Compositions and methods for transgene expression in the eye

Abstract

Provided herein are compositions and methods that include non-naturally occurring nucleic acid sequences encoding biologics. Also provided are methods of utilizing the provided compositions and methods as ocular therapeutics for the prevention and treatment of various diseases and conditions.

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 173,280, filed April 9, 2021, the entire contents of which are incorporated by reference herein.

[0002] Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on April 8, 2022, is named 59561-703_601_SL.txt and is 159,002 bytes in size. [Background technology]

[0003]

[0003] Aflibercept (VEGF-Trap) is a recombinant fusion protein that acts as a decoy receptor for vascular endothelial growth factor subtype A (VEGF-A), VEGF-B, and placental growth factor (PIGF). By binding to these ligands, aflibercept can prevent these ligands from binding to the vascular endothelial growth factor receptors (VEGFRs), VEGFR-1 and VEGFR-2, inhibit angiogenesis, and reduce vascular permeability. Aflibercept is a fusion of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 with the Fc fragment of IgG1. Aflibercept is commercially available under the trade name EYLEA® (aflibercept), which is an intravitreal injection of aflibercept fusion protein for ophthalmic use. Summary of the Invention [Problem to be solved by the invention]

[0004]

[0004] Current treatment using aflibercept has the problem of short duration and repeated monthly injections to suppress angiogenesis. Several gene therapy trials using AAV vectors carrying sequences encoding VEGF-Trap have been conducted for long-term treatment of angiogenesis. However, these trials suffer from low expression levels of the drug and therefore low efficacy of treatment. Therefore, it is becoming increasingly clear that the full potential of this technology will only be realized after modifications are made to improve the delivery and expression of VEGF-Trap and other antiangiogenic agents. [Means for solving the problem]

[0005]

[0005] In some aspects, described herein is an isolated non-naturally occurring nucleic acid comprising a sequence encoding a biologic comprising an anti-angiogenic agent, the sequence comprising a modification in the coding region of the sequence compared to an otherwise equivalent sequence lacking the modification in the coding region, the modification comprising replacement of at least four non-AGG arginine codons with AGG. In some embodiments, the sequence encoding the anti-angiogenic agent further comprises a second modification. In some embodiments, the second modification is within at least one codon in the coding region of the sequence, and the second modification is selected from the group consisting of (a) replacement of at least one non-CCC proline codon with CCC, (b) replacement of at least one non-TCC serine codon with TCC, (c) replacement of at least one non-CCG proline codon with CCG, and (d) any combination of (a)-(c). In some embodiments, the second modification comprises (a). In some embodiments, the at least one non-CCC proline codon is CCT. In some embodiments, the second modification comprises (b). In some embodiments, at least one non-TCC serine codon is AGC. In some embodiments, the second modification comprises (c). In some embodiments, at least one non-CCG proline codon is CCC. In some embodiments, the second modification comprises (d). In some embodiments, at least one non-CCC proline codon in (a) is CCT, at least one non-TCC serine codon in (b) is AGC, and at least one non-CCG proline codon in (c) is CCC. In some embodiments, the anti-angiogenic agent is selected from the group consisting of a VEGF inhibitor, a multi-tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, an inhibitor of Akt phosphorylation, a PDGF-1 inhibitor, a PDGF-2 inhibitor, an NP-1 inhibitor, an NP-2 inhibitor, a Del 1 inhibitor, and an integrin inhibitor. In some embodiments, the anti-angiogenic agent comprises a VEGF inhibitor, and the VEGF inhibitor is a non-antibody inhibitor. In some embodiments, the non-antibody inhibitor is a fusion protein comprising human VEGF receptors 1 and 2. In some embodiments, the fusion protein comprises VEGF-Trap or a modified version thereof.In some embodiments, the isolated non-naturally occurring nucleic acid further comprises a signal peptide. In some embodiments, the signal peptide is selected from the group consisting of a human antibody heavy chain (Vh), a human antibody light chain (Vl), and a VEGF-Trap. In some embodiments, the signal peptide is from a human antibody heavy chain. In some embodiments, the signal peptide is from a VEGF-Trap. In some embodiments, the isolated non-naturally occurring nucleic acid further comprises an intron sequence. In some embodiments, the intron sequence is selected from the group consisting of hCMV intron A, an adenovirus tripartite leader sequence intron, an SV40 intron, a hamster EF-1α gene intron 1, an intervening sequence intron, a human growth hormone intron, and a human β-globin intron. In some embodiments, the intron sequence is an SV40 intron. In some embodiments, the isolated non-naturally occurring nucleic acid further comprises a promoter. In some embodiments, the promoter is selected from the group consisting of cytomegalovirus (CMV) promoter, elongation factor 1 alpha (EF1α) promoter, simian vacuolar virus (SV40) promoter, phosphoglycerate kinase (PGK1) promoter, ubiquitin C (Ubc) promoter, human beta actin promoter, CAG promoter, tetracycline response element (TRE) promoter, UAS promoter, actin 5c (Ac5) promoter, polyhedron promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKIIa) promoter, GAL1 promoter, GAL10 promoter, TEF1 promoter, glyceraldehyde 3-phosphage dehydrogenase (GDS) promoter, ADH1 promoter, CaMV35S promoter, Ubi promoter, human polymerase III RNA (H1) promoter, U6 promoter, polyadenylation constructs thereof, and any combination thereof. In some embodiments, the promoter is a CMV promoter.In some embodiments, the sequence is modified to replace non-AGG arginine codons with AGG at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace all non-AGG arginine codons with AGG in the coding region of the sequence. In some embodiments, the sequence is modified to replace non-AGG arginine codons with AGG at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace all non-AGG arginine codons with AGG compared to SEQ ID NO: 70. In some embodiments, the non-AGG arginine codon comprises CGT, CGC, CGA, CGG, or AGA. In some embodiments, the non-AGG arginine codon is AGA. In some embodiments, the sequence is modified to replace AGA with AGG at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace all AGA with AGG in the coding region of the sequence. In some embodiments, the sequence is modified to replace AGA with AGG at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace all AGA with AGG, as compared to SEQ ID NO:70.In some embodiments, the sequence is modified to replace non-CCC proline codons with CCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or at least 30 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace all non-CCC proline codons with CCC in the coding region of the sequence. In some embodiments, the sequence is modified to replace non-CCC proline codons with CCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace all non-CCC proline codons with CCC compared to SEQ ID NO: 70. In some embodiments, the non-CCC proline codons include CCT or CCA. In some embodiments, the non-CCC proline codon is CCT. In some embodiments, the sequence is modified to replace CCT with CCC at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or at least 30 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace all CCT with CCC in the coding region of the sequence. In some embodiments, the sequence is modified to replace CCT with CCC at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or at least 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace all CCT with CCC compared to SEQ ID NO: 70.In some embodiments, the sequence is modified to replace non-TCC serine codons with TCC at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or at least 36 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace all non-TCC serine codons with TCC in the coding region of the sequence. In some embodiments, the sequence is modified to replace non-TCC serine codons with TCC at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or at least 36 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace all non-TCC serine codons with TCC compared to SEQ ID NO: 70. In some embodiments, the non-TCC serine codon comprises TCT, TCA, TCG, AGT, or AGC. In some embodiments, the non-TCC serine codon is AGC. In some embodiments, the sequence is modified to replace AGC with TCC at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or at least 36 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace all AGC with TCC in the coding region of the sequence. In some embodiments, the sequence is modified to replace AGC with TCC at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or at least 36 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace all AGC with TCC, as compared to SEQ ID NO:70.In some embodiments, the sequence is modified to replace non-CCG proline codons with CCG in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, or at least 30 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace all non-CCG proline codons with CCG in the coding region of the sequence. In some embodiments, the sequence is modified to replace at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, compared to SEQ ID NO:70. At least 24, at least 27, at least 30 codon positions are modified to replace non-CCG proline codons with CCG. In some embodiments, the sequence is modified to replace all non-CCG proline codons with CCG compared to SEQ ID NO: 70. In some embodiments, the non-CCG proline codons include CCC or CCA. In some embodiments, the non-CCG proline codons are CCC. In some embodiments, the sequence is modified to replace CCC with CCG in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, or at least 30 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace all CCC with CCG in the coding region of the sequence. In some embodiments, the sequence is modified to replace CCC with CCG at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, or at least 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace all CCC with CCG compared to SEQ ID NO: 70. In some embodiments, the nucleic acid comprises a viral vector sequence. In some embodiments, the viral vector sequence is a scAAV vector sequence. In some embodiments, the AAV vector sequence is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV vector sequence is of AAV2 serotype. In some embodiments, the viral vector sequence comprises sequences of at least two AAV serotypes. In some embodiments, at least two serotypes are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV11, and AAV12.In some embodiments, the isolated non-naturally occurring nucleic acid comprises at least about 60% sequence identity or similarity to any one of SEQ ID NOs: 13-19, 21-27, 31, 62, 64, 66, or 68. In some embodiments, the sequence identity is about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100% sequence identity to SEQ ID NO: 31. In some embodiments, the isolated non-naturally occurring nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the isolated non-naturally occurring nucleic acid consists of the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the isolated non-naturally occurring nucleic acid comprises about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100% sequence identity to SEQ ID NO: 66. In some embodiments, the isolated non-naturally occurring nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the isolated non-naturally occurring nucleic acid consists of the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the viral vector sequence is single-stranded. In some embodiments, the viral vector sequence is double-stranded. In some embodiments, the isolated non-naturally occurring nucleic acid is single-stranded. In some embodiments, the isolated non-naturally occurring nucleic acid is double-stranded. In some embodiments, the isolated non-naturally occurring nucleic acid, when contacted with a plurality of cells, increases expression of the biologic following transfection or transduction in the plurality of cells compared to an otherwise equivalent isolated non-naturally occurring nucleic acid lacking the modification in the otherwise equivalent plurality of cells. In some embodiments, increased expression includes an increase of at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, or at least 500-fold as determined by an enzyme-linked immunosorbent assay (ELISA) assay.

[0006]

[0006] In some aspects, described herein are isolated non-naturally occurring nucleic acids that comprise at least about 60% sequence identity or similarity to any one of the nucleic acid sequences of SEQ ID NOs: 13-19, 21-27, 31, 62, 64, 66, or 68. In some embodiments, the isolated non-naturally occurring nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the isolated non-naturally occurring nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 66.

[0007]

[0007] In some aspects, described herein are biologics encoded by the isolated non-naturally occurring nucleic acids described herein. In some embodiments, the biologic comprises at least about 60% sequence identity to SEQ ID NO: 12. In some embodiments, the sequence identity is about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, up to about 100%.

[0008]

[0008] In some aspects, described herein are engineered cells that contain the isolated non-naturally occurring nucleic acids described herein.

[0009]

[0009] In some aspects, described herein are a plurality of adeno-associated virus (AAV) particles that comprise an isolated non-naturally occurring nucleic acid described herein.

[0010] In some aspects, a composition is described herein that includes a plurality of AAV particles described herein in a unit dosage form. In some embodiments, the composition is cryopreserved.

[0011]

[0011] In some aspects, described herein is a container comprising an isolated non-naturally occurring nucleic acid described herein, a biologic described herein, an engineered cell described herein, or a plurality of AAV particles described herein.

[0012]

[0012] In some aspects, described herein is a method for modifying cells comprising: (a) contacting a plurality of cells with an isolated non-naturally occurring nucleic acid described in any one of claims 1-88; (b) contacting a plurality of cells with a plurality of adeno-associated virus (AAV) particles described in claim 93; or (c) both steps (a) and (b).

[0013] In some aspects, described herein is a pharmaceutical composition comprising an isolated non-naturally occurring nucleic acid described herein, a biologic described herein, or a plurality of AAV particles described herein. In some embodiments, the pharmaceutical composition is for treating an ophthalmic disease or condition. In some embodiments, the ophthalmic disease or condition is selected from the group consisting of color vision deficiency, age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, Bardet-Biedl syndrome, Best disease, total choroidal atrophy, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, Oguchi disease, Levantin disease (familial dominant drusen), and blue-cone monochromatism. In some embodiments, the ophthalmic disease or condition is AMD.

[0014]

[0014] In some aspects, described herein is a method of treating a disease or condition in a subject in need of such treatment, the method comprising administering to the subject an effective amount of a pharmaceutical composition described herein, thereby treating the disease.

[0015]

[0015] In some aspects, described herein are methods for treating a disease or condition in a subject in need of such treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an isolated non-naturally occurring nucleic acid comprising a sequence encoding a biologic comprising an anti-angiogenic agent, wherein the sequence has been modified to replace non-AGG arginine codons with AGG in at least four codons in the coding region of the sequence compared to an otherwise equivalent sequence lacking the modifications in the coding region, thereby treating the disease or condition in a subject in need of such treatment.

[0016]

[0016] In some aspects, described herein is a method for treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an isolated non-naturally occurring nucleic acid comprising a sequence encoding a biologic comprising an anti-angiogenic agent, wherein the sequence is modified to replace non-AGG arginine codons with AGG in at least four codons in the coding region of the sequence, compared to an otherwise equivalent sequence lacking the modification in the coding region, and the modification is effective to increase the level of the biologic in the subject in need of treatment of the disease or condition, compared to an otherwise equivalent subject administered the otherwise equivalent isolated non-naturally occurring nucleic acid lacking the modification. In some embodiments, the increased level of the biologic in the subject is an increase of at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, or at least 500-fold, as determined by a diagnostic assay. In some embodiments, the sequence encoding the anti-angiogenic agent further comprises a second modification. In some embodiments, the second modification is within at least one codon of the coding region of the sequence, and the second modification is selected from the group consisting of: (a) replacement of at least one non-CCC proline codon with CCC, (b) replacement of at least one non-TCC serine codon with TCC, (c) replacement of at least one non-CCG proline codon with CCG, and (d) any combination of (a)-(c). In some embodiments, the second modification comprises (a). In some embodiments, the second modification comprises (b). In some embodiments, the second modification comprises (c). In some embodiments, the second modification comprises (d).

[0017]

[0017] In some aspects, described herein are methods for treating a disease or condition in a subject in need of such treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an isolated non-naturally occurring nucleic acid comprising a sequence encoding a biologic comprising an anti-angiogenic agent, wherein the sequence has been modified to replace AGA with AGG in at least four codons in the coding region of the sequence compared to an otherwise equivalent sequence lacking the modifications in the coding region, thereby treating the disease or condition in a subject in need of such treatment.

[0018]

[0018] In some aspects, described herein are methods for treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an isolated non-naturally occurring nucleic acid comprising a sequence encoding a biologic comprising an anti-angiogenic agent, wherein the sequence is modified to replace AGA with AGG in at least four codons in the coding region of the sequence, compared to an otherwise equivalent sequence lacking the modification in the coding region, and the modification is effective to increase the level of the biologic in the subject in need thereof, compared to an otherwise equivalent subject administered the otherwise equivalent isolated non-naturally occurring nucleic acid lacking the modification. In some embodiments, the increased level of the biologic in the subject is an increase of at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, or at least 500-fold, as determined by a diagnostic assay. In some embodiments, the sequence encoding the anti-angiogenic agent further comprises a second modification. In some embodiments, the second modification is in at least one codon of the coding region of the sequence, and the second modification is selected from the group consisting of (a) CCT to CCC, (b) AGC to TCC, (c) CCC to CCG, and (d) any combination of (a)-(c). In some embodiments, the second modification comprises (a). In some embodiments, the second modification comprises (b). In some embodiments, the second modification comprises (c). In some embodiments, the second modification comprises (d). In some embodiments, the antiangiogenic agent comprises a VEGF inhibitor, a multi-tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, or an inhibitor of Akt phosphorylation. In some embodiments, the antiangiogenic agent comprises a VEGF inhibitor. In some embodiments, the VEGF inhibitor is a non-antibody inhibitor. In some embodiments, the non-antibody inhibitor comprises a fusion protein comprising human VEGF receptors 1 and 2, and the fusion protein comprises VEGF-Trap or a modified version thereof. In some embodiments, the isolated non-naturally occurring nucleic acid comprises at least about 60% sequence identity or similarity to any one of SEQ ID NOs: 13-19, 21-27, 31, 62, 64, 66, or 68.In some embodiments, the sequence identity is about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100%. In some embodiments, the isolated non-naturally occurring nucleic acid comprises about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100% sequence identity to SEQ ID NO:31. In some embodiments, the isolated non-naturally occurring nucleic acid comprises the nucleic acid sequence of SEQ ID NO:31. In some embodiments, the isolated non-naturally occurring nucleic acid consists of the nucleic acid sequence of SEQ ID NO:31. In some embodiments, the isolated non-naturally occurring nucleic acid comprises about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100% sequence identity to SEQ ID NO:66. In some embodiments, the isolated non-naturally occurring nucleic acid comprises the nucleic acid sequence of SEQ ID NO:66. In some embodiments, the isolated non-naturally occurring nucleic acid consists of the nucleic acid sequence of SEQ ID NO:66. In some embodiments, the nucleic acid comprises a viral vector sequence. In some embodiments, the viral vector sequence is a scAAV vector sequence. In some embodiments, the AAV vector sequence is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV vector sequence is of the AAV2 serotype. In some embodiments, administration is via intravitreal injection, subretinal injection, microinjection, or supraocular injection. In some embodiments, administration is via intravitreal injection. In some embodiments, the ocular disease or condition is selected from the group consisting of color vision deficiency, age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, Bardet-Biedl syndrome, Best disease, total choroidal atrophy, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, Oguchi disease, Levantin disease (familial dominant drusen), and blue-cone monochromatism. In some embodiments, the ocular disease or condition is AMD. In some embodiments, the AMD is wet AMD.In some embodiments, the AMD is dry AMD. In some embodiments, the administration is sufficient to at least alleviate symptoms of a disease or condition, treat a disease or condition, and / or eliminate a disease or condition. In some embodiments, the administration is about 1.0×10 9 vg, approx. 1.0×10 10 vg, approx. 1.0×10 11 vg, approx. 3.0×10 11 vg, approx. 6×10 11 vg, approx. 8.0×10 11 vg, approx. 1.0×10 12 vg, approx. 1.0×10 13 vg, approx. 1.0×10 14 vg, or approximately 1.0 × 10 15 vg of the isolated non-naturally occurring nucleic acid. In some embodiments, the administration is repeated. In some embodiments, the administration is twice daily, twice weekly, twice monthly, three times monthly, monthly, bimonthly, twice yearly, annually, or biennially. In some embodiments, prior to administration, the subject undergoes genetic testing. In some embodiments, the genetic testing detects mutations in a gene sequence compared to an otherwise equivalent wild-type sequence. In some embodiments, the method further comprises administering a secondary therapy. In some embodiments, the secondary therapy comprises at least one of photodynamic therapy (PDT), an anti-inflammatory agent, an antimicrobial agent, and laser photocoagulation therapy (LPT).

[0019]

[0019] Disclosed herein are isolated non-naturally occurring nucleic acids that may include sequences encoding a biologic that may include an anti-angiogenic agent. In some embodiments, the sequence may be modified to replace AGA with AGG in at least one codon of the coding region of the sequence, compared to an otherwise equivalent sequence lacking the modification in the coding region. In some embodiments, the sequence encoding the anti-angiogenic agent may further include a second modification. In some embodiments, the second modification may be within at least one codon of the coding region of the sequence. In some embodiments, the second modification may be selected from the group including a) CCT to CCC, b) AGC to TCC, c) CCC to CCG, or d) any combination thereof. In some embodiments, the second modification may include CCT to CCC. In some embodiments, the second modification may include AGC to TCC. In some embodiments, the second modification may include CCC to CCG. In some embodiments, the second modification may include a combination of CCT to CCC, AGC to TCC, or CCC to CCG. In some embodiments, the antiangiogenic agent may include a VEGF inhibitor, a multi-tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, an inhibitor of Akt phosphorylation, a PDGF-1 inhibitor, a PDGF-2 inhibitor, an NP-1 inhibitor, an NP-2 inhibitor, a Del 1 inhibitor, or an integrin inhibitor. In some embodiments, the antiangiogenic agent may include a VEGF inhibitor, and the VEGF inhibitor may be a non-antibody inhibitor. In some embodiments, the non-antibody inhibitor may be a fusion protein that may include human VEGF receptors 1 and 2. In some embodiments, the fusion protein may include VEGF-Trap or a modified version thereof. In some embodiments, the isolated non-naturally occurring nucleic acid may further include a signal peptide. In some embodiments, the signal peptide may be selected from the group consisting of a human antibody heavy chain (Vh), a human antibody light chain (Vl), and a VEGF-Trap. In some embodiments, the signal peptide may be derived from a human antibody heavy chain. In some embodiments, the signal peptide can be derived from VEGF-Trap.In some embodiments, the isolated non-naturally occurring nucleic acid can further comprise an intron sequence.In some embodiments, the intron sequence may be selected from the group consisting of CMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, hamster EF-1 alpha gene intron 1, intervening sequence intron, human growth hormone intron, and human beta globin intron. In some embodiments, the intron sequence may be an SV40 intron. In some embodiments, the isolated non-naturally occurring nucleic acid may further comprise a promoter. In some embodiments, the promoter may be selected from the group consisting of cytomegalovirus (CMV) promoter, elongation factor 1 alpha (EF1α) promoter, simian vacuolar virus (SV40) promoter, phosphoglycerate kinase (PGK1) promoter, ubiquitin C (Ubc) promoter, human β-actin promoter, CAG promoter, tetracycline response element (TRE) promoter, UAS promoter, actin 5c (Ac5) promoter, polyhedron promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKIIa) promoter, GAL1 promoter, GAL10 promoter, TEF1 promoter, glyceraldehyde 3-phosphate dehydrogenase (GDS) promoter, ADH1 promoter, CaMV35S promoter, Ubi promoter, human polymerase III RNA (H1) promoter, U6 promoter, polyadenylation constructs thereof, and any combination thereof. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the sequence may be modified to replace AGA with AGG in at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or up to 20 codons in the coding region of the sequence. In some embodiments, the sequence may be modified to replace AGA with AGG in 16 codons in the coding region of the sequence.

[0020] In some embodiments, the sequence may be modified to replace AGA with AGG at positions X1-X16 compared to SEQ ID NO:28. In some embodiments, the sequence may be modified to replace CCT with CCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codons of the coding region of the sequence. In some embodiments, the sequence may be modified to replace CCT with CCC in 30 codons of the coding region of the sequence. In some embodiments, the sequence may be modified to replace CCT with CCC in positions X1-X30 compared to SEQ ID NO:28. In some embodiments, the sequence may be modified to replace AGC with TCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or up to 36 codons of the coding region of the sequence. In some embodiments, the sequence may be modified to replace AGC with TCC in 36 codons of the coding region of the sequence. In some embodiments, the sequence may be modified to replace AGC with TCC in positions X1-X36 compared to SEQ ID NO:28. In some embodiments, the sequence may be modified to replace CCC with CCG in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codons of the coding region of the sequence. In some embodiments, the sequence may be modified to replace CCC with CCG in 29 codons of the coding region of the sequence. In some embodiments, the sequence may be modified to replace CCC with CCG at positions X1 to X29 compared to SEQ ID NO:28. In some embodiments, the nucleic acid may comprise a viral vector sequence. In some embodiments, the viral vector sequence may be a self-complementary AAV (scAAV) vector sequence.In some embodiments, the AAV vector sequence may be of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV vector sequence may be of AAV2 serotype. In some embodiments, the viral vector sequence may comprise sequences of at least two AAV serotypes. In some embodiments, the at least two serotypes may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV11, and AAV12. In some embodiments, the isolated non-naturally occurring nucleic acid may comprise a sequence having at least 60% sequence identity or similarity to any one of SEQ ID NO:13-SEQ ID NO:19. In some embodiments, the sequence identity can be from about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100%. In some embodiments, the isolated non-naturally occurring nucleic acid, when contacted with a plurality of cells, can increase expression of the biologic after transfection or transduction in a plurality of cells compared to an otherwise equivalent isolated non-naturally occurring nucleic acid that can lack the modification in the otherwise equivalent sequence in the equivalent plurality of cells. In some embodiments, increased expression can include an increase of at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, or at least 500-fold as determined by enzyme-linked immunosorbent assay (ELISA) assay.

