Nucleic acid encoding Anti-VEGF protein, polynucleotide expression cassette and recombinant adeno-associated virus

Abstract

Provided are a nucleic acid encoding an anti-VEGF protein, a polynucleotide expression cassette, a recombinant adeno-associated virus, and the related use thereof. By means of further optimizing and designing an AAV vector for encoding an anti-VEGF protein, the expression level of the protein is improved, and the potential clinical value thereof is better exerted.

Description

Nucleic acid encoding anti-VEGF protein, polynucleotide expression cassette and recombinant adeno-associated virus Technical Field

[0001] The present invention relates to the field of genetic engineering and biomedicine, and in particular to nucleic acids, polynucleotide expression cassettes, and recombinant adeno-associated viruses encoding anti-VEGF proteins and related applications thereof. Background Art

[0002] Neovascularization is key to the development and spread of many diseases. Numerous ocular diseases involve angiogenesis, including age-related macular degeneration (AMD), retinal vein occlusion (RVO), diabetic retinopathy (DR), and pathological myopia. VEGF is a highly specific vascular endothelial growth factor that promotes increased vascular permeability, extracellular matrix degeneration, endothelial cell migration and proliferation, and angiogenesis. Numerous studies have demonstrated that excessive VEGF expression can induce pathological neovascular eye diseases. Given the importance of VEGF signaling in angiogenesis, blocking VEGF or VEGF receptors to inhibit angiogenesis holds important therapeutic potential for angiogenesis-related diseases, including cancer and retinal vascular lesions. Over the past decade, several anti-VEGF drugs have been developed for the treatment of ocular neovascular diseases, including pegaptanib (Macugen), bevacizumab (Avastin), ranibizumab (Lucentis), and aflibercept (Eylea).

[0003] However, the short half-life of currently marketed drugs necessitates repeated dosing, placing an increased burden on both physicians and patients. Therefore, gene therapy approaches, such as adeno-associated virus (AAV) gene therapy tools, are highly anticipated due to their ability to sustain drug expression and maintain efficacy over time. However, the application of AAV currently faces challenges, such as injection site irritation and immune responses caused by dosage issues. Therefore, further optimization of the AAV vector design is desired.

[0004] Summary of the Invention

[0005] To address the above-mentioned issues, the present invention has extensively optimized the coding sequence for expressing anti-VEGF proteins, including codon optimization, CpG removal, and intron insertion within the coding sequence region, with the goal of increasing the expression level of the anti-VEGF protein. For example, in some preferred embodiments, the coding sequence is codon-optimized. The present invention provides codon-optimized coding sequences in which codons are modified using methods known in the art without altering the amino acid sequence of the encoded product, such as replacing codons with codons that are frequently expressed in target cells. In some preferred embodiments, the coding sequence is CpG-depleted. Modifying the composition of CpG islands or the GC content of the coding region may affect codon usage preference. In other preferred embodiments, the coding sequence is CpG-depleted and codon-optimized. In some preferred embodiments, the coding sequence encoding the anti-VEGF protein described herein includes codon optimization and / or CpG removal and / or intron insertion.

[0006] One aspect of the present invention is to provide a nucleic acid comprising a coding sequence encoding an anti-VEGF protein, wherein the coding sequence is codon-optimized.

[0007] In some embodiments, the anti-VEGF protein of the present invention comprises extracellular domain 2 of VEGF receptor 1 and extracellular domain 3 of VEGF receptor 2. In certain embodiments, the anti-VEGF protein of the present invention further comprises extracellular domain 4 of VEGF receptor 2. In certain embodiments, the anti-VEGF protein of the present invention further comprises an immunoglobulin Fc fragment (such as human immunoglobulin IgG1, IgG2, or IgG4).

[0008] In some embodiments, the anti-VEGF protein of the present invention comprises extracellular domain 2 of VEGF receptor 1, extracellular domain 3 of VEGF receptor 2, and an immunoglobulin Fc fragment. In other embodiments, the anti-VEGF protein of the present invention comprises extracellular domain 2 of VEGF receptor 1, extracellular domain 3 of VEGF receptor 2, extracellular domain 4 of VEGF receptor 2, and an immunoglobulin Fc fragment.

[0009] In some specific embodiments, the anti-VEGF protein of the present invention comprises SEQ ID NO: 1, SEQ ID NO: 2, or a protein having at least 85% homology (e.g., 85%, 90%, 95%, 98%, 99% or 100%) to any of these sequences.

[0010] In some specific embodiments, the codon-optimized nucleic acid comprises the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence that is at least 85% homologous thereto (e.g., 85%, 90%, 95%, 98%, 99% or 100%).

[0011] In some preferred embodiments, the nucleic acid molecule of the coding sequence further has CpGs removed. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 CpGs removed compared to the non-CpG-removed coding sequence. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the CpGs removed compared to the non-CpG-removed coding sequence. In some preferred embodiments, the nucleic acid of the coding sequence comprises the nucleotide sequence of SEQ ID NO: 8 or a nucleic acid sequence having at least 85% homology thereto (e.g., 85%, 90%, 95%, 98%, 99%, or 100%).

[0012] In some embodiments, the nucleic acid molecules encoding the sequences of the present invention further comprise an intron insertion. In some specific embodiments, the intron is selected from the group consisting of a VH4 intron, an SV40 intron, a Chi intron, and a Chimeric intron. In some specific embodiments, the VH4 intron, the SV40 intron, the Chi intron, and the Chimeric intron have the nucleotide sequences set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 20, respectively.

[0013] In some specific embodiments, the coding sequence encoding the anti-VEGF protein is codon-optimized, CpG-removed, and intron-inserted. In some preferred embodiments, the coding sequence encoding the anti-VEGF protein comprises the nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 22, or a nucleic acid sequence having at least 85% homology thereto.

[0014] Another aspect of the present invention is to provide a polynucleotide expression cassette comprising the nucleic acid of the present invention and an expression control element (wherein, as mentioned above, the nucleic acid of the present invention comprises a coding sequence encoding an anti-VEGF protein).

[0015] In a specific embodiment, the polynucleotide expression cassette of the present invention comprises, from 5' to 3', the following sequence:

[0016] 1) 5'ITR;

[0017] 2) Promoter;

[0018] 3) introns;

[0019] 4) the aforementioned nucleic acid encoding the anti-VEGF protein of the present invention;

[0020] 5) polyadenylation signal sequence;

[0021] 6) 3'ITR.

[0022] The promoter is selected from β-actin promoter (CBA), cytomegalovirus promoter (CMV), elongation factor 1α promoter (EF1α), EFS promoter, NA65P promoter, MNT promoter, UB6 promoter, CAG promoter, RPE65 promoter, UBC promoter or opsin promoter, preferably CBA, CMV, EF1α or CAG promoter.

[0023] The intron is selected from VH4 intron, SV40 intron, Chi intron, chicken β-actin intron (Chickenβ-action), U12 intron, RHD intron, PI intron, MBL intron, etc., preferably VH4 intron, SV40 intron, Chi intron, chicken β-actin intron.

[0024] The polyadenylation signal includes simian vacuolating virus 40 (SV40), human growth hormone (HGH), bovine growth hormone (BGH), or beta-globin (RGB).

[0025] In some specific embodiments, the nucleic acid encoding the anti-VEGF protein encodes SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 85% homology thereto.

[0026] In some more specific embodiments, the nucleic acid encoding the anti-VEGF protein comprises SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:22, or a nucleotide sequence at least 85% homologous thereto.

[0027] In some more specific embodiments, the polynucleotide expression cassette of the present invention comprises, from 5' to 3',:

[0028] 1) 5'ITR;

[0029] 2) CBA promoter;

[0030] 3) chicken β-actin intron;

[0031] 4) Kozak sequence;

[0032] 5) a nucleic acid encoding an anti-VEGF protein, wherein the nucleic acid encodes SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence having at least 85% homology thereto;

[0033] 6) RGB polyadenylation signal sequence;

[0034] 7) 3'ITR.

[0035] In some more specific embodiments, the polynucleotide expression cassette of the present invention comprises, from 5' to 3',:

[0036] 1) 5'ITR;

[0037] 2) CBA promoter;

[0038] 3) chicken β-actin intron;

[0039] 4) Kozak sequence;

[0040] 5) a nucleic acid encoding an anti-VEGF protein, wherein the nucleic acid comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 22, or a nucleotide sequence having at least 85% homology thereto;

[0041] 6) RGB polyadenylation signal sequence;

[0042] 7) 3'ITR.

[0043] In some specific embodiments, the nucleic acid encoding the anti-VEGF protein in the polynucleotide expression cassette is codon-optimized. In some more specific embodiments, the polynucleotide expression cassette (the nucleic acid encoding the anti-VEGF protein is codon-optimized) comprises, from 5' to 3', the nucleotide sequence of SEQ ID NO: 27 or a sequence having at least 85% homology thereto (e.g., 85%, 90%, 95%, 98%, 99% or 100%).

