for incorporating a carrier into the backbone, such as a phosphate or modified phosphate (e.g., sulfur-containing) backbone of ribonucleic acid. In some embodiments, "tethered attachment point" (TAP) refers to a constituent ring atom of a cyclic carrier that attaches to a selected moiety, such as a carbon atom or a heteroatom (other than the atom providing the backbone attachment point). The moiety may be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected moiety is linked to the cyclic carrier via an intermediate tether. Thus, the cyclic carrier will typically comprise a functional group, such as an amino group, or will typically provide a bond suitable for binding or tethering of another chemical entity (e.g., a ligand that makes up a ring).

The RNAi agent can be conjugated to the ligand via a carrier, wherein the carrier can be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl and decalinyl; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

In certain particular embodiments, the RNAi agent used in the methods of the present disclosure is an agent selected from the group of agents listed in any one of tables 2-7. These agents may further include ligands. The ligand may be linked to the sense strand, the antisense strand, or both at the 3 'end, the 5' end, or both. For example, the ligand may be conjugated to the sense strand, particularly the 3' end of the sense strand.

Irna conjugates

The iRNA agents disclosed herein may be in the form of conjugates. The conjugate may be attached at any suitable position in the iRNA molecule, for example, at the 3 'or 5' end of the sense or antisense strand. The conjugates are optionally linked by a linker.

In some embodiments, an iRNA agent described herein is chemically linked to one or more ligands, moieties, or conjugates, which can confer a function, e.g., by affecting (e.g., enhancing) the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al, 1989Proc. Natl. Acid. Sci. U.S. A.86:6553-6556), cholic acids (Manoharan et al, 1994Biorg. Med. Chem. Let., 4:1053-1060), thioethers, e.g., andalusite-S-triphenylmethyl mercaptan (Manoharan et al, 1992Ann. N. Y. Acad. Sci.,660:306-309; manoharan et al, 1993Biorg. Med. Chem. Lett. 3:2765-2770), thiocholesterol (Oberhauser et al, 1992Nucl.Acids Res.20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al, 1991EMBO J10:1111-1118; kabanov et al 1990FEBS Lett.259:327-330; svinarchuk et al 1993Biochimie 75:49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1, 2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al 1995Tetrahedron Lett.36:3651-3654; shea et al 1990Nucl.Acids Res.18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al 1995Nucleosides&Nucleotides 14:969-973) or adamantaneacetic acid (Manoharan et al 1995Tetrahedron Lett.36:3651-3654), palmitoyl moieties (Mishra et al 1995Biochim.Biophys.Acta 1264:229-237) or octadecylamine or hexylamino-carbonyloxy cholesterol moieties (Crooke et al 1996J. Pharmacol. Exp. Ther. 277:923-937).

In some embodiments, the ligand alters the distribution, targeting, or lifetime of the iRNA agent into which it is incorporated. In some embodiments, the ligand provides enhanced affinity for a selected target (e.g., a molecule, cell, or cell type), compartment (e.g., a cell or organ compartment, tissue, organ, or body region), as compared to a species in which such ligand is not present, for example. Typical ligands will not participate in duplex pairing in duplex nucleic acids.

The ligand may comprise naturally occurring substances, such as proteins (e.g., human Serum Albumin (HSA), low Density Lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or a lipid. The ligand may also be a recombinant molecule or a synthetic molecule, such as a synthetic polymer, e.g. a synthetic polyamino acid. Examples of the polyamino acid include polyamino acids, namely Polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacrylic acid), N-isopropylacrylamide polymer or polyphosphazine. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salts of polyamines, or alpha helical peptides.

The ligand may also comprise a targeting group, such as a cell or tissue targeting agent, e.g. a lectin, glycoprotein, lipid or protein, e.g. an antibody that binds to a specific cell type (e.g. kidney cells). The targeting group may be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein a, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin or RGD peptide mimetic.

Other examples of ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralene, mitomycin C), porphyrins (TPPC, texas porphyrin, expandaphyrins), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxyhexyl, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholanic acid, dimethoxytrityl or phenoxazine), peptide conjugates (e.g., antennapedia mutant peptides, tat peptides), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [ MPEG ] ₂, polyamino groups, alkyl groups, substituted alkyl groups, radiolabelled markers, enzymes, haptens (e.g., biotin), transport/absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, eu3+ complexes of tetraazamacrocycles), dinitrophenyl groups, HRP, or AP.

The ligand may be a protein (e.g., glycoprotein) or peptide (e.g., a molecule having a specific affinity for the co-ligand) or an antibody (e.g., an antibody that binds to a particular cell type, such as an ocular cell). The ligand may also comprise a hormone and a hormone receptor. The ligand may also comprise non-peptide substances such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose or multivalent fucose. The ligand may be, for example, lipopolysaccharide, an activator of p38MAP kinase or an activator of NF- κB.

The ligand may be a substance, such as a drug, which may increase uptake of the iRNA agent into the cell, for example, by disrupting the cytoskeleton of the cell, such as by disrupting microtubules, microfilaments and/or intermediate filaments of the cell. The drug may be, for example, taxane, vincristine, vinblastine, cytochalasin, nocodazole, jestictone (japlakinolide), langchun line A (latrunculin A), parachutein (phalloidin), s Wen Heli A (swinholide A), yin Dannuo octyl (indanocine) or mesenchyme (myoservin).

In some embodiments, the ligand linked to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin. Serum protein-binding oligonucleotides comprising a number of phosphorothioate linkages are also known, and thus short oligonucleotides comprising a number of phosphorothioate linkages in the backbone (e.g., oligonucleotides having about 5 bases, 10 bases, 15 bases, or 20 bases) are also suitable for use in the present disclosure as ligands (e.g., as PK modulating ligands). In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

The ligand-conjugated oligonucleotides of the present disclosure may be synthesized by using oligonucleotides with side-reactive functions, such as those derived from linking molecules attached to the oligonucleotides (as described below). Such reactive oligonucleotides may be reacted directly with commercially available ligands, synthetic ligands with any of a variety of protecting groups, or ligands having a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present disclosure may be conveniently and routinely prepared by well known solid phase synthesis techniques. Devices for such synthesis are sold by several suppliers, including Applied Biosystems (Foster City, calif.). Any other means known in the art for such synthesis may additionally or alternatively be employed. Other oligonucleotides (e.g., phosphorothioates and alkylated derivatives) are also known to be prepared using similar techniques.

In the ligand-conjugated oligonucleotides and ligand molecules with sequence-specific linked nucleosides of the present disclosure, the oligonucleotides and oligonucleotides may be assembled on a suitable DNA synthesizer using standard nucleotides or nucleoside precursors or nucleotides or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotides or nucleoside conjugate precursors already bearing a ligand molecule or non-nucleoside ligands bearing a building block.

When using nucleotide conjugate precursors that already carry a linking moiety, synthesis of the sequence-specific linked nucleoside is typically accomplished, and then the ligand molecule reacts with the linking moiety to form a ligand conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present disclosure are synthesized by an automated synthesizer using a phosphoramidite derived from a ligand-nucleoside conjugate as well as standard and non-standard phosphoramidites commercially available and conventionally used for oligonucleotide synthesis.

1. Lipophilic moiety

In certain embodiments, the lipophilic moiety is an aliphatic, cycloaliphatic (e.g., alicyclic), or polycyclic (e.g., polycycloaliphatic) compound, such as a steroid (e.g., a sterol) or a straight or branched chain aliphatic hydrocarbon. The lipophilic moiety may generally comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may include various substituents or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic moieties include, but are not limited to, saturated or unsaturated C ₄-C₃₀ hydrocarbons (e.g., C ₆-C₁₈ hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C ₁₀ terpenes, C ₁₅ sesquiterpenes, C ₂₀ diterpenes, C ₃₀ triterpenes, and C ₄₀ tetraterpenes), and other multi-alicyclic hydrocarbons. For example, the lipophilic moiety may contain a C ₄-C₃₀ hydrocarbon chain (e.g., a C ₄-C₃₀ alkyl or alkenyl group). In some embodiments, the lipophilic moiety contains a saturated or unsaturated C ₆-C₁₈ hydrocarbon chain (e.g., a straight C ₆-C₁₈ alkyl or alkenyl group). In some embodiments, the lipophilic moiety contains a saturated or unsaturated C ₁₆ hydrocarbon chain (e.g., a straight C ₁₆ alkyl or alkenyl group).

The lipophilic moiety may be attached to the RNAi agent by any means known in the art, including by functional groups already present in the lipophilic moiety or incorporated into the RNAi agent, such as hydroxyl groups (e.g., -CO-CH ₂ -OH). Functional groups that are already present in the lipophilic moiety or introduced into the RNAi agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

Conjugation of the RNAi agent to the lipophilic moiety can occur, for example, by forming an ether or carboxylic acid or carbamoyl ester linkage between the hydroxyl and alkyl R-, alkanoyl RCO-, or substituted carbamoyl RNHCO-. The alkyl group R may be cyclic (e.g., cyclohexyl) or acyclic (e.g., linear or branched; and saturated or unsaturated). The alkyl R may be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, or the like.

In some embodiments, the lipophilic moiety is conjugated to the double stranded RNAi agent by a linker comprising: ethers, thioethers, ureas, carbonates, amines, amides, maleimide-thioethers, disulfides, phosphodiesters, sulfonamide linkages, click reaction products (e.g., triazoles from azide-alkyne cycloaddition), or carbamates.

In another embodiment, the lipophilic moiety is a steroid, such as a sterol. Steroids are polycyclic compounds containing a perhydro-1, 2-cyclopentaphenanthrenering system. Steroids include, but are not limited to, bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxin, testosterone, cholesterol, and cationic steroids (e.g., cortisone). "cholesterol derivative" refers to a compound derived from cholesterol, for example, by substitution, addition or removal of substituents.

In another embodiment, the lipophilic moiety is an aromatic moiety. In this context, the term "aromatic" refers broadly to mono-and poly-aromatic hydrocarbons. Aromatic groups include, but are not limited to, C ₆-C₁₄ aryl moieties including one to three aromatic rings, which may be optionally substituted; "aralkyl" or "arylalkyl" including aryl groups covalently linked to alkyl groups, either of which may independently be optionally substituted or unsubstituted; a "heteroaryl" group. As used herein, the term "heteroaryl" refers to a group having 5 to 14 ring atoms, preferably 5, 6, 9 or 10 ring atoms; having 6, 10 or 14 pi electrons shared in a cyclic array and having one to about three heteroatoms selected from nitrogen (N), oxygen (O) and sulfur (S) in addition to carbon atoms.

As used herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl is a group having one to about four, preferably one to about three, more preferably one or two non-hydrogen substituents. Suitable substituents include, but are not limited to, halo, hydroxy, nitro, haloalkyl, alkyl, alkylaryl, aryl, aralkyl, alkoxy, aryloxy, amino, amido, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonylamino, arenesulfonylamino, aralkylsulfonylamino, alkylcarbonyl, acyloxy, cyano and ureido.

In some embodiments, the lipophilic moiety is an aralkyl moiety, e.g., a 2-aryl propionyl moiety. The structural characteristics of the aralkyl group are selected such that the lipophilic moiety will bind to at least one protein in vivo. In certain embodiments, the structural characteristics of the aralkyl group are selected such that the lipophilic moiety binds to serum, blood vessels, or cellular proteins. In certain embodiments, the structural features of the aralkyl group promote binding to albumin, immunoglobulin, lipoprotein, alpha-2-macroglobulin, or alpha-1-glycoprotein.

In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. Synthetic procedures for naproxen can be found in U.S. patent No.3,904,682 and U.S. patent No.4,009,197, which are hereby incorporated by reference in their entirety. Naproxen has the chemical name (S) -6-methoxy-alpha-methyl-2-naphthyridine acetic acid and the structure

In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. The synthetic procedure for ibuprofen can be found in US3,228,831, which is incorporated herein by reference for the methods provided therein. The structure of ibuprofen is

Further exemplary aralkyl groups are described in US 7,626,014, which is incorporated herein by reference for the methods provided therein.

In another embodiment, suitable lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxyhexanethiol, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) bile acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

In certain embodiments, more than one lipophilic moiety may be incorporated into the double stranded RNAi agent, particularly when the lipophilic moiety has low lipophilicity or hydrophobicity. In some embodiments, two or more lipophilic moieties are incorporated into the same strand of the double stranded RNAi agent. In some embodiments, each strand of the double stranded RNAi agent has one or more lipophilic moieties incorporated. In some embodiments, two or more lipophilic moieties are incorporated at the same position (i.e., the same nucleobase, the same sugar moiety, or the same internucleoside linkage) of the double stranded RNAi agent. This may be achieved, for example, by conjugating two or more lipophilic moieties via an overload agent, or by conjugating two or more lipophilic moieties via a branched linker, or by conjugating two or more lipophilic moieties via one or more linkers, wherein one or more linkers continuously link the lipophilic moieties.

The lipophilic moiety may be conjugated to the RNAi agent via a direct linkage to the ribose of the RNAi agent. Alternatively, the lipophilic moiety may be conjugated to the double stranded RNAi agent via a linker or carrier.

In certain embodiments, the lipophilic moiety may be conjugated to the RNAi agent via one or more linkers (tethers).

In some embodiments, the lipophilic moiety is conjugated to the double stranded RNAi agent by a linker comprising: ethers, thioethers, ureas, carbonates, amines, amides, maleimide-thioethers, disulfides, phosphodiesters, sulfonamide linkages, products of click reactions (e.g., triazoles from azide-alkyne cycloadditions), or carbamates.

2. Lipid conjugates

In some embodiments, the ligand is a lipid or lipid-based molecule. Such lipids or lipid-based molecules may typically bind to serum proteins, such as Human Serum Albumin (HSA). HSA binding ligands allow vascularization of the conjugate to the target tissue. For example, the target tissue may be an eye. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin may be used. The lipid or lipid-based ligand may (a) increase resistance to conjugate degradation, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) may be used to modulate binding to a serum protein, such as HSA.

Lipid-based ligands can be used to modulate, e.g., inhibit, the binding of the conjugate to a target tissue. For example, lipids or lipid-based ligands that bind more strongly to HSA will be less likely to be targeted to the kidneys and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the conjugate to the kidney.

In some embodiments, the lipid-based ligand binds to HSA. For example, the ligand may bind HSA with sufficient affinity to enhance the distribution of the conjugate in non-kidney tissue. However, affinity is generally not so strong that HSA ligand binding cannot be reversed.

In some embodiments, the lipid-based ligand binds weakly to HSA or not at all, such that the distribution of the conjugate in the kidney is enhanced. Other moieties targeted to kidney cells may also be used instead of or in addition to lipid-based ligands.

In another aspect, the ligand is a moiety, such as a vitamin, that is taken up by the target cell (e.g., a proliferating cell). These are particularly useful for treating diseases characterized by undesired cell proliferation, such as malignant or non-malignant types of diseases, such as cancer cells. Exemplary vitamins include vitamins A, E and K. Other exemplary vitamins that are included are B vitamins such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells. HSA and Low Density Lipoprotein (LDL) are also included.

3. Cell penetrating agent

In another aspect, the ligand is a cell penetrating agent, such as a helical cell penetrating agent. In some embodiments, the agent is amphiphilic. Exemplary agents are peptides, such as tat or antennapedia mutant peptides. If the agent is a peptide, it may be modified, including peptidomimetics, inversion bodies, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helices are typically alpha-helices and may have a lipophilic and lipophobic phase.

The ligand may be a peptide or a peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptide mimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic profile of iRNA, such as by enhancing cell recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, for example about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

The peptide or peptidomimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., consisting essentially of Tyr, trp, or Phe). The peptide moiety may be a dendrimer peptide, a constraint peptide or a cross-linked peptide. In another alternative, the peptide moiety may comprise a hydrophobic Membrane Translocation Sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). RFGF analogs (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) containing a hydrophobic MTS may also be targeting moieties. The peptide moiety may be a "delivery" peptide that can carry a large polar molecule including peptides, oligonucleotides and proteins across the cell membrane. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and drosophila (Drosophila Antennapedia) antennapedia mutein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been found to function as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al, nature,354:82-84,1991). Typically, examples of peptides or peptidomimetics that are linked to the dsRNA agent via an incorporated monomer unit are cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD) -peptides or RGD mimetics. The peptide portion may range in length from about 5 amino acids to about 40 amino acids. The peptide moiety may have structural modifications, such as increased stability or direct conformational properties. Any of the structural modifications described below may be used.

RGD peptides for use in the compositions and methods of the present disclosure may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a particular tissue. The RGD-containing peptides and peptide mimetics may comprise D-amino acids and synthetic RGD mimetics. In addition to RGD, other moieties that target integrin ligands can be used. In some embodiments, the conjugate of this ligand targets PECAM-1 or VEGF.

The RGD peptide moiety may be used to target specific cell types, for example tumor cells, such as endothelial tumor cells or breast Cancer tumor cells (Zitzmann et al, cancer Res.,62:5139-43,2002). RGD peptides can promote targeting of dsRNA agents to tumors of a variety of other tissues, including lung, kidney, spleen, or liver (Aoki et al CANCER GENE THERAPY 8:783-787,2001). Generally, RGD peptides will promote targeting of iRNA agents to the kidneys. RGD peptides may be linear or cyclic and may be modified, e.g. glycosylated or methylated, to facilitate targeting to a particular tissue. For example, glycosylated RGD peptides can deliver iRNA agents to tumor cells expressing αVβ3 (Haubner et al, journal. Nucl. Med.,42:326-336,2001).

"Cell penetrating peptide" is capable of penetrating a cell, such as a microbial cell (e.g., a bacterial or fungal cell) or a mammalian cell (e.g., a human cell). The microbial cell penetrating peptide may be, for example, an alpha-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide-containing peptide (e.g., an alpha-defensin, a beta-defensin, or a bacteriocin), or a peptide containing only one or two major amino acids (e.g., PR-39 or indomethacin). Cell penetrating peptides may also comprise Nuclear Localization Signals (NLS). For example, the cell penetrating peptide may be a bipartite amphiphilic peptide, such as MPG, derived from the fusion peptide domain of HIV-1gp41 and NLS of the SV40 large T antigen (Simeoni et al, 2003Nucl.Acids Res.31:2717-2724).

4. Carbohydrate conjugates and ligands

In some embodiments of the compositions and methods of the present disclosure, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate conjugated iRNA is advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use as described herein. As used herein, "carbohydrate" refers to a compound that is a carbohydrate that itself is comprised of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched, or cyclic), wherein an oxygen, nitrogen, or sulfur atom is bound to each carbon atom; or a compound having as part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched or cyclic), wherein an oxygen, nitrogen or sulfur atom is bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; disaccharides and trisaccharides include saccharides having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, the compositions and methods of the present disclosure comprise a C16 ligand. In exemplary embodiments, the C16 ligands of the present disclosure have the following structure (exemplified below for uracil bases, however, for nucleotides exhibiting any base (C, G, A, etc.) or having any other modification presented herein, ligation of the C16 ligand is contemplated, provided that a2 'ribose linkage is retained), and is linked at the 2' position of the thus modified residue, the core sugar:

as indicated above, the C16 ligand modified residue presents a linear alkyl group at the 2' -ribose position of the exemplary residue so modified (uracil here).

In some embodiments, the carbohydrate conjugates of RNAi agents of the present disclosure further comprise one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell penetrating peptide.

Additional carbohydrate conjugates and linkers suitable for the present disclosure include those described in WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

In certain embodiments, the compositions and methods of the present disclosure comprise Vinyl Phosphonate (VP) modifications of RNAi agents as described herein. In an exemplary embodiment, the vinyl phosphonate of the present disclosure has the following structure:

For example, when the phosphate mimic is 5 '-Vinyl Phosphonate (VP), the 5' -terminal nucleotide may have the following structure,

Wherein indicates the position of the bond at the 5' position adjacent to the nucleotide;

R is hydrogen, hydroxy, methoxy, fluoro (e.g., hydroxy or methoxy), or another modification described herein; and

B is a nucleobase or a modified nucleobase, optionally wherein B is adenine, guanine, cytosine, thymine or uracil.

The vinyl phosphonate of the present disclosure can be linked to the antisense strand or sense strand of the dsRNA of the present disclosure. In certain embodiments, a vinylphosphonate of the present disclosure is linked to the antisense strand of a dsRNA, optionally at the 5' end of the antisense strand of a dsRNA. The dsRNA agent may include a phosphorus-containing group at the 5' end of the sense strand or the antisense strand. The 5 'terminal phosphorus-containing group may be a 5' terminal phosphate (5 '-P), a 5' terminal phosphorothioate (5 '-PS), a 5' terminal phosphorothioate diester (5 '-PS 2), a 5' terminal vinylphosphonate (5 '-VP), a 5' terminal methylphosphonate (MePhos), or a 5 '-deoxy-5' -C-malonyl. When the 5' -terminal phosphorus-containing group is a 5' -terminal vinyl phosphonate (5 ' -VP), the 5' -VP may be the 5' -E-VP isomer (i.e., trans-vinyl phosphonate)) The 5' -Z-VP isomer (i.e., cis-vinyl phosphonate) Or a mixture thereof.

Vinyl phosphonate modifications are also contemplated for use in the compositions and methods of the present disclosure. Exemplary vinyl phosphate phosphonate structures are:

For example, when the phosphate mimic is a 5 '-vinyl phosphonate, the 5' terminal nucleotide may have a direct structure in which the phosphonate group is replaced with a phosphate.

In some embodiments, the carbohydrate conjugate comprises a monosaccharide. In some embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in U.S. patent No. 8,106,022, the entire contents of which are hereby incorporated by reference. In some embodiments, galNAc conjugates are used as ligands to target iRNA to a particular cell. In some embodiments, galNAc conjugates target iRNA to liver cells, e.g., by acting as a ligand for an asialoglycoprotein receptor of a liver cell (e.g., a liver cell).

In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. GalNAc derivatives may be linked by a linker, for example, a divalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3' end of the sense strand) via a linker, such as the linkers described herein.

In some embodiments, the GalNAc conjugate is

In some embodiments, the RNAi agent is linked to the carbohydrate conjugate by a linker, as shown in the following schematic, wherein X is O or S:

In some embodiments, the RNAi agent is conjugated to L96 as defined in table 1, and is as follows:

in some embodiments, the carbohydrate conjugates used in the compositions and methods of the present disclosure are selected from the following:

Another representative carbohydrate conjugate for use in embodiments described herein includes, but is not limited to:

When one of X or Y is an oligonucleotide, the other is hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell penetrating peptide.

In some embodiments, an iRNA of the present disclosure is conjugated to a carbohydrate through a linker. Non-limiting examples of linkers of iRNA carbohydrate conjugates with the compositions and methods of the present disclosure include, but are not limited to:

When one of X or Y is an oligonucleotide, the other is hydrogen.

5. Thermal instability modification

In certain embodiments, RNA interference of dsRNA molecules can be optimized by incorporating thermally labile modifications in the seed region of the antisense strand. As used herein, "seed region" refers to positions 2-9 of the 5' end of the reference chain. For example, thermally labile modifications can be incorporated into the seed region of the antisense strand to reduce or inhibit off-target gene silencing.

The term "thermally labile modification" includes modifications that will result in a dsRNA having a lower total melting temperature (Tm) than dsRNA without such modification. For example, the thermally labile modification can reduce the Tm of the dsRNA by 1-4 ℃, e.g., 1,2,3, or 4 ℃. Also, the term "thermally labile nucleotide" refers to a nucleotide containing one or more thermally labile modifications.

It has been found that dsRNA with an antisense strand comprises a thermally labile modification of at least one duplex within the first 9 nucleotide positions of the antisense strand (counted from the 5' end), which reduces off-target gene silencing activity. Thus, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermostable modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more thermally labile modifications of the duplex are located at positions 2-9, or preferably positions 4-8, of the 5' end of the antisense strand. In some further embodiments, the thermostable modification of the duplex is located at position 6, 7 or 8 of the 5' end of the antisense strand. In yet further embodiments, the thermostable modification of the duplex is located at position 7 of the 5' end of the antisense strand. In some embodiments, the thermostable modification of the duplex is located at position 2,3,4, 5 or 9 of the 5' end of the antisense strand.

The thermally labile modification may include, but is not limited to, an abasic modification; mismatches with the opposite nucleotide in the opposite strand; and sugar modifications, such as 2' -deoxy modifications or acyclic nucleotides, e.g., unlocking Nucleic Acids (UNA) or diol nucleic acids (GNA).

Exemplary abasic modifications include, but are not limited to, the following:

Wherein r=h, me, et or OMe; r' =h, me, et or OMe; r "=h, me, et or OMe

Wherein B is a modified or unmodified nucleobase.

Exemplary sugar modifications include, but are not limited to, the following:

Wherein B is a modified or unmodified nucleobase.

In some embodiments, the thermally labile modification of the duplex is selected from the group consisting of:

Wherein B is a modified or unmodified nucleobase and each structurally asterisk represents R, S or a racemate.

The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose, e.g., where any bond between ribose carbons (e.g., C1' -C2', C2' -C3', C3' -C4', C4' -O4', or C1' -O4 ') is absent or at least one ribose carbon or oxygen (e.g., C1', C2', C3', C4', or O4 ') is absent in the nucleotide, either independently or in combination. In some embodiments, the acyclic nucleotide is

Wherein B is a modified OR unmodified nucleobase, R ¹ and R ² are independently H, halogen, OR ₃ OR alkyl; r ₃ is H, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar). The term "UNA" refers to an unlocked acyclic nucleic acid in which any bonds of the sugar have been removed, forming an unlocked "sugar" residue. In one example, UNA also encompasses monomers where the bond between C1'-C4' is removed (i.e., covalent carbon-oxygen-carbon bonds between C1 'and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2 'and C3' carbons) is removed (see Mikhailov et al, 1985Tetrahedron Letters 26 (17): 2059; and Fluiter et al, 2009mol. Biosystem., 10:1039, which is hereby incorporated by reference in its entirety). Acyclic derivatives provide greater backbone flexibility without affecting Watson-Crick pairing. The acyclic nucleotides may be linked by a 2'-5' or 3'-5' linkage.

The term "GNA" refers to a diol nucleic acid, which is a polymer similar to DNA or RNA, but whose "backbone" is of different composition, consisting of repeating glycerol units linked by phosphodiester bonds:

The thermally labile modification of the duplex may be a mismatch (i.e., a non-complementary base pair) between a thermally labile nucleotide and an opposite nucleotide in an opposite strand in the dsRNA duplex. Exemplary mismatched base pairs include G G, G: A, G: U, G: T, A: A, A: C, C: C, C: U, C: T, U: U, T: T, U:T or combinations thereof. Other mismatched base pairs known in the art are also suitable for use in the present invention. Mismatches may occur between the nucleotides of naturally occurring or modified nucleotides, i.e., mismatched base pairs may occur between nucleobases from each nucleotide, regardless of the modification on the ribose of the nucleotide. In certain embodiments, the dsRNA molecule comprises at least one nucleobase, i.e., a 2' -deoxynucleobase, in a mismatch pair; for example, the 2' -deoxynucleobase is located in the sense strand.

In some embodiments, the thermally labile modification of the duplex in the seed region of the antisense strand includes a nucleotide with impaired W-C H binding to a complementary base on the target mRNA, for example:

further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO 2011/133876, which is incorporated herein by reference in its entirety.

Thermally labile modifications can also include universal bases whose ability to form hydrogen bonds with opposing bases is reduced or eliminated, as well as phosphate modifications.

In some embodiments, the thermally labile modification of the duplex includes a nucleotide having a non-standard base, such as, but not limited to, a nucleobase modification having the ability to damage or completely eliminate hydrogen bond formation with bases in the opposite strand. Destabilization of the central region of dsRNA duplex by these nucleobase modifications has been evaluated as described in WO 2010/0011895, which is incorporated herein by reference in its entirety. Exemplary nucleobase modifications are:

in some embodiments, the thermally labile modification of the duplex in the seed region of the antisense strand includes one or more α -nucleotides complementary to a base on the target mRNA, such as:

Wherein R is H, OH, OCH ₃、F、NH₂、NHMe、NMe₂ or O-alkyl.

Exemplary phosphate modifications that are known to reduce the thermal stability of dsRNA duplex compared to native phosphodiester linkages are:

The alkyl of the R group may be a C ₁-C₆ alkyl. Specific alkyl groups for the R group include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

As will be appreciated by those of skill in the art, whereas the functional role of nucleobases is to define the specificity of RNAi agents of the present disclosure, while nucleobase modifications may be made as described herein in various ways, e.g., to introduce destabilizing modifications to RNAi agents of the present disclosure, e.g., to enhance targeting effects relative to off-target effects, the range of modifications available to non-nucleobase modifications, e.g., modifications to the glycosyl or phosphate backbone of a polyribonucleotide, and typically present on RNAi agents of the present disclosure, tends to be greater. Such modifications are described in more detail in other parts of the disclosure, and RNAi agents of the disclosure are specifically contemplated having a natural nucleobase or a modified nucleobase as described above or elsewhere herein.

In addition to the antisense strand comprising a heat labile modification, the dsRNA may also comprise one or more stabilizing modifications. For example, a dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stable modifications. Without limitation, stable modifications may be present in one strand. In some embodiments, both the sense strand and the antisense strand comprise at least two stable modifications. The stabilizing modification may occur on any nucleotide of the sense strand or the antisense strand. For example, the stabilizing modification may occur on each nucleotide on the sense strand or the antisense strand; each stabilizing modification may occur on the sense strand or the antisense strand in an alternating pattern; or both the sense and antisense strands comprise an alternating pattern of stable modifications. The alternating pattern of stable modifications on the sense strand may be the same or different than the alternating pattern of stable modifications on the antisense strand, and the alternating pattern of stable modifications on the sense strand may be offset relative to the alternating pattern of stable modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stable modifications. Without limitation, stable modifications in the antisense strand may be present at any position.

In some embodiments, the antisense comprises stable modifications at positions 2, 6, 8, 9, 14 and 16 starting from the 5' end. In some other embodiments, the antisense comprises stable modifications at positions 2, 6, 14 and 16 starting from the 5' end. In yet other embodiments, the antisense comprises stable modifications at positions 2, 14 and 16 starting from the 5' end.

In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to a destabilizing modification. For example, the stabilizing modification may be a 5 'or 3' nucleotide of the destabilizing modification, i.e. a nucleotide at position-1 or +1 starting from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5 'and 3' ends of the destabilizing modification, i.e., from the position of the destabilizing modification, -1 and +1.

In some embodiments, the antisense strand comprises at least two stabilizing modifications 3' to the destabilizing modification, i.e., at positions +1 and +2 of the destabilizing modification.

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stable modifications. Without limitation, stable modifications in the sense strand may be present at any position. In some embodiments, the sense strand comprises stable modifications at positions 7, 10, and 11 starting from the 5' end. In some other embodiments, the sense strand comprises stable modifications at positions 7, 9, 10 and 11 starting from the 5' end. In some embodiments, the sense strand comprises a stable modification at a position opposite or complementary to positions 11, 12 and 15 of the antisense strand counted from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises a stable modification at a position opposite or complementary to positions 11, 12, 13 and 15 on the antisense strand counted from the 5' end of the antisense strand. In some embodiments, the sense strand comprises a set of two, three, or four stable modifications.

In some embodiments, the sense strand does not comprise a stabilizing modification at a position opposite or complementary to the thermally labile modification of the duplex in the antisense strand.

Exemplary thermostable modifications include, but are not limited to, 2' -fluoro modifications. Other thermostable modifications include, but are not limited to, LNA.

