Pharmaceutical compositions comprising recombinant adeno-associated viral vectors carrying an expression cassette encoding a transgene for suprachoroidal administration

Abstract

Provided herein is a pharmaceutical composition for administration to the suprachoroidal space of the eye of a subject. The pharmaceutical composition may comprise a recombinant adeno-associated virus (AAV) encoding a transgene. Also provided herein is a method for treating or preventing a disease in a subject by administering a therapeutically effective amount of the pharmaceutical composition to a subject in need thereof.

Description

[Technical field]

[0001] Priority This application claims the benefit of priority to U.S. Patent Application No. 63 / 328,249, filed April 6, 2022, the entirety of which is incorporated herein by reference.

[0002] Description of Electronically Submitted Sequence Listing This application contains a computer readable sequence listing that has been submitted herewith in the format of an XML file, the entire contents of which are incorporated herein by reference in their entirety. The sequence listing XML file submitted herewith is titled "12656-162-228_SEQLISTING.xml", was created on Mar. 30, 2023, and is 75,663 bytes in size. [Background technology]

[0003] 1. Background of the invention The human eye is a highly complex and highly developed sensory organ that is subject to numerous diseases and disorders. Approximately 285 million people worldwide are visually impaired, of which 39 million are blind and 246 million have moderate to severe vision impairments (World Health Organization, 2012, Global Data On Visual Impairments 2010, Geneva: World Health Organization). Some of the leading causes of blindness are cataracts (47%), glaucoma (12%), age-related macular degeneration (AMD) (9%), and diabetic retinopathy (5%) (World Health Organization, 2007, Global Initiative For The Elimination Of Avoidable Blindness: Action Plan 2006-2011, Geneva: World Health Organization).

[0004] Gene therapy has been employed in treating certain eye diseases (see, e.g., International Patent Application PCT / US2017 / 027650 (International Publication WO2017 / 181021A1)). Adeno-associated viruses (AAVs) are attractive tools for gene therapy due to their non-pathogenic properties, their broad host and cell type tropism range, including both dividing and non-dividing cells, and their ability to establish long-term transgene expression (e.g., Goncalves, 2005, Virology Journal, 2:43).

[0005] Current methods used for ocular gene therapy (e.g., by intravitreal or subretinal administration) are invasive and suffer from serious drawbacks, such as increased risk of cataracts, retinal detachment, and detachment of photoreceptors from the retinal pigment epithelium (RPE) at the fovea. There is a significant unmet medical need for therapies that improve or eliminate the drawbacks to current ocular gene therapy.

[0006] Adeno-associated viruses (AAVs), members of the Parvoviridae family named Dependoviruses, are small, non-enveloped, icosahedral viruses with a single-stranded linear DNA genome of approximately 4.7 kilobases (kb) to 6 kb. Due to their non-pathogenic properties, their broad host and cell type tropism range, including both dividing and non-dividing cells, and their ability to establish long-term transgene expression, AAVs are attractive tools for gene therapy (e.g., Goncalves, 2005, Virology Journal, 2:43). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2017 / 181021 [Non-patent literature]

[0008] [Non-Patent Document 1] World Health Organization, 2012, “Global Data On Visual Impairments 2010”, Geneva: World Health Organization. [Non-Patent Document 2] World Health Organization, 2007, “Global Initiative For The Elimination Of Avoidable Blindness: Action Plan 2006-2011”, Geneva: World Health Organization. [Non-Patent Document 3] Goncalves, 2005, Virology Journal, 2:43 Summary of the Invention [Problem to be solved by the invention]

[0009] Construct II is being investigated as a treatment delivered by injection into the suprachoroidal space. The suprachoroidal space (SCS) is the area between the sclera and choroid that expands upon injection of a drug solution (Habot-Wilner, 2019). The SCS space returns to its pre-injection size as the injected solution is cleared by physiological processes. The drug solution diffuses within the SCS and is absorbed into adjacent tissues. The capillaries in the choroid are permeable to low molecular weight osmolytes. The present disclosure addresses the unmet need to provide pharmaceutical compositions that provide longer residence times in the suprachoroidal space and consequently improved efficacy. [Means for solving the problem]

[0010] 2. Summary of the Invention In one embodiment, a pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of the eye of a human subject, the pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, the pharmaceutical composition, when administered to the eye of a pig, comprises: such that the clearance time of the pharmaceutical composition is about 5 days to about 15 days; Such that the thickness of the SCS at the injection site is about 400 μm to about 800 μm within 1 hour of administration; The pharmaceutical composition spreads circumferentially from the injection site to about one-eighth or less of the surface of the choroid within about one hour of administration. Provided herein are pharmaceutical compositions having a viscosity and / or elastic modulus.

[0011] In some embodiments, the composition has a gel temperature of about 27-32° C. In some embodiments, the composition has a gel time of about 15-90 seconds. -1 ~about 1000s -1 In some embodiments, the composition has a viscosity of about 183 mPas at 5° C. when measured at a shear rate of about 1 s -1 ~1000s -1 In some embodiments, the composition has a viscosity of less than about 183 mPas at 5° C. when measured at a shear rate of about 1 s -1 ~1000s -1 In some embodiments, the composition has a viscosity of about 183 mPas at 20° C. when measured at a shear rate of about 1 s -1 ~1000s -1 At 20° C., the viscosity is less than about 183 mPas when measured at a shear rate of 1.0 μm.

[0012] In some embodiments, the elastic modulus of the pharmaceutical compositions provided herein at less than 27° C. is less than or about 0.1 Pa, less than or about 0.01 Pa, less than or about 0.001 Pa, or zero. In some embodiments, the elastic modulus of the pharmaceutical compositions provided herein at about 32° C.-35° C. is about or at least about 0.1 Pa, about or at least about 1 Pa, about or at least about 10 Pa, about or at least about 100 Pa, about or at least about 1000 Pa, about or at least about 10,000 Pa, or about or at least about 10,000 Pa, or about or at least about 100,000 Pa.

[0013] In some embodiments, the clearance time after suprachoroidal administration is equal to or longer than the clearance time after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprises a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35 ° C. than the pharmaceutical composition.

[0014] In some embodiments, the clearance time is the clearance time from the SCS or from the eye. In some embodiments, the clearance time is the time required for the pharmaceutical composition and / or recombinant adeno-associated virus (AAV) vector to become undetectable in the SCS by any standard method. In some embodiments, the clearance time is about 2% or less or about 5% or less of the amount of the pharmaceutical composition and / or recombinant adeno-associated virus (AAV) vector detectable by any standard method when present in the SCS. In some embodiments, the clearance time is the amount of time after injection required to reduce the thickness of the injection site to about 1 nm or less, about 2 nm or less, about 5 nm or less, about 10 nm or less, about 25 nm or less, about 50 nm or less, about 100 nm or less, about 200 nm or less, or about 500 nm or less. In some embodiments, the clearance time is the amount of time after injection required for the thickness at the injection site to decrease to about 500 nm or less, about 200 nm or less, about 100 nm or less, about 50 nm or less, about 25 nm or less, about 10 nm or less, or become undetectable. In some embodiments, the clearance time is the amount of time after injection required for the pharmaceutical composition to spread circumferentially from the injection site to cover about one-sixteenth or more, about one-eighth or more, about one-quarter or more, about one-half or more, about three-quarters or more, or all of the circumference of the choroid of the eye.

[0015] In some embodiments, the circumferential spread after suprachoroidal administration is smaller than the circumferential spread after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprises a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35 ° C. than the pharmaceutical composition. In some embodiments, the circumferential spread after suprachoroidal administration is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 30 times, at least 35 times, at least 40% at least 45% at least 50% at least 55% at least 60% at least 65% at least 70% at least 75% at least 80% at least 85% at least 90% at least 95% at least 100% at least 150% or at least 200% at least 250% or at least 300% at least 400% or at least 500% smaller.

[0016] In some embodiments, the thickness of the injection site after suprachoroidal administration is compared to or is equal to or greater than the thickness of the injection site after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies being the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35 ° C. than the pharmaceutical composition.

[0017] In some embodiments, the expression level of the transgene is detected in the eye for a longer period of time after suprachoroidal administration compared to the period of time during which the expression level of the transgene is detected in the eye after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprises a recombinant AAV comprising an expression cassette encoding the transgene, the amount of recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35 ° C. than the pharmaceutical composition.

[0018] In some embodiments, the concentration of the transgene product in the eye after suprachoroidal administration is equal to or greater than the concentration of the transgene product in the eye after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus than the pharmaceutical composition at about 32-35 ° C. In some embodiments, the concentration of the transgene product in the back of the eye (e.g., retina) after suprachoroidal administration of the pharmaceutical composition is equal to or greater than the concentration of the transgene product in the back of the eye after suprachoroidal administration of the reference pharmaceutical composition, and / or the concentration of the transgene product in the outer layer of the eye (e.g., sclera) after suprachoroidal administration of the pharmaceutical composition is lower than the concentration of the transgene product in the outer layer of the eye after suprachoroidal administration of a reference pharmaceutical composition disclosed herein.

[0019] In some embodiments, the transduction rate at the injection site after suprachoroidal administration is compared to or is equal to or greater than the transduction rate at the injection site after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprises a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35 ° C. than the pharmaceutical composition.

[0020] In some embodiments, the level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration is equal to or reduced compared to the level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprises a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35 ° C. than the pharmaceutical composition.

[0021] In some embodiments, the viscosity and / or elastic modulus of the pharmaceutical composition and the viscosity and / or elastic modulus of the reference pharmaceutical composition are measured at the same shear rate. In some embodiments, the viscosity and / or elastic modulus of the pharmaceutical composition is at least about 1,000 s -1 , 2,000s -1 , 3,000s -1 , 4,000s -1 , 5,000s -1 , 6,000s -1 , 7,000s -1 , 8,000s -1 , 9,000s -1 , 10,000s -1 , 15,000s -1 , 20,000s -1 , or 30,000s -1 The shear rate is measured.

[0022] In some embodiments, the recombinant AAV is construct II. In some embodiments, the transgene is an anti-human vascular endothelial growth factor (anti-VEGF) antibody. In some embodiments, the transgene is not an anti-human vascular endothelial growth factor (anti-VEGF) antibody. In some embodiments, the recombinant AAV is 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, AAV.PHP.eB, AAV2.5, AAV2tY In some embodiments, the recombinant AAV comprises components from one or more adeno-associated virus serotypes selected from the group consisting of AAV.F, 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. In some embodiments, the recombinant AAV is AAV8. In some embodiments, the recombinant AAV is AAV9.

[0023] In some embodiments, the pharmaceutical composition comprises sucrose.In some embodiments, the pharmaceutical composition does not comprise sucrose.

[0024] In some embodiments, the clearance time following suprachoroidal administration of the pharmaceutical composition is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% longer. In some embodiments, the clearance time after suprachoroidal administration of the pharmaceutical composition is about 6 to about 15 days, about 7 to about 15 days, about 8 to about 15 days, about 9 to about 15 days, about 10 to about 15 days, about 11 to about 15 days, about 12 to about 15 days, about 13 to about 15 days, about 14 to about 15 days, about 5 to about 14 days, about 5 to about 13 days, about 5 to about 12 days, about 5 to about 11 days, about 5 to about 10 days, about 5 to about 9 days, about 5 to about 8 days, about 5 to about 7 days, or about 5 to about 6 days. In some embodiments, the clearance time after suprachoroidal administration of the pharmaceutical composition is not earlier than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In some embodiments, the clearance time after suprachoroidal administration of the reference pharmaceutical composition is less than about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the clearance time is the clearance time from the SCS or from the eye.

[0025] In some embodiments, the thickness of the injection site after suprachoroidal administration of the pharmaceutical composition is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% thicker. In some embodiments, the thickness of the injection site after suprachoroidal administration of the pharmaceutical composition is about 500 μm to about 3.0 mm, 750 μm to about 2.8 mm, about 750 μm to about 2.5 mm, about 750 μm to about 2 mm, or about 1 mm to about 2 mm.

[0026] In some embodiments, the thickness of the injection site after suprachoroidal administration of the pharmaceutical composition is at least about 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm thick. In some embodiments, the thickness of the injection site after suprachoroidal administration of the reference pharmaceutical composition is about 1 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm or less in thickness. In some embodiments, the thickness at the injection site following suprachoroidal administration of the pharmaceutical composition lasts for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 2 days, at least 3 days, at least 5 days, at least 10 days, at least 21 days, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 3 years, or at least 5 years.

[0027] In some embodiments, the thickness of the SCS at the injection site is about 400 μm to about 700 μm, about 400 μm to about 600 μm, about 400 μm to about 500 μm, about 500 μm to about 800 μm, about 600 μm to about 800 μm, or 700 μm to about 800 μm within 1 hour of administration. In some embodiments, the thickness of the SCS at the injection site is about 400 μm to about 700 μm, about 400 μm to about 500 μm, about 500 μm to about 800 μm, about 600 μm to about 800 μm, or 700 μm to about 800 μm within 1 hour of administration. In some embodiments, the thickness of the SCS at the injection site is about 5 minutes to about 10 minutes to about 15 minutes to about 20 minutes to about 30 minutes to about 45 minutes to about 60 minutes to about 5 minutes.

[0028] In some embodiments, the pharmaceutical composition spreads circumferentially from the injection site to about one-eighth or less of the choroidal surface at a time within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, or about 60 minutes after administration. In some embodiments, the pharmaceutical composition spreads circumferentially from the injection site to about one-sixteenth or less of the choroidal surface at a time within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, or about 60 minutes after administration.

[0029] In some embodiments, the concentration of the transgene in the eye following suprachoroidal administration of the pharmaceutical composition is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% greater. In some embodiments, the transgene is detectable in the eye for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days after suprachoroidal administration of the pharmaceutical composition.In some embodiments, the transgene is detected in the eye about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, or 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days or less after suprachoroidal administration of the reference pharmaceutical composition.

[0030] In some embodiments, the longer period of time following suprachoroidal administration of the pharmaceutical composition is at least 4 hours, 8 hours, 12 hours, 16 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, 92 days, 93 days, 94 days, 95 days, 96 days, 97 days, 98 days 7 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days longer.

[0031] In some embodiments, the level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration of the pharmaceutical composition is equal to or reduced compared to the level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration of a reference pharmaceutical composition. In some embodiments, the level of VEGF-induced vasodilation and / or vascular leakage following suprachoroidal administration of the pharmaceutical composition is reduced by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold, or by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500%.

[0032] In some embodiments, the transduction rate at the injection site following suprachoroidal administration of the pharmaceutical composition is at least about 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% greater.

[0033] In some embodiments, the stability of the recombinant AAV in the pharmaceutical composition is higher than that in the reference pharmaceutical composition. In some embodiments, the stability of the recombinant AAV is determined by the infectivity of the recombinant AAV. In some embodiments, the stability of the recombinant AAV is determined by the level of aggregation of the recombinant AAV. In some embodiments, the stability of the recombinant AAV is determined by the level of free DNA released by the recombinant AAV. In some embodiments, the pharmaceutical composition comprises about 50% more, about 25% more, about 15% more, about 10% more, about 5% more, about 4% more, about 3% more, about 2% more, about 1% more, about 0% more, about 1% less, about 2% less, about 5% less, about 7% less, about 10% less, about 2 times more, about 3 times more, about half, about one third of the free DNA compared to the level of free DNA in the reference pharmaceutical composition.

[0034] In some embodiments, the recombinant AAV in the pharmaceutical composition has 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 greater infectivity than the infectivity of the recombinant AAV in a reference pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 100% less, or at least 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less recombinant AAV aggregates than the level of recombinant AAV aggregates in the reference pharmaceutical composition.

[0035] In some embodiments, the transgene is a transgene suitable for treating or otherwise alleviating, preventing, or slowing the progression of a disease of interest.

[0036] In some embodiments, the human subject has been diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), x-linked disease, or Batten disease. In some embodiments, the human subject has been diagnosed with glaucoma or non-infectious uveitis.

[0037] In some embodiments, the AAV encodes palmitoyl protein thioesterase 1 (PPT1) or tripeptidyl-peptidase 1 (TPP1). In other embodiments, the AAV encodes an anti-VEGF fusion protein, an anti-VEGF antibody or antigen-binding fragment thereof, an anti-kallikrein antibody or antigen-binding fragment, an anti-TNF fusion protein, an anti-TNF antibody or antigen-binding fragment, an anti-C3 antibody or antigen-binding fragment, or an anti-C5 antibody or antigen-binding fragment.

[0038] In some embodiments, the amount of recombinant AAV genome copies is based on vector genome concentration.In some embodiments, the amount of recombinant AAV genome copies is based on genome copies per administration.In some embodiments, the amount of recombinant AAV genome copies is based on the total genome copies administered to a human subject.In some embodiments, the genome copies per administration are the genome copies of recombinant AAV per suprachoroidal administration.

[0039] In some embodiments, the total genome copies administered are the total genome copies of the recombinant AAV administered to the suprachoroid. In some embodiments, the vector genome concentration (VGC) is about 3×10 9 GC / mL, approximately 1×10 10 GC / mL, approximately 1.2×10 10 GC / mL, approximately 1.6×10 10 GC / mL, approximately 4×10 10 GC / mL, approximately 6×10 10 GC / mL, approximately 2×10 11 GC / mL, approximately 2.4×10 11 GC / mL, approximately 2.5×10 11 GC / mL, approximately 3×10 11GC / mL, approximately 6.2×10 11 GC / mL, approximately 1×10 12 GC / mL, approximately 2.5×10 12 GC / mL, approximately 3×10 12 GC / mL, approximately 5×10 12 GC / mL, approximately 6×10 12 GC / mL, approximately 1.5×10 13 GC / mL, approximately 2×10 13 GC / mL, or approximately 3 x 10 13 In some embodiments, the total genome copies administered is about 6.0×10 10 Genome copies, approximately 1.6 × 10 11 Genome copies, approximately 2.5 × 10 11 Genome copies, approximately 3.0 × 10 11 Genome copies, approximately 5.0 × 10 11 Genome copies, approximately 6.0 × 10 11 Genome copies, approximately 1.5 × 10 12 Genome copies, approximately 3 × 10 12 Genome copies, approximately 1.0 × 10 12 GC / mL, approximately 2.5×10 12 GC / mL, or approximately 3.0 x 10 13 In some embodiments, the total genome copies administered is about 6.0×10 10 Genome copies, approximately 1.6 × 10 11 Genome copies, approximately 2.5 × 10 11 Genome copies, approximately 5.0 × 10 11 Genome copies, approximately 1.5 × 10 12 Genome copies, approximately 3 × 10 12 Genome copies, approximately 1.0 × 10 12 Genome copies, approximately 2.5 × 10 12 Genome copies, or approximately 3.0 × 10 13 It is a genome copy.

[0040] In some embodiments, the pharmaceutical composition is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 times. In some embodiments, the reference pharmaceutical composition is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 times. In some embodiments, the pharmaceutical composition is administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, or seven times a day. In some embodiments, the reference pharmaceutical composition is administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, or seven times a day.

[0041] In some embodiments, the pharmaceutical composition contains poloxamer 407 and poloxamer 188. In some embodiments, the composition contains 16-22% poloxamer 407. In some embodiments, the composition contains 0-16% poloxamer 188. In some embodiments, the composition contains 19% poloxamer 407 and 6% poloxamer 188. In some embodiments, the composition contains 18% poloxamer 407 and 6.5% poloxamer 188. In some embodiments, the composition contains 17.5% poloxamer 407 and 7% poloxamer 188.

[0042] In some embodiments, the composition comprises modified Dulbecco's phosphate buffered saline solution, and optionally comprises a surfactant. In some embodiments, the pharmaceutical composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium dihydrogen phosphate, 5.84 mg / mL sodium chloride, 1.15 mg / mL anhydrous sodium phosphate dibasic, 40.0 mg / mL (4% w / v) sucrose, and optionally a surfactant. In some embodiments, the composition comprises potassium chloride, potassium dihydrogen phosphate, sodium chloride, anhydrous sodium phosphate dibasic, sucrose, and optionally a surfactant. In some embodiments, the composition comprises 18.0% w / v poloxamer 407 and 6.5% w / v poloxamer 188 mixed with a pH 7.4 solution containing 5.84 mg / mL sodium chloride, 0.201 mg / mL potassium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 0.200 mg / mL potassium phosphate dibasic, 40.0 mg / mL sucrose (4% w / v), 0.001% (0.01 mg / mL) poloxamer 188. In some embodiments, the composition comprises 18.0% w / v poloxamer 407 and 6.5% w / v poloxamer 188 in a pH 7.4 solution containing 5.84 mg / mL sodium chloride, 0.201 mg / mL potassium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 0.200 mg / mL potassium phosphate dibasic, 40.0 mg / mL (4% w / v) sucrose, 0.001% (0.01 mg / mL) poloxamer 188.

[0043] In one aspect, provided herein is a pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of the eye of a human subject, the pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, the pharmaceutical composition comprising 5.84 mg / mL sodium chloride, 0.201 mg / mL potassium chloride, 1.15 mg / mL anhydrous dibasic sodium phosphate, 0.200 mg / mL potassium dihydrogen phosphate, 40.0 mg / mL (4% w / v) sucrose, 18.0% w / v poloxamer 407 and 6.5% w / v poloxamer 188 in a solution of pH 7.4 containing 0.001% (0.01 mg / mL) poloxamer 188.

[0044] In another aspect, provided herein is a method of treating a disease in a subject, comprising administering to the subject a pharmaceutical composition provided herein. In some embodiments, the pharmaceutical composition is at a temperature of about 2-10° C. when administered. In some embodiments, the pharmaceutical composition is at a temperature of about 20-25° C. when administered.

[0045] In some embodiments, the pharmaceutical composition is administered at an injection pressure of less than about 43 PSI. In some embodiments, the pharmaceutical composition is administered at an injection pressure of less than about 65 PSI. In some embodiments, the pharmaceutical composition is administered at an injection pressure of less than about 100 PSI.

[0046] In some embodiments, the pharmaceutical composition is administered using a 29 gauge needle. In some embodiments, the pharmaceutical composition is administered using a 30 gauge needle.

[0047] In some embodiments, the pharmaceutical composition is administered with an injection time of about 10-15 seconds, hi some embodiments, the pharmaceutical composition is administered with an injection time of about 5-30 seconds.

[0048] In some embodiments, the expression cassette has a sequence comprising SEQ ID NO:56.

[0049] In some embodiments, the subject is a human.

[0050] In some embodiments, the disease is selected from the group consisting of nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), Batten disease, glaucoma, and non-infectious uveitis.

[0051] In one aspect, provided herein is a method of preparing a pharmaceutical composition, the method comprising: (a) providing a solution at pH 7.4 comprising 5.84 mg / mL sodium chloride, 0.201 mg / mL potassium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 0.200 mg / mL potassium dihydrogen phosphate, 40.0 mg / mL (4% w / v) sucrose, 0.001% poloxamer 188, and (b) mixing with the solution 18.0% w / v poloxamer 407 and 6.5% w / v poloxamer 188. In some embodiments, after the mixing step, the pH of the composition is about pH 6.0 to about pH 7.9.

[0052] 2.1 Exemplary Embodiments 1. A pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of the eye of a human subject, the pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, the pharmaceutical having an increasing viscosity and / or a higher elastic modulus as temperature increases, the viscosity and / or elastic modulus of the composition when administered to a porcine eye being: a. the clearance time of the pharmaceutical composition is about 5 days to about 15 days; b. The thickness of the SCS at the injection site is about 400 μm to about 800 μm within 1 hour of administration; c. The pharmaceutical composition spreads circumferentially from the injection site to about one-eighth or less of the surface of the choroid within about one hour of administration; The viscosity and / or elastic modulus of the pharmaceutical composition.

[0053] 2. The pharmaceutical composition of paragraph 1, having a gelling temperature of about 27 to 32°C.

[0054] 3. The pharmaceutical composition according to paragraph 1 or 2, having a gelation time of about 15 to 90 seconds.

[0055] 4. About 1 s -1 ~about 1000s -1 4. The pharmaceutical composition of any one of paragraphs 1 to 3, having a viscosity of about 183 mPas at 5° C. when measured at a shear rate of

[0056] 5. About 1 s -1 ~1000s -1 4. The pharmaceutical composition of any one of paragraphs 1 to 3, having a viscosity of less than about 183 mPas at 5° C. when measured at a shear rate of

[0057] 6. About 1 s -1 ~1000s -1 4. The pharmaceutical composition of any one of paragraphs 1 to 3, having a viscosity of about 183 mPas at 20° C. when measured at a shear rate of

[0058] 7. About 1 s -1 ~1000s -1 4. The pharmaceutical composition of any one of paragraphs 1 to 3, having a viscosity of less than about 183 mPas at 20° C. when measured at a shear rate of

[0059] 8. The pharmaceutical composition of any one of paragraphs 1-7, wherein the elastic modulus of the pharmaceutical composition provided herein at less than 8.27° C. is less than or about 0.1 Pa, less than or about 0.01 Pa, less than or about 0.001 Pa, or zero.

[0060] 9. The pharmaceutical composition of any one of paragraphs 1-7, wherein the elastic modulus of the pharmaceutical composition provided herein at 32° C. to 35° C. is about or at least about 0.1 Pa, about or at least about 1 Pa, about or at least about 10 Pa, about or at least about 100 Pa, about or at least about 1000 Pa, about or at least about 1000 Pa, about or at least about 10,000 Pa, or about or at least about 10,000 Pa.

[0061] 10. The pharmaceutical composition according to any one of paragraphs 1 to 8, wherein the clearance time after suprachoroidal administration is equal to or greater than the clearance time after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies being the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32 to 35 ° C. than the pharmaceutical composition.

[0062] 11. The pharmaceutical composition according to any one of paragraphs 1 to 8, wherein the circumferential spread after suprachoroidal administration is smaller than the circumferential spread after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies being the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32 to 35 ° C. than the pharmaceutical composition.

[0063] 12. The pharmaceutical composition according to paragraph 11, wherein the circumferential spread after suprachoroidal administration is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% smaller.

[0064] 13. The thickness of the injection site after suprachoroidal administration is equal to or greater than the thickness of the injection site after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies being the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35 ° C. than the pharmaceutical composition.

[0065] 14. The pharmaceutical composition of any one of paragraphs 1 to 8, wherein the expression level of the transgene is detected in the eye for a longer period of time after suprachoroidal administration compared to the period of time during which the expression level of the transgene is detected in the eye after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding the transgene, the amount of recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32 to 35 ° C. than the pharmaceutical composition.

[0066] 15. The concentration of the transgene product in the eye after suprachoroidal administration is equal to or greater than the concentration of the transgene product in the eye after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies being the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35 ° C. than the pharmaceutical composition; Optionally, the concentration of the transgene product (TP) in the back of the eye (e.g., retina) after suprachoroidal administration of the pharmaceutical composition is equal to or greater than the concentration of the transgene product in the back of the eye after suprachoroidal administration of a reference pharmaceutical composition, and / or the concentration of TP in the outer layer of the eye (e.g., sclera) after suprachoroidal administration of the pharmaceutical composition is lower than the concentration of TP in the outer layer of the eye after suprachoroidal administration of a reference pharmaceutical composition.

[0067] 16. The transduction rate at the injection site after suprachoroidal administration is compared to or is equal to or greater than the transduction rate at the injection site after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies being the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35 ° C. than the pharmaceutical composition.

[0068] 17. The level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration is equal to or reduced compared to the level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32 to 35 ° C. than the pharmaceutical composition.