[0021] Also described herein are isolated non-naturally occurring nucleic acids that may comprise at least 60% sequence identity or similarity to any one of the nucleic acid sequences of SEQ ID NO:13-SEQ ID NO:19 or SEQ ID NO:21-SEQ ID NO:27. In some embodiments, the biologic may be encoded by an isolated non-naturally occurring nucleic acid. In some embodiments, the biologic may comprise at least 60% sequence identity to SEQ ID NO:12. In some embodiments, the sequence identity may be from about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100%.

[0022]

[0022] Also described herein are engineered cells that contain the isolated non-naturally occurring nucleic acids described herein.

[0023]

[0023] Also described herein are multiple adeno-associated virus (AAV) particles that can be isolated from the engineered cells.

[0024]

[0024] Also described herein are compositions that can include AAV particles in unit dosage form. In some embodiments, the compositions can be cryopreserved.

[0025]

[0025] Also described herein are containers that can contain a) an isolated non-naturally occurring nucleic acid, b) a biologic, c) an engineered cell, or d) a plurality of AAV particles.

[0026]

[0026] Also described herein is a method for modifying cells, which may include the steps of a) contacting a plurality of cells with an isolated non-naturally occurring nucleic acid, and / or b) contacting a plurality of cells with a plurality of adeno-associated virus (AAV) particles.

[0027] Also described herein is a pharmaceutical composition that may include a) an isolated non-naturally occurring nucleic acid, b) a biologic, or c) a plurality of AAV particles. In some embodiments, the pharmaceutical may be for treating an ophthalmic disease or condition. In some embodiments, the ophthalmic disease or condition may be selected from the group consisting of color vision deficiency, age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, Bardet-Biedl syndrome, Best disease, total choroidal atrophy, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, Oguchi disease, Levantin disease (familial dominant drusen), and blue-cone monochromatism. In some embodiments, the ophthalmic disease or condition may be AMD.

[0028]

[0028] Also described herein is a method for treating a disease or condition in a subject in need of such treatment, which may include a step of administering to the subject an effective amount of a pharmaceutical composition, thereby treating the disease.

[0029]

[0029] Also described herein are methods for treating a disease or condition in a subject in need of such treatment. In some embodiments, the method may include administering a therapeutically effective amount of a pharmaceutical composition that may include an isolated non-naturally occurring nucleic acid that may include a sequence encoding a biologic that may include an anti-angiogenic agent. In some embodiments, the sequence may be modified to replace AGA with AGG in at least one codon of the coding region of the sequence, compared to an otherwise equivalent sequence lacking the modification in the coding region. In some embodiments, the method may treat a disease or condition in a subject in need of such treatment.

[0030]

[0030] Also described herein are methods for treating a disease or condition in a subject in need of such treatment. In some embodiments, the method may include administering a therapeutically effective amount of a pharmaceutical composition that may include an isolated non-naturally occurring nucleic acid that may include a sequence encoding a biologic that may include an anti-angiogenic agent. In some embodiments, the sequence may be modified to replace AGA with AGG in at least one codon of the coding region of the sequence, as compared to an otherwise equivalent sequence lacking the modification in the coding region. In some embodiments, the modification may be effective to increase the level of the biologic in a subject in need of increased levels of the biologic, as compared to an otherwise equivalent subject administered an otherwise equivalent isolated non-naturally occurring nucleic acid that lacks the modification. In some embodiments, the increased level of the biologic in the subject may be an increase of at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, or at least 500-fold, as determined by a diagnostic assay. In some embodiments, the sequence that may encode an anti-angiogenic agent may further include a second modification. In some embodiments, the second modification can be within at least one codon of the coding region of the sequence. In some embodiments, the second modification can be selected from the group including a) CCT to CCC, b) AGC to TCC, c) CCC to CCG, or d) any combination thereof. In some embodiments, the second modification can include CCT to CCC. In some embodiments, the second modification can include AGC to TCC. In some embodiments, the second modification can include CCC to CCG. In some embodiments, the second modification can include any combination of CCT to CCC, AGC to TCC, or CCC to CCG. In some embodiments, the antiangiogenic agent can include a VEGF inhibitor, a multi-tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, or an inhibitor of Akt phosphorylation. In some embodiments, the antiangiogenic agent can include a VEGF inhibitor. In some embodiments, the antiangiogenic agent can include a VEGF inhibitor. In some embodiments, the VEGF inhibitor can be a non-antibody inhibitor.In some embodiments, the non-antibody inhibitor may comprise a fusion protein that may comprise human VEGF receptors 1 and 2. In some embodiments, the fusion protein may comprise VEGF-Trap or a modified version thereof. In some embodiments, the isolated non-naturally occurring nucleic acid may comprise at least 60% sequence identity or similarity to any one of SEQ ID NO:13-SEQ ID NO:19 or SEQ ID NO:21-SEQ ID NO:27. In some embodiments, the sequence identity may be from about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100%. In some embodiments, the nucleic acid may comprise a viral vector sequence. In some embodiments, the viral vector sequence may be a scAAV vector sequence. In some embodiments, the AAV vector sequence may be of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV vector sequence may be of the AAV2 serotype. In some embodiments, administration may be via intravitreal injection, subretinal injection, microinjection, or supraocular injection. In some embodiments, administration may be via intravitreal injection. In some embodiments, the ocular disease or condition may be selected from the group consisting of color vision deficiency, age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, Bardet-Biedl syndrome, Best disease, total choroidal atrophy, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, Oguchi disease, Levantin disease (familial dominant drusen), and blue-cone monochromacy. In some embodiments, the ocular disease or condition may be AMD.

[0031] In some embodiments, the AMD can be wet AMD. In some embodiments, the AMD can be dry AMD. In some embodiments, the administration can be sufficient to reduce at least a symptom of a disease or condition, treat a disease or condition, and / or eliminate a disease or condition. In some embodiments, the administration can be about 1.0×10 9 vg, approx. 1.0×10 10 vg, approx. 1.0×10 11 vg, approx. 3.0×10 11 vg, approx. 6×10 11 vg, approx. 8.0×10 11 vg, approx. 1.0×10 12 vg, approx. 1.0×10 13 vg, approx. 1.0×10 14 vg, or approximately 1.0 × 10 15 vg of the isolated non-naturally occurring nucleic acid. In some embodiments, the administration can be repeated. In some embodiments, the administration can be twice daily, twice weekly, twice monthly, three times monthly, monthly, bimonthly, twice yearly, annually, or biennially. In some embodiments, prior to administration, the subject can undergo genetic testing. In some embodiments, the genetic testing can detect mutations in the gene sequence compared to an otherwise equivalent wild-type sequence. In some embodiments, the method can further include administering a secondary therapy. In some embodiments, the secondary therapy can include at least one of photodynamic therapy (PDT), an anti-inflammatory agent, an antimicrobial agent, or laser photocoagulation therapy (LPT).

[0032] Incorporation by Reference

[0032] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0033]

[0033] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0034] [Figure 1]

[0034] Figure 1 shows scAAV constructs carrying CMV enhancer / promoter, SV40 intron (INT), Kozak sequence (K), native (Af) or human IgG1 heavy chain (Vh) signal peptide (Sig), coding sequence, and synthetic polyadenylation signal (pA), flanked by full-length AAV2 ITR (F-ITR) and truncated AAV2 ITR (d-ITR). AMI059, AMI066, AMI067, AMI068, AMI119, AMI120, and AMI130 show different VEGF-Trap coding sequences with different signal peptides. AMI059 has an Af signal peptide and a coding sequence with low GC content. AMI066 has a Vh signal peptide and a coding sequence with low GC content. AMI067 has an Af signal peptide and a coding sequence with high GC content. AMI068 has a Vh signal peptide and a coding sequence with high GC content. AMI119 has an Af signal peptide and a coding sequence with high GC content, containing 16 Arg codon changes from AGA to AGG and 29 Pro codon changes from CCC to CCT. AMI120 has an Af signal peptide and a coding sequence with high GC content, containing 16 Arg codon changes from AGA to AGG and 36 Ser codon changes from AGC to TCC. AMI130 has an Af signal peptide and a coding sequence with high GC content, containing 16 Arg codon changes from AGA to AGG and 29 Pro codon changes from CCC to CCG. [Diagram 2]

[0035] Figure 2 shows a Western blot analysis of VEGF-Trap expression in HEK293 cells transfected with plasmid DNA. Lanes 1-5 are shown on a non-reducing gel, lanes 6-10 are shown on a reducing gel, lanes 1 and 6 were loaded with supernatant from cells transfected with plasmid AMI059, lanes 2 and 7 were loaded with supernatant from cells transfected with plasmid AMI066, lanes 3 and 8 were loaded with supernatant from cells transfected with plasmid AMI067, and lanes 4, 5, 9 and 10 were loaded with supernatant from cells transfected with plasmid AMI068. [Diagram 3]

[0036] Figures 3A and 3B show SDS-PAGE and Simply Blue staining analysis of low-GC and high-GC AAV vector production in Sf9 cells. In both figures, M indicates the lane loaded with size markers, and sizes are shown in kilodaltons. In Figure 3A, lane 1 shows AAV5 vector loaded as a control, and lanes 2, 3, 4, and 5 show AMI059, AMI066, AMI067, and AMI068, respectively. In Figure 3B, lane 1 shows AAV2 vector loaded as a control, and lanes 2, 3, 4, and 4 show AMI119, AMI120, and AMI130, respectively. [Figure 4]

[0037] Figures 4A and 4B show Western blot analysis of VEGF-Trap expression in HEK293 cells transduced with scAAV2 variant (AAV2.N53) vectors (AMI059, AMI066, AMI067, AMI068, AMI120, AMI119, AMI067, and AMI130). Twenty microliters of supernatant from HEK293 cells transduced with AAV2 variants was loaded into each lane. In Figure 4A, lanes 1, 2, 3, and 4 show AMI059, AMI066, AMI067, and AMI068, respectively. In Figure 4B, lanes 1, 2, 3, and 4 show AMI120, AMI119, AMI067, and AMI130, respectively. [Figure 5A]

[0038] Figure 5A shows SDS-PAGE and Simply Blue staining analysis of VEGF-Trap purified from the supernatant of HEK293 cells transduced with scAAV2-AMI067. Commercially available aflibercept (A) was used as a control. M indicates the lane loaded with size markers, sizes are given in kilodaltons. 1.5 μg / lane of purified protein was loaded in the lane marked V. [Figure 5B]

[0039] Figures 5B and 5C depict the binding affinity of VEGF-Trap from AAV2.N54.120 (AVMX-110) to VEGF-A165. [Figure 5C] Figures 5B and 5C depict the binding affinity of VEGF-Trap from AAV2.N54.120 (AVMX-110) to VEGF-A165. [Figure 6]

[0040] 6 shows the amino acid sequence (SEQ ID NO:61) and nucleic acid sequence (SEQ ID NO:62) of AMI068-pFB-scCMV-SV40-intron-kozak-Vh-VEGF-Trap-GC. Bold indicates the human antibody heavy chain signal peptide. [Figure 7]

[0041] 7 shows the amino acid sequence (SEQ ID NO:63) and nucleic acid sequence (SEQ ID NO:64) of AMI119-pFB-scCMV-SV40-intron-kozak-Af-VEGF-Trap-GCRP(CCT). Bold indicates the VEGF-Trap signal peptide, underlined indicates 16 changes from AGA to AGG, and italics indicates 30 changes from CCC to CCT. [Figure 8]

[0042] 8 shows the amino acid sequence (SEQ ID NO:65) and nucleic acid sequence (SEQ ID NO:66) of AMI120-pFB-scCMV-SV40-intron-kozak-Af-VEGF-Trap-GCRS (TCC). Bold indicates the VEGF-Trap signal peptide, underlined indicates 16 changes from AGA to AGG, and italics indicates 36 changes from AGC to TCC. [Figure 9]

[0043] 9 shows the amino acid sequence (SEQ ID NO:67) and nucleic acid sequence (SEQ ID NO:68) of AMI130-pFB-scCMV-SV40-intron-kozak-Af-VEGF-Trap-GCRP (CCG). Bold indicates the VEGF-Trap signal peptide, underlined indicates 16 changes from AGA to AGG, and italics indicates 29 changes from CCC to CCG. [Figure 10A]

[0044] FIG. 10A illustrates the measurement of AVMX-110 DNA and protein concentrations. [Figure 10B]

[0045] FIG. 10B shows a time course experiment of AAV2.VEGF-Trap expression levels in HEK93 cell cultures. [Figure 11]

[0046] FIG. 11 shows the persistence of AAV2.N53-VEGF-Trap and AAV2.N54-VEGF-Trap expression in HEK293 cells (upper panel) and human retinal cells (ARPE-19, lower panel). [Figure 12]

[0047] Figure 12 shows that VEGF-Trap from AAV2.054-AMI120 was glycosylated similarly to aflibercept on reducing or non-reducing gels (reducing gel: with or without treatment with PNGaseF; non-reducing gel: with or without treatment with PNGaseF). Lanes 1 and 3 show AAV2-VEGF-Trap, and lane 2 represents commercial aflibercept. Multiple bands in the PNGaseF-treated lane indicate that deglycosylation was not complete. [Figure 13]

[0048] FIG. 13 shows the binding affinity of VEGF-Trap derived from AAV2.054-AMI120 to human VEGF-A165 as detected by Biacore assay. [Figure 14]

[0049] FIG. 14 shows the binding affinity of VEGF-Trap expressed by the vector to rhVEGF-A165. [Figure 15]

[0050] FIG. 15 shows a comparison of vector-derived VEGF-Trap and VEGF-Trap in inhibiting HUVEC cell proliferation. [Figure 16]

[0051] FIG. 16 illustrates GFP fundus images on day 7 before fundus imaging in Example 11. [Figure 17]

[0052] Figure 17 illustrates fluorescein fundus angiography on day 7 in Example 11. Representative images from each group are shown. [Figure 18]

[0053] Figure 18 illustrates quantification of fluorescein leakage on day 7 after laser treatment plotted as mean (top) and individual values ​​(bottom). * represents p=0.0113, *** represents p=0.0002. [Figure 19]

[0054] FIG. 19 illustrates representative lesion images from flat mounts on day 7 after laser treatment. [Figure 20]

[0055] FIG. 20 illustrates quantification of flat-mount lesion area on day 7 after laser treatment, plotted as mean (top) and individual values ​​(bottom). [Figure 21]

[0056] FIG. 21 illustrates fundus images in Example 12. [Figure 22]

[0057] FIG. 22 illustrates IHC images of mouse eyes administered with constructs selected in Example 12. [Figure 23]

[0058] FIG. 23 illustrates fundus images of mouse eyes administered selected AAV constructs performed on day 24 for testing. [Figure 24]

[0059] FIG. 24 illustrates IHC images of pig eyes administered with constructs selected in Example 12. [Diagram 25]

[0060] FIG. 25 illustrates fundus images of pig eyes administered selected constructs taken 24 days after injection. [Figure 26]

[0061] FIG. 26 illustrates immunohistochemistry images from day 28 of the porcine study. [Figure 27]

[0062] FIG. 27 illustrates confocal microscopy images comparing the capsid permeability of AMI054 and V226. [Figure 28A]

[0063] FIG. 28A illustrates details of the AAV construct for AVMX-110 / 116. [Figure 28B]

[0064] FIG. 28B illustrates an exemplary chromatogram showing the production of a vector described herein (AVMX-110), as indicated by a single sharp fraction (arrow). [Figure 28C]

[0065] FIG. 28C illustrates an exemplary SDS-PAGE showing expression of AAV VP1, VP2, and VP3. [Figure 28D]

[0066] Figure 28D illustrates the difference in retention of empty and loaded AAV in the separation column process. [Figure 28E]

[0067] FIG. 28E illustrates the separation of empty capsids for purified AVMX-110. [Figure 28F]

[0068] Figure 28F illustrates an exemplary SDS-PAGE detected with a mouse anti-AAV2 antibody. Samples in lanes 1 and 2 are CsCl purified AVMX-110l, lanes 3 and 4 are samples from 2 L shakers, 5-8 are samples from a 2 L bioreactor, and lanes 9 and 10 were 10 μL and 20 μL of final purified drug substance per lane. Western blot with AAV2-specific antibody reactions demonstrates that AVMX-110 is an AAV2-specific serotype. [Figure 28G]

[0069] Figure 28G illustrates an exemplary silver stained image of AVMX-110 separated by 10% SDS-PAGE. Lanes 1 and 5: capture eluate samples. Lanes 2 and 6: blank. Lanes 3 and 7: non-reduced sample from peak 2 of column chromatography. The purity of AVMX-110 process intermediates was analyzed by SDS-PAGE stained with a silver stained gel. AAV VP1, VP2 and VP3 were clearly visualized and not a single impurity was found above 4%. [Figure 29]

[0070] Figure 29 illustrates representative images of fluorescein angiography (FA) data from the various test groups, and a bar graph showing efficacy. Statistical analysis: One-way ANOVA followed by Tukey's multiple comparison (*=0.0332; ***=0.0002). [Diagram 30]

[0071] FIG. 30 illustrates an in vitro cell-based assay for aflibercept expression in various serotypes. [Diagram 31]

[0072] Figure 31 illustrates the FA data for various AAV serotypes. ΔAflibercept was a sham vector that did not produce protein. Statistics were performed by one-way ANOVA followed by Dunnett's multiple comparisons (*=0.0332; **=0.0021; ***=0.0002). [Diagram 32]

[0073] Figure 32 illustrates the ELISA to quantify aflibercept expression in eye and serum samples. Statistics were performed using one-way ANOVA followed by Dunnett's multiple comparison (***=0.0002; ****≦0.0001). [Diagram 33]

[0074] FIG. 33 illustrates FA data for mouse CNV studies with various AAV6.N54-aflibercept vectors. [Diagram 34]

[0075] FIG. 34 illustrates a comparison of AAV1, AAV2 and AAV6-GFP expression using IHC images. [Diagram 35]

[0076] FIG. 35 illustrates a comparison of GFP expression in various serotypes injected into pig eyes. [Diagram 36]

[0077] FIG. 36 illustrates an in vitro comparison of wild-type (wt) AAV2 and N54-AAV2. [Figure 37A]

[0078] FIG. 37A illustrates a comparison of GFP expression in HEK293 cells transduced with different lots of N54-GFP. [Figure 37B]

[0079] FIG. 37B illustrates GFP expression in HEK293 cells transduced with different lots of N54-GFP. [Figure 38A]

[0080] FIG. 38A illustrates the comparison and statistical analysis of FA measurements. [Figure 38B]

[0081] Figure 38B illustrates a representative image of FA in Example 13. Arrows indicate the leaser lesions (bubbles). [Figure 39A]

[0082] FIG. 39A illustrates a comparison of VEGF-Trap levels in serum samples. [Figure 39B]

[0083] FIG. 39B illustrates a comparison of VEGF-Trap levels in ocular samples. [Figure 40A]

[0084] FIG. 40A illustrates the correlation between VEGF-Trap expression and lesion area. [Figure 40B]

[0085] FIG. 40B illustrates a comparison of AAV2 capsid protein levels in ocular samples. [Diagram 41]

[0086] FIG. 41A illustrates that expression of aflibercept in animals treated with and without sham vector control was comparable. In the third group of animals injected with AAV6.N54-aflibercept, approximately 2.3 pg aflibercept / mg of ocular homogenate was measured (FIG. 41A). Mid-range levels for AAV2 and AAV6.N54-aflibercept were approximately 8.8 and approximately 25 pg aflibercept / mg of ocular homogenate, respectively, with a three-fold increase in expression in animals treated with AAV6. Animals treated with high-dose AAV6 had the highest levels of aflibercept, approximately 400 pg aflibercept / mg of ocular homogenate. Serum samples followed a similar trend to ocular tissue samples. Groups 1 (Sham), 2 (AAV2-aflibercept) and 3 (AAV6-low dose) had no detectable aflibercept. Animals treated with medium and high doses of AAV6-aflibercept had 2-3 ng / mL of aflibercept in serum (Figure 41B). Data were analyzed with GraphPad Prism software using one-way ANOVA followed by Dunnett's multiple comparisons (**=0.005 and ****≦0.0001). [Diagram 42]

[0087] FIG. 42 illustrates representative images from day 0 fundus images (Groups 6-8). [Diagram 43]

[0088] FIG. 43 illustrates representative images from fluorescein fundus angiography on day 7. [Diagram 44]

[0089] FIG. 44 illustrates quantification of mean fluorescein leakage on day 7. [Diagram 45]

[0090] FIG. 45 illustrates representative images of isolectin lesion areas. [Diagram 46]

[0091] FIG. 46 illustrates the isolectin area measurements. [Figure 47]

[0092] FIG. 47 illustrates representative images of IHC on day 0 (Groups 6-8 only). [Figure 48A]

[0093] FIG. 48A illustrates a flat mount analysis of images obtained from a dose response study (Example 14). [Figure 48B]

[0094] FIG. 48B illustrates FA analysis of images obtained from a dose-response study (Example 14). [Figure 49A]

[0095] FIG. 49A illustrates the laser-induced lesion area and AVMX-110 dose-response curve. [Figure 49B]

[0096] FIG. 49B illustrates FA analysis of images from an AVMX-110 dosing study. [Figure 50]

[0097] FIG. 50 illustrates fluorescein angiography images of AVMX-110 in a mouse LCNV model. [Figure 51A]

[0098] FIG. 51A illustrates VEGF-Trap levels in the serum of animals (n=6 per group). [Figure 51B]

[0099] Figure 51B illustrates that VEGF-Trap levels in retinal tissue were also measured in retinal homogenates using ELISA. In retinal tissue suspensions, VEGF-Trap expression was in a dose-dependent manner for intravitreally injected AVMX-110. In the high dose group of 1.6e10vg / eye, an average of 13ng / mL of VEGF-Trap was detected. The highest expression level reached more than 40ng / mL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035]

[0100] Provided herein are compositions and methods for use in ocular therapy.In one aspect, provided herein are isolated non-naturally occurring nucleic acids.Nucleic acid can be utilized in compositions for use in ocular therapy for various ocular diseases and conditions.In some cases, nucleic acid can include one or more modifications that provide certain advantages over otherwise equivalent unmodified nucleic acid.

[0036]

[0101] In one aspect, isolated non-naturally occurring nucleic acids encoding biologics are provided herein. Biologics include a wide range of products, such as vaccines, blood and blood components, allergens, cells, gene therapy drugs, tissues, and recombinant therapeutic proteins. Biologics may be composed of sugars, proteins, nucleic acids, or complex combinations of these substances, or may be living entities, such as cells and tissues. Biologics can be isolated from a variety of natural sources (e.g., human, animal, microbial) and produced using a variety of methods. Gene-based and cell-based biologics, for example, are often at the forefront of biomedical research and can be used to treat a variety of medical conditions for which no other treatments are available.

[0037]

[0102] The biologics disclosed herein may include antiangiogenic agents. Angiogenesis refers to the formation of new blood vessels and / or the maintenance of existing vasculature. This process involves the migration, proliferation, and / or differentiation of endothelial cells that line the inner walls of blood vessels. The process of angiogenesis is regulated, at least in part, by chemical signals in the body. Some of these signals, for example, vascular endothelial growth factor (VEGF), bind to receptors on the surface of normal endothelial cells. When VEGF and other endothelial growth factors bind to receptors on endothelial cells, signals are initiated within these cells that promote the growth and survival of new blood vessels. Other chemical signals, for example, antiangiogenic agents, can interfere with blood vessel formation. Normally, the angiogenic stimulating and angiogenic inhibitory effects of these signals are balanced, so that blood vessels form only when and where they are needed, for example, during growth and healing. In the case of disease or various conditions, these signals can become unbalanced, causing increased or abnormal blood vessel growth that can lead to abnormal conditions or diseases. For example, abnormal angiogenesis contributes to wet age-related macular degeneration.

[0038]

[0103] Antiangiogenic agents can reduce or eliminate angiogenesis. Antiangiogenic agents can reduce or eliminate angiogenesis. Antiangiogenic agents can also reduce or eliminate existing vasculature. Angiogenesis inhibitors interfere with various stages in blood vessel growth in several ways. Some are biologics, such as monoclonal antibodies, that specifically recognize and bind to VEGF and / or other antiangiogenic agents. For example, when VEGF binds to these agents, it is unable to activate the VEGF receptor. Other antiangiogenic agents bind to VEGF and / or its receptor, as well as other receptors on the surface of endothelial cells, or other proteins in downstream signaling pathways, blocking their activity. Some antiangiogenic agents are immunomodulatory agents (e.g., agents that stimulate or suppress the immune system) that also have antiangiogenic properties.

