Gene therapy for SIRT1-related eye disorders

Abstract

A recombinant adeno-associated viral vector comprising a nucleic acid molecule containing a sequence encoding SIRT1 is provided. In one embodiment, a composition and a method for treating ocular neuropathy in a subject are provided. In a preferred embodiment, the subject is a human, cat, dog, sheep, or non-human primate.

Description

Background Technology

[0001] Statements regarding federal government-supported research and development

[0002] This invention was carried out with government support under EY019014 granted by the National Institutes of Health. The government holds specific rights to this invention.

[0003] Inclusion by reference in sequence list

[0004] A sequence listing file titled "UPN-24-10561.PCT", created on October 28, 2024, with a size of 85,020 bytes, is incorporated herein by reference in its entirety.

[0005] Background of the Invention

[0006] Retinal ganglion cells (RGCs) are critical mediating targets in neurodegenerative diseases of the eye, including optic neuritis, glaucoma, ischemic optic neuropathy, and Leber hereditary optic neuropathy. RGC apoptosis is the underlying cause of irreversible blindness in these and other optic neuropathyes. Damage to RGCs is commonly linked to various etiologies, such as inflammation of the optic nerve, reduced trophic factors, ischemia, increased oxidative stress resulting from the detrimental effects of reactive oxygen species exposure, and impaired axonal transport and electroactivity. There is currently an unmet therapeutic need to prevent or minimize RGC alterations and / or regenerate functioning RGCs under disease conditions. Neuroprotective strategies currently under active investigation include viral vector-mediated delivery of therapeutic recombinant transplanted genes to RGCs, as well as cell- and non-cell-based neuroprotective approaches. 7 Among these, AAV-based gene therapy models are being tested in preclinical studies and clinical trials. 8 There are more than 130 different naturally occurring AAV serotypes and genotypes that vary in capsid protein sequence, structure, and organization. 9This capsid diversity results in affinity for various cells and tissues, making AAV vectors a highly attractive gene delivery vehicle. Accordingly, AAVs are effectively used to treat numerous genetic and acquired human diseases; more specifically, monogenetic diseases caused by mutations in a single gene. 10-12 Today, countless AAV-based gene therapies are being studied, and some have been fully approved by regulatory agencies for use in humans to improve vision loss in patients with inherited retinal diseases. 13-15 However, there is still a need for improved vector designs that can maximize the targeting of highly efficient neuronal cells such as RGCs.

[0007] SIRT1 is a widely expressed histone deacetylase that alters the stability, activity, subcellular localization, DNA-binding ability, and protein-protein interactions of targeted histones and transcription factors by removing acetyl groups from lysine residues. 29 As such, SIRT1 plays an extensive role in the regulation of various fundamental processes related to metabolism, inflammation, stress, apoptosis, and DNA repair. 30 The SIRT1 / PGC-1α signaling pathway plays an endogenous neuroprotective role against neuronal damage in the brain and retina. 31, 32 In addition, SIRT1 has also been shown to deacetylate several apoptosis-associated proteins, such as p53, Smad7, FOXO3, and FOXO4, and protect cells from cell death. 30, 33 .

[0008] In RGC-targeted gene therapy, an optimized vector is required to maximize the expression of the transplanted gene. Such AAV-based gene therapies are useful, for example, in the treatment of ocular diseases, particularly optic neuropathy, where a therapeutic gene such as SIRT1 can be optimally introduced directly from the RGC.

[0009] Summary of the Invention

[0010] The present invention provides a highly efficient AAV vector having excellent transduction efficiency and specificity for RGC.

[0011] In one embodiment, a recombinant adeno-associated virus (rAAV) is provided, said rAAV comprises an AAV capsid and a vector genome packaged therein, said vector genome comprising (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) a CMV enhancer; (c) a human SNCG promoter; (d) an SV40 intron; (e) a coding sequence encoding human SIRT1; (f) a WPRE; (g) a bGH poly-A; and (h) an AAV 3' ITR sequence.

[0012] In one embodiment, the vector genome comprises: (a) an AAV 5' inversion terminal repeat (ITR) sequence having SEQ ID NO: 4 or a sequence having at least 90% sequence identity therewith; (b) a CMV enhancer sequence having SEQ ID NO: 5 or 29 or a sequence having at least 90% sequence identity therewith; (c) a human SNCG promoter sequence having SEQ ID NO: 6 or a sequence having at least 90% sequence identity therewith; (d) an SV40 intron sequence having SEQ ID NO: 7 or a sequence having at least 90% sequence identity therewith; (e) a coding sequence encoding a human SIRT1 sequence having SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith; (f) a WPRE sequence having SEQ ID NO: 8 or a sequence having at least 90% sequence identity therewith; (g) a bGH poly-A sequence having SEQ ID NO: 9 or 30 or a sequence having at least 90% sequence identity therewith; and (h) comprises one or more of the AAV 3' ITR sequence having SEQ ID NO: 10 or 31 or sequences having at least 90% sequence identity therewith. Preferably, the coding sequence encoding the human SIRT1 sequence is SEQ ID NO: 1.

[0013] In a preferred embodiment, the vector genome comprises (a) an AAV 5' ITR sequence, preferably having SEQ ID NO: 4; (b) a CMV enhancer sequence, having SEQ ID NO: 5 or 29; (c) a human SNCG promoter sequence, having SEQ ID NO: 6; (d) an SV40 intron sequence, having SEQ ID NO: 7; (e) a coding sequence encoding a human SIRT1 sequence, having SEQ ID NO: 1; (f) a WPRE sequence, having SEQ ID NO: 8; (g) a bGH poly-A sequence, having SEQ ID NO: 9 or 30; and (h) an AAV 3' ITR sequence, preferably having SEQ ID NO: 10 or 31.

[0014] In a particularly preferred embodiment, the vector genome comprises sequence number: 11, or a sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0015] In another embodiment of the present invention, rAAV is provided, said rAAV comprises an AAV capsid and a vector genome packaged therein, said vector genome comprising (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) an SV40 enhancer; (c) a human SNCG promoter; (d) an SV40 intron; (e) a coding sequence encoding human SIRT1; (f) a WPRE; (g) a bGH poly-A; and (h) an AAV 3' ITR.

[0016] In one embodiment, the vector genome comprises: (a) an AAV 5' inversion terminal repeat (ITR) sequence having SEQ ID NO: 4 or a sequence having at least 90% sequence identity therewith; (b) an SV40 enhancer sequence having SEQ ID NO: 12 or a sequence having at least 90% sequence identity therewith; (c) a human SNCG promoter sequence having SEQ ID NO: 6 or a sequence having at least 90% sequence identity therewith; (d) an SV40 intron sequence having SEQ ID NO: 7 or a sequence having at least 90% sequence identity therewith; (e) a coding sequence encoding a human SIRT1 sequence having SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith; (f) a WPRE sequence having SEQ ID NO: 8 or a sequence having at least 90% sequence identity therewith; (g) a bGH poly-A sequence having SEQ ID NO: 9 or 30 or a sequence having at least 90% sequence identity therewith; and (h) comprises one or more of the AAV 3' ITR sequence having SEQ ID NO: 10 or 31 or sequences having at least 90% sequence identity therewith. Preferably, the coding sequence encoding the human SIRT1 sequence is SEQ ID NO: 1.

[0017] In a preferred embodiment, the vector genome comprises (a) an AAV 5' ITR sequence, preferably having SEQ ID NO: 4; (b) an SV40 enhancer sequence, having SEQ ID NO: 12; (c) a human SNCG promoter sequence, having SEQ ID NO: 6; (d) an SV40 intron sequence, having SEQ ID NO: 7; (e) a coding sequence encoding a human SIRT1 sequence, having SEQ ID NO: 1; (f) a WPRE sequence, having SEQ ID NO: 8; (g) a bGH poly-A sequence, having SEQ ID NO: 9 or 30; and (h) an AAV 3' ITR sequence, preferably having SEQ ID NO: 10 or 31.

[0018] In a particularly preferred embodiment, the vector genome comprises sequence number: 13, or a sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0019] In another embodiment, rAAV is provided, said rAAV comprises an AAV capsid and a vector genome packaged therein, said vector genome comprising (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) a CAG promoter; (c) a coding sequence encoding human SIRT1; (d) a WPRE; (e) a bGH poly-A; and (f) an AAV 3' ITR.

[0020] In one embodiment, the vector genome comprises one or more of: (a) an AAV 5' inversion terminal repeat (ITR) sequence having SEQ ID NO: 4 or a sequence having at least 90% sequence identity therewith; (b) a CAG promoter having SEQ ID NO: 28, 33, or 35 or a sequence having at least 90% sequence identity with SEQ ID NO: 28, 33, or 35; (c) a coding sequence encoding a human SIRT1 sequence having SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith; (d) a WPRE sequence having SEQ ID NO: 8 or a sequence having at least 90% sequence identity therewith; (e) a bGH poly A sequence having SEQ ID NO: 9 or 30 or a sequence having at least 90% sequence identity therewith; and (f) an AAV 3' ITR sequence having SEQ ID NO: 10 or 31 or a sequence having at least 90% sequence identity therewith. Preferably, the coding sequence encoding the human SIRT1 sequence is SEQ ID NO: 1.

[0021] In a preferred embodiment, the vector genome comprises (a) an AAV 5' ITR sequence, preferably having SEQ ID NO: 4; (b) a CAG promoter sequence, having SEQ ID NO: 28, 33, or 35; (c) a coding sequence encoding a human SIRT1 sequence, having SEQ ID NO: 1; (d) a WPRE sequence, having SEQ ID NO: 8; (e) a bGH poly-A sequence, having SEQ ID NO: 9 or 30; and (f) an AAV 3' ITR sequence, preferably having SEQ ID NO: 10 or 31.

[0022] In a particularly preferred embodiment, the vector genome comprises sequence number: 18, or a sequence that shares at least 90% identity with it.

[0023] In another embodiment, rAAV is provided, said rAAV comprises an AAV capsid and a vector genome packaged therein, said vector genome comprising (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) a CAG promoter; (c) a coding sequence encoding human SIRT1; (d) a bGH poly-A; and (e) an AAV 3' ITR.

[0024] In one embodiment, the vector genome comprises one or more of: (a) an AAV 5' inversion terminal repeat (ITR) sequence having SEQ ID NO: 4 or a sequence having at least 90% sequence identity therewith; (b) a CAG promoter sequence having SEQ ID NO: 37 or a sequence having at least 90% sequence identity therewith; (c) a coding sequence encoding a human SIRT1 sequence having SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith; (d) a bGH poly-A sequence having SEQ ID NO: 9 or 30 or a sequence having at least 90% sequence identity therewith; and (e) an AAV 3' ITR sequence having SEQ ID NO: 10 or 31 or a sequence having at least 90% sequence identity therewith.

[0025] In one embodiment, the vector genome comprises (a) an AAV 5' inversion terminal repeat (ITR) sequence, preferably having SEQ ID NO: 4; (b) a CAG promoter sequence, having SEQ ID NO: 37; (c) a coding sequence encoding a human SIRT1 sequence, having SEQ ID NO: 1; (d) a bGH poly-A sequence, having SEQ ID NO: 9 or 30; and (e) an AAV 3' ITR sequence, preferably having SEQ ID NO: 10 or 31.

[0026] In one embodiment, the vector genome comprises sequence number: 23, or a sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0027] In one embodiment, the rAAV capsid is an AAV2 capsid or an AAV8 capsid.

[0028] In another aspect, cells transfected with the AAV vector of the present invention are provided.

[0029] In another aspect, a pharmaceutical composition is provided comprising rAAV or cells according to the present invention; and a pharmaceutically acceptable carrier or excipient.

[0030] In one embodiment, an aqueous suspension suitable for administration to a subject is provided, said suspension comprising an aqueous suspension and recombinant adeno-associated virus (rAAV). In one embodiment, the suspension comprises a surfactant, a preservative, and / or a buffer dissolved in the aqueous suspension. In a preferred embodiment, the pharmaceutical composition or aqueous suspension is suitable for delivery to the eye.

[0031] In another aspect of the present invention, a method for treating or preventing eye disorders in a subject requiring treatment is provided, comprising administering rAAV, cells, or a pharmaceutical composition according to the present invention to the subject.

[0032] In one embodiment, the disorder is an optic neuropathy, such as optic neuritis or glaucoma.

[0033] In a preferred embodiment, the disorder is glaucoma.

[0034] In one embodiment, the rAAV, cell, or pharmaceutical composition of the present invention is administered into the eye.

[0035] In a preferred embodiment, rAAV, cells, or a pharmaceutical composition are administered by subretinal, supracorbital, or intravitreal injection.

[0036] In one embodiment, rAAV is about 1 x 10 9 to about 1 x 10 13 It is delivered in a dose of vector genome / eye (vg / eye). In one embodiment, rAAV is delivered in a volume containing about or at least 100 microliters of 1 x 10⁶ 9 Up to 1 x 10 13 rAAV is administered in a dosage amount to prevent vision loss in the subject. In one embodiment, rAAV is administered in a volume comprising about 100 to 250 microliters. In one embodiment, rAAV is administered in a volume comprising 150 to 800 microliters. In one embodiment, rAAV is administered in a volume comprising 250 to 500 microliters. In one embodiment, rAAV is administered in a volume comprising about 500 microliters.

[0037] In a preferred embodiment, the object is a human.