[0069] 18. The pharmaceutical composition according to any one of paragraphs 10, 11 and 13 to 17, wherein the viscosity and / or elastic modulus of the pharmaceutical composition and the viscosity and / or elastic modulus of the reference pharmaceutical composition are measured at the same shear rate.

[0070] 19. The viscosity and / or elastic modulus of the pharmaceutical composition is at least about 1,000 s -1 , 2,000s -1 , 3,000s -1 , 4,000s -1 , 5,000s -1 , 6,000s -1 , 7,000s -1 , 8,000s -1 , 9,000s -1 , 10,000s -1 , 15,000s -1 , 20,000s -1 , or 30,000s -1 18. The pharmaceutical composition according to any one of paragraphs 4 to 11 and 13 to 17, measured at a shear rate of

[0071] 20. The pharmaceutical composition according to any one of paragraphs 1 to 19, wherein the recombinant AAV is construct II.

[0072] 21. The pharmaceutical composition according to any one of paragraphs 1 to 20, wherein the introduced gene is an anti-human vascular endothelial growth factor (anti-VEGF) antibody.

[0073] 22. The pharmaceutical composition according to any one of paragraphs 1 to 19, wherein the transgene is not an anti-human vascular endothelial growth factor (anti-VEGF) antibody.

[0074] 23. Recombinant AAV is 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, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03 23. The pharmaceutical composition of any one of paragraphs 1 to 22, comprising a component from one or more adeno-associated virus serotypes selected from the group consisting of 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.

[0075] 24. The pharmaceutical composition according to any one of paragraphs 1 to 23, wherein the recombinant AAV is AAV8.

[0076] 25. The pharmaceutical composition according to any one of paragraphs 1 to 19 and 21 to 23, wherein the recombinant AAV is AAV9.

[0077] 26. A pharmaceutical composition according to any one of paragraphs 1 to 25, comprising sucrose.

[0078] 27. A pharmaceutical composition according to any one of paragraphs 1 to 25, which does not contain sucrose.

[0079] 28. The pharmaceutical composition according to any one of paragraphs 1 to 27, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% longer.

[0080] 29. The pharmaceutical composition according to any one of paragraphs 1 to 28, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is about 6 to about 15 days, about 7 to about 15 days, about 8 to about 15 days, about 9 to about 15 days, about 10 to about 15 days, about 11 to about 15 days, about 12 to about 15 days, about 13 to about 15 days, about 14 to about 15 days, about 5 to about 14 days, about 5 to about 13 days, about 5 to about 12 days, about 5 to about 11 days, about 5 to about 10 days, about 5 to about 9 days, about 5 to about 8 days, about 5 to about 7 days, or about 5 to about 6 days.

[0081] 30. The pharmaceutical composition according to any one of paragraphs 1 to 28, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is not earlier than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days.

[0082] 31. The pharmaceutical composition according to any one of paragraphs 1 to 28, wherein the clearance time after suprachoroidal administration of the reference pharmaceutical composition is about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days or less.

[0083] 32. The pharmaceutical composition according to any one of paragraphs 1 to 31, wherein the clearance time is from the SCS or from the eye.

[0084] 33. The pharmaceutical composition according to any one of paragraphs 1 to 31, wherein the clearance time is the time required for the pharmaceutical composition and / or recombinant adeno-associated virus (AAV) vector to become undetectable in the SCS by any standard method.

[0085] 34. A pharmaceutical composition according to any one of paragraphs 1 to 31, wherein the clearance time is about 2% or less, or about 5% or less, of the amount detectable by any standard method when the pharmaceutical composition and / or recombinant adeno-associated virus (AAV) vector is present in the SCS.

[0086] 35. The pharmaceutical composition of any one of paragraphs 1-31, wherein the clearance time is the amount of time after injection required for the thickness at the injection site to decrease to about 500 nm or less, about 200 nm or less, about 100 nm or less, about 50 nm or less, about 25 nm or less, about 10 nm or less, or to become undetectable.

[0087] 36. The pharmaceutical composition of any one of paragraphs 1-31, wherein the clearance time is the amount of time after injection required for the pharmaceutical composition to spread circumferentially from the injection site to cover about one-sixteenth or more, about one-eighth or more, about one-quarter or more, about one-half or more, about three-quarters or more, or all of the circumference of the choroid of the eye.

[0088] 37. The pharmaceutical composition according to any one of paragraphs 13 and 20 to 36, wherein the thickness of the injection site after suprachoroidal administration of the pharmaceutical composition is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% thicker.

[0089] 38. The pharmaceutical composition according to any one of paragraphs 13 and 20 to 36, wherein the thickness of the injection site after suprachoroidal administration of the pharmaceutical composition is about 500 μm to about 3.0 mm, 750 μm to about 2.8 mm, about 750 μm to about 2.5 mm, about 750 μm to about 2 mm, or about 1 mm to about 2 mm.

[0090] 39. The pharmaceutical composition according to any one of paragraphs 13 and 20 to 36, wherein the thickness of the injection site after suprachoroidal administration of the pharmaceutical composition is at least about 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm thick.

[0091] 40. The pharmaceutical composition according to any one of paragraphs 13 and 20 to 36, wherein the thickness of the injection site after suprachoroidal administration of the reference pharmaceutical composition is about 1 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm or less.

[0092] 41. The pharmaceutical composition of any one of paragraphs 1 to 40, wherein the thickness of the SCS at the injection site is about 400 μm to about 700 μm, about 400 μm to about 600 μm, about 400 μm to about 500 μm, about 500 μm to about 800 μm, about 600 μm to about 800 μm, or 700 μm to about 800 μm within 1 hour of administration.

[0093] 42. The pharmaceutical composition of paragraph 41, wherein the time within 1 hour of administration is within about 5 minutes of administration, within about 10 minutes of administration, within about 15 minutes of administration, within about 20 minutes of administration, within about 30 minutes of administration, within about 45 minutes of administration, or within about 60 minutes of administration.

[0094] 43. The pharmaceutical composition according to any one of paragraphs 13 and 20 to 42, wherein the thickness of the injection site after suprachoroidal administration of the pharmaceutical composition lasts for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 2 days, at least 3 days, at least 5 days, at least 10 days, at least 21 days, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 3 years, or at least 5 years.

[0095] 44. The pharmaceutical composition of any one of paragraphs 1 to 43, wherein the pharmaceutical composition spreads circumferentially from the injection site to about one-eighth or less of the surface of the choroid within about 5 minutes after administration, within about 10 minutes after administration, within about 15 minutes after administration, within about 20 minutes after administration, within about 30 minutes after administration, within about 45 minutes after administration, or within about 60 minutes after administration.

[0096] 45. The pharmaceutical composition according to paragraph 44, having a circumferential extension from the injection site of about 1 / 16 or less of the surface of the choroid.

[0097] 46. ​​The pharmaceutical composition according to any one of paragraphs 15 and 20 to 45, wherein the concentration of the transgene in the eye after suprachoroidal administration of the pharmaceutical composition is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% greater.

[0098] 47. The longer period of time following suprachoroidal administration of the pharmaceutical composition is at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 47. The pharmaceutical composition of any one of paragraphs 14 and 20-46, wherein the period of administration is 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days longer.

[0099] 48. The transgene is administered for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 5 ... 48. The pharmaceutical composition of any one of paragraphs 1-47, wherein the effect is visually detectable for 0 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

[0100] 49. The transgene is administered for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 49. The pharmaceutical composition of any one of paragraphs 10-48, which is visually detectable after 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, or 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days or less.

[0101] 50. The pharmaceutical composition according to paragraph 21, wherein the level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration of the pharmaceutical composition is equal to or reduced compared to the level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration of a reference pharmaceutical composition.

[0102] 51. The level of VEGF-induced vasodilation and / or vascular leakage following suprachoroidal administration of the pharmaceutical composition is reduced by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold, or by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% 51. The pharmaceutical composition of any one of paragraphs 17 to 50, wherein the amount of erythrocyte / erythrocyte saturation is reduced by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500%.

[0103] 52. The transduction rate at the injection site after suprachoroidal administration of the pharmaceutical composition is at least about 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% greater.

[0104] 53. A pharmaceutical composition described in any one of paragraphs 1 to 52, wherein the stability of the recombinant AAV in the pharmaceutical composition is higher compared to the stability of the recombinant AAV in a reference pharmaceutical composition.

[0105] 54. The pharmaceutical composition according to paragraph 53, wherein the stability of the recombinant AAV is determined by the infectivity of the recombinant AAV.

[0106] 55. The pharmaceutical composition described in paragraph 53, wherein the stability of the recombinant AAV is determined by the level of aggregation of the recombinant AAV.

[0107] 56. The pharmaceutical composition according to paragraph 53, wherein the stability of the recombinant AAV is determined by the level of free DNA released by the recombinant AAV.

[0108] 57. The pharmaceutical composition according to paragraph 56, wherein the pharmaceutical composition comprises about 50% more, about 25% more, about 15% more, about 10% more, about 5% more, about 4% more, about 3% more, about 2% more, about 1% more, about 0% more, about 1% less, about 2% less, about 5% less, about 7% less, about 10% less, about twice as much, about three times as much, about half as much, or about a third as much free DNA compared to the level of free DNA in a reference pharmaceutical composition.

[0109] 58. The pharmaceutical composition according to paragraph 54, wherein the recombinant AAV in the pharmaceutical composition has 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 greater infectivity compared to the infectivity of the recombinant AAV in a reference pharmaceutical composition.

[0110] 59. The pharmaceutical composition of paragraph 55, comprising at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 100% less, or half-, third-, fifth-, tenth-, hundredth-, or thousandth-fold less recombinant AAV aggregation compared to the level of recombinant AAV aggregation in a reference pharmaceutical composition.

[0111] 60. The pharmaceutical composition according to any one of paragraphs 1 to 59, wherein the transgene is a transgene suitable for treating or otherwise alleviating, preventing or slowing the progression of a disease of interest.

[0112] 61. The pharmaceutical composition of any one of paragraphs 1 to 60, wherein the human subject has been diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), x-linked disease, or Batten disease.

[0113] 62. The pharmaceutical composition of any one of paragraphs 1 to 60, wherein the human subject has been diagnosed with glaucoma or non-infectious uveitis.

[0114] 63. The pharmaceutical composition of any one of paragraphs 1 to 19 and 23 to 62, wherein the AAV encodes palmitoyl protein thioesterase 1 (PPT1), tripeptidyl-peptidase 1 (TPP1), an anti-VEGF fusion protein, an anti-VEGF antibody or antigen-binding fragment thereof, an anti-kallikrein antibody or antigen-binding fragment, an anti-TNF fusion protein, an anti-TNF antibody or antigen-binding fragment, an anti-C3 antibody or antigen-binding fragment, or an anti-C5 antibody or antigen-binding fragment.

[0115] 64. The pharmaceutical composition of any one of paragraphs 1 to 63, wherein the amount of recombinant AAV genome copies is based on vector genome concentration.

[0116] 65. The pharmaceutical composition of any one of paragraphs 1 to 63, wherein the amount of recombinant AAV genome copies is based on genome copies per administration.

[0117] 66. The pharmaceutical composition of any one of paragraphs 1 to 63, wherein the amount of recombinant AAV genome copies is based on the total genome copies administered to the human subject.

[0118] 67. The pharmaceutical composition of paragraph 65, wherein the genome copies per administration are genome copies of recombinant AAV per suprachoroidal administration.

[0119] 68. The pharmaceutical composition of paragraph 66, wherein the total genome copies administered are the total genome copies of the recombinant AAV administered to the suprachoroid.

[0120] 69. The vector genome concentration (VGC) is approximately 3 × 10 9 GC / mL, approximately 1×10 10 GC / mL, approximately 1.2×10 10 GC / mL, approximately 1.6×10 10 GC / mL, approximately 4×10 10 GC / mL, approximately 6×10 10 GC / mL, approximately 2×10 11 GC / mL, approximately 2.4×10 11 GC / mL, approximately 2.5×10 11 GC / mL, approximately 3×10 11 GC / mL, approximately 6.2×10 11 GC / mL, approximately 1×10 12 GC / mL, approximately 2.5×10 12 GC / mL, approximately 3×10 12 GC / mL, approximately 5×10 12 GC / mL, approximately 6×10 12 GC / mL, approximately 1.5×10 13 GC / mL, approximately 2×10 13 GC / mL, or approximately 3 x 10 13 65. The pharmaceutical composition of paragraph 64, wherein the VGC is GC / mL.

[0121] 70. The total number of genome copies administered is approximately 6.0 × 10 10 Genome copies, approximately 1.6 × 10 11 Genome copies, approximately 2.5 × 10 11 Genome copies, approximately 3 × 10 11 Genome copies, approximately 5.0 × 10 11 Genome copies, approximately 6 × 10 11 Genome copies, approximately 3 × 10 12 Genome copies, approximately 1.0 × 10 12 Genome copies, approximately 1.5 × 10 12Genome copies, approximately 2.5 × 10 12 Genome copies, or approximately 3.0 × 10 13 69. The pharmaceutical composition according to any one of paragraphs 66 and 68, which is a genomic copy.

[0122] 71. The total number of genome copies administered is approximately 6.0 × 10 10 Genome copies, approximately 1.6 × 10 11 Genome copies, approximately 2.5 × 10 11 Genome copies, approximately 3 × 10 11 Genome copies, approximately 5.0 × 10 11 Genome copies, approximately 6 × 10 11 Genome copies, approximately 3 × 10 12 Genome copies, approximately 1.0 × 10 12 Genome copies, approximately 1.5 × 10 12 Genome copies, approximately 2.5 × 10 12 Genome copies, or approximately 3.0 × 10 13 68. The pharmaceutical composition according to any one of paragraphs 65 and 67, which is a genomic copy.

[0123] 72. The pharmaceutical composition of any one of paragraphs 1 to 71, which is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 times.

[0124] 73. The pharmaceutical composition according to any one of paragraphs 10 to 72, wherein the reference pharmaceutical composition is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 times.

[0125] 74. The pharmaceutical composition of any one of paragraphs 1 to 73, which is administered once daily, twice daily, three times daily, four times daily, five times daily, six times daily, or seven times daily.

[0126] 75. The pharmaceutical composition according to any one of paragraphs 10 to 73, wherein the reference pharmaceutical composition is administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, or seven times a day.

[0127] 76. A pharmaceutical composition according to any one of paragraphs 1 to 75, comprising poloxamer 407 and poloxamer 188.

[0128] 77. The pharmaceutical composition of any one of paragraphs 1 to 76, comprising 16 to 22% poloxamer 407.

[0129] 77. The pharmaceutical composition according to any one of paragraphs 1 to 76, comprising 78.0 to 16% poloxamer 188.

[0130] 77. The pharmaceutical composition according to any one of paragraphs 1 to 76, comprising 79.19% poloxamer 407 and 6% poloxamer 188.

[0131] 77. The pharmaceutical composition according to any one of paragraphs 1 to 76, comprising 80.18% poloxamer 407 and 6.5% poloxamer 188.

[0132] 81. The pharmaceutical composition according to any one of paragraphs 1 to 76, comprising 17.5% poloxamer 407 and 7% poloxamer 188.

[0133] 82. A pharmaceutical composition according to any one of paragraphs 1 to 81, comprising a modified Dulbecco's phosphate buffered saline solution, and optionally a surfactant.

[0134] 83. The pharmaceutical composition of any one of paragraphs 1 to 81, comprising 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium dihydrogen phosphate, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL sucrose (4% w / v), and optionally a surfactant.

[0135] 84. A pharmaceutical composition according to any one of paragraphs 1 to 81, comprising potassium chloride, potassium dihydrogen phosphate, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant.

[0136] 85. A method for treating a disease in a subject, comprising administering a pharmaceutical composition described in any one of paragraphs 1 to 81.

[0137] 86. A method for treating a disease in a subject, comprising administering to the subject a pharmaceutical composition described in paragraph 4 or 5, wherein the pharmaceutical composition is at a temperature of about 2 to 10°C when administered.

[0138] 87. A method for treating a disease in a subject, comprising administering to the subject a pharmaceutical composition described in paragraph 6 or 8, wherein the pharmaceutical composition is at a temperature of about 20-25°C when administered.

[0139] 88. The method of any one of paragraphs 85-87, wherein the pharmaceutical composition is administered at an injection pressure of less than about 43 PSI.

[0140] 89. The method of any one of paragraphs 85-87, wherein the pharmaceutical composition is administered at an injection pressure of less than about 65 PSI.

[0141] 90. The method of any one of paragraphs 85-87, wherein the pharmaceutical composition is administered at an injection pressure of less than about 100 PSI.

[0142] 91. The method of any one of paragraphs 85-90, wherein the pharmaceutical composition is administered using a 29 gauge needle.

[0143] 92. The method of any one of paragraphs 85-90, wherein the pharmaceutical composition is administered using a 30 gauge needle.

[0144] 93. The method of any one of paragraphs 85-92, wherein the pharmaceutical composition is administered with an injection time of about 10-15 seconds.

[0145] 94. The method of any one of paragraphs 85-92, wherein the pharmaceutical composition is administered with an injection time of about 5 to 30 seconds.

[0146] 95. The method of any one of paragraphs 85 to 94, wherein the subject is a human.

[0147] 96. The method of any one of paragraphs 85 to 95, wherein the disease is selected from the group consisting of nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), Batten disease, glaucoma and non-infectious uveitis.

[0148] 82. The pharmaceutical composition of any one of paragraphs 1 to 81, comprising 18.0% w / v poloxamer 407 and 6.5% w / v poloxamer 188 in a solution at pH 7.4 containing 97.5.84 mg / mL sodium chloride, 0.201 mg / mL potassium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 0.200 mg / mL potassium phosphate dibasic, 40.0 mg / mL (4% w / v) sucrose, 0.001% (0.01 mg / mL) poloxamer 188.

[0149] 98. A pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of the eye of a human subject, comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, comprising 5.84 mg / mL sodium chloride, 0.201 mg / mL potassium chloride, 1.15 mg / mL anhydrous dibasic sodium phosphate, 0.200 mg / mL potassium dihydrogen phosphate, 40.0 mg / mL (4% w / v) sucrose, 18.0% w / v poloxamer 407 and 6.5% w / v poloxamer 188 in a pH 7.4 solution containing 0.001% (0.01 mg / mL) poloxamer 188.

[0150] 99. The pharmaceutical composition described in any one of paragraphs 1 to 81, 97 or 98, wherein the expression cassette has a sequence comprising SEQ ID NO:56.

[0151] 100. A method for preparing a pharmaceutical composition according to any one of paragraphs 97 to 99, comprising the steps of: (a) providing a solution having a pH of 7.4 comprising 5.84 mg / mL sodium chloride, 0.201 mg / mL potassium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 0.200 mg / mL potassium phosphate dibasic, 40.0 mg / mL (4% w / v) sucrose, and 0.001% poloxamer 188; and (b) mixing with the solution 18.0% w / v poloxamer 407 and 6.5% w / v poloxamer 188.

[0152] 101. The method of paragraph 100, wherein after the mixing step, the pH of the composition is from about pH 6.0 to about pH 7.9.

[0153] 3. Brief description of the drawings [Brief description of the drawings]

[0154] [Figure 1] Overview of localization of Construct II in the choroidal space using a thermoresponsive gel formulation. The thermoresponsive gel formulation is expected to change from a liquid state to a gel during injection in the choroidal space, thus retaining the administered AAV locally in the choroidal space for longer with greater therapeutic effect. [Diagram 2] Calculated injection pressure as a function of viscosity for different 30 and 29 gauge needles. Panel A has scales up to a limit of 100 PSI, Panel B has scales up to a limit of 65 PSI, and Panel C has scales up to a limit of 45 PSI. [Diagram 3] External eye temperature measurement using a thermal camera. [Figure 4] Gel temperature as a function of formulation composition surface plot from an experimental design study. [Diagram 5] Viscosity at 20° C. as a function of formulation composition surface plot from a design of experiments study. [Figure 6] Viscosity at 5° C. as a function of formulation composition surface plot from a design of experiments study. [Figure 7]Summary of gel temperature rheology data from the Design of Experiments (DOE) study. Samples are labeled by P407-P188 level (e.g., sample #1 is labeled "16-0," which has 16% P407 and 0% P188). [Figure 8] FIG. 13 is an example of the gelation temperature profile for sample #9 in the DOE study (19% P407 / 8% P188) showing how the crossover of G′ and G″ is used to determine the gelation temperature. [Figure 9] Summary of gel time jumps from rheological data from DOE studies from 20° C. to 34° C. Samples are labeled with P407-P188 levels (e.g., sample #1 has 16% P407 and 0% P188). [Figure 10] Summary of gel time jumps from rheological data from DOE studies from 5° C. to 34° C. Samples are labeled with P407-P188 levels (e.g., sample #1 has 16% P407 and 0% P188). [Figure 11] Example of gel time jump from 20° C. to 34° C. profile for Sample #9 (19% P407 / 8% P188) showing how G' and G" crossovers are used to determine gel time. [Figure 12] Summary of viscosity vs. shear rate sweep at 20° C. from the DOE study. Samples are labeled by P407-P188 levels (e.g., sample #1 has 16% P407 and 0% P188). Note that samples 2 and 4 are already gelled at 20° C., thus demonstrating the effect of shear on the breakdown of the gel structure. All other samples show Newtonian behavior (constant viscosity as a function of shear rate). [Figure 13]Summary of viscosity vs. shear rate sweeps at 5° C. from a DOE study. Samples are labeled with levels of P407-P188 (e.g., sample #1 has 16% P407 and 0% P188). All samples show Newtonian behavior (constant viscosity as a function of shear rate). [Figure 14] Thermoresponsive gel formulation design space with limits of 27-32°C for gel temperature (white area). This design space can represent a scenario where a dose is prepared at 2-8°C and administered while still cooled or refrigerated. Limits: gel time of 15-90 seconds, viscosity at 5°C ≦183 mPas, duration of injection = 10 seconds, and needle inner diameter ≧220 μm). The shaded area corresponds to the region beyond the lower and upper limits defined for each element. The contour lines indicate the gelation temperature. [Figure 15] Thermoresponsive gel formulation design space (white area) with limits for gel temperature of 27-32°C (pink shading) and additional limits for viscosity at 20°C of ≦183 mPas (area >183 mPas is shown in green shading). In this scenario, the dose is administered at controlled room temperature (20°C) with a 10 second injection time. Limits: 15-90 second gel time (blue shading), viscosity at 20°C ≦183 mPas (green shading), duration of injection = 10 seconds, and needle inner diameter ≧220 μm). The shaded areas correspond to the areas beyond the lower and upper limits defined for each element. The contour lines indicate the gelation temperature. The crosses indicate three formulations that were further evaluated: A=6% / 19%, B=6.5% / 18%, and C=7%17.5% w / v of poloxamer 188 and 407, respectively. [Figure 16] Example of preparation of clinical drug product using autoclave sterilization. [Figure 17] Example of preparation of clinical drug product using sterile filtration. [Figure 18]Thermal camera images showing the setup for gelation time for droplet flow on a pre-warmed (31.3 °C) surface (bottle containing warm water for thermal mass). A 50 μL volume of Formulations A (left), B (center), and C (right) at 20 °C was dispensed onto the warm surface and a video of the droplet flow was used to determine the time when the droplets stopped flowing. [Figure 19] Gel temperature profile of formulation A. [Figure 20] Gel temperature profile of formulation B. [Figure 21] Gel temperature profile of formulation C. [Figure 22] Gel time jump for Formulation A from 20°C to 34°C. [Figure 23] Gel time jump for Formulation B from 20°C to 34°C. [Figure 24] Gel time jump for Formulation C from 20°C to 34°C. [Diagram 25] Gel time jump for Formulation A from 5°C to 34°C. [Figure 26] Gel time jump for Formulation B from 5°C to 34°C. [Figure 27] Gel time jump for Formulation C from 5°C to 34°C. [Figure 28] Viscosity vs. shear rate of Formulation A at 20° C. [Figure 29] Viscosity vs. shear rate of Formulation B at 20° C. [Diagram 30] Viscosity vs. shear rate of Formulation C at 20° C. [Diagram 31] Viscosity vs. shear rate of Formulation A at 5° C. [Diagram 32] Viscosity vs. shear rate of Formulation B at 5° C. [Diagram 33] Viscosity vs. shear rate of Formulation C at 5° C. [Diagram 34] Gelling profile of Formulation A diluted 10%. [Diagram 35] Gelling profile of Formulation B diluted 10%. [Diagram 36] Gelling profile of Formulation C diluted 10%. [Figure 37] Differential scanning fluorimetry profiles of the control (S-0DGN) and formulations A (S-0DGO), B (S-0DGP), and C (S-0DGQ). [Figure 38] Injection pressure profile of 0.1 mL of Formulation B injected into an enucleated porcine eye at approximately 35° C. using a CLSD device equipped with a 30 Ga needle. [Figure 39] Injection time profiles of 0.1 mL of Formulations A, B and C injected into air using a 1 mL syringe with a 30 Ga TW needle (pressure / force was kept approximately constant by the operator resulting in longer injection times being achieved with the higher viscosity formulations). [Diagram 40] Image of the SCS cavity following administration of Gel B formulation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0155] 4. Detailed Description of the Invention Provided herein is a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene suitable for administration to the suprachoroidal space (SCS) of the eye of a subject. The subject may be a subject diagnosed with one or more of the diseases described in Section 4.5. AAV vectors are described in Section 4.4, and dosages of such vectors are described in Section 4.3. In some embodiments, the pharmaceutical compositions provided in Section 4.1 are formulated such that they have one or more functional properties described in Section 4.2. In certain embodiments, the pharmaceutical compositions provided herein have various advantages, such as, for example, increased or slower clearance time after the pharmaceutical composition is administered to the SCS (Section 4.2.1); reduced circumferential spread (Section 4.2.2); increased SCS thickness (Section 4.2.3); reduced vasodilation and / or vascular leakage (Section 4.2.4); increased AAV levels and increased transduction rates at the injection site (Section 4.2.5); and increased concentration of the transgene. Without being bound by theory, functional properties can be achieved using the thermoresponsive formulations disclosed in Section 4.1. Assays that can be used in related studies (Section 4.6) are also provided herein.

[0156] 4.1 Formulation of pharmaceutical compositions The present disclosure provides a pharmaceutical composition suitable for suprachoroidal administration comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene. In some embodiments, a number of pharmaceutical compositions having different viscosity (or "loss modulus (G")) value properties at extraocular temperatures (about 32-35°C) are used to administer the AAV encoding the transgene. In some embodiments, a number of pharmaceutical compositions having different elastic modulus / storage modulus (G') properties at extraocular temperatures (about 32-35°C) are used to administer the AAV encoding the transgene.

[0157] In some embodiments, the pharmaceutical composition is thermoresponsive. The term "thermoresponsive" is known in the art to generally describe a substance that exhibits different physical properties at different temperatures. In certain embodiments, the pharmaceutical compositions provided herein have a lower viscosity, lower loss modulus (G"), and / or lower elastic modulus / storage (G') modulus at room temperature (e.g., about 20-25°C) than at external eye temperature (about 32-35°C). In certain embodiments, the pharmaceutical compositions provided herein have a lower viscosity and / or lower elastic modulus (G') when cooled (e.g., about 2-10°C) than at external eye temperature (about 32-35°C). The pharmaceutical compositions provided herein may be administered to the subject's eye at a temperature (e.g., chilled or at room temperature) at which the viscosity of the pharmaceutical composition is lower than the viscosity at external eye temperature (about 32-35°C). Without wishing to be bound by any particular theory, a change in temperature when administered to the subject's eye (e.g., suprachoroidal administration) can result in an increase in viscosity and / or elastic modulus (G'), resulting in an increased retention time of the composition near the injection site compared to a reference pharmaceutical composition, in which case the reference pharmaceutical composition has a lower viscosity at external eye temperature (about 32-35°C).

