WO2023196835A1 - Formulations pour administration suprachoroïdienne, telles que formulations de gel - Google Patents

Formulations pour administration suprachoroïdienne, telles que formulations de gel Download PDF

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Publication number
WO2023196835A1
WO2023196835A1 PCT/US2023/065358 US2023065358W WO2023196835A1 WO 2023196835 A1 WO2023196835 A1 WO 2023196835A1 US 2023065358 W US2023065358 W US 2023065358W WO 2023196835 A1 WO2023196835 A1 WO 2023196835A1
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WIPO (PCT)
Prior art keywords
days
pharmaceutical composition
aav
hours
administration
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PCT/US2023/065358
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English (en)
Inventor
Jared BEE
Tristan James MARSHALL
Sherri VAN EVEREN
Stephen Joseph Pakola
Nicholas Alexander Piers Sascha BUSS
Anthony Ray O'berry
Jesse I. Yoo
Ewa BUDZYNSKI
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Regenxbio Inc.
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Publication of WO2023196835A1 publication Critical patent/WO2023196835A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/10—Ophthalmic agents for accommodation disorders, e.g. myopia

Definitions

  • the human eye is a highly intricate and highly developed sensory organ, which is prone to a host of diseases and disorders. About 285 million people in the world are visually impaired, of whom 39 million are blind and 246 million have moderate to severe visual impairment (World Health Organization, 2012, “Global Data On Visual Impairments 2010,” Geneva: World Health Organization). Some of the leading causes of blindness are cataract (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).
  • Adeno-associated viruses are an attractive tool for gene therapy due to properties of non- pathogenicity, broad host and cell type tropism range of infectivity, including both dividing and non-dividing cells, and ability to establish long-term transgene expression (e.g., Gonçalves, 2005, Virology Journal, 2:43).
  • Adeno-associated virus a member of the Parvoviridae family designated Dependovirus, is a small nonenveloped, icosahedral virus with single-stranded linear DNA genomes of approximately 4.7 -kilobases (kb) to 6 kb.
  • the suprachoroidal space is a region between the sclera and the choroid that expands upon injection of the drug solution (Habot-Wilner, 2019).
  • the SCS space recovers to its pre-injection size as the injected solution is cleared by physiologic processes.
  • the drug solution diffuses within SCS and is absorbed into adjacent tissues.
  • Capillaries in the choroid are permeable to low molecular weight osmolytes.
  • a pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, and wherein the pharmaceutical composition has a viscosity and/or elastic modulus such that when administered to an eye of a pig: the clearance time of the pharmaceutical composition is between about 5 days and about 15 days; and the thickness of the SCS at the site of injection is between about 400 ⁇ m and about 800 ⁇ m at a time within one hour of administration; and the circumferential spread of the pharmaceutical composition from the site of injection is about one-eighth or less of a surface of the choroid at a time within about one hour of administration
  • the composition has a gelation temperature of about 27-32°C. In some embodiments, the composition has a gelation time of about 15-90 seconds. In some embodiments, the composition has a viscosity of about 183 mPas at 5°C as measured at a shear rate of about 1s -1 to about 1000s -1 . In some embodiments, the composition has a viscosity of less than about 183 mPas at 5°C as measured at a shear rate of about 1s -1 to 1000 s -1 .
  • the composition has a viscosity of about 183 mPas at 20°C as measured at a shear rate of at about 1s -1 to 1000 s -1 . In some embodiments, the composition has a viscosity of less than about 183 mPas at 20°C as measured at a shear rate of at about 1s -1 to 1000 s -1 [0010] In some embodiments, the elastic modulus of a pharmaceutical composition provided herein at under 27°C is less than about or about 0.1 Pa, less than about or about 0.01 Pa, less than about or about 0.001 Pa or zero.
  • the elastic modulus of a pharmaceutical composition 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 100,000 Pa..
  • the clearance time after suprachoroidal administration is equal to or greater than the clearance time of a reference pharmaceutical composition after suprachoroidal administration, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the reference pharmaceutical composition has a lower viscosity and/or lower elastic modulus than the pharmaceutical composition at about 32-35°C.
  • the clearance time is from the SCS or from the eye.
  • the clearance time is the time required for the pharmaceutical composition and/or the recombinant adeno-associated virus (AAV) vector to not be detectable in the SCS by any standard method. In some embodiments, the clearance time when the pharmaceutical composition and/or the recombinant adeno-associated virus (AAV) vector is present in the SCS in an amount that is at most about 2% or at most about 5% of the amount detectable by any standard method.
  • the clearance time is the amount of time required following injection for the thickness at the site of injection to decrease 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 required following injection for the thickness at the site of injection 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 is undetectable.
  • the clearance time is the amount of time required following injection for the pharmaceutical composition to spread circumferentially from the site of injection to cover about one-sixteenth or more, about one-eighth or more, about one- fourth or more, about one-half or more, about three-fourths or more, or all of the circumference of the choroid of the eye.
  • a circumferential spread after suprachoroidal administration is smaller as compared to a circumferential spread of a reference pharmaceutical composition after suprachoroidal administration, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the reference pharmaceutical composition has a lower viscosity and/or lower elastic modulus than the pharmaceutical composition at about 32-35°C.
  • the circumferential spread after suprachoroidal administration is smaller by 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 by at least 500%.
  • a thickness at a site of injection after suprachoroidal administration is equal to or higher as compared to a thickness at a site of injection after suprachoroidal administration of a reference pharmaceutical composition
  • the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the reference pharmaceutical composition has a lower viscosity and/or lower elastic modulus than the pharmaceutical composition at about 32-35°C.
  • an expression level of the transgene is detected in the eye for a longer period of time after suprachoroidal administration as compared to a period of time that an expression level of the transgene is detected in the eye after suprachoroidal administration of a reference pharmaceutical composition
  • the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the reference pharmaceutical composition has a lower viscosity and/or lower elastic modulus than the pharmaceutical composition at about 32-35°C.
  • the concentration of the transgene product in the eye after suprachoroidal administration is equal to or higher as compared to the concentration of the transgene product in the eye after suprachoroidal administration of a reference pharmaceutical composition
  • the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the reference pharmaceutical composition has a lower viscosity and/or lower elastic modulus than the pharmaceutical composition at about 32-35°C.
  • 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 higher as compared to 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 disclosed herein.
  • the rate of transduction at a site of injection after suprachoroidal administration is equal to or higher as compared to the rate of transduction at a site of injection after suprachoroidal administration of a reference pharmaceutical composition
  • the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the reference pharmaceutical composition has a lower viscosity and/or lower elastic modulus than the pharmaceutical composition at about 32-35°C.
  • the viscosity and/or elastic modulus of the pharmaceutical composition and the viscosity and/or elastic modulus of the reference pharmaceutical composition is measured at the same shear rate. In some embodiments, the viscosity and/or elastic modulus of the pharmaceutical composition is measured at a shear rate of at least about 1,000 s-1, 2,000 s-1, 3,000 s-1, 4,000 s-1, 5,000 s-1, 6,000 s-1, 7,000 s-1, 8,000 s-1, 9,000 s-1, 10,000 s-1, 15,000 s-1, 20,000 s-1, or 30,000 s-1.
  • the transgene is not an anti-human vascular endothelial growth factor (anti-VEGF) antibody.
  • the recombinant AAV comprises components from one or more adeno-associated virus serotypes selected from the group consisting of AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, 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, A
  • the recombinant AAV is AAV8. In some embodiments, the recombinant AAV is AAV9. [0020] In some embodiments, the pharmaceutical composition comprises sucrose. In some embodiments, the pharmaceutical composition does not comprise sucrose.
  • the clearance time after suprachoroidal administration of the pharmaceutical composition is greater by 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%.
  • the clearance time after suprachoroidal administration of the pharmaceutical composition is of about 6 days to about 15 days days, about 7 days to about 15 days, about 8 days to about 15 days, about 9 days to about 15 days, about 10 days to about 15 days, about 11 days to about 15 days, about 12 days to about 15 days, about 13 days to about 15 days, about 14 days to about 15 days, about 5 days to about 14 days, about 5 days to about 13 days, about 5 days to about 12 days, about 5 days to about 11 days, about 5 days to about 10 days, about 5 days to about 9 days, about 5 days to about 8 days, about 5 days to about 7 days, or about 5 days to about 6 days.
  • the clearance time after suprachoroidal administration of the pharmaceutical composition is not prior to about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.
  • the clearance time of the reference pharmaceutical composition after suprachoroidal administration is of at most 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.
  • the clearance time is from the SCS or from the eye.
  • the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is higher by 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 by at least 500%.
  • the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is about 500 ⁇ m to about 3.0 mm, 750 ⁇ m to about 2.8 mm, about 750 ⁇ m to about 2.5 mm, about 750 ⁇ m to about 2 mm, or about 1 mm to about 2 mm.
  • the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is of at least about 50 ⁇ m, 100 ⁇ m, 200 ⁇ m, 300 ⁇ m, 400 ⁇ m, 500 ⁇ m, 600 ⁇ m, 700 ⁇ m, 800 ⁇ m, 900 ⁇ m, 1000 ⁇ m, 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.
  • the thickness at the site of injection after the suprachoroidal administration of the reference pharmaceutical composition is of at most 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 ⁇ m, 5 ⁇ m, 10 ⁇ m, 15 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 35 ⁇ m, 40 ⁇ m, 50 ⁇ m, 100 ⁇ m, 200 ⁇ m, 300 ⁇ m, 400 ⁇ m, 500 ⁇ m, 600 ⁇ m, 700 ⁇ m, 800 ⁇ m, 900 ⁇ m, or 1000 ⁇ m.
  • the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition persists for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, at least twenty-four hours, at least two days, at least three days, at least five days, at least ten days, at least twenty-one days, at least one month, at least six weeks, at least two months, at least three months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least one year, at least three years, or at least five years.
  • the thickness of the SCS at the site of injection is about 400 ⁇ m to about 700 ⁇ m, about 400 ⁇ m to about 600 ⁇ m, about 400 ⁇ m to about 500 ⁇ m, about 500 ⁇ m to about 800 ⁇ m, about 600 ⁇ m to about 800 ⁇ m, 700 ⁇ m to about 800 ⁇ m at a time within one hour of administration.
  • the time within one 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.
  • the circumferential spread of the pharmaceutical composition from the site of injection is about one-eighth or less of a surface of the choroid at a time 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. In some embodiments, the circumferential spread from the site of injection is about one-sixteenth or less of a surface of the choroid.
  • the concentration of the transgene in the eye after suprachoroidal administration of the pharmaceutical composition is higher by 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 by at least 500%.
  • the transgene is detected in the eye after suprachoroidal administration of the pharmaceutical composition 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.
  • the transgene is detected in the eye after suprachoroidal administration of the reference pharmaceutical composition for at most 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 after.
  • the longer period of time after suprachoroidal administration of the pharmaceutical composition is longer by 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, 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.
  • the rate of transduction at the site of injection after suprachoroidal administration of the pharmaceutical composition is higher by at least about 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 by at least 500%.
  • the recombinant AAV stability is higher in the pharmaceutical composition as compared to the recombinant AAV stability in the reference pharmaceutical composition. In some embodiments, the recombinant AAV stability is determined by infectivity of the recombinant AAV. In some embodiments, the recombinant AAV stability is determined by a level of aggregation of the recombinant AAV. In some embodiments, the recombinant AAV stability is determined by a level of free DNA released by the recombinant AAV.
  • 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 2 times less, about 3 times less, free DNA as compared to a level of free DNA in the reference pharmaceutical composition.
  • the recombinant AAV in the pharmaceutical composition has an infectivity that is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher as compared to the infectivity of the recombinant AAV in the reference pharmaceutical composition.
  • the pharmaceutical composition comprises at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times less recombinant AAV aggregation as compared to a level of the recombinant AAV aggregation in the reference pharmaceutical composition.
  • the transgene is a transgene suitable to treat, or otherwise ameliorate, prevent or slow the progression of a disease of interest.
  • a human subject is diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), x-linked or Batten disease.
  • the human subject is diagnosed with glaucoma, non-infectious uveitis, or kallikrein-related disease.
  • the AAV encodes Palmitoyl-Protein Thioesterase 1 (PPT1) or Tripeptidyl-Peptidase 1 (TPP1).
  • the AAV encodes anti-kallikrein antibody or antigen-binding fragment, anti-TNF fusion protein, anti-TNF antibody or antigen-binding fragment, anti-C3 antibody or antigen-binding fragment, or anti-C5 antibody or antigen-binding fragment.
  • the amount of the recombinant AAV genome copies is based on a vector genome concentration. In some embodiments, the amount of the recombinant AAV genome copies is based on genome copies per administration. In some embodiments, the amount of the recombinant AAV genome copies is based on total genome copies administered to the human subject. In some embodiments, the genome copies per administration is the genome copies of the recombinant AAV per suprachoroidal administration.
  • the total genome copies administered is the total genome copies of the recombinant AAV administered suprachoroidally.
  • the vector genome concentration (VGC) is of 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/m
  • the total genome copies administered is about 6.0 ⁇ 10 10 genome copies, about 1.6 ⁇ 10 11 genome copies, about 2.5 ⁇ 10 11 genome copies, about 3.0 ⁇ 10 11 genome copies, about 5.0 ⁇ 10 11 genome copies, about 6.0 ⁇ 10 11 genome copies, about 1.5 ⁇ 10 12 genome copies, about 3 ⁇ 10 12 genome copies, about 1.0 ⁇ 10 12 GC/mL, about 2.5 ⁇ 10 12 GC/mL, or about 3.0 ⁇ 10 13 genome copies.
  • the total genome copies administered is about 6.0 ⁇ 10 10 genome copies, about 1.6 ⁇ 10 11 genome copies, about 2.5 ⁇ 10 11 genome copies, about 5.0 ⁇ 10 11 genome copies, about 1.5 ⁇ 10 12 genome copies, about 3 ⁇ 10 12 genome copies, about 1.0 ⁇ 10 12 genome copies, about 2.5 ⁇ 10 12 genome copies, or about 3.0 ⁇ 10 13 genome copies.
  • the pharmaceutical composition is administered 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.
  • the reference pharmaceutical composition is administered 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. In some embodiments, the pharmaceutical composition is administered once in one day, twice in one day, three times in one day, four times in one day, five times in one day, six times in one day, or seven times in one day. In some embodiments, the reference pharmaceutical composition is administered once in one day, twice in one day, three times in one day, four times in one day, five times in one day, six times in one day, or seven times in one day. [0037] In some embodiments, the pharmaceutical composition contains poloxamer 407 and poloxamer 188.
  • the composition comprises 16-22% poloxamer 407. In some embodiments, the composition comprises 0-16% poloxamer 188. In some embodiments, the composition comprises 19% poloxamer 407 and 6% poloxamer 188. In some embodiments, the composition comprises 18% poloxamer 407 and 6.5% poloxamer 188. In some embodiments, the composition comprises 17.5% poloxamer 407 and 7% poloxamer 188 [0038] In some embodiments, the composition comprises modified Dulbecco’s phosphate- buffered saline solution, and optionally a surfactant.
  • the pharmaceutical composition comprises 0.2 mg/mL potassium chloride, 0.2 mg/mL potassium phosphate monobasic, 5.84 mg/mL sodium chloride, 1.15 mg/mL sodium phosphate dibasic anhydrous, 40.0 mg/mL (4% w/v) sucrose, and optionally a surfactant.
  • the composition comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant.
  • the composition comprises 18.0% w/v poloxamer 407 and 6.5% w/v poloxamer 188 admixed with a solution 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 monobasic, 40.0 mg/mL (4% w/v) sucrose, 0.001% (0.01 mg/mL) poloxamer 188, pH 7.4.
  • the composition comprises 18.0% w/v poloxamer 407 and 6.5% w/v poloxamer 188 in a solution 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 monobasic, 40.0 mg/mL (4% w/v) sucrose, 0.001% (0.01 mg/mL) poloxamer 188, pH 7.4.
  • a pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, wherein the pharmaceutical composition comprises 18.0% w/v poloxamer 407 and 6.5% w/v poloxamer 188 in a solution 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 monobasic, 40.0 mg/mL (4% w/v) sucrose, 0.001% (0.01 mg/mL) poloxamer 188, pH 7.4.
  • AAV adeno-associated virus
  • a method of treating a disease in a subject comprising administering a pharmaceutical composition provided herein to the subject.
  • the pharmaceutical composition is at a temperature of about 2-10°C when being administered. In some embodiments, the pharmaceutical composition is at a temperature of about 20-25°C when being administered.
  • the pharmaceutical composition is administered with an injection pressure of less than about 43 PSI. In some embodiments, the pharmaceutical composition is administered with an injection pressure of less than about 65 PSI. In some embodiments, the pharmaceutical composition is administered with an injection pressure of less than about 100 PSI.
  • the pharmaceutical composition is administered using a 29 gauge needle.
  • the pharmaceutical composition is administered using a 30 gauge needle. [0043] In some embodiments, the pharmaceutical composition is administered in an injection time of about 10-15 seconds. In some embodiments, the pharmaceutical composition is administered in an injection time of about 5-30 seconds. [0044] In some embodiments, the subject is human. [0045] 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, non-infectious uveitis, and kallikrein-related disease.
  • nAMD wet AMD
  • RVO retinal vein occlusion
  • DME diabetic macular edema
  • DR diabetic retinopathy
  • Batten disease glaucoma
  • non-infectious uveitis non-infectious uveitis
  • kallikrein-related disease 2.1 Illustrative Em
  • a pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, and wherein the pharmaceutical has a viscosity and/or higher elastic modulus that increases with increasing temperature, and wherein the viscosity and/or elastic modulus of the composition is such that when administered to an eye of a pig: a. the clearance time of the pharmaceutical composition is between about 5 days and about 15 days; and b. the thickness of the SCS at the site of injection is between about 400 ⁇ m and about 800 ⁇ m at a time within one hour of administration; and c.
  • AAV adeno-associated virus
  • the circumferential spread of the pharmaceutical composition from the site of injection is about one-eighth or less of a surface of the choroid at a time within about one hour of administration.
  • the pharmaceutical composition of paragraph 1 wherein the composition has a gelation temperature of about 27-32°C.
  • the pharmaceutical composition of paragraph 1 or 2 wherein the composition has a gelation time of about 15-90 seconds.
  • 4. The pharmaceutical composition of any one of paragraphs 1-3, wherein the composition has a viscosity of about 183 mPas at 5°C as measured at a shear rate of about 1s -1 to about 1000s -1 . 5.
  • compositions of any one of paragraphs 1-3 wherein the composition has a viscosity of less than about 183 mPas at 5°C as measured at a shear rate of about 1s -1 to 1000 s -1 .
  • the elastic modulus of a pharmaceutical composition provided herein at under 27°C is less than about or about 0.1 Pa, less than about or about 0.01 Pa, less than about or about 0.001 Pa or zero.
  • the elastic modulus of a 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 10,000 Pa or about or at least about 100,000 Pa. 10.
  • a circumferential spread after suprachoroidal administration is smaller as compared to a circumferential spread of a reference pharmaceutical composition after suprachoroidal administration
  • the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the reference pharmaceutical composition has a lower viscosity and/or lower elastic modulus than the pharmaceutical composition at about 32-35°C. 12.
  • a thickness at a site of injection after suprachoroidal administration is equal to or higher as compared to a thickness at a site of injection after suprachoroidal administration of a reference pharmaceutical composition
  • the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the reference pharmaceutical composition has a lower viscosity and/or lower elastic modulus than the pharmaceutical composition at about 32-35°C.
  • the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the reference pharmaceutical composition has a lower viscosity and/or lower elastic modulus than the pharmaceutical composition at about 32-35°C.
  • the viscosity and/or elastic modulus of the pharmaceutical composition is measured at a shear rate of at least about 1,000 s -1 , 2,000 s -1 , 3,000 s -1 , 4,000 s -1 , 5,000 s -1 , 6,000 s -1 , 7,000 s -1 , 8,000 s -1 , 9,000 s -1 , 10,000 s -1 , 15,000 s -1 , 20,000 s -1 , or 30,000 s -1 .
  • the transgene is not an anti-human vascular endothelial growth factor (anti-VEGF) antibody.
  • the recombinant AAV comprises components from one or more adeno-associated virus serotypes selected from the group consisting of AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, 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
  • the pharmaceutical composition of any one of paragraphs 1-25, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is of about 6 days to about 15 days days, about 7 days to about 15 days, about 8 days to about 15 days, about 9 days to about 15 days, about 10 days to about 15 days, about 11 days to about 15 days, about 12 days to about 15 days, about 13 days to about 15 days, about 14 days to about 15 days, about 5 days to about 14 days, about 5 days to about 13 days, about 5 days to about 12 days, about 5 days to about 11 days, about 5 days to about 10 days, about 5 days to about 9 days, about 5 days to about 8 days, about 5 days to about 7 days, or about 5 days to about 6 days. 27.
  • the clearance time is the amount of time required following injection for the thickness at the site of injection 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 is undetectable.
  • clearance time is the amount of time required following injection for the pharmaceutical composition to spread circumferentially from the site of injection to cover about one-sixteenth or more, about one- eighth or more, about one-fourth or more, about one-half or more, about three-fourths or more, or all of the circumference of the choroid of the eye.
  • 35 The pharmaceutical composition of any one of paragraphs 13 and 19-33, wherein the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is about 500 ⁇ m to about 3.0 mm, 750 ⁇ m to about 2.8 mm, about 750 ⁇ m to about 2.5 mm, about 750 ⁇ m to about 2 mm, or about 1 mm to about 2 mm. 36.
