Methods and compositions for retinal gene therapy

Abstract

Methods and compositions for retinal gene therapy are described herein. In one aspect, a method of treating a patient in need thereof is described. In some embodiments, the method comprises performing a vitrectomy on the eye of the patient, and delivering a payload, such as a gene therapy vector, to the retina of the eye of the patient. In some instances, the method further comprises removing the inner limiting membrane (ILM) of the eye of the patient before delivering the payload to the retina of the eye of the patient. In one aspect, compositions for retinal gene therapy are described. In such embodiments, a composition comprises a payload and an adjuvant.

Description

METHODS AND COMPOSITIONS FOR RETINAL GENE THERAPYRELATED APPLICATION DATA

[0001] The present application claims priority to United States Provisional Patent Application Number 63 / 573,155 filed April 02, 2024 which is incorporated herein by reference in its entirety.FIELD

[0002] The present invention relates to the field of treating inherited retinal degeneration diseases, specifically using methods and compositions for retinal gene therapy.BACKGROUND

[0003] Currently, treatments for inherited retinal diseases are limited. For some inherited retinal diseases, gene therapies have emerged as potential treatment options. However, difficulties have arisen with these gene therapies. For example, it has been found that adeno- associated viral (AAV) gene therapy vector delivery does not penetrate the anatomical barriers to reach the retina efficiently when applied through an intravitreal injection, and can cause significant inflammation or low eye pressure, even resulting in the suspension of a clinical trial. Thus, improved methods and compositions for retinal gene therapies to treat retinal disorders are needed.SUMMARY

[0004] Described herein are methods and compositions for retinal gene therapy. In one aspect, methods of treating a patient in need thereof are described. In some implementations, such a method comprises performing a vitrectomy on the eye of the patient, and delivering a gene therapy vector to the retina of the eye of the patient. In some instances, the method further comprises removing the inner limiting membrane (ILM) of the eye of the patient before delivering the gene therapy vector to the retina of the eye of the patient. In some embodiments, the patient has neuronal ceroid lipofuscinosis. However, it is to be understood that the patient may have other retinal disorders. In some cases, the gene therapy vector comprises an adeno- associated viral (AAV) vector.

[0005] In another aspect, disclosed herein are compositions for retinal gene therapy. In some embodiments, such a composition comprises a gene therapy vector and an adjuvant. In some implementations, the gene therapy vector comprises an adeno-associated viral (AAV) vector.

[0006] Disclosed herein are methods of treating a patient in need thereof. In such implementations, a method comprises performing a vitrectomy on the eye of the patient, and delivering the compositions to the retina of the eye of the patient.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1A is a representative image of the ILM of the eye being removed.

[0008] FIG. IB is a schematic illustration of insulin binding to the insulin receptor and being taken up by retinal cells by induced endocytosis.

[0009] FIG. 2 is a schematic illustration of an AAV-CLN8 construct that includes an endogenous promoter driving the expression of human CLN8 cDNA, modified SV40 intron, and bGH PolyA terminator. CLN8 expression cassette is flanked by AAV2 ITRs to ensure packaging the complete flanked AAV9 capsid sequence. mITR: mutated inverted terminal repeats; SV40: Simian Virus 40 modified intron, Promoter 1 : optimized endogenous promoter; CLN8 cDNA: CLN8 complementary DNA; bGH pA, bovine growth hormone poly A terminator, ITR: inverted terminal repeats.

[0010] FIG. 3A is a representative image of a pig being prepared for vitrectomy with AAV retinal treatment. Live pig under general anesthesia before sterile prep and draping for surgery.

[0011] FIG. 3B is a confocal microscope data slide demonstrating successful pig vitrectomy with AAV retinal transduction. AAV transduction of porcine retina 4 weeks after vitrectomy and ILM peel with only a 30 minute AAV residence time on retina prior to removal and wash out, leaving no residual AAV in the eye to avoid inflammation after surgery. GFP is expressed throughout layers of retina, verified with anti-GFP antibody (red) to ensure signal is not autofluorescent in the green channel.

[0012] FIG. 3C is an confocal microscope data slide demonstrating successful pig vitrectomy with AAV retinal transduction, now with cell nuclei stained with DAPI to demonstrate transduction in both superficial and deep photoreceptor layers. AAV transduction of porcine retina 4 weeks after vitrectomy and ILM peel with only a 30 minute AAV residence time on retina prior to removal and wash out, leaving no residual AAV in the eye to avoid inflammationafter surgery. GFP is expressed throughout layers of retina, verified with anti-GFP antibody (red) to ensure signal is not autofluorescent in the green channel. Scale bar represents 100 pm.

[0013] FIG. 4A is an imaging representation of insulin increasing the transduction of AAV into retina. Murine retinal explants are incubated with AAV with insulin for 30 minutes with subsequent removal of AAV. After 5 days, explants were imaged with anti-GFP-647 (red).

[0014] FIG. 4B is an imaging representation of insulin increasing the transduction of AAV into retina. Murine retinal explants are incubated with AAV with insulin for 30 minutes with subsequent removal of AAV. After 5 days, explants were imaged with anti-GFP-647 (red) and DAPI to demonstrate layers of the retina and that the AAV penetrated the entire retina from anterior (top) to posterior (bottom) where the critical photoreceptors are located.

[0015] FIG. 4C is an imaging representation without insulin increasing the transduction of AAV into retina. Murine retinal explants are incubated with AAV without insulin for 30 minutes with subsequent removal of AAV. After 5 days, explants were imaged with anti-GFP-647.

[0016] FIG. 4D is an imaging representation without insulin increasing the transduction of AAV into retina. Murine retinal explants are incubated with AAV without insulin for 30 minutes with subsequent removal of AAV. After 5 days, explants were imaged with anti-GFP-647 and DAPI. Scale bar represents 100 pm.

[0017] FIG. 5A is a representative image of a suite of large animal retinal function equipment. A pig is shown under general anesthesia with ERG electrode setup.

[0018] FIG. 5B is a representative image of a suite of large animal retinal function equipment. A live ERG recording with Diagnosys system is shown.

[0019] FIG. 5C is a representative image of a suite of large animal retinal function equipment. A live OCT measurement is shown demonstrating thickness of the retina.

[0020] FIG. 5D is a representative image of a suite of large animal retinal function equipment. A live fundus photo of a porcine retina is shown four weeks after vitrectomy, ILM peel, and AAV administration with no inflammation noted.

[0021] FIG. 6 is an imaging representation of treatment of AAV9.CBA.GFP vector with insulin, which causes greater transduction compared to vector alone and salt-balance in cryosections of murine retinal explants after 3rdday of culturing. Representative confocal images of cryosections of mice retinal explants treated as described as follows: with salt-balance (top left image), vector alone (middle left image), vector with 10 ng of insulin (bottom left image), vectorwith 20 ng of insulin (top right image), and vector with 30 ng of insulin (middle right image). Images are shown labeled with anti-GFP (highlighting genetically delivered GFP with more green color) and insulin receptor (red) antibodies marking retinal ganglion cells. Nuclei are counterstained with DAPI. Representative individual channels are shown along with a merged image for each experimental dose. Scale bar = 100 pm. Montage made with ImageJ.

[0022] FIG. 7 shows graphical representations of treatment of AAV9.CBA.GFP vector with insulin, which causes greater transduction compared to vector alone and salt-balance in cryosections of mice retinal explants after 3rdday of culturing. In the left panel, GFP -488 mean intensity is shown for mice retinal explants treated with: salt-balance (control), vector alone (vec), vector with 10 ng of insulin (vec + 10), vector with 20 ng of insulin (vec + 20), and vector with 30 ng of insulin (vec + 30). In the right panel, GFP-647 mean intensity is shown for mice retinal explants treated with: salt-balance (control), vector alone (vec), vector with 10 ng of insulin (vec + 10), vector with 20 ng of insulin (vec + 20), and vector with 30 ng of insulin (vec + 30).

