Adeno-associated virus vector pharmaceutical compositions and methods

Abstract

Provided herein are pharmaceutical compositions comprising a recombinant adeno-associated virus (AAV), a salt excipient or buffer, a sugar, and a surfactant. Also provided herein are methods for treating or preventing a disease in a subject by administering to the subject in need thereof a therapeutically effective amount of the pharmaceutical composition.

Description

[0001] This application is a divisional application. The original application was filed on October 6, 2020, with application number 202080079668.3 (international application number PCT / US2020 / 054400), entitled "Adeno-associated virus vector pharmaceutical composition and method".

[0002] Cross-referencing related applications This application claims the benefit of U.S. Provisional Application No. 62 / 911,968, filed on October 7, 2019, the contents of which are incorporated herein by reference in their entirety.

[0003] References to sequence lists submitted electronically This application incorporates by reference a sequence list entitled "Sequence_Listing_12656-124-228.TXT", which was created on September 28, 2020 and is 97,652 bytes in size, and was filed together with this application as a text file. Background Technology

[0004] As a member of the parvoviridae family designated as a virus-dependent virus, adeno-associated virus (AAV) is a small, non-enveloped icosahedral virus with a single-stranded linear DNA genome ranging from approximately 4.7 kilobases (kb) to 6 kb. Its non-pathogenicity, broad host and cell-type tropism, including both dividing and non-dividing cells, and the ability to establish long-term transgenic expression make AAV an attractive tool for gene therapy (e.g., Gonçalves, 2005, *Virology Journal*, 2:43).

[0005] AAV products are typically stored in buffer solutions composed of various excipients to stabilize them during manufacturing, transport, storage, and administration. However, AAV biotherapeutic agents are distributed at -80°C to prevent degradation and the negative effects of potential material thawing, even though adequate refrigeration may not be possible at these temperatures when transported to certain regions. Maintaining cryogenic temperatures at ≤ -60°C is challenging, and from a logistical perspective, there is a need for a formulation that is robust to higher freezing temperatures (e.g., up to -20°C) and stable against repeated freeze-thaw shifts. Not all clinical sites have -80°C cryogenic facilities, and this requirement negatively impacts the ability to distribute products to a wide range of clinical locations. Therefore, there is a need for a formulation that is stable under refrigerated conditions for a short period (up to 12 months) to allow clinical sites to thaw and store the product in cryogenic facilities until patients are scheduled for dosing.

[0006] Maintaining various buffer properties within target specifications to ensure product stability is crucial, but storage at -80°C can impact the supply chain and distribution. Crystallization of water during slow freezing can lead to excipient concentration, potentially affecting the stability of biologics. Phase separation or pH changes may also occur, which can affect the stability of biologics. For the commercialization of any pharmaceutical product, identifying formulations that provide stability over extended periods would be advantageous. Further advantageous would be the identification of formulations stable under frozen storage at -20°C, taking into account freezing temperature deviations, variability, or temporary storage (up to 18 months) in a -20°C freezer, refrigerated conditions to allow short-term clinic storage prior to administration (up to 12 months at 2–8°C), room temperature to allow manufacturing and labeling, or under multiple free-thaw cycles to allow thawing of pharmaceutical substances and products for filling and labeling operations. Summary of the Invention

[0007] This disclosure provides a pharmaceutical composition comprising recombinant adeno-associated virus (AAV), a buffer, an ionic salt, sucrose, and a surfactant such as poloxamer 188. The sucrose is provided to prevent crystallization of the composition and to maintain a pH concentration between 6 and 9 during freezing and liquidization.

[0008] In some embodiments, the recombinant AAV comprises components from one or more adeno-associated virus serotypes selected from the group consisting of: AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7m8, AA V.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16. In some embodiments, the rAAV comprises the capsid protein of serotype AAV8 or AAV9.

[0009] In some embodiments, the pharmaceutical composition further comprises amino acids.

[0010] In some embodiments, this disclosure provides a pharmaceutical composition comprising recombinant adeno-associated virus (AAV), an ionic salt excipient or buffer, sucrose, and poloxamer 188. In some embodiments, the ionic salt excipient or buffer may be one or more components derived from the group consisting of: potassium dihydrogen phosphate, potassium phosphate, sodium chloride, anhydrous disodium hydrogen phosphate, sodium phosphate hexahydrate, sodium dihydrogen phosphate monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), amino acids, histidine, histidine hydrochloride (histidine-HCl), sodium succinate, sodium citrate, sodium acetate, and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), sodium sulfate, magnesium sulfate, magnesium chloride hexahydrate, calcium sulfate, potassium chloride, calcium chloride, and calcium citrate.

[0011] In some embodiments, the ionic strength of the pharmaceutical composition is not greater than about 150 mM, about 145 mM, about 140 mM, about 135 mM, about 130 mM, about 125 mM, about 120 mM, about 115 mM, or about 110 mM. In some embodiments, the buffer ionic strength of the pharmaceutical composition is not greater than about 150 mM, about 145 mM, about 140 mM, about 135 mM, about 130 mM, about 125 mM, about 120 mM, about 115 mM, or about 110 mM.

[0012] In some embodiments, the ionic strength of the pharmaceutical composition is not greater than 150 mM, 145 mM, 140 mM, 135 mM, 130 mM, 125 mM, 120 mM, 115 mM, or 110 mM. In some embodiments, the buffer ionic strength of the pharmaceutical composition is not greater than 150 mM, 145 mM, 140 mM, 135 mM, 130 mM, 125 mM, 120 mM, 115 mM, or 110 mM.

[0013] In some embodiments, the ionic strength of the pharmaceutical composition is from about 60 mM to 115 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 60 mM to 100 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 60 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 65 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 70 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 75 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 80 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 85 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 90 mM.

[0014] In some embodiments, the ionic strength of the pharmaceutical composition is from about 30 mM to 100 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 30 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 35 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 40 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 45 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 50 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 55 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 60 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 65 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 70 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 75 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 80 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 85 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 90 mM. In a specific embodiment, the ionic strength of the pharmaceutical composition is about 95 mM. In another specific embodiment, the ionic strength of the pharmaceutical composition is about 100 mM.

[0015] In some embodiments, the ionic strength of the pharmaceutical composition is from about 60 mM to 115 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 65 mM to 95 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 70 mM to 90 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 75 mM to 85 mM.

[0016] In some embodiments, the ionic strength of the pharmaceutical composition is from about 30 mM to 100 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 35 mM to 95 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 40 mM to 90 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 45 mM to 85 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 50 mM to 80 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 55 mM to 75 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 60 mM to 70 mM.

[0017] In some embodiments, the pharmaceutical composition comprises potassium chloride at a concentration of 0.2 g / L.

[0018] In some embodiments, the pharmaceutical composition comprises potassium dihydrogen phosphate at a concentration of 0.2 g / L.

[0019] In some embodiments, the pharmaceutical composition comprises sodium chloride at a concentration of 5.84 g / L.

[0020] In some embodiments, the pharmaceutical composition comprises anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L.

[0021] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 3% (w / v, 30 g / L) to 18% (w / v, 180 g / L). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 4% (w / v, 30 g / L) to 6% (w / v, 180 g / L). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 3% (w / v, 30 g / L). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 3% (w / v, 30 g / L). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 4% (w / v, 40 g / L). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 5% (w / v, 50 g / L). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 6% (w / v, 60 g / L). In some embodiments, the pharmaceutical composition comprises 7% (w / v, 70 g / L) sucrose. In some embodiments, the pharmaceutical composition comprises 8% (w / v, 80 g / L) sucrose. In some embodiments, the pharmaceutical composition comprises 9% (w / v, 90 g / L) sucrose. In some embodiments, the pharmaceutical composition comprises 10% (w / v, 100 g / L) sucrose. In some embodiments, the pharmaceutical composition comprises 11% (w / v, 110 g / L) sucrose. In some embodiments, the pharmaceutical composition comprises 12% (w / v, 120 g / L) sucrose. In some embodiments, the pharmaceutical composition comprises 13% (w / v, 130 g / L) sucrose. In some embodiments, the pharmaceutical composition comprises 14% (w / v, 140 g / L) sucrose. In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 15% (weight / volume, 150 g / L). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 16% (weight / volume, 160 g / L). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 17% (weight / volume, 170 g / L). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 18% (weight / volume, 180 g / L).

[0022] In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.001% (weight / volume, 0.01 g / L).

[0023] In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0005% (w / v, 0.005 g / L) to 0.05% (w / v, 0.5 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0001% (w / v, 0.001 g / L) to 0.01% (w / v, 0.1 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0005% (w / v, 0.005 g / L) to 0.001% (w / v, 0.01 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.001% (w / v, 0.01 g / L) to 0.05% (w / v, 0.5 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0005% (w / v, 0.005 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0006% (w / v, 0.006 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0007% (w / v, 0.007 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0008% (w / v, 0.008 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0009% (w / v, 0.009 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.001% (w / v, 0.01 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.002% (w / v, 0.02 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.003% (w / v, 0.03 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.004% (w / v, 0.04 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.005% (w / v, 0.05 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.01% (w / v, 0.1 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.05% (w / v, 0.5 g / L).

[0024] In some embodiments, this disclosure provides a pharmaceutical composition comprising recombinant adeno-associated virus (AAV), an ionic salt excipient or buffer, sucrose, and a surfactant. In some embodiments, the ionic salt excipient or buffer may be one or more components from the group consisting of: potassium dihydrogen phosphate, potassium phosphate, sodium chloride, anhydrous disodium hydrogen phosphate, sodium phosphate hexahydrate, sodium dihydrogen phosphate monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), amino acids, histidine, histidine hydrochloride (histidine-HCl), sodium succinate, sodium citrate, sodium acetate, and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), sodium sulfate, magnesium sulfate, magnesium chloride hexahydrate, calcium sulfate, potassium chloride, calcium chloride, and calcium citrate. In some embodiments, the surfactant may be one or more components from the group consisting of poloxamer 188, polysorbate 20, and polysorbate 80.

[0025] In some embodiments, the pharmaceutical composition comprises polysorbate 20 at a concentration of 0.0005% (w / v, 0.005 g / L) to 0.05% (w / v, 0.5 g / L). In some embodiments, the pharmaceutical composition comprises polysorbate 20 at a concentration of 0.0001% (w / v, 0.001 g / L) to 0.01% (w / v, 0.1 g / L). In some embodiments, the pharmaceutical composition comprises polysorbate 20 at a concentration of 0.0005% (w / v, 0.005 g / L) to 0.001% (w / v, 0.01 g / L). In some embodiments, the pharmaceutical composition comprises polysorbate 20 at a concentration of 0.001% (w / v, 0.01 g / L) to 0.05% (w / v, 0.5 g / L). In some embodiments, the pharmaceutical composition comprises 0.0005% (w / v, 0.005 g / L) of polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.0006% (w / v, 0.006 g / L) of polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.0007% (w / v, 0.007 g / L) of polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.0008% (w / v, 0.008 g / L) of polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.0009% (w / v, 0.009 g / L) of polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.001% (w / v, 0.01 g / L) of polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.002% (w / v, 0.02 g / L) of polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.003% (w / v, 0.03 g / L) of polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.004% (w / v, 0.04 g / L) of polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.005% (w / v, 0.05 g / L) of polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.01% (w / v, 0.1 g / L) of polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.05% (w / v, 0.5 g / L) of polysorbate 20.

[0026] In some embodiments, the pharmaceutical composition comprises polysorbate 80 at a concentration of 0.0005% (w / v, 0.005 g / L) to 0.05% (w / v, 0.5 g / L). In some embodiments, the pharmaceutical composition comprises polysorbate 80 at a concentration of 0.0001% (w / v, 0.001 g / L) to 0.01% (w / v, 0.1 g / L). In some embodiments, the pharmaceutical composition comprises polysorbate 80 at a concentration of 0.0005% (w / v, 0.005 g / L) to 0.001% (w / v, 0.01 g / L). In some embodiments, the pharmaceutical composition comprises polysorbate 80 at a concentration of 0.001% (w / v, 0.01 g / L) to 0.05% (w / v, 0.5 g / L). In some embodiments, the pharmaceutical composition comprises 0.0005% (w / v, 0.005 g / L) of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.0006% (w / v, 0.006 g / L) of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.0007% (w / v, 0.007 g / L) of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.0008% (w / v, 0.008 g / L) of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.0009% (w / v, 0.009 g / L) of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.001% (w / v, 0.01 g / L) of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.002% (w / v, 0.02 g / L) of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.003% (w / v, 0.03 g / L) of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.004% (w / v, 0.04 g / L) of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.005% (w / v, 0.05 g / L) of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.01% (w / v, 0.1 g / L) of polysorbate 80. In some embodiments, the pharmaceutical composition comprises 0.05% (w / v, 0.5 g / L) of polysorbate 80.

[0027] In some embodiments, the pH of the pharmaceutical composition is about 7.4.

[0028] In some embodiments, the pH of the pharmaceutical composition is about 6.0 to 8.8. In some embodiments, the pH of the pharmaceutical composition is about 6.0 to 9.0. In some embodiments, the pH of the pharmaceutical composition is about 6.0. In some embodiments, the pH of the pharmaceutical composition is about 6.1. In some embodiments, the pH of the pharmaceutical composition is about 6.2. In some embodiments, the pH of the pharmaceutical composition is about 6.3. In some embodiments, the pH of the pharmaceutical composition is about 6.4. In some embodiments, the pH of the pharmaceutical composition is about 6.5. In some embodiments, the pH of the pharmaceutical composition is about 6.6. In some embodiments, the pH of the pharmaceutical composition is about 6.7. In some embodiments, the pH of the pharmaceutical composition is about 6.8. In some embodiments, the pH of the pharmaceutical composition is about 6.9. In some embodiments, the pH of the pharmaceutical composition is about 7.0. In some embodiments, the pH of the pharmaceutical composition is about 7.1. In some embodiments, the pH of the pharmaceutical composition is about 7.2. In some embodiments, the pH of the pharmaceutical composition is about 7.3. In some embodiments, the pH of the pharmaceutical composition is about 7.4. In some embodiments, the pH of the pharmaceutical composition is about 7.5. In some embodiments, the pH of the pharmaceutical composition is about 7.6. In some embodiments, the pH of the pharmaceutical composition is about 7.7. In some embodiments, the pH of the pharmaceutical composition is about 7.8. In some embodiments, the pH of the pharmaceutical composition is about 7.9. In some embodiments, the pH of the pharmaceutical composition is about 8.0. In some embodiments, the pH of the pharmaceutical composition is about 8.1. In some embodiments, the pH of the pharmaceutical composition is about 8.2. In some embodiments, the pH of the pharmaceutical composition is about 8.3. In some embodiments, the pH of the pharmaceutical composition is about 8.4. In some embodiments, the pH of the pharmaceutical composition is about 8.5. In some embodiments, the pH of the pharmaceutical composition is about 8.6. In some embodiments, the pH of the pharmaceutical composition is about 8.7. In some embodiments, the pH of the pharmaceutical composition is about 8.8. In some embodiments, the pH of the pharmaceutical composition is about 8.9. In some embodiments, the pH of the pharmaceutical composition is about 9.0.

[0029] In some embodiments, the pH of the pharmaceutical composition is 7.4.

[0030] In some embodiments, the pH of the pharmaceutical composition is 6.0 to 8.8. In some embodiments, the pH of the pharmaceutical composition is 6.0 to 9.0. In some embodiments, the pH of the pharmaceutical composition is 6.0. In some embodiments, the pH of the pharmaceutical composition is 6.1. In some embodiments, the pH of the pharmaceutical composition is 6.2. In some embodiments, the pH of the pharmaceutical composition is 6.3. In some embodiments, the pH of the pharmaceutical composition is 6.4. In some embodiments, the pH of the pharmaceutical composition is 6.5. In some embodiments, the pH of the pharmaceutical composition is 6.6. In some embodiments, the pH of the pharmaceutical composition is 6.7. In some embodiments, the pH of the pharmaceutical composition is 6.8. In some embodiments, the pH of the pharmaceutical composition is 6.9. In some embodiments, the pH of the pharmaceutical composition is 7.0. In some embodiments, the pH of the pharmaceutical composition is 7.1. In some embodiments, the pH of the pharmaceutical composition is 7.2. In some embodiments, the pH of the pharmaceutical composition is 7.3. In some embodiments, the pH of the pharmaceutical composition is 7.4. In some embodiments, the pH of the pharmaceutical composition is 7.5. In some embodiments, the pH of the pharmaceutical composition is 7.6. In some embodiments, the pH of the pharmaceutical composition is 7.7. In some embodiments, the pH of the pharmaceutical composition is 7.8. In some embodiments, the pH of the pharmaceutical composition is 7.9. In some embodiments, the pH of the pharmaceutical composition is 8.0. In some embodiments, the pH of the pharmaceutical composition is 8.1. In some embodiments, the pH of the pharmaceutical composition is 8.2. In some embodiments, the pH of the pharmaceutical composition is 8.3. In some embodiments, the pH of the pharmaceutical composition is 8.4. In some embodiments, the pH of the pharmaceutical composition is 8.5. In some embodiments, the pH of the pharmaceutical composition is 8.6. In some embodiments, the pH of the pharmaceutical composition is 8.7. In some embodiments, the pH of the pharmaceutical composition is 8.8. In some embodiments, the pH of the pharmaceutical composition is 8.9. In some embodiments, the pH of the pharmaceutical composition is 9.0.

[0031] As used herein and unless otherwise stated, the term “about” means within plus or minus 10% of a given value or range.

[0032] In some embodiments, the pharmaceutical composition is contained in a hydrophobically coated glass vial.

[0033] In some embodiments, the pharmaceutical composition is contained in a cyclic olefin polymer (COP) vial.

[0034] In some embodiments, the pharmaceutical composition is contained in a Daikyo Crystal Zenith® (CZ) vial.

[0035] In some embodiments, the pharmaceutical composition is contained in a TopLyo-coated vial.

[0036] In some embodiments, a pharmaceutical composition is disclosed herein comprising: (a) the recombinant AAV; (b) potassium chloride at a concentration of 0.2 g / L; (c) potassium dihydrogen phosphate at a concentration of 0.2 g / L; (d) sodium chloride at a concentration of 5.84 g / L; (e) anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L; (f) sucrose at a concentration of 4% by weight / volume (40 g / L); (g) poloxamer 188 at a concentration of 0.001% by weight / volume (0.01 g / L); and (h) water, wherein the recombinant AAV is AAV8.

[0037] In some embodiments, the vector genome concentration (VGC) of the pharmaceutical composition is 3 × 10⁻⁶. 9 GC / mL, 1× 10 10 GC / mL, 1.2 × 10 10 GC / mL, 1.6 × 10 10 GC / mL, 4 × 10 10 GC / mL, 6 × 10 10 GC / mL, 2 × 10 11 GC / mL, 2.4 × 10 11 GC / mL, 2.5 × 10 11 GC / mL, 3 × 10 11 GC / mL, 3.2 × 10 11 GC / mL, 6.2 × 10 11 GC / mL, 6.5 × 10 11 GC / mL, 1 × 10 12 GC / mL, 3 × 10 12 GC / mL, 2× 10 13 GC / mL or 3 × 10 13 GC / mL.

[0038] In some embodiments, the vector genome concentration (VGC) of the pharmaceutical composition is 3 × 10⁻⁶. 9 GC / mL, 4 × 10 9 GC / mL, 5 × 10 9 GC / mL, 6 × 10 9GC / mL, 7 × 10 9 GC / mL, 8 × 10 9 GC / mL, 9 × 10 9 GC / mL, 1 × 10 10 GC / mL, 2 × 10 10 GC / mL, 3 × 10 10 GC / mL, 4 × 10 10 GC / mL, 5 × 10 10 GC / mL, 6 × 10 10 GC / mL, 7 × 10 10 GC / mL, 8 × 10 10 GC / mL, 9 × 10 10 GC / mL, 1 × 10 11 GC / mL, 2 × 10 11 GC / mL, 3 × 10 11 GC / mL, 4 × 10 11 GC / mL, 5 × 10 11 GC / mL, 6 × 10 11 GC / mL, 7 × 10 11 GC / mL, 8 × 10 11 GC / mL, 9 × 10 11 GC / mL, 1 × 10 12 GC / mL, 2 × 10 12 GC / mL, 3 × 10 12 GC / mL, 4 × 10 12 GC / mL, 5 × 10 12 GC / mL, 6 × 10 12 GC / mL, 7 × 10 12 GC / mL, 8 × 10 12 GC / mL, 9 × 10 12 GC / mL, 1 × 10 13 GC / mL, 1 × 10 13 GC / mL, 2 × 10 13 GC / mL or 3 × 10 13 GC / mL.

[0039] In some embodiments, the vector genome concentration (VGC) of the pharmaceutical composition is approximately 3 × 10⁻⁶. 9 GC / mL, approximately 1 × 10 10GC / mL, approximately 1.2 × 10⁻⁶ 10 GC / mL, approximately 1.6 × 10⁻⁶ 10 GC / mL, approximately 4 × 10⁻⁶ 10 GC / mL, approximately 6 × 10⁻⁶ 10 GC / mL, approximately 2 × 10 11 GC / mL, approximately 2.4 × 10⁻⁶ 11 GC / mL, approximately 2.5 × 10⁻⁶ 11 GC / mL, approximately 3 × 10 11 GC / mL, approximately 3.2 × 10⁻⁶ 11 GC / mL, approximately 6.2 × 10⁻⁶ 11 GC / mL, approximately 6.5 × 10 11 GC / mL, approximately 1 × 10 12 GC / mL, approximately 3 × 10 12 GC / mL, approximately 2 × 10 13 GC / mL or approximately 3 × 10⁻⁶ 13 GC / mL.

