WO2021050649A1 - Methods of treating ocular neovascular diseases using aav2 variants encoding aflibercept - Google Patents

Methods of treating ocular neovascular diseases using aav2 variants encoding aflibercept Download PDF

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Publication number
WO2021050649A1
WO2021050649A1 PCT/US2020/050079 US2020050079W WO2021050649A1 WO 2021050649 A1 WO2021050649 A1 WO 2021050649A1 US 2020050079 W US2020050079 W US 2020050079W WO 2021050649 A1 WO2021050649 A1 WO 2021050649A1
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Prior art keywords
eye
unit dose
raav particles
weeks
administration
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PCT/US2020/050079
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English (en)
French (fr)
Inventor
Mehdi Gasmi
Szilard Kiss
Aaron Osborne
Adam TURPCU
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Adverum Biotechnologies Inc
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Adverum Biotechnologies Inc
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Priority claimed from PCT/US2019/062066 external-priority patent/WO2021050094A1/en
Priority to FIEP20780467.5T priority Critical patent/FI4027983T3/fi
Priority to MX2022002961A priority patent/MX2022002961A/es
Priority to LTEPPCT/US2020/050079T priority patent/LT4027983T/lt
Priority to AU2020345915A priority patent/AU2020345915A1/en
Priority to KR1020227011745A priority patent/KR20220062353A/ko
Priority to CN202080063266.4A priority patent/CN114375195A/zh
Priority to CA3148376A priority patent/CA3148376A1/en
Priority to EP25180005.8A priority patent/EP4696318A1/en
Priority to HRP20260137TT priority patent/HRP20260137T1/hr
Priority to BR112022004027A priority patent/BR112022004027A2/pt
Priority to ES20780467T priority patent/ES3063491T3/es
Priority to DK20780467.5T priority patent/DK4027983T3/da
Application filed by Adverum Biotechnologies Inc filed Critical Adverum Biotechnologies Inc
Priority to RS20260196A priority patent/RS67774B1/sr
Priority to EP20780467.5A priority patent/EP4027983B1/en
Priority to SM20260070T priority patent/SMT202600070T1/it
Priority to JP2022515692A priority patent/JP2022547541A/ja
Publication of WO2021050649A1 publication Critical patent/WO2021050649A1/en
Priority to IL291182A priority patent/IL291182A/en
Anticipated expiration legal-status Critical
Priority to JP2025077233A priority patent/JP2025118798A/ja
Ceased legal-status Critical Current

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    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
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Definitions

  • a method for reducing retinal fluid in an eye of an individual with an ocular neovascular disease comprising administering a unit dose of rAAV particles to one eye of the individual, wherein the individual is a human, and wherein the rAAV particles comprise: (a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs), and (b) an AAV2 capsid protein comprising, or consisting of, an amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein.
  • ITRs AAV2 inverted terminal repeats
  • the method comprises administering the unit dose of rAAV particles to the one eye of the individual about 1 week or about 7 days after administration of the anti-VEGF agent. In some embodiments, the method comprises administering the unit dose of rAAV particles to the one eye of the individual about 1 week to about 2 weeks after administration of the anti- VEGF agent. In some embodiments, the method comprises administering the unit dose of rAAV particles to the one eye of the individual about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days after administration of the anti-VEGF agent.
  • the topical steroid treatment comprises about four administrations of topical steroid per day for about one month, followed by about three administrations of topical steroid per day for about one month, followed by about two administrations of topical steroid per day for about one month, and followed by about one administration of topical steroid per day for about one month; timing starting with and following administration of the anti-VEGF agent.
  • the unit dose of rAAV particles administered to the contralateral eye of the individual comprises the same or less vector genomes per eye (vg/eye) than the unit dose of rAAV particles administered to the one eye of the individual.
  • the administering the unit dose of rAAV particles to the contralateral eye is at least about 2 weeks after administering the unit dose of rAAV particles to the one eye.
  • the unit dose of rAAV particles administered to the contralateral eye of the individual comprises more vector genomes per eye (vg/eye) than the unit dose of rAAV particles administered to the one eye of the individual.
  • the pharmaceutical formulation comprises about 150 to about 200 mM sodium chloride, about 1 to about 10 mM monobasic sodium phosphate, about 1 to about 10 mM dibasic sodium phosphate, about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188, and about 6 c 10 13 to about 6 c 10 10 vector genomes (vg) per mL (vg/mL) of the rAAV particles, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.5.
  • the pharmaceutical formulation comprises about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, about 6 c 10 11 vg/mL of the rAAV particles, and about 0.001% (w/v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3.
  • the topical steroid treatment comprises about four administrations of topical steroid per day for about 3 weeks after administration of the unit dose of rAAV particles, followed by about 3 administrations of topical steroid per day for about 1 week, followed by about 2 administrations of topical steroid per day for about 1 week, and followed by about 1 administration of topical steroid per day for about 1 week.
  • AAV2.7m8-aflibercept is a recombinant, replication-deficient adeno-associated viral (rAAV) vector containing the AAV2.7m8 protein capsid and a vector genome containing an expression cassette of a codon-optimized version of the aflibercept cDNA under the control of a ubiquitous chimeric promoter (Cl 1).
  • the AAV2.7m8-aflibercept vector genome also contains two AAV2 inverted terminal repeat sequences (ITR) flanking the aflibercept cDNA expression cassette.
  • FIG. IB is a diagram summarizing the study design for the phase I study described in Examples 1 and 2.
  • FIG. 2C provides OCT images and retinal thickness maps derived from OCT images taken from Subject 2 at five office visits at the times indicated prior to the Screening aflibercept injection. OCT images were taken immediately prior to treatment with aflibercept standard of care. Subject 2 required six aflibercept IVT treatments in the 8 months prior to AAV2.7m8-aflibercept treatment to maintain retinal anatomy.
  • FIG. 2C provides OCT images and retinal thickness maps derived from OCT images taken from Subject 2 at five office visits at the times indicated prior to the Screening aflibercept injection. OCT images were taken immediately prior to treatment with aflibercept standard of care. Subject 2 required six aflibercept IVT treatments in the 8 months prior to AAV2.7m8-aflibercept treatment to maintain retinal anatomy.
  • FIG. 2F provides OCT images and retinal thickness maps derived from OCT images taken from Subject 3 at the Screening aflibercept injection (Day -7), the AAV2.7m8-aflibercept injection (Day 1), and at follow-up visits at the times indicated. Subject 3 did not require any rescue injections after the AAV2.7m8- aflibercept injection. Subject 3 exhibited resolution of refractory subretinal fluid by week 8, and stable retinal anatomy through week 24.
  • FIG. 2G provides OCT images and retinal thickness maps derived from OCT images taken from Subject 4 at five office visits at the times indicated prior to the Screening aflibercept injection.
  • FIG. 6 shows plots of anterior chamber cell and vitreous cell counts following treatment with AAV2.7m8-aflibercept for subjects 1-6 of the study described in Example 1.
  • the steroid treatment administered to each patient is indicated below each plot.
  • Aqueous cell count categories were based on the Standardization of Uveitis Nomenclature (SUN) criteria (Jabs, DA etal., J Ophthalmol. 2005; 140:509- 516). Vitreous cell count categories were based on National Institutes of Health (NIH) guidelines.
  • SUN Uveitis Nomenclature
  • FIGS. 9A-9B show optical coherence tomography (OCT) images and retinal thickness maps derived from OCT images taken from subjects 1-6 in Cohort 1 of the study described in Example 1 at a median follow-up time of 44 weeks.
  • OCT optical coherence tomography
  • FIGS. 9A-9B show optical coherence tomography (OCT) images and retinal thickness maps derived from OCT images taken from subjects 1-6 in Cohort 1 of the study described in Example 1 at a median follow-up time of 44 weeks.
  • the change in BCVA ETDRS letters and CST from Baseline are also provided.
  • the actual weeks during which the OCT images and retinal thickness maps were obtained for each subject are indicated.
  • the asterisk indicates that Subject 4 underwent retinal detachment repair with gas bubble injection in order to repair a spontaneous pseudophakic inferior macula off rhegmatogenous retinal detachment (RRD) (FIG. 9B).
  • Subject 4 remains under follow-up; OCT images and BC
  • FIG. 17A shows the mean BCVA (ETDRS letters) from baseline up to Week 36.
  • FIG. 17B shows the mean CST (pm) from baseline up to Week 36.
  • FIG. 19 is a Swimmer’s Lane Plot showing the number of anti-VEGF injections administered to subjects in Cohorts 1, 2 and 3 of the study described in Examples 1-5.
  • the x-axis represents time in weeks relative to the time AAV2.7m8-aflibercept was administered.
  • the y-axis shows each individual subject in Cohorts 1-3.
  • the circular shapes represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept.
  • the vertical line bisecting the plot indicates Day 1, when AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical line are each of the subsequent study visits.
  • FIG. 26 is a Swimmer’s Lane Plot showing the number of anti-VEGF injections administered to subjects in Cohorts 1, 2, 3, and 4 of the study described in Examples 1-5 and 7.
  • the x-axis represents time in weeks relative to the time AAV2.7m8-aflibercept was administered.
  • the y-axis shows each individual subject in Cohorts 1-4.
  • the circular shapes represent anti-VEGF IVT injections administered before and after treatment with AAV2.7m8-aflibercept.
  • the vertical line bisecting the plot indicates Day 1, when AAV2.7m8-aflibercept was administered. To the right of the bisecting vertical line are each of the subsequent study visits.
  • pharmaceutically acceptable can refer to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of a compound disclosed herein, and is relatively nontoxic (i.e., when the material is administered to an individual it does not cause undesirable biological effects nor does it interact in a deleterious manner with any of the components of the composition in which it is contained).
  • polypeptide can encompass both naturally occurring and non-naturally occurring proteins (e.g., a fusion protein), peptides, fragments, mutants, derivatives and analogs thereof.
  • a polypeptide may be monomeric, dimeric, trimeric, or polymeric. Further, a polypeptide may comprise a number of different domains, each of which has one or more distinct activities. For the avoidance of doubt, a “polypeptide” may be any length greater two amino acids.
  • a variant can have, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence homology to its counterpart reference protein. In some embodiments, a variant can have at least about 90% overall sequence homology to the wild-type protein. In some embodiments, a variant exhibits at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% overall sequence identity.
  • expression vector or “expression construct” or “cassette” or “plasmid” or simply “vector” can include any type of genetic construct, including AAV or rAAV vectors, containing a nucleic acid or polynucleotide coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and is adapted for gene therapy.
  • the transcript can be translated into a protein. In some embodiments, the transcript is partially translated or not translated.
  • expression includes both transcription of a gene and translation of mRNA into a gene product. In other aspects, expression only includes transcription of the nucleic acid encoding genes of interest.