[0044] In some specific embodiments, the nucleic acid encoding the anti-VEGF protein in the polynucleotide expression cassette is CpG-depleted. In some more specific embodiments, the polynucleotide expression cassette (the nucleic acid encoding the anti-VEGF protein is codon-optimized and CpG-depleted) comprises, from 5' to 3', the nucleotide sequence of SEQ ID NO: 29 or a sequence having at least 85% homology thereto (e.g., 85%, 90%, 95%, 98%, 99% or 100%).

[0045] In some specific embodiments, the nucleic acid encoding the anti-VEGF protein in the polynucleotide expression cassette further comprises an intron. Exemplary introns include, but are not limited to, a VH4 intron, an SV40 intron, a Chi intron, and a Chimeric intron. In some preferred embodiments, the nucleic acid encoding the anti-VEGF protein comprises an insertion of a VH4 intron, and the polynucleotide expression cassette comprises, from 5' to 3', the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 31, or a nucleic acid sequence having at least 85% homology thereto. In some preferred embodiments, the nucleic acid encoding the anti-VEGF protein comprises an insertion of an SV40 intron, and the polynucleotide expression cassette comprises, from 5' to 3', the nucleotide sequence of SEQ ID NO: 23, or a nucleic acid sequence having at least 85% homology thereto. In some preferred embodiments, the nucleic acid encoding the anti-VEGF protein comprises an insertion of a Chimeric intron, and the polynucleotide expression cassette comprises, from 5' to 3', the nucleotide sequence of SEQ ID NO: 24, or a nucleic acid sequence having at least 85% homology thereto.

[0046] In some more specific embodiments, the polynucleotide expression cassette of the present invention comprises, from 5' to 3',:

[0047] 1) 5'ITR;

[0048] 2) CBA promoter;

[0049] 3) chicken β-actin intron;

[0050] 4) Kozak sequence;

[0051] 5) a nucleic acid encoding an anti-VEGF protein, wherein the nucleic acid encodes SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence having at least 85% homology thereto;

[0052] 6) SV40 polyadenylation signal sequence;

[0053] 7) 3'ITR.

[0054] In some more specific embodiments, the polynucleotide expression cassette of the present invention comprises, from 5' to 3',:

[0055] 1) 5'ITR;

[0056] 2) CBA promoter;

[0057] 3) chicken β-actin intron;

[0058] 4) Kozak sequence;

[0059] 5) a nucleic acid encoding an anti-VEGF protein, wherein the nucleic acid comprises SEQ ID NO: 6 or a nucleotide sequence having at least 85% homology thereto;

[0060] 6) SV40 polyadenylation signal sequence;

[0061] 7) 3'ITR.

[0062] In some more specific embodiments, the polynucleotide expression cassette comprises the nucleotide sequence of SEQ ID NO: 33 from 5' to 3' or a sequence at least 85% homologous thereto (eg, 85%, 90%, 95%, 98%, 99% or 100%).

[0063] Another aspect of the present invention is to provide a plasmid comprising the aforementioned nucleic acid or polynucleotide expression cassette encoding the anti-VEGF protein of the present invention.

[0064] In some preferred embodiments, the plasmid of the present invention has a nucleotide sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, or a sequence having at least 85% homology thereto.

[0065] Another aspect of the present invention is to provide a recombinant adeno-associated virus (rAAV), which comprises a capsid protein and the aforementioned polynucleotide expression cassette of the present invention.

[0066] In some preferred embodiments, the capsid protein is a wild-type or modified capsid protein selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some more preferred embodiments, the capsid protein is an AAV8 capsid protein or a modified AAV8 capsid protein. In some preferred embodiments, the modified capsid protein includes a heterologous polypeptide substitution of about 5-14 amino acids at amino acids 588 to 592 of the parent AAV8 (amino acids 588 to 592 of the parent / wild-type AAV8 capsid protein are "QQNTA"), such as the polypeptide sequence RGNQQNTARQ (SEQ ID NO: 11).

[0067] Another aspect of the present invention is to provide a host cell, wherein the host cell comprises the aforementioned nucleic acid or polynucleotide expression cassette or plasmid or recombinant adeno-associated virus encoding the anti-VEGF protein of the present invention.

[0068] Another aspect of the present invention is to provide a pharmaceutical composition. The pharmaceutical composition comprises the polynucleotide expression cassette, plasmid, or recombinant virus of the present invention, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises the recombinant virus of the present invention and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is prepared as a formulation for intravitreal injection, subretinal injection, suprachoroidal injection, intravenous injection, intratumoral injection, or intramuscular injection.

[0069] Another aspect of the present invention provides the use of the polynucleotide expression cassette, plasmid, or recombinant virus of the present invention in the preparation of a medicament for treating a disease associated with VEGF. In certain embodiments, the disease associated with VEGF is an ocular neovascular disease. In certain embodiments, the ocular neovascular disease is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema due to branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, wet age-related macular degeneration with extreme low vision, and choroidal neovascularization secondary to pathological myopia.

[0070] Another aspect of the present invention is to provide a method for treating a VEGF-related disease in a mammalian subject, the method comprising delivering to the eye a therapeutically effective amount of an expression cassette, plasmid, or recombinant virus comprising a polynucleotide of the present invention. In certain embodiments, the VEGF-related disease is an ocular neovascular disease. In certain embodiments, the ocular neovascular disease is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema due to branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, wet age-related macular degeneration with extreme low vision, and choroidal neovascularization secondary to pathological myopia.

[0071] In some embodiments, the recombinant virus or pharmaceutical composition of the present invention is injected intravitreally, subretinally, suprachoroidally, intravenously, intratumorally, or intramuscularly.

[0072] In some embodiments, the recombinant adeno-associated virus is administered at a dose of E8 / eye to E16 / eye. In some preferred embodiments, the recombinant adeno-associated virus is administered at a dose of 5E11 / eye to 2E12 / eye. In some more preferred embodiments, the recombinant adeno-associated virus is administered at a dose of 2E12 / eye.

[0073] Detailed description

[0074] In the present invention, the following terms have the following definitions.

[0075] The term "polynucleotide expression cassette" as used herein refers to a polynucleotide sequence comprising two or more functional nucleotide sequences operably linked to each other, such as expression regulatory elements, translation initiation sequences, coding sequences, and termination sequences, and is typically composed of DNA.

[0076] As used herein, a "coding sequence" refers to a nucleotide sequence that encodes a gene product in vivo or in vitro. A coding sequence or gene can encode a polypeptide or protein molecule.

[0077] As used herein, "promoter" refers to a DNA sequence that directs RNA polymerase binding and thereby promotes RNA synthesis. Promoters and corresponding protein expression can be universal (meaning strongly active in cells, tissues, and species) or cell-specific, tissue-specific, or species-specific.

[0078] As used herein, "intron" refers to any sequence that can be transcribed but not translated. In some cases, an intron can refer to any sequence that is transcribed and removed from the mature RNA transcript in the cell.

[0079] The term "operably linked" refers to the juxtaposition of genetic elements in a relationship that allows them to operate in the intended manner. For example, if a promoter helps to initiate transcription of a coding sequence, it is operably linked to a coding region. Intervening residues can exist between the promoter and the coding region as long as this function is maintained.

[0080] The term "inverted terminal repeats (ITRs)" refers to the ITR sequences used for replication and packaging of AAV viruses.

[0081] The term "AAV" is an abbreviation for adeno-associated virus and is used to refer to the virus itself or its derivatives. The term includes all viral subtypes and viruses that exist in natural and recombinant forms. It includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), type 9 (AAV-9), type 10 (AAV-10), type 11 (AAV-11), type 12 (AAV-12), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primates, etc.

[0082] The term "variant" refers to a mutant of a reference nucleotide or amino acid, for example, one that differs from a naturally occurring nucleotide or amino acid sequence by at least one nucleotide or amino acid (e.g., an insertion, substitution, or deletion of an amino acid or nucleotide). Mutants of the nucleotides or amino acids described herein may also be described as modified amino acids or nucleotides.

[0083] The term "comprising" or "including" means that in addition to the necessary elements in the subject matter, elements not mentioned in the subject matter may also be included. For example, a polynucleotide expression cassette including a promoter may include other elements (such as ITRs, enhancers, introns, coding genes, polyadenylation sequences, etc.) in addition to the promoter.

[0084] The terms "polyadenylation sequence," "polyadenylation region," and "polyadenylation signal" include the recognition region required for endonucleases to cleave RNA transcripts, followed by the polyadenylation consensus sequence AATAAA. The polyadenylation sequence provides a Poly A site, a site on the RNA transcript to which adenine residues are added by post-transcriptional polyadenylation.

[0085] The term "Kozak sequence" refers to a nucleic acid sequence located after the 5' cap structure of eukaryotic mRNA, typically GCCACCAUGG. It can bind to translation initiation factors and mediate the initiation of translation of mRNA containing the 5' cap structure. It plays a role in the initiation of protein translation.

