CN118108862A - Anti-angiogenic fusion protein and application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine, and relates to fusion proteins of a plurality of VEGF family proteins and application thereof. The N-terminal of the fusion protein is the extracellular domain 2 and extracellular domain 3 of VEGF receptor 2, namely VEGFR2D2D3. The invention realizes the high expression and high activity of the multifunctional fusion protein in target tissues by the combination of different structural domains, the optimization of carrier elements and the optimization of codons and the delivery of an expression cassette for encoding the multifunctional fusion protein of an anti-VEGF family through AAV. Can be applied to the inhibition of VEGF-A and/or VEGF-C, in particular to the preparation of Sub>A preparation or Sub>A formulSub>A or Sub>A pharmaceutical composition for treating Vascular Endothelial Growth Factor (VEGF) related diseases, and has remarkable treatment effect on treating neovascular related eye diseases.

Description

Anti-angiogenic fusion protein and application thereof

Technical field:

The invention belongs to the field of biological medicine, and relates to an anti-angiogenesis fusion protein and application thereof.

The background technology is as follows:

vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) is a key factor in embryonic development and vascular repair, and is capable of inducing the regeneration of existing blood vessels (revascularization) or the growth of new blood vessels (angiogenesis). The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factors 1, 2 (PIGF-1, PIGF-2). VEGF-A is by far the most effective and most studied vascular growth inducing factor. Family members of VEGF are encoded by multiple exons, which, upon alternative splicing, can produce different subtypes, affecting solubility and receptor binding. For example, VEGF-A has 7 subtypes and VEGF-B has 2 subtypes. Family members of VEGF activate signal transduction by binding to VEGF receptors (VEGFR). VEGFR is a tyrosine kinase receptor with an extracellular region consisting of 7 immunoglobulin-like (immunoglobulin, IG) domains. VEGFR-1 (Flt-1) binds VEGF-A, VEGF-B, as well as PIGF, and may act as decoy receptors for VEGF or modulators of VEGFR-2. VEGFR-2 (KDR/Flk-1) binds VEGF-A, VEGF-C and VEGF-D and is the primary mediator of VEGF-induced angiogenic pathways. VEGFR-3 (Flt-4) binds VEGF-C with VEGF-D, but not VEGF-A, and is the primary mediator of lymphangiogenesis.

The formation of new blood vessels is Sub>A key factor in the development of many diseases, including eye-related diseases such as age-related macular degeneration (AMD), retinal Vein Occlusion (RVO), diabetic Retinopathy (DR), etc., and the current main therapy is anti-VEGF-Sub>A drugs (aflibercept, ranibizumab, etc.), which remain poorly effective in clinical treatment for Sub>A part of the population. And it has been reported that there is Sub>A rise in VEGF-C after administration of an anti-VEGF-A drug. In recent years, opthea company OPT-302 drugs against VEGF-C show good curative effect and safety in the secondary clinical experiments of nAMD. Inhibition of VEGF-C has been shown to be effective in the treatment of nAMD, and it has also been demonstrated that multiple proteins in the VEGF family are involved in the formation of choroidal neovascularization CNV, and inhibition of multiple proteins in the VEGF family helps to further enhance the therapeutic effects of nAMD.

Currently, for the treatment of the aforementioned various vascular neogenetic fundus diseases (AMD, RVO, DR), approved protein drugs are required to be treated by repeated intravitreal injections, which both cause inconvenience to the patient and burden the medical institution, and delivery of anti-VEGF genes by gene therapy has entered clinical late-stage development, but the current technology still has the main of delivering antagonists encoding anti-VEGF-Sub>A, failing to address the clinical needs faced by patients resistant to VEGF-Sub>A. In response to this problem, delivery of antagonists encoding proteins that are resistant to multiple VEGF families by gene therapy has become a breakthrough therapeutic regimen. The invention constructs a fusion protein for resisting a plurality of VEGF family proteins, and further realizes the inhibition of the plurality of VEGF family proteins through screening and optimizing the fusion protein and an expression element, and shows good curative effect in animal models.

The invention comprises the following steps:

At present, the medicaments for treating the ocular diseases related to neovascular diseases are mainly medicaments (Aflibercept, R1D2-R2D 3-Fc) such as anti-VEGF-A or anti-VEGF-A/PIGF/VEGF-B, but in recent years, VEGF-C/D is found to be important in the ocular diseases related to neovascular diseases, so that the invention provides fusion proteins for resisting various VEGF family proteins, and the binding activity and the inhibiting activity of the fusion proteins on VEGF-A and VEGF-C are mainly studied.

One of the technical schemes provided by the invention is Sub>A fusion protein with VEGF-A and VEGF-C resisting activities, wherein the N end of the fusion protein is an extracellular domain 2 and an extracellular domain 3 of VEGF receptor 2, namely VEGFR2D2D3;

Further, the fusion protein is VEGFR2D2D3-Fc, and the amino acid sequence is shown in a sequence table SEQ ID NO. 1;

further, the fusion protein is VEGFR2D2D3- (GGGGS) ₄ G-Fc, and the amino acid sequence is shown in a sequence table SEQ ID NO. 2;

Further, the fusion protein is VEGFR2D2D3- (GGGGS) ₄ G-VEGFR1D2-Fc, and the amino acid sequence is shown in a sequence table SEQ ID NO. 3.

Further, in the first technical scheme, the Fc fragment of the fusion protein is an engineered human IgG Fc fragment (mFc), the mutation site is H90E, and the amino acid sequence is shown in a sequence table SEQ ID NO. 7.

Further, the molecule with the modified human IgG Fc is VEGFR2D2D3- (GGGGS) ₄ G-VEGFR 1D2-mFc, and the amino acid sequence is shown in a sequence table SEQ ID NO. 8.

The second technical scheme provided by the invention is the first technical scheme of the encoding gene of the fusion protein;

Further, the nucleotide sequence of the fusion protein VEGFR2D2D3-Fc is shown in a sequence table SEQ ID NO. 4;

Further, the nucleotide sequence of the fusion protein VEGFR2D2D3- (GGGGS) ₄ G-Fc is shown in a sequence table SEQ ID NO. 5;

Further, the fusion protein VEGFR2D2D3- (GGGGS) ₄ G-VEGFR1D2-Fc has a nucleotide sequence shown in a sequence table SEQ ID NO. 6.

Further, the nucleotide sequence of the fusion protein VEGFR2D2D3- (GGGGS) ₄ G-VEGFR1D2-mFc is shown in a sequence table SEQ ID NO. 9.

Furthermore, the nucleotide sequence of the coding gene is subjected to codon optimization, and the nucleotide sequence carries out random substitution and screening of codons on the basis of not changing the amino acid sequence of the coding product, so that the nucleotide sequence has increased expression quantity relative to the original codons in target cells, and the expression level of the coding product in the target cells can be improved.

The third technical scheme provided by the invention is an expression cassette comprising the fusion protein coding gene of the second technical scheme, wherein the expression cassette comprises a structure shown in the following formula I from a 5'-3' end:

E1-E2-E3-E4 (formula I)

Wherein:

E1 is a promoter;

e2 is a signal peptide;

e3 is a nucleotide sequence for encoding the fusion protein of the technical scheme I;

E4 is a Poly A sequence.

