WO2020163250A1

WO2020163250A1 - Cx3cl1 compositions and methods for the treatment of degenerative ocular diseases

Info

Publication number: WO2020163250A1
Application number: PCT/US2020/016470
Authority: WO
Inventors: Sean K. WANG; Constance L. Cepko
Original assignee: President And Fellows Of Harvard College
Priority date: 2019-02-05
Filing date: 2020-02-04
Publication date: 2020-08-13

Abstract

The present invention provides CX3CL1 compositions, e.g., pharmaceutical compositions, which include a recombinant adeno-associated viral (AAV) expression construct, AAV vectors, AAV particles, and methods of treating a subject having a degenerative ocular disorder, e.g., retinitis pigmentosa.

Description

CX3CL1 COMPOSITIONS AND METHODS FOR THE TREATMENT OF

DEGENERATIVE OCULAR DISEASES

RELATED APPLICATIONS

The present application claims the benefit of priority to U.S. Provisional Application No. 62/801,184, filed on February 5, 2019, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 29, 2020, is named 117823-19420_SL.txt and is 106,174 bytes in size.

BACKGROUND OF THE INVENTION

Retinitis pigmentosa (RP) is a disease of the eye that presents with progressive degeneration of rod and cone photoreceptors, the light-sensing cells of the retina (Hartong DT, et al. (2006) Lancet 368(9549): 1795-1809). The disease can result from mutations in any of over 60 different genes and is the most common inherited form of blindness in the world, affecting an estimated 1 in 4000 individuals (Daiger SP, et al. (2013) Clin Genet 84(2):132-141 ; Berson EL (1996) Proc Natl Acad Sci U S A 93(10):4526-8; Haim M (2002) Acta Ophthalmol Scand Suppl (233):l-34). One approach to treat this disease is gene therapy, e.g. using adeno-associated vectors (AAVs) to deliver a wild-type allele to complement a mutated gene (Ali RR, et al. (1996) Hum Mol Genet 5(5):591-4; Murata T, et al.

(1997) Ophthalmic Res 29(5):242-251). While this approach has proven successful in other conditions, even leading to the approval of a gene therapy for RPE65-associated Leber’s congenital amaurosis (Maguire AM, et al. (2008) N Engl J Med 358(21):2240-2248), it is difficult to implement for the majority of RP patients, given the extensive heterogeneity of genetic lesions (Daiger SP, et al. (2013) Clin Genet 84(2):132-141). A broadly applicable gene therapy that is agnostic to the genetic lesion would provide a treatment option for a greater number of RP patients. Presently, there is no effective therapy of any kind for RP, and despite more than a dozen randomized clinical trials to date, none have been able to demonstrate an improvement in visual function (Sacchetti M, et al. 2015) J Ophthalmol 2015:737053).

In patients with RP, there is an initial loss of rods, the photoreceptors that mediate vision in dim light. Clinically, this results in the first manifestation of RP, poor or no night vision, which usually occurs between birth and adolescence (Hartong DT, et al. (2006) Lancet 368(9549): 1795-1809). Daylight vision in RP is largely normal for decades, but eventually deteriorates beginning when most of the rods have died. This is due to dysfunction, and then death, of the cone photoreceptors, which are essential for high acuity and color vision. Loss of cone function is the major source of morbidity in the disease (Hartong DT, et al. (2006) Lancet 368(9549): 1795-1809). Importantly, while the vast majority of genes implicated in RP are expressed in rods, few actually exhibit expression in cones, suggesting the existence of one or more common mechanisms by which diverse mutations in rods trigger non- autonomous cone degeneration (Narayan DS, et al. (2016) Acta Ophthalmol 94(8):748-754; Wang W, et al. (2016) Cell Rep 15(2):372-85; Komeima K, et al. (2006) Proc Natl Acad Sci U S A

103(30):11300-5). Attempts to elucidate these mechanisms have been made with the goal of developing therapies for RP that preserve cone vision regardless of the underlying mutation (Punzo C, et al.

(2009) Nat Neurosci 12(l):44-52; Xiong W, et al. (2015) J Clin Invest 125(4):1433-1445; Venkatesh A, et al. (2015) J Clin Invest 125 (4): 1446-58; Ait-Ali N, et al. (2015) Cell 161(4):817-832;

Murakami Y, et al. (2012) Proc Natl Acad Sci 109(36): 14598-14603).

One possible contributor to nonautonomous cone degeneration in RP that has yet to be closely examined is the body’s own immune system. As they die, many cells, including photoreceptors in RP, release damage-associated molecular patterns (DAMPs) that act as endogenous danger signals and incite inflammation ( Zitvogel L, et al. (2010) Cell. 140:798-804; Murakami Y, et al. (2015) Cell Death Dis. 6:e2038). By numerous pathways, DAMPs can then stimulate proinflammatory cytokine activity or recruit immune cells, such as neutrophils and T cells, to the site of cell death (Zitvogel L, et al. (2010) Cell. 140:798-804). Even in homeostatic conditions, the retina is continuously surveyed by microglia, resident macrophages of the central nervous system (CNS) derived from myeloid progenitors in the embryonic yolk sac (Silverman SM, et al. (2018) Anna Rev Vis Sci. 4:45-77; Salter MW, et al. (2017) Nat Med. 23:1018-1027). Following injury or exposure to noxious stimuli, microglia may become activated, a state characterized by acquisition of an ameboid morphology, up-regulation of cytokines, and increased phagocytosis of cell debris (Block ML, et al. (2005) Prog Neurobiol 76:77- 98; Lynch MA. (2009) Mol Neurobiol. 40:139-156; Liddelow SA, et al. (2017) Nature. 541 :481- 487). Notably, activation of microglia can also be modulated by various regulatory factors from the CNS, allowing for manipulation of these cells in both experimental models and humans (Hoek RM, et al. (2000) Science. 290:1768-1771 ; Biber K, et al. (2007) Trends Neurosci. 30:596-602; Cardona AE, et al. (2006) Nat Neurosci. 9:917-924).

Accordingly, there is a need in the art for therapies, such as mutation-independent therapies, to treat and prevent vision loss that results from degenerative ocular diseases, such as retinitis pigmentosa.

SUMMARY OF THE INVENTION

The present invention is based, at least in part on the discovery of mutation-independent compositions and methods of treatment for subjects having RP. More specifically, it has been discovered that microglia, resident macrophages of the central nervous system (CNS) derived from myeloid progenitors in the embryonic yolk sac (Silverman SM, Wong WT (2018) Annu Rev Vis Sci 4(1):45— 77; Salter MW, Stevens B (2017) Nat Med 23(9): 1018-1027), are activated throughout the period of cone death in mouse models of RP.

The present invention is also based, at least in part on the discovery that intraocular delivery of AAV vectors comprising genes that target retinal microglia, such as CX3CL1, also referred to as fractalldne or neurotactin, e.g., soluble CX3CL1 (sCX3CL1), significantly prolonged cone survival in three different mouse models of RP and that this rescue of cones was accompanied by improvements in visual function.

Accordingly, the present invention provides compositions, e.g., pharmaceutical compositions, which include a recombinant adeno-associated virus (AAV) vector, and methods of treating a subject having a degenerative ocular disorder, e.g., retinitis pigmentosa.

In one aspect, the present invention provides a composition, comprising an adeno-associated virus (AAV) expression cassette, the expression cassette comprising a retinal pigmented epithelium- specific (RPE- specific) promoter and a nucleic acid molecule encoding C-X3-C Motif Chemokine Ligand 1 (CX3CL1), e.g., soluble CX3CL1 (sCX3CL 1).

In another aspect, the present invention provides a composition, comprising an adeno- associated virus (AAV) expression cassette, the expression cassette comprising a photoreceptor-specific (PR- specific) promoter and a nucleic acid molecule encoding C-X3-C Motif Chemokine Ligand 1 (CX3CL1), e.g., soluble CX3CL1 (sCX3CL 1).

In one aspect, the present invention provides a composition, comprising an adeno-associated virus (AAV) expression cassette, the expression cassette comprising a bipolar cell-specific promoter and a nucleic acid molecule encoding C-X3-C Motif Chemokine Ligand 1 (CX3CL1), e.g., soluble CX3CL1 (sCX3CL 1).

In one embodiment, the RPE-specific promoter is a human bestrophin 1 (hBestl) promoter.

In one embodiment, the hBestl promoter comprises nucleotides -585 to +38 of the hBestlgene; nucleotides -595 to +30 of the hBestl gene; nucleotides -154 to +38 of the hBestl gene; or nucleotides -104 to +38 bp of the hBestl gene, or or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides -585 to +38 of the hBestlgene; nucleotides -595 to +30 of the hBestl gene; nucleotides -154 to +38 of the hBestl gene; or nucleotides -104 to +38 bp of the hBestl gene.

In another embodiment, the hBestl promoter comprises nucleotides 211-788 of SEQ ID NO:l or SEQ ID NO:2, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 211-788 of SEQ ID NO:l or SEQ ID NO:2.

In one embodiment, the PR-specific promoter is a human red opsin (hRO) promoter.

In one embodiment, the hRO promoter comprises nucleotides 210-2265 of SEQ ID NO:3 or SEQ ID NO:4, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 210-2265 of SEQ ID NO:3 or SEQ ID NO:4.

In one embodiment, the bipolar cell-specific promoter is a glutamate ionotropic receptor kainate type subunit 1 (Grikl) promoter. In one embodiment, the Grikl promoter comprises the nucleotide sequence of SEQ ID NO:15, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of SEQ ID NO:15.

In one embodiment, the nucleic acid molecule encoding CX3CL1 comprises nucleotides 80- 1102 of SEQ ID NO:6, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 80-1102 of SEQ ID NO:6.

In another embodiment, the nucleic acid molecule encoding CX3CL1 comprises nucleotides 80- 1273 of SEQ ID NO:6, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 80-1102 of SEQ ID NO:6.

In one embodiment, the nucleic acid molecule encoding CX3CL1 comprises nucleotides 246- 1013 of SEQ ID NO:7, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 246-1013 of SEQ ID NO:7.

In another embodiment, the nucleic acid molecule encoding CX3CL1 comprises nucleotides 246-1184 of SEQ ID NO:7, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 246-1184 of SEQ ID NO:7.

In one embodiment, the expression cassette further comprises an intron between the promoter and the nucleic acid molecule encoding CX3CL1, e.g., soluble CX3CL1 (CX3CL1 ), such as an SV- 40 intron, or a chimeric intron comprising a 5' -donor site from the first intron of the human b-globin gene and the branch and 3' -acceptor site from the intron that is between the leader and the body of an immunoglobulin gene heavy chain variable region.

In one embodiment, the expression cassette further comprises a post-transcriptional regulatory region.

In one embodiment, the expression cassette further comprises a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

In one embodiment, the expression cassette further comprises a post-transcriptional regulatory region comprising the nucleotide sequence of SEQ ID NO: 11, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of the nucleotide sequence of SEQ ID NO: 11.

In one embodiment, the expression cassette further comprises a polyadenylation signal, such as a bovine growth hormone polyadenylation signal.

In one embodiment, the expression cassette is present in a vector. In one embodiment, the vector is an AAV vector selected from the group consisting of AAV2, AAV 8, AAV2/5, and AAV 2/8.

The present invention also provides AAV vector particles comprising the compositions of the invention, isolated cells comprising the AAV particles of the invention, and pharmaceutical compositions comprising the AAV composition of the invention.

In one embodiment, the pharmaceutical compostions of the invention, further compre a viscosity inducing agent.

In one embodiment, the pharmaceutical compostions of the invention are for intraocular administration, such as intravitreal or subretinal, subvitreal, subconjuctival, sub-tenon, periocular, retrobulbar, suprachoroidal, and/or intrascleral administration.

In one aspect, the present invention provides a method for prolonging the viability of a photoreceptor cell, such as a cone cell, compromised by a degenerative ocular disorder. The method includes, contacting the cell with a composition, AAV viral particle, or pharmaceutical compos tion of the invention, thereby prolonging the viability of the photoreceptor cell compromised by the degenerative ocular disorder.

In one aspect, the present invention provides a method for treating or preventing a degenerative ocular disorder in a subject. The methods include administering to the subject a therapeutically effective amount of a composition, AAV viral particle, or pharmaceutical compostion of the invention, thereby treating or preventing said degenerative ocular disorder.

In one aspect, the present invention provides a method for delaying loss of functional vision in a subject having a degenerative ocular disorder. The methods include administering to the subject a therapeutically effective amount of a composition, AAV viral particle, or pharmaceutical compostion of the invention, thereby treating or preventing said degenerative ocular disorder.

In one aspect, the present invention provides a method for improving functional vision in a subject having a degenerative ocular disorder. The methods include administering to the subject a therapeutically effective amount of a composition, AAV viral particle, or pharmaceutical compostion of the invention, thereby treating or preventing said degenerative ocular disorder.

In one embodiment, the degenerative ocular disorder is associated with decreased viability of cone cells and/or decreased viability of rod cells.

In another embodiment, the degenerative ocular disorder is selected from the group consisting of retinitis pigmentosa, age related macular degeneration, cone rod dystrophy, and rod cone dystrophy.

In one embodiment, the degenerative ocular disorder is a genetic disorder.

In one embodiment, the degenerative ocular disorder is not associated with blood vessel leakage and/or growth.

In one embodiment, the degenerative ocular disorder is retinitis pigmentosa.

In one aspect, the present invention provides a method for treating or preventing retinitis pigmentosa in a subject. The methods inlcude administering to the subject a therapeutically effective amount of the a composition, AAV viral particle, or pharmaceutical compostion of the invention, thereby treating or preventing retinitis pigmentosa in said subject.

Other features and advantages of the invention will be apparent from the following detailed description and claims

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1H depict the expression of immune response genes and microglia localization during cone photoreceptor degeneration. (1A-1D) Whole retina RNA expression levels of immune response genes during onset (P20, P40) and peak (P35, P70) of cone degeneration in two RP mouse models (albino rd10 and pigmented rd10 ) versus two WT strains (albino CD1 and pigmented B6). (IE, 1G) Retinal cross-sections from RP and WT mice depicting Cx3cr1^GFP-labeled microglia during cone degeneration. Scale bar, 100 pm. (IF, 1H) Quantification of percent of total retinal microglia residing in the ONL during cone degeneration in RP and WT mice. Data shown as mean ± SEM. n = 4-6 animals per condition. * P<0.05, ** P<0.01 , *** P<0.001, **** P<0.0001 by two-tailed Student’s t- test with Bonferroni correction for (A-D), two-tailed Student’s t-test for (F, H). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; N.S., not significant.

Figures 2A-2F depict the effect of CD200 and CX3CL1 overexpression on cone survival. (2A, 2B) Schematics of AAV-GFP vector and delivery. (2C) Flat-mounted P50 rd10 retina infected at P0-P1 with AAV-GFP at P0-P1. Scale bar, 1 mm. (2D) Schematics of CD200 and CX3CL1 AAV vectors. (2E) Flat-mounted P50 rd10 retinas infected at P0-P1 with indicated AAVs at P0-P1. Scale bar, 1 mm. (2F) Quantification of cone survival in central retina of P50 rd10 retinas infected with indicated AAVs. Data shown as mean ± SEM. n = 7-18 animals per condition. **** P<0.0001 by two-tailed Student’s t- test with Bonferroni correction.

Figures 3A-3D” depict the effect of AAV-sCX3CL 1 on long-term cone survival in RP mouse models. (3A-3D’) Flat-mounted P75 rd10 (3A, 3A’), P100 rd10 (3B, 3B’), P100 rd10 (3C, 3C’), and P150 Rho ^; (3D, 3D’) retinas infected at P0-P1 with AAV-GFP alone or AAV-GFP plus AAV- sCX3CLl. Scale bar, 1 mm. (3A”-3D”) Quantification of cone survival in central retina of P75 rd10 (3A”), P100 rd10 (3B”), P100 rd10 (3C”), and P150 Rho ^; (3D”) retinas. Data shown as mean ± SEM. n = 7-9 animals per condition. ** P<0.01, *** P<0.001 by two-tailed Student’s t-test.