[0023] FIG. 8 is an imaging representation of treatment of AAV9.CBA.GFP vector with insulin, which causes greater transduction compared to vector alone and salt-balance in mice retinal explants after 3rd day of culturing. Representative confocal images of mice whole mount retinal explants treated as described from left to right: with salt-balance (leftmost image), vector alone (second-left image), vector with 10 ng of insulin (center image), vector with 20 ng of insulin (second-right image), and vector with 30 ng of insulin (rightmost image). Images are shown labeled with anti-GFP and Brn3a antibodies marking retinal ganglion cells. Nuclei counterstained with DAPI. Representative individual channels are shown along with a merged image for each experimental dose. Scale bar = 100 pm. Montage made with ImageJ.

[0024] FIG. 9 is a quantification of fluorescence of treatment of AAV9.CBA.GFP vector with insulin, which causes greater transduction compared to vector alone and salt-balance in mice retinal explants after 3rd day of culturing. GFP-647 mean intensity is shown for mice whole mount retinal explants treated with salt-balance (control), vector alone (vec), vector with 10 ng of insulin (vec + 10), vector with 20 ng of insulin (vec + 20), and vector with 30 ng of insulin (vec + 30).

[0025] FIG. 10 is a graphical representation of GFP protein quantification of mice retinal explants after 3rd day of culturing and transducing with AAV9-CBA-GFP vector by ELISA.GFP concentration is shown for mice retinal explants treated with salt-balance (control), vector alone (vec), vector with 10 ng of insulin (vec + 10), vector with 20 ng of insulin (vec + 20), and vector with 30 ng of insulin (vec + 30).

[0026] FIG. 11 is an imaging representation of an exemplary non-viral vehicle composition applied in vitro. Lipid nanoparticles deliver mRNA to cultured retinal pigment epithelium (RPE) cells. After 48 hours of incubation with 5 pL of a control or lipid nanoparticles and with the indicated doses of insulin (0 ng, 10 ng, and 30 ng via continued exposure), GFP expression is shown. DAPI staining and phase contrast are also shown.

[0027] Further implementations, features, and aspects of the disclosed technology, and the advantages offered thereby, are described in greater detail hereinafter, and can be understood with reference to the following detailed description, accompanying drawings, and claims.DETAILED DESCRIPTION

[0028] Embodiments described herein can be understood more readily by reference to the following detailed description, examples, claims, and attached Appendix. Elements, apparatus and methods described herein, however, are not limited to the specific embodiments presented in the detailed description, examples, claims, and attached Appendix. In particular, these embodiments are merely illustrative of the principles of the present invention. Accordingly, this disclosure is not intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the specification and in view of the claims.

[0029] All publications, patents and patent applications mentioned in this specification are incorporated herein in their entirety by reference, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

[0030] In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9. All ranges disclosedherein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10,” “from 5 to 10,” or “5-10” should generally be considered to include the end points 5 and 10.

[0031] Further, when the phrase “up to” is used in connection with an amount or quantity, it is to be understood that the amount is at least a detectable amount or quantity. For example, a material present in an amount “up to” a specified amount can be present from a detectable amount and up to and including the specified amount.

[0032] Additionally, in any disclosed embodiment, the terms “substantially,” “approximately,” and “about” may be substituted with “within [a percentage] of’ what is specified, where the percentage includes 0.1, 1, 5, and 10 percent.

[0033] It is also to be understood that the article “a” or “an” refers to “at least one,” unless the context of a particular use requires otherwise.

[0034] The present disclosure is directed to methods and compositions for retinal gene therapy. Such methods and compositions, in some cases, can provide one or more advantages compared to other methods and compositions for other retinal gene therapies. For example, in some embodiments, a method or composition described herein provides a novel combination of a surgical pre-retinal approach with an adjuvant to provide a treatment for retinal diseases without making subretinal blebs or detachments of the retina. In some instances, the method or composition allows for the removal of the payload.

[0035] In one aspect, methods of treating a patient in need thereof are described. In one implementation, such a method comprises performing a vitrectomy on the eye of the patient, and delivering a payload to the retina of the eye of the patient. In some instances, the method further comprises removing the inner limiting membrane (ILM) of the eye of the patient before delivering the payload to the retina of the eye of the patient.

[0036] In some instances, the patient has neuronal ceroid lipofuscinosis. Neuronal ceroid lipofuscinosis is a group of neurogenerative disorders, including CLN1 disease, CLN2 disease, CLN3 disease, CLN4 disease, CLN5 disease, CLN6 disease, and CLN8 disease. However, it is to be understood that the patient may have other retinal disorders. For example, a patient may have congenital cataracts, congenital glaucoma, retinal degeneration, optic atrophy, eye malformations, glaucoma, wet age-related macular degeneration, dry age-related macular degeneration, retinitis pigmentosa, choroideremia, Leber congenital amaurosis, Leber’shereditary optic neuropathy, early onset retinal dystrophy, achromatopsia, x-linked retinoschisis, Usher Syndrome IB, neovascular age-related macular degeneration, Stargardt’s macular degeneration, diabetic macular degeneration, or diabetic macular edema.

[0037] In some instances, the payload for delivery to the retina of the eye of a patient in need of treatment is a viral vehicle, a non-viral vehicle, or a combination thereof. In some cases, the viral vehicle is a gene therapy vector. In some cases, the non-viral vehicle is a liposome, a lipid nanoparticle, a gold nanoparticle, an antisense oligonucleotide, or a microvesicle.

[0038] In some instances, the dosage of payload administered via the disclosed methods is effective for treatment at approximately 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of a typical dosage of the same or equivalent payload administered via standard clinical pars plana injection. In some instances, the dosage of payload administered via the disclosed methods is effective for treatment at 75% or less, 50% or less, 40% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less.

[0039] In some instances, the payload delivered or administered is about 1 x 106to about 1 x 1015vg / eye. In some instances, the payload delivered or administered is about 2 x 109to about 6 x 1014vg / eye. In some instances, the payload of AAV delivered or administered is up to about 1 x 106vg / eye, up to about 1 x 107vg / eye, up to about 1 x 108vg / eye, up to about 1 x 109vg / eye, up to about 1 x 1010vg / eye, up to about 1 x 1011vg / eye, up to about 1 x 1012vg / eye, up to about 1 x 1013vg / eye, up to about 1 x 1014vg / eye, or up to about 1 x 1015vg / eye. In some instances, the payload delivered or administered is about 0.3 mL / eye to about 6 mL / eye. In some instances, the payload delivered or administered is about 0.3 mL / eye to about 5 mL / eye. In some instances, the payload of AAV delivered or administered is up to about 0.3 mL / eye, up to about 0.5 mL / eye, up to about 0.7 mL / eye, up to about 0.9 mL / eye, up to about 1 mL / eye, up to about 1.5 mL / eye, up to about 2 mL / eye, up to about 2.5 mL / eye, up to about 3 mL / eye, up to about 4 mL / eye, up to about 5 mL / eye, or up to about 6 mL / eye. In some instances, the amount or volume of AAV or other viral vehicle delivered or administered depends on the size of the patient’s eye, the surface area of the patient’s eye, the size of the patient’s retina, the surface area of the patient’s retina, the volume of vitreous removed from the patient’s eye, the age of the patient, the sex of the patient, the identity of the disease or condition to be treated, the progression of the disease or condition to be treated, or a combination thereof.

[0040] In some instances, the payload delivered or administered is about 0.2 mL per eye up to 8 mL per eye, depending on the desired retinal area of gene therapy treatment (macular only treatment in posterior pole of retina to entire retinal periphery treatment when the eye is in the supine surgical position). Importantly, depositing the payload when the eye is filled with air after removal of vitreous permits the payload to contact only the retina, titrating volume of delivery to each patient’s eye, which can change considerably during first two years of life and vary from person to person as an adult. These volumes result in non-viral lipid nanoparticle vehicle doses of 10 pg in 0.2 mL up to 24 mg in 8 mL per human eye. In some instances, the amount or volume of lipid nanoparticles or non-viral vehicle delivered or administered depends on the size of the patient’s eye, the surface area of the patient’s eye, the size of the patient’s retina, the surface area of the patient’s retina, the volume of vitreous removed from the patient’s eye, the age of the patient, the sex of the patient, the identity of the disease or condition to be treated, the progression of the disease or condition to be treated, or a combination thereof.