[0038] Other aspects and advantages of the present invention will be readily apparent from the following detailed description of the present invention. Brief explanation of the drawing

[0039] Figures 1a through 1h illustrate the AAV transduction potential. (a) Schematic representation of AAV vectors V1.G and V2.G. AAV2 and AAV8BP2 were modified to drive the expression of the transplant gene (eGFP) under the control of the CBA-CMV promoter. The transplant gene is positioned between two internal terminal repeats (ITRs). (b) Administration of V1.G and V2.G was achieved via a single injection of the AAV vector into the vitreous humor of the mouse eye. (c) Fluorescence confocal scanning laser ophthalmoscopy (CSLO) images obtained from mice 2 weeks after increasing doses of intravitreal injection of V1.G or V2.G. (d) 1 x 10⁶ of the V1.G or V2.G vector 9 Representative fluorescence micrograph of the RGC layer from a flat-mounted specimen of a mouse retina 2 weeks after vg / intravitreal injection into the eye. RGCs were labeled with a monoclonal antibody against Brn3a. Converged eGFP (green) and Brn3a (red) signals are shown. Transduction efficiency (E to F) at 1 and 2 weeks after intravitreal injection of V1.G and V2.G was determined as the percentage of Brn3a+ eGFP+ cells relative to total Brn3a+ positive cells averaged across the entire retinal area. Data are presented as mean ± SEM (*, p<0.05, n=4). (g to h) show the mean percentage ± SEM of brn3a+ RGCs expressing eGFP 1 (Fig. 1g) or 2 (Fig. 1h) weeks after intravitreal injection of the two vectors at three different concentrations (n ​​= 4 retinas / vector dose / time point). Figure 2 illustrates the time- and dose-dependent transduction potential of the AAV2 vector. Representative fluorescence micrographs of flat-mounted retinas were generated at weeks 1, 2, and 4 following the intravitreal administration of increasing doses of the V2.G vector, immunostained with Brn3a antibodies (not shown). The transduction efficiency at weeks 1, 2, and 4 of V2.G intravitreal injection was the total Brn3a averaged across the entire retinal area. +Regarding positive cells, Brn3a+ eGFP + It was determined as the percentage of cells. (Data is presented as mean ± SEM, n=4). FIGS. 3a to 3f In vivo Illustrates the optimization of AAV2 vectors for the high-efficiency delivery of transplanted genes to RGCs. (a) Diagram of AAV vectors containing various promoters and regulatory sequences to drive eGFP reporter expression. CMV.CBA: Cytomegalovirus enhancer with chicken β-actin promoter; hSNCG: Human gamma-synuclein promoter; WPRE: Woodchuck hepatitis post-transcriptional regulatory element; iSV40: SV40 intron; eCMV: CMV-immediate early enhancer; eSV40: SV40 enhancer; Arc: Active-regulatory cytoskeletal promoter; PolyA: Polyadenine nucleotide tail; ITR: Internal terminal repeat; eGFP: Enhanced green fluorescent protein. (b) 3 x 10 9(c) Representative fundus fluorescence images of eyes injected with vg / eye V3.G, V4.G, V5.G, V6.G, V7.G, and V8.G vectors and Brn3a immunostained flat-mount retinas (inset panel). (c) Transduction efficiency at 2 weeks after intravitreal injection with V2.G, V3.G, V4.G, V5.G, V6.G, V7.G, and V8.G. The percentage of GFP-positive Brn3a-positive cells (RGCs) in the retinal flat is plotted. Data are presented as the mean ± SEM of the percentage of EGP+ RGCs counted across all retinal regions (*p<0.05, n=3-4). (d) Quantification of eGFP protein levels in retinal lysates from mouse eyes injected intravitreally with V2.G, V3.G, V4.G, V5.G, V6.G, V7.G, or V8.G. The plotted values ​​are expressed as mean ± SEM. *p<0.05 (n=4). ***p<0.001 (n=4). Two weeks after injection (1E10 vg / eye), six out of eight constructs demonstrated equivalent transduction efficiencies of 35 to 40% of brn3a+ RGCs expressing eGFP (Fig. 3e; n = 6 eyes / vector). Only the hSNCG.eGFP construct had significantly lower transduction (*p<0.05) compared to the CMV and SV40 enhancer / SNCG promoter constructs, while the Arc enhancer constructs showed a tendency toward lower transduction. ELISA of whole retinal protein extracts (Fig. 3f; n = 3-4 retinas / vector) showed a non-significant trend toward higher expression with the CMV / CBA, CMV / SNCG, and SV40 / SNCG enhancer / promoter combinations. Figures 4a through 4c illustrate the RGC selectivity of the AAV2 vector. (a) 3 x 10 of V2.G, V4.G, V6.G, or V7.G 9Representative fluorescence micrographs of retinal cross-sections after vg / intravitreal administration and immunostaining with Brn3a antibody. Fluorescence images of eGFP and Brn3a are shown separately and superimposed. (b) Quantification of the population of eGFP+ RGCs transduced with V2.G, V4.G, V6.G, or V7.G. Selectivity of the promoter inserted in the AAV2 vector was calculated as the number of eGFP+ Brn3a+ cells out of the total number of eGFP+ cells. Data shown are mean ± SEM (*p<0.05 for V2.G, ANOVA using Turkey multiple comparison test, n=). (c) Two weeks after intravitreal injection (1E10 vg / eye), immunostaining of retinal cross-sections showed significantly fewer eGFP+ cells (60%) co-localized with the RGC marker brn3a in the eye injected with the CMV.CBA enhancer / promoter construct compared to the eye injected with either SNCG promoter-containing construct (90% each). ***p<0.001, n = 6 retinas / vector. A representative image (right) illustrates selective eGFP within the RGC layer in the eye injected with the SNCG promoter-containing construct. Figures 5a through 5e illustrate the AAV2-mediated expression of hSIRT1 in RGCs. (a) Schematic representation of an AAV2 capsid engineered to contain the hSIRT1 gene under the control of a CMV-CBA or hSNCG promoter combined with different regulatory sequences. In vectors V2.G, V6.G, and V7.G (shown in Figure 3), the eGFP reporter gene was replaced with the codon-optimized sequence of the human SIRT1 gene in AR0020, AR0018 (AR0020 with WPRE elements), AR0012, and AR0013 labeled AAV vectors, respectively. (b) 3 x 10 of the AR0020, AR0018, AR0012, or AR0013 vectors 9Fluorescence micrograph of flat-mounted specimens in the mouse retina after vg / intravitreal injection into the eye. RGCs were labeled with monoclonal antibodies against Brn3a and human-specific SIRT1. Separate and merged images of hSIRT1 (green) and Brn3a (red) are shown. (c) SIRT1 2 weeks after vector injection + Quantification of the RGC population. Transduction efficiency was SIRT1 among the total number of Brn3a+ cells counted in 12 representative visual fields including standardized regions across the central, mesoperiphery, and far peripheral retinas (n=6). + Brn3a + It was calculated as a percentage of cells. (d) hSIRT1 + The transduction efficiency in the central retinal region containing only the highest number of cells is SIRT1 among the number of Brn3a+ cells counted in the most intensely stained retinal region. + Brn3a + Calculated as a percentage of cells. Data shown are mean ± SEM (n=6). (e) Quantitative (q)PCR analysis of the relative amount of hSIRT1 mRNA in retinal lysates from mouse eyes injected intravitreally with AR0020, AR0018, AR0012, or AR0013 vectors. Data shown are mean ± SEM (n=6). Figures 6a and 6b illustrate the results from a mouse model of optic neuropathy. The EAE scores in Figure 6a showed that mild ascending paralysis occurred in all treatment groups. The EAE scores in Figure 6b showed more significant ascending paralysis reaching a moderate level in all treatment groups. Figure 6c demonstrates that the EAE score shows a slow, mild increase in ascending paresis from the first treatment. Mice were sham-injected with PBS into one eye 14 days before EAE induction and injected with a vector into the contralateral eye, or injected with a vector into one eye 9 days after EAE induction and into the other eye 15 days after EAE induction (n = 10 mice / group). Figure 6d demonstrates that the OKR score shows a similar decrease in visual function regardless of SIRT1 gene therapy (n = 10 eyes / treatment). Figure 6e demonstrates that RGC / retina attenuated by AR0012 or AR0018 treatment (n = 7-10 retinas / treatment) shows significant RGC loss induced by EAE. Although the effects of AR0012 or AR0018 did not reach statistical significance when compared by ANOVA, when each treatment group was compared to the untreated group by t-test, all groups showed a strong trend toward increased RGC survival, with RGC survival significantly improved in all three groups. Figure 6f demonstrates that the EAE score indicates typical moderate ascending paralysis. Mice were injected with a sham injection in one eye 14 days prior to EAE induction and with a vector in the contralateral eye, or injected in one eye 9 days after EAE induction and in the other eye 15 days after (n = 10 mice / group). OKR (Figure 6g) shows similar visual impairment regardless of SIRT1 gene therapy (n = 10 eyes / treatment). RGC / retina (n = 10 retina / treatment, Fig. 6h) shows significant RGC loss induced by EAE, which is significantly attenuated by AR0012 or AR0018 treatment when compared by ANOVA. Figures 7a through 7c illustrate the results of Experiment 1 as described in Example 4. IOP measurements (Figure 7a left) show average IOP levels exceeding double in all MB-injected treatment groups. Eyes were sham-injected or vector-injected 14 days prior to MB injection, or vector-injected 3 or 10 days after MB injection (n = 10 eyes / group). OKR (Figure 7b) includes only eyes with documented IOP elevation and no severe corneal staining, showing similar visual impairment regardless of SIRT1 gene therapy (n = 4-10 eyes / treatment). RGC / retina (Figure 7c) showed a trend toward increased RGC survival in all groups, with RGC survival significantly improved in one group when compared to the untreated group by t-test (*p < 0.05) (n = 6-9 eyes / treatment). Data is displayed only from the eye with the elevated IOP. Figures 8a through 8d illustrate the results of Experiment 2 as described in Example 4. IOP (Figure 8a) measurements show average IOP levels increased by 1.5 to 2.0-fold in all lower concentration microbead (MB) injected treatment groups. Eyes were sham-injected or vector-injected 14 days prior to MB injection, or vector-injected 10 days after MB injection (n = 10 eyes / group). OKR (upper right) shows significant visual impairment in MB-injected eyes without SIRT1 gene therapy (#p<0.0001 for control eyes, ANOVA with LS mean post-hoc analysis), including only eyes with documented IOP elevation and no severe corneal staining on the day of OKR measurement (n = 5-10 eyes / treatment). All treatments significantly improved the OKR response (for the invalid vector; LS mean post-hoc ANOVA; *p<0.05, **p<0.01 ***p<0.001). RGC / retina (inferior right) shows significant RGC loss in MB-injected eyes treated with the invalid vector (#p<0.05 for control eyes), and treatment with both V.6S and V2.wpre.S before and after MB injection resulted in increased RGC survival in all groups compared to the control vector (n = 7-10 eyes / treatment; LS mean post-hoc ANOVA; *p<0.05, **p<0.01, data including only eyes with elevated IOP). Cross-sections of the optic nerves of 4 randomly selected mice from each treatment group (n=4 nerves / treatment) were stained with toluidine blue, and the count of RGC axons by a shielded investigator showed a strong trend toward increased RGC axon density in treated versus ineffective-treated MB-injected mice (inferior left). Figures 9a through 9d illustrate the results of the experiment described in Example 5. The EAE score (Figure 9a) shows typical moderate ascending paresis. The eyes were injected with either a sham or therapeutic vector(s) 14 days prior to EAE induction. OKR (Figure 9b) shows similar visual impairment regardless of SIRT1 gene therapy (n = 14-16 eyes / treatment). RGC / retina (Figure 9c; n = 10 retinas / treatment) shows significant RGC loss induced by EAE (p<0.001 for EAE-sham) that is only attenuated by AR0012 treatment alone (p<0.05 for EAE-sham) when compared by t-test. Optic nerve inflammation induced in EAE-sham mice was not significantly altered by treatment (Figure 9d). Specific details for implementing the invention

[0040] Sirtuins are NAD-dependent protein deacetylases. Intravitreal delivery of sirtuin activators, such as resveratrol, has been shown to reduce defects from optic neuritis and optic nerve compression injury in mouse models. (Shindler et al. Invest Ophthalmol Vis Sci 48(8):3602 (2007); Zuo et al. Invest Ophthalmol Vis Sci 54(7):5097-102 (2013)). (Coding sequences and expression products interchangeably used herein) SIRT1Sirtuin-1 encoded by [substance] directly links transcriptional regulation to intracellular energy metabolism and participates in the coordination of several distinct cellular functions, such as the cell cycle, response to DNA damage, metabolism, apoptosis, and autophagy. Two isoforms are known: Transcript variant 1, comprising a protein of all 747 amino acids (Sequence No. 2), and Transcript variant 2, lacking amino acids 454–639 (using numbering from Transcript variant 1). The use of Transcript variant 1 is exemplified herein. However, similar constructs utilizing Transcript variant 2 are also considered herein. Natural [substances] encoding Sirtuin-1 SIRT1 The sequence is represented by sequence number: 3. Codon-optimized encoding sirtuin-1 SIRT1 The sequence is denoted by sequence number: 1. It was shown that an activator of SIRT1 significantly attenuated retinal ganglion cells (RGCs) in a dose-dependent manner. Shindler, KS et al., Invest Ophthalmol Vis Sci. 2007 Aug;48(8):3602-9, incorporated herein by reference.

[0041] rAAV vectors capable of delivering Sirtuin-1 to affected tissues, including retinal cells in the eye, are described herein. The vectors described herein are useful in the treatment of a wide range of genetic and acquired optic nerve disorders that affect the structure or function of the optic nerve. In one embodiment, any of these optic nerve disorders or conditions are referred to as optic neuropathy. Optic neuropathy includes glaucoma, optic neuritis, retinocytosis, ischemic optic neuropathy, compressive optic neuropathy, infiltrative optic neuropathy, traumatic optic neuropathy, mitochondrial optic neuropathy, trophic optic neuropathy, and toxic optic neuropathy. Optic neuropathy further includes hereditary optic neuropathy, including Leber hereditary optic neuropathy, dominant optic atrophy, Baer syndrome, and Burke-Tabatsky syndrome. In one embodiment, the ocular disorder negatively affects retinal ganglion cells. In another embodiment, the disorder negatively affects any of the neuronal cells.

[0042] Glaucoma is a neurodegenerative disease of the eye and is one of the leading causes of irreversible blindness. It is estimated that by 2020, more than 80 million people worldwide will be affected, and at least 6 to 8 million of them will become bilaterally blind. Glaucoma is characterized by damage to the optic nerve and the progressive degeneration of retinal ganglion cells (RGCs), which are critical factors in vision loss. Factors associated with the pathogenesis of glaucoma include high intraocular pressure (IOP), increased oxidative stress, aging, glutamate neurotoxicity, and susceptibility genes such as optineurin and myocillin. In one embodiment, the ocular disorder or condition treated using the rAAV vector described herein is glaucoma. In one embodiment, the ocular disorder or condition treated using the rAAV vector described herein is MS.

[0043] MS is a central nervous system disease characterized by chronic inflammation and demyelination. Approximately 2.5 million people worldwide are affected, with an average age of onset of 30. About 50% of patients diagnosed after the age of 25 require mobility aids. This disease is primarily considered an autoimmune condition involving the infiltration of the CNS by autoreactive immune effector cells. The central "triggering factors" for the disease's development remain largely unknown, and it is believed that complex genetic and environmental factors are at play. See Dendrou et al. (Nat Rev Imm, 2009), incorporated herein by reference. See McDougald et al., Investigative Ophthalmology & Visual Science, March 2018, Vol. 59, 1212-1220, the entire text incorporated herein by reference.

[0044] Optic neuritis is a demyelinating inflammation of the optic nerve that typically affects young adults in the age range of 18 to 45. Patients usually present with rapid visual decline, orbital pain aggravated by eye movements, color vision abnormalities, and afferent pupillary defects, with or without optic disc edema. There is a strong association between optic neuritis and multiple sclerosis (MS), an acute inflammatory demyelinating disease of the central nervous system (CNS), where optic neuritis is an early indication of MS in approximately 20% of MS patients, and the risk of developing MS up to 15 years after the onset of optic neuritis is 50%. In one embodiment, the ocular condition treated using the rAAV vector described herein is optic neuritis. See Kimura et al. cited above. In another embodiment, the ocular condition is autoimmune encephalomyelitis (EAE).

[0045] The rAAV vector of the present invention is suitable for targeting gene therapy, such as hSIRT1 gene therapy, to retinal ganglion cells.

[0046] In one embodiment, the rAAV vector is useful for preventing the loss of axons / myelin in the optic nerve when facing inflammatory, autoimmune, inherited, or acquired diseases.

[0047] The rAAV vector of the present invention is delivered to retinal ganglion cells, the axons of which include the optic nerve. In one embodiment, rAAV may be delivered via subretinal, intravitreal, or supracorbital injection. Preferably, rAAV is administered directly to retinal cells via intravitreal or supracorbital injection, thereby allowing for in-clinical treatment.

[0048] In one embodiment, the rAAV vector, method, and composition described herein are used to deliver a nucleic acid sequence encoding the SIRT1 protein to a subject requiring treatment for the treatment of optic neuropathy, preferably optic neuritis or glaucoma.

[0049] In one embodiment, the rAAV, composition, and method of the present invention comprises the delivery of a engineered SIRT1 coding sequence. In one embodiment, the coding sequence is represented by SEQ ID NO: 1. A composition comprising a natural SIRT1 coding sequence, as represented by SEQ ID NO: 3, is also incorporated herein. Preferably, the coding sequence is represented by SEQ ID NO: 1.