[0158] In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition comprise a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same amount of genome copies. In some embodiments, the pharmaceutical composition at about 32-35°C has a viscosity and / or elastic modulus (G') value higher than the viscosity of water at about 32-35°C. In some embodiments, the pharmaceutical composition at about 32-35°C has a viscosity and / or elastic modulus (G') value higher than the viscosity and / or elastic modulus (G') value of a control at about 32-35°C. In some embodiments, the pharmaceutical composition at about 32-35°C has a viscosity and / or elastic modulus (G') value higher than the viscosity of a solution typically used for subretinal injection at about 32-35°C. In some embodiments, the pharmaceutical composition at about 32-35°C has a viscosity and / or elastic modulus (G') value higher than the viscosity of PBS or dPBS at about 32-35°C. In some embodiments, the pharmaceutical composition at about 32-35° C. has a viscosity and / or elastic modulus (G') value higher than the viscosity of Hank's Balanced Saline (HBSS) at about 32-35° C. In some embodiments, the reference pharmaceutical composition at about 32-35° C. has a lower viscosity and / or elastic modulus (G') than the pharmaceutical composition at about 32-35° C. In some embodiments, the reference pharmaceutical composition has the same or similar viscosity and / or elastic modulus (G') as the pharmaceutical composition at about 20-25° C. In some embodiments, the reference pharmaceutical composition is a control solution (e.g., PBS, water, or HBSS). In some embodiments, the reference pharmaceutical composition includes sucrose. In some embodiments, the reference pharmaceutical composition is a pharmaceutical composition commonly used for AAV subretinal injection. In some embodiments, the reference pharmaceutical composition is not thermoresponsive, e.g., the reference pharmaceutical composition has substantially the same viscosity and / or elastic modulus (G') at about 20-25° C. as it has at about 32-35° C., or does not have a higher viscosity and / or elastic modulus (G') at increased temperature.

[0159] In some embodiments, the pharmaceutical composition can be administered at about 32-35° C., e.g., at a concentration of zero, 0.001, 0.01, 0.1, or 1 s. -1 or about 1000 s -1 Or at least about 1000s -1 at a shear rate of about, at least about, or at most about 10 cP, 15 cP, 20 cP, 25 cP, 30 cP, 35 cP, 40 cP, 45 cP, 50 cP, 60 cP, 70 cP, 80 cP, 90 cP, 100 cP, 150 cP, 200 cP, 250 cP, 300 cP, 350 cP, 400 cP, 450 cP, 500 cP, In some embodiments, the viscosity is about 550 cP, 600 cP, 650 cP, 700 cP, 800 cP, 900 cP, 1000 cP, 2,000 cP, 3,000 cP, 4,000 cP, 5,000 cP, 6,000 cP, 7,000 cP, 8,000 cP, 9,000 cP, 10,000 cP, 12,000 cP, or 15,000 cP. In some embodiments, the shear rate is about 100 s -1 , 50s -1 , 10s -1 , 1s -1 , 0.1s -1 , 0.01s -1 , 0.001s -1 , or 0.0001s -1 or about 100s -1 , 50s -1 , 10s -1 , 1s -1 , 0.1s -1 , 0.01s -1 , 0.001s -1 , or 0.0001s -1 In some embodiments, the viscosity of the pharmaceutical composition or the reference pharmaceutical composition is, for example, less than about 100 s -1 , 50s -1 , 10s -1 , 1s -1 , 0.01s -1 , 0.001s -1 , or 0.0001s -1 or about 100s -1 , 50s -1 , 10s -1 , 1s -1 , 0.01s -1, 0.001s -1 , or 0.0001s -1 Any viscosity disclosed herein at a shear rate of less than 100 nm.

[0160] In some embodiments, the pharmaceutical composition at about 32-35° C. or the reference pharmaceutical composition at about 32-35° C. (or control pharmaceutical composition or equivalent pharmaceutical composition) has a β-amino acid concentration of about or at least about 5 cP, about or at least about 10 cP, about or at least about 15 cP, about or at least about 20 cP, about or at least about 25 cP, about or at least about 30 cP, about or at least about 35 cP, about or at least about 40 cP, about or at least about 45 cP, about or at least about 50 cP, about or at least about 50 cP, about or at least about 60 cP, about or at least about 65 cP, about or at least about 70 cP, about or at least about 75 cP, about or at least about 80 cP, about or at least about 85 cP, about or at least about 90 cP, about or at least about 95 cP, about or at least about 100 cP, about or at least about 100 cP, about or at least about 150 cP, about or at least about 100 cP, about or at least about 150 cP, about or at least about 200 cP, about or at least about 25 cP, about or at least about 30 cP, about or at least about 35 cP, about or at least about 40 cP, about or at least about 45 cP, about or at least about ... All of the above are about 60 cP, about or at least about 70 cP, about or at least about 80 cP, about or at least about 90 cP, 100 cP, about or at least about 115 cP, about or at least about 120 cP, about or at least about 125 cP, about or at least about 130 cP, about or at least about 135 cP, about or at least about 140 cP, about or at least about 145 cP, about or at least about 150 cP, about or at least about 160 cP, about or at least about 170 cP, about or at least about 180 cP, about or at least about 190 cP, about 180 cP, about or at least about 190 cP, about or at least about 200 cP, about or at least about 300 cP, about or at least about 400 cP, about or at least about 500 cP, about or at least about 600 cP, about or at least about 700 cP, about or at least about 800 cP, about or at least about 900 cP, about or at least about 1000 cP, about or at least about 1500 cP, about or at least about 2000 cP, about or at least about 2500 cP, about or at least about 3000 cP, about or at least about 3500 cP, about or at least about 4000 cP, about or at least about 4500 cP, about or at least about 5000 cP, about or at least about 5500 cP, about or at least about 6000 cP, about or at least about 6500 cP, about or at least about 7000 cP, about or at least about 7500 cP, about or at least about 8000 cP, about or at least about 9000 cP, about or at least about 10000 cP, about or at least about 1 x 10 3 cP, about or at least about 3×103 cP, about or at least about 1 × 10 4 cP, about or at least about 3×10 4 cP, about or at least about 1 × 10 5 cP, about or at least about 1.7×10 5 cP, about or at least about 3×10 5 cP, about or at least about 1 × 10 6 cP, about or at least about 3×10 6 cP, about or at least about 1 × 10 7 cP, about or at least about 3×10 7 cP, about or at least about 1 × 10 8 cP, about or at least about 3×10 8 cP (e.g., about or at least about 1000 s -1 In some embodiments, the viscosity at about 32-35° C. (e.g., about or at least about 1000 s -1 (measured at a shear rate of about 25 cP to about 1 × 10 6 cP, approx. 25cP ~ approx. 1×10 4 cP, approx. 25cP ~ approx. 5,000cP, approx. 25cP ~ approx. 1×10 3 cP, approx. 100cP ~ approx. 1×10 6 cP, approx. 100cP ~ approx. 1×10 4 cP, approx. 100cP ~ approx. 5,000cP, approx. 100cP ~ approx. 1×10 3 In some embodiments, the viscosity at about 32-35° C. (e.g., about or at least about 1000 s -1 (measured at a shear rate of about 25 cP to about 3 × 10 6 cP, approx. 10cP ~ approx. 3×10 8 cP, about 50cP to about 5000cP, about 10cP to about 15000cP, about 25cP to about 1500cP, about 50cP to about 1500cP, about 25cP to about 3×10 4 In some embodiments, the pharmaceutical composition at about 32-35° C. has a concentration of at least about 25 cP to about 3×10 6 cP, at least about 10 cP to about 3 × 10 8cP, at least about 50 cP to about 5000 cP, at least about 10 cP to about 15000 cP, at least about 25 cP to about 1500 cP, at least about 50 cP to about 1500 cP, or at least about 25 cP to about 3×10 4 Viscosity in cP (e.g., about or at least about 1000 s -1 In some embodiments, the equivalent pharmaceutical composition, or reference pharmaceutical composition, or control at about 32-35° C. has a shear rate of about or at most about 1 cP, about or at most about 2 cP, about or at most about 3 cP, about or at most about 4 cP, about or at most about 5 cP, about or at most about 6 cP, about or at most about 7 cP, about or at most about 8 cP, about or at most about 9 cP, about or at most about 10 cP, about or at most about 15 cP, about or at most about 20 cP, about or at most about 25 cP, about or at most about 30 cP, about or at most about 35 cP, about or at most about A viscosity of about 40 cP, about or at most about 45 cP, about or at most about 50 cP, about or at most about 55 cP, about or at most about 60 cP, about or at most about 65 cP, about or at most about 70 cP, about or at most about 75 cP, about or at most about 80 cP, about or at most about 85 cP, about or at most about 90 cP, about or at most about 95 cP, about or at most about 100 cP, about or at most about 200 cP, about or at most about 300 cP, about or at most about 400 cP, about or at most about 500 cP (e.g., about or at least about 1000 s -1 In some embodiments, the equivalent pharmaceutical composition, or the reference pharmaceutical composition, or the control at about 32-35° C. has a viscosity of about 1 cP to about 25 cP, about 1 cP to about 20 cP, about 1 cP to about 24 cP, about 1 cP to about 10 cP, about 1 cP to about 50 cP, about 1 cP to about 100 cP, about 5 cP to about 50 cP, about 1 cP to about 5 cP, or about 1 cP to about 200 cP.

[0161] In some embodiments, the reference pharmaceutical composition at about 32-35° C. has a viscosity of about 1 cP or less than about 1 cP (e.g., about or at least about 1000 s -1 In some embodiments, the reference pharmaceutical composition at about 32-35° C. has a viscosity of less than about 1 cP (e.g., at a shear rate of at least about 1000 s -1(at a shear rate of 100 .mu.m).

[0162] In some embodiments, the pharmaceutical compositions provided herein have a viscosity of ≦183 mPas at 20° C. In some embodiments, the pharmaceutical compositions provided herein have a viscosity of ≦183 mPas at 5° C. Since viscosity is dependent on shear rate, the “viscosity” of a pharmaceutical composition is measured by the shear rate of 0.01 s -1 ~100,000s -1 In some embodiments, the units of viscosity can be defined as cP or mPas. In some cases, cP and mPas are used interchangeably.

[0163] In some embodiments, the pharmaceutical compositions provided herein have a viscosity of less than 265-655 mPas at 32-35°C.

[0164] In some embodiments, the viscosity of the pharmaceutical composition at about 32-35° C. is at least about 10 cP, or at least about 100 cP, or at least about 1000 cP, or at least about 10,000 cP, or at least about 70,000 cP, or up to about 200,000 cP, or up to about 250,000 cP, or up to about 300,000 cP or higher. In some embodiments, the shear rate is about or at least about a shear rate of 1000 / sec. In some embodiments, the formulation is characterized by a zero shear viscosity of at least 300,000 mPas. In some embodiments, the formulation is characterized by a zero shear viscosity of at least 1000 s. -1 The composition is further characterized by a viscosity of about 400 mPas or less at a shear rate of 100 .mu.m.

[0165] In some embodiments, the pharmaceutical compositions provided herein remain in the SCS (or in the eye) for a longer period of time following injection (measured at different time points) compared to a reference pharmaceutical formulation, or a formulation having a lower viscosity and / or elastic modulus (G') at about 32-35° C. In some embodiments, the pharmaceutical compositions provided herein enhance the thickness of the SCS or injection site (e.g., compared to a reference pharmaceutical composition, or a formulation having a lower viscosity and / or elastic modulus (G') at about 32-35° C.) (see Section 4.2.3).

[0166] In some embodiments, the elastic modulus of the pharmaceutical compositions provided herein at less than 27° C. is less than or about 0.1 Pa, less than or about 0.01 Pa, less than or about 0.001 Pa, or zero. In some embodiments, the elastic modulus of the pharmaceutical compositions provided herein at 32° C.-35° C. is about or at least about 0.1 Pa, about or at least about 1 Pa, about or at least about 10 Pa, about or at least about 100 Pa, about or at least about 1000 Pa, about or at least about 10,000 Pa, or about or at least about 10,000 Pa, or about or at least about 100,000 Pa.

[0167] In some embodiments, the pharmaceutical compositions provided herein have a gelation temperature greater than 27° C. In some embodiments, the pharmaceutical compositions provided herein have a gelation temperature less than 32° C. In some embodiments, the pharmaceutical compositions provided herein have a gelation temperature greater than about 27-32° C. In some embodiments, the pharmaceutical compositions provided herein have a gelation temperature of about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35° C.

[0168] In some embodiments, the pharmaceutical compositions provided herein have a gelation time of greater than about 10 seconds. In some embodiments, the pharmaceutical compositions provided herein have a gelation time of greater than about 15 seconds. In some embodiments, the pharmaceutical compositions provided herein have a gelation time of about 10-15 seconds, about 15-20 seconds, about 20-25 seconds, about 25-30 seconds, about 30-35 seconds, about 35-40 seconds, about 40-45 seconds, about 45-50 seconds, about 50-55 seconds, about 55-60 seconds, about 60-65 seconds, about 65-70 seconds, about 70-75 seconds, about 75-80 seconds, about 80-85 seconds, or about 85-90 seconds. In some embodiments, the pharmaceutical compositions provided herein have a gelation time of less than 90 seconds. In some embodiments, the gelation time is determined at about 34° C. In some embodiments, the gelation time is determined at about 32-34° C. In some embodiments, the gelation time of the pharmaceutical composition is longer than the injection time of said composition, hi some embodiments, the gelation time is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% longer or greater than 90% longer than the injection time.

[0169] In some embodiments, the pharmaceutical composition at about 32-35° C. has a viscosity sufficient to expand at least a portion of the injection site (e.g., the SCS) to a thickness of at least 500 μm or about 500 μm to about 3 mm for at least 2 hours after administration. In some embodiments, the viscosity of the pharmaceutical composition at about 32-35° C. is sufficient to expand the injection site (e.g., the SCS) to a thickness of about 750 μm to about 2.8 mm, about 750 μm to about 2.5 mm, about 750 μm to about 2 mm, or about 1 mm to about 2 mm. In some embodiments, the viscosity of the pharmaceutical composition at about 32-35° C. is sufficient to expand the injection site (e.g., the SCS) to a thickness of about 500 μm to about 3.0 mm for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 2 days, at least 3 days, at least 5 days, at least 10 days, at least 21 days, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 3 years, or at least 5 years after administration. In some embodiments, the viscosity of the pharmaceutical composition at about 32-35° C. is sufficient to expand the injection site (e.g., the SCS) to a thickness of about 1 mm to about 3 mm for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, or at least 24 hours after administration.In some embodiments, the viscosity of the pharmaceutical composition at about 32-35° C. is sufficient to expand the injection site (e.g., the SCS) to a thickness of about 1 mm to about 2 mm for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 2 days, at least 3 days, at least 5 days, at least 10 days, at least 21 days, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 3 years, or at least 5 years after administration. In some embodiments, the viscosity of the pharmaceutical composition at about 32-35° C. is sufficient to expand the injection site (e.g., SCS) to a thickness of about 2 mm to about 3 mm for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 2 days, at least 3 days, at least 5 days, at least 10 days, at least 21 days, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 3 years, or at least 5 years after administration. In some embodiments, the viscosity of the pharmaceutical composition at about 32-35° C. is sufficient to expand the injection site (e.g., SCS) to a thickness of about 750 μm to about 2.8 mm, about 750 μm to about 2.5 mm, about 750 μm to about 2 mm, or about 1 mm to about 2 mm indefinitely. The indefinite duration can be achieved, at least in part, due to the stability of the pharmaceutical composition in the injection site (eg, the SCS).

[0170] In some embodiments, a pharmaceutical composition at about 32-35° C. that has a viscosity sufficient to expand an injection site (e.g., an SCS) to a thickness of at least 500 μm, or from about 500 μm to about 3 mm, has a viscosity greater than the viscosity of water at about 32-35° C. (i.e., about 1 cP). In some embodiments, the pharmaceutical composition at about 32-35° C. has sufficient viscosity to expand the injection site (e.g., SCS) to a thickness of at least about 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, or greater. In some embodiments, the reference pharmaceutical composition at about 32-35° C. may be used to inject a fluororesin into the injection site at a concentration of about 1 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, 100 μm, 20 and has sufficient viscosity to expand to a thickness of 0 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm or less.

[0171] Also provided herein are methods of treating diseases (e.g., ocular diseases) described in section 4.5 using the pharmaceutical compositions disclosed herein. In some embodiments, the method of treating ocular diseases comprises administering an effective amount of a pharmaceutical composition (e.g., a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene) to a subject (e.g., a human). In some embodiments, the pharmaceutical composition is administered to the suprachoroidal space (SCS) of the subject's eye. In some embodiments, the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered to the SCS is less than the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered subretinally. In some embodiments, the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered to the SCS is less than the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered intravitreally. In some embodiments, the pharmaceutical composition has the same vector genome concentration when administered to the SCS as when administered via subretinal or intravitreal administration. In some embodiments, the pharmaceutical composition has the same amount of genome copies when administered to the SCS as when administered via subretinal or intravitreal administration. In some embodiments, the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response in a subject is lower than the effective amount of the reference pharmaceutical composition sufficient to elicit a therapeutic response in a subject when administered to the SCS. In some embodiments, the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered to the SCS is less than the effective amount of the reference pharmaceutical composition sufficient to elicit a therapeutic response when administered subretinally. In some embodiments, the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered to the SCS is less than the effective amount of the reference pharmaceutical composition sufficient to elicit a therapeutic response when administered intravitreally. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same amount of genome copies. In some embodiments, the pharmaceutical composition has a viscosity and / or elastic modulus (G') higher than the viscosity and / or elastic modulus (G') of the reference pharmaceutical composition.

[0172] In some embodiments, the pharmaceutical composition is localized substantially near the insertion site (see Sections 4.2.1 and 4.2.2). In some embodiments, the pharmaceutical composition provides a higher level of transgene expression (concentration) when the pharmaceutical composition is administered to the SCS compared to when the pharmaceutical composition is administered subretinal or intravitreal (see Section 4.2.6). In some embodiments, the pharmaceutical composition provides a higher level of transgene expression (concentration) when the pharmaceutical composition is administered to the SCS compared to when a reference pharmaceutical composition is administered subretinal, intravitreal, or SCS (see Section 4.2.6). In some embodiments, the pharmaceutical composition provides a higher level of AAV when the pharmaceutical composition is administered to the SCS compared to when the pharmaceutical composition is administered subretinal or intravitreal (see Section 4.2.5). In some embodiments, the pharmaceutical composition provides a higher level of AAV when the pharmaceutical composition is administered to the SCS compared to when a reference pharmaceutical composition is administered subretinal, intravitreal, or SCS (see Section 4.2.5). In some embodiments, the pharmaceutical composition provides a greater transduction rate (or infection rate) at the injection site when the pharmaceutical composition is administered to the SCS compared to when the pharmaceutical composition is administered subretinal or intravitreal (see Section 4.2.5). In some embodiments, the pharmaceutical composition provides a greater transduction rate (or infection rate) at the injection site when the pharmaceutical composition is administered to the SCS compared to when a reference pharmaceutical composition is administered subretinal, intravitreal, or SCS (see Section 4.2.5). In some embodiments, the pharmaceutical composition provides reduced vasodilation and / or vascular leakage when the pharmaceutical composition is administered to the SCS compared to when the pharmaceutical composition is administered subretinal or intravitreal (see Section 4.2.4). In some embodiments, the pharmaceutical composition provides reduced vasodilation and / or vascular leakage when the pharmaceutical composition is administered to the SCS compared to when a reference pharmaceutical composition is administered subretinal, intravitreal, or SCS (see Section 4.2.4). In some embodiments, the reference pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene.In some embodiments, the pharmaceutical composition at about 32-35° C. has a higher viscosity and / or elastic modulus (G') than a reference pharmaceutical composition at about 32-35° C. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same amount of genome copies.

[0173] 4.1.1 Viscosity manipulation In some embodiments, the viscosity and / or elastic modulus (G') of the pharmaceutical compositions provided herein increases to a value that sufficiently exceeds the viscosity of water (e.g., at least about 100 cP at a shear rate of 0.1 / sec) when the formulation is warmed to about 32-35° C., resulting in a formulation that is highly effective for placement, e.g., injection, into the eye (e.g., SCS) of a subject. In some embodiments, the relatively high viscosity and / or elastic modulus of the formulation at about 32-35° C. enhances the ability of such formulations to maintain a therapeutic component (e.g., an AAV comprising an expression cassette that includes a transgene) substantially homogenously suspended in the formulation for extended periods of time, and may also aid in the storage stability of the formulation.

[0174] The pharmaceutical compositions provided herein (e.g., thermoresponsive pharmaceutical compositions) may include a liquid dispersion medium ("solvent") and a gelling agent ("gelator"). The solvent molecules can penetrate the hydrocolloid network formed by the gelator. In some embodiments, the pharmaceutical compositions provided herein include a hydrophilic polymer in an aqueous system. In some embodiments, the pharmaceutical compositions provided herein include a natural polymer (e.g., xanthan gum, starch, gellan, konjac, carrageenan, collagen, fibrin, silk fibroin, hyaluronic acid, or gelatin). In some embodiments, the pharmaceutical compositions provided herein include a synthetic polymer (e.g., chitosan-β-glycerophosphate, poly(N-isopropylacrylamide) (pNIPAAm), Pluronic F127, methylcellulose, or PEG-PCL). See, e.g., Taylor et al., Gels. 2017 March;3(1):4.

[0175] Non-limiting examples of solutions having higher viscosity and / or elastic modulus (G') at about 32-35°C compared to lower temperatures and that can be used in the pharmaceutical compositions of the present disclosure include solutions containing various concentrations of poloxamer 407 (P407, CAS No.: 9003-11-6) and poloxamer 188 (P188, CAS No.: 9003-11-6). In some embodiments, the pharmaceutical compositions provided herein contain 16% P407 and 0% P188. In some embodiments, the pharmaceutical compositions provided herein contain 22% P407 and 0% P188. In some embodiments, the pharmaceutical compositions provided herein contain 16% P407 and 16% P188. In some embodiments, the pharmaceutical compositions provided herein contain 22% P407 and 16% P188. In some embodiments, the pharmaceutical compositions provided herein contain 19% P407 and 0% P188. In some embodiments, the pharmaceutical compositions provided herein comprise 16% P407 and 8% P188. In some embodiments, the pharmaceutical compositions provided herein comprise 22% P407 and 8% P188. In some embodiments, the pharmaceutical compositions provided herein comprise 19% P407 and 8% P188.

[0176] 4.1.2 Other components of the formulation In some embodiments, the present disclosure provides a pharmaceutical composition (e.g., a liquid formulation) comprising a recombinant adeno-associated virus (AAV) and at least one of potassium dihydrogen phosphate, sodium chloride, dibasic sodium phosphate anhydrous, sucrose, and a surfactant. In some embodiments, the pharmaceutical composition (e.g., a liquid formulation) does not comprise sucrose. Assays such as those described in Section 4.6 and / or Section 5 can be used to determine that the presence of additional components does not interfere with the properties of the formulations of the invention, such as higher viscosity and / or elastic modulus (G') at increased temperature.

[0177] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) and at least one of an ionic salt excipient or buffer, sucrose, and a surfactant. In some embodiments, the ionic salt excipient or buffer may be one or more members from the group consisting of potassium dihydrogen phosphate, potassium phosphate, sodium chloride, dibasic sodium phosphate anhydrous, sodium phosphate hexahydrate, monobasic sodium 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-piperazineethanesulfonic 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 members from the group consisting of poloxamer 188, polysorbate 20, and polysorbate 80.

[0178] In some embodiments, the pharmaceutical composition comprises about, at least about, or at most about 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1 mg / mL, 1.05 mg / mL, 1.10 mg / mL, 1.15 mg / mL, 1.20 mg / mL, 1.25 mg / mL, 1.30 mg / mL, 1.35 mg / mL, 1.40 mg / mL, 1.45 mg / mL, 1.50 mg / mL, or greater than 1.50 mg / mL sodium phosphate dibasic anhydrous (or equivalent). In some embodiments, the pharmaceutical composition comprises a dilution of about, at least about, or at most about 4.5 mg / mL, 4.55 mg / mL, 4.6 mg / mL, 4.65 mg / mL, 4.7 mg / mL, 4.75 mg / mL, 4.8 mg / mL, 4.85 mg / mL, 4.9 mg / mL, 4.95 mg / mL, 5 mg / mL, 5.05 mg / mL, 5.10 mg / mL, 5.15 mg / mL, 5.20 mg / mL, 5.25 mg / mL, 5.30 mg / mL, 5.35 mg / mL, 5.40 mg / mL, 5.45 mg / mL, / mL, 5.50 mg / mL, 5.55 mg / mL, 5.60 mg / mL, 5.65 mg / mL, 5.70 mg / mL, 5.75 mg / mL, 5.80 mg / mL, 5.81 mg / mL, 5.82 mg / mL, 5.83 mg / mL, 5.84 mg / mL, 5.85 mg / mL, 5.86 mg / mL, 5.87 mg / mL, 5.88 mg / mL, 5.89 mg / mL, 5.90 mg / mL, 5.95 mg / mL, 6 mg / mL, or greater than 6 mg / mL sodium chloride (or equivalent). In some embodiments, the pharmaceutical composition comprises about, at least about, or at most about 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, or greater than 1 mg / mL of potassium chloride and / or potassium dihydrogen phosphate (or equivalents thereof).

[0179] In certain embodiments, the pharmaceutical composition has an ionic strength of about 60 mM to about 115 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 60 mM to about 100 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 65 mM to about 95 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 70 mM to about 90 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 75 mM to about 85 mM.

[0180] In certain embodiments, the pharmaceutical composition has an ionic strength of about 30 mM to about 100 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 35 mM to about 95 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 40 mM to about 90 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 45 mM to about 85 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 50 mM to about 80 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 55 mM to about 75 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 60 mM to about 70 mM.

[0181] In certain embodiments, the pharmaceutical composition comprises potassium chloride (e.g., at a concentration of 0.2 g / L). In certain embodiments, the pharmaceutical composition comprises potassium dihydrogen phosphate (e.g., at a concentration of 0.2 g / L). In certain embodiments, the pharmaceutical composition comprises sodium chloride (e.g., at a concentration of 5.84 g / L). In certain embodiments, the pharmaceutical composition comprises sodium phosphate dibasic anhydrous (e.g., at a concentration of 1.15 g / L). In certain embodiments, the pharmaceutical composition comprises potassium chloride, potassium dihydrogen phosphate, sodium chloride, and sodium phosphate dibasic anhydrous.

[0182] In certain embodiments, the pharmaceutical composition contains sucrose in a concentration of 3% (weight / volume, 30 g / L) to 18% (weight / volume, 180 g / L).In certain embodiments, the pharmaceutical composition contains sucrose in a concentration of 4% (weight / volume, 40 g / L).

[0183] In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.001% (weight / volume, 0.01 g / L). In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0005% (weight / volume, 0.005 g / L) to 0.05% (weight / volume, 0.5 g / L). In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.001% (weight / volume, 0.01 g / L). In certain embodiments, the pharmaceutical composition comprises polysorbate 20 at a concentration of 0.0005% (weight / volume, 0.05 g / L) to 0.05% (weight / volume, 0.5 g / L). In certain embodiments, the pharmaceutical composition comprises polysorbate 80 at a concentration of 0.0005% (weight / volume, 0.05 g / L) to 0.05% (weight / volume, 0.5 g / L). In some embodiments, the pharmaceutical composition comprises a surfactant (e.g., poloxamer 188, polysorbate 20, and / or polysorbate 80) at a concentration of about, at least about, or at most about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003 ... Contains at concentrations of 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 0.1% or greater.

