WO2024238853A1 - Adeno-associated viruses for ocular delivery of gene therapy - Google Patents

Abstract

The present invention relates to recombinant adeno-associated viruses (rAAVs) having capsid proteins that have a tropism for ocular tissue. The rAAVs have capsids that have enhanced or increased transduction of retinal tissue or cell or a retinal pigment epithelium (RPE)-choroid tissue or cell as compared to sclera. Such rAAVs may be useful in delivering transgenes encoding therapeutic proteins for the treatment of ocular disease.

Description

ADENO-ASSOCIATED VIRUSES FOR OCULAR DELIVERY OF GENE THERAPY

REFERENCE TO SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (38013_0036Pl.xml; Size: 3,196,141 bytes; and Date of Creation: May 16, 2024) is herein incorporated by reference in its entirety.

1. FIELD OF THE INVENTION

[0002] Disclosed herein are recombinant adeno-associated viruses (rAAVs) having capsid proteins that target or have a tropism for, ocular tissue, and have enhanced delivery to ocular tissue, for example, to retinal tissue or cell or a retinal pigment epithelium (RPE)-choroid tissue or cell. In particular, provided are rAAV vectors having a capsid which is an AAV3B, AAV8, or AAV9 or other capsid demonstrated to target retinal tissue or cell or a retinal pigment epithelium (RPE)-choroid tissue or cell. Also provided capsid proteins that direct rAAVs to target tissues, and/or improve transduction of ocular tissues, including retinal tissue and RPE choroidal tissue, and deliver therapeutics for treating retinal diseases, in particular non- infectious uveitis, glaucoma, dry age-related macular degeneration (AMD) and dry AMD with geographic atrophy (GA).

2. BACKGROUND

[0003] The use of adeno-associated viruses (AAV) as gene delivery vectors is a promising avenue for the treatment of many unmet patient needs. Dozens of naturally occurring AAV capsids have been reported, and mining the natural diversity' of AAV sequences in primate tissues has identified over a hundred variants, distributed in clades. AAVs belong to the parvovirus family and are single-stranded DNA viruses with relatively small genomes and simple genetic components. Without a helper virus, AAV establishes a latent infection. An AAV genome generally has a Rep gene and a Cap gene, flanked by inverted terminal repeats (ITRs), which sene as replication and packaging signals for vector production. The capsid proteins form capsids that carry genome DNA and can determine tissue tropism to deliver DNA into target cells.

[0004] Due to low pathogenicity' and the promise of long-term, targeted gene expression, recombinant AAVs (rAAVs) have been used as gene transfer vectors, in which therapeutic sequences are packaged into various capsids. Such vectors have been used in preclinical gene therapy studies and over twenty gene therapy products are currently in clinical development. Recombinant AAVs, such as AAV2, have demonstrated desirable retinal cell transduction properties and clinical trials using recombinant AAV2 for treatment of ocular diseases are underway. Tropism for other ocular tissues is desirable depending upon the indication to be treated. Attempts to enhance ocular tissue tropism of rAAVs in human subjects have met with limited success.

[0005] There remains a need for rAAV vectors with enhanced tropism for ocular tissues or cells, including e.g., retinal tissues or cells or a retinal pigment epithelium (RPE)-choroid tissues or cells, to deliver therapies in treating disorders associated with the eye, e.g. non- infectious uveitis, glaucoma, dry age-related macular degeneration (AMD) and dry AMD with geographic atrophy (GA). There also is a need for rAAV vectors with enhanced tissue-specific targeting and/or enhanced tissue-specific transduction to deliver therapies.

3. SUMMARY OF THE INVENTION

[0006] Provided are recombinant AAV particles that have capsid proteins that direct the rAAVs to target tissues. The capsid proteins promote ocular tissue targeting and/or cellular uptake and/or integration of the rAAV genome, including targeting the rAAV particles to posterior segment tissue (such as retinal or RPE-choroid tissue), or the optic nerve (orbital segment or cranial segment), and deliver therapeutics for treating ocular disorders. The rAAVs may have a transgene encoding a therapeutic protein for treating ocular disorders, and provided are methods of administering the rAAV for delivery to ocular tissue for treatment of an ocular disease or disorder. In embodiments, the rAAV has a capsid of an AAV serot pe 3B (AAV3B; SEQ ID NO:74); AAV serotype 8 (AAV8; SEQ ID NO:66); or AAV serotype 9 (AAV9; SEQ ID NO:67) or the capsid is an engineered capsid having an insertion and/or one or more amino acid substitutions relative to one of the capsids disclosed herein, including, AAV serotype 3B (AAV3B; SEQ ID NO:74); AAV serotype 8 (AAV8; SEQ ID NO:66); or AAV serotype 9 (AAV9; SEQ ID NO:67).

[0007] In certain embodiments, the rAAV has a capsid of an AAV3B type. In embodiments, the capsid has a VP1 protein that is at least 90%, at least 95% or at least 99% identical to the AAV3B capsid protein having an amino acid sequence of SEQ ID NO: 74, and which retains the ocular tropism of AAV3B. Certain rAAV capsids have a tropism for specific ocular tissue and may be used to target specific ocular tissues. In embodiments, rAAVs having an AAV3B capsid may be administered to target the iris, retina, RPE choroid, and in certain embodiments. the ciliary body, Schlemm’s canal, trabelcular meshwork or optic nerve (orbital and/or cranial segment). In embodiments, rAAVs having an AAV3B capsid may be used to target the retina and/or RPE choroid tissue. The rAAV may be delivered by intravitreal, suprachoroidal, or intracameral administration and in certain embodiments the administration may be to a specific ocular tissue, such as to the, retina, retinal pigment epithelium, choroid, sclera or ciliary body. Provided are methods of delivering a transgene to ocular tissue, including retina and/or RPE choroid by suprachoroi dally administering an rAAV particle comprising an AAV3B capsid and and artificial genome comprising the transgene operably linked to regulatory sequences that promote transgene expression in ocular tissue flanked by AAV ITRs. In embodiments, administration of the AAV3B rAAV has reduced transduction and/or transgene expression in the sclera relative to the expression and/or transgene expressionin the retina and/or RPE choroid or relative to the transduction and/or transgene expression in the sclera of a comparable AAV8 rAAV administered in the same manner.

[0008] Also provided are engineered capsid proteins that promote transduction of the rAAV in one or more tissues, including one or more cell types, upon systemic, intravenous, intracameral, suprachoroidal or intravitreal administration, wherein the capsid proteins comprise a peptide that is inserted into a surface-exposed variable region (VR) of the capsid, e.g. VR-I, VR-IV or VR-VIII, or after the first amino acid of VP2, e.g.. immediately after residue 138 of the AAV9 capsid (amino acid sequence of SEQ ID NO:67) or immediately after the corresponding residue of another AAV capsid, or alternatively is engineered with one or more of the amino acid substitutions described herein, and transduction of the AAV having the engineered capsid in the at least one tissue, for example the anterior segment or the posterior segment, or both, is increased upon said administration compared to the transduction of the AAV having the corresponding unengineered capsid. In certain embodiments, transduction is measured by detection of transgene, such as GFP fluorescence. In particular embodiments, the rAAV having the engineered capsid transduced ocular tissue, including anterior segment or posterior segment tissues transduced ocular tissue, including anterior or posterior segments, by 1.1 fold, 1.5 fold, 2 fold, 3 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold greater than transduction by the reference AAV (the parental AAV serotype without the insertion).

[0009] In certain embodiments, provided are rAAVs incorporating the engineered capsids described herein, including rAAVs with genomes comprising a transgene of therapeutic interest. Packaging cells for producing the rAAVs described herein are provided. Method of treatment by delivery of, and pharmaceutical compositions comprising, the engineered rAAV s described herein are also provided. Also provided are methods of manufacturing the rAAVs with the engineered capsids described herein.

[0010] The invention is illustrated by way of examples infra describing the construction of engineered capsids and screening of capsids for tropism for ocular tissues after SCS or IVT administration using barcoded rAAVs in mice and NHPs.

3.1. Embodiments

[0011] Embodiment 1. A method of delivering a trans gene to an ocular tissue or ocular tissue target cell or cellular matrix thereof, said method comprising contacting said cell with an rAAV vector comprising a transgene encoding an ocular disease therapeutic operably linked to one or more regulatory elements that promote expression of the ocular disease therapeutic in the ocular tissue cell, wherein the rAAV has a capsid of AAV3B, having a capsid protein having an amino acid sequence of SEQ ID NO: 74 or a capsid having a capsid protein that is at least 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 74 and has tropism for ocular issue, wherein the ocular tissue or ocular tissue target cell is a retinal tissue or cell or a retinal pigment epithelium (RPE)-choroid tissue or cell.

[0012] Embodiment 2. A method of delivering atransgene to ocular tissue, or an ocular tissue target cell or cellular matrix thereof, of a subject in need thereof, said method comprising administering to said subject an rAAV vector comprising a transgene encoding an ocular disease therapeutic operably linked to one or more regulatory elements that promote expression of the ocular disease therapeutic in the ocular tissue, wherein the rAAV has a capsid AAV3B having a capsid protein having an amino acid sequence of SEQ ID NO: 74 or a capsid having a capsid protein that is at least 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 74 and has tropism for ocular issue.

[0013] Embodiment 3. The method of embodiment 1 or embodiment, in which the ocular tissue or ocular tissue target cell is a retinal tissue or cell.

[0014] Embodiment 4. The method of embodiment 1 or embodiment 2, wherein the ocular tissue or ocular tissue target cell is an RPE-choroid tissue or cell.

[0015] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the ocular disease is non-infectious uveitis.

[0016] Embodiment 6. The method of any one of embodiments 1 to 4, wherein the ocular disease is glaucoma. [0017] The Embodiment 7. The method of any one of embodiments 1 to 4, wherein the ocular disease is dry age-related macular degeneration (AMD).

[0018] Embodiment 8. The method of any one of embodiments 1 to 4, wherein the ocular disease is dry AMD with geographic atrophy (GA).

[0019] Embodiment 9. The method of any one of embodiments 1 to 8, wherein said rAAV vector is administered intravitreally, suprachoroidally, or intracamerally.

[0020] Embodiment 10. The method of embodiment 9, wherein said rAAV vector is administered systemically.

[0021] Embodiment 11. The method of embodiment 9, wherein said rAAV vector is administered suprachoroidally.

[0022] Embodiment 12. A pharmaceutical composition for use in delivering a transgene to an ocular tissue cell, said composition comprising an rAAV vector comprising a transgene encoding an ocular disease therapeutic operably linked to one or more regulatory elements that promote expression of the ocular disease therapeutic in the ocular tissue cell, wherein the rAAV has a capsid of AAV3B having a capsid protein having an amino acid sequence of SEQ ID NO: 74 or a capsid having a capsid protein that is at least 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 74 and has tropism for ocular issue, wherein the ocular tissue cell is a retinal tissue or cell or a retinal pigment epithelium (RPE)-choroid tissue or cell. [0023] Embodiment 13. The pharmaceutical composition of embodiment 12, wherein the ocular tissue or ocular tissue target cell is a retinal tissue or cell.

[0024] Embodiment 14. The pharmaceutical composition of embodiment 12. wherein the ocular tissue or ocular tissue target cell is a retinal pigment epithelium (RPE)-choroid tissue or cell.

[0025] Embodiment 15. The pharmaceutical composition of any one of embodiments 12 to 14, wherein the ocular disease is non-infectious uveitis.

[0026] Embodiment 16. The pharmaceutical composition of any one of embodiments 12 to 14. wherein the ocular disease is glaucoma.

[0027] Embodiment 17. The pharmaceutical composition of any one of embodiments 12 to 14, wherein the ocular disease is dry AMD.

[0028] Embodiment 18. The pharmaceutical composition of any one of embodiments 12 to 14, wherein the ocular disease id dry AMD with GA.

[0029] Embodiment 19. The pharmaceutical composition of any of embodiments 12 to 18, wherein said rAAV vector is administered intravitreally, suprachoroidally, or intracamerally. [0030] Embodiment 20. The pharmaceutical composition of embodiment 19, wherein said rAAV vector is administered systemically.

[0031] Embodiment 21. The pharmaceutical composition of embodiment 19, wherein said rAAV vector is administered suprachoroidally.

[0032] Embodiment 22. The method or pharmaceutical composition of any of embodiments 1 to 21 wherein the rAAV exhibits at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, or 10-fold greater transduction in the target tissue, compared to a reference AAV capsid.

[0033] Embodiment 23. The method or pharmaceutical composition of any of embodiments 1 to 22 wherein the abundance of transgene RNA is 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater in the target tissue compared to the abundance of transgene RNA from the reference AAV capsid.

[0034] Embodiment 24. The method or pharmaceutical composition of embodiment 22 or embodiment 23 where the reference AAV capsid is AAV2, AAV8 or AAV9

[0035] Embodiment 25. A nucleic acid comprising a nucleotide sequence encoding the rAAV capsid protein of any of the above embodiments, or encoding an amino acid sequence sharing at least 80% identity therewith.

[0036] Embodiment 26. A packaging cell capable of expressing the nucleic acid of embodiment 25 to produce AAV vectors comprising the capsid protein encoded by said nucleotide sequence.