[0086] The term "homology" refers to the degree of similarity between amino acid sequences or base sequences when two or more nucleotide sequences or amino acid sequences are aligned, expressed as a percentage, such as 85%, 90%, 95%, 99%, 100%.

[0087] The term "host cell" refers to a cell that is transduced, infected, transfected, or transformed with a vector. The vector can be a plasmid, a viral particle, a bacteriophage, or the like. It should be understood that the term "host cell" refers to the original transduced, infected, transfected, or transformed cell and its progeny.

[0088] The terms "transduced," "infected," "transfected," or "transformed" generally refer to a method for administering, introducing, or inserting exogenous DNA (vector) into a cell. When DNA is introduced into a cell by a virus or viral vector, the cell is transduced with exogenous DNA. When DNA is introduced into a cell by non-viral methods, the cell is transfected with exogenous DNA. The terms "transduced" and "infected" are used interchangeably herein to refer to a cell that has received exogenous DNA or polynucleotides from a virus or viral vector.

[0089] The term "VEGF-related disease" refers to a related disease caused by abnormal expression (eg, excess or deficiency) of VEGF.

[0090] The term "expression control element" refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence to which it is operably linked. An expression control element is "operably linked" to a nucleotide sequence when it controls and regulates the transcription and / or translation of the nucleotide sequence. Thus, an expression control element may include a promoter, an enhancer, an internal ribosome entry site (IRES), a transcription terminator, a start codon preceding a protein-coding gene, intron splicing signals, and a stop codon. The term "expression control element" is intended to include, at a minimum, a sequence that exists to affect expression, but may also include other advantageous components. The term may also include nucleic acid sequence designs that remove unwanted potential start codons in and out of frame from the sequence. It may also include nucleic acid sequence designs that remove unwanted potential splice sites. It may also include sequences that direct the addition of polyadenylation or polyA.

[0091] The term "codon optimization" refers to the modification of a nucleic acid sequence encoding the same product with synonymous codon sequences, for example, replacing less frequently expressed codons with codons that are frequently expressed in target cells or species, with the goal of enhancing the expression level of the target gene in the target cell, tissue, or species (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more). Any nucleic acid modification that can help improve the expression of the target gene in the target cell or species encoding the same product falls within the scope of codon optimization of the present invention.

[0092] carrier

[0093] To increase the level of therapeutic protein or antibody encoded or expressed by the target gene, the present invention has extensively optimized the coding sequence for expressing the anti-VEGF protein, such as codon optimization, CpG removal, and intron insertion within the coding sequence region, so that the expression vector (e.g., nucleic acid, polynucleotide expression cassette, plasmid, or recombinant adeno-associated virus) has increased infectivity or expression capacity in target tissues or target cells (e.g., retina). For example, in some embodiments, the expression level of the anti-VEGF protein in the target cell or target tissue by the expression vector is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or more, compared to the non-optimized vector.

[0094] Therefore, one aspect of the present invention is to provide a nucleic acid vector comprising a codon-optimized coding sequence encoding an anti-VEGF protein. The present invention provides a codon-optimized coding sequence in which codons are modified using methods known in the art without changing the amino acid sequence of the encoded product, such as replacing less frequently expressed codons with codons that are frequently expressed in target cells.

[0095] The anti-VEGF proteins of the present invention target (e.g., specifically bind) the human VEGF receptor. The VEGF receptor is a receptor for vascular endothelial growth factor (VEGF). There are three major subtypes of VEGF receptors: VEGFR-1, VEGFR-2, and VEGFR3. The VEGF receptor has an extracellular portion consisting of seven immunoglobulin-like domains (e.g., extracellular domains 1-7), a single transmembrane spanning region, and an intracellular portion containing a split tyrosine kinase domain.

[0096] In some embodiments, the anti-VEGF protein of the present invention is a human VEGF receptor or a portion of a receptor. It is capable of recognizing and binding to a VEGF ligand but is unable to activate the receptor complex, exerting an inhibitory effect, binding to the ligand and preventing the ligand from binding to a conventional receptor. For example, the anti-VEGF protein of the present invention is any one of the extracellular domains 1-7 of human VEGF receptor 1, or a combination of multiple domains. For another example, the anti-VEGF protein of the present invention is any one of the extracellular domains 1-7 of human VEGF receptor 2, or a combination of multiple domains. For another example, the anti-VEGF protein of the present invention is any one of the extracellular domains 1-7 of human VEGF receptor 3, or a combination of multiple domains. For another example, the anti-VEGF protein of the present invention is a combination of one or more extracellular domains from domains 1-7 of human VEGF receptor 1, and / or VEGF receptor 2, and / or VEGF receptor 3.

[0097] In some embodiments, the anti-VEGF protein of the present invention comprises extracellular domain 2 of VEGF receptor 1 and extracellular domain 3 of VEGF receptor 2. In certain embodiments, the anti-VEGF protein of the present invention further comprises extracellular domain 4 of VEGF receptor 2. In certain embodiments, the anti-VEGF protein of the present invention further comprises an immunoglobulin Fc fragment (such as human immunoglobulin IgG1, IgG2, or IgG4).

[0098] In some embodiments, the anti-VEGF protein of the present invention comprises extracellular domain 2 of VEGF receptor 1, extracellular domain 3 of VEGF receptor 2, and an immunoglobulin Fc fragment. In other embodiments, the anti-VEGF protein of the present invention comprises extracellular domain 2 of VEGF receptor 1, extracellular domain 3 of VEGF receptor 2, extracellular domain 4 of VEGF receptor 2, and an immunoglobulin Fc fragment.

[0099] The protein of the present invention includes polypeptides, fusion proteins, antibodies or functional fragments thereof.

[0100] In some specific embodiments, the anti-VEGF protein of the present invention comprises SEQ ID NO: 1, SEQ ID NO: 2, or a protein having at least 85% homology (e.g., 85%, 90%, 95%, 98%, 99% or 100%) to any of these sequences.

[0101] In some specific embodiments, the codon-optimized nucleic acid comprises the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence that is at least 85% homologous thereto (e.g., 85%, 90%, 95%, 98%, 99% or 100%).

[0102] In some preferred embodiments, the coding sequence of the nucleic acid vector further removes CpG coding sequences. Changing the composition of the CpG island or the GC content of the coding region may affect the choice of codon usage preference. In some embodiments, compared to the coding sequence without CpG removal, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 CpGs can be removed. In some embodiments, compared to the coding sequence without CpG removal, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the CpGs can be removed. In other preferred embodiments, the coding sequence is a coding sequence with CpG removed and codon optimized. In some specific embodiments, the codon-optimized and CpG-removed nucleic acid comprises the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 85% homology thereto (e.g., 85%, 90%, 95%, 98%, 99% or 100%).

[0103] In some preferred embodiments, the coding sequence encoding the anti-VEGF protein further comprises an intron insertion. In some preferred embodiments, the coding sequence encoding the anti-VEGF protein is simultaneously codon-optimized, CpG-removed, and intron-inserted. The intron in the coding sequence of the present invention is selected from the group consisting of a VH4 intron, an SV40 intron, a Chi intron, and a Chimeric intron. In some specific embodiments, the VH4 intron, the SV40 intron, the Chi intron, and the Chimeric intron have the nucleotide sequences set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:20, respectively. The present invention unexpectedly discovered that introducing an intron at a specific position in the coding sequence encoding the anti-VEGF protein can effectively increase protein expression levels, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%.

[0104] In a specific embodiment, the nucleic acid with a codon-optimized, CpG-removed, VH4 intron-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 13, or a nucleic acid sequence at least 85% homologous thereto (e.g., 85%, 90%, 95%, 98%, 99%, or 100%). In another specific embodiment, the nucleic acid with a codon-optimized, CpG-removed, SV40 intron-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 21, or a nucleic acid sequence at least 85% homologous thereto (e.g., 85%, 90%, 95%, 98%, 99%, or 100%). In another specific embodiment, the nucleic acid with a codon-optimized, CpG-removed, Chimeric intron-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a nucleic acid sequence at least 85% homologous thereto (e.g., 85%, 90%, 95%, 98%, 99%, or 100%).

[0105] Therefore, another aspect of the present invention is to provide a polynucleotide expression cassette comprising the nucleic acid of the present invention and an expression control element (as mentioned above, the nucleic acid of the present invention comprises a coding sequence encoding an anti-VEGF protein).

[0106] For example, the expression control element comprises a promoter, which is operably linked to the nucleic acid molecule coding sequence. The promoter includes a promoter sequence or a functional fragment thereof. The promoter is specific for eukaryotic cells or mammalian cells. The promoter is selected from the group consisting of: β-actin promoter (CBA), cytomegalovirus promoter (CMV), elongation factor 1α promoter (EF1α), CAG promoter, EFS promoter, NA65P promoter, MNT promoter, UB6 promoter, CAG promoter, RPE65 promoter, and opsin promoter.