[0050] The technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art and in the text disclosed by reference, which provides a general guide to the many terms used in this application and to those skilled in the art to which the present invention belongs. The definitions contained herein are provided to clarify the components and compositions herein and are not intended to limit the claimed invention.

[0051] In the context of nucleic acid sequences, the terms “percent (%) identity,” “sequence identity,” “percent sequence identity,” or “percent identical” refer to residues in two sequences that are identical when aligned for correspondence. The length of the sequence identity comparison may be the whole length of the genome, or exceed the whole length of the gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides is preferred. However, identity in smaller fragments, e.g., at least about 9 nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, or at least about 36 or more nucleotides, may also be preferred.

[0052] Percent identity can be easily determined for an amino acid sequence across the whole length of the protein, polypeptide, about 32 amino acids, about 330 amino acids, or its peptide fragment or corresponding nucleic acid sequence coding sequence. A suitable amino acid fragment may be at least about 8 amino acids in length and up to about 700 amino acids. Generally, when referring to "identity," "homology," or "similarity" between two different sequences, the "identity," "homology," or "similarity" is determined with respect to the "aligned" sequence. An "aligned" sequence or "alignment" refers to multiple nucleic acid sequences or protein (amino acid) sequences that often include corrections for missing or additional bases or amino acids when compared to a reference sequence.

[0053] Identity can be determined by preparing for the alignment of sequences and by using various algorithms and / or computer programs known in the art or commercially available [e.g., BLAST, ExPASy; ClustalO; FASTA; e.g., using the Needleman-Wunsch algorithm, Smith-Waterman algorithm]. Alignment is performed using any of the various publicly or commercially available multiple sequence alignment programs. Sequence alignment programs for amino acid sequences, e.g., "Clustal Omega", "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs, are available. Generally, any of these programs are used in the initial setup, although a person skilled in the art may change these settings as needed. Alternatively, a person skilled in the art may utilize another algorithm or computer program that provides at least the same level of identity or alignment as provided by the referenced algorithms and programs. For example, see JD Thomson et al., Nucl. Acids. Res., "Comprehensive Comparison of Multiple Sequence Alignments", 27(13):2682-2690 (1999).

[0054] Multiple sequence alignment programs for nucleic acid sequences are also available. Examples of such programs include "Clustal Omega," "Clustal W," "CAP Sequence Assembly," "BLAST," "MAP," and "MEME," which are accessible via web servers on the Internet. Other sources of these programs are known to those skilled in the art. Alternatively, vector NTI utilities are also used. There are also numerous algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program of GCG version 6.1. Fasta™ provides alignment of the region with the greatest overlap between the query sequence and the search sequence, and percent sequence identity. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its initial parameters (word size of 6 and NOPAM factor for the scoring matrix) as provided in GCG version 6.1 incorporated herein by reference.

[0055] A engineered nucleic acid sequence encoding human SIRT1 is useful herein. Preferably, the engineered SIRT1 coding sequence is SEQ ID NO: 1 or has about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or greater than 90% identity with the full-length SIRT1 coding sequence represented by SEQ ID NO: 1.

[0056] Sequence Number: 1 -

[0057]

[0058] “Engineered” means that the nucleic acid sequence encoding the SIRT1 protein described herein is assembled and placed on any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the SIRT1 sequence carried thereon into a host cell for delivery from a target to a host cell, for example, to form a non-viral delivery system (e.g., RNA-based system, naked DNA, or others) or to generate a viral vector within a host cell for packaging and / or delivery from a target to a host cell. In one embodiment, the genetic element is a plasmid. The methods used to construct such engineered constructs are known to those proficient in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. For example, see Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0059] As used herein, the term “host cell” may refer to a packaging cell line in which recombinant AAV is produced from a production plasmid. Alternatively, the term “host cell” may refer to any target cell in which expression of the coding sequence is desired. Thus, “host cell” refers to a prokaryotic or eukaryotic cell containing exogenous or heterologous DNA introduced into a cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposomal delivery, membrane fusion techniques, high-speed DNA-coating pellets, viral infection, and protoplast fusion. In certain embodiments herein, the term “host cell” refers to a cell used to produce and package a viral vector or recombinant virus. In other embodiments herein, the term “host cell” refers to the composition described herein In vitroIt refers to cultures of ocular cells or other neuronal cells of various mammalian species for evaluation.

[0060] In a further embodiment, the term "host cell" refers to optic neuropathy In vivo It is intended to designate the target cell of the subject being treated. In one embodiment, the host cell or target cell is an ocular cell. As used herein, the term “ocular cell” refers to any cell in the eye or in relation to the function of the eye. The term may refer to any of photoreceptor cells, including rod photoreceptors, cone photoreceptors, and photosensitive ganglion cells; retinal pigment epithelial (RPE) cells; Müller cells; choroidal cells; bipolar cells; horizontal cells; and amacrine cells. In another embodiment, the host cell or target cell is a neuronal cell. In one embodiment, the host cell or target cell is a retinal ganglion cell.

[0061] In one embodiment, the nucleic acid sequence encoding SIRT1 further comprises a nucleic acid encoding a tag polypeptide covalently bound thereto. The tag polypeptide may be selected, without limitation, from known “epitope tags” including myc tag polypeptide, glutathione-S-transferase tag polypeptide, green fluorescent protein tag polypeptide, myc-pyruvate kinase tag polypeptide, His6 tag polypeptide, influenza virus hemagglutinin tag polypeptide, flag tag polypeptide, and maltose-binding protein tag polypeptide. The use of the FLAG tag polypeptide is exemplified herein. Another example is the HA tag.

[0062] As used herein, "expression cassette" refers to a nucleic acid molecule containing a coding sequence for the SIRT1 protein, which is a promoter, and may contain other regulatory sequences thereof, and this cassette may be packaged in the capsid of a viral vector (e.g., a viral particle). Typically, such an expression cassette for producing a viral vector contains the SIRT1 sequence described herein and other expression control sequences, such as those described herein, adjacent to the packaging signal of the viral genome. For example, in the case of an AAV viral vector, the packaging signal is the 5' inversion terminal repeat (ITR) and the 3' ITR. When packaged in an AAV capsid, the ITR together with the expression cassette may be referred to herein as the "recombinant AAV (rAAV) genome" or "vector genome." In one embodiment, the expression cassette comprises a nucleic acid sequence encoding the SIRT1 protein. In one embodiment, the cassette provides SIRT1, which is functionally associated with an expression control sequence that induces the expression of a codon-optimized nucleic acid sequence encoding SIRT1 in a host cell.

[0063] In another embodiment, an expression cassette or vector genome for use in an AAV vector is provided. In that embodiment, the AAV vector genome comprises at least one AAV inversion terminal repeat (ITR) sequence. In another embodiment, the vector genome comprises a 5' ITR sequence and a 3' ITR sequence. In one embodiment, the 5' and 3' ITRs adjoin a nucleic acid sequence encoding SIRT, optionally as an additional sequence that induces the expression of a nucleic acid sequence encoding SIRT1 in a host cell. Thus, as described herein, the AAV expression cassette means the expression cassette described above, with the 5' end of the 5' AAV inversion terminal repeat sequence (ITR) and the 3' end of the 3' AAV ITR. Thus, this rAAV genome contains the minimum sequences required to package the expression cassette into an AAV viral particle, namely the AAV 5' and 3' ITRs. AAV ITRs may be obtained from the ITR sequences of any AAV, such as those described herein. These ITRs may be of the same AAV origin as the capsid used in the generated recombinant AAV, or of a different AAV origin (for producing the AAV pseudotype). In one preferred embodiment, an ITR sequence from AAV2, or a deleted version thereof (△ITR), is used for convenience and to accelerate regulatory approval. However, an ITR may be selected from other AAV sources. If the source of the ITR is AAV2 and the AAV capsid is from another AAV source, the generated vector may be designated as pseudotyped. Typically, the AAV vector genome comprises an AAV 5' ITR, a SIRT1 coding sequence and any regulatory sequence, and an AAV 3' ITR. However, other configurations of these elements may be suitable. A shortened version of the 5' ITR, designated as △ITR, in which the D-sequence and terminal resolution regions (trs) are deleted, has been described.In another embodiment, full-length AAV 5' and 3' ITRs are used. Each rAAV genome can then be introduced into a production plasmid.

[0064] As used herein, the terms “regulatory sequence,” “transcriptional control sequence,” or “expression control sequence” refer to DNA sequences that induce, repress, or otherwise control the transcription of a protein encoding a nucleic acid sequence to which they are operably linked, such as initiator sequences, enhancer sequences, and promoter sequences.

[0065] As used herein, the terms "operably linked" or "operably associated" refer to an expression control sequence adjacent to the nucleic acid sequence encoding SIRT1 and / or distal to control the transcription and expression thereof or Transro It refers to both of the active expression control sequences.

[0066] In one embodiment, a vector comprising any of the expression cassettes or vector genomes described herein is provided. As described herein, such a vector may be a plasmid of various origin and is useful for generating recombinant replication-deficient viruses as further described herein in certain embodiments.

[0067] A “vector” is a nucleic acid molecule into which an exogenous, heterologous, or engineered nucleic acid transfer gene may be inserted, as used herein, and which may then be introduced into a suitable host cell. A vector preferably has one or more replication origins and one or more sites into which recombinant DNA may be inserted. Vectors often have means for cells containing the vector to be selected from those without them, for example, they encode drug resistance genes. General vectors include plasmids, viral genomes, and “artificial chromosomes” (primarily in yeast and bacteria). Specific plasmids are described herein.

[0068] In one embodiment, the vector is a non-viral plasmid comprising the expression cassette described therein, e.g., “Naked DNA”, “Naked Plasmid DNA”, RNA, and mRNA; and is coupled with various compositions and nanoparticles, e.g., micelles, liposomes, cationic lipid-nucleic acid compositions, polyglycan compositions and other polymers, lipid and / or cholesterol-based nucleic acid conjugates, and other constructs, e.g., those described herein. See, e.g., X. Su et al., Mol. Pharmaceutics, 2011, 8 (3), pp 774-787; Web publication: March 21, 2011; see WO2013 / 182683, WO 2010 / 053572 and WO 2012 / 170930, all incorporated herein by reference. Such non-viral SIRT1 vectors may be administered by the routes described herein. Viral or non-viral vectors can be formulated with physiologically acceptable carriers for use in gene transport and gene therapy applications.

[0069] In another embodiment, the vector is a viral vector comprising an expression cassette described therein. A “viral vector” is defined as a replication-deficient virus containing an exogenous or heterologous SIRT1 nucleic acid transfer gene. In one embodiment, as described herein, the expression cassette may be engineered into a plasmid used for drug delivery or for the production of a viral vector. A suitable viral vector is preferably selected from those that are replication-deficient and target ocular cells. The viral vector may comprise any virus suitable for gene therapy, including, but not limited to, adenoviruses; herpesviruses; lentiviruses; retroviruses; parvoviruses, etc. In a preferred embodiment, the viral vector is an adeno-associated viral vector.

[0070] "Replication-deficient virus" or "viral vector" refers to a synthetic or recombinant viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, wherein any viral genomic sequence also packaged within the viral capsid or envelope is replication-deficient; that is, they cannot produce progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes necessary for replication (the genome can be manipulated to be "incapable"—containing only the transplanted gene of interest adjacent to signals necessary for the amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, since replication and infection by progeny virions cannot occur in the presence of viral enzymes necessary for replication, it is considered safe for use in gene therapy.

[0071] In another embodiment, a recombinant adeno-associated virus (rAAV) vector is provided. rAAV impairs an AAV capsid and a vector genome packaged therein. In one embodiment, the vector genome comprises (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) a promoter; (c) a coding sequence encoding a human SIRT; and (d) an AAV 3' ITR. In another embodiment, the vector genome is the expression cassette described herein. In one embodiment, the SIRT1 sequence encodes a full-length protein. In one embodiment, the SIRT1 sequence is the protein sequence of SEQ ID NO: 2. In another preferred embodiment, the coding sequence is SEQ ID NO: 1 or a variant thereof that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0072] Adeno-associated viruses (AAVs), members of the parvovirus family, are small, unenveloped, icosahedral viruses with a single-stranded linear DNA genome of 4.7 to 6 kb. Known AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and others. ITRs or other AAV components can be easily isolated or manipulated from AAVs using techniques available to those skilled in the art. Such AAVs may be isolated, manipulated, or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, Virginia). Alternatively, AAV sequences may be manipulated through synthesis or other suitable means by referring to publicly available sequences, such as those in the literature, or those available in databases, such as GenBank, PubMed, or others. AAV viruses can be manipulated by conventional molecular biology techniques, allowing these particles to be optimized for cell-specific delivery of nucleic acid sequences, minimization of immunogenicity, fine-tuning of stability and particle lifetime, efficient degradation, and accurate delivery to the nucleus.

[0073] Fragments of AAV can be readily utilized in various vector systems and host cells. Among the preferred AAV fragments are cap proteins containing vp1, vp2, vp3, and a hypervariable region, rep proteins containing rep 78, rep 68, rep 52, and rep 40, and sequences encoding these proteins. These fragments may be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements of other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, without limitation, AAVs having non-naturally occurring capsid proteins. Such artificial capsids may be generated by any suitable technique using the novel AAV sequence of the present invention (e.g., a fragment of the vp1 capsid protein) in combination with another AAV serotype (known or novel), a discontinuous portion of the same AAV serotype, a non-AAV viral source, or a heterogeneous sequence that may be obtained from a non-viral source. Artificial AAV serotypes may be, without limitation, chimeric AAV capsid, recombinant AAV capsid, or "humanized" AAV capsid.

[0074] The terms “AAV” or “AAV serotype” refer to dozens of naturally occurring and available adeno-associated viruses as used herein, as well as artificial AAVs. Among AAVs isolated from or engineered from human or non-human primates (NHP) and well-characterized, human AAV2 was the first AAV developed as a gene transport vector; it has been widely used in efficient gene transport experiments in different target tissues and animal models. Unless otherwise specified, the AAV capsid, ITR, and other selected AAV components described herein may be readily selected, without limitation, from any AAV including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, and AAVAnc80, variants of any known or mentioned AAV or AAV not yet discovered, or mixtures thereof. For example, see WO 2005 / 033321 incorporated herein by reference. Any AAV capsid having an affinity for ocular cells may be used. Preferably, the AAV capsid has an affinity for retinal ganglion cells.

[0075] Of all known AAVs, AAV2 is the dominant serotype used in preclinical and clinical trials because 50 to 96% of the human population is seropositive for it. 16 However, it was not linked to any disease in humans or other species. The AAV2 genome is approximately 4.7 kb of single-stranded DNA containing rep (replicator) and cap (capsid) genes, lateralized by an inversion terminal repeat (ITR), both packed into an icosahedral, non-encapsulated capsid. 17 AAV2 is the primary receptor for heparan sulfate proteoglycan (HSPG). 18 As, as well as fibroblast growth factor receptor 19 , hepatocyte growth factor receptor 20, integrin 21 , and CD9 22 It uses other helper receptors, including [it]. AAV2 has broad tissue affinity for the tissues of rodents, humans, and non-human primates. 23 Broader tissue affinity for AAV2 has been extended to most tissues by cross-packaging and cross-typing the AAV2 genome onto the capsids of other serotypes. 24 In addition, synthetic AAV vectors are In vivo It has been derived from AAV2 serotypes to improve transduction efficiency or immunological profiles. Recombinant AAV8BP2 is an AAV2 derivative that has been shown to target RGCs after intravitreal administration. 25

[0076] In one embodiment, the AAV capsid is an AAV2 capsid. In another embodiment, the AAV capsid is an AAV8 capsid. In another embodiment, the AAV capsid is an AAV8bp capsid. See WO 2014 / 024282, incorporated herein by reference. In another embodiment, the AAV capsid is an AAV7m8 capsid, which has demonstrated preferential delivery to the external retina. See Dalkara et al., In vivo-induced evolution of a novel adeno-associated virus for therapeutic external retinal gene delivery from the vitreous humor, Sci Transl Med 5, 189ra76 (2013), incorporated herein by reference. In another embodiment, the rAAV capsid is selected from the AAV8 capsid or a variant thereof, the AAV6 capsid or a variant thereof, the AAV9 capsid or a variant thereof, the AAV7 capsid or a variant thereof, the AAV5 capsid or a variant thereof, the AAV2 capsid or a variant thereof, the AAV1 capsid or a variant thereof, the AAV3 capsid or a variant thereof, and the AAV4 capsid or a variant thereof.

