Treatment of AMD using AAV2 variant with aflibercept

Abstract

To provide treatment of AMD using an AAV2 variant with aflibercept.SOLUTION: The present disclosure provides pharmaceutical compositions and methods thereof for treatment of age-related macular degeneration (AMD) in a subject by administering intravitreally a pharmaceutical composition adapted for gene therapy, comprising a vector having a nucleic acid sequence that encodes aflibercept. The present disclosure relates to pharmaceutical compositions and methods for prevention or treatment of neovascular (wet) age-related macular degeneration (AMD), macular edema following retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR) in patients with DME, retinal vein occlusion, and related eye diseases or conditions, in a primate or human subject by administering intravitreally or subretinally a pharmaceutical composition comprising a pharmaceutically effective amount of a vector or viral particles (e.g., rAAV) comprising a nucleic acid encoding aflibercept or a functional fragment or variant thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 351,234, filed June 16, 2016, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Aflibercept is a recombinant fusion protein that acts as a decoy receptor for vascular endothelial growth factor subtypes A and B (VEGF-A and VEGF-B) and placental growth factor (PIGF). By binding to these ligands, aflibercept can block these ligands from binding to vascular endothelial growth factor receptors (VEGFR), VEGFR-1, and VEGFR-2, suppressing angiogenesis and reducing vascular permeability. Aflibercept consists of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 fused to the Fc fragment of IgG1. Aflibercept is marketed under the trade name EYLEA® (aflibercept), which is an intravitreal aflibercept fusion protein injection for ophthalmic use. Summary of the Invention [Means for solving the problem]

[0003] While EYLEA® (aflibercept) is the current standard of care for treating wet AMD in patients, gene therapy methods that deliver aflibercept to the eye can offer patients improved treatment options. This is because gene therapy can provide long-term or sustained release of aflibercept in vivo without the need for repeated injections, which can increase the risk of inflammation, infection, and other adverse effects in some patients. Furthermore, because non-compliance can lead to vision loss and worsening of eye diseases or conditions, gene therapy addresses the challenges of patient compliance and adherence associated with therapies that require repeated injections by eliminating the need for repeated injections. The rate of 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. Delivering aflibercept to a patient's eye through gene therapy may therefore provide patients with a safer, potentially more cost-effective, and more convenient treatment option, and may improve patient outcomes by addressing the issues of non-compliance and non-adherence.

[0004] The present disclosure relates to pharmaceutical compositions and methods for the prevention or treatment of neovascular (wet) age-related macular degeneration (AMD), macular edema following retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR) in DME patients, retinal vein occlusion, and related ocular diseases or conditions in a primate or human subject by intravitreal or subretinal administration of a pharmaceutical composition comprising a pharmaceutically effective amount of a vector or viral particle (e.g., rAAV) comprising a nucleic acid encoding aflibercept, a functional fragment or variant thereof.

[0005] In some embodiments, disclosed herein are methods for treating an ocular condition or disease, comprising administering a unit dose of a pharmaceutical composition to the eye of a primate subject in need thereof via intravitreal injection, wherein the pharmaceutical composition comprises: (a) an rAAV2 variant comprising an amino acid sequence LGETTRP inserted between positions 587 and 588 of the capsid protein VP1, and a nucleic acid sequence encoding a polypeptide, functional fragment, or variant thereof having at least 80% homology to aflibercept, and (b) a pharmaceutically acceptable excipient. 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), or diabetic retinopathy associated with DME. In some cases, the ocular condition or disease is choroidal neovascularization or wet AMD. In some cases, the unit dose comprises between 1E12 and 1E13 vector genomes. In some cases, the unit dose contains between 2E12 and 6E12 vector genomes. In some cases, the unit dose contains a volume of 100 μL or less. In some cases, the unit dose contains a volume of 50 μL or less. In some cases, the subject is a non-human primate. In some cases, the subject is a human. In some cases, the subject is responsive to aflibercept. In some cases, the subject has been pre-treated with aflibercept. In some cases, the administration by intravitreal injection is performed no more than once every two years. In some cases, the administration by intravitreal injection is performed no more than once every five years. In some cases, the administration by intravitreal injection is a one-time administration. In some cases, the pharmaceutical composition is a suspension or a refrigerated suspension. In some cases, the method further comprises agitating the suspension prior to the administering step to ensure uniform distribution of the suspension. In some cases, the method further comprises warming the pharmaceutical composition to room temperature prior to the administering step.

[0006] In another embodiment, the pharmaceutical composition comprises a suspension further comprising an rAAV2 variant comprising the amino acid sequence LGETTRP inserted between positions 587 and 588 of the capsid protein VP1 and a nucleic acid sequence encoding a polypeptide, functional fragment, or variant thereof having at least 80% homology to aflibercept. In some cases, the nucleic acid sequence comprises SEQ ID NO: 2. In some cases, a unit dose of the composition comprises between 1E12 and 1E13 of the vector genome. In some cases, a unit dose of the pharmaceutical composition comprises between 2E12 and 6E12 of the vector genome. In some cases, the kit comprises a suspension pharmaceutical composition and a solution for diluting the pharmaceutical composition. In some cases, the pharmaceutical composition comprises 1 mL or less. In some cases, the pharmaceutical composition comprises 0.5 to 1.0 mL or less. In some cases, the pharmaceutical composition comprises less than or equal to 0.5 mL. In some cases, the solution of the kit comprises a buffer, salt, alcohol, surfactant, or any combination thereof. In some cases, the kit comprises a syringe.

[0007] In another aspect, a method for treating an ocular condition or disease includes agitating a refrigerated suspension, wherein the suspension contains an rAAV2 variant comprising the amino acid sequence LGETTRP inserted between positions 587 and 588 of the capsid protein VP1 and a nucleic acid sequence encoding a polypeptide, functional fragment, or variant thereof having at least 80% homology to aflibercept; and administering a volume of the refrigerated suspension to the eye of a human subject via intravitreal injection. In some cases, the subject is characterized by having been pretreated with aflibercept. In some cases, the subject is responsive to aflibercept. In some cases, the refrigerated suspension contains 1 mL or less of solution. In some cases, the refrigerated suspension contains 0.5 mL or less. 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 contains between 1E12 and 1E13 unit doses of vector genome. In some cases, the volume contains between 2E12 and 6E12 unit doses of vector genome. In some cases, the administering step occurs no more than once every two years. In some cases, the administering step is a one-time injection. In some cases, the method further includes assaying the subject for responsiveness to aflibercept prior to administering the composition. In some cases, the suspension includes a pharmaceutically 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 alcohols, polyoxyethylene sorbitan, octoxynol, Brij, Pluronic, and polyoxyl castor oil. In some cases, the pharmaceutically acceptable excipient includes 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), or diabetic retinopathy associated with DME. In some cases, the ocular condition or disease is choroidal neovascularization or wet AMD.In some cases, the method further comprises warming the suspension to room temperature prior to administration. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method of treating an ocular condition or disease, comprising administering to the eye of a primate subject in need thereof by intravitreal injection a unit dose of a pharmaceutical composition, wherein the pharmaceutical composition: (a) an rAAV2 variant comprising the amino acid sequence LGETTRP inserted between positions 587 and 588 of the capsid protein VP1, and a nucleic acid sequence encoding a polypeptide, functional fragment, or variant thereof having at least 80% homology to aflibercept; and (b) a pharmaceutically acceptable excipient A method comprising: (Item 2) 2. The method of item 1, wherein the ocular condition or disease is neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion, diabetic macular edema (DME), or diabetic retinopathy associated with DME. (Item 3) Item 10. The method of item 1, wherein the ocular condition or disease is choroidal neovascularization or wet AMD. (Item 4) 4. The method according to any one of items 1 to 3, wherein the unit dose comprises between 1E12 and 1E13 vector genomes. (Item 5) 4. The method according to any one of items 1 to 3, wherein the unit dose comprises between 2E12 and 6E12 vector genomes. (Item 6) 2. The method of claim 1, wherein the unit dose comprises a volume of 100 μL or less. (Item 7) 2. The method of claim 1, wherein the unit dose comprises a volume of 50 μL or less. (Item 8) Item 10. The method of item 1, wherein the subject is a non-human primate. (Item 9) Item 10. The method of item 1, wherein the subject is a human. (Item 10) Item 10. The method of item 1, wherein the subject is responsive to aflibercept. (Item 11) Item 10. The method of item 1, wherein the subject has been pretreated with aflibercept. (Item 12) Item 10. The method of item 1, wherein the administration by intravitreal injection is performed no more than once every two years. (Item 13) Item 10. The method of item 1, wherein the administration by intravitreal injection is performed no more than once every five years. (Item 14) 2. The method of claim 1, wherein the administration by intravitreal injection is a one-time administration. (Item 15) Item 10. The method of claim 1, wherein the pharmaceutical composition is a suspension. (Item 16) 16. The method of claim 15, further comprising the step of agitating the suspension prior to the administering step to ensure uniform distribution of the suspension. (Item 17) 10. The method of claim 1, further comprising warming the pharmaceutical composition to room temperature prior to the administering step. (Item 18) A pharmaceutical composition comprising a suspension containing an rAAV2 variant comprising the amino acid sequence LGETTRP inserted between positions 587 and 588 of the capsid protein VP1, and a nucleic acid sequence encoding a polypeptide, functional fragment or variant thereof having at least 80% homology to aflibercept. (Item 19) 19. The pharmaceutical composition of item 18, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO: 2. (Item 20) 19. The pharmaceutical composition of item 18, wherein a unit dose of the composition comprises between 1E12 and 1E13 vector genomes. (Item 21) 19. The pharmaceutical composition according to item 18, wherein a unit dose of the pharmaceutical composition contains 2E12 to 6E12 of the vector genome. (Item 22) 19. A kit comprising the pharmaceutical composition according to item 18 and a solution for diluting the pharmaceutical composition. (Item 23) 23. The kit of item 22, wherein the pharmaceutical composition comprises 1 mL or less. (Item 24) 23. The kit according to item 22, wherein the pharmaceutical composition comprises 0.5 to 1.0 mL or less. (Item 25) 23. The kit of item 22, wherein the pharmaceutical composition comprises less than or equal to 0.5 mL. (Item 26) 23. The kit of item 22, wherein the solution comprises a buffer, a salt, an alcohol, a surfactant, or any combination thereof. (Item 27) 23. The kit of item 22, further comprising a syringe. (Item 28) 1. A method of treating an ocular condition or disease, comprising: (a) stirring a refrigerated suspension, wherein the suspension contains an rAAV2 variant comprising an amino acid sequence LGETTRP inserted between positions 587 and 588 of capsid protein VP1, and a nucleic acid sequence encoding a polypeptide having at least 80% homology to aflibercept, a functional fragment, or a variant thereof; (b) administering a volume of the refrigerated suspension to the eye of a human subject via intravitreal injection; A method comprising: (Item 29) 29. The method of claim 28, wherein the subject has been pretreated with aflibercept. (Item 30) 29. The method of item 28, wherein the subject is responsive to aflibercept. (Item 31) 29. The method of claim 28, wherein the refrigerated suspension contains 1 mL or less of solution. (Item 32) 29. The method of claim 28, wherein the refrigerated suspension contains 0.5 mL or less. (Item 33) 29. The method of claim 28, wherein the volume administered to the subject is 50 μL or less. (Item 34) Item 35. The method of item 28, wherein the volume administered to the subject is 100 μL or less. 29. The method of item 28, wherein the volume comprises between 1E12 and 1E13 unit doses of vector genome. (Item 36) 29. The method of item 28, wherein the volume comprises between 2E12 and 6E12 unit doses of vector genome. (Item 37) 29. The method of claim 28, wherein the administering step is performed no more than once every two years. (Item 38) 29. The method of claim 28, wherein the administering step is a one-time injection. (Item 39) 29. The method of claim 28, further comprising assaying the subject for responsiveness to aflibercept prior to administering the composition. (Item 40) 29. The method of claim 28, wherein the suspension comprises a pharmaceutically acceptable excipient. (Item 41) 41. The method of claim 40, wherein the excipient comprises a surfactant or stabilizer. (Item 42) 42. The method of claim 41, 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 43) 41. The method of claim 40, wherein the pharmaceutically acceptable excipient comprises phenol, mannitol, sorbitol, or sodium chloride. (Item 44) 29. The method of item 28, wherein the ocular condition or disease is neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion, diabetic macular edema (DME), or diabetic retinopathy associated with DME. (Item 45) 29. The method of item 28, wherein the ocular condition or disease is choroidal neovascularization or wet AMD. (Item 46) 29. The method of claim 28, further comprising warming the suspension to room temperature prior to administration.

