WO2024078345A1 - snRNA核酸分子及其应用 - Google Patents

snRNA核酸分子及其应用 Download PDF

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WO2024078345A1
WO2024078345A1 PCT/CN2023/122201 CN2023122201W WO2024078345A1 WO 2024078345 A1 WO2024078345 A1 WO 2024078345A1 CN 2023122201 W CN2023122201 W CN 2023122201W WO 2024078345 A1 WO2024078345 A1 WO 2024078345A1
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snrna
sequence
nucleic acid
seq
acid molecule
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PCT/CN2023/122201
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French (fr)
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梁峻彬
欧家裕
徐辉
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广州瑞风生物科技有限公司
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a snRNA nucleic acid molecule and an application thereof.
  • Usher syndrome is a genetic disease, also known as deafness-retinitis pigmentosa syndrome, which is characterized by varying degrees of congenital sensorineural deafness and progressive vision loss caused by retinitis pigmentosa (RP).
  • Usher syndrome can be divided into three types: (1) Type I Usher syndrome: congenital severe profound sensorineural hearing loss, loss of vestibular response, pigmentary retinitis before puberty, and gradual blindness. The associated genes include MYO7A, CDH23, USH1C, PCHD15, etc.; (2) Type II Usher syndrome: congenital moderate to severe sensorineural hearing loss, normal vestibular response, pigmentary retinitis during puberty, and gradual blindness.
  • the associated genes include USH2A, GPR98, WHRN, etc.; (3) Type III Usher syndrome: progressive sensorineural hearing loss, normal vestibular response, pigmentary retinitis at the end of puberty, and gradual blindness.
  • the associated genes include CLRN1, etc.
  • USH2A gene mutation is the most common cause of Usher syndrome type II, covering more than 50% of Usher syndrome patients.
  • mutation of the USH2A gene is also one of the important causes of non-syndromic retinitis pigmentosa (NSRP).
  • NSRP non-syndromic retinitis pigmentosa
  • the USH2A gene is located at 1q41, spanning more than 800kb in the genome. It encodes a large transmembrane protein, Usherin, which is anchored on the plasma membrane of retinal photoreceptor cells and inner ear hair cells and is an essential component for the development and maintenance of cilia. In the retina, Usherin is an important part of the USH2 complex and is believed to play a role in stabilizing the outer segments of photoreceptors. USH2A has two subtypes. The main subtype in retinal cells contains 72 Exons and the coding region is about 15.6kb long.
  • the extracellular part of the Usherin protein contains many repeated domains, including 10 Laminin EGF-like (LE) domains and 35 Fibronectin type 3 (FN3) domains.
  • Human USH2A exon 13 is 642bp long, encoding amino acids 723 to 936 of Usherin, constituting 4 of the 10 LE domains in the Usherin protein.
  • Mutations in exon 13 of the USH2A gene include c.2802T>G (p.Cys934Trp), c.2299delG (p.Glu767Serfs*21, the most frequent mutation in European and American patients), c.2276G>T (p.cys759phe, the most common mutation site in non-syndromic RP), C.2522C>A (p.S841Y), c.2242C>T (p.Gln748X), c.2541C> A(C847X), c.2761delC(Leu921fs) and C.2776C>T(p.R926C), C.2209C>T, C.2310delA, c.2391_2392deITG, c.2431A>T, C.2431_2432delAA, c.2440C>T, c.2525dup, C.2610C>A, C.27
  • the USH2A coding region is approximately 15.6kb in length.
  • Conventional gene therapy delivery methods (such as recombinant lentivirus, recombinant adeno-associated virus, etc.) are difficult to package such a large coding sequence, so it is difficult to treat by directly delivering USH2A.
  • Exon 12 of mouse USH2A is homologous to exon 13 of human USH2A, both of which are 642bp in length. Removal of this exon did not cause subsequent frameshift mutations. Studies have shown that after knocking out exon 12 of mouse USH2A, Usherin can still be correctly positioned and perform normal functions.
  • Existing technologies can also promote exon skipping by using single-base editors to modify the key bases of the above-mentioned splicing-related sites.
  • existing single-base editors cannot be loaded through a single AAV vector, and are limited by PAM, editing window, and base conversion type, and may not have suitable gRNA near the splicing-related sites.
  • RPOQR (CN109804069A) discloses antisense oligonucleotides for treating eye diseases. Based on US10131910B2 and US10745699B2, this patent found that three sites Ex13-1, Ex13-2 and Ex13-3 seemed to give stronger single (exon 13) skipping signals.
  • AON promotes human exon 13 skipping, it also promotes human exon 12 and human exon 13 skipping.
  • Some AON treatments even cause double skipping, or there are mixed bands with abnormal/complete exon 13 skipping (possibly exon 12 single skipping).
  • the full length of human exon 12 is 196bp, which is not an integer multiple of 3. Its deletion will lead to frameshift mutation and inactivation of USH2A protein after double splicing skipping.
  • the effect of AON is not long-lasting and the frequency of medication is high; the efficiency of induced splicing skipping is not high; and the dosage of AON is very large.
  • snRNAs small nuclear RNAs
  • snRNPs small nuclear RNAs
  • Their length is about 100-215 nucleotides in mammals and are divided into 7 categories. Because they are rich in U, they are numbered U1 to U7. However, U7 snRNP does not participate in splicing, but is a key factor in the unique 3' end processing of replication-dependent histone (RDH) pre-mRNA.
  • RDH replication-dependent histone
  • the modified U7SnRNA is obtained by replacing the non-canonical Sm binding site of U7 snRNA with a common sequence derived from the major spliceosome U snRNP, and changing the histone binding sequence in the 5' region of U7 snRNA to the complementary sequence of the gene to be modified. It can induce splicing skipping of exons by targeting exons.
  • modified U7 snRNA since the development of modified U7 snRNA in 1998, its related research and application have not been extensive and are limited to a few targets.
  • the number of vectors used to administer U7 snRNP is limited.
  • modified U7 snRNA delivered by viruses such as AAV requires very high viral doses. High doses of viruses may be toxic or induce immune responses, which limits the application of viral delivery of U7-snRNA.
  • Other long-term delivery methods of gene integration have genome safety risks, and the effect of transient delivery is short-lived.
  • the technical problem to be solved by the present invention is to overcome the defect of low efficiency of inducing splicing skipping of USH2A exon 13 in the prior art, and provide a snRNA nucleic acid molecule and its application.
  • the present invention promotes the efficiency of single exon skipping reading by targeting pre-mRNA splicing of USH2A exon 13 with snRNA, significantly improving efficiency while ensuring safety.
  • the present invention solves the above technical problems through the following technical solutions.
  • the first aspect of the present invention provides a snRNA nucleic acid molecule, the snRNA nucleic acid molecule comprising: a recognition domain, a stem-loop sequence and an Sm sequence; wherein the number of the recognition domains is at least two;
  • each recognition domain is reverse complementary to the targeting sequence fragment from the 3′ end to the 5′ end of the pre-mRNA from the 5′ end to the 3′ end;
  • the pre-mRNA is the pre-mRNA corresponding to the USH2A gene.
  • each of the recognition domains is reverse complementary to the targeting sequence fragments from the 3′ end to the 5′ end of the pre-mRNA in sequence from the 5′ end to the 3′ end, that is, the recognition domains from the 5′ end to the 3′ end of the snRNA nucleic acid molecule are arranged from the 3′ end to the 5′ end according to the position of the target site corresponding to the recognition domain in the USH2A pre-mRNA.
  • each of the recognition domains is not reverse complementary to the targeting sequence fragments from 3′ to 5′ of the pre-mRNA in sequence from 5′ to 3′, that is, the order of the recognition domains from 5′ to 3′ of the snRNA nucleic acid molecule is not based on the position of the target site corresponding to the recognition domain in the USH2A pre-mRNA from 3′ to 5′.
  • the length of the recognition domain is at least 16 bp.
  • the length of the recognition domain is 18 to 40 bp.
  • the length of the recognition domain is 20 to 27 bp.
  • the length of the recognition domain is 22 to 27 bp.
  • the number of the recognition domains is two; preferably, the two recognition domains are adjacent to each other.
  • the number of the stem-loop sequences may be 1-2.
  • the snRNA nucleic acid molecule includes, from the 5′ end to the 3′ end, two adjacent recognition domains, an Sm sequence and a stem-loop sequence.
  • the pre-mRNA is all or part of the pre-mRNA corresponding to intron 12 to intron 13 of the USH2A gene.
  • the pre-mRNA is all or part of the pre-mRNA corresponding to exon 13 of the USH2A gene.
  • the genomic location of the pre-mRNA is Chr1:216246563-216247246; the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:1 and its mutant sequence.
  • the genomic location of the pre-mRNA is Chr1:216246563-216246753; the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:3 and its mutant sequence.
  • the genomic location of the pre-mRNA is Chr1:216246563-216246649; the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:4 and its mutant sequence.
  • the genomic location of the pre-mRNA is Chr1:216246563-216246626; the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:9 and its mutant sequence.
  • the genomic location of the pre-mRNA is Chr1:216246616-216246649; the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:34 and its mutant sequence.
  • the genomic location of the pre-mRNA is Chr1:216247130-216247246; the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:2 and its mutant sequence.
  • the genomic location of the pre-mRNA is Chr1:216247142-216247185; the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:32 and its mutant sequence.
  • the genomic location of the pre-mRNA is Chr1:216247130-216247161; the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:33 and its mutant sequence.
  • the genomic location of the pre-mRNA is Chr1:216247210-216247246; the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:36 and its mutant sequence.
  • the genomic location of the pre-mRNA is Chr1:216247204-216247232; the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:37 and its mutant sequence.
  • the genomic location of the pre-mRNA is Chr1:216247187-216247220; and the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:38 and its mutant sequence.
  • the genomic location of the pre-mRNA is Chr1:216247169-216247202; the targeting sequence fragment is selected from the nucleotide sequence shown in SEQ ID NO:39 and its mutant sequence.
  • the two recognition domains are respectively the first recognition domain and the second recognition domain from the 5′ end to the 3′ end; the first recognition domain and the second recognition domain can be RNA sequences that recognize different target sites, or can recognize RNA sequences of the same target site.
  • the reverse complementary RNA sequence that binds to the 3′ end of USH2A pre-mRNA serves as the second recognition domain of the snRNA
  • the reverse complementary RNA that binds to the 5′ end of USH2A pre-mRNA serves as the first recognition domain of the snRNA
  • the targeting sequence fragment that is reverse complementary to the first recognition domain or the second recognition domain is selected from the nucleotide sequence and its mutant sequence as shown in SEQ ID NO:34 and the nucleotide sequence and its mutant sequence as shown in SEQ ID NO:9; the corresponding targeting sequence fragment that is reverse complementary to the second recognition domain or the first recognition domain is selected from the nucleotide sequence and its mutant sequence as shown in SEQ ID NO:32, the nucleotide sequence and its mutant sequence as shown in SEQ ID NO:33, the nucleotide sequence and its mutant sequence as shown in SEQ ID NO:36, the nucleotide sequence and its mutant sequence as shown in SEQ ID NO:37, the nucleotide sequence and its mutant sequence as shown in SEQ ID NO:38, and the nucleotide sequence and its mutant sequence as shown in SEQ ID NO:39.
  • the targeting sequence fragment that is reverse complementary to the first recognition domain is selected from the nucleotide sequence shown in SEQ ID NO:34 and its mutant sequence and the nucleotide sequence shown in SEQ ID NO:9 and its mutant sequence; the targeting sequence fragment that is reverse complementary to the second recognition domain is selected from the nucleotide sequence shown in SEQ ID NO:32 and its mutant sequence, the nucleotide sequence shown in SEQ ID NO:33 and its mutant sequence, the nucleotide sequence shown in SEQ ID NO:36 and its mutant sequence, the nucleotide sequence shown in SEQ ID NO:37 and its mutant sequence, the nucleotide sequence shown in SEQ ID NO:38 and its mutant sequence, and the nucleotide sequence shown in SEQ ID NO:39 and its mutant sequence.
  • the mutant sequence is a sequence obtained by substitution, addition or deletion of one or more nucleotides based on the starting sequence.
  • the nucleotide sequence of the first recognition domain is shown in any one of SEQ ID NOs: 12 to 22, 59 to 61, and the nucleotide sequence of the second recognition domain is shown in any one of SEQ ID NOs: 40 to 58.
  • the nucleotide sequence of the first recognition domain is shown as SEQ ID NO: 12, 13, 15 or 17, and the nucleotide sequence of the second recognition domain is shown as SEQ ID NO: 48, 54, 56 or 58.
  • the nucleotide sequence of the first recognition domain is shown in SEQ ID NO: 12, 13 or 17, and the nucleotide sequence of the second recognition domain is shown in SEQ ID NO: 48, 54 or 58.
  • nucleotide sequence of the first recognition domain is shown as SEQ ID NO:12
  • nucleotide sequence of the second recognition domain is shown as SEQ ID NO:48.
  • nucleotide sequence of the first recognition domain is as shown in SEQ ID NO: 12
  • nucleotide sequence of the second recognition domain is shown in SEQ ID NO:54.
  • nucleotide sequence of the first recognition domain is shown as SEQ ID NO:12
  • nucleotide sequence of the second recognition domain is shown as SEQ ID NO:58.
  • nucleotide sequence of the first recognition domain is shown as SEQ ID NO: 13
  • nucleotide sequence of the second recognition domain is shown as SEQ ID NO: 48.
  • nucleotide sequence of the first recognition domain is shown as SEQ ID NO:13
  • nucleotide sequence of the second recognition domain is shown as SEQ ID NO:54.
  • nucleotide sequence of the first recognition domain is shown as SEQ ID NO: 13
  • nucleotide sequence of the second recognition domain is shown as SEQ ID NO: 58.
  • nucleotide sequence of the first recognition domain is shown as SEQ ID NO: 17
  • nucleotide sequence of the second recognition domain is shown as SEQ ID NO: 48.
  • nucleotide sequence of the first recognition domain is shown as SEQ ID NO: 17
  • nucleotide sequence of the second recognition domain is shown as SEQ ID NO: 54.
  • nucleotide sequence of the first recognition domain is shown as SEQ ID NO: 17
  • nucleotide sequence of the second recognition domain is shown as SEQ ID NO: 58.
  • nucleotide sequence of the first recognition domain is shown as SEQ ID NO:16
  • nucleotide sequence of the second recognition domain is shown as SEQ ID NO:42.
  • nucleotide sequence of the first recognition domain is shown as SEQ ID NO:18
  • nucleotide sequence of the second recognition domain is shown as SEQ ID NO:43.
  • nucleotide sequence of the first recognition domain is shown as SEQ ID NO:14
  • nucleotide sequence of the second recognition domain is shown as SEQ ID NO:55.
  • the mutant sequence refers to the substitution, addition or deletion of one or more nucleotides in the nucleotide sequence, preferably substitution.
  • the mutation is selected from natural pathogenic mutations and natural non-pathogenic mutations.
  • the natural pathogenic mutation is selected from one or more of c.2242C>T, c.2276G>T, c.2299delG, c.2522C>A, c.2541C>A, c.2761delC, c.2776C>T, c.2802T>G, c.2209C>T, c.2310delA, c.2391_2392deITG, c.2431A>T, c.2431_2432delAA, c.2440C>T, c.2525dup, c.2610C>A, c.2755C>T, c.2176T>C, c.2236C>G, c.2296T>C and c.2332G>T.
  • the natural pathogenic mutation is selected from one or more of c.2802T>G, c.2299delG and c.2276G>T; for example, the nucleotide mutation is c.2802T>G.
  • the Sm sequence is a consensus sequence
  • the stem-loop sequence may be a stem-loop sequence of U1, U2, U3, U4, U5, U6 or U7.
  • the stem-loop sequence is the stem-loop sequence of U7.
  • the stem-loop sequence is the stem-loop sequence of U1.
  • the Sm sequence is shown as SEQ ID NO:6.
  • the stem-loop sequence is shown in SEQ ID NO:7.
  • the snRNA nucleic acid molecule comprises modified nucleotides or their analogs monomers.
  • the modification is selected from: 2'-O-alkyl modification, 2'-O-methoxy modification and 2'-O-methoxyethyl modification.
  • the 2′-O alkyl modification is a 2′-O-methyl modification.
  • the nucleotide analog monomer is selected from 6'-modified bicyclic nucleosides, 5'-modified bicyclic nucleosides, 6'-disubstituted bicyclic nucleosides, tetrahydropyranoside analogs and 2'-deoxy 2'-fluoro- ⁇ -D-arabinonucleotides.
  • the nucleotides of the snRNA nucleic acid molecule are connected by chemical bonds, and the chemical bonds are selected from phosphate bonds, methylene bonds, amide bonds, methylphosphonate bonds and 3'-thioformal bonds.
  • the phosphate bond is selected from a phosphorothioate bond, a phosphorodithioate bond, an alkyl phosphonate bond, a phosphoroamidate bond, a boranophosphate bond, and a chiral linkage phosphorus.
  • the phosphate bond is a phosphorothioate bond.
  • the snRNA nucleic acid molecule comprises a modified nucleotide or its analog monomer at positions 1 to 80 from the 5′ end and/or the 3′ end.
  • the snRNA nucleic acid molecule comprises a modified nucleotide or its analog monomer at positions 3 to 40 from the 5' end and/or the 3' end.
  • the snRNA nucleic acid molecule comprises a modified nucleotide or its analog monomer at position 6 to 10 from the 5' end and/or the 3' end.
  • the snRNA nucleic acid molecule comprises a modified nucleotide or its analog monomer at positions 20 to 27 from the 5' end and/or the 3' end.
  • the snRNA nucleic acid molecule comprises at least one phosphate bond starting from the 5′ end or the 3′ end.
  • the snRNA nucleic acid molecule comprises one or more modifications as described above.
  • the snRNA nucleic acid molecule when chemically synthesized, all nucleotides are interconnected by phosphorothioate bonds and are 2′-O-methoxy modified. In some embodiments, only 3 nucleotides on both sides of the snRNA are connected by phosphorothioate bonds and are 2′-O-methoxy modified. In some chemically synthesized and modified U7 snRNA embodiments, there may be 0-5 mismatched nucleotides in the reverse complementary pairing between the recognition domain and the target site, preferably 0-1. In some embodiments, 3-40 bases on both sides of the chemically synthesized snRNA are modified and connected by phosphate bonds.
  • the snRNA nucleic acid molecule comprises 1 to 3 phosphate bonds starting from the 5′ end.
  • the snRNA nucleic acid molecule comprises 1 to 3 phosphate bonds starting from the 3′ end.
  • the snRNA nucleic acid molecule further comprises a unidirectional extension sequence or a bidirectional extension sequence at the nucleotides at the 5′ end and/or the 3′ end of the recognition domain.
  • the unidirectional extension sequence is an RNA sequence added to the 5' end or 3' end of the targeting sequence of the snRNA nucleic acid molecule;
  • the bidirectional extension sequence is an RNA sequence added to the 5' end and 3' end of the targeting sequence of the snRNA nucleic acid molecule, respectively.
  • the snRNA nucleic acid molecule further comprises a free tail sequence, wherein the tail sequence comprises a motif of a splicing regulatory protein and can bind to the splicing regulatory protein.
  • the splicing regulatory protein is selected from hnRNP A1 (Heterogeneous Nuclear Ribonucleoprotein A1), SRSF1 (Serine And Arginine Rich Splicing Factor 1), RBM4 (RNA Binding Motif Protein 4), DAZAP1 (DAZ Associated Protein 1) and SR (Serine And Arginine-Rich Protein).
  • the tail sequence is as shown in SEQ ID NO:35.
  • the second aspect of the present invention provides a combination of snRNA nucleic acid molecules, wherein the combination comprises one or more snRNA nucleic acid molecules as described in the first aspect.
  • At least two recognition domains are located on the same or different snRNA nucleic acid molecules.
  • the third aspect of the present invention provides a DNA molecule, wherein the DNA molecule encodes the snRNA nucleic acid molecule as described in the first aspect or the combination as described in the first aspect.
  • the fourth aspect of the present invention provides a gene expression cassette, which comprises a promoter and the DNA molecule according to the third aspect.
  • the promoter is U7 promoter.
  • the promoter is a mouse-derived U7 promoter.
  • the 3′ end of the gene expression cassette comprises a tailing sequence, and the tailing sequence is involved in the processing of the snRNA.
  • the length of the tailing sequence is 28 to 131 bp, for example, 106 bp.
  • the tailing sequence is the gene sequence after the 3′ end of the U7 snRNA gene, for example, as shown in SEQ ID NO:8.
  • the gene expression cassette includes a recognition domain and a backbone sequence; the backbone sequence is shown in SEQ ID NO:62.
  • the fifth aspect of the present invention is a recombinant expression vector, which comprises the snRNA nucleic acid molecule as described in the first aspect, the combination as described in the second aspect, or the gene expression cassette as described in the third aspect.
  • the expression vector of the recombinant expression vector is selected from the group consisting of plasmid, bacteriophage, minicircle DNA, linear DNA and virus.
  • the expression vector is a lentivirus or an adeno-associated virus.
  • the capsid protein of the adeno-associated virus is a capsid protein of natural origin or a mutant thereof, and the plasmid of the adeno-associated virus is single-stranded (ssAAV, single-stranded AAV) or double-stranded (scAAV, Self-complementary AAV) complementary to the single-stranded.
  • the AAV capsid protein of natural origin may be derived from an animal or a plant.
  • the AAV capsid protein derived from an animal may be derived from a human (such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9, etc.), or may be derived from a non-human primate (such as AAVrh.8, AAVrh.10 and AAVrh.43), or may be derived from vertebrates such as mice and pigs, or may be derived from insects.
  • AAV serotypes that are tropistic to retinal tissues of the eye are preferred, such as AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10 or AAV2.7m8.
  • the AAV ITR serotype should be consistent with the Rep gene serotype, but may be inconsistent with the Cap gene serotype.
  • the AAV can be a single-stranded AAV (ssAAV) or a double-stranded AAV (scAAV) formed by self-complementarity.
  • the capsid protein of natural origin is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10 and AAVrh.43;
  • the mutant is selected from AAV2.5, AAV2i8, AAV-TT, AAV9.HR and CAM130.
  • the sixth aspect of the present invention provides a virus particle, comprising a capsid protein and a nucleic acid, wherein the nucleic acid comprises the snRNA nucleic acid molecule as described in the first aspect, the combination as described in the second aspect, or the DNA molecule as described in the third aspect.
  • the capsid protein is a capsid protein from an adeno-associated virus.
  • the capsid protein from adeno-associated virus is as defined in the fifth aspect.
  • the seventh aspect of the present invention provides a pharmaceutical composition, which comprises the snRNA nucleic acid molecule as described in the first aspect, the combination as described in the second aspect, the DNA molecule as described in the third aspect, the gene expression cassette as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, or the viral particle as described in the sixth aspect.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the eighth aspect of the present invention provides a method for inducing the production of Usherin protein lacking exon 13, the method comprising introducing into a host cell the snRNA nucleic acid molecule as described in the first aspect, the combination as described in the second aspect, the DNA molecule as described in the third aspect, the gene expression cassette as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, the viral particle as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect, so that splicing skipping of exon 13 occurs.
  • the host cell is selected from retinal tissue cells, inner ear cells, cells with the potential to differentiate into retinal tissue cells and/or inner ear cells, and cells that can perform functions corresponding to retinal tissue cells and/or inner ear cells.
  • the retinal tissue cells are retinal photoreceptor cells
  • the inner ear cells are inner ear hair cells.
  • the stem cells are selected from induced pluripotent stem cells and embryonic stem cells.
  • the potential cells are selected from induced pluripotent stem cells, embryonic stem cells, neural precursor cells, retinal progenitor cells, retinal progenitor cells and mesenchymal stromal cells.
  • the ninth aspect of the present invention provides a method for inhibiting the expression and/or function of USH2A pre-mRNA exon 13, the method comprising administering the snRNA nucleic acid molecule as described in the first aspect, the combination as described in the second aspect, the DNA molecule as described in the third aspect, the gene expression cassette as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, the viral particle as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect.
  • the tenth aspect of the present invention provides a method for inducing splicing skipping of USH2A pre-mRNA exon 13, the method comprising administering the snRNA nucleic acid molecule as described in the first aspect, the combination as described in the second aspect, the DNA molecule as described in the third aspect, the gene expression cassette as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, the viral particle as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect.
  • the eleventh aspect of the present invention provides a method for reducing abnormal Usherin protein expression, the method comprising introducing into a host cell the snRNA nucleic acid molecule as described in the first aspect, the combination as described in the second aspect, the DNA molecule as described in the third aspect, the gene expression cassette as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, the viral particle as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect.