[0039]

[0104] In one embodiment, the antiangiogenic agent binds to a component of the VEGF signaling pathway, including, but not limited to, PLC, VRAP, Sck, Src, PI3K, PIP3, Akt, Bad, caspase, eNOS, FAK, paxillin, Cdc42, p38 MAPK, Hsp27, GRB2, SOS, SHC, Ras, Raf, MEK1 / 2, ERK1 / 2, PKC, cPLA2, PGI2, IP3, and combinations thereof.

[0040]

[0105] In one embodiment, the biologic is selected from macromolecules such as proteins, peptides, aptamers, and / or non-translated RNA such as antisense RNA, ribozymes, RNAi and / or siRNA.

[0041]

[0106] The biologic provided herein may include an antiangiogenic agent.In some cases, the biologic is a protein or polypeptide.In some cases, the biologic includes a polypeptide.The polypeptide can enhance and / or reduce one or more functions of ocular cells, such as rod or cone photoreceptor cells, retinal ganglion cells, Muller cells, bipolar cells, amacrine cells, horizontal cells, and / or retinal pigment epithelial cells.

[0042]

[0107] Exemplary classes of polypeptides include, but are not limited to, neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF, and NTN), anti-angiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptors, VEGF-binding antibodies, VEGF-binding antibody fragments (e.g., single chain anti-VEGF antibodies), endostatin, tumstatin, angiostatin, soluble Flt polypeptides (Lai et al. (2005) Mol. Ther. 12:659), Fc fusion proteins comprising soluble Flt polypeptides (see, e.g., Pechan et al. (2009) Gene Ther. 16:10), pigment epithelium-derived factor (PEDF), soluble Tie-2 receptor, and the like), tissue inhibitor of metalloproteinase-3 (TIMP-3), light-responsive opsins, e.g., rhodopsin, anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-X1), and the like. Exemplary polypeptides include, but are not limited to, glial-derived neurotrophic factor (GDNF), fibroblast growth factor 2, neurturin (NTN), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), neurotrophin-4 (NT4), brain-derived neurotrophic factor (BDNF, epidermal growth factor, rhodopsin, X-linked inhibitor of apoptosis, and sonic hedgehog.

[0043]

[0108] In some cases, the polypeptide is selected from the group consisting of retinoschisin, retinitis pigmentosa GTPase regulator (RGPR) interacting protein-1 (e.g., GenBank Accession Nos. Q96KN7, Q9EPQ2, and Q9GLM3; peripherin-2 (Prph2) (e.g., GenBank Accession No. NP_000313; peripherin, retinal pigment epithelium specific protein (RPE65), (see, e.g., GenBank AAC39660, and Morimura et al. (1998) Proc. Natl. Acad. Sci. USA 95:3088); CHM (total choroidermia, (Rab escort protein 1)), a polypeptide that, when defective or deleted, causes total choroidal atrophy (see, e.g., Donnelly et al. (1994) Hum. Mol. Genet. 3:1017, and van (see, e.g., Bokhoven et al. (1994) Hum. Mol. Genet. 3:1041), and Crumbs homolog 1 (CRB1), a polypeptide that, when deficient or deleted, causes Leber congenital amaurosis and retinitis pigmentosa (see, e.g., den Hollander et al. (1999) Nat. Genet. 23:217, and GenBank Accession No. CAM23328). Suitable polypeptides also include polypeptides that, when deficient or deleted, cause color vision defects, such as cone photoreceptor cGMP-gated channel subunit alpha (CNGA3) (see, e.g., GenBank Accession No. NP_001289, and Booij et al. (2011) Ophthalmology 118:160-167), cone photoreceptor cGMP-gated cation channel beta subunit (CNGB3) (see, e.g., Kohl et al. (2005) Eur J Hum Genet. 13(3):302), guanine nucleotide-binding protein (G protein), alpha transducing activity polypeptide 2 (GNAT2) (ACHM4), and ACHM5, as well as polypeptides that, when deficient or deleted, cause various forms of color vision deficiency.

[0044]

[0109] In some cases, the biologic comprises a protein or polypeptide that encodes a site-specific endonuclease that results in site-specific knockdown or knockout of gene function, for example, the endonuclease knocks out an allele associated with a retinal disease. For example, if the dominant allele encodes a defective copy of a gene that is a retinal structural protein when wild type and / or results in normal retinal function, the site-specific endonuclease can be targeted to the defective allele to knock out the defective allele. In addition to knocking out the defective allele, the site-specific nuclease can also be used to stimulate homologous recombination with donor DNA that encodes a functional copy of the protein encoded by the defective allele. Thus, a nucleic acid of non-natural origin can be used to deliver a site-specific endonuclease that knocks out the defective allele, and can also deliver a functional copy of the defective allele to result in the repair of the defective allele, thereby resulting in the production of a functional retinal protein (e.g., functional retinoschisin, functional RPE65, functional peripherin, etc.). See, e.g., Li et al. (2011) Nature 475:217. In some embodiments, the non-naturally occurring nucleic acid comprises a transgene encoding a site-specific endonuclease and a heterologous nucleotide sequence encoding a functional copy of a defective allele, where the functional copy encodes a functional retinal protein.

[0045]

[0110] Exemplary functional retinal proteins include, for example, retinoschisin, RPE65, retinitis pigmentosa GTPase regulator (RGPR) interacting protein-1, peripherin, peripherin-2, and the like. Suitable site-specific endonucleases for use include, for example, CRISPR, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), which are non-naturally occurring and modified to target specific genes. Such site-specific nucleases can be engineered to cleave specific locations in the genome, and then repair the cleavage by non-homologous end joining, inserting or deleting several nucleotides. Such site-specific endonucleases (also referred to as "INDELs") then displace the protein out of frame, effectively knocking out the gene. See, for example, US Patent Application Publication No. 2011 / 0301073. In some cases, the protein or polypeptide biologic is selected from lipoprotein lipase, retinoid isomerohydrolase RPE65, or complement H. In some cases, the biologic is a polypeptide, such as a fusion protein, such as aflibercept.

[0046]

[0111] In one embodiment, the biologic is aflibercept. Aflibercept is also known as VEGF Trap-eye (VTE) and EYLEA®, which may be used interchangeably herein. Aflibercept is a recombinant fusion protein that includes the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG. In contrast to the antibody-based VEGF binding strategy used by ranibizumab and bevacizumab, aflibercept incorporates the second binding domain of the VEGFR-1 receptor and the third domain of the VEGFR-2 receptor. Aflibercept acts as a soluble decoy receptor that binds to VEGF-A and PDGF with higher affinity than the native receptors. SEQ ID NO: 30 illustrates the amino acid sequence of aflibercept aligned with the DNA coding sequence of aflibercept (SEQ ID NO: 31). For SEQ ID NO:31, the nucleic acid contains an Af signal peptide and a coding sequence with high GC content, including 16 Arg codon changes from AGA to AGG and 36 Ser codon changes from AGC to TCC compared to SEQ ID NO:70.

[0047]

[0112] The approved dose of aflibercept intravitreal injection is 2.0 mg, and its dosing varies depending on the indication. Aflibercept is indicated for the treatment of neovascular (wet) age-related macular degeneration, macular edema associated with retinal vein occlusion, diabetic macular edema, and diabetic retinopathy. In some cases, the signal peptide provided herein may be from aflibercept or derived from aflibercept. In one embodiment, the signal peptide comprises a percent homology of about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100% identical to the sequence (e.g., signal peptide sequence) derived from aflibercept. In one embodiment, the signal peptide is derived from a human antibody heavy chain from aflibercept. In one embodiment, the signal peptide is derived from a human antibody light chain from aflibercept.

[0048]

[0113] In some cases, the biologic is an aptamer. The aptamer can be a DNA aptamer or an RNA aptamer. An aptamer is an oligonucleotide (single-stranded or double-stranded) that folds into a defined structure and binds to a target such as a protein. Unlike some protein-based biologics, aptamers generally contain modified sugars (e.g., at their 2' position), so aptamers do not elicit antibodies or elicit fewer antibodies compared to protein-based biologics. In addition, Toll-like receptor-mediated innate immune responses are also inhibited or reduced. Aptamer therapeutics can be developed against intracellular, extracellular, or cell surface targets. In some embodiments, the biologic therapeutic is an aptamer. Exemplary aptamers of interest include aptamers against vascular endothelial growth factor (VEGF). See, for example, Ng et al. (2006) Nat. Rev. Drug Discovery 5:123, and Lee et al. (2005) Proc. Natl. Acad. Sci. USA 102:18902. For example, the VEGF aptamer can comprise the nucleotide sequence 5'-cgcaaucagugaaugcuuauacauccg-3' (SEQ ID NO:69). Also suitable for use is the PDGF-specific aptamer, for example E10030, see, for example, Ni and Hui (2009) Ophthalmologica 223:401, and Akiyama et al. (2006) J. Cell Physiol. 207:407). Exemplary aptamers include pegaptanib. Pegaptanib is a 50kDa aptamer that is a specific nucleic acid ligand that binds to VEGF165. Exemplary aptamers include, but are not limited to, pegaptanib or fovista.

[0049]

[0114] In one embodiment, the biologic is a nucleic acid. The nucleic acid may include, but is not limited to, non-translated RNA, such as antisense RNA, ribozyme, RNAi and / or siRNA. In one embodiment, the biologic therapeutic is an interfering RNA (RNAi). Suitable RNAi include RNAi that can reduce the level of apoptotic or angiogenic factors in a cell. For example, the RNAi can be an shRNA or siRNA that reduces the level of a gene product that induces or promotes apoptosis in a cell. Genes whose gene products induce or promote apoptosis are referred to herein as "pro-apoptotic genes" and the products (mRNA, protein) of those genes are referred to as "pro-apoptotic gene products". Pro-apoptotic gene products include, for example, Bax, Bid, Bak, and Bad gene products. See, for example, U.S. Patent No. 7,846,730. The interfering RNA can also be directed against angiogenesis products, such as VEGF (e.g., Cand5, see, e.g., U.S. Patent Publication No. 2011 / 0143400, U.S. Patent Publication No. 2008 / 0188437, and Reich et al. (2003) Mol. Vis. 9:210), VEGFR1 (e.g., Sirna-027, see, e.g., Kaiser et al. (2010) Am. J. Ophthalmol. 150:33, and Shen et al. (2006) Gene Ther. 13:225), or VEGFR2 (Kou et al. (2005) Biochem. 44:15064). See also U.S. Patent Nos. 6,649,596, 6,399,586, 5,661,135, 5,639,872, and 5,639,736, as well as U.S. Patent Nos. 7,947,659 and 7,919,473.

[0050]

[0115] In some cases, the biologic includes an siRNA that targets VEGF-A, such as bevasiranib.

[0051]

[0116] In some embodiments, a cell type-specific or tissue-specific promoter may be operably linked to a transgene encoding a subject therapeutic agent, such that the gene product is selectively or preferentially produced in a particular cell type or tissue. In some embodiments, an inducible promoter may be operably linked to a transgene sequence. In some cases, the promoter may be operably linked to a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), e.g., a regulatory element that confers selective expression of the operably linked gene in photoreceptor cells. Suitable photoreceptor-specific regulatory elements include, for example, the rhodopsin promoter, the rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076), the β-phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015), the retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra), the interphotoreceptor retinoid binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra), the IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225), and the like.

[0052]

[0117] In some embodiments, the biologic delivered by the subject modified AAV can act to inhibit angiogenesis. In some embodiments, the biologic comprises an antiangiogenic agent. Exemplary antiangiogenic agents can include VEGF inhibitors, multi-tyrosine kinase inhibitors, receptor tyrosine kinase inhibitors, inhibitors of Akt phosphorylation, PDGF-1 inhibitors, PDGF-2 inhibitors, NP-1 inhibitors, NP-2 inhibitors, Del 1 inhibitors, and / or integrin inhibitors. In one embodiment, the antiangiogenic agent comprises a VEGF inhibitor. The VEGF inhibitor can target VEGF or VEGF receptors.

[0053]

[0118] The VEGF family includes placental growth factor (PLGF), VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGF-E. These agents are important regulators of angiogenesis and vascular permeability, with VEGF-A in particular playing a key role in pathological ocular angiogenesis. The VEGF-A gene is located on chromosome 6p12.3 and consists of eight exons and eight intermediate introns. There are nine isoforms of VEGF-A, including VEGF121, VEGF145, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF189 and VEGF206. These isoforms differ from each other in the number of amino acids and heparin binding affinity. Any of the aforementioned VEGF family members and / or their receptors can be targeted by inhibitors.

[0054]

[0119] In certain preferred embodiments, the biologic delivered by the subject modified AAV can act to inhibit the activity of one or more mammalian VEGF proteins selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and / or PDGF. In particularly preferred embodiments, the biologic delivered by the subject AAV variant inhibits the activity of VEGF-A. There are nine isoforms of VEGF-A that are generated by alternative splicing, of which the most physiologically relevant is VEGF165. VEGF-A levels have been found to be elevated in the vitreous of patients with wet age-related macular degeneration, diabetic macular edema, and retinal vein occlusion. Gene products that are effective in inhibiting the activity of VEGF-A in the eye and thus treating patients with increased vitreous VEGF-A include, but are not limited to, aflibercept, ranibizumab, brolucizumab, bevacizumab, and soluble fms-like tyrosine kinase 1 (sFLTl) (GenBank Accession No. U01134). In some embodiments, an infectious AAV virion is provided that includes (i) a variant AAV capsid protein as described herein and (ii) a transgene that includes a VEGF inhibitor. In one embodiment, the transgene includes multiple sequences, each of which encodes a different VEGF-A inhibitor. In one embodiment, the antiangiogenic agent is ranibizumab. Ranibizumab is a humanized monoclonal antibody Fab fragment that inhibits all human isoforms of VEGF-A. In one embodiment, the transgene can be aflibercept.

[0055]

[0120] In yet another embodiment, the isolated non-naturally occurring nucleic acid provided herein comprises modification.Various modifications are envisioned and can be used to improve the introduction, expression, persistence and / or function of biologics, including antiangiogenic agents, compared to otherwise equivalent biologics.In some cases, the otherwise equivalent biologics can be aflibercept.

[0056]

[0121] In one embodiment, the isolated non-naturally occurring nucleic acid contains a modification that confers enhanced expression of a biologic, including an anti-angiogenic agent. For example, some biologics known in the art contain unmodified sequences that are derived from natural gene sequences and are not optimized for introduction and expression in target cells. In one embodiment, the isolated non-naturally occurring nucleic acid is codon-optimized. Codon optimization can be specific to cell type-specific codon usage. Various organisms and cell types show biases that tend to use certain codons over others for the same amino acid. Some species are known to almost completely avoid certain codons. Similarly, there is a bias that some cell types tend to use certain codons over others for the same amino acid. In one embodiment, the method of optimizing the codons of a non-naturally occurring nucleic acid can include reassigning codon usage based on the usage of each codon in the target cell. In some cases, the target cell can be of a specific tissue or organ.

[0057]

[0122] In some cases, modifications are made to increase the guanine and / or cytosine content of the sequence, as described in Grzegorz Kudla et al., "High guanine and cytosine contents increases mRNA levels in mammalian cells," PLoS Biol 4(6):e180. DOI:10.1371 page / journal.pbio.0040180, which is incorporated by reference in its entirety.

[0058] [Table 1]

[0059]

[0123] In one embodiment, the non-naturally occurring nucleic acid sequence may be modified to replace at least one codon with another codon that codes for the same amino acid. In some cases, the codon is modified within the coding region of the sequence. In some cases, the codon is modified in a non-coding region of the sequence. In some cases, the codon is modified within about 100, about 50, about 25, about 15, or about 5 bases from a stop codon. The E-CAI can be utilized to estimate the value of the codon adaptation index as provided in Puigbo, P., Bravo, IG, and Garcia-Vallve, S., "E-CAI: a novel server to estimate an expected value of Codon Adaptation Index (eCAI)," BMC Bioinformatics 9, 65, doi:10.1186 / 1471-2105-9-65 (2008).

[0060]

[0124] Various modifications are envisioned herein. In some cases, codons can be exchanged. For example, sequences can be modified to replace AGA with AGG. In other cases, CCC is replaced with CCT. In other cases, AGC is replaced with TCC. In other cases, CCC is replaced with CCG. Any of the non-limiting replacements provided in Table 1 can be applied to modify nucleic acid. Any number of codons can be exchanged in nucleic acid. In some cases, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 42, at least 44, at least 46, at least 48, or up to 50 codons may be replaced. In one embodiment, the non-naturally occurring nucleic acid comprises 3 codon modifications. In one embodiment, the non-naturally occurring nucleic acid comprises 16 codon modifications. In one embodiment, the non-naturally occurring nucleic acid comprises 3-5, 5-10, 5-15, 10-15, 10-20, 15-20, 1-20, 12-20, 12-25, 15-30, or 15-25 codon modifications. In one embodiment, the non-naturally occurring nucleic acid comprises 2 codon modifications, AGA to AGG and at least one of the following: CCT to CCC, AGC to TCC, or CCC to CCG. In one embodiment, the non-naturally occurring nucleic acid comprises 3 codon modifications, AGA to AGG and at least two of the following: CCT to CCC, AGC to TCC, or CCC to CCG.In one embodiment, the non-naturally occurring nucleic acid comprises four codon modifications, which are AGA to AGG, CCT to CCC, AGC to TCC, and CCC to CCG. Further modifications may comprise any of the codon modifications presented in Table 1 in combination with any of the codons listed above and / or any of the further modifications possible from Table 1. In one embodiment, the nucleic acid is modified such that AGA is replaced with AGG and CCT is replaced with CCC. In one embodiment, the nucleic acid is modified such that AGA is replaced with AGG and AGC is replaced with TCC. In one embodiment, the nucleic acid is modified such that AGA is replaced with AGG and CCC is replaced with CCG.

[0061]

[0125] In one embodiment, the non-naturally occurring nucleic acid sequence is modified to replace AGA with AGG in 16 codons of the sequence. In one embodiment, the non-naturally occurring nucleic acid sequence is modified to replace AGA with AGG in 16 codons of the coding sequence. In some cases, the sequence is modified to replace CCT with CCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codons of the coding region of the nucleic acid sequence. In some cases, the sequence is modified to replace CCT with CCC in at least 30 codons of the coding region of the sequence. In some cases, the sequence is modified to replace AGC with TCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or up to 36 codons of the nucleic acid sequence region. In some cases, the sequence is modified to replace AGC with TCC in 36 codons of the coding region of the nucleic acid sequence. In some cases, the non-naturally occurring nucleic acid sequence is modified to replace CCC with CCG in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codons of the coding region of the sequence. In some cases, the non-naturally occurring nucleic acid sequence is modified to replace CCC with CCG in 29 codons of the coding region of the non-naturally occurring nucleic acid sequence.

[0062]

[0126] In some embodiments, the isolated non-naturally occurring nucleic acid described herein comprises a sequence encoding a biologic comprising an antiangiogenic agent, the sequence comprising a modification in the coding region of the sequence compared to an otherwise equivalent sequence lacking the modification in the coding region, the modification comprising the replacement of at least one, at least two, at least three, or at least four non-AGG arginine codons with AGG. In some embodiments, the non-AGG arginine codon is AGA. In some embodiments, the sequence is modified to replace non-AGG arginine codons with AGG in at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace non-AGG arginine codons with AGG in 16 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace non-AGG arginine codons with AGG in any one of the 16 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace non-AGG arginine codons with AGG in at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace non-AGG arginine codons with AGG in 16 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace non-AGG arginine codons with AGG in any one of the 16 codon positions compared to SEQ ID NO: 70. In some embodiments, the non-AGG arginine codon is AGA.In some embodiments, the sequence is modified to replace AGA with AGG at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 codons of the coding region of the sequence. In some embodiments, the sequence is modified to replace AGA with AGG at 16 codons of the coding region of the sequence. In some embodiments, the sequence is modified to replace AGA with AGG at any one of the 16 codons of the coding region of the sequence. In some embodiments, the sequence is modified to replace AGA with AGG at at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace AGA with AGG at 16 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace AGA with AGG at any one of the 16 codon positions compared to SEQ ID NO: 70.

[0063]

[0127] In some embodiments, the isolated non-naturally occurring nucleic acid described herein comprises a sequence encoding a biologic comprising an antiangiogenic agent, said sequence comprising a modification in the coding region of the sequence compared to an otherwise equivalent sequence lacking the modification in the coding region, said modification comprising replacement of at least one non-CCC proline codon with CCC. In some embodiments, at least one non-CCC proline codon is CCT. The sequence is modified to replace a non-CCC proline codon with CCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace a non-CCC proline codon with CCC in 30 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace a non-CCC proline codon with CCC in any one of the 30 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace a non-CCC proline codon with CCC at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace a non-CCC proline codon with CCC at 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace a non-CCC proline codon with CCC at any one of the 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the non-CCC proline codon is CCT. In some embodiments, the sequence is modified to replace CCT with CCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codons in the coding region of the sequence.In some embodiments, the sequence is modified to replace CCT with CCC in 30 codons of the coding region of the sequence. In some embodiments, the sequence is modified to replace CCT with CCC in any one of the 30 codons of the coding region of the sequence. In some embodiments, the sequence is modified to replace CCT with CCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace CCT with CCC in 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace CCT with CCC in any one of the 30 codon positions compared to SEQ ID NO: 70.

[0064]

[0128] In some embodiments, the isolated non-naturally occurring nucleic acid described herein comprises a sequence encoding a biologic comprising an antiangiogenic agent, the sequence comprising a modification in the coding region of the sequence compared to an otherwise equivalent sequence lacking the modification in the coding region, the modification comprising replacing at least one non-TCC serine codon with TCC. In some embodiments, the sequence is modified to replace a non-TCC serine codon with TCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or up to 36 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace a non-TCC serine codon with TCC in 36 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace a non-TCC serine codon with TCC in any one of the 36 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace non-TCC serine codons with TCC at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or up to 36 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace non-TCC serine codons with TCC at 36 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace non-TCC serine codons with TCC at any one of the 36 codon positions compared to SEQ ID NO: 70. In some embodiments, the non-TCC serine codon is AGC. In some embodiments, the sequence is modified to replace AGC with TCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or up to 36 codons in the coding region of the sequence.In some embodiments, the sequence is modified to replace AGC with TCC in 36 codons of the coding region of the sequence. In some embodiments, the sequence is modified to replace AGC with TCC in any one of the 36 codons of the coding region of the sequence. In some embodiments, the sequence is modified to replace AGC with TCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or up to 36 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace AGC with TCC in 36 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace AGC with TCC in any one of the 36 codon positions compared to SEQ ID NO: 70.

[0065]

[0129] 39. The isolated nucleic acid of non-natural origin according to any one of claims 3 to 38, wherein the sequence is modified to replace non-CCG proline codons with CCG in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, or up to 30 codons in the coding region of the sequence.

[0066]

[0130] In some embodiments, the isolated non-naturally occurring nucleic acid described herein comprises a sequence encoding a biologic comprising an antiangiogenic agent, the sequence comprising a modification in the coding region of the sequence compared to an otherwise equivalent sequence lacking the modification in the coding region, the modification comprising the replacement of at least one non-CCG proline codon with CCG. In some embodiments, the sequence is modified to replace a non-CCG proline codon with CCG in 30 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace a non-CCG proline codon with CCG in any one of the 30 codons in the coding region of the sequence. In some embodiments, the sequence is modified to replace a non-CCG proline codon with CCG in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, or up to 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace a non-CCG proline codon with CCG at 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace a non-CCG proline codon with CCG at any one of 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the non-CCG proline codon is CCC. In some embodiments, the sequence is modified to replace CCC with CCG at at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, or up to 30 codons of the coding region of the sequence. In some embodiments, the sequence is modified to replace CCC with CCG at 30 codons of the coding region of the sequence. In some embodiments, the sequence is modified to replace CCC with CCG at any one of 30 codons of the coding region of the sequence.In some embodiments, the sequence is modified to replace CCC with CCG at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, or up to 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace CCC with CCG at 30 codon positions compared to SEQ ID NO: 70. In some embodiments, the sequence is modified to replace CCC with CCG at any one of the 30 codon positions compared to SEQ ID NO: 70.

