Suprachoroidal injection methods for delivery of AAV vectors for treating ocular pathologies

Abstract

The disclosure includes a method of delivering a pharmaceutical composition comprising a rAAV vector to an eye of a subject, the method comprising: administering the pharmaceutical composition to the eye with an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the pharmaceutical composition is injected through the needle into the suprachoroidal space.

Description

SUPRACHOROID AL INJECTION METHODS FOR DELIVERY OF AAV VECTORS FOR TREATING OCULAR PATHOLOGIES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of U.S. Provisional Application No. 63 / 765,091, filed February 28, 2025; which is hereby incorporated by reference in its entirety.FIELD OF DISCLOSURE

[0001] The present disclosure pertains to the medical field and gene delivery, including AAV gene therapy for treating ocular pathologies. The disclosure includes apparatuses, systems, and methods for ocular injection of one or more substances (e.g., rAAV vectors or pharmaceutical compositions comprising rAAV vectors).BACKGROUND

[0002] Ocular pathologies significantly affect the quality of life of millions of people each year, and can lead to severe discomfort, impaired vision, or blindness. For example, posterior segment diseases, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), noninfectious uveitis, or central serous chorioretinopathy, can result in permanent vision loss once retinal architecture and physiology are disrupted. Chiang et al., Adv Drug Deliv Rev. 2018 Mar 12;126:58-66.

[0003] Successful treatment of ocular pathologies depends not only on the development of new therapeutic agents, but also on the methods of delivering these therapeutic agents. Traditional delivery methods for ocular therapeutic agents have many limitations and complications.

[0004] Current routes of drug delivery for therapeutics targeting the posterior segment of the eye include topical, subtenon, subretinal, and IVT injections. Each type of administration carries its own advantages and disadvantages. Topical administration is the least invasive but is often ineffective due to poor penetrance and low therapeutic levels at the posterior segment structures (Barar, J., et al. (2016). "Advanced drug delivery and targeting technologies for the ocular diseases." Bioimpacts 6(1): 49-67). Subretinalinjections are very targeted yet require an invasive surgical procedure that also carries significant risks (Kim, H. M. and S. J. Woo (2021). "Ocular Drug Delivery to the Retina: Current Innovations and Future Perspectives." Pharmaceutics 13(1)). IVT injection is easy to administer and can be performed in an office setting; however, it does not target a specific area and, although considered relatively safe, may have adverse effects, including endophthalmitis, elevated intraocular pressure (IOP), and cataract progression (Del Amo, E. M. and A. Urtti (2008). "Current and future ophthalmic drug delivery systems. A shift to the posterior segment." Drug Discov Today 13(3-4): 135-143).

[0005] Current techniques for delivery of therapies to the suprachoroidal space (SCS) (e.g., microneedles) have certain safety issues and limitations in the injectable volume, drug distribution, and particle size.

[0006] Drug delivery with minimal adverse effects and maximal efficacy to the target tissue is one of the biggest challenges faced during drug development. Due to the structural and metabolic properties of the ophthalmic regions, different methods of administration (i.e., topical, systemic, ocular, and periocular administration) can result in vastly different concentrations of the therapeutic delivered to the site of action, which can present challenges to be within a given therapeutic window. Thus, effectively delivering therapeutics to the ophthalmic tissues, particularly into the eye (e.g., to the posterior segment of the eye, such as to the retina), is an important step in developing successful therapies for ocular pathologies.

[0007] Topical instillation, the least invasive route of administration, is also one of the least efficacious, e.g., bioavailability of topical therapeutics is generally low and penetration into the cornea and conjunctival epithelium is inefficient.

[0008] Systemic delivery can be accomplished through oral administration in the form of a tablet or liquid consumable or through parenteral routes, such as intravenous, intramuscular, subcutaneous, and intradermal administration in an injectable form. Due to low ocular bioavailability and high systemic exposure, these methods are atypical for therapeutics delivery in ophthalmology. Systemic metabolism is another hurdle for systemic administration, and greatly reduces the concentration of therapeutics that can reach the ophthalmic region.

[0009] Periocular delivery is another method to introduce therapeutics into the ophthalmic region. The sub-conjunctival space is the most commonly used in clinical practice, but the therapeutic bioavailability is typically poor. Upon sub-conjunctivalinjection, therapeutic bioavailability has been determined to be roughly similar to topical administration and much lower than intravitreal administration (Subrizi, A. et al., Design Principles of Ocular Drug Delivery Systems: Importance of Drug Payload, Release Rate, and Material Properties. Drug Discov. Today 2019, 24, 1446-1457).

[0010] Intraocular administration includes the injection or implantation of a sterile solution or device within the eye, for example, intravitreal, subretinal, or suprachoroidal delivery routes.

[0011] Known methods of subretinal delivery include those in which a sharp injection device — for example, a syringe having a sharpened hollow needle — is used to penetrate the sclera from outside the eye to the subretinal space where the composition is injected. A major drawback of this method is that the composition remains localized in the subretinal space near the injection site and does not reach the macula.

[0012] Other methods, which are intended to deliver compositions to the macula, include inserting a thin flexible catheter from an incision site in the front sclera through the subretinal space from the incision site until the distal end of the catheter is near the macula to deliver the composition near the macula. Disadvantages of such methods include the risk of severe detachment of the retina from the sclera caused by the catheter, and risk of damaging the retina during the procedure.

[0013] In another known method, an incision is made in the frontal part of the sclera and a sharp rigid cannula is inserted into the incision across the eye and through the vitreous humor chamber to pierce the sensory retina across the incision site and enter the subretinal space near the macula where the composition is delivered. In addition to the fact that the injected compound remains localized in the subretinal space near the injection site, other drawbacks of this vitrectomy-like surgery include increased chance of cataract development, high ocular pressure, and bleeding in the eye. Moreover, the need for repeated injections may require several incisions in the frontal part of the sclera.

[0014] There is a need for the development of delivery strategies to the ophthalmic region, particularly into the eye (e.g., to the posterior segment of the eye, such as to the retina), that allow efficient delivery of therapeutics (e.g., vector-based therapeutics, such as rAAV-based therapeutics) while minimizing iatrogenic complications.BRIEF SUMMARY

[0015] Certain aspects of the disclosure are directed to a method of delivering a pharmaceutical composition comprising a rAAV vector to an eye of a subject, the method comprising: administering the pharmaceutical composition to the eye with an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the pharmaceutical composition is injected through the needle into the suprachoroidal space.

[0016] Certain aspects of the disclosure are directed to a method of delivering a pharmaceutical composition comprising a rAAV vector to an eye of a subject, the method comprising: (i) fixating a target portion of the sclera of the eye with a fixation device, (ii) penetrating the sclera portion with an injection device comprising a needle and tissue separator, (iii) creating a channel through the choroid of the eye with the tissue separator, and (iv) injecting the pharmaceutical composition through the needle into the suprachoroidal space.

[0017] Certain aspects of the disclosure are directed to a use of an injection device comprising a needle and a tissue separator for delivery of a pharmaceutical composition comprising a rAAV vector to an eye of a subject, wherein the tissue separator creates a channel through the choroid of the eye, and the pharmaceutical composition is injected through the needle into the suprachoroidal space.

[0018] Certain aspects of the disclosure are directed to a method of treating an ocular pathology in a subject comprising administering a pharmaceutical composition comprising a rAAV vector into the suprachoroidal space of an eye of the subject with an injection device comprising a needle and a tissue separator, wherein the injection device creates a channel through the choroid of the eye and the pharmaceutical composition is injected into the suprachoroidal space.

[0019] Certain aspects of the disclosure are directed to a method of treating an ocular pathology in a subject comprising administering a pharmaceutical composition comprising a rAAV vector to an eye of a subject, the method comprising: (i) fixating a target portion of the sclera of the eye with an injection device comprising a needle and a tissue separator, (ii) penetrating the sclera portion with the injection device, (iii) creating a channel through the choroid of the eye with the tissue separator, and (iv) injecting the pharmaceutical composition through the needle into the suprachoroidal space.

[0020] Certain aspects of the disclosure are directed to a use of an injection device comprising a needle and a tissue separator for treating an ocular pathology in a subject comprising administering a pharmaceutical composition comprising a rAAV vector to an eye of a subject, wherein the tissue separator creates a channel through the choroid of the eye, and the pharmaceutical composition is injected through the needle into the suprachoroidal space.

[0021] In some aspects, the method comprises insertion of the needle at a shallow angle, optionally, the shallow angle is 0° to 30° (e.g., 0° to 10°) relative to the sclera portion of the eye. In some aspects, the insertion of the needle penetrates the sclera at an approximately tangential angle. In some aspects, the insertion of the needle penetrates the sclera at an approximately 0° to 45°.

[0022] In some aspects, the separator and / or needle is extended about 1 mm to 5 mm, optionally, about 2 mm to 4 mm, in length within the sclera layer before penetrating the choroid.

[0023] In some aspects, the tissue separator is within the lumen of the needle In some aspects, the distal tip of the tissue separator penetrates into the sclera portion and into the choroid to create the channel through the choroid.

[0024] In some aspects, the tissue separator causes blunt dissection of the sclera and choroid thereby creating the channel through the choroid.

[0025] In some aspects, the pharmaceutical composition flows through the channel in the choroid into the posterior segment of the eye. In some aspects, the pharmaceutical composition flows through the channel in the choroid to the retina, to the retinal pigmented epithelium (RPE, e.g., choroid-RPE), and / or to the ciliary bodies.

[0026] In some aspects, the delivery or administration comprises a volume of about 0.05 mL to about 1 mL, about 0.1 mL to about 0.5 mL, about 0.1 mL to about 0.3 mL, about 0.15 mL to about 0.5 mL, about 0.15 mL to about 0.3 mL, or about 0.1 mL to about 0.2 mL of the pharmaceutical composition per injection into the suprachoroidal space.

[0027] In some aspects, the delivery or administration comprises a maximum volume of 0.5 mL. In some aspects, the delivery or administration comprises a volume greater than 0.1 mL. In some aspects, the delivery or administration comprises a volume of greater than 0.1 mL up to about 0.5 mL of the pharmaceutical composition per injection into the suprachoroidal space.

[0028] In some aspects, the delivery or administration comprises a volume of about 0.1 mL to about 0.5 mL, about 0.15 mL to about 0.5 mL, about 0.2 mL to about 0.5 mL, about 0.25 mL to about 0.5 mL, about 0.3 mL to about 0.5 mL, about 0.35 mL to about 0.5 mL, about 0.4 mL to about 0.5 mL, or about 0.45 mL to about 0.5 mL of the pharmaceutical composition per injection into the suprachoroidal space.

[0029] In some aspects, the delivery or administration comprises a total volume of about 0.1 mL to about 0.5 mL (e.g., about 0.1 mL to about 0.25 mL) of the pharmaceutical composition into the suprachoroidal space. In some aspects, the delivery or administration comprises a total volume of 0.075 mL to 0.125 mL (e.g., about 0.1 mL) of the pharmaceutical composition into the suprachoroidal space. In some aspects, the delivery or administration comprises a total volume of 0.2 mL to 0.3 mL (e.g., about 0.24 mL) of the pharmaceutical composition into the suprachoroidal space.

[0030] In some aspects, the delivery or administration comprises a pharmaceutical composition comprising at least 1E10 vector genomes (vg), of the rAAV vector.

[0031] In some aspects, the delivery or administration comprises a pharmaceutical composition comprising at least 1E10 vg and at most 1E13 vg of the rAAV vector.

[0032] In some aspects, the delivery or administration comprises a pharmaceutical composition comprising at least 1E10 vg and at most 1E12 vg of the rAAV vector.

[0033] In some aspects, the delivery or administration comprises a pharmaceutical composition comprising about 1E10 vg, about 2E10 vg, about 3E10 vg, about 4E10 vg, about 5E10 vg, about 6E10 vg, about 7E10 vg, about 8E10 vg, about 9E10 vg, about 1E11 vg, about 2E11 vg, about 3E11 vg, about 4E11 vg, about 5E11 vg, about 6E11 vg, about 7E11 vg, about 8E11 vg, about 9E11 vg, about 1E12 vg for the rAAV vector.

[0034] In some aspects, the delivery or administration is to one eye. In some aspects, the delivery or administration is to both eyes.

[0035] In some aspects, the delivery or administration is a single injection per eye. In some aspects, the delivery or administration comprises multiple injections per eye.

[0036] In some aspects, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 % of the rAAV vector contacts an ocular tissue.

[0037] In some aspects, the ocular tissue is selected from cornea, sclera, choroid, Brunch’s membrane, pupil, iris, lens, optic disc, optic nerve, macula (e.g., centralmacula), retina, retinal pigmented epithelium (RPE, e.g., choroid-RPE), ciliary bodies, fovea, or any combination thereof.

[0038] In some aspects, the ocular tissue comprises a retina. In some aspects, the ocular tissue comprises a retina and the rAAV vector contacts the inner limiting membrane, the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the middle limiting membrane, the outer plexiform layer, the outer nuclear layer, the external limiting membrane, the photoreceptor inner / outer segments, the retinal pigmented epithelium (RPE, e.g., choroid-RPE), or any combination thereof.

[0039] In some aspects, the rAAV vector contacts one or more ocular cells. In some aspects, the one or more ocular cells are a retinal cell selected from a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, a Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof.

[0040] In some aspects, the rAAV vector transduces the one or more ocular cells. In some aspects, the one or more ocular cells include retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells, retinal ganglion (RG) cells, or a combination thereof. In some aspects, cell transduction is circumferential at the posterior pole of the eye. In some aspects, the rAAV vector transduces ocular cells from the retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells through ganglion cells. In some aspects, the rAAV vector transduces RPE cells. In some aspects, the rAAV vector transduces ganglion cells. In some aspects, the rAAV vector transduces RPE cells and ganglion cells. In some aspects, the rAAV vector transduces one or more ocular cells in the macula. In some aspects, the rAAV vector transduces one or more ocular cells in the central macula. In some aspects, the rAAV vector transduces RPE cells, e.g., to the central macula.

[0041] In some aspects, the rAAV vector transduction efficiency is at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells and / or retinal ganglion (RG) cells. In some aspects, the rAAV vector transduction efficiency is at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal ganglion (RG) cells. In some aspects, the rAAV vector transduction efficiency is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells.

[0042] In some aspects, the rAAV vector does not readily transduce photoreceptor cells. In some aspects, the photoreceptor cells are a rod cells, a cone cells, or any combination thereof. In some aspects, the rAAV vector transduction efficiency is at less 5% (e.g., less than 4%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or less than 0.5%) of the photoreceptor cells.

[0043] In some aspects, the rAAV vector comprises a capsid, a vector genome, and an expression cassette. In some aspects, the vector genome comprises an inverted terminal repeat (ITR). In some aspects, the expression cassette comprises a nucleic acid sequence encoding a therapeutic agent.

[0044] In some aspects, the therapeutic agent is a therapeutic protein (e.g., a fusion protein). In some aspects, the therapeutic agent is an antibody.

[0045] In some aspects, the therapeutic agent is selected from an anti-TNF antibody (e.g., Adalimumab), a CR2-CR1 fusion protein, an inflammasome pathway inhibitor (e.g., tatCARD fusion proteins), a Vascular Endothelial Growth Factor (VEGF) inhibitor (e.g., FLT01, Aflibercept), an anti- VEGF antibody and antibody fragments (e.g. bevacizumab, ranibizumab, brolucizumab), Fibroblast Growth Factor 21 (FGF21) (GenBank ID:NC 000019.10, Uniprot ID: Q9NSA1), soluble membrane-independent form of CD59 (sCD59) (GenBank ID: NC 000011.10; Uniprot ID: P27274), soluble-fms like tyrosine kinase-1 (sFLT-1) (Uniprot ID: S5TRK2), Complement factor I (CFI) (GenBank ID: NC 000004.12, Uniprot ID: P05156), Rab Escort Protein 1 (REP1) (Uniprot ID:P24386), choroideremia / Rab Escort Protein 1 (CHM) (GenBank ID: NC 000023.11), Retinal Pigment Epithemium specific 65kDa protein (RPE65) (GenBank ID:NC_000001.ll, Uniprot ID: Q16518), Retinal Pigment Epithemium specific 65kDa protein v2 (RPE65v2), NADH ubiquinone oxidoreductase core subunit 4 (ND4) (Uniprot ID: P03905), human MER-proto oncogene Tyrosine Kinase (hMERTK) (GenBank ID: NC 000002.12, Uniprot ID: Q12866), Retinitis Pigmentosa GTPase Regulator (RPGR) (GenBank ID: NC 000023.11, Uniprot ID: Q92834), Phosphodiesterase 6A (PDE6A) (GenBank ID: NC 000005.10, Uniprot ID: P16499), Phosphodiesterase 6B (PDE6B) (GenBank ID: NC 000004.12, Uniprot ID: P35913), cyclic nucleotide gated channel subunit alpha 3 (CNGA3) (GenBank ID: NC_000002.12, UniProt: Q16281), cyclic nucleotide gated channel subunit beta 3 (CNGB3) (GenBank ID: NC_000008.ll, Uniprot ID: Q9NQW8), retinaldehyde binding protein 1 (RLBP1) (GenBank ID: NC 000015.10, Uniprot ID: P12271), retinoschisin 1 (RSI) (GenBank ID: NC 000023.11, Uniprot ID:015537), gene editing factors (e.g., SaCas9, gRNAs), Optogenetics factors (e.g., ChrimsonR-tdTomato, Channelrhodopsin-2, Multi-Characteristic Opsin (MCO)), or functional fragments thereof, or any fusion protein comprising one or more of the same, or any combination thereof.

[0046] In some aspects, the therapeutic agent is an anti-TNF antibody (e.g., Adalimumab).

[0047] In some aspects, the therapeutic agent is a CR2-CR1 fusion protein. In some aspects, the CR2-CR1 fusion protein comprises a ligand-binding region of complement receptor 2 (CR2) and a ligand-binding region of complement receptor 1 (CR1).

[0048] In some aspects, the therapeutic agent is an inflammasome pathway inhibitor (e.g., tatCARD fusion proteins).

[0049] In some aspects, the therapeutic agent is a Vascular Endothelial Growth Factor (VEGF) inhibitor (e.g., FLT01, Aflibercept)

[0050] In some aspects, the therapeutic agent is an anti-VEGF antibody or antibody fragment thereof (e.g. bevacizumab, ranibizumab, brolucizumab).

[0051] In some aspects, the therapeutic agent is the Fibroblast Growth Factor 21.

[0052] In some aspects, the rAAV vector comprises a capsid of or derived from a serotype selected from AAV1 (e.g. UniProt ID: Q9WBP8), AAV2 (e.g. UniProt ID: Q4KWI8), AAV3, AAV4 (e g. UniProt ID: 041855), AAV5 (e g. UniProt ID: Q5XXZ6), AAV6, AAV7 (e g. UniProt ID: Q8JQG0), AAV8 (e g. UniProt ID: Q8JQF8), AAVRh8 (e.g. UniProt ID: Q808Y3), AAVrh9, AAV9 (e.g. UniProt ID: Q6JC40), AAVrhlO (e.g. UniProt ID: Q8JQF8), AAV10, AAV11, AAV12 (e.g. UniProt ID: A9RAI0), AAV13, AAV14, AAV15, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B (e.g. UniProt ID: Q6JC40), AAV2.5, AAV2tYF, AAV2quadYF, AAV3B (e.g. UniProt ID: 056139), AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV2.5T, AAV2.5T.LSV1, or a modified version thereof. In some aspects, the capsid serotype is AAV2 or a modified version thereof or AAV8 or a modified version thereof. In some aspects, the capsid serotype is AAV2 or AAV8. In some aspects, the capsid serotype is AAV2QuadYF (Petrs-Silva H, et al., Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina. Mol Ther. 2011 Feb;19(2):293-301).

[0053] In some aspects, the ocular pathology for treatment is selected Achromatopsia, Behcet's Disease, Best's disease, Bietti's Crystalline Dystrophy, Blepharitis, Blepharospasm, Central Areolar Choroidal Dystrophy, Central Serous Chorioretinopathy, Choroideremia, Choroidal Melanoma, Coloboma, Corneal Conditions, Coat's Disease, Cone-Rod Dystrophy, Corneal Dystrophy, Cystoids Macular Edema, Diabetic Retinopathy, Doyne Honeycomb Retinal Dystrophy, Dry Eye, Fuch's Dystrophy, Glaucoma, Hypertensive Retinopathy, Idiopathic Intracranial Hypertension, Lattice Degeneration, Leber Congenital Amaurosis, Leber Hereditary Optic Neuropathy, Leber's Miliarly Aneurism, Macular Degeneration (including Age-Related Macular Degeneration, Juvenile Macular Degeneration, Atrophic Macular Degeneration), Macular Edema, Ocular Histoplasmosis (including Ocular Histoplasmosis Syndrome), Ocular Ischemic Syndrome, Papillophlebitis, Pink Eye, Polypoidal Choroidal Vasculopathy, Retinitis Pigmentosa, Retinoblastoma, Retinopathy of Prematurity, Retinoschisis (including Juvenile Retinoschi sis), Sorsby's Disease, Stargardt Disease, Toxoplasmosis, Thyroid Eye Disease (TED), Usher Syndrome, and Uveitis (e.g., Noninfectious Uveitis), Vascular Occlusions, Inflammations (such as uveitis, choroiditis and retinistis), various Tumors (including neoplasms), or any combination thereof. In some aspects, the ocular pathology for treatment is Geographic Atrophy (GA) secondary to Age-related Macular Degeneration (AMD).

[0054] In some aspects, the injection into the suprachoroidal space is a single dose. In some aspects, the single dose is administered at a single injection site. In some aspects, the single dose is administered at multiple injection sites. In some aspects, the injection into the suprachoroidal space is multiple doses. In some aspects, each dose of the multiple doses is administered at a single injection site. In some aspects, each dose of the multiple doses is administered at different injection sites.

[0055] In some aspects, the injection device comprises:

[0056] (a) the needle comprising a needle lumen extending between a needle proximal end and a needle distal end, said needle distal end including a size transitional portion terminating in a sharp needle distal tip;

[0057] (b) the tissue separator having a separator distal tip, at least a portion of said tissue separator being shiftable in the needle lumen; and

[0058] (c) an actuator configured to shift said tissue separator in said needle lumen between a first position, wherein said separator distal tip is fixedly positioned proximallyto said needle distal tip, and a second position, wherein said separator distal tip is fixedly positioned a first predetermined distance distally from said needle distal tip.

[0059] In some aspects, the injection / injector device further comprises a reservoir (e.g., a syringe).

[0060] In some aspects, the injection / injector device further comprises a cannula fluidly connectable between a syringe and said needle lumen, so that a fluid (e.g., the pharmaceutical composition) injected from said syringe is directly flowable to said needle distal tip via said cannula and said needle lumen.

[0061] In some aspects, the syringe is connectable to a separator hub of said separator.

[0062] In some aspects, the needle has a length of about 1 mm to about 5 mm. In some aspects, the needle has a length of 2 mm. In some aspects, the needle has a length of 1.5 mm. In some aspects, the needle has an internal diameter of about 0.1 mm to about 0.6 mm. In some aspects, the needle has an internal diameter of 0.1 mm. In some aspects, the needle has an internal diameter of 0.2 mm. In some aspects, the needle has an internal diameter of 0.3 mm. In some aspects, the tissue separator has a blunt tip. In some aspects, the tissue separator comprises a slender probe.

[0063] Certain aspects of the disclosure are directed to a method of delivering a pharmaceutical composition comprising a rAAV vector to the suprachoroidal space in an eye of a subject, the method comprising:

[0064] a. providing device for injection into the suprachoroidal space of the subject, the device comprising:

[0065] a needle comprising a needle lumen extending between a needle proximal end and a needle distal end, said needle distal end including a size transitional portion terminating in a sharp needle distal tip;

[0066] an elongated tissue separator having a separator distal tip, at least a portion of said tissue separator being shiftable in the needle lumen; and

[0067] an actuator configured to shift said tissue separator in said needle lumen between a first position, wherein said separator distal tip is fixedly positioned proximally to said needle distal tip, and a second position, wherein said separator distal tip is fixedly positioned a first predetermined distance distally from said needle distal tip;

[0068] b. penetrating outer surface (i.e., the sclera and optionally the conjunctiva) of the eye at a penetration point into the eye using said needle distal tip until contacting an outer surface layer (i.e., the sclera and optionally the conjunctiva) of the eye. In some aspectsthe method includes injecting the pharmaceutical composition into the suprachoroidal space.

[0069] In some aspects, the method further comprises using said actuator to shift said tissue separator longitudinally in said needle lumen such that said separator distal tip is repositioned to a distal-most position, wherein said separator distal tip is located a second predetermined length distally to said needle distal tip, thereby advancing in said outer surface layer (i.e., the sclera and optionally the conjunctiva) and penetrating through said outer surface layer into the interlayer (i.e., the sclera and optionally the conjunctiva) of the eye and forming a passage in said interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) with said separator distal tip.

[0070] In some aspects, the method comprises injecting the pharmaceutical composition into the suprachoroidal space.

[0071] In certain aspects, provided herein is a kit comprising: (i) a rAAV vector comprising a capsid, a vector genome, and an expression cassette, (ii) an injection device disclosed herein, and (iii) instructions for using the injection device to administer the rAAV to the eye. In some aspects, the injection device can be provided to a user disassembled, readily assembled in a form of a system, or provided as an assembled device. In some aspects, the injection device can be provided with a preloaded reservoir, e.g., a syringe, comprising the rAAV vector.

[0072] In certain aspects, provided herein is a kit comprising: (i) a recombinant adeno- associated virus (rAAV) vector comprising a capsid, a vector genome, and an expression cassette, (ii) an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the rAAV vector can be injected through the needle into the suprachoroidal space, and (iii) instructions for using the injection device to administer the rAAV to the suprachoroidal space of the eye.

[0073] In some aspects, the injection device is provided to a user disassembled, readily assembled in a form of a system, or provided as an assembled device.

[0074] In some aspects, the injection device is provided with a preloaded reservoir, e.g., a syringe, comprising the rAAV vector.

[0075] In some aspects, the rAAV vector is formulated for administration of at least 1E10 vg to at most 1E12 vg / eye.

[0076] In certain aspects, provided herein is a kit for administering at least 1E10 vg to at most 1E12 vg of a recombinant adeno-associated viral (rAAV) vector to an eye, whereinthe kit comprises: (i) a reservoir comprising a pharmaceutical composition comprising the rAAV vector which comprises a capsid, a vector genome, and an expression cassette; and (ii) an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the pharmaceutical composition can be injected through the needle into the suprachoroidal space.

[0077] In some aspects, the injection device is provided disassembled, readily assembled in a form of a system, or provided as an assembled device.

[0078] In certain aspects, provided herein is a system for administering a recombinant adeno-associated viral (rAAV) vector to the eye of a subject, wherein the system comprises: (i) a reservoir comprising a pharmaceutical composition comprising the rAAV vector which comprises a capsid, a vector genome, and an expression cassette; and (ii) an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the pharmaceutical composition can be injected through the needle into the suprachoroidal space, wherein the reservoir is preloaded with the pharmaceutical composition for administration of at least 1E10 vg / eye to at most 1E12 vg / eye of the rAAV vector.

[0079] In some aspects, the reservoir comprises at least 1E10 vector genomes (vg) of the rAAV vector.

[0080] In some aspects, the reservoir comprises at least 1E10 vg to at most 1E13 vg of the rAAV vector.

[0081] In some aspects, the reservoir comprises a total volume of at least 0.1 mL of the pharmaceutical composition

[0082] In some aspects, the reservoir comprises a total volume of at least 0.1 mL to about 0.5 mL (e.g., about 0.1 mL to about 0.25 mL) of the pharmaceutical composition.

[0083] In some aspects, the reservoir is for a single administration.

[0084] In some aspects, the kit or system comprises two or more reservoirs.BRIEF DESCRIPTION OF DRAWINGS

[0085] The accompanying drawings, which are incorporated herein and form part of the specification, illustrate the aspects and, together with the description, further serve to explain the principles of the aspects and to enable a person skilled in the relevant art(s) to make and use the aspects.

[0086] FIGs. 1A-1D show a schematic representation of a suprachoroidal injection by the Suprachoroidal Injector described herein. FIG 1A shows tangential insertion of the Suprachoroidal Injector's into the sclera; FIG IB shows the non-sharp tissue separator extended to create a path into the suprachoroidal space; FIG. 1C shows the channel created through the choroid after retraction of the non-sharp tissue separator; FIG. ID shows injection of a therapeutic agent (e.g., a rAAV or a pharmaceutical composition comprising rAAV vectors) and its distribution throughout the posterior segment.

[0087] FIG. IE shows a schematic representation of a suprachoroidal injection of a viral particle using the Suprachoroidal Injector described herein.