[0077] In one embodiment, a recombinant adeno-associated virus (rAAV) vector comprising the AAV capsid and expression cassette described herein is provided, wherein the expression cassette comprises a nucleic acid sequence encoding SIRT1, an inversion terminal repeat sequence, and an expression control sequence driving the expression of SIRT1 in a host cell.

[0078] In one embodiment, the vector genome comprises a 5' ITR, a CMV enhancer, a human SNCG promoter, an SV40 intron, the hSIRT1 coding sequence of SEQ ID NO: 1, a WPRE, a bGH polyA, and a 3' ITR. In a specific example, this construct is referred to as AR0012.In one embodiment, the vector genome comprises (a) an AAV 5' inversion terminal repeat (ITR) sequence having SEQ ID NO: 4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (b) a CMV enhancer sequence having SEQ ID NO: 5 or 29 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (c) A human SNCG promoter sequence having SEQ ID NO: 6 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (d) An SV40 intron sequence having SEQ ID NO: 7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (e) A coding sequence encoding a human SIRT1 sequence having SEQ ID NO: 1 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (f) a WPRE sequence having SEQ ID NO: 8 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (g) a bGH poly A sequence having SEQ ID NO: 9 or 30 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and (h) an AAV 3' ITR sequence having SEQ ID NO: 10 or 31 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith, comprising one or more of these.

[0079] In certain embodiments, the vector genome has the sequence of SEQ ID NO: 11, or a sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In certain embodiments, the expression cassette has the sequence of SEQ ID NO: 24, or a sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0080] In one embodiment, the vector genome comprises a 5' ITR, an SV40 enhancer, a human SNCG promoter, an SV40 intron, the hSIRT1 coding sequence of SEQ ID NO: 1, a WPRE, a bGH polyA, and a 3' ITR. In a specific example, this construct is referred to as AR0013.In one embodiment, the vector genome comprises: (a) an AAV 5' inversion terminal repeat (ITR) sequence having SEQ ID NO: 4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (b) an SV40 enhancer sequence having SEQ ID NO: 12 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (c) a human SNCG promoter sequence having SEQ ID NO: 6 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (d) an SV40 intron sequence having SEQ ID NO: 7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (e) a coding sequence encoding a human SIRT1 sequence having SEQ ID NO: 1 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (f) a WPRE sequence having SEQ ID NO: 8 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (g) a bGH poly A sequence having SEQ ID NO: 9- or 30 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and (h) one or more of an AAV 3' ITR sequence having SEQ ID NO: 10 or 31 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0081] In certain embodiments, the vector genome has the sequence of SEQ ID NO: 13, or a sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In certain embodiments, the expression cassette has the sequence of SEQ ID NO: 25, or a sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0082] In one embodiment, the vector genome comprises a 5' ITR, a CAG promoter, the hSIRT1 coding sequence of SEQ ID NO: 1, a WPRE, a bGH poly-A, and a 3' ITR. In certain examples, this construct is referred to as AR0018. In one embodiment, the vector genome comprises (a) an AAV 5' inversion terminal repeat (ITR) sequence having SEQ ID NO: 4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (b) a CAG promoter sequence having SEQ ID NO: 28, 33, or 35, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 28, 33, or 35; (c) a coding sequence encoding a human SIRT1 sequence having SEQ ID NO: 1, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the same; (d) a WPRE sequence having SEQ ID NO: 8 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (e) a bGH poly A sequence having SEQ ID NO: 9 or 30 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and (f) an AAV 3' ITR sequence having SEQ ID NO: 10 or 31 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith, comprising one or more of these.

[0083] In certain embodiments, the vector genome has the sequence of SEQ ID NO: 18, or a sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In certain embodiments, the expression cassette has the sequence of SEQ ID NO: 26, or a sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0084] In one embodiment, the vector genome comprises a 5' ITR, a CAG promoter, the hSIRT1 coding sequence of SEQ ID NO: 1, a bGH poly-A, and a 3' ITR. In certain examples, this construct is referred to as AR0020. In one embodiment, the vector genome comprises (a) an AAV 5' inversion terminal repeat (ITR) sequence having SEQ ID NO: 4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (b) a CAG promoter sequence having SEQ ID NO: 37 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (c) a coding sequence encoding a human SIRT1 sequence having SEQ ID NO: 1 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; (d) a bGH poly A sequence having SEQ ID NO: 9 or 30 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and (e) an AAV 3' ITR sequence having SEQ ID NO: 10 or 31 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith, comprising one or more of these.

[0085] In certain embodiments, the vector genome has the sequence of SEQ ID NO: 23, or a sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In certain embodiments, the expression cassette has the sequence of SEQ ID NO: 27, or a sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0086] As used herein with respect to AAV, the term variant means any AAV sequence derived from a known AAV sequence, comprising sharing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or excess sequence identity with respect to an amino acid or nucleic acid sequence. In another embodiment, the AAV capsid comprises a variant that may contain up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with the AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with the AAV capsid. When determining the percent identity of the AAV capsid, the comparison may be performed for any of the variable proteins (e.g., vp1, vp2, or vp3). In one embodiment, the AAV capsid shares at least 95% identity with AAV7m8 for vp1, vp2, or vp3. In one embodiment, the AAV capsid shares at least 95% identity with AAV2 for vp1, vp2, or vp3. In another embodiment, the AAV capsid shares at least 95% identity with AAV8BP2 for vp1, vp2, or vp3. In another embodiment, the capsid is, by reference to Kay et al., novel photoreceptor targeting via intravitreal delivery using a capsid-mutated AAV vector published online on April 26, 2013, incorporated herein by reference, PLoS One. 2013; As described by 8(4): e62097, it is an AAV8 capsid with Y447F, Y733F, and T494V mutations (also referred to as "AAV8(C&G+T494V)" and "rep2-cap8(Y447F+733F+T494V)").

[0087] In one embodiment, it is preferable to use an AAV capsid that exhibits affinity for a desired target cell, e.g., retinal ganglion cells or other ocular cells. In one embodiment, the AAV capsid is a tyrosine capsid-mutant in which a specific surface-exposed tyrosine residue is substituted with phenylalanine (F). Such an AAV variant is described, for example, by Mowat et al., Tyrosine Capsid-Mutant AAV Vector for Gene Delivery to Canine Retina from Subretinal or Intravitreal Approach, Gene Therapy 21, 96-105 (January 2014), which is incorporated herein by reference.

[0088] In one embodiment, the AAV capsid is selected from those that effectively transduce neuronal cells. In one embodiment, the AAV capsid is selected from AAV1, AAV2, AAV7, AAV8, AAV9, AAVrh.10, AAV5, AAVhu.11, AAV8DJ, AAVhu.32, AAVhu.37, AAVpi.2, AAVrh.8, AAVhu.48R3, and variants thereof. Each of these is incorporated herein by reference from Royo, et al., Brain Res, 2008 Jan, 1190:15-22; Petrosyan et al., Gene Therapy, 2014 Dec, 21(12):991-1000; Holehonnur et al., BMC Neuroscience, 2014, 15:28; and Cearley et al., Mol Ther. 2008 Oct; See 16(10): 1710-1718.

[0089] As used herein, “artificial AAV” means, without limitation, an AAV having a non-naturally occurring capsid protein. Such artificial capsid may be generated by any suitable technique using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a different selected AAV, a discontinuous portion of the same AAV, a non-AAV viral source, or a heterogeneous sequence that may be obtained from a non-viral source. Artificial AAV may, without limitation, be a detyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid. A detyped vector in which the capsid of one AAV is replaced with a heterogeneous capsid protein is useful in the present invention. In one embodiment, AAV2 / 8 is an exemplary detyped vector.

[0090] In one embodiment, a single-stranded AAV is used. In another embodiment, a self-complementary AAV is used. "Self-complementary AAV" refers to a plasmid or vector having an expression cassette designed such that a coding region carried by a recombinant AAV nucleic acid sequence forms an intramolecular double-stranded DNA template. Upon infection, instead of waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV will combine to form a single double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. For example, see DM McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independent of DNA synthesis," Gene Therapy (August 2001), Vol. 8, No. 16, pp. 1248–1254. Self-complementary AAVs are, for example, U.S. Patent No. 6,596,535, the entirety of which is incorporated herein by reference; It is listed in 7,125,717; and 7,456,683.

[0091] When used to describe nucleic acid sequences or proteins, the term "exogenous" means that the nucleic acid or protein does not occur naturally at a location existing on a chromosome or in a host cell. Exogenous nucleic acid sequences also refer to sequences that originate from and are inserted into the same host cell or target, but exist in a non-natural state, for example, at different copy numbers or under the control of different regulatory elements.

[0092] When used to describe a nucleic acid sequence or protein, the term "heterogeneous" means that the nucleic acid or protein originates from an organism different from the host cell or target in which it is expressed, or from a different species of the same organism. When the term "heterogeneous" is used in relation to a protein or nucleic acid in a plasmid, expression cassette, or vector, it indicates the presence of the protein or nucleic acid with another sequence or subsequence that is not found in nature in the same relationship to each other.

[0093] In yet another embodiment, an expression cassette comprising any of those described herein is used to generate a recombinant AAV genome.

[0094] In one embodiment, the expression cassette described herein is engineered into a suitable genetic element (vector) useful for delivery to a host cell, e.g., naked DNA, phage, transposon, cosmid, episome, etc., that carries the SIRT1 sequence carried therein, and / or for the generation of a viral vector. The selected vector may be delivered by any suitable method including transfection, electroporation, liposomal delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. The methods used to construct such constructs are known to those proficient in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0095] In the case of packaging an expression cassette, rAAV genome, or production plasmid into a virion, the ITR is the only AAV component required as the cis in the same construct as the expression cassette. In one embodiment, the coding sequences for the replica (rep) and / or capsid (cap) are removed from the AAV genome to generate an AAV vector. Translo Or supplied by the packaging cell line.

[0096] Methods for generating and isolating AAV viral vectors suitable for delivery to a target are known in the art. for example, [U.S. Patent 7790449; U.S. Patent 7282199; WO 2003 / 042397; WO 2005 / 033321, WO 2006 / 110689; and US 7588772 B2] , referenceIn one system, the production factor cell line is transiently transfected with a construct encoding a transplant gene attached by an ITR and construct(s) encoding rep and cap. In a second system, the packaging cell line stably supplying rep and cap is transiently transfected with a construct encoding a transplant gene attached by an ITR. In each of these systems, the AAV virion is generated in response to infection with a helper adenovirus or herpesvirus, requiring the isolation of rAAV from the contaminating virus. More recently, a system has been developed that does not require infection with a helper virus to recover AAV—required helper function ( in other words, Adenoviruses E1, E2a, VA, and E4 or herpesviruses UL5, UL8, UL52, and UL29, and herpesvirus polymerase) also, Translo , is supplied by the system. In these newer systems, the helper function can be supplied by transient transfection of cells with a construct encoding the required helper function, or the cells can be engineered to stably contain a gene encoding the helper function, and its expression can be controlled at the transcriptional or post-transcriptional level.

[0097] The term "isolated" means that a substance is removed from its original environment (e.g., the natural environment in cases of natural occurrence). For example, naturally occurring polynucleotides or polypeptides present in living animals are not isolated, but the same polynucleotides or polypeptides separated from some or all of a substance coexisting in nature are isolated, even if they are subsequently reintroduced into nature. Such polynucleotides may be part of a vector and / or may be part of a composition, and such vector or composition may still be isolated in that it is not part of its natural environment.

[0098] In yet another system, expression cassettes associated with the ITR and rep / cap genes are introduced into insect cells by infection with a baculovirus-based vector. In the case of a review of these production systems, generally, for example See Zhang et al., 2009, "Adenovirus-Adeno-Related Virus Hybrid for Large-Scale Recombinant Adeno-Related Virus Production," Human Gene Therapy 20:922-929, the contents of which are incorporated herein by reference in their entirety. Methods for constructing and using these and other AAV production systems are also described in the following U.S. Patents, the contents of which are incorporated herein by reference in their entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065. Generally, for example , Grieger & Samulski, 2005, "Adeno-associated viruses as gene therapy vectors: vector development, production, and clinical application", Adv. Biochem. Engin / Biotechnol. 99: 119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology", J. Gene Med. 10:717-733; and the references cited below, reference , each of these is incorporated herein by reference in its entirety.

[0099] The method used to construct any embodiment of the present invention is known to those skilled in nucleic acid manipulation and includes genetic engineering, recombinant engineering, and synthesis techniques. for example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012); referenceSimilarly, methods for generating rAAV virions are well known, and the selection of a suitable method is not limited to the present invention. See, for example, K. Fisher et al., (1993) J. Virol., 70:520-532 and U.S. Patent No. 5,478,745.

[0100] "Plasmid" is generally designated herein by a lowercase p followed by an uppercase letter and / or a number, in accordance with standard naming conventions familiar to those skilled in the art. Many plasmids and other cloning and expression vectors that can be used according to the present invention are well known and readily available to those skilled in the art. Furthermore, those skilled in the art can readily construct many other plasmids suitable for use in the present invention. The characteristics, construction, and uses of such plasmids, as well as other vectors in the present invention, will be readily apparent to those skilled in the art from this disclosure.

[0101] In one embodiment, the production plasmid is as described herein or as described in WO2012 / 158757, which is incorporated herein by reference. Various plasmids are known in the art and are available herein for use in the production of rAAV vectors. The production plasmid is cultured in host cells expressing AAV cap and / or rep proteins. In the host cells, each rAAV genome is rescued and packaged into a capsid protein or envelope protein to form an infectious viral particle.

[0102] In one embodiment, the rAAV vector genome is about 3 kilobases (kb) to about 6 kb, about 4.7 kb to about 6 kb, about 3 kb to about 5.5 kb, or about 4.7 kb to 5.0 kb in size. In a specific embodiment, the vector genome is about 5 kb.

[0103] The expression cassette, vector, and plasmid comprise other components that can be optimized for a specific species using techniques known in the art, including codon optimization, for example, as described herein. The components of the cassette, vector, plasmid, and virus or other composition described herein comprise a promoter sequence as part of an expression control sequence. In another embodiment, the promoter is cell-specific. The term "cell-specific" means that a specific promoter selected for a recombinant vector can drive the expression of the SIRT1 coding sequence in a specific cell type. In one embodiment, the promoter is specific for the expression of the transplant gene in ocular cells. In one embodiment, the promoter is specific for the expression of the transplant gene in retinal ganglion cells. In one embodiment, the promoter is specific for the expression of the transplant gene in photoreceptor cells. In another embodiment, the promoter is specific for expression in rods and cones. In another embodiment, the promoter is specific for expression in rods. In another embodiment, the promoter is specific for expression in cones. In one embodiment, the promoter is modified to add one or more restriction sites to facilitate cloning.

