Recombinant adeno-associated virus for delivery of kh902 (conbercept) and uses thereof

Abstract

Aspects of the disclosure relate to recombinant adenoviruses (e.g., rAAV2.7m8-KH902) encoding anti-vascular endothelial cell growth factor (VEGF) agents in a cell or subject. In some embodiments, the compositions described herein can be used to treat a subject having a disease associated with angiogenesis or aberrant VEGF activity / signaling.

Description

[0001] Related applications

[0002] This application is entitled to the benefit of U.S. Provisional Application Serial No. 62 / 940,288, filed November 26, 2019, entitled “RECOMBINANTADENO-ASSOCIATED VIRUS FOR DELIVERY OF KH902(CONBERCEPT) AND USES THEREOF”, filed pursuant to 35 USC119(e), the entire contents of which are incorporated herein by reference. Background of the Invention

[0004] KH902 is a vascular endothelial growth factor (VEGF) receptor fusion protein currently undergoing clinical trials for anti-VEGF therapy. Current challenges in anti-VEGF therapy include the need for repeated injections to maintain the efficacy of anti-VEGF drugs and the development of long-acting formulations. Therefore, there is a need to develop novel methods for the long-term delivery of anti-VEGF agents to targeted cells and / or tissues. Summary of the Invention

[0005] Several aspects of this disclosure relate to recombinant adeno-associated virus (rAAV) for delivering an anti-VEGF agent (e.g., KH902) to cells and / or tissues (e.g., cells of a subject). This disclosure is partially based on rAAV engineered for delivering an anti-VEGF agent (e.g., KH902).

[0006] In some aspects, the rAAV disclosed herein comprises an AAV capsid (e.g., AAV2.7m8 capsid) containing a nucleic acid encoding a transgenic expression cassette, said expression cassette containing a nucleic acid sequence flanking an anti-vascular endothelial growth factor (e.g., anti-VEGF) agent. In some embodiments, the anti-VEGF agent is a human VEGF decoy receptor. In some embodiments, the human VEGF decoy receptor comprises an extracellular domain 2 of human VEGF receptor 1. In some embodiments, the human VEGF decoy receptor comprises extracellular domains 3 and 4 of human VEGF receptor 2.

[0007] In some embodiments, the anti-VEGF agent is a human VEGF receptor fusion protein. In some embodiments, the human VEGF receptor fusion protein comprises an extracellular domain 2 of human VEGF receptor 1 fused to extracellular domains 3 and 4 of human VEGF receptor 2. In some embodiments, the human VEGF receptor fusion protein comprises an extracellular domain 2 of human VEGF receptor 1 fused to the Fc portion of an immunoglobulin. In some embodiments, the human VEGF receptor fusion protein comprises extracellular domains 3 and 4 of human VEGF receptor 3 fused to the Fc portion of an immunoglobulin. In some embodiments, the human VEGF receptor fusion protein comprises an extracellular domain 2 of human VEGF receptor 1 fused to extracellular domains 3 and 4 of human VEGF receptor 2 and further fused to the Fc portion of an immunoglobulin. In some embodiments, the anti-VEGF agent comprises an amino acid sequence, or a portion thereof, having at least 50%, at least 60%, at least 70%, at least 80%, 90%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:5.

[0008] In some embodiments, the human VEGF receptor fusion protein comprises a human VEGF receptor fused to the Fc portion of an immunoglobulin. In some embodiments, the anti-VEGF agent is KH902. In some embodiments, the transgene comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, 90%, 99%, or 100% identity with the nucleic acid sequence shown in SEQ ID NO:1, or a codon-optimized variant thereof.

[0009] In some implementations, anti-VEGF receptors (e.g., VEGF decoy receptors) are able to bind to both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF).

[0010] In some embodiments, the isolated nucleic acid (e.g., expression cassette) further comprises a promoter that is effectively linked to the transgene. In some embodiments, the promoter comprises a cytomegalovirus (CMV) early enhancer. In some embodiments, the promoter is a chimeric cytomegalovirus (CMV) / chicken β-actin (CB) promoter.

[0011] In some embodiments, the expression cassette (e.g., the transgene within the expression cassette) contains one or more introns. In some embodiments, at least one intron is located between the promoter and a nucleic acid sequence encoding an anti-vascular endothelial growth factor (anti-VEGF) agent. In some embodiments, the expression cassette (e.g., the transgene within the expression cassette) contains a Kozak sequence. In some embodiments, the Kozak sequence is located between the intron and the transgene encoding an anti-vascular endothelial growth factor (anti-VEGF) agent.

[0012] In some embodiments, the expression cassette (e.g., the transgene within the expression cassette) includes a 3' untranslated region (3'UTR). In some embodiments, the expression cassette (e.g., the transgene within the expression cassette) further includes one or more miRNA binding sites. In some embodiments, one or more miRNA binding sites are located within the 3'UTR of the transgene. In some embodiments, at least one miRNA binding site is an immune cell-associated miRNA binding site. In some implementations, the immune cell-associated miRNAs are selected from: miR-15a, miR-16-1, miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, miR-21, miR-29a / b / c, miR-30b, miR-31, miR-34a, miR-92a-1, miR-106a, miR-125a / b, miR-142-3p, miR-146a, miR-150, miR-155, miR-181a, miR-223 and miR-424, miR-221, miR-222, let-7i, miR-148 and miR-152.

[0013] In some implementations, the ITR is an adeno-associated virus ITR having a serotype selected from AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, and AAV6 ITR.

[0014] In some embodiments, the isolated nucleic acid described herein includes a nucleic acid sequence, or a portion thereof, having at least 80%, 90%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO:2.

[0015] In some embodiments, the isolated nucleic acid, as described in this disclosure, is located on a plasmid. In some embodiments, the plasmid comprises a nucleic acid sequence, or a portion thereof, having at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO:3.

[0016] In some aspects, this disclosure provides a recombinant adeno-associated virus (rAAV) comprising: (i) an rAAV capsid protein, which is AAV2.7m8, and (ii) isolated nucleic acid comprising, in the 5' to 3' sequence: (a) a 5'AAV ITR; (b) a CMV enhancer; (c) a CBA promoter; (d) a chicken β-actin intron; (e) a Kozak sequence; (f) a transgene encoding an anti-VEGF agent, wherein the anti-VEGF agent is encoded by the nucleic acid sequence in SEQ ID NO:1; (g) a rabbit β-globin polyA signal tail; and (h) a 3'AAV ITR.

[0017] In some embodiments, AAV2.7m8 exhibits tropism for ocular tissues. In some embodiments, ocular tissues include ocular neurons, retina, sclera, choroid, vitreous body, macula, fovea, optic disc, lens, pupil, iris, aqueous humor, cornea, conjunctival ciliary body, or optic nerve.

[0018] In some implementations, rAAV is a single-chain AAV (ssAAV).

[0019] Host cells containing the rAAV described herein are also within the scope of this disclosure. In some embodiments, the host cell is a mammalian cell, yeast cell, bacterial cell, or insect cell.

[0020] Another aspect of this disclosure relates to pharmaceutical compositions comprising rAAV as described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for intravitreal injection, intravenous injection, intratumoral injection, or intramuscular injection.

[0021] In some aspects, this disclosure relates to methods for inhibiting VEGF activity in subjects who require it, comprising administering to the subject a therapeutically effective amount of the rAAV, host cells, or pharmaceutical composition described herein.

[0022] In some aspects, this disclosure relates to a method of delivering an anti-VEGF agent to a subject in need, the method comprising administering to the subject a therapeutically effective amount of the rAAV, host cell, or pharmaceutical composition described herein.

[0023] In some aspects, this disclosure relates to methods for treating neovascularization-associated disease, angiogenesis-associated disease, or VEGF-associated disease in subjects with such need, said methods comprising administering to the subject a therapeutically effective amount of the rAAV, host cells, or pharmaceutical composition described herein.

[0024] In some respects, delivery of rAAV results in at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% inhibition of VEGF activity.

[0025] In some implementations, the subjects are non-human mammals. In some implementations, non-human mammals are mice, rats, cats, dogs, sheep, rabbits, horses, cattle, goats, pigs, guinea pigs, hamsters, chickens, turkeys, or non-human primates. In some implementations, the subjects are humans.

[0026] In some embodiments, the subject is diagnosed with or suspected of having angiogenesis-related disease or VEGF-related disease. In some embodiments, the disease is a tumor, cancer, retinopathy, wet age-related macular degeneration (wAMD), macular edema, choroidal angiogenesis, or corneal angiogenesis. In some embodiments, administration is systemic, such as intravenous injection. In some embodiments, administration is direct application to ocular tissue, such as intravitreal injection, intraocular injection, or topical application.

[0027] In some implementations, the application results in the delivery of the transgene to ocular tissues. In some implementations, ocular tissues include ocular neurons, retina, sclera, choroid, vitreous body, macula, fovea, optic disc, lens, pupil, iris, aqueous humor, cornea, conjunctival ciliary body, or optic nerve.

[0028] Detailed description of the attached figures

[0029] Figure 1A-1C The rAAV-CBA-KH902 vector and sequence are shown. The expressed rAAV vector expresses secreted KH902 (Conbercept) and is driven by a CMV enhancer and a chicken β-actin promoter (CBA) cassette. A Kozak sequence was also engineered at the 5' of the start codon to enhance translation initiation. A map of the plasmid is shown. Figure 1A ) and readout sequence (Figure 1B, SEQ ID NO:3). The sequence containing and surrounded by 5'-ITR and 3'-ITR is packaged into the AAV virion ( Figure 1C ).

[0030] Figure 2 Western blot analysis of RPE-conditioned medium infected with AAV2.7m8-KH902 is shown. PAGE was performed on 15 μl of ARPE-19- (left) or hTERT-RPE1- (right) conditioned medium under the specified conditions indicated above each lane. After semi-dry transfer, the membranes were blotted with anti-VEGFR1 antibody (R&D Systems BAF321). Each blot included 20 ng of KH902 drug (last lane) as a reference.

[0031] Figures 3A-3CIn vitro functional validation of the AAV2.7m8-KH902 vector was demonstrated. The angiogenesis or proliferation capacity of HUVECs stimulated with VEGF (25 ng / mL) in the presence of KH902 was shown; or the conditioned medium (1:10 dilution) of RPE cells infected with AAV2.7m8-KH902 or control GFP vector was demonstrated. Results were obtained via tube formation assays. Figure 3A and 3B ) or through CCK-8 activity ( Figure 3C To quantify anti-VEGF activity. *, p<0.01; **, p<0.001; ***, p<0.0001.

[0032] Figures 4A-4B The evaluation of rAAV2.7m8-KH902 in an oxygen-induced retinopathy mouse model is shown. Figure 4A Bright-field images of eyes injected with rAAV2.7m8-Egfp (left column) and a 5:1 mixture of rAAV2.7m8-KH902:rAAV2-Egfp (right column) and imaged immediately after dissection are shown. Eyes in the same row are from the same animal; therefore, eyes injected with rAAV2.7m8-Egfp can serve as controls for the degree of pathological induction within individual animals. Figure 4B Fluorescence imaging from representative mouse eyes was shown, followed by flat-mounting and hemagglutinin-B4 staining. Positive transduction regions were marked by EGFP expression. rAAV2.7m8-KH902 did not reduce normal vascular development and only slightly affected aneurysm nodule formation.

[0033] Figure 5 The percentage of eyes treated with rAAV showing lesions is displayed. Figures 4A-4B Edema or salvage of the mouse eye was scored. Experimental group: n=7.

[0034] Figures 6A-6D The study showed that rAAV2.7m8-KH902 treatment followed laser-induced choroidal angiogenesis. Figure 6A The images show laser-induced CNV induced by treatment with different concentrations of rAAV2.7m8-KH902. Mouse eyes were damaged 5 days prior to rAAV injection. rAAV2.7m8-EGFP was used as a negative control. rAAV2.7m8-KH902 was injected at three different doses: undiluted (3E9 vg / eye), 1:10 dilution (3E8 vg / eye), and 1:20 dilution (1.5E8 vg / eye). Points represent mean ± SEM. Figure 6BThis study demonstrates the detection of inflammatory cell infiltration into the eye following AAV2.7m8-KH902 treatment. Mice were treated with rAAV2.7m8-EGFP (control) or a 5:1 ratio of rAAV.2.7m8-KH902 and rAAV-2.7m8-EGFP (rAAV2.7m8-KH902, 3E9 vg / eye). Cell markers of general immune cells (CD4, left column), platelets (CD41, middle column), or antigen-presenting cells (class II MHC, right column) were used to detect infiltrating immune cells. Heteroglucosin B4 (IB4) was used to detect endothelial cells; outer nuclear layer (ONL); inner nuclear layer (INL); and ganglion cell layer (GCL). Figure 6C The effects of different doses of AAV2.7m8-KH902 treatment on inflammatory cell infiltration were demonstrated. Mice were treated with AAV2.7m8 containing KH902 and EGFP via intravitreal injection of 3E9 vg / eye, or a 5:1 ratio of 3E8 vg / eye or 3E9 vg / eye. Cellular markers targeting general immune cells (CD4), platelets (CD41), antigen-presenting cells (class II MHC), EGFP, endothelial cells (IB4 and PECAM1), and KH902 (VEGFR1) were shown. Figure 6D This study shows the quantification of KH902 in the mouse retina following intravitreal injection of AAV2.7m7-KH902 at a dose of (3E9 vg / eye). Injected eyes were collected every two weeks for RNA extraction and cDNA library construction. Transcripts were quantified by ddPCR. Y-axis: relative KH902 transcription levels normalized to the housekeeping gene gusb. X-axis: week number post-injection. n = 2–3; mean ± SD. Invention Details

[0036] In some aspects, this disclosure relates to compositions and methods for the sustained long-term delivery of vascular endothelial growth factor (anti-VEGF) agents (e.g., VEGF receptor fusion proteins, such as KH902) to cells and / or tissues (e.g., cells and / or tissues of a subject). This disclosure is partly based on recombinant adeno-associated virus (rAAV) having an AAV capsid (e.g., AAV2.7m8) containing a genetically engineered nucleic acid or a variant thereof encoding an anti-VEGF agent (e.g., a VEGF receptor fusion protein, such as KH902).

[0037] Recombinant adeno-associated virus (rAAV)

[0038] In some respects, this disclosure provides isolated adeno-associated viruses (AAVs). As used herein with respect to AAV, the term "isolated" refers to an AAV that is artificially produced or obtained. Isolated AAVs can be produced using recombinant methods. Such AAVs are referred to herein as "recombinant AAVs". Recombinant AAVs (rAAVs) preferably have tissue-specific targeting capabilities, thereby enabling transgene-specific delivery of rAAVs to one or more predetermined tissues (e.g., eye tissue). The AAV capsid is an important factor in determining these tissue-specific targeting capabilities (e.g., tissue tropism). Therefore, rAAVs with capsids suitable for the targeted tissues can be selected.

