Compositions and methods for treating eye diseases

Abstract

The present invention relates to a system for treating eye diseases and a method for treating eye diseases using the system.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits under Chinese Patent Application No. 202010706658.X (Agent Reference Number 57837-709.711), filed on 21 July 2020, and Chinese Patent Application No. 202010706505.5 (Agent Reference Number 57837-712.711), filed on 21 July 2020, all of which are incorporated herein by reference. [Background technology]

[0002] Neovascularization refers to the formation or growth of new blood vessels within tissues, or the further formation or growth of existing capillaries or blood vessels, and plays an important role in disease and health. Pathological neovascularization or neoangiogenesis of the eye can occur in the retina, choroid, and cornea and can cause severe visual impairment. Neovascularization of the eye is associated with a wide range of conditions, including wet age-related macular degeneration (wet AMD), diabetic retinopathy, and macular edema.

[0003] Many drugs have been developed to treat angiogenesis-related disorders, such as anti-VEGF antibodies (e.g., Lucentis) or fusion proteins (e.g., Eylea). However, these drugs have short half-lives, requiring frequent injections to maintain their effectiveness. Therefore, novel therapies that target angiogenesis are needed to treat angiogenesis-related eye diseases.

[0004] Sequence List This application includes a sequence listing filed electronically in ASCII format and incorporated herein by reference. A copy of the ASCII file prepared on 19 July 2021 is named 57837-712_601_SL and has a size of 24,376 bytes. [Overview of the Initiative]

[0005] Currently, there is a need in this field for the development of compositions and methods that can effectively treat ocular diseases associated with neovascularization.

[0006] In some embodiments, the Disclosure provides a composition comprising a first polynucleotide comprising a first sequence operably linked to a first promoter and a second sequence operably linked to a second promoter, wherein the first sequence encodes an adeno-associated virus (AAV) capsid protein and the second sequence encodes an AAV rep protein; and a second polynucleotide comprising a third sequence operably linked to a third promoter, wherein the third sequence comprises a codon-optimized nucleic acid sequence encoding a vascular endothelial growth factor (VEGF) inhibitor.

[0007] In some embodiments, the VEGF inhibitor is a fusion protein, or a VEGF antibody or its antigen-binding fragment. In some embodiments, the codon-optimized nucleic acid sequence encodes a protein containing the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the codon-optimized nucleic acid sequence encodes a protein containing the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the codon-optimized nucleic acid sequence encodes a protein containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the codon-optimized nucleic acid sequence contains CpG dinucleotides with a changed number compared to SEQ ID NO: 13. In some embodiments, the codon-optimized nucleic acid sequence contains 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or fewer than 5 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence does not contain CpG dinucleotides. In some embodiments, the third sequence contains the sequence of SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some embodiments, the first and second promoters are preferred for expression in insect cells or mammalian cells. In some embodiments, the insect cells are Sf9 cells. In some embodiments, the mammalian cells are HEK293 cells or their derivative cells. In some embodiments, the derivative cells are HEK293T cells. In some embodiments, the first or second promoter is the p10 promoter or the polh promoter. In some embodiments, the third promoter is the CMV promoter, CAG promoter, MNDU3 promoter, PGK promoter, EF1a promoter, or an eye-specific promoter.In some embodiments, eye-specific promoters are selected from the group consisting of the RPE65 gene promoter, the human retina-binding protein gene promoter, the mouse 11-cisretinoid alcohol dehydrogenase gene promoter, the rhodopsin promoter, the rhodopsin kinase promoter, the metalloproteinase 3 promoter (tissue inhibitors), the photoreceptor retinol-binding protein promoter, the vitiligo macular dystrophy 2 promoter, and the photoreceptor-retinoid-binding protein promoter. In some embodiments, the 3' end of the first sequence, the second sequence, or the third sequence further comprises a poly(A) sequence. In some embodiments, the poly(A) sequence is hGH poly(A), SV40 poly(A), or β-globin poly(A). In some embodiments, the first and second sequences are linked by a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises a 2A peptide. In some embodiments, the linker comprises an internal ribosome entry site (IRES). In some embodiments, IRES is derived from foot and mouth disease virus (FMDV). In some embodiments, the second polynucleotide includes an intron or a regulatory element. In some embodiments, the intron includes a chimeric intron. In some embodiments, the regulatory element includes sequences of TPL (an adenovirus tripartite leader sequence) and eMLP (an enhancer element derived from the adenovirus major late promoter). In some embodiments, the second polynucleotide includes a Kozak sequence. In some embodiments, the second polynucleotide includes a human skeletal attachment region (SAR) sequence. In some embodiments, the second polynucleotide further includes an enhancer. In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the second polynucleotide further includes a filler sequence. In some embodiments, the second polynucleotide further includes a terminal inversion repeat (ITR) sequence. In some embodiments, the ITR is an AAV2 ITR. In some embodiments, the second polynucleotide further includes a fourth sequence encoding an additional therapeutic protein.In some embodiments, the additional therapeutic protein is selected from the group consisting of VEGF inhibitors, PDGF inhibitors, placental growth factor inhibitors, integrin inhibitors, mTOR inhibitors, angiopoietin inhibitors, and TGFβ inhibitors. In some embodiments, the third and fourth sequences are linked by a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker contains a 2A peptide.

[0008] In another embodiment, the Disclosure provides recombinant adeno-associated virus (rAAV) particles prepared by introducing any one of the polynucleotides disclosed herein or any one of the compositions disclosed herein into cells. In some embodiments, the cells are insect cells or mammalian cells. In some embodiments, the insect cells are Sf9 cells. In some embodiments, the mammalian cells are HEK293 cells or derivative cells. In some embodiments, the derivative cells are HEK293T cells.

[0009] In another embodiment, the disclosure provides a polynucleotide comprising a codon-optimized nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the codon-optimized nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the codon-optimized nucleic acid sequence comprises a number of CpG dinucleotides that have been changed compared to SEQ ID NO: 13. In some embodiments, the codon-optimized nucleic acid sequence comprises 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or fewer than 5 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence does not contain CpG dinucleotides. In some embodiments, the third sequence comprises the sequence of SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some embodiments, the polynucleotide further comprises a promoter. In some embodiments, the promoter is a CMV promoter, a CAG promoter, an MNDU3 promoter, a PGK promoter, an EF1a promoter, or an eye-specific promoter. In some embodiments, eye-specific promoters are selected from the group consisting of the RPE65 gene promoter, the human retina-binding protein gene promoter, the mouse 11-cisretinoid alcohol dehydrogenase gene promoter, the rhodopsin promoter, the rhodopsin kinase promoter, the metalloproteinase 3 promoter (tissue inhibitors), the photoreceptor retinol-binding protein promoter, the vitiligo macular dystrophy 2 promoter, and the photoreceptor-retinoid-binding protein promoter. In some embodiments, the polynucleotide further comprises a poly(A) sequence. In some embodiments, the poly(A) sequence is hGH poly(A), SV40 poly(A), or β-globin poly(A). In some embodiments, the polynucleotide further comprises an intron or regulatory element. In some embodiments, the intron comprises a chimeric intron. In some embodiments, the regulatory element comprises the sequences of TPL (an adenovirus tripertite leader sequence) and eMLP (an adenovirus major late promoter enhancer element). In some embodiments, the polynucleotide further comprises a Kozak sequence.

[0010] In another aspect, the Disclosure provides recombinant adeno-associated virus (rAAV) particles comprising any one of the polynucleotides disclosed herein.

[0011] In another aspect, the Disclosure provides a method for expressing a VEGF inhibitor in cells or tissue of interest, comprising the step of administering to the cells or tissue of interest one of the rAAV particles disclosed herein or one of the polynucleotides disclosed herein.

[0012] In another embodiment, the Disclosure provides a method for treating an eye disease of a subject requiring treatment of the eye disease, comprising the step of administering to the subject a therapeutically effective dose of any one of the rAAV particles disclosed herein or any one of the polynucleotides disclosed herein. In some embodiments, the eye disease is selected from the group consisting of wet age-related macular degeneration (wet AMD), diabetic retinopathy, diabetic macular edema, proliferative diabetic retinopathy, and macular edema.

[0013] In another embodiment, the Disclosure provides a method for preparing recombinant adeno-associated virus (rAAV) particles, comprising the step of introducing one of the compositions disclosed herein or one of the polynucleotides disclosed herein into cells. In some embodiments, the Method comprises the step of expressing one of the polynucleotides disclosed herein in cells. In some embodiments, the cells are insect cells or mammalian cells. In some embodiments, the insect cells are Sf9 cells. In some embodiments, the mammalian cells are HEK293 cells or derivative cells. In some embodiments, the derivative cells are HEK293T cells. In some embodiments, the Method comprises the step of generating bacmid DNA and / or baculovirus. In some embodiments, the Method comprises the step of generating bacmid DNA containing a sequence expressing a VEGF inhibitor (such as a polynucleotide disclosed herein). In some embodiments, the Method comprises the step of generating a sequence expressing bacmid DNA rAAV cap-rep. In some embodiments, the Method comprises the step of producing baculovirus by transfecting cells with bacmid DNA. In some embodiments, the method includes the step of producing a baculovirus by transfecting cells with bacmid DNA containing a sequence expressing a VEGF inhibitor. In some embodiments, the method includes the step of producing a baculovirus containing a sequence expressing rAAV cap-rep by transfecting cells with bacmid DNA. In some embodiments, the method further includes the step of infecting cells (such as Sf9 cells) by mixing baculoviruses to obtain packaged rAAV / VEGF inhibitor virus particles disclosed herein.

[0014] Reference All publications, patents, and patent applications referred to in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, this specification is intended to supersede and / or take precedence over such conflicting subject matter.

Brief Description of the Drawings

[0015] The novel features of the invention are specifically set forth in the appended claims. The features and advantages of the invention will be better understood from the following detailed description, which sets forth exemplary embodiments in which the principles of the invention are utilized, and from the appended drawings (also referred to herein as "Figure" and "FIG").

[0016] [Figure 1A] It is a figure exemplifying a fluorescence image showing the expression of green fluorescent protein (GFP) 48 hours after transfection of a second polynucleotide encoding GFP in 293T cells. [Figure 1B] It is a figure exemplifying the results of flow cytometry showing the proportion of GFP-expressing cells 48 hours after transfection.

Modes for Carrying Out the Invention

[0017] Although various embodiments of the present disclosure are shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art can envision many variations, modifications, and substitutions without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the invention described herein may be utilized.

[0018] Unless otherwise specified, the implementation of some of the embodiments disclosed in this specification employs conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. For example, Molecular Cloning: A Laboratory Manual, 4th Edition (2012) by Sambrook and Green, the series Current Protocols in Molecular Biology (edited by F.M. Ausubel et al.), the series Methods In Enzymology (Academic Press, Inc.), PC 2: A Practical Approach (edited by M.J. MacPherson, B.D. Hames and G.R. Taylor (1995)), Antibodies, A Laboratory Manual edited by Harlow and Lane (1988), and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (edited by R.I. Freshney (2010)).

[0019] Definitions As used in this specification and the claims, the singular forms "a", "an", and "said" include plural references unless the context clearly dictates otherwise. For example, the term "immunoactivator" includes one or more immunoactivating agents.

[0020] The terms “about” or “approximately” mean that a value is within an acceptable margin of error for a particular value as determined by those skilled in the art, and that this depends in part on how the value is measured or determined, i.e., on the limitations of the measuring system. For example, in practice in the art, “about” may mean within one or more standard deviations. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean that the value is within one order of magnitude, preferably five times, more preferably two times. Where a particular value is described in this application and claims, the term “about” should be assumed to mean that, unless otherwise specified, that the value is within an acceptable margin of error for that particular value.