[0041] In some cases, the gene therapy vector comprises an adeno-associated viral (AAV) vector. For example, in some instances, in any of the disclosed methods or compositions, the gene therapy vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT, AAV-B1, Anc80, AAV7m8, AAV.PhP.eB, AAV2tYF, 4D-R100 AAV2-7m8, or their derivatives.

[0042] In some implementations, the method further comprises filling the eye of the patient with air. In some embodiments, the method further comprises removing the payload after a period of time.

[0043] In some cases, the method further comprises treating the retina of the eye of the patient with an adjuvant. In some such embodiments, treating the retina of the eye of the patient with an adjuvant and delivering a payload to the retina of the eye of the patient are done at the same time. In some such embodiments, treating the retina of the eye of the patient with an adjuvant and delivering a payload to the retina of the eye of the patient are done sequentially. In some cases, the retina of the eye of the patient is treated first with an adjuvant and then a payload is delivered. In some cases, the retina of the eye of the patient is treated first with a payload and then with an adjuvant. In some implementations, the administration of an adjuvant to the eye of the patient includes administration directly to the eye or local administration to the eye.Moreover, in some cases, the administration of an adjuvant to the eye includes systemic administration, such as intravenous administration or oral administration.

[0044] In some embodiments, an adjuvant comprises insulin. However, it is to be understood other adjuvants may be used in other implementations. Non-limiting examples of adjuvants include dexamethasone, polybrene, prostaglandins, cyclosporin H, cyclosporin A, chloroquine diphosphate, eeyarestatin I, rapamycin, teniposide, etoposide, dasatinib, or combinations thereof. In some embodiments, an adjuvant facilitates the permeability of the retina of the eye of the patient for delivery of payload to the retina. In some embodiments, the adjuvant is insulin and is delivered at about 0.5 pg / eye to about 10 pg / eye for patients. In some embodiments, the adjuvant is insulin and is delivered at about 1.5 pg / eye for patients.

[0045] In another aspect, disclosed herein are compositions for gene therapy, such as retinal gene therapy. In some embodiments, such a composition comprises a payload and an adjuvant. In some embodiments, such a composition comprises an adjuvant and a viral vehicle, a non-viral vehicle, or a combination thereof. In some cases, the viral vehicle is a gene therapy vector. In some cases, the gene therapy vector comprises an adeno-associated viral (AAV) vector. Exemplary gene therapy vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT, AAV-B1, Anc80, AAV7m8, AAV.PhP.eB, AAV2tYF, 4D-R100, AAV2-7m8, and their derivatives. In some cases, the non-viral vehicle is a liposome, a lipid nanoparticle, a gold nanoparticle, an antisense oligonucleotide, a microvesicle, or a combination thereof.

[0046] In some implementations, the compositions comprising a payload and an adjuvant are formulated for local delivery, such as intraocular delivery, intravenous delivery, sub-retinal delivery, suprachoroidal delivery, or intravitreal delivery. In addition, in some embodiments, the compositions comprising a payload and an adjuvant are formulated for systemic delivery, such as intravenous delivery, intracerebroventricular delivery, intramuscular delivery, intraparenchymal delivery, or intrathecal delivery.

[0047] In the disclosed compositions, in some implementations, the payload and the adjuvant are admixed for administration simultaneously. For example, in some cases, the payload and the adjuvant are administered simultaneously using the same mode of administration, or the payload and the adjuvant are administered simultaneously each using a different mode of administration. In some embodiments, in an additional example, the payload and the adjuvant are administeredsequentially using the same mode of administration, or the payload and the adjuvant are administered sequentially each using a different mode of administration.

[0048] In some instances, the disclosed compositions are configured for delivery to a human patient. In some instances, the disclosed compositions are configured for delivery to a human patient in a supine position. In some cases, the human patient is in a supine position and is undergoing, has previously been subjected to, or will be subjected to, a surgical procedure at the time of composition delivery.

[0049] In some embodiments, the payload is administered simultaneously with the adjuvant. In other cases, the optimized gene therapy vector is administered immediately before or immediately after the adjuvant. In other implementations, the payload is administered within about 1 minute, within about 5 minutes, within about 10 minutes, within about 15 minutes, within about 20 minutes, within about 25 minutes, within about 30 minutes, within about 45 minutes, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 12 hours, within 24 hours, within 36 hours, or within 48 hours of administration of the adjuvant. In some instances, the adjuvant is administered before administration of the payload or after administration of the payload.

[0050] In some embodiments, the method further comprises removing the payload after a period of time. In some implementations, the payload is removed after about 1 minute, after about 5 minutes, after about 10 minutes, after about 15 minutes, after about 20 minutes, after about 25 minutes, after about 30 minutes, after about 45 minutes, after about 1 hour, after about 2 hours, after about 3 hours, after about 4 hours, after about 5 hours, after about 6 hours, after about 7 hours, after about 8 hours, after about 9 hours, after about 10 hours, after about 12 hours, or after about 24 hours. In some implementations, the payload is removed after about 1 minute to about 60 minutes. In preferred implementations, the payload is removed after about 15 minutes to about 30 minutes. In some implementations, the removal of payload is followed by one or more rinses.

[0051] In some embodiments, the method comprises removing the payload after a period of time. In some instances, the payload is removed using suction. In some cases, the suction is provided by a suction instrument, such as a cannula. The cannula may be a soft-tip cannula that is conventionally used to remove liquid in a patient’s eye during surgery, as practiced according to methods known in the art.

[0052] In some instances, the disclosed methods are performed during a surgical procedure. In some instances, disclosed methods comprise performing a vitrectomy on the eye of the patient, delivering a payload to the retina of the eye of the patient, and removing excess payload from the eye of the patient during the course of a surgical procedure. In some cases, the surgical procedure is less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour.

[0053] Disclosed herein are methods of treating a patient in need thereof. In such implementations, a method comprises performing a vitrectomy on the eye of the patient, and delivering the described compositions to the retina of the eye of the patient.

[0054] In some embodiments, the patient has neuronal ceroid lipofuscinosis. However, it is to be understood that the patient may have other retinal disorders.

[0055] In some implementations, the method further comprises removing the inner limiting membrane (ILM) of the eye of the patient before delivering the compositions to the retina of the eye of the patient.

[0056] In some instances, the method further comprises fdling the eye of the patient with air.

[0057] In some cases, the method further comprises removing the payload after a period of time.

[0058] The disclosure also provides for methods of delivering a transgene to a patient comprising administering i) a gene therapy vector encoding the transgene, and ii) an adjuvant to the patient. The disclosure further provides for methods of treating a patient in need thereof comprising administering i) a gene therapy vector encoding the transgene, and ii) an adjuvant to the patient.

[0059] In some embodiments, the gene therapy vector and / or an adjuvant are administered locally, such as intraocular delivery, intravenous delivery, sub-retinal delivery, suprachoroidal delivery, or intravitreal delivery. In other embodiments, the gene therapy vector and / or the adjuvant are administered systemically, such as intravenous delivery, intracerebroventricular delivery, intramuscular delivery, intraparenchymal delivery, or intrathecal delivery.

[0060] In some embodiments, the gene therapy vector and / or the adjuvant are administered simultaneously to the patient, such as the gene therapy vector and the adjuvant are admixed. In other embodiments, the gene therapy vector and the adjuvant are administered separately.I. MEDICAMENTS AND COMPOSITIONS

[0061] The present disclosure provides for payloads that treat retinal disorders or disease. The present disclosure also provides for adjuvants that facilitate the delivery of payload to the retina of an eye of a patient. A payload may comprise one or more of a viral vehicle and a non- viral vehicle. A viral vehicle may comprise a gene therapy vector, such as an adeno-associated viral (AAV) vector. Exemplary gene therapy vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT, AAV-B1, Anc80, AAV7m8, AAV.PhP.eB, AAV2tYF, 4D-R100, AAV2-7m8, and their derivatives. A non-viral vehicle may comprise a liposome, a lipid nanoparticle, a gold nanoparticle, an antisense oligonucleotide, or a microvesicle.