  • the pharmaceutical composition of any one of paragraphs 13 and 19-33, wherein the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is of at least about 50 ⁇ m, 100 ⁇ m, 200 ⁇ m, 300 ⁇ m, 400 ⁇ m, 500 ⁇ m, 600 ⁇ m, 700 ⁇ m, 800 ⁇ m, 900 ⁇ m, 1000 ⁇ m, 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. 37.
  • the pharmaceutical composition of any one of paragraphs 13 and 19-33, wherein the thickness at the site of injection after the suprachoroidal administration of the reference pharmaceutical composition is of at most 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 ⁇ m, 5 ⁇ m, 10 ⁇ m, 15 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 35 ⁇ m, 40 ⁇ m, 50 ⁇ m, 100 ⁇ m, 200 ⁇ m, 300 ⁇ m, 400 ⁇ m, 500 ⁇ m, 600 ⁇ m, 700 ⁇ m, 800 ⁇ m, 900 ⁇ m, or 1000 ⁇ m.
  • any one of paragraphs 1-37 wherein the thickness of the SCS at the site of injection is about 400 ⁇ m to about 700 ⁇ m, about 400 ⁇ m to about 600 ⁇ m, about 400 ⁇ m to about 500 ⁇ m, about 500 ⁇ m to about 800 ⁇ m, about 600 ⁇ m to about 800 ⁇ m, 700 ⁇ m to about 800 ⁇ m at a time within one hour of administration.
  • 39. The pharmaceutical composition of paragraph 38, wherein the time within one 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. 40.
  • compositions of any one of paragraphs 15 and 19-42 wherein the concentration of the transgene in the eye after suprachoroidal administration of the pharmaceutical composition is higher by 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 by at least 500%.
  • the pharmaceutical composition of paragraph 51 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 2 times more, about 3 times more, about 2 times less, about 3 times less, free DNA as compared to a level of free DNA in the reference pharmaceutical composition. 53.
  • the pharmaceutical composition of paragraph 49 wherein the recombinant AAV in the pharmaceutical composition has an infectivity that is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher as compared to the infectivity of the recombinant AAV in the reference pharmaceutical composition. 54.
  • the pharmaceutical composition of paragraph 50 wherein the pharmaceutical composition comprises at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times less recombinant AAV aggregation as compared to a level of the recombinant AAV aggregation in the reference pharmaceutical composition.
  • the transgene is a transgene suitable to treat, or otherwise ameliorate, prevent or slow the progression of a disease of interest.
  • nAMD wet AMD
  • dry AMD retinal vein occlusion
  • DME diabetic macular edema
  • DR diabetic retinopathy
  • nAMD wet AMD
  • dry AMD retinal vein occlusion
  • DME diabetic macular edema
  • DR diabetic retinopathy
  • x-linked or Batten disease 57.
  • AAV encodes Palmitoyl-Protein Thioesterase 1 (PPT1), Tripeptidyl-Peptidase 1 (TPP1), , anti-kallikrein antibody or antigen-binding fragment, anti-TNF fusion protein, anti-TNF antibody or antigen- binding fragment, anti-C3 antibody or antigen-binding fragment, or anti-C5 antibody or antigen- binding fragment.
  • PPT1 Palmitoyl-Protein Thioesterase 1
  • TPP1 Tripeptidyl-Peptidase 1
  • anti-kallikrein antibody or antigen-binding fragment anti-TNF fusion protein
  • anti-TNF antibody or antigen- binding fragment anti-C3 antibody or antigen-binding fragment
  • anti-C5 antibody or antigen- binding fragment antigen-bind fragment.
  • the vector genome concentration (VGC) is of 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, or about 3 ⁇ 10 13 GC
  • the pharmaceutical composition of any one of paragraphs 61 and 63, wherein the total number of genome copies administered is about 6.0 ⁇ 10 10 genome copies, about 1.6 ⁇ 10 11 genome copies, about 2.5 ⁇ 10 11 genome copies, about 3 ⁇ 10 11 genome copies, about 5.0 ⁇ 10 11 genome copies, about 6 ⁇ 10 11 genome copies, about 3 ⁇ 10 12 genome copies, about 1.0 ⁇ 10 12 genome copies, about 1.5 ⁇ 10 12 genome copies, about 2.5 ⁇ 10 12 genome copies, or about 3.0 ⁇ 10 13 genome copies.
  • the total number of genome copies administered is about 6.0 ⁇ 10 10 genome copies, about 1.6 ⁇ 10 11 genome copies, about 2.5 ⁇ 10 11 genome copies, about 3 ⁇ 10 11 genome copies, about 5.0 ⁇ 10 11 genome copies, about 6 ⁇ 10 11 genome copies, about 3 ⁇ 10 12 genome copies, about 1.0 ⁇ 10 12 genome copies, about 1.5 ⁇ 10 12 genome copies, about 2.5 ⁇ 10 12 genome copies, or about 3.0 ⁇ 10 13 genome copies.
  • the pharmaceutical composition of any one of paragraphs 60 and 62, wherein the total number of genome copies administered is about 6.0 ⁇ 10 10 genome copies, about 1.6 ⁇ 10 11 genome copies, about 2.5 ⁇ 10 11 genome copies, about 3 ⁇ 10 11 genome copies, about 5.0 ⁇ 10 11 genome copies, about 6 ⁇ 10 11 genome copies, about 3 ⁇ 10 12 genome copies, about 1.0 ⁇ 10 12 genome copies, about 1.5 ⁇ 10 12 genome copies, about 2.5 ⁇ 10 12 genome copies, or about 3.0 ⁇ 10 13 genome copies. 67.
  • the pharmaceutical composition of any one of paragraphs 1-66 wherein the pharmaceutical composition is administered 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.
  • the pharmaceutical composition of any one of paragraphs 10-67 wherein the reference pharmaceutical composition is administered 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.
  • the pharmaceutical composition of any one of paragraphs 1-68 wherein the pharmaceutical composition is administered once in one day, twice in one day, three times in one day, four times in one day, five times in one day, six times in one day, or seven times in one day.
  • the pharmaceutical composition of any one of paragraphs 1-76 wherein the pharmaceutical composition comprises 0.2 mg/mL potassium chloride, 0.2 mg/mL potassium phosphate monobasic, 5.84 mg/mL sodium chloride, 1.15 mg/mL sodium phosphate dibasic anhydrous, 40.0 mg/mL (4% w/v) sucrose, and optionally a surfactant.
  • the pharmaceutical composition of any one of paragraphs 1-76 wherein the composition comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant.
  • a method of treating a disease in a subject the method comprising administering the pharmaceutical composition of any one of paragraphs 1-76. 81.
  • a method of treating a disease in a subject comprising administering the pharmaceutical composition of paragraph 4 or 5 to the subject, wherein the pharmaceutical composition is at a temperature of about 2-10°C when being administered.
  • 82. A method of treating a disease in a subject, the method comprising administering the pharmaceutical composition of paragraph 6 or 8 to the subject, wherein the pharmaceutical composition is at a temperature of about 20-25°C when being administered.
  • 83. The method of any one of paragraphs 80-82, wherein the pharmaceutical composition is administered with an injection pressure of less than about 43 PSI.
  • 84. The method of any one of paragraphs 80-82, wherein the pharmaceutical composition is administered with an injection pressure of less than about 65 PSI. 85.
  • nAMD wet AMD
  • dry AMD retinal vein occlusion
  • DME diabetic macular edema
  • DR diabetic retinopathy
  • a pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, wherein the pharmaceutical composition comprises 18.0% w/v poloxamer 407 and 6.5% w/v poloxamer 188 in a solution 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 monobasic, 40.0 mg/mL (4% w/v) sucrose, 0.001% (0.01 mg/mL) poloxamer 188, pH 7.4. 94.
  • AAV adeno-associated virus
  • a method of preparing a pharmaceutical composition according to paragraph 92 or paragraph 93 comprising (a) providing a solution 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 monobasic, 40.0 mg/ml (4% w/v) sucrose, 0.001% poloxamer 188, ph 7.4, and (b) admixing 18.0% w/v poloxamer 407 and 6.5% w/v poloxamer 188 to the solution.
  • the method of paragraph 94 wherein following the admixing step, the pH of the composition is between about pH 6.0 and about pH 7.9. 3.
  • FIG.1 Overview of the localization of a Construct (e.g., a construct of the disclosure) in the suprachoroidal space using a thermoresponsive gel formulation.
  • the thermoresponsive gel formulation is expected to change from liquid state during injection to a gel in the suprachoroidal space and therefore retain the dosed AAV locally in the suprachoroidal space for longer with a greater therapeutic effect.
  • FIGs.2A-2C Calculated injection pressure as a function of viscosity for different 30 gauge and 29 gauge needles. Panel A is scaled to a limit of 100 PSI, panel B to 65 PSI, and panel C to 45 PSI.
  • FIG.4. Gelation temperature as a function of formulation composition surface plot from design of experiments study. [0050] FIG.5. Viscosity at 20°C as a function of formulation composition surface plot from design of experiments study. [0051] FIG.6. Viscosity at 5°C as a function of formulation composition surface plot from design of experiments study. [0052] FIG.7. Summary of gelation temperature rheology data from design of experiment (DOE) study. Samples are labelled with P407-P188 level (e.g. sample #1, labeled “16-0” has 16% P407 and 0% P188). [0053] FIG.8.
  • DOE design of experiment
  • FIG.9. Summary of gelation time jump from 20°C to 34°C rheology data from DOE Study. Samples are labelled with P407-P188 level (e.g. sample #1 has 16% P407 and 0% P188).
  • FIG.10. Summary of gelation time jump from 5°C to 34°C rheology data from DOE Study. Samples are labelled with P407-P188 level (e.g. sample #1 has 16% P407 and 0% P188).
  • FIG.12. Summary of viscosity versus shear rate sweep at 20°C from DOE Study. Samples are labelled with P407-P188 level (e.g. sample #1 has 16% P407 and 0% P188). Note, sample 2 and 4 had already gelled at 20°C, and therefore are showing the impact of shear on breaking the gel structure. All other samples show Newtonian behavior (constant viscosity as function of shear rate).
  • FIG.13 Summary of viscosity versus shear rate sweep at 5°C from DOE Study.
  • FIG.15 Thermoresponsive gel formulation design space (white area) with limits of 27 to 32°C for gel temperature (pink shade) and an additional limit on viscosity at 20°C of ⁇ 183 mPas (region >183 mPas is shown in green shade).
  • the dose is administered at controlled room temperature (20°C) with an injection time of 10 s.
  • FIG.16 Example preparation of Clinical Drug Product with Autoclave Sterilization.
  • FIG.17 Example Preparation of Clinical Drug Product with Sterile Filtration.
  • FIG.18 Thermal camera image showing the setup for gelation time for droplet flow on a pre-warmed (31.3°C) surface (bottle containing warm water for thermal mass).
  • FIG.19 Gelation temperature profile for Formulation A.
  • FIG.20 Gelation temperature profile for Formulation B.
  • FIG.21 Gelation temperature profile for Formulation C.
  • FIG.22 Gelation time jump from 20°C to 34°C for Formulation A.
  • FIG.23 Gelation time jump from 20°C to 34°C for Formulation B.
  • FIG.24 Gelation time jump from 20°C to 34°C for Formulation C.
  • FIG.25 Gelation time jump from 5°C to 34°C for Formulation A.
  • FIG.26 Gelation time jump from 5°C to 34°C for Formulation B.
  • FIG.27 Gelation time jump from 5°C to 34°C for Formulation C.
  • FIG.28 Viscosity versus shear rate at 20°C for Formulation A.
  • FIG.29 Viscosity versus shear rate at 20°C for Formulation B.
  • FIG.30 Viscosity versus shear rate at 20°C for Formulation C.
  • FIG.31 Viscosity versus shear rate at 5°C for Formulation A.
  • FIG.32 Viscosity versus shear rate at 5°C for Formulation A.
  • FIG.33 Viscosity versus shear rate at 5°C for Formulation B.
  • FIG.33 Viscosity versus shear rate at 5°C for Formulation C.
  • FIG.34 Gelation profile for Formulation A diluted by 10%.
  • FIG.35 Gelation profile for Formulation B diluted by 10%.
  • FIG.36 Gelation profile for Formulation C diluted by 10%.
  • FIG.37 Differential Scanning Fluorometry Profiles of Control (S-0DGN) and Formulations A (S-0DGO), B (S-0DGP), and C (S-0DGQ).
  • S-0DGN Differential Scanning Fluorometry Profiles of Control
  • S-0DGO Formulations A
  • B S-0DGP
  • C S-0DGQ
  • FIG.39 Injection time profile for 0.1 mL of formulation A, B and C injected into air using a 1 mL syringe with 30 Ga TW needle (pressure/force was held about constant by operator, resulting in longer injection time for higher viscosity formulations).
  • FIG.40 Images of SCS space following administration of Gel B Formulation. 4.
  • compositions comprising recombinant adeno- associated virus (AAV) vector comprising an expression cassette encoding a transgene suitable for administration to a suprachoroidal space (SCS) of an eye of a subject.
  • AAV adeno-associated virus
  • SCS suprachoroidal space
  • the subject can be a subject diagnosed with one of more diseases described in Section 4.5.
  • the AAV vectors are described in Section 4.4 and dosages of such vectors are described in Section 4.3.
  • pharmaceutical compositions provided in Section 4.1 are formulated such that they have one or more functional properties described in Section 4.2.
  • the pharmaceutical composition provided herein has various advantages, for example, increased or slower clearance time (Section 4.2.1); decreased circumferential spread (Section 4.2.2); increased SCS thickness (Section 4.2.3); increased AAV level and increased rate of transduction at site of injection (Section 4.2.4); and increased concentration of the transgene after the pharmaceutical composition is administered in the SCS.
  • the functional properties can be achieved using thermoresponsive formulations as disclosed in Section 4.1.
  • assays that may be used in related studies (Section 4.6).
  • the disclosure provides a pharmaceutical composition suitable for suprachoroidal administration comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene.
  • AAV adeno-associated virus
  • several pharmaceutical compositions having different viscosity (or ”loss modulus (G”)”) values properties at extraocular temperature (about 32-35°C) are used to administer an AAV encoding a transgene.
  • G viscosity
  • G elastic/storage modulus
  • the pharmaceutical composition is thermoresponsive.
  • the term “thermoresponsive” is generally known in the art to describe a substance that exhibits different physical properties at different temperatures.
  • a pharmaceutical composition provided herein has a lower viscosity, a lower loss modulus (G”), and/or a lower elastic/storage (G’) modulus at room temperature (e.g., about 20-25 °C) than at extraocular temperature (about 32-35°C).
  • a pharmaceutical composition provided herein has a lower viscosity and/or a lower elastic modulus (G’) when chilled (e.g., about 2-10 °C) than at extraocular temperature (about 32-35°C).
  • a pharmaceutical composition provided herein may be administered to the eye of a subject at a temperature where the viscosity of the pharmaceutical composition is lower (e.g., chilled or at room temperature) that at extraocular temperature (about 32-35°C).
  • a temperature where the viscosity of the pharmaceutical composition is lower e.g., chilled or at room temperature
  • extraocular temperature about 32-35°C
  • the change in temperature upon administration to the eye of a subject e.g., suprachoroidal administration
  • G viscosity and/or elastic modulus
  • the pharmaceutical composition and the reference pharmaceutical composition comprise a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene.
  • AAV adeno-associated virus
  • the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration.
  • the pharmaceutical composition and a reference pharmaceutical composition have the same amount of genome copies.
  • the pharmaceutical composition at about 32-35°C has a viscosity and/or elastic modulus (G’) value that is higher than the viscosity of water at about 32-35°C.
  • the pharmaceutical composition at about 32-35°C has a viscosity and/or elastic modulus (G’) value that is higher than the viscosity and/or elastic modulus (G’) of a control at about 32-35°C.
  • the pharmaceutical composition at about 32-35°C has a viscosity and/or elastic modulus (G’) value that is higher than the viscosity of a solution normally used for subretinal injection at about 32-35°C.
  • the pharmaceutical composition at about 32-35°C has a viscosity and/or elastic modulus (G’) value that is higher than the viscosity of PBS or dPBS at about 32-35°C.
  • the pharmaceutical composition at about 32-35°C has a viscosity and/or elastic modulus (G’) value that is higher than the viscosity of Hank’s Balanced Salt Solution (HBSS) at about 32-35°C.
  • the reference pharmaceutical composition at about 32-35°C has lower viscosity and/or elastic modulus (G’) than the pharmaceutical composition at about 32-35°C.
  • the reference pharmaceutical composition has the same or similar viscosity and/or elastic modulus (G’) as the pharmaceutical composition at about 20-25°C.
  • the reference pharmaceutical composition is a control solution (e.g., PBS, water, or HBSS).
  • the reference pharmaceutical composition comprises sucrose.
  • the reference pharmaceutical composition is a pharmaceutical composition commonly used for AAV subretinal injection.
  • 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 does at about 32- 35°C or does not have a higher viscosity and/or elastic modulus (G’) at increased temperatures.
  • the pharmaceutical composition has viscosity 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, 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 c, 9,000 cP, 10,000 cP, 12,000 cP, or 15,000 cP at about 32-35°C, e.g.,
  • the shear rate is about or less than about 100 s -1 , 50 s -1 , 10 s -1 , 1 s -1 , 0.1 s -1 , 0.01 s -1 , 0.001 s -1 , or 0.0001 s -1 .
  • the viscosity of the pharmaceutical composition or the reference pharmaceutical composition is any viscosity disclosed herein at a shear rate of e.g., about or less than about 100 s -1 , 50 s -1 , 10 s -1 , 1 s -1 , 0.01 s -1 , 0.001 s -1 , or 0.0001 s -1 .
  • the pharmaceutical composition at about 32-35°C or the reference pharmaceutical composition (or a control pharmaceutical composition or a comparable pharmaceutical composition) at about 32-35°C has a viscosity (e.g., as measured at a shear rate of about or at least about 1000s -1 ) that is 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 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 c
  • a viscosity
  • the viscosity (e.g., as measured at a shear rate of about or at least about 1000s -1 ) at about 32-35°C is between about 25 cP to about 1 ⁇ 10 6 cP, between about 25 cP to about 1 ⁇ 10 4 cP, between about 25 cP to about 5,000 cP, between about 25 cP to about 1 ⁇ 10 3 cP, between about 100 cP to about 1 ⁇ 10 6 cP, between about 100 cP to about 1 ⁇ 10 4 cP, between about 100 cP to about 5,000 cP, between about 100 cP to about 1 ⁇ 10 3 cP.
  • the viscosity (e.g., as measured at a shear rate of about or at least about 1000s -1 ) at about 32-35°C is between about 25 cP to about 3 ⁇ 10 6 cP, between about 10 cP to about 3 ⁇ 10 8 cP, between about 50 cP to about 5000 cP, between about 10 cP to about 15000 cP, between about 25 cP to about 1500 cP, between about 50 cP to about 1500 cP, between about 25 cP to about 3 ⁇ 10 4 cP.
  • the pharmaceutical composition at about 32-35°C has a viscosity (e.g., as measured at a shear rate of about or at least about 1000s -1 ) that is at least between about 25 cP to about 3 ⁇ 10 6 cP, at least between about 10 cP to about 3 ⁇ 10 8 cP, at least between about 50 cP to about 5000 cP, at least between about 10 cP to about 15000 cP, at least between about 25 cP to about 1500 cP, at least between about 50 cP to about 1500 cP, or at least between about 25 cP to about 3 ⁇ 10 4 cP.
  • a viscosity e.g., as measured at a shear rate of about or at least about 1000s -1
  • a comparable pharmaceutical composition, or a reference pharmaceutical composition, or a control at about 32-35°C has a viscosity (e.g., as measured at a shear rate of about or at least about 1000s -1 ) 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 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
  • a comparable pharmaceutical composition, or a reference pharmaceutical composition, or a control at about 32-35°C has a viscosity of between about 1 cP to about 25 cP, between about 1 cP to about 20 cP, between about 1 cP to about 24 cP, between about 1 cP to about 10 cP, between about 1 cP to about 50 cP, between about 1 cP to about 100 cP, between about 5 cP to about 50 cP, between about 1 cP to about 5 cP, or between about 1 cP to about 200 cP.
  • a reference pharmaceutical composition at about 32-35°C has a viscosity of about 1 cP or less than about 1 cP (e.g., at a shear rate of about or at least about 1000 s ⁇ 1 ). In some embodiments, a 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 ). [0093] In some embodiments, a pharmaceutical composition provided herein has a viscosity of ⁇ 183 mPas at 20°C.
  • a pharmaceutical composition provided herein has a viscosity of ⁇ 183 mPas at 5°C. Because viscosity depends on shear rate, the “viscosity” of the pharmaceutical composition is the viscosity at any point between a shear rate of 0.01 s ⁇ 1 to 100,000 s ⁇ 1.
  • the unit for viscosity can be defined as cP or mPas. In some cases, cP and mPas are used interchangeably.
  • a pharmaceutical composition provided herein has a viscosity of less than 265 to 655 mPas 32-35°C.
  • 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 more.
  • a shear rate is a shear rate of about or at least about 1000/second.
  • a formulation is characterized by a zero shear viscosity of at least 300,000 mPas.
  • the formulation is further characterized by a viscosity of not more than about 400 mPas at 1000 s ⁇ 1 shear rate.
  • a pharmaceutical composition provided herein remains in the SCS (or in the eye) for a longer period of time after injection (measured at different time points) as compared to a reference pharmaceutical formulation, or a formulation having lower viscosity and/or elastic modulus (G’) at about 32-35°C.