Further, the structure of formula I further includes ITR sequences at both ends, specifically: ITR-E1-E2-E3-E4-ITR; the ITR sequence (inverted terminal repeat) is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9; preferably from AAV2;

Preferably, the promoter is a DNA sequence that can initiate transcription of the gene of interest, which sequence can be recognized by RNA polymerase and initiates transcription of the synthetic RNA. Such promoters include, but are not limited to, natural, optimized, or combined promoters;

Still further, the promoter elements have ocular cell tissue specificity, including but not limited to NA65p, hGRK1, GNAT2, PR1.7, SNCG, IRBP, etc. promoters;

still further, the promoter has the property of being widely expressed in eukaryotic cells or mammalian cells, preferably including but not limited to CMV, CBA, EF a, SV40, PGK1, ubc, CAG or miniCAG;

preferably, the signal peptide is from Human OSM, gaussia luc, human IL-2 or Albumin (HSA);

preferably, the Poly A sequence is selected from bGH polyA, SV40 polyA, HSV-TK polyA or hGH polyA;

Further, the above expression cassette also includes other expression regulatory elements including, but not limited to, regulatory elements for the following functions: (1) Enhancers, which may be derived from SV40 virus, CMV virus or adenovirus, etc.; (2) Regulatory elements for stabilizing RNA and enhancing expression, including human actin beta (ActB) 5` UTR、human hemoglobin beta (HBB) 5` UTR、artificial HBB 5` UTR、artificial ActB 5` UTR、HBB 3` UTR、AES-mtRNR13` UTR, etc.; (3) Introns, including human β-globin intron、chicken beta actin intron、rabbit β-globin intron、truncated SV40 late 16S intron、MVM intron-1, etc.; (4) The regulatory element may also be part of a kozak sequence GNCNCN, e.g. GCCACC, etc.; (5) The regulatory element may be the woodchuck hepatitis virus posttranscriptional element WPRE sequence or the human hepatitis B virus posttranscriptional element HPRE sequence.

Preferably, the expression cassette comprises from 5 'to 3': 5 'itrs, enhancers, promoters, introns, UTR sequences, kozak sequences, signal peptides, target protein coding sequences, RNA export signals, polyadenylation signal sequences, and 3' itrs;

more preferably, the expression cassette comprises from 5 'to 3': AAV2 ITR sequences at the 5' end, CMV enhancers, CMV promoters, actB ' UTR-HBB 2nd intron, kozak sequences, gaussia luc signal peptide, target protein coding sequences, WPRE, bGH polyadenylation signal sequences, and AAV2 ITR sequences at the 3' end;

Preferably, the expression cassette comprising CMV enhancer-CMV promoter-ActB' UTR-HBB 2nd intron-kozak-Gaussia luc signal peptide-RRG 004-027-WPRE-bGH polyA is shown in sequence table SEQ ID No. 10;

preferably, the fusion protein expression cassette is linked to a transient transfection vector; the vector is introduced into the host cell by a transfection reagent to express the fusion protein.

Further, the transient transfection vectors include, but are not limited to, PTT5, pCDNA3.1 (-), pCDNA3.1 (+), pPICZ alpha A, pGAPZ α A, PYES 2.0.0, pAAV-MCS, etc.; preferably, the transient transfection vector is pCDNA3.1 (+), pAAV-MCS.

Further, the transfection reagents used include Lipo2000, lipo3000, PEI, 293fectin ™, cellfectin, calcium phosphate, etc.; preferably, the transfection reagent is PEI.

Further, the host cells used include prokaryotic cells or eukaryotic cells; preferably, the host cell is selected from E.coli, yeast cells or mammalian cells; more preferably, the host cell is an Expi293 cell, ARPE-19 cell.

The fourth technical scheme of the invention is an AAV virus comprising the coding gene of the second technical scheme or the expression cassette of the third technical scheme, wherein the coding gene or the expression cassette is cloned between two terminal repeat ITRs in an AAV skeleton of the adeno-associated virus, a target gene plasmid GOI is constructed and applied to an AAV packaging vector system, and the packaging vector system comprises: the target gene plasmid GOI, a vector carrying AAV rep and cap genes and an auxiliary vector are packaged into AAV virus by three-plasmid transient transfection production cells.

The fifth technical scheme of the invention is to provide the application of the multifunctional fusion protein of the first technical scheme, the coding gene of the second technical scheme, the expression cassette of the third technical scheme or the AAV virus of the fourth technical scheme; further, in inhibiting VEGF-A, and/or VEGF-C;

further, the application of the compound in preparing medicines for inhibiting VEGF-A and/or VEGF-C is provided;

Further, the application of the composition in preparation of preparations or formulations or pharmaceutical compositions for treating Vascular Endothelial Growth Factor (VEGF) related diseases, including but not limited to ocular diseases, inflammatory diseases, autoimmune diseases or tumors, and the like, particularly in preparation of medicaments for treating age-related macular degeneration, diabetic retinopathy, diabetic macular edema and tumor-related diseases;

further, the formulation or recipe or medicament may be any dosage form including, but not limited to, injection dosage forms and ointment dosage forms;

Further, the preparation, formulation or medicament comprises the fusion protein, the expression cassette or the AAV virus as the only active ingredient.

Further, the administration mode is an AAV subretinal space injection mode;

Still further, the total dose administered was 1×10 ⁶-1×10¹³ viral genomes/eye per single dose throughout the life;

Further, the administration mode is intravitreal injection of protein, and the injection dosage is 1 mug-10 mg/eye.

The beneficial effects are that:

the invention provides Sub>A multifunctional fusion protein with high expression, high activity and stable property for resisting VEGF family (VEGF-A and VEGF-C) through optimized combination and molecular modification. Further through optimization and combination of vector elements, high expression and high activity of the multifunctional fusion protein in target tissues are realized through AAV delivery of recombinant adeno-associated viruses.

VEGFR2D2D3 was used in the amino acid structure composition of the three fusion proteins VEGFR2D2D3-Fc, VEGFR2D2D3- (GGGGS) ₄ G-Fc and VEGFR2D2D3- (GGGGS) ₄ G-VEGFR 1D2-Fc, the literature reports that VEGF-A has 1/10 of the affinity with VEGFR2 as VEGFR1, but in the present invention, VEGFR2D2D3 has the same expression level and anti-VEGF-A activity as that of the prior art VEGFR1D2-R2D 3; meanwhile, in the aspect of VEGF-C resistance, compared with the VEGFR3D1D2D3 in the prior art, the expression quantity and activity of the VEGFR2D2D3 are obviously improved. In addition, the invention also discovers that VEGFR2D2D3 needs to be put at the N end of the protein, otherwise, the expression quantity of the protein can be seriously influenced.

The invention improves the eye pharmacokinetics behavior of the target protein through the engineering modification (H90E) of Fc, reduces the protein exposure amount of blood entering the eye, greatly increases the accumulation of the target protein in the eye and prolongs the half life period.

According to the invention, through vector element optimization and codon optimization, an expression cassette for encoding the anti-VEGF family multifunctional fusion protein is delivered through AAV, the target protein is efficiently expressed in vivo, a better inhibition effect is shown in a mouse laser-induced choroidal neovascularization model, and a better treatment effect is provided for treating the neovascularization related eye diseases.

