Compositions and methods for reducing ocular neovascularization

Abstract

To provide compositions and methods for reducing ocular neovascularization.SOLUTION: The present disclosure provides pharmaceutical compositions and methods thereof for the prevention or treatment of ocular neovascularization, such as AMD, in a subject, by administering to the subject a pharmaceutical composition comprising a rAAV vector having a nucleic acid sequence that encodes an anti-VEGF agent. The present disclosure relates to pharmaceutical compositions and methods of prevention or treatment of ocular neovascularization, such as AMD and CNV, in a subject (e.g., a human subject), by administering subretinally or intravitreally a pharmaceutical composition comprising a pharmaceutically effective amount of a vector or viral particles comprising a nucleic acid encoding an anti-VEGF agent, such as sFlt-1, ranibizumab, or bevacizumab.SELECTED DRAWING: None

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 351,231, filed June 16, 2016, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Vascular endothelial growth factor (VEGF) is a signaling protein produced by cells that stimulates vasculogenesis and angiogenesis. VEGF may be part of a system that restores oxygen supply to tissues when blood circulation is inadequate. The normal function of VEGF may be to create new blood vessels in embryonic development, new blood vessels after injury, muscles after exercise, and new blood vessels to bypass blocked blood vessels.

[0003] Overexpression of VEGF can contribute to various disease states and conditions in mammals. Expression of VEGF in certain cancers can cause cancer cells to grow and metastasize. Overexpression of VEGF can cause vascular disease in the retina of the eye and other parts of the body.

[0004] VEGF and VEGF receptors (VEGFR) are implicated in a number of diseases, including choroidal neovascularization (CNV) and the development of age-related macular degeneration. Examples of eye diseases or conditions associated with VEGF and / or VEGFR activity include neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR) in DME patients, ischemic retinopathy, intraocular neovascularization, dry AMD, retinal neovascularization, diabetic retinal ischemia, diabetic retinal edema, proliferative diabetic retinopathy, central retinal vein occlusion and branch retinal vein occlusion. Summary of the Invention [Means for solving the problem]

[0005] Although some protein or antibody-based injection therapies, such as ranibizumab and bevacizumab, are available for the treatment of AMD, gene therapy methods for delivering anti-VEGF agents to the eye can provide improved treatment options for patients, since gene therapy can provide extended or sustained release of therapeutic agents in vivo without requiring repeated injections, which can increase the risk of inflammation, infection and other adverse effects in some patients. Moreover, by not requiring repeated injections, gene therapy addresses the patient compliance and adherence challenges associated with therapies that require repeated injections, since non-compliance can lead to vision loss and exacerbation of eye diseases or conditions. The rate of non-compliance and non-adherence to treatment regimens that require repeated or frequent visits to the clinic for administration is higher among elderly patients, who are most affected by AMD. Thus, delivery of therapeutic agents to a patient's eye via gene therapy may provide a safer, potentially more cost-effective and more convenient treatment option for patients and improve patient outcomes by addressing the issues of medication non-compliance and non-adherence.

[0006] The present disclosure relates to pharmaceutical compositions and methods of preventing or treating ocular neovascularization, such as AMD and CNV, in a subject (e.g., a human subject) by subretinal or intravitreal administration of a pharmaceutical composition comprising a pharma- ceutical effective amount of a vector or viral particle comprising a nucleic acid encoding an anti-VEGF agent, such as sFlt-1, ranibizumab, or bevacizumab.

[0007] In some aspects, disclosed herein is a method of treating an ocular disease or condition, comprising administering a unit dose of a pharmaceutical suspension to a primate subject by injection into the eye, the unit dose of the pharmaceutical suspension comprising a rAAV of between 1E12 and 1E13 vector genomes, the rAAV having a variant capsid protein comprising an insertion of an amino acid sequence selected from LGETTRP, NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF and STGKVPN at a position corresponding to amino acids 570-611 of capsid protein VP1 in AAV2; and a heterologous sequence encoding an anti-vascular endothelial growth factor (anti-VEGF) polypeptide. In some cases, the unit dose comprises between 2E12 and 6E12 vector genomes. In some cases, the subject is a non-human primate. In some cases, the subject is a human. In some cases, the ocular condition or disease is neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion, diabetic macular edema (DME), retinal vein occlusion or diabetic retinopathy associated with DME. In some cases, the ocular condition or disease is choroidal neovascularization or AMD. In some cases, administration of the suspension results in at least a 5% reduction in the percentage of grade IV lesions compared to vehicle control as measured by color fundus photography. In some cases, the reduction in the percentage of grade IV lesions is at least 10%. In some cases, the unit dose comprises a volume of 100 μL or less. In some cases, the unit dose comprises a volume of 50 μL or less. In some cases, the insertion is LGETTRP at a position between amino acids 587 and 588 in AAV2. In some cases, the subject is responsive to at least one of ranibizumab, bevacizumab and sVEGFR-1. In some cases, the subject is pre-treated with ranibizumab or bevacizumab. In some instances, the injection is intravitreal. In some instances, the injection is subretinal. In some cases, the injection is administered no more than once every two years. In some cases, the injection is administered no more than once every five years. In some cases, the administration is a one-time administration.In some cases, the method further comprises stirring the suspension to ensure uniform distribution prior to the administration step. In some cases, the method further comprises warming the suspension to room temperature prior to the administration step. In some cases, the suspension further comprises a surfactant. In some cases, the surfactant is selected from polysorbates, sodium dodecyl sulfate, sodium lauryl sulfate, lauryl dimethylamine oxide, polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol, Brij, pluronic, and polyoxyl castor oil. In some cases, the suspension further comprises phenol, mannitol, sorbitol, or sodium chloride. In some cases, the method further comprises administering an antibiotic solution or atropine sulfate ointment after injection. In some cases, the antibiotic solution comprises ciprofloxacin. In some cases, the anti-VEGF polypeptide is a humanized monoclonal antibody. In some cases, the anti-VEGF polypeptide is an antibody fragment or Fab. In some cases, the humanized monoclonal antibody is ranibizumab or bevacizumab. In some cases, the anti-VEGF polypeptide is a soluble truncated VEGF receptor 1 (sVEGFR-1).

[0008] In another aspect, a method of treating an ocular condition or disease is also disclosed herein, comprising stirring a suspension composition comprising a variant capsid protein comprising an insertion of an amino acid sequence selected from LGETTRP, NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF and STGKVPN at a position corresponding to amino acids 570-611 of capsid protein VP1 in AAV2; and a rAAV having a heterologous sequence encoding an anti-vascular endothelial growth factor (anti-VEGF) polypeptide; and administering the suspension composition to an eye of a human subject by injection. In some cases, the insertion is LGETTRP between amino acids 587 and 588 of AAV2. In some cases, the subject is characterized by being pretreated with ranibizumab or bevacizumab. In some cases, the subject is responsive to at least one of ranibizumab and bevacizumab. In some cases, the anti-VEGF polypeptide is a humanized monoclonal antibody. In some cases, the anti-VEGF polypeptide is an antibody fragment or Fab. In some cases, the humanized monoclonal antibody is ranibizumab or bevacizumab. In some cases, the anti-VEGF polypeptide is a soluble truncated VEGF receptor 1 (sVEGFR-1). In some cases, the volume administered to the subject is 50 μL or less. In some cases, the volume administered to the subject is 100 μL or less. In some cases, the volume comprises between 1E12 and 1E13 unit doses of vector genome. In some cases, the volume comprises between 2E12 and 6E12 unit doses of vector genome. In some cases, the administering step is performed no more than once in at least two years. In some cases, the administering step is a one-time injection. In some cases, the method further comprises assaying the subject for responsiveness to at least one approved therapy prior to administering the composition. In some cases, the approved treatment includes ranibizumab and bevacizumab. In some cases, the suspension includes a pharma- ceutically acceptable excipient. In some cases, the excipient includes a surfactant or stabilizer.In some cases, the surfactant is selected from polysorbate, sodium dodecyl sulfate, sodium lauryl sulfate, lauryl dimethylamine oxide, polyethoxylated alcohol, polyoxyethylene sorbitan, octoxynol, Brij, pluronic and polyoxyl castor oil. In some cases, the pharmaceutically acceptable excipient comprises phenol, mannitol, sorbitol or sodium chloride. In some cases, the ocular condition or disease is neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion, diabetic macular edema (DME), retinal vein occlusion or diabetic retinopathy associated with DME. In some cases, the ocular condition or disease is choroidal neovascularization or AMD. In some cases, the injection is intravitreal. In some instances, the injection is subretinal. In some cases, the insertion is LGETTRP at a position between amino acids 587 and 588 in AAV2. In some cases, the method further comprises warming the suspension to room temperature prior to administration.

[0009] In another aspect, also disclosed herein is a pharmaceutical composition comprising a unit dose suspension comprising a variant capsid protein comprising an insertion of an amino acid sequence selected from LGETTRP, NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF and STGKVPN at a position corresponding to amino acids 570-611 of capsid protein VP1 in AAV2; and a rAAV having a heterologous sequence encoding an anti-vascular endothelial growth factor (anti-VEGF) polypeptide. In some cases, the unit dose is between 1E12 and 1E13 of the vector genome. In some cases, the unit dose is between 2E12 and 6E12 of the vector genome. In some cases, the suspension is refrigerated. In some cases, the kit comprises a pharmaceutical composition and a solution for diluting the pharmaceutical composition. In some cases, the solution comprises a buffer, a salt, an alcohol, a surfactant, or any combination thereof. In some cases, the kit further comprises a syringe. In some cases, the anti-VEGF polypeptide is a humanized monoclonal antibody. In some cases, the anti-VEGF polypeptide is an antibody fragment or Fab. In some cases, the humanized monoclonal antibody is ranibizumab or bevacizumab. In some cases, the anti-VEGF polypeptide is a soluble truncated VEGF receptor 1 (sVEGFR-1). In some cases, the insertion is a LGETTRP at the position between amino acids 587 and 588 in AAV2.

[0010] Incorporation by Reference 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.

[0011] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: In certain embodiments, for example, the following items are provided: (Item 1) 1. A method of treating an ocular disease or condition, comprising administering to a primate subject by ocular injection a unit dose of a pharmaceutical suspension, said unit dose comprising: A method comprising: a variant capsid protein comprising an insertion of an amino acid sequence selected from LGETTRP, NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF and STGKVPN at a position corresponding to amino acids 570 to 611 of capsid protein VP1 in AAV2; and an rAAV with a vector genome between 1E12 and 1E13 having a heterologous sequence encoding an anti-vascular endothelial growth factor (anti-VEGF) polypeptide. (Item 2) 2. The method of claim 1, wherein the unit dose comprises between 2E12 and 6E12 vector genomes. (Item 3) 2. The method of claim 1, wherein the subject is a non-human primate. (Item 4) 2. The method of claim 1, wherein the subject is a human. (Item 5) 2. The method of claim 1, wherein the ocular condition or disease is neovascular (wet) age-related macular degeneration (AMD), macular edema following retinal vein occlusion, diabetic macular edema (DME), retinal vein occlusion or diabetic retinopathy associated with DME. (Item 6) 2. The method of claim 1, wherein the ocular condition or disease is choroidal neovascularization or AMD. (Item 7) 2. The method of claim 1, wherein administering the suspension results in at least a 5% reduction in the percentage of Grade IV lesions compared to a vehicle control as measured by color fundus photography. (Item 8) 8. The method of claim 7, wherein the reduction in the percentage of grade IV lesions is at least 10%. (Item 9) 2. The method of claim 1, wherein the unit dose comprises a volume of 100 μL or less. (Item 10) 2. The method of claim 1, wherein the unit dose comprises a volume of 50 μL or less. (Item 11) 2. The method of claim 1, wherein the insertion is a LGETTRP at a position between amino acids 587 and 588 in AAV2. (Item 12) 2. The method of claim 1, wherein the subject is responsive to at least one of ranibizumab, bevacizumab, and sVEGFR-1. (Item 13) The method of claim 1, wherein the subject has been pretreated with ranibizumab or bevacizumab. (Item 14) 2. The method of claim 1, wherein the injection is intravitreal. (Item 15) 2. The method of claim 1, wherein the injection is subretinal. (Item 16) The method according to claim 1, wherein the administration by injection is performed no more than once every two years. The method according to claim 1, wherein the administration by injection is performed no more than once every five years. 2. The method of claim 1, wherein the administration is a one-time administration. (Item 19) 13. The method of claim 1, further comprising the step of agitating the suspension prior to said administering step to ensure uniform distribution. (Item 20) 2. The method of claim 1, further comprising the step of warming the suspension to room temperature prior to the administering step. (Item 21) 2. The method of claim 1, wherein the suspension further comprises a surfactant. (Item 22) 22. The method according to claim 21, wherein the surfactant is selected from polysorbates, sodium dodecyl sulfate, sodium lauryl sulfate, lauryl dimethylamine oxide, polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol, Brij, pluronic and polyoxyl castor oil. (Item 23) 2. The method of claim 1, wherein the suspension further comprises phenol, mannitol, sorbitol or sodium chloride. (Item 24) 2. The method of claim 1, further comprising administering an antibiotic solution or atropine sulfate ointment after said injection. (Item 25) 25. The method of claim 24, wherein the antibiotic solution comprises ciprofloxacin. (Item 26) 2. The method of claim 1, wherein the anti-VEGF polypeptide is a humanized monoclonal antibody. (Item 27) 28. The method of claim 1, wherein the anti-VEGF polypeptide is an antibody fragment or a Fab. The method of claim 1, wherein the humanized monoclonal antibody is ranibizumab or bevacizumab. (Item 29) 2. The method of claim 1, wherein the anti-VEGF polypeptide is a soluble truncated VEGF receptor 1 (sVEGFR-1). (Item 30) 1. A method of treating an ocular condition or disease, comprising: (a) stirring a suspension composition comprising a variant capsid protein comprising an insertion of an amino acid sequence selected from LGETTRP, NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF, and STGKVPN at a position corresponding to amino acids 570 to 611 of the capsid protein VP1 in AAV2; and a rAAV having a heterologous sequence encoding an anti-vascular endothelial growth factor (anti-VEGF) polypeptide; (b) administering the suspension composition to the eye of a human subject by injection; A method comprising: (Item 31) 31. The method of claim 30, wherein the insertion is a LGETTRP between amino acids 587 and 588 of AAV2. (Item 32) 31. The method of claim 30, wherein the subject is pretreated with ranibizumab or bevacizumab. (Item 33) 31. The method of claim 30, wherein the subject is responsive to at least one of ranibizumab and bevacizumab. (Item 34) 31. The method of claim 30, wherein the anti-VEGF polypeptide is a humanized monoclonal antibody. (Item 35) 31. The method of claim 30, wherein the anti-VEGF polypeptide is an antibody fragment or Fab. (Item 36) 35. The method of claim 34, wherein the humanized monoclonal antibody is ranibizumab or bevacizumab. (Item 37) 31. The method of claim 30, wherein the anti-VEGF polypeptide is a soluble truncated VEGF receptor 1 (sVEGFR-1). (Item 38) 31. The method of claim 30, wherein the volume administered to the subject is 50 μL or less. (Item 39) 31. The method of claim 30, wherein the volume administered to the subject is 100 μL or less. (Item 40) 31. The method of claim 30, wherein the volume comprises between 1E12 and 1E13 unit doses of the vector genome. (Item 41) 31. The method of claim 30, wherein the volume comprises between 2E12 and 6E12 unit doses of the vector genome. (Item 42) The method according to claim 30, wherein the administration step is performed no more than once every two years. 31. The method of claim 30, wherein the administering step is a one-time injection. (Item 44) 31. The method of claim 30, further comprising the step of assaying the subject for responsiveness to at least one approved therapy prior to administering the composition. (Item 45) 45. The method of claim 44, wherein the approved treatments include ranibizumab and bevacizumab. (Item 46) 31. The method of claim 30, wherein the suspension comprises a pharma- ceutically acceptable excipient. (Item 47) 47. The method of claim 46, wherein the excipient comprises a surfactant or a stabilizer. (Item 48) 48. The method of claim 47, wherein the surfactant is selected from polysorbates, sodium dodecyl sulfate, sodium lauryl sulfate, lauryl dimethylamine oxide, polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol, Brij, Pluronic and polyoxyl castor oil. (Item 49) 47. The method of claim 46, wherein the pharma- ceutically acceptable excipient comprises phenol, mannitol, sorbitol or sodium chloride. (Item 50) 31. The method of item 30, wherein the ocular condition or disease is neovascular (wet) age-related macular degeneration (AMD), macular edema following retinal vein occlusion, diabetic macular edema (DME), retinal vein occlusion or diabetic retinopathy associated with DME. (Item 51) 31. The method of claim 30, wherein the ocular condition or disease is choroidal neovascularization or AMD. (Item 52) 31. The method of claim 30, wherein the injection is intravitreal. (Item 53) 31. The method of claim 30, wherein the injection is subretinal. (Item 54) 31. The method of claim 30, wherein the insertion is a LGETTRP at a position between amino acids 587 and 588 in AAV2. (Item 55) 31. The method of claim 30, further comprising the step of warming the suspension to room temperature prior to administration. (Item 56) A pharmaceutical composition comprising a unit dose of a suspension comprising: a variant capsid protein comprising an insertion of an amino acid sequence selected from LGETTRP, NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF and STGKVPN at a position corresponding to amino acids 570 to 611 of the capsid protein VP1 in AAV2; and an rAAV having a heterologous sequence encoding an anti-vascular endothelial growth factor (anti-VEGF) polypeptide. (Item 57) 57. The pharmaceutical composition of item 56, wherein the unit dose is between 1E12 and 1E13 of the vector genome. (Item 58) 57. The pharmaceutical composition of item 56, wherein the unit dose is between 2E12 and 6E12 of the vector genome. (Item 59) 57. The pharmaceutical composition of claim 56, wherein the suspension is refrigerated. (Item 60) 57. A kit comprising the pharmaceutical composition according to item 56 and a solution for diluting said pharmaceutical composition. (Item 61) 61. The kit of item 60, wherein the solution comprises a buffer, a salt, an alcohol, a surfactant, or any combination thereof. (Item 62) 61. The kit of item 60, further comprising a syringe. (Item 63) 57. The pharmaceutical composition of claim 56, wherein the anti-VEGF polypeptide is a humanized monoclonal antibody. (Item 64) 57. The pharmaceutical composition of item 56, wherein the anti-VEGF polypeptide is an antibody fragment or Fab. (Item 65) 57. The pharmaceutical composition of item 56, wherein the humanized monoclonal antibody is ranibizumab or bevacizumab. (Item 66) 57. The pharmaceutical composition of item 56, wherein the anti-VEGF polypeptide is a soluble truncated VEGF receptor 1 (sVEGFR-1). (Item 67) 57. The pharmaceutical composition of claim 56, wherein the insertion is a LGETTRP at a position between amino acids 587 and 588 in AAV2. [Brief description of the drawings]

[0012] [Figure 1] Figure 1 illustrates laser choroidal neovascularization (CNV) in a non-human primate model (African green monkey). Nine lesions were induced by a single laser application using 750 mW, 50 μm, 100 ms laser irradiation for all spots except the central spot, which was treated with 400 mW. Color fundus photography was performed immediately after laser treatment to document the laser lesions.

[0013] [Diagram 2] FIG. 2 illustrates the nucleic acid sequence of sVEGFR-1.