[0104] In one embodiment, the hSIRT1 coding sequence is placed under the control of a hybrid chicken β-actin (CBA) promoter, sometimes referred herein as a CAG promoter, such as that shown in SEQ ID NOs. 28, 33, or 35. This promoter consists of a cytomegalovirus (CMV) immediate early enhancer, a proximal chicken β-actin promoter, and optionally, a CBA exon 1 adjacent to an intron 1 sequence.

[0105] In one embodiment, the CMV enhancer is SEQ ID NO: 14

[0106]

[0107] It has the sequence of

[0108] In another embodiment, the CMV enhancer is SEQ ID NO: 32

[0109]

[0110] It has the sequence of

[0111] In one embodiment, the CBA promoter is SEQ ID NO: 15

[0112]

[0113] It has the sequence of

[0114] In one embodiment, CBA exon 1 is SEQ ID NO: 16

[0115]

[0116] It has the sequence of

[0117] In one embodiment, CBA intron 1 is SEQ ID NO: 17

[0118]

[0119] It has the sequence of

[0120] In one embodiment, CBA intron 1 is SEQ ID NO: 38

[0121]

[0122] It has the sequence of

[0123] In another embodiment, the promoter is the human gamma-synuclein gene (SNCG) promoter, e.g., SEQ ID NO: 6.

[0124]

[0125] Also, for example, Chaffiol A et al. A novel promoter allows optogenetic vision restoration with enhanced sensitivity in macaque retinas. Mol Ther. Nov 1, 2017;25(11):2546-2560. doi: 10.1016 / j.ymthe.2017.07.011. Epub July 20, 2017, see reference.

[0126] In other embodiments, the expression cassettes, vectors, plasmids, and viruses described herein include other suitable transcription initiation, termination, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; TATA sequences; sequences for stabilizing cytoplasmic mRNA; sequences for improving translation efficiency ( in other words It includes a Kozak common sequence; an intron; a sequence that enhances protein stability; and, if necessary, a sequence that enhances the secretion of the encoded product. The expression cassette or vector may not contain any of the elements described herein and may contain one or more.

[0127] Examples of suitable poly-A sequences are, for example , includes synthetic polyA or bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (RGB), or modified RGB (mRGB). In one embodiment, polyA is SEQ ID NO: 9

[0128]

[0129] or Sequence No.: 30

[0130]

[0131] It is a bGH poly A having an arbitrary nucleic acid sequence.

[0132] In one embodiment, the expression cassette, vector, plasmid, and virus described herein comprise a post-transcriptional regulatory element. In one embodiment, the post-transcriptional regulatory element is a Woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). In one embodiment, the WPRE is SEQ ID NO: 8

[0133]

[0134] It has the sequence of

[0135] Examples of suitable enhancers are, for example , includes a CMV enhancer and an SV40 enhancer. In one embodiment, the CMV enhancer is SEQ ID NO: 5

[0136]

[0137] It has the sequence of

[0138] In one embodiment, the CMV enhancer is SEQ ID NO: 29

[0139]

[0140] It has the sequence of

[0141] In one embodiment, the SV40 enhancer is SEQ ID NO: 12

[0142]

[0143] It has the sequence of

[0144] In one embodiment, an SV40 intron, e.g., SEQ ID NO: 7

[0145]

[0146] It includes having the sequence of

[0147] In one embodiment, the expression cassette, vector, plasmid, and virus contain a CMV enhancer, an hSNCG promoter, an SV40 intron, a human SIRT1 coding sequence, a WPRE, and a bGH poly-A. In a specific embodiment, the expression cassette comprises nts 181 through 4717 of SEQ ID NO: 11, or nts 238 through 4717 of SEQ ID NO: 11. In another embodiment, the vector genome comprises an expression cassette coupled by an ITR. In a specific embodiment, the 5' ITR has the nucleic acid sequences nts 1 through nts 130 of SEQ ID NO: 11, and the 3' ITR has the nucleic acid sequences nts 4767 through nts 4896 of SEQ ID NO: 11.

[0148] In one embodiment, the expression cassette, vector, plasmid, and virus contain an SV40 enhancer, an hSNCG promoter, an SV40 intron, a human SIRT1 coding sequence, a WPRE, and a bGH poly-A. In a specific embodiment, the expression cassette comprises nt 181 to 4648 of SEQ ID NO: 13. In another embodiment, the vector genome comprises an expression cassette coupled by an ITR. In a specific embodiment, the 5' ITR has the nt 1 to nt 130 nucleic acid sequence of SEQ ID NO: 13 and the 3' ITR has the nt 4698 to nt 4827 nucleic acid sequence of SEQ ID NO: 13.

[0149] In one embodiment, the expression cassette, vector, plasmid, and virus contain a CMV enhancer, a CBA promoter, exon and intron sequences, a human SIRT1 coding sequence, a WPRE, and a bGH poly-A. In a specific embodiment, the expression cassette contains nt 191 to 4236 of SEQ ID NO: 18 or 193 to 4236 of SEQ ID NO: 18. In another embodiment, the vector genome comprises an expression cassette attached by an ITR. In a specific embodiment, the 5' ITR has the nt 1 to nt 130 nucleic acid sequence of SEQ ID NO: 18 and the 3' ITR has the nt 4286 to nt 4415 nucleic acid sequence of SEQ ID NO: 18.

[0150] In some embodiments, when a WPRE sequence is included in the vector genome, an alternative embodiment without WPRE is considered.

[0151] In another aspect, a method for treating ocular neuropathy and / or restoring visual function in a subject having a need for treatment comprises delivering a vector encoding hSIRT1 (e.g., rAAV) or a cell or composition as described herein to the subject having a need for treatment, as described herein. In one embodiment, a method for treating a subject having ocular neuropathy with the rAAV described herein is provided. In one embodiment, a method for treating optic neuritis in a subject having a need for treatment comprises delivering a vector encoding hSIRT1 (e.g., rAAV) as described herein to the subject having a need for treatment. In a specific embodiment, rAAV is AR0012. In another embodiment, rAAV is AR0013. In another embodiment, rAAV is AR0018. In another embodiment, rAAV is AR0020.

[0152] In another embodiment, a method for treating glaucoma in a subject having a need for treatment comprises delivering a vector encoding hSIRT1 (e.g., rAAV) as described herein to the subject having a need for treatment. In a specific embodiment, rAAV is AR0012. In another embodiment, rAAV is AR0013. In another embodiment, rAAV is AR0018. In another embodiment, rAAV is AR0020.

[0153] In another aspect, a method for preserving retinal ganglion cell (RGC) function in a subject is provided, comprising administering rAAV as described herein. In a specific embodiment, rAAV is AR0012. In another embodiment, rAAV is AR0013. In another embodiment, rAAV is AR0018. In another embodiment, rAAV is AR0020.

[0154] In another embodiment, i) a method for improving or restoring vision or visual acuity, ii) a method for preventing the loss of vision or visual acuity, or iii) a method for slowing or reducing the rate of the loss of vision or visual acuity comprises delivering a vector encoding hSIRT1 (e.g., rAAV) as described herein to a subject requiring treatment. In a specific embodiment, rAAV is AR0012. In another embodiment, rAAV is AR0013. In another embodiment, rAAV is AR0018. In another embodiment, rAAV is AR0020.

[0155] As used in the present method, "administering" means delivering the composition to a target cell. In one embodiment, the present method comprises delivering the composition to a ganglion or other ocular cell by subretinal injection. In another embodiment, intravitreal injection is used on the subject. In another embodiment, supracorbital administration is used. In another embodiment, subretinal injection is used on the subject. In yet another method, intravascular injection, such as injection through the eyelid vein, may be used. Further methods of administration may be selected by those skilled in the art as given in the present disclosure.

[0156] “Administering” or “Route of administration” means the delivery of the composition described herein to a subject, with or without a pharmaceutical carrier or excipient. Routes of administration may be combined if desired. In some embodiments, administration is repeated periodically. The pharmaceutical compositions described herein are designed for delivery to subjects requiring treatment by any suitable route or a combination of different routes. In some embodiments, delivery directly to the eye (optional ocular delivery, subretinal injection, intraretinal injection, intravitreal, supracoradiochoroidal, or via the local area) or delivery via a systemic route, e.g., intravascular, intra-arterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral administration routes are utilized. The nucleic acid molecules, expression cassettes, and / or vectors described herein may be delivered as a single composition or multiple compositions. Optionally, two or more different AAVs, or multiple viruses, may be delivered [see, e.g., WO20 2011 / 126808 and WO 2013 / 049493]. In another embodiment, several viruses are different replication-defective viruses, either alone or in combination with proteins ( for example, It may contain AAV and adenovirus.

[0157] According to the present invention, pharmaceutical compositions comprising rAAV or cells and pharmaceutically acceptable carriers or excipients are also provided herein. In a preferred embodiment, the pharmaceutical composition is suitable for administration to human subjects and contains a therapeutically effective amount of rAAV according to the present invention. The pharmaceutical compositions described herein are designed for delivery to subjects requiring treatment by any suitable route or a combination of different routes. In some embodiments, these delivery means are designed to avoid direct systemic delivery of suspensions containing the AAV composition(s) described herein. Suitably, this may have the advantage of reduced dose, reduced toxicity, and / or reduced undesirable immune responses to the AAV and / or transplanted gene products when compared to systemic administration. The pharmaceutical compositions described herein are suitable for intraocular injection.

[0158] In a further embodiment, these nucleic acid sequences, vectors, expression cassettes, and rAAV viral vectors of the present invention are useful in pharmaceutical compositions that also comprise pharmaceutically acceptable carriers, excipients, buffers, diluents, surfactants, preservatives, and / or adjuvants, etc. Such pharmaceutical compositions are used to express SIRT1 in host cells through delivery by such recombinant engineered AAV or artificial AAV.

[0159] To prepare these pharmaceutical compositions containing nucleic acid sequences, vectors, expression cassettes, and rAAV viral vectors, the sequences or vectors or viral vectors are preferably evaluated for contamination by conventional methods and then formulated into pharmaceutical compositions suitable for administration to the eye. Such formulations include the use of pharmaceutically and / or physiologically acceptable vehicles or carriers, particularly suitable for administration to the eye, such as buffered saline or other buffers, e.g., HEPES, and, optionally, other medicinal preparations, formulations, pharmaceutical preparations, stabilizing preparations, buffers, carriers, adjuvants, diluents, surfactants, or excipients, etc., to maintain the pH at an appropriate physiological level. For injection, the carrier will typically be a liquid. Exemplary physiologically acceptable carriers include sterile, pyrogenic, pyrogen-removed water and sterile, pyrogenic, phosphate-buffered saline. Various such known carriers are provided in U.S. Patent Publication No. 7,629,322, incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is an equilibrium salt solution. In one embodiment, the carrier comprises a tween. If the virus needs to be stored for a long period, it may be frozen in the presence of glycerol or Tween20.

[0160] In certain embodiments, a pharmaceutically acceptable aqueous solution comprising an aqueous suspension and a therapeutically effective amount of recombinant adeno-associated virus (rAAV) is provided. The aqueous solution is suitable for administration to human patients. In certain embodiments, the suspension further comprises a surfactant, a preservative, and / or a buffer dissolved in the aqueous suspension. In certain embodiments, for administration to human patients, rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or a mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH in the range, for example, pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. As the pH of cerebrospinal fluid is about 7.28 to about 7.32, a pH within this range may be desirable for intrathecal delivery; On the other hand, for intravitreal or subretinal delivery, a pH of 6.8 to about 7.2 may be desirable. However, other pH values ​​within the widest range and these sub-ranges may be selected for other delivery routes.

[0161] A suitable surfactant, or a combination of surfactants, may be selected from non-toxic non-ionic surfactants. In one embodiment, a difunctional block copolymer surfactant terminated at a primary hydroxyl group, for example, Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH and an average molecular weight of 8400, is selected. Other surfactants and other poloxamers may be selected, namely, nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) adjacent to two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains poloxamers. These copolymers are generally named by appending a three-digit number following the letter "P" (in the case of poloxamers): the first two digits x 100 provide the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 provides the percentage polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount of up to about 0.0005% to about 0.001% of the suspension.

[0162] In one example, the formulation may contain a buffered saline solution comprising, for example, one or more of sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., magnesium sulfate·7H2O), potassium chloride, calcium chloride (e.g., calcium chloride·2H2O), dibasic sodium phosphate, and mixtures thereof in water. In another embodiment, the formulation may contain one or more permeation enhancers. Examples of suitable permeation enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0163] In another embodiment, the composition comprises a carrier, a diluent, an excipient, and / or an adjuvant. A suitable carrier can be readily selected by a person skilled in the art in terms of the indication for which the transport virus is driven. For example, one suitable carrier comprises a saline solution that can be formulated into various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers comprise sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer / carrier must include a component that does not interfere with in vivo rAAV binding activity while preventing rAAV from adhering to the infusion tube.

[0164] Optionally, the composition of the present invention may contain other conventional pharmaceutical components, such as preservatives or chemical stabilizers, in addition to rAAV and carrier(s). Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0165] According to the present invention, the composition may comprise a pharmaceutically acceptable carrier, such as that defined above. Suitably, the composition described herein comprises an effective amount of one or more AAVs suspended in a pharmaceutically acceptable carrier and / or mixed with a suitable excipient, designed for delivery to a subject via injection, an osmotic pump, an intrathecal catheter, or delivery via another device or route. In one example, the composition is formulated for intravitreal delivery. In one example, the composition is formulated for subretinal delivery. In one example, the composition is formulated for choroidal delivery.

[0166] In one exemplary specific embodiment, the composition of the carrier or excipient contains 180 mM NaCl, 10 mM NaPi, and pH 7.3 with 0.0001% to 0.01% Pluronic F68 (PF68). The exact composition of the saline component of the buffer is in the range of 160 mM to 180 mM NaCl. Optionally, a different pH buffer (potentially HEPES, sodium bicarbonate, TRIS) is used instead of the specifically described buffer. More alternatively, a buffer containing 0.9% NaCl is useful.

[0167] According to the present invention, a composition is formulated to deliver a therapeutically effective dose of rAAV to a subject. In the case of an AAV viral vector, the quantification of genomic copies (“GC”), vector genomes (“VG”), or viral particles may be used as a measure of the dose contained in the formulation or suspension. Any method known in the art may be used to determine the genomic copy (GC) number of the replication-defective viral composition of the present invention. One method for performing AAV GC number titration is as follows: A purified AAV vector sample is first treated with DNase to remove unencapsulated AAV genomic DNA or contaminating plasmid DNA from the production process. DNase-resistant particles are then heat-treated to release the genome from the capsid. The released genome is then quantified by real-time PCR using a primer / probe set that targets a specific region of the viral genome (usually a poly-A signal). In another method, an effective dose of recombinant adeno-associated virus carrying a nucleic acid sequence encoding a SIRT1 coding sequence is measured as described in SK McLaughlin et al., 1988 J. Virol., 62:1963, the entirety of which is included by reference.