[0040] In some embodiments, the vector genome concentration (VGC) of the pharmaceutical composition is approximately 3 × 10⁻⁶. 9 GC / mL, 4 × 10 9 GC / mL, 5 × 10 9 GC / mL, 6 × 10 9 GC / mL, 7 × 10 9 GC / mL, 8 × 10 9 GC / mL, 9 × 10 9 GC / mL, approximately 1 × 10 10 GC / mL, approximately 2 × 10 10 GC / mL, approximately 3 × 10 10 GC / mL, approximately 4 × 10⁻⁶ 10 GC / mL, approximately 5 × 10 10 GC / mL, approximately 6 × 10 10 GC / mL, approximately 7 × 10 10 GC / mL, approximately 8 × 10 10 GC / mL, approximately 9 × 10⁻⁶ 10 GC / mL, approximately 1 × 10 11 GC / mL, approximately 2 × 10 11 GC / mL, approximately 3 × 10 11 GC / mL, approximately 4 × 10⁻⁶ 11GC / mL, approximately 5 × 10 11 GC / mL, approximately 6 × 10 11 GC / mL, approximately 7 × 10 11 GC / mL, approximately 8 × 10 11 GC / mL, approximately 9 × 10 11 GC / mL, approximately 1 × 10 12 GC / mL, approximately 2 × 10 12 GC / mL, approximately 3 × 10 12 GC / mL, approximately 4 × 10 12 GC / mL, approximately 5 × 10 12 GC / mL, approximately 6 × 10 12 GC / mL, approximately 7 × 10 12 GC / mL, approximately 8 × 10 12 GC / mL, approximately 9 × 10 12 GC / mL, approximately 1 × 10 13 GC / mL, approximately 1 × 10 13 GC / mL, approximately 2 × 10 13 GC / mL, approximately 3 × 10⁻⁶ 13 GC / mL.

[0041] In some embodiments, the recombinant AAV in the pharmaceutical composition exhibits at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher stability to freeze / thaw cycles compared to the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0042] In some embodiments, the infectivity of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the viral infectivity of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, infectivity is measured before or after a freeze / thaw cycle.

[0043] In some embodiments, the aggregation of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the aggregation of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, the aggregation is measured before or after a freeze / thaw cycle.

[0044] In some embodiments, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold more stable than the same recombinant AAV in a reference pharmaceutical composition over a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, or about 4 years. In some embodiments, the stability of the recombinant AAV over a period of time is determined by one or more assays disclosed in Sections 4.5 and 5.

[0045] In some embodiments, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold more stable than the same recombinant AAV in a reference pharmaceutical composition over a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, or about 4 years. In some embodiments, the stability of the recombinant AAV over a period of time is determined by one or more assays disclosed in Sections 4.5 and 5.

[0046] In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, the in vitro relative potency (IVRP) is measured before or after a freeze / thaw cycle.

[0047] In some embodiments, the cell-free DNA of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold lower than that of the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the cell-free DNA of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5. In some embodiments, aggregation is measured before or after freeze / thaw cycles.

[0048] In some embodiments, the size variation of the recombinant AAV in the pharmaceutical composition over time periods such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years is up to 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, the size is measured before or after a freeze / thaw cycle.

[0049] In some embodiments, the size of the recombinant AAV in the pharmaceutical composition varies by up to 20%, 15%, 10%, 8%, 5%, 4%, 3%, 4%, 2%, or 1% over a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, the size is measured before or after a freeze / thaw cycle.

[0050] In some embodiments, when stored at -20°C, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0051] In some embodiments, the infectivity of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition when stored at -20°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years. In some embodiments, the viral infectivity of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0052] In some embodiments, the infectivity of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition when stored at -20°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years. In some embodiments, the viral infectivity of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0053] In some embodiments, when stored at -20°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the aggregation of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the aggregation of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0054] In some embodiments, when stored at -20°C for a period of time of at least, for example, at least about 11 weeks, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the aggregation of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the aggregation of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0055] In some embodiments, when stored at -20°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV over the time period is determined by one or more assays disclosed in Sections 4.5 and 5.

[0056] In some embodiments, when stored at -20°C for a duration of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV over a period of time is determined by one or more assays disclosed in Sections 4.5 and 5.

[0057] In some embodiments, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2, 3, 5, 10, 100, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition when performed by: (i) storage at -80°C for a first time period; (ii) subsequent thawing; and (iii) storage at 4°C for a second time period after thawing. In some embodiments, the stability of the recombinant AAV over a time period is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, the first time period is about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months. 28. In some embodiments, the second time period is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, or about 2 months.

[0058] In some embodiments, the recombinant AAV is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition when the following are performed: (i) storage at -80°C for a first time period; (ii) subsequent thawing; and (iii) storage at 4°C for a second time period after thawing. In some embodiments, the first time period is about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months. In some embodiments, the second time period is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, or about 2 months.

[0059] In some embodiments, the vector genome concentration of the recombinant AAV after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at -80°C for the said period of time. In some embodiments, the vector genome concentration of the recombinant AAV after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at -20°C for the said period of time. In some embodiments, the vector genome concentration of the recombinant AAV after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at 4°C for the said period of time.

[0060] In some embodiments, when stored at -20°C for a period of time, such as 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0061] In some embodiments, when stored at -20°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0062] In some embodiments, when stored at -20°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0063] In some embodiments, when stored at -20°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0064] In some embodiments, when stored at -20°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the cell-free DNA of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the cell-free DNA of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0065] In some embodiments, when stored at -20°C for a duration of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the cell-free DNA of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the cell-free DNA of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0066] In some embodiments, when stored at -20°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the size variation of the recombinant AAV in the pharmaceutical composition is at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0067] In some embodiments, when stored at -20°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the size variation of the recombinant AAV in the pharmaceutical composition is at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0068] In some embodiments, when stored at 37°C, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0069] In some embodiments, the infectivity of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition when stored at 37°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years. In some embodiments, the viral infectivity of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0070] In some embodiments, the infectivity of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition when stored at 37°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years. In some embodiments, the viral infectivity of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0071] In some embodiments, when stored at 37°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the aggregation of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the aggregation of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0072] In some embodiments, when stored at 37°C for a duration of at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than the same recombinant AAV in a reference pharmaceutical composition, after being stored at 37°C for a duration of at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, respectively, the aggregation of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower. In some embodiments, the aggregation of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0073] In some embodiments, when stored at 37°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV over the duration is determined by one or more assays disclosed in Sections 4.5 and 5.

[0074] In some embodiments, when stored at 37°C for a duration of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV over the time period is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, the vector genome concentration of the recombinant AAV after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at -80°C for the said period of time. In some embodiments, the vector genome concentration of the recombinant AAV after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at -20°C for the said period of time. In some embodiments, the vector genome concentration of the recombinant AAV after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at 4°C for the said period of time. In some embodiments, the in vitro potency of the recombinant AAV after storage at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of the recombinant AAV before storage at -80°C for the specified period of time. In some embodiments, the in vitro potency of the recombinant AAV after storage at -20°C for the specified period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of the recombinant AAV before storage at -20°C for the specified period of time. In some embodiments, the in vitro potency of the recombinant AAV after storage at -20°C for the specified period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of the recombinant AAV before storage at -20°C for the specified period of time.In some embodiments, the size distribution of the recombinant AAV after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of the recombinant AAV before being stored at -80°C for that period of time. In some embodiments, the size distribution of the recombinant AAV after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of the recombinant AAV before being stored at -20°C for that period of time. In some embodiments, the size distribution of the recombinant AAV after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the size distribution of the recombinant AAV before being stored at 4°C for that period of time. In some embodiments, the time period is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0075] In some embodiments, when stored at 37°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0076] In some embodiments, when stored at 37°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0077] In some embodiments, when stored at 37°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the cell-free DNA of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the cell-free DNA of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0078] In some embodiments, when stored at 37°C for a duration of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the cell-free DNA of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the cell-free DNA of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0079] In some embodiments, when stored at 37°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the size variation of the recombinant AAV in the pharmaceutical composition is at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0080] In some embodiments, when stored at 37°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the size variation of the recombinant AAV in the pharmaceutical composition is at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0081] On the other hand, this article provides a method for treating a subject diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising (ii) preparing a pharmaceutical composition described herein, storing the pharmaceutical composition at -80°C for a first time period; (iii) thawing the pharmaceutical composition; and (iii) after thawing, storing the pharmaceutical composition at 4°C for a second time period. In some embodiments, the first time period is about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months. 61. In some embodiments, the second time period is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, or about 2 months.

[0082] In some embodiments, this disclosure provides a pharmaceutical composition or formulation comprising recombinant adeno-associated virus (AAV), potassium dihydrogen phosphate, sodium chloride, anhydrous disodium hydrogen phosphate, sucrose, and poloxamer 188. In some embodiments, the AAV comprises a component derived from AAV8. In some embodiments, the AAV is a viral vector provided herein comprising the following elements in the following order: a) a constitutive or hypoxia-inducible promoter sequence, and b) a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion). In some embodiments, the transgene is a fully human post-translational modified (HuPTM) antibody against VEGF. Antibodies comprise, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-heavy chain pairs, intracellular antibodies, heteroconjugated antibodies, monovalent antibodies, antigen-binding fragments of full-length antibodies, and fusion proteins thereof. Such antigen-binding fragments include, but are not limited to, single-domain antibodies (variable domains (VHH) of heavy chain antibodies or nanobodies), Fab, F(ab')2, and scFv (single-chain variable fragments) of full-length anti-VEGF antibodies (preferably, full-length anti-VEGF monoclonal antibodies (mAbs)) (collectively referred to herein as “antigen-binding fragments”). In a preferred embodiment, the fully human post-translational modified antibody against VEGF is a fully human post-translational modified antigen-binding fragment of a monoclonal antibody (mAb) against VEGF (“HuPTMFabVEGFi”). In another preferred embodiment, the HuPTMFabVEGFi is a fully human glycosylated antigen-binding fragment of an anti-VEGF mAb (“HuGlyFabVEGFi”). In alternative embodiments, a full-length mAb may be used. In a preferred embodiment, the AAV used for delivery of the transgene should be tropism-prone to human retinal cells or photoreceptor cells. Such AAVs may comprise non-replicating recombinant adeno-associated virus vectors (“rAAVs”), particularly those carrying an AAV8 capsid. In a specific embodiment, the viral vector or other DNA expression construct described herein is construct I, wherein construct I comprises the following components: (1) an AAV8 inverted terminal repeat sequence side-connected to an expression cassette; (2) a control element comprising: a) a CB7 promoter comprising a CMV enhancer / chicken β-actin promoter; b) a chicken β-actin intron; and c) a rabbit β-globin poly A signal; and (3) a nucleic acid sequence encoding a heavy and light chain of an anti-VEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker, thereby ensuring the expression of equal amounts of heavy chain polypeptide and light chain polypeptide.In another specific embodiment, the viral vector or other DNA expression construct described herein is construct II, wherein construct II comprises the following components: (1) an AAV2 inverted terminal repeat sequence side-attached to an expression cassette; (2) a control element comprising: a) a CB7 promoter including a CMV enhancer / chicken β-actin promoter; b) a chicken β-actin intron; and c) a rabbit β-globin poly A signal; and (3) a nucleic acid sequence encoding a heavy and light chain of an anti-VEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker, thereby ensuring the expression of equal amounts of the heavy chain polypeptide and the light chain polypeptide. In a specific embodiment, the construct described herein is... Figure 4 As shown in the figure. In some embodiments, the anti-hVEGF antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, and the light chain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.

[0083] In some embodiments, the pharmaceutical composition comprises: (a) Construct II encoding an anti-human vascular endothelial growth factor (hVEGF) antibody; (b) potassium chloride at a concentration of 0.2 g / L; (c) potassium dihydrogen phosphate at a concentration of 0.2 g / L; (d) sodium chloride at a concentration of 5.84 g / L; (e) anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L; (f) sucrose at a concentration of 4% by weight / volume (40 g / L); poloxamer 188 at a concentration of 0.001% by weight / volume (0.01 g / L); and (h) water, wherein the anti-hVEGF antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, and the light chain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.

[0084] In some embodiments, the pharmaceutical composition is a liquid composition. In some embodiments, the pharmaceutical composition is a frozen composition. In some embodiments, the pharmaceutical composition is a lyophilized composition derived from the liquid compositions disclosed herein. In some embodiments, the pharmaceutical composition is a reconstituted lyophilized formulation.

[0085] In some embodiments, the pharmaceutical composition is a lyophilized composition comprising a residual moisture content between about 1% and about 7%.

[0086] In some respects, this document discloses a method for treating or preventing a disease in a subject, the method comprising administering a pharmaceutical composition to the subject.

[0087] In a specific aspect, this document discloses a method for treating or preventing a disease in a subject, the method comprising administering a pharmaceutical composition to the subject by: intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with microneedles), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device comprising a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a microneedle injected into the subretinal space at the posterior pole), and / or a posterior proximal scleral reservoir procedure (e.g., via a proximal scleral drug delivery device comprising a cannula, the tip of which can be inserted and held directly juxtaposed to the scleral surface).

[0088] In some respects, the pharmaceutical composition is suitable for ocular administration. In some respects, the pharmaceutical composition is suitable for suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or posterior near-scleral reservoir procedures.

[0089] In some embodiments, the pharmaceutical composition is suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via suprachoroidal drug delivery devices, such as microinjectors with microneedles), subretinal injection via the vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0090] In some embodiments, the pharmaceutical composition has a desired density suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0091] In some embodiments, the pharmaceutical composition has a desired osmotic pressure suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted posteriorly and tunnel through the suprachoroidal space, with a microneedle injected into the subretinal space at the posterior pole), and / or a posterior proximal scleral reservoir procedure (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed with the scleral surface). In a specific embodiment, the desired osmotic pressure for subretinal administration is 160-430 mOsm / kg H2O. In other specific embodiments, the desired osmotic pressure for suprachoroidal administration is less than 600 mOsm / kg H2O.

[0092] In some embodiments, the pharmaceutical composition has a desired viscosity suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0093] In some embodiments, the osmotic pressure of the pharmaceutical composition ranges from 200 mOsm / L to 660 mOsm / L. In some embodiments, the osmotic pressure ranges from 200 mOsm / L to 660 mOsm / L. In some embodiments, the osmotic pressure is less than 600 mOsm / kg H₂O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 200 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 250 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 300 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 350 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 400 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 450 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 500 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 550 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 600 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 650 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 660 mOsm / L. In some aspects, this document discloses a method for treating a subject diagnosed with mucopolysaccharidosis type IVA (MPS IVA), mucopolysaccharidosis type I (MPS I), or mucopolysaccharidosis type II (MPS II), the method comprising administering a pharmaceutical composition to the subject.

[0094] In some embodiments, the vector genome concentration of construct II after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of construct II before being stored at -80°C for the said period of time. In some embodiments, the vector genome concentration of construct II after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of construct II before being stored at -20°C for the said period of time. In some embodiments, the vector genome concentration of construct II after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the construct. In some embodiments, the time period is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0095] In some embodiments, the in vitro potency of construct II after storage at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of construct II before storage at -80°C for the said period of time. In some embodiments, the in vitro potency of construct II after storage at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of construct II before storage at -20°C for the said period of time. In some embodiments, the in vitro potency of construct II after storage at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of construct II before storage at 4°C for the said period of time. In some embodiments, the time period is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0096] In some embodiments, the size distribution of construct II after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of construct II before being stored at -80°C for the same period of time. In some embodiments, the size distribution of construct II after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of construct II before being stored at -20°C for the same period of time. In some embodiments, the size distribution of construct II after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the size distribution of construct II before being stored at 4°C for the same period of time. In some embodiments, the time period is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, or about 10 months. In some embodiments, the pharmaceutical composition can be stored at 4°C for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or 2 months after having been previously stored at -80°C for about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, or 24 months.

[0097] In some respects, this article discloses a method for treating a subject diagnosed with mucopolysaccharidosis type IVA (MPS IVA), mucopolysaccharidosis type I (MPS I), or mucopolysaccharidosis type II (MPS II), the method comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject by intravenous, subcutaneous, or intramuscular injection.

[0098] In some respects, this document discloses methods for treating or preventing diseases in subjects, including treating subjects diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), including administering a therapeutically effective amount of a pharmaceutical composition to the subject.

[0099] In some respects, this document discloses methods for treating subjects diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the methods comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject by: intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device comprising a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), or a posterior proximal scleral reservoir procedure (e.g., via a proximal scleral drug delivery device comprising a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0100] In some respects, this article describes a method for treating human subjects diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising delivering a therapeutically effective amount of anti-hVEGF antigen-binding fragments produced by human retinal cells to the retina of the human subject by means of: administering a pharmaceutical composition to the suprachoroidal space, subretinal space, or outer surface of the sclera in the human subject's eye (e.g., via suprachoroidal injection (e.g., via a suprachoroidal drug delivery device such as a microinjector with microneedles), via subretinal injection through the vitreous pathway (surgical procedure), via suprachoroidal administration (e.g., via a surgical procedure using a subretinal drug delivery device comprising a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), or via a posterior proximal scleral reservoir procedure (e.g., via a proximal scleral drug delivery device comprising a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface)).

[0101] In some aspects, this document provides a method for treating a subject diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising (ii) preparing a pharmaceutical composition described herein, storing the pharmaceutical composition at -80°C for a first time period; (iii) thawing the pharmaceutical composition; and (iii) after thawing, storing the pharmaceutical composition at 4°C for a second time period. In some embodiments, the first time period is about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months. In some embodiments, the second time period is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, or about 2 months.

[0102] This article describes antibodies against human vascular endothelial growth factor (hVEGF) produced by human retinal cells, such as anti-hVEGF antigen-binding fragments. Human VEGF (hVEGF) is produced by... VEGF (VEGFA, VEGFB, VEGFC) or VEGFD The human protein encoded by the hVEGF gene. An exemplary amino acid sequence of hVEGF can be found in GenBank accession number AAA35789.1. An exemplary nucleic acid sequence of hVEGF can be found in GenBank accession number M32977.1.

[0103] In some aspects of the methods described herein, the antigen-binding fragment comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO.2 or SEQ ID NO.4; and a light chain comprising the amino acid sequence of SEQ ID NO.1 or SEQ ID NO.3.

[0104] In some aspects of the methods described herein, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 17-19 or SEQ ID NO: 20, 18 and 21.

[0105] In specific embodiments of the method described herein, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamylation (pyro Glu). In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the eighth and eleventh amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO. 14)) each carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu). In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18, and 21, wherein the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) is unacetylated. In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18, and 21, wherein the eighth and eleventh amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO. 14)) each carry one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) is unacetylated. In a preferred embodiment, the chemical modifications or absence of chemical modifications described herein (as the case may be) are determined by mass spectrometry.

[0106] In specific embodiments of the method described herein, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamylation (pyro Glu). In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO. 20)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamate (pyro Glu), the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO. 18)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamate (pyro Glu), and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu). In a specific embodiment, the antigen-binding fragment includes light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) is unacetylated. In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO. 20)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyroGlu), the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO. 18)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyroGlu), and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) is unacetylated.In a preferred embodiment, the chemical modifications or absence of chemical modifications described herein (as the case may be) are determined by mass spectrometry.

[0107] In specific embodiments of the method described herein, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamylation (pyro Glu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamylation (pyro Glu). In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein: (1) the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO. 20)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyro Glu), the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO. 18)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyro Glu), and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamylation (pyro Glu). (2) The eighth and eleventh amino acid residues of the light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO.14)) each carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu), and the second amino acid residue of the light chain CDR3 (i.e. the second Q in QQYSTVPWTF (SEQ ID NO.16)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu).In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) is unacetylated, and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) is unacetylated. In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein: (1) the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO. 20)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyro Glu), the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO. 18)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyro Glu), and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) is not acetylated; and (2) the eighth and eleventh amino acid residues of light chain CDR1 (i.e., SASQDISNYLN (SEQ ID NO. 20)) The two N's in NO. 14 each carry one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyro Glu), and the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) is unacetylated. In a preferred embodiment, the chemical modifications described herein or the absence of such modifications (as applicable) are determined by mass spectrometry.

[0108] In some respects, this article describes a method for treating human subjects diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising: delivering to the eye of the human subject a therapeutically effective amount of an antigen-binding fragment of a mAb against hVEGF, the antigen-binding fragment containing an α2,6-sialylated glycan. In more respects, this article describes a method for treating human subjects diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising: delivering to the eye of the human subject a therapeutically effective amount of an antigen-binding fragment of a mAb against hVEGF by means of: the suprachoroidal space, subretinal space, or outer surface of the sclera in the human subject's eye (e.g., via suprachoroidal injection (e.g., via a suprachoroidal drug delivery device such as a microinjector with microneedles), via a transvitreal pathway. Subretinal injection (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), or posterior proximal scleral reservoir procedure (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed with the scleral surface) administers a drug composition comprising an expression vector encoding an antigen-binding fragment against an hVEGF mAb, the antigen-binding fragment containing an α2,6-sialylated glycan.

[0109] In some respects, this article describes a method for treating human subjects diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising: delivering to the eye of the human subject a therapeutically effective amount of a glycosylated antigen-binding fragment of a mAb against hVEGF, wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens (i.e., as used herein, "detectable" means a level detectable by standard assays described below). In specific embodiments, this document describes a method for treating human subjects diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising: delivering a therapeutically effective amount of a glycosylated antigen-binding fragment of a mAb against hVEGF to the eye of the human subject by means of: delivery to the suprachoroidal space, subretinal space, or outer scleral surface of the human subject's eye (e.g., via intrachoroidal injection (e.g., via an intrachoroidal drug delivery device such as a microinjector with microneedles), or via the retinal via the vitreous pathway. Subretinal injection (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), or posterior proximal scleral reservoir procedure (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed with the scleral surface) administers a pharmaceutical composition comprising an expression vector encoding a glycosylated antigen-binding fragment against hVEGF, wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens.

[0110] Further details regarding the antigen-binding fragments of anti-hVEGF antibodies or mAbs targeting hVEGF are provided in International Publication No.: WO2019 / 067540 (incorporated hereinously by reference).

[0111] Specifically, the anti-hVEGF antibody comprises a heavy chain, the heavy chain comprising the amino acid sequence of SEQ ID NO.2 or SEQ ID NO.4; and a light chain, the light chain comprising the amino acid sequence of SEQ ID NO.1 or SEQ ID NO.3. Specifically, the expression vector is an AAV8 vector.

[0112] In some aspects of the methods described herein, the antigen-binding fragment transgene encodes a leader peptide. The leader peptide may also be referred to herein as a signal peptide or leader sequence.

[0113] In some aspects of the methods described herein, delivery to the eye includes delivery to the retina, choroid, and / or vitreous fluid of the eye. In some aspects of the methods described herein, the antigen-binding fragment includes a heavy chain comprising one, two, three, or four additional amino acids at its C-terminus.

[0114] In certain embodiments, the method encompasses treating a patient diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) and identified as responding to treatment with an anti-VEGF antibody. In more specific embodiments, the patient responds to treatment with an anti-VEGF antigen-binding fragment. In some embodiments, the patient has shown a response to treatment with an anti-VEGF antigen-binding fragment injected intravitreal prior to gene therapy. In specific embodiments, the patient has previously been treated with LUCENTIS® (ranibizumab), EYLEA® (aflibercept), and / or AVASTIN® (bevacizumab) and has been found to respond to one or more of LUCENTIS®, EYLEA®, and / or AVASTIN®.