  • the host cell is as defined in the eighth aspect.
  • the twelfth aspect of the present invention provides a method for preparing the snRNA nucleic acid molecule as described in the first aspect or the combination as described in the second aspect, the method comprising the steps of biosynthesis or chemical synthesis of the snRNA nucleic acid molecule as described in the first aspect or the combination as described in the second aspect.
  • the thirteenth aspect of the present invention provides a use of the snRNA nucleic acid molecule as described in the first aspect, the combination as described in the second aspect, the DNA molecule as described in the third aspect, the gene expression cassette as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, the viral particle as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect in the preparation of a drug for treating diseases associated with USH2A exon 13 mutations.
  • the USH2A exon 13 mutation is a pathogenic mutation or a non-pathogenic mutation.
  • the disease is selected from eye diseases and ear diseases.
  • the methods described in the eighth, ninth, tenth and eleventh aspects are for non-therapeutic purposes, such as laboratory research and kit development for drug development.
  • the fourteenth aspect of the present invention provides a method for treating a disease associated with USH2A exon 13 mutation, the method comprising administering to a patient in need thereof an effective amount of the snRNA nucleic acid molecule as described in the first aspect, the combination as described in the second aspect, the DNA molecule as described in the third aspect, the gene expression cassette as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, the viral particle as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect.
  • the disease associated with USH2A exon 13 mutation is as described in the thirteenth aspect.
  • the reagents and raw materials used in the present invention are commercially available.
  • the snRNA nucleic acid molecule provided by the present invention targets USH2A exon 13 and target regions on both sides thereof.
  • the snRNA nucleic acid molecule comprises a recognition domain, a stem-loop sequence and an Sm sequence.
  • the number of the recognition domains is at least two, and at least two recognition domains form a "tandem" structure in the snRNA.
  • the U7-snRNA tandem at different target sites can efficiently induce splicing jumps of USH2A exon 13, and the frequency of single jumps is higher; especially for pathogenic or non-pathogenic USH2A exon 13, the snRNA of the present invention can safely and effectively perform single jumps of USH2A exon 13 at a low dose, and has important clinical value in preventing and/or treating eye diseases and ear diseases related to abnormal expression of Usherin protein.
  • FIG1 is a schematic diagram showing the structure and effect of U7-snRNA targeting exon 13 of USH2A using a single recognition domain as an example.
  • FIG. 2 is a schematic diagram showing the location of U7-snRNA targeting target region 8 on the genome.
  • Figures 3A-3B show the splicing skipping effect of USH2A pre-mRNA exon 13 induced by U7 snRNA at different target sites in reporter gene cells.
  • Figure 4 is a schematic diagram of the efficiency of snRNA with different targets inducing separate splicing skipping of USH2A pre-mRNA exon 13.
  • FIG5 is a schematic diagram showing the location of U7-snRNA targeting target region 1 on the genome.
  • Figure 6 shows the results of the proportion of cells in which U7-snRNA targeting target region 1 induced splicing skipping of USH2A pre-mRNA exon 13 in reporter vector cells.
  • Figure 7 is a bar graph of the average FITC intensity of GFP-positive cells induced by U7-snRNA targeting target region 1 (USH2A pre-mRNA exon 13 splicing skipping).
  • FIG8 is a schematic diagram showing the location of U7-snRNA targeting target region 2 on the genome.
  • Figure 9 shows the results of the proportion of cells in which U7-snRNA targeting target region 2 induced splicing skipping of USH2A pre-mRNA exon 13 in reporter vector cells.
  • Figure 10 is a bar graph of the average FITC intensity of GFP-positive cells induced by U7-snRNA targeting target region 2 (USH2A pre-mRNA exon 13 splicing skipping).
  • FIG. 11 is a schematic diagram showing the location of U7-snRNA targeting target region 3 on the genome.
  • Figure 12 shows the results of the proportion of cells in which U7-snRNA targeting target region 3 induced splicing skipping of USH2A pre-mRNA exon 13 in reporter vector cells.
  • FIG. 13 shows (USH2A pre-mRNA exon 13 splicing skipping) induced by U7-snRNA targeting target region 3 Histogram of mean FITC intensity of GFP-positive cells.
  • FIG. 14 is a schematic diagram showing the location of U7-snRNA targeting target region 4 on the genome.
  • Figure 15 shows the results of the proportion of cells in which U7-snRNA targeting target region 4 induced splicing skipping of USH2A pre-mRNA exon 13 in reporter vector cells.
  • FIG. 16 is a schematic diagram showing the location of U7-snRNA targeting target region 5 on the genome.
  • Figure 17 shows the results of the proportion of cells in which U7-snRNA targeting target region 5 induced splicing skipping of USH2A pre-mRNA exon 13 in reporter vector cells.
  • FIG. 18 is a schematic diagram showing the location of U7-snRNA targeting target region 6 on the genome.
  • Figure 19 shows the results of the proportion of cells in which U7-snRNA targeting target region 6 induced splicing skipping of USH2A pre-mRNA exon 13 in reporter vector cells.
  • FIG. 20 is a schematic diagram showing the location of U7-snRNA targeting target region 7 on the genome.
  • Figure 21 shows the results of the proportion of cells in which U7-snRNA targeting target region 7 induced splicing skipping of USH2A pre-mRNA exon 13 in reporter vector cells.
  • Figure 22 shows the average FITC intensity results of U7 snRNA targeting different regions inducing USH2A pre-mRNA exon 13 splicing skipping cells in reporter gene cells.
  • Figure 23 is a schematic diagram of the structure of the tandem U7 snRNA of the present invention.
  • Figure 24A is a schematic diagram 1 of the tandem U7 snRNA and USH2A pre-mRNA targeting method of the present invention
  • Figure 24B is a schematic diagram 2 of the tandem U7 snRNA and USH2A pre-mRNA targeting method of the present invention.
  • Figure 25 shows the splicing skipping efficiency of USH2A pre-mRNA exon 13 induced by pUC57-U7-snRNA with tandem recognition domains in Example 8-1.
  • Figure 26 shows the splicing skipping efficiency of USH2A pre-mRNA exon 13 induced by pUC57-U7-snRNA with tandem recognition domains in Example 8-2.
  • Figure 27 shows the efficiency of chemically synthesized U7 snRNA in inducing USH2A pre-mRNA exon 13 splicing skipping in WERI cells;
  • lane 1 50pmol chemically synthesized and modified U7-snRNA#30+#4
  • lane 2 50pmol chemically synthesized and modified U7-snRNA#26+#15
  • lane 3 50pmol AON1
  • lane 4 50pmol AON2
  • lane 5 EGFP
  • lane 6 GL DNA Marker 2000.
  • FIG28 is a bar graph showing quantitative analysis of RT-PCR electrophoresis bands in Example 9;
  • ⁇ E12-E13 indicates USH2A mRNA that splices skip exon 12 and exon 13 simultaneously
  • total ⁇ indicates the sum of USH2A mRNA that splices skip exon 13 or splices skip exon 12 and exon 13 simultaneously.
  • Figure 29A shows a schematic diagram of the structure of the tandem U7 snRNA.
  • FIG. 29B compares the induction of USH2A pre-mRNA exon 13 by U7-hnRNP A1-snRNA and U7-snRNA tandem Splice jumping efficiency.
  • Figure 30 shows that 3 ⁇ U7 snRNA tandem induced USH2A exon 13 splicing as tested by in vitro gradient climbing experiment.
  • Figure 31 shows that the in vitro dose-escalation experiment verified that the splicing skipping effect induced by AAV-U7 snRNA tandem is significantly better than that of AON.
  • Figure 32 shows that snRNA induces splicing skipping of USH2A pre-mRNA exon 13 in humanized mouse retinal cells better than AON.
  • Figure 33 shows that injection of AAV-U7 snRNA of different serotypes induces splicing skipping of USH2A pre-mRNA exon 13 in rabbit eye cells better than AON.
  • Figure 34 shows that U7 snRNA delivered by AAV can still maintain the induction of USH2A pre-mRNA exon 13 splicing skipping after 22 weeks.
  • Figure 35 shows the long-term maintenance ability of the induced splicing skipping efficiency of AAV-1 ⁇ U7 snRNA tandem and AAV-4 ⁇ U7 snRNA combination.
  • the wild-type U7 snRNA includes a stem-loop structure (scafford), a U7-specific Sm sequence (AAUUUGUCUAG, SEQ ID NO: 5) and a recognition domain (complementary to replication-dependent histone pre-mRNA).
  • the U7 snRNA of the present application can be based on the gene sequence of the mouse wild-type U7 snRNA on NCBI (NCBI Reference Sequence: NR_024201.3), wherein the U7-specific Sm binding site is replaced with an optimized common Sm sequence, namely SmOPT (AAUUUUUGGAG, SEQ ID NO: 6), the original recognition domain at the 5′ end of the SmOPT sequence is replaced with a recognition domain that is reverse complementary to the specific target site of USH2A pre-mRNA, and the 3′ end of the SmOPT sequence retains the U7 original stem-loop structure sequence (CAGGUUUUCUGACUUCGG UCGGAAAACCCCU, SEQ ID NO: 7).
  • NCBI NCBI Reference Sequence: NR_024201.3
  • the non-tandem U7 snRNA recognition domain sequence targeting USH2A pre-mRNA exon 13 is reversely complementary paired with a target sequence selected from USH2A pre-mRNA intron 12-exon 13-intron 13, and the target sequence can be selected from the 3′ sequence target region of USH2A pre-mRNA exon 13.
  • U7-snRNA gene expression cassette skeleton (5′-mouse U7 promoter-smOPT sequence-U7 snRNA scafford-snRNA gene specific 3′ cassette-3′) was synthesized by whole gene synthesis.
  • Two Type II restriction endonuclease recognition sites (such as BsaI, AarI, BsmBI, etc.) were added between the U7 promoter and smOPT to facilitate subsequent excision, replacement, and insertion of other recognition domain sequences.
  • the snRNA gene specific 3′ cassette is the sequence "GTCTACAATGAAA (SEQ ID NO: 8)" after the 3′ end of the U7 snRNA gene in the mouse genome (GenBank: X54748.1), which is involved in the processing of pre-snRNA.
  • SEQ ID NO: 1 The 3′ region sequence of USH2A pre-mRNA exon 13 (SEQ ID NO: 1) in this application corresponds to the human genome location Chr1: 216246563-216246753 (corresponding to the NCBI database GRch38 version), and the sequence of SEQ ID NO: 1 is as follows:
  • pre-mRNA exon 13 and the adjacent target regions on both sides can be target regions containing natural pathogenic/non-pathogenic mutations in addition to the non-mutation sequences listed above.
  • the mutation sites include at least one of the following mutation sites: c.2242C>T, c.2276G>T, c.2299delG, c.2522C>A, c.2541C> A, c.2761delC, c.2776C>T, c.2802T>G, c.2209C>T, c.2310delA, c.2391_2392deITG, c.2431A>T, c.2431_2432delAA, c.2440C>T, c.2525dup, c.2610C>A, c.2755C>T, c.2176T>C, c.2236C>G, c.2296T>C, c.2332G>T.
  • sequence of the 5′ target region of USH2A pre-mRNA exon 13 (the pre-mRNA region corresponding to chr1:216247130-216247246) (SEQ ID NO:2) is as follows:
  • sequence of the 3′ target region of USH2A pre-mRNA exon 13 (the pre-mRNA region corresponding to chr1:216246563-216246753) (SEQ ID NO:3) is as follows:
  • the preferred target region of the 3′ segment of USH2A pre-mRNA exon 13 (the pre-mRNA region corresponding to chr1:216246563-216246649) (SEQ ID NO:4) is as follows:
  • the sequence of the 3′ region of USH2A pre-mRNA exon 13 (the pre-mRNA region corresponding to Chr1: 216246563-216246626) (region 8, SEQ ID NO: 9) is: UGCCUAAUCGUCAAGGAAGAAGGUGUAAUCAGUGTCAACCAGGUAAGAAAGAAAUGUAUUACAU, or it can be the 3′ region sequence of USH2A pre-mRNA exon 13 containing a natural mutation, such as UGCCUAAUCGUCAAGGAAGAAGGUGUAAUCAGUGGCAACCAGGUAAGAAAGAAAUGUAUUACAU (the underlined one is the natural pathogenic mutation c.2802T>G, SEQ ID NO: 10).
  • the corresponding Oligo DNA is synthesized.
  • the sense strand of the Oligo DNA is the DNA sequence corresponding to the recognition domain sequence, and CCGCA is added to the 5′, and the antisense strand is the antisense complementary sequence of the recognition domain sequence, AATT is added to the 5′ and T is added to the 3′.
  • the recognition domain sequence is 5′-NNN-3′
  • the synthesized sense strand of the Oligo DNA is 5′-CCGCANNN-3′
  • the antisense strand is 5′-AATTNNNT-3′.
  • the synthesized Oligo DNA sense and antisense chains were mixed according to the annealing reaction system (total reaction volume 20 ⁇ l: Oligo-F (100 ⁇ M) 2 ⁇ l + Oligo-R (100 ⁇ M) 2 ⁇ l + 10 ⁇ NEB Cutter smart buffer 2 ⁇ l + deionized water 16 ⁇ l), incubated at 95°C for 5 minutes, and then placed on ice to cool and anneal to form double-stranded DNA with sticky ends. After diluting 100 times, 1 ⁇ l was taken and connected with 10ng of the linearized pUC57-U7 snRNA backbone plasmid recovered by BsaI enzyme digestion for T4 ligase ligation.
  • the ligation product was further transformed into Escherichia coli competent cells, single clones were selected, PCR and sequencing were verified to obtain the U7 snRNA vector for inducing USH2A exon 13 splicing skipping.
  • the plasmid was purified and stored at -20°C for use.
  • snRNA#24, snRNA#25, snRNA#27 and snRNA#29 are homologous to humans and monkeys.
  • U7 snRNA can also be produced by direct chemical synthesis to produce RNA containing a guide sequence, smOPT, and U7 snRNA scafford.
  • U7 snRNA synthesized in vitro can be specifically modified to make it resistant to nuclease degradation or to increase its affinity for the target sequence.
  • U7 snRNA was chemically synthesized, and the three bases at the 5' and 3' ends were each modified with 2' methoxy (2'-OME) and thiolation to increase nuclease resistance.
  • 2' methoxy (2'-OME) and thiolation to increase nuclease resistance.
  • snRNA#25 and snRNA#26 as examples, the chemically synthesized snRNA sequences and modifications are as follows (* indicates a thiophosphorylated backbone, m indicates a 2'-methoxy modification, the underline indicates a recognition domain that is reverse complementary to the target sequence, and italics indicate smOPT sequences):
  • the chemically synthesized and modified U7-snRNA#25 bidirectional extension sequence is as follows:
  • the chemically synthesized and modified U7-snRNA#26 unidirectional extension sequence is as follows:
  • Example 2 Construction of a reporter vector for quantitative evaluation of USH2A exon 13 splicing skipping efficiency
  • the RGleft-USH2A Exon13mut-RGright sequence (with AgeI and EcoRI restriction sites added to the 5′ and 3′ ends, respectively) was obtained by total gene synthesis.
  • the synthetic sequence and pX601 plasmid (Addgene, 61591) were digested with restriction endonucleases AgeI and EcoRI, electrophoresed, gel-cut and ligated.
  • the synthesized sequence was inserted between the AgeI and EcoRI restriction sites of the pX601 vector to replace the SaCas9 gene sequence of the original vector and obtain the reporter vector.
  • the purified reporter vector plasmid was further obtained by transforming E. coli competent cells, picking single clones, PCR and sequencing verification, and stored at -20°C for future use.
  • the reporter vector structure is: pCMV-RGleft-USH2A EXON13mut-RGright, RG represents a reporter gene, RGleft represents the first half of the 5′ end of the reporter gene without a reporter function, and RGright represents the second half of the 3′ end of the reporter gene without a reporter function.
  • the tandem expression of RGleft and RGright can normally perform the complete reporter gene function.
  • the reporter gene is the green fluorescent gene EGFP
  • the vector structure is pCMV-EGFPleft-Exon13mut-EGFPright.
  • Exon13mut represents USH2A exon 13 containing a pathogenic mutation, and its upstream and downstream intron sequences (the upstream intron sequence is a gene sequence composed of 204bp at the 5′ end and 490bp at the 3′ end of intron 12 of the human USH2A gene; the downstream intron sequence is a gene sequence composed of 703bp at the 5′ end and 216bp at the 3′ end of intron 13 of the human USH2A gene).
  • the pathogenic mutation of USH2A exon 13 described in the embodiments of the present application may be c.2299delG or c.2802T>G or any mutation.
  • the obtained vector structures are pCMV-EGFPleft-Exon13c.2299delG-EGFPright and pCMV-EGFPleft-Exon13c.2802T>G-EGFPright.
  • the mutations in some embodiments may also be or include c.2276G>T, C.2522C>A, c.2242C>T, c.2541C>A, c.2761delC and C.2776C>T, etc.
  • RGleft for example, the sequence of EGFPleft is:
  • RGright for example, the sequence of EGFPright is:
  • Example 3 Effects of U7-snRNA-mediated USH2A exon 13 splicing skipping at different target sites in the 3′ region (region 8) of USH2A pre-mRNA exon 13
  • 293T cells were seeded into 24-well plates in a certain amount so that the cell confluence reached about 80% after 24 hours.
  • Lipofectamine2000 was used to co-transfect 293T cells with pCMV-EGFPleft-Exon13mut-EGFPright and pUC57-U7 snRNA plasmid targeting USH2A pre-mRNA (the vector mass ratio was 100ng:400ng).
  • 293T cells transfected with reporter plasmid alone (Reporter, reporter group) and co-transfected with reporter plasmid and pUC57-U7Scramble (SC group) were used as two negative controls, and 293T cells without any plasmid were used as blank controls.
  • the transfected cells were cultured for 48-72 hours, digested into single cells with trypsin, and then the GFP positive rate of different U7 snRNA groups (i.e., the proportion of cells in which USH2A exon 13 was induced to splice jump) was detected by flow cytometry.
  • This example detected the average FITC intensity of different experimental groups (as shown in FIG. 2 ), that is, the average FITC fluorescence intensity of GFP-positive cells, as well as the GFP-positive rate, the GFP protein expression level of positive cells.
  • Example 4 Chemically synthesized snRNA efficiently induces USH2A pre-mRNA exon 13 single splicing skipping
  • Human host cells were inoculated into 24-well plates at 6 ⁇ 10 5 /well.
  • the human retinal neural cells selected in this example were WERI-Rb-1 cells (retinal neural cell line).
  • 100 pmol of U7-snRNA #24, #25, #26, #27, #28, #29, #30, #33, and #34 synthesized in vitro were transfected into WERI cells using Lipofectamine 2000.
  • the transfected cells were cultured for 72 hours, and then the RNA of each experimental group of cells was extracted and reverse transcribed to obtain cDNA.
  • 21 target sites are set for the 7 target regions of USH2A pre-mRNA.
  • the 7 target regions of USH2A Pre-mRNA are shown below.
  • Exon 13 region 1 (SEQ ID NO:32) (Chr1:216247142-216247185): CGAAGCUUUAAUGAUGUUGGAUGUGAGCCCUGCCAGUGUAACCU;
  • Exon 13 region 2 (SEQ ID NO:33) (Chr1:216247130-216247161): GAGCCCUGCCAGUGUAACCUCCAUGGCUCAGU;
  • Exon 13 region 3 (SEQ ID NO: 34) (Chr1: 216246616-216246649): AAUCAGUGGCCAGUGCCUGUGUGUGCCUAAUCGU;
  • Exon 13 region 4 (SEQ ID NO:36) (Chr1:216247210-216247246): UAAAUAUAUUUUAUCUUUAGGGCUUAGGUGUGAUCAU;
  • Exon 13 region 5 (SEQ ID NO:37) (Chr1:216247204-216247232): CUUUAGGGCUUAGGUGUGAUCAUUGCAAU;
  • Exon 13 region 6 (SEQ ID NO:38) (Chr1:216247187-216247220): GGUGUGAUCAUUGCAAUUUUGGAUUUAAAUUUCU;
  • Exon 13 region 7 (SEQ ID NO:39) (Chr1:216247169-216247202): UUGGAUUUAAAUUUCUCCGAAGCUUUAAUGAUGU.
  • the 21 target sites are shown in the following table.
  • snRNA#9, snRNA#15, snRNA#17 and snRNA#19 are homologous to humans and monkeys.
  • Example 6 Detection of the effect of USH2A exon 13 splicing skipping mediated by U7-snRNA targeting different regions
  • the GFP positivity and average FITC intensity of the U7 snRNA group targeting different regions were detected in the reporter cell system.
  • U7-snRNA targeting target region 1 The position of U7-snRNA targeting target region 1 on the genome (the picture from left to right corresponds to the genome from 5' to 3') is shown in Figure 5.
  • the experimental results of U7-snRNA targeting target region 1 are shown in Figures 6 to 7 and Table 4 below. All U7-snRNA targeting target region 1 can induce splicing jump of USH2A exon 13 in reporter gene cells.
  • the AON targeting region 1 in the prior art cannot induce splicing jump of exon 13, but the snRNA targeting region 1 can efficiently induce splicing jump of exon 13.
  • Table 4 The proportion of cells in which U7-snRNA targeting target region 1 induced USH2A pre-mRNA exon 13 splicing skipping in reporter gene cells
  • U7-snRNA targeting target region 2 The position of U7-snRNA targeting target region 2 on the genome (the picture from left to right corresponds to the genome from 5' to 3') is shown in Figure 8.
  • the experimental results of U7-snRNA targeting target region 2 are shown in Figures 9 to 10 and Table 5 below. All U7-snRNA targeting target region 2 can induce splicing jump of USH2A exon 13 in reporter gene cells.
  • the AON targeting region 2 in the prior art has a low effect on inducing splicing jump of exon 13, but the snRNA targeting region 2 can efficiently induce splicing jump of exon 13.
  • Table 5 The proportion of cells in which U7-snRNA targeting region 2 induced USH2A pre-mRNA exon 13 splicing skipping in reporter gene cells
  • U7-snRNA targeting target region 3 The position of U7-snRNA targeting target region 3 on the genome (the picture from left to right corresponds to the genome from 5' to 3') is shown in Figure 11.
  • the experimental results of U7-snRNA targeting target region 3 are shown in Figures 12 to 13 and Table 6 below. All U7-snRNA targeting target region 3 can induce splicing jump of USH2A exon 13 in reporter gene cells.
  • the AON targeting region 3 in the prior art has a low effect on inducing splicing jump of exon 13, but the snRNA targeting region 3 can effectively induce splicing jump of exon 13.
  • target regions 1, 2, and 3 were non-sensitive regions for AON targeting, i.e., targeting these regions was unable/low-efficiency to induce splicing skipping of exon 13, snRNA targeting these regions could significantly induce splicing skipping of exon 13. Therefore, although both snRNA and AON can induce splicing skipping, their mechanisms of action are different, and the sensitivity of target sites (target regions and target sites to which they are applicable) is also different.
  • U7-snRNA targeting region 4 The location of U7-snRNA targeting region 4 on the genome (the picture from left to right corresponds to the genome from 5' to 3') is shown in Figure 14.
  • the experimental results of U7-snRNA targeting region 4 are shown in Figure 15 and Table 8 below. All U7-snRNAs targeting target region 4 can induce splicing skipping of USH2A exon 13 in reporter gene cells.
  • Table 8 The proportion of cells in which U7-snRNA targeting region 4 induces USH2A pre-mRNA exon 13 splicing skipping in reporter gene cells
  • U7-snRNA targeting target region 5 The location of U7-snRNA targeting target region 5 on the genome (the picture from left to right corresponds to the genome from 5' to 3') is shown in Figure 16.
  • the experimental results of U7-snRNA targeting target region 5 are shown in Figure 17 and Table 9 below. All U7-snRNA targeting target region 5 can induce splicing jump of USH2A exon 13 in reporter gene cells.
  • U7-snRNA targeting target region 6 The location of U7-snRNA targeting target region 6 on the genome (the picture from left to right corresponds to the genome from 5' to 3') is shown in Figure 18.
  • the experimental results of U7-snRNA targeting target region 6 are shown in Figure 19 and Table 10 below. All U7-snRNA targeting target region 6 can induce splicing jump of USH2A exon 13 in reporter gene cells.
  • U7-snRNA targeting target region 7 The location of U7-snRNA targeting target region 7 on the genome (the picture from left to right corresponds to the genome from 5' to 3') is shown in Figure 20.
  • the experimental results of U7-snRNA targeting target region 7 are shown in Figure 21 and Table 11 below. All U7-snRNA targeting target region 7 can induce splicing jump of USH2A exon 13 in reporter gene cells.
  • Table 11 The proportion of cells in which U7-snRNA targeting region 7 induces USH2A pre-mRNA exon 13 splicing skipping in reporter gene cells
  • the average FITC intensity of GFP-positive cells induced by U7-snRNA in different regions is shown in Figure 22 and the following Table 12. Although the GFP% (proportion of cells induced to splice jump) targeting the same region is close, different target snRNAs targeting the same region induce different levels of splice jump mRNA and its protein (average FITC intensity) in the same cell.
  • Targeting region 2 not only obtained a higher proportion of cells induced to undergo splicing jumps (GFP%), but also induced higher levels of mRNA and protein (average FITC intensity) of splicing jumps in the same cells.
  • the target site #2 and its adjacent sites #1 and #3 (region 4) with higher efficiency of inducing splicing jumps by AON in the prior art have lower levels of mRNA that induce splicing jumps in the same cell in the snRNA system.
  • the efficiency of AON targeting region 7 in the prior art is higher than that of region 5, but the efficiency of targeting region 5 in the snRNA system is higher than that of region 7.
  • the efficiency of AON targeting region 3 in the prior art is higher than that of region 2, but the efficiency of targeting region 2 in the snRNA system is higher than that of region 3. Therefore, although both snRNA and AON can induce splicing jumps, their mechanisms of action are different and the sensitivity of target sites is also different.
  • Table 12 Average FITC intensity of U7-snRNA targeting different regions inducing USH2A pre-mRNA exon 13 splicing skipping cells in reporter gene cells
  • the U7-snRNA targeted to induce splicing skipping of USH2A pre-mRNA exon 13 in the present application is not limited to the U7-snRNA listed in Examples 3 and 4.
  • the target site recognized by the recognition domain of the U7-snRNA in the present application is selected from USH2A pre-mRNA intron 12-exon 13-intron 13, preferably selected from exon 13 and the adjacent target regions on both sides (SEQ ID NO: 1).
  • Example 7 USH2A exon 13 splicing skipping mediated by U7-snRNA combinations at different target sites
  • the Golden Gate Assembly product was further transformed into E. coli competent cells, single clones were selected, PCR and sequencing were performed to obtain the U7 snRNA multi-target combination vector for inducing splicing skipping of USH2A exon 13.
  • the plasmid was purified and stored at -20°C for future use.
  • Example 8-1 Recognition domain tandem U7 snRNA induces USH2A exon 13 splicing skipping in reporter gene cells