[0107] The expression control element of the present invention can also include an intron, which is located after the promoter. In some cases, an intron can refer to any sequence that can be transcribed but not translated. In some cases, an intron can refer to any sequence that is transcribed and removed from the mature RNA transcript in the cell. The intron is selected from VH4 intron, SV40 intron, Chi intron, chicken β-actin intron (Chicken β-action intron), U12 intron, RHD intron, etc.

[0108] The expression control element of the present invention may also include a polyadenylation signal (polyA). The polyadenylation signal protects mRNA from exonuclease attack and is important for transcription termination, export of mRNA from the cell nucleus, and translation. The polyadenylation signal comprises multiple consecutive adenosine monophosphates, typically containing AAUAAA repeats. The polyadenylation signal of the present invention is located downstream of the coding sequence encoding the VEGF antagonist. In some embodiments, the polyadenylation signal comprises simian vacuolating virus 40 (SV40), human growth hormone (HGH), bovine growth hormone (BGH), or beta-globin (RGB).

[0109] In certain embodiments, the polynucleotide expression cassettes of the present invention are flanked at the 5' and 3' ends by functional adenoviral inverted terminal repeats (ITRs). Functional adenoviral inverted terminal repeats (ITRs) are, for example, ITR sequences used for integration, replication, and packaging of AAV virions. The inverted terminal repeats are adeno-associated virus ITRs of a serotype selected from the group consisting of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAV9 ITR, AAV10 ITR, AAV11 ITR, and AAV12 ITR.

[0110] In some embodiments, the polynucleotide expression cassette comprises, in order from 5' to 3':

[0111] 1) 5'ITR;

[0112] 2) Promoter;

[0113] 3) introns;

[0114] 4) the aforementioned nucleic acid encoding the anti-VEGF protein of the present invention;

[0115] 5) polyadenylation signal sequence;

[0116] 6) 3'ITR.

[0117] The promoter is selected from the group consisting of β-actin promoter (CBA), cytomegalovirus promoter (CMV), elongation factor 1α promoter (EF1α), UBC promoter, EFS promoter, NA65P promoter, MNT promoter, UB6 promoter, CAG promoter, RPE65 promoter, and opsin promoter.

[0118] The intron is selected from VH4 intron, SV40 intron, Chi intron, chicken β-actin intron, U12 intron, RHD intron and the like.

[0119] The polyadenylation signal includes simian vacuolating virus 40 (SV40), human growth hormone (HGH), bovine growth hormone (BGH), or beta-globin (RGB).

[0120] In more specific embodiments, the nucleic acid encoding the anti-VEGF protein encodes SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence at least 85% homologous thereto.

[0121] In some more specific embodiments, the nucleic acid encoding the anti-VEGF protein comprises SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:22, or a nucleotide sequence at least 85% homologous thereto.

[0122] In some more specific embodiments, the polynucleotide expression cassette of the present invention comprises, from 5' to 3',:

[0123] 1) 5'ITR;

[0124] 2) CBA promoter;

[0125] 3) chicken β-actin intron;

[0126] 4) Kozak sequence;

[0127] 5) a nucleic acid encoding an anti-VEGF protein, wherein the nucleic acid encodes SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence having at least 85% homology thereto;

[0128] 6) RGB polyadenylation signal sequence;

[0129] 7) 3'ITR.

[0130] In some more specific embodiments, the polynucleotide expression cassette of the present invention comprises, from 5' to 3',:

[0131] 1) 5'ITR;

[0132] 2) CBA promoter;

[0133] 3) chicken β-actin intron;

[0134] 4) Kozak sequence;

[0135] 5) a nucleic acid encoding an anti-VEGF protein, wherein the nucleic acid comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 22, or a nucleotide sequence having at least 85% homology thereto;

[0136] 6) RGB polyadenylation signal sequence;

[0137] 7) 3'ITR.

[0138] In some more specific embodiments, the polynucleotide expression cassette of the present invention comprises, from 5' to 3',:

[0139] 1) 5' ITR having the nucleotide sequence set forth in SEQ ID NO: 14;

[0140] 2) CBA promoter having the nucleotide sequence as set forth in SEQ ID NO: 15;

[0141] 3) chicken β-actin intron having the nucleotide sequence as set forth in SEQ ID NO: 16;

[0142] 4) a kozak sequence having the nucleotide sequence set forth in SEQ ID NO: 17;

[0143] 5) a nucleic acid encoding an anti-VEGF protein, wherein the nucleic acid comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 22, or a nucleotide sequence having at least 85% homology thereto;

[0144] 6) RGB polyadenylation signal sequence having the nucleotide sequence set forth in SEQ ID NO: 18;

[0145] 7) 3' ITR having the nucleotide sequence set forth in SEQ ID NO: 19.

[0146] In some specific embodiments, the nucleic acid encoding the anti-VEGF protein in the polynucleotide expression cassette is codon-optimized. In some more specific embodiments, the polynucleotide expression cassette (the nucleic acid encoding the anti-VEGF protein is codon-optimized) comprises, from 5' to 3', the nucleotide sequence of SEQ ID NO: 27 or a sequence having at least 85% homology thereto (e.g., 85%, 90%, 95%, 98%, 99% or 100%).

[0147] In some specific embodiments, the nucleic acid encoding the anti-VEGF protein in the polynucleotide expression cassette is CpG-depleted. In some more specific embodiments, the polynucleotide expression cassette (the nucleic acid encoding the anti-VEGF protein is codon-optimized and CpG-depleted) comprises, from 5' to 3', the nucleotide sequence of SEQ ID NO: 29 or a sequence having at least 85% homology thereto (e.g., 85%, 90%, 95%, 98%, 99% or 100%).

[0148] In some specific embodiments, the nucleic acid encoding the anti-VEGF protein in the polynucleotide expression cassette further comprises an intron. Exemplary introns include, but are not limited to, a VH4 intron, an SV40 intron, a Chi intron, and a Chimeric intron. In some preferred embodiments, the nucleic acid encoding the anti-VEGF protein comprises an intron, and the polynucleotide expression cassette comprises, from 5' to 3', the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 31, or a nucleic acid sequence having at least 85% homology thereto (e.g., 85%, 90%, 95%, 98%, 99%, or 100%). In some preferred embodiments, the nucleic acid encoding the anti-VEGF protein comprises an intron of SV40, and the polynucleotide expression cassette comprises, from 5' to 3', the nucleotide sequence of SEQ ID NO: 23, or a nucleic acid sequence having at least 85% homology thereto. In some preferred embodiments, the nucleic acid encoding the anti-VEGF protein comprises an insertion of a Chimeric intron, and the polynucleotide expression cassette comprises the nucleotide sequence of SEQ ID NO: 24 from 5' to 3' or a nucleic acid sequence having at least 85% homology thereto (e.g., 85%, 90%, 95%, 98%, 99% or 100%).

[0149] In some more specific embodiments, the polynucleotide expression cassette of the present invention comprises, from 5' to 3',:

[0150] 1) 5'ITR;

[0151] 2) CBA promoter;

[0152] 3) chicken β-actin intron;

[0153] 4) Kozak sequence;

[0154] 5) a nucleic acid encoding an anti-VEGF protein, wherein the nucleic acid encodes SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence having at least 85% homology thereto;

[0155] 6) SV40 polyadenylation signal sequence;

[0156] 7) 3'ITR.

[0157] In some more specific embodiments, the polynucleotide expression cassette of the present invention comprises, from 5' to 3',:

[0158] 1) 5'ITR;

[0159] 2) CBA promoter;

[0160] 3) chicken β-actin intron;

[0161] 4) Kozak sequence;

[0162] 5) a nucleic acid encoding an anti-VEGF protein, wherein the nucleic acid comprises SEQ ID NO: 6 or a nucleotide sequence having at least 85% homology thereto;

[0163] 6) SV40 polyadenylation signal sequence;

[0164] 7) 3'ITR.

[0165] In some more specific embodiments, the polynucleotide expression cassette comprises the nucleotide sequence of SEQ ID NO: 33 from 5' to 3' or a sequence at least 85% homologous thereto (eg, 85%, 90%, 95%, 98%, 99% or 100%).

[0166] Another aspect of the present invention is to provide a plasmid comprising the aforementioned expression cassette comprising a nucleic acid or polynucleotide encoding an anti-VEGF protein. In some preferred embodiments, the plasmid comprises the nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34, or a sequence having at least 85% (e.g., 85%, 90%, 95%, 98%, 99%, or 100%) homology thereto.

[0167] The expression vectors of the present invention have been shown to increase anti-VEGF protein expression levels in target cells or target tissues. For example, after transient transfection of 293 cells with 2 μg of a single plasmid, protein expression was increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or more compared to a non-optimized plasmid.

[0168] For example, after transient transfection of APRE cells with 2.5 μg of a single plasmid, protein expression was increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more compared to the unoptimized plasmid.

[0169] Another aspect of the present invention provides a recombinant virus comprising: a capsid protein and a polynucleotide expression cassette according to the present invention. In some embodiments, the recombinant virus is a recombinant adeno-associated virus (rAAV), wherein the recombinant adeno-associated virus comprises rAAV capsid protein and a polynucleotide expression cassette according to the present invention.