[0062] An adjuvant may comprise insulin. Other non-limiting examples of adjuvants include dexamethasone, polybrene, prostaglandin E2, cyclosporin H, cyclosporin A, chloroquine diphosphate, eeyarestatin I, rapamycin, teniposide, etoposide, dasatinib, or combinations thereof.

[0063] Compositions of the present disclosure may comprise one or more payload and adjuvant as described above. The compositions disclosed may comprise one or more of such compounds, in combination with a pharmaceutically acceptable carrier. To form an acceptable composition suitable for administration, such compositions will contain a therapeutically effective amount of a payload.

[0064] The compositions of the disclosure may be used in the treatment methods of the present disclosure. Such compositions are administered to a patient in amounts sufficient to deliver a therapeutically effective amount of the payload so as to be effective in the treatment methods disclosed herein. The therapeutically effective amount may vary according to a variety of factors such as, but not limited to, the patient’s condition, weight, sex and age. Other factors include the mode and site of administration. The compositions may be provided to the patient in any method known in the art. Exemplary routes of administration include, but are not limited to, systemic, subcutaneous, intravenous, topical, epicutaneous, oral, intraosseous, intramuscular, intranasal, pulmonary, sub-retinal, suprachoroidal, intravitreal, or intrathecal. The compositions of the present disclosure may be administered only one time to the patient or more than one time to the patient. The compositions may be administered to the patient more than one time per day, or more than one time per surgical procedure. The therapeutically effective amount of the payload and appropriate dosage may be identified by routine testing in order to obtain optimaltreatment, while minimizing any potential side effects. In addition, co-admini strati on or sequential administration of other agents may be desirable.

[0065] The compositions of the present disclosure may further comprise agents which improve the solubility, half-life, absorption, etc. of the payload and / or adjuvant. Furthermore, the compositions of the present disclosure may further comprise agents that attenuate undesirable side effects and / or or decrease inflammatory responses.

[0066] The compositions of the present disclosure can be administered in a wide variety of dosage forms for administration. For example, the compositions can be administered in forms, such as, but not limited to, tinctures, solutions, suspensions, elixirs, syrups, ointments, creams, pastes, emulsions, or solutions for intravenous administration or injection.

[0067] In the present disclosure, the compositions may further comprise a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, vehicles, adjuvants, surfactants, suspending agents, emulsifying agents, inert fillers, diluents, excipients, wetting agents, binders, lubricants, and buffering agents. Typically, the pharmaceutically acceptable carrier is chemically inert to the payload and / or adjuvant and has no detrimental side effects or toxicity under the conditions of use. The pharmaceutically acceptable carriers can include polymers and polymer matrices. The nature of the pharmaceutically acceptable carrier may differ depending on the particular dosage form employed and other characteristics of the composition.

[0068] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the patient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The payload and / or adjuvant may be administered in a physiologically acceptable diluent, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as, but not limited to, a soap, an oil or a detergent, suspending agent, such as, but not limited to, pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.II. METHODS OF TREATMENT

[0069] The teachings of the present disclosure provide for the treatment of retinal diseases or conditions in a patient in need of such treatment. Inherited retinal diseases and conditions include neuronal ceroid lipofuscinosis. Neuronal ceroid lipofuscinosis is a group of neurogenerative disorders, including CLN1 disease, CLN2 disease, CLN3 disease, CLN4 disease, CLN5 disease, CLN6 disease, and CLN8 disease. Other retinal diseases and conditions include, but are not limited to congenital cataracts, congenital glaucoma, retinal degeneration, optic atrophy, eye malformations, glaucoma, wet age-related macular degeneration, dry age-related macular degeneration, retinitis pigmentosa, choroideremia, Leber congenital amaurosis, Leber’s hereditary optic neuropathy, early onset retinal dystrophy, achromatopsia, x-linked retinoschisis, Usher Syndrome IB, neovascular age-related macular degeneration, Stargardt’s macular degeneration, diabetic macular degeneration, or diabetic macular edema.

[0070] The methods of treatment comprise administering to the patient any of the payloads disclosed herein. The method may further comprise administering to the patent any of the adjuvants disclosed herein. The methods of treatment comprise administering to the patient any composition disclosed herein. The method will often further comprise identifying a subject in need of such treatment.

[0071] The following Examples are exemplary of methods and compositions described herein, and should not be considered limiting unless expressly stated.EXAMPLE 1

[0072] Not intending to be bound by theory, it is believed that intraocular delivery of viral transgenes will transduce the retina and decrease vision loss in NCL-associated retinopathy. However, it is also to be understood that such a method described herein is applicable to other retinal disorders.

[0073] The following disclosure and non-limiting specific Example describes comparing retinal transduction efficiency via intravitreal injection to novel pre-retinal application after surgical removal of the vitreous and retinal inner limiting membrane (ILM), the acceleration ofthe transduction of the retina with the addition of insulin to encourage cellular endocytosis of AAV particles, and characterizing retinal function and host immunologic response in response to viral transduction.The physical barriers of the vitreous and ILM limiting AAV transduction into the retina

[0074] Not intending to be bound by theory, it is believed that transduction will be superior with a technique allowing for vitrectomy, surgical removal of the ILM, and intravitreal injection. This technique is demonstrated using a porcine model with the ILM being removed (FIG. 1A). The efficacy of transgene expression is tested with an AAV vector with green fluorescent protein (GFP) in two experimental groups: (1) intravitreal injection and (2) intraoperative application under air following vitrectomy and ILM peel. Outcome measures include: % cells transduced by GFP, location of transduction within the retina, thickness of the retina measured with retinal cross section, and immunohistochemistry.Addition of physiologic insulin to the AAV and the effects on endocytosis and uptake of vector from the extracellular space to increase transduction efficiency

[0075] Found richly in the retina, insulin receptors are internalized within minutes of binding to insulin. Not intending to be bound by theory, it is believed that with AAV present in the extracellular space, physiological insulin will dramatically increase transduction of AAV when applied to the pre-retinal space (FIG. IB). The efficacy of transgene expression is tested with a dose response curve of insulin concentrations and AAV vector with green fluorescent protein (GFP) following vitrectomy and ILM peel as above. Outcome measures include: % cells transduced by GFP, location of transduction within the retina, thickness of the retina measured with retinal cross section, and immunohistochemistry.AAV-delivered CLN8 safely prevents retinal degradation without intraocular inflammation

[0076] Determining the functional change of gene therapy on retinal function with electroretinography. Not intending to be bound by theory, it is believed that transduction will prevent the degradation of electroretinography (ERG) signal. Retina function may be tested with electroretinography (ERG) in a CLN8mndmouse, which has been established with a truncated neuronal ceroid lipofuscinosis type 8 (CLN8) protein from an insertion mutation and readingframe shift. Eyes are randomized to intervention with intravitreal injection of established GMP- grade AAV9.CLN8 verses carrier control in the contralateral eye. Outcome measures include: a- and b-wave ERG amplitudes, scotopic rod, and flicker response.

[0077] Evaluating the safety of AAV with fluorescein angiography and inflammatory cytokine analysis. Intraocular inflammation is assessed following intraocular viral vector transduction using an immunocompetent porcine model and a mice model. A control cohort of pigs do not receive preoperative and perioperative steroids for their intravitreal injections. Fluorescein angiography is undertaken and quantified. Following sacrifice and retinal dissection, infiltration of polymorphonuclear neutrophils (PMN) is quantified. Outcome measures include: quantified area of vascular leakage by fluorescein angiography and a count of infiltrated lymphocytes. The aqueous humor is used for inflammatory cytokine analysis post-operatively.

[0078] This study represents a paradigm shift in retinal gene therapy delivery. Rather than subject a thin and atrophic retina to the challenging stretching forces of large subretinal bleb delivery, this approach leaves the fragile photoreceptors intact adjacent to their retinal pigment epithelium. Further, not intending to be bound by theory, by removing the central and often cited barrier, the inner limiting membrane, as well as incorporating insulin to trigger rapid uptake of viral particles, it is believed that this approach results in substantially less virus needed for treatment as well as virtually no remaining virus in the extracellular space to cause inflammation.

