WO2020136384A1

WO2020136384A1 - Methods of retinal administration

Info

Publication number: WO2020136384A1
Application number: PCT/IB2018/001567
Authority: WO
Inventors: Ian Jeffery CONSTABLE
Original assignee: Constable Ian Jeffery
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2020-07-02

Abstract

Provided herein are methods of administering a therapeutic agent to a retina of an eye of an individual. Methods herein comprise contacting an area of the retina of an eye from which a vitreous and at least a portion of an internal limiting membrane (ILM) have been removed to expose under an atmospheric gas at least a portion of a retina to a composition comprising a therapeutic agent or nucleic acid encoding a therapeutic protein.

Description

METHODS OF RETINAL ADMINISTRATION

BACKGROUND

[0001] The unique anatomy and physiology of the eye creates a challenge in ocular delivery of therapeutics to treat diseases of the eye. These include static barriers including layers of cornea, sclera, and retina (blood aqueous and blood retinal barriers) as well as dynamic barriers such as choroidal and conjunctival blood flow, lymphatic clearance, and tear dilution. Delivery of therapeutics to the posterior segment and the retina is particularly challenging.

SUMMARY

[0002] Provided herein are methods of expressing a therapeutic protein in a retina of an eye of an individual. In some embodiments, the method comprises: contacting an area of the retina of an eye from which a vitreous and at least a portion of an internal limiting membrane (ILM) have been removed to expose at least a portion of a retina with a composition comprising a nucleic acid encoding the therapeutic protein. In some embodiments, the therapeutic protein comprises RPE65, a VEGF inhibitor such as aflibercept or Ranibizumab, REP-1, MERTK, ABC4, ND4, CD59, RSI, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B, RLBPl, channelrhodopsin-2, or MY07A. In some embodiments, the VEGF inhibitor is aflibercept or Ranibizumab. In some embodiments, the nucleic acid comprises a viral vector. In some embodiments, the composition comprises a viral capsid protein. In some embodiments, the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus. In some embodiments, expression of the therapeutic protein is effective to reduce at least one symptom of an eye disease. In some embodiments, the eye disease is selected from age-related macular degeneration, choroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion. In some embodiments, the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual. In some embodiments, the method further comprises maintaining the individual in a substantially supine position for at least 30 minutes. In some embodiments, the contacting occurs in the presence of an atmospheric gas or a therapeutic gas. In some embodiments, the therapeutic gas is sulphur hexafluoride or octofluorocy cl obutane .

[0003] Also provided herein are methods of expressing a therapeutic protein in a retina of an eye of an individual. In some embodiments, the method comprises: a) removing a vitreous from a vitreal chamber of the eye; b) removing at least a portion of an internal limiting membrane (ILM) from an area of the eye to expose at least a portion of a retina; and c) contacting a composition comprising a nucleic acid encoding the therapeutic protein to at least a portion of the retina. In some embodiments, the therapeutic protein comprises RPE65, a VEGF inhibitor such as aflibercept or Ranibizumab, REP-1, MERTK, ABC4, ND4, CD59, RSI, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B, RLBPl, channelrhodopsin-2, or MY07A. In some embodiments, the VEGF inhibitor is aflibercept or Ranibizumab. In some embodiments, the nucleic acid comprises a viral vector. In some embodiments, the composition comprises a viral capsid protein. In some embodiments, the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus. In some embodiments, expression of the therapeutic protein is effective to reduce at least one symptom of an eye disease. In some embodiments, the eye disease is selected from age-related macular degeneration, choroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion. In some embodiments, the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual. In some embodiments, the ILM is visualized with a dye prior to step b). In some embodiments, the method further comprises maintaining the individual in a substantially supine position for at least 30 minutes, at least 45 minutes, or at least 60 minutes. In some embodiments, the individual is maintained in a substantially supine position for about 30 to about 90 minutes. In some embodiments, the individual is maintained in a substantially supine position for about 45 to about 75 minutes. In some embodiments, the method further comprises injecting an atmospheric gas or a therapeutic gas into the vitreal chamber prior to step c). In some embodiments, the therapeutic gas is sulphur hexafluoride or octofluorocyclobutane.

[0004] A method of topical administration to a retina in an individual of a gene therapy product, the method comprising: a) removing a vitreous from a vitreal chamber of an eye of the individual; b) removing at least a portion of an internal limiting membrane (ILM) of the eye to expose at least a portion of a retina; and c) contacting a composition comprising a nucleic acid encoding a therapeutic protein to at least a portion of the retina. In some embodiments, the therapeutic protein comprises RPE65, a VEGF inhibitor such as aflibercept or Ranibizumab, REP-1, MERTK, ABC4, ND4, CD59, RSI, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B,

RLBPl, channelrhodopsin-2, or MY07A. In some embodiments, the VEGF inhibitor is aflibercept or Ranibizumab. In some embodiments, the nucleic acid comprises a viral vector. In some embodiments, the composition comprises a viral capsid protein. In some embodiments, the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus. In some embodiments, the gene therapy product is effective to reduce at least one symptom of an eye disease. In some embodiments, the eye disease is selected from age-related macular degeneration, choroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic

Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion. In some embodiments, the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual. In some embodiments, the ILM is visualized with a dye prior to step b). In some embodiments, the method further comprises maintaining the individual in a substantially supine position for at least 30 minutes, at least 45 minutes, or at least 60 minutes. In some embodiments, the individual is maintained in a substantially supine position for about 30 to about 90 minutes. In some embodiments, the individual is maintained in a substantially supine position for about 45 to about 75 minutes. In some embodiments, the method further comprises injecting an atmospheric gas or a therapeutic gas into the vitreal chamber prior to step c). In some embodiments, the therapeutic gas is sulphur hexafluoride or octofluorocyclobutane.

[0005] Also provided herein are methods of treating an eye disease in an individual. In some embodiments, the method comprises: a) removing a vitreous from a vitreal chamber the eye; b) removing at least a portion of an internal limiting membrane (ILM) of the eye to expose at least a portion of a retina; and c) contacting a composition comprising a nucleic acid encoding a therapeutic protein effective in treating the eye disease to at least a portion of the retina. In some embodiments, the therapeutic protein comprises RPE65, a VEGF inhibitor such as aflibercept or Ranibizumab, REP-1, MERTK, ABC4, ND4, CD59, RSI, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B, RLBPl, channelrhodopsin-2, or MY07A. In some embodiments, the VEGF inhibitor is aflibercept or Ranibizumab. In some embodiments, the nucleic acid comprises a viral vector. In some embodiments, the composition comprises a viral capsid protein. In some embodiments, the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus. In some embodiments, expression of the therapeutic protein is effective to reduce at least one symptom of an eye disease. In some embodiments, the eye disease is selected from age-related macular degeneration, choroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion. In some embodiments, the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual. In some embodiments, the ILM is visualized with a dye prior to step b). In some embodiments, the method further comprises maintaining the individual in a substantially supine position for at least 30 minutes, at least 45 minutes, or at least 60 minutes. In some embodiments, the individual is maintained in a substantially supine position for about 30 to about 90 minutes. In some embodiments, the individual is maintained in a substantially supine position for about 45 to about 75 minutes. In some embodiments, the method further comprises injecting an atmospheric gas or a therapeutic gas into the vitreal chamber prior to step c). In some embodiments, the therapeutic gas is sulphur hexafluoride or octofluorocyclobutane.

[0006] Also provided herein are methods of topical administration to a retina in an individual of a therapeutic agent. In some embodiments, the method comprises: a) removing a vitreous from a vitreal chamber an eye of the individual; b) removing at least a portion of an internal limiting membrane (ILM) of the eye to expose at least a portion of a retina; and c) contacting a composition comprising the therapeutic agent to at least a portion of the retina. In some embodiments, the therapeutic agent comprises a protein, a peptide, an antibody, or a small molecule. In some embodiments, the therapeutic agent has a molecular weight greater than or equal to 100 kilodaltons. In some embodiments, the therapeutic agent is a biologically active protein having a molecular weight greater than or equal to 100 kilodaltons. In some

embodiments, the therapeutic agent is a humanized biologically active antibody having a molecular weight greater than or equal to 100 kilodaltons. In some embodiments, the therapeutic agent comprises a VEGF antagonist, a steroid, an anti-fungal agent, an anti-viral agent, or an antibiotic. In some embodiments, the composition comprises a pharmaceutically acceptable buffer or excipient. In some embodiments, the therapeutic agent is effective to reduce at least one symptom of an eye disease. In some embodiments, the eye disease is selected from age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, infection, endophthalmitis, retinitis, choroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion. In some embodiments, the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual. In some embodiments, the ILM is visualized with a dye prior to step b). In some embodiments, the method further comprises maintaining the individual in a

substantially supine position for at least 30 minutes, at least 45 minutes, or at least 60 minutes.

In some embodiments, the individual is maintained in a substantially supine position for about 30 to about 90 minutes. In some embodiments, the individual is maintained in a substantially supine position for about 45 to about 75 minutes. In some embodiments, the method further comprises injecting an atmospheric gas or a therapeutic gas into the vitreal chamber prior to step c). In some embodiments, the therapeutic gas is sulphur hexafluoride or octofluorocyclobutane. [0007] Also provided herein are methods of treating an eye disease in an individual. In some embodiments, the method comprising: a) removing a vitreous from a vitreal chamber the eye; b) removing at least a portion of an internal limiting membrane (ILM) of the eye to expose at least a portion of a retina; and c) contacting a composition comprising a therapeutic agent effective in treating the eye disease to at least a portion of the retina. In some embodiments, the therapeutic agent comprises a protein, a peptide, an antibody, or a small molecule. In some embodiments, the therapeutic agent has a molecular weight greater than or equal to 100 kilodaltons. In some embodiments, the therapeutic agent is a biologically active protein having a molecular weight greater than or equal to 100 kilodaltons. In some embodiments, the therapeutic agent is a humanized biologically active antibody having a molecular weight greater than or equal to 100 kilodaltons. In some embodiments, the therapeutic agent comprises a VEGF antagonist, a steroid, an anti-fungal agent, an anti-viral agent, or an antibiotic. In some embodiments, the composition comprises a pharmaceutically acceptable buffer or excipient. In some

embodiments, the therapeutic agent is effective to reduce at least one symptom of an eye disease. In some embodiments, the eye disease is selected from age-related macular

degeneration, choroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic

[0008] Also provided herein are gene therapy compositions for use in topical administration to a retina of an eye of an individual. In some embodiments, a vitreous and at least a portion of an internal limiting membrane (ILM) have been removed from the eye of the individual comprising a nucleic acid encoding a therapeutic protein and a pharmaceutically acceptable excipient. In some embodiments, the therapeutic protein comprises RPE65, a VEGF inhibitor such as aflibercept or Ranibizumab, REP-1, MERTK, ABC4, ND4, CD59, RSI, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B, RLBPl, channelrhodop sin-2, or MY07A. In some embodiments, the VEGF inhibitor is aflibercept or Ranibizumab. In some embodiments, the nucleic acid comprises a viral vector. In some embodiments, the composition comprises a viral capsid protein. In some embodiments, the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus. In some embodiments, the gene therapy composition is suitable for reducing at least one symptom of an eye disease. In some embodiments, the eye disease is selected from age-related macular degeneration, choroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa,

Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion. In some embodiments, the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual.