  • a pharmaceutical composition provided herein expands the SCS or the thickness at the site of injection (e.g., as compared to a reference pharmaceutical composition, or formulations having lower viscosity and/or elastic modulus (G’) at about 32-35°C) (see Section 4.2.3).
  • the elastic modulus of a pharmaceutical composition provided herein at under 27°C is less than about or about 0.1 Pa, less than about or about 0.01 Pa, less than about or about 0.001 Pa or zero.
  • the elastic modulus of a 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 10,000 Pa or about or at least about 100,000 Pa.
  • a pharmaceutical composition provided herein has a gelation temperature of over 27°C. In some embodiments, a pharmaceutical composition provided herein has a gelation temperature of less than 32°C. In some embodiments, a pharmaceutical composition provided herein has a gelation temperature over about 27-32°C.
  • a pharmaceutical composition provided herein has a gelation temperature of about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35°C. [0099] In some embodiments, a pharmaceutical composition provided herein has a gelation time of longer than about 10 seconds. In some embodiments, a pharmaceutical composition provided herein has a gelation time of longer than about 15 seconds.
  • a pharmaceutical composition provided herein has 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.
  • a pharmaceutical composition provided herein is a gelation time of less than 90 seconds.
  • the gelation time is determined at about 34 °C.
  • the gelation time is determined at about 32-34°C.
  • the gelation time of a pharmaceutical composition is longer than the injection time of said composition.
  • the gelation time is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% longer than the injection time.
  • the pharmaceutical composition at about 32-35°C has a viscosity sufficient to expand at least a portion of the site of injection (e.g. SCS) to a thickness of at least 500 ⁇ m or about 500 ⁇ m to about 3 mm, for at least two hours after administration. In some embodiments, the viscosity of the pharmaceutical composition at about 32-35°C is sufficient to expand the site of injection (e.g.
  • the viscosity of the pharmaceutical composition at about 32-35°C is sufficient to expand the site of injection (e.g.
  • SCS to a thickness of about 500 ⁇ m to about 3.0 mm for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, at least twenty-four hours, at least two days, at least three days, at least five days, at least ten days, at least twenty-one days, at least one month, at least six weeks, at least two months, at least three months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least one year, at least three years, or at least five years after the administration.
  • the viscosity of the pharmaceutical composition at about 32-35°C is sufficient to expand the site of injection (e.g. SCS) to a thickness of about 1 mm to about 3 mm for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, or at least twenty-four hours after administration.
  • the viscosity of the pharmaceutical composition at about 32- 35°C is sufficient to expand the site of injection (e.g.
  • SCS to a thickness of about 1 mm to about 2 mm for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, at least twenty-four hours, at least two days, at least three days, at least five days, at least ten days, at least twenty-one days, at least one month, at least six weeks, at least two months, at least three months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least one year, at least three years, or at least five years after the administration.
  • the viscosity of the pharmaceutical composition at about 32-35°C is sufficient to expand the site of injection (e.g. SCS) to a thickness of about 2 mm to about 3 mm for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, at least twenty-four hours, at least two days, at least three days, at least five days, at least ten days, at least twenty-one days, at least one month, at least six weeks, at least two months, at least three months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least one year, at least three years, or at least five years after the administration.
  • SCS site of injection
  • the viscosity of the pharmaceutical composition at about 32-35°C is sufficient to expand the site of injection (e.g. SCS) to a thickness of about 750 ⁇ m to about 2.8 mm, about 750 ⁇ m to about 2.5 mm, about 750 ⁇ m to about 2 mm, or about 1 mm to about 2 mm for an indefinite period.
  • An indefinite period may be achieved due, at least in part, to the stability of the pharmaceutical composition in the site of injection (e.g. SCS).
  • a pharmaceutical composition at about 32-35°C having a viscosity sufficient to expand the site of injection e.g.
  • a pharmaceutical composition at about 32-35°C has a viscosity sufficient to expand the site of injection (e.g.
  • SCS to a thickness of at least about 50 ⁇ m, 100 ⁇ m, 200 ⁇ m, 300 ⁇ m, 400 ⁇ m, 500 ⁇ m, 600 ⁇ m, 700 ⁇ m, 800 ⁇ m, 900 ⁇ m, 1000 ⁇ m, 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 larger than 10 mm.
  • a reference pharmaceutical composition at about 32-35°C has a viscosity sufficient to expand the site of injection to a thickness of at most 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 ⁇ m, 5 ⁇ m, 10 ⁇ m, 15 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 35 ⁇ m, 40 ⁇ m, 50 ⁇ m, 100 ⁇ m, 200 ⁇ m, 300 ⁇ m, 400 ⁇ m, 500 ⁇ m, 600 ⁇ m, 700 ⁇ m, 800 ⁇ m, 900 ⁇ m, 1000 ⁇ m, 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
  • a method of treating an ocular disease includes administering an effective amount of the pharmaceutical composition (e.g., recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene) to a subject (e.g., human).
  • the pharmaceutical composition is administered in the suprachoroidal space (SCS) of an eye of the subject.
  • 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 intravitreously. In some embodiments, the pharmaceutical composition has the same vector genome concentration when administered to the SCS as when administered via subretinal administration or via intravitreous administration. In some embodiments, the pharmaceutical composition has the same amount of genome copies when administered to the SCS as when administered via subretinal administration or via intravitreous administration.
  • the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response in a subject is lower as compared to the effective amount of a reference pharmaceutical composition sufficient to elicit a therapeutic response in the 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 a 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 a reference pharmaceutical composition sufficient to elicit a therapeutic response when administered intravitreously. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration.
  • the pharmaceutical composition and the reference pharmaceutical composition have the same amount of genome copies.
  • the pharmaceutical composition has a viscosity and/or elastic modulus (G’) that is higher than the viscosity and/or elastic modulus (G’) of the reference pharmaceutical composition.
  • the pharmaceutical composition is substantially localized near the insertion site (see Section 4.2.1 and Section 4.2.2).
  • the pharmaceutical composition results in a higher level of transgene expression (concentration) when the pharmaceutical composition is administered in the SCS as compared to when the pharmaceutical composition is administered subretinally or intravitreously (see Section 4.2.5).
  • the pharmaceutical composition results in a higher level of transgene expression (concentration) when the pharmaceutical composition is administered in the SCS as compared to when a reference pharmaceutical composition is administered subretinally, intravitreously, or in the SCS (see Section 4.2.5).
  • the pharmaceutical composition results in a higher level of AAV when the pharmaceutical composition is administered in the SCS as compared to when the pharmaceutical composition is administered subretinally or intravitreously (see Section 4.2.5).
  • the pharmaceutical composition results in a higher level of AAV when the pharmaceutical composition is administered in the SCS as compared to when a reference pharmaceutical composition is administered subretinally, intravitreously, or in the SCS (see Section 4.2.5).
  • the pharmaceutical composition results in a higher rate of transduction (or rate of infection) at a site of injection when the pharmaceutical composition is administered in the SCS as compared to when the pharmaceutical composition is administered subretinally or intravitreously (see Section 4.2.5). In some embodiments, the pharmaceutical composition results in a higher rate of transduction (or rate of infection) at a site of injection when the pharmaceutical composition is administered in the SCS as compared to when a reference pharmaceutical composition is administered subretinally, intravitreously, or in the SCS (see Section 4.2.5). In some embodiments, the pharmaceutical composition results in reduced vasodilation and/or vascular leakage when the pharmaceutical composition is administered in the SCS as compared to when the pharmaceutical composition is administered subretinally or intravitreously.
  • the pharmaceutical composition results in reduced vasodilation and/or vascular leakage when the pharmaceutical composition is administered in the SCS as compared to when a reference pharmaceutical composition is administered subretinally, intravitreously, or in the SCS.
  • the reference pharmaceutical composition includes the recombinant adeno-associated virus (AAV) vector comprising the expression cassette encoding the transgene.
  • AAV adeno-associated virus
  • the pharmaceutical composition at about 32-35°C has higher viscosity and/or elastic modulus (G’) than the reference pharmaceutical composition at about 32-35°C.
  • the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration.
  • the pharmaceutical composition and the reference pharmaceutical composition have the same amount of genome copies.
  • the viscosity and/or elastic modulus (G’) of a pharmaceutical composition provided herein increase to values well in excess of the viscosity of water (for example, at least about 100 cP at a shear rate of 0.1/second) as the formulation warms to at about 32-35°C, resulting in formulations that are highly effective for placement, e.g., injection, into an eye of a subject (e.g., to the SCS).
  • the relatively high viscosity and/or elastic modulus of the formulation at about 32-35°C enhances the ability of such formulations to maintain the therapeutic component (e.g., AAV comprising an expression cassette comprising a transgene) in substantially uniform suspension in the formulation for prolonged periods of time, and can also aid in the storage stability of the formulation.
  • a pharmaceutical composition provided herein e.g., a thermoresponsive pharmaceutical composition
  • the solvent molecules may penetrate a hydrocolloidal network formed by the gelator.
  • a pharmaceutical composition provided herein comprises hydrophilic polymers in an aqueous system.
  • a pharmaceutical composition provided herein comprises natural polymers (e.g., xanthan gum, starch, gellan, konjac, carrageenans, collagen, fibrin, silk fibroin, hyaluronic acid or gelatin).
  • a pharmaceutical composition provided herein comprises synthetic polymers (e.g., chitosan- ⁇ - glycerophosphate, poly (N-Isopropylacrylamide) (pNIPAAm), pluronic F127, methylcellulose or PEG-PCL). See, e.g., Taylor et al., Gels.2017 Mar; 3(1): 4.
  • Non-limiting examples of solutions that have a higher viscosity and/or elastic modulus (G’) at about 32-35°C compared to lower temperatures and that can be used in a pharmaceutical composition of the present disclosure include solutions comprising varying concentrations of poloxamer 407 (P407, CAS Number: 9003-11-6) and poloxamer 188 (P188, CAS Number: 9003- 11-6).
  • a pharmaceutical composition provided herein comprises 16% P407 and 0% P188.
  • a pharmaceutical composition provided herein comprises 22% P407 and 0% P188.
  • a pharmaceutical composition provided herein comprises 16% P407 and 16% P188.
  • a pharmaceutical composition provided herein comprises 22% P407 and 16% P188. In some embodiments, a pharmaceutical composition provided herein comprises 19% P407 and 0% P188. In some embodiments, a pharmaceutical composition provided herein comprises 16% P407 and 8% P188. In some embodiments, a pharmaceutical composition provided herein comprises 22% P407 and 8% P188. In some embodiments, a pharmaceutical composition provided herein comprises 19% P407 and 8% P188.
  • the disclosure provides a pharmaceutical composition (e.g., liquid formulation) comprising a recombinant adeno-associated virus (AAV) and at least one of: potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and surfactant.
  • the pharmaceutical composition e.g., liquid formulation
  • the pharmaceutical composition 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 present formulations such as higher viscosity and/or elastic modulus (G’) at increased temperatures.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) and at least one of: an ionic salt excipient or buffering agent, sucrose, and surfactant.
  • the ionic salt excipient or buffering agent can be one or more components from the group consisting of potassium phosphate monobasic, potassium phosphate, sodium chloride, sodium phosphate dibasic anhydrous, sodium phosphate hexahydrate, sodium phosphate monobasic monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), amino acid, 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 6-hydrate, calcium
  • the surfactant can be one or more components from the group consisting of poloxamer 188, polysorbate 20, and polysorbate 80.
  • 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 more than 1.50 mg/mL of sodium phosphate dibasic anhydrous (or an equivalent).
  • the pharmaceutical composition comprises 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, 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/m
  • 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 more than 1 mg/mL of potassium chloride and/or potassium phosphate monobasic (or equivalents thereof).
  • 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. [00111] In certain embodiments, the pharmaceutical composition has an ionic strength of about 30 mM to about 100 mM.
  • 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.
  • the pharmaceutical composition comprises potassium chloride (e.g., at a concentration of 0.2 g/L). In certain embodiments, the pharmaceutical composition comprises potassium phosphate monobasic (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 phosphate monobasic, sodium chloride, and sodium phosphate dibasic anhydrous.
  • the pharmaceutical composition comprises sucrose at a concentration of 3% (weight/volume, 30 g/L) to 18% (weight/volume, 180 g/L). In certain embodiments, the pharmaceutical composition comprises sucrose at a concentration of 4% (weight/volume, 40 g/L). [00114] 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).
  • 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).
  • 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%, 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 more than 0.1%.
  • a surfactant e.g., poloxamer 188, polysorbate 20, and/or polysorbate 80
  • 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. [00116] 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.
  • COP Cyclo Olefin Polymer
  • CZ Daikyo Crystal Zenith®
  • 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 phosphate monobasic 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, and wherein the recombinant AAV is AAV8.
  • the pharmaceutical composition does not comprise sucrose.
  • a pharmaceutical composition comprising a recombinant AAV, 18.0% w/v poloxamer 407, and 6.5% w/v poloxamer 188, each of which is in a solution 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 monobasic, 40.0 mg/mL (4% w/v) sucrose, 0.001% (0.01 mg/mL) poloxamer 188, pH 7.4.
  • the pharmaceutical composition comprises (a) an AAV8 or AAV9 that encodes Tripeptidyl-Peptidase 1 and at least one of: (b) potassium chloride at a concentration of 0.2 g/L, (c) potassium phosphate monobasic 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.
  • an AAV8 or AAV9 that encodes Tripeptidyl-Peptidase 1 and at least one of: (b) potassium chloride at a concentration of 0.2 g/L, (c) potassium phosphate monobasic at a concentration of 0.2 g/L, (d) sodium chloride at a concentration
  • the pharmaceutical composition does not comprise sucrose.
  • the pharmaceutical composition has desired viscosity, elastic modulus (G’), density, and/or osmolality that is suitable for suprachoroidal injection (for example, via a suprachoroidal drug delivery device such as a microinjector with a microneedle).
  • the pharmaceutical composition is a liquid composition.
  • the pharmaceutical composition is a frozen composition.
  • the pharmaceutical composition is a gel.
  • the pharmaceutical composition has a osmolality range of 200 mOsm/L to 660 mOsm/L.
  • the pharmaceutical composition has a osmolality of about, of at least about, or of 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.
  • gene therapy constructs are supplied as a frozen sterile, single use solution of the AAV vector active ingredient in a formulation buffer.
  • the pharmaceutical compositions suitable for suprachoroidal administration comprise a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients.
  • the composition comprises modified Dulbecco’s phosphate- buffered saline solution, and optionally a surfactant.
  • the pharmaceutical composition comprises 0.2 mg/mL potassium chloride, 0.2 mg/mL potassium phosphate monobasic, 5.84 mg/mL sodium chloride, 1.15 mg/mL sodium phosphate dibasic anhydrous, 40.0 mg/mL (4% w/v) sucrose, and optionally a surfactant.
  • the composition comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant.
  • a pharmaceutical composition provided herein is not a composition described in Zeinab et al. (European Journal of Pharmaceutics and Biopharmaceutics 114 (2017) 119–13).
  • the disclosure provides a pharmaceutical composition (e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene) resulting in a delayed clearance time from the SCS.
  • 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 results in delayed clearance time from the SCS as compared to a pharmaceutical composition which is non- viscous or low viscosity and/or less elastic and/or is not gelled at about 32-35°C.
  • 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 results in delayed clearance time from the eye as compared to a pharmaceutical composition which is non-viscous or low viscosity and/or less elastic and/or is not gelled at about 32-35°C.
  • a more viscous and/or elastic and/or gelled pharmaceutical composition results in delayed clearance time from the eye as compared to a less viscous and/or elastic and/or gelled pharmaceutical composition.
  • a pharmaceutical composition that is more viscous and/or elastic and/or gelled at about 32-35°C has a viscosity value that is higher than the viscosity of water at about 32-35°C.
  • a pharmaceutical composition that is more viscous and gelled at about 32-35°C has a viscosity value and/or an elastic modulus value that is higher than the viscosity and/or elastic modulus of a solution normally used for subretinal injection at about 32-35°C.
  • the clearance time of the pharmaceutical composition after the pharmaceutical composition is administered to the SCS is equal to or higher than the clearance time of a reference pharmaceutical composition after the reference pharmaceutical composition is administered subretinally or intravitreously.
  • a pharmaceutical composition results in a clearance time from the SCS 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 7 days, about 4
  • the clearance time from the SCS is of 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.
  • the “clearance time from the SCS” is the time required for substantially all of the pharmaceutical composition, the pharmaceutical agent, or the AAV to escape the SCS.
  • the “clearance time from the SCS” is the time required for the pharmaceutical composition, the pharmaceutical agent, or the AAV to not be detected in the SCS by any standard method (such as those described in Section 4.6 and Section 5). In some embodiments, the “clearance time from the SCS” is when the pharmaceutical composition, the pharmaceutical agent, or the AAV is present in the SCS in an amount that is at most about 2% or at most about 5% as detected by any standard method (such as those described in Section 4.6 and Section 5).
  • the pharmaceutical composition results in a clearance time from the eye 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
  • the clearance time from the eye is of 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.
  • the “clearance time from the eye” is the time required for substantially all of the pharmaceutical composition, the pharmaceutical agent, or the AAV to escape the eye.
  • the “clearance time from the eye” is the time required for the pharmaceutical composition, the pharmaceutical agent, or the AAV to not be detected in the eye by any method (such as those described in Section 4.6 and Section 5).
  • the “clearance time from the eye” is when the pharmaceutical composition, the pharmaceutical agent, or the AAV is present in the eye in an amount that is at most about 2% or at most about 5% as detected by any standard method (such as those described in Section 4.6 and Section 5).
  • the clearance time is not prior to (e.g., the clearance time from the SCS or the eye does not occur before) 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, 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,
  • the clearance 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, 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 administration of the pharmaceutical composition.
  • a pharmaceutical composition e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene
  • a clearance time that 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 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
  • a suprachoroidal administration of a pharmaceutical composition results in a clearance time that 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 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
  • a suprachoroidal administration of a pharmaceutical composition results in a clearance time that 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 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
  • a suprachoroidal administration of a pharmaceutical composition which is viscous results in a clearance time that 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 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,
  • viscous e.g., relatively viscous, medium to super high viscosity, or more viscous than water, or more viscous than a control solution, or more viscous than a solution commonly used for subretinal administration
  • a clearance time results in a clearance time that is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at
  • the clearance time of a pharmaceutical composition is greater than the clearance time of the same pharmaceutical composition administered via subretinal administration or via intravitreous administration.
  • the clearance time of a pharmaceutical composition e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene
  • a pharmaceutical composition which is comparably less viscous and/or less elastic and/or non-gelled at about 32-35°C administered by suprachoroidal injection.
  • the clearance time of a pharmaceutical composition e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene
  • a pharmaceutical composition which is comparable less viscous and/or less elastic and/or non-gelled at about 32-35°C administered via subretinal administration or via intravitreous administration.
  • the clearance time of a pharmaceutical composition e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene
  • a pharmaceutical composition that is viscous and/or elastic and/or gelled at about 32-35°C administered by suprachoroidal injection is greater than a pharmaceutical composition that is comparably viscous and/or elastic and/or gelled at about 32-35°C administered via subretinal administration or via intravitreous administration.
  • the clearance time of a pharmaceutical composition is greater than the same pharmaceutical composition administered via subretinal administration or via intravitreous administration by 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, 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,
  • the clearance time of a pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) which is more viscous and/or more elastic and/or gelled at about 32-35°C administered by suprachoroidal injection is greater than a pharmaceutical composition which is comparably less viscous and/or less elastic and/or not gelled at about 32-35°C administered by suprachoroidal injection by 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, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days,
  • the clearance time of a pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) which is more viscous and/or more elastic and/or gelled at about 32-35°C administered by suprachoroidal injection is greater than a pharmaceutical composition that is comparably less viscous and/or less elastic and/or not gelled at about 32-35°C administered via subretinal administration or via intravitreous administration by 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, 21 days, 23 days, 25 days, 27 days, 30 days, 35
  • the clearance time of the pharmaceutical composition administered via intravitreous injection or via subretinal injection is of at most about 30 minutes, 1 hours, 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, 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 at most
  • the clearance time of a reference pharmaceutical composition administered by intravitreous injection, subretinal injection, or to the SCS is of at most about 30 minutes, 1 hours, 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, 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,
  • the clearance time is the clearance time from the eye. In some embodiments, the clearance time is the clearance time from the SCS. In some embodiments, the clearance time is the clearance time from the site of injection. [00140] In some embodiments, the “clearance time from the SCS” is the amount of time required following injection of the pharmaceutical composition, the pharmaceutical agent, or the AAV required for the SCS thickness at or near the site of injection to decrease 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.
  • the “clearance time from the SCS” is the amount of time required following injection of the pharmaceutical composition, the pharmaceutical agent, or the AAV required for the SCS thickness at or near the site of injection to decrease 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 is 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).
  • the pharmaceutical composition e.g., diluted pharmaceutical composition
  • the pharmaceutical composition has a viscosity sufficient to make the clearance time 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, 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,
  • the “clearance time from the SCS” is the amount of time required following injection for the pharmaceutical composition, the pharmaceutical agent, or the AAV required to spread circumferentially from the site of injection to cover about one-sixteenth or more, about one-eighth or more, about one-fourth or more, about one-half or more, about three-fourths 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).
  • standard techniques e.g. in-vivo imaging techniques such as OCT imaging.
  • the pharmaceutical composition (e.g., diluted pharmaceutical composition) has a viscosity sufficient to make the clearance time 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, 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.
  • the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig (e.g., minipig, such as Yucatan minipig).
  • a pig e.g., minipig, such as Yucatan minipig.
  • the pig is a minipig.
  • the minipig can be Goettingen, Yucatan, Bama Xiang Zhu, Wuzhishan, and/or Xi Shuang Banna.