Description of the drawings:

figure 1 vector map: vector map of expression RRG004-027 in pCDNA3.1.

Figure 2 vector map: vector map of pAAV-MCS vector expression RRG 004-027.

Figure 3 vector map: c12 vector map.

FIG. 4 fusion protein expression levels.

1,3,24 … In the abscissa of the figure represents RRG004-001,003,024 …, respectively, and so on.

FIG. 5 inhibitory activity of fusion proteins against VEGFA.

FIG. 6 inhibitory Activity of fusion proteins against VEGFC.

FIG. 7 fusion protein inhibits HUVEC cell growth under VEGFA and VEGFC stimulation.

The specific embodiment is as follows:

The present application will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the present application.

Unless defined otherwise herein, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art.

In some embodiments of the invention, fusion proteins of the invention having anti-VEGF-A and VEGF-C activity employ R2D2D3 as Sub>A moiety capable of simultaneously resisting VEGF-A and VEGF-C, such as RRG004-024/025/027 and the like. There is evidence that VEGFR2 can bind VEGF-A and VEGF-C, but it is generally believed that VEGFR2 binds VEGFA less than VEGFR1, whereas the present invention finds that R2D2D3 binds VEGF-A as Sub>A moiety of VEGF-A with no less activity than or even more than R1D2-R2D3, while R2D2D3 binds VEGF-C with very strong binding activity; in addition, the invention also discovers that R2D2D3 needs to be placed at the N end of the protein, otherwise, the expression quantity of the protein can be seriously influenced. From the preclinical results, the efficacy of the RRG004-027, namely the VEGFR2D2D3- (GGGGS) ₄ G-VEGFR1D2-mFc is better than that of the anti-VEGFA (RRG 004-001) or the anti-VEGFC (RRG 004-063) singly.

In some embodiments, the fusion proteins of the invention having anti-VEGF-Sub>A and VEGF-C activity use an engineered human IgG Fc fragment, such as H90E, that can further improve the metabolic properties of the drug by mutating the binding site of FcRn, increase drug accumulation in the eye, and reduce systemic exposure to drug blood penetration through the eye.

The invention also provides a polynucleotide expression cassette comprising the structure of formula I:

E1-E2-E3-E4（I）

Wherein:

E1 is a promoter (including natural, optimized or combined promoters); e2 is a signal peptide; e3 is a nucleotide sequence encoding a fusion protein against a plurality of VEGF family proteins; e4 is a Poly A sequence.

In some embodiments, the structures described by formula I are: ITR-E1-E2-E3-E4-ITR; ITR sequences (inverted terminal repeats) are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9; preferably from AAV2;

In some embodiments, the expression cassette nucleic acid sequence has a promoter for promoting expression of the encodable nucleic acid sequence linked thereto, which may be constitutive or inducible, and which may be specifically expressed in cells of ocular tissue, or which may be ubiquitous. Preferably, the promoter is a DNA sequence that can initiate transcription of the gene of interest, which sequence can be recognized by RNA polymerase and initiates transcription of the synthetic RNA. Such promoters include, but are not limited to, natural, optimized, or combined promoters;

further, the promoter element has ocular cell tissue specificity, and comprises promoters such as NA65p, hGRK1, GNAT2, PR1.7, SNCG, IRBP and the like;

Still further, the promoter has a characteristic of being widely expressed in eukaryotic cells or mammalian cells, and preferably comprises a promoter of CMV, CBA, EF a, SV40, PGK1, ubc, CAG or miniCAG, etc.;

The signal peptide is a short peptide chain (5-30 amino acids in length) that directs the transfer of a newly synthesized protein to the secretory pathway, often referred to as the N-terminal amino acid sequence (sometimes not necessarily at the N-terminus) in the newly synthesized polypeptide chain that directs the transmembrane transfer (localization) of the protein. After the start codon there is an RNA region encoding a hydrophobic amino acid sequence, called a signal peptide sequence, responsible for directing the protein into subcellular organelles containing different membrane structures of the cell. Comprising three zones: one positively charged N-terminus, called the basic amino terminus; an intermediate hydrophobic sequence, based on neutral amino acids, capable of forming an alpha helix, which is the main functional region of the signal peptide; a longer negatively charged C-terminal, small amino acid containing, is the signal sequence cleavage site, also known as the processing region. When the signal peptide sequence is synthesized, it is recognized by Signal Recognition Particles (SRPs) that carry the ribosome to the endoplasmic reticulum and protein synthesis resumes, and protein synthesis is suspended or slowed down. Under the guidance of signal peptide, the newly synthesized protein enters the lumen of endoplasmic reticulum. The signal peptide sequence is excised under the action of signal peptidase. If the termination transport sequence is present at the C-terminus of the nascent peptide chain, it may be not cleaved by a signal peptidase.

In some embodiments of the invention, a commonly used eukaryotic expression signal peptide is selected, preferably, the signal peptide is Human OSM, gaussia luc, human IL-2 or Albumin (HSA);

Polyadenylation (Poly a) signals protect mRNA from exonuclease attack and are important for transcription termination, export of mRNA from the nucleus, and translation. Poly A signals contain multiple consecutive adenosine monophosphates, typically containing AAUAAA repeats. The Poly A sequence of the present invention is located downstream of the nucleotide sequence encoding the fusion protein against multiple VEGF family proteins. Preferably, the Poly A sequence is selected from bGH polyA, SV40 polyA, HSV-TK polyA or hGH polyA;

In some embodiments of the invention, the above-described expression cassette further comprises expression regulatory elements including, but not limited to, regulatory elements that function to: (1) Enhancers, which may be derived from SV40 virus, CMV virus or adenovirus, etc.; (2) Regulatory elements for expressing miRNA and siRNA sequences, including human actin beta (ActB) 5` UTR、human hemoglobin beta (HBB) 5` UTR、artificial HBB 5` UTR、artificial ActB 5` UTR、HBB 3` UTR、AES-mtRNR13` UTR, and the like; (3) Introns, including human β-globin intron、chicken beta actin intron、rabbit β-globin intron、truncated SV40 late 16S intron、MVM intron-1, etc.; (4) The regulatory element may also be part of a Kozak sequence, a nucleic acid sequence located behind the 5 'cap structure of eukaryotic mRNA, which may bind to a translation initiation factor to mediate translation initiation of mRNA containing the 5' cap structure, corresponding to the SD sequence of prokaryotes, which is a sequence present in eukaryotic mRNA and has an important role in translation initiation. The kozak sequence is G/N-C/N-C/N-ANNAUGG, such as GCCACCAUGG; (5) The regulatory element may be the woodchuck hepatitis virus posttranscriptional element WPRE sequence or the human hepatitis B virus posttranscriptional element HPRE sequence.

In some embodiments of the invention, the composition of the above expression cassette is optimized, preferably the expression cassette comprises from 5 'to 3': AAV2 ITR sequences at the 5' end, CMV enhancers, CMV promoters, actB ' UTR-HBB 2nd intron, kozak sequences, gaussia luc signal peptide, target protein coding sequences, WPRE, bGH polyadenylation signal sequences, and AAV2 ITR sequences at the 3' end. After optimization, the protein expression quantity of RRG004-027 reaches 1771.4ng/ml, which is far higher than the expression quantity obtained by other expression element combinations, and belongs to unexpected technical effects.