[0088] FIGs. 2A-2C show fundus autofluorescence by confocal scanning laser ophthalmoscopy (FIGs. 2A-2B) and immunohistochemistry for GFP (nucleic acids were also stained by hematoxylin) (red) (FIG. 2C) of non-human primates (NHPs) 28 Days after suprachoroidal administration of AAV2-GFP with Suprachoroidal Injector (for a representative NHP). Stain for nucleic acids by hematoxylin is also shown (blue). A temporal view is shown for each eye of a representative animal. FIGs. 2A-2B:arrowheads mark slight GFP expression visible in the parafoveal ring; FIG. 2C: stars indicate exemplary GFP staining; (OD = right eye; OS = left eye; GFP = green fluorescent protein).

[0089] FIGs. 3A-3B show biodistribution (DNA and RNA) of AAV2-GFP following Bilateral Suprachoroidal Administration to NHPs using Suprachoroidal Injector, vg = vector genomes.

[0090] FIGs. 3C-3D show biodistribution (DNA and RNA) of AAV2Q-GFP following Bilateral Suprachoroidal Administration to NHPs using Suprachoroidal Injector. VCN = vector copy number.

[0091] FIGs. 3E-3F show biodistribution (DNA and RNA) of AAV8-GFP following Bilateral Suprachoroidal Administration to NHPs using Suprachoroidal Injector. VCN = vector copy number.

[0092] FIGs. 4A-H show exemplary immunohistochemistry images detecting GFP in the eye of an NHP treated with AAV2-GFP (FIGs. 4A-4F), AAV2Q-GFP (FIG. 4G), or AAV8-GFP (FIG. 4H).

[0093] FIG. 5A is an isometric illustration of a kit for assembling a system, according to an exemplary aspect and including an optional docking device. In some exemplary aspects, the exemplary, non-limiting docking device shown may not be included, or analternative device be used. Other alternatives for fixating a sclera portion may be used, for example those shown and described with reference to FIGs. 11-18B. In some aspects, fixating the sclera portion may be omitted.

[0094] FIG. 5B is an isometric illustration of the assembled system shown in FIG. 5A, according to an exemplary aspect.

[0095] FIG. 5C is an isometric illustration of elements of an organ affecting device, according to an exemplary aspect.

[0096] FIG. 6A is a cross-section illustration of a side of the device shown in FIG. 5C assembled with a syringe, according to an exemplary aspect.

[0097] FIG. 6B is an exploded illustration of a side of the device shown in FIG. 5C assembled with a syringe, according to an exemplary aspect.

[0098] FIG. 7A is a cross-section illustration of a tissue separator, according to an exemplary aspect.

[0099] FIG. 7B is an exploded illustration of a tissue separator, according to an exemplary aspect.

[0100] FIG. 8A is a first illustration of a side of a distal portion of the device shown in FIG. 5C, according to an exemplary aspect.

[0101] FIG. 8B is a second illustration of a side of a distal portion of the device shown in FIG. 5C, according to an exemplary aspect.

[0102] FIG. 8C is a third illustration of a side of a distal portion of the device shown in FIG. 5C, according to an exemplary aspect.

[0103] FIGS. 9A-9O illustrate different configurations of the system shown in FIG. 5B for fixating a sclera portion, directing needle penetration into the sclera portion, and facilitating fluid injection into an interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region in an eye, according to exemplary aspects.

[0104] FIG. 10A is a cross-section illustration of the side of a system unit, according to an exemplary aspect.

[0105] FIG. 10B is an isometric illustration of elements of the system unit shown in FIG.10A, according to an exemplary aspect.

[0106] FIG. 10C is an illustration of different configurations of the elements of the system unit shown in FIG. 10B, according to exemplary aspects.

[0107] FIG. 11 is an illustration of a fixation tool, constructed and operative in accordance with a non-limiting embodiment of the invention, with a protector member covering fixation members, according to an exemplary aspect.

[0108] FIG. 12 is an illustration of the fixation tool with the protector member removed, exposing the fixation members, according to an exemplary aspect.

[0109] FIG. 13 is a planar-view illustration of the fixation tool, accordingly to an exemplary aspect.

[0110] FIG. 14A is a planar- view illustration of a distal end of the fixation tool, with the protector member removed, exposing the fixation members, according to an exemplary aspect.

[0111] FIG. 14B is an enlarged view of the distal end of the fixation tool with the fixation members as shown in FIG. 14 A, according to an exemplary aspect.

[0112] FIG. 15 is an illustration of the fixation tool being used together with a needle and separator tool in an ophthalmic procedure, such as creating a suprachoroidal passage for introducing a substance therein, according to an exemplary aspect.

[0113] FIG. 16A is an illustration of a tip angle and tip length of the fixation member, according to an exemplary aspect.

[0114] FIG. 16B is an illustration of a support angle of support structure of the fixation member, according to an exemplary aspect.

[0115] FIG. 16C is an illustration of a width W of the fixation member, FIG. 16C being rotated 90° relative to FIG. 16B, according to an exemplary aspect.

[0116] FIGs. 17A-17C are illustration of different fixation members and their support structure, in according with different non-limiting embodiments of the invention.

[0117] FIGs. 18A-18B are illustrations of the fixation tool, places a distance from the limbus and placed at the limbus, according to an exemplary aspect.DETAILED DESCRIPTION OF THE DISCLOSURE

[0118] Certain aspects of the disclosure are directed to methods of suprachoroidal administration of a rAAV vector, or a pharmaceutical composition comprising a rAAV, to the eye of a subject suffering from an ophthalmic pathology (e.g., an ocular pathology), wherein the methods use an injection device which creates a channel through the choroid of the eye for delivery of the pharmaceutical composition into the suprachoroidal space.In some aspects, the methods of the disclosure allow for increased volumes (e.g., greater than 100 pL per single injection) of the rAAV pharmaceutical composition to be delivered to the suprachoroidal space compared to microneedle injection methods (e.g., Yiu et al., Methods & Clinical Development Vol. 16: 179-191, March 2020). In some aspects, the methods of the disclosure allow for rAAV (e.g., AAV2) transduction of retinal pigmented epithelium (RPE, (e.g., choroid-RPE) and retinal ganglion (RG) cells without substantial transduction of photoreceptor cells.I. Definitions

[0119] In order that the present disclosure can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed disclosure.

[0120] It is to be noted that the term "a" or "an" entity refers to one or more of that entity;for example, "a nucleic acid sequence," is understood to represent one or more nucleic acid sequences, unless stated otherwise. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0121] Furthermore, "and / or," where used herein, is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0122] Numerical values presented herein may be written out, or expressed in scientific notation, i.e. x * 1CF, or scientific E notation xEy.

[0123] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of' and / or "consisting essentially of' are also provided.

[0124] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower).

[0125] The term "at least" prior to a number or series of numbers is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that could logically be included, as clear from context. When at least is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures).

[0126] As used herein, "no more than" or "less than" is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. When "no more than" is present before a series of numbers or a range, it is understood that "no more than" can modify each of the numbers in the series or range.

[0127] The term "derived from," as used herein, refers to a component that is isolated from or made using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from the specified molecule or organism. For example, a nucleic acid sequence (e.g., an AAV vector) that is derived from a second nucleic acid sequence (e.g., another AAV vector) can include a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In the case of a polynucleotide disclosed herein, the derived species can be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis or artificial random mutagenesis.

[0128] The term "expression vector" or "expression construct" or "expression cassette" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed.

[0129] As used herein, the term "delivery vector" or "vector" refers to any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc. A vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "delivery vector" or "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. In some aspects, insertion of a polynucleotide into a suitable vector canbe accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini. Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), P-galactosidase (LacZ), P-glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters. In some aspects, the delivery vector is selected from the group consisting of a viral vector (e.g., an AAV vector), a plasmid, a lipid, a protein particle, a bacterial vector, and a lysosome.

[0130] Some aspects of the disclosure are directed to biological vectors, which can include viruses (e.g., AAV vectors), particularly attenuated and / or replication-deficient viruses.

[0131] A "viral vector" refers to a viral derived component that can be used to carry and / or deliver a payload (e.g., a nucleic acid sequence that comprises one or more polynucleotide regions encoding or comprising a molecule of interest, e.g., a protein, a peptide, and an oligonucleotide or a plurality thereof). Viral vectors can be used to deliver genetic materials into cells. Viral vectors can be modified for specific applications. In some aspects, the delivery vector of the disclosure is a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

[0132] The term "adeno-associated virus vector" or "AAV vector" or "adeno-associated viral vector" as used herein refers to any vector that comprises or derives from components of an adeno-associated vector and is suitable to infect mammalian cells, preferably human cells (e.g., ocular cells). In some aspects, the term AAV vector can designate an AAV-type viral particle or virion comprising a payload. The AAV vector can be derived from various serotypes, including combinations of serotypes (i.e., "pseudotyped" AAV) or from various genomes (e.g., single stranded or self-complementary). In addition, the AAV vector can be replication defective and / or targeted. As used herein, an adeno-associated virus (AAV) can include, but is not limited to, AAV serotypes such as AAV type 1, AAV type 2, AAV type 3 (including types 3 A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV type 14, AAV type 15, AAVrh8, AAVrh9, AAVrhlO, AAV.rh20, AAV.rh39, AAVrh.74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV2quadYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV2.5T, AAV2.5T.LSV1, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, those AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (Virol. 33:375 (2004)), and any other AAV now known or later discovered. See, e.g., FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some aspects, an AAV vector includes a derivative of a known AAV vector. In some aspects, an AAV vector can include a modified or an artificial AAV vector. In some aspects, an AAV vector includes a hybrid vector (e.g., AAV-DJ, AAV-PHP.B, AAV2-ESGHGYF, AAVM41, AAV-LK03, AAV2-BR1, AAV587MTP, AAV-Anc80L65, AAV2-7m8, AAV2HBKO, AAV2YF, AAV6-RGD or AAV6.2). In some aspects, the AAV vector is modified relative to the wild-type AAV serotype sequence. In some aspects, the modified AAV vector is a modified AAV6, e.g., an AAV6 vector comprising the RGD peptide (an AAV6-RGD vector) or an AAV6 vector comprising mutations of surface exposed tyrosine residues as described, for example, in Sayroo et al. Gene Ther. 2016 Jan;23(1): 18-25. In some aspects, the AAV6-RGD vector further comprises modified amino acids corresponding to Y705, Y731, T492, and K531, (e.g., Y705, Y731F, T492V, and K531E; also referred to as AAV-RGD-Y705-731F+T492V+K531E). In some aspects, the AAV vector is an AAV2 vector (i.e., an AAV2 serotype vector). In some aspects, the AAV vector is an AAV2QuadYF vector. In some aspects, the AAV vector is an AAV8 vector (i.e., an AAV8 serotype vector).

[0133] As used herein, "self-complementary adeno-associated virus" or "scAAV" is a viral vector engineered from the naturally occurring AAV such that the coding region has been designed to form an intra-molecular double-stranded DNA template. Thus, uponinfection of a cell with a scAAV, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for replication and transcription. In some aspects, the coding capacity found in a typical rAAV (e.g., about 4.7-6kb) is reduced in by about half in a scAAV (e.g., 2.4kb). In some aspects, the rAAV is a scAAV.

[0134] As used herein, a "recombinant AAV particle", "recombinant AAV vector", "rAAV particle", or "rAAV vector" refers to an AAV vector that comprises an AAV capsid protein and a vector genome (or an AAV genome, e.g., at least one AAV inverted terminal repeat (ITR) region) comprising at least one payload region (e.g., an expression cassette including a polynucleotide encoding a therapeutic agent). In some aspects, the "AAV vectors of the present disclosure" or "AAV vectors" refer to AAV vectors comprising a polynucleotide encoding a therapeutic agent and a vector genome comprising a pair of AAV ITRs. In some aspects, the AAV vector is encapsulated in an AAV particle.

[0135] As used herein, a "therapeutic agent" refers to a compound (e.g., a chemical compound), a peptide, a protein (e.g., an antibody), a lipid, a carbohydrate, a nucleic acid, or any other molecule or compound capable of treating, ameliorating, or reducing the symptoms of a disease, a disorder, a condition, or a syndrome, or otherwise having a beneficial effect, upon administration to a subject suffering from the disease, disorder, condition, or syndrome.

[0136] The phrase "contacting a cell" (e.g., contacting a cell with a rAAV vector or pharmaceutical composition comprising the rAAV vector) as used herein, includes contacting a cell directly or indirectly. In some aspects, contacting a cell with a rAAV vector, or pharmaceutical composition comprising the rAAV vector, includes contacting a cell in vitro or contacting a cell in vivo with the rAAV vector, or pharmaceutical composition comprising the rAAV vector. Thus, for example, the rAAV vector, or pharmaceutical composition comprising the rAAV vector, can be put into physical contact with the cell by the individual performing the method, or alternatively, the rAAV vector, or pharmaceutical composition comprising the rAAV vector, can be put into a situation that will permit or cause it to subsequently come into contact with the cell.

[0137] In some aspects, contacting a cell in vitro can be done, for example, by incubating the cell with the rAAV vector or the pharmaceutical composition comprising the rAAV vector.

[0138] In some aspects, contacting a cell in vivo can be done, for example, by injecting a rAAV vector, or a pharmaceutical composition comprising the rAAV vector, into or near the tissue where the cell is located (e.g., into the eye), or by injecting the rAAV vector, or pharmaceutical composition comprising the rAAV vector, into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell can be contacted in vitro with a rAAV vector, or a pharmaceutical composition comprising the rAAV vector, and subsequently transplanted into a subject.

[0139] In some aspects, contacting a cell with a rAAV vector, or a pharmaceutical composition comprising the rAAV vector, includes "introducing" or "delivering" (directly or indirectly) the rAAV vector, or a pharmaceutical composition comprising the rAAV vector into the cell by facilitating or effecting uptake or absorption into the cell. In some aspects, introduction into a cell includes a method know in the art such as electroporation and lipofection.

[0140] As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0141] As used herein, the term "in vivo" refers to events that occur within an organism (e.g., animal, plant, or microbe, or cell or tissue thereof).

[0142] As used herein, the term "transfection" refers to methods to introduce exogenous nucleic acids into a cell by non-viral methods. Methods of transfection include, but are not limited to, chemical methods, physical treatments and cationic lipids or mixtures.

[0143] As used herein, the term "transduction" refers to methods to introduce exogenous nucleic acids into a cell by viral methods (i.e., by viral vector, such as a rAAV vector).

[0144] As used herein, "off target" refers to any unintended effect on any one or more target, gene, RNA transcript, protein, signaling mechanisms.

[0145] As used herein, the term "subject" refers to any organism to which a composition, e.g., a rAAV vector, or a pharmaceutical composition comprising a rAAV vector, can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes, for example, according to the methods disclosed herein. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject can seek or be in need of treatment, require treatment, be receivingtreatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease, disorder, syndrome, or condition.

[0146] As used herein, the term "administration" or "administering" refers to the process of giving to or delivery of a therapeutic agent (e.g., a rAAV vector, or a pharmaceutical composition comprising a rAAV vector) to a subject or system. Administration to an animal subject (e.g., to a human) can be by any appropriate route (e.g., ocular injection, such as suprachoroidal injection).

[0147] As used herein, the terms "re-administration," "repeat administration," and "redosing" refer to an administration of one or more additional dose(s) of a therapeutic agent, e.g., a rAAV vector, or a pharmaceutical composition comprising a rAAV vector, following a first administration.

[0148] As used herein, the terms "effective amount" or "therapeutically effective amount,", e.g., a rAAV vector, or a pharmaceutical composition comprising a rAAV vector, refer to a quantity sufficient to, when administered to the subject, including a human, effect beneficial or desired results, including clinical results, and, as such, an "effective amount" or synonym thereto depends on the context in which it is being applied. In some aspects, a therapeutically effective amount of an agent (e.g., a rAAV vector, or a pharmaceutical composition comprising a rAAV vector) is an amount that results in a beneficial or desired result in a subject as compared to a control.

[0149] The amount of a given agent to be administered (e.g., a rAAV vector, or a pharmaceutical composition comprising a rAAV vector) will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease, disorder, syndrome, or condition, the identity of the subject (e.g., age, sex, and / or weight) or host being treated, and the like.

[0150] The term "prophylactically effective amount," as used herein, includes the amount of an agent, (e.g., a rAAV vector, or a pharmaceutical composition comprising a rAAV vector) that, when administered to a subject having or predisposed to have a disease, disorder, syndrome, or condition (e.g., an ocular disease, disorder, syndrome, or condition) is sufficient to prevent, reduce the symptoms of, or ameliorate the disease, disorder, syndrome, or condition or one or more symptoms of the disease, disorder, syndrome, or condition. Ameliorating the disease, disorder, syndrome, or condition includes slowing the course of the disease, disorder, syndrome, or condition, or reducing the severity of later-developing disease, disorder, syndrome, or condition. The"prophylactically effective amount" can vary depending on the characteristics of the agent, e.g., a rAAV vector, or pharmaceutical composition comprising a rAAV vector, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

[0151] As used herein, the terms "treat," "treated," and "treating", unless indicated otherwise, mean both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disease, disorder, syndrome, or condition, or obtain beneficial or desired clinical results. In some aspects, treating reduces or lessens the symptoms associated disease, disorder, syndrome, or condition. In some aspects, the treating results in a beneficial or desired clinical result. For example, treating an ocular disease, disorder, syndrome, or condition can reduce or lessen the symptoms associated with the ocular disease, disorder, syndrome, or condition, reduces or lessens the severity of at least one indicator of the an ocular disease, disorder, syndrome, or condition.

[0152] Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a disease, disorder, syndrome, or condition; stabilized (i.e., not worsening) state of disease, disorder, syndrome, or condition; delay in onset or slowing of disease, disorder, syndrome, or condition progression; amelioration of the disease, disorder, syndrome, or condition state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of disease, disorder, syndrome, or condition . In some aspects, treatment includes eliciting a clinically significant response without excessive levels of side effects. In some aspects, treatment includes prolonging survival as compared to expected survival if not receiving treatment.

[0153] As used herein, the term "amelioration" or "ameliorating" refers to a lessening of severity of at least one indicator of a disease, disorder, syndrome, or condition.

[0154] As used herein, the term "preventing" or "prevention" refers to delaying or forestalling the onset, development or progression of a disease, disorder, syndrome, or condition for a period of time, including weeks, months, or years.

[0155] The term "pharmaceutical composition," as used herein, represents a composition comprising a compound or molecule, e.g., a rAAV vector, formulated with apharmaceutically acceptable excipient. In some aspects, the pharmaceutical composition can be manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease, disorder, syndrome, or condition in a mammal. In some aspects, the pharmaceutical composition can be a solution.

[0156] A "pharmaceutically acceptable excipient," as used herein, refers to any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient.

[0157] The term a "therapeutic agent" or "therapeutic molecule" includes a compound or molecule that, when present in an effective amount, produces a desired therapeutic effect, pharmacologic and / or physiologic effect on a subject in need thereof. It includes any compound, e.g., a small molecule drug, or a biologic (e.g., a polypeptide drug or a nucleic acid drug) that when administered to a subject has a measurable or conveyable effect on the subject, e.g., it alleviates or decreases a symptom of a disease, disorder, syndrome, or condition. In some aspects, the therapeutic agent is encoded by a nucleic acid sequence delivered via a rAAV vector. In some aspects, the rAAV comprises an expression cassette encoding the therapeutic agent; such therapeutic agent can be a protein, e.g., an antibody or functional fragment thereof; a functional protein that can replace a nonfunctional protein in a gene replacement therapy; a protein that interferers with gene expression to alter or restore defective gene expression; or a protein that interferers a signaling pathway, etc.

[0158] As used herein, the term "pathology" refers to any deviations from the healthy state. As used herein, the term "pathology" is to be understood as encompassing a "disease," a "disorder," a "syndrome," a "condition," or any combination thereof.

[0159] As used herein, the term "ocular" is to be understood as meaning of the eye. Thus, for example, an "ocular pathology" is a pathology affecting the eye.

[0160] As used herein, the term "suprachoroidal" refers to the region of the eye situated between the choroid and sclera. As used herein, "suprachoroidal space" refers to the space situated between the sclera and choroid.

[0161] As used herein, the term "sclera" refers to the outer tunic of the eyeball: an outer membrane layer, white, tough, opaque, and fibrous, which covers the eye entirely excepting the segment covered anteriorly by the cornea.

[0162] As used herein, the term "choroid" refers to a layer of vascular tissue of the outer wall of the eye, between the sclera and the retina. The choroid is filled with blood vessels that bring oxygen and nutrients to the eye.

[0163] As used herein, the term "ophthalmic" is to be understood as meaning of the eye, relating to eye, or situated near the eye, including surrounding the eye (e.g., within the orbit). Thus, the term ophthalmic is to be understood as encompassing both ocular and periocular.

[0164] As used herein, an "ocular pathology" is a pathology affecting the eye. Nonlimiting examples of ocular pathologies include, but are not limited to, Achromatopsia, , Behcet's Disease, Best's disease, Bietti's Crystalline Dystrophy, Blepharitis, Blepharospasm, Central Areolar Choroidal Dystrophy, Central Serous Chorioretinopathy, Choroideremia, Choroidal Melanoma, Coloboma, Corneal Conditions, Coat's Disease, Cone-Rod Dystrophy, Corneal Dystrophy, Cystoids Macular Edema, Diabetic Retinopathy, Doyne Honeycomb Retinal Dystrophy, Dry Eye, Fuch's Dystrophy, Glaucoma, Hypertensive Retinopathy, Idiopathic Intracranial Hypertension, Lattice Degeneration, Leber Congenital Amaurosis, Leber Hereditary Optic Neuropathy, Leber's Miliarly Aneurism, Macular Degeneration (including Age-Related Macular Degeneration, Juvenile Macular Degeneration, Atrophic Macular Degeneration), Macular Edema, Ocular Histoplasmosis (including Ocular Histoplasmosis Syndrome), Ocular Ischemic Syndrome, Papillophlebitis, Pink Eye, Polypoidal Choroidal Vasculopathy, Retinitis Pigmentosa, Retinoblastoma, Retinopathy of Prematurity, Retinoschisis (including Juvenile Retinoschi sis), Sorsby's Disease, Stargardt Disease, Toxoplasmosis, Thyroid Eye Disease (TED), Usher Syndrome, and Uveitis (e.g., Noninfectious Uveitis), Vascular Occlusions, Inflammations (such as uveitis, choroiditis and retinistis), various Tumors (including neoplasms), or any combination thereof. In some aspects, the ocular pathology for treatment is Geographic Atrophy (GA) secondary to Age-related Macular Degeneration (AMD).

[0165] As used herein, the terms "intraocular" refers to a location that is within or occurring through the eye.

[0166] As used herein, the term "gene therapy" includes the delivery of nucleic acid sequences (e.g., a polynucleotide comprising a promoter operably linked to a nucleic acid encoding a therapeutic molecule) into an individual's cells and / or tissues to treat, reduce the symptoms of, or reduce the likelihood of a pathology or disorder. Gene therapy alsoincludes delivery of transgene that are inhibitory in nature, i.e., that inhibit, decrease or reduce expression, activity or function of an endogenous gene or protein, such as an undesirable or aberrant (e.g., pathogenic) gene or protein. Such transgenes can be exogenous. An exogenous molecule or sequence is understood to be molecule or sequence not normally occurring in the cell, tissue and / or individual to be treated. Both acquired and congenital diseases are amenable to gene therapy.

[0167] As used herein, the term "channel" or "passage" refers to a space or an opening that is created, e.g., by a tissue separator, in an organ, between tissues (or layers) of an organ, and / or through a tissue (or layer) of an organ, and that can be filled with a substance (e.g., a rAAV vector or a pharmaceutical composition comprising a rAAV vector), such as a fluid. For example, a tissue separator can be inserted into an organ (e.g., into an eye, for example between the sclera and the choroid) to create an opening (i.e., a space) into the organ (e.g., through the choroid, and / or between the sclera and the choroid). A "channel" or "passage" created in the eye (e.g., created by a tissue separator through the choroid, and / or between the sclera and the choroid) can be a "virtual channel" or a "virtual passage." The term "virtual" indicates that the channel or passage remains "closed" (i.e., it is not an actual, real, empty space), due to intraocular pressure (IOP) and / or to the presence of attaching fibers. A "virtual channel" or a "virtual passage" can be opened upon injection of a desired substance (e.g., a rAAV vector or a pharmaceutical composition comprising a rAAV vector), such as a fluid. The channel can then channel and disperse the injected substance (e.g., a rAAV vector or a pharmaceutical composition comprising a rAAV vector), such as a fluid throughout a region of the organ (such as the posterior segment of the eye).II. Structure and Physiology of the Eye

[0168] The eye comprises three main layers: an outer layer of tough, white, opaque membrane known as the sclera; a middle layer known as the choroid, the front of which is the iris; and an inner layer, known as the retina, which lines the back two-thirds of the eye.

[0169] The retina can be subdivided into two sublayers: the sensory retina, which contains photoreceptor cells (rods and cones) that convert light images into electrochemical signals, and the retinal pigment epithelium (RPE). The retina can be further subdivided into ten distinct layers that are, in order from the innermost layerscloser to the pupil to the layers further towards the posterior and periphery of the eyeball: Inner Limiting Membrane, Retinal Nerve Fiber Layer, Ganglion Cell Layer, Inner Plexiform Layer, Inner Nuclear Layer, Outer Plexiform Layer, Outer Nuclear Layer, External Limiting Membrane, Photoreceptor Layer, Retinal Pigment Epithelium.

[0170] The retina contains several cell types, the retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cells of the RPE, photoreceptor cells (rods and cones), ganglion cells, bipolar cells, horizontal cells, amacrine cells, and Muller glia cells. Cells of the RPE absorb excess light and transport oxygen, nutrients, and cellular waste between the photoreceptors and the choroid. The RPE is separated from the photoreceptor outer segments by the subretinal space. The macula is an oval-shaped, highly pigmented yellow spot near the center of the retina. Near the center of the macula is the fovea, a small pit that contains the largest concentration of cone cells in the eye and is responsible for central vision, and also contains the parafovea and perifovea.

[0171] The suprachoroidal space (SCS) lies within the transition zone of the outermost border of the choroid and the internal edge of the sclera. The SCS is an expandable uveoscleral fluid channel transversing the posterior segment of the eye from the scleral spur anteriorly to the optic nerve posteriorly.III. Adeno-Associated Virus (AAV) Vectors

[0172] In some aspects, the disclosure is directed to administration of a vector comprising a DNA of interest (e.g., a nucleic acid encoding a therapeutic agent of interest), wherein the vector is a rAAV vector, to the suprachoroidal space of an eye.

[0173] AAV, a parvovirus belonging to the genus Dependovirus, has several features not found in other viruses. For example, AAV can infect a wide range of host cells, including non-dividing cells. Furthermore, AAV can infect cells from different species. AAV has not been associated with any human or animal disease, and does not appear to alter the physiological properties of the host cell upon integration. Finally, AAV is stable at a wide range of physical and chemical conditions, which lends itself to production, storage, and transportation requirements.

[0174] The AAV genome, a linear, single-stranded DNA molecule containing approximately 4700 nucleotides (the AAV-2 genome includes 4681 nucleotides), generally comprises an internal non-repeating segment flanked on each end by inverted terminal repeats (ITRs). The ITRs are approximately 145 nucleotides in length (AAV-1has ITRs of 143 nucleotides) and have multiple functions, including serving as origins of replication, and as packaging signals for the viral genome.

[0175] The internal non-repeated portion of the genome includes two large open reading frames (ORFs), known as the AAV replication (rep) and capsid (cap) regions. These ORFs encode replication and capsid gene products, respectively: replication and capsid gene products (i.e., proteins) allow for the replication, assembly, and packaging of a complete AAV virion. More specifically, a family of at least four viral proteins are expressed from the AAV rep region: Rep 78, Rep 68, Rep 52, and Rep 40, all of which are named for their apparent molecular weights. The AAV cap region encodes at least three proteins: VP1, VP2, and VP3.

[0176] AAV is a helper-dependent virus, requiring co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) in order to form functionally complete AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome or exists in an episomal form, but infectious virions are not produced. Subsequent infection by a helper virus "rescues" the integrated genome, allowing it to be replicated and packaged into viral capsids, thereby reconstituting the infectious virion. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV will replicate in canine cells that have been co-infected with a canine adenovirus.