[0079] Moreover, this surgical and insulin-adjuvant hybrid approach may be applied to many inherited retinal degenerations. This approach may effectively increase the therapeutic window of retinal gene therapy, allowing for a few copies of AAV to be delivered to restore cellular function while mitigating structural trauma and inflammation. The both novel and gene-agnostic implications of this treatment method is wide-sweeping for the entire field of inherited retinal disease.EXAMPLE 2

[0080] Some patients are challenged by ongoing chronic retinal atrophy in the area of required subretinal bleb formation during AAV administration. It is unclear why this occurs, but it may be due to direct toxicity from the viral vector, the subsequent immune response to said vector, or physical sharing forces at the time of initial application. Now with most surgeons avoiding direct sub-macular treatment but rather allowing the blebs placed more peripherally tocoalesce gently in the macula, the subretinal application of any therapy cannot escape the reality of intentional iatrogenic retinal detachment through the fovea. In addition to being technically difficult, as bleb formation can proceed anteriorly rather than posteriorly as intended, macular hole formation and ripping the retinal pigment epithelium are a concern. As with all intraocular therapy, inflammation remains of significant concern, especially if a viral vector is applied intravitreally through standard clinical pars plana injection.

[0081] Instead of inducing retinal detachment for subretinal access, herein, not intending to be bound by theory, it is proposed to apply gene therapy directly to the anterior surface of the retina after vitrectomy, leaving the delicate photoreceptors and retinal pigment epithelium untouched. Not intending to be bound by theory, the removal of the vitreous before filling the eye with air prior to pre-retinal application allows for vector to be placed in direct contact with the retina and the retina alone, preventing cellular contact and off target transduction of the ciliary, both lens, and other intraocular structures. It has been observed that transduction in the CNS after intrathecal injection of AAV occurs after only minutes rather than hours. Thus, not intending to be bound by theory, it is believed the pre-retinal application of AAV in an air filled eye after vitrectomy only requires 30 minutes to transduce the retina effectively. This provides the opportunity to remove the residual vector from the eye, washing inside the eye thoroughly, before the end of surgery, thus preventing any off-target transduction to anterior structures and thereby resulting inflammation.

[0082] Currently, no active clinical trial is utilizing pre-retinal application of gene therapy after vitrectomy in the air-filled eye to allow for direct AAV retinal contact and then removal and prevention of inflammation in the eye. No active clinical trial or published pre-clinical investigation is presently peeling the inner limiting membrane away prior to the application of gene therapy. No current therapy, clinical trial, or published pre-clinical investigation utilizes an endocytosis-inducing trigger, such as insulin, to help increase transduction efficiency in the retina. This will allow fewer viral copies dosed for the same treatment effect, and transduction can be accelerated to the order of minutes to allow for complete application and cellular uptake.

[0083] This new approach that combines novel surgical technique and molecular adjuvant therapy makes a substantial impact on the entire inherited retinal disorder field. This novel approach can be applied to inherited retinal disease patients who presently face a future condemned to blindness without any effective therapy available currently.EXAMPLE 3

[0084] The following disclosure describes embodiments of a novel method of retinal gene therapy. In this non-limiting Example, embodiments comprising a vitrectomy, followed by the removal of the ILM barrier, with AAV injection of the eye with and without air fill of the eye are described. Additionally, embodiments of vitrectomy, followed by removal of the ILM barrier, with AAV injection and concurrent insulin application of the eye with and without air fill of the eye are also described. In this particular embodiment, NCL is the focus. However, it is also to be understood that such methods described herein are applicable to other retinal disorders.General Materials and Methods

[0085] Animals. Homozygous CLN8mndmice are from Jackson Laboratories, where they are bred on a C57BL / 6J background. Juvenile porcine models are used for their similar size and immunologic response to the human eye, both of which are lacking in the murine model, in order to conduct more representative safety trials.

[0086] Transgenes. Initially, AAV vector with green fluorescent protein (GFP) is used to test for safety, surgical technique, and GFP expression. After optimization, GMP-grade AAV9.CLN8 construct is used (FIG. 2). 5 x 1012particles per porcine eye is used.

[0087] Survival Surgery. For the treatment of porcine eyes, AAV vector with green fluorescent protein (GFP) is delivered to the retina following general anesthesia and sterile preparation of the eye, as well as standard three port 25-gauge vitrectomy (Bausch and Lomb Stellaris and Zeiss operating microscope) and subsequent staining and removal of the ILM followed by air fluid exchange. This leaves the posterior segment of the eye with only air and a puddle of AAV vector lying on top of a macula that has had its inner limiting membrane mechanically removed to facilitate diffusion of AAV. 5 x 1012viral particles per porcine eye is applied. Based upon transduction times seen in the CNS on the order of minutes with the AAV9.CLN8 vector, the residual time is 30 minutes before removal of the vector and then instillation of balanced salt solution and closure of the eye. Treatment eyes are compared to carrier contralateral control eyes and evaluated with nonparametric paired statistical analysis.

[0088] The standard of care presently requires steroid treatment for any vitrectomy for any surgical indication. Thus, testing the surgical group with ILM peel without steroid treatment would be of little value. Those pigs which undergo vitrectomy and steroid treatment aremonitored for signs of inflammation. Fluorescein angiography is undertaken and quantified. Following sacrifice and retinal dissection, infiltration of polymorphonuclear neutrophils is quantified. Outcome measures include quantified area of vascular leakage by fluorescein angiography count of infiltrated lymphocytes. These data are evaluated nonparametrically with paired contralateral controls.

[0089] Cytokine Analysis. The aqueous humor may be tested with 42 cytokine array semiquantitative ELISA from Abeam (abl33997), including ENA-78, GCSF, GM-CSF, GRO, GRO-alpha, 1-309, IL-lalpha, IL-lbeta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p40 / p70, IL-13, IL-15, IFN-gamma, MCP-1, MCP-2, MCP-3, MCSF, MDC, MIG, MIP-ldelta, RANTES, SCF, SDF-1, TARC, TGF-betal, TNF-alpha, TNF-beta, EGF, IGF-I, Angiogenin, Oncostatin M, Thrombopoietin, VEGF-A, PDGF BB, and Leptin.

[0090] Intravitreal Injections. Both juvenile pigs and mice receive intravitreal injections per the groups described via the pars plana of the eye. After general anesthesia and sterile preparation of the eye, 5 x 1012viral particles in 0.05 mL is injected with a 30-gauge needle through the pars plana of the porcine eye, with a similar procedure is used with a 33 -gauge Hamilton needle and 1.5 x 1010viral particles in 3 microliters per mouse eye, owing to its volume being approximating 150x smaller than that of a pig eye.

[0091] Electroretinography . Mice and juvenile pigs are evaluated with two different ERG systems after careful evaluation for electrical noise and monitored dark adaptation under IACUC protocol. Two separate ERG systems by Diagnosys, one for mice and one for pigs, are used.

[0092] Fluorescein Angiography. After anesthesia, pigs have intravenous line placed and fluorescein dye injected for live imaging of their retinal vasculature. Mice have intraperitoneal fluorescein placed prior to their retinal imaging.

[0093] Rigor and reproducibility. Eyes are randomized to receive either control or a treatment such that no one animal receives potentially deleterious treatment bilaterally. Eyes are compared with nonparametric paired statistical analysis using Graphpad Prism with Wilcoxan matched pairs.MethodsRemoval of vitreous and inner limiting membrane (ILM) barriers facilitates AAV transduction of retinal cells

[0094] A barrier to AAV entry into the retina from the pre-retinal surface is the inner limiting membrane. Electron microscopy structural analysis reveals that the ILM limits passive diffusion to particles from 10-25 nm. However, the ILM is routinely removed after vitrectomy during surgical repair of epiretinal membranes and macular holes. By performing vitrectomy and ILM peel prior to AAV administration, the macula is prepared to receive the AAV, directly presenting extracellular spaces between the Mueller cells and allowing for deeper penetration of the AAV into the retina without inducing subretinal detachment required of subretinal delivery.