[0009] Also provided herein are gene therapy compositions for use in treating an eye disease. In some embodiments, the composition is suitable for topical administration to a retina of an eye of an individual, wherein a vitreous and at least a portion of an internal limiting membrane (ILM) have been removed from the eye of the individual comprising a nucleic acid encoding a therapeutic protein and a pharmaceutically acceptable excipient. In some embodiments, the therapeutic protein comprises RPE65, a VEGF inhibitor such as aflibercept or Ranibizumab, REP-1, MERTK, ABC4, ND4, CD59, RSI, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B,

RLBPl, channelrhodopsin-2, or MY07A. In some embodiments, the VEGF inhibitor is aflibercept or Ranibizumab. In some embodiments, the nucleic acid comprises a viral vector. In some embodiments, the composition comprises a viral capsid protein. In some embodiments, the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus. In some embodiments, the gene therapy composition is suitable for reducing at least one symptom of an eye disease. In some embodiments, the eye disease is selected from age-related macular degeneration, choroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion. In some embodiments, the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual.

[0010] Also provided herein are kits for topical administration of a nucleic acid encoding a therapeutic protein to a retina of an eye of an individual. In some embodiments, the system/kit comprises: a vitreous removal tool; an ILM removal tool; and an applicator tool configured to apply a composition comprising a nucleic acid encoding the therapeutic protein to the retina of the individual. In some embodiments, at least one of the vitreous removal tool and the ILM removal tool comprise a forceps, a scissors, a needle holder, a retractor, a hook, a pick, a fiber optic light pipe, or any combination thereof. In some embodiments, the forceps comprises a smooth forceps, a dressing forceps, a tying forceps, a straight forceps, a curved forceps, a utility forceps, a smooth jaw forceps, a plug forceps, a cross action forceps, a suturing forceps, a sleeve spreading forceps, a membrane peeling vitreous forceps, a membrane peeling microforcep, a blocked tip microforcep, a tapered, non-toothed microforcep, an asymmetrical microforcep, a maxi-grip ILM forcep, or any combination thereof. In some embodiments, the scissors comprise an angled scissors, a blunt scissors, or both. In some embodiments, the needle holder comprises a delicate needle holder, an extra-delicate needle holder, a straight needle holder, a curved needle holder, a locking needle holder, a non-locking needle holder, or any combination thereof. In some embodiments, the retractor comprises an orbital retractor. In some embodiments, the hook comprises a muscle hook. In some embodiments, the pick comprises an ILM pick, a rice pick, a membrane spatula, a round ball pick, a Michaels’s pick, a diamond dusted membrane scraper, a nitinol loop, or any combination thereof. In some embodiments, the kit further comprises a speculum, a stopcock, an infuser adapter, an irrigating contact lens, a depressor, or any combination thereof. In some embodiments, the applicator tool comprises a syringe, a tube, a bottle, a vial, a swab, a brush, a tissue, a pipette, or any combination thereof.

INCORPORATION BY REFERENCE

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0013] FIG. 1 illustrates a comparison of effectiveness of transfection as indicated by GFP signal between vitrectomy-only and vitrectomy and ILM peeled eyes in vivo. The left column shows an example of an eye that undergoes vitrectomy and ILM peel prior to GFP transfection. Color fundus photograph with the excitation filter overlay (top left) shows fluorescent signal of the transfected area of the retina. Transfection is more obviously seen on the fundus

autofluorescence (FAF) image (middle left). The bottom left image shows

immunohistochemistry for GFP on a 7-pm retinal section showing transfection in all layers of the retina. The images on the right column show the level of transfection to be substantially less in the vitrectomy-only eye with only vitrectomy and layering of the AAV-GFP under air. GFP fluorescence is confined only to the foveal area.

[0014] FIG. 2 illustrates a comparison of effectiveness of transfection as indicated by GFP signal between vitrectomy-only and vitrectomy and ILM peeled eyes over time. Serial FAF images of eyes over time from 2 to 16 weeks. Top row shows an example of an eye that underwent vitrectomy and ILM peel. The bottom row shows an example of an eye that underwent only vitrectomy. In both cases, the area of fluorescence does not change throughout the course of the study, with maximal transfection already present at 2 weeks.

[0015] FIG. 3 illustrates a comparison of the extent of transfection between vitrectomy-only and vitrectomy and ILM peeled eye. Top row shows transfected areas of the fovea region of four eyes that undergoes ILM peel. Bottom row shows transfected area in eyes that does not undergo ILM peel.

[0016] FIG. 4 illustrates localization of GFP signal in vitrectomy and ILM peeled eye. Top row shows that calretinin (CR)-stained amacrine cells are not transfected (white arrowheads). Middle row shows that glutamine synthetase (GS)-stained Miiller cells are transfected as indicated by GFP signal (white arrowheads). Bottom row shows that cone arrestin (CS)-stained cone photoreceptors are not transfected. However, distinct rod-like photoreceptors display GFP expression where rods cells are located (arrows, inset).

[0017] FIG. 5 illustrates histological comparison of effectiveness of transfection vitrectomy- only and vitrectomy and ILM peeled eye. Top two rows show images from eyes that undergo vitrectomy and ILM peel. The top two images on the left column show the color fundus photograph with the excitation filter and the FAF image. The dotted line represents the corresponding histology section (top row, second and third column from the left) passing through the fovea center and the solid line shows the corresponding histology section (middle row, second and third column from the left) passing through an area just outside the ILM peeled area). The bottom row shows image of a vitrectomy-only eye with the solid line in the FAF image (bottom left image) representing the histology sections (bottom row, second and third columns from the left. A transfected area as indicated by GFP signal within the ILM peeled area is observed compared with the area outside (middle column top image). Red arrow indicates areas of discrete retinal pigment epithelium (RPE) clumping. The image of control, vitrectomy- only eye indicates absence of transfection (middle column bottom image) and correspondingly normal retina architecture on hematoxylin-eosin staining (third column from the left, bottom image).

[0018] FIG. 6 illustrates anatomical integrity of vitrectomy and ILM peeled eye post transfection. Left image shows intraoperative area of ILM peel as seen as an area of retina not stained by membrane blue duo (dotted line). The corresponding area is marked on the FAF image (middle image) (dotted line). The solid line on the FAF image represents the histological section (left image). The dotted black line on the histology image (right) represents the edge of the ILM peel. The black arrow indicates a blood vessel corresponding to the enface FAF image. Qualitatively, there is minimal disruption of the retinal layers within the peeled area within no obvious thinning (left of black dotted line) as compared with the retina in the non-peeled area (right of black dotted line). The vitrectomy and ILM peeled eye does not show any obvious changes in the retina or RPE layers.

[0019] FIG. 7 shows the use of an ultrasound needle to disrupt an internal limiting membrane (ILM), in which the ultrasound needle is placed parallel to the membrane. The vibrating ultrasound needle creates gradient fields and radiant forces that disrupt the membrane.

[0020] FIG. 8A shows an embodiment of the use of microfluidics to apply a therapeutic agent to the exposed retina. Shown is a capillary tube using the capillary action of microchannels to draw fluid containing molecules of interest (e.g., a therapeutic agent) from a vial onto the exposed retina.

[0021] FIG. 8B shows the use of capillary fibers to wick material onto the exposed retina. The figure shows a cross sectional view (5 pm) of a fiber (40 g or the like) having multiple deep grooves running the length of the fiber to increase the surface area, which improves transport and uptake of the material delivered.

DETAILED DESCRIPTION

[0022] Provided herein are methods of administration of therapeutics to the retina of the eye. In some embodiments, methods herein comprise contacting an area of the retina of an eye from which the vitreous and at least a portion of an internal limiting membrane (ILM) have been removed to expose at least a portion of a retina with a composition comprising a therapeutic agent.

[0023] Provided herein in some embodiments are methods of delivery of gene therapy constructs to the retina. In some embodiments, the methods are suitable for treating target retinal tissue of a patient by injection of predetermined quantities of a substance into the vitreous cavity of the eye which has been temporarily filled with air. In some embodiments, these methods are applied to the delivery of therapeutic substances to the retina to replace defective genes and upregulate production of a specific disease associated protein or antagonist.

[0024] In some embodiments, the methods comprise removing the vitreous of the eye of a subject, detaching the posterior vitreous from the retinal surface, peeling of the Internal Limiting Membrane (ILM) off the retinal surface after staining with trypan and brilliant blue or other vital dye, air / fluid exchange of the vitreous cavity then layering of the therapeutic gene construct solution on the retinal surface under air for 30 minutes to two hours while the subject lies supine.

[0025] In some embodiments, the methods comprise removing the vitreous of the eye of a subject, detaching the posterior vitreous from the retinal surface, air / fluid exchange of the vitreous cavity, disrupting the Internal Limiting Membrane (ILM) with an ultrasound needle and layering of the therapeutic agent onto the disrupted ILM under air for 30 minutes to two hours while the subject lies supine.

Ocular Diseases

[0026] Provided herein are methods of treatment of diseases of the eye or ocular diseases comprising administering a therapeutic agent to the retina. In some embodiments, methods of treatment comprise contacting an area of the retinal of an eye from which a vitreous and at least a portion of an internal limiting membrane (ILM) have been removed to expose at least a portion of the retina with a composition comprising a therapeutic agent. In some embodiments, removal of the vitreous and internal limiting membrane provides direct access for topical administration of therapeutic agents, such as gene therapy vectors, therapeutic proteins, antibodies, nucleic acids, and small molecule therapeutics that may not reach effective concentrations when merely injected into the vitreous of the eye. In some embodiments, atmospheric gas or a therapeutic gas is injected into the vitreal chamber to further improve this effect. In some embodiments, the therapeutic gas is sulphur hexafluoride or octofluorocyclobutane.

[0027] Treatment of diseases requiring treatment of the retina is especially improved. However, any disease of a tissue inside the vitreal chamber is contemplated to be treated by methods herein. Specifically diseases of the vasculature, retina, and neurons of the eye are suitable diseases contemplated herein include but are not limited to age-related macular degeneration, chroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy,

Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, retinal dystrophy, uveitis, proliferative vitreoretinopathy, diabetic

retinopathy, retinal vein occlusion, bacterial infection, viral infection, fungal infection, chorioretinal inflammation, retinoschisis, retinal vascular occlusions, retinal hemorrhage, optic neuritis, papilloedema, and optic atrophy.

Methods of Administration

[0028] Provided herein are methods of treatment of diseases of the eye and methods of delivering therapeutics to the retina of the eye. In some embodiments, methods herein comprise preparation of the eye prior to administering the therapeutic to the retina. In some embodiments, the vitreous is removed. In some embodiments, at least a portion of the internal limiting membrane (ILM) is removed. In some embodiments, the vitreous and at least a portion of the internal limiting membrane are removed. In some embodiments, removal of the vitreous and/or the internal limiting membrane exposes the retina allowing for topical administration of the therapeutic agent. In some embodiments, atmospheric gas or a therapeutic gas is injected into the vitreal chamber after removal of the at least a portion of the internal limiting membrane. Once the eye has been prepared, the therapeutic agent is layered onto the retina. In some embodiments, the therapeutic gas is sulphur hexafluoride or octofluorocyclobutane.

[0029] In some embodiments of methods of treatment provided herein, the vitreous is removed from the eye. In some embodiments, removal of the vitreous is conducted by vitrectomy.

Methods of vitrectomy include but are not limited to posterior pars plana vitrectomy, core vitrectomy, and anterior vitrectomy. In some embodiments, the eye is entered through the pars plana in the sclera of the eye. In some embodiments, the gel-like vitreous is removed by cutting and removing the vitreous using a vitrectomy probe or vitrector. In some embodiments, a core vitrectomy is performed. In some embodiments, core vitrectomy is performed followed by posterior vitreous detachment. In some embodiments the vitrectomy is performed using a Constellation or Millenium (Alcon) vitrectomy machine. In some embodiments, the vitrectomy is performed using an operating microscope (OPMI MDO; Carl Zeiss).