In some embodiments, the dsRNA of the disclosure comprises at least four (e.g., four, five, six, seven, eight, nine, ten, or more) 2' -fluoro nucleotides. Without limitation, 2' -fluoro nucleotides may all be present in one strand. In some embodiments, both the sense strand and the antisense strand comprise at least two 2' -fluoro nucleotides. The 2' -fluoro modification may occur on any nucleotide of the sense strand or the antisense strand. For example, a 2' -fluoro modification can occur on each nucleotide on the sense strand or the antisense strand; each 2' -fluoro modification may occur on the sense strand or the antisense strand in an alternating pattern; either the sense or antisense strand comprises an alternating pattern of 2' -fluoro modifications. The alternating pattern of 2' -fluoro modifications on the sense strand may be the same or different than the alternating pattern of stable modifications on the antisense strand, and the alternating pattern of 2' -fluoro modifications on the sense strand may be offset relative to the alternating pattern of 2' -fluoro modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2' -fluoro modifications. Without limitation, the 2' -fluoro modification in the antisense strand may be present at any position. In some embodiments, the antisense comprises 2 '-fluoro modifications at positions 2, 6, 8, 9, 14 and 16 starting from the 5' end. In some embodiments, the antisense comprises 2 '-fluoro modifications at positions 2, 6, 14 and 16 starting from the 5' end. In yet other embodiments, the antisense comprises 2 '-fluoro modifications at positions 2, 14 and 16 starting from the 5' end.

In some embodiments, the antisense strand comprises at least one 2' -fluoro modification adjacent to a destabilizing modification. For example, the 2' -fluoro modified nucleotide may be a destabilized modified 5' or 3' nucleotide, i.e., a nucleotide at position-1 or +1 from the position of the destabilization modification. In some embodiments, the antisense strand comprises 2' -fluoro nucleotides at each of the 5' and 3' ends of the destabilization modification, i.e., -1 and +1 from the position of the destabilization modification.

In some embodiments, the antisense strand comprises at least two 2 '-fluoro nucleotides at the 3' end of the destabilization modification, i.e., at positions +1 and +2 of the destabilization modification.

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2' -fluoro nucleotides. Without limitation, the 2' -fluoro modification in the sense strand may be present at any position. In some embodiments, the antisense comprises 2 '-fluoro nucleotides at positions 7, 10, and 11 starting from the 5' end. In other embodiments, the sense strand comprises 2 '-fluoro nucleotides at positions 7, 9, 10 and 11 starting from the 5' end. In some embodiments, the sense strand comprises 2 '-fluoro nucleotides at positions opposite or complementary to positions 11, 12 and 15 of the antisense strand counted from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises 2 '-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13 and 15 of the antisense strand counted from the 5' end of the antisense strand. In some embodiments, the sense strand comprises a set of two, three, or four 2' -fluoro nucleotides.

In some embodiments, the sense strand does not comprise a 2' -fluoro nucleotide at a position opposite or complementary to the thermally labile modification of the duplex in the antisense strand.

In some embodiments, a dsRNA molecule of the present disclosure comprises a sense strand of 21 nucleotides (nt) and an antisense strand of 23 nucleotides (nt), wherein the antisense strand comprises at least one thermally labile nucleotide, wherein the at least one thermally labile nucleotide occurs in a seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand), wherein one end of the dsRNA is blunt and the other end comprises a 2nt overhang, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) antisense comprises 2, 3,4, 5 or 6 2' -fluoro modifications; (ii) Antisense comprises 1,2, 3,4, or 5 phosphorothioate internucleotide linkages; (iii) conjugation of the sense strand to the ligand; (iv) the sense strand comprises 2, 3,4 or 5 2' -fluoro modifications; (v) The sense strand comprises 1,2, 3,4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2' -fluoro modifications; and (vii) the dsRNA comprises a blunt end at the 5' end of the antisense strand. Preferably, the 2nt overhang is at the antisense 3' end.

In some embodiments, each nucleotide on the sense and antisense strands of the dsRNA molecule can be modified. Each nucleotide may be modified with the same or different modifications, which may include one or two or one or more changes in one or more of the non-linked phosphate oxides; altering the composition of ribose, such as the 2' hydroxyl group on ribose; a substantial replacement of the phosphate moiety with a "dephosphorylation" linker; modification or substitution of natural bases; substitution or modification of the ribose-phosphate backbone.

Since nucleic acids are polymers of subunits, many modifications occur at repeated positions within the nucleic acid, such as modifications of bases or phosphate moieties, or non-linking O of phosphate moieties. In some cases, the modification will occur at all positions in the nucleic acid, but in many cases will not. For example, the modification may occur only at the 3 'or 5' end position, may occur only at a terminal region, such as a position on a terminal nucleotide of one strand or the last 2, 3, 4, 5 or 10 nucleotides. Modification may occur in the double-stranded region, the single-stranded region, or both. Modification may occur only in the double-stranded region of the RNA or in the single-stranded region of the RNA. For example, phosphorothioate modifications at non-linked O positions may occur at only one or both ends, may occur at only the end region, e.g. at the end nucleotide position or last 2, 3, 4, 5 or 10 nucleotides of one strand, or may occur at double-and single-stranded regions, particularly at the ends. The 5' end or ends may be phosphorylated.

For example, stability may be improved, including a specific base at the overhang, or including a modified nucleotide or nucleotide substitute in a single stranded overhang, such as in a 5 'or 3' overhang, or both. For example, it may be desirable to include purine nucleotides at the overhangs. In some embodiments, all or some of the bases in the 3 'or 5' overhangs may be modified, for example with modifications described herein. Modifications may include, for example, modifications at the 2' position of the ribose using those known in the art, such as ribose modification using deoxyribonucleotides, 2' -deoxy-2 ' -fluoro (2 ' -F), or 2' -O-methyl groups instead of nucleobases, and modifications of phosphate groups, such as phosphorothioate modifications. The overhangs need not be homologous to the target sequence.

In some embodiments, each residue of the sense and antisense strands is independently Locked Nucleic Acid (LNA), unlocked Nucleic Acid (UNA), cyclohexene nucleic acid (CeNA), 2 '-methoxyethyl, 2' -O-methyl, 2 '-O-allyl, 2' -C-allyl, 2 '-deoxy, or 2' -fluoro. The chain may comprise more than one modification. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with 2 '-O-methyl or 2' -fluoro. It will be appreciated that these modifications are complementary to at least one thermally labile modification of the duplex present in the antisense strand.

There are typically at least two different modifications on the sense and antisense strands. The two modifications may be 2' -deoxy, 2' -O-methyl or 2' -fluoro modifications, acyclic nucleotides or others. In some embodiments, the sense strand and the antisense strand each comprise two different modified nucleotides selected from 2 '-O-methyl or 2' -deoxy. In some embodiments, the sense strand and the antisense strand are each independently surrounded by 2' -O-methyl nucleotides, 2' -deoxynucleotides, 2' -deoxy-2 ' -fluoro nucleotides, 2' -O-N-methylacetamido (2 ' -O-NMA) nucleotides, 2' -O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE) nucleotides, 2' -O-aminopropyl (2 ' -O-AP) nucleotides, or 2' -aza-F nucleotides. Likewise, it is understood that these modifications are complementary to at least one thermally labile modification of the duplex present in the antisense strand.

In some embodiments, the dsRNA of the present disclosure comprises an alternating pattern of modification, particularly in the B1, B2, B3, B1', B2', B3', B4' regions. The term "alternating motif" or "alternating pattern" as used herein refers to a motif with one or more modifications, each modification occurring on alternating nucleotides of one strand. Alternate nucleotides may refer to one or one every three nucleotides for each other nucleotide, or a similar pattern. For example, if A, B and C represent a modification to a nucleotide, respectively, the alternative motif may be "ABABABABABAB …", "AABBAABBAABB …", "AABAABAABAAB …", "AAABAAABAAAB …", "AAABBBAAABBB …" or "ABCABCABCABC …", etc.

The types of modifications contained in the alternating motifs may be the same or different. For example, if A, B, C, D each represents a modification on a nucleotide, then the alternating pattern, i.e., the modifications on every other nucleotide, may be identical, but each sense strand or antisense strand may be selected from several modification possibilities within the alternating motif, e.g., "ABABAB …", "ACACAC …", "BDBDBD …", or "CDCDCD …", etc.

In some embodiments, the dsRNA molecules of the present disclosure comprise a modification pattern of an alternating motif on the sense strand that is displaced relative to a modification pattern of an alternating motif on the antisense strand. The shift may be such that the modified nucleotide set of the sense strand corresponds to a different modified nucleotide set of the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand may begin with "ABABAB" starting from the 5'-3' of the strand and the alternating motif in the antisense strand may begin with "BABABA" starting from the 3'-5' within the duplex region. As another example, the alternating motif in the sense strand may start with "AABBAABB" starting from the 5'-3' of the strand and the alternating motif in the antisense strand may start with "BBAABBAA" starting from the 3'-5' within the duplex region, such that a complete or partial shift in the modification pattern between the sense and antisense strands occurs.

The dsRNA of the present disclosure further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. Phosphorothioate or methylphosphonate internucleotide linkage modifications may occur on any nucleotide of the sense or antisense strand or at any position of the strand. For example, internucleotide linkage modifications may occur on each nucleotide of the sense or antisense strand; each internucleotide linkage modification may occur on either the sense strand or the antisense strand in an alternating pattern; either the sense or antisense strand comprises an alternating pattern of two internucleotide linkage modifications. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may be shifted relative to the alternating pattern of internucleotide linkage modifications on the antisense strand.

In some embodiments, the dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide linkage modifications on the overhang region. For example, the overhang region comprises two nucleotides having phosphorothioate or methylphosphonate internucleotide linkages. Internucleotide linkage modifications may also be made to link the overhanging nucleotides to terminal pairing nucleotides within the duplex region. For example, at least 2,3,4, or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages, and optionally, additional phosphorothioate or methylphosphonate internucleotide linkages can be present to link the overhang nucleotide to the paired nucleotide immediately adjacent to the overhang nucleotide. For example, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are the overhang nucleotides and the third is the pairing nucleotide immediately adjacent to the overhang nucleotide. Preferably, these terminal three nucleotides may be 3' to the antisense strand.

In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphointernucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphoester internucleotide linkages, or an antisense strand comprising a phosphorothioate or methylphosphonate or phosphoester linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphointernucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphoester internucleotide linkages, or an antisense strand comprising a phosphorothioate or methylphosphonate or phosphoester linkage.

In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphointernucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphoester internucleotide linkages, or an antisense strand comprising a phosphorothioate or methylphosphonate or phosphoester linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphointernucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising a phosphorothioate or methylphosphonate or phosphate ester linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphointernucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphoester internucleotide linkages, or an antisense strand comprising a phosphorothioate or methylphosphonate or phosphoester linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1,2, 3,4,5, 6, 7, 8, 9, or 10 phosphointernucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising a phosphorothioate or methylphosphonate or phosphate ester linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphointernucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1,2, 3, 4, 5, or 6 phosphointernucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising a phosphorothioate or methylphosphonate or phosphate ester linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1,2, 3, or 4 phosphointernucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

In some embodiments, the dsRNA of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications at 1-10 of the terminal position of the sense strand or antisense strand. For example, at least 2, 3,4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked by phosphorothioate or methylphosphonate internucleotide linkages at one or both ends of the sense or antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-10 of the duplex interior region of each sense strand or antisense strand. For example, at least 2, 3, 4,5, 6, 7, 8, 9, or 10 nucleotides may be linked by phosphorothioate methylphosphonate internucleotide linkages at positions 8-16 counted from the 5' end of the sense strand through the duplex region; the dsRNA molecule may optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications at terminal positions 1-10.

In some embodiments, the dsRNA molecules of the present disclosure further comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 of the sense strand counted from the 5' end and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the sense strand counted from the 5' end, and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 and 18-23 of the antisense strand counted from the 5' end.

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification in positions 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification in positions 18-23, counting from the 5 'end of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2, counting from the 5' end of the antisense strand, and two phosphorothioate or methylphosphonate internucleotide linkage modifications in positions 18-23.

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications in positions 1-5 and one phosphorothioate internucleotide linkage modification in positions 18-23, counted from the 5 'end of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications in positions 18-23, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications in positions 1-5 and two phosphorothioate internucleotide linkage modifications in positions 18-23, counted from the 5 'end of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications in positions 18-23, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications in positions 1-5 and two phosphorothioate internucleotide linkage modifications in positions 18-23, counted from the 5 'end of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification in positions 18-23, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23, counted from the 5 'end of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23, counted from the 5 'end of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 of the sense strand counting from the 5 'end, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand counting from the 5' end.

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand counting from the 5 'end, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand counting from the 5' end.

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications in positions 1-5 and one phosphorothioate internucleotide linkage modification in positions 18-23, counted from the 5 'end of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification in positions 18-23, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications in positions 1-5 and one phosphorothioate internucleotide linkage modification in positions 18-23, counted from the 5 'end of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications in positions 18-23, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications in positions 1-5 and one phosphorothioate internucleotide linkage modification in positions 18-23, counted from the 5 'end of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications in positions 18-23, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21, counted from the 5 'end of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21, counted from the 5 'end of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22, counted from the 5 'end of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21, counted from the 5 'end of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 22 and 23, counted from the 5 'end of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21, counted from the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21, counted from the 5 'end of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23, counted from the 5' end of the antisense strand.

In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the common mode of the chiral centers of the main chains comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 8 internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 7 internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 6 internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 5 internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 4 internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 3 internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 2 internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 1 internucleotide linkage in the Rp configuration. in some embodiments, the common pattern of backbone chiral centers comprises no more than 8 achiral internucleotide linkages (as a non-limiting example, phosphodiester). In some embodiments, the common pattern of backbone chiral centers comprises no more than 7 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises no more than 6 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises no more than 5 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises no more than 4 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises no more than 3 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises no more than 2 achiral internucleotide linkages. In some embodiments, the common mode of the backbone chiral center comprises no more than 1 achiral internucleotide linkage, in some embodiments, the common mode of the backbone chiral center comprises at least 10 Sp configured internucleotide linkages, and no more than 8 achiral internucleotide linkages. In some embodiments, the common mode of the chiral centers of the main chain comprises at least 11 Sp-configured internucleotide linkages, and no more than 7 achiral internucleotide linkages. In some embodiments, the common mode of the chiral centers of the main chain comprises at least 12 Sp-configured internucleotide linkages, and no more than 6 achiral internucleotide linkages. In some embodiments, the common mode of the chiral centers of the main chain comprises at least 13 Sp-configured internucleotide linkages, and no more than 6 achiral internucleotide linkages. In some embodiments, the common mode of the chiral centers of the main chain comprises at least 14 Sp-configured internucleotide linkages, and no more than 5 achiral internucleotide linkages. In some embodiments, the common mode of the chiral centers of the main chain comprises at least 15 Sp-configured internucleotide linkages, and no more than 4 achiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration are optionally continuous or discontinuous. In some embodiments, the internucleotide linkages in the Rp configuration are optionally continuous or discontinuous. In some embodiments, achiral internucleotide linkages are optionally continuous or discontinuous.

In some embodiments, the compound-containing blocks of the present disclosure are stereochemical blocks. In some embodiments, the block is an Rp block because each internucleotide linkage of the block is Rp. In some embodiments, the 5' block is an Rp block. In some embodiments, the 3' block is an Rp block. In some embodiments, the block is an Sp block in that each internucleotide linkage of the block is Sp. In some embodiments, the 5' block is an Sp block. In some embodiments, the 3' block is an Sp block. In some embodiments, provided oligonucleotides comprise Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but do not comprise an Sp block. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, oligonucleotides are provided comprising one or more PO blocks, wherein each internucleotide linkage is a natural phosphate linkage.

In some embodiments, the compounds of the present disclosure comprise a 5 'block that is an Sp block, wherein each sugar moiety comprises a 2' -F modification. In some embodiments, the 5 'block is an Sp block, wherein each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2' -F modification. In some embodiments, the 5 'block is an Sp block, wherein each internucleotide linkage is a phosphorothioate internucleotide linkage and each sugar moiety comprises a 2' -F modification. In some embodiments, the 5' block comprises 4 or more nucleoside units. In some embodiments, the 5' block comprises 5 or more nucleoside units. In some embodiments, the 5' block comprises 6 or more nucleoside units. In some embodiments, the 5' block comprises 7 or more nucleoside units. In some embodiments, the 3 'block is an Sp block, wherein each sugar moiety comprises a 2' -F modification. In some embodiments, the 3 'block is an Sp block, wherein each internucleotide linkage is a modified nucleotide and each sugar moiety comprises a 2' -F modification. In some embodiments, the 3 'block is an Sp block, wherein each internucleotide linkage is a phosphorothioate internucleotide linkage and each sugar moiety comprises a 2' -F modification. In some embodiments, the 3' block comprises 4 or more nucleoside units. In some embodiments, the 3' block comprises 5 or more nucleoside units, in some embodiments, the 3' block comprises 6 or more nucleoside units, in some embodiments, the 3' block comprises 7 or more nucleoside units.

In some embodiments, a compound of the present disclosure comprises one type of nucleotide or oligonucleotide followed by a specific type of internucleotide linkage in the region, such as a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, and the like. In some embodiments, a is followed by Sp. In some embodiments, a is followed by Rp. In some embodiments, a is followed by a natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, a and G are followed by Sp. In some embodiments, a and G are followed by Rp.

In some embodiments, the dsRNA molecules of the disclosure comprise mismatches with a target, duplex, or combination thereof. Mismatches may occur in the overhang region or duplex region. Base pairs may be ordered according to their propensity to promote dissociation or melting (e.g., according to the binding or dissociation free energy of a particular pairing, the simplest approach being to examine the pairing on a single pairing basis, although the next adjacent or similar analysis may also be used). In promoting dissociation: a is better than G and C; g is better than G and C; and I: C is better than G: C (i=inosine). Mismatches, e.g., pairs that are non-standard or different than standard (as described elsewhere herein) are better than standard (A: T, A: U, G: C) pairs; and pairing comprising universal bases is preferred over standard pairing.

In some embodiments, the dsRNA molecules of the present disclosure comprise at least one of the first 1, 2-7 base pairs within a duplex region at the 5' end of the antisense strand independently selected from the group consisting of: a U, G: U, I, C, and a mismatch pair, e.g., a non-standard or different-standard pairing or pairing comprising a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

In some embodiments, the 1-nucleotide within the duplex region starting from the 5' -end of the antisense strand is selected from A, dA, dU, U and dT. Or at least one of the 1 st, 2 nd or 3 rd base pairs within the duplex region starting from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region starting at the 5' end of the antisense strand is an AU base pair.

It was found that the introduction of a 4' -modified or 5' -modified nucleotide at the 3' -end of a dinucleotide Phosphate (PO), phosphorothioate (PS) or phosphorodithioate (PS 2) linkage at any position of a single-or double-stranded oligonucleotide can produce a steric effect on the internucleotide linkage, thus protecting or stabilizing it from the nuclease.

In some embodiments, the 5 'modified nucleoside is introduced 3' of the dinucleotide at any position of the single-stranded or double-stranded siRNA. For example, a 5 '-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position of a single-stranded or double-stranded siRNA. The alkyl group at the 5' position of ribose may be a racemic or chiral pure R or S isomer. An exemplary 5 '-alkylated nucleoside is a 5' -methyl nucleoside. The 5' -methyl group may be a racemate or a chiral pure R or S isomer.

In some embodiments, the 4 'modified nucleoside is introduced 3' of the dinucleotide at any position of the single-stranded or double-stranded siRNA. For example, a4 '-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position of a single-stranded or double-stranded siRNA. The alkyl group at the 4' position of ribose may be a racemic or chiral pure R or S isomer. An exemplary 4 '-alkylated nucleoside is a 4' -methyl nucleoside. The 4' -methyl group may be a racemate or a chiral pure R or S isomer. Or a4 '-O-alkylated nucleoside can be introduced at the 3' end of the dinucleotide at any position of the single-stranded or double-stranded siRNA. The 4' -O-methyl group of ribose may be a racemic or chiral pure R or S isomer. An exemplary 4 '-O-alkylated nucleoside is 4' -O-methyl nucleoside. The 4' -O-methyl group may be the racemic or chiral pure R or S isomer.

In some embodiments, the 5' -alkylated nucleoside is introduced at any position of the sense strand or antisense strand of the dsRNA, and such modification maintains or increases the efficacy of the dsRNA. The 5' -alkyl group may be a racemic or chirally pure R or S isomer. An exemplary 5 '-alkylated nucleoside is a 5' -methyl nucleoside. The 5' -methyl group may be a racemate or a chiral pure R or S isomer.

In some embodiments, the 4' -alkylated nucleoside is introduced at any position of the sense strand or antisense strand of the dsRNA, and such modification maintains or increases the efficacy of the dsRNA. The 4' -alkyl group may be a racemic or chirally pure R or S isomer. An exemplary 4 '-alkylated nucleoside is a 4' -methyl nucleoside. The 4' -methyl group may be a racemate or a chiral pure R or S isomer.

In some embodiments, the 4' -O-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of the dsRNA, and such modification maintains or increases the efficacy of the dsRNA. The 5' -alkyl group may be a racemic or chirally pure R or S isomer. An exemplary 4 '-O-alkylated nucleoside is 4' -O-methyl nucleoside. The 4' -O-methyl group may be the racemic or chiral pure R or S isomer.

In some embodiments, dsRNA molecules of the present disclosure may comprise 2' -5' linkages (having 2' -H, 2' -OH, and 2' -OMe and having p=o or p=s). For example, 2' -5' bond modifications can be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to avoid RISC activation of the sense strand.

In another embodiment, the dsRNA molecules of the present disclosure can comprise an L-sugar (e.g., L-ribose, L-arabinose with 2' -H, 2' -OH, and 2' -OMe). For example, these L sugar modifications may be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or may be used at the 5' end of the sense strand to avoid RISC activation of the sense strand.

Various publications describe multimeric siRNA that can all be used with the dsRNA of the present disclosure. Such publications include WO2007/091269, US 7858769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520, which are all incorporated herein.

In some embodiments, the dsRNA molecules of the present disclosure are 5 'phosphorylated or comprise a phosphoryl analog at the 5' primer end. The 5' -phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include: 5 '-monophosphate ((HO) ₂ (O) P-O-5'); 5 '-diphosphate ((HO) ₂ (O) P-O-P (HO) (O) -O-5'); 5 '-triphosphate ((HO) ₂ (O) P-O- (HO) (O) P-O-P (HO) (O) -O-5'); 5' -guanosine caps (7-methylated or unmethylated) (7 m-G-O-5' - (HO) (O) P-O-P (HO) (O) -O-5 '); 5' -adenosine caps (Appp) and any modified or unmodified nucleotide cap structures (N-O-5 ' - (HO) (O) P-O- (HO) (O) P-O-P (HO) (O) -O-5 '); 5 '-monothiophosphate (phosphorothioate) (HO) ₂ (S) P-O-5'); 5 '-mono-dithiophosphate (dithiophosphate, (HO) (HS) (S) P-O-5'), 5 '-thiophosphate ((HO ₂ (O) P-S-5'); Any additional combination of oxygen/sulfur substituted mono-, di-, and tri-phosphates (e.g., 5' -alpha-thiophosphoric acid, 5' -gamma-thiophosphoric acid, etc.), 5' -phosphoramidates ((HO) ₂(O)P-NH-5'、(HO)(NH₂) (O) P-O-5 '), 5' -alkylphosphonates (=alkyl = methyl, ethyl, isopropyl, propyl, etc.), e.g., RP (OH) (O) -O-5' -, 5' -alkenylphosphonates (i.e., vinyl, substituted vinyl), (OH) ₂(O)P-5'-CH₂ -), 5 '-alkyl ether phosphonates (r=alkyl ether=methoxymethyl (MeOCH ₂ -), ethoxymethyl, etc., such as RP (OH) (O) -O-5' -). in one example, the modification may be placed in the antisense strand of the dsRNA molecule.

6. Joint

In some embodiments, the conjugates or ligands described herein can be linked to iRNA oligonucleotides having various cleavable or non-cleavable linkers.

The term "linker" or "linking group" refers to an organic moiety that connects two moieties of a compound, e.g., covalently connects two moieties of a compound. The linker typically comprises a direct bond or atom such as oxygen, sulfur, a unit such as NR8, C (O) NH, SO ₂、SO₂ NH, or a chain of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaryl alkyl, alkylaryl alkynyl, alkenylarylalkyl, alkenylaryl, alkenylarylalkynyl, alkynylalkyl, alkynylalkynyl, alkylheteroarylalkyl, heterocyclylalkynyl, alkynylalkyl, alkynylalkynyl, alkylaryl, alkynylalkyl, alkynylalkynyl, alkylaryl, alkynylalkyl, alkylaryl, or alkynylalkyl, or alkynylaalternatively, or in addition, in the present invention, or in addition, in the present invention, or in the present, or in, in or in, to, in or in, to, in or to, and to, to alkyl heteroarylalkyl, alkyl heteroarylalkynyl, alkenyl heteroarylalkyl, alkenyl heteroarylalkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkynyl, alkyl heterocyclylalkyl, alkyl heroylalkynyl, alkenyl heterocyclylalkyl, alkynyl heterocyclylalkenyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynyl, alkyl heteroaryl, alkenyl heteroaryl, alkynyl, in general, one or more methylene groups may be interrupted or terminated by O, S, S (O), SO ₂, N (R8), C (O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle; wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

In some embodiments, the dsRNA of the present disclosure is conjugated to a divalent or trivalent branched linker selected from the structures shown in any one of formulas (XXXI) to (XXXIV):

Wherein:

wherein q2A, q2B, q3A, q3B, q4A, q4B, q5A, q B and q5C independently represent 0 to 20 for each occurrence and wherein the repeating units may be the same or different;

P^2A、P^2B、P^3A、P^3B、P^4A、P^4B、P^5A、P^5B、P^5C、T^2A、T^2B、T^3A、T^3B、T^4A、T^4B、T^4A、T^5B、T^5C Independently for each occurrence, represents absence, CO, NH, O, S, OC (O), NHC (O), CH ₂、CH₂ NH, or CH ₂ O;

Q ^2A、Q^2B、Q^3A、Q^3B、Q^4A、Q^4B、Q^5A、Q^5B、Q^5C independently represents for each occurrence, an absent, alkylene, substituted alkylene, wherein one or more methylene groups may be interrupted or terminated by one or more of O, S, S (O), SO ₂, N (RN), C (R')=c (R), c≡c, or C (O);

R ^2A、R^2B、R^3A、R^3B、R^4A、R^4B、R^5A、R^5B、R^5C independently for each occurrence represents absent, NH, O, S, CH ₂、C(O)O、C(O)NH、NHCH(R^a)C(O)、-C(O)-CH(R^a) -NH-, CO, CH=N-O, Or a heterocyclic group;

L ^2A、L^2B、L^3A、L^3B、L^4A、L^4B、L^5A、L^5B and L ^5C represent ligands; that is, each occurrence is independently a monosaccharide (e.g., galNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R ^a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly useful for use with RNAi agents to inhibit target gene expression, such as those of formula (XXXV):

Of formula XXXV

Wherein L ^5A、L^5B and L ^5C represent monosaccharides such as GalNAc derivatives.

Examples of suitable divalent and trivalent branched linker conjugated GalNAc derivatives include, but are not limited to, the structures cited above as formulas II, VII, XI, X and XIII.

The cleavable linking group is one that is sufficiently stable outside the cell, but which cleaves after entry into the target cell to release the two parts of the linker that remain together. In some embodiments, cleavage of the cleavable linking group in the target cell or under a first reference condition (which may, for example, be selected to mimic or represent an intracellular condition) is at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least about 100-fold greater than cleavage rate in the subject's blood or under a second reference condition (which may, for example, be selected to mimic or represent a condition found in blood or serum).

Cleavable linking groups are susceptible to cleavage by a cleavage agent (e.g., pH, redox potential, or the presence of a degrading molecule). Generally, cleavage agents are more prevalent inside cells than in serum or blood, or are found at higher levels or activities. Examples of such degradation agents include: redox agents, selected for a particular substrate or not having substrate specificity, comprising, for example, an oxidation or reduction enzyme or reducing agent present in the cell, such as a thiol, which can cleave the redox cleavable linking group by reductive degradation; an esterase; endosomes or agents that can produce an acidic environment, for example, those that produce a pH of five or less; enzymes that hydrolyze or degrade acid cleavable linkers can be used as broad acids, peptidases (which may be substrate specific), and phosphatases.

Cleavable linkage groups, such as disulfide linkages, may be pH sensitive. The pH of human serum was 7.4, while the average intracellular pH was slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5-6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers will have cleavable linking groups that cleave at the appropriate pH, thereby releasing the cationic lipid from the ligand within the cell, or into the desired compartment of the cell.

The linker may comprise a cleavable linking group cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the cell targeted.

In general, the suitability of a candidate cleavable linking group can be assessed by testing the ability of the degrading agent (or condition) to cleave the candidate linking group. It would also be desirable to test candidate cleavable linking groups for their ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, a relative susceptibility to cleavage between a first condition and a second condition may be determined, wherein the first condition is selected to indicate cleavage in a target cell and the second condition is selected to indicate cleavage in other tissue or biological fluid, such as blood or serum. The evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. Initial evaluation was performed under cell-free or culture conditions and confirmed to be useful by further evaluation in whole animals. In some embodiments, the cleavage of a useful candidate compound in a cell (or under in vitro conditions selected to mimic intracellular conditions) is at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold faster than the cleavage rate in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

Redox cleavable linking groups

In certain embodiments, the cleavable linking group is a redox cleavable linking group that cleaves upon reduction or oxidation. An example of a reducible cleavable linking group is a disulfide linking group (-S-S-). To determine whether a candidate cleavable linker group is a suitable "reducible cleavage linker" or is suitable for use with a particular iRNA moiety and a particular targeting agent, for example, reference may be made to the methods described herein. For example, candidates may be evaluated by incubation with Dithiothreitol (DTT) or other reducing agent, using agents known in the art to mimic the cleavage rates observed in cells such as target cells. Candidates may also be evaluated under selection of simulated blood or serum conditions. One approach is that the candidate compound cuts up to about 10% in the blood. In other embodiments, the useful candidate compound degrades at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions mimicking intracellular conditions) than in blood or serum (or under in vitro conditions mimicking extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzymatic kinetic assays under conditions selected to mimic the intracellular medium and compared with conditions selected to mimic the extracellular medium.

Phosphate-based cleavable linking groups

In some embodiments, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that cleave phosphate groups in cells are enzymes, such as phosphatase in cells. An example of a phosphate-based linking group is -O-P(O)(ORk)-O-、-O-P(S)(ORk)-O-、-O-P(S)(SRk)-O-、-S-P(O)(ORk)-O-、-O-P(O)(ORk)-S-、-S-P(O)(ORk)-S-、-O-P(S)(ORk)-S-、-S-P(S)(ORk)-O-、-O-P(O)(Rk)-O-、-O-P(S)(Rk)-O-、-S-P(O)(Rk)-O-、-S-P(S)(Rk)-O-、-S-P(O)(Rk)-S-、-O-P(S)(Rk)-S-, where Rk at each occurrence may independently be C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. In some other embodiments, the phosphate-based linking group is -O-P(O)(OH)-O-、-O-P(S)(OH)-O-、-O-P(S)(SH)-O-、-S-P(O)(OH)-O-、-O-P(O)(OH)-S-、-S-P(O)(OH)-S-、-O-P(S)(OH)-S-、-S-P(S)(OH)-O-、-O-P(O)(H)-O-、-O-P(S)(H)-O-、-S-P(O)(H)-O、-S-P(S)(H)-O-、-S-P(O)(H)-S-、-O-P(S)(H)-S-., and in some embodiments, the phosphate-based linking group is-O-P (O) (OH) -O-. These candidates can be evaluated using methods similar to those described above.

Acid cleavable linking groups

In some embodiments, the cleavable linker comprises an acid-cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In certain embodiments, the acid-cleavable linking group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or less), or by a reagent (e.g., an enzyme that can function as a generalized acid). In cells, specific low pH organelles (e.g., endosomes or lysosomes) can provide a cleavage environment for acid cleavable linkers. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. The acid cleavable group may have the general formula-c=nn-, C (O) O or-OC (O). In some embodiments, the carbon attached to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group (e.g., dimethylpentyl or tertiary butyl). These candidates can be evaluated using methods similar to those described above.