[0039] Methods for obtaining recombinant AAV with the desired capsid protein are well known in the art. (See, for example, US 2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). Typically, these methods involve culturing a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector containing an AAV inverted terminal repeat (ITR) and transgenes; and a host cell with sufficient helper function to allow the recombinant AAV vector to be packaged into the AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the AAV cap gene. AAV contains three capsid proteins, namely virosomal proteins 1 to 3 (named VP1, VP2, and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2, and VP3 are approximately 87 kDa, approximately 72 kDa, and approximately 62 kDa, respectively. In some embodiments, after translation, the capsid protein forms a spherical 60-mer protein shell around the viral genome. In some embodiments, the capsid protein functions to protect the viral genome, deliver the genome, and interact with the host. In some respects, capsid proteins deliver the viral genome to the host in a tissue-specific manner.

[0040] In some embodiments, the AAV capsid protein exhibits tropism for ocular or muscular tissues. In some embodiments, ocular tissues include ocular neurons, retina, sclera, choroid, vitreous body, macula, fovea, optic disc, lens, pupil, iris, aqueous humor, cornea, conjunctival ciliary body, or optic nerve. In some embodiments, the AAV capsid protein targets specific ocular cell types (e.g., photoreceptor cells, retinal cells, etc.).

[0041] In some embodiments, the AAV capsid protein exhibits tropism for photoreceptors (e.g., photoreceptor cells). In some embodiments, the AAV capsid protein exhibiting tropism for photoreceptor cells is the AAV7m8 capsid protein. "7m8 capsid protein" refers to an AAV capsid protein with an amino acid insertion comprising the 7-mer amino acid sequence "LGETTRP" (SEQ ID NO:11) located in the solvent-exposed GH ring of the capsid protein. Typically, the 7m8 capsid protein can be a capsid protein containing a 7-mer amino acid insertion of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, etc. Within the scope of this disclosure, a 7-mer amino acid sequence can be inserted into any AAV serotype capsid protein to form a "7m8" capsid protein. In some embodiments, the 7m8 capsid protein, in addition to the "LGETTRP" (SEQ ID NO:11) amino acid insertion, also comprises one or more amino acid substitutions, insertions, deletions, or any combination thereof.

[0042] In some embodiments, a 7-mer amino acid sequence is inserted between the GH loops of the AAV2 capsid protein (e.g., between amino acid positions 587 and 588 of the AAV2 capsid protein, as shown in, for example, NCBI reference sequence number). In some embodiments, the AAV2 capsid protein with the amino acid insertion is referred to as AAV2.7m8 and is described, for example, by Dakara et al. (2013) Science Translational Medicine, 5(189):189RA76. An exemplary amino acid sequence of AAV2.7m8 is shown in SEQ ID NO:13. An exemplary nucleic acid sequence encoding AAV2.7m8 is shown in SEQ ID NO:14.

[0043] An exemplary amino acid sequence of the AAV2 capsid protein is shown in SEQ ID NO:12:

[0044]

[0045] An exemplary amino acid sequence of the AAV2.7m8 capsid protein is shown in SEQ ID NO:13 (7-mer insertion is shown in bold):

[0046]

[0047]

[0048] An exemplary nucleic acid coding sequence for the AAV2.7m8 capsid protein is shown in SEQ ID NO:14:

[0049]

[0050]

[0051] In some embodiments, the AAV capsid protein has an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.hr, AAVrh8, AAVrh10, AAVrh39, AAVrh43, AAV.PHP, and variants of any of the above. In some embodiments, the AAV capsid protein has a serotype derived from a non-human primate, such as serotype AAVrh8. In some embodiments, the capsid protein has AAV serotype 6 (e.g., AAV6 capsid protein), AAV serotype 8 (e.g., AAV8 capsid protein), AAV serotype 2 (e.g., AAV2 capsid protein), AAV serotype 5 (e.g., AAV5 capsid protein), or AAV serotype 9 (e.g., AAV9 capsid protein). In some embodiments, the AAV capsid protein with the desired tissue tropism may be selected from AAV capsid proteins isolated from mammals (e.g., tissues from a subject).

[0052] In some embodiments, the rAAV of this disclosure comprises a capsid protein containing nucleic acid encoding a transgene encoding an anti-VEGF agent (e.g., KH92). In some embodiments, the rAAV of this disclosure comprises a nucleotide sequence as shown in SEQ ID NO:2. In some embodiments, the rAAV of this disclosure comprises a nucleotide sequence having 100% identity, at least 99% identity, at least 98% identity, at least 97% identity, at least 96% identity, at least 95% identity, at least 94% identity, at least 93% identity, at least 92% identity, at least 91% identity, at least 90% identity, at least 85% identity, at least 80% identity, at least 75% identity, at least 70% identity, at least 65% identity, at least 60% identity, at least 55% identity, or at least 50% identity with the nucleotide sequence shown in SEQ ID NO:2.

[0053] In some implementations, the rAAV described herein is a single-stranded AAV (ssAAV). As used herein, ssAAV refers to rAAV that has the coding sequence and complementary sequence of the transgenic expression cassette on a separate strand and is packaged into a different viral capsid.

[0054] Components to be cultured in host cells to package the rAAV vector in an AAV capsid can be trans-provided to the host cells. Alternatively, any one or more desired components (e.g., recombinant AAV vector, rep sequence, cap sequence, and / or helper function) can be provided by stable host cells engineered to contain one or more desired components using methods known to those skilled in the art. Most preferably, such stable host cells contain the desired components under the control of an inducible promoter. However, one or more desired components may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein when discussing regulatory elements suitable for transgenic applications. In another alternative, selected stable host cells may contain one or more selected components under the control of a constitutive promoter and one or more other selected components under the control of one or more inducible promoters. For example, stable host cells derived from 293 cells (which contain E1 helper function under the control of a constitutive promoter) can be generated, but which contain rep and / or cap proteins under the control of an inducible promoter. Other stable host cells can also be generated by those skilled in the art.

[0055] In some embodiments, this disclosure relates to host cells containing nucleic acids comprising the coding sequence for a transgene (e.g., KH902) or rAAV (e.g., AAV2.7m8-KH902) delivering an anti-VEGF agent. "Host cell" refers to any cell that contains or is capable of containing the substance of interest. Host cells are typically mammalian cells. In some embodiments, host cells are photoreceptor cells, retinal pigment epithelial cells, keratinocytes, corneal cells, and / or tumor cells. Host cells can serve as recipients of AAV helper constructs, AAV small gene plasmids, accessory functional vectors, or other transferred DNA associated with the production of recombinant AAV. The term includes the progeny of the original transfected cell. Therefore, as used herein, "host cell" can refer to a cell that has been transfected with a foreign DNA sequence. It should be understood that the progeny of a single parental cell may not necessarily be identical to the original parent morphologically or in terms of genome or total DNA complementation due to natural, accidental, or intentional mutations. In some embodiments, host cells are mammalian cells, yeast cells, bacterial cells, insect cells, plant cells, or fungal cells. In some implementations, the host cell is a neuron, photoreceptor cell, pigmented retinal epithelial cell, or glial cell.

[0056] The recombinant AAV vector, rep sequence, cap sequence, and helper function required to generate the rAAV of this disclosure can be delivered to the packaging host cell using any suitable genetic element (vector). The selected genetic element can be delivered by any suitable method, including those described herein. Methods used to construct any embodiment of this disclosure are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV virions are well known, and the choice of suitable method is not a limitation of this disclosure. See, for example, K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0057] In some embodiments, a triple transfection method (described in detail in U.S. Patent No. 6,001,650) can be used to generate recombinant AAV. Typically, recombinant AAV is generated by transfecting host cells with an AAV vector (containing a transgene flanked by ITR elements) to be packaged into AAV particles, an AAV helper vector, and an accessory function vector. The AAV helper vector encodes “AAV helper function” sequences (e.g., rep and cap) that function in trans for productive AAV replication and encapsulation. Preferably, the AAV helper vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use in this disclosure include pHLP19, described in U.S. Patent No. 6,001,650, and the pRep6cap6 vector, described in U.S. Patent No. 6,156,303, the entire contents of which are incorporated herein by reference. Auxiliary function vectors encode nucleotide sequences for non-AAV-derived viral and / or cellular functions that AAV depends on for replication (e.g., "auxiliary functions"). Auxiliary functions include those required for AAV replication, including but not limited to those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and AAV capsid assembly. Virus-based auxiliary functions can be derived from any known helper virus, such as adenovirus, herpesvirus (except herpes simplex virus type 1), and vaccinia virus.

[0058] In some respects, this disclosure provides transfected host cells. The term “transfection” is used to refer to the uptake of exogenous DNA by a cell, and a cell is “transfected” when the exogenous DNA has been introduced into the cell membrane. Many transfection techniques are generally known in the art. 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 integrators and other nucleic acid molecules, into a suitable host cell.

[0059] As used herein, the term “recombinant cell” refers to a cell in which a foreign DNA fragment, such as a DNA fragment that leads to the transcription of a bioactive polypeptide or the production of a bioactive nucleic acid like RNA, is introduced.

[0060] As used herein, the term "vector" includes any genetic element, such as plasmids, bacteriophages, transposons, granules, chromosomes, artificial chromosomes, viruses, virions, etc., that is capable of replicating when bound to suitable control elements and of transferring gene sequences between cells. In some embodiments, the vector is a viral vector, such as an rAAV vector, lentiviral vector, adenoviral vector, retroviral vector, anellovirus vector (e.g., the anellovirus vector described in US20200188456A1), etc. Therefore, the term includes cloning and expression media, as well as viral vectors. In some embodiments, a useful vector is contemplated as one in which the nucleic acid fragment to be transcribed is under the transcriptional control of a promoter.

[0061] Nucleic acid encoding genetic modification

[0062] Several aspects of this disclosure relate to anti-VEGF agents. Part of this disclosure relates to nucleic acids encoding anti-vascular endothelial growth factor (anti-VEGF) proteins. Vascular endothelial growth factor (VEGF), originally called vascular permeability factor (VPF), is a cell-produced signaling protein that stimulates angiogenesis. VEGF is a subfamily of growth factors, namely the platelet-derived growth factor family of cystine junction growth factors. They are important signaling proteins involved in angiogenesis (de novo formation of the embryonic circulatory system) and angiogenesis (growth of blood vessels from a pre-existing vascular system). The normal function of VEGF is to generate new blood vessels during embryonic development, after injury, after exercise, and to generate new blood vessels (collateral circulation) that bypass obstructed vessels. However, abnormal VEGF activity / signaling can lead to various diseases, such as vascular diseases.

[0063] Anti-vascular endothelial growth factor therapy, also known as anti-VEGF therapy or anti-VEGF drug therapy, involves the use of drugs that block the activity of vascular endothelial growth factor. Non-limiting examples of anti-VEGF agents include VEGF receptor fusion proteins (e.g., KH902), monoclonal antibodies (e.g., bevacizumab), antibody derivatives (e.g., ranibizumab, Lucentis), or orally available small molecules that inhibit VEGF-stimulated tyrosine kinases (e.g., lapatinib, sunitinib, sorafenib, axitinib, and pazopanib).

[0064] In some embodiments, the nucleic acid encoding the anti-VEGF agent is an isolated nucleic acid. In some embodiments, the isolated nucleic acid of this disclosure contains a transgene encoding an anti-VEGF agent. In some embodiments, the anti-VEGF agent targets (e.g., specifically binds to) the human VEGF receptor. The VEGF receptor is the receptor for vascular endothelial growth factor (VEGF). There are three major subtypes of the VEGF receptor, numbered 1, 2, and 3. Vascular endothelial growth factor (VEGF) is an important signaling protein involved in many biological pathways (e.g., angiogenesis and vascularization). The VEGF receptor has an extracellular portion consisting of seven immunoglobulin-like domains (e.g., extracellular domains 1-7), a single transmembrane region, and an intracellular portion containing a dividing tyrosine kinase domain. In some embodiments, the anti-VEGF agent targets (e.g., specifically binds to) placental-derived growth factor (PlGF).

[0065] In some embodiments, the anti-VEGF agent is a human VEGF decoy receptor or a portion thereof. A "decoy receptor" is a receptor capable of recognizing and binding ligands (e.g., VEGF) but structurally unable to signal or activate a target receptor complex. It acts as an inhibitor, binding the ligand and preventing it from binding to its conventional receptor. In some embodiments, the VEGF decoy receptor comprises one or more extracellular domains of VEGF receptor 1 and / or VEGF receptor 2. In some embodiments, the anti-VEGF agent is a human VEGF decoy receptor fusion protein. In some embodiments, the human VEGF decoy receptor fusion protein comprises more than one extracellular domain selected from fused together VEGF receptor 1 and / or VEGF receptor 2. In some embodiments, the human VEGF decoy receptor fusion protein comprises a first portion comprising VEGF receptor 1 fused to VEGF receptor 2, which is further fused to a second portion comprising a different protein (e.g., the Fc portion of an immunoglobulin). VEGF decoy receptors and VEGF decoy receptor fusion proteins have been previously described, see, for example, WO2007112675 and EP1767546B1, the entire contents of which are incorporated herein by reference.

[0066] In some embodiments, the human VEGF decoy receptor includes an extracellular domain of a VEGF-binding protein. In some embodiments, the human VEGF decoy receptor includes an extracellular domain of human VEGF receptor 1. In some embodiments, the human VEGF decoy receptor includes extracellular domain 2 of human VEGF receptor 1. In some embodiments, the human VEGF decoy receptor includes an extracellular domain of human VEGF receptor 2. In some embodiments, the human VEGF decoy receptor includes extracellular domains 3 and 4 of human VEGF receptor 2.

[0067] In some embodiments, the human VEGF decoy receptor is a human VEGF receptor fusion protein. In some embodiments, the VEGF receptor fusion protein comprises an extracellular domain selected from VEGF receptor 1 or VEGF receptor 2, and one or more second extracellular domains selected from VEGF receptor 1 or VEGF receptor 2. In some embodiments, the VEGF receptor fusion protein comprises extracellular domain 2 of VEGF receptor 1 and extracellular domain 3 of VEGF receptor 2. In some embodiments, the VEGF receptor fusion protein comprises extracellular domain 2 of VEGF receptor 1 and extracellular domains 3 and 4 of VEGF receptor 2. In some embodiments, the VEGF receptor fusion protein comprises an extracellular domain 2 of VEGF receptor 1 fused to extracellular domain 3 of VEGF receptor 2 and further fused to extracellular domain 4 of VEGF receptor 1. In some embodiments, the VEGF receptor fusion protein comprises an extracellular domain 1 of VEGF receptor 2 fused to extracellular domain 2 of VEGF receptor 1 and further fused to extracellular domain 3 of VEGF receptor 2. In some embodiments, the VEGF receptor fusion protein comprises an extracellular domain 2 of VEGF receptor 1 fused to an extracellular domain 3 of VEGF receptor 2, and further fused to an extracellular domain 4 of VEGF receptor 2, and further fused to an extracellular domain 5 of VEGF receptor 2. In some embodiments, the VEGF receptor fusion protein comprises an extracellular domain 2 of VEGF receptor 1 fused to an extracellular domain 3 of VEGF receptor 2, and further fused to an extracellular domain 4 of VEGF receptor 2, and further fused to an extracellular domain 5 of VEGF receptor 1. In some embodiments, the fused extracellular domains of the VEGF decoy receptors are interconnected via adapters. In some embodiments, the fused extracellular domains of the VEGF decoy receptors are directly interconnected.