  • the minipig is Yucatan.
  • the pharmaceutical composition (e.g., diluted pharmaceutical composition) has a viscosity and/or elastic modulus such that, when administered to the SCS of the eye of a pig, the clearance time of the pharmaceutical composition is between about 5 days and about 15 days, about 6 days to about 15 days days, about 7 days to about 15 days, about 8 days to about 15 days, about 9 days to about 15 days, about 10 days to about 15 days, about 11 days to about 15 days, about 12 days to about 15 days, about 13 days to about 15 days, about 14 days to about 15 days, about 5 days to about 14 days, about 5 days to about 13 days, about 5 days to about 12 days, about 5 days to about 11 days, about 5 days to about 10 days, about 5 days to about 9 days, about 5 days to about 8 days, about 5 days to about 7 days, or about 5 days to about 6 days.
  • the clearance time of the pharmaceutical composition is between about 5 days and about 15 days, about 6 days to about 15 days days, about 7 days to about 15 days, about 8 days to about 15 days, about 9
  • the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig.
  • the pharmaceutical composition e.g., diluted pharmaceutical composition
  • a pharmaceutical composition localizes at the site of injection.
  • a pharmaceutical composition localizes at the site of injection for a longer period of time than a comparable pharmaceutical composition which has a lower viscosity and/or elastic modulus (G’) and/or is not gelled at extraocular temperature (about 32-35°C).
  • a pharmaceutical composition localizes at the site of injection for a longer period of time when injected in the SCS as compared to when the pharmaceutical composition is administered by subretinal injection or intravitreous injection.
  • the pharmaceutical composition can have different viscosity and/or elastic modulus values.
  • 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 as compared to a pharmaceutical composition that is a non-viscous or has low viscosity and/or is not gelled at about 32-35°C.
  • localization can be determined by evaluating circumferential spread (e.g., 2D circumferential spread).
  • circumferential spread is determined by analyzing SCS expansion or opening in the quadrant where the injection was made (in some cases, in 2D this space adjacent to the choroid appears linear).
  • a pharmaceutical composition results in a circumferential spread that is at least 2 times less, at least 3 times less, at least 4 times less, at least 5 times less, at least 6 times less, at least 7 times less, at least 8 times less, at least 9 times less, at least 10 times less, at least 15 times less, at least 20 times less, at least 50 times less, at least 100 times less, 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
  • a suprachoroidal administration of a pharmaceutical composition results in a circumferential spread that is at least 2 times less, at least 3 times less, at least 4 times less, at least 5 times less, at least 6 times less, at least 7 times less, at least 8 times less, at least 9 times less, at least 10 times less, at least 15 times less, at least 20 times less, at least 50 times less, at least 100 times less, 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%
  • a suprachoroidal administration of a pharmaceutical composition e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene
  • a pharmaceutical composition e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene
  • viscous e.g., relatively viscous, medium to super high viscosity, or more viscous than water, or more viscous than a control solution, or more viscous than a solution commonly used for subretinal administration
  • elastic and/or gelled at about 32-35°C results in a circumferential spread that is at least 2 times less, at least 3 times less, at least 4 times less, at least 5 times less, at least 6 times less, at least 7 times less, at least 8 times less, at least 9 times less, at least 10 times less, at least 15 times less, at least 20 times less, at least 50 times less, at least 100 times less, at least 5% less, at least 10% less, at least 15% less, at least 20%
  • the circumferential spread can be determined 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, 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 the pharmaceutical composition or the reference pharmaceutical composition is administered.
  • the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig.
  • the pharmaceutical composition e.g., diluted pharmaceutical composition
  • the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig.
  • the pharmaceutical composition e.g., diluted pharmaceutical composition
  • the pharmaceutical composition (e.g., diluted pharmaceutical composition) has a visocity and/or elastic modulus such that, when administered to the SCS of the eye of a pig, the circumferential spread of the pharmaceutical composition from the site of injection is about one-eighth or less of a surface of the choroid at a time within about one hour of administration.
  • SCS Thickness [00149]
  • localization can be determined by evaluating SCS thickness after a pharmaceutical composition is administered to a subject.
  • a pharmaceutical composition increases the thickness of the SCS after the pharmaceutical composition is injected in the SCS.
  • an SCS expands to accommodate the infusion of a pharmaceutical composition that has low viscosity and/or elastic modulus (G’) and/or is not gelled at about 32- 35°C.
  • the infusion of a greater volume of the low-viscosity and/or low- elastic modulus and/or non-gelled pharmaceutical composition does not cause further expansion of the SCS.
  • the greater volume of the low-viscosity and/or low elastic modulus fluid formulation is accommodated by increasing the area of fluid spread in the SCS without further expanding the SCS.
  • the infusion into the SCS of a pharmaceutical composition that is viscous and/or elastic and/or gelled at about 32-35°C can expand SCS thickness beyond the SCS thickness achieved when a low-viscosity and/or low elastic modulus and/or non- gelled pharmaceutical composition is infused into the SCS.
  • increasing the SCS thickness with a pharmaceutical composition which is viscous and/or gelled at about 32-35°C may ease access to the SCS, thereby easing or permitting the disposal of a device in the SCS.
  • expanding the SCS thickness allows for the pharmaceutical composition and/or the AAV encoded transgene to remain at the site of injection (localized) for a longer period of time.
  • a pharmaceutical composition that is viscous and/or elastic and/or gelled at about 32-35°C increases the thickness at or near the site of injection for a longer period of time as compared to a pharmaceutical composition that is non-viscous or has low viscosity and/or low elastic modulus and/or is not gelled at about 32-35°C.
  • a pharmaceutical composition that is more viscous and/or more elastic and/or gelled at about 32-35°C increases the thickness at or near the site of injection for a longer period of time as compared to a pharmaceutical composition that is less viscous and/or less elastic and/or not gelled at about 32-35°C.
  • the thickness at the site of injection after the pharmaceutical composition is administered to the SCS is equal to or higher than the thickness at the site of injection of a reference pharmaceutical composition after the reference pharmaceutical composition is administered subretinally or intravitreously. In some embodiments, the thickness at the site of injection of the pharmaceutical composition after the pharmaceutical composition is administered to the SCS is equal to or higher than the thickness at the site of injection of a reference pharmaceutical composition after the reference pharmaceutical composition is administered to the SCS.
  • a suprachoroidal administration of a pharmaceutical composition that is viscous results in an increase in the SCS thickness that 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 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%
  • a suprachoroidal administration of a pharmaceutical composition results in an increase in thickness at or near the site of injection that 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 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
  • a suprachoroidal administration of a pharmaceutical composition e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene
  • a pharmaceutical composition e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene
  • viscous e.g., relatively viscous, medium to super high viscosity, or more viscous than water, or more viscous than a control solution, or more viscous than a solution commonly used for subretinal administration
  • elastic and/or gelled at about 32-35°C results in an increase in thickness at or near the site of injection that 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 10% greater, at least 15%
  • the thickness obtained at the site of injection after a pharmaceutical composition e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene
  • a pharmaceutical composition e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene
  • the thickness obtained at the site of injection after a pharmaceutical composition is greater than after a pharmaceutical composition that is comparably less viscous and/or less elastic and/or not gelled at about 32-35°C is administered by suprachoroidal injection.
  • the thickness obtained at the site of injection after a pharmaceutical composition e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene
  • a pharmaceutical composition e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene
  • the thickness obtained at the site of injection after a pharmaceutical composition is greater than after a pharmaceutical composition that is comparably less viscous and/or less elastic and/or not gelled at about 32-35°C administered by subretinal injection or by intravitreous injection.
  • the thickness obtained at the site of injection after a pharmaceutical composition e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene
  • a pharmaceutical composition e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene
  • the thickness obtained at the site of injection after a pharmaceutical composition is greater than after the same pharmaceutical composition administered by subretinal administration or by intravitreous administration.
  • the thickness at or near the site of injection can be determined 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, 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
  • the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig.
  • the pharmaceutical composition (e.g., diluted pharmaceutical composition) has a visocity and/or elastic modulus such that, when administered to the SCS of the eye of a pig, the thickness of the SCS at the site of injection is between about 400 and about 800 ⁇ m, about 400 ⁇ m to about 700 ⁇ m, about 400 ⁇ m to about 600 ⁇ m, about 400 ⁇ m to about 500 ⁇ m, about 500 ⁇ m to about 800 ⁇ m, about 600 ⁇ m to about 800 ⁇ m, 700 ⁇ m to about 800 ⁇ m at a time at a time within about 5 minutes, about 10 minutes, 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about one hour, about 2 hours, about 4 hours, about 8 hours, or about 24 hours of administration.
  • the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig.
  • the pharmaceutical composition e.g., diluted pharmaceutical composition
  • the pharmaceutical composition (e.g., diluted pharmaceutical composition) has a visocity and/or elastic modulus such that, when administered to the SCS of the eye of a pig, the thickness of the SCS at the site of injection is between about 400 and about 800 ⁇ m at a time at a time within about one hour of administration.
  • 4.2.4 Rate of Transduction (Or Rate of Infection) At Site of Injection [00156] In some embodiments, the rate of infection at the site of transduction (or rate of injection) after a pharmaceutical composition is administered in the SCS is equal to or higher as compared to the rate of transductions (or rate of infection) at a site of injection after the same pharmaceutical composition is administered via a subretinal administration or via an intravenous administration.
  • the rate of transduction (or rate of infection) at the site of injection after a pharmaceutical composition is administered in the SCS is equal to or higher as compared to the rate of transduction (or rate of infection) at the site of injection after a comparable (e.g., less viscous and/or not gelled at about 32-35°C) pharmaceutical composition is administered via a subretinal, or intravenous administration, or to the SCS.
  • the pharmaceutical composition has a higher viscosity and/or elastic modulus (G’) and/or is gelled at about 32-35°C than the reference pharmaceutical composition (a pharmaceutical composition that is comparably less viscous and/or less elastic and/or not gelled at about 32-35°C).
  • 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.
  • the increase in the rate of transduction (or rate of infection) at the site of injection is an increase of at least about 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%
  • a level of AAV at the site of injection after the pharmaceutical composition is administered suprachoroidally is equal to or higher as compared to a level of AAV at the site of injection after a comparable (e.g., less viscous and/or less elastic and/or not gelled at about 32-35°C) pharmaceutical composition is administered via a subretinal, or intravenous administration, or to the SCS.
  • the pharmaceutical composition has a higher viscosity and/or elastic modulus (G’) and/or is gelled at about 32-35°C than the reference pharmaceutical composition.
  • the pharmaceutical composition and the reference pharmaceutical composition (a pharmaceutical composition that is comparably less viscous and/or less elastic and/or not gelled at about 32-35°C) have the same vector genome concentration. In some embodiments, the pharmaceutical composition and a reference pharmaceutical composition have the same amount of genome copies.
  • the increase in the level of AAV at the site of injection is an increase of at least about 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 by at least 500%.
  • the AAV level or the rate of transduction is determined 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, 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 at most about 400 days after administration.
  • the concentration of a transgene product is at least equal to or higher after a pharmaceutical composition is injected in the SCS as compared to after a reference (e.g., less viscous and/or less elastic and/or not gelled at about 32-35°C) pharmaceutical composition is injected in the SCS.
  • a reference e.g., less viscous and/or less elastic and/or not gelled at about 32-35°C
  • the concentration of a transgene product is at least equal to or higher after a pharmaceutical composition is injected in the SCS as compared to after a reference (less viscous and/or less elastic and/or not gelled at about 32-35°C) pharmaceutical composition is injected by subretinal injection or by intravitreous injection.
  • the concentration of a transgene product is at least equal to or higher after a pharmaceutical composition is injected in the SCS as compared to after the same pharmaceutical composition is injected by subretinal injection or by intravitreous injection.
  • a transgene product e.g., concentration of the transgene product
  • an eye e.g., vitreous humor
  • a comparable (less viscous and/or less elastic and/or not gelled at about 32-35°C) pharmaceutical composition is injected in the SCS.
  • a transgene product e.g., concentration of the transgene product
  • an eye e.g., vitreous humor
  • a pharmaceutical composition is injected in the SCS as compared to after a reference (less viscous and/or less elastic and/or not gelled at about 32-35°C) pharmaceutical composition is injected by subretinal injection or by intravitreous administration.
  • a transgene product e.g., concentration of the transgene product
  • an eye e.g., vitreous humor
  • a pharmaceutical composition is injected in the SCS as compared to after the same (or similar viscosity and/or elastic modulus (G’) at about 32-35°C) pharmaceutical composition is injected by subretinal injection or by intravitreous injection.
  • the longer period of time 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, 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.
  • 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, 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.
  • the transgene is detected in an eye (e.g., vitreous humor) for period of time, after the pharmaceutical composition is administered in the SCS, that is at least about or 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, 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
  • the transgene is detected in an eye (e.g., vitreous humor) for a period of time (e.g., after the reference pharmaceutical composition is administered via subretinal administration or via intravitreous administration or to the SCS; or after the pharmaceutical composition is administered via subretinal or via intravitreous administration) that is at most 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, 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,
  • the concentration of a transgene product in an eye can be determined 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, 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
  • a suprachoroidal administration of a pharmaceutical composition e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene
  • a pharmaceutical composition e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene
  • viscous e.g., relatively viscous, medium to super high viscosity, or more viscous than water, or more viscous than a control solution, or more viscous than a solution commonly used for subretinal administration
  • elastic and/or gelled at about 32-35°C results in a higher concentration of the transgene that 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 10% greater, at least 15% greater,
  • a suprachoroidal administration of a pharmaceutical composition results in a higher concentration of the transgene that 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 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
  • a suprachoroidal administration of a pharmaceutical composition e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene
  • a pharmaceutical composition e.g., a composition comprising an AAV comprising an expression cassette encoding a transgene
  • viscous e.g., relatively viscous, medium to super high viscosity, or more viscous than water, or more viscous than a control solution, or more viscous than a solution commonly used for subretinal administration
  • elastic and/or gelled at about 32-35°C results in a higher concentration of the transgene that 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 10% greater, at least 15% greater,
  • the concentration of the transgene product after a pharmaceutical composition is greater than after a pharmaceutical composition that is comparably less viscous and/or less elastic and/or not gelled at about 32-35°C is administered by suprachoroidal injection.
  • the concentration of the transgene product after a pharmaceutical composition is greater than after a pharmaceutical composition that is comparably less viscous and/or less elastic and/or not gelled at about 32-35°C is administered by subretinal administration or via intravitreous administration.
  • the concentration of the transgene product after a pharmaceutical composition is greater than after the same pharmaceutical composition is administered by subretinal administration or via intravitreous administration.
  • a pharmaceutical composition e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene
  • the pharmaceutical compositions disclosed herein provide greater transgene expression and/or tissue/cell transduction at the back of the eye (e.g., retina) than in the outer layer of the eye (e.g., sclera) through SCS delivery.
  • the concentration of a transgene product (TP) is equal to or higher in the retina after a presently disclosed pharmaceutical composition comprising AAV encoding the TP is injected in the SCS than a reference pharmaceutical composition comprising the same AAV is injected in the SCS.
  • the concentration of a TP is equal to or higher in the retina and the concentration of the TP is lower in the sclera after a pharmaceutical composition comprising AAV encoding the TP is injected in the SCS as compared to a reference pharmaceutical composition comprising the same AAV is injected in the SCS.
  • the pharmaceutical composition described herein has a desired viscosity and/or elastic modulus (G’) that is suitable for suprachoroidal injection.
  • the recombinant AAV in the pharmaceutical composition is at least as stable as the recombinant AAV in a reference pharmaceutical composition (or a comparable pharmaceutical composition).
  • the recombinant AAV in the pharmaceutical composition is at least 50% as stable as the recombinant AAV in a reference pharmaceutical composition (or a comparable pharmaceutical composition). In some embodiments, the recombinant AAV in the pharmaceutical composition has at least the same or a comparable aggregation level as the recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has at least the same or a comparable infectivity level as the recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has at least the same or a comparable free DNA level as the recombinant AAV in a reference pharmaceutical composition.
  • the recombinant AAV in the pharmaceutical composition has at least the same or a comparable in vitro relative potency (IVRP) as the recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has at least the same or a comparable change in size level as the recombinant AAV in a reference pharmaceutical composition.
  • IVRP in vitro relative potency
  • the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable to freeze/thaw cycles than the same recombinant AAV in a reference pharmaceutical composition.
  • the recombinant AAV in the pharmaceutical composition is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as stable to freeze/thaw cycles as the same recombinant AAV in a reference pharmaceutical composition.
  • the stability of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6 and Section 5.
  • 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 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more infectivity than the same recombinant AAV in a reference pharmaceutical composition.
  • the recombinant AAV in the pharmaceutical composition has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% the infectivity of the same recombinant AAV in a reference pharmaceutical composition.
  • the virus infectivity of the recombinant AAV is determined by an assay or assays disclosed in the present disclosure.
  • the size of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6 and Section 5. In certain embodiments, the size is measured prior to or after freeze/thaw cycles.
  • 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 times, 3 times, 5 times, 10 times, 100 times, or 1000 times less aggregation than the same recombinant AAV in a reference pharmaceutical composition.
  • the aggregation of the recombinant AAV is determined by an assay or assays disclosed in the present disclosure. In certain embodiments, the aggregation is measured prior to or after freeze/thaw cycles.
  • the aggregation of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6.
  • the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable over a period of time (e.g., when stored at -20oC or at 37 oC), 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, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years than the same recombinant AAV in a reference pharmaceutical composition.
  • the recombinant AAV in the pharmaceutical composition is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as stable over a period of time as the same recombinant AAV in a reference pharmaceutical composition.
  • the stability over a period of time of the recombinant AAV is determined by an assay or assays disclosed in the present disclosure.
  • the stability over a period of time of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6 and Section 5.
  • the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 higher in in vitro relative potency (IVRP) than the same recombinant AAV in a reference pharmaceutical composition (e.g., when stored at -20oC or at 37 oC).
  • the recombinant AAV in the pharmaceutical composition has about the same in vitro relative potency (IVRP) as the same recombinant AAV in a reference pharmaceutical composition.
  • the recombinant AAV in the pharmaceutical composition has about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in vitro relative potency (IVRP) as the same recombinant AAV in a reference pharmaceutical composition.
  • the in vitro relative potency (IVRP) of the recombinant AAV is determined by an assay or assays disclosed in the present disclosure.
  • the in vitro relative potency (IVRP) is measured prior to or after freeze/thaw cycles.
  • the in vitro relative potency (IVRP) of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6.
  • 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 times, 3 times, 5 times, 10 times, 100 times, or 1000 times less free DNA than the same recombinant AAV in a reference pharmaceutical composition.
  • the recombinant AAV in the pharmaceutical composition has about the same amount of free DNA as the same recombinant AAV in a reference pharmaceutical composition.
  • the recombinant AAV in the pharmaceutical composition has about not more than two times the amount of free DNA as the same recombinant AAV in a reference pharmaceutical composition.
  • the recombinant AAV in the pharmaceutical composition has about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% the amount of free DNA as the same recombinant AAV in a reference pharmaceutical composition.
  • 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 2 times less, or about 3 times less free DNA than the same recombinant AAV in a reference pharmaceutical composition.
  • the free DNA of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6 and Section 5.
  • the recombinant AAV in the pharmaceutical composition has at most 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, or 1% change in size over a period of time (e.g., when stored at -20oC or at 37 oC), 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.
  • the size of the recombinant AAV is determined by an assay or assays disclosed in the present disclosure. In certain embodiments, the size is measured prior to or after freeze/thaw cycles. In certain embodiments, the size of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6.
  • the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition (e.g., when stored at -20oC or at 37 oC).
  • the recombinant AAV in the pharmaceutical composition is about as stable as the same recombinant AAV in a reference pharmaceutical composition (e.g., when stored at -20oC or at 37 oC).
  • the recombinant AAV in the pharmaceutical composition is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as stable as the same recombinant AAV in a reference pharmaceutical composition (e.g., when stored at -20oC or at 37 oC).
  • the stability of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6.
  • a pharmaceutical composition provided herein is capable of being stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months without loss of stability as determined, e.g.by an assay or assays disclosed in Section 4.6.
  • a pharmaceutical composition provided herein is capable of being stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at 4 °C without loss of stability.
  • a pharmaceutical composition provided herein is capable of being stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at ⁇ 60 °C without loss of stability.
  • a pharmaceutical composition provided herein is capable of being stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at -80 °C without loss of stability. In certain embodiments, a pharmaceutical composition provided herein is capable of being stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at 4 °C after having been stored at -20 °C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months without loss of stability.
  • a pharmaceutical composition provided herein is capable of being first 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 -80 °C, then being thawed and, after thawing, being 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 additional months without loss of stability as determined, e.g., by an assay or assays disclosed in Section 4.6.
  • a pharmaceutical composition provided herein is capable of being first 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 -80 °C, then being thawed and, after thawing, being stored at about 4 °C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 additional months without loss of stability as determined, e.g., by an assay or assays disclosed in Section 4.6.
  • a pharmaceutical composition provided herein is capable of being first 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 ⁇ 60 °C, then being thawed and, after thawing, being stored at about 4 °C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 additional months without loss of stability as determined, e.g., by an assay or assays disclosed in Section 4.6.
  • Effects of the methods or pharmaceutical compositions provided herein may be monitored by measuring signs of vision loss, infection, inflammation and other safety events, including retinal detachment.
  • different pharmaceutical compositions having different viscosity and/or elastic modulus (G’) can be used to deliver the vector in the SCS.