[0177] In some aspects, to produce recombinant AAV (rAAV) virions containing the DNA of interest, a suitable host cell line is transfected with an AAV vector containing the DNA, but lacking rep and cap. The host cell is then infected with wild type (wt) AAV and a suitable helper virus to form rAAV virions. Alternatively, wt AAV genes (known as helper function genes, comprising rep and cap) and helper virus function genes (known as accessory function genes) can be provided in one or more plasmids, thereby eliminating the need for wt AAV and helper virus in the production of rAAV virions. The helper and accessory function gene products are expressed in the host cell where they act in trans on the rAAV vector containing the heterologous gene. The heterologous gene is then replicated and packaged as though it were a wt AAV genome, forming a recombinant AAV virion. When a patient's cells are transduced with the resulting rAAV virion, the DNA enters and is expressed in the patient's cells. Because the patient's cells lack the rep and cap genes, as well as the accessory function genes, the rAAV virion cannot furtherreplicate and package its genomes. Moreover, without a source of rep and cap genes, wt AAV virions cannot be formed in the patient's cells. See e.g., U.S. Appl. Publ. No.2003 / 0147853.

[0178] In some aspects, AAV vectors to be administered according to the methods of the present disclosure can comprise or be derived from any natural or recombinant AAV serotype. According to the present disclosure, the AAV serotype can be, but is not limited to, AAV1 (e g. UniProt ID: Q9WBP8), AAV2 (e g. UniProt ID: Q4KWI8), AAV3, AAV4 (e.g. UniProt ID: 041855), AAV5 (e.g. UniProt ID: Q5XXZ6), AAV6, AAV7 (e g. UniProt ID: Q8JQG0), AAV8 (e g. UniProt ID: Q8JQF8), AAVrh8 (e g. UniProt ID: Q808Y3), AAVrh9, AAV9 (e.g. UniProt ID: Q6JC40), AAV10, AAVrhlO (e.g. UniProt ID: Q8JQF8), AAV11, AAV12, (e.g. UniProt ID: A9RAI0), AAV13, AAV14, AAV15, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B (e.g. UniProt ID: Q6JC40), AAV2.5, AAV2tYF, AAV2quadYF, AAV3B (e.g. UniProt ID: 056139), AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV2.5T, and AAV2.5T.LSV1. In some aspects, the AAV vector is an AAV2 serotype. In some aspects, the AAV vector is an AAV8 serotype. In some aspects, the AAV vector is an AAV9 serotype. In some aspect, the AAV vector is modified relative to the wild-type AAV serotype sequence. In some aspects, the modified AAV vector is a modified AAV6 vector. In some aspects, the modified AAV6 is an AAV6-RGD vector. In some aspects, the AAV6-RGD vector further comprises modified amino acids corresponding to Y705, Y731, T492, and K531, (e.g., Y705, Y731F, T492V, and K531E; also referred to as AAV-RGD-Y705-731F+T492V+K531E). In some aspects, the AAV serotype is a synthetic serotype (e g., AAV-DJ, AAV-PHP.B, AAV2-ESGHGYF, AAVM41, AAV- LK03, AAV2-BR1, AAV587MTP, AAV-Anc80L65, AAV2-7m8, AAV2HBK0, AAV2YF, AAV6-RGD, or AAV6.2 capsid). In some aspects, the AAV vector is an AAV2 serotype. In some aspects, the AAV vector is an AAV2QuadYF serotype. In some aspects, the AAV serotype is AAV8. In some aspects, the AAV serotype is AAV9. In some aspects, the AAV serotype is AAV6. In some aspects, the AAV serotype is a modified AAV6, e.g. AAV6 comprising the RGD peptide (AAV6-RGD) or AAV6 comprising mutations of surface exposed tyrosine residues as described, for example, inSayroo et al. Gene Ther. 2016 Jan;23(l): 18-25. In some aspects, the AAV serotype is AAV-DJ.

[0179] In some aspects, the rAAV vectors, or pharmaceutical compositions comprising the rAAV vectors, to be administered according to the methods of the present disclosure, are suitable for delivery to the ophthalmic region (i.e., ocular region, e.g., into the eye). In some aspects, the rAAV vectors, or pharmaceutical compositions comprising the rAAV vectors, to be administered according to the methods of the present disclosure, are suitable for delivery to the ophthalmic region (i.e., ocular region, e.g., into the eye) by injection (e.g., suprachoroidal injection).

[0180] In some aspects, the rAAV vector, or pharmaceutical compositions comprising the rAAV vectors, to be administered according to the methods of the present disclosure, can be administered in combination with one or more additional therapeutic agents.

[0181] In some aspects, the rAAV vectors, or pharmaceutical compositions comprising the rAAV vectors, to be administered according to the methods of the present disclosure, can be delivered in combination (e.g., simultaneously or sequentially) with one or more additional therapeutic agents. In some aspects, the one or more additional therapeutic agents, can be administered prior to, at the same time, or after the administration of the rAAV vectors, or pharmaceutical compositions comprising the rAAV vectors, administered according to the methods of the present disclosure.IV. Therapeutic Agents

[0182] The present disclosure provides rAAV vectors and pharmaceutical compositions comprising AAV vectors, to be administered according to the methods of the present disclosure to the suprachoroidal space of the eye, for treating an ocular pathology in a subject in need thereof.

[0183] In some aspects, the rAAV vectors to be administered according to the methods of the present disclosure comprise a polynucleotide comprising an expression cassette. In some aspects, the expression cassette comprises a nucleic acid molecule encoding a therapeutic agent. In some aspects, the therapeutic agent is suitable for the treatment of an ocular pathology.

[0184] In some aspects, the therapeutic agent can be an immunotherapy agent, such as an antibody or an antigen binding fragment thereof. In some aspects, the therapeutic agent can be a gene therapy agent, such as a gene replacement therapy agent. In some aspects,the therapeutic agent can be a gene editing agent. In some aspects, the therapeutic agent can be a modulator (e.g., an inhibitor, such as a pathway inhibitor agent). In some aspects, the therapeutic agent is an agent that can be used in optogenetics.

[0185] Non-limiting examples of therapeutic agents that can be encoded by a polynucleotide comprised in the AAV vectors to be administered according to the methods of the present disclosure include an anti-TNF antibody (e.g., Adalimumab), a CR2-CR1 fusion protein, an inflammasome pathway inhibitor (e.g., tatCARD fusion proteins), a Vascular Endothelial Growth Factor (VEGF) inhibitor (e.g., FLT01, Aflibercept), an anti-VEGF antibody and antibody fragments (e.g. bevacizumab, ranibizumab, brolucizumab), Fibroblast Growth Factor 21 (FGF21) (GenBank ID:NC 000019.10, Uniprot ID: Q9NSA1), soluble membrane-independent form of CD59 (sCD59) (GenBank ID: NC 000011.10; Uniprot ID: P27274), soluble-fms like tyrosine kinase-1 (sFLT-1) (Uniprot ID: S5TRK2), Complement factor I (CFI) (GenBank ID: NC 000004.12, Uniprot ID: P05156), Rab Escort Protein 1 (REP1) (Uniprot ID:P24386), choroideremia / Rab Escort Protein 1 (CHM) (GenBank ID: NC 000023.11), Retinal Pigment Epithemium specific 65kDa protein (RPE65) (GenBank ID:NC_000001.ll, Uniprot ID: Q16518), Retinal Pigment Epithemium specific 65kDa protein v2 (RPE65v2), NADH ubiquinone oxidoreductase core subunit 4 (ND4) (Unitprot ID: P03905), human MER-proto oncogene Tyrosine Kinase (hMERTK) (GenBank ID: NC 000002.12, Uniprot ID: Q12866), Retinitis Pigmentosa GTPase Regulator (RPGR) (GenBank ID: NC 000023.11, Uniprot ID: Q92834), Phosphodiesterase 6A (PDE6A) (GenBank ID: NC 000005.10, Uniprot ID: P16499), Phosphodiesterase 6B (PDE6B) (GenBank ID: NC 000004.12, Uniprot ID: P35913), cyclic nucleotide gated channel subunit alpha 3 (CNGA3) (GenBank ID: NC_000002.12, UniProt: Q16281), cyclic nucleotide gated channel subunit beta 3 (CNGB3) (GenBank ID: NC_000008.ll, Uniprot ID: Q9NQW8), retinaldehyde binding protein 1 (RLBP1) (GenBank ID: NC 000015.10, Uniprot ID: P12271), retinoschisin 1 (RSI) (GenBank ID: NC 000023.11, Uniprot ID: 015537), gene editing factors (e.g., SaCas9, gRNAs), Optogenetics factors (e.g., ChrimsonR-tdTomato, Channelrhodopsin-2, Multi-Characteristic Opsin (MCO)), or functional fragments thereof, or any fusion protein comprising one or more of the same, or any combination thereof.

[0186] In some aspects, the therapeutic agent is an anti-TNF antibody (e.g.,Adalimumab).

[0187] In some aspects, the therapeutic agent is a CR2-CR1 fusion protein. In some aspects, the CR2-CR1 fusion protein comprises a ligand-binding region of complement receptor 2 (CR2) and a ligand-binding region of complement receptor 1 (CR1).

[0188] In some aspects, the therapeutic agent is an inflammasome pathway inhibitor (e.g., tatCARD fusion proteins).

[0189] In some aspects, the therapeutic agent is a Vascular Endothelial Growth Factor (VEGF) inhibitor (e.g., FLT01, Aflibercept)

[0190] In some aspects, the therapeutic agent is an anti-VEGF antibody or antibody fragment thereof (e.g. bevacizumab, ranibizumab, brolucizumab).

[0191] In some aspects, the therapeutic agent is the Fibroblast Growth Factor 21.

[0192] For example, VEGF-neutralizing proteins (e.g., FLT01), sFLT-1, sCD59, Complement factor CFI, Anti-VEGF AFab, Anti-VEGF protein, and VEGF inhibitors (e.g., Aflibercept) can be used for treating Age-related Macular Degeneration; REP1 and CHM genes can be used in gene replacement therapies for treating Choroideremia;RPE65v2 and RPE65 genes can be used in gene replacement therapies for treating Leber Congenital Amaurosis; ND4 gene can be used in gene replacement therapies for treating Leber Hereditary Optic Neuropathy; hMERTK, RPGR, PDE6B, PDE6A, RLBP1 genes can be used in gene replacement therapies for treating Retinitis Pigmentosa; CNGA3 and CNGB3 genes can be used in gene replacement therapies for treating Achromatopsia; RSI gene can be used in gene replacement therapies for treating Retinoschisis.V. Pharmaceutical Compositions

[0193] In some aspects, a pharmaceutical composition disclosed herein comprises (i) a rAAV vector of the present disclosure (e.g., comprising an AAV capsid and an expression cassette comprising a promoter operably linked to a nucleic acid sequence that encodes therapeutic agent) and (ii) a pharmaceutically-acceptable excipient or carrier, which is suitable for administration according to the methods of the disclosure.

[0194] Pharmaceutically acceptable excipients or carriers are determined in part by the particular pharmaceutical composition being administered, as well as by the particular method used to administer the pharmaceutical composition.

[0195] Accordingly, there can be a variety of suitable formulations of pharmaceutical compositions comprising a rAAV vector (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18th ed. (1990)). The pharmaceutical compositions aregenerally formulated sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. In some aspects, the pharmaceutical composition comprises more than one rAAV vector.

[0196] In some aspects, a pharmaceutical composition can comprise (i) one or more rAAV vectors, and (ii) one or more additional therapeutic agents for the treatment of a pathology (e.g., an ocular pathology).

[0197] In some aspects, the one or more rAAV vectors and the one or more additional therapeutic agents for the treatment of a pathology (e.g., an ocular pathology) are coadministered in a single pharmaceutical composition.

[0198] In some aspects, the one or more rAAV vectors and the one or more additional therapeutic agents for the treatment of a pathology (e.g., an ocular pathology) are coadministered as separate pharmaceutical compositions.

[0199] In some aspects, a pharmaceutical composition comprising one or more rAAV vectors is administered prior to, after, or concurrently with the administration of a pharmaceutical composition comprising one or more additional therapeutic agents for the treatment of a pathology (e.g., an ocular pathology).

[0200] In some aspects, the pharmaceutical composition of the disclosure is formulated for administration to the ophthalmic region (i.e., the suprachoroidal space (SCS) of an eye (e.g., one or both eyes)). In some aspects, the pharmaceutical composition of the disclosure is formulated for injection into the suprachoroidal space (SCS) of one or both eyes. In some aspects, the pharmaceutical composition of the disclosure is formulated for injection into the suprachoroidal space (SCS) of one eye. In some aspects, the pharmaceutical composition of the disclosure is formulated for injection into the suprachoroidal space (SCS) both eyes.

[0201] In some aspects, the pharmaceutical composition of the disclosure is formulated for administration by suprachoroidal injection as a single dose. In some aspects, the single dose administration comprises a single injection per eye (at a single injection site). In some aspects, the single dose administration comprises multiple suprachoroidal injections. In some aspects, the single dose administration comprises multiple injections per eye. In some aspects, the multiple injections per eye are at a single injection site. In some aspects, the multiple injections per eye are at multiple injection sites. In some aspects, the pharmaceutical composition of the disclosure is formulated for administration by suprachoroidal injection as multiple doses. In some aspects, each one of the multipledoses administration comprises multiple suprachoroidal injections. In some aspects, the multiple suprachoroidal injections are at a single injection site. In some aspects, the multiple suprachoroidal injections are at different injection sites.

[0202] In some aspects, the pharmaceutical composition of the disclosure is formulated for suprachoroidal administration providing for delivery of the rAAV to an ocular tissue. In some aspects, the ocular tissue is located in the anterior segment of the eye, in the posterior segment of the eye, or a combination thereof. In some aspects, the ocular tissue is located in the anterior segment of the eye. In some aspects, the ocular tissue is located in the posterior segment of the eye. In some aspects, the ocular tissue is selected from cornea, sclera, choroid, Brunch’s membrane, pupil, iris, lens, optic disc, optic nerve, macula (e.g., central macula), retina, retinal pigmented epithelium (RPE, e.g., choroid- RPE), ciliary bodies, fovea, and any combination thereof. In some aspects, the ocular tissue is the optic disc. In some aspects, the ocular tissue is the optic nerve. In some aspects, the ocular tissue is the macula. In some aspects, the ocular tissue is the retina. In some aspects, the ocular tissue is the fovea. In some aspects, the ocular tissue is the retinal pigmented epithelium (RPE, e.g., choroid-RPE). In some aspects, the ocular tissue is the ciliary bodies. In some aspects, the ocular tissue is the sclera. In some aspects, the ocular tissue is the peripheral retina. In some aspects, the ocular tissue is the nerve fiber layer. In some aspects, the ocular tissue is the choroid capillaries.

[0203] In some aspects, the ocular tissue is the retina and delivery is to a region of the retina selected from the group consisting of: the inner limiting membrane, the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the middle limiting membrane, the outer plexiform layer, the outer nuclear layer, the external limiting membrane, the photoreceptor inner / outer segments, the retinal pigmented epithelium (e.g., choroid-RPE), and any combination thereof. In some aspects, the ocular tissue is the retina and delivery is to the retinal pigmented epithelium (RPE, e.g., choroid- RPE). In some aspects, the ocular tissue is the retina and delivery is to the ganglion cell layer. In some aspects, the ocular tissue is the retina and delivery is to the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and to the ganglion cell layer. In some aspects, the ocular tissue is the retina and delivery is to the retinal pigmented epithelium (RPE, e.g., choroid-RPE) through the ganglion cell layer. In some aspects, the ocular tissue is the retina and delivery is to the retinal pigmented epithelium (RPE, e.g., choroid- RPE) and / or the ganglion cell layer in the macula. In some aspects, the ocular tissue is theretina and delivery is to the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and / or the ganglion cell layer in the central macula. In some aspects, the pharmaceutical composition of the disclosure is formulated for suprachoroidal administration for delivery to an ocular cell. In some aspects, the ocular cell is a retinal cell. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, photoreceptor cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, a Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof. In some aspects, the photoreceptor cell is a rod cell, a cone cell, or any combination thereof. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell. In some aspects, the retinal cell is ganglion cell. In some aspects, the pharmaceutical composition of the disclosure is formulated for suprachoroidal administration for delivery to retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cells and ganglion cells. In some aspects, the retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cells and / or ganglion cells are in the macula. In some aspects, the retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cells and / or ganglion cells are in the central macula.

[0204] In some aspects, delivery is to the optic nerve, the nerve fiber layer, choroid capillaries, or the peripheral retina. In some aspects, delivery is to the optic nerve. In some aspects, delivery is to nerve fiber layer. In some aspects, delivery is to the peripheral retina. In some aspects, delivery is to the choroid capillaries.

[0205] Also provided herein are pharmaceutical compositions comprising rAAV vectors having the desired degree of purity, and a pharmaceutically acceptable carrier or excipient, in a form suitable for suprachoroidal administration to an eye of a subject. Pharmaceutically acceptable excipients or carriers can be determined in part by the particular pharmaceutical composition being administered, as well as by the particular method used to administer the pharmaceutical composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions comprising a plurality of vectors, e.g., rAAV vectors described herein. (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 21st ed. (2005)). The pharmaceutical compositions are generally formulated sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0206] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g., animals or humans) at the dosages and concentrations employed.

[0207] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Except insofar as any conventional media or compound is incompatible with the rAAV vectors disclosed herein, use thereof in the pharmaceutical compositions is contemplated. In some aspects, a pharmaceutical composition is formulated to be compatible with its intended route of administration. The rAAV vectors can be administered to the ophthalmic region (i.e., the suprachoroidal space of an eye (e.g., one or both eyes)), for example, the rAAV vector, or the pharmaceutical composition, is suitable for suprachoroidal injection using the injection device disclosed herein.

[0208] The rAAV vectors can be formulated using one or more excipients to (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release; or (4) alter the biodistribution (e.g., target the rAAV vector to specific tissues or cell types).

[0209] In some aspects, the pharmaceutical compositions comprising a rAAV vector administered by the methods of the disclosure provide a rAAV vector transduction efficiency of at least 1% (e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the target ocular cells. In some aspects, the rAAV vector transduction efficiency is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the target ocular cells. In some aspects, the target ocular cell is a cell of the cornea, sclera, choroid, Brunch’s membrane, pupil, iris, lens, optic disc, optic nerve, macula (e.g., central macula), retina, retinal pigmented epithelium (RPE, e.g., choroid- RPE), ciliary bodies, fovea, or any combination thereof. In some aspects, the ocular cell is a retinal cell. In some aspects, the retinal cell is a cell of a region of the retina selected from the group consisting of: the inner limiting membrane, the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the middle limiting membrane, the outer plexiform layer, the outer nuclear layer, the external limiting membrane, the photoreceptor inner / outer segments, the retinal pigmented epithelium (RPE, e.g., choroid-RPE), and any combination thereof. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, photoreceptor cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, a Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof.In some aspects, the photoreceptor cell is a rod cell, a cone cell, or any combination thereof. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, a ganglion cell, or a combination thereof. In some aspects, the retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell and / or the ganglion cell are in the macula. In some aspects, the retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell and / or the ganglion cell are in the central macula.

[0210] In some aspects, the pharmaceutical compositions comprising a rAAV vector administered by the methods of the disclosure provide a rAAV vector transduction efficiency of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal ganglion (RG) cells an of the retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells. In some aspects, the pharmaceutical compositions comprising a rAAV vector administered by the methods of the disclosure provide a rAAV vector transduction efficiency of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal ganglion (RG) cells. In some aspects, the pharmaceutical compositions comprising a rAAV vector administered by the methods of the disclosure provide a rAAV vector transduction efficiency of at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal pigmented epithelium (RPE, e.g., choroid- RPE) cells. In some aspects, the pharmaceutical compositions comprising a rAAV vector administered by the methods of the disclosure provide a rAAV vector transduction efficiency of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal ganglion (RG) cells and of at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells.

[0211] In some aspects, the pharmaceutical compositions comprising a rAAV vector administered by the methods of the disclosure provide a rAAV vector transduction that does not readily transduce photoreceptor cells. In some aspects, the pharmaceutical compositions comprising a rAAV vector administered by the methods of the disclosure provide an rAAV vector transduction efficiency is at less 5% (e.g., less than 4%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or less than 0.5%) of the photoreceptor cells. In some aspects, the photoreceptor cells are a rod cells, cone cells, or any combination thereof.VI. Injection Device

[0212] In some aspects, the injection device used in the methods disclosed herein (e.g., for suprachoroidal administration of a rAAV vector, or a pharmaceutical composition comprising a rAAV) comprises a needle. In some aspects, the needle may include a needle hub. In some aspects, the needle may further include a needle shaft. In some aspects, the needle may further include an external stopper. In some aspects, a needle lumen may extend between a proximal end of the needle and a distal end of the needle. In some aspects, the needle distal end may further include a size transitional portion and a needle distal tip. In some aspects, the needle may penetrate into a portion of an outer layer of an eye, such as the sclera of an eye and optionally the conjunctiva. In some aspects, the needle may penetrate through a portion of an outer layer of an eye, such as the sclera of an eye and optionally the conjunctiva.

[0213] In some aspects, optionally, the injection device used in the methods disclosed herein (e.g., for suprachoroidal administration of a rAAV vector, or a pharmaceutical composition comprising a rAAV) comprises an eye docking device. In some aspects, optionally, the eye docking device may direct the injection device with a needle into a sclera portion of an eye. As used herein, the term "sclera portion" refers to a region of the sclera where the injection device is targeted. In some aspects, a distal tip of the needle may be directed along a predetermined penetration path into the sclera portion. In some aspects, a tissue separator may be shifted within a lumen of the needle such that a distal tip of the tissue separator is positioned at a distal tip of the needle. In some aspects, the distal tip of the tissue separator may penetrate into the sclera portion and into the choroid to create a channel through the choroid. In some aspects, the channel through the choroid may be a closed space such that the channel may be filled with fluid.

[0214] In some aspects, the needle has a length of about 0.2 mm to about 5 mm. In some aspects, the needle has a length of 3 mm. In some aspects, the needle has an internal diameter of about 1 mm to about 3 mm. In some aspects, the needle has an internal diameter of 2 mm. In some aspects, the tissue separator is a non-sharp tissue separator. In some aspects, the tissue separator comprises a slender probe.

[0215] In some aspects, the needle has a length of about 1 mm to about 5 mm. In some aspects, the needle has a length of 2 mm. In some aspects, the needle has a length of 1.5 mm. In some aspects, the needle has an internal diameter of about 0.1 mm to about 0.6mm. In some aspects, the needle has an internal diameter of 0.1 mm. In some aspects, the needle has an internal diameter of 0.2 mm. In some aspects, the needle has an internal diameter of 0.3 mm. In some aspects, the tissue separator has a blunt tip. In some aspects, the tissue separator comprises a slender probe.

[0216] In some aspects, the injection device comprises:

[0217] (a) the needle comprising a needle lumen extending between a needle proximal end and a needle distal end, said needle distal end including a size transitional portion terminating in a sharp needle distal tip;

[0218] (b) the tissue separator having a separator distal tip, at least a portion of said tissue separator being shiftable in the needle lumen; and

[0219] (c) an actuator configured to shift said tissue separator in said needle lumen between a first position, wherein said separator distal tip is fixedly positioned proximally to said needle distal tip, and a second position, wherein said separator distal tip is fixedly positioned a first predetermined distance distally from said needle distal tip.

[0220] In some aspects, the separator distal tip is in said first position, said separator distal tip protrudes from a laterally uncovered section of said size transitional portion.

[0221] In some aspects, the injector device further comprises a reservoir (e.g., a syringe).

[0222] In some aspects, the injector device further comprises a cannula fluidly connectable between a syringe and said needle lumen, so that a fluid (e.g., the pharmaceutical composition) injected from said syringe is directly flowable to said needle distal tip via said cannula and said needle lumen.

[0223] In some aspects, the syringe is connectable to a separator hub of said separator.

[0224] In some aspects, the injector device comprises the features described in one or more of FIGs. 5A-5C, 6A-6B, 7A-7B, 8A-8C, 9A-9O, and / or 10A-10C

[0225] In some aspects, the injection device used in the methods disclosed herein (e.g., for suprachoroidal administration of a rAAV vector, or a pharmaceutical composition comprising a rAAV) comprises a needle distal tip. In some aspects, the needle distal tip may penetrate the sclera portion at a predetermined length. In some aspects, the needle distal tip may penetrate the sclera portion at a steep angle. In some aspects, the needle distal tip may further penetrate the sclera portion at a tangential angle.

[0226] In some aspects, the injection device to be used in the methods disclosed herein (e.g., for suprachoroidal administration of a rAAV vector, or a pharmaceutical composition comprising a rAAV) may be configured to inject one or more substances(e.g., a rAAV vector, or a pharmaceutical composition comprising a rAAV) into an eye (i.e., an ocular or suprachoroidal space of an eye (e.g., one or both eyes)) at a predetermined volume and / or flow rate (e.g., 5 l / sec to lOOpl / sec).

[0227] In some aspects, the predetermined volume is about 0.05 mL to about 0.50 mL, about 0.10 mL to about 0.50 mL, about 0.11 mL to about 0.50 mL, about 0.12 mL to about 0.50 mL, about 0.13 mL to about 0.50 mL, about 0.14 mL to about 0.50 mL, about 0.15 mL to about 0.50 mL, about 0.16 mL to about 0.50 mL, about 0.17 mL to about 0.50 mL, about 0.18 mL to about 0.50 mL, about 0.19 mL to about 0.50 mL, about 0.20 mL to about 0.50 mL, about 0.25 mL to about 0.50 mL, about 0.30 mL to about 0.50 mL, about 0.35 mL to about 0.50 mL, about 0.40 mL to about 0.50 mL, about 0.45 mL to about 0.50 mL, about 0.01 mL to about 0.30 mL, about 0.02 mL to about 0.30 mL, about 0.03 mL to about 0.30 mL, about 0.04 mL to about 0.30 mL, about 0.05 mL to about 0.30 mL, about 0.06 mL to about 0.30 mL, about 0.07 mL to about 0.30 mL, about 0.08 mL to about 0.30 mL, about 0.09 mL to about 0.30 mL, about 0.10 mL to about 0.30 mL, about 0.11 mL to about 0.30 mL, about 0.12 mL to about 0.30 mL, about 0.13 mL to about 0.30 mL, about 0.14 mL to about 0.30 mL, about 0.15 mL to about 0.30 mL, about 0.16 mL to about 0.30 mL, about 0.17 mL to about 0.30 mL, about 0.18 mL to about 0.30 mL, about 0.19 mL to about 0.30 mL, about 0.20 mL to about 0.30 mL, about 0.21 mL to about 0.30 mL, about 0.22 mL to about 0.30 mL, about 0.23 mL to about 0.30 mL, about 0.24 mL to about 0.30 mL, about 0.25 mL to about 0.30 mL, about 0.26 mL to about 0.30 mL, about 0.27 mL to about 0.30 mL, about 0.28 mL to about 0.30 mL, or about 0.29 mL to about 0.30 mL.

[0228] In some aspects, the predetermined volume is greater than 100 pl, greater than 105 pl, greater than 110 pl, greater than 115 pl, greater than 120 pl, greater than 125 pl, greater than 130 pl, greater than 135 pl, greater than 140 pl, greater than 145 pl, greater than 150 pl, greater than 155 pl, greater than 160 pl, greater than 165 pl, greater than 170 pl, greater than 175 pl, greater than 180 pl, greater than 185 pl, greater than 190 pl, greater than 195 pl, greater than 200 pl, greater than 205 pl, greater than 210 pl, greater than 215 pl, greater than 220 pl, greater than 225 pl, greater than 230 pl, greater than 235 pl, greater than 240 pl, greater than 245 pl, greater than 250 pl, greater than 255 pl, greater than 260 pl, greater than 265 pl, greater than 270 pl, greater than 275 pl, greater than 280 pl, greater than 285 pl, greater than 290 pl, greater than 295 pl, greater than 300 pl, greater than 305 pl, greater than 310 pl, greater than 315 pl, greater than 320 pl,greater than 325 pl, greater than 330 pl, greater than 335 pl, greater than 340 pl, greater than 345 pl, greater than 350 pl, greater than 355 pl, greater than 360 pl, greater than 365 pl, greater than 370 pl, greater than 375 pl, greater than 380 pl, greater than 385 pl, greater than 390 pl, greater than 395 pl, greater than 400 pl, greater than 405 pl, greater than 410 pl, greater than 415 pl, greater than 420 pl, greater than 425 pl, greater than 430 pl, greater than 435 pl, greater than 440 pl, greater than 445 pl, greater than 450 pl, greater than 455 pl, greater than 460 pl, greater than 465 pl, greater than 470 pl, greater than 475 pl, greater than 480 pl, greater than 485 pl, greater than 490 pl, or greater than 495 pl, and optionally, the predetermined volume is less than 500 pl, less than 505 pl, less than 510 pl, less than 515 pl, less than 520 pl, less than 525 pl, less than 530 pl, less than 535 pl, less than 540 pl, less than 545 pl, or less than 550 pl.