[0095] Experimental. Using a juvenile Gottingen minipigs, a self-complementary adeno- associated viral vector version of AAV (FIG. 2) is applied, but modified to contain chicken beta actin promoter and green fluorescent protein (GFP), abbreviated scAAV9.CBA.GFP. Not intending to be bound by theory, it is believed that transduction will be superior with the present ILM peel technique in the porcine model when compared to carrier controls, with more expression and deeper penetration into retina. These groups, with carrier controls, are designed to ascertain the transduction efficiency of different variations in surgical technique, linked directly to presence or absence of vitreous and ILM. Following surgery or injection, pigs are evaluated in vivo every two weeks for 3 months prior to sacrifice and retinal processing. Experimental cohorts (4 juvenile Gottingen minipigs per cohort) may be divided into the following groups: (1) vitrectomy followed by ILM removal and AAV application when eye is filled with air (“ILM peel”), followed by removal of the AAV after 30 minutes with rinsing with balanced salt solution; (2) vitrectomy without ILM peel and AAV application in air-filled eye with removal 30 minutes later; (3) vitrectomy and AAV injection without air fill; and (4) AAV injection through the pars plana into the vitreous without vitrectomy. Outcome measures include: % cells transduced by GFP, location of transduction within the retina, thickness of the retina, measured both in vivo with optical coherence tomography (OCT), electroretinography (ERG), and fundus photography, as well as ex vivo analysis of retinal cross section, immunohistochemistry, and nucleus quantification.

[0096] Results. Not intending to be bound by theory, it is believed that removal of vitreous and also ILM will confer increase in transduction of AAV into the retina in an independent manner. A photograph of a live pig under general anesthesia before sterile prep and draping for surgery is shown in FIG. 3A. The minipigs were followed closely postoperatively with topical antibiotics, as well as topical and systemic steroids. Clinical diagnostic tools were used toperform live fundus photography, OCT, and ERG on the pigs. Following euthanasia, the retinas were carefully dissected and prepared for cryo-sectioning and subsequent immunohistochemistry, noting successful transduction of GFP for experimental group (1) (FIG. 3B and FIG. 3C). As the retina is prone to auto-fluorescence in the 488 channel anti-GFP-Alexa- 647 was used to amplify the signal and be certain that indeed transduction had occurred.Determining how the coadministration of insulin and AAV impacts transduction via endocytosis of vector from the extracellular space.

[0097] The second independent physical barrier to transduction of AAV particles is the cell membrane of each cellular subtype within the retina. Central to this overall approach with accelerated vector uptake by the retina on the order of minutes rather than hours or days is rapid acceleration through the cell membrane. Therefore, not intending to be bound by theory, adjuvant assisted transduction through the cell membrane may allow for the required short residence time of AAV on the retina prior to removal surgically to prevent further inflammation. Given the delicate nature of this vital CNS tissue, in some preferred embodiments, a method that utilizes an endogenous adjuvant that still operates on a timescale of endocytosis within minutes is desired. Insulin is such an adjuvant.

[0098] Experimental. The impact of adjuvant insulin versus AAV alone in the carrier control eye may be contralaterally assessed using Gottingen minipigs. Experiments (4 minipigs per group) may be divided into the following groups: (1) vitrectomy followed by ILM removal and AAV and insulin application when the eye is filled with air (ILM peel), followed by removal of the AAV / insulin after 30 minutes under air before refilling eye with balanced salt solution; (2) vitrectomy and AAV injection with and without insulin in paired air-filled eyes with removal after 30 minutes; (3) vitrectomy and AAV injection with and without insulin in paired fluid-filled eyes; and (4) simple AAV injection through the pars plana with and without insulin. Outcome measures include: % cells transduced by GFP, location of transduction within the retina, thickness of the retina, measured both in vivo with optical coherence tomography (OCT), electroretinography (ERG), fundus photography, ex vivo analysis of the retinal cross section, immunohistochemistry, and nucleus quantification. RNAseq is used to analyze transduction of non-GFP vector.

[0099] Murine retinal explants are also examined. A murine retinal explant was selected to approximate most closely the avitric retinal configuration of the surgical minipigs. Similar experimental groups to porcine experiments above with 30 minute residence time of the AAV with and without insulin are used, and then retinal explants are analyzed at 5 days prior to cellular degradation. Similarly, RNAseq is used to analyze transduction of non-GFP AAV. When applying the AAV9.CLN8 vector, retinal transduction is verified. Also, which cells in particular express the transgene (bipolar cells) and at which levels are also verified.

[0100] Results. Not intending to be bound by theory, it is believed that insulin increases the transduction throughout the retinal layers of both porcine in vivo experiments and also ex vivo murine retinal explants. Murine explants with 1.8 xlO11vg AAV9-GFP and 25 ng of insulin residence of 30 minutes, followed by analysis 5 days later, strongly suggested the influence of insulin on AAV9 transduction in murine retina explants (FIG. 4A-D). After 5 days, explants with insulin adjuvant incubation (FIG. 4A and FIG. 4B) demonstrated more robust transduction through the retina than those with AAV alone (FIG. 4C and FIG. 4D).How novel AAV delivery impacts retinal neural function and inflammation

[0101] Intraocular inflammation is of concern when considering any new AAV therapy within the eye.Experimental

[0102] Functional Assays. CLN8mndmice may be used with the AAV9.CLN8 vector as described previously. Mice may be broken into two groups, those receiving intravitreal injection at 6 weeks of age, and those undergoing sacrifice and retinal explant with subsequent ex vivo testing. Murine retinal explant is selected to approximate most closely the avitric retinal configuration of the surgical minipigs. These explants will be evaluated for cell count analysis compared to carrier control. Additional functional CLN8mndmouse in vivo experiments in which the mice are treated with AAV9.CLN8 will be conducted with simple intravitreal injection as well.Safety Assays.

[0103] Murine studies. Intraocular inflammation following intraocular viral vector transduction is assessed using immunocompetent juvenile porcine and CLN8mndmouse models using both cytokine analysis and also clinical live imaging with OCT, fundus photography, andfluorescein angiography. In vivo, optical coherence demography is also utilized to look for cystoid macular edema between the carrier and control side as well as evidence of vitreous cell on both the optical coherence demography and fundus photography. Ex vivo, matched locations in the retina are examined from the experimental to control side, comparing cell counts and looking for evidence of atrophy.

[0104] Porcine studies. Minipigs are injected with GMP grade AAV9.CLN8 with and without insulin adjuvant after vitrectomy, ILM peel, and air fill, as described previously. All murine and porcine subjects may be evaluated with in vivo OCT, fluorescein angiography, ERG, and fundus photography, as well as serial aqueous humor cytokine analysis, prior to sacrifice and sectioning for histological analysis.

[0105] Cytokine analysis. At one week and one month after surgical AAV administration in minipigs, aqueous samples from the anterior chamber may be taken and submitted for cytokine analysis. Prior to sacrifice and retinal dissection, infiltration of polymorphonuclear neutrophils (PMN) may be quantified. Outcome measures include quantified area of vascular leakage by fluorescein angiography, degree of vitritis as evidenced on fundus photography, and a count of infiltrated lymphocytes.Results

[0106] Functional Assays. Not intending to be bound by theory, it is believed that AAV9 transduction of the CLN8mndmouse retina will prevent subsequent ERG degradation measured over the following months, compared to carrier control eyes. Further, not intending to be bound by theory, it is believed that eyes treated with AAV9 vector retain normal retinal thickness and do not demonstrate characteristic thinning or inflammation compared to carrier controls, both for in vivo intravitreal injections as well as ex vivo explant analysis.