  • the U7 snRNA with tandem recognition domains means a U7 snRNA stem-loop structure and a smOPT sequence, which are connected to two or more recognition domains, and its structure is 5′-recognition domain B-recognition domain A-smOPT sequence-stem-loop structure-3′, as shown in Figure 23.
  • the recognition domains A and B of the tandem U7 snRNA recognize RNA sequences at different target sites.
  • the corresponding oligo DNAs were synthesized respectively.
  • the positive chain of the oligo DNA is the DNA corresponding to the recognition domain sequence, and CCGCA is added to the 5′, and the antisense chain is the antisense complementary sequence of the recognition domain sequence, AATT is added to the 5′ and T is added to the 3′.
  • the recognition domain sequences of snRNA#15 and snRNA#25 to be concatenated are ACACUGGCAGGGCUCACAUCCA (SEQ ID NO: 54) and AUUACACCUUCUUCCUUGACGAUU (SEQ ID NO: 13), respectively, and the synthesized positive chain of the oligo DNA is:
  • the antisense strand is:
  • the synthesized Oligo DNA sense and antisense strands were mixed according to the annealing reaction system (total reaction volume 20 ⁇ l: Oligo-F (100 ⁇ M) 2 ⁇ l + Oligo-R (100 ⁇ M) 2 ⁇ l + 10 ⁇ NEB Cutter smart buffer 2 ⁇ l + deionized water 16 ⁇ l), incubated at 95°C for 5 minutes, and then placed on ice to cool and anneal to form double-stranded DNA with sticky ends. After diluting 100 times, 1 ⁇ l was taken and ligated with 10ng BsaI-digested and recovered linearized pUC57-U7 snRNA backbone plasmid by T4 ligase.
  • the ligation product was further transformed into Escherichia coli competent cells, single clones were selected, PCR and sequencing were verified to obtain the U7 snRNA vector for inducing USH2A exon 13 splicing skipping.
  • the plasmid was purified and stored at -20°C for use.
  • the constructed vectors were named pUC57-U7 snRNA#B-#A, where A and B represent the recognition domain numbers, corresponding to the recognition domain lists and sequences in Tables 1 and 3.
  • pUC57-snRNA#25-#15 For example, pUC57-snRNA#25-#15, pUC57-snRNA#24-#9, pUC57-snRNA#24-#19, pUC57-snRNA#25-#9, pUC57-snRNA#25-#19, pUC57-snRNA#29-#9, and pUC57-snRNA#25-#15.
  • the recognition domain tandem U7 snRNA can also be chemically synthesized and modified according to the method described in Example 2.
  • its specific sequence and modification are as follows (* represents the phosphorothioate backbone, m represents 2′-methoxy modification, underline represents the recognition domain that is reverse complementary to the target sequence, and italics represent the smOPT sequence):
  • the total length of the chemically synthesized snRNA sequence is preferably greater than or equal to 96 bp.
  • 293T cells were seeded into 24-well plates in a certain amount so that the cell confluence reached about 80% after 24 hours.
  • Lipofectamine2000 was used to co-transfect 293T cells with pCMV-EGFPleft-Exon13mut-EGFPright and U7 snRNA plasmids expressing dual recognition domains or U7 snRNA plasmids expressing single recognition domains (the vector mass ratio was 100ng:400ng), and 293T cells transfected with the reporter plasmid alone (Report, report group), co-transfected with the reporter plasmid and pUC57-U7Scramble (SC group) were used as two negative controls, and 293T cells without any plasmid transfection were used as blank controls.
  • transfected cells were cultured for 48-72 hours, digested into single cells with trypsin, and then the GFP positive rate of different snRNA groups was detected by flow cytometry.
  • Table 13 and Figure 25 below show the efficiency of U7-snRNA with different target sites in inducing splicing skipping of USH2A pre-mRNA exon 13.
  • This embodiment uses U7-snRNAs that target different target sites in series, and finds that the efficiency of inducing splicing jumps of USH2A exon 13 can be improved in reporter gene cells.
  • the splicing jump efficiency induced by the series connection of different targets in this embodiment is higher than the effect of a single target, and is superior to the known splicing jump technology of USH2A exon 13.
  • the present invention constructs AONs with different target sites in series, and attempts to induce splicing jumps of USH2A pre-mRNA exon 13, but finds that there is almost no splicing jump efficiency, which further verifies that the mechanism of action of AON and snRNA inducing splicing jumps is different.
  • RNA sequences that recognize different target sites are concatenated in the recognition region of U7 snRNA to construct concatenated U7 snRNAs targeting different target sites.
  • the snRNA with concatenated recognition domains in this embodiment comprises two or more recognition domains, which are concatenated in series at the 5′ end of the snRNA (as shown in Figure 23).
  • two concatenated recognition domains can recognize the same target site, and under the same expression vector or the same snRNA drive, the number of targeted recognition domains is increased, thereby improving the efficiency of inducing splicing skipping of USH2A pre-mRNA exon 13.
  • the recognition domains from the 5′ end to the 3′ end of the U7 snRNA are ordered according to the position of the target site corresponding to the recognition domain in the USH2A pre-mRNA, and are ordered from the 3′ end to the 5′ end.
  • Example 8-2 Recognition domain tandem U7 snRNA induces USH2A exon 13 splicing skipping in reporter gene cells
  • the U7 snRNA with recognition domains in series means a U7 snRNA stem-loop structure and a smOPT sequence, which are connected to two or more recognition domains, and its structure is 5′-recognition domain B-recognition domain A-smOPT sequence-stem-loop structure-3′, as shown in Figure 23.
  • the recognition domains A and B of the tandem U7 snRNA recognize RNA sequences at different target sites.
  • the targeting method of the U7 snRNA with recognition domains in series and USH2A pre-mRNA is shown in Figure 24A or Figure 24B.
  • the corresponding oligo DNAs were synthesized respectively.
  • the positive chain of the oligo DNA is the DNA corresponding to the recognition domain sequence, and CCGCA is added to the 5′, and the antisense chain is the antisense complementary sequence of the recognition domain sequence, AATT is added to the 5′ and T is added to the 3′.
  • the method is the same as that of Example 8-1.
  • the recognition domain sequences of snRNA#15 and snRNA#25 to be concatenated are ACACUGGCAGGGCUCACAUCCA (SEQ ID NO: 54) and AUUACACCUUCUUCCUUGACGAUU (SEQ ID NO: 13), respectively, and the synthesized positive chain of the oligo DNA is:
  • the antisense strand is:
  • the synthesized oligo DNA sense strand and antisense strand were annealed according to the reaction system (total reaction volume 20 ⁇ l: Oligo-F (100 ⁇ M) 2 ⁇ l + Oligo-R (100 ⁇ M) 2 ⁇ l + 10 ⁇ NEB Cutter smart buffer 2 ⁇ l + deionized water 16 ⁇ l) were mixed, incubated at 95°C for 5 minutes, and then placed on ice to cool and anneal to form double-stranded DNA with sticky ends. After diluting 100 times, 1 ⁇ l was taken and ligated with 10ng BsaI digested and recovered linearized pUC57-U7 snRNA backbone plasmid by T4 ligase.
  • the ligation product was further transformed into Escherichia coli competent cells, single clones were picked, PCR and sequencing were verified to obtain the U7 snRNA vector for inducing USH2A exon 13 splicing skipping.
  • the plasmid was purified and stored at -20°C for use.
  • the constructed vector was named pUC57-U7 snRNA#B-#A, A and B represent the recognition domain numbers, corresponding to the recognition domain lists and sequences in Tables 1 and 3. For example, pUC57-snRNA#24-#9, pUC57-snRNA#25-#9, pUC57-snRNA#9-#24, and pUC57-snRNA#9-#25.
  • FIG24A In pUC57-snRNA#24-#9 and pUC57-snRNA#25-#9, the targeting pattern of U7 snRNA and USH2A pre-mRNA is shown in FIG24A ; in pUC57-snRNA#9-#24 and pUC57-snRNA#9-#25, the targeting pattern of U7 snRNA and USH2A pre-mRNA is shown in FIG24B .
  • the recognition domain tandem U7 snRNA can also be chemically synthesized and modified according to the method described in Example 2, the same as Example 8-1, for example, its specific sequence and modification are as follows (* represents the phosphorothioate backbone, m represents 2′-methoxy modification, underline represents the recognition domain that is reverse complementary to the target sequence, and italics represent the smOPT sequence):
  • the total length of the chemically synthesized snRNA sequence is preferably greater than or equal to 96 bp.
  • 293T cells were inoculated into 24-well plates in a certain amount so that the cell confluence reached about 80% after 24 hours.
  • Lipofectamine2000 was used to co-transfect 293T cells with pCMV-EGFPleft-Exon13mut-EGFPright and U7snRNA plasmids expressing dual recognition domains (the vector mass ratio was 100ng:400ng), and 293T cells transfected with the reporter plasmid alone (Report, report group), co-transfected with the reporter plasmid and pUC57-U7Scramble (SC group) were used as two negative controls, and 293T cells without any plasmid transfection were used as blank controls.
  • transfected cells were cultured for 48-72 hours, digested into single cells with trypsin, and then the GFP positive rate of different snRNA groups was detected by flow cytometry.
  • Table 14 and Figure 26 below show the efficiency of U7-snRNAs tandem with different target sites to induce splicing skipping of USH2A pre-mRNA exon 13.
  • Example 9 Chemically synthesized U7 snRNA induces USH2A exon 13 splicing skipping in WERI cells
  • WERI-Rb-1 cell line was used. WERI cells were transfected with 50 pmol of in vitro synthesized snRNA combination 1 (U7-snRNA#30 and U7-snRNA#4) and combination 2 (U7-snRNA#26 and U7-snRNA#15) using Lipofectamine2000, and the same dose (50 pmol) of antisense oligonucleotide AON1 (5′- MA*MG*MC*MU*MU*MC*MG*MG*MA*MG*MA*MA*MA*MU*MU*MA*MA*MA*MU*MC*-3′, "M” indicates 2′-O-methoxy modification, "*” indicates phosphorothioation, SEQ ID NO:77) and AON2 (5′-MU*MG*MA*MU*MC*MA*MC*MC*MC*MU*MA*MA*MG*MC*MC*MU*MAMA*MG*MC*MC*MU*MAMA
  • RT-PCR experiments were performed using primers AGCCTTTTCCGCCAAGGTGATC (SEQ ID NO: 30) and CACAACGTTGCCCAGCAATGG (SEQ ID NO: 31) to detect whether mature USH2A mRNA had exon splicing skipping, and the electrophoresis results were shown in Figure 27.
  • the rt-PCR electrophoresis bands were further quantitatively analyzed using ImageJ software, and the proportion of mature USH2A mRNA that spliced skipped exon 13 or spliced skipped exons 12 and 13 was statistically analyzed, as shown in Figure 28.
  • snRNA combination 2 targets an AON site adjacent to exons 12 and 13 in the prior art where the probability of double splicing skipping is extremely high; however, the probability of double exon splicing skipping of snRNA combination 2 is very low.
  • Example 10 Splicing skipping effect of U7 snRNA with a motif that can recruit splicing regulatory proteins
  • U7 snRNA connected to hnRNP A1 binding motif According to the pre-transcription DNA sequences corresponding to the sequences in the table, the corresponding oligo DNAs were synthesized respectively.
  • the sense strand of the oligo DNA is the reverse complementary sequence of the target sequence (DNA sequence corresponding to the recognition domain sequence), and CCGCAATATGATAGGGACTTAGGGTG (SEQ ID NO: 67) is added to the 5′ of the target sequence, and the antisense strand is the target sequence 5′ plus AATT and 3′ plus CACCCTAAGTCCCTATCATATT (SEQ ID NO: 68).
  • the recognition domain sequence is NNN (the length of the recognition domain is preferably greater than 16 nucleotides)
  • the synthesized sense strand of the oligo DNA is The antisense strand is (The underline indicates the DNA double-stranded sequence corresponding to the recognition domain sequence, and the bold italics indicate the DNA double-stranded sequence corresponding to the binding motif "UAGGGU" or "UAGGGA” of the hnRNP A1 protein).
  • the synthesized oligo DNA sense strand and antisense strand were annealed according to the reaction system (total reaction volume 20 ⁇ l: Oligo-F (100 ⁇ M)2 ⁇ l+Oligo-R(100 ⁇ M)2 ⁇ l+10 ⁇ NEB Cutter smart buffer 2 ⁇ l+deionized water 16 ⁇ l), incubate at 95°C for 5 minutes, and place on ice to cool and anneal to form double-stranded DNA with sticky ends. After diluting 100 times, take 1 ⁇ l and connect it with 10ng BsaI-digested and recovered linearized pUC57-U7 snRNA backbone plasmid.
  • FIG. 29A shows a schematic diagram of the snRNA vector with hnRNP A1.
  • U7-hnRNP A1-snRNA can also be chemically synthesized and modified according to the method described in the examples of this application. Taking snRNA#15 and snRNA#25 as examples, the chemically synthesized U7-hnRNP A1-snRNA sequence and modification are as follows (* represents the phosphorothioate backbone, m represents 2′-methoxy modification, underline represents the recognition domain that is reverse complementary to the target sequence, italics represent the smOPT sequence, and bold represents the hnRNP A1 protein binding motif):
  • U7 snRNA linked to the hnRNP A1 binding motif induces USH2A exon 13 splicing skipping in reporter cells.
  • 293T cells were seeded into 24-well plates at a certain amount so that the cell confluence reached about 80% after 24 hours.
  • Lipofectamine 2000 was used to co-transfect 293T cells with pCMV-EGFPleft-Exon13mut-EGFPright and pUC57-U7-hnRNP A1-snRNA#15 plasmid, pUC57-U7-hnRNP A1-snRNA#25 plasmid, pUC57-U7 snRNA#15 plasmid, pUC57-U7 snRNA#25 plasmid, and pUC57-U7 snRNA#25-#15 plasmid (the vector mass ratio was 100ng:400ng).
  • 293T cells transfected with the reporter plasmid alone (reporter group) and co-transfected with the reporter plasmid and pUC57-U7Scramble (SC group) were used as two negative controls, and 293T cells not transfected with any plasmid were used as blank controls.
  • the transfected cells were cultured for 48-72 hours, digested into single cells using trypsin, and then flow cytometry was used to detect the splicing skipping efficiency induced by different snRNA groups.
  • Table 15 and Figure 29B below show the splicing skipping efficiency of U7-hnRNP A1-snRNA USH2A pre-mRNA exon 13.
  • tandem snRNA inducing exon 13 splicing skipping was significantly better than the introduction of hnRNP A1 binding motif at the 5′ end of U7 snRNA, suggesting that splicing skipping of USH2A pre-mRNA exon 13 may be more sensitive to tandem snRNA.
  • a free tail is introduced at the 5′ end of U7 snRNA, and the free tail sequence includes the binding motif "UAGGGU” or "UAGGGA” of hnRNP A1 protein.
  • the free tail sequence may contain 1, 2 or more binding motifs of hnRNP A1 protein, preferably 2, and the free tail sequence is preferably "UAUGAUAGGGACUUAGGGUG (SEQ ID NO: 35)", which can recruit hnRNP A1 protein and promote splicing skipping of USH2A exon 13. And this structure is not suitable for snRNA with tandem recognition domains.
  • the free tail introduced into the 5′ end of the U7 snRNA is a motif that can recruit splicing regulatory proteins
  • the splicing regulatory proteins are hnRNP A1 (Heterogeneous Nuclear Ribonucleoprotein A1), SRSF1 (Serine And Arginine Rich Splicing Factor 1), RBM4 (RNA Binding Motif Protein 4), DAZAP1 (DAZ Associated Protein 1), SR (Serine And Arginine-Rich Protein), etc.
  • Example 11 Construction of AAV-U7 snRNA-related plasmid vector and viral packaging for targeted induction of USH2A pre-mRNA exon 13 splicing skipping
  • the U7 snRNA gene that targets and induces splicing skipping of USH2A pre-mRNA exon 13 is inserted and replaced in the middle gene sequence of the two ITR domains in the pAAV-CMV vector to construct the pAAV-U7 snRNA vector, and the host cells are co-transfected with the AAV packaging plasmid: the serotype pRC plasmid (containing the Rep gene of AAV2 and the Cap gene of each serotype), the pHelper plasmid (a vector plasmid containing the E2A, E4 and VA genes of adenovirus), and the AAV-U7 snRNA virus that targets splicing skipping of USH2A pre-mRNA exon 13 is packaged.
  • the specific operation process is as follows:
  • the gene sequence was synthesized by whole gene synthesis - U7-snRNA gene expression box skeleton (excluding recognition domain): 5'-mouse U7 promoter-smOPT sequence-U7 snRNA scafford-snRNA gene specific 3'box-3'.
  • Two Type IIs restriction endonuclease recognition sites (such as BsaI, AarI, BsmBI, etc.) were added between the U7 promoter and smOPT to facilitate subsequent excision, replacement and insertion of other recognition domain sequences.
  • the whole gene synthesized sequence was inserted into and replaced the pAAV-CMV plasmid ( Helper Free System (AAV5) kit, TAKARA, Code No. 6650) was used to obtain the gene sequence between the two AAV2-ITR domains to obtain the pAAV-U7 snRNA backbone vector.
  • the corresponding Oligo DNA positive chain and antisense chain are synthesized respectively, and sticky ends similar to those cut by the Type IIs restriction endonuclease recognition site are added to both ends.
  • Annealing forms a double-stranded DNA of the recognition domain (single/tandem) with sticky ends, and T4 ligase is ligated into the linearized pAAV-U7 snRNA backbone plasmid recovered by the corresponding Type IIs restriction endonuclease digestion to form a pAAV-U7 snRNA plasmid targeting the specific site of USH2A pre-mRNA exon 13 to induce splicing jump, and it is named according to the snRNA number corresponding to the recognition domain sequence, such as pAAV-U7 snRNA#25, etc.
  • the target gene (U7-snRNA gene expression cassette that targets and induces splicing skipping of USH2A pre-mRNA exon 13) was inserted into and replaced the gene sequence between the AAV2-ITR domains of the pAAV-CMV plasmid to obtain the pAAV-U7 snRNA plasmid vector.
  • the Helper Free System (AAV5) kit instructions and standard cell operation procedures are packaged to obtain AAV-U7 snRNA virus that targets and induces splicing skipping of USH2A pre-mRNA exon 13.
  • HEK293/293T cells were inoculated into 100mm cell culture dishes with 10% FBS DMEM culture medium. Transfection was performed when the confluence reached 80%-90%. 3 hours before transfection, the old culture medium was discarded and replaced with fresh culture medium.
  • pAAV-U7 snRNA plasmid, pRC plasmid, pHelper plasmid and PEI (polyethyleneimine) transfection reagent were prepared according to the following system and added dropwise to the culture dish. After the PEI transfection mixture was added, the culture dish was gently shaken to evenly distribute the transfection reagent, and the culture medium was placed in a 37°C, 5% CO2 incubator for culture.
  • PEI transfection system pAAV plasmid (1 ⁇ g/ ⁇ l), 6 ⁇ L; pRC1/2/5/6 plasmid (1 ⁇ g/ ⁇ l); (pRC plasmid capsid gene determines serotype), 6 ⁇ L; pHelper plasmid (1 ⁇ g/ ⁇ l), 6 ⁇ L; serum-free DMEM medium, 500 ⁇ L; PEI (1mg/mL), 110 ⁇ L.
  • Treatment method vortex mix several times and incubate at room temperature for 5 minutes.
  • the target gene fragment inserted between the AAV2-ITR domains of the pAAV-U7 snRNA plasmid should be less than 2.5kb
  • multiple U7-snRNA gene expression cassettes (5′-mouse U7 promoter-smOPT sequence, U7 snRNA scafford-snRNA gene specific 3′ cassette-3′) can be inserted to ensure that the expression level of U7snRNA is increased under the same number of AAV virus particles.
  • the gene sequence length is about 450bp, so it is preferred that the pAAV-U7 snRNA plasmid carries 1-5 U7-snRNA gene expression cassettes.
  • the multiple U7-snRNA gene expression cassettes in the pAAV-U7 snRNA plasmid can have the same recognition domain or recognition domain combination, or can have different or non-completely the same recognition domain combination.
  • the present application induces USH2A pre-mRNA exon 13 splicing skipping by delivering U7 snRNA through AAV.
  • the capsid protein of the AAV can be of natural origin, or a variant of the capsid protein based on natural origin, or subjected to directed evolution, or rational amino acid/peptide modification (codon optimization, chimeric functional peptides of different serotypes, etc.), etc., to enhance tissue and organ tropism, immunogenicity, and transfection efficiency, such as AAV2.5, AAV2i8, AAV-TT, AAV9.HR, CAM130, etc.
  • AAV2-3 ⁇ U7 snRNA #9-#25 (3 ⁇ U7 snRNA tandem), AAV2-2 ⁇ U7 snRNA #9-2 ⁇ U7 snRNA #25 (4 ⁇ U7 snRNA separate) and AAV2-2 ⁇ U7-hnRNP A1-snRNA #9-2 ⁇ U7-hnRNP A1-snRNA #25 (4 ⁇ U7 snRNA separate-motif) vectors of AAV2 serotype were constructed and transfected into HEK293/293T cells to package the viruses.
  • AAV2 viruses 3 ⁇ U7 snRNA tandem, 4 ⁇ U7 snRNA separate and 4 ⁇ U7 snRNA separate-motif were obtained by collection and purification, respectively.
  • the results show that the AAV2 virus transduced 3 ⁇ U7 tandem and 4 ⁇ U7-separate.
  • the RT-PCR results showed that the efficiency of 4 ⁇ U7 separate was poor, but the efficiency was improved after adding the hnRNPA1 binding motif, which was comparable to the efficiency of 3 ⁇ U7 tandem.
  • the qRT-PCR test results were consistent with the RT-PCR results.
  • the maximum capacity of the target gene of the SsAAV vector is 4.7kb
  • the maximum capacity of the target gene of the ScAAV vector is 2.5kb
  • the size of the U7 snRNA expression cassette is about 450bp. Therefore, a scAAV vector can accommodate up to 9 U7 snRNA expression cassettes, and a scAAV vector can accommodate up to 5 U7 snRNA expression cassettes. From the results of Example 14, the effect of 3 ⁇ U7 snRNA tandem in vitro is similar to the splicing jump induced by the 2 ⁇ U7 snRNA-hnRNP A1 combination, and is better than the 2 ⁇ U7 snRNA combination.
  • Example 13 AAV-U7 snRNA induces splicing skipping significantly better than AON - in vitro dose escalation
  • AAV2-U7 snRNA #9-#25 (1 ⁇ U7 snRNA tandem) vector of AAV2 serotype was constructed and HEK293/293T cells were transfected to package the virus.
  • AAV2 virus 1 ⁇ U7snRNA tandem i.e., AAV2-RM-101, was obtained by collection and purification, and the virus titer was tested for later use.
  • WERI-Rb-1 cells were seeded into 24-well plates at 6 ⁇ 10 5 /well, and AAV2-RM-101 virus was added to WERI-Rb-1 cells at MOI values of 3 ⁇ 10 5 , 1 ⁇ 10 5 , 3 ⁇ 10 4 , 1 ⁇ 10 4 , 3 ⁇ 10 3 , 1 ⁇ 10 3 , and 3 ⁇ 10 2 as experimental groups with different MOIs.
  • 50 nM and 200 nM PROQR EX13-3 (AON1 in Example 9) were used as positive control groups, and AAV2-U7-SCR (Scramble) and AAV2-U7-LUC (Luciferase recognition) at MOI of 3 ⁇ 10 5 were used as negative controls.
  • the WERI-Rb-1 cells after different treatments were cultured for 72 hours, and then RNA of each experimental group of cells was extracted, reverse transcribed to obtain cDNA, and RT-PCR and qRT-PCR experiments were performed using the corresponding primers/probes in Table 16 to detect the efficiency of splicing skipping of USH2A pre-mRNA exon 13. As shown in Figure 31, the results showed that the splicing skipping efficiency induced by AAV2-U7 snRNA #9-#25 was significantly better than that of AON1.
  • Example 14 snRNA induces splicing skipping of USH2A pre-mRNA exon 13 in humanized mouse retinal cells
  • the exon 12 of the USH2A gene of C57/BL6J mice + part of the flanking sequence [about 1670 bp upstream of the 12th exon (3′ end of the 11th intron of mice) to about 1600 bp downstream of the 12th exon (5′ end of the 12th intron of mice)] was replaced with the exon 13 of the human USH2A gene + part of the flanking sequence [about 1611 bp upstream of the 13th exon (3′ end of the 12th intron of humans) to about 1599 bp downstream of the 13th exon (5′ end of the 13th intron of humans)] + the insertion sequence, while c.2208T to G was introduced into human USH2A exon 13 to obtain USH2A exon 13 humanized mice carrying the c.2802T>G mutation (USH2A EXON13 c.2802T>G ).
  • AAV2-U7 snRNA #9-#25 AAV5-1 ⁇ U7
  • AAV5-2 ⁇ U7 snRNA #9-2 ⁇ U7 snRNA #25 AAV5-4 ⁇ U7 seperate viruses with a total volume of 1E+10 vg/eye were injected into the eyes of hUSH2A EXON13 c.2802T>G gene knock-in humanized mice by subretinal injection.
  • mice injected with AAV5-U7-scramble virus served as the negative control group, PROQR-AON (AON1 in Example 9) with a vitreous injection dose of 15 ⁇ g/eye (1 ⁇ L) served as the positive control group, and untreated mice served as the blank group (nontreated). Both eyes of each mouse were injected. Three weeks after the injection, the experimental mice were killed, the mouse retinal tissue was taken, RNA was extracted, and reverse transcribed into cDNA, and RT-PCR and qRT-PCR experiments were performed by the corresponding primers/probes in Table 16 to detect the efficiency of splicing jump of USH2A pre-mRNA exon 13.
  • the results show that although it is a 1 ⁇ U7 snRNA tandem, its effect of inducing splicing jump in the retina of the eye is better than that of the 2 ⁇ U7 snRNA combination, and both are better than PROQR AON (AON1 in Example 9) (according to the results of the non-treated group, human USH2A exon 13 containing mutations has a certain amount of spontaneous jump, which is consistent with existing research reports).
  • Example 15 Injection of AAV-U7 snRNA of different serotypes induces splicing skipping of exon 12 of USH2A pre-mRNA in rabbit ocular cells
  • AAV5-U7 snRNA #9-#25 (AAV5-1 ⁇ U7) and AAV8-U7 snRNA #9-# 25 (AAV8-1 ⁇ U7) were injected into the rabbit eyes at an MOI of 5 ⁇ 10 10 and 2 ⁇ 10 11 , respectively.
  • AAV5-CMV-GFP virus was injected into the rabbit subretinal cavity as a negative control group, and AON (AON1 in Example 9) with a dose of 50 ⁇ g (50 ⁇ L) injected into the vitreous cavity was used as a positive control group.
  • the USH2A gene in rabbits is wild-type rabbit USH2A, without exon 12 (equivalent to human USH2A exon 13) mutation.
  • human exon 13 containing mutations in hUSH2A EXON13c.2802T>G gene knock-in humanized mice is more susceptible to induced splicing skipping.
  • the effect of AAV-U7 snRNA and AON in inducing target exon splicing skipping in rabbits is significantly lower than that in humanized mice, suggesting that the effect of AON is more easily affected by exon sequence and mutation.
  • Example 16 Long-term effect of AAV-delivered U7 snRNA inducing USH2A pre-mRNA exon 13 splicing skipping.
  • AAV5-3 ⁇ U7 snRNA #9-#25 (AAV5- 3 ⁇ U7), AAV5-U7 snRNA #9-#25 (AAV5-1 ⁇ U7), AAV5-2 ⁇ U7 snRNA #9-2 ⁇ U7 snRNA #25 (AAV5-4 ⁇ U7 seperate) viruses were injected into the eyes of hUSH2A EXON13 c.2802T>G gene knock-in humanized mice, AAV5-U7-scramble virus injected mice were used as negative control group, PROQR-ASO (AON1 in Example 9) injected into the vitreous at a dose of 15 ⁇ g (1 ⁇ L) was used as positive control group, and mice without treatment were used as blank group (NTC).
  • NTC blank group
  • this example further performs qRT-PCR experiments using the corresponding probes in Table 16 to detect and compare the differences in the long-term maintenance of USH2A pre-mRNA exon 13 splicing jump efficiency between AAV5-1 ⁇ U7 and AAV5-4 ⁇ U7seperate in the retina.
  • the results show (as shown in Figure 34) that after 22 weeks, AAV-1 ⁇ U7 snRNA tandem still has the best splicing jump efficiency, and the effect is improved compared to 3 weeks, and there may be a cumulative effect of therapeutic effect.
  • the persistence of AAV-1 ⁇ U7 snRNA tandem is significantly better than that of AAV-4 ⁇ U7seperate.