[0030] In further embodiments of methods provided herein, the internal limiting membrane (ILM) is removed or peeled from the eye. In some embodiments, a dye is used to visualize the internal limiting membrane prior to removal. In some embodiments membrane blue-DUAL visualization is used. In some embodiments, at least 5% of the ILM is removed. In some embodiments, at least 10% of the ILM is removed. In some embodiments, at least 20% of the ILM is removed. In some embodiments, at least 25% of the ILM is removed. In some embodiments, at least 30% of the ILM is removed. In some embodiments, at least 40% of the ILM is removed. In some embodiments, at least 50% of the ILM is removed. In some embodiments, at least 60% of the ILM is removed. In some embodiments, at least 70% of the ILM is removed. In some embodiments, at least 75% of the ILM is removed. In some embodiments, at least 80% of the ILM is removed. In some embodiments, at least 90% of the ILM is removed. In some embodiments, at least 95% of the ILM is removed. In some embodiments, at least 99% of the ILM is removed. In some embodiments, the ILM over the macula is removed. In some embodiments the ILM peel is performed using a membrane peeling forcep, pic, or diamond dusted membrane scraper. In some embodiments, the ILM peel is performed using an operating microscope (OPMI MDO; Carl Zeiss).

[0031] In some embodiments, the methods comprise disrupting the ILM using an ultrasound needle rather than removing all or a portion of the ILM. In such embodiments, the ultrasound needle is placed parallel to the ILM, and the vibrating ultrasound needle creates gradient fields and radiant forces that disrupts the ILM to facilitate delivery of the therapeutic agent through the ILM to the retina.

[0032] In additional embodiments of methods provided herein, a therapeutic agent is layered onto the retina of the prepared eye. In some embodiments, the therapeutic agent is layered onto the retina with an applicator tool. In some embodiments, the applicator tool comprises a syringe, a tube, a bottle, a vial, a swab, a brush, a tissue, a pipette, a microcapillary, a capillary fiber, a needle with a dispersive tip, or any combination thereof. In some embodiments, the applicator tool is a microfluidic device comprising a capillary tube or capillary fiber. In some embodiments, a therapeutic agent is applied to the exposed retina using microfluidics. In some instances, a capillary tube using the capillary action of microchannels draws fluid containing a therapeutic agent from a vial and deposits it onto the exposed retina (see, e.g., Fig. 8A). In some embodiments, capillary fibers are used to wick a therapeutic agent from a vial or other container holding the therapeutic agent and deposits it onto the exposed retina. In some instances, a fiber (40 g or the like) multiple deep grooves running the length of the fiber (see, e.g., Fig. 8B or a 4DG fiber) is used. For example, Fig. 8B shows a cross sectional view (5 pm) of a capillary fiber having such grooves which increase the surface area of the fiber, improving the transport and uptake of the material delivered. In some embodiments, the applicator tool is a microfluidic device comprising a capillary tube or capillary fiber. In some embodiments, the applicator tool comprises a microneedle having a dispersive tip capable of providing a fine spray to coat the exposed retina with the therapeutic agent. Therapeutic agents useful in such methods, further detailed elsewhere in the application, include gene therapy constructs, therapeutic proteins, therapeutic nucleic acids, therapeutic antibodies, and small molecule therapeutics. A dose suitable for treating the disease is administered using methods herein. In some embodiments, the individual is left in a supine position for at least 10 minutes after administration of the therapeutic agent. In some embodiments, the individual is left in a supine position for at least 20 minutes after administration of the therapeutic agent. In some embodiments, the individual is left in a supine position for at least 30 minutes after administration of the therapeutic agent. In some embodiments, the individual is left in a supine position for at least 40 minutes after administration of the therapeutic agent. In some embodiments, the individual is left in a supine position for at least 50 minutes after administration of the therapeutic agent. In some embodiments, the individual is left in a supine position for at least 60 minutes after

administration of the therapeutic agent. In some embodiments, the individual is left in a supine position for at least 70 minutes after administration of the therapeutic agent. In some embodiments, the individual is left in a supine position for at least 80 minutes after

administration of the therapeutic agent. In some embodiments, the individual is left in a supine position for at least 90 minutes after administration of the therapeutic agent. In some embodiments, the individual is left in a supine position for at least 100 minutes after

administration of the therapeutic agent. In some embodiments, the individual is left in a supine position for at least 110 minutes after administration of the therapeutic agent. In some embodiments, the individual is left in a supine position for at least 120 minutes after

administration of the therapeutic agent.

[0033] In some embodiments of the methods provided herein, an additional therapeutic is administered to treat adverse symptoms experienced following the surgical procedures. Adverse symptoms contemplated herein include but are not limited to infection, inflammation, and pain. In some embodiments, an antibiotic is administered to the eye. In some embodiments, a steroid is administered to the eye. In some embodiments, dexamethasone or triamcinolone is administered to the eye. In some embodiments, a non-steroidal anti-inflammatory drug is administered to the eye. In some embodiments, flubiprofen is administered to the eye. In some embodiments, an analgesic is administered to the eye. In some embodiments, an analgesic is administered orally.

[0034] In some embodiments of the methods provided herein, the retina of the individual is examined after the treatment. In some embodiments fundus auto-fluorescence or optical coherence tomography is used to visualize the retina. In some embodiments intraoperative optical coherence tomography is used to visualize the retina. In some embodiments, the retina is examined about one week after the treatment. In some embodiments, the retina is examined about two weeks after the treatment. In some embodiments, the retina is examined about three weeks after the treatment. In some embodiments, the retina is examined about four weeks after the treatment. In some embodiments, the retina is examined about five weeks after the treatment. In some embodiments, the retina is examined about six weeks after the treatment. In some embodiments, the retina is examined about seven weeks after the treatment. In some embodiments, the retina is examined about eight weeks after the treatment. In some

embodiments, the retina is examined about nine weeks after the treatment. In some

embodiments, the retina is examined about ten weeks after the treatment. In some embodiments, the retina is examined about 12 weeks after the treatment. In some embodiments, the retina is examined about 16 weeks after the treatment. In some embodiments, the retina is examined about 20 weeks after the treatment. In some embodiments, the retina is examined about 24 weeks after the treatment. In some embodiments, the retina is examined about 30 weeks after the treatment. In some embodiments, the retina is examined about 40 weeks after the treatment.

[0035] In some embodiments of the methods provided herein, treatment of the eye using methods provided herein results in increased efficacy compared with treatment of the eye using conventional methods. In some embodiments, conventional methods do not include the step of removing the ILM before administering the therapeutic agent. In some embodiments, methods herein are at least about 10% more effective. In some embodiments, methods herein are at least about 20% more effective. In some embodiments, methods herein are at least about 30% more effective. In some embodiments, methods herein are at least about 40% more effective. In some embodiments, methods herein are at least about 50% more effective. In some embodiments, methods herein are at least about 60% more effective. In some embodiments, methods herein are at least about 70% more effective. In some embodiments, methods herein are at least about 80% more effective. In some embodiments, methods herein are at least about 90% more effective. In some embodiments, methods herein are at least about 100% more effective. In some embodiments, methods herein are at least about 125% more effective. In some

embodiments, methods herein are at least about 150% more effective. In some embodiments, methods herein are at least about 175% more effective. In some embodiments, methods herein are at least about 200% more effective.

[0036] In some embodiments of the methods provided herein, treatment of the eye using methods provided herein results in increased numbers of cells receiving the therapeutic agent compared with treatment of the eye using conventional methods. In some embodiments, conventional methods do not include the step of removing the ILM before administering the therapeutic agent. In some embodiments, methods herein deliver the therapeutic agent to at least about 10% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 20% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 30% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 40% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 50% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 60% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 70% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 80% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 90% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 100% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 125% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 150% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 175% more cells. In some embodiments, methods herein deliver the therapeutic agent to at least about 200% more cells. [0037] In some embodiments, the methods achieve a rapid high level of gene transfection and target protein production in all layers of the retina without subretinal injection. In some embodiments, the method comprises prior vitrectomy which removes any neutralising antibodies, ILM peeling then full air / fluid exchange, and layering of the gene therapy construct on the peeled retinal surface under air for 30 minutes to 2 hours leading to intense target protein production by both superficial and deep retinal cells within one week. In some embodiments, the area of ILM peeled is varied depending on the area of transfection and dose response of the target protein required. In some embodiments, repeat gene therapy administration is done if necessary as neither the vitreous nor the ILM regenerate. Both air/fluid exchange and layering of the gene product can be carried out in an outpatient day surgery setting. In some embodiments, the vitreous cavity is cleared of the gene therapy viral construct any time after 1 hour to eliminate the known risk of an immune- based inflammatory reaction in the vitreous chamber. Therapeutic agents

[0038] Provided herein are methods of treatment of diseases of the eye or ocular diseases comprising administering a therapeutic agent to the retina. Therapeutic agents contemplated herein include but are not limited to therapeutic agents suitable for treating diseases of the eye, such as diseases of the eye described herein, for example age-related macular degeneration, retinopathy, chroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion, bacterial infection, viral infection, fungal infection, chorioretinal inflammation, retinoschisis, retinal vascular occlusions, retinal dystrophy, retinal hemorrhage, optic neuritis, papilloedema, and optic atrophy. Therapeutic agents for use in methods of treatment herein are selected by a medical provider based on efficacy for treating the disease of the individual.

Thereapeutic agents include but are not limited to therapeutic proteins, therapeutic antibodies, therapeutic nucleic acids, small molecule therapeutics, and gene therapy constructs.

Therapeutic Proteins

[0039] Provided herein are methods of treatment of diseases of the eye or ocular diseases comprising administering a therapeutic protein to the retina. Methods herein provide therapeutic proteins directly to the interior of the eye, such as the retina, where they are needed. In some embodiments, the therapeutic protein is a biologically active protein having a molecular weight greater than or equal to 100 kilodaltons. Suitable therapeutic proteins include proteins replacing a missing or defective protein in the eye of the individual and inhibitory proteins that bind to or otherwise inhibit an overactive protein in the eye of the individual. A non-exhaustive list of therapeutic proteins include but is not limited to RPE65, VEGF inhibitory peptides, REP-1, MERTK, ABC4, ND4, CD59, RSI, aflibercept, Ranibizumab, sFLTOl, CNGB3, CNGA3, RPGR, PDE6, RLBPl, chanelrhodopsin-2, etanercept, and MY07A.

[0040] Therapeutic proteins contemplated herein are formulated for administration to the interior of the eye using pharmaceutically acceptable buffers or excipients. In some

embodiments, excipients for use with the compositions disclosed herein include maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and surfactant polyoxyethylene- sorbitan monooleate. In some embodiments, the composition further comprises a carrier.

Therapeutic Antibodies

[0041] Provided herein are methods of treatment of diseases of the eye or ocular diseases comprising administering a therapeutic antibody to the retina. Methods herein provide therapeutic antibodies directly to the interior of the eye, such as the retina, where they are needed. In some embodiments, the therapeutic antibody is a humanized biologically active antibody having a molecular weight greater than or equal to 100 kilodaltons. Suitable therapeutic antibodies include inhibitory antibodies and fragments thereof that bind to or otherwise inhibit an overactive protein in the eye of the individual. A non-exhaustive list of therapeutic antibodies include but is not limited to an anti-TNF antibody, an anti-EGFR antibody, an anti-VEGF antibody, an anti-bFGFR antibody, an anti-PDGF antibody, an anti- CD20 antibody, an anti-CD52 antibody, an anti-CDl la antibody, and an anti-IL-2 antibody. Exemplary antibodies include but are not limited to bevacizumab, ranibizumab, infliximab, adalimumab, rituximab, daclizumab, efalizumab, and alemtuzumab.