  • the amino acid sequence FAFGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 570-611 of the AAV2 capsid protein, e.g., between positions 587 and 588 of the AAV2 capsid protein, VP1.
  • the amino acid sequence FAFGETTRPA (SEQ ID NO: 1) is inserted into the GH loop of the AAV2 capsid protein, e.g., between positions 587 and 588 of AAV2 VP1 comprising the sequence of SEQ ID NO: 13.
  • the amino acid sequence LALGETTRPA (SEQ ID NO: 1) is inserted into the GH loop within amino acids 571-612 of the AAV1 capsid protein, e.g., between amino acids 590 and 591 of the AAV1 capsid protein.
  • AAV vectors to efficiently transduce target retinal cells following IVT injection has been exploited to successfully transfer therapeutic genes into photoreceptors, retinal pigment epithelium, and the inner retina to treat a variety of retinal diseases.
  • administration of rAAV particles encoding an anti-VEGF agent can provide prolonged and/or sustained release of the anti-VEGF agent in vivo.
  • the present disclosure provides methods of treating an ocular neovascular disease in an individual by administering a single unit dose of 6 c 10 11 vg/eye or less of rAAV particles encoding an anti-VEGF agent (e.g., aflibercept).
  • an anti-VEGF agent e.g., aflibercept
  • the present disclosure provides methods for reducing retinal fluid in the eye of an individual with an ocular neovascular disease by administering a single unit dose of rAAV particles encoding an anti-VEGF agent (e.g., aflibercept).
  • the methods disclosed herein reduce or eliminate the need for repeated IVT injections while providing long-term efficacy, thereby addressing the non-compliance and non-adherence problem.
  • the methods provided herein reduce the adverse effects associated with multiple IVT injections.
  • the individual exhibited a meaningful response in central subfield thickness to the prior treatments with an anti-VEGF agent in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles, for example, at least a 10% reduction in central subfield thickness.
  • the individual did not experience an adverse reaction to the prior treatments with an anti-VEGF agent prior to administration of the unit dose of rAAV particles.
  • the individual does not have neutralizing antibodies to AAV2.7m8 prior to administration of the unit dose of rAAV particles.
  • the individual has not initiated intensive insulin treatment, e.g., with an insulin pump or multiple daily insulin injections, prior to administration of the unit dose of rAAV particles.
  • the individual does not plan to initiate intensive insulin treatment, e.g., with an insulin pump or multiple daily insulin injections, within about 3 months after administration of the unit dose of rAAV particles.
  • the individual does not have a history of systemic autoimmune disease that requires treatment with systemic steroids or immunosuppressive treatments, e.g., methotrexate or adalimumab, prior to administration of the unit dose of rAAV particles.
  • the individual has not received an anti-VEGF therapy (e.g., aflibercept IVT injections) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles.
  • an anti-VEGF therapy e.g., aflibercept IVT injections
  • the individual has not received an anti-VEGF therapy (e.g., aflibercept IVT injections) in the eye administered the rAAV particles for at least 60 days prior to administration of the unit dose of rAAV particles.
  • the individual has not received more than two anti-VEGF treatments (e.g., aflibercept IVT injections) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles.
  • the individual does not have a history of intraocular or periocular steroid treatment for any ocular condition (e.g., IVT Triesence, Iluvien or Ozurdex) in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles.
  • the individual has not had refractive surgery in the eye administered the rAAV particles within at least about 90 days prior to administration of the unit dose of rAAV particles.
  • the individual does not have previous penetrating keratoplasty, endothelial keratoplasty, or ocular radiation in the eye administered the rAAV particles prior to administration of the unit dose of rAAV particles.
  • the unit dose of rAAV particles is administered to one eye of the individual.
  • the one eye of the individual is the right eye or the left eye.
  • the one eye of the individual is the right eye.
  • the one eye of the individual is the left eye.
  • the methods provided herein further comprise administering a unit dose of rAAV particles to the contralateral eye of the individual.
  • the one eye of the individual is the right eye and the contralateral eye is the left eye.
  • the one eye of the individual is the left eye and the contralateral eye is the right eye.
  • the rAAV particles comprise a nucleic acid comprising a codon-optimized sequence encoding an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs). In some embodiments, the rAAV particles comprise a nucleic acid comprising a codon-optimized sequence encoding an amino acid sequence with 100% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs).
  • the rAAV particles comprise an AAV2 capsid protein comprising the amino acid sequence LALGETTRPA (SEQ ID NO: 1) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein.
  • the pharmaceutical formulation comprises about 6 10 12 vg/mL of rAAV particles, about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, and about 0.001% (w/v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3.
  • the pharmaceutical formulation comprises about 6 10 11 vg/mL of rAAV particles, about 180 mM sodium chloride, about 5 mM monobasic sodium phosphate, about 5 mM dibasic sodium phosphate, and about 0.001% (w/v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.3.
  • the ophthalmic steroid treatment is a topical steroid treatment (e.g., a drop), a periocular steroid treatment (e.g., subtenons, subconjunctival), an intravitreal steroid treatment, or a superchoroidal steroid treatment.
  • a topical steroid treatment e.g., a drop
  • a periocular steroid treatment e.g., subtenons, subconjunctival
  • an intravitreal steroid treatment e.g., intravitreal steroid treatment
  • a superchoroidal steroid treatment e.g., a superchoroidal steroid treatment.
  • the steroid treatment is administered before administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and during administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during, and after administration of the unit dose of rAAV particles.
  • the topical steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the topical steroid is any of about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% difluprednate. In some embodiments, the topical steroid is difluprednate 0.05%. In some embodiments, a dose of difluprednate 0.05% is one drop of ophthalmic solution.
  • one drop is about 50 m ⁇ (e.g., about 25 m ⁇ to about 50 pi, about 50 m ⁇ to about 100 m ⁇ ).
  • a dose of difluprednate comprises about 1 pg to about 5 pg, or about 2 pg to about 3 pg, or about 2.5 pg difluprednate. In some embodiments, a dose of difluprednate comprises about 2.5 pg difluprednate.
  • the retinal fluid is subretinal fluid (SRF) or intraretinal fluid (IRF). In some embodiments, the retinal fluid is subretinal fluid (SRF). In some embodiments, the retinal fluid is intraretinal fluid (IRF).
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 5 pm to about 20 pm (e.g., any of about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, or about 20 pm).
  • CRT central retinal thickness
  • CST central subfield thickness
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 10 pm to about 200 pm (e.g., any of about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, or about 200 pm).
  • CRT central retinal thickness
  • CST central subfield thickness
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 90 pm to about 200 pm (e.g., any of about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, or about 200 pm).
  • CRT central retinal thickness
  • CST central subfield thickness
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 150 mih to about 200 mth (e.g., any of about 150 mih, about 155 mth, about 160 mth, about 165 mth, about 170 mih, about 175 mih, about 180 mth, about 185 mih, about 190 mth, about 195 mth, or about 200 mth).
  • CRT central retinal thickness
  • CST central subfield thickness
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of between about 160 pm to about 200 pm (e.g., any of about 160 pm, about 165 pm, about 170 pm, about 175 pm, about 180 pm, about 185 pm, about 190 pm, about 195 pm, or about 200 pm).
  • CRT central retinal thickness
  • CST central subfield thickness
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 8.3 pm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles.
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a decrease of central retinal thickness (CRT) or central subfield thickness (CST) of about 26.2 mth compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles.
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -30.0 pm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles.
  • CRT central retinal thickness
  • CST central subfield thickness
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a change in the central retinal thickness (CRT) or central subfield thickness (CST) of about -149.8 pm compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the unit dose of rAAV particles.
  • CRT central retinal thickness
  • CST central subfield thickness
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in an increase of central retinal thickness (CRT) or central subfield thickness (CST) of about 32 pm or less compared to the retinal thickness prior to administration of the unit dose of rAAV particles.
  • CRT central retinal thickness
  • CST central subfield thickness
  • the maintenance, the decrease, or the increase of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles is present at about 30 weeks or more after administration of the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual.
  • the maintenance, the decrease, or the increase of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles is present at any of about 30 weeks, about 34 weeks, about 44 weeks, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 5 years, about 10 years, or more, after administration of the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual.
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a decrease in macular volume of more than any of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% compared to the macular volume prior to administration of the unit dose of rAAV particles.
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a decrease in macular volume of at least about 10% compared to the macular volume prior to administration of the unit dose of rAAV particles.
  • the macular volume is determined by OCT or SD-OCT.
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in maintenance or an improvement of visual acuity compared to the visual acuity prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in an improvement of visual acuity compared to the visual acuity prior to administration of the unit dose of rAAV particles.
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in an improvement of visual acuity of more than any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or more, compared to the visual acuity prior to administration of the unit dose of rAAV particles.
  • visual acuity is best corrected visual acuity (BCVA).
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in an improvement of BCVA of at least 15 ETDRS letters (Vitale etal., (2016) JAMA Opthalmol 134(9): 1041: 1047) (e.g., at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, or about 70 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles.
  • ETDRS letters Vitale etal., (2016) JAMA Opthalmol 134(9): 1041: 1047
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in an improvement of BCVA of about 6.4 ETDRS letters or more compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in an improvement of BCVA of about 6.8 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles.
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in an improvement of BCVA of about 8.8 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in an improvement of BCVA of about 2.3 ETDRS letters compared to the BCVA prior to administration of the unit dose of rAAV particles.
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses 0 letters compared to the BCVA prior to administration of the unit dose of rAAV particles. In some embodiments, the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in maintenance of BCVA, wherein the individual loses about 1 letter compared to the BCVA prior to administration of the unit dose of rAAV particles.
  • treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye is assessed based on macular volume in the one eye and/or the contralateral eye.
  • macular volume is determined by SD-OCT.
  • treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye is determined if the macular volume assessed by SD-OCT is decreased after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye is determined if the CST and macular volume assessed by SD-OCT are decreased after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye is determined if the CST and macular volume assessed by SD-OCT are maintained after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye compared to prior to administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye is determined if an individual requires less than one rescue therapy treatment (e.g., aflibercept injection) any of every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, or more, after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • rescue therapy treatment e.g., aflibercept injection
  • treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye is determined if an individual does not require any rescue therapy treatment (e.g., aflibercept injection) for any of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks, at least 60 weeks, at least 70 weeks, at least 80 weeks, at least 90 weeks, at least 100 weeks, at least 110 weeks, or more, after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • rescue therapy treatment e.g., aflibercept injection
  • the individual does not require any rescue therapy treatment (e.g., aflibercept injection) for at least about 12 months after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye. In some embodiments, the individual does not require any rescue therapy treatment (e.g., aflibercept injection) for at least about 10 months after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye. In some embodiments, the individual does not require any rescue therapy treatment (e.g., aflibercept injection) for at least about 7 months after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • any rescue therapy treatment e.g., aflibercept injection
  • administration of a single unit dose of rAAV particles to the one eye and/or to the contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection).