Figures 4A- 4C depict the effect of AAV-sCX3CL 1 on cone-mediated visual function. (4A) Photopic ERG responses in P40 rd10 mice infected at P0-P1 with AAV-GFP in one eye only (n = 12) or AAV-GFP in one eye and AAV-GFP plus AAV-sCX3CL 1 in the contralateral eye ( n = 17). (4B) Representative photopic ERG traces from a P40 rd10 animal injected with AAV-GFP in one eye and AAV-GFP plus AAV-sCX3CL 1 in the contralateral eye. (4C) Optomotor assessments of visual acuity in rd10 mice at the indicated ages infected at P0-P1 with AAV-GFP {n = 20) or AAV-GFP plus AAV- sCX3CL 1 (n = 21) compared to contralateral uninjected eyes. Data shown as mean ± SEM. * P<0.05, ** P<0.01 by two-tailed two-way ANOVA. Figures 5A-5J depict the effect of AAV-CX3CL1 on microglia localization and expression of immune response genes during cone degeneration. (5 A) Mid-peripheral retinal cross-sections from rd10 andrd10 mice infected at P0-P1 with AAV-mCherry or AAV-mCherry plus AAV-CX3CL1 . Scale bar, 50 pm. (5B) Quantification of ONL thickness in rd10 and rd r1e0tinas during onset of cone degeneration. (5C, E) Cx3cr1^GFP-labeled microglia in rd10 (C) and (r5dE10) retinal cross-sections infected with AAV-mCherry or AAV-mCherry plus AAV-CX3CL1 . Scale bar, 100 pm. (5D, 5F) Quantification of microglia residing in the ONL during cone degeneration with or without AAV- CX3CL1 . (5G-5J) Whole retina RNA expression levels of immune response genes during the onset (5G, 51) and peak (5H, 5J) of cone degeneration with and without AAV-CX3CL1 . Data shown as mean ± SEM. n = 4-6 animals per condition. * P<0.05, ** P<0.01 , *** P<0.001 by two-tailed two-way ANOVA for (5B), two-tailed Student’s t-test for (5D, 5F), two-tailed Student’s t-test with Bonferroni correction for (5G-5J). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; N.S., not significant.

Figures 6A-6C depict the transcriptional profiling of retinal microglia during cone degeneration following CX3CL1 overexpression. (6 A) Volcano plot of upregulated and downregulated genes from P70rd10 retinal microglia following infection with AAV-GFP or AAV-GFP plus AAV-CX3CL1 . Dotted lines indicate adjusted P<0.05 and magnitude of fold-change >2. See Tables 2 and 3 for full gene lists. (6B) RT-PCR validation of gene expression changes in P70 r rdet1i0nal microglia with AAV-CX3CL1 . (6C) Gene set enrichment analysis comparing P70 rd m1i0croglia from retinas infected with AAV-GFP or AAV-GFP plus AAV-CX3CL1 . Gene sets with family-wise error rate (FWER) <0.05 are shown. Data shown as mean ± SEM. n = 7-8 animals per condition. * P<0.05, ** P<0.01, **** P<0.0001 by two-tailed Student’s t-test.

Figures 7A-7D depict the effect of microglia depletion on AAV-CX3CL1 cone rescue. (7 A) Representative flow cytometry gating of microglia (CDl lb+ Ly6G/Ly6C-) and CD l ib- Ly6G/Ly6C+ populations in P50 rd10 retinas with or without PLX3397 treatment from P20 to P49. Panels are gated on live cells (DAPI-) following doublet exclusion. (7B) Fraction of microglia and CD 11b- Ly6G/Ly6C+ cells remaining in P50 rd10 retinas infected with AAV-GFP alone or AAV-GFP plus AAV-CX3CL1 after 30 days of PLX3397 treatment. Retinas from littermates without PLX3397 treatment were used as controls. (7C) Flat-mounted P50 rd10 retinas from PLX3397 treated mice infected with AAV-GFP or AAV-GFP plus AAV-CX3CL1 . Scale bar, 1 mm. (7D) Quantification of cone survival in P50 rd10 retinas from PLX3397 treated mice infected with AAV-GFP or AAV-GFP plus AAV-CX3CL1 . Data shown as mean ± SEM. n = 3-4 animals per condition for (7 A), n = 9-18 animals per condition for (7D). **** P<0.0001 by two-tailed Student’s t-test.

Figures 8A-8C depict the expression of AAV-GFP in cone photoreceptors. (8A, 8B) Cross- section from a P50 WT (CD1) retina infected at P0-P1 with AAV-GFP and stained with peanut agglutinin lectin (PNA), a marker of cone inner and outer segments (1). Scale bars, 500 pm (8A), 50 pm (8B). (8C) High-magnification image of a flat-mounted P50 WT retina infected at P0-P1 with AAV-GFP and stained with PNA. Scale bar, 20 pm. Figures 9A-9C depict the validation of CX3CL1 overexpression with AAV-CX3CL1 . (9 A) Cross-section from a P30 WT (CD1) retina infected at P0-P1 with AAV-GFP or AAV-GFP plus AAV-CX3CL1 and stained with anti-CX3CLl antibody. Scale bar, 50 pm. (9B) Schematic of RPE explant culture. The RPE-choroid-sclera complex was isolated from the rest of the eye and placed on a cell culture insert resting on culture media. (9C) Quantification of secreted CX3CL1 from P40 WT RPE extracts infected at P0-P1 with AAV-GFP or AAV-GFP plus AAV-CX3CL1 . Media was collected 48 hours after explantation and assayed by ELISA. RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.

Figures 10A-10B’ depict the cone quantification methodology. (10A) Representative image of a P50 flat-mounted RP retina infected at P0-P1 with AAV-GFP to label cones. (10A’) A line was drawn from the optic nerve head to the edge of each of the four retinal leaflets. (10A”) An ImageJ module then subjected the image to an automatic threshold, connected the midpoints of these four lines to form a region defined as the central retina, and quantified the number of GFP-positive cones in the central retina. (10B, 10B’) Comparison of raw image from a flat-mounted RP retina infected with AAV-GFP versus the same retina after automatic thresholding.

Figures 11A-11B depict flow cytometry gating of retinal microglia. (11A) P35 rd10 ;Cx3cr1^GFP/+ retinas were dissociated and gated for DAPI-negative single cells, from which three populations were isolated. (1 IB) Histograms of Cx3cr1^GFP signal in each population. CDl lb+ Ly6G/Ly6C- cells were defined as microglia while CD1 lb- Ly6G/Ly6C- and CD1 lb- Ly6G/Ly6C+ cells were defined as non microglia. Data shown as mean ± SEM. n = 4 animals per condition.

Figures 12A-12B depict microglia and retinal cell markers in sorted cell populations. (12A) Comparison of microglia and non-microglia cell populations sorted from r rdet1i0nas for expression of 20 microglia-specific genes by RNA-seq (Daiger SP, et al. (2013) Clin Genet 84(2):132-141). (12B) Comparison of microglia and non-microglia cell populations sorted from r rdet1i0nas for expression of indicated retinal cell-type markers by RNA-seq (Berson EL (1996) Proc Natl Acad Sci U S A

93(10):4526-8; Haim M (2002) Acta Ophthalmol Scand Suppl (233):l-34; Ali RR, et al. (1996) Hum Mol Genet 5(5):591-4;Murata T, et al. (1997) Ophthalmic Res 29(5):242-251 ;Maguire AM, et al. (2008 ) N Engl J Med 358(21):2240-2248; Sacchetti M, et al. 2015) J Ophthalmol 2015:737053).

Data shown as mean ± SEM. n = 24 animals for microglia, n = 6 animals for non-microglia. RGC, retinal ganglion cell; AC, amacrine cell; HC, horizontal cell.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part on the discovery of mutation-independent compositions and methods of treatment for subjects having RP. More specifically, it has been discovered that microglia, resident macrophages of the central nervous system (CNS) derived from myeloid progenitors in the embryonic yolk sac (Silverman SM, Wong WT (2018) Anna Rev Vis Sci 4(l):45-77; Salter MW, Stevens B (2017) Nat Med 23(9): 1018-1027), are activated throughout the period of cone death in mouse models of RP. The present invention is also based, at least in part on the discovery that intraocular delivery of AAV vectors comprising genes that target retinal microglia, such as CX3CL1, also referred to as fractafkine or neurotactin, e.g., soluble CX3CL1 (CX3CL1 ), significantly prolonged cone survival in three different mouse models of RP and that this rescue of cones was accompanied by improvements in visual function.

Various aspects of the invention are described in further detail in the following subsections:

I. DEFINITIONS

As used herein, each of the following terms has the meaning associated with it in this section.

The articles“a” and“an” are used herein to refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element, e.g., a plurality of elements.

The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.

As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA. A nucleic acid molecule used in the methods of the present invention can be isolated using standard molecular biology techniques. Using all or portion of a nucleic acid sequence of interest as a hybridization probe, nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning. A Laboratory Manual. 2nd, ed., Cold Spring Flarbor Laboratory,

Cold Spring Flarbor Laboratory Press, Cold Spring Flarbor, N.Y., 1989).

An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term "isolated" includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid molecule is free of sequences which naturally flank the nucleic acid molecule (i.e., sequences located at the 5' and 3' ends of the nucleic acid molecule) in the genomic DNA of the organism from which the nucleic acid molecule is derived.

A nucleic acid molecule for use in the methods of the invention can also be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of a nucleic acid molecule of interest. A nucleic acid molecule used in the methods of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. Furthermore, oligonucleotides corresponding to nucleotide sequences of interest can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

The nucleic acids for use in the methods of the invention can also be prepared, e.g., by standard recombinant DNA techniques. A nucleic acid of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which has been automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein).

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes or nucleic acid molecules to which they are operatively linked and are referred to as“expression vectors” or "recombinant expression vectors.” Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. In some embodiments, "expression vectors" are used in order to permit pseudotyping of the viral envelope proteins.

Expression vectors are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, adeno-associated viruses, lentiviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells, those which are constitutively active, those which are inducible, and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). The expression vectors of the invention can be introduced into host cells to thereby produce proteins or portions thereof, including fusion proteins or portions thereof, encoded by nucleic acids as described herein.

The terms "transformation," "transfection," and“transduction” refer to introduction of a nucleic acid, e.g., a viral vector, into a recipient cell.

As used herein, the term "subject” includes warm-blooded animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the primate is a human.

As used herein, the various forms of the term "modulate" are intended to include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).

As used herein, the term "contacting" (i.e., contacting a cell with an agent) is intended to include incubating the agent and the cell together in vitro (e.g., adding the agent to cells in culture) or administering the agent to a subject such that the agent and cells of the subject are contacted in vivo. The term "contacting" is not intended to include exposure of cells to an agent that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process).

As used herein, the term“administering” to a subject includes dispensing, delivering or applying a composition of the invention to a subject by any suitable route for delivery of the composition to the desired location in the subject, including delivery by intraocular administration or intravenous administration. Alternatively or in combination, delivery is by the topical, parenteral or oral route, intracerebral injection, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route.

As used herein, the term“degenerative ocular disorder” refers generally to a disorder of the retina. In one embodiment, the degenerative ocular disorder is associated with death, of cone cells, and / or rod cells. Moreover, in a particular embodiment, a degenerative ocular disorder is not associated with blood vessel leakage and/or growth, for example, as is the case with diabetic retinopathy, but, instead is characterized primarily by reduced viability of cone cells and / or rod cells. In certain embodiments, the degenerative ocular disorder is a genetic or inherited disorder. In a particular embodiment, the degenerative ocular disorder is retinitis pigmentosa. In another embodiment, the degenerative ocular disorder is age-related macular degeneration. In another embodiment, the degenerative ocular disorder is cone-rod dystrophy. In another embodiment, the degenerative ocular disorder is rod-cone dystrophy. In other embodiments, the degenerative ocular disorder is not associated with blood vessel leakage and/or growth. In certain embodiments, the degenerative ocular disorder is not associated with diabetes and/or diabetic retinopathy. In further embodiments, the degenerative ocular disorder is not NARP (neuropathy, ataxia, and retinitis pigmentosa). In yet further embodiments, the degenerative ocular disorder is not a neurological disorder. In certain embodiments, the degenerative ocular disorder is not a disorder that is associated with a compromised optic nerve and/or disorders of the brain. In the foregoing embodiments, the degenerative ocular disorder is associated with a compromised photoreceptor cell, and is not a neurological disorder.

As used herein, the term“retinitis pigmentosa” or“RP” is known in the art and encompasses a disparate group of genetic disorders of rods and cones. Retinitis pigmentosa generally refers to retinal degeneration often characterized by the following manifestations: night blindness, progressive loss of peripheral vision, eventually leading to total blindness; ophthalmoscopic changes consist in dark mosaic-like retinal pigmentation, attenuation of the retinal vessels, waxy pallor of the optic disc, and in the advanced forms, macular degeneration. In some cases there can be a lack of pigmentation.

Retinitis pigmentosa can be associated to degenerative opacity of the vitreous body, and cataract. Family history is prominent in retinitis pigmentosa; the pattern of inheritance may be autosomal recessive, autosomal dominant, or X-linked; the autosomal recessive form is the most common and can occur sporadically.

As used herein, the terms“Cone-Rod Dystrophy” or“CRD” and“Rod-Cone Dystrophy” or “RCD” refer to art recognized inherited progressive diseases that cause deterioration of the cone and rod photoreceptor cells and often result in blindness. CRD is characterized by reduced viability or death of cone cells followed by reduced viability or death of rod cells. By contrast, RCD is characterized by reduced viability or death of rod cells followed by reduced viability or death of cone cells.

As used herein, the term "age-related macular degeneration" also referred to as“macular degeneration” or“AMD”, refers to the art recognized pathological condition which causes blindness amongst elderly individuals. Age related macular degeneration includes both wet and dry forms of AMD. The dry form of AMD, which accounts for about 90 percent of all cases, is also known as atrophic, nonexudative, or drusenoid (age-related) macular degeneration. With the dry form of AMD, drusen typically accumulate in the retinal pigment epithelium (RPE) tissue beneath/within the Bruch's membrane. Vision loss can then occur when drusen interfere with the function of photoreceptors in the macula. The dry form of AMD results in the gradual loss of vision over many years. The dry form of AMD can lead to the wet form of AMD. The wet form of AMD, also known as exudative or neovascular (age-related) macular degeneration, can progress rapidly and cause severe damage to central vision. The macular dystrophies include Stargardt Disease, also known as Stargardt Macular Dystrophy or Fundus Flavimaculatus, which is the most frequently encountered juvenile onset form of macular dystrophy.

“Preventing” or“prevention” refers to a reduction in risk of acquiring a disease or disorder (i.e. , causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).

As used herein, the terms“treating” or“treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms, diminishing the extent of infection, stabilized (i.e., not worsening) state of infection, amelioration or palliation of the infectious state, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

Various additional aspects of the methods of the invention are described in further detail in the following subsections.

II. COMPOSITIONS OF THE INVENTION

The present invention provides adeno-associated viral (AAV) expression cassettes, AAV expression cassettes present in AAV vectors, and AAV vectors comprising a recombinant viral genome which include an expression cassette.

Accordingly, in one aspect the present invention provides compositions comprising an adeno- associated virus (AAV) expression cassette, the expression cassette comprising a retinal pigmented epithelium-specific (RPE-specific) promoter operably linked to a nucleic acid molecule encoding C-X3- C Motif Chemokine Ligand 1 (CX3CL1).

In another aspect, the present invention provides compositions comprising an adeno-associated virus (AAV) expression cassette, the expression cassette comprising a photoreceptor-specific (PR- specific) promoter operably linked to a nucleic acid molecule encoding C-X3-C Motif Chemokine Ligand 1 (CX3CL1).