[0008] 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.

[0009] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 illustrates exemplary fundus images of a primate (African green monkey) eye after induction of CNV lesions by laser irradiation without treatment. 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 irradiation to document the laser lesions.

[0011] [Figure 2]Figure 2 illustrates representative fundus images from fluorescein angiography at day 70 after intravitreal (IVT) injection with either a vehicle control containing buffer alone or AAV2.7m8-aflibercept. Monkeys treated with AAV2.7m8-aflibercept showed fewer grade IV lesions and more lower-graded lesions than monkeys injected with the vehicle control alone.

[0012] [Figure 3] 3 illustrates the percent Grade IV CNV lesions in monkeys after intravitreal injection of EYLEA® (aflibercept), used as a positive control, compared to a vehicle control containing buffer only. EYLEA® (aflibercept) treatment demonstrated a significant reduction in the amount of Grade IV lesions compared to the vehicle control, based on fundus images collected on days 14 (light gray bars) and 28 (dark gray bars).

[0013] [Figure 4] Figure 4 illustrates the percent grade IV CNV lesions in monkeys treated with vehicle control, AAV2.7m8-aflibercept, or intravitreal injection of AAV2.7m8-sVEGFR-1 on days 14 (light gray bars) and 28 (dark gray bars) after intravitreal injection. Monkeys treated with intravitreal AAV2.7m8-aflibercept injections showed a significant reduction in the amount of grade IV lesions compared to monkeys treated with vehicle control on days 14 and 28, similar to the positive control results shown in Figure 3. Treatment with intravitreal injection of AAV2.7m8-sVEGFR-1 did not show any significant reduction in grade IV lesions compared to vehicle control.

[0014] [Figure 5] FIG. 5 depicts the nucleic acid sequence of aflibercept.

[0015] [Figure 6]6 depicts the nucleic acid sequence of soluble fms-like tyrosine kinase-1 (sFlt-1 or sVEGFR-1). sVEGFR-1 is a splice variant of VEGF receptor 1. DETAILED DESCRIPTION OF THE INVENTION

[0016] Some aspects are described below with reference to example applications for illustrative purposes. It should be understood that numerous specific details, relationships, and methods are set forth to provide a thorough understanding of the features described herein. However, a person skilled in the relevant art will readily recognize that the features described herein can be implemented without one or more of these specific details or by using other methods. The features described herein are not limited by the illustrated ordering of acts or events, as some acts may be performed in a different order and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to perform the methodology in accordance with the features described herein.

[0017] The present disclosure relates to pharmaceutical compositions and methods for the treatment or prevention of ocular diseases or conditions, comprising administering a gene therapy, vector, or construct comprising a nucleic acid sequence (e.g., cDNA) encoding aflibercept, a functional fragment, or variant thereof, via intravitreal injection into the eye of a primate (e.g., monkey or human). Upon intravitreal injection of the gene therapy, vector, or construct comprising the nucleic acid sequence for aflibercept, a functional fragment, or variant thereof, the nucleic acid sequence is expressed in vivo, for example, in retinal cells, to produce an aflibercept fusion protein, or a functional fragment or variant thereof, which confers a therapeutic effect.

[0018] In some embodiments, aflibercept-containing gene therapy, vector, or construct 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), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME) and / or diabetic retinopathy (DR) in DME patients, retinal vein occlusion, 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 aflibercept (e.g., EYLEA®). In some embodiments, the methods described herein are used to treat ocular diseases or conditions that are responsive to current standard therapy or to at least one approved therapy for AMD, RVO, DME, or DR in DME patients, such as aflibercept injection, ranibizumab injection, or bevacizumab injection.

[0019] In some embodiments, the pharmaceutical composition suitable for gene therapy comprises a nucleic acid sequence encoding a protein or polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to aflibercept, or its functional fragment or variant, for example, a recombinant adeno-associated virus (rAAV) comprising a variant or modified capsid protein VP1, such as the 7m8 capsid variant, is administered intravitreally or subretinally. Such homology can be based on the nucleic acid sequence, amino acid sequence, spatial conformation or protein structure of aflibercept.

[0020] The protein sequence of aflibercept is published in the DrugBank database, accession number DB08885. In some embodiments, aflibercept refers to the nucleic acid sequence encoding the fusion protein as disclosed in U.S. Patent Application Publication No. 2014 / 0371438 (FIG. 5).

[0021] One advantage of gene therapy compared to protein injection is that gene therapy provides long-term or sustained release of therapeutic agent (such as aflibercept), and in some embodiments, does not require multiple or repeated injections.This long-term or sustained release of aflibercept results from the delivery of a nucleic acid sequence encoding aflibercept fusion protein, which is expressed in vivo and produces a therapeutic effect.In some embodiments, the expression of aflibercept from the heterologous nucleic acid delivered to retinal cells can continue for at least 1 year, for more than 1 year, for at least 2, 3, 4, 5, 10 years or more.

[0022] In some embodiments, the rAAV can include a capsid variant protein that increases its infectivity of 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, such as an aflibercept fusion protein or a functional fragment or variant thereof, to target cells or tissues that can express the therapeutic transgene over a period of time (e.g., at least 1, 1.5, 2, 3, 4, 5, 10, or more years). The gene therapy methods disclosed herein can target specific tissues or cell types of interest, such as photoreceptor cells, which can help minimize off-target effects or provide more targeted delivery of a therapeutic transgene, such as aflibercept, in vivo.

[0023] The long-term or sustained delivery of aflibercept by in vivo gene therapy may allow pharmaceutical compositions comprising a nucleic acid sequence encoding aflibercept, its functional fragments, mutants or variants to be administered at lower doses within a certain period compared to current standard treatments (e.g., protein injections or treatments not based on gene therapy).In some embodiments, the total number of doses administered of the gene therapy comprising a nucleic acid sequence encoding aflibercept, its functional fragments or variants is one unit dose or less for 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 gene therapy comprising a nucleic acid sequence encoding aflibercept, its functional fragments or variants is administered once or once in the patient's lifetime. In some embodiments, a one-time administration of a gene therapy comprising a nucleic acid sequence encoding aflibercept, its functional fragment or variant can produce a therapeutic effect in patients that lasts for more than 1 year, or for more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 years. In some embodiments, a gene therapy comprising a nucleic acid sequence encoding aflibercept, its functional fragment or variant is administered to a patient no more than once 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, a gene therapy comprising a nucleic acid sequence encoding aflibercept is administered to a patient who is responsive to aflibercept or has been pretreated with EYLEA® before receiving the gene therapy disclosed herein.In some embodiments, patients receiving a gene therapy disclosed herein (e.g., AAV2.7m8-aflibercept) can optionally begin therapy with aflibercept, ranibizumab, and / or bevacizumab at least 2, 3, 4, 5, 10, or more years after receiving gene therapy. In some embodiments, patients do not require anti-VEGF therapy after receiving a gene therapy disclosed herein.

[0024] In some embodiments, a one-time administration of gene therapy comprising a nucleic acid sequence encoding aflibercept, a functional fragment, or variant thereof, eliminates the need for the patient to receive EYLEA® injections for more than 1 year, more than 1.5 years, or more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, a patient receiving an intravitreal injection of gene therapy comprising a nucleic acid sequence encoding aflibercept, a functional fragment, or variant thereof, does not require any additional injections of aflibercept for the rest of the patient's life. In other embodiments, a patient receiving a one-time intravitreal injection of 7m8-aflibercept gene therapy can optionally begin therapy with any one of aflibercept, ranibizumab, and / or bevacizumab at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after receiving gene therapy.

[0025] The terminology used in this disclosure is for the purpose of describing particular cases only and is not intended to be limiting of the compositions, methods, and compositions of the disclosure.

[0026] The compositions and methods of the present disclosure described herein may employ, unless otherwise indicated, conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, immunochemistry, and ophthalmology techniques within the skill of one of ordinary skill in the art. Such conventional techniques include methods for retinal or visual observation and analysis in a subject, cloning and propagation of recombinant viruses, formulation of pharmaceutical compositions, and biochemical purification and immunochemistry. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, it will be appreciated that equivalent conventional procedures can also be used. Such conventional techniques and descriptions are well known from Green et al. Eds., Genome Analysis: A Laboratory Manual Series (Volumes 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 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.) W.H. Freeman, NY (1995); Gait, "Oligonucleotide Synthesis: A Practical 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). No. 6,229,693, all of which are incorporated herein by reference in their entirety for all purposes.

[0027] In some embodiments, the present disclosure provides a pharmaceutical preparation comprising: (a) (i) a variant AAV2 capsid protein comprising an LGETTRP insertion between positions 587 and 588, and conferring increased infectivity of ocular cells compared with the AAV virion comprising the 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 aflibercept, its functional fragment or variant; and (b) a pharmaceutically acceptable excipient. In some embodiments, the encoded gene product is a fusion protein or polypeptide with at least 80%, 85%, 90%, 95% or 99% homology with aflibercept.

[0028] Also disclosed herein are methods of treating an ocular condition or disease for which aflibercept is indicated or approved for treatment, comprising administering a pharmaceutical formulation adapted for gene therapy, i.e., for in vivo delivery of a nucleic acid sequence encoding aflibercept as described herein to the eye of a subject by intravitreal injection.

