RU2846105C1 - Adeno-associated virus for kh902 (conbercept) delivery and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: what is described is a recombinant adeno-associated virus (rAAV) for transgene expression, an anti-vascular endothelial growth factor (VEGF) coding agent containing: (i) an AAV capsid protein, where the capsid protein is an AAV8 capsid protein or a variant thereof, an AAV2 capsid protein, or an AAV2/3 hybrid capsid protein; and (ii) an isolated nucleic acid comprising a transgene encoding an anti-vascular endothelial growth factor (anti-VEGF) agent, wherein transgene is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITR), the transgene comprising the nucleic acid sequence SEQ ID NO: 1. Invention provides high efficiency of biological process in bioreactor system. Disclosed is a recombinant adeno-associated virus for the expression of a transgene encoding an anti-VEGF agent comprising: (i) an rAAV capsid protein, where the capsid protein is a variant of the AAV8 capsid protein, the AAV2 capsid protein and/or the AAV2/3 hybrid capsid protein or a variant thereof; and (ii) a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid comprising, in sequence from 5' to 3': (a) 5' AAV ITR; (b) a CMV enhancer; (c) a CBA promoter; (d) a chicken beta-actin intron; (d) a Kozak sequence; (e) a transgene encoding an anti-VEGF agent, where the anti-VEGF agent is encoded by the nucleic acid sequence represented in SEQ ID NO: 1; (f) a rabbit beta-globin polyA signal tail; and (g) 3' ITR AAV. Also described is a host cell containing rAAV, for production of said rAAV. Disclosed is a pharmaceutical composition for inhibiting VEGF activity containing rAAV. Disclosed is the use of rAAV or a pharmaceutical composition for inhibiting VEGF activity in a subject in need thereof or for delivering an anti-VEGF agent to a subject in need thereof or for treating a disease associated with neovascularisation, a disease associated with angiogenesis, or a disease associated with VEGF in a subject in need thereof.

EFFECT: invention widens the range of agents for delivering the anti-VEGF agent.

55 cl, 22 dwg, 3 tbl, 8 ex

Description

Link to related applications

This application claims the benefit of 35 U.S.C. 119(e) by the filing dates of U.S. Provisional Application Ser. No. 63/179,700, filed April 26, 2021, entitled "ADENO-ASSOCIATED VIRUS FOR DELIVERY OF KH902 (CONBERCEPT) AND USES THEREOF," and U.S. Provisional Application Ser. No. 63/074,361, filed September 3, 2020, entitled "ADENO-ASSOCIATED VIRUS FOR DELIVERY OF KH902 (CONBERCEPT) AND USES THEREOF," the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION KH902 is a vascular endothelial growth factor (VEGF) receptor fusion protein that is currently undergoing clinical trials for anti-VEGF therapy. Current problems in anti-VEGF therapy include the need for repeated injections to maintain efficacy and the need for long-acting anti-VEGF drugs. Thus, there is a need to develop new methods for delivering a long-acting anti-VEGF agent to target cells and/or tissues.

Brief summary of the present invention

Aspects of the present invention relate to compositions and methods for delivering an anti-VEGF agent (e.g., KH902) to cells and/or tissues (e.g., cells of a subject). The present invention is based in part on rAAVs engineered to express a transgene encoding an anti-VEGF agent (e.g., KH902).

In some aspects, the present invention relates to a recombinant adeno-associated virus (rAAV) comprising: (i) an AAV capsid protein, wherein the capsid protein is a variant of an AAV2 capsid protein, a hybrid AAV2/3 capsid protein and/or an AAV8 capsid protein; and (ii) an isolated nucleic acid comprising a transgene encoding an anti-vascular endothelial growth factor (anti-VEGF) agent, wherein the transgene is flanked by inverted terminal repeats (ITRs) of an adeno-associated virus (AAV).

In some embodiments of the present invention, the anti-VEGF agent is a human VEGF receptor decoy. In some embodiments, the human VEGF receptor decoy comprises the extracellular domain 2 of human VEGF receptor 1. In some embodiments, the human VEGF receptor decoy comprises the extracellular domains 3 and 4 of human VEGF receptor 2. In some embodiments, the VEGF receptor decoy is capable of binding to and acting against vascular endothelial growth factor (VEGF) and/or placental growth factor (P1GF).

In some embodiments of the present invention, the anti-VEGF agent is a human VEGF receptor fusion protein. In some embodiments of the present invention, the human VEGF receptor fusion protein comprises the extracellular domain 2 of human VEGF receptor 1 fused to the extracellular domains 3 and 4 of human VEGF receptor 2. In some embodiments of the present invention, the human VEGF receptor fusion protein comprises the extracellular domain 2 of human VEGF receptor 1 fused to an Fc portion of an immunoglobulin. In some embodiments of the present invention, the human VEGF receptor fusion protein comprises the extracellular domains 3 and 4 of human VEGF receptor 2 fused to an Fc portion of an immunoglobulin. In some embodiments of the present invention, the human VEGF receptor fusion protein comprises the extracellular domain 2 of human VEGF receptor 1 fused to the extracellular domain 3 and 4 of human VEGF receptor 2, and further fused to an Fc portion of an immunoglobulin. In some embodiments of the present invention, the anti-VEGF agent is KH902. In some embodiments of the present invention, the anti-VEGF agent comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5, or a portion thereof. In some embodiments, the transgene comprises a nucleic acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 1 or a codon-optimized variant thereof. In some embodiments of the present invention, the anti-VEGF agent is capable of binding to and acting against vascular endothelial growth factor (VEGF) and/or placental growth factor (P1GF).

In some embodiments, the isolated nucleic acid further comprises a promoter operably 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. In some embodiments, the transgene comprises one or more introns. In some embodiments, at least one intron is located between the promoter and the nucleic acid sequence encoding an anti-vascular endothelial growth factor (anti-VEGF) agent.

In some embodiments of the present invention, the transgene comprises a Kozak sequence. In some embodiments of the present invention, the Kozak sequence is located between an intron and a transgene encoding an anti-vascular endothelial growth factor (anti-VEGF) agent.

In some embodiments, the transgene comprises a 3' untranslated region (3'UTR). In some embodiments, the transgene further comprises one or more microRNA binding sites. In some embodiments, the one or more microRNA binding sites are located in the 3'UTR of the transgene. In some embodiments, the at least one microRNA binding site is an immune cell-associated microRNA binding site. In some embodiments of the present invention, the immune cell-associated microRNA is 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-l, 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 embodiments of the present invention, the inverted terminal repeats (ITRs) are ITRs of an adeno-associated virus serotype that are selected from the group consisting of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, and AAV6 ITR.

In some embodiments of the present invention, the isolated nucleic acid comprises a nucleic acid sequence that is at least 80%, at least 90%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 2.

In some embodiments of the present invention, the capsid protein comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of capsid protein v224, capsid protein v326, capsid protein v358, capsid protein v46, capsid protein v56, capsid protein v66, capsid protein v67, capsid protein v81, capsid protein v439, capsid protein v453, capsid protein v513, capsid protein v551, capsid protein v556, capsid protein v562, or capsid protein v598. In some embodiments, the capsid protein comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of the v224 capsid protein, the v326 capsid protein, or the v56 capsid protein. In some embodiments, the capsid protein has affinity for ocular tissue. In some embodiments, the ocular tissue comprises ocular neurons, the retina, the sclera, the choroid, the retina, the vitreous, the macula, the fovea, the optic disc, the lens, the pupil, the iris, the aqueous humor, the cornea, the conjunctiva, the ciliary body, or the optic nerve.

In some embodiments of the present invention, the rAAV is a single-chain AAV (ssAAV) or a self-complementary AAV (scAAV).

In some embodiments of the present invention, the variant capsid protein is capable of increasing the packaging efficiency of rAAV compared to the wild-type capsid protein from which it is derived. In some embodiments of the present invention, the variant AAV2 capsid protein is capable of increasing the packaging efficiency of rAAV by at least 1-fold, 2-fold, 3-fold, 4-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to the wild-type AAV2 capsid protein. In some embodiments of the present invention, the variant AAV2/3 fusion capsid protein is capable of increasing the packaging efficiency of rAAV by at least 1-fold, 2-fold, 3-fold, 4-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to the wild-type AAV3b capsid protein.

In some aspects, the present invention relates to a recombinant adeno-associated virus comprising: (i) an rAAV capsid protein, wherein the capsid protein is a variant of an AAV8 capsid protein, an AAV2 capsid protein and/or an AAV2/3 hybrid capsid protein; and (ii) a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid comprising in a 5' to 3' sequence: (a) an AAV 5' ITR; (b) a CMV enhancer; (c) a CBA promoter; (d) a chicken beta-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 set forth in SEQ ID NO: 1; (g) a rabbit poly-A beta globin signal tail (poly-A RGB); and (h) 3' ITR AAV.

In some aspects, the present invention relates to a host cell comprising an rAAV as described herein. In some embodiments, the host cell is a mammalian cell, a yeast cell, a bacterial cell, or an insect cell.

In some aspects, the present invention relates to a pharmaceutical composition comprising rAAV as described herein. In some embodiments, the pharmaceutical 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.

In some aspects, the present invention provides a method for inhibiting VEGF or P1GF activity in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of rAAV or a pharmaceutical composition as described herein.

In some aspects, the present invention provides a method of delivering an anti-VEGF agent to a subject in need thereof, wherein the method comprises administering to the subject an effective amount of rAAV or a pharmaceutical composition as described herein.

In some aspects, the present invention relates to a method of treating a neovascularization (NV)-associated disease, an angiogenesis-associated disease, or a VEGF-associated disease in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of rAAV or a pharmaceutical preparation as described herein.

In some aspects, the present invention relates to rAAV or a composition comprising rAAV for use in inhibiting VEGF activity in a subject in need thereof, wherein the rAAV comprises an adeno-associated virus (AAV) capsid protein (e.g., AAV 2 variants or AAV2/3 hybrid variants) and an isolated nucleic acid comprising a transgene encoding an anti-VEGF agent (e.g., KH902).

In some aspects, the present invention relates to an rAAV or a composition comprising rAAV for use in delivering an anti-VEGF agent to a subject in need thereof, wherein the rAAV comprises an adeno-associated virus (AAV) capsid protein (e.g., AAV 2 or AAV2/3 hybrid variants) and an isolated nucleic acid comprising a transgene encoding an anti-VEGF agent (e.g., KH902).

In some aspects, the present invention relates to rAAV or a composition comprising rAAV for use in the treatment of a neovascularization-associated disease, an angiogenesis-associated disease, or a VEGF-associated disease in a subject in need thereof, wherein the rAAV comprises an adeno-associated virus (AAV) capsid protein (e.g., AAV 2 variants or AAV2/3 hybrid variants) and an isolated nucleic acid comprising a transgene encoding an anti-VEGF agent (e.g., KH902).

In some embodiments of the present invention, delivery of an anti-VEGF agent results in inhibition of VEGF activity by 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%.

In some embodiments of the present invention, the subject is a non-human mammal. In some embodiments of the present invention, the non-human mammal is a mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate. In some embodiments of the present invention, the subject is a human.

In some embodiments of the present invention, the subject has or is suspected of having an angiogenesis-related disease or a VEGF-related disease. In some embodiments of the present invention, the VEGF-related disease is a tumor, cancer, retinopathy, wet age-related macular degeneration (wAMD), macular edema, choroidal neovascularization, or corneal neovascularization.

In some embodiments of the present invention, the administration is systemic administration, and the systemic administration is optionally intravenous injection. In some embodiments of the present invention, the administration is direct administration to the ocular tissue, and the direct administration is optionally intravitreal injection, intraocular injection, or topical administration.

In some embodiments of the present invention, the administration results in delivery of the transgene to the ocular tissue. In some embodiments of the present invention, the ocular tissue comprises ocular neurons, the retina, the sclera, the choroid, the retina, the vitreous, the macula, the fovea, the optic disc, the lens, the pupil, the iris, the aqueous humor, the cornea, the conjunctiva, the ciliary body, or the optic nerve.

In some embodiments of the present invention, administration results in inhibition of VEGF in the subject for at least 5 days, 10 days, 15 days, 20 days, 1 month, two months, or more after administration.

In some aspects, the present invention relates to a method of treating corneal neovascularization (CNV) in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of rAAV or a pharmaceutical composition as described herein. In some embodiments, the rAAV comprises an AAV8 capsid protein. In some aspects, the present invention relates to a method of reducing corneal neovascularization (CNV) in a subject in need thereof (e.g., reducing CNV compared to an untreated subject or in the subject prior to administration), wherein the method comprises administering an effective amount of rAAV or a pharmaceutical composition as described herein. In some embodiments, the rAAV comprises an AAV8 capsid protein.

In some embodiments of the present invention, the administration results in delivery of the anti-VEGF agent to corneal cells. In some embodiments of the present invention, the administration results in delivery of the anti-VEGF agent to corneal keratocytes.

In some embodiments, the rAAV is administered once. In some embodiments, the administration results in expression of the anti-VEGF agent in corneal cells for more than three months, six months, a year, or more. In some embodiments, the administration results in inhibition of VEGF (e.g., VEGF expression or activity) in the subject for 1 month, two months, three months, six months, a year, or more after administration. In some embodiments, the administration is an intrastromal injection. In some embodiments, the subject is a human. In some embodiments, the corneal neovascularization is acute corneal neovascularization or chronic corneal neovascularization.

Brief description of the figures

Figures 1A-1C show the rAAV-CBA-KH902 vector and sequences. The rAAV expression vector expresses secreted KH902 (conbercept) and is driven by a CMV and chicken β-actin (CBA) enhancer promoter cassette. A Kozak sequence is also included at the 5' end of the start codon to enhance translation initiation. A map diagram (Figure 1A) and read sequence of the plasmid strand (Figure 1B, SEQ ID NO: 3) are shown. Sequences including and encompassed by the 5' ITR and 3' ITR are packaged into AAV virions (Figure 1C).

Figure 2 shows a Western blot of RPE-conditioned medium infected with AAV-KH902. Fifteen microliters of ARPE-19 (left) or hTERT-RPE1 (right) conditioned medium were subjected to PAGE analysis under the conditions indicated above each lane. After semi-dry transfer, membranes were blotted with anti-VEGFR1 antibody (R&D Systems BAF321). For each blot, 20 ng of KH902 preparation was included as a control (last lanes).

Figures 3A-3C show the results of in vitro functional testing of AAV-KH902 vectors. Shown are the results of angiogenesis or proliferative capacity of VEGF-stimulated (25 ng/ml) HUVECs in the presence of KH902 or conditioned medium (diluted 1:10) from AAV2-KH902-infected RPE cells or in the presence of the control vector GFP. Anti-VEGF activity was quantified by tube formation assays (Figures 3A and 3B) or CCK-8 activity assay (Figure 3C), respectively. *: p<0.01; **: p<0.001; ***: p<0.0001.

Figure 4 shows that intravitreal injection of rAAV2-KH902 prevents normal retinal vascular development. Neonatal mice (P0-P3) were injected intravitreally with rAAV2-KH902. Mice were raised under normoxic conditions (~21% O ₂ ) and sacrificed at >P18. Retinas were mounted and stained with anti-PECAM antibodies (endothelial cells) or with DAPI (DNA) and PNA (photoreceptors) and then imaged from the ganglion cell side (upper panels) or the photoreceptor side (lower panels).

Figures 5A-5C show that intravitreal injection of rAAV2-KH902 prevents retinal edema in retinopathy of prematurity. Neonatal mice (RO-P3) were injected with rAAV2-KH902 and raised for approximately 4 days under normoxic conditions (~21% O ₂ ) and then exposed to hyperoxia (75% O ₂ ) for approximately 1 week. Between P12 and P18, mice were returned to normoxic conditions for 6 days and then sacrificed. (Figure 5A) Retinas were mounted and stained with anti-isolectin B4 (vascular staining) and anti-PECAM (endothelial cell) antibodies. Each treatment group (n=6) was analyzed, and the results for edema occurrence (Figure 5B) and edema scores (Figure 5C) were summarized.

Figures 6A-6B show the evaluation of rAAV2-KH902 in a mouse model of oxygen-induced retinopathy. Figure 6A shows brightfield images of eyes injected with rAAV2-EGFP (left column) and a 5:1 mixture of rAAV2-KH902:rAAV2-EGFP (right column), obtained immediately after necropsy. Eyes in a row are from the same animal, so the rAAV2-Egfp-injected eyes served as a control for the degree of pathology induction in individual animals. Figure 6B shows fluorescence staining of eyes from a representative mouse mounted in flat mount and stained for isolectin B4. Areas of positive transduction are characterized by EGFP expression. rAAV2-KH902 administration reduces normal vascular development and aneurysmal nodules; i.e., strong EGFP expression reduces the development of retinal vasculature. Examples of aneurysmal nodules are indicated in the lower panel (arrows).

Figures 7A-7B show the evaluation of rAAV8-KH902 in a mouse model of oxygen-induced retinopathy. Figure 7A shows brightfield images of eyes injected with rAAV8-EGFP (left column) and a 5:1 mixture of rAAV8-KH902:rAAV8-EGFP (right column), obtained immediately after necropsy. Eyes in a row are from the same animal, so the rAAV8-Egfp-injected eyes served as a control for the degree of pathology induction in individual animals. Figure 7B shows an image of fluorescent staining of eyes from a representative mouse mounted in flat mount and stained for isolectin-B4. Areas of positive transduction are characterized by EGFP expression. Administration of rAAV8-KH902 does not reduce normal vascular development and has only a minor effect on aneurysm nodule formation.

Fig. 8 shows the percentage of pathologies for rAAV-treated eyes. The results of swelling or recovery assessment of the eyes of mice shown in Figs. 6A-6B and 7A-7B are shown. Experimental groups: rAAV2, n=10; rAAV8, n=10.

Figures 9A-9B are fundoscopic images showing fundus images of mice injected with rAAVs containing AAV2 capsid protein variants and AAV2/3 fusion variants and EGFP-encoding nucleic acid. Eight AAV2 variants (v224, v326, v358, v46, v56, v66, v67, and v81) and seven AAV2/3 fusion variants (v439, v453, v513, v551, v556, v562, and v598) packaging CB6-EGFP were administered by intravitreal injection. Representative eyes were imaged after injection at two weeks (Figure 9A) and four weeks (Figure 9B). Three capsids were selected for packaging KH902: v56, v224, v326 (marked with asterisks). The number of eyes scored (number of EGFP-positive eyes/number of total eyes) is indicated in the lower right corner of each micrograph.

Figure 10 shows the results of treatment of laser injury-induced CNV with rAAV.v224-packaged KH902 vector. Mice were laser-injured to induce choroidal neovascularization (CNV) manifestations. Five days after injury, rAAV was injected intravitreally. Results of longitudinal analysis of CNV after administration of a control capsid encoding GFP and V224-KH902 are shown. It is shown that rAAV v224-KH902 is able to reduce the extent of laser injury CNV to less than 80 percent 20 days after injury. Data represent mean ± ME at the 90% significance level.

Figure 11 shows that rAAV v224-KH902 does not cause ocular lesions associated with immune cell infiltration into the ocular vasculature.

Figure 12 shows the in vitro evaluation of packaging yield by PCR of crude lysate. The histogram shows the relative packaging yields for the AAV2 variants (upper panel), AAV2/3 variants (middle panel), and AAV8 variants (lower panel). The packaging yields for each capsid are expressed as a percentage of the yield provided by their prototypical forms: AAV2, AAV3b, and AAV8, respectively. The v56 capsid variant showed a 9.42-fold increase in yield compared to AAV2; the v224 capsid variant showed an 8.96-fold increase in yield compared to AAV2; and the v326 capsid variant showed a 9.79-fold increase in yield compared to AAV2. The total number of capsids is shown on the x-axis. The AAV2/3 hybrid variants also showed a 2- to 8-fold increase compared to AAV3b.

Figures 13A-13F show a comparison of transduction into corneal cells following intrastromal injection and subconjunctival injection of rAAV8-eGFP. Figures 13A-13C show images of mouse corneas following intrastromal injection. Figures 13D-13F show images following subconjunctival injection. Figures 13B and 13E show that eGFP signal was detected in images from living animals two weeks following intrastromal injection of rAAV8 (1.6×10 ¹⁰ GC in 4 μL per cornea). The dotted circle represents the edge of the mouse cornea. Figures 13C and 13F show fluorescence microscopy results for eGFP expression in representative cross-sections of the corneas from Figures 1 and 2. 13B and 13E, respectively. Arrows indicate injection sites. GC: genomic copies.

Figures 14A–14C show the kinetics of rAAV2- and rAAV8-mediated KH902 expression and cell tropism. Figure 14A shows that rAAV2- and rAAV8-mediated eGFP expression occurs at similar levels as determined by real-time imaging microscopy at different time points up to three months (12 weeks) after intrastromal injection. The dotted circle represents the edge of the mouse cornea. Figure 14B shows the relative expression of KH902 mRNA in the corneas of mice treated with rAAV8 and rAAV2 vectors (***: p<0.001, ****: p<0.0001). Data are presented as mean±SEM, n=3. Figure Figure 14C shows histological analysis of cell specificity in corneal sections with rAAV2 and rAAV8. Panels (i, ii): anti-vimentin staining of mouse cornea two weeks after intrastromal injection with rAAV2- and rAAV8-mediated eGFP expression. The eGFP signal in the corneal stroma is superimposed on vimentin-labeled keratocytes. Panels (iii, v): distribution of anti-human IgG (H+L) antibody labeling KH902 protein in a corneal section treated with intrastromal administration of rAAV8 or PBS, respectively. Panel (iv): higher magnification of the areas outlined by the dotted box in panel iii is shown, co-stained with anti-vimentin (red). Panels b and d: magnifications 400X; scale bar 25 μm. Panel c: magnification 200X; scale bar 500 µm. Key: Epi - epithelial layer; Stroma - stroma; Endo - endothelial layer; Anti-human IgG - antibody against human IgG; Merge - fusion. The dose of each rAAV vector for all experiments described above was 1.6×10 ¹⁰ GC in 4 µl PBS per cornea.