[0014] [Diagram 3] FIG. 3 illustrates CNV reduction following intravitreal injection of AAV2.7m8-sVEGFR-1. AAV2.7m8-sVEGFR-1 or vehicle control containing formulation buffer was administered to monkey eyes by intravitreal injection at a dose of 2.1×1012 vg. A reduction in percent grade IV CNV lesions was observed with AAV2.7m8-sVEGFR-1 compared to administration of vehicle alone for fundus images collected on day 14 (light gray bar). No significant difference in percent grade IV CNV lesions was observed with AAV2.7m8-sVEGFR-1 compared to administration of vehicle alone as measured by fundus images collected on day 28 (dark gray bar).

[0015] [Figure 4]Figure 4 illustrates that intravitreally administered AAV2.7m8-ranibizumab prevented the development of laser-induced grade IV CNV lesions. AAV2.7m8-ranibizumab, ranibizumab alone (positive control) or vehicle control containing formulation buffer was administered to monkey eyes by intravitreal injection at a dose of 2x1012vg. AAV2.7m8-ranibizumab significantly reduced grade IV CNV lesions to levels comparable to ranibizumab alone, as measured by fundus images collected on days 14 (light gray bars) and 28 (dark gray bars). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Some aspects are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships and methods are shown to allow a thorough understanding of the features described herein. However, a person skilled in the relevant art will immediately recognize that the features described herein can be implemented without one or more of such specific details or by using other methods. The features described herein are not limited by the illustrated ordering of acts or events, since some acts may be performed in different orders and / or simultaneously with other acts or events. Moreover, not all illustrated acts or events are required to perform the methodology according to the features described herein.

[0017] The present disclosure relates to pharmaceutical compositions and methods of treating or preventing ocular diseases or conditions comprising administering a gene therapy, vector or construct comprising a nucleic acid sequence (e.g., cDNA) encoding an anti-VEGF agent by intravitreal or subretinal injection into the eye of a primate (e.g., monkey or human). Upon intravitreal or subretinal injection of a gene therapy, vector or construct comprising a nucleic acid sequence encoding an anti-VEGF agent or transgene, the anti-VEGF gene is expressed in vivo in target cells or tissues, e.g., retinal cells, to produce an anti-VEGF protein or gene product, resulting in a therapeutic effect.

[0018] In some embodiments, gene therapy, vector or construct comprising anti-VEGF agent is used to treat or prevent one or more ocular diseases or conditions in primates or human subjects, including but not limited to neovascular (wet) age-related macular degeneration (AMD), retinal vein occlusion (RVO), macular edema after RVO, diabetic macular edema (DME) and / or diabetic retinopathy (DR) in DME patients, or any other related ocular disease or condition involving neovascularization (e.g., choroidal neovascularization (CNV)). In some embodiments, the methods described herein are used to treat ocular diseases or conditions that are responsive to standard of care therapies or existing treatments, such as ranibizumab or bevacizumab injections. In some embodiments, the methods described herein are used to treat ocular diseases or conditions that are responsive to at least one current standard of care for AMD, RVO, DME, DR, or DR in DME patients, such as ranibizumab or bevacizumab injections.

[0019] The present disclosure relates to compositions and methods for the prevention or treatment of ocular neovascularization in a subject (e.g., a non-human primate or a human) by administering, either subretinal or intravitreal, a pharmaceutical composition adapted for gene therapy comprising a pharma- ceutical effective amount of a vector, e.g., a viral vector, such as an adeno-associated virus (AAV) comprising a nucleic acid sequence encoding an anti-VEGF agent, a therapeutic transgene, or a polypeptide having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to sVEGFR-1, ranibizumab, bevacizumab, or any other known anti-(ant-)VEGF agent, or a functional fragment, mutant, or variant of any thereof. Such homology can be based on nucleic acid sequence (eg, cDNA), amino acid sequence, spatial conformation or protein structure (eg, secondary, tertiary or quaternary structure).

[0020] In some aspects, the vectors disclosed herein are adeno-associated viruses (AAV) of any serotype that contain a mutation, such as an insertion of 5-11 amino acids, at a site in the solvent-exposed GH loop or loop IV of the capsid protein. In some embodiments, a 7-mer amino acid sequence is inserted into the GH loop or loop IV of the AAV capsid protein. The GH loop / loop IV of the AAV capsid is described, for example, in van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79: 5047; and Shen et al. (2007) Mol. Ther. 15:1955. In some embodiments, an amino acid sequence comprising any one of the following: LGETTRP, NETITRP, KAGQANN, KDPKTTN, KDTDTR, RAGGSVG, AVDTTKF, and STGKVPN is inserted into the GH loop / loop IV of an AAV capsid protein (e.g., VP1 capsid protein), thereby generating an AAV variant having a variant capsid protein, respectively. In some embodiments, the amino acid insertion is made at the following positions in each AAV serotype: between 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 589 and 590 of AAV10.

[0021] In some embodiments, the amino acid can be inserted between two adjacent amino acids at the position between amino acids 570 and 611 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype. In some embodiments, an AAV2 vector comprising an LGETTRP amino acid insertion between amino acids 587 and 588 of VP1 of AAV2 is used in the gene therapy disclosed herein. In some embodiments, the treatment method described herein comprises subretinal or intravitreal administration of a pharmaceutical composition or formulation comprising any serotype of AAV comprising a nucleic acid sequence encoding an anti-VEGF agent (e.g., sVEGFR-1, ranibizumab or bevacizumab). In some aspects, subretinal or intravitreal injection of the pharmaceutical composition disclosed herein results in expression of the anti-VEGF agent in target cells, e.g., retinal cells, in the subject's eye, which results in reduced neovascularization or VEGF expression and / or inhibition of VEGF expression or activity in vivo, or disruption of VEGF-VEGFR interaction in vivo. In some instances, expression of an anti-VEGF agent sequesters endogenous VEGF in vivo, preventing VEGF binding to or interaction with endogenous VEGF receptors in vivo.

[0022] One advantage of gene therapy over protein injections is that gene therapy provides extended or sustained release of a therapeutic agent (e.g., an anti-VEGF agent) without the need for repeated injections. Such extended or sustained release of a therapeutic agent results from the delivery of a nucleic acid sequence encoding a transgene that is expressed in vivo to provide a therapeutic effect.

[0023] In some embodiments, the rAAV can include a capsid variant protein that increases its infectivity to target cells or tissues in the eye (e.g., retinal cells), allowing for more efficient delivery of a nucleic acid sequence encoding a therapeutic transgene to the target cells or tissues, where the therapeutic transgene can be expressed for a period of time, e.g., at least 1, 1.5, 2, 3, 4, 5, 10 years or longer. The gene therapy disclosed herein can be targeted to a specific tissue or cell type of interest, e.g., photoreceptor cells, which can help minimize off-target effects or provide a more targeted delivery of the therapeutic transgene in vivo.

[0024] The extended or sustained delivery of anti-VEGF agents in vivo by gene therapy may allow the administration of pharmaceutical compositions comprising nucleic acid sequences encoding anti-VEGF agents in a single dose or one-time dose. In some embodiments, the total number of doses of gene therapy administered to a subject is no more than once every at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years. In some embodiments, the administration of gene therapy comprising nucleic acid sequences encoding anti-VEGF agents is only once or once in the patient's lifetime. In some embodiments, the administration of gene therapy comprising nucleic acid sequences encoding anti-VEGF agents once can produce a therapeutic effect in a patient that lasts for more than 1 year or more than 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer. In some embodiments, gene therapy comprising nucleic acid sequence encoding anti-VEGF agent is administered to patient once or less for at least 2 years or more, at least 3 years or more, at least 4 years or more, at least 5 years or more, at least 6 years or more, at least 7 years or more, at least 8 years or more, at least 9 years or more, or at least 10 years or more.In some embodiments, gene therapy comprising nucleic acid sequence encoding anti-VEGF agent is administered to patient who is responsive to at least one current standard treatment or at least one existing treatment, such as ranibizumab or bevacizumab.In some embodiments, gene therapy is administered to patient who has been pretreated with ranibizumab or bevacizumab before receiving gene therapy.

[0025] In some embodiments, a one-time administration of gene therapy comprising a nucleic acid sequence encoding an anti-VEGF agent prevents a patient from having to receive ranibizumab, bevacizumab, or any other protein-based therapeutic or standard treatment for neovascularization in the eye for more than 1 year, more than 1.5 years, or more than 2, 3, 4, 5, 6, 7, 8, 9, 10 years. In some embodiments, a patient who receives an injection of gene therapy comprising a nucleic acid sequence encoding an anti-VEGF agent does not need any additional injections of ranibizumab, bevacizumab, or any other protein-based therapeutic or standard treatment for neovascularization in the eye for the rest of the patient's life. In other embodiments, a patient who receives a one-time injection of anti-VEGF gene therapy can begin treatment with ranibizumab, bevacizumab, and / or any other approved therapeutic if necessary after at least 1.5, 2, 5, 10 years or more have passed since receiving gene therapy.

[0026] The terminology used in the present disclosure is for the purpose of describing particular instances only and is not intended to be limiting of the compositions, methods, and compositions of the present disclosure.

[0027] The compositions and methods of the present disclosure described herein can use conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, immunochemistry and ophthalmology techniques within the skill of a person skilled in the art, unless otherwise stated. Such conventional techniques include retina or vision observation and analysis in subjects, recombinant virus cloning and propagation, pharmaceutical composition formulation, and biochemical purification and immunochemistry methods. Specific illustrations of suitable techniques can be obtained by reference to the examples herein. However, it is of course possible to use equivalent conventional procedures.Such conventional techniques and descriptions can be found in Green et al., eds., Genome Analysis: A Laboratory Manual Series (Vols. I-IV) (1999); Weiner et al., eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach and Dveksler, eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics: Sequence and Genome Analysis (2004); Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2006); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (all from Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4th ed.) WH Freeman, NY (1995); Gait, "Oligonucleotide Synthesis: A Practical Techniques for determining the molecular structure of a molecule can be found in standard laboratory manuals such as, for example, "A Novel Approach," IRL Press, London (1984); Nelson and Cox, Lehninger, Principles of Biochemistry, 3rd ed., W.H. Freeman Pub., New York (2000); and Berg et al., Biochemistry, 5th ed., W.H. Freeman Pub., New York (2002), all of which are incorporated by reference in their entirety for all purposes.

[0028] Disclosed herein in some embodiments is a pharmaceutical formulation comprising: (a) (i) a variant AAV2 capsid protein, wherein the variant AAV2 capsid protein comprises an LGETTRP insertion between positions 587 and 588, wherein the variant capsid protein confers increased infectivity to retinal cells relative to an AAV virion comprising a corresponding non-variant AAV2 capsid protein; and (ii) a recombinant adeno-associated virus (rAAV2) virion adapted for gene therapy, comprising a heterologous nucleic acid sequence encoding an anti-VEGF agent; and (b) a pharmaceutically acceptable excipient. In some embodiments, the encoded gene product is a polypeptide having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to ranibizumab, bevacizumab or any other known anti-VEGF agent.

[0029] Also disclosed herein is a method of treating an ocular condition or disease for which an anti-VEGF gene product (e.g., ranibizumab or bevacizumab) is indicated or approved for treatment, comprising administering a pharmaceutical composition adapted for gene therapy to the subject's eye by subretinal or intravitreal injection, i.e., delivering a nucleic acid sequence encoding an anti-VEGF gene product in vivo, as described herein. In some embodiments, the gene therapy is administered by intravitreal injection. In some embodiments, the anti-VEGF agent is ranibizumab, bevacizumab, sVEGFR-1, or a variant or functional fragment of any of these.

[0030] Also disclosed herein are pharmaceutical compositions comprising gene therapy or vectors encoding fusion proteins or polypeptides having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to known anti-VEGF proteins or fusion proteins (e.g., ranibizumab or bevacizumab), which may be lyophilized or provided in lyophilized form. In some embodiments, the pharmaceutical compositions in lyophilized form are provided in a kit together with a solution or buffer for reconstituting the pharmaceutical composition prior to administration. In some embodiments, the pharmaceutical compositions disclosed herein are provided as a solution, homogenous solution, suspension or refrigerated suspension.

[0031] Also disclosed herein is a recombinant adeno-associated virus (rAAV) virion adapted for gene therapy to reduce choroidal neovascularization, comprising: (a) a variant AAV capsid protein that confers increased infectivity to retinal cells compared to AAV virions that contain the corresponding non-variant or unmodified AAV capsid protein; (b) a heterologous nucleic acid sequence that encodes a polypeptide or therapeutic transgene having anti-VEGF activity. In some embodiments, the rAAV used in gene therapy is rAAV2.

[0032] Also disclosed herein is a method of treating an ocular condition or disease, comprising administering to the eye of a human subject an rAAV virion adapted for gene therapy and in vivo delivery of a nucleic acid sequence for expressing an anti-VEGF agent or a protein having anti-VEGF activity as described herein, wherein the human subject has previously been diagnosed with an ocular condition associated with neovascularization. In some embodiments, the gene therapy is administered to a patient who is responsive to at least one of approved anti-VEGF therapies, such as ranibizumab or bevacizumab. In some embodiments, the gene therapy is administered to a patient who has been pretreated with at least one of approved therapies, such as ranibizumab or bevacizumab. In some embodiments, the gene therapy disclosed herein is administered to a patient who has been pretreated with at least one of approved therapies, such as ranibizumab or bevacizumab injections, and has failed to show improvement. In some embodiments, patients undergoing the gene therapy methods disclosed herein have one or more risk factors that militate against treating the patient with a therapy that requires multiple repeated injections into the eye, e.g., increased risk of inflammation, infection, elevated intraocular pressure, and / or other adverse effects.

[0033] In some embodiments, disclosed herein are methods and pharmaceutical formulations comprising: (a) (i) a variant AAV capsid protein comprising an amino acid insertion selected from LGETTRP, NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF and STGKVPN at a position corresponding to amino acids 570-611 of capsid protein VP1 in AAV2, the variant AAV capsid protein conferring increased infectivity to retinal cells compared to an AAV virion comprising the corresponding non-variant AAV2 capsid protein; and (ii) a recombinant adeno-associated virus (rAAV) virion adapted for gene therapy comprising a heterologous nucleic acid sequence encoding an anti-VEGF agent; and (b) a pharmaceutically acceptable excipient. In some embodiments, the encoded gene product is a fusion protein, an antibody or an antibody fragment. In some embodiments, the pharmaceutically acceptable excipient comprises a surfactant that prevents aggregation in the pharmaceutical compositions disclosed herein.

[0034] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0035] The terminology used herein is for the purpose of describing particular instances only and is not intended to be limiting. 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 variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising." The term "comprising" is synonymous herein with "including" or "containing," and is inclusive or open-ended.

[0036] Any reference herein to "or" is intended to encompass "and / or" unless otherwise indicated. As used herein, a number followed by the term "about" refers to that number plus or minus 10%. A range followed by the term "about" refers to a range from minus 10% of its minimum value to plus 10% of its maximum value.

[0037] The terms "subject," "patient," or "individual" refer to primates, including non-human primates, such as African green and rhesus monkeys, as well as humans. In preferred embodiments, the subject is a human or a human patient.

[0038] The terms "treat", "treating", "treatment", "ameliorate" or "ameliorating" and other grammatical equivalents are intended herein to include alleviating, relieving or reversing disease or condition symptoms, preventing additional symptoms, reversing or preventing the underlying metabolic causes of symptoms, inhibiting a disease or condition, e.g., arresting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating conditions resulting from a disease or condition, or halting the symptoms of a disease or condition, including prophylaxis. The term further includes achieving a therapeutic benefit and / or a prophylactic benefit. Therapeutic benefit refers to the eradication or reversal of the underlying disease being treated. Therapeutic benefit is also achieved in some embodiments by eradicating or reversing one or more of the physiological symptoms associated with the underlying disease, such that an improvement is observed in the patient, even though the patient still suffers from the underlying disease. For prophylactic benefit, the pharmaceutical composition is administered to a patient at risk of developing a particular disease or who reports one or more of the physiological symptoms of the disease, even if a diagnosis of the disease has not been made.

[0039] The terms "administer," "administering," "administration," and the like, as used herein, can refer to methods used to enable delivery of a therapeutic agent or pharmaceutical composition to a desired site of biological action. Such methods include intravitreal or subretinal injection into the eye.

[0040] The terms "effective amount," "therapeutically effective amount," or "pharmaceutical effective amount," as used herein, can refer to a sufficient quantity of at least one pharmaceutical composition or compound being administered that will relieve to some extent one or more of the symptoms of the disease or condition being treated.

[0041] The term "pharmacologically acceptable" as used herein can refer to a material, such as a relatively non-toxic carrier or diluent, that does not abrogate the biological activity or properties of the compounds disclosed herein (i.e., the material does not elicit undesirable biological effects when administered to an individual, nor does it interact in a deleterious manner with any of the components of the composition in which it is contained).

[0042] The term "pharmaceutical composition" or simply "composition," as used herein, can refer to a biologically active compound optionally mixed with at least one pharma- ceutically acceptable chemical component, including but not limited to a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, and the like.

[0043] "AAV vector" or "rAAV vector" as used herein refers to an adeno-associated virus (AAV) vector or recombinant AAV (rAAV) vector that typically contains a polynucleotide sequence of non-AAV origin (i.e., a polynucleotide heterologous to AAV, such as a nucleic acid sequence encoding a therapeutic transgene, e.g., ranibizumab), which is a sequence of interest for genetic transformation of a cell. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeats (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV).

[0044] "AAV virus" or "AAV virus particle" or "rAAV vector particle" refers to a virus particle composed of at least one AAV capsid protein (typically all of the capsid proteins of wild-type AAV) and a polynucleotide rAAV vector. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector". Thus, the production of rAAV particles necessarily includes the production of rAAV vectors, since such vectors are contained within the rAAV particles.

[0045] The term "packaging" as used herein can refer to a series of intracellular events that can result in the assembly and encapsidation of rAAV particles.

[0046] AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0047] The term "polypeptide" can encompass both naturally occurring and non-naturally occurring proteins (e.g., fusion proteins), peptides, fragments, variants, derivatives and analogs thereof. A polypeptide can be a monomer, dimer, trimer or polymer. Furthermore, a polypeptide can contain multiple distinct domains, each having one or more distinct activities. For the avoidance of doubt, a "polypeptide" can be any length greater than two amino acids.

[0048] As used herein, "polypeptide variant" or simply "variant" refers to a polypeptide whose sequence contains amino acid modifications. In some instances, the modifications can be insertions, duplications, deletions, rearrangements or substitutions of one or more amino acids compared to the amino acid sequence of a reference protein or polypeptide, such as a native or wild-type protein. A variant can have a single amino acid at a position in the sequence of a reference protein changed to another amino acid, one or more amino acid point substitutions, one or more amino acids inserted or deleted, respectively, one or more insertions and / or deletions, and / or truncations of the amino acid sequence at either or both of the amino or carboxy termini. A variant can have the same or different biological activity compared to a reference protein or unmodified protein.

[0049] In some embodiments, the variant can have, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% overall sequence homology to its counterpart reference protein, and the reference protein can be naturally occurring or non-naturally occurring, or a derivative or variant of a naturally occurring protein. In some embodiments, the variant can have at least about 90% overall sequence homology to the wild-type protein. In some embodiments, the variant exhibits at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or at least about 99.9% overall sequence identity.

[0050] As used herein, "recombinant" can refer to a biological molecule, e.g., a gene or protein, that is (1) removed from its naturally occurring environment, (2) not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) operably linked to a polynucleotide to which it is not linked in nature, or (4) not occurring in nature. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant if, for example, it is synthesized from an mRNA synthesized from a recombinant gene present in the cell.