[0037] Embodiment 27. The pharmaceutical composition of embodiments 12 to 18, wherein said rAAV vector is formulated for suprachoroidal administration.

4. BRIEF DESCRIPTION OF THE FIGURES

[0038] FIG. 1 depicts alignment of AAVs l-9e, AAV3B, rhlO, rh20, rh39, rh73, and rh74 version 1 and version 2, hul2, hu21, hu26, hu37, hu51 and hu53 capsid sequences with insertion sites for heterologous peptides after the initiation codon of VP2, and within or near variable region 1 (VR-I), variable region 4 (VR-IV), and variable region 8 (VR-VIII). all highlighted in grey; a particular insertion site within variable region eight (VR-VIII) of each capsid protein is shown by the symbol

(after amino acid residue 588 according to the amino acid numbering of AAV9). FIG. 1, top to bottom, shows the sequence of SEQ ID NOs:59, 60, 61, 94, 74. 62, 95, 63, 64, 65, 66, 67. 68, 69, 70, 73, 75, 72, 96, 78, 77, 79, 71, 76, 80, respectively. [0039] FIGs. 2A and B depict the anatomy of the eye. FIG. 2A depicts a cross section of the anterior of the eye and FIG. 2B depicts the anatomy of the entire eye.

[0040] FIG. 3 shows the composition of the 6-member AAV library⁷, wherein the pool of vectors contained a mixture of 28.3% AAV3B.CAG.tdTom.BC (red fluorescent marker), 28.3% AAV8.CAG.GFP.BC (green fluorescent marker). 28.3% AAV9.CAG.iRFP.BC (magenta fluorescent marker), 5% AAV3B.CAG.ApoE.BC, 5% AAV8.CAG.ApoE.BC, and 5% AAV8.CAG.ApoE.BC.

[0041] FIG. 4 shows a representation of suprachoroidal delivery using a transcl eral microneedle.

[0042] FIG. 5 shows a representation of frozen posterior segment sample collection scheme.

[0043] FIG. 6 shows a representation of a right eye posterior segment dissection, sectionining, and imaging scheme.

[0044] FIGs. 7A-7D are bar graphs showing total DNA and RNA biodistribution (FIG. 7A), DNA and RNA biodistribution reported separately for peripheral (P) and central (C) punches (FIG. 7B), total DNA biodistribution in peripheral tissues (FIG. 7C), and fluorescent reporter DNA barcode distribution in peripheral tissues as determined by NGS (FIG. 7D). Note: each data point represents a single punch/sample from a single animal (n=up to 3 per location).

[0045] FIGs. 8A and 8B are bar graphs showing relative abundance of capsids. FIG. 8A shows total DNA and RNA relative abundance adjusted for input (RAAFI) as determined by NGS for AAV3B-, AAV8-. and AAV9.ApoE.BC, compiled from all punches from a given tissue. Each data point represents a single punch from a single animal (n=3 per location). FIG. SB shows total and location-specific (peripheral vs. central) RNA:DNA ratios. Ratios were calculated as RNA RAAFI/DNA RAAFL

[0046] FIGs. 9A-F are bar graphs showing relative abundance of DNA in retina (A), RNA in retina (B). DNA in RPE-choroid (C), RNA in RPE-choroid (D). DNA in sclera (E) and RNA in sclera (F). Note: each data point represents a single punch from a single animal (n=up to 3 per location).

[0047] FIGs. 10A-C are bar graphs showing the quantification of GFP or TdT pixels in ROIs in RPE (A), choroid (B), and sclera (C) in posterior segment sections from eyes transduced with 6-member AAV library. Data are reported as % positive area from one of nine regions of interest (based on location) in each tissue. 5. DETAILED DESCRIPTION

[0048] The inventors have identified capsids of adeno-associated viruses (AAVs) that promote targeting of recombinant AAV (rAAV) particles to ocular tissue, including transduction, cellular uptake, integration of the rAAV genome, and expression of transgenes delivered in the rAAV particle to a greater extent than an rAAV with a reference capsid, such as an AAV2, AAV8 or AAV9 capsid. Accordingly, provided are recombinant AAV particles that have capsid proteins that direct the rAAVs to target tissues. The capsid proteins promote ocular tissue targeting and/or cellular uptake and/or integration of the rAAV genome, including targeting the rAAV particles to anterior segment tissue (cornea, iris, ciliary' body, Schlemm’s canal and/or the trabecular meshwork), or posterior segment tissue (such as retinal or RPE- choroid tissue), or the optic nerve (orbital segment or cranial segment), and deliver therapeutics for treating ocular disorders. In embodiments, retinal and/or RPE-choroid tissue is transduced at a higher level than the sclera tissue. Included are rAAVs having capsid proteins engineered to include amino acid sequences that confer and/or enhance desired properties, such as ocular tissue targeting, transduction and integration of the rAAV genome relative to the parent, unengineered capsid or a reference capsid. The rAAVs may have a transgene encoding a therapeutic protein for treating ocular disorders, and provided are methods of administering the rAAV for delivery⁷ to ocular tissue for treatment of an ocular disease or disorder.

[0049] In embodiments, the rAAV has a capsid of an AAV serotype 3B (AAV3B) (SEQ ID NO: 74). In embodiments, the raAAV has a capsid which has a capsid protein (VP I) which has an amino acid sequence that is at least 95%, 90%, 95%, or 99% identical to SEQ ID NO: 74 and has ocular tropism.

[0050] Recombinant vectors comprising the capsid proteins also are provided, along with pharmaceutical compositions thereof, nucleic acids encoding the capsid proteins, and methods of making and using the capsid proteins and rAAV vectors having the ocular targeting capsids for targeted delivery, improved transduction and/or treatment of ocular disorders associated with the target ocular tissue. In particular, provided are compositions comprising rAAVs and methods of using capsid proteins to target rAAVs to ocular tissues, including the RPE-choroid and the retina, including by suprachoroidal delivery, including a lower level of transduction in the sclera relative to the level in the retina and/or RPE choroid and/or a reference rAAV, such as AAV8, and facilitate delivery⁷ of therapeutic agents for treating disorders of the eye.

[0051] In other embodiments, provided are rAAV vectors comprising a transgene which is an ophthalmic disease therapeutic and methods of treating an ocular disease or disorder in which the capsid is an AAV3B serotype capsid or other capsid shown herein to have tropism to an ocular tissue, including, the retina and/or RPE-choroid and, in embodiments, reduced tropism for sclera tissue. In an embodiment, the eye disorder is non-infectious uveitis. In an embodiment, the eye disorder is glaucoma. In an embodiment, the eye disorder is dry age- related macular degeneration (AMD). In an embodiment, the eye disorder is dry AMD with geographic atrophy (GA). Also provided are compositions comprising rAAVs comprising peptide insertions that target or home on target tissues, such as retina as well as methods of using same. In embodiments, the rAAV is delivered suprachoroidally.

[0052] As used throughout, AAV "serotype” refers to an AAV having an immunologically distinct capsid, a naturally-occurring capsid, or an engineered capsid.

5.1. Definitions

[0053] The term “AAV” or “adeno-associated virus” refers to a Dependoparvovirus within the Parvoviridae genus of viruses. The AAV can be an AAV derived from a naturally occurring “wild-type” virus, an AAV derived from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a naturally occurring cap gene and/or from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a non-naturally occurring capsid cap gene. An example of the latter includes a rAAV having a capsid protein comprising a peptide insertion into the amino acid sequence of the naturally-occurring capsid.

[0054] The term “rAAV” refers to a “recombinant AAV.” In some embodiments, a recombinant AAV has an AAV genome in which part or all of the rep and cap genes have been replaced with heterologous sequences.

[0055] The term “rep-cap helper plasmid” refers to a plasmid that provides the viral rep and cap gene function and aids the production of AAVs from rAAV genomes lacking functional rep and/or the cap gene sequences.

[0056] The term “cap gene” refers to the nucleic acid sequences that encode capsid proteins that form or help form the capsid coat of the virus. For AAV. the capsid protein may be VP1, VP2, or VP3.

[0057] The term “rep gene” refers to the nucleic acid sequences that encode the non- structural protein needed for replication and production of virus.

[0058] As used herein, the terms “nucleic acids” and “nucleotide sequences” include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules. Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases. Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as. for example, nuclease resistance or an increased ability to cross cellular membranes. The nucleic acids or nucleotide sequences can be single-stranded, doublestranded, may contain both single-stranded and double-stranded portions, and may contain triple-stranded portions, but preferably is double-stranded DNA.

[0059] As used herein, the terms ‘‘subject”, “host”, and “patient” are used interchangeably. As used herein, a subject is a mammal such as a non-primate (e.g., cows. pigs, horses, cats, dogs, rats etc.) or a primate (e.g., monkey and human), or, in certain embodiments, a human. [0060] As used herein, the terms “therapeutic agent” refers to any agent which can be used in treating, managing, or ameliorating symptoms associated with a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene. As used herein, a “therapeutically effective amount” refers to the amount of agent, (e.g., an amount of product expressed by the transgene) that provides at least one therapeutic benefit in the treatment or management of the target disease or disorder, when administered to a subject suffering therefrom. Further, a therapeutically effective amount with respect to an agent of the invention means that amount of agent alone, or when in combination with other therapies, that provides at least one therapeutic benefit in the treatment or management of the disease or disorder.

[0061] As used herein, the term “prophylactic agent” refers to any agent which can be used in the prevention, delay, or slowing down of the progression of a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene. As used herein, a “prophylactically effective amount” refers to the amount of the prophylactic agent (e.g., an amount of product expressed by the transgene) that provides at least one prophylactic benefit in the prevention or delay of the target disease or disorder, when administered to a subject predisposed thereto. A prophylactically effective amount also may refer to the amount of agent sufficient to prevent or delay the occurrence of the target disease or disorder; or slow the progression of the target disease or disorder; the amount sufficient to delay or minimize the onset of the target disease or disorder; or the amount sufficient to prevent or delay the recurrence or spread thereof. A prophylactically effective amount also may refer to the amount of agent sufficient to prevent or delay the exacerbation of symptoms of a target disease or disorder. Further, a prophylactically effective amount with respect to a prophylactic agent of the invention means that amount of prophylactic agent alone, or when in combination with other agents, that provides at least one prophylactic benefit in the prevention or delay of the disease or disorder.

[0062] A prophylactic agent of the invention can be administered to a subj ect “pre-disposed” to a target disease or disorder. A subject that is “pre-disposed” to a disease or disorder is one that shows symptoms associated with the development of the disease or disorder, or that has a genetic makeup, environmental exposure, or other risk factor for such a disease or disorder, but where the symptoms are not yet at the level to be diagnosed as the disease or disorder. For example, a patient with a family history of a disease associated with a missing gene (to be provided by a transgene) may qualify as one predisposed thereto. Further, a patient with a dormant tumor that persists after removal of a primary tumor may qualify as one predisposed to recurrence of a tumor.

[0063] The “central nervous system” (“CNS”) as used herein refers to neural tissue reaches by a circulating agent after crossing a blood-brain barrier, and includes, for example, the brain, optic nerves, cranial nerves, and spinal cord. The CNS also includes the cerebrospinal fluid, which fills the central canal of the spinal cord as well as the ventricles of the brain.

[0064] As used throughout, AAV “serotype” refers to an AAV having an immunologically distinct capsid, a naturally-occurring capsid, or an engineered capsid.

5.2. Ocular Targeting Capsids and rAAVs

5.2.1 AAV Capsids with Tropism for Ocular Tissue

[0065] Identified herein are capsids that have a tropism for transduction and expression of transgenes in ocular tissue, including particular ocular tissues of the posterior segments of the eye, including, the retina and/or the RPE-Choroid. The target tissue may also be a “retinal cell” type which include one or more of the cell types found in or near the retina, including amacrine cells, bipolar cells, horizontal cells, Muller glial cells, photoreceptor cells (e.g., rods and cones), retinal ganglion cells, retinal pigmented epithelium, and the like, and in particular, human photoreceptor cells (e.g., human cone cells and/or human rod cells), human horizontal cells, human bipolar cells, human amacrine cells, as well as human retina ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retina ganglion cells, photosensitive ganglion cells, and/or Muller glia), endothelial cells in the inner limiting membrane, and/or human retinal pigment epithelial cells in the external limiting membrane. In embodiments, the capsid has a reduced tropism for the sclera relative to a reference rAAV, such as AAV8.

[0066] In particular embodiments, provided are methods of delivering a transgene to ocular tissues, methods of treating an ocular disease and pharmaceutical compositions comprising an rAAV comprising a transgene encoding an ocular therapeutic, where the AAV has a capsid of AAV serotype 3B (AAV3B) (SEQ ID NO:74), AAV8 capsid (SEQ ID NO:66) or AAV serotype 9 (SEQ ID NO:67).

[0067] In specific embodiments, the capsid is an AAV3B serotype. In embodiments the capsid comprises a VP1 capsid protein having an amino acid sequence of SEQ ID NO:74 or has a VP1 capsid protein having an amino acid sequence that is at least 90%, at least 95% or at least 99% identical to the amino acid sequence of SEQ ID NO: 74 and has tropism for ocular tissue.