[0042] In some embodiments, the antibodies disclosed herein are monoclonal antibodies. In some embodiments, the antibodies disclosed herein are polyclonal antibodies. In some embodiments, the antibodies disclosed herein are IgM antibodies, IgG antibodies, IgA antibodies, IgE antibodies, IgD antibodies, or any subclass thereof. In some embodiments, the antibodies disclosed herein are IgM antibodies. In some embodiments, the antibodies are IgG antibodies. In some embodiments, the antibodies are IgA antibodies. In some embodiments, the antibodies are IgE antibodies. In some embodiments, the antibodies are IgD antibodies.

[0043] In some embodiments, the antibodies comprise an IgG constant domain, or variant thereof. In some embodiments, IgG constant domain variants herein comprise constant domains with reduced binding to complement proteins such as Clq. In some embodiments, the antibodies comprise an IgGl, IgG2, IgG3, or IgG4 constant domain, or variant thereof. In some embodiments, the antibodies are IgGl antibodies. In some embodiments, the antibodies are IgG2 antibodies. In some embodiments, the antibodies are IgG3 antibodies. In some embodiments, the antibodies are IgG4 antibodies.

[0044] In some embodiments, antibodies herein have kappa or lambda light chain sequences, either full length as in naturally occurring antibodies, mixtures thereof (i.e., fusions of kappa and lambda chain sequences), and subsequences/fragments thereof. Naturally occurring antibody molecules contain two kappa or two lambda light chains.

[0045] In some embodiments, the antibodies are antibody subsequences or antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab' and F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (sdFv), Cov-X-Body, Diabody, Triabody, dsDb, DART, scDb, tandAbs, triple body, triple heads, Fab-scFv, Fab’)2-scFv2, dAb-CHl/CL, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, 2 in 1-IgG, mAb2, Tandemab common LC, taFv-Fc, diabody, Di-diabody, scDbFc, scDb-CH3, scFv-Fc-scFV, HCAb-VHH, kih IgG, kih IgG common LC, scFv-kih-Fc, kih scFab-IgG, scFv-kih-CH3, CrossMab, mAb-Fv, kih-IgG-scFab, kih scFab-IgG-scFv, kih scFab-IgG-scFv, k/l-body common HC, SEED-body, CH3 charge pairs, hinge charge pairs, asymetric IgG, Duobody, nanobody, minibody, VL, and VH domain fragments.

[0046] In some embodiments, the antibody subsequences and antibody fragments have the binding affinity of a full length antibody, the binding specificity of a full length antibody, or one or more activities or functions of a full length antibody, e.g., a function or activity of antagonist antibody.

[0047] In some embodiments, the antibodies are human. In some embodiments, the antibody is humanized. In some embodiments, the antibody is chimeric.

[0048] Therapeutic antibodies contemplated herein are formulated for administration to the interior of the eye using pharmaceutically acceptable buffers or excipients. In some

Therapeutic Nucleic Acids

[0049] Provided herein are methods of treatment of disease of the eye or ocular diseases comprising administering a therapeutic protein to the retina. Therapeutic nucleic acids contemplated herein include but are not limited to inhibitory nucleic acids (siRNA, shRNA, miRNA), therapeutic transgenes, and guide RNA molecules. [0050] In some embodiments, siRNA and shRNA are small RNA molecules implicated in gene silencing. Introduction of dsRNA into an organism can cause specific interference of gene expression. This phenomenon, known as RNA interference (RNAi), results from a specific targeting of mRNA for degradation by cellular machinery in plant, invertebrate, and mammalian cells. Exemplary RNAi techniques known in the art include, without limitation, siRNA, shRNA and piRNA. Components of the RNAi machinery include the dsRNA targeting the target gene(s) (either siRNA or shRNA), Dicer, the Argonaute family of proteins (Ago-2 in particular),

Drosha, RISC, TRBP, and PACT. Small interfering RNA (siRNA) is generally recognized as dsRNA with 2 nt 3' end overhangs that activate RNAi, leading to the degradation of mRNAs in a sequence-specific manner dependent upon complimentary binding of the target mRNA.

shRNA is generally recognized as short hairpin RNA (shRNA) that contains a loop structure that is processed to siRNA and also leads to the degradation of mRNAs in a sequence-specific manner dependent upon complimentary binding of the target mRNA. Drosha is generally recognized as an RNase III enzyme that processes pri-miRNAs and shRNAs in the nucleus. Dicer is generally recognized as a ribonuclease (RNase) III enzyme which processes dsRNAs into 20-25 bp siRNAs leaving a 2 nt overhangs at the 3' end. Drosophila Dicer-2 cleaves long dsRNAs, while Dicer- 1 is important for miRNA processing. RISC is generally recognized as the minimal RNA-induced silencing complex (RISC) consists of the Argonaute protein and an associated siRNA. In some embodiments, it also contains PACT, TRBP, and Dicer. It should be noted that the exact composition of RISC has yet to be described. TRBP is generally recognized as needed for dsRNA cleavage by Dicer and subsequent passage to the RISC. Protein R (PKR)- activating protein (PACT) is generally recognized as associating with Dicer and TRBP for dsRNA cleavage. Along with the single-stranded siRNA, argonaute family of proteins assemble to form the RISC, bind 21-35 nt RNAs including miRNAs and siRNAs, and their associated target mRNA and then cleaves them through its endonucleolytic function.

[0051] In some embodiments, the guide nucleic acid molecule is a guide RNA molecule. In some cases the guide RNA molecule or other guide nucleic acid molecule directs

endonucleolytic cleavage of the DNA molecule to which it is bound, for example by recruiting a protein having endonuclease activity such as Cas9 protein. Zinc Finger Nucleases (ZFN), Transcription activator like effector nucleases and Clustered Regulatory Interspaced Short palindromic Repeat/Cas based RNA guided DNA nuclease (CRISPR/Cas9), among others, are compatible with some embodiments of the disclosure herein.

[0052] Guide RNA molecules or other guide nucleic acid molecules contemplated herein comprise sequences that base-pair with target sequence that is to be removed from sequencing (non-target sequence within the target sequence region). In some embodiments the base-pairing is complete, while in some embodiments the base pairing is partial or comprises bases that are unpaired along with bases that are paired to non-target sequence.

[0053] In some embodiments, guide RNA molecules or other guide nucleic acid molecules comprise a region or regions that form a‘hairpin’ structure. Such region or regions comprise partially or completely palindromic sequence, such that, in some embodiments, 5' and 3' ends of the region hybridize to one another to form a double-strand‘stem’ structure, which in some embodiments is capped by a non-palindromic loop tethering each of the single strands in the double strand loop to one another.

[0054] In some embodiments the guide RNA molecule or other guide nucleic acid molecule comprises a stem loop such as a tracrRNA stem loop. In some embodiments, a stem loop such as a tracrRNA stem loop complexes with or binds to a nucleic acid endonuclease such as Cas9 DNA endonuclease. In alternate embodiments, a stem loop complexes with an endonuclease other than Cas9 or with a nucleic acid modifying enzyme other than an endonuclease, such as a base excision enzyme, a methyltransferase, or an enzyme having other nucleic acid modifying activity that interferes with one or more DNA polymerase enzymes.

[0055] Therapeutic nucleic acids provided herein are contemplated to be formulated for administration. Formulations include but are not limited to colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). In some embodiments, therapeutic nucleic acids are formulated with targeted nanoparticles or other suitable sub-micron sized delivery systems.

Small Molecular Therapeutics

[0056] Provided herein are methods of treatment of disease of the eye or ocular diseases comprising administering a therapeutic protein to the retina. Suitable small molecules for administration to the eye include but are not limited to small molecules effective in treating diseases of the eye such as angiogenesis inhibitors, antibiotics, antifungals, antivirals, and anti inflammatories.

[0057] Suitable angiogenesis inhibitors include but are not limited to itraconazole,

carboxyamidotriazole, TNP-470, CM101, suramin, SU5416, angiostatin, endostatin, 2- methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, linomide, tasquinimod, Sorafenib, sunitinib, pazopanib, and everolimus.

[0058] Suitable antibiotics for treatment of diseases of the eye include but are not limited to amoxicillin, doxycycline, cephalexin, ciprofloxacin, cindamycin, metronidazole, azithromycin, sulfamethoxazole, amoxicillin, and levofloxacin. [0059] Suitable antifungals for treatment of diseases of the eye include but are not limited to amphotericin, candicin, filipin, hamycin, natamycin, nystatin, rimocidin, imidazoles, triazoles, and thiazoles.

[0060] Suitable antivirals for treatment of diseases of the eye include but are not limited to acyclovir, ganciclovir, and trifluridine.

[0061] Suitable anti-inflammatory drugs for treatment of diseases of the eye include but are not limited to non-steroidal anti-inflammatory drugs such as ketorolac, Acular, Acuvail, Bromday, Ilevro, Nevanac, Ocufen, Prolensa, Voltaren, and Xibrom as well as steroidal drugs such as fluocinolone, dexamethasone, loteprednol, difluprednate, prednisolone, fluorometholone, trimcinolone, and rimexolone.

Gene Therapy Constructs

[0062] Provided herein are gene therapy constructs comprising vectors in which a nucleic acid, such as a DNA, encoding a therapeutic protein are inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they are capable of transducing non-proliferating cells, such as hepatocytes and neurons. They also have the added advantage of low immunogenicity.

[0063] In some embodiments, the vector comprising the nucleic acid encoding the desired therapeutic protein provided herein is an adenoviral vector (A5/35). In some embodiments, the expression of nucleic acids encoding the desired therapeutic protein is accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases (See, June et al. 2009 Nature Reviews Immunol. 9.10: 704-716, incorporated herein by reference).

[0064] In some embodiments, the nucleic acid encoding the therapeutic protein is cloned into a number of types of vectors. For example, in some embodiments, the nucleic acid is cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0065] Further, the expression vector encoding the therapeutic protein, in some embodiments, is provided to a cell in the form of a viral vector. Viral vector technology is described, e.g., in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

[0066] A number of virally based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene, in some embodiments, is inserted into a vector and packaged in retroviral particles using suitable techniques. The recombinant virus is then isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are suitable for gene therapy. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are suitable for gene therapy. In some embodiments, adeno-associated virus vectors are used. A number of adeno-associated viruses are suitable for gene therapy. In one embodiment, lentivirus vectors are used.

[0067] Gene therapy constructs provided herein comprise a vector (or gene therapy expression vector) into which the gene of interest is cloned or otherwise which includes the gene of interest in a manner such that the nucleotide sequences of the vector allow for the expression

(constitutive or otherwise regulated in some manner) of the gene of interest. The vector constructs provided herein include any suitable gene expression vector that is capable of being delivered to a tissue of interest and which will provide for the expression of the gene of interest in the selected tissue of interest.

[0068] In some embodiments, the vector is an adeno-associated virus (AAV) vector because of the capacity of AAV vectors to cross the blood-brain barrier and transduction of neuronal tissue. In methods provided herein, AAV of any serotype is contemplated to be used. The serotype of the viral vector used in certain embodiments is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhS, AAVrhlO, AAVrh33, AAVrh34, AAVrh74, AAV Anc80, AAVPHP.B, AAV-DJ, and others suitable for gene therapy.