In some embodiments of the invention, the fusion protein expression cassette is linked to a transient transfection vector; the vector is introduced into the host cell by a transfection reagent to express the fusion protein. Further, the transient transfection vectors include, but are not limited to, PTT5, pCDNA3.1 (-), pCDNA3.1 (+), pPICZ alpha A, pGAPZ α A, PYES 2.0.0, pAAV-MCS, etc., preferably the transient transfection vectors are pCDNA3.1 (+), pAAV-MCS; further, the transfection reagents used include Lipo2000, lipo3000, PEI, 293fectin ™, cellfectin, calcium phosphate, etc.; preferably the transfection reagent is PEI; further, the host cells used include prokaryotic cells or eukaryotic cells; preferably the host cell is selected from E.coli, yeast cells or mammalian cells, more preferably the host cell is an Expi293 cell, ARPE-19 cell.

In some embodiments of the invention, the fusion protein is transiently expressed by an Expi293 cell and purified from transfected cell culture supernatants by recombinant protein G affinity chromatography. The further molecular sieve purification process yields a product with a purity of more than 90% and all fusion proteins are correctly formed and expressed.

In some embodiments of the invention, the binding capacity of the fusion protein to VEGF is verified by ELISA assays, and the fusion protein exhibits strong binding capacity to VEGF-A and VEGF-C.

In some embodiments of the invention, an assay is provided for testing the ability of Sub>A fusion protein to inhibit VEGF-A or VEGF-C induced transcriptional activation of the VEGFR2 signaling pathway; in another embodiment, an assay is provided for testing the ability of Sub>A fusion protein to inhibit VEGF-A and VEGF-C to co-stimulate proliferation of Human Umbilical Vein Endothelial Cells (HUVECs). The fusion proteins of the invention are proved to have biological activities of inhibiting VEGF-A and VEGF-C by the methods, and the inhibition effect is superior to that of Sub>A monomer molecule. In one embodiment, there is provided the use of a fusion protein for the treatment of a laser induced mouse Choroidal Neovascularization (CNV) mouse model (wet AMD model), the fusion protein provided by the present invention having superior therapeutic efficacy over the prior art.

In some embodiments of the invention, AAV viruses comprising the genes encoding the recombinant proteins or expression cassettes of the invention against multiple VEGF families are also provided, as well as methods of delivering genes encoding fusion proteins via AAV for the treatment of Vascular Endothelial Growth Factor (VEGF) related diseases. AAV viruses can be prepared using standard methods disclosed in the art, see "AAV Production Everywhere: A Simple, Fast, and Reliable Protocol for In-house AAV Vector Production Based on Chloroform Extraction".

Replication-defective recombinant AAV can be prepared by three plasmid cotranslative cell lines: the target nucleic acid sequence contained is flanked by two plasmids of the AAV Inverted Terminal Repeat (ITR) region, a helper packaging plasmid, and a plasmid carrying AAV encapsidation genes (rep and cap genes), which are packaged into AAV virus by three-plasmid transient transfection producer cells. The resulting AAV recombinant virus is then purified by standard techniques. Preferably, the recombinant adeno-associated virus is a single stranded AAV.

In some embodiments of the invention, the above recombinant proteins against multiple VEGF families are preferably expressed on AAV viral vectors, such as self-complementary AAV (scAAV), single-stranded AAV (ssAAV), pAAV-CMV, pAAV-MCS, and the like; preferably, the AAV viral vector is pAAV-MCS.

In some embodiments of the invention, the gene segments encoding recombinant proteins against multiple VEGF families are packaged into viral particles (e.g., AAV serotype viral particles including, but not limited to AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15、AAV16、AAV7m8、AAV PHP.eB, etc.). Thus, the present disclosure includes recombinant viral particles comprising any of the vectors described herein. Preferably, the recombinant adeno-associated virus serotype is AAV2, AAV8. More preferably, the recombinant adeno-associated virus serotype is AAV8.

The host cell used for assembling the viruses is used for AAV vector transduction cells to express recombinant proteins resisting a plurality of VEGF families. Preferably, the host cell is a mammalian cell (preferably a human-derived cell, more preferably a human optic nerve cell or photoreceptor cell), and the expression level of the recombinant protein against a plurality of VEGF families is increased, and the specific expression of the AAV vector molecule of the present invention is embodied. Such host cells include, but are not limited to, cells of Hela-S3, HEK-293T, HEK-293FT, expi293F, A549, and Sf 9; in some embodiments of the invention, the preferred host cell is HEK 293, HEK-293T cell or an Expi293F cell.

In some embodiments of the invention, the invention discloses the following recombinant viruses: AAV8-024. The recombinant virus is AAV8 serotype virus, the carrier carrying the transgene comprises a sequence shown in SEQ ID NO. 4, and the carried transgene is the encoding gene of VEGF receptor 2 extracellular domain 2 and domain 3 and human IgG Fc segment.

In some embodiments of the invention, the invention discloses the following recombinant viruses: AAV8-027. The recombinant virus is AAV8 serotype virus, the vector carrying the transgene comprises a sequence described in SEQ ID NO. 9, and the carried transgene is a coding gene of VEGF receptor 2 extracellular domain 2 and domain 3, VEGF receptor 1 extracellular domain 2 and H90E engineered human IgG Fc segment.

The recombinant virus constructed in the present invention is suitable for in vivo (in vivo) or in vitro (ex vivo), and preferably suitable for one-time administration in the vitreous, subretinal space or suprachoroidal space of a patient suffering from angiogenesis-related fundus diseases. Related ocular diseases include age-related macular degeneration, diabetic retinopathy, diabetic retinal edema, diabetic macular edema, post-retinal fibrosis, central retinal occlusion, retinal vein occlusion, ischemic retinopathy, hypertensive retinopathy, uveitis (e.g., anterior, middle, posterior or panuveitis), behcet's disease, bietti crystalline-like corneal retinal dystrophy, blepharitis, open angle glaucoma, neovascular glaucoma, corneal neovascularization, choroidal Neovascularization (CNV), subretinal neovascularization, corneal inflammation, and corneal graft complications. Wherein the age-related macular degeneration comprises wet age-related macular degeneration or dry age-related macular degeneration. Preferably, the safe and effective amount for mouse administration is in the range of 1×10 ⁶-1×10¹¹ viral genome/eye, the safe and effective amount for NHP administration is in the range of 1×10 ⁸-1×10¹³ viral genome/eye, and the safe and effective amount for human administration is in the range of 1×10 ⁸-1×10¹³ viral genome/eye (GC for short).

In some embodiments of the invention, the invention provides compositions comprising a polynucleotide or viral vector or adeno-associated virus of the invention.

In some embodiments of the invention, the invention provides methods of making compositions comprising the polynucleotides or viral vectors or adeno-associated viruses of the invention.

In some embodiments of the invention, the invention provides a method of administering a composition comprising a polynucleotide or viral vector or adeno-associated virus of the invention to the subretinal space.

In some embodiments of the invention, the invention provides methods of post-administration observation of mice/NHP/humans, including Fluorescein Fundus Angiography (FFA) and Optical Coherence Tomography (OCT).