[0068] Certain rAAV capsids have a tropism for specific ocular tissue and may be used to target specific ocular tissues. In embodiments, rAAVs having an AAV3B capsid may be administered to target the iris, retina, or RPE-choroid. In embodiments, rAAVs having an AAV3B capsid may be used to target the retina and/or RPE choroid tissue. In embodiments, the rAAV has reduced tropism for the sclera relative to its tropism for the retina and/or RPE choroid tissue and/or relative to the tropism for the sclera for a reference capsid such as an AAV8.

[0069] In some embodiments, the concentration of a transgene product (TP), or vector genomes, or transgene transcript is equal to or higher in the retina after a presently disclosed pharmaceutical composition comprising AAV3B having a genome with a transgene encoding the TP is injected in the SCS than a reference pharmaceutical composition comprising a different AAV (serotype), including AAV8, with the same recombinant genome comprising the same transgene is injected in the SCS. In other embodiments, the concentration of a TP, vector genomes or transgene transcript is equal to or higher in the retina and the concentration of the TP, vector genomes or transgene transcript is lower in the sclera after a pharmaceutical composition comprising AAV3B comprising a recombinant genome comprising a transgene encoding the TP is injected in the SCS as compared to a reference pharmaceutical composition comprising a different AAV, including AAV8, comprising the same recombinant genome comprising the same transgene is injected in the SCS.

[0070] In some embodiments, the concentration of the transgene product (TP) (or vector genomes or transgene transcripts) in the back of the eye (e.g., retina) after suprachoroidal administration of the AAV3B vector is equal to or higher as compared to the concentration of the transgene product (or vector genomes or transgene transcripts) in the back of the eye after suprachoroidal administration of a reference AAV vector, including AAV8, comprising the same transgene and/or the concentration of the TP (or vector genomes or transgene transcripts) in the outer layer of the eye (e.g., sclera) after suprachoroidal administration of the pharmaceutical composition is lower than the concentration of the TP (or vector genomes or transgene transcripts) in the outer layer of the eye after suprachoroidal administration of a reference AAV vector, including AAV8 comprising the same transgene.

[0071] The rAAV particles that have the ocular tissue targeting capsids described herein have enhanced targeting, transduction, genome integration, transgene mRNA transcription and/or transgene expression in ocular tissue compared to a reference rAAV particle having a reference capsid, for example an AAV2, AAV8 or AAV9 capsid and comprising a comparable recombinant genome. The enhancement may be in the ocular tissue overall or may be specifically the anterior segment tissue, posterior segment tissue or the optic nerve. In embodiments, the enhancement is in the iris, retina, RPE choroid, sclera, the ciliary body, Schl emm’s canal, trabelcular meshwork, or optic nerve. The enhancement may be assessed as known in the art, for example in Examples 15 to 18 herein. In embodiments, the rAAV particles with an ocular tissue targeting capsid exhibit at least 1.1-fold, 1.5-fold, 2-fold. 3-fold. 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater transduction or genome copy in the target tissue, compared to a reference AAV capsid, which may be AAV2, AAV8 or AAV9, and where the target tissue is ocular tissue, anterior ocular tissue, posterior ocular tissue, iris, retina, RPE choroid, sclera, the ciliary body, Schlemm’s canal, trabelcular meshwork, or optic nerve. In embodiments, rAAV particles with an ocular tissue targeting capsid exhibit at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater transgene mRNA or transgene protein expression in the target tissue compared to the abundance of transgene RNA or protein from the reference AAV capsid, which may be AAV2, AAV8 or AAV9. where the target tissue is ocular tissue, anterior ocular tissue, posterior ocular tissue, iris, retina, RPE choroid, sclera, the ciliary body, Schlemm’s canal, trabelcular meshwork, or optic nerve. In embodiments, the rAAV particles with an ocular tissue targeting capsid exhibit at least 1.1-fold. 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, or 10-fold reduced transduction or genome copy in the sclera, compared to the target tissue (e.g., retina or RPE choroid) or a reference AAV capsid, which may be AAV2, AAV8 or AAV 9. In embodiments, rAAV particles with an ocular tissue targeting capsid exhibit at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold reduced transgene rnRNA or transgene protein expression in sclera compared to the abundance of transgene RNA or protein in the target tissue (e.g., retina or RPE choroid) or a reference AAV capsid, which may be AAV2, AAV 8 orAAV9.

5.2.2 Engineered rAAV Vectors with Peptide Insertions

[0072] Another aspect relates to capsid proteins, and rAAV particles comprising the capsid proteins which are modified by insertion of a peptide and/or one or more amino acid substitutions to confer or enhance ocular cell-homing properties, including enhanced transduction, AAV genome copy abundance or integration, transgene mRNA levels, or transgene protein expression. The modified capsid may target cells of the retina, including amacrine cells, bipolar cells, horizontal cells, Muller glial cells, photoreceptor cells (e.g., rods and cones), retinal ganglion cells, retinal pigmented epithelium, and the like, and in particular, human photoreceptor cells (e.g., human cone cells and/or human rod cells), human horizontal cells, human bipolar cells, human amacrine cells, as well as human retina ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retina ganglion cells, photosensitive ganglion cells, and/or Muller glia), endothelial cells in the inner limiting membrane, and/or human retinal pigment epithelial cells in the external limiting membrane. The modified capsid maytarget other ocular tissues, including anterior segment tissues, including the iris, cornea, ciliary body, Schl emm’s canal, trabecular meshwork, and posterior segment tissues, such as the retina or RPE-choroid, and optic nerve.

[0073] In particular embodiments, the peptide insertion for targeting ocular tissue is at least or consists of 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids of RTIGPSV (SEQ ID NO: 12). In one embodiment of particular interest, the peptide insertion comprises or consists of the amino acid sequence RTIGPSV (SEQ ID NO: 12). The peptide may be inserted into the AAV3B capsid protein.

[0074] One aspect relates to a capsid protein of a recombinant adeno-associated virus (rAAV), the capsid protein engineered to target ocular tissue cells. In some embodiments the rAAV can comprise a peptide insertion, where the peptide insertion is surface exposed when packaged as an AAV particle. For example, the peptide insertion can be RTIGPSV (SEQ ID NO: 12) or LALGETTRPA (SEQ ID NOV) or any other peptide, which include SEQ ID NOs: 2-9, 13-17. and 20, at least or consists of 4, 5, 6, 7. 8, 9, or 10 contiguous amino acids of RTIGPSV (SEQ ID NO: 12) or LALGETTRPA (SEQ ID NOV) or any other peptide of SEQ ID NO: 2-9, 13-17, and 20. In some embodiments, the peptide insertion occurs within (i.e., between two amino acids without deleting any capsid amino acids) variable region IV (VR IV) of an AAV9 (SEQ ID NO: 118) capsid, or a corresponding region for another type AAV capsid, in particular, AAV3B, AAVrh73, AAV.hu.26, AAVhu.51, or AAVrh64Rl (see Table 5 and alignment in FIG. 1). In some embodiments, the peptide insertion occurs within (i.e.. between two amino acids without deleting any capsid amino acids) variable region VIII (VR-VIII) of an AAV9 capsid, or a corresponding region of a capsid for another AAV type (see exemplary⁷ alignments in FIG. 1).

[0075] In the various embodiments, the rAAV capsids and/or insertion peptides direct the rAAV particles to target tissues, more specifically, the eye, including the anterior segment tissues or the posterior segment tissues, and/or promote rAAV uptake, transduction and/or genome integration. Also provided are nucleic acids encoding the engineered capsid proteins and variants thereof, packaging cells for expressing the nucleic acids to produce rAAV vectors, rAAV vectors further comprising a transgene, and pharmaceutical compositions of the rAAV vectors, as well as methods of using the rAAV vectors to deliver the transgene to a target cell type or target tissue of a subject in need thereof.

[0076] In the various embodiments, the rAAV capsid specifically recognizes and/or promotes transduction of ocular tissue, or for example, one or more specific cell types, such as within the target tissue, or cellular matrix thereof. In particular, the capsids target rAAVs to ocular tissues, including the iris, cornea, ciliary body, Schlemm’s canal, trabecular meshwork, RPE-choroid, and optic nerve, and particularly, the retina.

[0077] Provided are capsids with the peptide inserted at positions amenable to peptide insertions within and near the AAV9 capsid VR-IV loop (see FIG. 2) and corresponding regions on the VR-IV loop of capsids of other AAV types. Though previous studies analyzed potential positions in various AAVs, none identified the AAV9 VR-IV as amenable for this purpose (consider, e.g., Wu et al, 2000, “Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism/’ J of Virology 74(18): 8635-8647; Lochrie et al, 2006, “Adeno-associated virus (AAV) capsid genes isolated from rat and mouse liver genomic DNA define two new AAV species distantly related to AAV-5,” Virology 353:68-82; Shi and Bartlett, 2003, “RGD Inclusion in VP3 Provides Adeno- Associated Virus Type 2 (AAV2)-Based Vectors with a Heparan Sulfate-Independent Cell Entry Mechanism,” Molecular Therapy 7(4):515525-; Nicklin et al., 2001, “Efficient and Selective AAV2-Mediated Gene Transfer Directed to Human Vascular Endothelial Cells” Molecular Therapy 4(2): 174-181; Grifman et al., 2001, “Incorporation of Tumor-Targeting Peptides into Recombinant Adeno-associated Virus Capsids,” Molecular Therapy 3(6):964- 975; Girod et al. 1999, “Genetic capsid modifications allow efficient re-targeting of adeno- associated virus type 2,” Nature Medicine 3(9): 1052-1056; Douar et al., 2003, “Deleterious effect of peptide insertions in a permissive site of the AAV2 capsid. “Virology 309:203-208; and Ponnazhagan, et al. 2001, 7. of Virology! 75(19):9493-9501).

[0078] Accordingly, provided are rAAV vectors carry ing a RTIGPSV (SEQ ID NO: 12), LGETTRP (SEQ ID NO: 8) or LALGETTRPA (SEQ ID NO:9) or other peptide, for example, SEQ ID Nos: 2-20, peptide insertion at insertion points, in particular, within surface-exposed variable regions in the capsid coat, particularly within or near the variable region IV of the capsid protein. In some embodiments, the rAAV capsid protein comprises a peptide insertion immediately after (i.e., connected by a peptide bond C-terminal to) an amino acid residue corresponding to one of amino acids 451 to 461 of AAV9 capsid protein (amino acid sequence SEQ ID NO:67 and see FIG. 1 for alignment of capsid protein amino acid sequence of other AAV serotypes with amino acid sequence of the AAV9 capsid), where said peptide insertion is surface exposed when the capsid protein is packaged as an AAV particle. For example, in a particular embodiment, an AAV3B capsid protein comprises the RTIGPSV (SEQ ID NO: 12)peptide insertion immediately after (i.e., connected by a peptide bond C-terminal to) an amino acid residue corresponding to one of amino acids 449 to 459 of the AAV3B (SEQ ID NO:74)or amino acids 452 to 461 of AAVrh73 capsid protein (SEQ ID NO:75), where said peptide insertion is surface exposed when the capsid protein is packaged as an AAV particle. The peptide insertions should not delete any residues of the AAV capsid protein. Generally, the peptide insertion occurs in a variable (poorly conserved) region of the capsid protein, compared with other serotypes, and in a surface exposed loop.