[0021] As used herein, the term “treatment” refers to an effort to modify the natural course of treatment for a disease in an individual, and may be a clinical intervention for prevention or implementation during the course of clinicopathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or reducing the disease state, and / or improving the prognosis.

[0022] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to amino acid polymers of any length. These polymers may be linear, cyclic, or branched, may contain modified amino acids, and may be inhibited by non-amino acids. These terms also include amino acid polymers modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodization, methylation, oxidation, proteolysis, phosphorylation, isoprenylation, racemization, selenization, transfer RNA-mediated addition of amino acids to proteins (e.g., arginine), ubiquitination, or conjugation with labeled components. As used herein, the term “amino acid” refers to natural and / or unnatural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimetic compounds. A polypeptide or amino acid sequence “derived” from a particular protein refers to the origin of the polypeptide. Preferably, the polypeptide has substantially the same amino acid sequence as the polypeptide encoded in the sequence or a portion thereof, the portion of which consists of at least 10 to 20 amino acids, at least 20 to 30 amino acids, or at least 30 to 50 amino acids, or can be immunologically identified by the polypeptide encoded in the sequence. This term also includes polypeptides expressed from specific nucleic acid sequences. As used herein, the term “domain” refers to a portion of a protein that is physically or functionally distinct from other parts of a protein or peptide. Physically defined domains include highly hydrophobic or hydrophilic amino acid sequences, such as membrane-bound or cytoplasm-bound sequences. Domains can also be defined by internal homology, for example, resulting from gene replication. Functionally defined domains have different biological functions. For example, an antigen-binding domain refers to an antigen-binding unit, or a portion of an antibody that binds to an antigen.Functionally defined domains do not need to be encoded by a continuous amino acid sequence, and may contain one or more physically defined domains.

[0023] As used herein, the term “amino acid” refers to natural and / or unnatural or synthetic amino acids, including but not limited to D or L optical isomers, as well as amino acid analogs and peptide mimetic compounds. Standard one- or three-letter codes are used to designate amino acids. In this context, amino acids are generally represented by one- and three-letter abbreviations well known in the art. For example, alanine can be represented as A or Ala.

[0024] As used herein, in the case of a polypeptide, “sequence” is the sequence of amino acids in the polypeptide from the amino terminus to the carboxyl terminus, where adjacent residues in the sequence are present in the polypeptide. The primary structure is continuous. The sequence can also be a linear sequence of a subset of a polypeptide known to contain additional residues in one or two directions.

[0025] As used herein, “identity,” “homologousity,” or “sequence identity” refers to the sequence similarity between two or more polynucleotide sequences or two or more polypeptide sequences. When sequence identity, similarity, or homology between two different amino acid sequences is determined using a program such as Emboss Needle or BestFit, default settings can be used, or an appropriate scoring matrix such as bloom45 or bloom80 can be selected to optimize the identity, similarity, or homology scores. Preferably, homologous polynucleotides hybridize under tight conditions and have sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% compared to these sequences. When sequences of equivalent length are optimally aligned, homologous polypeptides preferably have sequence identity of at least 80%, at least 90%, at least 95%, at least 97%, or at least 98%, or at least 99%.

[0026] With respect to the antigen-binding units disclosed herein, “percentage of sequence identity (%)” is defined as the percentage of identical amino acids between a query sequence and another reference polypeptide sequence after obtaining the maximum percentage of sequence identity by aligning the sequences and introducing gaps as necessary, without performing any conservative substitutions as part of sequence identity. Alignment for determining the percentage of amino acid sequence identity can be achieved by various methods conceivable to those skilled in the art, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR). Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithms necessary to obtain the maximum alignment beyond the full length of the comparison sequence. The percentage of identity can be measured against the length of a given entire polypeptide sequence, or against the length of a fragment obtained from a larger given polypeptide sequence, such as a shorter length, e.g., a fragment of at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, or at least 200 consecutive residues. These lengths are for illustrative purposes only, and it should be understood that any fragment length supported by the sequences shown in the tables, drawings, or sequence listings herein can be used to describe lengths for which the proportion of identity can be measured.

[0027] The proteins described herein may have one or more modifications to the reference sequence. These modifications may be deletions, insertions, additions, or substitutions of amino acid residues. "Deletion" refers to a change in the amino acid sequence resulting from the removal of one or more amino acid residues. "Insertion" or "addition" refers to a change in the amino acid sequence resulting from the addition of one or more amino acid residues compared to the reference sequence. "Substitution" or "substituted" means that one or more amino acids are replaced with different amino acids. In this specification, mutations in an antigen-binding fragment compared to a reference sequence can be determined by comparing the antigen-binding fragment with the reference sequence. Optimal sequence alignment for comparison can be performed according to any method known in the art.

[0028] As used herein, the term “isolated” refers to the separation of polynucleotides, peptides, polypeptides, proteins, antibodies, or their fragments from cellular components or other components where they naturally occur. Those skilled in the art will understand that non-spontaneously occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or their fragments do not need to be “isolated” to distinguish them from their spontaneously occurring counterparts. In addition, “enriched,” “isolated,” or “diluted” polynucleotides, peptides, polypeptides, proteins, antibodies, or their fragments are distinguishable from their spontaneously occurring counterparts by having a higher (“enriched”) or lower (“isolated”) concentration or number of molecules per unit volume than those of their spontaneously occurring counterparts. Enrichment can be measured based on an absolute amount, such as the weight of solution per unit volume, or against another potentially interfering substance present in the source mixture.

[0029] The terms “polynucleotide,” “nucleic acid,” “nucleotide,” and “oligonucleotide” are used interchangeably. These refer to polymeric forms of nucleotides (whether deoxyribonucleotides or ribonucleotides) or their analogues of any length. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA, isolated RNA, nucleic acid probes, primers, oligonucleotides, or synthetic DNA. Polynucleotides may include modified nucleotides such as methylated nucleotides or nucleotide analogs. Modifications to the nucleotide structure can be conferred before or after polymer formation. The sequence of nucleotides may be blocked by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeling components.

[0030] When applied to polynucleotides, "recombinant" means that the polynucleotide is a product of cloning, restriction digestion and / or ligation, and / or other procedures, and that this product is different from naturally occurring polynucleotides.

[0031] The terms “gene” and “gene fragment” are used interchangeably herein. These refer to polynucleotides containing at least one open reading frame that can encode a particular protein after transcription and translation. A gene or gene fragment may be a genome, cDNA, or synthetic product, insofar as the polynucleotide contains at least one open reading frame, and may cover an entire coding region or a segment thereof.

[0032] The terms "operably linked" or "effectively connected" refer to the juxtaposition of components in a way that allows them to function as intended. For example, if a promoter sequence facilitates the transcription of a coding sequence, the promoter sequence is operably linked to the coding sequence.

[0033] As used herein, “expression” refers to the process by which a polynucleotide is transcribed into mRNA, and / or the process by which the transcribed mRNA (also referred to as a “transcript”) is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, expression may involve splicing of mRNA in eukaryotic cells.

[0034] As used herein, the term “vector” refers to a nucleic acid vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it is capable of expressing a protein encoded by the inserted polynucleotides. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material carried by the vector can be expressed in the host cell. Vectors well known to those skilled in the art include, but are not limited to, plasmids, phagemids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as lambda phages or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillae polyoma vacuolar viruses (e.g., SV40). A vector may contain numerous elements that regulate expression, including but not limited to promoters, transcription initiators, enhancers, selection elements, and reporter genes. In addition, a vector may also contain a replication origin.

[0035] The term "transfection" is used to refer to the uptake of foreign DNA by cells, and a cell is "transfected" when exogenous DNA is introduced into the cell membrane. Numerous transfection techniques are publicly known in the art as a whole. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as nucleotide integration vectors or other nucleic acid molecules, into a suitable host cell.

[0036] As used herein, the term “antibody” generally refers to an immunoglobulin molecule consisting of two pairs of polypeptide chains (each pair having one “light” (L) chain and one “heavy” (H) chain). The light chains of antibodies can be classified into κ light chains and λ light chains. The heavy chains can be classified as μ, δ, γ, α, or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by “J” regions of about 12 or more amino acids, and the heavy chain also contains a “D” region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of the domain CL. The constant region of an antibody can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can also be subdivided into highly denatured regions (also called complementarity-determining regions (CDRs)), interspersed among them are highly conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, which are positioned from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form antibody-binding sites, respectively. The assignment of amino acids to each region or domain follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or J.Mol.Biol.196:901-917 by Chothia & Lesk (1987), or Nature 342:878-883 by Chothia et al. (1989). The term “antibody” is not limited by any particular method of antibody production.Examples of antibodies include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies may also be antibodies of different isotypes, such as IgG (e.g., subtypes of IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM.

[0037] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to an antigen to which a full-length antibody binds, and / or the ability to compete with a longer antibody for specific binding to an antigen. The antigen-binding fragment is also known as the “antigen-binding moiety.” In general, for all purposes, refer to Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989)), which is incorporated herein by reference in its entirety. Recombinant DNA technology allows for the production of antibody antigen-binding fragments by enzymatic or chemical fragmentation of intact antibodies. Antigen-binding fragments may include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides containing at least a portion of the antibody sufficient to confer specific antigen-binding ability. In some cases, the antigen-binding fragment is a single-chain antibody (e.g., scFv), in which the VL and VH domains are paired to form a monovalent molecule by enabling the production of a linker, which is a single polypeptide chain (e.g., Science 242:423 by Bird et al.). See 426 (1988) and Proc. Natl. Acad. Sci. USA 85:5879 5883 (1988) by Huston et al. Such scFv molecules may have NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH as common structures. Suitable linkers include, but are not limited to, repeating GGGGS amino acid sequences or their variants. For example, linkers having amino acid sequence (GGGGS)4 and its variants can be used (see Proc Natl. Acad. Sci. USA 90:6444-6448 by Holliger et al. (1993)).Other available linkers are listed in Protein Eng. 8:725-731 by Alfthan et al. (1995), Eur. J Immunol. 31:94-106 by Choi et al. (2001), Cancer Res. 56:3055-3061 by Hu et al. (1996), J. Mol. Biol. 293:41-56 by Kipriyanov et al. (1999), and Cancer Immunol. by Roovers et al. (2001). All references are incorporated herein by reference in their entirety for all purposes.

[0038] Antibody antigen-binding fragments (e.g., antibody fragments as described above) can be obtained by conventional techniques (e.g., recombinant DNA technology, enzymatic fragmentation, or chemical fragmentation) and can be specifically screened in the same way as intact antibodies. Unless otherwise explicitly stated in the context, the term "antibody" refers not only to the entire antibody but also to the antigen-binding fragment of the antibody.

[0039] As used herein, the term “host cell” refers to, but is not limited to, prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast and Aspergillus, insect cells such as Drosophila S2 cells and Sf9 insect cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or derivatives thereof.

[0040] The terms “antagonist” and “inhibitor” are used interchangeably herein and refer to molecules capable of inhibiting the biological function of a target protein by inhibiting its activity or expression. Therefore, the terms “antagonist” and “inhibitor” are defined in the context of the biological effects of the target protein. Preferred antagonists herein interact specifically with (e.g., bind to) the target, but molecules that inhibit the biological activity of a target protein by interacting with other members of a signaling pathway in which the target protein is a member are also included in this definition.

[0041] As used herein, “effective dose” refers to the minimum amount necessary to achieve a measurable improvement or prevention of a particular disease or illness. The effective dose may vary based on the patient’s disease state, age, sex, and weight. The effective dose is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the treatment. In the treatment of cancer or tumors, the effective dose of a drug may have the effect of reducing the number of cancer cells, shrinking the size of the tumor, inhibiting the invasion of cancer cells into peripheral organs, inhibiting tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating one or more symptoms associated with the disease to some extent. The effective dose may be administered for one or more uses.