[0184] In certain embodiments, the pH of the pharmaceutical composition is about 7.4. In certain embodiments, the pH of the pharmaceutical composition is about 6.0 to 9.0. In certain embodiments, the pH of the pharmaceutical composition is 7.4. In certain embodiments, the pH of the pharmaceutical composition is 6.0 to 9.0.

[0185] In certain embodiments, the pharmaceutical composition is in a hydrophobically coated glass vial. In certain embodiments, the pharmaceutical composition is in a Cyclo Olefin Polymer (COP) vial. In certain embodiments, the pharmaceutical composition is in a Daikyo Crystal Zenith® (CZ) vial. In certain embodiments, the pharmaceutical composition is in a TopLyo coated vial.

[0186] In certain embodiments, disclosed herein is a pharmaceutical composition comprising a recombinant AAV and at least one of: (a) potassium chloride at 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, (d) sodium phosphate dibasic anhydrous at a concentration of 1.15 g / L, (e) sucrose at a concentration of 4% weight / volume (40 g / L), (f) poloxamer 188 at a concentration of 0.001% weight / volume (0.01 g / L), and (g) water, wherein the recombinant AAV is AAV8. In some embodiments, the pharmaceutical composition does not comprise sucrose.

[0187] In certain embodiments, disclosed herein is a pharmaceutical composition comprising recombinant AAV, 18.0% w / v poloxamer 407, and 6.5% w / v poloxamer 188, each of which is in a solution of 5.84 mg / mL sodium chloride, 0.201 mg / mL potassium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 0.200 mg / mL potassium phosphate dibasic, 40.0 mg / mL sucrose (4% w / v), and 0.001% (0.01 mg / mL) poloxamer 188, pH 7.4.

[0188] In some embodiments, the pharmaceutical composition comprises (a) a construct II encoding an anti-human vascular endothelial growth factor (hVEGF) antibody, and at least one of: (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) sodium phosphate dibasic anhydrous at a concentration of 1.15 g / L, (f) sucrose at a concentration of 4% weight / volume (40 g / L), (g) poloxamer 188 at a concentration of 0.001% weight / volume (0.01 g / L), and (h) water, wherein the anti-hVEGF antibody 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. In some embodiments, the pharmaceutical composition does not comprise sucrose.

[0189] In some embodiments, the pharmaceutical composition comprises a viral vector comprising a vector genome comprising SEQ ID NO:56 (equivalent to SEQ ID NO:14 of US 11,197,937) and at least one of: (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) sodium phosphate dibasic anhydrous at a concentration of 1.15 g / L, (f) sucrose at a concentration of 4% weight / volume (40 g / L), (g) poloxamer 188 at a concentration of 0.001% weight / volume (0.01 g / L), (h) water, (i) poloxamer 407 at 18% w / v, and (j) poloxamer 188 at 6.5% w / v. In some embodiments, the pharmaceutical composition does not comprise sucrose.

[0190] In some embodiments, the pharmaceutical composition comprises (a) AAV8 or AAV9 encoding tripeptidyl-peptidase 1, and at least one of: (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) sodium phosphate dibasic anhydrous at a concentration of 1.15 g / L, (f) sucrose at a concentration of 4% weight / volume (40 g / L), (g) poloxamer 188 at a concentration of 0.001% weight / volume (0.01 g / L), and (h) water. In some embodiments, the pharmaceutical composition does not comprise sucrose.

[0191] In some embodiments, the pharmaceutical composition has a desired viscosity, elastic modulus (G'), density, and / or osmolality suitable for injection into the choroid (e.g., via a drug delivery device into the choroid, such as a microinjector with a fine needle). 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 gel.

[0192] In certain embodiments, the pharmaceutical composition has an osmolality range of 200 mOsm / L to 660 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about, at least about, or at most about 200 mOsm / L, 250 mOsm / L, 300 mOsm / L, 350 mOsm / L, 400 mOsm / L, 450 mOsm / L, 500 mOsm / L, 550 mOsm / L, 600 mOsm / L, 650 mOsm / L, or 660 mOsm / L.

[0193] In certain embodiments, the gene therapy construct is provided as a freeze-sterilized, single-use solution of AAV vector active ingredients in a formulation buffer. In a specific embodiment, a pharmaceutical composition suitable for suprachoroidal administration comprises a suspension of a recombinant (e.g., rHuGlyFabVEGFi) vector in a formulation buffer that includes a physiologically compatible aqueous buffer, a surfactant, and optional excipients. In some embodiments, the construct is formulated in Dulbecco's phosphate buffered saline and 0.001% poloxamer 188, pH=7.4.

[0194] In a specific embodiment, the composition comprises modified Dulbecco's phosphate buffered saline solution, and optionally a surfactant. In another specific embodiment, the pharmaceutical composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium dihydrogen phosphate, 5.84 mg / mL sodium chloride, 1.15 mg / mL anhydrous sodium phosphate dibasic, 40.0 mg / mL (4% w / v) sucrose, and optionally a surfactant. In another specific embodiment, the composition comprises potassium chloride, potassium dihydrogen phosphate, sodium chloride, anhydrous sodium phosphate dibasic, sucrose, and optionally a surfactant.

[0195] In some embodiments, the pharmaceutical compositions provided herein are not compositions described in Zeinab et al. (European Journal of Pharmaceutics and Biopharmaceutics 114 (2017) 119-13).

[0196] 4.2 Functional characteristics 4.2.1 Clearance Time The present disclosure provides pharmaceutical compositions (e.g., compositions comprising an AAV comprising an expression cassette encoding a transgene) that provide a delayed clearance time from the SCS. In some embodiments, a pharmaceutical composition that is viscous (or more viscous), and / or elastic, and / or gelled (or more elastic, and / or more gelled) at about 32-35° C. provides a delayed clearance time from the SCS compared to a pharmaceutical composition that is non-viscous or has a low viscosity and / or has a low elasticity and / or is not gelled at about 32-35° C. In some embodiments, a pharmaceutical composition that is viscous (or more viscous), and / or elastic, and / or gelled (or more elastic, and / or more gelled) at about 32-35° C. provides a delayed clearance time from the eye compared to a pharmaceutical composition that is non-viscous or has a low viscosity and / or has a low elasticity and / or is not gelled at about 32-35° C. In some embodiments, a more viscous and / or more elastic and / or more gelled pharmaceutical composition results in a delayed clearance time from the eye compared to a less viscous and / or less elastic and / or less gelled pharmaceutical composition. In some embodiments, a more viscous and / or more elastic and / or more gelled pharmaceutical composition at about 32-35° C. has a viscosity value higher than the viscosity of water at about 32-35° C. In some embodiments, a more viscous, more gelled pharmaceutical composition at about 32-35° C. has a viscosity value and / or elastic modulus value higher than the viscosity and / or elastic modulus of a solution typically used for subretinal injection at about 32-35° C. In some embodiments, the clearance time of the pharmaceutical composition after the pharmaceutical composition is administered to the SCS is equal to or longer than the clearance time of the reference pharmaceutical composition after the reference pharmaceutical composition is administered subretinal or intravitreal. In some embodiments, the clearance time of the pharmaceutical composition after the pharmaceutical composition is administered to the SCS is equal to or longer than the clearance time of the reference pharmaceutical composition after the reference pharmaceutical composition is administered to the SCS.

[0197] In some embodiments, a pharmaceutical composition (e.g., a composition comprising an AAV that includes an expression cassette encoding a transgene) may be administered for about 30 minutes to about 20 hours, about 2 hours to about 20 hours, about 30 minutes to about 24 hours, about 1 hour to about 2 hours, about 30 minutes to about 90 days, about 30 minutes to about 60 days, about 30 minutes to about 30 days, about 30 minutes to about 21 days, about 30 minutes to about 14 days, about 30 minutes to about 7 days, about 30 minutes to about 3 days, about 30 minutes to about 2 days, about 30 minutes to about 1 day, about 4 hours to about 90 days, about 4 hours to about 60 days, about 4 hours to about 30 days, about 4 hours to about 21 days, The clearance time from the SCS is about 4 hours to about 14 days, about 4 hours to about 7 days, about 4 hours to about 3 days, about 4 hours to about 2 days, about 4 hours to about 1 day, about 4 hours to about 8 hours, about 4 hours to about 16 hours, about 4 hours to about 20 hours, about 1 day to about 90 days, about 1 day to about 60 days, about 1 day to about 30 days, about 1 day to about 21 days, about 1 day to about 14 days, about 1 day to about 7 days, about 1 day to about 3 days, about 2 days to about 90 days, about 3 days to about 90 days, about 3 days to about 60 days, about 3 days to about 30 days, about 3 days to about 21 days, about 3 days to about 14 days, or about 3 days to about 7 days. In some embodiments, the clearance time from the SCS is about 3 days to about 365 days, about 3 days to about 300 days, about 3 days to about 200 days, about 3 days to about 150 days, about 3 days to about 125 days, about 7 days to about 365 days, about 7 days to about 300 days, about 7 days to about 200 days, about 7 days to about 150 days, about 7 days to about 125 days. "Clearance time from the SCS" is the time required for substantially all of the pharmaceutical composition, medicinal substance, or AAV to be eliminated from the SCS. In some embodiments, "clearance time from the SCS" is the time required for the pharmaceutical composition, medicinal substance, or AAV to become undetectable in the SCS by any standard method (e.g., those described in Sections 4.6 and 5). In some embodiments, the "clearance time from the SCS" is when the pharmaceutical composition, drug substance, or AAV is present in the SCS in an amount of about 2% or less, or about 5% or less, as detected by any standard method (e.g., those described in Sections 4.6 and 5).

[0198] In some embodiments, a pharmaceutical composition (e.g., a composition comprising an AAV that includes an expression cassette encoding a transgene) is administered in an amount of about 30 minutes to about 20 hours, about 2 hours to about 20 hours, about 30 minutes to about 24 hours, about 1 hour to about 2 hours, about 30 minutes to about 90 days, about 30 minutes to about 60 days, about 30 minutes to about 30 days, about 30 minutes to about 21 days, about 30 minutes to about 14 days, about 30 minutes to about 7 days, about 30 minutes to about 3 days, about 30 minutes to about 2 days, about 30 minutes to about 1 day, about 4 hours to about 90 days, about 4 hours to about 60 days, about 4 hours to about 30 days, about 4 hours to about 21 days. , about 4 hours to about 14 days, about 4 hours to about 7 days, about 4 hours to about 3 days, about 4 hours to about 2 days, about 4 hours to about 1 day, about 4 hours to about 8 hours, about 4 hours to about 16 hours, about 4 hours to about 20 hours, about 1 day to about 90 days, about 1 day to about 60 days, about 1 day to about 30 days, about 1 day to about 21 days, about 1 day to about 14 days, about 1 day to about 7 days, about 1 day to about 3 days, about 2 days to about 90 days, about 3 days to about 90 days, about 3 days to about 60 days, about 3 days to about 30 days, about 3 days to about 21 days, about 3 days to about 14 days, or about 3 days to about 7 days. In some embodiments, the clearance time from the eye is about 3 days to about 365 days, about 3 days to about 300 days, about 3 days to about 200 days, about 3 days to about 150 days, about 3 days to about 125 days, about 7 days to about 365 days, about 7 days to about 300 days, about 7 days to about 200 days, about 7 days to about 150 days, about 7 days to about 125 days. "Clearance time from the eye" is the time required for substantially all of the pharmaceutical composition, medicinal substance, or AAV to be eliminated from the eye. In some embodiments, "clearance time from the eye" is the time required for the pharmaceutical composition, medicinal substance, or AAV to become undetectable in the eye by any method (e.g., those described in Sections 4.6 and 5). In some embodiments, the "clearance time from the eye" is when the pharmaceutical composition, drug substance, or AAV is present in the eye in an amount of about 2% or less, or about 5% or less, as detected by any standard method (e.g., those described in Sections 4.6 and 5).

[0199] In some embodiments, the clearance time is within about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, Not earlier than 23, 25, 27, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 days (e.g., clearance time from the SCS or eye does not occur prior to that). In some embodiments, the clearance time is within about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17th, 18th, 19th, 20th, 21st, 23rd, 25th, 27th, 30th, 35th, 40th, 50th, 55th, 60th, 65th, 70th, 75th, 80th, 85th, 90th, 95th, 100th, 120th, 140th, 160th, 180th, 200th, 220th, 240th, 260th, 280th, 300th, 320th, 340th, 360th, 380th, or 400 days.

[0200] In some embodiments, a more viscous and / or more elastic and / or more gelled pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) will last at least 2 times longer, at least 3 times longer, at least 4 times longer, at least 5 times longer, at least 6 times longer, at least 7 times longer, at least 8 times longer, at least 9 times longer, at least 10 times longer, at least 15 times longer, at least 20 times longer, at least 50 times longer than when a less viscous and / or less elastic and / or less gelled pharmaceutical composition is used to administer the AAV comprising the expression cassette encoding a transgene (e.g., via subretinal administration, intravitreal administration, or into the SCS). , at least 100 times longer, at least 5% longer, at least 10% longer, at least 15% longer, at least 20% longer, at least 25% longer, at least 30% longer, at least 35% longer, at least 40%, at least 45% longer, at least 50% longer, at least 55% longer, at least 60% longer, at least 65% longer, at least 70% longer, at least 75% longer, at least 80% longer, at least 85% longer, at least 90% longer, at least 95% longer, at least 100% longer, at least 150% longer, or at least 200% longer, at least 250% longer, or at least 300%, at least 400% longer, or at least 500% longer.

[0201] In some embodiments, suprachoroidal administration of a more viscous and / or more elastic and / or more gelled pharmaceutical composition at about 32-35 ° C. (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) is at least 2 times longer, at least 3 times longer, at least 4 times longer, at least 5 times longer, at least 6 times longer, at least 7 times longer, at least 8 times longer, at least 9 times longer, at least 10 times longer, at least 15 times longer, at least 20 times longer, at least 50 times longer than when a less viscous and / or less elastic and / or less gelled pharmaceutical composition at about 32-35 ° C. is used to administer, for example, an AAV comprising an expression cassette encoding a transgene by suprachoroidal administration. , at least 100 times longer, at least 5% longer, at least 10% longer, at least 15% longer, at least 20% longer, at least 25% longer, at least 30% longer, at least 35% longer, at least 40%, at least 45% longer, at least 50% longer, at least 55% longer, at least 60% longer, at least 65% longer, at least 70% longer, at least 75% longer, at least 80% longer, at least 85% longer, at least 90% longer, at least 95% longer, at least 100% longer, at least 150% longer, or at least 200% longer, at least 250% longer, or at least 300%, at least 400% longer, or at least 500% longer.

[0202] In some embodiments, suprachoroidal administration of a more viscous and / or more elastic and / or more gelled pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) at about 32-35° C. may be at least 2 times longer, at least 3 times longer, at least 4 times longer, at least 5 times longer, at least 6 times longer, at least 7 times longer, at least 8 times longer, at least 9 times longer, at least 10 times longer, at least 15 times longer, at least 20 times longer, at least 50 times longer, at least 100 times longer than when a less viscous pharmaceutical composition at about 32-35° C. is used to administer, for example, an AAV comprising an expression cassette encoding a transgene by subretinal or intravitreal administration. , resulting in a clearance time that is at least 5% longer, at least 10% longer, at least 15% longer, at least 20% longer, at least 25% longer, at least 30% longer, at least 35% longer, at least 40%, at least 45% longer, at least 50% longer, at least 55% longer, at least 60% longer, at least 65% longer, at least 70% longer, at least 75% longer, at least 80% longer, at least 85% longer, at least 90% longer, at least 95% longer, at least 100% longer, at least 150% longer, or at least 200% longer, at least 250% longer, or at least 300%, at least 400% longer, or at least 500% longer.

[0203] In some embodiments, suprachoroidal administration of a pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) that is viscous (e.g., relatively viscous, moderately to very highly viscous, or more viscous than water, or more viscous than a control solution, or more viscous than a solution typically used for subretinal administration) at about 32-35° C. is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, when the same pharmaceutical composition is used to administer, e.g., an AAV comprising an expression cassette encoding a transgene via subretinal or intravitreal administration. The clearance time may be at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater.

[0204] In some embodiments, the clearance time of a relatively viscous and / or elastic and / or gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) administered by injection into the suprachoroidal layer at about 32-35° C. is longer than the clearance time of the same pharmaceutical composition administered via subretinal or intravitreal administration. In some embodiments, the clearance time of a more viscous and / or more elastic and / or more gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) administered by injection into the suprachoroidal layer at about 32-35° C. is longer than a comparably less viscous and / or less elastic and / or less gelled pharmaceutical composition administered by injection into the suprachoroidal layer at about 32-35° C. In some embodiments, the clearance time of a more viscous and / or more elastic and / or more gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) administered by injection into the suprachoroidal layer is longer than a comparable less viscous and / or less elastic and / or less gelled pharmaceutical composition administered via subretinal or intravitreal administration at about 32-35° C. In some embodiments, the clearance time of a viscous and / or elastic and / or gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) administered by injection into the suprachoroidal layer is longer than a comparable viscous and / or elastic and / or gelled pharmaceutical composition administered via subretinal or intravitreal administration at about 32-35° C.

[0205] In some embodiments, the clearance time of a viscous and / or elastic and / or gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) administered by injection into the suprachoroidal layer at about 32-35° C. is at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 30 minutes, 35 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 120 minutes, 140 minutes, 160 minutes, 180 minutes, 200 minutes ...5 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 120 minutes, 140 minutes, 160 minutes, 180 minutes, 200 minutes, 22 hours, 35 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 120 minutes, 140 minutes, 160 minutes, 180 minutes, 200 minutes, 22 hours, 35 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 120 minutes, 14 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days longer.

[0206] In some embodiments, the clearance time of a more viscous and / or more elastic and / or more gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) administered by injection into the suprachoroidal membrane at about 32-35° C. is at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, or less than a comparably less viscous and / or less elastic and / or non-gelled pharmaceutical composition administered by injection into the suprachoroidal membrane at about 32-35° C. between, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days longer.

[0207] In some embodiments, the clearance time of a more viscous and / or more elastic and / or more gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) administered by injection into the suprachoroidal layer is at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours ... 4 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days longer.

[0208] In some embodiments, the clearance time of a pharmaceutical composition administered via intravitreal or subretinal injection is about 30 minutes or less, 1 hour or less, 2 hours or less, 3 hours or less, 4 hours or less, 5 hours or less, 6 hours or less, 7 hours or less, 8 hours or less, 9 hours or less, 10 hours or less, 12 hours or less, 14 hours or less, 16 hours or less, 18 hours or less, 20 hours or less, 22 hours or less, 24 hours or less, 1 day or less, 2 days or less, 3 days or less, 4 days or less, 5 days or less, 6 days or less, 7 days or less, 8 days or less, 9 days or less, 10 days or less, 11 days or less, 12 days or less, 13 days or less, 14 days or less, 15 days or less after administration. , 16 days or less, 17 days or less, 18 days or less, 19 days or less, 20 days or less, 21 days or less, 23 days or less, 25 days or less, 27 days or less, 30 days or less, 35 days or less, 40 days or less, 50 days or less, 55 days or less, 60 days or less, 65 days or less, 70 days or less, 75 days or less, 80 days or less, 85 days or less, 90 days or less, 95 days or less, 100 days or less, 120 days or less, 140 days or less, 160 days or less, 180 days or less, 200 days or less, 220 days or less, 240 days or less, 260 days or less, 280 days or less, 300 days or less, 320 days or less, 340 days or less, 360 days or less, 380 days or less, or about 400 days or less.

[0209] In some embodiments, the clearance time of a reference pharmaceutical composition administered by intravitreal injection, subretinal injection, or into the SCS is about 30 minutes or less, 1 hour or less, 2 hours or less, 3 hours or less, 4 hours or less, 5 hours or less, 6 hours or less, 7 hours or less, 8 hours or less, 9 hours or less, 10 hours or less, 12 hours or less, 14 hours or less, 16 hours or less, 18 hours or less, 20 hours or less, 22 hours or less, 24 hours or less, 1 day or less, 2 days or less, 3 days or less, 4 days or less, 5 days or less, 6 days or less, 7 days or less, 8 days or less, 9 days or less, 10 days or less, 11 days or less, 12 days or less, 13 days or less, 14 days ... or 5 days or less, 16 days or less, 17 days or less, 18 days or less, 19 days or less, 20 days or less, 21 days or less, 23 days or less, 25 days or less, 27 days or less, 30 days or less, 35 days or less, 40 days or less, 50 days or less, 55 days or less, 60 days or less, 65 days or less, 70 days or less, 75 days or less, 80 days or less, 85 days or less, 90 days or less, 95 days or less, 100 days or less, 120 days or less, 140 days or less, 160 days or less, 180 days or less, 200 days or less, 220 days or less, 240 days or less, 260 days or less, 280 days or less, 300 days or less, 320 days or less, 340 days or less, 360 days or less, 380 days or less, or about 400 days or less.

[0210] In some embodiments, the clearance time is from the eye. In some embodiments, the clearance time is from an SCS. In some embodiments, the clearance time is from an injection site.

[0211] In some embodiments, "clearance time from the SCS" is the amount of time required following injection of a pharmaceutical composition, pharmaceutical agent, or AAV necessary to reduce the thickness of the SCS at or near the injection site to about 1 nm or less, about 2 nm or less, about 5 nm or less, about 10 nm or less, about 25 nm or less, about 50 nm or less, about 100 nm or less, about 200 nm or less, or about 500 nm or less, as measured by standard techniques (e.g., in vivo imaging techniques such as OCT imaging, ultra-high resolution OCT (UHR-OCT), ultrasound, and three-dimensional (3D) cryo-reconstruction). In some embodiments, "clearance time from the SCS" is the amount of time required following injection of a pharmaceutical composition, pharmaceutical agent, or AAV required to reduce the thickness of the SCS at or near the injection site to 500 nm or less, about 200 nm or less, about 100 nm or less, about 50 nm or less, about 25 nm or less, about 10 nm or less, or to become undetectable, as measured by standard techniques (e.g., in vivo imaging techniques such as OCT imaging, UHR-OCT, ultrasound, and three-dimensional (3D) cryo-reconstruction). In some embodiments, the thickness of the SCS is measured using Heidelberg optical coherence tomography (OCT). In some embodiments, the pharmaceutical composition (e.g., the diluted pharmaceutical composition) has a clearance time of at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 22 hours, 24 hours, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days and has sufficient viscosity to last for 6, 17, 18, 19, 20, 21, 23, 25, 27, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 days.

[0212] In some embodiments, "clearance time from the SCS" is the amount of time required after injection for the pharmaceutical composition, pharmaceutical agent, or AAV to spread circumferentially from the injection site to cover about one-sixteenth or more, about one-eighth or more, about one-quarter or more, about one-half or more, about three-quarters or more, or all of the surface of the choroid as measured by standard techniques (e.g., in vivo imaging techniques such as OCT imaging). In some embodiments, the pharmaceutical composition (e.g., the diluted pharmaceutical composition) has a clearance time of at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 22 hours, 24 hours, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days and has sufficient viscosity to last for 6, 17, 18, 19, 20, 21, 23, 25, 27, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 days.

[0213] In some embodiments, the pharmaceutical composition (e.g., the diluted pharmaceutical composition) is administered to the suprachoroidal space of the eye of a pig (e.g., a minipig, such as a Yucatan minipig). In some embodiments, the pig is a minipig. In some embodiments, the minipig can be a Gottingen, Yucatan, Bama Xiang Zhu, Wuzhishan, and / or Xi Shuang Banna. In some embodiments, the minipig is a Yucatan. In some embodiments, the pharmaceutical composition (e.g., a diluted pharmaceutical composition) has a viscosity and / or elastic modulus such that, when administered to the SCS of a pig's eye, the clearance time of the pharmaceutical composition is about 5 to about 15 days, about 6 to about 15 days, about 7 to about 15 days, about 8 to about 15 days, about 9 to about 15 days, about 10 to about 15 days, about 11 to about 15 days, about 12 to about 15 days, about 13 to about 15 days, about 14 to about 15 days, about 5 to about 14 days, about 5 to about 13 days, about 5 to about 12 days, about 5 to about 11 days, about 5 to about 10 days, about 5 to about 9 days, about 5 to about 8 days, about 5 to about 7 days, or about 5 to about 6 days. In some embodiments, the pharmaceutical composition (e.g., a diluted pharmaceutical composition) is administered to the suprachoroidal space of a pig's eye. In some embodiments, the pharmaceutical composition (e.g., the diluted pharmaceutical composition) has a viscosity and / or elastic modulus such that, when administered to the SCS of a pig eye, the clearance time of the pharmaceutical composition is about 5 days to about 15 days.

[0214] 4.2.2 Circumferential spread In some embodiments, the pharmaceutical composition localizes at the injection site. In some embodiments, the pharmaceutical composition localizes at the injection site for a longer period of time than a comparable pharmaceutical composition that has a lower viscosity and / or elastic modulus (G') at extraocular temperature (about 32-35°C) and / or is not gelled. In some embodiments, the pharmaceutical composition localizes at the injection site for a longer period of time when injected into the SCS compared to when the pharmaceutical composition is administered by subretinal or intravitreal injection. The pharmaceutical composition may have different viscosity and / or elastic modulus values. In some embodiments, a pharmaceutical composition that is viscous and / or elastic and / or gelled (or more viscous, more elastic and / or more gelled) at about 32-35°C remains localized in the SCS for a longer period of time compared to a pharmaceutical composition that is non-viscous or has a lower viscosity and / or is not gelled at about 32-35°C.

[0215] In some embodiments, localization can be determined by assessing circumferential spread (e.g., 2D circumferential spread). In some embodiments, circumferential spread is determined by analyzing the expansion or opening of the SCS in the quadrant where the injection is made (in some cases, in 2D, this space adjacent to the choroid appears linear). In some embodiments, the pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) has a viscosity that is at least 5%, at least 10%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 5% less than that of a reference pharmaceutical composition having a lower viscosity at external eye temperature (about 32-35° C.) when used to administer the AAV comprising the expression cassette encoding a transgene (e.g., by suprachoroidal injection, by subretinal injection, or by intravitreal injection). resulting in a circumferential extent that is 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40%, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, at least 100% less, at least 150% less, or at least 200% less, at least 250% less, or at least 300%, at least 400% less, or at least 500% less.

[0216] In some embodiments, suprachoroidal administration of a pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) is at least 5% less, at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 10% less, at least 10% less, at least 15% less, at least 20% less, at least 1 ... % less, at least 25% less, at least 30% less, at least 35% less, at least 40%, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, at least 100% less, at least 150% less, or at least 200% less, at least 250% less, or at least 300%, at least 400% less, or at least 500% less circumferential extent.

[0217] In some embodiments, suprachoroidal administration of a viscous (e.g., relatively viscous, moderately to very highly viscous, or more viscous than water, or more viscous than a control solution, or more viscous than a solution typically used for subretinal administration) or elastic and / or gelled pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) at about 32-35° C. may be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, when the same pharmaceutical composition is used to administer an AAV comprising an expression cassette encoding a transgene by subretinal or intravitreal administration. resulting in a circumferential extent that is at least 5% less, at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40%, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, at least 100% less, at least 150% less, or at least 200% less, at least 250% less, or at least 300%, at least 400% less, or at least 500% less.