[0079] A peptide insertion described as inserted “at” a given site refers to insertion immediately after, that is having a peptide bond to the carboxy group of, the residue normally found at that site in the wild type virus. For example, insertion at Q588 in AAV9 means that the peptide insertion appears between Q588 and the consecutive amino acid (A589) in the AAV9 wildtype capsid protein sequence (SEQ ID NO:67). In embodiments, there is no deletion of amino acid residues at or near (within 5, 10, 15 residues or within the structural loop that is the site of the insertion) the point of insertion. In particular embodiments, the capsid protein is an AAV3B capsid protein or an AAVrh73 capsid protein and the insertion occurs immediately after at least one of the amino acid residues 449 to 459 or 451 to 461 , respectively. In particular embodiments, the peptide insertion occurs immediately after amino acid residues Q449, G450, T451, T452, S453, G454, T455, T456, N457, Q458, or S459 of the AAV3B capsid or Q452, S453, T454, G455, G456, T457, A458, G459, T460, or Q461 of the AAVrh73 capsid. In certain embodiments, the peptide is inserted between residues S454 and G455 of

AAV9 capsid protein, between residues G454 and T455 of AAV3B capsid protein, between residues G457 and T458 of AAVrh73, or between the residues corresponding to S454 and G455 of an AAV capsid protein other than an AAV9 capsid protein (amino acid sequence SEQ ID NO:67). In other embodiments, the capsid protein is from at least one AAV type selected from AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3). serotype 3B (AAV3B) serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype rh73 (AAVrh73), serotype hu.37 (AAVhu.37). serotype rh74 (AAVrh74, versions 1 and 2), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hu!2 (AAV.hul2), or serotype hu26 (AAV.hu26), (see FIG. 1), and the insertion occurs immediately after an amino acid residue corresponding to at least one of the amino acid residues 451 to 461 of AAV9. The alignments of these different AAV serotypes, as shown in FIG. 1, indicates “corresponding"’ amino acid residues in the different capsid amino acid sequences such that a “corresponding” amino acid residue is lined up at the same position in the alignment as the residue in the reference sequence. In some particular embodiments, the peptide insertion occurs immediately after one of the amino acid residues within: 450-459 of AAV1 capsid (SEQ ID NO: 59); 449-458 of AAV2 capsid (SEQ

ID NO:60); 449-459 of AAV3 capsid (SEQ ID NO:61); 449-459 of AAV3B capsid (SEQ ID

NO:74), 443-453 of AAV4 capsid (SEQ ID NO:62); 442-445 of AAV5 capsid (SEQ ID

NO:63); 450-459 of AAV6 capsid (SEQ ID NO:64); 451-461 of AAV7 capsid (SEQ ID

NO:65); 451-461 of AAV8 capsid (SEQ ID NO:66); 451-461 of AAV9 capsid (SEQ ID

NO: 67); 452-461 of AAV9e capsid (SEQ ID NO: 68); 452-461 of AAVrhlO capsid (SEQ ID

NO:69); 452-461 of AAVrh20 capsid (SEQ ID NO:70); 452-461 of AAVhu.37 (SEQ ID NO:71); 452-461 of AAVrh73; 452-461 of AAVrh74 (SEQ ID NO:72 or SEQ ID NO: 1); 452- 461 of AAVrh39 (SEQ ID NO:73), 449-458 of AAVhul2 (SEQ ID NO:78), 449-458 of AAVhu21 (SEQ ID NO:77), 449-458 of AAVhu26 (SEQ ID NO:79), or 449-458 of AAVhu51 (SEQ ID NO:76) in the sequences depicted in FIG. 1. In certain embodiments, the rAAV capsid protein comprises a peptide insertion immediately after (z.e., C-terminal to) amino acid 588 of AAV9 capsid protein (having the amino acid sequence of SEQ ID NO:67 and see FIG. 1), where said peptide insertion is surface exposed when the capsid protein is packaged as an AAV particle. In specific embodiments, the rAAV capsid protein comprises a peptide insertion, in particular, LALGETTRPA (SEQ ID NOV), immediately after amino acid 588 of AAV3B capsid protein or immediately after amino acid 590 of AAVrh73 capsid protein. In other embodiments, the rAAV capsid protein has a peptide insertion that is not immediately after amino acid 588 of AAV9 or corresponding to amino acid 588 of AAV9.

[0080] In other embodiments, when the peptide is a targeting peptide, including, at least 4 contiguous amino acids, or at least 10 contiguous amino acids, or is exactly 10 contiguous amino acids, or functional fragments thereof, of RTIGPSV (SEQ ID NO: 12), the capsid protein is from at least one AAV type selected from AAV serotype 1 (AAV1), serotype 2 (AAV 2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV 6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), serotype rh73 (AAVrh73), serotype rh74 (AAVrh74, versions 1 and 2), serotype hu51 (AAV.hu51), serotype hu21 (AAV.hu21), serotype hul2 (AAV.hul2), or seroty pe hu26 (AAV.hu26) (see FIG. 1), and the peptide is inserted in the capsid protein at any point such that the peptide is surface exposed when incorporated into the AAV vector. In specific embodiments, the peptide is inserted after 138; 262-272; 450-459; or 585-593 of AAV 1 capsid (SEQ ID NO:59); 138: 262-272; 449-458; or 584-592 of AAV2 capsid (SEQ ID NO:60); 138; 262-272; 449-459; or 585-593 of AAV3 capsid (SEQ ID NO:61); 138; 262-272; 449-459; or 585-593 of AAV3B capsid (SEQ ID NO:74); 137; 256-262; 443-453; or 583-591 of AAV4 capsid (SEQ ID NO:62); 137; 252-262; 442-445; or 574-582 of AAV5 capsid (SEQ ID NO:63); 138; 262-272; 450-459; 585-593 of AAV6 capsid (SEQ ID NO:64); 138; 263-273;

451-461; 586-594 of AAV7 capsid (SEQ ID NO:65); 138; 263-274; 452-461; 587-595 of AAV8 capsid (SEQ ID NO:66); 138; 262-273; 452-461; 585-593 of AAV9 capsid (SEQ ID NO:67); 138; 262-273; 452-461; 585-593 of AAV9e capsid (SEQ ID NO;68); 138; 263-274;

452-461; 587-595 of AAVrhlO capsid (SEQ ID NO:69); 138; 263-274; 452-461; 587-595 of AAVrh20 capsid (SEQ ID NO:70); 138; 263-274; 452-461; 587-595 of AAVrh73 capsid (SEQ ID NO:75); 138; 263-274; 452-461; 587-595 of AAVrh74 capsid (SEQ ID NO:72 or SEQ ID NO: 1) , 138; 263-274; 452-461; 587-595 of AAVhu37 capsid (SEQ ID NO:71); 138; 263-274; 452-461; 587-595 of AAVrh39 capsid (SEQ ID NO:734); 138; 264-271; 449-458; 584-592 of AAVhul2 capsid (SEQ ID NO:78); 449-458; 584-592 of AAVhu21 capsid (SEQ ID NO:77); 449-458; 584-592 of AAVhu26 capsid (SEQ ID NO:79); and 449-458; 584-592 of AAVhu51 capsid (SEQ ID NO:76) (as numbered in FIG. 1).

[0081] In some embodiments, the capsid protein is from an AAV other than serotj pe AAV2. In some embodiments, the peptide insertion does not occur immediately after an amino acid residue corresponding to amino acid 570 or 611 of AAV2 capsid protein. In some embodiments, the peptide insertion does not occur between amino acid residues corresponding to amino acids 587-588 of AAV2 capsid protein (see US 2014/0294771 to Schaffer et al).

[0082] Also provided are AAV vectors comprising the engineered capsids. In some embodiments, the AAV vectors are non-replicating and do not include the nucleotide sequences encoding the rep or cap proteins (these are supplied by the packaging cells in the manufacture of the rAAV vectors). In some embodiments, AAV-based vectors comprise components from one or more serotypes of AAV. In some embodiments, AAV based vectors provided herein comprise capsid components from one or more of AAV1. AAV2, AAV3, AAV3B. AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh73, AAV.rh74, AAV.RHM4- 1, AAV.hu.26, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV. PHP. eB, AAV2.5, AAV21YF, AAV3B. AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3. AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC 10, AAV.HSC11 , AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particles, or combinations of two or more thereof. In some embodiments, AAV based vectors provided herein comprise components from one or more of AAV1. AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh73, AAV.rh74, AAV.RHM4-1, AAV.hu.26, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7. AAV.HSC8. AAV.HSC9. AAV.HSC10, AAV.HSC11. AAV.HSC12. AAV.HSC 13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particles, or combinations of two or more thereof serotypes. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%. 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., VP1, VP2 and/or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh73, AAV.rh74, AAV.RHM4-1, AAV.hu.26, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7. AAV.HSC8. AAV.HSC9. AAV.HSC10, AAV.HSC11. AAV.HSC12. AAV.HSC 13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof. These engineered AAV vectors may comprise a genome comprising a transgene encoding a therapeutic protein.

[0083] In particular embodiments, the recombinant AAV for use in compositions and methods herein is Anc80 or Anc80L65 (see, e.g., Zinn etal., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety ). In particular embodiments, the recombinant AAV for use in compositions and methods herein is AAV.7m8 (including variants thereof) (see, e.g., US 9,193,956; US 9,458.517; US 9,587,282; US 2016/0376323, and WO 2018/075798, each of which is incorporated herein by reference in its entirety ). In particular embodiments, the AAV for use in compositions and methods herein is any AAV disclosed in US 9,585,971, such as AAV-PHP.B. In particular embodiments, the AAV for use in compositions and methods herein is an AAV2/Rec2 or AAV2/Rec3 vector, which has hybrid capsid sequences derived from AAV8 and serotypes cy5, rh20 or rh39 (see, e.g.. Issa et al., 2013, PLoS One 8(4): e60361 , which is incorporated by reference herein for these vectors). In particular embodiments, the AAV for use in compositions and methods herein is an AAV disclosed in any of the following, each of which is incorporated herein by reference in its entirety: US 7,282,199; US 7,906.111; US 8,524,446; US 8,999,678; US 8.628,966; US 8,927,514; US 8,734,809; US9,284,357; US 9,409,953; US 9,169,299; US 9,193,956; US 9,458,517; US 9,587,282; US 2015/0374803; US 2015/0126588; US 2017/0067908; US 2013/0224836; US 2016/0215024; US 2017/0051257; PCT/US2015/034799; and PCT/EP2015/053335. In some embodiments. rAAV particles have a capsid protein at least 80% or more identical, e.g. 85%. 85%. 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7.906,111; 8.524,446; 8,999.678; 8,628.966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9,458,517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application Nos. PCT/US2015/034799; PCT/EP2015/053335.

[0084] In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsids of AAVLK03 or AAV3B, as described in Puzzo etal.. 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16. each of which is incorporated by reference in its entirety . [0085] In some embodiments. rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2 of '051 publication), WO 2005/033321 (see, e.g, SEQ ID NOs: 123 and 88 of '321 publication), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397 publication). WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of '888 publication), WO 2006/110689, (see, e.g. SEQ ID NOs: 5-38 of '689 publication) W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of '964 publication), WO 2010/127097 (see, e.g., SEQ ID NOs: 5-38 of '097 publication), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294 of '508 publication), and U.S. Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of '924 publication), the contents of each of which is herein incorporated by reference in its entirety. In some embodiments. rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g, SEQ ID NO: 2 of '051 publication). WO 2005/033321 (see, e.g. SEQ ID NOs: 123 and 88 of '321 publication), WO 03/042397 (see, e.g, SEQ ID NOs: 2, 81, 85, and 97 of '397 publication), WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of '888 publication), WO 2006/110689 (see, e.g, SEQ ID NOs: 5-38 of '689 publication) W02009/104964 (see, e.g, SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of 964 publication), W0 2010/127097 (see, e.g, SEQ ID NOs: 5-38 of '097 publication), and WO 2015/191508 (see, e.g, SEQ ID NOs: 80-294 of '508 publication), and U.S. Appl. Publ. No. 20150023924 (see, e.g, SEQ ID NOs: 1, 5-10 of '924 publication).

[0086] In additional embodiments, rAAV particles comprise a pseudotyped AAV capsid. In some embodiments, the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g, Duan etal.. J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74: 1524-1532 (2000); Zolotukhin et al., Methods 28: 158-167 (2002); and Auricchio et al.. Hum. Molec. Genet. 10:3075-3081, (2001).

[0087] In certain embodiments, a single-stranded AAV (ssAAV) may be used. In certain embodiments, a self-complementary’ vector, e.g, scAAV, may be used (see. e.g., Wu, 2007, Human Gene Therapy, 18(2): 171-82; McCarty et al, 2001, Gene Therapy, 8(16): 1248-1254; US 6,596,535; US 7,125,717; and US 7,456,683, each of which is incorporated herein by reference in its entirety ).

[0088] Generally, the peptide insertion is sequence of contiguous amino acids from a heterologous protein or domain thereof. The peptide to be inserted typically is long enough to retain a particular biological function, characteristic, or feature of the protein or domain from which it is derived. The peptide to be inserted typically is short enough to allow the capsid protein to form a coat, similarly or substantially similarly to the native capsid protein without the insertion. In preferred embodiments, the peptide insertion is from about 4 to about 30 amino acid residues in length, about 4 to about 20, about 4 to about 15, about 5 to about 10, or about 7 amino acids in length. The peptide sequences for insertion are at least 4 amino acids in length and may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the peptide sequences are 16, 17, 18, 19, or 20 amino acids in length. In embodiments, the peptide is no more than 7 amino acids, 10 amino acids or 12 amino acids in length.

[0089] A “peptide insertion from a heterologous protein” in an AAV capsid protein refers to an amino acid sequence that has been introduced into the capsid protein and that is not native to any AAV seroty pe capsid. Non-limiting examples include a peptide of a human protein in an AAV capsid protein.

[0090] The present inventors also have surprisingly discovered particular peptides that can be used to re-target AAV vectors to specific tissues, organs, or cells; in particular, providing peptides that cause rAAV vectors to target ocular tissue. Without being bound by any one theory, a peptide, e.g., the RTIGPSV (SEQ ID NO: 12) peptide, inserted in an AAV capsid variable region loop, was demonstrated to enhance transduction efficiency in ocular tissues. Such peptides can provide enhanced transport of AAV particles encapsidating a transgene across an endothelial cellular matrix.

[0091] The follow summarizes insertion sites for the peptides described herein, immediately after amino acid residues of AAV capsids as set forth below (see also, FIG. 1):

AAV3B: 138; 262-272; 449-459; 585-593; and in particular embodiment, between 452-453 (SEQ ID NO:74).

AAV8: 138; 263-274; 451-461; 587-595; and in particular embodiment, between 453-

454 (SEQ ID NO: 66).

AAV9: 138; 262-273; 452-461; 585-593; and in particular embodiment, between 454-

455 (SEQ ID NO:67).