[0029] Also disclosed herein are pharmaceutical compositions comprising gene therapies or vectors encoding fusion proteins or polypeptides having at least 80%, 85%, 90%, 95%, or 99% homology to aflibercept, which can be lyophilized or provided in suspension form. In some embodiments, the lyophilized form or suspension of the pharmaceutical composition is provided in a kit together with a buffer for reconstituting or diluting the pharmaceutical composition, respectively.

[0030] Also disclosed herein are pharmaceutical compositions comprising a gene therapy or vector encoding a fusion protein or polypeptide having at least 80%, 85%, 90%, 95%, or 99% homology to aflibercept, provided as a refrigerated suspension. In some embodiments, the refrigerated suspension of the pharmaceutical composition is provided in a kit along with a buffer for diluting the pharmaceutical composition.

[0031] Also disclosed herein is a recombinant adeno-associated virus (rAAV) virion adapted for gene therapy to reduce choroidal neovascularization, comprising: (a) a variant AAV2 capsid protein comprising a peptide insertion of the amino acid sequence LGETTRP inserted between amino acid positions 587 and 588 of AAV2, wherein the peptide insertion confers increased infectivity of ocular cells compared to an AAV virion comprising a corresponding non-variant or unmodified AAV2 capsid protein; and (b) a heterologous nucleic acid sequence encoding a polypeptide or therapeutic transgene having at least 80% homology to aflibercept, or a functional fragment or variant thereof.

[0032] Also disclosed herein is a method for treating ocular conditions or diseases, comprising administering an rAAV virion adapted for gene therapy and in vivo delivery of a nucleic acid sequence expressing aflibercept or its functional fragment or variant as described herein to the eye of a human subject, wherein the human subject has previously been diagnosed with an ocular condition associated with neovascularization.In some embodiments, aflibercept gene therapy is administered to patients who are responsive to aflibercept or who have been pre-treated with EYLEA (Aflibercept).

[0033] In some embodiments, methods and pharmaceutical formulations are disclosed herein that include: (i) a variant AAV capsid protein containing 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 the AAV2 capsid protein VP1, conferring increased infectivity of retinal cells (e.g., photoreceptor cells or retinal pigment epithelium) compared to an AAV virion containing the corresponding non-variant AAV2 capsid protein; and (ii) a recombinant adeno-associated virus (rAAV) virion adapted for gene therapy containing a heterologous nucleic acid sequence encoding aflibercept; and (b) a pharmaceutically acceptable excipient. In some embodiments, the encoded gene product is a fusion protein or polypeptide having at least 80% homology to aflibercept, or a functional fragment or variant thereof. In some embodiments, the pharmaceutically acceptable excipient comprises a surfactant (e.g., a non-ionic surfactant, pluronic, poloxamer, or polysorbate) 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. and "the" includes the plural as well, unless the context clearly indicates otherwise. Furthermore, the terms "including" and "includes" are intended to include "having," "has," "with," or To the extent that these variations are used in either the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising." The term "comprising" is used herein synonymously with "including" or "containing" and is intended to be inclusive or open-ended. It is a nd.

[0036] Any reference herein to "or" is intended to encompass "and / or" unless expressly stated otherwise. As used herein, a number preceded by the term "about" refers to that number plus or minus 10%. A range preceded 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 monkeys, e.g., African green monkeys 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, as used herein, include alleviating, relieving, or ameliorating disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic cause of symptoms, inhibiting a disease or condition, e.g., halting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating conditions caused by a disease or condition, or halting the symptoms of a disease or condition, and are intended to include prophylaxis. The term further includes achieving a therapeutic benefit and / or a prophylactic benefit. Therapeutic benefit refers to the eradication or amelioration of the underlying disease being treated. Additionally, therapeutic benefit is achieved, in some embodiments, by the eradication or amelioration of one or more physiological symptoms associated with the underlying disease, such that an improvement is observed in the patient despite the patient still suffering from the underlying disease. For prophylactic benefit, the pharmaceutical composition is administered to patients at risk of developing a particular disease or who report one or more 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 effect. Such methods include intravitreal or subretinal injection into the eye.

[0040] The terms "effective amount," "therapeutically effective amount," or "pharmaceutically 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 "pharmaceutically 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., it does not cause any undesired 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 pharmaceutically acceptable chemical component, including but not limited to a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, etc.

[0043] As used herein, "AAV vector" or "rAAV vector" refers to an adeno-associated virus (AAV) vector or recombinant AAV (rAAV) vector that contains a polynucleotide sequence that is not of AAV origin (i.e., a polynucleotide heterologous to AAV, such as a nucleic acid sequence encoding a therapeutic transgene, e.g., aflibercept), which is typically the 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 viral particle" or "rAAV vector particle" refers to a viral 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 a 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, production of rAAV particles necessarily includes 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 lead to 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 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 different domains, each with one or more distinct activities. For the avoidance of doubt, a "polypeptide" can be anything greater than two amino acids in length.

[0048] As used herein, "polypeptide variant" or simply "variant" refers to a polypeptide whose sequence contains an amino acid modification. In some instances, the modification can be an insertion, duplication, deletion, rearrangement, or substitution 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 the reference protein changed to another amino acid, one or more amino acid point substitutions, one or more amino acid insertions or deletions, one or more insertions and / or deletions, and / or truncations of the amino acid sequence at either or both the amino or carboxy terminus. A variant can have the same or different biological activity compared to the reference protein or unmodified protein.

[0049] In some embodiments, a variant can have, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% overall sequence homology with its corresponding reference protein. In some embodiments, a variant can have at least about 90% overall sequence homology with the wild-type protein. In some embodiments, a 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 biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or part of a polynucleotide in which the gene is found in nature, (3) is operably linked to a polynucleotide to which it is not linked in nature, or (4) does not occur 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 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, wherein the elements are in a relationship permitting them to operate in a predicted manner. For example, a promoter can be operably linked to a coding region if it serves to initiate transcription of the coding sequence. Intervening residues can be present between the promoter and the coding region, so long as this functional relationship is maintained.

[0052] The terms "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, into which part or all of the nucleic acid encoding sequence can be transcribed and adapted for gene therapy. The transcript can be translated into protein. In some cases, the transcript can be partially or not translated. In certain embodiments, expression includes both transcription of the gene and translation of 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 protein expression in target cells. The combination of control elements and the gene(s) to which the control elements are 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 rest 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 removed from its native coding sequence and operably linked to a coding sequence not found in association with it in nature can be a heterologous promoter.

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

[0055] "7m8 variant" refers to an rAAV, which can 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 VP1, 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, for example, 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, for example, 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, for example, 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] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0058] vector 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 aflibercept, a functional fragment, or a variant thereof, to retinal cells in a human subject or patient in need thereof (e.g., a patient diagnosed with AMD, RVO, or DME). The delivery of a therapeutic transgene nucleic acid to a patient using a delivery system such as an rAAV or viral vector is also referred to as gene therapy.

[0059] In some embodiments, delivery of the aflibercept nucleic acid sequence can be carried out using any suitable "vector" (also called "gene delivery" or "gene transfer vehicle"). The vector (e.g., rAAV), delivery vehicle, gene delivery vehicle, or gene transfer vehicle can include any suitable polymer or molecular complex containing a polynucleotide to be delivered to target cells, such as retinal cells, for example, photoreceptors, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells. 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 the coding sequence of a therapeutic transgene, such as an aflibercept transgene.

[0060] The compositions and methods of the present disclosure provide any suitable method for delivering anti-aflibercept nucleic acid sequences to the ocular or retinal cells of non-human primates or human subjects. In some cases, the delivery of nucleic acid molecules, polynucleotides or gene therapies is formulated or adapted for intravitreal injection into the eye of non-human primates or human subjects.

[0061] In some embodiments, suitable vectors include, but are not limited to, viral vectors such as adenoviruses, adeno-associated viruses (AAV) and retroviruses, retroviruses, lentiviruses, liposomes, lipid-containing complexes, nanoparticles, and other macromolecular complexes capable of delivering 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.

[0062] 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 the expression of aflibercept. In some embodiments, the RNA can comprise a transcript of a gene of interest (e.g., aflibercept), introns, untranslated regions (UTRs), termination sequences, etc. In other embodiments, the DNA can comprise, but is not limited to, sequences such as a promoter sequence, a gene of interest (e.g., aflibercept), UTRs, termination sequences, etc. In some cases, a combination of DNA and RNA can be used.

[0063] In some embodiments, the present disclosure provides a recombinant virus, such as a recombinant adeno-associated virus (rAAV), as a vector for delivery and expression of aflibercept in a subject.

[0064] In some embodiments, any suitable viral vector can be engineered or optimized for use in 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 so that they are replication-deficient in human or primate subjects.In some embodiments, a hybrid viral vector system can be obtained using methods known to those skilled in the art and used to deliver the nucleic acid encoding aflibercept to retinal cells.In some embodiments, viral delivery system or gene therapy can incorporate the nucleic acid sequence comprising the aflibercept gene into target cell genome (for example, the genome of retinal cells), resulting in stable gene expression of the gene over time.In some embodiments, the aflibercept gene is not incorporated into target cell genome, but is expressed from a plasmid or vector introduced into target cells.

[0065] In some embodiments, the viral vector suitable for delivering the nucleic acid sequence of aflibercept 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 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 delivering therapeutic transgenes, such as aflibercept, can be any serotype. In some embodiments, the pharmaceutical compositions and methods of the present disclosure utilize any suitable AAV serotype, including AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, AAV-DJ, and any hybrid or chimeric AAV thereof. In some embodiments, the serotype used is based on the tropism of the virus or the infectivity of the intended target cell. In some embodiments, AAV2 or rAAV2 is used to deliver the nucleic acid sequence encoding aflibercept to the eye or retinal cells of a subject through intravitreal or subretinal injection. In some embodiments, rAAV2.7m8 is used to deliver the nucleic acid sequence of aflibercept to the retinal cells of a subject.

[0066] In some embodiments, AAV or rAAV viruses, particles, or virions comprising variant capsid proteins with increased infectivity for target cells, e.g., retinal cells, are used to increase transduction of retinal cells in a subject or to increase the targeting of gene delivery to retinal cells. 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 modification site is a solvent-exposed portion of the GH loop / loop IV of the AAV capsid protein. For a description of the GH loop / loop IV of the AAV capsid, see, e.g., van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2007 .... 005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 1 5:1955. Several AAV capsid variants are known, including the 7m8 variant. In some embodiments, a rAAV virion comprises a variant AAV capsid protein that includes an insertion of a sequence of 5 to 11 amino acids, e.g., 7 amino acids, in the GH loop of the capsid protein compared to the corresponding parent AAV capsid protein, and the variant capsid protein confers increased infectivity of retinal cells compared to the infectivity of retinal cells by AAV virions containing the corresponding parent or unmodified AAV capsid protein. In some embodiments, 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, any one of the amino acid sequences LGETTRP(7m8), NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF, and STGKVPN is inserted into the solvent-exposed GH loop of the VP1 capsid protein of rAAV. In some embodiments, rAAV.7m8 containing aflibercept is used for gene therapy.

[0067] In some embodiments, any one of the following amino acid sequences: NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF, and STGKVPN can be inserted into 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.