[0115] Subjects to whom such viral vectors or other DNA expression constructs are delivered should respond to an anti-hVEGF antigen-binding fragment encoded by the transgene in the viral vector or expression construct. To determine responsiveness, the anti-VEGF antigen-binding fragment transgene product (e.g., produced in cell cultures, bioreactors, etc.) can be administered directly to the subject, for example, via intravitreal injection.

[0116] In some aspects of the methods described herein, the antigen-binding fragment includes a heavy chain that does not include additional amino acids at the C-terminus.

[0117] In certain aspects of the methods described herein, a population of antigen-binding fragment molecules is generated, wherein said antigen-binding fragment molecules comprise a heavy chain, and wherein 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, or 20% or less of said antigen-binding fragment molecule population comprises one, two, three, or four additional amino acids at the C-terminus of said heavy chain.

[0118] In certain aspects of the methods described herein, a population of antigen-binding fragment molecules is generated, wherein the antigen-binding fragment molecules comprise a heavy chain, and wherein 0.5-1%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.5%-5%, 0.5%-10%, 0.5%-20%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%-10%, 1%-20%, 2%-3%, 2%-4%, 2%-5%, 2%-10%, 2%-20%, 3%-4%, 3%-5%, 3%-10%, 3%-20%, 4%-5%, 4%-10%, 4%-20%, 5%-10%, 5%-20%, or 10%-20% of the antigen-binding fragment molecule population comprises one, two, three, or four additional amino acids at the C-terminus of the heavy chain.

[0119] HuPTMFabVEGFi encoded by a transgene, such as HuGlyFabVEGFi, may include, but is not limited to: an antigen-binding fragment of an antibody that binds to hVEGF, such as bevacizumab; an anti-hVEGF Fab moiety, such as ranibizumab; or such bevacizumab or ranibizumab Fab moiety engineered to contain additional glycosylation sites on the Fab domain (e.g., see Courtois et al., 2016, Monoclonal Antibodies 8: 99-112, which is incorporated herein by reference in its entirety by reference to a description of a derivative of bevacizumab with high glycosylation on the Fab domain of a full-length antibody).

[0120] The recombinant vector used for delivering the transgene should be tropism-positive for human retinal cells or photoreceptor cells. Such vectors may contain non-replicating recombinant adeno-associated virus vectors (“rAAV”), with those carrying an AAV8 capsid being particularly preferred. However, other viral vectors may be used, including, but not limited to, lentiviral vectors, vaccinia virus vectors, or non-viral expression vectors known as “naked DNA” constructs. Preferably, HuPTMFabVEGFi, such as HuGlyFabVEGFi, should be controlled by appropriate expression control elements, such as CB7 promoters (chicken β-actin promoter and CMV enhancer), RPE65 promoters, or opsin promoters, and may contain other expression control elements that enhance the expression of the vector-driven transgene (e.g., introns, such as chicken β-actin introns, mouse parvovirus (MVM) introns, human factor IX introns (e.g., FIX truncated intron 1), β... - Introns of globin splice donors / immunoglobulin heavy chain spice acceptors, introns of adenovirus splice donors / immunoglobulin splice acceptors, introns of SV40 late splice donors / splicing acceptors (19S / 16S), and introns of hybrid adenovirus splice donors / IgG splice acceptors, and polyA signals such as rabbit β-globin polyA signaling, human growth hormone (hGH) polyA signaling, SV40 late polyA signaling, synthetic polyA (SPA) signaling, and bovine growth hormone (bGH) polyA signaling. See, for example, Powell and Rivera-Soto, 2015, *Discov. Med.*, 19(102):49-57.

[0121] Gene therapy constructs are designed to express both the heavy and light chains. More specifically, the heavy and light chains should be expressed in approximately equal amounts, in other words, at a heavy-to-light chain ratio of approximately 1:1. The coding sequences for the heavy and light chains can be engineered in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES, such that the heavy-chain polypeptide and light-chain polypeptide are expressed individually. For specific leader sequences, see, for example, section 5.2.4, and for specific IRES, 2A, and other linker sequences that can be used with the methods and compositions provided herein, see section 5.2.5.

[0122] In some embodiments, the gene therapy construct is provided as a frozen, sterile, single-use solution of the AAV carrier active ingredient in a formulation buffer. In specific embodiments, pharmaceutical compositions suitable for subretinal administration comprise a suspension of a recombinant (e.g., rHuGlyFabVEGFi) carrier in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant, and optional excipients.

[0123] In some embodiments, the gene therapy construct is provided as a frozen, sterile, single-use solution of the AAV carrier active ingredient in a formulation buffer. In specific embodiments, pharmaceutical compositions suitable for choroidal, subretinal, juxtascleral, and / or intraretinal administration comprise a suspension of a recombinant (e.g., rHuGlyFabVEGFi) carrier in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant, and optional excipients.

[0124] The therapeutically effective dose of the recombinant carrier should be administered subretinal and / or intraretinal (e.g., by means of subretinal injection via a transvitreal route (surgical procedure) or subretinal administration via the suprachoroidal space) in a volume ranging from ≥ 0.1 mL to ≤ 0.5 mL, preferably from 0.1 to 0.30 mL (100–300 µl), and most preferably 0.25 mL (250 µl). The therapeutically effective dose of the recombinant carrier can be administered in one or more injections during the same visit.

[0125] Therapeutic doses of the recombinant carrier should be administered onto the choroid (e.g., by means of choroidal injection) in volumes of 100 μl or less, such as 50-100 μl. Therapeutic doses of the recombinant carrier should also be administered onto the outer surface of the sclera in volumes of 500 μl or less, such as 10-20 μl, 20-50 μl, 50-100 μl, 100-200 μl, 200-300 μl, 300-400 μl, or 400-500 μl (e.g., by means of a posterior proximal scleral reservoir procedure). Subretinal injection is a surgical procedure performed by a trained retinal surgeon involving a vitrectomy performed on the subject under local anesthesia and the subretinal injection of gene therapy into the retina (see, for example, Campochiaro et al., 2017, Human Gene Therapy 28(1):99-111, which is incorporated herein by reference in its entirety). In a specific embodiment, the subretinal administration is performed using a suprachoroidal catheter that injects the drug into the subretinal space, such as a subretinal drug delivery device including the catheter, which can be inserted and tunneled through the suprachoroidal space to the posterior pole, where a small needle is injected into the subretinal space (see, for example, Baldassarre et al., 2017, Subretinal Delivery of Cells via the Suprachoroidal Space: Janssen Trial. in the following references: Schwartz (ed.) Cell Therapy for Retinopathy, Springer, Cam; International Patent Application Publication No. WO 2016 / 040635 A1; each of the references is incorporated herein by reference in its entirety). The procedure of administration to the choroid involves the application of a drug to the suprachoroidal space of the eye and is typically performed using a choroidal drug delivery device such as a microinjector with microneedles (see, for example, Hariprasad, 2016, Retinal Physician 13: 20-23; Goldstein, 2014, Retina Today 9(5): 82-87; each of the references is incorporated herein by reference in its entirety).Intrachoroidal drug delivery devices that can be used to deposit expression vectors in the suprachoroidal space of the invention as described herein include, but are not limited to, intrachoroidal drug delivery devices manufactured by Clearside® Biomedical, Inc. (see, for example, Hariprasad, 2016, Retinal Surgeon 13: 20-23) and the MedOne suprachoroidal catheter. Subretinal drug delivery devices that can be used to deposit expression vectors in the subretinal space via the suprachoroidal space of the invention as described herein include, but are not limited to, subretinal drug delivery devices manufactured by Anssen Pharmaceuticals, Inc. (see, for example, International Patent Application Publication No. WO 2016 / 040635 A1). In a particular embodiment, administration to the outer surface of the sclera is performed by a proximal scleral drug delivery device comprising a cannula, the tip of which can be inserted and held directly juxtaposed to the scleral surface. For further details on the different administration modalities, see section 5.3.2. Application to the choroid, subretinal, juxtascleral, and / or intraretinal regions should result in the delivery of the soluble transgenic product to the retina, vitreous fluid, and / or aqueous solution. Delivery and maintenance of the transgenic product in the retina, vitreous fluid, and / or aqueous solution are caused by the expression of the transgenic product (e.g., an encoded anti-VEGF antibody) by retinal cells, such as rods, cones, retinal pigment epithelium, horizontal, bipolar, aprocessile, ganglion, and / or Miller cells. In specific embodiments, it is desirable to maintain the concentration of the transgenic product at a Cmin of at least 0.330 µg / mL in the vitreous fluid or 0.110 µg / mL in the aqueous solution (anterior chamber of the eye) for three months; thereafter, a vitreous Cmin concentration ranging from 1.70 to 6.60 µg / mL and / or an aqueous Cmin concentration ranging from 0.567 to 2.20 µg / mL should be maintained. However, since the transgenic product is continuously produced, maintaining a lower concentration may be effective. The concentration of the transgenic product can be measured in vitreous fluid and / or aqueous patient samples from the anterior chamber of the treated eye. Alternatively, the vitreous fluid concentration can be estimated and / or monitored by measuring the patient's serum concentration of the transgenic product—the ratio of systemic exposure to vitreous exposure is approximately 1:90,000.(For example, see, “vitreous humor and serum concentrations of ranibizumab reported”, Xu L et al., 2013, Invest. Opthal. Vis. Sci. 54: 1616-1624, pp. 1621 and 1623, Table 5, which is incorporated herein by reference in its entirety.)

[0126] In some embodiments, the dosage is measured by the number of genome copies per ml or the number of genome copies administered to the patient's eye (e.g., via choroidal injection (e.g., via a choroidal drug delivery device such as a microinjector with microneedles), subretinal injection via the vitreous route (surgical procedure), or subretinal administration via the suprachoroidal space). In some embodiments, 2.4 × 10⁻⁶ ppm is administered per ml. 11 One genome copy per ml, 1 × 10⁻⁶ 13 One genome copy. In a specific embodiment, 2.4 × 10⁶ copies were administered per ml. 11 One genome copy per ml, 5 × 10 11 One genome copy. In another specific embodiment, 5 × 10⁸ per ml is administered. 11 One genome copy per ml, 1 × 10⁻⁶ 12 One genome copy. In another specific embodiment, 1 × 10⁻⁶ per ml is administered. 12 One genome copy per ml, 5 × 10⁵ 12 One genome copy. In another specific embodiment, 5 × 10⁸ per ml is administered. 12 One genome copy per ml, 1 × 10⁻⁶ 13 One genome copy. In another specific embodiment, approximately 2.4 × 10⁻⁶ per ml is administered. 11 One genome copy. In another specific embodiment, approximately 5 × 10⁸ copies are administered per ml. 11 One genome copy. In another specific embodiment, approximately 1 × 10⁻⁶ copies are administered per ml. 12 One genome copy. In another specific embodiment, approximately 5 × 10⁸ copies are administered per ml. 12 One genome copy. In another specific embodiment, approximately 1 × 10⁻⁶ copies are administered per ml. 13 One genome copy. In some embodiments, 1 × 10⁻⁶ is applied. 9 One to 1 × 10 12 One genome copy. In a specific embodiment, 3 × 10⁻⁶ copies are applied.9 Up to 2.5 × 10 11 One genome copy. In a specific embodiment, 1 × 10⁻⁶ copies are applied. 9 Up to 2.5 × 10 11 One genome copy. In a specific embodiment, 1 × 10⁻⁶ copies are applied. 9 One to 1 × 10 11 One genome copy. In a specific embodiment, 1 × 10⁻⁶ copies are applied. 9 5 × 10 9 One genome copy. In a specific embodiment, 6 × 10⁻⁶ copies are applied. 9 Up to 3 × 10 10 One genome copy. In a specific embodiment, 4 × 10⁻⁶ copies are applied. 10 One to 1 × 10 11 One genome copy. In a specific embodiment, 2 × 10⁻⁶ copies are applied. 11 One to 1× 10 12 One genome copy. In a specific embodiment, approximately 3 × 10⁻⁶ copies are applied. 9 One genome copy (which corresponds to approximately 1.2 × 10⁻⁶ per ml in a 250 μl volume) 10 (One genome copy). In another specific embodiment, approximately 1 × 10⁻⁶ copies are applied. 10 One genome copy (which corresponds to approximately 4 × 10⁻⁶ per ml in a 250 μl volume). 10 (One genome copy). In another specific embodiment, approximately 6 × 10⁻⁶ copies are applied. 10 One genome copy (which corresponds to approximately 2.4 × 10⁻⁶ per ml in a 250 μl volume) 11 (One genome copy). In another specific embodiment, approximately 1.6 × 10⁻⁶ copies were administered. 11 One genome copy (which corresponds to approximately 6.2 × 10⁻⁶ per ml in a 250 μl volume). 11 (One genome copy). In another specific embodiment, approximately 1.6 × 10⁻⁶ copies were administered. 11 One genome copy (which corresponds to approximately 6.4 × 10⁻⁶ per ml in a 250 μl volume). 11 (One genome copy). In another specific embodiment, approximately 1.55 × 10⁻⁶ copies were administered. 11 One genome copy (which corresponds to approximately 6.2 × 10⁻⁶ per ml in a 250 μl volume). 11 (One genome copy). In another specific embodiment, approximately 2.5 × 10⁻⁶ copies are applied. 11 One genome copy (which corresponds to approximately 1.0 × 10⁻⁶ in a 250 μl volume) 12 indivual).

[0127] As used herein and unless otherwise stated, the term “about” means within plus or minus 10% of a given value or range.

[0128] This invention offers several advantages over standard care treatment involving repeated ocular injections of high-dose boluses of VEGF inhibitors, which dissipate over time, resulting in peak and trough levels. The sustained expression of transgenic antibodies allows for more consistent antibody levels at the site of action compared to repeated antibody injections, and is less risky and convenient for patients due to fewer injections required, leading to fewer physician visits. Consistent protein production can lead to better clinical outcomes because rebound edema in the retina is less likely to occur. Furthermore, due to the different microenvironments during and after translation, antibodies expressed by transgenes are post-translationally modified in a manner different from those delivered directly. Unbound by any particular theory, this results in antibodies with different diffusion, biological activity, distribution, affinity, pharmacokinetics, and immunogenicity properties, making antibodies delivered to the site of action "biologically superior" compared to those delivered directly by injection.

[0129] Furthermore, antibodies expressed in vivo by transgenes are unlikely to contain degradation products associated with antibodies produced by recombinant technologies such as protein aggregation and protein oxidation. Aggregation is a problem associated with protein production and storage due to high protein concentrations, interactions with the surfaces of manufacturing equipment and containers, and purification using certain buffer systems. These conditions that promote aggregation are not present in transgene expression in gene therapy. Oxidation, such as the oxidation of methionine, tryptophan, and histidine, is also associated with protein production and storage and is caused by stressful cell culture conditions, contact with metals and air, and impurities in buffers and excipients. Proteins expressed in vivo by transgenes can also be oxidized under stress conditions. However, humans and many other organisms possess antioxidant defense systems that not only reduce oxidative stress but also sometimes repair and / or reverse oxidation. Therefore, proteins produced in vivo are unlikely to be in oxidized form. Both aggregation and oxidation can affect potency, pharmacokinetics (clearance), and immunogenicity.

[0130] Unbound by theory, the methods and compositions presented in this paper are partly based on the following principles: (i) Human retinal cells are secretory cells with cellular mechanisms for post-translational processing of secreted proteins—including glycosylation and tyrosine-O-sulfation—which are powerful processes in retinal cells. (See, for example, Wang et al., 2013, Analytical Biochemistry 427: 20-28 and Adamis et al., 1993, BBRC 193: 631-638, which report the production of glycoproteins by retinal cells; and Kanan et al., 2009, Experimental Eye Research 89: 559-567 and Kanan and Al-Ubaidi, 2015, Experimental Eye Research 133: 126-131, which report the production of tyrosine-sulfated glycoproteins secreted by retinal cells, each of which is incorporated herein by reference in its entirety by reference to post-translational modifications performed by human retinal cells.)

[0131] (ii) Contrary to the prior art understanding, anti-VEGF antigen-binding fragments such as ranibizumab (and the Fab domains of full-length anti-VEGF mAbs, such as bevacizumab) do indeed have N-linked glycosylation sites. See, for example, [link to previous section]. Figure 1 It identified C H Structure Domain (TVSWN) 165 SGAL) and C L Structure Domain (QSGN) 158 The non-common asparagine (“N”) glycosylation site in SQE and the V as ranibizumab H Structure domain (Q) 115 GT) and V L TFQ (Transcription Domain) 100 Glutamine (“Q”) residues at glycosylation sites in GT (and corresponding sites in bevacizumab). (See, for example, Valliere-Douglass et al., 2009, Journal of Biochemistry 284: 32493-32506, and Valliere-Douglass et al., 2010, Journal of Biochemistry 285: 16012-16022, each of which is incorporated herein by reference in its entirety by reference to the identification of N-linked glycosylation sites in antibodies).

[0132] (iii) Although such non-canonical sites typically result in low levels of glycosylation in the antibody population (e.g., about 1-5%), functional benefits may be significant in immune-exempt organs such as the eye (see, for example, van de Bovenkamp et al., 2016, J. Immunol. 196:1435-1441). For example, Fab glycosylation may affect antibody stability, half-life, and binding properties. To determine the effect of Fab glycosylation on the antibody's affinity for its target, any technique known to those skilled in the art can be used, such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR). To determine the effect of Fab glycosylation on the antibody's half-life, any technique known to those skilled in the art can be used, for example, by measuring the level of radioactivity in the blood or organs (e.g., the eye) of a subject who has been administered a radiolabeled antibody. To determine the effect of Fab glycosylation on antibody stability, such as aggregation or protein unfolding levels, any technique known to those skilled in the art can be used, such as differential scanning calorimetry (DSC), high-performance liquid chromatography (HPLC), such as size exclusion HPLC (SEC-HPLC), capillary electrophoresis, mass spectrometry, or turbidity measurements. The HuPTMFabVEGFi described herein, such as HuGlyFabVEGFi, induced the generation of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more of Fab glycosylated at non-canonical sites. In some embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more of Fab from the Fab population are glycosylated at non-canonical sites. In some embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more of non-canonical sites are glycosylated. In some embodiments, the glycosylation of Fab at these non-canonical sites is 25%, 50%, 100%, 200%, 300%, 400%, 500%, or more greater than the amount of glycosylation at these non-canonical sites in Fab produced in HEK293 cells.

[0133] (iv) In addition to glycosylation sites, anti-VEGF Fabs such as ranibizumab (and bevacizumab Fab) contain tyrosine (“Y”) sulfated sites in or near the CDR; see also Figure 1 It identified the V of ranibizumab. H (EDTAVY) 94 Y 95 ) and V L (EDFATY) 86The tyrosine-O-sulfation site in the domain (and the corresponding site in the Fab of bevacizumab). (See, for example, Yang et al., 2015, Molecules 20:2138-2164, especially on page 2154, the literature is incorporated in its entirety by citing an analysis of the amino acids surrounding the tyrosine residues undergoing tyrosine sulfate in the protein. The “rule” can be summarized as follows: Y residues having E or D in the +5 to -5 positions of Y, and where position -1 of Y is a neutral or acidic charged amino acid—but not a basic amino acid, e.g., an eliminated sulfated R, K, or H). Human IgG antibodies can exhibit many other post-translational modifications, such as N-terminal modifications, C-terminal modifications, degradation or oxidation of amino acid residues, cysteine-related variants, and glycosylation (see, for example, Liu et al., 2014, Monoclonal Antibodies 6(5):1145-1154).

[0134] (v) Glycosylation of anti-VEGF Fab fragments such as ranibizumab or bevacizumab by human retinal cells results in the addition of glycans, which can improve the stability and half-life of transgenic products and reduce unwanted aggregation and / or immunogenicity. (For a review of emerging significance of Fab glycosylation, see, for example, Bovenkamp et al., 2016, Journal of Immunology 196: 1435-1441). Notably, the glycans presented herein that can be added to HuPTMFabVEGFi, such as HuGlyFabVEGFi, are highly processed complex-type biantennary N-glycans containing 2,6-sialic acid (see, for example, [link to article]). Figure 2The figure illustrates the incorporation of glycans into HuPTMFabVEGFi (e.g., HuGlyFabVEGFi) and bipartite GlcNAc, instead of NGNA (N-hydroxyacetylneuraminic acid, Neu5Gc). Such glycans are not present in ranibizumab (manufactured in E. coli and without any glycosylation) or bevacizumab (manufactured in CHO cells that do not possess the 2,6-sialyltransferase required for this post-translational modification), nor in CHO cell products that bipartite GlcNAc, but do contain added Neu5Gc (NGNA) because sialic acid is atypical for humans but not for Neu5Ac (NANA). See, for example, Dumont et al., 2015, Crit. Rev. Biotechnol. (early online, published online September 18, 2015, pp. 1–13, on page 5). Furthermore, CHO cells can also produce anti-α-Gal antibody responses present in most individuals and, at high concentrations, immunogenic glycans, namely α-Gal antigens, that can trigger allergic reactions. See, for example, Bosques, 2010, Nature Biotechnology 28: 1153-1156. The human glycosylation patterns of HuPTMFabVEGFi, such as HuGlyFabVEGFi, presented in this article should reduce the immunogenicity of transgenic products and improve their efficacy.

[0135] (vi) Tyrosine sulfation of anti-VEGF Fab fragments, such as those of ranibizumab or bevacizumab—a potent post-translational process in human retinal cells—can produce transgenic products with increased affinity for VEGF. Indeed, tyrosine sulfation of Fab fragments of therapeutic antibodies targeting other targets has shown significantly increased affinity and activity for antigens. (See, for example, Loos et al., 2015, Proceedings of the National Academy of Sciences 112: 12675-12680, and Choe et al., 2003, Cell 114: 161-170). This post-translational modification is not present in ranibizumab (which is produced in E. coli, a host lacking the enzymes required for tyrosine sulfation) and is at best underrepresented in bevacizumab—a CHO cell product. Unlike human retinal cells, CHO cells are not secretory cells and have limited capacity for post-translational tyrosine sulfation. (See, for example, Mikkelsen and Ezban, 1991, Biochemistry 30: 1533-1537, especially the discussion on page 1537).