Figures 15A-15D show the results of central corneal thickness (CCT) measurements and immune response assessment to rAAV2 and rAAV8 vectors. Figure 15A shows optical coherence tomography (OCT) images of corneas before injection, immediately after injection, and 1, 2, and 12 weeks after injection of PBS, rAAV2-KH902, and rAAV8-eGFP/KH902 at a dose of 1.6×10 ¹⁰ GC in 4 μL per cornea. Figure 15B shows the results of quantitative analysis of central corneal thickness obtained by measuring the corneas shown in Figure 15A. Figure 15C shows the results of analysis of corneal immune responses to high (1.6×10 ¹⁰ GC/cornea) and low doses (8×10 ⁸ GC/cornea) of rAAV2- or rAAV8-eGFP/KH902 with immunofluorescence staining of monocytes/macrophages (CDllb, F4/80, red). Magnification 200×. Scale bar 50 μm. Fig. 15D shows the calculated percentages of CD11b+ cells and F4/80+ cells in the groups indicated in Fig. 15C. ***: p<0.001; ****: p<0.0001. Data are presented as mean±SEM, n=5.

Figures 16A-16E show the results of long-term inhibition of corneal neovascularization (CoNV) by rAAV8-KH902 via single-dose intrastromal delivery in the caustic burn-induced CoNV model. Figure 16A shows representative images of CoNV from caustic-treated corneas injected with PBS, rAAV8-eGFP, conbercept (10 mg/mL, 4 μL), rAAV2-KH902, rAAV8-KH902, and rAAV8-KH902 in combination with conbercept (10 mg/mL, 4 μL) on days 5, 10, and weeks 2, 3, 4, 8, and 12. Figures 16B-16C show histograms of the CoNV area quantification results for each event shown in the panels of Figure 1. 16A. significant difference from the PBS group; *: significant difference from the rAAV8-eGFP or KH902 groups; #: significant difference from the rAAV2-KH902 group; * and #: p<0.05; ** and ##: p<0.01; *** and ###: p<0.001; **** and ####: p<0.0001. Data are presented as mean±SEM, n=5~7. Fig. 16D shows immunofluorescence images of mouse corneal flat mounts. For each case shown in Fig. 16A, corneas taken 12 weeks after alkali burn were double stained. The area covered by CD31 ⁺⁺⁺ /LYVE-1 represents blood vessels, and the area covered by CD31 ⁺ /LYVE-1 ⁺⁺⁺ represents lymphatic vessels (+++ indicates strong positive; ++, moderate positive; +, moderate positive). Magnification is 100X. Scale bar is 50 μm. In Fig. 16E shows the results of corneal angiogenesis and lymphangiogenesis assessments by measuring the stained areas covered by CD31 ⁺⁺⁺ and LYVE-1 ⁺⁺⁺ , respectively, for each case shown in Fig. 16D. *: significant difference from the PBS group; •: significant difference from the rAAV8-eGFP group; #: significant difference from the KH902 group; significant difference from the rAAV2-KH902 group. ###: p<0.001; ****, ••••, #### And p<0.0001.

Data are presented as mean±SEM, n=4. The dose of each rAAV vector for the experiments described above was 8× ¹⁰⁸ GC in 4 µl PBS or conbercept solution per cornea.

Figures 17A-17F show that AAV8-delivered KH902 downregulates D114/Notch signaling and ERK activation in an alkali burn-induced CoNV model. Figure 17A shows the results of immunofluorescence analysis of D114 expression in CD31-costained mouse corneal flat mounts two weeks after alkali burn of corneas treated with PBS, rAAV8-eGFP, and rAAV8-KH902 (8×10 ⁸ GC/cornea). Magnification 200X. Scale bar 100 μm. Figure 17B, 17C, 17D show the Western blots and the results of semiquantitative analysis of D114 and NICD expression in the cornea of mice two weeks after alkali burn in each of the indicated treatment groups. Figs. 17E and 17F show the Western blots and the results of semiquantitative analysis of ERK activation. The results are expressed as the ratio of phosphorylated ERK (pERK) to total ERK (pERK/ERK) in the indicated treatment groups eight days after alkali burn. ***: p<0.001; ****: p<0.0001.

Figures 18A–18C show that TAAV8-KH902 prevents the progression of pre-existing neovascularization in the alkali burn-induced CoNV model. Figure 18A shows corneas of mice following intrastromal injection of PBS, rAAV8-eGFP, and rAAV8-KH902 (8 × 10 ⁸ GC in 4 μL per cornea) at day 10 post-alkali burn (baseline). Representative CoNV images recorded weekly for four weeks are shown. Figure 18B shows the results of weekly CoNV area quantification in each group shown in Figure 18A. Asterisk indicates significant differences between the observation endpoint (week 4) and the corresponding baseline (**: p < 0.01). Data are expressed as mean ± SEM, n = 5. Figure 18C shows the results of weekly side-by-side comparison of CoNV area quantification between the experimental groups shown in Fig. 18A over four weeks. (*: p<0.05, **: p<0.01, ***: p<0.001). Data are presented as mean±SEM, n=5.

Figures 19A–19C show that TAAV8-KH902 prevents the progression of pre-existing neovascularization in the suture-induced CoNV model. Figure 19A shows corneas from mice subjected to five days of suture placement (baseline) that received an intrastromal injection of PBS, rAAV8-eGFP, and rAAV8-KH902 (8× ¹⁰⁸ GC in 4 μL per cornea). Representative weekly images of CoNVs obtained during four weeks of observation are shown. Figure 19B shows the results of weekly comparative quantification of CoNV areas for the experimental groups shown in Figure 19A obtained over four weeks (****: p<0.0001). Data are presented as mean±SEM, n=4~7. Figure 19C shows the results of weekly comparative quantitative analysis of CoNV areas for the experimental groups shown in Fig. 19A. Asterisks indicate significant differences between the endpoint (week 4) and baseline (****: p<0.0001).

Figures 20A-20C show the results of corneal transduction following intrastromal injection and subconjunctival injection of eGFP delivered by the rAAV2 vector. Figure 20A shows representative images of eGFP expression in a mouse corneal section obtained by fluorescence microscopy 2 weeks after intrastromal injection of the rAAV2 vector (1.6×10 ¹⁰ GC /cornea). Figures 20B and 20C show representative images of the eGFP signal from live animal imaging 2 weeks after intrastromal or subconjunctival injection of rAAV2, respectively. The dotted circle represents the edge of the mouse cornea observed by microscopy.

Figures 21A-21B show the histological analysis of rAAV2 vector-mediated KH902 expression in the cornea after intrastromal injection. Figure 21A shows representative images of the eyeball upon expression of KH902 labeled with anti-human IgG antibody (H+L). Magnification is 200X. Scale bar is 500 μm. Figure 21B shows a magnified view of the areas boxed in Figure 21A co-stained with anti-vimentin. The images show that KH902 expression is mainly distributed in the corneal stromal layer. Magnification is 400X. Scale bar is 100 μm. The dose of rAAV2 vectors was 1.6×10 ¹⁰ GC in 4 μL of PBS per cornea.

Figs. 22A-22C show representative data and results related to fundoscopy and fluorescein angiography (FP and FFA) of non-human primates (NHPs) injected with rAAVs encoding KH902. Fig. 22A shows representative data showing that injection of rAAVs encoding KH902 reduces grade IV CNV lesions compared to control injections; Conbercept was used as a positive treatment control. Fig. 22B shows representative data showing that injection of rAAVs encoding KH902 reduces fluorescein leakage area compared to control injections. Conbercept was used as a positive treatment control. Fig. 22C shows representative FFA images showing regression of bright spots at day 29 post-injection.

Detailed disclosure of the present invention

Aspects of the present invention relate to compositions and methods for expressing anti-VEGF agents (e.g., KH902) in a cell or subject. The present invention is based in part on rAAVs comprising a capsid protein (e.g., AAV2/3 capsid protein, AAV8 capsid protein, etc.) and an rAAV vector comprising a nucleic acid encoding an anti-VEGF agent flanked by adeno-associated inverted terminal repeats (ITRs) of a virus (AAV). In some embodiments of the present invention, the nucleic acid comprises a promoter, such as a CMV promoter or a chicken beta-actin (CBA) promoter.

In some aspects, the rAAV described herein comprises an AAV capsid (e.g., an AAV2 variant capsid protein or an AAV2/3 fusion variant capsid protein) comprising an isolated nucleic acid encoding a transgene expression cassette that comprises a nucleic acid sequence encoding an anti-vascular endothelial growth factor agent (e.g., anti-VEGF) flanked by AAV inverted terminal repeats (ITRs). The present invention is based, in part, on rAAVs engineered to express transgenes encoding an anti-VEGF agent (e.g., a VEGF receptor fusion protein such as KH902) or variants thereof. In some embodiments, the compositions described herein (e.g., rAAV compositions) are useful for treating ocular diseases, such as treating corneal vascularization.

Recombinant adeno-associated viruses (rAAV)

In some aspects, the present invention relates to isolated adeno-associated viruses (AAVs). As used herein in relation to AAVs, the term "isolated" refers to an AAV that has been artificially produced or manufactured. Isolated AAVs can be produced using recombinant techniques. Such AAVs are referred to herein as "recombinant AAVs". Recombinant AAVs (rAAVs) preferably have tissue-specific targeting capabilities such that the rAAV transgene will be specifically delivered to one or more predetermined tissues (e.g., eye tissues). The AAV capsid is an important element in generating these tissue-specific targeting capabilities (e.g., tissue tropism capabilities). Thus, an rAAV can be selected that has a capsid that is suitable for a particular target tissue.

The present invention relates at least in part to a recombinant adeno-associated virus (rAAV) comprising: (i) an AAV capsid protein, wherein the capsid protein is an AAV2 variant or an AAV2/3 hybrid, and (ii) an isolated nucleic acid comprising a transgene encoding an anti-vascular endothelial growth factor (anti-VEGF) agent, wherein the transgene is flanked by inverted terminal repeats (ITRs). The present invention also relates at least in part to a recombinant adeno-associated virus (rAAV) comprising: (i) an AAV capsid protein, wherein the capsid protein is of the AAV8 serotype, and (ii) an isolated nucleic acid comprising a transgene encoding an anti-vascular endothelial growth factor (anti-VEGF) agent, wherein the transgene is flanked by inverted terminal repeats (ITRs).

Methods for producing recombinant AAVs containing a desired capsid protein are well known in the art (see, for example, U.S. Patent Application Publication No. US 2003/0138772, the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing a host cell that contains a nucleic acid sequence encoding an AAV capsid protein, a functional rep gene, a recombinant AAV vector comprising AAV inverted terminal repeats (ITRs) and a transgene, and sufficient auxiliary elements to enable packaging of the recombinant AAV vector into AAV capsid proteins. In some embodiments, the capsid proteins are structural proteins encoded by the AAV cap gene. AAVs contain three capsid proteins, virion proteins 1 through 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, the capsid proteins, when translated, form a spherical 60-mer protein coat around the viral genome. In some embodiments, the capsid proteins function to protect the viral genome, deliver the genome, and interact with the host. In some aspects, the capsid proteins deliver the viral genome to the host in a tissue-specific manner.

In some embodiments of the present invention, the AAV capsid protein has affinity for ocular tissue or muscle tissue. In some embodiments of the present invention, the AAV capsid protein targets specific types of ocular cells (e.g., photoreceptor cells, retinal cells, etc.).

In some embodiments of the present invention, the AAV capsid protein is an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.hr, AAVrh8, AAVrh10, AAVrh39, AAVrh43, AAV.PHP, and variants of any of the foregoing. In some embodiments of the present invention, the AAV capsid protein has a serotype derived from an AAV of a non-human primate, such as serotype AAVrh8. In some embodiments, the capsid protein is 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 is AAV1. In some embodiments, the AAV capsid is AAV2. In some embodiments, the AAV capsid protein with the desired tissue affinity can be selected from AAV capsid proteins isolated from mammals (e.g., tissue of a subject) (see, e.g., WO2010138263A2 and WO2018071831, the entire contents of which are incorporated herein by reference). In some embodiments, the AAV capsid is AAV8.

In some embodiments, the AAV capsid is a variant or homolog of a known AAV capsid protein. In some embodiments, combinations of variant capsid proteins and KH902 provide advantages in rAAV-based therapy (e.g., better packaging efficiency, effective inhibition of VEGF, or less toxicity associated with KH902 overexpression) than previously described capsids for delivering KH902. A variant capsid typically contains at least one amino acid substitution, insertion, or deletion compared to the wild-type capsid(s) from which it is derived. In some embodiments of the present invention, the AAV variant comprises from about 1 to about 100 amino acids (e.g., from 1 to 10 amino acids, from 1 to 20 amino acids, from 1 to 30 amino acids, from 20 to 50 amino acids, from 20 to 60 amino acids, from 50 to 80 amino acids, from 50 to 100 amino acids, from 60 to 100 amino acids, etc.), 1 substitution, insertion, or deletion compared to a known AAV capsid (e.g., AAV serotype 2 or AAV2/3 (e.g., AAV2/3 hybrid), etc.). In some embodiments of the present invention, the AAV variant comprises more than 100 amino acids (e.g., 100 to 200 amino acids, 200 to 300 amino acids, 100 to 500 amino acids, 500 to 1000 amino acids or more) substitution, insertion, or deletion compared to a known AAV capsid (e.g., AAV serotype 2 or AAV2/3 (e.g., AAV2/3 hybrid), etc.). In some embodiments of the present invention, an AAV variant may comprise from about 5 to about 50 amino acids (e.g., from 5 to 10 amino acids, from 5 to 20 amino acids, from 5 to 30 amino acids, from 5 to 40 amino acids, from 10 to 20 amino acids, from 10 to 30 amino acids, from 10 to 40 amino acids, from 10 to 50 amino acids, or from 30 to 50 amino acids), substitution, insertion, or deletion compared to a known AAV serotype (e.g., AAV 2 or AAV2/3 serotype (e.g., AAV2/3 hybrid), etc.). In some embodiments of the present invention, an AAV variant may comprise from about 10 to about 30 amino acids (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids), substitution, insertion, or deletion compared to a known AAV serotype (e.g., AAV serotype 2 or AAV2/3 (e.g., an AAV2/3 hybrid)). In some embodiments, the AAV variant may comprise 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 amino acid substitutions, insertions, or deletions compared to a known AAV serotype (e.g., AAV serotype 2 or AAV2/3 (e.g., an AAV2/3 hybrid)). In some embodiments, the AAV variant comprises an amino acid sequence that is 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% identical to an amino acid sequence of a known AAV capsid (e.g., AAV serotype 2 or AAV2/3 (e.g., AAV2/3 hybrid), etc.).

In some embodiments, the capsid variant may be a chimeric capsid variant. The chimeric capsid variant sequence may comprise portions of two or more AAV capsid serotypes or variants thereof. In some embodiments, the chimeric capsid comprises portions of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different capsid protein serotypes. In some embodiments, the chimeric capsid proteins have other properties, such as tissue tropism, etc., that are greater than the AAV capsid proteins from which they are derived. The fragments may be incorporated by any suitable method, such as recombinant DNA cloning.

In some embodiments, the AAV variants described herein are variants of AAV2, AAV2/3 (e.g., an AAV2/3 hybrid), or AAV8. AAV2 has been found to efficiently transduce ocular tissue (e.g., photoreceptor cells and retinal pigment epithelium (RPE)), human central nervous system (CNS) tissue, kidney tissue, and other tissues. In some embodiments, the AAV capsid described herein is an AAV2 variant. Accordingly, in some embodiments, the AAV2 variants described herein may be useful for delivering gene therapy agents to ocular tissue (e.g., the retina). AAV3 has been observed to efficiently transduce human hepatocyte cancer cells. In some embodiments, the AAV variant described herein is AAV2/3 (e.g., an AAV2/3 hybrid).

In some embodiments, the capsid variant (e.g., an AAV2 variant, an AAV 2/3 variant, or an AAV8 variant) is any of the capsid variants described in WO2018071831, the entire contents of which are incorporated herein by reference. In some embodiments, the AAV2 variant is v224, v326, v358, v46, v56, v66, v67, or v81. In some embodiments, the AAV2 variant is v224. In some embodiments, the AAV2 variant is v326. In some embodiments, the AAV2 variant is v56. In some embodiments, the AAV2/3 hybrid is v439, v453, v513, v551, v556, v562, or v598. In some embodiments of the present invention, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of wild-type AAV2/3. In some embodiments of the present invention, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of wild-type AAV8. In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the wild-type AAV2 amino acid sequence set forth in SEQ ID NO: 11. An exemplary wild-type AAV2 amino acid sequence is set forth in SEQ ID NO: 11:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2 v224 variant, at least in SEQ ID NO: 12. An exemplary amino acid sequence of v224 is set forth in SEQ ID NO: 12:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2 v326 variant shown in SEQ ID NO: 13. An exemplary amino acid sequence of v326 is shown in SEQ ID NO: 13:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2 v56 variant shown in SEQ ID NO: 14. An exemplary amino acid sequence of v56 is shown in SEQ ID NO: 14:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2 v358 variant shown in SEQ ID NO: 15. An exemplary amino acid sequence of v358 is shown in SEQ ID NO: 15:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2 v46 variant shown in SEQ ID NO: 16. An exemplary amino acid sequence of v46 is shown in SEQ ID NO: 16:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2 v66 variant shown in SEQ ID NO: 17. An exemplary amino acid sequence of v66 is shown in SEQ ID NO: 17:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2 v67 variant shown in SEQ ID NO: 18. An exemplary amino acid sequence of v67 is shown in SEQ ID NO: 18:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2 v81 variant set forth in SEQ ID NO: 19. An exemplary amino acid sequence of v81 is set forth in SEQ ID NO: 19:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2/3 v439 hybrid variant shown in SEQ ID NO: 20. An exemplary amino acid sequence of v439 is shown in SEQ ID NO: 20:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2/3 v453 hybrid variant set forth in SEQ ID NO: 21. An exemplary amino acid sequence of v453 is set forth in SEQ ID NO: 21:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2/3 v513 hybrid variant shown in SEQ ID NO: 22. An exemplary amino acid sequence of v513 is shown in SEQ ID NO: 22:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2/3 v551 hybrid variant shown in SEQ ID NO: 23. An exemplary amino acid sequence of v551 is shown in SEQ ID NO: 23:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2/3 v556 hybrid variant shown in SEQ ID NO: 24. An exemplary amino acid sequence of v556 is shown in SEQ ID NO: 24:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2/3 v562 hybrid variant shown in SEQ ID NO: 25. An exemplary amino acid sequence of v562 is shown in SEQ ID NO: 25:

In some embodiments, the capsid protein comprises an amino acid sequence that is 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% identical to the amino acid sequence of the AAV2/3 v598 hybrid variant shown in SEQ ID NO: 26. An exemplary amino acid sequence of v598 is shown in SEQ ID NO: 26:

In some embodiments of the present invention, the rAAV described herein is a single-stranded AAV (ssAAV). The term ssAAV as used herein refers to an rAAV with a coding sequence and a complementary sequence of a transgene expression cassette on separate strands packaged into separate viral capsids.

To package the rAAV vector into the AAV capsid in a host cell, additional components may be simultaneously introduced into the host cell while culturing the host cell. Alternatively, one or more of any desired components (e.g., the recombinant AAV vector, rep, cap, and/or helper sequences) may be provided by the stable host cell itself, which has been engineered to contain one or more of the desired components, using methods known to those skilled in the art. Most preferably, such a stable host cell will contain the desired component(s) under the control of an inducible promoter. However, the desired component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein in the discussion of regulatory elements suitable for use with a transgene. In another embodiment of the present invention, the selected stable host cell may comprise the selected component(s) under the control of a constitutive promoter and another selected component or components under the control of one or more inducible promoters. For example, a stable host cell may be obtained that is derived from 293 cells (which contain E1 helpers operating under the control of a constitutive promoter) but which contain rep and/or cap proteins under the control of inducible promoters. Other stable host cells may be obtained by one skilled in the art.

In some embodiments of the present invention, the AAV capsid protein described herein provides better packaging efficiency than a reference AAV capsid protein. The term AAV packaging efficiency, as used herein, refers to the percentage of AAV virions with encapsidated intact genomes in a batch of AAV virions produced. Packaging efficiency can be determined by any known method suitable for determining packaging efficiency (e.g., by PCR of crude lysate or by infecting cells and assessing transgene expression as described in Zhou et al., In Vitro Packaging of Adeno-Associated Virus DNA, J Virol. 1998 Apr; 72(4): 3241-3247). In some embodiments of the present invention, better packaging efficiency means that an increase of 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%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or more is obtained in the number of AAV virions with encapsulated intact genome than in the case of a reference capsid protein. In some embodiments of the present invention, improved packaging efficiency refers to at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 10-fold, 50-fold (e.g., 10-, 20-, 30-, 40-, or 50-fold), at least 50-100-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold) or more increase in the number of AAV virions with encapsulated intact genome than a reference capsid protein. In some embodiments, the reference capsid protein is a prototype capsid protein from which the capsid variants (e.g., AAV2 or AAV3b capsid proteins) are derived. In some embodiments, the capsid variants described herein provide improved packaging efficiency compared to the prototype capsid from which they are derived. In some embodiments, the AAV2 variants described herein (e.g., v224, v56, or v326) have a packaging efficiency of at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, or higher than AAV2. In some embodiments of the present invention, the AAV2/3 hybrid capsid proteins described herein (e.g., v439, v453, v513, v551, v556, v562, or v598) have a packaging efficiency that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, or more higher than AAV3b.