[0068] Furthermore, any VEGF receptor fusion protein described herein can be fused with another protein. In some embodiments, the VEGF receptor fusion protein comprises a portion of a VEGF receptor (e.g., any VEGF decoy receptor or VEGF decoy receptor fusion protein described herein) fused with another protein to provide dimerizing or multimerizing properties. A non-limiting example of a protein providing dimerizing or multimerizing properties for the fusion protein is the Fc portion of an immunoglobulin. In some embodiments, the VEGF receptor fusion protein comprises a portion of a VEGF receptor (e.g., any VEGF decoy receptor or VEGF decoy receptor fusion protein described herein) fused to the Fc portion of an immunoglobulin. In some embodiments, the VEGF receptor fusion protein (e.g., the VEGF decoy receptor or VEGF decoy receptor fusion protein described herein) is directly fused with other portions (e.g., Fc domains). In some embodiments, the VEGF receptor fusion protein (e.g., a VEGF receptor decoy) is fused with other portions via a linker.

[0069] Suitable linkers are known in the art. (See, for example, Chen et al., Fusion protein linkers: property, design and functionality, Adv Drug Deliv Rev. 2013 Oct; 65(10):1357-69). In some embodiments, the VEGF receptor fusion protein is further fused to the Fc region of an immunoglobulin. In some embodiments, the VEGF receptor fusion protein is KH902. KH902, also known as conbercept (e.g., US20100272719A1, the entire contents of which are incorporated herein by reference), is a decoy receptor protein constructed by fusing the extracellular domains of vascular endothelial growth factor (VEGF) receptor 1 and VEGF receptor 2 to the Fc region of a human immunoglobulin. KH902 is approximately 142 kDa in size. Conbercept-mediated VEGF and placental growth factor (PIGF) (which can induce angiogenesis) blockade has been shown to be effective in treating wet age-related macular degeneration (wAMD) in clinical trials (including phase 3 trials), see, for example, Liu et al., AJO, August 17, 2019, the entire contents of which are incorporated herein by reference.

[0070] In some embodiments, the anti-VEGF agent comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:5. An exemplary amino acid sequence of KH902 is shown in SEQ ID NO:5 (extracellular domain 2 of VEGF receptor 1 is shown in bold; extracellular domain 3 of VEGF receptor 2 is shown in underline; extracellular domain 4 of VEGF receptor 2 is shown in italics and bold; Fc domain is shown in italics and underline).

[0071]

[0072]

[0073] In some embodiments, the anti-VEGF agent comprises the portion of SEQ ID NO:5. In some embodiments, the anti-VEGF agent comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of the extracellular domain 2 of the VEGF receptor 1 as shown in SEQ ID NO:6. In some embodiments, the anti-VEGF agent comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of extracellular domains 3 and 4 of VEGF receptor 2 as shown in SEQ ID NO:7. In some embodiments, the anti-VEGF agent comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of the extracellular domain 2 of VEGF receptor 1 fused with extracellular domains 3 and 4 of VEGF receptor 2 as shown in SEQ ID NO:8. In some embodiments, the anti-VEGF agent comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of the extracellular domain 2 of a VEGF receptor 1 fused to the Fc portion of an immunoglobulin as shown in SEQ ID NO:9. In some embodiments, the anti-VEGF agent comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of the extracellular domains 3 and 4 of the VEGF receptor 2 fused to the Fc portion of an immunoglobulin as shown in SEQ ID NO:10.

[0074] An exemplary amino acid sequence of the extracellular domain 2 of VEGF receptor 1 is shown in SEQ ID NO:6:

[0075]

[0076] An exemplary amino acid sequence of extracellular domains 3 and 4 of VEGF receptor 2 is shown in SEQ ID NO:7:

[0077]

[0078] An exemplary amino acid sequence of extracellular domain 2 of VEGF receptor 1, fused with extracellular domains 3 and 4 of VEGF receptor 2, is shown in SEQ ID NO:8:

[0079]

[0080] An exemplary amino acid sequence of the extracellular domain 2 of VEGF receptor 1 fused with the Fc portion is shown in SEQ ID NO:9:

[0081]

[0082]

[0083] An exemplary amino acid sequence of extracellular domains 3 and 4 of VEGF receptor 2 fused with the Fc portion is shown in SEQ ID NO:10:

[0084]

[0085] In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleic acid sequence shown in SEQ ID NO:1. An exemplary coding sequence of KH902 is shown in SEQ ID NO:1.

[0086]

[0087]

[0088] Any anti-VEGF agents and / or combinations thereof described herein can be expressed by the isolated nucleic acids described herein. In some embodiments, the isolated nucleic acids comprise a first region encoding an extracellular domain 2 of VEGF receptor 1 and a second region encoding extracellular domains 3 and 4 of VEGF receptor 2. In some embodiments, the isolated nucleic acids comprise a first region encoding an extracellular domain 2 of VEGF receptor 1 fused to the Fc portion of an immunoglobulin, and a second region encoding extracellular domains 3 and 4 of VEGF receptor 2 fused to the Fc portion of an immunoglobulin. In some embodiments, the first region can be located at any suitable location. The first region can be located upstream of the second region. For example, the first region can be located between the first codon of the second region and 2000 nucleotides upstream of said first codon. The first region can be located between the first codon of the second region and 1000 nucleotides upstream of said first codon. The first region can be located between the first codon of the second region and 500 nucleotides upstream of said first codon. The first region can be located between the first codon of the second region and 250 nucleotides upstream of said first codon. The first region may be located between the first codon of the second region and 150 nucleotides upstream of the first codon. In other embodiments, the first region may be located downstream of the second region. The first region may be located between the last codon of the second region and 2000 nucleotides downstream of the last codon. The first region may be located between the last codon of the second region and 1000 nucleotides downstream of the last codon. The first region may be located between the last codon of the second region and 500 nucleotides downstream of the last codon. The first region may be located between the last codon of the second region and 250 nucleotides downstream of the last codon. The first region may be located between the last codon of the second region and 150 nucleotides downstream of the last codon.

[0089] In some embodiments, the nucleic acid may further include a third region. In some embodiments, the isolated nucleic acid includes a first region encoding an extracellular domain 2 of VEGF receptor 1, a second region encoding extracellular domains 3 and 4 of VEGF receptor 2, and a third region encoding an extracellular domain 2 of VEGF receptor 1 fused with extracellular domains 3 and 4 of VEGF receptor 2. In some embodiments, the isolated nucleic acid includes a first region encoding an extracellular domain 2 of VEGF receptor 1 fused with the Fc portion of immunoglobulin, a second region encoding extracellular domains 3 and 4 of VEGF receptor 2 fused with the Fc portion of immunoglobulin, and a third region encoding an extracellular domain 2 of VEGF receptor 1 fused with extracellular domains 3 and 4 of VEGF receptor 2 and further fused with the Fc portion of immunoglobulin. In some embodiments, the third region is located upstream of the first codon of the first region. In some embodiments, the third region is located between the last codon of the first region and the first codon of the second region. In some embodiments, the third region is located downstream of the last codon of the second region.

[0090] In some embodiments, the regions of the isolated nucleic acids disclosed herein are expression cassettes for expressing the anti-VEGF agents or combinations of anti-VEGF agents described herein. In some embodiments, the polycistronic expression construct comprises two or more expression cassettes encoding one or more of the anti-VEGF agents or combinations of anti-VEGF agents described herein.

[0091] In some embodiments, the polycistronic expression construct includes expression cassettes positioned in different ways. For example, in some embodiments, a polycistronic expression construct is provided in which a first expression cassette (e.g., an expression cassette encoding a first anti-VEGF agent or a portion thereof) is positioned adjacent to a second expression cassette (e.g., an expression cassette encoding a second anti-VEGF agent or a portion thereof). In some embodiments, a polycistronic expression construct is provided in which the first expression cassette contains an intron, and the second expression cassette is located within the intron of the first expression cassette. In some embodiments, the second expression cassette located within the intron of the first expression cassette contains a promoter and a nucleic acid sequence encoding a gene product effectively linked to said promoter.

[0092] In different implementations, a polycistronic expression construct is provided in which the expression boxes are oriented in different ways. For example, in some implementations, a polycistronic expression construct is provided in which the first expression box and the second expression box are in the same orientation. In some implementations, a polycistronic expression construct is provided which includes a first expression box and a second expression box in opposite orientations.

[0093] As used herein, the term "orientation" in relation to expression cassettes refers to the directional characteristics of a given cassette or structure. In some embodiments, the expression cassette contains a 5' promoter encoding a nucleic acid sequence, and transcription of the nucleic acid sequence runs from the 5' end of the sense strand to the 3' end, making it an oriented cassette (e.g., 5'-promoter / (intron) / coding sequence-3'). Since virtually all expression cassettes are oriented in this sense, those skilled in the art can readily determine the orientation of a given expression cassette relative to a second nucleic acid structure (e.g., a second expression cassette, a viral genome), or, if the cassette is contained within an AAV construct, relative to an AAV ITR.

[0094] For example, if a given nucleic acid construct contains two expression cassettes in configuration 5'-promoter 1 / coding sequence 1---promoter 2 / coding sequence 2-3',

[0095] >>> ...

[0096] The expression cassettes are oriented in the same direction, with arrows indicating the transcriptional direction of each cassette. For another example, if a given nucleic acid construct contains two expression cassettes in the sense strand at conformation 5'-promoter 1 / coding sequence 1---coding sequence 2 / promoter 2-3',

[0097] >>>>>>>>>>>>>>>>>>>>>>>>> <<<<<<<<<<<<<<<<<<<<<<

[0098] The expression cassettes are positioned opposite each other, and as the arrows indicate, the transcription of the expression cassettes is in the opposite direction. In this example, the strand shown contains the antisense strands of promoter 2 and coding sequence 2.

[0099] For another example, if the expression cassette is contained within the AAV construct, the cassette can be oriented in the same or opposite direction as the AAV ITR. The AAV ITR is oriented. For instance, if both the ITR and the expression cassette are located on the same nucleic acid strand, the 3' ITR will be oriented in the same direction as the promoter 1 / coding sequence 1 expression cassette in the example above, but in the opposite direction to the 5' ITR.

[0100] Extensive evidence suggests that polycistronic expression constructs typically fail to achieve optimal expression levels compared to expression systems containing only one cistron. One possible reason for the suboptimal expression levels achieved using polycistronic expression constructs containing two or more promoter elements is promoter interference (see, for example, Curtin JA, Dane AP, Swanson A, Alexander IE, Ginn SL. Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct. Gene Ther. 2008 Mar; 15(5):384-90; and Martin-Duque P, Jezzard S, Kaftansis L, Vassaux G. Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes. Hum Gene Ther. 2004 Oct; 15(10):995-1002; both references are incorporated herein by reference to disclose promoter interference). Several strategies have been proposed to overcome promoter interference, such as generating polycistronic expression constructs containing only a single promoter to drive transcription of multiple coding nucleic acid sequences separated by internal ribosome entry sites, or separating cistrons containing their own promoters from transcriptional insulator elements. However, all proposed strategies for overcoming promoter interference face their own set of problems. For example, polycistronic expression driven by a single promoter often results in uneven cistron expression levels. Furthermore, some promoters cannot be effectively separated, and the separated elements are incompatible with some gene transfer vectors, such as some retroviral vectors.

[0101] In some embodiments of the present invention, a polycistronic expression construct is provided that allows efficient expression of a first coding nucleic acid sequence driven by a first promoter and a second coding nucleic acid sequence driven by a second promoter, without the use of transcriptional insulator elements. Various configurations of such polycistronic expression constructs are provided herein, for example, expression constructs containing a first expression cassette comprising an intron and a second expression cassette located within the intron and in the same or opposite orientation to the first cassette. Other configurations are described in more detail elsewhere herein.

[0102] In some embodiments, a polycistronic expression construct is provided that allows for the efficient expression of two or more coding nucleic acid sequences. In some embodiments, the polycistronic expression construct comprises two expression cassettes. In some embodiments, the first expression cassette of the polycistronic expression construct provided herein contains a first RNA polymerase II promoter, and the second expression cassette contains a second RNA polymerase II promoter. In some embodiments, the first expression cassette of the polycistronic expression construct provided herein contains an RNA polymerase II promoter, and the second expression cassette contains an RNA polymerase III promoter.

[0103] In some implementations, the provided polycistronic expression construct is a recombinant AAV (rAAV) construct.

[0104] In some embodiments, the isolated nucleic acid described herein comprises a codon-optimized nucleic acid sequence of an anti-VEGF agent (e.g., KH902). Codon optimization of the nucleic acid coding sequence can be achieved using methods known in the art to optimize expression in target cells (e.g., mammalian cells).

[0105] A “nucleic acid” sequence refers to a DNA or RNA sequence. In some embodiments, the proteins and nucleic acids disclosed herein are isolated. As used herein, the term “isolated” means artificially produced. As used herein, with respect to nucleic acids, the term “isolated” means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) produced by clonal recombination; (iii) purified, such as by lysis and gel separation; (iv) synthesized by, for example, chemical synthesis. Isolated nucleic acids are nucleic acids that are readily manipulated using recombinant DNA techniques known in the art. Thus, a nucleotide sequence contained in a vector in which the 5' and 3' restriction sites are known or the polymerase chain reaction (PCR) primer sequences are disclosed is considered isolated, but a nucleic acid sequence present in its natural host in its native state is not. Isolated nucleic acids may be substantially purified, but are not required to be. For example, nucleic acids isolated in a cloning or expression vector are not pure because they may only constitute a small percentage of the material in the cell in which they reside. However, such nucleic acids are isolated, as the term is used herein, because they are readily manipulated using standard techniques known to those skilled in the art. As used in this article with respect to proteins or peptides, the term "isolated" refers to proteins or peptides that have been isolated from their natural environment or artificially produced (e.g., through chemical synthesis, through recombinant DNA technology, etc.).