  • an anti-VEGF rescue treatment e.g., aflibercept injection
  • administration of a single unit dose of rAAV particles to the one eye and/or to the contralateral eye of a plurality of individuals results in at least about 50% (e.g., any of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%) of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 4 weeks after administration of the rAAV particles, e.g., any of at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about
  • administration of a single unit dose of rAAV particles to the one eye and/or to the contralateral eye of a plurality of individuals results in 100% of the individuals in the plurality not requiring an anti-VEGF rescue treatment (e.g., aflibercept injection) for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more, after administration of the rAAV particles.
  • an anti-VEGF rescue treatment e.g., aflibercept injection
  • administration of a unit dose of rAAV particles in the one eye and/or the contralateral eye of a plurality of individuals results in about 50% or less (e.g., any of about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2.5% or less, about 1% or less, or about 0.5% or less) of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and/or the contralateral eye.
  • any rescue treatment e.g., aflibercept injection
  • administration of a unit dose of rAAV particles in the one eye and/or the contralateral eye of a plurality of individuals results in less than about 30% (e.g., less than any of about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 2.5%, about 1%, or about 0.5%) of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and/or the contralateral eye.
  • any rescue treatment e.g., aflibercept injection
  • administration of a unit dose of rAAV particles in the one eye and/or the contralateral eye of a plurality of individuals results in less than about 30% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and/or the contralateral eye. In some embodiments, administration of a unit dose of rAAV particles in the one eye and/or the contralateral eye of a plurality of individuals results in less than about 20% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and/or the contralateral eye.
  • rescue treatment e.g., aflibercept injection
  • administration of a unit dose of rAAV particles in the one eye and/or the contralateral eye of a plurality of individuals results in less than about 30% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and/or the contralateral eye for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more, after administration of the rAAV particles.
  • any rescue treatment e.g., aflibercept injection
  • administration of a unit dose of rAAV particles in the one eye and/or the contralateral eye of a plurality of individuals results in 0% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and/or the contralateral eye for at least about 20 weeks after administration of the rAAV particles, e.g., any of at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 66 weeks, or more after administration of the rAAV particles.
  • any rescue treatment e.g., aflibercept injection
  • administration of a unit dose of rAAV particles in the one eye and/or the contralateral eye of a plurality of individuals results in 0% of the individuals in the plurality requiring any rescue treatment (e.g., aflibercept injection) in the one eye and/or the contralateral eye for at least about 4 weeks after administration of the rAAV particles, e.g., any of at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 76 weeks, at least about 80 weeks, at least about 84 weeks, at least about 88 weeks, at least about 92 weeks, at least about 96 weeks, at least about 100 weeks,
  • administration of the unit dose of rAAV particles to the one eye and/or to the contralateral eye of a plurality of individuals results in a reduction in the mean annualized anti-VEGF injection rate of any of at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 99%, or 100%, compared to the mean annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles.
  • administration of the unit dose of rAAV particles to the one eye and/or to the contralateral eye of a plurality of individuals results in a reduction in the mean annualized anti-VEGF injection rate of about 87% or more compared to the mean annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles.
  • administration of the unit dose of rAAV particles to the one eye and/or to the contralateral eye of a plurality of individuals results in a reduction in the mean annualized anti-VEGF injection rate of 100% compared to the mean annualized anti-VEGF injection rate prior to administration of the unit dose of rAAV particles.
  • the mean annualized anti-VEGF injection rate after administration of the unit dose of rAAV particles is calculated according to the formula:
  • an individual is determined to require a rescue treatment (e.g., anti-VEGF intravitreal injection, such as aflibercept injection) after administration of the rAAV particles if the individual exhibits loss of 10 or more letters in BCVA (e.g., using the ETDRS protocol) in the one eye and/or the contralateral eye administered the rAAV particles that is attributed to intraretinal or subretinal fluid (e.g., as determined by SD-OCT) compared to the BCVA in the one eye and/or the contralateral eye administered the rAAV particles prior to administration of the rAAV particles.
  • a rescue treatment e.g., anti-VEGF intravitreal injection, such as aflibercept injection
  • an individual is determined to require a rescue treatment (e.g., anti-VEGF intravitreal injection, such as aflibercept injection) after administration of the rAAV particles if the individual exhibits an increase in central subfield thickness (e.g., CST or CRT) greater than 75 pm in the one eye and/or the contralateral eye administered the rAAV particles compared to the central subfield thickness in the one eye and/or the contralateral eye administered the rAAV particles prior to administration of the rAAV particles, e.g., as determined by SD-OCT.
  • a rescue treatment e.g., anti-VEGF intravitreal injection, such as aflibercept injection
  • a rescue treatment comprises administration of a standard of care anti- VEGF therapy.
  • Such standard of care anti-VEGF therapy comprises one or more anti-VEGF treatments (e.g., anti-VEGF intravitreal injections).
  • a rescue treatment comprises one or more aflibercept IVT injections.
  • a rescue treatment comprises one or more aflibercept IVT injections comprising about 2 mg of aflibercept.
  • treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye is assessed based on the level of retinal fluid compared the level of retinal fluid prior to administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye is determined if a reduction in retinal fluid is observed after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye compared to the level of retinal fluid prior to administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • the ocular neovascular disease is wAMD.
  • treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye is assessed based on the resolution of pigment epithelial detachment (PED) compared to PED prior to administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • PED pigment epithelial detachment
  • treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye is determined if CNV lesions do not grow (e.g., grow less than any of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%) after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye compared to CNV lesions present prior to administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • the ocular neovascular disease is wAMD.
  • treatment of an ocular neovascular disease in an individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye is assessed based on the anatomical features of the one eye and/or the contralateral eye based on any methods known in the art (e.g., SD-OCT, OCT, fluorescein angiography, digital color fundus photography, etc.).
  • the unit dose of rAAV particles is administered in combination with steroid treatment.
  • the steroid treatment is a corticosteroid treatment.
  • corticosteroids include, without limitation, aclometasone, amcinomide, beclometasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortivazol, deflazacort, deoxycorticosterone, desonide desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumetasone, fhmisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluoromethol
  • the topical steroid treatment is a difluprednate treatment, a medrysone treatment, a loteprednol treatment, a prednisolone treatment, a fluocinolone treatment, a triamcinolone treatment, a rimexolone treatment, a dexamethasone treatment, a fluorometholone treatment, a fluocinolone treatment, a rimexolone treatment, or a prednisone treatment.
  • the ophthalmic steroid treatment is a difluprednate treatment.
  • the steroid treatment is a prednisone treatment.
  • the steroid treatment is a difluprednate treatment.
  • the steroid is administered during and after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered before and/or after administration of the unit dose of rAAV particles. In some embodiments, the steroid is administered before and after administration of the unit dose of rAAV particles.
  • the steroid treatment is an oral prednisone treatment.
  • the oral prednisone treatment is initiated prior to administration of the unit dose of rAAV particles.
  • an initial oral prednisone treatment is administered at a dose of any of about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, or about 70 mg of prednisone per day any of about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, or 0 days before administration of the unit dose of rAAV particles, and is continued for any of about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days, or more.
  • an initial oral prednisone treatment is administered at a dose of about 60 mg of prednisone per day about 3 days before administration of the unit dose of rAAV, and is continued for about 3 days
  • the steroid treatment is administered before, during, and/or after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and during administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before and after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered during, and after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before, during, and after administration of the unit dose of rAAV particles. In some embodiments, the steroid treatment is administered before, during, and after administration of the unit dose of rAAV particles.
  • the steroid treatment is an ophthalmic steroid treatment, e.g., a topical steroid treatment.
  • the ophthalmic steroid treatment e.g., a topical steroid treatment
  • the methods of treatment provided herein comprise administering an anti- VEGF agent (e.g., an aflibercept IVT injection) to one eye of the individual prior to administration of the unit dose of rAAV particles to the one eye of the individual.
  • an anti- VEGF agent e.g., an aflibercept IVT injection
  • the anti-VEGF agent is administered about 7 days or about 1 week prior to administration of the unit dose of rAAV particles.
  • the anti-VEGF agent is administered on about Day 1 and the unit dose of rAAV particles is administered on about Day 8.
  • the topical steroid treatment comprises a 7-week topical steroid treatment, e.g., 0.05% difluprednate.
  • the topical steroid treatment comprises about four administrations of topical steroid per day (i.e., QID) on Day 1 to about Day 30, followed by about three administrations of topical steroid per day (i.e., TID) on about Day 31 to about Day 60, followed by about two administrations of topical steroid per day (i.e., BID) on about Day 61 to about Day 90, and followed by about one administration of topical steroid per day (i.e., QD) on about Day 91 to about Day 120; timing starting at Day 1.
  • the topical steroid treatment is continued if inflammation is present.
  • AAV or rAAV are small non-enveloped single -stranded DNA viruses.
  • rAAVs are non- pathogenic human parvoviruses and can be made to be dependent on helper viruses, including adenovirus, herpes simplex virus, vaccinia virus and CMV, for replication.
  • AAV and rAAV used for gene therapy for delivery of an anti-VEGF agent can be of any serotype.
  • the methods of the disclosure provide for use of any suitable AAV serotype, including AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
  • AAV 10 AAV 10, AAV 11, AAV 12, rhlO, AAV-DJ, and any hybrid or chimeric AAV thereof.
  • the serotype used is based on tropism of the virus, or infectivity of a target cell of interest.
  • several AAV vectors may be generated to allow selection of the most optimal serotype for use with an anti-VEGF agent transgene (e.g., aflibercept transgene).
  • the rAAV particles comprise a nucleic acid, e.g., a heterologous nucleic acid.
  • the nucleic acid encodes a transgene, e.g., an anti-VEGF agent (e.g., aflibercept).
  • the encoded transgene, e.g., anti-VEGF agent is under the transcriptional control of a promoter that initiates transcription of the nucleic acid.
  • the promoter is a “ubiquitous” promoter.
  • the promoter is a “strong” or constitutively active promoter, e.g., a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EFla) promoter, a glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) promoter, or a connexin36 (or “Cx36”) promoter.
  • CMV cytomegalovirus
  • EFla elongation factor 1 alpha
  • GPDH glyceraldehyde 3 -phosphate dehydrogenase
  • Cx36 connexin36
  • the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOs: 28-32, or a sequence with at least 85% identity thereto.
  • the 5' arm of the polynucleotide cassette comprises or consists of SEQ ID NO: 33 or a sequence with at least 85% identity thereto.
  • the 3' arm of the polynucleotide cassette comprises or consists of SEQ ID NO: 34 or a sequence with at least 85% identity thereto.