[0107] Safety Assays. Not intending to be bound by theory, it is believed that mice that receive intravitreal injection of CLN8.AAV9 may demonstrate mild inflammation as the vector will reside in the eye longer than that in the murine explant or the porcine surgical eyes, as assayed with cytokine analysis. Not intending to be bound by theory, it is believed that porcine retinas receiving CLNA8.AAV9 administration will not demonstrate inflammatory cytokine increase or vascular leakage on fluorescein angiography compared to controls, being well controlled with perioperative steroids. There was no inflammation noted at one week and onemonth in the first four minipigs as examined with indirect ophthalmoscopy and fundus photography or degradation of signal on ERG (FIG. 5A-D).EXAMPLE 4Insulin assisted AAV9.CBA.GFP gene therapy shows enhanced transduction in murine retinal explant cultures

[0108] Retinal explant cultures are a powerful intermediate model system between in vitro and in vivo models to evaluate retina’s development, pharmacology, and toxicology. AAV transduction in animal models often requires high viral titers. Here, we show that insulin therapy can improve adeno-associated virus (AAV) transduction in mice retinal explant cultures after 30 min exposure only, validating the results of the ILM peel technique used with larger animals in above Examples.

[0109] Methods: Without the intent of being bound to any theory, it is believed that insulin can enhance AAV transduction. Retinal explant cultures were treated with AAV9.CBA.GFP having various doses of insulin (10 ng, 20 ng and 30 ng respectively) for 30 min only.Thereafter, cultured media was replaced with fresh ones. Transduction was determined immunohistochemically on 3rd, 5th and 7th day of culturing from both whole mounting retinal explants (FIG. 8) as well as from cryosections (FIG. 6). Keeping in mind retinal autofluorescence, anti-GFP with Alexa fluor 647 were used to determine true GFP signals. All images were acquired using confocal microscope at 20X magnification and analyzed with Image J for further quantification (FIG. 7 and FIG. 9). GFP concentrations from retinal explants were also measured using a GFP -ELISA kit (FIG. 10).

[0110] Results: After 3rd day of transfection, an increase in mean pixel GFP intensity was observed in retinal explants with insulin dose response when compared with controls showing negative GFP (FIG. 7 and FIG. 9). GFP concentrations were also higher in the 30 ng insulin dose samples with ELISA kit (FIG. 10). The number of transduced RGC cells showed significant increase in insulin response, as shown by co-localization with antibody markers compared with the control.

[0111] Without the intention of being bound by any theory, it was inferred that dose response insulin assisted AAV transduction enhanced in mice retinal explants after 30 min exposure.EXAMPLE 5Non-viral vehicles deliver mRNA expressing GFP in vitro

[0112] In FIG. 11, retinal pigment epithelium (RPE) cells were cultured and treated with either a control solution (top row of images) or 0.25 pg lipid nanoparticles / 5 pL in each well with mRNA expressing GFP. For the lipid nanoparticle samples, delivery also included increasing dosage of insulin, with images taken when the dosage was 0 ng, 10 ng, and 30 ng of insulin (upper middle row, lower middle row, and bottom row of images, respectively). GFP expression is shown in the GFP fluorescence channel, with DAPI staining and phase contrast images also displayed individually and merged with the GFP fluorescence images.References1. Santavuori, P., Neuronal ceroid-lipofuscinoses in childhood. Brain Dev, 1988. 10(2): p. 80-3.2. Rider, J. A. and D.L. Rider, Batten disease: past, present, and future. Am J Med Genet Suppl, 1988. 5: p. 21-6.3. Isolation of a novel gene underlying Batten disease, CLN3. The International Batten Disease Consortium. Cell, 1995. 82(6): p. 949-57.4. Preising, M.N., et al., Ocular morphology and function in juvenile neuronal ceroid lipofuscinosis (CLN3) in the first decade of life. Ophthalmic Genet, 2017. 38(3): p. 252-259.5. Johnson, T.B., et al., AAV9 Gene Therapy Increases Lifespan and Treats Pathological and Behavioral Abnormalities in a Mouse Model of CLN8-Batten Disease. Mol Ther, 2021. 29(1): p. 162-175.6. Comander, J., et al., 29. Novel Surgical Method for Intravitreal AAV Administration Overcomes Transduction Barriers in Non-Human Primates. Molecular Therapy, 2016. 24: p. S13-S14.7. Teo, K.Y.C., et al., Surgical Removal of Internal Limiting Membrane and Layering of AAV Vector on the Retina Under Air Enhances Gene Transfection in a Nonhuman Primate. Invest Ophthalmol Vis Sci, 2018. 59(8): p. 3574-3583.8. Reiter, C.E. and T.W. Gardner, Functions of insulin and insulin receptor signaling in retina: possible implications for diabetic retinopathy. Prog Retin Eye Res, 2003. 22(4): p. 545-62.9. Hall, C., H. Yu, and E. Choi, Insulin receptor endocytosis in the pathophysiology ofinsidin resistance. Experimental & Molecular Medicine, 2020. 52(6): p. 911-920.10. Reinhardt, K., et al., Novel CLN8 mutations confirm the clinical and ethnic diversity of late infantile neuronal ceroid lipofuscinosis. Clin Genet, 2010. 77(1): p. 79-85.11. Mendel, T. A., et al., Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy. PLoS One, 2013. 8(5): p. e65691.12. Gange, W.S., et al., Perifoveal Chorioretinal Atrophy after Subretinal Voretigene Neparvovec-rzyl for RPE65-Mediated Leber Congenital Amaurosis. Ophthalmol Retina, 2022. 6(1): p. 58-64.13. Baumal, C.R., et al., Retinal Vasculitis and Intraocular Inflammation after Intravitreal Injection of Brolucizumab. Ophthalmology, 2020. 127(10): p. 1345-1359.14. Bucher, K., et al., Immune responses to retinal gene therapy using adeno-associated viral vectors - Implications for treatment success and safety. Prog Retin Eye Res, 2021. 83: p. 100915.15. Park, T.K., et al., Intravitreal delivery ofAAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the R 1 -KO mouse. Gene Therapy, 2009. 16(7): p. 916-926.16. Yiu, G., et al., Suprachoroidal and Subr etinal Injections of AAV Using Transscleral Microneedles for Retinal Gene Delivery in Nonhuman Primates. Mol Ther Methods Clin Dev, 2020. 16: p. 179-191.17. Zhang, K.Y. and T.V. Johnson, The internal limiting membrane: Roles in retinal development and implications for emerging ocular therapies. Exp Eye Res, 2021. 206: p. 108545.18. Kleine Holthaus, S.M., et al., Prevention of Photoreceptor Cell Loss in a Cln6(nclf) Mouse Model of Batten Disease Requires CLN6 Gene Transfer to Bipolar Cells. Mol Ther, 2018. 26(5): p. 1343-1353.EMBODIMENTS

[0113] According to an aspect of the invention, there is provided a method of treating a patient in need thereof. The method comprises delivering a payload to a biological compartment of the patient. The method may comprise delivering a payload to an eye of the patient. The method may further comprise performing a vitrectomy on an eye of the patient and / or delivering a payload to a retina of the eye of the patient and / or treating the retina of the eye of the patient with an adjuvant. The payload delivery results in the treatment of a condition or disease, with the vitrectomy providing more direct application of the payload to the retina, and / or with the adjuvant facilitating the permeability of the retina of the eye for payload delivery to the retina. Removal of the vitreous allows for payload to be placed in direct contact with the retina, preventing cellular contact and off target transduction of other intraocular structures. Application of payload to the eye after vitrectomy may reduce transduction time to the retina, such as allowing transduction under an hour.

[0114] In some embodiments, the method further comprises removing an inner limiting membrane (ILM) of the eye of the patient before delivering the payload to the retina of the eye of the patient. By removing the ILM, substantially less payload is required for treatment, reducinginflammation of the eye in comparison to other methods requiring more payload for effective treatment.

[0115] In some embodiments, the method further comprises filling the eye of the patient with air. Filling the eye with air after vitrectomy allows for payload to be placed in direct contact with the retina and time for payload transduction of the retina, such as allowing transduction to occur within 30 minutes after payload application.

[0116] In some embodiments, the method further comprises removing the payload after a period of time. Removal of the residual payload from the eye, and optionally washing inside the eye thoroughly prevents or reduces off-target transduction to anterior structures and thereby reduces or eliminates resulting inflammation.