[0106] In some embodiments, the isolated nucleic acids and rAAVs described herein comprise one or more of the following structural features (e.g., control or regulatory sequences): a long chicken β-actin (CBA) promoter, an extended CBA intron, a Kozak sequence, a nucleic acid sequence encoding an anti-VEGF agent (e.g., KH902) or a codon-optimized variant of an anti-VEGF agent (e.g., KH902), one or more microRNA binding sites, and a rabbit β-globin (RBG) poly A sequence. In some embodiments, one or more of the aforementioned control sequences are efficiently linked to the nucleic acid sequence encoding an anti-VEGF agent (e.g., KH902).

[0107] As used herein, when nucleic acid sequences (e.g., coding sequences) and regulatory sequences are covalently linked in a manner that places the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequence, they are referred to as “effectively linked.” Two DNA sequences are considered effectively linked if the goal is to translate the nucleic acid sequence into a functional protein, if the induction of a promoter in the 5' regulatory sequence leads to the transcription of the coding sequence, and if the nature of the link between the two DNA sequences does not (1) introduce a frameshift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Therefore, a promoter region is effectively linked to a nucleic acid sequence if it can influence the transcription of the DNA sequence, resulting in a transcript that can be translated into the desired protein or polypeptide. Similarly, two or more coding regions are effectively linked when their transcription from a common promoter leads to the expression of two or more proteins already translated within their reading frames.

[0108] In some implementations, the transgene contains a nucleic acid sequence encoding an anti-VEGF agent (e.g., KH902) that is effectively linked to the promoter. A “promoter” is a DNA sequence recognized by or introduced into the cell’s synthetic mechanisms for the specific transcription of a gene. The phrases “effectively linked,” “effectively positioned,” “under control,” or “under transcriptional control” mean that the promoter is in the correct position and orientation relative to the nucleic acid to control the initiation of RNA polymerase and gene expression.

[0109] Typically, promoters can be constitutive promoters, inducible promoters, or tissue-specific promoters.

[0110] Examples of constitutive promoters include, but are not limited to, the retroviral Rouss sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, for example, Boshart et al., Cell, 41:521-530 (1985)], the chimeric cytomegalovirus (CMV) / chicken β-actin (CB) promoter (CBA promoter), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the glycerol phosphokinase (PGK) promoter, and the EF1α promoter [Invitrogen]. In some embodiments, the promoter is the RNApol II promoter. In some embodiments, the promoter is the chimeric cytomegalovirus (CMV) / chicken β-actin (CB) promoter (CBA promoter). In some embodiments, the promoter is the RNApol III promoter, such as U6 or H1.

[0111] Examples of inducible promoters regulated by exogenously provided promoters include the zinc-inducible sheep metallothioneine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, and the T7 polymerase promoter system (WO). 98 / 10088); Ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), tetracycline inhibition system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), tetracycline induction system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), RU486 induction system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997) and the rapamycin induction system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Other types of inducible promoters that may be useful in this context are those regulated by specific physiological states, such as temperature, acute phase, specific differentiation state of the cell, or only in replicating cells.

[0112] In some implementations, regulatory sequences confer tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory sequences bind to tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: retinoschisin proximal promoter, interphotoreceptor retinoid-binding protein enhancer (RS / IRBPa), rhodopsin kinase (RK), liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synaptic protein-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, α-myosin heavy chain (α-MHC) promoter, or cardiac troponin T (cTnT) promoter. Other exemplary promoters include the β-actin promoter, the hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002-9 (1996)); the alpha-fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)); the osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); the bone salivary protein promoter (Chen et al., J. BoneMiner. Res., 11:654-64 (1996)); the CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998)); the immunoglobulin heavy chain promoter; the T cell receptor α-chain promoter; and neuron-specific enolases. The neuron-specific (NSE) promoter (Andersen et al., Cell.Mol.Neurobiol., 13:503-15 (1993)), the neurofilament light chain gene promoter (Piccioli et al., Proc.Natl.Acad.Sci.USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), as well as other promoters that are obvious to a person skilled in the art.

[0113] In some implementations, the tissue-specific promoter is an eye-specific promoter. Examples of eye-specific promoters include the retinoschistosome proximal promoter, the interretinoid retinol-binding protein enhancer (RS / IRBPa), rhodopsin kinase (RK), RPE65, and the human cone opsin promoter.

[0114] In some embodiments, the promoter is a chicken β-actin (CB) promoter. The chicken β-actin promoter can be a short chicken β-actin promoter or a long chicken β-actin promoter. In some embodiments, the promoter (e.g., a chicken β-actin promoter) includes an enhancer sequence, such as a cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence can be a short CMV enhancer sequence or a long CMV enhancer sequence. In some embodiments, the promoter includes a long CMV enhancer sequence and a long chicken β-actin promoter. In some embodiments, the promoter includes a short CMV enhancer sequence and a short chicken β-actin promoter. However, those skilled in the art will recognize that a short CMV enhancer can be used with a long CB promoter, and a long CMV enhancer can be used with a short CB promoter (and vice versa).

[0115] The isolated nucleic acids described herein may also contain one or more introns. In some embodiments, at least one intron is located between the promoter / enhancer sequence and the transgene. In some embodiments, the introns are synthetic or artificial (e.g., heterologous) introns. Examples of synthetic introns include intron sequences derived from SV-40 (referred to as SV-40T intron sequences) and intron sequences derived from the chicken β-actin gene. In some embodiments, the transgenes described herein contain one or more (1, 2, 3, 4, 5 or more) artificial introns. In some embodiments, one or more artificial introns are located between the promoter and the nucleic acid sequence encoding an anti-VEGF agent (e.g., KH902).

[0116] In some embodiments, the transgene described herein comprises a Kozak sequence. The Kozak sequence is a nucleic acid motif containing the concordant sequence GCC(A / G)CC (SEQ ID NO:4), which is present in eukaryotic mRNA and plays a role in the initiation of protein translation. In some embodiments, the Kozak sequence is located between an intron and a transgene encoding an anti-VEGF agent (e.g., KH902).

[0117] The isolated nucleic acid described in this disclosure can encode a transgene that further comprises a polyadenylated (poly A) sequence. In some embodiments, the transgene comprising the poly A sequence is the rabbit β-globin (RBG) poly A sequence.

[0118] In some embodiments, the transgene includes a 3'-untranslated region (3'-UTR). In some embodiments, this disclosure relates to isolated nucleic acids containing a transgene encoding an anti-VEGF agent (e.g., KH902) and one or more miRNA binding sites. Without wishing to be bound by any particular theory, incorporating miRNA binding sites into gene expression constructs allows for the regulation (e.g., suppression) of transgene expression in cells and tissues expressing the corresponding miRNA. In some embodiments, incorporating one or more miRNA binding sites into the transgene allows for off-target transgene expression in a cell-type-specific manner. In some embodiments, one or more miRNA binding sites are located within the 3'-UTR of the transgene, for example, between the last codon and the poly A sequence of the nucleic acid sequence encoding an anti-VEGF agent (e.g., KH902).

[0119] In some embodiments, the transgene includes one or more (e.g., 1, 2, 3, 4, 5 or more) miRNA binding sites that cause the expression of an anti-VEGF agent (e.g., KH902) to be off-target from immune cells (e.g., antigen-presenting cells (APCs), such as macrophages, dendritic cells, etc.). The inclusion of miRNA binding sites with immune-associated miRNAs can cause transgene (e.g., KH902) expression to be off-target from antigen-presenting cells, thereby reducing or eliminating the (cellular and / or humoral) immune response in a subject to the transgene product, as described, for example, in US 2018 / 0066279, the entire contents of which are incorporated herein by reference.

[0120] In some aspects, this disclosure relates to isolated nucleic acids containing a transgene encoding an anti-VEGF agent (e.g., KH902) and one or more miRNA binding sites. Without being bound by any particular theory, incorporating miRNA binding sites into gene expression constructs allows for the regulation (e.g., suppression) of transgene expression in cells and tissues expressing the corresponding miRNA. In some embodiments, incorporating one or more miRNA binding sites into the transgene allows for off-target transgene expression in a cell-type-specific manner. In some embodiments, the one or more miRNA binding sites are located in the 3' untranslated region (3'UTR) of the transgene, for example, between the last codon and the polyA sequence of a nucleic acid sequence encoding one or more GM3S proteins.

[0121] In some embodiments, the transgene contains one or more (e.g., 1, 2, 3, 4, 5 or more) miRNA binding sites that cause anti-VEGF agent (e.g., KH902) expression to be off-target from hepatocytes. For example, in some embodiments, the transgene contains one or more miR-122 binding sites.

[0122] In some embodiments, the transgene includes one or more (e.g., 1, 2, 3, 4, 5 or more) miRNA binding sites that cause the expression of one or more GM3S proteins to be off-target from immune cells (e.g., antigen-presenting cells (APCs), such as macrophages, dendritic cells, etc.). The incorporation of miRNA binding sites for immune-associated miRNAs can cause transgene expression to be off-target from antigen-presenting cells, thereby reducing or eliminating (cellular and / or humoral) immune responses in subjects to the transgene product, as described in, for example, US2018 / 0066279, the entire contents of which are incorporated herein by reference.

[0123] As used herein, “immune cell-associated miRNA” is a miRNA preferentially expressed in cells of the immune system, such as antigen-presenting cells (APCs). In some embodiments, the immune cell-associated miRNA is a miRNA expressed in immune cells at a level at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than that expressed in non-immune cells (e.g., control cells, such as HeLa cells, HEK293 cells, mesenchymal cells, etc.). In some embodiments, the immune system cells (immune cells) in which the immune cell-associated miRNA is expressed are B cells, T cells, cytotoxic T cells, helper T cells, γδ T cells, dendritic cells, macrophages, monocytes, vascular endothelial cells, or other immune cells. In some implementations, the cells of the immune system are B cells expressing one or more of the following markers: B220, BLAST-2 (EBVCS), Bu-1, CD19, CD20 (L26), CD22, CD24, CD27, CD57, CD72, CD79a, CD79b, CD86, chB6, D8 / 17, FMC7, L26, M17, MUM-1, Pax-5 (BSAP), and PC47H. In some implementations, the cells of the immune system are T cells expressing one or more of the following markers: ART2, CD1a, CD1d, CD11b (Mac-1), CD134 (OX40), CD150, CD2, CD25 (interleukin-2 receptor α), CD3, CD38, CD4, CD45RO, CD5, CD7, CD72, CD8, CRTAM, FOXP3, FT2, GPCA, HLA-DR, HML-1, HT23A, Leu-22, Ly-2, Ly-m22, MICG, MRC OX8, MRCOX-22, OX40, PD-1 (programmed cell death-1), RT6, TCR (T cell receptor), Thy-1 (CD90), and TSA-2 (thymic shared Ag-2). In some implementations, the immune cell-associated miRNAs are selected from: miR-15a, miR-16-1, miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, miR-21, miR-29a / b / c, miR-30b, miR-31, miR-34a, miR-92a-1, miR-106a, miR-125a / b, miR-142-3p, miR-146a, miR-150, miR-155, miR-181a, miR-223 and miR-424, miR-221, miR-222, let-7i, miR-148 and miR-152.In some implementations, the transgene described herein contains one or more binding sites targeting miR-142.

[0124] In some embodiments, the isolated nucleic acid contains inverted terminal repeats. The isolated nucleic acid of this disclosure may be a recombinant adeno-associated virus (AAV) vector (rAAV vector). In some embodiments, the isolated nucleic acid as described in this disclosure contains a region containing a first adeno-associated virus (AAV) inverted terminal repeat (ITR) (e.g., a first region), or a variant thereof. The isolated nucleic acid (e.g., a recombinant AAV vector) may be packaged into a capsid protein and administered to a subject and / or delivered to selected target cells. A “recombinant AAV (rAAV) vector” typically consists of at least a transgene and its regulatory sequence, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The transgene may contain a region encoding, for example, a protein (e.g., an anti-VEGF agent, such as KH902) and / or an expression control sequence (e.g., a poly-A tail), as described elsewhere in this disclosure.

[0125] Typically, the ITR sequence is about 145 bp in length. Preferably, a substantially complete sequence encoding the ITR is used in the molecule, although some degree of minor modification to these sequences is permitted. The ability to modify these ITR sequences is within the scope of the art. (See, for example, textbooks such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). One example of such a molecule used in this disclosure is a transgenic "cis-acting" plasmid in which the selected transgenic sequence and the associated regulatory element are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequence can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the isolated nucleic acid further comprises a region containing a second AAV ITR (e.g., a second region, a third region, a fourth region, etc.). In some embodiments, the flanking element of the isolated nucleic acid encoding the transgene is an AAV ITR (e.g., at the 5'-ITR-transgene-ITR-3' direction). In some embodiments, the AAV ITR is selected from AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, and AAV6 ITR.

[0126] In some embodiments, the isolated nucleic acid (e.g., an rAAV vector) as described herein comprises, from 5' to 3', the following sequence: 5' AAV ITR, CMV enhancer, CBA promoter, intron (e.g., chicken β-actin intron), Kozak sequence, transgene encoding an anti-VEGF agent (e.g., KH902), rabbit β-globin poly A, and 3' AAV ITR. An exemplary sequence of the isolated nucleic acid is shown in SEQ ID NO:2. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleic acid sequence shown in SEQ ID NO:2 (Kozak sequence is underlined; KH902 encoding sequence is bold).

[0127]

[0128]

[0129]

[0130]

[0131] Furthermore, plasmids containing the isolated nucleic acids described herein are also within the scope of this disclosure. An exemplary full-length plasmid sequence of pAAV-CBA-KH902 is shown in SEQ ID NO:3. In some embodiments, the plasmid contains a nucleic acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleic acid sequence shown in SEQ ID NO:3.

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] In some embodiments, the anti-VEGF agent described herein (e.g., KH902) can be delivered to subjects via a non-viral platform. In some embodiments, the anti-VEGF agent described herein (e.g., KH902) can be delivered to subjects via closed-end linear double-stranded DNA (ceDNA). Delivery of transgenes (e.g., anti-VEGF agents, such as KH902) has been previously described; see, for example, WO2017152149, the entire contents of which are incorporated herein by reference. In some embodiments, nucleic acids having asymmetric terminal sequences (e.g., asymmetric discontinuous self-complementary sequences) form closed-end linear double-stranded DNA structures (e.g., ceDNA), which in some embodiments exhibit reduced immunogenicity compared to currently available gene delivery vectors. In some embodiments, ceDNA behaves identically to linear double-stranded DNA under natural conditions and transforms into single-stranded circular DNA under denaturing conditions. Not wishing to be bound by any particular theory, in some embodiments, ceDNA can be used to deliver transgenes (e.g., anti-VEGF agents, such as KH902) to subjects.