In some embodiments, the present invention relates to a host cell comprising a nucleic acid that comprises a sequence encoding a transgene (e.g., KH902). A "host cell" refers to any cell that comprises or is capable of comprising a substance of interest. Often, the host cell is a mammalian cell. In some embodiments, the host cell is a photoreceptor cell, a retinal pigment epithelial cell, a keratocyte, a corneal cell, and/or a tumor cell. The host cell can be used as a recipient of an AAV helper construct, an AAV vector, a vector with additional function, or other transfer DNA associated with the production of recombinant AAVs. The term also encompasses the progeny of the original cell that has been transfected. Thus, the term "host cell" as used herein can refer to a cell that has been transfected with an exogenous DNA sequence. It is understood that the progeny of one parent cell may not necessarily be completely identical in morphology, genome, or have completely complementary DNA as the original parent cell, due to natural, accidental, or deliberate mutation. In some embodiments of the present invention, the host cell is a mammalian cell, a yeast cell, a bacterial cell, an insect cell, a plant cell, or a fungal cell. In some embodiments of the present invention, the host cell is a neuron, a photoreceptor cell, a retinal pigmented epithelial cell, or a glial cell.

The recombinant AAV vector, rep sequences, cap sequences, and auxiliary functional sequences necessary to produce the rAAV of the present invention 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 the present invention are known to those skilled in the art of nucleic acid manipulation, and such methods include genetic engineering, recombinant engineering, and synthetic methods. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Likewise, methods for producing rAAV virions are well known, and the choice of an appropriate method is not limiting to the present invention. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. №5478745.

In some embodiments of the present invention, recombinant AAVs can be produced using a triple transfection method (described in detail in U.S. Patent No. 6,001,650). Typically, recombinant AAVs are produced by transfecting a host cell with an AAV vector (containing a transgene flanked by ITR elements) for packaging into AAV particles, a functional AAV helper vector, and a helper vector. A functional AAV helper vector encodes "AAV helper function" sequences (e.g., rep and cap sequences) that simultaneously function to ensure productive replication and encapsidation of AAV. Preferably, a functional AAV helper vector supports efficient production of the AAV vector without the formation of 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 the present invention include pHLP19 described in U.S. Patent No. 6,001,650 and the pRep6 cap6 vector described in U.S. Patent No. 6,156,303 (both of which are incorporated herein by reference). A vector providing auxiliary functions encodes viral and/or cellular nucleotide functional sequences not of AAV origin on which AAV replication depends (e.g., providing "auxiliary functions"). Auxiliary or additional functions include functional fragments necessary for AAV replication, including, but not limited to, functional fragments that participate in AAV gene transcriptional activation specific to the AAV mRNA splicing step, AAV DNA replication, cap gene expression product synthesis, and AAV capsid assembly. Helper viral functional fragments can be obtained from any of the known helper viruses, such as adenovirus, herpes virus (except herpes simplex virus type 1) and vaccinia virus.

In some aspects, the present invention relates to transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA into a cell, and a cell is said to be "transfected" when exogenous DNA has been introduced into the cell membrane. A number of transfection techniques are well 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 a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.

As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment has been introduced, such as a DNA segment that results in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid, such as RNA.

As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, and the like, that is capable of replication when linked to appropriate control elements and that can transfer gene sequences between cells. In some embodiments, the vector is a viral vector, such as an rAAV vector, a lentiviral vector, an adenoviral vector, a retroviral vector, an anellovirus vector (e.g., an anellovirus vector as described in U.S. Patent Application US 2020/0188456 A1), and the like. Thus, the term encompasses cloning and expression vehicles as well as viral vectors. In some embodiments, useful vectors are those in which the transcribed nucleic acid segment is located under the control of a transcription promoter.

Isolated Nucleic Acids Encoding a Transgene In some aspects, the present invention relates to isolated nucleic acids encoding an anti-vascular endothelial growth factor (anti-VEGF) protein. Vascular endothelial growth factor (VEGF), originally known as vascular permeability factor (VPF), is a signaling protein produced by cells that stimulates the formation of blood vessels. VEGF is a member of a subfamily of growth factors, the platelet-derived growth factor family with a cystine knot. It is an important signaling protein involved in both vasculogenesis (the formation of the embryonic blood system de novo) and angiogenesis (the growth of blood vessels from a pre-existing vasculature). The normal function of VEGF is to create new blood vessels during embryonic development, new blood vessels after injury, muscle after exercise, and new vessels to bypass blocked vessels (collateral circulation). However, aberrant VEGF activity/signaling contributes to the development of various diseases, such as vascular disease.

Anti-vascular endothelial growth factor therapy, also known as anti-VEGF therapy or anti-VEGF treatment, 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 protein (eg, KH902), monoclonal antibodies such as bevacizumab, antibody derivatives such as ranibizumab (Lucentis), or orally available small molecules that inhibit VEGF-stimulated tyrosine kinases (eg, lapatinib, sunitinib, sorafenib, axitinib, and pazopanib).

In some embodiments, the isolated nucleic acids described herein comprise a transgene encoding an anti-VEGF agent. In some embodiments, the anti-VEGF agent targets (e.g., specifically binds to) a human VEGF receptor. VEGF receptors are vascular endothelial growth factor (VEGF) receptors. There are three major subtypes of VEGF receptors, numbered 1, 2, and 3. VEGFR-1, VEGFR-2, and VEGFR-3 belong to the receptor tyrosine kinase family (Figure 1A). VEGFR-1 and VEGFR-2 are primarily involved in angiogenesis, while VEGFR-3 is involved in hematopoiesis and lymphangiogenesis. These VEGFRs contain approximately 750 amino acid residues of an extracellular domain that is organized into seven immunoglobulin-like folds. The extracellular domain is flanked by a single transmembrane region, followed by a juxtamembrane domain, a cleaved tyrosine kinase domain that is interrupted by a 70-amino acid kinase insertion, and a C-terminal tail. Activation of the VEGF receptor requires dimerization. To provide ligand-binding properties, VEGFRs form both homodimers and heterodimers. Dimerization of VEGFRs is accompanied by activation of the receptor kinase, resulting in autophosphorylation. Signal transduction is propagated when activated VEGF receptors phosphorylate SH2 domain-containing protein substrates. Vascular endothelial growth factor (VEGF) is an important signaling protein involved in many biological pathways (e.g., vasculogenesis and angiogenesis). VEGF receptors have an extracellular portion consisting of 7 immunoglobulin-like domains (extracellular domains 1-7), one transmembrane region, and an intracellular portion containing a cleaved tyrosine kinase domain. In some embodiments of the present invention, human VEGF receptor 1 comprises an amino acid sequence that is set forth for protein with NCBI accession number NP_001153392.1, protein with NCBI accession number NP 001153502.1, protein with NCBI accession number NP_001153503.1, or protein with NCBI accession number NP_002010.2. In some embodiments, human VEGF receptor 2 comprises an amino acid sequence set forth in NCBI accession number NP_002244.1. In some embodiments, the human VEGF receptor 3 comprises an amino acid sequence set forth for protein with NCBI accession number NP_002011.2, protein with NCBI accession number NP 001341918.1, or protein with NCBI accession number NP_891555.2. Vascular endothelial growth factor (VEGF) is an important signaling protein involved in many biological pathways (e.g., vasculogenesis and angiogenesis). VEGF receptors have an extracellular portion consisting of 7 immunoglobulin-like domains (such as extracellular domains 1-7), one transmembrane region, and an intracellular portion containing a cleaved tyrosine kinase domain. In some embodiments, an anti-VEGF agent targets and specifically binds to placenta-derived growth factor (P1GF), for example.

In some embodiments of the present invention, the anti-VEGF agent is a human VEGF decoy receptor or a portion thereof. "Decoy receptor" refers to a receptor that is capable of recognizing and binding a ligand (e.g., VEGF), but is structurally incapable of signaling or activating the cognate receptor complex of the ligand. The VEGF decoy receptor acts as an inhibitor by binding the ligand and keeping it from binding to its normal 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 VEGF receptor 1 and/or VEGF receptor 2 fused together. In some embodiments of the present invention, a 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 another protein (e.g., an Fc portion of an immunoglobulin). VEGF decoy receptors and VEGF decoy receptor fusion proteins have been described previously, see, for example, WO 2007/112675 and EP 1 767 546 B1, the entire contents of which are incorporated herein by reference.

In some embodiments of the present invention, a human VEGF decoy receptor comprises an extracellular domain of a protein that binds VEGF. In some embodiments, a human VEGF decoy receptor comprises an extracellular domain of human VEGF receptor 1. In some embodiments, a human VEGF decoy receptor comprises an extracellular domain of human VEGF receptor 1. In some embodiments, a human VEGF decoy receptor comprises an extracellular domain of human VEGF receptor 2. In some embodiments, a human VEGF decoy receptor comprises extracellular domains 3 and 4 of human VEGF receptor 2.

In some embodiments of the present invention, the human VEGF decoy receptor is a human VEGF receptor fusion protein. In some embodiments of the present invention, 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 of the present invention, the VEGF receptor fusion protein comprises extracellular domain 2 of VEGF receptor 1 and extracellular domain 3 of VEGF receptor 2. In some embodiments of the present invention, 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 of the present invention, the VEGF receptor fusion protein comprises VEGF receptor 1 extracellular domain 2 fused to VEGF receptor 2 extracellular domain 3 and further fused to VEGF receptor 1 extracellular domain 4. In some embodiments of the present invention, the VEGF receptor fusion protein comprises VEGF receptor 2 extracellular domain 1 fused to VEGF receptor 1 extracellular domain 2 further fused to VEGF receptor 2 extracellular domain 3. In some embodiments of the present invention, the VEGF receptor fusion protein comprises VEGF receptor 1 extracellular domain 2 fused to VEGF receptor 2 extracellular domain 3 and further fused to VEGF receptor 2 extracellular domain 4, further fused to VEGF receptor 2 extracellular domain 5. In some embodiments of the present invention, the VEGF receptor fusion protein comprises VEGF receptor 1 extracellular domain 2 fused to VEGF receptor 2 extracellular domain 3 and further fused to VEGF receptor 2 extracellular domain 4, further fused to VEGF receptor 5 extracellular domain. 1. In some embodiments of the present invention, the fused extracellular domains of the VEGF decoy receptor are connected to each other by a linker. In some embodiments of the present invention, the fused extracellular domains of the VEGF decoy receptor are directly linked to each other.

In addition, any of the VEGF receptor fusion proteins described herein can be fused to another protein. In some embodiments, the VEGF receptor fusion protein comprises a VEGF receptor moiety (e.g., any VEGF decoy receptor or VEGF decoy receptor fusion protein described herein) fused to another protein that provides the necessary properties for dimerization or multimerization. Non-limiting examples of a protein that provides dimerization or multimerization properties to the fusion protein include an Fc portion of an immunoglobulin. In some embodiments, the VEGF receptor fusion protein comprises a VEGF receptor moiety (e.g., any VEGF decoy receptor or VEGF decoy receptor fusion protein described herein) fused to an Fc portion of an immunoglobulin. In some embodiments of the present invention, a VEGF receptor fusion protein (e.g., a VEGF decoy receptor or a VEGF decoy receptor fusion protein described herein) is fused directly to another portion (e.g., an Fc domain). In some embodiments of the present invention, a VEGF receptor fusion protein (e.g., a VEGF decoy receptor) is fused to another portion via a linker.

Suitable linkers are known in the art (see, e.g., 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 an Fc portion of an immunoglobulin. In some embodiments, the VEGF receptor fusion protein is KH902. KH902, also known as conbercept (see, e.g., U.S. Patent Publication No. US 2010/0272719 A1, the entire contents of which are incorporated herein by reference), is a decoy protein that is 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 blockade of VEGF and placental growth factor (PIGF), which can induce neovascularization, has been shown to effectively treat wet age-related macular degeneration (wAMD) in clinical trials, including phase 3 trials (see, e.g., Liu et al., AJO, August 17, 2019, the entire contents of which are incorporated herein by reference).

In some embodiments, the anti-VEGF agent comprises an amino acid sequence that is 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% identical to the amino acid sequence set forth in SEQ ID NO: 5. An exemplary amino acid sequence for KH902 is set forth in SEQ ID NO: 5:

(SEQ ID NO: 5)

In some embodiments, the anti-VEGF agent comprises a portion of the sequence of SEQ ID NO: 5. In some embodiments, the anti-VEGF agent comprises an amino acid sequence that is 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% identical to the amino acid sequence of the extracellular domain 2 of VEGF receptor 1 as set forth in SEQ ID NO: 6. In some embodiments, the anti-VEGF agent comprises an amino acid sequence that is 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% identical to the amino acid sequence of VEGF receptor 1 extracellular domain 2 as set forth in SEQ ID NO: 6. In some embodiments, the anti-VEGF agent comprises an amino acid sequence that is 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% identical to the amino acid which is 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% identical to the amino acid sequence of the extracellular domain 3 and 4 of VEGF receptor 2 as set forth in SEQ ID NO: 7. In some embodiments, the anti-VEGF agent comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 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% identical to the amino acid sequence of VEGF receptor 1 extracellular domain 2 fused to VEGF receptor 1 extracellular domain 2 as set forth in SEQ ID NO: 8. In some embodiments, the anti-VEGF agent comprises an amino acid sequence that is 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% identical to the amino acid sequence of the extracellular domain 2 of VEGF receptor 1 fused to an immunoglobulin Fc region as set forth in SEQ ID NO:9. In some embodiments of the present invention, an anti-VEGF agent comprises an amino acid sequence that is 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% identical to the amino acid sequence of the extracellular domain 3 and 4 of VEGF receptor 2 fused to an immunoglobulin Fc region as set forth in SEQ ID NO: 10.

A typical amino acid sequence of the extracellular domain 2 of VEGF receptor 1 is shown in SEQ ID NO: 6:

(SEQ ID NO: 6)

A typical amino acid sequence of the extracellular domain 3 and 4 of VEGF receptor 2 is shown in SEQ ID NO: 7:

A typical amino acid sequence of the extracellular domain 2 of VEGF receptor 1 fused to the extracellular domains 3 and 4 of VEGF receptor 2 is shown in SEQ ID NO: 8:

A typical amino acid sequence of the extracellular domain 2 of VEGF receptor 1 fused to the Fc portion is shown in SEQ ID NO: 9:

A typical amino acid sequence of the extracellular domain 3 and 4 of VEGF receptor 2 fused to the Fc portion is shown in SEQ ID NO: 10:

In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence that is 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% identical to the nucleic acid sequence set forth in SEQ ID NO: 1. An exemplary coding sequence for KH902 is set forth in SEQ ID NO: 1:

Any anti-VEGF agent described herein and/or a combination thereof can be expressed by an isolated nucleic acid of the invention. In some embodiments, the isolated nucleic acid comprises a first region encoding the extracellular domain 2 of VEGF receptor 1 and a second region encoding the extracellular domains 3 and 4 of VEGF receptor 2. In some embodiments, the isolated nucleic acid comprises a first region encoding the extracellular domain 2 of VEGF receptor 1 fused to an Fc portion of an immunoglobulin and a second region encoding the extracellular domain 3 and 4 of VEGF receptor 2 fused to an Fc portion of an immunoglobulin. In some embodiments, the first region can be located in 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 the first codon. The first region can be located between the first codon of the second region and 1000 nucleotides upstream of the first codon. The first region can be located between the first codon of the second region and 500 nucleotides upstream of the first codon. The first region can be located between the first codon of the second region and 250 nucleotides upstream of the first codon. The first region can be located between the first codon of the second region and 150 nucleotides upstream of the first codon. In other embodiments, the first region can be located below the second region. The first region can be between the last codon of the second region and a position 2000 nucleotides downstream of the last codon. The first region can be between the last codon of the second region and a position 1000 nucleotides downstream of the last codon. The first region can be between the last codon of the second region and a position 500 nucleotides downstream of the last codon. The first region may be between the last codon of the second region and a position 250 nucleotides downstream of the last codon. The first region may be between the last codon of the second region and a position 150 nucleotides downstream of the last codon.

In some embodiments of the present invention, the nucleic acid may also comprise a third region. In some embodiments of the present invention, the isolated nucleic acid comprises a first region encoding the extracellular domain 2 of VEGF receptor 1, a second region encoding the extracellular domain 3 and 4 of VEGF receptor 2, and a third region encoding the extracellular domain 2 of VEGF receptor 1 fused to the extracellular domain 3 and 4 of VEGF receptor 2. In some embodiments, the isolated nucleic acid comprises a first region encoding the extracellular domain 2 of VEGF receptor 1 fused to an Fc region of immunoglobulin, a second region encoding the extracellular domain 3 and 4 of VEGF receptor 2 fused to an Fc region of immunoglobulin, and a third region encoding the extracellular domain 2 of VEGF receptor 1 fused to the extracellular domain 3 and 4 of VEGF receptor 2 and further fused to an Fc region 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.

In some embodiments of the present invention, the various regions of the isolated nucleic acid disclosed herein are expression cassettes for expressing an anti-VEGF agent or a combination of anti-VEGF agents described herein. In some embodiments of the present invention, the multicistronic expression construct comprises two or more expression cassettes encoding one or more anti-VEGF agents or a combination of anti-VEGF agents described herein.

In some embodiments, the multicistronic expression constructs comprise expression cassettes that are arranged differently. For example, in some embodiments, the present invention provides a multicistronic expression construct in which a first expression cassette (e.g., an expression cassette encoding a first anti-VEGF agent or a portion thereof) is located adjacent to a second expression cassette (e.g., an expression cassette encoding a second anti-VEGF agent or a portion thereof). In some embodiments, the first anti-VEGF agent is provided in a multicistronic expression construct in which the first expression cassette comprises an intron and the second expression cassette is located within an intron of the first expression cassette. In some embodiments, the second expression cassette located within an intron of the first expression cassette comprises a promoter and a nucleic acid sequence encoding a gene product operably linked to the promoter.

In various embodiments of the present invention, multicistronic expression constructs are provided in which the expression cassettes are oriented differently. For example, in some embodiments of the present invention, a multicistronic expression construct is provided in which the first expression cassette has the same orientation as the second expression cassette. In some embodiments of the present invention, a multicistronic expression construct is provided comprising the first expression cassette and the second expression cassette in opposite orientations.

The term "orientation" as used herein in connection with expression cassettes refers to the directional characteristic of the cassette or structure. In some embodiments of the invention, the expression cassette comprises a promoter at the 5' end of the coding nucleic acid sequence, and transcription of the coding nucleic acid sequence proceeds from the 5' end to the 3' end of the sense strand, rendering the cassette directional (e.g., 5' promoter/(intron)/coding sequence-3'). Since virtually all expression cassettes are directional in this sense, those skilled in the art can readily determine the orientation of a given expression cassette with respect to a second nucleic acid structure, e.g., a second expression cassette, a viral genome, or, if the cassette is contained in an AAV construct, with respect to the AAV ITR.

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

then the expression cassettes have the same orientation, and the arrows indicate the direction of transcription of each cassette. In another example, if a given nucleic acid construct contains a sense strand containing two expression cassettes in the configuration 5'-promoter 1/coding sequence 1 - coding sequence 2/promoter 2-3',

then the expression cassettes are in opposite orientation to each other and, as shown by the arrows, the direction of transcription of the expression cassettes is opposite. In this example, the strand shown contains the antisense strand of promoter 2 and coding sequence 2.

In another example, if the expression cassette is contained in an AAV construct, the cassette may be in either the same orientation as the AAV ITR or in the opposite orientation. AAV ITRs are directional. For example, the 3'ITR would be in the same orientation as the promoter 1/coding sequence 1 expression cassette in the examples above, but in the opposite orientation to the 5'ITR if the ITR and expression cassette were on the same nucleic acid strand.

A large body of evidence suggests that multicistronic expression constructs often do not achieve optimal expression levels compared to expression systems containing only one cistron. One of the proposed reasons for the low expression levels achieved with multicistronic expression constructs containing two or more promoter elements is the phenomenon of 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 publications are incorporated herein by reference for their disclosure of the phenomenon of promoter interference). Various strategies have been proposed to overcome the problem of promoter interference, such as by generating multicistronic expression constructs containing only one promoter driving the transcription of multiple nucleic acid coding sequences separated by internal ribosomal entry sites, or by separating cistrons containing their own promoter with transcriptional isolation elements. However, all proposed strategies to overcome promoter interference are burdened with their own set of problems. For example, expression of several cistrons driven by a single promoter usually results in uneven expression levels among the cistrons. In addition, some promoters cannot be efficiently isolated, and isolation elements are incompatible with some gene transfer vectors, such as some retroviral vectors.