[0051] "Operably linked" or "operably linked" or "coupled" can refer to the proximity of genetic elements, which are in a relationship that allows them to operate in a predictable manner. For example, a promoter can be operably linked to a coding region if it serves to initiate transcription of the coding sequence. There can be intervening residues between the promoter and the coding region, so long as this functional relationship is maintained.

[0052] The term "expression vector" or "expression construct" or "cassette" or "plasmid" or simply "vector" can include any type of genetic construct, including AAV or rAAV vectors, containing a nucleic acid or polynucleotide encoding a gene product, where a portion or the entire nucleic acid encoding sequence can be transcribed and adapted for gene therapy. The transcript can be translated into a protein. In some cases, the transcript can be partially translated or not translated. In certain embodiments, expression includes both transcription of the gene and translation of the mRNA into a gene product. In other embodiments, expression includes only transcription of the nucleic acid encoding the gene of interest. An expression vector can also include control elements operably linked to the coding region to facilitate expression of the protein in the target cell. The combination of the control element and the gene(s) to which the control element is operably linked for expression is sometimes referred to as an "expression cassette," many of which are known and available in the art or can be readily constructed from components available in the art.

[0053] The term "heterologous" can refer to an entity that is genotypically distinct from the remainder of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species can be a heterologous polynucleotide. A promoter that has been removed from its native coding sequence and operably linked to a coding sequence that is not naturally found in association with it can be a heterologous promoter.

[0054] As used herein, "7m8" refers to the 7-mer amino acid sequence LGETTRP.

[0055] "7m8 variant" refers to a rAAV, which may be of any serotype, with the amino acid sequence LGETTRP inserted into the solvent-exposed GH loop of the capsid protein.

[0056] When 7m8 is inserted into rAAV2 (also referred to as AAV2.7m8), the 7-mer amino acid sequence LGETTRP is inserted into the GH loop of the AAV2 capsid protein, e.g., between positions 587 and 588 of the AAV2 capsid protein. When 7m8 is inserted into rAAV1 (also referred to as AAV1.7m8), the 7-mer amino acid sequence LGETTRP is inserted into the GH loop of the AAV1 capsid protein, e.g., between amino acids 590 and 591 of the AAV1 capsid protein. When 7m8 is inserted into rAAV5 (also referred to as AAV5.7m8), the 7-mer amino acid sequence LGETTRP is inserted into the GH loop of the AAV5 capsid protein, e.g., between amino acids 575 and 576 of the AAV5 capsid protein. When 7m8 is inserted into rAAV6 (also referred to as AAV6.7m8), the 7-mer amino acid sequence LGETTRP is inserted into the GH loop of the AAV6 capsid protein, e.g., between amino acids 590 and 591 of the AAV6 capsid protein. When 7m8 is inserted into rAAV7 (also referred to as AAV7.7m8), the 7-mer amino acid sequence LGETTRP is inserted into the GH loop of the AAV7 capsid protein, e.g., between amino acids 589 and 590 of the AAV7 capsid protein. When 7m8 is inserted into rAAV8 (also referred to as AAV8.7m8), the 7-mer amino acid sequence LGETTRP is inserted into the GH loop of the AAV8 capsid protein, e.g., between amino acids 590 and 591 of the AAV8 capsid protein. When 7m8 is inserted into rAAV9 (also referred to as AAV9.7m8), the 7-mer amino acid sequence LGETTRP is inserted into the GH loop of the AAV9 capsid protein, e.g., between amino acids 588 and 589 of the AAV9 capsid protein. When 7m8 is inserted into rAAV10 (also referred to as AAV10.7m8), the 7-mer amino acid sequence LGETTRP is inserted into the GH loop of the AAV10 capsid protein, e.g., between amino acids 589 and 590 of the AAV10 capsid protein.

[0057] In some embodiments, disclosed herein is a recombinant adeno-associated virus (rAAV) virion for reducing neovascularization comprising: (a) a variant AAV capsid protein comprising an amino acid modification in a solvent-exposed region of the capsid protein and exhibiting increased infectivity for retinal cells relative to a corresponding non-variant AAV capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product or therapeutic transgene, wherein administration of an effective amount of the rAAV by intravitreal or subretinal injection in the eye of a primate or human subject results in reduced neovascularization in the eye.

[0058] Also disclosed herein is a recombinant adeno-associated virus (rAAV) virion for reducing neovascularization comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises an amino acid modification in a solvent-exposed region of the AAV capsid protein, wherein the variant capsid protein confers an increased ability to cross the inner limiting membrane (ILM) in the eye; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product, wherein administration of an effective amount of the rAAV by intravitreal or subretinal injection in the eye of a primate or human subject results in reduced neovascularization in the eye.

[0059] Also disclosed herein is a recombinant adeno-associated virus (rAAV) virion comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion of LGETTRP after the amino acid position corresponding to 587 in AAV2, wherein the variant capsid protein confers an increased ability to deliver a gene product across the inner limiting membrane (ILM) of the eye in a primate; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding the gene product.

[0060] Also disclosed herein is a gene therapy composition in a unit dose form for treating an ocular condition or disease, comprising: (a) (i) a variant AAV capsid protein comprising an amino acid modification in a solvent-exposed region of the capsid protein and exhibiting increased infectivity for retinal cells relative to a corresponding non-variant AAV capsid protein; and (ii) a recombinant adeno-associated virus (rAAV) virion comprising a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product that, when transduced, reduces neovascularization in the eye of a primate or human subject; and (b) a pharmaceutically acceptable excipient, wherein the rAAV virion is in an amount sufficient to at least partially reduce neovascularization when administered as a unit dose by intravitreal or subretinal injection in the eye of a primate.

[0061] Also disclosed herein are methods of treating an ocular condition or disease comprising administering to a subject, e.g., a human subject, a unit dose of the rAAV gene therapy described herein.

[0062] The term "anti-VEGF agent" includes any therapeutic agent, including a protein, polypeptide, peptide, fusion protein, multimeric protein, gene product, antibody, human monoclonal antibody, antibody fragment, aptamer, small molecule, kinase inhibitor, receptor or receptor fragment, or nucleic acid molecule, that can reduce, interfere with, disrupt, block, and / or inhibit the activity or function of endogenous VEGF and / or endogenous VEGF receptor (VEGFR), or VEGF-VEGFR interaction or pathway in vivo. The anti-VEGF agent can be any one of known therapeutic agents, such as ranibizumab or bevacizumab, that can reduce new blood vessel growth or formation and / or edema or swelling when delivered to a cell, tissue, or subject in vivo. In some embodiments, the anti-VEGF agent can be naturally occurring, non-naturally occurring, or synthetic. In some embodiments, the anti-VEGF agent can be derived from a naturally occurring molecule that has subsequently been modified or mutated to confer anti-VEGF activity. In some embodiments, the anti-VEGF agent is a fusion or chimeric protein. In such proteins, functional domains or polypeptides are artificially fused to moieties or polypeptides to produce fusion or chimeric proteins that can sequester VEGF in vivo or function as VEGFR decoys, hi some embodiments, the anti-VEGF agent is a fusion or chimeric protein that blocks endogenous VEGFR from interacting with its ligand.

[0063] As used herein, unless otherwise required, "VEGF" can refer to any isoform of VEGF, including but not limited to VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or any combination thereof, or any functional fragment or variant. Unless otherwise required, "VEGF" can refer to any member of the VEGF family, including the members: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D, or any combination thereof, functional fragment or variant.

[0064] As used herein, "VEGF receptor" or "VEGFR" or "VEGF-R" can be used to refer to any one of the receptors for VEGF, including, but not limited to, VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR) and VEGFR-3 (or Flt-4). A VEGFR can be a membrane-bound or soluble form of the receptor or a functional fragment or truncation of the receptor.

[0065] Examples of anti-VEGF agents include, but are not limited to, ranibizumab, bevacizumab, or any combination, variant, or functional fragment thereof.

[0066] The section headings used herein are for organizational purposes only and are not to be construed as limitations on the subject matter described.

[0067] vector A variety of viral vectors can be used in gene therapy, including adenoviruses, adeno-associated viruses, retroviruses and lentiviruses.

[0068] In some embodiments, the pharmaceutical compositions and methods of the present disclosure provide for the delivery of a nucleic acid sequence (e.g., a cDNA sequence) encoding an anti-VEGF agent, a functional fragment or variant thereof, to a retinal cell in a human subject or patient in need thereof (e.g., a patient diagnosed with AMD, RVO, DME). The delivery of a therapeutic transgene nucleic acid to a patient using a delivery system such as a rAAV or viral vector is also referred to as gene therapy.

[0069] In some embodiments, delivery of the anti-VEGF agent nucleic acid sequence can be performed using any suitable "vector" (also referred to as "gene delivery" or "gene transfer vehicle"). The vector (e.g., rAAV), delivery vehicle, gene delivery vehicle or gene transfer vehicle can include any suitable macromolecular or molecular complex that includes a polynucleotide to be delivered to a target cell, e.g., a retinal cell, including a photoreceptor, a retinal ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell or a retinal pigment epithelial cell. In some cases, the target cell can be any cell to which a nucleic acid molecule or gene is delivered. The polynucleotide to be delivered can include a coding sequence for a therapeutic transgene, such as a sequence encoding ranibizumab.

[0070] The compositions and methods of the present disclosure provide any suitable method for delivery of anti-VEGF (e.g., ranibizumab) nucleic acid sequences to the eye or retinal cells of a non-human primate or human subject. In some cases, the delivery of the nucleic acid molecule, polynucleotide, or gene therapy is formulated or adapted for intravitreal injection into the eye of the non-human primate or human subject.

[0071] In some embodiments, suitable vectors include, but are not limited to, viral vectors such as adenovirus, adeno-associated virus (AAV) and retrovirus, retrovirus, lentivirus, liposomes, lipid-containing complexes, nanoparticles, and other macromolecular complexes that can deliver polynucleotides to retinal cells. In some embodiments, the viral vector comprises a strong eukaryotic promoter, such as a cytomegalovirus (CMV) promoter or a constitutive promoter, operably linked to the polynucleotide.

[0072] In some embodiments, the vector comprises a recombinant viral vector incorporating one or more nucleic acid molecules. As described herein, nucleic acid refers to polynucleotide. Nucleic acid and polynucleotide can be used interchangeably. In some embodiments, the nucleic acid comprises DNA or RNA. In some cases, the nucleic acid comprises DNA (e.g., cDNA) or RNA for expression of anti-VEGF agent or therapeutic transgene. In some embodiments, the RNA can comprise a transcript of a gene of interest (e.g., ranibizumab), introns, untranslated regions (UTRs), termination sequences, and other sequences. In other embodiments, the DNA can include, but is not limited to, sequences such as promoter sequences, gene of interest (e.g., ranibizumab), UTRs, termination sequences, and other sequences. In some cases, a combination of DNA and RNA can be used.

[0073] In some embodiments, the present disclosure provides recombinant viruses, such as recombinant adeno-associated viruses (rAAV), as vectors for delivery and expression of ranibizumab, bevacizumab, sFLT-1, or a functional fragment or variant of any of these in a subject.

[0074] In some embodiments, any suitable viral vector can be engineered or optimized for use with the compositions and methods of the present disclosure. For example, recombinant viral vectors derived from adenovirus (Ad) or adeno-associated virus (AAV) can be modified to be replication-deficient in human or primate subjects. In some embodiments, hybrid viral vector systems can be obtained using methods known to those skilled in the art and used to deliver nucleic acids encoding anti-VEGF agents to retinal cells. In some embodiments, viral delivery systems or gene therapy can integrate nucleic acid sequences comprising anti-VEGF genes into target cell genomes (e.g., the genome of retinal cells) to provide stable gene expression of the genes over time. In some embodiments, the anti-VEGF genes are not integrated into the target cell genome and are expressed from a plasmid or vector introduced into the target cells.

[0075] In some embodiments, the viral vector suitable for delivery of anti-VEGF nucleic acid sequence to retinal cells is AAV or rAAV, which is a small non-enveloped single-stranded DNA virus.rAAV is a non-pathogenic human parvovirus and can be made to depend on helper viruses, including adenovirus, herpes simplex virus, vaccinia virus and CMV, for replication.Exposure to wild type (wt) AAV is not known to be associated with or cause any human pathology, and is common in the general population, making AAV or rAAV a suitable delivery system for gene therapy.The AAV and rAAV used in gene therapy for delivery of therapeutic transgenes, such as ranibizumab, can be of any serotype. In some embodiments, the pharmaceutical compositions and methods of the present disclosure provide for the use of any suitable AAV serotype, including AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, AAV-DJ, and any of these hybrid or chimeric AAVs. In some embodiments, the serotype used is based on the tropism of the virus or the infectivity of the target cell of interest. In some embodiments, AAV2 or rAAV2 is used to deliver the nucleic acid sequence encoding ranibizumab to the subject's eye or retinal cells by intravitreal or subretinal injection. In some embodiments, rAAV2.7m8 is used to deliver the nucleic acid sequence of ranibizumab to the subject's retinal cells.

[0076] In some embodiments, AAV or rAAV viruses, particles or virions comprising variant capsid proteins with increased infectivity to target cells, e.g., retinal cells, are used to increase transduction of retinal cells or increase targeting of gene delivery to retinal cells in a subject. In some embodiments, the rAAV virion comprises an amino acid modification in the capsid protein GH loop / loop IV of the AAV capsid protein. In some cases, the site of the modification is a solvent accessible portion of the GH loop / loop IV of the AAV capsid protein. Several AAV capsid variants are known, including the 7m8 variant. In some embodiments, the rAAV virion comprises a variant AAV capsid protein comprising an insertion of 5 amino acids to 11 amino acids, e.g., a 7 amino acid sequence, in the GH loop of the capsid protein compared to the corresponding parent AAV capsid protein, and the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of retinal cells by AAV virions comprising the corresponding parent or unmodified AAV capsid protein. In some embodiments, an insertion of any one of the following amino acid sequences can be inserted into the GH loop of the capsid protein: LGETTRP(7m8), NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF, and STGKVPN. In some embodiments, rAAV.7m8 containing ranibizumab is used for gene therapy.

[0077] In some embodiments, any one of the following amino acid sequences: NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF, and STGKVPN can be inserted at the following positions to generate rAAV variants for use in gene therapy: between positions 587 and 588 of the AAV2 capsid protein; between amino acids 590 and 591 of the AAV1 capsid protein; between amino acids 575 and 576 of the AAV5 capsid protein; between amino acids 590 and 591 of the AAV6 capsid protein; between amino acids 589 and 590 of the AAV7 capsid protein; between amino acids 590 and 591 of the AAV8 capsid protein; between amino acids 588 and 589 of the AAV9 capsid protein; or between amino acids 589 and 590 of the AAV10 capsid protein.

[0078] In some embodiments, the nucleic acid encoding a gene product, such as ranibizumab, can be under transcriptional control by a promoter that initiates transcription of the gene. In some embodiments, the promoter is a "strong" or constitutively active promoter, such as the CMV promoter. In some embodiments, the connexin 36 promoter is used to drive expression of a therapeutic transgene, such as ranibizumab. In some embodiments, a tissue-specific promoter can be used to provide transcription in specific tissues or cells, such as retinal cells, to reduce potential toxicity or undesirable effects on non-targeted cells. In some embodiments, the recombinant virus and / or plasmid used to generate the rAAV virus can include other transcriptional or regulatory elements, such as polyA (polyadenylation) sequences, untranslated regions (UTRs), 3'UTRs, or termination sequences. In some embodiments, two or more genes can be expressed from a vector or plasmid using an internal ribosome entry site (IRES) or similar element that allows for simultaneous expression of two or more proteins or results in a multigene or polycistronic mRNA.

[0079] In some embodiments, the rAAV and / or plasmid used to generate the rAAV virus comprises the following nucleic acid elements: a first ITR sequence; a promoter sequence; an intron sequence; a first UTR sequence; a sequence encoding an anti-VEGF transgene; a second UTR sequence; a polyA sequence; and a second ITR sequence. In some embodiments, a linker sequence is used between each of these nucleic acid elements. In some embodiments, the sequence encoding the anti-VEGF transgene comprises a sequence encoding an anti-VEGF transgene fusion protein or a functional fragment thereof.

[0080] In some embodiments, the viral vectors of the present disclosure are measured as vector genomes. In some cases, the unit dose of the recombinant virus of the present disclosure is 1×10 10 ~2×10 10 Between 2×10 10 ~3×10 10 Between 3×10 10 ~4×10 10 Between 4×10 10 ~5×10 10 Between 5×10 10 ~6×10 10 Between 6×10 10 ~7×10 10 Between 7×10 10 ~8×10 10 Between 8×10 10 ~9×10 10 Between 9×10 10 ~10×10 10 Between 1×10 11 ~2×10 11 Between 2×10 11 ~3×10 11 Between 3×10 11 ~4×10 11 Between 4×10 11 ~5×10 11 Between 5×10 11 ~6×10 11 Between 6×10 11 ~7×10 11 Between 7×10 11 ~8×10 11 Between 8×10 11 ~9×10 11Between 9×10 11 ~10×10 11 Between 1×10 12 ~2×10 12 Between 2×10 12 ~3×10 12 Between 3×10 12 ~4×10 12 Between 4×10 12 ~5×10 12 Between 5×10 12 ~6×10 12 Between 6×10 12 ~7×10 12 Between 7×10 12 ~8×10 12 Between 8×10 12 ~9×10 12 Between 9×10 12 ~10×10 12 Between 1×10 13 ~2×10 13 Between 2×10 13 ~3×10 13 Between 3×10 13 ~4×10 13 Between 4×10 13 ~5×10 13 Between 5×10 13 ~6×10 13 Between 6×10 13 ~7×10 13 Between 7×10 13 ~8×10 13 Between 8×10 13 ~9×10 13 Between or 9×10 13 ~10×10 13 In some embodiments, the rAAV of the present disclosure comprises a vector genome of between about 2.1 x 10 12 In some embodiments, the rAAV of the present disclosure is a vector genome of 10 10 ~10 13 Between 10 10 ~10 11 Between 10 11 ~10 12 Between 10 12 ~10 13 Between 10 13 ~10 14 Between 2×10 11~4×10 11 Between 3×10 11 ~5×10 11 Between 4×10 11 ~6×10 11 Between 5×10 11 ~7×10 11 Between 6×10 11 ~8×10 11 Between 7×10 11 ~9×10 11 Between 8×10 11 ~10×10 11 Between 1×10 12 ~3×10 12 Between 2×10 12 ~4×10 12 Between 3×10 12 ~5×10 12 Between 4×10 12 ~6×10 12 Between 5×10 12 ~7×10 12 Between 6×10 12 ~8×10 12 Between 7×10 12 ~9×10 12 Between 8×10 12 ~10×10 12 Between 1×10 13 ~5×10 13 Between 5×10 13 ~10×10 13 Between 10 12 ~5×10 12 Between or 5×10 12 ~1×10 13 It is a vector genome between.