[0168] In one embodiment, the cells are prepared in suitable cell culture (e.g., HEK 293) cells. The method for preparing a gene therapy vector described herein comprises methods well known in the art, such as the generation of plasmid DNA used in the production of the gene therapy vector, the generation of a vector, and the purification of the vector. In some embodiments, the gene therapy vector is an AAV vector, and the generated plasmids are an AAV cis-plasmid encoding the AAV genome and the gene of interest, an AAV trans-plasmid containing the AAV rep and cap genes, and an adenovirus helper plasmid. The vector generation process may include method steps, such as the initiation of cell culture, passage of cells, seeding of cells, transfection of cells with plasmid DNA, exchange of medium after transfection with serum-free medium, and harvesting of the vector-containing cells and culture medium. The harvested vector-containing cells and culture medium are referred herein as unpurified cell harvest. In yet another system, the gene therapy vector is introduced into insect cells by infection with a baculovirus-based vector. For a review of these production systems, generally, see, for example, Zhang et al., 2009, "Adenovirus-Adeno-Related Virus Hybrid for Large-Scale Recombinant Adeno-Related Virus Production", Human Gene Therapy 20:922-929, the contents of which are incorporated herein by reference in their entirety. Methods for constructing and using these and other AAV production systems are also described in the following U.S. Patents, the contents of which are incorporated herein by reference in their entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0169] Unpurified cell harvesters can then be subjected to method steps, such as concentration of vector harvesters, dialysis filtration of vector harvesters, microfluidization of vector harvesters, nuclease digestion of vector harvesters, filtration of microfluidized intermediates, adjustment by chromatography, adjustment by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to produce a bulk vector.

[0170] Two-step affinity chromatography purification at high salt concentrations followed by anion exchange resin chromatography is used to purify vector drug products and remove empty capsids. These methods are described in more detail in International Patent Publication No. WO 2017 / 160360, which is incorporated herein by reference. A purification method for AAV8, International Patent Publication No. WO 2017 / 100676, a purification method for rh10, International Patent Publication No. WO 2017 / 100704, and a purification method for AAV1, International Patent Publication No. WO 2017 / 100674 are all incorporated herein by reference.

[0171] To calculate the empty and filled particle content, selected samples ( for example In the example herein, the VP3 band volume for the iodixanol gradient (purified formulation) where GC # = particle # is plotted against the loaded GC particles. The generated linear equation (y = mx+c) is used to calculate the number of particles from the band volume of the test peak. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to provide particles (pt) / mL. pt / mL divided by GC / mL provides the ratio of particles to genome copies (pt / GC). pt / mL minus GC / mL provides empty pt / mL. Empty pt / mL divided by pt / mL and multiplied by 100 provides the percentage of empty particles.

[0172] In general, methods for verifying genome-packaged AAV vector particles and empty capsids are known in the art. For example, Grimm et al. Gene Therapy (1999) 6:1322-1330; see Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsid, the present method comprises applying a treated AAV stock, consisting of any gel capable of separating three capsid proteins, e.g., a gradient gel containing 3 to 8% Tris-acetate in buffer, to SDS-polyacrylamide gel electrophoresis, then running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. An anti-AAV capsid antibody is then used as a primary antibody that binds to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used, comprising a means capable of binding to the primary antibody and detecting the binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound thereto, most preferably a sheep anti-mouse IgG antibody covalently bound to mustard bean peroxidase. A method for detecting the binding, preferably a detection method capable of detecting radioisotope emission, electromagnetic radiation, or a colorimetric change, most preferably a chemiluminescent detection kit, is used to semi-quantitatively determine the binding between the primary antibody and the secondary antibody. For example, in the case of SDS-PAGE, a sample may be obtained from the column fraction and a reducing agent ( for example The capsid protein can be heated in an SDS-PAGE loading buffer containing , DTT, and the pre-template gradient polyacrylamide gel ( for example,It was digested on Novex. Silver staining can be performed using SilverXpress (Invitrogen, CA) or other suitable staining methods, namely SYPRO Ruby or Coomassie staining, according to the manufacturer's instructions. In one embodiment, the concentration of AAV vector genome (vg) in the column fraction can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After nuclease inactivation, the sample is further diluted and amplified using a TaqMan™ fluorescence probe specific to the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (critical cycle, Ct) is measured for each sample using an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the vector genome titer by normalizing it to the Ct value of the plasmid standard curve. Endpoint assays based on digital PCR can also be used.

[0173] In one embodiment, an optimized q-PCR method utilizing a broad-spectrum serine protease, e.g., Protease K (commercially available from Qiagen, e.g.), is used. More specifically, the optimized q-PCR genomic titer assay is similar to a standard assay, except that after DNase I digestion, the sample is diluted with Proteinase K buffer, treated with Proteinase K, and then heat-inactivated. Suitablely, the sample is diluted with Proteinase K buffer in an amount equal to the sample size. The Proteinase K buffer may be concentrated by more than twofold. Typically, the Proteinase K treatment is about 0.2 mg / mL, but can be varied from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally performed at about 55°C for about 15 minutes, but can be performed at a lower temperature (e.g., from about 37°C to about 50°C) for a longer period (e.g., from about 20 minutes to about 30 minutes), or at a higher temperature (e.g., up to about 60°C) for a shorter period (e.g., from about 5 to 10 minutes). Similarly, thermal inactivation is generally performed at about 95°C for about 15 minutes, but the temperature can be lowered (e.g., from about 70°C to about 90°C) and the time can be extended (e.g., from about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and applied to TaqMan analysis as described in the standard assay.

[0174] Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genomic titers by ddPCR are described. For example, see M. Lock et al., Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi: 10.1089 / hgtb.2013.131. Epub Feb. 14, 2014.

[0175] As used herein, the term “dosage” may refer to the total dose delivered to the subject during the course of treatment, or the amount delivered in a single unit (or multiple units or divided doses) administration. The pharmaceutical viral composition contains approximately 1.0 x 10⁶ per dose, comprising any integer or decimal amount within the range. 9 GC to about 1.0 x 10 15 It may be formulated in dose units to contain an amount of replication-defective virus carrying a nucleic acid sequence encoding SIRT1 as described herein within the GC range. In one embodiment, the composition comprises at least 1 x 10⁻¹⁰ per dose containing any integer or fractional amount within the range. 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , or 9x10 9 It is formulated to contain GC. In another embodiment, the composition comprises at least 1 x 10 per dose containing any integer or fractional amount within a range. 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 , or 9x10 10 It is formulated to contain GC. In another embodiment, the composition comprises at least 1 x 10 per dose containing any integer or fractional amount within a range. 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11, or 9x10 11 It is formulated to contain GC. In another embodiment, the composition comprises at least 1 x 10 per dose containing any integer or fractional amount within a range. 12 , 2x10 12 , 3x10 12 , 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 , or 9x10 12 It is formulated to contain GC. In another embodiment, the composition comprises at least 1 x 10 per dose containing any integer or fractional amount within a range. 13 , 2x10 13 , 3x10 13 , 4x10 13 , 5x10 13 , 6x10 13 , 7x10 13 , 8x10 13 , or 9x10 13 It is formulated to contain GC. In another embodiment, the composition comprises at least 1 x 10 per dose containing any integer or fractional amount within a range. 14 , 2x10 14 , 3x10 14 , 4x10 14 , 5x10 14 , 6x10 14 , 7x10 14 , 8x10 14 , or 9x10 14 It is formulated to contain GC. In another embodiment, the composition comprises at least 1 x 10 per dose containing any integer or fractional amount within a range. 15 , 2x10 15 , 3x10 15 , 4x10 15 , 5x10 15 , 6x10 15 , 7x10 15 , 8x10 15 , or 9x10 15It is formulated to contain GC. In one embodiment, for human application, the dose is 1 x 10⁻¹⁰ per dose comprising any integer or fractional amount within a range. 10 to about 1x10 12 The GC range may be. All dosages may be measured by any known method, including those measured by oqPCR or digital droplet PCR (ddPCR) as described in M. Lock et al., Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi: 10.1089 / hgtb.2013.131, incorporated herein by reference.

[0176] In one embodiment, an aqueous suspension suitable for administration to a patient with an ocular disorder is provided. The suspension contains approximately 1 x 10⁶ per eye of the recombinant adeno-associated virus (rAAV) described herein, which is useful as a therapeutic agent for the ocular disorders described herein. 9 GC or viral particles to about 1 x 10 13 It includes GC or viral particles and an aqueous suspension. In one embodiment, the eye disorder is optic neuritis. In another embodiment, the eye disorder is glaucoma.

[0177] It may also be desirable to administer multiple "booster" doses of the pharmaceutical composition of the present invention. For example, depending on the duration of the transplanted gene within ocular target cells, booster doses may be delivered at 6-month intervals or annually after the first administration. The fact that AAV-neutralizing antibodies are not produced by the administration of the rAAV vector should allow for additional booster administration.

[0178] The dosage of such boosters and the need for them may be monitored by the attending physician, for example, using the retinal and visual function tests and visual behavior tests described in the examples below. Other similar tests may be used to determine the condition of the treated subject over time. The selection of appropriate tests may be carried out by the attending physician. More alternatively, the method of the present invention may also involve the injection of larger volumes of the virus-containing solution in single or multiple injections to allow for a level of visual function close to that found in a wild-type retina.

[0179] In another embodiment, the pharmaceutical composition of the present invention is administered only once, that is, without the need for repeated administration.

[0180] In another embodiment, the amounts of the vector, virus, and replication-defective virus described herein carrying a codon-optimized nucleic acid sequence encoding SIRT1 are about 1.0 x 10⁶ per eye, comprising any integer or decimal amount within the range. 7 VG to about 1.0 x 10 per eye 15 It is a range of VG. In one embodiment, the amount thereof is at least 1x10 per eye, comprising all integer or fractional amounts within the range. 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , or 9x10 7 It is VG. In one embodiment, the amount thereof is at least 1x10 per eye, comprising all integer or fractional amounts within a range. 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8, or 9x10 8 It is VG. In one embodiment, the amount thereof is at least 1x10 per eye, comprising all integer or fractional amounts within a range. 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , or 9x10 9 It is VG. In one embodiment, the amount thereof is at least 1x10 per eye, comprising all integer or fractional amounts within a range. 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 , or 9x10 10 It is VG. In one embodiment, the amount thereof is at least 1x10 per eye, comprising all integer or fractional amounts within a range. 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11 , or 9x10 11 It is VG. In one embodiment, the amount thereof is at least 1x10 per eye, comprising all integer or fractional amounts within a range. 12 , 2x10 12 , 3x10 12 , 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 , or 9x10 12 It is VG. In one embodiment, the amount thereof is at least 1x10 per eye, comprising all integer or fractional amounts within a range.13 , 2x10 13 , 3x10 13 , 4x10 13 , 5x10 13 , 6x10 13 , 7x10 13 , 8x10 13 , or 9x10 13 It is VG. In one embodiment, the amount thereof is at least 1x10 per eye, comprising all integer or fractional amounts within a range. 14 , 2x10 14 , 3x10 14 , 4x10 14 , 5x10 14 , 6x10 14 , 7x10 14 , 8x10 14 , or 9x10 14 It is VG. In one embodiment, the amount thereof is at least 1x10 per capacity comprising all integer or fractional amounts within a range. 15 , 2x10 15 , 3x10 15 , 4x10 15 , 5x10 15 , 6x10 15 , 7x10 15 , 8x10 15 , or 9x10 15 It is VG. In a preferred embodiment, the method is 1 x 10 per eye per dose. 9 to about 1x10 13 It involves administering a dose in the VG range. In another embodiment, the method involves the delivery of a vector in an aqueous suspension. In another embodiment, the method involves 1 x 10⁶ per volume of about or at least 150 microliters. 9 Up to 1 x 10 13The method includes administering rAAV as described herein in the dosage of GC, thereby restoring visual function in the subject. All dosages may be measured by any known method, including, for example, by oqPCR or digital droplet PCR (ddPCR) as described in M. Lock et al., Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi: 10.1089 / hgtb.2013.131, incorporated herein by reference.

[0181] These above doses may be administered in various volumes of carrier, excipient, or buffer formulations ranging from about 25 to about 1000 microliters, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume of the carrier, excipient, or buffer is at least about 25 μL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 75 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In yet another embodiment, the volume is about 225 μL. In yet another embodiment, the volume is about 250 μL. In yet another embodiment, the volume is about 275 μL. In yet another embodiment, the volume is about 300 μL. In yet another embodiment, the volume is about 325 μL. In yet another embodiment, the volume is about 350 μL. In yet another embodiment, the volume is about 375 μL. In yet another embodiment, the volume is about 400 μL. In yet another embodiment, the volume is about 450 μL. In yet another embodiment, the volume is about 500 μL. In yet another embodiment, the volume is about 550 μL. In yet another embodiment, the volume is about 600 μL. In yet another embodiment, the volume is about 650 μL. In yet another embodiment, the volume is about 700 μL. In yet another embodiment, the volume is about 800 μL. In yet another embodiment, the volume is about or at least 100 μL. In another embodiment, the volume is about 100 to 250 μL. In another embodiment, the volume is about 150 to 800 μL. In another embodiment, the volume is about 700 to 1000 μL.In another embodiment, the volume is about 250 to 500 μL.

[0182] In one embodiment, the viral construct is at least 1 x 10⁻⁶ in a volume of about 1 μL to about 3 μL for small animal subjects, e.g., mice. 9 to about at least 1x10 11 It can be administered in GC doses. For large veterinary subjects with eyes approximately the same size as human eyes, the higher human dosages and volumes stated above are useful. For example, for a discussion of best practices regarding the administration of the substance to various veterinary animals, see Diehl et al., J. Applied Toxicology, 21:15-23 (2001). This literature is incorporated herein by reference.

[0183] It is desirable to utilize the lowest effective concentration of the virus or other delivery vehicle to reduce the risk of undesirable effects, such as toxicity, retinal dysplasia, and detachment. Further dosages within this range may be selected by the attending physician, taking into account the subject being treated, preferably the human physical condition, the subject's age, and the degree of development of ocular pathology and disorders, if progressive.

[0184] A further aspect described herein is a method for treating or preventing optic neuropathy in a subject in need of treatment, comprising administering rAAV, cells, or a pharmaceutical composition to a subject according to the present invention. In a preferred embodiment, the optic neuropathy is optic neuritis or glaucoma. In a preferred embodiment, the subject to be treated is a mammalian subject, preferably a human subject. In one embodiment, rAAV suspended in a physiologically compatible carrier, diluent, excipient, and / or adjuvant carrying a SIRT1 sequence may be administered to a preferred subject, including a human subject. The method comprises administering any of a nucleic acid sequence, an expression cassette, an rAAV genome, a plasmid, a vector, or an rAAV vector, or a composition containing these, to a subject in need of treatment. In one embodiment, the composition is delivered subretinally. In another embodiment, the composition is delivered into the vitreous humor. In yet another embodiment, the composition is delivered using a combination of administration routes suitable for the treatment of optic neuropathy or optic neuritis, and may also include administration through the eyelid vein or other intravenous vessels or conventional administration routes.

[0185] The method of the present invention delivers a therapeutically effective dose of rAAV to a subject. For use in these methods, the volume and viral titer of each dose are determined individually, as further described herein, and may be the same or different from other treatments performed on the same or opposite eye. The dosage, administration, and method of use may be determined by the attending physician in consideration of the teachings of this specification. In one embodiment, the composition is administered in a single dose selected from those listed above to the diseased eye. In another embodiment, the composition is administered as a single dose selected from those listed above to two diseased eyes, either simultaneously or sequentially. Sequential administration may imply a time gap between administrations from one eye to another at intervals of minutes, hours, days, weeks, or months. In another embodiment, the method comprises administering the composition to the eye in two or more doses (e.g., divided doses). In another embodiment, multiple injections are performed on different parts of the same eye. In another embodiment, a second administration of rAAV containing a selected expression cassette (e.g., a SIRT1-containing cassette) is performed at a later time. This time may be weeks, months, or years after the first administration. In one embodiment, this second administration is performed with rAAV having a different capsid from the rAAV from the first administration. In another embodiment, the rAAV from the first and second administrations has the same capsid.

