Engineered viral capsids for therapeutic delivery

Abstract

Provided herein are compositions and methods of utilizing engineered viral capsid for therapeutic delivery. Also provided herein are methods of using the provided compositions and methods for delivering therapeutic for treating diseases.

Description

WSGR Docket No. 59561-717.601ENGINEERED VIRAL CAPSIDS FOR THERAPEUTIC DELIVERY CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application Number 63 / 721,200 filed on November 15, 2024; U.S. Provisional Patent Application Number 63 / 738,353 filed on December 23, 2024; U.S. Provisional Patent Application Number 63 / 753,868 filed on February 4, 2025; U.S. Provisional Patent Application Number 63 / 721,207 filed on November 15, 2024; U.S. Provisional Patent Application Number 63 / 738,356 filed on December 23, 2024; and U.S. Provisional Patent Application Number 63 / 753,870 filed on February 4, 2025, each of which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Adeno-associated virus (AAV) is a small, single-stranded DNA-containing, non-pathogenic parvovirus with a non-enveloped protein capsid that has gained significant attention as an efficient and safe vector for gene transfer or therapeutic. Recombinant AAV vectors have been being used in various clinical trials with limited success. For example, AAV vectors have been used to deliver aflibercept for treating ocular diseases. Aflibercept (VEGF-Trap) is a recombinant fusion protein that acts as a decoy receptor for vascular endothelial growth factor subtypes A (VEGF-A), VEGF-B, and placental growth factor (PIGF). By binding to these ligands, aflibercept is able to prevent these ligands from binding to vascular endothelial growth factor receptors (VEGFR), VEGFR-1 and VEGFR-2, to suppress neovascularization and decrease vascular permeability. Aflibercept consists of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 fused with the Fc fragment of IgGl. Aflibercept is commercially marketed under the trade name EYLEA® (aflibercept), which is an ophthalmic intravitreal aflibercept fusion protein injection. However, the use of virus for therapeutic delivery (e.g., gene therapy) is limited by challenges such as off-targeting, poor penetrance, and limited cargo size.SUMMARY

[0003] Current treatments employing aflibercept suffer from short durability and repeated monthly injections in order to suppress the neovascularization. Several gene therapy studies using AAV vectors carrying the coding sequence for VEGF-Trap has been investigated for long term treatment of the neovascularization. However, these studies suffer from low expression levels of the agent and therefore reduced treatment efficacy. Accordingly, it has become increasingly clear that the full potential of this technology can be realized after modifications are made for improved delivery and expression of VEGF-Trap and other anti-angiogenic agents. Furthermore,WSGR Docket No. 59561-717.601there remains a need to effectively deliver VEGF-Trap or a comparable anti-angiogenic agent to a target cell to increase therapeutic efficacy and decrease deleterious side effects.

[0004] Accordingly, there remains a need for development of an engineered virus for effective therapeutic delivery. There also remains a need for such engineered virus, where the efficacy of therapeutic delivery of the engineered virus results in an improvement of treatment regimen or outcome for a subject treated with such engineered virus.

[0005] Provided herein, in some aspects is a method for treating a disease or condition in a subject, comprising: administering a viral particle comprising an engineered capsid encapsulating a vector to the subject, the engineered capsid comprising a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A, and wherein the polypeptide sequence increases expression of the vector in a cell, and wherein the expression of the vector treats the disease or condition in the subject. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof. In some embodiments, the cell comprises a macula cell. In some embodiments, the cell comprises a retinal cell. In some embodiments, the cell comprises an ocular cell. In some embodiments, the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or a combination thereof. In some embodiments, the engineered capsid comprises an engineered adeno-associated virus (AAV) capsid. In some embodiments, the engineered AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the engineered AAV capsid comprises anWSGR Docket No. 59561-717.601engineered AAV2 capsid. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 13). In some embodiments, the polypeptide sequence is the amino acid sequence of LALGQTTKPA (SEQ ID NO: 13). In some embodiments, the polypeptide sequence is inserted in the VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the engineered capsid further comprises a mutation. In some embodiments, the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain. In some embodiments, the mutation is in a GH loop. In some embodiments, the mutation is at a residue at position 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 1. In some embodiments, the vector comprises one or more expression cassettes for expressing one or more transgene. In some embodiments, the vector encodes an anti-angiogenic agent. In some embodiments, the anti-angiogenic agent comprises a VEGF-Trap. In some embodiments, the anti-angiogenic agent consists of the VEGF-Trap. In some embodiments, the anti -angiogenic agent is encoded in the vector comprising one or more codon modifications. In some embodiments, wherein the one or more codon modifications comprises one or more replacements of non-AGG arginine codon to AGG; non-CCC proline codon to CCC; non-TCC serine codon with TCC; non-CCG proline codon with CCG; or a combination thereof. In some embodiments, the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or any combination thereof to the subject. In some embodiments, the engineered capsid delivers the vector to the cell in the subject. In some embodiments, the engineered capsid delivering the vector decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell. In some embodiments, the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence. In some embodiments, the engineered capsid delivering the vector increases delivery of the vector to a cell that is in a deeper cell layer in the subject compared to a comparable non-engineered capsid without the polypeptide sequence delivering the vector to a cell in a cell layer. In some embodiments, the cell layer comprises a ganglion cellWSGR Docket No. 59561-717.601layer, an inner plexiform layer, an inner nuclear layer, an outer plexiform cell, an outer nuclear layer, an inner segment layer, an outer segment layer, a retinal pigment epithelium layer, or any combination thereof. In some embodiments, the engineered capsid delivering the vector results in a decreased retinal leakage compared to a comparable capsid without the polypeptide sequence delivering a comparable vector encoding a comparable VEGF inhibitor. In some embodiments, the decreased retinal leakage is resulted from one or more administrations. In some embodiments, the decreased retinal leakage is resulted from one administration. In some embodiments, the engineered capsid delivering the vector decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the engineered capsid delivering the vector decreases a dose of the anti-angiogenic agent administered to the subject compared to a second dose of a comparable anti-angiogenic agent administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable anti -angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not increase inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not decrease ERG in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not increase intraocular pressure in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not increase an expression of anti-drug antibody (ADA) in the subject compared to a second expression of the ADA in the subject resulted from directly administering the subject with a comparable anti-angiogenic agent. In some embodiments, an expression of the anti-angiogenic agent delivered by the engineered capsid is increased in the subject compared to a second expression of a comparable anti-angiogenic agent resulted from treatment by the comparable anti-angiogenic agent. In some embodiments, the anti -angiogenic agent delivered by the engineered capsid is expressed for a longer duration in the subject compared to a second duration of expressing a comparable anti-angiogenic agent by administering the comparable anti-angiogenic agent to the subject. In some embodiments, the comparable anti-angiogenic agent is Elyea. In some embodiments, theWSGR Docket No. 59561-717.601comparable capsid is an unmodified AAV capsid. In some embodiments, the unmodified AAV capsid is an AAV2 capsid. In some embodiments, the subject is administered a dose comprising the viral particle from about 1.0 x 109vg / eye to about 10 x IO10vg / eye. In some embodiments, the dose is from about 1.0 x IO10vg / eye to about 10 x IO10vg / eye. In some embodiments, the dose is from about 2.0 x 1010vg / eye to about 5 x 1010vg / eye. In some embodiments, the dose is about 2.1 x 1010vg / eye, about 2.2 x 1010vg / eye, about 2.3 x 1010vg / eye, about 2.4 x 1010vg / eye, about 2.5 x 1010vg / eye, about 2.6 x 1010vg / eye, about 2.7 x 1010vg / eye, about 2.8 x 1010vg / eye, or about 2.9 x 1010vg / eye. In some embodiments, the dose is about 2.6 x 1010vg / eye. In some embodiments, the dose is about 3.1 x 1010vg / eye, about 3.2 x 1010vg / eye, about 3.3 x 1010vg / eye, about 3.4 x 1010vg / eye, about 3.5 x 1010vg / eye, about 3.6 x 1010vg / eye, about 3.7 x 1010vg / eye, about 3.8 x 1010vg / eye, or about 3.9 x 1010vg / eye. In some embodiments, the dose is about 3.7 x 1010vg / eye. In some embodiments, the dose is about 8.1 x 1010vg / eye, about 8.2 x 1010vg / eye, about 8.3 x 1010vg / eye, about 8.4 x 1010vg / eye, about 8.5 x 1010vg / eye, about 8.6 x 1010vg / eye, about 8.7 x 1010vg / eye, about 8.8 x 1010vg / eye, or about 8.9 x 1010vg / eye. In some embodiments, the dose is about 8.1 x 1010vg / eye. In some embodiments, a single administrating of the viral particle comprising engineered capsid and the vector is curative of the disease or condition in the subject.

[0006] Provided herein, in some aspects is a method for treating a disease or condition in a subject, comprising: administering a dose of a viral particle comprising an engineered capsid encapsulating a vector to the subject, wherein the vector comprises a nucleic acid sequence encoding an anti-angiogenic agent, said nucleic acid sequence comprises a modification in a coding region of the nucleic acid sequence as compared to an otherwise comparable nucleic acid sequence lacking the modification in the coding region, said modification comprises replacing at least four non-AGG arginine codons to AGG, wherein the anti-angiogenic agent treats the disease or condition, and wherein the dose is from about 1.0 x 109vg / eye to about 10 x 1010vg / eye. In some embodiments, the nucleic acid sequence that encodes 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 nucleic acid sequence, and wherein the second modification is selected from the group consisting of replacement of at least one non-CCC proline codon with CCC; replacement of at least one non-TCC serine codon with TCC; replacement of at least one non-CCG proline codon with CCG; and any combination of (a)-(c). 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 ofWSGR Docket No. 59561-717.601Akt phosphorylation, a PDGF-1 inhibitor, a PDGF-2 inhibitor, a NP-1 inhibitor, a NP-2 inhibitor, a Del 1 inhibitor, and an integrin inhibitor. In some embodiments, the anti-angiogenic agent comprises the VEGF inhibitor, and wherein the VEGF inhibitor is a non-antibody inhibitor. In some embodiments, the non-antibody inhibitor is a fusion protein that comprises human VEGF receptors 1 and 2. In some embodiments, the fusion protein comprises VEGF-Trap or a modified version thereof. In some embodiments, the nucleic acid sequence is modified to replace non-AGG arginine codon with AGG in at least 4 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCC proline codon with CCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-TCC serine codon with TCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCG proline codon with CCG in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid comprises a viral vector sequence. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to any one of the nucleic acid sequences of SEQ ID NOS: 43-49, 51-57, 61, 62, 64, 66, 68, 71, or 72. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or any combination thereof. In some embodiments, the engineered capsid delivers the vector to a cell in the subject. In some embodiments, the cell comprises a macula cell. In some embodiments, the cell comprises a retinal cell. In some embodiments, the cell comprises an ocular cell. In some embodiments, the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or any combination thereof. In some embodiments, the engineered capsid comprises an engineered adeno-associated virus (AAV) capsid. In some embodiments, the engineered AAV capsid is of serotype AAV1,WSGR Docket No. 59561-717.601AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the engineered AAV capsid comprises an engineered AAV2 capsid. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is the amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is inserted in a VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the engineered capsid further comprises a mutation. In some embodiments, the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain. In some embodiments, the mutation is in a GH loop. In some embodiments, the mutation is at a residue at position of 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 1. In some embodiments, the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or any combination thereof to the subject. In some embodiments, the engineered capsid delivering the vector decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell. In some embodiments, the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence. In some embodiments, the engineered capsid delivering the vector increases delivery of the vector to a cell that is in a deeper cell layer in the subject compared to a comparable non-engineered capsid without the polypeptide sequence delivering the vector to a cell in a cell layer. In some embodiments, the cell layer comprises a ganglion cell layer, an inner plexiform layer, an inner nuclear layer, an outer plexiform cell, an outer nuclear layer, an inner segment layer, an outer segment layer, a retinal pigment epithelium layer, or any combination thereof. In some embodiments, the engineered capsid delivering the vector results in a decreased retinal leakage compared to a comparable capsid without the polypeptide sequence delivering a comparable vector encoding a comparable VEGF inhibitor. In some embodiments, the decreased retinal leakage is resulted from one or more administrations. In some embodiments, the decreased retinal leakage is resulted from one administration. In someWSGR Docket No. 59561-717.601embodiments, the engineered capsid delivering the vector decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the comparable capsid is an unmodified AAV capsid. In some embodiments, the unmodified AAV capsid is an AAV2 capsid. In some embodiments, the anti-angiogenic agent decreases retinal leakage in the subject compared to a second retinal leakage resulted from treatment by a comparable anti-angiogenic agent. In some embodiments, the retinal leakage is resulted from one or more administrations. In some embodiments, the retinal leakage is resulted from one administration. In some embodiments, the anti-angiogenic agent decreases retinal lesion in the subject compared to a second retinal lesion resulted from treatment by a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent decreases a dose of the anti-angiogenic agent administered to the subject compared to a second dose of a comparable anti-angiogenic agent administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the anti-angiogenic agent decreases inflammation of the cell, or an environment associated with the cell compared to contacting the cell with a comparable anti -angiogenic agent. In some embodiments, the anti -angiogenic agent does not increase inflammation of the cell, or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent does not decrease ERG in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent does not increase intraocular pressure in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent does not increase an expression of anti-drug antibody (ADA) in the subject compared to a second expression of the ADA in the subject resulted from directly administering the subject with a comparable anti-angiogenic agent. In some embodiments, an expression of the anti -angiogenic agent is increased in the subject compared to a second expression of a comparable anti -angiogenic agent resulted from treatment by the comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent is expressed for a longer duration in the subject compared to a second duration of expressing a comparable anti -angiogenic agent by administering the comparable anti -angiogenic agent to the subject. In some embodiments, the comparable anti -angiogenic agent is Elyea. In some embodiments, the dose is about 2.1 x IO10vg / eye, about 2.2 x IO10vg / eye, about 2.3 x IO10vg / eye, about 2.4 x IO10vg / eye, about 2.5 x IO10WSGR Docket No. 59561-717.601vg / eye, about 2.6 x IO10vg / eye, about 2.7 x IO10vg / eye, about 2.8 x IO10vg / eye, or about 2.9 x IO10vg / eye. In some embodiments, the dose is about 2.6 x IO10vg / eye. In some embodiments, the dose is about 3.1 x IO10vg / eye, about 3.2 x IO10vg / eye, about 3.3 x IO10vg / eye, about 3.4 x IO10vg / eye, about 3.5 x IO10vg / eye, about 3.6 x IO10vg / eye, about 3.7 x IO10vg / eye, about 3.8 x 1010vg / eye, or about 3.9 x IO10vg / eye. In some embodiments, the dose is about 3.7 x IO10vg / eye. In some embodiments, the dose is about 8.1 x IO10vg / eye, about 8.2 x IO10vg / eye, about 8.3 x IO10vg / eye, about 8.4 x IO10vg / eye, about 8.5 x IO10vg / eye, about 8.6 x IO10vg / eye, about 8.7 x IO10vg / eye, about 8.8 x IO10vg / eye, or about 8.9 x IO10vg / eye. In some embodiments, the dose is about 8.1 x IO10vg / eye. In some embodiments, a single administrating the dose is curative of the disease or condition in the subject.

[0007] Provided herein, in some aspects is a method for treating a disease or condition in a subject, comprising: administering to the subject a viral particle comprising an engineered capsid comprising a polypeptide encapsulating a vector encoding VEGF-Trap to the subject, wherein the polypeptide sequence increases expression of the vector in a cell of the subject, and wherein treating the subject with the viral particle increases therapeutic efficacy or decreases side effect compared to if the subject is treated with a comparable anti-angiogenic agent. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof. In some embodiments, the cell comprises a macula cell. In some embodiments, the cell comprises a retinal cell. In some embodiments, the cell comprises an ocular cell. In some embodiments, the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or a combination thereof. In some embodiments, the engineered capsid comprises an engineered adeno-associated virus (AAV) capsid. In some embodiments, the engineered AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,WSGR Docket No. 59561-717.601AAV11, AAV12, or any combination thereof. In some embodiments, the engineered AAV capsid comprises an engineered AAV2 capsid. In some embodiments, the polypeptide sequence is inserted in the VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1 In some embodiments, the engineered capsid further comprises a mutation. In some embodiments, the mutation is in a VP1 domain, a VP2 domain, a VP3 domain or a GH loop. In some embodiments, the mutation is at a residue at position 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 1. In some embodiments, the VEGF-Trap is encoded in the vector comprising one or more codon modifications. In some embodiments, the one or more codon modifications comprises one or more replacements of non-AGG arginine codon to AGG; non-CCC proline codon to CCC; non-TCC serine codon with TCC; non-CCG proline codon with CCG; or a combination thereof. In some embodiments, the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or any combination thereof to the subject. In some embodiments, the engineered capsid delivers the vector to the cell in the subject. In some embodiments, the engineered capsid delivering the vector decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell. In some embodiments, the engineered capsid delivering the vector increases delivery of the vector to a cell that is in a deeper cell layer in the subject compared to a comparable non-engineered capsid without the polypeptide sequence delivering the vector to a cell in a cell layer. In some embodiments, the cell layer comprises a ganglion cell layer, an inner plexiform layer, an inner nuclear layer, an outer plexiform cell, an outer nuclear layer, an inner segment layer, an outer segment layer, a retinal pigment epithelium layer, or any combination thereof. In some embodiments, the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence. In some embodiments, the engineered capsid delivering the vector results in a decreased retinal leakage compared to a comparable capsid without the polypeptide sequence delivering a comparable vector encoding a comparable VEGF inhibitor. In some embodiments, the decreased retinalWSGR Docket No. 59561-717.601leakage is resulted from one or more administrations. In some embodiments, the decreased retinal leakage is resulted from one administration. In some embodiments, the engineered capsid delivering the vector decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the engineered capsid delivering the vector decreases a dose of the VEGF-Trap administered to the subject compared to a second dose of the comparable anti-angiogenic agent administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the side effect inflammation of the cell or an environment associated with the cell. In some embodiments, the engineered capsid delivering the vector does not increase inflammation of the cell or an environment associated with the cell compared to contacting the cell with the comparable anti -angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not decrease ERG in the subject compared to if the subject is directly administered with the comparable anti-angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not increase intraocular pressure in the subject compared to if the subject is directly administered with the comparable anti-angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not increase an expression of antidrug antibody (ADA) in the subject compared to a second expression of the ADA in the subject resulted from directly administering the subject with the comparable anti-angiogenic agent. In some embodiments, an expression of the VEGF-Trap is increased in the subject compared to a second expression of the comparable anti-angiogenic agent resulted from treatment by the comparable anti-angiogenic agent. In some embodiments, the VEGF-Trap is expressed for a longer duration in the subject compared to a second duration of expressing the comparable anti-angiogenic agent by administering the comparable anti-angiogenic agent to the subject. In some embodiments, the comparable capsid is an unmodified AAV capsid. In some embodiments, the unmodified AAV capsid is an AAV2 capsid. In some embodiments, the subject is administered a dose comprising the viral particle from about 1.0 x 109vg / eye to about 10 x IO10vg / eye. In some embodiments, the dose is from about 1.0 x IO10vg / eye to about 10 x IO10vg / eye. In some embodiments, the dose is from about 2.0 x 1010vg / eye to about 5 x 1010vg / eye. In some embodiments, the dose is about 2.1 x 1010vg / eye, about 2.2 x 1010vg / eye, about 2.3 x 1010vg / eye, about 2.4 x 1010vg / eye, about 2.5 x 1010vg / eye, about 2.6 x 1010vg / eye, about 2.7 x 1010vg / eye, about 2.8 x 1010vg / eye, or about 2.9 x 1010vg / eye. In some embodiments, the dose isWSGR Docket No. 59561-717.601about 2.6 x IO10vg / eye. In some embodiments, the dose is about 3.1 x IO10vg / eye, about 3.2 x IO10vg / eye, about 3.3 x IO10vg / eye, about 3.4 x IO10vg / eye, about 3.5 x IO10vg / eye, about 3.6 x IO10vg / eye, about 3.7 x IO10vg / eye, about 3.8 x IO10vg / eye, or about 3.9 x IO10vg / eye. In some embodiments, the dose is about 3.7 x IO10vg / eye. In some embodiments, the dose is about 8.1 x IO10vg / eye, about 8.2 x IO10vg / eye, about 8.3 x IO10vg / eye, about 8.4 x IO10vg / eye, about 8.5 x IO10vg / eye, about 8.6 x IO10vg / eye, about 8.7 x IO10vg / eye, about 8.8 x IO10vg / eye, or about 8.9 x IO10vg / eye. In some embodiments, the dose is about 8.1 x IO10vg / eye. In some embodiments, the engineered capsid comprises at least one post translational modification. In some embodiments, a single administrating of the viral particle comprising the engineered capsid and the vector is curative of the disease or condition in the subject. In some embodiments, the polypeptide comprises L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-XI -X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183).

[0008] Described herein, in some aspects, is a method for treating a disease or condition in a subject, comprising: administering a viral particle comprising an engineered capsid encapsulating a vector to the subject, the engineered capsid comprising a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A, and wherein the polypeptide sequence increases expression of the vector in a cell, and wherein the expression of the vector treats the disease or condition in the subject. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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,WSGR Docket No. 59561-717.601Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof. In some embodiments, the cell comprises a macula cell. In some embodiments, the cell comprises a retinal cell. In some embodiments, the cell comprises an ocular cell. In some embodiments, the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or a combination thereof. In some embodiments, the engineered capsid comprises an engineered adeno-associated virus (AAV) capsid. In some embodiments, the engineered AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the engineered AAV capsid comprises an engineered AAV2 capsid. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 13). In some embodiments, the polypeptide sequence is the amino acid sequence of LALGQTTKPA (SEQ ID NO: 13). In some embodiments, the polypeptide sequence is inserted in the VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the engineered capsid further comprises a mutation. In some embodiments, the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain. In some embodiments, the mutation is in a GH loop. In some embodiments, the mutation is at a residue at position 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 1. In some embodiments, the vector comprises one or more expression cassettes for expressing one or more transgene. In some embodiments, the vector encodes an anti -angiogenic agent. In some embodiments, the anti-angiogenic agent comprises a VEGF-Trap. In some embodiments, the anti-angiogenic agent consists of the VEGF-Trap. In some embodiments, the anti -angiogenic agent is encoded in the vector comprising one or more codon modifications. In some embodiments, the one or more codon modifications comprises one or more replacements of non-AGG arginine codon to AGG; non-CCC proline codon to CCC; non-TCC serine codon with TCC; non-CCG proline codon with CCG; or a combination thereof. In some embodiments, the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or any combination thereof to the subject. In some embodiments, the engineered capsid delivers the vector to the cell in theWSGR Docket No. 59561-717.601subject. In some embodiments, the engineered capsid delivering the vector decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell. In some embodiments, the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence. In some embodiments, the engineered capsid delivering the vector increases delivery of the vector to a cell that is in a deeper cell layer in the subject compared to a comparable non-engineered capsid without the polypeptide sequence delivering the vector to a cell in a cell layer. In some embodiments, the cell layer comprises a ganglion cell layer, an inner plexiform layer, an inner nuclear layer, an outer plexiform cell, an outer nuclear layer, an inner segment layer, an outer segment layer, a retinal pigment epithelium layer, or any combination thereof. In some embodiments, the engineered capsid delivering the vector results in a decreased retinal leakage compared to a comparable capsid without the polypeptide sequence delivering a comparable vector encoding a comparable VEGF inhibitor. In some embodiments, the decreased retinal leakage is resulted from one or more administrations. In some embodiments, the decreased retinal leakage is resulted from one administration. In some embodiments, the engineered capsid delivering the vector decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the engineered capsid delivering the vector decreases a dose of the anti-angiogenic agent administered to the subject compared to a second dose of a comparable anti-angiogenic agent administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable anti -angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not increase inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not decrease ERG in the subject compared to if the subject is directly administered with a comparable anti -angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not increase intraocular pressure in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not increase an expressionWSGR Docket No. 59561-717.601of anti-drug antibody (ADA) in the subject compared to a second expression of the ADA in the subject resulted from directly administering the subject with a comparable anti-angiogenic agent. In some embodiments, an expression of the anti-angiogenic agent delivered by the engineered capsid is increased in the subject compared to a second expression of a comparable anti-angiogenic agent resulted from treatment by the comparable anti-angiogenic agent. In some embodiments, the anti -angiogenic agent delivered by the engineered capsid is expressed for a longer duration in the subject compared to a second duration of expressing a comparable anti-angiogenic agent by administering the comparable anti-angiogenic agent to the subject. In some embodiments, the comparable anti-angiogenic agent is Elyea. In some embodiments, the comparable capsid is an modified AAV capsid. In some embodiments, the unmodified AAV capsid is an AAV2 capsid. In some embodiments, the subject is administered a dose comprising the viral particle from about 1.0 x 109vg / eye to about 10 x IO10vg / eye. In some embodiments, the dose is from about 1.0 x IO10vg / eye to about 10 x IO10vg / eye. In some embodiments, the dose is from about 2.0 x 1010vg / eye to about 5 x 1010vg / eye. In some embodiments, the dose is about 2.1 x 1010vg / eye, about 2.2 x 1010vg / eye, about 2.3 x 1010vg / eye, about 2.4 x 1010vg / eye, about 2.5 x 1010vg / eye, about 2.6 x 1010vg / eye, about 2.7 x 1010vg / eye, about 2.8 x 1010vg / eye, or about 2.9 x 1010vg / eye. In some embodiments, the dose is about 2.6 x 1010vg / eye. In some embodiments, the dose is about 3.1 x 1010vg / eye, about 3.2 x 1010vg / eye, about 3.3 x 1010vg / eye, about 3.4 x 1010vg / eye, about 3.5 x 1010vg / eye, about 3.6 x 1010vg / eye, about 3.7 x 1010vg / eye, about 3.8 x 1010vg / eye, or about 3.9 x 1010vg / eye. In some embodiments, the dose is about 3.7 x 1010vg / eye. In some embodiments, the dose is about 8.1 x 1010vg / eye, about 8.2 x 1010vg / eye, about 8.3 x 1010vg / eye, about 8.4 x 1010vg / eye, about 8.5 x 1010vg / eye, about 8.6 x 1010vg / eye, about 8.7 x 1010vg / eye, about 8.8 x 1010vg / eye, or about 8.9 x 1010vg / eye. In some embodiments, the dose is about 8.1 x 1010vg / eye. In some embodiments, a single administrating of the viral particle comprising engineered capsid and the vector is curative of the disease or condition in the subject.

[0009] Described herein, in some aspects, is a method for treating a disease or condition in a subject, comprising: administering an engineered capsid to the subject, the engineered capsid comprising a vector and a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A, and wherein the polypeptide sequence increasesWSGR Docket No. 59561-717.601expression of the vector in a cell, and wherein the expression of the vector treats the disease or condition in the subject. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof. In some embodiments, the cell comprises a macula cell. In some embodiments, the cell comprises a retinal cell. In some embodiments, the cell comprises an ocular cell. In some embodiments, the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or a combination thereof. In some embodiments, the engineered capsid comprises an engineered adeno-associated virus (AAV) capsid. In some embodiments, the engineered AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the engineered AAV capsid comprises a modified AAV2 capsid. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 13).In some embodiments, the polypeptide sequence is the amino acid sequence of LALGQTTKPA (SEQ ID NO: 13). In some embodiments, the polypeptide sequence is inserted in the VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the engineered capsid further comprises a mutation. In some embodiments, the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain. In some embodiments, the mutation is in a GH loop. In some embodiments, the mutation is at a residue at position 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 1. In some embodiments, the vector comprises one or more expression cassettes for expressing one or more transgene. In some embodiments, the vector encodes an anti -angiogenic agent. In some embodiments, the anti -angiogenic agent comprises a VEGF-Trap. In some embodiments, the anti-angiogenic agent consists of the VEGF-Trap. In some embodiments, the anti-angiogenic agent is encoded in the vector comprising one orWSGR Docket No. 59561-717.601more codon modifications. In some embodiments, the one or more codon modifications comprises one or more replacements of non-AGG arginine codon to AGG; non-CCC proline codon to CCC; non-TCC serine codon with TCC; non-CCG proline codon with CCG; or a combination thereof. In some embodiments, the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or any combination thereof to the subject. In some embodiments, the engineered capsid delivers the vector to the cell in the subject. In some embodiments, the engineered capsid delivering the vector decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell. In some embodiments, the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence. In some embodiments, the engineered capsid delivering the vector increases delivery of the vector to a cell that is in a deeper cell layer in the subject compared to a comparable non-engineered capsid without the polypeptide sequence delivering the vector to a cell in a cell layer. In some embodiments, the cell layer comprises a ganglion cell layer, an inner plexiform layer, an inner nuclear layer, an outer plexiform cell, an outer nuclear layer, an inner segment layer, an outer segment layer, a retinal pigment epithelium layer, or any combination thereof. In some embodiments, the engineered capsid delivering the vector results in a decreased retinal leakage compared to a comparable capsid without the polypeptide sequence delivering a comparable vector encoding a comparable VEGF inhibitor. In some embodiments, the decreased retinal leakage is resulted from one or more administrations. In some embodiments, the decreased retinal leakage is resulted from one administration. In some embodiments, the engineered capsid delivering the vector decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the engineered capsid delivering the vector decreases a dose of the anti -angiogenic agent administered to the subject compared to a second dose of a comparable anti -angiogenic agent administered to the subject, wherein the doseWSGR Docket No. 59561-717.601and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not increase inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not decrease ERG in the subject compared to if the subject is directly administered with a comparable anti -angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not increase intraocular pressure in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. In some embodiments, the engineered capsid delivering the vector does not increase an expression of anti-drug antibody (ADA) in the subject compared to a second expression of the ADA in the subject resulted from directly administering the subject with a comparable anti-angiogenic agent. In some embodiments, an expression of the anti-angiogenic agent delivered by the engineered capsid is increased in the subject compared to a second expression of a comparable anti-angiogenic agent resulted from treatment by the comparable anti-angiogenic agent. In some embodiments, the anti -angiogenic agent delivered by the engineered capsid is expressed for a longer duration in the subject compared to a second duration of expressing a comparable anti-angiogenic agent by administering the comparable anti-angiogenic agent to the subject. In some embodiments, the comparable anti-angiogenic agent is Elyea. In some embodiments, the comparable capsid is an unmodified AAV capsid. In some embodiments, the modified AAV capsid is an AAV2 capsid. In some embodiments, the subject is administered a dose of the engineered capsid from about 1.0 x 109vg / eye to about 10 x IO10vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid from about 1.0 x IO10vg / eye to about 10 x IO10vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid from about 2.0 x 1010vg / eye to about 5 x 1010vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 2.1 x 1010vg / eye, at about 2.2 x 1010vg / eye, at about 2.3 x 1010vg / eye, at about 2.4 x 1010vg / eye, at about 2.5 x 1010vg / eye, at about 2.6 x 1010vg / eye, at about 2.7 x 1010vg / eye, at about 2.8 x 1010vg / eye, or at about 2.9 x 1010vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 2.6 x 1010vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 3.1 x 1010vg / eye, at about 3.2 x 1010vg / eye, at about 3.3 x 1010vg / eye, at about 3.4 x 1010vg / eye, at about 3.5 x 1010vg / eye, at about 3.6 x 1010WSGR Docket No. 59561-717.601vg / eye, at about 3.7 x IO10vg / eye, at about 3.8 x IO10vg / eye, or at about 3.9 x IO10vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 3.7 x IO10vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 8.1 x IO10vg / eye, at about 8.2 x IO10vg / eye, at about 8.3 x IO10vg / eye, at about 8.4 x IO10vg / eye, at about 8.5 x IO10vg / eye, at about 8.6 x IO10vg / eye, at about 8.7 x IO10vg / eye, at about 8.8 x IO10vg / eye, or at about 8.9 x IO10vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 8.1 x IO10vg / eye. In some embodiments, a single dose of administrating the engineered capsid comprising the vector is curative of the disease or condition in the subject.