[0081] In some cases, the recombinant virus of the disclosure may be about 1E10, about 1.5E10, about 2E10, about 2.5E10, about 3E10, about 3.5E10, about 4E10, about 4.5E10, about 5E10, about 5.5E10, about 6E10, about 6.5E10, about 7E10, about 7.5E10, about 8E10, about 8.5E10, about 9E10, about 9.5E10, about 10E10, about 1E11, about 1.5E11, about 2E11, about 2.5E11, about 3E11, about 3.5E11, about 4E11, about 4.5E11, about 5E11, about 5.5E11, about 6E11, about 6.5E11, about 7E11, about 7.5E11, about 8E11, about 8.5E11, about 9E11, about 9.5E11, about 10E11, about 1E12, about 1.3E12, about 1.5E 12, about 2E12, about 2.1E12, about 2.3E12, about 2.5E12, about 2.7E12, about 2.9E12, about 3E12, about 3.1E1 2, about 3.3E12, about 3.5E12, about 3.7E12, about 3.9E12, about 4E12, about 4.1E12, about 4.3E12, about 4.5E 12, about 4.7E12, about 4.9E12, about 5E12, about 5.1E12, about 5.3E12, about 5.5E12, about 5.7E12, about 5.9E 12, about 6E12, about 6.1E12, about 6.3E12, about 6.5E12, about 6.7E12, about 6.9E12, about 7E12, about 7.1E1 2, about 7.3E12, about 7.5E12, about 7.7E12, about 7.9E12, about 8E12, about 8.1E12, about 8.3E12, about 8.5E 12, about 8.7E12, about 8.9E12, about 9E12, about 9.1E12, about 9.3E12, about 9.5E12, about 9.7E12, about 9.9 E12, about 10E12, about 10.1E12, about 10.3E12, about 10.5E12, about 10.7E12, about 10.9E12, about 11E12 , about 11.5E12, about 12E12, about 12.5E12, about 13E12, about 13.5E12, about 14E12, about 14.5E12, about 15 E12, about 15.5E12, about 16E12, about 16.5E12, about 17E12, about 17.5E12, about 18E12, about 18.5E12, Approx. 19E12, Approx. 19.5E12, Approx. 20E12, Approx. 20.5E12, Approx. 30E12, Approx. 30.5E12, Approx. 40E12, Approx. 40.5E 12, about 50E12, about 50.5E12, about 60E12, about 60.5E12, about 70E12, about 70.5E12, about 80E12, about 80.5E12, approximately 90E12, approximately 95E12 or approximately 100E12, where E is an abbreviation for the base 10 exponent, and xEy refers to x multiplied by the power / exponent of base 10.

[0082] In some embodiments, the pharmaceutical compositions disclosed herein have a cytotoxicity of at least 5E11, at least 5.5E11, at least 6E11, at least 6.5E11, at least 7E11, at least 7.5E11, at least 8E11, at least 8.5E11, at least 9E11, at least 9.5E11, at least 10E11, at least 1E12, at least 1.3E12, at least 1.5E12, at least 2E12, at least 2.1E12, at least 2.3E12, at least 2.5E12, at least 2.7E12, at least Also 2.9E12, at least 3E12, at least 3.1E12, at least 3.3E12, at least 3.5E12, at least 3.7E12, at least 3.9E12, at least 4E12, at least 4.1E12, at least 4.3E12, at least 4.5E12, at least 4.7E12, at least 4.9E12, at least 5E12, at least 5.1E12, at least 5.3E12, at least 5.5E12, at least 5.7E12, at least 5.9E12, at least 6E12, at least 6.1E12, at least 6.3E12, at least 6.5E12, at least 6.7E12, at least 6.9E12, at least 7E12, at least 7.1E12, at least 7.3E12, at least 7.5E12, at least 7.7E12, at least 7.9E12, at least 8E12, at least 8.1E12, at least 8.3E12, at least 8.5E12, at least 8.7E12, at least 8.9E12, at least 9E12, at least 9.1E12, at least 9.3E12, at least 9.5E12, at least 9.7E12, at least 9.9E12, at least 10E12, at least 10.1E12, at least 10.3E12, at least 10.5E12, at least 10.7E12, at least 10.9E12, at least 11E12, at least 11.5E12, at least 12E12, at least 12.5E12, at least 13E12, at least 13.5E12, at least 14E12, at least 14.5E12, at least 15E12, at least 15.5E12, at least 16E12, at least 16.5E12, at least 17E12, at least 17.The recombinant virus comprises a vector genome of at least 5E12, at least 18E12, at least 18.5E12, at least 19E12, at least 19.5E12, at least 20E12, at least 20.5E12, at least 30E12, at least 30.5E12, at least 40E12, at least 40.5E12, at least 50E12, at least 50.5E12, at least 60E12, at least 60.5E12, at least 70E12, at least 70.5E12, at least 80E12, at least 80.5E12, at least 90E12, at least 95E12, or at least 100E12, where E is an abbreviation for exponentiation base 10 and xEy refers to x multiplied by the power / exponent of base 10.

[0083] In some embodiments, the viral vectors of the present disclosure are measured using a multiplicity of infection (MOI). In some cases, MOI refers to the ratio or multiple of vector or viral genome to cells to which the nucleic acid can be delivered. In some cases, MOI is 1×10 6 In some cases, the recombinant virus of the disclosure is at least 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1 × 10 18 In some cases, the recombinant viruses of the present disclosure may be at an MOI of 1×10 8 ~1×10 15 In some cases, the recombinant virus of the present disclosure may be at an MOI of at most 1×10 1 , 1×10 2 , 1×103 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1 × 10 18 It can be the MOI.

[0084] In some embodiments, the nucleic acid can be delivered without the use of a virus (i.e., using a non-viral vector) and can be measured as the amount of nucleic acid. Generally, any suitable amount of nucleic acid can be used with the pharmaceutical compositions and methods of the present disclosure. In some cases, the nucleic acid can be at least 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900μg, 1ng, 10ng, 100ng, 200ng, 300ng, 400ng, 500ng, 600ng, 700ng, 800ng, 900ng, 1mg, 10mg, 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1g, 2g, 3g, 4g or 5g. In some cases, the nucleic acid may be at most about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 pg, The amount of the active ingredient may be 00 μg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g or 5 g.

[0085] In some embodiments, self-complementary vectors (sc) can be used. As shown by Wu, Hum Gene Ther. 2007, 18(2):171-82, incorporated herein by reference, the use of self-complementary AAV vectors can bypass the need for viral second strand DNA synthesis and can result in better expression rates of transgene proteins.

[0086] In some embodiments, several AAV vectors can be generated to allow selection of the optimal serotype and promoter for use with the anti-VEGF transgene.

[0087] In some cases, the vector may be a targeted vector, particularly a targeted rAAV (e.g., AAV2.7m8), that exhibits higher infectivity to specific cells, such as retinal cells or photoreceptors, retinal ganglion cells, Muller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells. Viral vectors for use in the present disclosure can include viral vectors that exhibit low toxicity and / or low immunogenicity in a subject and express a therapeutically effective amount of an anti-VEGF transgene in a subject, e.g., a human patient.

[0088] Disclosed herein are pharmaceutical compositions and methods for delivering nucleic acids encoding anti-VEGF agents to target retinal cells of subjects using rAAVs comprising 7m8 variant capsid proteins, or rAAV2.7m8, and nucleic acid sequences encoding anti-VEGF transgenes in non-human primates or human subjects. In some instances, delivery of anti-VEGF agents by gene therapy can be used to at least partially reverse or prevent ocular diseases or conditions disclosed herein.

[0089] In some embodiments, the increase in retinal cell infectivity of the rAAV variant (e.g., the 7m8 variant) is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% compared to AAV virions containing the corresponding parent or unmodified AAV capsid proteins. In some embodiments, the increase in infectivity of retinal cells is between 5%-100%, between 5%-95%, between 5%-90%, between 5%-85%, between 5%-80%, between 5%-75%, between 5%-70%, between 5%-65%, between 5%-60%, between 5%-55%, between 5%-50%, between 5%-45%, between 5%-40%, between 5%-35%, between 5%-30%, between 5%-25%, between 5%-20%, between 5%-15%, between 5%-10%, compared to AAV virions containing the corresponding parent or unmodified AAV capsid proteins.

[0090] In some embodiments, the increase in infectivity of the rAAV variant to retinal cells is at least 1-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, or at least 2-fold, compared to AAV virions containing the corresponding parental or unmodified AAV capsid proteins. In some embodiments, the increase in infectivity is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, compared to AAV virions containing the corresponding parental AAV capsid proteins. In some embodiments, the increase in infectivity is at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, or at least 100-fold compared to AAV virions containing the corresponding parent or unmodified AAV capsid proteins.

[0091] In some embodiments, the increase in retinal cell infectivity is between 10-fold and 100-fold, between 10-fold and 95-fold, between 10-fold and 90-fold, between 10-fold and 85-fold, between 10-fold and 80-fold, between 10-fold and 75-fold, between 10-fold and 70-fold, between 10-fold and 65-fold, between 10-fold and 60-fold, between 10-fold and 55-fold, between 10-fold and 50-fold, between 10-fold and 45-fold, between 10-fold and 40-fold, between 10-fold and 35-fold, between 10-fold and 30-fold, between 10-fold and 25-fold, between 10-fold and 20-fold, or between 10-fold and 15-fold, compared to AAV virions comprising the corresponding parent or unmodified AAV capsid proteins.

[0092] In some embodiments, the increase in retinal cell infectivity is between 2-fold and 20-fold, between 2-fold and 19-fold, between 2-fold and 18-fold, between 2-fold and 17-fold, between 2-fold and 16-fold, between 2-fold and 15-fold, between 2-fold and 14-fold, between 2-fold and 13-fold, between 2-fold and 12-fold, between 2-fold and 11-fold, between 2-fold and 10-fold, between 2-fold and 9-fold, between 2-fold and 8-fold, between 2-fold and 7-fold, between 2-fold and 6-fold, between 2-fold and 5-fold, between 2-fold and 4-fold, or between 2-fold and 3-fold, compared to AAV virions comprising the corresponding parent or unmodified AAV capsid proteins.

[0093] In some embodiments, the amino acid modifications of the capsid proteins described herein can confer an increased ability to cross the inner limiting membrane (ILM) in the eye of a primate or human subject, compared to the ability of an AAV virion containing the corresponding parent or unmodified AAV capsid protein to cross the ILM in the eye of the subject. In some embodiments, the increased ability to cross the ILM is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase, compared to an AAV virion containing the corresponding parent or unmodified AAV capsid protein. In some embodiments, the increase in ability to cross the ILM is an increase of between 5%-100%, between 5%-95%, between 5%-90%, between 5%-85%, between 5%-80%, between 5%-75%, between 5%-70%, between 5%-65%, between 5%-60%, between 5%-55%, between 5%-50%, between 5%-45%, between 5%-40%, between 5%-35%, between 5%-30%, between 5%-25%, between 5%-20%, between 5%-15%, or between 5%-10%, compared to the parent or unmodified AAV capsid protein.

[0094] In some embodiments, the increase in ability to cross the ILM is at least 1-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, or at least 2-fold, compared to an AAV virion comprising the corresponding parental AAV capsid protein. In some embodiments, the increase in ability to cross the ILM is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, compared to an AAV virion comprising the corresponding parental AAV capsid protein. In some embodiments, the increase in ability to cross the ILM is at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, or at least 100-fold, compared to AAV virions comprising the corresponding parent or unmodified AAV capsid proteins.

[0095] In some embodiments, the increase in ability to cross the ILM is between 10-fold and 100-fold, between 10-fold and 95-fold, between 10-fold and 90-fold, between 10-fold and 85-fold, between 10-fold and 80-fold, between 10-fold and 75-fold, between 10-fold and 70-fold, between 10-fold and 65-fold, between 10-fold and 60-fold, between 10-fold and 55-fold, between 10-fold and 50-fold, between 10-fold and 45-fold, between 10-fold and 40-fold, between 10-fold and 35-fold, between 10-fold and 30-fold, between 10-fold and 25-fold, between 10-fold and 20-fold, or between 10-fold and 15-fold, compared to AAV virions comprising the corresponding parent or unmodified AAV capsid proteins.

[0096] In some embodiments, the increase in ability to cross the ILM is between 2-fold and 20-fold, between 2-fold and 19-fold, between 2-fold and 18-fold, between 2-fold and 17-fold, between 2-fold and 16-fold, between 2-fold and 15-fold, between 2-fold and 14-fold, between 2-fold and 13-fold, between 2-fold and 12-fold, between 2-fold and 11-fold, between 2-fold and 10-fold, between 2-fold and 9-fold, between 2-fold and 8-fold, between 2-fold and 7-fold, between 2-fold and 6-fold, between 2-fold and 5-fold, between 2-fold and 4-fold, or between 2-fold and 3-fold, compared to AAV virions comprising the corresponding parent or unmodified AAV capsid proteins.

[0097] In some embodiments, the vector can be a retroviral vector. The retroviral vector can include a Moloney murine leukemia virus and a virus based on HIV. In some embodiments, a viral vector based on HIV can be used, and the viral vector based on HIV includes at least two vectors, the gag and pol genes are derived from the HIV genome, and the env gene is derived from another virus. In some embodiments, a DNA viral vector can be used. Such vectors include pox vectors, such as orthopox or avipox vectors, herpes virus vectors, such as herpes simplex virus I (HSV) vectors [Geller, AI et al., J. Neurochem, vol. 64:487 (1995); Lim, F. et al., in DNA Cloning: Mammalian Systems, edited by D. Glover (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci. USA: vol. 90, pp. 7603 (1993); Geller, AI et al., Proc Natl. Acad. Sci. USA: vol. 87:1149 (1990)], adenovirus vectors [LeGal LaSalle et al., Science, vol. 259:988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol. 69:2004 (1995)] and adeno-associated viral vectors [Kaplitt, MG et al., Nat. Genet. 8:148 (1994)], the entireties of which are incorporated herein by reference.

[0098] In some embodiments, the vector can be a lentiviral vector.The lentiviral vector for use in the present disclosure can be derived from human and non-human (including SIV) lentivirus.An example of a lentiviral vector can include a tissue-specific promoter operably linked to an anti-VEGF protein gene, together with the nucleic acid sequence required for vector propagation.The nucleic acid sequence can include a viral LTR, a primer binding site, a polypurine tract, an att site and an encapsidation site.

[0099] In some embodiments, the vector can be an alphavirus vector. Vectors based on alphaviruses, such as those made from Semliki Forest Virus (SFV) and Sindbis Virus (SIN), can also be used in the present disclosure. The use of alphaviruses is described in Lundstrom, K., Intervirology 43:247-257, 2000 and Perri et al., Journal of Virology 74:9802-9807, 2000, which are incorporated herein by reference in their entirety.

[0100] In some embodiments, the vector can be a poxvirus vector. Poxvirus vectors can introduce genes into the cytoplasm of cells. Avian poxvirus vectors can only provide short-term expression of genes or nucleic acids. Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors can be used with the compositions and methods of the present disclosure. Adenovirus vectors can provide shorter-term expression (e.g., less than about one month) than adeno-associated virus, and in some aspects can show much longer expression. The particular vector selected can depend on the target cell and the condition being treated.

[0101] Disclosed herein are compositions and methods for delivering nucleic acids encoding gene products of interest to target cells of a subject.In some instances, the gene products of interest are delivered to a subject after administration of a vector containing the gene products.In some instances, delivery of the gene products can be used to at least partially ameliorate or treat diseases or conditions disclosed herein.In some instances, the compositions can be used as gene therapy or are adapted for gene therapy or delivery of anti-VEGF in vivo.

[0102] In some instances, a vector, delivery vehicle, gene delivery vehicle, or gene transfer vehicle refers to any suitable macromolecular or molecular complex that contains a polynucleotide to be delivered to a target cell, tissue, or subject. In some cases, a target cell can be any cell to which a nucleic acid or gene is delivered.

[0103] In some embodiments, vectors, such as naked DNA or plasmids, can be delivered to cells, tissues, or subjects using micelles; microemulsions; liposomes; nanospheres; nanoparticles; nanocapsules; solid lipid nanoparticles; dendrimers; polyethyleneimine derivatives and single-walled carbon nanotubes; and other polymeric complexes that can mediate the delivery of polynucleotides to target cells. In some cases, vectors can be organic or inorganic molecules. In some cases, vectors are small molecules (i.e., <5kD) or polymers (i.e., >5kD).

[0104] In some embodiments, the vector comprises a recombinant viral vector (e.g., a rAAV vector) incorporating one or more nucleic acids. As described herein, the nucleic acid can comprise a polynucleotide. In some instances, the nucleic acid comprises DNA or RNA. In some cases, the nucleic acid comprises DNA or RNA for expression of a gene product or an aptamer. In some cases, the RNA molecule can comprise a transcript of a gene of interest, introns, non-translated regions, termination sequences, and other sequences. In other cases, the DNA molecule can comprise sequences such as hybrid promoter gene sequences, strong constitutive promoter sequences, gene of interest, non-translated regions, termination sequences, and other sequences. In some cases, any combination of DNA and RNA can be used.

[0105] In some embodiments, the present disclosure provides recombinant virus as a vector for mediating the expression of gene products or gene therapy for delivering gene products to target cells or subjects in vivo, for example, to the eye or the vitreous of the eye.Any suitable recombinant virus vector can be engineered to be optimized for use with the compositions and methods of the present disclosure.For example, recombinant virus vectors derived from adenovirus (Ad) or adeno-associated virus (AAV) can be used.

[0106] Both human and non-human viral vectors can be used, and recombinant viral vectors can be modified to be replication-deficient in humans or subjects. In some embodiments, the vector can be a replication-deficient adenovirus or rAAV that includes a polynucleotide having a promoter operably linked to a therapeutic transgene that encodes a gene product or therapeutic agent, such as an anti-VEGF agent.

[0107] In some embodiments, the vector can be a retroviral vector. The retroviral vector can include a Moloney murine leukemia virus and a virus based on HIV. In some embodiments, a viral vector based on HIV can be used, and the viral vector based on HIV includes at least two vectors, the gag and pol genes are derived from the HIV genome, and the env gene is derived from another virus. In some embodiments, a DNA viral vector can be used. Such vectors include pox vectors, such as orthopox or avipox vectors, herpes virus vectors, such as herpes simplex I virus (HSV) vectors [Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F. et al., in DNA Cloning: Mammalian Systems, edited by D. Glover (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci. USA: 90, 7603 (1993); Geller, AI et al., Proc Natl. Acad. Sci. USA: 87:1149 (1990)], adenovirus vectors [LeGal LaSalle et al., Science, 259:988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol. 69:2004 (1995)] and adeno-associated viral vectors [Kaplitt, MG et al., Nat. Genet. 8:148 (1994)], the entireties of which are incorporated herein by reference.

[0108] In some embodiments, the vector can be a lentiviral vector.The lentiviral vector for use in the present disclosure can be derived from human and non-human (including SIV) lentivirus.An example of a lentiviral vector can include a tissue-specific promoter operably linked to an anti-VEGF protein gene, together with the nucleic acid sequence required for vector propagation.The nucleic acid sequence can include a viral LTR, a primer binding site, a polypurine tract, an att site and an encapsidation site.

[0109] In some embodiments, adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors can be used with the compositions and methods of the present disclosure. The particular vector (e.g., lentivirus, adenovirus or AAV) used can depend on the target cell, the size of the therapeutic transgene or drug to be expressed from the vector, and / or the condition being treated.

[0110] In some embodiments, the vector is an adeno-associated virus (AAV) vector or is derived from AAV. AAV is a small, non-enveloped, single-stranded DNA virus. AAV is a non-pathogenic human parvovirus and can depend on helper viruses for replication, including adenovirus, herpes simplex virus, vaccinia virus and CMV. Exposure to wild-type AAV may not be associated with or known to cause any human pathology, and is common in the general population, usually occurring within the first decade of life in association with adenovirus infection.

[0111] In some instances, the rAAV can be a native or wild-type AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or DJ. In some instances, the rAAV can be a chimeric AAV comprising capsid proteins from at least two serotypes. In some embodiments, the rAAV virus or virion can comprise a variant AAV capsid protein. In some cases, the variant AAV capsid protein can comprise an amino acid modification selected from the group consisting of a substitution, an insertion, a deletion, and any combination thereof, compared to the corresponding parent AAV capsid protein.