[0068] In some embodiments, a nucleic acid encoding a gene product, such as aflibercept, 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 a CMV promoter. In some embodiments, the connexin 36 promoter is used to drive expression of a therapeutic transgene, such as aflibercept. In some embodiments, a tissue-specific promoter can be used to direct 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 a polyadenylation sequence, untranslated region (UTR), 3'UTR, or termination sequence. 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, which allows for simultaneous expression of two or more proteins or results in a multigene or polycistronic mRNA.

[0069] 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; an aflibercept-encoding sequence; 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 aflibercept-encoding sequence comprises a sequence encoding an aflibercept fusion protein or a functional fragment thereof.

[0070] In some embodiments, the viral vectors of the present disclosure are measured as vector genomes. In some cases, the unit dose of a recombinant virus of this disclosure is 1 x 10 10 ~2×10 10 Between 2 x 10 10 ~3×10 10 Between 3 x 10 10 ~4×10 10 Between 4 x 10 10 ~5×10 10 Between 5×10 10 ~6×10 10 Between 6 x 10 10 ~7×10 10 Between 7 x 10 10 ~8×10 10 Between 8 x 10 10 ~9×10 10 Between 9 x 10 10 ~10×10 10 Between 1×10 11 ~2×10 11 Between 2 x 10 11 ~3×10 11 Between 3 x 10 11 ~4×10 11 Between 4 x 10 11 ~5×10 11 Between 5×10 11 ~6×10 11 Between 6 x 10 11 ~7×10 11 Between 7 x 10 11 ~8×10 11 Between 8 x 10 11 ~9×10 11Between 9 x 10 11 ~10×10 11 Between 1×10 12 ~2×10 12 Between 2 x 10 12 ~3×10 12 Between 3 x 10 12 ~4×10 12 Between 4 x 10 12 ~5×10 12 Between 5×10 12 ~6×10 12 Between 6 x 10 12 ~7×10 12 Between 7 x 10 12 ~8×10 12 Between 8 x 10 12 ~9×10 12 Between 9 x 10 12 ~10×10 12 Between 1×10 13 ~2×10 13 Between 2 x 10 13 ~3×10 13 Between 3 x 10 13 ~4×10 13 Between 4 x 10 13 ~5×10 13 Between 5×10 13 ~6×10 13 Between 6 x 10 13 ~7×10 13 Between 7 x 10 13 ~8×10 13 Between 8 x 10 13 ~9×10 13 Between or 9 x 10 13 ~10×10 13 In some embodiments, the rAAV of this disclosure comprises a vector genome of between about 2.1 x 10 12 In some embodiments, the rAAV of the present disclosure comprises a vector genome of 2E12 to 6E12. In some embodiments, the rAAV of the present disclosure comprises a vector genome of 10 10 ~10 13 Between 10 10 ~10 11 Between 10 11 ~10 12 Between 10 12 ~10 13 Between 1013 ~10 14 Between 2 x 10 11 ~4×10 11 Between 3 x 10 11 ~5×10 11 Between 4 x 10 11 ~6×10 11 Between 5×10 11 ~7×10 11 Between 6 x 10 11 ~8×10 11 Between 7 x 10 11 ~9×10 11 Between 8 x 10 11 ~10×10 11 Between 1×10 12 ~3×10 12 Between 2 x 10 12 ~4×10 12 Between 3 x 10 12 ~5×10 12 Between 4 x 10 12 ~6×10 12 Between 5×10 12 ~7×10 12 Between 6 x 10 12 ~8×10 12 Between 7 x 10 12 ~9×10 12 Between 8 x 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 x 10 12 ~1×10 13 It is a vector genome between.

[0071] In some embodiments, a lower amount or range of vector genome is selected for the unit dose to avoid aggregation. In some embodiments, a higher amount or range of vector genome is selected for the 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 changes 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.

[0072] In some cases, the recombinant viruses 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.The number is 5E12, approximately 90E12, approximately 95E12, or approximately 100E12, where E is an abbreviation for base 10 exponentiation, and xEy refers to x multiplied by the power y of base 10.

[0073] 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 at least 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 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 y of base 10.

[0074] In some embodiments, the unit dose comprises between 2E12 and 6E12 vector genomes, hi 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 ... Contain vector genomes between 0.5E12 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, a 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, a 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.

[0075] In some embodiments, the viral vectors of the present disclosure are measured using multiplicity of infection (MOI). In some cases, MOI refers to the ratio or multiple of vector or viral genome to cells to which nucleic acid can be delivered. In some cases, MOI is 1×10 6 In some cases, the recombinant viruses of the disclosure are 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 x 10 8 ~1×10 15 In some cases, the recombinant viruses of the present disclosure may be at an MOI of at most 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 It can be MOI.

[0076] In some embodiments, nucleic acids can be delivered without the use of viruses (i.e., using non-viral vectors) 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.

[0077] In some embodiments, self-complementary vectors (sc) can be used. As demonstrated 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 the transgene protein.

[0078] In some embodiments, several AAV vectors can be generated to allow for selection of the optimal serotype and promoter for use with the aflibercept transgene.

[0079] 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, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells. Viral vectors for use in the present disclosure may include those that exhibit low toxicity and / or low immunogenicity in a subject and express a therapeutically effective amount of aflibercept protein in a subject, such as a human patient.

[0080] Disclosed herein is a pharmaceutical composition and method for using 7m8 variant capsid protein or rAAV2.7m8 and rAAV that comprises the nucleic acid sequence of encoding aflibercept to deliver the nucleic acid of encoding aflibercept to target retinal cells of non-human primates or human subjects.In some cases, the delivery of aflibercept by gene therapy can be used to at least partially improve or prevent the ocular diseases or conditions disclosed herein.

[0081] 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 parental or unmodified AAV capsid proteins. In some embodiments, the increase in infectivity of retinal cells is between 5% and 100%, between 5% and 95%, between 5% and 90%, between 5% and 85%, between 5% and 80%, between 5% and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5% and 15%, or between 5% and 10%, compared to AAV virions containing the corresponding parent or unmodified AAV capsid proteins.

[0082] In some embodiments, the increase in infectivity of the rAAV variant to retinal cells compared to AAV virions comprising the corresponding parental or unmodified AAV capsid proteins 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. 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 comprising 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 comprising the corresponding parent or unmodified AAV capsid proteins.

[0083] 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 containing the corresponding parental or unmodified AAV capsid proteins.

[0084] 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 containing the corresponding parental or unmodified AAV capsid proteins.

[0085] 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 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 subject's eye. 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% increased 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 between 5% and 100%, between 5% and 95%, between 5% and 90%, between 5% and 85%, between 5% and 80%, between 5% and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5% and 15%, or between 5% and 10% increase compared to the parent or unmodified AAV capsid protein.

[0086] 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.

[0087] 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 parental or unmodified AAV capsid proteins.

[0088] 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 parental or unmodified AAV capsid proteins.

[0089] Aflibercept In some embodiments, gene therapy is used to deliver a therapeutic transgene comprising a nucleic acid sequence that encodes or expresses aflibercept when administered to the eye or vitreous of non-human primates or human subjects.In some embodiments, rAAV comprising capsid variants (such as AAV2.7m8) described herein comprises a heterologous nucleic acid sequence that encodes aflibercept, and is used to deliver the sequence of aflibercept gene to retinal cells during intravitreal or subretinal injection into a subject.In some embodiments, the rAAV comprising aflibercept gene is formulated for gene therapy and intravitreal injection.In some embodiments, the aflibercept gene refers to its functional fragment or variant.In some embodiments, the nucleic acid sequence of aflibercept is derived from its amino acid sequence.In some embodiments, the nucleic acid sequence of aflibercept is codon-optimized to improve its expression in a subject.

[0090] 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, such as 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. See, 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 Codon usage tables are readily available, including "Sequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000).

[0091] Aflibercept is a 115 kDa fusion protein, which may be glycosylated. Aflibercept contains an IgG backbone fused to the extracellular VEGF receptor sequences of human VEGFR-1 and VEGFR-2, and functions like a soluble decoy receptor by binding to VEGF-A with greater affinity than its native or endogenous receptor. See, for example, Stewart MW. Aflibercept (VEGF Trap-eye): the newest anti-VEGF drug. Br. J. Ophthalmol. September 2012; 96 (9): 1157-8. V Aflibercept's high affinity for EGF prevents or disrupts the subsequent binding and activation of native or endogenous VEGF receptors. Reduced VEGF activity can lead to decreased angiogenesis and vascular permeability. Aflibercept's inhibition of placental growth factors PIGF and VEGF-B can also contribute to the treatment of neovascular conditions. PIGF is associated with angiogenesis and can be elevated in several conditions, such as wet AMD. VEGF-B overexpression can be associated with blood-retinal barrier breakdown and retinal neovascularization. Therefore, the inhibition of VEGF-A, VEGF-B, and PIGF can all contribute to the efficacy of aflibercept.

[0092] The gene product disclosed herein consists of aflibercept, a functional fragment or mutant or variant thereof. The amino acid sequence of aflibercept is known in the art: 4318 H 6788 N 1164 O 1304 S 32 The FDA Unique Ingredient Identifier (UNII) is 15C2VL427D. The amino acid sequence of aflibercept is available in DrugBank, accession number DB08885: [ka]

[0093] The nucleic acid sequence of aflibercept (SEQ ID NO: 2) is depicted in Figure 5. In some embodiments, the nucleic acid sequence of aflibercept is codon-optimized for expression in a primate or human subject. Construction of a synthetic gene corresponding to the aflibercept amino acid sequence has been described in the literature, e.g., Kanda A, Noda K, Saito W, Ishida S. Aflibercept Traps Galectin-1, an Angiogenic Factor Associated with Diabetic Retinopathy. Scientific Reports 5:17946 (2015) ("VEGF-Trap"). R1R2(Afribe Aflibercept cDNA corresponding to aflibercept was generated as a synthetic gene by IDT (Coralville, IA) as described in ( ). Given the available amino acid sequence of aflibercept, any method known in the art can be used to generate aflibercept cDNA for use in gene therapy or rAAV as described herein.

[0094] As used herein, "aflibercept" refers to a polypeptide or protein sequence 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 more, or 100% homology with the above-identified aflibercept amino acid sequence, or a functional fragment, variant, or mutant thereof. Homology refers to the percent conservation of residues in the alignment between two sequences, including but not limited to functional fragments, sequences containing insertions, deletions, substitutions, pseudofragments, pseudogenes, splice variants, or artificially optimized sequences.

[0095] In some cases, the amino acid sequence of aflibercept is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% homologous to the aflibercept amino acid sequence of SEQ ID NO:1. 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 aflibercept amino acid sequence identified above and exhibit at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% sequence homology between the aflibercept nucleic acid sequence (e.g., SEQ ID NO: 2). In some cases, aflibercept is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% spatially homologous to aflibercept (e.g., in terms of its secondary, tertiary and quaternary structure or conformation).In some cases, the aflibercept of the pharmaceutical compositions and methods disclosed herein is at most 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% spatially homologous to the aflibercept used in standard treatment (e.g., in terms of its secondary, tertiary and quaternary structure or conformation).