[0186] In a further embodiment, the compositions described herein may be delivered as a single composition or multiple compositions. Optionally, two or more different AAVs, or multiple viruses, may be delivered [ for example , WO 2011 / 126808 and WO 2013 / 049493, reference ]. In another embodiment, several viruses are different replication-defective viruses ( for example,It may contain AAV and adenovirus.

[0187] In certain embodiments of the present invention, it is desirable to perform non-invasive retinal imaging and functional studies to identify the area of ​​ganglion cells to be targeted for therapy as well as to examine the efficacy of the treatment. In these embodiments, clinical diagnostic tests are used to determine the precise location(s) for one or more subretinal injections. These tests may include electroretinography (ERG), visual field testing, topography of retinal layers and measurement of their thickness using confocal scanning laser ophthalmoscopy (cSLO) and optical coherence tomography (OCT), topography of cone density using adaptive optics (AO), functional eye examination, multi-electrode array (MEA), pupillary photoreaction, etc., depending on the species of the subject to be treated, their physical condition and health, and the dosage. In terms of imaging and functional studies, in some embodiments of the present invention, one or more injections are performed in the same eye to target different areas of the diseased eye. The volume and viral titer of each injection are determined individually, as further described herein, and may be the same or different from other injections performed in the same or opposite eye. In another embodiment, a single, large-volume injection is administered to treat the entire eye. In one embodiment, the volume and concentration of the rAAV composition are selected so that only the area of ​​damaged ocular cells is affected. In another embodiment, the volume and concentration of the rAAV composition are larger amounts to reach a wider portion of the eye, including non-damaged ganglion cells.

[0188] In another embodiment, the method comprises performing additional studies, e.g., functional and imaging studies, to determine the efficacy of the treatment. In the case of tests in animals, these tests include evaluation of retinal and visual function via electroretinography (ERG) to examine rod and cone photoreceptor function, optic nystagmus, pupillary measurement, water maze test, light-to-dark preference test, optical coherence tomography (to measure the thickness of various layers of the retina), histology (immunofluorescence to document retinal thickness, nuclear arrangement of the outer layer, and transplant gene expression, cone photoreceptor count, and staining of retinal cross-sections with peanut agglutinin to identify cone photoreceptor shells).

[0189] Specifically, for human subjects, after administration of the dosage of the composition described herein, the subject is tested for the efficacy of the treatment using electroretinography (ERG) to test rod and cone photoreceptor function, pupillometry visual acuity, contrast sensitivity color vision test, visual field test (Humphrey field / Goldman field), visual field mobility test (obstacle course), and reading speed test. Other useful post-treatment efficacy tests to which the subject is exposed after treatment with the pharmaceutical composition described herein include functional magnetic resonance imaging (fMRI), full-field photosensitivity test, retinal structural studies including optical coherence tomography, fundus photography, fundus autofluorescence, adaptive optical laser scanning ophthalmoscopy, mobility test, reading speed and accuracy test, microvisametry and / or ophthalmoscopy. These and other efficacy tests are described by reference in U.S. Patent No. 8,147,823; and co-pending international patent application publication WO 2014 / 011210 or WO 2014 / 124282.

[0190] In one embodiment of the method described herein, a single intraocular delivery of the composition as described herein, for example, delivery of rAAV according to the present invention, is useful for treating optic neuropathy, preferably optic neuritis or glaucoma in a subject. In another embodiment of the method described herein, a single intraocular delivery of the composition as described herein, for example, delivery of rAAV according to the present invention, is useful for treating optic neuropathy, preferably optic neuritis or glaucoma in a subject at risk. In a specific embodiment, rAAV is AR0012. In another embodiment, rAAV is AR0013. In another embodiment, rAAV is AR0018. In another embodiment, rAAV is AR0020.

[0191] Thus, in one embodiment, the composition is administered before the onset of the disease. In another embodiment, the composition is administered before the onset of visual impairment or loss. In another embodiment, the composition is administered after the onset of visual impairment or loss. In yet another embodiment, the composition is administered when less than 90% of the RGC is functioning or remaining compared to the non-affected eye.

[0192] In another embodiment, the composition is re-administered at a later date. Optionally, more than one re-administration is permitted. Such re-administration may be performed as described herein with the same type of vector, with a different viral vector, or through non-viral delivery. In one embodiment, the vector is re-administered to the patient to a different part of the retina that was initially injected. In one embodiment, the vector is re-administered to the patient to the same part of the retina that was initially injected.

[0193] In yet another embodiment, any of the methods described above are performed in combination with another or secondary therapy. The secondary therapy may be any currently known or yet unknown therapy that helps prevent, stop, or improve any of these mutations or defects or associated effects. The secondary therapy may be administered before, simultaneously with, or after the administration of the composition described above. In one embodiment, the secondary therapy comprises a non-specific approach for maintaining the health of retinal cells, such as the administration of neurotrophic factors, antioxidants, or anti-apoptotic agents. The non-specific approach is achieved through the injection of proteins, recombinant DNA, recombinant viral vectors, stem cells, fetal tissue, or genetically modified cells. The latter may include genetically modified cells that are encapsulated.

[0194] In one embodiment, a method for generating recombinant rAAV comprises obtaining a plasmid containing an AAV expression cassette as described above and culturing packaging cells carrying the plasmid in the presence of sufficient viral sequences to package the AAV viral genome into an infectious AAV envelope or capsid. A specific method for generating an rAAV vector may be used to generate an rAAV vector capable of delivering SIRT1 coding sequences from the expression cassette and genome described above and in the examples below.

[0195] In certain embodiments of the present invention, the subject has the optic neuropathy or optic neuritis or other ocular condition described herein, which is designed to be treated by the components, compositions, and methods of the present invention. As used herein, the term “subject” means a mammal, including humans, veterinary or farm animals, livestock or pets, and generally animals used in clinical research. In one embodiment, the subject of these methods and compositions is a human. Other suitable subjects include, without limitation, murine, rat, dog, cat, pig, cattle, sheep, non-human primates, and others. As used herein, the term “subject” is used interchangeably with “patient.”

[0196] As used herein, the terms “treatment” or “to treat” are defined as including administering one or more of the compounds or compositions described herein to a subject for the purpose of improving one or more of the symptoms of the ocular disorders described herein. “Treatment” may thus include one or more of the following: reducing the onset or progression of the ocular disorders described herein, preventing the disease, reducing the severity of disease symptoms, or delaying their progression, including the progression of blindness, eliminating disease symptoms, delaying the onset of the disease, or monitoring the progression of the disease or the efficacy of the therapy in a given subject.

[0197] It should be noted that the term "one" or "one" refers to one or more. As such, the terms "one" (or "one"), "one or more," and "at least one" are used interchangeably herein.

[0198] The words "include," "include," and "include" should be interpreted inclusively rather than exclusively. The words "constitute," "constituted," and variants thereof should be interpreted inclusively rather than exclusively. Although various embodiments are presented in this specification using the language of "include," under other circumstances, related embodiments are also intended to be interpreted and described using the language of "constituted by" or "essentially composed by."

[0199] As used herein, "disease," "disorder," and "pathological condition" are used interchangeably to denote an abnormal state in a subject.

[0200] As used herein, the terms “about” or “~” mean a variability of 10% from the given reference unless otherwise specified.

[0201] The term “modulation” or variation thereof refers to the ability of a composition to inhibit one or more components of a biological pathway as used herein.

[0202] Unless otherwise defined in this specification, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art by reference to the published text, which provides a general guide to many of the terms used in this application.

[0203]

[0204]

[0205] Examples

[0206] The following examples are merely illustrative and are not intended to limit the invention.

[0207] Example 1: Material and Method

[0208] animal

[0209] C57Bl / 6J mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and housed on a 12-hour light / dark cycle. Two male and female mice were used without discrimination in all experiments. Mice were housed at the University of Pennsylvania animal facility in compliance with the ARVO statement on the use of animals in ophthalmic and vision research, as well as institutional and federal regulations.

[0210] AAV Vector Creation and Production

[0211] The following AAV vectors were produced and packaged by the Research Vector Core of the Center for Advanced Retina and Ocular Therapeutics at the University of Pennsylvania: AAV2.CMV.CBA.eGFP (i.e., V2.G), AAV2.hSNCG.eGFP (i.e., V3.G), AAV2.hSNCG.eGFP.WPRE (i.e., V4.G), AAV2.hSNCG.iSV40.eGFP.WPRE (i.e., V5.G), AAV2.eCMV.hSNCG.iSV40.eGFP.WPRE (i.e., V6.G), AAV2.iSV40.hSNCG.iSV40.eGFP.WPRE (i.e., V7.G), AAV2.Arc.hSNCG.iSV40.eGFP.WPRE (i.e., V8.G), AAV2.CMV.CBA.hSIRT1 (i.e., AR0020), AAV2.CMV.CBA.hSIRT1.WPRE (i.e., AR0018), AAV2.eCMV.hSNCG.iSV40.hSITRT1.WPRE (i.e., AR0012), and AAV2.eSV40.hSNCG.iSV40.hSITRT1.WPRE (i.e., AR0013). The original proviral vector consisted of a CMV enhancer / CBA promoter derived from the Invivogen pDRIVE CAG plasmid (San Diego, CA, USA), or codon-optimized SNCG (gamma-synuclein promoter) 34, cDNA encoding enhanced GFP (eGFP) protein, WPRE elements (i.e., nucleotides 1093-1685, GenBank deposit number J04514), and bovine growth hormone (bGH) polyadenylation signal. A human (h)SIRT1 (transcript variant 1) cDNA clone was obtained from OriGene (Rockville, MD, USA). An AAV expression cassette was lateralized to an AAV2 inversion terminal repeat. The sequence was amplified with DNA polymerase (New England Biolabs, Ipswich, MA, USA) and cloned into an AAV expression plasmid using a commercial cloning kit (Clontech Laboratories, Mountain View, CA, USA).All AAV vectors were packaged using the previously described method and purified with a CsCl gradient of 35.

[0212] Alternatively, the AAV8BP2 capsid plasmid from the inventors' previous study 25 was used as indicated. The vector was stored in 0.001% Pluronic F-68 at -80°C until immediately before use, and then diluted to the concentrations indicated in the text.

[0213] Intravitreal injection of AAV vector

[0214] Four-week-old mice were anesthetized by isoflurane inhalation. Proparacaine (0.5% alkyne; Alcon Laboratories, Belgium) was applied topically for local anesthesia. A small incision was made at the limbus using a 33½-gauge needle. A 10-µL Hamilton syringe (701 RN; Hamilton Company, Reno, NV, USA) attached to a 33-gauge blunt-tipped needle was inserted into the vitreous cavity 1 mm posterior to the serration to avoid lens damage. 2 µL of the AAV product (at the concentration indicated in the text) was injected into each eye. The injection was administered slowly to allow for the diffusion of the viral solution. The vehicle-treated eye was injected with an equivalent volume of vector dilution buffer (0.001% Pluronic F68 in PBS).

[0215] Vivid retinal fundus imaging

[0216] In vivo imaging was performed using a confocal scanning laser ophthalmoscope to visualize GFP-positive cells following intravitreal injection of an AAV vector expressing an eGFP reporter. Mice were anesthetized systemically with xylazine and ketamine and locally with 0.5% proparacaine eye drops. The pupils were dilated with one drop of 0.5% tropamide and one drop of 2.5% phenylephrine hydrochloride. Ophthalmic gel and custom-made polymethyl methacrylate planocontact lenses were used to maintain corneal hydration and improve image quality. Fluorescence images were then acquired using a 488 nm excitation laser and a bandpass filter at 505 to 545 nm.

[0217] Immunohistochemistry and quantification of eGFP or hSIRT1-expressing cells and RGC density

[0218] Mouse eyes were enucleated and fixed in 4% paraformaldehyde at 4°C for 30 minutes. The retinas were incised, translucent, and blocked with 2% Triton X-100, 10% normal donkey serum, and PBS. For RGC and hSIRT1 staining, tissues were incubated at 4°C with a 1:1000 diluted rabbit anti-Brn3a antibody (Synaptic systems, Cat# 411 003) and a 1:1000 diluted goat anti-hSIRT1 antibody (ProSci, Poway, CA, USA). The retinas were then washed three times and incubated at room temperature for 1 hour with secondary antibody solutions containing donkey anti-rabbit AlexaFluor 488 and anti-goat AlexaFluor 594 (1:1000 dilutions, respectively). After several washes, the retina was mounted on a glass slide in aqueous mounting medium (SouthernBiotech, Birmingham, AL, USA), covered with a coverslip, and sealed. Immunofluorescence was detected using a Nikon fluorescence microscope. Retinal images were taken at 12 standard fields of view (1 / 6, 3 / 6, and 5 / 6 of the retinal radius from the center of the retina in each quadrant) using a 40x objective lens. Total RGCs counted in the 12 fields of view were recorded in the experimental and control groups using ImageJ software (http: / / imagej.nih.gov / ij / ; available in the public domain by the National Institutes of Health, Bethesda, Maryland, USA). Mice injected with an AAV vector expressing an eGFP reporter were stained with anti-Brn3a antibody alone for RGC colocalization with the eGFP signal. The number of retinal eGFP+ and / or Brn3+ cells was determined as described above for Brn3a+ SIRT1+ cells.

[0219] Quantitative real-time PCR (qRT-PCR) for hSIRT1 expression

[0220] The retina was excised and immediately flash-frozen in liquid nitrogen. Total RNA was extracted using the RNeasy kit (Qiagen, Valenca, CA, USA) and converted to cDNA using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara Biotechnology, CA, USA). GAPDH and hSIRT1 mRNA were analyzed using a pre-designed master mix containing primers and probes for hSIRT1 or GAPDH obtained from Integrated DNA Technologies (Coralville, Iowa, USA). Relative mRNA levels were determined by calculating Ct and ΔΔCt values.

[0221] Enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of eGFP in retinal lysates

[0222] eGFP protein levels were determined in retinal lysates using a GFP ELISA kit (Abcam, Waltham, MA, USA) according to the manufacturer's instructions.

[0223] Statistical analysis

[0224] Differences among treatment groups regarding the number of Brn3a+, hSIRT1+, and eGFP+ cells were compared using a one-way ANOVA followed by Turkey's test for straight-line significance. Data were entered and analyzed using GraphPad Prism 5.0 software (GraphPad Software Company, Boston, MA, USA). Differences were considered statistically significant at P < 0.05. The data satisfy the assumption of a normal distribution of the test, with variance between the groups being compared.