[0229] In some aspects, the predetermined volume is about 0.05 mL to about 1 mL, about 0.1 mL to about 0.5 mL, about 0.1 mL to about 0.3 mL, about 0.15 mL to about 0.5 mL, about 0.15 mL to about 0.3 mL, or about 0.1 mL to about 0.2 mL.

[0230] In some aspects, the predetermined volume is a volume of greater than 0.1 mL up to about 0.5 mL. In some aspects, the predetermined volume is about 0.1 mL to about 0.5 mL, about 0.15 mL to about 0.5 mL, about 0.2 mL to about 0.5 mL, about 0.25 mL to about 0.5 mL, about 0.3 mL to about 0.5 mL, about 0.35 mL to about 0.5 mL, about 0.4 mL to about 0.5 mL, or about 0.45 mL to about 0.5 mL.

[0231] In some aspects, in some aspects the predetermined volume is a total volume of about 0.1 mL to about 0.5 mL (e.g., about O.lmL to about 0.25 mL or about 0.15mL to about 0.25 mL). In some aspects, the predetermined volume is 0.075 mL to 0.125 mL (e.g., about 0.1 mL). In some aspects, the predetermined volume is 0.2 mL to 0.3 mL (e.g., about 0.24 mL).

[0232] In some aspects the flow rate is about 5 pl / sec to about 100 pl / sec, about 10 pl / sec to about 100 pl / sec, about 15 pl / sec to about 100 pl / sec, about 20 pl / sec to about 100 pl / sec, about 25 pl / sec to about 100 pl / sec, about 30 pl / sec to about 100 pl / sec, about 35 pl / sec to about 100 pl / sec, about 40 pl / sec to about 100 pl / sec, about 45 pl / sec to about 100 pl / sec, about 50 pl / sec to about 100 pl / sec, about 55 pl / sec to about 100 pl / sec, about 60 pl / sec to about 100 pl / sec, about 65 pl / sec to about 100 pl / sec, about 70 pl / sec to about 100 pl / sec, about 75 pl / sec to about 100 pl / sec, about 80 pl / sec to about 100 pl / sec,about 85 pl / sec to about 100 pl / sec, about 90 pl / sec to about 100 pl / sec, about 95 pl / sec to about 100 pl / sec.

[0233] In some aspects, the injection device to be used in the methods disclosed herein (e.g., for suprachoroidal administration of a rAAV vector, or a pharmaceutical composition comprising a rAAV) may be configured to inject one or more substances (e.g., a rAAV vector, or a pharmaceutical composition comprising a rAAV) into an eye (i.e., an ocular or suprachoroidal space of an eye (e.g., one or both eyes)) at a predetermined volume of 0.1 mL to about 0.5 mL (e.g., about 0. ImL to about 0.25 mL or about 0.15mL to about 0.25 mL) and at flow rate of about 5 pl / sec to about 100 pl / sec, about 10 pl / sec to about 100 pl / sec, about 15 pl / sec to about 100 pl / sec, about 20 pl / sec to about 100 pl / sec, about 25 pl / sec to about 100 pl / sec, about 30 pl / sec to about 100 pl / sec, about 35 pl / sec to about 100 pl / sec, about 40 pl / sec to about 100 pl / sec, about 45 pl / sec to about 100 pl / sec, about 50 pl / sec to about 100 pl / sec, about 55 pl / sec to about 100 pl / sec, about 60 pl / sec to about 100 pl / sec, about 65 pl / sec to about 100 pl / sec, about 70 pl / sec to about 100 pl / sec, about 75 pl / sec to about 100 pl / sec, about 80 pl / sec to about 100 pl / sec, about 85 pl / sec to about 100 pl / sec, about 90 pl / sec to about 100 pl / sec, about 95 pl / sec to about 100 pl / sec..VII. Methods of Treatment and Use

[0234] Certain aspects of the disclosure are directed to methods of treatment and uses comprising administration of a rAAV vector (e.g., pharmaceutical compositions comprising rAAV vectors) comprising injection of the rAAV vector into the suprachoroidal space of an eye using an injection device disclosed herein for treating an ocular pathology in a subject in need thereof.

[0235] In some aspects, the methods comprise delivery or administration of a rAAV vector, or pharmaceutical composition comprising a rAAV vector to the suprachoroidal space of the eye with an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the rAAV vector, or pharmaceutical composition comprising a rAAV vector is injected through the needle into the suprachoroidal space.

[0236] Certain aspects of the disclosure are directed to a method of delivering a pharmaceutical composition comprising a rAAV vector to the suprachoroidal space in an eye of a subject, the method comprising:

[0237] a. providing device for injection into the suprachoroidal space of the subject, the device comprising:

[0238] a needle comprising a needle lumen extending between a needle proximal end and a needle distal end, said needle distal end including a size transitional portion terminating in a sharp needle distal tip;

[0239] an elongated tissue separator having a separator distal tip, at least a portion of said tissue separator being shiftable in the needle lumen; and

[0240] an actuator configured to shift said tissue separator in said needle lumen between a first position, wherein said separator distal tip is fixedly positioned proximally to said needle distal tip, and a second position, wherein said separator distal tip is fixedly positioned a first predetermined distance distally from said needle distal tip;

[0241] b. positioning said separator distal tip at the first position between ends of said size transitional portion,

[0242] c. positioning said separator distal tip at a first predetermined distance proximally to said needle distal tip, the separator distal tip protruding from the size transitional portion;

[0243] d. penetrating outer surface of the eye (i.e., the sclera and optionally the conjunctiva) at a penetration point into the eye using said needle distal tip until contacting an outer surface layer of the eye (i.e., the sclera and optionally the conjunctiva) with said separator distal tip, wherein said first predetermined length is shorter than a thickness of said outer surface layer (i.e., the sclera and optionally the conjunctiva) adjacent to said penetration point; and

[0244] e. injecting the pharmaceutical composition into the suprachoroidal space.

[0245] In some aspects, the administration is suprachoroidal administration of a rAAV vector, or a pharmaceutical composition comprising a rAAV, to a subject suffering from an ocular pathology as described herein.

[0246] In some aspects, the ocular pathology is a pathology affecting the anterior segment of the eye, the posterior segment of the eye, or a combination thereof. In some aspects, the ocular pathology is a pathology affecting the anterior segment of the eye. In some aspects, the ocular pathology is a pathology affecting the posterior segment of the eye.

[0247] In some aspects, the ocular pathology is a pathology affecting the optic disc, the optic nerve, the macula, the retina, the fovea, the retinal pigmented epithelium (RPE, e.g., choroid-RPE), the ciliary bodies, or any combination thereof.

[0248] In some aspects, the ocular pathology is a pathology affecting the optic disc. In some aspects, the ocular pathology is a pathology affecting the optic nerve. In some aspects, the ocular pathology is a pathology affecting the macula. In some aspects, the ocular pathology is a pathology affecting the retina. In some aspects, the ocular pathology is a pathology affecting the fovea. In some aspects, the ocular pathology is a pathology affecting the retinal pigmented epithelium (RPE, e.g., choroid-RPE). In some aspects, the ocular pathology is a pathology affecting the ciliary bodies.

[0249] In some aspects, the ocular pathology is a pathology affecting a region of the retina selected from the group consisting of: the inner limiting membrane, the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the middle limiting membrane, the outer plexiform layer, the outer nuclear layer, the external limiting membrane, the photoreceptor inner / outer segments, the retinal pigmented epithelium (RPE, e.g., choroid-RPE), and any combination thereof.

[0250] In some aspects, the ocular pathology is a pathology affecting a retinal cell. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, photoreceptor cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, a Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof. In some aspects, the photoreceptor cell is a rod cell, a cone cell, or any combination thereof. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell. In some aspects, the retinal cell is a ganglion cell.

[0251] In some aspects, the rAAV vector comprises a polynucleotide encoding a therapeutic agent.

[0252] In some aspects, the therapeutic agent can be an immunotherapy agent, such as an antibody or an antigen binding fragment thereof; a gene therapy agent, such as a gene replacement therapy agent; a gene editing agent; a modulator (e.g., an inhibitor, such as a pathway inhibitor agent); or an agent that can be used in optogenetics; or any combination thereof.

[0253] Non-limiting examples of therapeutic agents that can be encoded by a polynucleotide comprised in the AAV vectors to be administered according to the methods of the present disclosure include an anti-TNF antibody (e.g., Adalimumab), aCR2-CR1 fusion protein, an inflammasome pathway inhibitor (e.g., tatCARD fusion proteins), a Vascular Endothelial Growth Factor (VEGF) inhibitor (e.g., FLT01, Aflibercept), an anti-VEGF antibody and antibody fragments (e.g. bevacizumab, ranibizumab, brolucizumab), Fibroblast Growth Factor 21 (FGF21) (GenBank ID:NC 000019.10, Uniprot ID: Q9NSA1), soluble membrane-independent form of CD59 (sCD59) (GenBank ID: NC 000011.10; Uniprot ID: P27274), soluble-fms like tyrosine kinase-1 (sFLT-1) (Uniprot ID: S5TRK2), Complement factor I (CFI) (GenBank ID: NC 000004.12, Uniprot ID: P05156), Rab Escort Protein 1 (REP1) (Uniprot ID:P24386), choroideremia / Rab Escort Protein 1 (CHM) (GenBank ID: NC 000023.11), Retinal Pigment Epithemium specific 65kDa protein (RPE65) (GenBank ID:NC_000001.ll, Uniprot ID: Q16518), Retinal Pigment Epithemium specific 65kDa protein v2 (RPE65v2), NADH ubiquinone oxidoreductase core subunit 4 (ND4) (Unitprot ID: P03905), human MER-proto oncogene Tyrosine Kinase (hMERTK) (GenBank ID: NC 000002.12, Uniprot ID: Q12866), Retinitis Pigmentosa GTPase Regulator (RPGR) (GenBank ID: NC 000023.11, Uniprot ID: Q92834), Phosphodiesterase 6A (PDE6A) (GenBank ID: NC 000005.10, Uniprot ID: P16499), Phosphodiesterase 6B (PDE6B) (GenBank ID: NC 000004.12, Uniprot ID: P35913), cyclic nucleotide gated channel subunit alpha 3 (CNGA3) (GenBank ID: NC_000002.12, UniProt: Q16281), cyclic nucleotide gated channel subunit beta 3 (CNGB3) (GenBank ID: NC_000008.ll, Uniprot ID: Q9NQW8), retinaldehyde binding protein 1 (RLBP1) (GenBank ID: NC 000015.10, Uniprot ID: P12271), retinoschisin 1 (RSI) (GenBank ID: NC 000023.11, Uniprot ID: 015537), gene editing factors (e.g., SaCas9, gRNAs), Optogenetics factors (e.g., ChrimsonR-tdTomato, Channelrhodopsin-2, Multi-Characteristic Opsin (MCO)), or functional fragments thereof, or any fusion protein comprising one or more of the same, or any combination thereof.

[0254] In some aspects, the therapeutic agent is an anti-TNF antibody (e.g., Adalimumab).

[0255] In some aspects, the therapeutic agent is a CR2-CR1 fusion protein. In some aspects, the CR2-CR1 fusion protein comprises a ligand-binding region of complement receptor 2 (CR2) and a ligand-binding region of complement receptor 1 (CR1).

[0256] In some aspects, the therapeutic agent is an inflammasome pathway inhibitor (e.g., tatCARD fusion proteins).

[0257] In some aspects, the therapeutic agent is a Vascular Endothelial Growth Factor (VEGF) inhibitor (e.g., FLT01, Aflibercept)

[0258] In some aspects, the therapeutic agent is an anti-VEGF antibody or antibody fragment thereof (e.g. bevacizumab, ranibizumab, brolucizumab).

[0259] In some aspects, the therapeutic agent is the Fibroblast Growth Factor 21.

[0260] In some aspects, the rAAV vectors can be comprised in a pharmaceutical composition, such as a pharmaceutical composition provided for herein.

[0261] In some aspects, the subject suffers from an ocular pathology (i.e., disease, disorder, condition, syndrome, or any combination thereof). In some aspects, the ocular pathology is selected from the group consisting of Achromatopsia, Behcet's Disease, Best's disease, Bietti's Crystalline Dystrophy, Blepharitis, Blepharospasm, Central Areolar Choroidal Dystrophy, Central Serous Chorioretinopathy, Choroideremia, Choroidal Melanoma, Coloboma, Corneal Conditions, Coat's Disease, Cone-Rod Dystrophy, Corneal Dystrophy, Cystoids Macular Edema, Diabetic Retinopathy, Doyne Honeycomb Retinal Dystrophy, Dry Eye, Fuch's Dystrophy, Glaucoma, Hypertensive Retinopathy, Idiopathic Intracranial Hypertension, Lattice Degeneration, Leber Congenital Amaurosis, Leber Hereditary Optic Neuropathy, Leber's Miliarly Aneurism, Macular Degeneration (including Age-Related Macular Degeneration, Juvenile Macular Degeneration, Atrophic Macular Degeneration), Macular Edema, Ocular Histoplasmosis (including Ocular Histoplasmosis Syndrome), Ocular Ischemic Syndrome, Papillophlebitis, Pink Eye, Polypoidal Choroidal Vasculopathy, Retinitis Pigmentosa, Retinoblastoma, Retinopathy of Prematurity, Retinoschisis (including Juvenile Retinoschisis), Sorsby's Disease, Stargardt Disease, Toxoplasmosis, Thyroid Eye Disease (TED), Usher Syndrome, and Uveitis (e.g., Noninfectious Uveitis), Vascular Occlusions, Inflammations (such as uveitis, choroiditis and retinistis), various Tumors (including neoplasms), or any combination thereof.

[0262] Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD) characterized by atrophy of retinal tissues and typically occurs in individuals over the age of 50 years. In some aspects, the ocular pathology for treatment is Geographic Atrophy (GA). In some aspects, the ocular pathology for treatment is Geographic Atrophy (GA) secondary to Age-related Macular Degeneration (AMD).

[0263] In some aspects, the delivery or administration is to the suprachoroidal space via the injection system (e.g., included in a kit) as described herein. In some aspects, the administration provides for delivery of the rAAV to an ocular tissue or cell.

[0264] In some aspects, the ocular tissue or ocular cell is located in the anterior segment of the eye, in the posterior segment of the eye, or a combination thereof. In some aspects, the ocular tissue or ocular cell is located in the anterior segment of the eye. In some aspects, the ocular tissue or ocular cell is located in the posterior segment of the eye.

[0265] In some aspects, the ocular tissue is selected from the group consisting of: cornea, sclera, choroid, Brunch’s membrane, pupil, iris, lens, optic disc, optic nerve, macula (e.g., central macula), retina, retinal pigmented epithelium (RPE, e.g., choroid-RPE), ciliary bodies, fovea, and any combination thereof. In some aspects, the ocular tissue is the optic disc. In some aspects, the ocular tissue is the optic nerve. In some aspects, the ocular tissue is the macula. In some aspects, the ocular tissue is the retina. In some aspects, the ocular tissue is the fovea. In some aspects, the ocular tissue is the retinal pigmented epithelium (RPE, e.g., choroid-RPE). In some aspects, the ocular tissue is the ciliary bodies.

[0266] In some aspects, the ocular tissue is the retina and the rAAV contacts a region of the retina selected from the inner limiting membrane, the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the middle limiting membrane, the outer plexiform layer, the outer nuclear layer, the external limiting membrane, the photoreceptor inner / outer segments, the retinal pigmented epithelium (RPE, e.g., choroid-RPE), or any combination thereof.

[0267] In some aspects, the ocular cell is a retinal cell. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, photoreceptor cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, a Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof. In some aspects, the photoreceptor cell is a rod cell, a cone cell, or any combination thereof. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell. In some aspects, the retinal cell is a ganglion cell. In some aspects, the retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell and / or the ganglion cell are in the macula. In some aspects, the retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell and / or the ganglion cell are in the central macula.

[0268] In some aspects, the administration is by suprachoroidal injection as a single dose. In some aspects, the single dose administration comprises a single injection per eye (at a single injection site). In some aspects, the single dose suprachoroidal administration comprises multiple injections. In some aspects, the single dose administration comprises multiple injections per eye. In some aspects, the multiple injections per eye are at a single injection site. In some aspects, the multiple injections per eye are at multiple injection sites. In some aspects, the suprachoroidal administration is by injection as multiple doses. In some aspects, each one of the multiple doses administration comprises multiple suprachoroidal injections. . In some aspects, the multiple suprachoroidal injections are at a single injection site. In some aspects, the multiple suprachoroidal injections are at different injection sites.

[0269] In some aspects, administration by suprachoroidal injection using the injection device (e.g., comprised in a kit) as described herein.

[0270] In some aspects, treating the subject according to the methods of the disclosure treats or ameliorates an ocular pathology in a subject suffering an ocular pathology. In some aspects, treating the subject according to the methods of the disclosure treats or reduces symptoms of an ocular pathology in a subject suffering an ocular pathology. In some aspects, the ocular pathology is selected from the group consisting of Achromatopsia, Behcet's Disease, Best's disease, Bietti's Crystalline Dystrophy, Blepharitis, Blepharospasm, Central Areolar Choroidal Dystrophy, Central Serous Chorioretinopathy, Choroideremia, Choroidal Melanoma, Coloboma, Corneal Conditions, Coat's Disease, Cone-Rod Dystrophy, Corneal Dystrophy, Cystoids Macular Edema, Diabetic Retinopathy, Doyne Honeycomb Retinal Dystrophy, Dry Eye, Fuch's Dystrophy, Glaucoma, Hypertensive Retinopathy, Idiopathic Intracranial Hypertension, Lattice Degeneration, Leber Congenital Amaurosis, Leber Hereditary Optic Neuropathy, Leber's Miliarly Aneurism, Macular Degeneration (including Age-Related Macular Degeneration, Juvenile Macular Degeneration, Atrophic Macular Degeneration), Macular Edema, Ocular Histoplasmosis (including Ocular Histoplasmosis Syndrome), Ocular Ischemic Syndrome, Papillophlebitis, Pink Eye, Polypoidal Choroidal Vasculopathy, Retinitis Pigmentosa, Retinoblastoma, Retinopathy of Prematurity, Retinoschisis (including Juvenile Retinoschi sis), Sorsby's Disease, Stargardt Disease, Toxoplasmosis, Thyroid Eye Disease (TED), Usher Syndrome, and Uveitis (e.g., Noninfectious Uveitis), Vascular Occlusions, Inflammations (such as uveitis, choroiditis and retinistis), various Tumors(including neoplasms), or any combination thereof. In some aspects, the ocular pathology for treatment is Geographic Atrophy (GA) secondary to Age-related Macular Degeneration (AMD).VIII. Suprachoroidal Administration

[0271] The suprachoroidal space (SCS) lies within the transition zone of the outermost border of the choroid and the internal edge of the sclera. The SCS is an expandable uveoscleral fluid channel transversing the posterior segment of the eye from the scleral spur anteriorly to the optic nerve posteriorly.

[0272] The SCS, being located between the sclera and the choroid, is a target for pharmacotherapy due to its proximity to the choroid, retinal pigment epithelium, and retina. The introduction of fluid into the space and / or mechanical cannulation can atraumatically separate the tissues, creating a true space between them. This space is often referred to as a "virtual space" as it remains "closed" due to intraocular pressure (IOP) and the presence of attaching fibers.

[0273] As disclosed herein, rAAV vectors and pharmaceutical compositions comprising rAAV vectors can be administered by suprachoroidal injection, e.g., using the injection device described herein, thereby resulting in an improved therapeutically effective outcome. In some aspects, the methods disclosed herein provide improved results over methods previously disclosed, e.g., microneedle injection methods, for delivery of rAAV to the SCS.

[0274] In some aspects, the administration by suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, can be for delivery of the rAAV vector to the ocular region, into an eye (e.g., one or both eyes). For example, administration by suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, can be for delivery of a rAAV vector to any ocular tissue or cell (e.g., to retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells and / or to ganglion cells).

[0275] In some aspects, the ocular tissue is located in the anterior segment of the eye, in the posterior segment of the eye, or a combination thereof. In some aspects, the ocular tissue is located in the anterior segment of the eye. In some aspects, the ocular tissue is located in the posterior segment of the eye.

[0276] In some aspects, the ocular tissue is selected from the group consisting of: cornea, sclera, choroid, Brunch’s membrane, pupil, iris, lens, optic disc, optic nerve, macula (e.g., central macula), retina, retinal pigmented epithelium (RPE, e.g., choroid-RPE), ciliary bodies, fovea, and any combination thereof. In some aspects, the ocular tissue is the optic disc. In some aspects, the ocular tissue is the optic nerve. In some aspects, the ocular tissue is the macula. In some aspects, the ocular tissue is the retina. In some aspects, the ocular tissue is the fovea. In some aspects, the ocular tissue is the retinal pigmented epithelium (RPE, e.g., choroid-RPE). In some aspects, the ocular tissue is the ciliary bodies.

[0277] In some aspects, the ocular tissue is the retina and administration is to a region of the retina selected from the group consisting of: the inner limiting membrane, the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the middle limiting membrane, the outer plexiform layer, the outer nuclear layer, the external limiting membrane, the photoreceptor inner / outer segments, the retinal pigmented epithelium (RPE, e.g., choroid-RPE), and any combination thereof. In some aspects, the ocular tissue is the retina and administration is to the retinal pigmented epithelium (RPE, e.g., choroid-RPE). In some aspects, the ocular tissue is the retina and administration is to the ganglion cell layer. In some aspects, the ocular tissue is the retina and administration is to the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and to the ganglion cell layer. In some aspects, the ocular tissue is the retina and administration is to the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and / or to the ganglion cell layer in the macula. In some aspects, the ocular tissue is the retina and administration is to the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and / or to the ganglion cell layer in the central macula.

[0278] In some aspects, the suprachoroidal administration delivers the rAAV to an ocular cell. In some aspects, the ocular cell is a retinal cell. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, photoreceptor cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof. In some aspects, the photoreceptor cell is a rod cell, a cone cell, or any combination thereof. In some aspects, the retinal cell is a retinal pigmented epithelial cell (e.g., a choroid-RPE cell). In some aspects, the retinal cell is a ganglion cell. In some aspects, the suprachoroidal administration delivers the rAAV to a retinal pigmented epithelial cell(e.g., a choroid-RPE cell) and to a ganglion cell. In some aspects, the retinal pigmented epithelial cell (e.g., a choroid-RPE cell) and / or the ganglion cell are in the macula. In some aspects, the retinal pigmented epithelial cell (e.g., a choroid-RPE cell) and / or the ganglion cell are in the central macula. In some aspects, administration is by suprachoroidal injection as a single dose. In some aspects, the single dose administration comprises a single injection per eye (at a single injection site). In some aspects, the single dose administration comprises multiple injections. In some aspects, the single dose administration comprises multiple injections per eye. In some aspects, the multiple injections per eye are at a single injection site. In some aspects, the multiple injections per eye are at multiple injection sites. In some aspects, administration is by suprachoroidal injection as multiple doses. In some aspects, each one of the multiple doses administration comprises multiple suprachoroidal injections. In some aspects, the multiple suprachoroidal injections are at a single injection site. In some aspects, the multiple suprachoroidal injections are at different injection sites.

[0279] In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter an ocular tissue of the subject.

[0280] In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the anterior segment of the eye, the posterior segment of an eye, or a combination thereof. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the anterior segment of an eye of the subject. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the posterior segment of an eye of the subject.

[0281] In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates therAAV vectors to enter the cornea, sclera, choroid, Brunch’s membrane, pupil, iris, lens, optic disc, optic nerve, macula (e.g., central macula), retina, retinal pigmented epithelium (RPE, e.g., choroid-RPE), ciliary bodies, fovea of the subject, or any combination thereof.

[0282] In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the optic disc. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the optic nerve. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the macula. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the retina. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the fovea. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the retinal pigmented epithelium (RPE, e.g., choroid-RPE). In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the ciliary bodies.

[0283] In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter a region of the retina selected from the group consisting of: the inner limiting membrane, the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the middle limiting membrane, the outerplexiform layer, the outer nuclear layer, the external limiting membrane, the photoreceptor inner / outer segments, the retinal pigmented epithelium (RPE, e.g., choroid- RPE), and any combination thereof. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the retinal pigmented epithelium (RPE, e.g., choroid-RPE). In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the ganglion cell layer. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and the ganglion cell layer. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and the ganglion cell layer in the macula. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and the ganglion cell layer in the central macula.

[0284] In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the optic nerve, the nerve fiber layer, choroid capillaries, or the peripheral retina. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter the optic nerve. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methodsdisclosed herein, in a way which facilitates the rAAV vectors to enter the nerve fiber layer. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to the peripheral retina. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to the choroid capillaries.

[0285] In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the cornea, sclera, choroid, Brunch’s membrane, pupil, iris, lens, optic disc, optic nerve, macula (e.g., central macula), retina, retinal pigmented epithelium (RPE, e.g., choroid-RPE), ciliary bodies, fovea of the subject, or any combination thereof. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the optic disc. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the optic nerve. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the macula. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the retina. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methodsdisclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the fovea. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the retinal pigmented epithelium (RPE, e.g., choroid-RPE). In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the ciliary bodies.

[0286] In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the optic nerve, the nerve fiber layer, choroid capillaries, or the peripheral retina. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the optic nerve. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the nerve fiber layer. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the peripheral retina. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the choroid capillaries.

[0287] In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device describedherein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in a region of the retina selected from the group consisting of: the inner limiting membrane, the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the middle limiting membrane, the outer plexiform layer, the outer nuclear layer, the external limiting membrane, the photoreceptor inner / outer segments, the retinal pigmented epithelium (RPE, e.g., choroid-RPE), and any combination thereof. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the retinal pigmented epithelium (RPE, e.g., choroid-RPE). In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) a cell of the subject located in the ganglion cell layer. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) cells of the subject located in the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and in the ganglion cell layer. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) cells of the subject located in the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and / or in the ganglion cell layer in the macula. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) cells of the subject located in the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and / or in the ganglion cell layer in the central macula.

[0288] In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates therAAV vectors to enter (e.g., transduce) a retinal cell. In some aspects, the retinal cell is a retinal pigmented epithelial cell (e.g., a choroid-RPE cell), photoreceptor cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, a Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof. In some aspects, the photoreceptor cell is a rod cell, a cone cell, or any combination thereof. In some aspects, the retinal cell is a retinal pigmented epithelial cell (e.g., a choroid-RPE cell). In some aspects, the retinal cell is a ganglion cell. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroi dally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cells and ganglion cells. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cells and / or ganglion cells in the macula. In some aspects, rAAV vectors, or of pharmaceutical compositions comprising rAAV vectors, can be suprachoroidally administered, by the injection device described herein and according to the methods disclosed herein, in a way which facilitates the rAAV vectors to enter (e.g., transduce) retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cells and / or ganglion cells in the central macula.

[0289] In some aspects, suprachoroi dal administration of rAAV vectors, by the injection device described herein and according to the methods disclosed herein, results in a rAAV vector transduction efficiency of at least 1% (e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the ocular cells. In some aspects, suprachoroi dal administration of rAAV vectors, by the injection device described herein and according to the methods disclosed herein, results in a rAAV vector transduction efficiency of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells and / or retinal ganglion (RG) cells. In some aspects, suprachoroi dal administration of rAAV vectors, by the injection device described herein and according to the methods disclosed herein, results in a rAAV vector transduction efficiency of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal ganglion (RG) cells. In some aspects,suprachoroidal administration of rAAV vectors, by the injection device described herein and according to the methods disclosed herein, results in a rAAV vector transduction efficiency of at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells. In some aspects, suprachoroidal administration of rAAV vectors, by the injection device described herein and according to the methods disclosed herein, results in a rAAV vector transduction efficiency of at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal ganglion (RG) cells and of at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells.

[0290] In some aspects, suprachoroidal administration of rAAV vectors, by the injection device described herein and according to the methods disclosed herein, does not results in readily rAAV vector transduction of photoreceptor cells. In some aspects, suprachoroidal administration of rAAV vectors, by the injection device described herein and according to the methods disclosed herein, results in a rAAV vector transduction efficiency of less 5% (e.g., less than 4%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or less than 0.5%) of the photoreceptor cells. In some aspects, the photoreceptor cells are a rod cells, cone cells, or any combination thereof.

[0291] In some aspects, the delivery vectors (e.g., AAV vectors) are introduced into the ocular tissue in vivo, e.g., by suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, which can be accomplished by perfusion (e.g., continuous injection), or by a single, discontinuous injection, or a combination thereof. In some aspects, the delivery vectors (e.g., AAV vectors) are introduced into the ocular tissue in vivo, e.g., by suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, by perfusion (e.g., continuous injection). In some aspects, the delivery vectors (e.g., AAV vectors) are introduced into the ocular tissue in vivo, e.g., by suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, by a single, discontinuous injection.