[0096] In some cases, the aflibercept gene product or aflibercept transgene to be included in rAAV-based gene therapy comprises a capsid variant (e.g., a 7m8 variant) disclosed herein and 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 with the amino acid sequence of SEQ ID NO: 1 above or the corresponding cDNA sequence of aflibercept (e.g., the cDNA of the aflibercept sequence used in gene therapy compared to SEQ ID NO: 2). In some embodiments, the methods and pharmaceutical compositions disclosed herein comprise a functional fragment of aflibercept, or a variant or mutant thereof. In some embodiments, the nucleic acid sequence of aflibercept is modified or codon-optimized to enhance its activity, expression, stability and / or solubility in vivo.

[0097] In some embodiments, AAV2.7m8 is used as a gene therapy or delivery system for aflibercept. AAV2.7m8-aflibercept refers to rAAV2 that contains a 7m8 insertion between positions 587 and 588 of the capsid protein VP1 of rAAV2 and a nucleic acid sequence encoding aflibercept.

[0098] 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 aflibercept fusion protein or a fragment or variant thereof, and 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.

[0099] In some embodiments, a pharmaceutical composition comprising rAAV or AAV2.7m8 and a nucleic acid sequence encoding an aflibercept fusion protein, or a fragment or variant thereof, is supplied as a reconstituted homogenous solution. In some embodiments, the solution may be a suspension. In some embodiments, the solution is isotonic. In other embodiments, a pharmaceutical composition comprising rAAV or AAV2.7m8 and a nucleic acid sequence encoding an aflibercept fusion protein, or a fragment or variant thereof, is supplied in lyophilized form and reconstituted prior to administration to a patient. In some embodiments, the methods for treating or preventing ocular diseases or conditions disclosed herein include first reconstituting, dissolving, or solubilizing a lyophilized pharmaceutical composition comprising rAAV (e.g., AAV2.7m8) and a nucleic acid sequence encoding an aflibercept fusion protein, or a functional fragment or variant thereof, in a buffer solution. In some embodiments, such a lyophilized pharmaceutical composition may further comprise a cryoprotectant, a surfactant, a salt, a stabilizer, or any combination thereof. In some embodiments, the homogenous solution containing the pharmaceutical composition is supplied as a pre-filled syringe.

[0100] In some embodiments, the pharmaceutical compositions disclosed herein are supplied as a suspension. In some embodiments, the suspension is a solution. In some embodiments, the suspension is refrigerated. In some embodiments, the methods of treating or preventing ocular diseases or conditions disclosed herein include warming the refrigerated suspension to room temperature and / or stirring the suspension prior to administration to a patient or intravitreal injection to ensure uniform distribution of the suspension. In some embodiments, the suspension is diluted prior to administration to a patient. In some embodiments, such pharmaceutical compositions include a surfactant, a salt, a stabilizer, or any combination thereof. In some embodiments, the suspension containing the pharmaceutical composition is supplied as a pre-filled syringe.

[0101] In some embodiments, the gene therapy or pharmaceutical compositions described herein are provided as suspensions or refrigerated suspensions. In some embodiments, the suspensions include pharmaceutically acceptable excipients, such as surfactants, glycerol, non-ionic surfactants, buffers, glycols, salts, 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 suspensions are at a neutral pH or a pH between 6.5 and 7.5. In some embodiments, the pH of the refrigerated suspensions is slightly basic (e.g., a pH of about 7.5, 8, 8.2, 8.4, 8.5, or 9). In some embodiments, the pH of the suspensions or solutions is slightly acidic (e.g., a pH of about 6.5, 6.3, 6.1, 6, 5.5, or 5). In some embodiments, the suspensions are solutions. In some embodiments, the suspensions include micelles. In some embodiments, the suspensions are stirred and / or warmed to room temperature prior to administration.

[0102] In some embodiments, gene therapy comprising a pharmaceutical composition comprising an rAAV (e.g., AAV2.7m8) and an aflibercept nucleic acid sequence is supplied as a kit containing a lyophilized or freeze-dried pharmaceutical composition disclosed herein (e.g., a single unit dose in a vial) and a solution for dissolving, diluting, and / or reconstituting the lyophilized pharmaceutical composition. In some embodiments, the solution for reconstitution or dilution may be supplied as a pre-filled syringe. In some embodiments, the kit includes 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 includes a buffer that helps prevent aggregation upon reconstitution of the pharmaceutical composition disclosed herein. In some embodiments, the pharmaceutical composition is provided as a pre-filled syringe. In some embodiments, the kit includes a dual-chamber syringe or container, one of the chambers containing a buffer for dissolving or diluting the pharmaceutical composition. 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 reconstituted pharmaceutical composition prior to administration to a patient.

[0103] In some embodiments, for storage stability and ease of handling, pharmaceutical compositions containing rAAV (e.g., AAV2.7m8) and a nucleic acid sequence encoding an aflibercept fusion protein can be formulated as a lyophilized, freeze-dried, or vacuum-dried powder that can be reconstituted with saline, buffer, or water before administration to a subject. Alternatively, the pharmaceutical composition can be formulated as an aqueous solution, such as a suspension or homogeneous solution. The pharmaceutical composition can contain rAAV virions or particles containing a nucleic acid sequence encoding aflibercept. 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 aflibercept. Various excipients, such as phosphate, PBS or Tris buffer, glycol, glycerol, saline, surfactants (e.g., Pluronic or polysorbate), or any combination thereof, can be used to stabilize the pharmaceutical composition. Additionally, cryoprotectants, such as alcohol, can be used as stabilizers under the freezing or drying conditions of lyophilization. In some embodiments, the gene therapy is provided as a suspension or refrigerated suspension.

[0104] In some embodiments, a suspension or reconstituted form of a lyophilized pharmaceutical composition comprising an aflibercept gene therapy disclosed herein has a 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 suspension of a pharmaceutical composition comprising an aflibercept gene therapy disclosed herein has a volume of between 0.1 and 0.5 mL, between 0.1 and 0.2 mL, between 0.3 and 0.5 mL, between 0.5 and 1.0 mL, between 0.5 and 0.7 mL, between 0.6 and 0.8 mL, between 0.8 and 1 mL, between 0.9 and 1.1 mL, between 1.0 and 1.2 mL, or between 1.0 and 1.5 mL. In other embodiments, the volume is no more than 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.

[0105] 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 aflibercept is formulated for intravitreal injection into the eye of a subject. In some embodiments, the pharmaceutical composition is formulated or reconstituted to a concentration that allows for intravitreal injection in a volume of about or less than 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. In some embodiments, a unit dose of the pharmaceutical composition comprises a volume of about or less than 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. In some embodiments, the treatment methods disclosed herein comprise intravitreal injection of a solution comprising an rAAV (e.g., AAV2.7m8) and a nucleic acid sequence encoding aflibercept in 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, or 150 μL.

[0106] In some cases, AAV2.7m8 virions containing the nucleic acid sequence of the aflibercept transgene described herein may be a component of a gene therapy pharmaceutical composition. In some embodiments, rAAV virions of any serotype containing the 7m8 variant capsid protein described herein can be used to prepare freeze-dried or lyophilized pharmaceutical compositions or suspensions thereof. In some embodiments, the gene therapy is formulated as a refrigerated suspension. In some embodiments, the rAAV virions are rAAV2. In some embodiments, the lyophilized pharmaceutical composition or suspension of the pharmaceutical composition contains rAAV2 having a 7m8 variant capsid protein and a DNA sequence encoding aflibercept, a functional fragment or variant thereof. In some embodiments, the suspension is refrigerated.

[0107] In some embodiments, the pharmaceutical compositions disclosed herein are indicated for gene therapy or intravitreal delivery of aflibercept as a therapeutic agent in human patients or non-human primates. In some embodiments, the unit dose of the pharmaceutical composition is 1 x 10 10 ~1×10 13 In some embodiments, the unit dose comprises between about 2.1 x 10 viral genomes (vg). 11 , about 2.1×10 12 or approximately 2.1 x 10 13 In some embodiments, the unit dose of the pharmaceutical composition of the present disclosure comprises 1 x 10 vector genome. 10 ~3×10 12 In some cases, the unit dose of the pharmaceutical composition of the present disclosure is 1 x 10 9 ~3×10 13 In some cases, the unit dose of the pharmaceutical composition of the present disclosure is 1 x 10 10 ~1×10 11 In some cases, the unit dose of the pharmaceutical composition of the present disclosure is 1 x 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 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 or 1×10 18 In some cases, the unit dose of the pharmaceutical composition of the present disclosure is 1 x 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.

[0108] In some cases, a unit dose of a pharmaceutical composition of the present disclosure can be measured in pfu (plaque-forming units). In some cases, the pfu of a unit dose of a pharmaceutical composition of the present disclosure is about 1 x 10 8 ~Approx. 1×10 12 In some cases, the pfu of a unit dose of a 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 , 1×109 , 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 × 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 in a unit dose of a pharmaceutical composition of the present disclosure may be 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 , 8×1010 , 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 It may be pfu.

[0109] 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 1 x 10 10 ~1×10 13 In some cases, the unit dose of the pharmaceutical composition of the present disclosure may be 1 x 10 vector genome. 9 ~1×10 14 In some cases, the unit dose of the pharmaceutical composition of the present disclosure may be 1 x 10 vector genome. 10 ~1×10 11 In some cases, the unit dose of the pharmaceutical composition of the present disclosure may be 1 x 10 vector genome. 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 x 108 ~1×10 15 In 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 20It is a vector genome between.

[0110] In some embodiments, the unit dose of the pharmaceutical composition of the present disclosure is 1 x 10 10 ~2×10 10 Between 2 x 10 10 ~3×10 10 Between 3 x 10 10 ~4×10 10 Between 4 x 10 10 ~5×10 10 Between 5 x 10 10 ~6×10 10 Between 6 x 10 10 ~7×10 10 Between 7 x 10 10 ~8×10 10 Between 8 x 10 10 ~9×10 10 Between 9 x 10 10 ~10×10 10 Between 1×10 11 ~2×10 11 Between 2 x 10 11 ~3×10 11 Between 2 x 10 11 ~2.5×10 11 Between, 2.5 x 10 11 ~3×10 11 Between 3 x 10 11 ~4×10 11 Between 4 x 10 11 ~5×10 11 Between 5 x 10 11 ~6×10 11 Between 6 x 10 11 ~7×10 11 Between 7 x 10 11 ~8×10 11 Between 8 x 10 11 ~9×10 11 Between 9 x 10 11 ~10×10 11 Between 1×10 12 ~2×10 12 Between 2 x 10 12 ~3×10 12 Between, 2.5 x 10 12 ~3×10 12 Between 3 x 10 12 ~4×10 12Between 4 x 10 12 ~5×10 12 Between 5 x 10 12 ~6×10 12 Between 6 x 10 12 ~7×10 12 Between 7 x 10 12 ~8×10 12 Between 8 x 10 12 ~9×10 12 Between 9 x 10 12 ~10×10 12 Between 1×10 13 ~2×10 13 Between 2 x 10 13 ~3×10 13 Between 3 x 10 13 ~4×10 13 Between 4 x 10 13 ~5×10 13 Between 5 x 10 13 ~6×10 13 Between 6 x 10 13 ~7×10 13 Between 7 x 10 13 ~8×10 13 Between 8 x 10 13 ~9×10 13 Between or 9 x 10 13 ~10×10 13 It is a vector genome between.