[0136] For the aforementioned reasons, the generation of HuPTMFabVEGFi, such as HuGlyFabVEGFi, should result in the treatment of nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) achieved through gene therapy—for example, by administering the treatment to the suprachoroidal space, subretinal space, or sclera of the eye of a patient (human subject) diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR). Extracellular surface application of HuPTMFabVEGFi, such as a viral vector or other DNA expression construct of HuGlyFabVEGFi (e.g., via choroidal injection (e.g., via an on-choroidal drug delivery device such as a microinjector with microneedles), subretinal injection via the vitreous pathway (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior near-scleral reservoir procedure) – to generate in the eye a continuous supply of a fully human, post-translationally modified, such as human glycosylated, sulfated transgenic product, produced by transduced retinal cells, to a “bio-better” molecule. The cDNA construct of FabVEGFi should contain a signal peptide that ensures proper co-translation and post-translational processing (glycosylation and protein sulfate) by transduced retinal cells. Such signal sequences used by retinal cells may include, but are not limited to: ●MNFLLSWVHW SLALLLYLHH AKWSQA (VEGF-A signal peptide) (SEQ ID NO: 5) ●MERAAPSRRV PLPLLLLGGL ALLAAGVDA (Peronein-1 signal peptide) (SEQ ID NO: 6) ●MAPLRPLLIL ALLAWVALA (Bel-linkin signal peptide) (SEQ ID NO: 7) ●MRLLAKIICLMLWAICVA (Complement Factor H Signal Peptide) (SEQ ID NO: 8) ●MRLLAFLSLL ALVLQETGT (Optical Signal Peptide) (SEQ ID NO: 9) ●MKWVTFISLLFLFSSAYS (Albumin Signal Peptide) (SEQ ID NO: 22) ●MAFLWLLSCWALLGTTFG (chymotrypsinogen signal peptide) (SEQ ID NO: 23) ●MYRMQLLSCIALILALVTNS (Interleukin-2 signal peptide) (SEQ ID NO: 24) ●MNLLLILTFVAAAVA (trypsinogen-2 signal peptide) (SEQ ID NO: 25) ●MYRMQLLLLIALSLALVTNS (mutated interleukin-2 signal peptide) (SEQ ID NO: 52).

[0137] ●See, for example, Stern et al., 2007, Trends in Cell and Molecular Biology, 2:1-17 and Dalton and Barton, 2014, Protein Science, 23:517-525, each of which is incorporated herein by reference in its entirety by way of reference to the signal peptide that may be used.

[0138] As an alternative or adjunctive treatment to gene therapy, HuPTMFabVEGFi products, such as HuGlyFabVEGFi glycoprotein, can be generated in human cell lines using recombinant DNA technology and administered via intravitreal or subretinal injection to patients diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR). HuPTMFabVEGFi products, such as glycoproteins, can also be administered to patients with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR). Human cell lines that can be used for the production of such recombinant glycoproteins include, but are not limited to, human embryonic kidney 293 cells (HEK293), fibrosarcoma HT-1080, HKB-11, CAP, HuH-7, and retinal cell line PER.C6, or RPE, to name just a few (see, for example, Dumont et al., 2015, Key Reviews of Biotechnology (early online, published online on September 18, 2015, pp. 1–13) “Human cell lines for biopharmaceutical manufacturing: history, status and future prospects,” which is cited in its entirety as a review of human cell lines that can be used for the recombinant production of HuPTMFabVEGFi products, such as HuGlyFabVEGFi glycoproteins). To ensure complete glycosylation, especially sialylation and tyrosine sulfation, the cell lines used for production can be enhanced by engineering the host cells to co-express α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferase) and / or the TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation in retinal cells.

[0139] The methods described herein cover the delivery of HuPTMFabVEGFi, such as HuGlyFabVEGFi, to the eye / retina in combination with the delivery of other available treatments. Additional treatments can be administered before, concurrently with, or after gene therapy. Available treatments for nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) that can be combined with the gene therapy described herein include, but are not limited to, laser photocoagulation, photodynamic therapy with verteporfen, and intravitreal (IVT) injections with antiVEGF agents including, but not limited to, pilgartanib, ranibizumab, aflibercept, or bevacizumab. Additional treatment with antiVEGF agents such as biological agents may be referred to as “rescue” therapy.

[0140] Unlike small molecule drugs, biopharmaceuticals typically comprise mixtures of many variants with different modifications or forms, exhibiting varying potency, pharmacokinetics, and safety profiles. Each molecule produced in gene therapy or protein therapy approaches does not need to be fully glycosylated and sulfated. Instead, the resulting glycoprotein population should have sufficient glycosylation (from approximately 1% to approximately 10% of the population), including 2,6-sialylation, and sulfation to demonstrate efficacy. The gene therapy treatments presented in this article aim to slow or halt the progression of retinal degeneration and to slow or prevent vision loss with minimal intervention / invasive procedures. Efficacy can be monitored by measuring BCVA (best corrected visual acuity), intraocular pressure, slit-lamp biopsy, indirect ophthalmoscopy, SD-OCT (SD-optical coherence tomography), and electroretinography (ERG). Signs of vision loss, infection, inflammation, and other safety events, including retinal detachment, can also be monitored. Retinal thickness can be monitored to determine the efficacy of the treatments presented in this article. Without being bound by any particular theory, retinal thickness can be used as a clinical reading, where greater reduction in retinal thickness or a longer period before retinal thickening indicates a more effective treatment. For example, retinal thickness can be determined using SD-OCT. SD-OCT is a three-dimensional imaging technique that uses low-coherence interferometry to determine the echo time delay and magnitude of backscattered light reflected from an object of interest. OCT can be used to scan layers of tissue samples (e.g., the retina) at axial resolutions from 3 to 15 μm, and SD-OCT improves axial resolution and scan speed compared to previous forms of the technique (Schuman, 2008, *Proceedings of the American Academy of Ophthalmology* (Am. Opthamol. Soc.) 106:426-458). Retinal function can also be determined using ERG. ERG is an FDA-approved non-invasive electrophysiological test for retinal function in humans that examines the eye's light-sensitive cells (rods and cones) and their connecting ganglion cells, specifically examining the response to flashing stimuli.

[0141] In a preferred embodiment, the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal. As used herein, “detectable NeuGc and / or α-Gal” refers to the NeuGc and / or α-Gal moiety that can be detected by standard assays known in the art. For example, NeuGc can be detected by HPLC according to Hara et al., 1989, “Highly Sensitive Determination of N-Acetyl-and N-Glycolylneuraminic Acids in Human Serum and Urine and Rat Serum by Reversed-Phase Liquid Chromatography with Fluorescence Detection”, *Journal of Chromatography B: Biomed.* 377: 111–119, which is incorporated herein by reference to the method for detecting NeuGc. Alternatively, NeuGc can be detected by mass spectrometry.α-Gal can be detected using ELISA, see, for example, Galili et al., 1998, “A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody”, *Transplantation* 65(8):1129-32, or by mass spectrometry, see, for example, Ayoub et al., 2013, “Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques”, *Landes Bioscience* 5(5): 699–710. See also Platts-Mills et al., 2015, “Anaphylaxis to the Carbohydrate Side-Chain Alpha-gal”, Immunol Allergy Clin North Am. 35(2): 247–260.

[0142] In some respects, this document also provides anti-VEGF antigen-binding fragments (i.e., antigen-binding fragments that immunize against VEGF), said antigen-binding fragments comprising light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamylation (pyro Glu). In a specific embodiment, the antigen-binding fragment comprises the light chain CDR1-3 of SEQ ID NO: 14-16 and the heavy chain CDR1-3 of SEQ ID NO: 20, 18 and 21, wherein the eighth and eleventh amino acid residues of the light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO. 14)) each carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu), and the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu). In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18, and 21, wherein the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) is unacetylated. In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18, and 21, wherein the eighth and eleventh amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO. 14)) each carry one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyro Glu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) is unacetylated. The anti-VEGF antigen-binding fragments provided herein can be used in any of the methods of the invention described herein. In a preferred embodiment, the chemical modifications described herein, or the absence of such modifications (as applicable), are determined by mass spectrometry.

[0143] In some respects, this document also provides anti-VEGF antigen-binding fragments comprising light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamylation (pyro Glu). In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO. 20)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamate (pyro Glu), the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO. 18)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamate (pyro Glu), and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu). In a specific embodiment, the antigen-binding fragment includes light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) is unacetylated. In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO. 20)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyro Glu), the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO. 18)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyro Glu), and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) is unacetylated.The anti-VEGF antigen-binding fragments provided herein can be used in any of the methods of the invention described herein. In a preferred embodiment, the chemical modifications described herein, or the absence of such modifications (as applicable), are determined by mass spectrometry.

[0144] In some respects, this document also provides an anti-VEGF antigen-binding fragment comprising light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu). In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein: (1) the ninth amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO. 20)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyro Glu), the third amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO. 18)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyro Glu), and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamylation (pyro Glu). (2) The eighth and eleventh amino acid residues of the light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO.14)) each carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu), and the second amino acid residue of the light chain CDR3 (i.e. the second Q in QQYSTVPWTF (SEQ ID NO.16)) does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation and pyroglutamate (pyro Glu).In a specific embodiment, the antigen-binding fragment comprises light chain CDRs 1-3 of SEQ ID NO: 14-16 and heavy chain CDRs 1-3 of SEQ ID NO: 20, 18 and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) is unacetylated, and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) is unacetylated. In a specific embodiment, the antigen-binding fragment comprises the light chain CDR1-3 of SEQ ID NO: 14-16 and the heavy chain CDR1-3 of SEQ ID NO: 20, 18 and 21, wherein: (1) the ninth amino acid residue of the heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO. 20)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyro Glu), the third amino acid residue of the heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO. 18)) carries one or more of the following chemical modifications: acetylation, deamidation and pyroglutamylation (pyro Glu), and the last amino acid residue of the heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO. 20)) is not acetylated; and (2) the eighth and eleventh amino acid residues of the light chain CDR1 (i.e., SASQDISNYLN (SEQ ID NO. 20)) The two N's in NO. 14 each carry one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyro Glu), and the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO. 16)) is unacetylated. The anti-VEGF antigen-binding fragment provided herein can be used in any method of the invention described herein. In preferred embodiments, the chemical modifications described herein, or the absence of such modifications (as applicable), are determined by mass spectrometry.

[0145] Another proposed administration route is subretinal drug delivery via the suprachoroidal space using a subretinal drug delivery device with a catheter inserted posteriorly and tunneling through the suprachoroidal space to inject into the subretinal space, with a small needle inserted into the subretinal space at the posterior pole. This route allows the vitreous to remain intact, thus reducing the risk of complications (lower risk of gene therapy outflow and complications such as retinal detachment and macular holes), and the resulting bubble may diffuse more widely without vitrectomy, allowing transduction of a larger retinal surface area with a smaller volume. The risk of subsequent cataract induction is minimized, which is desirable in younger patients. Furthermore, this procedure allows for safer delivery of the bubble in the fovea compared to the standard transvitreal approach, which is desirable for patients with inherited retinal diseases affecting central vision, especially when the target cells for transduction are located in the macula. This procedure is also advantageous for patients with neutralizing antibodies (Nab) against AAV in systemic circulation, which could affect other delivery routes. In addition, this method has been shown to produce fewer air bubbles at the retinal incision site compared to the standard transvitreal approach.

[0146] Near-scleral application provides an additional route of administration that avoids the risks of intraocular infection and retinal detachment, side effects typically associated with injecting therapeutic agents directly into the eye.

[0147] In some embodiments, a kit is described herein comprising one or more containers and instructions for use, wherein the one or more containers comprise a pharmaceutical composition. In some embodiments, at least one of the one or more containers is made of a hydrophobically coated glass vial. In some embodiments, at least one of the one or more containers is made of a Daikyo Crystal Zenith® (CZ) vial. In some embodiments, at least one of the one or more containers is made of a TopLyo-coated vial. In some embodiments, at least one of the one or more containers is made of a cyclic olefin polymer (COP).

[0148] On the other hand, this article provides a single-unit dosage form comprising 3.2 × 10 11 GC / mL, 6.5 × 10 11 GC / mL, 2.5 × 10 12 GC / mL, 3 × 10 13The single-unit dosage form comprises GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, pH 7.4, and 0.001% P188, and is contained in a cyclic olefin polymer (COP) vial with a volume of at least about 0.5 mL, at least about 0.8 mL, about 0.6 mL, and about 0.95 mL. In some embodiments, the single-unit dosage form can be stored at 4°C for 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, and about 2 months. In some embodiments, the single-unit dosage form can be stored at -80°C for about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, and about 24 months. In some embodiments, the single-unit dosage form is capable of being stored at 4°C for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or 2 months after being previously stored at -80°C for approximately 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, or 24 months. In some embodiments, the vector genome concentration of construct II after being stored at -80°C, -20°C, or 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of construct II before being stored at -80°C, -20°C, or 4°C for the said period of time. In some embodiments, the in vitro potency of construct II after storage at -80°C, -20°C, or 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of construct II before storage at -80°C, -20°C, or 4°C for the said period of time. In some embodiments, the size distribution of construct II after storage at -80°C, -20°C, or 4°C for the said period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the size distribution of construct II before storage at -80°C, -20°C, or 4°C for the said period of time. In some embodiments, the time period is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0149] On the other hand, this article provides a single-unit dosage form comprising 3.2 × 10 11 GC / mL, 6.5 × 10 11 GC / mL, 2.5 × 10 12 GC / mL, 3 × 1013 The single-unit dosage form comprises GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, pH 7.4, 4% sucrose, and 0.001% P188, and is contained in COP vials with volumes of at least about 0.5 mL, at least about 0.8 mL, about 0.6 mL, and about 0.95 mL. In some embodiments, the single-unit dosage form can be stored at 4°C for 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, and about 2 months. In some embodiments, the single-unit dosage form can be stored at -80°C for about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, and about 24 months. In some embodiments, the single-unit dosage form is capable of being stored at 4°C for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or 2 months after being previously stored at -80°C for approximately 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, or 24 months. In some embodiments, the vector genome concentration of construct II after being stored at -80°C, -20°C, or 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of construct II before being stored at -80°C, -20°C, or 4°C for the said period of time. In some embodiments, the in vitro potency of construct II after storage at -80°C, -20°C, or 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of construct II before storage at -80°C, -20°C, or 4°C for the said period of time. In some embodiments, the size distribution of construct II after storage at -80°C, -20°C, or 4°C for the said period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the size distribution of construct II before storage at -80°C, -20°C, or 4°C for the said period of time. In some embodiments, the time period is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0150] On the other hand, this article provides a pre-filled syringe containing the single-unit dosage form provided herein. On the other hand, this article provides a kit comprising a pre-filled syringe containing the single-unit dosage form provided herein.

[0151] 2.1 Illustrative Examples 1. A pharmaceutical composition comprising: (a) Recombinant adeno-associated virus (AAV); (b) Potassium chloride; (c) Potassium dihydrogen phosphate; (d) Sodium chloride; (e) Anhydrous disodium hydrogen phosphate; (f) sucrose; and (e) Poloxamer 188, polysorbate 20 or polysorbate 80.

[0152] 2. The pharmaceutical composition according to paragraph 1, wherein the recombinant AAV comprises a component from one or more adeno-associated virus serotypes selected from the group consisting of: AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7 m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV .HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 and AAV.HSC16.

[0153] 3. The pharmaceutical composition according to any one of paragraphs 1 to 2, wherein the recombinant AAV is AAV8.

[0154] 4. The pharmaceutical composition according to any one of paragraphs 1 to 2, wherein the recombinant AAV is AAV9.

[0155] 5. The pharmaceutical composition according to any one of paragraphs 1 to 4, wherein the pharmaceutical composition further comprises one or more amino acids.

[0156] 6. The pharmaceutical composition according to any one of paragraphs 1 to 5, wherein the ionic strength of the pharmaceutical composition is in the range of about 60 mM to about 115 mM.

[0157] 7. The pharmaceutical composition according to paragraph 4, wherein the ionic strength of the pharmaceutical composition is in the range of about 30 mM to about 100 mM.

[0158] 8. The pharmaceutical composition according to any one of paragraphs 1 to 3, wherein the pharmaceutical composition comprises: (a) Potassium chloride with a concentration of 0.2 g / L; (b) Potassium dihydrogen phosphate at a concentration of 0.2 g / L; (c) Sodium chloride at a concentration of 5.84 g / L; and (d) Anhydrous disodium hydrogen phosphate with a concentration of 1.15 g / L.

[0159] 9. The pharmaceutical composition according to any one of paragraphs 1 to 8, wherein the pharmaceutical composition comprises sucrose in a concentration ranging from 3% (weight / volume, 30 g / L) to 18% (weight / volume, 180 g / L).

[0160] 10. The pharmaceutical composition according to any one of paragraphs 1 to 8, wherein the pharmaceutical composition comprises sucrose at a concentration of 4% (w / v, 40 g / L).

[0161] 11. The pharmaceutical composition according to any one of paragraphs 1 to 10, wherein the pharmaceutical composition comprises poloxamer 188, polysorbate 20, or polysorbate 80; and wherein the concentration of said poloxamer 188, polysorbate 20, or polysorbate 80 is in the range of 0.0005% (w / v, 0.005 g / L) to 0.05% (w / v, 0.5 g / L).

[0162] 12. The pharmaceutical composition according to any one of paragraphs 1 to 10, wherein the pharmaceutical composition comprises poloxamer 188, polysorbate 20 or polysorbate 80; and wherein the concentration of said poloxamer 188, polysorbate 20 or polysorbate 80 is 0.001% (weight / volume, 0.01 g / L).

[0163] 13. The pharmaceutical composition according to any one of paragraphs 48 to 54, wherein the pH of the pharmaceutical composition is in the range of about 6.0 to about 9.0.

[0164] 14. The pharmaceutical composition according to any one of paragraphs 1 to 12, wherein the pH of the pharmaceutical composition is about 7.4.

[0165] 15. The pharmaceutical composition according to any one of paragraphs 1 to 14, wherein the osmotic pressure of the pharmaceutical composition is in the range of about 200 mOsm / L to about 660 mOsm / L.

[0166] 16. The pharmaceutical composition according to any one of paragraphs 1 to 15, wherein the pharmaceutical composition is contained in a hydrophobically coated glass vial.

[0167] 17. The pharmaceutical composition according to any one of paragraphs 1 to 15, wherein the pharmaceutical composition is contained in a cyclic olefin polymer (COP) vial.

[0168] 18. The pharmaceutical composition according to any one of paragraphs 1 to 15, wherein the pharmaceutical composition is contained in a DaikyoCrystal Zenith® (CZ) vial.

[0169] 19. The pharmaceutical composition according to any one of paragraphs 1 to 15, wherein the pharmaceutical composition is contained in a TopLyo-coated vial.

[0170] 20. The pharmaceutical composition according to any one of paragraphs 1 to 19, wherein the pharmaceutical composition comprises: (a) The recombinant AAV; (b) Potassium chloride with a concentration of 0.2 g / L; (c) Potassium dihydrogen phosphate at a concentration of 0.2 g / L; (d) Sodium chloride with a concentration of 5.84 g / L; (e) Anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L; (f) Sucrose at a concentration of 4% by weight / volume (40 g / L); (g) Poloxamer 188, polysorbate 20, or polysorbate 80 at a concentration of 0.001% by weight / volume (0.01 g / L); and (h) water, and The recombinant AAV mentioned above is AAV8.

[0171] 21. The pharmaceutical composition according to any one of paragraphs 1 to 20, wherein the vector genome concentration (VGC) of the pharmaceutical composition is about 3 × 10⁻⁶. 9 GC / mL, approximately 1 × 10 10 GC / mL, approximately 1.2 × 10⁻⁶ 10 GC / mL, approximately 1.6 × 10 10 GC / mL, approximately 4 × 10⁻⁶ 10 GC / mL, approximately 6 × 10 10 GC / mL, approximately 2 × 10 11 GC / mL, approximately 2.4 × 10⁻⁶ 11 GC / mL, approximately 2.5 × 10⁻⁶ 11 GC / mL, approximately 3 × 1011 GC / mL, approximately 3.2 × 10⁻⁶ 11 GC / mL, approximately 6.2 × 10⁻⁶ 11 GC / mL, approximately 6.5 × 10 11 GC / mL, approximately 1 × 10 12 GC / mL, approximately 3 × 10 12 GC / mL, approximately 2 × 10 13 GC / mL or approximately 3 × 10⁻⁶ 13 GC / mL.

[0172] 22. The pharmaceutical composition according to any one of paragraphs 1 to 21, wherein the recombinant AAV has a stability to freeze / thaw cycles that is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition.

[0173] 23. The pharmaceutical composition according to any one of paragraphs 1 to 22, wherein when stored at -20°C for a sustained period of time, the recombinant AAV is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in the reference pharmaceutical composition. The period of time is approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 12 months, approximately 15 months, approximately 18 months, or approximately 24 months.

[0174] 24. The pharmaceutical composition according to any one of paragraphs 1 to 23, wherein when stored at -80°C for a sustained period of time, the recombinant AAV is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in the reference pharmaceutical composition. The period of time is approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 12 months, approximately 15 months, approximately 18 months, or approximately 24 months.

[0175] 25. The pharmaceutical composition according to any one of paragraphs 1 to 24, wherein when stored at room temperature for a sustained period of time, the recombinant AAV is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. The period of time is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0176] 26. The pharmaceutical composition according to any one of paragraphs 1 to 25, wherein the recombinant AAV is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in the reference pharmaceutical composition when performed by: (i) storage at -80°C for a first time period; (ii) subsequent thawing; and (iii) storage at 4°C for a second time period after thawing.

[0177] 27. The pharmaceutical composition according to paragraph 26, wherein the first time period is about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0178] 28. The pharmaceutical composition according to paragraph 26, wherein the second time period is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, or about 2 months.

[0179] 29. The pharmaceutical composition according to any one of paragraphs 1 to 22, wherein the vector genome concentration of the recombinant AAV after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at -80°C for the period of time.

[0180] 30. The pharmaceutical composition according to any one of paragraphs 1 to 22, wherein the vector genome concentration of the recombinant AAV after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at -20°C for the period of time.

[0181] 31. The pharmaceutical composition according to any one of paragraphs 1 to 22, wherein the vector genome concentration of the recombinant AAV after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at 4°C for the period of time.

[0182] 32. The pharmaceutical composition according to any one of paragraphs 1 to 22, wherein the in vitro potency of the recombinant AAV after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of the recombinant AAV before being stored at -80°C for the period of time.

[0183] 33. The pharmaceutical composition according to any one of paragraphs 1 to 22, wherein the in vitro potency of the recombinant AAV after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of the recombinant AAV before being stored at -20°C for the period of time.