[0042] As used herein, the terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably and refer to any mammalian subject, preferably human, being diagnosed or treated.

[0043] As used herein, the terms “treatment,” “treating,” and their equivalents are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely stabilizes or cures a disease and / or adverse reactions resulting from the disease. As used herein, “treatment” encompasses all treatments for diseases in mammals, including mice, rats, rabbits, pigs, humans and other apes, and primates, preferably humans. The term includes (a) preventing a disease or symptom from occurring in an object that may be susceptible to the disease or symptom but has not yet been diagnosed, (b) inhibiting the symptoms of the disease, (c) preventing the onset of the disease, (d) alleviating the symptoms of the disease, (e) causing a recurrence of the disease or symptom, or any combination of (a) to (e).

[0044] Where used herein, the term “kit” refers to a combination packaged for general use or commercial purposes. For example, a kit of the Disclosure may comprise the Compositions of the Disclosure and instructions for the use of the Compositions or the Kit. The term “instructions” refers to the explanatory leaflet typically included in the commercial packaging of a therapeutic agent, which includes information regarding indications, use, dosage, administration, combination therapy, contraindications, and / or warnings relating to the use of such therapeutic agent.

[0045] The term "codon optimization" refers to the modification of codons that construct a nucleic acid sequence so that the codons are best suited for expression in a particular system (e.g., a particular species or group of species). For example, a nucleic acid sequence is optimized for more efficient expression in mammalian cells. Codon optimization does not alter the amino acid sequence of the encoded protein, as synonymous codons exist. Various codon optimization methods are known in the art, including those disclosed in U.S. Patents 5,786,464 and 6,114,148, which are incorporated herein by whole reference for all purposes. "Synonymous codons" refer to codons that encode the same amino acid.

[0046] Proteins consist of 20 amino acids and 64 codons that code for them. Each amino acid corresponds to at least one codon, and one amino acid can correspond to up to six codons (degenerate codons). The frequency of use of degenerate codons differs across different organisms, and even across different protein-coding genes in the same organism, indicating specific preferences. Among these, frequently used codons are called preferred codons, while rarely used codons are called rare or low-frequency codons. Gene codon optimization can increase protein expression levels by utilizing preferred codons, avoiding rare or low-frequency codons with low utilization rates, simplifying the secondary structure of mRNA after gene transcription, incorporating motifs that contribute to high-efficiency expression while reducing motifs undesirable for expression, and adjusting GC content. Many common codon optimization principles exist, but these general principles cannot be uniformly applied to a single gene therapy vector. Various general optimization principles can be contradictory. For example, changing the composition of CpG islands or the GC content of coding regions may affect the selection of codon preference. In addition, various codon optimizations can lead to various post-translational modifications and diverse biological activities.

[0047] In the context of this invention, "active" or "active" refers to a form of therapeutic protein that retains the bioactivity of the corresponding naturally occurring polypeptide. The activity may be higher, equal to, or lower than the activity observed in the corresponding naturally occurring polypeptide.

[0048] Wet age-related macular degeneration Macular degeneration, also known as retinal degeneration, is an eye disease characterized by macular degeneration.

[0049] Age-related macular degeneration (AMD) is one of the most important causes of irreversible vision loss in people over 50 years of age. Clinically, AMD is divided into two types: "dry" and "wet." In the wet form of AMD, new blood vessels form, altering the blood supply to retinal tissue, particularly the submacular tissue. However, these new vessels are fragile, and when they rupture, it leads to bleeding and damage to surrounding tissue, scarring of retinal tissue, and rapid loss of vision. This disease progresses rapidly and often results in blindness. Wet macular degeneration usually begins with distortion of the central field of vision and accounts for about 90% of macular degeneration-related blindness.

[0050] Several cytokines have been found to play a crucial role in regulating angiogenesis, including, but are not limited to, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), placental growth factor, platelet-derived growth factor (PDGF), hypoxia-inducible factor (HIF), angiopoietin (Ang), and other cytokines, as well as mitogen-activated protein kinase (MPK).

[0051] Vascular endothelial growth factor (VEGF) is a 46 kDa glycoprotein expressed in ophthalmic cells, including pigment epithelial cells, pericytes, vascular endothelial cells, glial cells, and ganglion cells. VEGF is known to be associated with a variety of eye diseases, including but not limited to ischemic retinopathy, intraocular neovascularization, age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, diabetic macular edema, diabetic retinal ischemia, diabetic retinal edema, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, and retinal branch vein occlusion.

[0052] Eylea®, a VEGF-binding fusion protein (aflibercept), is an approved drug for the treatment of wet AMD. It can treat wet AMD by preventing neovascularization in the eye. Lucentis®, an anti-VEGF antibody (ranibizumab), is another approved drug for the treatment of wet AMD. This also treats wet AMD by preventing neovascularization in the eye. Clinical trials have shown that approximately 95% of patients treated with Lucentis® exhibited improvement or stabilization of their visual field. However, these drugs are very expensive and have short half-lives, requiring frequent injections to maintain effectiveness. Therefore, there is a need for novel therapies that target VEGF to treat related eye diseases.

[0053] Recombinant AAV vectors Adeno-associated viruses (AAVs) belong to the Parvoviridae family and are single-stranded DNA (ssDNA) viruses. The AAV genome is approximately 4.7 kilobases long and can contain terminal inversion repeats (ITRs) at both ends of the DNA strand, as well as two open reading frames (ORFs) called rep and cap.

[0054] AAV terminal inverted repeat (ITR) sequences are approximately 145-nucleotide sequences located at both ends of the natural single-stranded AAV genome. ITRs are symmetrical nucleic acid sequences for efficient replication in the adeno-associated virus genome, can be used as origins for viral DNA synthesis, and are essential structural components of recombinant AAV vectors.

[0055] "Rep" contains polynucleotide sequences encoding four rep proteins necessary for the AAV lifecycle: rep78, rep68, rep52, and rep40. "Cap" contains polynucleotide sequences encoding AAV capsid proteins VP1, VP2, and VP3, which interact with each other to form 24 symmetric AAV capsids.

[0056] AAV can effectively infect both dividing and non-dividing human cells, and its genome can be integrated into a single chromosomal region within the host cell genome. Most importantly, while AAV is present in the human body, current research suggests that AAV is not associated with any disease. Based on its high safety, low immunogenicity, broad host region, and ability to mediate the long-term, stable expression of exogenous genes in animals, AAV has become the most promising vector system for gene therapy.

[0057] Based on AAV serotype or infected tissue or cells, thirteen different AAVs, namely AAV1 to AAV13, have been identified to date. Furthermore, as shown in Table 1 below, many advantageous vector systems using AAVs have been developed for transfection of specific cell types. Among the AAV serotypes, serotype 2 (AAV2) is the most widely studied and used. It can infect retinal epithelium, photoreceptor cells, skeletal muscle, the central nervous system, and hepatocytes, among others. It has also been used as a carrier in numerous clinical trials.

[0058] [Table 1]

[0059] As used herein, the term “recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector containing one or more heterologous sequences (i.e., non-AAV-derived nucleic acid sequences) adjacent to two AAV terminal inversion repeats (ITRs). When present in host cells expressing AAV rep and cap proteins, rAAV vectors can be replicated and packaged into AAV virus particles.

[0060] "Recombinant AAV (rAAV) virus" or "rAAV virus particle" refers to an AAV virus particle consisting of at least one AAV capsid protein that encapsulates an rAAV vector. Host cells currently used for rAAV virus particle production include all mammalian cell types, such as 293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines. rAAV virus particles can be produced in mammalian cell culture systems given an rAAV plasmid. However, the output of most mammalian cell culture systems is insufficient to meet the requirements for clinical trial and commercial-scale production. Therefore, rAAV virus particle production systems using insect cells, such as Sf9 cells, have been developed in recent years. However, producing AAV in insect cells requires several modifications to obtain the precise theoretical mixing ratio of AAV capsid proteins.

[0061] Baculoviruses belong to the family Baculoviridae and are double-stranded circular DNA viruses. Their genome size is between 90kb and 230kb. Baculoviruses primarily parasitize arthropods and are known to infect over 600 species of insects. In 1983, Smith et al. successfully created the first baculovirus expression system by using Autographa Californica Multicapsid Nuclear Polyhedrosis Virus (AcMNPV) to express human β-interferon in the Spodoptera frugiperda cell line Sf9 (Mol Cell Biol, 1983, 3:2156-2165). Since then, baculovirus expression systems have been continuously improved and developed, becoming widely used eukaryotic expression systems. In 2002, Urabe et al. confirmed that Sf9 insect cells infected with baculovirus can support AAV replication. Urabe et al. successfully prepared rAAV virus particles by co-infecting Sf9 cells with three recombinant baculoviruses carrying the AAV rep gene, cap gene, and ITR core expression elements, respectively. Since then, researchers have been continuously developing systems better suited for large-scale preparation of rAAV virus particles.

[0062] Currently, there are two main baculovirus expression systems for the large-scale preparation of rAAV virus particles: the 2-baculovirus system (Two Bac system) and the 1-baculovirus system (One Bac system), which is dependent on the packaging cell line. The main process for preparing rAAV virus particles using the 2-baculovirus system involves integrating the AAV rep and cap genes into one baculovirus genome and integrating the ITR core expression elements and the target gene of interest into another baculovirus genome. Then, by co-infecting host cells with these two recombinant baculoviruses, rAAV virus particles carrying the target gene of interest are produced. The main process for preparing rAAV virus particles using the 1-baculovirus system begins with establishing a packaging cell line that induces the expression of the rep and cap genes. This packaging cell line integrates the expression elements of the rep and cap genes so that they are under the control of the polh promoter, a potent promoter of late baculovirus gene expression. The hr2 enhancer sequence and / or the AAV rep protein binding sequence can be added further upstream of the polh promoter. After infection with recombinant baculovirus containing the AAV ITR and target gene, the rep and cap genes in the packaging cell line are induced to express the protein, thereby generating rAAV virus particles that integrate the target gene.

[0063] In some embodiments, the rAAV vector used to carry the gene of interest in the rAAV virus particle may further include one or more “expression regulators.” The term “expression regulator,” as used herein, refers to a nucleic acid sequence that affects the expression of a manipulably linked polynucleotide, including a polynucleotide sequence that facilitates the transcription and translation of heterologous polynucleotides. Non-limiting examples of expression regulators include, but are not limited to, promoters, enhancers, intron splicing signals, polyadenylations (poly(A)), and terminal inversion repeats (ITRs). In some embodiments, the poly(A) sequence may be hGH poly(A), SV40 poly(A), or β-globin poly(A).

[0064] A "promoter" is a DNA sequence positioned adjacent to a heterologous polynucleotide sequence encoding a target product, and is typically manipulatively ligated to adjacent sequences such as the heterologous polynucleotide. Promoters generally increase the expression level of the heterologous polynucleotide compared to the expression level of the heterologous polynucleotide without the promoter.

[0065] An "enhancer" is a sequence that enhances the activity of a promoter. Unlike promoters, enhancers do not possess promoter activity and can usually function independently of their position relative to the promoter (i.e., upstream or downstream of the promoter). Non-restrictive examples of enhancer elements (or parts thereof) include baculovirus enhancers and enhancer elements found in insect cells.

[0066] A "filler sequence" refers to a nucleotide sequence contained within a larger nucleic acid molecule, such as a vector, and is typically used to create the necessary space between two nucleic acid sequences, such as between a promoter sequence and a coding sequence, or to extend a nucleic acid sequence to a desired length. Filler sequences do not contain protein coding information. Filler sequences may be of unknown or synthetic origin and / or may be unrelated to other nucleic acid sequences within the larger nucleic acid molecule.