[0010] Described herein, in some aspects, is a method of delivering a vector to a cell, comprising: contacting a cell with an engineered capsid, the engineered capsid comprising a vector and a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-XI -X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A, and wherein the polypeptide sequence increases expression of the vector in the cell. Also described herein, in some aspects, is a method for treating a disease or condition in a subject, comprising: administering an engineered capsid to the subject, the engineered capsid comprising a vector and a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A, and wherein the polypeptide sequence increases expression of the vector in the cell, and wherein the expression of the vector treats the disease or condition in the subject. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber Congenital Amaurosis, macular degeneration, polypoidal choroidal vasculopathy (PCV), retinitis pigmentosa, Refsum disease, Stargardt disease, Usher syndrome, X-linked retinoschisis (XLRS),WSGR Docket No. 59561-717.601rod-cone dystrophy, Cone-rod dystrophy, Oguchi disease, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof. In some embodiments, the cell comprises a macula cell. In some embodiments, the cell comprises a retinal cell. In some embodiments, the cell comprises an ocular cell. In some embodiments, the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or a combination thereof. In some embodiments, the engineered capsid comprises a, engineered adeno-associated virus (AAV) capsid. In some embodiments, the engineered AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the engineered AAV capsid comprises an engineered AAV2 capsid. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 13).In some embodiments, the polypeptide sequence is the amino acid sequence of LALGQTTKPA (SEQ ID NO: 13). In some embodiments, the polypeptide sequence is inserted in the VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the engineered capsid further comprises a mutation. In some embodiments, the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain. In some embodiments, the mutation is in a GH loop. In some embodiments, the mutation is at a residue at position of 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 1. In some embodiments, the vector comprises one or more expression cassettes for expressing one or more transgene. In some embodiments, the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or a combination thereof to the subject. In some embodiments, the engineered capsid decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell. In some embodiments, the engineered capsid decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence. In some embodiments, the cell is a cell in a ganglion cell layer, a cell in an inner plexiform layer, a cell in an inner nuclear layer, a cell in an outer plexiform cell, a cell in an outer nuclear layer, a cell inWSGR Docket No. 59561-717.601an inner segment layer, a cell in an outer segment layer, or a cell in a retinal pigment epithelium layer. In some embodiments, the engineered capsid decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the comparable capsid is an unmodified AAV capsid. In some embodiments, the unmodified AAV capsid is an AAV2 capsid.

[0011] Described herein, in some aspects, is an engineered capsid comprising: a vector and a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A; and wherein the polypeptide sequence increases expression of the vector in the cell.

[0012] Described herein, in some aspects, is a method for treating a disease or condition in a subject, comprising: administering a dose of a viral particle comprising an engineered capsid encapsulating a vector to the subject, wherein the vector comprises a nucleic acid sequence encoding an anti-angiogenic agent, said nucleic acid sequence comprises a modification in a coding region of the nucleic acid sequence as compared to an otherwise comparable nucleic acid sequence lacking the modification in the coding region, said modification comprises replacing at least four non-AGG arginine codons to AGG, wherein the anti-angiogenic agent treats the disease or condition, and wherein the dose is from about 1.0 x 109vg / eye to about 10 x 1010vg / eye. In some embodiments, the nucleic acid sequence that encodes 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 nucleic acid sequence, and wherein the second modification is selected from the group consisting of: replacement of at least one non-CCC proline codon with CCC; replacement of at least one non-TCC serine codon with TCC; replacement of at least one non-CCG proline codon with CCG; and any combination thereof. 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, a NP-1 inhibitor, a NP-2 inhibitor, a Del 1 inhibitor, and an integrin inhibitor. In some embodiments, the anti-angiogenic agent comprises the VEGF inhibitor, and wherein the VEGF inhibitor is a non-antibody inhibitor. InWSGR Docket No. 59561-717.601some embodiments, the non-antibody inhibitor is a fusion protein that comprises human VEGF receptors 1 and 2. In some embodiments, the fusion protein comprises VEGF-Trap or a modified version thereof. In some embodiments, the nucleic acid sequence is modified to replace non-AGG arginine codon with AGG in at least 4 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCC proline codon with CCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-TCC serine codon with TCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCG proline codon with CCG in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid comprises a viral vector sequence. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to any one of the nucleic acid sequences of SEQ ID NOS: 43-49, 51-57, 61, 62, 64, 66, 68, 71, or 72. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or any combination thereof. In some embodiments, the engineered capsid delivers the vector to a cell in the subject. In some embodiments, the cell comprises a macula cell. In some embodiments, the cell comprises a retinal cell. In some embodiments, the cell comprises an ocular cell. In some embodiments, the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or any combination thereof. In some embodiments, the engineered capsid comprises an engineered adeno-associated virus (AAV) capsid. In some embodiments, the engineered AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the engineered AAV capsid comprises an engineered AAV2 capsid. In some embodiments, the polypeptide sequences comprise an aminoWSGR Docket No. 59561-717.601acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is the amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is inserted in a VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the engineered capsid further comprises a mutation. In some embodiments, the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain. In some embodiments, the mutation is in a GH loop. In some embodiments, the mutation is at a residue at position of 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 1. In some embodiments, the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or any combination thereof to the subject. In some embodiments, the engineered capsid delivering the vector decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell. In some embodiments, the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence. In some embodiments, the engineered capsid delivering the vector increases delivery of the vector to a cell that is in a deeper cell layer in the subject compared to a comparable non-engineered capsid without the polypeptide sequence delivering the vector to a cell in a cell layer. In some embodiments, the cell layer comprises a ganglion cell layer, an inner plexiform layer, an inner nuclear layer, an outer plexiform cell, an outer nuclear layer, an inner segment layer, an outer segment layer, a retinal pigment epithelium layer, or any combination thereof. In some embodiments, the engineered capsid delivering the vector results in a decreased retinal leakage compared to a comparable capsid without the polypeptide sequence delivering a comparable vector encoding a comparable VEGF inhibitor. In some embodiments, the decreased retinal leakage is resulted from one or more administrations. In some embodiments, the decreased retinal leakage is resulted from one administration. In some embodiments, the engineered capsid delivering the vector decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result inWSGR Docket No. 59561-717.601comparable therapeutic efficacy in the subject. In some embodiments, the comparable capsid is an unmodified AAV capsid. In some embodiments, the unmodified AAV capsid is an AAV2 capsid. In some embodiments, the anti-angiogenic agent decreases retinal leakage in the subject compared to a second retinal leakage resulted from treatment by a comparable anti-angiogenic agent. In some embodiments, the retinal leakage is resulted from one or more administrations. In some embodiments, the retinal leakage is resulted from one administration. In some embodiments, the anti-angiogenic agent decreases retinal lesion in the subject compared to a second retinal lesion resulted from treatment by a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent decreases a dose of the anti-angiogenic agent administered to the subject compared to a second dose of a comparable anti-angiogenic agent administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the anti-angiogenic agent decreases inflammation of the cell, or an environment associated with the cell compared to contacting the cell with a comparable anti -angiogenic agent. In some embodiments, the anti -angiogenic agent does not increase inflammation of the cell, or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent does not decrease ERG in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent does not increase intraocular pressure in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent does not increase an expression of anti-drug antibody (ADA) in the subject compared to a second expression of the ADA in the subject resulted from directly administering the subject with a comparable anti-angiogenic agent.

[0013] Described herein, in some aspects, is a method for treating a disease or condition in a subject, comprising: administering an engineered capsid to the subject, the engineered capsid comprising a vector, wherein the vector comprises a nucleic acid sequence encoding an anti-angiogenic agent, said nucleic acid sequence comprises a modification in a coding region of the nucleic acid sequence as compared to an otherwise comparable nucleic acid sequence lacking the modification in the coding region, said modification comprises replacing at least four non-AGG arginine codons to AGG, and wherein the anti-angiogenic agent treats the disease or condition. In some embodiments, the nucleic acid sequence that encodes 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 nucleic acid sequence, and wherein the secondWSGR Docket No. 59561-717.601modification is selected from the group consisting of replacement of at least one non-CCC proline codon with CCC; replacement of at least one non-TCC serine codon with TCC; replacement of at least one non-CCG proline codon with CCG; and any combination of (a)-(c). 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, a NP-1 inhibitor, a NP-2 inhibitor, a Del 1 inhibitor, and an integrin inhibitor. In some embodiments, the anti-angiogenic agent comprises the VEGF inhibitor, and wherein the VEGF inhibitor is a non-antibody inhibitor. In some embodiments, the non-antibody inhibitor is a fusion protein that comprises human VEGF receptors 1 and 2. In some embodiments, the fusion protein comprises VEGF-Trap or a modified version thereof. In some embodiments, the nucleic acid sequence is modified to replace non-AGG arginine codon with AGG in at least 4 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCC proline codon with CCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-TCC serine codon with TCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCG proline codon with CCG in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid comprises a viral vector sequence. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to any one of the nucleic acid sequences of SEQ ID NOS: 43-49, 51-57, 61, 62, 64, 66, or 68. In some embodiments, disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or any combination thereof. In some embodiments, the engineered capsid delivers the vector to a cell in the subject. In some embodiments, the cell comprises a macula cell. In some embodiments, the cellWSGR Docket No. 59561-717.601comprises a retinal cell. In some embodiments, the cell comprises an ocular cell. In some embodiments, the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or any combination thereof. In some embodiments, the engineered capsid comprises a modified adeno-associated virus (AAV) capsid. In some embodiments, the modified AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the modified AAV capsid comprises a modified AAV2 capsid. In some embodiments, the polypeptide sequences comprise an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is the amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is inserted in a VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the engineered capsid further comprises a mutation. In some embodiments, the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain. In some embodiments, the mutation is in a GH loop. In some embodiments, the mutation is at a residue at position of 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 1. In some embodiments, the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or any combination thereof to the subject. In some embodiments, the engineered capsid delivering the vector decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell. In some embodiments, the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence. In some embodiments, the engineered capsid delivering the vector increases delivery of the vector to a cell that is in a deeper cell layer in the subject compared to a comparable non-engineered capsid without the polypeptide sequence delivering the vector to a cell in a cell layer. In some embodiments, the cell layer comprises a ganglion cell layer, an inner plexiform layer, an inner nuclear layer, an outer plexiform cell, an outer nuclear layer, an inner segment layer, an outer segment layer, a retinal pigment epithelium layer, or anyWSGR Docket No. 59561-717.601combination thereof. In some embodiments, the engineered capsid delivering the vector results in a decreased retinal leakage compared to a comparable capsid without the polypeptide sequence delivering a comparable vector encoding a comparable VEGF inhibitor. In some embodiments, the decreased retinal leakage is resulted from one or more administrations. In some embodiments, the decreased retinal leakage is resulted from one administration. In some embodiments, the engineered capsid delivering the vector decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the comparable capsid is an unmodified AAV capsid. In some embodiments, the unmodified AAV capsid is an AAV2 capsid. In some embodiments, the anti-angiogenic agent decreases retinal leakage in the subject compared to a second retinal leakage resulted from treatment by a comparable anti-angiogenic agent. In some embodiments, the retinal leakage is resulted from one or more administrations. In some embodiments, the retinal leakage is resulted from one administration. In some embodiments, the anti-angiogenic agent decreases retinal lesion in the subject compared to a second retinal lesion resulted from treatment by a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent decreases a dose of the anti-angiogenic agent administered to the subject compared to a second dose of a comparable anti-angiogenic agent administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the anti-angiogenic agent decreases inflammation of the cell, or an environment associated with the cell compared to contacting the cell with a comparable anti -angiogenic agent. In some embodiments, the anti -angiogenic agent does not increase inflammation of the cell, or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent does not decrease ERG in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent does not increase intraocular pressure in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent does not increase an expression of anti-drug antibody (ADA) in the subject compared to a second expression of the ADA in the subject resulted from directly administering the subject with a comparable anti-angiogenic agent. In some embodiments, an expression of the anti -angiogenic agent is increased in the subject compared to a second expression of a comparable anti -angiogenic agent resulted from treatment by the comparable anti-angiogenicWSGR Docket No. 59561-717.601agent. In some embodiments, the anti-angiogenic agent is expressed for a longer duration in the subject compared to a second duration of expressing a comparable anti -angiogenic agent by administering the comparable anti -angiogenic agent to the subject. In some embodiments, the comparable anti -angiogenic agent is Elyea. In some embodiments, the subject is administered a dose of the engineered capsid from about 1.0 x 109vg / eye to about 10 x IO10vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid from about 1.0 x IO10vg / eye to about 10 x IO10vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid from about 2.0 x 1010vg / eye to about 5 x 1010vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 2.1 x 1010vg / eye, at about 2.2 x 1010vg / eye, at about 2.3 x 1010vg / eye, at about 2.4 x 1010vg / eye, at about 2.5 x 1010vg / eye, at about 2.6 x 1010vg / eye, at about 2.7 x 1010vg / eye, at about 2.8 x 1010vg / eye, or at about 2.9 x 1010vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 2.6 x 1010vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 3.1 x 1010vg / eye, at about 3.2 x 1010vg / eye, at about 3.3 x 1010vg / eye, at about 3.4 x 1010vg / eye, at about 3.5 x 1010vg / eye, at about 3.6 x 1010vg / eye, at about 3.7 x 1010vg / eye, at about 3.8 x 1010vg / eye, or at about 3.9 x 1010vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 3.7 x 1010vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 8.1 x 1010vg / eye, at about 8.2 x 1010vg / eye, at about 8.3 x 1010vg / eye, at about 8.4 x 1010vg / eye, at about 8.5 x 1010vg / eye, at about 8.6 x 1010vg / eye, at about 8.7 x 1010vg / eye, at about 8.8 x 1010vg / eye, or at about 8.9 x 1010vg / eye. In some embodiments, the subject is administered the dose of the engineered capsid at about 8.1 x 1010vg / eye. In some embodiments, a single dose of administrating the engineered capsid comprising the vector is curative of the disease or condition in the subject.

[0014] Described herein, in some aspects, is a method of delivering a vector to a cell, comprising: contacting a cell with an engineered capsid comprising a vector, wherein the vector comprises a nucleic acid sequence encoding an anti -angiogenic agent, said nucleic acid sequence comprises a modification in a coding region of the nucleic acid sequence as compared to an otherwise comparable nucleic acid sequence lacking the modification in the coding region, said modification comprises replacing at least four non-AGG arginine codons to AGG. Also described herein, in some aspects, is a method for treating a disease or condition in a subject, comprising: administering an engineered capsid to the subject, the engineered capsid comprising a vector, wherein the vector comprises a nucleic acid sequence encoding an anti-angiogenicWSGR Docket No. 59561-717.601agent, said nucleic acid sequence comprises a modification in a coding region of the nucleic acid sequence as compared to an otherwise comparable nucleic acid sequence lacking the modification in the coding region, said modification comprises replacing at least four non-AGG arginine codons to AGG, and wherein the anti-angiogenic agent treats the disease or condition. In some embodiments, the nucleic acid sequence that encodes 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 nucleic acid sequence, and wherein the second modification is selected from the group consisting of replacement of at least one non-CCC proline codon with CCC; replacement of at least one non-TCC serine codon with TCC; replacement of at least one non-CCG proline codon with CCG; and any combination thereof. 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, a NP-1 inhibitor, a NP-2 inhibitor, a Del 1 inhibitor, and an integrin inhibitor. In some embodiments, the anti-angiogenic agent comprises the VEGF inhibitor, and wherein the VEGF inhibitor is a non-antibody inhibitor. In some embodiments, the non-antibody inhibitor is a fusion protein that comprises human VEGF receptors 1 and 2. In some embodiments, the fusion protein comprises VEGF-Trap or a modified version thereof. In some embodiments, the nucleic acid sequence is modified to replace non-AGG arginine codon with AGG in at least 4 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCC proline codon with CCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-TCC serine codon with TCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCG proline codon with CCG in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid comprises a viral vector sequence. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to any one of the nucleic acid sequences of SEQ ID NOS: 43-49, 51-57, 61, 62, 64, 66, or 68. In some embodiments, the disease or condition comprises an ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR),WSGR Docket No. 59561-717.601proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof. In some embodiments, the cell comprises a macula cell. In some embodiments, the cell comprises a retinal cell. In some embodiments, the cell comprises an ocular cell. In some embodiments, a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or a combination thereof. In some embodiments, the engineered capsid comprises a modified adeno-associated virus (AAV) capsid. In some embodiments, the modified AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the modified AAV capsid comprises a modified AAV2 capsid. In some embodiments, the polypeptide sequences comprise an amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is the amino acid sequence of LALGQTTKPA. In some embodiments, the polypeptide sequence is inserted in the VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the engineered capsid further comprises a mutation. In some embodiments, the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain. In some embodiments, the mutation is in a GH loop. In some embodiments, the mutation is at a residue at position of 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 1. In some embodiments, the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or a combination thereof to the subject. In some embodiments, the engineered capsid decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell. In some embodiments, the engineered capsid decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence. In someWSGR Docket No. 59561-717.601embodiments, the cell is a cell in a ganglion cell layer, a cell in an inner plexiform layer, a cell in an inner nuclear layer, a cell in an outer plexiform cell, a cell in an outer nuclear layer, a cell in an inner segment layer, a cell in an outer segment layer, or a cell in a retinal pigment epithelium layer. In some embodiments, the engineered capsid decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the comparable capsid is an unmodified AAV capsid. In some embodiments, the unmodified AAV capsid is an AAV2 capsid. In some embodiments, the anti-angiogenic agent decreases retinal lesion in the subject compared to a second retinal lesion resulted from treatment by a comparable anti -angiogenic agent. In some embodiments, the anti -angiogenic agent decreases retinal in the subject to a second retinal lesion resulted from treatment by a comparable anti-angiogenic agent. In some embodiments, the anti-angiogenic agent decreases a dose of the anti-angiogenic agent administered to the subject compared to a second dose of a comparable anti-angiogenic agent administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the anti-angiogenic agent decreases inflammation of the cell, or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent. In some embodiments, the comparable anti-angiogenic agent is Elyea.

[0015] Described herein, in some aspects, is an engineered capsid comprising: a vector and a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A; and wherein the vector comprises a nucleic acid sequence encoding an anti-angiogenic agent, said nucleic acid sequence comprises a modification in a coding region of the nucleic acid sequence as compared to an otherwise comparable nucleic acid sequence lacking the modification in the coding region, said modification comprises replacing at least four non-AGG arginine codons to AGG. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to any one of the nucleic acid sequences of SEQ ID NOS: 43-49, 51-57, 61, 62, 64, 66, or 68.WSGR Docket No. 59561-717.601INCORPORATION BY REFERENCE

[0016] 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. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein).

[0018] Fig. 1 illustrates increased expression in a macula cell, where the macula cell was contacted a vector encoding a transgene delivered by an engineered capsid described herein. Reporter construct was delivered to the eye by: R100 (an adeno-associated virus or AAV capsid with a ten amino acid insertion), 7m8 (AAV.7m8 capsid), and the engineered capsid described herein. The delivery of the reporter construct by the engineered capsid described here yielded the highest expression in the targeted macula cell at a comparable dose or at a lower dose compared to R100 and 7m8.

[0019] Fig. 2A illustrates increased delivery efficiency of the engineered capsid described herein compared to an AAV2 capsid.

[0020] Fig. 2B illustrates increased transduction of the transgene delivered by the engineered capsid described herein compared to AAV2 and 7m8.

[0021] Fig. 2C illustrates increased penetration and expression of the transgene delivered by the engineered capsid described herein into multiple cell layers of monkey retina compared to delivery of the transgene by AAV2 and 7m8.

[0022] Fig. 3A illustrates a non-limiting example of an engineered capsid described herein comprising a vector encoding a VEGF-Trap.

[0023] Fig. 3B illustrates a mechanism of action for delivering the VEGF-Trap into a cell to decrease expression of VEGF in the cell. Such decrease can lead to decrease of angiogenesis.

[0024] Fig. 4A illustrates a smaller retinal lesion in the mouse eyes treated with the engineered capsid and VEGF-Trap described herein compared to Eylea.WSGR Docket No. 59561-717.601

[0025] Fig. 4B illustrates decreased retinal leakage in the eyes treated by with the engineered capsid and VEGF-Trap described herein compared to Eylea.

[0026] Fig. 5 illustrates a non-limiting example of a clinical trial, where the engineered capsid and the VEGF-Trap described herein can be administered at various dose.

[0027] Fig. 6 illustrates a comparison of clinical stage therapies illustrating the increased therapeutic efficacy of the engineered capsid and the vector described herein.

[0028] Fig. 7A illustrates an example of best corrected visual acuity (BCVA) testing worksheet for the right eye.

[0029] Fig. 7B illustrates an example of best corrected visual acuity (BCVA) testing worksheet for the left eye.

[0030] Fig. 8A illustrates a schematic representation of Vector AAT. The vector genome elements are listed: A: ITR, B: CMV enhancer, C: CMV promotor, D: UTR, E: Intron, F: Kozak sequence, G: VEGF-Rl / Fltl leader sequence, H: VEGF-Trap coding sequence, I; synthetic poly A sequence, and J : truncated ITR.

[0031] Fig. 8B illustrates a vector map of Vector AAT.

[0032] Fig. 9 illustrates representative fundus images, cobalt blue (fluorescein-leakage), from each group on Day 7 post-laser are shown. Measurement of lesions in the AAV2-GFP dosed group was not feasible as the GFP expressed likely drove inflammation such that lesions were so large and precluded analysis. Graphical representation of the area of fluorescein leakage after the laser bums and quantitative analysis of the other groups are shown in the lower panel.AAV2.N54-Aflibercept is later named AAV2.N54-VEGF-Trap (Vector AAT). Mean±SD.

[0033] Fig. 10 illustrates representative lesion images from flat mounts on Day 7 post-laser. Quantitative analysis is also shown in the lower panel. Mean±SD. Protection against laser CNV in the mouse (C57BL / 6) was shown from increasing single IVT doses of Vector AAT. Note that some data points had to be excluded due to technical issues so the numbers of lesions scored are listed out of the 16 possible for each group: Vehicle 12 / 16; Elyea 11 / 16; Vector AAT 2 x 107vg 12 / 16; Vector AAT 4 x 108vg 15 / 16; Vector AAT 1.6 x 1010vg 5 / 16.

[0034] Fig. 11 illustrates images of retinal leakage in rabbit DL-a-AAA challenge retinal neovascularization Model.

[0035] Fig. 12 illustrates change of retinal leakage over time in corrected total regional fluorescence over time. Mean±SEM.

[0036] Fig. 13 illustrates aflibercept expression in the eye at different timepoints. ** p<0.01.WSGR Docket No. 59561-717.601

[0037] Fig. 14 illustrates aflibercept concentration in VH, Retina, RPE / CHR, and serum, ns, not significant; * p<0.05; **** pO.OOOl.

[0038] Fig. 15 illustrates Vector AAT DNA Level in retina, RPE / CHR, or VH. The total DNA levels in AH and VH samples were too low, so the data was normalized with 100 mg tissue, ns, not significant, * p < 0.05.

[0039] Fig. 16 illustrates RNA Level in retina, RPE / CHR, or VH. The total RNA levels in AH and VH samples were too low, so the data was normalized with 100 mg tissue, ns, not significant.

[0040] Fig. 17 illustrates ocular examination scores and intraocular pressures in rabbit efficacy study.

[0041] Fig. 18 illustrates ERG data from GLP toxicity study (representative data as ERG oscillatory potentials. B Amplitude data: male (top panel) and female (bottom panel).

[0042] Fig. 19 illustrates ADA results in GLP NHP toxicity study in serum. Serum samples for each animal were collected multiple times during the study that is 2-, 4-, 8-, 13-, and 26-weeks. The Vector AAT ADA values were plotted as the concentration in ng / mL units. Error bars are mean with standard deviation. Few animals in group 3 showed low ADA concentration; therefore, the standard deviation was high. Control animals mostly were below the cut point, and low-dose animals started showing ADA higher than the cut-point but still were at the lower level but high-dose animals showed a very high concentration of ADA. Two-way ANOVA was used to perform the statistics for the screening assay. The data reported here had QC samples which met the acceptance criteria and r2 value of >0.975.

[0043] Fig. 20 illustrates ADA results in GLP NHP toxicity study in vitreous humor.

[0044] Fig. 21 illustrates IFN-y response to Vector AAT capsid peptides. The data for the IFN-y had signals higher than LoQ when treated with peptides. The statistical analysis was performed after subtracting the baseline. IFN-y level was below baseline for the control group and low dose group at 6 months’ time point but it was positive for low dose at 3 months’ time point and high dose at both 3 months and 6 months’ time point, showing that Vector AAT-encoded peptide elicited cellular immune response albeit at a very low level of 2-3 pg / mL with 2.0E+05 PBMCs / well. Figure shows the IFN-y production with two-way ANOVA statistical analysis with data represented as mean±SEM.

[0045] Fig.22A and Fig. 22B illustrate improved penetration of AAV2.N54 capsid across the ILM after IVT injection in mice. A total of 18 engineered capsids together with AAV2 and AAV2.7m8, encoding GFP, were comparatively evaluated in mice (n=4 in each group). Fig. 22AWSGR Docket No. 59561-717.601illustrates fundus images of the mouse (C57BL / 6) retina of example animals from key groups: AAV2.N54, AAV2, AAV2.7m8. Day 23 Fundus images of the posterior section of the eye in color (left-hand panel) and cobalt blue (GFP-right-hand panel). Imaging acquisition settings for GFP expression were calibrated on AAV2.7m8 animals, and levels were kept consistent across the study. In AAV2, GFP expression was inconsistent across animals; the image shown represents the eye with the highest expression level. Fig. 22B illustrates representative images from the IHC analysis of Day 28 eyes. Two different antibody cocktails, (anti -Rhodopsin & anti-Laminin: left-hand panels; anti-RPE65 & PNA Lectin: right-hand panels) were utilized to evaluate the localization of GFP expression with DAPI-staining to highlight the inner (INL) and outer nuclear layers (ONL). Merged color images for each cocktail are shown on the left and grayscale pullouts of the GFP channel are shown on the right.

[0046] Figs. 23A-23C illustrate improved penetration of AAV2.N54 capsid across the ILM after IVT injection in pigs. A total of 5 engineered capsids (AAV2.N53, AAV2.N54, AAV2.N101, AAV2.N104, AAV2.N105) and AAV2.7m8 were comparatively evaluated in pigs (weanling farm pigs -Hampshire cross, n=2 each group). Fig. 23A illustrates fundus images of the pig retina of example animals from key groups: AAV2.N54 and AAV2.7m8. Day 21 fundus images of the posterior section of the eye - imaged in both color (left-hand panel) and cobalt blue (GFP) channels (right-hand panel). Imaging acquisition settings for GFP expression were calibrated on AAV2.7m8 animals and levels were kept consistent across the study. Fig. 23B illustrates representative images from the IHC analysis of Day 28 eyes; cryosections (14 pm) were evaluated for the localization of GFP (green), RPE65 (red), phalloidin (magenta), and DAPI (blue), the latter to highlight the INL and ONL; merged images are shown on the left-hand panel and pullouts of the GFP channel (green only) are shown on the right-hand panel. Fig. 23C illustrates confocal microscopic image of the pig (no. 26) retina IHC results. Each layer from vitreous humor to choroid is the ganglion cell layer (GCL), inner plexiform layer (IPL), INL, ONL, inner segment layer (ISL), outer segment layer (OSL), retina pigment epithelium (RPE).

[0047] Figs. 24A-24C illustrate safety parameters in the pig capsid screening study. Fig. 24A illustrates change in IOP throughout the study for eyes in all treatment groups. Mean±SD. Fig.24B illustrates body weight remained stable for all animals over the course of the study. Data are presented as the individual weight for each animal at each time point. Mean±SD. Fig. 24C illustrates retinal thicknesses and volumes for each treatment group, as measured by OCT within the EDTRS grid. Mean±SD.WSGR Docket No. 59561-717.601

[0048] Fig. 25A and Fig. 25B illustrate representative fundus and cSLO images of retinas demonstrated improved tropism of AAV2.N54 in NHPs. Two engineered capsids (AAV2.N54, AAV6.N54) and controls (AAV2, AAV6, AAV2.7m8, vehicle) were comparatively evaluated.Fig. 25A illustrates representative optic nerve head color (left panel) and fluorescence (right panel) raw images obtained on Day 35 for eyes treated with vehicle (n=l), AAV2-GFP (n=2), AAV2.N54-GFP (n=2), AAV6-GFP (n=2), AAV6.N54-GFP (n=2), and AAV2.7m8-GFP (n=l).Fig. 25B illustrates representative infrared (left panel) and autofluorescence (right panel) cSLO images were obtained on Day 42 for each group. Two engineered capsids (AAV2.N54, AAV6.N54) and controls (AAV2, AAV6, AAV2.7m8, vehicle) were comparatively evaluated.