  • vectors delivered using a pharmaceutical composition that has medium to high viscosity and/or elastic modulus (G’) at about 32-35°C are more effective than vectors delivered using a pharmaceutical composition that has a low viscosity and/or elastic modulus (G’) at about 32-35°C (e.g., when administered in the SCS).
  • vectors delivered using a formulation that has medium to high viscosity and/or elastic modulus (G’) at about 32-35°C results in improved vision as compared to vectors delivered using a formulation which has low viscosity and/or elastic modulus (G’) at about 32-35°C.
  • Effects of the methods or pharmaceutical compositions provided herein may also be measured by a change from baseline in National Eye Institute Visual Functioning Questionnaire, the Rasch-scored version (NEI-VFQ-28-R) (composite score; activity limitation domain score; and socio-emotional functioning domain score).
  • effects of the methods provided herein may also be measured by a change from baseline in National Eye Institute Visual Functioning Questionnaire 25-item version (NEI-VFQ-25) (composite score and mental health subscale score). In some embodiments, effects of the methods provided herein may also be measured by a change from baseline in Macular Disease Treatment Satisfaction Questionnaire (MacTSQ) (composite score; safety, efficacy, and discomfort domain score; and information provision and convenience domain score).
  • the efficacy of a method or vector (vector formulation) described herein is reflected by an improvement in vision at about 4 weeks, 12 weeks, 6 months, 12 months, 24 months, 36 months, or at other desired timepoints.
  • the improvement in vision is characterized by an increase in BCVA, for example, an increase by 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.
  • the improvement in vision is characterized by a 5%, 10%, 15%, 20%, 30%, 40%, 50% or more increase in visual acuity from baseline.
  • there is no inflammation in the eye after treatment or little inflammation in the eye after treatment for example, an increase in the level of inflammation by 10%, 5%, 2%, 1% or less from baseline).
  • a method of suprachoroidal administration for treating a pathology of the eye comprising administering to the suprachoroidal space in the eye of a human subject in need of treatment a recombinant viral vector comprising a nucleotide sequence encoding a therapeutic product such that the therapeutic product is expressed and results in treatment of the pathology of the eye.
  • the administering step is by injecting the recombinant viral vector into the suprachoroidal space using a suprachoroidal drug delivery device.
  • the suprachoroidal drug delivery device is a microinjector.
  • a pharmaceutical composition or a reference pharmaceutical composition provided herein is suitable for administration by one, two or more routes of administration (e.g., suitable for suprachoroidal and subretinal administration).
  • the vector genome concentration (VGC) of the pharmaceutical composition is 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 3.2 ⁇ 10 11 GC/mL, about 6.2 ⁇ 10 11 GC/mL, about 6.5 ⁇ 10 11 GC/mL, about 1 ⁇ 10 12 GC/mL, about 2.5
  • the vector genome concentration (VGC) of the pharmaceutical composition is about 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, about 1 ⁇ 10 10 GC/mL, about 2 ⁇ 10 10 GC/mL, about 3 ⁇ 10 10 GC/mL, about 4 ⁇ 10 10 GC/mL, about 5 ⁇ 10 10 GC/mL, about 6 ⁇ 10 10 GC/mL, about 7 ⁇ 10 10 GC/mL, about 8 ⁇ 10 10 GC/mL, about 9 ⁇ 10 10 GC/mL, about 1 ⁇ 10 11 GC/mL, about 2 ⁇ 10 11 GC/mL, about 3 ⁇ 10 11 GC/mL, about 4 ⁇
  • the volume of the pharmaceutical composition is any volume capable of reducing the minimum force to separate the sclera and choroid.
  • the volume of the pharmaceutical composition is about 50 ⁇ L to about 1000 ⁇ L, 50 ⁇ L to about 500 ⁇ L, 50 ⁇ L to about 400 ⁇ L, 50 ⁇ L to about 350 ⁇ L, 50 ⁇ L to about 300 ⁇ L, about 50 ⁇ L to about 275 ⁇ L, about 50 ⁇ L to about 250 ⁇ L, about 50 ⁇ L to about 225 ⁇ L, about 50 ⁇ L to about 200 ⁇ L, about 50 ⁇ L to about 175 ⁇ L, about 50 ⁇ L to about 150 ⁇ L, about 60 ⁇ L to about 140 ⁇ L, about 70 ⁇ L to about 130 ⁇ L, about 80 ⁇ L to about 120 ⁇ L, about 90 ⁇ L to about 110 ⁇ L, or about 100 ⁇ L.
  • SC suprachoroidal space
  • scleral flap technique catheters and standard hypodermic needles
  • microneedles A hollow-bore 750 um-long microneedle (Clearside Biomedical, Inc.) can be inserted at the pars, and has shown promise in clinical trials.
  • a microneedle designed with force-sensing technology can be utilized for SC injections, as described by Chitnis, et al. (Chitnis, G.D., et al. A resistance-sensing mechanical injector for the precise delivery of liquids to target tissue. Nat Biomed Eng 3, 621–631 (2019).
  • Oxular Limited is developing a delivery system (Oxulumis) that advances an illuminated cannula in the suprachoroidal space.
  • the Orbit device (Gyroscope) is a specially-designed system enabling cannulation of the suprachoroidal space with a flexible cannula.
  • a microneedle inside the cannula is advanced into the subretinal space to enable targeted dose delivery.
  • Ab interno access to the SCS can also be achieved using micro-stents, which serve as minimally-invasive glaucoma surgery (MIGS) devices.
  • MIGS minimally-invasive glaucoma surgery
  • 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., 29 gauge needle).
  • a microneedle or syringe is selected based on the viscosity of a pharmaceutical composition.
  • a microneedle is selected based on the pressure resulted in the eye (e.g., in the SCS) when a pharmaceutical composition is administered.
  • a pharmaceutical composition having medium or high viscosity and/or elastic modulus (G’) at about 32-35°C may benefit from the use of a wider microneedle for injection.
  • the pressure in the SCS is lower when a wider microneedle is used as compared to the pressure obtained when a narrower microneedle is used.
  • 10 gauge needle, 11 gauge needle, 12 gauge needle, 13 gauge needle, 14 gauge needle, 15 gauge needle, 16 gauge needle, 17 gauge needle, 18 gauge needle, 19 gauge needle, 20 gauge needle, 21 gauge needle, 22 gauge needle, 23 gauge needle, 24 gauge needle, 25 gauge needle, 26 gauge needle, 27 gauge needle, 28 gauge needle, 29 gauge needle, 30 gauge needle, 31 gauge needle, 32 gauge needle, 33 gauge needle, or 34 gauge needle is used.
  • a 27 gauge needle is used.
  • a 28 gauge needle is used.
  • a 29 gauge needle is used.
  • a 30 gauge needle is used.
  • a 31 gauge needle is used.
  • the pressure during administration of a 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.
  • the pressure during administration of a 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.
  • the pressure to open the SCS during administration of a 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.
  • the pressure during administration of a pharmaceutical composition is between 20 PSI and 50 PSI, 20 PSI and 75 PSI, 20 PSI and 40 PSI, 10 PSI and 40 PSI, 10 PSI and 100 PSI, or 10 PSI and 80 PSI.
  • a pharmaceutical composition provided herein is administered to the human eye with an injection pressure of less than 43 PSI. In some embodiments, a pharmaceutical composition provided herein is administered to the human eye with an injection pressure of a about 43 PSI. In some embodiments, a pharmaceutical composition provided herein is administered to the human eye with an injection pressure of about 43-65 PSI. In some embodiments, a pharmaceutical composition provided herein is administered to the human eye with an injection pressure of about 65 PSI.
  • a pharmaceutical composition provided herein is administered to the human eye with an injection pressure of less than 65 PSI. In some embodiments, a pharmaceutical composition provided herein is administered to the human eye with an injection pressure of about 65-100 PSI. In some embodiments, a pharmaceutical composition provided herein is administered to the human eye with an injection pressure of about 100 PSI. In some embodiments, a pharmaceutical composition provided herein is administered to the human eye with an injection pressure of less than 100 PSI. [00195] In some embodiments, a pharmaceutical composition provided herein is administered to the human eye in an injection time of about 5-10 seconds. In some embodiments, a pharmaceutical composition provided herein is administered to the human eye in an injection time of about 10-15 seconds.
  • a pharmaceutical composition provided herein is administered to the human eye in an injection time of about 15-20 seconds. In some embodiments, a pharmaceutical composition provided herein is administered to the human eye in an injection time of about 20-25 seconds. In some embodiments, a pharmaceutical composition provided herein is administered to the human eye in an injection time of about 25-30 seconds. In some embodiments, a pharmaceutical composition provided herein is administered to the human eye in an injection time of less than 30 seconds. In some embodiments, a pharmaceutical composition provided herein is administered to the human eye in an injection time which is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the length of the gelation time of the composition at extraocular temperature (about 32-35°C).
  • a concentration of the transgene product at a Cmin of at least 0.330 ⁇ g/mL in the eye (e.g., Vitreous humor), or 0.110 ⁇ g/mL in the Aqueous humour (the anterior chamber of the eye) for three months are desired; thereafter, Vitreous Cmin concentrations of the transgene product ranging from 1.70 to 6.60 ⁇ g/mL, and/or Aqueous Cmin concentrations ranging from 0.567 to 2.20 ⁇ g/mL should be maintained.
  • the transgene product is continuously produced (under the control of a constitutive promoter or induced by hypoxic conditions when using an hypoxia-inducible promoter), maintenance of lower concentrations can be effective.
  • Transgene concentrations can be measured directly in patient samples of fluid collected from a bodily fluid, ocular fluid, vitreous humor, or the anterior chamber, or estimated and/or monitored by measuring the patient’s serum concentrations of the transgene product – the ratio of systemic to vitreal exposure to the transgene product is about 1:90,000. (e.g., see, Xu L, et al., 2013, Invest. Opthal. Vis. Sci.54: 1616-1624, at p.1621 and Table 5 at p.1623, which is incorporated by reference herein in its entirety).
  • dosages are measured by genome copies per ml (GC/mL) or the number of genome copies administered to the eye of the patient (e.g., administered suprachoroidally).
  • GC/mL genome copies per ml
  • 2.4 ⁇ 10 11 GC/mL to 1 ⁇ 10 13 GC/mL are administered
  • 2.4 ⁇ 10 11 GC/mL to 5 ⁇ 10 11 GC/mL are administered
  • 5 ⁇ 10 11 GC/mL to 1 ⁇ 10 12 GC/mL are administered
  • 1 ⁇ 10 12 GC/mL to 5 ⁇ 10 12 GC/mL are administered
  • 5 ⁇ 10 12 GC/mL to 1 ⁇ 10 13 GC/mL are administered.
  • 1.5 ⁇ 10 13 GC/mL to 3 ⁇ 10 13 GC/mL are administered.
  • about 2.4 ⁇ 10 11 GC/mL, about 5 ⁇ 10 11 GC/mL, about 1 ⁇ 10 12 GC/mL, about 2.5 ⁇ 10 12 GC/mL, about 5 ⁇ 10 12 GC/mL, about 1 ⁇ 10 13 GC/mL or about 1.5 ⁇ 10 13 GC/mL are administered.
  • 1 ⁇ 10 9 to 1 ⁇ 10 12 genome copies are administered.
  • 3 ⁇ 10 9 to 2.5 ⁇ 10 11 genome copies are administered.
  • 1 ⁇ 10 9 to 2.5 ⁇ 10 11 genome copies are administered.
  • 1 ⁇ 10 9 to 1 ⁇ 10 11 genome copies are administered. In specific embodiments, 1 ⁇ 10 9 to 5 ⁇ 10 9 genome copies are administered. In specific embodiments, 6 ⁇ 10 9 to 3 ⁇ 10 10 genome copies are administered. In specific embodiments, 4 ⁇ 10 10 to 1 ⁇ 10 11 genome copies are administered. In specific embodiments, 2 ⁇ 10 11 to 1.5 ⁇ 10 12 genome copies are administered. In a specific embodiment, about 3 ⁇ 10 9 genome copies are administered (which corresponds to about 1.2 ⁇ 10 10 GC/mL in a volume of 250 ⁇ l). In another specific embodiment, about 1 ⁇ 10 10 genome copies are administered (which corresponds to about 4 ⁇ 10 10 GC/mL in a volume of 250 ⁇ l).
  • about 6 ⁇ 10 10 genome copies are administered (which corresponds to about 2.4 ⁇ 10 11 GC/mL in a volume of 250 ⁇ l).
  • about 6.4 ⁇ 10 10 genome copies are administered (which corresponds to about 3.2 ⁇ 10 11 GC/mL in a volume of 200 ⁇ l).
  • about 1.3 ⁇ 10 11 genome copies are administered (which corresponds to about 6.5 ⁇ 10 11 GC/mL in a volume of 200 ⁇ l).
  • about 6.4 ⁇ 10 10 genome copies are administered per eye, or per dose, or per route of administration.
  • about 6.4 ⁇ 10 10 genome copies is the total number of genome copies administered.
  • about 1.3 ⁇ 10 11 genome copies are administered per eye, or per dose, or per route of administration. In some embodiments, about 1.3 ⁇ 10 11 genome copies is the total number of genome copies administered. In some embodiments, about 2.5 ⁇ 10 11 genome copies are administered per eye, or per dose, or per route of administration. In some embodiments, about 2.5 ⁇ 10 11 genome copies is the total number of genome copies administered. In some embodiments, about 5 ⁇ 10 11 genome copies are administered per eye, or per dose, or per route of administration. In some embodiments, about 5 ⁇ 10 11 genome copies is the total number of genome copies administered. In some embodiments, about 3 ⁇ 10 12 genome copies are administered per eye, or per dose, or per route of administration.
  • about 3 ⁇ 10 12 genome copies is the total number of genome copies administered.
  • about 1.6 ⁇ 10 11 genome copies are administered (which corresponds to about 6.2 ⁇ 10 11 GC/mL in a volume of 250 ⁇ l).
  • about 1.55 ⁇ 10 11 genome copies are administered (which corresponds to about 6.2 ⁇ 10 11 GC/mL in a volume of 250 ⁇ l).
  • about 1.6 ⁇ 10 11 genome copies are administered (which corresponds to about 6.4 ⁇ 10 11 GC/mL in a volume of 250 ⁇ l).
  • about 2.5 ⁇ 10 11 genome copies (which corresponds to about 1.0 ⁇ 10 12 in a volume of 250 ⁇ l) are administered.
  • about 2.5 ⁇ 10 11 genome copies are administered (which corresponds to about 2.5 ⁇ 10 12 GC/mL in a volume of 100 ⁇ l).
  • about 5 ⁇ 10 11 genome copies are administered (which corresponds to about 5 ⁇ 10 12 GC/mL in a volume of 200 ⁇ l).
  • about 1.5 ⁇ 10 12 genome copies are administered (which corresponds to about 1.5 ⁇ 10 13 GC/mL in a volume of 100 ⁇ l).
  • about 3 ⁇ 10 11 genome copies are administered (which corresponds to about 3 ⁇ 10 12 GC/mL in a volume of 100 ⁇ l).
  • about 6 ⁇ 10 11 genome copies are administered (which corresponds to about 3 ⁇ 10 12 GC/mL in a volume of 200 ⁇ l). In another specific embodiment, about 6 ⁇ 10 11 genome copies are administered (which corresponds to about 6 ⁇ 10 12 GC/mL in a volume of 100 ⁇ l). [00198] In certain embodiments, about 6.0 ⁇ 10 10 genome copies per administration, or per eye are administered. In certain embodiments, about 6.4 ⁇ 10 10 genome copies per administration, or per eye are administered. In certain embodiments, about 1.3 ⁇ 10 11 genome copies per administration, or per eye are administered. In certain embodiments, about 1.5 ⁇ 10 11 genome copies per administration, or per eye are administered.
  • about 1.6 ⁇ 10 11 genome copies per administration, or per eye are administered. In certain embodiments, about 2.5 ⁇ 10 11 genome copies per administration, or per eye are administered. In certain embodiments, about 3 ⁇ 10 11 genome copies per administration, or per eye are administered. In certain embodiments, about 5.0 ⁇ 10 11 genome copies per administration, or per eye are administered. In certain embodiments, about 6 ⁇ 10 11 genome copies per administration, or per eye are administered. In some embodiments, about 1.5 ⁇ 10 12 genome copies are administered per eye, or per dose, or per route of administration. In some embodiments, about 1.5 ⁇ 10 12 genome copies is the total number of genome copies administered. [00199] In certain embodiments, about 3 ⁇ 10 12 genome copies per administration, or per eye are administered.
  • about 1.0 ⁇ 10 12 GC/mL per administration, or per eye are administered. In certain embodiments, about 2.5 ⁇ 10 12 GC/mL per administration, or per eye are administered. In certain embodiments, about 3 ⁇ 10 12 GC/mL per administration, or per eye are administered. In certain embodiments, about 3.0 ⁇ 10 13 genome copies per administration, or per eye are administered. In certain embodiments, up to 3.0 ⁇ 10 13 genome copies per administration, or per eye are administered. [00200] In certain embodiments, about 1.5 ⁇ 10 11 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 2.5 ⁇ 10 11 genome copies per administration, or per eye are administered by suprachoroidal injection.
  • about 3 ⁇ 10 11 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 5.0 ⁇ 10 11 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 6 ⁇ 10 11 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 1.5 ⁇ 10 12 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 3 ⁇ 10 12 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 2.5 ⁇ 10 11 genome copies per eye are administered by a single suprachoroidal injection. In certain embodiments, about 3 ⁇ 10 11 genome copies per administration, or per eye are administered by a single suprachoroidal injection.
  • about 3 ⁇ 10 11 genome copies per administration, or per eye are administered by a single suprachoroidal injection in a volume of 100 ⁇ l. In certain embodiments, about 3 ⁇ 10 11 genome copies per administration, or per eye are administered by a single suprachoroidal injection in a volume of 200 ⁇ l. In certain embodiments, about 3 ⁇ 10 11 genome copies per administration, or per eye are administered by double suprachoroidal injections. In certain embodiments, about 3 ⁇ 10 11 genome copies per administration, or per eye are administered by double suprachoroidal injections, wherein each injection is in a volume of 50 ⁇ l.
  • about 3 ⁇ 10 11 genome copies per administration, or per eye are administered by double suprachoroidal injections, wherein each injection is in a volume of 100 ⁇ l. In certain embodiments, about 5.0 ⁇ 10 11 genome copies per administration, or per eye are administered by double suprachoroidal injections. In certain embodiments, about 6 ⁇ 10 11 genome copies per administration, or per eye are administered by a single suprachoroidal injection. In certain embodiments, about 6 ⁇ 10 11 genome copies per administration, or per eye are administered by a single suprachoroidal injection in a volume of 100 ⁇ l. In certain embodiments, about 6 ⁇ 10 11 genome copies per administration, or per eye are administered by a single suprachoroidal injection in a volume of 200 ⁇ l.
  • about 6 ⁇ 10 11 genome copies per administration, or per eye are administered by double suprachoroidal injections. In certain embodiments, about 6 ⁇ 10 11 genome copies per administration, or per eye are administered by double suprachoroidal injections, wherein each injection is in a volume of 50 ⁇ l. In certain embodiments, about 6 ⁇ 10 11 genome copies per administration, or per eye are administered by double suprachoroidal injections, wherein each injection is in a volume of 100 ⁇ l. In certain embodiments, about 3.0 ⁇ 10 13 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, up to 3.0 ⁇ 10 13 genome copies per administration, or per eye are administered by suprachoroidal injection.
  • about 2.5 ⁇ 10 12 GC/mL per eye are administered by a single suprachoroidal injection in a volume of 100 ⁇ l. In certain embodiments, about 2.5 ⁇ 10 12 GC/mL per eye are administered by double suprachoroidal injections, wherein each injection is in a volume of 100 ⁇ l. In certain embodiments, about 1.5 ⁇ 10 13 GC/mL per eye are administered by a single suprachoroidal injection in a volume of 100 ⁇ l. [00201] In certain embodiments, the recombinant viral vector is administered by double suprachoroidal injections.
  • the first injection in the right eye is administered in the superior temporal quadrant (i.e., between the 10 o’clock and 11 o’clock positions), and the second injection in the same eye is administered in the inferior nasal quadrant (i.e., between the 4 o’clock and 5 o’clock positions).
  • the first injection in the right eye is administered in the inferior nasal quadrant (i.e., between the 4 o’clock and 5 o’clock positions), and the second injection in the same eye is administered in the superior temporal quadrant (i.e., between the 10 o’clock and 11 o’clock positions).
  • the first injection in the left eye is administered in the superior temporal quadrant (i.e., between the 1 o’clock and 2 o’clock positions), and the second injection in the same eye is administered in the inferior nasal quadrant (i.e., between the 7 o’clock and 8 o’clock positions).
  • the first injection in the left eye is administered in the inferior nasal 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).
  • the recombinant viral vector is administered by a single suprachoroidal injection.
  • the single injection in the right eye is administered in the superior temporal quadrant (i.e., between the 10 o’clock and 11 o’clock positions). In certain embodiments, the single injection in the right eye is administered in the inferior nasal quadrant (i.e., between the 4 o’clock and 5 o’clock positions). In certain embodiments, the single injection in the left eye is administered in the superior temporal quadrant (i.e., between the 1 o’clock and 2 o’clock positions). In certain embodiments, the single injection in the left eye is administered in the inferior nasal quadrant (i.e., between the 7 o’clock and 8 o’clock positions).
  • the pharmaceutical composition or the reference pharmaceutical composition is administered to a human subject (e.g., suprachoroidally, subretinally, or intravitreously) 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.
  • the pharmaceutical composition or the reference pharmaceutical composition is administered to a human subject once in one day, twice in one day, three times in one day, four times in one day, five times in one day, six times in one day, or seven times in one day.