In one aspect, the present invention provides compositions comprising an adeno-associated virus (AAV) expression cassette, the expression cassette comprising a bipolar cell-specific promoter and a nucleic acid molecule encoding C-X3-C Motif Chemokine Ligand 1 (CX3CL1).

In one embodment, the PR-specific promoter is a human red opsin (hRO) promoter.

In one embodiment, the bipolar cell-specific promoter is a glutamate ionotropic receptor kainate type subunit 1 (Grikl) promoter.

In one embodiment, the nucleic acid molecule encodes a substantially full-length CX3CL1 protein (i.e., membrane-bound protein). In another embodiment, the nucleic acid molecule encodes soluble CX3CL1 (CX3CL1 ).

In some embodiments, the expression cassettes of the invention further comprise an intron, such as an intron between the promoter and the nucleic acid molecule encoding CX3CL1.

In some embodiments of the invention, the expression cassettes of the invention further comprise expression control sequences including, but not limited to, appropriate transcription sequences (i.e. initiation, termination, and enhancer), efficient RNA processing signals (e.g. splicing and polyadenylation (poly A) signals), sequences that stabilize cytoplasmic mRNA, sequences that code for a transcriptional enhancer, sequences that code for a posttranscriptional enhancer, sequences that enhance translation efficiency (i.e. Kozak consensus sequence), sequences that enhance protein stability, and when desired, sequences that enhance secretion of the encoded product.

The terms "adeno-associated virus", "AAV virus", "AAV virion", "AAV viral particle", and "AAV particle", as used interchangeably herein, refer to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a particular AAV serotype) and an encapsidated polynucleotide AAV genome. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell) flanked by the AAV inverted terminal repeats (ITRs), it is typically referred to as an "AAV vector particle.”

AAV viruses belonging to the genus Dependovirus of the Parvoviridae family and, as used herein, include any serotype of the over 100 serotypes of AAV viruses known. In general, serotypes of AAV viruses have genomic sequences with a significant homology at the level of amino acids and nucleic acids, provide an identical series of genetic functions, produce virions that are essentially equivalent in physical and functional terms, and replicate and assemble through practically identical mechanisms.

The AAV genome is approximately 4.7 kilobases long and is composed of single-stranded deoxyribonucleic acid (ssDNA) which may be either positive- or negative-sensed. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The rep frame is made of four overlapping genes encoding Rep proteins required for the AAV life cycle. The cap frame contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry. See Carter B, Adeno-associated virus and adeno- associated virus vectors for gene delivery, Lassie D, et al, Eds., "Gene Therapy: Therapeutic Mechanisms and Strategies" (Marcel Dekker, Inc., New York, NY, US, 2000) and Gao G, et al, J. Virol. 2004; 78(12):6381 -6388.

The term "AAV vector" or“AAV construct” refers to a vector derived from an adeno- associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, AAV7, AAV8, and AAV9. "AAV vector" refers to a vector that includes AAV nucleotide sequences as well as heterologous nucleotide sequences. AAV vectors require only the 145 base terminal repeats in cis to generate virus. All other viral sequences are dispensable and may be supplied in trans (Muzyczka (1992) Curr. Topics Microbiol. Immunol. 158:97-129). Typically, the rAAV vector genome will only retain the inverted terminal repeat (ITR) sequences so as to maximize the size of the transgene that can be efficiently packaged by the vector. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, as long as the sequences provide for functional rescue, replication and packaging.

In particular embodiments, the AAV vector is an AAV8, AAV2, AAV2.7m8, AAV2/5, or AAV2/8 vector. Suitable AAV vectors are described in, for example, U.S. Patent No. 7,056,502 and Yan et al. (2002) J. Virology 76(5):2043-2053, the entire contents of which are incorporated herein by reference.

Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products (i.e. AAV Rep and Cap proteins), and wherein the host cell has been transfected with a vector which encodes and expresses a protein from the adenovirus open reading frame E4orf6.

The term "cap gene" or "AAV cap gene", as used herein, refers to a gene that encodes a Cap protein. The term "Cap protein", as used herein, refers to a polypeptide having at least one functional activity of a native AAV Cap protein (e.g. VP1, VP2, VP3). Examples of functional activities of Cap proteins (e.g. VP1, VP2, VP3) include the ability to induce formation of a capsid, facilitate

accumulation of single-stranded DNA, facilitate AAV DNA packaging into capsids (i.e. encapsidation), bind to cellular receptors, and facilitate entry of the virion into host.

The term "capsid", as used herein, refers to the structure in which the viral genome is packaged. A capsid consists of several oligomeric structural subunits made of proteins. For instance, AAV have an icosahedral capsid formed by the interaction of three capsid proteins: VP1, VP2 and VP3.

The term "genes providing helper functions", as used herein, refers to genes encoding polypeptides which perform functions upon which AAV is dependent for replication (i.e. "helper functions"). The helper functions include those functions required for AAV replication including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus. Helper functions include, without limitation, adenovirus El, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase. In one embodiemtn, a helper function does not include adenovirus El.

The term "rep gene" or "AAV rep gene", as used herein, refers to a gene that encodes a Rep protein. The term "Rep protein", as used herein, refers to a polypeptide having at least one functional activity of a native AAV Rep protein (e.g. Rep 40, 52, 68, 78). A "functional activity" of a Rep protein (e.g. Rep 40, 52, 68, 78) is any activity associated with the physiological function of the protein, including facilitating replication of DNA through recognition, binding and nicking of the AAV origin of DNA replication as well as DNA helicase activity. Additional functions include modulation of transcription from AAV (or other heterologous) promoters and site- specific integration of AAV DNA into a host chromosome.

The term "adeno-associated virus ITRs" or "AAV ITRs", as used herein, refers to the inverted terminal repeats present at both ends of the DNA strand of the genome of an adeno-associated virus. The ITR sequences are required for efficient multiplication of the AAV genome. Another property of these sequences is their ability to form a hairpin. This characteristic contributes to its self-priming which allows the primase- independent synthesis of the second DNA strand. The ITRs have also shown to be required for efficient encapsidation of the AAV DNA combined with generation of fully assembled, deoxyribonuclease- resistant AAV particles.

The term "expression cassette", as used herein, refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit transcription of a particular nucleic acid in a target cell.

The expression cassettes of the invention include a promoter that is operably linked to a nucleic acid molecule encoding C-X3-C Motif Chemokine Ligand 1 (CX3CL1). Exemplary expression cassettes of the invention are depicted in Figure 2D.

The term "promoter" as used herein refers to a recognition site of a DNA strand to which the RNA polymerase binds. The promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity. The complex can be modified by activating sequences termed "enhancers" or inhibitory sequences termed "silencers".

Suitable promoters for use in the expression cassetees of the invention may be ubiquitous promoters, such as a CMV promoter or an SV40 promoter, but are preferably tissue-specific promoters, i.e., promoters that direct expression of a nucleic acid molecule preferentially in a particular cell type.

In one embodiment, a tissue-specific promoter for use in the present invention is a retinal pigmented epithelium-specific (RPE-specific) promoter. In another embodiment, a tissue-specific promoter for use in the present invention is a photoreceptor-specific (PR-specific) promter. The PR- specific promoter may be a rod-specific promoter; a cone-specific promoter; or a rod- and cone-specific promoter. In yet another embodiment, a tissue-specific promoter for use in the present invention is a bipolar cell-specific promter. The bipolar cell-specific promoter may be a rod bipolar cell-specific promoter or a ceon bipolar cell-specific promoter. In one embodiment, the bipolar cell-specific promoter is an OFF bipolar cell-specific promoter. In another embodiment, the bipolar cell-specific promoter is an ON bipolar cell-specific promoter.

Suitable RPE-specific promoters are known in the art and include, for example, bestrophin 1 and retinal pigment epithelium-specific 65 kDa protein, also known as retinoid isomerohydrolase (RPE65).

In certain embodiments, a suitable RPE-specific promoter is a human bestrophin 1 (hBestl) promoter.

As used interchangeably herein, the terms“bestrophin 1,”“hBestl,” and“hBESTl” refer to bestrophin- 1, also known as Bestrophin 1 ; Vitelliform Macular Dystrophy Protein 2; Best Disease; TU15B; VMD2; Vitelliform Macular Dystrophy 2; BestlVlDelta2; Bestrophin-1 ; BEST; RP50; ARB; and BMD refers to the gene that is highly and preferentially expressed in the RPE. There are four transcript variants of hBest, the nucleotide and amino acid sequences of which are known and may be found in, for example, GenBank Reference Sequences NM_001139443.1 ; NM_001300786.1 ;

NM_001300787.1 ; and NM_004183.3. The nucleotide sequence of the genomic region containing the hBestl gene (including the region upstream of the coding region of hBestl which includes the hBestl promoter region) is also known and may be found in, for example, GenBank Reference Sequence NG_009033.1 (SEQ ID NO: 10, the entire contents of which is incorporated herein by reference).

Suitable hBestl promoters for use in the present invention include nucleic acid molecules which include nucleotides -585 to +38 of the hBestlgene, (i.e., nucleotides 4885-5507 of SEQ ID NO:10); nucleotides -585 to +39 of the hBestlgene, (i.e., nucleotides 4885-5508 of SEQ ID NO:10);

nucleotides -154 to +38 of the hBestl gene (i.e., nucleotides 5316-5507 of SEQ ID NO:10); or nucleotides -104 to +38 bp of the hBestl gene (i.e., nucleotides 5366-5507 of SEQ ID NO:10), or or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 4885-5507 of SEQ ID NO: 10, nucleotides 4885-5508 of SEQ ID NO: 10, nucleotides 5316-5507 of SEQ ID NO:10, or nucleotides 5366-5507 of SEQ ID NO:10. In one embodiement, an hBestl promoter comprises nucleotides -585 to +38 of the hBestlgene, (i.e., nucleotides 4885-5507 of SEQ ID NO:10), or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 4885-5507 of SEQ ID NO: 10. In one embodiement, an hBestl promoter comprises nucleotides -585 to +39 of the hBestlgene, (i.e., nucleotides 4885-5507 of SEQ ID NO:10), or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 4885-5507 of SEQ ID NO: 10. In another embodiment, an hBestl promoter comprises nucleotides 211-788 of SEQ ID NO:l or SEQ ID NO:2, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 211-788 of SEQ ID NO:l or SEQ ID NO:2.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions total # of positions (e.g., overlapping positions) xlOO).

The determination of percent identity between two sequences may be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sol.

USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Nati. Accid Sci. USA 90:5873- 5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the BLASTN program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTP program, score— 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, a newer version of the BLAST algorithm called Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res 25:3389-3402, which is able to perform gapped local alignments for the programs BLASTN, BLASTP and BLASTX.

Suitable PR-specific promoters are known in the art and include, for example, a human red opsin promoter, a human rhodopsin promoter, a human rhodopsin kinase (RK) promoter, and a G protein-coupled receptor kinase 1 (GRK1) promoter.

In certain embodiments, a suitable PR-specific promoter is a human red opsin (hRO) promoter.

As used interchangeably herein, the terms“red opsin,”“RO,” and“hRO” refer to Opsin 1, Long Wave Sensitive, also known as Red Cone Photoreceptor Pigment, Opsin 1 (Cone Pigments), Long-Wave-Sensitive, Cone Dystrophy 5 (X-Linked), Red-Sensitive Opsin, RCP, ROP, Long-Wave- Sensitive Opsin, Color Blindness, Protan, Red Cone Opsin, COD5, CBBm, and CBP. The nucleotide sequence of the genomic region containing the hRO gene (including the region upstream of the coding region of hRO which includes the hRO promoter region) is also known and may be found in, for example, GenBank Reference Sequence NG_009105.2 (SEQ ID NO: 12, the entire contents of which is incorporated herein by reference).

Suitable RO promoters for use in the present invention include nucleic acid molecules which include nucleotides 452-2017 of SEQ ID NO:12 directly linked, /^'. e. , no intervening nucleotide sequences, to nucleotides 4541-5032 of SEQ ID NO:12; or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 452-2017 of SEQ ID NO: 12 directly linked to nucleotides 4541-5032 of SEQ ID NO:12.

Suitable bipolar cell-specific promoters for use in the present invention are known in the art and include, for example, a glutamate ionotropic receptor kainate type subunit 1 (Grikl) promoter.

As used interchangeably herein, the terms“glutamate ionotropic receptor kainate type subunit 1” and“Grikl” refer to an ionotropic glutamate receptor (GluR) subunit that functions as a ligand gated ion channel. The specific GluR subunit encoded by this gene is of the kainate receptor subtype and mediates excitatory neurotransmission during normal retinal synaptic function. The nucleotide sequence of a suitable Grikl promoter for use in the present invention is provided in SEQ ID NO:15.

In one embodiment, the Grikl promoter comprises SEQ ID NO:15, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of SEQ ID NO:15.

As used herein, the term“CX3CL1” refers to the protein C-X3-C Motif Chemokine Ligand 1, a chemokine which is a member of the CX₃C chemokine family. CX3CL1 is also known as Small Inducible Cytokine Subfamily D (Cys-X3-Cys), Member 1 (Fractalkine, Neurotactin), Chemokine (C- X3-C Motif) Ligand, CX3C Membrane-Anchored Chemokine, Small-Inducible Cytokine D, C-X3-C Motif Chemokine 1, Neurotactin, Fractalkine, SCYD1, NTT, Small Inducible Cytokine Subfamily D (Cys-X3-Cys), Member-, C3Xkine 3, ABCD-, CXC3C, CXC3, NTN, and FKN. The encoded protein contains an extended mucin-like stalk with a chemokine domain on top, and exists in both a membrane- anchored form where it acts as a binding molecule, or, in soluble form, as a chemotactic cytokine. The mature form of this protein can be cleaved at the cell surface, yielding different soluble forms that can interact with the G-protein coupled receptor, C-X3-C motif chemokine receptor 1 gene product. There are two transcript variants of CX3CL1, the nucleotide and amino acid sequences of which are known and may be found in, for example, GenBank Reference Sequences NM_002996.6 and

NM_001304392.2 Homo (SEQ ID NOs:6 and 7, respectively). In one embodiment, a nucleic acid molecule encoding CX3CL1 comprises nucleotides 80-1102 of SEQ ID NO:6, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 80-1102 of SEQ ID NO:6. In another embodiment, the nucleic acid molecule encoding CX3CL1 comprises nucleotides 80-1273 of SEQ ID NO:6, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 80-1102 of SEQ ID NO:6. In one embodiment, the nucleic acid molecule encoding CX3CL1 comprises nucleotides 246-1013 of SEQ ID NO:7, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 246-1013 of SEQ ID NO:7. In another embodiment, the nucleic acid molecule encoding CX3CL1 comprises nucleotides 246-1184 of SEQ ID NO:7, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 246-1184 of SEQ ID NO:7.

The invention further encompasses nucleic acid molecules that differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acids encoding a CX3CL1 polypeptide, and, thus, encode the same protein.

In some embodiments, the expression cassettes of the invention further comprise an intron between the promoter and the nucleic acid molecule endoing CX3CL1. As used herein,“an intron” refers to a non-coding nucleic acid molecule which is removed by RNA splicing during maturation of a final RNA product.

In one embodiment, the intron is an SV40 intron. In another embodiment, the intron is a the human beta-globin intron (SEQ ID NO: 13). In another embodiment, the intron is a chimeric intron.

A“chimeric intron” is an artificial (or non-naturally occurring intron that enhances mRNA processing and increases expression levels of a downstream open reading frame.

In some embodiments, the expression cassettes of the invention further comprise a post- transcriptional regulatory region.

The term "post-transcriptional regulatory region", as used herein, refers to any polynucleotide that facilitates the expression, stabilization, or localization of the sequences contained in the cassette or the resulting gene product.

In one embodiment, a post-transcriptional regulatory region suitable for use in the expression cassettes of the invention includes a Woodchuck hepatitis virus post-transcriptional regulatory element.