  • the nucleic acid sequences of SEQ ID NOs: 22-34 are provided below:
  • the increase in retinal cell infectivity is between 2-fold to 20-fold, between 2-fold to 19-fold, between 2-fold to 18-fold, between 2-fold to 17-fold, between 2-fold to 16- fold, between 2-fold to 15 -fold, between 2-fold to 14-fold, between 2-fold to 13 -fold, between 2-fold to 12-fold, between 2-fold to 11-fold, between 2-fold to 10-fold, between 2-fold to 9-fold, between 2-fold to 8-fold, between 2-fold to 7-fold, between 2-fold to 6-fold, between 2-fold to 5-fold, between 2-fold to 4- fold, or between 2-fold to 3-fold, as compared to an AAV particle comprising the corresponding parental or unmodified AAV capsid protein.
  • an amino acid modification of a capsid protein described herein can confer an increase in an ability to cross an internal limiting membrane (ILM) in an eye of an individual, e.g., a human, as compared to the ability of an AAV particle comprising the corresponding parental or unmodified AAV capsid protein to cross the ILM in the eye of the subject.
  • ILM internal limiting membrane
  • rAAV2.7m8 is used to deliver the nucleic acid sequence of the anti- VEGF agent (e.g., aflibercept) into the retinal cells of a subject.
  • the heterologous nucleic acid e.g., a nucleic acid that encodes an anti-VEGF agent such as aflibercept
  • integrates into the target cell genome e.g., retinal cell genome
  • the anti-VEGF agent such as aflibercept
  • the rAAV particles comprise a nucleic acid with any of at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 100% sequence homology to the nucleic acid sequence of aflibercept (e.g., SEQ ID NO: 36), and wherein the nucleic acid is flanked by AAV2 inverted terminal repeats (ITRs).
  • ITRs AAV2 inverted terminal repeats
  • the rAAV particles are manufactured using two baculoviruses in Sf9 cells, wherein a first baculovirus encodes the genes for AAV2 Rep and AAV2.7m8 Cap proteins and a second baculovirus comprises a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and flanked by AAV2 inverted terminal repeats (ITRs).
  • the polypeptide comprises the amino acid sequence of SEQ ID NO: 35.
  • the polypeptide is aflibercept.
  • the unit dose of rAAV particles is administered to one eye of the individual.
  • the one eye of the individual is the right eye or the left eye.
  • the one eye of the individual is the right eye.
  • the one eye of the individual is the left eye.
  • the methods provided herein further comprise administering a unit dose of rAAV particles to the contralateral eye of the individual.
  • the one eye of the individual is the right eye and the contralateral eye is the left eye.
  • the one eye of the individual is the left eye and the contralateral eye is the right eye.
  • the administering the unit dose of rAAV particles to the contralateral eye is at least about 2 weeks after administering the unit dose of rAAV particles to the one eye and the unit dose of rAAV particles administered to the contralateral eye of the individual is higher (e.g., any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or more, higher) than the unit dose of rAAV particles administered to the one eye of the individual.
  • the unit dose of rAAV particles administered to the one eye of the individual is higher (e.g., any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%
  • the unit dose is about 6 c 10 10 vector genomes (vg) to about 2 c 10 11 vg of the rAAV particles. In some embodiments, the unit dose is about 6 c 10 10 vg to about 2 c 10 11 vg, about 7 c 10 10 vg to about 2x 10 11 vg, about 8 c 10 10 vg to about 2 c 10 11 vg, about 9 c 10 10 vg to about 2 c 10 11 vg, about 10x 10 10 vg to about 2x 10 11 vg, or about 1 c 10 11 vg to about 2 c 10 11 vg of the rAAV particles.
  • the unit dose is about 6 c 10 10 vg to about 2 c 10 11 vg of the rAAV particles. In some embodiments, the unit dose is about 6 c 10 10 vg to about 7 c 10 10 vg, about 7 c 10 10 vg to about 8 c 10 10 vg, about 8x 10 10 vg to about 9 c 10 10 vg, about 9 c 10 10 vg to about 10x 10 10 vg, about 10x 10 vg to about 1 c 10 11 vg, or about 1 c 10 11 vg to about 2 c 10 11 vg of the rAAV particles.
  • the unit dose is about 1 c 10 10 to about 2 c 10 10 , between about 2x 10 10 to about 3 c 10 10 , between about 3 c 10 10 to about 4 c 10 10 , between about 4 c 10 10 to about 5 x 10 10 , between about 5 c 10 10 to about 6 c 10 10 , between about 6 c 10 10 to about 7 c 10 10 , between about 7x 10 10 to about 8 c 10 10 , between about 8 c 10 10 to about 9 c 10 10 , between about 9 c 10 10 to about 10x 10 10 , between about 1 c 10 11 to about 2 c 10 11 , between about 2 c 10 11 to about 3 c 10 11 , between about 3 c 10 11 to about 4x 10 11 , between about 4 c 10 11 to about 5 c 10 11 , or between about 5 c 10 11 to about 6 c 10 11 vg/eye of the rAAV particles, including any value within these ranges, of the rAAV particles
  • the unit dose is about 6 c 10 10 vg/eye, about 2 c 10 11 vg/eye, or about 6x 10 11 vg/eye. In some embodiments, the unit dose is about 6 c 10 10 vg/eye. In some embodiments, the unit dose is about 2 c 10 11 vg/eye. In some embodiments, the unit dose is about 6 c 10 11 vg/eye.
  • the unit dose is about 6E 10 vector genomes (vg) to about 2E 11 vg of the rAAV particles. In some embodiments, the unit dose is about 6E 10 vg to about 2E 11 vg, about 7E 10 vg to about 2E 11 vg, about 8E 10 vg to about 2E 11 vg, about 9E 10 vg to about 2E 11 vg, about 10E 10 vg to about 2E 11 vg, or about IE 11 vg to about 2E 11 vg of the rAAV particles. In some embodiments, the unit dose is about 6E 10 vg to about 2E 11 vg of the rAAV particles.
  • the unit dose is about 6E 10 vg to about 7E 10 vg, about 7E 10 vg to about 8E 10 vg, about 8E 10 vg to about 9E 10 vg, about 9E 10 vg to about 10E 10 vg, about 10E 10 vg to about IE 11 vg, or about IE 11 vg to about 2E 11 vg of the rAAV particles.
  • the unit dose is about 6E 10 vg, about 7E 10 vg, about 8E 10 vg, about 9E 10 vg, about 10E 10 vg, about IE 11 vg, or about 2E 11 vg of the rAAV particles.
  • the unit dose of rAAV particles is administered to one eye and/or to the contralateral eye of the individual.
  • the unit dose is expressed as the number of vector genomes (vg) per eye (vg/eye). In some embodiments, the unit dose is about 6E 11 vg/eye or less of the rAAV particles.
  • the unit dose is about IE 10 to about 2E 10 , between about 2E 10 to about 3E 10 , between about 3E 10 to about 4E 10 , between about 4E 10 to about 5E 10 , between about 5E 10 to about 6E 10 , between about 6E 10 to about 7E 10 , between about 7E 10 to about 8E 10 , between about 8E 10 to about 9E 10 , between about 9E 10 to about 10E 10 , between about IE 11 to about 2E 11 , between about 2E 11 to about 3E 11 , between about 3E 11 to about 4E 11 , between about 4E 11 to about 5E 11 , or between about 5E 11 to about 6E 11 vg/eye of the rAAV particles, including any value within these ranges, of the rAAV particles.
  • the unit dose is about 6E 10 vg/eye to about 2E 11 vg/eye of the rAAV particles. In some embodiments, the unit dose is about 6E 10 vg/eye to about 7E 10 vg/eye, about 7E 10 vg/eye to about 8E 10 vg/eye, about 8E 10 vg/eye to about 9E 10 vg/eye, about 9E 10 vg/eye to about 10E 10 vg/eye, about 10E 10 vg/eye to about IE 11 vg/eye, or about IE 11 vg/eye to about 2E 11 vg/eye of the rAAV particles.
  • the unit dose of rAAV particles is a unit dose sufficient to cause expression of aflibercept in the aqueous fluid. In some embodiments, the unit dose of rAAV particles is a unit dose sufficient to achieve a concentration of aflibercept of at least about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0 pg/ml, or more, including any range in between these values, in the aqueous fluid.
  • the unit dose of rAAV particles is administered to one eye and/or to the contralateral eye of the individual.
  • the unit dose of rAAV particles is a unit dose sufficient to cause expression of the therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) in the choroid.
  • the unit dose of rAAV particles is a unit dose sufficient to achieve a concentration of the therapeutic protein (e.g., an anti-VEGF agent such as aflibercept) at about any one of 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10 pg/g, or more, including any range in between these values, in the choroid.
  • the unit dose of rAAV particles is a unit dose sufficient to cause expression of aflibercept in the choroid.
  • the unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause maintenance or a decrease of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, the unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause a decrease of retinal thickness compared to the retinal thickness prior to administration of the unit dose of rAAV particles. In some embodiments, retinal thickness is central subfield thickness (CST) or central retinal thickness (CRT).
  • CST central subfield thickness
  • CRT central retinal thickness
  • the unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause an improvement of visual acuity of more than any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or more compared to the visual acuity prior to administration of the unit dose of rAAV particles.
  • visual acuity is best corrected visual acuity (BCVA).
  • the unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause an improvement of BCVA compared to the BCVA prior to administration of the unit dose of rAAV particles.
  • BCVA is expressed as an ETDRS score, which corresponds to the number of letters correctly read (Vitale et al., (2016) JAMA Opthalmol 134(9): 1041: 1047).
  • the unit dose of rAAV particles is a therapeutically effective dose if the unit dose is sufficient to cause an improvement of BCVA of at least 15 ETDRS letters (Vitale et al., (2016) JAMA Opthalmol 134(9): 1041: 1047) (e.g., at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, or about 70 letters) compared to the BCVA prior to administration of the unit dose of rAAV particles.
  • ETDRS letters Vitale et al., (2016) JAMA Opthalmol 134(9): 1041: 1047
  • the unit dose of rAAV particles is a therapeutically effective dose if, after administration of the unit dose of rAAV particles, the retinal thickness (e.g., central retinal thickness (CRT) or central subfield thickness (CST)) and macular volume are decreased compared to prior to administration of the unit dose of rAAV particles.
  • the unit dose of rAAV particles is a therapeutically effective dose if, after administration of the unit dose of rAAV particles, the retinal thickness (e.g., central retinal thickness (CRT) or central subfield thickness (CST)) and macular volume are maintained compared to prior to administration of the unit dose of rAAV particles.
  • the unit dose of rAAV particles is a therapeutically effective dose if, after administration of the unit dose of rAAV particles, the individual is determined to have a reduction in retinal fluid compared to the level of retinal fluid prior to administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • the unit dose of rAAV particles is a therapeutically effective dose if, after administration of the unit dose of rAAV particles, the individual is determined to have maintenance in retinal fluid compared to the level of retinal fluid prior to administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye.