In some embodiments, the present invention provides a multicistronic expression construct that provides for efficient expression of a first nucleic acid coding sequence driven by a first promoter and a second nucleic acid coding sequence driven by a second promoter without the use of a transcriptional insulator element. Various configurations of such multicistronic expression constructs are provided herein, for example, expression constructs comprising a first expression cassette comprising an intron and a second expression cassette positioned within the intron in either the same or opposite orientation as the first cassette. Other configurations are described in more detail elsewhere herein.

In some embodiments, the present invention provides multicistronic expression constructs that efficiently express two or more nucleic acid coding sequences. In some embodiments, the multicistronic expression construct comprises two expression cassettes. In some embodiments, the first expression cassette of the multicistronic expression construct provided herein comprises a first RNA polymerase II promoter and the second expression cassette comprises a second RNA polymerase II promoter. In some embodiments, the first expression cassette of the multicistronic expression construct provided herein comprises an RNA polymerase II promoter and the second expression cassette comprises an RNA polymerase III promoter.

In some embodiments of the present invention, the provided multicistronic expression construct is a recombinant AAV (rAAV) construct.

In some embodiments of the present invention, 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 coding sequence of the nucleic acid for optimized expression in target cells (e.g., mammalian cells) can be achieved by methods known in the art.

A "nucleic acid" sequence refers to a DNA or RNA sequence. In some embodiments, the proteins and nucleic acids of the invention are isolated. As used herein, the term "isolated" means artificially produced. As used herein, the term "isolated" with respect to nucleic acids means: (i) amplified in vitro, such as by polymerase chain reaction (PCR); (ii) produced recombinantly by cloning; (iii) purified by digestion and gel separation; or (iv) synthesized, such as by chemical synthesis. An isolated nucleic acid is a nucleic acid that is readily manipulated by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which the 5' and 3' restriction sites are known, or for which polymerase chain reaction (PCR) primer sequences have been disclosed, is said to be isolated, but such a nucleic acid sequence is not present in a native state in its natural host. An isolated nucleic acid may necessarily be substantially purified. For example, a nucleic acid isolated in a cloning or expression vector is not pure in the sense that it may contain only a small percentage of the target material in the cell in which it is contained. However, such a nucleic acid is isolated as the term is used herein because it is readily manipulated by standard techniques known to those skilled in the art. As used herein in relation to proteins or peptides, the term “isolated” refers to a protein or peptide that has been isolated from its natural environment or that has been produced artificially (e.g., by chemical synthesis, recombinant DNA technology, etc.).

In some embodiments, the isolated nucleic acid and rAAV described herein comprise one or more of the following structural elements (e.g., control or regulatory sequences): a chicken beta-actin (CBA) long promoter, an extended CBA intron, a Kozak sequence, an anti-VEGF agent encoding sequence (e.g., KH902) or a codon-optimized anti-VEGF agent encoding sequence (e.g., KH902), one or more microRNA binding sites, and a rabbit beta globin poly A (RBG) sequence. In some embodiments, one or more of the above control sequences are operably linked to an anti-VEGF agent encoding nucleic acid sequence (e.g., KH902).

As used herein, a nucleic acid sequence (e.g., a coding sequence) and regulatory sequences are said to be "operably linked" when they are covalently linked such that expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequences. If it is desired that the nucleic acid sequences be translated into a functional protein, the two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequences results in transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) introduce a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to translate into a protein. Thus, a promoter region may be operably linked to a nucleic acid sequence if the promoter region is capable of transcribing the DNA sequence such that the resulting transcript is translated into a desired protein or polypeptide. Similarly, two or more coding regions are functionally linked when they are linked such that their transcription from a common promoter results in the expression of two or more proteins translated out of frame.

In some embodiments of the present invention, the transgene comprises a nucleic acid sequence encoding an anti-VEGF agent (e.g., KH902) operably linked to a promoter. "Promoter" refers to a DNA sequence recognized by the cellular or introduced cellular synthesis machinery necessary for the initiation of specific transcription of a gene. The phrases "operably linked," "operably located," "under the control of," or "under transcriptional control" mean that the promoter is in the correct location and orientation relative to the nucleic acid to control RNA polymerase initiation and gene expression.

Typically, a promoter can be a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

Examples of constitutive promoters include, but are not limited to, the Rous sarcoma virus (RSV) retroviral LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, e.g., 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 phosphoglycerol kinase (PGK) promoter, and the EFla promoter [from Invitrogen]. In some embodiments of the present invention, the promoter is the RNA pol II promoter. In some embodiments of the present invention, the promoter is a chimeric cytomegalovirus (CMV)/chicken β-actin (CB) promoter (CBA promoter). In some embodiments of the present invention, the promoter is an RNA pol III promoter, such as U6 or H1.

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

In some embodiments of the present invention, the regulatory sequences allow expression of tissue-specific genes. In some cases, the tissue-specific regulatory sequences bind 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. Examples of tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: retinochisin proximal promoter, retinoid-binding protein interphotoreceptor enhancer (RS/IRBPa), rhodopsin kinase (RK), liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, creatine kinase (CK) promoter, mammalian desmin (DES) promoter, alpha-myosin heavy chain (a-MHC) promoter, or cardiac troponin T (cTnT) promoter. Other typical promoters include the beta-actin promoter, the hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654–64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063–8 (1998); immunoglobulin heavy chain promoter, T-cell receptor a-chain promoter, neuronal promoter such as the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503–15 (1993)), 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)), the use of which is obvious to a person skilled in the art.

In some embodiments of the present invention, the tissue-specific promoter is an eye-specific promoter. Examples of eye-specific promoters include the retinoschisin proximal promoter, the interphotoreceptor enhancer of retinoid binding protein (RS/IRBPa), rhodopsin kinase (RK), RPE65, and the human cone opsin promoter.

In some embodiments, the promoter is a chicken beta-actin (CB) promoter. The chicken beta-actin promoter can be a short chicken beta-actin or a long chicken beta-actin. In some embodiments, the promoter (e.g., the chicken beta-actin promoter) comprises 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 comprises a long CMV enhancer sequence and a long chicken beta-actin promoter. In some embodiments, the promoter comprises a short CMV enhancer sequence and a short chicken beta-actin promoter. However, one of skill 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).

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

In some embodiments, the transgene described herein comprises a Kozak sequence. A Kozak sequence is a nucleic acid motif containing the consensus sequence GCC(A/G)CC (SEQ ID NO: 4), which is found in eukaryotic mRNA and plays an important 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).

The isolated nucleic acid described herein may encode a transgene that further comprises a polyadenylation (poly-A) sequence. In some embodiments of the present invention, the transgene comprises a poly-A sequence that is a rabbit beta-globin (RBG) poly-A sequence.

In some embodiments, the transgene comprises a 3' untranslated region (3' UTR). In some embodiments, the invention provides isolated nucleic acids comprising a transgene encoding an anti-VEGF agent (e.g., KH902) and one or more microRNA (miRNA) binding sites. Without wishing to be bound by any particular theory, inclusion of miRNA binding sites in expression gene constructs allows for the regulation of transgene expression (e.g., inhibition of transgene expression) in cells and tissues where the corresponding miRNA is expressed. In some embodiments, inclusion of one or more miRNA binding sites in a transgene allows for the retargeting of transgene expression in a cell type-specific manner. In some embodiments of the present invention, one or more miRNA binding sites are located in the 3' untranslated region (3' UTR) of the transgene, such as between the last codon of the nucleic acid sequence encoding the anti-VEGF agent (e.g., KH902) and the poly A sequence.

In some embodiments of the present invention, the transgene comprises one or more (e.g., 1, 2, 3, 4, 5 or more) siRNA binding sites that redirect expression of an anti-VEGF agent (e.g., KH902) from immune cells (e.g., antigen-presenting cells (APCs) such as macrophages, dendrites, etc.). Inclusion of siRNA binding sites for immune-associated siRNAs can deactivate expression of the transgene (e.g., KH902) from antigen-presenting cells and, thus, reduce or eliminate immune responses (cellular and/or humoral) generated in a subject against the products of the transgene, such as described in U.S. Patent Application US 2018/0066279, the entire contents of which are incorporated herein by reference.

In some aspects, the present invention provides isolated nucleic acids comprising a transgene encoding an anti-VEGF agent (e.g., KH902) and one or more microRNA binding sites. Without wishing to be bound by any particular theory, it should be noted that the inclusion of microRNA (miRNA) binding sites in gene expression constructs allows for the regulation of transgene expression (e.g., inhibition of transgene expression) in cells and tissues where the corresponding miRNA is expressed. In some embodiments of the present invention, the inclusion of one or more miRNA binding sites in the transgene allows for retargeting the expression of the transgene in a cell type-specific manner. In some embodiments of the present invention, the one or more miRNA binding sites are located in the 3' untranslated region (3' UTR) of the transgene, such as between the last codon of the nucleic acid sequence encoding one or more GM3S proteins and the poly A sequence.

In some embodiments of the present invention, the transgene comprises one or more (e.g., 1, 2, 3, 4, 5, or more) miRNA binding sites that redirect expression of an anti-VEGF agent (e.g., KH902) to liver cells. For example, in some embodiments of the present invention, the transgene comprises one or more miR-122 binding sites.

In some embodiments of the present invention, the transgene comprises one or more (e.g., 1, 2, 3, 4, 5 or more) siRNA binding sites that redirect expression of one or more GM3S proteins from immune cells (e.g., antigen-presenting cells (APCs), such as macrophages, dendrites, etc.). Inclusion of siRNA binding sites for immune-associated siRNAs can redirect expression of the transgene from antigen-presenting cells, and thus reduce or eliminate immune responses (cellular and/or humoral) produced in a subject against transgene products, such as described in U.S. Patent Application US 2018/0066279, the entire contents of which are incorporated herein by reference.

As used herein, the term "immune cell-associated miRNA" is an miRNA that is preferentially expressed in cells of the immune system, such as antigen-presenting cells (APCs). In some embodiments of the present invention, the immune cell-associated miRNA is an miRNA expressed in immune cells, while exhibiting a higher expression level in an immune cell, which is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold higher than the expression level in a non-immune cell (e.g., a control cell such as a HeLa cell, a HEK293 cell, a mesenchymal cell, etc.). In some embodiments, the immune cell (immune cell) expressing the immune cell-associated miRNA is a B cell, a T cell, a killer T cell, a T helper cell, a y5 T cell, a dendritic cell, a macrophage, a monocyte, a vascular endothelial cell, or another immune cell. In some embodiments, the immune cell is a B cell 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 embodiments of the present invention, the immune system cell is a T cell expressing one or more of the following markers: ART2, CDla, CDld, CD11b (Mac-1), CD134 (OX40), CD150, CD2, CD25 (interleukin receptor 2 alpha), 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 OX 8, MRC OX-22, OX40, PD-1 (programmed death factor-1), RT6, TCR (T cell receptor), Thy-1 (CD90) and TSA-2 (total thymic Ag-2). In some embodiments of the present invention, the immune cell-associated miRNA is selected from: miR-31, miR-106a, miR-125a/b, miR-146a, miR-150, miR-155, miR-181a, miR-223, miR-221, miR-222, miR-7i, miR-148, miR-152, miR-126a, miR-142, miR-15, miR-150, miR-155, miR-16, miR-17, miR-18, miR-181a, miR-19a, miR-19b, miR-20, miR-21a, miR-223, miR-24-3p, miR-29a, miR-29b, miR-29c, miR-302a-3p, miR-30b, miR-33-5p, miR-34a, miR-424, miR-652-3p, miR-652-5p, miR-9-3p, miR-9-5p, miR-92a and miR-99b-5. In some embodiments, the transgene described herein comprises one or more miR-142 binding sites.

In some embodiments, the isolated nucleic acid comprises inverted terminal repeats. The isolated nucleic acids of the invention can be recombinant adeno-associated virus (AAV) vectors (rAAV vectors). In some embodiments, the isolated nucleic acid described herein comprises a region (e.g., a first region) comprising the first inverted terminal repeat (ITR) of an adeno-associated virus (AAV) or a variant thereof. The isolated nucleic acid (e.g., a recombinant AAV vector) can be packaged into a capsid protein and administered to a subject and/or delivered to a selected target cell. "Recombinant AAV (rAAV) vectors" typically consist of at least a transgene and its regulatory sequences, as well as the 5' and 3' terminal inverted repeats (ITRs) of an AAV. The transgene may comprise 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 document.

Typically, the ITR sequences are approximately 145 bp in length. Preferably, substantially all of the ITR coding sequences are utilized in the molecule, although some degree of minor modification of these sequences is permissible. The possibility of modifying these ITR sequences is within the skill of the art. See, for example, 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). An example of such a molecule useful in the present invention is a "cis-acting" plasmid containing a transgene, wherein the selected transgene sequence and associated regulatory elements are flanked 5' and 3' by AAV ITR sequences. The AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) comprising a second AAV ITR. In some embodiments, the isolated nucleic acid encoding the transgene is flanked by an AAV ITR (e.g., in a 5'-ITR-transgene-ITR-3' orientation). In some embodiments, the AAV ITRs are selected from the group consisting of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, and AAV6 ITR. In some embodiments, the second ITR is a mutant ITR that lacks a functional terminal resolution site (TRS). The term "lacking a terminal resolution site" can refer to an AAV ITR that contains a mutation (e.g., a sense mutation, such as a non-synonymous mutation or a missense mutation) that abolishes the function of the terminal resolution site (TRS) of the ITR, or a truncated AAV ITR that lacks a nucleic acid sequence encoding a functional TRS (e.g., an ATRS or AITR ITR). Without wishing to be bound by any particular theory, an rAAV vector containing an ITR lacking a functional TRS produces a self-complementary rAAV vector, such as described in McCarthy (2008) Molecular Therapy 16(10): 1648-1656. In some embodiments, the vectors described herein contain one or more AAV ITRs, and wherein at least one ITR is an ITR variant of a known ITR of an AAV serotype. In some embodiments of the present invention, the AAV ITR variant is a synthetic AAV ITR (e.g., an AAV ITR that is not found in nature). In some embodiments of the present invention, the AAV ITR variant is a hybrid ITR (e.g., a hybrid ITR comprising sequences derived from the ITRs of two or more different AAV serotypes).

In some embodiments of the present invention, an isolated nucleic acid (e.g., an rAAV vector) as described herein comprises in sequence from 5' to 3': an AAV 5' ITR, a CMV enhancer, a CBA promoter, an intron (e.g., a chicken beta-actin intron), a Kozak sequence, a transgene encoding an anti-VEGF agent (e.g., KH902), rabbit poly-A beta-globin, and an AAV 3' ITR. An exemplary isolated nucleic acid sequence is set forth in SEQ ID NO: 2. In some embodiments, the rAAV comprises an isolated nucleic acid comprising a nucleic acid sequence that is 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% identical to the nucleic acid sequence set forth in SEQ ID NO: 2 (Kozak sequence underlined; KH902 coding sequence in bold):

Also included within the scope of the present invention are vectors comprising an isolated nucleic acid as described herein. In some embodiments, the vector is a plasmid. In some embodiments, the plasmid comprising the rAAV vector further comprises one or more selection markers. Selection markers are known in the art and include antibiotic resistance markers. In some embodiments, the selection marker comprises a kanamycin resistance marker (e.g., a neomycin phosphotransferase II (nptII) gene). In some embodiments, the selection marker comprises an ampicillin resistance marker (e.g., a beta-lactamase gene).

An example of the complete sequence of the pAAV-CBA0KH902 vector is set forth in SEQ ID NO: 3. In some embodiments of the present invention, the rAAV vector comprises a nucleic acid sequence that is 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% identical to the nucleic acid sequence set forth in SEQ ID NO: 3:

In some embodiments, the rAAV comprises an isolated nucleic acid comprising a nucleic acid sequence that is 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% identical to the nucleic acid sequence set forth in SEQ ID NO: 30:

In some embodiments, the rAAV comprises an isolated nucleic acid comprising a nucleic acid sequence that is 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% identical to the nucleic acid sequence set forth in SEQ ID NO: 31:

In some embodiments, the rAAV comprises an isolated nucleic acid comprising a nucleic acid sequence that is 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% identical to the nucleic acid sequence set forth in SEQ ID NO: 32:

In some embodiments, an anti-VEGF agent (e.g., KH902) described herein can be delivered to a subject via a non-viral platform. In some embodiments, an anti-VEGF agent (e.g., KH902) described herein can be delivered to a subject via closed-ended double-stranded linear DNA (ccDNA). Transgene delivery (e.g., an anti-VEGF agent such as KH902) has been described previously, see, e.g., WO2017152149, the entire contents of which are incorporated herein by reference. In some embodiments, nucleic acids having asymmetric terminal sequences (e.g., asymmetric interrupted self-complementary sequences) form closed-ended double-stranded linear DNA structures (e.g., ccDNA), which in some embodiments have reduced immunogenicity compared to currently available gene delivery vectors. In some embodiments of the present invention, the ccDNA behaves as double-stranded linear DNA under native conditions, and it is transformed into single-stranded circular DNA under denaturing conditions. Without wishing to be bound by any particular theory, it should be noted that in some embodiments of the present invention, the ccDNA can be used to deliver a transgene (e.g., an anti-VEGF agent such as KH902) to a subject.

AAV-mediated transgene delivery to ocular tissue

Aspects of the present invention relate to compositions comprising a recombinant AAV that comprises a capsid protein 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 of the present invention, the nucleic acid further comprises an AAV ITR.

Isolated nucleic acids, vectors, rAAV, and compositions comprising an isolated nucleic acid described herein, a vector described herein, or a rAAV described herein can be delivered to a subject in compositions according to any suitable methods known in the art. For example, rAAV, preferably suspended in a physiologically compatible carrier (i.e., as a composition), can be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, 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.

In some embodiments of the present invention, administration of an isolated nucleic acid and/or rAAV as described herein delivers a transgene (e.g., KH902) to ocular tissue. Delivery of rAAV to a mammalian subject can be accomplished, for example, by intraocular injection, by subretinal injection, by topical administration to the eye (e.g., as eye drops), or by injection into the eye of a mammal directly into the ocular tissue (e.g., by intravitreal injection or intrastromal injection). As used herein, the term "ocular tissue" refers to any tissue of or contained within the eye. Non-limiting examples of ocular tissues include neurons, the retina (e.g., photoreceptor cells), the sclera, the choroid, the retina, the vitreous, the macula, the fovea, the optic disc, the lens, the pupil, the iris, the aqueous humor, the cornea (e.g., corneal cells such as keratocytes, corneal endothelial cells, corneal basal cells, corneal wing cells, and corneal squamous cells), the ciliary body of the conjunctiva, and the optic nerve. The retina is located at the back of the eye and is made up of photoreceptor cells. These photoreceptor cells (e.g., rods, cones) provide visual acuity by detecting color as well as contrast within the visual field. In some embodiments of the present invention, administration of an isolated nucleic acid and/or rAAV as described herein results in delivery of a transgene (e.g., KH902) to the cornea. In some embodiments of the present invention, administration of an isolated nucleic acid and/or rAAV as described herein results in delivery of a transgene (e.g., KH902) to corneal keratocytes.

Alternatively, delivery of rAAV to a mammalian subject may be by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, artery, or any other vascular channel. Non-limiting exemplary methods of intramuscular administration of rAAV include intramuscular (IM) injection and intravascular infusion into the limbs. In some embodiments of the present invention, rAAV is administered into the bloodstream by isolated limb perfusion, a technique well known in the art, wherein the method essentially involves isolating the limb from the systemic circulation prior to administration of the rAAV virions. A variation of the isolated limb perfusion method described in U.S. Patent No. 6,177,403 may also be used by one of skill in the art to administer virions into the vasculature of an isolated limb to potentially enhance transduction in muscle cells or tissues. In some embodiments, the rAAV or composition (e.g., a composition comprising an isolated nucleic acid or rAAV) described herein is administered by intravitreal injection. In some embodiments, the rAAV or composition (e.g., a composition comprising an isolated nucleic acid or rAAV) described herein is administered by intraocular injection. In some embodiments, the rAAV or composition (e.g., a composition comprising an isolated nucleic acid or rAAV) described herein is administered by subretinal injection. In some embodiments, the rAAV or composition (e.g., a composition comprising an isolated nucleic acid or rAAV) described herein is administered by intravenous injection. In some embodiments, the rAAV or composition (e.g., a composition comprising an isolated nucleic acid or rAAV) described herein is administered by intramuscular injection. In some embodiments, the rAAV or composition (e.g., a composition comprising an isolated nucleic acid or rAAV) described herein is administered by intratumoral injection.