  • the same amount of AAV genome copies are administered per administration.
  • the same genome copies are administered suprachoroidally, subretinally, or intravitreously.
  • the same total amount of AAV genome copies are administered.
  • the same total amount of AAV genome copies are administered suprachoroidally, subretinally, or intravitreously regardless of the number of total administrations (e.g., if subretinal administration is performed once and suprachoroidal administration is performed twice, the genome copies in the one subretinal administration is the same as the genome copies in both suprachoroidal administrations combined).
  • the term “about” means within plus or minus 10% of a given value or range.
  • the recombinant vectors provided herein comprise the following elements in the following order: a) a constitutive or a hypoxia-inducible promoter sequence, and b) a sequence encoding the transgene (e.g., therapeutic product).
  • 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 a 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 the transgene, i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, l) a fifth linker sequence, and m) a second ITR sequence.
  • 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 a 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 the transgene, i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, l) a fifth linker sequence, and m) a second ITR sequence, wherein the transgene encodes a light chain and a heavy chain sequence separated by a cleavable F/F2A sequence.
  • the AAV (AAV viral vectors) provided herein comprise the following elements in the following order: a) a constitutive or a hypoxia-inducible promoter sequence, and b) a sequence encoding the transgene.
  • the AAV used for delivering the transgene should have a tropism for human retinal cells or photoreceptor cells.
  • Such AAV can include non-replicating recombinant adeno-associated virus vectors (“rAAV”), particularly those bearing an AAV8 capsid are preferred.
  • the viral vector comprises a signal peptide.
  • the signal peptide is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55).
  • the signal peptide is derived from IL-2 signal sequence.
  • the viral vector comprises a signal peptide from any signal peptide disclosed in Table 1, such as 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: 5); M
  • the viral vector or other expression construct suitable for packaging in an AAV capsid comprises (1) AAV inverted terminal repeats (ITRs) flank the expression cassette; (2) regulatory control elements, consisting essentially of one or more enhancers and/or promoters, d) a poly A signal, and e) optionally an intron; and (3) a transgene providing (e.g., coding for) one or more RNA or protein products of interest.
  • ITRs AAV inverted terminal repeats
  • regulatory control elements consisting essentially of one or more enhancers and/or promoters, d) a poly A signal, and e) optionally an intron
  • a transgene providing (e.g., coding for) one or more RNA or protein products of interest.
  • the disclosure provides for a nucleic acid for use, wherein the nucleic acid encodes a therapeutic product operatively linked to a promoter or enhancer-promoter described herein.
  • the disclosure provides for a nucleic acid for use, wherein the nucleic acid encodes a transgeneoperatively linked to a promoter selected from the group consisting of: the CB7 promoter (a chicken ⁇ -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.
  • the transgene is operatively linked to the CB7 promoter.
  • recombinant vectors that comprise one or more nucleic acids (e.g. polynucleotides).
  • the nucleic acids may comprise DNA, RNA, or a combination of DNA and RNA.
  • the DNA comprises one or more of the sequences selected from the group consisting of promoter sequences, the sequence encoding the therapeutic product of interest (the transgene), untranslated regions, and termination sequences.
  • recombinant vectors provided herein comprise a promoter operably linked to the sequence encoding the therapeutic product of interest.
  • nucleic acids e.g., polynucleotides
  • nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59:149-161).
  • the recombinant vectors provided herein comprise modified mRNA encoding for the therapeutic product of interest (e.g., the transgene).
  • the recombinant vectors provided herein comprise a nucleotide sequence encoding for a therapeutic product that is an shRNA, siRNA, or miRNA.
  • the vectors provided herein comprise components that modulate protein delivery.
  • the viral vectors provided herein comprise one or more signal peptides.
  • signal peptides include, but is 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), mutant interleukin-2 signal peptide (SEQ ID NO: 55).
  • the viral vectors provided herein are AAV based viral vectors.
  • the viral vectors provided herein are AAV8 based viral vectors.
  • the AAV8 based viral vectors provided herein retain tropism for retinal cells.
  • the AAV-based vectors provided herein encode the AAV rep gene (required for replication) and/or the AAV cap gene (required for synthesis of the capsid proteins). Multiple AAV serotypes have been identified.
  • AAV-based vectors provided herein comprise components from one or more serotypes of AAV.
  • AAV based vectors provided herein comprise capsid components from one or more 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
  • AAV based vectors provided herein comprise components from one or more of AAV8, AAV9, AAV10, AAV11, or AAVrh10 serotypes.
  • the recombinant viral vectors provided herein are altered such that they are replication-deficient in humans.
  • the recombinant viral vectors are hybrid vectors, e.g., an AAV vector placed into a “helpless” adenoviral vector.
  • provided herein are recombinant viral vectors comprising a viral capsid from a first virus and viral envelope proteins from a second virus.
  • the second virus is vesicular stomatitis virus (VSV).
  • the envelope protein is VSV-G protein.
  • AAV8 vectors comprising a viral genome comprising an expression cassette for expression of the transgene, under the control of regulatory elements and flanked by ITRs and a viral capsid that has the amino acid sequence of the AAV8 capsid protein or is 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 the biological function of the AAV8 capsid.
  • 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 the biological function of the AAV8 capsid.
  • the AAV that is used in the methods described herein is Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep.12(6): 1056-1068, which is incorporated by reference in its entirety.
  • the AAV that is used in the methods described herein comprises one of the following amino acid insertions: LGETTRP or LALGETTRP, as described in United States Patent Nos.9,193,956; 9458517; and 9,587,282 and US patent application publication no.2016/0376323, each of which is incorporated herein by reference in its entirety.
  • the AAV that is used in the methods described herein is AAV.7m8, as described in United States Patent Nos.9,193,956; 9,458,517; and 9,587,282 and US patent application publication no.2016/0376323, each of which is incorporated herein by reference in its entirety.
  • the AAV that is used in the methods described herein is any AAV disclosed in United States Patent No.9,585,971, such as AAV.PHP.B.
  • the AAV that is used in the methods described herein is an AAV disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos.7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282 US patent application publication nos.2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application Nos.
  • AAV8-based viral vectors are used in certain of the methods described herein. Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent No.7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No.8,318,480 B2, United States Patent No.8,962,332 B2 and International Patent Application No. PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety.
  • AAV e.g., AAV8-based viral vectors encoding a transgene.
  • recombinant AAV viral vectors have been shown to have particular tropism for ocular tissues and are efficient at transducing and expressing the transgene product of such recombinant AAV.
  • ocular-tropic vectors are useful in the methods and pharmaceutical compositions disclosed herein.
  • the method or pharmaceutical composition disclosed herein comprises an AAV viral vector having enhanced tropism for posterior segments of the eye, such as retina and RPE/choroid.
  • the method or pharmaceutical composition disclosed herein comprises a recombinant AAV3B viral vector.
  • the AAV vector is an AAV vector disclosed in PCT International Application No. PCT/US2021/054008 (PCT International Publication No. WO2022076711A2, published April 14, 2022), which is incorporated herein by reference in its entirety.
  • a single-stranded AAV ssAAV
  • a self-complementary vector e.g., scAAV
  • scAAV may be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82, McCarty et al, 2001, Gene Therapy, Vol 8, Number 16, Pages 1248- 1254; and U.S.
  • the viral vectors used in the methods described herein are adenovirus based viral vectors.
  • a recombinant adenovirus vector may be used to transfer in the transgene.
  • the recombinant adenovirus can be a first generation vector, with an E1 deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region.
  • the recombinant adenovirus can be a second generation vector, which contains full or partial deletions of the E2 and E4 regions.
  • a helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi).
  • the transgene is inserted between the packaging signal and the 3’ITR, with or without stuffer sequences to keep the genome close to wild-type size of approx.36 kb.
  • An exemplary protocol for production of adenoviral vectors may be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12:S18-S27, which is incorporated by reference herein in its entirety.
  • a vector for use in the methods described herein is one that encodes a transgene 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 sulfated variant of the transgene product or fragment is expressed by the cell.
  • a relevant cell e.g., a retinal cell in vivo or in vitro
  • the expressed transgene product or fragment comprises a glycosylation and/or tyrosine sulfation pattern.
  • the therapeutic products can be, for example, therapeutic proteins (for example, antibodies), therapeutic RNAs (for example, shRNAs, siRNAs, and miRNAs), or therapeutic aptamers.
  • the disclosure provides a pharmaceutical composition comprising recombinant AAV encoding a transgene.
  • rAAV viral vectors which do not encode an anti-VEGF Fab or anti-VEGF antibody.
  • rAAV8-based viral vectors which do not encode an anti-VEGF Fab or anti-VEGF antibody.
  • rAAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein.
  • TPP1 tripeptidyl peptidase 1
  • rAAV9-based viral vectors encoding TPP1.
  • rAAV8- based viral vectors encoding microdystrophin.
  • rAAV9- based viral vectors encoding microdystrophin.
  • rAAV viral vectors encoding anti- kallikrein (anti-pKal) protein encoding anti- kallikrein (anti-pKal) protein.
  • 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 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 are rAAV8-based or rAAV9-based viral vectors encoding CLN2. In some embodiments, provided herein are rAAV8-based or rAAV9-based viral vectors encoding CLN3.
  • rAAV8-based or rAAV9-based viral vectors encoding CLN6.
  • rAAV viral vectors encoding etanercept (an anti-TNF fusion protein).
  • rAAV viral vectors encoding adalimumab antibody (an anti-TNF antibody) or an antigen-binding fragment thereof.
  • rAAV viral vectors encoding a full-length anti-C3 or anti- C5 antibody, an anti-C3 or anti-C5 Fab, a complement factor H (CFH), or a complement factor H- like (CFHL-1) protein.
  • rAAV viral vectors encoding eculizumab, ravulizumab, tesidolumab, crovalimab, NGM621 or BB5.1 antibody, or an antigen- binding fragment thereof.
  • the therapeutic product e.g., transgene
  • the therapeutic product is: (1) Palmitoyl- Protein Thioesterase 1 (PPT1); (2) Tripeptidyl-Peptidase 1 (TPP1); (3) Battenin (CLN3); and (4) CLN6 Transmembrane ER Protein (CLN6).
  • the disclosure provides a pharmaceutical composition comprising recombinant AAV encoding a transgene.
  • rAAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein.
  • TPP1 tripeptidyl peptidase 1
  • rAAV9-based viral vectors encoding TPP1.
  • rAAV viral vectors encoding anti- kallikrein (anti-pKal) protein, such as lanadelumab.
  • provided herein are rAAV viral vectors encoding CLN2.
  • provided herein are rAAV viral vectors encoding CLN3.
  • rAAV viral vectors encoding CLN6 are provided herein.
  • the rAAV vector is an AAV vector encoding an antibody transgene disclosed in PCT International Application No. PCT/US2020/029802 (PCT International Publication No. WO2020219868A1, published October 29, 2020), which is incorporated herein by reference in its entirety.
  • the vectors provided herein can be used for (1) the pathology of the eye associated with Batten-CLN1 and the therapeutic product is Palmitoyl-Protein Thioesterase 1 (PPT1); (2) the pathology of the eye associated with Batten-CLN2 and the therapeutic product is Tripeptidyl-Peptidase 1 (TPP1); (3) the pathology of the eye associated with Batten-CLN3 and the therapeutic product is Battenin (CLN3); (4) the pathology of the eye associated with Batten-CLN6 and the therapeutic product is CLN6 Transmembrane ER Protein (CLN6); (5) the pathology of the eye associated with Batten-CLN7 and the therapeutic product is Major Facilitator Superfamily Domain Containing 8 (MFSD8); and (6) the pathology of the eye associated with Batten-CLN1 and the therapeutic product is Palm
  • the pharmaceutical composition or the reference pharmaceutical composition provided herein 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.
  • nAMD wet AMD
  • RVO retinal vein occlusion
  • DME diabetic macular edema
  • DR diabetic retinopathy
  • nAMD wet AMD
  • dry AMD retinal vein occlusion
  • DME diabetic macular edema
  • DR diabetic retinopathy
  • Batten administering to the subject a therapeutically effective amount of the pharmaceutical composition by suprachoroidal injection (for example, via a suprachoroidal drug delivery device such as a microinjector with a microneedle).
  • the pharmaceutical composition described herein is administered to a subject diagnosed with dry AMD, wherein the pharmaceutical composition comprises a rAAV vector encoding a transgene product that ameliorate sdisease pathology in the posterior segments of the eye.
  • Such pharmaceutical composition can slow or arrest the progression of or relieve one or more symptoms of dry AMD, such as to reduce the rate of geographic atrophy or improve visual acuity (or reduce the rate of loss of visual acuity).
  • the pharmaceutical composition can be administered suprachoroidally as a method to deliver transgene product to the retina and/or RPE- choroid or other posterior segments of the eye.
  • physical changes to the eye, including changes in geographic atrophy can be measured Optical Coherence Tomography using methods known in the art.
  • the compositions and methods described herein may be assessed for efficacy using in vitro complement inhibition assays, such as membrane attack complex (“MAC”) formation, C5a generation and hemolysis.
  • MAC membrane attack complex
  • Complement inhibition assays can be performed in any appropriate cell type, such as ARPE19 cells (MAC and C5a assays), iPSC-derived RPE cells (MAC and C5a assays) or sheep/rabbit erythrocytes (hemolysis assay).
  • MAC formation assays measure the deposition of MAC on the surface of RPE cells (% relative inhibition of MAC formation).
  • Hemolysis assays allow the comparison of complement inhibition among different complement inhibitors (50% complement inhibition dose (ng/ml) (CH50; AH50).
  • Animal models may be administered vectors described herein, for example, suprachoroidally, and then assessed for geographic atrophy (or change therein) by OCT, retinal pathology (damage or repair to RPE), and other assessments of dry AMD pathology, as well as reduction in C3a or C5a, cleavage of C3 or C5 or other markers of complement activation.
  • Animals and animal models can be administered the pharmaceutical compositions described herein, for example, suprachoroidally, and then assessed for concentration of the transgene product in target tissues (see e.g., Examples 2-7).
  • the patient has diabetic retinopathy.
  • the patient has dry AMD.
  • a pharmaceutical composition containing about 2.5 ⁇ 10 11 GC/eye, about 5 ⁇ 10 11 GC/eye, or about 1.5 ⁇ 10 12 GC/eye of a Construct (e.g., a construct of the disclosure) of a pharmaceutical composition comprising 10% w/v sucrose is administered to a patient via suprachoroidal administration.
  • the patient has diabetic retinopathy.
  • the patient has dry AMD.
  • the pharmaceutical composition has a tonicity/osmolality equal to or greater than 240 mOsm/kg.
  • disclosed herein are pharmaceutical compositions suitable for treating a subject diagnosed with kallikrein-related disease.
  • the pharmaceutical composition or the reference pharmaceutical composition provided herein can be administered to a subject diagnosed with (1) Batten-CLN2 and the therapeutic product is Tripeptidyl-Peptidase 1 (TPP1); (2) Usher’s-Type 1 and the therapeutic product is Myosin VIIA (MYO7A); (3) Usher’s-Type 1 and the therapeutic product is Cadherin Related 23 (CDH23); (4) Usher’s-Type 2 and the therapeutic product is Protocadherin Related 15 (PCDH15); (5) Usher’s-Type 2 and the therapeutic product is Usherin (USH2A); (6) Usher’s-Type 3 and the therapeutic product is Clarin 1 (CLRN1); (7) Stargardt’s and the
  • the skilled artesian may use the assays as described herein and/or techniques known in the art to study the composition and methods described herein, for example to test the formulations provided herein.
  • a skilled person may use any assay and/or techniques described in, for example, Chiang et a. “Clearance Kinetics and Clearance Routes of Molecules From the Suprachoroidal Space After Microneedle Injection”, Invest Ophthalmol Vis Sci.2017 Jan; 58(1): 545–554; Gu et al.
  • a high-frequency ultrasound (U/S) probe (UBM Plus; Accutome, Malvern, PA, USA) can be used to determine SCS thickness by generating 2D cross-sectional images of the SCS in animal eyes ex vivo after injecting different volumes ranging in viscosity and/or elastic modulus (G’) (e.g., from 25 ⁇ L to 500 ⁇ L ranging from low viscosity to high viscosity) at about 32-35°C.
  • G viscosity and/or elastic modulus
  • An U/S probe cover (Clearscan, Eye-Surgical-Instruments, Plymouth, MN) can be attached to the UBM Plus to facilitate U/S image acquisition.
  • the U/S probe can be used to acquire sagittal views around the eye (e.g., eight sagittal views).
  • Postprocessing of the U/S B-scans can be performed to find the thickness from the outer sclera to the inner retina at, for example, 1, 5, and 9 mm posterior to the scleral spur. The mean, median, and standard deviation for each eye can be calculated.
  • High-frequency (50 MHz) ultrasound E-technologies, Bettendorf, IA
  • E-technologies, Bettendorf, IA can be used to image the effect and distension of the injections on the SCS in real time.
  • the ultrasound probe can be positioned immediately over the injection site so that the adjacent sclera, SCS, and ciliary body/choroid can be imaged in real time during the injection. Images can be collected, and the maximal distance that the SCS is distended when injected with PBS or the pharmaceutical composition can be measured with internal calipers of the ultrasound.
  • This technique can also be used to determine the effect of physiologic intraocular pressure (IOP) on SCS distension after injection and to determine the changes of IOP after SCS injection.
  • a needle e.g., 27-gauge
  • cyano-acrylate tissue adhesive can be used to seal the limbal incision.
  • the needle can be connected with 0.9% saline-filled tubing to a pressure transducer (MedEx LogiCal Transducer, Model MX960; MedExSupply Medical Supplies, Monsey, NY) and an electronic monitor, allowing for continuous measurement of IOP. Maximal distension of the SCS can be measured and reported using internal calipers of the ultrasound. The IOP (in mm Hg) at the time of maximal SCS distension can be recorded for each injection volume.
  • a pressure transducer MedEx LogiCal Transducer, Model MX960; MedExSupply Medical Supplies, Monsey, NY
  • IOP in mm Hg
  • the porcine eye can be placed in a water bath, and microbubble contrast medium can be injected through catheters placed into the anterior SCS.
  • Dynamic contrast medium inflow can be observed in a midsagittal plane, followed by 3D imaging of the entire globe to assess spatial distribution of the contrast agent.
  • Percentage of maximal distribution in the SCS of contrast agent can be determined slice by slice using image analysis software (Elements 4.0 [Adobe Photoshop]; ImageJ 1.42q). 4.6.5 Measuring SCS Thickness Based on Liquid Volume [00244] 3D cryo-reconstruction imaging can be used to measure SCS thickness.
  • High- frequency ultrasound B-scan can be used to determine the rate of SCS collapse.
  • Eight sagittal views over the pars plana can be acquired: (a) supranasal, over the injection site; (b) superior; (c) nasal; (d) supratemporal; (e) temporal; (f) infratemporal; (g) inferior; and (h) infranasal.
  • Off-line post processing can be performed on the U/S views to measure the SCS thickness.
  • the U/S probe can have a minimum axial resolution of 15 ⁇ m. For each U/S view, a line segment 5 mm posterior to the scleral spur and perpendicular to the sclera can be created.
  • a line can start at the outer surface of the sclera and end at the inner surface of the retina.
  • the sclera and chorioretina can be included in the measurement to ensure the line is perpendicular.
  • SCS thickness is then calculated by subtracting the tissue thickness from the measured line length. Curve fitting is done to determine the rate of SCS collapse.
  • U/S B-scan can be used to determine SCS thickness at multiple locations over time and the rate of SCS collapse can be calculated.
  • the approximate clearance rate of injected fluorescent material from the SCS can be found by taking fluorescence fundus images in the animal eyes in vivo over time until fluorescence is no longer detected.
  • Topical eye drops of tropicamide and phenylephrine can be administered prior to each imaging session to dilate the eye.
  • a RetCam II (Clarity Medical Systems, Pleasanton, CA) with the 130° lens attachment and the built-in fluorescein angiography module can be used to acquire the images.
  • Multiple images can be taken with the blue light output from the RetCam II set at, for example, 0.0009, 1.6, and 2.4 W/m2.
  • nine images can be captured: central, supranasal, superior, supratemporal, temporal, infratemporal, inferior, infranasal, and nasal. This allows imaging into the far periphery.
  • Imaging can be done immediately after injection, at 1 h, every 3 h for 12 h, and every two days post-injection.
  • the total clearance time which can be defined as the first time point in which fluorescence is not detectable by visual observation, is determined for all eyes injected.
  • Fluorescein isothiocyanate-conjugated AAV (FITC-AAV), or FITC Conjugated-AAV capsid Protein-specific monoclonal antibody may be utilized in analogous experiments to track movement and clearance of AAV particles in the SCS.
  • Methods for fluorescent labeling of AAV are known in the art (Shi, et al. Sci. Adv.2020; 6 : eaaz3621; and Tsui, T. Y., et al.
  • compositions of the present disclosure containing fluorescein, or fluorescently labeled AAV are injected into the SCS. After SCS injection and freezing, eyes can be prepared to assess the 2D spread of particles and fluorescein. The frozen eye are sliced open from the limbus to the posterior pole to generate equidistant scleral flaps. The resulting scleral flaps are splayed open and the frozen vitreous humor, lens, and aqueous humor are removed.
  • a digital SLR camera (Canon 60D, Canon, Melville, N.Y.) with a 100 mm lens (Canon) can be used to acquire brightfield and fluorescence images. Camera parameters are held constant.