Ester-based linking groups

In some embodiments, the cleavable linker comprises an ester-based cleavable linking group. The cleavable ester-based linking group is cleaved by enzymes such as esterases and amidases in the cell. Examples of ester-based cleavable linking groups include, but are not limited to, alkylene, alkenylene, and alkynylene esters. The ester cleavable linking group has the general formula-C (O) O-, or-OC (O) -. These candidates can be evaluated using methods similar to those described above.

Peptide-based cleavable groups

In some embodiments, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by an enzyme (e.g., a peptidase or protease in a cell). Peptide-based cleavable linkers are peptide bonds formed between amino acids to create oligopeptides (e.g., dipeptides, tripeptides, etc.) as well as polypeptides. The peptide-based cleavable group does not include an amide group (-C (O) NH-). The amide groups may be formed between any alkylene, alkenylene or alkynylene groups. Peptide bonds are specific types of amide bonds formed between amino acids to produce peptides as well as proteins. The peptide-based cleavage groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides as well as proteins, and do not include the entire amide functionality. The peptide-based cleavable linking group has the general formula-NHCHRAC (O) NHCHRBC (O) -, where RA and RB are R groups of these two contiguous amino acids. These candidates can be evaluated using methods similar to those described above. Representative U.S. patents teaching the preparation of RNA conjugates include, but are not limited to, U.S. patent nos. 4,828,979;4,948,882;5,218,105;5,525,465;5,541,313;5,545,730;5,552,538;5,578,717,5,580,731;5,591,584;5,109,124;5,118,802;5,138,045;5,414,077;5,486,603;5,512,439;5,578,718;5,608,046;4,587,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904,582;4,958,013;5,082,830;5,112,963;5,214,136;5,082,830;5,112,963;5,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241,5,391,723;5,416,203,5,451,463;5,510,475;5,512,667;5,514,785;5,565,552;5,567,810;5,574,142;5,585,481;5,587,371;5,595,726;5,597,696;5,599,923;5,599,928 and 5,688,941;6,294,664;6,320,017;6,576,752;6,783,931;6,900,297;7,037,646;8,106,022, the entire contents of each of which are incorporated herein by reference.

It is not necessary to modify all positions in a given compound uniformly, and in fact, more than one of the modifications described above may be incorporated into a single compound or even at a single nucleoside within an iRNA. The present disclosure also includes iRNA compounds as chimeric compounds.

In the context of the present disclosure, a "chimeric" iRNA compound or "chimera" is an iRNA compound, e.g., a dsRNA, comprising two or more chemically distinct regions, each region being composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These irnas typically contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity to the target nucleic acid. The additional region of the iRNA can serve as a substrate for an enzyme capable of cleaving RNA: DNA or RNA: RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves RNA strands of RNA: DNA duplex. Thus, activation of RNase H cleaves the RNA target, thereby greatly enhancing the efficiency of iRNA repressor gene expression. Thus, comparable results are generally obtained with shorter irnas when chimeric dsRNA is used, as compared to phosphorothioate deoxydsrna that hybridizes to the same target region. Cleavage of an RNA target can be routinely detected by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art.

In certain examples, the RNA of the iRNA can be modified by a non-ligand group. Some non-ligand molecules have been conjugated to iRNA to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugation are available in the scientific literature. Such non-ligand moieties have included lipid moieties such as cholesterol (Kubo, T. Et al, biochem. Biophys. Res. Comm.,2007,365 (1): 54-61; letsinger et al, proc. Natl. Acad. Sci. USA,1989, 86:6553), cholic acid (Manoharan et al, biorg. Med. Chem. Lett.,1994, 4:1053), thioethers, for example hexyl-S-tritylthiol (Manoharan et al, ann. N. Acad. Sci.,1992,660:306; Manoharan et al, bioorg.med.chem.let.,1993, 3:2765), thiocholesterol (Oberhauser et al, nucleic acids res.,1992, 20:533), fatty chains, e.g., dodecyl glycol or undecyl residues (Saison-Behmoaras et al, EMBO j.,1991,10:111; kabanov et al, FEBS Lett.,1990,259:327; svinarchuk et al, biochimie,1993, 75:49), phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1, 2-di-O-hexadecyl-rac-glycerol-3-H-phosphate (Manoharan et al, tetrahedron lett.,1995,36:3651; Shea et al, nucleic acids Res.,1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al, nucleic & nucleic oxides, 1995, 14:969) or adamantylacetic acid (Manoharan et al, tetrahedron letters, 1995, 36:3651), palmityl moieties (Mishra et al, biochim. Biophys. Acta,1995, 1264:229) or octadecylamine or hexylamine-carbonyl-hydroxycholesterol moieties (Crooke et al, pharmacol. Exp. Ther.,1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of RNA that has an amino linker at one or more positions in the sequence. The amino group is then reacted with the conjugated molecule using a suitable coupling or activating agent. The conjugation reaction may be performed with the RNA still bound to the solid support or in solution after cleavage of the RNA. Purification of the RNA conjugate typically by HPLC provides the pure conjugate.

Delivery of iRNA

Delivery of iRNA to a subject in need thereof may be accomplished in a number of different ways. In vivo delivery may be directly performed by administering a composition (e.g., dsRNA) comprising iRNA to a subject. Alternatively, delivery may be effected indirectly by administration of one or more vectors encoding and directing expression of the iRNA. These alternatives are discussed further below.

1. Direct delivery

In general, any method of delivering a nucleic acid molecule may be suitable for use with an iRNA (see, e.g., akhtar S. And Julian RL.,1992Trends Cell.Biol.2 (5): 139-144 and WO94/02595, which are incorporated herein by reference in their entirety). However, for successful delivery of iRNA molecules in vivo, there are three important factors to consider: (a) biostability of the delivered molecule; (2) preventing nonspecific effects; and (3) accumulation of the delivered molecule in the target tissue. The nonspecific effects of iRNA can be minimized by local administration, for example by direct injection or implantation into tissue (as a non-limiting example, the eye) or by topical administration of a formulation. Local administration to the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may be damaged by the agent or that may degrade the agent, and allows for administration of lower total doses of the iRNA molecule. Several studies have shown successful knockdown of gene products when iRNA is administered locally. For example, prevention of neovascularization in experimental models of age-related macular degeneration has been shown in cynomolgus monkeys by both intravitreal injection (Tolentino, MJ. et al, 2004Retina 24:132-138) and in mice by subretinal injection (Reich, SJ. et al, 2003mol. Vis. 9:210-216) for intraocular delivery VEGF DSRNA. In addition, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J. Et al, 2005mol. Ther.11:267-274) and could extend survival of tumor-bearing mice (Kim, WJ. et al, 2006mol. Ther.14:343-350; li, S. Et al, 2007mol. Ther.15:515-523). RNA interference has also shown success in local delivery to the CNS by direct injection (Dorn, g. Et al 2004Nucleic Acids 32:e49; Tan, PH., et al, 2005Gene Ther.12:59-66; makimura, h. et al 2002BMC Neurosci.3:18; SHISHKINA, GT., etc., 2004Neuroscience 129:521-528; thakker, ER. et al, 2004Proc.Natl. Acad.Sci.U.S. A.101:17270-17275; akaneya, y, et al, 2005j. Neurophysiol. 93:594-602), and to the lungs by intranasal administration (Howard, ka, et al, 2006mol. Ter. 14:476-484; Zhang, x. Et al, 2004j.biol. Chem.279:10677-10684; bitko, V.et al, 2005Nat. Med. 11:50-55). To administer iRNA systemically to treat a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods are used to prevent rapid degradation of dsRNA by endo-and exonucleases in vivo.

Modification of the RNA or drug carrier can also allow the iRNA composition to target the target tissue and avoid undesirable off-target effects. The iRNA molecule can be modified by conjugation to other groups (e.g., lipid or carbohydrate groups as described herein). Such conjugates can be used to target iRNA to a particular cell, such as a liver cell, e.g., a liver cell. For example, galNAc conjugates or lipid (e.g., LNP) formulations can be used to target iRNA to a particular cell, such as a liver cell, e.g., a liver cell.

IRNA molecules can also be modified by chemical conjugation to lipophilic groups (e.g., cholesterol) to enhance cellular uptake and prevent degradation. For example, iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety is injected systemically into mice and results in knockdown of ApoB mRNA in both liver and jejunum (Soutschek, j. Et al, 2004Nature 432:173-178). In a mouse model of prostate cancer, conjugation of iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression (McNamara, jo et al, 2006nat. Biotechnol.24:1005-1015). In alternative embodiments, the iRNA can be delivered using a drug delivery system (e.g., nanoparticle, dendrimer, polymer, liposome, or cationic delivery system). The positively charged cation delivery system promotes binding of (negatively charged) iRNA molecules and also enhances interactions at negatively charged cell membranes to allow efficient uptake of iRNA by cells. The cationic lipid, dendrimer, or polymer can bind to or be induced to form vesicles or micelles that encapsulate the iRNA (see, e.g., kim SH. et al, 2008Journal of Controlled Release 129 (2): 107-116). When administered systemically, the formation of vesicles or micelles further prevents degradation of the iRNA. Methods for preparing and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., sorensen, DR., et al, 2003J. Mol. Biol.327:761-766; verma, UN. Et al, 2003Clin.Cancer Res.9:1291-1300; arnold, AS, et al, 2007J. Hypertens.25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems that can be used for systemic delivery of iRNA include DOTAP (Sorensen, DR. et al, 2003 supra; verma, UN. et al, 2003 supra), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al, 2006Nature 441:111-114), cardiolipin (Chien, PY. et al, 2005Cancer Gene Ther.12:321-328; pal, a. Et al, 2005Int J.Oncol.26:1087-1091), polyethyleneimine (Bonnet ME. et al, 2008Pharm.Res.Aug 16 are first published electronically before printing; Aigner, A.,2006 J.biomed.Biotechnol.71659), arg-Gly-Asp (RGD) peptide (Liu, S.,2006 mol.Pharm.3:472-487), and polyamidoamine (Tomalia, DA. et al, 2007biochem.Soc.Trans.35:61-67; yoo, H.et al, 1999 Pharm.Res.16:1799-1804). In some embodiments, the iRNA forms a complex with cyclodextrin for systemic administration. Methods of administration, as well as pharmaceutical compositions of iRNA and cyclodextrin, can be found in U.S. patent No. 7,427,605, which is incorporated herein by reference in its entirety.

2. Vectors encoding iRNA

In another aspect, RNAi agents targeting the ANGPTL7 gene may be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., couture, A et al, TIG. (1996), 12:5-10; WO 00/22113, WO 00/22114 and US 6,054,299). Depending on the particular construct used and the target tissue or cell type, expression is preferably sustained (months or longer). These transgenes may be introduced as linear constructs, circular plasmids, or viral vectors, which may be integrating or non-integrating vectors. Transgenes can also be constructed so that they inherit as extrachromosomal plasmids (Gassmann et al, (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The single strand or multiple strands of the RNAi agent can be transcribed from the promoter on the expression vector. Where two separate strands are to be expressed to produce, for example, dsRNA, the two separate expression vectors can be co-introduced (e.g., by transfection or infection) into the target cell. Alternatively, each individual strand of dsRNA may be transcribed from a promoter located on the consent expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

The iRNA expression vector is typically a DNA plasmid or viral vector. Expression vectors compatible with eukaryotic cells, such as vertebrate cells, can be used to produce recombinant constructs for expression of an iRNA as described herein. Eukaryotic expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors contain convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of the iRNA expression vector can be systemic, such as by intravenous or intramuscular administration, by administration of target cells transplanted from a patient, followed by reintroduction into the patient, or by any other means that allows for the introduction of the desired target cells.

The iRNA expression plasmid can be transfected into the target cell as a complex with a cationic lipid carrier (e.g., oligofectamine) or a non-cationic lipid-based carrier (e.g., transit-TKOTM). The present disclosure also contemplates multi-lipid transfection for iRNA-mediated knockdown that targets different regions of target RNA for one week or more. The successful introduction of the vector into the host cell may be monitored using a variety of known methods. For example, transient transfection may be signaled by a reporter gene, such as a fluorescent marker, e.g., green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide transfected cells with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems that may be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) Retroviral vectors, including but not limited to lentiviral vectors, moronella leukemia virus, and the like; (c) an adeno-associated viral vector; (d) a herpes simplex virus vector; (e) SV40 vector; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) a picornaviral vector; (i) Poxvirus vectors such as orthopoxes (e.g., vaccinia virus vectors) or fowlpox (e.g., canary pox or fowlpox); and (j) helper-dependent or entero-free adenoviruses. Replication-defective viruses may also be advantageous. The different vectors will or will not be incorporated into the genome of the cell. If desired, the construct may comprise viral sequences for transfection. Alternatively, the construct may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA will typically require regulatory elements, such as promoters, enhancers, and the like, to ensure expression of the iRNA in the target cell. Other aspects to be considered for vectors and constructs are described further below.

Vectors useful for delivering iRNA will contain regulatory elements (promoters, enhancers, etc.) sufficient to express the iRNA in the desired target cell or tissue. Regulatory elements may be selected to provide constitutive or regulated/inducible expression.

Expression of iRNA can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to certain physiological regulatory factors, such as circulating glucose levels or hormones (Docherty et al, 1994FASEB J.8:20-24). Such inducible expression systems suitable for controlling dsRNA expression in a cell or in a mammal include, for example, modulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl- β -D1-thiogalactoside (IPTG). One skilled in the art will be able to select appropriate regulatory/promoter sequences based on the intended use of the iRNA transgene.

In particular embodiments, viral vectors containing nucleic acid sequences encoding irnas may be used. For example, retroviral vectors can be used (see Miller et al, 1993Meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for proper packaging of the viral genome and integration into the host cell DNA. Cloning the nucleic acid sequence encoding the iRNA into one or more vectors facilitates delivery of the nucleic acid into a patient. More details on retroviral vectors can be found, for example, in Boesen et al, 1994 biotheraphy 6:291-302, which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to render the stem cells more resistant to chemotherapy. Other references describing the use of retroviral vectors in gene therapy are: clowes et al, 1994J.Clin.Invest.93:644-651; kiem et al, 1994Blood 83:1467-1473; salmons and Gunzberg,1993Human Gene Therapy 4:129-141; and Grossman and Wilson,1993Curr.Opin.in Genetics and Devel.3:110-114. Lentiviral vectors contemplated for use include, for example, U.S. patent No. 6,143,520;5,665,557; and 5,981,276, which are incorporated herein by reference.

Adenoviruses are also contemplated for delivery of iRNA. Adenoviruses are particularly attractive mediators, for example, for delivering genes to airway epithelial cells. Adenovirus naturally infects airway epithelial cells, which cause mild disease therein. Other targets of adenovirus-based delivery systems are liver, central nervous system, endothelial cells and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson,1993Current Opinion in Genetics and Development 3:499-503 review adenovirus-based gene therapy. Bout et al 1994Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the airway epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in the following: rosenfeld et al, 1991Science 252:431-434; rosenfeld et al, 1992cell 68:143-155; MASTRANGELI et al, 1993J.Clin. Invest.91:225-234; PCT publication WO94/12649; and Wang et al, 1995Gene Therapy2:775-783. Suitable AV vectors for expressing the iRNAs proposed in the present disclosure, methods of constructing recombinant AV vectors, and methods of delivering vectors into target cells are described in Xia H et al, 2002Nat. Biotech.20:1006-1010.

The use of adeno-associated virus (AAV) vectors is also contemplated (Walsh et al, 1993Proc. Soc. Exp. Biol. Med.204:289-300; U.S. Pat. No. 5,436,146). In some embodiments, the iRNA can be expressed as two separate complementary single stranded RNA molecules from a recombinant AAV vector having, for example, a U6 or H1 RNA promoter or a Cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA set forth in the present disclosure, methods for constructing recombinant AV vectors, and methods of delivering the vectors into target cells are described in the following: samulski R et al, 1987J.Virol.61:3096-3101; fisher K J et al, 1996J.Virol.70:520-532; samulski R et al, 1989J.Virol.63:3822-3826; U.S. Pat. nos. 5,252,479; U.S. Pat. nos. 5,139,941; international patent application number WO 94/13788; and international patent application number WO 93/24641, the entire disclosures of which are incorporated herein by reference.

Another typical viral vector is a poxvirus, such as vaccinia virus, e.g., an attenuated vaccinia, such as Modified Virus Ankara (MVA) or NYVAC, fowl pox, such as fowl pox or canary pox.

The tropism of viral vectors may be modified by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by appropriately substituting different viral capsid proteins. For example, lentiviral vectors may be pseudotyped with surface proteins from Vesicular Stomatitis Virus (VSV), rabies, ebola, mokola (Mokola), and the like. AAV vectors can be engineered to express different capsid protein serotypes, thereby targeting different cells; see, e.g., rabinowitz j.e. et al, 2002J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

The pharmaceutical formulation of the carrier may comprise the carrier in an acceptable diluent or may comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, such as retroviral vectors, the pharmaceutical product may comprise one or more cells that produce the gene delivery system.

Pharmaceutical compositions comprising iRNA

In some embodiments, the present disclosure provides pharmaceutical compositions containing an iRNA as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with the expression or activity of ANGPTL7 (e.g., glaucoma or conditions associated with glaucoma). Such pharmaceutical compositions are formulated based on the mode of delivery. In some embodiments, the composition may be formulated for topical delivery, e.g., by intraocular delivery (e.g., intravitreal administration, e.g., intravitreal injection, transscleral administration, e.g., transscleral injection, subconjunctival administration, e.g., subconjunctival injection, retrobulbar administration, e.g., retrobulbar injection, intracameral administration, e.g., intracameral injection, or subretinal administration, e.g., subretinal injection). In other embodiments, the compositions may be formulated for topical delivery. In other embodiments, the compositions may be formulated for systemic administration by parenteral delivery, such as by Intravenous (IV) delivery. In some embodiments, the compositions provided herein (e.g., compositions comprising GalNAc conjugates or LNP formulations) are formulated for intravenous delivery.

The pharmaceutical compositions presented herein are administered in a dose sufficient to inhibit the expression of ANGPTL 7. Typically, a suitable dose of iRNA will be in the range of 0.01 milligrams to 200.0 milligrams per kilogram of body weight of the recipient per day. The pharmaceutical composition may be administered once daily, or the iRNA may be administered at appropriate intervals in two, three or more sub-doses throughout the day, or even delivered using continuous infusion or by a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller to achieve a total daily dose. Dosage units may also be mixed for delivery over several days, for example, using conventional slow release formulations that provide slow release of the iRNA over a period of several days. Sustained release formulations are well known in the art and are particularly useful for delivering agents at specific sites, as may be used with the agents of the present disclosure. In this embodiment, the dosage unit comprises a corresponding multiple of the daily dose.

The effect of a single dose on ANGPTL7 levels may be durable such that subsequent doses are administered no more than 3 days, 4 days, or 5 days apart, or no more than 1 week, 2 weeks, 3 weeks, 4 weeks, 12 weeks, 24 weeks, or 36 weeks apart.

Those of skill in the art will appreciate that certain factors may affect the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatments, the overall health and/or age of the subject, and other diseases present. Furthermore, the treatment of a subject with a therapeutically effective amount of the composition may comprise a single treatment or a series of treatments. The estimation of effective doses and in vivo half-life of the individual irnas encompassed by the present disclosure can be performed using conventional methods or based on in vivo testing using a suitable animal model.

Suitable animal models, such as mice or cynomolgus monkeys, e.g., animals containing transgenes expressing human ANGPTL7, can be used to determine therapeutically effective doses and/or effective dose regimens of administration of an ANGPTL7 siRNA.

The present disclosure also includes pharmaceutical compositions and formulations comprising the iRNA compounds set forth herein. The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (local) (e.g., by intraocular injection), external (topical) (e.g., by eye drop solution), or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous (e.g., by implanted device); or intracranial (e.g., by intraparenchymal, intrathecal, or intraventricular administration).

Pharmaceutical compositions and formulations for topical administration may comprise transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous bases, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the iRNA proposed in the present disclosure is admixed with a topical delivery agent, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoyl phosphatidyl DOPE ethanolamine, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), negative (e.g., dimyristoyl phosphatidyl glycerol DMPG), and cationic (e.g., dioleoyl tetramethyl aminopropyl DOTAP and dioleoyl phosphatidyl ethanolamine DOTMA). The iRNA proposed by the present disclosure may be encapsulated in liposomes or may form complexes with them, in particular with cationic liposomes. Alternatively, the iRNA may be complexed with a lipid, in particular a cationic lipid. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glycerol monooleate, glycerol dilaurate, glycerol 1-monocaprate, 1-dodecylazepan-2-one, acylcarnitine, acylcholine, or C1-20 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. patent No. 6,747,014, incorporated herein by reference.

A. liposome preparation

In addition to microemulsions, there are a number of organized surfactant structures that have been studied and used in drug formulation. These include unilamellar, micellar, bilayer and vesicle. Vesicles (e.g., liposomes) are of great concern due to their specificity and duration of action provided from a drug delivery perspective. As used in this disclosure, the term "liposome" refers to vesicles composed of amphiphilic lipids arranged in one or more spherical bilayers.

Liposomes are unilamellar or multilamellar vesicles having a membrane formed of a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes, although not as effective as cell walls, are taken up by macrophages in the body.

In order to pass through intact mammalian skin, lipid vesicles must pass through a series of pores with a diameter of less than 50nm under the influence of a suitable transdermal gradient. Thus, it is desirable to use liposomes that are highly deformable and capable of passing through such pores.

Additional advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a variety of water-soluble and lipid-soluble drugs; liposomes can protect the encapsulated drug in its internal compartment from metabolism and degradation (Rosoff, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editorial) 1988,Marcel Dekker,Inc, new York, n.y., volume 1, p.245). Important considerations for the preparation of liposomal formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes can be used to transfer and deliver active ingredients to the site of action. Because the liposome membrane is similar in structure to a biological membrane, when the liposome is applied to a tissue, the liposome begins to merge with the cell membrane, and as the liposome merges and the cell progresses, the liposome contents are emptied into the cell where the active agent can function.

Liposome formulations have been the focus of extensive research as a means of delivery for many drugs. There is increasing evidence that liposomes present several advantages over other formulations for topical administration. Such advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target site, and the ability to administer multiple drugs (hydrophilic and hydrophobic) into the skin.

Several reports detail the ability of liposomes to deliver agents comprising high molecular weight DNA into the skin. Compounds comprising analgesics, antibodies, hormones and high molecular weight DNA have been applied to the skin. Most applications are targeting the epidermis.

Liposomes fall into two broad categories. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form stable complexes. The positively charged DNA/liposome complex binds to the negatively charged cell surface and internalizes in endosomes. Due to the acidic pH in the endosome, the liposomes burst, releasing their contents into the cytoplasm (Wang et al, 1987biochem. Biophys. Res. Commun. 147:980-985).

Liposomes that are pH sensitive or negatively charged entrap DNA rather than complex with it. Since both DNA and lipids carry similar charges, rejection rather than complex formation occurs. However, some DNA is embedded within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the foreign gene was detected in the target cells (Zhou et al 1992Journal of Controlled Release 19,269-274).

One major type of liposome composition comprises phospholipids other than phosphatidylcholine of natural origin. Neutral liposome compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are typically formed from dimyristoyl phosphatidylglycerol, whereas anionic fusogenic liposomes are formed predominantly from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from Phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed by a mixture of phospholipids and/or phosphatidylcholine and/or cholesterol.

Several studies evaluated the local delivery of liposomal pharmaceutical formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpetic ulcers, whereas delivery of interferon by other means (e.g., as a solution or emulsion) was ineffective (Weiner et al 1992Journal of Drug Targeting 2:405-410). Further, additional studies tested the efficacy of interferon administered as part of a liposome formulation for administration of interferon using an aqueous system, and concluded that: liposome formulations are preferred over aqueous administration (du Plessis et al, 1992Antiviral Research,18:259-265).

Nonionic liposome systems have also been examined to determine their utility in delivering drugs to the skin, particularly systems that include nonionic surfactants and cholesterol. Cyclosporin a was delivered into the dermis of the mouse skin using a non-ionic liposome formulation comprising Novasome ^TM I (glycerol dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome ^TM II (glycerol distearate/cholesterol/polyoxyethylene-10-stearyl ether). The results indicate that such a nonionic liposome system is effective in promoting cyclosporin A deposition into different layers of the skin (Hu et al S.T.P.1994Pharma. Sci.4; 6:466).

Liposomes also include "sterically stabilized" liposomes, as used herein, which comprise liposomes of one or more specific lipids, which when incorporated into liposomes, result in an extended circulation lifetime relative to liposomes lacking such specific lipids. Examples of sterically stabilized liposomes are those wherein a portion of the vesicle-forming lipid fraction of the liposome (a) comprises one or more glycolipids, such as monosialoganglioside G _M1, or (B) is derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. While not wishing to be bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes comprising gangliosides, sphingomyelins, or PEG-derived lipids, the circulation half-life of these sterically stabilized liposomes is prolonged by reduced uptake by reticuloendothelial system (RES) cells (Allen et al, (1987) FEBS Letters,223:42; wu et al, (1993) CANCER RESEARCH, 53:3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjoulous et al (Ann.N.Y. Acad.Sci., (1987), 507:64) report the ability of monosialoganglioside G _M1, galactosylsulfate and phosphatidylinositol to increase the blood half-life of liposomes. Gabizon et al (Proc. Natl. Acad. Sci. U.S. A., (1988), 85:6949) set forth these findings. U.S. Pat. No. 4,837,028 to Allen et al and WO 88/04924 disclose liposomes comprising (1) sphingomyelin and (2) ganglioside G _M1 or galactocerebroside sulfate. U.S. patent No. 5,543,152 (Webb et al) discloses liposomes comprising sphingomyelin. Liposomes comprising 1, 2-sn-dimyristoyl phosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

A number of liposomes including lipids derivatized with one or more hydrophilic polymers and methods of making the same are known in the art. Sunamoto et al (Bull. Chem.1980Soc. Jpn. 53:2778) describe liposomes comprising the nonionic detergent 2C1215G (containing a PEG moiety). Illum et al (1984FEBS Lett.167:79) noted that hydrophilic coatings of polystyrene particles with polymeric glycols can significantly increase blood half-life. Carboxyl modified synthetic phospholipids by attachment of polyalkylene glycols (e.g., PEG) are described by Sears (national patent nos. 4,426,330 and 4,534,899). Klibanov et al (1990FEBS Lett.268:235) describe experiments demonstrating that liposomes comprising Phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significantly increased blood circulation half-life. Blume et al (1990Biochimica et Biophysica Acta,1990 1029:91) extend this observation to other PEG-derivatized phospholipids, such as DSPE-PEG formed from a combination of distearoyl phosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their outer surface are described in Fisher's European patent numbers EP 0 445 131 B1 and WO 90/04384. The following describes liposome compositions containing 1 to 20 mole percent of PEG-derivatized PE and methods of use thereof: woodle et al (U.S. Pat. No. 5,013,556 and 5,356,633) and Martin et al (U.S. Pat. No. 5,213,804 and European patent No. EP 0496 813B 1). Liposomes comprising many other lipopolymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both from Martin et al) and WO 94/20073 (Zalipsky et al). Liposomes comprising PEG modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. patent No. 5,540,935 (Miyazaki et al) and U.S. patent No. 5,556,948 (Tagawa et al) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surface.

A number of liposomes including nucleic acids are known in the art. WO 96/40062 (from Thierry et al) discloses a method for encapsulating high molecular weight nucleic acids in liposomes. U.S. patent No. 5,264,221 (from Tagawa, etc.) discloses protein-bound liposomes and states that the contents of such liposomes may comprise dsRNA. U.S. patent No. 5,665,710 (from Rahman, et al) describes certain methods for encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 (from Love et al) discloses liposomes comprising dsRNA targeting the raf gene.

The carrier is yet another type of liposome and is a highly deformable lipid aggregate, an attractive candidate for drug delivery vehicles. The transfer body may be described as a lipid droplet, which is highly deformable such that it readily penetrates through pores smaller than the droplet. The delivery body is able to adapt to the environment in which it is used, e.g. it is self-optimizing (adapts to the shape of the pores in the skin), self-repairing, often reaches its target without fracturing, and often self-loading. To prepare the transfer body, a surface edge activator, typically a surfactant, may be added to the standard liposome composition. Transfer bodies have been used to deliver serum albumin to the skin. The carrier-mediated delivery of serum albumin has been demonstrated to be as effective as subcutaneous injections of serum albumin-containing solutions.

Surfactants find wide use in formulations such as those described herein, particularly in emulsions (including microemulsions) and liposomes. The most common method of classifying and ordering the characteristics of many different types of surfactants (natural and synthetic) is to use the hydrophilic/lipophilic balance (HLB). The nature of the hydrophilic groups (also referred to as "heads") provides the most useful method of classifying the different surfactants used in the formulation (Rieger, pharmaceutical Dosage Forms, MARCEL DEKKER, inc., new York, n.y.,1988, p.285).

If the surfactant molecules are not ionized, they are classified as nonionic surfactants. Nonionic surfactants are widely used in pharmaceuticals and cosmetics and can be used over a wide range of pH values. Typically, they have HLB values ranging from 2 to about 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glycerol esters, polyglycerol esters, sorbitan esters, sucrose esters and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols and ethoxylated/propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are the most popular members of the class of nonionic surfactants.

Surfactants are classified as anionic if they are negatively charged when dissolved or dispersed in water. Anionic surfactants include carboxylic acid esters such as soaps, acyl lactylates, amides of amino acids, sulfates such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the class of anionic surfactants are alkyl sulfates and soaps.

Surfactants are classified as cationic surfactants if the surfactant molecules are positively charged when dissolved or dispersed in water. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

Surfactants are classified as amphoteric if they have the ability to carry a positive or negative charge. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines and phospholipids.

The use of surfactants in pharmaceuticals, formulations and emulsions has been examined (Rieger, pharmaceutical Dosage Forms, MARCEL DEKKER, inc., new York, n.y.,1988, p.285).

1. Nucleic acid lipid particles

In some embodiments, an ANGPTL7 dsRNA as set forth in the present disclosure is fully encapsulated in a lipid formulation, e.g., to form SPLP, pSPLP, SNALP or other nucleic acid lipid particles. SNALP and SPLP typically contain cationic lipids, non-cationic lipids, and lipids that prevent aggregation of the particles (e.g., PEG-lipid conjugates). SNALP and SPLP are very useful for systemic applications because they exhibit extended cycle life following intravenous (i.v.) injection and accumulate at distant sites (e.g., sites physically separated from the site of administration). SPLP comprises "pSPLP", which comprises an encapsulated condensing agent-nucleic acid complex as shown in PCT publication No. WO 00/03683. The particles of the present disclosure typically have an average diameter of about 50nm to about 150nm, more typically about 60nm to about 130nm, more typically about 70nm to about 110nm, most typically about 70nm to about 90nm, and are substantially non-toxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present disclosure resist degradation by nucleases in aqueous solutions. Nucleic acid-lipid particles and methods of making the same are disclosed, for example, in U.S. patent No. 5,976,567;5,981,501;6,534,484;6,586,410;6,815,432; and PCT publication number WO 96/40964.