[0049] Fig. 25C illustrates regional fluorescence, quantified by GFP scoring of cSLO images as shown in Fig. 25A and Fig. 25B for perifoveal, peripheral, and perivascular regions. Mean±SD.

[0050] Figs. 26A-26D illustrate ocular inflammation assessment in the NHP capsid screening study. Ocular inflammation ranged from absent to moderate as revealed by total clinical score (Fig. 26A), vitreous cell (Fig. 26B), aqueous cell (Fig. 26C), and keratic precipitates (Fig. 26D), over the course of the study. Mean±SD.

[0051] Figs. 27A-27D illustrate GFP transgene expression in various tissues in NHPs: GFP levels in serum (Fig. 27A); retina and RPE / choroid (Fig. 27B); AH and VH (Fig. 27C); or optic nerve, LGN, and visual cortex (Fig. 27D).

[0052] Fig. 28 illustrates IHC analysis of retinas in the NHP capsid screening study. Slides were screened for GFP expression and sections with the brightest signal at the levels of optic nerve head (ONH), fovea and in the periphery were selected for imaging. One section from each level / eye was imaged. A 4x tiled overview and a 20x image at the site of highest GFP expression was acquired for each section using an Olympus Bx63 upright fluorescent microscope with 4x and 20x plan apochromatic objectives. Images were post-processed using the Cell Sens (Olympus) deconvolution software to bring out the structures of the retinal layers and reduce autofluorescence.

[0053] Figs. 29A-29B illustrate fluorescein angiography and flatmount analysis of retinas demonstrated the efficacy of AAV2.N54-Aflibercept in the mouse LCNV model. AAV2.N54-Aflibercept (2 x 107, 4 x 108, 1.6 x 1010vg / eye), AAV2-GFP (1.6 x 1010vg / eye), and Eylea (40 pg) were comparatively evaluated in the LCNV mouse model (n=8 each group). Fig. 29A.Representative fundus images, cobalt blue (fluorescein-leakage), from each group on Day 7 postlaser are shown. Measurement of lesions in the AAV2-GFP group was not feasible as the GFP expression likely drove inflammation such that lesions were so large and precluded analysis.WSGR Docket No. 59561-717.601Graphical representation of the area of fluorescein leakage after the laser burns and quantitative analysis of the other groups are shown in the lower panel. Mean±SD. Fig. 29B. Representative lesion images from flatmounts on Day 7 post-laser. Quantitative analysis is also shown in the lower panel. Mean±SD. Protection against laser CNV in the mouse (C57BL / 6) is shown from increasing single IVT doses of AAV2.N54-Aflibercept. Note that some data points had to be excluded due to technical issues so the numbers of lesions scored are listed out of the 16 possible for each group.

[0054] Figs. 30A-30C illustrate AAV2.N54-Aflibercept achieved significant efficacy in the DL-AAA rabbit model. Model induction was performed on Day 1 through IVT injection of DL-AAA, while AAV2.N54-Aflibercept injection was performed on Day 22 (n=6 in each group). Fig. 30A. Representative images from Day 106 IHC analysis. Fig. 30B. Images indicated retina leakage from treated and control animals. Fig. 30C. Change in corrected total regional fluorescence over time. Mean±SD.

[0055] Fig. 31A and Fig. 31B illustrate ocular examination (OE) scores and lOPs in the rabbit efficacy study. Fig. 31A illustrates change in ocular examination scores over time. At baseline and on Days 15, 23, 29, 37, 43, 50, 64, 78, 92, and 106, pupils were dilated for OEs using topical 1% tropicamide HC1. OEs were performed using a slit lamp biomicroscope and indirect ophthalmoscope to evaluate ocular surface morphology and anterior and posterior segment inflammation. The modified Hackett and McDonald ocular grading system with additional parameters for the posterior segment was used to score inflammation. Animals were not tranquilized for examinations. Fig. 31B illustrates change in intraocular pressure (IOP) over time. At baseline and on Days 15, 23, 29, 37, 43, 50, 64, 78, 92, and 106, IOP was measured in both eyes of all animals with a Tonovet probe (iCare Tonometer, Espoo, Finland). The IOP measurements were performed with the animal manually restrained and maintained in an upright position. The tip of the probe was directed to gently contact the central cornea. Six consecutive measurements were obtained and the average IOP shown on the display was recorded. Three independent measurements were obtained and recorded for each eye.

[0056] Fig. 32A and Fig. 32B illustrate vector shedding and IOP data from the GLP toxicity study in NHPs. Fig. 32A. IOP was measured at designated timepoints. Mean±SD. Fig. 32B. Vector copy number was measured by QPCR. Blood and feces were reported as copies per pg. Urine, saliva, and tears were reported as copies per mL urine, per saliva swab, and per tear strip, respectively. Saliva was reported as copies per swab. Tears were reported as copies per strip.WSGR Docket No. 59561-717.601

[0057] Fig. 33 Illustrates graphical representation of GFP expression from AAV2.N54 demonstrated improved tropism across species of mouse, pig, and non-human primate (NHP). GFP expression was scored across all retinal and extra-retinal layers and across both antibody cocktails, for a survey of ocular GFP expression across all eyes enrolled in cryosection analysis. Dark green cells (+++) indicate where GFP expression was the highest; Medium green cells (++) indicate where there was moderate GFP expression; Light green cells (+) indicate where there was weak GFP expression; Red cells (-) indicate where GFP was absent. Abbreviations:GCL / ILM - ganglion cell layer / Inner limiting membrane; IPL - inner plexiform layer; INL -inner nuclear layer; OPL - outer plexiform layer; ONL - outer nuclear layer; IS - inner segment; OS - outer segment; RPE - retinal pigmented epithelium; C - choroid; S - sclera; M - muscle; ONH - optic nerve head.

[0058] Fig. 34 illustrates post-translational modification (PTM) position detected in AAV2.N54 engineered capsid. Single capital letter on the top of amino acid (aa) sequence indicates the type of PTM. The highlighted regions were confirmed aa sequences by LC-MS / MS. The detected PTMs were A-acetylation (A); C-carbamidomethyl (C); D-deamidation (Q, N); G-N (6) glycylglycyl (GG) (K); M-methylation (R, K,Y); O-oxidation (M); P-phosphorylation (S,T,Y); Q-propionamide (K); the red sharp triangles are pointing to the glycosylation sites (K, S).

[0059] Fig. 35 illustrates that K595 acetylation increased AAV macular tropism to retinal ganglion cell.

[0060] Fig. 36 illustrates increased penetration of delivery of a reporter construct (GFP) by the AAV2.N54 engineered capsid into a deeper macular cell layer compared to a comparable wild type AAV capsid or other comparable modified AAV capsid.

[0061] Fig. 37 shows example of scAAV constructs carrying the CMV enhancer / promoter, SV40 intron (INT), Kozak sequence (K), original (Af) or human IgGl heavy chain (Vh) signal peptide (Sig), coding sequence, and synthetic polyadenylation signal (pA) flanked by a full AAV2 ITR (F-ITR) and a truncated AAV2 ITR (d-ITR). Vector TKR, Vector TMM, Vector TMP, Vector TMQ, Vector AAR, Vector ACT, and Vector AET show different VEGF-Trap coding sequences with different signal peptides. Vector TKR has an Af signal peptide and a low GC content coding sequence. Vector TMM has a Vh signal peptide and a low GC content coding sequence. Vector TMP has an Af signal peptide and a high GC content coding sequence. Vector TMQ has a Vh signal peptide and a high GC content coding sequence. Vector AAR has an Af signal peptide and a high GC content coding sequence containing 16 Arg codon changes from AGA to AGG and 29 Pro codon changes from CCC to CCT. Vector ACT has an Af signal peptide and a high GCWSGR Docket No. 59561-717.601content coding sequence containing 16 Arg codon changes from AGA to AGG and 36 Ser codon changes from AGC to TCC. Vector AET has an Af signal peptide and a high GC content coding sequence containing 16 Arg codon changes from AGA to AGG and 29 Pro codon changes from CCC to CCG.

[0062] Fig. 38 shows amino acid sequence (SEQ ID NO: 63) and nucleic sequence (SEQ ID NO: 62) of non-limiting example of Vector TMQ-pFB-scCMV-SV40-intron-kozak-Vh-VEGF-Trap-GC. The bold letters indicate the human antibody heavy chain signal peptide.

[0063] Fig. 39 shows amino acid sequence (SEQ ID NO: 65) and nucleic acid sequence (SEQ ID NO: 64) of non-limiting example of Vector AAR-pFB-scCMV-SV40-intron-kozak-Af-VEGF-Trap-GCRP (CCT). The bold letters indicate the VEGF-Trap signal peptide, underlines indicate the 16 AGA to AGG changes and italics indicates the 30 CCC to CCT changes.

[0064] Fig. 40 shows amino acid sequence (SEQ ID NO: 67) and nucleic acid sequence (SEQ ID NO: 66) of non-limiting example of Vector ACT-pFB-scCMV-SV40-intron-kozak-Af-VEGF-Trap-GCRS (TCC). The bold letters indicate the VEGF-Trap signal peptide, the underlines indicate the 16 AGA to AGG changes and italics indicate the 36 AGC to TCC changes.

[0065] Fig. 41 shows amino acid sequence (SEQ ID NO: 69) and nucleic acid sequence (SEQ ID NO: 68) of non-limiting example of Vector AET-pFB-scCMV-SV40-intron-kozak-Af-VEGF-Trap-GCRP (CCG). The bold letters indicate the VEGF-Trap signal peptide, the underlines indicate the 16 AGA to AGG changes and italics indicates the 29 CCC to CCG changes.

[0066] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments.DETAILED DESCRIPTION

[0067] Described herein are engineered capsids and methods for using such engineered capsids. In some embodiments, the engineered capsid comprises a vector. In some embodiments, the vector encodes one or more transgene. In some embodiments, the vector is encapsulated by an engineered capsid described herein. In some embodiments, the engineered capsid and the vector are part of a viral particle. In some embodiments, the viral particle comprising the engineered capsid encapsulating the vector is administered to a subject to treat a disease or condition. In some embodiments, the viral particle, the engineered capsid, or the vector can be administered toWSGR Docket No. 59561-717.601a subject for treating a disease or condition in the subject. In some embodiments, a dose of a viral particle comprising an engineered capsid encapsulating a vector is administered to the subject. In some embodiments, the vector comprises a nucleic acid sequence encoding an anti-angiogenic agent. In some embodiments, the nucleic acid sequence comprises a modification in a coding region of the nucleic acid sequence as compared to an otherwise comparable nucleic acid sequence lacking the modification in the coding region. For example, the modification can include replacing at least four non-AGG arginine codons to AGG.

[0068] In some embodiments, the engineered capsid comprises a polypeptide sequence. In some embodiments, the polypeptide sequence is inserted into an amino acid sequence of any one of SEQ ID NOs: 1-6 (Table 1). In some embodiments, the polypeptide sequence is inserted in the VP domain of the AAV capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the engineered capsid further comprises a mutation. In some embodiments, the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain. In some embodiments, the mutation is in a GH loop. In some embodiments, the mutation is at a residue at position of 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 1.

[0069] In some embodiments, the polypeptide sequence comprises an amino acid sequence of any one of the amino acid sequence in Table 2. In some embodiments, the polypeptide sequence comprises an amino acid sequence that is identical to at least 5, 6, 7, or 8 contiguous amino acids of any one of the amino acid sequence in Table 2. In some embodiments, the polypeptide sequence comprises an amino acid sequence of: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4. In some embodiments, XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H). In some embodiments, X3 is E, S, or Q. In some embodiments, X4 is K, R, E, or A. In some embodiments, the engineered capsid comprises an engineered adeno-associated virus (AAV) capsid. In some embodiments, the engineered AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the engineered AAV capsid comprises an engineered AAV2 capsid. In some embodiments, the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 13). In some embodiments, the polypeptide sequence is the amino acid sequence of LALGQTTKPA (SEQ ID NO: 13). In some embodiments, the AAV capsid is part of a viral particle.WSGR Docket No. 59561-717.601

[0070] In some embodiments, the engineered capsid comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence of any one of the amino acid sequence in Table 3. In some embodiments, the engineered capsid comprises an amino acid sequence that is any one of the amino acid sequence in Table 3. In some embodiments, the engineered capsid comprises a vector comprising one or more expression cassettes. In some embodiments, the one or more expression cassettes encode one or more transgenes (e.g., one or more therapeutics). In some embodiments, the one or more transgenes can treat a disease or condition in a subject. In some embodiments, the engineered capsid comprising the polypeptide sequence increases the expression of the vector in a targeted cell. Fig. 3A illustrates a non-limiting example of an engineered capsid described herein comprising a vector encoding a VEGF-Trap. Table 4 illustrates non-limiting examples of the VEGF-Trap sequence or vector sequences encoding the VEGF-Trap. Fig. 3B illustrates a mechanism of action for delivering the VEGF-Trap into a cell to decrease expression of VEGF in the cell. Such decrease can lead to decrease of angiogenesis.

[0071] In some embodiments, described herein is a vector encoding the anti-angiogenesis agent, where the expression of the anti-angiogenesis agent in a cell treats a disease or condition associated with the cell. In some embodiments, the vector also encodes an engineered capsid described herein. 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, a NP-1 inhibitor, a NP-2 inhibitor, a Del 1 inhibitor, and an integrin inhibitor. In some embodiments, the anti -angiogenic agent comprises the VEGF inhibitor, and wherein the VEGF inhibitor is a nonantibody inhibitor. In some embodiments, the non-antibody inhibitor is a fusion protein that comprises human VEGF receptors 1 and 2. In some embodiments, the fusion protein comprises VEGF-Trap or a modified version thereof. Fig. 37 illustrates non-limiting examples of vector constructs for encoding VEGF-Trap (aflibercept). Figs. 38-41 illustrate non-limiting examples of nucleic acid sequence comprising modified codons for encoding VEGF-Trap (aflibercept).

[0072] In some embodiments, the vector comprises a nucleic acid sequence for expressing the anti -angiogenic agent. In some embodiments, the nucleic acid sequence is modified to replace non-AGG arginine codon with AGG in at least 4 codon positions as compared to SEQ ID NO:70. In some embodiments, the nucleic acid sequence is modified to replace non-CCC proline codon with CCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-TCC serine codon with TCCWSGR Docket No. 59561-717.601in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCG proline codon with CCG in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid comprises a viral vector sequence. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to any one of the nucleic acid sequences of SEQ ID NOS: 43-49, 51-57, 61, 62, 64, 66, 68, 71, or 72. In some embodiments, the isolated, nucleic acid sequence comprises the nucleic acid sequence identity is from about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100% to SEQ ID NO: 61. In some embodiments, the isolated, nucleic acid sequence comprises a nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the isolated, nucleic acid sequence is 100% identical to a nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the isolated, nucleic acid sequence comprises the nucleic acid sequence identity is from about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100% to SEQ ID NO: 66. In some embodiments, the isolated, nucleic acid sequence comprises a nucleic acid sequence of SEQ ID NO: 66 In some embodiments, the isolated, nucleic acid sequence is 100% identical to a nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to the nucleic acid sequence of SEQ ID NO: 71 or 72. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is SEQ ID NO: 71 or 72.

[0073] In some embodiments, the vector is encapsulated by an engineered capsid described herein. In some embodiments, the vector is encapsulated by a viral particle described herein. In some embodiments, the vector is encapsulated by a viral particle comprising the engineered capsid described herein.

[0074] Described herein, in some aspects, is a method for of delivering a vector to a cell. In some embodiments, the method comprises contacting a cell with an engineered capsid described herein. In some embodiments, the engineered capsid comprises a vector and a polypeptide sequence described herein. In some embodiments, described herein is a for treating a disease or condition in a subject. In some embodiments, the method comprises administering an engineered capsid described herein to the subject. In some embodiments, the polypeptide sequence increases expression of the vector in the cell. In some embodiments, the expression of the vector treats the disease or condition in the subject. In some embodiments, the disease or condition comprises anWSGR Docket No. 59561-717.601ocular disease. In some embodiments, the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

[0075] In some embodiments, the engineered capsid delivers the vector to a cell. In some embodiments, the cell comprises a macula cell. In some embodiments, the cell comprises a retinal cell. In some embodiments, the cell comprises an ocular cell. In some embodiments, the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or a combination thereof. In some embodiments, the engineered capsid can be delivered or administered intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or a combination thereof to the subject.

[0076] In some embodiments, the engineered capsid decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell. In some embodiments, the engineered capsid decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence. In some embodiments, the cell is a cell in a ganglion cell layer, a cell in an inner plexiform layer, a cell in an inner nuclear layer, a cell in an outer plexiform cell, a cell in an outer nuclear layer, a cell in an inner segment layer, a cell in an outer segment layer, or a cell in a retinal pigment epithelium layer. In some embodiments, the engineered capsid decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result inWSGR Docket No. 59561-717.601comparable therapeutic efficacy in the subject. In some embodiments, the comparable capsid is an unmodified AAV capsid. In some embodiments, the unmodified AAV capsid is an AAV2 capsid. In some embodiments, the AAV capsid is part of a viral particle.

[0077] Post-translational modifications (PTMs) in viral capsid proteins are chemical or protein modifications introduced by the host cell’s enzymatic machinery after translation. PTMs regulate the viral vector’s structural stability, functionality, and interactions of the capsid with host cells. PTMs refer to covalent modifications that occur on specific amino acid residues following protein translation. In certain embodiments, the PTMs include, but are not limited to, phosphorylation, ubiquitination, glycosylation, SUMOylation, acetylation, or any combination thereof (see Mary B. et al., Post-translational modifications in capsid proteins of recombinant adeno-associated virus (AAV)l-rhlO serotypes, FEBS J., 2019 Aug 1 ;286(24):4964- -4981. doi: 10.111 l / febs.15013). Such PTMs may influence viral tropism, immunogenicity, and therapeutic efficacy.

[0078] Accordingly, assessment of viral capsid PTMs is performed. Detection and characterization of PTMs can be conducted using methods such as liquid chromatography (LC), mass spectrometry (MS), or other methods known in the art. Non-limiting examples of PTMs are provided in Table 18. In some embodiments, the PTM comprises acetylation, as described in Table 19. In some embodiments, the PTM comprises deamidation, as described in Table 20. In some embodiments, the PTM comprises methylation, as described in Table 21. In some embodiments, the PTM comprises oxidation, as described in Table 22. In some embodiments, the PTM comprises O-glycosylation, as described in Table 23. In some embodiments, the PTM comprises phosphorylation, as described in Table 24. In some embodiments, the PTM comprises propionylation, as described in Table 25.Method

[0079] Provided herein are also methods of treating a disease or condition. A method of treatment can comprise introducing to a subject in need a virion (e.g., comprising an engineered capsid described herein) or vector coding for a biologic provided herein. In some cases, a method of treatment comprises introducing a plurality of virions or vectors that code for the biologic that comprises an anti-angiogenic agent. Also provided is a method of treating the disease or condition that comprises administering a pharmaceutical composition to a subject in need thereof. A pharmaceutical composition can comprise a nucleic sequence that encodes a biologic such as an anti-angiogenic agent or virions that code for the anti-angiogenic agent.WSGR Docket No. 59561-717.601

[0080] In an embodiment, a method of treatment comprises administering a therapeutically effective amount of a pharmaceutical composition that comprises a nucleic acid encoding an anti-angiogenic agent. A sequence can be or can comprise any of the nucleic acids provided herein. For example, the sequence can comprise a nucleic acid sequence encoding an anti-angiogenesis agent described herein. In some embodiments, the sequence is single stranded. In some embodiments, the sequence is double stranded. In some embodiments, the sequence can comprise a nucleic acid sequence that is at least about 60% sequence identity or similarity with any one SEQ ID NOS: 43-49, 51-57, 61, 62, 64, 66, 68, 71, or 72. 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%, and up to about 100%. In some embodiments, the sequence comprises the sequence identity is from about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100% to SEQ ID NO: 61. In some embodiments, the sequence comprises a nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the sequence is 100% identical to a nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the sequence comprises the sequence identity is from about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, and up to about 100% to SEQ ID NO: 66. In some embodiments, the iso sequence comprises a nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the sequence is 100% identical to a nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to the nucleic acid sequence of SEQ ID NOs: 71 or 72. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is the nucleic acid sequence of SEQ ID NOs: 71 or 72. In some embodiments, the sequence is single stranded. In some embodiments, the sequence is double stranded.

[0081] In some cases, a sequence is modified according to the disclosure, for example the sequence is modified to replace AGA with AGG in at least one codon of a coding region of the sequence as compared to an otherwise comparable sequence lacking the modification in the coding region. As provided herein, the modifications of the disclosure can have certain benefits such as increasing a level of the biologic in a subject as compared to an otherwise comparable subject administered an otherwise comparable isolated non-naturally occurring nucleic acid lacking a modification. Increasing levels of biologies in subjects can have therapeutic effects and can reduce or eliminate any of the diseases or conditions provided herein.WSGR Docket No. 59561-717.601

[0082] In some cases, an increased level of a biologic in a subject is at least a 5-fold, a 10-fold, a 20-fold, a 50-fold, a 100-fold, a 200-fold, or a 500-fold increased, as determined by a diagnostic assay. Suitable diagnostic assays can include ocular diagnostic assays. Ocular diagnostic assays can include ophthalmic testing such as refraction testing, ocular scans, Ocular coherence tomography, Farnworth-Munsell 100 Hue Test, Computerized Optic Disc Imaging and Nerve Fiber Layer Analysis (GDX, HRT, OCT), Corneal Topography, Electroretinography (ERG), electro-oculography (EOG), visual evoked potentials (VEP), visual evoked response (VER), Fluorescein Angiography, Ocular Coherence Tomography (OCT), retinal photography, fundus photography, Specular Microscopy, Goldmann, Humphrey, FDT, Octopus, Biometry / IOL calculation, A-Scan, B-Scan, and combinations thereof.

[0083] In some cases, a retinal test can be utilized. Nonlimiting methods for assessing retinal function and changes thereof include assessing visual acuity (e.g. best-corrected visual acuity [BCVA], ambulation, navigation, object detection and discrimination), assessing visual field (e.g. static and kinetic visual field perimetry), performing a clinical examination (e.g. slit lamp examination of the anterior and posterior segments of the eye), assessing electrophysiological responsiveness to all wavelengths of light and dark (e.g. all forms of electroretinography (ERG) [full-field, multifocal and pattern], all forms of visual evoked potential (VEP), electrooculography (EOG), color vision, dark adaptation and / or contrast sensitivity). Nonlimiting methods for assessing anatomy and retinal health and changes thereof include Optical Coherence Tomography (OCT), fundus photography, adaptive optics scanning laser ophthalmoscopy (AO-SLO), fluorescence and / or autofluorescence; measuring ocular motility and eye movements (e.g. nystagmus, fixation preference, and stability), measuring reported outcomes (patient-reported changes in visual and non-visually-guided behaviors and activities, patient-reported outcomes [PRO], questionnaire-based assessments of quality-of-life, daily activities and measures of neurological function (e.g. functional Magnetic Resonance Imaging (MRI)).

[0084] Relevant ocular diseases and conditions can include but are not limited to: blindness, Achromatopsia, Age-related macular degeneration (AMD), Diabetic retinopathy (DR), Glaucoma, Bardet-Biedl Syndrome, Best Disease, Choroideremia, Leber 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, Malattia Leventinese (Familial Dominant Drusen), and Blue-cone monochromacy. In an embodiment, the ocular disease or condition is AMD. AMD can be wet AMD or dry AMD.WSGR Docket No. 59561-717.601

[0085] In some cases, an administration of a pharmaceutical is sufficient to reduce at least a symptom of a disease or condition, treat the disease or condition, and / or eliminate the disease or condition. In some cases, improvements of diseases or conditions can be ascertained by any of the provided diagnostic assays. In other cases, an improvement can be obtained via an interview with the treated subject. For example, a subject may be able to communicate to an attending physician that their vision is improved as compared to their vision prior to administration of a subject pharmaceutical. In other cases, an in vivo animal model may be used to ascertain reduction of a disease or condition after treatment. Suitable animal models include mouse models, primate models, rat models, canine models, and the like.

[0086] Pharmaceutical compositions can be administered to a subject using various techniques, such as: intravitreally, intramuscular, intravenous, subcutaneous, and / or intraperitoneal injection.

[0087] For in vivo delivery, subject nucleic acids and / or AAV virions can be formulated into pharmaceutical compositions and can generally be administered intravitreally or parenterally (e.g., administered via an intramuscular, subcutaneous, intratumoral, transdermal, intrathecal, etc., route of administration). In some aspects, a pharmaceutical composition can be used to treat a subject such as a human or mammal, in need thereof. In some cases, a subject can be diagnosed with a disease, e.g., ocular disease. In some aspects, subject pharmaceutical compositions are coadministered with secondary therapies. A secondary therapy can comprise any therapy for ocular use. In some cases, a secondary therapy comprises nutritional therapy, vitamins, laser treatment, such as laser photocoagulation, photodynamic therapy, Visudyne, anti-VEGF therapy, eye-wear, eye drops, numbing agents, Orthoptic vision therapy, Behavioral / perceptual vision therapy, and the like. In some aspects, any of the previously described biologies can be considered a secondary therapy.

[0088] Any of the pharmaceutical compositions can also comprise 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 can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein.WSGR Docket No. 59561-717.601

[0089] In some embodiments, an effective amount of the subject rAAV virion results in a decrease in the rate of loss of retinal function, anatomical integrity, or retinal health, e.g. a 2-fold, 3-fold, 4-fold, or 5-fold or more decrease in the rate of loss and hence progression of disease, for example, a 10-fold decrease or more in the rate of loss and hence progression of disease. In some embodiments, the effective amount of the subject rAAV virion results in a gain in visual function, retinal function, an improvement in retinal anatomy or health, and / or an improvement in ocular motility and / or improvement in neurological function, e.g. a 2-fold, 3-fold, 4- fold or 5 -fold improvement or more in retinal function, retinal anatomy or health, and / or improvement in ocular motility, e.g. a 10-fold improvement or more in retinal function, retinal anatomy or health, and / or improvement in ocular motility. As can be readily appreciated by the ordinarily skilled artisan, the dose required to achieve the desired treatment effect can typically be in the range of 1 x 108to about 1 x 1015recombinant virions, typically referred to by the ordinarily skilled artisan as 1 x 108to about 1 x 1015vector genomes.

[0090] In some aspects, compositions provided herein, such as pharmaceutical compositions are administered to a subject in need thereof. In some cases, an administration comprises delivering a dosage of a viral particle comprising an engineered capsid and a vector described herein at about 0.5 x 109vg, 1.0 x 109vg, 1.0 x 1010, 1.0 x 1011vg, 3.0 x 1011vg, 6 x 1011vg, 8.0 x 1011vg, 1.0 x 1012vg, 1.0 x 1013vg, 1.0 x 1014vg, 1.0 x 1015vg, 1.5 x 1015vg. For example, for in vivo injection, e.g., injection directly into the eye, a therapeutically effective dose can be on the order of from about 106to about 1015of subject AAV virions, e.g., from about 108to 1012engineered AAV virions. For in vitro transduction, an effective amount of engineered AAV virions to be delivered to cells can be on the order of from about 108to about 1013of the engineered AAV virions. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.

[0091] Administrations can be repeated for any amount of time. In some aspects, administering is performed: twice daily, every other day, twice a week, bimonthly, trimonthly, once a month, every other month, semiannually, annually, or biannually.

[0092] Dosage treatment may be a single dose schedule or a multiple dose schedule. Moreover, the subject may be administered as many doses as appropriate. One of skill in the art can readily determine an appropriate number of doses. In some aspects, a pharmaceutical composition is administered via intravitreal injection, subretinal injection, microinjection, or supraocular injection.WSGR Docket No. 59561-717.601

[0093] In some aspects, a subject can be screened via genetic testing for a mutation before, during, and / or after administration of a pharmaceutical composition provided herein. Relevant genes that can be screened for mutations comprise: RPE65, CRB1, AIPL1, CFH, or RPGRIP.

[0094] Also provided are kits comprising any of the compositions provided herein. Provided is also a container that comprises a) a subject engineered adeno-associated virus (AAV) capsid; b) a subject vector; or c) a subject engineered virion. In an aspect, the container is a vial, syringe, or needle. In some cases, the container is configured for ocular delivery.

[0095] Kits may comprise a suitably aliquoted composition. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits can generally include at least one vial, test tube, flask, bottle, syringe, or another container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also can generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits also can 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.

[0096] In some instances, a packaged product comprising a composition described herein can be properly labeled. In some instances, the pharmaceutical composition described herein can be manufactured according to good manufacturing practice (cGMP) and labeling regulations. In some cases, a pharmaceutical composition disclosed herein can be aseptic.

[0097] Described herein, in some embodiments, is a method for treating a disease or condition in a subject. In some embodiments, the method comprises administering an engineered capsid to the subject, where the engineered capsid comprises a vector described herein. In some embodiments, the vector comprises a nucleic acid sequence encoding an anti -angiogenic agent. In some embodiments, said nucleic acid sequence comprises a modification in a coding region of the nucleic acid sequence as compared to an otherwise comparable nucleic acid sequence lacking the modification in the coding region. In some embodiments, said modification comprises replacing at least four non-AGG arginine codons to AGG. Fig. 37 illustrates non-limiting examples of vector constructs for encoding VEGF-Trap (aflibercept). Figs. 38-41 illustrate non-limiting examples of nucleic acid sequence comprising modified codons for encoding VEGF-Trap (aflibercept). In some embodiments, expression of the anti-angiogenic agent treats the disease or condition such as an ocular disease. Non-limiting example of the disease or condition can includeWSGR Docket No. 59561-717.601ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

[0098] In some embodiments, the nucleic acid sequence that encodes the anti -angiogenic agent further comprises a second modification, where the second modification is in at least one codon of the coding region of the nucleic acid sequence, and wherein the second modification is selected from the group consisting of: replacement of at least one non-CCC proline codon with CCC; replacement of at least one non-TCC serine codon with TCC; replacement of at least one non-CCG proline codon with CCG; and any combination thereof. 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, aPDGF-2 inhibitor, aNP-1 inhibitor, aNP-2 inhibitor, a Del 1 inhibitor, and an integrin inhibitor. In some embodiments, the anti-angiogenic agent comprises the VEGF inhibitor, and wherein the VEGF inhibitor is a non-antibody inhibitor. In some embodiments, the non-antibody inhibitor is a fusion protein that comprises human VEGF receptors 1 and 2. In some embodiments, the nucleic acid sequence is modified to replace non-AGG arginine codon with AGG in at least 4 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCC proline codon with CCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-TCC serine codon with TCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCG proline codon with CCG in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the fusion protein comprises VEGF-Trap (e.g., aflibercept) or a modified version thereof. In some embodiments, the VEGF-Trap comprises an amino acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to any one of the amino acid sequence of any one of SEQ ID NOs: 42, 60, 63, 65, 67, 69, or 73. InWSGR Docket No. 59561-717.601some embodiments, the VEGF-Trap comprises an amino acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to SEQ ID NO: 73. In some embodiments, the VEGF-Trap consists an amino acid sequence that is SEQ ID NO: 73.