As used herein, the term "Woodchuck hepatitis virus posttranscriptional regulatory element" or "WPRE,” refers to a DNA sequence that, when transcribed, creates a tertiary structure capable of enhancing the expression of a gene. See Lee Y, et al, Exp. Physiol. 2005; 90(l):33-37 and Donello J, et al, J. Virol. 1998; 72(6):5085-5092.

In one embodiment, a WPRE includes the nucleotide sequence of SEQ ID NO: 11 (See, e.g., J Virol. 1998 Jun; 72(6): 5085-5092), or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of the nucleotide sequence of SEQ ID NO: 11.

In some embodiments, the expression cassettes of the invention further comprises a polyadenylation signal.

As used herein, a“polyadenylation signal” or“polyA signal,” as used herein refers to a nucleotide sequence that terminates transcription. Suitable polyadenylation signals for use in the AAV vectors of the invention are known in the art and include, for example, a bovine growth hormone polyA signal (BGH pA), or an SV40 polyadenylation signal (SEQ ID NO: 14).

In some embodiments, the expression cassettes of the invention further comprise an enhancer.

The term "enhancer", as used herein, refers to a DNA sequence element to which transcription factors bind to increase gene transcription.

The AAV vectors of the invention may also include cis- acting 5' and 3' inverted terminal repeat (ITR) sequences. In some embodiments, the ITR sequences are about 145 bp in length. In some embodiments, substantially the entire sequences encoding the ITRs are used in the molecule. In other embodiments, the ITRs include modifications. Procedures for modifying these ITR sequences are known in the art. See Brown T, "Gene Cloning" (Chapman & Hall, London, GB, 1995), Watson R, et al, "Recombinant DNA", 2nd Ed. (Scientific American Books, New York, NY, US, 1992), Alberts B, et al, "Molecular Biology of the Cell" (Garland Publishing Inc., New York, NY, US, 2008), Innis M, et al, Eds., "PCR Protocols. A Guide to Methods and Applications" (Academic Press Inc., San Diego, CA, US, 1990), Erlich H, Ed., "PCR Technology. Principles and Applications for DNA Amplification" (Stockton Press, New York, NY, US, 1989), Sambrook J, et al, "Molecular Cloning. A Laboratory Manual" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, US, 1989), Bishop T, et al, "Nucleic Acid and Protein Sequence. A Practical Approach" (IRL Press, Oxford, GB, 1987), Reznikoff W, Ed., "Maximizing Gene Expression" (Butterworths Publishers, Stoneham, MA, US, 1987), Davis L, et al, "Basic Methods in Molecular Biology" (Elsevier Science Publishing Co., New York, NY, US, 1986), and Schleef M, Ed., "Plasmid for Therapy and Vaccination" (Wiley- VCH Verlag GmbH, Weinheim, DE, 2001).

The AAV vectors of the invention may include ITR nucleotide sequences derived from any one of the AAV serotypes. In a preferred embodiment, the AAV vector comprises 5' and 3' AAV ITRs. In one embodiment, the 5' and 3' AAV ITRs derive from AAV2. AAV ITRs for use in the AAV vectors of the invention need not have a wild- type nucleotide sequence (See Kotin, Hum. Gene Ther. , 1994, 5:793-801). As long as ITR sequences function as intended for the rescue, replication and packaging of the AAV virion, the ITRs may be altered by the insertion, deletion or substitution of nucleotides or the ITRs may be derived from any of several AAV serotypes or its mutations.

In one embodiment, a 5’ ITR includes nucleotides 1-130 of any one of the nucleotide sequences of SEQ ID NOs:l-4, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 1-130 of any one of the nucleotide sequences of SEQ ID NOs:l-4.

In one embodiment, a 3’ ITR includes nucleotides 2856-2985 of the nucleotide sequence of SEQ ID NO:l ; nucleotides 2562-2691 of SEQ ID NO:2; nucleotides4327-4456 of SEQ ID NOG; or nucleotides 3976-4105 of SEQ ID NO:4, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 2856-2985 of the nucleotide sequence of SEQ ID NO:l ; nucleotides 2562-2691 of SEQ ID NOG; nucleotides 4327-4456 of SEQ ID NOG; or nucleotides 3976-4105 of SEQ ID NOG.

In addition, an AAV vector can contain one or more selectable or screenable marker genes for initially isolating, identifying, or tracking host cells that contain DNA encoding the ithe AAV vector (and/or rep, cap and/helper genes), e.g., antibiotic resistance, as described herein.

As indicated above, the AAV vectors of the invention may be packaged into AAV viral particles for use in the methods, e.g., gene therapy methods, of the invention (discussed below) to produce AAV vector particles using methods known in the art.

Such methods generally include packaging the AAV vectors of the invention into infectious AAV viral particles in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products (i.e. AAV Rep and Cap proteins), and with a vector which encodes and expresses a protein from the adenovirus open reading frame E4orf6.

Suitable AAV Caps may be derived from any serotype. In one embodiment, the capsid is derived from the AAV of the group consisting on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9. In another embodiment, the AAV of the invention comprises a capsid derived from the AAV5 or AAV8 serotypes.

In some embodiments, an AAV Cap for use in the method of the invention can be generated by mutagenesis (i.e. by insertions, deletions, or substitutions) of one of the aforementioned AAV Caps or its encoding nucleic acid. In some embodiments, the AAV Cap is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more similar to one or more of the aforementioned AAV Caps.

In some embodiments, the AAV Cap is chimeric, comprising domains from two, three, four, or more of the aforementioned AAV Caps. In some embodiments, the AAV Cap is a mosaic of VP1, VP2, and VP3 monomers originating from two or three different AAV or a recombinant AAV. In some embodiments, a rAAV composition comprises more than one of the aforementioned Caps.

Suitable rep may be derived from any AAV serotype. In one embodiment, the rep is derived from any of the serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In another embodiment, the AAV rep is derived from the serotype AAV2.

Suitable helper genes may be derived from any AAV serotype and include adenovirus E4, E2a and VA.

The AAV rep, AAV cap and genes providing helper functions can be introduced into the cell by incorporating the genes into a vector such as, for example, a plasmid, and introducing the vector into a cell. The genes can be incorporated into the same plasmid or into different plasmids. In one, the AAV rep and cap genes are incorporated into one plasmid and the genes providing helper functions are incorporated into another plasmid.

The AAV vectors of the invention and the polynucleotides comprising AAV rep and cap genes and genes providing helper functions may be introduced into a host cell using any suitable method well known in the art. See Ausubel F, et al, Eds., "Short Protocols in Molecular Biology", 4th Ed. (John Wiley and Sons, Inc., New York, NY, US, 1997), Brown (1995), Watson (1992), Alberts (2008), Innis (1990), Erlich (1989), Sambrook (1989), Bishop (1987), Reznikoff (1987), Davis (1986), and Schleef (2001), supra. Examples of transfection methods include, but are not limited to, co-precipitation with calcium phosphate, DEAE-dextran, polybrene, electroporation, microinjection, liposome-mediated fusion, lipofection, retrovirus infection and biolistic transfection. When the cell lacks the expression of any of the AAV rep and cap genes and genes providing adenoviral helper functions, said genes can be introduced into the cell simultaneously with the AAV vector. Alternatively, the genes can be introduced in the cell before or after the introduction of the AAV vector of the invention.

Methods of culturing packaging cells and exemplary conditions which promote the release of AAV vector particles, such as the producing of a cell lysate, are known in the art. Producer cells are grown for a suitable period of time in order to promote release of viral vectors into the media.

Generally, cells may be grown for about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, up to about 10 days. After about 10 days (or sooner, depending on the culture conditions and the particular producer cell used), the level of production generally decreases significantly. Generally, time of culture is measured from the point of viral production. For example, in the case of AAV, viral production generally begins upon supplying helper virus function in an appropriate producer cell as described herein. Generally, cells are harvested about 48 to about 100, preferably about 48 to about 96, preferably about 72 to about 96, preferably about 68 to about 72 hours after helper virus infection (or after viral production begins).

The AAV vector particles of the invention can be obtained from both: i) the cells transfected with theforegoing and ii) the culture medium of the cells after a period of time post-transfection, preferably 72 hours. Any method for the purification of the AAV vector particles from the cells or the culture medium can be used for obtaining the AAV vector particles of the invention. In a particular embodiment, the AAV vector particles of the invention are purified following an optimized method based on a polyethylene glycol precipitation step and two consecutive cesium chloride (CsC1) or iodixanol density gradient ultracentrifugation. See Ayuso et al., 2014, Zolotukhin S, et al , Gene Ther. 1999; 6; 973-985. Purified AAV vector particles of the invention can be dialyzed against an appropriate formulation buffer such as PBS, filtered and stored at -80°C. Titers of viral genomes can be determined by quantitative PCR following the protocol described for the AAV2 reference standard material using linearized plasmid DNA as standard curve. See Aurnhammer C, et al , Hum Gene Ther Methods, 2012, 23, 18-28, D’Costa S, et al , Mol Ther Methods Clin Dev. 2016, 5, 16019.

In some embodiments, the methods further comprise purification steps, such as treatment of the cell lysate with benzonase, purification of the cell lysate with the use of affinity chromatography and/or ion-exchange chromotography. See Halbert C, et al, Methods Mol Biol 2004; 246:201-212, Nass S, et al , Mol Ther Methods Clin Dev. 2018 Jun 15; 9: 33-46.

AAV Rep and Cap proteins and their sequences, as well as methods for isolating or generating, propagating, and purifying such AAV, and in particular, their capsids, suitable for use in producing AAV are known in the art. See Gao, 2004, supra, Russell D, et al, US 6,156,303, Hildinger M, et al, US 7,056,502, Gao G, et al, US 7,198,951, Zolotukhin S, US 7,220,577, Gao G, et al, US 7,235,393, Gao G, et al, US 7,282,199, Wilson J, et al, US 7,319,002, Gao G, et al, US 7,790,449, Gao G, et al, US 20030138772, Gao G, et al, US 20080075740, Hildinger M, et al, WO 2001/083692, Wilson J, et al, WO 2003/014367, Gao G, et al, WO 2003/042397, Gao G, et al, WO 2003/052052, Wilson J, et al, WO 2005/033321, Vandenberghe L, et al, WO 2006/110689, Vandenberghe L, et al, WO

2007/127264, and Vandenberghe L, et al, WO 2008/027084.

III. PHARMACEUTICAL COMPOSITIONS OF THE INVENTION

In one aspect of the invention, an AAV viral particle of the invention will be in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier. As used herein

"pharmaceutically acceptable carrier" refers to any substantially non-toxic carrier conventionally useable for administration of pharmaceuticals in which the isolated polypeptide of the present invention will remain stable and bioavailable. The pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the mammal being treated. It further should maintain the stability and bioavailability of an active agent. The

pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition. Suitable pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutically acceptable carriers also include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the gene therapy vector, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Pharmaceutical compositions of the invention may be formulated for delivery to animals for veterinary purposes (e.g. livestock (cattle, pigs, dogs, mice, rats), and other non-human mammalian subjects), as well as to human subjects.

In a particular embodiment, the pharmaceutical compositions of the present invention are in the form of injectable compositions. The compositions can be prepared as an injectable, either as liquid solutions or suspensions. The preparation may also be emulsified. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, phosphate buffered saline or the like and combinations thereof. In addition, if desired, the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH-buffering agents, adjuvants, surfactant or

immunopotentiator s .

In a particular embodiment, the AAV particles of the invention are incorporated in a composition suitable for intraocular administration. For example, the compositions may be designed for intravitreal, subretinal, subconjuctival, sub-tenon, periocular, retrobulbar, suprachoroidal, and/or intrascleral administration, for example, by injection, to effectively treat the retinal disorder.

Additionally, a sutured or refillable dome can be placed over the administration site to prevent or to reduce "wash out", leaching and/or diffusion of the active agent in a non-preferred direction.

Relatively high viscosity compositions, as described herein, may be used to provide effective, and preferably substantially long-lasting delivery of the nucleic acid molecules and/or vectors, for example, by injection to the posterior segment of the eye. A viscosity inducing agent can serve to maintain the nucleic acid molecules and/or vectors in a desirable suspension form, thereby preventing deposition of the composition in the bottom surface of the eye. Such compositions can be prepared as described in U.S. Patent No. 5,292,724, the entire contents of which are hereby incorporated herein by reference.

Sterile injectable solutions can be prepared by incorporating the compositions of the invention in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Toxicity and therapeutic efficacy of nucleic acid molecules described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the EDso (the dose therapeutically effective in 50% of the population). Data obtained from cell culture assays and/or animal studies can be used in formulating a range of dosage for use in humans. The dosage typically will lie within a range of concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.

IV. METHODS OF THE INVENTION

The present invention also provides methods of use of the compositions of the invention, which generally include contacting an ocular cell with an AAV viral particle or pharmaceutical composition comprising an AAV particle of the invention.

Accordingly, in one aspect, the present invention provides methods for prolonging the viability of a photoreceptor cell, e.g., a photoreceptor cell, e.g., a cone cell, compromised by degenerative ocular disorder, e.g., retinitis pigmentosa, age related macular degeneration, cone rod dystrophy, and rod cone dystrophy. The methods generally include contacting the cell with an AAV viral particle or pharmaceutical composition comprising an AAV particle of the invention.

The present invention further provides methods for treating a degenerative ocular disorder in a subject having a degenerative ocular disorder, e.g., retinitis pigmentosa, age related macular degeneration, cone rod dystrophy, and rod cone dystrophy. The methods inlcude administering to the subject a therapeutically effective amount of an AAV viral particle or pharmaceutical composition comprising an AAV particle of the invention.

The present invention also provides methods for preventing a degenerative ocular disorder in a subject having a degenerative ocular disorder, e.g., retinitis pigmentosa, age related macular degeneration, cone rod dystrophy, and rod cone dystrophy. The methods inlcude administering to the subject a prohylatically effective amount of an AAV viral particle or pharmaceutical composition comprising an AAV particle of the invention.

In another aspect, the present invention provides methods of treating a subject having retinitis pigmentosa. The methods inlcude administering to the subject a therapeutically effective amount of an AAV viral particle or pharmaceutical composition comprising an AAV particle of the invention. In another aspect, the present invention provides methods of treating a subject having age- related macular degeneration. The methods inlcude administering to the subject a therapeutically effective amount of an AAV viral particle or pharmaceutical composition comprising an AAV particle of the invention.

Generally, methods are known in the art for viral infection of the cells of interest. The virus can be placed in contact with the cell of interest or alternatively, can be injected into a subject suffering from a degenerative ocular disorder.

Guidance in the introduction of the compositions of the invention into subjects for therapeutic purposes are known in the art and may be obtained in the above-referenced publications, as well as in U.S. Patent Nos. 5,631,236, 5,688,773, 5,691,177, 5,670,488, 5,529,774, 5,601,818, and PCT Publication No. WO 95/06486, the entire contents of which are incorporated herein by reference.

The compositions of the invention may be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470), stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91 :3054-3057), or by in vivo electroporation (see, e.g., Matsuda and Cepko (2007) Proc. Natl. Acad. Sci. U.S.A. 104:1027-1032).

Preferably, the compositions of the invention are administered to the subject locally. Local administration of the compositions described herein can be by any suitable method in the art including, for example, injection (e.g., intravitreal or subretinal, subvitreal, subconjuctival, sub-tenon, periocular, retrobulbar, suprachoroidal, and/or intrascleral injection), gene gun, by topical application of thecomposition in a gel, oil, or cream, by electroporation, using lipid-based transfection reagents, transcleral delivery, by implantation of scleral plugs or a drug delivery device, or by any other suitable transfection method.

Application of the methods of the invention for the treatment and/or prevention of a disorder can result in curing the disorder, decreasing at least one symptom associated with the disorder, either in the long term or short term or simply a transient beneficial effect to the subject.

Accordingly, as used herein, the terms“treat,”“treatment” and“treating” include the application or administration of compositions, as described herein, to a subject who is suffering from a degenerative ocular disease or disorder, or who is susceptible to such conditions with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving or affecting such conditions or at least one symptom of such conditions. As used herein, the condition is also“treated” if recurrence of the condition is reduced, slowed, delayed or prevented.