In some embodiments, the ratio of lipid to drug (mass/mass ratio) (e.g., ratio of lipid to dsRNA) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

For example, the cationic lipid may be N, N-dioleyl-N, N-dimethyl ammonium chloride (DODAC), N, N-distearyl-N, N-dimethyl ammonium bromide (DDAB), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethyl ammonium chloride (DOTAP), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethyl ammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleyloxy) propylamine (DODMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-Dihydrolinoleyloxy-N, N-dimethylaminopropane (DLenDMA), 1, 2-dialkylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-dialkylcarbamoyloxy-3- (dimethylamino) acetoxypropane (DLin-DAC), 1, 2-dialkylcarbamoyloxy-3-morpholinopropane (DLin-MA), 1, 2-dihydrooleoyl-3-dimethylaminopropane (DLinDAP), 1, 2-dihydrooleoyl thio-3-dimethylaminopropane (DLin-S-DMA), and, 1-linoleoyl-2-linoleoyl-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleoyloxy-3-trimethylaminopropane chloride (DLin-TMA. Cl), 1, 2-dioleoyloxy-3-trimethylaminopropane chloride (DLin-TAP. Cl), 1, 2-dioleoyloxy-3- (N-methylpiperazine) propane (DLin-MPZ) or 3- (N, N-dioleylamino) -1, 2-propanediol (DLinAP), 3- (N, N-dioleylamino) -1, 2-propanediol (DOAP), 1, 2-dioleyloxy-3- (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DMA), 1, 2-Dihydrooleoyloxy-N, N-dimethylaminopropane (DLinDMA), 2-dihydrooleoyl-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA) or analogues thereof, (3 aR,5s,6 aS) -N, N-dimethyl-2, 2-di ((9Z, 12Z) -octadecyl-9, 12-dienyl) tetrahydro-3 aH-cyclopentene [ d ] [1,3] dioxan-5-amine (ALN 100), (6Z, 9Z,28Z, 31Z) -4- (dimethylamino) butanoic acid trilatene-6,9,28,31-tetraen-19-yl ester (MC 3), 1,1' - (2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethylazanediyl) docosan-2-ol (Tech G1) or a mixture thereof. The cationic lipid may comprise from about 20mol% to about 50mol% or about 40mol% of the total lipid present in the particle.

In some embodiments, the compound 2, 2-dihydrooleoyl-4-dimethylaminoethyl- [1,3] -dioxolane may be used to prepare lipid siRNA nanoparticles. The synthesis of 2, 2-dioleoyl-4-dimethylaminoethyl- [1,3] -dioxolane is described in U.S. provisional patent application No. 61/107,998, filed on 10/23 of 2008, which is incorporated herein by reference.

In some embodiments, the lipid siRNA particles comprise 40% 2, 2-dioleoyl-4-dimethylaminoethyl- [1,3] -dioxolane: 10% DSPC:40% cholesterol: 10% PEG-C-DOMG (mole percent), wherein the particle size was 63.0.+ -. 20nm and the siRNA/lipid ratio was 0.027.

The non-cationic lipid may be a lipid comprising an anion or neutral lipid, including but not limited to distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl Oleoyl Phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid may comprise from about 5mol% to about 90mol%, about 10mol% or about 58mol% (if cholesterol is included) of the total lipid present in the particle.

Conjugated lipids that inhibit aggregation of particles may be, for example, polyethylene glycol (PEG) lipids, including but not limited to PEG-Diacylglycerol (DAG), PEG-Dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be, for example, PEG-dilauroxypropyl (Ci 2), PEG-dimyristoxypropyl (Ci 4), PEG-dipalmitoxypropyl (Ci 6) or PEG-distearxypropyl (C ] 8). The conjugated lipid that prevents aggregation of the particles may be from 0mol% to about 20mol% or about 2mol% of the total lipid present in the particles.

In some embodiments, the nucleic acid lipid particle further comprises cholesterol, which is, for example, about 10mol% to about 60mol% or about 48mol% of the total lipids present in the particle.

In some embodiments, the iRNA is formulated in Lipid Nanoparticles (LNPs).

LNP01

In some embodiments, lipid ND 98.4 HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed on even 26 at 3/2008, which is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids) can be used to make lipid dsRNA nanoparticles (e.g., LNP01 particles). Stock solutions of each substance in ethanol can be prepared as follows: ND98, 133mg/ml; cholesterol, 25mg/ml, PEG-ceramide C16, 100mg/ml. The ND98, cholesterol, and PEG-ceramide C16 stock solutions may then be combined at a molar ratio of, for example, 42:48:10. The combined lipid solution may be mixed with an aqueous dsRNA solution (e.g., in sodium acetate at pH 5) such that the final ethanol concentration is about 35% to 45%, and the final sodium acetate concentration is about 100mM to 300mM. Lipid dsRNA nanoparticles typically spontaneously form upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate film (e.g., 100nm cutoff) using, for example, a hot barrel extruder, such as a Lipex extruder (Northern lips, inc). In some cases, the extrusion step may be omitted. Ethanol removal and simultaneous buffer exchange can be achieved by, for example, dialysis or tangential flow filtration. The buffer may be exchanged with, for example, phosphate Buffered Saline (PBS) at about pH7, e.g., about pH 6.9, about pH7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, for example, in international application publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid dsRNA formulations are provided in table a below.

Table a: exemplary lipid formulations

DSPC: distearoyl lecithin

DPPC: dipalmitoyl phosphatidylcholine

PEG-DMG: PEG-Dimethylglycerol (C14-PEG or PEG-C14) (PEG with average molar weight of 2000)

PEG-DSG: PEG-stilbene glycerol (C18-PEG or PEG-C18) (PEG with average molar weight of 2000)

PEG-cDMA: PEG-carbamoyl-1, 2-dipyridamole (PEG with average molar weight of 2000)

Formulations comprising SNALP (1, 2-dithioenoxy-N, N-dimethylaminopropane (DLinDMA)) are described in WO 2009/127060 filed 4/15/2009, which is incorporated herein by reference.

Formulations comprising XTC are described, for example, in the following: U.S. provisional serial nos. 61/148,366 submitted on 1 month 29 of 2009; U.S. provisional serial nos. 61/156,851 submitted on 3/2 2009; U.S. temporary serial numbers 61/185,712 submitted on 6/10 2009; U.S. provisional serial No. 61/228,373 submitted 24 in 7 months 2009; U.S. provisional serial No. 61/239,686 filed on 9/3/2009 and international application number PCT/US2010/022614 filed on 29/1/2010, which are hereby incorporated by reference.

Formulations comprising MC3 are described in, for example, U.S. provisional serial No. 61/244,834, submitted at month 22 of 2009, U.S. provisional serial No. 61/185,800, submitted at month 10 of 2009, and International application No. PCT/US10/28224, submitted at month 10 of 2010, which documents are hereby incorporated by reference.

Formulations comprising ALNY-100 are described, for example, international patent application number PCT/US09/63933 filed on 11/10 2009, which is hereby incorporated by reference.

Formulations comprising C12-200 are described in U.S. provisional serial No. 61/175,770 filed 5/2009 and international application No. PCT/US10/33777 filed 5/2010, which are hereby incorporated by reference.

2. Synthesis of cationic lipids

Any of the compounds, e.g., cationic lipids, etc., used in the nucleic acid lipid particles proposed in the present disclosure can be prepared by known organic synthesis techniques. Unless otherwise indicated, all substituents are defined below.

"Alkyl" means a straight or branched, acyclic or cyclic saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, while saturated branched chain alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; and unsaturated cyclic alkyl groups include cyclopentenyl, cyclohexenyl, and the like.

"Alkenyl" means an alkyl group, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyl groups include both cis and trans isomers. Representative straight and branched alkenyl groups include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2, 3-dimethyl-2-butenyl and the like.

"Alkynyl" means any alkyl or alkenyl group as defined above that additionally contains at least one triple bond between adjacent carbons. Representative straight and branched chain alkynyl groups include ethynyl, alkynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

"Acyl" means any alkyl, alkenyl or alkynyl group in which the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, -C (=o) alkyl, -C (=o) alkenyl, and-C (=o) alkynyl are acyl groups.

"Heterocycle" means a saturated, unsaturated or aromatic 5-to 7-membered monocyclic or 7-to 10-membered bicyclic, heterocyclic ring, and which contains 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, including bicyclic rings, wherein any of the above heterocyclic rings is fused to a benzene ring. The heterocycle may be attached through any heteroatom or carbon atom. The heterocyclic ring comprises heteroaryl as defined below. Heterocycles include morpholinyl, pyrrolidonyl, pyrrolidinyl, piperidinyl, piperazinynyl, hydantoin, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms "optionally substituted alkyl", "optionally substituted alkenyl", "optionally substituted alkynyl", "optionally substituted acyl" and "optionally substituted heterocycle" mean that at least one hydrogen atom is replaced by a substituent when substituted. In the case of an oxo substituent (=o), two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocyclic ring 、-OR^x、-NR^xR^y、-NR^xC(＝O)R^y、-NR^xSO₂R^y、-C(＝O)R^x、-C(＝O)OR^x、-C(＝O)NR^xR^y、–SO_nR^x, and —so _nNR^xR^y, where n is 0,1, or 2, R ^x and R ^y are the same or different and are independently hydrogen, alkyl, or heterocyclic, and each of the alkyl and heterocyclic substituents may be further substituted with one or more of: oxo, halogen, -OH, -CN, alkyl, -OR ^x, heterocycle 、-NR^xR^y、-NR^xC(＝O)R^y、-NR^xSO₂R^y、-C(＝O)R^x、-C(＝O)OR^x、-C(＝O)NR^xR^y、-SO_nR^x and-SO _nNR^xR^y.

"Halogen" means fluorine, chlorine, bromine or iodine.

In some embodiments, the methods presented in the present disclosure may require the use of protecting groups. Protecting group methods are well known to those skilled in the art (see, e.g., protective Groups in Organic Synthesis, green, T.W., et al, wiley-Interscience, new York City, 1999). Briefly, within the context of the present disclosure, a protecting group is any group that reduces or eliminates the undesired reactivity of a functional group. Protecting groups may be added to the functional groups to mask their reactivity during certain reactions, and then removed to reveal the original functional groups. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates the undesirable reactivity of an alcohol functional group. The protecting groups may be added and removed using techniques well known in the art.

Synthesis of formula A

In some embodiments, the nucleic acid lipid particles set forth in the present disclosure are formulated using a cationic lipid of formula a:

Wherein R ₁ and R ₂ are independently alkyl, alkenyl or alkynyl, each optionally substituted, and R ₃ and R ₄ are independently lower alkyl, or R ₃ and R ₄ may together form an optionally substituted heterocycle. In some embodiments, the cationic lipid is XTC (2, 2-dioleoyl-4-dimethylaminoethyl- [1,3] -dioxolane). In general, the lipids of formula a above can be prepared by the following reaction schemes 1 or 2, wherein all substituents are as defined above unless otherwise indicated.

Scheme 1

Lipid a, wherein R ₁ and R ₂ are independently alkyl, alkenyl or alkynyl, each of which may be optionally substituted, and R ₃ and R ₄ are independently lower alkyl, or R ₃ and R ₄ may together form an optionally substituted heterocycle, may be prepared according to scheme 1. Ketone 1 and bromide 2 may be purchased or prepared according to methods known to those of ordinary skill in the art. The reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula a. The lipid of formula a may be converted to the corresponding ammonium salt using an organic salt of formula 5, wherein X is an anionic counterion selected from the group consisting of halogen, hydroxide, phosphate, sulfate, and the like.

Scheme 2

Alternatively, the ketone 1 starting material may be prepared according to scheme 2. The grignard reagent 6 and cyanide 7 may be purchased or prepared according to methods known to those of ordinary skill in the art. The reaction of 6 and 7 yields ketone 1. The conversion of ketone 1 to the corresponding lipid of formula a is depicted in scheme 1.

Synthesis of MC3

DLin-M-C3-DMA (i.e., (6Z, 9Z,28Z, 31Z) -4- (dimethylamino) butanoic acid heptadecan-6,9,28,31-tetraen-19-yl ester) was prepared as follows. A solution of (6Z, 9Z,28Z, 31Z) -heptadecan-6,9,28,31-tetraen-19-ol (0.53 g), 4-N, N-dimethylaminobutyrate hydrochloride (0.51 g), 4-N, N-dimethylaminopyridine (0.61 g) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid and then with dilute aqueous sodium bicarbonate. The organic fraction was dried over anhydrous magnesium sulfate, filtered and the solvent was removed on a rotary evaporator. The residue was passed down a silica gel column (20 g) using a 1% to 5% methanol/dichloromethane elution gradient. Fractions containing purified product were combined and solvent removed to give a colorless oil (0.54 g).

ALNY-100 Synthesis

The synthesis of ketal 519[ ALNY-100] was performed using scheme 3 below:

515 synthesis

To a stirred suspension of LiAlH ₄ (3.74 g, 0.09850 mol) in 200mL of double necked RBF (1L) of anhydrous THF under nitrogen atmosphere was slowly added a solution of 514 (10 g,0.04926 mol) in 70mL of THF at 0 ℃. After complete addition, the reaction mixture was warmed to room temperature and then heated to reflux for 4 hours. The progress of the reaction was monitored by TLC. After the reaction was complete (by TLC), the mixture was cooled to 0 ℃ and quenched by careful addition of saturated Na ₂SO₄ solution. The reaction mixture was stirred at room temperature for 4 hours and filtered off. The residue was washed thoroughly with THF. The filtrate and washings were mixed and diluted with 400mL dioxane and 26mL concentrated HCl and stirred at room temperature for 20 minutes. The volatiles were stripped under vacuum to provide 515 hydrochloride as a white solid. Yield: 7.12g. ¹ H-NMR (DMSO, 400 MHz) delta=9.34 (width, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

516 Synthesis

To a stirred solution of compound 515 in 100mL of dry DCM in 250mL of a two-necked RBF was added NEt ₃ (37.2 mL,0.2669 mol) and cooled to 0℃under a nitrogen atmosphere. After slow addition of N- (benzyloxy-carbonyloxy) -succinimide (20 g,0.08007 mol) in 50mL dry DCM, the reaction mixture was warmed to room temperature. After the reaction was completed (2 to 3 hours by TLC), the mixture was washed with 1N HCl solution (1×100 mL) and saturated NaHCO ₃ solution (1×50 mL) in this order. The organic layer was then dried over anhydrous Na ₂SO₄ and the solvent evaporated to give a crude material which was purified by silica gel column chromatography to give 516 as a viscous material. Yield rate ：11g.¹H-NMR(CDCl₃,400MHz):δ＝7.36-7.27(m,5H),5.69(s,2H),5.12(s,2H),4.96(br.,1H)2.74(s,3H),2.60(m,2H),2.30-2.25(m,2H).LC-MS[M+H]-232.3(96.94％).

Synthesis of 517A and 517B

Cyclopentene 516 (5 g,0.02164 mol) was dissolved in a solution of 220mL of acetone and water (10:1) in a single neck 500mL RBF, and N-methylmorpholine-N-oxide (7.6 g,0.06492 mol) was added thereto followed by 4.2mL of a solution of 7.6% OsO ₄ (0.275 g,0.00108 mol) in t-butanol at room temperature. After the reaction was completed (about 3 hours), the mixture was quenched by addition of solid Na ₂SO₃, and the resulting mixture was stirred at room temperature for 1.5 hours. The reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL), then with saturated NaHCO ₃ (1×50 mL) solution, water (1×30 mL) and finally brine (1×50 mL). The organic phase was dried over anhydrous Na ₂SO₄ and the solvent was removed in vacuo. Silica gel column chromatography purification of the crude material gave a mixture of diastereomers, which were separated by preparative HPLC. Yield: -6g of crude material.

517A-peak 1 (white solid ),5.13g(96％).¹H-NMR(DMSO,400MHz):δ＝7.39-7.31(m,5H),5.04(s,2H),4.78-4.73(m,1H),4.48-4.47(d,2H),3.94-3.93(m,2H),2.71(s,3H),1.72-1.67(m,4H).LC-MS-[M+H]-266.3,[M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry was confirmed by X-ray.

518 Synthesis

Using a procedure analogous to that described for the synthesis of compound 505, the compound was obtained as a colourless oil 518(1.2g,41％).¹H-NMR(CDCl₃,400MHz):δ＝7.35-7.33(m,4H),7.30-7.27(m,1H),5.37-5.27(m,8H),5.12(s,2H),4.75(m,1H),4.58-4.57(m,2H),2.78-2.74(m,7H),2.06-2.00(m,8H),1.96-1.91(m,2H),1.62(m,4H),1.48(m,2H),1.37-1.25(br m,36H),0.87(m,6H).HPLC-98.65％.

General procedure for the synthesis of compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added dropwise to an ice-cold solution of LAH in THF (1 m,2 eq). After complete addition, the mixture was heated at 40 ℃ for 0.5 hours and then cooled again in an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na ₂SO₄, then filtered through celite and reduced to an oil. Column chromatography afforded pure 519 (1.3 g, 68%) as a colorless oil .¹³C NMR＝130.2,130.1(x2),127.9(x3),112.3,79.3,64.4,44.7,38.3,35.4,31.5,29.9(x2),29.7,29.6(x2),29.5(x3),29.3(x2),27.2(x3),25.6,24.5,23.3,226,14.1; electrospray MS (+ve): c ₄₄H₈₀NO₂ (m+h) + calculated molecular weight is 654.6, found 654.6.

Formulations prepared by standard methods or extrusion-free methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. It should be a whitish translucent solution without aggregates or deposits. The particle size and particle size distribution of the lipid nanoparticles can be measured by using light scattering such as Malvern Zetasizer Nano ZS (Malvern, USA). The size of the particles should be about 20nm to 300nm, such as 40nm to 100nm. The particle size distribution should be unimodal. Total dsRNA concentration in the formulation was estimated using dye exclusion assay and embedded fractions. Samples of formulated dsrnas can be incubated with RNA binding dye (Ribogreen (Molecular Probes)) in the presence or absence of a surfactant that disrupts the formulation (e.g., 0.5% Triton-X100). The total dsRNA in the formulation can be determined by the signal from the surfactant-containing sample (relative to a standard curve). The embedded fraction was determined by subtracting the "free" dsRNA content (as measured by signal in the absence of surfactant) from the total dsRNA content. The percentage of embedded dsRNA is typically >85%. For SNALP formulations, the particle size is at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, at least 90nm, at least 100nm, at least 110nm, and at least 120nm. Suitable ranges are typically from about at least 50nm to about at least 110nm, from about at least 60nm to about at least 100nm, or from about at least 80nm to about at least 90nm.

Compositions and formulations for oral administration comprise powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which the dsRNA set forth in the present disclosure is administered with one or more tonicity enhancing agent surfactants and chelating agents. Suitable surfactants comprise fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucuric acid, glycocholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, niu Huangji-24, 25-dihydro-sodium Fuxidate and sodium Ganod-dihydro-Fuxidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glyceryl monooleate, glyceryl dilaurate, glyceryl 1-monocaprate, 1-dodecylazepan-2-one, acylcarnitine, acylcholine or monoglyceride, diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, a combination of permeation enhancers is used, such as a combination of fatty acids/salts and bile acids/salts. An exemplary combination is the sodium salts of lauric acid, capric acid, and UDCA. Additional permeation enhancers include polyoxyethylene-9-dodecyl ether, polyoxyethylene-20-cetyl ether. the dsRNA presented in the present disclosure may be delivered orally, in particulate form comprising spray-dried particles, or complexed to form micro-or nanoparticles. The dsRNA complexing agent comprises a polyamino acid; a polyimine; a polyacrylate; polyalkylacrylates, polyoxyethylenes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylate, polyethylene glycol (PEG) and starch; polyalkylcyanoacrylates; DEAE-derived polyamines, pullulan, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyproteins, protamine, polyvinylpyridine, polythiodiethylaminomethyl ethylene P (TDAE), polyaminostyrene (e.g., para-amino), poly (methyl cyanoacrylate), poly (ethyl cyanoacrylate), poly (butyl cyanoacrylate), poly (isobutyl cyanoacrylate), poly (isohexide), DEAE-methacrylate, DEAE-hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethacrylate, polyhexyl acrylate, poly (D, L-lactic acid), poly (DL-lactic-co-glycolic acid) (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are described in detail in U.S. patent No. 6,887,906, U.S. publication No. 20030027780, and U.S. patent No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (access), intrathecal, intravitreal, subretinal, transscleral, subconjunctival, retrobulbar, intracameral, intraventricular, or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives such as, but not limited to, tonicity agents, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be produced from a variety of components including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids.

The pharmaceutical formulations set forth in the present disclosure may conveniently be presented in unit dosage form and may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of associating the active ingredient with a pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and fully associating the active ingredient with a liquid carrier or a fine solid carrier or both and then shaping the product if necessary.

The compositions presented in the present disclosure may be formulated into any of a number of possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions may also be formulated as suspensions in aqueous, non-aqueous or mixed media. The aqueous suspension may further contain substances which increase the viscosity of the suspension, including for example sodium carboxymethyl cellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

B. other formulations

1. Emulsion

The compositions of the present disclosure may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another in the form of droplets (usually over 0.1 μm in diameter) (see, e.g., ansel's Pharmaceutical Dosage Forms and Drug DELIVERY SYSTEMS, allen, LV., popovich ng. And Ansel HC.,2004,Lippincott Williams&Wilkins (8 th edition), new York, NY; Idson, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editions), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, page 199; rosoff, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editions), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, page 245; Block in Pharmaceutical Dosage Forms, lieberman, rieger and Banker (editions), 1988,Marcel Dekker,Inc, new York, volume 2, page 335; higuchi et al, remington's Pharmaceutical Sciences, mack Publishing Co., easton, pa.,1985, p.301). Emulsions are generally biphasic systems comprising two mutually immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or oil-in-water (o/w). When the aqueous phase is finely divided and dispersed as tiny droplets into the bulk oil phase, the resulting composition is referred to as a water-in-oil (w/o) emulsion. Alternatively, when the oil phase is finely divided as tiny droplets and dispersed into the bulk aqueous phase, the resulting composition is referred to as an oil-in-water (o/w) emulsion. The emulsion may contain other components in addition to the dispersed phase and the active agent, which may be as a solution in the aqueous phase, the oil phase, or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes and antioxidants may also be present if desired. The pharmaceutical emulsion may also be a multiple emulsion comprising more than two phases, as is the case, for example, for oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations generally provide certain advantages over simple binary emulsions. When each oil droplet of the o/w emulsion in the multiple emulsion is also coated with a small water droplet, the multiple emulsion forms a w/o/w emulsion. Likewise, a system of encapsulating oil droplets in stabilized water droplets in an oil continuous phase constitutes an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Typically, the dispersed or discontinuous phase of the emulsion is well dispersed in the external or continuous phase and maintained in this form by the viscosity of the emulsifier or formulation. Other ways of stabilizing emulsions require the use of emulsifiers that can be incorporated into any of the phases of the emulsion. Emulsifiers can be broadly divided into 4 categories: synthetic surfactants, naturally occurring emulsifiers, absorbent matrices, and finely divided solids (see, e.g., ansel's Pharmaceutical Dosage Forms and Drug DELIVERY SYSTEMS, allen, LV., popovich NG. And Ansel HC.,2004,Lippincott Williams&Wilkins (8 th edition), new York, NY; idson, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editions), 1988,Marcel Dekker,Inc, new York, N.Y. volume 1, page 199).

Synthetic surfactants, also known as surfactants, have been widely used in the preparation of emulsions and have been reviewed in the literature (see, e.g., ansel's Pharmaceutical Dosage Forms and Drug DELIVERY SYSTEMS, allen, LV., popovich ng. And Ansel HC.,2004,Lippincott Williams&Wilkins (8 th edition), new York, NY; rieger, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editorial), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, page 285; idson, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editorial), MARCEL DEKKER, inc., new York, n.y.,1988, volume 1, page 199). Surfactants are typically amphiphilic molecules and include a hydrophilic portion and a hydrophobic portion. The ratio of the Hydrophile and Lipophile Balance (HLB) of a surfactant is defined as the hydrophile/lipophile balance (HLB), which is a valuable tool for classifying and selecting surfactants in the preparation of formulations. The surface-active substance may be based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphiphilic groups are divided into different classes (see, e.g., ansel's Pharmaceutical Dosage Forms and Drug DELIVERY SYSTEMS, allen, LV., popovich ng. And Ansel HC.,2004,Lippincott Williams&Wilkins (8 th edition), new York, NY Rieger, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editions), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, page 285).

Natural emulsifiers for emulsion formulations include lanolin, beeswax, phospholipids, lecithins and acacia. Absorbent matrices are hydrophilic in that they can absorb water to form w/o emulsions while maintaining their semi-solid consistency, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids are also used as good emulsifiers, especially in combination with surfactants and in viscous formulations. These include polar inorganic solids such as heavy metal oxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and non-polar solids such as carbon or glycerol tristearate.

Also included in the emulsion formulation are a variety of non-emulsifying materials which contribute to the characteristics of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrocolloids, preservatives and antioxidants (Block, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editions), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, page 335; idson, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editions), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, page 199).

Hydrocolloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carboxypolymers, cellulose ethers and carboxyvinyl polymers). They disperse or swell in water to form a colloidal solution, stabilizing the emulsion by forming a strong interfacial film around the dispersed phase droplets and enhancing the viscosity of the external phase.

Since emulsions typically contain a variety of ingredients, such as carbohydrates, proteins, sterols, and phospholipids, which can readily support microbial growth, these formulations typically incorporate preservatives. Preservatives commonly used in emulsion formulations include methyl parahydroxybenzoate, propyl parahydroxybenzoate, quaternary ammonium salts, benzalkonium chloride, parabens and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. The antioxidants used may be free radical scavengers such as tocopherol, alkyl gallate, butyl hydroxyanisole, butyl hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant potentiators such as citric acid, tartaric acid and lecithin.

The literature reviews the use of emulsion formulations and methods of making them by dermatological, oral and parenteral routes (see, e.g., ansel's Pharmaceutical Dosage Forms and Drug DELIVERY SYSTEMS, allen, LV., popovich NG. And Ansel HC.,2004,Lippincott Williams&Wilkins (8 th edition), new York, NY; idson, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editions), 1988,Marcel Dekker,Inc, new York, N.Y., volume 1, p.199). Emulsion formulations for oral delivery have been very widely used due to ease of formulation and efficacy from the standpoint of absorption and bioavailability (see, e.g., ansel's Pharmaceutical Dosage Forms and Drug DELIVERY SYSTEMS, allen, LV., popovich ng. And Ansel HC.,2004,Lippincott Williams&Wilkins (8 th edition), new York, NY, rosoff, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editorial), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, p.245; idson, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editorial), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, p.199). Mineral oil matrix laxatives, oil-soluble microorganisms and high fat nutritional formulations are materials that are commonly administered orally as o/w emulsions.

In some embodiments of the disclosure, the composition of iRNA and nucleic acid is formulated as a microemulsion. Microemulsions can be defined as systems of water, oil and amphiphilic molecules which are optically isotropic and thermodynamically stable single liquid solutions (see, e.g., ansel' sPharmaceutical Dosage Forms and Drug DELIVERY SYSTEMS, allen, LV., popovich ng. And Ansel HC.,2004,Lippincott Williams&Wilkins (8 th edition), new York, NY; rosoff, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editions), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, stage 245). Typically, microemulsions are systems prepared by the following methods: the oil is first dispersed into an aqueous surfactant solution and then a sufficient amount of a fourth component, typically a medium chain length alcohol, is added to form a transparent system. Microemulsions are therefore also described as thermodynamically stable isotropic clear dispersions of two immiscible liquids stabilized by interfacial films of surface active molecules (Leung and Shah, controlled Release of Drugs: polymers AND AGGREGATE SYSTEMS, rosoff, M.J., eds., 1989,VCH Publishers,New York, pages 185-215). Microemulsions are typically prepared by a combination of three to five components including oil, water, surfactants, cosurfactants and electrolytes. Microemulsions are of the water-in-oil (w/o) or oil-in-water (o/w) type, depending on the nature of the oil and surfactant used, and the structural and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, remington's Pharmaceutical Sciences, mack Publishing co., easton, pa.,1985, p.271).

The phenomenological manner of using phase diagrams has been widely studied and a full knowledge has been developed to the person skilled in the art about how to formulate microemulsions (see, for example, ,Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems,Allen,LV.,Popovich NG.,and Ansel HC.,2004,Lippincott Williams&Wilkins( th edition 8), new York, NY; rosoff, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editions), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, p.245; block, pharmaceutical Dosage Forms, lieberman, rieger and Banker (editions), 1988,Marcel Dekker,Inc, new York, N.Y., volume 1, p.335). Microemulsions generally have the advantage over traditional emulsions of dissolving water-insoluble drugs in spontaneously formed thermodynamically stable droplets.

Surfactants for microemulsion preparation include, but are not limited to, ionic surfactants, nonionic surfactants, brij 96, polyoxyethylene oleyl ether, polyglyceryl fatty acid esters, tetraglyceryl laurate (ML 310), tetraglyceryl monooleate (MO 310), hexaglyceryl monooleate (PO 310), hexaglyceryl pentaoleate (PO 500), glyceryl monocaprylate (MCA 750), decaglyceryl monooleate (MO 750), decaglyceryl linoleate (SO 750), decaglyceryl caprate (DAO 750), alone or in combination with a co-surfactant. Cosurfactants, typically short chain alcohols such as ethanol, 1-propanol and 1-butanol, typically penetrate into the surfactant film to increase interfacial mobility, creating disordered films due to the void spaces created between the surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, aqueous solutions of drugs, glycerol, PEG300, PEG400, polyglycerol, propylene glycol and derivatives of ethanol. The oil phase may include, but is not limited to, materials such as Captex 300, captex355, capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-, and triglycerides, polyoxyethylated glycerol fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

Microemulsions are of particular interest from the standpoint of drug dissolution and enhancing drug absorption. Lipid-based microemulsions (o/w and w/o) have been proposed to improve the oral bioavailability of drugs including peptides (see, e.g., U.S. Pat. Nos. 6,191,105;7,063,860;7,070,802;7,157,099; constantinides et al Pharmaceutical Research,1994,11,1385-1390; ritschel, meth.find.exp.Clin.Pharmacol.,1993,13,205). The microemulsion has the following advantages: improving drug solubility, protecting the drug from enzymatic hydrolysis, possibly enhancing drug absorption due to membrane fluidity and permeability changes caused by surfactants, ease of manufacture, ease of oral administration compared to solid dosage forms, improved clinical efficacy and reduced toxicity (see, e.g., U.S. Pat. nos. 6,191,105;7,063,860;7,070,802;7,157,099; constantanides et al Pharmaceutical Research,1994,11,1385; ho et al, j. Pharm. Sci.,1996,85,138-143). When their components are mixed together at ambient temperature, microemulsions are typically formed spontaneously. This may be particularly advantageous when formulating heat resistant drugs, peptides or iRNA agents. Microemulsions are also effective in the transdermal delivery of active ingredients for cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present disclosure are expected to promote increased systemic absorption of iRNA agents and nucleic acids from the gastrointestinal tract, as well as improve local cellular uptake of iRNA agents and nucleic acids.

The microemulsions of the present disclosure may also contain additional components and additives, such as sorbitan monostearate (Grill 3), labrasol, and permeation enhancers to improve the properties of the formulations and enhance the absorption of the iRNA agents and nucleic acids of the present disclosure. Permeation enhancers for microemulsions of the present disclosure can be categorized as belonging to one of five broad classes-surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al CRITICAL REVIEWS IN Therapeutic Drug CARRIER SYSTEMS,1991, p.92). Each of these categories has been discussed above.

2. Penetration enhancer

In some embodiments, the present disclosure uses various permeation enhancers to achieve efficient delivery of nucleic acids, particularly iRNA agents, to the skin of an animal. Most drugs exist in solution in ionized and non-ionized forms. However, only lipid-soluble or lipophilic drugs are generally easy to permeate the cell membrane. It has been found that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a permeation enhancer. In addition to helping the non-lipophilic drug diffuse across the cell membrane, the permeation enhancer also enhances the permeability of the lipophilic drug.

Penetration enhancers can be divided into one of five classes, namely surfactants, fatty acids, bile salts, chelating agents and non-chelating non-surfactants (see, e.g., malmsten, m.surfactants and polymers in Drug delivery, informa HEALTH CARE, new York, NY,2002; lee et al, CRITICAL REVIEWS IN Therapeutic Drug CARRIER SYSTEMS,1991, p.92). Each of the above-described classes of permeation enhancers is described in more detail below.

And (2) a surfactant: in connection with the present disclosure, a surfactant (or "surfactant") is a chemical entity that, when dissolved in an aqueous solution, reduces the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, thereby enhancing the absorption of the RNAi agent through the mucosa. In addition to bile salts and fatty acids, such permeation enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, e.g., malmsten, m. Surfactants and polymers in Drug delivery, informa HEALTH CARE, new York, NY,2002; lee et al, CRITICAL REVIEWS IN Therapeutic Drug CARRIER SYSTEMS,1991, p.92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al, J.Pharm.Pharmacol.,1988,40,252).