[0292] In some aspects suprachoroidal administration can be accomplished by insertion of a needle, as described with reference to the injection device (e.g., comprised in a kit) described herein.

[0293] In some aspects, suprachoroidal administration, by the injection device (e.g., comprised in a kit) described herein and according to the methods disclosed herein, provides advantages, e.g., because the vector is presented to the cells in a generally immune privileged microenvironment, the immunological and inflammatory reactions that are commonly observed as a result of the administration of transforming formulations and their adjuvants into blood and interstitial fluid can be avoided.

[0294] In some aspects, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, is performed in a chosen spatial direction relative to the target tissue portion.

[0295] In some aspects, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, is performed at a shallow angle (e.g., 0° to 30°, or 0° to 10°), or substantially tangential, to the target tissue portion (e.g., the sclera and / or the choroid) and / or to the interlayer (i.e., the choroid layers, or / and between choroid and retina of the eye) adjacent to the point of entry of the needle into the target tissue portion.

[0296] In some aspects, the needle of the injection device described herein is inserted at a shallow angle. In some aspects, the shallow angle is 0° to 30° relative to the sclera portion of the eye. In some aspects, the shallow angle is 0° to 10° relative to the sclera portion of the eye. In some aspects, the insertion of the needle penetrates the sclera at an approximately tangential angle. In some aspects, the needle is extended about 1 mm to 5 mm in length within the sclera layer before penetrating the choroid. In some aspects, the needle is extended about 2 mm to 4 mm in length within the sclera layer before penetrating the choroid.

[0297] In some aspects, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, is performed by creating, prior to injection, a directional spaced passage into and through the outer most or anterior organ layer (i.e., the sclera optionally with the conjunctiva) and an interlayer region (i.e., within choroid layers, or / and between choroid and retina of the eye). This artificial passage effectively contains, channels, and disperses fluids therefrom to distant and / or large areas throughout the suprachoroidal space.

[0298] In some aspect, in the methods disclosed herein suprachoroidal delivery is achieved by using the injection device described herein. In some aspect, the injection device described herein creates a channel through the choroid such that thepharmaceutical composition may be administered to the suprachoroidal space. In some aspect, the injection device described herein creates a channel through the choroid via blunt dissection of the sclera and choroid. In some aspect, the channel through the choroid is created with a tissue separator. In some aspect, the tissue separator is a nonsharp tissue separator. In some aspect, the tissue separator is within the lumen of the needle. In some aspect, the tissue separator is positioned proximal to distal tip of the needle during insertion. In some aspect, the distal tip of the tissue separator penetrates into the sclera portion and into the choroid to create the channel through the choroid. In some aspect, the tissue separator causes blunt dissection of the sclera and choroid thereby creating the channel through the choroid.

[0299] In some aspect, creating a channel through the choroid allows distribution to the posterior segment of the eye. In some aspect, creating a channel through the choroid allows rapid and extensive distribution to the posterior segment of the eye. In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows distribution to the posterior segment. In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows rapid and extensive distribution to the posterior segment.

[0300] In some aspect, creating a channel through the choroid allows distribution to the posterior segment of the eye and macula (e.g., central macula). In some aspect, creating a channel through the choroid allows rapid and extensive distribution to the posterior segment of the eye and macula (e.g., central macula). In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows distribution to the posterior segment of the eye and macula (e.g., central macula). In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows rapid and extensive distribution to the posterior segment of the eye and macula (e.g., central macula).

[0301] In some aspect, creating a channel through the choroid allows distribution to the retina, to the RPE (e.g., choroid RPE), to the ganglion cells, and to the ciliary bodies. In some aspect, creating a channel through the choroid allows rapid and extensive distribution to the retina, to the RPE (e.g., choroid RPE), to the ganglion cells, and to the ciliary bodies. In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows distribution to the retina, to the RPE (e.g., choroid RPE), to the ganglion cells, and to the ciliary bodies. In some aspect, creating a channel throughthe choroid via blunt dissection of the sclera and choroid allows rapid and extensive distribution to the retina, to the RPE (e.g., choroid RPE), to the ganglion cells, and to the ciliary bodies.

[0302] In some aspect, creating a channel through the choroid allows distribution to the retina. In some aspect, creating a channel through the choroid allows rapid and extensive distribution to the retina. In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows distribution to the retina. In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows rapid and extensive distribution to the retina.

[0303] In some aspect, creating a channel through the choroid allows distribution to the RPE (e.g., choroid RPE). In some aspect, creating a channel through the choroid allows rapid and extensive distribution to the RPE (e.g., choroid RPE). In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows distribution to the RPE (e.g., choroid RPE). In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows rapid and extensive distribution to the RPE (e.g., choroid RPE).

[0304] In some aspect, creating a channel through the choroid allows distribution to the ganglion cell layer. In some aspect, creating a channel through the choroid allows rapid and extensive distribution to the ganglion cell layer. In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows distribution to the ganglion cell layer. In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows rapid and extensive distribution to the ganglion cell layer.

[0305] In some aspect, creating a channel through the choroid allows distribution to the RPE (e.g., choroid RPE) and ganglion cell layer. In some aspect, creating a channel through the choroid allows rapid and extensive distribution to the RPE (e.g., choroid RPE) and ganglion cell layer. In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows distribution to the RPE (e.g., choroid RPE) and ganglion cell layer. In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows rapid and extensive distribution to the RPE (e.g., choroid RPE) and ganglion cell layer.

[0306] In some aspect, creating a channel through the choroid allows distribution to the ciliary bodies. In some aspect, creating a channel through the choroid allows rapid andextensive distribution to the ciliary bodies. In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows distribution to the ciliary bodies. In some aspect, creating a channel through the choroid via blunt dissection of the sclera and choroid allows rapid and extensive distribution to the ciliary bodies.

[0307] In some aspect, the tissue separator causes blunt dissection of the sclera and choroid thereby creating the channel through the choroid and the pharmaceutical composition flows through the channel in the posterior segment of the eye.

[0308] In some aspect, the tissue separator causes blunt dissection of the sclera and choroid thereby creating the channel through the choroid and the pharmaceutical composition flows through the channel in the posterior segment of the eye and macula (e.g., central macula).

[0309] In some aspect, the tissue separator causes blunt dissection of the sclera and choroid thereby creating the channel through the choroid and the pharmaceutical composition flows through the channel in the retina, RPE (e.g., choroid RPE), ganglion cells, and ciliary bodies.

[0310] In some aspect, the tissue separator causes blunt dissection of the sclera and choroid thereby creating the channel through the choroid and the pharmaceutical composition flows through the channel in the RPE (e.g., choroid RPE).

[0311] In some aspect, the tissue separator causes blunt dissection of the sclera and choroid thereby creating the channel through the choroid and the pharmaceutical composition flows through the ganglion cell layer.

[0312] In some aspect, the tissue separator causes blunt dissection of the sclera and choroid thereby creating the channel through the choroid and the pharmaceutical composition flows through the channel in the RPE (e.g., choroid RPE) and ganglion cell layer.

[0313] In some aspect, the tissue separator causes blunt dissection of the sclera and choroid thereby creating the channel through the choroid and the pharmaceutical composition flows through the channel in ciliary bodies.

[0314] In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of the posterior segment of the eye. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in circumferential transduction of the posterior segment of the eye. In some aspect, the circumferentialtransduction occurs at the posterior pole. In some aspect, the circumferential transduction occurs past the posterior pole. In some aspect, the circumferential transduction occurs past the posterior pole and across layers of the retina beyond the RPE. In some aspect, the circumferential transduction occurs from the injection site, around to the posterior pole, past the macula, and into the nasal retina.

[0315] In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the retina, the RPE (e.g., choroid RPE), the ganglion cell layer, the ciliary bodies, choroid capillaries, the optic nerve, nerve fiber layer, peripheral retina, the macula, and the fovea. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the retina. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the RPE (e.g., choroid RPE). In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the ganglion cell layer. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the RPE (e.g., choroid RPE) and ganglion cell layer. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the RPE (e.g., choroid RPE) and ganglion cell layer in the macula. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the RPE (e.g., choroid RPE) and ganglion cell layer in the central macula. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the ciliary bodies. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the macula. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the fovea.

[0316] In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located inthe choroid capillaries. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the optic nerve. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the nerve fiber layer. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the peripheral retina.

[0317] In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, does not result in readily transduction of photoreceptor cells. In some aspect, the photoreceptor cells are a rod cells, cone cells, or any combination thereof.

[0318] In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the retina. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of the retina from the RPE through ganglion cells. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, achieves transduction of RPE cells and of other layers of the retina including transduction through ganglion cells. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the macula. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the central macula. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the fovea. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the ciliary bodies. In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of cells located in the RPE.

[0319] In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, results in transduction of the retina layers through the posterior pole superior to the results reported in literature with other injection devices. In some aspect, suprachoroidal injection, by the injection device described hereinand according to the methods disclosed herein, results in transduction of the retina layers through the posterior pole superior to the results reported in literature with microneedles.

[0320] In some aspect, transduction of retinal pigmented epithelium (RPE, e.g., choroid- RPE) and retinal ganglion cells (RGC) (the layers above and below the photoreceptors) is advantageous, e.g., because it allows to avoid overburdening the photoreceptors when transduction of photoreceptors is not necessary for the therapy.

[0321] In some aspect, rAVV vectors and of pharmaceutical compositions comprising rAVV vectors are compatible with the injection device described herein. In some aspect, extended contact with the injection device described herein, does not alter the biological properties of the rAVV vectors and pharmaceutical compositions comprising rAVV vectors.

[0322] In some aspect, extended contact with the injection device described herein, does not affect vector genome titers and / or potency of rAVV vectors and of pharmaceutical compositions comprising rAVV vectors.

[0323] In some aspect, after extended contact the injection device described herein, vector genome titers of pharmaceutical compositions comprising rAVV vectors are within + / - 35%, + / - 30%, + / - 25%, + / - 20%, + / - 15%, + / - 10%, + / - 5%, or + / - 1%, of preexposure vector genome titers. In some aspect, after extended contact the injection device described herein, potency of the rAW vectors or pharmaceutical compositions comprising rAVV vectors, are within + / - 40%, + / - 35%, + / - 30%, + / - 25%, + / - 20%, + / - 15%, + / - 10%, + / - 5%, or + / - 1%, of pre-exposure potency.

[0324] In some aspects, suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, comprises a volume of an effective amount of an rAAV vector or of a pharmaceutical compositions comprising a rAAV vector that is reduced compared to a volume of an effective amount of a rAAV vector or of a pharmaceutical compositions comprising the rAAV vector administered via other methods such as intravenous (IV), intralymphatic, subconjuntival, or peribulbar injection.

[0325] In some aspects, suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, comprises a volume of an effective amount of an rAAV vector or of a pharmaceutical compositions comprising a rAAV vector that is reduced compared to a volume of an effective amount of a rAAV vector or of a pharmaceutical compositions comprising the rAAV vector administered via an injection device that is not the injection device described herein.

[0326] In some aspects, suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, comprises a volume of an effective amount of an rAAV vector or of a pharmaceutical compositions comprising a rAAV vector that is increased compared to a volume of an effective amount of a rAAV vector or of a pharmaceutical compositions comprising the rAAV vector administered via a microneedle injection device.

[0327] In some aspect, suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, allows single injections of rAAV volumes higher than the volumes that can be injected with a microneedle injection device.

[0328] In some aspects, the volume of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered suprachoroidally, by the injection device described herein and according to the methods disclosed herein, is about 0.05 mL to about 0.50 mL, about 0.10 mL to about 0.50 mL, about 0.11 mL to about 0.50 mL, about 0.12 mL to about 0.50 mL, about 0.13 mL to about 0.50 mL, about 0.14 mL to about 0.50 mL, about 0.15 mL to about 0.50 mL, about 0.16 mL to about 0.50 mL, about 0.17 mL to about 0.50 mL, about 0.18 mL to about 0.50 mL, about 0.19 mL to about 0.50 mL, about 0.20 mL to about 0.50 mL, about 0.25 mL to about 0.50 mL, about 0.30 mL to about 0.50 mL, about 0.35 mL to about 0.50 mL, about 0.40 mL to about 0.50 mL, about 0.45 mL to about 0.50 mL, about 0.01 mL to about 0.30 mL, about 0.02 mL to about 0.30 mL, about 0.03 mL to about 0.30 mL, about 0.04 mL to about 0.30 mL, about 0.05 mL to about 0.30 mL, about 0.06 mL to about 0.30 mL, about 0.07 mL to about 0.30 mL, about 0.08 mL to about 0.30 mL, about 0.09 mL to about 0.30 mL, about 0.10 mL to about 0.30 mL, about 0.11 mL to about 0.30 mL, about 0.12 mL to about 0.30 mL, about 0.13 mL to about 0.30 mL, about 0.14 mL to about 0.30 mL, about 0.15 mL to about 0.30 mL, about 0.16 mL to about 0.30 mL, about 0.17 mL to about 0.30 mL, about 0.18 mL to about 0.30 mL, about 0.19 mL to about 0.30 mL, about 0.20 mL to about 0.30 mL, about 0.21 mL to about 0.30 mL, about 0.22 mL to about 0.30 mL, about 0.23 mL to about 0.30 mL, about 0.24 mL to about 0.30 mL, about 0.25 mL to about 0.30 mL, about 0.26 mL to about 0.30 mL, about 0.27 mL to about 0.30 mL, about 0.28 mL to about 0.30 mL, or about 0.29 mL to about 0.30 mL.

[0329] In some aspects, the volume of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered suprachoroidally by the injection devicedescribed herein and according to the methods disclosed herein is about 100 pL to about 300 pL.

[0330] In some aspects, the volume of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered suprachoroidally, by the injection device described herein and according to the methods disclosed herein, is greater than 100 pl, greater than 105 pl, greater than 110 pl, greater than 115 pl, greater than 120 pl, greater than 125 pl, greater than 130 pl, greater than 135 pl, greater than 140 pl, greater than 145 pl, greater than 150 pl, greater than 155 pl, greater than 160 pl, greater than 165 pl, greater than 170 pl, greater than 175 pl, greater than 180 pl, greater than 185 pl, greater than 190 pl, greater than 195 pl, or greater than 200 pl, and optionally, the predetermined volume is less than 500 pl, less than 505 pl, less than 510 pl, less than 515 pl, less than 520 pl, less than 525 pl, less than 530 pl, less than 535 pl, less than 540 pl, less than 545 pl, or less than 550 pl.

[0331] In some aspects the total injection volume would be administered by a single injection or multiple injections. In some aspects, the multiple injections is two injections. In some aspects, the administration comprises a first injection (e.g., about 100 pL to 250 pL) and a second injection (e.g., about 100 pL to 250 pL).

[0332] In some aspects, the delivery or administration comprises a pharmaceutical composition comprising at least 1E10 vector genomes (vg), of the rAAV vector.

[0333] In some aspects, the delivery or administration comprises a pharmaceutical composition comprising at least 1E10 vg and at most 1E13 vg of the rAAV vector (i.e., at least 1E10 vg to at most 1E13 vg of the rAAV vector).

[0334] In some aspects, the delivery or administration comprises a pharmaceutical composition comprising at least 1E10 vg and at most 1E12 vg of the rAAV vector (i.e., at least 1E10 vg to at most 1E12 vg of the rAAV vector).

[0335] In some aspects, the delivery or administration comprises a pharmaceutical composition comprising about 1E10 vg, about 2E10 vg, about 3E10 vg, about 4E10 vg, about 5E10 vg, about 6E10 vg, about 7E10 vg, about 8E10 vg, about 9E10 vg, about 1E11 vg, about 2E11 vg, about 3E11 vg, about 4E11 vg, about 5E11 vg, about 6E11 vg, about 7E11 vg, about 8E11 vg, about 9E11 vg, or about 1E12 vg for the rAAV vector.

[0336] In some aspects, the total injection volume of about 100 pL to about 300 pL is administered in multiple injections (e.g., two injections to one eye). In some aspects, thefirst injection is about 50 pL to about 150 pL (e.g., about 50 pL, about 100 pL, or about 150 pL) and the second injection is about 50 pL to about 150 pL (e.g., about 50 pL, about 100 pL, or about 150 pL).

[0337] In some aspects, the volume of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered suprachoroidally, by the injection device described herein and according to the methods disclosed herein, is 100 uL to 250 uL per injection (e.g., two injections per eye), 100 uL to 225 uL per injection (e.g., two injections per eye), 100 uL to 200 uL per injection (e.g., two injections per eye), 100 uL to 175 uL per injection (e.g., two injections per eye), 100 uL to 150 uL per injection (e.g., two injections per eye), 115 uL to 250 uL per injection (e.g., two injections per eye), 115 uL to 225 uL per injection (e.g., two injections per eye), 115 uL to 200 uL per injection (e.g., two injections per eye), 115 uL to 175 uL per injection (e.g., two injections per eye), or 115 uL to 150 uL per injection (e.g., two injections per eye). .

[0338] In some aspects, the volume of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered suprachoroidally, by the injection device described herein and according to the methods disclosed herein, is about 0.05 mL to about 1 mL, about 0.1 mL to about 0.5 mL, about 0.1 mL to about 0.3 mL, or about 0.1 mL to about 0.2 mL.

[0339] In some aspects, the volume of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered suprachoroidally, by the injection device described herein and according to the methods disclosed herein, is greater than 0.1 mL up to about 0.5 mL, greater than 0.1 mL up to about 0.3 mL, or greater than 0.1 mL up to about 0.2 mL.

[0340] In some aspects, the volume of the AAV vector or pharmaceutical compositions comprising the AAV vector administered suprachoroidally, by the injection device described herein and according to the methods disclosed herein, to the subject is about 0.05 mL to about 0.5 mL.

[0341] In some aspects, the suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, comprises a volume of about 0.05 mL to about 1 mL of the pharmaceutical composition per suprachoroidal injection.

[0342] In some aspects, the suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, comprises a volume ofabout 0.1 mL to about 0.5 mL of the pharmaceutical composition per suprachoroidal injection.

[0343] In some aspects, the suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, comprises a volume of about 0.1 mL to about 0.3 mL of the pharmaceutical composition per suprachoroidal injection.

[0344] In some aspects, the suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, comprises a volume of about 0.1 mL to about 0.2 mL of the pharmaceutical composition per suprachoroidal injection.

[0345] In some aspects, the suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, comprises a volume of about 0.25 mL to about 0.4 mL of the pharmaceutical composition per suprachoroidal injection.

[0346] In some aspects, the suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, comprises a volume of about 0.2 mL to about 0.3 mL of the pharmaceutical composition per suprachoroidal injection.

[0347] In some aspects, the volume of the rAAV vector or pharmaceutical compositions comprising the AAV vector administered via suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, is at least %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, or at least 95%, reduced compared to the volume of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered via other methods such as intravenous (IV), intralymphatic, subconjuntival, or peribulbar injection.

[0348] In some aspects, the volume of the rAAV vector or pharmaceutical compositions comprising the AAV vector administered via suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, atleast 90%, or at least 95%, reduced compared to the volume of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered via an injection device that is not the injection device described in the kit section herein.

[0349] In some aspects, the volume of the rAAV vector or pharmaceutical compositions comprising the AAV vector administered via suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, or at least 95%, increased compared to the volume of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered via an injection device that is not the injection device described in the kit section herein.

[0350] In some aspects, at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered to the subject via suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, contacts an ocular tissue. In some aspects, the ocular tissue is selected from the group consisting of: cornea, sclera, choroid, Brunch’s membrane, pupil, iris, lens, optic disc, optic nerve, macula (e.g., central macula), retina, retinal pigmented epithelium (RPE, e.g., choroid-RPE), ciliary bodies, the nerve fiber layer, choroid capillaries, the peripheral retina, and any combination thereof. In some aspects, the ocular tissue is the optic disc. In some aspects, the ocular tissue is the optic nerve. In some aspects, the ocular tissue is the nerve fiber layer. In some aspects, the ocular tissue is the peripheral retina. In some aspects, the ocular tissue is the choroid capillaries. In some aspects, the ocular tissue is the macula. In some aspects, the ocular tissue is the retina. In some aspects, the ocular tissue is the fovea. In some aspects, the ocular tissue is the retinal pigmented epithelium (RPE, e.g., choroid-RPE). In some aspects, the ocular tissue is the ciliary bodies. In some aspects, the ocular tissue is a region of the retina selected from the group consisting of: the inner limiting membrane, the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the middle limiting membrane, the outer plexiform layer, the outer nuclear layer,the external limiting membrane, the photoreceptor inner / outer segment, the retinal pigmented epithelium (RPE, e.g., choroid-RPE), and any combination thereof.

[0351] In some aspects, at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered to the subject via suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, contacts an ocular cell. In some aspects, the ocular cell is a retinal cell. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, photoreceptor cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, a Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof. In some aspects, the photoreceptor cell is a rod cell, a cone cell, or any combination thereof.

[0352] In some aspects, at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered to the subject via suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, transduces an ocular cell. In some aspects, the ocular cell is a retinal cell. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, photoreceptor cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, a Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof. In some aspects, the photoreceptor cell is a rod cell, a cone cell, or any combination thereof.

[0353] In some aspects, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered via suprachoroidal injection, by the injection device described herein and according to themethods disclosed herein, to the subject does not contact and / or transduce a tissue which is not an ocular tissue.

[0354] In some aspects, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the rAAV vector or pharmaceutical compositions comprising the rAAV vector administered via suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, to the subject does not contact and / or transduce a cell which is not an ocular cell.

[0355] In some aspects, the administration via suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, comprises a dose within the range of about 1 x 108vg to about 5 x 1013vg, e.g., about 1 x 108vg to about 1 x 1013vg. In some aspects, the administration comprises a dose within the range of about 1 x 108vg to about 5 x 1013vg, about 1 x 108vg to about 1 x 1013vg, about 1 x 109vg to about 1 x 1013vg, about 1 x 1010vg to about 1 x 1013vg, about 1 x 1011vg to about 1 x 1013vg, about 1 x 1012vg to about 1 x 1013vg, about 1 x 108vg to about 1 x 1012vg, about 1 x 108vg to about 1 x 1011vg, about 1 x 108vg to about 1 x 1010vg, about 1 x 108vg to about 1 x 109vg, about 1 x 1010vg to about 1 x 1012vg, about 1 x 109vg to about 1 x 1012vg, or about 1 x 1010vg to about 1 x 1011vg.

[0356] In some aspects, the administration via suprachoroidal injection, by the injection device described herein and according to the methods disclosed herein, comprises a dose within the range of about 1 x 106vg / kg to about 1 x 1011vg / kg. In some aspects, the administration comprises a dose within the range of about 1 x 106vg / kg to about 1 x 1011vg / kg, about 1 x 107vg / kg to about 1 x 1011vg / kg, about 1 x 108vg / kg to about 1 x 1011vg / kg, about 1 x 109vg / kg to about 1 x 1011vg / kg, about 1 x 1010vg / kg to about 1 x 1011vg / kg, about 1 x 106vg / kg to about 1 x 1010vg / kg, about 1 x 106vg / kg to about 1 x 109vg / kg, about 1 x 106vg / kg to about 1 x 108vg / kg, about 1 x 106vg / kg to about 1 x 107vg / kg, about 1 x 108vg / kg to about 1 x 1010vg / kg, about 1 x 107vg / kg to about 1 x 1010vg / kg, or about 1 x 108vg / kg to about 1 x 109vg / kg.

[0357] The amount of nucleic acid to transform a sufficient number of cells and provide for expression of therapeutic levels of the therapeutic agent (e.g., a protein, such as an antibody or antigen binding fragment thereof) can be assessed using an animal model(e.g., a rodent (mouse or rat) or other mammalian animal model, such as a non-human primate (NHP)) to assess factors such as the efficiency of transformation, the levels of expression achieved, the susceptibility of the targeted cells to transformation, and the amounts of vector and / or nucleic acid required to transform target cells.

[0358] The precise amount of vector and / or nucleic acid administered will vary greatly according to a number of factors including the susceptibility of the target cells to transformation, the size and weight of the subject, the levels of protein expression desired, and the condition to be treated.

[0359] The rAAV vector or pharmaceutical compositions comprising the rAAV vector can be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for liquid solution or suspension in a liquid solution.

[0360] In some aspects, suprachoroidal administration, by the injection device described herein and according to the methods disclosed herein, provides an rAAV vector transduction efficiency of at least 1% (e.g., 1% to 95%, 5% to 95%, 10% to 95%, 15% to 95%, 20% to 95%, 25% to 95%, 30% to 95%, 40% to 95%, or 50% to 95%) of the target ocular cells. In some aspects, the ocular cell is a retinal cell. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, photoreceptor cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, a Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof. In some aspects, the photoreceptor cell is a rod cell, a cone cell, or any combination thereof.

[0361] In some aspects, the rAAV vector transduction efficiency is at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the target ocular cells. In some aspects, the ocular cell is a retinal cell. In some aspects, the retinal cell is a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, photoreceptor cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, a Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof. In some aspects, the photoreceptor cell is a rod cell, a cone cell, or any combination thereof.

[0362] The administration may be carried out via one or more kits as described herein.

[0363] In some aspects the suprachoroidal injection disclosed herein can be exemplified by the methods disclosed below:

[0364] Example 1. A method of delivering a rAAV vector or a pharmaceutical composition comprising a rAAV vector to a back of an eye, comprising:a. injecting tangentially said rAAV vector or pharmaceutical composition comprising the rAAV vector into an ocular layer in said eye at a flow rate of from 5 pL / sec to 100 pL / sec (e.g., about 5 pL / sec, 10 pL / sec, 15 pL / sec, 20 pL / sec, 25 pL / sec, 30 pL / sec, 35 pL / sec, 40 pL / sec, 45 pL / sec, 50 pL / sec, 55 pL / sec, 60 pL / sec, 65 pL / sec, 70 pL / sec, 75 pL / sec, 80 pL / sec, 85 pL / sec, 90 pL / sec, 95 pL / sec, 100 pL / sec)b. maintaining a level of intra-ocular pressure (IOP) of said eye within a normal range within 30 minutes after said injecting.

[0365] Example 2. The method according to example 1, further comprising inserting tangentially a needle cannula of no more than 2 mm into an ocular layer in said eye before said injecting.

[0366] Example 3. The method according to example 1 or example 2, further comprising inserting tangentially a needle cannula a length of from about 1.5 mm to about 2mm into an ocular layer in said eye before said injecting.

[0367] Example 4. The method according to any one of examples 1-3, further comprising extending a tissue separator from within said needle cannula into said ocular layer a distance of from 0.3 mm to 10 mm before said injecting.

[0368] Example 5. The method according to any one of examples 1-4, further comprising requesting from said patient to look in a certain direction before said inserting.

[0369] Example 6. The method according to any one of examples 1-5, wherein said inserting is performed at a quadrant of the eye exposed by said patient looking at said certain direction.

[0370] Example 7. The method according to any one of examples 1-6, wherein said certain direction is up in relation to said patient.

[0371] Example 8. The method according to any one of examples 1-7, wherein said normal range of IOP is from 11 mmHg to 21 mmHg.

[0372] Example 9. The method according to any one of examples 1-8, wherein said injecting is painless or almost painless under topical anesthetic.

[0373] Example 10. The method according to any one of examples 1-9, further comprising assessing a correct position of said needle cannula after said inserting.

[0374] Example 11. The method according to any one of examples 1-10, wherein said assessing comprises lifting said needle cannula parallel to a longitudinal axis of said needle cannula and away from said eye.

[0375] Example 12. The method according to any one of examples 1-11, wherein said inserting is performed at an angle from 0° to 45° in relation to the surface of the eye.

[0376] Example 13. The method according to any one of examples 1-12, wherein said extending generates an artificial passage configured for containing, channeling and dispersing said rAAV vector or pharmaceutical composition comprising the rAAV vector to distant and / or large areas throughout the interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region of said eye.

[0377] Example 14. A method of painlessly or almost painlessly delivering rAAV vector or pharmaceutical composition comprising the rAAV vector to a back of an eye under topical anesthetic, comprising:a. inserting tangentially a needle cannula of no more than 2mm into an ocular layer in said eye;b. extending a tissue separator from within said sharp tip into said ocular layer a distance of from 0.3 mm to 10 mm ;c. injecting said rAAV vector or pharmaceutical composition comprising the rAAV vector into an ocular layer in said eye at a flow of from lOpl / sec to lOOpl / sec.

[0378] Example 15. The method according to example 14, wherein said inserting tangentially a needle cannula is to a length of from about 1.5 mm to about 2 mm into an ocular layer in said eye before said injecting.

[0379] Example 16. The method according to example 14 or example 15, further comprising maintaining a level of intra-ocular pressure (IOP) of said eye within a normal range within 30 minutes after said injecting.

[0380] Example 17. The method according to any one of examples 14-16, wherein said normal range of IOP is from 11 mmHg to 21 mmHg.