[0111] In some embodiments, the unit dose of rAAV of this disclosure is 2.1 x 10 11 ~2.1×10 12 In some embodiments, the unit dose of the rAAV of this 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.

[0112] In some embodiments, the unit dose of rAAV of the present disclosure is 1 x 10 10 ~2×1010 Between 2 x 10 10 ~4×10 10 Between 3 x 10 10 ~5×10 10 Between 4 x 10 10 ~6×10 10 Between 5 x 10 10 ~7×10 10 Between 6 x 10 10 ~8×10 10 Between 7 x 10 10 ~9×10 10 Between 8 x 10 10 ~10 11 Between 1×10 11 ~2×10 11 Between 2 x 10 11 ~4×10 11 Between 3 x 10 11 ~5×10 11 Between 4 x 10 11 ~6×10 11 Between 5 x 10 11 ~7×10 11 Between 6 x 10 11 ~8×10 11 Between 7 x 10 11 ~9×10 11 Between 8 x 10 11 ~10×10 11 Between 1×10 12 ~3×10 12 Between 2 x 10 12 ~4×10 12 Between 3 x 10 12 ~5×10 12 Between 4 x 10 12 ~6×10 12 Between 5 x 10 12 ~7×10 12 Between 6 x 10 12 ~8×10 12 Between 7 x 10 12 ~9×10 12 Between 8 x 10 12 ~10×10 12 Between 1×10 13 ~5×10 13 Between 5 x 10 13 ~10×10 13 Between 10 12 ~5×10 12Between 5 x 10 12 ~1×10 13 Between 7 x 10 12 ~1×10 13 Between 8 x 10 12 ~2×10 13 Between 9 x 10 12 ~2×10 13 Between 9 x 10 12 ~2×10 13 Between 9 x 10 12 ~4×10 13 Between 1×10 13 ~3×10 13 Between 1×10 13 ~2×10 13 Between 2 x 10 13 ~3×10 13 Between 3 x 10 13 ~4×10 13 Between 4 x 10 13 ~5×10 13 Between 5 x 10 13 ~6×10 13 Between 6 x 10 13 ~7×10 13 Between 7 x 10 13 ~8×10 13 Between 8 x 10 13 ~9×10 13 Between or 8 x 10 13 ~1×10 14 It is a vector genome between.

[0113] In some embodiments, a lower concentration (e.g., vector genome) is used for the unit dose to prevent aggregation that may occur at higher concentrations.In some embodiments, a higher concentration (e.g., higher vector genome) is selected for the unit dose to increase the efficacy of gene therapy or maximize the delivery of aflibercept transgene in one injection or one-time administration of gene therapy.In some embodiments, the higher concentration of the pharmaceutical compositions disclosed herein allows for a smaller injection volume, which can reduce the adverse effects associated with intravitreal injection, such as high intraocular pressure, inflammation, irritation or pain.

[0114] 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 is the number of times a nucleic acid can be delivered. It can refer to the ratio or multiple of vector or viral genome to cells. In some cases, MOI is 1 x 10 6 In some cases, the MOI can be about 1 x 10 5 ~Approx. 1×10 7 In some cases, the MOI can be between 1 x 10 4 ~1×10 8 In some cases, the recombinant viruses of the present disclosure can 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 viruses of the present disclosure may be at an MOI of about 1 x 10 8 ~Approx. 1×10 15 In some cases, the recombinant viruses 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 x 10 10 ~4×10 10 Between 3 x 10 10 ~5×10 10 Between 4 x 10 10 ~6×10 10 Between 5 x 10 10 ~7×10 10 Between 6 x 10 10 ~8×10 10 Between 7 x 10 10 ~9×10 10 Between 8 x 10 10 ~10 11 Between 1×10 11 ~2×10 11 Between 2 x 10 11 ~4×10 11 Between 3 x 10 11 ~5×10 11 Between 4 x 10 11 ~6×10 11 Between 5 x 10 11 ~7×10 11 Between 6 x 10 11 ~8×10 11 Between 7 x 10 11 ~9×10 11 Between 8 x 10 11 ~10×10 11 Between 1×10 12 ~3×10 12 Between 2 x 10 12 ~4×10 12 Between 3 x 10 12 ~5×10 12 Between 4 x 10 12 ~6×10 12 Between 5 x 10 12 ~7×10 12 Between 6 x 10 12 ~8×10 12 Between 7 x 10 12 ~9×10 12 Between 8 x 10 12 ~10×10 12 Between 1×1013 ~5×10 13 Between 5 x 10 13 ~10×10 13 Between 10 12 ~5×10 12 Between 5 x 10 12 ~1×10 13 Between 7 x 10 12 ~1×10 13 Between 8 x 10 12 ~2×10 13 Between 9 x 10 12 ~2×10 13 Between 9 x 10 12 ~2×10 13 Between 9 x 10 12 ~4×10 13 Between 1×10 13 ~3×10 13 Between 1×10 13 ~2×10 13 Between 2 x 10 13 ~3×10 13 Between 3 x 10 13 ~4×10 13 Between 4 x 10 13 ~5×10 13 Between 5 x 10 13 ~6×10 13 Between 6 x 10 13 ~7×10 13 Between 7 x 10 13 ~8×10 13 Between 8 x 10 13 ~9×10 13 Between or 8 x 10 13 ~1×10 14 It is between.

[0115] 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. Pharmaceutical compositions 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, pharmaceutical compositions can include an isotonic agent, such as a salt or glycerol. In some embodiments, surfactants or stabilizers are added to the pharmaceutical composition to prevent aggregation.

[0116] In some cases, 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, and liquid polyethylene glycol), and any combination thereof. Proper fluidity can be achieved, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using polysorbates (e.g., Tween™, polysorbate 20, polysorbate 80), sodium dodecyl sulfate (sodium lauryl sulfate), lauryl dimethylamine oxide, cetyltrimethylammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol (Triton X100™), N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide (HTAB), polyoxyl 10 lauryl ether, Brij Prevention of the action of microorganisms can be achieved by the use of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, cresol, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, polyalcohols, e.g., malic acid, methylparaben ... It is preferable to include ethanol, sorbitol, and sodium chloride in the composition.The agent that delays absorption, such as aluminum monostearate and gelatin, can be included in the composition to bring about prolonged absorption of the internal composition.In some embodiments, pharmaceutical carriers include sodium phosphate, sodium chloride, polysorbate, and sucrose.In some embodiments, pharmaceutical compositions include surfactants, such as non-ionic surfactants, for example, polysorbate, poloxamer, or pluronic.In some embodiments, the addition of non-ionic surfactants reduces aggregation in pharmaceutical compositions.

[0117] In some embodiments, pharmaceutical compositions useful for the present disclosure can be packaged into kits to facilitate application of the present disclosure. In some aspects, the present method provides a kit containing a recombinant nucleic acid of the present disclosure (e.g., rAAV or rAAV2.7m8 containing the nucleic acid sequence of aflibercept). In some aspects, the present method provides a kit containing a lyophilized form of the recombinant virus of the present disclosure and a solution for reconstituting the virus before administration to a patient. In some embodiments, the kit includes the recombinant virus provided herein and instructions for administering a therapeutically effective amount of the recombinant virus to a subject's eye or retinal cells. In some aspects, the kit includes a pharmaceutically acceptable salt or solution for administering the recombinant virus. Optionally, the kit can further include instructions for suitable operating parameters in the form of a label or a separate insert. For example, the kit can have standard instructions informing a physician or laboratory technician of the information for preparing a unit dose of the recombinant virus and / or reconstituting the lyophilized composition. In some embodiments, the kit optionally further comprises a device for administration, such as a syringe, filter needle, extension tubing, cannula, or subretinal syringe.

[0118] In some embodiments, the pharmaceutical composition is provided as a suspension or a refrigerated suspension. In some embodiments, the suspension or refrigerated suspension is provided in a kit, which may include a syringe or buffer for dilution. In some embodiments, the suspension or refrigerated suspension is provided as a pre-filled syringe.

[0119] In some embodiments, any suitable method can be used for the biochemical purification of recombinant virus (e.g., rAAV) for use in the pharmaceutical compositions described herein.Recombinant AAV virus can be directly recovered from cells or from the culture medium containing cells.Before freeze-drying or before formulation as suspension, virus can be purified using various biochemical means, such as gel filtration, filtration, chromatography, affinity purification, gradient ultracentrifugation or size exclusion method.

[0120] Indications In some cases, rAAV virions of any serotype, including a 7m8 variant (e.g., rAAV2.7m8) as described herein, or pharmaceutical compositions thereof, can at least partially ameliorate an ocular condition or disease associated with ocular neovascularization or associated with CNV. In some embodiments, rAAV virions comprising capsid variant proteins are used to deliver aflibercept, a functional fragment or variant thereof, to the eye of a human subject.

[0121] Approved indications for aflibercept fusion proteins include neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), 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 aflibercept is 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 aflibercept, including, but not limited to, CNV, wet AMD, dry AMD, DME, RVO, macular edema after RVO, 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 AMD, DME, RVO, angiogenesis-related diseases, cancer, autoimmune diseases, infectious disease organisms, etc.

[0122] In some embodiments, the ocular condition may be diabetic macular edema. Diabetic macular edema (DME) is 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 small area rich in cones, specialized nerve endings that detect color; daytime vision relies on cones. As macular edema develops, blurring occurs in the center or just to the side of central vision. Blindness due to diabetic macular edema can progress over a period of several months, making it impossible to focus clearly. Common symptoms of DME are blurred vision, floaters, and double vision, which, if left untreated, eventually lead to blindness. In some embodiments, the methods and pharmaceutical compositions disclosed herein are used to treat DME.

[0123] 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 eye that converts light images into nerve signals and sends them to the brain. Retinal vein occlusion is most often caused by hardening of the arteries (atherosclerosis) and the formation of blood clots. Blockage of smaller veins in the retina (branch veins or BRVOs) often occurs where retinal arteries thickened or hardened by atherosclerosis cross and put pressure on the retinal veins. Symptoms of retinal vein occlusion 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.

[0124] 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 can be a major cause of blindness. CNV can cause a sudden worsening of central vision, noticeable within a few weeks. Other symptoms may include impaired color vision and metamorphopsia (distortion of straight lines that appear wavy). Bleeding of new blood vessels can accelerate the onset of CNV symptoms. 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.

[0125] 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 of plasma and / or bleeding into the subretinal space, so if it occurs in the macula, there may be severe sudden loss of central vision. The term "AMD" can refer to 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.