[0092] In particular embodiments, the peptide insertion occurs between amino acid residues 588-589 of the AAV9 capsid, or between corresponding residues of another AAV type capsid as determined by an amino acid sequence alignment (for example, as in FIG. 1). In particular embodiments, the peptide insertion occurs immediately after amino acid residue 1451 to L461, S268 and Q588 of the AAV9 capsid sequence, or immediately after corresponding residues of another AAV capsid sequence (FIG. 1).

[0093] In some embodiments, one or more peptide insertions from one or more homing domains can be used in a single system. In some embodiments, the capsid is chosen and/or further modified to reduce recognition of the AAV particles by the subject's immune system, such as avoiding pre-existing antibodies in the subject. In some embodiments. In some embodiments, the capsid is chosen and/or further modified to enhance desired tropism/targeting.

5.3. Methods of Making rAAV Molecules

[0094] Another aspect of the present invention involves making molecules disclosed herein. In some embodiments, a molecule according to the invention is made by providing a nucleotide comprising the nucleic acid sequence encoding any of the capsid protein molecules herein; and using a packaging cell system to prepare corresponding rAAV particles with capsid coats made up of the capsid protein. In some embodiments, the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of a capsid protein molecule described herein, and retains (or substantially retains) biological function of the capsid protein (including in some embodiments having an inserted peptide from a heterologous protein or domain thereof). In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99% or 99.9%. identity to the sequence of the AAV9 capsid protein (SEQ ID NO: 67 and see FIG. 1), while retaining (or substantially retaining) biological function of the AAV9 capsid protein. In some embodiments, the nucleic acid encodes a sequence having at least 60%. 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99% or 99.9%, identity to the sequence of the AAV3B capsid protein (SEQ ID NO:74) or AAV8 capsid protein (SEQ ID NO:66) while retaining (or substantially retaining) biological function of the AAV3B or AAV8 capsid protein.

[0095] The capsid protein, coat, and rAAV particles may be produced by techniques known in the art. In some embodiments, the viral genome comprises at least one inverted terminal repeat to allow packaging into a vector. In some embodiments, the viral genome further comprises a cap gene and/or a rep gene for expression and splicing of the cap gene. In other embodiments, the cap and rep genes are provided by a packaging cell and not present in the viral genome.

[0096] In some embodiments, the nucleic acid encoding the engineered capsid protein is cloned into an AAV Rep-Cap helper plasmid in place of the existing capsid gene. When introduced together into host cells, this plasmid helps package an rAAV genome into the engineered capsid protein as the capsid coat. Packaging cells can be any cell type possessing the genes necessary to promote AAV genome replication, capsid assembly, and packaging. Nonlimiting examples include 293 cells or derivatives thereof, HELA cells, or insect cells.

[0097] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See. e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press. Cold Spring Harbor. N.Y. (1989)), which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery’, and treatment of patients. Nucleic acid sequences of AAV-based viral vectors, and methods of making recombinant AAV and AAV capsids, are taught, e g., in US 7,282,199; US 7,790,449; US 8,318,480; US 8,962,332; and PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety.

[0098] In embodiments, the rAAVs provided herein comprise a recombinant AAV genome that comprises an expression cassette, flanked by ITR sequences, such as AAV2 or AAV9 ITR sequences, where the expression cassette comprises a nucleotide sequence encoding a therapeutic protein for treatment of an ocular indication. In embodiments, the therapeutic protein is a VEGF fusion protein, such as aflibercept, an anti-VEGF antibody, or antigenbinding fragment thereof, such as, sevacizumab. ranibizumab, bevacizumab, or brolucizumab, an anti-kallikrein antibody, or antigen binding fragment thereof, such as lanadelumab, an anti- IL6 or anti-IL6R antibody, or antigen binding fragment thereof, such as satralizumab, sarilumab, siltuximab, clazakizumab, sirukumab, olokizumab, gerilimzumab, or tocilizumab, an anti-TNF antibody, or antigen binding fragment thereof, such as, adalimumab, infliximab, golimumab, or certolizumab-pegol, a TNF Receptor fusion protein, such as etanercept, an anti- C3 antibody, or antigen binding fragment thereof, such as, NGM621 , eculizumab, ravulizumab, or tesidolumab, or an anti-C5 antibody, or antigen binding fragment thereof, such as crovalimab.

[0099] In some embodiments, the r AAV s provide transgene delivery vectors that can be used in therapeutic and prophylactic applications, as discussed in more detail below. In some embodiments, the rAAV vector also includes regulatory control elements known to one skilled in the art to influence the expression of the RNA and/or protein products encoded by nucleic acids (transgenes) within target cells of the subject. Regulatory’ control elements and may be tissue-specific, that is. active (or substantially more active or significantly more active) only in the target cell/tissue. In specific embodiments, the AAV vector comprises a regulatory sequence, such as a promoter, operably linked to the transgene that allows for expression in target tissues. The promoter may be a constitutive promoter, for example, the CB7 promoter. Additional promoters include: cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter, or opsin promoter. In some embodiments, particularly where it may be desirable to turn off transgene expression, an inducible promoter is used, e.g., hypoxia-inducible or rapamycin-inducible promoter.

[00100] Provided in particular embodiments are AAV3B serotype capsid vectors comprising a viral genome comprising an expression cassette for expression of the transgene, under the control of regulatory elements, and flanked by ITRs and an engineered viral capsid as described herein or is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAV3B (SEQ ID NO: 74), while retaining the biological function of the AAV3B serotype. In certain embodiments, the encoded AAV3B serotype capsid has the sequence of AAV3B serotype with, in addition, 1, 2, 3. 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17. 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions with respect to the AAV3B serotype.

[00101] The recombinant adenovirus can be a first-generation vector, with an El deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region. The recombinant adenovirus can be a second-generation vector, which contains full or partial deletions of the E2 and E4 regions. A helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi). The transgene generally is inserted between the packaging signal and the 3 TTR, with or without stuffer sequences to keep the genome close to wild-type size of approximately 36 kb. An exemplary protocol for production of adenoviral vectors may be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12:S18-S27, which is incorporated by reference herein in its entirety

[00102] The rAAV vector for delivering the transgene to target tissues, cells, or organs, has a tropism for that particular target tissue, cell, or organ, in particular the eye and tissues within the eye. Tissue-specific promoters may also be used. The construct further can include expression control elements that enhance expression of the transgene driven by the vector (e.g., introns such as the chicken (3-actin intron, minute virus of mice (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), |3-globin splice donor/immunoglobulin heavy chain spice acceptor intron, adenovirus splice donor /immunoglobulin splice acceptor intron, SV40 late splice donor /splice acceptor (19S/16S) intron, and hybrid adenovirus splice donor/IgG splice acceptor intron and polyA signals such as the rabbit (3-globin polyA signal, human growth hormone (hGH) polyA signal. SV40 late polyA signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal. See, e.g., Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57. [00103] In certain embodiments, nucleic acids sequences disclosed herein may be codon- optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149-161).

[00104] In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) control elements, which include a constitutive promoter or an ocular tissue specific promoter, optionally, an intron sequence, such as a chicken p-actin intron and a poly A signal; and (3) trans gene providing (e.g., coding for) a nucleic acid or protein product of interest. In embodiments, the protein of interest is an ocular therapeutic protein, including, for example, a VEGF fusion protein, such as aflibercept, an anti-VEGF antibody, or antigen-binding fragment thereof, such as, sevacizumab, ranibizumab, bevacizumab, or brolucizumab, an anti-kallikrein antibody, or antigen binding fragment thereof, such as lanadelumab, an anti-IL6 or anti-IL6R antibody, or antigen binding fragment thereof, such as satralizumab, sarilumab, siltuximab, clazakizumab, sirukumab, olokizumab, gerilimzumab, or tocilizumab, an anti-TNF antibody, or antigen binding fragment thereof, such as, adalimumab, infliximab, golimumab, or certolizumab-pegol, a TNF Receptor fusion protein, such as etanercept, an anti-C3 antibody, or antigen binding fragment thereof, such as, NGM621, eculizumab, ravulizumab. or tesidolumab, or an anti-C5 antibody, or antigen binding fragment thereof, such as crovalimab. [00105] The viral vectors provided herein may be manufactured using host cells, e.g., mammalian host cells, including host cells from humans, monkeys, mice, rats, rabbits, or hamsters. Nonlimiting examples include: A549, WEHI, 10T1/2, BHK, MDCK, COS1. COS7, BSC 1, BSC 40, BMT 10. VERO. W138. HeLa. 293, Saos. C2C12, L. HT1080, HepG2. primary fibroblast, hepatocyte, and myoblast cells. Typically, the host cells are stably transformed with the sequences encoding the transgene and associated elements (i.e., the vector genome), and genetic components for producing viruses in the host cells, such as the replication and capsid genes (e.g. , the rep and cap genes of AAV). For a method of producing recombinant AAV vectors with AAV8 capsids, see Section IV of the Detailed Description of U.S. Patent No. 7,282,199 B2, which is incorporated herein by reference in its entirety. Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis. Virions may be recovered, for example, by CsCh sedimentation. Alternatively, baculovirus expression systems in insect cells may be used to produce AAV vectors. For a review, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102: 1045-1054, which is incorporated by reference herein in its entirety for manufacturing techniques. [00106] In vitro assays, e.g., cell culture assays, can be used to measure transgene expression from a vector described herein, thus indicating, e.g., potency of the vector. For example, the PER.C6® Cell Line (Lonza), a cell line derived from human embry onic retinal cells, or retinal pigment epithelial cells, e.g, the retinal pigment epithelial cell line hTERT RPE-1 (available from ATCC®), can be used to assess transgene expression. Alternatively, cell lines derived from liver or other cell types may be used, for example, but not limited, to HuH-7, HEK293, fibrosarcoma HT-1080, HKB-11, and CAP cells. Once expressed, characteristics of the expressed product (z.e., transgene product) can be determined, including determination of the glycosylation and tyrosine sulfation patterns, using assays known in the art.

5.4. Therapeutic and Prophylactic Uses

[00107] Another aspect relates to therapies which involve administering a transgene via a rAAV vector according to the invention to a subject in need thereof, for delaying, preventing, treating, and/or managing an ocular disease or disorder, and/or ameliorating one or more symptoms associated therewith. A subject in need thereof includes a subject suffering from the disease or disorder, or a subject pre-disposed thereto, e.g, a subject at risk of developing or having a recurrence of the disease or disorder. Generally, a rAAV carrying a particular transgene will find use with respect to a given disease or disorder in a subject where the subject’s native gene, corresponding to the transgene, is defective in providing the correct gene product, or correct amounts of the gene product. The transgene then can provide a copy of a gene that is defective in the subject.

[00108] The transgene may comprise cDNA that restores protein function to a subject having a genetic mutation(s) in the corresponding native gene. In some embodiments, the cDNA comprises associated RNA for performing genomic engineering, such as genome editing via homologous recombination. In some embodiments, the transgene encodes a therapeutic RNA, such as a shRNA, artificial miRNA, or element that influences splicing.

[00109] As described herein, the AAV vector may be selected or engineered as described herein to target the appropriate tissue or cell type, including ocular tissue, including posterior ocular tissue, such as retina or RPE choroid, for delivery of the transgene to effect the therapeutic or prophylactic use. In embodiments, the capsid is an AAV3B capsid.

[00110] In particular aspects, the rAAVs described herein find use in delivery to target ocular tissues, or target ocular tissue cell types, including cell matrix associated with the target cell t pes, associated with the disorder or disease to be treated/prevented. A disease or disorder associated with a particular tissue or cell type is one that largely affects the particular tissue or cell type, in comparison to other tissue of cell types of the body, or one where the effects or symptoms of the disorder appear in the particular tissue or cell type. Methods of delivering a transgene to a target tissue of a subject in need thereof involve administering to the subject an rAAV where the capsid has a tropism for the tissue cell type, including enhanced transduction, genome integration, transgene mRNA and protein expression in ocular tissue, including as compared to an rAAV having a reference capsid, such as AAV2, AAV8 or AAV9.

[00111] For a disease or disorder associated with the retina or eye, the rAAV vector has a capsid with ocular tropism, directing the rAAV to target the eye or ocular tissues of the subject, including, in embodiments, crossing the blood-eye barrier. The term “retinal cell” refers to one or more of the cell types found in or near the retina, including amacrine cells, bipolar cells, horizontal cells, Muller glial cells, photoreceptor cells (e.g., rods and cones), retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retina ganglion cells, and photosensitive ganglion cells), retinal pigmented epithelium, endothelial cells of the inner limiting membrane, and the like. Ocular tissues include anterior segment tissues, including the iris, cornea, lens, ciliary body, Schl emm’s canal, and trabecular meshwork, and posterior segment tissues, such as the retina or RPE-choroid, and optic nerve (see FIGS. 2A and 2B).

[00112] In additional embodiments, methods and compositions are provided in which an rAAV comprising a recombinant genome comprising a transgene encoding an ocular therapeutic have a capsid with a tropism for transduction and/or transgene expression in ocular tissue, including anterior and/or posterior segments, with a capsid of an AAV serotype 3B (AAV3B; SEQ ID NO:74), or has a VP1 capsid protein that is at least 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 74 and has ocular tropism.