Fatty acid: various fatty acids and derivatives thereof useful as permeation enhancers include, for example, oleic acid, lauric acid, capric acid (n-capric acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-racemic glycerol), dilaurate, caprylic acid, arachidonic acid, glycerol 1-monocaprylate, 1-dodecylcycloheptan-2-one, acylcarnitines, acylcholines, their C _1-20 alkyl esters (e.g., methyl, isopropyl and t-butyl), and monoglycerides and diglycerides thereof (i.e., oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, and the like) (see, e.g., touitou, e.etc., ENHANCEMENT IN Drug Delivery, CRC Press, danvers, MA,2006; lee et al ,Critical Reviews in Therapeutic Drug Carrier Systems,1991,p.92;Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems,1990,7,1-33;El Hariri, and the like, j.rm.pharmacol., 1992,44,651-654).

Bile salts: physiological effects of bile include promoting the dispersion and absorption of lipids and fat-soluble vitamins (see, e.g., malmsten, M.surfactants and polymers in drug delivery, informa HEALTH CARE, new York, N.Y., 2002; brunton, chapter 38: goodman & Gilman's The Pharmacological Basis of Therapeutics, 9 th edition, hardman et al, eds., mcGraw-Hill, new York,1996, pp.934-935). Various natural bile salts and synthetic derivatives thereof are used as permeation enhancers. The term "bile salts" therefore includes any naturally occurring bile component as well as any synthetic derivative thereof. Suitable bile salts include, for example, cholic acid (or a pharmaceutically acceptable sodium salt thereof, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucocholic acid (sodium glycocholate), glycocholic acid (sodium glycodeoxycholate), taurocholate (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (UDCA), taurine-24, 25-dihydrofusidic acid Sodium (STDHF), sodium ethanoldihydrogen melt and polyoxyethylene-9-lauryl ether (POE) (see, e.g., malmsten, M.surfactants and polymers in Drug delivery, informa HEALTH CARE, new York, NY,2002; lee et al, CRITICAL REVIEWS IN thermo Drug CARRIER SYSTEMS,1991, page 92; swinyard, chapter 39, remington's Pharmaceutical Sciences, 18, cheno, mack Publ, eastton, 7825, 1993-35, U.S. Pat. No. 3, U.M. 2, U.S. Pat. No. 3, U.S. 2, U.S. No. 3, U.S. 5, U.S. Pat. No. 2, U.S. 3, U.S. 5, U.S. 2, J. Pat. No. 3, J. 5, U.S. 3, J. No. 5, U.S. 3, J. 5, U.S. 3, J. Pat. No. 5, whereby, J. 5, whereby there is provided by the present invention.

Chelating agent: chelating agents used in combination with the present disclosure may be defined as compounds that remove metal ions from solution by forming complexes therewith, thereby enhancing the uptake of RNAi agents through the mucosa. With respect to their use as permeation enhancers in the present disclosure, chelators have the additional advantage of also acting as DNase inhibitors, as most characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelators (Jarrett, j. Chromatogr.,1993,618,315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediamine tetraacetate (EDTA), citric acid, salicylic acid (e.g., sodium salicylate, 5-methoxysalicylic acid and homovanadate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines) (see, e.g., katdare, A. Et al ,Excipient development for pharmaceutical,biotechnology,and drug delivery,CRC Press,Danvers,MA,2006;Lee et al, CRITICAL REVIEWS IN Therapeutic Drug CARRIER SYSTEMS,1991, page 92; muranishi, CRITICAL REVIEWS IN Therapeutic Drug CARRIER SYSTEMS,1990,7,1-33; buur et al J. Control Rel.,1990,14,43-51).

Non-chelating non-surfactant: as used herein, a non-chelating non-surfactant penetration enhancing compound may be defined as a compound that exhibits insignificant activity as a chelating agent or as a surfactant but still enhances the absorption of RNAi agents through the mucosa of the digestive tract (see, e.g., muranishi, CRITICAL REVIEWS IN Therapeutic Drug CARRIER SYSTEMS,1990,7,1-33). Such permeation enhancers include, for example, unsaturated cyclic ureas, 1-alkyl and 1-alkenyl azacycloalkanone derivatives (Lee et al CRITICAL REVIEWS IN Therapeutic drugs CARRIER SYSTEMS,1991, page 92); and non-steroidal anti-inflammatory drugs such as sodium diclofenac, indomethacin, and phenylbutazone (Yamashita et al, j.pharm.pharmacol.,1987,39,621-626).

Agents that enhance uptake of iRNA at the cellular level may also be added to the medicaments and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al, PCT application WO 97/30731), are also known to enhance cellular uptake of dsRNA. Examples of commercially available transfection reagents include, for example, Lipofectamine^TM(Invitrogen;Carlsbad,CA)、Lipofectamine 2000^TM(Invitrogen;Carlsbad,CA)、293fectin^TM(Invitrogen;Carlsbad,CA)、Cellfectin^TM(Invitrogen;Carlsbad,CA)、DMRIE-C^TM(Invitrogen;Carlsbad,CA)、FreeStyle^TMMAX(Invitrogen;Carlsbad,CA)、Lipofectamine^TM2000CD(Invitrogen;Carlsbad,CA)、Lipofectamine^TM(Invitrogen;Carlsbad,CA)、RNAiMAX(Invitrogen;Carlsbad,CA)、Oligofectamine^TM(Invitrogen;Carlsbad,CA)、Optifect^TM(Invitrogen;Carlsbad,CA)、X-tremeGENE Q2 transfection reagent (Roche; grenzacherstrasse, switzerland), DOTAP liposome transfection reagent (Grenzacherstrasse, switzerland), DOSPER liposome transfection reagent (Grenzacherstrasse, switzerland) or Fugene (Grenzacherstrasse, switzerland),Reagents (Promega; madison, wis.), transFast ^TM transfection reagent (Promega; madison, wis.), tfx ^TM -20 reagent (Promega; madison, wis.), tfx ^TM -50 reagent (Promega;Madison,WI)、DreamFect^TM(OZ Biosciences;Marseille,France)、EcoTransfect(OZ Biosciences;Marseille,France)、TransPass^a D1 transfection reagent (New England Biolabs;Ipswich,MA,USA)、LyoVec^TM/LipoGen^TM(Invivogen;San Diego,CA,USA)、PerFectin transfection reagent (Genlantis; san Diego, calif., USA), neuroPORTER transfection reagent (Genlantis; san Diego, calif., USA), genePORTER transfection reagent (Genlantis; san Diego, calif., USA), genePORTER transfection reagent (Genlantis; san Diego, calif., USA), cytofectin transfection reagent (Genlantis; san Diego, calif., USA), baculoPORTER transfection reagent (Genlantis; san Diego, calif., USA), troganPORTER ^TM transfection reagent (Genlantis;San Diego,CA,USA)、RiboFect(Bioline;Taunton,MA,USA)、PlasFect(Bioline;Taunton,MA,USA)、UniFECTOR(B-Bridge International;Mountain View,CA,USA)、SureFECTOR(B-Bridge International;Mountain View,CA,USA) or HiFect ^TM (B-Bridge International; mountaine View, calif., USA), and the like.

Other agents may be used to enhance penetration of the administered nucleic acid, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azones and terpenes such as limonene and menthone.

C. Carrier body

Certain compositions of the present disclosure also incorporate carrier compounds in the formulation. As used herein, a "carrier compound" may refer to a nucleic acid or analog thereof that is inert (i.e., does not itself have biological activity), but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of the biologically active nucleic acid by, for example, degrading the biologically active nucleic acid or facilitating its removal from the circulation. Co-administration of the nucleic acid and the carrier compound (typically with an excess of the latter substance) can result in a significant reduction in the amount of nucleic acid recovered in the liver, kidney or other extra-circulatory reservoir, possibly due to competition for co-receptors between the carrier compound and the nucleic acid. For example, when a portion of phosphorothioate dsRNA is co-administered with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4 '-isothiocyanato-stilbene-2, 2' -disulfonic acid, recovery of a portion of phosphorothioate dsRNA in liver tissue may be reduced (Miyao et al, 1995DsRNA Res.Dev.5:115-121; takakura et al, 1996DsRNA&Nucl.Acid Drug Dev.6:177-183).

1. Excipient

In contrast to carrier compounds, a pharmaceutical carrier or excipient is a pharmaceutically acceptable solvent, suspending agent, or other pharmacologically inert carrier for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid, and upon combination with the nucleic acid and other components of a given pharmaceutical composition, is selected according to the intended mode of administration to provide a desired volume, consistency, etc. Typical drug carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, and the like); disintegrants (e.g., starch, sodium starch glycolate, etc.); and a wetting agent (e.g., sodium dodecyl sulfate, etc.).

Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration and which do not adversely react with nucleic acids may also be used to formulate compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone and the like.

Formulations for topical application of nucleic acids may include sterile or non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of nucleic acids in liquid or solid oil bases. The solution may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration and which do not adversely react with nucleic acids may also be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone and the like.

D. Other components

The compositions of the present disclosure may additionally comprise other auxiliary components conventionally found in pharmaceutical compositions, at established levels of use in the art. Thus, for example, the compositions may comprise additional, compatible pharmaceutically active materials, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may comprise various acute additional materials useful in physically formulating the compositions of the present disclosure, such as dyes, fragrances, preservatives, antioxidants, opacifying agents, thickening agents, and stabilizers. However, these materials should not unduly interfere with the biological activity of the components of the compositions of the present disclosure when added. The formulation may be sterilized and, if desired, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavoring or aromatic substances and the like which do not deleteriously interact with the nucleic acids of the formulation.

The aqueous suspension may contain substances that increase the viscosity of the suspension, including for example sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may also contain stabilizers.

In some embodiments, the pharmaceutical compositions presented in the present disclosure comprise (a) one or more iRNA compounds and (b) one or more biological agents that act through a non-RNAi machinery. Examples of such biological agents include agents that interfere with the interaction of ANGPTL7 and at least one ANGPTL7 binding partner.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD ₅₀ (the dose lethal to 50% of the population) and ED ₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio of LD ₅₀/ED₅₀. Compounds exhibiting high therapeutic indices are preferred.

The data obtained from cell culture experiments and animal studies can be used to formulate a range of dosages for humans. The dosage of the compositions characterized herein in this disclosure is generally within a circulating concentration range, including ED ₅₀, with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration employed. For any compound used in the methods of the features of the present disclosure, a therapeutically effective dose can be initially estimated from a cell culture assay. Dosages may be formulated in animal models to achieve a circulating plasma concentration range of the compound, or where appropriate, of the polypeptide product of the target sequence (e.g., to achieve a reduced polypeptide concentration), including IC ₅₀ as determined in cell culture (i.e., the concentration of the test compound that achieves half of the maximum symptom inhibition). Such information may be used to more accurately determine useful doses in humans. For example, the level in plasma may be measured by high performance liquid chromatography.

In addition to their administration, the irnas set forth in the present disclosure may also be administered in combination with other known drugs effective in treating diseases or disorders associated with ANGPTL7 expression (e.g., glaucoma or conditions associated with glaucoma), as discussed above. In any event, the administering physician can adjust the amount and time of iRNA administration based on the results observed using standard measurements of efficacy known in the art or described herein.

Methods of treating conditions associated with ANGPTL7 expression

The present disclosure relates to the use of iRNA that target ANGPTL7 to inhibit ANGPTL7 expression and/or to treat a disease, disorder, or pathological process associated with ANGPTL7 expression (e.g., glaucoma or a condition associated with glaucoma).

In some aspects, a method of treating a disorder associated with ANGPTL7 expression is provided, the method comprising administering to a subject in need thereof an iRNA (e.g., dsRNA) disclosed herein. In some embodiments, the iRNA inhibits (reduces) ANGPTL7 expression.

In some embodiments, the subject is an animal that is a model of a disorder associated with ANGPTL7 expression (e.g., glaucoma or a disorder associated with glaucoma).

A. Glaucoma or conditions associated with glaucoma

In some embodiments, the condition associated with ANGPTL7 expression is glaucoma or a condition associated with glaucoma. Non-limiting examples of glaucoma or glaucoma-related conditions that can be treated using the methods described herein include glaucoma, open-angle glaucoma, primary open-angle glaucoma, closed-angle glaucoma, ocular inflammation, systemic inflammation, anterior uveitis, acute retinal necrosis, sturge-Weber syndrome, xenfeld-Rieger syndrome, marfan syndrome, homocystinuria, weill-MARCHESANI syndrome, and autoimmune diseases, such as juvenile rheumatoid arthritis and Marie Strumpell ankylosing spondylitis.

Glaucoma is a group of ocular disorders characterized by progressive optic nerve damage, usually caused by a relative rise in intraocular pressure. Clinical and pathological features of glaucoma or conditions related to glaucoma include, but are not limited to, increased intraocular pressure, decreased vision, decreased visual acuity (e.g., characterized by floating spots, blurred vision at the edges or around the center (e.g., dark spots), ocular inflammation, ocular pain, headache, and/or optic nerve damage.

Open angle glaucoma is the most common glaucoma, with normal angle between iris and cornea, caused by slow blockage of aqueous humor drainage tubes. Primary open-angle glaucoma is open-angle glaucoma with no identifiable cause. Open angle glaucoma generally progresses gradually.

Closed angle glaucoma, also known as acute glaucoma or narrow angle glaucoma, presents a closed or narrow angle between the iris and cornea, caused by obstruction of the aqueous humor drainage tube. Angle-closure glaucoma generally progresses rapidly and has obvious symptoms, requiring immediate treatment.

In some embodiments, the subject suffering from glaucoma or a condition associated with glaucoma is less than 18 years old. In some embodiments, the subject suffering from glaucoma or a glaucoma-associated condition is an adult. In some embodiments, the subject has or is identified as having an elevated ANGPTL7 mRNA or protein level relative to a reference level (e.g., an ANGPTL7 level greater than the reference level).

In some embodiments, glaucoma or a condition associated with glaucoma is diagnosed by analyzing a sample (e.g., an optic nerve sample) from a subject. In some embodiments, the sample is analyzed using one or more methods selected from the group consisting of: fluorescence In Situ Hybridization (FISH), immunohistochemistry, ANGPTL7 immunoassay, electron microscopy, laser microdissection and mass spectrometry. In some embodiments, glaucoma or conditions associated with glaucoma are diagnosed by any suitable diagnostic test or technique, e.g., intraocular pressure measurement, thickness measurement, retinal evaluation, gonioscopy, angiography (e.g., fluorescein angiography or indocyanine green angiography), electroretinography, ultrasonography, optical Coherence Tomography (OCT), computed Tomography (CT), and Magnetic Resonance Imaging (MRI), color vision testing, visual field testing, slit lamp inspection, ophthalmoscopy, and physical examination (e.g., evaluating sensitivity (e.g., by ophthalmoscopy or Optical Coherence Tomography (OCT)).

B. Combination therapy

In some embodiments, an iRNA (e.g., dsRNA) disclosed herein is administered in combination with a second therapy (e.g., one or more additional therapies) known to be effective in treating a disorder associated with ANGPTL7 expression (glaucoma) or symptoms of such disorder. The iRNA may be administered before, after, or concurrently with the second therapy. In some embodiments, the iRNA is administered prior to the second therapy. In some embodiments, the iRNA is administered after the second therapy. In some embodiments, the iRNA is administered concurrently with the second therapy.

The second therapy may be an additional therapeutic agent. The iRNA and the additional therapeutic agent may be administered in combination in the same composition, or the additional therapeutic agent may be administered as part of separate compositions.

In some embodiments, the second therapy is a non-iRNA therapeutic agent effective to treat the disorder or symptoms of the disorder.

In some embodiments, the iRNA is administered with a therapy.

Exemplary combination therapies include, but are not limited to, drugs that reduce intraocular pressure, laser therapy, surgery, or trabeculectomy. In some embodiments, the additional therapeutic agent comprises a prostaglandin analog, a beta blocker, an alpha adrenergic agonist, a carbonic anhydrase inhibitor, a Rho kinase inhibitor (ROCK), an iRNA agent directed against ROCK, an inhibitor of Rho GTPase, an anti-Rho GTPase agent, or an anti-ANGPTL 7 agent.

In some embodiments, the other therapeutic agent is a prostaglandin analog. In some embodiments, the prostaglandin analog comprises bimatoprostLatanoprostTafluprost (Zioptan ^TM), latanoprost nitrate (Vyzulta ^TM) or travoprost (Travatan))。

In some embodiments, the other therapeutic agent is a beta blocker. In some embodiments, the beta blocker comprises betaxolol (Betoptic) Or timololTimoptic)。

In some embodiments, the other therapeutic agent is an alpha adrenergic agonist. In some embodiments, the alpha adrenergic agonist comprises brimonidine @P) or alcalidine

In some embodiments, the other therapeutic agent is a carbonic anhydrase inhibitor. In some embodiments, the carbonic anhydrase inhibitor comprises dorzolamideBrinzolamideAcetazolamide (Diamox) or methazolamide

In some embodiments, the other therapeutic agent is a ROCK inhibitor or a ROCK iRNA agent. In some embodiments, the ROCK inhibitor is nettadil

In some embodiments, the therapeutic agent is an anti-Rho GTPase agent. In some embodiments, the anti-Rho GTPase agent is an antibody molecule. In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the other therapeutic agent is an anti-ANGPTL 7 agent. In some embodiments, the anti-ANGPTL 7 agent is an antibody molecule. In some embodiments, the antibody is a monoclonal antibody.

C. dosage, route and timing of administration

A therapeutic amount of an iRNA can be administered to a subject (e.g., a human subject, e.g., a patient). The therapeutic amount may be, for example, 0.05-50mg/kg.

In some embodiments, the iRNA is formulated for delivery to a target organ, e.g., to the eye.

In some embodiments, the iRNA is formulated as a lipid formulation, e.g., an LNP formulation as described herein. In some such embodiments, the therapeutic amount is 0.05mg/kg to 5mg/kg dsRNA. In some embodiments, the lipid formulation, e.g., LNP formulation, is administered intravenously.

In some embodiments, the iRNA is in the form of a GalNAc conjugate, e.g., as described herein. In some such embodiments, the therapeutic amount is 0.5mg to 50mg dsRNA. In some embodiments, for example, the GalNAc conjugate is administered subcutaneously.

In some embodiments, for example, the administration is repeated periodically, such as daily, every two weeks (i.e., every two weeks), for one month, two months, three months, four months, six months, or more. After the initial treatment regimen, the treatment may be administered on a less frequent basis. For example, after administration every two weeks for three months, administration may be repeated once a month for six months or one year or more.

In some embodiments, the iRNA agent is administered in two or more doses. In some embodiments, the number or amount of subsequent doses depends on the achievement of the desired effect, e.g., (a) inhibiting or lowering intraocular pressure; (b) inhibiting or reducing expression or activity of ANGPTL 7; (c) increasing drainage of aqueous humor; (d) inhibiting or reducing optic nerve damage; or (e) inhibit or reduce retinal ganglion cell death, or achieve a therapeutic or prophylactic effect, e.g., reducing or preventing one or more symptoms associated with a disorder.

In some embodiments, the iRNA agent is administered according to a schedule. For example, the iRNA agent may be administered once a week, twice a week, three times a week, four times a week, or five times a week. In some embodiments, the schedule involves administration at regular intervals, e.g., every hour, every four hours, every six hours, every eight hours, every twelve hours, daily, every 2 days, every 3 days, every 4 days, every 5 days, weekly, every two weeks, or monthly. In some embodiments, the iRNA agent is administered at a frequency necessary to achieve the desired effect.

In some embodiments, the schedule involves closely spaced administrations followed by a longer period of time during which no agent is administered. For example, the schedule may involve an initial dose group administered over a relatively short period of time (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours), followed by a longer period of time (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) during which no iRNA agent is administered. In some embodiments, the iRNA agent is administered initially hourly, followed by administration at longer intervals (e.g., daily, weekly, biweekly, or monthly). In some embodiments, the iRNA agent is administered initially daily, followed by longer intervals (e.g., weekly, biweekly, or monthly). In some embodiments, the longer interval increases over time or is determined based on the achievement of the desired effect.

A smaller dose, such as a 5% infusion dose, may be administered to the patient prior to administration of the full dose of iRNA, and adverse effects, such as allergic reactions, or elevated lipid levels or blood pressure, monitored. In another example, the patient may be monitored for undesirable effects.

V. methods for modulating ANGPTL7 expression

In some aspects, the present disclosure provides a method for modulating (e.g., inhibiting or activating) ANGPTL7 expression, e.g., in a cell, in a tissue, or in a subject. In some embodiments, the cell or tissue is ex vivo, in vitro, or in vivo. In some embodiments, the cells or tissue are in the eye (e.g., optic nerve cells, trabecular meshwork cells, schlemm's canal cells (e.g., including endothelial cells), proximal tubular tissue cells, ciliary muscle cells, retinal cells, astrocytes, pericytes, muller cells, ganglion cells (e.g., including retinal ganglion cells), endothelial cells, photoreceptor cells, retinal blood vessels (e.g., including endothelial cells and vascular smooth muscle cells), suprascleral vein or choroidal tissue, e.g., choroidal blood vessels). In some embodiments, the cell or tissue is in a subject (e.g., a mammal, such as, for example, a human). In some embodiments, a subject (e.g., a human) is at risk of, or diagnosed with, a disorder associated with expression of ANGPTL7 expression, as described herein.

In some embodiments, the method comprises contacting the cell with an iRNA as described herein in an amount effective to reduce expression of ANGPTL7 in the cell. In some embodiments, contacting the cell with the RNAi agent comprises contacting the cell with the RNAi agent in vitro or contacting the cell with the RNAi agent in vivo. In some embodiments, the RNAi agent is physically contacted with the cell by an individual performing the method, or the RNAi agent can be brought into conditions that allow or bring it into subsequent contact with the cell. In vitro contacting of cells can be performed, for example, by incubating the cells with an RNAi agent. In vivo contacting of cells may be performed, for example, by injecting the RNAi agent into or near the tissue in which the cells are located, or by injecting the RNAi agent into another area, such as ocular tissue. For example, an RNAi agent can contain or be conjugated to a ligand, e.g., a lipophilic moiety or moieties, as described herein and in further detail, e.g., in PCT/US2019/031170, which is incorporated herein by reference in its entirety, including paragraphs in which lipophilic moieties are described that guide or otherwise stabilize the RNAi agent at the site of interest. Combinations of in vitro and in vivo contact methods are also possible. For example, the cells may also be contacted with an RNAi agent in vitro and subsequently transplanted into a subject.

The expression of ANGPTL7 may be assessed based on the level of ANGPTL7 mRNA, the expression of ANGPTL7 protein, or the level of another parameter functionally related to the level of ANGPTL7 expression. In some embodiments, expression of ANGPTL7 is inhibited by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, IC ₅₀ of the iRNA is in the range of 0.001nM to 0.01nM, 0.001nM to 0.10nM, 0.001nM to 1.0nM, 0.001nM to 10nM, 0.01nM to 0.05nM, 0.01nM to 0.50nM, 0.02nM to 0.60nM, 0.01nM to 1.0nM, 0.01nM to 1.5nM, 0.01nM to 10 nM. IC ₅₀ values can be normalized to an appropriate control value (e.g., non-iRNA-targeted IC ₅₀).

In some embodiments, the method comprises introducing an iRNA as described herein into a cell or tissue, and maintaining the cell or tissue for a time sufficient to obtain degradation of mRNA transcripts of ANGPTL7, thereby inhibiting expression of ANGPTL7 in the cell or tissue.

In some embodiments, the methods comprise administering to the mammal a composition described herein, e.g., a composition comprising iRNA that binds to ANGPTL7, such that expression of the target ANGPTL7 is reduced, e.g., for an extended duration, e.g., for at least two days, three days, four days, or more, e.g., one week, two weeks, three weeks, or four weeks, or more. In some embodiments, a decrease in expression of ANGPTL7 is detectable within 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours of the first administration.

In some embodiments, the method comprises administering to a mammal a composition as described herein such that expression of target ANGPTL7 is increased, e.g., by at least 10% compared to untreated animals. In some embodiments, the activation of ANGPTL7 occurs over an extended duration, e.g., for at least two days, three days, four days, or more, e.g., one week, two weeks, three weeks, four weeks, or more. Without wishing to be bound by theory, iRNA may activate ANGPTL7 expression by stabilizing ANGPTL7 mRNA transcripts, interacting with promoters in the genome, or inhibiting inhibitors of ANGPTL7 expression.

The iRNA useful in the methods and compositions presented in the present disclosure specifically targets the RNA (virgin or processed) of ANGPTL 7. Compositions and methods of using iRNA to inhibit expression of ANGPTL7 can be prepared and performed as described elsewhere herein.

In some embodiments, the method comprises administering a composition comprising an iRNA, wherein the iRNA comprises a nucleotide sequence that is complementary to at least a portion of an RNA transcript of ANGPTL7 of a subject (e.g., mammal, e.g., human) to be treated. The composition may be administered by any suitable means known in the art, including but not limited to ocular (e.g., intraocular), topical, and intravenous administration.

In certain embodiments, the composition is administered intra-ocularly (e.g., by intravitreal administration, e.g., intravitreal injection, transscleral administration, e.g., transscleral injection, subconjunctival administration, e.g., subconjunctival injection, retrobulbar administration, e.g., retrobulbar injection, intracameral administration, e.g., intracameral injection, or subretinal administration, e.g., subretinal injection). In other embodiments, the composition is administered topically. In other embodiments, the composition is administered by intravenous infusion or injection.

In certain embodiments, the composition is administered by intravenous infusion or injection. In some such embodiments, the composition comprises a lipid-formulated siRNA for intravenous infusion (e.g., an LNP formulation, such as an LNP11 formulation).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNA and methods set forth in the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Detailed Description

Specific embodiments of the disclosure are provided below.

(1) DsRNA agent

In one aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 7 (ANGPTL 7), wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1,2, or 3 mismatches with a portion of a mouse ANGPTL7 coding strand, and the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1,2, or 3 mismatches with a corresponding portion of a non-coding strand of mouse ANGPTL7, such that the sense strand is complementary to at least 15 consecutive nucleotides in the antisense strand. In one embodiment, the coding strand of mouse ANGPTL7 comprises the sequence SEQ ID No. 1. In a further embodiment, the non-coding strand of mouse ANGPTL7 comprises the sequence of SEQ ID No. 2.

In another aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ANGPTL7, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1,2 or 3 mismatches with a portion of the nucleotide sequence of SEQ ID NO:2 such that the sense strand is complementary to at least 15 consecutive nucleotides in the antisense strand. In one embodiment, the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0 or 1,2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 1.

In certain embodiments, the dsRNA of any of the preceding embodiments comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 consecutive nucleotides having 0, 1,2, or 3 mismatches with a portion of the nucleotide sequence of SEQ ID No.2 such that the sense strand is complementary to at least 17 consecutive nucleotides in the antisense strand. In a further embodiment, the sense strand comprises a nucleotide sequence comprising at least 17 consecutive nucleotides having 0 or 1,2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 1.

In certain embodiments, the dsRNA of any of the preceding embodiments comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides having 0, 1,2, or 3 mismatches with a portion of the nucleotide sequence of SEQ ID No.2 such that the sense strand is complementary to at least 19 contiguous nucleotides in the antisense strand. In a further embodiment, the sense strand comprises a nucleotide sequence comprising at least 19 consecutive nucleotides having 0 or 1,2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 1.

In certain embodiments, the dsRNA of any of the preceding embodiments comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides having 0, 1,2, or 3 mismatches with a portion of the nucleotide sequence of SEQ ID No.2 such that the sense strand is complementary to at least 21 contiguous nucleotides in the antisense strand. In a further embodiment, the sense strand comprises a nucleotide sequence comprising at least 21 consecutive nucleotides having 0 or 1,2 or 3 mismatches with the corresponding portion of the nucleotide sequence of SEQ ID NO. 1.

In certain embodiments, a dsRNA agent of any of the preceding embodiments is provided, wherein the portion of the sense strand is a portion within the sense strand in any of tables 2-7.

In certain embodiments, a dsRNA agent of any of the preceding embodiments is provided, wherein the portion of the antisense strand is a portion within the antisense strand in any of tables 2-7.

In certain embodiments, a dsRNA agent of any one of the preceding embodiments is provided, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides that is mismatched with 0, 1, 2, or 3 of one of the antisense sequences listed in any one of tables 2-7.

In certain embodiments, a dsRNA agent of any of the preceding embodiments is provided, wherein the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1,2, or 3 mismatches with a sense sequence set forth in any of tables 2-7 corresponding to the antisense sequence.

In certain embodiments, a dsRNA agent of any one of the preceding embodiments is provided, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 consecutive nucleotides having 0, 1,2, or 3 mismatches with one of the antisense sequences set forth in any one of tables 2-7.

In certain embodiments, a dsRNA agent of any of the preceding embodiments is provided, wherein the sense strand comprises a nucleotide sequence comprising at least 17 consecutive nucleotides having 0, 1,2, or 3 mismatches with a sense sequence set forth in any of tables 2-7 corresponding to the antisense sequence.

In certain embodiments, a dsRNA agent of any one of the preceding embodiments is provided, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 consecutive nucleotides having 0, 1,2, or 3 mismatches with one of the antisense sequences set forth in any one of tables 2-7.

In certain embodiments, a dsRNA agent of any of the preceding embodiments is provided, wherein the sense strand comprises a nucleotide sequence comprising at least 19 consecutive nucleotides having 0, 1,2, or 3 mismatches with a sense sequence set forth in any of tables 2-7 corresponding to the antisense sequence.

In certain embodiments, a dsRNA agent of any one of the preceding embodiments is provided, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 consecutive nucleotides having 0, 1,2, or 3 mismatches with one of the antisense sequences set forth in any one of tables 2-7.

In certain embodiments, a dsRNA agent of any of the preceding embodiments is provided, wherein the sense strand comprises a nucleotide sequence comprising at least 21 consecutive nucleotides having 0, 1,2, or 3 mismatches with a sense sequence set forth in any of tables 2-7 corresponding to the antisense sequence.

In certain embodiments, the dsRNA agent of any of the preceding embodiments is provided, wherein the sense strand is at least 23 nucleotides in length, e.g., 23-30 nucleotides in length.

In certain embodiments, the dsRNA agent of any one of the preceding embodiments is provided, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties. In a further embodiment, the lipophilic moiety is conjugated to one or more positions in the double stranded region of the dsRNA agent. In a further embodiment, the lipophilic moiety is conjugated through a linker or carrier. In a further embodiment, the lipophilic moiety has a lipophilicity of greater than 0 as measured by logKow.

In certain embodiments, the dsRNA agent of any one of the preceding embodiments is provided, wherein the hydrophobicity of the double stranded RNAi agent measured by unbound fraction in a plasma protein binding assay of the double stranded RNAi agent is greater than 0.2. In a further embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin.

In certain embodiments, the dsRNA agent of any of the preceding embodiments comprises at least one modified nucleotide. In a further embodiment, no more than five of the sense strand nucleotides and no more than five of the antisense strand nucleotides are unmodified nucleotides. In an alternative further embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise modifications. In a further embodiment of any of these embodiments, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 3' -terminal deoxythymine (dT) nucleotides, 2' -O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxymodified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic nucleotides, 2' -amino modified nucleotides, 2' -O-allyl modified nucleotides, 2' -C-alkyl modified nucleotides, 2' -C-methoxyethyl modified nucleotides, 2' -O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising a non-natural base, tetrahydrofuran modified nucleotides, 1, 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5' -phosphate esters, nucleotides comprising 5' -phosphate ester mimetics, diol modified nucleotides and 2-O- (N-methylacetamide) modified nucleotides, and combinations thereof. 33. The dsRNA agent of any one of embodiments 29-31, wherein no more than five of the sense strand nucleotides and no more than five of the antisense strand nucleotides comprise a modification other than a2' -O-methyl modified nucleotide, a2' -fluoro modified nucleotide, a2' -deoxy modified nucleotide, an Unlocked Nucleic Acid (UNA) or a Glycol Nucleic Acid (GNA).