[0069] AAV vectors are derived from single stranded DNA parvoviruses that are nonpathogenic for mammals. Briefly, AAV-based vectors have the rep and cap viral genes that account for 96% of the viral genome removed, leaving the two flanking 145 basepair inverted terminal repeats (ITR) which are used to initiate viral DNA replication, packaging, and integration. In the absence of helper virus, wild-type AAV integrates into the human host-cell genome with preferential site-specificity at chromosome 19q 13.3 or in some embodiments, it remains expressed episomally. A single AAV particle is capable of accommodating up to 5 kb of ssDNA.

[0070] In an illustrative embodiment, the AAV backbone, comprising sequences between two AAV ITRs is pseudotyped using the serotype 2 capsid to create an AAV2 vector. Optionally, the AAV viral capsid is AAV2/9, AAV9, AAVrhS, AAVrhlO, AAVAnc80, or AAV PHP.B. [0071] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements is often increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements function either cooperatively or independently to activate transcription.

[0072] An example of a promoter that is capable of expressing a therapeutic fusion protein, such as a vIGF fusion or a signal sequence fusion, optionally having an internal ribosomal entry sequence, transgene in a mammalian T-cell is the EFla promoter. The native EFla promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving expression from transgenes cloned into a lentiviral vector (see, e.g., Milone et ak, Mol. Ther. 17(8): 1453-1464 (2009)). Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences are sometimes also used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, gene therapy vectors are not contemplated to be limited to the use of constitutive promoters. Inducible promoters are also contemplated here. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline-regulated promoter.

[0073] In order to assess the expression of a therapeutic fusion protein, such as a vIGF fusion or a signal sequence fusion, optionally having an internal ribosomal entry sequence, or portions thereof, the expression vector to be introduced into a cell often contains either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker is often carried on a separate piece of DNA and used in a co transfection procedure. Both selectable markers and reporter genes are sometimes flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0074] Methods of introducing and expressing genes into a cell are suitable for methods herein. In the context of an expression vector, the vector is readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector is transferred into a host cell by physical, chemical, or biological means.

[0075] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are suitable for methods herein (see, e.g., Sambrook et ah, 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0076] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors, in some embodiments, are derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like.

[0077] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.

[0078] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid is associated with a lipid. The nucleic acid associated with a lipid, in some embodiments, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, in some embodiments, they are present in a bilayer structure, as micelles, or with a“collapsed” structure. Alternately, they are simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which are, in some embodiments, naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0079] Lipids suitable for use are obtained from commercial sources. For example, in some embodiments, dimyristyl phosphatidylcholine (“DMPC”) is obtained from Sigma, St. Louis, Mo.; in some embodiments, dicetyl phosphate (“DCP”) is obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”), in some embodiments, is obtained from Calbiochem- Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids are often obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol are often stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol.“Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes are often characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when

phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids, in some embodiments, assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine- nucleic acid complexes.

[0080] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the therapeutic protein, provided herein, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays are contemplated to be performed. Such assays include, for example,“molecular biological” assays suitable for methods herein, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope herein.

[0081] The present disclosure further provides a vector comprising a therapeutic protein encoding nucleic acid molecule. In one aspect, a therapeutic fusion protein vector is capable of being directly transduced into a cell. In one aspect, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the therapeutic protein construct in mammalian cells. In one aspect, the mammalian cell is a human cell.

Kits

[0082] Provided herein are kits for topical administration of a therapeutic agent to a retina of an eye of an individual. In some embodiments kits herein are contemplated to comprise a vitreous removal tool, an ILM removal tool, and an applicator tool configured to apply the therapeutic agent to the retina of the individual. In some embodiments kits herein are contemplated to comprise a vitreous removal tool, an ILM disrupting tool, and an applicator tool configured to apply the therapeutic agent to the retina of the individual. In some embodiments, at least one of the vitreous removal tool and the ILM removal tool comprise a forceps, a scissors, a needle holder, a retractor, a hook, a pick, a fiber optic light pipe, or any combination thereof. In some embodiments, the forceps comprises a smooth forceps, a dressing forceps, a tying forceps, a straight forceps, a curved forceps, a utility forceps, a smooth jaw forceps, a plug forceps, a cross action forceps, a suturing forceps, a sleeve spreading forceps, a membrane peeling vitreous forceps, a membrane peeling microforcep, a blocked tip microforcep, a tapered, non-toothed microforcep, an asymmetrical microforcep, a maxi-grip ILM forcep, or any combination thereof. In some embodiments, the scissors comprise an angled scissors, a blunt scissors, or both. In some embodiments, the needle holder comprises a delicate needle holder, an extra-delicate needle holder, a straight needle holder, a curved needle holder, a locking needle holder, a non locking needle holder, or any combination thereof. In some embodiments, the retractor comprises an orbital retractor. In some embodiments, the hook comprises a muscle hook. In some embodiments, the pick comprises an ILM pick, a rice pick, a membrane spatula, a round ball pick, a Michaels’s pick, diamond dusted membrane scraper, a nitinol loop, or any combination thereof. In some embodiments, the kit further comprises a speculum, a stopcock, an infuser adapter, an irrigating contact lens, a depressor, or any combination thereof. In some embodiments, the applicator tool comprises a syringe, a tube, a bottle, a vial, a swab, a brush, a tissue, a pipette, a microcapillary, capillary fiber, a needle with a dispersive tip, or any combination thereof. In some embodiments, a therapeutic agent is applied to the exposed retina using microfluidics. In some instances, a capillary tube using the capillary action of microchannels draws fluid containing a therapeutic agent from a vial and deposits it onto the exposed retina (see, e.g., Fig. 8A). In some embodiments, capillary fibers are used to wick a therapeutic agent from a vial or other container holding the therapeutic agent and deposits it onto the exposed retina. In some instances, a fiber (40 g or the like) multiple deep grooves running the length of the fiber (see, e.g., Fig. 8B or a 4DG fiber) is used. For example, Fig. 8B shows a cross sectional view (5 pm) of a capillary fiber having such grooves which increase the surface area of the fiber, improving the transport and uptake of the material delivered. In some embodiments, the applicator tool is a microfluidic device comprising a capillary tube or capillary fiber. In some embodiments, the applicator tool comprises a microneedle having a dispersive tip capable of providing a spray to coat the exposed retina. In some embodiments, the ILM disrupting tool is an ultrasound needle. In some embodiments the ultrasound needle is a microneedle of a size appropriate for use in the eye.

[0083] In some embodiments, kits herein comprise a therapeutic agent. Therapeutic agents contemplated herein for inclusion in kits include but are not limited to therapeutic agents suitable for treating diseases of the eye, such as diseases of the eye described herein, for example age-related macular degeneration, retinopathy, chroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa,

Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion, bacterial infection, viral infection, fungal infection, chorioretinal inflammation, retinoschisis, retinal vascular occlusions, retinal dystrophy, retinal hemorrhage, optic neuritis, papilloedema, and optic atrophy.

Therapeutic agents for use in methods of treatment herein are selected by a medical provider based on efficacy for treating the disease of the individual. Thereapeutic agents include but are not limited to therapeutic proteins, therapeutic antibodies, therapeutic nucleic acids, small molecule therapeutics, and gene therapy constructs. In some embodiments, the therapeutic agent is contained in a viscous formulation. In some embodiments, the viscous formulation comprises an ophthalmically acceptable excipient. In some embodiments, the viscous formulation comprises sodium hyaluronate.

[0084] In some embodiments, an instrument to facilitate delivery of a gene construct precisely to an exposed area of retina with the internal limiting membrane removed is used. In some embodiments, the instrument comprises and injection system comprising a 1 ml syringe, a connecting acrylic tube which is shortened to 5 cm so that it has a dead space internal volume of 0.2 ml, connected in turn to a hand held injection device with an internal thin walled metal tube of 25 gauge external diameter and an internal dead space volume of 0. lmL. The tip, which is inserted through the scleral entry port used for standard vitrectomy, is curved, rounded and blunt with an opening on the convex surface. This allows even dispersion of the gene therapy product onto the posterior retinal surface exposed under air after peeling of the internal limiting membrane.

[0085] In some embodiments, an instrument to facilitate delivery of therapeutic agent comprises a preloaded microsyringe with a precise volume and concentration of a gene therapy construct or other therapeutic agent appropriate for a specific ocular disease. The microsyringe is attached to fine 30-42 gauge cannula or needle which can be placed manually or by an automated syringe driver onto a bared retinal surface under air after surgical or enzymatic removal of the internal limiting membrane. The extruded dose layers on the exposed retinal surface. In some embodiments the instrument contains a sensor to confirm that the needle or cannula is in the appropriate ocular space for application of the therapeutic agent.

Definitions

[0086] As used herein, the terms "therapeutic protein," "therapeutic polypeptide," "therapeutic peptide,"“protein,” polypeptide,” and“peptide” are used interchangeably to refer to two or more amino acids linked together.

[0087] As used herein, the terms“polynucleotide”,“nucleotide”,“nucleotide sequence”,

“nucleic acid” and“oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides herein are contemplated to have any three dimensional structure, and to perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. In some embodiments, a polynucleotide comprises one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure is imparted before or after assembly of the polymer. In some embodiments, the sequence of nucleotides is interrupted by non-nucleotide components. In some embodiments, a polynucleotide is further modified after polymerization, such as by conjugation with a labeling component.

[0088] Pharmaceutical formulations include“pharmaceutically acceptable” and“physiologically acceptable” carriers, diluents or excipients. The terms“pharmaceutically acceptable” and “physiologically acceptable” include solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration to a mammal, for example a human. In some embodiments, such formulations are contained in a liquid, e.g., emulsion, suspension, syrup or elixir; or solid form, i.e., tablet (e.g., coated or uncoated, immediate, delayed, continuous, or pulsatile release), capsule (e.g., hard or soft, immediate, delayed, continuous, or pulsatile release), powder, granule, crystal, or microbead. In some embodiments, supplementary compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) are also incorporated into the formulations.

[0089] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000 fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.

[0090] As used herein, singular forms“a,”“and,” and“the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to“a polypeptide” includes a plurality of polypeptides and reference to“a treatment or therapy” in some embodiments includes multiple, sequential or simultaneous treatments or therapies, and so forth.

EXAMPLES

[0091] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

[0092] Example: Surgical removal of internal limiting membrane and layering of AAV vector on the retina under air.

[0093] Ten non-human primate eyes (NHP) (macaque) were studied to determine if the surgical removal of the ILM and layering of the gene product on the exposed retinal surface under air would markedly improve transfection of adeno-associated viral (AAV) vectors in retinal cells using green fluorescent protein (GFP) as a reporter. [0094] Five adult Macaca fascicularis non-human primates (NHPs) were used, each weighing approximately 6 kg with ages ranging from 3 to 11 years. All eyes including retina were examined and ascertained as normal at baseline.

[0095] The AAV vector and gene payload were obtained commercially (Vector Biolabs, Burlingame, CA, USA). Briefly, AAV vectors were packaged and purified by standard methods. The virus used contained both AAV serotype 2 capsid and inverted terminal repeat-containing genomes packaged inside AAV2 capsid carrying enhanced GFP (eGFP). The eGFP expression was under the control of a cytomegalovirus (CMV) promoter. Physical titer of the vector was 1.7 x 10¹³ genome copies (GC)/mL. Efficacy of the AAV-GFP was confirmed by preliminary in vitro transfection of both endothelial and Miiller cell lines.