The invention is further illustrated below in conjunction with specific examples.

Example 1 design of multifunctional fusion protein Structure composition

In this example, 12 proteins were constructed, the structures of which are shown in Table 1 below, respectively. Wherein:

R1D2 represents VEGFR1 receptor domain 2, R2D3 represents VEGFR2 receptor domain 3, R2D2D3 represents VEGFR2 receptor domain 2-3, R3D1D2D3 represents VDGFR receptor domain 1-3, (GGGGS) ₄ G represents GS linker, fc is the Fc region of IgG1, and mFc is the engineered IgG1 Fc region (mutation site is H90E).

The natural nucleotide sequence of the above protein structure (fusion protein shown in Table 1) was used as the coding gene (wherein, in NCBI database, VEGFR1 nucleotide sequence was derived from NM-002019.4, VEGFR2 nucleotide sequence was derived from NM-002253.4, VEGFR3 nucleotide sequence was derived from NM-182925.5, fc sequence was derived from AJ294730.1, (GGGGS) ₄ G nucleotide sequence was GGTGGCGGAGGCTCAGGCGGAGGTGGCTCTGGCGGTGGCGGTTCCGGCGGAGGTGGGTCCGGA), and a GCCGCCACC kozak sequence and a Gaussia luc signal peptide sequence were added at the 5' end, a BamHI cleavage site was added at the 5' end, a XhoI cleavage site was added at the 3' end, and a fragment of the gene was synthesized by the Nanjin Biotech limited and was incorporated into a pCDNA3.1 (+) vector by BamHI and BamHI cleavage, and the protein coding cassette contained in the vector comprised from 5' to 3 ': CMV enhancer-CMV promoter-kozak sequence-Gaussia luc signal peptide sequence-fusion protein gene sequence-bGH polyA sequence encoding multiple VEGF families.

TABLE 1 structural design of fusion proteins

EXAMPLE 2 expression and purification of multifunctional fusion proteins of different structural compositions

In the pcdna3.1 (+) vector (an example of a partially recombinant vector, as shown in fig. 1) containing the protein-encoding gene prepared in example 1, a plasmid vector was transferred into an Expi293 cell using PEI MAX transfection reagent (purchased from the next holothurian) according to the method described in the transfection reagent instructions. After 5 days of cell culture, the supernatant was collected and preliminary isolation and purification of the protein was performed using recombinant protein G affinity chromatography medium (purchased from Kirsrui). Filling 3-5ml recombinant protein G into a column, balancing with 0.02M PBS buffer solution (pH 7.4), and slowly loading the cell culture supernatant to fully combine protein with chromatography medium; non-specific heteroproteins that did not bind were washed with PBS buffer, and finally eluted with 0.1M glycine buffer, pH3.0, and the pH of the eluate was adjusted to neutral with pH9.0 Tris buffer. Reference was made to the instructions for the use of the packing, fine purification was carried out using Superdex 200 prep grade gel filtration packing (commercially available from Cytiva). The purified protein was concentrated by Millipore Amicon Ultra-15 30kD ultrafiltration concentration tube and exchanged with GE PD-10 desalting column into 20mM PBS buffer (20 mM sodium phosphate, 150mM NaCl, pH 7.4). Filtering with 0.22 μm filter membrane to obtain purified multifunctional fusion protein shown in Table 1, packaging under aseptic condition, and storing at ultralow temperature.

Example 3 Effect of different Structure compositions on expression level of multifunctional fusion proteins

The amount of protein expressed in the culture supernatant of the plasmid transient cells in example 2 was measured by ELISA. Anti-IgG Fc (purchased from Sigma) was diluted to 200ng/mL with carbonate buffer and coated at 2-8deg.C overnight at 100 μl per well. The following day, plates were washed and blocked with 1% BSA. The samples were diluted with 1% BSA to a range of 0.5-32ng/mL, 100. Mu.L of standard curve, quality control sample and diluted test sample were added to each well, and incubated at room temperature for 1 hour. Plates were washed, HRP conjugated goat anti-human IgG Fc antibody (purchased from Sigma) at 1:4000 dilution was added and incubated for 1 hour at room temperature. The plate was washed, developed by adding TMB, and after 10 minutes the reaction was stopped and the OD at 450nm was read with a microplate reader. And drawing a standard curve by using software, calculating the concentration of the sample to be detected, and carrying out normalization treatment according to the molecular weight.

The results are shown in FIG. 4: it can be seen that the combination of different domains, and the different positions of the same domain in the protein, have a large impact on expression of fusion proteins against multiple VEGF families.

The multifunctional fusion protein molecules RRG004-024, 025, 026, 027, 028, etc. have higher transient expression levels compared to RRG 004-001.

Comparison of RRG004-028, 029 and 032 revealed that VEGFR2D2D3 was required to be N-terminally placed in the protein, otherwise the expression level of the fusion protein was reduced. A portion of the protein was selected for protein binding experiments.

Example 4 Effect of different structural compositions on binding Activity of multifunctional fusion proteins to VEGF-A

The binding activity of multifunctional fusion protein molecules to VEGF-A was examined, VEGF-A (available from R & D systems) was diluted to 200ng/mL with carbonate buffer and coated overnight at 2-8deg.C at 100 μl per well. The following day, plates were washed and blocked with 1% BSA. Samples were diluted with 1% BSA, which was the cell supernatant obtained by the culture of example 2, each sample was diluted from 20nM, diluted 3-fold in a gradient to 0.00034nM, the plate was washed after the end of blocking, 100. Mu.L of diluted sample to be tested was added to each well, and incubated at room temperature for 1 hour. Plates were washed, HRP conjugated goat anti-human IgG Fc antibody (purchased from Sigma) at 1:4000 dilution was added and incubated for 1 hour at room temperature. The plate was washed, developed by adding TMB, and after 10 minutes the reaction was stopped and the OD at 450nm was read with a microplate reader. Finally, curve fitting is carried out by using a four-parameter regression mode, and the binding activity EC ₅₀ of each sample is calculated.

The results of the comparison of the activity of VEGF-A binding with RRG004-001 are shown in Table 2, and indicate that:

Fusion protein molecules RRG004-024, 025, 027 and 028 have strong binding capacity to VEGF-A.

TABLE 2 fusion protein binding Activity in cell supernatants to VEGF-A

Example 5 Effect of different structural compositions on binding Activity of multifunctional fusion proteins to VEGF-C

Similar to the method in example 4, the binding substrate was replaced with VEGF-C protein. The results are shown in Table 3, with RRG004-003 protein as a control. The results show that:

The activity of VEGF-C binding by RRGs 004-024, 025, 027, 028 containing the VEGFR2D2D3 domain was significantly increased compared to RRG 004-003.

TABLE 3 fusion protein and VEGF-C binding Activity in cell supernatants

Example 6 Effect of different structural compositions on inhibition of the VEGFR2 Signal pathway by purified multifunctional fusion proteins

We validated the inhibition experiments of the purified fusion proteins on the VEGFR2 signaling pathway using VEGFR2-luciferase as test cells (bezoby, C1C 42) with 10ng/mL VEGF-Sub>A or 100ng/mL VEGF-C followed by the addition of gradient diluted fusion protein drugs, the inhibition of fluorescent signals is shown in fig. 5, 6 and table 4.