  • the unit dose of rAAV particles administered to the one eye and/or to the contralateral eye of the individual is therapeutically effective if administration of the dose to the one eye and/or to the contralateral eye of the individual results in complete resolution, partial resolution or maintenance of the ocular neovascular disease as assessed by best corrected visual acuity (BCVA) (e.g., based on an ETDRS score; Vitale etal., (2016) JAMA Opthalmol 134(9): 1041: 1047), central retinal thickness as determined by SD-OCT, the number of rescue therapy treatments (e.g., aflibercept injections) required by the individual after administration of the unit dose of rAAV particles in the one eye and/or the contralateral eye, the presence of intraretinal fluid (IRF) and/or subretinal fluid (SRF), the resolution of pigment epithelial detachment (PED), choroidal neovascularization (CNV) lesion growth, anatomical features based on any methods known
  • BCVA
  • the unit dose of rAAV particles administered to the one eye of the individual is the same as the unit dose of rAAV particles administered to the contralateral eye of the individual. In some embodiments, the unit dose of rAAV particles administered to the one eye of the individual is different from the unit dose of rAAV particles administered to the contralateral eye of the individual.
  • the unit dose of rAAV particles administered to the one eye of the individual is higher, e.g., more than any of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300% or more, than the unit dose of rAAV particles administered to the contralateral eye of the individual.
  • the unit dose of rAAV particles is about 6 c 10 10 vg/eye, about 2 c 10 11 vg/eye, or about 6 c 10 11 vg/eye. In some embodiments, the unit dose of rAAV particles is about 6 c 10 10 vg/eye. In some embodiments, the unit dose of rAAV particles is about 2 c 10 11 vg/eye. In some embodiments, the unit dose of rAAV particles is about 6 x 10 11 vg/eye .
  • the unit dose of rAAV particles administered to the one eye of the individual and the unit dose of rAAV particles administered to the contralateral eye of the individual are administered at the same time. In some embodiments, the unit dose of rAAV particles administered to the one eye of the individual and the unit dose of rAAV particles administered to the contralateral eye of the individual are administered at different times.
  • a single unit dose of rAAV particles is administered to the one eye and/or the contralateral eye of the individual.
  • the single unit dose of rAAV particles administered to the one eye and/or to the contralateral eye is a therapeutically effective dose.
  • more than one dose of rAAV particles e.g., more than any of about 2, 3, 4, 5, or more unit doses
  • the more than one doses of rAAV particles administered to the one eye and/or to the contralateral are therapeutically effective doses.
  • the pharmaceutical formulation comprises about 1 c 10 10 vg/mL to about 1 x 10 13 vg/mL of rAAV particles, about 150 mM to about 200 mM sodium chloride, about 1 mM to about 10 mM monobasic sodium phosphate, about 1 mM to about 10 mM dibasic sodium phosphate, and about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.5.
  • the pharmaceutical formulation comprises about 6x 10 11 vg/mL to about 6x 10 12 vg/mL of rAAV particles, about 150 mM to about 200 mM sodium chloride, about 1 mM to about 10 mM monobasic sodium phosphate, about 1 mM to about 10 mM dibasic sodium phosphate, and about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.5.
  • the pharmaceutical formulation comprises about 6 10 12 vg/mL of rAAV particles, about 150 mM to about 200 mM sodium chloride, about 1 mM to about 10 mM monobasic sodium phosphate, about 1 mM to about 10 mM dibasic sodium phosphate, and about 0.0005% (w/v) to about 0.005% (w/v) poloxamer 188, wherein the pharmaceutical formulation has a pH of about 7.0 to about 7.5.
  • the rAAV particles in the pharmaceutical formulation are present at a concentration of about 1 c 10 09 vg/ml to about 2 / 10 09 vg/ml, about 2 / 10 09 vg/ml to about 3 c 10 09 , about 3 x 10 09 vg/ml to about 4 c 10 09 , about 4 c 10 09 vg/ml to about 5 c 10 09 , about 5 c 10 09 vg/ml to about 6 c 10 09 , about 6 x 10 09 vg/ml to about 7x 10 09 , about 7 c 10 09 vg/ml to about 8x 10 09 , about 8 c 10 09 vg/ml to about 9x 10 09 , about 9 c 10 09 vg/ml to about 10x 10 09 , about 10x 10 09 vg/ml to about 1 c 10 10 , about 1 c 10 10 vg/ml to about 2x 10 10 , about 2 / 10 09
  • the sodium phosphate dibasic is present in the pharmaceutical formulation at a concentration of about 1 mM to about 10 mM. In some embodiments, the sodium phosphate dibasic is present in the pharmaceutical formulation at a concentration of any of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM. In certain embodiments, the sodium phosphate dibasic is present in the pharmaceutical formulation at a concentration of about 5 mM.
  • the pharmaceutical formulations are suitable for administration to the one eye and/or the contralateral eye of the individual, e.g., a human patient, via intravitreal (IVT) injection to achieve a desired therapeutic or prophylactic effect.
  • the pharmaceutical formulation is supplied as a reconstituted homogenous solution.
  • the solution is a suspension.
  • the pharmaceutical formulation is supplied as a frozen suspension, and is thawed prior to administration to the one eye and/or the contralateral eye of the individual.
  • the solution is isotonic.
  • the pharmaceutical composition comprising e.g., an AAV2.7m8 vector that comprises a nucleic acid sequence encoding the anti-VEGF agent (e.g., aflibercept or a functional fragment or variant thereof), is supplied in a lyophilized form, and is reconstituted prior to administration to the one eye and/or the contralateral eye of the individual.
  • the anti-VEGF agent e.g., aflibercept or a functional fragment or variant thereof
  • the pharmaceutical formulation is a homogenous solution.
  • the homogenous solution is supplied in a pre-fdled syringe.
  • the pharmaceutical formulation is supplied as a suspension.
  • a suspension is a solution.
  • the suspension is refrigerated.
  • the suspension is frozen.
  • methods provided herein further comprise the step of warming the refrigerated suspension to room temperature and/or agitating the suspension to ensure that the active ingredient(s) are dissolved and/or evenly distributed in solution prior to administering to the one eye and/or the contralateral eye of the individual (e.g., via IVT injection).
  • methods provided herein further comprise the step of thawing the frozen suspension and warming to room temperature and/or agitating the suspension to ensure that the active ingredient(s) are dissolved and/or evenly distributed in solution prior to administering to the one eye and/or the contralateral eye of the individual (e.g., via IVT injection).
  • the suspension is diluted prior to administration to the subject (e.g., via IVT injection).
  • the suspension is supplied as a pre-fdled syringe.
  • the pharmaceutical formulation is provided as a frozen suspension.
  • the suspension comprises a pharmaceutically acceptable excipient, e.g., surfactant, glycerol, non-ionic surfactant, buffer, glycol, salt, and any combination thereof.
  • the suspension is a solution. In some embodiments, the suspension comprises micelles.
  • excipients such as phosphate, PBS, or Tris buffer, glycol, glycerol, saline, surfactant (e.g., pluronic or polysorbate), or any combination thereof, can be used to stabilize a pharmaceutical formulation.
  • cryoprotectants such as alcohols can be used as a stabilizer under freezing or drying conditions.
  • the gene therapy is provided as a suspension, a refrigerated suspension, or a frozen suspension.
  • a suspension of the pharmaceutical formulation as disclosed herein has a volume of any of about 20 pL, 30 pL, 40 pL, 50 pL, 60 pL, 70 pL, 80 pL, 90 pL, 100 pL, 200 pL, 300 pL, 400 pL, 500 pL, 600 pL, 700 pL, 800 pL, 900 pL, or 1000 pL. In some embodiments, a suspension of the pharmaceutical formulation as disclosed herein has a volume of about 250 pL.
  • the suspension of the pharmaceutical formulation as disclosed herein has a volume of between 0.1 to 0.5 mL, between 0.1 to 0.2 mL, between 0.3 to 0.5 mL, between 0.5-1.0 mL, between 0.5- 0.7 mL, between 0.6 to 0.8 mL, between 0.8 to 1 mL, between 0.9 to 1.1 mL, between 1.0 to 1.2 mL, or between 1.0 to 1.5 mL.
  • a suspension of the pharmaceutical formulation as disclosed herein is provided as a sterile -fdtered, frozen suspension in a sterile, ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial) with a ready-to-use stopper (e.g., a stopper made of chlorobutyl), and sealed (e.g., with a sterile aluminum tear-off seal).
  • a sterile, ready-to-use vial e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial
  • a ready-to-use stopper e.g., a stopper made of chlorobutyl
  • sealed e.g., with a sterile aluminum tear-off seal
  • a suspension of the pharmaceutical formulation as disclosed herein is provided as a sterile-filtered, frozen suspension in a sterile, ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial) with, a ready-to-use stopper (e.g., a stopper made of chlorobutyl), and sealed (e.g., with a sterile aluminum tear-off seal), wherein the vial contains a volume of between 0.1 to 0.5 mL, between 0.1 to 0.2 mL, between 0.2 to 0.3 mL, between 0.3 to 0.4 mL, or between 0.4 mL to 0.5 mL of the suspension of the pharmaceutical formulation.
  • a sterile, ready-to-use vial e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial
  • a ready-to-use stopper e.g.,
  • pharmaceutical formulations disclosed herein are designed, engineered, or adapted for administration to a primate (e.g., non-human primate and human subjects) via intravitreal or subretinal injection.
  • a pharmaceutical formulation comprising rAAV particles comprising a nucleic acid sequence that encodes the anti-VEGF agent (e.g., aflibercept) is formulated for intravitreal injection into an eye of an individual.
  • the pharmaceutical composition is formulated to or reconstituted to a concentration that allows intravitreal injection of a volume not more than about or not more than any of 25 pL, 30 pL, 35 pL, 40 pL, 45 pL, 50 pL, 55 pL, 60 pL, 65 pL, 70 pL, 75 pL, 80 pL, 85 pL, 90 pL, 95 pL, 100 pL, 110 pL, 120 pL, 130 pL, 140 pL, 150 pL, 160 pL, 170 pL, 180 pL, 190 pL, 200 pL, 210 pL, 220 pL, 230 pL, 240 pL, or 250 pL.
  • methods disclosed herein comprise intravitreal injection of a volume of any of about 25 pL. 30 pL. 35 pL. 40 pL. 45 pL. 50 pL. 55 pL. 60 pF, 65 mE, 70 mE, 75 mE, 80 mE, 85 mE, 90 mE, 95 mE, 100 mE, 110 mE, 120 mE, 130 mE, 140 mE, 150 mE, 160 mE, 170 mE, 180 mE, 190 mE, 200 mE, 210 mE, 220 mE, 230 mE, 240 mE, or 250 mE of a solution or suspension of a pharmaceutical formulation comprising a rAAV (e.g., AAV2.7m8) and a nucleic acid sequence that encodes the anti-VEGF agent (e.g., aflibercept).