[00113] Generally, where the rAAV vector has a tropism for ocular tissues, the vector is administered by in vivo inj ection, such as inj ection directly into the eye. F or example, the rAAV comprising a peptide insertion for increasing tropism for ocular, retinal or RPE-choroid tissue may be injected intravitreally. intracamerally or suprachoroi dally. In some embodiments, the rAAV with ocular tissue tropism is administered by intraocular injection, e.g., through the pars plana into the vitreous body or aqueous humor of the eye. In some embodiments, the rAAV for increasing ocular tissue tropism is administered peribulbar injection or subconjunctival injection. In some embodiments, the rAAV with ocular tissue tropism is administered by suprachoroidal injection, that is in the space between the sclera and the choroid. One advantage of rAAV vectors with ocular tissue tropism, is that the subject may avoid surgery', e.g., avoiding surgery to implant the therapeutic instead delivered by injection. In certain embodiments, the therapeutic is delivered by a rAAV vector described herein by intracameral, intravitreal or suprachoroidal injection, to provide a therapeutically effective amount for treating a disease or disorder associated with the eye, particularly, a disease or disorder associated with the eye of the subject. In more embodiments, treatment is achieved following a single intracameral. intravitreal or suprachoroidal injection, not more than two intracameral, intravitreal or suprachoroidal injections, not more than three intracameral, intravitreal or suprachoroidal injections, not more than four intracameral, intravitreal or suprachoroidal injections, not more than five intracameral, intravitreal or suprachoroidal injections, or not more than six intracameral, intravitreal or suprachoroidal injections.

[00114] Diseases/disorders associated with the eye or retina are referred to as "‘ocular diseases.'’ Nonlimiting examples of ocular diseases include anterior ischemic optic neuropathy; acute macular neuroretinopathy; Bardet-Biedl syndrome; Behcet's disease; branch retinal vein occlusion; central retinal vein occlusion; choroideremia; choroidal neovascularization; chorioretinal degeneration; cone-rod dystrophy; color vision disorders (e.g., achromatopsia, protanopia, deuteranopia, and tritanopia); congenital stationary night blindness; diabetic uveitis; epiretinal membrane disorders; inherited macular degeneration; histoplasmosis; macular degeneration (e.g., acute macular degeneration, non-exudative age related macular degeneration, exudative age related macular degeneration, dry age related macular degeneration or dry age related macular degeneration with geographic atrophy); diabetic retinopathy; edema (e.g., macular edema, cystoid macular edema, diabetic macular edema); glaucoma; Leber congenital amaurosis; Leber's hereditary optic neuropathy; macular telangiectasia; multifocal choroiditis; non-retinopathy diabetic retinal dysfunction; ocular trauma; ocular tumors; proliferative vitreoretinopathy (PVR); retinopathy of prematurity; retinoschisis; retinitis pigmentosa; retinal arterial occlusive disease, retinal detachment, Stargardt disease (fundus flavimaculatus); sympathetic opthalmia; uveal diffusion; uveitic retinal disease; Usher syndrome; Vogt Koyanagi-Harada (VKH) syndrome; or a posterior ocular condition associated with ocular laser or photodynamic therapy.

[00115] In particular embodiments, the disease or disorder is non-infectious uveitis, neuromyelitis optica, macular degeneration, including dry age-related macular degeration, dry age-related macular degeration with geographic atrophy’, macular edema, diabetic retinopathy or glaucoma. [00116] In particular embodiments, the rAAV targets (including, transduction and transgene expression) one or more specific ocular tissues, including the anterior segment tissues or the posterior segment tissues and, in more specific embodiments, the rAAV targets the cornea, iris or lens, or ciliary’ body, Schlemm’s canal or trabecular meshwork, or retinal, retinal pigment epithelium (RPE-) choroid or sclera, or the optic nerve. In particular embodiments, rAAVs having an AAV3B may be administered to target the iris, retina, RPE choroid or sclera, and in certain embodiments, the ciliary’ body, Schlemm’s canal, trabelcular meshwork or optic nene (orbital and/or cranial segment). In embodiments, rAAVs having an AAV3B capsid may be used to target the retina and/or RPE choroid tissue, including where there is reduced transduction and/or expression in the sclera relative to the transduction and/or expression in the retina and/or RPE or relative transduction and/or expression in the sclera of a reference capsid such as AAV8. In embodiments, the recombinant AAV capsid is administered in the absence of hyaluronic acid.

[00117] In certain embodiments, the transgene comprises a nucleotide sequence which encodes an ocular disease therapeutic which is a VEGF fusion protein, such as aflibercept, an anti-VEGF antibody, or antigen-binding fragment thereof, such as, sevacizumab, ranibizumab, bevacizumab, or brolucizumab, an anti-kallikrein antibody, or antigen binding fragment thereof, such as lanadelumab, an anti-IL6 or anti-IL6R antibody, or antigen binding fragment thereof, such as satralizumab, sarilumab, siltuximab, clazakizumab, sirukumab, olokizumab, gerilimzumab, or tocilizumab, an anti-TNF antibody, or antigen binding fragment thereof, such as, adalimumab, infliximab, golimumab, or certolizumab-pegol. a TNF Receptor fusion protein, such as etanercept, an anti-C3 antibody, or antigen binding fragment thereof, such as, NGM621, eculizumab, ravulizumab, or tesidolumab, or an anti-C5 antibody, or antigen binding fragment thereof, such as, or crovalimab, LKA-651, solanezumab, GSK933776, lecanemab, ascrinvacumab, carotuximab, AND-007, inebilizumab, sFlt-1, sFlt2, Endostatin, Angiostatin, TIMP3, PEDF, Prph2, Rho, BPDE, Bcl2, FGF-2, Epo. CNTF, Mertk, GUCY2D, AIPL1, RPGROP, RPE65. GNA2, ABCA4, Rs l. BDNF, GDNF, IL-10, ILRa. IFNB, TK. L-opsin. amd GABP. Gene therapy constructs encoding antibodies, or antigen binding fragments thereof, are designed such that both the heavy’ and light chains are expressed. The coding sequences for the heavy and light chains can be engineered in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES, such as a furin-T2A linker or the like, so that separate heavy and light chain polypeptides are expressed. In certain embodiments, the coding sequences encode for a Fab or F(ab’)2 or an scFv. In certain embodiments the full length heavy and light chains of the antibody are expressed. In other embodiments, the constructs express an scFv in which the heavy and light chain variable domains are connected via a flexible, non-cleavable linker. The nucleotide sequence coding for the therapeutic protein is operably linked to regulatory elements to promote expression of the therapeutic protein in the target ocular tissue.

[00118] The rAAV vectors of the invention also can facilitate delivery, in particular, targeted delivery, of oligonucleotides, drugs, imaging agents, inorganic nanoparticles, liposomes, antibodies to target cells or tissues. The rAAV vectors also can facilitate delivery, in particular, targeted delivery, of non-coding DNA, RNA, or oligonucleotides to target tissues.

[00119] The agents may be provided as pharmaceutically acceptable compositions as known in the art and/or as described herein. Also, the rAAV molecule of the invention may be administered alone or in combination with other prophylactic and/or therapeutic agents.

[00120] The dosage amounts and frequencies of administration provided herein are encompassed by the terms therapeutically effective and prophy lactically effective. The dosage and frequency will typically vary according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity' and type of disease, the route of administration, as well as age, body weight, response, and the past medical history of the patient, and should be decided according to the judgment of the practitioner and each patient's circumstances. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician 's Desk Reference (56^th ed., 2002). Prophylactic and/or therapeutic agents can be administered repeatedly. Several aspects of the procedure may vary such as the temporal regimen of administering the prophylactic or therapeutic agents, and whether such agents are administered separately or as an admixture.

[00121] The amount of an agent of the invention that will be effective can be determined by standard clinical techniques. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the ICso (z.e., the concentration of the test compound that achieves a half- maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. [00122] Prophylactic and/or therapeutic agents, as well as combinations thereof, can be tested in suitable animal model systems prior to use in humans. Such animal model systems include, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in the art may be used. Such model systems are widely used and well known to the skilled artisan. In some embodiments, animal model systems for a ocular condition are used that are based on rats, mice, or other small mammal other than a primate. [00123] Once the prophylactic and/or therapeutic agents of the invention have been tested in an animal model, they can be tested in clinical trials to establish their efficacy. Establishing clinical trials will be done in accordance with common methodologies known to one skilled in the art, and the optimal dosages and routes of administration as well as toxicity profiles of agents of the invention can be established. For example, a clinical trial can be designed to test a rAAV molecule of the invention for efficacy and toxicity in human patients.

[00124] Toxicity and efficacy of the prophylactic and/or therapeutic agents of the instant invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00125] A rAAV generally will be administered for a time and in an amount effective for obtain a desired therapeutic and/or prophylactic benefit. The data obtained from the cell culture assays and animal studies can be used in formulating a range and/or schedule for dosage of the prophylactic and/or therapeutic agents for use in humans. The dosage of such agents lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[00126] A therapeutically effective dosage of an rAAV vector for patients is generally from about 0.1 ml to about 100 ml of solution containing concentrations of from about IxlO⁹ to about IxlO¹⁶ genomes, or about IxlO¹⁰ to about IxlO¹⁵ genomes, about IxlO¹² to about IxlO¹⁶ genomes, about IxlO¹⁴ to about IxlO¹⁶ genomes, about IxlO¹¹ to about IxlO¹³ genomes, or about IxlO¹² to about IxlO¹⁴ genomes. Levels of expression of the transgene can be monitored to determine/adjust dosage amounts, frequency, scheduling, and the like.

[00127] Treatment of a subject with a therapeutically or prophy tactically effective amount of the agents of the invention can include a single treatment or can include a series of treatments. For example, pharmaceutical compositions comprising an agent of the invention may be administered once or may be administered 2, 3 or 4 times, for example, separated by a week, month, 2 months or three months.

[00128] The rAAV molecules of the invention may be administered alone or in combination with other prophylactic and/or therapeutic agents. Each prophylactic or therapeutic agent may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. Each therapeutic agent can be administered separately, in any appropriate form and by any suitable route.

[00129] In various embodiments, the different prophylactic and/or therapeutic agents are administered less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 1 1 hours apart, at about 11 hours to about 12 hours apart, no more than 24 hours apart, or no more than 48 hours apart. In certain embodiments, two or more agents are administered within the same patient visit.

[00130] Methods of administering agents described herein include, but are not limited to, parenteral administration (e.g, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous, including infusion or bolus injection), epidural, and by absorption through epithelial or mucocutaneous or mucosal linings (e.g., intranasal, oral mucosa, rectal, and intestinal mucosa, etc.). In particular embodiments, such as where the transgene is intended to be expressed in the eye, the vector is administered via intravitreal, intraocular, suprachoroidaL or intracameral injection. In particular embodiments, the vector is administered directly to the target tissue, for example, is administered directly to the retina or ciliary body.

[00131] In certain embodiments, the agents of the invention are administered intravenously and may be administered together with other biologically active agents.

[00132] In another specific embodiment, agents of the invention may be delivered in a sustained release formulation, e.g., where the formulations provide extended release and thus extended half-life of the administered agent. Controlled release systems suitable for use include, without limitation, diffusion-controlled, solvent-controlled, and chemically-controlled systems. Diffusion controlled systems include, for example reservoir devices, in which the molecules of the invention are enclosed within a device such that release of the molecules is controlled by permeation through a diffusion barrier. Common reservoir devices include, for example, membranes, capsules, microcapsules, liposomes, and hollow fibers. Monolithic (matrix) device are a second type of diffusion controlled system, wherein the dual antigenbinding molecules are dispersed or dissolved in an rate-controlling matrix (e.g., a polymer matrix). Agents of the invention can be homogeneously dispersed throughout a rate-controlling matrix and the rate of release is controlled by diffusion through the matrix. Polymers suitable for use in the monolithic matrix device include naturally occurring polymers, synthetic polymers and synthetically modified natural polymers, as well as polymer derivatives.

[00133] Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more agents described herein. See, e.g. U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCT publication WO 96/20698; Ning et al., “Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained- Release Gel,'’ Radiotherapy & Oncology, 39: 179 189, 1996; Song et al., “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology, 50:372 397, 1995; Cleek et al., “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro. Inti. Symp. Control. Rel. Bioact. Mater., 24:853 854, 1997; and Lam et al., “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater., 24:759 760, 1997, each of which is incorporated herein by reference in its entirety’. In one embodiment, a pump may be used in a controlled release system (see Langer, supra,- Sefton, CRC Crit. Ref. Biomed. Eng., 14:20, 1987; Buchwald et al., Surgery, 88:507, 1980; and Saudek et al., N. Engl. J. Med., 321 :574, 1989). In another embodiment, polymeric materials can be used to achieve controlled release of agents comprising dual antigen-binding molecule, or antigen-binding fragments thereof (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.. 23:61, 1983; see also Levy et al., Science, 228: 190, 1985; During et al., Ann. Neurol., 25:351, 1989; Howard et al., J. Neurosurg., 7 1 : 105, 1989); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target (e.g., an affected joint), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 138 (1984)). Other controlled release systems are discussed in the review by Langer, Science, 249:1527 1533, 1990.