The results show that: RRGs 004-024, 025, 027, 028 each inhibit VEGF-A or VEGF-C induced activation of the VEGFR2 signaling pathway. RRG004-027 and 028 have higher inhibition activity on VEGF-A or C than RRG004-001 and RRG004-003 in the prior art.

Table 4 fusion proteins inhibit VEGFR2 Signal pathway Activity

Example 7 Effect of different structural compositions on multifunctional fusion proteins inhibiting VEGF-A/C costimulatory HUVEC cell proliferation Activity

Whether the biological activity of the fusion protein molecules in inhibiting VEGF-A/VEGF-C co-stimulating the proliferation of HUVEC cells would be affected was examined. 6X 10 ³ HUVEC cells (available from Promcell) were seeded per well in 96-well cell culture plates and attached overnight. The next day, VEGF165-A to 200 ng/mL, VEGF-C to 100 ng/mL was diluted with DMEM+2% FBS medium dilution, and the final concentration of multifunctional fusion protein was added to 20nM, which resulted in a VEGFA+C-fusion protein mixture. The original culture solution was aspirated from the cell wells, and 100. Mu.L of the VEGFA+C-fusion protein mixture was added to each well, taking care not to use the limbic wells. The treated cells were incubated at 37℃in a 5% CO ₂ incubator, after 96 hours the cell plates were removed and 10. Mu.L of CCK-8 chromogenic solution (available from Dojindo) was added to each well; incubated at 37℃in a 5% CO ₂ incubator for 3h, and the OD at 450nm was read with a microplate reader. Finally, the inhibition rate of the fusion protein on HUVEC cells was calculated by taking the group without adding the fusion protein as a control. The results are shown in FIG. 7:

The results show that the inhibition of the multifunctional fusion proteins RRG004-024, 025, 027 and 028 on cell growth is higher than that of the prior art RRG004-001 and RRG004-003. The inhibition of cell growth by RRG004-025, 027 and 028 was greater than the combined use of RRG004-001 and RRG004-003 (20 nM RRG 004-001+20nM RRG 004-003).

Example 8 Fc Effect of engineering on ocular pharmacokinetics of multifunctional fusion proteins

The PK behaviour and blood ingress in ocular tissues after single injection of rabbit binocular vitreous was examined for Fc H90E engineered RRG004-027 versus control RRG 004-028.

2.5 Kg-3.5 kg of New Zealand rabbits were selected and were subjected to binocular examination, wherein 21 New Zealand rabbits were selected for each group. After the new zealand rabbit is anesthetized by pentobarbital sodium, the eye to be injected is disinfected by povidone iodine solution, and after 1-2 drops of obucaine hydrochloride eye drops are dropped into the eye to be injected for surface anesthesia, 1 mg/eye fusion protein is respectively injected into both eyes through the vitreous body of the eye according to the volume of 50 mu L/eye.

After the vitreous injection, about 1-2 drops of ofloxacin eye ointment are added to both eyes of each group of rabbits so as to keep cornea moist and resist infection. Taking eye tissue at different time points 1-672 h after administration, separating retina/choroid plexus, aqueous humor and vitreous humor, homogenizing tissue, and taking supernatant; and collecting a venous blood sample; protein concentrations in ocular tissues and serum were measured by ELISA.

The average concentration of each group of animals was plotted against time for 3 tissue samples of vitreous, aqueous and retinochoroid plexus, and PK analysis was performed using an atrioventricular model, and the results are shown in table 5. Comparing the elimination half-life t _1/2 of each protein with the area under the curve AUC _0-t when the protein is exposed, the half-life t _1/2 of RRG004-027 is 1.23-1.40 times of RRG004-028, and the exposure AUC is 1.15-1.46 times of RRG 004-028.

The average value of serum sample concentration is used for plotting a pharmaceutical time curve against time point, and a non-atrioventricular model is used for calculating the drug exposure AUC (see Table 6) of each fusion protein after ocular bleeding, and the average AUC of RRG004-027 is reduced by 58.6 percent compared with RRG 004-028.

Table 5 fusion proteins each eye tissue PK parameters (one chamber model)

Table 6 serum Exposure AUC (non-atrioventricular model) of fusion proteins

Example 9 multifunctional fusion protein inhibition laser induced mouse CNV model

SPF grade C57BL/6J male mice of about 2 months of age were purchased and kept in the laboratory for 3-5 days. Before molding, anesthetizing animals by using injection of sultai (25-50 mg/kg, i.p.) and cyromazine hydrochloride (5 mg/kg, i.p.), checking whether the fundus is abnormal by using an ophthalmoscope, and selecting normal animals into groups; binocular modeling was performed using a YAG laser photocoagulation instrument (VITRA, quantel Medical) at 1-1.5PD from the optic disc around the disk at 3 points (wavelength 532 nm,250 mw,50 μm,100 ms) with the laser spot position avoiding the retinal macrovessels and injection points. On day 3 post-molding, 2. Mu.l PBS (vehicle) or 0.15mM RRG004-001, 025, 027, 064 was injected through the vitreous cavity (IVT). On day 10 post-administration, a fluorescein sodium fundus angiography (FFA) examination was performed to evaluate the Choroidal Neovascularization (CNV) leak area.

The results are shown in Table 7, with respect to the PBS group, RRG004-025 and RRG004-027 were each able to significantly inhibit CNV leakage area at this dose, whereas partial inhibition was observed for RRG004-001 or RRG004-064, but no statistical differences were seen.

Table 7 fusion protein inhibits mouse CNV leakage area

Note that: * P <0.05, single factor analysis of variance, mean±sem.

Example 10 Effect of Polynucleotide expression cassettes with different expression regulatory elements on protein expression Capacity

The pCDNA3.1 (+) recombinant vector expressing RRG004-027 constructed in example 1 was digested with BamHI and XhoI, and the pAAV-CMS plasmid was digested with BamHI and XhoI, ligated using T4 ligase. And using ampicillin resistance screening according to a conventional molecular cloning method, pAAV-RRG004-027 (as shown in C01 in Table 8, see FIG. 2) recombinant plasmids were obtained.

The recombinant plasmid of C02-C12 was obtained by optimizing a part of the expression elements on C01 as shown in Table 8 based on pAAV-RRG004-027 (C01). The specific method comprises the following steps:

For the 027 molecule, a sequence from the enhancer to the Bsu36I cleavage site of the 027 molecule (Bsu 36I cleavage site is located in the signal peptide) was synthesized according to the sequence of the expression cassette provided in Table 8, and the MluI cleavage site was added at the 5' end. The C01 and the synthesized fragment were digested with MluI and Bsu36I, and ligated using T4 ligase. And screening with ampicillin resistance according to a conventional molecular cloning method to obtain a part or all of the sequence of C02-C12.

WPRE of C12 is modified by XhoI single enzyme cutting homologous recombination. SV40 late polyA and HSV TK polyA of C10 and C11 are modified by XhoI and BstEII digestion.