[0099] In some embodiments, the nucleic acid sequence is modified to replace non-AGG arginine codon with AGG in at least 4 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCC proline codon with CCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-TCC serine codon with TCC in at least 3 codon positions as compared to SEQ ID NO: 70. In some embodiments, the nucleic acid sequence is modified to replace non-CCG proline codon with CCG in at least 3 codon positions as compared to SEQ ID NO: 70 In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to any one of the nucleic acid sequence of any one of SEQ ID NOs: 43-49, 51-57, 61, 62, 64, 66, 68, 71, or 72. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to the nucleic acid sequence of SEQ ID NOs: 71 or 72. In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence that is the nucleic acid sequence of SEQ ID NOs: 71 or 72.

[0100] In some embodiments, the nucleic acid comprises a viral vector sequence. In some embodiments, the vector is capsulated by an engineered capsid described herein. In some embodiments, the engineered capsid is part of viral particle described herein. In some embodiments, the engineered capsid delivers the vector to a cell in the subject. For example, the engineered capsid can deliver the vector to a macula cell, a retinal cell, an ocular cell, or a combination thereof. In some embodiments, the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or a combination thereof. In some embodiments, the engineered capsid or the vector can be administered intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or a combination thereof to the subject.

[0101] In some embodiments, the engineered capsid comprises a modified adeno-associated virus (AAV) capsid. In some embodiments, the modified AAV capsid is of serotype AAV1,WSGR Docket No. 59561-717.601AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof. In some embodiments, the modified AAV capsid comprises a modified AAV2 capsid. In some embodiments, the modification to the AAV capsid includes insertion of an amino acid sequence of LALGQTTKPA (SEQ ID NO: 13) in the VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the engineered capsid further comprises a mutation, the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain. In some embodiments, the mutation is in a GH loop or is at a residue at position of 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1. In some embodiments, the mutation is R to A at position 585 or 588 of SEQ ID NO: 1.

[0102] In some embodiments, the engineered capsid comprises an engineered AAV1 capsid (e.g., AV1.N54), where LALGQTTKPA (SEQ ID NO: 13) is inserted after S588 amino acid residue of the AAV1 VP1 capsid. In some embodiments, the engineered capsid comprises an engineered AAV2 capsid (e.g., AAV2.N54), where LALGQTTKPA (SEQ ID NO: 13) is inserted after N587 amino acid residue of the AAV2 VP1 capsid (e.g., as illustrated in SEQ ID NO: 1). In some embodiments, the engineered capsid comprises an engineered AAV6 capsid (AAV6.N54), where LALGQTTKPA (SEQ ID NO: 13) is inserted after N587 amino acid residue of the AAV6 VP1 capsid (e.g., as illustrated in SEQ ID NO: 4).

[0103] In some embodiments, the engineered capsid comprises a motif of X-X-X-K-P, where X can be any hydrophilic or uncharged amino acid residue (e.g., Q, S, or T), and K is acetylated. In some embodiments, the motif can be a polypeptide sequence described herein. For example, the motif can include the polypeptide sequence of any one of SEQ ID NOs: 8-13 or 18. In some embodiments, the motif is LALGQTTKPA (SEQ ID NO: 13), where X-X-X is Q-T-T, and K is acetylated. In some embodiments, the acetylation of the lysine of the X-X-X-K-P motif in the engineered capsid increases specificity or efficacy of delivering the vector described herein to a cell (e.g., a macular cell). In some embodiments, the acetylation of the lysine of the X-X-X-K-P motif in the engineered capsid increases expression of the vector in the cell that is contacted with the engineered capsid. In some embodiments, the increased expression of the vector (e.g., due to the acetylation of the lysine of the X-X-X-K-P motif in the engineered capsid) increases therapeutic efficacy of treating a disease or condition such as wet age-related macular degeneration (wAMD) or polypoidal choroidal vasculopathy (PCV). In some embodiments, the increased expression of the vector (e.g., due to the acetylation of the lysine of the X-X-X-K-P motif in the engineered capsid) decreases toxicity or unwanted side effects due to using the vector for treating the disease or condition, because, at least partially, the increased expression ofWSGR Docket No. 59561-717.601the vector allows administering a decreased dose of the viral particle or the engineered capsid to achieve a comparable therapeutic efficacy compared to other therapeutic options. For example, a decreased dose of the viral particle or the engineered capsid is needed to obtain comparable VEGF-Trap expression and therapeutic efficacy compared to direct administration of VEGF-Trap or administration of a comparable gene delivery system (e.g., other AAV gene delivery system in absence of the N54 modification or the acetylation).

[0104] In some embodiments, the method of using the engineered capsid for delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence. Fig. 21 illustrates such decrease in inflammation.

[0105] In some embodiments, using the engineered capsid to deliver a vector described herein can increase delivery efficiency and expression of the vector. Fig. 2A illustrates increased delivery efficiency of the engineered capsid described herein compared to an AAV2 capsid. Fig.2B illustrates increased transduction of the transgene delivered by the engineered capsid described herein compared to AAV2 and 7m8. In some embodiments, the engineered capsid delivering the vector increases delivery of the vector to a cell that is in a deeper cell layer in the subject compared to a comparable non-engineered capsid without the polypeptide sequence delivering the vector to a cell in a cell layer. For example, the cell layer comprises a ganglion cell layer, an inner plexiform layer, an inner nuclear layer, an outer plexiform cell, an outer nuclear layer, an inner segment layer, an outer segment layer, or a retinal pigment epithelium layer. Fig.2C illustrates the increased penetration in the optic cell layers resulted from the use of the engineered capsid.

[0106] In some embodiments, the engineered capsid delivering the vector results in a decreased retinal leakage compared to a comparable capsid without the polypeptide sequence delivering a comparable vector encoding a comparable VEGF inhibitor. Fig. 4B and Fig. 12 illustrate such decreased retinal leakage. In some embodiments, the decreased retinal leakage is resulted from one or more administrations. In some embodiments, the decreased retinal leakage is resulted from one administration. In some embodiments, the engineered capsid delivering the vector decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the comparable capsid is an unmodified AAV capsid such as an AAV2 capsid. In some embodiments, the anti-angiogenic agent decreases retinal leakage in the subject comparedWSGR Docket No. 59561-717.601to a second retinal leakage resulted from treatment by a comparable anti-angiogenic agent (e.g., Elyea). In some embodiments, the anti-angiogenic agent decreases retinal leakage in the subject compared to a second retinal leakage resulted from treatment by a comparable anti-angiogenic agent (e.g., Elyea) by at least at least 10%, at least 20%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0107] In some embodiments, the engineered capsid delivering the vector decreases retinal lesion in the subject compared to a second retinal lesion resulted from treatment by a comparable anti -angiogenic agent. Fig. 4A, Fig. 9, Fig. 10, and Fig. 11 illustrate such decreased retinal lesion. In some embodiments, the-angiogenic agent decreases the retinal lesion compared to the second retinal lesion by at least 10%, at least 20%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0108] In some embodiments, the engineered capsid delivering the vector decreases a dose of the anti-angiogenic agent administered to the subject compared to a second dose of a comparable anti -angiogenic agent administered to the subject. In some embodiments, the dose and the second dose result in comparable therapeutic efficacy in the subject. In some embodiments, the dose increases therapeutic efficacy compared to the second doe. In some embodiments, the dose, when compared to the second dose, is decreased by at least 10%, at least 20%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0109] In some embodiments, the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent (e.g., Elyea). Fig. 21 illustrates such decreases in inflammation. In some embodiments, the anti-angiogenic agent does not increase inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent. In some embodiments, contacting the cell or the environment associated with the cell with the anti-angiogenic agent decreases inflammation compared to contacting the cell with a comparable anti-angiogenic agent (e.g., Elyea) by at least 10%, at least 20%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0110] In some embodiments, the engineered capsid delivering the vector does not decrease ERG in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. Fig. 18 illustrates the lack of ERG in subjects administered with the anti-angiogenic agent compared to if the subjects were administered with a comparable anti-angiogenic agent (e.g., Elyea).WSGR Docket No. 59561-717.601

[0111] In some embodiments, , the engineered capsid delivering the vector does not increase intraocular pressure in the subject compared to if the subject is directly administered with a comparable anti -angiogenic agent. Fig. 17 illustrates the lack of increases in the intraocular pressure in subjects administered with the anti-angiogenic agent compared to if the subjects were administered with a comparable anti -angiogenic agent (e.g., Elyea).

[0112] In some embodiments, , the engineered capsid delivering the vector does not increase an expression of anti-drug antibody (ADA) in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent. Fig. 19 and Fig. 20 illustrate the lack of increases in the expression of anti-drug antibody (ADA) in subjects administered with the anti-angiogenic agent compared to if the subjects were administered with a comparable anti-angiogenic agent (e.g., Elyea).

[0113] In some embodiments, an expression of the anti-angiogenic agent delivered by the engineered capsid described herein is increased in the subject compared to a second expression of a comparable anti-angiogenic agent (e.g., Elyea). Figs. 11-14 illustrate such expression of the anti -angiogenic agent. In some embodiments, the expression of the anti-angiogenic agent is increased compared to expression of the comparable anti -angiogenic agent (e.g., Elyea) by at least 10%, at least 20%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0114] In some embodiments, the anti-angiogenic agent is expressed for a longer duration in the subject compared to a second duration of expressing a comparable anti -angiogenic agent (e.g., Elyea) by administering the comparable anti-angiogenic agent to the subject. Fig. 16 illustrates the increased duration of expression of the anti-angiogenic agent. In some embodiments, the duration of the expression of the anti -angiogenic agent is increased by at least 10%, at least 20%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500% compared to the duration of expressing the comparable anti-angiogenic agent.

[0115] In some embodiments, the subject is administered a dose of a viral particle comprising an engineered capsid described herein. In some embodiments, the engineered capsid encapsulates a vector described herein. In some embodiments, the dose of the viral particle comprising the engineered capsid or the vector is from about 1.0 x 109vg / eye to about 10 x 1011vg / eye. In some embodiments, the dose of the viral particle is from about 1.0 x 109vg / eye to about 10 x 1010vg / eye. In some embodiments, the dose of the viral particle is from about 1.0 x 1010vg / eye to about 10 x 1010vg / eye. In some embodiments, the dose of the viral particle is from about 2.0 x 1010vg / eye to about 5 x 1010vg / eye. In some embodiments, the dose of the viral particle is about 2.1 x 1010vg / eye, about 2.2 x 1010vg / eye, about 2.3 x 1010vg / eye, about 2.4 x 1010vg / eye, aboutWSGR Docket No. 59561-717.6012.5 x IO10vg / eye, about 2.6 x IO10vg / eye, about 2.7 x IO10vg / eye, about 2.8 x IO10vg / eye, or about 2.9 x IO10vg / eye. In some embodiments, the dose of the viral particle is about 2.6 x IO10vg / eye. In some embodiments, the dose of the viral particle is about 3.1 x IO10vg / eye, about 3.2 x IO10vg / eye, about 3.3 x IO10vg / eye, about 3.4 x IO10vg / eye, about 3.5 x IO10vg / eye, about 3.6 x IO10vg / eye, about 3.7 x IO10vg / eye, about 3.8 x IO10vg / eye, or about 3.9 x IO10vg / eye. In some embodiments, the dose of the viral particle is about 3.7 x IO10vg / eye. In some embodiments, the dose of the viral particle is about 8.1 x IO10vg / eye, about 8.2 x IO10vg / eye, about 8.3 x IO10vg / eye, about 8.4 x IO10vg / eye, about 8.5 x IO10vg / eye, about 8.6 x IO10vg / eye, about 8.7 x IO10vg / eye, about 8.8 x IO10vg / eye, or about 8.9 x IO10vg / eye. In some embodiments, the dose of the viral particle is about 8.1 x IO10vg / eye. In some embodiments, a single administrating of the dose of the viral particle comprising the engineered capsid and the vector encoding the anti-angiogenic agent is curative of the disease or condition in the subject. In some embodiments, administrating the engineered capsid comprising the vector encoding the anti -angiogenic agent results in a decreased number of dosing needed compared to administration of a comparable anti-angiogenic agent (e.g., Elyea).

[0116] In some embodiments, the subject is administered a dose of the engineered capsid from about 1.0 x 109vg / eye to about 10 x 1011vg / eye. In some embodiments, the subject is administered a dose of the engineered capsid from about 1.0 x 109vg / eye to about 10 x 1010vg / eye. In some embodiments, the subject is administered a dose of the engineered capsid from about 1.0 x 1010vg / eye to about 10 x 1010vg / eye. In some embodiments, the subject is administered a dose of the engineered capsid from about 2.0 x 1010vg / eye to about 5 x 1010vg / eye. In some embodiments, the subject is administered a dose of the engineered capsid at about 2.1 x 1010vg / eye, at about 2.2 x 1010vg / eye, at about 2.3 x 1010vg / eye, at about 2.4 x 1010vg / eye, at about 2.5 x 1010vg / eye, at about 2.6 x 1010vg / eye, at about 2.7 x 1010vg / eye, at about 2.8 x 1010vg / eye, or at about 2.9 x 1010vg / eye. In some embodiments, the subject is administered a dose of the engineered capsid at 2.6 x 1010vg / eye. In some embodiments, the subject is administered a dose of the engineered capsid at about 3.1 x 1010vg / eye, at about 3.2 x 1010vg / eye, at about 3.3 x 1010vg / eye, at about 3.4 x 1010vg / eye, at about 3.5 x 1010vg / eye, at about 3.6 x 1010vg / eye, at about 3.7 x 1010vg / eye, at about 3.8 x 1010vg / eye, or at about 3.9 x 1010vg / eye. In some embodiments, the subject is administered a dose of the engineered capsid at 3.7 x 1010vg / eye. In some embodiments, the subject is administered a dose of the engineered capsid at about 8.1 x 1010vg / eye, at about 8.2 x 1010vg / eye, at about 8.3 x 1010vg / eye, at about 8.4 x 1010vg / eye, at about 8.5 x 1010vg / eye, at about 8.6 x 1010vg / eye, at about 8.7 x 1010WSGR Docket No. 59561-717.601vg / eye, at about 8.8 x IO10vg / eye, or at about 8.9 x IO10vg / eye. In some embodiments, the subject is administered a dose of the engineered capsid at 8.1 x IO10vg / eye. In some embodiments, a single dose of administrating the engineered capsid comprising the vector encoding the anti-angiogenic agent is curative of the disease or condition in the subject. In some embodiments, administrating the engineered capsid comprising the vector encoding the anti-angiogenic agent results in a decreased number of dosing needed compared to administration of a comparable anti-angiogenic agent (e.g., Elyea).Table 1. Wild type AAV polypeptide (VP1) sequence<<<<WSGR Docket No. 59561-717.601<<<<<WSGR Docket No. 59561-717.601<<<<<<WSGR Docket No. 59561-717.601Table 2. AAV insertion location and sequence of the polypeptide sequenceWSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601Table 3. Non-limiting example of engineered capsid amino acid sequenceWSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601Table 4. Sequences of VEGF-Trap and vector encoding the VEGF-TrapWSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601<<<

[0117] Use of absolute or sequential terms, for example, “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” are not meant to limit scope of the present embodiments disclosed herein but as exemplary.

[0118] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0119] As used herein, the phrases “at least one”, “one or more”, and “and / or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and / or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.WSGR Docket No. 59561-717.601

[0120] As used herein, “or” may refer to “and”, “or,” or “and / or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[0121] Any systems, methods, software, and platforms described herein are modular.Accordingly, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts.

[0122] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term “about” refers to ±10% of a stated number or value.

[0123] The terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount. In some aspects, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

[0124] The terms “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some aspects, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.

[0125] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way ofWSGR Docket No. 59561-717.601example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. 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 be covered thereby.EXAMPLES

[0126] The following illustrative examples are representative of embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way.Example 1. Increased expression in targeted cell due to the engineered capsid

[0127] Different capsids containing reporter construct were delivered to the eyes of test subjects for determination of the expression in the targeted cells (e.g., macula or retina cells). Fig. 1 illustrates increased expression in a macula cell using a vector encoding a transgene delivered by an engineered capsid described herein. Reporter construct was delivered to the eye by: R100 (an adeno-associated virus or AAV capsid with a ten amino acid insertion), 7m8 (AAV.7m8 capsid), and an engineered capsid described herein. The delivery of the reporter construct by the engineered capsid described here yielded the highest expression in the targeted macula cell at a comparable dose or at a lower dose compared to R100 and 7m8. Fig. 2A illustrates increased delivery efficiency of the engineered capsid described herein compared to an AAV2 capsid. Fig.2B illustrates increased transduction of the transgene delivered by the engineered capsid described herein compared to AAV2 and 7m8. Fig. 2C illustrates increased penetration and expression of the transgene delivered by the engineered capsid described herein into the multiple cell layers of monkey retina compared to delivery of the transgene by AAV2 and 7m8.Example 2. Clinical study protocol

[0128] Example 2 illustrates a Phase l / 2a, open-label, multiple-cohort, dose-escalation study to evaluate the safety, tolerability, and preliminary efficacy of intravitreal (IVT) injection of Vector AAT encapsulated in AAV2.N54 (for expression of VEGF-Trap) in subjects with neovascularWSGR Docket No. 59561-717.601(wet) Age-Related Macular Degeneration (wAMD) or with symptomatic macular polypoidal choroidal vasculopathy (PCV). The primary objective includes determining the safety and tolerability of IVT injection of Vector AAT encapsulated in AAV2.N54 (for expression of VEGF-Trap) in subjects with wAMD, including symptomatic macular PCV. The secondary objective includes assessing the preliminary efficacy of IVT injection of Vector AAT encapsulated in AAV2.N54 (for expression of VEGF-Trap) in subjects with wAMD, including symptomatic macular PCV. Other secondary objective includes evaluation of the pharmacokinetics (PK) / pharmacodynamics (PD), immunogenicity, or optimal dose of IVT injection of Vector AAT encapsulated in AAV2.N54 (for expression of VEGF-Trap). Table 5 lists abbreviations as shown in this example.Table 5. List of abbreviationsWSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601Study design

[0129] This Phase l / 2a, open-label, multiple-cohort, dose-escalation study is designed to evaluate the safety, tolerability, and preliminary efficacy of a single IVT injection of Vector AAT encapsulated in AAV2.N54 (for expression of VEGF-Trap) in subjects previously treated with at least two intravitreal injections of an anti-VEGF therapy for wAMD, including symptomatic macular PCV. Vector AAT uses an AAV2.N54 vector that contains a gene encoding a recombinant protein composed of the binding domains of two human vascular endothelial growth factor (VEGF) receptors fused with the Fc region of human immunoglobulin gamma 1 (IgGl ) which binds to and neutralizes VEGF activity. Safety evaluation and monitoring can be the primary focus for the initial 52 weeks after a single Vector AAT administration (primary endpoint). This trial can include a screening period, confirmation of response to EYLEA (aflibercept), single administration of Vector AAT, and follow-up period of 104 weeks after IVT Vector AAT injection. The screening period can be up to 45 days prior to investigational product administration. Screening can begin upon completion of the informed consent process. Once consent has been granted by each subject, a thorough screening process can take place, including detailed medical history, physical and ophthalmology examination, vital signs, concomitant medications, safety labs, urinalysis, serology panel, and assessment of inclusion and exclusion criteria. Ophthalmology examinations can include safety and tolerability assessments and morphological evaluations utilizing spectral domain optical coherence tomography (SD-OCT), color fundus photography (CFP), and fluorescein angiography (FA). If a subject’s screening SD-OCT does not demonstrate SRF, IRF, or IRC, the site may repeat the Screening SD-OCT one time during the screening period. Upon completion of screening, qualified subjects can be enrolled and receive a single IVT dose of EYLEA (aflibercept 2 mg). After confirmation of anti-VEGF response to EYLEA (aflibercept) at Week 1 by the Central Reading Center, subjects who have a response to aflibercept (2 mg) can be enrolled into one of three sequential dosing cohorts. Subjects who do not have a response to EYLEA (aflibercept) can be excluded from the study. Individuals who do not meet the criteria for participation in this study (screen failure) may be rescreened one time if there is cause to believe that the subject may be eligible upon rescreen; allWSGR Docket No. 59561-717.601screening procedures must be repeated. Each qualified subject can receive a single dose of Vector AAT administered by IVT injection in the study eye at Week 2. If both eyes qualify, the study eye can be the worse seeing eye. During the study, subjects can be evaluated for safety and tolerability, PK / PD, immunogenicity, and preliminary efficacy of Vector AAT. In each dose cohort, a sentinel group consisting of one subject can be dosed first. The remaining subjects of the dose cohort can be staggered by least 14 days after the sentinel subject dosing, with a 3 -day staggering period between consecutive subjects within a dose cohort. The staggering interval between enrollment in each subsequent dose cohort is 14 days - to clarify, before enrolling a new cohort, there can be at least 14 days between the dosing of the last subject in the prior cohort before dosing the first subject in the subsequent cohort.

[0130] Three dose levels can be studied in up to 18 subjects. Subjects who meet the inclusion and no exclusion criteria and have a response to EYLEA (aflibercept 2 mg) can receive a single dose of Vector AAT administered by a 50 pL intravitreal injection in the study eye. For Cohort 1, Vector AAT can be administered at 5.27 El 1 (or 5.27 x 1011) vg / mL, 50 pL = 2.6 E10 (or 2.6 x 1010) vg / eye for 3 to 6 subjects. For Cohort 2, Vector AAT can be administered at 7.41 El 1 (or 7.41 x 1011) vg / mL, 50 pL = 3.7 E10 (or 3.7 x 1010) vg / eye for 3 to 6 subjects. For Cohort 3, Vector AAT can be administered at 1.63 E12 (or 1.63 x 1012) vg / mL, 50 pL = 8.1 E10 (or 8.1 x 1010) vg / eye for 6 subjects. Enrollment can begin with the lowest dose and progress to the next higher dose only after review of safety data. If a dose-limiting toxicity (DLT) occurs within a dose level cohort, 3 more subjects can be added to the dose level cohort for a total of 6 subjects in the dose level cohort. If a DLT occurs in two subjects of a dosing cohort, then the maximum tolerated dose (MTD) has been exceeded, and the dose cannot continue to be escalated. The MTD can be the dose level below the dose level with 2 to 6 subjects having DLTs. Following completion of the primary endpoint, subjects can continue to be assessed for 104 weeks following treatment with Vector AAT in this treatment study. Subjects can be followed for a total of 5 years after Vector AAT treatment in the long term follow up study.Stopping rules

[0131] Further enrollment can be stopped if any of the following conditions are met: more than one subject has been diagnosed with endophthalmitis; any subject develops an intraocular tumor; or any subject develops an adverse event (AE) that results in enucleation. Further enrollment can be suspended temporarily pending the outcome of additional testing or investigation as to the cause of the event if any of the following conditions are met: any evidence of intraocular or intravitreal inflammation greater than 3+ACC / 3+ ACF / 3 VH; any vascular intraocular event; or aWSGR Docket No. 59561-717.601significant abrupt increase of IOP which is clinically significant based on the investigator’s opinion. Stopping rules are independent of attribution (e.g., relationship to product).Safety review committee

[0132] The Sponsor can establish a Safety Review Committee (SRC), which can consist of retinal specialist investigators who have enrolled at least one subject in the cohort and retinal specialists. The SRC meetings can occur after all subjects in each dose cohort have completed at least 14 days of safety follow-up. The SRC can review the collected safety data from the lower dose cohort, before allowing dose escalation to the subsequent cohort. The safety reviews can include all necessary safety and other data, including events qualifying as DLTs. SRC meetings can be minuted. Ad hoc SRC meetings may be convened upon the occurrence of events that meet study Stopping Rules or otherwise warranted to discuss emerging safety findings. Data reviewed by the SRC can be unmasked.Dose limiting toxicity

[0133] DLTs can be defined as toxicities that have not resolved within 14 days after Vector AAT administration. Suspected, unanticipated, serious adverse reactions (SUSAR)s or Systemic AEs that are grade 2 and have persisted past two weeks can also be considered as a potential DLT. DLTs can also include treatment-emergent ophthalmic adverse events (AEs) and systemic AEs. Ophthalmic AEs can be graded according to a 3-point scale (mild, moderate, or severe) and uveitis can be graded according to the Standardization of Uveitis Nomenclature (SUN) system. Systemic AEs can be graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events Version 5.0 (CTCAE v5.0). All Grade 3 toxicities for vital signs and laboratory parameters must be confirmed with a repeat measurement obtained within 1 hour for vital signs and as soon as laboratory retest is possible for laboratory parameters. Additional ophthalmic DLTs can include, but are not limited to: any Severe grade ophthalmic AE according to a 3-point scale (mild, moderate, or severe); severe intraocular inflammation according to the SUN system (4+ anterior chamber cell / flare or vitreous haze score 4); decrease in BCVA score of > 15 letters Early Treatment Diabetic Retinopathy Study (ETDRS); or IOP > 35 mmHg that cannot be medically managed. In addition, systemic DLTs can include, but are not limited to: any Grade > 3 systemic AE or laboratory abnormality as defined in CTCAE (version 5.0); or any death not clearly due to extraneous causes.Mandatory steroid prophylaxisWSGR Docket No. 59561-717.601

[0134] Topical steroid (prednisolone 1%) drops can be administered in the study eye QID starting one day before the Vector AAT intravitreal injection and continuing for 7 days, followed by TID for 1 week, BID for 1 week, and then QD for 1 week.Rescue treatment

[0135] Anti-VEGF rescue (EYLEA or aflibercept at 2 mg) can be administered beginning 6 weeks post-treatment with Vector AAT and every 8 weeks thereafter, as needed, if one or more of the following criteria apply: CNV-related increased, new or persistent fluid > 75 microns; vision loss of > 10 letters associated with accumulation of fluid; or new retinal hemorrhage. Inclusion criteria

[0136] Subject must be willing and able to provide written, signed informed consent. The age of the subject can be between 50 to 89 years of age at the time of signing the informed consent. Other inclusion criteria can include: diagnosis of wAMD, including symptomatic macular PCV, confirmed by the Central Reading Center; the macular neovascularization lesion can be confirmed as one of the following types as determined by the Central Reading Center based on SD-OCT imaging at screening: type 1 macular neovascularization (vascular complex originates in the choroid and breaks through the RPE to reach the subretinal space); type 2 macular neovascularization (vascular complex originates from choroid and remains under RPE); type 3 macular neovascularization (vascular complex originates in the retina); mixed Type macular neovascularization (prominent vascular complex present in subretinal and sub-RPE compartments); BCVA < 20 / 32 and > 20 / 250 (< 78L and > 30L ETDRS letters); History of > 2 anti-VEGF injections prior to trial entry with meaningful response to anti-VEGF and continued need for anti-VEGF treatment (must demonstrate SRF, IRF, or IRC at Screening SD-OCT); response to anti-VEGF at trial entry (must demonstrate a response, defined as a central retinal thickness reduction >50 pm or central fluid reduction >30% from the initial value at Week 1 SD-OCT); must be pseudophakic (status post cataract surgery): or sex and contraceptive / barrier requirement of male or female. Male subjects must use a contraceptive method that is highly effective with a failure rate <_2% (i.e., condoms or vasectomy) during the study intervention period and for at least 30 days after the study intervention. Female subjects must be of nonchildbearing potential.Exclusion criteria

[0137] Exclusion criteria can include, for study or fellow eye: prior gene therapy in either eye; any active ocular / intraocular infection or inflammation (e.g., blepharitis, infectious conjunctivitis, keratitis, scleritis, or uveitis) or history of idiopathic or autoimmune-associated uveitis in eitherWSGR Docket No. 59561-717.601eye; history of retinal disease other than wAMD or PCV in study eye; any condition preventing visual acuity improvement (e.g., fibrosis, atrophy, or retinal epithelial tear in the center of the fovea) in study eye; history of (or active) retinal detachment in study eye; uncontrolled glaucoma (e.g., IOP > 25 mm Hg despite treatment) in study eye; history of steroid-response ocular hypertension / glaucoma in either eye; history of intravitreal therapy (other than anti-VEGF therapy) such as intravitreal steroid injection or investigational product in the 6 months prior to screening in study eye; any prior treatment with photodynamic therapy or laser photocoagulation in study eye; history of glaucoma filtration, vitrectomy, or other procedure that could affect drug distribution or clearance in study eye;presence of a transplant or implant (e.g., corneal or RPE transplants, glaucoma, or retinal implants) at screening (excluding intraocular lens implant) in study eye; or history of (or current) ophthalmic conditions which may affect safe participation in the study or interpretation of study results in study eye. Exclusion criteria can include, for medical or systemic condition: prior gene therapy; myocardial infarction, cerebrovascular accident, or transient ischemic attacks within the past 6 months; uncontrolled hypertension (e.g., systolic blood pressure (BP) > 180 mm Hg or diastolic BP > 100 mm Hg) despite maximal medical treatment; or history of (or current) medical or psychiatric reasons that may affect safe participation in the study or interpretation of study result.Study termination criteria

[0138] The sponsor may terminate the study prematurely at any time. Reasons for the closure of an investigational site or termination of a study may include but are not limited to: the investigator fails to comply with the protocol or ICH / GCP guidelines; inadequate recruitment of subjects by the investigator; completion of the study; lack of funding; the sponsor requests termination of the study on the premise that the subjects’ rights and safety are not sufficiently guaranteed; drug regulatory authorities or the Ethics Committee orders to terminate the study for some reason; or principal investigator (PI) request to withdraw from participation.Study assessment

[0139] Study assessment can include: informed consent; inclusion criteria; exclusion criteria; demographics; medical and ophthalmic history; physical exam; vital signs; lab assessment of hematology, chemistry, or urinalysis; best corrected visual acuity (BCVA) or early treatment diabetic retinopathy study (ETDRS); ophthalmic exam (e.g., SLE, IOP, or dilated ophthalmoscopy); SD-OCT; color fundus photography (CFP); intravenous fluoresceinWSGR Docket No. 59561-717.601angiography (IVFA); adverse events (ocular and non-ocular); concomitant medications; or concurrent procedures. Pharmacodynamic or pharmacokinetics evaluations or biomarkers

[0140] Examples of pharmacodynamic or pharmacokinetics evaluations or biomarkers can include peripheral blood mononuclear cell (PBMC) collection; peripheral blood / tear qPCR; blood for PK / PD (plasma VEGF-Trap); blood for ADA (serum anti-AAV2 antibodies and anti-VEGF-Trap antibodies); or CST, IRF, SRF, or PED as measured by SD-OCT.Objective or endpoint

[0141] Primary objective can include safety and tolerability (e.g., incidence of ocular and nonocular AEs and serious adverse events (SAEs) at Week 52 after Vector AAT treatment).Secondary endpoint can include preliminary efficacy (e.g., change from baseline to Week 52 after Vector AAT treatment in BCVA as assessed by ETDRS chart at a starting distance of 4 M); PK / PD (e.g., changes in plasma VEGF-Trap SD-OCT); immunogenicity (e.g., changes in Anti-AAV2 antibodies (ADA) and anti-VEGF-Trap antibodies in serum); or optimal dose (e.g., number and frequency of rescue injections)Statistical Methods

[0142] Data can be summarized using descriptive statistics (e.g., number of subjects, mean, median, standard deviation, minimum, or maximum for continuous variables and frequencies and percentages for discrete variables). Data can be presented by dose level of Vector AAT. Key listings can be created. Exploratory analyses of the data can be conducted as deemed appropriate.Vector AAT

[0143] Vector AAT (AAV2.N54-VEGF-Trap) is a recombinant adeno-associated virus vector (rAAV) serotype 2 (AAV2) carrying a transgene coding for the same protein sequence as aflibercept, which functions as a vascular endothelial growth factor (VEGF) trap, whose capsid protein (VP1) is modified for IVT delivery and transduction of retinal cells near blood vessels and macular. This engineered capsid is named N54. Once Vector AAT crosses the inner limiting membrane (ILM) and penetrates all layers of the retina, the VEGF-Trap protein encoded from the transgene binds VEGF and blocks VEGF -associated angiogenesis, relieving the symptoms of wAMD.