[0067] composition In one embodiment, the present disclosure provides a composition comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a first sequence operably linked to a first promoter and a second sequence operably linked to a second promoter.

[0068] In some embodiments, the first sequence is the cap protein of adeno-associated virus (AAV). The cap protein may be any structural protein known in the art that is capable of forming a functional AAV capsid (i.e., capable of packaging DNA to infect target cells). In some embodiments, the cap protein comprises VP1, VP2, and VP3. In some embodiments, the cap protein does not need to contain all of VP1, VP2, and VP3, as long as it can produce a functional AAV capsid. In some embodiments, the cap protein comprises VP1 and VP2. In some embodiments, the cap protein comprises VP1 and VP3. In some embodiments, the cap protein comprises VP2 and VP3. In some embodiments, the cap protein comprises VP1. In some embodiments, the cap protein comprises VP2. In some embodiments, the cap protein comprises VP3.

[0069] VP1, VP2, and VP3 may be derived from any AAV serotype. In some embodiments, VP1 may be derived from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3 including serotypes 3A and 3B), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV-Rh10, AAV-Rh74, AAV-2i8, and any other known AAV. In some embodiments, VP1 is derived from wild-type VP1 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has at least 75%, 80%, 85%, 90%, 95%, or more identity with these wild-type VP1 proteins. In some embodiments, VP1 is derived from wild-type VP1 from AAV1, AAV2, AAV2 variants (such as AAV2.7m8, AAV2(quad YF), and AAV2tYF), AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8 and has one or more amino acid substitutions, deletions, and / or additions.

[0070] In some embodiments, VP2 may originate from AAV1, AAV2, AAV2 variants (such as AAV2.7m8, AAV2(quad YF), and AAV2tYF), AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, as well as any other known AAV. In some embodiments, VP2 is derived from wild-type VP2 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has at least 75%, 80%, 85%, 90%, 95%, or more identity with these wild-type VP1 proteins. In some embodiments, VP2 is derived from wild-type VP2 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has one or more amino acid substitutions, deletions, and / or additions.

[0071] VP3 may originate from AAV1, AAV2, AAV2 variants (such as AAV2.7m8, AAV2(quad YF), and AAV2tYF), AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, as well as any other known AAV. In some embodiments, VP3 is derived from wild-type VP3 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has at least 75%, 80%, 85%, 90%, 95%, or more identity with these wild-type VP3 proteins. In some embodiments, VP3 is derived from wild-type VP3 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has one or more amino acid substitutions, deletions, and / or additions.

[0072] In some embodiments, the cap includes VP1, VP2, and / or VP3 derived from the same cellotype AAV, for example, the cap may include VP1, VP2, and / or VP3 all derived from AAV2. In some embodiments, the cap includes VP1, VP2, and / or VP3 derived from different cellotype AAVs. For example, the cap may include one or more of VP1, VP2, and / or VP3 derived from any of AAV1, AAV2, AAV2 variants (such as AAV2.7m8, AAV2(quad YF), and AAV2tYF), AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, and AAV-2i8.

[0073] In some embodiments, a first sequence encoding cap is operably linked to a first promoter. The first promoter can be any suitable promoter known in the art that is capable of inducing cap expression in cells. In some embodiments, the first promoter can be a tissue-specific promoter, a constitutive promoter, or a regulated promoter. In some embodiments, the first promoter may be selected from a different source; for example, the first promoter may be a viral promoter, a plant promoter, or a mammalian promoter.

[0074] Examples of primary promoters include, but are not limited to, human cytomegalovirus (CMV) enhancer / promoter (e.g., CMV IE enhancer / promoter), SV40 enhancer / promoter (e.g., SV40 early enhancer / promoter), JC polyomavirus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latent-associated promoter (LAP), Roussarcoma virus (RSV) long-terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP), chicken β-actin promoter, CAG, MNDU3, PGK, and EF1a promoter.

[0075] In some embodiments, the first promoter is a promoter suitable for expression in mammalian cells. In some embodiments, the mammalian cell is a HEK293 cell or a derivative cell. In some embodiments, the derivative cell is a HEK293T cell. In some embodiments, the first promoter is a promoter suitable for expression in insect cells. In some embodiments, the insect cell is an Sf9 cell. In some embodiments, suitable promoters for expression in insect cells include, but are not limited to, the polh promoter, the p10 promoter, the basic promoter, the inducible promoter, the E1 promoter, or the ΔE1 promoter. In some embodiments, the first promoter is the polh promoter. In some embodiments, the first promoter is the p10 promoter.

[0076] In some embodiments, the 3' end of the first sequence further includes a polyadenylated sequence (i.e., a poly(A) sequence). In some embodiments, the length of the polyadenylated sequence may range from about 1 bp to 500 bp. In some embodiments, the length of the polyadenylated sequence may be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 10, 200, or 500 nucleotides. In some embodiments, the poly(A) sequence is hGH poly(A), SV40 poly(A), or β-globin poly(A).

[0077] In some embodiments, the second sequence encodes an AAV rep protein, in which case the rep protein can be any replication protein necessary for the replication and packaging of rAAV virus particles. In some embodiments, the rep protein includes rep78, rep68, rep52, and rep40. In some embodiments, the rep protein does not need to include all of rep78, rep68, rep52, and rep40, as long as it enables the replication and packaging of rAAV virus particles. In some embodiments, the rep protein includes any three of rep78, rep68, rep52, and rep40. In some embodiments, the rep protein includes any two of rep78, rep68, rep52, and rep40. In some embodiments, the rep protein includes any one of rep78, rep68, rep52, and rep40. In some embodiments, the rep protein includes rep78 and rep52. In some embodiments, the rep protein includes rep78 and rep40. In some embodiments, the rep protein includes rep68 and rep52. In some embodiments, the rep protein includes rep68 and rep40.

[0078] rep78, rep68, rep52, and rep40 may originate from any AAV 00type. In some embodiments, rep78 may originate from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, as well as any other known AAV. In some embodiments, rep78 is derived from wild-type rep78 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has at least 75%, 80%, 85%, 90%, 95%, or more identity to this wild-type rep78 protein. In some embodiments, rep78 is derived from wild-type rep78 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has one or more amino acid substitutions, deletions, and / or additions.

[0079] In some embodiments, rep68 may originate from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, as well as any other known AAV. In some embodiments, rep68 is derived from wild-type rep68 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has at least 75%, 80%, 85%, 90%, 95%, or more identity to this wild-type rep68 protein. In some embodiments, rep68 is derived from wild-type rep68 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has one or more amino acid substitutions, deletions, and / or additions.

[0080] In some embodiments, rep52 may originate from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, as well as any other known AAV. In some embodiments, rep52 is derived from wild-type rep52 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has at least 75%, 80%, 85%, 90%, 95%, or more identity to this wild-type rep52 protein. In some embodiments, rep52 is derived from wild-type rep52 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has one or more amino acid substitutions, deletions, and / or additions.

[0081] In some embodiments, rep40 may originate from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, and any other known AAV. In some embodiments, rep40 is derived from wild-type rep52 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has at least 75%, 80%, 85%, 90%, 95%, or more identity with this wild-type rep52 protein. In some embodiments, rep40 is derived from wild-type rep52 from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or AAV-2i8, and has one or more amino acid substitutions, deletions, and / or additions.

[0082] In some embodiments, rep includes rep78, rep68, rep52, and / or rep40 derived from the same serotype AAV. For example, rep may include rep78, rep68, rep52, and / or rep40 derived only from AAV2. In some embodiments, rep includes rep78, rep68, rep52, and / or rep40 derived from different serotype AAVs. For example, rep may include AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, and any other known AAV rep78, rep68, rep52, and / or rep40.

[0083] In some embodiments, a second sequence encoding the rep protein is operably ligated to a second promoter. The second promoter can be any suitable promoter known in the art that is capable of inducing cap expression within cells. In some embodiments, the second promoter can be a tissue-specific promoter, a constitutive promoter, or a regulated promoter. In some embodiments, the second promoter can be selected from different sources. For example, the second promoter can be a viral promoter, a plant promoter, or a mammalian promoter.

[0084] Examples of second promoters include, but are not limited to, human cytomegalovirus (CMV) enhancer / promoter (e.g., CMV IE enhancer / promoter), SV40 enhancer / promoter (e.g., SV40 early enhancer / promoter), JC polyomavirus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latent-associated promoter (LAP), Roussarcoma virus (RSV) long-terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP), chicken β-actin promoter, CAG, MNDU3, PGK, and EF1a promoter.

[0085] In some embodiments, the second promoter is a promoter suitable for expression in mammalian cells. In some embodiments, the mammalian cell is a HEK293 cell or a derivative cell. In some embodiments, the derivative cell is a HEK293T cell. In some embodiments, the second promoter is a promoter suitable for expression in insect cells. In some embodiments, the insect cell is an Sf9 cell. In some embodiments, suitable promoters for expression in insect cells include, but are not limited to, the polh promoter, the p10 promoter, the basic promoter, the inducible promoter, the E1 promoter, or the ΔE1 promoter. In some embodiments, the second promoter is the polh promoter. In some embodiments, the second promoter is the p10 promoter.

[0086] In some embodiments, the 3' end of the second sequence further includes a polyadenylated sequence (i.e., a poly(A) sequence). In some embodiments, the length of the polyadenylated sequence may range from about 1 bp to 500 bp. In some embodiments, the length of the polyadenylated sequence may be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 10, 200, or 500 nucleotides. In some embodiments, the poly(A) sequence is hGH poly(A), SV40 poly(A), or β-globin poly(A).

[0087] In some embodiments, cap and rep may originate from the same AAV serotype. For example, cap and rep may both originate from the same AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, or any other known AAV.

[0088] In some embodiments, cap and rep may originate from different AAV serotypes. For example, cap may originate from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, or any other known AAV, while rep may originate from any of the aforementioned AAVs other than the AAV from which cap originates. For example, cap may originate from AAV2, while rep may originate from AAV5.

[0089] In some embodiments, the first promotor and the second promotor may be the same promotor. For example, the first promotor and the second promotor may be the same, selected from the group consisting of polh promotor, p10 promotor, basic promotor, inductive promotor, E1 promotor, and ΔE1 promotor. For example, in some embodiments, both the first and second promotors are polh promotors. In some embodiments, both the first and second promotors are p10 promotors.

[0090] In some embodiments, the first and second promotors may be different promotors. For example, the first and second promotors may be two different promotors selected from the polh promotor, p10 promotor, basic promotor, inductive promotor, E1 promotor, and ΔE1 promotor. For example, in some embodiments, the first promotor is a polh promotor and the second promotor is a p10 promotor. In some embodiments, the first promotor is a p10 promotor and the second promotor is a polh promotor.

[0091] In some embodiments, the first and second sequences are linked by a sequence encoding a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is a sequence containing a 2A peptide. In some embodiments, the 2A peptide may be selected from 2A peptides derived from the genera Aphthora or Cardiovirus, for example, 2A peptides derived from foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thoseaasigna virus (Tav), or porcine Jieshen virus peptide (PTV-1). In some embodiments, the sequence encoding the linker further includes a promoter sequence. In some embodiments, the promoter is an FMDV promoter.

[0092] In some embodiments, the second polynucleotide of the composition disclosed herein comprises a third sequence and includes a codon-optimized nucleic acid sequence encoding a VEGF inhibitor. In some embodiments, the composition comprises an scAAV vector, the scAAV vector comprises the second polynucleotide. In some embodiments, the composition comprises an ssAAV vector, the ssAAV vector comprises the second polynucleotide.