[0112] In some embodiments, a rAAV virion can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acid deletions in a capsid protein compared to the corresponding parent AAV capsid protein. In some embodiments, a rAAV virion can comprise at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 amino acid deletions in a capsid protein. In some embodiments, a rAAV virion can comprise at most about 100 amino acids, at most about 200, at most about 300, or at most about 400 amino acid deletions in a capsid protein. In some embodiments, the rAAV virion can include a deletion of about 1 to about 100, about 1 to about 90, about 1 to about 80, about 1 to about 70, about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5 amino acids in the capsid protein. In some embodiments, the rAAV virion can comprise a deletion of about 5 to about 20 amino acids, about 5 to about 19 amino acids, about 5 to about 18 amino acids, about 5 to about 17 amino acids, about 5 to about 16 amino acids, about 5 to about 15 amino acids, about 5 to about 14 amino acids, about 5 to about 13 amino acids, about 5 to about 12 amino acids, about 5 to about 11 amino acids, about 5 to about 10 amino acids, about 5 to about 9 amino acids, about 5 to about 8 amino acids, about 5 to about 7 amino acids, or about 5 to about 6 amino acids in the capsid protein.

[0113] In some embodiments, a rAAV virion can comprise an insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids in a capsid protein compared to a corresponding parent AAV capsid protein. In some embodiments, a rAAV virion can comprise an insertion of at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 amino acids in a capsid protein. In some embodiments, a rAAV virion can comprise an insertion of at most about 100 amino acids, at most about 200, at most about 300, or at most about 400 amino acids in a capsid protein. In some embodiments, the rAAV virion can include an insertion of about 1 to about 100, about 1 to about 90, about 1 to about 80, about 1 to about 70, about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5 amino acids in the capsid protein. In some embodiments, the rAAV virion can comprise an insertion of about 5 to about 20 amino acids, about 5 to about 19 amino acids, about 5 to about 18 amino acids, about 5 to about 17 amino acids, about 5 to about 16 amino acids, about 5 to about 15 amino acids, about 5 to about 14 amino acids, about 5 to about 13 amino acids, about 5 to about 12 amino acids, about 5 to about 11 amino acids, about 5 to about 10 amino acids, about 5 to about 9 amino acids, about 5 to about 8 amino acids, about 5 to about 7 amino acids, or about 5 to about 6 amino acids in the capsid protein.

[0114] In some embodiments, the rAAV virion can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acid substitutions in the capsid protein compared to the corresponding parent AAV capsid protein. In some embodiments, the rAAV virion can include substitutions of about 1 to about 20 amino acids, about 1 to about 19 amino acids, about 1 to about 18 amino acids, about 1 to about 17 amino acids, about 1 to about 16 amino acids, about 1 to about 15 amino acids, about 1 to about 14 amino acids, about 1 to about 13 amino acids, about 1 to about 12 amino acids, about 1 to about 11 amino acids, about 1 to about 10 amino acids, about 1 to about 9 amino acids, about 1 to about 8 amino acids, about 1 to about 7 amino acids, about 1 to about 6 amino acids, about 1 to about 5 amino acids, about 1 to about 4 amino acids, about 1 to about 3 amino acids, or about 1 to about 2 amino acids in the capsid protein.

[0115] In some embodiments, the rAAV virion can comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 total amino acid insertions, deletions, or substitutions in the capsid protein compared to the corresponding parent, unmodified capsid protein. In some embodiments, the rAAV virions can comprise at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 total amino acid insertions, deletions, or substitutions in the capsid protein compared to the corresponding parent, unmodified capsid protein. In some embodiments, the rAAV virions can comprise at least about 100, at least about 200, at least about 300, or at least about 400 total amino acid insertions, deletions, or substitutions in the capsid protein compared to the corresponding parent, unmodified capsid protein.

[0116] In some embodiments, the rAAV virion comprises a variant capsid protein having an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homologous to the capsid protein of the parent, unmodified AAV capsid protein.

[0117] In some cases, the modification may be after amino acid 587 of AAV2 or the corresponding residue of the capsid subunit of another AAV serotype. Note that residue 587 is based on the AAV2 capsid protein. The modification may be incorporated into the corresponding site in an AAV serotype other than AAV2 (e.g., AAV8, AAV9, etc.). A person skilled in the art will know the location of the modification site corresponding to amino acid 587 of AAV2 in the capsid protein of any given AAV serotype based on a comparison of the amino acid sequences of the capsid proteins of various AAV serotypes. See, e.g., GenBank accession number NP_049542 for AAV1; GenBank accession number AAD13756 for AAV5; GenBank accession number AAB95459 for AAV6; GenBank accession number YP_077178 for AAV7; GenBank accession number YP_077180 for AAV8; GenBank accession number AAS99264 for AAV9 and GenBank accession number AAT46337 for AAV10.

[0118] In some embodiments, the amino acid modification is an insertion of about 5 amino acids to about 11 amino acids in the protein GH loop or loop IV. In some embodiments, the amino acid modification is an insertion that disrupts the solvent exposed region of the capsid protein to include one or more amino acids that include the GH loop. In some specific embodiments, the modification includes an insertion of the amino acid sequence LGETTRP between residues 587 and 588 in VP1 of AAV2. In some embodiments, other insertions or AAV2 variants can be used as vectors or gene therapy to deliver anti-VEGF agents, such as sFLT-1, ranibizumab, or bevacizumab, to a subject.

[0119] In some embodiments, the amino acid modifications of the capsid proteins described herein can confer increased infectivity of ocular cells compared to infectivity of retinal cells by AAV virions containing the corresponding parent or unmodified AAV capsid proteins. In some cases, the ocular cells can be photoreceptor cells (e.g., rods; cones). In some cases, the ocular cells can be retinal ganglion cells (RGCs). In some cases, the retinal cells can be retinal pigment epithelial (RPE) cells. In some cases, the ocular cells can be Müller cells. In some cases, the ocular cells can be astrocytes. In some cases, the retinal cells can include amacrine cells, bipolar cells, or horizontal cells.

[0120] In some embodiments, the increase in infectivity is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to AAV virions containing the corresponding parent AAV capsid protein. In some embodiments, the increase in infectivity is an increase of between about 5% and about 100%, between about 5% and about 95%, between about 5% and about 90%, between about 5% and about 85%, between about 5% and about 80%, between about 5% and about 75%, between about 5% and about 70%, between about 5% and about 65%, between about 5% and about 60%, between about 5% and about 55%, between about 5% and about 50%, between about 5% and about 45%, between about 5% and about 40%, between about 5% and about 35%, between about 5% and about 30%, between about 5% and about 25%, between about 5% and about 20%, between about 5% and about 15%, or between about 5% and about 10%, compared to AAV virions containing the corresponding parent AAV capsid protein.

[0121] In some embodiments, the increase in infectivity is at least about 1-fold, at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, or at least about 2-fold, as compared to AAV virions comprising the corresponding parent AAV capsid proteins. In some embodiments, the increase in infectivity is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold, as compared to AAV virions comprising the corresponding parent AAV capsid proteins. In some embodiments, the increase in infectivity is at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, or at least about 100-fold, compared to AAV virions containing the corresponding parent AAV capsid protein.

[0122] In some embodiments, the increase in infectivity is between about 10-fold and about 100-fold, between about 10-fold and about 95-fold, between about 10-fold and about 90-fold, between about 10-fold and about 85-fold, between about 10-fold and about 80-fold, between about 10-fold and about 75-fold, between about 10-fold and about 70-fold, between about 10-fold and about 65-fold, between about 10-fold and about 60-fold, between about 10-fold and about 55-fold, between about 10-fold and about 50-fold, between about 10-fold and about 45-fold, between about 10-fold and about 40-fold, between about 10-fold and about 35-fold, between about 10-fold and about 30-fold, between about 10-fold and about 25-fold, between about 10-fold and about 20-fold, or between about 10-fold and about 15-fold, compared to AAV virions containing the corresponding parent AAV capsid protein.

[0123] In some embodiments, the increase in infectivity is between about 2-fold and about 20-fold, between about 2-fold and about 19-fold, between about 2-fold and about 18-fold, between about 2-fold and about 17-fold, between about 2-fold and about 16-fold, between about 2-fold and about 15-fold, between about 2-fold and about 14-fold, between about 2-fold and about 13-fold, between about 2-fold and about 12-fold, between about 2-fold and about 11-fold, between about 2-fold and about 10-fold, between about 2-fold and about 9-fold, between about 2-fold and about 8-fold, between about 2-fold and about 7-fold, between about 2-fold and about 6-fold, between about 2-fold and about 5-fold, between about 2-fold and about 4-fold, or between about 2-fold and about 3-fold, compared to AAV virions comprising the corresponding parent AAV capsid protein.

[0124] In some embodiments, the amino acid modifications of the capsid proteins described herein can confer increased ability to cross the inner limiting membrane (ILM) in the eye of a subject compared to the ability of AAV virions containing the corresponding parent or unmodified AAV capsid proteins to cross the ILM in the eye of the subject.

[0125] In some embodiments, the increased ability to cross the ILM is an increase of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, compared to an AAV virion containing the corresponding parent AAV capsid protein. In some embodiments, the increased ability to cross the ILM is an increase of between about 5% and about 100%, between about 5% and about 95%, between about 5% and about 90%, between about 5% and about 85%, between about 5% and about 80%, between about 5% and about 75%, between about 5% and about 70%, between about 5% and about 65%, between about 5% and about 60%, between about 5% and about 55%, between about 5% and about 50%, between about 5% and about 45%, between about 5% and about 40%, between about 5% and about 35%, between about 5% and about 30%, between about 5% and about 25%, between about 5% and about 20%, between about 5% and about 15%, or between about 5% and about 10%, compared to an AAV virion comprising the corresponding parent AAV capsid protein.

[0126] In some embodiments, the increase in ability to cross the ILM is at least about 1-fold, at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, or at least about 2-fold, as compared to an AAV virion comprising the corresponding parental AAV capsid protein. In some embodiments, the increase in ability to cross the ILM is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold, as compared to an AAV virion comprising the corresponding parental AAV capsid protein. In some embodiments, the increase in ability to cross the ILM is at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, or at least about 100-fold, compared to an AAV virion comprising the corresponding parent AAV capsid protein.

[0127] In some embodiments, the increase in ability to cross the ILM is between about 10-fold and about 100-fold, between about 10-fold and about 95-fold, between about 10-fold and about 90-fold, between about 10-fold and about 85-fold, between about 10-fold and about 80-fold, between about 10-fold and about 75-fold, between about 10-fold and about 70-fold, between about 10-fold and about 65-fold, between about 10-fold and about 60-fold, between about 10-fold and about 55-fold, between about 10-fold and about 50-fold, between about 10-fold and about 45-fold, between about 10-fold and about 40-fold, between about 10-fold and about 35-fold, between about 10-fold and about 30-fold, between about 10-fold and about 25-fold, between about 10-fold and about 20-fold, or between about 10-fold and about 15-fold, as compared to an AAV virion comprising the corresponding parent AAV capsid protein.

[0128] In some embodiments, the increase in ability to cross the ILM is between about 2-fold and about 20-fold, between about 2-fold and about 19-fold, between about 2-fold and about 18-fold, between about 2-fold and about 17-fold, between about 2-fold and about 16-fold, between about 2-fold and about 15-fold, between about 2-fold and about 14-fold, between about 2-fold and about 13-fold, between about 2-fold and about 12-fold, between about 2-fold and about 11-fold, between about 2-fold and about 10-fold, between about 2-fold and about 9-fold, between about 2-fold and about 8-fold, between about 2-fold and about 7-fold, between about 2-fold and about 6-fold, between about 2-fold and about 5-fold, between about 2-fold and about 4-fold, or between about 2-fold and about 3-fold, as compared to an AAV virion comprising the corresponding parent AAV capsid protein.

[0129] One advantage of gene therapy is that it requires less frequent administration of therapeutic agents, such as anti-VEGF agents disclosed herein, and also provides extended or sustained release of therapeutic agents, compared to conventional methods of administering proteins.Gene therapy utilizing vectors, such as AAV2.7m8, that target specific tissues or cell types of interest can also minimize off-target effects or provide more targeted delivery of therapeutic agents, such as anti-VEGF agents.Extended or sustained delivery of anti-VEGF agents in vivo by gene therapy may allow the pharmaceutical composition to be administered no more than once every 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer.

[0130] Therapeutic Agents In some embodiments, gene therapy is used to deliver a therapeutic transgene having anti-VEGF activity suitable or adapted for administration to the eye or vitreous of a non-human primate or human subject. In some embodiments, rAAV, including capsid variants described herein (e.g., AAV2.7m8), includes a heterologous nucleic acid sequence encoding an anti-VEGF agent and is used to deliver the sequence of the anti-VEGF gene to retinal cells upon intravitreal or subretinal injection into a subject. In some embodiments, the rAAV containing the anti-VEGF gene is formulated for gene therapy and intravitreal injection. In some embodiments, the anti-VEGF gene refers to a functional fragment or variant thereof. In some embodiments, the nucleic acid sequence of the anti-VEGF agent, such as sFLT-1, ranibizumab, or bevacizumab, is derived from its readily available amino acid sequence. In some embodiments, the nucleic acid sequence of the anti-VEGF agent, such as sFLT-1, ranibizumab, or bevacizumab, is further codon-optimized to improve its expression in a subject. In some embodiments, the nucleic acid and / or amino acid sequence of the anti-VEGF agent is modified to enhance its activity, expression, stability and / or solubility in vivo.

[0131] Codon optimization can be achieved by any method known in the art. Codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression of a gene in a target or host cell of interest, for example, a human retinal cell, by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100 or more codons) of the native sequence with a codon that is more frequently or most frequently used in the host cell, while maintaining the native amino acid sequence. Codon usage tables are readily available, including, for example, GenScript Codon Usage Frequency Table Tool, http: / / www.genscript.com / tools / codon-frequency-table; Codon Usage Database, http: / / www.kazusa.or.jp / codon / ; and Nakamura, Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000).

[0132] In some embodiments, the amino acid sequence of the anti-VEGF agent encoded in the gene therapy is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% homologous to the amino acid sequence of any one of the following anti-VEGF agents: sFLT-1, ranibizumab, or bevacizumab. In some embodiments, the nucleic acid sequences used in the gene therapy or rAAV disclosed herein are compared to the corresponding cDNA sequence of the amino acid sequence of any one of sFLT-1, ranibizumab, or bevacizumab and exhibit at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% sequence homology between the nucleic acid sequence of any one of sFLT-1, ranibizumab, or bevacizumab. In some cases, the anti-VEGF expressed from the gene therapy is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% spatially homologous (e.g., in terms of its secondary, tertiary and quaternary structure or conformation) to any one of sFLT-1, ranibizumab or bevacizumab. In some cases, the anti-VEGF agents of the pharmaceutical compositions and methods disclosed herein are at most 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% spatially homologous (e.g., secondary, tertiary and quaternary structure or conformation) to any one of sFLT-1, ranibizumab or bevacizumab used in standard therapy.

[0133] In some instances, the anti-VEGF agent included in the rAAV-based gene therapy comprising a capsid variant (e.g., 7m8 variant) disclosed herein encodes a protein, fusion protein, or polypeptide having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homology to the corresponding cDNA sequence of the anti-VEGF agent (e.g., sFLT-1, ranibizumab, or bevacizumab). In some embodiments, the methods and pharmaceutical compositions disclosed herein comprise sFLT-1, ranibizumab, or bevacizumab, or a functional fragment or variant or mutant thereof. In some embodiments, the nucleic acid sequence of any of sFLT-1, ranibizumab, or bevacizumab is modified or codon optimized to enhance its activity, expression, and / or solubility in vivo.

[0134] In some embodiments, AAV2.7m8 is used as a gene therapy or delivery system for any one of sFLT-1, ranibizumab or bevacizumab. AAV2.7m8-sVEGFR-1 refers to rAAV2 that contains a 7m8 insertion between positions 587 and 588 of the capsid protein VP1 of AAV2, and the nucleic acid sequence that codes for sVEGFR-1. AAV2.7m8-ranibizumab refers to rAAV2 that contains a 7m8 insertion between positions 587 and 588 of the capsid protein VP1 of AAV2, and the nucleic acid sequence that codes for ranibizumab.

[0135] The present disclosure contemplates methods and pharmaceutical compositions disclosed herein that include one or more therapeutic agents. In some embodiments, the therapeutic agent is an anti-VEGF agent. In some embodiments, the anti-VEGF agent is expressed from an rAAV vector or gene therapy, or delivered to a target cell, tissue, or subject in vivo. Gene therapy has the advantage of providing a therapeutic agent, e.g., an anti-VEGF agent, in vivo for an extended period of time, which reduces the need for repeated injections compared to administration of protein-based therapy. Such advantages of gene therapy can result in more sustained delivery of the therapeutic agent in vivo, which provides an improvement over current standard treatments. Moreover, gene therapy can also result in more targeted delivery of the therapeutic agent in vivo, e.g., to target cells, minimizing off-target effects.

[0136] In some embodiments, the gene product disclosed herein can be a polypeptide that, when expressed, can result in reduced neovascularization in the eye of a subject. In some cases, the expressed polypeptide can be an anti-vascular endothelial growth factor (VEGF) protein or peptide or an angiogenesis inhibitor.

[0137] In some embodiments, the gene product disclosed herein can be an anti-VEGF antibody or fragment thereof that can target or at least partially inhibit VEGF. In some instances, the antibody can be a full-length antibody that includes both variable and Fc regions. In some instances, the antibody can be a single-chain fv fragment. In some instances, the antibody can have a defined binding affinity to a VEGF epitope. In some instances, the antibody can have a Kd of at least about 1 mM, at least about 100 μM, at least about 10 μM, at least about 1 μM, at least about 100 nM, at least about 10 nM, at least about 1 nM, at least about 100 pM, at least about 10 pM, or at least about 1 pM. In some instances, the anti-VEGF agent binds to endogenous VEGF or VEGFR more strongly than the corresponding endogenous VEGFR or VEGF. The stronger binding of the anti-VEGF agent allows the anti-VEGF agent to sequester endogenous VEGF or block endogenous proteins from interacting with endogenous VEGFR.

[0138] In some embodiments, the anti-VEGF antibody may be a humanized monoclonal antibody. In some instances, the humanized monoclonal antibody may be rhuMab. In some embodiments, the anti-VEGF antibody may be ranibizumab, a monoclonal antibody fragment, or a variant or fragment thereof. In some embodiments, the anti-VEGF antibody may be bevacizumab, a recombinant humanized monoclonal antibody, or a variant or fragment thereof. In some embodiments, the anti-VEGF agent is PAN-90806 or a variant or fragment thereof. In some embodiments, the therapeutic agent is a nucleic acid sequence encoding one or more polypeptides including the anti-VEGF agent disclosed herein, such as an antibody, an antibody fragment, a monoclonal antibody, a humanized monoclonal antibody, a fusion protein, an aptamer, etc. In some embodiments, the anti-VEGF agent is a soluble receptor decoy that binds to VEGF or is a soluble form of one or more VEGF receptors that can sequester VEGF in vivo.

[0139] In some embodiments, the therapeutic agent disclosed herein may be a nucleic acid, such as an aptamer, an interfering RNA, an mRNA, or the like. In some cases, the aptamer may be pegaptanib. In some embodiments, the therapeutic agent may be a steroid or a small molecule. In some instances, the steroid may be a corticosteroid. Examples of corticosteroids may include triamcinolone, dexamethasone, fluocinolone acetonide, cortisone, prednisolone, flumetholone, and derivatives thereof. In some instances, the steroid may be an anti-inflammatory steroid.