The term“prophylactic” or“therapeutic” treatment refers to administration to the subject of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g. , disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e. , it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e. , it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom). "Therapeutically effective amount," as used herein, is intended to include the amount of a composition of the invention that, when administered to a patient for treating a degenerative ocular disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective amount" may vary depending on the composition, how the composition is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by the disease expression, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of a composition that, when administered to a subject who does not yet experience or display symptoms of e.g., a degenerative ocular disorder, but who may be predisposed to the disease, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The

"prophylactically effective amount" may vary depending on the composition, how the composition is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A "therapeutically-effective amount" or“prophylacticaly effective amount” also includes an amount of a composition that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. A composition employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

Subjects suitable for treatment using the regimens of the present invention should have or are susceptible to developing a degenerative ocular disease or disorder. For example, subjects may be genetically predisposed to development of the disorders. Alternatively, abnormal progression of the following factors including, but not limited to visual acuity, the rate of death of cone and / or rod cells, night vision, peripheral vision, attenuation of the retinal vessels, and other ophthalmoscopic factors associated with degenerative ocular disorders such as retinitis pigmentosa may indicate the existence of or a predisposition to a retinal disorder.

In one embodiment, the disorder includes, but are not limited to, retinitis pigmentosa, age related macular degeneration, cone rod dystrophy, and rod cone dystrophy. In other embodiments, the disorder is not associated with blood vessel leakage and/or growth. In certain embodiments, the disorder is not associated with diabetes. In another embodiment, the disorder is not diabetic retinopathy. In further embodiments, the disorder is not NARP (neuropathy, ataxia and retinitis pigmentosa). In one embodiment, the disorder is a disorder associated with decreased viability of cone and/or rod cells. In yet another embodiment, the disorder is a genetic disorder.

The compositions, as described herein, may be administered as necessary to achieve the desired effect and depend on a variety of factors including, but not limited to, the severity of the condition, age and history of the subject and the nature of the composition, for example, the identity of the genes or the affected biochemical pathway.

The pharmaceutical compositions of the invention may be administered in a single dose or, in particular embodiments of the invention, multiples doses (e.g. two, three, four, or more administrations) may be employed to achieve a therapeutic effect.

The therapeutic or preventative regimens may cover a period of at least about 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks, or be chronically administered to the subject.

In one embodiment, the viability or survival of photoreceptor cells, such as cones cells, is, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 years, about 4 years, about 5 years, about 10 years, about 15, years, about 20 years, about 25 years, about 30 years, about 40 years, about 50 years, about 60 years, about 70 years, and about 80 years.

In general, the nucleic acid molecules and/or the vectors of the invention are provided in a therapeutically effective amount to elicit the desired effect, e.g. , increase CX3CL1, e.g., soluble CX3CL1, expression. The quantity of the viral particle to be administered, both according to number of treatments and amount, will also depend on factors such as the clinical status, age, previous treatments, the general health and/or age of the subject, other diseases present, and the severity of the disorder. Precise amounts of active ingredient required to be administered depend on the judgment of the gene therapist and will be particular to each individual patient. Moreover, treatment of a subject with a therapeutically effective amount of the nucleic acid molecules and/or the vectors of the invention can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result from the results of diagnostic assays as described herein. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

In some embodiments, a therapeutically effective amount or a prophylactically effective amount of a viral particle of the invention (or pharmaceutical composition of the invention) is in titers ranging from about lxlO⁵ , about 1.5x10^s, about 2x10 ^s, about 2.5x10^s, about 3x10^s, about 3.5x10^s, about 4x10^s, about 4.5x10^s, about 5x10^s, about 5.5x10^s, about 6x10^s, about 6.5x10^s, about 7x10^s, about 7.5x10^s, about 8x10^s, about 8.5x10^s, about 9x10^s, about 9.5x10^s, about lxlO⁶, about 1.5xl0⁶, about 2xl0⁶, about 2.5xl0⁶, about 3xl0⁶, about 3.5xl0⁶, about 4xl0⁶, about 4.5xl0⁶, about 5xl0⁶, about 5.5xl0⁶, about 6xl0⁶, about 6.5xl0⁶, about 7xl0⁶, about 7.5xl0⁶, about 8xl0⁶, about 8.5x10, about 9xl0⁶, about 9.5xl0⁶, about lxlO⁷, about 1.5xl0⁷, about 2xl0⁷, about 2.5xl0⁷, about 3xl0⁷, about 3.5xl0⁷, about 4xl0⁷, about 4.5xl0⁷, about 5xl0⁷, about 5.5xl0⁷, about 6xl0⁷, about 6.5xl0⁷, about 7xl0⁷, about 7.5xl0⁷, about 8xl0⁷, about 8.5xl0⁷, about 9xl0⁷, about 9.5xl0⁷, about 1x10^s, about 1.5x10^s, about 2x10^s, about 2.5x10^s, about 3x10^s, about 3.5x10^s, about 4x10^s, about 4.5x10^s, about 5x10^s, about 5.5x10^s, about 6x10^s, about 6.5x10^s, about 7x10^s, about 7.5x10^s, about 8x10^s, about 8.5x10^s, about 9x10^s, about 9.5x10^s, about lxlO⁹, about 1.5xl0⁹, about 2xl0⁹, about 2.5x109^s, about 3xl0⁹, about 3.5xl0⁹, about 4xl0⁹, about 4.5xl0⁹, about 5xl0⁹, about 5.5xl0⁹, about 6xl0⁹, about 6.5xl0⁹, about 7xl0⁹, about 7.5xl0⁹, about 8xl0⁹, about 8.5xl0⁹, about 9xl0⁹, about 9.5xl0⁹, about lxlO¹⁰, about 1.5xl0¹⁰, about 2xl0¹⁰, about 2.5xl0¹⁰, about 3xl0¹⁰, about 3.5xl0¹⁰, about 4xl0¹⁰, about 4.5xl0¹⁰, about 5xl0¹⁰, about 5.5xl0¹⁰, about 6xl0¹⁰, about 6.5xl0¹⁰, about 7xl0¹⁰, about 7.5xl0¹⁰, about 8xl0¹⁰, about 8.5xl0¹⁰, about 9xl0¹⁰, about 9.5xl0¹⁰, about lxlO¹¹, about 1.5xl0ⁿ, about 2xlOⁿ, about 2.5xlOⁿ, about 3xl0ⁿ, about 3.5xl0ⁿ, about 4xlOⁿ, about 4.5xlOⁿ, about 5xl0ⁿ, about 5.5xl0ⁿ, about 6xl0ⁿ, about 6.5xl0ⁿ, about 7xlOⁿ, about 7.5xlOⁿ, about 8xl0ⁿ, about 8.5xl0ⁿ, about 9xlOⁿ, about 9.5xl0ⁿ, about lxlO¹² viral particles (vp).

In some embodiments, a therapeutically effective amount or a prophylactically effective amount of a viral particle of the invention (or pharmaceutical composition of the invention) is in genome copies (“GC”), also referred to as“viral genomes” ("vg") ranging from about 1x10^s , about 1.5x10^s, about 2x10^s, about 2.5x10^s, about 3x10^s, about 3.5x10^s, about 4x10^s, about 4.5x10^s, about 5x10^s, about 5.5x10^s, about 6x10^s, about 6.5x10^s, about 7x10^s, about 7.5x10^s, about 8x10^s, about 8.5x10^s, about 9x10^s, about 9.5x10^s, about lxlO⁶, about 1.5xl0⁶, about 2xl0⁶, about 2.5xl0⁶, about 3xl0⁶, about 3.5xl0⁶, about 4xl0⁶, about 4.5xl0⁶, about 5xl0⁶, about 5.5xl0⁶, about 6xl0⁶, about 6.5xl0⁶, about 7xl0⁶, about 7.5xl0⁶, about 8xl0⁶, about 8.5x10, about 9xl0⁶, about 9.5xl0⁶, about lxlO⁷, about 1.5xl0⁷, about 2xl0⁷, about 2.5xl0⁷, about 3xl0⁷, about 3.5xl0⁷, about 4xl0⁷, about 4.5xl0⁷, about 5xl0⁷, about 5.5xl0⁷, about 6xl0⁷, about 6.5xl0⁷, about 7xl0⁷, about 7.5xl0⁷, about 8xl0⁷, about 8.5xl0⁷, about 9xl0⁷, about 9.5xl0⁷, about 1x10^s, about 1.5x10^s, about 2x10^s, about 2.5x10^s, about 3x10^s, about 3.5x10^s, about 4x10^s, about 4.5x10^s, about 5x10^s, about 5.5x10^s, about 6x10^s, about 6.5x10^s, about 7x10^s, about 7.5x10^s, about 8x10^s, about 8.5x10^s, about 9x10^s, about 9.5x10^s, about lxlO⁹, about 1.5xl0⁹, about 2xl0⁹, about 2.5x109^s, about 3xl0⁹, about 3.5xl0⁹, about 4xl0⁹, about 4.5xl0⁹, about 5xl0⁹, about 5.5xl0⁹, about 6xl0⁹, about 6.5xl0⁹, about 7xl0⁹, about 7.5xl0⁹, about 8xl0⁹, about 8.5xl0⁹, about 9xl0⁹, about 9.5xl0⁹, about lxlO¹⁰, about 1.5xl0¹⁰, about 2xl0¹⁰, about 2.5xl0¹⁰, about 3xl0¹⁰, about 3.5xl0¹⁰, about 4xl0¹⁰, about 4.5xl0¹⁰, about 5xl0¹⁰, about 5.5xl0¹⁰, about 6xl0¹⁰, about 6.5xl0¹⁰, about 7xl0¹⁰, about 7.5xl0¹⁰, about 8xl0¹⁰, about 8.5xl0¹⁰, about 9xl0¹⁰, about 9.5xl0¹⁰, about lxlO¹¹, about 1.5xl0ⁿ, about 2xlOⁿ, about 2.5xlOⁿ, about 3xl0ⁿ, about 3.5xl0ⁿ, about 4xlOⁿ, about 4.5xlOⁿ, about 5xl0ⁿ, about 5.5xl0ⁿ, about 6xl0ⁿ, about 6.5xl0ⁿ, about 7xlOⁿ, about 7.5xlOⁿ, about 8xl0ⁿ, about 8.5xl0ⁿ, about 9xlOⁿ, about 9.5xl0ⁿ, about lxlO¹² vg.

Any method known in the art can be used to determine the genome copy (GC) number of the viral compositions of the invention. One method for performing AAV GC number titration is as follows: purified AAV viral particle samples are first treated with DNase to eliminate un-encapsidated AAV genome DNA or contaminating plasmid DNA from the production process. The DNase resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genomes are then quantitated by real-time PCR using primer/probe sets targeting specific region of the viral genome. In various embodiments, the methods of the present invention further comprise monitoring the effectiveness of treatment. For example, visual acuity, the rate of death of cone and / or rod cells, night vision, peripheral vision, attenuation of the retinal vessels, and other ophthalmoscopic changes associated with retinal disorders such as retinitis pigmentosa may be monitored to assess the effectiveness of treatment. Additionally, the rate of death of cells associated with the particular disorder that is the subject of treatment and/or prevention may be monitored. Alternatively, the viability of such cells may be monitored, for example, as measured by phospholipid production. The assays described in the Examples section below may also be used to monitor the effectiveness of treatment (e.g., electroretinography - ERG).

In certain embodiments of the invention, a composition of the invention is administered in combination with an additional therapeutic agent or treatment. The compositions and an additional therapeutic agent can be administered in combination in the same composition or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.

Examples of additional therapeutic agents suitable for use in the methods of the invention include those agents known to treat retinal disorders, such as retinitis pigmentosa and age-related macular degeneration and include, for example, fat soluble vitamins (e.g., vitamin A, vitamin E, and ascorbic acid), calcium channel blockers (e.g., diltiazem) carbonic anhydrase inhibitors (e.g., acetazol amide and methazolamide), anti-angiogenics (e.g., anti VEGF antibodies), growth factors (e.g., rod-derived cone viability factor (RdCVF), BDNF, CNTF, bFGF, and PEDF), antioxidants, other gene therapy agents (e.g., optogenetic gene threrapy, e.g., channelrhodopsin, melanopsin, and

halorhodopsin), and compounds that drive photoreceptor regeneration by, e.g., reprogramming Muller cells into photoreceptor progenitors (e.g., alpha-aminoadipate). Exemplary treatments for use in combination with the treatment methods of the present invention include, for example, retinal and/or retinal pigmented epithelium transplantation, stem cell therapies, retinal pros theses, laser

photocoagulation, photodynamic therapy, low vision aid implantation, submacular surgery, and retinal translocation.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures, are hereby incorporated by reference. EXAMPLES

Example 1: Soluble CX3CL1 gene therapy improves cone survival and function in mouse models of retinitis pigmentosa.

The following Materials and Methods were used in this Example.

Mice

CD-I (#022), rd10 (FVB/N) (#207), and C57BL/6 (#027) mice were purchased from Charles River Laboratories. Cx3cr1^GFP (#005582) (Jung S, et al. (2000) Mol Cell Biol 20(11):4106-14) and rd10 (#004297) (Chang B, et al. (2002) Vision Res 42(4):517-25) mice on a C57BL/6 background were purchased from The Jackson Laboratory. Rhodopsin null (Rho ^; ) mice were a gift (Lem J, et al. (1999) Proc Natl Acad Sci U S A 96(2):736-41). Animals were subsequently bred and maintained at Harvard University on a 12-hour alternating light and dark cycle. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Harvard University.

Plasmids

The AAV-human red opsin-GFP-WPRE-bGH (AAV-GFP) vector plasmid was a gift (Busskamp V, et al. (2010) Science (80) 329(5990) :413-417) and used the promoter region originally developed by Wang et al. (Wang Y, et al. (1992) Neuron 9(3):429-40). The AAV-mCherry vector was generated by replacing the GFP coding sequence with that of mCherry flanked by Notl and Agel restriction sites. AAV-fCD200 and AAV-fCX3CLl were then cloned by digesting AAV-mCherry with Notl and Hind!!! restriction enzymes and replacing the mCherry coding sequence with the

GCCGCCACC Kozak sequence followed by full-length mouse cDNA for CD200 (NM_010818.3) or CX3CL1 (NM_009142.3), respectively. A vector backbone for human Bestl promoter AAVs was created by replacing the CMV promoter of the AAV-CMV-PI-EGFP-WPRE-bGH plasmid, a gift, with the -585/+39 base pair region of the human Bestl promoter (Esumi N, et al. (2004) J Biol Chem 279(18): 19064-19073). Vector plasmids for AAV-sCD200 and AAV-CX3CL1 were subsequently cloned by digesting the human Bestl promoter backbone with Notl and Hind!!! restriction enzymes and replacing the EGFP coding sequence with the GCCGCCACC Kozak sequence followed by the first 714 base pairs (amino acids 1-238) of CD200 or first 1008 base pairs (amino acids 1-336) of CX3CL1, respectively, followed by a stop codon.

AAV production and purification

Recombinant AAV8 viruses were generated as previously described (Grieger JC, et al. (2006) Nat Protoc 1 (3) : 1412—28 ; Xiong W, et al. (2015) J Clin Invest 125(4):1433-1445). Briefly,

HEK293T cells were transfected using polyethylenimine with a mixture of the AAV plasmid, rep2/cap8 packaging plasmid, and adenovirus helper plasmid. Seventy-two hours post-transfection, the supernatant was harvested and viral particles precipitated by overnight PEGylation followed by centrifugation. To remove cell debris, viruses were then subjected to centrifugation through an iodixanol gradient. The recovered AAV was washed three times with PBS and concentrated to a final volume of 100-200 pi. The titer of purified AAVs was semi-quantitatively determined by staining of viral capsid proteins VP1, VP2, and VP3 using SYPRO Ruby (Molecular Probes) and relating the staining intensity to a standard AAV titered using qPCR of genome sequences.