[0126] In some embodiments, the methods and pharmaceutical compositions disclosed herein are used to prevent or treat ocular diseases or conditions that are responsive to aflibercept in vivo.

[0127] In some embodiments, the methods and pharmaceutical compositions disclosed herein, i.e., AAV gene therapy comprising aflibercept, a functional fragment or variant thereof, 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 color fundus photography as a percentage of grade IV lesions following CNV formation.

[0128] In some embodiments, the methods and pharmaceutical compositions disclosed herein, i.e., AAV gene therapy comprising aflibercept, its functional fragments, or variants, result in a reduction in neovascularization or CNV equivalent to that of aflibercept or aflibercept not based on gene therapy, as measured by color fundus photography of the percentage of grade IV lesions after CNV formation. In some embodiments, the reduction in CNV or therapeutic effect persists longer with the administration of aflibercept-containing gene therapy compared to the administration of aflibercept or aflibercept protein solution not based on gene therapy. In some embodiments, the therapeutic effect of aflibercept gene therapy persists for at least 1 year, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 years, or longer, after a single intravitreal injection. In some embodiments, the pharmaceutical compositions disclosed herein inhibit or sequester endogenous VEGF and / or PIGF.

[0129] How to use In some embodiments, the present disclosure provides a method for treating a pathological angiogenesis-associated ocular disease, comprising administering a pharmaceutically effective amount of a pharmaceutical composition provided herein to a human subject in need of such treatment. 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.

[0130] In some embodiments, a pharmaceutical composition comprising an rAAV (e.g., rAAV.7m8) comprising a variant capsid protein and a nucleic acid sequence encoding aflibercept is used to treat or prevent AMD, including dry AMD and wet AMD. In some embodiments, a pharmaceutical composition comprising an rAAV (e.g., rAAV.7m8) comprising a variant capsid protein and a nucleic acid sequence encoding aflibercept 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 aflibercept 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.

[0131] In some embodiments, the method for treating AMD, DME, RVO or DR comprises pretreating the patient with EYLEA® before administering to the patient a gene therapy comprising aflibercept nucleic acid sequence.In some embodiments, the patient is pretreated with EYLEA® before receiving a one-time dose of the aflibercept gene therapy disclosed herein.In some embodiments, the patient is responsive to aflibercept before receiving a one-time dose of the aflibercept gene therapy disclosed herein. In some embodiments, patients who are aflibercept-responsive or have been pre-treated with aflibercept are treated with an aflibercept gene therapy disclosed herein, followed by at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years, or more than 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years, during which the patient does not receive either EYLEA® or aflibercept gene therapy. In some cases, after a patient receives an intravitreal injection of aflibercept gene therapy, the patient does not begin receiving injections of EYLEA® or another standard of care, or another approved therapy (e.g., ranibizumab or bevacizumab injections), until at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years have passed since the gene therapy treatment. In some embodiments, the treatment method includes assessing or evaluating the patient for responsiveness to aflibercept (e.g., by immunoassay or blood test) before administering aflibercept gene therapy to the patient.

[0132] In some embodiments, the aflibercept gene therapy disclosed herein is a one-time administration.In some embodiments, after patient receives a unit dose of the aflibercept gene therapy disclosed herein, patient does not need to use any other aflibercept-based therapy.

[0133] In some cases, patients who experience adverse effects associated with repeated intravitreal injections of EYLEA® or other approved therapies, such as inflammation, high intraocular pressure, or bacterial infection, may be candidates for treatment with the aflibercept gene therapy disclosed herein.In some cases, such risks are lower in gene therapy, because gene therapy only requires one injection in the patient's life, or is administered no more than once in at least 2, 5, 10, 20, 30, 40, 50 years or more.In some cases, the therapeutic effect of gene therapy can last longer, and the cost of a one-time gene therapy injection can be lower than the combined cost of multiple repeated protein injections, so treatment with the aflibercept gene therapy disclosed herein can be more cost-effective than protein-based injections.

[0134] In addition, because non-compliance (for example, when a patient forgets or misses one or more scheduled injections) can lead to vision loss and the worsening of eye diseases or conditions, gene therapy addresses the patient compliance and adherence issues associated with treatments that require repeated injections by not requiring repeated injections.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.Therefore, for example, gene therapy delivery of aflibercept to patient eyes as a one-time intravitreal injection can provide patients with more convenient treatment options and improve patient outcomes by addressing the problem of non-compliance and non-adherence.

[0135] In some embodiments, the method of use comprises: pretreating a human patient or subject with a drug that is currently considered standard treatment, such as aflibercept protein injection, ranibizumab injection or bevacizumab injection; determining the patient's responsiveness to aflibercept; and administering the aflibercept gene therapy described herein to the patient who is responsive to aflibercept.Determining the patient's responsiveness to aflibercept can include, but is not limited to, blood tests, immunoassays, ex vivo experiments, or administering aflibercept protein injection to the patient and testing the patient for responsiveness to aflibercept.

[0136] In some embodiments, the method of using the aflibercept gene therapy described herein comprises reconstituting a lyophilized form of the pharmaceutical composition described herein (i.e., rAAV2.7m8 comprising an aflibercept nucleic acid sequence) according to the drug label and administering the reconstituted aflibercept gene therapy to a subject or human patient. In some embodiments, the aflibercept gene therapy is provided as a suspension. In some embodiments, the suspension is stirred before administration. In some embodiments, the suspension is refrigerated. In some embodiments, the refrigerated suspension is warmed to room temperature before administration. In some embodiments, the human patient has been pretreated with an aflibercept injection or another protein drug injection, such as a ranibizumab injection or a bevacizumab injection. In some embodiments, such patients receive no more than one injection or administration of rAAV2.7m8-aflibercept gene therapy for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years; or no more than one injection or administration of rAAV2.7m8-aflibercept gene therapy for more than 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.

[0137] In some embodiments, the present disclosure also provides a method for preventing or treating an ocular condition or disease, comprising administering an effective amount of an rAAV virion comprising a nucleic acid sequence encoding aflibercept as described herein, or a pharmaceutical composition thereof, to an individual in need thereof, for example, an individual with an ocular condition or disease responsive to aflibercept. In some embodiments, the rAAV2.7m8-aflibercept virion can be administered to the eye of an individual via intraocular injection, intravitreal injection, subretinal injection, or any other convenient administration mode or route. Other convenient administration modes or routes can include, for example, intravenous, topical, eye drops, etc. In some embodiments, the methods and pharmaceutical compositions disclosed herein include administration via intravitreal injection.

[0138] In some embodiments, the gene therapy or pharmaceutical compositions described herein are provided as refrigerated suspensions. In some embodiments, the refrigerated suspensions contain pharmaceutically acceptable excipients, such as surfactants, glycerol, non-ionic surfactants, buffers, glycols, salts, 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 suspensions are neutral pH or between 6.5 and 7.5. In some embodiments, the pH of the suspensions is slightly basic (e.g., a pH of about 7.5, 8, 8.2, 8.4, 8.5, or 9). In some embodiments, the pH of the suspensions or solutions is slightly acidic (e.g., a pH of about 6.5, 6.3, 6.1, 6, 5.5, or 5). In some embodiments, the suspensions are solutions. In some embodiments, the refrigerated suspensions contain micelles. In some embodiments, the refrigerated suspensions are stirred prior to administration. In some embodiments, the refrigerated suspension is stored at a temperature between 35°F and 46°F (2°C and 8°C). In some embodiments, the refrigerated suspension is warmed to room temperature prior to administration to a patient.

[0139] The "therapeutically effective amount" described herein may be a relatively broad range that can be determined through clinical trials. For direct injection into the eye or intravitreal injection, a therapeutically effective dose is 10 times that of 7m8-aflibercept. 11 ~10 12 or 10 12 ~10 13 In some embodiments, the unit dose or therapeutically effective amount of 7m8-aflibercept may be about 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 In some embodiments, the unit dose of a pharmaceutical composition comprising 7m8-aflibercept of the present disclosure is between 1 x 10 10 ~2×10 10 Between 2 x 10 10 ~3×10 10 Between 3 x 10 10 ~4×10 10 Between 4 x 10 10 ~5×10 10Between 5 x 10 10 ~6×10 10 Between 6 x 10 10 ~7×10 10 Between 7 x 10 10 ~8×10 10 Between 8 x 10 10 ~9×10 10 Between 9 x 10 10 ~10×10 10 Between 1×10 11 ~2×10 11 Between 2 x 10 11 ~3×10 11 Between 2 x 10 11 ~2.5×10 11 Between, 2.5 x 10 11 ~3×10 11 Between 3 x 10 11 ~4×10 11 Between 4 x 10 11 ~5×10 11 Between 5 x 10 11 ~6×10 11 Between 6 x 10 11 ~7×10 11 Between 7 x 10 11 ~8×10 11 Between 8 x 10 11 ~9×10 11 Between 9 x 10 11 ~10×10 11 Between 1×10 12 ~2×10 12 Between 2 x 10 12 ~3×10 12 Between, 2.5 x 10 12 ~3×10 12 Between 3 x 10 12 ~4×10 12 Between 4 x 10 12 ~5×10 12 Between 5 x 10 12 ~6×10 12 Between 6 x 10 12 ~7×10 12 Between 7 x 10 12 ~8×10 12 Between 8 x 10 12 ~9×10 12 Between 9 x 10 12 ~10×10 12Between 1×10 13 ~2×10 13 Between 2 x 10 13 ~3×10 13 Between 3 x 10 13 ~4×10 13 Between 4 x 10 13 ~5×10 13 Between 5 x 10 13 ~6×10 13 Between 6 x 10 13 ~7×10 13 Between 7 x 10 13 ~8×10 13 Between 8 x 10 13 ~9×10 13 Between or 9 x 10 13 ~10×10 13 In some embodiments, the unit dose of 7m8-aflibercept of the present disclosure is between 2.1 x 10 11 or 2.1 x 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.

[0140] In some embodiments, the unit dose of 7m8-aflibercept of the present disclosure is 1×10 10 ~2×10 10 Between 2 x 10 10 ~4×10 10 Between 3 x 10 10 ~5×10 10 Between 4 x 10 10 ~6×10 10 Between 5 x 10 10 ~7×10 10 Between 6 x 10 10 ~8×10 10 Between 7 x 10 10 ~9×10 10 Between 8 x 1010 ~10 11 Between 1×10 11 ~2×10 11 Between 2 x 10 11 ~4×10 11 Between 3 x 10 11 ~5×10 11 Between 4 x 10 11 ~6×10 11 Between 5 x 10 11 ~7×10 11 Between 6 x 10 11 ~8×10 11 Between 7 x 10 11 ~9×10 11 Between 8 x 10 11 ~10×10 11 Between 1×10 12 ~3×10 12 Between 2 x 10 12 ~4×10 12 Between 3 x 10 12 ~5×10 12 Between 4 x 10 12 ~6×10 12 Between 5 x 10 12 ~7×10 12 Between 6 x 10 12 ~8×10 12 Between 7 x 10 12 ~9×10 12 Between 8 x 10 12 ~10×10 12 Between 1×10 13 ~5×10 13 Between 5 x 10 13 ~10×10 13 Between 10 12 ~5×10 12 Between 5 x 10 12 ~1×10 13 Between 7 x 10 12 ~1×10 13 Between 8 x 10 12 ~2×10 13 Between 9 x 10 12 ~2×10 13 Between 9 x 10 12 ~2×10 13 Between 9 x 10 12 ~4×10 13 Between 1×10 13~3×10 13 Between 1×10 13 ~2×10 13 Between 2 x 10 13 ~3×10 13 Between 3 x 10 13 ~4×10 13 Between 4 x 10 13 ~5×10 13 Between 5 x 10 13 ~6×10 13 Between 6 x 10 13 ~7×10 13 Between 7 x 10 13 ~8×10 13 Between 8 x 10 13 ~9×10 13 Between or 8 x 10 13 ~1×10 14 It is a vector genome between.