[0093] A VEGF inhibitor can be any polypeptide or protein capable of inhibiting the biological function of the VEGF protein by inhibiting the activity or expression of the VEGF protein. In some embodiments, the VEGF inhibitor is an anti-VEGF antibody or its antigen-binding fragment. In some embodiments, the antigen-binding fragment may be, but is not limited to, Fab, Fab', F(ab')2, Fd, Fv, dAb, as well as complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, and diabodies. In some embodiments, the anti-VEGF inhibitor may be selected from ranibizumab, bevacizumab, or aflibercept.

[0094] In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 1, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 1. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 1. In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 2, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 2. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 2. In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 3, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 3. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 3. In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 4, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 4. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 4.

[0095] In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 5, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 5. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 5. In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 6, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 6. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 6. In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 7, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 7. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 7. In some embodiments, the VEGF inhibitor includes the sequences of SEQ ID NOs: 5, 6, and 7. In some embodiments, the VEGF inhibitor includes sequences having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NOs. 5, 6, and 7. In some embodiments, the VEGF inhibitor includes sequences having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NOs. 5, 6, and 7.

[0096] In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 8, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 8. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 8. In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 9, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 9. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 9. In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 10, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 10. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 10. In some embodiments, the VEGF inhibitor includes the sequences of SEQ ID NOs: 8, 9, and 10. In some embodiments, the VEGF inhibitor includes a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NOs. 8, 9, and 10. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NOs. 8, 9, and 10.

[0097] In some embodiments, the codon-optimized nucleic acid sequence encodes ranibizumab. In some embodiments, the codon-optimized nucleic acid sequence encodes bevacizumab. In some embodiments, the codon-optimized nucleic acid sequence encodes aflibercept. In some embodiments, the codon-optimized nucleic acid sequence encoding a protein includes the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 1. In some embodiments, the codon-optimized nucleic acid sequence encoding a protein includes the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 2. In some embodiments, the codon-optimized nucleic acid sequence encoding a protein includes the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 3. In some embodiments, the codon-optimized nucleic acid sequence encoding the protein includes the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 4.

[0098] In some embodiments, the codon-optimized nucleic acid sequence encoding a protein includes the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 5. In some embodiments, the codon-optimized nucleic acid sequence encoding a protein includes the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 6. In some embodiments, the codon-optimized nucleic acid sequence encoding the protein includes the amino acid sequence of SEQ ID NO: 7, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 7. In some embodiments, the codon-optimized nucleic acid sequence encoding a protein includes the amino acid sequences of SEQ ID NOs. 5, 6, and 7, or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NOs. 5, 6, and 7.

[0099] In some embodiments, the codon-optimized nucleic acid sequence encoding a protein includes the amino acid sequence of SEQ ID NO: 8, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 8. In some embodiments, the codon-optimized nucleic acid sequence encoding a protein includes the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 9. In some embodiments, the codon-optimized nucleic acid sequence encoding the protein includes the amino acid sequence of SEQ ID NO: 10, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 10. In some embodiments, the codon-optimized nucleic acid sequence encoding a protein includes the amino acid sequences of SEQ ID NOs. 8, 9, and 10, or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NOs. 8, 9, and 10.

[0100] In some embodiments, the codon-optimized nucleic acid sequence encoding a protein includes the amino acid sequence of SEQ ID NO: 11, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 11. In some embodiments, the codon-optimized nucleic acid sequence encoding a protein includes the amino acid sequence of SEQ ID NO: 12, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 12.

[0101] In some embodiments, the codon-optimized nucleic acid sequence contains a number of CpG dinucleotides that has changed compared to SEQ ID NO: 13. In some embodiments, the codon-optimized nucleic acid sequence contains fewer CpG dinucleotides than SEQ ID NO: 13. In some embodiments, the codon-optimized nucleic acid sequence contains more CpG dinucleotides than SEQ ID NO: 13. In some embodiments, the codon-optimized nucleic acid sequence contains 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or fewer than 5 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 0 to 80 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 5 to 75 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 10 to 70 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 15 to 65 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 20 to 60 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 25 to 55 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 30 to 50 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 35 to 45 CpG dinucleotides. In some embodiments, the nucleic acid sequence of the present invention includes the CpG dinucleotides 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, and 30. In some embodiments, the codon-optimized nucleic acid sequence does not contain CpG dinucleotides. In some embodiments, the third sequence includes the sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0102] In some embodiments, a third sequence is operably linked to a third promoter. In some embodiments, the third promoter is a CMV promoter, a CAG promoter, an MNDU3 promoter, a PGK promoter, an EF1a promoter, or an eye-specific promoter. In some embodiments, the eye-specific promoter is a promoter specific to retinal pigment epithelium (RPE) cells. Examples of RPE cell-specific promoters include, but are not limited to, the RPE65 gene promoter, the human retina-binding protein (CRALBP) gene promoter, the mouse 11-cis-retinol dehydrogenase (RDH) gene promoter, the rhodopsin promoter, the rhodoposin kinase promoter, the tissue inhibitor metalloproteinase 3 (Timp3) promoter, the photoreceptor retina-binding protein promoter, and the vitreomacular dystrophy 2 (vitiligo-like macular dystrophy 2) promoter and the photoreceptor-retinoid-binding protein (IRBP) promoter.

[0103] In some embodiments, the second polynucleotide further includes other regulatory sequences, including but not limited to terminal inversion repeats (ITRs), enhancers, splicing signals, polyadenylation signals (Poly(A)), stuffing sequences, terminators, proteolytic signals, internal ribosome entry elements (IRESs), and 2A sequences. In some embodiments, the Poly(A) sequence is hGH Poly(A), SV40 Poly(A), or β-globin Poly(A).

[0104] In some embodiments, the second polynucleotide further comprises an enhancer region. In some embodiments, the enhancer region may be an SV40 enhancer, a cytomegalovirus enhancer, an IRBP enhancer, or an enhancer derived from an immunoglobulin gene. In some embodiments, the enhancer region is located upstream of the CMV, CAG, MNDU3, PGK, or EF1a promoter. In some embodiments, the enhancer is located upstream of an eye-specific promoter. In some embodiments, the enhancer region is located downstream of the CMV, CAG, MNDU3, PGK, or EF1a promoter. In some embodiments, the enhancer is located downstream of an eye-specific promoter.

[0105] In some embodiments, the second polynucleotide further comprises terminal inverted repeat sequences (ITRs). In some embodiments, the second polynucleotide comprises at least one ITR. In some embodiments, the second polynucleotide comprises two ITRs. In some embodiments, the two ITRs are the same. In some embodiments, the two ITRs are different from each other. In some embodiments, the ITR is an ITR derived from an AAV. In some embodiments, the ITR may be derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, and any other known AAV. In some embodiments, the ITR has one or more nucleotide mutations, insertions, or deletions compared to wild-type ITRs derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, and any other known AAV, but retains desired terminal repeat sequence functions such as target gene replication, viral packaging, and / or integration.

[0106] In some embodiments, the second polynucleotide further comprises one or more filler sequences. In some embodiments, the filler sequences are located upstream of the CMV, CAG, MNDU3, PGK, or EF1a promoter sequence. In some embodiments, the filler sequences are located downstream of the CMV, CAG, MNDU3, PGK, or EF1a promoter sequence. In some embodiments, the filler sequences are located upstream of an eye-specific promoter. In some embodiments, the filler sequences are located downstream of an eye-specific promoter. In some embodiments, the filler sequences are located at the 5' end of the 5' ITR sequence. In some embodiments, the filler sequences are located at the 3' end of the 5' ITR sequence. In some embodiments, the filler sequences are located at the 5' end of the 3' ITR sequence. In some embodiments, the filler sequences are located at the 3' end of the 3' ITR sequence.

[0107] In some embodiments, the length of the filler array is not limited to 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1kb, 1.1kb, 1.2kb, 1.3kb, 1.4kb, 1.5kb, 1.6kb, 1.7kb, 1.8kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2. It can range from approximately 0.1kb to 5kb, such as 5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3kb, 3.1kb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4.0kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8kb, 4.9kb, or 5.0kb.

[0108] In some embodiments, the second polynucleotide further comprises an intron. In some cases, an intron may refer to any sequence that can be transcribed but not translated. In some cases, an intron may refer to any sequence that is transcribed and removed from mature RNA transcripts in cells. In some cases, an intron may contain at least about 1 bp, 50 bp, 100 bp, 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, or 5000 bp. In some cases, an intron may be about 300 bp. In some cases, an intron may be about 200–400 bp. In some cases, an intron may be about 100–500 bp. In some cases, an intron may be about 50–200 bp. Depending on the case, the intron may be either a naturally occurring intact intron or a chimeric intron. In some embodiments, the intron is located upstream of the third sequence. In some embodiments, the intron is located downstream of the promoter. In some embodiments, the second polynucleotide further includes a regulatory element. In some embodiments, the regulatory element includes sequences of TPL (an adenovirus tripertite leader sequence) and eMLP (an enhancer element derived from the adenovirus major late promoter). In some embodiments, the regulatory element is located upstream of the third sequence. In some embodiments, the regulatory element is located downstream of the promoter. In some embodiments, the second polynucleotide includes a Kozak sequence. In some embodiments, the Kozak sequence is located upstream of the third sequence. In some embodiments, the Kozak sequence is located downstream of the intron. In some embodiments, the second polynucleotide includes a human skeleton attachment region (SAR) sequence. In some embodiments, the SAR sequence is located downstream of the third sequence. In some embodiments, the SAR sequence is located upstream of the polyA signal.

[0109] In some embodiments, the second polynucleotide comprises 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or less than 10 CpG dinucleotides. In some embodiments, the second polynucleotide contains 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more CpG dinucleotides. In some embodiments, the second polynucleotide contains 100-300, 100-200, 100-150, 150-200, 150-250, 150-300, 200-250, 200-300, or 250-300 CpG CpG dinucleotides.

[0110] In some embodiments, the second polynucleotide further comprises a fourth sequence encoding a different therapeutic protein. In some embodiments, the different therapeutic protein is a VEGF inhibitor, a PDGF inhibitor, an integrin inhibitor, an mTOR inhibitor, an angiopoietin inhibitor, or a TGFβ inhibitor.

[0111] In some embodiments, the fourth and third sequences are linked by a sequence encoding a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker includes a sequence of 2A peptides. In some embodiments, the 2A peptide may be selected from 2A peptides derived from the genera Aphthora or Cardiovirus, such as the 2A peptides of foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thoseaasigna virus (Tav), or porcine rhinitis virus (PTV-1).

[0112] Recombinant AAV virus particles In another embodiment, the Disclosure provides recombinant adeno-associated virus (rAAV) particles prepared by introducing the compositions or polynucleotides of the Disclosure into cells. In some embodiments, the cells are insect cells or mammalian cells. In some embodiments, the insect cells are Sf9 cells. In some embodiments, the cells are mammalian cells, and are HEK293 cells or derivative cells. In some embodiments, the derivative cells are HEK293T cells.

[0113] In some embodiments, the compositions of the present disclosure can be delivered to cells by any method known in the art. In some embodiments, the method includes, but is not limited to, electroporation, calcium phosphate precipitation, and liposome-mediated methods. In some embodiments, the compositions are stably transfected to cells. In some embodiments, the compositions are transiently transfected to cells. In some embodiments, cells are used to produce rAAV virus particles.

[0114] rAAV virus particles can be isolated and purified from cells by methods known to those skilled in the art. For example, rAAV can be purified using centrifugation, HPLC, hydrophobic interaction chromatography (HIC), anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, ultrafiltration, gel electrophoresis, affinity chromatography, and / or other purification techniques for virus particles.

[0115] In another embodiment, the Disclosure provides rAAV particles comprising any of the polynucleotides disclosed herein.