[0184] 34. The pharmaceutical composition according to any one of paragraphs 1 to 22, wherein the in vitro potency of the recombinant AAV after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of the recombinant AAV before being stored at 4°C for the period of time.

[0185] 35. The pharmaceutical composition according to any one of paragraphs 1 to 22, wherein the size distribution of the recombinant AAV after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of the recombinant AAV before being stored at -80°C for the period of time.

[0186] 36. The pharmaceutical composition according to any one of paragraphs 1 to 22, wherein the size distribution of the recombinant AAV after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of the recombinant AAV before being stored at -20°C for the period of time.

[0187] 37. The pharmaceutical composition according to any one of paragraphs 1 to 22, wherein the size distribution of the recombinant AAV after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of the recombinant AAV before being stored at 4°C for the period of time.

[0188] 38. The pharmaceutical composition according to any one of paragraphs 29 to 37, wherein the time period is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0189] 39. The pharmaceutical composition according to any one of paragraphs 1 to 21, wherein the stability of the recombinant AAV is determined by the infectivity of the recombinant AAV.

[0190] 40. The pharmaceutical composition according to any one of paragraphs 1 to 21, wherein the stability of the recombinant AAV is determined by the aggregation level of the recombinant AAV.

[0191] 41. The pharmaceutical composition according to any one of paragraphs 1 to 21, wherein the stability of the recombinant AAV is determined by the level of free DNA released by the recombinant AAV particles.

[0192] 42. The pharmaceutical composition according to any one of paragraphs 1 to 41, wherein the pharmaceutical composition is a liquid composition.

[0193] 43. The pharmaceutical composition according to any one of paragraphs 1 to 41, wherein the pharmaceutical composition is a frozen composition.

[0194] 44. The pharmaceutical composition according to any one of paragraphs 1 to 41, wherein the pharmaceutical composition is a lyophilized composition or a reconstituted lyophilized composition.

[0195] 45. The pharmaceutical composition according to any one of paragraphs 1 to 44, wherein the pharmaceutical composition is suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or posterior proximal scleral reservoir procedures.

[0196] 46. ​​The pharmaceutical composition according to any one of paragraphs 1 to 44, wherein the pharmaceutical composition has a desired density suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or posterior proximal scleral reservoir procedures.

[0197] 47. The pharmaceutical composition according to any one of paragraphs 1 to 44, wherein the pharmaceutical composition has a desired osmotic pressure suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or posterior proximal scleral reservoir procedures.

[0198] 48. The pharmaceutical composition according to paragraph 47, wherein the osmotic pressure is 160-230 mOsm / kg H2O.

[0199] 49. The pharmaceutical composition according to paragraph 47, wherein the osmotic pressure is less than 600 mOsm / kg H2O.

[0200] 50. The pharmaceutical composition according to any one of paragraphs 1 to 44, wherein the pharmaceutical composition has a desired viscosity suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or posterior proximal scleral reservoir procedures.

[0201] 51. The pharmaceutical composition according to any one of paragraphs 1 to 44, wherein the pharmaceutical composition is suitable for application to the eye.

[0202] 52. The pharmaceutical composition according to paragraph 45 is suitable for suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or posterior near-scleral reservoir procedures.

[0203] 53. The pharmaceutical composition according to any one of paragraphs 1 to 52, wherein the pharmaceutical composition is capable of being stored at 4°C for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or 2 months after having been previously stored at -80°C for about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0204] 54. A method of treating or preventing a disease in a subject, the method comprising administering to the subject a pharmaceutical composition according to any one of paragraphs 1 to 53.

[0205] 55. A method of treating a subject diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising administering to the subject a pharmaceutical composition according to any one of paragraphs 1 to 53.

[0206] 56. A method of treating a subject diagnosed with: mucopolysaccharidosis type IVA (MPS IVA), mucopolysaccharidosis type I (MPS I), mucopolysaccharidosis type II (MPS II), familial hypercholesterolemia (FH), homozygous familial hypercholesterolemia (HoFH), coronary artery disease, cerebrovascular disease, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, and sporadic inclusion body myositis or kallikrein-related disease, said method comprising administering to said subject a pharmaceutical composition according to any one of paragraphs 1 to 53.

[0207] 57. The method according to any one of paragraphs 54 to 56, wherein the pharmaceutical composition is administered via intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or posterior proximal scleral reservoir procedure.

[0208] 58. The method according to any one of paragraphs 54 to 57, wherein the subject is a human subject.

[0209] 59. A method of treating a subject diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising (ii) preparing a pharmaceutical composition according to any one of paragraphs 1 to 53, storing the pharmaceutical composition at -80°C for a first time period; (ii) thawing the pharmaceutical composition; and (iii) after thawing, storing the pharmaceutical composition at 4°C for a second time period.

[0210] 60. The method according to paragraph 59, wherein the first time period is about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0211] 61. The method according to paragraph 59, wherein the second time period is approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 1 month, or approximately 2 months.

[0212] 62. A kit comprising one or more containers and instructions for use, wherein the one or more containers comprise a pharmaceutical composition according to any one of paragraphs 1 to 53.

[0213] 63. The kit according to paragraph 62, wherein at least one of the one or more containers is made of a hydrophobically coated glass vial.

[0214] 64. The kit according to paragraph 62, wherein at least one of the one or more containers is made of Daikyo Crystal Zenith® (CZ) vials.

[0215] 65. The kit according to paragraph 62, wherein at least one of the one or more containers is made of a TopLyo-coated vial.

[0216] 66. The kit according to paragraph 62, wherein at least one of the one or more containers is made of a cyclic olefin polymer (COP).

[0217] 67. A pharmaceutical composition comprising: (a) Construct II encoding an antibody against human vascular endothelial growth factor (hVEGF); (b) Potassium chloride; (c) Potassium dihydrogen phosphate; (d) Sodium chloride; (e) Anhydrous disodium hydrogen phosphate; (f) sucrose; and (e) Poloxamer 188, polysorbate 20 or polysorbate 80, and The anti-hVEGF antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, and the light chain comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.

[0218] 68. The pharmaceutical composition according to paragraph 67, wherein the ionic strength of the pharmaceutical composition is in the range of about 60 mM to about 100 mM.

[0219] 69. The pharmaceutical composition according to any one of paragraphs 67 to 68, wherein the pharmaceutical composition comprises: (a) Potassium chloride with a concentration of 0.2 g / L; (b) Potassium dihydrogen phosphate at a concentration of 0.2 g / L; (c) Sodium chloride at a concentration of 5.84 g / L; and (d) Anhydrous disodium hydrogen phosphate with a concentration of 1.15 g / L.

[0220] 70. The pharmaceutical composition according to any one of paragraphs 67 to 69, wherein the pharmaceutical composition comprises sucrose in a concentration ranging from 3% (weight / volume, 30 g / L) to 18% (weight / volume, 180 g / L).

[0221] 71. The pharmaceutical composition according to any one of paragraphs 67 to 69, wherein the pharmaceutical composition comprises sucrose at a concentration of 4% (by weight / volume, 40 g / L).

[0222] 72. The pharmaceutical composition according to any one of paragraphs 67 to 71, wherein the pharmaceutical composition comprises poloxamer 188, polysorbate 20, or polysorbate 80; and wherein the concentration of said poloxamer 188, polysorbate 20, or polysorbate 80 is in the range of 0.0005% (w / v, 0.005 g / L) to 0.05% (w / v, 0.5 g / L).

[0223] 73. The pharmaceutical composition according to any one of paragraphs 67 to 71, wherein the pharmaceutical composition comprises poloxamer 188, polysorbate 20 or polysorbate 80 at a concentration of 0.001% (w / v, 0.01 g / L).

[0224] 74. The pharmaceutical composition according to any one of paragraphs 67 to 73, wherein the pH of the pharmaceutical composition is in the range of about 6.0 to about 9.0.

[0225] 75. The pharmaceutical composition according to any one of paragraphs 67 to 73, wherein the pH of the pharmaceutical composition is about 7.4.

[0226] 76. The pharmaceutical composition according to any one of paragraphs 67 to 75, wherein the osmotic pressure of the pharmaceutical composition is in the range of about 200 mOsm / L to about 660 mOsm / L.

[0227] 77. The pharmaceutical composition according to any one of paragraphs 67 to 76, wherein said construct II comprises a capsid from AAV8.

[0228] 78. The pharmaceutical composition according to any one of paragraphs 67 to 77, wherein the construct II has at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher stability to freeze / thaw cycles compared to the same construct II in the reference pharmaceutical composition.

[0229] 78. The pharmaceutical composition according to any one of paragraphs 67 to 77, wherein when stored at –20°C for a sustained period of time, said construct II is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same construct II in the reference pharmaceutical composition. The period of time is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0230] 79. The pharmaceutical composition according to any one of paragraphs 67 to 77, wherein when stored at –80°C for a sustained period of time, said construct II is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same construct II in the reference pharmaceutical composition. The period of time is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0231] 80. The pharmaceutical composition according to any one of paragraphs 67 to 77, wherein, when stored at room temperature for a sustained period of time, said construct II is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same construct II in a reference pharmaceutical composition. The period of time is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0232] 81. The pharmaceutical composition according to any one of paragraphs 67 to 77, wherein the recombinant AAV is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same construct II in the reference pharmaceutical composition when performed: (i) when stored at -80°C for a first time period; (ii) subsequently thawed; and (iii) after thawing, stored at 4°C for a second time period.

[0233] 82. The pharmaceutical composition according to paragraph 81, wherein the first time period is about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0234] 83. The pharmaceutical composition according to paragraph 81, wherein the second time period is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, or about 2 months.

[0235] 84. The pharmaceutical composition according to any one of paragraphs 67 to 79, wherein the vector genome concentration after the construct II has been stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration before the construct II has been stored at -80°C for the period of time.

[0236] 85. The pharmaceutical composition according to any one of paragraphs 67 to 79, wherein the vector genome concentration after the construct II has been stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration before the construct II has been stored at -20°C for the period of time.

[0237] 86. The pharmaceutical composition according to any one of paragraphs 67 to 79, wherein the vector genome concentration after the construct II is stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration before the construct II is stored at 4°C for the period of time.

[0238] 87. The pharmaceutical composition according to any one of paragraphs 67 to 79, wherein the in vitro potency of said construct II after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of said construct II before being stored at -80°C for the period of time.

[0239] 88. The pharmaceutical composition according to any one of paragraphs 67 to 79, wherein the in vitro potency of said construct II after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of said construct II before being stored at -20°C for the period of time.

[0240] 89. The pharmaceutical composition according to any one of paragraphs 67 to 79, wherein the in vitro potency of said construct II after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of said construct II before being stored at 4°C for the period of time.

[0241] 90. The pharmaceutical composition according to any one of paragraphs 67 to 79, wherein the size distribution of said construct II after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of said construct II before being stored at -80°C for the period of time.

[0242] 91. The pharmaceutical composition according to any one of paragraphs 67 to 79, wherein the size distribution of said construct II after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of said construct II before being stored at -20°C for the said period of time.

[0243] 92. The pharmaceutical composition according to any one of paragraphs 67 to 79, wherein the size distribution of said construct II after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of said construct II before being stored at 4°C for the said period of time.

[0244] 93. The pharmaceutical composition according to any one of paragraphs 84 to 92, wherein the time period is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0245] 94. The pharmaceutical composition according to any one of paragraphs 77 to 83, wherein the stability of said construct II is determined by the infectivity of recombinant AAV.

[0246] 95. The pharmaceutical composition according to any one of paragraphs 77 to 83, wherein the stability of said construct II is determined by the aggregation level of recombinant AAV.

[0247] 96. The pharmaceutical composition according to any one of paragraphs 77 to 83, wherein the stability of said construct II is determined by the level of free DNA released by the recombinant AAV particles.

[0248] 97. The pharmaceutical composition according to any one of paragraphs 67 to 96, wherein the pharmaceutical composition is contained in a hydrophobically coated glass vial.

[0249] 98. The pharmaceutical composition according to any one of paragraphs 67 to 96, wherein the pharmaceutical composition is contained in a cyclic olefin polymer (COP) vial.

[0250] 99. The pharmaceutical composition according to any one of paragraphs 67 to 96, wherein the pharmaceutical composition is contained in a Daikyo Crystal Zenith® (CZ) vial.

[0251] 100. The pharmaceutical composition according to any one of paragraphs 67 to 96, wherein the pharmaceutical composition is in a TopLyo-coated vial.

[0252] 101. The pharmaceutical composition according to any one of paragraphs 67 to 100, wherein the pharmaceutical composition comprises: (a) Construct II encoding the antibody against human vascular endothelial growth factor (hVEGF); (b) Potassium chloride with a concentration of 0.2 g / L; (c) Potassium dihydrogen phosphate at a concentration of 0.2 g / L; (d) Sodium chloride with a concentration of 5.84 g / L; (e) Anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L; (f) Sucrose at a concentration of 4% by weight / volume (40 g / L); (g) Poloxamer 188, polysorbate 20, or polysorbate 80 at a concentration of 0.001% by weight / volume (0.01 g / L); and (h) water, and The anti-hVEGF antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, and the light chain comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.

[0253] 102. The pharmaceutical composition according to paragraph 101, wherein the vector genome concentration (VGC) of the pharmaceutical composition is about 3 × 10⁻⁶. 9 GC / mL, approximately 1 × 10 10 GC / mL, approximately 1.2 × 10⁻⁶ 10 GC / mL, approximately 1.6 × 10⁻⁶ 10 GC / mL, approximately 4 × 10⁻⁶ 10 GC / mL, approximately 6 × 10 10 GC / mL, approximately 2 × 10 11 GC / mL, approximately 2.4 × 10⁻⁶ 11 GC / mL, approximately 2.5 × 10⁻⁶ 11 GC / mL, approximately 3 × 10 11 GC / mL, approximately 3.2 × 10⁻⁶ 11 GC / mL, approximately 6.2 × 10⁻⁶ 11 GC / mL, approximately 6.5 × 10 11 GC / mL, approximately 1 × 10 12 GC / mL, approximately 3 × 10 12 GC / mL, approximately 2 × 10 13 GC / mL or approximately 3 × 10 13 GC / mL.

[0254] 103. The pharmaceutical composition according to any one of paragraphs 67 to 102, wherein the pharmaceutical composition is suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or posterior proximal scleral reservoir procedures.

[0255] 104. The pharmaceutical composition according to any one of paragraphs 67 to 102, wherein the pharmaceutical composition has a desired density suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or posterior proximal scleral reservoir procedures.

[0256] 105. The pharmaceutical composition according to any one of paragraphs 67 to 102, wherein the pharmaceutical composition has a desired osmotic pressure suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or posterior proximal scleral reservoir procedures.

[0257] 106. The pharmaceutical composition according to paragraph 105, wherein the osmotic pressure is 160-230 mOsm / kg H2O.

[0258] 107. The pharmaceutical composition according to paragraph 105, wherein the osmotic pressure is less than 600 mOsm / kg H2O.

[0259] 108. The pharmaceutical composition according to any one of paragraphs 67 to 102, wherein the pharmaceutical composition has a desired viscosity suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or posterior proximal scleral reservoir procedures.

[0260] 109. The pharmaceutical composition according to any one of paragraphs 67 to 108, wherein the pharmaceutical composition is a liquid composition.

[0261] 110. The pharmaceutical composition according to any one of paragraphs 67 to 108, wherein the pharmaceutical composition is a frozen composition.

[0262] 111. The pharmaceutical composition according to any one of paragraphs 67 to 108, wherein the pharmaceutical composition is a lyophilized composition or a reconstituted lyophilized composition.

[0263] 112. The pharmaceutical composition according to any one of paragraphs 67 to 111, wherein the pharmaceutical composition is capable of being stored at 4°C for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or 2 months after having been previously stored at -80°C for about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0264] 113. A method of treating a subject diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising administering to the subject a pharmaceutical composition according to any one of paragraphs 67 to 111.

[0265] 114. The method according to paragraph 113, wherein the pharmaceutical composition is administered via suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, or a posterior proximal scleral reservoir procedure.

[0266] 115. The method according to any one of paragraphs 113 or 114, wherein the subject is a human subject.

[0267] 116. A method of treating a subject diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising (ii) preparing a pharmaceutical composition according to any one of paragraphs 67 to 108, storing the pharmaceutical composition at -80°C for a first time period; (iii) thawing the pharmaceutical composition; and (iv) after thawing, storing the pharmaceutical composition at 4°C for a second time period.

[0268] 117. The method according to paragraph 119, wherein the first time period is about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0269] 118. The method according to paragraph 119, wherein the second time period is approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 1 month, or approximately 2 months.

[0270] 119. A kit comprising one or more containers and instructions for use, wherein the one or more containers comprise a pharmaceutical composition according to any one of paragraphs 67 to 102.

[0271] 120. The kit according to paragraph 119, wherein at least one of the one or more containers is made of a cyclic olefin polymer (COP).

[0272] 121. A stable liquid pharmaceutical composition comprising: (a) Recombinant adeno-associated virus (rAAV); (b) Buffers that include ionic salts and have an ionic strength between 60 mM and 150 mM; (d) sucrose; and (e) Surfactants.

[0273] 122. The composition according to paragraph 121, wherein the rAAV comprises AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7m8, AAV.PHP.B, A AV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, A AV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 or AAV.HSC16.

[0274] 123. The composition according to paragraph 121 or 122, wherein the composition comprises 3-16% sucrose.

[0275] 124. The composition according to any one of paragraphs 121 to 123, wherein the buffer maintains the pH between about pH 6 and about pH 9 in a temperature range of -20°C to room temperature.

[0276] 125. The composition according to any one of paragraphs 121 to 124, wherein the composition comprises 4-6% sucrose.

[0277] 126. The composition according to any one of paragraphs 121 to 125, wherein the ionic strength of the buffer is not greater than about 150 mM, about 145 mM, about 140 mM, about 135 mM, about 130 mM, about 125 mM, about 120 mM, about 115 mM or about 110 mM.

[0278] 127. The composition according to any one of paragraphs 121 to 126, wherein the ionic strength of the buffer is between about 60 mM and about 115 mM.

[0279] 128. The combination of any one of paragraphs 121 to 127, wherein the ionic strength of the buffer is between about 60 mM and about 110 mM.

[0280] 129. The composition according to any one of paragraphs 121 to 128, wherein the composition comprises NaCl between 60 mM and 100 mM.

[0281] 130. The composition according to any one of paragraphs 121 to 129, wherein the composition comprises 4-6% sucrose.

[0282] 131. The combination according to any one of paragraphs 121 to 130, wherein the composition is frozen to a temperature of about -20°C.

[0283] 132. The composition according to any one of paragraphs 121 to 131, wherein the freezing composition maintains the pH between about pH 6 and about pH 9.

[0284] 133. The composition according to any one of paragraphs 121 to 132, wherein the buffer comprises one or more components selected from the group consisting of: potassium dihydrogen phosphate, potassium phosphate, sodium chloride, anhydrous disodium hydrogen phosphate, sodium phosphate hexahydrate, sodium dihydrogen phosphate monohydrate, sodium phosphate hexahydrate, sodium dihydrogen phosphate monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), amino acids, histidine, histidine hydrochloride (histidine-HCl), sodium succinate, sodium citrate, sodium acetate, and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), sodium sulfate, magnesium sulfate, magnesium chloride 6-hydrate, calcium sulfate, potassium chloride, calcium chloride, and calcium citrate.

[0285] 134. The composition according to any one of paragraphs 121 to 132, wherein the buffer comprises potassium chloride, potassium dihydrogen phosphate, sodium chloride, and anhydrous disodium hydrogen phosphate.

[0286] 135. The composition according to any one of paragraphs 121 to 132, wherein the buffer comprises sodium chloride and Tris hydrochloride.

[0287] 136. The composition according to any one of paragraphs 121 to 133, wherein the surfactant is poloxamer 188, polysorbate 20, or polysorbate 80.

[0288] 137. The composition according to any one of paragraphs 121 to 133, wherein the surfactant is poloxamer 188, polysorbate 20, or polysorbate 80; and wherein the concentration of said poloxamer 188, polysorbate 20, or polysorbate 80 is in the range of 0.005% (w / v, 0.005 g / L) to 0.05% (w / v, 0.5 g / L).

[0289] 138. The composition according to any one of paragraphs 121 to 133, wherein the surfactant is poloxamer 188, polysorbate 20 or polysorbate 80; and wherein the concentration of said poloxamer 188, polysorbate 20 or polysorbate 80 is 0.001% (w / v, 0.01 g / L).

[0290] 139. The composition according to any one of paragraphs 121 to 138, wherein the liquid pharmaceutical composition is further lyophilized.

[0291] 140. The composition according to any one of paragraphs 121 to 138, wherein the liquid pharmaceutical composition is a reconstituted lyophilized powder.

[0292] 141. The pharmaceutical composition according to any one of paragraphs 121 to 140, wherein the vector genome concentration of the recombinant AAV after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at -80°C for the period of time.

[0293] 142. The pharmaceutical composition according to any one of paragraphs 121 to 140, wherein the vector genome concentration of the recombinant AAV after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at -20°C for the period of time.

[0294] 143. The pharmaceutical composition according to any one of paragraphs 121 to 140, wherein the vector genome concentration of the recombinant AAV after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before being stored at 4°C for the said period of time.

[0295] 144. The pharmaceutical composition according to any one of paragraphs 121 to 140, wherein the in vitro potency of the recombinant AAV after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of the recombinant AAV before being stored at -80°C for the period of time.

[0296] 145. The pharmaceutical composition according to any one of paragraphs 121 to 140, wherein the in vitro potency of the recombinant AAV after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of the recombinant AAV before being stored at -20°C for the period of time.

[0297] 146. The pharmaceutical composition according to any one of paragraphs 121 to 140, wherein the in vitro potency of the recombinant AAV after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of the recombinant AAV before being stored at 4°C for the period of time.

[0298] 147. The pharmaceutical composition according to any one of paragraphs 121 to 140, wherein the size distribution of the recombinant AAV after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of the recombinant AAV before being stored at -80°C for the period of time.

[0299] 148. The pharmaceutical composition according to any one of paragraphs 121 to 140, wherein the size distribution of the recombinant AAV after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of the recombinant AAV before being stored at -20°C for the period of time.

[0300] 149. The pharmaceutical composition according to any one of paragraphs 121 to 140, wherein the size distribution of the recombinant AAV after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of the recombinant AAV before being stored at 4°C for the period of time.