[0096] All non-human primates underwent surgery with general anesthesia provided by an expert veterinary anesthesiologist using a combination of ketamine and xylazine. A total of 10 eyes in five NHPs were studied. All eyes underwent standard 25G three-port pars plana vitrectomy under sterile operative conditions. Core vitrectomy was performed, then posterior vitreous detachment was achieved in all eyes. This was clearly evident as a ring of vitreous detached in a wave extending out from the optic disc to the equator (Fig. 1). Six eyes in five animals then underwent an ILM peel under membrane blue-DUAL visualization (Dutch

Ophthalmic, USA, Exeter, NH, USA). All eyes then underwent a complete fluid-air exchange before the delivery of AAV-GFP. All eyes received 40 pL of 1.7 x 10¹³ GC/mL AAV-GFP. This was delivered via a soft tip mounted on a microinjector into the air-filled eye. The AAV-GFP was pooled over the peeled ILM region of the macula or on the normal macula in non-peeled eyes, respectively. The animal was subsequently left in a supine position for 1 hour to maximize contact of AAV-GFP with the macula. The vitrectomy and ILM peel were performed using a Constellation (Alcon, Fort Worth, TX, USA) vitrectomy machine and operating microscope (OPMI MDO; Carl Zeiss, Oberkochen, Germany). An intraoperative optical coherence tomography (OCT) was also available for visualization of the ILM and retina layers.

[0097] Fundus color photographs and cross-section OCT of the retina were obtained. Two imaging modalities were used to ascertain the extent of GFP transfection in vivo. Fluorescent images of GFP transfection were obtained using a modified fundus camera with an excitation bandpass filter of 457 to 487 nm (FF01-472/30; Semrock, Rochester, NY, USA) and a barrier filter with a bandpass of 502.5 to 537.5 nm (FF01-520/35; Semrock). Fundus autofluorescence (FAF) images were acquired using infrared imaging by confocal scanning laser ophthalmoscopy (cSLO) (Heidelberg Retinal Angiograph [HRA]; Heidelberg Engineering, Dossenheim,

Germany). All imaging was performed at baseline and weeks 2, 4, 8, and 16 (before euthanasia) after surgery for each eye. [0098] The non-human primate test subjects were euthanatized 16 weeks after surgery and AAV vector administration for histological analysis. The eyes were enucleated and fixed with 4% paraformaldehyde in PBS overnight at 4°C. The cornea and lens were then removed and fixed in 4% paraformaldehyde in PBS overnight at 4°C. The eyes were soaked sequentially in 10% sucrose for 4 hours, 20% sucrose overnight, and 30% sucrose overnight, after which they were frozen in optimum cutting temperature compound at 80°C. 10-mm-thick sections were cut using a cryostat (HYRAX C 50, Carl Zeiss Microimaging GmbH, Germany). Hematoxylin and Eosin staining and Toluidine Blue O (TB) staining were performed to detect the area of peeled ILM.

[0099] After blocking thin sections (7 pm) in 5% BSA in PBS containing 0.1% Triton X-100 (PBS), the sections were incubated in a humidified chamber overnight at 4°C with a rabbit anti- GFP IgG antibody (1 : 1000 dilution; Invitrogen, Carlsbad, CA, USA). The sections were then washed three times with PBS and incubated with Alexa 488 goat anti-rabbit IgG (1 :500 dilution; Invitrogen) for 90 minutes at room temperature. To analyze GFP expression in each seven slides containing sections 50 pm apart from the center of the macula were examined. Two images of corresponding regions were taken from each slide using the same camera gain and time settings.

[00100] Double immunofluorescence staining on 7-pm sections for both GFP and either calretinin (ganglion cells), glutamine synthetase (Miiller cells), arrestin (cone

photoreceptor cells), or RPE 65 (RPE cells) were used to examine the extent of GFP localization in individual retinal cell layers and RPE.

[00101] Each cSLO FAF image and GFP expression image were compared for GFP expression with ImageJ (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). The extent of green fluorescence of GFP imaging was determined qualitatively. For quantitative measurements cSLO, FAF imaging was used to assess the transfected area as measured as pixel area of fluorescence on cSLO FAF images and intensity of fluorescence, which was determined by calculating the ratio of the mean pixel intensity of the fluorescent area over the mean pixel intensity of blood vessels of the retina. This ratio was presented as a fold increase of fluorescent intensity. A c2 test and two-tailed t-test were performed to ascertain significance in proportions and means, respectively. A P < 0.05 was used as the level of significance. All statistical analysis was performed with R version 3.2.2.

[00102] Details of 5 NHPs receiving the surgeries are summarized in Table 1.

Vitrectomy and posterior vitreous detachment were achieved in all 10 eyes. One control eye had localized retinal trauma nasal to the optic disc, but this did not lead to retinal detachment. There were no further complications associated with the surgical trauma.

Table 1. Non-human Primate Study Details.

[00103] Both eyes of five NHPs were used in this study. Four eyes underwent vitrectomy with retinal surface pooling of AAV-GFP under air, and six eyes underwent vitrectomy and ILM peel with pooling of AAV-GFP under air. AAV-GFP was allowed to pool on the retina postsurgery with the NHP left in a supine position for 1 hour.

[00104] Inflammation was noted in four eyes (three ILM peeled eyes and one non- peeled eye). In two eyes (ILM peeled eyes, MK2445 right eye and MK5338 right eye), transient inflammation was noted at 4 weeks postsurgery, which was suppressed with the use of topical anti-inflammatory drops and subsequently resolved by 10 weeks postsurgery. In the other two eyes (one ILM peeled eye and one non-peeled eye of the same NHP, MK7048 left and right eye, respectively), inflammation was noted at 4 weeks postsurgery and was persistent despite topical treatment. Intravitreal dexamethasone, 1 mg in 0.05 mL was administered at week 8.

Inflammation subsequently resolved in both eyes by week 14 with no further intervention. The resolution of inflammation was determined by clinical slit lamp biomicroscopy, which confirmed the resolution of anterior chamber and vitreous cells and flare.

[00105] FAF on cSLO was found to be more sensitive for imaging GFP

transfection, detecting green fluorescence as early as 2 weeks postsurgery as compared with fluorescent imaging by the modified fundus camera, which only detected green fluorescence at 4 to 6 weeks in the same eyes. In vivo imaging by FAF on cSLO allowed for better comparison of the area transfected and intensity of fluorescence and showed significant difference in fluorescence between ILM peeled and non-peeled eyes. In ILM peeled eyes, the AAV2 transfection occurred up to the boundaries of the ILM peel with very obvious drop off in fluorescence outside of the peeled area. In eyes that did not undergo ILM peel, the transfection was noted only in the foveal center and in sparsely scattered spots throughout the peripheral retina (Fig. 1).

[00106] GFP fluorescence was quantified at 16 weeks before euthanasia, where the six eyes that underwent ILM peel showed a much larger mean area (confidence interval [Cl]) transfected 50.7 (33.1-58.4) pixel² as compared with 5.1 (0.6-7.6) pixel² in the non-ILM peeled eyes (P < 0.01). The fluorescence intensity ratio was also higher (10.3 [2.2-18.5]) in the ILM peeled eyes as compared with the non-peeled eyes (1.9 [0.6-4.4]), P = 0.05 (Table 2). Expression stabilized at 6 to 8 weeks postsurgery and was sustained throughout the course of the study (Fig. 2).

[00107] Two modalities were used to compare the amount of transfection between the two groups (Table 2). FAF imaging allowed for in vivo area measurement of hyperfluorescence (HF) of the retina representing the area of transfection. Intensity of fluorescence was determined by obtaining a ratio of the mean hyperfluorescent intensity over the fluorescent intensity of a reference point (blood vessel). Histology was obtained at 16 weeks and transfected cells visualized. Area of green signifying transfection was measured and compared between the two different groups. Eyes that underwent ILM peel showed significantly more area of transfection and intensity of fluorescence on histology than those that underwent vitrectomy only.

Table 2. Comparison of measures of AAV-GFP transfection in vivo between vitrectomy - only and vitrectomy with ILM Peel at 16 weeks before enucleation.

[00108] Histological sections of each eye through the foveal area and also through the non- peeled areas of the more peripheral retina were analyzed for extent of GFP expression.

Expression of GFP was noted in most layers of the retina in all ILM peeled eyes as compared with expression confined to the inner retinal layers around the fovea in non-peeled eyes (Fig. 3). This explained the increase intensity of fluorescence noted in the ILM peeled eyes. Staining to identify retina cell layers and co-localization with GFP fluorescence was performed. In the ILM peeled eyes, there was evident transfection in the deeper retinal layers as seen in the qualitative analysis of the immunohistochemical sections. In these eyes, there was co-localization in Miiller cell layer (glutamine synthetase antibody stain). In the photoreceptor layer, although there was no co-localization with cone photoreceptors (cone arrestin antibody stain), adjacent cells with distinctive shape and location suggestive of rod photoreceptors expressed strong transfection. Amacrine cells (calretinin antibody stain), however, did not show any co-localization (Fig. 4).

[00109] An alternating hyper-hypofluorescent halo immediately adjacent to the ILM peeled area was noted in four of the six ILM peeled eyes on FAF (MK2445 right eye, MK5229 right eye, MK5338 right eye, and MK7048 left eye). Histology section through this area showed diffused RPE clumping (Fig. 5); however, the rest of the retina layers appeared intact with no obvious thinning and relatively normal architecture (Figs. 5, 6).

[00110] These results demonstrated a markedly increased efficacy of AAV-mediated GFP transfection when delivered with a combination of an ILM peel, vitrectomy and intraoperative pooling of the AAV-GFP on the retina under air compared with eyes that had no ILM peel. Improved GFP transfection was seen in terms of a large increase in area as well as intensity on multimodal in vivo imaging and this was confirmed histologically. The broad transfected area in eyes that underwent ILM peel correlated well with the area of ILM peel, whereas in eyes without ILM peel, transfection occurred only at the foveal center with minor scattered spots throughout the peripheral retina. More cell layers were transfected in ILM peel eyes as compared with non-peeled eyes as seen on histological examination. This also correlated well with the increased intensity of fluorescence noted.

[00111] The imaging techniques in this study included the use of FAF on cSLO, which provided much more obvious in vivo evidence of transfection compared with traditional FAF systems. In this study, imaging was also obtained using traditional FAF systems, which used the addition of excitation and barrier filters on a fundus camera. FAF images acquired by cSLO on the HRA system uses an excitation wavelength of 488 nm and a barrier filter with a cutoff at 500 nm, which blocks the excitation wavelength and allows transmission only of the autofluorescent light. The advantage in the cSLO-acquired FAF images is the ability to overcome the low- intensity signal of traditional FAF systems and the lens interference. In addition, the HRA system uses image averaging, in which a series of FAF images are captured and combined to reduce background noise, increase contrast, and improve the quality of the image captured. Although these features of the FAF cSLO system allowed for more subtle transfection to be appreciated, quantification of the intensity of the fluorescence image between subjects could not be reliably performed because of the intrinsic image manipulations for improved contrast.

Hence, the level of intensity of fluorescence reported in these findings was a ratio of the average hyperfluorescence over the background fluorescence level of the same image. This relative increase of fluorescent intensity allowed for a quantitative interimage comparison and tracking of the change in intensity over time.