[0381] Example 18. The method according to any one of examples 14-17, further comprising requesting from said patient to look to a certain direction before said inserting.

[0382] Example 19. The method according to any one of examples 14-18, wherein said inserting is performed at a quadrant of the eye exposed by said patient looking at said certain direction.

[0383] Example 20. The method according to any one of examples 14-19, wherein said certain direction is up in relation to said patient.

[0384] Example 21. The method according to any one of examples 14-20, further comprising assessing a correct position of said needle cannula after said inserting.

[0385] Example 22. The method according to any one of examples 14-21, wherein said assessing comprises lifting said needle cannula parallel to a longitudinal axis of said needle cannula and away from said eye.

[0386] Example 23. The method according to any one of examples 14-22, wherein said inserting is performed at an angle from 0° to 45° in relation to the surface of the eye.

[0387] Example 24. The method according to any one of examples 14-23, wherein said extending generates an artificial passage configured for containing, channeling and dispersing said rAAV vector or pharmaceutical composition comprising the rAAV vector to distant and / or large areas throughout the interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region of said eye.

[0388] Example 25. A method of delivering a rAAV vector or a pharmaceutical composition comprising a rAAV vector to a back of an eye of a patient, comprising: a. requesting from said patient to look to a certain direction;b. inserting tangentially a needle cannula to the sclera of the patient;c. injecting tangentially said rAAV vector or pharmaceutical composition comprising the rAAV vector into an ocular layer in said eye.

[0389] Example 26. The method according to example 25, wherein said inserting is performed at a quadrant of the eye exposed by said patient looking at said certain direction.

[0390] Example 27. The method according to example 25 or example 26, wherein said certain direction is up in relation to said patient.

[0391] Example 28. The method according to any one of examples 25-27, wherein said injecting tangentially said rAAV vector or pharmaceutical composition comprising the rAAV vector into said ocular layer is characterized by injecting at a flow of from 5pL / sec to lOOpL / sec or lOpL / sec to lOOpL / sec.

[0392] Example 29. The method according to any one of examples 25-28, further comprising maintaining a level of intra-ocular pressure (IOP) of said eye within a normal range within 30 minutes after said injecting.

[0393] Example 30. The method according to any one of examples 25-29, wherein said normal range of IOP is from 11 mmHg to 21 mmHg.

[0394] Example 31. The method according to any one of examples 25-30, wherein said inserting tangentially comprises inserting said needle cannula no more than 2mm into said sclera.

[0395] Example 32. The method according to any one of examples 25-31, wherein said inserting tangentially comprises inserting tangentially said needle cannula a length of from about 1.5 mm to about 2mm into an ocular layer in said eye before said injecting.

[0396] Example 33. The method according to any one of examples 25-32, further comprising extending a tissue separator from within said needle cannula into said sclera a distance of from 0.3 mm to 10 mm before said injecting.

[0397] Example 34. The method according to any one of examples 25-33, wherein said injecting is painless or almost painless under topical anesthetic.

[0398] Example 35. The method according to any one of examples 25-34, further comprising assessing a correct position of said needle cannula after said inserting.

[0399] Example 36. The method according to any one of examples 25-35, wherein said assessing comprises lifting said needle cannula parallel to a longitudinal axis of said needle cannula and away from said eye.

[0400] Example 37. The method according to any one of examples 25-36, wherein said inserting is performed at an angle from 0° to 45° in relation to the surface of the eye.

[0401] Example 38. The method according to any one of examples 25-37, wherein said extending generates an artificial passage configured for containing, channeling and dispersing said rAAV vector or pharmaceutical composition comprising the rAAV vector to distant and / or large areas throughout the interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region of said eye.

[0402] Example 39. A method for ensuring correct positioning of an injector for suprachoroidal delivery of materials, comprising:a. tangentially inserting into an eye a needle cannula having a length of no more than 2 mm;b. extending a tissue separator a distance of from 1 mm to 5 mm from a distal end of said needle canula.

[0403] Example 40. The method according to example 39, further comprising providing a suprachoroidal delivery system comprising said needle cannula having a length of no more than 2 mm.

[0404] Example 41. The method according to example 39 or example 40, wherein said needle cannula comprises a length of from 1.5 mm to 2 mm.

[0405] Example 42. The method according to any one of examples 39-41, wherein said tangentially inserting comprises tangentially inserting until reaching a sleeve stopper.

[0406] Example 43. The method according to any one of examples 39-42, wherein said suprachoroidal delivery system comprises a sleeve stopper.

[0407] Example 44. The method according to any one of examples 39-43, wherein said inserting is performed at an angle from 0° to 45° in relation to the surface of the eye.

[0408] Example 45. The method according to any one of examples 39-44, wherein said tissue separator is flexible.

[0409] Example 46. The method according to any one of examples 39-45, wherein said inserting comprises reaching a sclera layer.

[0410] Example 47. The method according to any one of examples 39-46, wherein said inserting comprises not reaching a choroid layer.

[0411] Example 48. The method according to any one of examples 39-47, wherein said extending comprises reaching a choroid layer.

[0412] Example 49. The method according to any one of examples 39-48, wherein said extending comprises not reaching a retina layer.

[0413] Example 50. The method according to any one of examples 39-49, wherein said extending generates an artificial passage configured for containing, channeling and dispersing said rAAV vector or pharmaceutical composition comprising the rAAV vector to distant and / or large areas throughout the interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region of said eye.

[0414] Example 51. A method of providing a rapid posterior flow suprachoroidal delivery of rAAV vector or pharmaceutical composition comprising the rAAV vector, comprising:a. tangentially inserting into an eye a needle cannula having a length of no more than 2 mm;b. extending a tissue separator a distance of from 1 mm to 5 mm from a distal end of said needle canula;c. injecting said rAAV vector or pharmaceutical composition comprising the rAAV vector into an ocular layer in said eye at a flow of from lOpl / sec to lOOpl / sec.

[0415] Example 52. The method according to example 51, further comprising providing a suprachoroidal delivery system comprising said needle cannula having a length of no more than 2 mm.

[0416] Example 53. The method according to example 51 or example 52, wherein said needle cannula comprises a length of from 1.5 mm to 2 mm.

[0417] Example 54. The method according to any one of examples 51-53, wherein said tangentially inserting comprises tangentially inserting until reaching a sleeve stopper.

[0418] Example 55. The method according to any one of examples 51-54, wherein said suprachoroidal delivery system comprises a sleeve stopper.

[0419] Example 56. The method according to any one of examples 51-55, wherein said inserting is performed at an angle from 0° to 45° in relation to the surface of the eye.

[0420] Example 57. The method according to any one of examples 51-56, wherein said tissue separator is flexible.

[0421] Example 58. The method according to any one of examples 51-57, wherein said inserting comprises reaching a sclera layer.

[0422] Example 59. The method according to any one of examples 51-58, wherein said inserting comprises not reaching a choroid layer.

[0423] Example 60. The method according to any one of examples 51-59, wherein said extending comprises reaching a choroid layer.

[0424] Example 61. The method according to any one of examples 51 -60, wherein said extending comprises not reaching a retina layer.

[0425] Example 62. The method according to any one of examples 51-61, wherein said extending generates an artificial passage configured for containing, channeling and dispersing said rAAV vector or pharmaceutical composition comprising the rAAV vector to distant and / or large areas throughout the interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region of said eye.IX. Kits

[0426] The present disclosure also provides kits, or products of manufacture, comprising (i) a rAAV vector or a pharmaceutical composition comprising a rAAV vector suitable for suprachoroidal administration, and optionally, (ii) the injection device describedherein, and (iii) instructions for use (e.g., a package insert with instructions to perform any of the methods for suprachoroidal administration described herein).

[0427] In some aspects, the kit or product of manufacture comprises a rAAV vector or a pharmaceutical composition comprising a rAAV vector suitable for suprachoroidal administration, and optionally, (ii) the injection device described herein, and optionally (iii) an additional therapeutic agent, and optionally (iv) instructions for use (e.g., a package insert with instructions to perform any of the methods for suprachoroidal administration described herein).

[0428] In some aspects, the components of a kit or product of manufacture disclosed herein are in one or more containers. In some aspects, the kit or product of manufacture comprises (i) a rAAV vector or a pharmaceutical composition comprising a rAAV vector suitable for suprachoroidal administration, and optionally, (ii) the injection device described herein, and optionally (iii) a brochure with instructions to perform any of the methods for suprachoroidal administration described herein.

[0429] In some aspects, a kit or product of manufacture of the present disclosure comprises at least (i) a rAAV vector or a pharmaceutical composition comprising a rAAV vector suitable for suprachoroidal administration and (ii) the injection device described herein.

[0430] In certain aspects, provided herein is a kit comprising: (i) a recombinant adeno- associated virus (rAAV) vector comprising a capsid, a vector genome, and an expression cassette, (ii) an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the rAAV vector can be injected through the needle into the suprachoroidal space, and (iii) instructions for using the injection device to administer the rAAV to the suprachoroidal space of the eye.

[0431] In some aspects, the injection device is provided to a user disassembled, readily assembled in a form of a system, or provided as an assembled device.

[0432] In some aspects, the injection device is provided with a preloaded reservoir, e.g., a syringe, comprising the rAAV vector.

[0433] In some aspects, the rAAV vector is formulated for administration of at least 1E10 vg to at most 1E12 vg / eye.

[0434] In certain aspects, provided herein is a kit for administering at least 1E10 vg to at most 1E12 vg of a recombinant adeno-associated viral (rAAV) vector to an eye, wherein the kit comprises: (i) a reservoir comprising a pharmaceutical composition comprising therAAV vector which comprises a capsid, a vector genome, and an expression cassette; and (ii) an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the pharmaceutical composition can be injected through the needle into the suprachoroidal space.

[0435] In some aspects, the injection device is provided disassembled, readily assembled in a form of a system, or provided as an assembled device.

[0436] In certain aspects, provided herein is a system for administering a recombinant adeno-associated viral (rAAV) vector to the eye of a subject, wherein the system comprises: (i) a reservoir comprising a pharmaceutical composition comprising the rAAV vector which comprises a capsid, a vector genome, and an expression cassette; and (ii) an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the pharmaceutical composition can be injected through the needle into the suprachoroidal space, wherein the reservoir is preloaded with the pharmaceutical composition for administration of at least 1E10 vg / eye to at most 1E12 vg / eye of the rAAV vector.

[0437] In some aspects, the reservoir comprises at least 1E10 vector genomes (vg) of the rAAV vector.

[0438] In some aspects, the reservoir comprises at least 1E10 vg to at most 1E13 vg of the rAAV vector.

[0439] In some aspects, the reservoir comprises a total volume of at least 0.1 mL of the pharmaceutical composition

[0440] In some aspects, the reservoir comprises a total volume of at least 0.1 mL to about 0.5 mL (e.g., about 0.1 mL to about 0.25 mL) of the pharmaceutical composition.

[0441] In some aspects, the reservoir is for a single administration.

[0442] In some aspects, the kit or system comprises two or more reservoirs.

[0443] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature.

[0444] Additional details of the kits and devices applicable to this disclosure are described below.

[0445] Reference is made to FIGS. 5A-5C. In some aspects, FIG. 5A illustrates an exemplary kit 10 for assembling an exemplary system 11, which is configured forfacilitating and performing injections to an interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region in an organ (e.g., eye) of a subject. In some aspects, FIG. 5B illustrates system 11 fully assembled. Kit 10 or system 11 includes a fluid reservoir, such as a syringe 12, an organ affecting device 100, and an organ docking device 200.

[0446] In some aspects, optionally, organ docking device 200 is configured for fixating a target tissue portion (e.g., target portion of a sclera) of an organ (e.g., an eye of a subject) and for directing needle penetration into the target tissue portion, such as by physically confining a path for sliding thereon to organ affecting device 100 in a chosen spatial direction relative to the target tissue portion. Docking device 200 is optional, and is not required in the kits, systems, etc. disclosed. Additionally, other variations described are contemplated for potential fixating of a sclera portion.

[0447] Optionally, organ affecting device 100 is configured for facilitating low to high- volume fluid dispersions in an organ's interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region, by creating, prior to injection, a directional spaced passage into and through the outer most or anterior organ layer (e.g., the sclera optionally with the conjunctiva) and an interlayer region (e.g., within choroid layers, or / and between choroid and retina of the eye), in an accurate anatomical location, chosen alignment, and predefined dimensions. This artificial passage is configured for effectively containing, channeling and dispersing fluids therefrom to distant and / or large areas throughout the interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region of the organ. In some embodiments, the passage is oriented at a shallow angle (e.g., 0° to 30°), or substantially tangential, to the target tissue portion and / or to the interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region adjacent to point of entry of the needle into the target tissue portion.

[0448] In some aspects, FIG. 5C illustrates elements forming organ affecting device 100, which include an actuator 101, a switch 102, a tissue separator 103, a sealed connector member 104 and a needle 105. Needle 105 is configured to penetrate into and / or through a target portion of an outer layer of the organ, such as the sclera of an eye (optionally including also the conjunctiva), tissue separator 103 is configured for passing with distal tip thereof via lumen of needle 105 and is selectively positionable at different fixed positions aimed in accordance with different functions, including at least one of: (i) forming a directional spaced passage, by piercing into and separating (spacing-apart)adjacent layers in an interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region portion, (ii) assist in restricting penetration length of needle 105 in the target tissue portion, and (iii) channeling a fluid from syringe 12 into lumen of needle 105. Sealed connector member 104 facilitates fluid tight connection between tissue separator 103 at one end, and needle 105 at an opposing end.

[0449] In some aspects, actuator 101 forms the handheld body of organ affecting device 100 and configured for housing area of connection of syringe 12 with tissue separator 103. It is equipped with switch 102 for shifting distal tip of tissue separator 103 between preset fixed positions. Switch 102 may be a toggle type switch as illustrated, or it may be of any other type, such as a push button, a revolving knob, or a slide switch, configured with at least two (optionally three) switch positions.

[0450] In some aspects, optionally, actuator 101 also includes an aligning element 130 configured for fastening into a mating recess region in docking device 200 for facilitating alignment and predetermined travel length along a directing surface of docking device 200.

[0451] FIGS. 6A-6B respectively illustrate a side cut view and a magnified portion of device 100 assembled with syringe 12. Syringe 12 includes a piston 13, a barrel 14 in which piston 13 slides, and a syringe connector 15. Syringe connector 15 is configured for connecting with device 100 in a dedicated hollow of actuator 101, to a separator hub 106 of tissue separator 103, which may be equipped with a Luer connection 114 or other type of leak-free connector. Tissue separator 103 (shown separately in FIG. 7A-7B) also includes a cannula 107 connecting between separator hub 106 and an elongated probe 108. Probe 108 has a probe fixed portion 109 fixedly connected to an inner cannula wall 110 of cannula 107, and a probe protruding portion 111 which extends distally from a cannula distal end 112 of cannula 107 and ending with a separator distal tip 113. This way of connection facilitates fluid flow from cannula 107 to pass across probe fixed portion 109 towards needle 105. Probe protruding portion 111 has length sufficient to facilitate the preset fixed positions of separator distal tip 113 relative to needle 105. In other embodiments, and as will be described below, a drug reservoir (optionally in a form of a syringe or including a piston) may be provided as an integral part of the tissue affecting device, thus eliminating the need for connectors and hub, which are required for assembling parts but possess significant space and design limitations in the overall design.

[0452] Switch 102 engages with separator hub 106 via a switch arm 115 that is connected to a switch button 116 across a switch pivot connection 117, such that moving switch button 116 about switch pivot connection 117 results in shifting tissue separator 103 by pushing it between preset fixed positions via switch arm 115.

[0453] Sealed connector member 104 includes a separator port 118, configured for accommodating length and diameter of cannula 107 therethrough, a needle connector 119 (optionally in a form of a male Luer connection, as shown), configured for leak-free connecting with needle 105, and a seal 120 configured for sealing around cannula 107 while allowing cannula 107 slide therein without compromising sealing properties.

[0454] Needle 105 includes needle hub 121, optionally comprising a female Luer connection member as shown, a needle shaft 122, and an external stopper 123. A needle lumen 124 extends between a needle proximal end 125 and a needle distal end 126; needle distal end 126 includes a size transitional portion 127 ending with a sharp needle distal tip 128. Size transitional portion 127 refers to the distal end portion of needle 105 along which there is a change, sudden or gradual, in dimensions relative to general shaft 122 dimensions, such as change in diameter or change in circumference length, for example a bevel or a chamfer. Size transitional portion is at least 1 mm in length, optionally about 3 mm. External stopper 123 is configured to physically suppress or block penetration of needle distal tip 128 in a penetrated organ beyond a predetermined length. External stopper 123 is in a form of a tube extending externally along at least part of needle shaft 122, with a stopper distal end 129 thereof located proximally to needle distal tip 128. Stopper distal end 129 is optionally fixedly positioned, although it may be selectively shiftable along length of needle shaft 122 proximally to size transitional portion 127.

[0455] Reference is now made to FIGS. 8A-8C which, in some aspects, illustrate side views of a distal portion of device 100, showing different preset positions of separator distal tip 128 relative to needle distal end 126. The elongated tissue separator 103 is sized and configured to extend in and throughout needle lumen 124 such that separator distal tip 113 is in juxtaposition with needle distal end 126. Actuator 101 is configured to facilitate selective shifting of tissue separator 103 in needle lumen 124 such that separator distal tip 113 is movable between several preset fixed positions relative to needle distal tip 128. Device 100 is configured such that cannula distal end 112 is positioned within, ordistally to, seal 120 in each of the preset fixed positions in order to prevent leakage during the entire use of the device.

[0456] In some aspects, one of the preset fixed positions is an intermediate position (shown in FIG. 8 A, for example), wherein switch 102 is provided in a nominal position and separator distal tip 113 is fixedly positioned and located a 'first' predetermined length LI proximally to needle distal tip 128, optionally within a range of about 0.2 mm to about 1.5 mm, optionally about 0.5 mm. In the intermediate position, separator distal tip 113 protrudes from a laterally uncovered section 131 of transitional portion 127. Separator distal tip 113 is blunt, optionally rounded shaped, and configured to physically suppress or block penetration of size transitional portion 127 in the penetrated organ being greater than the first predetermined length, when it is fixed in the intermediate position.

[0457] Another preset fixed position is a distal-most position (shown in FIG. 8C, for example), wherein switch 102 is provided in a 'first' pressed position and separator distal tip 113 is fixedly positioned distally to needle distal tip 128. When in the distal-most position, separator distal tip 113 is located a 'second' predetermined length L2 distally to needle distal tip 128, optionally about 1 mm or more, optionally about 2 mm.

[0458] A third optional preset fixed position is a proximal-most position (shown in FIG.8B, for example), wherein switch 102 is provided in a 'second' pressed position and separator distal tip 113 is entirely concealed in needle lumen 124 proximally to laterally uncovered section 131 of size transitional portion 127.

[0459] External stopper 123 is configured to physically suppress or block penetration of the needle distal tip in the organ over a 'third' predetermined length L3 (shown in FIG. 8C), which equals the longitudinal distance between needle distal tip 128 and stopper distal end 129, and is optionally at least 1 mm, optionally about 3 mm in length.

[0460] In some aspects, FIGS. 9A-9O illustrate views of kit 10, system 11 organ affecting device 100 or / and (optional) organ docking device 200 in different configurations representing exemplary steps in a method for fixating a sclera portion, directing needle penetration into the sclera portion, and facilitating fluid injection to an interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region in an eye of a subject. In some aspects, the first group of steps is intended for fixating a target tissue portion (in this example a sclera portion SP) of an organ (eyeball) and directing needle penetration into the target tissue portion, using (optional) organ docking device 200. Docking device 200 may also not be used, or an alternative be used as described.

[0461] As illustrated in FIG. 9A, optionally, organ docking device is positioned in a location chosen by the user (an ophthalmologist, for example) and organ contact surface 202 is positioned against and in contact with target sclera portion SP. Placing of organ docking device 200 is optionally performed symmetrically around the limbus (which is the area of transition from clear cornea to opaque sclera) in a chosen eye quadrant.

[0462] In some aspects, since the sclera is considered structurally anisotropic, although tangential penetration of the needle 105 is desired for facilitating the requested injection direction, an initial perpendicular penetration is preferred in order to minimize forces resistive to needle puncture and travel in the sclera. After initial perpendicular penetration, the needle is shifted and advanced in the sclera in a path tangential (or close to tangential) to the choroid until reaching the target injection site in the interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region within the choroid layers. Penetration to the sclera is done using the needle tip, while penetration into the choroid is done using the blunt tissue separator, for avoiding or minimizing potential (unintentional) damage to blood vessels located within the choroid.

[0463] In some aspects, the point of penetration from the sclera to the choroid is distanced from the point of penetration into the sclera relative to organ contact surface 202 (see FIG. 9L). Such a distance is important to the sealing effect created by injecting medication through a passage PSG in choroid CR: After injection, hydrostatic pressure in the eye would tend to force the injected medication to flow back, however, the sclera portion proximal (relative to eye globe) to the penetration to the choroid is intact, so that back flow is prevented, and the entire passage PSG thus acts a one-way valve preventing the medication to reflux from the perpendicular penetration to the sclera portion.

[0464] In some aspects, optionally, when the organ docking device 200 is positioned symmetrically around the limbus, the user is indicated by the (optional) organ docking device to perpendicularly penetrate the needle 105 into the sclera portion 2 mm to 4 mm posterior to limbus (See FIG. 9E, needle tip 128 should be inserted in the opening between anchors 207).

[0465] Distances from the limbus are: pars plicata 1-2 mm, pars plana about 4 mm anterior-posteriorly. This ensures that the perpendicular penetration to the sclera portion will be given proximally (relative to eye globe) to the pars plana, and that the tangential penetration will result in penetration of the separator into the choroid.

[0466] In some aspects, although perpendicular penetration into the sclera portion is partial (not all the way through the sclera), perpendicular penetration proximally into the pars plana relative to the eye globe is a safety measure. Perpendicular penetration into the sclera portion more anteriorly may result in hemorrhage due to trauma to the highly vascularized pars plicata. A perpendicular penetration to the sclera portion more posteriorly may result in retinal detachment.

[0467] In some aspects, anchoring member 205 is provided in a withdrawn position (FIG.9B) so, after positioning, anchoring actuator 206 is then applied to shift anchoring member 205 to anchoring position (FIG. 9C) for anchoring to the target sclera portion. As shown, anchors 207 penetrate approximately perpendicularly into the sclera layer, at the chosen target portion thereof, about 0.5 mm deep, until the blunt restrictor tips 217 contact the outer surface of the sclera and suppresses further advance (at least in a magnitude noticeable by the user, as a sign for him to stop advancing forward). While organ contact surface 202 is in contact with sclera portion SP, tissue pressing member 214 presses against an upper periphery of the target sclera portion and tightens the conjunctiva CJ adjacent to target sclera portion SP.

[0468] In some aspects, once deployed in a chosen position against target sclera portion SP, organ docking device 200 can serve for directing organ affecting device 100 with needle 105 for penetrating (piercing) with needle distal tip 128 into sclera portion SP along predetermined needle penetration path 204, while countering forces generated between (optional) organ docking device body 201 and the eyeball (for preventing revolving of the eyeball) during sclera penetration.

[0469] In some aspects, the next group of steps is intended for facilitating fluid injection to an interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region in an eye of a subject. The requested result, as previously described, is an artificially formed passage or channel, oriented at a shallow angle, approximately tangentially to the target sclera portion SP adjacent to needle entry point, and extends about 2 mm to 4 mm in length from within sclera layer into the choroid. The passage, once cleared from objects, creates a directional space in the interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region of the choroid and adjacent layers, which can then be filled with fluid (medication) which then disperses throughout the interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) region space including posterior portions remote from the formed passage.

[0470] In some aspects, before applying organ affecting device 100, the user has to verify that switch 102 is in its nominal position so that separator distal tip 113 is fixedly positioned at the intermediate position, with first predetermined length LI proximally to needle distal tip 128, and protrudes from a laterally uncovered section 131 of size transitional portion 127 (as shown in FIG. 8A). First predetermined length LI is shorter than a thickness of said sclera, optionally within a range of 0.2 mm to 1.5 mm, optionally about 0.5 mm in length. With separator distal tip 113 at the intermediate position, organ affecting device 100 is applied (FIG. 9D) in order to penetrate into (but not throughout) the sclera portion SP using needle distal tip 128 until contacting the eye outer surface (i.e., the sclera and optionally the conjunctiva) with separator distal tip 113 (FIGS. 9E- 9G). As shown, sclera penetration is performed at a penetration angle within a range of 30° to 120°, optionally perpendicularly, relative to eye outer surface (i.e., the sclera and optionally the conjunctiva) adjacent to needle penetration point, between the two anchors 207.

[0471] In some aspects, once needle distal tip 128 is at a depth equal to the first predetermined length LI in sclera portion SP, it is then maneuvered from the steep penetration angle to a shallow injection angle, within a range of 0° to 30° s, optionally tangentially, to the eye outer surface (i.e., the sclera and optionally the conjunctiva) adjacent to the penetration point (as shown in FIGS. 9H-9J).

[0472] Optionally, organ docking device 200 is configured with needle directing surface 203 angled to organ contacting surface 202, such that needle distal tip 128 maneuvering is accomplished by positioning actuator 101 on top of and along needle directing surface, with the needle distal tip 128 pointing towards the sclera portion SP in the direction of predetermined needle penetration path 204 (with the beveled portion of needle 105 positioned towards the eye).

[0473] In some aspects, optionally, to secure orientation of needle 105 along predetermined needle penetration path 204, aligning element 130 of actuator 101 is fitted in elongated recess 211 of organ docking device body 201.

[0474] In some aspects, following needle distal tip 128 maneuvering to the injection angle, separator distal tip 113 is withdrawn to proximal-most position (FIG. 9K), so that it is entirely concealed in needle lumen 124 proximally to laterally uncovered section of size transitional portion 127. Needle distal tip 128 can then be pushed forward in sclera (FIG. 9L) without being restricted to maximal travel length of first predetermined lengthLI. Since the injection angle is shallow, optionally approximately tangential to the sclera layer, the needle can advance a few millimeters deep in the sclera without penetrating through into the choroid, for example along a length within a range of 1 mm to 5 mm, optionally of about 3 mm. Passing an allowed penetration length is suppressed or blocked with external stopper 123.

[0475] In some aspects, in order to create the directional passage into the choroid, use of a sharp needle is avoided in order to prevent damage to blood vessels in the choroid, so the passage is created instead with the blunt separator distal tip 113. Therefore, tissue separator 107 is shifted distally within the needle lumen 124 such that separator distal tip 113 repositions to the distal-most position (FIG. 9M), along second predetermined length L2 of at least 1 mm, optionally about 2 mm, distally to needle distal tip 128, thereby advancing in sclera portion SP, penetrating through into choroid CR and forming a passage PSG in choroid CR (shown in FIG. 9N).

[0476] In some aspects, passage PSG may have a length and orientation in the eye, so as to pass in the extra-vascular choroid layer of choroid CR. Optionally, additionally or alternatively, passage PSG may have a length and orientation in the eye, so as to enlarge a suprachoroidal space distally to sclera portion SP. Optionally, additionally or alternatively, passage PSG may have a length and orientation in the eye, so as to extend between choroid CR and a retinal pigment epithelium layer adjacent thereto.

[0477] In some aspects, after forming passage PSG, separator distal tip 113 is withdrawn again, optionally to the proximal-most position so it is entirely concealed in needle lumen 124 (FIG. 9N), and medication can then be injected into passage PSG using syringe 12. The methods and devices described herein may be used to deliver a therapeutic composition (e.g., pharmaceutical composition, especially a liquid pharmaceutical composition) to the subretinal space (or to the extra-vascular choroidal layer of choroid, or the suprachoroidal space or between choroid and a retinal pigment epithelium layer adjacent thereto) of an animal (especially mammalian eye) such as a pharmaceutical composition including a pharmaceutically effective amount of an active ingredient (e.g., an active pharmaceutical ingredient and / or a cell and / or a gene) in a suitable carrier.

[0478] Reference is now made to FIG. 10A which, in some aspects illustrates a side cut view and rear view of an exemplary system unit 500 configured for facilitating injection into an inter-layer region in the eye of a subject. System unit 500 includes a unit body 501 housing an elongated channel 502 configured for accommodating a length of a piston of asyringe for pressurizing a fluid therethrough. Elongated channel 502 is fluidly connected via a distal end thereof, within unit body 501, with a cannula 503 of a tissue separator 504 (being similar in function and structure to tissue separator 103). Tissue separator 504 further includes a slender probe 505 configured for fitting and sliding along a lumen of a needle 506 (being similar in function and structure to needle 105).