[00134] In addition, rAAVs can be used for in vivo delivery' of transgenes for scientific studies such as optogenetics, gene knock-down with miRNAs. recombinase delivery for conditional gene deletion, gene editing with CRISPRs, and the like.

5.5. Pharmaceutical Compositions and Kits

[00135] Further provided herein are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions disclosed herein (e.g., a pharmaceutical composition comprising an AAV3B comprising an expression cassette encoding a transgene) provide greater transgene expression and/or tissue/cell transduction at the back of the eye (e.g., retina) than in the outer layer of the eye (e.g., sclera) through SCS delivery. Such features of the presently disclosed pharmaceutical compositions have advantages because subretinal delivery is not required to achieve higher expression of the ocular transgenes at the back of the eye than the outer layer of the eye by administering the presently disclosed pharmaceutical compositions suprachoroi dally, which is an injection by in-office procedure. Further advantages are foreseen for a gene therapy that reduces AAV transduction and transgene expression at the outer layer of the eye, e.g. sclera, thus lowering the likelihood of inflammation such as episcleritis.

[00136] The pharmaceutical compositions disclosed herein comprise a pharmaceutically acceptable carrier and an agent of the invention, said agent comprising a rAAV molecule of the invention. In some embodiments, the pharmaceutical composition comprises rAAV combined with a pharmaceutically acceptable carrier for administration to a subject. In one embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, e.g.. peanut oil. soybean oil, mineral oil. sesame oil and the like. Water is a common carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Additional examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; saltforming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™ as known in the art. The pharmaceutical composition of the present invention can also include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[00137] In certain embodiments of the invention, pharmaceutical compositions are provided for use in accordance with the methods of the invention, said pharmaceutical compositions comprising a therapeutically and/or prophy tactically effective amount of an agent of the invention along with a pharmaceutically acceptable earner.

[00138] In certain embodiments, the agent of the invention is substantially purified (z.e., substantially free from substances that limit its effect or produce undesired side-effects). In a specific embodiment, the host or subject is an animal, e.g., a mammal such as non-primate (e.g, cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey such as, a cynomolgus monkey and a human). In a certain embodiment, the host is a human.

[00139] The invention provides further kits that can be used in the above methods. In one embodiment, a kit comprises one or more agents of the invention, e.g., in one or more containers. In another embodiment, a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of a condition, in one or more containers.

[00140] The invention also provides agents of the invention packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent or active agent. In one embodiment, the agent is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline, to the appropriate concentration for administration to a subject. Typically, the agent is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg. more often at least 10 mg. at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized agent should be stored at between 2 and 8°C in its original container and the agent should be administered within 12 hours, usually within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In an alternative embodiment, an agent of the invention is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of agent or active agent. Typically, the liquid form of the agent is supplied in a hermetically sealed container at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, or at least 25 mg/ml.

[00141] The comositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) as well as pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient). Bulk drug compositions can be used in the preparation of unit dosage forms, e.g., comprising a prophylactically or therapeutically effective amount of an agent disclosed herein or a combination of those agents and a pharmaceutically acceptable carrier.

[00142] The invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the agents of the invention. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of the target disease or disorder can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.

[00143] Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of agent or active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[00144] In some embodiments, a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, hyaluronic acid in an amount of about 0.7% w/v, sucrose in an amount of about 4% w/v, sodium chloride in an amount of about 5.8 mg/mL, potassium chloride in an amount of about 0.20 mg/rnL, sodium phosphate dibasic anhydrous in an amount of about 1.2 mg/mL. potassium phosphate monobasic in an amount of about 0.20 mg/mL. and poloxamer 188 in an amount of about 0.001% w/v is administered to a patient via SCS administration. In some embodiments, the patient has an ocular disease.

[00145] In some embodiments, the pharmaceutical composition comprising: (a) a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, (b) potassium chloride, (c) potassium phosphate monobasic, (d) sodium chloride, (e) sodium phosphate dibasic anhydrous, (I) sucrose, and (e) poloxamer 188, polysorbate 20, or polysorbate 80.

[00146] In other embodiments, the pharmaceutical composition comprising: (a) a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, (b) potassium chloride at a concentration of 0.2 g/L, (c) potassium phosphate monobasic at a concentration of 0.2 g/L, (d) sodium chloride at a concentration of 5.84 g/L, (e) sodium phosphate dibasic anhydrous at a concentration of 1.15 g/L, (f) sucrose at a concentration of 4% weight/volume (40 g/L), and (g) poloxamer 188, polysorbate 20, or polysorbate 80 at a concentration of 0.001% weight/volume (0.01 g/L).

[00147] In some embodiements, the pharmaceutical composition comprises an ionic strength of at most about 200 rnM and at least about 3% aggregated recombinant AAV prior to suprachoroidal administration.

[00148] In other embodiments, the pharmaceutical composition has viscosity of between about 25 cP to about 3 x 106 cP as measured at a shear rate of at most about 1 s-1 and comprises at least one of sucrose, 4% sucrose, 6% sucrose, 10% sucrose, 2% carboxy methyl cellulose sodium salt, 1% carboxymethyl cellulose sodium salt, carboxymethyl cellulose (CMC), 0.5% CMC, 1% CMC, 2% CMC, 4% CMC. polyvinyl alcohol, hydroxy ethyl cellulose, carboxymethyl cellulose sodium salt, and hydroxypropyl methylcellulose. [00149] In other embodiments, the pharmaceutical composition has a viscosity and/or higher elastic modulus that increases with increasing temperature and optionally the pharmaceutical composition contains poloxamer 407 and poloxamer 188.

[00150] In some embodiments, the pharmaceutical composition is formulated for suprachoroidal administration.

6. EXAMPLES

6.1. Example 1- Analysis of AAV capsids for peptide insertion points

[00151] FIG. 1 dpicts alignment of AAVs l-9e, 3B, rhlO, rh20, rh39, rh73, rh74 version 1 and version 2, hul2, hu21, hu26, hu37, hu51 and hu53 sequences within insertion sites for peptides that enhance ocular tissue tropism within or near the initiation codon of VP2, variable region 1 (VR-I), variable region 4 (VR-IV), and variable region 8 (VR-VIII) highlighted in grey; a particular insertion site within variable region eight (VR-VIII) of each capsid protein is shown by the symbol

(after amino acid residue 588 according to the amino acid numbering of AAV9).

6.2. Example 2- High Level Biodistribution Analysis Following Suprachoroidal Administration of a Pool of AAV3B, AAV8, and AAV9 Vectors to Non-Human Primates Reveals Spatial and Cell-Based Tropism Differences

[00152] Delivery of adeno-associated viral (AAV) vectors to the eye has the potential to address unmet needs associated with many ocular disorders. While subretinal (SR) delivery of AAV can be an effective therapy due to significant transduction of the retina and retinal pigment epithelium (RPE), it is a specialized procedure requiring surgery in an operating room. Intravitreal (IVT) delivery , while less invasive, has been associated with significant ocular inflammation. Targeted delivery to the suprachoroidal space (SCS) using transscleral microneedles can be performed in-office and may cause less inflammation than IVT delivery. AAV transduction following SCS delivery is not well-characterized compared to SR and IVT. In this study, we assessed biodistribution and transduction efficiency of a small pool of AAVs following SCS delivery in cynomolgus macaques.

[00153] Based on the results of pilot studies assessing a 118-member AAV library intraocularly in primates, we selected AAV3B, AAV8, and AAV9 for further evaluation in a small, 6-member library, assessing DNA, RNA, and protein expression in multiple ocular tissues and regions following SCS delivery to three cynomolgus macaques.

[00154] Vector production and library formulation: 6 AAV vector preps were produced by triple transfection in HEK-293T cells and purified by iodixanol gradient. Each contained a 20bp barcode between the transgene and poly A; three encoded fluorescent reporter genes (AAV3B.CAG.tdTomato.BC, AAV8.CAG.GFP.BC, and AAV9.CAG.iRFP670.BC), and three encoded an identical endogenous primate gene (AAV3B.CAG.hApoE.BC, AAV8.CAG.hApoE.BC, and AAV9.CAG.hApoE.BC). This 6-member library wasformulated to be comprised of 85% fluorescent reporterpreps and 15% endogenous gene preps (FIG. 3).

[00155] Study design and vector administration: Male cynomolgus macaques (aged 2.5- 3.5yo;negative for AAV2/8 NAbs and AAV8/9 TAbs, except for 1002, which was low AAV2 NAb+)Received 5.3el 1 GC/eye in O. lmL weredelivered suprachoroidally using a custom MedOne transscleral microneedle (0.7mm, 29G) approximately 4-5mm posterior to the limbus in the superior temporal quadrant (FIG. 4). Animals were sacrificed 3 weeks postadministration; tissues from the left eye as well as non-ocular tissues were collected frozen for biodistribution and next-generation sequencing (NGS). while the right eye was collected and fixed whole for histological analysis.

[00156] Biodistribution and NGS analysis: Left eyes were enucleated, and anterior segment samples were collected. The posterior segment was then cut into quadrants, and 2x6mm punches collected from each quadrant. Each punch was then dissected into separate RET, RPE/CH, and SC samples. DNA/RNA was extracted from each punch; total biodistribution for all samples was determined by ddPCR, and relative abundance adjusted for input (RAAFI) of barcodes in each sample was determined by NGS of barcode amplicons using the Illumina MiSeq platform.

[00157] FIG. 7 shows that delivery of an AAV library to the superior temporal suprachoroidal space results in location-dependent transduction of retina, RPE-choroid, and sclera with limited distribution to peripheral tissues.

[00158] FIG. 8 shows AAV3B, AAV8, and AAV9 relative abundance is dependent on tissue t pe, with limited impactof sample location; AAV3B transduces sclera less efficiently than AAV8 and AAV9, with morefavorable RNA:DNA in both RPE and sclera.

[00159] FIGs. 9A-F show that sample location has a limited impact on AAV3B, AAV8, and AAV9 biodistribution and relative abundance. [00160] Analysis of right eyes for fluorescent reporter protein expression: Following enucleation, the optic nerve and extraocular muscles were removed. A slit was made at the level of the ora serrata, and the entire globe was fixed in 4% PFA in 0. IM phosphate buffer pH 7.2 for 36-48h. The anterior and posterior segments were then separated, washed with additional phosphate buffer, and incubated in 15% w/v sucrose followed by 30% w/v sucrose. The posterior segment was further divided into superior (1), equatorial (2), and inferior blocks (3). Tissues were then embedded in OCT and cryo-sectioned on the horizontal plane. Immunoflourescent staining was performed using antibodies against GFP and tdTomato and a DAPI co-stain; iRFP670 staining was not performed. Images were collected using the Zeiss Axioscan 7 and analyzed in ImageJ by manually tracing regions of interest (RET, RPE, CH, SC) and separately determining percentage of GFP and TdT (+) pixels in these ROIs, which were further subdivided into temporal, central, and nasal regions.

[00161] FIG. 10 shows expression of fluorescent reporter proteins from AAV3B. tdTomato (TdT) and AAV8.GFP is similar in RPE; expression from AAV3B is markedly reduced in sclera relative to AAV8.

CONCLUSIONS

[00162] The magnitude of transduction following SCS AAV library administration to the primate eye was dependent on location within posterior segment; peripheral regions were more highly transduced in all tissues analyzed.

[00163] NGS data suggest: (1) Increased RNA expression in retina from AAV3B and AAV9 as compared to AAV8; (2) Similar RNA expression in RPE-choroid from AAV3B, AAV8, and AAV9; (3) Markedly less AAV3B transduction in sclera when compared to AAV8 and AAV9; and (4) RNA:DNA is comparable across capsids in retina, while AAV3B has the highest RNA:DNA in RPE-choroid and low est in sclera

[00164] Immunofluorescent imaging of posterior segments supports findings by NGS that (1) Similar expression from AAV3B. TdT and AAV8.GFP in RPE; and (2) AAV3BTdT expression is nearly absent in sclera.

6.3. Example 3 - Investigational Study of AAV8 and AAV3B Test Articles Following Suprachoroidal Administration in NHP

[00165] The objective of this study is to evaluate the ocular tropism of up to two different adeno-associated virus (AAV) pools/libranes following suprachoroidal administration to female cynomolgus monkeys. See Table 1. Following dosing on Day 1, animals will be observed for 3 to 12 weeks for biodistribution sample collection to investigate the transduction protocol.

Table 1 - Group Assignment and Dose Levels

GC: Genome copies a: Dose levels are based on a dose volume of 100 pl/eye b: Animals in Groups 1 and 2 will be designated as terminal sacrifice animals (based on survival).

Procedures

[00166] Animals will be dosed lOOpl/eye via suprachoroidal injection on Day 1 of the dosing phase. The right eye will be dosed first. All post-dose collection times will be based on the time of the dosing of the left eye.

Opthalmic Examinations

[00167] Opthalmic Examinations will be performed at least twice pre-dose. During the dosing phase, ophthalmic examinations will be performed on days 3, 8, 15. 17, 29, 31, 42, 57, 59, and 85.