In some embodiments of the present invention, administration of the isolated nucleic acid and/or rAAV described herein results in inhibition of VEGF (e.g., VEGF activity). In some embodiments of the present invention, administration of the isolated nucleic acid and/or rAAV described herein results in inhibition of VEGF (e.g., VEGF activity) in ocular tissue. 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 corneal vascular neovascularization (CNV) assessment in an alkali burn model or in a suture-induced CoNV model), etc.). In some embodiments, VEGF activity (e.g., VEGF activity) in subjects receiving an anti-VEGF agent (e.g., receiving an injection of the isolated nucleic acid and/or rAAV described herein) is 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 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 a subject who did not receive the injection, or compared to the same subject before receiving the anti-VEGF agent. In some embodiments, the inhibition of VEGF (e.g., VEGF activity) in a subject who has not previously received an injection or in a subject 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%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 100% of baseline, or at least 1-fold, at least 2-fold, at least 3-fold, at least less than 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 10-50-fold (e.g., 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold), at least 50-100-fold (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold) from baseline or more, compared to a subject previously administered an anti-VEGF agent (e.g., injected with isolated nucleic acid and/or rAAV as described herein). In some embodiments, administration of an anti-VEGF agent (e.g., an isolated nucleic acid and/or rAAV described herein) results in inhibition of VEGF (e.g., VEGF activity) for greater than 1 day, greater than 2 days, greater than 3 days, greater than 4 days, greater than 5 days, greater than 6 days, greater than 7 days, greater than 1 week (e.g., 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days), greater than 2 weeks (e.g., 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days), greater than 3 weeks (e.g., 22 days, 23 days, 24 days, 25 days, 25 days, 27 days, or 28 days), greater than 4 weeks (e.g., 29 days, 30 days, 40 days, 50 days, 60 days, 100 days or more), more than 1 month (e.g. 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more), more than 2 months (e.g. 2 months to 2.5 months, 2 months to 3 months, 2 months to 4 months, 2 months to 5 months, 2 months to 6 months, 2 months to 7 months, 2 months to 8 months, 2 months to 9 months, 2 months to 10 months, 2 months to 11 months, 2 months to 12 months), more than 3 months (e.g. 3 months to 4 months, 3 months to 5 months, 3 months to 6 months, 3 months to 7 months, 3 months to 8 months, 3 months to 9 months, 3 months to 10 months, 3 months to 11 months, 3 months to 12 months), more than 4 months (e.g. 4 months to 5 months, 4 months to 6 months, 4 months to 7 months, 4 months to 8 months, 4 months to 9 months, 4 months to 10 months, 4 months to 11 months, 4 months to 12 months), more than 5 months (e.g. 5 months to 6 months, 5 months to 7 months, 5 months to 8 months, 5 months to 9 months, 5 months to 10 months, 5 months to 11 months, 5 months to 12 months), more than 6 months (e.g. 6 months to 7 months, 6 months to 8 months, 6 months to 9 months, 6 months to 10 months, 6 months to 11 months, 6 months to 12 months), more than 7 months (e.g. 7 months to 8 months, 7 months to 9 months, 7 months to 10 months, 7 months to 11 months, 7 months to 12 months), more than 8 months (e.g. 8 months to 9 months, 8 months to 10 months, 8 months to 11 months, 8 months to 12 months), more than 9 months (e.g. 9 months to 10 months, 9 months to 11 months, 9 months to 12 months), more than 10 months (e.g. 10 months to 11 months, 11 months to 12 months), more than 11 months (e.g. 11 months to 12 months), more than 12 months (e.g. 12 to 15 months, 12 to 18 months, 12 to 21 months, 12 to 22 months), more than 1 year (e.g. 1 to 1.5 years), more than 2 years, more than 3 years, more 4 years, more than 5 years, more than 10 years, more than 15 years, more than 20 years and more.

The compositions of the present invention may comprise rAAV alone or 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 comprising one or more different transgenes.

In some embodiments of the present invention, the composition further comprises a pharmaceutically acceptable carrier. Suitable carriers can be readily selected by one of skill in the art based on the indication for which the rAAV is intended to be treated. For example, one suitable carrier includes saline, which can be prepared with various buffer solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The choice of carrier is not limiting to the present invention.

The compositions of the invention, in addition to rAAV and the carrier(s), may optionally contain other conventional pharmaceutical ingredients such as preservatives or chemical stabilizers. Examples of suitable preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, parachlorophenol, and poloxamers (non-ionic surfactants) such as Pluronic® F-68. Suitable chemical stabilizers include gelatin and albumin.

rAAV or compositions (e.g., compositions comprising isolated nucleic acid or rAAV as described herein) are administered in amounts sufficient to transfect cells of the desired tissue and to provide sufficient levels of gene transfer and expression without undesirable side effects. Common and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the organ of choice (e.g., intravitreal delivery to the eye), intraocular injection, subretinal injection, oral administration, inhalation (including intranasal and intratracheal delivery), intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intratumoral administration, and other parenteral routes of administration. If desired, routes of administration can be combined.

The dose of rAAV virions required to achieve a certain "therapeutic effect", for example, a unit dose expressed in the number of genome copies per kilogram of body weight (GC/kg), will vary depending on several factors, including, but not limited to: the route of administration of the rAAV virion, the level of gene or RNA expression required to achieve the therapeutic effect, the particular disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine the range of doses of rAAV virion required to treat a patient suffering from a particular disease or disorder based on the aforementioned factors, as well as other factors well known in the art.

An effective amount of an rAAV or composition (e.g., a composition comprising an isolated nucleic acid or rAAV as described herein) is an amount sufficient to target a particular animal, to target a desired tissue (e.g., muscle tissue, eye tissue, etc.). In some embodiments, an effective amount of an rAAV is administered to a subject at a pre-symptomatic stage of a degenerative disease. In some embodiments, an rAAV or composition is administered to a subject after one or more signs or symptoms of a degenerative disease have occurred. In some embodiments, an effective amount will depend primarily on factors such as the species of the subject, its age, body weight, the health of the subject, and the type of tissue being targeted, and thus, an effective amount may vary depending on the animal and tissue. For example, an effective amount of rAAV is typically in the range of about 10 ⁶ to 10 ¹⁶ genome copies (e.g., 1 x 10 ⁶ to 1 x 10 ^6, inclusive) in a solution of about 1 ml to about 100 ml. In some embodiments, the effective amount of rAAV is in the range of 1 x 10 ⁹ to 1 x 10 ¹⁴ rAAV genome copies. In some cases, a suitable dose is about 10 ¹¹ to 10 ¹² rAAV genome copies. In some embodiments, a suitable dose is about 10 ¹¹ to 10 ¹³ rAAV genome copies. In some embodiments, a suitable dose is about 10 ¹¹ to 10 ¹⁴ rAAV genome copies. In some embodiments, a suitable dose is about 10 ¹¹ to 10 ¹⁵ copies of the rAAV genome. In some embodiments, a suitable dose is about 10 ¹² to 10 ¹⁴ copies of the rAAV genome. In some embodiments, a suitable dose is about 10 ¹³ to 10 ¹⁴ copies of the rAAV genome. In some embodiments, the dose is about 1×0 ¹² , about 1.1×10 ¹² , about 1.2×10 ¹² , about 1.3×10 ¹² , about 1.4×10 ¹² , about 1.5×10 ¹² , about 1.6×10 ¹² , about 1.7×10 ¹² , about 1.8×10 ¹² , about 1.9×10 ¹² , about 1×10 ¹³ , about 1.1×10 ¹³ , about 1.2×10 ¹³ , about 1.3×10 ¹³ , about 1.4×10 ¹³ , about 1.5×10 ¹³ , about 1.6×10 ¹³ , about 1.7×10 ¹³ , about 1.8×10 ¹³ , about 1.9×10 ¹³ , or about 2.0×10 ¹⁴ vector genome copies (vg) per kilogram (kg) of body weight. In some embodiments, a suitable dose is about 4×10 ¹² to 2×10 ¹³ rAAV genome copies. In some embodiments, a suitable dose is about 1.5×10 ¹³ vg/kg when administered intravenously. In some embodiments, a suitable effective dose for target tissues (e.g., the eye) is 10 ¹² to 10 ¹³ rAAV genome copies.

In some embodiments of the present invention, the effective dose for target tissues (e.g., the eye) is 10 ¹³ - 10 ¹⁴ copies of the rAAV genome.

In some embodiments, the subject is administered rAAV by injection. In other embodiments, the subject is administered rAAV by topical administration (e.g., eye drops). In some embodiments, the effective amount of rAAV is an amount sufficient to express an effective amount of an anti-VEGF agent (e.g., KH902) in a target tissue (e.g., the eye) of the subject.

In some embodiments of the present invention, the delivery of an effective amount of rAAV by injection (e.g., the delivery of rAAV encoding an anti-VEGF agent (e.g., KH902) is in an amount sufficient to express an effective amount of the anti-VEGF agent (e.g., KH902) in the target tissue). In some embodiments of the present invention, the delivery of an effective amount of rAAV encoding an anti-VEGF agent (e.g., KH902) is sufficient to deliver from 10 μg to 10 mg of the anti-VEGF agent (e.g., KH902; or any value in between) to a subject in the eye by suitable routes of administration (e.g., by intraocular injection, intravenous injection, intraperitoneal injection, and intramuscular injection). In some embodiments of the present invention, rAAV encoding an anti-VEGF agent (e.g., KH902) is sufficient to deliver 20 μg to 5 mg, or any value in between, of the anti-VEGF agent (e.g., KH902) to a subject per eye. In some embodiments, the amount of rAAV encoding an anti-VEGF agent (e.g., KH902) administered to a subject 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 an anti-VEGF agent (eg, KH902).

In some embodiments of the present invention, rAAV encoding an anti-VEGF agent (e.g., KH902) is administered to a subject once daily, once weekly, once every two weeks, once monthly, once every 2 months, once every 3 months, once every 6 months, once annually, or once per lifetime.

In some embodiments of the present invention, delivery of an effective amount of rAAV is performed by local administration, such as eye drops (e.g., delivery of rAAV encoding an anti-VEGF agent (e.g., KH902) is performed in an amount sufficient to express an effective amount of the anti-VEGF agent (e.g., KH902) in the target tissue). In some embodiments of the present invention, eye drops containing the encoding rAAV are administered to a subject once a week, once a month, once every 3 months, once every 6 months, or once a year.

In some embodiments of the present invention, the eye drops comprise rAAV encoding an anti-VEGF agent (e.g., KH902) in an amount sufficient to deliver the anti-VEGF agent at a concentration of 1 mg/mL to 20 mg/mL. In some embodiments of the present invention, the eye drops comprise rAAV encoding an anti-VEGF agent (e.g., KH902) in an amount sufficient to deliver the anti-VEGF agent at a concentration of 2.5 mg/mL to 10 mg/mL. In some embodiments of the present invention, the eye drops comprise rAAV encoding an anti-VEGF agent (e.g., KH902) in an amount sufficient to deliver the anti-VEGF agent at a concentration 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 embodiments, the eye drops are administered in a volume 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.

An effective amount of rAAV or a composition (e.g., a composition comprising an isolated nucleic acid or rAAV described herein) may also depend on the route of administration. For example, exposure to ocular tissue (e.g., cornea) via intrastromal administration or subcutaneous injection may, in some cases, require different (e.g., higher or lower) doses than exposure to ocular tissue (e.g., cornea) via other routes of administration (e.g., systemic administration, local administration). In some embodiments, intrastromal injection (IS) of certain rAAV serotypes (e.g., AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.39, and AAVrh.43) mediates efficient transduction into ocular cells (e.g., corneal cells, retinal cells, etc.). Thus, in some embodiments, the injection is an intrastromal injection (IS). In some embodiments of the present invention, the injection is a local administration (eg, local administration to the eye). In some cases, multiple doses of rAAV are administered.

In some embodiments of the present invention, rAAV compositions of the present invention are formulated to reduce aggregation of AAV particles in the composition, particularly where high concentrations of rAAV are present (e.g., ~10 ¹³ GC/mL or more). 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).

The use of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable dosage and treatment regimens using the specific compositions described herein for various treatment regimens.

Typically, these compositions may contain at least about 0.1% of the active compound or more, although the percentage of active ingredient(s) may, of course, vary, and may be from about 1% or 2% to about 70% or 80% or even more by weight or volume of the total composition. Naturally, the amount of active compound in each therapeutically useful composition may be such as to provide a suitable dosage of the compound for any given unit dose. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations will be taken into account by one skilled in the art of preparing pharmaceutical compositions, and it may also be desirable to take into account various dosage requirements and requirements associated with a treatment regimen.

In some cases, it will be desirable to deliver the rAAV therapeutic constructs in the suitable pharmaceutical compositions described herein intravitreally, intraocularly, subretinally, intrastromally, subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, orally, intraperitoneally, or by inhalation. In some embodiments, the routes of administration described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363 (each of which is incorporated herein by reference in its entirety) can be used to deliver rAAV. In some embodiments, a preferred route of administration is portal vein injection.

The pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions as well as sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases, the pharmaceutical form is sterile and fluid to the extent that it is easy to syringe. It must be stable under the conditions of manufacture and storage, and it must also be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and/or vegetable oils. Proper fluidity can be maintained, for example, by using a particle coating such as lecithin, and by maintaining the required particle size in the case of dispersion and by using surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, for example sugars or sodium chloride. Prolonged absorption of the injectable compositions can be ensured by using agents in the compositions that delay absorption, for example aluminum monostearate and gelatin.

For administration of an aqueous injection solution, the solution, for example, may be suitably buffered if necessary, and the liquid diluent first rendered isotonic with a sufficient amount of saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Such sterile aqueous media are known to those skilled in the art and can be used. For example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or infused into the fluid (see, for example, "Remington's Pharmaceutical Sciences" ^15th Edition, pp.1035-1038, 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. In any event, the person responsible for administration determines the appropriate dose for the particular host.

Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in an appropriate solvent with various other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from among those enumerated above. In the case of sterile powders used for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization techniques, which yield a powder of the active ingredient with any additional desired ingredient from a previously sterile-filtered solution.

The rAAV compositions disclosed herein can be prepared using rAAV in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein) which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids, such as, acetic, oxalic, tartaric, mandelic, etc. Salts formed with free carboxyl groups can also be prepared using inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc. After preparation of solutions, they will be administered in a manner compatible with the resulting composition of the compositions, and in such an amount that is therapeutically effective. The compositions can be easily administered in various dosage forms, such as injection solutions, drug-release capsules, etc.

As used herein, the term "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such vehicles and agents for pharmaceutically active substances is well known in the art. Additional active ingredients may also be included in the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not cause an allergic or other untoward reaction when administered to a host.

For introducing the compositions of the present invention into suitable host cells, delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used. In particular, rAAV vectors delivering transgenes can be encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc.

Such formulations may be preferred for administering the pharmaceutically acceptable nucleic acid compositions or rAAV constructs disclosed herein. The formation and use of liposomes is generally well known to those skilled in the art. Recently, liposomes with improved serum stability and long half-lives have been developed (see U.S. Patent No. 5,741,516). In addition, various methods for using liposomes and liposome-like formulations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

Liposomes have been successfully used with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free from the DNA length limitations typical of viral-based delivery systems. Liposomes have been effectively used to deliver genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials have been completed to study the efficacy of liposome-mediated drug delivery.

Liposomes are formed from phospholipids that are dispersed in an aqueous medium, and they spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs). MLVs typically have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with a diameter of 200 to 500 Å, which contain an aqueous solution in the core.

Alternatively, rAAV nanocapsule formulations can be used. Nanocapsules can generally entrap substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) should be produced using polymers that are degradable in vivo. Biodegradable polyalkyl cyanoacrylate nanoparticles that meet these requirements are proposed.

In addition to the delivery methods described above, the following methods are also contemplated as alternative methods for delivering rAAV compositions to the host. Sonophoresis (i.e., ultrasound), as described in U.S. Patent No. 5,656,016, has been used as a means to enhance the rate and efficiency of drug penetration into and through the circulatory system. Other contemplated alternatives for drug delivery include intraosseous injection (U.S. Patent No. 5,779,708), microarray devices (U.S. Patent No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Patents Nos. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

In some embodiments, the anti-VEGF agent (e.g., KH902) described herein is delivered to the subject via ccDNA. Any compositions comprising ccDNA encoding the anti-VEGF agent (e.g., KH902) are also within the scope of the present invention. In some embodiments, the ccDNA encoding the anti-VEGF agent (e.g., KH902) and compositions thereof can be administered to the subject using any suitable method described herein. In some embodiments, the delivery of an effective amount of ccDNA encoding the anti-VEGF agent (e.g., KH902) by injection is in an amount sufficient to express an effective amount of the anti-VEGF agent (e.g., KH902) in the target tissue. In some embodiments of the present invention, delivery of an effective amount of ccDNA encoding an anti-VEGF agent (e.g., KH902) is sufficient to deliver 10 μg to 10 mg of the anti-VEGF agent (e.g., KH902), or any other amount in the specified range, to the eye of a subject is carried out by suitable routes of administration (e.g., intraocular injection, intravenous injection, intraperitoneal injection, and intramuscular injection). In some aspects of the present invention, it is recognized that one potential side effect of administering an AAV to a subject is a response of the subject to the AAV, including inflammation. In some embodiments of the present invention, the subject is immunosuppressed prior to administration of one or more rAAVs as described herein.

As used herein, the term "immunosuppression" or "suppression of an immune response" refers to a decrease in the activation or effectiveness of an immune response in a subject. Immunosuppression can be induced in a subject using one or more (e.g., several, such as 2, 3, 4, 5, or more) agents, including, but not limited to, rituximab, methylprednisolone, prednisolone, sirolimus, immune globulin injection, prednisolone, solu-medrol, lansoprazole, trimethoprim/sulfamethoxazole, methotrexate, and any combination thereof. In some embodiments, the immunosuppressive regimen comprises the administration of sirolimus, prednisolone, lansoprazole, trimethoprim/sulfamethoxazole, or any combination thereof.

In some embodiments, the methods described herein further comprise the step of inducing immunosuppression in the subject (e.g., administering one or more immunosuppressants) prior to administering the rAAV (e.g., the rAAV or pharmaceutical composition described herein) to the subject. In some embodiments, the subject is immunosuppressed (e.g., the subject is induced to be immunosuppressed) for a period of between about 30 days and about 0 days (e.g., any time between 30 days prior to administration of the rAAV and the day of administration of the rAAV, inclusive) prior to administration of the rAAV to the subject. In some embodiments, the subject is pretreated with immunosuppressive agents (e.g., rituximab, sirolimus, and/or prednisone) for at least 7 days.

In some embodiments, the methods described herein further comprise co-administering or pre-administering an additional agent to a subject to which the rAAV or a pharmaceutical composition comprising the rAAV of the invention is administered. In some embodiments, the additional agent is selected from the group consisting of miglustat, keppra, prevacid, clonazepam, and any combination thereof. In some embodiments, the rAAV (e.g., the rAAV of KH902) and the additional agent can be administered to the subject in any order. In some embodiments, the rAAV (e.g., the rAAV of KH902) and the additional agent (e.g., miglustat, keppra, prevacid, clonazepam) are administered to the subject concurrently. In some embodiments, the rAAV (e.g., the rAAV for KH902) and the additional agent (e.g., miglustat, keppra, prevacid, clonazepam) are co-administered to the subject, such as in the same composition or in different compositions. In some embodiments, the rAAV (e.g., the rAAV for KH902) is administered prior to the additional agent (e.g., miglustat, keppra, prevacid, clonazepam). In some embodiments, the rAAV (e.g., the rAAV for KH902) is administered after the additional agent (e.g., miglustat, keppra, prevacid, clonazepam) is administered. In some embodiments of the present invention, the rAAV (e.g., the rAAV for KH902) and the additional agent (e.g., miglustat, keppra, prevacid, clonazepam) are administered to the subject at different frequencies, such as the subject is administered the rAAV (e.g., the rAAV for KH902) every month, every two months, every six months, every year, every two years, every three years, every 5 years or more, but is administered the additional agent (e.g., miglustat, keppra, prevacid, clonazepam) daily, weekly, biweekly, monthly, twice daily, three times daily, or twice weekly.

In some embodiments of the present invention, the immunosuppressed state of the subject is maintained during and/or after administration of the rAAV or pharmaceutical composition. In some embodiments of the present invention, the subject is immunosuppressed (e.g., is administered one or more immunosuppressants) for a period of 1 day to 1 year after administration of the rAAV or pharmaceutical composition.

Treatment options for VEGF and/or angiogenesis-related diseases

A number of aspects of the present invention relate to methods of delivering a transgene encoding an anti-VEGF agent (e.g., KH902) to a subject (e.g., to a cell of the subject). In some embodiments of the present invention, the subject is a human. In some embodiments of the present invention, the subject is a non-human mammal. Non-limiting examples of non-human mammals include a mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate.

In some embodiments, the present invention relates to a method of inhibiting VEGF activity in a subject in need thereof. In some embodiments, the methods described herein are useful for treating a subject having or suspected of having a VEGF-related disease. As used herein, the term "VEGF-related diseases" refers to a group of diseases associated with aberrant VEGF activity/signaling. VEGF is a signaling protein produced by cells that stimulates the formation of blood vessels. VEGF is a known angiogenesis-inducing factor. In some embodiments, the methods described herein are useful for treating a subject having or suspected of having a disease associated with angiogenesis. Angiogenesis-related disease, as used herein, refers to diseases associated with abnormal angiogenesis. Non-limiting examples of angiogenesis-related diseases include angiogenesis-dependent cancer, 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), blood-borne tumors such as leukemias, metastatic tumors, benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas), rheumatoid arthritis, psoriasis, rubeosis, Auxier-Webber syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasias, hemophilic joints, or angiofibromas.

In some embodiments of the present invention, angiogenesis-related ocular diseases include, but are not limited to, corneal neovascularization (CoNV), diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear syndrome, atopic keratitis, superior limbal keratitis, pterygium sicca, Sjogren's disease, acne rosacea, phylectenulosis, syphilis, mycobacterial infections, lipid dystrophy, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoal infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson disease, pemphigoid radial keratotomy and corneal graft rejection, sickle cell anemia, sarcoid, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, obstructive carotid disease, chronic uveitis/vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, Eales disease, Behcet's disease, infections causing retinitis or choroiditis, presumed ocular histoplasmosis, Best disease, myopia, optic pits, Stargardt disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma or post-laser complications.