TABLE 8 Polynucleotide expression cassettes 5' -3' of different expression regulatory elements '

The nucleotide sequences of the different expression regulatory elements in table 8 are as follows:

CMV enhancer ：CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG

CMV promoter:

GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATC

CBA promoter ：TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

NA65p promoter ：AGATCTTCGAAATACTCTCAGAGTGCCAAACATATACCAATGGACAAGAAGGTGAGGCAGAGAGCAGACAGGCATTAGTGACAAGCAAAGATATGCAGAATTTCATTCTCAGCAAATCAAAAGTCCTCAACCTGGTTGGAAGAATATTGGCACTGAATGGTATCAATAAGGTTGCTAGAGAGGGTTAGAGGTGCACAATGTGCTTCCATAACATTTTATACTTCTCCAATCTTAGCACTAATCAAACATGGTTGAATACTTTGTTTACTATAACTCTTACAGAGTTATAAGATCTGTGAAGACAGGGACAGGGACAATACCCATCTCTGTCTGGTTCATAGGTGGTATGTAATAGATATTTTTAAAAATAAGTGAGTTAATGAATGAGGGTGAGAATGAAGGCACAGAGGTATTAGGGGGAGGTGGGCCCCAGAGAATGGTGCCAAGGTCCAGTGGGGTGACTGGGATCAGCTCAGGCCTGACGCTGGCCACTCCCACCTAGCTCCTTTCTTTCTAATCTGTTCTCATTCTCCTTGGGAAGGATTGAGGTCTCTGGAAAACAGCCAAACAACTGTTATGGGAACAGCAAGCCCAAATAAAGCCAAGCATCAGGGGGATCTGAGAGCTGAAAGCAACTTCTGTTCCCCCTCCCTCAGCTGAAGGGGTGGGGAAGGGCTCCCAAAGCCATAACTCCTTTTAAGGGATTTAGAAGGCATAAAAAGGCCCCTGGCTGAGAACTTCCTTCTTCATTCTGCAGTTGG

HGRK1 promoter:

GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGAAGGGGCCGGGCAGAATGATCTAATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAGCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGGCTCCCAGGGGCTTCCCAGTGGTCCCCAGGGAACCCTCGACAGGGCCAGGGCGTCTCTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCAAGGGC

human β-globin intron：

CGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACATCGATTGAATTCATCCTCTAGA

truncated SV40 late 16S intron：

AACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCCCCGGGATCC

chimeric intron：

GTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAG

rabbit β-globin intron：

GTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATGGACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTAATCACTTTTTTTTCAAGGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAGAGAACAATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAACTACATCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAG

MVM intron-1 intron：

AAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGG

Gaussia luc signal peptide:

ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGTATCGCCGTGGCCGAGGCC

human OSM signal peptide:

ATGGGGGTACTGCTCACACAGAGGACGCTGCTCAGTCTGGTCCTTGCACTCCTGTTTCCAAGCATGGCGAGCATG

Human IL-2 signal peptide:

ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGT

bGH polyA：

CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

SV40 late polyA：

AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA

HSV TK polyA：

ATGACGGCAATAAAAAGACAGAATAAAACCCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGT

Kozak：

GCCGCCACC

ActB 5’ UTR：

ACCGCCGAGACCGCGTCCGCCCCGCGAGCACAGAGCCTCGCCTTTGCCGATCCGCCGCCCGTCCACACCCGCCGCCAG

HBB 2^ndintron：

GTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAG

HBB 5’ UTR：

ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCC

arti HBB 5’ UTR：

TCGTTTACTAGTATCGTATCCGTGTTAACTGTCACTGTTTCTCATTGAAG

arti ActB 5’ UTR：

ACCGCATAGACCGCGTAAGCACCGTGAACACTGTGTTTCGCATTATCCGATACGCAGACCGCAAAATTGCACAGTCACGCAACC

CMV 5’ UTR：

TCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAG

exin21：

CAACCGCGGTTCGCGGCCGCT

WPRE：

AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC

HBB 1^stINTRON：

GTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAG

HBB 3' UTR：

GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAA

AES-mtRNR1 3' UTR：

CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC

ARPE-19 cells (purchased from ATCC) were seeded at 1E 6/well in 6-well plates and placed in a 5% CO ₂ incubator at 37℃overnight. The following day, 1. Mu.g of the C01-C12 AAV plasmid vector constructed as described above was transferred into ARPE-19 cells, respectively, using PEI MAX transfection reagent (purchased from the following holothurian). After 4 days of cell culture, the supernatant was collected, and the protein expression amount in the cell culture supernatant was measured by the method of example 3. As shown in Table 9, the results indicate that the expression level of the target protein can be improved by adopting the combination of the regulatory elements in the C12 expression cassette, and the expression level is far higher than that obtained by other expression element combinations, and the expression level belongs to unexpected technical effects. The vector map of C12 is shown in FIG. 3.

Wherein the expression cassette containing CMV enhancer-CMV promoter-ActB' UTR-HBB 2nd intron-kozak-Gaussia luc signal peptide-RRG 004-027-WPRE-bGH polyA is shown in a sequence table SEQ ID No. 10.

TABLE 9 protein expression Capacity of different nucleotide expression cassettes

Example 11 AAV8 Virus preparation carrying the target protein expression cassette

Packaging AAV virus by adopting a method of co-transfecting VPC2.0 cells by three plasmids, wherein the three plasmids are respectively helper packaging plasmid, AAV8 rep-cap plasmid and each transgenic plasmid GOI (AAV vector containing target protein expression cassette).

According to the method of example 10, an expression cassette containing target proteins such as RRG004-001, 024, 027, 064 and the like is inserted into a pAAV-MCS vector to construct a GOI plasmid, and the expression regulatory element adopts the corresponding element in C12 and only replaces the target protein. The constructed GOI plasmid, AAV8 rep-cap plasmid and pHelper helper plasmid are subjected to large-scale extraction, the concentration is more than 1 mug/mu L, and A260/280 is between 1.8 and 2.0 for packaging viruses. VPC2.0 cells were cultured in serum-free suspension to a cell density of 1E+6 cells/ml. The three plasmids extracted are mixed according to the mol ratio of 1:1:1, then plasmid DNA is uniformly mixed with PEIpro transfection reagent according to the mass ratio of 1:2, and after incubation for 20 minutes at room temperature, the three plasmids are slowly added into a cell suspension (the ratio of the cells to the three plasmids is 1mL:1 mug) and uniformly mixed. Shaking culture was carried out at 37℃with 8% CO ₂ for 3 days, and the cell suspension was collected.

Centrifuging 10,000 g of cell suspension for 10min, transferring the obtained centrifugal supernatant into a new centrifuge tube, re-suspending the obtained cell precipitate with a small amount of PBS solution, and repeatedly freezing and thawing to lyse cells; after freeze thawing, the cells were centrifuged at 10,000 g for 10min, and the supernatant obtained by centrifugation was collected. The supernatants collected by the two centrifugation were mixed together and filtered with a 0.45 μm filter to remove impurities. 1/2 volume of 1M NaCl,10% PEG8000 solution was added and mixed well at 4℃overnight. Centrifugation at 12,000 rpm for 2h, discarding the supernatant, dissolving the viral pellet with an appropriate amount of PBS solution, and filtering and sterilizing with 0.22 μm filter after complete dissolution. The residual plasmid DNA (final concentration 50U/ml) was removed by digestion with Benzonase nuclease. The tube cap was closed and inverted several times to mix thoroughly. Incubation at 37 ℃ for 30 min; filtering with 0.45 μm needle filter, and collecting filtrate to obtain concentrated AAV virus.