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Abstract

提供一种snRNA核酸分子及其应用。snRNA核酸分子包括:识别结构域、茎环序列和Sm序列;其中,所述识别结构域的数量为至少两个;其中,各识别结构域自5'端至3'端与pre-mRNA的3'端至5'端的靶向序列片段反向互补;所述pre-mRNA为USH2A基因对应的pre-mRNA。snRNA核酸分子能够高效诱导USH2A外显子13的剪接跳跃,且单跳读的频率更高;尤其对于发生致病或非致病变的USH2A外显子13,本能够以低剂量安全、有效进行USH2A外显子13单跳读,在预防和/或治疗Usherin蛋白表达异常相关的眼病和耳病中具有重要的临床价值。

Description

snRNA核酸分子及其应用 技术领域
本发明属于生物技术领域,具体涉及一种snRNA核酸分子及其应用。
背景技术
Usher综合征(Usher Syndrome)是一类遗传性疾病,又称耳聋-色素性视网膜炎综合征,其特征是不同程度的先天性感音神经性耳聋,以及色素性视网膜炎(Retinitis Pigmentosa,RP)引起的进行性视力丧失。在临床上Usher综合征可分为3种类型:(1)I型Usher综合征:听力先天性重深度感音神经性耳聋,前庭反应消失,青春期前出现色素性视网膜炎,逐渐致盲,关联基因涉及MYO7A、CDH23、USH1C、PCHD15等;(2)II型Usher综合征:听力先天性中重度感音神经性耳聋,前庭反应正常,青春期出现色素性视网膜炎,逐渐致盲,关联基因涉及USH2A、GPR98、WHRN等;(3)III型Usher综合征:听力进行性感音神经性耳聋,前庭反应正常,青春期末出现色素性视网膜炎,逐渐致盲,关联基因涉及CLRN1等。
其中,II型占Usher综合症的比例超过50%。而USH2A基因突变是Usher综合征II型的最常见原因,涵盖超过50%的Usher综合征患者。同时,USH2A基因的突变也是导致非综合征性视网膜色素变性(NSRP)的重要原因之一。
USH2A基因定位于1q41,其在基因组中的跨度超过800kb,编码一个大型跨膜蛋白Usherin,其锚定在视网膜感光细胞和内耳毛细胞的质膜上,是纤毛发育和维持必不可少的组分。在视网膜中,Usherin是USH2复合物的重要部分,被认为在稳定光感受器的外节段发挥作用。USH2A具有2个亚型,在视网膜细胞中主要的亚型含有72个Exon,编码区长度约为15.6kb。Usherin蛋白的胞外部分包含许多重复的结构域,包括10个Laminin EGF-like(LE)结构域和35个Fibronectin type 3(FN3)结构域。人USH2A外显子13长度为642bp,编码Usherin第723~936位氨基酸,构成Usherin蛋白中10个LE结构域中的4个。
USH2A基因的第13号外显子、第50号外显子和第40号内含子的突变会引发Usher综合征。迄今为止已经鉴定出超过1000个分布在整个USH2A基因中的致病性突变,其中的外显子13是USH2A基因中突变最频繁的外显子,约占35%。USH2A基因第13号外显子的突变,包括c.2802T>G(p.Cys934Trp)、c.2299delG(p.Glu767Serfs*21,欧美患者频率最高突变)、c.2276G>T(p.cys759phe,导致非综合征性RP中最常见的突变位点)、C.2522C>A(p.S841Y)、c.2242C>T(p.Gln748X)、c.2541C>A(C847X)、c.2761delC(Leu921fs)和C.2776C>T(p.R926C)、C.2209C>T、C.2310delA、c.2391_2392deITG、c.2431A>T、C.2431_2432delAA、c.2440C>T、c.2525dup、C.2610C>A、C.2755C>T、C.2176T>C、C.2236C>G、 c.2296T>C、C.2332G>T、c.2339G>T(PMID:31904091)。
USH2A编码区长度约为15.6kb,常规的基因治疗递送方法(如重组慢病毒、重组腺相关病毒等)难以包装如此庞大的编码序列,因此难以通过直接递送USH2A进行治疗。小鼠USH2A的外显子12与人USH2A外显子13同源,长度均为642bp,移除该外显子并没有造成后续的移码突变。有研究显示,在敲除了小鼠USH2A的外显子12后,Usherin依然能够正确定位并且行使正常的功能。以此类推,对于包含致病性突变的人USH2A外显子13,我们可以利用一系列手段使其发生跳读进行治疗。
现有技术通过CRISPR/Cas系统进行基因组DNA的编辑直接删除外显子13,或者破坏RNA剪接相关的位点。使用片段删除存在风险,如染色体重排、病毒整合、反向重新整合,以及长时间表达CAS系统或者基于相对庞大的基因组背景进行两个gRNA诱导的双切的脱靶概率高。
现有技术通过使用单碱基编辑器修改上述剪接相关位点的关键碱基,亦可促进外显子跳读。但是现有的单碱基编辑器无法通过单个AAV载体装载,并且受PAM、编辑窗口以及碱基转换类型的限制,可能在剪接相关位点附近没有合适的gRNA。
现有技术显示通过反义寡核苷酸(AONs,Antisense oligonucleotides)靶向干扰pre-mRNA剪接,外显子跳读的效率较高。然而,AON在促进人外显子13跳读的同时,也促进人外显子12与人外显子13共同跳读,甚至有的AON处理导致的全是双跳读,而没有单跳读。而人外显子12全长196bp,非3整数倍,缺失会导致移码突变,USH2A蛋白失活。
天主教大学基金US10131910B2和US10745699B2公开了用于治疗2型Usher综合征的反义寡核苷酸。该等专利通过反义寡核苷酸(AON)靶向人USH2A外显子13(内含子12)、PE40、外显子50,诱导外显子跳跃和内含子12的保留。
该专利虽然利用靶向内含子12的AON4a,有效保留了外显子12,但是使用多个AON,则脱靶风险增大,同时,AON4a需求量大,则总体AON给药量则更大,大剂量AON存在细胞毒性风险。
RPOQR(CN109804069A)公开了治疗眼部疾病的反义寡核苷酸。该专利在US10131910B2和US10745699B2的基础上,发现了三个位点Ex13-1、Ex13-2和Ex13-3似乎给出更强的单(外显子13)跳读的信号。
然而,AON在促进人外显子13跳读的同时,也促进人外显子12与人外显子13共同跳读,甚至有的AON处理导致的大部分是双跳读,或者存在非正常/完整外显子13跳读(可能为外显子12单跳读)的杂条带。而人外显子12全长196bp,非3整数倍,缺失会导致移码突变,剪接双跳后的USH2A蛋白失活。AON效果持续性不长,用药频率高;诱导剪接跳跃效率不高;AON给药剂量非常大。
细胞内有核小RNA(small nuclear RNA,snRNA),它是真核生物转录后加工过程中RNA剪接体(spliceosome)的主要成分,通过与snRNP蛋白结合参与mRNA前体的加工过程。其长度在哺乳动物中约为100-215个核苷酸,共分为7类,由于含U丰富,故编号为U1~U7。但U7 snRNP,不参与剪接,而是复制依赖型组蛋白(RDH)pre-mRNA独特3'末端加工的关键因素。修饰后的U7SnRNA是通过将U7 snRNA的非规范Sm结合位点替换为衍生自主要剪接体U snRNP的共有序列,将U7 snRNA的5'区的组蛋白结合序列改变为待修饰基因的互补序列,可以通过靶向外显子来诱导外显子的剪接跳跃。
然而,自1998年修饰的U7 snRNA研发以来,其相关研究和应用并不广泛,仅局限于少数几个靶点,受限于U7 snRNP施用的载体数量,如使用病毒如AAV递送的修饰U7 snRNA需要非常高的病毒剂量,高剂量的病毒可能会存在毒性或引发免疫反应,这限制了病毒递送U7-snRNA的应用。而其它基因整合的长期递送方式存在基因组安全风险,瞬时递送的效果短暂。
发明内容
本发明所要解决的技术问题是为了克服现有技术中USH2A外显子13剪接跳跃诱导效率不高的缺陷,提供了一种snRNA核酸分子及其应用。本发明通过snRNA靶向干扰USH2A外显子13的pre-mRNA剪接,促进外显子单跳读的效率,在显著提升效率的同时保证了安全性。
本发明通过以下技术方案解决上述技术问题。
本发明的第一方面提供一种snRNA核酸分子,所述snRNA核酸分子包括:识别结构域、茎环序列和Sm序列;其中,所述识别结构域的数量为至少两个;
其中,各识别结构域自5′端至3′端与pre-mRNA的3′端至5′端的靶向序列片段反向互补;所述pre-mRNA为USH2A基因对应的pre-mRNA。
本发明一些实施方案中,各所述识别结构域自5′端至3′端依次与pre-mRNA的3′端至5′端的靶向序列片段反向互补,即snRNA核酸分子的5′端到3′端的识别结构域排序为依据识别结构域对应的靶位点在USH2A pre-mRNA中的位置按3′端到5′端排序。
本发明一些实施方案中,各所述识别结构域自5′端至3′端非依次与pre-mRNA的3′端至5′端的靶向序列片段反向互补,即snRNA核酸分子的5′端到3′端的识别结构域排序并非依据识别结构域对应的靶位点在USH2A pre-mRNA中的位置按3′端到5′端排序。
本发明一些实施方案中,所述识别结构域的长度为至少16bp。
本发明另一些实施方案中,所述识别结构域的长度为18~40bp。
本发明另一些实施方案中,所述识别结构域的长度为20~27bp。
本发明另一些实施方案中,所述识别结构域的长度为22~27bp。
本发明一些实施方案中,所述识别结构域的数量为两个;优选地,所述两个识别结构域相邻分布。
本发明一些实施方案中,所述茎环序列可为1-2个。
本发明一些具体实施方案中,所述snRNA核酸分子自5′端至3′端依次包括:两个相邻的识别结构域、Sm序列和茎环序列。
本发明一些实施方案中,所述pre-mRNA为USH2A基因第12号内含子至第13号内含子对应的全部或部分pre-mRNA。
本发明一些较佳实施方案中,所述pre-mRNA为USH2A基因第13号外显子对应的全部或部分pre-mRNA。
本发明一些实施方案中,所述pre-mRNA的基因组定位为Chr1:216246563-216247246;所述靶向序列片段选自如SEQ ID NO:1所示的核苷酸序列及其突变序列。
本发明另一些实施方案中,所述pre-mRNA的基因组定位为Chr1:216246563-216246753;所述靶向序列片段选自如SEQ ID NO:3所示的核苷酸序列及其突变序列。
本发明一些较佳实施方案中,所述pre-mRNA的基因组定位为Chr1:216246563-216246649;所述靶向序列片段选自如SEQ ID NO:4所示的核苷酸序列及其突变序列。
本发明一些具体实施方案中,所述pre-mRNA的基因组定位为Chr1:216246563-216246626;所述靶向序列片段选自如SEQ ID NO:9所示的核苷酸序列及其突变序列。
本发明一些具体实施方案中,所述pre-mRNA的基因组定位为Chr1:216246616-216246649;所述靶向序列片段选自如SEQ ID NO:34所示的核苷酸序列及其突变序列。
本发明一些具体实施方案中,所述pre-mRNA的基因组定位为Chr1:216247130-216247246;所述靶向序列片段选自如SEQ ID NO:2所示的核苷酸序列及其突变序列。
本发明一些具体实施方案中,所述pre-mRNA的基因组定位为Chr1:216247142-216247185;所述靶向序列片段选自如SEQ ID NO:32所示的核苷酸序列及其突变序列。
本发明一些具体实施方案中,所述pre-mRNA的基因组定位为Chr1:216247130-216247161;所述靶向序列片段选自如SEQ ID NO:33所示的核苷酸序列及其突变序列。
本发明一些具体实施方案中,所述pre-mRNA的基因组定位为Chr1:216247210-216247246;所述靶向序列片段选自如SEQ ID NO:36所示的核苷酸序列及其突变序列。
本发明一些具体实施方案中,所述pre-mRNA的基因组定位为Chr1:216247204-216247232;所述靶向序列片段选自如SEQ ID NO:37所示的核苷酸序列及其突变序列。
本发明一些具体实施方案中,所述pre-mRNA的基因组定位为Chr1:216247187-216247220;所述靶向序列片段选自如SEQ ID NO:38所示的核苷酸序列及其突变序列。
本发明一些具体实施方案中,所述pre-mRNA的基因组定位为Chr1:216247169-216247202;所述靶向序列片段选自如SEQ ID NO:39所示的核苷酸序列及其突变序列。
本发明中,两个所述识别结构域自5′端向3′分别为第一识别结构域和第二识别结构域;所述第一识别结构域和第二识别结构域可以是识别不同靶位点的RNA序列,也可以识别同一靶位点的RNA序列。
本发明一些具体实施方案中,识别不同靶位点的RNA序列中,与USH2A pre-mRNA 3′端结合的反向互补RNA序列作为snRNA的第二识别结构域,与USH2A pre-mRNA 5′端结合的反向互补RNA作为snRNA第一识别结构域。
本发明一些实施方案中,与所述第一识别结构域或第二识别结构域反向互补的靶向序列片段选自如SEQ ID NO:34所示的核苷酸序列及其突变序列和如SEQ ID NO:9所示的核苷酸序列及其突变序列;相应与所述第二识别结构域或第一识别结构域反向互补的靶向序列片段选自如SEQ ID NO:32所示的核苷酸序列及其突变序列、如SEQ ID NO:33所示的核苷酸序列及其突变序列、如SEQ ID NO:36所示的核苷酸序列及其突变序列、如SEQ ID NO:37所示的核苷酸序列及其突变序列、如SEQ ID NO:38所示的核苷酸序列及其突变序列和如SEQ ID NO:39所示的核苷酸序列及其突变序列。
本发明一些实施方案中,与所述第一识别结构域反向互补的靶向序列片段选自如SEQ ID NO:34所示的核苷酸序列及其突变序列和如SEQ ID NO:9所示的核苷酸序列及其突变序列;与所述第二识别结构域反向互补的靶向序列片段选自如SEQ ID NO:32所示的核苷酸序列及其突变序列、如SEQ ID NO:33所示的核苷酸序列及其突变序列、如SEQ ID NO:36所示的核苷酸序列及其突变序列、如SEQ ID NO:37所示的核苷酸序列及其突变序列、如SEQ ID NO:38所示的核苷酸序列及其突变序列和如SEQ ID NO:39所示的核苷酸序列及其突变序列。
本发明中,所述突变序列为基于出发序列发生一个或多个核苷酸的替换、增加或缺失后得到的序列。
本发明一些具体实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:12~22、59~61任一项所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:40~58任一项所示。
本发明一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:12、13、15或17所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:48、54、56或58所示。
本发明一些具体实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:12、13或17所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:48、54或58所示。
本发明一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:12所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:48所示。
本发明另一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:12所示, 所述第二识别结构域的核苷酸序列如SEQ ID NO:54所示。
本发明另一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:12所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:58所示。
本发明另一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:13所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:48所示。
本发明另一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:13所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:54所示。
本发明另一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:13所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:58所示。
本发明另一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:17所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:48所示。
本发明另一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:17所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:54所示。
本发明另一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:17所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:58所示。
本发明另一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:16所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:42所示。
本发明另一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:18所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:43所示。
本发明另一些实施方案中,所述第一识别结构域的核苷酸序列如SEQ ID NO:14所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:55所示。
本发明中,所述突变序列为在所述核苷酸序列上存在一个或多个核苷酸的替换、增加或缺失,优选地为替换。
本发明中,所述突变选自天然致病突变和天然非致病突变。所述天然致病突变选自c.2242C>T、c.2276G>T、c.2299delG、c.2522C>A、c.2541C>A、c.2761delC、c.2776C>T、c.2802T>G、c.2209C>T、c.2310delA、c.2391_2392deITG、c.2431A>T、c.2431_2432delAA、c.2440C>T、c.2525dup、c.2610C>A、c.2755C>T、c.2176T>C、c.2236C>G、c.2296T>C和c.2332G>T中的一种或多种。
本发明一些具体实施方案中,所述天然致病突变选自c.2802T>G、c.2299delG和c.2276G>T中的一种或多种;例如,所述核苷酸突变为c.2802T>G。
本发明一些实施方案中,所述Sm序列为共有序列,所述茎环序列可为U1、U2、U3、U4、U5、U6或U7的茎环序列。
本发明一些具体实施方案中,所述茎环序列为U7的茎环序列。
本发明一些具体实施方案中,所述茎环序列为U1的茎环序列。
本发明一些具体实施方案中,所述Sm序列如SEQ ID NO:6所示。
本发明一些具体实施方案中,所述茎环序列如SEQ ID NO:7所示。
本发明一些实施方案中,所述snRNA核酸分子包含修饰的核苷酸或其类似物单体。
本发明中,所述修饰选自:2′-O-烷基修饰、2′-O-甲氧基修饰和2′-O-甲氧基乙基修饰。
本发明一些具体实施方案中,所述2′-O烷基修饰为2′-O-甲基修饰。
本发明一些实施方案中,所述核苷酸类似物单体选自6′-修饰的双环核苷、5′-修饰的双环核苷、6′-双取代双环核苷、四氢吡喃核苷类似物和2'-脱氧2'-氟-β-D-阿拉伯糖核苷酸。