  • a rAAV e.g., AAV2.7
  • methods disclosed herein comprise intravitreal injection of a volume of about 30 pL or about 100 pF of a solution or suspension of a pharmaceutical formulation comprising a rAAV (e.g., AAV2.7m8) and a nucleic acid sequence that encodes the anti-VEGF agent (e.g., aflibercept).
  • methods disclosed herein comprise intravitreal injection of a volume of about 30 pL of a solution or suspension of a pharmaceutical formulation comprising a rAAV (e.g., AAV2.7m8) and a nucleic acid sequence that encodes the anti-VEGF agent (e.g., aflibercept).
  • the administration of the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual is by intravitreal (IVT) injection.
  • IVT intravitreal
  • the rAAV particles can be delivered in the form of a suspension of a pharmaceutical formulation (e.g., as described herein).
  • topical anesthetic is applied to the surface of the eye followed by an ophthalmic antiseptic solution.
  • the eye is held open, with or without instrumentation, and the rAAV particles are injected through the sclera with a short, narrow needle, e.g., a 30-gauge needle, into the vitreous cavity of the one eye and/or the contralateral eye of the individual under direct observation.
  • a short, narrow needle e.g., a 30-gauge needle
  • the pharmaceutical formulation is a unit dose (e.g., a therapeutically effective dose) to be administered to the one eye and/or the contralateral eye of an individual (e.g., a human or non-human primate) via IVT injection for the treatment of an ocular disease or disorder characterized by abnormal (e.g., excessive) angiogenesis or neovascularization.
  • the pharmaceutical formulation comprises a unit dose (e.g., a therapeutically effective dose) as described in further detail elsewhere herein.
  • the volume of the unit dose (e.g., a therapeutically effective dose) of a viral vector (e.g., an rAAV vector disclosed herein) administered to the subject is no more than any one of about 25 pL, 30 pL, 35 pL, 40 pL, 45 pL, 50 pL, 55 pL, 60 pL, 65 pL, 70 pL, 75 pL, 80 pL, 85 pL, 90 pL, 95 pL, 100 pL, 110 pL, 120 pL, 130 pL, 140 pL, 150 pL, 160 pL, 170 pL, 180 pL, 190 pL, 200 pL, 210 pL, 220 pL, 230 pL, 240 pL, or 250 pL, including any range in between these values.
  • Minimizing the volume of the unit dose to be administered to the subject may obviate or mitigate changes in ocular
  • compositions suitable for ocular use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions, suspension, or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, phosphate buffered saline (PBS), and/or an isotonic agent, e.g., glycerol.
  • the pharmaceutical formulation is sterile and fluid to the extent that easy syringability or injectability exists.
  • the pharmaceutical formulation is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the pharmaceutical composition can include an isotonic agent, such as a salt or glycerol.
  • a surfactant or a stabilizer is added to the pharmaceutical composition to prevent aggregation.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants such as polysorbates (e.g., TweenTM, polysorbate 20, polysorbate 80), sodium dodecyl sulfate (sodium lauryl sulfate), lauryl dimethyl amine oxide, cetyltrimethylammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol (Triton X100TM), N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide (HTAB), polyoxyl 10 lauryl ether, Brij 721TM, bile salts (sodium deoxycholate, sodium cholate), pluronic acids (F-68, F-127), polyoxyl castor oil (Cremo
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, cresol, thimerosal, and the like.
  • isotonic agents are included in the pharmaceutical formulation, for example, sugars, polyalcohols such as mannitol, sorbitol, and/or sodium chloride. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
  • the pharmaceutical carrier includes sodium phosphate, sodium chloride, polysorbate, and sucrose.
  • kits comprising at least one pharmaceutical formulation described herein.
  • the kit comprises a frozen suspension of a pharmaceutical formulation (e.g., one unit dose in a vial).
  • the kit comprises a lyophilized or freeze-dried pharmaceutical formulation (e.g., one unit dose in a vial) disclosed herein and a solution for dissolving, diluting, and/or reconstituting the lyophilized pharmaceutical composition.
  • the solution for reconstituting or dilution is supplied as a pre-filled syringe.
  • a kit comprises a freeze-dried or lyophilized pharmaceutical composition comprising rAAV (e.g., AAV2.7m8) and a solution for reconstituting the pharmaceutical composition to a desired concentration or volume.
  • the kit includes a buffer that helps to prevent aggregation upon reconstituting the pharmaceutical composition disclosed herein.
  • the pharmaceutical composition is provided in a pre-filled syringe.
  • a kit comprises a dual -chamber syringe or container wherein one of the chambers contains a buffer for dissolving or diluting the pharmaceutical composition.
  • the kit comprises a syringe for injection.
  • the reconstituted solution is filtered before administration.
  • the kit comprises a filter or a filter syringe for filtering the reconstituted pharmaceutical composition before administration to a patient.
  • the kit comprises a suspension of the pharmaceutical formulation comprising the rAAV particles as disclosed herein provided as a sterile-filtered, frozen suspension in a sterile, ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial) with a ready-to-use stopper (e.g., a stopper made of chlorobutyl), and sealed (e.g., with a sterile aluminum tear-off seal).
  • a sterile-filtered, frozen suspension in a sterile, ready-to-use vial e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial
  • a ready-to-use stopper e.g., a stopper made of
  • the kit comprises a suspension of the pharmaceutical formulation comprising the rAAV particles as disclosed herein provided as a sterile-filtered, frozen suspension in a sterile, ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial) with a ready-to-use stopper (e.g., a stopper made of chlorobutyl), and sealed (e.g., with a sterile aluminum tear-off seal), wherein the vial contains a volume of between 0.1 to 0.5 mL, between 0.1 to 0.2 mL, between 0.2 to 0.3 mL, between 0.3 to 0.4 mL, or between 0.4 mL to 0.5 mL of the suspension of the pharmaceutical formulation.
  • a sterile, ready-to-use vial e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial
  • the kit comprises a suspension of the pharmaceutical formulation comprising the rAAV particles as disclosed herein provided as a sterile-filtered, frozen suspension in a sterile, ready-to-use vial (e.g., a 0.5 mL vial; e.g., a Crystal Zenith® vial) with a ready-to-use stopper (e.g., a stopper made of chlorobutyl), and sealed (e.g., with a sterile aluminum tear-off seal), wherein the vial contains a volume of about 0.25 mL of the suspension of the pharmaceutical formulation.
  • the kit further comprises instructions for use, e.g., instructions for treating an ocular neovascular disease with the rAAV particles disclosed herein.
  • the present disclosure provides methods for treating an ocular neovascular disease in an individual. In another aspect, the present disclosure provides methods for reducing retinal fluid in the eye of an individual with an ocular neovascular disease.
  • the ocular neovascular disease is age-related macular degeneration (AMD), wet-AMD, retinal neovascularization, choroidal neovascularization diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, or any combination thereof.
  • the ocular neovascular disease is active choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD).
  • the ocular neovascular disease is recurrent and/or persistent wAMD.
  • the ocular neovascular disease is active subfoveal CNV secondary to AMD.
  • the active subfoveal CNV secondary to AMD occupies > 50% of the total lesion size.
  • the active subfoveal CNV secondary to AMD occupies > 50% of the total lesion size with evidence of leakage on fluorescein angiogram (FA), fluid on spectral domain optical coherence tomography (SD-OCT), and/or subretinal hemorrhage on color fundus photography.
  • FA fluorescein angiogram
  • SD-OCT fluid on spectral domain optical coherence tomography
  • subretinal hemorrhage on color fundus photography subretinal hemorrhage on color fundus photography.
  • the active subfoveal CNV secondary to AMD occupies > 50% of the total lesion size with evidence of leakage on fluorescein angiogram (FA), fluid on spectral domain optical coherence tomography (SD-OCT), and/or subretinal hemorrhage on color fundus photography, and the entire dimension of the lesion does not exceed 12 macular photocoagulation study disc areas.
  • FA fluorescein angiogram
  • SD-OCT fluid on spectral domain optical coherence tomography
  • subretinal hemorrhage on color fundus photography and the entire dimension of the lesion does not exceed 12 macular photocoagulation study disc areas.
  • the one eye and/or the contralateral eye of the individual exhibited best corrected visual acuity (BCVA) based on an ETDRS letters assessment of 78-25 (e.g., less than any of about 78, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, or about 25) prior to administration of the unit dose of rAAV particles of the present disclosure.
  • BCVA visual acuity
  • the one eye and/or the contralateral eye of the individual exhibited best corrected visual acuity (BCVA) based on an ETDRS letters assessment of more than any of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 prior to administration of the unit dose of rAAV particles of the present disclosure.
  • BCVA visual acuity
  • the individual had polypoidal choroidal vasculopathy (PCV) in the one eye and/or the contralateral eye prior to administration of the unit dose of rAAV particles.
  • PCV polypoidal choroidal vasculopathy
  • ETDRS letters assessment is done at about 0.5 meters, about 1 meter, about 2 meters, about 3 meters, or about 4 meters. In some embodiments, ETDRS letters assessment is done at about 4 meters.
  • the individual received at least one prior treatment (e.g., at least one, at least two, at least three, at least four, at least 5 or more treatments) with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and/or aflibercept) in about the last 12 weeks (e.g., about 3 or about 4 months) prior to administration of the unit dose of rAAV particles.
  • an anti-VEGF agent e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and/or aflibercept
  • the individual received 2 or 3 prior treatments with an anti-VEGF agent e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and/or aflibercept
  • an anti-VEGF agent e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and/or aflibercept
  • an anti-VEGF agent e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunit
  • the individual received at least about 1, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, or more prior treatments with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and/or aflibercept) in the one eye and/or the contralateral eye.
  • an anti-VEGF agent e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806
  • the individual had a calculated anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, and/or aflibercept) injection interval in the one eye and/or the contralateral eye of about 2 weeks, about 3 weeks, 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, or more.
  • a calculated anti-VEGF agent e.g., bevacizumab, brolucizumab, ranibizumab, and/or aflibercept
  • the individual received about 9 or about 10 prior treatments with an anti-VEGF agent (e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and/or aflibercept) in the one eye and/or in the contralateral eye during the last about 12 months prior to administration of the unit dose of rAAV particles to the one eye and/or the contralateral eye.
  • an anti-VEGF agent e.g., bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, injectable sunitinib maleate (GB-102), PAN-90806 (PanOptica), and/or aflibercept
  • the individual demonstrated a meaningful response to a prior anti-VEGF treatment (e.g., aflibercept, a functional variant thereof, or a functional fragment thereof) for the ocular neovascular disease in the one eye and/or in the contralateral eye prior to administration of the unit dose of rAAV particles to one eye and/or the contralateral eye.