[0144] Traditional pharmacokinetics (PK) studies have not been performed for Vector AAT, as the IVT injection of AAV2 vector carrying VEGF-trap (aflibercept) into retinal tissue prevents absorption, metabolism, and excretion. However, tissue distribution studies were conducted to evaluate biodistribution of VEGF-Trap and AAV vector post IVT injection. The pharmacokinetics of Vector AAT had been investigated in mice and rabbits following intravitrealWSGR Docket No. 59561-717.601administration. Vector AAT showed protection against laser-induced CNV in a mouse model and neutralization of induced VEGF via the production of VEGF-Trap from a single dose of Vector AAT, in a dose-dependent manner. Vector AAT also demonstrated attenuation of retinal leakage through Day 106 in the Rabbit DL-a-AAA Challenge Retinal Neovascularization model.Nonclinical Toxicology Findings

[0145] Nonclinical studies of Vector AAT administered by intravitreal injection in cynomolgus monkeys demonstrated that Vector AAT was well-tolerated based on the lack of clinical effects, anticipated ocular inflammation, and minimal severity of microscopic findings. Benefits of receiving Vector AAT in this study can include a decrease in the number of recurrent anti-VEGF injections, thorough medical evaluations, possible improvement in BCVA, or contribution to scientific understanding and the drug development process.Primary or secondary objective

[0146] Primary objective can include determining the safety and tolerability of IVT of Vector AAT in subjects with wAMD, including symptomatic macular PCV. Secondary object can include assessing the preliminary efficacy of IVT Vector AAT injection in subjects with wAMD, including symptomatic macular PCV, as measured by BCVA; evaluating the PK / PD of IVT Vector AAT injection; evaluating the immunogenicity of IVT Vector AAT injection; or determining the optimal dose of IVT Vector AAT injection for later phases of study.Trial design

[0147] This is an open-label, multiple-cohort, dose-escalation study to evaluate the safety, tolerability, and preliminary efficacy of IVT Vector AAT (AAV2.N54-VEGF-Trap) injection in subjects with wAMD), including symptomatic macular PCV. Vector AAT uses an AAV2.N54 vector that contains a gene encoding a recombinant protein composed of the binding domains of two human VEGF receptors fused with the Fc region of human immunoglobulin gamma 1 (IgGl) which binds to and neutralizes VEGF activity. Safety can be the primary focus for the initial 52 weeks after a single Vector AAT administration (primary endpoint). This trial can include a screening period, confirmation of response to EYLEA (aflibercept), single administration of Vector AAT, and follow-up period of 104 weeks after IVT Vector AAT injection.

[0148] The screening period can be up to 45 days prior to investigational product administration. Screening can begin upon completion of the informed consent process. Once consent has been granted by each subject, a thorough screening process can take place, including detailed medical history, physical and ophthalmology examination, vital signs, concomitant medications, safety labs, urinalysis, serology panel, and assessment of inclusion and exclusionWSGR Docket No. 59561-717.601criteria. If a subject’s screening SD-OCT does not demonstrate SRF, IRF, or IRC, the site may repeat the Screening SD-OCT one time during the screening period. Individuals who do not meet the criteria for participation in this study (screen failure) may be rescreened one time if there is cause to believe that the subject may be eligible upon rescreen; all screening procedures must be repeated. Ophthalmology examinations can include safety and tolerability assessments and morphological evaluations utilizing spectral domain optical coherence tomography (SD-OCT), color fundus photography (CFP), and fluorescein angiography (FA). Upon completion of screening, qualified subjects can receive a dose of EYLEA (aflibercept 2 mg). After confirmation of anti-VEGF response to EYLEA (aflibercept) at Week 1 by the Central Reading Center, subjects who have a response to EYLEA (aflibercept 2 mg) can be enrolled into one of three sequential dosing cohorts. Anti-VEGF response is defined as absence of intraretinal fluid, intraretinal cysts, and subretinal fluid on SD-OCT. Subjects who do not have a response to EYLEA (aflibercept) can be excluded from the study.

[0149] Each qualified subject can receive a single dose of Vector AAT administered by IVT injection in the study eye at Week 2. If both eyes qualify, the study eye can be the worse seeing eye. During the study, subjects can be evaluated for safety and tolerability, PK / PD, immunogenicity, and preliminary efficacy of Vector AAT as measured by BCVA. In each dose cohort, a sentinel group consisting of one subject can be dosed first. The remaining subjects of the dose cohort can be staggered by least 14 days after the sentinel subject dosing, with a 3 -day staggering period between consecutive subjects within a dose cohort. The staggering interval between enrollment in each subsequent dose cohort is 14 days. Before enrolling a new cohort, there can be at least 14 days between the dosing of the last subject in the prior cohort before dosing the first subject in the subsequent cohort.Dose escalation

[0150] Three dose levels can be studied in up to 18 subjects. Subjects who meet all inclusion and no exclusion criteria and have a response to EYLEA (aflibercept 2 mg) can receive a single dose of Vector AAT administered by a 50 pL intravitreal injection in the study eye: Cohort 1: Vector AAT 5.27 El 1 (or 5.27 xlO11) vg / mL, 50 pL, dose per eye: 2.6 E10 (or 2.6 xlO10) vg: N = 3-6 subjects; Cohort 2: Vector AAT 7.41 Ell (or 7.41 xlO11) vg / mL, 50 pL, dose per eye: 3.7 E10 (or 3.7 xlO10) vg: N = 3-6 subjects; and Cohort 3: Vector AAT 1.63 E12 (or 1.63 xlO12) vg / mL, 50 pL, dose per eye: 8.1 E10 (or 8.1 xlO10) vg: N = 6 subjects. Fig. 5 illustrates a trial design schematic. Enrollment can begin with the lowest dose cohort and can progress to the next higher dose cohort only after review of safety data. Safety reviews can be performed after theWSGR Docket No. 59561-717.601first three subjects of the first two cohorts have been followed for 14 days. Dose Limiting Toxicities are defined as toxicities that have not resolved within 14 days. If a Dose-Limiting Toxicity (DLT) occurs within a dose level cohort, 3 more subjects can be added to the dose level cohort for a total of 6 subjects in the dose level cohort. If a DLT occurs in two subjects of a dosing cohort, the dose would not continue to be escalated. The Maximum Tolerated Dose (MTD) can be the dose level below the dose level with 2 / 6 subjects having DLTs. Subjects who discontinue from the study prematurely may be replaced, upon the Sponsor’s discretion.Stopping Rules

[0151] Further enrollment can be stopped if any of the following conditions is met: more than 1 subject has been diagnosed with endophthalmitis; any subject develops an intraocular tumor; or any subject develops an AE that results in enucleation. Further enrollment can be suspended temporarily pending the outcome of additional testing or investigation as to the cause of the event if any of the following conditions are met: any evidence of intraocular or intravitreal inflammation greater than 3+ACC / 3+ ACF / 3 VH; any vascular intraocular event; or a significant abrupt increase of IOP which is clinically significant based on the investigator’s opinion.Stopping rules are independent of attribution (e.g., relationship to product).Safety Review Committee

[0152] The Sponsor can establish a Safety Review Committee (SRC) which can consist of retinal specialist investigators who have enrolled at least one subject in the cohort and retinal specialists. The SRC meetings can occur after all subjects in each dose cohort have completed at least 14 days of safety follow-up. The SRC can review the collected safety data from the lower dose cohort, before allowing dose escalation to the subsequent cohort. The safety reviews can include all necessary safety and other data including events qualifying as DLTs. SRC meetings can be minuted. Ad hoc SRC meetings may be convened upon the occurrence of events that meet study Stopping Rules or otherwise warranted to discuss emerging safety findings. Data reviewed by the SRC can be unmasked. Following completion of the primary endpoint at Week 54, subjects can continue to be assessed for a total of 104 weeks following treatment with Vector AAT in this treatment study. Subjects can be followed for a total of 5 years after Vector AAT treatment in the Long Term Follow Up study.Trial treatment

[0153] Vector AAT (AAV2.N54-VEGF-Trap) is a recombinant adeno-associated virus vector (rAAV) serotype 2 (AAV2) carrying a transgene coding for the same protein sequence as aflibercept, which functions as a VEGF-trap. Vector AAT can be supplied as a sterile, frozen,WSGR Docket No. 59561-717.601liquid stored in a 2 mL, 13 mm Daikyo Crystal Zenith vial (CZV). The route of administration and dosing regimen can be a single 50 pL IVT injection. Vector AAT is a recombinant adeno-associated virus vector (rAAV) serotype 2 (AAV2) whose capsid protein (VP1) is modified for intravitreal (IVT) delivery and contains a DNA fragment coding the same protein sequence as aflibercept. This engineered capsid is called N54. Vector AAT drug product can be defined as AAV2.N54-VEGF-Trap vector as the active pharmaceutical ingredient (API). Vector AAT can be formulated in 10 mM sodium phosphate buffer, 180 mM sodium chloride, 0.001% (w / v) Poloxamer 188, pH 7.3 ± 0.2. Vector AAT can be supplied as a sterile, frozen, liquid formulation stored at < -60 degrees C in 2 mL, 13 mm Daikyo Crystal Zenith® vials (CZV). The product can be administered as a single intravitreal injection (IVT) at 50 pL per study eye per patient as maintenance therapy administered up to 1 month following anti-VEGF protein injection loading doses.Starting Dose

[0154] The starting dose of Vector AAT was selected based of the results of preclinical efficacy and toxicology studies. The no observed adverse effect level (NOAEL) of Vector AAT was determined to be 5.7xlOn. The human equivalent dose (HED) is adjusted for the 2X size difference of the smaller monkey eye compared to the larger human eye to be 11.4xlOn(1.14xl012). The clinical starting dose of Vector AAT is 2.6xlO10which is much lower than the safety factor of 10 recommended by the FDA. Example 3 illustrates information on the preclinical efficacy and toxicology studies of Vector AAT.Rescue Treatment

[0155] Anti-VEGF rescue treatment EYLEA (aflibercept 2 mg) may be administered beginning 6 weeks post-treatment with Vector AAT and every 8 weeks thereafter, as needed, if one or more of the following criteria apply: CNV-related increased, new, or persistent fluid > 75 microns; vision loss of > 10 letters associated with accumulation of fluid; or new retinal hemorrhage. Selection and withdrawal of subject

[0156] The target population consists of men and women diagnosed with wAMD, including symptomatic PCV, who have been previously treated and have had a clinical response to IVT anti-VEGF injections. Subjects must meet all of the inclusion criteria to be eligible to participate in the trial: must be willing and able to provide written, signed informed consent; and age between 50 and 89 years of age at the time of signing the informed consent. Study eye for inclusion criteria include diagnosis of wAMD, including symptomatic macular PCV, confirmed by the Central Reading Center; the macular neovascularization lesion must be confirmed as oneWSGR Docket No. 59561-717.601of the following types as determined by the Central Reading Center based on SD-OCT imaging at screening (Type 1 macular neovascularization, where vascular complex originates in the choroid and breaks through the RPE to reach the subretinal space; Type 2 macular neovascularization, where vascular complex originates from choroid and remains under RPE; Type 3 macular neovascularization, where vascular complex originates in the retina; or mixed Type macular neovascularization, where prominent vascular complex present in subretinal and sub-RPE compartments); BCVA < 20 / 32 and > 20 / 250 (<78 L and >30 L ETDRS letters); history of > 2 anti-VEGF injections prior to trial entry with meaningful response to anti-VEGF and continued need for anti-VEGF treatment (must demonstrate SRF, IRF, or IRC at Screening SD-OCT); response to anti-VEGF at trial entry (must demonstrate a response, defined as a central retinal thickness CRT reduction >50 pm or central fluid reduction >30% from the initial value at Week 1 SD-OCT); or must be pseudophakic (status post cataract surgery). Male subjects must use a contraceptive method that is highly effective with a failure rate < 2% (i.e., condoms or vasectomy) during the study intervention period and for at least 30 days after the study intervention. Female subjects must be of non-childbearing potential.

[0157] Subjects can be excluded from the trial if any of the exclusion criteria apply: prior gene therapy in either eye; any active ocular / intraocular infection or inflammation (e.g., blepharitis, infectious conjunctivitis, keratitis, scleritis, uveitis) or history of idiopathic or autoimmune-associated uveitis in either eye; history of retinal disease other than wAMD or PCV in study eye; any condition preventing visual acuity improvement (e.g., fibrosis, atrophy, or retinal epithelial tear in the center of the fovea) in study eye; history of (or active) retinal detachment in study eye; uncontrolled glaucoma (defined as IOP > 25 mm Hg despite treatment), study eye, or history of steroid-response ocular hypertension / glaucoma in either eye; history of intravitreal therapy, (other than anti-VEGF therapy) such as intravitreal steroid injection or investigational product, in the 6 months prior to screening in study eye; any prior treatment with photodynamic therapy or laser photocoagulation in study eye; history of glaucoma filtration, vitrectomy, or other procedure that could affect drug distribution or clearance in study eye; presence of a transplant or implant (e.g., corneal or RPE transplants, glaucoma or retinal implants) at screening (excluding intraocular lens implant) in study eye; or history of (or current) ophthalmic conditions which may affect safe participation in the study or interpretation of study results in study eye. Subjects are excluded from the trial if any of the exclusion criteria apply: prior gene therapy; myocardial infarction, cerebrovascular accident, or transient ischemic attacks within the past 6 months; uncontrolled hypertension [systolic blood pressure (BP) >180 mm Hg, diastolic BP >100 mmWSGR Docket No. 59561-717.601Hg] despite maximal medical treatment; or history of (or current) medical or psychiatric reasons that may affect safe participation in the study or interpretation of study results.Subject withdrawal from treatment or trial

[0158] Subjects (or their legally authorized representative) may choose to withdraw from the trial for any reason at any time without prejudice. Because Vector AAT is administered as a single IVT injection, subjects should be encouraged to complete all safety evaluation visits. Subjects withdrawn from the trial before administration of Vector AAT may be replaced with a qualified subject. Subjects withdrawn from the trial after administration of Vector AAT would not be replaced. The study assessments for Early Termination Visit should be conducted in the event a subject discontinues from the study prematurely. If a subject withdraws from the trial, he or she may not reenter the study. The investigator or sponsor may withdraw a subject from the trial for any of the following reasons: adverse event; prohibited therapy (subject required concomitant medication or medical procedure prohibited by the protocol); subject noncompliance (subject does not adhere to the requirements specified by the protocol); subject improper entry (subject was enrolled in the study but did not meet all eligibility criteria); subject withdrawal of consent lost to follow-up; or death.

[0159] At the time of withdrawal, the investigator should advise the subject of other available treatment options. When a subject is withdrawn from the trial for any reason, the reason(s) for withdrawal can be recorded in the electronic case report form (eCRF). Whenever possible, subjects who withdraw from the study prematurely can undergo assessments listed for the Early Termination Visit. If a subject fails to return for a scheduled visit, it is the responsibility of the investigator or designee to document all efforts to contact the subject and to determine the reason the subject did not return. If subject cannot be contacted with 3 documented telephone call attempts, followed by a certified letter, and does not have a known cause for discontinuation (e.g., withdrawal of consent or an AE), the reason for discontinuation can be recorded as “lost to follow-up”. The date the certified letter was mailed can be recorded as the date of study withdrawal. In the event of a subject death during the study, the date of death (as listed on the death certificate) can be recorded as the date of study withdrawal. If a subject is withdrawn due to an AE, every effort must be made to complete protocol-specified safety follow-up procedures. The Medical Monitor should be notified promptly when a subject is withdrawn.Study termination criteria

[0160] The sponsor may terminate the study prematurely at any time. Reasons for the closure of an investigational site or termination of a study may include, but are not limited to: theWSGR Docket No. 59561-717.601investigator fails to comply with the protocol or ICH / GCP guidelines; inadequate recruitment of subjects by the investigator; completion of the study; lack of funding; the sponsor requests termination of the study on the premise that the subject’s rights and safety are sufficiently guaranteed; drug regulatory authorities or the Ethics Committee orders to terminate the study for some reason; or PI request to withdraw from participationTreatment of subject

[0161] Before any study-specific procedures are performed, the investigator or designee can provide the subject or the subject’s legally authorized representative with a copy of the current IRB-approved Informed Consent Form (ICF) and allow adequate time for review and the opportunity to ask any questions regarding the study and / or ICF. The investigator should answer all questions to the best of his / her ability and to the satisfaction of the subject or the subject’s legally authorized representative before performing any study visit assessments. The ICF should be signed and dated by the subject or subject’s legally authorized representative, the investigator or his / her designee, and witness (where applicable) before any study-specific procedures are performed. Screening assessments and Week 1 SD-OCT can be confirmed by the Central Reading Center to determine eligibility before enrolling a subject in a dosing level cohort for inclusion or exclusion criteria. Demographic data can be recorded including date of birth, gender, race, and ethnicity. Medical history can be recorded and should elicit all major illnesses, diagnoses, and surgeries for the subject. Ocular history can be recorded and should be specific to each eye as appropriate. The physical examination should include review of the following systems, with any abnormalities noted by qualified site’s staff: head, ears, eyes, nose, and throat; cardiopulmonary; endocrine; gastrointestinal; musculoskeletal / rheumatic; neurologic / psychiatric; dermatologic; hepatic / renal systems; and others as applicable. If any clinically significant abnormalities are found at screening, the investigator must perform (or refer the subject for) a directed physical examination prior to enrollment. In the event of newly noted abnormalities or worsening of previously noted abnormities, findings should be recorded on the Adverse Event eCRF, and the investigator should use his or her clinical judgment for appropriate treatment and / or medical referral. Vital signs can include measurements of pulse and systolic and diastolic blood pressure while the subject is in a seated position, after resting for 5 minutes. Vital signs should be taken before IVT injection. BCVA can be measured by trained and certified personnel at the study sites. The following is required to conduct the BCVA examination: certified visual acuity examiner; certified examination lane of adequate dimensions to allow testing at required distances, starting at a distance of 4 meters; standard chair with a firm back; set of three PrecisionWSGR Docket No. 59561-717.601Vision or Lighthouse distance visual acuity charts (modified Early Treatment Diabetic Retinopathy Study Charts 1, 2 and R); retro-illuminated box; trial frame; trial lens set. Slit lamp biomicroscopy can be performed for each eye by qualified site personnel. Slit lamp biomicroscopy should be conducted consistent with standard clinical practice in the same manner for all subjects and can include an assessment of each of the following as normal or abnormal (lids, conjunctiva, cornea, anterior chamber, iris); all abnormal findings that are clinically significant can be described. IOP measurement should be performed prior to dilation, using a Goldmann tonometry throughout the study. Tonometers should be cleaned and disinfected before each use and calibrated monthly. Dilated Ophthalmoscopy can be performed by qualified site personnel, conducted consistent with standard clinical practice in the same manner for all subjects and can include an assessment of each of the following as normal or abnormal (vitreous, macula, and peripheral retina, choroid, optic nerve with C / D ratio; all abnormal findings that are clinically significant can be described.Study assessment by visitTable 6 illustrates the schedule of event for year 1 of the study. Table 7 illustrates the schedule of event for year 1 of the study.Table 6. Schedule of Events for Year 1WSGR Docket No. 59561-717.601X*, XPRN*: Treatment with EYLEA (aflibercept) or Vector AAT must be administered after all other assessments are completeTable 7. Schedule of Events for Year 2WSGR Docket No. 59561-717.601

[0162] Screening Assessments, Visit 1 (Day -45 to Day-5), can include assessment for informed consent; inclusion criteria or exclusion criteria; demographics; medical and ophthalmic history; physical exam; vital signs; central labs; BCVA (ETDRS); SLE; IOP; dilated ophthalmoscopy; SD-OCT (if a subject’s screening SD-OCT does not demonstrate SRF, IRF, IRC, the site may repeat the screening SD-OCT one time during the screening period); CFP; IVFA; or concomitant medications. Visit 2 (Day 1) can include assessment for vital signs; PBMC collection; peripheral blood / tear PCR; blood for PK / PD; blood for ADA; BCVA (ETDRS); SLE; IOP; dilated ophthalmoscopy; SD-OCT; adverse events; concomitant medications; concurrent procedures; or EYLEA (afhbercept) 2 mg (after all other assessments). Visit 3 (Week 1) can include assessment for inclusion criteria or exclusion criteria; vital signs; BCVA (ETDRS); SLE; IOP; dilated ophthalmoscopy; SD-OCT; adverse events; concomitant medications; or concurrent procedures. Visit 4 (Week 2) can include assessment for vital signs; BCVA (ETDRS); SLE; IOP; dilatedWSGR Docket No. 59561-717.601ophthalmoscopy; SD-OCT; adverse events; concomitant medications; concurrent procedures; or Vector AAT administration (after all other assessments). Visit 5 (Week 2 + 2 days) can include assessment for vital signs; BCVA (ETDRS); SLE; IOP; dilated ophthalmoscopy; SD-OCT; adverse events; concomitant medications; or concurrent procedures. Visit 6 (Week 4) can include lab visit for central labs; peripheral blood / tear PCR; blood for PK / PD; or blood for ADA. Visit 7 (Week 8) can include assessment for vital signs; PBMC collection; peripheral blood / tear PCR; blood for PK / PD; blood for ADA; BCVA (ETDRS); SLE; IOP; dilated ophthalmoscopy; SD-OCT; adverse events; concomitant medications; concurrent procedures; or PRN EYLEA (aflibercept) 2 mg administration (after all other assessments). Visit 8 (Week 16) can include assessment for vital signs; PBMC collection; peripheral blood / tear PCR; Blood for PK / PD;Blood for ADA; Central Labs; BCVA (ETDRS); SLE; IOP; dilated ophthalmoscopy; SD-OCT; adverse events; concomitant medications; concurrent procedures; or PRN EYLEA (aflibercept) 2 mg administration (after all other assessments). Visit 9 (Week 24) to Visit 12 (Week 48) can include assessment for BCVA (ETDRS); SLE; IOP; dilated ophthalmoscopy; SD-OCT; adverse events; concomitant medications; concurrent procedures; or PRN EYLEA (aflibercept) 2 mg administration (after all other assessments). Visit 13 (Week 54) or early termination before Week 54 can include assessment for physical Exam; vital signs; PBMC collection; peripheral blood / tear PCR; Blood for PK / PD; Blood for ADA; BCVA (ETDRS); SLE; IOP; dilated ophthalmoscopy; SD-OCT; CFP; IVFA; adverse events; concomitant medications; concurrent procedures; or PRN EYLEA (aflibercept) 2 mg administration (Visit 13, only). Visit 14 (Week 62) to Visit 19 (Week 102) can include assessment for BCVA (ETDRS); SLE; IOP; dilated ophthalmoscopy; SD-OCT; adverse events; concomitant medications; concurrent procedures; or PRN EYLEA (aflibercept) 2 mg administration (after all other assessments). Visit 16 (Week 78) can include assessment for blood for PK / PD. Visit 20 (Week 106) or early termination before Week 106 can include assessment for physical exam; vital signs; Central Labs; PBMC collection; peripheral blood / tear PCR; blood for PK / PD; blood for ADA; BCVA (ETDRS); SLE; IOP; dilated ophthalmoscopy; SD-OCT; CFP; IVFA; adverse events; concomitant medications; or concurrent procedures.Assessment for efficacy

[0163] The primary objective of this phase l / 2a study is to determine the safety and tolerability of IVT Vector AAT injection in subjects with wAMD, including symptomatic macular PCV. Secondary objectives can include: to assess the preliminary efficacy of IVT Vector AAT injection in subjects with wAMD, including symptomatic macular PCV; to evaluate the PK / PDWSGR Docket No. 59561-717.601of IVT Vector AAT injection; to evaluate the immunogenicity of IVT Vector AAT injection; or to determine the optimal dose of IVT of Vector AAT for later phases of study injection. Efficacy assessments can include: BCVA; SD-OCT; color fundus; IVFA; or PK / PD. The frequency of testing can be found in the schedule of events (e.g., as shown in Table 6 or Table 7).Assessment of safety

[0164] The safety of Vector AAT can be assessed through the collection and analysis of adverse events (AEs), baseline medical conditions, results of physical exams, eye examination results, laboratory test results, incidence of serum antibodies to Vector AAT, or vital sign data. The assessment of AE can be performed by the investigator. Any events that occur after the informed consent form is signed but before treatment (before the EYLEA (aflibercept) injection at Day 1) can be considered part of medical history. The frequency of safety testing can be found in the schedule of events (e.g., as shown in Table 6 or Table 7) but can be more frequent at the discretion of the investigator based on symptoms.