[0479] In some aspects, FIG. 10B illustrates an isometric view of some of the assembled components of system unit 500 (while illustratively concealing other components assembled in system unit 500, for sake of simplicity) and FIG. 10C illustrates views of the same assembled components in different configurations (I), (II) and (III). As shown, a retractable drawer mechanism 507, comprising a distal outer drawer 508 slidably connected to needle 506 and a proximal inner drawer 509 fixedly connected to tissue separator 504, is provided in unit body 501 and configured for facilitating (e.g., by confining or marking, for example) relative movement between tissue separator 504 and needle 506 to axial sliding of probe 505 in and through needle 506.

[0480] In some aspects, in Configuration (I), shown in FIG. 10C, unit body 501 with needle 506 is oriented at a steep penetration angle into target sclera portion of the eye within a range of 30° to 120°, optionally perpendicularly, relative to eye outer surface (i.e., the sclera and optionally the conjunctiva) adjacent to needle penetration point (similarly to as shown in FIGS. 9F and 9G). In this configuration, tissue separator 504 can slide freely within needle 506.

[0481] In some aspects, in Configuration (II), shown in FIG. 10C, unit body 501 with needle 506 is oriented at an intermediate angle within a range of 10° to 30° s, relative to the eye outer surface (i.e., the sclera and optionally the conjunctiva) adjacent to the penetration point. In this configuration, tissue separator is confined to a withdrawn position relative to distal tip of needle 506 and is entirely concealed in the needle lumen.

[0482] In some aspects, in Configuration (III), shown in FIG. 10C, unit body 501 with needle 506 is oriented at a shallow injection angle within a range of 0° to 10°, optionally tangentially, relative to the eye outer surface (i.e., the sclera and optionally the conjunctiva) adjacent to the penetration point (similarly to as shown in FIG. 9K). In this configuration, tissue separator 504 can slide freely within needle 506.

[0483] In some aspects, distal outer drawer 508 connects with outer arms 510 thereof unit body 501 to an organ docking member 511 by way of pivot connection, thereby facilitating a range of angular orientations of needle 506 relative to an organ contactsurface 512 provided at a distal end of (optional) organ docking member 511 and configured to fit or conform to a shape imposed by outer surface of a target organ (e.g., eyeball (e.g., the sclera and optionally the conjunctiva)).

[0484] Optionally, in some aspects, organ docking member 511 is similar in function and structure to organ docking device 200 and further includes anchors 513 for fixating a target tissue portion, prior to needle 506 penetration therethrough, and needle directing surfaces 514 configured to facilitate a predetermined needle penetration path across organ contact surface 512 when outer arms 510 rest thereon and needle 506 travels distally towards organ contact surface 512.

[0485] In some aspects, optionally, organ docking member 511 further includes curved track surfaces 515 configured to effect linear motion of tissue separator 504 relative to needle 506 during rotation of outer arms 510 from a far position relative to needle directing surfaces 514 (as shown in Configuration (I) in FIG. 10C, for example) to near position relative to needle directing surfaces 514 (as shown in Configuration (II) in FIG.10C, for example).

[0486] In some aspects, proximal inner drawer 509 includes inner arms 516 each having track follower surface 517 configured and arranged for continuous sliding on a respective curved track surface 515 during outer arms 510 rotation from the far position to the near position, such that tissue separator 504 is withdrawn during outer arms 510 motion from an intermediate position (e.g., similar to intermediate position shown in FIG. 8A) to a withdrawn position (e.g., similar to proximal-most position shown in FIG. 8B). This motion of tissue separator 504 relative to needle 510 is considered advantageous as it effects a precise shift along a very short travel (e.g., 1 mm or less, optionally about 0.5 mm) during unit body 510 rotation and allows the user to concentrate on other aspects of the process in this delicate maneuver.

[0487] Reference is now made to FIGS. 11-13, which illustrate an optional surgical fixation tool 1110, which may be used in place of the described docking devices, or not used, constructed and operative in accordance with a non-limiting embodiment of the invention.

[0488] The surgical fixation tool 1110 includes at least one fixation member 1112, whose structure is described hereinbelow. Fixation tool 1110 may include a protector member 1114 that can cover the one or more fixation members 1112 before use, to protect thesharp end of the fixation member 1112 from being damaged or contaminated and protect the user from accidentally being injured by the fixation member 1112.

[0489] Although the invention can be carried out with just one fixation member 1112, in the illustrated and preferred embodiment there are more than one fixation members 1112, such as a pair of fixation members 1112, each of which protrudes from a leg 1116.Accordingly in the illustrated embodiment, there are a pair of legs 1116 separated by a (e.g., U-shaped) channel 1118. The legs 1116 extend from a proximal grasping portion 1120 of the tool 1110.

[0490] The protector member 1114 may be formed with at least one hollow receiving member 1122, one for each of the fixation members 1112. A tongue 1124 may protrude axially outwards between the hollow receiving members 1122 and may be arranged to move into channel 1118 of the tool 1110. The protector member 1114 may be formed with at least one window 1126 (also seen in FIG. 14A) for viewing the at least one fixation member 1112 while the protector member 1114 covers the fixation members 1112. In this manner, even before removing protector member 1114, the user can visually inspect the integrity of the fixation members 1112 before use.

[0491] Reference is now made to FIG. 14A. Each fixation member 1112 includes a needle 1128 that includes a needle shaft 1127 with a sharp tip 1129 at a distal end of the needle shaft 1127. Needle 1128 may or may not be hollow and can be generally round / tubular or have any other cross-section, size and shape. Needle 1128 protrudes distally from support structure 1130, which has one or more (most preferably multiple) sloped support surfaces as explained below. The support surfaces 1130 are sloped with respect to the needle shaft 1127. Referring again to FIG. 3, the tips 1129 of the fixation members 1112 may be separated by a distance G, which may be, without limitation, 7 mm to 10 mm, or 2 mm to 24 mm. The separation or gap G between the fixation members 1112 may enable the fixation members 1112 to resist rotational movement of the eye and apply tension to the conjunctiva and sclera. Additionally, the separation G between the fixation members 1112 may serve as a slot to indicate the correct access point for a procedure. Additionally, the separation G between the fixation members 1112 may provide clearance to manipulate the fixation tool in vicinity of surgical tools or structures working within the separation or gap G.

[0492] Reference is now made to FIG. 15. The fixation tool 1110 may be used in surgical procedures, such as ophthalmic procedures, such as but not limited to, injection of asubstance into an interlayer, such as the suprachoroidal layer of the eye, such as in the procedure described in PCT Application WO 2019 / 202603, or any other ophthalmic procedure that requires scleral or conjunctival fixation. FIG. 15 shows fixation tool fixing the conjunctiva (CJ) so that a needle and separator tool 1140 (described in PCT Application WO 2019 / 202603) can create a suprachoroidal tunnel between the choroid CH and the sclera SC. (RE is the retina.) The fixation tool 1110 can apply counter forces to multiple forces of varying directions during the ophthalmic procedure. For example, the separator tool 1140 can apply forces tangential to the ocular tissue, or forces at any acute or obtuse angle.

[0493] Reference is now made to FIG. 16A. The needle 1128 may be a pencil point that subtends an angle A, which is preferably but not necessarily 18° to 22° or 7° to 150°. This shape helps penetrate and hold tissue without cutting or tearing. The needle 1128 may alternatively be a chisel point. The length D of needle 1128 that protrudes outwards from the support structure 1130 is intended to anchor the tissue without perforating the target tissue, and is preferably but not necessarily 0.4 mm, or 0.2 mm to 1.2 mm. The full diameter of needle 1128 is preferably but not necessarily 0.3 mm or 0.05 mm or 2 mm.

[0494] The gap G between tips 1129 of needles 1128 may be fixed, flexible or adjustable, such as by mounting legs 1116 on a gear rack or groove and the like, so the legs 1116 can be moved towards and away from each other.

[0495] The needles 1128 may include a sensor 1132 (FIG. 14B) that senses contact with the tissue or a parameter of the tissue. Sensor 1132 may be, without limitation, an optical, force, impedance, temperature, resistance sensor and others. Sensor 1132 may be adjusted to measure intraocular pressure.

[0496] The needle 1128 may be fixed with respect to support structure 1130, or alternatively, may be spring loaded, so that the needles 1128 may be actuated or adjusted relative to support structure 1130.

[0497] Support structure 1130 may be fixed with respect to legs 1116, or alternatively, may be spring loaded, so that the support structure 1130 may be actuated or adjusted relative to legs 1116.

[0498] Support structure 1130 may be actuated by an actuator to shield or expose needle 1128.

[0499] Reference is now made to FIG. 16B and to FIGS. 17A-17C, which illustrate examples of the support structure 1130. The support structure 1130 includes two or moresloping shoulders 1134 that are joined at a curved portion 1136. The angle S (FIG. 16B) between the sloping shoulders may be, but is not necessarily, 80°, or 10° to 180°. The sloping shoulders 1134 enable manipulating the angle of the fixation tool relative to the ocular tissue after the needle tips have engaged the ocular tissue, without having to remove the needle tips and re-position them and without losing the grip force to the tissue. This minimizes injuring the ocular tissue (only one placement of the needle tips is necessary). This also provides greater and easier access to, and view of, the ocular surface between the tips of the tool. In this manner, the tool can apply counter forces to multiple forces of varying directions during the ophthalmic procedure.

[0500] The support structure 1130 also provides control over tip penetration depth into the ocular tissue.

[0501] There are many ways to produce the sharp tip 1129 and the support structure 1130. For example, the tips can be produced as an independent component or integral to the support structure 1130. The support structure 1130 can be produced from a tube (FIG.17A), in which the tube longitudinal central axis is concentric and parallel to the longitudinal central axis of the needle. Angled surfaces may be ground or otherwise formed on the tube wall. In FIG. 17B, the longitudinal central axis of the tube is perpendicular to the needle. Holes are formed in the tube to accommodate the needle tip. The round shape of the tube provides the sloping shoulders 1134. The protective shoulder can be produced from any other shape and suitable material.

[0502] Reference is now made to FIGS. 18A and 18B. The total length of the projections (support structure 1130 and needles 1128 that protrude from the legs 1116) may be used as an indicator to measure distances; e.g., 3 mm distance from the limbus (FIG. 18B) and afterwards placing the needles 3mm from the limbus (FIG. 18 A).

[0503] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0504] Additionally, in some aspects, injection of suprachoroidally-administered AAV gene therapy into the eye can be performed using the methods and / or devices described and disclosed in International Publication Nos. WO 2019 / 202603, WO 2015 / 015467, and WO 2022 / 053912, U.S. Patent No. 10,285,852 to Rotenstreich, and U.S. Patent Publication No. 2021 / 0236743, each of which is incorporated by reference in its entirety, for all purposes.

[0505] The following examples are offered by way of illustration and not by way of limitation.ExamplesExample 1: AA V vector compatibility with the Suprachoroidal Injector

[0506] Compatibility assessment of vector and the Suprachoroidal Injector indicated that after extended contact with the injector, vector genome titers were within + / - 20% (Table 1) and potency results were within + / - 30% (Table 2) of pre-exposure values, supporting that the Suprachoroidal Injector and vector were suitable for use together.Table 1: GFP and ITR Vector Genome Titer Results (GFP = green fluorescent protein; ITR = inverted terminal repeat; Percent Difference: ((Post-Pre) / Pre))).Table 2: Potency Results Measured by Cell Transduction Efficiency (MOI = multiplicity of infection; Percent Difference: ((Post-Pre) / Pre))).Example 2: AA V GFP Constructs Administered via a Single Suprachoroidal Injection in Cynomolgus Monkeys

[0507] A study was conducted to evaluate ocular and potential other target biodistribution of AAV GFP constructs, when administered via a single suprachoroidal injection in Cynomolgus monkeys followed by a 28-day observation period. Specifically, the ability of AAV2, AAV2Quad+l (AAV2Q), or AAV8 to transduce the non-human primate (NHP) retina four weeks after SCS delivery was evaluated. The Suprachoroidal Injector (described in FIGs. 5A-10C) was used to deliver the AAV vector. The SuprachoroidalInjector employed achieves suprachoroidal delivery by creating a channel within the choroid via blunt dissection of the sclera and choroid, leading to rapid and extensive distribution to the posterior segment and macula.1. Study design:

[0508] All animals received suprachoroidal administration of 5E11 vector genomes per eye (vg / eye) in a volume of 120 pl per eye of AAV composition using the Suprachoroidal Injector (FIGs. 5A-10C) on day 1 followed by a 28-day observation period. The study design is shown in Table 3. Dose scaling for retinal gene therapy can be accomplished by scaling based on the retinal area of each species or, alternatively, based on the surface area of the eye globe (area=4 *7i * r2). In each case the human equivalent dose in VG / eye is approximately 2x a give NHP dose in VG / eye.Table 3: Study Design.Animal Identification Dose DoseGroup Test Material Dose Level Concentration Volume Males Females 1 AAV2Q-GFP 5.0 E+ll vg / eye 4.17 E12 vg / mL 120 pL 1 4 2 AAV2-GFP 5.0 E+ll vg / eye 4.17 E12 vg / mL 120 pL 2 5 3 AAV8-GFP 5.0 E+ll vg / eye 4.17 E12 vg / mL 120 pL 3 6

[0509] For simplicity, in some instances, AAV2Quad+l-GFP is referred to as AAV2Q, AAV2-GFP is referred to as AAV2, AAV8-GFP is referred to as AAV8, respectively.

[0510] Animals were confirmed negative for AAV2 or AAV8 respectively neutralizing antibodies before dosing. Animals were monitored for intraocular pressure (IOP), confocal scanning laser ophthalmoscopy (cSLO) was utilized to measure GFP expression and optical coherence tomography (OCT) allowed monitoring for retinal or choroidal changes. At 4 weeks post-dosing, animals were terminated, and ocular and systemic tissues were evaluated for vector genomes (qPCR), mRNA (RT-qPCR) and GFP protein by immunohistochemistry (IHC). Fixed globes were sagittally sectioned from above to below the optic nerve and the slides were counterstained for hematoxylin and GFP.2. Suprachoroidal injection procedure:

[0511] Suprachoroidal delivery was performed with the Suprachoroidal Injector (FIG. 1).The area surrounding the eye was swabbed with 5% Betadine ophthalmic solution and the conjunctive fornices of the eye were flushed with a 0.1% Betadine solution using a sterile syringe and cannula prior to dose administration. Drops of topical ocular anesthetic (Proparacaine) were placed into each eye. A sterile drape was placed over the eye to be dosed with a hole made with sterile scissors. An eyelid speculum was placed to hold the eyelid open for the eye to be dosed. The conjunctiva was grasped with a tissue forceps near the limbus and the eye was rotated to expose the area to be dosed (superior / temporal, inferior / temporal). A caliper was used to mark spots at 3-5 mm posterior to the limbus on the superotemporal or inferotemporal sclera. A sterile skin marker could have been used to label the caliper in order to mark the injection site. The globe was rotated to expose the area to be dosed. The injection device needle was placed approximately 3 to 4 mm posterior to the limbus. The initial device entry was approximately 30 degrees to the sclera and then flattened tangential to the sclera, advancing to the needle stopper to create a scleral tunnel. This was followed by extension and retraction of the tissue separator and slow injection of the dose formulations (120 pL was administered over 5 to 10 seconds, achieved by depression of the syringe plunger by an assistant through an extension set). After completion of dosing, the device was held in place for 20 seconds prior to withdrawing the needle from the eye. Upon withdrawal of the needle from the eye, a sterile, cotton-tipped applicator was placed over the injection site for approximately 15 to 20 seconds. Ophthalmic ointment was placed in the eye to prevent it from drying out while the animal was recovering from anesthesia.3. Endpoints of the study:

[0512] Endpoints of the study included mortality, clinical observations, food evaluation, body weight, intraocular pressure, and ophthalmology (including Confocal Scanning Laser Ophthalmoscopy (cSLO) and Optical Coherence Tomography (OCT) imaging for Green Fluorescent Protein (GFP) expression). Tissue samples were collected during necropsy on Day 29 for evaluation of vector and mRNA biodistribution, and the evaluation of the humoral and cellular response to AAV2 and AAV8 capsid was done. Samples of ocular tissue were collected for immunohistochemistry on Day 29 as well.4. Summary of the results of the Study:

[0513] There were no unscheduled deaths. There were no AAV GFP construct (AAV2Q- GFP, AAV2-GFP, and AAV8-GFP)-related effects on food consumption, body weight, body weight changes, or intraocular pressure. No retinal or choroidal changes were observed in all animals’ eyes in OCT scans.

[0514] AAV GFP construct-related clinical observations consisted of a cloudy eye, a constricted pupil, and / or partially closed eye in 1 male administered AAV2Q-GFP and 1 female administered AAV8-GFP; however, based on the duration, severity, and frequency of these observations, these responses were neither construct specific or sex dependent.

[0515] Based on cSLO and OCT imaging, AAV2Q-, AAV2- and AAV8-GFP given once via suprachoroidal injection were associated with minimal to mild transduction and expression of constructs observed as retinal fluorescence at Day 8 and / or Day 28.Fluorescensce from the AAV2Q-GFP construct was detected as early as Day 8, and slightly increased at Day 28. Multifocal areas of discoloration in retina and pigmented vitreous cells did not resolve and were considered AAV GFP construct related. Transient, AAV8-GFP-related findings included vitreous haze.

[0516] Eye tissues close to the injection site that were positive with abundant levels of vector genomes included sclera, lens, choroid, iris and ciliary body, cornea and retina. Optic nerve samples from all dosed groups were positive; however, the vector levels in optic nerve samples were much higher with administration of the AAV8-GFP. One AAV2Q-GFP dosed optic chiasm sample was positive with vector genomes and the rest of the optic chiasm samples were negative. Outside of the eye tissues, the test article vectors were consistently found in all dosed spleen, mandibular lymph node, liver, and lung tissues at various levels. Quantifiable vectors were found in some animals, but not all for each of the following tissues: spinal cord (cervical, midthoracic, lumbar), occipital lobe, brain, thalamus, kidney and heart. All sex tissues (ovary and testes) were negative.

[0517] AAV GFP construct mRNA levels were mainly detected in the eye tissues adjacent to the injection sites including cornea, iris and ciliary body, retina, choroid and sclera, but not the lens. In contrast to the detection of vector genomes, not all eye tissues had detectable and abundant levels of construct mRNA. The optic nerve from AAV8- GFP-dosed animals had quantifiable construct mRNA. Outside of these tissues, only 1AAV2-GFP-dosed cervical spinal cord sample and 1 spleen sample from an AAV8-GFP- dosed animal contained mRNA copies which were below the limit of quantification.

[0518] In conclusion, biodistribution was evidenced via fluorescence, quantification of the DNA vector constructs, and expression of the AAV GFP constructs-derived mRNA following a single suprachoroidal injection of AAV2Q-GFP, AAV2-GFP, and AAV8- GFP at 5.0 E+l 1 vg / eye to cynomolgus monkeys (followed by a 28-day observation period). Clinical observations, nonspecific to construct, were limited to cloudy eye, a constricted pupil, and / or partially closed eye in 2 animals.

[0519] Fluorescence in the retina was observed on Day 28 for the AAV2- and AAV8- GFP constructs, and as early as Day 8 for AAV2Q-GFP with increased florescence of this construct on Day 28. Exemplary fluorescenent fundus images taken on Day 28 of an animal treated with AAV2-GFP are shown in FIG. 2A-2B.

[0520] Persistent multifocal areas of discoloration in retina and pigmented vitreous cells were considered AAV GFP construct related, as well as the transient AAV8-GFP-related findings of vitreous haze.

[0521] All animals dosed with AAV2Q-GFP, AAV2-GFP, or AAV8-GFP constructs via suprachoroidal administration generated NAb and BAb to the vectors by study Day 29 to varying degrees.

[0522] Abundant levels of DNA vector for all constructs were observed in most eye tissues evaluated, and the vector levels in optic nerve samples were much higher with administration of the AAV8-GFP. AAV2Q-GFP was also detected in 1 sample of the optic chiasm. Vectors were consistently found in spleen, mandibular lymph node, liver, and lung tissues and less consistently in the spinal cords (cervical, midthoracic, lumbar), occipital lobe, brain, thalamus, kidney and heart. AAV GFP-construct mRNA levels were quantifiable in the eye tissues adjacent to the injection sites including cornea, iris and ciliary body, retina, choroid, sclera, and AAV8-GFP optic nerve, but not the lens. There was minimal mRNA in tissues outside of the eye (see FIGs. 3A-3F).A. In-life Examinations:a. Clinical Observations:

[0523] There were no unscheduled deaths, all animals survived until scheduled euthanasia on Day 29. One male administered AAV2Q-GFP and one female administeredAAV8-GFP were observed to have a mildly cloudy right eye on Days 3 to 7, and a constricted right pupil from Days 3 to 29 and 23, respectively. The female also had a constricted left pupil on Days 20 to 23, and its left eye appeared partially closed on Days 20 to 29. All other animals had no abnormal clinical signs. All animals seroconverted.b. Food Consumption:

[0524] There were no effects on food consumption related to administration with the AAV GFP constructs (AAV2Q-GFP, AAV2-GFP, and AAV8-GFP), when compared with baseline values and across the dose groups.c. Body Weights and Body Weight Gain:

[0525] There were no effects on body weights and body weights gains related to administration with the AAV GFP constructs, when compared with pretest values and across the dose groups.d. Ophthalmology:

[0526] A complete ophthalmology examination including slit-lamp and binocular indirect ophthalmoscopy was performed.

[0527] Confocal Scanning Laser Ophthalmoscopy (cSLO) and Optical Coherence Tomography (OCT) images were obtained from both eyes once prior to dosing initiation, and on Days 8 and 28 using the Spectralis HRA / OCT. Attempts were made to capture images from Green Fluorescent Protein (GFP) expression or any pathology that could be observed. The data was reviewed by the veterinary ophthalmologist to discern any group differences that may be considered a test article-related effect.

[0528] Fundus images were obtained from both eyes once prior to dosing initiation, and on Days 8 and 28 using the RetCam Shuttle. Attempts were made to capture images from any pathology that could be observed. The data was reviewed by the veterinary ophthalmologist to discern any group differences that may be considered a test article- related effect.

[0529] No retinal or choroidal changes were observed in all animals’ eyes in Optical Coherence Tomography (OCT) scans at any time point. See FIG. 2A-2B.

[0530] Fluorescence observed at the parafoveal, nerve fiber layer (NFL) bundle between fovea / optic nerve head (ONH) and at the retinal ganglion cells (RGC) / NFL areas wasconsidered test-article related corroborating the transduction and expression of constructs. Comparisons by the veterinary ophthalmologist based on the cSLO / OCT images included minimal fluorescence at the parafoveal area was observed in 1 of 4 eyes injected with AAV2Q-GFP at Day 8, and in 2 of 4 eyes injected with AAV2-GFP at Day 28. Minimal fluorescence increased to mild in the eye dosed with AAV2Q-GFP at Day 28. The single eye injected with AAV2Q-GFP presenting fluorescence at parafoveal area was observed with minimal fluorescence at the NFL bundle area between fovea / ONH and at the RGC / NFL at Day 28. One of 4 eyes injected with AAV2-GFP presenting parafoveal fluorescence was observed with minimal fluorescence only at the RGC / NFL at Day 28. In the group injected with AAV8-GFP, 1 of 4 eyes presented minimal fluorescence only at the RGC / NFL at Day 8, which was not detected at Day 28. In animals treated with AAV2-GFP, in-life assessments detected GFP expression in the parafoveal ring, where the ganglion cell layer is thickest.

[0531] Multifocal areas of discoloration at superior and inferior retina were observed in 2 of 4 eyes injected with AAV2Q, in 1 of 4 eyes injected with AAV2, and in 3 of 4 eyes injected with AAV8 at Day 28. Areas of discoloration in the retina did not resolve and were considered test article-related. Moderate (2+) pigmented vitreous cells were observed in 3 of 4 eyes injected with AAV2Q at Day 8 and persisted through Day 28. Moderate (2+) pigmented vitreous cells were observed in 2 of 4 eyes injected with AAV2 at Day 8, which persisted through Day 28 in 1 of 2 affected eyes and decreased to trace (0.5+) in 1 of 2 affected eyes.

[0532] Mild to moderate pigmented vitreous cells (1 to 2+) were observed in 2 of 4 eyes injected with AAV8 at Day 8; moderate scoring persisted through Day 28 in 1 of 2 affected eyes and increased from mild to moderate (1 to 3+) in 1 of 2 affected eyes.Pigmented vitreous cells were considered test article-related. A trace of vitreous haze (0.5+) was observed in 1 of 4 eyes injected with AAV8 at Day 8. It resolved uneventfully at Day 28 following treatment with 0.2 mL Triamcinolone subconjunctival in the right eye on Day 8, and eye lubricant on Days 21 to 23. Vitreous haze was transient and considered test article-related.

[0533] Transient and injection procedure-related findings included: aqueous flare, aqueous cells, fibrin clot, and corneal edema, all of which resolved by Day 8.cSLO / OCT:AAV2Q:Minimal fluorescence at parafoveal area was observed in 1 of 4 eyes at Day 8 which increased to mild at Day 28. Same eye presented minimal fluorescence at the NFL bundle area between fovea / ONH and at the RGC / NFL superior / nasal at Day 28.No fluorescence was observed in 3 of 4 eyes at any time point.No retinal / choroidal changes were observed in all 4 eyes in OCT scans at any time point.AAV2:Minimal fluorescence at parafoveal area was observed in 2 of 4 eyes at Day 28. One eye presenting parafoveal fluorescence also presented minimal fluorescence at the RGC / NFL superiorly at Day 28.No fluorescence was observed in 4 of 4 eyes at Day 8 and in 2 of 4 eyes at Day 28.No retinal / choroidal changes were observed in all 4 eyes in OCT scans at any time point.AAV8:Minimal fluorescence at the RGC / NFL superior / nasal was observed in 1 of 4 eyes at Day 8, which was not detected at Day 28.No fluorescence was observed in 3 of 4 eyes at Day 8 and in 4 of 4 eyes at Day 28.No retinal / choroidal changes were observed in all 4 eyes in OCT scans at any timepoint.

[0534] Fluorescence observed at the parafoveal, NFL bundle between fovea / ONH and at the RGC / NFL areas was considered test article-related corroborating the transduction and expression of constructs.

[0535] Test Articles related findings included areas of discoloration in retina and retinal fluorescence.

[0536] Multifocal areas of discoloration in retina were observed in 2, 1 and 3 of 4 eyes injected with AAV2Q, AAV2 and AAV 8, respectively at Day 28.

[0537] There were no changes in the data to indicate administration with the AAV GFP constructs had an effect on intraocular pressure, when compared with baseline values and across the dose groups.B. Tissue Bioanalysis

[0538] The objective of this sample analysis study was to evaluate the biodistribution of AAV GFP constructs which carried the bovine growth hormone (bGH) sequence, when administered via a single suprachoroidal injection in Cynomolgus monkeys followed by a 28-day observation period.1. Sample analysis by qPCR:

[0539] AAV GFP constructs carried the bovine growth hormone (bGH) polyA signal sequence. DNA samples were analyzed for recombinant vector (rAAV-bGH) copy numbers. Almost all eye tissues close to the injection site were positive with abundant levels of vector genomes: sclera, lens, choroid, iris and ciliary body, cornea and retina. AAV2Q and AAV2 animals had consistently higher vector genomes across eye tissues. All optic nerve samples from the dosed groups were positive; however, the vector levels in optic nerve were 2-log higher in AAV8-GFP administered eyes compared with levels in eyes administered AAV2Q- and AAV2-GFP constructs. Only 1 AAV2Q-GFP-dosed optic chiasm sample was positive with vector genomes and the rest of the optic chiasm samples were negative. Outside of the eye tissues, variable levels of vector genome were detected in all spleen, mandibular lymph node, and liver tissues from dosed animals. Only 1 lung sample from an AAV2Q-GFP-dosed lung contained detectable but unquantifiable levels of vector. Quantifiable vector was found in some animals, but not all, for each of the following tissues: spinal cords (cervical, midthoracic, lumbar), occipital lobe, brain, thalamus, kidney, and heart. Vector was undetectable in all sex tissues (ovary and testes).2. Sample analysis by RT-qPCR:

[0540] RNA samples were analyzed for the bovine growth hormone (bGH) polyA signal sequence copy numbers by RT-qPCR. AAV GFP constructs-derived bGH-containing mRNA was mainly detected in the eye tissues adjacent to the injection sites including cornea, iris and ciliary body, retina, choroid and sclera, but not the lens. However, not alleye tissues had detectable bGH-containing mRNA. This contrasts with the detection of vector genomes in all eye tissues and at abundant levels in most eye tissues. In addition, the optic nerve from AAV8-GFP-dosed animals had quantifiable bGH-containing mRNA. Outside of these tissues, only 1 tissue from an AAV2-GFP-dosed animal (cervical spinal cord sample) and 1 tissue from an AAV8-GFP-dosed animal (spleen sample) contained unquantifiable bGH RNA copies, and the rest of the tissues, including those tissues with quantifiable vector genomes, were all negative for bGH-containing mRNA.3. Immunohi stochemi stry

[0541] Tissues collected for immunohistochemistry were fixed in freshly prepared neutral buffered formalin for 24 hours at room temperature. After fixation tissues were placed in 70% ethanol. Tissues collected for qPCR and RT-qPCR were preserved in RNAlater.