[0301] 150. The pharmaceutical composition according to any one of paragraphs 141 to 149, wherein the time period is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0302] 151. The pharmaceutical composition according to any one of paragraphs 121 to 140, wherein the pharmaceutical composition is capable of being stored at 4°C for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or 2 months after having been previously stored at -80°C for about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0303] 152. A method of treating a disease of interest, the method comprising administering to a subject a pharmaceutical composition according to any one of paragraphs 121 to 140, wherein the rAAV encodes a transgene that treats or otherwise improves, prevents or slows the progression of the disease of interest.

[0304] 153. A method of treating a subject diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), the method comprising (ii) preparing a pharmaceutical composition according to any one of paragraphs 121 to 140, storing the pharmaceutical composition at -80°C for a first time period; (ii) thawing the pharmaceutical composition; and (iii) after thawing, storing the pharmaceutical composition at 4°C for a second time period.

[0305] 154. The method composition according to paragraph 153, wherein the first time period is about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0306] 155. The method described in paragraph 153, wherein the second time period is approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 1 month, or approximately 2 months.

[0307] 156. A kit comprising one or more containers and instructions for use, wherein the one or more containers comprise a pharmaceutical composition according to any one of paragraphs 121 to 140.

[0308] 157. A stable liquid formulation comprising a pharmaceutical composition according to any one of paragraphs 1 to 53, 67 to 111, and 121 to 140.

[0309] 158. A single-unit dosage form comprising 3.2 × 10⁻⁶ units at a pH of 7.4. 11The single-unit formulation of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, and 0.001% P188 is contained in a cycloolefin polymer (COP) vial with a volume of approximately 0.95 mL.

[0310] 159. A single-unit dosage form comprising 3.2 × 10⁻⁶ units at a pH of 7.4. 11 The single unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, and 0.001% P188, in a COP vial with a volume of at least 0.8 mL.

[0311] 160. A single-unit dosage form comprising 3.2 × 10⁻⁶ units at a pH of 7.4. 11 The single-unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, 4% sucrose, and 0.001% P188, in a COP vial with a volume of approximately 0.95 mL.

[0312] 161. A single-unit dosage form comprising 3.2 × 10⁻⁶ units at a pH of 7.4. 11 The single-unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, 4% sucrose, and 0.001% P188, in a COP vial with a volume of at least 0.8 mL.

[0313] 162. A single-unit dosage form comprising 6.5 × 10⁻⁶ units at a pH of 7.4. 11 The single-unit formulation of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, and 0.001% P188 is contained in a COP vial with a volume of approximately 0.95 mL.

[0314] 163. A single-unit dosage form comprising 6.5 × 10⁻⁶ units at a pH of 7.4. 11 The single unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, and 0.001% P188, in a COP vial with a volume of at least 0.8 mL.

[0315] 164. A single-unit dosage form comprising 6.5 × 10⁻⁶ units at a pH of 7.4. 11 The single-unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, 4% sucrose, and 0.001% P188, in a COP vial with a volume of approximately 0.95 mL.

[0316] 165. A single-unit dosage form comprising 6.5 × 10⁻⁶ units at a pH of 7.4. 11 The single-unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, 4% sucrose, and 0.001% P188, in a COP vial with a volume of at least 0.8 mL.

[0317] 166. A single-unit dosage form comprising 2.5 × 10⁻⁶ units at a pH of 7.4. 12 The single-unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, and 0.001% P188, in a COP vial with a volume of approximately 0.6 mL.

[0318] 167. A single-unit dosage form comprising 2.5 × 10⁻⁶ units at a pH of 7.4. 12 The single unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, and 0.001% P188, in a COP vial with a volume of at least 0.5 mL.

[0319] 168. A single-unit dosage form comprising 3 × 10⁻⁶ units at a pH of 7.4. 13 The single-unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, 4% sucrose, and 0.001% P188, in a COP vial with a volume of approximately 0.6 mL.

[0320] 169. A single-unit dosage form comprising 3 × 10⁻⁶ units at a pH of 7.4. 13The single-unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, 4% sucrose, and 0.001% P188, in a COP vial with a volume of at least 0.5 mL.

[0321] 170. A single-unit dosage form according to any one of paragraphs 158 to 169, which can be stored at 4°C for 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, or about 2 months.

[0322] 171. A single-unit dosage form according to any one of paragraphs 158 to 169, which can be stored at -80°C for about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0323] 172. The single-unit dosage form according to any one of paragraphs 158 to 169, which can be stored at 4°C for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or 2 months after having been previously stored at -80°C for about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0324] 173. The single-unit dosage form according to any one of paragraphs 158 to 169, wherein the vector genome concentration after the construct II has been stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration before the construct II has been stored at -80°C for the period of time.

[0325] 174. The single-unit dosage form according to any one of paragraphs 158 to 169, wherein the vector genome concentration after the construct II has been stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration before the construct II has been stored at -20°C for the period of time.

[0326] 175. The single-unit dosage form according to any one of paragraphs 158 to 169, wherein the vector genome concentration after the construct II has been stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration before the construct II has been stored at 4°C for the period of time.

[0327] 176. A single-unit dosage form according to any one of paragraphs 158 to 169, wherein the in vitro potency of said construct II after being stored at -80°C for a period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of said construct II before being stored at -80°C for the period of time.

[0328] 177. A single-unit dosage form according to any one of paragraphs 158 to 169, wherein the in vitro potency of said construct II after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of said construct II before being stored at -20°C for the period of time.

[0329] 178. A single-unit dosage form according to any one of paragraphs 158 to 169, wherein the in vitro potency of said construct II after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the in vitro potency of said construct II before being stored at 4°C for the period of time.

[0330] 179. A single-unit dosage form according to any one of paragraphs 158 to 169, wherein the size distribution of said construct II after being stored at -80°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of said construct II before being stored at -80°C for the period of time.

[0331] 180. A single-unit dosage form according to any one of paragraphs 158 to 169, wherein the size distribution of said construct II after being stored at -20°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of said construct II before being stored at -20°C for the period of time.

[0332] 181. A single-unit dosage form according to any one of paragraphs 158 to 169, wherein the size distribution of said construct II after being stored at 4°C for a certain period of time is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% the same as the size distribution of said construct II before being stored at 4°C for the said period of time.

[0333] 182. A single-unit dosage form according to any one of paragraphs 173 to 181, wherein the time period is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

[0334] 183. A prefilled syringe containing a single-unit dosage form according to any one of paragraphs 158 to 183.

[0335] 184. A kit comprising a pre-filled syringe as described in paragraph 183.

[0336] 2.2 Common words and abbreviations Attached Figure Description

[0337] Figure 1 The amino acid sequence of ranibizumab (top) shows five distinct residues in bevacizumab Fab (bottom). Variable heavy chain and constant heavy chain (V H and C H ) as well as variable light chains and constant light chains (V L and C L The beginning of the ) is indicated by an arrow (), and the CDR is not underlined. Non-common glycosylation sites (“Gsite”) and tyrosine-O-sulfation sites (“Ysite”) are indicated.

[0338] Figure 2 Glycans that can attach to HuGlyFabVEGFi. (Adapted from Bondt et al., 2014, Molecular & Cell Proteomics 13.1: 3029-3039).

[0339] Figure 3 Amino acid sequences of highly glycosylated variants of ranibizumab (top) and bevacizumab Fab (bottom). Variable heavy chain and constant heavy chain (V H and C H ) as well as variable light chains and constant light chains (V L and C L The beginning of the symbol is indicated by an arrow (), and the CDR is not underlined. Non-common glycosylation sites (“Gsite”) and tyrosine-O-sulfation sites (“Ysite”) are indicated. The four highly glycosylated variants are indicated by an asterisk (*).

[0340] Figure 4 A schematic diagram of the AAV8-antiVEGFfab genome.

[0341] Figure 5 A choroidal drug delivery device manufactured by Clearside® Biopharmaceuticals.

[0342] Figure 6 A subretinal drug delivery device manufactured by Janssen Pharmaceuticals, Inc., comprising a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, and a small needle at the posterior pole is injected into the subretinal space.

[0343] Figures 7A-7D A diagram illustrating the procedure for the posterior scleral reservoir.

[0344] Figure 8 Cluster multiple sequence alignment of AAV capsids 1-9 (SEQ ID NO: 41-51). Amino acid substitutions can be performed on AAV9 and AAV8 capsids by “recruiting” amino acid residues from corresponding positions in other aligned AAV capsids (shown in bold in the bottom row). The sequence region specified by “HVR” = highly variable region.

[0345] Figure 9 Rapid freezing / slow thawing (FF / ST) temperature profiles.

[0346] Figure 10 A magnified view of the SEC result curve of construct II in .DPBS formulation A. The leading peak represents free DNA and aggregates. The trailing peaks contain buffer and excipient species.

[0347] Figure 11 A magnified view of the SEC result curve of construct II in formulation B. The back peaks contain both buffers and excipients. The back peak caused by sucrose in formulation B is relatively large.

[0348] Figure 12 Results of dynamic light scattering accumulation of construct II in formulation A (dark gray) and formulation B (light gray) controls, and after five rapid and slow freeze / thaw cycles.

[0349] Figure 13 Low-temperature DSC thermogram of DPBS formulation A buffer of construct II.

[0350] Figure 14 Low-temperature DSC thermogram of construct II in formulation B buffer containing 4% sucrose and 0.001% poloxamer 188, pH 7.4.

[0351] Figure 15 At 37°C, at 1.0 × 10 12Stability trends of construct II potency in formulations A (dark gray circle) and B (light gray square) at GC / mL.

[0352] Figure 16 At 37°C, at 1.0 × 10 12 Stability trends of free DNA in construct II in formulations A (dark gray circles) and B (light gray squares) at GC / mL, as measured by dye fluorescence.

[0353] Figure 17 At -80℃ and -20℃, formulation B has a viscosity of 1.0 × 10⁻⁶. 12 Stability trend of construct II potency at GC / mL.

[0354] Figure 18 At -80℃ and -20℃, formulation B has a growth rate of 2.1 × 10⁻⁶. 11 Stability trend of construct II potency at GC / mL.

[0355] Figure 19A Temperature profiles were measured in Nalgene HDPE BDS bottles with two different filling volumes.

[0356] Figure 19B Record the temperature profile of 0.6 mL of filler in a 2 mL cryogenic tube that is circulated between -80°C and room temperature or -20°C.

[0357] Figure 20A Rapid freezing / rapid thawing (FF / FT) temperature profiles.

[0358] Figure 20B Rapid freezing / rapid thawing (FF / FT) temperature profiles (left axis) and shelf and probe rates (right axis).

[0359] Figure 21 Rapid freezing / slow thawing (FF / ST) temperature profiles.

[0360] Figure 22 Slow freezing / rapid thawing (SF / FT) temperature profile.

[0361] Figure 23 Slow freezing / slow thawing (SF / ST) temperature profile.

[0362] Figure 24 Slow freezing / slow thawing (SF / ST) temperature profiles (left axis) and shelf and probe rates (right axis).

[0363] Figure 25 A magnified view of the SEC result curve for the control formulation buffer.

[0364] Figure 26 A magnified view of the SEC result curve of construct II in .dPBS formulation buffer.

[0365] Figure 27 A magnified view of the SEC result curve of construct II in modified dPBS with sucrose buffer.

[0366] Figure 28 DLS diameter results for construct II in .dPBS freeze-thawed samples.

[0367] Figure 29 DLS diameter results for construct II in modified dPBS with sucrose freeze-thawed samples.

[0368] Figure 30 Comparison of the diameter results of DLS accumulation in construct II in dPBS with modified dPBS containing sucrose freeze-thawed samples.

[0369] Figure 31 Comparison of the DLS regularized diameter results of construct II in dPBS with modified dPBS containing sucrose freeze-thawed samples.

[0370] Figure 32 Low-temperature DSC thermogram of the dPBS formulation buffer of construct II.

[0371] Figure 33 Low-temperature DSC thermogram of the formulation buffer of builder II “modified dPBS with sucrose”.

[0372] Figure 34 Formula B contains an amorphous excipient that inhibits crystallization / eutectic transformation, thereby improving stability against freezing / thawing stress.

[0373] Figure 35 For formulation A, the amount of free DNA increases with each freeze / thaw cycle.

[0374] Figure 36 The potency of formulation a decreased with >5x freeze / thaw cycles, while the potency of formulation b remained stable for 30x freeze / thaw cycles. Formulation B (dark gray bar), a modified dPBS with 4% sucrose, maintained its potency after 30 freeze-thaw cycles. In contrast, the potency of the reference formulation (dPBS, light gray bar) decreased to between 66% and 72% after 15 to 30 freeze-thaw cycles. An example is AAV8 carrying the gene for green fluorescent protein. Freeze-thaw cycles are used to simulate temperature variations in transport and storage logistics and can also be used as "accelerated" stress to force AAV degradation for formulation optimization.

[0375] Figure 37 Adsorption loss occurred in the glass vials, but was not detected in the COP vials.

[0376] Figure 38 Formulas A and B exhibit similar long-term freezing stability at -80°C; formula B is also stable at -20°C. The modified dPBS formula with 4% sucrose maintained its potency for 12 months at both -20°C (round) and -80°C (square). The reference formula (dPBS) is shown for storage at -80°C as a comparison.

[0377] Figure 39 Real-time monitoring of the pH and temperature of formulation #2, a modified dPBS with 4% sucrose, in a -20°C AD freezer showed an acidification of approximately 3 pH units during freezing (top trajectory, left axis). The temperature profile (bottom curve, right axis) shows temperature fluctuations during the freezer's automatic defrost cycle. The pH of the frozen solution, measured directly with a freezer pH electrode, showed fluctuations between approximately 4.3 and 5.5 during freezing, which correlated with the temperature of the frozen formulation.

[0378] Figure 40 pH comparison of different buffers after multiple defrost cycles of pressure. Upon freezing, the DPBS formulation changed from 7.4 to approximately 4.3. The formulation with sucrose exhibited a lower pH upon freezing, changing from 7.4 to approximately 6.2. The TRIS formulation initially changed relative to room temperature but then remained stable upon freezing.

[0379] Figure 41 Comparison of buffer pH as the temperature decreased from 0°C to -20°C. Phosphate-based formulation #1 exhibited a large pH change upon freezing. Formulations 2-7 showed much lower acidification changes upon freezing, which is preferred for product stability. Formulation 8 showed a slight change in alkalinity, within the acceptable limits of formulation stability.

[0380] Figure 42 The magnitude of pH change in different buffer solutions after stabilization.

[0381] Figure 43 Low-temperature DSC thermogram of formulation #1: dPBS formulation buffer.

[0382] Figure 44 .Formulation #2: Low-temperature DSC thermogram of modified dPBS with 4% sucrose formulation buffer.

[0383] Figure 45Comparison of low-temperature DSC thermograms of phase transition behavior of different formulations. Formulation 1 (dPBS) did not show a glass transition (trace indicated by text). Formulation 2 (formulation B) and variants (formulations #3-7), as well as formulation 8 using TRIS buffer, showed phase transition behavior similar to formulation #2, with glass transitions between -40 and -45 °C.

[0384] Figure 46 Virus particle aggregation is affected by ionic strength.

[0385] Figure 47 Minimum ionic strength to prevent aggregation. Prepared at different NaCl concentrations, 1.8 × 10⁻⁶ ions. 13 The effective diameter of AAV8 particles at GC / mL. As indicated by the particle diameter, an ionic strength of ≥ 90 mM appears to be required to prevent particle aggregation.

[0386] Figure 48 The minimum ionic strength is serum-type dependent. Effective diameters of AAV8 (hollow square) and AAV9 (hollow triangular) prepared at different NaCl concentrations. The carrier concentration was 6 × 10⁻⁶. 11 GC / mL.

[0387] Figure 49 Formula C is a variant of "modified dPBS with sucrose" containing 60 mM NaCl and 6% sucrose (light gray triangles), and is stable for 2 years at -20°C. The reference formula (dPBS) is shown as a comparator (dark gray squares) stored at -20°C and is unstable at -20°C. Formulas B and C show comparable and excellent long-term stability at -20°C.

[0388] Figure 50 The efficacy trend of component II in formulation B at 2-8°C.

[0389] Figure 51 .Constructor II FDP batch 200320-314-DL7 at controlled room temperature at 3.0 × 10 13 Efficacy trend at GC / mL.

[0390] Figure 52 At -80℃ and -20℃, formulation B has a viscosity of 1.0 × 10⁻⁶. 12 Stability trend of construct II potency at GC / mL.

[0391] Figure 53 At -80℃ and -20℃, formulation B has a growth rate of 2.1 × 10⁻⁶. 11Stability trend of construct II potency at GC / mL.

[0392] Figure 54 Formulas A and B exhibit similar long-term freezing stability at -80°C; formula B is also stable at -20°C. The modified dPBS formula with 4% sucrose maintained its potency for 12 months at both -20°C (round) and -80°C (square). The reference formula (dPBS) is shown for storage at -80°C as a comparison. Detailed Implementation

[0393] This document provides pharmaceutical compositions, treatment methods associated with said pharmaceutical compositions, and kits associated with said pharmaceutical compositions. In some embodiments, the compositions provided in Section 4.1 are formulated to have one or more functional properties described in Section 4.2. In some embodiments, the pharmaceutical compositions provided herein have various advantages, such as improved stability after free / thaw cycles and improved long-term stability under various conditions. Assays that can be used in related studies are also provided herein (Section 4.5).

[0394] 4.1 Formulation of pharmaceutical compositions This disclosure provides a pharmaceutical composition comprising recombinant adeno-associated virus (AAV), potassium dihydrogen phosphate, sodium chloride, anhydrous disodium hydrogen phosphate, sucrose, and a surfactant.

[0395] In some embodiments, the pharmaceutical composition further comprises amino acids.

[0396] In some embodiments, this disclosure provides a pharmaceutical composition comprising recombinant adeno-associated virus (AAV), an ionic salt excipient or buffer, sucrose, and poloxamer 188. In some embodiments, the ionic salt excipient or buffer may be one or more components derived from the group consisting of: potassium dihydrogen phosphate, potassium phosphate, sodium chloride, anhydrous disodium hydrogen phosphate, sodium phosphate hexahydrate, sodium dihydrogen phosphate monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), amino acids, histidine, histidine hydrochloride (histidine-HCl), sodium succinate, sodium citrate, sodium acetate, and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), sodium sulfate, magnesium sulfate, magnesium chloride hexahydrate, calcium sulfate, potassium chloride, calcium chloride, and calcium citrate.

[0397] In some embodiments, the ionic strength of the pharmaceutical composition is from about 60 mM to 115 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 60 mM to 100 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 65 mM to 95 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 70 mM to 90 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 75 mM to 85 mM.

[0398] In some embodiments, the ionic strength of the pharmaceutical composition is from about 30 mM to 100 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 35 mM to 95 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 40 mM to 90 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 45 mM to 85 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 50 mM to 80 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 55 mM to 75 mM. In some embodiments, the ionic strength of the pharmaceutical composition is from about 60 mM to 70 mM.

[0399] In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 60 mM to 115 mM. In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 60 mM to 100 mM. In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 65 mM to 95 mM. In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 70 mM to 90 mM. In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 75 mM to 85 mM.

[0400] In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 30 mM to 100 mM. In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 35 mM to 95 mM. In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 40 mM to 90 mM. In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 45 mM to 85 mM. In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 50 mM to 80 mM. In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 55 mM to 75 mM. In some embodiments, the ionic strength of the pharmaceutical composition is in the range of 60 mM to 70 mM.

[0401] In some embodiments, the pharmaceutical composition comprises potassium chloride at a concentration of 0.2 g / L.

[0402] In some embodiments, the pharmaceutical composition comprises potassium dihydrogen phosphate at a concentration of 0.2 g / L.

[0403] In some embodiments, the pharmaceutical composition comprises sodium chloride at a concentration of 5.84 g / L.

[0404] In some embodiments, the pharmaceutical composition comprises anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L.

[0405] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 3% (w / v, 30 g / L) to 18% (w / v, 180 g / L). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 4% (w / v, 40 g / L).

[0406] In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.001% (weight / volume, 0.01 g / L).

[0407] In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0005% (w / v, 0.005 g / L) to 0.05% (w / v, 0.5 g / L). In some embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.001% (w / v, 0.01 g / L).

[0408] In some embodiments, this disclosure provides a pharmaceutical composition comprising recombinant adeno-associated virus (AAV), an ionic salt excipient or buffer, sucrose, and a surfactant. In some embodiments, the ionic salt excipient or buffer may be one or more components from the group consisting of: potassium dihydrogen phosphate, potassium phosphate, sodium chloride, anhydrous disodium hydrogen phosphate, sodium phosphate hexahydrate, sodium dihydrogen phosphate monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), amino acids, histidine, histidine hydrochloride (histidine-HCl), sodium succinate, sodium citrate, sodium acetate, and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), sodium sulfate, magnesium sulfate, magnesium chloride hexahydrate, calcium sulfate, potassium chloride, calcium chloride, and calcium citrate. In some embodiments, the surfactant may be one or more components from the group consisting of poloxamer 188, polysorbate 20, and polysorbate 80.

[0409] In some embodiments, the pharmaceutical composition comprises polysorbate 20 at a concentration of 0.0005% (w / v, 0.05 g / L) to 0.05% (w / v, 0.5 g / L).

[0410] In some embodiments, the pharmaceutical composition comprises polysorbate 80 at a concentration of 0.0005% (w / v, 0.05 g / L) to 0.05% (w / v, 0.5 g / L).

[0411] In some embodiments, the pH of the pharmaceutical composition is about 7.4.

[0412] In some embodiments, the pH of the pharmaceutical composition is about 6.0 to 9.0.

[0413] In some embodiments, the pH of the pharmaceutical composition is 7.4.

[0414] In some embodiments, the pH of the pharmaceutical composition is from 6.0 to 9.0.

[0415] As used herein and unless otherwise stated, the term “about” means within plus or minus 10% of a given value or range.

[0416] In some embodiments, the pharmaceutical composition is contained in a hydrophobically coated glass vial.

[0417] In some embodiments, the pharmaceutical composition is contained in a cyclic olefin polymer (COP) vial.

[0418] In some embodiments, the pharmaceutical composition is contained in a Daikyo Crystal Zenith® (CZ) vial.

[0419] In some embodiments, the pharmaceutical composition is contained in a TopLyo-coated vial.

[0420] In some embodiments, a pharmaceutical composition is disclosed herein comprising: (a) the recombinant AAV; (b) potassium chloride at a concentration of 0.2 g / L; (c) potassium dihydrogen phosphate at a concentration of 0.2 g / L; (d) sodium chloride at a concentration of 5.84 g / L; (e) anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L; (f) sucrose at a concentration of 4% by weight / volume (40 g / L); (g) poloxamer 188 at a concentration of 0.001% by weight / volume (0.01 g / L); and (h) water, wherein the recombinant AAV is AAV8.