[0218] In some embodiments, the circumferential spread is measured at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 18 days, 20 days ... In one embodiment, the period can be determined as follows: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 27, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 days.

[0219] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachoroidal space of a pig's eye. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has a viscosity and / or elastic modulus such that when administered to the SCS of a pig's eye, the pharmaceutical composition spreads circumferentially from the injection site to about 1 / 16 or less, about 1 / 8 or less, about 1 / 4 or less, or about 1 / 2 or less of the choroidal surface within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, or about 24 hours after administration. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachoroidal space of a pig's eye. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has a viscosity and / or elastic modulus such that when administered to the SCS of a pig's eye, the pharmaceutical composition spreads circumferentially from the injection site to about one-eighth or less of the choroidal surface within about 5 minutes, about 10 minutes, 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, or about 24 hours after administration. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has a viscosity and / or elastic modulus such that when administered to the SCS of a pig's eye, the pharmaceutical composition spreads circumferentially from the injection site to about one-eighth or less of the choroidal surface within about 1 hour after administration.

[0220] 4.2.3 Thickness of the SCS In some embodiments, localization can be determined by assessing the thickness of the SCS after the pharmaceutical composition is administered to the subject. In some embodiments, the pharmaceutical composition increases the thickness of the SCS after the pharmaceutical composition is injected into the SCS. In some embodiments, the SCS expands to accommodate injection of a pharmaceutical composition having a low viscosity and / or elastic modulus (G') and / or that is not gelled at about 32-35°C. In some embodiments, injection of a larger volume of a pharmaceutical composition having a low viscosity and / or elastic modulus and / or that is not gelled does not cause further expansion of the SCS. In some embodiments, a larger volume of a fluid formulation having a low viscosity and / or elastic modulus is accommodated by increasing the area of ​​fluid spread in the SCS without further expanding the SCS. In some embodiments, injection of a pharmaceutical composition having a viscosity and / or elasticity and / or gelled at about 32-35°C into the SCS can expand the thickness of the SCS beyond the thickness of the SCS achieved when a pharmaceutical composition having a low viscosity and / or a low elastic modulus and / or that is not gelled is injected into the SCS. In some embodiments, increasing the thickness of the SCS with a viscous and / or gelled pharmaceutical composition at about 32-35° C. can facilitate access to the SCS, thereby facilitating or allowing removal of the device in the SCS. In some embodiments, expanding the thickness of the SCS allows the pharmaceutical composition and / or the AAV encoding the transgene to remain (localize) at the injection site for a longer period of time. In some embodiments, a viscous and / or elastic and / or gelled pharmaceutical composition at about 32-35° C. increases the thickness at or near the injection site for a longer period of time compared to a pharmaceutical composition that is non-viscous or has a low viscosity and / or a low elastic modulus and / or is not gelled at about 32-35° C. In some embodiments, a more viscous and / or more elastic and / or more gelled pharmaceutical composition at about 32-35° C. increases the thickness at or near the injection site for a longer period of time compared to a less viscous and / or less elastic and / or not gelled pharmaceutical composition at about 32-35° C.In some embodiments, the thickness of the injection site of the pharmaceutical composition after administration to the SCS is equal to or greater than the thickness of the injection site of the reference pharmaceutical composition after the reference pharmaceutical composition is administered subretinal or intravitreal. In some embodiments, the thickness of the injection site of the pharmaceutical composition after administration to the SCS is equal to or greater than the thickness of the injection site of the reference pharmaceutical composition after the reference pharmaceutical composition is administered to the SCS.

[0221] In some embodiments, suprachoroidal administration of a viscous (e.g., relatively viscous, moderately to very highly viscous, or more viscous than water, or more viscous than a control solution, or more viscous than a solution commonly used for subretinal administration) and / or elastic and / or gelled pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) at about 32-35 ° C. is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater than when a less viscous and / or less elastic and / or non-gelled pharmaceutical composition is used to administer, for example, an AAV comprising an expression cassette encoding a transgene by suprachoroidal administration. or at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater.

[0222] In some embodiments, suprachoroidal administration of a more viscous and / or more elastic and / or more gelled pharmaceutical composition at about 32-35 ° C. (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, or at least 5% greater than when a less viscous and / or less elastic and / or non-gelled pharmaceutical composition at about 32-35 ° C. is used to administer, for example, an AAV comprising an expression cassette encoding a transgene by subretinal or intravitreal administration. , resulting in an increase in thickness at or near the injection site of at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater.

[0223] In some embodiments, administration of a viscous (e.g., relatively viscous, moderately to very highly viscous, or more viscous than water, or more viscous than a control solution, or more viscous than a solution typically used for subretinal administration) and / or elastic and / or gelled pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) at about 32-35 ° C. is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, than when the same pharmaceutical composition is used to administer, for example, an AAV comprising an expression cassette encoding a transgene by subretinal or intravitreal administration. resulting in an increase in thickness at or near the injection site of at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater.

[0224] In some embodiments, the thickness achieved at the injection site after a more viscous and / or more elastic and / or more gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) is administered by injection into the suprachoroidal fold at about 32-35° C. is thicker than after a comparably less viscous and / or less elastic and / or ungelled pharmaceutical composition is administered by injection into the suprachoroidal fold at about 32-35° C. In some embodiments, the thickness achieved at the injection site after a more viscous and / or more elastic and / or more gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) is administered by injection into the suprachoroidal fold at about 32-35° C. is thicker than after a comparably less viscous and / or less elastic and / or ungelled pharmaceutical composition is administered by subretinal or intravitreal injection at about 32-35° C. In some embodiments, the thickness achieved at the injection site after a viscous and / or gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV containing an expression cassette encoding a transgene) is administered by injection into the suprachoroidal fold is greater than the thickness achieved after the same pharmaceutical composition is administered by subretinal or intravitreal administration at about 32-35°C.

[0225] In some embodiments, the thickness at or near the injection site (e.g., thickness at or near the SCS) is measured at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 16 days, 18 days, 19 days, 20 days, 22 days, 24 hours, 24 hours, 28 days, 29 days, 30 days, 30 minutes, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 days, 11 days, 12 days, 16 days, 18 days, 20 days, 22 days, 24 hours, 24 hours, 28 days, 29 days, 30 minutes, 30 minutes, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 days, 11 days, 12 days, 24 days, 24 hours ... The period may be determined as 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 27, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 days.

[0226] In some embodiments, the pharmaceutical composition (e.g., a diluted pharmaceutical composition) is administered to the suprachoroidal space of a pig's eye. In some embodiments, the pharmaceutical composition (e.g., a diluted pharmaceutical composition) has a viscosity and / or elastic modulus such that, when administered to the SCS of a pig's eye, the thickness of the SCS at the injection site is about 400 to about 800 μm, about 400 μm to about 700 μm, about 400 μm to about 600 μm, about 400 μm to about 500 μm, about 500 μm to about 800 μm, about 600 μm to about 800 μm, or 700 μm to about 800 μm within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, or about 24 hours after administration. In some embodiments, the pharmaceutical composition (e.g., a diluted pharmaceutical composition) is administered to the suprachoroidal space of a pig's eye. In some embodiments, the pharmaceutical composition (e.g., a diluted pharmaceutical composition) has a viscosity and / or elastic modulus such that when administered to the SCS of a pig's eye, the thickness of the SCS at the injection site is about 400 to about 800 μm within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, or about 24 hours after administration. In some embodiments, the pharmaceutical composition (e.g., a diluted pharmaceutical composition) has a viscosity and / or elastic modulus such that when administered to the SCS of a pig's eye, the thickness of the SCS at the injection site is about 400 to about 800 μm within about 1 hour after administration.

[0227] 4.2.4 Vasodilatation and vascular leakage In some embodiments, the level of VEGF-induced vasodilation and / or vascular leakage after the pharmaceutical composition is administered to the SCS is equal to or less than the level of VEGF-induced vasodilation and / or vascular leakage after the reference pharmaceutical composition is administered subretinal or intravitreal. In some embodiments, the level of VEGF-induced vasodilation and / or vascular leakage after the pharmaceutical composition is administered to the SCS is equal to or less than the level of VEGF-induced vasodilation and / or vascular leakage after the reference pharmaceutical composition is administered to the SCS. In some embodiments, the pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) results in a reduction in the level of VEGF-induced vasodilation and / or vascular leakage after the pharmaceutical composition is administered to the SCS compared to after the pharmaceutical composition is administered via subretinal or intravitreal administration. In some embodiments, the pharmaceutical composition provides a reduced level of VEGF-induced vasodilation and / or vascular leakage after the pharmaceutical composition is administered into the SCS, as compared to a comparable pharmaceutical composition (having a lower viscosity at about 32-35° C.) administered via subretinal administration, via intravitreal administration, or into the SCS. In some embodiments, VEGF-induced vasodilation and / or vascular leakage is reduced by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold, or by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500%. In some embodiments, the transgene is an anti-human vascular endothelial growth factor (anti-VEGF) antibody.

[0228] In some embodiments, VEGF-induced vasodilation and / or vascular leakage is measured at about 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 20 days, 22 hours, 24 hours, 24 hours, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, The period may be determined as follows: 19, 20, 21, 23, 25, 27, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or less than about 400 days.

[0229] 4.2.5 Transduction rate (or infection rate) at the injection site In some embodiments, the infection rate (or rate of injection) at the site of transduction after the pharmaceutical composition is administered to the SCS is equal to or greater than the transduction rate (or infection rate) at the injection site after the same pharmaceutical composition is administered via subretinal or intravenous administration. In some embodiments, the transduction rate (or infection rate) at the injection site after the pharmaceutical composition is administered to the SCS is equal to or greater than the transduction rate (or infection rate) at the injection site after a comparable (e.g., less viscous and / or non-gelled) pharmaceutical composition is administered via subretinal or intravenous administration or to the SCS. In some embodiments, the pharmaceutical composition has a higher viscosity and / or elastic modulus (G') at about 32-35°C and / or is gelled than a reference pharmaceutical composition (a pharmaceutical composition that is equally less viscous and / or less elastic and / or non-gelled at about 32-35°C). In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same amount of genome copies. In some embodiments, the increase in transduction rate (or infection rate) at the injection site is at least about 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% increase.

[0230] In some embodiments, the level of AAV at the injection site after the pharmaceutical composition is administered suprachoroidally is equal to or greater than the level of AAV at the injection site after the same pharmaceutical composition is administered via subretinal or intravenous administration. In some embodiments, the level of AAV at the injection site after the pharmaceutical composition is administered suprachoroidally is equal to or greater than the level of AAV at the injection site after a comparable (e.g., less viscous and / or less elastic and / or ungelled) pharmaceutical composition is administered via subretinal or intravenous administration or to the SCS. In some embodiments, the pharmaceutical composition has a higher viscosity and / or elastic modulus (G') at about 32-35°C and / or is gelled than a reference pharmaceutical composition. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition (a pharmaceutical composition with an equivalently lower viscosity and / or less elastic and / or ungelled pharmaceutical composition at about 32-35°C) have the same vector genome concentration. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same amount of genome copies. In some embodiments, the increase in the level of AAV at the injection site is at least about a 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% increase.

[0231] In some embodiments, AAV levels or transduction rates (or infection rates) are measured at about 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 hours, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days The period will be determined as follows: 9, 20, 21, 23, 25, 27, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380 days, or approximately 400 days or less.

[0232] 4.2.6 Transgene Expression In some embodiments, the concentration of the transgene product is at least equal to or greater after the pharmaceutical composition is injected into the SCS compared to after a reference (e.g., less viscous and / or less elastic and / or ungelled at about 32-35° C.) pharmaceutical composition is injected into the SCS. In some embodiments, the concentration of the transgene product is at least equal to or greater after the pharmaceutical composition is injected into the SCS compared to after a reference (e.g., less viscous and / or less elastic and / or ungelled at about 32-35° C.) pharmaceutical composition is injected by subretinal or intravitreal injection. In some embodiments, the concentration of the transgene product is at least equal to or greater after the pharmaceutical composition is injected into the SCS compared to after the same pharmaceutical composition is injected by subretinal or intravitreal injection.

[0233] In some embodiments, the transgene product (e.g., concentration of the transgene product) is detected in the eye (e.g., vitreous humor) for a longer period of time after the pharmaceutical composition is injected into the SCS compared to after a comparable (less viscous and / or less elastic and / or ungelled at about 32-35° C.) pharmaceutical composition is injected into the SCS. In some embodiments, the transgene product (e.g., concentration of the transgene product) is detected in the eye (e.g., vitreous humor) for a longer period of time after the pharmaceutical composition is injected into the SCS compared to after a reference (less viscous and / or less elastic and / or ungelled at about 32-35° C.) pharmaceutical composition is injected by subretinal injection or intravitreal administration. In some embodiments, the transgene product (e.g., concentration of the transgene product) is detectable in the eye (e.g., vitreous humor) for a longer period of time after the pharmaceutical composition is injected into the SCS as compared to after the same (or of a similar viscosity and / or elastic modulus (G') at about 32-35° C.) pharmaceutical composition is injected by subretinal or intravitreal injection.

[0234] In some embodiments, the longer time period is at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 20 days, 22 days, 24 hours ... 19, 20, 21, 23, 25, 27, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or more than 400 days. In some embodiments, the longer period of time is about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, A period longer than 19, 20, 21, 23, 25, 27, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 days.

[0235] In some embodiments, the transgene is administered to the SCS at least about, or at least about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, or 20 days after administration of the pharmaceutical composition to the SCS. , 18, 19, 20, 21, 23, 25, 27, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 days of time.

[0236] In some embodiments, the transgene is administered within about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 24 hours, 26 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 is detected in the eye (e.g., vitreous humor) for a period of time of 3 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, or 100 days or less (e.g., after a reference pharmaceutical composition is administered via subretinal or intravitreal administration, or into the SCS; or after a pharmaceutical composition is administered via subretinal or intravitreal administration).

[0237] In some embodiments, the concentration of the transgene product in the eye (e.g., vitreous humor) is measured at about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 22 hours, 24 hours, 24 hours, 26 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days In one embodiment, the period can be determined as follows: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 27, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 days.

[0238] In some embodiments, suprachoroidal administration of a viscous (e.g., relatively viscous, moderately to very highly viscous, or more viscous than water, or more viscous than a control solution, or more viscous than a solution typically used for subretinal administration) and / or more elastic and / or gelled pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) at about 32-35 ° C. may have a concentration at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater than the concentration of a comparably less viscous and / or less elastic and / or non-gelled pharmaceutical composition at about 32-35 ° C. used to administer, for example, an AAV comprising an expression cassette encoding a transgene by suprachoroidal administration. , at least 100-fold greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater.

[0239] In some embodiments, suprachoroidal administration of a more viscous and / or more elastic and / or more gelled pharmaceutical composition at about 32-35 ° C. (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, or at least 5% greater than a comparable less viscous and / or less elastic and / or non-gelled pharmaceutical composition at about 32-35 ° C. (reference pharmaceutical composition) used to administer, for example, an AAV comprising an expression cassette encoding a transgene by subretinal or intravitreal administration. In some embodiments, the transgene concentration may be greater than 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% greater concentration.

[0240] In some embodiments, administration of a viscous (e.g., relatively viscous, moderately to very highly viscous, or more viscous than water, or more viscous than a control solution, or more viscous than a solution typically used for subretinal administration) and / or elastic and / or gelled pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) at about 32-35° C. is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, at least 5% greater, at least 5% greater, at least 5% greater, at least 6% greater, at least 7% greater, at least 8% greater, at least 9% ...5% greater, at least 5% greater, at least 5% greater, at least 5% greater, at least 5% greater, at least 5% greater, at least 5% greater, at least 5% greater, at least 5% greater, at least 5% greater, at least 5% greater, at least 5% greater, at least 5% greater, at least 5% greater, at In some embodiments, the transgene concentration may be greater than 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% greater.

[0241] In some embodiments, the concentration of the transgene product after a more viscous and / or more elastic and / or more gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) is administered by injection into the suprachoroidal layer at about 32-35° C. is greater than after a comparably less viscous and / or less elastic and / or ungelled pharmaceutical composition is administered by injection into the suprachoroidal layer at about 32-35° C. In some embodiments, the concentration of the transgene product after a more viscous and / or more elastic and / or more gelled pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) is administered by injection into the suprachoroidal layer at about 32-35° C. is greater than after a comparably less viscous and / or less elastic and / or ungelled pharmaceutical composition is administered by subretinal administration or via intravitreal administration at about 32-35° C. In some embodiments, the concentration of the transgene product after a pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV containing an expression cassette encoding a transgene) viscous at about 32-35° C. is administered by injection into the suprachoroidal membrane is greater than after the same pharmaceutical composition is administered by subretinal administration or via intravitreal administration.

[0242] In some embodiments, the pharmaceutical compositions disclosed herein (e.g., pharmaceutical compositions comprising an AAV containing an expression cassette encoding a transgene) provide greater transgene expression and / or tissue / cell transduction in the back of the eye (e.g., the retina) than in the outer layers of the eye (e.g., the sclera) via SCS delivery. This feature of the pharmaceutical compositions disclosed herein is advantageous because by using the pharmaceutical compositions disclosed herein suprachoroidally, subretinal delivery is not required to achieve higher ocular transgene expression in the back of the eye than in the outer layers of the eye.

[0243] In some embodiments, the concentration of the transgene product (TP) is equal to or greater than the concentration in the retina when a pharmaceutical composition disclosed herein comprising an AAV encoding the TP is injected into the SCS, followed by a reference pharmaceutical composition comprising the same AAV. In other embodiments, the concentration of the TP is equal to or greater than the concentration in the retina and the concentration of the TP is less than the concentration in the sclera when a pharmaceutical composition comprising an AAV encoding the TP is injected into the SCS, compared to a reference pharmaceutical composition comprising the same AAV being injected into the SCS.

[0244] 4.2.7 Other Functional Characteristics In some embodiments, the pharmaceutical compositions described herein have a desired viscosity and / or elastic modulus (G') suitable for injection into the suprachoroid. In some embodiments, the recombinant AAV in the pharmaceutical composition is at least as stable as the recombinant AAV in a reference pharmaceutical composition (or an equivalent pharmaceutical composition). In some embodiments, the recombinant AAV in the pharmaceutical composition is at least 50% as stable as the recombinant AAV in a reference pharmaceutical composition (or an equivalent pharmaceutical composition). In some embodiments, the recombinant AAV in the pharmaceutical composition has an aggregation level at least the same or equivalent to the recombinant AAV in the reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has an infectivity level at least the same or equivalent to the recombinant AAV in the reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has a free DNA level at least the same or equivalent to the recombinant AAV in the reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has an in vitro relative potency (IVRP) at least the same or equivalent to the recombinant AAV in the reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has at least the same or a comparable level of size alteration as the recombinant AAV in the reference pharmaceutical composition.

[0245] In certain 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% or 2-fold, 3-fold, 5-fold, 10-fold, 100-fold or 1000-fold more stable to freeze / thaw cycles than the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more stable to freeze / thaw cycles than the same recombinant AAV in the reference pharmaceutical composition. In certain embodiments, the stability of the recombinant AAV is determined by the assay(s) disclosed in Sections 4.6 and 5.

[0246] In certain 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 more infectivity than the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% infectivity of the same recombinant AAV in the reference pharmaceutical composition. In certain embodiments, the viral infectivity of the recombinant AAV is determined by the assay(s) disclosed in the present disclosure. In certain embodiments, the size of the recombinant AAV is determined by the assay(s) disclosed in Section 4.6 and Section 5. In certain embodiments, the size is measured before or after a freeze / thaw cycle.

[0247] In certain 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%, or 100% less aggregation, or 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less aggregation, than the same recombinant AAV in a reference pharmaceutical composition. In certain embodiments, the aggregation of the recombinant AAV is determined by the assay(s) disclosed in the present disclosure. In certain embodiments, the aggregation is measured before or after a freeze / thaw cycle. In certain embodiments, the aggregation of the recombinant AAV is determined by the assay(s) disclosed in section 4.6.

[0248] In certain embodiments, the recombinant AAV in a 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, or 1000-fold more stable over a period of time (e.g., when stored at -20°C or 37°C), e.g., at least about or 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, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, or about 4 years, than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% stable over a period of time compared to the same recombinant AAV in a reference pharmaceutical composition. In certain embodiments, the stability of the recombinant AAV over a period of time is determined by the assay(s) disclosed in the present disclosure. In certain embodiments, the stability of the recombinant AAV over a period of time is determined by the assay(s) disclosed in Sections 4.6 and 5.

[0249] In certain embodiments, the recombinant AAV in the pharmaceutical composition has an in vitro relative potency (IVRP) that 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 greater than the same recombinant AAV in a reference pharmaceutical composition (e.g., when stored at -20°C or 37°C). In some embodiments, the recombinant AAV in the pharmaceutical composition has about the same in vitro relative potency (IVRP) as the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has an in vitro relative potency (IVRP) of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the same recombinant AAV in the reference pharmaceutical composition. In certain embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by the assay(s) disclosed in the present disclosure. In certain embodiments, the in vitro relative potency (IVRP) is measured before or after a freeze / thaw cycle. In certain embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by the assay(s) disclosed in section 4.6.

[0250] In certain embodiments, the recombinant AAV in the pharmaceutical composition has at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 100% less free DNA than the same recombinant AAV in the reference pharmaceutical composition, or less than 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less free DNA. In some embodiments, the recombinant AAV in the pharmaceutical composition has about the same amount of free DNA as the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has about 2-fold or less free DNA than the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the amount of free DNA of the same recombinant AAV in the reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has at least about 50% more, about 25% more, about 15% more, about 10% more, about 5% more, about 4% more, about 3% more, about 2% more, about 1% more, about 0% more, about 1% less, about 2% less, about 5% less, about 7% less, about 10% less, about 2 times more, about 3 times more, about half, or about a third less free DNA than the same recombinant AAV in the reference pharmaceutical composition. In certain embodiments, the free DNA of the recombinant AAV is determined by the assay(s) disclosed in Sections 4.6 and 5.

[0251] In certain embodiments, the recombinant AAV in the pharmaceutical composition has a size change of 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1% or less over a period of time (e.g., when stored at -20°C or 37°C), for example, at least about or 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 certain embodiments, the size of the recombinant AAV is determined by the assay(s) disclosed in the present disclosure. In certain embodiments, the size is measured before or after a freeze / thaw cycle. In certain embodiments, the size of the recombinant AAV is determined by the assay(s) disclosed in Section 4.6.

[0252] In certain 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 (e.g., when stored at -20°C or 37°C). In some embodiments, the recombinant AAV in the pharmaceutical composition is approximately as stable as the same recombinant AAV in a reference pharmaceutical composition (e.g., when stored at -20°C or 37°C). In some embodiments, the recombinant AAV in the pharmaceutical composition is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% stable of the same recombinant AAV in a reference pharmaceutical composition (e.g., when stored at -20° C. or 37° C.). In certain embodiments, the stability of the recombinant AAV is determined by the assay(s) disclosed in Section 4.6.

[0253] In certain embodiments, the pharmaceutical compositions provided herein can be stored 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, e.g., as determined by the assay(s) disclosed in Section 4.6. In certain embodiments, the pharmaceutical compositions provided herein can be stored 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 at 4° C. without loss of stability. In certain embodiments, the pharmaceutical compositions provided herein can be stored at or below 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, or 24 months without loss of stability. In certain 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, or 24 months without loss of stability. In certain embodiments, the pharmaceutical compositions provided herein can be stored at -20°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months, followed by storage 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.

[0254] In certain embodiments, the pharmaceutical compositions provided herein can be initially 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, or 24 months, and then thawed and stored at 2-10°C, 4-8°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, or 9°C for an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months after thawing without loss of stability, e.g., as determined by the assay(s) disclosed in Section 4.6. In certain embodiments, the pharmaceutical compositions provided herein can be initially 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, or 24 months, and then thawed and stored at about 4°C after thawing for an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months without loss of stability, e.g., as determined by the assay(s) disclosed in Section 4.6. In certain embodiments, the pharmaceutical compositions provided herein can be initially stored at or below 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, or 24 months, and then thawed and stored after thawing at about 4° C. for an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months without loss of stability, e.g., as determined by the assay(s) disclosed in Section 4.6.

[0255] The effect of the methods or pharmaceutical compositions provided herein can also be monitored by measuring signs of other safety events, including vision loss, infection, inflammation, and retinal detachment. In some embodiments, different pharmaceutical compositions having different viscosities and / or elastic moduli (G') (e.g., ranging from low to very high viscosities) at about 32-35°C can be used to deliver vectors to the SCS. In some embodiments, vectors delivered using pharmaceutical compositions having a medium to high viscosity and / or elastic modulus (G') at about 32-35°C are more effective (e.g., when administered to the SCS) than vectors delivered using pharmaceutical compositions having a low viscosity and / or elastic modulus (G') at about 32-35°C. In some embodiments, vectors delivered using formulations having a medium to high viscosity and / or elastic modulus (G') at about 32-35°C result in improved vision compared to vectors delivered using formulations having a low viscosity and / or elastic modulus (G') at about 32-35°C.

[0256] The effect of the methods or pharmaceutical compositions provided herein can also be measured by the change from baseline in the National Eye Institute Visual Functioning Questionnaire, Rasch scored version (NEI-VFQ-28-R) (composite score; activity limitation domain score; and socio-emotional functioning domain score). In some embodiments, the effect of the methods provided herein can also be measured by the change from baseline in the National Eye Institute Visual Functioning Questionnaire, 25 item version (NEI-VFQ-25) (composite score and mental health subscale score). In some embodiments, the effect of the methods provided herein can also be measured by the change from baseline in the Macular Disease Treatment Satisfaction Questionnaire (MacTSQ) (composite score; safety, efficacy, and discomfort domain scores; and informational and convenience domain scores).

[0257] In specific embodiments, efficacy of the methods or vectors (vector formulations) described herein is reflected by improved visual acuity at about 4 weeks, 12 weeks, 6 months, 12 months, 24 months, 36 months, or other desired time points. In specific embodiments, improved visual acuity is characterized by an increase in BCVA, e.g., an increase of 1 letter, 2 letters, 3 letters, 4 letters, 5 letters, 6 letters, 7 letters, 8 letters, 9 letters, 10 letters, 11 letters, or 12 letters, or more. In specific embodiments, improved visual acuity is characterized by a 5%, 10%, 15%, 20%, 30%, 40%, 50% or more increase in visual acuity from baseline.

[0258] In specific embodiments, there is no inflammation in the eye following treatment or there is only minimal inflammation in the eye following treatment (e.g., a 10%, 5%, 2%, 1% or less increase in the level of inflammation from baseline).

[0259] 4.3 Dosage and Mode of Administration In one aspect, provided herein is a method of suprachoroidal administration for treating an ocular condition, comprising administering a recombinant viral vector comprising a nucleotide sequence encoding a therapeutic product to the suprachoroidal space in the eye of a human subject in need of treatment, such that the therapeutic product is expressed to provide treatment for the ocular condition. In certain embodiments, the administering step is by injecting the recombinant viral vector into the suprachoroidal space using a suprachoroidal drug delivery device. In certain embodiments, the suprachoroidal drug delivery device is a microinjector. In some embodiments, the pharmaceutical compositions provided herein or reference pharmaceutical compositions are suitable for administration by one, two or more routes of administration (e.g., suitable for suprachoroidal and subretinal administration).