[0170] In some embodiments, the rAAV of the present invention comprises capsid proteins, which are structural proteins encoded by the cap gene of AAV. rAAV contains three capsid proteins, designated VP1, VP2, and VP3, all of which are transcribed from a single cap gene through alternative splicing. VP1, VP2, and VP3 have molecular weights of approximately 87 kDa, approximately 72 kDa, and approximately 62 kDa, respectively. After translation, the capsid proteins form a spherical, 60-mer protein shell around the viral genome. The functions of the capsid proteins are to protect the viral genome, deliver the genome, and interact with the host.

[0171] The capsid protein of the present invention can be derived from any adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, etc., and any of the adeno-associated virus serotypes can serve as a gene delivery vector. For example, the AAV capsid can be a wild-type capsid or a natural capsid. Wild-type AAV capsids of particular interest include AAV2, AAV5, AAV8, and AAV9. Like ITR, the capsid does not have to be a wild-type capsid, but as long as the capsid is capable of transducing specific cells or tissues, the wild-type VP1, VP2, or VP3 sequence can be changed by insertion, deletion, or substitution of nucleotides. In other words, the AAV capsid can be a variant AAV capsid comprising one or more amino acid substitutions, deletions, or insertions relative to the parent capsid protein or AAV capsid protein.

[0172] In some embodiments, the AAV capsid variant comprises about 1 to about 100 amino acid substitutions, insertions, or deletions (e.g., between 1-10 amino acids, between 1-20 amino acids, between 1-30 amino acids, between 20-50 amino acids, between 20-60 amino acids, between 50-80 amino acids, between 50-100 amino acids, between 60-100 amino acids, etc.) relative to a known parental AAV capsid (e.g., AAV serotype 2, AAV2 / 3 (e.g., AAV2 / 3 hybrid), AAV serotype 8). In some embodiments, the AAV capsid variant comprises more than 100 amino acid substitutions, insertions, or deletions (e.g., between 100-200 amino acids, between 200-300 amino acids, between 100-500 amino acids, between 500-1000 amino acids, or more) relative to a parental capsid. In some embodiments, the AAV capsid variant can comprise substitutions, insertions, or deletions of about 5 to about 50 amino acids (e.g., between 5-10 amino acids, between 5-20 amino acids, between 5-30 amino acids, between 5-40 amino acids, between 10-20 amino acids, between 10-30 amino acids, between 10-40 amino acids, between 10-50 amino acids, or between 30-50 amino acids) relative to a parental capsid (e.g., AAV serotype 2, AAV2 / 3 (e.g., AAV2 / 3 hybrid), AAV serotype 8). In some embodiments, the AAV capsid variants may comprise about 10 to about 30 amino acid substitutions, insertions, or deletions relative to the parental capsid (e.g., AAV serotype 2, AAV2 / 3 (e.g., AAV2 / 3 hybrid), AAV serotype 8). In some embodiments, the AAV capsid variants may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In some embodiments, the AAV capsid variants may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, insertions, or deletions relative to the parental capsid (e.g., AAV serotype 2, AAV2 / 3 (e.g., AAV2 / 3 hybrid), AAV serotype 8).

[0173] In some embodiments, the capsid variant can be a chimeric capsid variant. The chimeric capsid variant sequence can comprise portions of two or more AAV capsid serotypes or variants thereof. In some embodiments, the chimeric capsid comprises portions of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different capsid protein serotypes. In some embodiments, the chimeric capsid protein has different properties, such as tissue tropism of the AAV capsid protein from which it is derived. Fragments can be incorporated by any appropriate method, such as recombinant DNA cloning.

[0174] In some embodiments, the AAV capsid variants described herein are variants of AAV2, AAV2 / 3 (e.g., AAV2 / 3 hybrids), or AAV8. AAV2 has been observed to effectively transduce ocular tissues (e.g., photoreceptor cells and retinal pigment epithelium (RPE)), human central nervous system (CNS) tissues, kidney tissues, and other tissues. In some embodiments, the AAV capsids described herein are AAV2 variants, and the AAV2 variants can be used to deliver gene therapy to ocular tissues (e.g., retina). AAV3 has been observed to effectively transform cancer cells. In some embodiments, the AAV variants described herein are AAV2 / 3 (e.g., AAV2 / 3 hybrids). AAV8 has also been observed to transduce ocular tissues (e.g., retinal ganglion cells, stem cells).

[0175] In one embodiment, the modified AAV capsid protein is a modified AAV8 capsid protein. The applicant has studied and applied for the relevant patent CN202211691417.8 or PCT / CN2022 / 142185 (invention name: modified AAV capsid protein and its use) of AAV8 capsid variants, the entire content of which is introduced here. In some preferred embodiments, the capsid protein is a modified AAV8 capsid protein, and the modified capsid protein includes a heterologous polypeptide substitution of about 5-14 amino acids at amino acids 588 to 592 of the parent AAV8 (amino acids 588 to 592 of the parent / wild-type AAV8 capsid protein are "QQNTA"), such as the polypeptide sequence RGNQQNTARQ (SEQ ID NO: 11).

[0176] In some embodiments, the rAAV described herein is single-stranded AAV (ssAAV) or double-stranded AAV (scAAV). ssAAV refers to an rAAV that has the coding sequence and complementary sequence of the target gene expression cassette on separate strands and is packaged in separate viral capsids. scAAV is an rAAV with its coding region engineered into double-stranded DNA.

[0177] Another aspect of the present invention is to provide a host cell comprising the aforementioned nucleic acid or polynucleotide expression cassette or plasmid encoding the anti-VEGF protein of the present invention. The components to be cultured in the host cell to encapsulate the rAAV vector in the AAV capsid are reversely provided to the host cell. Any one or more of the required components (e.g., recombinant AAV vector, rep sequence, cap sequence and / or auxiliary function) can be provided by a stable host cell that has been designed to contain one or more of the required components using methods known in the art. Preferably, the stable host cell will contain the required components under the control of an inducible promoter. The present invention has previously described in detail the selectable promoter types of the present invention.

[0178] "Host cell" refers to any cell that contains or is capable of containing the target substance. Typically, the host cell is a mammalian cell. In some embodiments, the host cell is a photoreceptor cell, a retinal pigment epithelial cell, a keratinocyte, a corneal cell, and / or a tumor cell. The host cell can be used as a recipient of AAV helper constructs, AAV vectors, helper function vectors, or other transfer DNA associated with the production of recombinant AAV. The term includes the descendants of the original cell that has been transfected. Therefore, as used herein, "host cell" can refer to a cell that has been transfected with an exogenous DNA sequence. It is understood that the descendants of a single parent cell may not be completely identical to the original parent in morphology or genomic or total DNA complement due to natural, accidental, or deliberate mutations. In some embodiments, the host cell is a mammalian cell, a yeast cell, a bacterial cell, an insect cell, a plant cell, or a fungal cell. In some embodiments, the host cell is a neuron, a photoreceptor cell, a pigmented retinal epithelial cell, or a glial cell. The recombinant AAV vector, rep sequence, cap sequence, and helper function required to produce the rAAV of the present invention can be delivered to the packaging host cell using any appropriate genetic element (vector). The genetic elements can be delivered by any method known and suitable to those skilled in the art, such as genetic engineering, recombinant engineering and synthetic techniques.

[0179] Pharmaceutical composition

[0180] Another aspect of the present invention is to provide a pharmaceutical composition comprising the polynucleotide expression cassette, plasmid or recombinant virus of the present invention and a pharmaceutically acceptable excipient, carrier or diluent.

[0181] Those skilled in the art can easily select suitable diluents, carriers or excipients. As used herein, carriers or excipients include any solvents, dispersion media, carriers, coatings, diluents, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, buffers, carrier solutions, suspensions, colloids, preservatives or chemical stabilizers, etc.

[0182] Exemplary carriers or excipients include sterile physiological saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water. For example, a suitable excipient or carrier includes saline, which can be formulated with a variety of buffer solutions (e.g., phosphate buffered saline). The effects of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is best to include an isotonic agent, such as sugar or sodium chloride. The absorption of the injectable composition can be prolonged by using an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0183] For administration of injectable aqueous solutions, the solution may be suitably buffered as necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0184] Sterile injectable solutions are prepared by mixing the required amount of active rAAV with the various other ingredients listed herein in an appropriate solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the dispersion medium and the required other ingredients.

[0185] The rAAV compositions of the present invention can also be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts and are formed by inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid). Salts formed by free carboxyl groups can also come from inorganic bases, such as sodium, potassium, ammonium, calcium or ferric hydroxide, or organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc. After preparation, the solution will be administered in a manner compatible with the dosage form and administered in a therapeutically effective amount. The preparation is easy to administer in various dosage forms, such as injectable solutions, drug release capsules, etc.

[0186] The compositions of the present invention can be delivered to suitable host cells via delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like.