[0067]

[0131] In some embodiments, the non-naturally occurring nucleic acid sequences described herein comprise a nucleic acid sequence having at least about 60% sequence identity or similarity to any one of SEQ ID NOs: 13-19, 21-27, 31, 62, 64, 66, or 68. In some embodiments, the sequence identity is from about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, up to about 100%. In some embodiments, the isolated non-naturally occurring nucleic acid comprises a sequence identity from about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, up to about 100% to SEQ ID NO: 31. In some embodiments, the isolated non-naturally occurring nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the isolated non-naturally occurring nucleic acid is 100% identical to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the isolated non-naturally occurring nucleic acid comprises a sequence identity that is about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, up to about 100% to SEQ ID NO: 66. In some embodiments, the isolated non-naturally occurring nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the isolated non-naturally occurring nucleic acid is 100% identical to the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the isolated non-naturally occurring nucleic acid is single-stranded. In some embodiments, the isolated non-naturally occurring nucleic acid is double-stranded.

[0068]

[0132] In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace AGA with AGG at positions X1-X16 compared to SEQ ID NO:70. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCT with CCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codons of the coding region of the nucleic acid sequence of non-natural origin. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCT with CCC in 30 codons of the coding region of the nucleic acid sequence of non-natural origin. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCT with CCC in positions X1-X30 compared to SEQ ID NO:70. In some embodiments, the non-naturally occurring nucleic acid sequence may be modified to replace AGC with TCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or up to 36 codons of the coding region of the non-naturally occurring nucleic acid sequence. In some embodiments, the non-naturally occurring nucleic acid sequence may be modified to replace AGC with TCC in 36 codons of the coding region of the non-naturally occurring nucleic acid sequence. In some embodiments, the non-naturally occurring nucleic acid sequence may be modified to replace AGC with TCC at positions X1 to X36 compared to SEQ ID NO:70. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCC with CCG in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codons of the coding region of the nucleic acid sequence of non-natural origin. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCC with CCG in 29 codons of the coding region of the nucleic acid sequence of non-natural origin. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCC with CCG in positions: X1 to X29 compared to SEQ ID NO:70.In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace AGA with AGG at positions X1-X16 compared to SEQ ID NO:28. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCT with CCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codons of the coding region of the nucleic acid sequence of non-natural origin. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCT with CCC in 30 codons of the coding region of the nucleic acid sequence of non-natural origin. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCT with CCC in positions X1-X30 compared to SEQ ID NO:28. In some embodiments, the non-naturally occurring nucleic acid sequence may be modified to replace AGC with TCC in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or up to 36 codons in the coding region of the non-naturally occurring nucleic acid sequence. In some embodiments, the non-naturally occurring nucleic acid sequence may be modified to replace AGC with TCC in 36 codons in the coding region of the non-naturally occurring nucleic acid sequence. In some embodiments, the non-naturally occurring nucleic acid sequence may be modified to replace AGC with TCC at positions X1 to X36 compared to SEQ ID NO:28. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCC with CCG in at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 21, at least 24, at least 27, at least 29, or up to 30 codons of the coding region of the nucleic acid sequence of non-natural origin. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCC with CCG in 29 codons of the coding region of the nucleic acid sequence of non-natural origin. In some embodiments, the nucleic acid sequence of non-natural origin may be modified to replace CCC with CCG in positions X1 to X29 compared to SEQ ID NO:28.In some embodiments, the non-naturally occurring nucleic acid sequence may comprise a viral vector sequence. In some embodiments, the viral vector sequence may be a scAAV vector sequence. In some embodiments, the AAV vector sequence may be of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the AAV vector sequence may be of the AAV2 serotype. In some embodiments, the viral vector sequence may comprise sequences of at least two AAV serotypes. In some embodiments, the at least two serotypes may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV11, and AAV12. In some embodiments, the isolated non-naturally occurring nucleic acid sequence comprises a sequence having at least about 60% sequence identity or similarity to any one of SEQ ID NOs: 13-19 or 21-27. In some embodiments, the AAV vector sequence is single-stranded. In some embodiments, the AAV vector sequence is double-stranded.

[0069]

[0133] In some cases, modification can also include chemical modification. Modified nucleic acids can include their backbone, sugar, or nucleobase modifications, and even novel bases or base pairs. Modified nucleic acids can have improved chemical and / or biological stability. Decoration with various chemical substituents (e.g., hydrophobic groups) can also produce improved properties and functions, such as new structural motifs and enhanced target binding.

[0070]

[0134] Exemplary chemical modifications include, but are not limited to, 2'F, 2'-fluoro, 2'OMe, 2'-O-methyl, LNA, locked nucleic acid, FANA, 2'-fluoroarabinose nucleic acid, HNA, hexitol nucleic acid, 2'MOE, 2'-O-methoxyethyl, riboNA, (1'-3')-β-L-riboNA, TNA, α-L-threose nucleic acid, tPhoNa, 3'-2'phosphonomethyl-threosyl nucleic acid, dXNA, 2'-deoxyxylonucleic acid, PS, phosphorothioate, phNA, alkylphosphonate nucleic acid, PNA, and peptide nucleic acid.

[0071]

[0135] In some cases, the nucleic acid comprises additional features, which may include sequences such as tags, signal peptides, intron sequences, promoters, stuffer sequences, and the like.

[0072]

[0136] In some cases, the nucleic acid comprises a signal peptide. A signal peptide, which may be referred to as a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide, is a short peptide present at the N-terminus of most newly synthesized proteins destined for the secretory pathway. These proteins include those that reside within specific organelles (endoplasmic reticulum, Golgi apparatus, endosomes), those that are secreted outside the cell, or those that are inserted into most cell membranes. In some cases, the nucleic acid provided herein may comprise a signal peptide. The signal peptide may be of any length, but typically is 15-30 amino acids long. The signal peptide may be about 10-15, 10-20, 10-30, 15-20, 15-25, 15-30, 20-30, or 25-30 amino acids long. A variety of signal peptides may be utilized, including, but not limited to, human antibody heavy chain (Vh), human antibody light chain (Vl), and aflibercept.

[0073]

[0137] In some cases, the intron comprises an intron sequence. An intron is any nucleotide sequence within a sequence that can be removed by RNA splicing during maturation of the final RNA product. In other words, an intron is a non-coding region of an RNA transcript or its encoding DNA that is removed by splicing before translation. Introns do not code for protein products, but are involved in regulating gene expression. Some introns themselves code for functional RNAs and are further processed after splicing to generate non-coding RNA molecules. Alternative splicing is widely used to generate multiple proteins from a single gene. In addition, some introns play an essential role in a wide range of gene expression regulation functions, such as nonsense-mediated decay and mRNA export. In one embodiment, the intron sequence is included in the nucleic acid of the present disclosure and can be selected from hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, hamster EF-1α gene intron 1, intervening sequence intron, human growth hormone intron, and / or human β-globin intron. Any number of intron sequences are envisioned. In one embodiment, the intron sequence is SV40. In some cases, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or up to 10 intron sequences may be included in the nucleic acid.

[0074]

[0138] In one embodiment, further features include a promoter. A promoter is a sequence of DNA to which a protein binds that initiates transcription of a single RNA from the downstream DNA. This RNA may code for a protein or may have a function in itself, such as tRNA, mRNA or rRNA. The promoter is located upstream of the DNA (towards the 5' region of the sense strand) near the transcription start site of the gene. Promoters are about 100-1000 base pairs in length. A variety of promoters are contemplated and may be used with the non-naturally occurring nucleic acids of the present disclosure. In one embodiment, the promoter is a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter, a simian vacuolar virus (SV40) promoter, a phosphoglycerate kinase (PGK1) promoter, a ubiquitin C (Ubc) promoter, a human β-actin promoter, a CAG promoter, a tetracycline response element (TRE) promoter, a UAS promoter, an actin 5c (Ac5) promoter, a polyhedron promoter, a Ca2+ / calmodulin-dependent protein kinase II (CaMKIIa) promoter, a GAL1 promoter, a GAL10 promoter, a TEF1 promoter, a glyceraldehyde 3-phosphate dehydrogenase (GDS) promoter, an ADH1 promoter, a CaMV35S promoter, a Ubi promoter, a human polymerase III RNA (H1) promoter, a U6 promoter, a polyadenylation construct thereof, and any combination thereof. In some cases, the promoter is a CMV promoter.

[0075]

[0139] Any of the nucleic acid sequences provided may include viral vector sequences. The viral vector may be, but is not limited to, lentivirus, retrovirus, or adeno-associated virus. The viral vector may be an adeno-associated virus (AAV) vector. In some cases, the viral vector is an adeno-associated virus vector. Many serotypes of AAV vectors are envisioned, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and / or AAV12. Based on these initial serotypes, the AAV capsid of each serotype can be engineered to be more suitable for biological function, tissue, or cell selection. In some embodiments, the AAV vector is AAV2 and variants AAV2.N53 and AAV2.N54, which are used in the examples of the present invention. Chimeric AAV vectors are also envisioned, which may contain at least two AAV serotypes. In some cases, at least three, at least four, at least five, at least six, at least seven, or up to eight serotypes are combined in a chimeric AAV vector.In some cases, only a portion of AAV is chimeric.For example, suitable portions can include capsid, VP1, VP2, or VP3 domains and / or Rep.In some cases, at least one of VP1, VP2, and VP3 has at least one amino acid substitution compared to otherwise equivalent wild-type AAV capsid protein.In some cases, mutations can occur in VP1 and VP2, VP1 and VP3, VP2 and VP3, or VP1, VP2, and VP3. In some embodiments, at least one of VP1, VP2, and VP3 has about 1 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3, e.g., about 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, or about 20 to about 25 amino acid substitutions compared to wild-type AAV VP1, VP2, and VP3. In some cases, the VPs may be deleted.For example, in some embodiments, the mutant AAV does not include at least one of VP1, VP2, or VP3.

[0076]

[0140] In some cases, AAV vector can be modified.For example, AAV vector can include modification, such as insertion, deletion, chemical modification or synthetic modification.In some cases, a single nucleotide is inserted into AAV vector.In other cases, multiple nucleotides are inserted into vector.The nucleotide that can be inserted can range from about 1 nucleotide to about 5 kb.

[0077]

[0141] In some cases, the mutations may occur at any of the aforementioned AAV capsid positions and may include any number of mutations. In some cases, the mutations may be from one amino acid to another. Any combination or permutation of standard amino acids may be made. Any of the following amino acid modifications may be made in any of VP1, VP2 and VP3: A to R, A to N, A to D, A to C, A to Q, A to E, A to G, A to H, A to I, A to L, A to K, A to M, A to F, A to P, A to S, A to T, A to W, A to Y, A to V, R to N, R to D, R to C, R to Q, R to E, R to G, R to H, R to I, R to L, R to I ... to K, R to M, R to F, R to P, R to S, R to T, R to W, R to Y, R to V, N to D, N to C, N to Q, N to E, N to G, N to H, N to I, N to L, N to K, N to M, N to F, N to P, N to S, N to T, N to W, N to Y, N to V, D to C, D to Q, D to E, D to G, D to H, D to I, D to L, D to K, D to M, D to F, D to P, D to S, D to T, D to W, D to Y, D to V, C to Q, C to E, C to G, C to H, C to I, C to L, C to K, C to M, C to F, C to P, C to S, C to T, C to W, C to Y, C to V, Q to E, Q to G, Q to H, Q to I, Q to L, Q to K, Q to M, Q to F, Q to P, Q to S, Q to T, Q to W, Q to Y, Q to V, E to G, E to H, E to I, E to L, E to K, E to M, E to F, E to P, E to S, E to T, E to W, E to Y, E to V, G to H, G to I, G to L, G to K, G to M, G to F, G to P, G to S, G to T, G to W, G to Y, G to V, H to I, H to L, H to K, H to M, H to F, H to P, H to S, H to T, H to W, H to Y, H to V, I to L, I to K, I to M, I to F,I to P, I to S, I to T, I to W, I to Y, I to V, L to K, L to M, L to F, L to P, L to S, L to T, L to W, L to Y, L to V, K to M, K to F, K to P, K to S, K to T, K to W, K to Y, K to V, M to F, M to P, M to S, M to T, M to W, M to Y, M to V, F to P, F to S, F to T, F to W, F to Y, F to V, P to S, P to T, P to W, P to Y, P to V, S to T, S to W, S to Y, S to V, T to W, T to Y, T to V, W to Y, W to V, Y to V, and the reverse of any of the preceding mutations.

[0078]

[0142] The mutations may be conservative mutations or substitutions. For example, the 20 naturally occurring amino acids may share similar properties. The aliphatic amino acids may be glycine, alanine, valine, leucine, or isoleucine. The hydroxyl or sulfur / selenium-containing amino acids may be serine, cysteine, selenocysteine, threonine, or methionine. The cyclic amino acids may be proline. The aromatic amino acids may be phenylalanine, tyrosine, or tryptophan. The basic amino acids may be histidine, lysine, and arginine. The acidic amino acids may be aspartic acid, glutamic acid, asparagine, or glutamine. The conservative mutations may be serine to glycine, serine to alanine, serine to serine, serine to threonine, or serine to proline. Conservative mutations can be arginine to asparagine, arginine to lysine, arginine to glutamine, arginine to arginine, arginine to histidine. Conservative mutations can be leucine to phenylalanine, leucine to isoleucine, leucine to valine, leucine to leucine, leucine to methionine. Conservative mutations can be proline to glycine, proline to alanine, proline to serine, proline to threonine, proline to proline. Conservative mutations can be threonine to glycine, threonine to alanine, threonine to serine, threonine to threonine, threonine to proline. Conservative mutations can be alanine to glycine, alanine to threonine, alanine to proline, alanine to alanine, alanine to serine. Conservative mutations can be valine to methionine, valine to phenylalanine, valine to isoleucine, valine to leucine, valine to valine. Conservative mutations can be glycine to alanine, glycine to threonine, glycine to proline, glycine to serine, glycine to glycine. Conservative mutations can be isoleucine to phenylalanine, isoleucine to isoleucine, isoleucine to valine, isoleucine to leucine, isoleucine to methionine.Conservative mutations can be phenylalanine to tryptophan, phenylalanine to phenylalanine, phenylalanine to tyrosine. Conservative mutations can be tyrosine to tryptophan, tyrosine to phenylalanine, tyrosine to tyrosine. Conservative mutations can be cysteine ​​to serine, cysteine ​​to threonine, cysteine ​​to cysteine. Conservative mutations can be histidine to asparagine, histidine to lysine, histidine to glutamine, histidine to arginine, histidine to histidine. Conservative mutations can be glutamine to glutamic acid, glutamine to asparagine, glutamine to aspartic acid, glutamine to glutamine. Conservative mutations can be asparagine to glutamic acid, asparagine to asparagine, asparagine to aspartic acid, asparagine to glutamine. Conservative mutations can be lysine to asparagine, lysine to lysine, lysine to glutamine, lysine to arginine, lysine to histidine. Conservative mutations can be aspartic acid to glutamic acid, aspartic acid to asparagine, aspartic acid to aspartic acid, aspartic acid to glutamine. Conservative mutations can be glutamine to glutamine, glutamine to asparagine, glutamine to aspartic acid, glutamine to glutamine. Conservative mutations can be methionine to phenylalanine, methionine to isoleucine, methionine to valine, methionine to leucine, methionine to methionine. Conservative mutations can be tryptophan to tryptophan, tryptophan to phenylalanine, tryptophan to tyrosine.

[0079]

[0143] In some embodiments, the modified AAV vector comprises a modified AAV2 serotype vector.In some embodiments, the modified AAV vector comprises modified VP1, VP2, VP3, or a combination thereof.In some embodiments, the modified AAV2 serotype vector comprises modified VP1, VP2, VP3, or a combination thereof.

[0080]

[0144] In one aspect, the isolated non-naturally occurring nucleic acid comprises at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and up to about 100% sequence identity or similarity to any one of SEQ ID NOs: 13-19. In one embodiment, the isolated non-naturally occurring nucleic acid comprises at least about 60% sequence identity or similarity to any one of SEQ ID NOs: 13-19. In some cases, the isolated non-naturally occurring nucleic acid is any one of SEQ ID NOs: 13-19. In one aspect, the isolated non-naturally occurring nucleic acid comprises at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and up to about 100% sequence identity or similarity to any one of SEQ ID NOs: 21-27. In one embodiment, the isolated non-naturally occurring nucleic acid comprises at least 60% sequence identity or similarity to any one of SEQ ID NOs: 21-27. In some cases, the isolated non-naturally occurring nucleic acid is any one of SEQ ID NOs: 21-27. In some cases, the nucleic acid encodes a polypeptide having 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to SEQ ID NO: 12. In some cases, the nucleic acid encodes a polypeptide of SEQ ID NO: 12.

[0081]

[0145] In one aspect, a method of modifying a cell and thereby producing an engineered cell is also provided herein. The cell can refer to a primary cell, a recombinant cell, or a cell line. In some cases, the cell is a packaging cell. The packaging cell can be any one of HEK293 cells, HeLa cells, and Vero cells, to name a few. The engineered cell can be a primary cell. In some cases, the engineered cell can be an ocular cell. Suitable ocular cells include, but are not limited to, photoreceptor cells, ganglion cells, RPE cells, amacrine cells, horizontal cells, Müller cells, and the like.

[0082]

[0146] In some cases, the cell is a packaging cell used to generate viral particles. To generate AAV virions or viral particles, an AAV expression vector is introduced into a suitable host cell using known techniques, such as transfection. In some cases, transfection techniques, such as CaPO 4 Transfection or electroporation, and / or infection of cell lines such as human embryonic kidney cell line HEK293 (a human kidney cell line that contains a functional adenovirus E1 gene that provides trans-acting E1 protein) with hybrid adenovirus / AAV vectors are used. Transfection techniques are 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. Suitable transfection methods include calcium phosphate coprecipitation, direct microinjection, electroporation, liposome-mediated gene transfer, and nucleic acid delivery using high-velocity particle bombardment, which are known in the art.

[0083]

[0147] To engineer the cells, a plurality of cells can be contacted with the isolated non-naturally occurring nucleic acid. The contact can include any length of time, including from about 5 minutes to about 5 days. The contact can last for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In some cases, the contact can last for 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, or up to about 5 days.

[0084]

[0148] In some cases, the packaging cell line supernatant is treated by PEG precipitation to concentrate the virus. In other cases, a centrifugation step can be used to concentrate the virus. For example, a column can be used to concentrate the virus during centrifugation. In some embodiments, precipitation occurs at about 4°C or lower (e.g., about 3°C, about 2°C, about 1°C, or about 1°C) for at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours, or at least about 24 hours. In some embodiments, the recombinant AAV is isolated from the PEG precipitated supernatant by low-speed centrifugation followed by a CsCl gradient. The low-speed centrifugation can be at about 4000 rpm, about 4500 rpm, about 5000 rpm, or about 6000 rpm for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes. In some cases, recombinant AAV is isolated from PEG-precipitated supernatant by centrifugation at about 5000 rpm for about 30 minutes followed by CsCl gradient.In some cases, CsCl purification can be replaced by IDX gradient ultracentrifugation.Supernatant can be collected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours after transfection, or between any two of these time points.Supernatant can also be purified, concentrated, or a combination thereof.For example, concentration or virus titer can be determined by qPCR or silver staining.

[0085]

[0149] In one embodiment, a plurality of AAV particles isolated from the engineered cells is also provided. The viral titer is about 10 2 vp / mL, approx. 10 3 vp / mL, approx. 10 4 vp / mL, approx. 10 5 vp / mL, approx. 10 6 vp / mL, approx. 10 7 vp / mL, approx. 10 8 vp / mL or up to about 10 9 The viral titer can be approximately 10 2 GC / mL, approx. 10 3 GC / mL, approx. 10 4 GC / mL, approx. 10 5 GC / mL, approx. 10 6 GC / mL, approx. 10 7 GC / mL, approx. 10 8 From GC / mL or up to about 10 9 In some cases, the viral titer may be about 10 2 TU / mL, approximately 10 3 TU / mL, approximately 10 4 TU / mL, approximately 10 5 TU / mL, approximately 10 6 TU / mL, approximately 10 7 TU / mL, 10 8 From TU / mL or up to about 10 9 The optimal viral titer may be in TU / mL. Optimal viral titers may vary depending on the cell type being transduced. The viral range may be from about 1,000 MOI to about 2000 MOI, from about 1500 MOI to about 2500 MOI, from about 2000 MOI to about 3000 MOI, from about 3000 MOI to about 4000 MOI, from about 4000 MOI to about 5000 MOI, from about 5000 MOI to about 6000 MOI, from about 6000 MOI to about 7000 MOI, from about 7000 MOI to about 8000 MOI, from about 8000 MOI to about 9000 MOI, from about 9000 MOI to about 10,000 MOI. For example, to infect 1 million cells using an MOI of 10,000, 10,000 x 1,000,000 = 10 10 GC is required.

[0086]

[0150] In some cases, multiple AAV particles can be formulated into a unit dosage form. Various formulations are envisioned for delivery to adults or children, including 0.5×10 9 vg, 1.0×10 9 vg, 1.0×10 10 vg, 1.0×10 11 vg, 3.0×10 11 vg, 6×10 11 vg, 8.0×10 11 vg, 1.0×10 12 vg, 1.0×10 13 vg, 1.0×10 14 vg, 1.0×10 15 vg, or up to 1.5×10 15 vg. The viral particle composition can be frozen or otherwise stored in a suitable container.

[0087]

[0151] The compositions and methods provided herein may be sufficient to enhance delivery and / or expression of a subject biologic by at least about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100% over an otherwise equivalent unmodified nucleic acid. In some cases, the otherwise equivalent unmodified nucleic acid encodes VEGF-Trap. In some cases, the modifications enhance delivery and / or expression of a subject biologic at least about 1 fold, about 6 fold, about 11 fold, about 16 fold, about 21 fold, about 26 fold, about 31 fold, about 36 fold, about 41 fold, about 46 fold, about 51 fold, about 56 fold, about 61 fold, about 66 fold, about 71 fold, about 76 fold, about 81 fold, about 86 fold, about 91 fold, about 96 fold, about 101 fold, about 106 fold, about 111 fold, about 116 fold, about 121 fold, about 126 fold, about 131 fold, about 136 fold, about 141 fold, about 146 fold, about 151 fold, about 156 fold, about 161 fold, about 166 fold , about 171-fold, about 176-fold, about 181-fold, about 186-fold, about 191-fold, about 196-fold, about 201-fold, about 206-fold, about 211-fold, about 216-fold, about 221-fold, about 226-fold, about 231-fold, about 236-fold, about 241-fold, about 246-fold, about 251-fold, about 256-fold, about 261-fold, about 266-fold, about 271-fold, about 276-fold, about 281-fold, about 286-fold, about 291-fold, about 296-fold, about 301-fold, about 306-fold, about 311-fold, about 316-fold, about 321-fold, about 326-fold, about 331-fold, about 336-fold, about 341-fold, about 346-fold, or about 350-fold. In one embodiment, increased expression comprises an increase of at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, or at least 500-fold as determined by in vitro assays. Suitable in vitro assays include ELISA, Western blot, Luminex, microscopy, imaging, and / or flow cytometry.

[0088]

[0152] A subject AAV virion can exhibit an increase in infectivity of retinal cells (e.g., photoreceptor cells, ganglion cells, RPE cells, amacrine cells, horizontal cells, Muller cells, etc.) of at least 1-fold, at least 6-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or greater than 50-fold, as compared to the infectivity of retinal cells (e.g., photoreceptor cells, ganglion cells, RPE cells, amacrine cells, horizontal cells, Muller cells, etc.) by an AAV virion containing an otherwise equivalent WT AAV capsid protein.

[0089]

[0153] Methods of treating a disease or condition are also provided herein. The methods of treatment may include introducing a virion or vector encoding a biologic provided herein into a subject in need thereof. In some cases, the methods of treatment include introducing a plurality of virions or vectors encoding a biologic comprising an antiangiogenic agent. Methods of treating a disease are also provided, comprising administering a pharmaceutical composition to a subject in need of disease treatment. The pharmaceutical composition may include a sequence encoding a biologic comprising an antiangiogenic agent and / or a virion encoding an antiangiogenic agent.