Subretinal AAV delivery

Subretinal injection of AAVs was performed on P0-P1 neonatal mice as previously described (Grieger JC, et al. (2006) Nat Protoc 1 (3) : 1412—28 ; Xiong W, et al. (2015) J Clin Invest 125(4):1433-1445). Following anesthetization of the mouse on ice, the palpebral fissure was carefully cut using a 30-gauge needle and the eye exposed with dull forceps. A glass needle controlled by a FemtoJet microinjector (Eppendorf) was then used to deliver 10⁸-10⁹ genome copies of each AAV into the subretinal space.

Histology

Enucleated eyes for retinal cross-sections were dissected in PBS. Following removal of the cornea, iris, lens, and ciliary body, the remaining eye cup was fixed in 4% paraformaldehyde for two hours at room temperature, cryoprotected in 10%, 20%, and 30% sucrose in PBS, and embedded in a 1 :1 mixture of 30% sucrose in PBS and optimal cutting temperature (OCT) compound (Tissue-Tek) on dry ice. Frozen eye cups were cut on a Leica CM3050S cryostat (Leica Microsystems) into 50 pm sections for Cx3cr1^GFP retinas or 20 pm sections otherwise and stained with 4',6-diamidino-2- phenylindole (DAPI) (Thermo Fisher Scientific) for five minutes at room temperature before mounting with Fluoromount-G (SouthernBiotech). For flat-mounted retinas, isolated retinas were fixed in 4% paraformaldehyde for 30 minutes at room temperature. Four radial incisions were made to relax the retina into four leaflets, which were flattened onto a microscope slide with the ganglion cell layer facing up using a fine-haired brush. To perform antibody staining of retinal cross-sections or whole retinas, tissues were blocked with 5% goat serum or 5% bovine serum albumin in PBS with 0.1% Triton X-100 for one hour at room temperature, after which tissues were incubated with primary antibodies in block solution at 4oC overnight followed by secondary antibodies in PBS for two hours at room temperature. Primary antibodies included rabbit anti-CX3CFl (Abeam, ab25088, 1 :500) and rhodamine-conjugated peanut agglutinin (PNA) (Vector Faboratories, RF-1072, 1 :1000). Goat anti-rabbit Alexa Fluor 594 (Jackson ImmunoResearch, 111-585-144, 1 :1000) was used as a secondary antibody.

RPE explants

Enucleated eyes were dissected in PBS to remove the cornea, iris, lens, ciliary body, retina, and connective tissue. Four relaxing radial incisions were made to the remaining RPE-choroid-sclera complex. Each complex was then placed on a 12 mm Millicell cell culture insert (Millipore) resting on 3 mL of pre- warmed culture media with the RPE side facing up. Culture media consisted of a 1 :1 ratio of DMEM and F-12 supplemented with L-glutamine, B27, N2, and penicillin-streptomycin. Explants were maintained in humidified incubators at 37°C and 5% C02 for 48 hours, after which the media was collected and assayed for CX3CL1 protein using a commercial ELISA kit (R&D Systems) according to manufacturer’s instructions. ELISA reactions were performed in duplicate using 50 pi of media as input.

Image acquisition and analysis

Images of microglia in retinal cross-sections and of flat-mounted retinas were acquired on a Keyence BZ-9000 widefield fluorescent microscope using a 1 Ox air objective. All other images were acquired on a Zeiss LSM710 scanning confocal microscope using a lOx air, 20x air, or 40x oil objective. Image analysis was performed using ImageJ. To calculate the percentage of microglia in the ONL, a mask was drawn around the ONL following the outlines of DAPI-labeled nuclei. Each microglia was determined to reside in the ONL if 50% or more of its cell body was located within the mask. To assay cone survival in flat-mounted retinas, a custom ImageJ module was created. First, a line was drawn from the optic nerve head to the edge of each of the four retinal leaflets as depicted in Fig. 9. The image was next subjected to an automatic threshold to separate GFP-positive cells from any background signal. The four aforementioned lines were then connected at their midpoints to form the boundaries of the central retina. Finally, GFP-positive particles located within these boundaries and of the appropriate size were quantified by the module to calculate the number of GFP-positive cones in the central retina.

Electroretinography (ERG)

In vivo ERG recordings were performed using the Espion E3 System (Diagonsys) as previously described (Xiong W, et al. (2015) J Clin Invest 125(4):1433-1445; Xue Y, et al. (2015) J Clin Invest 125(2):727-738). Briefly, mice were dark-adapted overnight and anesthetized with an intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Following dilation of the pupils with 1% tropicamide, gold- wire electrodes were applied to the surface of both eyes and hydrated with a drop of PBS. Reference and ground electrodes were placed subcutaneously near the scalp and tail, respectively. The animal was then light-adapted for 12 minutes under a 30 cd/m² background light. Upon completion of light adaptation, photopic vision was assessed using multiple flashes of 1, 10, and 100 cd/m² light. The average amplitude of the photopic b-wave in response to each flash intensity was subsequently measured by an observer blinded to the treatment assignment.

Optomotor assay

Visual acuity was measured using the OptoMotry System (CerebralMechanics) as previously described (Xiong W, et al. (2015) J Clin Invest 125(4):1433-1445; Xue Y, et al. (2015) J Clin Invest 125(2):727-738). Briefly, mice were placed on a platform within a virtual-reality chamber in which the spatial frequency of a displayed sine wave grating could be altered using a computer program. A bright background luminance setting was used to saturate rod responses to provide a measure of cone vision. During each test, an observer blinded to the treatment assignment assessed for reflexive head-tracking movements in response to the grating stimulus. Right and left eyes were tested independently using counterclockwise and clockwise gratings, respectively, as only motion in the temporal-to-nasal direction evokes the optomotor response in mice (Douglas RM, et al. (2005) Vis Neurosci 22(5):677-84). For each eye, the highest spatial frequency at which the animal tracked the grating was determined to be the visual acuity.

RT-PCR

For RT-PCR of whole retinas, freshly dissected retinas were homogenized using a handheld pellet pestle (Kimble Chase) in 350 m1 of RLT buffer containing 1% beta-mercaptoethanol. One retina was used per sample. For RT-PCR of microglia, approximately 1000 microglia per retina were sorted into 10 mΐ of Buffer TCL (Qiagen) to lyse cells, to which 70 mΐ of RLT buffer containing 1% beta- mercaptoethanol was added. RNA extractions were performed using an RNeasy Micro Kit (Qiagen) followed by cDNA synthesis using the Superscript III First-Strand Synthesis System (Invitrogen). RT- PCR reactions were conducted in triplicate using the Power SYBR Green PCR Master Mix (Applied Biosystems) on a CFX96 real-time PCR detection system (BioRad) to determine cycle threshold (Ct) values. Expression levels were quantified by normalizing to the housekeeping gene Gapdh with fold changes relative to age-matched WT (CD-I or B6) retinas. P-values were calculated using AACt values. Primers for RT-PCR were designed using PrimerBank (Wang X, et al. (2012) Nucleic Acids Res 40(D 1 ) :D 1144-D 1149) with sequences available in T able 1.

Table 1. RT-PCR primers.

Flow cytometry and cell sorting

Retinal microglia were isolated using fluorescence-activated cell sorting (FACS) and data analyzed on FlowJo 10 (Tree Star). To dissociate cells, freshly dissected retinas were incubated in 400 mΐ of cysteine-activated papain solution (Worthington) for 5 minutes at 37°C, followed by gentle trituration with a micropipette. Dissociated cells were subsequently blocked with rat anti-mouse CD16/32 (BD Pharmingen, 1 :100) for 5 minutes on ice followed by staining with PE-Cy5 -conjugated anti-CDl lb (BioLegend, Ml/70, 1 :200), APC-Cy7-conjugated anti-Ly6G (BioLegend, 1A8, 1 :200), and APC-Cy7-conjugated anti-Ly6C (BioLegend, F1K1.4, 1 :200) for 20 minutes on ice. After washes, cells were resuspended in FACS buffer (2% fetal bovine serum, 2mM EDTA in PBS) containing 0.5 pg/mL DAPI to label non-viable cells and passed through a 40 mm filter. Sorting was performed on a BD FACS Aria II using a 70 pm nozzle according to the gating shown in Fig. 11.

RNA sequencing of microglia

For each retina, 1000 microglia (CDl lb+ Ly6G/Ly6C-) were sorted into 10 pi of Buffer TCL (Qiagen) containing 1 % beta-mercaptoethanol and immediately frozen on dry ice. For a subset of retinas, 1000 non-microglia cells (CDl lb-) were also sorted. Upon collection of all samples, frozen microglia and non-microglia lysates were thawed on wet ice and loaded into a 96-well plate for cDNA library synthesis and sequencing. A modified Smart-Seq2 protocol was performed on samples by the Broad Institute Genomics Platform (Picelli S, et al. (2013) Nat Methods 10(11): 1096— 8). Libraries from 96 samples with unique barcodes were combined and sequenced on a NextSeq 500 Sequencing System (Illumina) to an expected coverage of about 6 million reads per sample. Following

demultiplexing, reads were subjected to quality control measures and mapped to the GRCm38.p6 reference genome. Reads assigned to each gene were quantified using featureCounts (Liao Y, et al. (2014) Bioinformatics 30(7):923-30), and samples with fewer than 30% assigned reads were excluded from further analysis. Count data were analyzed using DESeq2 to identify differentially expressed genes with an adjusted P- value less than 0.05 considered significant (Anders S, Huber W (2010) Genome Biol 11(10):R106). Gene set enrichment analysis (GSEA) was performed using GSEA v3.0 software from the Broad Institute under default settings on the GO Cellular Component Ontology collection (580 gene sets) from the Molecular Signatures Database (MSigDB). Gene sets with a family- wise error rate (FWER) less than 0.05 were considered significantly enriched.

Microglia Depletion

Microglia were depleted using PLX3397 (SelleckChem), also known as pexidartinib, an orally available CSF1R inhibitor. PLX3397 was incorporated into AIN-76A rodent chow (Research Diets) at 290 mg/kg and provided ad libitum for 30 days from P20 to P49 followed by harvesting of the animal on P50. Statistics

Unpaired two-tailed Student’s t-tests were used for comparisons between two groups. For comparisons of three or more groups, a Bonferroni correction was added to the test by multiplying the resulting P-value by the number of hypotheses tested. Two-tailed two-way ANOVA was used to analyze ERG, optomotor, and ONL thickness experiments in which the same subjects are compared over a series of conditions or time points. In all cases, a P-value less than 0.05 was considered

statistically significant.

Microglia reside in the photoreceptor layer throughout cone degeneration.

The rd10 and rd10 mouse lines are commonly used models of RP ( Chang B, et al. (2002)

Vision Res 42(4):517-25). Each strain harbors a different mutation in the rod-specific

phosphodiesterase beta subunit, with rd10 exhibiting more rapid photoreceptor degeneration than rd10 (Pennesi ME, et al. (2012) Investig Opthalmology Vis Sci 53(8):4644). To characterize immune activity during non-autonomous cone degeneration, RT-PCR was first performed on retinas from albino rd10 and pigmented rd10 mice versus those from albino CD-I and pigmented C57BL/6 (B6) mice, two strains with wild-type (WT) vision. Primers were designed to assay for RNAs representing both innate and adaptive immunity components, including inflammatory cytokines (Ilia, Illb, 116, Tnf), the complement system ( Clqa ), neutrophils ( Ly6g ), T cells (Cd4, Cd8a), and microglia ( Tmemll9 , Cd68). In each strain, two distinct time points were examined corresponding to the onset (postnatal day 20

[P20] for rd10, P40 for rd10 ) and peak (P35 for rd10, P70 for r )d1 o0f cone degeneration (Punzo C, et al. (2009) Nat Neurosci 12(l):44-52; Xiong W, et al. (2015) J Clin Invest 125(4):1433-1445).

Compared to age-matched CD-I and B6 retinas, rd10 and rd1 r0etinas demonstrated significant upregulation of Ilia, Tnf, and Clqa at both time points, as well as Illb specifically in rd10 mice (Fig. 1A-1D). Upregulation of these factors was also associated with higher expression levels of Tmemll9, a microglia-specific marker (Bennett ML, et al. (2016) Proc Natl Acad Sci U S A 113(12):E1738- E1746), and Cd68, a marker of lysosomal activity and microglia activation (Bodea L-G, et al. (2014) J Neurosci 34(25):8546-56), but not Ly6g, Cd4, or Cd8a. As activated microglia have been previously shown to produce and secrete ILIA, TNF, and C1Q (Liddelow SA, et al. (2017) Nature

541(7638):481-487), these data pointed to a proinflammatory role of microglia during non-autonomous cone death.

Rods in mice, and in humans, are far more abundant than cones, representing -95% of photoreceptors (Carter-Dawson LD, et al. (1979) J Comp Neurol 188(2):245-262; Curcio CA, et al. (1990) J Comp Neurol 292(4):497-523). In the retina, rod and cone cell bodies form a structure called the outer nuclear layer (ONL), which dramatically shrinks with rod death in RP until only a single row of cells remains, comprised of the surviving cones. Pathologic infiltration of microglia into the ONL has been described during the initial rod death phase of RP (Peng B, et al. (2014) J Neurosci

34(24):8139-8150; Zhao L, et al. (2015) EMBO Mol Med 7(9): 1179-97). However, less is known about how microglia behave during the subsequent period of cone dysfunction and death. To aid in visualizing microglia within the retina, RP and WT animals were bred with Cx3cr1^GFP reporter mice, in which microglia are labeled with GFP (Jung S, et al. (2000) Mol Cell Biol 20(11):4106-14). Only about 10% of retinal microglia were normally located in the ONL in cross-sections from

CDl ;Cx3cr1^GFP/+ and B6;Cx3cr1^GFP/+ WT eyes (Fig. 1 E- 1 H). Conversely, about 40-50% of retinal microglia could be seen in the ONL in rd10 ;Cx3cr1^GFP/+ and ; Crdx130cr1^GFP/+ mice throughout the period of cone degeneration. Thus, even after the disappearance of rods, the period of cone degeneration had both cytokine upregulation and continued localization of microglia to the photoreceptor layer.

Overexpression of soluble CX3CL1 prolongs cone survival in RP mice

It was hypothesized that during the later stages of RP, activated microglia may create a proinflammatory environment deleterious to nearby cones. It was further postulated that overexpression of factors opposing microglia activation might alleviate this damage, favoring cone survival. To test this idea, a previously characterized AAV expressing GFP under the human red opsin promotor (AAV- GFP) was chosen to label cones and aid in their quantification (Fig. 2A) (Li Q, T et al. (2008) Vision Res 48(3):332-338). Subretinal administration of AAV-GFP in neonatal mice brightly labeled cones throughout the entire retina, allowing for visualization of these cells in adult animals (Fig. 2 B and Fig. 8). AAVs were then designed expressing either CD200 or CX3CL1, membrane-bound proteins reported to suppress proinflammatory activity via their respective receptors on microglia, CD200R and CX3CR1 (Hoek RM, et al. (2000) Science (80- ) 290(5497):1768-71 ; Biber K, et al. (2007) Trends Neurosci 30(l l):596-602; Cardona AE, et al. (2006) Nat Neurosci 9(7):917-924.). Given the hypothesized interaction between microglia and degenerating cones, full-length variants of CD200 (fCD200) and CX3CL1 (fCX3CLl) were expressed under the cone-specific human red opsin promoter (Fig. 2D). Because soluble variants of both proteins have also been described (Wong KK, et al. (2016) PLoS One l l(4):e0152073; Bazan JF, et al. (1997) Nature 385(6617):640-644), additional AAVs were generated for soluble CD200 (sCD200) and CX3CL1 (CX3CL1 ) using the human Bestl promoter to drive expression in the retinal pigment epithelium (RPE), a cell layer adjacent to the photoreceptors (Fig. 9) (Young RW (1967) J Cell Biol 33(l):61-72).