[0165] Safety assessments can include monitoring and recording protocol-defined AEs and serious adverse events (SAEs), measurement of protocol-specified hematology, clinical chemistry, coagulation, urinalysis variables, measurement of protocol-specified vital signs, or other protocol-specified tests that are deemed critical to the safety evaluation of the study drug.Adverse event

[0166] An adverse event (AE) can be any unfavorable and unintended sign, symptom, or disease in a clinical trial subject that is temporally associated with the use of an investigational product or other protocol -imposed intervention, regardless of attribution. An example of the AE can include: exacerbation of pre-existing illness; an increase in frequency or intensity of a preexisting episodic event or condition; or continuous persistent disease or symptoms present at baseline that worsen following the start of the study. An adverse event does not include a preexisting disease or conditions present or detected at the start of the study that does not worsen; medical or surgical procedure (e.g., surgery, tooth extraction, transfusion); the medical condition that leads to the procedure may be an AE; or elective surgery or routine diagnostic procedures are not considered AEs.Dose-limiting toxicity

[0167] Dose limiting toxicities (DLT) can be defined as toxicities that have not resolved within 14 days. DLTs can include treatment emergent ophthalmic AEs and systemic AEs. Ophthalmic AEs can be graded according to a 3-point scale (mild, moderate, or severe), and uveitis can be graded according to the Standardization of Uveitis Nomenclature (SUN) system. Systemic AEsWSGR Docket No. 59561-717.601can be graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events Version 5.0 (CTCAE v5.0). All Grade 3 toxicities for vital signs and laboratory parameters can be confirmed with a repeat measurement obtained within 1 hour for vital signs and as soon as laboratory retest is possible for laboratory parameters. Additional ophthalmic DLTs can include, but not limited to: severe grade ophthalmic AE according to a 3-point scale (mild, moderate, or severe); severe intraocular inflammation according to the SUN system (4+ anterior chamber cell / flare or vitreous haze score 4); decrease in BCVA score of > 15 letters ETDRS; or IOP > 35 mm Hg that cannot be medically managed. In addition, systemic DLTs can include, but not limited to: any Grade > 3 systemic AE or laboratory abnormality as defined in CTCAE (version 5.0); or any death not clearly due to extraneous causes. Suspected, unanticipated, serious adverse reactions (SUSAR)s or Systemic AEs that are grade 2 and have persisted past two weeks can also be considered as a potential DLT.Serious adverse event

[0168] A serious adverse event (SAE) can be any untoward medical occurrence that: results in death; is life-threatening; any AE that places the subject, in the view of the investigator, at immediate risk of death; requires in-patient hospitalization or prolongation of existing hospitalization (elective hospitalization is not an SAE); results in persistent or significant disability or incapacity; results in a congenital anomaly / birth defect; is an event that required intervention to prevent permanent impairment or damage (events for which elective interventions are performed for reversible impairment, e.g., cataract surgery, are not considered SAEs); or is considered a significant medical event by the investigator (e.g., may jeopardize the subject or require medical surgical intervention to prevent one of the outcomes listed above). Severity refers to the intensity of an AE (as in mild, moderate, or severe pain); the event itself may be of relatively minor medical significance (such as a severe headache). “Serious” is a regulatory definition and is based on event outcome or action criteria usually associated with events that pose a threat to a subject’s life or vital functions. Seriousness (not severity) serves as the guide for defining regulatory reporting obligations.Adverse event severity

[0169] Adverse event (AE) severity can include ophthalmic adverse event grading. For grading of ophthalmic adverse events, Table 8 can be used.Table 8. Ophthalmic adverse events gradingWSGR Docket No. 59561-717.601<

[0170] Tables 9-11 illustrate standardization of uveitis nomenclature for the schema for grading intraocular inflammation (anterior chamber cell, anterior chamber flare, or vitreous flare). All AEs can be assessed by the investigator using National Cancer Institute Common Terminology Criteria for Adverse Events Version 5.0 (CTCAE v5.0) to quantify severity (intensity) as illustrated in Table 12. Systemic or laboratory abnormalities can be individually graded using CTCAE v5.0 specific to that abnormality.Table 9. Anterior chamber cell<>*Field size is a 1 mm by 1 mm slit beamTable 10. Anterior chamber flareTable 11. Vitreous hazeWSGR Docket No. 59561-717.601Table 12. CTCAE for systemic event gradingAbbreviations: AE = adverse event; ADL = activities of daily livinginstrumental ADL refer to preparing meals, shopping for groceries or clothes, using the telephone, managing money, etc.bSelf-care ADL refer to bathing, dressing, and undressing, feeding self, using the toilet, taking medications, and not bedridden.Study drug causality

[0171] An event is considered related if there is a reasonable possibility that there is a causal relationship between event and the study intervention. Any AE judged by the investigator or sponsor representatives as having a reasonably suspected, causal relationship to the study intervention qualifies as a related AE. In addition, this study collects information on AEs that are related to the intervention procedure (e.g., IVT injection procedure). The investigator can be obligated to assess the relationship between study intervention and each occurrence of each AE / SAE. The investigator may change opinion of causality in light of follow-up information and send a follow-up report with the updated causality assessment. Adverse events can be categorized as not related if any of the following exists: another cause of the event is most plausible; aWSGR Docket No. 59561-717.601clinically plausible temporal sequence is inconsistent with the onset of the event and the study intervention; or a causal relationship is considered biologically implausible. Adverse events can be categorized as related if any of the following exists: a “reasonable possibility” of a relationship conveys that there are facts, evidence, and / or arguments to suggest a causal relationship, rather than a relationship cannot be ruled out; the event is uncommon in the general population and follows a reasonable, temporal sequence from study intervention; the event cannot readily have been produced by the clinical state of the patient, environmental factors, concomitant medications, or other modes of concomitant therapy; or the event improves on stopping study intervention and reappears upon subsequent injection of study intervention.Relationship assessment for individual AEs is part of investigator responsibilities. Regardless of relationship, sponsor representatives review all AEs against cumulative experience to identify and communicate new safety findings to investigators and regulatory authorities, as applicable.Adverse event reporting procedures

[0172] All AEs / SAEs can be collected from the start of intervention until the final study visit. Events occurring after signing the Informed Consent Form (ICF) but prior to the first dose should be documented as medical history, unless the event is study procedure-related, in which case it should be reported as an AE. Events observed during or following the first injection procedure of the protocol (EYLEA (aflibercept)) until the final study visit, are to be recorded as AEs. Site personnel can provide subjects with investigator contact information for reporting SAEs. All SAEs can be recorded and reported to the sponsor or designee immediately and under no circumstance should this exceed 24 hours. The primary mechanism for reporting an SAE to the sponsor can be a paper-based SAE form. If an ongoing SAE that is assessed as related to study intervention remains unresolved at the conclusion of the study, additional follow-up must continue until resolution or stabilization of the SAE. All initial and follow-up information regarding SAEs or suspected unexpected serious adverse reactions (SUSARs), including those related to protocol mandated procedures, must be reported by the investigator immediately (within 24 hours of discovery) to a sponsor representative. Reporting must not be delayed by waiting for additional information. Additional information can be provided in a follow-up report. Forms and instructions for completion and submission of SAE report forms are provided as part of the study instructions.Reporting serious adverse events to regulatory agencies (SUSAR)

[0173] A SUSAR can be any AE for which there is evidence to suggest a causal relationship between the study intervention and the AE, and which can be assessed as both unexpected andWSGR Docket No. 59561-717.601serious. An unexpected adverse reaction (e.g., any untoward and unintended response to the study intervention) is one for which the nature and severity is inconsistent with the applicable reference safety information (per the Investigator’s Brochure). SUSARs are subject to expedited reporting to applicable regulatory authorities.Pregnancy

[0174] Pregnancies that occur during study participation, while not considered AEs, can be reported by the investigator to sponsor representatives within 1 business day of awareness. The pregnancy should be followed until there is a pregnancy outcome. Following delivery or termination of the pregnancy, the investigator can report the pregnancy outcome within 1 business day of awareness. Any AEs observed during pregnancy or any abnormality in the newborn are evaluated for potential SAE reporting. Newborns can be followed for at least 6 months whenever possible, for any potential congenital anomalies. Sponsor representatives ensure due diligence to collect the outcome of the pregnancy (e.g., term or preterm delivery, induced abortion, stillbirth, or spontaneous abortion), details of the birth if available, and additional relevant information.Follow up of adverse events

[0175] The investigator should follow all AEs and SAEs until the events are resolved or stabilized, the subject is lost to follow-up, or it has been determined that the study treatment or participation is not the cause of the AE / SAE. Resolution of AEs and SAEs (with dates) should be documented on the appropriate AE / SAE eCRF and the subject’s medical record to facilitate source data verification (SDV). Signs or symptoms of a suspected overdose of investigational product should be reported as an AE / SAE (i.e., “Overdose” per se should not be reported as an AE / SAE).Statistics

[0176] Data can be summarized using descriptive statistics (number of subjects, mean, median, standard deviation, minimum and maximum for continuous variables and frequencies and percentages for discrete variables. Data can be presented by dose level of Vector AAT. Key listings can be created. This study is descriptive in nature, and no formal comparisons or hypothesis testing can be performed. Exploratory analyses of the data can be conducted as deemed appropriate.Sample size and powerWSGR Docket No. 59561-717.601

[0177] Sample size for this study can be determined by clinical and practical rather that statistical considerations. Approximately 18 subjects are planned for this study as described in the description of the study.Analysis sets

[0178] The safety analysis set can include all subjects who received Vector AAT and can be the basis of analysis for safety and efficacy endpoints. The pharmacokinetic analysis set includes all subjects who received Vector AAT and have a baseline and at least 1 post-baseline blood sample to measure PK parameters. The pharmacokinetic analysis set can be the basis of the analysis of PK results. The pharmacodynamic analysis set can include all subjects who received Vector AAT and have a baseline and at least 1 post-baseline blood sample to measure PD results. The pharmacodynamic analysis set can be the basis of the analysis of pharmacodynamic results. A per protocol set may be the basis of a selection of analyses and would include all subjects who received Vector AAT and do not have important protocol deviations. Important protocol deviations may include but are not limited to violations of eligibility criteria and non-compliance with study assessments.Subject disposition

[0179] Subject disposition, including receipt of Vector AAT, duration on study, early discontinuation, and reason for discontinuation can be summarized.Baseline and demographic characteristics

[0180] Subject demographics (gender, age, ethnicity, and race) and baseline characteristics (ophthalmic examination results as applicable) can be summarized using the safety analysis set.Safety analysis

[0181] The safety analyses outlined below can focus on the treatment-emergent period, which is defined as the time from the first administration of any study treatment (Vector AAT or EYLEA (aflibercept) and occur before termination of the study (or date of study discontinuation) or were present before study drug administration and worsened after dose administration. Safety data analysis can primarily be conducted on the safety analysis set. Adverse events can be mapped to system organ class (SOCs) and preferred terms (PTs) using the Medical Dictionary for Regulatory Activities (MedDRA). Adverse events can be monitored during the study and the data analyzed with respect to overall incidence as well as severity and potential relationship of AEs to study treatments. Treatment-emergent adverse event (TEAE) incidence tables can present the number (n) and percentage (%) of participants experiencing at least one TEAE by SOC and PT. Multiple occurrences of the same event in the same participant can be counted only once in theWSGR Docket No. 59561-717.601tables. The denominator for computation of percentages is the safety population within each group. TEAE incidence tables can be provided by dose level for: TEAEs; TEAE by severity; TEAEs by relationship (related or not); treatment-emergent SAEs; or TEAEs leading to study discontinuation. TEAE summaries can be presented by study eye, fellow eye, and for non-ocular adverse events. Laboratory parameters can be summarized using descriptive statistics. Shift tables reflecting changes from baseline may be presented in lieu of descriptive statistics of changes from baseline. Vital sign measurements can be summarized by changes from baseline values at each Vector AAT dose level using descriptive statistics. Safety results (including DLTs) cam be listed for all subjects. Subject deaths can be listed.Pharmacokinetics Analysis

[0182] Pharmacokinetics analyses can be conducted on the PK analysis Set. Individual cellular PK parameters, including but not limited to maximum concentration (Cmax), time to maximum concentration (Tmax), area under the curve (AUC), or duration of persistence of Vector AAT in the peripheral blood can be estimated using non-compartmental models.Pharmacodynamic Analysis

[0183] Changes in PD parameters by Vector AAT dose level can be provided.Immunogenicity analysis

[0184] Summary statistics of SD-OCT results by Vector AAT dose level can be provided.Efficacy Analysis

[0185] BCVA by time point and changes in BCVA from baseline to each time point can be summarized by Vector AAT dose level.Rescue treatment

[0186] Anti-VEGF rescue treatment may be administered in the study eye. Subjects who received rescue treatment can be summarized by Vector AAT dose level and time point.Source data documentation

[0187] Study monitors can perform ongoing source document verification to confirm that critical protocol data (i.e., source data) entered into the eCRFs by authorized site personnel are accurate, complete, and verifiable from source documents.

[0188] Source documents are where subject data are recorded and documented for the first time. They include, but are not limited to: hospital records, clinical and office charts, laboratory notes, memoranda, subject diaries or evaluation checklists, pharmacy dispensing records, recorded data from automated instruments, copies of transcription that are certified after verification as being accurate and complete, microfiche, photographic negatives, microfilm or magnetic media, x-rays,WSGR Docket No. 59561-717.601patient files, and records kept at the pharmacy, laboratories, and medico-technical departments involved in a clinical trial.

[0189] Source documents that are required to verify the validity and completeness of data entered into the eCRFs must never be obliterated or destroyed.

[0190] Use of computerized systems

[0191] When clinical observations are entered directly into an investigational site’s computerized medical record system (i.e., in lieu of original hardcopy records), the electronic record can serve as the source document if the system has been validated in accordance with FDA requirements pertaining to computerized systems used in clinical research. If a site’s computerized medical record system is not adequately validated for the purposes of clinical research, applicable hardcopy source documents must be maintained to ensure that critical protocol data entered in the eCRFs can be verified.Study medication accountability

[0192] All study drug required for completion of this study can be provided. The recipient can acknowledge receipt of the drug, indicating shipment content and condition. Damaged supplies can be replaced. Accurate records of all study drug received at, dispensed from, and returned should be recorded.Quality control and quality assurance

[0193] Prior to participation, sponsor representatives evaluate potential study sites and investigators for appropriate qualifications and ability to conduct the study. Study training must take place at each study site before any participants may be enrolled at that site. The study initiation visit may include a review of GCP guidelines, study protocol and procedures review, management of AEs, preparation and injection of Vector AAT, and data collection and participant eligibility requirements.

[0194] All clinical work conducted under this protocol is subject to GCP regulations. This includes an inspection by sponsor and competent-authority representatives at any time. The investigator must agree to the inspection of study-related records by competent-authority representatives and audits by sponsor representatives. Sponsor representatives may conduct a quality assurance audit at the study site at any time before, during, or after completion of the study. Sponsor representatives inform the investigator if an audit is to take place and advise him or her as to the scope of the audit. Representatives of competent authorities may also conduct an audit of the study site. If informed of such an inspection, the investigator should notify sponsorWSGR Docket No. 59561-717.601representatives immediately. The investigator must ensure that auditors have access to study supplies, site facilities, original source documentation, and all study records.Ethics

[0195] The investigator can ensure that this study is conducted in conformity with the protocol, all local and country regulatory requirements, ICH GCP regulations and guidelines, and the Declaration of Helsinki, whichever affords the greater protection to the subject.Data handling and record keeping

[0196] The investigator can agree to maintain accurate source documentation as part of the case history of each subject. Recorded data should be updated / corrected in a manner that does not obliterate, destroy, or render illegible the previous entry (e.g., by drawing a single line through the incorrect entry and writing the revision next to the corrected data). An individual who has corrected an entry should make clear who made the correction and when by adding his / her initials and the date of the correction. Subject medical information obtained by this study is confidential and may only be disclosed to third parties as permitted by the Informed Consent Form (or separate authorization to use and disclose personal health information) signed by the subject or unless permitted or required by law. Medical information may be given to a subject’s personal physician or other appropriate medical personnel responsible for the subject’s welfare for treatment purposes. Data generated by this study must be available for inspection upon request by representative of the US FDA and other regulatory agencies, national, or local health authorities. US FDA regulations [21 CFR 312.62(c)] and the ICH Guideline for GCP (see Section 4.9 of the guideline) require that records and documents pertaining to the conduct of this study and the distribution of investigational drug, including the eCRFs, consent forms, laboratory test results, and medication inventory records, must be retained by the principal investigator for 2 years after the last marketing application approval in an ICH region or after at least 2 years have elapsed since formal discontinuation of clinical development of the investigational product. All state and local laws for retention of records also apply. No records should be disposed of without the written approval of Avirmax Biopharma Inc. Written notification should be provided to Avirmax Biopharma Inc. for transfer of any records to another party or moving them to another location. For studies conducted outside the United States under a US IND, the principal investigator must comply with the record retention requirements set forth in the US FDA regulations and the relevant national and local health authorities, whichever is longer.Procedure for Vector AAT injectionWSGR Docket No. 59561-717.601

[0197] Preparation of Vector AAT for injection can include: withdraw the entire volume of Vector AAT (approximately 0.12 mL) from the sterile vial, using the sterile (0.25 mL) syringe with an 18-gauge x 1-1 / 2-inch, 5-micron, filter needle; replace the filter needle with a 30-gauge x 1 / 2-inch injection needle; or expel the excess volume of Vector AAT from the syringe just prior to injection, leaving only the required injection volume (50 pL) in the syringe.

[0198] For intravitreal injection, the study eye can be first verified. Additional procedure can include: apply 1 drop of topical phenylephrine hydrochloride ophthalmic solution 2.5% to the study eye 30 minutes before the injection to allow visualization of the posterior pole after the injection, if necessary; have the participant lie back in the examination chair with the neck well supported; apply topical proparacaine 0.5% to the study eye; apply povidone-iodine 10% to the eyelashes and eyelid margins. Avoid extensive massage of the eyelids either pre- or postinjection to avoid meibomian gland expression; retract the eyelids away from the intended injection site for the duration of the procedure; or apply povidone-iodine 5% to the conjunctival surface, including the intended injection site. For injection with Vector AAT, the needle can be inserted perpendicularly to the sclera, 3.5 to 4 mm posterior to the limbus, between the vertical and horizontal rectus muscles. A sterile cotton-tip applicator can be applied over the injection site immediately following removal of the needle to reduce vitreous reflux.Post-intravitreal injection

[0199] Procedure for post-intravitreal injection can include: assess hand motion vision or central retinal artery perfusion immediately and rule out other causes of vision loss such as vitreous hemorrhage. If hand motion vision or central retinal artery perfusion is not observed and no other cause of vision loss is found, perform digital massage and administer topical / oral IOP lowering medications until hand motion vision or central retinal artery perfusion is observed; or obtain IOP measurement at 30 minutes post-injection and then every 15 minutes until IOP < 25 mm Hg. Intraocular pressure in excess of 30 mm Hg for more than 15 minutes should be treated at the physician’s discretion. Topical antibiotics are not required.Best corrected visual acuity (BCVA) testing

[0200] The Example 2 involves the assessment of Best Corrected Visual Acuity (BCVA). The BCVA testing can be measured according to standard procedures developed for the Early Treatment Diabetic Retinopathy Study (ETDRS). The BCVA assessments can be performed using previously certified visual acuity lanes and visual acuity examiners. Appropriate and relevant documentation of previous certification procedures should be available for the study file. No additional equipment or examiner certifications are required for the study. The followingWSGR Docket No. 59561-717.601visual acuity testing equipment is used: a set of three charts - the modified ETDRS Charts 1, 2, and R; and a retro-illuminated box (light box) providing standardized chart illumination.

[0201] Visual acuity testing cam be performed in a visual acuity testing room (“visual acuity lane”) and is required at a distance of 4 meters and, for patients with reduced vision, at a 1 -meter distance. The 4-meter distance should be marked clearly and permanently in the room; the 1-meter distance is measured with a 1 -meter stick with the patient sitting in a chair.Visual acuity charts

[0202] Charts 1 and 2 are used for testing the right and left eye, respectively, and Chart R is used for refraction. The features of the charts are high-contrast Sloan letters of equal difficulty, 5 letters in each of the 14 lines, and a geometric progression of letter size [and, thus, an arithmetic progression of the logarithm of minimum angle of resolution (LogMAR) from line to line]. Charts 1, 2, and R have different letter sequences. Patients should be prevented from seeing Charts 1 and 2 until refraction has been completed and the visual acuity test begins.Visual Acuity Light Box

[0203] The dimensions of the light box are 62.9 cm (24.75 inches) by 65.4 cm (25.75 inches) by 17.8 cm (7 inches). The box can be mounted on a wall, on a countertop, or on a stand mounted on a wheelbase. The light box should be mounted or placed at a height such that the top of the third row of letters (0.8 LogMAR) is 124.5 ± 5.1 cm (49 ± 2 inches) from the floor. The rear of the box provides storage space for the two charts not being used.

[0204] The light box is equipped with two daylight 20-watt fluorescent tubes. Given that the illumination of fluorescent tubes diminishes by 5 percent during the first 100 hours and by another 5 percent during the next 2,000 hours, new tubes should be kept on for 96 hours (does not have to be continuous), and all tubes should be replaced every 12 months. Clinical centers are required to maintain a written log with the date (month, day, and year) of the last change of tubes. This log should be best kept on the back of the light box. The status of tubes can be checked periodically.

[0205] The fluorescent tubes should be checked periodically for proper functioning.Replacement tubes can be purchased at a local store or from specialized providers. Each tube is partially covered by a 35.6 cm (14-inch) fenestrated sleeve, open in the back, which serves as a baffle to reduce illumination. Each sleeve should be centered on the tube such that an equal length of tube, approximately 10.6 cm (4.25 inches) is left uncovered to the right and left of the sleeve. The openings in the backs of the sleeves should be oriented to point directly toward the back of the box (i.e., the sleeves should not be tilted up or down). Light tubes with built-inWSGR Docket No. 59561-717.601fenestrated sleeves are the most currently available and are interchangeable with all ETDRS light boxes without regard to manufacture date.

[0206] The appropriate working status of both charts and light boxes is required in the documentation for the study, although the above procedures could have been completed as part of other studies that use the same charts and light boxes at the clinical site. If the site has completed these preparation procedures, it does not need to repeat them if documentation is available for the study file.

[0207] The BCVA required testing distance in the study is 4 meters, and 1 meter for patients with reduced vision. The distance of 4 meters (13 feet and 1.5 inches, or 157.5 inches) is required between the patient's eyes and the visual acuity chart for the 4-meter test, and a distance of 1 meter (393 / s inches) is required for the 1 -meter test. Therefore, the room for visual acuity testing must have, in addition to the 4-meter lane, space for the visual acuity box (for a wall-mounted box, an additional 17.8 cm or 7 inches; for a stand-mounted box, an additional 33 cm or 13 inches) and space for the patient and the chair. Most of the room lights should be turned off during the visual acuity test. The box itself provides sufficient illumination for the examiner to record the test results.

[0208] The study does not require a specific refraction protocol for the BCVA testing. The refraction Chart R or another standard clinic refraction chart in any room and at any distance is permitted to determine the best correction in each eye. This minimizes the patient’s waiting time and the impact on the clinical center. The best lenses correction obtained are then placed in the lens trial frame and adjusted on the patient’s face so that the lenses are parallel with the orbits and are centered in front of the pupils.4-Meter Visual Acuity Testing

[0209] Visual acuity testing is performed with the trial frames containing the best correction lenses. One eye is tested at a time, with the other eye occluded. The right eye is tested first using Chart l(as illustrated in Fig. 7A), and the left eye is tested second using Chart 2 (as illustrated in Fig. 7B). The testing procedure for visual acuity is driven by the objective to test visual acuity and not ability to concentrate or follow instructions, or intelligence (although all of these factors are influencing the test). The patient should be told that the chart has letters only and no numbers. If the patient reads a number, he or she should be reminded that the chart contains no numbers, and the examiner should request a letter instead.

[0210] The patient should be asked to read slowly, at a rate of about one letter per second in order to achieve the best identification of each letter and to only proceed when the patient hasWSGR Docket No. 59561-717.601given a definite response. If the patient loses his or her place in reading or the examiner loses his or her place (possibly because the letters are read too quickly), the examiner should ask the patient to go back to where the place was lost. Examiners should not point to the chart or to specific letters on the chart or read any of the letters during the test.

[0211] Each letter is scored as right or wrong. Once a patient has identified a letter with a definite single-letter response and has read the next letter, a correction of the previous letter cannot be accepted. If the patient changes a response aloud (e.g., "That was a “C,” not an “O") before he or she has read aloud the next letter, then the change should be accepted; if the response is changed after beginning to read the next letter, the change is not accepted.

[0212] When the patient states that he or she cannot read a letter, they should be encouraged to guess. If the patient identifies a letter as one of two or more letters, he or she should be asked to choose one letter and, if necessary, to guess even if the next letter has already been read. The examiner may encourage the patient to move his or her head from side to side or up and down without moving forward or backward and if they do this, the fellow eye should remain covered. When it becomes evident that no further meaningful readings can be made, despite urgings to read or guess, the examiner should stop the test for that eye.

[0213] The reasons for encouraging patients to guess: (1) patients' statements that they cannot identify a letter are often unreliable; (2) encouraging them to guess helps to maximize their effort; (3) it helps assure uniformity among procedures performed in different clinical centers; and (4) it may help prevent patient bias (malingering).1 -meter visual acuity testing

[0214] If fewer than 19 letters are read correctly at 4 meters with an eye, that eye should be tested at 1 meter. Before testing at 1 meter, a +0.75 sphere should be added to the 4-meter correction already in the trial frame to compensate for the closer testing distance. The patient may stand or sit for the 4-meter test but can sit for the 1 -meter test and avoid head movement forward or backward. The patient is asked to read only the first 6 lines of the chart at 1 meter, for the maximum attainable score of 30 at that distance.Scoring BCVA

[0215] The examiner can record each letter identified correctly by circling the corresponding letter on the visual acuity worksheet as shown in Fig. 7A or Fig. 7B. Letters read incorrectly and letters for which no guesses are made are not marked on the form. Each letter read correctly is scored as one point. The score for each line and the total score for each eye are recorded on the visual acuity worksheet as shown in Fig. 7A or Fig. 7B after testing is completed. A line whereWSGR Docket No. 59561-717.601the patient attempted to guess letters but did not guess any letters correctly should have a zero (0) entered as the score for that line. Any line(s) on which the patient did not attempt to guess letters should be marked with a dash (-). If testing at 1-meter is not required, 30 points are automatically scored for the 1-meter test. The total combined score (i.e., the sum of the 4 and 1-meter scores) and the approximate Snellen acuity equivalent (i.e., the lowest line read with one or fewer mistakes), are recorded on the visual acuity worksheet.Count fingers, hand motion, light perception, and no light perception

[0216] If the patient cannot read the largest letter even at the 1-meter distance, the examiner holds his or her fingers before the patient’s eye in good light and the vision is recorded as the furthest distance at which the fingers can be counted (for example, count fingers (CF) at 3 feet or CF at 1 foot) on the visual acuity worksheet as shown in Fig. 7A or Fig. 7B. If the patient cannot distinguish fingers, the examiner should wave a hand in front of the eye; movements of the hand that are perceived by the patient, should be recorded as hand movement (HM) visual acuity on the visual acuity worksheet as shown in Fig. 7A or Fig. 7B. If visual acuity is so poor that the patient cannot read any of the largest letters at 1 meter or fails CF and HM test, light perception should be tested with the indirect ophthalmoscope as the light source. Room lighting should remain at the level of normal visual acuity testing. The patient should close the opposite eye and occlude it by making a tight seal with the palm around the orbit and the bridge of the nose. The indirect ophthalmoscope light should be in focus at 1 meter (approximately 3 feet) with the rheostat set at maximum voltage and from that distance the beam should be directed in and out of the eye at least 4 times, and the patient should be asked to respond when he or she sees the light. If the examiner is convinced that the patient perceives the light, vision should be recorded as "light perception (LP)"; if not, vision should be recorded as "no light perception (NLP).” Example 3. Vector AAT information and assessment

[0217] Example 3 illustrates examination of Vector AAT and its safety and therapeutic efficacy.Table 13 lists abbreviations as shown in Example 3. Vector AAT (AAV2.N54-VEGF-Trap) is a recombinant, self-complementary, adeno-associated virus vector (rAAV) serotype 2 (AAV2) carrying a transgene coding for the same protein sequence as aflibercept, which functions as a vascular endothelial growth factor (VEGF) trap. The capsid protein (VP1) of Vector AAT is modified for intravitreal (IVT) delivery and transduction of retinal cells near blood vessels and the macula. This engineered capsid is named as N54. Once Vector AAT crosses the inner limiting membrane (ILM) and penetrates all layers of the retina, the aflibercept protein encoded from the transgene binds VEGF and blocks VEGF-associated angiogenesis, relieving theWSGR Docket No. 59561-717.601symptoms of neovascular (wet) age-related macular degeneration (wAMD) or symptomatic macular polypoidal choroidal vasculopathy (PCV). Preclinical studies in this example can support the initiation of a phase 1 clinical trial of Vector AAT in wAMD patients. The preclinical program, including pharmacodynamics (PD), biodistribution (BD), or toxicology studies, is designed to evaluate the mechanism of action (MO A), dose-response relationships, pharmacokinetics (PK), tissue distribution, or potential target organ toxicity of Vector AAT. Table 13. List of abbreviationsWSGR Docket No. 59561-717.601WSGR Docket No. 59561-717.601

[0218] For Vector AAT, coding sequence for aflibercept was cloned into a series of AAV-based expression cassettes and lab-scale AAV were produced with the AAV2.N54 capsid. A total of 18 transgene expression cassettes were designed and screened. The expression profile of all the constructs was assessed after transfection into human embryonic kidney cells (HEK293) and ARPE-19 cells (human retinal pigment epithelial cells). Transgene expression was measured by ELISA after transfection into HEK293 and APRE-19 cells. The chosen cassette is shown schematically in Fig. 8, consisting of a double-stranded DNA or self-complementary DNA (scDNA) with the human cytomegalovirus immediate early enhancer / promoter driving VEGF-Trap expression with the SV40 intron, a synthetic Kozak sequence and a polyA tail and a truncated 3’ ITR. Vector AAT exhibited high transgene expression levels. Vector AAT is a purified rAAV vector composed of capsid proteins and enclosed dsDNA, which encodes VEGF-Trap protein after transduction into host cells. Vector AAT can be formulated in 10 mM sodium phosphate, 180 mM sodium chloride, 0.001% poloxamer 188, pH 7.3. Vector AAT can function through intravitreal administration of recombinant AAV encoding aflibercept protein, via transduction of retinal cells and sustained transgene expression of aflibercept. Vector AAT can be supplied as a sterile frozen liquid formulation stored in 2 mL, 13 mm Daikyo Crystal Zenith® vials (CZV). The vials can be stored in a <-60oC freezer. In terms of handling, the drug productWSGR Docket No. 59561-717.601can be taken out from the freezer, thawed at room temperature, cleaned on the surface of the vial, sealed with disinfectant, e.g., 70% alcohol wipe, and aspirated into a sterile StaClear® 0.25 mL Luer-Lock syringe with a BD blunt 18G 11 ” needle, 5 Micron filter needle and followed by a dosing intravitreally using a BD PrecisionGlideTM Needle, 30G x / i (0.3 mm xl3 mm) Dosing Needle at medical office settings.Evaluation of Vector AAT for efficacy in mouse laser choroidal neovascularization (LCNV) model

[0219] Aflibercept had already been shown to have sufficient cross-reactivity to the mouse form of VEGF which is one of the main drivers of the retinal leakage in the mouse LCNV model, to demonstrate some efficacy in this model, so it was decided to evaluate the efficacy of Vector AAT, compared to IVT dosing with Aflibercept (Eylea®) in this model initially. AAV2.N54-VEGF-Trap (Vector AAT) was injected at three different doses (2 x 107, 4 x 108, and 1.6 x 1010vg / eye), on Day -28 to ensure adequate expression prior to laser induction; vehicle and Eylea® were injected on Day -3. Both color and cobalt blue (EGFP expression) fundus imaging were performed on both eyes (OU) of Group 3 (AAV2-GFP; 1.6 x 1010vg / eye) animals on Day 7 after laser. On Day 7, after systemic dosing with fluorescein to enable the Fluorescein angiography (FA) analysis, animals were humanely euthanized, and ocular tissues were collected for flatmount analysis. The data from Day 7, both FA and flat-mount analysis is shown in Fig. 9 and Fig. 10. Fig. 9 illustrates representative fundus images, cobalt blue (fluorescein-leakage), from each group on Day 7 post-laser are shown. Measurement of lesions in the AAV2-GFP dosed group was not feasible as the GFP expressed likely drove inflammation such that lesions were so large and precluded analysis. Graphical representation of the area of fluorescein leakage after the laser bums and quantitative analysis of the other groups are shown in the lower panel.AAV2.N54-Aflibercept is later named AAV2.N54-VEGF-Trap (Vector AAT). Mean±SD.

[0220] Fig. 10 illustrates representative lesion images from flat mounts on Day 7 post-laser. Quantitative analysis is also shown in the lower panel. Mean±SD. Protection against laser CNV in the mouse (C57BL / 6) is shown from increasing single IVT doses of Vector AAT. Note that some data points had to be excluded due to technical issues so the numbers of lesions scored are listed out of the 16 possible for each group: Vehicle 12 / 16; Elyea 11 / 16; Vector AAT 2 x 107vg 12 / 16; Vector AAT 4 x 108vg 15 / 16; Vector AAT 1.6 x 1010vg 5 / 16.