[0542] IHC detection of EGFP expression was noted in a small percentage of cells in the retinal layers of all animals on study. In optic nerve samples, the percentage of EGFP expression was affected by the size of sections in optic nerves. Additional analysis was performed in nerve fiber layer (NFL), ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), retinal pigment epithelium (RPE) and choroid. The percentage of positive cells varied across retina layers with RPE showing the highest percentage of positive cells across all samples. Very few cells were positive in the INL and ONL compared to GCL. The percentage of positive cells in the choroid layer varied across samples. NFL also showed positivity in a limited number of cells across samples. Results are shown in Table 4. Exemplary IHC images are shown in FIGs. 2C and 4A- 4DTable 4.

[0543] LS Red IHC assay was performed to detect EGFP protein on a serial section in each of six formalin fixed NHP eye samples (Level 2 and 3). Anti-EGFP (Mouse monoclonal, catalog# abl84601, Abeam) 1:600 (1.67 pg / ml) was incubated for 30 minutes at room temperature and detected using an AP conjugated compact polymer system. Fast red was used as the chromogen. Anti-EGFP expression was noted in a small percentage of cells in retinal layers across both levels (L2 and L3) of all the samples. The percentage of EGFP positive was affected by the size of sections in Optic Nerves.Additional analysis was performed in NFL, GCL, INL, ONL, RPE, and choroid. The percentage of positive cells varied across various layers with RPE showing the highest percentage of positive cells across all samples. Very few cells were positive in the INL and ONL compared to GCL. The percentage of positive cells in the choroid layer varied across samples. NFL also showed positivity in a limited number of cells across samples.

[0544] H&E staining was performed on separate tissue sections, apart from the marker evaluation tissue slides, to aid in identifying various cell types and review of the tissue morphology in general.

[0545] Anti-EGFP positive cells were seen in both retina, choroid and optic nerve in varying percentage of cells.

[0546] In conclusion, biodistribution was evidenced via fluorescence, quantification of the DNA vector constructs, and expression of the AAV GFP constructs-derived mRNA following a single suprachoroidal injection of AAV2Q-GFP, AAV2-GFP, and AAV8-GFP at 5.0 E+l 1 vg / eye to cynomolgus monkeys (followed by a 28-day observation period). Abundant levels of DNA vector for all constructs were observed in most eye tissues evaluated, and the vector levels in optic nerve samples were much higher with administration of the AAV8-GFP. AAV2Q-GFP was also detected in 1 sample of the optic chiasm. Vectors were consistently found in spleen, mandibular lymph node, liver, and lung tissues and less consistently in the spinal cords (cervical, midthoracic, lumbar), occipital lobe, brain, thalamus, kidney and heart. AAV GFP-construct mRNA levels were mainly detected in the eye tissues adjacent to the injection sites including cornea, iris and ciliary body, retina, choroid, sclera, and AAV8-GFP optic nerve, but not the lens. There was minimal mRNA in tissues outside of the eye. Protein expression of EGFP was noted in a small percentage of cells in the retinal layers and variable percentages in the optic nerve of all animals on study (AAV2Q-, AAV2- and AAV8-constructs).

[0547] Administration of AAV2-GFP into the SCS using the Suprachoroidal Injector was well tolerated. Circumferential transduction occurred past the posterior pole, and across layers of the retina beyond the RPE. After 4 weeks, vector genomes were detected in all ocular tissues tested and transgene mRNA was detected in the posterior eye cup layers, which corresponded to detectable GFP protein by cSLO and H4C. Overall, the Suprachoroidal Injector successfully dosed AAV to the SCS, and AAV2-GFP transduced layers of the retina from the RPE through ganglion cells, supporting the use of AAV2 as a SCS-delivered gene therapy vector for treating retinal diseases.

[0548] When injected using the Suprachoroidal Injector AAV vectors were able to transduce RPE cells consistently and intensely up to the central macula. This finding differs from previous studies utilizing different microneedle injection systems, which reported that transduction was concentrated around the injection site and faded in intensity posteriorly to the optic nerve (Ding et al., AAV8-vectored suprachoroidal gene transfer produces widespread ocular transgene expression. J Clin Invest.13; 129(11):490I-4911, August 2019 ; Yiu et al., 2019).

[0549] Previous evaluations of serotypes delivered by microneedles reported limited transduction efficiency of AAV2 across the retina (Ding et al., 2019). Unexpectedly, the combination of the Suprachoroidal Injector with AAV2 in the present study exhibited clear transduction of the retina layers through the posterior pole superior to the results reported in literature with microneedles.

[0550] Additionally, the Suprachoroidal Injector allowed for injection of sufficient AAV to reach the fovea too, and not only to transduce RPE cells, but also other layers of the retina including evidence of transduction through ganglion cells, a feature previously not reported with AAVs delivery by microneedle (Yiu et al., 2019).

[0551] Further, while previous reports on AAV suprachoroidal delivery with microneedles reported an upper limit volume of 100 |1L, the present study shows that higher volume single injections of AAV (e.g., 120 |1L) can be administered and tolerated, providing an additional benefit for total vg dosing over other microneedle systems and AAV vectors combinations.Example 3 (Prophetic): Suprachoroidal Administration of AAV-CR2-CR1 with the Injector for the Treatment of Geographic Atrophy Secondary to Age-related Macular Degeneration in Humans

[0552] This first-in-human (FIH) clinical study is designed to assess the safety, tolerability, and efficacy of a single suprachoroidal injection administration of a recombinant adeno-associated viral (rAAV) vector, which includes an AAV serotype 2 capsid containing a single-stranded encapsidated vector genome designed to express a recombinant CR2-CR1 fusion protein comprised of the ligand-binding regions of complement receptor 2 (CR2) and complement receptor 1 (CR1), referred to herein as AAV2-CR2-CR1. AAV2-CR2-CR1 is administered using the Suprachoroidal Injector, a sterile, single use, targeting to the posterior segment of the suprachoroidal space (SCS).

[0553] In this study, the Suprachoroidal Injector is used to deliver an AAV2-CR2-CR1 in adults diagnosed with geographic atrophy (GA) secondary to Age-related Macular Degeneration (AMD).

[0554] Since the Suprachoroidal Injector is designed for single use, no calibration or maintenance is required. The Injector is designed to be used together with a standard Luer-lock syringe.

[0555] The Suprachoroidal Injector is part of the Suprachoroidal Drug Delivery System (SDDS) (Everads Therapy Ltd.), which consists of the Injector and 2 accessories: a Fixation tool and an Extension kit. The Fixation tool is an accessory tool and is a singleuse sterile device designed for atraumatic eye fixation during injection. It holds the eye and limits movement to allow easy penetration of Injector into the sclera. The Extension kit is an optional accessory tool and is a single-use device that allows injection of thetherapy by an assistant without touching the plunger of the syringe in the device, thereby alleviating any potential interference of the procedure performed by the ophthalmologist.

[0556] This study will evaluate the safety of the Suprachoroidal Injector for suprachoroidal administration of an AAV2-CR2-CR1. Participants will be monitored for any signs of procedure-related or device-related adverse events (AEs). Frequency of any device-related AEs and serious adverse events (SAEs) in the study eye following the administration of AAV2-CR2-CR1 with the Suprachoroidal Injector is assessed.

[0557] In addition, performance of the Suprachoroidal Injector for suprachoroidal administration of AAV2-CR2-CR1 will be evaluated. Delivery of AAV2-CR2-CR1 into the suprachoroidal space is monitored by ocular thermography.

[0558] Investigational interventions in this study include an AAV2-CR2-CR1 (investigational medicinal product) and the Suprachoroidal Injector (investigational device). A summary of the administration details for these products is provided in Table 5.Table 5: Study Interventions Administered

[0559] The suprachoroidal injections are done by physicians who have been trained.

[0560] On Day 1, all participants are administered AAV2-CR2-CR1 by suprachoroidal injection. The delivery of AAV2-CR2-CR1 will be performed with the Suprachoroidal Injector and fixator tool. The study eye must have an IOP <25 mmHg prior to treatment. Topical antiseptic is used according to standard practice prior to sterile suprachoroidal injection. Prior to injection, anesthesia may be administered (2 to 3 drops of proparacaine 3X, 3 to 5 minutes apart plus lidocaine gel IX OR subconjunctival lidocaine injection) and a broad-spectrum microbicide may be applied to periocular skin, eyelid and ocular surface.

[0561] The Suprachoroidal Injector employs a delivery method which follows three (3) main steps:

[0562] 1) Controlled tangential insertion of needle into the sclera up to the stopper.

[0563] 2) Extension of the blunt tissue separator into the suprachoroidal space.

[0564] 3) Retraction of the tissue separator, leaving a channel into the suprachoroidal space through which the injectate flows into the SDS.

[0565] Once a channel is created, AAV2-CR2-CR1 is injected. The study eye is then assessed by indirect ophthalmoscopy immediately after the injection for safety evaluation. A non-contact thermographic camera is used to take thermal images of the study eye during suprachoroidal delivery of AAV2-CR2-CR1, in order to confirm injection into the SCS and not into the vitrious (IVT)

[0566] Participants will receive 40 mg / day prednisone (or equivalent) orally once daily starting on Day 7, for 7 days. This will be followed by a 14-day taper.

[0567] Participants will also take corticosteroid eye drops (Durezol or equivalent) starting on Day 1, followed by a taper off Durezol and a switch to prednisolone through Day 128.

[0568] Following treatment, with AAV2-CR2-CR1 by suprachoroidal injection using the Suprachoroidal Injector and fixator tool, the rate of GA lesion growth (as assessed by fundus autofluorescence (FAF), the rate of photoreceptor (PR) and retinal pigment epithelium (RPE) area loss (as assessed by optical coherence tomography (OCT)), best- corrected visual acuity (BCVA), low-luminance visual acuity (LLVA), are assessed periodically over the course of the study period.

Claims

- Ill -WHAT IS CLAIMED IS:

1. A method of delivering a pharmaceutical composition comprising a recombinant adeno- associated virus (rAAV) vector to an eye of a subject, the method comprising: administering the pharmaceutical composition to the eye with an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the pharmaceutical composition is injected through the needle into the suprachoroidal space.

2. A method of delivering a pharmaceutical composition comprising a rAAV vector to an eye of a subject, the method comprising: (i) fixating a target portion of the sclera of the eye, (ii) penetrating the sclera portion with an injection device comprising a needle and a tissue separator, (iii) creating a channel through the choroid of the eye with the tissue separator, and (iv) injecting the pharmaceutical composition through the needle into the suprachoroidal space.

3. A method of treating an ocular pathology in a subject comprising administering a pharmaceutical composition comprising a rAAV vector into the suprachoroidal space of an eye of the subject with an injection device comprising a needle and a tissue separator, wherein the injection device creates a channel through the choroid of the eye and the pharmaceutical composition is injected into the suprachoroidal space.

4. A method of treating an ocular pathology in a subject comprising administering a pharmaceutical composition comprising a rAAV vector to an eye of a subject, the method comprising: (i) fixating a target portion of the sclera of the eye with a fixation device, (ii) penetrating the sclera portion with an injection device comprising a needle and a tissue separator, (iii) creating a channel through the choroid of the eye with the tissue separator, and (iv) injecting the pharmaceutical composition through the needle into the suprachoroidal space.

5. The method of any one of claims 1-4, wherein the method comprises insertion of the needle at a shallow angle.

6. The method of claim 5, wherein the shallow angle is 0° to 30° relative to the sclera portion of the eye.

7. The method of claim 6, wherein the shallow angle is 0° to 10° relative to the sclera portion of the eye.

8. The method of any one of claims 5-7, wherein the insertion of the needle penetrates the sclera at an approximately tangential angle.

9. The method of any one of claims 1-8, wherein the needle is extended about 1 mm to 5 mm in length within the sclera layer before penetrating the choroid.

10. The method of claim 9, wherein the needle is extended about 1 mm to 2 mm in length within the sclera layer before penetrating the choroid.

11. The method of any one of claims 1-10, wherein the tissue separator is within the lumen of the needle.

12. The method of claim 11, wherein the distal tip of the tissue separator is positioned proximal to the distal tip of the needle during insertion.

13. The method of any one of claims 1-12, wherein the distal tip of the tissue separator penetrates into the sclera portion and into the choroid to create the channel through the choroid.

14. The method of any one of claims 1-13, wherein the tissue separator causes blunt dissection of the sclera and choroid thereby creating the channel through the choroid.

15. The method of any one of claims 1-14, wherein the pharmaceutical composition flows through the channel in the choroid into the posterior segment of the eye.

16. The method of any one of claims 1-15, wherein the pharmaceutical composition flows through the channel in the choroid to the retina, to the retinal pigmented epithelium (RPE, e.g., choroid-RPE), and / or to the ciliary bodies.

17. The method of any one of claims 1-16, wherein the delivery or administration comprises a volume of about 0.1 mL to about 0.5 mL, about 0.1 mL to about 0.3 mL, or about 0.1 mL to about 0.25 mL of the pharmaceutical composition per injection into the suprachoroidal space.

18. The method of any one of claims 1-17, wherein the delivery or administration comprises a volume of greater than 0.1 mL up to about 0.5 mL, greater than 0.1 mL up to about 0.4 mL, or greater than 0.1 mL up to about 0.3 mL of the pharmaceutical composition per injection into the suprachoroidal space.

19. The method of claim 17 or 18, wherein the delivery or administration comprises a total volume of about 0. ImL to about 0.5 mL of the pharmaceutical composition into the suprachoroidal space, optionally the total volume is 0.1 ml to 0.3 mL.

20. The method of any one of claims 1-19 wherein the pharmaceutical composition comprises at least 1E10 vector genomes (vg), of the rAAV vector.

21. The method of any one of claims 1-20, wherein the pharmaceutical composition comprises at least 1E10 vg and at most 1E13 vg of the rAAV vector.

22. The method of any one of claims 1-21, wherein the pharmaceutical composition comprises at least 1E10 vg and at most 1E12 vg of the rAAV vector.

23. The method of any one of claims 1-22, wherein the pharmaceutical composition comprises about 1E10 vg, about 2E10 vg, about 3E10 vg, about 4E10 vg, about 5E10 vg, about 6E10 vg, about 7E10 vg, about 8E10 vg, about 9E10 vg, about 1E11 vg, about 2E11 vg, about 3E11 vg, about 4E11 vg, about 5E11 vg, about 6E11 vg, about 7E11 vg, about 8E11 vg, about 9E11 vg, about 1E12 vg for the rAAV vector.

24. The method of any one of claims 1-23, wherein the delivery or administration is to one eye.

25. The method of any one of claims 1-23, wherein the delivery or administration is to both eyes.

26. The method of any one of claims 1-25, wherein the delivery or administration is a single injection per eye.

27. The method of any one of claims 1-25, wherein the delivery or administration is multiple injections per eye.

28. The method of any one of claims 1-27, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 % of the rAAV vector contacts an ocular tissue.

29. The method of claim 28, wherein the ocular tissue is selected from cornea, sclera, choroid, Brunch’s membrane, pupil, iris, lens, optic disc, optic nerve, macula (e.g., central macula), retina, retinal pigmented epithelium (RPE, e.g., choroid-RPE), ciliary bodies, fovea, or any combination thereof.

30. The method of claim 29, wherein the ocular tissue comprises a retina.

31. The method of claim 30, wherein the ocular tissue comprises a retina and the rAAV vector contacts the inner limiting membrane, the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the middle limiting membrane, the outer plexiform layer, the outer nuclear layer, the external limiting membrane, the photoreceptor inner / outer segments, the retinal pigmented epithelium (RPE, e.g., choroid- RPE), or any combination thereof.

32. The method of any one of claims 28-31, wherein the rAAV vector contacts one or more ocular cells.

33. The method of claim 32, wherein the one or more ocular cells are a retinal cell selected from a retinal pigmented epithelial (RPE, e.g., a choroid-RPE) cell, a ganglion cell, a bipolar cell, a horizontal cell, an amacrine cell, a Muller glia cell, a choroid epithelial cell, an optic nerve cell, or an optic fiber, or any combination thereof.

34. The method of claim 32 or 33, wherein the rAAV vector transduces the one or more ocular cells.

35. The method of claim 34, wherein the one or more ocular cells are retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells, retinal ganglion (RG) cells, or a combination thereof.

36. The method of claim 34 or 35, wherein the cell transduction is circumferential at the posterior pole of the eye.

37. The method of any one of claims 34-36, wherein the rAAV vector transduces ocular cells from the retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells through ganglion cells.

38. The method of any one of claims 34-37, wherein the rAAV vector transduces RPE cells.

39. The method of any one of claims 34-38, wherein the rAAV vector transduces ganglion cells.

40. The method of any one of claims 34-39, wherein the rAAV vector transduces one or more ocular cells in the macula.

41. The method of claim 40, wherein the rAAV vector transduces one or more ocular cells in the central macula.

42. The method of claim 41, wherein the rAAV vector transduces RPE cells to the central macula.

43. The method of any one of claims 34-42, wherein the rAAV vector transduction efficiency is at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal pigmented epithelium (RPE, e.g., choroid-RPE) and / or retinal ganglion (RG) cells.

44. The method of claim 43, wherein the rAAV vector transduction efficiency is at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal ganglion (RG) cells.

45. The method of claim 43 or 44, wherein the rAAV vector transduction efficiency is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the retinal pigmented epithelium (RPE, e.g., choroid-RPE) cells.

46. The method of any one of claims 34-45, wherein the rAAV vector does not readily transduce photoreceptor cells.

47. The method of claim 46, wherein the photoreceptor cells are a rod cells, cone cells, or any combination thereof.

48. The method of claim 46 or 47, wherein the rAAV vector transduction efficiency is at less 5% (e.g., less than 4%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or less than 0.5%) of the photoreceptor cells.

49. The method of any one of claims 1-48, wherein the rAAV vector comprises a capsid, a vector genome, and an expression cassette.

50. The method of claim 49, wherein the vector genome comprises an inverted terminal repeat (ITR).

51. The method of claim 49 or 50, wherein the expression cassette comprises a nucleic acid sequence encoding a therapeutic agent.

52. The method of claim 51, wherein the therapeutic agent is a fusion protein or an antibody.

53. The method of claim 51 or 52, wherein the therapeutic agent is selected from an anti-TNF antibody (e.g., Adalimumab), a CR2-CR1 fusion protein, Fibroblast Growth Factor 21 (FGF21), an inflammasome pathway inhibitor (e.g., a tatCARD fusion protein), a Vascular Endothelial Growth Factor (VEGF)-neutralizing protein (e.g., FLT01), an anti- VEGFAFab, an anti-VEGF protein, a VEGF inhibitor (e.g., Aflibercept), solublemembrane-independent form of CD59 (sCD59), soluble-fms like tyrosine kinase- 1 (sFLT-1), Complement factor CFI, Rab Escort Protein 1 (REP1), choroideremia / Rab Escort Protein 1 (CHM), Retinal Pigment Epithemium specific 65kDa protein (RPE65), RPE65v2, NADH ubiquinone oxidoreductase core subunit 4 (ND4), human MER-proto oncogene Tyrosine Kinase (hMERTK), Retinitis Pigmentosa GTPase Regulator (RPGR), Phosphodiesterase 6A (PDE6A), Phosphodiesterase 6B (PDE6B), cyclic nucleotide gated channel subunit alpha 3 (CNGA3), CNGB3, retinaldehyde binding protein 1 (RLBP1), retinoschisin 1 (RSI), a gene editing factor (e.g., SaCas9, gRNAs), a Optogenetics factor (e.g., ChrimsonR-tdTomato, Channelrhodopsin-2, Multi-Characteristic Opsin (MCO)), or functional fragments thereof, any fusion proteins comprising one or more of the same, or any combination thereof.

54. The method of any one of claims 49-53, wherein the rAAV vector comprises a capsid of or derived from a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRh8, AAVrh9, AAV9, AAVrhlO, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B (e.g. UniProtID: Q6JC40), AAV2.5, AAV2tYF, AAV2quadYF, AAV3B (e.g. UniProt ID: 056139), AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, or AAV2.5T, AAV2.5T.LSV1, or a modified version thereof.

55. The method of claim 54, wherein the capsid serotype is AAV2 or a modified version thereof or AAV8 or a modified version thereof.

56. The method of claim 55, wherein the capsid serotype is AAV2 or AAV8.

57. The method of any one of claims 3-56, wherein the ocular pathology for treatment is selected from Achromatopsia, Behcet's Disease, Best's disease, Bietti's Crystalline Dystrophy, Blepharitis, Blepharospasm, Central Areolar Choroidal Dystrophy, Central Serous Chorioretinopathy, Choroideremia, Choroidal Melanoma, Coloboma, Corneal Conditions, Coat's Disease, Cone-Rod Dystrophy, Corneal Dystrophy, Cystoids MacularEdema, Diabetic Retinopathy, Doyne Honeycomb Retinal Dystrophy, Dry Eye, Fuch's Dystrophy, Glaucoma, Hypertensive Retinopathy, Idiopathic Intracranial Hypertension, Lattice Degeneration, Leber Congenital Amaurosis, Leber Hereditary Optic Neuropathy, Leber's Miliarly Aneurism, Macular Degeneration (including Age-Related Macular Degeneration, Juvenile Macular Degeneration, Atrophic Macular Degeneration), Macular Edema, Ocular Histoplasmosis (including Ocular Histoplasmosis Syndrome), Ocular Ischemic Syndrome, Papillophlebitis, Pink Eye, Polypoidal Choroidal Vasculopathy, Retinitis Pigmentosa, Retinoblastoma, Retinopathy of Prematurity, Retinoschisis (including Juvenile Retinoschisis), Sorsby's Disease, Stargardt Disease, Toxoplasmosis, Thyroid Eye Disease (TED), Usher Syndrome, and Uveitis (e.g., Noninfectious Uveitis), Vascular Occlusions, Inflammations (such as uveitis, choroiditis and retinistis), various Tumors (including neoplasms), or any combination thereof, optionally the ocular pathology for treatment is Geographic Atrophy (GA) secondary to Age-related Macular Degeneration (AMD).

58. The method of any one of claims 1-57, wherein the injection into the suprachoroidal space is a single dose.

59. The method of claim 58, wherein the single dose is administered at a single injection site.

60. The method of claim 58, wherein the single dose is administered at multiple injection sites.

61. The method of any one of claims 1-57, wherein the injection into the suprachoroidal space is multiple doses.

62. The method of claim 61, wherein each dose of the multiple doses is administered at a single injection site.

63. The method of claim 61, wherein each dose of the multiple doses is administered at different injection sites.

64. The method of any one of claims 1-63, wherein the injection device comprises:(a) the needle comprising a needle lumen extending between a needle proximal end and a needle distal end, said needle distal end including a size transitional portion terminating in a sharp needle distal tip;(b) the tissue separator having a separator distal tip, at least a portion of said tissue separator being shiftable in the needle lumen; and(c) an actuator configured to shift said tissue separator in said needle lumen between a first position, wherein said separator distal tip is fixedly positioned proximally to said needle distal tip, and a second position, wherein said separator distal tip is fixedly positioned a first predetermined distance distally from said needle distal tip.

65. The method of claim 64, wherein the separator distal tip is in said first position, said separator distal tip protrudes from a laterally uncovered section of said size transitional portion.

66. The method of claim 64 or 65, wherein the injection device further comprises a reservoir (e.g., a syringe).

67. The method of any one of claims 64-66, wherein the injection device further comprises a cannula fluidly connectable between a syringe and said needle lumen, so that a fluid (e.g., the pharmaceutical composition) injected from said syringe is directly flowable to said needle distal tip via said cannula and said needle lumen.

68. The method of claim 66 or 67, wherein the syringe is connectable to a separator hub of said separator.

69. The method of any one of claims 1-68, wherein the needle has a length of about 1 mm to about 5 mm.

70. The method of claim 69, wherein the needle has a length of 1.5 mm.

71. The method of any one of claims 1-70, wherein the needle has an internal diameter of about 0.1 mm to about 0.3 mm72. The method of claim 71, wherein the needle has an internal diameter of 0.3.

73. The method of any one of claims 1-72, wherein the tissue separator has a blunt tip.

74. The method of claim 73, wherein the tissue separator comprises a slender probe.

75. A method of delivering a pharmaceutical composition comprising a rAAV vector to the suprachoroidal space in an eye of a subject, the method comprising:a. providing device for injection into the suprachoroidal space of the subject, the device comprising:a needle comprising a needle lumen extending between a needle proximal end and a needle distal end, said needle distal end including a size transitional portion terminating in a sharp needle distal tip;an elongated tissue separator having a separator distal tip, at least a portion of said tissue separator being shiftable in the needle lumen; andan actuator configured to shift said tissue separator in said needle lumen between a first position, wherein said separator distal tip is fixedly positioned proximally to said needle distal tip, and a second position, wherein said separator distal tip is fixedly positioned a first predetermined distance distally from said needle distal tip;b. inserting tangentially the needle cannula of no more than 2 mm into an ocular layer in said eye;c. extending a tissue separator from within said sharp tip to a second position, wherein said separator distal tip is fixedly positioned a first predetermined distance distally from said needle distal tip into an ocular layer a distance of from 0.3 mm to 10 mm; andd. injecting the pharmaceutical composition into the suprachoroidal space.

76. The method of claim 75, wherein the method further comprises using said actuator to shift said tissue separator longitudinally in said needle lumen such that said separator distal tip is repositioned to a distal-most position, wherein said separator distal tip is located a second predetermined length distally to said needle distal tip, thereby advancing in said outer surface layer and penetrating through said outer surface layer into the interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) of the eye and forming a passage in said interlayer (i.e., within choroid layers, or / and between choroid and retina of the eye) with said separator distal tip.

77. A kit comprising: (i) a recombinant adeno-associated virus (rAAV) vector comprising a capsid, a vector genome, and an expression cassette, (ii) an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the rAAV vector can be injected through the needle into the suprachoroidal space, and (iii) instructions for using the injection device to administer the rAAV to the suprachoroidal space of the eye.

78. The kit of claim 77, wherein the injection device is provided to a user disassembled, readily assembled in a form of a system, or provided as an assembled device.

79. The kit of claim 77 or 78, wherein the injection device is provided with a preloaded reservoir, e.g., a syringe, comprising the rAAV vector.

80. The kit of any one of claims 77-79, wherein the rAAV vector is formulated for administration of at least 1E10 vg to at most 1E12 vg / eye.

81. A kit for administering at least 1E10 vg to at most 1E12 vg of a recombinant adeno- associated viral (rAAV) vector to an eye, wherein the kit comprises: (i) a reservoir comprising a pharmaceutical composition comprising the rAAV vector which comprises a capsid, a vector genome, and an expression cassette; and (ii) an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the pharmaceutical composition can be injected through the needle into the suprachoroidal space.

82. The kit of claim 81, wherein the injection device is provided disassembled, readily assembled in a form of a system, or provided as an assembled device.

83. A system for administering a recombinant adeno-associated viral (rAAV) vector to the eye of a subject, wherein the system comprises: (i) a reservoir comprising a pharmaceutical composition comprising the rAAV vector which comprises a capsid, a vector genome, and an expression cassette; and (ii) an injection device comprising a needle and a tissue separator, wherein the tissue separator creates a channel through the choroid of the eye and the pharmaceutical composition can be injected through the needleinto the suprachoroidal space, wherein the reservoir is preloaded with the pharmaceutical composition for administration of at least 1E10 vg / eye to at most 1E12 vg / eye of the rAAV vector.

84. The kit or system of any one of claims 79-83, wherein the reservoir comprises at least IE 10 vector genomes (vg) of the rAAV vector.

85. The kit or system of any one of claims 79-84, wherein the reservoir comprises at least IE 10 vg to at most IE 13 vg of the rAAV vector.

86. The kit or system of any one of claims 79-85, wherein the reservoir comprises a total volume of at least 0.1 mL of the pharmaceutical composition.

87. The kit or system of any one of claims 79-86, wherein the reservoir comprises a total volume of at least 0.1 mL to about 0.5 mL (e.g., about 0.1 mL to about 0.25 mL) of the pharmaceutical composition.

88. The kit or system of any one of claims 79-87, wherein the reservoir is for a single administration.

89. The kit of any one of 79-88 which comprises two or more reservoirs.

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