[0140] The recombinant viruses, gene therapy, pharmaceutical compositions and methods of the present disclosure can include sequences encoding anti-VEGF proteins, including but not limited to VEGF binding proteins or functional fragments thereof, as disclosed in U.S. Patent Nos. 5,712,380, 5,861,484 and 7,071,159, and also described in U.S. Patent Application Publication No. 2014 / 0371438. In some embodiments, the anti-VEGF protein comprises the sFLT-1 protein, ranibizumab or bevacizumab, as described herein.

[0141] In some embodiments, the gene therapy, pharmaceutical compositions and methods of the disclosure can include sequences encoding anti-VEGF proteins, such as sFlt-1. In some embodiments, anti-VEGF agents include, but are not limited to, functional fragments of sFlt-1 including the sequence of sFlt-1 domain 2 or sequence. Anti-VEGF agents can include sequences or polypeptides expressed from DNA encoding such sequences, using the genetic code, standard techniques as understood by those skilled in the art.

[0142] As used herein, "sFlt-1 protein" or "sFlt" or "sVEGFR-1" are used interchangeably to refer to a polypeptide sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% homology to the human sFLT-1 sequence, such that the sFlt-1 protein or polypeptide binds to VEGF and / or a VEGF receptor in vivo, or a functional fragment or variant thereof. Figure 2 illustrates the nucleic acid sequence of sFlt-1. Homology refers to the percent conservation of residues in alignment between two sequences (e.g., a naturally occurring human sFLT-1 protein can include any suitable variant of sFLT-1, including but not limited to functional fragments, sequences containing insertions, deletions, substitutions, pseudofragments, pseudogenes, splice variants, or artificially optimized sequences). In some cases, the "sFLT-1 protein" is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to a naturally occurring human sFLT-1 protein sequence. In some embodiments, the "sFLT-1 protein" is at most about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to a naturally occurring human sFLT-1 protein sequence. In some cases, an "sFLT-1 protein" is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% spatially homologous to a naturally occurring human sFLT-1 protein conformation.In some cases, an "sFLT-1 protein" is at most about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% spatially homologous to a naturally occurring human sFLT-1 protein conformation.

[0143] In some cases, sFLT-1, a soluble truncated form of the VEGF receptor FLT-1, is the only known endogenous inhibitor of VEGF. sFLT-1 can be generated by alternative RNA splicing and lacks the membrane-proximal immunoglobulin-like domain, the transmembrane region, and the intracellular tyrosine kinase domain.

[0144] In some cases, administration of gene therapy or pharmaceutical compositions disclosed herein comprising sFLT-1 can inhibit or reduce VEGF or its activity in vivo by binding or sequestering endogenous VEGF or by forming inactive heterodimers with transmembrane isoforms of VEGF receptors FLTt-1 and FLK-1 / KDR. These properties of sFLT-1 are described in Kendall and Thomas, 1993; Proc Natl Acad. Sci. 90:10705-10709, which are incorporated herein by reference in their entirety. In some embodiments, functional fragments of sFLT-1 can be used instead of the full-length protein. In some embodiments, the VEGF binding domain (domain 2), or KDR or another family member can be used to bind and inactivate VEGF.

[0145] In some embodiments, the methods and pharmaceutical compositions of the disclosure include an anti-VEGF agent that is bevacizumab or ranibizumab, a functional fragment or variant thereof. Catt Research, Group; Martin, DF; Maguire, MG; Ying, GS; Grunwald, JE; Fine, SL; Jaffe, GJ (2011) "Ranibizumab and Bevacizumab for Neovascular Age-Related Macular Degeneration." New England Journal of Medicine. 364(20):1897-1908.

[0146] The ranibizumab light and heavy chain amino acid sequences are published in the DrugBank database, accession number DB01270: [ka]

[0147] The bevacizumab light and heavy chain amino acid sequences are published in the DrugBank database, accession number DB00112 (BTD00087, BIOD00087): [ka]

[0148] Bevacizumab is a recombinant humanized monoclonal IgG1 antibody that binds to all VEGF-A isoforms and blocks angiogenesis by inhibiting VEGF-A. Los, M.; Roodhart, JML; Voest, EE (2007) "Target Practice: Lessons from Phase III Trials with Bevacizumab and Vatalanib in the Treatment of Advanced Colorectal Cancer" The Oncologist. 12(4): 443-50; Shih, T; Lindley, C (November 2006) "Bevacizumab: an angiogenesis inhibitor for the treatment of solid malignancies." Clinical Therapeutics. 28(11): 1779-802.

[0149] Ranibizumab is a recombinant humanized IgG1 kappa isotype monoclonal antibody fragment, which binds to all VEGF-A isoforms with higher affinity than bevacizumab. Ranibizumab lacks the Fc region. In some embodiments, the anti-VEGF agent is bevacizumab or ranibizumab or a functional fragment or variant thereof. In some embodiments, the anti-VEGF agent is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% homologous to bevacizumab or ranibizumab in amino acid and / or nucleic acid (e.g., cDNA) sequence. In some embodiments, the anti-VEGF agent is at most about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% homologous to bevacizumab or ranibizumab in amino acid and / or nucleic acid (e.g., cDNA) sequences known in the art. In some cases, the anti-VEGF agent is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% spatially homologous to bevacizumab or ranibizumab protein conformations, including secondary, tertiary or quaternary structures.

[0150] Given the amino acid sequence, the corresponding cDNA or nucleic acid sequence can be readily generated and used in the gene therapy methods disclosed herein. Methods for back-translating amino acid sequences include the EMBOSS Protein Sequence Back-translation tool available at http: / / www.ebi.ac.uk / Tools / st / . In some embodiments, the nucleic acid sequence of the anti-VEGF agent is codon-optimized using any of the techniques known in the art, such as the GenScript Codon Usage Frequency Table Tool, http: / / www.genscript.com / tools / codon-frequency-table; Codon Usage Database, http: / / www.kazusa.or.jp / codon / ; and Nakamura, Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000).

[0151] Pharmaceutical Compositions In some embodiments, the pharmaceutical composition is a formulation containing one or more active ingredients, e.g., AAV2.7m8 comprising a nucleic acid sequence encoding an anti-VEGF agent or a fragment or variant thereof, together with one or more excipients, carriers, stabilizers or bulking agents suitable for administration to a human patient by intravitreal or subretinal injection to achieve a desired therapeutic or prophylactic effect.

[0152] In some embodiments, the pharmaceutical composition comprising rAAV or AAV2.7m8 and a nucleic acid sequence encoding an anti-VEGF agent is provided as a reconstituted solution or suspension. In other embodiments, the pharmaceutical composition comprising rAAV or AAV2.7m8 and a nucleic acid sequence encoding an anti-VEGF agent is provided in lyophilized form and is reconstituted prior to administration to a patient. In some embodiments, the method of treating or preventing an ocular disease or condition disclosed herein comprises first reconstituting, dissolving or solubilizing a lyophilized pharmaceutical composition comprising rAAV (e.g., AAV2.7m8) and a nucleic acid sequence encoding an anti-VEGF agent in a buffer. In some embodiments, such a lyophilized pharmaceutical composition comprising rAAV (e.g., AAV2.7m8) and a nucleic acid sequence encoding an anti-VEGF agent disclosed herein may further comprise a cryoprotectant, a surfactant, a salt, a stabilizer or any combination thereof.

[0153] In some embodiments, the pharmaceutical composition comprising rAAV or AAV2.7m8 and a nucleic acid sequence encoding an anti-VEGF agent is provided as a suspension. In some embodiments, the suspension is refrigerated. In some embodiments, the suspension is a solution. In some embodiments, a homogenous solution containing the pharmaceutical composition is provided as a pre-filled syringe. In some embodiments, the pharmaceutical composition disclosed herein is provided as a suspension. In some embodiments, the suspension is refrigerated. In some embodiments, the method of treating or preventing an ocular disease or condition disclosed herein comprises warming the refrigerated suspension to room temperature and / or stirring the suspension to ensure uniform distribution prior to administration to a patient or intravitreal injection. In some embodiments, the suspension is diluted prior to administration to a patient. In some embodiments, the pharmaceutical composition comprises a surfactant, a salt, a stabilizer, or any combination thereof. In some embodiments, the suspension containing the pharmaceutical composition is provided as a pre-filled syringe.

[0154] In some embodiments, the gene therapy or pharmaceutical compositions described herein are provided as a suspension or refrigerated suspension. In some embodiments, the suspension comprises a pharma- ceutically acceptable excipient, such as a surfactant, glycerol, a non-ionic surfactant, a buffer, a glycol, a salt, and any combination thereof. In some embodiments, hydrochloric acid and sodium hydroxide are used to adjust the pH of the solution. In some embodiments, the refrigerated suspension is at a neutral pH, or a pH between 6.5 and 7.5. In some embodiments, the pH of the refrigerated suspension is slightly basic (e.g., about pH 7.5, 8, 8.2, 8.4, 8.5, or 9). In some embodiments, the pH of the refrigerated suspension or solution is slightly acidic (e.g., about pH 6.5, 6.3, 6.1, 6, 5.5, or 5). In some embodiments, the refrigerated suspension is a solution. In some embodiments, the refrigerated suspension comprises micelles. In some embodiments, the refrigerated suspension is stirred prior to administration.

[0155] In some embodiments, the gene therapy comprising an rAAV (e.g., AAV2.7m8) and an anti-VEGF agent disclosed herein is supplied as a kit comprising a lyophilized or freeze-dried pharmaceutical composition disclosed herein and a buffer solution for dissolving, diluting or reconstituting the lyophilized pharmaceutical composition. In some embodiments, the kit comprises a freeze-dried or lyophilized pharmaceutical composition comprising an rAAV (e.g., AAV2.7m8) and a solution for reconstituting the pharmaceutical composition to a desired concentration or volume. In some embodiments, the kit comprises a buffer that helps prevent aggregation upon reconstitution of the pharmaceutical composition disclosed herein. In some embodiments, the gene therapy comprising an anti-VEGF agent is provided as a suspension. In some embodiments, the pharmaceutical composition is provided in a pre-filled syringe. In some embodiments, the kit comprises a dual-chamber syringe, one of the chambers containing a buffer for dissolving or diluting the pharmaceutical composition.

[0156] In some embodiments, the kit includes a syringe for injection. In some embodiments, the reconstituted solution is filtered before administration. In some embodiments, the kit includes a filter or filter syringe for filtering the pharmaceutical composition in the kit before administration to a patient.

[0157] In some embodiments, for storage stability and convenience of handling, pharmaceutical compositions comprising rAAV (e.g., AAV2.7m8) and a nucleic acid sequence encoding an anti-VEGF agent disclosed herein can be formulated as a lyophilized (i.e., freeze-dried) or vacuum-dried powder that can be reconstituted with saline, buffer, or water prior to administration to a subject. Alternatively, the pharmaceutical composition can be formulated as an aqueous solution or suspension. The pharmaceutical composition can contain rAAV virions or particles comprising a nucleic acid sequence encoding an anti-VEGF agent disclosed herein. In some embodiments, different viruses or delivery systems, such as nanoparticles or lipid-based complexes, can be used to deliver the nucleic acid sequence encoding an anti-VEGF agent disclosed herein. Various excipients can be used to stabilize the pharmaceutical composition, such as phosphate, PBS or Tris buffer, glycol, glycerol, saline, surfactants (e.g., pluronic or polysorbate), or any combination thereof. Additionally, cryoprotectants such as alcohol, DMSO, glycerol and PEG can be used as stabilizers under the freezing or drying conditions of lyophilization or to make refrigerated suspensions.

[0158] In some embodiments, a lyophilized pharmaceutical composition or suspension of a pharmaceutical composition comprising an anti-VEGF gene therapy disclosed herein has a volume (or reconstituted volume) of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μL. In some embodiments, a pharmaceutical composition in lyophilized or suspension form comprising an anti-VEGF gene therapy disclosed herein has a volume of between 0.1-0.5 mL, between 0.1-0.2 mL, between 0.3-0.5 mL, between 0.5-1.0 mL, between 0.5-0.7 mL, between 0.6-0.8 mL, between 0.8-1 mL, between 0.9-1.1 mL, between 1.0-1.2, or between 1.0-1.5 mL. In other embodiments, the reconstituted volume is less than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 mL.

[0159] In some embodiments, the pharmaceutical compositions disclosed herein are designed, engineered or adapted for administration to primates (e.g., non-human primates and human subjects) by intravitreal or subretinal injection. In some embodiments, the pharmaceutical composition comprising the rAAV virion comprising the nucleic acid sequence encoding the anti-VEGF agent is formulated for intravitreal injection into the eye of a subject. In some embodiments, the pharmaceutical composition is formulated to a concentration that allows intravitreal injection in a volume of about 2, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 μL or less. In some embodiments, the methods of treatment disclosed herein comprise intravitreal injection of a volume of about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150 μL of a solution or suspension comprising an rAAV (e.g., AAV2.7m8) comprising a nucleic acid sequence encoding an anti-VEGF agent disclosed herein.

[0160] In some instances, the AAV2.7m8 virion comprising the nucleic acid sequence of the anti-VEGF transgene described herein can be a component of a gene therapy pharmaceutical composition. In some embodiments, any serotype of rAAV virion comprising the 7m8 variant capsid protein described herein can be used to make a lyophilized pharmaceutical composition or a suspension of pharmaceutical composition. In some embodiments, the rAAV virion is rAAV2. In some embodiments, the pharmaceutical composition in lyophilized or suspension form comprises rAAV2 having a DNA sequence encoding the 7m8 variant capsid protein and the anti-VEGF agent disclosed herein.

[0161] In some embodiments, the pharmaceutical compositions disclosed herein are adapted for gene therapy or intravitreal delivery of anti-VEGF agents as therapeutic agents in human patients or non-human primates. In some embodiments, the unit dose of the pharmaceutical composition is 1×10 10 ~1×10 13 In some embodiments, the unit dose comprises between about 2.1 x 10 11 , about 2.1×10 12 or about 2.1 x 10 13 In some embodiments, the unit dose of the pharmaceutical composition of the present disclosure comprises 1×10 10 ~3×10 12 In some cases, the unit dose of the pharmaceutical composition of the present disclosure is 1×10 9 ~3×10 13 In some cases, the unit dose of the pharmaceutical composition of the present disclosure is 1×10 10 ~1×10 11 In some cases, the unit dose of the pharmaceutical composition of the present disclosure is 1×10 8 ~3×10 14 In some cases, the unit dose of the pharmaceutical composition of the present disclosure is at least 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×105 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 or 1×10 18 In some cases, the unit dose of the pharmaceutical composition of the present disclosure is 1×10 10 ~5×10 13 In some cases, the unit dose of the pharmaceutical composition of the present disclosure is at most about 1 x 10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1 × 10 18 This is the vector genome.

[0162] In some cases, the unit dose of the pharmaceutical composition of the present disclosure can be measured as pfu (plaque forming units). In some cases, the pfu of the unit dose of the pharmaceutical composition of the present disclosure is about 1×10 8 ~Approx. 1×10 12 In some cases, the pfu of a unit dose of the pharmaceutical composition of the present disclosure is at least about 1 x 10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×109 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 or 1×10 12 In some cases, the pfu of a unit dose of the pharmaceutical composition of the present disclosure is at most about 1 x 10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×1011 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 or 1×10 12 It may be pfu.

[0163] In some cases, the viral vectors of the present disclosure can be measured as vector genomes (vg). In some cases, the unit dose of the pharmaceutical composition of the present disclosure is about 1×10 10 ~1×10 13 In some cases, the unit dose of the pharmaceutical composition of the present disclosure may be 1×10 9 ~1×10 14 In some cases, the unit dose of the pharmaceutical composition of the present disclosure may be 1×10 10 ~1×10 11 In some cases, the unit dose of the pharmaceutical composition of the present disclosure may be 1×10 8 ~1×10 15 In some cases, the unit dose of the pharmaceutical composition of the present disclosure may be at least 1 x 10 vector genome. 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1 × 10 18 In some cases, the unit dose of the pharmaceutical composition of the present disclosure is 1×10 8 ~1×10 15In some cases, the unit dose of the pharmaceutical composition of the present disclosure is at most about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1 × 10 18 In some embodiments, the unit dose is 10 10 ~10 11 Between 10 11 ~10 12 Between 10 10 ~10 12 Between 10 12 ~10 13 Between 10 11 ~10 13 Between 10 12 ~10 13 Between 10 12 ~10 14 Between 10 11 ~10 14 Between 10 11 ~10 15 Between 10 12 ~10 15 Between 10 13 ~10 14 Between 10 14 ~10 15 Between 10 15 ~10 16 Between 10 16 ~10 17 Between 10 17 ~10 18 Between 10 18 ~10 19 Between or 10 19 ~10 20 It is a vector genome between.

[0164] In some embodiments, the unit dose of the pharmaceutical composition of the present disclosure is about 1×10 10 ~2×10 10 Between 2×10 10 ~3×10 10 Between 3×10 10 ~4×10 10 Between 4×10 10 ~5×10 10 Between 5×10 10 ~6×10 10 Between 6×10 10 ~7×10 10 Between 7×10 10 ~8×10 10 Between 8×10 10 ~9×10 10 Between 9×10 10 ~10×10 10 Between 1×10 11 ~2×10 11 Between 2×10 11 ~3×10 11 Between 2×10 11 ~2.5×10 11 Between, 2.5×10 11 ~3×10 11 Between 3×10 11 ~4×10 11 Between 4×10 11 ~5×10 11 Between 5×10 11 ~6×10 11 Between 6×10 11 ~7×10 11 Between 7×10 11 ~8×10 11 Between 8×10 11 ~9×10 11 Between 9×10 11 ~10×10 11 Between 1×10 12 ~2×10 12 Between 2×10 12 ~3×10 12 Between, 2.5×10 12 ~3×10 12 Between 3×10 12 ~4×10 12 Between 4×10 12 ~5×1012 Between 5×10 12 ~6×10 12 Between 6×10 12 ~7×10 12 Between 7×10 12 ~8×10 12 Between 8×10 12 ~9×10 12 Between 9×10 12 ~10×10 12 Between 1×10 13 ~2×10 13 Between 2×10 13 ~3×10 13 Between 3×10 13 ~4×10 13 Between 4×10 13 ~5×10 13 Between 5×10 13 ~6×10 13 Between 6×10 13 ~7×10 13 Between 7×10 13 ~8×10 13 Between 8×10 13 ~9×10 13 Between or 9×10 13 ~10×10 13 It is a vector genome between.

[0165] In some embodiments, the unit dose of the rAAV of the present disclosure is 2×10 11 ~8×10 11 Between or 2×10 12 ~8×10 12 In some embodiments, the unit dose of the rAAV of the present disclosure is between 10 10 ~10 13 Between 10 10 ~10 11 Between 10 11 ~10 12 Between 10 12 ~10 13 Between or 10 13 ~10 14 It is a vector genome between.