[0260] In certain embodiments, the vector genome concentration (VGC) of the pharmaceutical composition (or the reference pharmaceutical composition) is about 3×10 9 GC / mL, approximately 1×10 10 GC / mL, approximately 1.2×10 10 GC / mL, approximately 1.6×10 10 GC / mL, approximately 4×10 10 GC / mL, approximately 6×10 10 GC / mL, approximately 2×10 11 GC / mL, approximately 2.4×10 11 GC / mL, approximately 2.5×10 11 GC / mL, approximately 3×10 11 GC / mL, approximately 3.2×10 11 GC / mL, approximately 6.2×10 11 GC / mL, approximately 6.5×10 11 GC / mL, approximately 1×10 12 GC / mL, approximately 2.5×10 12 GC / mL, approximately 3×10 12 GC / mL, approximately 5×1012 GC / mL, approximately 1.5×10 13 GC / mL, approximately 2×10 13 GC / mL, or approximately 3×10 13 GC / mL.

[0261] In certain embodiments, the vector genomic concentration (VGC) of the pharmaceutical composition (or reference pharmaceutical composition) is approximately 3×10 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 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 10 GC / mL, approximately 1×10 11 GC / mL, approximately 2×10 11 GC / mL, approximately 3×10 11 GC / mL, approximately 4×10 11 GC / 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 1×1013 GC / mL, approximately 1.5×10 13 GC / mL, approximately 2×10 13 GC / mL, approximately 3×10 13 GC / mL.

[0262] In some embodiments, the volume of the pharmaceutical composition is any volume that can reduce the minimum force required to separate the sclera and the choroid. In some embodiments, the volume of the pharmaceutical composition is about 50 μL to about 1000 μL, 50 μL to about 500 μL, 50 μL to about 400 μL, 50 μL to about 350 μL, 50 μL to about 300 μL, about 50 μL to about 275 μL, about 50 μL to about 250 μL, about 50 μL to about 225 μL, about 50 μL to about 200 μL, about 50 μL to about 175 μL, about 50 μL to about 150 μL, about 60 μL to about 140 μL, about 70 μL to about 130 μL, about 80 μL to about 120 μL, about 90 μL to about 110 μL, or about 100 μL.

[0263] There are currently available technologies for suprachoroidal space (SCS) delivery. Preclinically, SC injections have been achieved using scleral flap techniques, catheters, and standard hypodermic needles, as well as with fine needles. A hollow bore, 750 um long fine needle (Clearside Biomedical, Inc.) can be inserted into the pars pars and has shown promise in clinical trials. Microneedles designed with force-sensing technology are available for SC injections, as described by Chitnis et al. (Chitnis, GD, et al., A resistance-sensing mechanical injector for the precise delivery of liquids to target tissue. Nat Biomed Eng 3, 621-631 (2019). https: / / doi.org / 10.1038 / s41551-019-0350-2). Oxular Limited is developing a delivery system (Oxulumis) that advances an illuminated cannula into the suprachoroidal space. The Orbit device (Gyroscope) is a specially designed system that allows cannulation of the suprachoroidal space with a flexible cannula. A fine needle inside the cannula is advanced into the subretinal space to allow targeted dose delivery. Access to the SCS from the inside can also be achieved using microstents, which serve as minimally invasive glaucoma surgery (MIGS) devices. Examples include CyPass® microstents (Alcon, Fort Worth, Texas, US) and iStent® (Glaukos), which are surgically implanted to provide a conduit from the anterior chamber to the SCS for drainage of aqueous humor without forming a filtering bleb. Other devices anticipated for suprachoroidal delivery include those described in GB 2531910 and US Pat. No. 10,912,883.

[0264] In some embodiments, the suprachoroidal drug delivery device is a syringe with a 1 millimeter 30 gauge needle. In some embodiments, the syringe has a larger circumference (e.g., a 29 gauge needle). During injection using this device, the needle penetrates to the base of the sclera, and fluid containing the drug enters the suprachoroidal space, causing expansion of the suprachoroidal space. As a result, there is tactile and visual feedback during injection. After injection, the fluid flows posteriorly and is absorbed predominantly in the choroid and retina. This results in the production of the transgene protein from all retinal cell layers and choroidal cells. The use of this type of device and procedure allows for a quick and simple in-office procedure with a low risk of complications.

[0265] In some embodiments, the fine needle or syringe is selected based on the viscosity of the pharmaceutical composition. In some embodiments, the fine needle is selected based on the pressure that is generated at the eye (e.g., SCS) when the pharmaceutical composition is administered. For example, pharmaceutical compositions having a medium or high viscosity and / or elastic modulus (G') at about 32-35°C may benefit from using a wider fine needle for injection. In some embodiments, the pressure at the SCS is lower when a wider fine needle is used compared to the pressure obtained when a narrower fine needle is used. In some embodiments, a 10 gauge needle, an 11 gauge needle, a 12 gauge needle, a 13 gauge needle, a 14 gauge needle, a 15 gauge needle, a 16 gauge needle, a 17 gauge needle, an 18 gauge needle, a 19 gauge needle, a 20 gauge needle, a 21 gauge needle, a 22 gauge needle, a 23 gauge needle, a 24 gauge needle, a 25 gauge needle, a 26 gauge needle, a 27 gauge needle, a 28 gauge needle, a 29 gauge needle, a 30 gauge needle, a 31 gauge needle, a 32 gauge needle, a 33 gauge needle, or a 34 gauge needle is used. In some embodiments, a 27 gauge needle is used. In some embodiments, a 28 gauge needle is used. In some embodiments, a 29 gauge needle is used. In some embodiments, a 30 gauge needle is used. In some embodiments, a 31 gauge needle is used. In some embodiments, a gauge smaller than a 27 gauge needle is used. In some embodiments, a gauge larger than a 27 gauge needle is used. In some embodiments, smaller than 30 gauge needles are used. In some embodiments, larger than 30 gauge needles are used.

[0266] In some embodiments, the pressure during administration of the pharmaceutical composition is about 10 PSI, 15 PSI, 20 PSI, 25 PSI, 30 PSI, 35 PSI, 40 PSI, 45 PSI, 50 PSI, 55 PSI, 60 PSI, 65 PSI, 70 PSI, 75 PSI, 80 PSI, 85 PSI, 90 PSI, 95 PSI, 100 PSI, 150 PSI, or 200 PSI. In some embodiments, the pressure during administration of the pharmaceutical composition is not greater than about 10 PSI, 15 PSI, 20 PSI, 25 PSI, 30 PSI, 35 PSI, 40 PSI, 45 PSI, 50 PSI, 55 PSI, 60 PSI, 65 PSI, 70 PSI, 75 PSI, 80 PSI, 85 PSI, 90 PSI, 95 PSI, 100 PSI, 150 PSI, or 200 PSI. In some embodiments, the pressure to open the SCS during administration of the pharmaceutical composition is no greater than about 10 PSI, 15 PSI, 20 PSI, 25 PSI, 30 PSI, 35 PSI, 40 PSI, 45 PSI, 50 PSI, 55 PSI, 60 PSI, 65 PSI, 70 PSI, 75 PSI, 80 PSI, 85 PSI, 90 PSI, 95 PSI, 100 PSI, 150 PSI, or 200 PSI. In some embodiments, the pressure (or pressure required to open the SCS) during administration of the pharmaceutical composition is between 20 PSI and 50 PSI, between 20 PSI and 75 PSI, between 20 PSI and 40 PSI, between 10 PSI and 40 PSI, between 10 PSI and 100 PSI, or between 10 PSI and 80 PSI. In some embodiments, the pressure decreases as the injection rate decreases (e.g., going from a 4 second injection rate to a 10 second injection rate, the pressure decreases.) In some embodiments, the pressure decreases as the needle size increases.

[0267] In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection pressure of less than 43 PSI. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection pressure of about 43 PSI. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection pressure of about 43-65 PSI. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection pressure of about 65 PSI. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection pressure of less than 65 PSI. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection pressure of about 65-100 PSI. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection pressure of about 100 PSI. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection pressure of less than 100 PSI.

[0268] In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection time of about 5-10 seconds. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection time of about 10-15 seconds. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection time of about 15-20 seconds. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection time of about 20-25 seconds. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection time of about 25-30 seconds. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection time of less than 30 seconds. In some embodiments, the pharmaceutical compositions provided herein are administered to the human eye with an injection time of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the length of the gelation time of the composition at external eye temperature (about 32-35° C.).

[0269] A dose that maintains a concentration of the transgene product at a Cmin of at least 0.330 μg / mL in the eye (e.g., vitreous humor) or 0.110 μg / mL in the aqueous humor (anterior chamber of the eye) over a period of three months is desirable; thereafter, a vitreous Cmin concentration of the transgene product in the range of 1.70-6.60 μg / mL and / or an aqueous Cmin concentration in the range of 0.567-2.20 μg / mL should be maintained. However, because the transgene product is produced continuously (either under the control of a constitutive promoter or induced by hypoxia when using a hypoxia-inducible promoter), maintaining a lower concentration may be effective. The transgene concentration may be measured directly in a patient sample of body fluid, ocular fluid, vitreous humor, or fluid collected from the anterior chamber, or may be estimated and / or monitored by measuring the patient's serum concentration of the transgene product, where the ratio of systemic to vitreous exposure to the transgene product is approximately 1:90,000. (See, e.g., the vitreous and serum concentrations of ranibizumab reported in Table 5 of Xu L et al., 2013, Invest. Opthal. Vis. Sci. 54:1616-1624, pages 1621 and 1623, which is incorporated by reference in its entirety.)

[0270] In certain embodiments, dosage is measured by genome copies per ml (GC / mL) or genome copy number administered to the patient's eye (e.g., administered to the suprachoroid). In some embodiments, dosage is measured by genome copies per ml (GC / mL) or genome copy number administered to the patient's eye (e.g., administered to the suprachoroid). 11 GC / mL ~ 1 × 10 13 GC / mL was administered, 2.4 × 10 11 GC / mL~5×10 11 GC / mL was administered, 5 × 10 11 GC / mL ~ 1 × 10 12 GC / mL was administered, 1 × 10 12 GC / mL~5×10 12 GC / mL administered or 5×10 12 GC / mL ~ 1 × 10 13 In some embodiments, 1.5×10 GC / mL is administered. 13 GC / mL~3×10 13In some embodiments, about 2.4×10 GC / mL is administered. 11 GC / mL, approximately 5×10 11 GC / mL, approximately 1×10 12 GC / mL, approximately 2.5×10 12 GC / mL, approximately 5×10 12 GC / mL, approximately 1×10 13 GC / mL or approximately 1.5 x 10 13 In some embodiments, 1×10 GC / mL is administered. 9 ~1×10 12 In some embodiments, 3×10 genome copies are administered. 9 ~2.5×10 11 In a specific embodiment, 1×10 genome copies are administered. 9 ~2.5×10 11 In a specific embodiment, 1×10 genome copies are administered. 9 ~1×10 11 In a specific embodiment, 1×10 genome copies are administered. 9 ~5×10 9 In a specific embodiment, 6×10 genome copies are administered. 9 ~3×10 10 In a specific embodiment, 4×10 genome copies are administered. 10 ~1×10 11 In a specific embodiment, 2×10 genome copies are administered. 11 ~1.5×10 12 In a specific embodiment, about 3×10 genome copies are administered. 9 Genome copies are administered (approximately 1.2 x 10 in a volume of 250 μl). 10 In another specific embodiment, about 1×10 10 Genome copies are administered (approximately 4×10 in a volume of 250 μl) 10 In another specific embodiment, about 6×10 10 Genome copies are administered (this is approximately 2.4 x 10 in a volume of 250 μl) 11 In another specific embodiment, about 6.4×1010 Genome copies are administered (approximately 3.2 x 10 in a volume of 200 μl). 11 In another specific embodiment, about 1.3×10 11 Genome copies are administered (this is approximately 6.5×10 in a volume of 200 μl) 11 In some embodiments, about 6.4×10 10 In some embodiments, about 6.4×10 genome copies are administered. 10 Genome copies is the total number of genome copies administered. In some embodiments, about 1.3×10 per eye, or per dose, or per route of administration. 11 In some embodiments, about 1.3×10 genome copies are administered. 11 Genome copies is the total number of genome copies administered. In some embodiments, about 2.5×10 per eye, or per dose, or per route of administration. 11 In some embodiments, about 2.5×10 genome copies are administered. 11 Genome copies is the total number of genome copies administered. In some embodiments, about 5×10 per eye, or per dose, or per route of administration. 11 In some embodiments, about 5×10 genome copies are administered. 11 Genome copies is the total number of genome copies administered. In some embodiments, about 3×10 per eye, or per dose, or per route of administration. 12 In some embodiments, about 3×10 genome copies are administered. 12 Genome copies is the total number of genome copies administered. In another specific embodiment, about 1.6×10 11 Genome copies are administered (this is approximately 6.2 x 10 in a volume of 250 μl) 11 In another specific embodiment, about 1.55×10 11 Genome copies are administered (this is approximately 6.2 x 10 in a volume of 250 μl) 11In another specific embodiment, about 1.6×10 11 Genome copies are administered (this is approximately 6.4 x 10 in a volume of 250 μl) 11 In another specific embodiment, about 2.5×10 11 Genome copies (this is approximately 1.0 × 10 in a volume of 250 μl) 12 In another specific embodiment, about 2.5×10 11 Genome copies are administered (this is approximately 2.5×10 in a volume of 100 μl) 12 In another specific embodiment, about 5×10 11 Genome copies are administered (approximately 5×10 in a volume of 200 μl) 12 In another specific embodiment, about 1.5×10 12 Genome copies are administered (this is approximately 1.5×10 in a volume of 100 μl) 13 In another specific embodiment, about 3×10 11 Genome copies are administered (approximately 3×10 in a volume of 100 μl) 12 In another specific embodiment, about 6×10 11 Genome copies are administered (approximately 3×10 in a volume of 200 μl) 12 In another specific embodiment, about 6×10 11 Genome copies are administered (approximately 6×10 in a volume of 100 μl) 12 (corresponding to GC / mL).

[0271] In certain embodiments, about 6.0 x 10 per dose or per eye. 10 In certain embodiments, about 6.4 x 10 genome copies are administered per administration or per eye. 10 In certain embodiments, about 1.3 x 10 genome copies are administered per administration or per eye. 11In certain embodiments, about 1.5 x 10 genome copies are administered per administration or per eye. 11 In certain embodiments, about 1.6 x 10 genome copies are administered per administration or per eye. 11 In certain embodiments, about 2.5 x 10 genome copies are administered per administration or per eye. 11 In certain embodiments, about 3×10 genome copies are administered per administration or per eye. 11 In certain embodiments, about 5.0 x 10 genome copies are administered per administration or per eye. 11 In certain embodiments, about 6×10 genome copies are administered per administration or per eye. 11 In some embodiments, about 1.5×10 genome copies are administered per eye, or per dose, or per route of administration. 12 In some embodiments, about 1.5×10 genome copies are administered. 12 Genome copies is the total number of genome copies administered.

[0272] In certain embodiments, about 3 x 10 per administration or per eye. 12 In certain embodiments, about 1.0 x 10 genome copies are administered per administration or per eye. 12 In certain embodiments, about 2.5×10 GC / mL is administered per administration or per eye. 12 In certain embodiments, about 3×10 GC / mL is administered per administration or per eye. 12 In certain embodiments, about 3.0×10 GC / mL is administered per administration or per eye. 13 In certain embodiments, up to 3.0 x 10 genome copies are administered per administration or per eye. 13 A copy of the genome is administered.

[0273] In certain embodiments, about 1.5 x 10 per administration or per eye. 11In certain embodiments, about 2.5 x 10 genome copies are administered by injection into the suprachoroid. 11 In certain embodiments, about 3×10 genome copies are administered per administration or per eye by injection into the suprachoroid. 11 The genome copies are administered by injection into the suprachoroid. In certain embodiments, about 5.0 x 10 genome copies are administered per administration or per eye. 11 In certain embodiments, about 6×10 genome copies are administered per administration or per eye by injection into the suprachoroid. 11 The genome copies are administered by injection into the suprachoroid. In certain embodiments, about 1.5×10 genome copies are administered per administration or per eye. 12 In certain embodiments, about 3×10 genome copies are administered per administration or per eye by injection into the suprachoroid. 12 The genome copies are administered by injection into the suprachoroid. In certain embodiments, about 2.5×10 genome copies are administered per eye. 11 The genome copies are administered by a single suprachoroidal injection. In certain embodiments, about 3×10 genome copies are administered per administration or per eye. 11 The genome copies are administered by a single suprachoroidal injection. In certain embodiments, about 3×10 genome copies are administered per administration or per eye in a volume of 100 μl. 11 The genome copies are administered by a single suprachoroidal injection. In certain embodiments, about 3×10 genome copies are administered per administration or per eye in a volume of 200 μl. 11 The genome copies are administered by a single suprachoroidal injection. In certain embodiments, about 3×10 genome copies are administered per administration or per eye. 11 The genome copies are administered by two suprachoroidal injections. In certain embodiments, about 3×10 genome copies are administered per administration or per eye. 11 The genome copies are administered by two suprachoroidal injections, each injection in a volume of 50 μl. In certain embodiments, about 3×10 genome copies are administered per administration or per eye. 11The genome copies are administered by two suprachoroidal injections, each injection in a volume of 100 μl. In certain embodiments, about 5.0×10 genome copies are administered per administration or per eye. 11 In certain embodiments, about 6×10 genome copies are administered per administration or per eye by two suprachoroidal injections. 11 The genome copies are administered by a single suprachoroidal injection. In certain embodiments, about 6×10 genome copies are administered per administration or per eye in a volume of 100 μl. 11 The genome copies are administered by a single suprachoroidal injection. In certain embodiments, about 6×10 genome copies are administered per administration or per eye in a volume of 200 μl. 11 The genome copies are administered by a single suprachoroidal injection. In certain embodiments, about 6×10 genome copies are administered per administration or per eye. 11 The genome copies are administered by two suprachoroidal injections. In certain embodiments, about 6×10 genome copies are administered per administration or per eye. 11 The genome copies are administered by two suprachoroidal injections, each injection in a volume of 50 μl. In certain embodiments, about 6×10 genome copies are administered per administration or per eye. 11 The genome copies are administered by two suprachoroidal injections, each injection in a volume of 100 μl. In certain embodiments, about 3.0×10 genome copies are administered per administration or per eye. 13 In certain embodiments, up to 3.0 x 10 genome copies are administered by suprachoroidal injection. 13 The genome copies are administered by injection into the suprachoroid. In certain embodiments, about 2.5×10 genome copies are administered per eye in a volume of 100 μl. 12 GC / mL is administered by a single suprachoroidal injection. In certain embodiments, about 2.5×10 GC / mL per eye is administered by a single suprachoroidal injection. 12 GC / mL is administered by two suprachoroidal injections, each injection in a volume of 100 μl. In certain embodiments, about 1.5×10 GC / mL is administered per eye in a volume of 100 μl. 13GC / mL is administered by a single suprachoroidal injection.

[0274] In certain embodiments, the recombinant viral vector is administered by two suprachoroidal injections. In certain embodiments, the first injection in the right eye is administered in the superior temporal quadrant (i.e., between the 10 o'clock position and the 11 o'clock position), and the second injection in the same eye is administered in the inferior nasal quadrant (i.e., between the 4 o'clock position and the 5 o'clock position). In certain embodiments, the first injection in the right eye is administered in the inferior nasal quadrant (i.e., between the 4 o'clock position and the 5 o'clock position), and the second injection in the same eye is administered in the superior temporal quadrant (i.e., between the 10 o'clock position and the 11 o'clock position). In certain embodiments, the first injection in the left eye is administered in the superior temporal quadrant (i.e., between the 1 o'clock position and the 2 o'clock position), and the second injection in the same eye is administered in the inferior nasal quadrant (i.e., between the 7 o'clock position and the 8 o'clock position). In certain embodiments, the first injection in the left eye is administered in the inferonasal quadrant (i.e., between the 7 o'clock and 8 o'clock positions) and the second injection in the same eye is administered in the superior temporal quadrant (i.e., between the 1 o'clock and 2 o'clock positions).

[0275] In certain embodiments, the recombinant viral vector is administered by a single suprachoroidal injection. In certain embodiments, the single injection in the right eye is administered in the superior temporal quadrant (i.e., between the 10 o'clock position and the 11 o'clock position). In certain embodiments, the single injection in the right eye is administered in the inferior nasal quadrant (i.e., between the 4 o'clock position and the 5 o'clock position). In certain embodiments, the single injection in the left eye is administered in the superior temporal quadrant (i.e., between the 1 o'clock position and the 2 o'clock position). In certain embodiments, the single injection in the left eye is administered in the inferior nasal quadrant (i.e., between the 7 o'clock position and the 8 o'clock position).

[0276] In some embodiments, the pharmaceutical composition or the reference pharmaceutical composition is administered to a human subject once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, twenty-five times, or thirty times (e.g., suprachoroidally, subretinally, or intravitreally). In some embodiments, the pharmaceutical composition or the reference pharmaceutical composition is administered to a human subject once a day, twice a day, three times a day, four times a day, five times a day, six times a day, or seven times a day. In some embodiments, the same amount of AAV genome copies is administered per administration. For example, the same genome copies are administered suprachoroidally, subretinally, or intravitreally. In some embodiments, the same total amount of AAV genome copies is administered. For example, the same total amount of AAV genome copies is administered regardless of the total number of doses administered suprachoroidally, subretinatally, or intravitreally (e.g., if one subretinal dose is administered and two suprachoroidal doses are administered, the genome copies in one subretinal dose are the same as the genome copies in both suprachoroidal doses combined).

[0277] As used herein, unless otherwise specified, the term "about" means within plus or minus 10% of a given value or range.

[0278] 4.4 Constructs and Formulations In some embodiments, the recombinant vectors provided herein comprise the following elements, in the following order: a) a constitutive or hypoxia-inducible promoter sequence, and b) a sequence encoding a transgene (e.g., a therapeutic product). In certain embodiments, the recombinant vectors provided herein comprise the following elements, in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or hypoxia-inducible promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion), i) a second UTR sequence, j) a fourth linker sequence, k) a polyA sequence, l) a fifth linker sequence, and m) a second ITR sequence.

[0279] In certain embodiments, a recombinant vector provided herein comprises the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or hypoxia inducible promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding a transgene (e.g., an anti-VEGF antigen binding fragment portion), i) a second UTR sequence, j) a fourth linker sequence, k) a polyA sequence, l) a fifth linker sequence, and m) a second ITR sequence, wherein the transgene comprises a signal peptide of VEGF (SEQ ID NO:5), and wherein the transgene encodes light and heavy chain sequences separated by a cleavable F / F2A sequence.

[0280] In some embodiments, the AAV (AAV viral vector) provided herein comprises 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 post-translationally modified fully human (HuPTM) antibody against VEGF. In some embodiments, the post-translationally modified fully human antibody against VEGF is a post-translationally modified fully human antigen-binding fragment of a monoclonal antibody (mAb) against VEGF ("HuPTMFabVEGFi"). In some embodiments, 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 some embodiments, the AAV used to deliver the transgene should have affinity for human retinal cells or photoreceptor cells. Such AAVs may include non-replicating recombinant adeno-associated viral vectors ("rAAVs"), particularly those with AAV8 capsids. In a specific embodiment, the viral vector or other DNA expression construct described herein is construct I, which comprises the following components: (1) AAV8 inverted terminal repeats flanked by expression cassettes; (2) control elements including a) a CMV enhancer / CB7 promoter including chicken β-actin promoter, b) a chicken β-actin intron, and c) a rabbit β-globin polyA signal; and (3) nucleic acid sequences encoding the heavy and light chains of an anti-VEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker to ensure expression of equal amounts of heavy and light chain polypeptides. In some embodiments, the viral vector comprises a vector genome (ITR-CB7-CI-aVEGFv3-rBG-ITR) comprising SEQ ID NO:56. In some embodiments, the vector genome comprises SEQ ID NO: 14 of US11197937 (hereby incorporated by reference). In some embodiments, the vector genome comprises any one of the sequences disclosed in US11197937 (hereby incorporated by reference).In some embodiments, the vector genome comprises a 5' AAV-2 inverted terminal repeat, an expression cassette consisting of contiguous nucleotides 198 to 3733 of SEQ ID NO:56 (equivalent to SEQ ID NO:14 of US11197937, which is incorporated herein by reference), and a 3' AAV-2 inverted terminal repeat. In some embodiments, the viral vector comprises a signal peptide. In some embodiments, the signal peptide is MYRMQLLLLIALSLALVTNS (SEQ ID NO:55). In some embodiments, the signal peptide is derived from the IL-2 signal sequence. In some embodiments, the viral vector comprises a signal peptide from any of the signal peptides disclosed in Table 1, for example, MNFLLSWVHW SLALLLYLHH AKWSQA (VEGF-A signal peptide) (SEQ ID NO: 5); MERAAPSRRV PLPLLLLGGL ALLAAGVDA (Fibulin-1 signal peptide) (SEQ ID NO: 6); MAPLRPLLIL ALLAWVALA (Vitronectin signal peptide) (SEQ ID NO: 7); MRLLAKIICLMLWAICVA (Complement factor H signal peptide) (SEQ ID NO: 8); MRLLAFLSLL ALVLQETGT (opticin 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); or MYRMQLLLLIALSLALVTNS (mutated interleukin-2 signal peptide) (SEQ ID NO: 55).In another specific embodiment, the viral vector or other DNA expression construct described herein is Construct II, which comprises the following components: (1) AAV2 inverted terminal repeats flanked by an expression cassette; (2) control elements including a) a CMV enhancer / CB7 promoter including chicken β-actin promoter, b) a chicken β-actin intron, and c) a rabbit β-globin polyA signal; and (3) nucleic acid sequences encoding the heavy and light chains of an anti-VEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker to ensure expression of equal amounts of heavy and light chain polypeptides. In some embodiments, the anti-hVEGF antibody 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.

[0281] In some embodiments, a viral vector or other expression construct suitable for packaging into an AAV capsid comprises: (1) AAV inverted terminal repeats (ITRs) flanked by expression cassettes; (2) regulatory control elements consisting essentially of one or more enhancers and / or promoters, d) a polyA signal, and e) optionally an intron; and (3) one or more transgenes that provide (e.g., encode) one or more RNA or protein products of interest.

[0282] In some aspects, the disclosure provides a nucleic acid for use, encoding a therapeutic product operably linked to a promoter or enhancer-promoter described herein.

[0283] In some aspects, the present disclosure provides a nucleic acid for use, the nucleic acid encoding a HuPTMFabVEGFi, for example, a HuGlyFabVEGFi operably linked to a promoter selected from the group consisting of CB7 promoter (chicken β-actin promoter and CMV enhancer), cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter, and opsin promoter. In a specific embodiment, the HuPTMFabVEGFi is operably linked to the CB7 promoter.

[0284] In certain embodiments, provided herein are recombinant vectors comprising one or more nucleic acids (e.g., polynucleotides). The nucleic acids may comprise DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA comprises one or more sequences selected from the group consisting of a promoter sequence, a sequence encoding a therapeutic product of interest (transgene, e.g., an anti-VEGF antigen-binding fragment), an untranslated region, and a termination sequence. In certain embodiments, the recombinant vectors provided herein comprise a promoter operably linked to a sequence encoding a therapeutic product of interest.

[0285] In certain embodiments, the nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein may be codon optimized, for example, via any codon optimization technique known to those of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59:149-161).

[0286] In certain embodiments, the recombinant vectors provided herein comprise a modified mRNA encoding a therapeutic product of interest (e.g., a transgene, e.g., an anti-VEGF antigen-binding fragment moiety). In certain embodiments, modified mRNA encoding an anti-VEGF antigen-binding fragment moiety is provided herein. In certain embodiments, the recombinant vectors provided herein comprise a nucleotide sequence encoding a therapeutic product that is an shRNA, siRNA, or miRNA.