[0187] It is advantageous to formulate injectable, oral or parenteral compositions in unit dosage form for ease of administration. As used herein, a unit dosage form refers to a physically discrete unit suitable for use as a single dose in a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present invention are determined by the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the art of mixing such active compounds for use in treating individuals. For example, a unit dose can be a certain amount of vector genomes, or a certain amount of vector genomes per milliliter, or a unit dose of a pharmaceutical composition measured using a multiplicity of infection (MOI), which refers to the ratio or number of cells to which a vector or viral genome can be delivered to a nucleic acid. The pharmaceutical composition can be contained in a container, package or dispenser together with instructions for administration, such as a syringe.

[0188] In some embodiments, the pharmaceutical composition is prepared as a formulation suitable for intravitreal injection, subretinal injection, suprachoroidal injection, intravenous injection, intratumoral injection, or intramuscular injection.

[0189] Delivery of recombinant adeno-associated virus (rAAV)

[0190] Another aspect of the present invention is to provide a use of the polynucleotide expression cassette, plasmid, or recombinant virus of the present invention in the preparation of a medicament for treating a disease associated with VEGF. Alternatively, another aspect of the present invention is to provide a method of treating a disease associated with VEGF in a mammalian subject, the method comprising delivering a therapeutically effective amount of the polynucleotide expression cassette, plasmid, or recombinant virus of the present invention to the eye.

[0191] The rAAV of the present invention can be delivered to a subject according to any suitable method known in the art. For example, it is preferred that the rAAV suspended in a physiologically compatible carrier (e.g., in a composition) be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cattle, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., macaque). In some embodiments, the host animal does not include humans. In some embodiments, the subject is a human.

[0192] A "therapeutically effective amount" means an amount effective at a dosage and for a period of time necessary to achieve the desired therapeutic effect. The therapeutically effective amount of the rAAV virus or pharmaceutical composition may vary depending on factors such as the disease state, age, sex, and weight of the subject to be treated, and the ability of the rAAV virus or pharmaceutical composition to elicit a desired response in the subject. The dosage regimen can be adjusted to provide the optimal therapeutic response. A therapeutically effective amount is also generally an amount in which any toxic or deleterious effects of the rAAV virus or pharmaceutical composition are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" means an amount effective at a dosage and for a period of time necessary to achieve the desired prophylactic effect, such as preventing or suppressing various conditions. Prophylactic doses can be used in subjects before or at an early stage of disease, and in some cases, the prophylactically effective amount may be greater than or less than the therapeutically effective amount. The dosage administered depends largely on the condition and size of the subject being treated, as well as the therapeutic formulation, the frequency of treatment, and the route of administration. The regimen for ongoing treatment, including dosage, formulation, and frequency, can be guided by the initial response and clinical judgment.

[0193] In some embodiments, the dosage of the recombinant adeno-associated virus is E8 / eye to E16 / eye. In some preferred embodiments, the dosage of the recombinant adeno-associated virus is E8 / eye to E15 / eye. In some preferred embodiments, the dosage of the recombinant adeno-associated virus is E9 / eye to E15 / eye. In some preferred embodiments, the dosage of the recombinant adeno-associated virus is E9 / eye to E14 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E10 / eye to E14 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E11 / eye to E14 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E12 / eye to E14 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E13 / eye to E14 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E8 / eye to E13 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E9 / eye to E13 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E10 / eye to E13 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E11 / eye to E13 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E12 / eye to E13 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E8 / eye to E12 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E9 / eye to E12 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E10 / eye to E12 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E11 / eye to E12 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E8 / eye to E11 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E9 / eye to E11 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E10 / eye to E11 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E8 / eye to E10 / eye. In other preferred embodiments, the dosage of the recombinant adeno-associated virus is E9 / eye to E10 / eye.In other specific embodiments, the dosage of the recombinant adeno-associated virus is E8 / eye, 2E8 / eye, 3E8 / eye, 4E8 / eye, 5E8 / eye, 6E8 / eye, 7E8 / eye, 8E8 / eye, 9E8 / eye, E9 / eye, 2E9 / eye, 3E9 / eye, 4E9 / eye, 5E9 / eye, 6E9 / eye, 7E9 / eye, 8E9 / eye, 9E9 / eye, E10 / eye, 2E10 / eye, 3E10 / eye, 4E10 / eye, 5E10 / eye, 6E10 / eye, 7E10 / eye, 8E10 / eye, 9E10 / eye, E11 / eye, 2E11 / eye, 3E11 / eye, 4E11 / eye , 5E11 / eye, 6E11 / eye, 7E11 / eye, 8E11 / eye, 9E11 / eye, E12 / eye, 2E12 / eye, 3E12 / eye, 4E12 / eye, 5E12 / eye, 6E12 / eye, 7E12 / eye, 8E12 / eye, 9E12 / eye, E13 / eye, 2E13 / eye, 3E13 / eye, 4E13 / eye, 5E13 / eye, 6E13 / eye, 7E13 / eye, 8E13 / eye, 9E13 / eye, E14 / eye, 2E14 / eye, 3E14 / eye, 4E14 / eye, 5E14 / eye, 6E14 / eye, 7E14 / eye, 8E14 / eye, 9E14 / eye. In some preferred embodiments, the dosage of the recombinant adeno-associated virus is 5E11 / eye to 2E12 / eye. In some more preferred embodiments, the dosage of the recombinant adeno-associated virus is 2E12 / eye.

[0194] In some embodiments, the rAAV virus or pharmaceutical composition is administered to the subject daily, weekly, biweekly, monthly, every 2 months, every 3 months, every 6 months, annually, every 2 years, every 5 years, or once a lifetime.

[0195] Exemplary routes of administration and delivery include intravenous (IV), intraarticular, intraperitoneal (IP), intraarterial, intramuscular, parenteral, subcutaneous, intrapleural, dermal, transdermal, parenteral, e.g., transmucosal, intracranial, intraspinal, oral (digestive), mucosal, respiratory, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavitary, iontophoresis, intraocular, intraglandular, intraorgan, intrafallopian tube.

[0196] In some embodiments, rAAV is delivered to a mammalian subject by, for example, intraocular injection, subretinal injection, choroidal injection (e.g., suprachoroidal injection), or topical administration (e.g., eye drops), or by injection into an affected ocular tissue to affect the eye of a mammal (e.g., intravitreal injection). "Ocular tissue" refers to any tissue derived from or contained in the eye. Non-limiting examples of ocular tissue include neurons, retina (e.g., photoreceptor cells), sclera, choroid, retina, vitreous body, macula, fovea, optic disc, lens, pupil, iris, aqueous solution, cornea, conjunctiva ciliary body, and optic nerve. The retina is located at the back of the eye and is composed of photoreceptor cells. These photoreceptor cells (e.g., rods, cones) impart vision by distinguishing colors and contrast in the visual field. In some embodiments, the rAAV or composition described in the present invention is administered by intraocular injection. In some embodiments, the rAAV or composition described in the present invention is administered by intravitreal injection. In some embodiments, the rAAV or composition described in the present invention is administered by subretinal injection. In some embodiments, the rAAV or composition described herein is administered by intrachoroidal (e.g., suprachoroidal) injection. In some embodiments, the rAAV or composition described herein is administered by intravenous injection. In some embodiments, the recombinant virus or pharmaceutical composition described herein is administered by intravitreal injection, subretinal injection, suprachoroidal injection, intravenous injection, intratumoral injection, or intramuscular injection.

[0197] "VEGF-related diseases" refers to a group of diseases associated with abnormal VEGF activity / signaling. Many studies have shown that if VEGF is abnormally excessive, it can stimulate and induce pathological angiogenesis, causing angiogenesis-related eye diseases. Non-limiting exemplary angiogenesis-related eye diseases include angiogenesis-dependent cancers, angiogenesis-related eye diseases, solid tumors (e.g., lung cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, head and neck cancer, colon cancer), melanoma, blood-derived tumors such as leukemia, metastatic tumors, benign tumors (e.g., hemangioma, acoustic neuroma, neurofibroma, tracheitis and pyogenic granuloma), rheumatoid arthritis, psoriasis, erythema, Osier-Webber syndrome, myocardial angiogenesis, plaques, telangiectasias, hemophilic joints or angiofibroma.

[0198] In some embodiments, angiogenesis-related eye diseases include, but are not limited to, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia, epidemic keratoconjunctivitis, vitamin A deficiency, excessive contact lens wear, atopic keratitis, bacterial keratitis, ulcer, ulcer, bacterial keratitis, ulcer, primary keratosis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Steven Johnson disease, pemphigoid keratoderma radialis Corneal incision, corneal transplant rejection, sickle cell anemia, sarcoidosis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid obstructive disease, chronic uveitis / vitritis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, Eales disease, Behçet disease, retinitis or choroiditis infection, presumed ocular histoplasmosis, Best's disease, myopia, optic pit, Stargardt disease, pars planitis, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, traumatic or post-laser complications.

[0199] In some embodiments, the ocular disease is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization, corneal neovascular eye disease, non-infectious uveitis, or glaucoma.