In certain embodiments, the dsRNA agent of any of the preceding embodiments comprises a non-nucleotide spacer between two consecutive nucleotides of the sense strand or between two consecutive nucleotides of the antisense strand (wherein optionally the non-nucleotide spacer comprises a C3-C6 alkyl group).

In certain embodiments, the dsRNA agent of any one of the preceding embodiments is provided, wherein each strand is no more than 30 nucleotides in length.

In certain embodiments, the dsRNA agent of any one of the preceding embodiments is provided, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

In certain embodiments, the dsRNA agent of any one of the preceding embodiments is provided, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

In certain embodiments, the dsRNA agent of any one of the preceding embodiments is provided, wherein the double-stranded region is 15-30 nucleotide pairs in length. In a further embodiment, the double stranded region is 17-23 nucleotide pairs in length. In yet a further embodiment, the double stranded region is 17-25 nucleotide pairs in length. In yet a further embodiment, the double stranded region is 23-27 nucleotide pairs in length. In yet a further embodiment, the double stranded region is 19-21 nucleotide pairs in length. In yet a further embodiment, the double stranded region is 21-23 nucleotide pairs in length.

In certain embodiments, the dsRNA agent of any one of the preceding embodiments is provided, wherein each strand has 19-30 nucleotides.

In certain embodiments, the dsRNA agent of any one of the preceding embodiments is provided, wherein each strand has 19-23 nucleotides.

In certain embodiments, the dsRNA agent of any one of the preceding embodiments is provided, wherein each strand has 21-23 nucleotides.

In certain embodiments, there is provided a dsRNA agent of any one of the preceding embodiments, wherein the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In a first particular embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand. In a further embodiment, the strand is an antisense strand. In an alternative further embodiment, the strand is the sense strand. 51. In a second particular embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand. In a further embodiment, the strand is an antisense strand. In an alternative further embodiment, the strand is the sense strand. In a third particular embodiment, each of the 5 'and 3' ends of one strand comprises phosphorothioate or methylphosphonate internucleotide linkages. In a further embodiment, the strand is an antisense strand. In certain embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

In certain embodiments, the dsRNA agent of any one of the preceding embodiments is provided, wherein the base pair at 1-position at the 5' -end of the duplex antisense strand is an AU base pair.

In certain embodiments, a dsRNA agent of any of the preceding embodiments is provided, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, wherein the one or more lipophilic moieties are conjugated to one or more internal positions on the at least one strand. In a further embodiment, one or more lipophilic moieties are conjugated to one or more internal positions on at least one chain via a linker or carrier. In a further embodiment, the internal locations include all but the terminal two locations of each end of at least one strand. In an alternative further embodiment, the internal positions include all but the terminal three positions of each end of at least one strand. In a particular embodiment of these further embodiments, the internal position does not include a cleavage site region of the sense strand. In a further embodiment, the internal positions include all positions except positions 9-12 from the 5' end of the sense strand. In an alternative further embodiment, the internal positions include all positions except positions 11-13 from the 3' end of the sense strand. In a second particular embodiment of these further embodiments, the internal position does not include a cleavage site region of the antisense strand. In a further embodiment, the internal positions include all positions except positions 12-14 from the 5' end of the antisense strand. In a third particular embodiment of these further embodiments, the internal positions include all positions except positions 11-13 from the 3 'end of the sense strand and positions 12-14 from the 5' end of the antisense strand.

In certain embodiments, there is provided a dsRNA agent of any one of the preceding embodiments, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, wherein the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of: positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counted from the 5' end of each strand. In a further embodiment, one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of: positions 5, 6, 7, 15 and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counted from the 5' end of each strand.

In certain embodiments, the dsRNA agent of the preceding embodiments is provided wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, wherein the one or more lipophilic moieties are conjugated to one or more positions in the double-stranded region of the dsRNA agent, wherein the position in the double-stranded region does not comprise the cleavage site region of the sense strand.

In certain embodiments, a dsRNA agent of any one of the preceding embodiments is provided, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, wherein the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand, or position 16 of the antisense strand. In a further embodiment, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1 or position 7 of the sense strand. In an alternative further embodiment, the lipophilic moiety is conjugated to position 21, position 20 or position 15 of the sense strand. In yet a further embodiment, the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand. In yet a further embodiment, the lipophilic moiety is conjugated to position 16 of the antisense strand. In yet a further embodiment, wherein the lipophilic moiety is conjugated to position 6 of the sense strand, counting from the 5' end.

In certain embodiments, a dsRNA agent of any of the preceding embodiments is provided, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, wherein the lipophilic moiety is an aliphatic, alicyclic, or polycycloaliphatic compound. In a further embodiment, the lipophilic moiety is selected from the following: lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxyhexanethiol, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) bile acid, dimethoxytrityl or phenoxazine. In a further embodiment, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and optionally a functional group selected from the group consisting of: hydroxyl, amine, carboxylic acid, sulfonate, phosphate, sulfhydryl, azide, and alkyne. In a further embodiment, the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain. In a further embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

In certain embodiments, a dsRNA agent of any of the preceding embodiments is provided, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, wherein the lipophilic moieties are conjugated by a carrier that replaces one or more nucleotides within the internal position or double-stranded region. In a further embodiment, the carrier is a cyclic group selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, tetrahydrothiazolyl, isothiazolyl, quinoxalinyl, pyridazinyl, tetrahydrofuran and decalin; or an acyclic moiety based on a serine backbone or a diethanolamine backbone.

In certain embodiments, there is provided a dsRNA agent of any one of the preceding embodiments, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, wherein the lipophilic moiety is conjugated to the double stranded iRNA agent by a linker comprising: ethers, thioethers, ureas, carbonates, amines, amides, maleimide-thioethers, disulfides, phosphodiesters, sulfonamide linkages, click reaction products, or carbamates.

In certain embodiments, there is provided a double-stranded dsRNA agent of any one of the preceding embodiments, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, wherein the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

In certain embodiments, there is provided a double-stranded dsRNA agent of any one of the preceding embodiments, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, wherein the lipophilic moiety or targeting ligand is conjugated through a bio-cleavable linker selected from the group consisting of: DNA, RNA, disulfides, amides, functionalized mono-or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

In certain embodiments, there is provided a dsRNA agent of any one of the preceding embodiments, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, wherein the 3' end of the sense strand is protected by a cap, the cap being a cyclic group having an amine, the cyclic group selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, tetrahydrothiazolyl, isothiazolyl, quinoxalinyl, pyridazinyl, tetrahydrofuran and decalin.

In certain embodiments, a dsRNA agent of any of the preceding embodiments is provided, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, which further comprises a targeting ligand, e.g., a ligand that targets ocular tissue. In a further embodiment, the ligand is conjugated to the sense strand. In a further embodiment, the ligand is conjugated to the 3 'end or the 5' end of the sense strand. In a further embodiment, the ligand is conjugated to the 3' end of the sense strand. In a further embodiment, the ocular tissue is an optic nerve, trabecular meshwork, proximal tubular tissue, ganglion (e.g., including retinal ganglion), suprascleral vein, or schlemm's canal (e.g., including endothelial cells). In a further embodiment, the targeting ligand comprises N-acetylgalactosamine (GalNAc). In yet a further embodiment, the targeting ligand is one or more GalNAc conjugates or one or more GalNAc derivatives. In a further embodiment, the one or more GalNAc conjugates or the one or more GalNAc conjugates are attached by a monovalent linker or a divalent, trivalent or tetravalent branching linker.

In a further embodiment, wherein the targeting ligand is one or more GalNAc conjugates or one or more GalNAc derivatives, the ligand is

In a further embodiment of the foregoing embodiments, the dsRNA agent is conjugated to a ligand, as shown in the following schematic diagram

Wherein X is O or S.

In a further embodiment of the foregoing embodiment, X is O.

In certain embodiments, there is provided a dsRNA agent of any one of the preceding embodiments, further comprising a terminal, chiral modification at a first internucleotide linkage present at the 3' end of the antisense strand, having a linking phosphorus atom of Sp configuration;

A terminal, chiral modification at the first internucleotide linkage present at the 5' end of the antisense strand, having a linking phosphorus atom of Rp configuration; and

A terminal, chiral modification at the first internucleotide linkage at the 5' end of the sense strand, having a linking phosphorus atom in Rp configuration or Sp configuration.

In certain embodiments, there is provided a dsRNA agent of any of the preceding embodiments, further comprising:

A terminal, chiral modification at the first and second internucleotide linkages present at the 3' end of the antisense strand, having a linking phosphorus atom of Sp configuration;

A terminal, chiral modification at the first internucleotide linkage present at the 5' end of the antisense strand, having a linking phosphorus atom of Rp configuration; and

A terminal, chiral modification at the first internucleotide linkage at the 5' end of the sense strand, having a linking phosphorus atom of Rp or Sp configuration.

Or in certain embodiments, there is provided a dsRNA agent of any of the preceding embodiments, further comprising:

Terminal, chiral modifications at the first, second and third internucleotide linkages present at the 3' end of the antisense strand, having the connecting phosphorus atom in Sp configuration;

A terminal, chiral modification at the first internucleotide linkage present at the 5' end of the antisense strand, having a linking phosphorus atom of Rp configuration; and

A terminal, chiral modification at the first internucleotide linkage at the 5' end of the sense strand, having a linking phosphorus atom of Rp or Sp configuration.

Additionally, or in certain embodiments, there is provided a dsRNA agent of any of the preceding embodiments, further comprising:

A terminal, chiral modification at the first and second internucleotide linkages present at the 3' end of the antisense strand, having a linking phosphorus atom of Sp configuration;

a terminal, chiral modification at the third internucleotide linkage present at the 3' end of the antisense strand, having a linking phosphorus atom of Rp configuration;

a terminal, chiral modification at the first internucleotide linkage occurring at the 5' -end of the antisense strand, having a linking phosphorus atom of Rp configuration, and

A terminal, chiral modification at the first internucleotide linkage at the 5' end of the sense strand, having a linking phosphorus atom of Rp or Sp configuration.

Additionally, or in certain embodiments, there is provided a dsRNA agent of any of the preceding embodiments, further comprising:

A terminal, chiral modification at the first and second internucleotide linkages present at the 3' end of the antisense strand, having a linking phosphorus atom of Sp configuration;

A terminal, chiral modification at the first and second internucleotide linkages present at the 5' end of the antisense strand, having a linking phosphorus atom of Rp configuration; and

A terminal, chiral modification at the first internucleotide linkage at the 5' end of the sense strand, having a linking phosphorus atom of Rp or Sp configuration.

In certain embodiments, a dsRNA agent of any of the preceding embodiments is provided that further comprises a phosphate or phosphate mimetic at the 5' end of the antisense strand. In a further embodiment, the phosphate mimic is a 5' -Vinyl Phosphonate (VP). In one embodiment, the phosphate mimic is 5' -Cyclopropyl Phosphonate (CP). In some embodiments, the 5 '-end of the antisense strand of the double-stranded iRNA agent does not comprise a 5' -Vinylphosphonate (VP).

In one embodiment, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 2 '-O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2 '-deoxymodified nucleotides, diol modified nucleotides (GNAs), e.g., ggn, cgn, tgn or Agn, nucleotides with 2' phosphates, e.g., G2p, C2p, A2p or U2p, and vinylphosphonate nucleotides; and combinations thereof. In other embodiments, each of the duplex of tables 3, 5, and 7 can be specifically modified to provide another double stranded iRNA agent of the present disclosure. In one example, the 3' end of each sense duplex can be modified by removing the 3' end L96 ligand and exchanging two phosphodiester internucleotide linkages between the three 3' end nucleotides with phosphorothioate internucleotide linkages. That is, three 3' terminal nucleotides (N) of the sense sequence of the formula:

5’-N₁-…-N_n-2N_n-1N_nL96 3’

Can be used as

5'-N ₁-…-N_n-2sN_n-1sN_n' substitution.

That is, for example, AD-1561710, sense sequence:

csusuggaAfgGfAfAfagcuauagguL96(SEQ ID NO:393)

Can be used as

CsusuggaAfgGfAfAfagcuauagsgsu (SEQ ID NO: 1473),

While the antisense sequence remains unchanged to provide another double stranded iRNA agent of the present disclosure.

(2) Cells

In a certain aspect, the present disclosure provides a cell comprising a dsRNA agent of any one of the preceding embodiments.

In certain embodiments, the disclosure provides human eye cells, e.g., (optic nerve cells, trabecular meshwork cells, schlemm's canal cells (e.g., endothelial cells), proximal tubular tissue cells, ciliary muscle cells, retinal cells, astrocytes, pericytes, muller cells, ganglion cells (e.g., including retinal ganglion cells), endothelial cells, photoreceptor cells, retinal blood vessels (e.g., including endothelial cells and vascular smooth muscle cells), suprascleral vein or choroidal tissue, e.g., choroidal blood vessels, comprising reduced levels of ANGPTL7 mRNA or reduced levels of ANGPTL7 protein as compared to other similar untreated cells, wherein optionally the levels are reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In a further embodiment, the human cells of the foregoing embodiments are produced by a method comprising contacting the human cells with a dsRNA agent of any of the foregoing embodiments.

(3) Pharmaceutical composition

In a certain aspect, the present disclosure provides a pharmaceutical composition for inhibiting ANGPTL7 expression comprising a dsRNA agent of any one of the previous embodiments.

In a certain aspect, the present disclosure provides a pharmaceutical composition comprising a dsRNA agent of any one of the preceding embodiments and a lipid formulation.

(4) Methods of inhibiting ANGPTL7 expression in cells

In a certain aspect, the present disclosure provides a method of inhibiting ANGPTL7 expression in a cell, the method comprising:

(a) Contacting a cell with the dsRNA agent of any of the preceding embodiments or the pharmaceutical composition of any of the preceding embodiments; and

(B) Maintaining the cell produced in step (a) for a time sufficient to obtain degradation of mRNA transcripts of ANGPTL7, thereby inhibiting ANGPTL7 expression in the cell.

In a certain aspect, the present disclosure provides a method of inhibiting ANGPTL7 expression in a cell, the method comprising:

(a) Contacting a cell with the dsRNA agent of any of the preceding embodiments or the pharmaceutical composition of any of the preceding embodiments; and

(B) Maintaining the cell produced in step (a) for a time sufficient to reduce the level of ANGPTL7mRNA, ANGPTL7 protein, or both ANGPTL7mRNA and protein, thereby inhibiting expression of ANGPTL7 in the cell.

In certain embodiments, there is provided a method of one of the preceding embodiments, wherein the cell is in a subject. In a further embodiment, the subject is a human.

In certain embodiments, there is provided a method of any one of the preceding embodiments, wherein the level of ANGPTL7 mRNA is inhibited by at least 50%.

In certain embodiments, there is provided a method of any one of the preceding embodiments, wherein the level of ANGPTL7 protein is inhibited by at least 50%.

In certain embodiments, the method of any one of the preceding embodiments is provided, wherein the cell is in a subject, wherein inhibiting expression of ANGPTL7 reduces ANGPTL7 protein levels by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in a biological sample (e.g., an optic nerve sample) from the subject. In a further embodiment, the subject has been diagnosed with an ANGPTL7 related disorder, e.g., glaucoma. In a particular embodiment, the ANGPTL 7-related disorder is glaucoma or a glaucoma-related condition.

In certain embodiments, a method of inhibiting ANGPTL7 expression in an eye cell or tissue is provided, the method comprising:

(a) Contacting a cell or tissue with a dsRNA agent that binds ANGPTL; and

(B) Maintaining the cell or tissue produced in step (a) for a time sufficient to reduce the level of ANGPTL7 mRNA, ANGPTL7 protein, or both ANGPTL7 mRNA and protein, thereby inhibiting expression of ANGPTL7 in the cell or tissue. In a further embodiment, the ocular cells or tissues comprise optic nerve cells, trabecular meshwork cells, schlemm's canal cells (e.g., including endothelial cells), proximal tubular tissue cells, ciliary muscle cells, retinal cells, astrocytes, pericytes, muller cells, ganglion cells (e.g., including retinal ganglion cells), endothelial cells, photoreceptor cells, retinal blood vessels (e.g., including endothelial cells and vascular smooth muscle cells), suprascleral veins, or choroidal tissues, e.g., choroidal blood vessels.

(5) Methods of treating a subject

In a certain aspect, the present disclosure provides a method of treating a subject diagnosed with an ANGPTL 7-related disorder, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any of the foregoing embodiments or the pharmaceutical composition of any of the foregoing embodiments, thereby treating the disorder. In a particular embodiment, the ANGPTL 7-related disorder is glaucoma or a glaucoma-related condition.

In certain embodiments, there is provided a method of treating a subject according to the preceding embodiments, wherein treating comprises ameliorating at least one sign or symptom of the disorder. In a further embodiment, the at least one sign or symptom of glaucoma includes an indication of one or more of intraocular pressure, vision loss, optic nerve injury, ocular inflammation, visual acuity, or the presence, level, or activity of ANGPTL7 (e.g., ANGPTL7 gene, ANGPTL7 mRNA, or ANGPTL7 protein).

In certain embodiments, there is provided a method of treating a subject according to the preceding embodiments, wherein treating comprises preventing progression of the disorder.

In certain embodiments, there is provided a method of treating a subject according to any of the preceding embodiments, wherein treating comprises ameliorating at least one sign or symptom of the disorder, or preventing progression of the disorder, wherein the treating comprises one or more of: (a) inhibiting or reducing intraocular pressure; (b) inhibiting or reducing expression or activity of ANGPTL 7; (c) increasing drainage of aqueous humor; (d) inhibiting or reducing optic nerve damage; or (e) inhibit or reduce retinal ganglion cell death. The treatment is a drug that reduces intraocular pressure, laser treatment, surgery, or trabeculectomy. In a further embodiment, the treatment results in an average decrease of at least 30% in ANGPTL7mRNA in optic nerve cells, trabecular meshwork cells, schlemm's canal cells (e.g., including endothelial cells), proximal tubular tissue cells, ciliary muscle cells, retinal Pigment Epithelium (RPE), retinal cells, astrocytes, pericytes, muller cells, ganglion cells (e.g., including retinal ganglion cells), endothelial cells, photoreceptor cells, retinal vessels (e.g., including endothelial cells and vascular smooth muscle cells), suprascleral veins, or choroidal tissue, e.g., choroidal blood vessels, from baseline. In a further embodiment, the treatment results in an average decrease of at least 60% in ANGPTL7mRNA in optic nerve cells, trabecular meshwork cells, schlemm's canal cells (e.g., including endothelial cells), proximal tubular tissue cells, ciliary muscle cells, retinal Pigment Epithelium (RPE), retinal cells, astrocytes, pericytes, muller cells, ganglion cells (e.g., including retinal ganglion cells), endothelial cells, photoreceptor cells, retinal vessels (e.g., including endothelial cells and vascular smooth muscle cells), suprascleral veins, or choroidal tissue, e.g., choroidal blood vessels, from baseline. In a further embodiment, the treatment results in an average decrease of at least 90% in ANGPTL7mRNA in optic nerve cells, trabecular meshwork cells, schlemm's canal cells (e.g., including endothelial cells), proximal tubular tissue cells, ciliary muscle cells, retinal Pigment Epithelium (RPE), retinal cells, astrocytes, pericytes, muller cells, ganglion cells (e.g., including retinal ganglion cells), endothelial cells, photoreceptor cells, retinal vessels (e.g., including endothelial cells and vascular smooth muscle cells), suprascleral veins, or choroidal tissue, e.g., choroidal blood vessels, from baseline.

In certain embodiments, there is provided a method of treating a subject according to any of the preceding embodiments, wherein treating comprises ameliorating at least one sign or symptom of the disorder, or preventing progression of the disorder, wherein after treatment, the subject experiences a knockdown duration of at least 8 weeks following a single dose of dsRNA, as assessed by an ANGPTL7 protein in the optic nerve. In a further embodiment, the treatment results in a knockdown duration of at least 12 weeks following a single dose of dsRNA, as assessed by ANGPTL7 protein in the optic nerve. In a further embodiment, the treatment results in a knockdown duration of at least 16 weeks following a single dose of dsRNA, as assessed by ANGPTL7 protein in the optic nerve.

(6) Delivery and administration methods

In certain embodiments, the present disclosure provides a method of any one of the preceding embodiments for inhibiting expression of ANGPTL7 in a cell of a subject or for treating a subject diagnosed with an ANGPTL7 related disease, wherein the subject is a human.

In a particular embodiment of the foregoing embodiments, the dsRNA agent is administered at a dose of about 0.01mg/kg to about 50 mg/kg.

In a particular embodiment of the foregoing embodiments, the dsRNA agent is administered to the subject intraocularly, intravenously, or topically. In a further embodiment, wherein the intraocular administration comprises intravitreal administration (e.g., intravitreal injection), transscleral administration (e.g., transscleral injection), subconjunctival administration (e.g., subconjunctival injection), retrobulbar administration (e.g., retrobulbar injection), intracameral administration (e.g., intracameral injection), or subretinal administration (e.g., subretinal injection).

In certain embodiments, there is provided a method of any one of the preceding embodiments, further comprising measuring the level of ANGPTL7 (e.g., an ANGPTL7 gene, an ANGPTL7 mRNA, or an ANGPTL7 protein) in the subject. In a further embodiment, measuring the level of ANGPTL7 in a subject comprises measuring the level of an ANGPTL7 gene, an ANGPTL7 protein, or an ANGPTL7 mRNA in a biological sample (e.g., an optic nerve sample) from the subject.

In certain embodiments, there is provided a method of any of the preceding embodiments, further comprising performing a blood test, an imaging test, an intraocular pressure measurement, or an optic nerve biopsy.

In a further embodiment, the ANGPTL7 (e.g., ANGPTL7 gene, ANGPTL7 mRNA, or ANGPTL7 protein) level in the subject is measured prior to treatment with the dsRNA agent or pharmaceutical composition. In a further embodiment, the dsRNA agent or pharmaceutical composition is administered to the subject after determining that the subject's ANGPTL7 level (e.g., ANGPTL7 gene, ANGPTL7 mRNA, or ANGPTL7 protein) is greater than a reference level. In a further embodiment, the ANGPTL7 (e.g., ANGPTL7 gene, ANGPTL7 mRNA, or ANGPTL7 protein) level in the subject is measured after treatment with the dsRNA agent or pharmaceutical composition.

In certain embodiments, there is provided a method of any one of the preceding embodiments, further comprising administering to the subject an additional agent and/or therapy suitable for treating or preventing an ANGPTL 7-related disorder. In a further embodiment, the other agent and/or therapy comprises one or more of the following: prostaglandin analogs, beta blockers, alpha adrenergic agonists, carbonic anhydrase inhibitors, ROCK iRNA agents, inhibitors of Rho GTPase, anti-Rho GTPase agents, or anti-ANGPTL 7 agents.

Examples

Example 1: ANGPTL7 siRNA

The nucleic acid sequences provided herein are represented using standard nomenclature. See abbreviations in table 1.

Table 1: abbreviations for nucleotide monomers used in the representation of nucleic acid sequences.

It will be appreciated that these monomers, when present in the oligonucleotide, are linked to each other by a 5' -3' -phosphodiester linkage, and that when the nucleotide contains a 2' -fluorine modification, the fluorine replaces the hydroxyl group at that position in the parent nucleotide (i.e., it is a 2' -deoxy-2 ' -fluoronucleotide).

¹ The chemical structure of L96 is as follows:

Experimental method

Bioinformatics

Transcripts

A targeting mouse ANGPTL7 "angiopoietin-like 7" (NCBI GeneID: 654812) siRNA was generated. Mouse NM-001039554.3 REFSEQ mRNA has a length of 2062 bases. The pairs of oligonucleotides were generated and ordered using bioinformatics methods, with exemplary pairs of oligonucleotides shown in tables 2,3, 4 and 5. The modified sequences are shown in tables 3 and 5. Unmodified sequences are shown in tables 2 and 4. The oligonucleotides in tables 2,3, 4 and 5 cross-react with rat ANGPTL7 and can cross-react with human and monkey ANGPTL 7.

SiRNA targeting human ANGPTL7 "angiopoietin-like 7" (NCBI GeneID: 10218) was generated. Human NM-021146.4 REFSEQ mRNA has a length of 2224 bases. Pairs of oligonucleotides were generated and ordered using bioinformatics methods, exemplary pairs of oligonucleotides are shown in tables 6 and 7. The modified sequences are shown in table 7. Unmodified sequences are shown in table 6. The oligonucleotides in tables 6 and 7 can cross-react with monkey, mouse and rat ANGPTL 7. It should be understood that throughout this application, a duplex name without a decimal is equivalent to a duplex name with a decimal that references only to duplex lot numbers. For example, AD-1094991 is equivalent to AD-1094991.1.

SiRNA synthesis

The siRNA is synthesized and annealed using conventional methods well known in the art.

Briefly, siRNA sequences were synthesized on a Mermade 192 synthesizer (BioAutomation) on a1 μmol scale using solid support-mediated phosphoramidite chemistry. The solid support is a controlled pore glass (500A) loaded with custom GalNAc ligands or a universal solid support (AM biochem). Auxiliary synthesis reagents 2'-F and 2' -O-methyl RNA and deoxyphosphoramides were obtained from Thermo Fisher (Waltham, mass.) and Hongene (China). The corresponding phosphoramidites were used to introduce 2' F2 ' -O-methyl, GNA (diol nucleic acid), 5' phosphate and other modifications. Synthesis of 3' GalNAc conjugated single strands was performed on GalNAc modified CPG supports. Custom CPG universal solid supports were used for synthesis of antisense single strands. The coupling time for all phosphoramidites (100 mM in acetonitrile) was 5min using 5-ethylthio-1H-tetrazole (ETT) as activator (0.6M in acetonitrile). Phosphorothioate linkages were produced using a 50mM solution of 3- ((dimethylaminomethylene) amino) -3H-1,2, 4-dithiazole-3-thione (DDTT, available from CHEMGENES (Wilmington, mass., USA)) in anhydrous acetonitrile/pyridine (1:1 v/v). The oxidation time was 3 minutes. All sequences were synthesized and eventually the DMT groups were removed ("DMT excision").

After completion of the solid phase synthesis, the oligonucleotides were cleaved from the solid support and deprotected in a sealed 96-well plate using 200 μl of aqueous ethylamine reagent at 60 ℃ for 20 minutes. For sequences containing 2 'ribose residues (2' -OH) protected with t-butyldimethylsilyl (TBDMS) groups, the second deprotection step was performed using TEA.3HF (triethylamine trihydrofluoride) reagent. 200. Mu.L of dimethyl sulfoxide (DMSO) and 300. Mu.L of TEA.3HF reagent were added to the methylamine deprotected solution, and the solution was incubated at 60℃for an additional 20min. At the end of the cleavage and deprotection steps, the synthetic plate was brought to room temperature and was prepared by adding 1mL of acetone: ethanol mixture (9:1) precipitated. The plates were cooled at-80 ℃ for 2hr and the supernatant carefully decanted by means of a multichannel pipette. The oligonucleotide pellet was resuspended in 20mM NaOAc buffer and desalted using a 5mL HiTrap size exclusion chromatography column (GE HEALTHCARE) on an AKTA purifier system equipped with an A905 autosampler and a Frac 950 fraction collector. Desalted samples were collected in 96-well plates. Samples of each sequence were analyzed by LC-MS to confirm their identity, quantified by UV (260 nm), and a set of samples was selected by IEX chromatography to determine purity.

Single strand annealing was performed on a Tecan liquid handling robot. Equimolar mixtures of sense and antisense single strands were combined and annealed in 96-well plates. After binding the complementary single strands, the 96-well plate was sealed and heated in an oven at 100 ℃ for 10 minutes and slowly brought to room temperature over 2-3 hours. The concentration of each duplex was normalized to 10 μm in 1X PBS and submitted to an in vitro screening assay.

Table 2: unmodified sense and antisense strand sequences of ANGPTL7 dsRNA agents

Table 3: modified sense and antisense strand sequences of ANGPTL7 dsRNA agents

Table 4: unmodified sense and antisense strand sequences of ANGPTL7 dsRNA agents

Table 5: modified sense and antisense strand sequences of ANGPTL7 dsRNA agents

Table 6: unmodified sense and antisense strand sequences of ANGPTL7 dsRNA agents

Table 7: modified sense and antisense strand sequences of ANGPTL7 dsRNA agents

Example 2: in vitro screening of mouse ANGPTL7 siRNA in COS-7 cells

Experimental method

Cloning of vector containing ANGPTL7 mRNA sequence and dual luciferase reporter

The entire sequence of mouse NM-001039554.3 REFSEQ mRNA was cloned into the double luciferase reporter construct (psiCHECK ^TM -2, promega) at the XhoI/NotI site. In cells transfected with this construct, a decrease in renilla luciferase activity would indicate an effect of RNAi. Firefly luciferase was used for normalization of renilla luciferase expression.

Cell culture and transfection

At the position ofANGPTL 7/reporter vector stock was diluted to 5 ng/. Mu.L in (Thermo Fisher). mu.L of carrier solution (5 ng/. Mu.L) was added to each well of 384-well plates. Then, 5 μl per well of each siRNA duplex (10 x final concentration) was added to the carrier solution. Dose experiments were performed at final siRNA duplex concentrations of 50nM, 10nM, 1nM and 0.1 nM.

Mu.L of Opti-MEM was added to each well at 0.1. Mu.L2000 (Thermo Fisher) was added to the vector/siRNA mixture. The mixture was incubated at room temperature for 15 minutes. Then 35. Mu.L of DMEM without antibiotics per well was added to the carrier/siRNA/liposome mixture, containing about 3X 10 ³ trypsin-digested COS-7 cells per well (ATCC, manassas, va.). Cells were incubated for 48 hours prior to dual luciferase reading.

Dual luciferase read

As shown below, use is made ofThe luciferase assay system (catalog # E2980, promega) performs a double luciferase read. The medium was removed from each well. By putting 1 bottleLuciferase buffer was added to 1 vial of lyophilizedPreparation in luciferase substrateLuciferase reagent. Preparation of 20. Mu.L per wellLuciferase reagent was added to a 20. Mu.L mixture of DMEM and added to each well. Plates were incubated on a shaker for 30 minutes. Firefly luciferase activity was measured with a photometer.

After firefly luciferase was read, 20. Mu.L per well was preparedStop&Buffer solution is added with 0.2 mu LStop&A mixture of substrates was added to each well. Kong Fuyo was carried out for about 10 minutes. Renilla luciferase activity was measured with a luminometer. Each siRNA duplex was tested at least twice. The normalized renilla luciferase activity of each well was compared to the activity of cells transfected with non-targeted control siRNA.

Results

Dose screening results for exemplary mouse ANGPTL7 siRNA in COS-7 cells are shown in table 8. Experiments were performed at final duplex concentrations of 50nM, 10nM, 1nM and 0.1nM, data expressed as percentage of remaining information relative to non-targeted controls.

Table 8: mouse ANGPTL7 siRNA dose selection in COS-7 cells

It is expressly contemplated that nucleotides 1562-1584, 546-568, 709-731, 862-884 and/or 232-256 of NM-001039554.3 comprise hot spot regions, which are targeted by AD-1094991, AD-1093984, AD-1094129, AD-1094262, AD-1093670 and AD-1093672, respectively.

Example 3: in vitro screening of human ANGPTL7 siRNA in Hepa1-6 cells

Experimental method

Cloning of vector comprising ANGPTL7 mRNA sequence and a bifluorescence enzyme reporter

Cloning the entire sequence of human NM-021146.4 REFSEQ mRNA into the double luciferase reporter constructV163 plasmid). In cells transfected with this construct, a decrease in renilla luciferase activity would indicate an effect of RNAi. Firefly luciferase was used for normalization of renilla luciferase expression.

Cell culture and transfection

100Ng of ANGPTL7/reporter plasmid (1000 ng/. Mu.L) was added to each well of a 96-well plate. The siRNA duplex mixture (1000 nM) was then added to the plasmid solution to provide a final siRNA duplex concentration of 10 nM.