[0287] In certain embodiments, the vectors provided herein include components that modulate protein delivery. In certain embodiments, the viral vectors provided herein include one or more signal peptides. Examples of signal peptides include, but are not limited to, VEGF-A signal peptide (SEQ ID NO:5), fibulin-1 signal peptide (SEQ ID NO:6), vitronectin signal peptide (SEQ ID NO:7), complement factor H signal peptide (SEQ ID NO:8), opticin signal peptide (SEQ ID NO:9), albumin signal peptide (SEQ ID NO:22), chymotrypsinogen signal peptide (SEQ ID NO:23), interleukin-2 signal peptide (SEQ ID NO:24), and trypsinogen-2 signal peptide (SEQ ID NO:25), mutated interleukin-2 signal peptide (SEQ ID NO:55).

[0288] 4.4.1 Viral vectors In some embodiments, the viral vector provided herein is an AAV-based viral vector. In a preferred embodiment, the viral vector provided herein is an AAV8-based viral vector. In certain embodiments, the AAV8-based viral vector provided herein retains tropism for retinal cells. In certain embodiments, the AAV-based vector provided herein encodes the AAV rep gene (required for replication) and / or the AAV cap gene (required for synthesis of capsid protein). Multiple AAV serotypes have been identified. In certain embodiments, the AAV-based vector provided herein comprises components from one or more serotypes of AAV. In certain embodiments, the AAV-based vectors provided herein are 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.7m8, 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. In a preferred embodiment, the AAV-based vector provided herein comprises components from one or more of the AAV8, AAV9, AAV10, AAV11, or AAVrhlO serotypes. In certain embodiments, the recombinant viral vector provided herein is modified such that it is replication-deficient in humans. In certain embodiments, the recombinant viral vector is a hybrid vector, for example an AAV vector disposed in a "helpless" adenoviral vector.In a specific embodiment, a recombinant viral vector is provided herein, comprising a viral capsid from a first virus and a viral envelope protein from a second virus.In a specific embodiment, the second virus is vesicular stomatitis virus (VSV).In a more specific embodiment, the envelope protein is VSV-G protein.

[0289] In certain embodiments, an AAV8 vector is provided that comprises a viral genome comprising an expression cassette for expression of a transgene under the control of regulatory elements and flanked by ITRs, and a viral capsid having the amino acid sequence of an AAV8 capsid protein or at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of the AAV8 capsid protein (SEQ ID NO: 48) while retaining a biological function of the AAV8 capsid. In certain embodiments, the encoded AAV8 capsid has the sequence of SEQ ID NO: 48 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions and retaining a biological function of the AAV8 capsid.

[0290] In certain embodiments, the AAV used in the methods described herein is Anc80 or Anc80L65, which are described in Zinn et al., 2015, Cell Rep. 12(6):1056-1068, which are incorporated by reference in their entireties. In certain embodiments, the AAV used in the methods described herein contains one of the following amino acid insertions: LGETTRP or LALGETTRP, which are described in U.S. Patent Nos. 9,193,956; 9458517; and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated by reference in its entirety. In certain embodiments, the AAV used in the methods described herein is AAV.7m8, which is described in U.S. Patent Nos. 9,193,956; 9,458,517; and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV used in the methods described herein is any AAV disclosed in U.S. Patent No. 9,585,971, such as AAV.PHP.B. In certain embodiments, the AAV used in the methods described herein is an AAV disclosed in any of the following patents and patent applications, each of which is incorporated by reference herein in its entirety: U.S. Pat. Nos. 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US Pat. Nos. 9,284,357; 9,409, 9,524,446; 9,628,966; 9,734,809; and US Pat. Nos. 9,284,357, 9,409, 9,524,446, 9,524,446, 9,999,678, 9,524,446, 9,524,446, 9,524,446, 9,999,678, 9,628,96 ...927,514, 9,734,809, and 9,284,357. ,953; 9,169,299; 9,193,956; 9458517; and 9,587,282, U.S. Patent Application Publication Nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335.

[0291] AAV8-based viral vectors are used in certain of the methods described herein. The nucleic acid sequences of AAV-based viral vectors and the methods of making recombinant AAV and AAV capsids are taught in, for example, U.S. Patent No. 7,282,199, U.S. Patent No. 7,790,449, U.S. Patent No. 8,318,480, U.S. Patent No. 8,962,332 and International Patent Application PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety. In one aspect, AAV (e.g., AAV8)-based viral vectors are provided herein that encode transgenes (e.g., anti-VEGF antigen-binding fragments). In a specific embodiment, AAV8-based viral vectors are provided herein that encode anti-VEGF antigen-binding fragments. In a more specific embodiment, AAV8-based viral vectors are provided herein that encode ranibizumab.

[0292] In certain embodiments, single-stranded AAV (ssAAV) can be used as above. In certain embodiments, self-complementary vectors, such as scAAV, can be used (see, for example, Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al., 2001, Gene Therapy, Vol. 8, No. 16, pp. 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0293] In certain embodiments, the viral vector used in the methods described herein is an adenovirus-based viral vector. Recombinant adenovirus vectors can be used to transfer in anti-VEGF antigen-binding fragments. The recombinant adenovirus can be a first-generation vector with an E1 deletion, with or without an E3 deletion, with an expression cassette inserted into any deleted region. The recombinant adenovirus can be a second-generation vector containing a complete or partial deletion of the E2 and E4 regions. Helper-dependent adenoviruses retain only the adenovirus inverted terminal repeats and packaging signal (phi). The transgene is inserted between the packaging signal and the 3'ITR, with or without a stuffer sequence to maintain the genome close to the wild-type size of approximately 36 kb. Exemplary protocols for producing adenoviral vectors can be found in Alba et al., 2005, "Gutless adenovirus: last generation adenovirus for gene therapy," Gene Therapy 12:S18-S27, which is incorporated herein by reference in its entirety.

[0294] In a specific embodiment, a vector for use in the methods described herein is a vector that encodes an anti-VEGF antigen-binding fragment (e.g., ranibizumab) such that upon introduction of the vector into a relevant cell (e.g., a retinal cell in vivo or in vitro), a glycosylated and / or tyrosine sulfation variant of the anti-VEGF antigen-binding fragment is expressed by the cell. In a specific embodiment, the expressed anti-VEGF antigen-binding fragment comprises a glycosylation and / or tyrosine sulfation pattern.

[0295] 4.4.2 Therapeutic Products or Transgenes The therapeutic product may be, for example, a therapeutic protein (eg, an antibody), a therapeutic RNA (eg, shRNA, siRNA, and miRNA), or a therapeutic aptamer.

[0296] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a recombinant AAV encoding a transgene. In some embodiments, provided herein is a rAAV viral vector encoding an anti-VEGF Fab or an anti-VEGF antibody. In some embodiments, provided herein is a rAAV viral vector that does not encode an anti-VEGF Fab or an anti-VEGF antibody. In some embodiments, provided herein is a rAAV8-based viral vector that encodes an anti-VEGF Fab or an anti-VEGF antibody. In some embodiments, provided herein is a rAAV8-based viral vector that does not encode an anti-VEGF Fab or an anti-VEGF antibody. In some embodiments, provided herein is a rAAV viral vector that encodes a tripeptidyl peptidase 1 (TPP1) protein. In some embodiments, provided herein is a rAAV9-based viral vector that encodes TPP1. In some embodiments, provided herein is a rAAV viral vector that encodes an anti-kallikrein (anti-pKal) protein. In some embodiments, provided herein is a rAAV8-based or rAAV9-based viral vector that encodes a lanadelumab Fab or full-length antibody. In some embodiments, provided herein is a rAAV viral vector that encodes CLN2. In some embodiments, provided herein is a rAAV viral vector encoding CLN3. In some embodiments, provided herein is a rAAV viral vector encoding CLN6. In some embodiments, provided herein is a rAAV8-based or rAAV9-based viral vector encoding CLN2. In some embodiments, provided herein is a rAAV8-based or rAAV9-based viral vector encoding CLN3. In some embodiments, provided herein is a rAAV8-based or rAAV9-based viral vector encoding CLN6.

[0297] In certain embodiments, the therapeutic product (e.g., a transgene) is: (1) an anti-human vascular endothelial growth factor (hVEGF) antibody or aptamer; (2) an anti-hVEGF antigen-binding fragment; (3) a Fab, F(ab')2, or single-chain variable fragment (scFv); (4) palmitoyl protein thioesterase 1 (PPT1); (5) tripeptidyl-peptidase 1 (TPP1); (6) Battenin (CLN3); and (7) CLN6 transmembrane ER protein (CLN6).

[0298] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a recombinant AAV encoding a transgene. In some embodiments, provided herein is a rAAV viral vector encoding an anti-VEGF Fab or an anti-VEGF antibody. In some embodiments, provided herein is a rAAV8-based viral vector encoding an anti-VEGF Fab or an anti-VEGF antibody. In further embodiments, provided herein is a rAAV8-based viral vector encoding ranibizumab. In some embodiments, provided herein is a rAAV viral vector encoding a tripeptidyl peptidase 1 (TPP1) protein. In some embodiments, provided herein is a rAAV9-based viral vector encoding TPP1. In some embodiments, provided herein is a rAAV viral vector encoding a micro-dystrophin protein. In some embodiments, provided herein is a rAAV8-based viral vector encoding micro-dystrophin. In some embodiments, provided herein is a rAAV9-based viral vector encoding micro-dystrophin. In some embodiments, provided herein is a rAAV viral vector encoding an anti-kallikrein (anti-pKal) protein. In some embodiments, provided herein are rAAV8-based or rAAV9-based viral vectors encoding lanadelumab Fab or full-length antibody. In some embodiments, provided herein are rAAV viral vectors encoding human-alpha-sarcoglycan-gamma-sarcoglycan. In some embodiments, provided herein are rAAV viral vectors encoding hu follistatin 344. In some embodiments, provided herein are rAAV viral vectors encoding human-alpha-sarcoglycan-gamma-sarcoglycan. In some embodiments, provided herein are rAAV viral vectors encoding CLN2. In some embodiments, provided herein are rAAV viral vectors encoding CLN3. In some embodiments, provided herein are rAAV viral vectors encoding CLN6.In some embodiments, provided herein is a rAAV8- or rAAV9-based viral vector encoding human-alpha-sarcoglycan-gamma-sarcoglycan. In some embodiments, provided herein is a rAAV8- or rAAV9-based viral vector encoding hu follistatin 344. In some embodiments, provided herein is a rAAV8- or rAAV9-based viral vector encoding human-alpha-sarcoglycan-gamma-sarcoglycan. In some embodiments, provided herein is a rAAV8- or rAAV9-based viral vector encoding CLN2. In some embodiments, provided herein is a rAAV8- or rAAV9-based viral vector encoding CLN3. In some embodiments, provided herein is a rAAV8- or rAAV9-based viral vector encoding CLN6.

[0299] In certain embodiments, the vectors provided herein can be used for: (1) eye conditions associated with Batten-CLN1, where the therapeutic product is palmitoyl protein thioesterase 1 (PPT1); (2) eye conditions associated with Batten-CLN2, where the therapeutic product is tripeptidyl-peptidase 1 (TPP1); (3) eye conditions associated with Batten-CLN3, where the therapeutic product is Battenin (CLN3); (4) eye conditions associated with Batten-CLN6, where the therapeutic product is CLN6 transmembrane ER protein (CLN6); (5) eye conditions associated with Batten-CLN7, where the therapeutic product is Major Facilitator Superfamily Domain Containing 8 (MFSD8); (6) eye conditions associated with Batten-CLN1, where the therapeutic product is palmitoyl protein thioesterase 1 (PPT1).

[0300] In some embodiments, the HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, encoded by the transgene includes, but is not limited to, an antigen-binding fragment of an antibody that binds VEGF, such as bevacizumab; an anti-VEGF Fab portion, such as ranibizumab; or such a bevacizumab or ranibizumab Fab portion that has been engineered to contain additional glycosylation sites on the Fab domain (see, e.g., Courtois et al., 2016, mAb 8:99-112, which is incorporated by reference in its entirety, for a description of a derivative of bevacizumab that is highly glycosylated in the Fab domain of its full-length antibody).

[0301] In certain embodiments, the vectors provided herein encode an anti-VEGF antigen-binding fragment transgene. In specific embodiments, the anti-VEGF antigen-binding fragment transgene is controlled by expression control elements suitable for expression in retinal cells: In certain embodiments, the anti-VEGF antigen-binding fragment transgene comprises the Fab portion of bevacizumab light and heavy chain cDNA sequences (SEQ ID NOs: 10 and 11, respectively). In certain embodiments, the anti-VEGF antigen-binding fragment transgene comprises the light and heavy chain cDNA sequences of ranibizumab (SEQ ID NOs: 12 and 13, respectively). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes a bevacizumab Fab comprising the light and heavy chains of SEQ ID NOs: 3 and 4, respectively. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a heavy chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 3, and a heavy chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes a highly glycosylated ranibizumab comprising the light and heavy chains of SEQ ID NOs: 1 and 2, respectively.In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a heavy chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:2. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:1, and a heavy chain comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO:2.

[0302] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes a highly glycosylated bevacizumab Fab comprising the light and heavy chains of SEQ ID NOs: 3 and 4 with one or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or Q160S (light chain). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes a highly glycosylated ranibizumab comprising the light and heavy chains of SEQ ID NOs: 1 and 2 with one or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or Q160S (light chain). The sequence of the cDNA of the antigen-binding fragment transgene can be found, for example, in Table 1. In certain embodiments, the sequence of the cDNA of the antigen-binding fragment transgene is obtained by replacing the signal sequence of SEQ ID NOs: 10 and 11 or SEQ ID NOs: 12 and 13 with one or more signal sequences.

[0303] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequence of six bevacizumab CDRs. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequence of six ranibizumab CDRs. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1-3 (SEQ ID NOs: 20, 18, and 21) of ranibizumab. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDRs 1-3 (SEQ ID NOs: 14-16) of ranibizumab. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1-3 (SEQ ID NOs: 17-19) of bevacizumab. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1-3 of bevacizumab (SEQ ID NOs: 14-16). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDR1-3 of ranibizumab (SEQ ID NOs: 20, 18, and 21) and a light chain variable region comprising light chain CDR1-3 of ranibizumab (SEQ ID NOs: 14-16). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDR1-3 of bevacizumab (SEQ ID NOs: 17-19) and a light chain variable region comprising light chain CDR1-3 of bevacizumab (SEQ ID NOs: 14-16).

[0304] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1-3 of SEQ ID NOs: 14-16, and the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (PyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1 to 3 of SEQ ID NO: 14 to 16, and the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the 2nd amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1 to 3 of SEQ ID NO: 14 to 16, and the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). The second amino acid residue of DR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) is not acetylated. In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1-3 of SEQ ID NO: 14-16, in which the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamination (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) is not acetylated. In a preferred embodiment, the chemical modifications or lack of chemical modifications (as the case may be) described herein are determined by mass spectrometry.

[0305] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1-3 of SEQ ID NOs: 20, 18, and 21, and wherein the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (PyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1-3 of SEQ ID NOs: 20, 18, and 21, in which the 9th amino acid residue of the heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamination (PyroGlu), and the 3rd amino acid residue of the heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamination (PyroGlu). and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamination (pyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated.In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising heavy chain CDRs 1-3 of SEQ ID NOs: 20, 18, and 21, wherein the 9th amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamination (pyroGlu), the 3rd amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamination (pyroGlu), and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated. In a preferred embodiment, the chemical modifications or lack of chemical modifications (as the case may be) described herein are determined by mass spectrometry.

[0306] In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1-3 of SEQ ID NOs: 14-16 and a heavy chain variable region comprising heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the last amino acid residue of the heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1 to 3 of SEQ ID NOs: 14 to 16 and a heavy chain variable region comprising heavy chain CDR1 to 3 of SEQ ID NOs: 20, 18, and 21, (1) the 9th amino acid residue of the heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), the 3rd amino acid residue of the heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the 4th amino acid residue of the heavy chain CDR2 (i.e., N in The last amino acid residue (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu); (2) the 8th and 11th amino acid residues of the light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu).In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising light chain CDR1-3 of SEQ ID NOs: 14-16 and a heavy chain variable region comprising heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, in which the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) is not acetylated, and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated. In a specific embodiment, the antigen-binding fragment comprises a heavy chain CDR1 of SEQ ID NO: 20, and (1) the 9th amino acid residue of the heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), the 3rd amino acid residue of the heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the last amino acid residue of the heavy chain CDR1 (i.e., GYDFT (2) the 8th and 11th amino acid residues of the light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamination (PyroGlu), and the 2nd amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) is not acetylated. In a preferred embodiment, the chemical modifications or lack of chemical modifications (as the case may be) described herein are determined by mass spectrometry.

[0307] In certain aspects, also provided herein are anti-VEGF antigen-binding fragments comprising light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, as well as transgenes encoding such antigen-VEGF antigen-binding fragments, wherein the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamination (PyroGlu). In a specific embodiment, the antigen-binding fragment comprises light chain CDR1 to 3 of SEQ ID NOs: 14 to 16 and heavy chain CDR1 to 3 of SEQ ID NOs: 20, 18, and 21, and the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the 2nd amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the antigen-binding fragment comprises light chain CDR1 to 3 of SEQ ID NOs: 14 to 16 and heavy chain CDR1 to 3 of SEQ ID NOs: 20, 18, and 21, and the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). The second amino acid residue (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) is not acetylated. In a specific embodiment, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, in which the 8th and 11th amino acid residues of light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamination (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any of the methods according to the invention described herein.In a preferred embodiment, the chemical modifications or lack of chemical modifications (as the case may be) described herein are determined by mass spectrometry.

[0308] In certain aspects, also provided herein are anti-VEGF antigen-binding fragments comprising light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, as well as transgenes encoding such antigen-VEGF antigen-binding fragments, wherein the last amino acid residue of heavy chain CDR1 (i.e., the N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamination (PyroGlu). In a specific embodiment, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, in which the 9th amino acid residue of the heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamination (PyroGlu), and the 3rd amino acid residue of the heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamination (PyroGlu). and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamination (pyroGlu). In a specific embodiment, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated.In a specific embodiment, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the 9th amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamination (pyroGlu), the 3rd amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamination (pyroGlu), and the last amino acid residue of heavy chain CDR1 (i.e., N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any of the methods according to the invention described herein. In a preferred embodiment, the chemical modifications or lack of chemical modifications (as the case may be) described herein are determined by mass spectrometry.

[0309] In certain aspects, also provided herein are anti-VEGF antigen-binding fragments comprising light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, as well as transgenes encoding such antigen-VEGF antigen-binding fragments, wherein the last amino acid residue of heavy chain CDR1 (i.e., the N in GYDFTHYGMN (SEQ ID NO: 20)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu). In a specific embodiment, the antigen-binding fragment comprises light chain CDR1 to 3 of SEQ ID NOs: 14 to 16 and heavy chain CDR1 to 3 of SEQ ID NOs: 20, 18, and 21, (1) the 9th amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), the 3rd amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the last amino acid residue of heavy chain CDR1 (i.e., GYDF (2) the 8th and 11th amino acid residues of the light chain CDR1 (i.e., the two Ns in SASQDISNYLN (SEQ ID NO: 14) each have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu), and the 2nd amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) does not have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamation (pyroGlu).In a specific embodiment, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16 and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, in which the last amino acid residue of heavy chain CDR1 (i.e., the N in GYDFTHYGMN (SEQ ID NO: 20)) is not acetylated, and the second amino acid residue of light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) is not acetylated. In a specific embodiment, the antigen-binding fragment comprises light chain CDR1 to 3 of SEQ ID NOs: 14 to 16 and heavy chain CDR1 to 3 of SEQ ID NOs: 20, 18, and 21, and (1) the 9th amino acid residue of heavy chain CDR1 (i.e., M in GYDFTHYGMN (SEQ ID NO: 20)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), the 3rd amino acid residue of heavy chain CDR2 (i.e., N in WINTYTGEPTYAADFKR (SEQ ID NO: 18)) has one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamation (pyroGlu), and the last amino acid residue of heavy chain CDR1 (i.e., (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 have one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamination (PyroGlu), and the second amino acid residue of the light chain CDR3 (i.e., the second Q in QQYSTVPWTF (SEQ ID NO: 16)) is not acetylated. The anti-VEGF antigen-binding fragments and transgenes provided herein can be used in any of the methods according to the invention described herein. In a preferred embodiment, the chemical modifications or lack of chemical modifications (as the case may be) described herein are determined by mass spectrometry.

[0310] [Table 1] TIFF2025512962000003.tif252168TIFF2025512962000004.tif251168TIFF2025512962000005.tif252168TIFF2025512962000006.tif252168 TIFF2025512962000007.tif252168TIFF2025512962000008.tif254168TIFF2025512962000009.tif251167TIFF2025512962000010.tif235167

[0311] 4.5 Disease The pharmaceutical compositions provided herein or the reference pharmaceutical compositions (e.g., Section 4.1) can be administered to a subject diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), or Batten Disease.

[0312] In some embodiments, disclosed herein are methods of treating a subject diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), or Batten disease by administering a therapeutically effective amount of a pharmaceutical composition to the subject by injection into the suprachoroid (e.g., via a suprachoroidal drug delivery device, such as a microinjector with fine needles).

[0313] In some embodiments, about 2.5×10 of a pharmaceutical composition comprising 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium dihydrogen phosphate, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL sucrose 4% w / v, and optionally a surfactant. 11 GC / eye, approx. 5 x 10 11 GC / item, or approximately 1.5 x 10 12 The pharmaceutical composition containing the GC / eye construct II is administered to the patient via suprachoroidal administration. In some embodiments, the patient has diabetic retinopathy.

[0314] In some embodiments, about 2.5×10 of a pharmaceutical composition comprising 10% w / v sucrose 11 GC / eye, approx. 5 x 10 11 GC / item, or approximately 1.5 x 10 12 The pharmaceutical composition containing the GC / eye construct II is administered to the patient via suprachoroidal administration. In some embodiments, the patient has diabetic retinopathy. In some embodiments, the pharmaceutical composition has a tonicity / osmolality equal to or greater than 240 mOsm / kg.

[0315] In some embodiments, the pharmaceutical compositions provided herein or the reference pharmaceutical compositions (e.g., Section 4.1) can be administered to: (1) a subject diagnosed with Batten-CLN2, where the therapeutic product is tripeptidyl-peptidase 1 (TPP1); (2) a subject diagnosed with Usher Type 1, where the therapeutic product is myosin VIIA (MYO7A); (3) a subject diagnosed with Usher Type 1, where the therapeutic product is cadherin-associated 23 (CDH23); or (4) a subject diagnosed with Usher Type 2. (5) may be administered to a subject diagnosed with Uscher 2 and the therapeutic product is Usherin (USH2A); (6) may be administered to a subject diagnosed with Uscher 3 and the therapeutic product is Clarin 1 (CLRN1); (7) may be administered to a subject diagnosed with Stargardt's disease and the therapeutic product is ATP-binding cassette subfamily A member 4 (ABCA4); (8) may be administered to a subject diagnosed with Stargardt's disease and the therapeutic product is (9) may be administered to a subject diagnosed with red-green color blindness, and the therapeutic product is L opsin (OPN1LW); (10) may be administered to a subject diagnosed with red-green color blindness, and the therapeutic product is M opsin (OPN1MW); (11) may be administered to a subject diagnosed with blue-cone monochromacy, and the therapeutic product is M opsin (OPN1MW); (12) may be administered to a subject diagnosed with Leber congenital amaurosis-1 (LCA1), and the therapeutic product is guanylate cyclase (13) may be administered to a subject diagnosed with Leber congenital amaurosis-2 (LCA2), where the therapeutic product is retinoid isomerohydrolase RPE65 (RPE65); (14) may be administered to a subject diagnosed with Leber congenital amaurosis-4 (LCA4), where the therapeutic product is aryl hydrocarbon receptor interacting protein-like 1 (AIPL1); (15) may be administered to a subject diagnosed with Leber congenital amaurosis-7 (LCA7), where the therapeutic product is cone-rod homeobox (CRX);(16) may be administered to a subject diagnosed with Leber congenital amaurosis-8 (LCA8), where the therapeutic product is Crumbs cell polarity complex component 1 (CRB1); (17) may be administered to a subject diagnosed with Leber congenital amaurosis-9 (LCA9), where the therapeutic product is nicotinamide nucleotide adenylyltransferase 1 (NMNAT1); (18) may be administered to a subject diagnosed with Leber congenital amaurosis-10 (LCA10), where the therapeutic product is centrosomal protein 290 (CEP290); (19) Leber congenital amaurosis (20) may be administered to a subject diagnosed with Leber congenital amaurosis-15 (LCA15), where the therapeutic product is inosine monophosphate dehydrogenase 1 (IMPDH1); (21) may be administered to a subject diagnosed with LHON, where the therapeutic product is mitochondrially encoded NADH dehydrogenase 4 (MT-ND4); (22) may be administered to a subject diagnosed with LHON, where the therapeutic product is mitochondrially encoded NADH dehydrogenase 4 (MT-ND4). (23) may be administered to a subject diagnosed with choroideremia, where the therapeutic product is Rab escort protein 1 (CHM); (24) may be administered to a subject diagnosed with X-linked retinoschisis (XLRS), where the therapeutic product is retinoschisin (RS1); (25) may be administered to a subject diagnosed with Bardet-Biedl syndrome 1, where the therapeutic product is Bardet-Biedl syndrome 1 (BBS1); (26) may be administered to a subject diagnosed with Bardet-Biedl syndrome 6, where the therapeutic product is (27) may be administered to a subject diagnosed with Bardet-Biedl syndrome 10, and the therapeutic product is Bardet-Biedl syndrome 10 (BBS10); (28) may be administered to a subject diagnosed with cone dystrophy, and the therapeutic product is guanylate cyclase activator 1A (GUCA1A); (29) may be administered to a subject diagnosed with optic nerve atrophy, and the therapeutic product is OPA1 mitochondrial dynamin-like GTPase (OPA1);(30) may be administered to a subject diagnosed with retinitis pigmentosa 1, where the therapeutic product is RP1 axonemal microtubule associated (RP1); (31) may be administered to a subject diagnosed with retinitis pigmentosa 2, where the therapeutic product is RP2 activator of ARL3 GTPase (RP2); (32) may be administered to a subject diagnosed with retinitis pigmentosa 7, where the therapeutic product is peripherin 2 (PRPH2); (33) may be administered to a subject diagnosed with retinitis pigmentosa 11, where the therapeutic product is pre-mRNA processing factor 31 (PRPF31); (34) may be administered to a subject diagnosed with retinitis pigmentosa 13, where the therapeutic product is pre-mRNA processing factor 8 (PRPF8); (35) may be administered to a subject diagnosed with retinitis pigmentosa 37, where the therapeutic product is (36) may be administered to a subject diagnosed with retinitis pigmentosa 38, where the therapeutic product is MER proto-oncogene, tyrosine kinase (MERTK); (37) may be administered to a subject diagnosed with retinitis pigmentosa 40, where the therapeutic product is phosphodiesterase 6B (PDE6B); (38) may be administered to a subject diagnosed with retinitis pigmentosa 41, where the therapeutic product is prominin 1 (PROM1); (39) may be administered to a subject diagnosed with retinitis pigmentosa 56, where the therapeutic product is interphotoreceptor matrix proteoglycan 2 (IMPG2); (40) may be administered to a subject diagnosed with retinitis pigmentosa (petinitis (41) may be administered to a subject diagnosed with retinitis pigmentosa 62, where the therapeutic product is male germ cell associated kinase (MAK); (41) may be administered to a subject diagnosed with retinitis pigmentosa 80, where the therapeutic product is intraflagellar transporter 140 (IFT140); or (42) may be administered to a subject diagnosed with Best disease, where the therapeutic product is bestrophin 1 (BEST1);

[0316] 4.6 Assay A skilled artisan can use the assays described herein and / or techniques known in the art to study the compositions and methods described herein, for example, to test the formulations provided herein. In some embodiments, a skilled artisan can use any assay and / or technique, such as those described in Chiang et al., "Clearance Kinetics and Clearance Routes of Molecules From the Suprachoroidal Space After Microneedle Injection," Invest Ophthalmol Vis Sci. January 2017; 58(1):545-554; Gu et al., "Real-Time Monitoring of Suprachoroidal Space (SCS) Following SCS Injection Using Ultra-High Resolution," Optical Coherence Tomography in Guinea Pig Eyes," Invest Ophthalmol Vis Sci. June 2015; 56(6):3623-34; and Seiler et al., "Effect and Distribution of Contrast Medium after Injection into the Anterior Suprachoroidal Space in Ex Vivo Eyes," Invest Ophthalmol Vis Sci. Sci. 2011 Jul. 29;52(8):5730-6, each of which is incorporated herein in its entirety. As detailed in Section 5, the following assays are also provided herein:

[0317] 4.6.1 Ultrasound B-scan A high frequency ultrasound (U / S) probe (UBM Plus; Accutome, Malvern, PA, USA) can be used to determine the thickness of the SCS by generating 2D cross-sectional images of the SCS in the animal's eye ex vivo after injection of different volumes of a given range (e.g., ranging from 25 μL to 500 μL from low to high viscosity) at a given viscosity and / or elastic modulus (G') at approximately 32-35 °C. A U / S probe cover (Clearscan, Eye-Surgical-Instruments, Plymouth, MN) can be attached to the UBM Plus to facilitate acquisition of the U / S images. The U / S probe can be used to acquire sagittal views (e.g., eight sagittal views) around the eye. Post-processing of the U / S B-scans can be performed to find the thickness from the outer sclera to the inner retina, e.g., at 1, 5, and 9 mm posterior to the scleral promontory. The mean, median, and standard deviation for each eye can be calculated.