[0116] Polynucleotides In another embodiment, the disclosure provides a polynucleotide comprising a codon-optimized nucleic acid sequence encoding a VEGF inhibitor. The VEGF inhibitor may be any polypeptide or protein capable of inhibiting the biological function of the VEGF protein by inhibiting the activity or expression of the VEGF protein. In some embodiments, the VEGF inhibitor is an anti-VEGF antibody or its antigen-binding fragment. In some embodiments, the antigen-binding fragment may be, but is not limited to, Fab, Fab', F(ab')2, Fd, Fv, dAb, as well as complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, and diabodies. In some embodiments, the anti-VEGF inhibitor may be selected from ranibizumab, bevacizumab, or aflibercept.

[0117] In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 1, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 1. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 1. In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 2, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 2. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 2. In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 3, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 3. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 3. In some embodiments, the VEGF inhibitor includes the sequence of SEQ ID NO: 4, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 4. In some embodiments, the VEGF inhibitor includes a sequence having at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology to SEQ ID NO: 4. In some embodiments, a codon-optimized nucleic acid sequence encoding a protein including the amino acid sequence of SEQ ID NO: 1. In some embodiments, the codon-optimized nucleic acid sequence includes a number of CpG dinucleotides that have changed compared to SEQ ID NO: 13. In some embodiments, the codon-optimized nucleic acid sequence includes fewer CpG dinucleotides than SEQ ID NO: 13.In some embodiments, the codon-optimized nucleic acid sequence contains more CpG dinucleotides than SEQ ID NO: 13. In some embodiments, the codon-optimized nucleic acid sequence contains 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or fewer than 5 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 0 to 80 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 5 to 75 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 10 to 70 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 15 to 65 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 20 to 60 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 25 to 55 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 30 to 50 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence contains 35 to 45 CpG dinucleotides. In some embodiments, the nucleic acid sequence of the present invention contains 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, and 30 CpG dinucleotides. In some embodiments, the codon-optimized nucleic acid sequence does not contain CpG dinucleotides. In some embodiments, the third sequence includes the sequence of sequence number 14, sequence number 15, sequence number 16, sequence number 17, or sequence number 18.

[0118] In some embodiments, the polynucleotide further comprises a promoter. In some embodiments, the promoter is the CMV promoter, CAG promoter, MNDU3 promoter, PGK promoter, EF1a promoter, or an eye-specific promoter. In some embodiments, the eye-specific promoter is selected from the group consisting of the RPE65 gene promoter, the human retina-binding protein gene promoter, the mouse 11-cisretinoid alcohol dehydrogenase gene promoter, the rhodopsin promoter, the rhodopsin kinase promoter, the metalloproteinase 3 promoter (a tissue inhibitor), the photoreceptor retinol-binding protein promoter, the vitiligo macular dystrophy 2 promoter, and the photoreceptor-retinoid-binding protein promoter.

[0119] In some embodiments, the polynucleotide further includes other regulatory sequences, including but not limited to terminal inversion repeats (ITRs), enhancers, splicing signals, polyadenylation signals (PolyA), stuffing sequences, terminators, proteolytic signals, internal ribosome entry elements (IRESs), and 2A sequences. In some embodiments, the PolyA sequence is hGH poly(A), SV40 poly(A), or β-globin poly(A).

[0120] In some embodiments, the polynucleotide further comprises an enhancer region. In some embodiments, the enhancer region may be an SV40 enhancer, a cytomegalovirus enhancer, an IRBP enhancer, or an immunoglobulin gene-derived enhancer. In some embodiments, the enhancer region is located upstream of the CMV, CAG, MNDU3, PGK, or EF1a promoter. In some embodiments, the enhancer is located upstream of an eye-specific promoter. In some embodiments, the enhancer region is located downstream of the CMV, CAG, MNDU3, PGK, or EF1a promoter. In some embodiments, the enhancer is located downstream of an eye-specific promoter.

[0121] In some embodiments, the polynucleotide further comprises terminal inverted repeats (ITRs). In some embodiments, the polynucleotide comprises at least one ITR. In some embodiments, the polynucleotide comprises two ITRs. In some embodiments, the two ITRs are the same. In some embodiments, the two ITRs are different from each other. In some embodiments, the ITR is an ITR derived from an AAV. In some embodiments, the ITR may be derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, and any other known AAV. In some embodiments, the ITR has one or more nucleotide mutations, insertions, or deletions compared to wild-type ITRs derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, and any other known AAV, but retains desired terminal repeat sequence functions such as target gene replication, viral packaging, and / or integration.

[0122] In some embodiments, the polynucleotide further comprises one or more filler sequences. In some embodiments, the filler sequence is located upstream of the CMV, CAG, MNDU3, PGK, or EF1a promoter sequence. In some embodiments, the filler sequence is located downstream of the CMV, CAG, MNDU3, PGK, or EF1a promoter sequence. In some embodiments, the filler sequence is located upstream of an eye-specific promoter. In some embodiments, the filler sequence is located downstream of an eye-specific promoter. In some embodiments, the filler sequence is located at the 5' end of the 5' ITR sequence. In some embodiments, the filler sequence is located at the 3' end of the 5' ITR sequence. In some embodiments, the filler sequence is located at the 5' end of the 3' ITR sequence. In some embodiments, the filler sequence is located at the 3' end of the 3' ITR sequence.

[0123] In some embodiments, the length of the filler array is not limited to 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1kb, 1.1kb, 1.2kb, 1.3kb, 1.4kb, 1.5kb, 1.6kb, 1.7kb, 1.8kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2. It can range from approximately 0.1kb to 5kb, such as 5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3kb, 3.1kb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4.0kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8kb, 4.9kb, or 5.0kb.

[0124] In some embodiments, the polynucleotide further comprises introns. In some cases, an intron may refer to any sequence that can be transcribed but not translated. In some cases, an intron may refer to any sequence that is transcribed and removed from mature RNA transcripts in cells. In some cases, an intron may contain at least about 1 bp, 50 bp, 100 bp, 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, or 5000 bp. In some cases, an intron may be about 300 bp. In some cases, an intron may be about 200–400 bp. In some cases, an intron may be about 100–500 bp. In some cases, an intron may be about 50–200 bp. In some cases, an intron may be either a naturally occurring intact intron or a chimeric intron. In some embodiments, the intron is located upstream of the codon-optimized nucleic acid sequence. In some embodiments, the intron is located downstream of the promoter. In some embodiments, the polynucleotide further includes a regulatory element. In some embodiments, the regulatory element includes sequences of TPL (an adenovirus tripertite leader sequence) and eMLP (an enhancer element derived from the adenovirus major late promoter). In some embodiments, the regulatory element is located upstream of the codon-optimized nucleic acid sequence. In some embodiments, the regulatory element is located downstream of the promoter. In some embodiments, the polynucleotide includes a Kozak sequence. In some embodiments, the Kozak sequence is located upstream of the codon-optimized nucleic acid sequence. In some embodiments, the Kozak sequence is located downstream of the intron. In some embodiments, the polynucleotide includes a human skeleton attachment region (SAR) sequence. In some embodiments, the SAR sequence is located downstream of the codon-optimized nucleic acid sequence. In some embodiments, the SAR sequence is located upstream of the polyA signal.

[0125] system In another aspect, the Disclosure provides a system for treating eye diseases of subjects requiring treatment of eye diseases, comprising rAAV particles and pharmaceutically acceptable carriers or excipients as disclosed herein.

[0126] As used herein, “pharmaceutically or therapeutically acceptable carrier or excipient” refers to a carrier medium that does not interfere with the efficacy of the bioactivity of the active ingredient and is non-toxic to the host or patient. The type of carrier used in a pharmaceutical formulation will depend on which method of administration of the therapeutic compound is used. Methods for preparing pharmaceutical compositions for multiple routes of administration are well known in the art. “pharmaceutically acceptable ophthalmic carrier” refers to a pharmaceutically acceptable carrier or excipient that can be used to deliver the rAAV virus particles disclosed herein directly to the eye, indirectly to the eye, or near the eye.

[0127] In some embodiments, the system is prepared by dissolving the rAAV virus particles disclosed herein in a suitable solvent. Suitable solvents include, but are not limited to, water, saline (e.g., NaCl), buffer solutions, or other solvents. In certain embodiments, the solvent is sterile.

[0128] The aqueous solutions and diluents used for the suspension in the preparation of this system may include distilled water or physiological saline. Various additives may be included. These additives may include additional components, additives, or carriers suitable for contact with or use around the eyes without excessive toxicity, incompatibility, instability, irritation, or allergies. Exemplary additives include solvents, bases, cosolvents, suspending agents, thickeners, emulsifiers, stabilizers, buffers, isotonic adjusters, pH adjusters, chelating agents, sedatives, preservatives, flavoring agents, colorants, excipients, binders, lubricants, surfactants, absorption enhancers, dispersants, preservatives, and solubilizers.

[0129] For example, buffers are added to maintain a constant pH and may include pharmaceutically acceptable buffers such as borate buffer, citrate buffer, tartaric acid buffer, phosphate buffer, acetate buffer, or Tris-HCl buffer (containing tris(hydroxymethyl)aminomethane and HCl).

[0130] In addition to the buffer solution, an isotonic agent may be added to this system to prepare a preparation that isotonic with tears. Examples of isotonic agents include, but are not limited to, sugars such as dextrose, glucose, sucrose, and fructose; sugar alcohols such as mannitol and sorbitol; polyols such as glycerin, polyethylene glycol, and propylene glycol; and salts such as sodium chloride, sodium citrate, benzalkonium chloride, phedrine chloride, potassium chloride, procaine chloride, chloramphenicol, and sodium succinate. The isotonic agent is added in an amount such that the osmotic pressure of the eye drop is equal to the osmotic pressure of tears.

[0131] In some embodiments, it is also desirable to use additional agents, including but not limited to stabilizers such as sodium sulfate, sodium carbonate, and propylene glycol; antioxidants such as ascorbic acid, sodium ascorbate, butylhydroxytoluene (BHT), butylhydroxyanisole (BHA), tocopherol, and sodium thiosulfate; and / or chelating agents such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis-(2-aminoethyl)-N,N,N,N-tetraacetic acid (EGTA), and sodium citrate.

[0132] The systems disclosed herein can be prepared by aseptic procedures, or alternatively, sterilized at a suitable stage of preparation. For example, the system can be prepared by aseptically mixing sterile components. Alternatively, the system can be prepared by first mixing the components and then sterilizing the final formulation. Sterilization methods include, but are not limited to, heat sterilization, radiation, and filtration.