[0166] In some embodiments, the unit dose of the rAAV of the present disclosure is 1×10 10~2×10 10 Between 2×10 10 ~4×10 10 Between 3×10 10 ~5×10 10 Between 4×10 10 ~6×10 10 Between 5×10 10 ~7×10 10 Between 6×10 10 ~8×10 10 Between 7×10 10 ~9×10 10 Between 8×10 10 ~10 11 Between 1×10 11 ~2×10 11 Between 2×10 11 ~4×10 11 Between 3×10 11 ~5×10 11 Between 4×10 11 ~6×10 11 Between 5×10 11 ~7×10 11 Between 6×10 11 ~8×10 11 Between 7×10 11 ~9×10 11 Between 8×10 11 ~10×10 11 Between 1×10 12 ~3×10 12 Between 2×10 12 ~4×10 12 Between 3×10 12 ~5×10 12 Between 4×10 12 ~6×10 12 Between 5×10 12 ~7×10 12 Between 6×10 12 ~8×10 12 Between 7×10 12 ~9×10 12 Between 8×10 12 ~10×10 12 Between 1×10 13 ~5×10 13 Between 5×10 13 ~10×10 13 Between 10 12 ~5×1012 Between 5×10 12 ~1×10 13 Between 7×10 12 ~1×10 13 Between 8×10 12 ~2×10 13 Between 9×10 12 ~2×10 13 Between 9×10 12 ~2×10 13 Between 9×10 12 ~4×10 13 Between 1×10 13 ~3×10 13 Between 1×10 13 ~2×10 13 Between 2×10 13 ~3×10 13 Between 3×10 13 ~4×10 13 Between 4×10 13 ~5×10 13 Between 5×10 13 ~6×10 13 Between 6×10 13 ~7×10 13 Between 7×10 13 ~8×10 13 Between 8×10 13 ~9×10 13 Between or 8×10 13 ~1×10 14 It is a vector genome between.

[0167] In some embodiments, a lower amount or range of vector genome is selected for unit dose to avoid aggregation. In some embodiments, a higher amount or range of vector genome is selected for unit dose so that a smaller volume can be used for injection. Injecting a smaller volume (e.g., less than 50, 40, 30, 20, 10 or 5 μL) can help reduce the change in intraocular pressure and other adverse effects associated with intravitreal injection. In some embodiments, a higher concentration of rAAV also helps ensure efficient delivery of therapeutic transgenes to target cells.

[0168] In some embodiments, the unit dose comprises between 2E12 and 6E12 vector genomes, in some embodiments, the unit dose comprises about 1E12, 1.5E12, 2E12, 2.5E12, 3E12, 3.5E12, 4E12, 4.5E12, 5E12, 5.5E12, 6E12, 6.5E12, 7E12, 7.5E12, 8E12, 8.5E12, 9E12, or 9.5E12 vector genomes. In some embodiments, the unit dose is between 1E12 and 1.5E12, between 1.5E12 and 2E12, between 2E12 and 2.5E12, between 2.5E12 and 3.0E12, between 3.0E12 and 3.5E12, between 3.5E12 and 4.0E12, between 4.0E12 and 4.5E12, between 4.5E12 and 5.0E12, between 5.0E12 and 5.5 ... Containing vector genomes between .5E12 and 6.0E12, between 6.0E12 and 6.5E12, between 6.5E12 and 7.0E12, between 7.0E12 and 7.5E12, between 7.5E12 and 8.0E12, between 8.0E12 and 8.5E12, between 8.5E12 and 9.0E12, between 9.0E12 and 9.5E12 or between 9.5E12 and 10E12. In some embodiments, the unit dose comprises at least 1E12, 1.5E12, 2E12, 2.5E12, 3E12, 3.5E12, 4E12, 4.5E12, 5E12, 5.5E12, 6E12, 6.5E12, 7E12, 7.5E12, 8E12, 8.5E12, 9E12, or 9.5E12 vector genomes. In some embodiments, the unit dose comprises no more than 1E12, 1.5E12, 2E12, 2.5E12, 3E12, 3.5E12, 4E12, 4.5E12, 5E12, 5.5E12, 6E12, 6.5E12, 7E12, 7.5E12, 8E12, 8.5E12, 9E12, or 9.5E12 vector genomes.

[0169] In some cases, the unit dose of the pharmaceutical composition of the present disclosure can be measured using the multiplicity of infection (MOI). In some cases, the MOI can refer to the ratio or multiple of vector or viral genome to cells to which the nucleic acid can be delivered. In some cases, the MOI is 1×10 6 In some cases, the MOI can be about 1×10 5 ~Approx. 1×107 In some cases, the MOI can be between 1×10 4 ~1×10 8 In some cases, the recombinant virus of the disclosure may be at least about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1 × 10 18 In some cases, the recombinant virus of the present disclosure can be at an MOI of about 1 x 10 8 ~Approx. 1×10 15 In some cases, the recombinant virus of the present disclosure may be at an MOI of at most about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1 × 10 18 In some embodiments, the MOI is 1×10 10 ~2×10 10 Between 2×10 10 ~4×10 10 Between 3×10 10 ~5×10 10 Between 4×1010 ~6×10 10 Between 5×10 10 ~7×10 10 Between 6×10 10 ~8×10 10 Between 7×10 10 ~9×10 10 Between 8×10 10 ~10 11 Between 1×10 11 ~2×10 11 Between 2×10 11 ~4×10 11 Between 3×10 11 ~5×10 11 Between 4×10 11 ~6×10 11 Between 5×10 11 ~7×10 11 Between 6×10 11 ~8×10 11 Between 7×10 11 ~9×10 11 Between 8×10 11 ~10×10 11 Between 1×10 12 ~3×10 12 Between 2×10 12 ~4×10 12 Between 3×10 12 ~5×10 12 Between 4×10 12 ~6×10 12 Between 5×10 12 ~7×10 12 Between 6×10 12 ~8×10 12 Between 7×10 12 ~9×10 12 Between 8×10 12 ~10×10 12 Between 1×10 13 ~5×10 13 Between 5×10 13 ~10×10 13 Between 10 12 ~5×10 12 Between 5×10 12 ~1×10 13 Between 7×10 12 ~1×10 13 Between 8×10 12~2×10 13 Between 9×10 12 ~2×10 13 Between 9×10 12 ~2×10 13 Between 9×10 12 ~4×10 13 Between 1×10 13 ~3×10 13 Between 1×10 13 ~2×10 13 Between 2×10 13 ~3×10 13 Between 3×10 13 ~4×10 13 Between 4×10 13 ~5×10 13 Between 5×10 13 ~6×10 13 Between 6×10 13 ~7×10 13 Between 7×10 13 ~8×10 13 Between 8×10 13 ~9×10 13 Between or 8×10 13 ~1×10 14 It is between.

[0170] Pharmaceutical compositions suitable for ophthalmic use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions, suspensions or dispersions. For intravitreal administration, suitable carriers include physiological saline, bacteriostatic water, phosphate buffered saline (PBS) and / or an isotonic agent, such as glycerol. In all cases, the pharmaceutical composition must be sterile. It should be fluid to the extent that easy syringability or injectability exists. The pharmaceutical composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. In some embodiments, the pharmaceutical composition can include an isotonic agent, such as salts or glycerol. In some embodiments, surfactants or stabilizers are added to the pharmaceutical composition to prevent aggregation.

[0171] In some instances, the excipient may be a carrier. The carrier may be, for example, a solvent or dispersion medium containing water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and any combination thereof. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using polysorbates (e.g., Tween™, polysorbate 20, polysorbate 80), sodium dodecyl sulfate (sodium lauryl sulfate), lauryl dimethylamine oxide, cetyl trimethyl ammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol (Triton X100™), N,N-dimethyldodecylamine-N-oxide, hexadecyl trimethyl ammonium bromide (HTAB), polyoxyl 10 lauryl ether, Brij Proper fluidity can be maintained by the use of surfactants such as 721™, bile salts (sodium deoxycholate, sodium cholate), pluronic acid (F-68, F-127), polyoxyl castor oil (Cremophor™), nonylphenol ethoxylate (Tergitol™), cyclodextrin, and ethylbenzethonium chloride (Hyamine™). Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, cresol, ascorbic acid, thimerosal, and others. In many cases, it may be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the internal composition can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. In some embodiments, the pharmaceutical carrier includes sodium phosphate, sodium chloride, polysorbates, and sucrose. In some embodiments, the pharmaceutical composition comprises a surfactant, e.g., a non-ionic surfactant, such as a polysorbate, a poloxamer, or a pluronic. In some embodiments, the addition of a non-ionic surfactant reduces aggregation in a suspension or solution.

[0172] In some embodiments, pharmaceutical compositions useful in the present disclosure can be packaged in a kit to facilitate application of the present disclosure. In some aspects, the method provides a kit comprising a recombinant nucleic acid of the present disclosure (e.g., an rAAV comprising a nucleic acid sequence of an anti-VEGF agent). In some aspects, the method provides a kit comprising a recombinant virus of the present disclosure in lyophilized form and a solution for reconstituting the virus prior to administration to a patient. In some embodiments, the kit comprises a recombinant virus of the present disclosure in suspension form and a solution for diluting the suspension. In some embodiments, the suspension is supplied as a pre-filled syringe. In some embodiments, the suspension or kit thereof is refrigerated. In some embodiments, the suspension is warmed to room temperature prior to administration. In some embodiments, the suspension is stirred to ensure uniform distribution prior to administration.

[0173] In some embodiments, the kit comprises a recombinant virus provided herein and instructions for administering a therapeutically effective amount of the recombinant virus to an ocular or retinal cell of a subject. In some aspects, the kit comprises a pharma- ceutically acceptable salt or solution for administering the recombinant virus. Optionally, the kit can further comprise instructions for suitable operational parameters in the form of a label or separate insert. For example, the kit can have standard instructions informing a physician or laboratory technician how to prepare a unit dose of the recombinant virus from a solution or suspension and / or reconstitute a lyophilized composition. In some embodiments, optionally, the kit further comprises a device for administration, such as a syringe, a filter needle, an extension tube, a cannula, or a subretinal syringe.

[0174] In some embodiments, the pharmaceutical composition is provided as a refrigerated suspension.In some embodiments, the refrigerated suspension is provided in a kit that may include a syringe and / or a dilution buffer.In some embodiments, the refrigerated suspension is provided as a pre-filled syringe.

[0175] In some embodiments, any suitable method can be used in the biochemical purification of recombinant virus (e.g., rAAV) for use in the pharmaceutical compositions described herein.Recombinant AAV virus can be collected directly from cells or from the culture medium containing cells.Virus can be purified using various biochemical means, such as gel filtration, filtration, chromatography, affinity purification, gradient ultracentrifugation or size exclusion methods, before freeze-drying or making suspension of rAAV virus.

[0176] Indications In some cases, rAAV virions of any serotype comprising a variant capsid protein and a therapeutic transgene as described herein, or pharmaceutical compositions thereof, can at least partially reverse an ocular condition or disease associated with ocular neovascularization or CNV. In some embodiments, rAAV virions comprising a capsid variant protein are used to deliver an anti-VEGF transgene to the eye of a human subject.

[0177] Indications for gene therapy or pharmaceutical compositions described herein include neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), retinal vein occlusion and diabetic retinopathy (DR) in DME patients. In some cases, the methods and pharmaceutical compositions disclosed herein can be used to prevent or treat ocular conditions or diseases for which an anti-VEGF transgene has been approved or indicated. In some embodiments, gene therapy (e.g., AAV2.7m8-based gene therapy) is used to treat or prevent ocular conditions or diseases that are responsive to at least one current standard of care for ocular conditions / diseases, including but not limited to CNV, wet AMD, dry AMD, macular edema after RVO, DME, and diabetic retinopathy in DME patients. In some embodiments, rAAV gene therapy is used to treat or prevent any ocular condition or disorder characterized by neovascularization or CNV. In another aspect, the present disclosure provides pharmaceutical compositions provided herein for the treatment of diseases such as, for example: AMD, DME, RVO, angiogenesis-related diseases, cancer, autoimmune diseases, infectious disease organisms, etc.

[0178] In some embodiments, the eye condition may be diabetic macular edema. Diabetic macular edema (DME) is the swelling of the retina in diabetes mellitus due to leakage of fluid from blood vessels in the macula. The macula is the central part of the retina, a sub-area rich in cones, specialized nerve endings that detect color, and daytime vision depends on the cones. As macular edema develops, blurring occurs in the middle or just to the side of the central vision. Blindness from diabetic macular edema can progress over a period of months, making it impossible to focus clearly. Common symptoms of DME are blurred vision, floaters, double vision, and ultimately blindness if left untreated. In some embodiments, the methods and pharmaceutical compositions disclosed herein are used to treat DME.

[0179] In some embodiments, the eye condition may be retinal vein occlusion. Retinal vein occlusion is the blockage of small veins that carry blood away from the retina. The retina is a layer of tissue at the back of the inner eye that converts light images into nerve signals and sends them to the brain. Retinal vein occlusion is most often caused by hardening of arteries (atherosclerosis) and the formation of blood clots. Blockage of smaller veins (branch veins or BRVO) in the retina often occurs where retinal arteries that have thickened or hardened due to atherosclerosis cross and put pressure on the retinal vein. Symptoms of retinal vein occlusion may include sudden blurring or loss of vision in all or part of one eye. In some embodiments, the methods and pharmaceutical compositions disclosed herein are used to treat retinal vein occlusion.

[0180] In some embodiments, the ocular condition may be choroidal neovascularization (CNV), also known as wet AMD. Choroidal neovascularization may involve the growth of new blood vessels originating from the choroid into the sub-retinal pigment epithelium (sub-RPE) or subretinal space through breaks in Bruch's membrane, which may be a major cause of blindness. CNV may result in a sudden worsening of central vision, which is noticeable within a few weeks. Other symptoms that may occur include color vision disorders and metamorphopsia (distortion of straight lines that appear wavy). Hemorrhage of new blood vessels may accelerate the onset of symptoms of CNV. CNV may also include a feeling of pressure at the back of the eye. In some embodiments, the methods and pharmaceutical compositions disclosed herein are used to treat CNV or ocular conditions associated with neovascularization.

[0181] The advanced "wet" form (neovascular or exudative) of AMD is less common, but can frequently cause rapid and often substantial loss of central vision in patients. In the wet form of AMD, choroidal neovascularization forms and develops a network of blood vessels that can grow under and through the retinal pigment epithelium. This is accompanied by leakage and / or hemorrhage of plasma into the subretinal space, so that if it occurs in the macula, there may be severe sudden loss of central vision. The term "AMD" can be either dry AMD or wet AMD, unless otherwise specified. The present disclosure contemplates the treatment or prevention of AMD, wet AMD and / or dry AMD. In some embodiments, the methods and pharmaceutical compositions disclosed herein are used to treat AMD.

[0182] In some embodiments, the methods and pharmaceutical compositions disclosed herein are used to prevent or treat ocular diseases or conditions that are responsive to at least one of the current standard or approved therapies, such as ranibizumab or bevacizumab. In some embodiments, the patient has been pretreated with any one of ranibizumab, bevacizumab, and any other approved therapeutic agent for ocular diseases or conditions, or any combination thereof, prior to receiving or being eligible for anti-VEGF gene therapy.

[0183] In some embodiments, the methods and pharmaceutical compositions disclosed herein, i.e., AAV gene therapy comprising an anti-VEGF agent, result in a reduction in neovascularization or CNV of at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to vehicle or buffer controls as measured by the percentage of grade IV lesions following CNV formation by color fundus photography.

[0184] In some embodiments, the methods and pharmaceutical compositions disclosed herein, i.e., AAV gene therapy comprising an anti-VEGF agent, result in a reduction in neovascularization or CNV that is comparable to approved therapies, as measured by the percentage of grade IV lesions following CNV formation by color fundus photography. In some embodiments, the reduction in CNV or therapeutic effect is longer-lasting with administration of gene therapy comprising an anti-VEGF agent, compared to non-gene therapy-based injections or protein injections.

[0185] In some cases, the rAAV virion or pharmaceutical composition thereof can at least partially ameliorate ocular conditions, diseases, or combinations thereof.In some instances, the ocular condition or disease can be associated with ocular neovascularization.In some cases, the ocular condition or disease is any condition or disease that is responsive to or treatable by the anti-VEGF agent of the present disclosure.

[0186] In some embodiments, the gene therapy described herein is used to treat any ocular disease or condition involving abnormal neovascularization, for example, as a result of abnormal VEGF and / or VEGFR activity or expression, AMD, diabetic retinopathy, and preeclampsia. In some embodiments, the anti-VEGF agent in the gene therapy is an agent that inhibits or interferes with members of the VEGF family in mammals, including VEGF-A, B, C, D, and placental growth factor (PIGF), or any combination or variant thereof. In some embodiments, the anti-VEGF agent in the gene therapy is an agent that inhibits or interferes with any one of the VEGF-related proteins, such as VEGF-E expressed by some viruses and VEGF-F found in the venom of some snakes, which can also have therapeutic properties for additional indications related to angiogenesis in vivo. In some embodiments, the anti-VEGF agent can also interfere with, bind to, or inhibit placental growth factor (PIGF) in vivo.

[0187] How to use In some embodiments, the present disclosure provides a method for treating pathological angiogenesis-associated ocular diseases, comprising administering to a human subject in need of such treatment a pharma- tically effective amount of a pharmaceutical composition provided herein. In some embodiments, the disease is selected from the group of ocular neovascular diseases including age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, and diabetic retinal edema, and any combination thereof.

[0188] In some embodiments, a pharmaceutical composition comprising a rAAV comprising a variant capsid protein (e.g., rAAV.7m8) and a nucleic acid sequence encoding an anti-VEGF agent is used to treat or prevent AMD, including dry AMD and wet AMD. In some embodiments, a pharmaceutical composition comprising a rAAV comprising a variant capsid protein (e.g., rAAV.7m8) and a nucleic acid sequence encoding an anti-VEGF agent is used to treat or prevent CNV or reduce grade IV CNV lesions. In some embodiments, a pharmaceutical composition comprising an rAAV comprising a variant capsid protein (e.g., rAAV.7m8) and a nucleic acid sequence encoding an anti-VEGF agent is used to treat or prevent any one of AMD, wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, RVO, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy and diabetic retinal edema, DR in DME patients, and any combination thereof.

[0189] In some embodiments, the method of treating AMD, DME, RVO or DR comprises pretreating the patient with an approved therapy, for example, ranibizumab or bevacizumab injection, before administering to the patient a gene therapy comprising an anti-VEGF agent, for example, ranibizumab or bevacizumab nucleic acid sequence. In some embodiments, the patient is pretreated with an approved therapy before receiving a one-time dose of the anti-VEGF gene therapy disclosed herein. In some embodiments, the patient is responsive to any one of ranibizumab or bevacizumab injection before receiving a one-time dose of the anti-VEGF gene therapy disclosed herein. In some embodiments, patients who are responsive to any one of ranibizumab or bevacizumab, or patients who have been pretreated with any one of ranibizumab or bevacizumab, are treated with ranibizumab or bevacizumab gene therapy disclosed herein for at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more or for a period of more than 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more during which the patient does not receive any of these treatments for AMD. In some cases, after the patient receives an intravitreal injection of ranibizumab or bevacizumab gene therapy, the patient does not begin receiving ranibizumab or bevacizumab protein injections or another approved therapy until at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more have passed.

[0190] In some embodiments, the ranibizumab or bevacizumab gene therapy or any other anti-VEGF gene therapy disclosed herein is a one-time administration. In some embodiments, after a patient receives a unit dose of the ranibizumab or bevacizumab gene therapy disclosed herein, the patient does not require the use of any other approved protein-based therapeutic agent.

[0191] In some cases, patients who experience adverse effects associated with repeated injections of approved therapies for CNV or AMD, such as inflammation or bacterial infection, may be candidates for treatment with anti-VEGF gene therapy or ranibizumab or bevacizumab gene therapy disclosed herein.In some cases, such risks are lower in gene therapy, because gene therapy requires only one injection in the patient's lifetime, or is given no more than once in at least 2, 5, 10, 20, 30, 40 or 50 years.In some cases, the therapeutic effect of gene therapy lasts longer, and the cost of one-time gene therapy injection can be lower than the combined cost of multiple repeated injections of protein, so treatment with anti-VEGF gene therapy or ranibizumab or bevacizumab gene therapy disclosed herein can be more cost-effective than protein-based injection.