Will be 0.5 mu L2000 (Thermo Fisher) was added to the plasmid/siRNA mixture. Culture medium containing about 2X 10 ⁴ trypsin digested Hepa1-6 cells (ATCC, manassas, va.) per well was then added to the vector/siRNA/liposome mixture. Cells were incubated for 24 hours prior to dual luciferase reading. Each siRNA duplex was tested in quadruplicates at a final concentration of 10 nM.

Dual luciferase read

As described in example 2, use is made ofThe luciferase assay system (catalog # E2980, promega) performs a double luciferase read. The activities of firefly and Renilla luciferases were measured with a photometer. For each well, renilla luciferase activity normalized to firefly luciferase activity was compared to the activity of cells transfected with non-targeted control siRNA.

Results

The results of single dose screening of exemplary human ANGPTL7 siRNA in Hepa1-6 cells are shown in table 9. The experiments were performed at a final duplex concentration of 10nM and the data are expressed as a percentage of the remaining information relative to the non-targeted control.

Table 9: single dose screening of human ANGPTL7 in Hepa1-6 cells

It is clearly contemplated that nucleotides 1993-2146、1910-1932、1726-1823、1628-1685、1591-1613、1551-1573、1420-1442、1380-1402、1243-1265、1195-1217、1096-1118、940-962 and/or 299-321 of NM-021146.4 comprise a hot spot region, which is targeted by AD-1565389、AD-1565368、AD-1565357、AD-1565345、AD-1565324、AD-1565303、AD-1565288、AD-1565212、AD-1565141、AD-1565126、AD-1565113、AD-1565091、AD-1565034、AD-1565015、AD-1565004、AD-1564969、AD-1094381、AD-1564428、AD-1564936、AD-1564823、AD-1564802、AD-1564666、AD-1564618 and AD-1563396, respectively.

Example 4: in vitro screening of ANGPTL7 siRNA in RPE-J cells

Experimental method

Cell culture and transfection

RPE-J cells (ATCC) were grown to near confluence in Dulbecco modified Eagle minimal essential medium (Gibco) supplemented with 10% FBS (ATCC) at 37 ℃, in an atmosphere of 5% co ₂, and then released from the plates by trypsin digestion. RPE-J cell transfection was performed by adding 14.8 μl of LOpti-MEM per well to each siRNA duplex (5 μl) in each well of a 96 well plate plus 0.2 μl L Lipofectamine RNAiMax (Invitrogen, carlsbad ca. Catalog # 13778-150). The mixture was then incubated at room temperature for 15 minutes. Then 80. Mu.L of antibiotic-free complete growth medium containing about 2X 10 ⁴ RPE-J cells or PMH was added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Dose experiments were performed at 50nM, 10nM, 1nM and 0.1nM final duplex concentrations.

Total RNA isolation Using DYNABEADS mRNA isolation kit

RNA was isolated using an automated protocol on the BioTek-EL406 platform using DYNABEADs (Invitrogen, catalog # 61012). Briefly, 70. Mu.L of lysis/binding buffer and 10. Mu.L of lysis buffer containing 3. Mu.L of magnetic beads were added to the plates with cells. Plates were incubated at room temperature for 10 minutes on an electromagnetic shaker, then the magnetic beads were captured and the supernatant removed. The bead-bound RNA was then washed 2 times with 150. Mu.L of wash buffer A and once with wash buffer B. The beads were then washed with 150 μl of elution buffer, recaptured and the supernatant removed.

CDNA Synthesis Using ABI Hi-Fi cDNA reverse transcription kit (AppliedBiosystems, fosterCity, CA, catalog # 4368813)

Mu.L of a premix containing 1. Mu.L of 10 Xbuffer, 0.4. Mu.L of 25 XdNTPs, 1. Mu.L of 10 Xrandom primer, 0.5. Mu.L of reverse transcriptase, 0.5. Mu.L of RNase inhibitor and 6.6. Mu. L H ₂ O was added to the above isolated RNA. The plates were sealed, mixed and incubated at room temperature for 10 minutes on a electromagnetic shaker, then at 37 ℃ for 2h.

Real-time PCR

Mu.L of cDNA and 5 mu L LIGHTCYCLER probe premix (Roche catalog # 04887301001) were added to 0.5. Mu.L of rat GAPDH TAQMAN probe (Rn 01775763_g1, thermo Fisher) or 0.5. Mu.L of mouse ANGPTL7 probe (Mm 01256626_m1, thermo Fisher) in each well of 384 well plates (Roche catalog # 04887301001). Real-time PCR was performed in a LightCycler480 real-time PCR system (Roche). Each duplex was tested at least twice and data normalized to cells transfected with non-targeted control siRNA. To calculate the relative fold change, the real-time data was analyzed using the ΔΔct method and normalized to the assay performed with cells transfected with non-targeted control siRNA.

Example 5: ANGPTL7 knockout inhibiting DEX-Ac-induced high intraocular pressure in mice

The weekly injection of dexamethasone-21-acetate (DEX-Ac) in the periocular Conjunctival Fornix (CF) in both eyes significantly increased intraocular pressure (IOP) in ANGPTL7WT mice compared to vehicle-dosed control WT mice. As shown in fig. 1, IOP measurements of DEX-Ac treated WT mice ("WT-DEX-Ac", n=18) produced an intraocular pressure elevation from week 1 to week 6 relative to vehicle treated WT mice ("WT-vehicle", n=6); * P <0.0001, p <0.01. In contrast, injection of DEX-Ac per periocular CF in both eyes of ANGPTL7 Knockout (KO) mice did not increase intraocular pressure compared to ANGPTL7 KO mice administered with vehicle. As shown in fig. 1, the IOP measurement of the DEX-Ac treated ANGPTL7 KO mice ("KO-DEX-Ac", n=20) did not produce IOP elevation from week 1 to week 6 relative to the vehicle treated ANGPTL7 KO mice ("KO vehicle", n=12).

Example 6: in vivo evaluation of ANGPTL7 siRNA in wild-type mice

The dsRNA agents designed and assayed in examples 1-4 were evaluated for their ability to reduce ANGPTL7 RNA levels and/or reduce intraocular pressure (IOP) in wild type mice.

Experimental method

Six different siRNAs targeting ANGPTL7 were tested in C57BL/6J wild-type mice (siRNA #1-6; see Table 10) and the change in IOP over time was monitored. C57BL/6J mice were each intravitreally injected with 15. Mu.g siRNA or PBS control. Animals in the natural group did not receive injections. Six weeks later, animals were sacrificed, eyes were collected, and limbal rings were carefully microdissected. qPCR was performed on limbal rings dissected from mice eyes expressing ANGPTL7 enriched in vascular network (TM). Data are expressed as percent information remaining relative to baseline values and as mean ± Standard Error of Mean (SEM).

Table 10: sense and antisense strand sequences of ANGPTL7 dsRNA agents for use in vivo studies

Results

The in vivo evaluation results are shown in fig. 2 and 3. As shown in fig. 2, mice treated with two of the six sirnas (siRNA #3 and #5, n=6-8/group) had significantly lower IOP at 2 weeks post injection compared to PBS-treated group (n=6) or natural group (no injection, n=5); * p <0.05. During the study period (weeks 0-6), natural and PBS-treated animals maintained their IOP at baseline levels. In contrast, in mice treated with siRNA #3 and #5, IOP was lowered by 2-4mmHg from week 2, compared to PBS-treated or naive mice, and remained lowered at the end of the study (i.e., 6 weeks); * P <0.01, # p <0.001, # p <0.0001, # p <0.01, # p <0.001, # p <0.0001.siRNA #3 and #5 represent AD-1094129 and AD-1094991, respectively.

As shown in figure 3, in qPCR of limbal ring tissue harvested at the end of the study (i.e., 6 weeks after siRNA administration), siRNA #3 and #5 observed the highest knockdown level (> 50%) of ANGPTL7 mRNA compared to PBS-treated or native mice. This mRNA knockdown was consistent with the IOP reduction observed in mice injected with one of the two sirnas. The results indicate that inhibiting ANGPTL7 expression also reduces IOP and demonstrate the ability of exemplary dsRNA agents to reduce ANGPTL7 expression and lower IOP in vivo.

Example 7: in vivo knock-down ANGPTL7 by siRNA inhibits steroid-induced elevation of intraocular pressure in wild-type mice

The ability of dsRNA agents targeting ANGPTL7 (siRNA #1- # 6) to reduce steroid induced IOP in wild-type mice was further assessed. siRNA #3 and #5 represent AD-1094129 and AD-1094991, respectively (see Table 10)

Mice were divided into four groups as shown in fig. 4: (a) a carrier (n=4), (b) a carrier+pbs (n=6), (c) DEX-Ac (n=12), (d) DEX-ac+sirna#3 (n=14) and (e) DEX-ac+sirna#5 (n=14). Starting on day 1, each group of mice was injected weekly with a DEX-Ac suspension or vehicle into both eyes with periocular CF and the intraocular pressure (IOP) was monitored over time.

On day 22, siRNA Intravitreal (IVT) targeting ANGPTL7 (# 3 and # 5) or PBS was injected into (d) (dexac+sirna # 3), (e) (dexac+sirna # 5) and (b) (vehicle+pbs), and IOP measurements were continuously recorded.

Periocular CF, where DEX-Ac suspension was injected weekly into both eyes, caused DEX-induced high intraocular pressure (OHT), with sustained and significant increases in intraocular pressure in WT mice. The IOP of DEX-Ac treated mice was elevated rapidly and significantly higher compared to vehicle treated mice starting 6 days after injection; * P <0.001, # p <0.0001, the # # p is <0.001, ++++ p <0.0001, ++p <0.01. (c) DEX-Ac treated mice in the group developed DEX-induced OHT with persistent and significant elevation of IOP throughout the study. After the administration of siRNA on day 22, IOP was significantly reduced in groups (d) and (e) and returned to baseline IOP within one week after the administration of siRNA, as compared to group (c) treated with DEX-Ac and not treated with siRNA. In groups (d) and (e), the IOP levels of the siRNA-treated mice remained at baseline levels, and for the remainder of the study (i.e., by day 70), even though the mice of groups (d) and (e) continued to receive weekly DEX-Ac treatment, their IOP levels were significantly lower than those of DEX-Ac treated and siRNA treated groups (c); # # p <0.0001, # p <0.001, ++++ p < the total weight of the composition was 0.0001, 0.0001 the process comprises.

ANGPTL7 sequence

SEQ ID NO:1

NM-001039554.3 mouse angiopoietin-like 7 (Angptl 7), mRNA

TCAGACTAAGGAAGGAAAGAGTTCCATTTCAGAATCTCTAGCTTTAAGAAAGGCTAAGCAAGCACACAGAGGAAGGAGATCACGGGGAAGGAAGAAAACTGCCAGTGTGGGTCAGAGAAAGAAGCTTCCTACTTCTCCAGGGACAGACTCTAAGGGGAACAGGCCTGCACACCATGCTGAGGGAGACCTGGCTATGTGTTATCCTTGTAGCCTTTGTCAGCCACCCAGTGTGGCTGCAGAAGCCTCATAAACGCAAGACACAGCTCAAAGCAGCGGGCTGCTGTGAGGAGATGAGGGAGCTCAAAGCCCAGGTGGCCAACCTCAGCAGTCTGCTGGGAGAGCTGAGCAGGAAGCAGGAGAGCGACTGGGTCAGTGTGGTCATGCAGGTGATGGAGCTGGAGAGCAGCAGCAAGCACATGGAGTCTCGGCTCAGCACTGCCGAGAGCAAGTACTCTGAGATGAACAACCAGATTGACATCATGCAGCTGCAGGCTGCGCAGACCGTCACGCAGACCTCGGCAGATGCCATCTATGACTGTTCTTCCCTGTACCAGAAGAACTACCGAATCTCTGGAGTGTACAAGCTTCCTCCTGACGAGTTCCTGGGGAGCCCTGAGCTAGAGGTGTTCTGTGACATGGAAACTTCAGGAGGAGGCTGGACCATCATCCAGAGACGTAAGAGTGGCCTTGTCTCCTTCTACCAAGACTGGAGACAGTATAAGCAAGGGTTTGGCAGCATCCGAGGTGACTTCTGGCTGGGGAATGAACATATCCACCGGCTCACCAGGCAGCCAAGCCGGCTTCGTGTGGAGCTGGAGGACTGGGAGGGCAATGCACGCTACGCAGAGTATAGCTACTTTGCGTTGGGCAATGAACTGAACAGCTACCGCCTCTTCCTGGGGAACTACAGTGGCAACGTGGGGAAGGACGCCCTCCTCTACCATAACAACACCGTCTTCAGCACCAAGGACAAGGACAACGACAACTGCTTGGACAAGTGCGCACAGCTCCGAAAAGGTGGCTACTGGTACAACTGCTGCACAGACTCCAACCTCAATGGGGTGTACTACCGCCTTGGCGAGCACCGAAAGCACATGGATGGCATCAGCTGGTATGGCTGGCATGGAGCCAACTATTCCCTCAAACGTGTGGAAATGAAGATCCGCCCAGAAGCCTTCAAGCCCTGAGAGAAGGCAGACACTGAGGAGGGAGAACAGCATGGGAGGAGGAGGTGGACACAGGGTAGGAGGGAACAGTTTATCATCCAGGAGCACAATATAACTTTACCTGTGTGAGCACACACACACAATAGAACCACACGTGCCAACAGTGCACACTAGCAGATGGAGCCAGGCGGACCCAGTGGGGCCTGCCACGGTGCCTCACGGGAGAACTCATGGACAACGGTAACCCTGAGGTCACTTAACCCATTTTCCCTAACTGAGGCTTAGATGACACGAGGGAAAAGAACAAATAAAAACCTGGTGTGATTCTCAGCGGAGAGGCTGTGAGAAATGAAAGAAAGCAGGTGGTGGAGAAGGGGCTTCCAAGTCTTACCCCGCGACACTTCCTTGTGTCTATAGTATTTGTTTTGTTTTTCTTTTTGAGACAGGGTCTCTCTACACAGCTCTTTCTGTCCTGGAACTCACTATGTAGACCAGGCTGACCTTGAACTCACAGAGATCTACCTGCTTCTGCCTCCCAAGTACAGGGATTAAAGGCATGTACCACCATACCCAGTATATATAATTTTTAAGACACAAAAAACATGGAGATAGAGAGCAGCTGCCCAGGTGTCTCCGGGGGGGCCTTGTTGTCAGAGTCCTGGGGGAGAGAGGAGCACTGGACAACATGCTGCGGGTCTGACGTGGCGAGAACACCAGCCGGAGGTGAGCACAGACTCTGGGTGATCACAATACTGCCTTCAAACATCCTCAGTCAAAAACCAAAAGATCCCCTTTAATAAAAATGCTTGGAAAATGAAGGTAGATGGCGCTGTGGTTTAAAACTTGTGATGTATATAGAAGCATCTTCCTTGTAAAAATAAAATATTGTAATTCCT

SEQ ID NO:2

Reverse complement of SEQ ID NO. 1

AGGAATTACAATATTTTATTTTTACAAGGAAGATGCTTCTATATACATCACAAGTTTTAAACCACAGCGCCATCTACCTTCATTTTCCAAGCATTTTTATTAAAGGGGATCTTTTGGTTTTTGACTGAGGATGTTTGAAGGCAGTATTGTGATCACCCAGAGTCTGTGCTCACCTCCGGCTGGTGTTCTCGCCACGTCAGACCCGCAGCATGTTGTCCAGTGCTCCTCTCTCCCCCAGGACTCTGACAACAAGGCCCCCCCGGAGACACCTGGGCAGCTGCTCTCTATCTCCATGTTTTTTGTGTCTTAAAAATTATATATACTGGGTATGGTGGTACATGCCTTTAATCCCTGTACTTGGGAGGCAGAAGCAGGTAGATCTCTGTGAGTTCAAGGTCAGCCTGGTCTACATAGTGAGTTCCAGGACAGAAAGAGCTGTGTAGAGAGACCCTGTCTCAAAAAGAAAAACAAAACAAATACTATAGACACAAGGAAGTGTCGCGGGGTAAGACTTGGAAGCCCCTTCTCCACCACCTGCTTTCTTTCATTTCTCACAGCCTCTCCGCTGAGAATCACACCAGGTTTTTATTTGTTCTTTTCCCTCGTGTCATCTAAGCCTCAGTTAGGGAAAATGGGTTAAGTGACCTCAGGGTTACCGTTGTCCATGAGTTCTCCCGTGAGGCACCGTGGCAGGCCCCACTGGGTCCGCCTGGCTCCATCTGCTAGTGTGCACTGTTGGCACGTGTGGTTCTATTGTGTGTGTGTGCTCACACAGGTAAAGTTATATTGTGCTCCTGGATGATAAACTGTTCCCTCCTACCCTGTGTCCACCTCCTCCTCCCATGCTGTTCTCCCTCCTCAGTGTCTGCCTTCTCTCAGGGCTTGAAGGCTTCTGGGCGGATCTTCATTTCCACACGTTTGAGGGAATAGTTGGCTCCATGCCAGCCATACCAGCTGATGCCATCCATGTGCTTTCGGTGCTCGCCAAGGCGGTAGTACACCCCATTGAGGTTGGAGTCTGTGCAGCAGTTGTACCAGTAGCCACCTTTTCGGAGCTGTGCGCACTTGTCCAAGCAGTTGTCGTTGTCCTTGTCCTTGGTGCTGAAGACGGTGTTGTTATGGTAGAGGAGGGCGTCCTTCCCCACGTTGCCACTGTAGTTCCCCAGGAAGAGGCGGTAGCTGTTCAGTTCATTGCCCAACGCAAAGTAGCTATACTCTGCGTAGCGTGCATTGCCCTCCCAGTCCTCCAGCTCCACACGAAGCCGGCTTGGCTGCCTGGTGAGCCGGTGGATATGTTCATTCCCCAGCCAGAAGTCACCTCGGATGCTGCCAAACCCTTGCTTATACTGTCTCCAGTCTTGGTAGAAGGAGACAAGGCCACTCTTACGTCTCTGGATGATGGTCCAGCCTCCTCCTGAAGTTTCCATGTCACAGAACACCTCTAGCTCAGGGCTCCCCAGGAACTCGTCAGGAGGAAGCTTGTACACTCCAGAGATTCGGTAGTTCTTCTGGTACAGGGAAGAACAGTCATAGATGGCATCTGCCGAGGTCTGCGTGACGGTCTGCGCAGCCTGCAGCTGCATGATGTCAATCTGGTTGTTCATCTCAGAGTACTTGCTCTCGGCAGTGCTGAGCCGAGACTCCATGTGCTTGCTGCTGCTCTCCAGCTCCATCACCTGCATGACCACACTGACCCAGTCGCTCTCCTGCTTCCTGCTCAGCTCTCCCAGCAGACTGCTGAGGTTGGCCACCTGGGCTTTGAGCTCCCTCATCTCCTCACAGCAGCCCGCTGCTTTGAGCTGTGTCTTGCGTTTATGAGGCTTCTGCAGCCACACTGGGTGGCTGACAAAGGCTACAAGGATAACACATAGCCAGGTCTCCCTCAGCATGGTGTGCAGGCCTGTTCCCCTTAGAGTCTGTCCCTGGAGAAGTAGGAAGCTTCTTTCTCTGACCCACACTGGCAGTTTTCTTCCTTCCCCGTGATCTCCTTCCTCTGTGTGCTTGCTTAGCCTTTCTTAAAGCTAGAGATTCTGAAATGGAACTCTTTCCTTCCTTAGTCTGA

SEQ ID NO:3

NM-021146.4 homo sapiens angiopoietin-like 7 (ANGPTL 7), mRNA

GGGCTTGGAAGGAAAGCTATAGGCTACCCATTCAGCTCCCCTGTCAGAGACTCAAGCTTTGAGAAAGGCTAGCAAAGAGCAAGGAAAGAGAGAAAACAACAAAGTGGCGAGGCCCTCAGAGTGAAAGCGTAAGGTTCAGTCAGCCTGCTGCAGCTTTGCAGACCTCAGCTGGGCATCTCCAGACTCCCCTGAAGGAAGAGCCTTCCTCACCCAAACCCACAAAAGATGCTGAAAAAGCCTCTCTCAGCTGTGACCTGGCTCTGCATTTTCATCGTGGCCTTTGTCAGCCACCCAGCGTGGCTGCAGAAGCTCTCTAAGCACAAGACACCAGCACAGCCACAGCTCAAAGCGGCCAACTGCTGTGAGGAGGTGAAGGAGCTCAAGGCCCAAGTTGCCAACCTTAGCAGCCTGCTGAGTGAACTGAACAAGAAGCAGGAGAGGGACTGGGTCAGCGTGGTCATGCAGGTGATGGAGCTGGAGAGCAACAGCAAGCGCATGGAGTCGCGGCTCACAGATGCTGAGAGCAAGTACTCCGAGATGAACAACCAAATTGACATCATGCAGCTGCAGGCAGCACAGACGGTCACTCAGACCTCCGCAGATGCCATCTACGACTGCTCTTCCCTCTACCAGAAGAACTACCGCATCTCTGGAGTGTATAAGCTTCCTCCTGATGACTTCCTGGGCAGCCCTGAACTGGAGGTGTTCTGTGACATGGAGACTTCAGGCGGAGGCTGGACCATCATCCAGAGACGAAAAAGTGGCCTTGTCTCCTTCTACCGGGACTGGAAGCAGTACAAGCAGGGCTTTGGCAGCATCCGTGGGGACTTCTGGCTGGGGAACGAACACATCCACCGGCTCTCCAGACAGCCAACCCGGCTGCGTGTAGAGATGGAGGACTGGGAGGGCAACCTGCGCTACGCTGAGTATAGCCACTTTGTTTTGGGCAATGAACTCAACAGCTATCGCCTCTTCCTGGGGAACTACACTGGCAATGTGGGGAACGACGCCCTCCAGTATCATAACAACACAGCCTTCAGCACCAAGGACAAGGACAATGACAACTGCTTGGACAAGTGTGCACAGCTCCGCAAAGGTGGCTACTGGTACAACTGCTGCACAGACTCCAACCTCAATGGAGTGTACTACCGCCTGGGTGAGCACAATAAGCACCTGGATGGCATCACCTGGTATGGCTGGCATGGATCTACCTACTCCCTCAAACGGGTGGAGATGAAAATCCGCCCAGAAGACTTCAAGCCTTAAAAGGAGGCTGCCGTGGAGCACGGATACAGAAACTGAGACACGTGGAGACTGGATGAGGGCAGATGAGGACAGGAAGAGAGTGTTAGAAAGGGTAGGACTGAGAAACAGCCTATAATCTCCAAAGAAAGAATAAGTCTCCAAGGAGCACAAAAAAATCATATGTACCAAGGATGTTACAGTAAACAGGATGAACTATTTAAACCCACTGGGTCCTGCCACATCCTTCTCAAGGTGGTAGACTGAGTGGGGTCTCTCTGCCCAAGATCCCTGACATAGCAGTAGCTTGTCTTTTCCACATGATTTGTCTGTGAAAGAAAATAATTTTGAGATCGTTTTATCTATTTTCTCTACGGCTTAGGCTATGTGAGGGCAAAACACAAATCCCTTTGCTAAAAAGAACCATATTATTTTGATTCTCAAAGGATAGGCCTTTGAGTGTTAGAGAAAGGAGTGAAGGAGGCAGGTGGGAAATGGTATTTCTATTTTTAAATCCAGTGAAATTATCTTGAGTCTACACATTATTTTTAAAACACAAAAATTGTTCGGCTGGAACTGACCCAGGCTGGACTTGCGGGGAGGAAACTCCAGGGCACTGCATCTGGCGATCAGACTCTGAGCACTGCCCCTGCTCGCCTTGGTCATGTACAGCACTGAAAGGAATGAAGCACCAGCAGGAGGTGGACAGAGTCTCTCATGGATGCCGGCACAAAACTGCCTTAAAATATTCATAGTTAATACAGGTATATCTATTTTTATTTACTTTGTAAGAAACAAGCTCAAGGAGCTTCCTTTTAAATTTTGTCTGTAGGAAATGGTTGAAAACTGAAGGTAGATGGTGTTATAGTTAATAATAAATGCTGTAAATAAGCATCTCACTTTGTAAAAATAAAATATTGTGGTTTTGTTTTAAACATTCAACGTTTCTTTTCCTTCTACAATAAACACTTTCAAAATGTGA

SEQ ID NO:4

Reverse complement of SEQ ID NO. 3 TCACATTTTGAAAGTGTTTATTGTAGAAGGAAAAGAAACGTTGAATGTTTAAAACAAAACCACAATATTTTATTTTTACAAAGTGAGATGCTTATTTACAGCATTTATTATTAACTATAACACCATCTACCTTCAGTTTTCAACCATTTCCTACAGACAAAATTTAAAAGGAAGCTCCTTGAGCTTGTTTCTTACAAAGTAAATAAAAATAGATATACCTGTATTAACTATGAATATTTTAAGGCAGTTTTGTGCCGGCATCCATGAGAGACTCTGTCCACCTCCTGCTGGTGCTTCATTCCTTTCAGTGCTGTACATGACCAAGGCGAGCAGGGGCAGTGCTCAGAGTCTGATCGCCAGATGCAGTGCCCTGGAGTTTCCTCCCCGCAAGTCCAGCCTGGGTCAGTTCCAGCCGAACAATTTTTGTGTTTTAAAAATAATGTGTAGACTCAAGATAATTTCACTGGATTTAAAAATAGAAATACCATTTCCCACCTGCCTCCTTCACTCCTTTCTCTAACACTCAAAGGCCTATCCTTTGAGAATCAAAATAATATGGTTCTTTTTAGCAAAGGGATTTGTGTTTTGCCCTCACATAGCCTAAGCCGTAGAGAAAATAGATAAAACGATCTCAAAATTATTTTCTTTCACAGACAAATCATGTGGAAAAGACAAGCTACTGCTATGTCAGGGATCTTGGGCAGAGAGACCCCACTCAGTCTACCACCTTGAGAAGGATGTGGCAGGACCCAGTGGGTTTAAATAGTTCATCCTGTTTACTGTAACATCCTTGGTACATATGATTTTTTTGTGCTCCTTGGAGACTTATTCTTTCTTTGGAGATTATAGGCTGTTTCTCAGTCCTACCCTTTCTAACACTCTCTTCCTGTCCTCATCTGCCCTCATCCAGTCTCCACGTGTCTCAGTTTCTGTATCCGTGCTCCACGGCAGCCTCCTTTTAAGGCTTGAAGTCTTCTGGGCGGATTTTCATCTCCACCCGTTTGAGGGAGTAGGTAGATCCATGCCAGCCATACCAGGTGATGCCATCCAGGTGCTTATTGTGCTCACCCAGGCGGTAGTACACTCCATTGAGGTTGGAGTCTGTGCAGCAGTTGTACCAGTAGCCACCTTTGCGGAGCTGTGCACACTTGTCCAAGCAGTTGTCATTGTCCTTGTCCTTGGTGCTGAAGGCTGTGTTGTTATGATACTGGAGGGCGTCGTTCCCCACATTGCCAGTGTAGTTCCCCAGGAAGAGGCGATAGCTGTTGAGTTCATTGCCCAAAACAAAGTGGCTATACTCAGCGTAGCGCAGGTTGCCCTCCCAGTCCTCCATCTCTACACGCAGCCGGGTTGGCTGTCTGGAGAGCCGGTGGATGTGTTCGTTCCCCAGCCAGAAGTCCCCACGGATGCTGCCAAAGCCCTGCTTGTACTGCTTCCAGTCCCGGTAGAAGGAGACAAGGCCACTTTTTCGTCTCTGGATGATGGTCCAGCCTCCGCCTGAAGTCTCCATGTCACAGAACACCTCCAGTTCAGGGCTGCCCAGGAAGTCATCAGGAGGAAGCTTATACACTCCAGAGATGCGGTAGTTCTTCTGGTAGAGGGAAGAGCAGTCGTAGATGGCATCTGCGGAGGTCTGAGTGACCGTCTGTGCTGCCTGCAGCTGCATGATGTCAATTTGGTTGTTCATCTCGGAGTACTTGCTCTCAGCATCTGTGAGCCGCGACTCCATGCGCTTGCTGTTGCTCTCCAGCTCCATCACCTGCATGACCACGCTGACCCAGTCCCTCTCCTGCTTCTTGTTCAGTTCACTCAGCAGGCTGCTAAGGTTGGCAACTTGGGCCTTGAGCTCCTTCACCTCCTCACAGCAGTTGGCCGCTTTGAGCTGTGGCTGTGCTGGTGTCTTGTGCTTAGAGAGCTTCTGCAGCCACGCTGGGTGGCTGACAAAGGCCACGATGAAAATGCAGAGCCAGGTCACAGCTGAGAGAGGCTTTTTCAGCATCTTTTGTGGGTTTGGGTGAGGAAGGCTCTTCCTTCAGGGGAGTCTGGAGATGCCCAGCTGAGGTCTGCAAAGCTGCAGCAGGCTGACTGAACCTTACGCTTTCACTCTGAGGGCCTCGCCACTTTGTTGTTTTCTCTCTTTCCTTGCTCTTTGCTAGCCTTTCTCAAAGCTTGAGTCTCTGACAGGGGAGCTGAATGGGTAGCCTATAGCTTTCCTTCCAAGCCC