[0221] Eyes dosed with AAV2-GFP, (a negative control, incorporating AAV injection and expression of an irrelevant transgene), had the largest lesion area, though this may have been impacted in part by an inflammatory response to the GFP transgene and the AAV2 capsidWSGR Docket No. 59561-717.601delivered IVT. The negative control of IVT injection of vehicle had the next largest CNV lesion size and leakage and the performance of both Eylea® and varying doses of Vector AAT in reducing these lesions are compared from this experiment in the prophylactic setting in Fig. 9 and Fig. 10. It is clear from the flat-mount pictures and the FA analysis shown in Fig. 9, that the highest dose of Vector AAT (AAV2.N54-VEGF-Trap at 1.6 x IO10vg / eye) had the smallest average lesion area, whilst the vehicle group had the largest average lesion area.

[0222] Animals injected with Eylea® (Aflibercept protein) had decreased lesion size at Day 7 post-laser when compared to animals in the vehicle group. In comparison to Eylea® (positive control; 40.5% reduction in fluorescein area), the two doses of Vector AAT (4 x 108 and 1.6 x 1010 vg / eye) demonstrated equivalence or superiority by at least one endpoint analysis, with the highest dose of Vector AAT (1.6 x 1010vg / eye) having a 68% reduction in vascular leakage for the angiography endpoint. These results demonstrated the prophylactic efficacy of Vector AAT in reducing CNV lesions induced by laser in the mouse on Day 7 post-laser.

[0223] The results show protection against LCNV in a mouse model and neutralization of induced VEGF via the production of aflibercept from a single dose of Vector AAT, in a dosedependent manner. Aflibercept protein exhibited cross-reactivity to mouse VEGF but only showed robust activity as a protein at high doses in mouse LCNV models, so this acted as a comparative positive control for high-level aflibercept expression from an AAV vector. The lower panel of Fig. 9 and Fig. 10 show the quantification of lesion areas in FA and flat-mount images of each group, and statistical analysis showed a significant reduction in FA lesion areas in treated mice (at least high dose).Evaluation of Vector AAT for efficacy in rabbit DL-q-AAA challenge retinal neovascularization model

[0224] Ocular vascular pathological conditions are characterized by increased vascular permeability and growth of new vessels which may bleed or leak exudates and cause retinal edema followed by fibrous scar that destroys the photoreceptor cells in the retina. One obstacle to the discovery and development of improved therapies for retinal vascular diseases is the lack of animal models with larger eyes that can mimic the chronic phenotype of human ocular vascular diseases. It has been shown that post laser treatment in the LCNV model, VEGF levels reach a peak on Day 5 but decline quickly thereafter causing the CNV lesion to completely heal by Day 14. A recent model has been developed and characterized and relies upon a DL-a-AAA (retinal glial cell toxin) induced retinopathy in pigmented Dutch Belted rabbits and it has been evaluated, and it provides a chronic or therapeutic model with a long duration of action. For AAV-basedWSGR Docket No. 59561-717.601gene therapy development, it was deemed important to demonstrate the efficacy of Vector AAT in a chronic, therapeutic preclinical model in addition to prophylaxis. The treatment efficacy of Vector AAT was performed using the Rabbit DL-a-AAA model and is detailed in Report 23-AVI-013. Two doses: high (1.9 x 1011vg / eye) and low (1.7 x IO10vg / eye) of Vector AAT were tested for efficacy against a single dose of Eylea® in a single eye (OS) where retinal leakage had already been induced with IVT DL-a-AAA and AAV was introduced 21 days post-induction with the impact on retinal leakage score by fluorescein angiography monitored at period intervals throughout the study until termination on Day 106, where samples were taken for biodistribution analysis.

[0225] A composite of all the imaging data measured as fluorescein leakage is shown in Fig. 11 and Fig. 12. Fig. 11 illustrates images of retinal leakage in rabbit DL-a-AAA challenge retinal neovascularization Model. Fig. 12 illustrates change of retinal leakage over time in corrected total regional fluorescence over time. Mean±SEM. The retinal leakage of all groups on Day 17 was 108-124 arbitrary unit (a.u.) On Day 29, following IVT administration on Day 22, the retinal leakage of animals treated with Eylea®, Vector AAT (1.9 x 1011vg / eye, high dose) decreased substantially compared to Day 17 retinal leakage. Retinal leakage in Vector AAT (1.7 x IO10vg / eye - low dose Vector AAT) also decreased, but less than in the other groups. Retinal leakage in animals treated with the vehicle remained high. Retinal leakage from animals treated with Eylea® increased substantially on Day 78 and returned to slightly above their Day 17 levels. The retinal leakage in Vector AAT (both high and low doses) remained low throughout the study up until the endpoint on Day 106. Retinal leakage data in this model indicated that IVT administration of Eylea began to lose effectiveness after Day 64, but that both low and high doses of Vector AAT (AAV2.N54-VEGF-Trap) were still effective at attenuating retinal leakage through Day 106. This finding indicates that Vector AAT has a long-acting efficacy from a single IVT dose while Eylea did not in a chronic model of retinal vascular leakage. The model had been utilized to validate and develop both therapies and retrospectively to validate previously licensed anti-VEGF approaches such as aflibercept for ocular vascular diseases over an extended period of time.Biodistribution in mice

[0226] The objective of this non-clinical study was to evaluate ocular and systemic pharmacokinetics and biodistribution of Vector AAT in male and female mice following a single bilateral intravitreal administration of a single dose of 1.14 x 1010vg / eye. In brief, 6 / sex / group C57BL / 6 mice were enrolled into each of the 7 groups and received a 1 pL IVT injection of 1.14WSGR Docket No. 59561-717.601x IO10vg / eye Vector in both eyes on Day 1. Each group was euthanized at different timepoints, and biofluids and ocular and systemic tissues were collected for pharmacokinetic (PK) analysis and biodistribution. Group 1 animals were euthanized 30 minutes post-dose on Day 1; Group 2 animals were euthanized at 24 hours post-dose on Day 2; Group 3 animals were euthanized on Day 8, 1 week post-dose, Group 4 animals were euthanized on Day 29, 1 month post-dose;Group 5 animals were euthanized on Day 57, 2 months post-dose, Group 6 animals were euthanized 3 months post-dose, and Group 7 animals were euthanized 6 months post-dose.Ocular examinations (OEs) were performed at baseline to determine enrollment into the study. The body weight of the animals was monitored throughout the study. All collections made at the time of necropsy were analyzed for the presence of viral DNA by PCR and aflibercept protein transgene by ELISA Aflibercept sandwich ELISA was performed using a GLP -validated assay. One week after injection of Vector AAT (1.14 x IO10vg / eye), the aflibercept expression was detected in the eye tissue. The expression reached its peak at the 4-week time point. The expression went down to 1.2±1.1 ng / eye (or 53.2±48.7 pg / mg tissue) at the 8-week time point and stayed constant thereafter till the last sacrifice at the 6-month time point (Fig. 13).

[0227] All non-ocular tissues showed very low aflibercept concentration (0-4.2 pg / mg tissue). Interestingly, they had the same pattern where the highest aflibercept concentration was observed at the 4-week timepoint and then the aflibercept concentration dropped significantly. Serum showed a very low concentration of 1.3±0.7 ng / mL at the 4-week timepoint. Preauricular lymph node and brain that are near to the site of injection also showed very low aflibercept concentration showing that most of the concentration was localized at the injection site.

[0228] DNA biodistribution of Vector AAT was performed using QPCR with primers targeting aflibercept. DNA levels in the eye were the highest, compared to other tissue or shedding samples. As expected, the highest reading was at 30 minutes, which is the first time point of sample collection. The DNA level kept on falling till the fourth time point (29 days), and then the titer remained stable. All the organs showed around 100 to 10,000 copies / pg DNA at earlier time points (starting from 8 days) and the level went down at every subsequent time point in most of the tissues. There were no significant differences between males and females. The biodistribution of Vector AAT was shown to be superior compared to other clinical stage therapies, ADVM-022, RGX-314, and 4D-150 (Fig. 6).

[0229] Shedding samples were also collected at all the time points (30 min, 24 h, 8 days, 29 days, 57 days, 3 months, and 6 months) for QPCR using primers targeting aflibercept. Shedding samples were whole blood, saliva, urine, and feces. DNA levels of Vector AAT decreased overWSGR Docket No. 59561-717.601time in shedding samples, with baseline levels (undetectable) observed starting from 29 or 57 days.Biodistribution in rabbit

[0230] The purpose of this study was to analyze aflibercept expression in the rabbit samples of ocular and non-ocular tissues after DL-a-AAA challenge and IVT injection of Vector AAT and its expressed transgene. Aflibercept expression in the AH was high in group 1 (Eylea) group at day 37- or 15-days post IVT and day 50- or 28-days post IVT but at day 92- or 70-days after injection was BLOD. Vehicle-injected group 2 animals didn’t show any aflibercept concentration as expected. Group 3 which was LD of Vector AAT (LD, 1.7 x IO10vg / eye), on Day 37, the aflibercept concentration in AH was around 6 ng / mL. But on Day 50 and till the end of the study, the concentration was around 20 ng / mL. Group 4 which was Vector AAT, HD the level of aflibercept was ~20 ng / mL throughout the study showing a quick onset of expression, unlike LD. VH level was BLOD for groups 1 and 2. Vector AAT LD and HD had a similar level that is >100 ng / mL. Retina, RPE / CHR, optic chiasm, LGN, and visual cortex tissues in group 1 and group 2 had a baseline measurement between 1-4 pg / mg tissue. Vector AAT LD and HD had 176.6±120.9 and 147±193.4 pg / mL respectively in retinal tissue. In RPE / CHR, Vector AAT LD and HD had 154±87.9 and 72.1±117.3 pg / mL respectively. Both these groups had comparable expression in ocular tissue samples. The expression was around 7-10 fold lower in the optic nerve, optic chiasm, and LGN in the respective groups when compared to their retinal or RPE / CHR tissue. The expression further went down in the visual cortex averaging around 4 pg / mg tissue. The non-ocular tissues in groups 3 and 4 had 10-20 fold lower aflibercept levels showing that the expression of aflibercept was localized to or around the site of injection. Group 1 injected with Eylea® showed very high Aflibercept concentration in AH and serum at Day 37-or 15-days post IVT injection. The concentration dropped at Day 50- or 28-days after injection and was BLOQ at Day 92- or 70-days after injection. None of the tissues collected showed any Aflibercept concentration at the time of sacrifice. Group 2 vehicle-injected animals didn’t show any Aflibercept concentration in any tissues as expected and it worked as a baseline measurement. In the Vector AAT low dose group 3 VH showed 101.5 ng / mL (Fig. 14). Retina and RPE / CHR ~0.2 ng / mg of tissue. Other tissues that showed Aflibercept expression were the optic nerve, optic chiasm, and LGN. In the Vector AAT high dose group 4 (HD, 1.9 x 1011vg / eye), the expression in AH was -20 ng / mL throughout the study. High dose showed a quick onset of detectable Aflibercept expression, unlike low dose Vector AAT. Animal #11856 had no aflibercept expression, and the aflibercept expression of animal #11858 dropped to zero fromWSGR Docket No. 59561-717.601Day 50. These were the animals that had high ocular observations (posterior cortical cataracts, elevated OE scores, and hazy fundus) during the in-life study. VH had a concentration of 116.04 ng / mL. Retina had 0.15 and RPE / CHR had 0.07 ng aflibercept / mg tissue.

[0231] Aflibercept concentration was very high in Group 1, animals injected with Eylea® protein at Day 37 or 15 days after IVT injection and it rapidly dropped at Day 50 or 28 days after IVT. At Day 92 or 70 days after IVT injection there was no detectable Aflibercept concentration in the serum samples. For the Vector AAT -injected Groups 3 and 4, serum levels of aflibercept were very low throughout the study. The expression stayed locally for these groups unlike Group 1. In addition, Other than ocular samples and tissue close to the site of injection, tissues like liver, kidney, adrenal, spleen, heart, colon, lung, mesenteric lymph node, mandibular lymph node, testicle, cerebral cortex, and spinal cord were assessed to check for the concentration of aflibercept at site far from the site of injection. All the above-mentioned tissues had far lower aflibercept concentration: around 20-100 fold lower expression than found in the eye.

[0232] DNA and RNA biodistribution of Vector AAT were measured using QPCR and RT-QPCR with primers targeting the Aflibercept sequence, respectively. As shown in Fig. 15, there was an increase in DNA copies of Vector AAT in the ocular tissues of rabbits challenged with DL-a-AAA. However, two animals from the high-dose group (animals #11856 and 11858) had lower DNA levels which affected the overall distribution and the average number of copies. AH data looks inconsistent because of the low DNA level in the AH sample. In-life samples were obtained by performing an AH tap into the selected eyes hence, the sample volume was low which is another reason that impacted the result for AH samples. In addition, there was a very low DNA level detected anywhere other than ocular tissues.

[0233] RNA analysis was performed using the reverse transcriptase (RT)-qPCR method. Retina, RPE / CHR (choroid), AH, and VH samples were analyzed for RNA level. Similar to the DNA analysis, retina and RPE / CHR showed an increase in RNA level after treatment (Fig. 16), however, two animals from Group 4 (animals #11856 and 11858) showed low RNA levels in turn possibly affecting the overall outcome of the results.Biodistribution in non-human primate (NHP)

[0234] The objective of this study phase was to evaluate the biodistribution and viral vector shedding of Vector AAT when administered as a single dose via intravitreal injection to Cynomolgus monkeys as part of the toxicity study. The intravitreal route of administration was chosen, because it was the intended human therapeutic route. The high dose was the maximum feasible dose (MFD) according to the current manufacturing capability and size of monkey eyes.WSGR Docket No. 59561-717.601The highest concentration of a stable drug product is 1.14 x 1013vg / mL, and the maximal volume to be delivered into the vitreous of a monkey was 50 pL / eye. The high dose was intended to provide a 64.7-fold margin to the intended clinical starting dose of 2 * IO10vg / eye / dose. This GLP study evaluated the toxicity of Vector AAT when administered as a single dose via IVT injection to cynomolgus monkeys with 4-week and 13-week interim terminations.Viral Transgene (aflibercept) analysis as DNA copy number

[0235] The Vector AAT vector genome was detected abundantly in all VH and retina tissues collected on Days 29, 92, and 183 post-administration of >1.49 x 1011vg / eye. AH, optic nerve (ON), and brain tissues connected to the optic nerve (optic chiasm, optic tract, and LGN, from animals administered >1.49 x 1011vg / eye contained less abundant vector levels on Day 29. The Vector AAT vector was cleared from some of these tissues by Day 92 or 183. Outside of the eye and brain tissues, the Vector AAT vector was noted in some spleen, liver, and mandibular and mesenteric lymph node tissues from Vector AAT -treated animals at low levels in general, but not all, up to Day 183. The lacrimal gland was also noted with vector genomes at low or non-quantifiable levels in some Vector AAT -treated animals up to Day 92, and the vector was cleared by Day 183.Viral transgene (aflibercept) Analysis as mRNA gene expression

[0236] In addition, mRNA levels were measured using a GLP -validated method. RNA samples were analyzed for both Vector AAT vector-derived mRNA (aflibercept transgene expression) copy numbers, and Ct values of monkey endogenous HPRT1 mRNA using the validated one-step duplex RT-qPCR method. The LOD, LLOQ, and ULOQ of the assay are 25, 50, and 108 copies of ss Aflib RNA Oligo per 100 ng of total monkey RNA. Gene expression data with undetectable values or results below 25 ss copies of vector-derived mRNA per RT-qPCR reaction were reported as BLOD. Gene expression data > 25 ss copies and < 50 ss copies of vector-derived mRNA per RT-qPCR reaction are reported as BLOQ. BLOD and BLOQ values were assigned 0 and 25 ss copies per reaction for group mean calculations and then were normalized prior to calculating group mean (i.e., 90 ng of RNA testing BLOQ would be given a value of 28 ss copies per 100 ng; 25 copies / 90 ng * 100 ng = 28 copies / 100 ng).

[0237] Vector-derived Vector AAT mRNA (indicating Aflibercept transgene expression) was detected in all retinal tissues of Vector AAT -treated animals, which were also the tissues with the most abundant vector genome. Outside the retinal tissues, a limited expression of Aflibercept mRNA was noted in some optic nerves, optic tract, and optic chiasm up to Day 183. For animals administered 5.70 x 1011vg / eye, one male had low quantifiable Vector AAT mRNA expressionWSGR Docket No. 59561-717.601noted in liver and spleen tissues, and one female had low quantifiable Vector AAT mRNA expression detected in lacrimal gland during interim sacrifices on Day 92 or 29, respectively. One male administered 1.49 x 1011vg / eye had Vector AAT mRNA BLOQ noted in liver tissue at the terminal sacrifice. The remaining liver, spleen, and lacrimal gland tissues were negative for Vector AAT mRNA. All LGN, mandibular, and mesenteric tissues were negative.Viral transgene (aflibercept) analysis as protein expression

[0238] Aflibercept protein levels were also measured by ELISA. Control animals (Group 1) showed no aflibercept expression at the site of injection and many other tissues far from the site of injection. Low-dose animals (Group 2) showed consistent expression of aflibercept throughout the study in the tissues at and around the site of injection that is VH, AH, and retina. Starting from Day 29, the aflibercept level increased, and maximum expression was observed on Day 92 and showed an almost similar expression on Day 183. Tissues close to the eye, brain, and spinal cord such as optic nerve, optic tract, optic chiasm, LGN, visual and cerebral cortex, spinal cord, and dorsal root ganglia didn’t show any Aflibercept level showing that Vector AAT concentrated in the retina and did not go beyond the retina. All the serum samples showed an Aflibercept level higher than LOQ (0.125 ng / mL) but still, the expression level was significantly lower than the VH. Animals sacrificed on Day 183 showed the highest level of Aflibercept in serum samples reaching around 2-3 ng / mL. High-dose animals (Group 3) showed expression of Aflibercept at the site of injection, especially AH, VH, and retina but the expression was high on Days 29 and 92 but on Day 183, the expression dropped significantly in all three tissues suggesting that there is a quick onset of expression but the durability of expression in the high dose compared to low dose animals is reduced.

[0239] The potential reason for the lack of dose response in the GLP Toxicity study could be immune responses against the higher dose of Vector AAT. This was supported by the DNA vector copy number in the retina decreasing over time, more prominently in the high dose group although the Aflibercept mRNA level in the retina did not seem to decrease over time in the high dose group; Aflibercept protein levels in the retina did appear to decrease over time, more prominently so in the higher dose group. The levels of ADA detected in serum from the higher dose group at all timepoints and high levels of ADA were detected in vitreous humor in the high dose group on Day 183. Though the detected ADA was to the capsid protein and not to Aflibercept. Additionally, the U-PLEX Multiplex assay could detect low levels of ZFN-y levels as a surrogate for increased cellular immunity against Vector AAT capsid and low levels remained detectable above the cut-off point for the assay at the latest time point in the higher dose group.WSGR Docket No. 59561-717.601

[0240] The GLP toxicity study in NHP used relatively high doses (1.49 x 1011and 5.7 x 1011vg / eye) to assess the maximal toxicity of Vector AAT, which may have resulted in dosedependent immune responses. This has created challenges in interpreting pharmacokinetics inferences in the higher dose group particularly. Yet, the clinical dose would be much lower than 5.7 x 1011vg / eye. It is important to note: (1) animal immune responses cannot be fully or directly extrapolated to humans; (2) the clinical dose can be titrated to decrease the risk of developing immunogenicity against Vector AAT at high dose, and (3) ADA / immunogenicity can be clinically monitored.Toxicology

[0241] A number of preclinical single-dose studies via IVT injection to the eye were performed. Studies were either performed as non-GLP studies with research-grade small-scale AAV production or with large-scale GLP AAV material that was produced to support the full singledose toxicology study. In addition to the ocular / whole body distribution or efficacy studies performed some potential safety findings were obtained.Single dose studies in mice.

[0242] AAV2.N54-VEGF-Trap (Vector AAT) was delivered by IVT in an LCNV mouse model. Both color and cobalt blue (GFP expression) fundus imaging were performed on both eyes dosed with AAV2-GFP; 1.6 x 1010vg / eye animals on Day 7 after laser and prior to FA, GFP was observed in all eyes, except 318 OS, dosed with AAV2-GFP which had a cataract that prevented imaging of the fundus. The cortical cataract, corneal scar, vitreous hemorrhage, and vitreous debris described may well have been caused due to nicking the lens during IVT injection - an issue with rodent models with a large relative lens size to vitreal chamber ratio and is likely unrelated to the use of the AAV2.N54 capsid. No unusual ocular observations were made in the Vector AAT dosed groups, although overall 6 animals died in the course of the study, 3 of these were in the positive control group dosed with Aflibercept protein so the deaths are likely due to the frequency of the anesthesia events during the study and unrelated to dosing with Vector AAT.

[0243] Finally, a mouse biodistribution study was performed with Vector AAT. Animal 12 died from anesthesia on the day of dosing and was replaced. Animal 38 was never enrolled in the study. This animal had bilateral cataracts on baseline ocular examination. This animal was euthanized and not dosed because of this finding. There are no unexpected deaths following dosing. In summary, no significant toxicity findings related to Vector AAT were observed in these mouse studies.Single dose studies in rabbitsWSGR Docket No. 59561-717.601

[0244] For each group, animals, (n=6) were dosed in both eyes with either 1.7 xlO10vg / eye (Vector AAT low dose) or 1.9 xlO11vg / eye (Vector AAT high dose). The OS eye had been predosed 21 days earlier with DL-a-AAA to induce retinal leakage as an efficacy model. OEs were performed using a slit lamp bio-microscope and an indirect ophthalmoscope to evaluate ocular surface morphology and anterior and posterior segment inflammation. All baseline OEs were unremarkable. In general, the OE scores in the oculus sinister (OS) were highest (note that these had two IVT injections within 21 days) and had the most variability in animals receiving the high dose of Vector AAT and Vector AAM (similar design to Vector AAT but for AAV serotype 6), respectively (Fig. 17). OE scores in the rest groups were lowest from Day 37 to 64; OE scores in these groups increased slightly from Day 78 to 106. The ocular findings in the OS eyes can also be attributed to the model. Inflammation in the ODs of most animals including the low dose Vector AAT was generally mild. Inflammation (vitreal haze) in animals dosed with high dose Vector AAT was observed in 2 animals (2 / 6), with a higher incidence of vitreal haze through Day 106. At baseline and on Days 15, 23, 29, 37, 43, 50, 64, 78, 92, and 106, IOP was measured in both eyes of all animals. The average baseline IOP of all eyes was 19.3±3.1 mmHg, indicated by the dashed gray line in Fig. 17. The lOPs of all groups generally remained within the normal range for this strain and species at all timepoints.26-week GLP toxicity study of Vector AAT after a single IVT dose in the cynomolgus monkey (NHP)

[0245] Cynomolgus monkeys historically have been used in safety evaluation studies and are recommended and accepted by appropriate regulatory agencies. The cynomolgus monkey was selected as the relevant species because of the similarity of monkeys to humans. A GLP toxicity study in cynomolgus monkey was performed, demonstrating the safety profile of intravitreal dosing of Vector AAT at doses of 1.49 x 1011vg / eye (low) and 5.70 x 1011vg / eye (high).

[0246] Naive cynomolgus monkeys of Asian origin were acclimated for 48-63 days prior to initiation. At initiation of dosing, animals were 33 - 48 months old, and body weights ranged from 1.9 to 2.9 kg for males and 2.0 to 2.8 kg for females. Prior to arrival, stock animals had been screened for anti-AAV2 neutralizing antibodies (NAb). Animals were selected for shipment to the test facility as potential study candidates based on the anti-AAV2 NAb screening results and animal health history.

[0247] The intravitreal route of administration was chosen because it is the intended human therapeutic route. The high dose was the maximum feasible dose (MFD) according to the current manufacturing capability and size of monkey eyes. The highest concentration of a stable drugWSGR Docket No. 59561-717.601product is 1.14 x io13vg / mL, and the maximal volume to be delivered into the vitreous of a monkey is 50 pL / eye / dose. The high dose was intended to provide a sufficient safety margin to the intended clinical starting dose of 2.6 IO10vg / eye._This GLP study evaluated the toxicity of Vector AAT when administered as a single dose via IVT injection to cynomolgus monkeys with 4-week and 13-week interim terminations. The experimental design of the study is shown in Table 14Table 14. Experimental design of single dose GLP toxicity study in NHPAnimals were divided into two cohorts. Cohort 1 consisted of all animals designated for the terminal sacrifice, and Cohort 2 consisted of all animals designated for the interim sacrifices.a:Group 1 was administered the control article only,b:Dose levels were based on a dose volume of 50 pL / eye / dose,c:One male / group and two females / group were designated as interim sacrifice I animals (Day 29), two males / group and one female / group were designated as interim sacrifice II animals (Day 92), and two males / group and two females / group were designated as terminal sacrifice animals (Day 183).

[0248] After dosing, animals were observed post-dosing for at least 29 days (interim sacrifice I), 92 days (interim sacrifice II), or 183 days (terminal sacrifice) to assess the reversibility or persistence of any effects. Male and female cynomolgus monkeys were assigned to three groups. Animals were dosed via IVT injection into both eyes once on Day 1 of the dosing phase at a volume of 50 pL / eye.

[0249] Pre-weighed tissues, blood, feces, urine, saliva swabs, and tear strip samples from all NHPs were shipped to NBS for qPCR shedding analysis. Samples were analyzed using a GLP-validated method. DNA was extracted from each sample for qPCR analysis. The limit of detection (LOD), lower limit of quantitation (LLOQ), and upper limit of quantitation (ULOQ) of the assay are 5, 25, and 108 copies of Afhbercept standard DNA or double-stranded (ds) Vector AAT vector DNA per collection respectively. Biodistribution data with undetectable values or testing below 5 copies of ds Vector AAT vector DNA per qPCR reaction are reported as belowWSGR Docket No. 59561-717.601limit of detection (BLOD). Data between > 5 copies and < 25 copies of ds Vector AAT vector DNA per qPCR reaction are reported as below limit of quantification (BLOQ). BLOD and BLOQ values were assigned 0 and 5 copies / reaction, respectively, for group mean calculations. Viral shedding analysis of tear, urine, feces, and saliva samples demonstrated complete clearance of the viral vector genome by Day 3 or 15 post-dosing.

[0250] Assessment of toxicity was based on clinical observations, body weights, qualitative food consumption, electrocardiographic (ECG) measurements, vital signs (body temperatures), and clinical and anatomic pathology. Assessment of ocular toxicity was based on ophthalmic observations, IOP measurements, spectral domain optical coherence tomography (OCT), ocular photography, and full-field electroretinography (ERG). For the biodistribution studies, some samples were taken during the in-life phase: saliva, fecal, tear, and urine samples were collected for viral shedding analysis; blood samples were collected for vector genome, mRNA level, and transgene expression analysis. At termination, frozen tissues were collected for vector genome and transgene expression analyses. Assessment of immunogenicity was based on the collection of blood samples, and ocular fluids (vitreous humor) for analysis of potential immunogenicity: (i) anti-drug antibody (ADA) responses to the AAV2.N54 capsid and (ii) expressed viral transgene: aflibercept analysis. Peripheral blood mononuclear cells (PBMCs) were isolated for cellular immunotoxicology analysis.Whole body (systemic) toxicology findings in the single dose GLP toxicity study

[0251] All animals survived until their scheduled sacrifices. No Vector AAT -related clinical observations, veterinary observations, body weight changes, or qualitative food consumption changes were noted. For data evaluation and statistical analysis, various models of calculators, computers, and computer programs were used to analyze data in this study. Values in some tables (e.g., means, standard deviations, or individual values) may differ slightly from those in other tables, from individually calculated data, or statistical analysis data, because different models round off or truncate numbers differently. Neither the integrity nor the interpretation of the data was affected by these differences. Data for each sex were analyzed separately; only data collected on or after the first day of dosing were analyzed statistically. Analysis of variance (ANOVA) and pairwise comparisons were used to analyze the following: Absolute body weight, Body weight change, ECG data, continuous clinical pathology values, Terminal body weight, Absolute organ weight, orgambody weight percentage, and orgambrain weight percentage.

[0252] Where only two groups were available for analysis, a two-sample t-test was performed. Data containing values above / below the limit of quantitation were not analyzed, and the tablesWSGR Docket No. 59561-717.601were footnoted accordingly. Where insufficient data were available for meaningful analysis, no analysis was performed, and the tables were footnoted accordingly. All statistical tests were evaluated at the 5.0% probability level.

[0253] No Vector AAT-related ECG changes were observed on Day 23, 57, 86 / 90, or 181 in animals administered 1.49 x 1011or 5.70 x 1011vg / eye. No abnormal waveforms or arrhythmias were observed during the qualitative assessment of the ECGs. In conclusion, no ECG changes were attributed to Vector A AT.

[0254] No Vector AAT-related clinical pathology (hematology, coagulation, clinical chemistry, or urinalysis) test results were noted for animals administered 1.49 x 1011or 5.70 x 1011vg / eye. No Vector AAT-110-related organ weight differences or macroscopic observations occurred at any sacrificeSlit lamp observations

[0255] The control article was well tolerated, with findings limited to mild (0.5 to 1+) vitreous cells through Day 22 of the dosing phase. No ocular abnormalities were noted after Day 22 of the dosing phase in this group. IVT administration of >1.49 x 1011 vg / eye Vector AAT was well tolerated, with findings attributable to the physical presence of the test article itself or sporadic instances of mild (0.5 to 1+) or, rarely, moderate (2+) vitreous cell that was not bright white and refractile. Findings considered to represent the test article in the vitreous included varying amounts of bright white and refractile cell-sized structures in the anterior vitreous; white vitreous floaters near the dose site; and sporadic instances of bright white and refractile structures in the anterior chamber. It was not possible to determine, based on the ophthalmic examination alone, whether these bright white and refractile structures represented test article precipitates, aggregates, or cellular responses. On Day 176 of the dosing phase (Week 26 and the last examination before the terminal sacrifice), no ocular abnormalities were noted in any of the remaining eight eyes administered 1.49 x 1011vg / eye.