[0090]

[0154] In one embodiment, the method of treatment comprises administering a therapeutically effective amount of a pharmaceutical composition comprising an isolated non-naturally occurring nucleic acid comprising a sequence encoding a biologic comprising an antiangiogenic agent. The sequence may be or may include any of the nucleic acids provided herein. For example, the sequence may comprise a nucleic acid sequence having at least about 60% sequence identity or similarity to any one of SEQ ID NOs: 13-19, 21-27, 31, 62, 64, 66, or 68. In some embodiments, the sequence identity is from about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, up to about 100%. In some embodiments, the sequence comprises a sequence identity from about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, up to about 100% to SEQ ID NO: 31. In some embodiments, the sequence comprises the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the sequence is 100% identical to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the sequence comprises about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, up to about 100% sequence identity to SEQ ID NO: 66. In some embodiments, the sequence comprises the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the sequence is 100% identical to the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the sequence is single stranded. In some embodiments, the sequence is double stranded.

[0091]

[0155] In some cases, the sequence is modified according to the present disclosure, for example, the sequence is modified to replace AGA with AGG in at least one codon in the coding region of the sequence, compared to an otherwise equivalent sequence that lacks modification in the coding region.As provided herein, the modification of the present disclosure may have certain advantages, such as increasing the level of a biologic in a subject, compared to an otherwise equivalent subject that is administered an otherwise equivalent isolated non-naturally occurring nucleic acid that lacks modification.Increasing the level of a biologic in a subject may have a therapeutic effect, and may alleviate or eliminate any of the diseases or conditions provided herein.

[0092]

[0156] In some cases, the increase in the level of a biologic in a subject is at least a 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 500-fold increase as determined by a diagnostic assay.

[0093]

[0157] Suitable diagnostic assays may include ophthalmic diagnostic assays, such as ophthalmic examinations, such as refraction tests, eye scans, ocular coherence tomography, Falnworth-Munsell 100 color tests, computerized optic disc imaging and retinal layer analysis (GDX, HRT, OCT), corneal topography, electroretinogram (ERG), electrooculogram (EOG), visual evoked potentials (VEP), visual evoked responses (VER), fluorescein fundus angiography, ocular coherence tomography (OCT), retinal photography, fundus photography, specular microscopy, Goldmann, Humphrey, FDT, Octopus biometry / IOL calculation, A-scans, B-scans, and combinations thereof.

[0094]

[0158] In some cases, retinal examinations can be used. Non-limiting methods for evaluating retinal function and its changes include evaluating visual acuity (e.g., best corrected visual acuity [BCVA], walking, navigation, object detection and identification), evaluating visual field (e.g., static and kinetic perimetry), performing a medical examination (e.g., slit lamp examination of the anterior and posterior parts of the eye), evaluating electrophysiological response to light and dark of all wavelengths (e.g., any form of electroretinogram (ERG) [full field, multifocal and pattern], any form of visual evoked potential (VEP), electrooculogram (EOG), color vision, dark adaptation and / or contrast sensitivity). Non-limiting methods for assessing anatomy and retinal health and changes therein include optical coherence tomography (OCT), fundus photography, adaptive optics scanning laser ophthalmoscopy (AO-SLO), fluorescence and / or autofluorescence, ocular activity and eye movement measurements (e.g., nystagmus, fixation preference, and stability), reported outcome measures (patient-reported changes in visually and non-visually guided behaviors and activities, patient-reported outcomes [PROs], quality of life, questionnaire-based assessments of daily activities, and measures of neurological function (e.g., functional magnetic resonance imaging (MRI)).

[0095]

[0159] Related eye diseases and conditions may include, but are not limited to, blindness, color vision deficiency, age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, Bardet-Biedl syndrome, Best disease, total choroidal atrophy, Leber's congenital amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS), rod-cone dystrophy, cone-rod dystrophy, Oguchi disease, Levantine disease (familial dominant drusen), and blue-cone monochromatism. In one embodiment, the eye disease or condition is AMD. AMD may be wet AMD or dry AMD.

[0096]

[0160] In some cases, administration of the pharmaceutical agent is sufficient to at least alleviate symptoms of the disease or condition, treat the disease or condition, and / or eliminate the disease or condition. In some cases, the improvement of the disease or condition can be confirmed by any of the diagnostic assays provided. In other cases, the improvement can be obtained through interviewing the treated subject. For example, the subject can tell his / her doctor that the subject's vision has improved compared to the vision before administration of the subject pharmaceutical agent. In other cases, an in vivo animal model can be used to confirm the alleviation of the disease or condition after treatment. Suitable animal models include mouse models, primate models, rat models, dog models, etc.

[0097]

[0161] The pharmaceutical compositions may be administered to the subject using a variety of techniques, such as intravitreal, intramuscular, intravenous, subcutaneous, and / or intraperitoneal injection.

[0098]

[0162] For in vivo delivery, the subject nucleic acid and / or AAV virion can be formulated into a pharmaceutical composition, which will generally be administered intravitreally or parenterally (e.g., via intramuscular, subcutaneous, intratumoral, transdermal, intrathecal, etc. routes of administration). In some embodiments, the pharmaceutical composition can be used to treat a subject, such as a human or mammal, in need of treatment. In some cases, the subject can be diagnosed with a disease, e.g., an eye disease. In some embodiments, the subject pharmaceutical composition can be administered simultaneously with a secondary therapy. The secondary therapy can include any therapy for use on the eye. In some cases, the secondary therapy includes nutritional therapy, vitamins, laser therapy such as laser photocoagulation, photodynamic therapy, visudyne, anti-VEGF therapy, eye patches, eye drops, paralyzing agents, corrective vision therapy, behavioral / sensory visual therapy, etc. In some embodiments, any of the biologics previously described can be considered a secondary therapy.

[0099]

[0163] Any of the pharmaceutical compositions may also include an excipient. Such excipients, carriers, diluents, and buffers include any pharmaceutical agent that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and salts of organic acids such as acetate, propionate, malonate, benzoate, can be included in such vehicles. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharma-ceutically acceptable excipients are known in the art and need not be discussed in detail here. Pharmaceutically acceptable excipients are well described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th Edition, Lippincott, Williams, and Wilkins, "Pharmaceutical Dosage Forms and Drug Delivery Systems" (1999) eds. H. C. Ansel et al., 7th Edition, Lippincott, Williams, and Wilkins, and "Handbook of Pharmaceutical Excipients" (2000) eds. A. H. Kibbe et al., 3rd Edition, Amer. Pharmaceutical Assoc.

[0100]

[0164] In some embodiments, an effective amount of a subject rAAV virion results in a reduction in the rate of loss of retinal function, anatomical integrity, or retinal health, e.g., a reduction in the rate of loss and therefore the rate of disease progression by 2-fold, 3-fold, 4-fold, or 5-fold or more, e.g., a reduction in the rate of loss and therefore the rate of disease progression by more than 10-fold. In some embodiments, an effective amount of a subject rAAV virion results in a gain of visual function, retinal function, improvement in retinal anatomy or health, and / or improvement in ocular activity and / or improvement in neurological function, e.g., a 2-fold, 3-fold, 4-fold, or 5-fold or more improvement in retinal function, retinal anatomy or health, and / or improvement in ocular activity, e.g., a 10-fold or more improvement in retinal function, retinal anatomy or health, and / or improvement in ocular activity. As will be readily appreciated by those skilled in the art, the dosage required to achieve the desired treatment effect is typically less than 1×10 8 From 1 x 10 15 recombinant virions, which is typically in the range of 1×10 8 From 1 x 10 15 This is called the "vector genome."

[0101]

[0165] In some aspects, the compositions, e.g., pharmaceutical compositions, provided herein are administered to a subject in need of the composition. In some cases, the administration is at least about 0.5×10 9 vg, 1.0×10 9 vg, 1.0×10 10 vg, 1.0×10 11 vg, 3.0×10 11 vg, 6×10 11 vg, 8.0×10 11 vg, 1.0×10 12 vg, 1.0×10 13 vg, 1.0×10 14 vg, 1.0×10 15 vg, 1.5×10 15 For example, in the case of in vivo injection, i.e., direct injection into the eye, a therapeutically effective dose is about 106 From 10 to 10 15 A subject AAV virion, e.g., on the order of about 10 8 pcs to 10 12 For in vitro transduction, the effective amount of engineered AAV virions delivered to cells can be about 10 8 From 10 to 10 13 Other effective dosages can be readily established by one of ordinary skill in the art through routine testing to establish dose-response curves.

[0102]

[0166] Administration can be repeated at any interval, hi some embodiments, administration occurs twice daily, every other day, twice weekly, twice monthly, three times monthly, monthly, every two months, twice yearly, annually, or every two years.

[0103]

[0167] Dosage treatment can be a single dose schedule or a multiple dose schedule. Moreover, subjects can be administered multiple doses as needed. Those skilled in the art can easily determine the appropriate number of doses. In some aspects, the pharmaceutical composition is administered via intravitreal injection, subretinal injection, microinjection, or supraocular injection.

[0104]

[0168] In some embodiments, subjects may be screened for mutations via genetic testing before, during, and / or after administration of the pharmaceutical compositions provided herein.Relevant genes that may be screened for mutations include RPE65, CRB1, AIPL1, CFH, or RPGRIP.

[0105]

[0169] Also provided is a kit that includes any of the compositions provided herein.Also provided is a container that includes a) a subject modified adeno-associated virus (AAV) capsid, b) a subject vector, or c) a subject engineered virion.In one aspect, the container is a vial, a syringe, or a needle.In some cases, the container is configured for intraocular delivery.

[0106]

[0170] The kit may include a suitably dispensed composition. The components of the kit may be packaged in either aqueous media or lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed and preferably suitably dispensed. Where multiple components are present in the kit, the kit will also generally include a second, third, or other additional container into which the additional components may be placed separately. However, various combinations of components may be included in the vial. The kit will also typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0107]

[0171] In some cases, the packaged product comprising the composition described herein can be appropriately labeled.In some cases, the pharmaceutical composition described herein can be manufactured in accordance with current Good Manufacturing Practice (cGMP) and labeling regulations.In some cases, the pharmaceutical composition disclosed herein can be sterile.

[0108]

[0172] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in implementing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby. EXAMPLES

[0109] Example 1: Codon optimization and cloning of nucleic acid constructs

[0173] A DNA sequence containing SV40 intron-VEGF-Trap open reading frame (ORF)-synthetic polyA signal was synthesized by a service company (Twist Bioscience, South San Francisco, CA) to contain 40% GC content in the VEGF-Trap ORF. This DNA sequence was PCR amplified with forward primer A024 and reverse primer A025, and cloned into the XbaI and SphI sites of pFB-scCMV-MIF using NEBuilder HiFi DNA assembly kit (New England Biolabs, Ipswich, MA) to generate AMI059-pFB-scCMV-SV40-intron-Af-VEGF-Trap, as shown in Figure 1. The pFB-scCMV-MIF plasmid, which contains one full-length AAV ITR and one truncated ITR for self-complementary AAV (scAAV), is a derivative of the pFastBac shuttle plasmid from the Bac-to-Bac baculovirus expression system (Invitrogen). In the cloning process, MIF was replaced by the VEGF-Trap gene. The VEGF-Trap gene contains its original signal peptide (Af). To replace Af with the human antibody heavy chain signal peptide (Vh), the SV40-intron-Vh-fragment was PCR amplified with forward primer A024 and reverse primer A082, and the plasmid AMI060 containing the SV40-intron-Vh sequence as a template. The VEGF-Trap ORF was amplified with forward primer A085 and reverse primer A025, and the plasmid AMI059-pFB-scCMV-SV40-intron-kozak-VEGF-Trap as a template. These two PCR fragments were cloned into the XbaI and SphI sites of AMI059 using the NEBuilder HiFi DNA assembly kit to generate AMI066-pFB-scCMV-SV40-intron-Vh-VEGF-Trap, as shown in Figure 1.

[0110]

[0174] To optimize the VEGF-Trap ORF, the VEGF-Trap protein sequence was reverse-translated into DNA by selecting the preferred codon output of homo sapiens using SnapGene Software (GSL Biotech, San Diego, CA). The sequence was synthesized by Twist Bioscience and PCR-amplified with forward primer A086 and reverse primer A087. This PCR fragment was cloned into the StuI and BstBI sites of AMI059 via HiFi reaction to generate AMI067-pFB-scCMV-SV40-intron-kozak-Af-VEGF-Trap-GC, as shown in Figure 1. To replace the signal peptide Af in AMI067 with Vh, the SV40-intron-Vh-fragment was PCR-amplified with forward primer A024 and reverse primer A089, and the plasmid AMI060 containing the SV40-intron-Vh sequence as a template. The Twist-synthesized VEGF-Trap ORF was PCR amplified with forward primer A088 and reverse primer A090. These two PCR fragments were cloned into the XbaI and BstBI sites of AMI059 using the NEBuilder HiFi DNA assembly kit to generate AMI068-pFB-scCMV-SV40-intron-kozak-Vh-VEGF-Trap-GC, as shown in Figures 1 and 6.

[0111]

[0175] To further optimize the VEGF-Trap DNA codons, the DNA sequence was manually adjusted to generate three more versions of variant VEGF-Trap DNA molecules. In version 1, 16 Arg codons were changed from AGA to AGG and 29 Pro codons were changed from CCC to CCT to generate AMI119-pFB-scCMV-SV40-intron-kozak-Af-VEGF-Trap-GCRP (CCT) (see Figure 1 and Figure 7). In version 2, in addition to the change in 16 Arg codons, 36 Ser codons were further changed from AGC to TCC to generate AMI120-pFB-scCMV-SV40-intron-kozak-Af-VEGF-Trap-GCRS (TCC) (see Figure 1 and Figure 8). In version 3, in addition to the change of 16 Arg codons, 29 Pro codons were further changed from CCC to CCG to generate AMI130-pFB-scCMV-SV40-intron-kozak-Af-VEGF-Trap-GCRP (CCG) (see Figure 1 and Figure 9). All variant versions of the VEGF-Trap DNA sequence were first synthesized by Twist Bioscience, followed by PCR amplified by forward and reverse primers, and finally cloned into the BstBI and StuI sites of the AMI067 plasmid using the NEBuilder HiFi DNA assembly kit. The plasmid constructs are listed in Table 2, and the information of the primers used for PCR amplification is listed in Table 3 and Table 4.

[0112] [Table 2]

[0113] [Table 3]

[0114] [Table 4]

[0115]

[0176] To clarify the relationship between codon optimization and protein expression levels in human cells, four plasmids were constructed that contain the same expression cassette but with variants for the VEGF-Trap DNA coding sequence. Two of the coding sequences have low GC content and two have high GC content. In each of the low and high GC content coding sequences, a human antibody heavy chain (Vh) secretory signal peptide was used to replace the VEGF-Trap secretory signal peptide (Af). All four plasmids have identical DNA sequences except for the VEGF-Trap open reading frame (ORF). The expression cassette contains a CMV promoter, an SV40 intron, a Kozak sequence located upstream of the start codon of the VEGF-Trap ORF, and a synthetic polyadenylation signal located downstream of the stop codon. To generate more variants of the VEGF-Trap codon, the AMI119, AMI120, and AMI130 plasmids were cloned with altered VEGF-Trap codons. A schematic diagram of these expression cassettes flanked by full-length and truncated AAV2 ITRs is shown in Figure 1. The full-length DNA sequence of each expression cassette is SEQ ID NO:13 to SEQ ID NO:19 or SEQ ID NO:21 to SEQ ID NO:27 (SEQ ID NO:20 is the control plasmid sequence, and SEQ ID NO:28 or SEQ ID NO:70 are the reference coding sequences for the reference VEGF-Trap).

[0116]

[0177] The relationship between the estimated eCAI value and GC content of each ORF for human cells was revealed. The predicted codon adaptation index (eCAI) value was estimated based on the web-based free E-CAI server. Briefly, the target DNA coding sequence was pasted into the dialogue box respectively. The human codon usage table was selected from the "Codon Usage Database" in the same server and pasted into the next dialogue box. The Poisson method was selected (very similar results were obtained from the Markov method) and the "Standard" genetic code was selected. The eCAI value was estimated by pressing the "Allow" button. The results are shown in Table 5. The GC content was positively correlated with the eCAI value, and the higher the GC content, the higher the eCAI value. AMI059 and AMI066 both contained a low GC content of 40% and contained eCAIs of 0.723 and 0.722. AMI067 and AMI068 both contained a high GC content of 62% and contained eCAI values ​​of 0.867 and 0.864. The native cDNA sequence of VEGF-Trap was compiled using the secretory signal peptide sequence and FLT1 domain from the native mRNA sequence of NM_001160031, the native mRNA KDR domain of NM_002252.3, and the human antibody IgG1 Fc domain of the native mRNA AK129809.1. This compiled native VEGF-Trap was estimated to contain 52% GC and have an eCAI of 0.790, which is much lower than the optimized codons of AMI067 and AMI068. Further changes in VEGF-Trap codons resulted in slight changes in GC content and eCAI values, as shown in Table 5. Higher eCAI values ​​indicate the presence of more commonly used codons in the ORF, which may suggest higher protein expression levels in these expression cassettes.

[0117] [Table 5]

[0118] Example 2: Increasing GC content levels enhance VEGF-Trap expression in transiently transfected mammalian cells

[0178] To clarify the effect of GC content on VEGF-Trap expression in mammalian cells, four plasmids (AMI059, AMI066, AMI067, and AMI068) were purified and transfected into HEK293 cells. For transfection, cells were plated on 100 mm cell culture dishes (Corning, NY) at approximately 2 × 10 cells in 10 mL medium. 6 The cells were seeded at 1000 cells / dish overnight. 14 μg of plasmid DNA and 22 μL of Lipofectamine 3000 (Thermo Fisher) were diluted in 0.5 mL of Opti-medium (Thermo Fisher) and mixed together. The mixture was added dropwise to the cells, and the cells were incubated with CO. 2 The cells were incubated in an incubator at 37° C. for 48 hours, and the medium was collected.

[0119]

[0179] The medium was collected 48 hours after transfection and either loaded directly (non-reduced) or mixed with loading buffer and heated at 90°C for 5 min (reduced) before loading onto SDS-gels. After blotting onto PVDF membranes, VEGF-Trap protein was detected with anti-human IgG Fc antibody.

[0120]

[0180] As shown in Figure 2, ORFs with high GC content in coding sequences resulted in higher protein expression than those with low GC content. Lanes 1-5 are shown on a non-reduced gel, and lanes 6-10 are shown on a reduced gel. Lanes 3, 4, 5, 8, 9, and 10 show high GC content (plasmid AMI067, lanes 3 and 8, and AMI068, lanes 4, 5, 9, and 10), whereas lanes 1, 2, 6, and 7 show low GC content (plasmid AMI059, lanes 1 and 6, and AMI066, lanes 2 and 7). Based on the intensity of the Western blot images measured with ImageJ (free software, NIH), ORFs with high GC content showed a 9- to 11-fold increase in protein expression over ORFs with low GC content. Differences in secretion signal peptides appeared to have only a minor effect on protein secretion into the medium. In ORFs with low GC content, the original VEGF-Trap secretory peptide showed slightly higher expression than the human antibody heavy chain secretory signal peptide, but in ORFs with high GC content, there was no difference in protein secretion between these two secretory peptides.

[0121] Example 3: GC content level does not affect production of AAV2.N53 and AAV2.N54 vectors

[0181] Expression cassettes were cloned into scAAV shuttle plasmid backbones and rBVs were generated to produce AAV2, AAV2.53 and AAV2.N54 vectors by co-infection of Sf9 cells.

[0122]

[0182] Sf9 cells (Expression Systems) were grown in ESF AF medium (Expression Systems) containing 100 units / mL penicillin and 100 μg / mL streptomycin (Thermo Fisher Scientific, Pleasanton, CA) in Corning bottles at 28° C. with gentle shaking at 150 rpm. When cells grew to approximately 1e+7 cells / mL, they were split 1:4 in fresh medium into new bottles and continuously grown for maintenance purposes.

[0123]

[0183] Recombinant baculovirus (rBV) was generated using the Bac-to-Bac baculovirus expression system according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Briefly, pFB shuttle plasmids containing the target genes were each diluted to 1 ng / uL in TE buffer, and 2 ng of each DNA was mixed with 20 μL of Δcath-DH10Bac competent bacteria (Virovek, Hayward, CA) containing bacmid DNA molecules with the cathepsin gene deleted, incubated on ice for 30 min, and then heat shocked at 42°C for 30 s. After incubation on ice for 2 min, the bacteria were cultured at 37°C for 4 h to recover and then plated on agar plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 40 μg / mL IPTG, and 100 μg / mL X-gal. After 48 hours of incubation at 37°C, white colonies containing recombinant bacmid DNA were picked and miniprep bacmid DNA was purified under sterile conditions. Approximately 5 μg of each bacmid DNA and 10 μL of GeneJet Reagent (SignaGen Laboratories, Fredrick, MD) were each diluted in 100 μL of ESFAF medium (Expression Systems, Davis, CA) and then mixed together for approximately 30 minutes to form the transfection mixture. Sf9 cells were plated in 6-well plates at 1.5e+6 cells / well in 2 mL of ESFAF medium and placed at 28°C for approximately 30 minutes. After removing the old medium from the Sf9 cells, each transfection mixture was diluted in 800 uL of ESFAF medium and then added to the Sf9 cells. After overnight incubation at 28°C, an additional 1 mL of ESFAF medium was added to each well. After a total incubation time of 4 days, the medium containing the rBV was collected and amplified at a ratio of 1:200 to generate sufficient quantities of rBV for use in the AAV production process.

[0124]

[0184] To produce and purify AAV vectors containing non-naturally occurring nucleic acids containing the modifications described herein (e.g., at least one codon modification), rBVs carrying the AAV2 Rep and capsid genes (rBV-Cap2-Rep, rBV-Cap2.N53-Rep, rBV-Cap2.N54-Rep) and a rBV carrying a VEGF-Trap expression cassette (rBV-VEGF-Trap) were used to co-infect Sf9 cells for AAV production. Briefly, 10 multiplicity of infection (moi) of rBV-Cap2-Rep (or rBV-Cap2.N53-Rep or rBV-Cap2.N54-Rep) and 5 moi of rBV-VEGF-Trap were used to co-infect Sf9 cell lines with 50% fresh ESFAF medium at a cell density of approximately 5e+6 cells / mL for 3 days at 28°C in a shaking incubator with a shaking speed of 180 revolutions per minute (rpm). At the end of the infection, cell pellets were collected by centrifugation at 3,000 rpm for 10 min. The cells were lysed in Sf9 lysis buffer containing 50 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 1% Sarkosyl, 1% Triton X-100, and 125 units / mL benzonase by vigorous vortexing, followed by shaking at 350 rpm at 37°C for 1 h. At the end of the shaking, the salt concentration was increased to 500 millimolar (mM) by vortexing, and the lysate was clarified by centrifugation at 8,000 rpm for 20 min at 4°C. The clarified lysate was transferred to an ultra-clear centrifuge tube for a SW28 swinging bucket rotor containing 5 mL of 1.50 g / cc and 10 mL of 1.30 g / cc cesium chloride solution. After centrifugation at 28,000 rpm and 15°C for approximately 18 hours, the AAV band was collected with a syringe and transferred to an ultra-clear centrifuge tube for a 70Ti centrifuge rotor. The centrifuge tube was filled with 1.38 g / cc cesium chloride solution and heat sealed. This AAV sample was subjected to a second ultracentrifugation at 65,000 rpm and 15°C for approximately 18 hours, and the AAV band was collected with a syringe. The purified AAV sample was buffer exchanged into PBS buffer containing 0.001% Pluronic F-68 and sterilized by filtration through a 0.22 μm syringe filter.Sterile AAV samples were stored at 4°C for no more than one month before being transferred to -80°C for long-term storage. AAV titers were determined using real-time PCR with the QuantStudio7 Flex Real-Time PCR System (Invitrogen).

[0125]

[0185] The titers of AAV2, AAV2.53, and AAV2.N54 vectors are listed in Table 6 below. The purity of AAV2.53 vectors is shown in Figure 3, which shows a Simply Blue stained SDS-PAGE of low GC and high GC AAV vector production in Sf9 cells. M indicates the size marker of the gel. Figure 3A shows the AAV5 vector loaded as a control (1e+11 vg / lane loaded) compared to AMI059 loaded in lane 2 (5e+10 viral genomes (vg) / lane loaded), AMI066 loaded in lane 3 (5e+10 vg / lane loaded), AMI067 loaded in lane 4 (5e+10 vg / lane loaded), and AMI068 loaded in lane 5 (5e+10 vg / lane loaded). Figure 3B shows AAV2 vector loaded as a control (loaded 1e+11 vg / lane) compared to AMI119 loaded in lane 2 (loaded 1e+11 vg / lane), AMI120 loaded in lane 3 (loaded 1e+11 vg / lane) and AMI130 loaded in lane 4 (loaded 1e+11 vg / lane). All AAV vectors were produced in the normal range and no differences related to GC content were observed.