[0192] In addition, by not requiring repeated injections, gene therapy addresses the patient compliance and adherence issues associated with therapies that require repeated injections, since non-compliance (e.g., when a patient forgets or misses one or more scheduled injections) can lead to vision loss and the worsening of eye diseases or conditions. The rate of non-compliance and non-adherence to treatment regimens that require repeated or frequent visits to the clinic for administration is higher among elderly patients who are most affected by AMD. Thus, for example, delivery of anti-VEGF agents to the patient's eye by gene therapy as a one-time intravitreal injection can provide patients with a more convenient treatment option and improve patient outcomes by addressing the problem of non-compliance and non-adherence.

[0193] In some embodiments, the method of use includes pretreating a human patient or subject with an approved drug that is considered the current standard of care, such as ranibizumab injections or bevacizumab injections, determining the patient's responsiveness to ranibizumab or bevacizumab, and administering the anti-VEGF gene therapy described herein to the patient who is responsive to the approved therapy. Determining the patient's responsiveness to the approved therapy or current standard of care can include, but is not limited to, blood tests, immunoassays, ex vivo experiments, or administering ranibizumab or bevacizumab protein injections to the patient and assaying the patient's responsiveness to ranibizumab or bevacizumab.

[0194] In some embodiments, a method of using an anti-VEGF gene therapy described herein comprises reconstituting a lyophilized form of a pharmaceutical composition described herein (i.e., rAAV2.7m8 comprising an anti-VEGF nucleic acid sequence) according to the label of the drug, and administering said reconstituted anti-VEGF gene therapy to a subject or human patient. In some embodiments, a method of using an anti-VEGF gene therapy described herein comprises administering a suspension of a pharmaceutical composition described herein according to the label of the drug, and administering said suspension of anti-VEGF gene therapy to a subject or human patient. In some embodiments, an additional step for administering the suspension comprises stirring the suspension and / or warming the suspension to room temperature prior to use.

[0195] In some embodiments, the human patient is pretreated with approved protein injections or current standard treatments, such as ranibizumab injections or bevacizumab injections.In some embodiments, the patient receives no more than one injection or administration of rAAV2.7m8-ranibizumab gene therapy for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer; or receives no more than one injection or administration of rAAV2.7m8-ranibizumab gene therapy for more than 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer.

[0196] Also disclosed herein in some embodiments is a method for preventing or treating an ocular condition or disease, comprising administering to an individual in need thereof, e.g., an individual having an ocular condition or disease responsive to an approved drug, an effective amount of a rAAV virion or pharmaceutical composition thereof comprising a nucleic acid sequence encoding an anti-VEGF agent described herein, e.g., ranibizumab or bevacizumab. In some embodiments, the rAAV2.7m8-ranibizumab virion can be administered by intraocular injection, intravitreal injection, subretinal injection, or any other convenient mode or route of administration to the eye of an individual. Other convenient modes or routes of administration can include, for example, intravenous, topical, eye drops, etc. In some embodiments, the methods and pharmaceutical compositions disclosed herein involve administration by intravitreal injection.

[0197] The "therapeutically effective amount" described herein may be in a relatively broad range that can be determined by clinical trials. For direct injection into the eye or intravitreal injection, the therapeutically effective dose is approximately 10 11 ~10 12 or 10 12 ~10 13 In some embodiments, the unit dose or therapeutically effective amount of 7m8-ranibizumab or any other anti-VEGF gene therapy may be 10 10 ~10 11 Between 10 11 ~1012 Between 10 10 ~10 12 Between 10 12 ~10 13 Between 10 11 ~10 13 Between 10 12 ~10 13 Between 10 12 ~10 14 Between 10 11 ~10 14 Between 10 11 ~10 15 Between 10 12 ~10 15 Between 10 13 ~10 14 Between 10 14 ~10 15 Between 10 15 ~10 16 Between 10 16 ~10 17 Between 10 17 ~10 18 Between 10 18 ~10 19 Between or 10 19 ~10 20 In some embodiments, the unit dose of a pharmaceutical composition comprising 7m8-ranibizumab or any other anti-VEGF gene therapy of the present disclosure is between 1×10 10 ~2×10 10 Between 2×10 10 ~3×10 10 Between 3×10 10 ~4×10 10 Between 4×10 10 ~5×10 10 Between 5×10 10 ~6×10 10 Between 6×10 10 ~7×10 10 Between 7×10 10 ~8×10 10 Between 8×10 10 ~9×10 10 Between 9×10 10 ~10×10 10 Between 1×10 11 ~2×10 11 Between 2×10 11 ~3×1011 Between 2×10 11 ~2.5×10 11 Between, 2.5×10 11 ~3×10 11 Between 3×10 11 ~4×10 11 Between 4×10 11 ~5×10 11 Between 5×10 11 ~6×10 11 Between 6×10 11 ~7×10 11 Between 7×10 11 ~8×10 11 Between 8×10 11 ~9×10 11 Between 9×10 11 ~10×10 11 Between 1×10 12 ~2×10 12 Between 2×10 12 ~3×10 12 Between, 2.5×10 12 ~3×10 12 Between 3×10 12 ~4×10 12 Between 4×10 12 ~5×10 12 Between 5×10 12 ~6×10 12 Between 6×10 12 ~7×10 12 Between 7×10 12 ~8×10 12 Between 8×10 12 ~9×10 12 Between 9×10 12 ~10×10 12 Between 1×10 13 ~2×10 13 Between 2×10 13 ~3×10 13 Between 3×10 13 ~4×10 13 Between 4×10 13 ~5×10 13 Between 5×10 13 ~6×10 13 Between 6×10 13 ~7×10 13 Between 7×10 13 ~8×1013 Between 8×10 13 ~9×10 13 Between or 9×10 13 ~10×10 13 In some embodiments, the unit dose of 7m8-ranibizumab or any other anti-VEGF gene therapy of the present disclosure is between 2.1 x 10 11 ~2.1×10 12 In some embodiments, the unit dose of the rAAV of the present disclosure is between 10 10 ~10 13 Between 10 10 ~10 11 Between 10 11 ~10 12 Between 10 12 ~10 13 Between or 10 13 ~10 14 It is a vector genome between.

[0198] In some embodiments, the unit dose of 7m8-ranibizumab or any other anti-VEGF gene therapy of the present disclosure is 1×10 10 ~2×10 10 Between 2×10 10 ~4×10 10 Between 3×10 10 ~5×10 10 Between 4×10 10 ~6×10 10 Between 5×10 10 ~7×10 10 Between 6×10 10 ~8×10 10 Between 7×10 10 ~9×10 10 Between 8×10 10 ~10 11 Between 1×10 11 ~2×10 11 Between 2×10 11 ~4×10 11 Between 3×10 11 ~5×10 11 Between 4×10 11 ~6×10 11 Between 5×10 11 ~7×10 11Between 6×10 11 ~8×10 11 Between 7×10 11 ~9×10 11 Between 8×10 11 ~10×10 11 Between 1×10 12 ~3×10 12 Between 2×10 12 ~4×10 12 Between 3×10 12 ~5×10 12 Between 4×10 12 ~6×10 12 Between 5×10 12 ~7×10 12 Between 6×10 12 ~8×10 12 Between 7×10 12 ~9×10 12 Between 8×10 12 ~10×10 12 Between 1×10 13 ~5×10 13 Between 5×10 13 ~10×10 13 Between 10 12 ~5×10 12 Between 5×10 12 ~1×10 13 Between 7×10 12 ~1×10 13 Between 8×10 12 ~2×10 13 Between 9×10 12 ~2×10 13 Between 9×10 12 ~2×10 13 Between 9×10 12 ~4×10 13 Between 1×10 13 ~3×10 13 Between 1×10 13 ~2×10 13 Between 2×10 13 ~3×10 13 Between 3×10 13 ~4×10 13 Between 4×10 13 ~5×10 13 Between 5×10 13 ~6×10 13 Between 6×1013 ~7×10 13 Between 7×10 13 ~8×10 13 Between 8×10 13 ~9×10 13 Between or 8×10 13 ~1×10 14 It is a vector genome between.

[0199] In some embodiments, the total amount of 7m8-ranibizumab or any other anti-VEGF gene therapy injected into a human patient or subject within a 5-10 year period is about 10 10 ~10 13 , 10 10 ~10 11 , 10 11 ~10 12 , 10 12 ~10 13 Or 10 13 ~10 14 or 1 × 10 10 ~2×10 10 , 2×10 10 ~4×10 10 , 3×10 10 ~5×10 10 , 4×10 10 ~6×10 10 , 5×10 10 ~7×10 10 , 6×10 10 ~8×10 10 , 7×10 10 ~9×10 10 , 8×10 10 ~10 11 , 1×10 11 ~2×10 11 , 2×10 11 ~4×10 11 , 3×10 11 ~5×10 11 , 4×10 11 ~6×10 11 , 5×10 11 ~7×10 11 , 6×10 11 ~8×10 11 , 7×10 11 ~9×10 11 , 8×10 11~10×10 11 , 1×10 12 ~3×10 12 , 2×10 12 ~4×10 12 , 3×10 12 ~5×10 12 , 4×10 12 ~6×10 12 , 5×10 12 ~7×10 12 , 6×10 12 ~8×10 12 , 7×10 12 ~9×10 12 , 8×10 12 ~10×10 12 , 1×10 13 ~5×10 13 , 5×10 13 ~10×10 13 , 10 12 ~5×10 12 , 5×10 12 ~1×10 13 , 7×10 12 ~1×10 13 , 8×10 12 ~2×10 13 , 9×10 12 ~2×10 13 , 9×10 12 ~2×10 13 , 9×10 12 ~4×10 13 , 1×10 13 ~3×10 13 , 1×10 13 ~2×10 13 , 2×10 13 ~3×10 13 , 3×10 13 ~4×10 13 , 4×10 13 ~5×10 13 , 5×10 13 ~6×10 13 , 6×10 13 ~7×10 13 , 7×10 13 ~8×10 13 , 8×10 13 ~9×10 13 or 8×10 13 ~1×10 14The vector genome is as follows:

[0200] In some embodiments, a therapeutically effective amount of a pharmaceutical composition disclosed herein comprises between 2E12 and 6E12 vector genomes. In some embodiments, a unit dose comprises about 1E12, 1.5E12, 2E12, 2.5E12, 3E12, 3.5E12, 4E12, 4.5E12, 5E12, 5.5E12, 6E12, 6.5E12, 7E12, 7.5E12, 8E12, 8.5E12, 9E12, or 9.5E12 vector genomes. In some embodiments, the unit dose is between 1E12 and 1.5E12, between 1.5E12 and 2E12, between 2E12 and 2.5E12, between 2.5E12 and 3.0E12, between 3.0E12 and 3.5E12, between 3.5E12 and 4.0E12, between 4.0E12 and 4.5E12, between 4.5E12 and 5.0E12, between 5.0E12 and 5.5 ... Containing vector genomes between .5E12 and 6.0E12, between 6.0E12 and 6.5E12, between 6.5E12 and 7.0E12, between 7.0E12 and 7.5E12, between 7.5E12 and 8.0E12, between 8.0E12 and 8.5E12, between 8.5E12 and 9.0E12, between 9.0E12 and 9.5E12 or between 9.5E12 and 10E12. In some embodiments, the unit dose comprises at least 1E12, 1.5E12, 2E12, 2.5E12, 3E12, 3.5E12, 4E12, 4.5E12, 5E12, 5.5E12, 6E12, 6.5E12, 7E12, 7.5E12, 8E12, 8.5E12, 9E12, or 9.5E12 vector genomes. In some embodiments, the unit dose comprises no more than 1E12, 1.5E12, 2E12, 2.5E12, 3E12, 3.5E12, 4E12, 4.5E12, 5E12, 5.5E12, 6E12, 6.5E12, 7E12, 7.5E12, 8E12, 8.5E12, 9E12, 9.5E12, or 10E12 vector genomes.

[0201] In some embodiments, a lower concentration (e.g., vector genome) is used for unit dose to prevent aggregation that may occur at higher concentrations. In some embodiments, a higher concentration, e.g., more vector genome, is selected for unit dose to increase the efficacy of gene therapy or maximize the delivery of anti-VEGF transgene in one injection or one-time administration of gene therapy. In some embodiments, a higher concentration of the pharmaceutical composition disclosed herein allows for injection of a smaller volume, which can reduce the adverse effects associated with intravitreal injection, such as elevated intraocular pressure, inflammation, irritation or pain.

[0202] In some embodiments, 7m8-ranibizumab or any other anti-VEGF gene therapy or pharmaceutical composition thereof can be administered as a single dose or a one-time dose.In some embodiments, more than one administration can be used to achieve the desired level of gene expression over a sustained period of time at various intervals, for example, not more than once for at least 2 years or at least 3, 4, 5, 6, 7, 8, 9, 10 years or longer.In some embodiments, intravitreal injection of 7m8-ranibizumab or any other anti-VEGF gene therapy can eliminate the need for patients to receive approved protein injections for at least 1 year or 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 years or longer. EXAMPLES

[0203] Example 1: Efficacy evaluation of 7m8-sVEGFR-1 in monkeys

[0204] Objective: To inhibit the development of choroidal neovascularization (CNV) induced by laser photocoagulation in African green monkeys using 2 × 10 12 To evaluate the efficacy of 7m8-sFLT-1 following intravitreal (IVT) administration in the vg. A further objective may be to evaluate localized sFLT-1 expression in ocular tissues.

[0205] The CNV lesion model in monkeys is generally accepted and widely used as the standard primate model for evaluating the potential efficacy of therapies to treat ocular diseases associated with neovascularization, such as wet AMD.

[0206] Subject Recruitment: Monkeys underwent baseline screening to assess ocular and general health by intraocular pressure measurement, slit-lamp biomicroscopy, fundus examination, color fundus photography (CFP), fluorescein angiography (FA), and optical coherence tomography (OCT). Thirty-nine animals with normal findings were enrolled in the study and randomized into four treatment groups by baseline weight and sex (Table 1). After baseline examination, atropine 1% ophthalmic ointment was applied. [Table 1]

[0207] Monkeys in groups 1-2 received IVT AAV2.7m8-sFLT-1 or vehicle OU on day 0 of the study according to the treatment schedule (Table 1). Prior to IVT dosing, topical local anesthesia was administered (0.5% proparacaine) and the eyes were disinfected with 5% Betadine and rinsed with sterile normal saline. IVT injections can be administered using a 31-gauge 0.5 inch needle placed 2 mm posterior to the limbus in the inferior temporal quadrant, targeting the central vitreous.

[0208] All IVT injections can be followed by topical administration of 0.3% ciprofloxacin or equivalent antibiotic ophthalmic solution and 1% atropine sulfate ointment.

[0209] On day 56, CNV was induced between the temporal vascular arcades by laser burn. Nine laser spots were placed symmetrically on each eye by an ophthalmologist using an Iridex Oculight TX 532 nm laser with a laser duration of 100 ms, spot size of 50 μm, and power of 750 mW. The laser spots were applied using a 0.9× contact laser lens. The target locations of the laser spots were mapped by a trained ophthalmologist onto color fundus images obtained before the laser procedure (and after bleb placement) for reference during laser spot placement. Color fundus photography was performed immediately after the laser procedure to document the laser lesion. Any spots showing severe retinal / subretinal hemorrhage immediately after the laser were excluded from the analysis. FIG. 1 illustrates an exemplary fundus photograph of a non-human primate eye after induction of a CNV lesion by laser irradiation.

[0210] Bilateral color fundus images of the retina were captured in a 50-degree field centered on the fovea using a Topcon TRC-50EX retinal camera with Canon 6D digital imaging hardware and New Vision Fundus Image Analysis System software. FA was performed with intravenous administration of 0.1 mL / kg of 10% sodium fluorescein. Fluorescein leakage in angiograms of CNV lesions was graded (I–IV; Table 2) by a masked ophthalmologist evaluating composites generated after uniform adjustment of image intensity. Lesion grading assessments were confirmed on fundus images by two other trained ophthalmologists. Image fluorescence densitometry analysis of late raw angiograms can also be performed using ImageJ software. [Table 2]

[0211] Subjects were assessed twice daily for general health. Detailed observations were performed once a week. Body weights were obtained at baseline screening and every two weeks during the in-life study.

[0212] On day 85, or shortly after if images were to be examined, all animals were euthanized with pentobarbital after checking the quality of fundus imaging. Animals were then euthanized with pentobarbital and the eyes enucleated. Excess orbital tissue was trimmed and both OD and OS eyes were snap frozen in liquid nitrogen and then dissected along the frozen tissue plane at room temperature to isolate the vitreous and retina along with the choroidal sub-tissue. After collection of the lamina, 5 mm punches of neural retina with RPE / choroid were taken from the macula and superior, inferior, temporal and nasal regions. Additional peripheral punches were made when room permitted. Retinas with underlying RPE / choroid tissue from each punch were transferred to pre-tared labeled cryotubes, weighed and flash frozen in liquid nitrogen. Before and after collection of punch biopsies, photographs of flat-mounted retinas were taken to document the areas from which punches were collected, along with an indication of orientation.

[0213] Statistical methods: Fisher's exact test was used to assess the incidence of different lesion grades. Two-way ANOVA with repeated measures followed by Tukey-Kramer test or contrast procedure was used to analyze OCT CNV complex area and angiogram image densitometry data. Nonparametric tests were applied when data were not normally distributed and had heterogeneous variance. P values ​​of 0.05 or less were considered statistically significant.

[0214] FIG. 3 illustrates plots of the percentage of grade IV lesions at days 14 and 28 for animals in groups 3 and 4 that were intravitreally injected with either AAV2.7m8-sVEGFR-1 or a vehicle control containing only the formulation buffer. CNV lesions were induced by laser irradiation immediately after injection in each group of test subjects, and color fundus photography was used to grade each lesion on a scale of I to IV. For fundus images collected at day 14 when administered intravitreally, monkeys treated with AAV2.7m8-sVEGFR-1 showed a slight reduction in the amount of grade IV lesions compared to administration of vehicle alone. At day 28, monkeys treated with intravitreous AAV2.7m8-sFLT-1 did not show a significant reduction in the amount of grade IV lesions compared to administration of vehicle alone.

[0215] Example 2: Efficacy evaluation of 7m8-ranibizumab in monkeys

[0216] Similar in vivo studies to those described in Example 1 were carried out in monkeys using the same protocol and AAV2.7m8-ranibizumab, an rAAV2 containing the 7m8 sequence and a nucleic acid sequence encoding ranibizumab inserted between positions 587 and 588 of the capsid protein VP1 of AAV2.

[0217] As illustrated in Figure 4, intravitreally administered AAV2.7m8-ranibizumab prevented the development of laser-induced grade IV CNV lesions. AAV2.7m8-ranibizumab, ranibizumab alone (positive control) or a vehicle control containing formulation buffer were administered at 2x10 12 vg into non-human primate eyes by intravitreal injection. CNV lesions were then induced in all groups by laser irradiation, and color fundus photography was used to grade each lesion on a scale of I-IV. Measurements of the percentage of grade IV lesions were then averaged and plotted. At days 14 (light gray bars) and 28 (dark gray bars), AAV2.7m8-ranibizumab significantly reduced CNV lesions in vivo to levels comparable to ranibizumab alone.

[0218] These in vivo studies in monkeys suggested that AAV2.7m8-ranibizumab may be a viable gene therapy option for humans.

Claims

[Claim 1] The invention described in this specification.

Images