本发明一些实施方案中,所述snRNA核酸分子的核苷酸间通过化学键连接,所述化学键选自磷酸键、亚甲基键、酰胺键、甲基膦酸酯键和3'-硫代甲缩醛键。
本发明一些实施方案中,所述磷酸键选自硫代磷酸酯键、二硫代磷酸酯键、烷基膦酸酯键、酰胺磷酸酯键(phosphoroamidate)、硼烷磷酸酯(boranophosphate)键、手性连接磷(chiral linkage phosphorus)。
本发明一些具体实施方案中,所述磷酸键为硫代磷酸酯键。
本发明一些实施方案中,所述snRNA核酸分子自5′端和/或3′端起的第1~80位包含修饰的核苷酸或其类似物单体。
本发明另一些实施方案中,所述snRNA核酸分子自5′端和/或3′端起的第3~40位包含修饰的核苷酸或其类似物单体。
本发明另一些实施方案中,所述snRNA核酸分子自5′端和/或3′端起的第6~10位包含修饰的核苷酸或其类似物单体。
本发明另一些实施方案中,所述snRNA核酸分子自5′端和/或3′端起的第20~27位包含修饰的核苷酸或其类似物单体。
本发明一些具体实施方案中,所述snRNA核酸分子自5′端或3′端起包含至少一个磷酸键。
本发明一些实施方案中,所述snRNA核酸分子包括上述一种或多种修饰。
本发明中,当所述snRNA核酸分子为化学合成时,所有的核苷酸均通过硫代磷酸酯键相互连接,且均进行了2′-O-甲氧基修饰。在一些实施方案中,仅snRNA两侧的3个核苷酸通过硫代磷酸酯键连接,并进行了2′-O-甲氧基修饰。在一些化学合成和修饰的U7 snRNA实施方案中,识别结构域与靶位点反向互补配对中可存在0-5个错配核苷酸,优选为0-1个。在一些实施方案中,化学合成的snRNA两侧的3-40个碱基为经过修饰并通过磷酸键连接。
本发明一些具体实施方案中,所述snRNA核酸分子自5′端起包含1~3个磷酸键。
本发明一些具体实施方案中,所述snRNA核酸分子自3′端起包含1~3个磷酸键。
本发明一些实施方案中,所述snRNA核酸分子进一步在所述识别结构域的5′端和/或3′端的核苷酸上包括单向延长序列或双向延长序列。
所述单向延长序列为在所述snRNA核酸分子的靶向序列的5′端或3′端增加的RNA序列;所述双向延长序列为在所述snRNA核酸分子的靶向序列的5′端和3′端分别增加的RNA序列。
本发明一些实施方案中,所述snRNA核酸分子进一步包括游离的尾部序列,所述尾部序列包含剪接调控蛋白的基序,可结合剪接调控蛋白。
本发明一些具体实施方案中,所述剪接调控蛋白选自hnRNP A1(Heterogeneous Nuclear Ribonucleoprotein A1)、SRSF1(Serine And Arginine Rich Splicing Factor 1)、RBM4(RNA Binding Motif Protein 4)、DAZAP1(DAZ Associated Protein 1)和SR(Serine And Arginine-Rich Protein)。
例如,所述剪接调控蛋白为hnRNP A1时,所述尾部序列如SEQ ID NO:35所示。
本发明的第二方面提供一种snRNA核酸分子的组合,所述组合包括一种或多种如第一方面所述的snRNA核酸分子。
本发明一些实施方案中,至少两个识别结构域位于相同或不同的snRNA核酸分子上。
本发明的第三方面提供一种DNA分子,所述DNA分子编码如第一方面所述的snRNA核酸分子或者如第一方面所述的组合。
本发明的第四方面提供一种基因表达盒,所述基因表达盒包含启动子和如第三方面所述的DNA分子。
本发明一些实施方案中,所述启动子为U7启动子。
本发明一些具体实施方案中,所述启动子为小鼠来源的U7启动子。
本发明一些实施方案中,所述基因表达盒的3′端包含加尾序列,所述加尾序列参与所述snRNA的加工。
本发明中,所述加尾序列的长度为28~131bp,例如为106bp。
本发明一些实施方案中,所述加尾序列为U7 snRNA基因3′端后的基因序列,例如如SEQ ID NO:8所示。
本发明一些实施方案中,所述基因表达盒包括识别结构域和骨架序列;所述骨架序列如SEQ ID NO:62所示。
本发明的第五方面一种重组表达载体,所述重组表达载体包含如第一方面所述的snRNA核酸分子、如第二方面所述的组合或者如第三方面所述的基因表达盒。
本发明一些实施方案中,所述重组表达载体的表达载体选自质粒、噬菌体、微环DNA、线性DNA和病毒。
本发明一些具体实施方案中,所述表达载体为慢病毒或腺相关病毒。
本发明中,所述腺相关病毒的衣壳蛋白为天然来源的衣壳蛋白或其突变体,所述腺相关病毒的质粒为单链(ssAAV,single-stranded AAV)或与所述单链互补的双链(scAAV,Self-complementary AAV)。
本发明中,所述天然来源的AAV衣壳蛋白可以是来源于动物体的,也可以是来源于植物的,所述来源于动物体的AAV衣壳蛋白可以是来源于人体的(如AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8和AAV9等),也可以是来源于非人灵长类动物(如AAVrh.8、AAVrh.10和AAVrh.43),也可以是来源于小鼠、猪等脊椎动物,也可以是来源于昆虫。优选对眼部视网膜组织具有亲嗜性的AAV血清型,如AAV1、AAV2、AAV4、AAV5、AAV7、AAV8、AAV9、AAVrh10或AAV2.7m8。本发明AAV质粒体系中,例如,AAV ITR血清型应与Rep基因血清型一致,与Cap基因血清型可不一致。所述AAV可以是单链AAV(ssAAV,single-stranded AAV),也可以是自互补形成的双链AAV(scAAV,Self-complementary AAV)。
本发明一些具体实施方案中,所述天然来源的衣壳蛋白选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAVrh.8、AAVrh.10和AAVrh.43;
所述突变体选自AAV2.5、AAV2i8、AAV-TT、AAV9.HR和CAM130。
本发明的第六方面提供一种病毒颗粒,包括衣壳蛋白和核酸,所述核酸包括如第一方面所述的snRNA核酸分子、如第二方面所述的组合或者如第三方面所述的DNA分子。
本发明一些实施方案中,所述衣壳蛋白为来自腺相关病毒的衣壳蛋白。
本发明一些实施方案中,所述来自腺相关病毒的衣壳蛋白如第五方面所定义。
本发明的第七方面提供一种药物组合物,所述药物组合物包含如第一方面所述的snRNA核酸分子、如第二方面所述的组合、如第三方面所述的DNA分子、如第四方面所述的基因表达盒、如第五方面所述的重组表达载体或者如第六方面所述的病毒颗粒。
本发明一些实施方案中,所述药物组合物还包含药学上可接受的载体。
本发明的第八方面提供一种诱导产生缺失外显子13的Usherin蛋白的方法,所述方法包括向宿主细胞中导入如第一方面所述的snRNA核酸分子、如第二方面所述的组合、如第三方面所述的DNA分子、如第四方面所述的基因表达盒、如第五方面所述的重组表达载体、如第六方面所述的病毒颗粒或者如第七方面所述的药物组合物,使外显子13发生剪接跳跃。
本发明一些实施方案中,所述宿主细胞选自视网膜组织细胞,内耳细胞,具有分化为视网膜组织细胞和/或内耳细胞的潜能细胞,以及可行使包含具有与视网膜组织细胞和/或内耳细胞对应功能的细胞。
本发明一些具体实施方案中,所述视网膜组织细胞为视网膜感光细胞,所述内耳细胞为内耳毛细胞。
本发明中,所述干细胞选自诱导多能干细胞和胚胎干细胞。
本发明中,所述潜能细胞选自诱导多能干细胞、胚胎干细胞、神经前体细胞、视网膜祖细胞、视网膜前体细胞和间充质基质细胞。
本发明的第九方面提供一种抑制USH2A pre-mRNA外显子13表达和/或功能的方法,所述方法包括施用如第一方面所述的snRNA核酸分子、如第二方面所述的组合、如第三方面所述的DNA分子、如第四方面所述的基因表达盒、如第五方面所述的重组表达载体、如第六方面所述的病毒颗粒或者如第七方面所述的药物组合物。
本发明的第十方面提供一种诱导USH2A pre-mRNA外显子13剪接跳跃的方法,所述方法包括施用如第一方面所述的snRNA核酸分子、如第二方面所述的组合、如第三方面所述的DNA分子、如第四方面所述的基因表达盒、如第五方面所述的重组表达载体、如第六方面所述的病毒颗粒或者如第七方面所述的药物组合物。
本发明的第十一方面提供一种降低异常Usherin蛋白表达的方法,所述方法包括向宿主细胞中导入如第一方面所述的snRNA核酸分子、如第二方面所述的组合、如第三方面所述的DNA分子、如第四方面所述的基因表达盒、如第五方面所述的重组表达载体、如第六方面所述的病毒颗粒或者如第七方面所述的药物组合物。
本发明一些实施方案中,所述宿主细胞如第八方面所定义。
本发明的第十二方面提供一种制备如第一方面所述的snRNA核酸分子或者如第二方面所述的组合的方法,所述方法包括生物合成或化学合成如第一方面所述的snRNA核酸分子或者如第二方面所述的组合的步骤。
本发明的第十三方面提供一种如第一方面所述的snRNA核酸分子、如第二方面所述的组合、如第三方面所述的DNA分子、如第四方面所述的基因表达盒、如第五方面所述的重组表达载体、如第六方面所述的病毒颗粒或者如第七方面所述的药物组合物在制备治疗USH2A外显子13突变相关的疾病的药物中的应用。
本发明一些实施方案中,所述USH2A外显子13突变为致病突变或非致病突变。
本发明一些实施方案中,所述疾病选自眼病和耳病。
本发明中,如第八方面、第九方面、第十方面和第十一方面所述的方法是非治疗目的的,例如用于药物研发的实验室研究、试剂盒开发。
本发明的第十四方面提供一种治疗USH2A外显子13突变相关的疾病的方法,所述方法包括向有需要的患者施用有效量的如第一方面所述的snRNA核酸分子、如第二方面所述的组合、如第三方面所述的DNA分子、如第四方面所述的基因表达盒、如第五方面所述的重组表达载体、如第六方面所述的病毒颗粒或者如第七方面所述的药物组合物。
本发明一些实施方案中,所述USH2A外显子13突变相关的疾病如第十三方面所述。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
本发明提供的snRNA核酸分子靶向USH2A外显子13及其两侧靶区域,snRNA核酸分子包括识别结构域、茎环序列和Sm序列,识别结构域的数量为至少两个,至少两个的识别结构域在snRNA中形成“串联”结构,不同靶位点的U7-snRNA串联能够高效诱导USH2A外显子13的剪接跳跃,且单跳读的频率更高;尤其对于发生致病或非致病变的USH2A外显子13,本发明的snRNA能够以低剂量安全、有效进行USH2A外显子13单跳读,在预防和/或治疗Usherin蛋白表达异常相关的眼病和耳病中具有重要的临床价值。
附图说明
图1为以单个识别结构域为例靶向USH2A的外显子13的U7-snRNA的结构和作用效果示意图。
图2为靶向靶区域8的U7-snRNA在基因组上的位置示意图。
图3A-3B显示的是,不同靶位点U7 snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃效果。
图4为不同靶点的snRNA高效诱导USH2A pre-mRNA外显子13单独剪接跳跃效率示意图。
图5为靶向靶区域1的U7-snRNA在基因组上的位置示意图。
图6为靶向靶区域1的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA外显子13剪接跳跃的细胞比例结果图。
图7为靶向靶区域1的U7-snRNA诱导的(USH2A pre-mRNA外显子13剪接跳跃)GFP阳性细胞的平均FITC强度柱状图。
图8为靶向靶区域2的U7-snRNA在基因组上的位置示意图。
图9为靶向靶区域2的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA外显子13剪接跳跃的细胞比例结果图。
图10为靶向靶区域2的U7-snRNA诱导的(USH2A pre-mRNA外显子13剪接跳跃)GFP阳性细胞的平均FITC强度柱状图。
图11为靶向靶区域3的U7-snRNA在基因组上的位置示意图。
图12为靶向靶区域3的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA外显子13剪接跳跃的细胞比例结果图。
图13为靶向靶区域3的U7-snRNA诱导的(USH2A pre-mRNA外显子13剪接跳跃) GFP阳性细胞的平均FITC强度柱状图。
图14为靶向靶区域4的U7-snRNA在基因组上的位置示意图。
图15为靶向靶区域4的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA外显子13剪接跳跃的细胞比例结果图。
图16为靶向靶区域5的U7-snRNA在基因组上的位置示意图。
图17为靶向靶区域5的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA外显子13剪接跳跃的细胞比例结果图。
图18为靶向靶区域6的U7-snRNA在基因组上的位置示意图。
图19为靶向靶区域6的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA外显子13剪接跳跃的细胞比例结果图。
图20为靶向靶区域7的U7-snRNA在基因组上的位置示意图。
图21为靶向靶区域7的U7-snRNA在报告载体细胞中诱导USH2A pre-mRNA外显子13剪接跳跃的细胞比例结果图。
图22为靶向不同区域的U7 snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃细胞的平均FITC强度结果图。
图23为本发明串联U7 snRNA的结构示意图。
图24A为本发明串联U7 snRNA与USH2A pre-mRNA靶向方式示意图1;
图24B为本发明串联U7 snRNA与USH2A pre-mRNA靶向方式示意图2。
图25为实施例8-1识别结构域串联的pUC57-U7-snRNA诱导USH2A pre-mRNA外显子13剪接跳跃效率。
图26为实施例8-2识别结构域串联的pUC57-U7-snRNA诱导USH2A pre-mRNA外显子13剪接跳跃效率。
图27显示在WERI细胞中检测化学合成的U7 snRNA诱导USH2A pre-mRNA外显子13剪接跳跃的效率;
图中,泳道1:50pmol化学合成和修饰的U7-snRNA#30+#4;泳道2:50pmol化学合成和修饰的U7-snRNA#26+#15;泳道3:50pmol AON1;泳道4:50pmol AON2;泳道5:EGFP;泳道6:GL DNA Marker 2000。
图28为实施例9的RT-PCR电泳条带定量分析柱状图;
图中,▲E12-E13表示表示同时剪接跳过外显子12和外显子13的USH2A mRNA,总▲表示剪接跳过外显子13或同时剪接跳过外显子12和外显子13的USH2A mRNA总和。
图29A显示串联U7 snRNA的结构示意图。
图29B对比U7-hnRNP A1-snRNA和U7-snRNA串联诱导USH2A pre-mRNA外显子13 剪接跳跃效率。
图30显示通过体外梯度爬坡实验检验,3×U7 snRNA串联诱导USH2A外显子13剪接。
图31显示通过体外剂量爬坡实验验证AAV-U7 snRNA串联诱导剪接跳跃效果显著优于AON。
图32显示snRNA诱导人源化小鼠视网膜细胞USH2A pre-mRNA外显子13剪接跳跃优于AON。
图33显示不同血清型的AAV-U7 snRNA注射诱导兔子眼部细胞USH2A pre-mRNA外显子13剪接跳跃优于AON。
图34显示22周AAV递送的U7 snRNA仍能维持诱导USH2A pre-mRNA外显子13剪接跳跃。
图35显示AAV-1×U7 snRNA串联与AAV-4×U7 snRNA组合的诱导剪接跳跃效率的长期维持能力。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1:合成U7-snRNA
1、snRNA的骨架合成
野生型U7 snRNA包括茎环结构(scafford)、U7特异性的Sm序列(AAUUUGUCUAG,SEQ ID NO:5)和识别结构域(与复制依赖型组蛋白pre-mRNA互补)。本申请的U7 snRNA可以在NCBI上小鼠野生型U7 snRNA的基因序列(NCBI Reference Sequence:NR_024201.3)的基础上,其中U7特异性Sm结合位点被替换为优化的共有Sm序列,即SmOPT(AAUUUUUGGAG,SEQ ID NO:6),SmOPT序列的5′端的原识别结构域更换为与USH2A pre-mRNA特定靶位点反向互补配对的识别结构域,SmOPT序列的3′端保留U7原茎环结构序列(CAGGUUUUCUGACUUCGG UCGGAAAACCCCU,SEQ ID NO:7)。
如图1所示,靶向USH2A pre-mRNA外显子13的非串联U7 snRNA识别结构域序列与选自USH2A pre-mRNA内含子12-外显子13-内含子13的靶序列反向互补配对,靶序列可以选自USH2A pre-mRNA外显子13的3′段序列靶区域。
具体操作过程:
首先,通过全基因合成的方式,合成包含基因序列——U7-snRNA基因表达盒骨架(5′-小鼠U7启动子-smOPT序列-U7 snRNA scafford-snRNA基因特异性3′盒-3′)的pUC57载体。其中U7启动子与smOPT之间加入2个Tpye II型限制性内切酶识别位点(如BsaI、AarI、BsmBI等),以方便后续切除、替换以及插入其他识别结构域序列。snRNA基因特异性3′盒为小鼠基因组(GenBank:X54748.1)U7 snRNA基因3′端后,包含“GTCTACAATGAAA(SEQ ID NO:8)”的序列,参与pre-snRNA的加工。
2、构建靶向靶区域的U7 snRNA载体
本申请USH2A pre-mRNA外显子13的3′段区域序列(SEQ ID NO:1)对应的人基因组定位为Chr1:216246563-216246753(对应于NCBI数据库GRch38版本),SEQ ID NO:1的序列如下:
UAAAUAUAUUUUAUCUUUAGGGCUUAGGUGUGAUCAUUGCAAUUUUGGAUUUAAAUUUCUCCGAAGCUUUAAUGAUGUUGGAUGUGAGCCCUGCCAGUGUAACCUCCAUGGCUCAGUGAACAAAUUCUGCAAUCCUCACUCUGGGCAGUGUGAGUGCAAAAAAGAAGCCAAAGGACUUCAGUGUGACACCUGCAGAGAAAACUUUUAUGGGUUAGAUGUCACCAAUUGUAAGGCCUGUGACUGUGACACAGCUGGAUCCCUCCCUGGGACUGUCUGUAAUGCUAAGACAGGGCAGUGCAUCUGCAAGCCCAAUGUUGAAGGGAGACAGUGCAAUAAAUGUUUGGAGGGAAACUUCUACCUACGGCAAAAUAAUUCUUUCCUCUGUCUGCCUUGCAACUGUGAUAAGACUGGGACAAUAAAUGGCUCUCUGCUGUGUAACAAAUCAACAGGACAAUGUCCUUGCAAAUUAGGGGUAACAGGUCUUCGCUGUAAUCAGUGUGAGCCUCACAGGUACAAUUUGACCAUUGACAAUUUUCAACACUGCCAGAUGUGUGAGUGUGAUUCCUUGGGGACAUUACCUGGGACCAUUUGUGACCCAAUCAGUGGCCAGUGCCUGUGUGUGCCUAAUCGUCAAGGAAGAAGGUGUAAUCAGUGUCAACCAGGUAAGAAAGAAAUGUAUUACAU。以上所述USH2A pre-mRNA外显子13及两侧临近靶区域(Chr1:216246563-216247246对应的pre-mRNA区域)除了以上列举的无突变序列外,还可以是含有天然致病/非致病突变的靶区域,所述突变位点包括以下突变位点中至少1个的靶区域:c.2242C>T、c.2276G>T、c.2299delG、c.2522C>A、c.2541C>A、c.2761delC、c.2776C>T、c.2802T>G、c.2209C>T、c.2310delA、c.2391_2392deITG、c.2431A>T、c.2431_2432delAA、c.2440C>T、c.2525dup、c.2610C>A、c.2755C>T、c.2176T>C、c.2236C>G、c.2296T>C、c.2332G>T。
USH2A pre-mRNA外显子13的5′段靶区域(chr1:216247130-216247246对应的pre-mRNA区域)(SEQ ID NO:2)序列如下:

USH2A pre-mRNA外显子13的3′段靶区域(chr1:216246563-216246753对应的pre-mRNA区域)(SEQ ID NO:3)序列如下:
USH2A pre-mRNA外显子13的3′段优选靶区域(chr1:216246563-216246649对应的pre-mRNA区域)(SEQ ID NO:4)序列如下:
USH2A pre-mRNA外显子13的3′段区域(Chr1:216246563-216246626对应的pre-mRNA区域)(区域8,SEQ ID NO:9)序列为:UGCCUAAUCGUCAAGGAAGAAGGUGUAAUCAGUGTCAACCAGGUAAGAAAGAAAUGUAUUACAU,也可以是含有天然突变的USH2A pre-mRNA外显子13的3′段区域序列,如UGCCUAAUCGUCAAGGAAGAAGGUGUAAUCAGUGGCAACCAGGUAAGAAAGAAAUGUAUUACAU(下划线标注的为天然致病突变c.2802T>G,SEQ ID NO:10)。
根据表1中的snRNA识别结构域序列对应的转录前DNA序列,分别合成对应的Oligo DNA。Oligo DNA正义链为识别结构域序列对应的DNA序列,并且5′加CCGCA,反义链为识别结构域序列的反义互补序列5′加AATT并且3′加T。例如,识别结构域序列为5′-NNN-3′,则合成的Oligo DNA正义链为5′-CCGCANNN-3′,反义链为5′-AATTNNNT-3′。
将合成的Oligo DNA正义链和反义链按照退火反应体系(反应总体积20μl:Oligo-F(100μM)2μl+Oligo-R(100μM)2μl+10×NEB Cutter smart buffer 2μl+去离子水16μl)混合,95℃孵育5分钟后放置在冰上冷却退火形成带粘性末端的双链DNA。稀释100倍后取1μl与10ng BsaI酶切回收的线性化pUC57-U7 snRNA骨架质粒进行T4连接酶连接。连接产物进一步通过转化大肠杆菌感受态细胞、挑单克隆、PCR和测序验证,获得用于诱导USH2A外显子13剪接跳跃的U7 snRNA载体。提纯质粒,保存于-20℃备用。
表1.snRNA的识别结构域序列

其中,snRNA#24、snRNA#25、snRNA#27和snRNA#29为人、猴同源。
3、U7-snRNA的化学合成和修饰
与寡核苷酸类似,U7 snRNA也可以通过直接化学合成的方式,产生包含引导序列、smOPT和U7 snRNA scafford的RNA。体外合成的U7 snRNA可以通过特定修饰使其耐受核酸酶降解,或者增加对靶序列的亲和力。
本实施例通过化学合成了U7 snRNA,其5'和3'末端的3个碱基各进行2'甲氧基(2'-OME)修饰和硫代修饰,以增加核酸酶抗性。以snRNA#25、snRNA#26为例,化学合成的snRNA序列和修饰如下(*表示硫代磷酸化骨架,m表示2′-甲氧基修饰,下划线表示与靶序列反向互补配对的识别结构域,斜体表示smOPT序列):
化学合成和修饰的U7-snRNA#25序列如下:
化学合成和修饰的U7-snRNA#25双向延长序列如下:
化学合成和修饰的U7-snRNA#26序列如下:
化学合成和修饰的U7-snRNA#26单向延长序列如下:
实施例2:构建用于定量评价USH2A外显子13剪接跳跃效率的报告载体
将RGleft-USH2A Exon13mut-RGright序列(5′端和3′端分别加入AgeI和EcoRI酶切位点)通过全基因合成的方式获取,通过对合成序列、pX601质粒(Addgene,61591)进行限制性内切酶AgeI和EcoRI酶切、电泳、切胶回收和连接,将合成的序列插入pX601载体的AgeI和EcoRI酶切位点之间,替换原载体的SaCas9基因序列,获得报告载体。进一步通过转化大肠杆菌感受态细胞、挑单克隆、PCR和测序验证,获得提纯报告载体质粒,保存于-20℃备用。
报告载体结构为:pCMV-RGleft-USH2A EXON13mut-RGright,RG表示报告功能基因(reporter gene),RGleft表示没有报告功能的报告基因5′端前半部分,RGright表示没有报告功能的报告基因3′端后半部分,RGleft和RGright串联表达可正常行使完整报告基因功能。本发明实施例中报告基因为绿色荧光基因EGFP,则载体结构为pCMV-EGFPleft-Exon13mut-EGFPright。Exon13mut表示包含致病突变的USH2A外显子13,及其上下游内含子序列(上游内含子序列为人USH2A基因内含子12的5′端204bp和3′端490bp组合的基因序列;下游内含子序列为人USH2A内含子13的5′端703bp和3′端216bp组合的基因序列)。本申请实施例中所述的USH2A外显子13的致病突变可以为c.2299delG或c.2802T>G或任意突变,则 获得的载体结构分别为pCMV-EGFPleft-Exon13c.2299delG-EGFPright、pCMV-EGFPleft-Exon13c.2802T>G-EGFPright。一些实施例中的突变还可以是或包括c.2276G>T、C.2522C>A、c.2242C>T、c.2541C>A、c.2761delC和C.2776C>T等。
RGleft,例如EGFPleft序列为:
RGright,例如EGFPright序列为:
实施例3:USH2A pre-mRNA外显子13的3′段区(区域8)不同靶位点的U7-snRNA介导的USH2A外显子13剪接跳跃效果
293T细胞按一定量接种至24孔板,使得24小时后细胞汇合度达到约80%。选用Lipofectamine2000将pCMV-EGFPleft-Exon13mut-EGFPright和靶向USH2A pre-mRNA的pUC57-U7 snRNA质粒共转染293T细胞(载体质量比例为100ng:400ng),使用单独转染报告质粒(Reporter,报告组)、共转染报告质粒和pUC57-U7Scramble(SC组)的293T细胞分别作为两种阴性对照,不转染任何质粒的293T细胞作为空白对照。转染后的细胞继续培养48-72小时,使用胰酶消化成单细胞,随后使用流式细胞仪检测不同U7 snRNA组的GFP阳性率(即USH2A外显子13被诱导剪接跳跃的细胞比例)。本实施例检测了不同实验组(如图2所示)的平均FITC强度,即GFP阳性细胞FITC荧光强度的平均值,以及GFP阳性率,阳性细胞的GFP蛋白表达量。
本实施例比对了不同靶位点的U7-snRNA诱导剪接跳跃的效率。下表2和图3A-3B显示了不同靶位点U7 snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃效果。结果显示所有靶向USH2A pre-mRNA外显子13的3′段序列的U7-snRNA(#24-#34)均能在报告基因细胞中诱导USH2A外显子13的剪接跳跃,USH2A诱导剪接跳跃的效率高。
表2不同靶位点U7 snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃细胞比例
实施例4:化学合成的snRNA高效诱导USH2A pre-mRNA外显子13单独剪接跳跃
人源宿主细胞按6×105/孔接种至24孔板,本实施例选用的人源视网膜神经细胞为WERI-Rb-1细胞(视网膜神经细胞系)。用Lipofectamine2000将体外合成的100pmol U7-snRNA#24、#25、#26、#27、#28、#29、#30、#33、#34转染WERI细胞。转染后的细胞继续培养72小时,随后提取每个实验组细胞的RNA,反转录获得cDNA,通过引物AGCCTTTCCGCCAAGGTGATC(SEQ ID NO:30)和CACAACGTTGCCCAGCAATGG(SEQ ID NO:31)进行RT-PCR实验,检测成熟的USH2A mRNA是否存在外显子的剪接跳跃,电泳结果如图4所示。结果显示,U7-snRNA#24-34均能高效诱导外显子13的剪接跳跃,且几乎未见外显子13和外显子12共同剪接跳跃,可知靶向3′端区域的U7 snRNA可高效诱导USH2A pre-mRNA外显子13的单独剪接跳跃,具有较高的安全性。
实施例5:构建靶向USH2A 外显子13及其附近不同位点的U7-snRNA
本实施例分别针对USH2A pre-mRNA的7个靶区域设置了21个靶位点,USH2A Pre-mRNA的7个靶区域如下所示。
外显子13区域1(SEQ ID NO:32)(Chr1:216247142-216247185):CGAAGCUUUAAUGAUGUUGGAUGUGAGCCCUGCCAGUGUAACCU;
外显子13区域2(SEQ ID NO:33)(Chr1:216247130-216247161):GAGCCCUGCCAGUGUAACCUCCAUGGCUCAGU;
外显子13区域3(SEQ ID NO:34)(Chr1:216246616-216246649): AAUCAGUGGCCAGUGCCUGUGUGUGCCUAAUCGU;
外显子13区域4(SEQ ID NO:36)(Chr1:216247210-216247246):UAAAUAUAUUUUAUCUUUAGGGCUUAGGUGUGAUCAU;
外显子13区域5(SEQ ID NO:37)(Chr1:216247204-216247232):CUUUAGGGCUUAGGUGUGAUCAUUGCAAU;
外显子13区域6(SEQ ID NO:38)(Chr1:216247187-216247220):GGUGUGAUCAUUGCAAUUUUGGAUUUAAAUUUCU;
外显子13区域7(SEQ ID NO:39)(Chr1:216247169-216247202):UUGGAUUUAAAUUUCUCCGAAGCUUUAAUGAUGU。
所述21个靶位点如下表所示。
表3.snRNA的识别结构域序列

其中,snRNA#9、snRNA#15、snRNA#17和snRNA#19为人、猴同源。
实施例6:检测靶向不同区域的U7-snRNA介导的USH2A外显子13剪接跳跃效果
根据实施例3所述方法,在报告细胞体系中检测靶向不同区域的U7 snRNA组的GFP阳性率和平均FITC强度。
靶向靶区域1的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5′端到3′端)如图5所示。靶向靶区域1的U7-snRNA实验结果如图6~7和下表4所示,靶向靶区域1的所有U7-snRNA均能在报告基因细胞中诱导USH2A外显子13的剪接跳跃。现有技术靶向该区域1的AON不能诱导外显子13剪接跳跃,但靶向该区域1的snRNA却可高效诱导外显子13剪接跳跃。
表4靶向靶区域1的U7-snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃细胞比例
靶向靶区域2的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5′端到3′端)如图8所示。靶向靶区域2的U7-snRNA实验结果如图9~10和下表5所示,靶向靶区域2的所有U7-snRNA均能在报告基因细胞中诱导USH2A外显子13的剪接跳跃。现有技术靶向该区域2的AON诱导外显子13剪接跳跃的效果较低,但靶向该区域2的snRNA却可高效诱导外显子13剪接跳跃。
表5靶向区域2的U7-snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃细胞比例

靶向靶区域3的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5′端到3′端)如图11所示。靶向靶区域3的U7-snRNA的实验结果如图12~13和下表6所示,靶向靶区域3的所有U7-snRNA均能在报告基因细胞中诱导USH2A外显子13的剪接跳跃。现有技术靶向该区域3的AON诱导外显子13剪接跳跃的效果较低,但靶向该区域3的snRNA却可高效诱导外显子13剪接跳跃。
表6靶向区域3的U7-snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃细胞比例
结合靶区域1、2、3结果分析发现,尽管现有技术中显示靶区域1、2、3是AON靶向非敏感区,即靶向该区域无法/低效率诱导外显子13剪接跳跃,但是靶向该区域的snRNA却可显著外显子13剪接跳跃。因此,尽管snRNA与AON都可以诱导剪接跳跃,但两者的作用机制不同,靶位点敏感性(所适用的靶区域靶位点)也不相同。
同时,如图6、9、12和下表7所示,尽管靶向同一区域的(诱导剪接跳跃的细胞比例)GFP%接近,但是靶向同一区域的不同靶点snRNA在同一细胞中诱导获得剪接跳跃的mRNA及其蛋白水平(平均FITC强度)有所区别。且靶区域1、靶区域2在同一细胞中诱导获得剪接跳跃的mRNA及其蛋白水平均优于靶区域3。
表7靶向靶区域1、2、3的U7-snRNA诱导的(USH2A pre-mRNA外显子13剪接跳跃)GFP阳性细胞的平均FITC强度

靶向区域4的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5′端到3′端)如图14所示。靶向区域4的U7-snRNA的实验结果如图15和下表8所示,靶向靶区域4的所有U7-snRNA均能在报告基因细胞中诱导USH2A外显子13的剪接跳跃。
表8靶向区域4的U7-snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃细胞比例
靶向靶区域5的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5′端到3′端)如图16所示。靶向靶区域5的U7-snRNA的实验结果如图17和下表9所示,靶向靶区域5的所有U7-snRNA均能在报告基因细胞中诱导USH2A外显子13的剪接跳跃。
表9靶向区域5的U7-snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃细胞比例
靶向靶区域6的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5′端到3′端)如图18所示。靶向靶区域6的U7-snRNA实验结果如图19和下表10所示,靶向靶区域6的所有U7-snRNA均能在报告基因细胞中诱导USH2A外显子13的剪接跳跃。
表10靶向区域6的U7-snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃细胞比例
靶向靶区域7的U7-snRNA在基因组上的位置(图片从左到右对应于基因组从5′端到3′端)如图20所示。靶向靶区域7的U7-snRNA的实验结果如图21和下表11所示,靶向靶区域7的所有U7-snRNA均能在报告基因细胞中诱导USH2A外显子13的剪接跳跃。
表11靶向区域7的U7-snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃细胞比例

不同区域的U7-snRNA诱导的GFP阳性细胞的平均FITC强度如图22和下表12所示。尽管靶向同一区域的(诱导剪接跳跃的细胞比例)GFP%接近,但是靶向同一区域的不同靶点snRNA在同一细胞中诱导获得剪接跳跃的mRNA及其蛋白水平(平均FITC强度)不同。
靶向区域2不仅获得较高的诱导剪接跳跃的细胞比例(GFP%),而且在同一细胞中诱导获得剪接跳跃的mRNA及其蛋白水平(平均FITC强度)较高。
而现有技术AON诱导剪接跳跃效率较高的靶位点#2以及其临近位点#1和#3(区域4),在snRNA体系中在同一细胞中诱导获得剪接跳跃的mRNA水平较低。现有技术靶向区域7的AON效率比区域5高,但在snRNA体系靶向区域5的效率比区域7更高。现有技术靶向区域3的AON效率比区域2更高,但是在snRNA体系靶向区域2的效率比区域3更高。因此,尽管snRNA与AON都可以诱导剪接跳跃,但两者的作用机制不同,靶位点敏感性也不相同。且结合实施例8结果分析,由于snRNA#24的效率与snRNA#2、AON1(SEQ ID NO:77)的效率近似,则可推知snRNA#3-#11效果均优于snRNA#2、snRNA#24,优于AON1。
表12靶向不同区域的U7-snRNA在报告基因细胞中诱导USH2A pre-mRNA外显子13剪接跳跃细胞的平均FITC强度
本申请靶向诱导USH2A pre-mRNA外显子13剪接跳跃的U7-snRNA并不限于实施例3和4所列举的U7-snRNA。本申请U7-snRNA识别结构域识别的靶位点选自USH2A pre-mRNA内含子12-外显子13-内含子13,优选地选自外显子13及两侧临近靶区域(SEQ ID NO:1)。
实施例7:不同靶位点的U7-snRNA组合介导的USH2A外显子13剪接跳跃
1、构建U7-snRNA多靶点组合载体
根据Golden Gate Assembly技术,以不同U7 snRNA质粒为模板,PCR扩增U7 snRNA cassette(表达盒)同时通过引物在扩增子的两端引入额外的5′侧翼碱基和正确方向的BsaI酶切位点,使得相邻的不同U7 snRNA cassette通过BsaI酶切后产生特异的互补粘性末端,首尾U7 snRNA cassette则通过BsaI酶切后产生与HindIII+NotI酶切线性化骨架载体相同的粘性末端。最后使用Golden Gate Assembly Kit(NEB#E1601)将上述PCR产物以及HindIII+NotI酶切回收的pUC57-U7 snRNA Backbone组装。组装方法如下所示:pUC57-U7 snRNA Backbone-HindIII+NotI、80ng;U7 snRNA#A cassette PCR product、20ng;U7 snRNA#B cassette PCR product、20ng;U7 snRNA#C cassette PCR product、20ng;T4 DNA Ligase Buffer(10X)、2μl;NEB Golden Gate Assembly Mix、1μl;反应过程:(37℃,5min→16℃,5min)×20→60℃,5min。
Golden Gate Assembly组装产物进一步通过转化大肠杆菌感受态细胞、挑选单克隆、PCR和测序验证,获得用于诱导USH2A外显子13剪接跳跃的U7 snRNA多靶点组合载体。提纯质粒,保存于-20℃备用。
实施例8-1:识别结构域串联U7 snRNA在报告基因细胞中诱导USH2A外显子13剪接跳跃
1、识别结构域串联U7 snRNA的制备
识别结构域串联的U7 snRNA意为一个U7 snRNA茎环结构、一个smOPT序列,连接着两个或者两个以上的识别结构域,其结构为5′-识别结构域B-识别结构域A-smOPT序列-茎环结构-3′,如图23所示。所述串联U7 snRNA的识别结构域A和B识别不同靶位点的RNA序列。
根据表1和表3中的snRNA识别结构域序列对应的转录前DNA序列,分别合成对应的Oligo DNA。Oligo DNA正义链为识别结构域序列对应的DNA,并且5′加CCGCA,反义链为识别结构域序列的反义互补序列5′加AATT并且3′加T。例如,待串联的snRNA#15和snRNA#25的识别结构域序列分别为ACACUGGCAGGGCUCACAUCCA(SEQ ID NO:54)和AUUACACCUUCUUCCUUGACGAUU(SEQ ID NO:13),则合成的Oligo DNA正义链为: 反义链为:
将合成的Oligo DNA正义链和反义链按照退火反应体系(反应总体积20μl:Oligo-F(100μM)2μl+Oligo-R(100μM)2μl+10×NEB Cutter smart buffer 2μl+去离子水16μl)混合,95℃孵育5分钟后放置在冰上冷却退火形成带粘性末端的双链DNA。稀释100倍后取1μl与10ng BsaI酶切、回收的线性化pUC57-U7 snRNA骨架质粒进行T4连接酶连接。连接产物进一步通过转化大肠杆菌感受态细胞、挑单克隆、PCR和测序验证,获得用于诱导USH2A外显子13剪接跳跃的U7 snRNA载体。提纯质粒,保存于-20℃备用。构建的载体命名为pUC57-U7 snRNA#B-#A,A和B分别表示识别结构域编号,对应于表1和表3中的识别结构域列表和序列。例如,pUC57-snRNA#25-#15、pUC57-snRNA#24-#9、pUC57-snRNA#24-#19、pUC57-snRNA#25-#9、pUC57-snRNA#25-#19、pUC57-snRNA#29-#9、pUC57-snRNA#25-#15。
识别结构域串联U7 snRNA还可以依据实施例2所述的方法进行化学合成和修饰,例如,其具体序列和修饰如下(*表示硫代磷酸化骨架,m表示2′-甲氧基修饰,下划线表示与靶序列反向互补配对的识别结构域,斜体表示smOPT序列):
化学合成和修饰的U7 snRNA #28-#3:
化学合成和修饰的U7 snRNA #30-#4:
化学合成和修饰的U7 snRNA #25-#15:
化学合成和修饰的U7 snRNA #26-#16:
上述序列中,“-”仅表示两段序列的连接顺序。
在一些实施例中,优选化学合成的snRNA序列总长度大于等于96bp。
2、识别结构域串联U7 snRNA在报告基因细胞中诱导USH2A外显子13剪接跳跃
293T细胞按一定量接种至24孔板,使得24小时后细胞汇合度达到约80%。使用Lipofectamine2000将pCMV-EGFPleft-Exon13mut-EGFPright分别和表达双识别结构域串联U7snRNA质粒或表达单识别结构域U7 snRNA质粒共转染293T细胞(载体质量比例为100ng:400ng),使用单独转染报告质粒(Report,报告组)、共转染报告质粒的和pUC57-U7Scramble(SC组)的293T细胞作为两种阴性对照,不转染任何质粒的293T细胞作为空白对照。转染后的细胞继续培养48-72小时,使用胰酶消化成单细胞,随后使用流式细胞仪检测不同snRNA组的GFP阳性率。下表13和图25显示的是不同靶位点串联的U7-snRNA诱导USH2A pre-mRNA外显子13剪接跳跃的效率。
本实施例通过将靶向不同靶位点串联的U7-snRNA应用,发现可以在报告基因细胞中提升诱导USH2A外显子13的剪接跳跃的效率。本实施例中的不同靶点串联诱导的剪接跳跃效率高于单个靶点的效果,而且优于已知的USH2A外显子13的剪接跳跃技术。同时,结合其它实施例的数据,让人意外的是,尽管单个U7 snRNA#25诱导外显子13剪接跳跃的效率不高,但是U7 snRNA#25串联其它U7 snRNA诱导外显子13剪接跳跃的效率却显著提升。此外,本发明构建了不同靶位点串联的AON,尝试诱导USH2A pre-mRNA外显子13剪接跳跃,但发现几乎没有剪接跳跃效率,进一步验证了AON与snRNA诱导剪接跳跃的作用机制不相同。
表13不同靶位点的U7-snRNA串联诱导USH2A pre-mRNA外显子13剪接跳跃效率