  • a prior anti-VEGF treatment e.g., aflibercept, a functional variant thereof, or a functional fragment thereof
  • the individual is determined to have a meaningful response to a prior treatment with anti-VEGF agent (e.g., aflibercept, a functional variant thereof, or a functional fragment thereof) for the ocular neovascular disease if a reduction of >20% (e.g., any of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) in central subfield thickness and/or central retinal thickness is observed compared to the central subfield thickness and/or central retinal thickness prior to administration of the prior treatment with the anti-VEGF agent.
  • anti-VEGF agent e.g., aflibercept, a functional variant thereof, or a functional fragment thereof
  • the individual is determined to have a meaningful response to a prior treatment with anti-VEGF agent (e.g., aflibercept, a functional variant thereof, or a functional fragment thereof) for the ocular neovascular disease if a reduction of >20% (e.g., any of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) in central subfield thickness and/or central retinal thickness is observed any of at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, or more, after administration of the prior treatment with an anti-VEGF agent, compared to the central subfield thickness and/or central retinal thickness prior to administration of the prior treatment with an anti-VEGF agent
  • the individual is determined to have a meaningful response to a prior treatment with an anti-VEGF agent (e.g., aflibercept, a functional variant thereof, or a functional fragment thereof) for the ocular neovascular disease if a reduction of >20% (e.g., any of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) in central subfield thickness and/or central retinal thickness is observed about 7 days, about 10 days, or about 14 days after administration of the prior treatment with the anti-VEGF agent, compared to the central subfield thickness and/or central retinal thickness prior to administration of the prior treatment with an anti-VEGF agent.
  • an anti-VEGF agent e.g., aflibercept, a functional variant thereof, or a functional fragment thereof
  • the central subfield thickness and/or central retinal thickness is determined by SD-OCT in the one eye and/or the contralateral eye.
  • the individual is determined to have a meaningful response to a prior treatment with an anti-VEGF agent (e.g., aflibercept, a functional variant thereof, or a functional fragment thereof) for the ocular neovascular disease if normalization of CST is observed with no observable vascular exudation after the anti-VEGF treatment in the one eye and/or the contralateral eye.
  • Normalization refers to a CST value that is normal for that class of patient (e.g., based on age, sex, etc.)
  • the individual is determined to have a meaningful response to a prior anti- VEGF treatment (e.g., aflibercept, a functional variant thereof, or a functional fragment thereof) for the ocular neovascular disease if a reduction of >20% (e.g., any of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) in central retinal thickness (CRT) or central subfield thickness (CST) is observed any of at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, or more, after the anti-VEGF treatment, compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the
  • the individual is determined to have a meaningful response to a prior anti-VEGF treatment (e.g., aflibercept, a functional variant thereof, or a functional fragment thereof) for the ocular neovascular disease if a reduction of >20% (e.g., any of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) in central retinal thickness (CRT) or central subfield thickness (CST) is observed about 7 days, about 10 days, or about 14 days after the anti- VEGF treatment, compared to the central retinal thickness (CRT) or central subfield thickness (CST) prior to administration of the anti-VEGF treatment, as determined by SD-OCT in the one eye and/or the contralateral eye.
  • a reduction of >20% e.g., any of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%
  • the individual has not received a prior treatment for an ocular neovascular disease. In some embodiments, the individual has not received a prior treatment in the one eye and/or the contralateral eye for an ocular neovascular disease. In some embodiments, the individual has not received a prior anti-VEGF treatment. In some embodiments, the individual has not received a prior anti-VEGF treatment in the one eye and/or the contralateral eye. In some embodiments, the individual has not received a prior aflibercept treatment. In some embodiments, the individual has not received a prior aflibercept treatment in the one eye and/or the contralateral eye.
  • the ocular disease or disorder treated according to the methods described herein is diabetic macular edema.
  • Diabetic macular edema is a swelling of the retina in diabetes mellitus due to leaking of fluid from blood vessels within the macula.
  • the macula is the central portion of the retina, a small area rich in cones, the specialized nerve endings that detect color and upon which daytime vision depends.
  • blurring occurs in the middle or just to the side of the central visual field. Visual loss from diabetic macular edema can progress over a period of months and make it impossible to focus clearly.
  • DME Common symptoms of DME are blurry vision, floaters, double vision, and eventually blindness if it goes untreated.
  • methods and pharmaceutical compositions as disclosed herein are used to treat DME.
  • treatment of DME is assessed by measuring refraction or visual acuity (e.g., BCVA using ETDRS letters).
  • visual acuity is measured starting at a distance of about 4 meters prior to dilation of the one eye and/or the contralateral eye.
  • visual acuity is considered to be maintained if the individual loses fewer than 15 letters in the ETDRS score after administration of the unit dose of rAAV particles compared to prior to administration of the unit dose of rAAV particles.
  • one or more aflibercept rescue treatments are administered after administration of the unit dose of rAAV particles if an increase in central subfield thickness (CST) of > 50 pm occurs, assessed by SD-OCT, compared to the lower of the two CST measurements recorded prior to administration of the unit dose of rAAV particles (e.g., on Day 1) and about 3 weeks after administration of the unit dose of rAAV particles (e.g., on Week 4).
  • CST central subfield thickness
  • one or more aflibercept rescue treatments are administered after administration of the unit dose of rAAV particles if loss of > 5 letters in BCVA occurs due to worsening DME disease activity compared to the higher of the two BCVA measurements recorded prior to administration of the unit dose of rAAV particles (e.g., on Day 1) and about 3 weeks after administration of the unit dose of rAAV particles (e.g., on Week 4).
  • treatment of DME is assessed using the Diabetic Retinopathy Severity Scale (DRSS), e.g., using ultra-wide field color fundus photography, compared to DRSS prior to administration of the unit dose of rAAV particles.
  • DRSS Diabetic Retinopathy Severity Scale
  • treatment of DME is assessed by the occurrence of vision threatening complications (e.g., anterior segment neovascularization, diabetic macular edema, high-risk PDR development, vitreous hemorrhage, ortractional retinal detachment).
  • vision threatening complications are assessed by ultra-wide field imaging and clinical examination.
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a 2-step or in a 3 -step improvement in Diabetic Retinopathy Severity Scale (DRSS).
  • DRSS Diabetic Retinopathy Severity Scale
  • the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a 2-step improvement in Diabetic Retinopathy Severity Scale (DRSS). In some embodiments, the administering the unit dose of rAAV particles to the one eye and/or to the contralateral eye of the individual results in a 3 -step improvement in Diabetic Retinopathy Severity Scale (DRSS).
  • DRSS Diabetic Retinopathy Severity Scale
  • the ocular disease or disorder treated according to the methods described herein is a retinal vein occlusion.
  • Retinal vein occlusion is a blockage of the small veins that carry blood away from the retina.
  • the retina is the layer of tissue at the back of the inner eye that converts light images to nerve signals and sends them to the brain.
  • Retinal vein occlusion is most often caused by hardening of the arteries (atherosclerosis) and the formation of a blood clot.
  • Blockage of smaller veins (branch veins or BRVO) in the retina often occurs in places where retinal arteries that have been thickened or hardened by atherosclerosis cross over and place pressure on a retinal vein.
  • Symptoms of retinal vein occlusion can include a sudden blurring or vision loss in all or part of one eye.
  • the ocular disease or disorder treated according to the methods described herein is choroidal neovascularization (CNV), also known as wet age-related macular degeneration (wAMD).
  • CNV choroidal neovascularization
  • wAMD wet age-related macular degeneration
  • Choroidal neovascularization can involve the growth of new blood vessels that originate from the choroid through a break in the Bruch membrane into the sub-retinal pigment epithelium (sub-RPE) or subretinal space, which can be a major cause of visual loss.
  • CNV can create a sudden deterioration of central vision, noticeable within a few weeks.
  • Other symptoms can include color disturbances, and metamorphopsia (distortions in which straight lines appears wavy). Hemorrhaging of the new blood vessels can accelerate the onset of symptoms of CNV.
  • CNV may also include feeling of pressure behind the eye.
  • the advanced “wet” form (neovascular or exudative) of AMD may frequently cause a rapid and often substantial loss of central vision in patients.
  • choroidal neovascularization forms and develops into a network of vessels that may grow under and through the retinal pigment epithelium. As this is accompanied by leakage of plasma and/or hemorrhage into the subretinal space, there could be severe sudden loss of central vision if this occurs in the macula.
  • the present disclosure contemplates treatment or prevention of AMD, wet AMD.
  • methods and pharmaceutical compositions as disclosed herein are used to treat AMD.
  • the individual was diagnosed with the ocular neovascular disease at least 1 day, at least 1 week, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, at least 54 months, at least 60 months, at least 66 months, at least 72 months, at least 78 months, at least 84 months, at least 90 months, 96 months, at least 102 months, at least 108 months, at least 114 months, at least 120 months, at least 126 months, at least 132 months, or more, prior to administration of the unit dose of rAAV particles to the one eye and/or the contralateral eye.
  • Example 1 An open label Phase 1 study of AA V2.7m8-aflibercept in neovascular (wet) age-related macular degeneration at a dose of 6 x lO ⁇ vg/eye.
  • This example describes an open label Phase 1 study of AAV2.7m8-aflibercept, a rAAV vector containing the VEGF inhibitor aflibercept and the AAV2.7m8 protein capsid, for the treatment of age- related macular degeneration (AMD) with choroidal neovascularization (wet AMD; wAMD).
  • AMD age-related macular degeneration
  • wAMD choroidal neovascularization
  • the primary objective of this study was to assess the safety and tolerability of a single intravitreal (IVT) injection of AAV2.7m8-aflibercept in subjects with wAMD.
  • CST central subfield thickness
  • CRT central retinal thickness
  • the secondary endpoints of this study were: • The mean change in BCVA from baseline over time, as assessed by ETDRS letters over time from Day 8 to Week 104, compared to baseline.
  • CNV choroidal neovascularization
  • AMD age-related macular degeneration
  • Subjects were under active anti-VEGF treatment for wAMD with a minimum of 2 injections within 4 months prior to Screening.
  • VEGF responsiveness was confirmed by the Investigator for purposes of confirmation of anti- VEGF response at Day 1 visit prior to dosing with AAV2.7m8-aflibercept. Subjects determined not to have a meaningful anti-VEGF response failed screening and were not enrolled in this study.
  • NAbs to AAVs in serum and vitreous fluid
  • subjects were screened for NAbs to the AAV2.7m8 vector as a precaution.
  • Subjects with documented anti-AAV2.7m8 neutralizing antibody titer levels > 1: 125 within 6 months prior to dosing with AAV2.7m8-aflibercept were excluded from this study.
  • CNV lesion characteristics o Fibrosis or atrophy, retinal epithelial tear in the center of the fovea in the study eye, or any condition preventing visual acuity improvement o Scarring or fibrosis making up > 50% of total lesion area.