[0318] 4.6.2 Inflation of the SCS High frequency (50 MHz) ultrasound (E-technologies, Bettendorf, IA) can be used to image the effect of injection and expansion into the SCS in real time. An ultrasound probe can be placed directly over the injection site so that the adjacent sclera, SCS, and ciliary body / choroid can be imaged in real time during injection. Images can be collected and the maximum distance the SCS expands upon injection of PBS or pharmaceutical composition can be measured using an internal ultrasound caliper.

[0319] This technique can also be used to determine the effect of physiological intraocular pressure (IOP) during SCS inflation after injection, and to determine the change in IOP after SCS injection. After the catheter is placed in the SCS, a needle (e.g., 27 gauge) can be placed through the limbus into the anterior chamber, and cyanoacrylate tissue adhesive can be used to seal the limbal incision. The needle can be connected to a pressure transducer (MedEx LogiCal transducer, model MX960; MedExSupply Medical Supplies, Monsey, NY) and electronic monitor using tubing filled with 0.9% saline, allowing continuous measurement of IOP. Maximum inflation of the SCS can be measured and reported using an internal ultrasonic caliper. IOP (in mmHg) at maximum SCS inflation can be recorded for each injection volume.

[0320] 4.6.3 2D ultrasound imaging of SCS Using contrast ultrasound (Mylab70; Biosound Esaote, Inc., Indianapolis, IN), microbubble ultrasound contrast agent (Targestar-P, Targeson Inc., San Diego, CA) can be injected into the anterior suprachoroidal space via the placed cannula. The percentage of maximum distribution of contrast agent in the SCS can be determined in the sagittal ultrasound plane using image analysis software (Elements 4.0 [Adobe Photoshop]; ImageJ 1.42q). Using contrast detection software (Qontrast; Biosound Esaote, Inc., Indianapolis, IN), regions of interest can be placed over the entire SCS and the posterior SCS, and contrast enhancement over time can be measured as mean pixel intensity.

[0321] 4.6.4 Three-dimensional ultrasound imaging of SCS Ex vivo porcine eyes can be imaged using a custom-built 3D imaging system that interfaces a computer-controlled linear motion axis with a clinical ultrasound scanner (Acuson Sequoia; Siemens Medical Solutions, Malvern, PA). A 4 MHz (4-C1) transducer and imaging (Cadence CPS; Siemens Medical Solutions, Malvern, PA) can be used. The porcine eye can be placed in a water bath and microbubble contrast can be injected via a catheter placed into the anterior SCS. Dynamic contrast influx can be observed in the midsagittal plane, followed by 3D imaging of the entire sphere to assess the spatial distribution of contrast. The percentage of maximum distribution of contrast in the SCS can be determined for each section using image analysis software (Elements 4.0 [Adobe Photoshop]; ImageJ 1.42q).

[0322] 4.6.5 Measuring SCS thickness based on liquid volume 3D cryo-reconstruction imaging can be used to measure the thickness of the SCS. For example, the eyes of...

Claims

1. A pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of the eye of a human subject, the pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, the pharmaceutical having an increasing viscosity and / or a higher elastic modulus as temperature increases, the viscosity and / or elastic modulus of the composition, when administered to a porcine eye, being: a. such that the clearance time of the pharmaceutical composition is from about 5 days to about 15 days; b. The thickness of the SCS at the injection site is about 400 μm to about 800 μm within 1 hour of administration; c. The pharmaceutical composition spreads circumferentially from the injection site to about one-eighth or less of the surface of the choroid within about one hour of administration; The viscosity and / or elastic modulus of the pharmaceutical composition.

2. 10. The pharmaceutical composition of claim 1, having a gelling temperature of about 27-32°C.

3. 3. The pharmaceutical composition of claim 1, having a gel time of about 15 to 90 seconds.

4. About 1 sec. －１ ~about 1000s －１ 4. The pharmaceutical composition according to any one of claims 1 to 3, having a viscosity of about 183 mPas at 5°C when measured at a shear rate of 1.

5. About 1 sec. －１ ~1000s －１ 4. The pharmaceutical composition of claim 1, having a viscosity of less than about 183 mPas at 5° C. when measured at a shear rate of 1.

6. About 1 sec. －１ ~1000s －１ 4. The pharmaceutical composition according to any one of claims 1 to 3, having a viscosity of about 183 mPas at 20°C when measured at a shear rate of 1.

7. About 1 sec. －１ ~1000s －１ 4. The pharmaceutical composition of claim 1, having a viscosity of less than about 183 mPas at 20° C. when measured at a shear rate of 1.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the elastic modulus of the pharmaceutical composition provided herein at less than 27°C is less than or about 0.1 Pa, less than or about 0.01 Pa, less than or about 0.001 Pa, or zero.

9. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the elastic modulus of the pharmaceutical composition provided herein at 32°C to 35°C is about or at least about 0.1 Pa, about or at least about 1 Pa, about or at least about 10 Pa, about or at least about 100 Pa, about or at least about 100 Pa, about or at least about 1000 Pa, about or at least about 10,000 Pa, or about or at least about 100,000 Pa.

10. The pharmaceutical composition according to any one of claims 1 to 8, wherein the clearance time after suprachoroidal administration is equal to or greater than the clearance time after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies being the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32 to 35 ° C. than the pharmaceutical composition.

11. The pharmaceutical composition according to any one of claims 1 to 8, wherein the circumferential spread after suprachoroidal administration is smaller than the circumferential spread after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprises a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32 to 35 ° C. than the pharmaceutical composition.

12. 12. The pharmaceutical composition of claim 11, wherein the circumferential spread after suprachoroidal administration is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% smaller.

13. The thickness of the injection site after suprachoroidal administration is equal to or greater than the thickness of the injection site after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies being the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35° C. than the pharmaceutical composition.

14. The pharmaceutical composition of any one of claims 1 to 8, wherein the expression level of the transgene is detected in the eye for a longer period of time after suprachoroidal administration compared to the period of time the expression level of the transgene is detected in the eye after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprises a recombinant AAV comprising an expression cassette encoding the transgene, the amount of recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35 ° C. than the pharmaceutical composition.

15. The concentration of the transgene product in the eye after suprachoroidal administration is equal to or greater than the concentration of the transgene product in the eye after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies being the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35° C. than the pharmaceutical composition; Optionally, the concentration of the transgene product in the back of the eye (e.g., retina) after suprachoroidal administration of the pharmaceutical composition is equal to or greater than the concentration of the transgene product in the back of the eye after suprachoroidal administration of a reference pharmaceutical composition, and / or the concentration of the transgene product in the outer layer of the eye (e.g., sclera) after suprachoroidal administration of the pharmaceutical composition is lower than the concentration of the transgene product in the outer layer of the eye after suprachoroidal administration of a reference pharmaceutical composition.

16. The transduction rate at the injection site after suprachoroidal administration is compared to or is equal to or greater than the transduction rate at the injection site after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies being the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35° C. than the pharmaceutical composition.

17. The level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration is equal to or reduced compared to the level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration of a reference pharmaceutical composition, the reference pharmaceutical composition comprising a recombinant AAV comprising an expression cassette encoding a transgene, the amount of recombinant AAV genome copies being the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and the reference pharmaceutical composition has a lower viscosity and / or a lower elastic modulus at about 32-35 ° C. than the pharmaceutical composition.

18. The pharmaceutical composition according to any one of claims 10, 11 and 13 to 17, wherein the viscosity and / or elastic modulus of the pharmaceutical composition and the viscosity and / or elastic modulus of the reference pharmaceutical composition are measured at the same shear rate.

19. The viscosity and / or elastic modulus of the pharmaceutical composition is at least about 1,000 s －１ , 2,000s －１ , 3,000s －１ , 4,000s －１ , 5,000s －１ , 6,000s －１ , 7,000s －１ , 8,000s －１ , 9,000s －１ , 10,000s －１ , 15,000s －１ , 20,000s －１ , or 30,000s －１ The pharmaceutical composition according to any one of claims 4 to 11 and 13 to 17, measured at a shear rate of

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the recombinant AAV is construct II.

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the transgene is an anti-human vascular endothelial growth factor (anti-VEGF) antibody.

22. The pharmaceutical composition according to any one of claims 1 to 19, wherein the transgene is not an anti-human vascular endothelial growth factor (anti-VEGF) antibody.

23. The recombinant AAV is 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. AAV. PHP. eB, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV.

23. The pharmaceutical composition of any one of claims 1 to 22, comprising components from one or more adeno-associated virus serotypes selected from the group consisting of 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.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the recombinant AAV is AAV8.

25. The pharmaceutical composition according to any one of claims 1 to 19 and 21 to 23, wherein the recombinant AAV is AAV9.

26. 26. The pharmaceutical composition of any one of claims 1 to 25, comprising sucrose.

27. The pharmaceutical composition of any one of claims 1 to 25, which is sucrose free.

28. The pharmaceutical composition according to any one of claims 1 to 27, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% longer.

29. 29. The pharmaceutical composition according to any one of claims 1 to 28, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is about 6 to about 15 days, about 7 to about 15 days, about 8 to about 15 days, about 9 to about 15 days, about 10 to about 15 days, about 11 to about 15 days, about 12 to about 15 days, about 13 to about 15 days, about 14 to about 15 days, about 5 to about 14 days, about 5 to about 13 days, about 5 to about 12 days, about 5 to about 11 days, about 5 to about 10 days, about 5 to about 9 days, about 5 to about 8 days, about 5 to about 7 days, or about 5 to about 6 days.

30. The pharmaceutical composition according to any one of claims 1 to 28, wherein the clearance time following suprachoroidal administration of the pharmaceutical composition is not earlier than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days.

31. The pharmaceutical composition according to any one of claims 1 to 28, wherein the clearance time after suprachoroidal administration of the reference pharmaceutical composition is about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days or less.

32. The pharmaceutical composition according to any one of claims 1 to 31, wherein the clearance time is from the SCS or from the eye.

33. The pharmaceutical composition according to any one of claims 1 to 31, wherein the clearance time is the time required for the pharmaceutical composition and / or recombinant adeno-associated virus (AAV) vector to become undetectable in the SCS by any standard method.

34. The pharmaceutical composition of any one of claims 1 to 31, wherein the clearance time is about 2% or less or about 5% or less of the amount detectable by any standard method when the pharmaceutical composition and / or recombinant adeno-associated virus (AAV) vector is present in the SCS.

35. 32. The pharmaceutical composition of any one of claims 1 to 31, wherein the clearance time is the amount of time after injection required for the thickness at the injection site to decrease to about 500 nm or less, about 200 nm or less, about 100 nm or less, about 50 nm or less, about 25 nm or less, about 10 nm or less, or to become undetectable.

36. 32. The pharmaceutical composition of any one of claims 1 to 31, wherein the clearance time is the amount of time after injection required for the pharmaceutical composition to spread circumferentially from the injection site to cover about one-sixteenth or more, about one-eighth or more, about one-quarter or more, about one-half or more, about three-quarters or more, or all of the circumference of the choroid of the eye.

37. 37. The pharmaceutical composition according to any one of claims 13 and 20 to 36, wherein the thickness of the injection site after suprachoroidal administration of the pharmaceutical composition is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% thicker.

38. The pharmaceutical composition according to any one of claims 13 and 20 to 36, wherein the thickness of the injection site after suprachoroidal administration of the pharmaceutical composition is from about 500 μm to about 3.0 mm, from 750 μm to about 2.8 mm, from about 750 μm to about 2.5 mm, from about 750 μm to about 2 mm, or from about 1 mm to about 2 mm.

39. 37. The pharmaceutical composition according to any one of claims 13 and 20 to 36, wherein the thickness of the injection site after suprachoroidal administration of the pharmaceutical composition is at least about 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm thick.

40. The pharmaceutical composition according to any one of claims 13 and 20 to 36, wherein the thickness at the injection site after suprachoroidal administration of the reference pharmaceutical composition is about 1 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm or less.

41. The thickness of the SCS at the injection site is about 400 μm to about 700 μm, about 400 μm to about 600 μm, about 400 μm to about 500 μm, about 500 μm to about 800 μm, about 600 μm to about 800 μm, or 700 μm to about 800 μm within 1 hour of administration. The pharmaceutical composition of any one of claims 1 to 40.

42. 42. The pharmaceutical composition of claim 41, wherein the time within 1 hour of administration is within about 5 minutes of administration, within about 10 minutes of administration, within about 15 minutes of administration, within about 20 minutes of administration, within about 30 minutes of administration, within about 45 minutes of administration, or within about 60 minutes of administration.

43. 43. The pharmaceutical composition according to any one of claims 13 and 20 to 42, wherein the thickness of the injection site following suprachoroidal administration of the pharmaceutical composition lasts for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 2 days, at least 3 days, at least 5 days, at least 10 days, at least 21 days, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 3 years, or at least 5 years.

44. 44. The pharmaceutical composition of any one of claims 1 to 43, wherein the pharmaceutical composition spreads circumferentially from the injection site to about one-eighth or less of the surface of the choroid within about 5 minutes after administration, within about 10 minutes after administration, within about 15 minutes after administration, within about 20 minutes after administration, within about 30 minutes after administration, within about 45 minutes after administration, or within about 60 minutes after administration.

45. 45. The pharmaceutical composition of claim 44, wherein the circumferential extent from the injection site is about 1 / 16 or less of the surface of the choroid.

46. 46. ​​The pharmaceutical composition of any one of claims 15 and 20 to 45, wherein the concentration of the transgene in the eye following suprachoroidal administration of the pharmaceutical composition is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% greater.

47. The longer period of time following suprachoroidal administration of the pharmaceutical composition is at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 47. The pharmaceutical composition of any one of claims 14 and 20-46, wherein the period is longer than 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

48. The transgene is administered for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 130 days, 140 days, 150 days, 160 days, 170 days, 180 days, 190 days, 200 days, 210 days, 230 ...

48. The pharmaceutical composition of any one of claims 1 to 47, wherein the anti-inflammatory effect is visually detectable for 1, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 days.

49. The transgene is administered for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days after suprachoroidal administration of the reference pharmaceutical composition.

49. The pharmaceutical composition of any one of claims 10 to 48, wherein the composition is visually detectable after no more than 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, or 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

50. 22. The pharmaceutical composition of claim 21, wherein the level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration of the pharmaceutical composition is equal to or reduced compared to the level of VEGF-induced vasodilation and / or vascular leakage after suprachoroidal administration of a reference pharmaceutical composition.

51. The level of VEGF-induced vasodilation and / or vascular leakage following suprachoroidal administration of the pharmaceutical composition is reduced by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold, or by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 51. The pharmaceutical composition of any one of claims 17 to 50, wherein the amount of IL-16 in the IL-16 antibody is reduced by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500%.

52. 52. The pharmaceutical composition according to any one of claims 16 and 20 to 51, wherein the transduction rate at the injection site following suprachoroidal administration of the pharmaceutical composition is at least about 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500% greater.

53. 53. The pharmaceutical composition of any one of claims 1 to 52, wherein the stability of the recombinant AAV in the pharmaceutical composition is higher compared to the stability of the recombinant AAV in a reference pharmaceutical composition.

54. The pharmaceutical composition of claim 53, wherein the stability of the recombinant AAV is determined by the infectivity of the recombinant AAV.

55. The pharmaceutical composition of claim 53, wherein the stability of the recombinant AAV is determined by the level of aggregation of the recombinant AAV.

56. The pharmaceutical composition of claim 53, wherein the stability of the recombinant AAV is determined by the level of free DNA released by the recombinant AAV.

57. 57. The pharmaceutical composition of claim 56, wherein the pharmaceutical composition contains about 50% more, about 25% more, about 15% more, about 10% more, about 5% more, about 4% more, about 3% more, about 2% more, about 1% more, about 0% more, about 1% less, about 2% less, about 5% less, about 7% less, about 10% less, about 2 times more, about 3 times more, about half, or about a third less free DNA compared to the level of free DNA in a reference pharmaceutical composition.

58. 55. The pharmaceutical composition of claim 54, wherein the recombinant AAV in the pharmaceutical composition has 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 greater infectivity compared to the infectivity of the recombinant AAV in a reference pharmaceutical composition.

59. 56. The pharmaceutical composition of claim 55, comprising at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 100% less, or at least half-, third-, fifth-, tenth-, hundredth-, or thousandth-fold less recombinant AAV aggregates compared to the level of recombinant AAV aggregates in a reference pharmaceutical composition.

60. 60. The pharmaceutical composition of any one of claims 1 to 59, wherein the transgene is a transgene suitable for treating or otherwise alleviating, preventing or slowing the progression of a disease of interest.

61. 61. The pharmaceutical composition of any one of claims 1 to 60, wherein the human subject has been diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), glaucoma, non-infectious uveitis, x-linked disease, or Batten disease.

62. The pharmaceutical composition of any one of claims 1 to 60, wherein the human subject has been diagnosed with glaucoma or non-infectious uveitis.

63. 63. The pharmaceutical composition of any one of claims 1 to 19 and 23 to 62, wherein the AAV encodes palmitoyl protein thioesterase 1 (PPT1), tripeptidyl-peptidase 1 (TPP1), an anti-VEGF fusion protein, an anti-VEGF antibody or antigen-binding fragment thereof, an anti-kallikrein antibody or antigen-binding fragment, an anti-TNF fusion protein, an anti-TNF antibody or antigen-binding fragment, an anti-C3 antibody or antigen-binding fragment, or an anti-C5 antibody or antigen-binding fragment.

64. 64. The pharmaceutical composition of any one of claims 1 to 63, wherein the amount of recombinant AAV genome copies is based on the vector genome concentration.

65. 64. The pharmaceutical composition of any one of claims 1 to 63, wherein the amount of recombinant AAV genome copies is based on genome copies per administration.

66. 64. The pharmaceutical composition of any one of claims 1 to 63, wherein the amount of recombinant AAV genome copies is based on the total genome copies administered to a human subject.

67. 66. The pharmaceutical composition of claim 65, wherein the genome copies per administration are genome copies of the recombinant AAV per suprachoroidal administration.

68. 67. The pharmaceutical composition of claim 66, wherein the total genome copies administered are total genome copies of a recombinant AAV administered to the suprachoroid.

69. ベクターゲノム concentration (VGC)が, about 3×10 ９ GC / mL, about 1×10 １０ GC / mL, about 1.2×10 １０ GC / mL, about 1.6×10 １０ GC / mL, about 4×10 １０ GC / mL, about 6×10 １０ GC / mL, about 2×10 １１ GC / mL, about 2.4×10 １１ GC / mL, about 2.5×10 １１ GC / mL, about 3×10 １１ GC / mL, about 6.2×10 １１ GC / mL, about 1×10 １２ GC / mL, about 2.5×10 １２ GC / mL, about 3×10 １２ GC / mL, about 5×10 １２ GC / mL, about 6×10 １２ GC / mL, about 1.5×10 １３ GC / mL, about 2×10 １３ GC / mL, about 3×10 １３ GC / mLのVGCである, the medicinal composition described in claim 64.

70. The total number of genome copies administered is approximately 6.0 × 10 １０ Genome copies, approximately 1.6 x 10 １１ Genome copies, approximately 2.5 x 10 １１ Genome copies: approximately 3 x 10 １１ Genome copies, approximately 5.0 x 10 １１ Genome copies: approximately 6 x 10 １１ Genome copies: approximately 3 x 10 １２ Genome copies, approximately 1.0 x 10 １２ Genome copies, approximately 1.5 x 10 １２ Genome copies, approximately 2.5 x 10 １２ Genome copies, or approximately 3.0 x 10 １３ 69. The pharmaceutical composition of any one of claims 66 and 68, which is a genomic copy.

71. The total number of genome copies administered is approximately 6.0 × 10 １０ Genome copies, approximately 1.6 x 10 １１ Genome copies, approximately 2.5 x 10 １１ Genome copies: approximately 3 x 10 １１ Genome copies, approximately 5.0 x 10 １１ Genome copies: approximately 6 x 10 １１ Genome copies: approximately 3 x 10 １２ Genome copies, approximately 1.0 x 10 １２ Genome copies, approximately 1.5 x 10 １２ Genome copies, approximately 2.5 x 10 １２ Genome copies, or approximately 3.0 x 10 １３ 68. The pharmaceutical composition of any one of claims 65 and 67, which is a genomic copy.

72. 72. The pharmaceutical composition of any one of claims 1 to 71, which is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 times.

73. 73. The pharmaceutical composition of any one of claims 10 to 72, wherein the reference pharmaceutical composition is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 times.

74. 74. The pharmaceutical composition of any one of claims 1 to 73, which is administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, or seven times a day.

75. 74. The pharmaceutical composition of any one of claims 10 to 73, wherein the reference pharmaceutical composition is administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, or seven times a day.

76. 76. The pharmaceutical composition according to any one of claims 1 to 75, comprising poloxamer 407 and poloxamer 188.

77. 77. A pharmaceutical composition according to any one of claims 1 to 76, comprising 16-22% poloxamer 407.

78. 77. A pharmaceutical composition according to any one of claims 1 to 76, comprising 0-16% poloxamer 188.

79. 77. The pharmaceutical composition of any one of claims 1 to 76, comprising 19% poloxamer 407 and 6% poloxamer 188.

80. 77. A pharmaceutical composition according to any one of claims 1 to 76, comprising 18% poloxamer 407 and 6.5% poloxamer 188.

81. 77. A pharmaceutical composition according to any one of claims 1 to 76, comprising 17.5% poloxamer 407 and 7% poloxamer 188.

82. 82. A pharmaceutical composition according to any one of claims 1 to 81, comprising modified Dulbecco's phosphate buffered saline solution, optionally comprising a surfactant.

83. 82. The pharmaceutical composition of any one of claims 1 to 81 comprising 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium dihydrogen phosphate, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL sucrose (4% w / v), and optionally a surfactant.

84. 82. The pharmaceutical composition of any one of claims 1 to 81, comprising potassium chloride, potassium dihydrogen phosphate, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant.

85. 82. A method of treating a disease in a subject, comprising administering a pharmaceutical composition according to any one of claims 1 to 81.

86. 10. A method of treating a disease in a subject, comprising administering to the subject a pharmaceutical composition according to claim 4 or 5, wherein the pharmaceutical composition is at a temperature of about 2-10°C when administered.

87. 10. A method of treating a disease in a subject, comprising administering to the subject a pharmaceutical composition according to claim 6 or 8, wherein the pharmaceutical composition is at a temperature of about 20-25°C when administered.

88. 88. The method of any one of claims 85 to 87, wherein the pharmaceutical composition is administered at an injection pressure of less than about 43 PSI.

89. 88. The method of any one of claims 85 to 87, wherein the pharmaceutical composition is administered at an injection pressure of less than about 65 PSI.

90. 88. The method of any one of claims 85 to 87, wherein the pharmaceutical composition is administered at an injection pressure of less than about 100 PSI.

91. 91. The method of any one of claims 85 to 90, wherein the pharmaceutical composition is administered using a 29 gauge needle.

92. 91. The method of any one of claims 85 to 90, wherein the pharmaceutical composition is administered using a 30 gauge needle.

93. 93. The method of any one of claims 85 to 92, wherein the pharmaceutical composition is administered with an injection time of about 10 to 15 seconds.

94. 93. The method of any one of claims 85 to 92, wherein the pharmaceutical composition is administered with an injection time of about 5 to 30 seconds.

95. The method of any one of claims 85 to 94, wherein the subject is a human.

96. 96. The method of any one of claims 85 to 95, wherein the disease is selected from the group consisting of nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), Batten disease, glaucoma, and non-infectious uveitis.

97. 82. The pharmaceutical composition of any one of claims 1 to 81, comprising 18.0% w / v of poloxamer 407 and 6.5% w / v of poloxamer 188 in a solution containing 5.84 mg / mL sodium chloride, 0.201 mg / mL potassium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 0.200 mg / mL potassium phosphate dibasic, 40.0 mg / mL (4% w / v) sucrose, 0.001% (0.01 mg / mL) poloxamer 188, at pH 7.

4.

98. A pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of the eye of a human subject, comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, comprising 5.84 mg / mL sodium chloride, 0.201 mg / mL potassium chloride, 1.15 mg / mL anhydrous dibasic sodium phosphate, 0.200 mg / mL potassium dihydrogen phosphate, 40.0 mg / mL (4% w / v) sucrose, 18.0% w / v poloxamer 407 and 6.5% w / v poloxamer 188 in a solution of pH 7.4 containing 0.001% (0.01 mg / mL) poloxamer 188.

99. 99. The pharmaceutical composition of any one of claims 1 to 81, 97 or 98, wherein the expression cassette has a sequence comprising SEQ ID NO:

56.

100. 100. A method of preparing the pharmaceutical composition of any one of claims 97 to 99, comprising the steps of: (a) providing a solution containing 5.84 mg / mL sodium chloride, 0.201 mg / mL potassium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 0.200 mg / mL potassium phosphate dibasic, 40.0 mg / mL sucrose (4% w / v), 0.001% poloxamer 188, at a pH of 7.4; and (b) mixing with the solution 18.0% w / v poloxamer 407 and 6.5% w / v poloxamer 188.

101. 101. The method of claim 100, wherein after the mixing step, the pH of the composition is from about pH 6.0 to about pH 7.9.

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