Adding solid CsCl to the virus concentrate until the density is 1.41 g/ml (refractive index 1.372); adding the sample into an overspeed centrifuge tube, and filling the residual space of the centrifuge tube with a pre-prepared 1.41 g/ml CsCl solution; centrifuge at 175,000 g for 24 hours to develop a density gradient. Samples of different densities were collected in sequential steps and sampled for titer determination. The fractions enriched in AAV particles were collected.

The above procedure was repeated once. The virus was placed in a 100 kDa dialysis bag and desalted overnight by 4 degree dialysis, the dialysis buffer composition being PBS containing 0.001% Pluronic F68, pH7.2. The obtained dialyzed sample is purified AAV virus, and can be used for in vivo drug efficacy verification.

AAV virus used in the invention is prepared by adopting the method, and AAV carrying different target genes, such as AAV8-001, AAV8-024, AAV8-027 or AAV8-064, can be prepared by packaging different GOI plasmids.

Example 12 AAV8 Virus-inhibiting laser-induced mouse CNV model carrying target Gene

SPF grade C57BL/6J male mice of about 2 months of age were purchased and kept in the laboratory for 3-5 days. Prior to grouping, animals were subjected to a general ophthalmic examination and qualified animals were screened for testing by subretinal injection at a dose of 1E8vg per eye with PBS, AAV8-024, AAV8-027, or AAV8-064, respectively. The injection is followed by application of atropine sulfate ophthalmic gel and tobramycin dexamethasone eye ointment 3 times daily for 4 consecutive days. The administration interval of different ointments is more than 10 min.

Fundus Angiography (FFA) was performed 10 days after dosing, and animals with no abnormalities in both eyes were screened. At 2 weeks post-dose, modeling was performed using a YAG laser photocoagulation instrument (VITRA, quantel Medical) at 3 points (wavelengths of 532nm,250mw,50 μm,100 ms) around the disk at 1-1.5 PD.

FFA detection was performed 7 days and 14 days after laser modeling, and FFA leakage areas were quantified.

As shown in Table 10, the results show that the multifunctional fusion protein AAV can significantly inhibit CNV facula leakage, and AAV8-024 and AAV8-027 are superior to AAV8-001 in the prior art.

TABLE 10 AAV 8-inhibited mouse CNV leakage area carrying fusion protein Gene

Note that: * P <0.05, < P <0.01, (using one-way anova).

The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Therefore, the protection scope of the patent is subject to the claims.

Claims (12)

1. A fusion protein, characterized in that the fusion protein is N-terminally ectodomain 2 and ectodomain 3 of VEGF receptor 2, VEGFR2D 3;

the fusion protein is VEGFR2D2D3-Fc, and the amino acid sequence is shown in a sequence table SEQ ID NO. 1;

The fusion protein is VEGFR2D2D3- (GGGGS) ₄ G-Fc, and the amino acid sequence is shown in a sequence table SEQ ID NO. 2;

The fusion protein is VEGFR2D2D3- (GGGGS) ₄ G-VEGFR1D2-Fc, and the amino acid sequence is shown in a sequence table SEQ ID NO. 3.

2. The fusion protein of claim 1, wherein the Fc fragment of the fusion protein is an engineered human IgG Fc fragment mFc, the mutation site is H90E, and the amino acid sequence is shown in sequence table SEQ ID No. 7;

The fusion protein is VEGFR2D2D3- (GGGGS) ₄ G-VEGFR1D2-mFc, and the amino acid sequence is shown in a sequence table SEQ ID NO. 8.

3. A gene encoding the fusion protein of claim 1 or 2.

4. The coding gene of claim 3, wherein the fusion protein VEGFR2D2D3-Fc has a nucleotide sequence shown in a sequence table SEQ ID NO. 4;

The nucleotide sequence of the fusion protein VEGFR2D2D3- (GGGGS) ₄ G-Fc is shown in a sequence table SEQ ID NO. 5;

The nucleotide sequence of the fusion protein VEGFR2D2D3- (GGGGS) ₄ G-VEGFR1D2-Fc is shown in a sequence table SEQ ID NO. 6;

The nucleotide sequence of the fusion protein VEGFR2D2D3- (GGGGS) ₄ G-VEGFR1D2-mFc is shown in a sequence table SEQ ID NO. 9.

5. An expression cassette comprising the coding gene of claim 3, wherein the expression cassette comprises from the 5'-3' end the structure of formula I:

E1-E2-E3-E4 (formula I)

Wherein: e1 is a promoter; e2 is a signal peptide; e3 is a nucleotide sequence encoding a fusion protein; e4 is a Poly A sequence.

6. The expression cassette of claim 5, wherein the promoter element has ocular cell tissue specificity, including but not limited to NA65p, hGRK1, GNAT2, PR1.7, SNCG, IRBP promoters;

the promoter has the property of being widely expressed in eukaryotic cells or mammalian cells, including but not limited to CMV, CBA, EF a, SV40, PGK1, ubc, CAG or miniCAG;

the signal peptide is from Human OSM, gaussia luc, human IL-2 or Albumin (HSA);

the Poly A sequence is selected from bGH polyA, SV40 polyA, HSV-TK polyA or hGH polyA.

7. The expression cassette of claim 5, further comprising other expression regulatory elements including, but not limited to, regulatory elements that function to: (1) Enhancers, which may be from SV40 virus, CMV virus or adenovirus; (2) Regulatory elements for stabilizing RNA and enhancing expression, including human actin beta (ActB) 5` UTR、human hemoglobin beta (HBB) 5` UTR、artificial HBB 5` UTR、artificial ActB 5` UTR、HBB 3` UTR、AES-mtRNR13` UTR;（3） introns, comprising the human β-globin intron、chicken beta actin intron、rabbit β-globin intron、truncated SV40 late 16S intron、MVM intron-1;（4）kozak sequence, kozak sequence GNCNCN; (5) WPRE sequence or HPRE sequence.

8. The expression cassette of claim 5, wherein said expression cassette is set forth in SEQ ID No. 10.

9. An AAV virus comprising the coding gene of claim 3 or the expression cassette of claim 5, wherein the coding gene or expression cassette is cloned between two terminal repeats ITR in an adeno-associated virus AAV backbone, a gene plasmid GOI of interest is constructed, and is applied to an AAV packaging vector system for packaging into an AAV virus.

10. Use of the fusion protein of claim 1 or 2, or the coding gene of claim 3, or the expression cassette of claim 5, or the AAV virus of claim 9.

11. Use according to claim 10, in inhibiting VEGF-Sub>A, and/or VEGF-C; or in the preparation of Sub>A medicament for inhibiting VEGF-A, and/or VEGF-C.

12. The use according to claim 10, in the preparation of a formulation or a pharmaceutical composition for the treatment of vascular endothelial growth factor related diseases;

Such diseases include, but are not limited to, ocular diseases, inflammatory diseases, autoimmune diseases or tumors;

the ocular diseases include, but are not limited to, age-related macular degeneration, diabetic retinopathy, and diabetic macular edema.