The ability of the four AAVs (AAV-fCD200, AAV-sCD200, AAV-fCX3CLl, AAV- CX3CL1 ) to prolong cone survival was initially tested in rd10 mice, which were injected at P0-P1 and evaluated at P50. In mouse RP, cone death proceeds from center to periphery starting from the optic nerve head. To assay cone survival during degeneration, the central retina was therefore interrogated. Using an ImageJ module, the number of GFP-positive cones in the central retina could be reliably quantified (Fig. 10). Compared to rd10 retinas infected with AAV-GFP alone (Fig. 2 C), there was no significant improvement in cone survival with the addition of AAV-fCD200, AAV-sCD200, or AAV- fCX3CLl (Fig. 2 E and F). In contrast, co-infection of AAV-GFP with AAV-CX3CL1 significantly increased the number of cones remaining in the central retina (PcO.0001 ), supporting a potential therapeutic effect of CX3CL1 in RP. Cone survival with AAV-CX3CL1 was also examined in older, more degenerated rd10 mice.

At P75, co-infection of AAV-GFP with AAV-CX3CL1 continued to prolong cone survival compared to AAV-GFP alone (PcO.001 ) (Fig. 3 A-A”). Even at P100, by which time the central retinas of AAV- GFP infected eyes were nearly devoid of cones, significantly greater cone survival with AAV- CX3CL1 was observed (PcO.01 ) (Fig. 3 B-B”). To determine if CX3CL1 might provide a generic therapy for non-autonomous cone death, AAV-CX3CL1 infection was trialed in r (dF1ig0. 3 C-C”) and Rho ¹ (Fig. 3 D-D”) mice. Rho ¹ mice lack rhodopsin, the photopigment gene in rods, which is also the most commonly mutated gene in humans with autosomal dominant RP (Sung CH, et al. (1991)

Proc Natl Acad Sci U S A 88(15):6481—5). Photoreceptors in the Rho ¹ strain degenerate at a slower rate than in the rd10 or rd10 strain (Gargini C, et al. (2007) J Comp Neurol 500(2):222-238; Lem J, et al. (1999) Proc Natl Acad Sci U S A 96(2):736-41). In rd a1n0d Rho ⁷ mice, AAV-GFP plus AAV- CX3CL1 again resulted in a higher number of cones in the central retina compared to AAV-GFP only (P<0.01 for rd10, P<0.001 for Rho ^/ ). Taken together, these findings demonstrate that AAV-CX3CL1 gene therapy promotes cone survival in RP patients harboring different genetic lesions.

AAV-sCX3CLl improves cone-mediated visual function.

As preservation of cones by AAV-CX3CL1 was observed using histological assays, it was possible that vision was also rescued. Electroretinography (ERG), a physiological measure of retinal activity in response to light, can be used to reveal rod or cone activity. ERG was first used to measure photopic b-wave responses, a cone-mediated signal from the inner retina known to decline relatively early in RP in both animals and humans (Hartong DT, et al. (2006) Lancet 368(9549): 1795-1809; Xiong W, et al. (2015) J Clin Invest 125(4):1433-1445). ERG recordings from P40 r md1ic0e showed no difference in photopic b-waves between AAV-GFP infected and untreated eyes, as expected (Fig. 4A). In contrast, in animals treated with AAV-GFP plus AAV-CX3CL1 in one eye and AAV- GFP in the other, a modest but significant increase in photopic b-wave amplitudes could be seen (P< 0.05) (Fig. 4A and B).

To evaluate vision using a behavioral test, the optomotor assay was used. This assay elicits a motor response to simulated motion, that of moving stripes. By varying the stripe width until the animal is no longer able track the stimulus, a spatial frequency threshold can be calculated, corresponding to the visual acuity in each eye (Prusky GT, et al. (2004) Investig Opthalmology Vis Sci 45(12):4611 ; Douglas RM, et al. (2005) Vis Neurosci 22(5):677-84). Mice were placed under bright light conditions to probe cone vision. In rd10 mice infected with AAV-GFP in one eye and untreated in the other, optomotor results from P45 to P60 showed a similar drop in visual acuity between the two eyes over time (Fig. AC). However, when animals were infected with AAV-GFP plus AAV-CX3CL1 instead of AAV-GFP alone, loss of visual acuity over the same interval was slowed in the AAV-CX3CL1 treated eye (PcO.01 ), showing that AAV-CX3CL1 gene therapy is able to preserve cone-mediated vision. AAV-sCX3CLl does not improve rod survival, microglia localization, or retinal inflammation.

Absence of CX3CL1 signaling during rod degeneration in RP mice has been shown to decrease rod survival, reduce the number of microglia in the ONL, and upregulate levels of TNF and IL1B in the retina (Peng B, et al. (2014) J Neurosci 34(24):8139-8150; Zabel MK, et al. (2016) Glia 64(9):1479- 91). Thus, the effect of AAV-CX3CL1 on these phenotypes was investigated to uncover possible mechanisms by which CX3CL1 gene therapy alleviates cone degeneration. Rods normally comprise -95% of cells in the ONL and are thought to support cone survival through several pathways, such as secretion of trophic factors and maintenance of a normoxic environment Ait-Ali N, et al. (2015) Cell 161(4):817— 832; Carter-Dawson LD, et al. (1979) J Comp Neurol 188(2):245-262; Yu DY, et al. (2000) Invest Ophthalmol Vis Sci 41(12):3999-4006). To examine how CX3CL1 treatment affected rods, the thickness of the ONL in RP retinas was measured. In P20 rd10 and P40 rd re1t0inas infected with AAV-GFP, only one to two rows of nuclei remained in the ONL (Fig. 5A), consistent with near completion of rod death by the onset of cone degeneration (Punzo C, et al. (2009) Nat Neurosci 12(l):44-52; Xiong W, et al. (2015) J Clin Invest 125(4):1433-1445 (12, 13). Relative to these retinas, co-infection with AAV-CX3CL1 did not significantly alter ONL thickness (Fig. 5A and B). This observation not only demonstrates a lack of rod preservation by AAV-CX3CL1 , but also shows that cone survival was not secondary to rescue of rods.

It was next determined how microglia responded to CX3CL1 therapy by comparing retinas from rd10 ;Cx3cr1^GFP/+ (Fig. 5 C) and rd100\ Cx3cr1^GFP/+ (Fig. 5 D) mice with and without AAV- CX3CL1 . As use of AAV-GFP in these animals complicated visualization of GFP-expressing microglia, an analogous AAV-mCherry virus was generated for control infections. During both the onset and peak of cone degeneration in eyes receiving only AAV-mCherry, -40% of retinal microglia could be found in the ONL, indicating continued localization of these cells to the vicinity of cones (Fig.

5 E and F). This percentage remained unchanged with the addition of AAV-CX3CL1 , arguing against a role of CX3CL1 in reducing microglial residence in the ONL.

Finally, given upregulation of inflammatory cytokines and complement during the period of non-autonomous cone death, the effect of AAV-CX3CL1 on these factors was evaluated by RT-PCR. For the majority of genes tested, including Ilia, Illb, Clqa, and Tmemll9, expression levels in the retina were similar regardless of treatment with AAV-GFP or AAV-GFP plus AAV-CX3CL1 (Fig. 5G-J). The notable exception was Cd68, a microglia activation marker which was upregulated with AAV-CX3CL1 throughout cone degeneration (Bodea L-G, et al. (2014) J Neurosci 34(25):8546-56). Collectively, these data challenged the notion that AAV-CX3CL1 might attenuate complement and inflammatory cytokine activity in the retina. Moreover, they showed that even with massive rod death, microglia in the ONL, and ongoing inflammation in the eye, AAV-CX3CL1 was still able to prolong cone survival.

AAV-sCX3CLl induces markers of microglia activation.

To probe for gene expression changes in microglia that might be brought about by AAV- CX3CL1 , RNA sequencing (RNA-seq) of retinal microglia from AAV-CX3CL1 infected eyes was performed. Flow cytometry of RP retinas carrying the Cx3cr1^GFP transgene indicated that microglia corresponded to a CD1 lb+ Ly6G/Ly6C- population in the retina (Fig. 11), consistent with earlier studies (Liyanage SE, et al. (2016) Exp Eye Res 151 :160-70; Murinello S, et al. (2016) J Vis Exp (116). doi:10.3791/54677). Using these cell surface markers, retinal microglia from mrdic1e0 infected with AAV-GFP or AAV-GFP plus AAV-CX3CL1 were sorted at P70 during the peak of cone degeneration. Sorted microglia were a highly purified population, expressing microglia-specific genes, such as Fcrls, P2ryl2, and Tmemll9, but not markers for other retinal cell types compared to non microglia (CDl lb- Ly6G/Ly6C- and CDl lb- Ly6G/Ly6C+) cells (Fig. 12) (Butovsky O, et al. (2014) Nat Neurosci 17(1):131— 143 ; Fiickman SE, et al. (2013 ) Nat Neurosci 16(12): 1896—1905; Akimoto M, et al. (2006) Proc Natl Acad Sci U S A 103(10):3890-5; Shekhar K, et al. (2016) Cell

166(5):1308-1323.e30; Roesch K, et al. (2008) J Comp Neurol 509(2):225-38; Chintalapudi SR, et al. (2017) J Vis Exp (125):e55785; Cherry TJ, et al. (2009) Proc Natl Acad Sci U S A 106(23):9495- 500; Dyer MA, et al. (2003) Nat Genet 34(1):53— 58).

RNA-seq analysis of sorted microglia from P70 rd1 r0etinas infected with AAV-CX3CL1 demonstrated significant (adjusted P<0.05, fold change >2) upregulation and downregulation of 50 and 40 genes, respectively (Fig. 6A and Tables 2 and 3) Four of these expression changes were validated by RT-PCR on independent samples (Fig. 6 B). Among the genes upregulated with AAV-CX3CL1 were known markers of microglia activation during neurodegeneration, including Cst7. Sppl, Igfl, Csfl, Lyz2, Cd63-ps, and Gpnmb (Keren-Shaul H, et al. (2017) Cell 169(7):1276-1290.el7; Song WM, et al. (2018) J Exp Med 215(3):745-760; Chiu IM, et al. (2013) Cell Rep 4(2):385-401 ; Iaccarino FiF, et al. (2016) Nature 540(7632):230-235) In support of this, gene set enrichment analysis (GSEA) of microglia with AAV-CX3CL1 revealed significant enrichment of lysosome components (Fig. 6 C), a prominent feature of activated microglia (Bodea L-G, et al. (2014) J Neurosci 34(25):8546-56; Zhao L, et al. (2015) EMBO Mol Med 7(9):1179-97; Neumann H, et al. (2009) Brain 132(Pt 2):288-95). Interestingly, low levels of multiple cone-specific genes such as Aipll, Chrnb4, and GnbS were observed in microglia from AAV-GFP treated retinas (Iribarne M, et al. (2017) Sci Rep 7(1):45962; Decembrini S, et al. (2017) Mol Ther 25(3):634-653; Tummala H, et al. (2006) Investig

Opthalmology Vis Sci 47(l l):4714(62-64), potentially due to phagocytosis of dying cones or cone fragments. AAV-CX3CL1 significantly downregulated expression of these transcripts, hinting that it might affect the digestion of phagocytosed materials.

Table 2. Significantly upregulated genes in P70 rd10 microglia following sCX3CLl

overexpression.

Table 3. Significantly downregulated genes in P70 rd10 microglia following sCX3CLl overexpression.

Normal numbers of microglia are not necessary for cone rescue with AAV-sCX3CLl.

In healthy eyes, CX3CR1, the only known receptor for CX3CL1, is thought to be specifically expressed by microglia (Combadiere C, et al. (2007) J Clin Invest 117(10):2920-8). This fact and the above RNA-seq data prompted the questions of what effect ablation of microglia might have on cone survival and if microglia were necessary for the rescue of cones by AAV-CX3CL1 . It is possible to pharmacologically deplete microglia using PLX3397, a potent colony stimulating factor 1 receptor (CSF1R) inhibitor (Liddelow SA, et al. (2017) Nature 541(7638):481-487; HI more MRP, et al. (2014) Neuron 82(2):380-97). To this end, rd10 mice were fed PLX3397 for 10 or 30 days, and depletion of retinal microglia was assessed using flow cytometry. PLX3397 treatment led to -95% depletion of microglia after 10 days and -99% after 30 days (Fig. 7 A and R). To determine if reduction in microglia preserved cones, and to test whether the activity of AAV-CX3CL1 in preserving cone survival required microglia, rd10 mice were infected with AAV-GFP with or without AAV-CX3CL1 and administered PLX3397 for 30 days during the period of cone degeneration. Depletion of microglia non-significantly (P>0.05) increased cone survival in both conditions (Fig. 1C and D). Moreover, depletion of microglia did not abrogate the ability of AAV-CX3CL1 to rescue cones (PcO.0001 ).

DISCUSSION

As described herein, a gene therapy vector, AAV-CX3CL1 , that prolonged cone survival in three different RP mouse models and delayed the loss of cone-mediated vision was developed.

Preservation of cones with AAV-CX3CL1 occurred despite elevated cytokine levels in the retina, and despite the continued presence of microglia in the ONL. Depletion of up to -99% of microglia during cone degeneration non-significantly improved cone survival and did not disrupt the rescue effect of AAV-CX3CL1 . These findings show that CX3CL1 gene therapy is beneficial for a wide range of RP patients, and for other patients with inflammatory processes that affect vision.

CX3CL1 is a 373-amino acid protein with a single transmembrane domain that can undergo proteolytic cleavage to release CX3CL1 into the extracellular environment (Bazan JF, et al. (1997) Nature 385(6617):640-644). In the CNS, both fCX3CLl and CX3CL1 are primarily produced by neurons and, by binding CX3CR1 on microglia, are thought to regulate key aspects of microglial physiology (Paolicelli RC ,et al. (2014) Front Cell Neurosci 8:129; Lauro C, et al. (2015) Ann N Y Acad Sci 1351 (1): 141— 148). One of the main responsibilities of CX3CL1 in neuron-microglia interactions is to suppress the activation of microglia (Zujovic Y,et al. (2000) Glia 29(4):305-15; Mizuno T, et al. (2003) Brain Res 979(l-2):65-70). Supporting this notion, exogenous delivery of CX3CL1 has been shown to decrease microglia activation as well as neurological deficits in animal models of Parkinson’s disease and stroke (Nash KR, et al. (2015) Mol Ther 23(l):17-23; Pabon MM, et al. (2011 J Neuroinflammation 8(1):9; Cipriani R, et al. (2011) J Neurosci 31(45):16327-35).