[0139] AAV-mediated transgene delivery to eye tissue

[0140] Several aspects of this disclosure relate to compositions comprising recombinant AAV containing a capsid protein (e.g., AAV2.7m8) and a nucleic acid encoding a transgene, wherein the transgene comprises a nucleic acid sequence encoding an anti-VEGF agent (e.g., KH902). In some embodiments, the nucleic acid further comprises an AAV ITR.

[0141] rAAV (e.g., rAAV2.7m8-KH902) and compositions comprising the rAAV described herein may be delivered to a subject in compositional form according to any suitable method known in the art. For example, rAAV (e.g., rAAV2.7m8-KH902), preferably suspended in a physiologically compatible carrier (e.g., in a composition), may be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cattle, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., macaque). In some embodiments, the host animal does not include a human. In some embodiments, the subject is a human.

[0142] In some implementations, administration of rAAV, as described herein, results in the delivery of the transgene (e.g., KH902) to ocular tissues. rAAV (e.g., rAAV2.7m8-KH902) can be delivered to the ocular tissues of a mammalian subject by, for example, intraocular injection, subretinal injection, topical application (e.g., eye drops), or by injection into the eye of a mammalian subject (e.g., intravitreal injection). As used herein, “ocular tissues” means any tissue derived from or contained within the eye. Non-limiting examples of ocular tissues include neurons, the retina (e.g., photoreceptor cells), sclera, choroid, vitreous body, macula, fovea, optic disc, lens, pupil, iris, aqueous humor, cornea (e.g., keratinocytes, corneal endothelial cells, corneal basal cells, corneal pterygoid cells, and corneal squamous cells), conjunctival ciliary body, and optic nerve. The retina is located at the back of the eye and contains photoreceptor cells. These photoreceptor cells (e.g., rod cells, cone cells) impart visual acuity and contrast in the visual field by distinguishing colors.

[0143] Alternatively, rAAV (e.g., rAAV2.7m8-KH902) can be delivered to a mammalian subject by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream can be achieved by injection into a vein, artery, or any other vascular catheter. Non-limiting exemplary methods of intramuscular administration of rAAV (e.g., rAAV2.7m8-KH902) include intramuscular (IM) injection and intravascular limb infusion. In some embodiments, rAAV is administered into the bloodstream via detached limb perfusion, a technique known in the surgical field that essentially allows a technician to detach the limb from the systemic circulation prior to administration of the rAAV virion. A variation of the detached limb perfusion technique is described in U.S. Patent No. 6,177,403, which can be used by a technician to administer the virion into the vascular system of a detached limb to potentially enhance transduction to muscle cells or tissues. In some embodiments, rAAV (e.g., rAAV2.7m8-KH902) or compositions (e.g., rAAV-containing compositions) as described in this disclosure are administered via intravitreal injection. In some embodiments, rAAV (e.g., rAAV2.7m8-KH902) or compositions (e.g., rAAV-containing compositions) as described in this disclosure are administered via intraocular injection. In some embodiments, rAAV (e.g., rAAV2.7m8-KH902) or compositions (e.g., rAAV-containing compositions) as described in this disclosure are administered via subretinal injection. In some embodiments, rAAV (e.g., rAAV2.7m8-KH902) or compositions (e.g., rAAV-containing compositions) as described in this disclosure are administered via intravenous injection. In some embodiments, rAAV (e.g., rAAV2.7m8-KH902) or compositions (e.g., rAAV-containing compositions) as described in this disclosure are administered via intramuscular injection. In some embodiments, rAAV (e.g., rAAV2.7m8-KH902) or compositions (e.g., compositions containing rAAV) as described in this disclosure are administered via intratumoral injection.

[0144] In some embodiments, application of isolated nucleic acids and / or rAAV as described herein results in inhibition of VEGF (e.g., VEGF activity). The degree of VEGF inhibition can be measured by any suitable known method (e.g., HUVEC angiogenesis assay, retinal vascular development assay, retinal edema assay, laser-induced choroidal neovascularization (CNV), etc.). In some embodiments, the VEGF (e.g., VEGF activity) activity in subjects who received an anti-VEGF agent (e.g., injection of the isolated nucleic acid and / or rAAV described herein) was inhibited by at least 2%, at least 5%, at least 10%, at least 15%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100% compared to subjects who did not receive the anti-VEGF agent or the same subjects before receiving the anti-VEGF agent. In some embodiments, the VEGF (e.g., VEGF activity) in uninjected subjects or subjects prior to receiving an anti-VEGF agent is at least 2%, at least 5%, at least 10%, at least 15%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 100%, at least 1x, at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, at least 10x, at least 10 to 50x (e.g., 10x, 20x, 30x, 40x, or 50x), at least 50 to 100x (e.g., 50x, 60x, 70x, 80x, 90x, or 100x). In some implementations, administration of an anti-VEGF agent (e.g., isolated nucleic acids and / or rAAV as described herein) results in inhibition of VEGF (e.g., VEGF activity) for longer than 1 day, longer than 2 days, longer than 3 days, longer than 4 days, longer than 5 days, longer than 6 days, longer than 7 days, longer than 1 week (e.g., 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days), longer than 2 weeks (e.g., 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days), longer than 3 weeks (e.g., 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days), longer than 4 weeks (e.g., 29 days, 30 days, 40 days, 50 days, 60 days, 100 days, or more), and longer than 1 month (e.g.,5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more), longer than 2 months (e.g., between 2 and 2.5 months, between 2 and 3 months, between 2 and 4 months, between 2 and 5 months, between 2 and 6 months, between 2 and 7 months, between 2 and 8 months, between 2 and 9 months, between 2 and 10 months, between 2 and 11 months, between 2 and 12 months), longer than 3 months (e.g., between 3 and 4 months, between 3 and 5 months, between 3 and 6 months, between 3 and 7 months, between 3 and 8 months, 3... Between 1 and 9 months, 3 to 10 months, 3 to 11 months, 3 to 12 months), longer than 4 months (e.g., between 4 and 5 months, 4 and 6 months, 4 and 7 months, 4 and 8 months, 4 and 9 months, 4 and 10 months, 4 and 11 months, 4 and 12 months), longer than 5 months (e.g., between 5 and 6 months, 5 and 7 months, 5 and 8 months, 5 and 8 months, 5 and 9 months, 5 and 10 months, 5 months). (e.g., 6 to 11 months, 5 to 12 months), longer than 6 months (e.g., 6 to 7 months, 6 to 8 months, 6 to 9 months, 6 to 10 months, 6 to 11 months, 6 to 12 months), longer than 7 months (e.g., 7 to 8 months, 7 to 9 months, 7 to 10 months, 7 to 11 months, 7 to 12 months), longer than 8 months (e.g., 8 to 9 months, 8 to 10 months, 8 to 11 months, 8 to 12 months), longer The following are considered acceptable timeframes: 9 months (e.g., between 9 and 10 months, 9 and 11 months, 9 and 12 months), longer than 10 months (e.g., between 10 and 11 months, 11 and 12 months), longer than 11 months (e.g., between 11 and 12 months), longer than 12 months (e.g., between 12 and 15 months, 12 and 18 months, 12 and 21 months, 12 and 2 months), longer than 1 year (e.g., between 1 and 1.5 years), longer than 2 years, longer than 3 years, longer than 4 years, longer than 5 years, longer than 10 years, longer than 15 years, longer than 20 years, or longer.

[0145] The compositions disclosed herein may comprise a single rAAV (e.g., rAAV2.7m8-KH902) or an rAAV (e.g., rAAV2.7m8-KH902) in combination with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs, each having one or more different transgenes.

[0146] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. Given the indications targeted by rAAV (e.g., rAAV2.7m8-KH902), those skilled in the art can readily select a suitable carrier. For example, a suitable carrier includes saline solution, which can be formulated with a variety of buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrose, agar, pectin, peanut oil, sesame oil, and water. The choice of carrier is not a limitation of the invention.

[0147] Optionally, in addition to rAAV (e.g., rAAV2.7m8-KH902) and the carrier, the compositions of this disclosure may also contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, p-chlorophenol, and poloxamer (a nonionic surfactant). F-68. Suitable chemical stabilizers include gelatin and albumin.

[0148] Administer rAAV (e.g., rAAV2.7m8-KH902) or a combination thereof (e.g., a composition containing rAAV) in an amount sufficient to transfect cells of the desired tissue and provide adequate levels of gene transfer and expression without excessive side effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., intravitreal delivery to the eye), intravitreal injection, subretinal injection, oral administration, inhalation (including intranasal and intratracheal delivery), intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parenteral routes. Routes of administration may be combined if desired.

[0149] The dose of rAAV virus required to achieve a specific "therapeutic effect," expressed as a unit of measurement in genome copy number per kilogram of body weight (GC / kg), will vary based on several factors, including but not limited to: the route of administration of the rAAV virus, the gene or RNA expression level required to achieve the therapeutic effect, the specific disease or condition being treated, and the stability of the gene or RNA product. Based on the above factors and other factors known in the art, those skilled in the art can readily determine the range of rAAV virus doses required to treat patients with a specific disease or condition.

[0150] An effective amount of rAAV or a composition (e.g., a composition containing the isolated nucleic acid or rAAV described herein) is sufficient to target and infect an animal and target a tissue (e.g., muscle tissue, eye tissue, etc.). In some embodiments, an effective amount of rAAV is administered to a subject in the pre-symptomatic stage of a degenerative disease. In some embodiments, rAAV or a composition is administered to a subject after one or more signs or symptoms of a degenerative disease have been observed. In some embodiments, the effective amount will depend primarily on factors such as species, age, weight, the health of the subject, and the tissue to be targeted, and therefore can vary between animals and tissues. For example, an effective amount of rAAV is typically in the range of about 1 ml to about 100 ml of solution containing about 10 6 Up to 10 16 One genome copy (e.g., 1 x 10^15) 6 Up to 1x 10 16 (including endpoints). In some implementations, the effective amount of rAAV ranges from 1x10. 9 Up to 1x10 14 Between 10 rAAV genome copies. In some cases, approximately 10 11 Up to 10 12 The dosage between rAAV genome copies is appropriate. In some implementations, approximately 10 11 Up to 10 13 The dosage between rAAV genome copies is appropriate. In some implementations, approximately 10 11 Up to 10 14 The dosage between rAAV genome copies is appropriate. In some implementations, approximately 10 11 Up to 10 15 The dosage between rAAV genome copies is appropriate. In some implementations, approximately 10 12 Up to 10 14 A dose of one rAAV genome copy is appropriate. In some implementations, approximately 10 13 Up to 10 14 A dose of one rAAV genome copy is appropriate. In some implementations, approximately 1 x 10^ ...12 Approximately 1.1 x 10 12 Approximately 1.2 x 10 12 Approximately 1.3 x 10 12 Approximately 1.4 x 10 12 Approximately 1.5 x 10 12 Approximately 1.6 x 10 12 Approximately 1.7 x 10 12 Approximately 1.8 x 10 12 Approximately 1.9 x 10 12 Approximately 1 x 10 13 Approximately 1.1 x 10 13 Approximately 1.2 x 10 13 Approximately 1.3 x 10 13 Approximately 1.4 x 10 13 Approximately 1.5 x 10 13 Approximately 1.6 x 10 13 Approximately 1.7 x 10 13 Approximately 1.8 x 10 13 Approximately 1.9 x 10 13 Or approximately 2.0 x 10 14 One vector genome (vg) copy / kilogram (kg) body weight is suitable. In some implementations, approximately 4 x 10^6 copies / kg is appropriate. 12 Up to 2x 10 13 The dosage between rAAV genome copies is appropriate. In some implementations, approximately 1.5 x 10^6 kilosporins are administered intravenously. 13 A dose of vg / kg is appropriate. In some embodiments, 10 12 -10 13 One rAAV genome copy is effective against target tissues (e.g., the eye). In some implementations, 10 13 -10 14 One rAAV genome copy is effective against target tissues (e.g., the eye).

[0151] In some embodiments, rAAV is injected into the subject. In other embodiments, rAAV is administered to the subject by topical application (e.g., eye drops). In some embodiments, the effective amount of rAAV is sufficient to express an effective amount of an antiVEGF agent (e.g., KH902) in the subject's target tissue (e.g., the eye).

[0152] In some embodiments, the effective amount of rAAV delivered by injection (e.g., delivery of rAAV encoding an anti-VEGF agent (e.g., rAAV2.7m8-KH902) is sufficient to express an effective amount of anti-VEGF agent (e.g., KH902) in the target tissue. In some embodiments, the effective amount of rAAV encoding an anti-VEGF agent (e.g., rAAV2.7m8-KH902) is sufficient to deliver 10 μg to 10 mg or any intermediate value of anti-VEGF agent (e.g., KH902) per eye to a subject via a suitable route of administration (e.g., intraocular injection, IV injection, intraperitoneal injection, and intramuscular injection). In some embodiments, the rAAV encoding an anti-VEGF agent (e.g., KH902) (e.g., rAAV2.7m8-KH902) is sufficient to deliver 10 μg to 10 mg or any intermediate value between these values ​​to a subject per eye. An anti-VEGF agent (e.g., KH902) ranging from 20 μg to 5 mg or any intermediate value in between. In some embodiments, the rAAV encoding the anti-VEGF agent (e.g., KH902) is sufficient to deliver 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg or more of the anti-VEGF agent (e.g., KH902) to the subject per eye.

[0153] In some implementations, rAAV encoding an anti-VEGF agent (e.g., rAAV2.7m8-KH902) is administered to the subject once daily, once weekly, once every two weeks, once monthly, once every two months, once every three months, once every six months, once a year, or once in the subject's lifetime.

[0154] In some embodiments, the effective amount of rAAV delivered by topical application, such as eye drops (e.g., delivering an effective amount of rAAV encoding an anti-VEGF agent (e.g., KH902) is sufficient to express an effective amount of the anti-VEGF agent (e.g., KH902) in the target tissue). In some embodiments, eye drops containing encoded rAAV are administered to the subject once weekly, once monthly, once every 3 months, once every 6 months, or once a year.

[0155] In some embodiments, the eye drops contain an rAAV encoding an anti-VEGF agent (e.g., rAAV2.7m8-KH902) sufficient to deliver an anti-VEGF agent at a concentration of 1 mg / ml to 20 mg / ml. In some embodiments, the eye drops contain an rAAV encoding an anti-VEGF agent (e.g., rAAV2.7m8-KH902) sufficient to deliver an anti-VEGF agent at a concentration of 2.5 mg / ml to 10 mg / ml. In some embodiments, the eye drops contain an rAAV encoding an anti-VEGF agent (e.g., rAAV2.7m8-KH902) sufficient to deliver an anti-VEGF agent at concentrations of 1 mg / ml, 2 mg / ml, 2.5 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml. In some implementations, the eye drops are applied in doses of 0.01 ml, 0.02 ml, 0.03 ml, 0.04 ml, 0.05 ml, 0.06 ml, 0.07 ml, 0.08 ml, 0.09 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, or 0.5 ml.