[0256] Intravitreal administration of 5.70 x 1011vg / eye of Vector AAT was associated with an intraocular inflammatory response that began on Day 22 of the dosing phase (and persisted through the last examination on Day 176 of the dosing phase), characterized by aqueous flare (up to 2+), predominantly white aqueous cell (up to 3+), keratic precipitates, incomplete pupil dilation following the topical application of a mydriatic, vitreous cell (up to 4+), vitreous haze (up to 2+), white retinal perivascular sheathing, and altered retinal reflectivity in areas of previous retinal perivascular sheathing. On Day 176 of the dosing phase (Week 26 and the last examination before the terminal sacrifice), findings in the remaining eight eyes administered 5.70WSGR Docket No. 59561-717.601x IO11vg / eye consisted of vitreous cells (0.5+ in one eye, 1+ in five eyes, and 2+ in two eyes) and white retinal perivascular sheathing (three eyes).Retinal fundus photography

[0257] Ocular photography was conducted with OCT once during the pre-dose phase and once during Weeks 4, 8, 13, and 26 of the dosing phase, as applicable. Animals were already anesthetized with the anesthetics used for other ophthalmic procedures on that same day (i.e., OCT and FA) and were fasted at least 10 hours before the procedure. Pupils were dilated with a mydriatic agent. Color photographs were taken of each eye with a digital fundus camera to include stereoscopic photographs of the posterior pole and non-stereoscopic photographs of two mid-peripheral fields (temporal and nasal). Assessment of test article-related effects on ocular structure for this portion of the study was based on fundus ocular photography (OP) and spectral domain OCT. No fundus photographic abnormalities were noted in any control eyes at any interval. No fundus photographic abnormalities were noted in any eyes of the group administered 1.49 x 1011vg / eye at any interval. One male was noted with small patches of depigmentation in the temporal peripheries of both eyes. These were noted during the predose phase and at all subsequent intervals. Such patches are a normal variant in this species.

[0258] Retinal fundus photography during Week 13 noted mild perivascular sheathing in two eyes of two animals administered 5.7 x 1011vg / eye. The perivascular sheathing is an indicator of retinal inflammation. One of these two animals had OPs obtained during Week 26, and the perivascular sheathing noted during Week 13 had resolved. One female was noted with small patches of depigmentation in the posterior pole and nasal periphery of the left eye. These were noted during the predose phase and at all subsequent intervals. Such patches are a normal variant in this species.SD-OCT findings

[0259] Spectral domain optical coherence tomography (SD-OCT) was conducted once during the predose phase and once during Weeks 4, 8, 13, and 26 of the dosing phase, as applicable. The SD-OCTs were conducted in a manner to obtain axial views of the retinal surface in the posterior fundus. The instruments were set to perform standard retinal scans (macular volume scans, line scans, or circle scans). SD-OCT demonstrated increased vitreous haze / floaters observed in some eyes from all groups, including the control group, so some aspects of these observations may be related to the physical trauma of IVT injection itself. The highest incidence was noted in eyes administered 5.7 x 1011vg / eye, where the finding was observed in at least one eye of all animals. These all resolved during the study duration. Two other findings were observed only in eyesWSGR Docket No. 59561-717.601administered 5.7 x IO11vg / eye and included small accumulations of reflective material in the fovea and multiple, small spots on the ILM, especially in the ONH. These two incidental findings, drusen-like spots in the interdigitation zone (IZ) / RPE and a few discontinuities / disorganizations in photoreceptors (PR) adjacent to the ONH, were observed during the predose phase and remained unchanged throughout the study and were therefore not article related.Microscopic finding

[0260] At interim sacrifice I, a Vector AAT-related microscopic finding of minimal mononuclear cell infiltrates of the vitreous was noted in one male administered 1.49 x 1011vg / eye._At interim sacrifice II, Vector AAT-related microscopic findings of minimal mononuclear cell infiltrates were noted in the vitreous of the right eye of males administered >1.49 x 1011vg / eye and in the optic nerve head and sclera of one male administered 5.70 x 1011vg / eye.

[0261] At the terminal sacrifice, Vector AAT-related microscopic findings in the right eye consisted of minimal mononuclear cell infiltrates of the vitreous of both sexes administered 1.49 x 1011vg / eye and minimal mononuclear cell infiltrates in the perivascular retina of males administered 5.70 x 1011vg / eye and in the sclera of one male administered 5.70 x 1011vg / eye. Lymphoplasmacytic infiltrates were noted in the ciliary body of two females administered 5.70 x 1011vg / eye, and plasma cell infiltrates were noted in one female administered 5...

Claims

WSGR Docket No. 59561-717.601CLAIMS WHAT IS CLAIMED IS:

1. A method for treating a disease or condition in a subject, comprising: administering a viral particle comprising an engineered capsid encapsulating a vector to the subject, the engineered capsid comprising a polypeptide sequence in a VP domain of the engineered capsid, the polypeptide sequence comprising: L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4; wherein: XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H); X3 is E, S, or Q; and X4 is K, R, E, or A, and wherein the polypeptide sequence increases expression of the vector in a cell, and wherein the expression of the vector treats the disease or condition in the subject.

2. The method of claim 1, wherein the disease or condition comprises an ocular disease.

3. The method of claim 2, wherein the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

4. The method of any one of claims 1-3, wherein the cell comprises a macula cell.

5. The method of any one of claims 1-3, wherein the cell comprises a retinal cell.

6. The method of any one of claims 1-3, wherein the cell comprises an ocular cell.

7. The method of claim 6, wherein the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or a combination thereof.

8. The method of any one of claims 1-7, wherein the engineered capsid comprises an engineered adeno-associated virus (AAV) capsid.

9. The method of claim 8, wherein the engineered AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof.WSGR Docket No. 59561-717.60110. The method of claim 9, wherein the engineered AAV capsid comprises an engineered AAV2 capsid.

11. The method of any one of claims 1-10, wherein the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 13).

12. The method of claim 11, wherein the polypeptide sequence is the amino acid sequence of LALGQTTKPA (SEQ ID NO: 13).

13. The method of any one of claims 1-12, wherein the polypeptide sequence is inserted in the VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO:

114. The method of any one of claims 1-13, wherein the engineered capsid further comprises a mutation.

15. The method of claim 14, wherein the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain.

16. The method of claim 14, wherein the mutation is in a GH loop.

17. The method of claim 14, wherein the mutation is at a residue at position 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1.

18. The method of claim 17, wherein the mutation is R to A at position 585 or 588 of SEQ ID NO:

119. The method of any one of claims 1-18, wherein the vector comprises one or more expression cassettes for expressing one or more transgene.

20. The method of claim 19, wherein the vector encodes an anti-angiogenic agent.

21. The method of claim 20, wherein the anti -angiogenic agent comprises a VEGF-Trap.

22. The method of claim 21, wherein the anti -angiogenic agent consists of the VEGF-Trap.

23. The method of any one of claims 20-22, wherein the anti -angiogenic agent is encoded in the vector comprising one or more codon modifications.

24. The method of claim 23, wherein the one or more codon modifications comprises one or more replacements of non-AGG arginine codon to AGG; non-CCC proline codon to CCC; non-TCC serine codon with TCC; non-CCG proline codon with CCG; or a combination thereof.

25. The method of any one of claims 1-24, wherein the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually,WSGR Docket No. 59561-717.601transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or any combination thereof to the subject.

26. The method of any one of claims 1-25, wherein the engineered capsid delivers the vector to the cell in the subject.

27. The method of any one of claims 1-26, wherein the engineered capsid delivering the vector decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell.

28. The method of any one of claims 1-27, wherein the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence.

29. The method of any one of claims 1-28, wherein the engineered capsid delivering the vector increases delivery of the vector to a cell that is in a deeper cell layer in the subject compared to a comparable non-engineered capsid without the polypeptide sequence delivering the vector to a cell in a cell layer.

30. The method of claim 29, wherein the cell layer comprises a ganglion cell layer, an inner plexiform layer, an inner nuclear layer, an outer plexiform cell, an outer nuclear layer, an inner segment layer, an outer segment layer, a retinal pigment epithelium layer, or any combination thereof.

31. The method of any one of claims 1-30, wherein the engineered capsid delivering the vector results in a decreased retinal leakage compared to a comparable capsid without the polypeptide sequence delivering a comparable vector encoding a comparable VEGF inhibitor.

32. The method of claim 31, wherein the decreased retinal leakage is resulted from one or more administrations.

33. The method of claim 32, wherein the decreased retinal leakage is resulted from one administration.

34. The method of any one of claims 1-33, wherein the engineered capsid delivering the vector decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject.

35. The method of any one of claims 1-34, wherein the engineered capsid delivering the vector decreases a dose of the anti-angiogenic agent administered to the subject compared to a secondWSGR Docket No. 59561-717.601dose of a comparable anti-angiogenic agent administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject.

36. The method of any one of claims 1-35, wherein the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent.

37. The method of any one of claims 1-36, wherein the engineered capsid delivering the vector does not increase inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent.

38. The method of any one of claims 1-37, wherein the engineered capsid delivering the vector does not decrease ERG in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent.

39. The method of any one of claims 1-38, wherein the engineered capsid delivering the vector does not increase intraocular pressure in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent.

40. The method of any one of claims 1-39, wherein the engineered capsid delivering the vector does not increase an expression of anti-drug antibody (ADA) in the subject compared to a second expression of the ADA in the subject resulted from directly administering the subject with a comparable anti-angiogenic agent.

41. The method of any one of claims 1-40, wherein an expression of the anti-angiogenic agent delivered by the engineered capsid is increased in the subject compared to a second expression of a comparable anti-angiogenic agent resulted from treatment by the comparable anti-angiogenic agent.

42. The method of any one of claims 1-41, wherein the anti-angiogenic agent delivered by the engineered capsid is expressed for a longer duration in the subject compared to a second duration of expressing a comparable anti -angiogenic agent by administering the comparable anti-angiogenic agent to the subject.

43. The method of any one of claims 34-42, wherein the comparable anti-angiogenic agent is Elyea.

44. The method of any one of claims 27-43, wherein the comparable capsid is an unmodified AAV capsid.

45. The method of claim 44, wherein the unmodified AAV capsid is an AAV2 capsid.

46. The method of any one of claims 1-45, wherein the subject is administered a dose comprising the viral particle from about 1.0 x 109vg / eye to about 10 x IO10vg / eye.WSGR Docket No. 59561-717.60147. The method of claim 46, wherein the dose is from about 1.0 x IO10vg / eye to about 10 x IO10vg / eye.

48. The method of claim 47, wherein the dose is from about 2.0 x IO10vg / eye to about 5 x IO10vg / eye.

49. The method of claim 48, wherein the dose is about 2.1 x IO10vg / eye, about 2.2 x IO10vg / eye, about 2.3 x IO10vg / eye, about 2.4 x IO10vg / eye, about 2.5 x IO10vg / eye, about 2.6 x IO10vg / eye, about 2.7 x IO10vg / eye, about 2.8 x IO10vg / eye, or about 2.9 x IO10vg / eye.

50. The method of claim 49, wherein the dose is about 2.6 x IO10vg / eye.

51. The method of claim 48, wherein the dose is about 3.1 x IO10vg / eye, about 3.2 x IO10vg / eye, about 3.3 x IO10vg / eye, about 3.4 x IO10vg / eye, about 3.5 x IO10vg / eye, about 3.6 x IO10vg / eye, about 3.7 x IO10vg / eye, about 3.8 x IO10vg / eye, or about 3.9 x IO10vg / eye.

52. The method of claim 51, wherein the dose is about 3.7 x IO10vg / eye.

53. The method of claim 47, wherein the dose is about 8.1 x IO10vg / eye, about 8.2 x IO10vg / eye, about 8.3 x IO10vg / eye, about 8.4 x IO10vg / eye, about 8.5 x IO10vg / eye, about 8.6 x IO10vg / eye, about 8.7 x IO10vg / eye, about 8.8 x IO10vg / eye, or about 8.9 x IO10vg / eye.

54. The method of claim 53, wherein the dose is about 8.1 x IO10vg / eye.

55. The method of any one of claims 1-54, wherein a single administrating of the viral particle comprising engineered capsid and the vector is curative of the disease or condition in the subject.

56. A method for treating a disease or condition in a subject, comprising: administering a dose of a viral particle comprising an engineered capsid encapsulating a vector to the subject, wherein the vector comprises a nucleic acid sequence encoding an anti -angiogenic agent, said nucleic acid sequence comprises a modification in a coding region of the nucleic acid sequence as compared to an otherwise comparable nucleic acid sequence lacking the modification in the coding region, said modification comprises replacing at least four non-AGG arginine codons to AGG, wherein the anti-angiogenic agent treats the disease or condition, and wherein the dose is from about 1.0 x 109vg / eye to about 10 x 1010vg / eye.

57. The method of claim 56, wherein the nucleic acid sequence that encodes the anti-angiogenic agent further comprises a second modification.

58. The method of claim 56 or 57, wherein the second modification is in at least one codon of the coding region of the nucleic acid sequence, and wherein the second modification is selected from the group consisting of: replacement of at least one non-CCC proline codon with CCC; replacement of at least one non-TCC serine codon with TCC; replacement of at least one non-CCG proline codon with CCG; and any combination of (a)-(c).WSGR Docket No. 59561-717.60159. The method of any one of claims 56-58, wherein 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, a NP-1 inhibitor, a NP-2 inhibitor, a Del 1 inhibitor, and an integrin inhibitor.

60. The method of claim 59, wherein the anti -angiogenic agent comprises the VEGF inhibitor, and wherein the VEGF inhibitor is a non-antibody inhibitor.

61. The method of claim 60, wherein the non-antibody inhibitor is a fusion protein that comprises human VEGF receptors 1 and 2.

62. The method of claim 61, wherein the fusion protein comprises VEGF-Trap or a modified version thereof.

63. The method of any one of claims 56-62, wherein the nucleic acid sequence is modified to replace non-AGG arginine codon with AGG in at least 4 codon positions as compared to SEQ ID NO: 70.

64. The method of any one of claims 56-63, wherein the nucleic acid sequence is modified to replace non-CCC proline codon with CCC in at least 3 codon positions as compared to SEQ ID NO: 70.

65. The method of any one of claims 56-64, wherein the nucleic acid sequence is modified to replace non-TCC serine codon with TCC in at least 3 codon positions as compared to SEQ ID NO: 70.

66. The method of any one of claims 56-65, wherein the nucleic acid sequence is modified to replace non-CCG proline codon with CCG in at least 3 codon positions as compared to SEQ ID NO: 70.

67. The method of any one of claims 56-66, wherein the nucleic acid comprises a viral vector sequence.

68. The method of any one of claims 56-62, wherein the nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, 85%, at least 90%, 95%, or at least 99%, identical to any one of the nucleic acid sequences of SEQ ID NOS: 43-49, 51-57, 61, 62, 64, 66, 68. 71, or 72.

69. The method of claim 56, wherein the disease or condition comprises an ocular disease.

70. The method of claim 69, wherein the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME),WSGR Docket No. 59561-717.601diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or any combination thereof.

71. The method of any one of claims 56-70, wherein the engineered capsid delivers the vector to a cell in the subject.

72. The method of claim 71, wherein the cell comprises a macula cell.

73. The method of claim 71, wherein the cell comprises a retinal cell.

74. The method of claim 71, wherein the cell comprises an ocular cell.

75. The method of claim 74, wherein the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or any combination thereof.

76. The method of any one of claims 56-75, wherein the engineered capsid comprises an engineered adeno-associated virus (AAV) capsid.

77. The method of claim 76, wherein the engineered AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof.

78. The method of claim 77, wherein the engineered AAV capsid comprises an engineered AAV2 capsid.

79. The method of any one of claims 56-78, wherein the polypeptide sequence comprises an amino acid sequence of LALGQTTKP A.

80. method of claim 79, wherein the polypeptide sequence is the amino acid sequence of LALGQTTKPA.

81. The method of any one of claims 56-80, wherein the polypeptide sequence is inserted in a VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1.

82. The method of any one of claims 56-81, wherein the engineered capsid further comprises a mutation.

83. The method of claim 82, wherein the mutation is in a VP1 domain, a VP2 domain, or a VP3 domain.

84. The method of claim 83, wherein the mutation is in a GH loop.WSGR Docket No. 59561-717.60185. The method of claim 81, wherein the mutation is at a residue at position of 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1.

86. The method of claim 85, wherein the mutation is R to A at position 585 or 588 of SEQ ID NO: 1.

87. The method of any one of claims 56-86, wherein the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or any combination thereof to the subject.

88. The method of any one of claims 56-87, wherein the engineered capsid delivering the vector decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell.

89. The method of any one of claims 56-88, wherein the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence.

90. The method of any one of claims 56-89, wherein the engineered capsid delivering the vector increases delivery of the vector to a cell that is in a deeper cell layer in the subject compared to a comparable non-engineered capsid without the polypeptide sequence delivering the vector to a cell in a cell layer.

91. The method of claim 90, wherein the cell layer comprises a ganglion cell layer, an inner plexiform layer, an inner nuclear layer, an outer plexiform cell, an outer nuclear layer, an inner segment layer, an outer segment layer, a retinal pigment epithelium layer, or any combination thereof.

92. The method of any one of claims 56-91, wherein the engineered capsid delivering the vector results in a decreased retinal leakage compared to a comparable capsid without the polypeptide sequence delivering a comparable vector encoding a comparable VEGF inhibitor.

93. The method of claim 92, wherein the decreased retinal leakage is resulted from one or more administrations.

94. The method of claim 93, wherein the decreased retinal leakage is resulted from one administration.WSGR Docket No. 59561-717.60195. The method of any one of claims 56-94, wherein the engineered capsid delivering the vector decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject.

96. The method of any one of claims 89-95, wherein the comparable capsid is an unmodified AAV capsid.

97. The method of claim 96, wherein the unmodified AAV capsid is an AAV2 capsid.

98. The method of any one of claims 56-97, wherein the anti-angiogenic agent decreases retinal leakage in the subject compared to a second retinal leakage resulted from treatment by a comparable anti -angiogenic agent.

99. The method of claim 98, wherein the retinal leakage is resulted from one or more administrations.

100. The method of claim 98, wherein the retinal leakage is resulted from one administration.

101. The method of any one of claims 56-100, wherein the anti -angiogenic agent decreases retinal lesion in the subject compared to a second retinal lesion resulted from treatment by a comparable anti-angiogenic agent.

102. The method of any one of claims 56-101, wherein the anti-angiogenic agent decreases a dose of the anti-angiogenic agent administered to the subject compared to a second dose of a comparable anti -angiogenic agent administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject.

103. The method of any one of claims 56-102, wherein the anti -angiogenic agent decreases inflammation of the cell, or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent.

104. The method of any one of claims 56-102, wherein the anti -angiogenic agent does not increase inflammation of the cell, or an environment associated with the cell compared to contacting the cell with a comparable anti-angiogenic agent.

105. The method of any one of claims 56-104, wherein the anti -angiogenic agent does not decrease ERG in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent.

106. The method of any one of claims 56-105, wherein the anti -angiogenic agent does not increase intraocular pressure in the subject compared to if the subject is directly administered with a comparable anti-angiogenic agent.WSGR Docket No. 59561-717.601107. The method of any one of claims 56-106, wherein the anti -angiogenic agent does not increase an expression of anti-drug antibody (ADA) in the subject compared to a second expression of the ADA in the subject resulted from directly administering the subject with a comparable anti-angiogenic agent.

108. The method of any one of claims 56-107, wherein an expression of the anti-angiogenic agent is increased in the subject compared to a second expression of a comparable anti-angiogenic agent resulted from treatment by the comparable anti-angiogenic agent.

109. The method of any one of claims 56-108, wherein the anti-angiogenic agent is expressed for a longer duration in the subject compared to a second duration of expressing a comparable anti-angiogenic agent by administering the comparable anti-angiogenic agent to the subject.

110. The method of any one of claims 98-109, wherein the comparable anti-angiogenic agent is Elyea.

111. The method of any one of claims 56-110, wherein the dose is about 2.1 x 1010vg / eye, about 2.2 x 1010vg / eye, about 2.3 x 1010vg / eye, about 2.4 x 1010vg / eye, about 2.5 x 1010vg / eye, about 2.6 x 1010vg / eye, about 2.7 x 1010vg / eye, about 2.8 x 1010vg / eye, or about 2.9 x 1010vg / eye.

112. The method of claim 111, wherein the dose is about 2.6 x 1010vg / eye.

113. The method of any one of claims 56-110, wherein the dose is about 3.1 x 1010vg / eye, about 3.2 x 1010vg / eye, about 3.3 x 1010vg / eye, about 3.4 x 1010vg / eye, about 3.5 x 1010vg / eye, about 3.6 x 1010vg / eye, about 3.7 x 1010vg / eye, about 3.8 x 1010vg / eye, or about 3.9 x 1010vg / eye.

114. The method of claim 113, wherein the dose is about 3.7 x 1010vg / eye.

115. The method of any one of claims 56-110, wherein the dose is about 8.1 x 1010vg / eye, about 8.2 x 1010vg / eye, about 8.3 x 1010vg / eye, about 8.4 x 1010vg / eye, about 8.5 x 1010vg / eye, about 8.6 x 1010vg / eye, about 8.7 x 1010vg / eye, about 8.8 x 1010vg / eye, or about 8.9 x 1010vg / eye.

116. The method of claim 115, wherein the dose is about 8.1 x 1010vg / eye.

117. The method of any one of claims 56-116, wherein a single administrating the dose is curative of the disease or condition in the subject.

118. A method for treating a disease or condition in a subject, comprising:administering to the subject a viral particle comprising an engineered capsid comprising a polypeptide encapsulating a vector encoding VEGF-Trap to the subject,wherein the polypeptide sequence increases expression of the vector in a cell of the subject, andWSGR Docket No. 59561-717.601wherein treating the subject with the viral particle increases therapeutic efficacy or decreases side effect compared to if the subject is treated with a comparable anti-angiogenic agent.

119. The method of claim 118, wherein the disease or condition comprises an ocular disease.

120. The method of claim 119, wherein the ocular disease comprises ocular ischemic syndrome, proliferative retinopathies, neovascular glaucoma (NG), glaucoma, traumatic glaucoma, uveitis, neovascular uveitis, achromatopsia, age-related macular degeneration (nAMD), geographic atrophy (GA), dry age-related macular degeneration (dAMD), diabetic macular edema (DME), diabetic macular retinopathy (DMR), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), Bardet-Biedl Syndrome, Best Disease, choroideremia, Leber 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, Malattia leventinese (Familial Dominant Drusen), blue-cone monochromacy, or a combination thereof.

121. The method of any one of claims 118-120, wherein the cell comprises a macula cell.

122. The method of any one of claims 118-120, wherein the cell comprises a retinal cell.

123. The method of any one of claims 118-120, wherein the cell comprises an ocular cell.

124. The method of claim 123, wherein the ocular cell comprises a photoreceptor cell, a ganglion cell, a retinal pigment epithelium (RPE) cell, an amacrine cell, a horizontal cell, a muller cell, or a combination thereof.

125. The method of any one of claims 118-124, wherein the engineered capsid comprises an engineered adeno-associated virus (AAV) capsid.

126. The method of claim 125, wherein the engineered AAV capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof.

127. The method of claim 126, wherein the engineered AAV capsid comprises an engineered AAV2 capsid.

128. The method of any one of claims 118-127, wherein the polypeptide sequence is inserted in the VP domain of the engineered capsid at position 452, 453, 585, 586, 587, or 588 of SEQ ID NO: 1129. The method of any one of claims 118-128, wherein the engineered capsid further comprises a mutation.WSGR Docket No. 59561-717.601130. The method of claim 129, wherein the mutation is in a VP1 domain, a VP2 domain, a VP3 domain or a GH loop.

131. The method of claim 129, wherein the mutation is at a residue at position 452, 453, 466, 467, 468, 471, 585, 586, 587, or 588 of SEQ ID NO: 1.

132. The method of claim 131, wherein the mutation is R to A at position 585 or 588 of SEQ ID NO: 1133. The method of any one of claims 118-132, wherein the VEGF-Trap is encoded in the vector comprising one or more codon modifications.

134. The method of claim 133, wherein the one or more codon modifications comprises one or more replacements of non-AGG arginine codon to AGG; non-CCC proline codon to CCC; non-TCC serine codon with TCC; non-CCG proline codon with CCG; or a combination thereof.

135. The method of any one of claims 118-134, wherein the administrating comprises administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, subretinally, suprachoroidally, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, topically, by eye-drop, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or any combination thereof to the subject.

136. The method of any one of claims 118-135, wherein the engineered capsid delivers the vector to the cell in the subject.

137. The method of any one of claims 118-136, wherein the engineered capsid delivering the vector decreases the expression of the vector in a non-macula cell compared to a second expression of the vector delivered by a comparable capsid without the polypeptide sequence in the non-macula cell.

138. The method of any one of claims 118-137, wherein the engineered capsid delivering the vector increases delivery of the vector to a cell that is in a deeper cell layer in the subject compared to a comparable non-engineered capsid without the polypeptide sequence delivering the vector to a cell in a cell layer.

139. The method of claim 138, wherein the cell layer comprises a ganglion cell layer, an inner plexiform layer, an inner nuclear layer, an outer plexiform cell, an outer nuclear layer, an inner segment layer, an outer segment layer, a retinal pigment epithelium layer, or any combination thereof.WSGR Docket No. 59561-717.601140. The method of any one of claims 118-139, wherein the engineered capsid delivering the vector decreases inflammation of the cell or an environment associated with the cell compared to contacting the cell with a comparable capsid without the polypeptide sequence.

141. The method of any one of claims 118-140, wherein the engineered capsid delivering the vector results in a decreased retinal leakage compared to a comparable capsid without the polypeptide sequence delivering a comparable vector encoding a comparable VEGF inhibitor.

142. The method of claim 141, wherein the decreased retinal leakage is resulted from one or more administrations.

143. The method of claim 142, wherein the decreased retinal leakage is resulted from one administration.

144. The method of any one of claims 118-143, wherein the engineered capsid delivering the vector decreases a dose of the engineered capsid administered to the subject compared to a second dose of a comparable capsid without the polypeptide sequence administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject.

145. The method of any one of claims 118-144, wherein the engineered capsid delivering the vector decreases a dose of the VEGF-Trap administered to the subject compared to a second dose of the comparable anti-angiogenic agent administered to the subject, wherein the dose and the second dose result in comparable therapeutic efficacy in the subject.

146. The method of any one of claims 118-145, wherein the side effect inflammation of the cell or an environment associated with the cell.

147. The method of any one of claims 118-146, wherein the engineered capsid delivering the vector does not increase inflammation of the cell or an environment associated with the cell compared to contacting the cell with the comparable anti -angiogenic agent.

148. The method of any one of claims 118-147, wherein the engineered capsid delivering the vector does not decrease ERG in the subject compared to if the subject is directly administered with the comparable anti-angiogenic agent.

149. The method of any one of claims 118-148, wherein the engineered capsid delivering the vector does not increase intraocular pressure in the subject compared to if the subject is directly administered with the comparable anti-angiogenic agent.

150. The method of any one of claims 118-149, wherein the engineered capsid delivering the vector does not increase an expression of anti-drug antibody (ADA) in the subject compared to aWSGR Docket No. 59561-717.601second expression of the ADA in the subject resulted from directly administering the subject with the comparable anti -angiogenic agent.

151. The method of any one of claims 118-150, wherein an expression of the VEGF-Trap is increased in the subject compared to a second expression of the comparable anti -angiogenic agent resulted from treatment by the comparable anti -angiogenic agent.

152. The method of any one of claims 118-151, wherein the VEGF-Trap is expressed for a longer duration in the subject compared to a second duration of expressing the comparable anti-angiogenic agent by administering the comparable anti-angiogenic agent to the subject.

153. The method of any one of claims 140-152, wherein the comparable capsid is an unmodified AAV capsid.

154. The method of claim 153, wherein the unmodified AAV capsid is an AAV2 capsid.

155. The method of any one of claims 118-154, wherein the subject is administered a dose comprising the viral particle from about 1.0 x 109vg / eye to about 10 x IO10vg / eye.

156. The method of claim 155, wherein the dose is from about 1.0 x 1010vg / eye to about 10 x 1010vg / eye.

157. The method of claim 156, wherein the dose is from about 2.0 x 1010vg / eye to about 5 x 1010vg / eye.

158. The method of claim 157, wherein the dose is about 2.1 x 1010vg / eye, about 2.2 x 1010vg / eye, about 2.3 x 1010vg / eye, about 2.4 x 1010vg / eye, about 2.5 x 1010vg / eye, about 2.6 x 1010vg / eye, about 2.7 x 1010vg / eye, about 2.8 x 1010vg / eye, or about 2.9 x 1010vg / eye.

159. The method of claim 158, wherein the dose is about 2.6 x 1010vg / eye.

160. The method of claim 157, wherein the dose is about 3.1 x 1010vg / eye, about 3.2 x 1010vg / eye, about 3.3 x 1010vg / eye, about 3.4 x 1010vg / eye, about 3.5 x 1010vg / eye, about 3.6 x 1010vg / eye, about 3.7 x 1010vg / eye, about 3.8 x 1010vg / eye, or about 3.9 x 1010vg / eye.

161. The method of claim 160, wherein the dose is about 3.7 x 1010vg / eye.

162. The method of claim 156, wherein the dose is about 8.1 x 1010vg / eye, about 8.2 x 1010vg / eye, about 8.3 x 1010vg / eye, about 8.4 x 1010vg / eye, about 8.5 x 1010vg / eye, about 8.6 x 1010vg / eye, about 8.7 x 1010vg / eye, about 8.8 x 1010vg / eye, or about 8.9 x 1010vg / eye.

163. The method of claim 162, wherein the dose is about 8.1 x 1010vg / eye.

164. The method of any one of claims 118-163, wherein the engineered capsid comprises at least one post translational modification.WSGR Docket No. 59561-717.601165. The method of any one of claims 118-164, wherein a single administrating of the viral particle comprising the engineered capsid and the vector is curative of the disease or condition in the subject.

166. The method of any one of claims 118-165, wherein the polypeptide comprises L-A-L-G-X3-X1-X1-X4, L-K-L-G-X3-X1-X1-X4, or V-K-L-G-X3-X1-X1-X4;wherein:XI is Alanine (A), Asparagine (N), Glutamine (Q), Serine (S), Threonine (T), Glutamic Acid (E), Aspartic Acid (D), Lysine (K), Arginine (R), or Histidine (H);X3 is E, S, or Q; andX4 is K, R, E, or A.

167. The method of claim 166, wherein the polypeptide sequence comprises an amino acid sequence of LALGQTTKPA (SEQ ID NO: 183).

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