As used herein, the term "treatment" refers to the use or administration of a composition comprising an anti-VEGF agent (e.g., KH902) to a subject who has a symptom or disease associated with aberrant VEGF activity or associated with angiogenesis, or who has a predisposition to a disease associated with aberrant VEGF activity or angiogenesis, to cure, eliminate, alleviate, reduce, modify, restore, improve, or affect the disorder, the symptom of the disease, or the predisposition to a disease associated with aberrant VEGF activity or angiogenesis. In some embodiments of the present invention, administration of an anti-VEGF agent results in a decrease in VEGF activity by 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a control value of activity. Methods for measuring VEGF activity are known in the art. A non-limiting exemplary reference value for VEGF activity may be the VEGF activity in the same subject before treatment with an anti-VEGF agent. In some embodiments of the present invention, administration of an anti-VEGF agent results in a decrease in angiogenesis by 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value. Methods for assessing the level of angiogenesis are known in the art. A non-limiting exemplary reference value may be the level of angiogenesis in the same subject before treatment with an anti-VEGF agent.

In some embodiments, the present invention relates to a method for reducing corneal neovascularization (CoNV) in a subject in need thereof (e.g., reducing CoNV when compared to an untreated subject or when compared to a subject before treatment). In some embodiments, the method reduces CoNV by at least 5%, 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 85%, at least 90%, at least 95%, at least 99%, or 100% when compared to an untreated subject or when compared to a subject before treatment. Methods for assessing CoNV are known in the art (e.g., optical coherence tomography angiography (OCTA), indocyanine green angiography (ICGA), etc.). Any suitable method for assessing CoNV can be used within the framework of the present invention.

Alleviation of a disease associated with aberrant VEGF activity or angiogenesis includes delaying the development or progression of the disease or reducing the severity of the disease. Alleviation of the disease does not necessarily require therapeutic results. As used herein, the term "delay" in the development of a disease (such as a disease associated with aberrant VEGF activity or angiogenesis) means postponing, preventing, slowing, arresting, stabilizing the disease and/or delaying the progression of the disease. This delay may be of varying duration, depending on the history of the disease and/or the subjects being treated. A method that "delays" or alleviates the development of a disease or delays the onset of a disease is a method that reduces the likelihood of developing one or more symptoms of the disease in a given period of time and/or reduces the severity of symptoms in a given period of time, compared to the case where the said method is not used. Such comparisons are typically based on clinical studies using a sufficient number of subjects to obtain a statistically significant result.

"Development" or "progression" of a disease means the manifestation of initial signs of a disease and/or subsequent progression of the disease. Development of a disease can be detected and assessed using standard clinical methods well known in the art. However, development also refers to progression, which may be unnoticeable. Within the framework of the present invention, reference to development or progression refers to the biological manifestation of symptoms. "Development" includes occurrence, manifestation and recurrence. As used herein, the term "onset" or "occurrence" of a disease associated with aberrant VEGF activity or angiogenesis includes the initial manifestation and/or recurrence of the disease.

EXAMPLES

Example 1: rAAV vector platform for delivery of Conbercept (KH902)

Here, an rAAV vector platform for the delivery of conbercept (KH902), an anti-VEGF therapeutic, to the retina by intravitreal administration (or other routes) is described. The unique construct is a single-stranded AAV vector genome that contains the KH902 transgene driven by a CMV/chicken β-actin enhancer promoter regulatory cassette (Figs. 1A-1C). A Kozak sequence is included upstream of the KH902 initiation codon to enhance translation (Fig. 1C). Following delivery of the cis plasmid (Fig. 1A) into packaging cell lines that express the AAV Rep and Cap genes and obligatory helper genes, via transplasmid cotransfection or stable integration, sequences that include and are flanked by inverted terminal repeat (ITR) sequences are packaged into AAV capsid virions.

Cells were infected or transduced with the resulting ssAAV-KH902 virions expressing secreted KH902, which was detected by standard Western blotting (Fig. 2). Transduction of retinal pigment epithelial (RPE) cell lines with ssAAV-KH902 packaged within an AAV2 capsid resulted in the expression of a KH902 protein with a molecular weight that matched that of the conbercept produced in Chinese hamster ovary (CHO) cell lines. These data demonstrate that the rAAV-KH902 vector can be packaged into a variety of AAV capsids and that when cells are infected, they can efficiently secrete KH902.

Conditioned media from rAAV-KH902-infected RPE cells strongly inhibited angiogenesis, as evidenced by a reduction in vascular endothelial growth factor (VEGF)-induced tubulogenesis (Figs. 3A and 3B) and proliferation (CCK-8 assay, Fig. 3C) of human umbilical vein endothelial cells (HUVECs), similar to conbercept. These data indicate that rAAV-KH902-infected cells can express and secrete functional antiangiogenic KH902 in vitro.

Intravitreal injection of rAAV2-CBA-KH902 in neonatal mice prevented normal retinal vascular development (Fig. 4). These data indicate that AAV-KH902 virions inhibit vascularization in vivo. To determine whether this vector construct could inhibit choroidal neovascularization, the rAAV2-CBA-KH902 vector was tested in a mouse retinopathy of prematurity (ROP) model (Fig. 5). Intravitreal injection of rAAV2-CBA-KH902 in mice and subsequent exposure to hyperoxic conditions resulted in a decrease in the percentage of eyes with detectable edema, as well as the amount of edema in the eyes of treated mice, compared to control eyes of uninjected mice. These data suggest that AAV-KH902 is a potential and promising platform for gene therapy to prevent, and possibly reverse, choroidal neovascularization.

Example 2: Intravitreal injection of AAV2 vector is effective in delivering KH902, preventing oxygen-induced retinopathy and vascularization in mice Neonatal mice were injected intravitreal with the vector at postnatal days (PN) 1-3. Each mouse was injected with a vector packaging the EGFP transgene (rAAV-EGFP) in one eye and with a mixture of vectors packaging the KH902 transgene (rAAV-KH902) and rAAV-EGFP in a 5:1 ratio, respectively, in the contralateral eye. In all cases, the total dose was 1.5E9 vector genome copies (vg) per eye in a volume of 1 μl. Mice were then maintained under 70% oxygen conditions until PN 7 and then placed in normoxic conditions (20-21% oxygen) until PN 11. Mice were sacrificed at PN 18, eyes were removed and imaged (Figs. 6A-6B and Figs. 7A-7B). The pathology of treated eyes was then assessed by visual inspection and scoring (Fig. 8). Eyes treated with rAAV-EGFP alone reflect the degree of induction under hyperoxic conditions and serve as an internal control for pathology assessment. It should be noted that the absence of edema does not mean that hyperoxia cannot induce retinopathy, and the presence of edema in rAAV-KH902-treated eyes does not mean that the vector is ineffective. Resolution of vascular pathology is determined by the presence or absence of aneurysmal nodules.

In rAAV2-Egfp-treated eyes, which serve as a negative control, vascular pathologies were observed resulting from excessive proliferation and nodule formation of vascular aneurysms (Fig. 6B, lower panel). Treatment with rAAV2-KH902 effectively prevented the eye pathologies (Figs. 6A–6B) and also reduced vascular development to some extent (Fig. 6B, right panels). In contrast, rAAV8-KH902 was very ineffective in preventing vascular pathologies (Figs. 7A–7B). This observation correlates with the low transduction of rAAV8-EGFP in retinal tissues (Fig. 7B, left panels). However, in areas where there is some transduction, rAAV8-KH902 is able to partially prevent the pathologies (Fig. 7B, right panels, and Fig. 8).

Overall, hyperoxia-induced retinopathy worked in all mice, even when the eyes did not develop edema (Fig. 8). Importantly, the KH902 transgene was able to reverse abnormal vascularization with rAAV2 but poorly with rAAV8. However, these results do not predict the outcome of treatment in adult animals, since normal vascular development is complete by this time.

Example 3: Human-derived AAV capsid variants for efficient and safe gene delivery to the retina

To assess whether AAV2 variant capsids and AAV2/3 hybrid capsids are capable of delivering the transgene to retinal cells, AAV2 variants and AAV2/3 hybrid capsids were packaged into rAAV carrying the EGFP transgene tag and injected into mice either intravitreally or subretinally. GFP expression in the retina reflects the ability of a particular capsid protein to transduce retinal cells.

In the initial study, eight AAV2 variants (v224, v326, v358, v46, v56, v66, v67, and v81) and seven AAV2/3 hybrid variants (v439, v453, v513, v551, v556, v562, and v598) were tested. rAAVs containing each capsid variant and the EGFP transgene tag were administered intravitreal to mice (three mice per group; 45 mice in total). Transduction efficiency was assessed by fundoscopy at two weeks (Fig. 9A) and four weeks (Fig. 9B) after injection. Among all the variants tested, v56, v224, and v326 had the highest transduction, as determined by fundoscopy. These three capsids were used to package KH902 for subsequent studies in mouse models of laser-induced choroidal neovascularization. This study found that the AAV2 variants performed better than the AAV2/3 hybrid variants in mice.

The efficacy of rAAV-KH902 was investigated in a model for the treatment of laser-induced ocular pathology. Laser injury induces choroidal neovascularization (CNV) manifestations, and KH902 can reduce CNV in eyes after laser injury. The number of CNV manifestations and the size of CNV can be measured as indicators of the effective delivery of KH902 to the eyes. In this study, the eyes of mice were laser-injured 5 days before rAAV injections. Mice were injected with GFP as a control or AAVv224-KH902. As shown in Fig. 10, the number of CNV manifestations was reduced to less than 80% in AAVv224-KH902-treated mice 20 days after laser eye injury, compared with the GFP-treated control group. In the same mouse model, AAVv56-KH902 and AAVv326-KH902 showed similar therapeutic efficacy to AAVv224-KH902. In addition, CNV area was also measured, and the results suggest that delivery of KH902 with either the AAV2 variant or the AAV2/3 hybrid variant may reduce CNV size.

Previously, it was shown that overexpression of KH902 causes ocular lesions in a dose-dependent manner due to accumulation of immune cells in the vasculature. Such lesions can be observed as white streaks in the eye using brightfield fundoscopy. To test whether v224-KH902 would cause similar ocular lesions, mice were injected intravitreally with v224-KH902 as well as control capsid-KH902, which was previously shown to cause ocular lesions, with control capsid-KH902 administered at a 1:10 dilution, a 1:20 dilution, a 1:50 dilution, or a 1:100 dilution. As shown in Fig. 11, undiluted control capsid-KH902 caused ocular lesions, while dilutions of control capsid-KH902 reduced the lesions. No lesions were observed when V224-KH902 was injected into the eyes of mice.

Furthermore, the capsid variants (e.g., AAV2 variants, AAV2/3 hybrid variants, and AAV8 variants) showed improved packaging efficiency compared to their respective capsid prototype proteins. As shown in Fig. 12, the in vitro packaging yield assessment performed by PCR of crude lysate is presented as a bar graph showing the relative packaging yields for AAV2 variants (upper panel), AAV2/3 variants (middle panel), and AAV8 variants (lower panel). The packaging yield values for each capsid are expressed as a percentage of the yield provided by their prototype forms: AAV2, AAV3b, and AAV8, respectively. Capsid variant v56 showed a 9.42-fold increase compared to AAV2; v224 showed an 8.96-fold increase compared to AAV2, and v326 showed a 9.79-fold increase compared to AAV2. The total number of capsids is shown on the x-axis.

Example 4:

The normal mammalian cornea is transparent and devoid of blood and lymphatic vessels. In pathological conditions where the balance of pro- and antiangiogenic factors is disrupted, such as inflammation, hypoxia, and limbal barrier dysfunction, corneal neovascularization (CoNV) may occur. CoNV reduces corneal transparency and leads to visual impairment. It affects over 1.4 million people per year in the United States. Current treatments for CoNV include topical steroids and nonsteroidal anti-inflammatory drugs, laser cauterization, fine-needle diathermy, and amniotic membrane transplantation. However, all of the above methods have limited efficacy and are accompanied by associated side effects. Vascular endothelial growth factor (VEGF) plays a critical role in corneal angiogenesis, but none of the above treatments specifically target this key molecule. The successful use of anti-VEGF agents in corneal neovascularization has led to a surge of interest in testing these agents for their ability to drive corneal angiogenesis in animal models and in clinical trials. For example, several early studies demonstrated the potential of bevacizumab in inhibiting CoNV via topical, subconjunctival, perilimbal, and intrastromal administration in multiple animal models and clinical trials. Since then, more anti-VEGF agents have been developed and applied in clinical therapy for corneal neovascularization, including ranibizumab and aflibercept. However, due to the short half-life of these VEGF-neutralizing proteins, their limited duration of action is an obvious obstacle to achieving sustainable and effective treatment of CoNV. Significant advances in gene therapy technologies have inspired efforts to enhance the resistance of anti-VEGF agents by packaging an expression cassette encoding a VEGF-neutralizing protein into recombinant adeno-associated virus (rAAV) vectors, which are very attractive vehicles for in vivo delivery of therapeutic transgenes in ocular diseases. The use of rAAV vectors is promising due to their low immunogenicity, genotoxicity, and high transduction profile. A single dose of rAAV vector can provide robust and sustained gene expression, which is important to achieve the treatment goal and reduce the patient burden in the treatment of chronic corneal diseases.

In this study, the aim was to develop a novel therapeutic approach using rAAV-mediated exogenous expression of KH902 with a single administration to sustainably prevent and inhibit angiogenesis in damaged corneas.

Intrastromal injection of rAAV2 and rAAV8 vectors ensures efficient transduction into corneal cells

To ensure successful rAAV-mediated gene therapy, the route of administration must ensure efficient delivery and expression of the therapeutic gene within the target tissue. Currently, therapeutic agents targeting the cornea are mainly administered via local instillation, subconjunctival injection, or intrastromal injection. Local administration of rAAV vectors without removal of the surface epithelium has shown relatively low transduction efficiency; therefore, the biodistribution of rAAV vectors was primarily compared after subconjunctival and intrastromal routes of administration. Mice were injected with rAAV2 or rAAV8 packaged with the eGFP reporter transgene under the universal chicken β-actin promoter (rAAV2-eGFP or rAAV8-eGFP) in equal doses (1.6×10 ¹⁰ genome copies (GC)/cornea) either via the intrastromal or subconjunctival route (Figs. 13A, 13D). Corneal eGFP expression levels were assessed two weeks after injection by direct detection of the eGFP signal using a live animal imaging system (Micron IV chamber). Importantly, eGFP signal was successfully detected in the corneas of mice dosed via the intrastromal route but not via the subconjunctival route (Figs. 13B, 13D, 20B, 20C). To further confirm the biodistribution pattern of eGFP in the cornea, eGFP fluorescence was analyzed in enucleated globe sections two weeks after administration. Consistent with the obtained real-time imaging data, eGFP was expressed throughout the cornea following intrastromal vector injection. In contrast, subconjunctival injections resulted in expression in limbal, conjunctival, and scleral tissues, while expression in the cornea was weak or absent (Figs. 13C, 13F, 20A). Collectively, these data indicate that intrastromal injection is a more efficient route of delivery of rAAV2 and rAAV8 vectors to achieve widespread transduction in the cornea.

Corneal cell tropism and kinetics of eGFP and KH902 expression mediated by rAAV2 and rAA V8

To test the kinetics of rAAV2- and rAAV8-mediated transgene expression upon intrastromal injection into the cornea, rAAV2-eGFP or rAAV8-eGFP were injected intrastromally at an equal dose of 1.6 × 10 ¹⁰ GC per cornea. The rAAV8-mediated eGFP signal was readily detectable as early as 28 h post-injection in live animal imaging (Fig. 14A). However, the eGFP signal in rAAV2-eGFP-treated corneas was not observed for up to one week post-injection and was then observed at a much weaker signal intensity than that observed with rAAV8-eGFP treatment (Fig. 14A). Then, rAAV2-KH902 or rAAV8-KH902 was injected intrastromally into the cornea of wild-type mice at a dose of 1.6 × 10 ¹⁰ GC/cornea, and the relative expression of KH902 mRNA was assessed at weeks 1 and 2, and months 1, 2, and 3 using droplet digital PCR (ddPCR). The rAAV8-KH902-treated group showed robust KH902 mRNA expression, which peaked at one week after injection. Meanwhile, rAAV2 also resulted in detectable KH902 expression, but at significantly lower levels and with a delayed peak at two weeks after injection. After reaching peak levels, rAAV2- and rAAV8-mediated KH902 mRNA expression gradually declined but remained detectable for three months after injection, which was the final time point in the experiment (Fig. 14B). Notably, the relative rAAV8-mediated KH902 mRNA expression was higher than that achieved by rAAV2 at each time point. This observation is consistent with the eGFP signals conveyed by the rAAV-eGFP vectors (Fig. 14A).

To investigate the transduction tropism of rAAV2 and rAAV8 in the mouse cornea, corneal tissue sections were analyzed after intrastromal injections of rAAV2-eGFP or rAAVS-eGFP two weeks after injection (Fig. 14C). The results showed that the eGFP signal mainly co-localized with vimentin, a keratocyte marker, indicating that rAAV2 and rAAV8 mainly transduce keratocytes. Sporadic expression in epithelial cells was also observed. On the other hand, neither rAAV2-eGFP nor rAAV8-eGFP transduced corneal endothelial cells (Fig. 14C: i, ii). Then, the distribution of KH902 protein was examined using anti-human IgG (H+L) antibody in corneas transduced with rAAV2-KH902 or rAAV8-KH902 (1.6×10 ¹⁰ GC/cornea). KH902 protein after rAAV2 and rAAV8 transduction was mainly detected in keratocytes and rarely in corneal epithelial cells (Fig. 14C: iii, iv, and Figs. 21A-21B), which is consistent with the expression pattern of eGFP after rAAV2-eGFP and rAAV8-eGFP transduction. However, the results clearly show that KH902 protein is not only distributed in the cell bodies of keratocytes but also diffuses throughout the corneal stromal layer. This is due to the fact that KH902 contains a secretory signal peptide at the N-terminus, which promotes the secretion of KH902 protein from keratocytes into the corneal stromal matrix.

Overall, our data showed that rAAV8 mediated a stronger and earlier onset of KH902 expression in the cornea after intrastromal delivery than rAAV2, suggesting that rAAV8-KH902 is superior to rAAV2-KH902 in the treatment of corneal neovascularization. Moreover, rAAV8-mediated KH902 expression was sustained for up to three months and distributed throughout the corneal stromal layer. This finding indicates the potential and robust ability of rAAVS-KH902 to neutralize VEGF and thus attenuate CoNV.

Characteristics of immunological responses in mouse cornea to rAAV2-KH902 or rAAV8-KH902

Given that central corneal thickness (CCT) is an indicator of corneal health and physiological function, CCT was analyzed at different time points by optical coherence tomography (OCT) after intrastromal injection of PBS, rAAV8-eGFP, rAAV2-KH902, and rAAV8-KH902 (1.6×10 ¹⁰ GC/cornea). PBS and rAAVS-eGFP were used as injection control and vehicle control in animal groups, respectively. Immediately after injection, the CCT was found to increase by 22.73±2.93 μm in the PBS group, 23.50±5.37 μm in the rAAV8-eGFP group, 22.12±3.43 μm in the rAAV2-KH902 group, and 22.75±3.12 μm in the rAAV8-KH902 group, and then gradually decreased to pre-injection levels at the end of week 12 (final data collection time point) with normal morphological and anatomical structure determined by OCT scanning (Fig. 15A, 15B). No significant difference in CCT was observed among all groups. Taken together, these data indicate that intrastromal injection of rAAV vectors and the KH902 transgene product does not alter central corneal thickness or disrupt the physiological structure of the cornea, with no local scar formation or Descemet membrane detachment observed at three months. In addition, the immune response was also assessed two weeks after rAAY-KH902 injection by assessing corneal monocyte/macrophage markers (CD11b, F4/80). The levels of CD11b+ or F4/80+ cells were significantly higher after administration of high doses (1.6× ¹⁰¹⁰ GC/cornea) of vAAV2-eGFP/KH902 and vAAV8-eGFP/KH902, whereas the percentage of CD11b+ or F4/80+ cells in their low dose counterparts (8× ¹⁰⁸ GC/cornea) were significantly lower compared to the control levels, which are at the same levels as the PBS control (Fig. 15C, 15D). Therefore, in subsequent in vivo CoNV therapy studies, a low dose (8× ¹⁰⁸ GC/cornea) administration regimen was used to deliver rAAY2-KH902 and rAAV8-KH902.