[0126] [Table 6]

[0127] Example 4: Increasing GC content levels enhance VEGF-Trap expression in scAAV2.N53-transduced mammalian cells

[0186] We generated and purified scAAV2.N53 vectors carrying a VEGF-Trap expression cassette, used them to transduce HEK293 cells, and the expression levels of VEGF-Trap were determined by Western blot analysis and an ELISA assay using a goat anti-human IgG-Fc antibody.

[0128]

[0187] For cell culture, human HEK293 cells were cultured in DMEM medium (Thermo Fisher) with 5% FBS (ATCC, Manassas, VA) in a Series II water-jacketed CO 2 The cells were cultured in an incubator (Thermo Forma) at 37° C. For maintenance, the cells were split 1:10 once a week.

[0129]

[0188] For AAV vector transduction, HEK293 cells (Figure 10) or hARPE-19 cells (Figure 11) were cultured at 2 x 10 6 Seed at a density of 100-150 cells / well and incubate at 37 °C in CO. 2 The cells were grown overnight in an incubator until they reached approximately 80% confluency. The next morning, the AAV vector was added to the cells at 1.0 × 10 4 Cells were added at an MOI of vg / cell and transduction was carried out for 48 h at 37° C. Cell-free supernatants were collected and analyzed for protein expression by Western blot or protein levels by ELISA. All transductions were performed at least three independent experiments.

[0130]

[0189] For ELISA, media collected from transient transfection or AAV transduction were centrifuged at 10,000 rpm for 5 min to remove particulates and used in the assay. First, 96-well plates were coated with 100 μL / well recombinant human endothelial growth factor-A (rhVEGF-A) in coating buffer overnight at room temperature. After removing the coating buffer, the plates were washed three times with 200 μL / well washing buffer (0.05% Tween-20 / PBS, pH 7.3 ± 0.1) and blocked with 200 μL / well blocking buffer (1% BSA / PBS, pH 7.3 ± 0.1) for a minimum of 1 h at room temperature. After removing the blocking buffer, the plates were washed three times with washing buffer. After removing the residual washing buffer, 100 μL / well test samples diluted in blocking buffer were added to the plates for 2 h at room temperature. After removing the test samples, the plates were washed three times again with washing buffer. After removing the residual wash buffer, 100 μL / well of biotinylated mouse monoclonal (H2) anti-human IgG Fc (biotin) detection antibody (Abcam, Cambridge, MA) was added to the plate and incubated at room temperature for 1 h protected from direct light. After the incubation was completed, the plate was washed three times and 100 μL / well of 1X HRP-streptavidin solution was added to the plate and incubated at room temperature for 45 min. After the incubation was completed, the plate was washed three times and 200 μL / well of substrate solution (tetramethylbenzidine) was added to the plate. After incubation at room temperature for about 10 to about 15 min to develop the color, 50 μL / well of stop solution (2N sulfuric acid) was added to the plate and the optical density value was determined at a wavelength of 450 nm using a Multimode Plate Reader EnVision (PerkinElmer, Santa Clara, CA).

[0131]

[0190] For Western blots, HEK293 cell supernatants were collected 48 hours after transfection. A total of 20 μl of supernatant was mixed with loading buffer, heated at 95° C. for 5 min, and loaded onto a NuPAGE 10% Tris-Glycine gel (Invitrogen) for electrophoresis. For non-reducing gels, the supernatant was mixed with non-reducing loading buffer and subsequently loaded onto the gel. Proteins on the gel were transferred to a PVDF membrane using the X Cell II™ Blot Module (Invitrogen, Carlsbad, CA, USA). The membranes were treated with casein blocker in PBS (Thermo Scientific, Waltham, MA, USA) for at least 1 hour at room temperature and probed with the appropriate primary antibody, followed by incubation with the appropriate anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (Amer Biosciences, Uppsala, Sweden). Proteins were detected using ECL™ Western blotting reagents (Amersham).

[0132]

[0191] The results are shown in Figure 4 and Table 7. In Figure 4A, lanes 1-4 were loaded with AMI059, AMI066, AMI067, and AMI068, respectively. In Figure 4B, lanes 1-4 were loaded with AMI120, AMI119, AMI067, and AMI130, respectively. As shown in Figure 4A, in lanes 3 and 4, which correspond to AMI067 and AMI068, respectively, increasing the GC content in the VEGF-Trap ORF increased the expression of the VEGF-Trap protein. At low GC content in the VEGF-Trap ORF, the Af signal peptide produced higher expression than the human Vh signal peptide (7-fold difference), as shown in Table 6. However, with the high GC content of the VEGF-Trap ORF, there is no difference between the Af signal peptide and the human Vh signal peptide, as shown in Table 7, and also by comparing lane 3 of Figure 4A (Af signal peptide) with lane 4 of Figure 4A and lanes 1, 2, 3 and 4 of Figure 4B (Vh signal peptide).

[0133] [Table 7]

[0134] Example 5: Highly pure VEGF-Trap purified from HEK293 cell culture medium

[0192] 500 mL of culture supernatant was harvested from HEK293 cells transduced with AAV2-VEGF-Trap (AMI067) and filtered through a 0.2 μm OptiScale filter (Millipore) by a peristaltic pump. A HiTrapTM 1 mL MabSelect™ PrismA Protein A column (GE Health Care Lifesciences) was first equilibrated with 5 column volumes (CV) of equilibration buffer (Tris-HCl pH 7.2, 150 mM NaCl) at a flow rate of 1 mL / min. The filtered supernatant was then loaded onto the column at the same flow rate using an AKTA explorer 100 system according to the instruction manual. The column was washed with 10 CV of equilibration buffer at the same flow rate before elution. The AAV-Eylea protein was eluted using 6 CV of elution buffer (0.1 M sodium citrate dihydrate pH 3.0) (Fisher Chemical) at a flow rate of 0.5 mL / min. Fractions were collected in tubes containing 0.1 volume of neutralization buffer (1 M Tris / HCl buffer pH 9.0). The eluted material was desalted with 1×PBS using a centrifugal filter (Millipore, Amico Ultra-4).

[0135]

[0193] The medium from HEK293 cells transduced with AAV2.53-AMI067 was subjected to column chromatography purification. A total of about 4.3 mg of pure VEGF-Trap was obtained from about 500 mL of medium harvested from HEK293 cells transduced with scAAV2.53-AMI067. SDS-PAGE and SimplyBlue staining show that the protein is pure and has the correct molecular weight of 66.5 kDa, which is the same size in the gel as the commercial product (Regeneron Tarrytown, NY) shown in Figure 5.

[0136]

[0194] The purified AAV2.VEGF-Trap vector was used to transduce HEK293 cells at an MOI of 500,000 vector genomes (vg) per cell. VEGF-Trap produced and released into the cell culture supernatant was transduced with commercially available VEGF-A as a coating antigen. 165 The assay was performed daily using the operating protocol. 165was expressed in HEK293 cells, purified to homogeneity (GeneScript, Cat. No. Z03073), and reconstituted in sterile Milli-Q water at 100 μg / mL. The ELISA procedure was as follows: 96-well plates were coated with VEGF at a final concentration of 0.1 μg / mL per well in a volume of 50 μL of coating buffer, the plates were sealed and covered, and the plates were placed at 4° C. overnight. The procedure for the next day's ELISA was as follows: the plates were removed and the solution discarded, the plates were tapped on a paper towel to remove excess solution, and the plates were washed three times with wash buffer (300 μL). After the final wash, the plates were tapped again on a fresh paper towel to remove as much buffer as possible. 300 μL of blocking buffer was added to each well using a multichannel pipette. The plates were covered with a sealing cover and placed in a 37° C. incubator for 2 hours. After incubation, the blocking buffer was discarded and the plate was tapped on a paper towel to remove excess buffer. Sample dilutions were prepared in blocking buffer by adding 50 μL to each well according to the experimental scheme. The plate was covered with a sealing cover and placed back in the incubator for 1 hour (hr). After 1 hour, the solution was discarded and the plate was washed 6 times with 300 μL of wash buffer. Excess solution was removed by tapping on a paper towel as above. Capture antibody was added at a 1:40,000 dilution in blocking buffer and incubated at 37°C for 1 hour. The solution was discarded and the wash procedure was repeated if necessary. Detection antibody was added at a 1:40,000 dilution in blocking buffer and placed at 37°C for 1 hour. The solution was discarded and the plate was further incubated (5 min in wash buffer) and washed as a final wash. The wash buffer was then discarded and one more wash with PBS was added. 50 μL of TMB was added. The plate was kept away from direct light for 15-20 min (can be less than 15 min) or until saturation of signal was observed in the highest concentration wells. Subsequently, 50 μL of stop solution was added and the plate was read at 450 nm with a reference of 620 nm within 15 min.

[0137]

[0195] As can be seen in Figure 10, cell culture supernatants from HEK293 cells transduced with AAV2.054-AMI120 secreted VEGF-Trap up to approximately 800 ng / mL, whereas non-transduced HEK93 cells did not secrete VEGF-Trap. Production continued over increasing culture days until the cells became senescent (Figure 11).

[0138]

[0196] VEGF-Trap expressed by transduction of HEK293 cells with the AAV2.N53-VEGF-Trap vector was purified by Protein A affinity column chromatography. The column eluate was neutralized and buffer-changed to 10 mM phosphate buffer (pH 7.0, 150 mM NaCl). The purity of vector-derived VEGF-Trap was determined by loading 1.5 μg / lane onto an SDS-PAGE gel (10%) and staining with Coomassie Blue R-250, which revealed a single band of 66.5 kDa in the gel, the size of which was identical to that of the commercially available VEGF-Trap (Figure 5A).

[0139]

[0197] The binding affinity of AVMX-110-derived VEGF-Trap was 165 The binding of VEGF-A to AVMX-110-derived VEGF-Trap and the commercially available aflibercept (Regeneron, Tarrytown, NY) was measured by two methods: ELISA and surface plasmon resonance (SPR). 165 Binding to AVMX-110-derived VEGF-Trap (A) and aflibercept (B) was performed in parallel under the same conditions. The results, shown in Figure 5B and Figure 5C, indicate that binding is the same for both, with IC 50were 159.1 ng / mL and 158.8 ng / mL, respectively. The binding constant (Kd) was calculated in nanomolar concentrations using a molecular weight of 115,000 Da. The affinity of AVMX-110-derived VEGF-Trap was identical to that of commercial aflibercept (1.38 nM for both AVMX-110-derived VEGF-Trap and commercial aflibercept). Thus, the purified product functioned similarly to that derived from AAV2.VEGF-Trap.

[0140]

[0198] Based on biochemical and biological properties, AVMX-110 is a qualified AAV vector carrying the VEGF-Trap gene for transduction of human cells, including HEK293 and retinal cell ARPE-19. The product of AAV2.N54-VEGF-Trap can produce VEGF-Trap in vivo, similar to the currently marketed aflibercept. Therefore, AVMX-110 (AAV2.N54-VEGF-Trap) can be used for long-term expression for the treatment of neovascular retinal disorders in vivo.

[0141]

[0199] AVMX-110-derived VEGF-Trap and human VEGF-A 165 The interaction between AVMX-110 VEGF-Trap and aflibercept was analyzed by surface plasmon resonance (SPR). 15 μL of 200 nM AVMX-110 VEGF-Trap or aflibercept in phosphate buffered saline containing 0.01% Tween 20 (PBS-T) was bound to the sensor chip protein A. AVMX-110 VEGF-Trap and aflibercept bind to human VEGF-A. 165 The antibodies bound tightly to HEK293 expressing IgG1 with Kds of 33 pM and 55 pM, respectively (Figure 13 and Table 8).

[0142]

[0200] The potency of AVMX-110 in vitro was determined using the inhibition of proliferation of HUVEC cells. Briefly, AVMX-110 was first used to transduce HEK293 cells and cultured at 37°C for 4 days. The cell culture supernatant was harvested and purified via affinity column chromatography. The purified VEGF-Trap was synthesized by immunohistochemistry using human VEGF-A. 165 The assay was compared to commercially available aflibercept (Eylea, Regeneron Pharmaceuticals, Tarrytown, NY) in blocking HUVEC cell proliferation induced by EGFR-1. The assay was repeated twice. Briefly, HUVEC cells (ATCC, Cat. No. CRL-1730, Manassas, VA) were seeded at 5000 cells / well in 100 μL of cell culture medium in 96-well microplates and incubated at 37 °C for 24 h at 5% CO. 2 and 90% humidified CO 2 The cells were allowed to attach for 24 hours, the cell culture medium was removed, and 20 ng / mL of human VEGF-A was added. 165 Replace with 100 mL / well of fresh assay medium (vascular cell basal medium with 1% dFBS) containing 1:0, 1:2, 1:5, 1:10 and 1:100 molar ratios of VEGF-A 165 AAV2.VEGF-Trap was replaced with 0, 101, 253, 506 and 5060 ng / mL purified VEGF-Trap (VEGF-A / VEGF-Trap), respectively. Both commercial aflibercept (40 mg / mL) and AAV2.VEGF-Trap were diluted in the same ratio. The plates were incubated for 72 hours before the measurement procedure. The assay medium in each well was removed and fresh assay medium (90 μL) was added with 10 μL WST-8 (Dojindo Molecular Technologies, Inc., Cat. No. CK04-11, Rockville, MD). The plates were read every hour after addition. The data demonstrated that the inhibition of HUVEC cell proliferation by AAV2.VEGF-Trap is dependent on the concentration of VEGF-Trap. Its potency is equally effective as aflibercept (Figure 12).

[0143] Example 6: Measurement of glycosylation of AAV2-derived VEGF-Trap

[0201] AAV2.N54-AMI120-derived VEGF-Trap protein was purified from cell culture supernatants of HEK293 cells transduced with AVMX-110 at an MOI of 100,000 and purified by affinity column chromatography. The purified VEGF-Trap was treated with recombinant Flavobacterium meningosepticum PNGase F enzyme, which is ideal for removing all N-linked carbohydrates from glycoproteins. Treatment was performed using commercially available aflibercept. The treatment mixture was separated by SDS-PAGE gel shift analysis. In the presence of NPGase F, carbohydrates were removed from glycoproteins via N-linkages, and the deglycosylated protein migrated faster due to the reduced molecular weight (Figure 12).

[0144] Example 7: VEGF-A using vector expressing VEGF-Trap and aflibercept (Elyea) 165 Biacore binding assay for binding affinity (KD) of

[0202] VEGF-Trap protein was expressed in HEK293 cells transfected with the expression vector AAV2.N54-AMI120 and purified by protein A affinity column chromatography. The eluate was neutralized and concentrated to a concentration of 4.31 mg / ml. Aflibercept was a commercially available product and was diluted to 4 mg / ml. VEGF-A 165 were obtained from Genescript (GeneScript, Cat. No. Z03073, Nanjing China) and reconstituted to 1 mg / ml. Working solutions of each stock protein were prepared at 200 nM prior to the experiment. Molar concentrations were calculated using MW: 115 kDa for VEGF-Trap and aflibercept, 115 kDa for VEGF-A 165The Biacore procedure consisted of coating a Protein A sensor chip with VEGF-Trap or aflibercept at 200 nM and injecting 15 μL at 5 μL / min, followed by serial dilutions of 200, 100, 50, 25, 12.5 and 0 nM VEGF-A. 165 The injection was started by injecting 90 μL of the solution at a flow rate of 30 μL / min. The binding kinetics simulation was analyzed using Biacore evaluation software. The KD of AAV2.N54-AMI120-derived VEGF-Trap was 1.0 times higher than that of VEGF-A. 165 The KD of aflibercept was 33 pM for binding to VEGF-A. 165 The binding kinetic parameters shown in Table 8 and Figure 13 indicate that AAV2.N54-AMI120 derived VEGF-Trap is similar to aflibercept.

[0145] [Table 8]

[0146] Example 8. rhVEGF-A Vector Expressing VEGF-Trap 165 Binding affinity with

[0203] Assays to determine binding affinity were performed. Briefly, purified VEGF-Trap expressed by AAV2.N54-VEGF-Trap was assayed for binding affinity to rhVEGF. 96-well plates were filled with 0.1 μg / mL rhVEGF-A. 165Plates were coated overnight at 2-8°C with 5% CO. The plates were washed 3x300μL with wash buffer (0.05% Tween-20 / PBS, pH 7.3±0.1) and blocked with blocking buffer containing 1% casein for 120 min. VEGF-Trap or aflibercept (same lot as above) were serially diluted at 200, 100, 50, 25, 12.5, 6.25 and 3.1ng / mL and added individually to each well at 100μL / well and incubated for 60 min at 37±1°C. Diluted (1:40,000) biotinylated rabbit anti-human IgG1 Fc antibody solution was added at 100μL / well after 3x300μL / well of wash buffer. Plates were further incubated for 60 min at 37±1°C and washed 3x300μL / well of wash buffer. Finally, streptavidin-HRP, 1:40,000, was added to each well at 100 μL / well and incubated for 60 min at 37±1° C. After 3×300 μL / well washes, substrate TMB solution was added and incubated for 15 min at 37±1° C. before being stopped with 100 μL / well stop solution. Plates were read at 600 nm wavelength in a microplate reader (Molecular Device, Sunnyvale, Calif.). The assay showed that rhVEGF-A 165 The binding affinities of VEGF-Trap and aflibercept to rhVEGF-A were 75 pM and 60 pM, respectively. 165 It was demonstrated that binding to IgG4 was comparable for both proteins (Figure 14 and Table 9).

[0147] [Table 9]

[0148] Example 9. VEGF-A 165 Inhibition of HUVEC cell proliferation stimulated by

[0204] Purified VEGF-Trap was compared to commercially available aflibercept (Eylea) (Regeneron Pharmaceuticals, Tarrytown, NY) for blocking human VEGF-A-induced HUVEC cell proliferation. Assays were repeated twice. Briefly, HUVEC cells (ATCC, Cat. No. CRL-1730, Manassas, VA) were seeded at 5000 cells / well in 100 μL of cell culture medium in 96-well microplates and incubated at 4°C for 1 h at 5% CO. 2 and 90% humidified CO 2 After 24 hours of incubation, the cell culture medium was removed and 20 ng / mL of human VEGF-A was added. 165 Replace with 100 mL / well of fresh assay medium containing 1:0, 1:2, 1:5, 1:10 and 1:100 molar ratios of VEGF-A 165 / VEGF-Trap) or purified VEGF-Trap at 0, 101, 253, 506 and 5060ng / mL, respectively. Commercially available aflibercept (40mg / mL) and AAV.VEGF-Trap were diluted in the same ratio. The assay medium was vascular basal medium with 1% dFBS, and the plates were incubated for 72 hours before the measurement procedure. The assay medium in each well was removed and fresh assay medium (90μL) was added along with 10μL of WST-8 (Dojindo Molecular Technologies,Inc., Catalog No. CK04-11, Rockville, MD). The plates were read every hour after addition. The data demonstrated that AAV.VEGF-Trap inhibited HUVEC cell proliferation in a VEGF-Trap concentration-dependent manner. Its potency was equally effective as aflibercept (Figure 15).

[0149] Example 10. In vivo evaluation of AAV2.N54-AMI120VEGF-Trap protection of choroidal damage caused by laser-induced neovascularization

[0205] The efficacy and pharmacokinetics of AAV2.N54-AMI120-VEGF-Trap were investigated using a mouse LCNV model. The cohorts of studies are shown in Table 10.

[0150] [Table 10]

[0151]

[0206] Mice (Mus Musculus) / C57BL / 6 (Charles River or Tacomic Farms) aged 8-12 weeks and weighing 15-25 g each are used for the study. Animals are randomly divided into groups of 5 animals per group. 28 days prior to laser irradiation, each animal is injected with the test article intravitreally (IVT) bilaterally, 10 eyes per group. AAV2.N54AMI120 test article is tested at three doses per microliter (μL): high (1.6e+10), medium (4e+8) and low (2e+7). As a control, AAV2.N54-GFP is used as a vector transduction control. AAV2.N54-AMI120 formulation buffer (vehicle) is used as a negative control. Mice are administered with test article and control (IVT administration) 28 days before laser injury, and control animals are administered with aflibercept and vehicle 3 days before laser irradiation. All groups are analyzed for fluorescein angiography, serum and eye cup VEGF-Trap levels 7 days after laser irradiation. VEGF-Trap concentrations are detected using ELISA. According to the results of the in vitro efficacy test described above, animals administered with AAV2.N54-AMI120 can obtain protection by angiogenesis in response to inflammation and wound healing, while AAV2.N54-GFP and vehicle control may not obtain any protection.

[0152] Example 11: Efficacy of a modified adeno-associated virus (AAV2) encoding aflibercept (AVMX-110) in a mouse laser-induced choroidal neovascularization (CNV) model

[0207] The purpose of this study was to evaluate the inhibition of angiogenesis using adeno-associated virus (AAV2) vectors in a laser-induced model of choroidal neovascularization (CNV) in mice. Table 11 illustrates the experimental design of this study.

[0153] [Table 11]

[0154]

[0208] This laser choroidal neovascularization (CNV) study was performed using mice (Mus Musculus) C57BL / 6 strain. The study met the criteria for passing a valid non-GLP study because the mean size of CNV lesions, assessed via fluorescein angiography (FA) and flat-mount immunofluorescence, was significantly reduced in animals treated with aflibercept after IVT injection compared to formulation buffer or control vector (GFP-AAV2).

[0155]

[0209] Test articles were provided ready for injection and were stored below -70°C until use. Aflibercept was stored refrigerated (2-8°C) and injected undiluted. Thirty minutes prior to injection, each test article was thawed in a 37°C water bath for 20 minutes and vortexed to reduce viral aggregation.

[0156]

[0210] Mice were administered buprenorphine 0.01-0.05 mg / kg subcutaneously (SQ) on days -28 (groups 3-6) and -3 (groups 1 and 2) prior to injection. Animals were then sedated by isoflurane inhalation for intravitreal injection, and one drop of 0.5% proparacaine HCL was applied to both eyes. The conjunctiva was gently grasped with Dumont #4 forceps, and injections were performed using a 33G needle and Hamilton Syringe. After dispensing the syringe contents, the syringe needle was slowly removed. After the injection procedure, one drop of ofloxacin ophthalmic solution was applied topically to the ocular surface along with an ocular lubricant.

[0157]

[0211] On day 0, mice were administered buprenorphine 0.01–0.05 mg / kg SQ and topical mydriatics (1.0% tropicamide HCL and 2.5% phenylephrine HCL) were applied at least 15 min before laser procedure. Mice were sedated with an intraperitoneal (IP) injection of ketamine / xylazine. Topical eyewash was used to keep the cornea moist, and a hot pad was used to maintain body temperature. A 532 nm diode laser delivered through a slit lamp was used to create four single laser spots surrounding the optic nerve. Both eyes of mice were lasered at predetermined time intervals. Ocular lubricant was applied post-laser (OU).

[0158]

[0212] Morbidity and mortality were observed daily and externally from the cage, with particular attention paid to both eyes. Four animals died after dosing but before laser irradiation in groups 2 (no. 210, 212, and 216) and 4 (no. 432). Two further animals died before final tissue collection, but after laser irradiation, in groups 4 (no. 430) and 6 (no. 644).

[0159] Fundus photography

[0213] Both color and cobalt blue (EGFP expression) fundus photography was performed on both eyes of animals in group 3 (AAV2-GFP, 1.6e10vg / eye) on day 7 post-laser and before fluorescein fundus angiography (FA). Animals were given a cocktail of tropicamide (1.0%) and phenylephrine (2.5%) topically to dilate and proptosis the eyes, and topical ocular anesthetic (0.5% proparacaine or similar) was applied to the eyes. Color fundus photography was followed by cobalt blue photography. Intensity settings were kept constant between animals during acquisition. As shown in Figure 16, GFP was observed in all eyes except for 318 OS, which had a cataract that prevented fundus photography. The observed cataract may have been caused by trauma to the lens during IVT injection.