本实施例在U7 snRNA的识别区域中将识别不同靶位点的RNA序列串联,构建靶向不同靶位点的串联U7SnRNA。本实施例中的识别结构域串联的snRNA,其包含两个或者两个以上的识别结构域,串联于snRNA的5′端(如图23)。在一些实施例中,两个串联的识别结构域可以识别同一个靶位点,在同一表达载体或者同一snRNA驱动的情况下,提升靶向识别结构域数量,提升诱导USH2A pre-mRNA外显子13的剪接跳跃效率。
当所述U7 snRNA是两个或者两个以上不同的识别结构域串联,则从U7 snRNA的5′端到3′端的识别结构域排序为依据识别结构域对应的靶位点在USH2A pre-mRNA中的位置,按3′端到5′端排序。
实施例8-2:识别结构域串联U7 snRNA在报告基因细胞中诱导USH2A外显子13剪接跳跃
1、识别结构域串联U7 snRNA的制备
识别结构域串联的U7 snRNA意为一个U7 snRNA茎环结构、一个smOPT序列,连接着两个或者两个以上的识别结构域,其结构为5′-识别结构域B-识别结构域A-smOPT序列-茎环结构-3′,如图23所示。所述串联U7 snRNA的识别结构域A和B识别不同靶位点的RNA序列。进一步的,识别结构域串联的U7 snRNA与USH2A pre-mRNA靶向方式如图24A或图24B。
根据表1和表3中的snRNA识别结构域序列对应的转录前DNA序列,分别合成对应的Oligo DNA。Oligo DNA正义链为识别结构域序列对应的DNA,并且5′加CCGCA,反义链为识别结构域序列的反义互补序列5′加AATT并且3′加T。方法同实施例8-1,例如,待串联的snRNA#15和snRNA#25的识别结构域序列分别为ACACUGGCAGGGCUCACAUCCA(SEQ ID NO:54)和AUUACACCUUCUUCCUUGACGAUU(SEQ ID NO:13),则合成的Oligo DNA正义链为: 反义链为:
将合成的Oligo DNA正义链和反义链按照退火反应体系(反应总体积20μl:Oligo-F(100 μM)2μl+Oligo-R(100μM)2μl+10×NEB Cutter smart buffer 2μl+去离子水16μl)混合,95℃孵育5分钟后放置在冰上冷却退火形成带粘性末端的双链DNA。稀释100倍后取1μl与10ng BsaI酶切、回收的线性化pUC57-U7 snRNA骨架质粒进行T4连接酶连接。连接产物进一步通过转化大肠杆菌感受态细胞、挑单克隆、PCR和测序验证,获得用于诱导USH2A外显子13剪接跳跃的U7 snRNA载体。提纯质粒,保存于-20℃备用。构建的载体命名为pUC57-U7 snRNA#B-#A,A和B分别表示识别结构域编号,对应于表1和表3中的识别结构域列表和序列。例如,pUC57-snRNA#24-#9、pUC57-snRNA#25-#9、pUC57-snRNA#9-#24、pUC57-snRNA#9-#25。pUC57-snRNA#24-#9和pUC57-snRNA#25-#9中,U7 snRNA与USH2A pre-mRNA靶向方式如图24A;pUC57-snRNA#9-#24和pUC57-snRNA#9-#25中,U7 snRNA与USH2A pre-mRNA靶向方式如图24B。
识别结构域串联U7 snRNA还可以依据实施例2所述的方法进行化学合成和修饰,同实施例8-1,例如,其具体序列和修饰如下(*表示硫代磷酸化骨架,m表示2′-甲氧基修饰,下划线表示与靶序列反向互补配对的识别结构域,斜体表示smOPT序列):
化学合成和修饰的U7 snRNA #28-#3:
化学合成和修饰的U7 snRNA #30-#4:
化学合成和修饰的U7 snRNA #25-#15:
化学合成和修饰的U7 snRNA #26-#16:
上述序列中,“-”仅表示两段序列的连接顺序。
在一些实施例中,优选化学合成的snRNA序列总长度大于等于96bp。
2、识别结构域串联U7 snRNA在报告基因细胞中诱导USH2A外显子13剪接跳跃
293T细胞按一定量接种至24孔板,使得24小时后细胞汇合度达到约80%。使用Lipofectamine2000将pCMV-EGFPleft-Exon13mut-EGFPright分别和表达双识别结构域串联U7snRNA质粒共转染293T细胞(载体质量比例为100ng:400ng),使用单独转染报告质粒(Report,报告组)、共转染报告质粒的和pUC57-U7Scramble(SC组)的293T细胞作为两种阴性对照,不转染任何质粒的293T细胞作为空白对照。转染后的细胞继续培养48-72小时,使用胰酶消化成单细胞,随后使用流式细胞仪检测不同snRNA组的GFP阳性率。下表14和图26显示的是不同靶位点串联的U7-snRNA诱导USH2A pre-mRNA外显子13剪接跳跃的效率。
本实施例通过将靶向不同靶位点串联的U7-snRNA应用,发现可以在报告基因细胞中提升诱导USH2A外显子13的剪接跳跃的效率。本实施例中,U7 snRNA#24-#9和U7 snRNA#9-#24诱导外显子13剪接跳跃的效率无明显区别,U7 snRNA#25-#9和U7 snRNA#9-#25诱导外显子13剪接跳跃的效率无明显区别,表明识别结构域串联U7 snRNA中的两个以上识别结构域满足各自识别结构域与其在USH2A pre-mRNA中的靶位点反向互补即可,无需从U7snRNA的5′端到3′端的识别结构域排序为依据识别结构域对应的靶位点在USH2A pre-mRNA中的位置按3′端到5′端排序。
表14不同靶位点的U7-snRNA串联诱导USH2A pre-mRNA外显子13剪接跳跃效率
实施例9:化学合成的U7 snRNA在WERI细胞中诱导USH2A外显子13剪接跳跃
人源宿主细胞按6×105/孔接种至24孔板,本实施例选用的是WERI-Rb-1细胞系。用Lipofectamine2000将体外合成的50pmol snRNA组合1(U7-snRNA#30和U7-snRNA#4)、组合2(U7-snRNA#26和U7-snRNA#15)分别转染WERI细胞,转染相同剂量(50pmol)的反义寡核苷酸AON1(5′- MA*MG*MC*MU*MU*MC*MG*MG*MA*MG*MA*MA*MA*MU*MU*MU*MA*MA*MA*MU*MC*-3′,“M”表示2′-O-甲氧基修饰,“*”表示硫代磷酸化,SEQ ID NO:77)和AON2(5′-MU*MG*MA*MU*MC*MA*MC*MA*MC*MC*MU*MA*MA*MG*MC*MC*MC*MU*MA*MA*MA*-3′,“M”表示2′-O-甲氧基修饰,“*”表示硫代磷酸化,SEQ ID NO:78)作为对照组,转染1μg EGFP质粒作为阴性对照,不转染任何质粒的WERI细胞作为空白对照。转染后的细胞继续培养72小时,随后提取每个实验组细胞的RNA,反转录获得cDNA,通过引物AGCCTTTCCGCCAAGGTGATC(SEQ ID NO:30)和CACAACGTTGCCCAGCAATGG(SEQ ID NO:31)进行RT-PCR实验,检测成熟的USH2A mRNA是否存在外显子剪接跳跃,电泳结果如图27所示。进一步通过ImageJ软件对rt-PCR电泳条带进行定量分析,并针对剪接跳过外显子13或剪接跳过外显子12和13的成熟USH2A mRNA的比例进行统计和分析,如图28所示。
在内源性表达Usherin蛋白的WERI细胞中,将不同靶位点U7 snRNA组合诱导USH2A pre-mRNA外显子13剪接跳跃的效果与现有技术优选AON技术方案进行比较,由RT-PCR试验数据和分析结果可知,snRNA组合1和snRNA组合2诱导外显子13单剪接跳跃的效果显著优于现有技术最优技术方案AON1和AON2,且snRNA组合1和snRNA组合2诱导外显子12和13双剪接跳跃mRNA占总剪接跳跃mRNA的比例却比AON1、AON2低。因此,可明确U7 snRNA在确保较低的双跳USH2A mRNA副产品的同时,显著提升外显子13单剪接跳跃的效率。
此外,snRNA组合2是靶向临近于现有技术外显子12和13双剪接跳跃概率极高的AON位点,然而,snRNA组合2的双外显子剪接跳跃的发生概率却非常低。
实施例10带有可募集剪接调控蛋白的基序的U7 snRNA的剪接跳跃效果
连接hnRNP A1结合基序的U7 snRNA的构建。根据表中的序列对应的转录前DNA序列,分别合成对应的Oligo DNA。Oligo DNA正义链为靶序列的反向互补序列(识别结构域序列对应的DNA序列),并且5′加CCGCAATATGATAGGGACTTAGGGTG(SEQ ID NO:67),反义链为靶序列5′加AATT并且3′加CACCCTAAGTCCCTATCATATT(SEQ ID NO:68)。例如,识别结构域序列为NNN(识别结构域长度优选大于16个核苷酸),则合成的Oligo DNA正义链为反义链为(下划线表示识别结构域序列对应的DNA双链序列,粗斜体表示hnRNP A1蛋白的结合基序“UAGGGU”或“UAGGGA”对应的DNA双链序列)。
将合成的Oligo DNA正义链和反义链按照退火反应体系(反应总体积20μl:Oligo-F(100 μM)2μl+Oligo-R(100μM)2μl+10×NEB Cutter smart buffer 2μl+去离子水16μl)混合,95℃孵育5分钟后放置在冰上冷却退火形成带粘性末端的双链DNA。稀释100倍后取1μl与10ng BsaI酶切、回收的线性化pUC57-U7 snRNA backbone质粒连接。进一步通过转化大肠杆菌感受态细胞、挑选单克隆、PCR和测序验证,获得含有hnRNP A1结合基序的用于诱导USH2A外显子13剪接跳跃的U7 snRNA载体,载体命名为pUC57-U7-hnRNP A1-snRNA#A。提纯质粒,保存于-20℃备用。图29A显示的是,带有hnRNP A1的snRNA载体示意图。
U7-hnRNP A1-snRNA还可以依据本申请实施例所述的方法进行化学合成和修饰。以snRNA#15、snRNA#25为例,化学合成的U7-hnRNP A1-snRNA序列和修饰如下(*表示硫代磷酸化骨架,m表示2′-甲氧基修饰,下划线表示与靶序列反向互补配对的识别结构域,斜体表示smOPT序列,粗体表示hnRNP A1蛋白结合基序):
U7-hnRNP A1-snRNA#15:
U7-hnRNP A1-snRNA#25:
连接hnRNP A1结合基序的U7 snRNA在报告基因细胞中诱导USH2A外显子13剪接跳跃。293T按一定量接种至24孔板,使得24小时后细胞汇合度达到约80%。使用Lipofectamine2000将pCMV-EGFPleft-Exon13mut-EGFPright分别和pUC57-U7-hnRNP A1-snRNA#15质粒、pUC57-U7-hnRNP A1-snRNA#25质粒、pUC57-U7 snRNA#15质粒、pUC57-U7 snRNA#25质粒、pUC57-U7 snRNA#25-#15质粒共转染293T细胞(载体质量比例为100ng:400ng),使用单独转染报告质粒(报告组)、共转染报告质粒的和pUC57-U7Scramble(SC组)的293T细胞作为两种阴性对照,不转染任何质粒的293T细胞作为空白对照。转染后的细胞继续培养48-72小时,使用胰酶消化成单细胞,随后使用流式细胞仪检测不同snRNA组诱导的剪接跳跃效率。下表15和图29B显示的是,U7-hnRNP A1-snRNAUSH2A pre-mRNA外显子13剪接跳跃效率。
数据显示,在U7 snRNA的5′端引入hnRNP A1结合基序可显著提升诱导USH2A pre-mRNA外显子13剪接跳跃的效果,不仅提升了外显子13剪接跳跃的细胞(GFP+)的比例, 而且提升了每个细胞中剪接跳过外显子的mRNA水平(平均FITC强度)。而串联snRNA诱导外显子13剪接跳跃效果特别是平均FITC强度,则显著优于U7 snRNA的5′端引入hnRNP A1结合基序,提示USH2A pre-mRNA外显子13的剪接跳跃可能对串联结构的snRNA更为敏感。
表15 U7-hnRNP A1-snRNA诱导USH2A pre-mRNA外显子13剪接跳跃效率
本实施例在U7 snRNA的5′端引入游离尾部,所述游离尾部序列包括hnRNP A1蛋白的结合基序“UAGGGU”或“UAGGGA”,所述游离尾部序列可以含有1个、2个或者2个以上的hnRNP A1蛋白的结合基序,优选为2个,游离尾部序列优选为“UAUGAUAGGGACUUAGGGUG(SEQ ID NO:35)”,可募集hnRNP A1蛋白,促进USH2A外显子13的剪接跳跃。且该结构并不适用于识别结构域串联的snRNA。
在一些实施例中,所述U7 snRNA的5′端引入游离尾部为可以募集剪接调控蛋白的基序,所述剪接调控蛋白为hnRNP A1(Heterogeneous Nuclear Ribonucleoprotein A1)、SRSF1(Serine And Arginine Rich Splicing Factor 1)、RBM4(RNA Binding Motif Protein 4)、DAZAP1(DAZ Associated Protein 1)、SR(Serine And Arginine-Rich Protein)等。
实施例11:靶向诱导USH2A pre-mRNA外显子13剪接跳跃的AAV-U7 snRNA相关质粒载体构建和病毒包装
本实施例将靶向诱导USH2A pre-mRNA外显子13剪接跳跃的U7 snRNA基因插入并替换pAAV-CMV载体中两个ITR结构域的中间基因序列,构建pAAV-U7 snRNA载体,与AAV包装质粒:血清型pRC质粒(包含AAV2的Rep基因和每个血清型各自的Cap基因)、pHelper质粒(包含腺病毒的E2A、E4和VA基因的载体质粒)共转染宿主细胞,包装获得靶向USH2A pre-mRNA外显子13剪接跳跃的AAV-U7 snRNA病毒。具体操作过程如下:
首先,通过全基因合成的方式,合成基因序列——U7-snRNA基因表达盒骨架(未包含识别结构域):5′-小鼠U7启动子-smOPT序列-U7 snRNA scafford-snRNA基因特异性3′盒-3′。其中U7启动子与smOPT之间加入2个Tpye IIs型限制性内切酶识别位点(如BsaI、AarI、BsmBI等),以方便后续切除、替换以及插入其他识别结构域序列。将全基因合成的序列插入并替换pAAV-CMV质粒(Helper Free System(AAV5)试剂盒,TAKARA公司,Code No.6650)两个AAV2-ITR结构域之间的基因序列,获得pAAV-U7 snRNA骨架载体。
依照上述实施例所述方法,根据本申请的snRNA识别结构域序列或识别结构域序列的串联对应的转录前DNA序列,分别合成对应的Oligo DNA正义链和反义链,两端加入类似于Tpye IIs型限制性内切酶识别位点切割后的粘性末端。退火形成带粘性末端的识别结构域(单独/串联)双链DNA,T4连接酶连接入经过对应Tpye IIs型限制性内切酶酶切回收的线性化pAAV-U7 snRNA骨架质粒中,形成靶向USH2A pre-mRNA外显子13特定位点诱导剪接跳跃的pAAV-U7 snRNA质粒,依据识别结构域序列对应的snRNA编号对其进行命名,如pAAV-U7 snRNA#25等。
将目的基因(靶向诱导USH2A pre-mRNA外显子13剪接跳跃的U7-snRNA基因表达盒子)插入并替换pAAV-CMV质粒AAV2-ITR结构域之间的基因序列后,获得pAAV-U7 snRNA质粒载体。依据Helper Free System(AAV5)试剂盒说明书和标准的细胞操作流程包装获取靶向诱导USH2A pre-mRNA外显子13剪接跳跃的AAV-U7 snRNA病毒。
在转染之前24小时,将HEK293/293T细胞接种到100mm细胞培养皿,培养基为10%FBS的DMEM培养基,汇合度达到80%-90%时转染。转染前3小时,弃去旧培养基,更换新鲜培养基。转染时,同时将pAAV-U7 snRNA质粒、pRC质粒、pHelper质粒和PEI(聚乙烯亚胺)转染试剂按照以下的体系配置好,逐滴加入培养皿中。PEI转染混合物添加完毕后,轻轻晃动培养皿使转染试剂分布均匀,将培养基放置于37℃,5%CO2培养箱中培养。
PEI转染体系:pAAV质粒(1μg/μl)、6μL;pRC1/2/5/6质粒(1μg/μl);(pRC质粒衣壳基因决定血清型)、6μL;pHelper质粒(1μg/μl)、6μL;无血清DMEM培养基、500μL;PEI(1mg/mL)、110μL处理方式:涡旋混合数次,室温孵育5min。
转染后24小时,更换新鲜2%FBS的DMEM培养基。转染48-72小时后,收集含AAV病毒的细胞,清洗、离心,收集细胞沉淀,涡旋振荡使细胞沉淀松散。随后,依照Helper Free System(AAV5)试剂盒说明书,在细胞沉淀中加入0.5mL的AAV Extraction Solution A,涡旋振荡15秒使细胞沉淀充分悬浮。室温静置5分钟后,再涡旋振荡15秒。4℃,2000-14000g离心10分钟,去除细胞碎片。收集上清液到新的无菌离心管中,加入50μL AAV Extraction Solution B,使用移液枪吸打混匀,获得不同识别结构域的AAV-U7 snRNA病毒溶液,取部分以qPCR法检测病毒滴度,保存于80℃备用。
由于插入pAAV-U7 snRNA质粒AAV2-ITR结构域之间插入的目的基因片段应小于2.5kb,因此,可通过插入多个U7-snRNA基因表达盒子(5′-小鼠U7启动子-smOPT序列、U7 snRNA scafford-snRNA基因特异性3′盒-3′),从而确保在相同AAV病毒颗粒数量的情况下,提升U7snRNA的表达量,基因序列长度约为450bp,则优选地pAAV-U7 snRNA质粒中携带1-5个U7-snRNA基因表达盒子,所述pAAV-U7 snRNA质粒中的多个U7-snRNA基因表达盒子可以是具有相同的识别结构域或识别结构域组合,也可以是具有不同或者不完全相同的识别结构域组合。
本申请通过AAV递送U7 snRNA诱导USH2A pre-mRNA外显子13剪接跳跃,所述AAV的衣壳蛋白可以是天然来源的,也可以是基于天然来源衣壳蛋白的变体、或进行定向进化、或进行氨基酸/肽段合理改造(密码子优化、嵌合不同血清型功能肽段等)等,提升组织器官亲嗜性、免疫原性、提升转染效率等特性,如AAV2.5、AAV2i8、AAV-TT、AAV9.HR、CAM130等。
实施例12:串联U7 snRNA诱导剪接跳跃效果显著优于组合U7—体外剂量爬坡
依据实施例11所述,构建了AAV2血清型的AAV2-3×U7 snRNA #9-#25(3×U7 snRNA串联)、AAV2-2×U7 snRNA #9-2×U7 snRNA #25(4×U7 snRNA seperate)以及AAV2-2×U7-hnRNP A1-snRNA #9-2×U7-hnRNP A1-snRNA #25(4×U7 snRNA seperate-motif)载体,转染HEK293/293T细胞包装病毒,收集、纯化等操作分别获得AAV2病毒3×U7 snRNA串联、4×U7 snRNA seperate和4×U7 snRNA seperate-motif。
检测病毒滴度,按照MOI值分别为3×105、1×105、3×104、1×104、3×103、1×103、3×102(公式MOI值=病毒滴度(TU/mL)×病毒体积(mL)/细胞个数),分别在24孔板的WERI-Rb-1细胞(6×105/孔)中加入相应体积的病毒液,感染后的细胞继续培养72小时,随后提取每个实验组细胞的RNA,反转录获得cDNA,并通过表16中的对应引物/探针进行RT-PCR和qRT-PCR实验,检测AAV-U7 snRNA诱导USH2A pre-mRNA外显子13剪接跳跃的效率。
表16 RT-PCR引物和qRT-PCR探针列表

如图30所示,结果显示,AAV2病毒转导3×U7串联和4×U7-separate的对比,RT-PCR结果显示4×U7 separate效率较差,但加了hnRNPA1结合基序后效率提升,与3×U7串联效率可比。qRT-PCR检测结果与RT-PCR结果一致。
SsAAV载体的目的基因最大容量为4.7kb,ScAAV载体的目的基因最大容量为2.5kb,U7 snRNA表达盒大小约为450bp,则一个scAAV载体中最高能容纳9个U7 snRNA表达盒,一个scAAV载体中最高能容纳5个U7 snRNA表达盒。从实施例14的结果看,体外3×U7snRNA串联效果与2×U7 snRNA-hnRNP A1组合的诱导剪接跳跃效果相似,且优于2×U7snRNA组合。从实施例15和实施例16的结果看,体内1×U7 snRNA串联诱导剪接跳跃略优于2×U7 snRNA组合,但1×U7 snRNA串联诱导剪接跳跃的长期持续更显著。在不引入hnRNP A1结合基序的情况下,串联U7 snRNA的效果是优于U7 snRNA组合的。
实施例13:AAV-U7 snRNA诱导剪接跳跃效果显著优于AON—体外剂量爬坡
依据实施例11所述,构建了AAV2血清型的AAV2-U7 snRNA #9-#25(1×U7 snRNA串联)载体,转染HEK293/293T细胞包装病毒,收集、纯化等操作分别获得AAV2病毒1×U7snRNA串联,即AAV2-RM-101,检测病毒滴度备用。
WERI-Rb-1细胞按6×105/孔接种至24孔板,AAV2-RM-101病毒按照MOI值分别为3×105、1×105、3×104、1×104、3×103、1×103、3×102加入到WERI-Rb-1细胞中,作为不同MOI的实验组。分别以50nM、200nM的PROQR EX13-3(实施例9中的AON1)作为阳性对照组,分别以MOI为3×105的AAV2-U7-SCR(Scramble)、AAV2-U7-LUC(识别Luciferase)作为阴性对照。不同处理后的WERI-Rb-1细胞继续培养72小时,随后提取每个实验组细胞的RNA,反转录获得cDNA,并通过表16中的对应引物/探针进行RT-PCR和qRT-PCR实验,检测USH2A pre-mRNA外显子13剪接跳跃的效率。如图31所示,结果显示,AAV2-U7 snRNA #9-#25诱导的剪接跳跃效率显著优于AON1。
实施例14:snRNA诱导人源化小鼠视网膜细胞USH2A pre-mRNA外显子13剪接跳跃
通过基因编辑技术将C57/BL6J小鼠USH2A基因外显子12+部分侧翼序列[第12外显子上游(小鼠第11内含子3′端)约1670bp至第12外显子下游(小鼠第12内含子5′端)约1600bp]替换为人类USH2A基因外显子13+部分侧翼序列[第13外显子上游(人第12内含子3′端)约1611bp至第13外显子下游(人第13内含子5′端)约1599bp]+插入序列,而c.2208T  to G被引入到人USH2A外显子13中,获得携带c.2802T>G突变的USH2A外显子13人源化小鼠(USH2A EXON13c.2802T>G)。
通过视网膜下腔注射,分别将1μL总量为1E+10vg/眼的AAV2-U7 snRNA #9-#25(AAV5-1×U7)、AAV5-2×U7 snRNA #9-2×U7 snRNA #25(AAV5-4×U7 seperate)病毒注射到hUSH2A EXON13c.2802T>G基因敲入人源化小鼠眼部,以AAV5-U7-scramble病毒注射小鼠作为阴性对照组,以玻璃体注射剂量为15μg/眼(1μL)的PROQR-AON(实施例9中的AON1)为阳性对照组,以不做处理的小鼠作为空白组(nontreated),每只注射两边眼睛。注射三周后,处死实验小鼠,取小鼠视网膜组织,提取RNA,并逆转录为cDNA,并通过表16中的对应引物/探针进行RT-PCR和qRT-PCR实验,检测USH2A pre-mRNA外显子13剪接跳跃的效率。如图32所示,结果显示,尽管是1×U7 snRNA串联,其在眼部视网膜中诱导剪接跳跃的效果优于2×U7 snRNA组合,且均优于PROQR AON(实施例9中的AON1)(根据non-treated组结果显示,包含突变的人USH2A外显子13存在一定的自发跳跃,与现有研究报道一致)。
实施例15:不同血清型的AAV-U7 snRNA注射诱导兔子眼部细胞USH2A pre-mRNA外显子12剪接跳跃
通过视网膜下腔注射,分别按照MOI为5×1010、2×1011将AAV5-U7 snRNA #9-#25(AAV5-1×U7)、按照MOI为5×1010将AAV8-U7 snRNA #9-#25(AAV8-1×U7)病毒注射到兔子眼部,以AAV5-CMV-GFP病毒视网膜下腔注射兔子作为阴性对照组,以玻璃体注射剂量为50μg(50μL)的AON(实施例9中的AON1)为阳性对照组。注射两周后,处死实验兔子,取兔子视网膜组织,提取RNA,并逆转录为cDNA,并通过表16中的对应引物/探针进行RT-PCR和qRT-PCR实验,检测USH2A pre-mRNA外显子13剪接跳跃的效率。
如图33所示,结果显示,AAV5递送的U7 snRNA诱导剪接跳跃的效果优于AAV8,且不同AAV血清型递送的U7 snRNA串联的效果均优于AON。
兔子中的USH2A基因为野生型的兔USH2A,不含外显子12(相当于人USH2A外显子13)突变。相对而言,hUSH2A EXON13c.2802T>G基因敲入人源化小鼠中含有突变的人外显子13更容易被诱导剪接跳跃)AAV-U7 snRNA和AON在兔子上诱导靶外显子剪接跳跃的效果显著低于其在人源化小鼠中的效率,提示AON的效果更容易受到外显子序列和突变的影响。
实施例16:AAV递送的U7 snRNA诱导USH2A pre-mRNA外显子13剪接跳跃的长期效果。
通过视网膜下腔注射,分别将剂量为1E+10vg(1μl)的AAV5-3×U7 snRNA #9-#25(AAV5- 3×U7)、AAV5-U7 snRNA #9-#25(AAV5-1×U7)、AAV5-2×U7 snRNA #9-2×U7 snRNA #25(AAV5-4×U7 seperate)病毒注射到hUSH2A EXON13c.2802T>G基因敲入人源化小鼠眼部,以AAV5-U7-scramble病毒注射小鼠作为阴性对照组,以玻璃体注射剂量为15μg(1μL)的PROQR-ASO(实施例9中的AON1)为阳性对照组,以不做处理的小鼠作为空白组(NTC)。注射22周后,处死实验小鼠,取小鼠视网膜组织,提取RNA,并逆转录为cDNA,并通过表7中的对应引物进行RT-PCR实验,检测USH2A pre-mRNA外显子13剪接跳跃的效率。结果显示(图34~图35),22周后,AAV递送的U7 snRNA串联仍具有最优的剪接跳跃和长期维持效果。为了进一步对比U7 snRNA串联与U7 snRNA组合之间的长期维持情况,本实施例进一步通过表16中的对应探针进行qRT-PCR实验,检测并对比AAV5-1×U7和AAV5-4×U7seperate在视网膜中长期维持USH2A pre-mRNA外显子13剪接跳跃效率的差异。结果显示(如图34),22周后,AAV-1×U7 snRNA串联仍具有最优的剪接跳跃效率,且相对于3周效果有所提升,可能存在疗效累计效应。而AAV-1×U7 snRNA串联的持续性显著优于AAV-4×U7seperate。