Treatment with intrastromal rAAV8-KH902 effectively inhibits CoNV in an alkali burn model

To test whether rAAV2-KH902 and rAAV8-KH902 delivered intrastromally would therapeutically inhibit corneal neovascularization, mice corneas were subjected to alkali burn to generate a CoNV model and then mice were injected with PBS, rAAV8-eGFP, rAAV2-KH902, or tAAV8-KH902 at a dose of 8 × ¹⁰ GC/cornea on day 1 post-alkali burn. CoNV progression was monitored at days 5, 10, and 2, 3, 4, 8, and 12 weeks post-corneal injury (Fig. 16A). At day 5 post-alkali burn, CoNV ingrowth and cleavage from the limbal choroid plexus were visible in the CoNV area in all groups, with no statistical differences between the groups. Importantly, the length and area of CoNV were not obviously increased in the rAAV8-KH902-treated group, and they remained relatively stable from day 5 to week 12 (experimental endpoint). In contrast, vAAV2-KH902 failed to inhibit further CoNV growth from the limbus to the center of the cornea approximately four weeks after the burn, which was observed in the rAAV8-eGFP and PBS-treated control groups (Figs. 16A, 16B). To compare the therapeutic efficacy of rAAV8-KH902 and conbercept, the CoNV area was quantified in both the conbercept (10 mg/mL) single dose and rAAV8-KH902-treated groups. The results showed that there was no significant difference in the early stages of CoNV growth at days 5 and 10. However, in the single-dose conbercept group, CoNV development was observed two weeks after injection and progressed further, penetrating the central cornea at four weeks, resulting in intense CoNV with sizes similar to those in the PBS-treated control group at twelve weeks. The CoNV area size in the rAAV8-KH902-transduced group was significantly smaller compared to that in the conbercept group from two to twelve weeks after injection, indicating that rAAV8-KH902 exhibits prolonged anti-VEGF efficacy. Furthermore, rAAV8-KH902 in combination with conbercept did not inhibit the CoNV region to a greater extent than TAAV8-KH902 alone throughout the observation period (Figs. 16A, 16C), indicating that at this dose, rAAV8-delivered KH902 expression was adequate to neutralize VEGF in a timely manner, thereby achieving the antiangiogenic effect. Additional conbercept did not further enhance the therapeutic effect of rAAV8-KH902. This was verified by immunostaining with anti-CD31 (also known as platelet-endothelial cell adhesion molecule 1, PECAM-1) to delineate blood vessels in whole-mount corneas. The results showed that CoNV size correlated with the results of macroscopic vascular pathology assessment at week 12 (Fig. 16D, left panels).

Lymphangiogenesis is also involved in the pathological process of CoNV. The formation of new lymphatic vessels from the corneal limbus is thought to be similar to vessel growth during angiogenesis and is mainly induced by the binding of VEGF-C and VEGF-D to VEGFR-2 and VEGFR-3. Given that VEGF-A also promotes lymphangiogenesis and conbercept blocks all VEGF-A isoforms, the effect of rAAV8-KH902 on lymphangiogenesis was also assessed. Since pathological lymphatic vessels infiltrating the cornea are not directly visible, corneas from mice were harvested at week 12 in each group. Whole-mount corneas were double-stained using CD31 as a pan-endothelial marker and LYVE-1 (lymphatic vascular endothelial receptor 1) as a lymphatic vessel-specific marker. In whole corneal mounts, the area of CD31 ⁺⁺⁺ /LYVE- ^1- coated blood vessels and the area of CD31 ⁺ /LYVE-1 ⁺⁺⁺ coated lymphatic vessels were measured. Twelve-week follow-up of animals in all experimental groups revealed that treatment with rAAV8-KH902 alone or in combination with conbercept did not reduce lymphangiogenesis size, and no significant differences were observed between the PBS control group and the various treatment groups (Fig. 16D, 16E).

rAAV8-KH902 suppresses Dll4/Notch signaling and ERK activation Sprouting during angiogenesis is led by endothelial tip cells that direct the sprouting process, while endothelial stalk cells extend new vessel processes. During this process, VEGF and Notch signaling pathways are involved in the selection of tip and stalk cells in the vascular endothelium. Specialized endothelial tip cells direct blood vessel processes toward the VEGF-A gradient. D114 and Notch signaling reporters are mosaically distributed among endothelial cells of actively sprouting vessels. VEGF stimulation induces tip cell filopodia formation in quiescent endothelial cells and increases D114 expression in tip cells. In turn, D114 ligand activates Notch signaling in stalk cells, resulting in the release of the active Notch intercellular domain (NICD) from the cell membrane, thereby allowing properly oriented branching and sprouting. However, previous studies have not addressed the role of D114/Notch signaling in CoNV. Therefore, we assessed the expression of D114/Notch signaling in the corneas of mice with actively growing vessels by immunostaining and Western blot analyses two weeks after alkali burn. In the PBS- and rAAV8-eGFP-treated control groups, D114 was ubiquitously expressed in corneal neovascular sprouts, suggesting that D114 is involved in the process of corneal angiogenesis. In contrast, in the rAAV8-KH902-treated group, D114 was rarely detected and tip cell filopodia were completely retracted (Fig. 17A). These results indicate that rAAVS-KH902 blocks VEGF-A stimulation, preventing tip cell migration and CoNV progression. These findings were supported by the Western blot results, which showed a significant decrease in D114 and NICD expression in the rAAV8-KH902-treated group compared with the control groups (Figs. 17B, 17C, 17D).

VEGF binding to VEGFR results in phosphorylation of VEGFR2, initiating a subsequent cascade of signaling pathways related to angiogenesis and causing several cellular responses in epithelial cells (ECs). Among these pathways, VEGF-induced ERK1/2 signaling has been well studied and shown to regulate microvascular endothelial differentiation and proliferation. Therefore, to evaluate the level of ERK activation under each condition, corneas of mice were collected eight days after alkali burn. According to the results of Western blot analysis, the ratio of phosphorylated ERK (pERK) to total ERK (pERK/ERK) was significantly decreased in the rAAV8-KH902-treated group compared with the PBS-treated group and the rAAV8-eGFP-treated group (Figs. 17E, 17F). This suggests that blocking VEGF with rAAV8-KH902 results in inhibition of ERK activation in alkali burn-induced CoNV mice.

rAAV8-KH902 prevents progression of existing neovascularization in alkali burn-induced CoNV models and suture-induced CoNV models.

Chemical burn is an acute ocular injury and a complex pathological condition with varying severity and specific lesions. In cases where immediate or intensive treatment is not achieved at the initial stage, CoNV progresses rapidly in the active stage. Therefore, it is of great interest to investigate whether rAAV8-KH902 can suppress or even regress the active spread of CoNV induced by alkali burn. For this purpose, mice were intrastromally injected with PBS, rAAV8-eGFP or yAAV8-KH902 (8×10 ⁸ GC/cornea) ten days after alkali burn, when CoNV had already invaded the cornea to varying degrees, i.e. it had reached the active stage and was constantly growing (Fig. 18A). Following CoNV induction by alkali burn, no significant regression was observed in any of the experimental groups using the self-monitoring assay (Fig. 18A, 18B). However, in the rAAV8-KH902-treated group, the developed CoNV was significantly suppressed and it was maintained at the pre-treatment level during the four-week observation period (Fig. 18A, 18C).

To confirm the effect of rAAV8-KH902 on CoNV inhibition in another injury model, additional studies were performed using a suture-induced CoNV mouse model by placing an intrastromal knotted suture on the mouse cornea. After 5 days of suture stimulation, CoNV was actively progressing and the lesions had invaded the cornea. At this time, mice were intrastromally injected with PBS, rAAV8-eGFP, or rAAV8-KH902 at a dose of ⁸ x 10 GC per cornea. The level of CoNV progression was monitored and quantified before and after injection. Compared with PBS and rAAV8-eGFP controls, CoNV progression was significantly inhibited by rAAV8-KH902 treatment, and the inhibitory effect was maintained until the endpoint (Figs. 19A, 19B). However, no regression of controlled corneal vessels was observed after rAAV8-KH902 treatment (Fig. 19A, 19C). Thus, these data confirmed that rAAV8-KH902 exerts a robust therapeutic effect against active CoNV.

Corneal neovascularization has a significant impact on visual function and can be a pathological consequence of various etiologies, such as contact lens wear, dry eye, trauma, chemical burn, limbal stem cell deficiency, ocular surface inflammation, and corneal infection with bacteria, fungi, and viruses. Current treatments are limited by efficacy and safety concerns. Intrastromal injection of conbercept can inhibit corneal neovascularization, but this treatment requires repeated administration of the drug and is associated with injection-related adverse events. To reduce the frequency of drug administration, a study was conducted using rAAV vectors to mediate corneal expression of KH902. The results showed that rAAV8-KH902 induced robust and long-lasting KH902 expression in the cornea and successfully inhibited CoNV with a single low-dose intrastromal injection without any noticeable side effects, and that treatment with rAAV8-KH902 alone was sufficient to suppress angiogenesis in established CoNV within the established time frame.

The therapeutic window for antiangiogenic treatment of CoNV is difficult to define because different cases have different pathological etiologies. The overall angiogenesis pattern and the correct therapeutic course strongly depend on the characteristics of the types of precursors and the underlying pathologies. For example, in herpes simplex virus (HSV) keratitis, CoNV may manifest itself as early as the first day and may then persist for up to three weeks after HSV-1 infection of the cornea. As the disease progresses, infection, inflammation, and CoNV will trigger each other in a positive feedback loop, leading to a protracted course. In other cases, in patients with severe chemical injuries, the disease may enter a chronic phase that may persist for more than six weeks, with the development of significant limbal stem cell deficiency and neovascularization-related complications. In addition, the data showed that a single dose of rAAV8-KH902 provided a therapeutic window of at least three months, while direct administration of conbercept could only last for 10-14 days. Therefore, rAAV8-KH902 exerts a persistent anti-VEGF effect, significantly extending the therapeutic window. This significantly reduces the need for repeated anti-VEGF drug administration in patients with chronic corneal diseases.

Angiogenesis is the formation of new vessels from existing blood vessels. It not only depends on endothelial cell (EC) proliferation and invasion but also requires subsequent envelopment of vessels by pericytes for their stabilization and maturation. In the absence of pericytes, newly formed ECs are unstable and are prone to regression without VEGF stimulation, suggesting that immature vessels are VEGF dependent for survival and growth. A study of anti-VEGF monotherapy showed that it was ineffective in inducing mature vessels to regress due to these vessels being less dependent on VEGF (49). Similarly, the successful inhibition and failure of CoNV regression found in the study also proved that VEGF is important for the maintenance and stimulation of newly formed vessels but is not required for the maintenance of mature blood vessels. Therefore, earlier treatment of CoNV with rAAV8-KH902 will lead to better outcomes.

The route of administration is a critical factor to consider when developing gene therapy for ocular diseases. Various routes of administration for rAAV gene transduction into corneal tissues are being explored. Topical administration is the simplest route of administration, but is not ideal for rAAV vectors as they have relatively low transduction efficiency and also exhibit potential adverse effects associated with transduction into non-target tissues when the vector is distributed through tears. Corneal biodistribution was compared between subconjunctival and intrastromal injections of rAAV2-eGFP and rAAV8-eGFP. The results showed that intrastromal delivery of rAAV2 or rAAV8 vectors provides more efficient and extensive transduction in the cornea compared to subconjunctival injection. Furthermore, intrastromal injection of rAAV2 and rAAV8 had similar tropism for corneal cells, mainly keratocytes, with interspersion in epithelial cells but not endothelial cells. rAAV8-mediated gene expression occurred with an earlier onset and higher efficiency compared to rAAV2. This explains why rAAV8-KH902 successfully inhibits CoNV, whereas rAAV2-KH902 does not.

The corneal wound healing cascade consists of angiogenesis, epithelialization, and abnormal deposition of various collagen types, which contribute to scar formation and corneal opacification. Intrastromal injection of rAAV-KH902 or rAAV-eGFP into healthy corneas did not cause scarring or opacification, indicating that corneal injury caused by alkali burn is the cause of scarring and decreased transparency during wound healing.

Thus, a single low-dose injection of TAAV8-KH902 into the corneal stroma results in effective inhibition of CoNV over a prolonged period of time. This study confirms the long-lasting potential and relative safety of rAAV-based anti-VEGF gene therapy for CoNV.

Methods

Obtaining vectors

Vectors were packaged with transgene cassettes encoding eGFP or KH902 under the control of the cytomegalovirus (CMV)/chicken β-actin promoter. The vector encoding KH902 was constructed using rabbit poly-A globin. Vectors were prepared using triple transfection as described below. Vectors were purified by CsCl gradient ultracentrifugation and titrated by both ddPCR and silver staining on sodium dodecyl sulfate (SDS)-polyacrylamide gels.

Animals

C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and bred and maintained under standard conditions with a 12-hour light/12-hour dark cycle in the animal facility of the University of Massachusetts Medical School. All experiments were approved by the Institutional Animal Care and Use Committees in accordance with the ARVO statement for the use of animals in ophthalmology and vision research.

Alkali burn-induced CoNV

The alkali burn-induced CoNV mouse model was performed as described previously (25) with some modifications. Mice were anesthetized by intraperitoneal injection of a combination of ketamine (5 mg/ml) and xylazine (2 mg/ml) (10 ml/kg body weight), and the local anesthetic proparacaine (0.5%) was applied to the corneal surface. Circular filter paper discs (2 mm in diameter) were pre-soaked in 1 M NaOH for 20 sec and then placed on the central cornea for approximately 40 sec, followed by copious rinsing with sterile saline (15 ml) for 1 min.

CoNV induced by suture

A modified suturing method described previously (55) was used to induce CoNV. Briefly, the right cornea of mice was sutured under general anesthesia [intraperitoneal injection of a combination of ketamine (5 mg/ml) and xylazine (2 mg/ml) (10 ml/kg body weight)] supplemented with local anesthesia (0.5% proparacaine). A single 10-0 nylon suture was placed intrastromally, with a knot in the temporal cornea 1 mm from the limbus. To ensure consistency and reproducibility of the procedure, the entire process was performed on each animal by the same investigator under a dissecting microscope.

Delivery of rAAV vector by intrastromal or subconjunctival injection

Intrastromal injections were performed using a previously published method (7). Briefly, an approximately 1.0 mm incision was first made in the corneal epithelium equidistant between the temporal limbus and the center of the cornea using the tip of a 30-gauge needle. Either 1.6 × 10 ¹⁰ or 8 × 10 ⁸ GC rAAV vectors in 4 μl PBS were then injected into the corneal stroma through the incision using a 5 μl Hamilton syringe with a 34-gauge needle (Hamilton, Reno, NV, USA; 30° bevel angle). Subconjunctival injection was also performed using a 5 μl Hamilton syringe. The superior, inferior, nasal and temporal subconjunctiva were injected with 1.6×10 ¹⁰ GC rAAV vectors in a volume of 1 μl (0.4×10 ¹⁰ GC) per site, respectively. Antibiotic ointment was applied after the injections.

In vivo fluorescence imaging

Animals in each group were observed weekly for twelve weeks after rAAV administration. Eye eGFP expression was recorded using a Micron IV camera (Phoenix Research Labs, Pleasanton, CA, USA).

Optical coherence tomography of the cornea

Optical coherence tomography (OCT) of the mouse cornea was performed using a Micron IV OCT imaging system (Phoenix Research Labs, Pleasanton, CA, USA). Mice were anesthetized by intraperitoneal injection as described above. The mouse cornea was positioned against the camera lens, and the entire anterior chamber was imaged. Central corneal thickness (CCT) was then measured from the resulting images using ImageJ software (available online at imagej.nih.gov/ij/). CCT was measured before injection, immediately after injection, and at 1, 2, and 12 weeks after injection.

Color imaging of the anterior region and quantification of corneal NV The procedures were performed under general anesthesia and topical anesthesia with eye drops as described above. The mouse eye was placed under an ophthalmic surgical microscope (WILD HEERBRUGG), and the cornea was photographed from different angles using a digital camera attached to the microscope. CoNV was analyzed at given time points using ImageJ software. The area of CoNV was calculated using the following formula: Area ( ^mm2 ) = CN/12 × 3.1416 × [ ^R2 - (R - VL) ² ], where CN is the NV developed at a given time; R is the corneal radius; and VL is the longest vessel length originating from the limbal vasculature (56). Color images of each cornea of a living mouse were acquired at eight different angles, and the area of corneal neovascularization (NV) was calculated in quadruplicate for each angle.

Immunohistochemistry of whole corneal flat mounts

The globes were enucleated and fixed with 4% PFA for 1 hour at room temperature after a small hole was made at the limbus with a needle. The excised globes were then prepared for whole-mount staining using a modified method as described in previous reports (31). Briefly, the cornea and sclera were separated by an incision along the limbus, followed by removal of the lens and iris. Four radial corneal incisions were made to ensure whole-mount flattening. The tissues were then washed with 0.3% Triton X-100 in PBS and then blocked for 1 hour with blocking buffer (0.3% Triton X-100/5% normal bovine serum albumin (BSA, Cell Signaling Technology)/1X PBS). Corneas were stained overnight at 4°C with rat anti-CD31 (PECAM-1, 1:400, sc-18916, Santa Cruz), rabbit anti-mouse LYVE-1 (1:200, 11-034, AngioBio Co), or goat anti-mouse D114 (1:40, AF1389, R&D Systems). Primary antibodies were detected with Alexa Fluor-conjugated goat anti-rabbit, anti-rat, or donkey anti-goat secondary antibodies. 488 or 594 (Thermo Fisher Scientific, Singapore). After a final rinse of corneal tissues with 0.3% Triton X-100 in PBS, they were mounted endothelial side down and imaged using a Leica DM6 microscope with a 16-bit monochrome camera. Image processing was performed using Adobe Photoshop CC 2019 to improve clarity. Areas covered by blood and lymphatic vessel markers were recorded and measured using ImageJ software. Whole corneas were analyzed by two independent observers blinded to treatment status to minimize sampling bias.

Histology and immunohistochemistry of corneal cryosection

In preparation for sectioning, freshly excised eyeballs were placed directly into an OCT machine (Fisher Scientific, Pittsburgh, PA). Frozen blocks were cryosectioned at 14 μm thickness (Leica CM3050 S, Leica Biosystems Inc., Buffalo Grove, IL). After fixation of sections with 4% PFA for 15 min at room temperature, tissue sections were washed with 0.3% Triton X-100 in PBS and blocked for 1 h with blocking buffer (11 PBS/1% BSA/0.3% Triton™ X-100). Sections were stained overnight at 4°C with primary antibodies. The following primary antibodies were used: rat anti-F4/80 (1:400, NB600-404, Novus), rat anti-mouse CD11b (1:50, #550282, BD Pharmingen), rabbit anti-vimentin (1:100, #5741, Cell Signaling Technology), and donkey anti-human IgG (H+L) conjugated with Alexa Fluor 488 (1:400, #144222, Jackson ImmunoResearch Laboratories Inc.), which were diluted in PBS with 0.3% Triton X-100 and 5% BSA. The secondary antibodies counterstained with DAPI (#9542, Sigma-Aldrich) used were labeled goat anti-rat IgG-Alexa Fluor 594 and labeled goat anti-rabbit IgG-Alexa Fluor 594. Fluorescence images were acquired using a Leica DM6 microscope. Image analysis was performed using Adobe Photoshop software.

CDllb+ or F4/80+ cells were recorded and counted using ImageJ software.

Analysis of mRNA expression of KH902

RNA from corneas of normal mice treated or not with rAAV2-KH902 or TAAV8-KH902 (4 corneas per group) at 1 and 2 weeks and 1, 2, and 3 months post-treatment was isolated using the RNeasy Plus Micro Kit and reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (both Qiagen, Hilden, Germany). Multiplex ddPCR was performed using the QX200 ddPCR System (Bio-Rad Laboratories, Hercules, CA, USA) with probes targeting KH902 and the reference transcript, beta-glucuronidase (GUSB) (#4448489; Thermo Fisher). Primer and probe sets for KH902 were designed and synthesized by Integrated DNA Technologies (Coralville, IA, USA) (forward primer: 5'-GGACATACACAACCAGAGAGAC-3' (SEQ ID NO: 27), reverse primer: 5'-GTGAGTGAAAGAGACACAGGAA-3 (SEQ ID NO: 28), probe: 5'-/56-FAM/CCCATTTCA/ZEN/AAGGAGAAGCAGAGCCA/3IABkfq/-3' (SEQ ID NO: 29)). KH902 mRNA copy numbers were normalized to GUSB copy numbers. ddPCR results were expressed as the ratio of KH902 to GUSB values.

Western blot

Total protein from pooled corneas in each group was extracted on ice in RIPA lysis buffer with fresh protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) after homogenization using the QIAGEN TissueLysis kit II. A total of 20 μg of protein extract per lane was loaded onto a Bis-Tris 4–12% precast gel (QP3510, SMOBIO) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Non-specific binding was blocked with 5% BSA in Tris-buffered saline with Tween-20 (TBST). The membranes were incubated overnight at 4°C with rabbit anti-Cleaved Notch1 antibody (#4147, Cell Signaling Technology), goat anti-mouse D114 antibody (AF1389, R&D Systems), rabbit anti-pERK1/2 antibody (#4370, Cell Signaling Technology), and anti-ERK1/2 antibody (#9102, Cell Signaling Technology). The membranes were incubated with rabbit anti-ERK antibody after abrupt removal of the membrane. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10000, G-21234; Invitrogen) or rabbit anti-goat IgG (1:1000; HAF017, R&D Systems) for one hour and half an hour. Protein detection was performed using enhanced chemiluminescence (ECL) Western blot substrate (cat. no. W1001; Promega, Madison, WI, USA) in combination with the Odyssey system. The intensity of specific bands was quantified using ImageJ software.

Statistics

Results were expressed as mean±SEM (standard error of the mean). Each data point represents the mean of three replicates. Analysis was performed using one-way or two-way analysis of variance for multiple variables, and Tukey's multiple comparison test was used to assess between-group differences using GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA). A p value of <0.05 was considered significant.

Example 5: Plasmid Analysis

Two plasmids containing the KH902 transgene were obtained. Plasmid 1 contained the rAAV vector containing the 5'AAV ITR, the CBA promoter, an intron, a Kozak sequence, the transgene encoding KH902, rabbit poly-A globulin and the 3'AAV ITR. The rAAV vector sequence extends from the 5'-ITR to the 3'-ITR of plasmid 1 and is presented in SEQ ID NO:3. The complete sequence of plasmid 1 is presented in SEQ ID NO:30.