[0156] The effective amount of rAAV (e.g., rAAV2.7m8-KH902) or a composition (e.g., a composition containing the rAAV described herein) can also depend on the method of administration. For example, in some cases, targeting ocular (e.g., corneal) tissue by intrastromal administration or subcutaneous injection may require a different (e.g., higher or lower) dose than by another method (e.g., systemic administration, topical administration). Thus, in some embodiments, the injection is an intrastromal injection (IS). In some embodiments, the injection is a topical administration (e.g., topical application to the eye). In some cases, multiple doses of rAAV (e.g., rAAV2.7m8-KH902) are administered.

[0157] In some embodiments, rAAV (e.g., rAAV2.7m8-KH902) compositions are formulated to reduce the aggregation of AAV particles in the composition, especially in the presence of high rAAV concentrations (e.g., ~10). 13 In cases where GC / mL or higher is used. Methods for reducing rAAV aggregation are well known in the art and include, for example, adding surfactants, adjusting pH, adjusting salt concentration, etc. (See, for example, Wright FR, et al., Molecular Therapy (2005) 12, 171–178, the contents of which are incorporated herein by reference.)

[0158] The formulation of pharmaceutically acceptable excipients and carrier solutions, as well as the development of suitable dosing and treatment regimens for the use of the specific compositions described herein in a variety of treatment regimens, are well known to those skilled in the art.

[0159] Typically, these formulations may contain at least about 0.1% or more of the active compound, although the percentage of the active ingredient can, of course, vary and can conveniently be between about 1 or 2% of the total weight or volume of the formulation and about 70% or 80% or more. Naturally, the amount of the active compound in each therapeutically useful composition can be prepared in a manner that would yield a suitable dose at any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be considered by those skilled in the art in preparing such pharmaceutical formulations; therefore, a variety of dosages and treatment regimens may be desirable.

[0160] In certain circumstances, it is desirable to deliver rAAV-based therapeutic constructs in suitably formulated pharmaceutical compositions disclosed herein via one of the following methods: intravitreal, intraocular, subretinal, subcutaneous, intrapancreatic, intranasal, parenteral, intravenous, intramuscular, intrathecal, oral, intraperitoneal, or inhalation. In some embodiments, rAAV can be delivered using administration methods described in U.S. Patent Nos. 5,543,158; 5,641,515 and 5,399,363 (each incorporated herein by reference in its entirety). In some embodiments, a preferred administration method is via portal vein injection.

[0161] Suitable drug forms for injection include sterile aqueous solutions or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and in oils. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. In many cases, the form is sterile and has a flowability sufficient for easy injection. It must remain stable under manufacturing and storage conditions, and its preservation must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Suitable flowability can be maintained, for example, by using coating agents such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Microbial action can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. Prolonged absorption of injectable compositions can be caused by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0162] For example, for the administration of injectable aqueous solutions, the solution may be appropriately buffered if necessary, and the liquid diluent should first be isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Sterile aqueous media that can be used in this regard are known to those skilled in the art. For example, a dose may be dissolved in 1 mL of isotonic NaCl solution and then added to 1000 mL of subcutaneous infusion or injected at the recommended infusion site (see, for example, "Remington's Pharmaceutical Sciences," 15th edition, pp. 1035-1038 and 1570-1580). Dosage variations will inevitably occur depending on the host. In any case, the person responsible for administration will determine the appropriate dose for the individual host.

[0163] A sterile injectable solution is prepared by incorporating the desired amount of active rAAV with various other ingredients listed herein (as needed) into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile medium containing a base dispersion medium and other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying techniques, which produce a powder from its previously sterile filtered solution containing the active ingredient plus any other desired ingredients.

[0164] The rAAV compositions disclosed herein can also be formulated into neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (forming with the free amino group of a protein), which form with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. Through formulation, the solution will be administered in a dosage form compatible with the dosage formulation and at a therapeutically effective amount. The formulation is readily applicable in various dosage forms, such as injectable solutions, drug-release capsules, etc.

[0165] As used herein, "carrier" includes any and all solvents, dispersion media, mediators, coating agents, diluents, antibacterial and antifungal agents, isotonic and absorption-retarding agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and reagents for pharmaceutically active substances is well known in the art. Supplemental active ingredients may also be incorporated into the composition. The phrase "pharmaceuticalally acceptable" refers to molecular entities and compositions that do not produce allergic reactions or similar adverse reactions when administered to a host.

[0166] Delivery media such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, and vesicles can be used to introduce the compositions of this disclosure into suitable host cells. In particular, transgenes delivered by rAAV vectors can be formulated for delivery encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles.

[0167] Such formulations are preferably used in pharmaceutically acceptable formulations that introduce the nucleic acid or rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Currently, liposomes with improved serum stability and circulating half-life have been developed (US Patent No. 5,741,516). Furthermore, various methods for using liposomes and liposome-like preparations as potential drug carriers have been described (US Patent Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868 and 5,795,587).

[0168] Liposomes have been successfully used in many cell types that are typically resistant to transfections performed via other steps. Furthermore, liposomes are not limited by DNA length, a limitation typically associated with virus-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapy agents, viruses, transcription factors, and allosteric effectors into a wide variety of cultured cell lines and animals. In addition, several successful clinical trials have been completed to examine the effectiveness of liposome-mediated drug delivery.

[0169] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously forming multilayered concentric bilayered vesicles (also known as multilayered vesicles (MLVs)). MLVs typically have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of vesicles with diameters ranging from 200 nm to... Small, single-layered vesicles (SUVs) within a range, whose cores contain an aqueous solution.

[0170] Alternatively, nanocapsule formulations of rAAV can be used. Nanocapsules typically capture substances in a stable and reproducible manner. To avoid side effects from intracellular polymer overload, these ultrafine particles (approximately 0.1 μm in size) should be designed using polymers that are biodegradable in vivo. Consider using biodegradable alkyl cyanoacrylate nanoparticles that meet these requirements.

[0171] In addition to the delivery methods described above, the following technologies are considered as alternatives for delivering rAAV compositions to the host. Ultrasound delivery (i.e., ultrasound) has been used and described in U.S. Patent No. 5,656,016 as a device to improve the rate and efficacy of drug penetration into and through the circulatory system. Other drug delivery alternatives considered are intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic preparations (Bourlais et al., 1998), transdermal matrix (U.S. Patent Nos. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0172] In some embodiments, the anti-VEGF agent described herein (e.g., KH902) is delivered to the subject via ceDNA. Any composition containing ceDNA encoding an anti-VEGF agent (e.g., KH902) is also within the scope of this disclosure. In some embodiments, ceDNA encoding an anti-VEGF agent (e.g., KH902) and compositions thereof may be administered to the subject using any suitable method described herein. In some embodiments, the effective amount of ceDNA encoding an anti-VEGF agent (e.g., KH902) delivered by injection is an amount sufficient to express an effective amount of the anti-VEGF agent (e.g., KH902) in the target tissue. In some embodiments, the delivery of an effective amount of ceDNA encoding an anti-VEGF agent (e.g., KH902) is sufficient to deliver 10 μg to 10 mg or any intermediate value between these amounts to the subject per eye via a suitable route of administration (e.g., intraocular injection, IV injection, intraperitoneal injection, and intramuscular injection). In some aspects, this disclosure relates to knowing that a potential side effect of administering AAV to a subject is an immune response to AAV in the subject, including inflammation. In some embodiments, the subject is immunosuppressed prior to administration of one or more rAAVs as described herein.

[0173] As used herein, “immunosuppressive” or “immunosuppression” refers to the activation or reduced efficacy of an immune response in a subject. Immunosuppression can be induced in a subject using one or more agents (e.g., multiple, such as 2, 3, 4, 5 or more), including but not limited to rituximab, methylprednisolone, prednisolone, sirolimus, immunoglobulin injection, prednisone, solu-Medrol, lansoprazole, trimethoprim / sulfamethoxazole, methotrexate, and any combination thereof. In some embodiments, the immunosuppressive regimen includes administration of sirolimus, prednisolone, lansoprazole, trimethoprim / sulfamethoxazole, or any combination thereof.

[0174] In some embodiments, the method described in this disclosure further includes the step of inducing immunosuppression in the subject (e.g., administering one or more immunosuppressants) prior to administration of rAAV (e.g., the rAAV or pharmaceutical composition described in this disclosure). In some embodiments, immunosuppression (e.g., inducing immunosuppression in the subject) is performed on the subject between approximately 30 days and approximately 0 days prior to administration of rAAV (e.g., any time within 30 days prior to administration of rAAV, including endpoints). In some embodiments, the subject is pretreated with an immunosuppressant (e.g., rituximab, sirolimus, and / or prednisone) for at least 7 days.

[0175] In some embodiments, the methods described in this disclosure further include co-administering or pre-administering the agent to a subject who has been given an rAAV of this disclosure (e.g., rAAV2.7m8-KH902) or a pharmaceutical composition containing rAAV. In some embodiments, the agent is selected from miglutar, Keppra, lansoprazole, clonazepam, and any combination thereof. In some embodiments, the rAAV (e.g., the rAAV for KH902) and the additional agent may be delivered to the subject in any order. In some embodiments, the rAAV (e.g., the rAAV for KH902) and the additional agent (e.g., miglutar, Keppra, lansoprazole, clonazepam) are delivered to the subject simultaneously. In some embodiments, rAAV (e.g., rAAV for KH902) and additional agents (e.g., miglitol, keppra, lansoprazole, clonazepam) are co-administered to the subject (e.g., in one composition or in different compositions). In some embodiments, rAAV (e.g., rAAV for KH902) is delivered before the additional agents (e.g., miglitol, keppra, lansoprazole, clonazepam). In some embodiments, rAAV (e.g., rAAV for KH902) is delivered after the additional agents (e.g., miglitol, keppra, lansoprazole, clonazepam). In some implementations, rAAV (e.g., rAAV for KH902) and additional agents (e.g., miglitol, keppra, lansoprazole, clonazepam) are delivered to the subject at different frequencies. For example, the subject receives rAAV (e.g., for KH902) every month, every two months, every six months, every year, every two years, every three years, every five years or longer, but receives additional agents (e.g., miglitol, keppra, lansoprazole, clonazepam) daily, weekly, every two weeks, monthly, twice daily, three times daily or twice weekly.

[0176] In some embodiments, immunosuppression in the subject is maintained during and / or after administration of rAAV (e.g., rAAV2.7m8-KH902) or the pharmaceutical composition. In some embodiments, immunosuppression in the subject is maintained for a period of 1 day to 1 year after administration of rAAV or the pharmaceutical composition (e.g., administration of one or more immunosuppressants).

[0177] Treatment methods for diseases related to VEGF and / or angiogenesis

[0178] Several aspects of this disclosure relate to rAAV (e.g., rAAV2.7m8-KH902)-mediated methods of delivering a transgene encoding an anti-VEGF agent (e.g., KH902) to a subject (e.g., cells in the subject). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. Non-limiting examples of non-human mammals are mice, rats, cats, dogs, sheep, rabbits, horses, cattle, goats, pigs, guinea pigs, hamsters, chickens, turkeys, or non-human primates.

[0179] In some embodiments, this disclosure relates to a method for inhibiting VEGF activity in a subject with this need. In some embodiments, the methods described in this disclosure can be used to treat subjects who have or are suspected of having VEGF-related diseases. As used herein, "VEGF-related diseases" refers to a group of diseases associated with abnormal VEGF activity / signaling. VEGF is a cell-produced signaling protein that stimulates angiogenesis. VEGF is a known angiogenesis-inducing factor. In some embodiments, the methods described in this disclosure can be used to treat subjects who have or are suspected of having angiogenesis-related diseases. As used herein, angiogenesis-related diseases refer to diseases associated with abnormal angiogenesis. Non-limiting exemplary angiogenesis-related diseases include angiogenesis-dependent cancers, including, for example, angiogenesis-related eye diseases, solid tumors (e.g., lung cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, head and neck cancer, colon cancer, melanoma), hematogenous tumors such as leukemia, metastatic tumors, benign tumors (e.g., hemangioma, acoustic neuroma, neurofibroma, granular conjunctivitis, and pyogenic granuloma), rheumatoid arthritis, psoriasis, rubeosis, Osier-Webber syndrome, myocardial angiogenesis, plaque angiogenesis, telangiectasia, hemophilic arthritis, or angiofibroma.

[0180] In some implementations, angiogenesis-related eye diseases include, but are not limited to, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal transplant rejection, neovascular glaucoma and retrolental fibrosis, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwearing, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, Sjögren's syndrome, rosacea, phylectenulosis, syphilis, mycobacterial infection, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infection, herpes zoster infection, protozoan infections, Kaposi's sarcoma, and mooren's keratitis. (ulcer), Terrien's marginal degeneration of the cornea, marginal keratolysis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson syndrome, pemphigoid radial keratotomy, and corneal transplant rejection, sickle cell anemia, sarcoma, pseudoxanthoma elastica, Paget's disease, venous occlusion, arterial occlusion, carotid atresia, chronic uveitis / hyalitis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, Eales' disease, Behcet's disease, infections causing retinitis or choroiditis, presumed ocular histoplasmosis, Bests' disease, myopia, congenital optic pits, Stargardt's disease, pars plana cyclitis, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, and complications after trauma or laser surgery.

[0181] As used herein, the term "treatment" refers to the application or administration of a composition comprising an anti-VEGF agent (e.g., KH902) to a subject suffering from symptoms or diseases associated with abnormal VEGF activity or angiogenesis, or who is susceptible to diseases associated with abnormal VEGF activity or angiogenesis, with the aim of treating, curing, alleviating, relieving, altering, remedying, improving, refining, or influencing the symptoms of the condition or disease, or the susceptibility to diseases associated with abnormal VEGF activity or angiogenesis. In some embodiments, administration of the anti-VEGF agent results in a 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in VEGF activity compared to a reference value. Methods for measuring VEGF activity are known in the art. A non-limiting exemplary reference value may be the VEGF activity of the same subject prior to treatment with the anti-VEGF agent. In some implementations, administration of an anti-VEGF agent results in a 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in angiogenesis compared to a reference value. Methods for measuring angiogenesis are known in the art. A non-limiting exemplary reference value could be the angiogenesis level of the same subject prior to receiving anti-VEGF agent treatment.