[0160] Fluorescein angiography (FA)

[0214] FA was performed on both eyes on the 7th day after laser. Mydriasis for FA was achieved using topical eye drops (1.0% tropicamide HCL and 2.5% phenylephrine HCL) in each eye 15 minutes before the examination. Mice were sedated with an IP injection of ketamine / xylazine. Retinal photography was performed approximately 1 minute after intravenous sodium fluorescein injection (1% stock solution, 12 mg / kg). Fluorescein leakage was analyzed with ImageJ software. Representative images are shown in FIG. 17, which illustrates fluorescein fundus angiography on day 7. Representative images of each group are shown. Measurement of lesions in group 3 could not be performed because GFP expressed by AAV interfered with the analysis. Fundus angiograms were used to quantify lesion area as pixels ± standard deviation (SD), and the results are presented in FIG. 18. On the 7th day post-laser examination, Group 6 (AVMX110, 1.6e10vg / eye) had the smallest mean lesion area, while the vehicle group had the largest.

[0161] Ocular tissue collection and processing

[0215] On day 7, animals were humanely euthanized, eyes were enucleated and immediately fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at 4°C. Using a dissecting microscope, each eye was cleared of extraneous tissue, and the anterior segment and lens were removed. The retina was peeled off and removed from the optic nerve head with fine curved scissors. Eyecups were rinsed in cold immunocytochemistry (ICC) buffer (PBS + 0.5% BSA and 0.2% Tween 20) and blocked in ICC buffer for at least 6 hours. Eyecups were then placed in cold ICC buffer containing 4',6-diamidino-2-phenylindole (DAPI, nuclei), phalloidin DyLight 550 (actin cytoskeleton) and isolectin IB4-DyLight 649 (blood vessels) and incubated for 2.5 hours at 4°C with gentle rotation. The tissue was washed and cut radially toward the optic nerve head avoiding the lesion, and the tissue was flat-mounted. Two-dimensional (2D) fluorescent microscopy images were acquired with an Olympus Bx63 fluorescent scope, and the newly formed blood vessels were quantified using isolectin IB4 signal area (μm2) and CellSens software. Representative images are shown in Figure 19. Quantification of isolectin area is shown in Figure 20. Lesion area was reduced by injection of aflibercept. Group 3 eyes had the largest lesion area, which may have been partially influenced by GFP mobilized by the AAV2 construct. Compared to AAV2-GFP from group 3, AVMX-110 reduced lesion size at all doses tested (groups 4-6).

[0162]

[0216] This study was conducted to demonstrate inhibition of retinal vascular leakage following IVT administration of test articles (groups 3-6) and positive control (aflibercept, group 2) to mice. AAV2 vectors (groups 3-6) were injected on day 28, and vehicle and aflibercept (groups 1 and 2) were injected on day 3 to ensure sufficient expression before induction by laser CNV. Based on the tests performed and the objectives of this study, the aflibercept control group had reduced lesion size on day 7 post-laser compared to the vehicle group. Compared to aflibercept (positive control, 40.5% reduction in fluorescein area), test articles in groups 5 and 6 demonstrated equivalence or superiority in at least one endpoint analysis, with the highest dose of AVMX-110 (1.6e10vg / eye) reducing vascular leakage by 68% at the angiographic endpoint. Analysis of lesion size in flat mounts showed that all eyes injected with AAV2 preparations had larger lesions than eyes injected with vehicle or aflibercept, but when AVMX-110 was compared with AAV2-GFP, all three dose levels of AVMX-110 reduced lesion size, with the highest dose of AVMX-100 producing a 40% reduction in isolectin area.

[0163] Example 12. Analysis of AVMX-110 and AVMX-116

[0217] Example 12 shows a comprehensive analysis of the data leading to the selection of candidates AVMX-110 and AVMX-116. The strategy included construct design, cloning, sequencing, production, purification and bioanalysis. The data was analyzed using one-way ANOVA. Candidates with the best expression, potency and penetration (in vivo) were selected based on the following criteria: gene of interest (GOI), capsid, and AAV serotype. Capsid Engineering and Selection

[0218] The wild-type AAV2 capsid has been engineered in an orderly manner to introduce mutations at specific sites to increase the permeability or expression of the GOI. The following list of mutations was introduced and screened in mouse and / or pig models to examine expression and permeability (Table 12). The sequences and positions are inserted with short peptide fragments into loop 4 of AAV2-VP1 (Table 12) and the plasmid names are listed in Table 13.

[0164] [Table 12]

[0165] [Table 13]

[0166]

[0219] The various constructs were injected into the mouse model at 2E+10vg / eye. Figure 21 shows fundus images of all groups. These images were recorded 23 days after injection of the constructs. V226 was a positive control and showed good expression. One eye from each group was further analyzed by immunohistochemistry (Figure 22), and eyes injected with constructs AMI-053, AMI-054, AMI104 and V226 showed high intensity GFP expression. AMI054 showed uniform expression especially in the ganglion cell layer (GCL), inner plexiform layer (IPL) and outer nuclear layer (ONL). In other mouse studies, the rest of the aforementioned constructs were analyzed. Similar to 20-AVI-002, constructs at 2E+10vg / eye were injected into the mouse eyes. Fundus photography was performed on day 24 (Figure 23).

[0167]

[0220] AMI101, AMI104 and AMI105 were subjected to immunohistochemistry (Figure 24) on day 28, where AMI104 showed uniform distribution of GFP expression in the GCL, IPL, inner nuclear layer (INL), outer plexiform layer (OPL), ONL, inner segment (IS) and outer segment (OS). From these mouse studies, several candidates (AMI053, AMI054, AMI101, AMI104, AMI105 and V226) were selected for additional porcine studies. Each porcine eye was injected with 1E+11vg / eye. Figure 25 shows fundus images of animals in the porcine study. AMI053 showed slight GFP expression, similar to the results observed in the mouse study. AMI054 showed uniform and high-brightness expression, similar to V226. To further confirm the expression and penetration of the constructs, immunohistochemistry (IHC) was performed on frozen sections (Figure 6) made on day 28 after injection, similar to the mouse study. According to the IHC data, AMI054 and V226 showed the brightest, most widespread, and consistent GFP expression. V226 showed strong GFP expression mainly in the GCL, while AMI054 showed strong GFP signal in the INL. From the mouse and pig studies, it can be concluded that AMI054 showed the brightest, most widespread, and consistent results. Therefore, AMI054 was selected and finally determined as the AAV capsid for insertion and screening of AVMX-110. To better visualize the different layers of the eye, confocal microscopy images were also recorded. These images showed the penetration of the capsid into the different ocular layers (Figure 27). AMI054 showed less GFP expression but deeper penetration into the INL. V226 showed more expression in the RGC layer. GOI screening

[0221] Several constructs were designed and screened before finalizing the candidates. The expression profile of all constructs was examined in human embryonic kidney (HEK293) cells. Some of the constructs were also examined in ARPE-19 cells (human retinal pigment epithelial cells) and hTERT-rpe1 (human telomerase reverse transcriptase-retinal pigment epithelial cells). hTERT-rpe1 cells were derived by transfecting the RPE-340 cell line (primary human RPE cell line) with the pGRN145 hTERT expression plasmid. This was used to mimic primary RPE cells. Table 14 shows the expression of these constructs in various cell lines. For example, AAV2.N54.120 viral particles (also designated AVMX-110) comprise a non-naturally occurring nucleic acid packaged within an AAV2.N54 capsid and contain a high GC content (indicated by "GC" in "GCRS"), the codon AGG encoding arginine (indicated by "R" in "GCRS"), and the codon TCC encoding serine (indicated by "S" in "GCRS"). As another example, AAV6.N54.120 viral particles (also designated AVMX-116) comprise a non-naturally occurring nucleic acid packaged within an AAV2.N54 capsid and contain a high GC content (indicated by "GC" in "GCRS"), the codon AGG encoding arginine (indicated by "R" in "GCRS"), and the codon TCC encoding serine (indicated by "S" in "GCRS").

[0168] [Table 14-1]

[0169] [Table 14-2]

[0170] [Table 14-3]

[0171] [Table 14-4]

[0172]

[0222] Three constructs (N54.120, N54.190 and N54.221) listed in Table 14 showed higher expression of aflibercept compared to other constructs. The AAV construct backbone for these three candidates is shown in Figure 28A. During the manufacturing process, samples were taken at different time points of the process steps for various testing purposes with the aim of achieving precise control of the process. Exemplary sample process chromatograms for two manufacturing runs are shown in Figure 28B. Throughout the capture step, a single and sharp fraction of AVMX-110 vector was obtained during the process (arrow). Nearly 100% particles of AVMX-110 were captured by the capture process, with more than 80% eluting in the process intermediate pool. Figure 28C Analysis of the purity of the eluted AVMX-110 process intermediate by SDS-PAGE. AAV VP1, VP2 and VP3 were clearly visualized, and no other major impurity proteins were found. Due to the difference in capsid charge, a performance profile of empty and filled AAV in the column process was established. Under this condition, empty capsids migrated through the column faster than filled capsids. The difference in retention time was sufficient to separate empty capsids from filled capsids (Figure 28D). The capture step eluate pool was treated to separate a portion of the empty capsids. The separation curve is shown in Figure 28E. Using this separation model, AVMX-110 was purified and enriched for filled capsid content. Two separate peaks were obtained in the separation chromatogram. Concentration and buffer exchange were successfully performed on the maximum capacity enriched fraction post-column chromatography to generate drug substance for further evaluation. For each development lot, samples of AVMX-110 manufacturing intermediates and drug substance were analyzed for specificity by Western blot detection of AAV2 antibodies. Antibodies against AAV2 VP1, VP2 and VP3 were the only protein bands observed on the gel and Western blot (Figure 28F). The purity of AVMX-110 was analyzed by gel electrophoresis stained with silver staining. The first peak was empty capsid and the second peak was filled capsid. The product was AAV particles containing VP1, VP2 and VP3.The purity of AVMX-110 process intermediates was analyzed by SDS-PAGE stained with a silver stained gel (Figure 28G). AAV VP1, VP2 and VP3 were clearly visualized, and no single impurity was found above 4%.

[0173] host cell contaminants

[0223] Sf9 host cell protein contaminants in AVMX-110 development products were measured by ELISA. Exemplary measurements of host cell protein contaminants are shown in Table 15. Host protein levels were very low (≦170 ng / 10 12 vg). The purification process was highly specific for the GOI with 1.5+10-fold removal of host protein from the starting cell culture lysate. Host protein levels exceeded the requirement of ≤100ng / dose for product release. For this lot, host cell protein levels were 4ng / dose when the dose was 2e+11vg. Another batch had host cell DNA detection the limits PCNA was ≤46.12pg / 1e+12vg. Host cell protein and DNA testing showed that AVMX-110 is highly purified and exceeds regulatory requirements.

[0174] [Table 15]

[0175]

[0224] Sf9 cell genomic DNA was purified with a Qiagen genomic DNA purification kit, and DNA was diluted to 1,000, 250, 63, 16, 4, and 1 ng / mL for use as standards. AVMX-110 process samples were diluted 2-fold and 10-fold in QPCR dilution buffer for use with qPCR primers and probes for Sf9 cells. In all tests performed with AVMX-110 process samples, Sf9 cell DNA was undetectable at ≤10 pg / reaction, which corresponds to 1 ng / mL ≤300 pg / dose. Determination of Baculovirus and Other Plasmid DNA Contaminants

[0225] Baculovirus DNA was purified according to the protocol provided by the Qiagen genomic DNA purification kit with minor modifications. Briefly, 7.5 mL of baculovirus in culture medium was mixed with an equal volume of cold 20% PEG8000 in 1N NaCl and left at room temperature for 30 min. The baculovirus particles were pelleted by centrifugation at 10,000 rpm for 10 min and dissolved in 5 mL of G2 buffer. 95 μL of proteinase K was added to digest the protein capsid for 60 min at 50°C. The column was equilibrated and the sample was loaded. After washing, the baculovirus DNA was eluted and subjected to qPCR testing using primers and probes complementary to the GP64 gene of the baculovirus genome. DNA was diluted to 1000, 250, 63, 16, 4, and 1 ng / mL and used as standards. AVMX-110 samples were diluted 2-fold and 10-fold in qPCR dilution buffer (without DNase digestion) and used in the qPCR assay. Baculovirus DNA levels were 10% of the AVMX-110 vector as shown in Table 16. 12 It was 8.58e+5 per vg.

[0176] [Table 16]

[0177]

[0226] Other plasmid selection marker genes, such as gentlemen, ampicillin (AMP), Rep, and host cell DNA markers, PCNA, and BacIE1 DNA contamination were below regulatory requirements, indicating that AVMX-110 is of high purity and meets regulatory safety requirements. The levels of these DNA markers evaluated were approximately 10-fold lower than the regulatory requirements.

[0178] Endotoxin levels

[0227] Endotoxin levels in AVMX-110 samples were measured by a chromogenic Limulus amebocyte lysate (LAL) release assay using a commercially available kit. In the presence of endotoxin, pNA is released by proteolytic cleavage of the colorless chromogenic substrate in pyrochrome LAL, resulting in a yellow solution that absorbs at 405 nm. The results of endotoxin levels in AVMX-110 preparations are shown in Table 17. Endotoxin levels in AVMX-110 samples were 0.0154 EU / mL or 0.005 EU / dose.

[0179] [Table 17]

[0180] sterility

[0228] Each batch of AVMX-110 was assayed for biological contamination, and no microbial contamination was detected in the drug substance used in animal studies.

[0181] Levels of replication-competent AAV

[0229] In the conventional method of using mammalian cells to produce AAV vectors by plasmid transfection or viral infection, replication-competent AAV (rcAAV) virus particles are generally generated to some extent due to non-homologous recombination between the AAV genome and the AAV rep-cap plasmid. Contamination of rcAAV is undesirable in GMP manufacturing, and the amount of contamination needs to be reported to the FDA. The AAV system described herein removes and destroys AAV promoters and replaces them with insect cell promoters that are not active in mammalian cells. In the Bac-to-AAV system, rcAAV cannot be produced during the manufacturing process. To establish an assay for monitoring rcAAV, 100 wild-type (wt) AAV particles were added into 1e+12vg AAV sample. The rcAAV assay was performed. Briefly, 1e+12vg AAV sample was mixed with 100vg wtAAV2 particles in the presence of adenovirus-type helper, and HEK293 cells were transduced for 3 days. Cells were harvested and lysates were prepared. After heat inactivation at 56°C for 30 min to inactivate adenovirus, 50% of the lysates were used to transduce HEK293 cells in the presence of adenovirus type 5 and amplify rcAAV or added wtAAV for 3 days. Cells were harvested and lysates were prepared for qPCR detection of rcAAV signal. The results showed that there was no positive signal for rcAAV without the addition of wtAAV2. A positive signal was detected when 100vg of wtAAV was added to the 1e+12vg AAV sample (Table 18).

[0182] [Table 18]

[0183] Estimation of empty / filled capsids

[0230] The ratio of filled capsids to empty capsids for each lot was estimated using vector-specific qPCR and AAV2-specific antibody ELISA. Product fractions from column chromatography were analyzed for vector copy number and protein levels. Empty capsids had no or only very low levels of vector DNA and only capsid protein, while filled vectors had both vector DNA and protein content. The data showed that eluate 1 from the polishing step had no or only low levels of the gene of interest. The eluate from peak 1 had very low or even undetectable levels of AAV2.N54-VEGF-Trap DNA, but not protein. The second peak in the profile (peak 2) showed both vector DNA and protein. These results demonstrated that the purified AVMX-110 and UFDF pools were all filled particles (Figure 10A).

[0184] Infectivity of AVMX-110

[0231] The infectivity of AVMX-110 was determined by TCID 50 The TCID50 assay (tissue culture infectious dose 50% / mL) was determined, which is the dilution at which the inoculum infects 50% of the target culture (i.e., the starting sample is converted to TCID 50 The TCID is the concentration of infectious organism in an inoculum determined by the addition of 1 mL aliquots to multiple target cultures (when diluted with an inoculum equivalent to 100 μg / mL, the addition of 1 mL aliquots to multiple target cultures will infect, on average, 50% of the cultures). 50 The assay was used to determine the potency of AAV vectors according to the manufacturer's protocol (ATCC). Briefly, a series of diluted AAV samples were used to transduce HelaRC32 cells in the presence of adenovirus 5 helper in 96-well plates for 3 days. Cell lysates were prepared and positive signals were detected by qPCR. TCID 50 Values ​​were calculated based on the Spearman-Carber formula, and the results are shown in Table 19.

[0185] [Table 19]

[0186] AVMX-110 expression of VEGF-Trap (AAV2.VEGF-Trap) in HEK293 cells

[0232] The purified AAV2-VEGF-Trap vector was used to transduce HEK293 cells using an MOI of 100,000 vector genomes (vg) per cell. VEGF-Trap produced and released into the cell culture supernatant was transduced with commercially available VEGF-A. 165 The VEGF-A was analyzed daily using an internal standard operating protocol (21-AD-VEGF-ELISA.01) using as the coating antigen. 165 was expressed in HEK293 cells, purified to homogeneity (GeneScript, Cat. No. Z03073), and reconstituted in sterile Milli-Q water at 100 μg / mL. The ELISA procedure was as follows: VGEF-A 165 was coated onto a 96-well ELISA plate and incubated O / N at 4°C. The next day the plate was washed 3 times with wash buffer and blocking buffer was added to each well. The plate was covered and incubated at 37°C for 2 hours. The plate was covered and returned to the incubator for 1 hour. The plate was then washed and anti-human Fc antibody was added to the plate and again incubated at 37°C for 1 hour. The plate was washed and streptavidin-HRP was added and incubated for 45 minutes. The plate was washed and TMB substrate was added. After approximately 15 minutes, stop solution was added. The wells were read at 450 nm on a microplate reader. As can be seen in Figure ​(B),10B, cell culture supernatants harvested from HEK293 cells transduced with AMVX-110 (AAV2 N53-VEGF-Trap) secreted VEGF-Trap at approximately 800 ng / mL, whereas non-transduced HEK293 cells did not secrete VEGF-Trap, and production continued to increase daily in culture until the cells became senescent.

[0187] Persistence of AAV2.VEGF-Trap expression in vitro

[0233] HEK293 and ARPE-19 (human retinal pigment epithelium) cells were cultured with 10% FBS and transduced with AAV2·N53-VEGF-Trap and AAV2·N54-VEGF-Trap at an MOI of 100,000 per cell. Samples were taken for ELISA quantification of VEGF-trap expression. AAV2·N54-VEGF-Trap showed better expression compared to AAV2·N53-VEGF-Trap (Figure 11). This difference was observed to be significantly lower in ARPE19 cells than in HEK293 cells. In ARPE19 cells, VEGF-Trap expression derived from transduction of the N53-VEGF-Trap construct was negligible from day 20 onwards. In contrast, VEGF-Trap expression derived from transduction of the N54-VEGF-Trap construct was still present in quantifiable amounts. VEGF-Trap expression was higher in HEK293 cells compared to ARPE19 cells.

[0188] Efficacy studies in mice

[0234] The construct AAV2.N54.120 (AVMX-110) was selected and administered to mice at three different doses to study efficacy. Briefly, the study included six groups: vehicle (formulation buffer), commercial Eylea (protein), AAV2.N54-GFP (-) administered at 1.6E+10vg / eye, low dose (2.0E+07vg / eye), medium dose (4.8E+08vg / eye) and high dose (1.6E+10vg / eye) of AVMX-110. Fluorescence fundus angiography (FA) data are shown in Figure 29. The medium dose of AAV2.N54-120 had similar efficacy to commercial aflibercept (Eylea) protein administered at 40μg / eye.

[0189]

[0235] Aflibercept ELISA was used to quantify the expression of aflibercept in ocular samples (local expression) and serum samples (systemic levels). Aflibercept expression was detected in ocular samples injected with the high dose of the construct. However, aflibercept expression was very low or not detectable in the other groups.

[0190] Serotype selection

[0236] It is well known that AAV (especially AAV2) neutralizing antibodies are widely present in humans. Another challenge in using AAV2 is the lack of tropism. For this reason, AAV1 and AAV6 serotypes were screened for in vitro expression level (Figure 30 and Table 30), in vivo efficacy (FA data, Figure 31) and aflibercept expression in ocular tissue (Figure 32 and Table 20).

[0191] [Table 20]

[0192]

[0237] Aflibercept ELISA was performed and the LoQ of the assay was determined to be 2ng / mL. Values ​​below 2ng / mL were designated as >LoQ. From Table 20, it could be concluded that aflibercept was not detected in serum samples. However, AAV6 and AAV1 detected aflibercept in ocular samples.

[0193]

[0238] A dose-response study was designed and conducted in which mice were administered AAV6.N54-aflibercept at 2.0E+07 (low dose), 4.8E+08 (medium dose) and 1.6E+10 high doses. Figure 33 shows the FA data as a bar graph to compare different groups.

[0194]

[0239] Animals were injected with AAV2, AAV1 and AAV6-GFP at 5E+09 vg / eye to examine expression and penetrance, and these animals showed no advantage over AAV2.N54-GFP (Figure 34).

[0195]

[0240] To compare the various serotypes, a study in pigs was performed in which pigs were injected with AAV1, AAV2 and AAV6-GFP at 1E+11 vg / eye. As in the previous pig study, V226 (+ control) was also included. Figure 35 shows representative fundus and IHC images. Fundus images were taken on days 21, 28 and 34, as the GFP signal was relatively low in animals injected with AAV1, AAV2 and AAV6-GFP. Finally, animals were sacrificed on day 34 and IHC was performed on selected eyes. An in vitro ELISA assay for GFP did not show a higher expression of GFP in AAV2.N54-GFP when compared to AAV2.wt-GFP (Figure 36).

[0196]

[0241] In GOI screening, lot-to-lot variations of AAV constructs were detected. For example, N54.120 constructs were purified multiple times and stored at -80°C. The purification process was varied by using different volumes of starting cell culture, purifying using either two cycles of ultracentrifugation (UC) or AAVx affinity columns, cell viability, and other culture condition variations. Table 21 shows an overview of small lots of N54.120.

[0197] [Table 21]

[0198]

[0242] Below are exemplary GFP expression captured in fluorescent microscope images (Figure 37A) and analyzed using a GFP quantification ELISA (Figure 37B and Table 22), showing lot-to-lot variation: GFP lot number 20-147 had lower intensity than the 20-06-30-N54 lot, but the ELISA showed no significant difference in GFP expression in vitro.

[0199] [Table 22]

[0200]

[0243] After testing in mice and pigs, capsid N54 was selected, with AMI120 optimized GOI. Animal efficacy testing showed significant laser damage recovery with AAV vector treatment, which was comparable to commercial aflibercept. In mouse efficacy CNV models, AAV2 serotypes appeared to be more consistent than AAV6 serotypes.

[0201] Example 13. Comparative bioanalysis results from animal studies

[0244] Example 13 shows a bioanalytical comparison of mouse eye choroidal neovascularization (CNV) or macular neovascularization (MNV) efficacy models from two different studies to examine the reproducibility of the models. Both studies used the same titer and amount of non-GLP AAV2 construct for intravitreal injection (IVT). The study protocols were also similar (Table 23). AAV2 capsid quantification was performed using a commercially available AAV2 capsid protein kit (Progen, Catalog No.: PRAAV2R and Lot No.: A20008). GraphPad Prism (v9.0.1) was used for statistical analysis.

[0202] [Table 23]

[0203] material and method

[0245] Table 24 shows the number of serum and eye samples obtained from the mouse studies.

[0204] [Table 24]

[0205]

[0246] A 532 nm diode laser was used to create four single laser spots surrounding the optic nerve. If the injection of the construct or the vehicle caused any inflammatory reaction, the laser irradiation would not form a lesion (air bubble). If no air bubble was present, the spot was not used for analysis. Four laser spots were induced in every eye, and the size of the lesion was estimated 7 days after the laser treatment. The lesion corresponded to wound healing due to the effect of the gene products of the various constructs. Eight animals were used in each group, corresponding to 16 eyes with four laser spots in each eye, resulting in a total of 64 laser spots. Table 25 summarizes the number of air bubbles not formed by each group injected with AVMX-110 or vehicle control.

[0206] [Table 25]

[0207]

[0247] Eyecup samples consisting of retina / RPE / choroid / sclera were homogenized using 1x PBS and BSA without protease inhibitors as described in the study protocols for both studies. Each eye sample was further homogenized using a sonicator with 21-second pulses and 30-second intervals repeated 5-6 times. When a clear supernatant was obtained that was visible to the naked eye, the samples were centrifuged at 13,000 rpm for 3 minutes. The supernatant was then collected and used to measure VEGF-Trap levels.