[0133] The rAAV virus particles disclosed herein can also be provided in combination with other therapeutic agents. In various embodiments, the compounds disclosed herein are also used in combination with Acular (ketoprofen tromethamine eye drops) 0.5%, Acuvail (ketrolactromethamine), AK-Con-A (naphazoline eye drops), Akten (lidocaine hydrochloride), Alamast, Alphagan (bromidine), Alrex, Astepro (hydrochloride) (azelastine nasal spray), AzaSite (azithromycin), Bepreve (besilicic acid Bepotastine eye drops, Besivance (besifloxacin eye drops), Betaxon, BSS sterile washing solution, Cosopt, Durezol (difluprednate), Eylea (averucept), Lotemax, Lucentis (ranibizumab), Lumigan (bimatoprost eye drops), Macugen (pigatinib), Ocuflox (oxyflurane) Saxine eye drops 0.3%, OcuHist, Oz Urdex (dexamethasone), Quixin (levofloxacin), Rescula (unoprostone isopropyl eye drops) 0.15%, Restasis (cyclosporine eye drops), Salagen tablets, Travatan (travoprost eye drops), Valcyte (valganciclovir hydrochloride), trifluorothymidine (Viroptic), Vistide (cidofovir), Visudyne (verteporfin for injection), Vitrasert implant, formamivir injection, ZADITOR, Zioptan (tafluprost eye drops), Zirgan (ganciclovir eye drops), Zymaxid (gatifloxacin eye drops), atropine, flurbiprofen, physiothymine, Azopt, gentamicin, propalacine, bacitracin, hypromellose eye drops (Goniosol), polymyxin B.Povidone-iodine (Betadine), gramicidin, prednisolone, betaxolol, fumorsol, promethaine, betaxolol eye drops (Betoptic), hyalartin, propine, brinzolamide, hypertonic sodium chloride, puralube, BSS, indocyanine green, rose bengal, carbachol, itraconazole, sodium hyaluronate, cefazolin, latanoprost, Sulofen, Xiao Celluvisc, mannitol, oxytetracycline, chloramphenicol, metazolamide, timolol, ciloxan, miconazole, tobramycin, ciprofloxacin, Miostat, triamcinolone, Cosopt, Muro128, trifluorouridine, Demecarium, neomycin, topiramate, dextran dexamethasone, Neptazane, Trusopt, Dipicolin, Ocuflox, adenosine arabinoside, dorzolamide, ofloxacin, Vira-A, epinephrine, oxytetracycline, trifluorothymidine, fluorescent, phenylephrine, and Xalatan may be provided in combination with ophthalmic therapeutic agents selected from the group.

[0134] Exemplary drugs include anti-angiogenic agents such as angiostatins, anecotastat, thrombospondin, and VEGF receptor tyrosine kinase inhibitors; anti-vascular endothelial growth factor (anti-VEGF) drugs such as ranibizumab, bevacizumab, pegaptanib, sunitinib, and sorafenib; and any other known small molecule and angiogenic transcription inhibitors; adrenergic antagonists (e.g., acetobutrol, atenolol, bisoprolol) Ophthalmic drugs including glaucoma drugs such as β-blockers (including dol, carvedilol, asmolol, labetalol, nadolol, penbutrol, pindolol, propranolol, metipranolol, betaxolol, carteolol, levobetaxolol, levobunolol, and timolol); adrenaline agonists such as epinephrine, dipivefrin, clonidine, apraclonidine, and brimonidine; pilocarpine, carbacol, yogurt Parasympathetic stimulants or cholinergic receptor agonists such as phosphorine uride, physostigmine, salicylic acid, acetylcholine chloride, esserine, diisopropyl fluorophosphate, and demepotassium bromide; muscarinic agonists; carbonic anhydrase inhibitors including topical and / or systemic agents such as acetozolamide, brinzolamide, dorzolamide, metazolamide, etoxzolamide, diamox, and dichlorfenamide; mydriatic-ciliary muscle paralysis agonists such as atropine, cyclopentolate, succinylcholine, homatropine, phenylephrine, scopolamine, and tropicamide; prostaglandins such as prostaglandin F2α, anti-prostaglandins, and prostaglandin precursors; or sometimes prostaglandin analogs such as bimatoprost, latanoprost, travoprost, and unoprostone.

[0135] Additional exemplary drugs also include, for example, betamethasone, cortisone, dexamethasone, dexamethasone 21-phosphate, methylprednisolone, prednisolone 21-phosphate, prednisolone acetate, prednisolone, flumilon, loteprednol, methylprednisolone, fluocinolone, triamcinolone, triamcinolone acetate, beclomethasone, budesonide, flunisolide, flumethasone, fluticasone, fludrocortisone, hydrocortisone, hydrocortisone acetate, loteprednol Glucocorticoids and corticosteroids such as limmetholone; aspirin, diclofenac, flurbiprofen, ibuprofen, bromfenac, nepafenac, ketoprofen, salicylate, indomethacin, napsoprene, piroxicam, nabumeton, diflunisal, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, chlorate, mefenamic acid, meloxicam, nabumeton, oxaprozin, piroxicam, disalicylate, sulindac, and tolme Nonsteroidal anti-inflammatory drugs such as tin; COX-2 inhibitors such as celecoxib, rofecoxib, and valdecoxib; antibiotics, such as tetracycline, chlortetracycline, bacitracin, neomycin, polymyxin, brevivacillin, cephalexin, oxytetracycline, chloramphenicol, rifampicin, ciprofloxacin, tobramycin, gentamicin, erythromycin, penicillin, sulfonamides, sulfadiazines, sulfacetoamides, sulfamethoxazoles Antiinfective or antibacterial agents such as sulfisoxazole, nitrofurazone, sodium propionate, aminoglycosides, e.g., gentamicin, tobramycin, amikacin, and streptomycin; fluoroquinolones such as ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, norfloxacin, and ofloxacin; bacitracin, erythromycin, fusidic acid, neomycin, polymyxin B, gramicidin, alpha-oxybenzidine, and sulfamethoxamide;Antifungal agents such as amphotericin B, caspofungin, clotrimazole, fluconazole, itraconazole, ketoconazole, voriconazole, terbinafine, nystatin, and miconazole; antimalarial agents such as chloroquine, atovaquone, mefloquine, primaquine, quinidine, and quinine; antimycobacterial agents such as ethambutol, isoniazid, pyrazinamide, rifampin, and rifabutin; and antiparasitic agents such as albendazole, mebendazole, thiobendazole, bisazolate suppositories, tiuracil, atovaquone, iodoquinaol, ivermectin, paromomycin, praziquantel, and trimatrexate may also be mentioned.

[0136] method In another embodiment, the Disclosure provides a method for expressing a VEGF inhibitor in cells or tissue of interest, comprising the step of administering to the cells or tissue of interest one of the compositions, rAAV particles, polynucleotides, or systems disclosed herein. In some embodiments, the compositions, rAAV particles, polynucleotides, or systems may be administered to the subject by any suitable method known in the art. In some embodiments, the cells or tissue are eye-related. In some embodiments, the compositions, rAAV particles, polynucleotides, or systems may be applied to the eye by subconjunctival, posterior-ocular, periorbital, subretinal, suprachoroidal, or intraocular routes.

[0137] In another aspect, the present disclosure provides a method for treating an eye disease, comprising the step of administering a composition, rAAV particles, or system disclosed herein to a subject in need of a therapeutically effective dose.

[0138] In some embodiments, the system can be administered to a subject by any suitable method known in the art. In some embodiments, the system can be applied to the eye via subconjunctival, posterior, periorbital, subretinal, suprachoroidal, or intraocular routes.

[0139] In some embodiments, eye diseases include, but are not limited to, age-related macular degeneration (AMD), wet AMD, dry AMD, retinal angiogenesis, choroidal angiogenesis, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, and diabetic retinal edema.

[0140] In some embodiments, a system comprising rAAV virus particles is provided in a therapeutically effective amount that achieves a desired biological effect at a medically acceptable toxicity level. The dosage can vary based on the route of administration and the severity of the disease. The dosage can also be adjusted according to the weight, age, gender, and / or degree of symptoms of each patient being treated. It is understood that conventional variations in dosage may need to be made according to the patient's age and weight, as well as the severity of the disease being treated.

[0141] In some embodiments, a therapeutically effective amount is generally about 1×10 5 ~1×10 13 rAAV virus particles. In some embodiments, a therapeutically effective amount is generally about 1×10 6 ~1×10 12 rAAV virus particles. In some embodiments, a therapeutically effective amount is generally about 1×10 7 ~1×10 12 rAAV virus particles. In some embodiments, a therapeutically effective amount is generally about 1×10 8 ~1×10 12 rAAV virus particles. In some embodiments, a therapeutically effective amount is generally about 1×10 9 ~1×10 12 rAAV virus particles. In some embodiments, a therapeutically effective amount is generally about 1×10 10 ~1×10 12 rAAV virus particles.

[0142] In some embodiments, the delivered volume is approximately 0.005 mL to 0.5 mL per eye. In some embodiments, the delivered volume is approximately 0.05 mL to 0.5 mL per eye. In some embodiments, the delivered volume is approximately 0.1 mL to 0.5 mL per eye. In some embodiments, the delivered volume is approximately 0.2 mL to 0.5 mL per eye.

[0143] In some embodiments, the administration frequency may be at least once a day, including twice, three, four, or five times a day. In some embodiments, the treatment is administered on days 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, 30, 31, 32, 33, 34, 35, It can last for 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 750, 1000 days, or longer than 1000 days.

[0144] In some embodiments, the administration of rAAV particles or polynucleotides may also include ex vivo administration. In some embodiments, ex vivo administration includes (1) isolation of the target cells or tissue from the subject, (2) contact of the cells or tissue with a sufficient amount of rAAV to transfect the cells or tissue to result in a sufficient level of gene transfer and expression without excessive side effects, and (3) returning the cells or tissue to the subject. In some embodiments, the cells or tissue may be cultured ex vivo for several days before and / or after transfection. In some embodiments, the cells or tissue are eye-related.

[0145] In another embodiment, the Disclosure provides a method for preparing recombinant adeno-associated virus (rAAV) particles, comprising the step of transfecting cells with any of the compositions, rAAV particles, polynucleotides, or systems disclosed herein. In some embodiments, the cells are insect cells or mammalian cells. In some embodiments, the insect cells are Sf9 cells. In some embodiments, the mammalian cells are HEK293 cells or their derivative cells. In some embodiments, the derivative cells are HEK293T cells. In some embodiments, the Method comprises the step of generating bacmid DNA and / or baculovirus. In some embodiments, the Method comprises the step of generating bacmid DNA containing a sequence expressing a VEGF inhibitor (such as a polynucleotide disclosed herein). In some embodiments, the Method comprises the step of generating a sequence expressing bacmid DNA rAAV cap-rep. In some embodiments, the Method comprises the step of producing baculovirus by transfecting cells with bacmid DNA. In some embodiments, the method includes the step of producing a baculovirus by transfecting cells with bacmid DNA containing a sequence expressing a VEGF inhibitor. In some embodiments, the method includes the step of producing a baculovirus containing a sequence expressing rAAV cap-rep by transfecting cells with bacmid DNA. In some embodiments, the method further includes the step of infecting cells (such as Sf9 cells) by mixing baculoviruses to obtain packaged rAAV / VEGF inhibitor virus particles disclosed herein.

[0146] In some embodiments, the compositions of the Disclosure, or the polynucleotides of the Disclosure, can be delivered to cells by any method known in the Art. In some embodiments, the methods include, but are not limited to, electroporation, calcium phosphate precipitation, and liposome-mediated methods. In some embodiments, the compositions or polynucleotides are stably transfected to cells. In some embodiments, the compositions or polynucleotides are transiently transfected to cells. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, and endoplasmic reticulum may be used to introduce the vectors, compositions, or polynucleotides into cells. Specifically, the vectors, compositions, or polynucleotides may be formulated for delivery encapsulated in any of the following: lipid particles, liposomes, endoplasmic reticulum, nanospheres, or nanoparticles.

[0147] kit On the other hand, this disclosure provides a kit for treating an eye disease, comprising rAAV particles or a system disclosed herein and instructions. In some embodiments, the instructions are used to indicate how to administer the rAAV particles or system to treat the eye disease.

[0148] In some embodiments, the kit further comprises a container. In some embodiments, the container is configured to deliver the system described herein. In some embodiments, the container includes a vial, a dropper, a bottle, a tube, and a syringe. In some embodiments, the container is a dropper for applying the system. In some embodiments, the container is a syringe for administering the system.

[0149] Some embodiments of this disclosure are further illustrated by the following embodiments, but these should not be construed as limiting. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to work well in carrying out the embodiments described herein, and that they can therefore be considered to construct those used to carry out these embodiments. However, those skilled in the art will understand that, based on this disclosure, many modifications can be made to the specific embodiments introduced herein without departing from the spirit and scope of this disclosure, and that the same or similar results can always be achieved. [Examples]

[0150] The present invention is further illustrated by the following embodiments. These embodiments are intended solely to illustrate the present invention and should not be construed as limiting it.