[0182] Relieving disease associated with abnormal VEGF activity or angiogenesis involves delaying disease development or progression, or reducing disease severity. Disease relief does not necessarily require a curative outcome. As used herein, “delaying” the development of a disease (e.g., a disease associated with abnormal VEGF activity or angiogenesis) means postponing, hindering, slowing, delaying, stabilizing, and / or postponing disease progression. This delay can be of varying lengths, depending on the disease history and / or the individual receiving treatment. Methods of “delaying” or mitigating disease development or delaying disease onset involve reducing the likelihood of developing one or more symptoms of the disease within a given timeframe and / or reducing the severity of symptoms within a given timeframe, compared to not using such methods. Such comparisons are typically based on clinical studies using a sufficient number of participants to yield statistically significant results.

[0183] The term "development" or "progression" of a disease refers to the initial presentation and / or subsequent progression of the disease. Disease development can be detected and assessed using standard clinical techniques known in the art. However, development also refers to progression that may be undetectable. For the purposes of this disclosure, development or progression refers to the biological process of symptoms. "Development" includes occurrence, relapse, and onset. As used herein, an "onset" or "occurrence" of a disease associated with abnormal VEGF activity or angiogenesis includes an initial onset and / or relapse. Example

[0184] Example 1: AAV 2.7m8 delivery of Conbercept (KH902)

[0185] Conbercept (KH902) is an anti-VEGF therapeutic agent delivered to the retina via intravitreal administration (or other routes) using recombinant adeno-associated virus. Its unique design comprises a single-stranded AAV vector genome containing the KH902 transgene driven by a CMV enhancer / chicken β-actin promoter regulatory cassette. Figure 1A-1C A Kozak sequence was designed upstream of the KH902 start codon to enhance translation. Figure 1C When cis plasmid ( Figure 1A When delivered to packaging cell lines expressing AAV Rep and Cap genes and mandatory helper genes via trans-plasmid co-transfection or by stable integration, the inverted terminal repeat (ITR) sequence and its side-joined sequence are packaged into AAV2.7m8 capsid virion.

[0186] Cells were infected or transduced with the resulting ssAAV2.7m8-KH902 virion expressing and secreting KH902, and the results were detected by standard Western blot analysis. Figure 2 Transduction of retinal pigment epithelium (RPE) cell lines with ssAAV-KH902 encapsulated in an AAV2.7m8 capsid resulted in KH902 protein expression with a molecular weight similar to that of conbercept produced in the Chinese hamster ovary (CHO) cell line. These data indicate that the rAAV-KH902 vector can be packaged into different AAV capsids and can efficiently secrete KH902 upon infection into cells.

[0187] Conditioned culture medium from RPE cells infected with rAAV2.7m8-KH902 strongly inhibited angiogenesis, such as vascular endothelial growth factor (VEGF)-induced renal tubule formation. Figure 3A and 3B The reduction of ) and the proliferation of human umbilical vein endothelial cells (HUVECs) in the same manner as conbercept (CCK-8, Figure 3C As shown in the figure. This data indicates that cells infected with rAAV-KH902 can express and secrete functional anti-angiogenic KH902 in vitro.

[0188] Example 2: Intravitreal injection of AAV2.7m8-KH902 effectively delivers KH902 to prevent oxygen-induced oxygen deficiency in mice. Retinopathy and angiogenesis

[0189] Newborn mice were treated with the vector via intravitreal injection 1–3 days after birth (PN). One eye of each mouse was treated with the vector (rAAV2.7m8-Egfp) packaging the Egfp transgene, and the other eye was treated with a 5:1 mixture of the vector (rAAV2.7m8-KH902) packaging the KH902 transgene and rAAV2.7m8-Egfp. In all cases, the total dose was 1.5E per eye. 9 The volume of the vaccine was 1 μL. Mice were then kept under 70% oxygen until PN 7, and then placed under normoxic conditions (20-21% oxygen) until PN 11. Mice were sacrificed at PN 18, and their eyes were harvested and observed. Figures 4A-4B Then, the pathology of the treated eye was scored and assessed through visual examination. Figure 5 Eyes treated solely with rAAV-Egfp can indicate the degree of hyperoxia induction and serve as an internal control for pathological changes. It should be noted that the absence of edema does not imply that hyperoxia cannot induce retinopathy, and the presence of edema in eyes treated with rAAV2.7m8-KH902 does not necessarily mean the carrier is ineffective. Salvage of vascular pathology depends on the presence of aneurysmal nodules.

[0190] In eyes treated with rAAV2.7m8-Egfp as a negative control, vascular pathology was observed due to excessive proliferation and the formation of vascular aneurysm nodules. Figure 4A Eyes treated with rAAV2-KH902 effectively prevented pathological changes. Figure 4B (See the small image on the right), and it also reduces vascular development to some extent. Figure 6B (See the small image on the right). Similarly, rAAV2.7m8-KH902 effectively prevented vascular pathology ( Figure 5 However, rAAV2.7m8-KH902 also appears to effectively inhibit normal angiogenesis, with transduction being the highest. Consequently, these mice exhibited a higher frequency of edema. Figure 5 It is speculated that strong inhibition of major blood vessels during development appears to lead to vascular sprouting in small localized areas.

[0191] This data suggests that AAV2.7m8-KH902 is a potentially viable gene therapy platform for the prevention and possible reversal of choroidal angiogenesis.

[0192] Example 3: Efficacy and toxicity of rAAV2.7m8-KH902

[0193] The efficacy of rAAV-KH902 was investigated in a laser-induced injury treatment model. Throughout a 15-day post-injection observation window, treatment with a 1:10 dilution (3E8 vg / eye) of AAV2.7m8-KH902 after laser injury reduced the percentage of choroidal angiogenesis (CNV) to a similar degree to that with the undiluted carrier (3E9 vg / eye). Importantly, this result was achieved without inducing a vasculitis-like phenotype induced by KH902 treatment.

[0194] The efficacy of rAAV-KH902 was quantified by the percentage of residual CNV in the eyes of mice that had been laser-damaged and subsequently treated with doses of rAAV2.7m8-KH902 at doses of 3E9 (undiluted), 3E8 (1:10 dilution), and 1.5E8 (1:20 dilution) vg / eye 5 days post-damage. Figure 6A At 20 days post-injury, the percentage of residual CNV was 49% after the 3E9 vg / eye dose, compared to 71% and 74% after the 3E8 and 1.5E8 doses, respectively. The 3E8 and 1.5E8 doses showed improved CNV retention at 15 days post-treatment (20 days post-injury). Figure 6A The ability to reduce CNV is relatively equal.

[0195] High doses of AAV-KH902 induce a “vasculitis” effect in the eye, manifested by the infiltration of immune cells into the retina. A dose of 3E9 vg / eye of AAV2.7m8-KH902 can induce vasculitis up to 4 weeks after injection. Cross-sections were taken and evaluated under an immunofluorescence microscope to identify the cell types infiltrating the transduced retina. Figure 6B The study showed immune cell infiltration and platelet aggregation in the retinal ganglion cell layer after treatment with AAV2.7m8-KH902. Subsequently, the ability of rAAV2.7m8-KH902 to induce vasculitis in a flat, mounted slide under immunofluorescence microscopy was assessed in parallel with a lower dose (1:10 dilution, 3E8 vg / eye) compared to a dose of 3E9 vg / eye. The retina was stained with a series of immune cell markers (…). Figure 6C As expected, 3E8 did not cause infiltration, consistent with the lack of vasculitis phenotype observed at this dose.

[0196] Furthermore, KH902 expression levels were assessed after rAAV2.7m8-KH902 injection to measure the kinetics of transgene efficacy, CNV reduction, and transgene-induced vascular phenotype. ddPCR was performed to quantify the relative expression of KH902 in eyes treated with AAV2.7m8-KH902 (3E9 vg / eye). Figure 6D KH902 expression approximately doubled between week 1 and week 8, and its level appeared to reach a steady state by week 6.

[0197] Equivalent scheme

[0198] Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages described herein, and each of these variations and / or modifications is considered to be within the scope of the invention. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are exemplary, and actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications in which the teachings of this invention are used. Those skilled in the art will recognize or be able to determine many equivalent embodiments of the specific embodiments of the invention described herein using experiments not exceeding conventional methods. Therefore, it should be understood that the foregoing embodiments are presented by way of example only, and that the invention may be practiced in ways other than those specifically described and claimed within the scope of the appended claims and their equivalents. The invention relates to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the invention if such features, systems, articles, materials, and / or methods do not contradict each other.

[0199] Unless explicitly stated otherwise, the indefinite article “a (type)” used herein in the specification and claims shall be understood to mean “at least one (type)”.

[0200] The phrase “and / or” as used herein in the specification and claims should be understood to mean “one or both” of the elements so combined, i.e., the elements coexist in some cases and exist separately in others. Other elements may optionally exist in addition to those specifically identified by the “and / or” clause, whether related to or unrelated to those specifically identified, unless explicitly stated otherwise. Thus, as a non-limiting example, when used in conjunction with open-ended language such as “comprising,” a reference to “A and / or B” may in one embodiment refer to A without B (optionally including elements other than B); in another embodiment, refer to B without A (optionally including elements other than A); in yet another embodiment, refer to both A and B (optionally including other elements); and so on.

[0201] As used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted as inclusive, i.e., including many elements or at least one of the elements in the list, but also including more than one, as well as optional other unlisted items. Only terms that explicitly indicate the opposite, such as “only one” or “solely one,” or when used in a claim, “consisting of…” will refer to including many elements or the sole element in the list. In general, before exclusive terms such as “any,” “one of,” “only one,” or “solely one,” the term “or” as used herein should only be interpreted as an alternative to exclusivity (i.e., “one or the other, not two”). “Substantially consisting of…” when used in a claim should have its ordinary meaning as used in the field of patent law.

[0202] As used herein in the specification and claims, the phrase “at least one” in relation to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one element from every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements referred to by the phrase “at least one,” whether related to or unrelated to those specifically identified elements. Thus, as a non-limiting example, “at least one of A and B” (or equivalently “at least one of A or B”; or equivalently “at least one of A and / or B”) in one embodiment may mean at least one, optionally including more than one A but without B (and optionally including elements other than B); in another embodiment, it means at least one, optionally including more than one B but without A (and optionally including elements other than A); in yet another embodiment, it means at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0203] In the claims and in the foregoing description, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” etc., shall be understood as open-ended, meaning including but not limited to. Only the transitional phrases “consisting of” and “consisting substantially of” shall be closed or semi-closed transitional phrases, respectively, as described in Section 2111.03 of the U.S. Patent Examination Procedure Manual.