  • a green optical band-pass filter (520 ⁇ 10 nm; Edmunds Optics, Barrington, N.J.) can be placed on the lens, and the sample can be illuminated by a lamp with the violet setting of a multicolor LED bulb (S Series RGB MR16/E26. HitLights, Baton Rouge, La.).
  • a red filter (610 ⁇ 10 nm; Edmunds Optics) can be placed on the lens, and the sample can be illuminated with the same lamp switched to green light.
  • the area of green and red fluorescence that are above threshold can be calculated for each eye using ImageJ (National Institutes of Health, Bethesda, Md.). Thresholding can be set manually based on visual inspection of background signal.
  • Thresholding can be set manually based on visual inspection of background signal.
  • ophthalmic tonometer Teono-Pen; AVIA, Reichert Technologies, Depew, NY, USA
  • the intraocular pressure measurement can be stopped when the intraocular pressure is returned to the baseline level.
  • 4.6.10 Temperature Stress Assay [00253] A temperature stress development stability study can be conducted at 1.0 ⁇ 10 12 GC/mL over 4 days at 37 °C to evaluate the relative stability of formulations provided herein. Assays can be used to assess stability include but are not limited to in vitro relative potency (IVRP), vector genome concentration (VGC by ddPCR), free DNA by dye fluorescence, dynamic light scattering, appearance, and pH.
  • an in vitro bioassay may be performed by transducing HEK293 cells and assaying the cell culture supernatant for transgeneprotein levels.
  • HEK293 cells are plated onto three poly-D-lysine-coated 96-well tissue culture plates overnight.
  • the cells are then pre-infected with wild-type human Ad5 virus followed by transduction with three independently prepared serial dilutions of AAV vector reference standard and test article, with each preparation plated onto separate plates at different positions.
  • the cell culture media is collected from the plates and measured for transgeneprotein levels via ELISA.
  • ELISA 96-well ELISA plates coated with receptor are blocked and then incubated with the collected cell culture media to capture transgeneproduced by HEK293 cells.
  • Fab- specific anti-human IgG antibody is used to detect the transgeneprotein.
  • HRP horseradish peroxidase
  • an in vitro bioassay may be performed by transducing HEK293 cells and assaying for transgene (e.g. enzyme) activity.
  • HEK293 cells are plated onto three 96-well tissue culture plates overnight.
  • the cells are then pre-infected with wild-type human adenovirus serotype 5 virus followed by transduction with three independently prepared serial dilutions of enzyme reference standard and test article, with each preparation plated onto separate plates at different positions.
  • the cells are lysed, treated with low pH to activate the enzyme, and assayed for enzyme activity using a peptide substrate that yields increased fluorescence signal upon cleavage by transgene (enzyme).
  • the fluorescence or RFU is plotted versus log dilution, and the relative potency of each test article is calculated relative to the reference standard on the same plate fitted with a four-parameter logistic regression model after passing the parallelism similarity test, using the formula: EC50 reference ⁇ EC50 test article.
  • the potency of the test article is reported as a percentage of the reference standard potency, calculated from the weighted average of the three plates.
  • FITC-AAV Fluorescein isothiocyanate-conjugated AAV
  • FITC Conjugated-AAV capsid Protein-specific monoclonal antibody may be utilized in analogous experiments to track movement and clearance of AAV particles in the SCS.
  • Free DNA can be determined by fluorescence of SYBR® Gold nucleic acid gel stain (‘SYBR Gold dye’) that is bound to DNA. The fluorescence can be measured using a microplate reader and quantitated with a DNA standard. The results in ng/ ⁇ L can be reported.
  • SEC Size Exclusion Chromatography
  • the mobile phase can be, for example, 20 mM sodium phosphate, 300 mM NaCl, 0.005% poloxamer 188, pH 6.5, with a flow rate of 0.35 mL/minute for 20 minutes, with the column at ambient temperature.
  • Data collection can be performed with 2 point/second sampling rate and 1.2 nm resolution with 25 point mean smoothing at 214, 260, and 280 nm.
  • the ideal target load can be 1.5x10 11 GC.
  • the samples can be injected with 50 ⁇ L, about 1/3 of the ideal target or injected with 5 ⁇ L.
  • Dynamic light scattering (DLS) can be performed on a Wyatt DynaProIII using Corning 3540384 well plates with a 30 ⁇ L sample volume. Ten acquisitions each for 10 s can be collected per replicate and there can be three replicate measurements per sample.
  • the solvent can be set according to the solvent used in the samples, for example ‘PBS’ for an AAV vector in dPBS. Results not meeting data quality criteria (baseline, SOS, noise, fit) can be ‘marked’ and excluded from the analysis.
  • Viscosity can be measured using methods known in the art, for example methods provide in the United States Pharmacopeia (USP) published in 2019 and previous versions thereof (incorporated by reference herein in their entirety). Viscosity at low shear was measured using a capillary viscometer, using methods described in USP ⁇ 911>. [00264] Viscosity versus shear rate can be determined using a cone and plate rotational rheometer. Rheometry measurements are described in the United States Pharmacopeia (USP) USP ⁇ 1911> and rotational viscometry is described in USP ⁇ 912>.
  • Rotational rheometry viscosity measurements can be collected with an AR-G2 rheometer equipped with a Peltier temperature control plate with a 60 mm 1° angle aluminum cone accessory (TA Instruments, New Castle, DE).
  • a viscosity versus shear rate sweep can be performed over the range starting at ⁇ 0.3 s-1 ramped up to 5000 s -1 with 5 points per decade collected.
  • the viscosity versus shear rate was collected at 20°C. Viscosity at 10,000 and 20,000 s -1 were extrapolated from the data.
  • the viscosity of the pharmaceutical composition or the reference pharmaceutical composition can be measured at zero, 0.1 s -1 , 1 s -1 , 1000 s -1 , 5000 s-1, 10,000 s -1 , 20,000 s -1 , or more than 20,000 s -1 .
  • 4.6.17 Rheology Measurements [00266] Rheometry measurements are described in the United States Pharmacopeia (USP) USP ⁇ 1911> and rotational viscometry is described in USP ⁇ 912>.
  • DSF data was collected using a Promethius NTPlex Nano DSF Instrument (NanoTemper technologies, Munich, Germany). Samples were loaded into the capillary cell at 20°C and the temperature ramped at a rate of 1°C/min to 95°C. The signal output ratio of emission at 350 nm (unfolded) and 330 nm (unfolded) was used to determine the T m .
  • 4.6.20 Injection Pressure Measurements [00273] Injection pressures were measured using either a Flow Screen and Fluid Sensor (Viscotec America, Kennesaw, GA) or a PressureMAT-DPG with single use pressure sensor S-N-000 (PendoTECH, Princeton, NJ).
  • compositions were either performed manually or using a Legato-100 syringe pump (Kd Scientific, Holliston, MA) to apply a consistent flow rate.
  • the eyes were mounted on a Mandell eye mount (Mastel) with applied suction to adjust the introcular pressure of the eye.
  • the viscosity of a composition provided herein may be evaluated by comparing the composition to a reference pharmaceutical composition.
  • the reference pharmaceutical composition is a pharmaceutical composition comprising the same type and amount of recombinant AAV as the composition being evaluated, but is not a thermoresponsive composition.
  • the reference pharmaceutical composition is a pharmaceutical composition comprising the same type and amount of recombinant AAV as the composition being evaluated, but has a lower viscosity and/or elastic modulus at extraocular temperature (about 32- 35°C) than the composition being evaluated.
  • the reference pharmaceutical composition is a pharmaceutical composition comprising the same recombinant AAV in the same concentration as the composition being evaluated in phosphate-buffered saline.
  • the reference pharmaceutical composition is a pharmaceutical composition comprising the same recombinant AAV in the same concentration as the composition being evaluated in Dulbecco’s phosphate buffered saline with 0.001% poloxamer 188, pH 7.4.
  • the reference pharmaceutical composition is a pharmaceutical composition comprising the same recombinant AAV in the same concentration as the composition being evaluated in Dulbecco’s phosphate buffered saline with 4% sucrose and 0.001% poloxamer 188, pH 7.4.
  • a reference pharmaceutical composition may be administered by the same route or a different route as the composition being evaluated. In some embodiments, the reference pharmaceutical composition is administered suprachoroidally.
  • a pharmaceutical composition described herein is prepared by a method comprising: a) preparing a pre-gel solution comprising between about 10% and about 30% poloxamer 407 and between about 2% and about 15% poloxamer 188, b) heating the pre-gel solution in an autoclave, c) optionally diluting the pre-gel solution with water, and d) mixing the pre-gel solution with a concentrated solution of a recombinant adeno-associated viral vector described herein.
  • the pre-gel solution in step a) comprises between about 18% and about 22% poloxamer 407.
  • the pre-gel solution in step a) comprises about 20% poloxamer 407. In some embodiments, the pre-gel solution in step a) comprises between about 5% and about 9% poloxamer 188. In one embodiment, the pre-gel solution in step a) comprises about 7.2% poloxamer 188. In one embodiment, the pre-gel solution in step a) comprises about 20% poloxamer 407 and about 7.2% poloxamer 188. [00279] In some embodiment, the pre-gel solution is heated to a temperature of between about 80 oC and about 150 oC in step b). In some embodiments, the pre-gel solution is heated to a temperature of between about 115 oC and about 125 oC in step b).
  • the pre-gel solution is heated to a temperature of about 120 oC in step b). In one embodiment, the pre-gel solution is heated to a temperature of about 121 oC in step b). [00280] In some embodiments, the heating in step b) results in loss of material (e.g. water) and/or volume from the pre-gel solution. In one embodiment, the heating in step b) results in a water loss of between about 1% and about 5% from the pre-gel solution. In one embodiment, the water loss in step b) is about 3%.
  • material e.g. water
  • the heating in step b) results in a water loss of between about 1% and about 5% from the pre-gel solution. In one embodiment, the water loss in step b) is about 3%.
  • the volumes of the pre-gel and concentrated recombinant adeno- associated viral vector solutions mixed in step d) are selected such that the concentration of the recombinant adeno-associated viral vector in the final mixture is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 10 times, about 12 times, about 15 times, or about 20 times lower than in the concentrated recombinant adeno-associated viral vector solution.
  • the concentration of the recombinant adeno-associated viral vector in the final mixture is about 10 times less than in the concentrated recombinant adeno- associated viral vector solution.
  • the volumes of the pre-gel and concentrated recombinant adeno- associated viral vector solutions mixed in step d) are selected such that the final mixture comprises between about 15% and about 25% poloxamer 407. In one embodiment, the final mixture comprises about 18% poloxamer 407. In some embodiments, the volumes of the pre-gel and concentrated recombinant adeno-associated viral vector solutions mixed in step d) are selected such that the final mixture comprises between about 3% and about 10% poloxamer 188. In one embodiment, the final mixture comprises about 6.5% poloxamer 188. In one embodiment, the final mixture comprises about 18% poloxamer 407 and about 6.5% poloxamer 188. 5.
  • the suprachoroidal space (SCS) is a region between the sclera and the choroid that expands upon injection of the drug solution (Habot-Wilner, 2019). See also FIG.1.
  • the SCS space recovers to its pre-injection size as the injected solution is cleared by physiologic processes.
  • the drug solution diffuses within SCS and is absorbed into adjacent tissues.
  • AAV adeno-associated virus
  • Initial design parameters for formulation feasibility are subject to adjustment with additional data and described here to demonstrate the starting point for formulation optimization.
  • Injection to the eye is a sensitive injection procedure as the eye is a critical organ.
  • the needle gauge cannot be too high when injecting a drug in the eye in order to avoid pain, tissue damage, or inflammation.
  • a 30 gauge can be selected and in other cases, a 29 gauge is selected.
  • For injection into the eye it must be possible to inject the solution through a very narrow bore needle within a specified time frame before it gels and plugs the needle and injection site.
  • the formulation should gel at the temperature of the eye.
  • the temperature of the surface of the eye has been reported as 34.5 ⁇ 0.8°C (Tkacova et al., 2011, , MEASUREMENT 2011, Proceedings of the 8th International Conference, Smolenice, Slovakia).
  • FIG.3. Shows a measured extraocular temperature at 33.1°C using a thermal camera (FLIR, model T530).
  • the temperature of the surface of the eye can be taken as a worst case for the suprachoroidal space temperature, which may be slightly warmer than the surface.
  • the limit for gelation temperature is the temperature of the eye (34.5 ⁇ C).
  • the average minus three standard deviations of the eye temperature is 32°C. Therefore, a preferred gelation temperature of ⁇ 32°C was used a design parameter to ensure that gelation will occur in the eye.
  • the thermoresponsive gel should be an injectable liquid when at a temperature between refrigerated conditions (2-8°C) and controlled room temperature (20°C). Ideally, for ease of dose preparation and manufacturing, the gel temperature should be sufficiently above room temperature to allow for variability in room temperature and for practical mixing, pumping and other operations to be performed.
  • a gelation temperature of ⁇ 27°C might be more easily handled.
  • the higher gelation temperature ( ⁇ 27°C) is preferred but is not a rigid requirement because the dose could be chilled to a lower temperature for dose preparation and if needed also chilled before administration.
  • the formulation should have an acceptable viscosity that will allow for an injection using commonly available syringe components (i.e. an injection pressure limit based on syringe pressure-rating limitations). There are conflicting design parameters to balance between gel-forming temperature and time and injectability.
  • the desired needle size is 30 or 29 gauge, and this will impact the pressure of the injection. Gelation should not occur before the injection is complete to avoid clogging the needle or injection site.
  • the gelation time should be greater than the injection time to avoid clogging the needle or injection site and should be less than the expected clearance time of 10 minutes (600 s) for pressure driven reflux reported in the literature (Chiang, IOVS, 2017, 58 (1) 545 – 554).
  • An initial design preferred target of ⁇ 90 seconds is considered (if feasible) for the formulation evaluation as a very conservative gelation time compared to the 600 s reflux clearance time expected.
  • Medium and longer-term clearance of AAV from the SCS spaces by blood flow, erosion of the gel, diffusion and convention is expected to be significantly reduced after gelation has occurred.
  • a conversion factor of PSI Pa/6894.76 was used to convert to PSI.
  • a combination of poloxamer 407 and poloxamer 188 was evaluated in a design of experiments approach to identify if there is a formulation composition with a gel temperature in desired target range while also limiting the viscosity to a level that can be injected through a 30 or 29 gauge needle.
  • the impact of the composition of the hydrogel formulation on the gelation temperature, and the gelation time and viscosity at both 5°C and 20°C was assessed using a response surface central composite design of experiments (DOE) approach utilizing IMP 15 software (Cary, NC).
  • DOE response surface central composite design of experiments
  • a DOE approach was needed based on the number of formulation design limitations required that have differing responses to composition changes and to enable to determination of one or more optimal target formulations.
  • Pol oxamer 407 was varied from 16% to 22 % w/v and pol oxamer 188 from 0% to 16 % w/v with a duplicate center point.
  • Gelation can be determined using different methods. Some method may measure the onset of gelation and others may measure an objective ‘crossover’ in material properties as the gelation temperature. For the purposes of the formulation design the crossover in G’ and G” as a function of temperature was taken as the gelation temperature for consistency and objectivity. The raw data shows that the onset of gelation occurs at a slightly lower temperature than the crossover.
  • Table 4 The data was fit in JMP DOE software to produce a response surface for the different measurements, FIG.4. shows the gelation temperature as a function of formulation composition response surface, Responses surfaces for viscosity as a function of composition are shown in FIG.5 (20°C) and FIG.6. (5°C) and a summary of the raw shear rate sweep data is shown in FIG.12. and FIG.13.. [00302] The raw data for gelation profiles are summarized in FIG.7, and a specific example for how the gelation temperature is determined as the crossover in G’ and G” for sample #9 shown in FIG.8. The raw data shows that the onset of gelation occurs at a slightly lower temperature than the crossover.
  • FIG.9. and FIG.10. show the G’ versus time used to determine the gelation times for the samples exposed to a temperature jump from 5°C and 20 ⁇ C to 34°C respectively.
  • FIG.11.. shows the specific example for sample #9.
  • the data were taken as a whole to evaluate and optimize the potential formulation design space for all the design parameters summarized in Table 1.
  • FIG.14. shows a thermoresponsive gel formulation design space (white area) with limits of 27 to 32°C for gel temperature.
  • Modified Dulbecco’s phosphate-buffered saline with sucrose solution was used as a control (100 mM sodium chloride, 2.70 mM potassium chloride, 8.10 mM sodium phosphate dibasic anhydrous, 1.47 mM potassium phosphate monobasic, 117 mM sucrose, 0.001% (0.01 mg/mL) poloxamer 188).
  • the spike solutions were prepared slightly more concentrated by a ratio of 10/9 or 1.11-fold so that they can be spiked at a ratio of 9/10 with a ratio of 1/10 of the AAV intermediate to achieve the desired final composition, for example. Other dilution ratios could also be used.
  • Viscous formulations can be difficult to sterile filter, so they were sterilized by autoclave at 121°C for 20 min (suitable for up to 200 mL; longer times may be used for larger volumes, such as 40 min for 2000 mL).
  • a ‘liquids’ cycle was used that gradually reduces the pressure when the temperature is reduced to prevent ‘boiling-over’ and a bottle with a cap equipped with a sterile filter was used to allow the steam to enter the bottle while maintaining sterility for then transferred to a sterile hood for filling (example caps include Chemglass CLS1484-12, or Sartorius MYCAPTM series of caps or equivalent).
  • the onset for gelation is also shown and ranged from 27 to 30° and the plateau for compete gelation ranged from 29°C to 33°C. These all fall within the design space for gelation to occur at or below the temperature of the eye ( ⁇ 34.5°C).
  • the time to gel ranged from 16 to 29 s for the three formulations, also within the initial design parameters.
  • the viscosity of the three formulations were ⁇ 183 mPas at 20°C, also within the design parameters.
  • the profiles for gelation of the formulations A, B, and C are shown in FIG.19, FIG, 20., and FIG.21. The onset of gelation occurs about 2°C below the crossover point and the change in G’ over the entire gelation covers 6 to 7 orders of magnitude increase in elastic modulus.
  • FIG.18. shows a different method of assessing gelation time.
  • a 50 ⁇ L volume of formulation A (left), B (middle) and C (right) at 20°C were dispensed on the warm surface and a video of the flow of the droplet used to determine the time that the droplet stopped flowing.
  • the times to gel using this approach were about 10s (A), 25s (B), and 45s (C).
  • the rheology profiles for gelation time starting at 20°C and 5°C with a jump to 34°C are shown in FIG.22 through to FIG 27.
  • Formulations may be slightly diluted after injection before they become fully gelled, especially at the periphery of the bolus injection.
  • Formulations A, B, and C were placed in a way towards the upper-right of the design space that will allow for a dilution (slight down and left movement in design space) along the isothermal lines for gelation (see FIG. 15). Therefore, the formulations gelation properties will be robust and maintained if there is some slight dilution after injection. The main integrity of the bolus for these formulations is expected to be maintained given how rapidly they will gel.
  • Table 5 Rheological and Thermal Properties of Formulations A, B, and C Table 6: Impact of Dilution on the Gel Temperature and Gel Time for Formulations A, B, and C Table 7: Impact of Autoclave Sterilization on the Gel Temperature and Time 5.
  • Stress Stability of a Construct in Formulations A, B and C [00311] Differential scanning fluorimetry (DSF) measures the intrinsic tryptophan and tyrosine emission of proteins as a function of temperature. The local environment of Trp and Tyr residues changes as the protein unfolds resulting in a large increase in fluorescence.
  • FIG.37. shows differential scanning fluorimetry thermal ramp data for a control compared to formulations A, B, and C. Top panel: raw melting curve signal.
  • Table 8 summarizes the results that the formulations A, B and C have a similar thermal stability to the control formulation and that the AAV is therefore stable.
  • Table 8 Differential Scanning Fluorometry Melting Temperature of the Construct AAV Capsid in the Different Gel Formulations Compared to the Control [00312]
  • Table 9 summarizes the impact of 37°C stress on the stability of AAV in formulations A, B and C compared to a control.
  • the free DNA detected which is a measure of AAV instability resulting in capsid disruption with release of the DNA payload was low and similar to the initial level accounting for method variability for all samples after 3 days at 37°C.
  • the vector genome concentration (by ddPCR) of the formulations A, B, and C was measured on samples diluted 3-fold to reduce the viscosity. The results had some variability, likely related to the fact they were diluted by volume rather than by mass (since they have high viscosity the dilution may have introduced variability).
  • the 3 day formulation A and B samples had similar vector genome concentrations to the control and overall indicate they were stable. The variability in dilution can be seen in the initial sample of formulation B being higher than the target and the 3 day sample of formulation C being lower.
  • FIG.38 shows the injection pressure for injection of formulation B into an enucleated porcine eye equilibrated at 35°C using a Clearside syringe device (CLS-HN001) and 30 gauge (160 ⁇ m ID, CLS-MN1100) needle. The formulation was easily injected into the eye, although the pressure was about 160 PSI.
  • FIG.39 shows injection into air using a BD 1 mL syringe (309628) with 30 GG*1/2 inch (0.3 * 13 mm) TW needle (Nipro, HN-3013-ET).
  • the Clearside device and needle are designed with a specific exposed needle length for suprachoroidal injection.
  • formulation C was injected over 12 s with about 120 PSI with the 30 GaTW (165 ⁇ m ID) needle, which will reduce to about 27 PSI if a 240 ⁇ m ID needle is used.
  • Formulation B was injected over 17 s with about 120 PSI, which will also reduce to about 27 PSI with a larger needle. If formulation B is injected more quickly, such as over 12 s , then a larger need ID pressure of about 38 PSI is expected. These are well within the injection pressure design space identified.