Here, CX3CL1 was overexpressed in RP mice with the hope that it would attenuate immune responses in the retina that were perpetuating non-autonomous cone death. Use of CX3CL1 indeed prolonged cone survival during degeneration, though it did so without reducing inflammation or the number of microglia in the ONL. Interestingly, cone rescue was seen when CX3CL1 was produced from the RPE using the human Bestl promoter, but not when full-length membrane-bound CX3CL1 was expressed on cones by the human red opsin promoter. This result could be due to differences in the level of expression, as the human Bestl promoter is quite strong relative to the human red opsin promoter. Alternatively, it could be that CX3CL1 acts on other cell types besides microglia and is better able to reach these cells when secreted. In contrast, overexpression of CD200, another repressor of microglia activation (Hoek RM, et al. (2000) Science (80- ) 290(5497): 1768-71), failed to rescue cones whether expressed as a sCD200 from the RPE or fCD200 on cones. Activated microglia are a hallmark of early RP, given their migration into the ONL, production of inflammatory cytokines, and phagocytosis of living photoreceptors (Peng B, et al. (2014) J Neurosci 34(24):8139-8150; Zabel MK, et al. (2016) Glia 64(9): 1479-91 ; Zhao L, et al. (2015) EMBO Mol Med 7(9): 1179-97 (33, 34, 45). In early RP, rods are degenerating, and these microglia activities are deleterious, as genetic ablation of microglia has been demonstrated to ameliorate rod death (Zhao L, et al. (2015) EMBO Mol Med 7(9): 1179-97). Acute retinal detachment, another condition causing photoreceptor loss, is similarly characterized by inflammatory cytokines and phagocytic microglia (Nakazawa T, et al. (2006) Mol Vis 12:867-78; Okunuki Y, et al. (2018) Proc Natl Acad Sci U S A 115(27):E6264-E627). Unlike with early RP, however, removal of microglia during retinal detachment was observed to accelerate photoreceptor demise, implying a protective role for activated microglia (Okunuki Y, et al. (2018) Proc Natl Acad Sci U S A 115(27):E6264-E6273). Here, evidence of microglia activation during cone death in RP, as illustrated by the presence of microglia in the ONL, and upregulation of Ilia, Tnf, Clqa, and Cd68, was found. It washypothesized that, as in early RP, these microglia might be detrimental, and consequently, the goal was to develop AAVs capable of suppressing retinal microglia activation. Interestingly, drug-induced depletion of microglia in rd10 retinas provided evidence for only a slight negative effect of activated microglia on cones; only a small increase in the number of cones was seen with microglia depletion, and this change did not reach statistical significance. One explanation for this could be that while activated microglia in RP do hinder cone survival, they may also provide some beneficial functions. One such benefit may be increased clearance of harmful cell debris. By RNA-seq, small amounts of cone-specific RNAs in microglia from AAV-GFP infected rd10 retinas, potentially from phagocytosis of cones or cone fragments, were detected. Thus, during cone degeneration in RP, cone debris might accumulate in microglia if digestion of these materials cannot keep up with engulfment. Inability of microglia to complete phagocytosis may then trigger the release of factors injurious to cones, akin to the model of“frustrated phagocytosis” experienced by microglia in Alzheimer’s disease (Sokolowski JD, Mandell JW (2011) Am J Pathol 178(4):1416-28). Conversely, upregulation of lysosomal pathways in microglia by AAV-CX3CL1 may enable these cells to more efficiently digest cone material, alleviating this frustration and favoring cone preservation.

Notably, depletion of up to -99% of microglia also failed to abrogate AAV-CX3CL1 cone rescue. Although this could indicate that CX3CL1 prolongs cone survival independently of microglia, the possibility that only a few microglia are needed to mediate the rescue effect cannot be ruled out. In a recent study by Liddelow et al., depletion of microglia by the same drug, PLX3397, was unable to eliminate a phenotype in astrocytes induced by microglia (Liddelow SA, et al. (2017) Nature

541(7638):481-487). Specifically, the authors found that inflammatory cytokines from activated microglia caused astrocytes to acquire a neurotoxic state that could go on to damage neurons. Astrocyte neurotoxicity could be blocked by using CSF1R null mice, which are devoid of microglia (Ginhoux F, et al. (2010) Science (80) 330(6005):841-845). However, in other strains, neurotoxicity was still observed despite pharmacologically depleting 95% of microglia (Liddelow SA, et al. (2017) Nature 541(7638):481-487). Thus, it could be that the about 1-5% of retinal microglia that survive PLX3397 treatment are sufficient to respond to CX3CL1 and preserve degenerating cones. For these remaining microglia, greater CX3CL1 signaling per cell may additionally account for the small additivity of AAV-CX3CL1 and microglia depletion on cone rescue.

An alternative model, given how modest the effect of microglia depletion was on cone survival, is that non-autonomous cone death is caused by mechanisms largely independent of microglia (Narayan DS, et al. (2016) Acta Ophthalmol 94(8):748-754; Xiong W, et al. (2015) J Clin Invest 125(4):1433- 1445; Venkatesh A, et al. (2015) J Clin Invest 125 (4): 1446-58; Ait-Ali N, et al. (2015) Cell

161(4):817— 832) For AAV-CX3CL1 , the reason for cone rescue might then be due to CX3CL1 acting on a CX3CR1 -expressing cell type other than microglia. This cell type would have to be external to the retina, since none of the non-microglia cells in our rd10 ;Cx3crl^GFP/+ retinas expressed CX3CR1 when analyzed by flow cytometry. Outside of the CNS, CX3CR1 is also present on several immune cell populations in the blood, including monocytes and certain subsets of T cells, natural killer cells, and dendritic cells (Jung S, et al. (2000) Mol Cell Biol 20(11):4106-14; Imai T, et al. (1997) Cell 91(4):521-30). In these populations, one of the roles of CX3CR1 is to mediate a chemotactic response to CX3CLl(Imai T, et al. (1997) Cell 91(4):521-30; Haskell CA, et al. (2000) J Biol Chem

275(44):34183-34189). It is therefore plausible that CX3CL1 secreted by the RPE might act on one of these cell types in the choroid, perhaps to induce migration into the subretinal space.

Recently, voretigene neparvovec-rzyl (Luxturna) became the first AAV gene therapy to be approved for an inherited retinal disorder (Russell S, et al. (2017) Lancet 390(10097):849-860).

Despite this achievement, there are still thousands of retinal disease mutations for which no effective treatment exists (Ran X, et al. (2014) Database (Oxford) 2014. doi:10.1093/database/bau047).

Addressing these lesions one by one would be prohibitively expensive and time-consuming, and specifically for RP, rods carrying the mutation have often died by the time of diagnosis (Hartong DT, et al. (2006) Lancet 368(9549): 1795-1809), making gene correction therapy infeasible. Mutation- independent gene therapies represent an alternative approach that, while not curative, may improve vision for a much larger number of patients. Previously, only two examples of mutation-independent gene therapies have been shown to rescue cones in animal models of RP (Fortuny C, Flannery JG (2018) Adv Exp Med Biol 1074:75-81). In 2015, Byrne et al. reported that two strains of RP mice had better cone survival and vision with viral-mediated expression of rod-derived cone viability factor (RdCVF), a protein normally secreted from rods that stimulates cones to uptake glucose (Ait-Ali N, et al. (2015) Cell 161(4):817— 832; Byrne LC, et al. (2015) J Clin Invest 125(1): 105— 116; Leveillard T, et al. (2004) Nat Genet 36(7):755-759). That same year, Xiong et al. found that cone numbers and function in three RP mouse lines could be improved with AAV-mediated delivery of antioxidants, particularly a master antioxidant transcription factor called NRF2 (Xiong W, et al. (2015) J Clin Invest 125(4): 1433-1445). Here, it was demonstrated that AAV-CX3CL1 is also a mutation- independent gene therapy capable of saving cones in different types of RP mice. List of Sequences

SEQ ID NO:l (AAV-SCX3CL1 : Nucleotides 1-130 = 5' -inverted terminal repeat; nucleotides 211-788 = human Bestl promoter; nucleotides 983-1993 = mouse soluble CX3CL1; nucleotides 2856-2985 = 3' -inverted terminal repeat)

SEQ ID NO:2 (AAV-sCD200: Nucleotides 1-130 = 5'-inverted terminal repeat; nucleotides 211-788 = human Bestl promoter; nucleotides 983-1699 = mouse soluble CD200; nucleotides 2562-2691 = 3' -inverted terminal repeat)

SEQ ID NO: 3 (AAV-fCX3CLl : Nucleotides 1-130 = 5' -inverted terminal repeat; nucleotides 210-2265 = human red opsin promoter; nucleotides 2283-3470 = mouse full-length CX3CL1; nucleotides 4327-4456 = 3' -inverted terminal repeat )

SEQ ID NO: 4 (AAV-fCD200: Nucleotides 1-130 = 5' -inverted terminal repeat; nucleotides 210-2265 = human red opsin promoter; nucleotides 2283-3119 = mouse full-length CD200; nucleotides 3976-4105 = 3' -inverted terminal repeat )

SEQ ID NO: 5 (AAV-GFP : Nucleotides 1-130 = 5' -inverted terminal repeat;

nucleotides 210-2265 = human red opsin promoter; nucleotides 2275-2994 = Green Flourescent Protein (GFP); nucleotides 3858-3987 = 3' -inverted terminal repeat)

SEQ ID NO: 6

>NM_002996.6 Homo sapiens C-X3-C motif chemokine ligand 1 (CX3CL1), transcript variant 1, mRNA

SEQ ID NO: 7

>NM_001304392.2 Homo sapiens C-X3-C motif chemokine ligand 1 (CX3CL1), transcript variant 2, mRNA

SEQ ID NO: 8

>NP_002987.1 fractalkine isoform 1 precursor [Homo sapiens]

SEQ ID NO: 9

>NP_001291321.1 fractalkine isoform 2 [Homo sapiens]

SEQ ID NO: 10

Homo sapiens bestrophin 1 (BEST1), RefSeqGene on chromosome 11

NCBI Reference Sequence: NG_009033.1 (Also see, Esumi et al 2004, figure 1 b at page 19066)

GenBank Graphics

>NG_009033. I Homo sapiens bestrophin 1 (BEST1), RefSeqGene on chromosome 11

SEQ ID NO: 11

WPRE

SEQ ID NO: : 12

>NG_009105.2 Homo sapiens opsin 1, long wave sensitive (OPN1LW) , RefSeqGene on chromosome X

SEQ ID NO: 13

HUMAN BETA-GLOBIN INTRON SEQ ID NO: 14

SV40 POLY-ADENYLATION (POLYA) SEQ ID NO: 15

GRIK-1 PROMOTER

SEQ ID NOs: 16-45

PCR PRIMERS

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A composition, comprising an adeno-associated virus (AAV) expression cassette, the expression cassette comprising a retinal pigmented epithelium-specific (RPE- specific) promoter and a nucleic acid molecule encoding C-X3-C Motif Chemokine Ligand 1 (CX3CL1).

2. A composition, comprising an adeno-associated virus (AAV) expression cassette, the expression cassette comprising a photoreceptor-specific (PR- specific) promoter and a nucleic acid molecule encoding C-X3-C Motif Chemokine Ligand 1 (CX3CL1).

3. A composition, comprising an adeno-associated virus (AAV) expression cassette, the expression cassette comprising a bipolar cell-specific promoter and a nucleic acid molecule encoding C- X3-C Motif Chemokine Ligand 1 (CX3CL1).

4. The composition of claim 1 , wherein the RPE-specific promoter is a human bestrophin 1 (hBestl) promoter.

5. The composition of claim 4, wherein the hBestl promoter comprises nucleotides -585 to +38 of the hBestl gene; nucleotides -595 to +30 of the hBestl gene; nucleotides -154 to +38 of the hBestl gene; or nucleotides -104 to +38 bp of the hBestl gene, or or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides -585 to +38 of the hBestlgene; nucleotides -595 to +30 of the hBestl gene; nucleotides -154 to +38 of the hBestl gene; or nucleotides -104 to +38 bp of the hBestl gene.

6. The composition of claim 4 or 5, wherein the hBestl promoter comprises nucleotides 211- 788 of SEQ ID NO:l or SEQ ID NO:2, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 211-788 of SEQ ID NO:l or SEQ ID NO:2.

7. The composition of claim 2, wherein the PR-specific promoter is a human red opsin (hRO) promoter.

8. The composition of claim 7, wherein the hRO promoter comprises nucleotides 210-2265 of SEQ ID NO:3 or SEQ ID NO:4, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 210-2265 of SEQ ID NO:3 or SEQ ID NO:4.

9. The composition of claim 3, wherein the bipolar cell-specific promoter is a glutamate ionotropic receptor kainate type subunit 1 (Grikl) promoter.

10. The composition of claim 9, wherein the Grikl promoter comprises the nucleotide sequence of SEQ ID NO:15, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of SEQ ID NO:15.

11. The composition of any one of claims 1-10, wherein the nucleic acid molecule encoding CX3CL1 comprises nucleotides 80-1102 of SEQ ID NO:6, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 80-1102 of SEQ ID NO:6.

12. The composition of any one of claims 1-11, wherein the nucleic acid molecule encoding CX3CL1 comprises nucleotides 80-1273 of SEQ ID NO:6, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 80-1102 of SEQ ID NO:6.

13. The composition of any one of claims 1-12, wherein the nucleic acid molecule encoding CX3CL1 comprises nucleotides 246-1013 of SEQ ID NO:7, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 246-1013 of SEQ ID NO:7.

14. The composition of any one of claims 1-11, wherein the nucleic acid molecule encoding CX3CL1 comprises nucleotides 246-1184 of SEQ ID NO:7, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of nucleotides 246-1184 of SEQ ID NO:7.

15. The composition of any one of claims 1-14, wherein the expression cassette further comprises an intron between the promoter and the nucleic acid molecule encoding CX3CL1.

16. The composition of claim 15, wherein the intron is an SV-40 intron.

17. The composition of claim 15, wherein the intron is a chimeric intron comprising a 5' -donor site from the first intron of the human b-globin gene and the branch and 3' -acceptor site from the intron that is between the leader and the body of an immunoglobulin gene heavy chain variable region.

18. The composition of any one of claims 1-17, wherein the expression cassette further comprises a post-transcriptional regulatory region.

19. The composition of any one of claims 1-18, wherein the expression cassette further comprises a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

20. The composition of any one of claims 1-19, wherein the expression cassette further comprises a post-transcriptional regulatory region comprising the nucleotide sequence of SEQ ID NO: 11, or a nucleotide sequence having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% nucleotide sequence identity to the entire nucleotide sequence of the nucleotide sequence of SEQ ID NO: 11.

21. The composition of any one of claims 1-20, wherein the expression cassette further comprises a polyadenylation signal.

22. The composition of claim 21, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal.

23. The composition of any one of claims 1-22, wherein the expression cassette is present in a vector.

24. The composition of claim 23, wherein the vector is an AAV vector selected from the group consisting of AAV2, AAV 8, AAV2/5, and AAV 2/8.

25. An AAV vector particle comprising the composition of any one of claims 1-24.

26. An isolated cell comprising the AAV particle of claim 25.

27. A pharmaceutical composition comprising the AAV composition of any one of claims 1-24 or the AAV particle of claim 25.

28. The pharmaceutical compostion of claim 27, further comprising a viscosity inducing agent.

29. The pharmaceutical compostion of claim 27 or 28, which is for intraocular administration.

30. The pharmaceutical composition of claim 29, wherein the intraocular administration is selected from the group consisting of intravitreal or subretinal, subvitreal, subconjuctival, sub-tenon, periocular, retrobulbar, suprachoroidal, and/or intrascleral administration.

31. A method for prolonging the viability of a photoreceptor cell compromised by a degenerative ocular disorder, comprising contacting said cell with the composition of any one of claims 1-24, the AAV viral particle of claim 25, or the pharmaceutical compostion of any one of claims 27-30, thereby prolonging the viability of the photoreceptor cell compromised by the degenerative ocular disorder.

32. A method for treating or preventing a degenerative ocular disorder in a subject, comprising administering to said subject a therapeutically effective amount of the composition of any one of claims 1-24, the AAV viral particle of claim 25, or the pharmaceutical compostion of any one of claims 27-30, thereby treating or preventing said degenerative ocular disorder.

33. A method for delaying loss of functional vision in a subject having a degenerative ocular disorder, comprising administering to said subject a therapeutically effective amount of the composition of any one of claims 1-24, the AAV viral particle of claim 25, or the pharmaceutical compostion of any one of claims 27-30, thereby treating or preventing said degenerative ocular disorder.

34. The method of any one of claims 31-33, wherein the degenerative ocular disorder is associated with decreased viability of cone cells and/or decreased viability of rod cells.

35. The method of any one of claims 31-34, wherein the degenerative ocular disorder is selected from the group consisting of retinitis pigmentosa, age related macular degeneration, cone rod dystrophy, and rod cone dystrophy.

36. The method of any one of claims 31-34, wherein the degenerative ocular disorder is a genetic disorder.

37. The method of any one of claims 31-36, wherein the degenerative ocular disorder is not associated with blood vessel leakage and/or growth.

38. The method of any one of claims 31-37, wherein the degenerative ocular disorder is retinitis pigmentosa.

39. A method for treating or preventing retinitis pigmentosa in a subject, comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1-24, the AAV viral particle of claim 25, or the pharmaceutical compostion of any one of claims 27-30, thereby treating or preventing retinitis pigmentosa in said subject.