[0141] In some embodiments, a therapeutically effective amount of a pharmaceutical composition disclosed herein comprises between 2E12 and 6E12 vector genomes, hi 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 ... Contain vector genomes between 0.5E12 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, a 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, a 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.

[0142] In some embodiments, the total amount of 7m8-aflibercept injected into a human patient or subject within a period of 2 to 5 years or 5 to 10 years is 10 10 ~10 13 , 10 10 ~10 11 , 10 11 ~10 12 , 1012 ~10 13 Or 10 13 ~10 14 or 1 x 10 vector genomes 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 ~10×1013 , 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 x 10 13 ~1×10 14 The vector genome is as follows:

[0143] In some embodiments, the rAAV.7m8-aflibercept virion 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 desired gene expression levels over a variable duration, for example, at least 2 years, or at least 3, 4, 5, 6, 7, 8, 9, 10 or more years. In some embodiments, intravitreal injection of 7m8-aflibercept eliminates the need for patients to receive aflibercept protein injections for at least 1 year, 1.5 years, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more years. [Example]

[0144] Example 1: Efficacy evaluation of rAAV2.7m8-aflibercept in monkeys

[0145] Delivery of therapeutic transgenes (or payloads) to target cells or tissues via gene therapy is hypothesized to be highly dependent on AAV capsid proteins and their role in targeting the AAV virus to relevant or target cells in primate or human subjects. It has also been reported that the 7m8 variant exhibits increased infectivity or targeting of retinal cells when injected intravitreally. Therefore, it would be expected that the 7m8 variant would perform similarly in targeting various therapeutic transgenes to retinal cells when injected intravitreally.

[0146] Objective: To inhibit the development of choroidal neovascularization (CNV) induced by laser photocoagulation in African green monkeys. 12 To evaluate the efficacy of rAAV2.7m8 containing a nucleic acid sequence encoding aflibercept along with the efficacy of rAAV2.7m8 containing a nucleic acid sequence encoding sVEGFR-1 following intravitreal (IVT) administration of each gene therapy at a dose of 1000 mg / kg. The nucleic acid sequence for sVEGFR-1 (also known as sFLT-1) has been published, for example, as described in U.S. Patent Application Publication No. 2014 / 0371438.

[0147] Figure 5 illustrates the nucleic acid sequence of aflibercept. Figure 6 illustrates the nucleic acid sequence of sFLT-1 (SEQ ID NO: 3).

[0148] 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 neovascularization-related eye diseases, such as wet AMD.

[0149] 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). Monkeys with normal findings were enrolled in the study and randomized into three treatment groups according to baseline weight and sex (Table 1). After the baseline examination, atropine 1% ophthalmic ointment was applied.

[0150] As used herein, rAAV2.7m8-sVEGFR-1 comprises rAAV2 containing a 7m8 insertion between positions 587 and 588 of the capsid protein VP1 of rAAV2 and a nucleic acid sequence encoding sVEGFR-1. [Table 1]

[0151] On day 0 of the study, monkeys in groups 1-3 received intravitreal (IVT) AAV2.7m8-aflibercept, vehicle, or AAV2.7m8-sVEGFR-1 OU according to the treatment schedule (Table 1). Prior to IVT administration, topical local anesthesia was administered (0.5% proparacaine), and the eyes were disinfected with 5% Betadine and rinsed with sterile normal saline. IVT injections were 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.

[0152] On day 56 of the study, monkeys in Group 4 received IVT aflibercept (EYLEA®, 30 μL of 40 mg / mL per eye) immediately after laser treatment, following an IVT injection procedure identical to that of Groups 1-3, except for a reduced injection volume (30 μL). IVT administration of aflibercept is the clinical standard of care for CNV and is therefore used as a positive control in this study. The dose was adjusted to accommodate the relatively smaller vitreous volume of African green monkeys (2.7 mL) compared to humans (4 mL).

[0153] All IVT injections were followed by topical administration of 0.3% ciprofloxacin or equivalent antibiotic ophthalmic solution and 1% atropine sulfate ointment.

[0154] On day 56, CNV was induced between the temporal vascular arcades by laser burn. Nine laser spots were placed symmetrically in each eye by an ophthalmologist using an Iridex Oculight TX 532 nm laser with a 100 ms laser duration, a 50 μm spot size, and a power output of 750 mW. Laser spots were applied using a 0.9× contact laser lens. A trained ophthalmologist mapped the target locations of the laser spots onto color fundus images obtained before laser treatment (and after bleb placement) for reference during laser spot placement. Color fundus photography was performed immediately after laser treatment to document the laser lesions. Any spots showing severe retinal / subretinal hemorrhage immediately after laser treatment were excluded from analysis. Figure 1 illustrates exemplary fundus photographs of a non-human primate eye after induction of CNV lesions by laser irradiation without treatment.

[0155] Bilateral color fundus images of the retina were captured using a Topcon TRC-50EX retinal camera with Canon 6D digital imaging hardware and New Vision Fundus Image Analysis System software, with a 50-degree field of view centered on the fovea. FA was performed with intravenous administration. Fluorescein leakage in the angiograms of CNV lesions was graded from I to IV by a masked ophthalmologist evaluating the composite generated after uniform adjustment of image intensity (Table 2). The lesion grading assessment was confirmed on fundus images by two other trained ophthalmologists. Image fluorescence densitometry analysis of the raw angiograms in the late phase can also be performed using ImageJ software. [Table 2]

[0156] 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.

[0157] On day 85, or slightly later if imaging was pending, all animals were euthanized with pentobarbital after confirming the quality of fundus imaging. The animals were then euthanized with pentobarbital and the eyes enucleated. Excess orbital tissue was trimmed, and both OD and OS eyes were flash-frozen in liquid nitrogen. Dissection along the frozen tissue plane at room temperature was then performed to isolate the vitreous and retina along with the choroidal subtissue. After vitreous collection, 5 mm punches of neural retina with RPE / choroid were taken from the macula and superior, inferior, temporal, and nasal regions. The retina with underlying RPE / choroidal tissue from each punch was transferred to pre-tared, labeled cryotubes, weighed, and flash-frozen in liquid nitrogen. Before and after punch biopsy collection, photographs of the flat-mounted retina were taken to document the area from which the punch was collected, along with an indication of orientation.

[0158] 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. Data were not normally distributed and unevenly distributed. If the data had homogeneous variances, non-parametric tests were applied. A P value of 0.05 or less was considered statistically significant.

[0159] CNV lesions were induced in each group of test subjects (monkeys) by laser irradiation immediately after injection, and color fundus photography was used to grade each lesion on a scale of I to IV. Figure 2 shows the effect of AAV2.7m8-aflibercept or vehicle control on CNV lesions of 2.1 x 10 12Figure 3 illustrates exemplary fundus photography used to assess monkey lesions at day 70 after intravitreal treatment with a dose of 0.05 mg / kg. Monkeys treated with AAV2.7m8-aflibercept exhibited grade II and III lesions but no grade IV lesions, while monkeys treated with vehicle control exhibited more grade IV lesions. Similar data from multiple monkeys were pooled and plotted for quantitative analysis of each study group, as further described below (Figures 3 and 4).

[0160] Figure 3 illustrates plots of the percentage of grade IV lesions on days 14 and 28 for monkeys in Group 4 that received intravitreal injections of either EYLEA® (or aflibercept fusion protein without a gene therapy approach) as a positive control, or a vehicle control containing formulation buffer alone. Following the same IVT injection procedure used in Groups 1-3 (Table 1), except for a reduced injection volume (30 μL) to accommodate the relatively smaller vitreous volume of African green monkeys (2.7 mL) compared to humans (4 mL), 30 μL of 40 mg / mL / eye of EYLEA® was administered immediately after laser irradiation. IVT administration of EYLEA® is the clinical standard of care for CNV and is therefore used as the positive control in this study. Data from multiple monkeys was averaged and plotted for quantitative analysis. Animals treated with intravitreal injection of EYLEA® showed a significant reduction in the amount of Grade IV lesions compared to administration of vehicle alone via intravitreal injection, as measured by fundus images collected 14 and 28 days after injection.

[0161] Figure 4 shows the results of 2.1 x 10 HIV-1 immunizations with either rAAV2.7m8-aflibercept (gene therapy comprising rAAV2 with the 7m8 variant capsid protein and an aflibercept nucleic acid sequence); or AAV2.7m8-sVEGFR-1 (gene therapy comprising rAAV2 with the 7m8 variant capsid protein and an sVEGFR-1 nucleic acid sequence). 12Figure 1 illustrates plots of the percentage of grade IV lesions on days 14 and 28 for monkeys (groups 1-3 in Table 1) intravitreally injected with rAAV2.7m8-aflibercept at doses of 100 mg / kg or with a vehicle control containing formulation buffer alone. CNV lesions were induced by laser irradiation, and color fundus photography was used to grade each lesion on a scale of I to IV. Measurements of the percentage of grade IV lesions were then pooled and plotted. Monkeys treated with intravitreal injections of rAAV2.7m8-aflibercept showed a significant reduction in the amount of grade IV lesions compared with vehicle control alone, based on fundus images collected on days 14 and 28. In contrast, and unexpectedly, monkeys treated with intravitreal injections of rAAV2.7m8-sVEGFR-1 showed little or no reduction in grade IV CNV lesions compared with vehicle control. This data suggests that, contrary to what many previously thought, capsid variation and / or administration route may not work for all transgenes, and that transgene characteristics may play an important role in the efficacy of AAV gene therapy. Such in vivo data in monkeys also showed that treatment of primates with rAAV2.7m8-aflibercept led to fewer grade IV lesions compared with monkeys receiving vehicle control alone, suggesting that rAAV2.7m8-aflibercept may be a viable gene therapy option for humans.

[0162] These in vivo studies of rAAV2.7m8-aflibercept demonstrated that rAAV2 containing the 7m8 variant capsid protein can be effective as a gene therapy for delivering a nucleic acid sequence encoding aflibercept to retinal cells of a subject by intravitreal injection, and that it resulted in in vivo expression of active aflibercept such that it exerted a therapeutic effect, i.e., reduction of CNV lesions, at a level similar to that of the EYLEA® positive control.

Claims

[Claim 1] A composition as described in the specification.

Images