[0200] In some embodiments, the eye disease is selected from age-related macular degeneration, macular edema, diabetic macular edema, macular edema secondary to retinal vein occlusion, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, macular edema caused by branch retinal vein occlusion, diabetic retinopathy, diabetic retinopathy, proliferative diabetic retinopathy, diabetic retinal ischemia, polypoidal choroidal vasculopathy, choroidal neovascularization secondary to degenerative myopia, or retinopathy of prematurity. Specific forms of macular degeneration can include acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration.

[0201] In some preferred embodiments, the eye disease is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema due to branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, wet age-related macular degeneration with extreme low vision, and choroidal neovascularization secondary to pathological myopia.

[0202] Figures in the specification

[0203] Figure 1 Schematic diagram of the polynucleotide expression cassettes in plasmids No.3, No.4, and No.5

[0204] Figure 2 Four-level spot diagram of buffer and high-dose group in Example 4 DETAILED DESCRIPTION

[0205] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Furthermore, the following examples do not represent that the following experiments are all or only experiments of the present invention.

[0206] Example 1: Optimization of anti-VEGF protein coding sequence and plasmid construction

[0207] The primary purpose of this example is to construct a plasmid capable of expressing the anti-VEGF protein described in SEQ ID NO: 1. Standard recombinant DNA cloning techniques or general molecular biology techniques were used to construct a series of polynucleotide expression cassettes containing various combinations of regulatory elements and coding sequences (Table 1). Conventional DNA recombination and cloning techniques were then used to construct and clone recombinant plasmids containing the polynucleotide expression cassettes described in Table 1 in Escherichia coli.

[0208] First, the optimization of the anti-VEGF protein coding sequence involves, for example, codon optimization, CpG removal, and / or intron insertion. For example, for codon optimization, a Homo sapiens codon optimization strategy was employed, aligning the CDS region with humanization preferences, reducing GC content, and reducing repetitive sequences. For CpG removal optimization, the codon-optimized CDS region was removed according to the humanization codon optimization preference strategy. For VH4 intron insertion, based on the characteristics of the primary and secondary amino acid structures and the characteristics of VH4 intron insertion, the insertion conformed to the characteristic "A / CAGGU...AGG" sequence of the pre-mRNA, where the first three bases are exons, the third and fourth bases are 5' splice sites, and the 3' splice site is between the penultimate and second bases at the 3' end. Two insertion methods were designed.

[0209] Polynucleotide expression cassettes and plasmids based on the optimized or non-optimized anti-VEGF protein coding sequences were then constructed. For example, the 5' to 3' sequence of expression cassettes or plasmids Nos. 1-7 includes: AAV 5' ITR, CBA promoter, chicken β-actin intron (denoted as Chi intron in FIG. 1 ), Kozak sequence, nucleic acid encoding the anti-VEGF protein coding sequence, rabbit globin (RGB) polyadenylation signal, and AAV 5' ITR.

[0210] Among the plasmids comprising the above-mentioned polynucleotide expression cassette, the plasmid whose coding sequence for the anti-VEGF protein has not been optimized is numbered No. 1, wherein the coding sequence nucleic acid for the anti-VEGF protein is such as SEQ ID NO: 12, the 5' to 3' nucleotide sequence is such as SEQ ID NO: 25, and the plasmid nucleotide sequence is such as SEQ ID NO: 26; the plasmid whose coding sequence for the anti-VEGF protein has been codon-optimized is numbered No. 2, wherein the coding sequence nucleic acid for the anti-VEGF protein is such as SEQ ID NO: 7, the 5' to 3' nucleotide sequence is such as SEQ ID NO: 27, and the plasmid nucleotide sequence is such as SEQ ID NO: 28; the plasmid whose coding sequence for the anti-VEGF protein has been codon-optimized and CpG removed is numbered No. 3, wherein the coding sequence nucleic acid for the anti-VEGF protein is such as SEQ ID NO: 8, the 5' to 3' nucleotide sequence is such as SEQ ID NO: 29, and the plasmid nucleotide sequence is such as SEQ ID NO:30; the VH4 intron is inserted into different positions of the anti-VEGF protein coding sequence in plasmids No.4 and No.5, respectively. The nucleic acid coding sequence of the anti-VEGF protein in plasmid No.4 is such as SEQ ID NO:6, the 5' to 3' nucleotide sequence is such as SEQ ID NO:9, and the plasmid nucleotide sequence is such as SEQ ID NO:10; the nucleic acid coding sequence of the anti-VEGF protein in plasmid No.5 is such as SEQ ID NO:13, the 5' to 3' nucleotide sequence is such as SEQ ID NO:31, and the plasmid nucleotide sequence is such as SEQ ID NO:32; the SV40 intron and the Chimeric intron are inserted into the coding sequence of the anti-VEGF protein in plasmids No.6 and No.7, respectively; plasmids No.8-19 contain different expression regulatory elements (such as promoters, introns and polyadenylation signals), the nucleic acid coding sequence of the anti-VEGF protein in plasmid No.8 is such as SEQ ID NO:6, the 5' to 3' nucleotide sequence is such as SEQ ID NO:33, and the plasmid nucleotide sequence is such as SEQ ID NO:34. The plasmid numbers and main components are shown in Table 1 below, and the main components of representative plasmids are shown in Figure 1.

[0211] Table 1

[0212] Example 2: Protein expression in 293 cells transiently transfected with plasmids

[0213] In order to evaluate the expression characteristics of each polynucleotide expression cassette or plasmid in vitro, each recombinant construct was transfected into suspended HEK293F cells using a transfection reagent. The specific method is as follows: select an appropriate cell generation (no more than P15), and 2 hours before transfection of the plasmid, carefully add 10 ml of DMEM (containing 10% FBS) culture medium without disturbing the cell monolayer; then use LipofectamineTM Single plasmid transfection was performed using the LiP2000 method, specifically comprising: adding 2 μg of a plasmid containing the target gene (endotoxin removed) to 1 mL of serum-free medium, and further adding 5 μL of LiP2000 to 1 mL of serum-free medium; allowing each to stand for 5 minutes; then gently mixing and allowing to stand for 15 minutes; adding the mixture obtained in the previous step to a cell culture dish, distributing the mixture evenly at various locations during addition, gently mixing the entire culture medium after completion, and incubating at 37°C, 5% CO2; 6 hours after transfection, carefully aspirating the culture medium and replacing it with fresh culture medium (10% FBS, 1% P / S), continuing to culture for 48 to 72 hours, and then collecting the cells and cell supernatant; preparing cell lysates containing the expressed protein by repeated freezing and thawing three times, and determining the protein content by ELISA (n=3), as shown in Table 2.

[0214] Table 2

[0215] Example 3: Protein expression in APRE19 cells transiently transfected with plasmids

[0216] Each recombinant construct was transfected into suspended APRE19 cells using a transfection reagent. The specific method was as follows: select the appropriate cell generation (no more than P15), and 2 hours before plasmid transfection, carefully add 10 ml of DMEM (containing 10% FBS) without disturbing the cell monolayer; then use Lipofectamine TM Single plasmid transfection was performed using the LiP2000 method, specifically comprising: adding 2.5 μg of a plasmid containing the target gene (endotoxin removed) to 1 mL of serum-free medium, and further adding 5 μL of LiP2000 to 1 mL of serum-free medium; allowing each to stand for 5 minutes; then gently mixing and allowing to stand for 15 minutes; adding the mixture obtained in the previous step to a cell culture dish, distributing the mixture evenly at various locations during addition, gently mixing the entire culture medium after completion, and incubating at 37°C, 5% CO2; 6 hours after transfection, carefully aspirating the culture medium and replacing it with fresh culture medium (10% FBS, 1% P / S), continuing to culture for 48 to 72 hours, and then collecting the cells and cell supernatant; preparing cell lysates containing the expressed protein by repeated freezing and thawing three times, and determining the protein content by ELISA (n=3), as shown in Table 3.

[0217] Table 3

[0218] Example 3: Protein expression in 293 cells infected with AAV virus

[0219] The AAV capsid used in this example is a modified AAV8 capsid protein, which includes a polypeptide sequence having a substitution of amino acids 588 to 592 of the parent AAV8 with a polypeptide sequence as described in SEQ ID NO: 11 (RGNQQNTARQ). The construction of a plasmid containing this capsid protein is described in detail in the related patent application PCT / CN2022 / 142185 (Invention Title: Modified AAV Capsid Protein and Uses Thereof), which is incorporated herein.

[0220] AAV viral packaging was generated by co-transfecting cells with three plasmids: a first plasmid (plasmid No. 4) containing the ITRs flanked by the anti-VEGF protein coding sequence constructed in Example 1; a second plasmid encoding the Rep / Cap genes as described above (encoding the modified AAV8 capsid protein); and a third plasmid containing adenoviral helper genes. After transfection, cells were harvested, lysed, and the virus released. Viral titers were determined by PCR and used for further analysis.