Claims (79)

  1. 一种snRNA核酸分子,其特征在于,所述snRNA核酸分子包括:识别结构域、茎环序列和Sm序列;其中,所述识别结构域的数量为至少两个;
    其中,各识别结构域自5′端至3′端与pre-mRNA的3′端至5′端的靶向序列片段反向互补;
    所述pre-mRNA为USH2A基因对应的pre-mRNA。
  2. 如权利要求1所述的snRNA核酸分子,其特征在于,各所述识别结构域自5′端至3′端依次与pre-mRNA的3′端至5′端的靶向序列片段反向互补。
  3. 如权利要求1所述的snRNA核酸分子,其特征在于,各所述识别结构域自5′端至3′端非依次与pre-mRNA的3′端至5′端的靶向序列片段反向互补。
  4. 如权利要求1~3任一项所述的snRNA核酸分子,其特征在于,所述识别结构域的长度为至少16bp。
  5. 如权利要求1~4任一项所述的snRNA核酸分子,其特征在于,所述识别结构域的长度为18~40bp。
  6. 如权利要求1~5任一项所述的snRNA核酸分子,其特征在于,所述识别结构域的长度为20~27bp。
  7. 如权利要求1~6任一项所述的snRNA核酸分子,其特征在于,所述识别结构域的数量为两个。
  8. 如权利要求7所述的snRNA核酸分子,其特征在于,所述snRNA核酸分子自5′端至3′端依次包括:两个相邻的识别结构域、Sm序列和茎环序列。
  9. 如权利要求1~8任一项所述的snRNA核酸分子,其特征在于,所述pre-mRNA为USH2A基因第12号内含子至第13号内含子对应的全部或部分pre-mRNA。
  10. 如权利要求9所述的snRNA核酸分子,其特征在于,所述pre-mRNA为USH2A基因第13号外显子对应的全部或部分pre-mRNA。
  11. 如权利要求9所述的snRNA核酸分子,其特征在于,所述pre-mRNA的基因组定位为Chr1:216246563-216247246;所述靶向序列片段选自如SEQ ID NO:1所示的核苷酸序列及其突变序列。
  12. 如权利要求11所述的snRNA核酸分子,其特征在于,所述pre-mRNA的基因组定位为Chr1:216246563-216246753;所述靶向序列片段选自如SEQ ID NO:3所示的核苷酸序列及其突变序列。
  13. 如权利要求12所述的snRNA核酸分子,其特征在于,所述pre-mRNA的基因组定位为Chr1:216246563-216246649;所述靶向序列片段选自如SEQ ID NO:4所示的核苷酸序列及其突变序列。
  14. 如权利要求13所述的snRNA核酸分子,其特征在于,所述pre-mRNA的基因组定位为Chr1:216246563-216246626;所述靶向序列片段选自如SEQ ID NO:9所示的核苷酸序列及其突变序列;或者,
    所述pre-mRNA的基因组定位为Chr1:216246616-216246649;所述靶向序列片段选自如SEQ ID NO:34所示的核苷酸序列及其突变序列。
  15. 如权利要求11所述的snRNA核酸分子,其特征在于,所述pre-mRNA的基因组定位为Chr1:216247130-216247246;所述靶向序列片段选自如SEQ ID NO:2所示的核苷酸序列及其突变序列。
  16. 如权利要求15所述的snRNA核酸分子,其特征在于,所述pre-mRNA的基因组定位为Chr1:216247142-216247185;所述靶向序列片段选自如SEQ ID NO:32所示的核苷酸序列及其突变序列;或者,
    所述pre-mRNA的基因组定位为Chr1:216247130-216247161;所述靶向序列片段选自如SEQ ID NO:33所示的核苷酸序列及其突变序列;或者,
    所述pre-mRNA的基因组定位为Chr1:216247210-216247246;所述靶向序列片段选自如SEQ ID NO:36所示的核苷酸序列及其突变序列;或者,
    所述pre-mRNA的基因组定位为Chr1:216247204-216247232;所述靶向序列片段选自如SEQ ID NO:37所示的核苷酸序列及其突变序列;或者,
    所述pre-mRNA的基因组定位为Chr1:216247187-216247220;所述靶向序列片段选自如SEQ ID NO:38所示的核苷酸序列及其突变序列;或者,
    所述pre-mRNA的基因组定位为Chr1:216247169-216247202;所述靶向序列片段选自如SEQ ID NO:39所示的核苷酸序列及其突变序列。
  17. 如权利要求1~16任一项所述的snRNA核酸分子,其特征在于,所述识别结构域的数量为两个,两个识别结构域相邻分布。
  18. 如权利要求17所述的snRNA核酸分子,其特征在于,两个所述识别结构域自5′端向3′分别为第一识别结构域和第二识别结构域;
    其中,与所述第一识别结构域或第二识别结构域反向互补的靶向序列片段选自如SEQ ID NO:34所示的核苷酸序列及其突变序列和如SEQ ID NO:9所示的核苷酸序列及其突变序列;相应与所述第二识别结构域或第一识别结构域反向互补的靶向序列片段选自如SEQ ID NO:32所示的核苷酸序列及其突变序列、如SEQ ID NO:33所示的核苷酸序列及其突变序列、如SEQ ID NO:36所示的核苷酸序列及其突变序列、如SEQ ID NO:37所示的核苷酸序列及其突变序列、如SEQ ID NO:38所示的核苷酸序列及其突变序列和如SEQ ID NO:39所示的核苷酸序列及其突变序列。
  19. 如权利要求18所述的snRNA核酸分子,其特征在于,与所述第一识别结构域反向互补的靶向序列片段选自如SEQ ID NO:34所示的核苷酸序列及其突变序列和如SEQ ID NO:9所示的核苷酸序列及其突变序列;与所述第二识别结构域反向互补的靶向序列片段选自如SEQ ID NO:32所示的核苷酸序列及其突变序列、如SEQ ID NO:33所示的核苷酸序列及其突变序列、如SEQ ID NO:36所示的核苷酸序列及其突变序列、如SEQ ID NO:37所示的核苷酸序列及其突变序列、如SEQ ID NO:38所示的核苷酸序列及其突变序列和如SEQ ID NO:39所示的核苷酸序列及其突变序列。
  20. 如权利要求19所述的snRNA核酸分子,其特征在于,所述第一识别结构域的核苷酸序列如SEQ ID NO:12~22、59~61任一项所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:40~58任一项所示。
  21. 如权利要求20所述的snRNA核酸分子,其特征在于,所述第一识别结构域的核苷酸序列如SEQ ID NO:12、13、15或17所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:48、54、56或58所示。
  22. 如权利要求21所述的snRNA核酸分子,其特征在于,所述第一识别结构域的核苷酸序列如SEQ ID NO:12、13或17所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:48、54或58所示。
  23. 如权利要求20所述的snRNA核酸分子,其特征在于,所述第一识别结构域的核苷酸序列如SEQ ID NO:12所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:48所示;或者,
    所述第一识别结构域的核苷酸序列如SEQ ID NO:12所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:54所示;或者,
    所述第一识别结构域的核苷酸序列如SEQ ID NO:12所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:58所示;或者,
    所述第一识别结构域的核苷酸序列如SEQ ID NO:13所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:48所示;或者,
    所述第一识别结构域的核苷酸序列如SEQ ID NO:13所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:54所示;或者,
    所述第一识别结构域的核苷酸序列如SEQ ID NO:13所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:58所示;或者,
    所述第一识别结构域的核苷酸序列如SEQ ID NO:17所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:48所示;或者,
    所述第一识别结构域的核苷酸序列如SEQ ID NO:17所示,所述第二识别结构域的核苷 酸序列如SEQ ID NO:54所示;或者,
    所述第一识别结构域的核苷酸序列如SEQ ID NO:17所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:58所示;或者,
    所述第一识别结构域的核苷酸序列如SEQ ID NO:16所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:42所示;或者,
    所述第一识别结构域的核苷酸序列如SEQ ID NO:18所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:43所示;或者,
    所述第一识别结构域的核苷酸序列如SEQ ID NO:14所示,所述第二识别结构域的核苷酸序列如SEQ ID NO:55所示。
  24. 如权利要求11~23任一项所述的snRNA核酸分子,其特征在于,所述突变序列为在所述核苷酸序列上存在一个或多个核苷酸的替换、增加或缺失。
  25. 如权利要求24所述的snRNA核酸分子,其特征在于,所述突变序列为在所述核苷酸序列上存在一个或多个核苷酸的替换。
  26. 如权利要求24所述的snRNA核酸分子,其特征在于,所述突变选自天然致病突变和天然非致病突变;所述天然致病突变选自c.2242C>T、c.2276G>T、c.2299delG、c.2522C>A、c.2541C>A、c.2761delC、c.2776C>T、c.2802T>G、c.2209C>T、c.2310delA、c.2391_2392deITG、c.2431A>T、c.2431_2432delAA、c.2440C>T、c.2525dup、c.2610C>A、c.2755C>T、c.2176T>C、c.2236C>G、c.2296T>C和c.2332G>T中的一种或多种。
  27. 如权利要求26所述的snRNA核酸分子,其特征在于,所述天然致病突变选自c.2802T>G、c.2299delG和c.2276G>T中的一种或多种。
  28. 如权利要求27所述的snRNA核酸分子,其特征在于,所述天然致病突变为c.2802T>G。
  29. 如权利要求1~28任一项所述的snRNA核酸分子,其特征在于,所述Sm序列为共有序列,所述茎环序列包括U1、U2、U3、U4、U5、U6或U7的茎环序列。
  30. 如权利要求29所述的snRNA核酸分子,其特征在于,所述茎环序列为U7的茎环序列。
  31. 如权利要求29所述的snRNA核酸分子,其特征在于,所述茎环序列为U1的茎环序列。
  32. 如权利要求29所述的snRNA核酸分子,其特征在于,所述茎环序列为1-2个。
  33. 如权利要求29所述的snRNA核酸分子,其特征在于,所述Sm序列如SEQ ID NO:6所示。
  34. 如权利要求29所述的snRNA核酸分子,其特征在于,所述茎环序列如SEQ ID NO:7所示。
  35. 如权利要求1~34任一项所述的snRNA核酸分子,其特征在于,所述snRNA核酸分子包含修饰的核苷酸或其类似物单体。
  36. 如权利要求35所述的snRNA核酸分子,其特征在于,其进一步在所述识别结构域的5′端和/或3′端的核苷酸上包括单向延长序列或双向延长序列。
  37. 如权利要求35所述的snRNA核酸分子,其特征在于,所述修饰选自:2′-O-烷基修饰、2′-O-甲氧基修饰和2′-O-甲氧基乙基修饰;所述2′-O-烷基修饰优选为2′-O-甲基修饰。
  38. 如权利要求37所述的snRNA核酸分子,其特征在于,所述类似物单体选自6′-修饰的双环核苷、5′-修饰的双环核苷、6′-双取代双环核苷、四氢吡喃核苷类似物和2'-脱氧2'-氟-β-D-阿拉伯糖核苷酸。
  39. 如权利要求1~38任一项所述的snRNA核酸分子,其特征在于,所述snRNA核酸分子的核苷酸间通过化学键连接,所述化学键选自磷酸键、亚甲基键、酰胺键、甲基膦酸酯键和3'-硫代甲缩醛键。
  40. 如权利要求39所述的snRNA核酸分子,其特征在于,所述磷酸键选自硫代磷酸酯键、二硫代磷酸酯键、烷基膦酸酯键、酰胺磷酸酯键、硼烷磷酸酯键和手性连接磷。
  41. 如权利要求40所述的snRNA核酸分子,其特征在于,所述磷酸键选自硫代磷酸酯键。
  42. 如权利要求35~41任一项所述的snRNA核酸分子,其特征在于,所述snRNA核酸分子自5′端和/或3′端起的第1~80位包含修饰的核苷酸或其类似物单体。
  43. 如权利要求42所述的snRNA核酸分子,其特征在于,所述snRNA核酸分子自5′端和/或3′端起的第3~40位包含修饰的核苷酸或其类似物单体。
  44. 如权利要求43所述的snRNA核酸分子,其特征在于,所述snRNA核酸分子自5′端和/或3′端起的第6~10位包含修饰的核苷酸或其类似物单体。
  45. 如权利要求35~44任一项所述的snRNA核酸分子,其特征在于,所述snRNA核酸分子自5′端或3′端起包含至少一个磷酸键。
  46. 如权利要求45所述的snRNA核酸分子,其特征在于,所述snRNA核酸分子自5′端起包含1~3个磷酸键;或者,
    所述snRNA核酸分子自3′端起包含1~3个磷酸键。
  47. 一种snRNA核酸分子的组合,其特征在于,所述组合包括一种或多种如权利要求1~46任一项所述的snRNA核酸分子。
  48. 如权利要求47所述的组合,其特征在于,至少两个识别结构域位于相同或不同的snRNA核酸分子上。
  49. 一种DNA分子,其特征在于,所述DNA分子编码如权利要求1~46任一项所述的 snRNA核酸分子或者如权利要求47或48所述的组合。
  50. 一种基因表达盒,其特征在于,所述基因表达盒包含启动子和如权利要求49所述的DNA分子。
  51. 如权利要求50所述的基因表达盒,其特征在于,所述启动子与所述DNA分子的Sm序列间还包括可剪切位点例如Tpye II型限制性内切酶识别位点。
  52. 如权利要求50或51所述的基因表达盒,其特征在于,所述启动子为U7启动子。
  53. 如权利要求52所述的基因表达盒,其特征在于,所述启动子为小鼠来源的U7启动子。
  54. 如权利要求50~53任一项所述的基因表达盒,其特征在于,所述基因表达盒包括识别结构域和骨架序列;所述骨架序列如SEQ ID NO:62所示。
  55. 一种重组表达载体,其特征在于,所述重组表达载体包含如权利要求1~46任一项所述的snRNA核酸分子、如权利要求47或48所述的组合或者如权利要求50~54任一项所述的基因表达盒。
  56. 如权利要求55所述的重组表达载体,其特征在于,所述重组表达载体的表达载体选自质粒、噬菌体、微环DNA、线性DNA和病毒。
  57. 如权利要求56所述的重组表达载体,其特征在于,所述表达载体为慢病毒或腺相关病毒。
  58. 如权利要求57所述的重组表达载体,其特征在于,所述腺相关病毒的衣壳蛋白为天然来源的衣壳蛋白或其突变体,所述腺相关病毒的质粒为单链或与所述单链互补的双链。
  59. 如权利要求58所述的重组表达载体,其特征在于,所述天然来源的衣壳蛋白选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAVrh.8、AAVrh.10和AAVrh.43;
    所述突变体选自AAV2.5、AAV2i8、AAV-TT、AAV9.HR和CAM130。
  60. 一种病毒颗粒,包括衣壳蛋白和核酸,其特征在于,所述核酸包括如权利要求1~46任一项所述的snRNA核酸分子、如权利要求47或48所述的组合或者如权利要求49所述的DNA分子。
  61. 如权利要求60所述的病毒颗粒,其特征在于,所述衣壳蛋白为来自腺相关病毒的衣壳蛋白。
  62. 如权利要求61所述的病毒颗粒,其特征在于,所述来自腺相关病毒的衣壳蛋白为天然来源的衣壳蛋白或其突变体。
  63. 如权利要求62所述的病毒颗粒,其特征在于,所述天然来源的衣壳蛋白选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAVrh.8、AAVrh.10和AAVrh.43; 所述突变体选自AAV2.5、AAV2i8、AAV-TT、AAV9.HR和CAM130。
  64. 一种药物组合物,其特征在于,所述药物组合物包含如权利要求1~46任一项所述的snRNA核酸分子、如权利要求47或48所述的组合、如权利要求49所述的DNA分子、如权利要求50~54任一项所述的基因表达盒、如权利要求55~59所述的重组表达载体或者如权利要求60~63任一项所述的病毒颗粒。
  65. 如权利要求64所述的药物组合物,其特征在于,所述药物组合物还包含药学上可接受的载体。
  66. 一种诱导产生缺失外显子13的Usherin蛋白的方法,其特征在于,所述方法包括向宿主细胞中导入如权利要求1~46任一项所述的snRNA核酸分子、如权利要求47或48所述的组合、如权利要求49所述的DNA分子、如权利要求50~54任一项所述的基因表达盒、如权利要求55~59所述的重组表达载体、如权利要求60~63任一项所述的病毒颗粒或者如权利要求64或65所述的药物组合物,使外显子13发生剪接跳跃。
  67. 如权利要求66所述的方法,其特征在于,所述宿主细胞选自视网膜组织细胞,内耳细胞,具有分化为视网膜组织细胞和/或内耳细胞的潜能细胞,以及可行使包含具有与视网膜组织细胞和/或内耳细胞对应功能的细胞。
  68. 如权利要求67所述的方法,其特征在于,所述视网膜组织细胞为视网膜感光细胞,所述内耳细胞为内耳毛细胞。
  69. 如权利要求67所述的方法,其特征在于,所述潜能细胞选自诱导多能干细胞、胚胎干细胞、神经前体细胞、视网膜祖细胞、视网膜前体细胞和间充质基质细胞。
  70. 一种抑制USH2A pre-mRNA外显子13表达和/或功能的方法,其特征在于,所述方法包括施用如权利要求1~46任一项所述的snRNA核酸分子、如权利要求47或48所述的组合、如权利要求49所述的DNA分子、如权利要求50~54任一项所述的基因表达盒、如权利要求55~59所述的重组表达载体、如权利要求58~61任一项所述的病毒颗粒或者如权利要求64或65所述的药物组合物。
  71. 一种诱导USH2A pre-mRNA外显子13剪接跳跃的方法,其特征在于,所述方法包括施用如权利要求1~46任一项所述的snRNA核酸分子、如权利要求47或48所述的组合、如权利要求49所述的DNA分子、如权利要求50~54任一项所述的基因表达盒、如权利要求55~59所述的重组表达载体、如权利要求60~63任一项所述的病毒颗粒或者如权利要求64或65所述的药物组合物。
  72. 一种降低异常Usherin蛋白表达的方法,其特征在于,所述方法包括向宿主细胞中导入如权利要求1~46任一项所述的snRNA核酸分子、如权利要求47或48所述的组合、如权利要求49所述的DNA分子、如权利要求50~54任一项所述的基因表达盒、如权利要求55~59 所述的重组表达载体、如权利要求60~63任一项所述的病毒颗粒或者如权利要求64或65所述的药物组合物。
  73. 如权利要求72所述的方法,其特征在于,所述宿主细胞所述宿主细胞选自视网膜组织细胞,内耳细胞,具有分化为视网膜组织细胞和/或内耳细胞的潜能细胞,以及可行使包含具有与视网膜组织细胞和/或内耳细胞对应功能的细胞。
  74. 如权利要求73所述的方法,其特征在于,所述视网膜组织细胞为视网膜感光细胞,所述内耳细胞为内耳毛细胞。
  75. 如权利要求74所述的方法,其特征在于,所述潜能细胞选自诱导多能干细胞、胚胎干细胞、神经前体细胞、视网膜祖细胞、视网膜前体细胞和间充质基质细胞。
  76. 一种制备如权利要求1~46任一项所述的snRNA核酸分子或者如权利要求47或48所述的组合的方法,所述方法包括生物合成或化学合成如权利要求1~44任一项所述的snRNA核酸分子或者如权利要求47或48所述的组合的步骤。
  77. 一种如权利要求1~46任一项所述的snRNA核酸分子、如权利要求47或48所述的组合、如权利要求49所述的DNA分子、如权利要求50~54任一项所述的基因表达盒、如权利要求55~59所述的重组表达载体、如权利要求60~63任一项所述的病毒颗粒或者如权利要求64或65所述的药物组合物在制备治疗USH2A外显子13突变相关的疾病的药物中的应用。
  78. 如权利要求77所述的应用,其特征在于,所述USH2A外显子13突变为致病突变或非致病突变。
  79. 如权利要求77所述的应用,其特征在于,所述疾病选自眼病和耳病。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080194027A1 (en) * 2006-08-30 2008-08-14 Trustees Of The University Of Pennsylvania Compositions, methods and kits based on small nuclear RNAs
US20130045538A1 (en) * 2010-03-17 2013-02-21 Association Institut De Myologie Modified u7 snrnas for treatment of neuromuscular diseases
CN109072239A (zh) * 2016-04-25 2018-12-21 ProQR治疗上市公司Ⅱ 治疗眼病的寡核苷酸
CN109804069A (zh) * 2016-09-23 2019-05-24 ProQR治疗上市公司Ⅱ 治疗眼部疾病的反义寡核苷酸
WO2021216853A1 (en) * 2020-04-22 2021-10-28 Shape Therapeutics Inc. Compositions and methods using snrna components
CN114787357A (zh) * 2019-12-09 2022-07-22 安斯泰来制药株式会社 用于编辑靶标rna的添加了功能性区域的反义型指导rna

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080194027A1 (en) * 2006-08-30 2008-08-14 Trustees Of The University Of Pennsylvania Compositions, methods and kits based on small nuclear RNAs
US20130045538A1 (en) * 2010-03-17 2013-02-21 Association Institut De Myologie Modified u7 snrnas for treatment of neuromuscular diseases
CN109072239A (zh) * 2016-04-25 2018-12-21 ProQR治疗上市公司Ⅱ 治疗眼病的寡核苷酸
CN109804069A (zh) * 2016-09-23 2019-05-24 ProQR治疗上市公司Ⅱ 治疗眼部疾病的反义寡核苷酸
CN114787357A (zh) * 2019-12-09 2022-07-22 安斯泰来制药株式会社 用于编辑靶标rna的添加了功能性区域的反义型指导rna
WO2021216853A1 (en) * 2020-04-22 2021-10-28 Shape Therapeutics Inc. Compositions and methods using snrna components

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAM, K.A. ET AL.: "Induction of Cryptic Pre‑mRNA Splice‑switching by Antisense Oligonucleotides", SCIENTIFIC REPORTS, vol. 11, 23 July 2021 (2021-07-23), XP093078469, DOI: 10.1038/s41598-021-94639-x *

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