[00112] This study reported two aspects pertinent to delivery of AAV-mediated gene therapy in the eye. The implications of these findings are important, as this form of therapy may be applicable to a wide range of therapies for ocular diseases, including genetic retinal disease, angiogenic disease, and glaucoma. First, it revealed the predominate nature of the ILM as a barrier to transfection, and second, it described and demonstrated an effective and practical method for AAV-mediated gene therapy delivery.

[00113] The ILM appeared to be the predominate barrier to AAV transfection as compared with other possible ocular barriers, such as the vitreous gel or a dilution effect on the injected dose. The ILM is a basement membrane that lies between the vitreous and retina. It has been shown to be a physical barrier to AAV transfection in prior studies using small animal and NHP models in which increased transfection of AAV-GFP was noted after the ILM was

enzymatically digested as well as in genetically modified rodent eyes with ILM compromise.

[00114] The other barrier to transfection is postulated to be the vitreous itself. Two mechanisms of action are postulated to contribute to the barrier function of the vitreous. First, compared with the subretinal space, which is a relatively immune-privileged area, the vitreous cavity has antibodies present that can result in the neutralization of AAV. Second, the dilution of AAV within the vitreous humor and its gel structure has also been suggested to result in poorer transfection of the retina.

[00115] The design of this study allowed a comparison of purely the effect of the ILM as a barrier to transfection. All eyes underwent vitrectomy, hence removing the barrier properties of the vitreous. AAV was also pooled directly on the retina surface in an air-filled eye

intraoperatively in both vitrectomized-only eyes and those with vitrectomy and ILM peel to ensure maximal contact and dose concentration. Despite these measures, the amount of transfection in the non-peeled eyes was similar to previous reports after intravitreal injections of AAV without vitrectomy, in which only a ring of transfection occurred around the fovea with minor scattered transfection in the peripheral retina. The transfected cell types seen on both histological and cSLO imaging were confined in these cases with ILM intact to the inner retinal ganglion cell layer. In contrast, the ILM peeled eyes in this study showed transfection of deeper retinal layers, including Miiller cells and Henle nerve fiber layer, when examined within the area of the peel. Within the photoreceptor layer, cone photoreceptors did not appear to be transfected; however, rod photoreceptors as inferred by their well-defined shape and location in proximity to cone cells showed strong transfection. These findings are consistent with a prior study, including the inference on rod transfection, on the efficacy and safety of AAV2 versus AAV8 transfection.

[00116] In this study, it was difficult to comment on the transfection of the RPE. Due to its inherent autofluorescent nature, examination of the RPE stain with a GFP filter showed positive co-localization regardless of the degree of transfection, hence a reliable comparison between ILM peeled and control eyes were not ascertained. In addition, the limitation of AAV2 and CMV promoter may also restrict the type of cells transfected.

[00117] In addition to showing the predominate function of ILM as a barrier to transfection, this study also demonstrated a surgically simple and safe technique of vector delivery.

Traditionally, AAV delivery is via a subretinal injection. This technique can be technically challenging and is complicated by the fact that subretinal injection is carried out in disease- compromised retinas, adding to the difficulty of the procedure. The subretinal injection induces a bleb of fluid where the AAV vector resides in an iatrogenic retinal detachment. Prolonged presence of subretinal fluid has been shown to affect eventual retinal function despite eventual anatomical reattachment. The other possible mechanism of damage could result from the direct toxic effects from the therapeutics injected. Even a subretinal injection of an iso-osmolar balanced salt solution, a relatively inert and harmless compound, has been shown to cause mild photoreceptor outer-segment damage over time.

[00118] Other strategies for vector delivery to circumvent the barrier properties of the ILM have shown promising results. One such method involves sub-ILM injections as opposed to subretinal injections. However, this may also prove to be even more technically challenging than a subretinal delivery. The advantage of sub-ILM injection is that there is minimal disruption to the anatomy of the eye and consequently less chance of iatrogenic damage. The disadvantage of this technique is that the ILM is left intact and repeated treatments will likely entail repeated complex surgery. AAV gene therapy may result in long-term expression of the transgene;

however, the therapeutic effects are unlikely to last forever and may still require repeat treatments at later dates. In the animal model studies exploring the efficacy of sFLT for long term anti-VEGF inhibition, a biological effect was noted to last up to 8 months in mice and 17 months in NHPs; however, expression beyond these time points is unknown. This may be particularly relevant in the treatment of nAMD, in which repeated gene therapy may be required to maintain therapeutic levels of anti-VEGF in an ongoing disease process.

[00119] The ideal method of vector delivery would still be via intravitreal injection without complex surgical intervention. This method of administration is technically simple and can be safely performed in the clinic setting. Studies have explored the delivery of AAV-mediated gene therapy vector via intravitreal injection; however, results have so far been disappointing and fail to show widespread transfection in terms of area and depth. An alternative approach has been to develop novel AAV variants that are able to partly overcome the barriers in the eye and achieve efficacious transfection through all layers of the retina. Specifically, AAV2-7m8 and AAV2 (quadY-F+T-V) have shown promise in transfection of deep retinal layers of the eye in rodent models. However, although more effective than prior AAV variants, these novel variants still display only patchy transfection when used in NHP models. Nonetheless, due to the therapeutic potential, especially in the treatment of nAMD, there is great interest both in the research and commercial space in developing a novel AAV variant that can effectively transfect deep retinal cells via intravitreal injection. These variants, however, are still in the early preclinical stages and require further research and proof of concept in early human trials.

[00120] In this study, the results demonstrated transfection using a simple safe and familiar method. Vitrectomy with ILM peel is a well-proven technique in the surgical treatment of vitreoretinal interface abnormalities, such as epiretinal membrane and full-thickness macular hole. This procedure is routine for vitreoretinal surgeons and requires minimal to no change to the technique when used for AAV-mediated gene therapy. This affords the practical advantage of delivering gene therapy with a procedure in which vitreoretinal surgeons are well versed, and it is well tolerated by patients. This technique also involves the direct pooling of AAV on the peeled retina under air for an hour, which is important for the ILM peel to achieve effective transfection. The fluid-air exchange during surgery and subsequent air fill postsurgery is also a well-recognized and common surgical step with air commonly left in the eye postoperatively as a tamponade during vitreoretinal surgery in humans with no known damage to the retina. Taken together, it was likely that because the ILM barrier was completely removed over the whole posterior pole, any further repeated treatments may require only an intravitreal injection of AAV gene product with or without fluid/air exchange and supine positioning for 1 hour.

[00121] The importance of the direct“pooling under air” technique should not be

underestimated, as evidenced by a study comparing transfection in eyes pretreated with either a vitrectomy and ILM peel or vitrectomy only, with the key difference in that AAV-GFP was administered as a delayed intravitreal injection as compared with the immediate and direct pooling of AAV under air during surgery in the present study. That study did not demonstrate obvious in vivo transfection after 16 weeks in either group; however, histological imaging revealed some transfection through the deep retinal cell layers at the corresponding peeled areas versus patchy and reduced transfection in vitrectomy-only eyes. The delayed intravitreal injection of AAV likely resulted in a weaker transfection efficacy as compared with the presently disclosed technique of direct“pooling under air.” The difference in methodology between this study and that one adds to the further understanding of the natural barriers of transfection in the eye. The removal of both the ILM and vitreous with direct contact of AAV- GFP on peeled retina resulted in transfection, which was seen by in vivo imaging as early as 2 weeks posttreatment. Compared with the results presented previously, the presently disclosed findings suggested that the barrier properties of the vitreous itself and dilution effect in the vitreous cavity should also not be underestimated. These differences in efficacy may also be due to the dilution effect of the AAV-GFP in the fluid-filled vitreous cavity despite a prior vitrectomy. There also could be a reaccumulation of antibodies within the vitrectomized eye, which could neutralize AAV and reduce transfection. Furthermore, in a vitrectomized eye, there also could be a higher washout of AAV-GFP. This phenomenon is seen in eyes post vitrectomy where the efficacy of anti-VEGF therapeutic agents is reduced; however, in terms of the larger AAV capsid, this has yet to be proven.

[00122] Although transfection appeared to be efficient using this surgical technique, it was noted in most ILM peeled eyes the presence of a halo around the ILM peeled areas evident on FAF. Histologically, this halo corresponded to areas of RPE clumping, but no obvious retinal layer thinning or disruption of cell architecture. This could have been a result of off toxicity of AAV. AAV-mediated transfection was shown to result in transgene-specific immune responses that resulted in histopathological evidence of retina damage by inflammation as a result of a transient breach of the retina-blood barrier.

[00123] In conclusion, the results have demonstrated that the ILM is the predominate barrier to transfection after vitreous injection of AAV gene constructs. The method of ILM peel has also suggested a practical and relatively simple method of AAV gene delivery, taking into account the potential need for repeat treatments.

[00124] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of expressing a therapeutic protein in a retina of an eye of an individual, the method comprising: contacting an area of the retina of an eye from which a vitreous and at least a portion of an internal limiting membrane (ILM) have been removed to expose at least a portion of a retina with a composition comprising a nucleic acid encoding the therapeutic protein.

2. The method of claim 1, wherein the therapeutic protein comprises RPE65, a VEGF inhibitor, REP-1, MERTK, ABC4, ND4, CD59, RSI, aflibercept, Ranibizumab, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B, RLBP1, channelrhodopsin-2, or MY07A.

3. The method of claim 1, wherein the nucleic acid comprises a viral vector.

4. The method of claim 1, wherein the composition comprises a viral capsid protein.

5. The method of claim 3 or claim 4, wherein the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus.

6. The method of any of claims 1-5, wherein expression of the therapeutic protein is effective to reduce at least one symptom of an eye disease.

7. The method of claim 6, wherein the eye disease is selected from age-related macular degeneration, chroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion, chorioretinal inflammation, retinoschisis, retinal vascular occlusions, retinal dystrophy, retinal hemorrhage, optic neuritis, papilloedema, and optic atrophy.

8. The method of any of claims 1-7, wherein the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual.

9. The method of any of claims 1-8, further comprising maintaining the individual in a substantially supine position for at least 30 minutes.

10. The method of any of claims 1-9, wherein the contacting occurs in the presence of an atmospheric gas or a therapeutic gas.

11. A method of expressing a therapeutic protein in a retina of an eye of an individual, the method comprising: a) removing a vitreous from a vitreal chamber of the eye; b) removing at least a portion of an internal limiting membrane (ILM) from an area of the eye to expose at least a portion of a retina; and c) contacting a composition comprising a nucleic acid encoding the therapeutic protein to at least a portion of the retina.

12. The method of claim 11, wherein the therapeutic protein comprises RPE65, a VEGF inhibitor, REP-1, MERTK, ABC4, ND4, CD59, RSI, aflibercept, Ranibizumab, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B, RLBP1, channelrhodopsin-2, or MY07A.

13. The method of claim 11, wherein the nucleic acid comprises a viral vector.

14. The method of claim 11, wherein the composition comprises a viral capsid protein.

15. The method of claim 13 or claim 14, wherein the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus.

16. The method of any of claims 11-15, wherein expression of the therapeutic protein is effective to reduce at least one symptom of an eye disease.

17. The method of claim 16, wherein the eye disease is selected from age-related macular degeneration, chroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X- linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion, chorioretinal inflammation, retinoschisis, retinal vascular occlusions, retinal dystrophy, retinal hemorrhage, optic neuritis, papilloedema, and optic atrophy.

18. The method of any of claims 11-17, wherein the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual.

19. The method of any of claims 11-18, wherein the ILM is visualized with a dye prior to step b).

20. The method of any of claims 11-19, further comprising maintaining the individual in a substantially supine position for at least 30 minutes.