[0151] Example 1: Design of recombinant AAV vectors By synthesizing the cap and rep coding sequences derived from AAV2 along with their corresponding promoters, and cloning them into pUC57, pFastBac1, modified pUC57, or modified pFastBac1, a first polynucleotide containing the coding sequences of the cap and rep proteins was obtained.

[0152] Nucleic acid sequences encoding green fluorescent protein (GFP) or the VEGF inhibitor aflibercept were synthesized together with their corresponding promoters and cloned into pUC57, pFastBac1, modified pUC57, or modified pFastBac1 to obtain second polynucleotides containing the GFP or aflibercept coding sequences, respectively. The structural designs of the second polynucleotides can be found in Table 2.

[0153] [Table 2]

[0154] "co" refers to "codon optimization." For example, "co1" refers to the codon-optimized sequence #1. "CMVep" refers to the CMV enhancer and promoter. "sv40i" refers to the SV40 intron.

[0155] Example 2 Plasmid transfection To determine the expression intensity of the designed structure, 2 × 10 5 HEK293T cells were seeded in a 24-well plate and cultured overnight. 0.5 μg of each expression construct plasmid was mixed with 1.5 μl of Mirus TransT-VirusGEN® transfection reagent per well in 50 μl of Opti-Mem medium (DNA(ug):Mirus reagent(ug) = 1:3). After 48 hours, GFP expression was detected by fluorescence microscopy and flow cytometry (Figures 1A and 1B), suggesting that the cells were successfully transfected by the expression cassette. Supernatant culture medium was collected after 48 hours for aflibercept detection.

[0156] Example 3: Preparation of recombinant AAV virus particles A composition was formed by mixing the first polynucleotide and the second polynucleotide obtained in Example 1. A helper plasmid was added to this composition, and HEK293T cells were transfected to obtain packaged rAAV2.7m8 / afliberceptvirus particles and rAAV2.7m8 / GFP virus particles. AAV particles could also be produced using the bac to AAV technique, i.e., by first generating two bacmids containing Rep-Cap and a transgene expression cassette, respectively, and then producing baculovirus against these two bacmids. rAAV could be produced by infecting Sf9 cells with both Rep-Cap and transgene expression baculovirus. Recombinant AAV2.7m8 / afliberceptvirus particles and AAV2.7m8 / GFP virus particles were isolated from HEK293T cells by gradient ultracentrifugation and purified.

[0157] Example 4: Expression levels of aflibercept from transfected cells The expression level of aflibercept in cell culture supernatant was measured by quantitative ELISA. ELISA plates were coated with 100 μL / well of recombinant human VEGFA (rhVEGFA) at a concentration of 1 μg / mL in coating buffer and incubated overnight at 4°C. After washing with wash buffer, the plates were blocked with 300 μL / well of protein-free blocking buffer. The plates were then washed, and the samples were added at a 1:1000 dilution (100 μL / well) and incubated at room temperature for 2 hours. The plates were then washed again, and 100 μL / well of the anti-human Fc domain of IgG(Fcγ)-specific antibody conjugated to horseradish peroxidase (HRP) was added to the wells at a concentration of 500 ng / mL in PBS with 1% BSA. After washing, 100 μL / well of Supersignal ELISA Pico chemiluminescent substrate was added to the wells, and the luminescence signal was measured using a microplate reader. The results are shown in Table 3.

[0158] [Table 3]

[0159] Design the data within the following range: Aflibercept concentration (ng / ml): A>15,000>B>10,000>C>1,000>D

[0160] Example 5. Activity level of aflibercept from rAAV The activity level of aflibercept expressed after rAAV transduction in 293T cells was measured using a HUVEC proliferation assay. Specifically, 100 μl of human umbilical vein endothelial cell (HUVEC) suspension was dispensed into basal medium (approximately 5000 cells / well) in a 96-well plate. The plate was incubated for 4 hours. A 100-fold dilution of the supernatant sample was prepared in basal medium with VEGF (80 ng / mL) and incubated for 1 hour. 10 μl of this dilution was added to the HUVEC cells. The plate was incubated for 4 days. 20 μl of cell counting kit-8 (CCK-8) solution was added to each well of the plate. The plate was incubated in an incubator for 4 hours. The plate was read at 450 nm.

[0161] The inhibitory activity of aflibercept against HUVEC was calculated based on the following formula: Inhibition % = (OD (GFP control) -OD (sample) ) / (OD (GFP control) -OD (blank) )*100%

[0162] As will be understood by those skilled in the art, CCK-8 is non-radioactive and enables highly sensitive colorimetric assays for determining the number of viable cells in cell proliferation, as well as cytotoxicity assays. Reduction of WST-8 with dehydrogenase in cells yields an orange product (formazan) that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of viable cells and is measured by absorbance at 460 nm. Cell Counting Kit 8 (WST-8 / CCK8) (ab228554) provides a convenient and robust method for performing cell viability assays. This kit uses a water-soluble tetrazolium salt to quantify the number of viable cells by producing an orange formazan dye upon in vivo reduction in the presence of electron carriers.

[0163] As a result, it was found that secreted aflibercept inhibited VEGF-induced HUVEC proliferation. In other words, secreted aflibercept retained its bioactivity. The results are shown in Table 4.

[0164] [Table 4]

[0165] Design the data within the following range: Inhibition of HUVEC cell proliferation: A>40>B>25>C>10>D

[0166] Example 6. In vitro AAV infection 6×10 5 293 T cells (6 × 10) cells / mL 4 100 μL of cells (cells / well) were seeded in DMEM complete medium in each well of a 96-well plate. After culturing the cells for 1 hour, the medium was discarded. MOI 5.56 × 10⁴ 3 and 1.67 × 10 4 30 μL of DMEM complete medium containing the 7m8AAV vector was added to each well and incubated overnight. MOI was calculated based on droplet digital PCR (ddPCR) titers. The following day, 70 μL of DMEM complete medium was added. The cells were cultured for a total of 48 hours.

[0167] Next, the expression level of aflibercept in the cell culture supernatant was measured by quantitative ELISA. ELISA plates were coated with 100 μL / well of recombinant human VEGFA (rhVEGFA) at a concentration of 1 μg / mL in coating buffer and incubated overnight at 4°C. After washing with wash buffer, the plates were blocked with 300 μL / well of protein-free blocking buffer. The plates were then washed, and the samples were added at a 1:1000 dilution (100 μL / well) and incubated at room temperature for 2 hours. The plates were then washed again, and 100 μL / well of the anti-human Fc domain of IgG(Fcγ)-specific antibody conjugated to horseradish peroxidase (HRP) was added to the wells at a concentration of 500 ng / mL in 1% BSA in PBS. After washing, 100 μL / well of Supersignal ELISA Pico chemiluminescent substrate was added to the wells, and the luminescence signal was measured using a microplate reader. The results are shown in Table 5.

[0168] [Table 5]

[0169] Design the data within the following range: In the left column (MOI=1.67E+4), the aflibercept concentrations (ng / ml) are: A>3,000>B>2,000>C>1,000>D, and in the right column (MOI=5.56E+3), the aflibercept concentrations (ng / ml) are: A>1,500>B>1,000>C>500>D.

[0170] Example 7. Measurement of aflibercept activity from rAAV 20 μL of aflibercept supernatant was mixed with 20 μL of DMEM complete medium (containing 2 μg / mL of VEGF) and incubated for 1 hour. 25 μL of the sample dilution was dispensed into 25 μL of pre-seeded cells according to the manufacturing instructions for the VEGF Bioassay (Promega GA2001). Aflibercept activity was calculated based on the following formula: Inhibition ratio = RLU (cells only - assay sample) / RLU (cells only - background) (RLU: Relative Luminescence)

[0171] Inhibition of VEGF by aflibercept expressed from rAAV confirmed that aflibercept secreted in the culture supernatant was biologically active. The data are shown in Table 6.

[0172] [Table 6]

[0173] Design the data within the following range: In the left column (MOI=1.67E+4), VEGF inhibition (luciferase reporter) is A>60>B>40>C>20>D, and in the right column (MOI=5.56E+3), VEGF inhibition (luciferase reporter) is A>35>B>25>C>15>D.

[0174] Example 8: Delivery and expression of VEGF inhibitors in mice The mice were divided into two groups: a control group and an experimental group. Viral particles of the AAV2.7m8 / VEGF inhibitor, purified in Example 3, were injected into the vitreous humor of the mice in both groups. Eye tissue was collected for measurement of aflibercept concentration by ELISA.

[0175] Example 9: In vivo efficacy of the composition of this application Laser-induced choroidal neovascularization (LCNV) is a model of choroidal neovascularization used as a preclinical model for wet age-related macular degeneration. To verify the effectiveness of the system described in this application, a two-group clinical trial was conducted using a control system containing rAAV2.7m8 / VEGF inhibitor virus particles.

[0176] Sequence List

[0177] [ka]

[0178] [ka]

[0179] [ka]

[0180] [ka]

[0181] [ka]

[0182] [ka]

[0183]

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[0184]

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[0185]

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[0186]

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[0187]

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[0188]

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Claims

1. A composition, (i) A first polynucleotide comprising a first sequence operably linked to a first promoter and a second sequence operably linked to a second promoter, The first sequence described above encodes an adeno-associated virus (AAV) capsid protein, The second sequence above encodes the AAV rep protein. The first polynucleotide, (ii) A second polynucleotide comprising a third sequence operably linked to a CAG promoter and β-globin poly(A), wherein the third sequence comprises a codon-optimized nucleic acid sequence encoding a vascular endothelial growth factor (VEGF) inhibitor, and the third sequence comprises the sequence of Sequence ID No.

14. Includes, A composition wherein the VEGF inhibitor comprises the amino acid sequence of SEQ ID NO:

1.

2. The composition according to claim 1, wherein the first promoter or the second promoter is a p10 promoter or a polh promoter.

3. The composition according to claim 2, wherein the 3' end of the first sequence or the second sequence independently comprises a poly(A) sequence, the poly(A) sequence being hGH poly(A), SV40 poly(A), or β-globin poly(A).

4. Recombinant adeno-associated virus (rAAV) particles prepared by transfecting cells with the composition described in claim 1, wherein the cells are Sf9 cells, HEK293 cells, or derivatives thereof.

5. A polynucleotide comprising a codon-optimized nucleic acid sequence encoding a protein containing the amino acid sequence of SEQ ID NO: 1, a CAG promoter, and β-globin poly(A), wherein the codon-optimized nucleic acid sequence contains SEQ ID NO:

14.

6. The polynucleotide according to claim 5, further comprising an intron containing a chimeric intron, or a regulatory element containing a sequence of TPL (a tripartite leader sequence derived from adenovirus) and eMLP (an enhancer element derived from the major late promoter of adenovirus), or a Kozak sequence.

7. Recombinant adeno-associated virus (rAAV) particles comprising the polynucleotide described in claim 5 or 6.

8. A composition according to claim 1 for use in the treatment of an eye disease requiring treatment of the eye disease, wherein the eye disease is selected from the group consisting of wet age-related macular degeneration (wet AMD), diabetic retinopathy, diabetic macular edema, proliferative diabetic retinopathy, and macular edema.

9. A polynucleotide according to claim 5 or 6 for use in the treatment of an eye disease requiring treatment of the eye disease, wherein the eye disease is selected from the group consisting of wet age-related macular degeneration (wet AMD), diabetic retinopathy, diabetic macular edema, proliferative diabetic retinopathy, and macular edema.

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