[0421] In some embodiments, the vector genome concentration (VGC) of the pharmaceutical composition is approximately 3 × 10⁻⁶. 9 GC / mL, approximately 1 × 10 10 GC / mL, approximately 1.2 × 10⁻⁶ 10 GC / mL, approximately 1.6 × 10⁻⁶ 10 GC / mL, approximately 4 × 10⁻⁶10 GC / mL, approximately 6 × 10⁻⁶ 10 GC / mL, approximately 2 × 10 11 GC / mL, approximately 2.4 × 10⁻⁶ 11 GC / mL, approximately 2.5 × 10⁻⁶ 11 GC / mL, approximately 3 × 10 11 GC / mL, approximately 6.2 × 10⁻⁶ 11 GC / mL, approximately 1 × 10 12 GC / mL, approximately 3 × 10 12 GC / mL, approximately 2 × 10 13 GC / mL or approximately 3 × 10¹³ GC / mL.

[0422] In some embodiments, this disclosure provides a pharmaceutical composition or formulation comprising recombinant adeno-associated virus (AAV), potassium dihydrogen phosphate, sodium chloride, anhydrous disodium hydrogen phosphate, sucrose, and poloxamer 188. In some embodiments, the AAV comprises a component derived from AAV8. In some embodiments, the AAV is a viral vector provided herein comprising the following elements in the following order: a) a constitutive or hypoxia-inducible promoter sequence, and b) a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion). In some embodiments, the transgene is a fully human post-translational modified (HuPTM) antibody against VEGF. Antibodies comprise, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-heavy chain pairs, intracellular antibodies, heteroconjugated antibodies, monovalent antibodies, antigen-binding fragments of full-length antibodies, and fusion proteins thereof. Such antigen-binding fragments include, but are not limited to, single-domain antibodies (variable domains (VHH) of heavy chain antibodies or nanobodies), Fab, F(ab')2, and scFv (single-chain variable fragments) of full-length anti-VEGF antibodies (preferably, full-length anti-VEGF monoclonal antibodies (mAbs)) (collectively referred to herein as “antigen-binding fragments”). In a preferred embodiment, the fully human post-translational modified antibody against VEGF is a fully human post-translational modified antigen-binding fragment of a monoclonal antibody (mAb) against VEGF (“HuPTMFabVEGFi”). In another preferred embodiment, the HuPTMFabVEGFi is a fully human glycosylated antigen-binding fragment of an anti-VEGF mAb (“HuGlyFabVEGFi”). In alternative embodiments, a full-length mAb may be used. In a preferred embodiment, the AAV used for delivery of the transgene should be tropism-prone to human retinal cells or photoreceptor cells. Such AAVs may comprise non-replicating recombinant adeno-associated virus vectors (“rAAVs”), particularly those carrying an AAV8 capsid. In a specific embodiment, the viral vector or other DNA expression construct described herein is construct I, wherein construct I comprises the following components: (1) an AAV8 inverted terminal repeat sequence side-connected to an expression cassette; (2) a control element comprising: a) a CB7 promoter comprising a CMV enhancer / chicken β-actin promoter; b) a chicken β-actin intron; and c) a rabbit β-globin poly A signal; and (3) a nucleic acid sequence encoding a heavy and light chain of an anti-VEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker, thereby ensuring the expression of equal amounts of heavy chain polypeptide and light chain polypeptide.In another specific embodiment, the viral vector or other DNA expression construct described herein is construct II, wherein construct II comprises the following components: (1) an AAV2 inverted terminal repeat sequence side-attached to an expression cassette; (2) a control element comprising: a) a CB7 promoter including a CMV enhancer / chicken β-actin promoter; b) a chicken β-actin intron; and c) a rabbit β-globin poly A signal; and (3) a nucleic acid sequence encoding a heavy and light chain of an anti-VEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker, thereby ensuring the expression of equal amounts of the heavy chain polypeptide and the light chain polypeptide. In some embodiments, the anti-hVEGF antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, and the light chain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.

[0423] In another embodiment, a viral vector or other expression construct suitable for packaging in an AAV capsid includes (1) an AAV inverted terminal repeat (ITR) sequence side-mounted to an expression cassette; (2) a regulatory control element consisting essentially of one or more enhancers and / or promoters, d) a poly A signal and e) optionally an intron; and (3) a transgene that provides (e.g., encodes) one or more RNA or protein products of interest.

[0424] In some embodiments, the pharmaceutical composition comprises: (a) Construct II encoding an anti-human vascular endothelial growth factor (hVEGF) antibody; (b) potassium chloride at a concentration of 0.2 g / L; (c) potassium dihydrogen phosphate at a concentration of 0.2 g / L; (d) sodium chloride at a concentration of 5.84 g / L; (e) anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L; (f) sucrose at a concentration of 4% by weight / volume (40 g / L); poloxamer 188 at a concentration of 0.001% by weight / volume (0.01 g / L); and (h) water, wherein the anti-hVEGF antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, and the light chain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.

[0425] In some embodiments, the pharmaceutical composition comprises: (a) an AAV capsid packaging carrier encoding the transgene of interest; (b) potassium chloride at a concentration of 0.2 g / L; (c) potassium dihydrogen phosphate at a concentration of 0.2 g / L; (d) sodium chloride at a concentration of 5.84 g / L; (e) anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L; (f) sucrose at a concentration of 4% by weight / volume (40 g / L); (g) poloxamer 188 at a concentration of 0.001% by weight / volume (0.01 g / L); and (h) water, wherein the transgene of interest encodes the RNA of interest or the protein of interest, such as an antibody or enzyme.

[0426] In some embodiments, the pharmaceutical composition comprises: (a) Construct II encoding an antibody against human vascular endothelial growth factor (hVEGF); (b) potassium chloride at a concentration of 0.2 g / L; (c) potassium dihydrogen phosphate at a concentration of 0.2 g / L; (d) sodium chloride at a concentration of 5.84 g / L; (e) anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L; (f) sucrose at a concentration of 4% by weight / volume (40 g / L); poloxamer 188 at a concentration of 0.001% by weight / volume (0.01 g / L); and (h) water, wherein the anti-hVEGF antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, and the light chain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4. The amino acid sequence of NO:3, wherein the pharmaceutical composition has a desired viscosity, density, and / or osmotic pressure suitable for: intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed with the scleral surface).

[0427] In some embodiments, the pharmaceutical composition comprises: (a) Construct II encoding an anti-human vascular endothelial growth factor (hVEGF) antibody; (b) potassium chloride at a concentration of 0.2 g / L; (c) potassium dihydrogen phosphate at a concentration of 0.2 g / L; (d) sodium chloride at a concentration of 5.84 g / L; (e) anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L; (f) sucrose at a concentration of 4% by weight / volume (40 g / L); poloxamer 188 at a concentration of 0.001% by weight / volume (0.01 g / L); and (h) water, wherein the anti-hVEGF antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, and the light chain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, wherein the ionic strength of the pharmaceutical composition is about 60 mM to 100 mM.

[0428] In some embodiments, the pharmaceutical composition comprises: (a) Construct II encoding an antibody against human vascular endothelial growth factor (hVEGF); (b) potassium chloride at a concentration of 0.2 g / L; (c) potassium dihydrogen phosphate at a concentration of 0.2 g / L; (d) sodium chloride at a concentration of 5.84 g / L; (e) anhydrous disodium hydrogen phosphate at a concentration of 1.15 g / L; (f) sucrose at a concentration of 4% by weight / volume (40 g / L); poloxamer 188 at a concentration of 0.001% by weight / volume (0.01 g / L); and (h) water, wherein the anti-hVEGF antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, and the light chain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4. The amino acid sequence of NO:3, wherein the pharmaceutical composition has a desired viscosity, density, and / or osmotic pressure suitable for: intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed with the scleral surface).

[0429] In some embodiments, the pharmaceutical composition is a liquid composition. In some embodiments, the pharmaceutical composition is a frozen composition. In some embodiments, the pharmaceutical composition is a lyophilized composition derived from the liquid compositions disclosed herein. In some embodiments, the pharmaceutical composition is a reconstituted lyophilized formulation.

[0430] In some embodiments, the pharmaceutical composition is a lyophilized composition comprising a residual moisture content between about 1% and about 7%. In some embodiments, the pharmaceutical composition is a lyophilized composition comprising a residual moisture content between about 2% and about 6%. In some embodiments, the pharmaceutical composition is a lyophilized composition comprising a residual moisture content between about 3% and about 4%. In some embodiments, the pharmaceutical composition is a lyophilized composition comprising a residual moisture content of about 5%.

[0431] In some aspects, this document discloses a method for treating or preventing a disease in a subject, the method comprising administering a pharmaceutical composition to the subject. In some embodiments, the pharmaceutical compositions provided herein are suitable for administration via one, two, or more routes of administration (e.g., suitable for suprachoroidal and subretinal administration).

[0432] The provided methods are applicable to producing pharmaceutical compositions comprising recombinant AAV encoding a transgene. In some embodiments, rAAV viral vectors encoding anti-VEGF Fab or anti-VEGF antibodies are provided herein. In some embodiments, rAAV8-based viral vectors encoding anti-VEGF Fab or anti-VEGF antibodies are provided herein. In further embodiments, rAAV8-based viral vectors encoding ranibizumab are provided herein. In some embodiments, rAAV viral vectors encoding iduronate (IDUA) are provided herein. In some embodiments, rAAV9-based viral vectors encoding IDUA are provided herein. In some embodiments, rAAV viral vectors encoding iduronate 2-sulfatase (IDS) are provided herein. In some embodiments, rAAV9-based viral vectors encoding IDS are provided herein. In some embodiments, rAAV viral vectors encoding low-density lipoprotein receptor (LDLR) are provided herein. In some embodiments, rAAV8-based viral vectors encoding LDLR are provided herein. In some embodiments, rAAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein are provided herein. In some embodiments, rAAV9-based viral vectors encoding TPP1 are provided herein. In some embodiments, this document provides an rAAV viral vector encoding micro-antidystrophin. In some embodiments, this document provides an rAAV8-based viral vector encoding micro-antidystrophin. In some embodiments, this document provides an rAAV9-based viral vector encoding micro-antidystrophin. In some embodiments, this document provides an rAAV viral vector encoding anti-kalkinase (anti-pKal) protein. In some embodiments, this document provides an rAAV8-based or rAAV9-based viral vector encoding landrumab Fab or a full-length antibody. In some embodiments, this document provides an rAAV viral vector encoding human α-caryogamicin-γ-caryogamicin. In some embodiments, this document provides an rAAV viral vector encoding huFollistatin344. In some embodiments, this document provides an rAAV viral vector encoding human α-caryogamicin-γ-caryogamicin. In some embodiments, this document provides an rAAV viral vector encoding CLN2. In some embodiments, this document provides an rAAV viral vector encoding CLN3. In some embodiments, this document provides an rAAV viral vector encoding CLN6. In some embodiments, this document provides rAAV8-based or rAAV9-based viral vectors encoding human α-caryogam-γ-caryogam. In some embodiments, this document provides rAAV8-based or rAAV9-based viral vectors encoding huFollistatin344. In some embodiments, this document provides rAAV8-based or rAAV9-based viral vectors encoding human α-caryogam-γ-caryogam.In some embodiments, this document provides an rAAV8-based or rAAV9-based viral vector encoding CLN2. In some embodiments, this document provides an rAAV8-based or rAAV9-based viral vector encoding CLN3. In some embodiments, this document provides an rAAV8-based or rAAV9-based viral vector encoding CLN6.

[0433] In a specific aspect, this document discloses a method for treating or preventing a disease in a subject, the method comprising administering a pharmaceutical composition to the subject by: intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with microneedles), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device comprising a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a microneedle injected into the subretinal space at the posterior pole), and / or a posterior proximal scleral reservoir procedure (e.g., via a proximal scleral drug delivery device comprising a cannula, the tip of which can be inserted and held directly juxtaposed to the scleral surface).

[0434] In some embodiments, the pharmaceutical compositions provided herein are suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via suprachoroidal drug delivery devices, such as microinjectors with microneedles), subretinal injection via the vitreous route (surgical procedures), subretinal administration via the suprachoroidal space (e.g., surgical procedures via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0435] In some embodiments, the pharmaceutical composition has a desired viscosity suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0436] In some embodiments, the pharmaceutical composition has a desired density suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0437] In some embodiments, the pharmaceutical composition has a desired osmotic pressure suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted posteriorly and tunnel through the suprachoroidal space, with a microneedle injected into the subretinal space at the posterior pole), and / or a posterior proximal scleral reservoir procedure (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed with the scleral surface). In a specific embodiment, the desired osmotic pressure for subretinal administration is 160-430 mOsm / kg H2O. In other specific embodiments, the desired osmotic pressure for suprachoroidal administration is less than 600 mOsm / kg H2O.

[0438] In some embodiments, the osmotic pressure of the pharmaceutical composition is about 100 to 500 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 130 to 470 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 160 to 430 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 200 to 400 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 240 to 340 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 280 to 300 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 295 to 395 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is less than 600 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition ranges from 200 mOsm / L to 660 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 200 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 250 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 300 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 350 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 400 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 450 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 500 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 550 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 600 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 650 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 660 mOsm / L. In some aspects, this document discloses a method for treating a subject diagnosed with: mucopolysaccharidosis type IVA (MPS IVA), mucopolysaccharidosis type I (MPS I), mucopolysaccharidosis type II (MPS II), familial hypercholesterolemia (FH), homozygous familial hypercholesterolemia (HoFH), coronary artery disease, cerebrovascular disease, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, and sporadic inclusion body myositis or kallikrein-related disease, the method comprising administering the pharmaceutical composition to the subject.

[0439] In some respects, this document discloses a method for treating subjects diagnosed with: mucopolysaccharidosis type IVA (MPS IVA), mucopolysaccharidosis type I (MPS I), mucopolysaccharidosis type II (MPS II), familial hypercholesterolemia (FH), homozygous familial hypercholesterolemia (HoFH), coronary artery disease, cerebrovascular disease, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, and sporadic inclusion body myositis or kallikrein-related disease, the method comprising administering the pharmaceutical composition to the subject.

[0440] In some respects, this article discloses methods for treating subjects diagnosed with: mucopolysaccharidosis type IVA (MPS IVA), mucopolysaccharidosis type I (MPS I), mucopolysaccharidosis type II (MPS II), familial hypercholesterolemia (FH), homozygous familial hypercholesterolemia (HoFH), coronary artery disease, cerebrovascular disease, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, and sporadic inclusion body myositis or kallikrein-related disease, the methods comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject via intravenous, subcutaneous, or intramuscular injection.

[0441] In some respects, this document discloses methods for treating or preventing diseases in subjects, including treating subjects diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), or Betton's disease, including administering a therapeutically effective amount of a pharmaceutical composition to the subject.

[0442] In some respects, this document discloses methods for treating subjects diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), or Bethany's disease, the methods comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject by: intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device comprising a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), or a posterior proximal scleral reservoir procedure (e.g., via a proximal scleral drug delivery device comprising a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0443] In some embodiments, compositions and methods for delivering pharmaceutical compositions comprising a fully human post-translational modified (HuPTM) antibody against VEGF to the retina / vitreous fluid of the eye of a patient (human subject) diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR). The antibodies comprise, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-heavy chain pairs, intracellular antibodies, heteroconjugated antibodies, monovalent antibodies, and antigen-binding fragments of full-length antibodies, as well as fusion proteins thereof. Such antigen-binding fragments include, but are not limited to, single-domain antibodies (variable domains (VHH) of heavy chain antibodies or nanobodies), Fab, F(ab')2, and scFv (single-chain variable fragments) of full-length anti-VEGF antibodies (preferably, full-length anti-VEGF monoclonal antibodies (mAbs)) (collectively referred to herein as “antigen-binding fragments”). In a preferred embodiment, the fully human post-translational modified antibody against VEGF is a fully human post-translational modified antigen-binding fragment of a monoclonal antibody (mAb) against VEGF (“HuPTMFabVEGFi”). In another preferred embodiment, the HuPTMFabVEGFi is a fully human glycosylated antigen-binding fragment of an anti-VEGF mAb (“HuGlyFabVEGFi”). See also International Patent Application Publication No. WO / 2017 / 180936 (International Patent Application No. PCT / US2017 / 027529, filed April 14, 2017), International Patent Application Publication No. WO / 2017 / 181021 (International Patent Application No. PCT / US2017 / 027650, filed April 14, 2017), and International Patent Application Publication No. WO2019 / 067540 (International Patent Application No. PCT / US2018 / 052855, filed September 26, 2018), each of which is incorporated herein by reference in its entirety with respect to compositions and methods that may be used according to the invention described herein. In alternative embodiments, full-length mAbs may be used.Delivery can be accomplished via gene therapy—for example, by administering a viral vector or other DNA expression construct encoding an anti-VEGF antigen-binding fragment or mAb (or a highly glycosylated derivative) to the suprachoroidal space, subretinal space (via a transvitreal approach or through a catheter across the suprachoroidal space), intraretinal space, and / or outer surface of the sclera (i.e., near-scleral application) of the eye to a patient (human subject) diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), to said eye, to create a permanent reservoir in said eye that continuously supplies human PTM, such as human glycosylated transgenic products. See, for example, the administration modalities described in Section 5.3.2.

[0444] In some embodiments, the patient has shown a response to treatment with an anti-VEGF antigen-binding fragment injected intravitreally prior to gene therapy. In specific embodiments, the patient has previously been treated with LUCENTIS® (ranibizumab), EYLEA® (aflibercept), and / or AVASTIN® (bevacizumab) and has been found to respond to one or more of these drugs.

[0445] Subjects to whom such viral vectors or other DNA expression constructs are delivered should respond to an anti-VEGF antigen-binding fragment encoded by the transgene in the viral vector or expression construct. To determine responsiveness, the anti-hVEGF antigen-binding fragment transgene product (e.g., produced in cell cultures, bioreactors, etc.) can be administered directly to the subject, for example, via intravitreal injection.

[0446] HuPTMFabVEGFi encoded by a transgene, such as HuGlyFabVEGFi, may include, but is not limited to: an antigen-binding fragment of an antibody that binds to hVEGF, such as bevacizumab; an anti-hVEGF Fab moiety, such as ranibizumab; or such bevacizumab or ranibizumab Fab moiety engineered to contain additional glycosylation sites on the Fab domain (e.g., see Courtois et al., 2016, Monoclonal Antibodies 8: 99-112, which is incorporated herein by reference in its entirety by reference to a description of a derivative of bevacizumab with high glycosylation on the Fab domain of a full-length antibody).

[0447] The recombinant vector used for delivery of the transgene should be tropism-positive for human retinal cells or photoreceptor cells. Such vectors may contain non-replicating recombinant adeno-associated virus vectors (“rAAV”), with those carrying an AAV8 capsid being particularly preferred. However, other viral vectors may be used, including, but not limited to, lentiviral vectors, vaccinia virus vectors, or non-viral expression vectors known as “naked DNA” constructs. Preferably, HuPTMFabVEGFi, such as HuGlyFabVEGFi, should be controlled by appropriate expression control elements, such as CB7 promoters (chicken β-actin promoter and CMV enhancer), RPE65 promoters, or opsin promoters, and may contain other expression control elements that enhance the expression of the vector-driven transgene (e.g., introns, such as chicken β-actin introns, mouse parvovirus (MVM) introns, human factor IX introns (e.g., FIX truncated intron 1), β... - Introns of globin splice donors / immunoglobulin heavy chain spice acceptors, introns of adenovirus splice donors / immunoglobulin splice acceptors, introns of SV40 late splice donors / splicing acceptors (19S / 16S), and introns of hybrid adenovirus splice donors / IgG splice acceptors, and polyA signals such as rabbit β-globin polyA signaling, human growth hormone (hGH) polyA signaling, SV40 late polyA signaling, synthetic polyA (SPA) signaling, and bovine growth hormone (bGH) polyA signaling. See, for example, Powell and Rivera-Soto, 2015, Medical Discovery, 19(102):49-57.

[0448] In a preferred embodiment, the gene therapy construct is designed such that both the heavy and light chains are expressed. More specifically, the heavy and light chains should be expressed in approximately equal amounts, in other words, in a heavy-to-light chain ratio of approximately 1:1. The coding sequences of the heavy and light chains can be engineered in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES, such that the heavy-chain polypeptide and light-chain polypeptide are expressed separately. For a specific leader sequence, see, for example, section 5.2.4, and for specific IRES, 2A, and other linker sequences that can be used with the methods and compositions provided herein, see section 5.2.5.

[0449] In some embodiments, the gene therapy construct is provided as a frozen, sterile, single-use solution of the AAV carrier active ingredient in a formulation buffer. In specific embodiments, pharmaceutical compositions suitable for subretinal administration comprise a suspension of a recombinant (e.g., rHuGlyFabVEGFi) carrier in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant, and optional excipients. In specific embodiments, the construct is formulated in Durbecco's phosphate-buffered saline and 0.001% poloxamer 188, pH 7.4.

[0450] 4.2 Functional Characteristics In some embodiments, the pharmaceutical compositions described herein are suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via suprachoroidal drug delivery devices, such as microinjectors with microneedles), subretinal injection via the vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0451] In some embodiments, the pharmaceutical composition has a desired density suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0452] In some embodiments, the pharmaceutical composition has a desired osmotic pressure suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0453] In some embodiments, the pharmaceutical composition has a desired viscosity suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device, such as a microinjector with a microneedle), subretinal injection via a vitreous route (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device including a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole), and / or posterior proximal scleral reservoir procedures (e.g., via a proximal scleral drug delivery device including a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface).

[0454] In some embodiments, the osmotic pressure of the pharmaceutical composition is about 100 to 500 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 130 to 470 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 160 to 430 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 200 to 400 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 280 to 300 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 240 to 340 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 295 to 395 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition is less than 600 mOsm / kg H2O. In some embodiments, the osmotic pressure of the pharmaceutical composition ranges from 200 mOsm / L to 660 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 200 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 250 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 300 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 350 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 400 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 450 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 500 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 550 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 600 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 650 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition is about 660 mOsm / L. In some aspects, this document discloses a method for treating a subject diagnosed with: mucopolysaccharidosis type IVA (MPS IVA), mucopolysaccharidosis type I (MPS I), mucopolysaccharidosis type II (MPS II), familial hypercholesterolemia (FH), homozygous familial hypercholesterolemia (HoFH), coronary artery disease, cerebrovascular disease, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, and sporadic inclusion body myositis or kallikrein-related disease, the method comprising administering the pharmaceutical composition to the subject.