SEQ ID NO:5

XM_005544804.2PREDICTED cynomolgus monkey angiopoietin-like 7 (ANGPTL 7), mRNA

AAGAAAGACTCGCCCCATCTCCCTCCTCCCCTCCTCTGGCCTAAGTTGCCGCTGACTTCACCCAACAGGCACCTGACCCTCCCAGATGAGCTGGGAGGGGCTAAAGCCCGGTGCGGCCATGGTGGGGGTGGAGGTACAGGCAGCAAACAATATTTAAGATGCTGACTTGTGGAGCATTCAGGCTTGGGAAGGAAAGCTATAGGCTATCCATTCAGCTCCCCTGTCAGAGACTCAAGCTTTGAGAAAGGCCAGCAAAGAGCAAGGAAAAGAGAGAAAACAACAAAGTGGCGAGGCCCTCAGAGTGAAAGCGTAAGGTTCAGTCAGCCTCCTGCAGCTTTGCAGACCTCAGCTGGGCATCTCCAGGCTCCCCTGGAGGAAGAGCCTTCCTCACCCAAACCCACAAAAGATGCTGAAAAAGCCTCTCTCAGCTGTGACCTGGCTCTGCATTTTCATCGTGGCCTTTGTCAGCCACCCAGCATGGCTGCAGAAGCCCTCTAAGCGCAAGACACCAGCACAGCTCAAAGCGGCCACCTGCTGTGAGGAGGTGAAGGAGCTCAAGGCCCAAGTCGCCAACCTCAGCAGCCTGCTGAGTGAACTGAACAAGAAGCAGGAAAGGGACTGGGTCAGTGTGGTCATGCAGGTGATGGAGCTGGAGAGCAACAGCAAGCGCATGGAGTCGCGGCTCACAGATGCCGAGAGCAAGTACTCTGAGATGAACAACCAAATCGACATCATGCAGCTGCAGGCGGCACAGACGGTCACTCAGACCTCCGCAGATGCCATCTACGACTGCTCTTCACTCTACCAGAAGAACTACCGCATCTCTGGAGTGTATAAGCTTCCTCCTGATGACTTCCTGGGCAGCCCTGAACTGGAGGTGTTCTGTGACATGGAGACTTCAGGTGGAGGCTGGACCATCATCCAGAGACGAAAAAGTGGCCTTGTCTCCTTCTACCAGGACTGGAAGCAGTACAAGCAGGGCTTTGGCAGCATCCGTGGGGACTTCTGGCTGGGGAATGAACACATCCACCGGCTCTCCAGACAGCCAACCCGGCTGCGTGTAGAGATGGAGGACTGGGAGGGCAACCTGCGCTACGCTGAGTATAGCCACTTTGTTCTGGGCAATGAACTCAACAGCTATCGCCTCTTCCTGGGGAACTACACTGGCAATGTGGGGAACGACGCCCTCCAGTATCATAACAACACAGCCTTCAGCACCAAGGACAAGGACAATGACAACTGCTTAGACAAGTGTGCACGGCTCCGCAAAGGTGGCTACTGGTACAACTGCTGCACAGACTCCAATCTCAATGGAGTGTACTACCGCCTGGGCGAGCACAACAAGCACTTGGATGGCATCACCTGGTACGGCTGGCATGGATCTACCTACTCCCTGAAACGGGTGGAGATGAAAATCCGCCCGGAAGACTTTAAGCCTTAAAAGGAGGCTGCCGTGGAGCACAGATGCAGACACTGAGACACCTGGAGATTAGATGAGGGCAGATGAGGACAGGAAGAGAGTATTAGAAAGGGTAGGGTTGAGAAACAGCCTATACTCTCCAAAGAAAGAATAAGTCTCCAAGGAACACAATAAAATCATATGTACCAAGGATGTTACAGTAAACAGGATGAACCATTTAAACCCACTGGGTCCTGCCACATCCTTCTTAAGGGGGTAGACTCAGTGGGGTCTCTCTGCCCAAGATCCCTGACATAGCAGTAGCTTGTCTTTTCCATATGATTTGTCTGTGTTTTCCATATGATTTGTCTGTGAAAGAAAATAACTTTGAGATCGCTTTATCTATTTTCTTTAAGGCTTAGGCTACATGAGGGCCAAAACACAAATCCCTTTGCTAAAAAGAACCATATTATTTTGATTCTCAAAGAAGAGGCCTTTGAGTGTTAGAGAAAGGAGTGAAGGAGGCAGGTGGGAGATGGGTATTTCTATTTTTAAATCCAGTGAAATTATCTTGAGTCTACATATTATTTTTAAAACACAAAAATTGTTCGGCTGTAGGTGAACTGACCCAGGCTGGACTTGCGAGGAGGAAACTCCAGGGCACTGGGTCTGGCAATCAGACTGAGCACTGCCCGTGCTCACCTTGGTCAGGTACAGCACTGAAAGGTATGAAGCACCGGCAGGAGGTGGACACAGTCTCTCATGAATGCTGGCACAAAACTGCCTTAAAATATTCATAGTTAATACAGGTATACCTATTTTTATTTACTTTGTAAGAAACAAGCTCAAGGGGCTTCCTTTTAAATTTTGTCTATAGGAAATGGCTGAAAACTGAAGGTAGATGGTGTTATAGTTAATAATGAATGCTGTATATAAGCATCTTGCTTTGTAAAAATAAAATATTGTGGTTTTGTTTTAAACATTTAACGTTTCTTTTCCTTCTACAATAAACACTTTCAAAA

SEQ ID NO:6

Reverse complement of SEQ ID NO. 5

TTTTGAAAGTGTTTATTGTAGAAGGAAAAGAAACGTTAAATGTTTAAAACAAAACCACAATATTTTATTTTTACAAAGCAAGATGCTTATATACAGCATTCATTATTAACTATAACACCATCTACCTTCAGTTTTCAGCCATTTCCTATAGACAAAATTTAAAAGGAAGCCCCTTGAGCTTGTTTCTTACAAAGTAAATAAAAATAGGTATACCTGTATTAACTATGAATATTTTAAGGCAGTTTTGTGCCAGCATTCATGAGAGACTGTGTCCACCTCCTGCCGGTGCTTCATACCTTTCAGTGCTGTACCTGACCAAGGTGAGCACGGGCAGTGCTCAGTCTGATTGCCAGACCCAGTGCCCTGGAGTTTCCTCCTCGCAAGTCCAGCCTGGGTCAGTTCACCTACAGCCGAACAATTTTTGTGTTTTAAAAATAATATGTAGACTCAAGATAATTTCACTGGATTTAAAAATAGAAATACCCATCTCCCACCTGCCTCCTTCACTCCTTTCTCTAACACTCAAAGGCCTCTTCTTTGAGAATCAAAATAATATGGTTCTTTTTAGCAAAGGGATTTGTGTTTTGGCCCTCATGTAGCCTAAGCCTTAAAGAAAATAGATAAAGCGATCTCAAAGTTATTTTCTTTCACAGACAAATCATATGGAAAACACAGACAAATCATATGGAAAAGACAAGCTACTGCTATGTCAGGGATCTTGGGCAGAGAGACCCCACTGAGTCTACCCCCTTAAGAAGGATGTGGCAGGACCCAGTGGGTTTAAATGGTTCATCCTGTTTACTGTAACATCCTTGGTACATATGATTTTATTGTGTTCCTTGGAGACTTATTCTTTCTTTGGAGAGTATAGGCTGTTTCTCAACCCTACCCTTTCTAATACTCTCTTCCTGTCCTCATCTGCCCTCATCTAATCTCCAGGTGTCTCAGTGTCTGCATCTGTGCTCCACGGCAGCCTCCTTTTAAGGCTTAAAGTCTTCCGGGCGGATTTTCATCTCCACCCGTTTCAGGGAGTAGGTAGATCCATGCCAGCCGTACCAGGTGATGCCATCCAAGTGCTTGTTGTGCTCGCCCAGGCGGTAGTACACTCCATTGAGATTGGAGTCTGTGCAGCAGTTGTACCAGTAGCCACCTTTGCGGAGCCGTGCACACTTGTCTAAGCAGTTGTCATTGTCCTTGTCCTTGGTGCTGAAGGCTGTGTTGTTATGATACTGGAGGGCGTCGTTCCCCACATTGCCAGTGTAGTTCCCCAGGAAGAGGCGATAGCTGTTGAGTTCATTGCCCAGAACAAAGTGGCTATACTCAGCGTAGCGCAGGTTGCCCTCCCAGTCCTCCATCTCTACACGCAGCCGGGTTGGCTGTCTGGAGAGCCGGTGGATGTGTTCATTCCCCAGCCAGAAGTCCCCACGGATGCTGCCAAAGCCCTGCTTGTACTGCTTCCAGTCCTGGTAGAAGGAGACAAGGCCACTTTTTCGTCTCTGGATGATGGTCCAGCCTCCACCTGAAGTCTCCATGTCACAGAACACCTCCAGTTCAGGGCTGCCCAGGAAGTCATCAGGAGGAAGCTTATACACTCCAGAGATGCGGTAGTTCTTCTGGTAGAGTGAAGAGCAGTCGTAGATGGCATCTGCGGAGGTCTGAGTGACCGTCTGTGCCGCCTGCAGCTGCATGATGTCGATTTGGTTGTTCATCTCAGAGTACTTGCTCTCGGCATCTGTGAGCCGCGACTCCATGCGCTTGCTGTTGCTCTCCAGCTCCATCACCTGCATGACCACACTGACCCAGTCCCTTTCCTGCTTCTTGTTCAGTTCACTCAGCAGGCTGCTGAGGTTGGCGACTTGGGCCTTGAGCTCCTTCACCTCCTCACAGCAGGTGGCCGCTTTGAGCTGTGCTGGTGTCTTGCGCTTAGAGGGCTTCTGCAGCCATGCTGGGTGGCTGACAAAGGCCACGATGAAAATGCAGAGCCAGGTCACAGCTGAGAGAGGCTTTTTCAGCATCTTTTGTGGGTTTGGGTGAGGAAGGCTCTTCCTCCAGGGGAGCCTGGAGATGCCCAGCTGAGGTCTGCAAAGCTGCAGGAGGCTGACTGAACCTTACGCTTTCACTCTGAGGGCCTCGCCACTTTGTTGTTTTCTCTCTTTTCCTTGCTCTTTGCTGGCCTTTCTCAAAGCTTGAGTCTCTGACAGGGGAGCTGAATGGATAGCCTATAGCTTTCCTTCCCAAGCCTGAATGCTCCACAAGTCAGCATCTTAAATATTGTTTGCTGCCTGTACCTCCACCCCCACCATGGCCGCACCGGGCTTTAGCCCCTCCCAGCTCATCTGGGAGGGTCAGGTGCCTGTTGGGTGAAGTCAGCGGCAACTTAGGCCAGAGGAGGGGAGGAGGGAGATGGGGCGAGTCTTTCTT

SEQ ID NO:7

XM_006225622.3PREDICTED Brown mouse angiopoietin-related protein 7-like (LOC 102552055), mRNA

GATTCCTTTATAGATGGTTGTGAGCCACCATGTGGTTGCTGGGATTTGAACTCAGGATGCCAGGACTGCTGAGCGGTCTCTCTGGTCCCTCCTTTTCATTTTACCATGTAGCTCTTGCTATGTAATACTTGCAGTGTAGCTCAGGTGGTCAAAAACTTACAACCCTCCTGCCTCTGCCTCTCAGTGTTGGGAGAAGAGGAGAGCCACCACACTGGCCCACACACCTTCATGTTTGTCCTGATGTTGGCACTTTGGGTGTCTGGCTTATCTCAAGATGCTGAAGGCAATCCCTACACCAATGCATTACGGTTTCGCCTTTCACCTCCAGAATTTAAATGTAGGACTGAGATTTTACATATAGTGTGAGGTGAAGATCAAAATGTGAACGTTGTCACCTGAATGTTACCCAGCTGGCTCACTACGCTCACTGCAAAGGCTGGCCTGCCCCAGTGCTGTCTTCTGCACAAGACACTGCAGTTCTAGGGCAGCCGGGTCAGCTAGGATGCTTCCTACATGATCATATGGCACACAGCGTCACTGCACAGACGTGAGACTACCTTAAAGAGCTCCACAGTTTAGCAGACATTGATGCTAAACACGAATAAACTTCATGGAACCAGCAAAGGCCATGATGCCCTGCTAAACTAACAAAGGCTAATGTAGCTGCTCACTGAGAAAACTAAGAAAAAGCTGGGACTAGCACAGTGCCACCTCCCGTCAGAGCCCCTGAGATGGTAACCCAGAAGATGTTAGTAACAACCTGATGCCCAAGGAGGCAAGCCCGACAGGAGGTGAGAGGGAACGAGCTGTGGCTTACCTGAGCGCCTACTGCTTTTCATTCAGAAATGTGACCCTGCTGAGTAACCCCAGCTGCCTCAACAGAAATCACTTTCCAAGAGCCAGAGACTCATCTGGGAAAGGCTGGGTGGGCGGGGCTAAGGGAGGAAGAGCCGTGGGACCCACACTATGTCAGGCAGAGCTCCTGCCGTAGGGAAGGAGGGAGCGTTGTACAGTTTTAGGGGAAAGTCCCATCTTTTACAGGGTGTTAGAAGAGCGGTGCCCTTCAAGTAAAGGTGTGAACACTACTTTTCTCAGACAGCATGTATGTAGGTACAGGGGGCTAGCAGCAAGTTGCAAACCACACTGCAGACAGAGAGAGAACCTAGATTTCTAATCTTCATTTATTAGGTGATGAGGAGTCTTTTCTGAGTTTGAACCTCGGGTAAAGGTCAAGTCCACTCACTGCTGCCCAGAAGCTGACAGAAGTGTGGATGACCAACTACAGACCACGTCTGGCATGGCTGAGTCTTCCTCTCTGCCCTGTGCTGTGACAACTCTTGTAAAATGCTGAGTGATCTTTTAGGAAGGAGAATGTGTAAAAAGGAAGACAGCTCCATAGATGCAAACCTCTAATTCAAGTTCAATAGGATTCTGTGGAACACTGAATGATGACCAGAAAGGACGTATCCAGTCCTGTGAGAACTCGGCTTAGCCCACATAAGCCAGAACCTTAAGTAAAGAGCACGGATTACCTTAGCCAGCCTCCCTGGCTCAGCAGTCTCCATTCCAGCTCCTTTTAGCTACCTCGCCCTCAGTCTTACATAACCTTCAGACAGGAGTTGGGAAAGCCTTTCACTTGGCCTGTCTGCTGACAGAGCTGAGCCAGTGGCTGGCAGGCCATAATCTACTAGGCACAACTGGAAACACGTTCACAGCTCCCGTCTGAGCACACAGACTGTACAGACAAGGAAACAAACACTCGCCCTGTCCATCCTCCTCCTCCTCTCCTCTGGCCTAGGTTTGTGCTGACTTCACCCAACAGGCACCTGACCCTCCCAGATGAGGTGGGAGGGGCTTTAGCACACAGGGCCCTAGGGGTGGAGGTACAGGCACCAACAATATTTAAGATGCTGCCTTGGTGGGGTATTCAGACTTGGGAAGGAAAAGTGTTTAAATACCCACCAAGCTCCGTTTCAGAAACTCCAGCTTTAAGAAAGGCACACAGAGGAAGGAGACCACGAGGAAGGAAGAAAACTGCCCTTGTGAGTCAAACACTAAGCTTCCTAGTTCTGCTGAATACAGACTCTAGGAGGAAGACCTGCCCCAGAGGCCTGCACACAATGCTGAGGACCACCTGGCTATGCATTCTCCTGGTAGCCTCTGTCAGTCGCCCCGTGTGGCTGCAGAAGCCTCATAAACGCAAGACACAGCTCAAAGCAGCCGGCTGCTGTGAGGAGATGAGGGAGCTCAAGGCCCAGGTCGCCAACCTCAGCAGTCTGCTGGGTGAGCTGAGCAGGAAGCAGGAGAGCGACTGGGTCAGTGTGGTCATGCAGGTGATGGAGCTGGAGAGCAGCAGCAAGCGCATGGAGTCTCGGCTCACCACTGCCGAGAGCAAGTACTCTGAGATGAACAACCAGATCGACATCATGCAGTTACAGGCTGCACAGACCGTCACACAGACCTCGGCAGATGCCATCTACGACTGTTCCTCCCTGTACCAGAAGAACTACCGAATCTCTGGAGTGTACAAGCTTCCTCCAGATGAGTTCCTGGGCAGCCCTGAGTTAGAGGTGTTCTGTGACATGGAAACTTCAGGAGGAGGCTGGACCATCATCCAGAGGCGCAAGAGTGGCCTAGTCTCCTTCTACCAAGACTGGAAACAGTATAAGCAAGGGTTTGGCAGCATTCGAGGCGACTTCTGGCTAGGGAATGAACATATTCACCGGCTTACCAGGCAGCCAACAAGGCTTCGTGTGGAGCTGGAGGACTGGGAGGGCAACGCACGCTACGCAGAGTACAGCTACTTTGCGTTGGGCAATGAACTGAACAGCTACCGCCTCTTCCTGGGGAACTACAGTGGCAACGTGGGGAAGGACGCTCTCCTCTATCATAACAACACCGTCTTCAGCACCAAGGACAAGGACAATGACAACTGCTTGGACAAGTGTGCACAGCTCCGAAAAGGTGGCTACTGGTACAACTGCTGCACAGACTCCAACCTCAATGGGGTGTACTACCGCCTGGGGGAGCACCGGAAGCACATGGATGGCATCAGCTGGTATGGCTGGCATGGAGCCAACTATTCCCTCAAACGGGTGGAGATGAAGATCCGTCCAGAAGCCTTCACGCCCTAGGAGAAGGTTGCTGCAGAGCTATGTGAGGCGGAGGCTGAGGAGGGAGAGCAGGATGGGAAGAGGGTGGACAAAAGGTAGGAAGGGGACAGTTTATCATCCAGGAGCGTGACACAACTTCACCTGTGCACACAAGAGCACATGCACACACAACAGAACCACACAGACCAAACAGTGCACATTAGCAGATGGCACCAGGCCAGTAGGTGCCATGGTGCCTCAGGGGAGGACTGAGTGGGCCCACAGAGCAGAAGCTCATCCTCCACACCCTTAGCTGTGCTCAACAGTGACCCTGAGGTCACGAAACCTGTTTCCCCTACCTGAGGCTCAGATGACATGAGGGAAAAGAAAAATAAAAGGAACTGTTGTGACCCTCCGTGGAGAGGCCATGAAAAATGAAAGCAGATGGTGGAGAAGGGGCTTCCCCTTCTTAGGTCCCATGACACTTCCTTGTGTCTATAGGATTTGTTTTGTTTTCCTTTGTGACACAGGGTCTCTCTACACAGCTCTTGCTGTCCTGGAACTTACTATGTAGACC

SEQ ID NO:8

Reverse complement of SEQ ID NO. 7

GGTCTACATAGTAAGTTCCAGGACAGCAAGAGCTGTGTAGAGAGACCCTGTGTCACAAAGGAAAACAAAACAAATCCTATAGACACAAGGAAGTGTCATGGGACCTAAGAAGGGGAAGCCCCTTCTCCACCATCTGCTTTCATTTTTCATGGCCTCTCCACGGAGGGTCACAACAGTTCCTTTTATTTTTCTTTTCCCTCATGTCATCTGAGCCTCAGGTAGGGGAAACAGGTTTCGTGACCTCAGGGTCACTGTTGAGCACAGCTAAGGGTGTGGAGGATGAGCTTCTGCTCTGTGGGCCCACTCAGTCCTCCCCTGAGGCACCATGGCACCTACTGGCCTGGTGCCATCTGCTAATGTGCACTGTTTGGTCTGTGTGGTTCTGTTGTGTGTGCATGTGCTCTTGTGTGCACAGGTGAAGTTGTGTCACGCTCCTGGATGATAAACTGTCCCCTTCCTACCTTTTGTCCACCCTCTTCCCATCCTGCTCTCCCTCCTCAGCCTCCGCCTCACATAGCTCTGCAGCAACCTTCTCCTAGGGCGTGAAGGCTTCTGGACGGATCTTCATCTCCACCCGTTTGAGGGAATAGTTGGCTCCATGCCAGCCATACCAGCTGATGCCATCCATGTGCTTCCGGTGCTCCCCCAGGCGGTAGTACACCCCATTGAGGTTGGAGTCTGTGCAGCAGTTGTACCAGTAGCCACCTTTTCGGAGCTGTGCACACTTGTCCAAGCAGTTGTCATTGTCCTTGTCCTTGGTGCTGAAGACGGTGTTGTTATGATAGAGGAGAGCGTCCTTCCCCACGTTGCCACTGTAGTTCCCCAGGAAGAGGCGGTAGCTGTTCAGTTCATTGCCCAACGCAAAGTAGCTGTACTCTGCGTAGCGTGCGTTGCCCTCCCAGTCCTCCAGCTCCACACGAAGCCTTGTTGGCTGCCTGGTAAGCCGGTGAATATGTTCATTCCCTAGCCAGAAGTCGCCTCGAATGCTGCCAAACCCTTGCTTATACTGTTTCCAGTCTTGGTAGAAGGAGACTAGGCCACTCTTGCGCCTCTGGATGATGGTCCAGCCTCCTCCTGAAGTTTCCATGTCACAGAACACCTCTAACTCAGGGCTGCCCAGGAACTCATCTGGAGGAAGCTTGTACACTCCAGAGATTCGGTAGTTCTTCTGGTACAGGGAGGAACAGTCGTAGATGGCATCTGCCGAGGTCTGTGTGACGGTCTGTGCAGCCTGTAACTGCATGATGTCGATCTGGTTGTTCATCTCAGAGTACTTGCTCTCGGCAGTGGTGAGCCGAGACTCCATGCGCTTGCTGCTGCTCTCCAGCTCCATCACCTGCATGACCACACTGACCCAGTCGCTCTCCTGCTTCCTGCTCAGCTCACCCAGCAGACTGCTGAGGTTGGCGACCTGGGCCTTGAGCTCCCTCATCTCCTCACAGCAGCCGGCTGCTTTGAGCTGTGTCTTGCGTTTATGAGGCTTCTGCAGCCACACGGGGCGACTGACAGAGGCTACCAGGAGAATGCATAGCCAGGTGGTCCTCAGCATTGTGTGCAGGCCTCTGGGGCAGGTCTTCCTCCTAGAGTCTGTATTCAGCAGAACTAGGAAGCTTAGTGTTTGACTCACAAGGGCAGTTTTCTTCCTTCCTCGTGGTCTCCTTCCTCTGTGTGCCTTTCTTAAAGCTGGAGTTTCTGAAACGGAGCTTGGTGGGTATTTAAACACTTTTCCTTCCCAAGTCTGAATACCCCACCAAGGCAGCATCTTAAATATTGTTGGTGCCTGTACCTCCACCCCTAGGGCCCTGTGTGCTAAAGCCCCTCCCACCTCATCTGGGAGGGTCAGGTGCCTGTTGGGTGAAGTCAGCACAAACCTAGGCCAGAGGAGAGGAGGAGGAGGATGGACAGGGCGAGTGTTTGTTTCCTTGTCTGTACAGTCTGTGTGCTCAGACGGGAGCTGTGAACGTGTTTCCAGTTGTGCCTAGTAGATTATGGCCTGCCAGCCACTGGCTCAGCTCTGTCAGCAGACAGGCCAAGTGAAAGGCTTTCCCAACTCCTGTCTGAAGGTTATGTAAGACTGAGGGCGAGGTAGCTAAAAGGAGCTGGAATGGAGACTGCTGAGCCAGGGAGGCTGGCTAAGGTAATCCGTGCTCTTTACTTAAGGTTCTGGCTTATGTGGGCTAAGCCGAGTTCTCACAGGACTGGATACGTCCTTTCTGGTCATCATTCAGTGTTCCACAGAATCCTATTGAACTTGAATTAGAGGTTTGCATCTATGGAGCTGTCTTCCTTTTTACACATTCTCCTTCCTAAAAGATCACTCAGCATTTTACAAGAGTTGTCACAGCACAGGGCAGAGAGGAAGACTCAGCCATGCCAGACGTGGTCTGTAGTTGGTCATCCACACTTCTGTCAGCTTCTGGGCAGCAGTGAGTGGACTTGACCTTTACCCGAGGTTCAAACTCAGAAAAGACTCCTCATCACCTAATAAATGAAGATTAGAAATCTAGGTTCTCTCTCTGTCTGCAGTGTGGTTTGCAACTTGCTGCTAGCCCCCTGTACCTACATACATGCTGTCTGAGAAAAGTAGTGTTCACACCTTTACTTGAAGGGCACCGCTCTTCTAACACCCTGTAAAAGATGGGACTTTCCCCTAAAACTGTACAACGCTCCCTCCTTCCCTACGGCAGGAGCTCTGCCTGACATAGTGTGGGTCCCACGGCTCTTCCTCCCTTAGCCCCGCCCACCCAGCCTTTCCCAGATGAGTCTCTGGCTCTTGGAAAGTGATTTCTGTTGAGGCAGCTGGGGTTACTCAGCAGGGTCACATTTCTGAATGAAAAGCAGTAGGCGCTCAGGTAAGCCACAGCTCGTTCCCTCTCACCTCCTGTCGGGCTTGCCTCCTTGGGCATCAGGTTGTTACTAACATCTTCTGGGTTACCATCTCAGGGGCTCTGACGGGAGGTGGCACTGTGCTAGTCCCAGCTTTTTCTTAGTTTTCTCAGTGAGCAGCTACATTAGCCTTTGTTAGTTTAGCAGGGCATCATGGCCTTTGCTGGTTCCATGAAGTTTATTCGTGTTTAGCATCAATGTCTGCTAAACTGTGGAGCTCTTTAAGGTAGTCTCACGTCTGTGCAGTGACGCTGTGTGCCATATGATCATGTAGGAAGCATCCTAGCTGACCCGGCTGCCCTAGAACTGCAGTGTCTTGTGCAGAAGACAGCACTGGGGCAGGCCAGCCTTTGCAGTGAGCGTAGTGAGCCAGCTGGGTAACATTCAGGTGACAACGTTCACATTTTGATCTTCACCTCACACTATATGTAAAATCTCAGTCCTACATTTAAATTCTGGAGGTGAAAGGCGAAACCGTAATGCATTGGTGTAGGGATTGCCTTCAGCATCTTGAGATAAGCCAGACACCCAAAGTGCCAACATCAGGACAAACATGAAGGTGTGTGGGCCAGTGTGGTGGCTCTCCTCTTCTCCCAACACTGAGAGGCAGAGGCAGGAGGGTTGTAAGTTTTTGACCACCTGAGCTACACTGCAAGTATTACATAGCAAGAGCTACATGGTAAAATGAAAAGGAGGGACCAGAGAGACCGCTCAGCAGTCCTGGCATCCTGAGTTCAAATCCCAGCAACCACATGGTGGCTCACAACCATCTATAAAGGAATC

Claims (43)

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of angiopoietin-like 7 (ANGPTL 7) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by NO more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs 1 or 3, and the antisense strand comprises at least 15 contiguous nucleotides differing by NO more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs 2 or 4.

2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ANGPTL7, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides having 0, 1, 2 or 3 mismatches with one of the antisense sequences listed in any of tables 2-7, and wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides having 0, 1, 2 or 3 mismatches with a sense sequence listed in any of tables 2-7 corresponding to the antisense sequence.

3. The dsRNA agent of claim 1 or claim 2, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.

4. The dsRNA agent of claim 3, wherein the lipophilic moiety is conjugated by a linker or carrier.

5. The dsRNA agent of claim 3 or claim 4, wherein one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.

6. The dsRNA agent of claim 5, wherein the one or more lipophilic moieties are conjugated to the one or more internal positions on at least one strand by a linker or carrier.

7. The dsRNA agent of any one of claims 3-6, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

8. The dsRNA agent of claim 7, wherein the lipophilic moiety comprises a saturated or unsaturated C16 hydrocarbon chain.

9. The dsRNA agent of any one of claims 3-8, wherein the lipophilic moiety is conjugated by a carrier that replaces one or more nucleotides in the internal position or the double-stranded region.

10. The dsRNA agent of any one of claims 3-8, wherein the lipophilic moiety is conjugated to the double stranded iRNA agent by a linker comprising: ethers, thioethers, ureas, carbonates, amines, amides, maleimide-thioethers, disulfides, phosphodiesters, sulfonamide linkages, click reaction products, or carbamates.

11. The double stranded iRNA agent of any one of claims 3-9, wherein the lipophilic moiety is conjugated to a nucleobase, a sugar moiety or an internucleoside linkage.

12. The dsRNA agent of any one of claims 1-11, wherein the dsRNA agent comprises at least one modified nucleotide.

13. The dsRNA agent of claim 12, wherein no more than five of all sense strand nucleotides and no more than five of the antisense strand nucleotides are unmodified nucleotides.

14. The dsRNA agent of claim 12, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

15. The dsRNA agent of any one of claims 12-14, wherein at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 3' -terminal deoxythymine (dT) nucleotides, 2' -O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxymodified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic nucleotides, 2' -amino modified nucleotides, 2' -O-allyl modified nucleotides, 2' -C-alkyl modified nucleotides, 2' -C-methoxyethyl modified nucleotides, 2' -O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising a non-natural base, tetrahydropyran modified nucleotides, 1, 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5' -phosphate esters, nucleotides comprising 5' -phosphate ester mimetics, diol modified nucleotides and 2-O- (N-methylacetamide) modified nucleotides, and combinations thereof.

16. The dsRNA agent of any one of claims 1-15, wherein each of the sense strand, the antisense strand, or both the sense and antisense strands comprises a 3' overhang of at least 2 nucleotides.

17. The dsRNA agent of any one of claims 1-16, wherein the double-stranded region is 15-30 nucleotide pairs in length.

18. The dsRNA agent of claim 17, wherein the double-stranded region is 17-23 nucleotide pairs in length.

19. The dsRNA agent of any one of claims 1-18, wherein the sense strand and the antisense strand each have 19-30 nucleotides.

20. The dsRNA agent of any one of claims 1-19, wherein the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

21. The dsRNA agent of any one of claims 3-20, further comprising a targeting ligand.

22. The dsRNA agent of claim 21, wherein the targeting ligand targets ocular tissue.

23. The dsRNA agent of claim 22, wherein the ocular tissue is an optic nerve, trabecular meshwork, proximal tubular tissue, ganglion (e.g., including retinal ganglion), episcleral vein, or schlemm's canal (e.g., including endothelial cells).

24. The dsRNA agent of any one of claims 1-23, further comprising a phosphate or phosphate mimetic at the 5' -end of the antisense strand.

25. The dsRNA agent of claim 24, wherein the phosphate mimic is 5' -Vinyl Phosphonate (VP).

26. The dsRNA of any one of claims 1-25, wherein said dsRNA agent targets a hot spot region of mRNA encoding ANGPTL 7.

27. The dsRNA agent of claim 26, wherein the hot spot region comprises nucleotides 1562-1584, 546-568, 709-731, 862-884 and/or 232-256 of SEQ ID No. 1, or nucleotides 1993-2146、1910-1932、1726-1823、1628-1685、1591-1613、1551-1573、1420-1442、1380-1402、1243-1265、1195-1217、1096-1118、940-962 and/or 299-321 of SEQ ID No. 3.

28. The dsRNA agent of claim 27, wherein the dsRNA agent is selected from the group consisting of ：AD-1094991、AD-1093984、AD-1094129、AD-1094262、AD-1093670、AD-1093672、AD-1565389、AD-1565368、AD-1565357、AD-1565345、AD-1565324、AD-1565303、AD-1565288、AD-1565212、AD-1565141、AD-1565126、AD-1565113、AD-1565091、AD-1565034、AD-1565015、AD-1565004、AD-1564969、AD-1094381、AD-1564428、AD-1564936、AD-1564823、AD-1564802、AD-1564666、AD-1564618 below and AD-1563396.

29. A dsRNA agent that targets a hot spot region of an angiopoietin-like 7 (ANGPTL 7) mRNA.

30. A cell comprising the dsRNA agent of any one of claims 1-29.

31. A pharmaceutical composition for inhibiting ANGPTL7 expression comprising the dsRNA agent of any one of claims 1-29.

32. A method of inhibiting ANGPTL7 expression in a cell, the method comprising:

(a) Contacting the cell with the dsRNA agent of any one of claims 1-29 or the pharmaceutical composition of claim 31; and

(B) Maintaining the cells produced in step (a) for a time sufficient to reduce the level of ANGPTL7 mRNA, ANGPTL7 protein, or both ANGPTL7 mRNA and protein, thereby inhibiting expression of ANGPTL7 in the cells.

33. The method of claim 32, wherein the cell is within a subject.

34. The method of claim 33, wherein the subject is a human.

35. The method of claim 34, wherein the subject has been diagnosed with an ANGPTL 7-related disorder.

36. A method of treating a subject diagnosed with an ANGPTL 7-related disorder, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-25 or the pharmaceutical composition of claim 27, thereby treating the disorder.

37. The method of claim 36, wherein the ANGPTL 7-related disorder is glaucoma.

38. The method of claim 37, wherein the glaucoma is primary open angle glaucoma.

39. The method of any one of claims 36-38, wherein the treating comprises ameliorating at least one sign or symptom of the disorder.

40. The method of any one of claims 36-39, wherein the treatment comprises one or more of: (a) Inhibiting or reducing intraocular pressure (b) inhibiting or reducing expression or activity of ANGPTL 7; (c) increasing drainage of aqueous humor; (d) inhibiting or reducing optic nerve damage; or (e) a drug that inhibits or reduces retinal ganglion cell death, reduces intraocular pressure, laser treatment, surgery, or trabeculectomy.

41. The method of any one of claims 33-40, wherein the dsRNA agent is administered to the subject intraocularly, intravenously, or topically.

42. The method of claim 41, wherein the intraocular administration comprises intravitreal administration (e.g., intravitreal injection), transscleral administration (e.g., transscleral injection), subconjunctival administration (e.g., subconjunctival injection), postbulbar administration (e.g., postbulbar injection), intracameral administration (e.g., intracameral injection), or subretinal administration (e.g., subretinal injection).

43. The method of any one of claims 33-42, further comprising administering to the subject an additional agent or therapy comprising one or more of the following suitable for treating or preventing an ANGPTL 7-related disorder: prostaglandin analogs, beta blockers, alpha adrenergic agonists, carbonic anhydrase inhibitors, ROCK inhibitors, ROCKiRNA agents, inhibitors of Rho GTPase, anti-Rho GTPase agents, or anti-ANGPTL 7 agents.