[00168] Briefly, Animals will be anesthetized with ketamine. Animals will be examined with a slit-lamp biomicroscope and indirect ophthalmoscope. The adnexa and anterior portion of both eyes will be examined using a slit-lamp biomicroscope. The ocular fundus of both eyes will be examined (where visible) using an indirect ophthalmoscope. Prior to examination with the indirect ophthalmoscope, pupils will be dilated with a mydriatic agent (e.g., 1% tropicamide).

Intraocular Pressure Measurements

[00169] Intraocular pressure measurements (lOPs) will be performed in conjunction with ophthalmic examinations (OEs). On days of OEs, intraocular pressure measurements (lOPs) will be conducted on eyes that have been previously dilated. Intraocular pressure measurements will be performed at least twice pre-dose. During the dosing phase, ophthalmic examinations will be performed on days 3, 8, 15, 17, 29, 31, 42, 57, 59, and 85.

[00170] Briefly, animals will be anesthetized with ketamine. The IOP measurements will be done using an applanation tonometer. A topical anesthetic (e.g.. 0.5% proparacaine) will be applied before IOP measurements. Spectral Domain Optical Coherence Tomography

[00171] Spectral domain optical coherence tomography (OCT or sdOCT) will be performed at least once pre-dose. During the dosing phase, OCT will be performed once during weeks 2, 4, 8, and 12.

[00172] Animals will be fasted (for at least 10 hours) before the procedure. Animals will be anesthetized with ketamine and maintained on sevoflurane. Pupils will be dilated with a mydriatic agent. OCT will be performed and the data will be evaluated. Imaging will be done in a manner to obtain axial views of the retinal surface in the posterior fundus. The instruments will be set to perform standard retinal scans (macular volume scans and/or line scans and/or circle scans). Additional methods or scans may be used. A 55 degree lens may be used, if necessary.

Ocular Photography - Fundus

[00173] Ocular photography will be performed at least once pre-dose. During the dosing phase, ocular photography will be performed once during weeks 2, 4. 8, and 12.

[00174] Animals will be fasted (for at least 10 hours) before the procedure. Animals will be anesthetized with ketamine and dexmedetomidine. Pupils will be dilated with a mydriatic agent. Photographs will be taken with a wide angle lens and a digital fundus camera. Color photographs will be taken of each eye to include stereoscopic photographs of the posterior pole and nonstereoscopic photographs of two midperipheral fields (temporal and nasal); additional images will also be taken superior temporal, if possible.

Fundus Autofluorescent Imaging

[00175] Fundus Autofluorescent Imagining will be performed at least once pre-dose. During the dosing phase, fundus autoflurorescent imaging will be performed once during weeks 2, 4, 8, and 12.

[00176] Animals will be fasted (for at least 10 hours) before the procedure. Animals will be anesthetized with ketamine and dexmedetomidine or ketamine and maintained on sevoflurane, as applicable. Pupils will be dilated with a mydriatic agent. Images will be taken with a Heidelberg SPECTRALIS® instrument. Fundus autofluorescence images, to include posterior pole and nonstereoscopic photographs of two midperipheral fields (temporal and nasal, if possible); additional images will also be taken superior temporal, if possible.

Anti-AAV2 and AAV8 Neutralizing Antibody (NAM) Analysis

[00177] Anti-AAV 2 and AAV8 neutralizing antibody analysis will be performed within 5 days of animal transfer pre-dose. During the dosing phase. anti-AAV 2 and AAV8 neutralizing antibody analysis will be performed prior to dosing on day 1 and on each day of scheduled sacrifice.

[00178] 2.4mL of blood will be taken from the femoral vein. An alternate site may be used if necessary’, and the site of blood collection will be documented. Blood samples will be held at room temperature and allowed to clot prior to centrifugation. Samples will be centrifuged within 1 hour of

[00179] collection, and serum will be harvested into two approximately equal aliquots. Following harvesting, samples w ill be placed on dry' ice until stored in a freezer.

Anti-AAV9 Total Antibody (TAB) Analysis

[00180] Anti-AAV 9 total antibody analysis will be performed within 5 days of animal transfer pre-dose.

[00181] 2.4mL of blood w ill be taken from the femoral vein. An alternate site may be used if necessary, and the site of blood collection will be documented. Blood samples will be held at room temperature and allowed to clot prior to centrifugation. Samples will be centrifuged within 1 hour of collection, and serum will be harvested into two approximately equal aliquots. Following harvesting, samples will be placed on dry' ice until stored in a freezer.

Peripheral Blood Monocuclear Cell Isolation for ELISPOT

[00182] Peripheral blood mononuclear cells (PBMCs) will be isolated for ELISPOT at least once pre-dose. During the dosing phase, PBMCs will be isolated on days 15, 29, 57, and 85. Briefly, a 3mL blood sample will be collected from the femoral vein. An alternate site may be used if necessary.

Whole Blood Collection

[00183] Whole blood will be collected during the dosing phase on days 3, 8, 15, 29, 57, and 85. Briefly, a blood sample will be collected from the femoral vein. An alternate site may be used if necessary.

[00184] Whole Blood: Blood samples for whole blood collection will be maintained on wet ice or chilled cryoracks following collection. Whole blood will be harvested and transferred into 3 approximately' equal aliquots.

[00185] Serum: Blood samples for serum collection will be held at room temperature and allowed to clot prior to centrifugation. Samples will be centrifuged within 1 hour of collection, and serum will be harvested into 3 approximately equal aliquots. Following harvesting, whole blood and serum samples will be placed on dry ice until stored in a freezer. Aqueous Humor Collection

[00186] Aqueous humor will be collected once during pre-dose. Aqueous humor will be collected once on days 15, 22, 29, 57 and 85. Briefly, a blood sample will be collected from the femoral vein. An alternate site may be used if necessary.

[00187] Aqueous humor samples from each eye will be placed into separate tubes with Watson barcoded labels, snap frozen in liquid nitrogen, and placed on dry ice until stored in a freezer. Aqueous humor samples will be analysed for transgene product.

Blood Collection for Clinical Chemistry

[00188] Blood will be collected at least twice during pre-dose. Blood will be collected during the dosing phase on days 8, 15, 29, 57, and 85. Briefly, a blood sample will be collected from the femoral vein. An alternate site may be used if necessary'. 1 mL will be collected for hematology, 1.8 mL will be collected for coagulation and 1 mL will be collected for clinical chemistry'. Tests are provided in Table 2.

Table 2 - Blood Tests

Ocular Fluid and Frozen Ocular Tissue Collection for Biodistribution and Transgene Product Analysis

[00189] The following ocular fluid/tissues from the left eye from 2 of 3 animals in Groups 1 and 2, and both eyes from 1 of 3 animals in Groups 1 and 2. Each eye (as applicable) will be enucleated. Immediately following enucleation, a sample of aqueous humor will be collected and divided into two approximately equal aliquots. Vitreous humor and the ocular tissues will then be collected. Vitreous humor will be collected and divided into two approximately equal aliquots. See Table 3.

[00190] The anterior segment of the eye will be removed and the eyes will be divided into four approximately equal quadrants (superior temporal [to include the area of the dose site], superior nasal, inferior temporal, and inferior nasal). Two approximately equal strips (located distal, and proximal to the optic disc) from each quadrant will be collected.

[00191] All other ocular tissues will be collected as single samples (one sample/tube). The tissues will be rinsed with saline and blotted dry, as appropriate. Following collection, samples will be placed in separate tubes and flash-frozen with liquid nitrogen and stored on dry ice (unless immediately stored in a freezer).

[00192] Ocular fluids and tissues will be collected using ultra-clean procedures, according to Labcorp SOPs, in order to minimize the risk for potential contamination. In addition, any w ork surfaces and non-disposable tools used will be cleaned with DNA Away Surface Decontaminant (Thermo Scientific, Catalog No. 7010 or equivalent) and RNAse decontamination solution (Invitrogen RNaseZap or equivalent) between animals.

Table 3 - Ocular Tissue Collection

Frozen Systemic Tissue Collection for Biodistribution and Transgene Product Analysis [00193] The following tissues (see Table 4) will be collected and frozen for analysis of biodistribution and transgene product analysis.

Table 4 - Tissue Collection

6.21. Capsid Amino Acid Sequences

[00194] Table 5 provides the amino acid sequences of certain engineered capsid proteins and unengineered capsid proteins described and/or used in studies described herein. Heterologous peptides and amino acid substitutions are indicated in gray shading.

Table 5. Capsid Amino Acid Sequences

7. Equivalents

[00195] Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[00196] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entireties.

[00197] The discussion herein provides a better understanding of the nature of the problems confronting the art and should not be construed in any way as an admission as to prior art nor should the citation of any reference herein be construed as an admission that such reference constitutes “prior art” to the instant application.

[00198] All references including patent applications and publications cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

We claim:

1. A method of delivering a transgene to an ocular tissue or ocular tissue target cell or cellular matrix thereof, said method comprising contacting said cell with an rAAV vector comprising a transgene encoding an ocular disease therapeutic operably linked to one or more regulatory elements that promote expression of the ocular disease therapeutic in the ocular tissue cell, wherein the rAAV has a capsid of AAV3B (SEQ ID NO:74), wherein the ocular tissue or ocular tissue target cell is a retinal tissue or cell or a retinal pigment epithelium (RPE)-choroid tissue or cell.

2. A method of delivering a transgene to ocular tissue, or an ocular tissue target cell or cellular matrix thereof, of a subject in need thereof, said method comprising administering to said subject an rAAV vector comprising a transgene encoding an ocular disease therapeutic operably linked to one or more regulatory elements that promote expression of the ocular disease therapeutic in the ocular tissue, wherein the rAAV has a capsid AAV3B (SEQ ID NO:74).

3. The method of claim 1 or claim 2, in which the ocular tissue or ocular tissue target cell is a retinal tissue or cell.

4. The method of claim 1 or claim 2, wherein the ocular tissue or ocular tissue target cell is an RPE-choroid tissue or cell.

5. The method of any one of claims 1 to 4, wherein the ocular disease is non- infectious uveitis.

6. The method of any one of claims 1 to 4, wherein the ocular disease is glaucoma.

7. The method of any one of claims 1 to 4, wherein the ocular disease is dry age- related macular degeneration (AMD).

8. The method of any one of claims 1 to 4, wherein the ocular disease is dry AMD with geographic atrophy (GA).

9. The method of any one of claims 1 to 8, wherein said rAAV vector is administered intravitreally, suprachoroidally, or intracamerally.

10. The method of claim 9. wherein said rAAV vector is administered systemically.

11. The method of claim 9, wherein said rAAV vector is administered suprachoroidally.

12. A pharmaceutical composition for use in delivering a transgene to an ocular tissue cell, said composition comprising an rAAV vector comprising a transgene encoding an ocular disease therapeutic operably linked to one or more regulatory elements that promote expression of the ocular disease therapeutic in the ocular tissue cell, wherein the rAAV has a capsid of AAV3B (SEQ ID NO:74). wherein the ocular tissue cell is a retinal tissue or cell or a retinal pigment epithelium (RPE)-choroid tissue or cell.

13. The pharmaceutical composition of claim 12, wherein the ocular tissue or ocular tissue target cell is a retinal tissue or cell.

14. The pharmaceutical composition of claim 12, wherein the ocular tissue or ocular tissue target cell is a retinal pigment epithelium (RPE)-choroid tissue or cell.

15. The pharmaceutical composition of any one of claims 12 to 14, wherein the ocular disease is non-infectious uveitis.

16. The pharmaceutical composition of any one of claims 12 to 14, wherein the ocular disease is glaucoma.

17. The pharmaceutical composition of any one of claims 12 to 14, wherein the ocular disease is dry AMD.

18. The pharmaceutical composition of any one of claims 12 to 14, wherein the ocular disease is dry AMD with GA.

19. The pharmaceutical composition of any of claims 12 to 18, wherein said rAAV vector is administered intravitreally, suprachoroidally, or intracamerally.

20. The pharmaceutical composition of claim 19, wherein said rAAV vector is administered systemically.

21. The pharmaceutical composition of claim 19, wherein said rAAV vector is administered suprachoroidally.

22. The method or pharmaceutical composition of any of claims 1 to 21 wherein the rAAV exhibits at least 1.1-fold, 1.5-fold, 2-fold. 3-fold, 4-fold, 5-fold, 6-fold, 7-fold. 8- fold, 9-fold, or 10-fold greater transduction in the target tissue, compared to a reference AAV capsid.

23. The method or pharmaceutical composition of any of claims 1 to 22 wherein the abundance of transgene RNA is 1.1-fold, 1.5-fold. 2-fold, 3-fold, 4-fold, 5-fold, 6-fold. 7- fold, 8-fold, 9-fold, or 10-fold greater in the target tissue compared to the abundance of transgene RNA from the reference AAV capsid.

24. The method or pharmaceutical composition of claim 22 or claim 23 where the reference AAV capsid is AAV2, AAV8 or AAV9

25. A nucleic acid comprising a nucleotide sequence encoding the rAAV capsid protein of any of the above claims, or encoding an amino acid sequence sharing at least 80% identity therewith.

26. A packaging cell capable of expressing the nucleic acid of claim 25 to produce AAV vectors comprising the capsid protein encoded by said nucleotide sequence.

27. The pharmaceutical composition of claim 12 to 18, wherein said rAAV vector is formulated for suprachoroidal administration.