Plasmid 2 contained the rAAV vector containing the 5'AAV ITR, CMV promoter, intron, Kozak sequence, transgene encoding KH902, SV40 poly-A and 3'AAV ITR. The KH902 transgene sequence was codon optimized. The complete plasmid sequence of plasmid 2 is presented in SEQ ID NO: 31.

Each plasmid has two ITR sequences, named ITR1 and ITR2 (e.g., 5' and 3' ITR of the rAAV vector, respectively). The two plasmids have multiple SmaI sites, two in ITR1 and two in ITR2. The number and size of theoretical DNA bands after SmaI digestion of the plasmid were calculated. When the plasmid is intact, the position of the SmaI cleavage can be determined according to the DNA sequence, and the number and size of DNA bands can be calculated after complete SmaI digestion. This is the band profile of the intact plasmid. After cleavage of ITR1 of the plasmid (which is equivalent to deletion of the SmaI sites in ITR1), the number and size of DNA bands of the plasmid after complete SmaI digestion were calculated. This is the band profile of the plasmid with ITR1 deletion. The same method was used to calculate the band profile of the ITR2 deletion plasmid and the ITR1+ITR2 deletion plasmid after complete digestion with SmaI. Plasmid 1 and plasmid 2 samples were completely digested with SmaI and then subjected to agarose gel electrophoresis. In the case of ITR deletions, the sample is expected to be a mixture of intact and deletion plasmids. The electrophoretic DNA bands were used to assess the possible type and extent of ITR deletions.

If plasmid 1 is intact, the gel electrophoresis spectrum will show bands at approximately 407 bp, 307 bp, 343 bp, 2868 bp, and 2817 bp. When ITR1 of plasmid 1 is deleted, a band at approximately 3171 bp appears. If ITR2 is absent, a band at approximately 5696 bp appears, and if both ITR1 and ITR2 are absent, a band at approximately 6050 bp appears. The experimental results showed that the gel electrophoresis spectrum of plasmid 1 digestion was consistent with the theoretical full spectrum of the plasmid, but there was no band at 3171, 5696, or 6050 bp, indicating that plasmid 1 did not have ITR deletions.

If plasmid 2 is intact, the full spectrum of gel electrophoresis will show bands at approximately 2817 bp and 2834 bp. When ITR1 and/or ITR2 are absent, a band at 5673 bp appears. The gel electrophoresis results of plasmid 2 digestion showed several bands >5000 bp, while no bands >5000 bp were detected in the normal plasmid digestion map, indicating that plasmid 2 contains deletions of ITR1 and/or ITR2.

Example 6: Protein expression after a single intravitreal injection in cyanotic blue rabbits

This example describes the distribution of KH902 protein expressed by different rAAV vectors in the ocular tissues of cyanotic blue rabbits following a single intravitreal injection. For this purpose, rAAV 7m8-CBA-KH902 was used, which contains the KH902 transgene driven by a regulatory cassette of the CMV promoter enhancer and chicken β-actin (e.g., plasmid 1 described in Example 5) encapsidated by the AAV7m8 capsid protein. For comparison, rAAV 7m8-CMV-KH902 was used, which contains an isolated nucleic acid containing the CMV promoter, an intron, a Kozak sequence, a codon-optimized transgene encoding KH902, WPRE, SV40 poly A, and the 3' AAV ITR encapsidated by the AAV7m8 capsid protein. The plasmid used to produce rAAV7m8-CMV-902 is shown in SEQ ID NO: 32.

Following intravitreal injection of rAAV 7m8-CBA-KH902 or rAAV 7m8-CMV-KH902 into the left and right eyes of cyanotic blue rabbits (2E11 vg/50 μl), animals were sacrificed at the indicated times (week 2 and week 4). The globes and optic nerves were then removed, the globes were dissected, the retinal-choroidal region and vitreous were separated, and conbercept expression (e.g., KH902 protein) was measured after homogenization.

The obtained results show that rAAV 7m8-CBA-KH902 had higher and more stable expression in the choroid plexus of the retina and vitreous compared with rAAV 7m8-CMV-KH902 (Table 1).

Example 7: Aqueous humor expression in cynomolgus monkeys with subretinal delivery

In this study, rAAV8-CBA-KH902 was used, containing the KH902 transgene driven by a CMV enhancer promoter regulatory cassette and chicken β-actin (e.g., plasmid 1 described in Example 5).

Cynomolgus macaques were injected subretinal with rAAV8-CBA-KH902 at a dose of 1E12 gv/100 μl/eye on the temporal side, just below the superior vascular arch. Anterior chamber aqueous humor samples were collected at 3, 7, 21, and 28 days post-injection, at a volume of approximately 50 μl/eye. The concentration of target protein in the aqueous humor was determined using ELISA. The results are presented in Table 2. It was found that the concentration of conbercept protein (e.g., KH902 protein) in the aqueous humor gradually increased over 28 days post-injection.

Example 8: Vector efficiency in rhesus monkeys for subretinal delivery

Non-human primates (NHPs) have a similar macula architecture to humans. The laser photocoagulation-induced choroidal neovascularization model in NHPs is one model of nAMD (Wang Q, et al. British Journal of Ophthalmology, 2015, 99(1): 119-24.). The rAAV8-CBA-KH902 described in Example 7 was used in this study. Rhesus macaques with healthy eyes were selected and positioned in a supine position on the operating table after pupil dilation and anesthesia. The periorbital skin was disinfected with povidone-iodine and the conjunctival sac was washed with povidone-iodine mucosal disinfectant. Using a WPI microinjection needle (36G), the vitreous cavity was penetrated through the stoma, and the retinoscope was placed on the cornea. The injection was performed in the superior vascular arch region of the posterior pole with a volume of 100 μL/eye. The day of administration was designated as day 1. The dosage and composition of the groups are shown in Table 3 below.

On day 15, rhesus monkeys were dilated and anesthetized, Carbomer ophthalmic drops were applied to the ocular surface, and a laser lens was used to examine the fundus. After the fundus was clearly visible, a site approximately 1.5 2 PD from the macular fovea was selected for photocoagulation, avoiding blood vessels. The laser parameters used were: wavelength 532 nm, power 450-550 MW, spot diameter 50 μm, exposure time 100 ms. In the positive control group, 50 μl of conbercept ophthalmic injection (0.5 mg/eye) was injected intravitreally immediately after laser photocoagulation.

Enhanced depth imaging optical coherence tomography (EDI-OCT) was used to visualize and characterize the lesions in the eyes of animals in groups 1–4 before injection, immediately after injection, on day 15 (before and after modeling), on days 29, 43, and 57. Eyes of animals in group 5 were examined with OCT before injection, on day 15 (after modeling), on days 29, 43, and 57. The laser control zone included the posterior pole, the injection zone, and all laser photocoagulation sites.

The hyperreflective material thickness (SHRM) on the OCT image corresponding to grade 4 fluorescence leakage lesion was measured using the software supplied with the instrument, and the mean SHRM thickness was calculated for each eye.

For the examination of the eyes of animals in groups 1-4, fundus photography and fluorescein angiography (FP and FFA) were performed before administration, immediately after administration (FP only), on day 15 (FP only, before and after modeling), on days 29, 43, 57. FA and FFA were used to examine the eyes of animals in group 5 on day 15 (FP only, after modeling), on days 29, 43, and 57. Before fundus fluorescein angiography, animals were intravenously injected with sodium fluorescein (10 mg/kg, 100 mg/ml).

Early and late fundus images obtained by fluorescein angiography were compared, and choroidal neovascularization and the presence of fluorescence leakage in the fundus of animals were determined. The degree of leakage was assessed by the fluorescence level, and the number and frequency of grade 4 lesions were calculated. The rating criteria are presented below:

Grade 1: There is no high fluorescence in the affected area;

Grade 2: There is high fluorescence in the affected area, but no fluorescence leakage;

Grade 3: high fluorescence in the affected area with slight fluorescence leakage, and the leakage does not extend beyond the edges of the affected area;

Grade 4: Highly fluorescent lesion with slight fluorescence leakage, with the leakage extending beyond the edge of the lesion area.

For grade 4 lesions, the leak area was measured (note: if the photocoagulation spot is assessed as a grade 4 lesion at one of the control time points after drug administration, then the fluorescence leak area for this lesion should be measured at all control time points; if there is no fluorescence leak, then measurement is not required).

The ratio of grade 4 lesions and grade 4 leakage areas at day 29 after administration (i.e., 14 days after the simulation) are shown in Fig. 22A and Fig. 22B, respectively. It can be seen that the ratio of grade 4 lesions and grade 4 leakage areas in the high, medium, and low dose groups were significantly reduced compared with the negative control group.

In addition, regression of the bright spot was observed by FFA at day 29 after administration (Fig. 22C). This figure shows the representative results obtained by FFA for each experimental group. As shown in Fig. 22C, grade 4 lesions in the high, medium, and low dose drug groups were significantly reduced compared with the negative control group.

EQUIVALENTS

Although several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily conceive of many other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such changes and/or modifications is considered to be within the scope of the present invention. More generally, those skilled in the art will understand that all parameters, dimensions, materials and configurations described herein are intended to be exemplary only, and that the actual parameters, dimensions, materials and/or configurations will depend on the particular application or applications in which the principles of the present invention are employed. Those skilled in the art will know, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It should therefore be understood that the above embodiments of the present invention are presented by way of example only, and that within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention may employ 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, if such features, systems, articles, materials and/or methods are not mutually incompatible, is within the scope of the present invention.

The terms used herein in the description and in the claims in the singular, unless expressly stated otherwise, shall be understood to mean "at least one".

The expression "and/or" as used in this specification and in the claims is to be understood to mean "one or both" of the elements combined such that the elements are in some instances present/used together and in other instances present/used separately as an alternative. Other elements other than the elements specifically recited using the expression "and/or" may optionally be present, whether related or unrelated to those specifically recited elements, unless explicitly stated otherwise. Thus, as a non-limiting example, a reference to "A and/or B" when used in combination with an open-ended expression such as "comprising" may refer in one embodiment to A without B (optionally including other elements other than B); in another embodiment, it may refer to B without A (optionally including other elements other than A); in yet another embodiment, it may refer to both A and B (optionally including other elements); and so on.

As used in this specification and in the claims, the reference "or" shall be understood to have the same meaning as "and/or" as defined above. For example, when separating elements in a list with the conjunction "or" or the expression "and/or", this shall be interpreted as an inclusive reference, i.e., including at least one, but also including more than one, of the number or list of elements, and optionally additional elements not included in the list. Only terms clearly indicating the contrary, such as "only one of" or "exactly one of" or, when used in the claims, "consisting of", refer to the inclusion of only one element of the number or list of elements. In general, the conjunction "or" as used herein shall be interpreted only as denoting exclusive alternatives (i.e., "one or the other, but not both") when preceded by exclusivity terms such as "any", "one of", "only one of" or "exactly one of". The expression "consisting essentially of..." when used in a claim has its ordinary meaning as used in the field of patent law.

As used in this specification and in the claims, the expression "at least one" with respect to a list of one or more elements shall be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each element specifically recited in the list of elements, and without excluding any combinations of elements in the list of elements. This definition also allows that elements other than the elements specifically recited in the list of elements to which the expression "at least one" refers may optionally be present, whether or not related to those specifically recited 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") may refer in one embodiment to at least one element A, optionally including more than one element, but without the presence of B (and thus optionally including elements other than B); in another embodiment, it may refer to at least one element B, optionally including more than one element but without the presence of A (and optionally including elements other than A); in yet another embodiment, it may refer to at least one element A, optionally including more than one element, and at least one element B, optionally including more than one element (and optionally including other elements); and so on.

In the claims and in the description above, all transitional words and phrases such as "comprising," "including," "carrying," "having," "containing," "encompassing," "holding," and the like are to be construed as open-ended, i.e., meaning inclusion but not limitation. Only the transitional phrases "consisting of" and "consisting essentially of" are to be considered closed or semi-closed transitional phrases, respectively, as provided in Section 2111.03 of the Patent Examining Manual of the United States Patent Office.

The use of ordinal numbers and corresponding terms such as "first", "second", "third", etc., in the claims to indicate an element or feature does not in itself imply any priority, precedence or order of one element or feature with respect to others, and does not determine the temporal order in which the actions of the method are performed, but is used simply as labels to distinguish one element having a certain name from another element having the same name (due to the difference determined by the indicated number).

Claims (68)

1. A recombinant adeno-associated virus (rAAV) for expression of a transgene encoding an anti-vascular endothelial growth factor (VEGF) agent, comprising: (i) an AAV capsid protein, wherein the capsid protein is an AAV8 capsid protein or variant thereof, an AAV2 capsid protein, or an AAV2/3 hybrid capsid protein; and (ii) an isolated nucleic acid comprising a transgene encoding an anti-vascular endothelial growth factor (anti-VEGF) agent, wherein the transgene is flanked by inverted terminal repeats (ITRs) of adeno-associated virus (AAV), wherein the transgene comprises the nucleic acid sequence SEQ ID NO: 1. 2. The rAAV of claim 1, wherein the anti-VEGF agent is capable of binding to and acting against vascular endothelial growth factor (VEGF) and/or placental growth factor (PIGF). 3. The rAAV of claim 1 or 2, wherein the isolated nucleic acid further comprises a promoter operably linked to the transgene. 4. The rAAV of claim 3, wherein the promoter comprises a cytomegalovirus (CMV) early enhancer. 5. The rAAV of claim 4, wherein the promoter comprises a chimeric cytomegalovirus (CMV)/chicken β-actin (CB) promoter. 6. The rAAV of any one of claims 1-5, wherein the transgene comprises one or more introns. 7. The rAAV of claim 6, wherein at least one intron is located between the promoter and a nucleic acid sequence encoding an anti-vascular endothelial growth factor (anti-VEGF) agent. 8. The rAAV of any one of claims 1-7, wherein the transgene comprises a Kozak sequence. 9. The rAAV of claim 8, wherein the Kozak sequence is located between the intron and the transgene encoding an anti-vascular endothelial growth factor (anti-VEGF) agent. 10. The rAAV of any one of claims 1-9, wherein the transgene comprises a 3' untranslated region (3'UTR). 11. The rAAV of any one of claims 1-10, wherein the transgene further comprises one or more microRNA binding sites. 12. The rAAV of claim 11, wherein the one or more microRNA binding sites are located in the 3'UTR of the transgene. 13. The rAAV of claim 11 or 12, wherein at least one microRNA binding site is an immune cell-associated microRNA binding site. 14. The rAAV of claim 13, wherein the immune cell-associated microRNA is 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. 15. The rAAV of any one of claims 1-14, wherein the ITRs are ITRs of an adeno-associated virus serotype that are selected from the group consisting of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR and AAV6 ITR. 16. The rAAV of any one of claims 1-15, wherein the isolated nucleic acid comprises a nucleic acid sequence that is at least 80%, at least 90%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 2. 17. The rAAV of any one of claims 1-16, wherein the isolated nucleic acid comprises a nucleic acid sequence that is at least 80%, at least 90%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 30. 18. The rAAV of any one of claims 1 to 17, wherein the capsid protein comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of capsid protein v224, capsid protein v326, capsid protein v358, capsid protein v46, capsid protein v56, capsid protein v66, capsid protein v67, capsid protein v81, capsid protein v439, capsid protein v453, capsid protein v513, capsid protein v551, capsid protein v556, capsid protein v562, or capsid protein v598. 19. The rAAV of claim 18, wherein the capsid protein comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of capsid protein v224, capsid protein v326, or capsid protein v56. 20. The rAAV of any one of claims 1-19, wherein the capsid protein has tropism for ocular tissue. 21. The rAAV of claim 20, wherein the ocular tissue comprises ocular neurons, the retina, sclera, choroid, retina, vitreous, macula, fovea, optic disc, lens, pupil, iris, aqueous humor, cornea, conjunctiva, ciliary body, or optic nerve. 22. The rAAV of any one of claims 1-21, wherein the rAAV is a single-chain AAV (ssAAV) or a self-complementary AAV (scAAV). 23. The rAAV of any one of claims 1-22, wherein the capsid protein variants are capable of increasing the packaging efficiency of the rAAV compared to the wild-type capsid protein from which they are derived. 24. The rAAV of claim 23, wherein the AAV2 capsid protein variant is capable of increasing the packaging efficiency of rAAV by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times compared to the wild-type AAV2 capsid protein. 25. The rAAV of claim 23, wherein the AAV2/3 hybrid capsid protein variant is capable of increasing the packaging efficiency of rAAV by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times compared to the wild-type AAV3b capsid protein. 26. A recombinant adeno-associated virus for expression of a transgene encoding an anti-VEGF agent comprising: (i) an rAAV capsid protein, wherein the capsid protein is a variant of an AAV8 capsid protein, an AAV2 capsid protein and/or an AAV2/3 hybrid capsid protein or variant thereof; and (ii) a recombinant adeno-associated virus (rAAV) vector containing a nucleic acid comprising in the sequence from 5' to 3': (a) 5' AAV ITR; (b) CMV enhancer; (c) CBA promoter; (d) chicken beta-actin intron; (d) Kozak sequence; (e) a transgene encoding an anti-VEGF agent, wherein the anti-VEGF agent is encoded by the nucleic acid sequence set forth in SEQ ID NO: 1; (f) the signal tail of rabbit polyA beta-globin; and (g) 3’ ITR AAV. 27. A host cell comprising the rAAV of any one of claims 1-26, for producing said rAAV. 28. The host cell of claim 27, wherein the host cell is a mammalian cell, a yeast cell, a bacterial cell, or an insect cell. 29. A pharmaceutical composition for inhibiting VEGF activity, comprising rAAV according to any one of claims 1-26. 30. The pharmaceutical composition according to claim 29, additionally containing a pharmaceutically acceptable carrier. 31. The pharmaceutical composition of claim 29 or 30, wherein the pharmaceutical composition is prepared for intravitreal injection, intravenous injection, intratumoral injection, intrastromal injection, or intramuscular injection. 32. The use of rAAV according to any one of claims 1-26 or a pharmaceutical composition according to any one of claims 29-31 for inhibiting VEGF activity in a subject in need thereof. 33. Use of the rAAV of any one of claims 1-26 or the pharmaceutical composition of any one of claims 29-31 for delivering an anti-VEGF agent to a subject in need thereof. 34. Use of rAAV according to any one of claims 1-26 or a pharmaceutical composition according to any one of claims 29-31 for the treatment of a disease associated with neovascularization, a disease associated with angiogenesis, or a disease associated with VEGF in a subject in need thereof. 35. The use according to claims 32-34, wherein delivery of the anti-VEGF agent results in inhibition of VEGF activity by 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%. 36. The use according to any one of paragraphs 32-35, wherein the subject is a mammal other than a human, a chicken, or a turkey. 37. The use according to claim 36, wherein the non-human mammal is a mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, or non-human primate. 38. Use according to any of paragraphs 32-35, where the subject is a human. 39. The use according to claim 38, wherein the subject is diagnosed with or suspected of having an angiogenesis-related disease or a VEGF-related disease. 40. The use according to claim 39, wherein the VEGF-associated disease is a tumor, cancer, retinopathy, wet age-related macular degeneration (wAMD), macular edema, choroidal neovascularization or corneal neovascularization. 41. The use according to any one of claims 32-40, wherein the administration is systemic administration, and the systemic administration is not necessarily intravenous injection. 42. The use according to any one of claims 32-41, wherein the administration is direct administration into the ocular tissue, and the direct administration is optionally intravitreal injection, intraocular injection, intrastromal injection or topical administration. 43. The use according to any one of claims 32-42, wherein the administration results in delivery of the transgene to the ocular tissue. 44. The use according to claim 43, wherein the ocular tissue comprises ocular neurons, the retina, the sclera, the choroid, the retina, the vitreous body, the macula, the fovea, the optic disc, the lens, the pupil, the iris, the aqueous humor, the cornea, the conjunctiva, the ciliary body, or the optic nerve. 45. The use according to any one of claims 32-44, wherein the administration results in inhibition of VEGF in the subject for at least 5 days, 10 days, 15 days, 20 days, 1 month, two months or more after administration. 46. Use of rAAV according to any one of claims 1-26 or pharmaceutical composition according to any one of claims 29-31 for treating corneal neovascularization (CoNV) in a subject in need thereof, comprising administering to the subject an effective amount of rAAV or pharmaceutical composition. 47. The use according to claim 46, wherein the rAAV comprises the AAV8 capsid protein. 48. The use according to claim 46 or 47, wherein the administration results in delivery of the anti-VEGF agent to the corneal cells. 49. The use according to claim 48, wherein the administration results in delivery of the anti-VEGF agent to corneal keratocytes. 50. The use according to any of paragraphs 46-49, wherein rAAV is administered once. 51. The use according to any one of claims 46-50, wherein the administration results in expression of the anti-VEGF agent in corneal cells for more than three months, six months, a year or more. 52. The use according to claim 51, wherein the administration results in inhibition of VEGF in the subject for 1 month, two months, three months, six months, a year or more after administration. 53. Use according to any one of claims 46-52, wherein the administration is by intrastromal injection. 54. Use according to any of paragraphs 46-53, where the subject is a human. 55. The use according to any one of claims 46-54, wherein the corneal neovascularization is acute corneal neovascularization or chronic corneal neovascularization.