  • o Lesion size > 12 Macular Photocoagulation Study disc areas (30.5 mm 2 ), including blood, scars, and neovascularization as assessed by Fluorescein Angiography (FA).
  • VMT vitreomacular traction
  • retinal disease in the study eye other than wAMD including diabetic retinopathy (in either eye), retinal vein occlusion, uveitis, suspected retinal angiomatous proliferation, polypoidal choroidopathy, or CNV due to other causes (e.g., ocular histoplasmosis, trauma, or pathologic myopia), or any other vascular disease in the eye (benign conditions of the vitreous or peripheral retina were non-exclusionary).
  • AAV2.7m8-aflibercept defined as intraocular pressure [IOP] > 22 mmHg despite treatment with anti -glaucoma medication) or use of > 2 IOP lowering medications at the time of screening.
  • the investigational medicinal product (IMP), AAV2.7m8-aflibercept was a recombinant, replication-deficient adeno-associated viral (rAAV) vector containing the AAV2.7m8 protein capsid derived by in vivo directed evolution on the AAV2 capsid (Dalkara et al. , (2013) Sci Transl Med 5(189): 189ra76; US2014/0364338).
  • rAAV adeno-associated viral
  • AAV2.7m8-aflibercept carried an expression cassette of a codon- optimized version of the aflibercept cDNA under the control of a ubiquitous chimeric promoter (FIG. 1 A) (See WO2018170473A1).
  • AAV2.7m8-aflibercept was manufactured using a baculovirus expression vector system in Sf9 cells where two different baculoviruses were used, one encoding the genes for AAV2 Rep and AAV2.7m8 Cap proteins, and the other encoding the human aflibercept cDNA expression cassette.
  • AAV2.7m8-aflibercept was supplied as a sterile-filtered, frozen suspension in a sterile, ready-to- use 0.5 mL Crystal Zenith vial which contained 0.25 mL of IMP, formulated as shown in Table 1.
  • Cataract Surgery in the study eye could be performed if clinically indicated and was scheduled > 90 days after IVT administration and/or >7 days after the last injection of aflibercept.
  • SD-OCT Spectral Domain Optical Coherence Tomosraphy
  • SD-OCT was used to obtain depth-resolved tissue structure information encoded in the magnitude and delay of the back-scattered light by spectral analysis of the interference fringe pattern.
  • AAV2.7m8-aflibercept The safety of AAV2.7m8-aflibercept was assessed through the collection of AEs, vital signs, physical and eye examinations, ECG, pregnancy testing, and laboratory evaluations.
  • Intense monitoring of subjects was done in the first 8 weeks of the study, followed by regular safety assessments for safety and efficacy thereafter. All subjects had their visual acuity tested by Early Treatment Diabetic Retinopathy Study (ETDRS) letters assessments at each study visit, and standard of care aflibercept IVT injections were used as a rescue treatment.
  • EDRS Early Treatment Diabetic Retinopathy Study
  • FA was performed to assess CNV lesions using a standard technique to evaluate leakage compared to Baseline.
  • Table 4 Baseline characteristics of subjects in Cohort 1.
  • Table 5 Disease characteristics and treatment histories of study subjects in Cohort 1.
  • FIGS. 2A-2L OCT images taken prior to and after treatment with AAV2.7m8-aflibercept indicated that a robust anatomical response was evident in all six subjects in Cohort 1.
  • Subject 1 e.g., compare FIGS. 2A and 2B
  • Subject 2 e.g., compare FIGS. 2C and 2D
  • Subject 3 e.g., compare FIGS. 2E and 2F
  • Subject 4 e.g., compare FIGS.
  • BCVA Best corrected visual acuity
  • a summary of safety and efficacy results evaluated at the median follow up time of 34 weeks is provided in Table 7.
  • a summary of efficacy results evaluated at the median follow up time of 44 weeks is provided in Table 8.
  • Table 8 Summary of efficacy results at median follow up time of 44 weeks.
  • Table 9 Summary of efficacy results at the follow up time of 40-52 weeks (median 44 weeks).
  • Subject 1 has stopped receiving topical steroid eye drops.
  • Subjects 2 and 6 exhibited early onset inflammation by the first month after administration of AAV2.7m8-aflibercept, with an AC response and some spillover to vitreous cells.
  • the inflammation in both subjects resolved following treatment with topical steroids.
  • Both subjects continued twice daily eye drops (BID).
  • Subjects 3 and 4 experienced more delayed cellular inflammation onset, after topical steroids were discontinued (by months 2-3 after administration of AAV2.7m8-aflibercept). The inflammation in these subjects was responsive to topical steroids.
  • Subject 3 has stopped receiving topical steroid eye drops.
  • Subject 4 experienced an unrelated retinal detachment and is receiving BID eye drops.
  • FIGS. 9A-9B show OCT imaging of Subjects 1-6 through follow-up weeks 40 to 52. No subjects exhibited signs of disease reactivation on OCT imaging, and BCVA and anatomic improvements were maintained. In addition, no subjects required any rescue anti-VEGF IVT injections or met retreatment criteria.
  • AAV2.7m8-aflibercept provided a clear benefit for improving retinal anatomy and stabilizing vision, while exhibiting an acceptable safety profile. Specifically, patients maintained vision during the study, and AAV2.7m8-aflibercept was shown to be safe and well tolerated, with observed inflammation generally being mild and responsive to steroid eye drops.
  • Example 2 An open label Phase 1 study of AA V2.7m 8-aflibercept in neovascular (wet) age-related macular degeneration at doses lower than 6 x 10 u vg/eye and with topical corticosteroids.
  • the investigational medicinal product is AAV2.7m8-aflibercept, as described in detail in Section III of Example 1, and as depicted in FIG. 1A, with the exception that the concentration of AAV2.7m8- aflibercept was varied in order to maintain a suitable injection volume as described in Table 10, below.
  • Table 10 Formulation of the A A V2.7m8-aflibercept investigational medicinal product for cohorts 2-4.
  • AAV2.7m8-aflibercept at the doses described below was administered as described in Section IV of Example 1. Study Cohorts 2-4
  • Cohort 2 Six subjects who are diagnosed with wAMD are enrolled into Cohort 2. Subjects in Cohort 2 are administered a single IVT injection of AAV2.7m8-aflibercept at Dose 2 of 2 c 10 n vg/eye with a prophylactic oral prednisone regimen.
  • Cohort 3 Nine subjects who are diagnosed with wAMD are enrolled into Cohort 3. Subjects in Cohort 3 are administered a single IVT injection of AAV2.7m8-aflibercept at Dose 2 of 2 c 10 n vg/eye with a prophylactic topical corticosteroid regimen.
  • Cohort 4 Nine subjects who are diagnosed with wAMD are enrolled into Cohort 4. If signs of choroidal neovascularization exudation requiring rescue therapy are observed in a majority of subjects in Cohorts 2 and 3, the subjects in Cohort 4 are administered a single IVT injection of AAV2.7m8- aflibercept at Dose 3 of 6 10 10 vg/eye with a topical corticosteroid regimen (Cohort 4b).
  • the subjects in Cohort 4 are administered a single IVT injection of AAV2.7m8- aflibercept at Dose 1 of 6 1 O' 1 vg/eye with a topical corticosteroid regimen (Cohort 4a).
  • Subjects in Cohort 2 are administered a prophylactic 13 -day oral corticosteroid regimen as described in detail for Cohort in Example 1 :
  • Cohort 2 Subjects in Cohort 2 are administered a single IVT injection of AAV2.7m8- aflibercept at Dose 2 of 2 c 10 n vg/eye with a prophylactic 13-day oral corticosteroid regimen, starting with 60 mg of prednisone 3 days before and 3 days after AAV2.7m8-aflibercept treatment for a total of 6 days. This is followed by a 7-day prednisone taper.
  • Table 2 of Example 1 A summary of the oral prednisone regimen for Cohort 2 is provided in Table 2 of Example 1.
  • Cohort 3 Subjects in Cohort 3 are administered a single IVT injection of AAV2.7m8- aflibercept at Dose 2 of 2 c 10 n vg/eye with a prophylactic topical 4-week tapering corticosteroid regimen.
  • Cohort 4 Subjects in Cohort 4b are administered a single IVT injection of AAV2.7m8- aflibercept at Dose 3 of 6 10 10 vg/eye with a prophylactic topical 4-week tapering corticosteroid regimen that can be extended at the discretion of the treating physician.
  • Subjects in Cohort 4a are administered a single IVT injection of AAV2.7m8-aflibercept at Dose 3 of 6 10 11 vg/eye with a prophylactic topical 4- week tapering difluprednate regimen that can be extended at the discretion of the treating physician.
  • Table 11 A Summary of topical difluprednate regimen.
  • Subjects in Cohorts 3 and 4 may also be administered a 6-week prophylactic corticosteroid regimen that can be extended at the discretion of the treating physician.
  • a summary of the 6-week prophylactic corticosteroid regimen is provided in Table 11B.
  • Table l IB Summary of the 6-week topical difluprednate regimen.
  • Initiating immunosuppression immediately after exposure to AAV2.7m8-aflibercept IVT injection is designed to limit the immune response upon exposure to capsid antigens.
  • Subjects self- administer one drop of difluprednate 0.05% solution (2.5 pg difluprednate) to the conjunctival sac of the treated eye 4 times per day for the first week beginning on the day of injection of AAV2.7m8-aflibercept (Day 1, post-injection). This is followed by 3 times per day for second week, 2 times per day for the third week, and 1 time per day for the fourth week.
  • the tapering corticosteroid regimen may be extended at the discretion of the treating physician.
  • Table 12 Summary of study design for Cohorts 2-4.
  • Aqueous humor samples are obtained at screening (prior to aflibercept injection) and on Day 1 (prior to AAV2.7m8-aflibercept injection), Weeks 12, 24, 36, 52, 76, 88, 104, and at any visit requiring the first aflibercept (Eylea) rescue treatment, or early termination.
  • Aqueous humor samples are analyzed for aflibercept, VEGF-A, and NAb concentrations.
  • Vitreous humor samples are obtained at any point during the study when a vitrectomy is performed. Vitreous humor samples are analyzed primarily for aflibercept concentrations. Remaining samples are analyzed for VEGF-A concentrations.
  • AAV2.7m8-aflibercept The safety of AAV2.7m8-aflibercept is assessed through the collection of vital signs, physical and eye examinations, ECG, pregnancy testing, and laboratory evaluations.

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HRP20260137TT HRP20260137T1 (hr) 2019-09-11 2020-09-10 Liječenje očnih neovaskularnih bolesti upotrebom aav2 varijanti koje kodiraju aflibercept
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BR112022004027A BR112022004027A2 (pt) 2019-09-11 2020-09-10 Métodos para tratar doenças neovasculares oculares usando variantes de aav2 que codificam aflibercepte
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