[0221] ARPE19 cells were seeded in 6-well plates and cultured to 70-80% cell confluency. The culture medium (10% FBS, 1% P / S) was aspirated and replaced with serum-free and dual-antibody-free culture medium per well. Virus was added at the desired ratio (6E10 vg AAV virus to 3.6E5 ARPE19 cells) to a total volume of 1000 μL of virus plus serum-free and dual-antibody-free culture medium. After 6 hours of infection, 2 mL of fresh culture medium (10% FBS, 1% P / S) was added to each well and cultured for 72 hours. The expression of the target protein was determined by ELISA. The results showed that the AAV virus expressed 3437.6 ng / mL of protein.

[0222] Example 4: Drug efficacy experiment after AAV virus infection of NHP

[0223] Animal models

[0224] A total of 16 cynomolgus monkeys were divided into 4 groups: 1 negative control group and 3 test groups (4 monkeys in each dose).

[0225] Test drug

[0226] rAAV dose groups: low-dose group—5E11 vg / eye, medium-dose group—1E12 vg / eye, high-dose group—2E12 vg / eye (rAAV was prepared according to Example 3 and dialyzed in buffer (20 mM Tris-Cl pH 8.0, 150 mM NaCl, 1 mM MgCl2, 0.003% Poloxamer 188)).

[0227] Negative control group (Vehicle): Buffer (20 mM Tris-Cl pH 8.0, 150 mM NaCl, 1 mM MgCl2, 0.003% Poloxamer 188)

[0228] Dosage

[0229] On day 0, the drug was administered to both the dose group and the negative control group via the suprachoroidal space, with a 100 μL injection per eye. On day 21, laser modeling was performed in each group, with six laser spots per eye (48 spots each in the negative control group, the medium- and low-dose groups, and one monkey in the high-dose group was not included in the analysis due to bleeding, resulting in a total of 42 spots). On days 35 and 49, 14 and 28 days after modeling, fundus fluorescein angiography (FFA) was used to assess the formation of grade 4 spots.

[0230] Test results

[0231] [Corrected 17.06.2024 according to Rule 91] As shown in Figure 2, 14 days after model establishment, the number of level 4 spots in the buffer group was 21; the number of level 4 spots in the low-dose rAAV group was 16; the number of level 4 spots in the medium-dose rAAV group was 11; and the number of level 4 spots in the high-dose rAAV group was 0. Twenty-eight days after model establishment, the number of level 4 spots in the buffer group was 34; the number of level 4 spots in the low-dose rAAV group was 20; the number of level 4 spots in the medium-dose rAAV group was 12; and the number of level 4 spots in the high-dose rAAV group was 0. The proportion of level 4 spots in each group is shown in Table 4. An example of the FFA results of the high-dose rAAV group is shown in Figure 2.

[0232] Table 4

Claims

1. A nucleic acid, characterized in that The nucleic acid comprises a coding sequence encoding an anti-VEGF protein, and the coding sequence is codon optimized.

2. The nucleic acid according to claim 1, characterized in that The anti-VEGF protein comprises the extracellular domain 2 of VEGF receptor 1 and the extracellular domain 3 of VEGF receptor 2; preferably, the anti-VEGF protein also comprises the extracellular domain 4 of VEGF receptor 2; more preferably, the anti-VEGF protein also comprises an immunoglobulin Fc fragment; more preferably, the anti-VEGF protein comprises SEQ ID NO: 1, SEQ ID NO: 2 or a protein having at least 85% homology with any of the sequences therein.

3. The nucleic acid according to claim 2, characterized in that The nucleic acid comprises SEQ ID NO: 7 or a sequence having at least 85% homology thereto.

4. The nucleic acid according to claim 1, characterized in that The coding sequence of the nucleic acid is further subjected to CPG removal.

5. The nucleic acid according to claim 4, characterized in that The nucleic acid comprises SEQ ID NO: 8 or a sequence having at least 85% homology thereto.

6. The nucleic acid according to any one of claims 1 to 5, characterized in that The coding sequence of the nucleic acid further comprises an insertion of an intron.

7. The nucleic acid according to claim 6, characterized in that The intron is selected from VH4 intron, SV40 intron, Chi intron or Chimeric intron; preferably, the VH4 intron, SV40 intron, Chi intron or Chimeric intron has a nucleotide sequence as described in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

20.

8. The nucleic acid according to claim 7, characterized in that The nucleic acid comprises the nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 13, SEQ ID NO: 21 or SEQ ID NO: 22, or a nucleic acid having at least 85% homology to any of the sequences.

9. A polynucleotide expression cassette, characterized in that: The polynucleotide expression cassette comprises the nucleic acid and expression control elements described in any one of claims 1 to 8.

10. The polynucleotide expression cassette according to claim 9, characterized in that The polynucleotide expression cassette comprises, in order from 5' to 3': 1) 5'ITR; 2) Promoter; 3) Introns; 4) The nucleic acid according to any one of claims 1 to 8; 5) polyadenylation signal sequence; 6) 3'ITR.

11. The polynucleotide expression cassette according to claim 10, characterized in that: The polynucleotide expression cassette comprises, in order from 5' to 3': 1) 5'ITR; 2) CBA promoter; 3) chicken β-actin intron; 4) Kozak sequence; 5) The nucleic acid according to any one of claims 1 to 8; 6) RGB polyadenylation signal sequence; 7) 3'ITR; or The polynucleotide expression cassette comprises, in order from 5' to 3': 1) 5'ITR; 2) CBA promoter; 3) chicken β-actin intron; 4) Kozak sequence; 5) The nucleic acid according to any one of claims 1 to 8; 6) SV40 polyadenylation signal sequence; 7) 3'ITR.

12. The polynucleotide expression cassette according to claim 11, characterized in that The polynucleotide expression cassette comprises the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, or a sequence having at least 85% homology thereto.

13. A plasmid comprising the nucleic acid of any one of claims 1-8 or the polynucleotide expression cassette of any one of claims 9-12.

14. The plasmid according to claim 13, characterized in that The plasmid comprises the nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or a sequence having at least 85% homology thereto.

15. A recombinant adeno-associated virus (rAAV), comprising a capsid protein and a polynucleotide expression cassette according to any one of claims 9 to 12; preferably, the capsid protein is a wild-type or modified capsid protein selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

16. The recombinant adeno-associated virus according to claim 15, characterized in that include: (i) rAAV capsid protein, wherein the capsid protein is AAV8 capsid protein or a modified AAV8 capsid protein; and (ii) The polynucleotide expression cassette of any one of claims 9 to 12.

17. The recombinant adeno-associated virus according to claim 16, characterized in that The capsid protein is a modified AAV8 capsid protein, wherein the modified AAV8 capsid protein includes a substitution of the polypeptide sequence as described in SEQ ID NO: 11 at amino acids 588 to 592 of the parent AAV8.

18. A host cell, characterized in that The host cell comprises the nucleic acid of any one of claims 1-8 or the polynucleotide expression cassette of any one of claims 9-12 or the plasmid of claim 13 or 14 or the recombinant adeno-associated virus of any one of claims 15-17.

19. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a nucleic acid according to any one of claims 1 to 8, a polynucleotide expression cassette according to any one of claims 9 to 12, a plasmid according to claim 13 or 14, or a recombinant adeno-associated virus according to any one of claims 15 to 17 and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is an intravitreal injection, a subretinal injection preparation, an intrachoroidal injection preparation, an intravenous injection preparation, an intratumoral injection preparation or an intramuscular injection preparation.

20. Use of the nucleic acid of any one of claims 1-8 or the polynucleotide expression cassette of any one of claims 9-12 or the plasmid described in claim 13 or 14 or the recombinant adeno-associated virus of any one of claims 15-17 in the preparation of a medicament for treating a disease associated with VEGF; preferably, the disease associated with VEGF is an ocular neovascular disease; more preferably, the ocular neovascular disease is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema caused by branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, wet age-related macular degeneration with very low vision, and choroidal neovascularization secondary to pathological myopia.

21. A method for preventing or treating a disease associated with VEGF, characterized in that: A therapeutically effective dose of the nucleic acid of any one of claims 1-8, or the polynucleotide expression cassette of any one of claims 9-12, or the plasmid of claim 13 or 14, or the recombinant adeno-associated virus of any one of claims 15-17 is administered to the subject; preferably, the disease associated with VEGF is an ocular neovascular disease; more preferably, the ocular neovascular disease is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, macular edema caused by branch retinal vein occlusion, macular edema secondary to retinal vein occlusion, polypoidal choroidal vasculopathy, wet age-related macular degeneration with extremely low vision, and choroidal neovascularization secondary to pathological myopia.

22. The method according to claim 21, characterized in that The method comprises administering the drug by intravitreal injection, subretinal injection or suprachoroidal injection, preferably by suprachoroidal injection.

23. The method according to claim 22, characterized in that The dosage of the recombinant adeno-associated virus is E8 / eye to E16 / eye, preferably 5E11 / eye to 2E12 / eye, and more preferably 2E12 / eye.