[0204] The use of sequential terms such as “first,” “second,” and “third” to modify a claim element does not imply any priority, order, or temporal sequence of actions of one claim element relative to another claim element. Rather, it serves merely as a label to distinguish one claim element with a specific name from another element with the same name (but used in sequential terms) to differentiate claim elements. sequence list <110> University of Massachusetts Chengdu Kanghong Biotechnology Co., Ltd. <120> Recombinant adeno-associated virus for delivering KH902 (Conbercept) and its uses <130> U0120.70127WO00 <140> Not specified <141> Provided in conjunction with the above content <150> US 62 / 940,288 <151> 2019-11-26 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 1659 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 1 atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc 60 acaggatcta gttccggagg tagacctttc gtagagatgt acagtgaaat ccccgaaatt 120 atacacatga ctgaaggaag ggagctcgtc attccctgcc gggttacgtc acctaacatc 180 actgttactt taaaaaagtt tccactgac actgatcc ctgatggaaa acccataatc 240 tgggacagta gaaagggctt catcatatca atgcaacgt aaagaat agggctctg 300 acctgtgaag actagacaa actactcac acatcgacaa 360 accaatacaa tcatagatgt ggttctgagt ccgtctcatg gattgaact atctgttgga 420 gaaagcttg tcttaatttg tacagcaga actgaacta atgtgggat tgactcaac 480 tgggaatacc cttcttcgaa gcatcagcat aagaaacttg taaaccgaga cctaaaaacc 540 cagtctggga gtgagatgaa gaattttg agcaccttaa ctatagatgg tgtaacccgg 600 agtgaccaag gattgtacac ctgtgcagca tccagtgggc tgatgaccaa gagaacagc 660 acatttgtca gggtccatga aaaacctttt gttgctttg gaagtggcat ggaatctctg 720 gtggaagcca cggtggggga gcgtgtcaga atccctgcga agtaccttgg ttacccaccc 780 ccagaaataa aatgtataa aaatgaata ccccttgagt ccaatcacac aattaagcg 840 gggcatgtac tgacgattat ggaagtgagt gaagagaca caggaatta cactgtcatc 900 cttaccaatc ccatttcaaa ggagaagcag agccatgtgg tctctctggt tgtgtatgtc 960 ccaccgggcc cgggcgacaa aactcacaca tgcccactgt gcccagcacc tgaactcctg 1020 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 1080 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 1140 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1200 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1260 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1320 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1380 gatgagctga ccaagaacca ggtcagcctg acctgcctag tcaaaggctt ctatcccagc 1440 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa ggccacgcct 1500 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1560 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1620 tacacgcaga agagcctctc cctgtctccg ggtaaatga 1659 <210> 2 <211> 4011 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 2 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180 atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600 gttctgcttc actctcccca tctcccccccc ctccccaccc ccaattttgt atttattat 660 tttttaatta ttttgtgcag cgatggggc gggggggggg ggggggcgcg cgccaggcgg 720 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780 gagcggcgcg ctccgaaagt ttcctttat ggcgaggcgg cggcggcggc ggccctataa 840 aaagcgaagc gcgcggcggg cggggagtcg ctgcgacgct gccttcgccc cgtgccccgc 900 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 960 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 1020 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1080 gggagcggct cgggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc 1140 gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt 1200 gtgcgcgagg ggagcgcggc cggggcggt gccccgcggt gcgggggggg ctgcgagggg 1260 aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg 1320 gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg 1380 ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg 1440 caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg 1500 cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt 1560 ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg 1620 aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc 1680 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc 1740 cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg 1800 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat 1860 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat 1920 tttggcaaag aattcgccac catggtcagc tactgggaca ccggggtcct gctgtgcgcg 1980 ctgctcagct gtctgcttct cacaggatct agttccggag gtagaccttt cgtagagatg 2040 tacagtgaaa tccccgaaat tatacacatg actgaaggaa gggagctcgt cattccctgc 2100 cgggttacgt cacctaacat cactgttact ttaaaaaagt ttccacttga cactttgatc 2160 cctgatggaa aacgcataat ctgggacagt agaaagggct tcatcatatc aaatgcaacg 2220 tacaaagaaa tagggcttct gacctgtgaa gcaacagtca atgggcattt gtataagaca 2280 aactatctca cacatcgaca aaccaataca atcatagatg tggttctgag tccgtctcat 2340 ggaattgaac tatctgttgg agaaaagctt gtcttaaatt gtacagcaag aactgaacta 2400 aatgtgggga ttgacttcaa ctgggaatac ccttcttcga agcatcagca taagaaactt 2460 gtaaaccgag acctaaaaac ccagtctggg agtgagatga agaaattttt gagcacctta 2520 actatagatg gtgtaacccg gagtgaccaa ggattgtaca cctgtgcagc atccagtggg 2580 ctgatgacca agaagaacag cacatttgtc agggtccatg aaaaaccttt tgttgctttt 2640 ggaagtggca tggaatctct ggtggaagcc acggtggggg agcgtgtcag aatccctgcg 2700 aagtaccttg gttacccacc cccagaaata aaatggtata aaaatggaat accccttgag 2760 tccaatcaca caattaaagc ggggcatgta ctgacgatta tggaagtgag tgaaagagac 2820 acaggaaatt acactgtcat ccttaccaat cccatttcaa aggagaagca gagccatgtg 2880 gtctctctgg ttgtgtatgt cccaccgggc ccgggcgaca aaactcacac atgcccactg 2940 tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 3000 gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 3060 gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 3120 acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 3180 ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 3240 ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 3300 tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgccta 3360 gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 3420 aacaactaca aggccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 3480 aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 3540 catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 3600 acgcgtggta cctctagagt cgacccgggc ggcctcgagg acggggtgaa ctacgcctga 3660 ggatccgatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat 3720 ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg 3780 tgtctctcac tcggaagcaa ttcgttgatc tgaatttcga ccacccataa tacccattac 3840 cctggtagat aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga 3900 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 3960 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca g 4011 <210> 3 <211> 6832 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 3 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc cgggcgtcg ggcgacctt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180 atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt attacggta 420 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600 gttctgcttc actctcccca tctcccccc ctccccaccc ccaatttgtgt atttatttat 660 ttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840 aaagcgaagc gcgcggcggg cggggagtcg ctgcgacgct gccttcgccc cgtgccccgc 900 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 960 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 1020 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1080 gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc 1140 gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt 1200 gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg 1260 aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg 1320 gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg 1380 ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg 1440 caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggaggctc gggggagggg 1500 cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt 1560 ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg 1620 aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc 1680 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc 1740 cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg 1800 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat 1860 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat 1920 tttggcaaag aattcgccac catggtcagc tactgggaca ccggggtcct gctgtgcgcg 1980 ctgctcagct gtctgcttct cacaggatct agttccggag gtagaccttt cgtagagatg 2040 tacagtgaaa tccccgaaat tatacacatg actgaaggaa gggagctcgt cattccctgc 2100 cgggttacgt cacctaacat cactgttact ttaaaaaagt ttccacttga cactttgatc 2160 cctgatggaa aacgcataat ctgggacagt agaagggct tcatcatatc aaatgcaacg 2220 tacaagaaaa taggctctt gacctgtgaa gcaacagtca atgggcatttt gtataagaca 2280 aactatctca cacatcgaca aaccataca atcatagatg tggttctgag tccgtctcat 2340 ggaattgaac tatctgttgg agaaagctt gtcttaaatt gtacagcaag aactgaacta 2400 aatgtgggga ttgactcaa ctgggaatac ccttctcga agcatcagca tagaaactt 2460 gtaaaccgag acctaaaaac ccagtctggg agtgagatga agaattttt gagcacctta 2520 actatagatg gtgtaacccg gagtgaccaa ggattgtaca cctgtgcagc atccagtggg 2580 ctgatgacca agaagacag cacattgtc agggtccatg aaaaaccttt tgttgctttt 2640 ggaagtggca tggaatctct ggtggaagcc acggtggggg agcgtcag aatccctgcg 2700 aagtaccttg gttaccacc cccagaata aaatgtata aaatggaat accccttgag 2760 tccaatcaca cattaagc ggggcatgta ctgacgatta tggagtgag tgaagagac 2820 acaggaaatt acactgtcat ccttaccaat cccattcaa aggagaagca gagccatgtg 2880 gtctctctgg ttgtgtatgt cccaccgggc ccgggcgaca aaactcacac atgcccactg 2940 tgcccagcac ctgaactcct gggggaccg tcagtcttcc tcttcccccc aaaacccaag 3000 gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 3060 gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 3120 acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 3180 ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 3240 ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 3300 tacaccctgc cccacatcccg ggatgagctg accaagaacc aggtcagcct gacctgccta 3360 gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 3420 aacaactaca aggccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 3480 aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 3540 catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 3600 acgcgtggta cctctagagt cgacccgggc ggcctcgagg acggggtgaa ctacgcctga 3660 ggatccgatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat 3720 ctgacttctg gctaataag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg 3780 tgtctctcac tcggaagcaa ttcgttgatc tgaatttcga ccaccataa tacccattac 3840 cctggtagat aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga 3900 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 3960 ccgacgccccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gccttaatta 4020 acctaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca 4080 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg 4140 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag 4200 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 4260 cgccctagcg cccgctcctt tcgctttctt cccttcctt ctcgccacgt tcgccggctt 4320 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg cttacggca 4380 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata 4440 gacggtttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 4500 aactggaaca acactcaacc ctatctcggt ctattcttttt gatttataag ggattttgcc 4560 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 4620 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct 4680 atttgttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 4740 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 4800 cttattccct ttttgcggc atttgcctt cctgtttttg ctcacccaga aacgctggtg 4860 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 4920 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 4980 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 5040 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 5100 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 5160 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 5220 5280 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 5340 5400 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 5460 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 5520 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 5580 gacgaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 5640 gaccaagttt actcatatat actttaagatt gatttaaaac ttcattttta atttaaaaagg 5700 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 5760 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 5820 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 5880 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 5940 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 6000 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 6060 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 6120 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 6180 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 6240 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 6300 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 6360 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 6420 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 6480 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 6540 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 6600 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 6660 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 6720 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 6780 ggaaacagct atgaccatga ttacgccaga tttaattaag gccttaatta gg 6832 <210> 4 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <220> <221> misc_feature <222> (4)..(4) <223> Where n is adenine or cytosine. <400> 4 gccncc 6 <210> 5 <211> 552 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 5 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Gly Arg Pro Phe Val Glu 20 25 30 Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu 35 40 45 Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu 50 55 60 Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile 65 70 75 80 Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu 85 90 95 Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys 100 105 110 Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val 115 120 125 Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val 130 135 140 Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn 145 150 155 160 Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg 165 170 175 Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr 180 185 190 Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys 195 200 205 Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg 210 215 220 Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu 225 230 235 240 Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu 245 250 255 Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu 260 265 270 Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu 275 280 285 Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro 290 295 300 Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val Val Tyr Val 305 310 315 320 Pro Pro Gly Pro Gly Asp Lys Thr His Thr Cys Pro Leu Cys Pro Ala 325 330 335 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 340 345 350 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 355 360 365 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 370 375 380 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 385 390 395 400 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 405 410 415 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 420 425 430 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 435 440 445 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 450 455 460 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 465 470 475 480 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 485 490 495 Lys Ala Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 500 505 510 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 515 520 525 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 530 535 540 Ser Leu Ser Leu Ser Pro Gly Lys 545 550 <210> 6 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 6 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Gly Arg Pro Phe Val Glu 20 25 30 Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu 35 40 45 Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu 50 55 60 Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile 65 70 75 80 Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu 85 90 95 Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys 100 105 110 Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val 115 120 125 <210> 7 <211> 195 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 7 Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu 1 5 10 15 Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe 20 25 30 Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn 35 40 45 Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser 50 55 60 Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr 65 70 75 80 Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val 85 90 95 Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser 100 105 110 Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr 115 120 125 Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro 130 135 140 Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr Ile Met 145 150 155 160 Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn 165 170 175 Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val Val Tyr 180 185 190 Val Pro Pro 195 <210> 8 <211> 322 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Gly Arg Pro Phe Val Glu 20 25 30 Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu 35 40 45 Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu 50 55 60 Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile 65 70 75 80 Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu 85 90 95 Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys 100 105 110 Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val 115 120 125 Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val 130 135 140 Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn 145 150 155 160 Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg 165 170 175 Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr 180 185 190 Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys 195 200 205 Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg 210 215 220 Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu 225 230 235 240 Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu 245 250 255 Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu 260 265 270 Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu 275 280 285 Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro 290 295 300 Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val Val Tyr Val 305 310 315 320 Pro Pro <210> 9 <211> 357 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 9 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Gly Arg Pro Phe Val Glu 20 25 30 Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu 35 40 45 Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu 50 55 60 Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile 65 70 75 80 Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu 85 90 95 Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys 100 105 110 Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Gly 115 120 125 Pro Gly Asp Lys Thr His Thr Cys Pro Leu Cys Pro Ala Pro Glu Leu 130 135 140 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 145 150 155 160 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 165 170 175 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 180 185 190 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 195 200 205 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 210 215 220 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 225 230 235 240 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 245 250 255 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 260 265 270 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 275 280 285 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala Thr 290 295 300 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 305 310 315 320 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 325 330 335 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 340 345 350 Leu Ser Pro Gly Lys 355 <210> 10 <211> 425 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 10 Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu 1 5 10 15 Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe 20 25 30 Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn 35 40 45 Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser 50 55 60 Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr 65 70 75 80 Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val 85 90 95 Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser 100 105 110 Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr 115 120 125 Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro 130 135 140 Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr Ile Met 145 150 155 160 Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn 165 170 175 Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val Val Tyr 180 185 190 Val Pro Pro Gly Pro Gly Asp Lys Thr His Thr Cys Pro Leu Cys Pro 195 200 205 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 210 215 220 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 225 230 235 240 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 245 250 255 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 260 265 270 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 275 280 285 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 290 295 300 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 305 310 315 320 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 325 330 335 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 340 345 350 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 355 360 365 Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 370 375 380 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 385 390 395 400 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 405 410 415 Lys Ser Leu Ser Leu Ser Pro Gly Lys 420 425 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 11 Leu Gly Glu Thr Thr Arg Pro 1 5 <210> 12 <211> 735 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 12 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190 Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445 Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 450 455 460 Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly 465 470 475 480 Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn 485 490 495 Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505 510 Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp 515 520 525 Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys 530 535 540 Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr 545 550 555 560 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575 Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr 580 585 590 Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp 595 600 605 Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr 610 615 620 Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys 625 630 635 640 His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn 645 650 655 Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln 660 665 670 Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys 675 680 685 Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr 690 695 700 Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr 705 710 715 720 Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 13 <211> 745 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 13 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190 Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Thr 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445 Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 450 455 460 Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly 465 470 475 480 Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn 485 490 495 Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505 510 Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp 515 520 525 Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys 530 535 540 Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr 545 550 555 560 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575 Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Leu Gly Glu 580 585 590 Thr Thr Arg Pro Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln 595 600 605 Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln 610 615 620 Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro 625 630 635 640 Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile 645 650 655 Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser 660 665 670 Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val 675 680 685 Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp 690 695 700 Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Ile Asn Val 705 710 715 720 Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile 725 730 735 Gly Thr Arg Tyr Leu Thr Arg Asn Leu 740 745 <210> 14 <211> 2238 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 14 atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60 cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120 gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180 aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctatgac 240 cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300 caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360 gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420 ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480 aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540 tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600 aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660 gtgggtaatt cctcgggaaa ttggcattgc gatccacat ggatgggcga cagagtcacc 720 accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaaacaaatt 780 tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840 tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900 aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960 aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020 caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080 tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140 aaaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200 cagatgctgc gtaccggaaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260 cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320 tacctgtatt acttgagcag aaaacact ccaagtggaa ccaccacgca gtcaaggctt 1380 cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440 ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500 tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560 ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620 atctttggga agcaaggctc agaaaaca aatgtggaca ttgaaaaggt catgattaca 1680 gacgaagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740 accaacctcc agagaggcaa cctagcactc ggcgaaacaa caagacctgc taggcaagca 1800 gctaccgcag atgtcaacac acaaggcgtt cttccaggca tggtctggca ggacagagat 1860 gtgtaccttc aggggcccat ctgggcaaag attccacaca cggacggaca ttttcacccc 1920 tctcccctca tgggtggatt cggacttaaa caccctcctc cccagattct catcaagaac 1980 accccggtac ctgcgaatcc ttcgaccacc ttcagtgcgg caaagtttgc ttccttcatc 2040 acacagtact ccacgggaca ggtcagcgtg gagatcgagt gggagctgca gaaggaaaac 2100 agcaaacgct ggaatcccga aattcagtac acttccaact acaacaagtc tattaatgtg 2160 gactttactg tggacactaa tggcgtgtat tcagagcctc gccccattgg caccagatac 2220 ctgactcgta atctgtaa 2238

Claims

1. A recombinant adeno-associated virus (rAAV) comprising: (i) rAAV capsid protein, wherein the capsid protein is AAV2.7m8; and (ii) Nucleic acids, which contain the following in the 5' to 3' sequence: (a) 5' AAV ITR; (b) CMV enhancer; (c) CBA starter; (d) Chicken β-actin introns; (e) Kozak sequence; (f) A transgene encoding an anti-VEGF agent, wherein the anti-VEGF agent is encoded by the nucleic acid sequence in SEQ ID NO: 1; (g) Rabbit β-globin polyA signal tail; and (h) 3' AAV ITR.

2. The rAAV of claim 1, wherein the AAV ITR has a serotype selected from AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR and AAV6 ITR.

3. The rAAV of claim 1, comprising the nucleic acid sequence shown in SEQ ID NO:

2.

4. The rAAV as described in claim 1, wherein the rAAV is a single-chain AAV (ssAAV).

5. A host cell comprising rAAV according to any one of claims 1-4, wherein the host cell is cultured in vitro and does not have the ability to develop into a complete plant or animal.

6. The host cell of claim 5, wherein the host cell is a mammalian cell, yeast cell, bacterial cell, or insect cell.

7. A pharmaceutical composition comprising rAAV according to any one of claims 1-4 or the host cell according to claim 5 or 6.

8. The pharmaceutical composition of claim 7, further comprising a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 7 or 8, wherein the pharmaceutical composition is formulated for intravitreal injection, intraocular injection or topical application.

10. Use of a therapeutically effective amount of the rAAV of any one of claims 1-4, the host cell of any one of claims 5 or 6, or the pharmaceutical composition of any one of claims 7-9 in the preparation of a medicament for treating angiogenesis-related diseases, vascularization-related diseases, or VEGF-related diseases in a subject with such need, wherein the angiogenesis-related disease, vascularization-related disease, or VEGF-related disease is retinopathy, wet age-related macular degeneration (wAMD), macular edema, choroidal angiogenesis, or corneal angiogenesis, and wherein the subject is a mouse or a human.

11. The use as described in claim 10, wherein the subject is a human.

12. The use as described in claim 10 or 11, wherein the drug is formulated for direct application to ocular tissue.

13. The use as described in claim 12, wherein the drug is formulated for direct application to ocular tissue via intravitreal injection, intraocular injection, or topical application.

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