21. The method of any of claims 11-20, further comprising injecting an atmospheric gas or therapeutic gas into the vitreal chamber prior to step c).

22. A method of topical administration to a retina in an individual of a gene therapy product, the method comprising: a) removing a vitreous from a vitreal chamber of an eye of the individual; b) removing at least a portion of an internal limiting membrane (ILM) of the eye to expose at least a portion of a retina; and c) contacting a composition comprising a nucleic acid encoding a therapeutic protein to at least a portion of the retina.

23. The method of claim 22, wherein the therapeutic protein comprises RPE65, a VEGF inhibitor, REP-1, MERTK, ABC4, ND4, CD59, RSI, aflibercept, Ranibizumab, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B, RLBP1, channelrhodopsin-2, or MY07A.

24. The method of claim 22, wherein the nucleic acid comprises a viral vector.

25. The method of claim 22, wherein the composition comprises a viral capsid protein.

26. The method of claim 24 or claim 25, wherein the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus.

27. The method of any of claims 22-26, wherein the gene therapy product is effective to reduce at least one symptom of an eye disease.

28. The method of claim 27, wherein the eye disease is selected from age-related macular degeneration, chroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X- linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion, bacterial infection, viral infection, fungal infection, chorioretinal inflammation, retinoschisis, retinal vascular occlusions, retinal dystrophy, retinal hemorrhage, optic neuritis, papilloedema, and optic atrophy.

29. The method of any of claims 22-28, wherein the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual.

30. The method of any of claims 22-29, wherein the ILM is visualized with a dye prior to step b).

31. The method of any of claims 22-30, further comprising maintaining the individual in a substantially supine position for at least 30 minutes.

32. The method of any of claims 22-31, further comprising injecting an atmospheric gas or a therapeutic gas into the vitreal chamber prior to step c).

33. A method of treating an eye disease in an individual, the method comprising: a) removing a vitreous from a vitreal chamber the eye; b) removing at least a portion of an internal limiting membrane (ILM) of the eye to expose at least a portion of a retina; and c) contacting a composition comprising a nucleic acid encoding a therapeutic protein effective in treating the eye disease to at least a portion of the retina.

34. The method of claim 33, wherein the therapeutic protein comprises RPE65, a VEGF inhibitor, REP-1, MERTK, ABC4, ND4, CD59, RSI, aflibercept, Ranibizumab, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B, RLBPl, channelrhodopsin-2, or MY07A.

35. The method of claim 33, wherein the nucleic acid comprises a viral vector.

36. The method of claim 33, wherein the composition comprises a viral capsid protein.

37. The method of claim 35 or claim 36, wherein the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus.

38. The method of any of claims 33-37, wherein expression of the therapeutic protein is effective to reduce at least one symptom of an eye disease.

39. The method of claim 38, wherein the eye disease is selected from age-related macular degeneration, chroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X- linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion, bacterial infection, viral infection, fungal infection, chorioretinal inflammation, retinoschisis, retinal vascular occlusions, retinal dystrophy, retinal hemorrhage, optic neuritis, papilloedema, and optic atrophy.

40. The method of any of claims 33-39, wherein the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual.

41. The method of any of claims 33-40, wherein the ILM is visualized with a dye prior to step b).

42. The method of any of claims 33-41, further comprising maintaining the individual in a substantially supine position for at least 30 minutes.

43. The method of any of claims 33-42, further comprising injecting an atmospheric gas or a therapeutic gas into the vitreal chamber prior to step c).

44. A method of topical administration to a retina in an individual of a therapeutic agent, the method comprising: a) removing a vitreous from a vitreal chamber an eye of the individual; b) removing at least a portion of an internal limiting membrane (ILM) of the eye to expose at least a portion of a retina; and c) contacting a composition comprising the therapeutic agent to at least a portion of the retina.

45. The method of claim 44, wherein the therapeutic agent comprises a protein, a peptide, an antibody, or a small molecule.

46. The method of claim 44 or claim 45, wherein the therapeutic agent comprises a VEGF antagonist, a steroid, an anti-fungal agent, an anti-viral agent, or an antibiotic.

47. The method of claim 44, wherein the composition comprises a pharmaceutically acceptable buffer or excipient.

48. The method of any of claims 44-47, wherein the therapeutic agent is effective to reduce at least one symptom of an eye disease.

49. The method of claim 48, wherein the eye disease is selected from age-related macular degeneration, chroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X- linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion, bacterial infection, viral infection, fungal infection, chorioretinal inflammation, retinoschisis, retinal vascular occlusions, retinal dystrophy, retinal hemorrhage, optic neuritis, papilloedema, and optic atrophy.

50. The method of any of claims 44-49, wherein the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual.

51. The method of any of claims 44-50, wherein the ILM is visualized with a dye prior to step b).

52. The method of any of claims 44-51, further comprising maintaining the individual in a substantially supine position for at least 30 minutes.

53. The method of any of claims 44-52, further comprising injecting an atmospheric gas or a therapeutic gas into the vitreal chamber prior to step c).

54. A method of treating an eye disease in an individual, the method comprising: a) removing a vitreous from a vitreal chamber the eye; b) removing at least a portion of an internal limiting membrane (ILM) of the eye to expose at least a portion of a retina; and c) contacting a composition comprising a therapeutic agent effective in treating the eye disease to at least a portion of the retina.

55. The method of claim 54, wherein the therapeutic agent comprises a protein, a peptide, an antibody, or a small molecule.

56. The method of claim 54 or claim 55, wherein the therapeutic agent comprises a VEGF antagonist, a steroid, an anti-fungal agent, an anti-viral agent, or an antibiotic.

57. The method of claim 54, wherein the composition comprises a pharmaceutically acceptable buffer or excipient.

58. The method of any of claims 54-57, wherein the therapeutic agent is effective to reduce at least one symptom of an eye disease.

59. The method of claim 58, wherein the eye disease is selected from age-related macular degeneration, chroideremia, Leber’s congenital amaurosis, Leber’s Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X- linked Retinoschisis, Usher’s syndrome, uveitis, proliferative vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion, bacterial infection, viral infection, fungal infection, chorioretinal inflammation, retinoschisis, retinal vascular occlusions, retinal dystrophy, retinal hemorrhage, optic neuritis, papilloedema, and optic atrophy.

60. The method of any of claims 54-59, wherein the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual.

61. The method of any of claims 54-60, wherein the ILM is visualized with a dye prior to step b).

62. The method of any of claims 54-61, further comprising maintaining the individual in a substantially supine position for at least 30 minutes.

63. The method of any of claims 54-62, further comprising injecting an atmospheric gas or a therapeutic gas into the vitreal chamber prior to step c).

64. A gene therapy composition for use in topical administration to a retina of an eye of an individual, wherein a vitreous and at least a portion of an internal limiting membrane (ILM) have been removed from the eye of the individual comprising a nucleic acid encoding a therapeutic protein and a pharmaceutically acceptable excipient.

65. The composition of claim 64, wherein the therapeutic protein comprises RPE65, a VEGF inhibitor, REP-1, MERTK, ABC4, ND4, CD59, RSI, aflibercept, Ranibizumab, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B, RLBP1, channelrhodopsin-2, or MY07A.

66. The composition of claim 64, wherein the nucleic acid comprises a viral vector.

67. The method of claim 64, wherein the composition comprises a viral capsid protein.

68. The composition of claim 66 or claim 67, wherein the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus.

69. The composition of claim 64, wherein the gene therapy composition is suitable for reducing at least one symptom of an eye disease.

70. The composition of claim 69, wherein the eye disease is selected from age- related macular degeneration, chroideremia, Leber’s congenital amaurosis, Leber’s

Hereditary Optic Neuropathy, Achromatopsia, retinitis pigmentosa, Stargardt’s macular dystrophy, X-linked Retinoschisis, Usher’s syndrome, uveitis, proliferative

vitreoretinopathy, diabetic retinopathy, and retinal vein occlusion, bacterial infection, viral infection, fungal infection, chorioretinal inflammation, retinoschisis, retinal vascular occlusions, retinal dystrophy, retinal hemorrhage, optic neuritis, papilloedema, and optic atrophy.

71. The composition of claim 64, wherein the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual

72. A gene therapy composition for use in treating an eye disease, wherein the composition is suitable for topical administration to a retina of an eye of an individual, wherein a vitreous and at least a portion of an internal limiting membrane (ILM) have been removed from the eye of the individual comprising a nucleic acid encoding a therapeutic protein and a pharmaceutically acceptable excipient.

73. The composition of claim 72, wherein the therapeutic protein comprises RPE65, a VEGF inhibitor, REP-1, MERTK, ABC4, ND4, CD59, RSI, aflibercept, Ranibizumab, sFLTOl, CNGB3, CNGA3, RPGR, PDE6B, RLBP1, channelrhodopsin-2, or MY07A.

74. The composition of claim 72, wherein the nucleic acid comprises a viral vector.

75. The composition of claim 72, wherein the composition comprises a viral capsid protein.

76. The composition of claim 74 or claim 75, wherein the virus is selected from a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus.

77. The composition of claim 72, wherein the gene therapy composition is suitable for reducing at least one symptom of an eye disease.

78. The composition of claim 77, wherein the eye disease is selected from age- related macular degeneration, chroideremia, Leber’s congenital amaurosis, Leber’s

79. The composition of claim 72, wherein the composition is administered to the retina using an applicator tool configured to apply the composition to the retina of the individual.

80. A kit for topical administration of a nucleic acid encoding a therapeutic protein to a retina of an eye of an individual, the system/kit comprising:

(i) a vitreous removal tool;

(ii) an ILM removal tool; and

(iii) an applicator tool configured to apply a composition comprising a nucleic acid encoding the therapeutic protein to the retina of the individual.

81. The kit of claim 80, wherein at least one of the vitreous removal tool and the ILM removal tool comprise a forceps, a scissors, a needle holder, a retractor, a hook, a pick, a fiber optic light pipe, or any combination thereof.

82. The kit of claim 81, wherein the forceps comprises a smooth forceps, a dressing forceps, a tying forceps, a straight forceps, a curved forceps, a utility forceps, a smooth jaw forceps, a plug forceps, a cross action forceps, a suturing forceps, a sleeve spreading forceps, a membrane peeling vitreous forceps, a membrane peeling microforcep, a blocked tip microforcep, a tapered, non-toothed microforcep, an asymmetrical microforcep, a maxi-grip ILM forcep, or any combination thereof.

83. The kit of claim 81, wherein the scissors comprise an angled scissors, a blunt scissors, or both.

84. The kit of claim 81, wherein the needle holder comprises a delicate needle holder, an extra-delicate needle holder, a straight needle holder, a curved needle holder, a locking needle holder, a non-locking needle holder, or any combination thereof.

85. The kit of claim 81, wherein the retractor comprises an orbital lid retractor.

86. The kit of claim 81, wherein the hook comprise a muscle hook.

87. The kit of claim 81, wherein the pick comprises an ILM pick, a rice pick, a membrane spatula, a round ball pick, a Michaels’s pick, a diamond dusted membrane scraper, a nitinol loop, or any combination thereof.

88. The kit of claim 81, further comprising a speculum, a stopcock, an infuser adapter, an irrigating contact lens, a depressor, or any combination thereof.

89. The kit of claim 80, wherein the applicator tool comprises a syringe, a tube, a bottle, a vial, a swab, a brush, a tissue, a pipette, a capillary tube, a capillary fiber, or any combination thereof.