[0117] In some embodiments, treating the retina of the eye of the patient with the adjuvant and delivering the payload to the retina of the eye of the patient are done at the same time. In some embodiments, treating the retina of the eye with the adjuvant includes administering the adjuvant locally to the eye. In some embodiments, treating the retina of the eye with the adjuvant includes administering the adjuvant systemically, intravenously, or orally. In some embodiments, the adjuvant comprises insulin.

[0118] In some embodiments, the payload is a viral vehicle. In some embodiments, the payload is a non-viral vehicle. In some embodiments, the payload is a combination of viral and non-viral vehicles. In some embodiments, the payload is selected from the group consisting of a gene therapy vector, a liposome, and a lipid nanoparticle. In some embodiments, the gene therapy vector an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is delivered at about 1 x 106to about 1 x 1015vg / eye.

[0119] According to another aspect of the invention, there is provided a composition including a payload and / or an adjuvant. The payload is configured to treat a condition or disease when delivered to a biological compartment of a patient. The adjuvant facilitates the payload delivery to the biological compartment by increasing biological compartment permeability. Where the biological compartment is an eye and the disease or condition effects the retina of the eye, adjuvant facilitating the permeability of the retina of the eye for payload delivery to the retina.

[0120] In some embodiments, the composition is formulated for local intravenous delivery, sub-retinal delivery, intravitreal delivery or intrathecal delivery. In some embodiments, the payload and the adjuvant are admixed for administration simultaneously.

[0121] In some embodiments, the adjuvant is insulin. Insulin acts as an endocytosis-inducing trigger, increasing transduction efficiency in the retina. The use of an adjuvant, such as insulin, allows less payload to be delivered for the same treatment effect as conventional treatment methods. Further, an adjuvant, such as insulin, can accelerate transduction to the timeframe of minutes to allow for complete application and cellular uptake of payload.

[0122] In some embodiments, the payload includes a gene therapy vector. In some embodiments, the payload is sufficient for delivery of about 1 x 106to about 1 x 1015vg / eye.

[0123] According to another aspect of the invention, there is provided a method of treating a patient in need thereof by delivering any of the disclosed compositions to a retina of the eye of the patient. The method may include performing a vitrectomy on an eye of the patient and delivering any of the disclosed compositions to a retina of the eye of the patient.

[0124] In some embodiments, the patient has a retinal disease or condition. In some embodiments, the patient has an inherited retinal disease or condition. In some embodiments, the patient has neuronal ceroid lipofuscinosis. In some embodiments, the patient has congenital cataracts, congenital glaucoma, retinal degeneration, optic atrophy, eye malformations, glaucoma, wet age-related macular degeneration, dry age-related macular degeneration, retinitis pigmentosa, choroideremia, Leber congenital amaurosis, Leber’s hereditary optic neuropathy, early onset retinal dystrophy, achromatopsia, x-linked retinoschisis, Usher Syndrome IB, neovascular age-related macular degeneration, Stargardt’s macular degeneration, diabetic macular degeneration, or diabetic macular edema.

[0125] In some embodiments, the method further comprises removing an inner limiting membrane (ILM) of the eye of the patient before delivering the disclosed composition to the retina of the eye of the patient. In some embodiments, method further comprises filling the eye of the patient with air. In some embodiments, the method further comprises removing the payload after a period of time.

[0126] According to another aspect of the invention, there is provided a method of delivering a transgene to a patient including administering i) a gene therapy vector encoding the transgene, and / or ii) an adjuvant to the patient.

[0127] According to another aspect of the invention, there is provided a method of treating a patient in need thereof including administering i) a gene therapy vector encoding a transgene, and / or ii) an adjuvant to the patient.

[0128] In some embodiments, the gene therapy vector is administered to the patient using local intravenous delivery, sub-retinal delivery, intravitreous delivery or intrathecal delivery. In some embodiments, the gene therapy vector encoding the transgene and the adjuvant are administered simultaneously to the patient. In some embodiments, the gene therapy vector encoding the transgene and the adjuvant are admixed. In some embodiments, the gene therapy vector encoding the transgene and the adjuvant are administered separately. In some embodiments, the adjuvant comprises insulin.

[0129] Features or steps which are described in the context of separate aspects and embodiments of the invention may be used together and / or be interchangeable. Similarly, features described in the context of a single embodiment may also be provided separately or in any suitable sub-combination.

Claims

What is claimed:

1. A method of treating a patient in need thereof, the method comprising: performing a vitrectomy on an eye of the patient, delivering a payload to a retina of the eye of the patient, and treating the retina of the eye of the patient with an adjuvant.

2. The method of claim 1, wherein the method further comprises removing an inner limiting membrane (ILM) of the eye of the patient before delivering the payload to the retina of the eye of the patient.

3. The method of claim 1 or claim 2, wherein the patient has neuronal ceroid lipofuscinosis.

4. The method of any one of claims 1-3, wherein the method further comprises filling the eye of the patient with air.

5. The method of any one of claims 1-4, wherein the method further comprises removing the payload after a period of time.

6. The method of claim 5, wherein treating the retina of the eye of the patient with the adjuvant and delivering the payload to the retina of the eye of the patient are done at the same time.

7. The method of claim 5 or 6, wherein treating the retina of the eye with the adjuvant comprises administering the adjuvant locally to the eye.

8. The method of claim 5 or 6, wherein treating the retina of the eye with the adjuvant comprises administering the adjuvant systemically, intravenously, or orally.

9. The method of any one of claims 5-8, wherein the adjuvant comprises insulin.

10. The method of any one of claims 5-9, wherein the payload is selected from the group consisting of a gene therapy vector, a liposome, an antisense oligonucleotide, a gold nanoparticle, and a lipid nanoparticle.11 The method of claim 10, wherein the gene therapy vector comprises an adeno-associated viral (AAV) vector.

12. The method of claim 11, wherein the AAV vector is delivered at about 1 x 106to about 1 x 1015vg / eye.

13. The method of claim 11 , wherein the AAV vector is delivered at a volume not to exceed 6 mL / eye.

14. A composition comprising: a payload; and an adjuvant.

15. The composition of claim 14, wherein the composition is formulated for local intravenous delivery, sub-retinal delivery, intravitreal delivery or intrathecal delivery.

16. The composition of claims 14 or 15, wherein the payload and the adjuvant are admixed for administration simultaneously.

17. The composition of any one of claims 14-16, wherein the adjuvant comprises insulin.

18. The composition of any one of claims 14-17, wherein the payload comprises a gene therapy vector.

19. The composition of any one of claims 14-18, wherein the payload is sufficient for delivery of about 1 x 106to about 1 x 1015vg / eye.

20. A method of treating a patient in need thereof, the method comprising: performing a vitrectomy on an eye of the patient, and delivering the composition of any one of claims 13-18 to a retina of the eye of the patient.

21. The method of claim 20, wherein the patient has neuronal ceroid lipofuscinosis.

22. The method of claim 20 or 21, wherein the method further comprises removing an inner limiting membrane (ILM) of the eye of the patient before delivering the composition of any one of claims 12-17 to the retina of the eye of the patient.

23. The method of any one of claims 20-22, wherein the method further comprises filling the eye of the patient with air.

24. The method of any one of claims 20-23, wherein the method further comprises removing the payload after a period of time.

25. A method of delivering a transgene to a patient comprising administering i) a gene therapy vector encoding the transgene, and ii) an adjuvant to the patient.

26. A method of treating a patient in need thereof comprising administering i) a gene therapy vector encoding a transgene, and ii) an adjuvant to the patient.

27. The method of claim 25 or 26, wherein the gene therapy vector is administered to the patient using local intravenous delivery, sub-retinal delivery, intravitreous delivery or intrathecal delivery.

28. The method of any one of claims 25-27, wherein the gene therapy vector encoding the transgene and the adjuvant are administered simultaneously to the patient.

28. The method of claim 27, wherein the gene therapy vector encoding the transgene and the adjuvant are admixed.

29. The method of any one of claims 24-26, wherein the gene therapy vector encoding the transgene and the adjuvant are administered separately.

30. The method of any one of claims 24-29, wherein the adjuvant comprises insulin.

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