[0455] In some embodiments, the osmotic pressure of the pharmaceutical composition ranges from 200 mOsm / L to 660 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition ranges from 250 mOsm / L to 600 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition ranges from 300 mOsm / L to 550 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition ranges from 350 mOsm / L to 500 mOsm / L. In some embodiments, the osmotic pressure of the pharmaceutical composition ranges from 400 mOsm / L to 500 mOsm / L.

[0456] In some embodiments, the recombinant AAV in the pharmaceutical composition exhibits at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher stability to freeze / thaw cycles compared to the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0457] In some embodiments, the infectivity of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the viral infectivity of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, infectivity is measured before or after a freeze / thaw cycle.

[0458] In some embodiments, the aggregation of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the aggregation of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, the aggregation is measured before or after a freeze / thaw cycle.

[0459] In some embodiments, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold more stable than the same recombinant AAV in a reference pharmaceutical composition over a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, or about 4 years. In some embodiments, the stability of the recombinant AAV over a period of time is determined by one or more assays disclosed in Sections 4.5 and 5.

[0460] In some embodiments, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold more stable than the same recombinant AAV in a reference pharmaceutical composition over a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, or about 4 years. In some embodiments, the stability of the recombinant AAV over a period of time is determined by one or more assays disclosed in Sections 4.5 and 5.

[0461] In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, the in vitro relative potency (IVRP) is measured before or after a freeze / thaw cycle.

[0462] In some embodiments, the cell-free DNA of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold lower than that of the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the cell-free DNA of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5. In some embodiments, aggregation is measured before or after freeze / thaw cycles.

[0463] In some embodiments, the size variation of the recombinant AAV in the pharmaceutical composition over time periods such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years is up to 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, the size is measured before or after a freeze / thaw cycle.

[0464] In some embodiments, the size of the recombinant AAV in the pharmaceutical composition varies by up to 20%, 15%, 10%, 8%, 5%, 4%, 3%, 4%, 2%, or 1% over a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5. In some embodiments, the size is measured before or after a freeze / thaw cycle.

[0465] In some embodiments, when stored at -20°C, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0466] In some embodiments, the infectivity of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition when stored at -20°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years. In some embodiments, the viral infectivity of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0467] In some embodiments, the infectivity of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition when stored at -20°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years. In some embodiments, the viral infectivity of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0468] In some embodiments, when stored at -20°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the aggregation of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the aggregation of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0469] In some embodiments, when stored at -20°C for a period of time of at least, for example, at least about 11 weeks, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the aggregation of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the aggregation of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0470] In some embodiments, when stored at -20°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV over the time period is determined by one or more assays disclosed in Sections 4.5 and 5.

[0471] In some embodiments, when stored at -20°C for a duration of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV over a period of time is determined by one or more assays disclosed in Sections 4.5 and 5.

[0472] In some embodiments, when stored at -20°C for a period of time, such as 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0473] In some embodiments, when stored at -20°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0474] In some embodiments, when stored at -20°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0475] In some embodiments, when stored at -20°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0476] In some embodiments, when stored at -20°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the cell-free DNA of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the cell-free DNA of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0477] In some embodiments, when stored at -20°C for a duration of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the cell-free DNA of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the cell-free DNA of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0478] In some embodiments, when stored at -20°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the size variation of the recombinant AAV in the pharmaceutical composition is at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0479] In some embodiments, when stored at -20°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the size variation of the recombinant AAV in the pharmaceutical composition is at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0480] In some embodiments, when stored at 37°C, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0481] In some embodiments, the infectivity of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition when stored at 37°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years. In some embodiments, the viral infectivity of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0482] In some embodiments, the infectivity of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition when stored at 37°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years. In some embodiments, the viral infectivity of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0483] In some embodiments, when stored at 37°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the aggregation of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the aggregation of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0484] In some embodiments, when stored at 37°C for a duration of at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than the same recombinant AAV in a reference pharmaceutical composition, after being stored at 37°C for a duration of at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, respectively, the aggregation of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower. In some embodiments, the aggregation of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0485] In some embodiments, when stored at 37°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV over the duration is determined by one or more assays disclosed in Sections 4.5 and 5.

[0486] In some embodiments, when stored at 37°C for a duration of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV over the time period is determined by one or more assays disclosed in Sections 4.5 and 5.

[0487] In some embodiments, when stored at 37°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0488] In some embodiments, when stored at 37°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the in vitro relative potency (IVRP) of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by one or more assays disclosed in sections 4.5 and 5.

[0489] In some embodiments, when stored at 37°C for a duration of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 months, 2 years, 3 years, and 4 years, the cell-free DNA of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the cell-free DNA of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0490] In some embodiments, when stored at 37°C for a duration of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the cell-free DNA of the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times lower than that of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the cell-free DNA of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0491] In some embodiments, when stored at 37°C for a period of time, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the size variation of the recombinant AAV in the pharmaceutical composition is at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0492] In some embodiments, when stored at 37°C for a period of time, such as at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years, the size variation of the recombinant AAV in the pharmaceutical composition is at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size of the recombinant AAV is determined by one or more assays disclosed in Sections 4.5 and 5.

[0493] In some embodiments, as determined by one or more assays disclosed, for example, in Sections 4.5 or 5, the pharmaceutical compositions provided herein are capable of being stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 months without loss of stability. In some embodiments, the pharmaceutical compositions provided herein are capable of being stored at 4°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months without loss of stability. In some embodiments, the pharmaceutical compositions provided herein can be stored at ≤ 60°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 months without losing stability. In some embodiments, the pharmaceutical compositions provided herein can be stored at -80°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 months without losing stability. In some embodiments, the pharmaceutical compositions provided herein are able to be stored at 4°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months after being stored at -20°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months without losing stability.

[0494] In some embodiments, as determined by one or more assays disclosed, for example, in Sections 4.5 or 5, the pharmaceutical compositions provided herein are capable of being stored at -80°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 months, then thawed, and subsequently stored at 2-10°C, 4-8°C, or 2, 3, 4, 5, 6, 7, 8, or 9°C for an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months without loss of stability. In some embodiments, as determined by one or more assays disclosed, for example, in Sections 4.5 or 5, the pharmaceutical compositions provided herein are capable of being stored at -80°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 months, then thawed, and subsequently stored at about 4°C for an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months without loss of stability. In some embodiments, as determined by one or more assays disclosed, for example, in Sections 4.5 or 5, the pharmaceutical compositions provided herein are capable of being stored at ≤ 60°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 months, then thawed, and subsequently stored at about 4°C for an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months without loss of stability.

[0495] On the other hand, this document provides single-unit dosage forms that include the recombinant AAV (e.g., construct II) provided herein. As used herein, the term "single-unit dosage form" refers to a dosage form that includes the amount of recombinant AAV (e.g., construct II) required for one patient's single visit. In some embodiments, the recombinant AAV (e.g., construct II) may be administered to one eye of a patient. In other embodiments, the recombinant AAV (e.g., construct II) may be administered to both eyes of a patient.

[0496] In some embodiments, this document provides a single-unit dosage form comprising 3.2 × 10 11 Construct II containing 1 genomic copy (GC) / mL, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, pH 7.4, and 0.001% P188, wherein the single-unit dosage form is in a vial with a volume of approximately 0.95 mL. In some embodiments, this document provides a single-unit dosage form comprising 3.2 × 10⁻⁶ genomic copies (GC) / mL, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, pH 7.4, and 0.001% P188, wherein the single-unit dosage form is in a vial with a volume of approximately 0.95 mL. 11 The single-unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, pH 7.4, and 0.001% P188, and is contained in a vial with a volume of at least 0.8 mL.

[0497] In some embodiments, this document provides a single-unit dosage form comprising 3.2 × 10 11 The single-unit dosage form comprises GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, pH 7.4, 4% sucrose, and 0.001% P188, and is contained in a vial with a volume of approximately 0.95 mL. In some embodiments, a single-unit dosage form comprising 3.2 × 10⁻⁶ ppm is provided herein. 11 The single-unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, pH 7.4, 4% sucrose, and 0.001% P188, and is contained in a vial with a volume of at least 0.8 mL.

[0498] In some embodiments, this document provides a single-unit dosage form comprising 6.5 × 10 11 The single-unit dosage form comprises GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, pH 7.4, and 0.001% P188, and is contained in a vial with a volume of approximately 0.95 mL. In some embodiments, a single-unit dosage form comprising 6.5 × 10 11 The single-unit dosage form consists of GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, pH 7.4, and 0.001% P188, and is contained in a vial with a volume of at least 0.8 mL.

[0499] In some embodiments, this document provides a single-unit dosage form comprising 6.5 × 10 11 The single-unit dosage form comprises GC / mL construct II, 0.2 g / L potassium chloride, 0.2 g / L potassium dihydrogen phosphate, 8.01 g / L sodium chloride, 1.15 g / L anhydrous disodium hydrogen phosphate, pH 7.4, 4% sucrose, and 0.001% P188, and is contained in a vial with a volume of approximately 0.95 mL. In some embodiments, a single-unit dosage form comprising 6.5 × 10 11 The single...

Claims

1. A pharmaceutical composition for treating Betton's disease, wherein the pharmaceutical composition comprises: (a) Recombinant adeno-associated virus (rAAV); (b) Potassium chloride; (c) Potassium dihydrogen phosphate; (d) Sodium chloride; (e) Anhydrous disodium hydrogen phosphate; (f) sucrose; and (e) poloxamer 188.

2. The pharmaceutical composition according to claim 1, wherein the recombinant AAV comprises a component of one or more adeno-associated virus serotypes selected from the group consisting of: AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.H SC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 and AAV.HSC16.

3. The pharmaceutical composition according to claim 1, wherein the rAAV is AAV9 or AAV10.

4. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises one or more amino acids.

5. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises an ionic strength of about 60 mM to about 115 mM.

6. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises: (a) Potassium chloride at a concentration of approximately 0.2 g / L; (b) Potassium dihydrogen phosphate at a concentration of approximately 0.2 g / L; (c) Sodium chloride at a concentration of about 3.50 g / L to about 8.01 g / L; and (d) Anhydrous disodium hydrogen phosphate at a concentration of approximately 1.15 g / L.

7. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises: (a) Potassium chloride at a concentration of approximately 0.2 g / L; (b) Potassium dihydrogen phosphate at a concentration of approximately 0.2 g / L; (c) Sodium chloride at a concentration of approximately 5.84 g / L; and (d) Anhydrous disodium hydrogen phosphate at a concentration of approximately 1.15 g / L.

8. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises sucrose at a concentration of about 3% (w / v, 30 g / L) to about 18% (w / v, 180 g / L).

9. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises sucrose at a concentration of about 4% (weight / volume, 40 g / L).

10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises poloxamer 188; and wherein the concentration of poloxamer 188 is from about 0.0005% (weight / volume, 0.005 g / L) to about 0.05% (weight / volume, 0.5 g / L).

11. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises poloxamer 188; and wherein the concentration of poloxamer 188 is about 0.001% (weight / volume, 0.01 g / L).

12. The pharmaceutical composition of claim 1, wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, and wherein the vector genome comprises: (a) AAV 5' inverted terminal repeat (ITR); (b) Promoter; (c) Genetically modified organisms (GMOs); as well as (d) AAV 3' ITR.

13. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises a concentration of about 3.0 × 10⁻⁶. 9 GC / mL to approximately 3 × 10⁻⁶ 13 Vector genome concentration (VGC) in GC / mL.

14. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a concentration of about 1.0 × 10⁻⁶. 11 Vector genome concentration (VGC) in GC / mL.

15. The pharmaceutical composition of claim 1, wherein the rAAV comprises a transgene associated with the treatment of Beddon's disease.

16. The pharmaceutical composition of claim 1, wherein the rAAV comprises a genetically modified organism (GMO), wherein the genetically modified organism is CLN2.

17. The pharmaceutical composition of claim 1, wherein the rAAV comprises a transgene, wherein the transgene encodes a tripeptidyl peptidase 1 (TPP1) protein.

18. The pharmaceutical composition of claim 1, wherein the AAV 5' ITR and / or the AAV 3' ITR are derived from AAV2.

19. The pharmaceutical composition according to claim 1, wherein the promoter is a chicken β-actin (CBA) promoter.

20. The pharmaceutical composition of claim 1, wherein the promoter is a hybrid promoter comprising a CBA promoter sequence and a cytomegalovirus enhancer element.

21. The pharmaceutical composition of claim 1, wherein the vector genome further comprises: (a) polyA; (b) Introns; and / or (c) Enhancer.

22. The pharmaceutical composition of claim 21, wherein the polyA is synthetic polyA, or derived from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin, or modified RGB.

23. The pharmaceutical composition of claim 21, wherein the intron is derived from CBA, human β-globin, IVS2, SV40, α-globin, β-globin, collagen, ovalbumin, or p53.

24. The pharmaceutical composition of claim 21, wherein the enhancer is a CMV enhancer, RSV enhancer, APB enhancer, ABPS enhancer, αmic / bik enhancer, TTR enhancer, en34, or ApoE.

25. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a pH of about 6.0 to about 9.

0.

26. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises an osmotic pressure of about 200 mOsm / L to about 660 mOsm / L.

27. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is suitable for application to the eye.

28. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered at a dose sufficient to produce a therapeutically effective concentration of TPP1 protein in the eye, wherein the therapeutically effective concentration is produced by retinal cells.

29. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is applied independently to more than one eye.

30. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered via one, two or more routes of administration.

31. The pharmaceutical composition of claim 1, wherein the treatment comprises intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, and / or posterior proximal scleral administration.

32. Use of the pharmaceutical composition according to any one of claims 1 to 31 for the preparation of a medicament for treating Bedton syndrome.

33. A stable liquid formulation comprising the pharmaceutical composition according to any one of claims 1 to 31.

34. A stable liquid formulation comprising a pharmaceutical composition according to any one of claims 1 to 31; wherein the vector genome concentration of the recombinant AAV is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV prior to a freeze / thaw cycle.

35. A stable liquid formulation comprising a pharmaceutical composition according to any one of claims 1 to 31; wherein, after storage at -20°C, -80°C, 4°C, or room temperature for a certain period of time, the vector genome concentration of the recombinant AAV is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the vector genome concentration of the recombinant AAV before storage at -20°C, -80°C, 4°C, or room temperature for the said period of time, wherein the period of time is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, or about 24 months.

36. A stable liquid formulation comprising a pharmaceutical composition according to any one of claims 1 to 31; wherein the recombinant AAV has a stability to freeze / thaw cycles that is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in a reference pharmaceutical composition.

37. A stable liquid formulation comprising a pharmaceutical composition according to any one of claims 1 to 31; wherein, when stored for a sustained period of time at -20°C, -80°C, 4°C, or at room temperature, the recombinant AAV exhibits stability at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher than that of the same recombinant AAV in a reference pharmaceutical composition. The period of time is approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 12 months, approximately 15 months, approximately 18 months, or approximately 24 months.

38. A single-unit dosage form comprising a pharmaceutical composition for treating Bethany's disease in a container, wherein the pharmaceutical composition comprises: i. (a) The concentration is approximately 1.0 × 10⁻⁶. 10 GC / mL to approximately 1.0 × 10⁻⁶ 13 Vector genome concentration (VGC) in GC / mL; (b) Potassium chloride at a concentration of approximately 0.2 g / L; (c) Potassium dihydrogen phosphate at a concentration of approximately 0.2 g / L; (d) Sodium chloride with a concentration of approximately 5.84 g / L; (e) Anhydrous disodium hydrogen phosphate at a concentration of approximately 1.15 g / L; (f) Sucrose with a concentration ranging from about 3% (by weight, 30 g / L) to about 18% (by weight, 180 g / L); and (g) Poloxamer 188 at a concentration of approximately 0.001% (w / v, 0.01 g / L); or ii. (a) The concentration is approximately 1.0 × 10⁻⁶. 11 Vector genome concentration (VGC) in GC / mL; (b) Potassium chloride at a concentration of approximately 0.2 g / L; (c) Potassium dihydrogen phosphate at a concentration of approximately 0.2 g / L; (d) Sodium chloride with a concentration of approximately 5.84 g / L; (e) Anhydrous disodium hydrogen phosphate at a concentration of approximately 1.15 g / L; (f) Sucrose with a concentration ranging from about 3% (by weight, 30 g / L) to about 18% (by weight, 180 g / L); and (g) Poloxamer 188 at a concentration of about 0.001% (w / v, 0.01 g / L).

39. A single-unit dosage form comprising a pharmaceutical composition for treating Bethany's disease in a container, wherein the pharmaceutical composition comprises: i. (a) The concentration is approximately 1.0 × 10⁻⁶. 10 GC / mL to approximately 1.0 × 10⁻⁶ 13 Vector genome concentration (VGC) in GC / mL; (b) Potassium chloride at a concentration of approximately 0.2 g / L; (c) Potassium dihydrogen phosphate at a concentration of approximately 0.2 g / L; (d) Sodium chloride with a concentration of approximately 5.84 g / L; (e) Anhydrous disodium hydrogen phosphate at a concentration of approximately 1.15 g / L; (f) Sucrose with a concentration range of approximately 4% (weight / volume, 40 g / L); and (g) Poloxamer 188 at a concentration of approximately 0.001% (w / v, 0.01 g / L); or ii. (a) The concentration is approximately 1.0 × 10⁻⁶. 11 Vector genome concentration (VGC) in GC / mL; (b) Potassium chloride at a concentration of approximately 0.2 g / L; (c) Potassium dihydrogen phosphate at a concentration of approximately 0.2 g / L; (d) Sodium chloride with a concentration of approximately 5.84 g / L; (e) Anhydrous disodium hydrogen phosphate at a concentration of approximately 1.15 g / L; (f) Sucrose with a concentration range of approximately 4% (weight / volume, 40 g / L); and (g) Poloxamer 188 at a concentration of about 0.001% (w / v, 0.01 g / L).

40. A single-unit dosage form comprising a pharmaceutical composition according to any one of claims 1 to 31 in a container.

41. A prefilled syringe containing a single-unit dosage form according to any one of claims 38 to 40.

42. A reagent kit comprising: (i) One or more containers and instructions for use, wherein the one or more containers comprise the pharmaceutical composition according to any one of claims 1 to 31; (ii) One or more containers and instructions for use, wherein the one or more containers comprise a stable liquid formulation according to any one of claims 33 to 37; (iii) One or more containers and instructions for use, wherein the one or more containers comprise a single-unit dosage form according to any one of claims 38 to 40; (iv) The pre-filled syringe and instruction manual according to claim 41; and Optionally, at least one of the one or more containers is (a) A hydrophobically coated glass vial, (b) Daikyo Crystal Zenith® (CZ) vial; (c) TopLyo coated vials; or (d) Made from cyclic olefin polymers (COP).

43. Use of a pharmaceutical composition for the preparation of a medicament for treating Bethany's disease, wherein the pharmaceutical composition comprises: (a) Recombinant adeno-associated virus (rAAV); (b) Potassium chloride; (c) Potassium dihydrogen phosphate; (d) Sodium chloride; (e) Anhydrous disodium hydrogen phosphate; (f) sucrose; and (e) poloxamer 188.

44. Use of a pharmaceutical composition in the preparation of a medicament for treating Bethany's disease, wherein the pharmaceutical composition comprises: (a) Recombinant adeno-associated virus (rAAV); (b) Potassium chloride; (c) Potassium dihydrogen phosphate; (d) Sodium chloride; (e) Anhydrous disodium hydrogen phosphate; (f) sucrose; and (e) poloxamer 188.

45. The use according to claim 43 or 44, wherein the agent is formulated for application to the eyes.

46. ​​The use according to claim 43 or 44, wherein the treatment comprises intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection, subretinal injection via the vitreous route, subretinal administration via the suprachoroidal space, and / or posterior proximal scleral administration.

47. The use according to claim 46, wherein (i) The choroidal injection includes the use of an on-choroidal drug delivery device, such as a microinjector with microneedles; (ii) The subretinal injection via the vitreous route includes a surgical procedure; (iii) The subretinal administration via the suprachoroidal space includes the use of a subretinal drug delivery device comprising a catheter that can be inserted toward the posterior pole and tunnel through the suprachoroidal space, with a small needle injected into the subretinal space at the posterior pole; and / or (iv) The posterior proximal scleral administration includes the use of a proximal scleral drug delivery device comprising a cannula, the tip of which can be inserted into and held directly juxtaposed with the scleral surface.

48. A pharmaceutical composition for treating Bethany's disease in a subject of need, wherein the pharmaceutical composition comprises: (a) Recombinant adeno-associated virus (rAAV); (b) Buffer; (d) Sucrose; as well as (e) Surfactants.

49. The pharmaceutical composition of claim 48, wherein the rAAV comprises AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7m8, AAV.PHP.B, and AAV. PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV .HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 or AAV.HSC16.

50. The pharmaceutical composition of claim 48, wherein the pharmaceutical composition comprises sucrose at a concentration of about 3% (w / v, 30 g / L) to about 18% (w / v, 180 g / L).

51. The pharmaceutical composition according to any one of claims 48 to 50, wherein the buffer has an ionic strength of about 60 mM to about 150 mM.

52. The pharmaceutical composition according to any one of claims 48 to 50, wherein the buffer comprises one or more components selected from the group consisting of: potassium dihydrogen phosphate, potassium phosphate, sodium chloride, anhydrous disodium hydrogen phosphate, sodium phosphate hexahydrate, sodium dihydrogen phosphate monohydrate, sodium phosphate hexahydrate, sodium dihydrogen phosphate monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), amino acids, histidine, histidine hydrochloride (histidine-HCl), sodium succinate, sodium citrate, sodium acetate, and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), sodium sulfate, magnesium sulfate, magnesium chloride 6-hydrate, calcium sulfate, potassium chloride, calcium chloride, and calcium citrate.

53. The pharmaceutical composition according to any one of claims 48 to 50, wherein the surfactant is poloxamer 188, polysorbate 20, or polysorbate 80.

54. The pharmaceutical composition according to any one of claims 48 to 50, wherein the concentration of the surfactant is from about 0.0005% (weight / volume, 0.005 g / L) to about 0.05% (weight / volume, 0.5 g / L).

55. The pharmaceutical composition of claim 48, wherein the pharmaceutical composition is further lyophilized.

56. The pharmaceutical composition of claim 48, wherein the pharmaceutical composition is a reconstituted lyophilized powder.

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