[0208] Comparison of FA analysis

[0248] Seven days after laser injury, the lesion area of ​​both eyes of each mouse was analyzed by FA. The gene product of AVMX-110 in treated mice was expected to reduce laser injury compared to vehicle control treated mice. Figure 38 and Table 26 show a comparison of the effectiveness of AVMX-110 in repairing laser lesions. In both studies, AVMX-110 treated mice showed equal laser wound repair. Although there was variation in baseline area in the two studies, animals treated with AVMX-110 showed nearly identical lesion repair. Representative images from FA image analysis also show a similar reduction in lesion area in Figure 39.

[0209] [Table 26]

[0210] Comparison of VEGF-Trap concentrations in serum samples

[0249] Serum samples were directly placed in 96-well plates without dilution. Figure 39A shows VEGF-Trap levels in serum samples. VEGF-Trap levels in serum samples were below the quantification limit of qualified VEGF-Trap ELISA. VEGF-Trap levels in ocular samples

[0250] VEGF-Trap in ocular samples was quantified by ELISA and plotted directly as ng / mL. VEGF-Trap concentration per mg of tissue weight was calculated by dividing the absolute amount of VEGF-Trap measured by ELISA by the volume of homogenized ocular tissue. The calculated amount was then divided by the tissue weight to obtain pg of VEGF-Trap per eyecup. VEGF-Trap data was plotted (Figure 39B) and VEGF-Trap levels in serum and ocular samples were recorded in Table 31 (vehicle samples were excluded from the table because their values ​​were below the LoQ). Taken together, VEGF-Trap expression in all serum and ocular samples was very low, with most animals below the quantification limit of VEGF-Trap ELISA.

[0211] [Table 27]

[0212] Correlation analysis between VEGF-Trap concentration and recovery area after laser treatment

[0251] The presence of lesions was measured as area in pixels. Lesion pixel data was plotted against pg of VEGF-Trap per eye cup, correlations were calculated using Pearson correlation, and two-sided p-values ​​were estimated for 95% confidence intervals using Graphpad Prism software. Figure 40A illustrates the comparison between the two experiments. In both analyses, a negative correlation was observed between VEGF-Trap concentration and lesion pixel area. The data show that the smaller the lesion area (pixels), the higher the VEGF-Trap concentration. The correlation coefficients and p-values ​​for both studies are shown in Table 32.

[0213] [Table 28]

[0214] Comparison of AAV2 capsid protein amounts in both studies

[0252] AAV2 capsid assay was performed using AAV2 capsid ELISA kit. AAV2 capsid protein amount in both studies was also similar (Figure 40B). However, it was important to note that the amount of ocular samples (in both studies) was significantly different between samples, which affected the overall estimate of capsid protein.

[0215]

[0253] The results of FA images and lesion area analysis showed consistency between both studies. There were more "no bubbles" in the vehicle group. For VEGF-Trap concentrations in ocular samples, many data points were below the limit of quantification. The LoQ of the assay was approximately 2ng / mL. This LoQ value was lower than that of the commercial kit (approximately 5-6ng / mL). In these studies, the ocular samples consisted of the ocular cup rather than the whole eyeball, which may have influenced the amount and concentration of VEGF-Trap in the samples. For this reason, we decided to include the whole eyeball in future studies. Similar to VEGF-Trap, the AAV2 capsid protein ELISA was also affected by the sample volume. As a control and positive control, commercially available aflibercept (40μg / eye) was injected. AVMX-110 at 4.0E+08vg / eye showed lesion recovery comparable to commercially available aflibercept. Given the similar results of these two studies, it could be concluded that this AVMX-110 dose showed results comparable to commercially available aflibercept. Taken together, the mouse MNV model was a suitable model for initial construct screening before progressing to larger animal models such as NHPs.

[0216] Example 14. Efficacy of a modified adeno-associated virus (AAV6.N54-120) encoding aflibercept (AVMX-116) in a laser-induced choroidal neovascularization (CNV) model

[0254] Example 14 shows that different serotypes of adeno-associated virus vectors were used in groups 1-5 to evaluate the inhibition of angiogenesis in a laser-induced model of choroidal neovascularization (LCNV) in mice, and groups 6-8 to evaluate the distribution of AAV6-N54-GFP, AAV1-N54-GFP, and AAV2.N54-GFP in mouse retinal tissue. Two untreated animals (group 9) were included for method development and bioanalysis efforts. Table 33 shows the experimental design.

[0217] [Table 29]

[0218] method

[0255] On day 28, mice were administered buprenorphine 0.01-0.05 mg / kg subcutaneously (SQ). Animals were then sedated by isoflurane inhalation for intravitreal injection, and one drop of 0.5% proparacaine HCl was applied to both eyes. The conjunctiva was gently grasped with Dumont #4 forceps, and the injection was performed using a 33G needle and Hamilton Syringe. After dispensing the syringe contents, the syringe needle was slowly removed. After the injection procedure, one drop of ofloxacin ophthalmic solution was applied topically to the ocular surface along with an ocular lubricant.

[0219]

[0256] On day 0, mice were administered buprenorphine 0.01–0.05 mg / kg SQ and topical mydriatics (1.0% tropicamide HCl and 2.5% phenylephrine HCl) were applied at least 15 min prior to laser procedure. Mice were sedated with an intraperitoneal (IP) injection of ketamine / xylazine. Topical eyewash was used to keep the cornea moist, and a hot pad was used to maintain body temperature. A 532 nm diode laser delivered through a slit lamp was used to create four single laser spots surrounding the optic nerve. Both eyes of mice were lasered according to the schedule of the experimental design. Ophthalmic lubricant was applied after laser (OU). Animals were observed daily for morbidity and mortality, as well as external cage observations, with particular attention paid to both eyes. Animal 436 (group 4) died before the imaging procedure on day 7. The baseline weight of all animals was 25.1 ± 1.5 g. Animals in all groups gained normal amounts of weight throughout the study.

[0220]

[0257] Samples from groups 1-5 and 9 were shipped or stored on dry ice. Animals randomized to pharmacokinetic analysis (n=2 / group) did not undergo LCNV induction for aflibercept quantification. Ocular samples (i.e., whole globes) were enucleated, frozen after isolation, and weighed. Table 34 shows a detailed record of tissue weight and the amount of 1×PBS containing protease inhibitors (PI) added to each sample prior to homogenization. Samples were placed on ice and homogenized using a sonicator with 3 cycles of 20-second pulses followed by 20-second pauses. After sonication, samples were placed on ice.

[0221] [Table 30]

[0222]

[0258] Aflibercept expression in ocular (whole eye) and serum samples was quantified using aflibercept ELISA. Briefly, 0.1 μg / mL VEGF was coated onto a 96-well plate and incubated O / N at 4°C. The plate was washed with washing buffer and blocked with blocking buffer. Ocular and serum samples were delivered directly to specific wells undiluted, except for group 5 samples (1:5, 1:10, and 1:100 dilutions). Samples were incubated for 1 hour. The plate was washed and detection antibody was added to each well at a 1:40,000 dilution. After the 1 hour incubation, the plate was washed and streptavidin-horseradish peroxidase (HRP) was added at a 1:40,000 dilution. After 45 min of incubation, the plates were washed and aflibercept was detected by addition of 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The reaction was stopped by adding stop solution and the plates were immediately read at 450 nm with a reference wavelength of 600 nm.

[0223]

[0259] Aflibercept expression in animals treated and untreated with the sham vector control was comparable. In group 3 animals injected with AAV6.N54-aflibercept, approximately 2.3 pg aflibercept / mg of ocular homogenate was measured (Figure 41A). Mid-range levels for AAV2 and AAV6.N54-aflibercept were approximately 8.8 and 25 pg aflibercept / mg of ocular homogenate, respectively, with a three-fold increase in expression in animals treated with AAV6. Animals treated with high-dose AAV6 had the highest levels of aflibercept, approximately 400 pg aflibercept / mg of ocular homogenate. Serum samples followed a similar trend to ocular tissue samples. Groups 1 (sham), 2 (AAV2-aflibercept), and 3 (AAV6-low dose) had no detectable aflibercept. Animals treated with both medium and high doses of AAV6-aflibercept had 2-3 ng / mL of aflibercept in serum (Figure 41B). Data were analyzed with GraphPad Prism software using one-way ANOVA followed by Dunnett's multiple comparisons (**=0.005 and ****≦0.0001). Aflibercept expression was as shown in Table 35 (AAV1.N54-aflibercept and AAV6.N54-aflibercept showed 30-fold higher expression of aflibercept compared to AAV2.N54-aflibercept) and Table 36.

[0224] [Table 31]

[0225] [Table 32]

[0226] Fundus photography (Groups 6 to 8, Day 0)

[0260] Color and cobalt blue fundus photography was performed for EGFP on both eyes of animals enrolled in groups 6-8. Animals were administered topical mydriatics (1.0% tropicamide HCl and 2.5% phenylephrine HCl) to dilate and proptose the eyes, and local anesthetic (proparacaine 0.5%) was applied to the eyes. Color fundus photography was followed by cobalt blue photography. Acquisition settings were set for group 7 (positive control) and were kept consistent for groups 6 and 8. Representative images are shown in Figure 42. GFP signal was observed in all three groups. GFP signal was most intense and bright in group 7 (positive control), followed by group 8 and then group 6. Expression was more limited than observed in previous studies, despite exposure times being kept constant throughout all studies.

[0227] Fluorescein angiography (FA)

[0261] FA was performed on both eyes on the 7th day after laser. Mydriasis for FA was achieved using topical eye drops (1.0% tropicamide HCl and 2.5% phenylephrine HCl) in each eye 15 minutes prior to testing. Mice were sedated with an IP injection of ketamine / xylazine. Retinal photography was performed approximately 1 minute after intravenous sodium fluorescein injection (1% stock solution, 12 mg / kg). Fluorescein leakage was analyzed with ImageJ software. Representative images showing the lesions are shown in Figure 43 below. Fundus angiograms were used to quantify lesion area as pixels, 2.5 standard deviations (SD), and the results are presented in Figure 44. When analyzing the size of the isolectin area, lesions in which Bruch's membrane did not rupture, lesions with vitreous hemorrhage, or lesions with data points greater than 2.5 standard deviations from the mean were excluded. Test material in group 5 (highest dose of AVMX-116) performed better than AVMX-110 (positive control) in group 2, with the smallest lesion area among all groups. Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparisons in GraphPad Prism software (*=0.0138).

[0228]

[0262] Comparing data from this study to previous studies, homogenization of the whole eyeball provided superior estimation of aflibercept expression when compared to the eyecup alone, improving the accuracy of quantification. Ocular samples with AAV6.aflibercept showed a roughly 10-fold increase in protein expression with a 20-fold increase in viral vector dose, and the AAV6 construct showed a 3-fold enhancement in aflibercept expression when compared to AAV2. Animals receiving either medium or high doses of AAV6.aflibercept showed substantial aflibercept expression in serum samples. This study demonstrated that animals receiving IVT injections of the positive control AVMX-110 had a significant reduction in the average size of CNV lesions as assessed by fluorescein angiography (FA), consistent with other studies utilizing the same construct.

[0229] Ocular tissue collection and processing

[0263] On day 7, animals were humanely euthanized by carbon dioxide asphyxiation and death was confirmed by cervical dislocation.

[0230] Tissue collection for flat mount analysis (Groups 1-5 only)

[0264] Eyes were enucleated and immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and stored overnight at 4°C. The next day, eyes were transferred to cold immunocytochemistry (ICC) buffer (PBS containing 0.5% BSA and 0.2% Tween 20) until processing. Using a dissecting microscope, the eyes were carefully cleared of extraneous tissue and the anterior segment and lens were removed. The retina was detached and removed from the optic nerve head with fine curved scissors. The eyecups were washed with cold ICC buffer. Eyecups were placed in cold ICC containing 4',6-diamidino-2-phenylindole (DAPI, nuclear stain), isolectin IB4 AlexaFluor649 (blood vessels, Vector Labs catalog number DL-1208-.5) and phalloidin AlexaFluor555 (f-actin, ThermoFisher A34055). The eyecups were incubated for 4 hours at 4°C with gentle rotation and washed with cold ICC buffer. A radial cut was then made toward the optic nerve head avoiding the lesion, and the sclera-choroid / RPE complex was flat mounted, covered and sealed. Two-dimensional (2D) fluorescence microscopy images were acquired, digitized and analyzed using an Olympus Bx63 upright fluorescence microscope and CellSens (Olympus) software. Post-acquisition analysis was performed using CellSens software. To quantify newly formed blood vessels, isolectin IB4 area was measured in μm2. Representative images from isolectin lesion area measurements are shown in Figure 45.

[0231]

[0265] When analyzing the size of the isolectin area (Figure 46), lesions that did not rupture Bruch's membrane, had vitreous hemorrhage, or had data points that were more than 2.5 standard deviations from the mean were excluded. Lesions that did not rupture Bruch's membrane, had vitreous hemorrhage, or had data points that were more than 2.5 standard deviations from the mean were excluded. In animals in group 1, the lesion area was 60,900 ± 38,500 μm 2 In group 5, the lesion area was 18,700 ± 7,200 μm 2The smallest was 70% smaller in lesion area. The positive control was performed similarly to the other studies and had a lesion area 30% smaller than the sham vector control. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons (***=0.0001 and ****≦0.0001).

[0232] Tissue collection for immunohistochemistry (Groups 6-8 only)

[0266] All eyes designated for immunohistochemistry (IHC) were enucleated and the tissue was placed in 1x PBS after marking the approximate injection sites. Eyes were fixed overnight at 4°C with 4% paraformaldehyde in separately labeled vials. Subsequently, eyes were transferred to 0.1M phosphate buffer (PB) and subjected to a serial sucrose gradient (10-30%, 1 h each) before being embedded in OCT medium and frozen on dry ice. Whole eyes were cryosectioned (14 μm sections) and stained with the following antibodies: 1 / 250 chicken anti-GFP, 1 / 250 rabbit anti-RPE65, followed by 1 / 200 anti-chicken Cy2, 1 / 200 anti-rabbit Cy3, 1 / 100 PNA lectin Cy5, and 1 / 1,000 DAPI.

[0233]

[0267] Groups 6 and 7 had the brightest and most widespread GFP signal, but Group 8 had GFP signal as well. In all groups, GFP expression tended to be localized in the central retina near the optic nerve, but there was variability with location. Expression was consistently observed from the ganglion cell layer through the inner segment, with less consistency observed in the outer segment and retinal pigment epithelium. The GFP signal observed in stained cryosections was consistent with the GFP signal observed in fundus images. While the RPE staining observed in 856 OD was due to AAV transduction into the RPE after standard IVT injection, the GFP expression observed in the RPE of mouse 652 OS may have been enhanced due to unintentional delivery of AAV to the subretinal space rather than the vitreous space. Representative images of IHC at day 0 are shown in FIG. 47. FIG. 48A illustrates a flat mount analysis of images obtained from the dose-response study of Example 14. FIG. 48B illustrates a fluorescein fundus angiography (FA) analysis of images obtained from the dose-response study of Example 14.

[0234] Example 15. Formulation and Efficacy Testing of AVMX

[0268] AVMX-110 was formulated in a liquid composition in sterile vials. AVMX-110 drug formulation concentrate for IVT injection solution was manufactured as a sterile frozen liquid formulation stored in 1 mL 13 mm cyclic olefin copolymer (COC) vials with 13 mm grey bromobutyl stoppers and 13 mm flip-off aluminum seals. Each vial was filled to a target weight of 100 μL to allow for a draw volume of at least 50 μL (density 1.01 g / mL). The product was 4.0×10 12 For clinical use, the drug formulation was thawed, aseptically aspirated using a provided filter needle, and delivered to patients via IVT by a retinal specialist.

[0235]

[0269] AVMX-110: Efficacy studies using AAV2·N54-VEGF-Trap were performed according to the protocol shown in Table 37. The efficacy of AVMX-110 in mice was evaluated by using a laser-induced choroidal neovascularization (LCNV) model.

[0236] [Table 33]

[0237]

[0270] Animals were allowed to acclimate to the study environment for a minimum of 3 days. At the end of the acclimation period, a physical examination was performed on each animal by a laboratory animal technician to determine suitability for study participation. Examinations included, but were not limited to, skin and external ear, eyes, abdominal, behavioral, and general condition. Animals deemed to be in good health were included in the study. Animal randomization, intravitreal injection, laser-induced CNV treatment, animal examination, fluorescein angiography (FA), ocular tissue collection, and flat mounts (n=6 / group) were described in the protocol. In addition, all mice were terminally bled to collect serum and retinal tissue after FA observation and flat mount collection.

[0238]

[0271] FA analysis showed that animals receiving 40 μg / eye aflibercept or AVMX-110 IVT injection showed a significant reduction in lesion area (Figure 49A). For the AVMX-110 group, the reduction in lesion area was inversely correlated with the dose of vector genome copies (vg) (P<0.01 for 4e+8vg / eye, P<0.001 for 1.6e+10vg / eye), and the more vector injected, the smaller the lesion area observed 7 days after laser irradiation (Table 38). With 1.6e+10vg / eye, complete healing of the laser lesion was achieved. Figure 49B illustrates the FA analysis of images from the AVMX-110 dosing study.

[0239] [Table 34]

[0240]

[0272] After euthanasia, blood was collected from animals in groups 1-5 and 9 for serum isolation, and the eyes were enucleated and flash frozen. Eyes were placed in appropriate pre-weighed, labeled analytical vials, immediately re-weighed to determine sample weight, and placed on dry ice until transfer to the freezer. Samples were weighed on a balance capable of measuring to four decimal points. After flash freezing and weighing, samples were returned to the -80°C freezer. VEFG-Trap concentrations in serum and retinal tissue were tested by ELISA assay. Eyes were enucleated, and retina and RPE / choroid sections were dissociated from fresh eyeballs and flash frozen. Tissues were placed in appropriate pre-weighed, labeled analytical vials, immediately re-weighed to determine sample weight, and placed on dry ice until transfer to the freezer. Samples were weighed on a balance capable of measuring to four decimal points.

[0241]

[0273] Samples were assayed using ELISA. VEGF-Trap was approximately 50ng / mL in the aflibercept control group, whereas in serum VEGF-Trap was detected at low levels of approximately 2ng / mL in the high dose group (1.6e10vg / eye) and was not detected in the low and mid dose groups (Figure 51A). VEGF-Trap levels in retinal tissue were also measured in retinal homogenates using ELISA (Figure 51B). In retinal tissue suspensions, VEGF-Trap expression was in a good dose-dependent manner for intravitreally injected AVMX-110. An average of 13ng / mL of VEGF-Trap was detected in the high dose group at 1.6e10vg / eye. Maximum expression levels reached more than 40ng / mL.

[0242]

[0274] Although the foregoing disclosure has been described in some detail for clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the present disclosure. For example, all of the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually and separately indicated to be incorporated by reference for all purposes.

[0243] [Table 35-1]

[0244] [Table 35-2]

[0245] [Table 35-3]

[0246] [Table 35-4]

[0247] [Table 35-5]

[0248] [Table 35-6]

[0249] [Table 35-7]

[0250] [Table 35-8]

[0251]

Table 35-9

[0252]

Table 35-10

[0253]

Table 35-11

[0254]

Table 35-12

[0255]

Table 35-13

[0256]

Table 35-14

[0257]

Table 35-15

[0258]

Table 35-16

[0259]

Table 35-17

[0260]

Table 35-18

[0261]

Table 35-19

[0262]

Table 35-20

[0263]

Table 35-21

[0264]

Table 35-22

[0265]

Table 35-23

[0266]

Table 35-24

[0267]

Table 35-25

[0268]

Table 35-26

[0269]

Table 35-27

[0270]

Table 35-28

[0271]

Table 35-29

[0272]

Table 35-30

[0273]

Table 35-31

[0274]

Table 35-32

[0275]

Table 35-33

[0276]

Table 35-34

[0277]

Table 35-35

[0278]

Table 35-36

[0279]

Table 35-37

[0280]

Table 35-38

[0281]

Table 35-39

Claims

1. An isolated non-naturally occurring nucleic acid comprising a sequence encoding a biologic comprising an anti-angiogenic agent, said sequence comprising a modification in the coding region of the sequence compared to an otherwise equivalent sequence lacking the modification in the coding region, said modification comprising replacement of at least four non-AGG arginine codons with AGG.

2. 2. The non-naturally occurring nucleic acid of claim 1, wherein the sequence encoding the anti-angiogenic agent further comprises a second modification.

3. The second modification is within at least one codon of the coding region of the sequence, and the second modification is (a) replacement of at least one non-CCC proline codon with CCC; (b) replacement of at least one non-TCC serine codon with TCC; (c) replacement of at least one non-CCG proline codon with CCG, and (d) any combination of (a) to (c) 3. The non-naturally occurring nucleic acid of claim 2, selected from the group consisting of:

4. 4. The non-naturally occurring nucleic acid of claim 3, wherein at least one non-CCC proline codon in (a) is CCT, at least one non-TCC serine codon in (b) is AGC, or at least one non-CCG proline codon in (c) is CCC.

5. 5. The non-naturally occurring nucleic acid according to any one of claims 1 to 4, wherein the anti-angiogenic agent is selected from the group consisting of VEGF inhibitors, multi-tyrosine kinase inhibitors, receptor tyrosine kinase inhibitors, inhibitors of Akt phosphorylation, PDGF-1 inhibitors, PDGF-2 inhibitors, NP-1 inhibitors, NP-2 inhibitors, Del 1 inhibitors and integrin inhibitors.

6. 6. The non-naturally occurring nucleic acid of claim 5, wherein the anti-angiogenic agent comprises a VEGF inhibitor, and the VEGF inhibitor is a non-antibody inhibitor.

7. 7. The non-naturally occurring nucleic acid of claim 6, wherein the non-antibody inhibitor is a fusion protein comprising human VEGF receptors 1 and 2.

8. The non-naturally occurring nucleic acid of claim 7, wherein the fusion protein comprises VEGF-Trap or a modified version thereof.

9. The isolated non-naturally occurring nucleic acid according to any one of claims 1 to 4, further comprising a signal peptide.

10. 10. The non-naturally occurring nucleic acid of claim 9, wherein the signal peptide is selected from the group consisting of human antibody heavy chain (Vh), human antibody light chain (Vl), and VEGF-Trap.

11. A nucleic acid of non-natural origin according to any one of claims 1 to 4, further comprising an intron sequence.

12. 12. The non-naturally occurring nucleic acid of claim 11, wherein the intron sequence is selected from the group consisting of hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, hamster EF-1α gene intron 1, an intervening sequence intron, a human growth hormone intron, and a human β-globin intron.

13. A nucleic acid of non-natural origin according to any one of claims 1 to 4, further comprising a promoter.

14. 14. The non-naturally occurring nucleic acid of claim 13, wherein the promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter, a simian vacuolar virus (SV40) promoter, a phosphoglycerate kinase (PGK1) promoter, a ubiquitin C (Ubc) promoter, a human β-actin promoter, a CAG promoter, a tetracycline response element (TRE) promoter, a UAS promoter, an actin 5c (Ac5) promoter, a polyhedron promoter, a Ca2+ / calmodulin-dependent protein kinase II (CaMKIIa) promoter, a GAL1 promoter, a GAL10 promoter, a TEF1 promoter, a glyceraldehyde 3-phosphate dehydrogenase (GDS) promoter, an ADH1 promoter, a CaMV35S promoter, a Ubi promoter, a human polymerase III RNA (H1) promoter, a U6 promoter, polyadenylation constructs thereof, and any combination thereof.

15. A non-naturally occurring nucleic acid according to any one of claims 1 to 4, wherein the non-AGG arginine codon is AGA.

16. A nucleic acid of non-natural origin according to any one of claims 1 to 4, comprising a viral vector sequence.

17. The nucleic acid of non-natural origin described in claim 16, wherein the viral vector sequence is an AAV viral vector sequence, the AAV vector sequence being of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof.

18. 5. A non-naturally occurring nucleic acid according to any one of claims 1 to 4, comprising at least about 60% sequence identity or similarity to any one of SEQ ID NOs: 13-19, 21-27, 31, 62, 64, 66, or 68.

19. An adeno-associated virus (AAV) particle comprising a non-naturally occurring nucleic acid according to any one of claims 1 to 4.

20. The use of a nucleic acid of non-natural origin comprising a sequence encoding a biological agent, including an anti-angiogenic agent, in the manufacture of a medicament for treating a disease or condition, wherein the sequence has been modified to replace at least four non-AGG arginine codons in the codon region of the sequence with AGG, compared to an otherwise equivalent sequence lacking the modifications in the coding region.