  • DSF measures the intrinsic tryptophan and tyrosine emission of proteins as a function of temperature.
  • the local environment of Trp and Tyr residues changes as the protein unfolds resulting in a large increase in fluorescence.
  • the temperature where 50% of proteins are unfolded is defined as the ‘melting’ temperature (T m ).
  • T m melting temperature
  • Fluorescence spectroscopy is described in the USP ⁇ 853> and USP ⁇ 1853>.
  • DSF data was collected using a Promethius NT.Plex Nano DSF Instrument (NanoTemper technologies, Kunststoff, Germany). Samples were loaded into the capillary cell at 20°C and the temperature ramped at a rate of 1°C/min.to 95°C.
  • Injection Pressure Measurements were measured using either a Flow Screen and Fluid Sensor (Viscotec America, Kennesaw, GA) or a PressureMAT-DPG with pressure sensor S-N-000 (PendoTECH, Princeton, NJ). [00324] Injections into air were either performed manually or using a Legato-100 syringe pump (Kd Scientific, Holliston, MA) to apply a consistent flow rate.
  • test articles and control articles are stored in a freezer between -60°C and -80°C and thawed at room temperature on the day of use.
  • the formulations are thawed at room temperature and stored on cold packs until used for syringe filling.
  • Animals are anesthetized with ketamine and dexmedetomidine prior to suprachoroidal injection.
  • two suprachoroidal injections of 50 ⁇ L (Groups 1 and 2) is administered to each eye (between 3 and 4 mm from the limbus) over 10 to 15 seconds.
  • the first injection is in the right eye administered in the superior temporal quadrant (i.e., between the 10 o’clock and 11 o’clock positions), and the second injection in the right eye (as applicable) is administered in the inferior nasal quadrant (i.e., between the 4 o’clock and 5 o’clock positions).
  • the first injection in the left eye is administered in the superior temporal quadrant (i.e., between the 1 o’clock and 2 o’clock positions), and the second injection in the left eye (as applicable) is administered in the inferior nasal quadrant (i.e., between the 7 o’clock and 8 o’clock positions).
  • the needle is kept in the eye for approximately 30 seconds before being withdrawn.
  • Aqueous Humor Collection approximately 50 ⁇ L is removed from each eye at least 2 weeks prior to administration, on Day 15, and on the day the scheduled sacrifice (Day 29). Aqueous humor samples from each eye are placed into separate tubes with Watson barcoded labels, snap frozen in liquid nitrogen, and placed on dry ice until stored between -60°C and -80°C. [00327] Termination of Study: animals are anesthetized with sodium pentobarbital and exsanguinated on Day 29.
  • Bone marrow smears were collected on Day 29, and collected (if possible) from animals sacrificed at an unscheduled interval.
  • Necropsy Collections of Aqueous Humor and Vitreous Humor Following exsanguination, eyes are enucleated and aqueous humor and vitreous humor samples are collected. Following collection, samples were flash-frozen and stored between -60°C and -80°C. Aqueous and vitreous samples are analyzed for transgene concentration by a validated method.
  • Ocular Tissue Collection for Biodistribution following exsanguination, the right eye from each animal and the left eye from the last two animals (depending on survival) are enucleated and tissues are collected.
  • Tissues are collected into separate tubes. Collected tissue include choroid with retinal pigmented epithelium, retina, and sclera. Tissues are collected using ultra-clean procedures as described above, and rinsed with saline and blotted dry. Samples are flash-frozen and stored between -60°C and -80°C. Samples are analyzed for vector DNA or RNA using a qPCR or qRT-PCR method.
  • Comparator study in a Cynomolgous monkey study conducted analogously to the protocols described in this Example, a control formulation (control article 1.5) is injected to the SCS of the each eye of the subjects (temporal superior and nasal inferior injection with microinjector). The aqueous control formulation does not form a gel.
  • Table 11 Preparation of Control Formulation [00331] Data Evaluation and Statistical Analysis: statistical data analyses is calculated using means and standard deviations. Transgene product (protein) in aqueous humor is assessed at 15 and 29 days, otherwise TP, DNA and RNA was assessed in vitreous humor at 29 days. 5.3
  • Example 3 Suprachoroidal Formulation: Pharmacodynamic/ Biodistribution, and Toxicity Study in Pigs [00332] This example relates to the evaluation of pharmacodynamic/biodistribution and toxicity of different formulations of a test article (e.g., AAV.GFP) in animals (e.g., minipigs, such as Yucatan minipig) after a single suprachoroidal injection.
  • a test article e.g., AAV.GFP
  • animals e.g., minipigs, such as Yucatan minipig
  • three (3) pigs receive each test article formulation via bilateral suprachoroidal injections into the superior temporal quadrant (performed once).
  • the animals are analyzed at least twice daily for signs of overt discomfort such as severe blepharospasm, severe conjunctival hyperemia, epiphora, excessive rubbing at the eye, and not eating. If these conditions persist for 12 hours then the pigs are humanely euthanized.
  • Preparation steps are as follows: 1. Attach vial adapter to Clustering Stock and Clustering Diluent vials 2. Withdraw active from Clustering Stock vial ( ⁇ 200 ⁇ L) 3. Prime clustering stock to 100 ⁇ L 4. Add 100 ⁇ L of clustering stock to Empty Vial and remove syringe. 5. Withdraw clustering diluent from Clustering Diluent vial ( ⁇ 1100 ⁇ L) 6. Prime clustering diluent to 900 ⁇ L 7. Add 900 ⁇ L of clustering diluent to Empty Vial (which has 100 ⁇ L of the clustering stock in it from step 4) and remove syringe. 8.
  • animals e.g., minipigs; 10 Yucatan (females 4-6 months old)
  • a topical mydriatic (1.0% tropicamide HCl is applied.
  • Buprenorphine 0.01-0.05 mg/kg
  • meloxicam 0.4 mg/kg
  • IM intramuscularly
  • Animals are anesthetized with ketamine (10 mg/kg) / dexmedetomidine (0.05 mg/kg) IM.
  • the area around the eye, including the eyelid, is cleaned with a 10% solution of baby shampoo using gauze, rinsed with sterile saline, topical application of 5% betadine solution, and rinsed again with sterile saline.
  • the animal receives one drop each of 0.5% proparacaine HCl and 10.0% phenylephrine HCl topically in both eyes.
  • a sterile cotton-tipped applicator soaked in 1% betadine the conjunctival cul-de-sacs in both eyes and under the third eyelid is swabbed, followed by rinsing with sterile saline.
  • test articles are administered by suprachoroidal space (SCS) injection using a 30-gauge needle approximately 900 or 1100 ⁇ m in length.
  • SCS suprachoroidal space
  • injections are delivered with 1 mL BD needle and MedOne 900 ⁇ m (or 1100 ⁇ m) needles are used first for injection.
  • needles selected based on the first animal dosed are used. Injections are performed bilaterally and delivered to supra-temporal quadrant 4 mm from the limbus between 10 and 11 o’clock in the right eye and between 1 and 2 o’clock in the left eye.
  • a topical antibiotic (neo-poly gramicidin or equivalent) is administered.
  • OCT with enhanced depth imaging (EDI) is performed acquiring approximately 15 b-scans spanning the dosing site.
  • An en-face blue autofluorescence image is then performed while the animal remains sedated and in the same position.
  • a second OCT is done spanning the visual streak (ON placed in the top 3rd of the view), followed by a blue auto-fluorescence image. Animals receive atipamezole IM, if needed, to reverse the effects of dexmedetomidine and are allowed to recover normally from the procedure.
  • a second drop of topical antibiotic is given following the imaging and also at 6 hours later.
  • Prophylactic antibiotics are administered BID for 2 additional days with 6-8 hours between doses.
  • Parameters Measured animals are assessed for mortality or morbidity twice daily, morning and afternoon, and body weights are acquired prior to dosing, once weekly, and at termination.
  • Ocular Eye Examinations (OEs) pupils are dilated for ocular examination using topical 1% tropicamide HCL (one drop in each eye 15 minutes prior to examination). Complete OEs using a slit lamp biomicroscope and indirect ophthalmoscope are used to evaluate ocular surface morphology, anterior and posterior segment at each timepoint as indicated in Table 12.
  • the modified Ralphett and McDonald ocular grading system with additional scoring parameters for the ocular posterior segment are used to grade inflammation (Hackett, R.B. and McDonald, T.O. Ophthalmic Toxicology and Assessing Ocular Irritation. Dermatoxicology, Fifth Edition. Ed. F.N. Marzulli and H.I. Maibach. Washington, D.C.: Hemisphere Publishing Corporation.1996; 299-305 and 557-566). If high levels of ocular inflammation are noted by the veterinarian ophthalmologist after Day 8 examinations, treatment of the eyes with difluprednate BID (4-8 hours) for the remainder of the study is considered. In some cases, difluprednate treatment is required.
  • Tonometry intraocular pressure (IOP) is measured in both eyes at the timepoints indicated in Table 12. Measurements are performed in non-sedated animals. The measurements are taken using a Tonovet probe (iCare Tonometer, Espoo, Finland) without use of topical anesthetic. The tip of the Tonovet probe is directed to gently contact the central cornea. The average IOP shown on the display is recorded, and three measurements are determined.
  • OCT Optical Coherence Tomography
  • Blood Collections for Serum serum collection for tab analysis: at baseline (prior to dosing) and just prior to euthanasia, at least 1.2 mL of whole blood is drawn from the jugular vein and deposited into non-anticoagulant tubes (1.3 mL) for serum collection from all animals. Following collection, the tubes are gently mixed by inverting the tubes 3-5 times. Blood samples are stored at room temperature for at least 30 minutes, but less than 60 minutes, prior to processing.
  • Euthanasia while remaining under sedation, animals are euthanized with an intravenous injection of an AVMA-approved barbiturate-based euthanasia agent (e.g., Euthasol). Death is ensured by auscultation.
  • Serum samples are split into 2 tubes per collection (2 mL polypropylene screw cap tube) 2.
  • Whole blood samples are split into 2 tubes per collection (2 mL DNAse/RNAse free polypropylene tubes) 3.
  • Aqueous humor (2 mL polypropylene tube) 4.
  • Vitreous humor (5-7 mL polypropylene tube) 5.
  • Sclera 1 tube per collection (5-7 mL polypropylene tube) 6.
  • Optic Nerve 2 tubes (2 mL polypropylene screw cap tube) 7.
  • Retina 1 tube per collection (2 mL polypropylene screw cap tube) 8.
  • RPE-Choroid 1 tube per collection (2 mL polypropylene screw cap tube) 9.
  • Iris-ciliary body 1 tube (2 mL polypropylene screw cap tube) 10.
  • Optic chiasm 2 tubes (2 mL polypropylene screw cap tube) 11.
  • Occipital lobe 3 tubes (3) 6-7 mm biopsy punches) per collection (2 mL polypropylene tubes) 12.
  • Frontal cortex 3 tubes (3) 6-7 mm biopsy punches) per collection (2 mL polypropylene tubes) [00375]
  • Group 0 Immediately after euthanasia, both eyes are enucleated, and the injection site is marked with a tissue marker. Aqueous humor from both eyes are removed via a 27 or 30-gauge syringe, weighed, and snap frozen by immersing in liquid nitrogen.
  • Vitreous humor is first collected using a 23–25-gauge needle with a 3 ml syringe. The needle is inserted 2 mm below the limbus into the central vitreous avoiding contact with the lens. VH is slowly collected and placed in a 2 ml polypropylene tube. Care is taken to not apply too much vacuum pressure. The Iris-ciliary body is then be collected. The lens is removed and the eye is cut into 4 quadrants based on the marker (superior temporal (dose site), inferior-temporal, superior nasal, and inferior nasal).
  • Iris-ciliary body 1 tube (2 mL polypropylene screw cap tube) 7.
  • Optic chiasm 2 tubes (2 mL polypropylene screw cap tube) 8.
  • Occipital lobe 3 tubes (3) 6-7 mm biopsy punches) per collection (2 mL polypropylene tubes) 9.
  • Non-Ocular Tissue Collections (All Groups): following enucleation, non-ocular tissues are collected and held for potential analysis. Clean collection techniques are utilized in order to avoid cross-contamination, and two samples of each tissue are taken. Samples are snap frozen and stored at -80°C prior to analysis.
  • List of Tissues for Collection 1. Liver (6-7 mm biopsy punch, left lateral lobe) (3 samples) (2 mL polypropylene tubes) 2. Lacrimal gland main (2) (2 mL polypropylene tubes) 3. Mandibular lymph nodes (2) (2 mL polypropylene tubes) 4.
  • Example 5 Suprachoroidal Formulation: Exploratory Study in Pigs
  • PK/PD pharmacokinetic/pharmacodynamic
  • a secreted transgene product e.g. IgG or Fab
  • animals e.g., minipigs
  • This example further aims to characterize PK/PD of full-length and fragment antibody in the eye and to assess TP concentrations in ocular compartments.
  • the study is performed analogously to previous Example, having the study design as illustrated in Table 16.
  • Table 16 Study Outline for Lanadelumab Dosing [00381] Administration of formulated AAV.Lanadelumab is administered either subretinally or suprachoroidally, as indicated in the above table.
  • Low dose SCS 3 ⁇ 10 11 GC/eye
  • High dose SCS 5 ⁇ 10 11 - 10 ⁇ 10 11 GC/eye or 7 ⁇ 10 11 GC/eye.
  • Low dose SR 1 ⁇ 10 10 - 3 ⁇ 10 10 GC/eye; High dose SR: 3 ⁇ 10 10 - 7 ⁇ 10 10 GC/eye.
  • Ophthalmic Exams/IOPs Predose (1 week after AH humor collection): ⁇ Day 3, and Weeks 1, 2, 4, 6, and 8 postdose On days of Aqueous humor tap, OEs is conducted prior to tap.
  • Aqueous Humor Collection Predose, and ⁇ Weeks 2, 4, 6, 8 postdose
  • Color Imaging/ Fluorescein Angiography Predose (all), Day 1 after dosing (SR animals only) and ⁇ Weeks 2, 4, 8 postdose. Area centralis, temporal and nasal fields. For SCS, attempts are made to image the temporal superior quadrant at all intervals (dose delivery site)
  • OCT Optical Coherence Tomography
  • the following tissues are collected: - Aqueous humor - Vitreous humor - Optic Nerve - Iris-ciliary body - Optic chiasm - Occipital lobe - Frontal cortex - Cornea - Lens Samples are collected fresh; for posterior segment 48-mm punches and remaining posterior eye cup are collected and separated into retina, ,RPE/choroid, and sclera. Non-ocular tissues are also collected and examined: o Liver (left lateral lobe; 3 samples) o Lacrimal gland main (2) o Mandibular lymph nodes (2) o Parotid lymph node (2) 5.6.
  • EXAMPLE 6 Pharmacodynamic, Biodistribution, And Tolerability Study In Cynomolgus Monkeys Using Different Suprachoroidal Formulations
  • the objective of this study was to evaluate the biodistribution (DNA and mRNA), pharmacodynamics (transgene concentration), and tolerability of Gel Formulation B comprising AAV8 construct when administered as a single dose via suprachoroidal injection to Cynomolgus monkeys. After dosing, animals were observed postdose for at least 4 weeks. Each group was administered two injections to achieve the same dose volume. The group assignment and dose levels were shown in Table 17.
  • the test article was an AAV8 construct expressing a transgene product (TP).
  • the control article was a placebo.
  • Table 17 Group Assignment and Dose Levels [00384] Dose Administration: the preparation of test articles and control articles are shown in Table 18. The test articles and control articles were stored in a freezer between -60°C and -80°C and thawed at room temperature on the day of use. The formulations were thawed at room temperature and stored on cold packs until used for syringe filling. Animals were anesthetized with ketamine and dexmedetomidine prior to suprachoroidal injection. In administration, two suprachoroidal injections of 50 ⁇ L (Groups 1 and 2) was administered to each eye (between 3 and 4 mm from the limbus) over 10 to 15 seconds. The syringe and microneedle size are shown in Table 18.
  • the first injection in the right eye was administered in the superior temporal quadrant (i.e., between the 10 o’clock and 11 o’clock positions), and the second injection in the right eye (as applicable) was administered in the inferior nasal quadrant (i.e., between the 4 o’clock and 5 o’clock positions).
  • the first injection in the left eye was administered in the superior temporal quadrant (i.e., between the 1 o’clock and 2 o’clock positions), and the second injection in the left eye (as applicable) was administered in the inferior nasal quadrant (i.e., between the 7 o’clock and 8 o’clock positions).
  • the needle was kept in the eye for approximately 30 seconds before being withdrawn.
  • TP Analysis blood samples were taken as discussed above once predose, and on the day of scheduled sacrifice (Day 29). Serum samples were analyzed for antibodies to TP using a validated antibody assay. For TP transgene analysis, blood samples were taken as described above at least two weeks prior to administration, on Day 15, and on the day of scheduled sacrifice (Day 29). Samples were then analyzed by a validated antibody assay. [00386] Aqueous Humor Collection: approximately 50 ⁇ L was removed from each eye at least 2 weeks prior to administration, on Day 15, and on the day the scheduled sacrificed (Day 29).
  • Aqueous humor samples from each eye were placed into separate tubes with Watson barcoded labels, snap frozen in liquid nitrogen, and placed on dry ice until stored between -60°C and -80°C. Samples were analyzed for TP concentration by a validated method.
  • Termination of Study animals were anesthetized with sodium pentobarbital and exsanguinated on Day 29. Bone marrow smears were collected on Day 29, and collected (if possible) from animals sacrificed at an unscheduled interval.
  • Necropsy Collections of Aqueous Humor and Vitreous Humor Following exsanguination, eyes were enucleated and aqueous humor and vitreous humor samples were collected.
  • Transgene product (protein) in aqueous humor was assessed at 15 and 29 days, otherwise TP, DNA and RNA was assessed in vitreous humor at 29 days.
  • Results Table 20 Aqueous Humor Transgene Product (ng/mL)
  • Two injections of Test article 1 (gel formulation) into the SCS of the eye at the temporal superior and nasal inferior resulted in greater transgene product (TP) concentration in the aqueous humor compared to the Control Formulation.
  • Table 21 Vitreous Humor Transgene Product (ng/mL)
  • Test Article 1 injected into the SCS at the temporal superior and nasal inferior location of the eye results in a greater TP concentration in the VH compared to Control Formulation.
  • Vitreous humor transgene product concentration was higher overall than TP found in aqueous humor at 15 days and 29 days following injection.
  • Table 22 Serum Transgene Product (ng/mL)
  • Test article 1 (gel) or Control formulation containing TP injected into the SCS produced minimal titers of transgene product in the serum.
  • Table 23 DNA or RNA (copies/ ⁇ g) Biodistribution in Tissues [00397] Test Article 1 (gel) had an impact on delivery to the retina and choroid, compared to the Control formulation.
  • Example 7 Suprachoroidal Formulation: Pharmacodynamic/ Biodistribution Study in Minipigs [00398] This example evaluated the pharmacodynamic and biodistribution of clustering formulation of AAV.GFP in minipigs after a single suprachoroidal injection. Methods and materials used in this example were substantially the same as the methods and materials disclosed in Example 3 of the present disclosure [00399] This example primarily evaluated whether the Gel B formulation was associated with increased protein levels and DNA in retina or RPE/choroid compared to the sucrose formulation. This example further evaluated the in vivo imaging techniques for monitoring the presence of formulation in SCS space, the in vivo imaging techniques for expression monitoring, and the distribution of transgene product (TP), vector DNA and RNA.
  • TP transgene product
  • Minipigs were chosen for this example because of the similarity to humans in eye size, sclera, blood supply, and retina of humans. Study design of this example is shown in Table 24. Table 24: Group Assignment and Dose Levels [00400] This experiment found that in vivo imaging techniques and SCS route of administrations were feasible in minipigs. No abnormalities were observed on imaging or during ophthalmic exams for Gel B formulation for SCS administration. All minipigs had either negative or low titers serum TAbs at pre-dose, which did not affect biodistribution (BD). [00401] SCS space was detected following administration of Gel B formulation (FIG.40) .
  • the BD pattern of vector DNA in different tissues was sclera > RPE/choroid > retina, which was consistent with the BD pattern in cynomolgus monkeys shown in Example 6.
  • the gel formulation had higher RPE/choroid BD than sucrose formulation.
  • the BD was comparable between formulations, except that the Gel B appeared to be higher in choroid for vector DNA.
  • the Gel B formulations had higher retina and choroid BD than sucrose formulation.
  • the BD in cynomolgus monkeys was higher than in minipigs, except for sucrose formulation in retina.
  • the GFP protein transgene product (TP) concentrations in tissues were also measured (cohorts a and b) and shown in Table 26. OD was collected for TP quantification. BLQ was 18.8 pg/ml/mg total protein. Data distribution and averages from cohorts a and b were comparable. Gel B had higher (8.5X) mean TP concentration in retina when compared to sucrose formulation. Transgene product of Gel B was also slightly higher in choroid based on these data.
  • TP transgene product

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Abstract

La présente invention concerne des compositions pharmaceutiques destinées à être administrées à un espace suprachoroïdien d'un œil d'un sujet. Ces compositions pharmaceutiques peuvent comprendre un virus adéno-associé (AAV) recombinant codant un transgène. La présente invention concerne également des méthodes de traitement ou de prévention d'une maladie chez un sujet par administration d'une quantité thérapeutiquement efficace desdites compositions pharmaceutiques au sujet dont l'état le nécessite.
PCT/US2023/065358 2022-04-06 2023-04-05 Formulations pour administration suprachoroïdienne, telles que formulations de gel WO2023196835A1 (fr)

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