[0225] Example 2: Results

[0226] AAV transduction potential in RGC

[0227] A synthetic AAV2-derived AAV8BP2 vector was previously designed to provide potent affinity for cells of the inner retina. 25,36 . The infection efficiency of retinal cells using AAV8BP2 and its parent AAV2 vector was evaluated after intravitreal injection into adult mice (Fig. 1a). The viral vector is the CBA-CMV promoter, a potent universal expression cassette. 37 Under control, eGFP markers were delivered to effectively infect retinal cells without inducing retinal detachment or other adverse effects. Increasing amounts of AAV2.CMV.CBA-eGFP or AAV8BP2.CMV.CBA.eGFP [1 x 10 8 , 1 x 10 9 , and 1 x 10 9The vector genome (vg) was delivered to each eye (Fig. 1b). Fundus photography was used to assess the degree of eGFP expression in the retinas of living animals. eGFP fluorescence was detectable in all mice 1 and 2 weeks after injection. An example of an image acquired 2 weeks after viral vector injection is shown in Fig. 1c. Since a dose-dependent increase in eGFP-expressing cells was observed at each time point, the density of eGFP-labeled cells for the two vectors followed a similar trend for the three tested amounts for the two AAV2.CMV.CBA-eGFP and AAV8BP2.CMV.CBA.eGFP vectors. Transduction efficiency was evaluated by double Brn3a immunohistochemical staining and eGFP fluorescence on flat-mounted retinas. Fig. 1d shows an example of a combined fluorescence micrograph of a Brn3a-immunostained retina from mice transduced with AAV2.CMV.CBA-eGFP or AAV8BP2.CMV.CBA.eGFP. Considering the non-selective activity of the universal promoter used, eGFP-positive cells were co-localized with two types of Brn3a-positive and Brn3a-negative cells. The percentage of Brn3a+ eGFP+ cells was determined relative to the total Brn3a+ positive cells averaged across all retinal areas. RGC transduction using the AAV2.CMV.CBA-eGFP vector showed a tendency toward higher transduction for all AAV amounts tested. The difference between the AAV2.CMV.CBA-eGFP and AAV8BP2.CMV.CBA.eGFP vectors was not statistically significant, primarily due to within-group variability in Brn3a-labeled and eGFP-positive cells. 1 x 10⁶ 9 The total infection efficacy of AAV2.CMV.CBA-eGFP and AAV8BP2.CMV.CBA.eGFP at clinically acceptable concentrations of vg / ocular was 25% and 15%, respectively, at 2 weeks post-injection.

[0228] Figures 1e to 1h illustrate the average transduction efficiency of the treatment groups. The data show that both AAV2 and AAV8BP2 serotypes transduce more RGCs at a concentration of 1e10 than at lower doses. AAV2 at a dose of 1e9 shows increased transduction 2 weeks versus 1 week after injection. Since AAV8BP2 does not appear to offer an advantage over AAV2 because its transduction rate is similar or shows a tendency toward deterioration, the AAV2 serotype was selected.

[0229] Time- and dose-dependent transduction potential of AAV2.CMV.CBA-eGFP vector

[0230] Transduction efficiency of AAV2.CMV.CBA-eGFP vector at clinically relevant concentrations of the vector at weeks 1, 2, or 4 post-injection (1 x 10⁻⁶ 9 , 3 x 10 9 , and 1 x 10 10 Further analysis was performed using vg / eye). At each time point, retinal flat mounts were prepared and immunolabeled with the RGC marker, Brn3a (data not shown). Cells with co-localized eGFP and Brn3a signals were counted. RGC transduction efficiency was determined by calculating the percentage of Brn3a+ cells co-expressing eGFP. The minimum transduction efficiency was 1 x 10⁻¹⁰ 9 Observations were made at week 1 in vg / eye. Although the difference was not statistically significant, transduction efficiency was higher at weeks 2 and 4 than at week 1 for all tested concentrations (Fig. 2).

[0231] Optimization of AAV vectors for efficient delivery of transplanted genes to RGCs

[0232] One potential disadvantage of AAV-based gene therapy is the tendency of these vectors to transduce RGCs with low efficiency when injected into the eye, for example, into the vitreous humor. 38Selective and successful transduction of RGCs can be enhanced by incorporating an RGC-specific promoter in front of the transplant gene. A minimal promoter was developed from the human gamma-synuclein gene (hSNCG), which has been shown to drive selective gene expression in RGCs. 26 . The expression of the eGFP reporter was driven by inserting the hSNCG promoter into the AAV2 backbone, and a vector containing a constitutive CMV enhancer and a CBA promoter (3 x 10 9 Expression in eyes injected into the vitreous humor using vg / eye) and in RGCs was compared (Fig. 3a). In the absence of any additional factors, the CBA-CMV promoter showed a tendency toward higher, though not significant, transduction efficiency when compared to the hSNCG promoter (Fig. 3d; AAV vector V2.G big V3.G; 31% and 25%, respectively). Normalized eGFP protein levels determined by ELISA were significantly higher in eyes transfected with vector V2.G compared to vector V3.G (Fig. 3d).

[0233] To improve the transduction efficiency of the hSNCG promoter, a series of recombinant AAV2 vectors were generated in which additional regulatory sequences were introduced as shown in Fig. 3. One vector (V4.G) has an hSNCG promoter that drives a reporter gene lateralized to the 3' end by a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The latter has additional sequences that substantially increase gene expression levels in a transplant-, promoter-, and vector-independent manner. Sis - Contains an action sequence 39 WPREs function by modifying RNA polyadenylation and effluxing mRNA partially bound to the CRM-1-dependent nuclear efflux pathway. 40 Furthermore, since introns can regulate gene expression in eukaryotic cells 41 The inventors of the SV40 intron element (iSV40)42 A vector positioned downstream of the hSNCG promoter (V5.G) was generated. The hSNCG promoter was further modified by placing enhancer elements from the human CMV immediate-early promoter (V6.G construct), SV40 (V7.G construct), or the active-regulatory cytoskeleton (Arc) (V8.G construct) upstream of this vector in the forward direction. These enhancer elements were shown to enhance cell type-specific or universal promoters in different tissues. 43, 44 . AAV vector (3 x 10 9 After intravitreal injection of vg / eye), eGFP fluorescence In vivo It was easily detectable by fundus imaging. eGFP fluorescence was evident in cells across most of the retinal surface upon inclusion of the WPRE sequence in the AAV2 vector (i.e., vector V4.G) (data not shown). However, among all tested vectors, the AAV construct with the hSNCG promoter lateralized to the CMV enhancer and SV40 intron (vector V6.G) showed the highest density of eGFP-expressing cells (p<0.05). The hSNCG promoter lateralized to the Arc enhancer and SV40 intron (i.e., vector V8.G) showed a lower density of eGFP-expressing cells. Immunohistochemical labeling of retinal flat mounts with the Brn3a antibody showed co-localization of eGFP-positive cells with Brn3a in the GCL (inset panel of Fig. 3b). A multiple comparison test of Turkey showed that transduction efficiency using AAV constructs with the hSNCG promoter coupled to SV40 introns and CMV (i.e., V6.G) or SV40 enhancers (i.e., V7.G) was significantly higher when compared to an AAV vector containing hSNCG alone (i.e., V3.G) (Fig. 3c). The average arithmetic mean of transduction efficiency was highest in the group that received the V6.G vector. ELISA-based measurements of eGFP levels in protein lysates confirmed the transduction efficiency levels (Fig. 3d).

[0234] AAV2 serotypes were selected for the construction of a vector containing an RGC-selective plasmid with a variable enhancer, using an eGFP transgene as a reporter to compare expression specificity and efficiency in mice. The construct was administered via intravitreal injection at an MOI of 1E10 vg / ocular In vivo Transduction efficiency was tested and evaluated using eGFP expression by immunohistochemistry and ELISA to quantify it (Figs. 3e and 3f).

[0235] RGC-selective expression of the eGFP reporter gene driven by the hSNCG promoter lateralized by the CMV enhancer and SV40 intron

[0236] To test the selectivity of AAV constructs and the efficiency of transduced RGCs, the inventors performed immunohistochemical staining of retinal cross-sections prepared from mice 2 weeks after intravitreal injection with AAV constructs containing a constitutive CMV-CBA promoter (V2.G), or an hSNCG promoter with or without a CMV enhancer (V6.G) or SV40 enhancer (V7.G). Co-localization of eGFP-positive cells with the Brn3a marker in the ganglion cell layer is illustrated in Fig. 4a. eGFP reporter expression driven by the CMV-CBA promoter (i.e., V2.G) was less selective for RGCs than constructs containing the hSNCG promoter (~62.5%; 13.0 Brn3a+ RGCs out of 20.8 eGFP+ cells per imaged retinal cross-section, n=5) (Fig. 4b). Conversely, eGFP expression driven by the hSNCG promoter and CMV enhancer was highly selective for RGCs (~90.4%; 15.67 Brn3a+ RGCs out of 17.33 eGFP+ cells per imaged retinal cross-section, n=5). Similarly, eGFP expression driven by the hSNCG promoter and SV40 enhancer was highly selective for RGCs (92.2%; 11.8 Brn3a+ RGCs out of 12.8 eGFP+ cells per imaged retinal cross-section, n=5).

[0237] The ability of the top three eGFP-expressing constructs to selectively transduce RGCs was also tested. As expected, the construct containing the SNCG promoter demonstrated highly selective expression within RGCs, whereas the construct containing the CBA promoter was significantly less RGC-selective (Fig. 4c).

[0238] AAV vector with optimal promoter-driven expression of the therapeutic gene hSIRT1

[0239] The maximum genome that can be packaged into an AAV vector is ~4.7 kb. 45A CMV enhancer (0.304 kb)-SNCG promoter (0.953 kb)-SV40 intron DNA fragment (0.097 kb) combined with a poly(A) tail (0.208 kb) and WPRE (0.589 kb) sequence is approximately 2.151 kb in length, so it can still carry up to 2.549 kb of therapeutic gene. An AAV2 vector version containing an SV40 enhancer instead of a CMV enhancer is 2.082 kb in length and can carry up to 2.618 kb of transplanted gene. The inventors generated new AAV vectors (AR0020, AR0012, and AR0013) in which the eGFP reporter gene in the V2.G, V6.G, and V7.G vectors was replaced with a codon-optimized sequence of the hSIRT1 gene (2.244 kb), as shown in FIG. 5a. Because the WPRE element showed a strong tendency for increased reporter gene expression, the inventors also generated a new AAV vector with a WPRE element added to the AR0020 vector (AR0018; FIG. 5a). Mice were administered intravitreal injections of the AR0020, AR0018, AR0012, or AR0013 vectors. After 2 weeks, retinas were harvested from all groups and treated for hSIRT1 expression at protein and mRNA levels. FIG. 5b shows a representative photograph of a flat-mounted retina prepared by immunolabeling with a SIRT1 antibody that recognizes only hSIRT1. With a slightly non-significant trend toward lower efficiency without the WPRE component when measured across the retina and in both the entire retina (Fig. 5c), as well as in the most highly transduced area of ​​the central retina (Fig. 5d), the AAV-mediated hSIRT1 transduction efficiency in RGC was similar between the hSNCG promoter-containing AAV construct and the CMV-CBA-promoter-containing AAV vector-containing WPRE.Similarly, the expression levels of the hSIRT1 gene were not significantly different between retinal lysates from eyes injected with a CMV-CBA promoter-containing vector compared to those injected with an hSNCG promoter-containing vector (Fig. 5e).

[0240] Example 3: Therapeutic Efficacy of SIRT1 Gene Therapy in Experimental Optic Neuropathy

[0241] EAE was induced in 8-week-old C57BL / 6 mice by immunization with MOG peptides, and disease severity was monitored using the EAE ascending paralysis score. This experiment was repeated twice, once with mildly induced EAE disease (Fig. 6a) and twice with moderately severe induced EAE disease (Fig. 6b).

[0242] In Experiment 1 (Figs. 6c to 6e), EAE scores, evaluated on a relative 5-point scale by a shielded investigator, showed that mild ascending paralysis occurred in all treatment groups with no significant difference. Visual loss also did not show a significant difference between treatment groups, similar to previous studies where SIRT1 therapy mediated no visual gain or only small improvement. The primary outcome of RGC survival showed that both AR0012 and AR0018 significantly reduced RGC loss when injected prior to disease induction, and that AR0018 significantly reduced RGC loss when administered at the peak of optic neuritis.

[0243] In Experiment 2 (Figs. 6f to 6h), EAE scores, evaluated on a relative 5-point scale by a shielded investigator, showed that in all treatment groups with no significant difference, more significant ascending paresis reaching a moderate level occurred. Visual loss also did not show a significant difference between treatment groups similar to Experiment 1 and previous studies where SIRT1 therapy mediated no visual gain or only small improvement. The primary outcome of RGC survival showed that both AR0012 and AR0018 significantly reduced RGC loss when injected prior to disease induction, and when administered at the onset and peak of optic neuritis.

[0244] Immunohistochemistry and quantification of RGC density

[0245] Mouse eyes were enucleated and fixed in 4% paraformaldehyde at 4°C for 30 minutes. The retina was incised, translucent, and blocked with 2% Triton X-100, 10% normal donkey serum, and PBS. For RGC staining, the tissue was incubated with a 1:1000 diluted rabbit anti-Brn3a antibody (Synaptic systems, Cat# 411 003) at 4°C. The retina was then washed three times and incubated with a secondary antibody solution containing donkey anti-rabbit AlexaFluor 488 (1:1000 dilution) for 1 hour at room temperature. After several washes, the retina was mounted on a glass slide in aqueous mounting medium (SouthernBiotech, Birmingham, AL, USA), covered with a coverslip, and sealed. Immunofluorescence was detected using a Nikon fluorescence microscope. Retinal images were taken with a 40x objective lens at 12 standard fields of view (1 / 6, 3 / 6, and 5 / 6 of the retinal radius from the center of the retina in each quadrant). Total RGCs counted at the 12 fields of view were recorded in the experimental and control groups using ImageJ software.

[0246] Optic nerve axon staining and quantification

[0247] The optic nerve was bisected at the level of the optic disc and fixed in 2.0% paraformaldehyde and 2.5% glutaraldehyde in 1 M sodium cacodilate buffer at pH 7.4. The optic nerve was additionally fixed in 2% osmium tetroxide, and the tissue was dehydrated in increasing concentrations of ethanol. After dehydration, the tissue was embedded in epoxy resin Embed 812 (Electron Microscopy Science, Hatfield, PA), sectioned into 0.75 µM cross-sections, and stained with 1% toluidine blue. Sections were obtained from approximately 1 mm from the proximal end of the optic disc. Most of each optic nerve section was captured as centrally oriented at 40X magnification, and axons were manually counted by a masked observer using ImageJ software. Three adjacent sections were counted for each optic nerve.

[0248] Derivation and Scoring of EAE

[0249] Eight-week-old female C57BL / 6J mice were anesthetized with isoflurane and injected subcutaneously into the superior and inferior dorsal regions with a total of 200 μg of myelin oligodendrocyte glycoprotein (MOG) peptides (MOG35-55; Genscript, Piscataway, NJ, USA) emulsified in complete Freund adjuvant containing 2.5 mg / ml Mycobacterium tuberculosis (Difco). Control mice were injected with the same volume of phosphate-buffered saline (PBS) instead of MOG and complete Freund adjuvant. All animals were administered 200 ng pertussis toxin in 0.1 ml PBS (List Biological, Campbell, CA, USA) by intraperitoneal injection at 0 and 48 hours post-immunization. The severity of EAE was scored using a 5-point scale: 0 = no disease; 0.5 = partial tail paralysis; 1.0=Tail paralysis or waddling gait; 1.5=Partial tail paralysis and waddling gait; 2.0=Tail paralysis and waddling gait; 2.5=Partial quadriplegia; 3.0=Unilateral quadriplegia; 3.5=Unilateral quadriplegia and partial quadriplegia on the other side; 4.0=Bilateral quadriplegia; 4.5=Dying state; 5.0=Death.

[0250] The EAE score (Fig. 6) demonstrates that EAE disease was successfully induced in mice, with milder and later-onset disease in the experiment on the upper left compared to the experiment on the upper right. The number of RGCs from control mice, untreated EAE mice, and EAE mice treated with the indicated vector at varying time points is shown for both experiments.

[0251] Example 4: Therapeutic efficacy of SIRT1 gene therapy in experimental glaucoma.

[0252] Magnetic microbead blockage model / microbead product

[0253] A publicly available magnetic MB mouse model of glaucoma was used to induce and sustain elevated intraocular pressure (IOP). Stock magnetic microbead (MB) solution (4.5 μm diameter, 4 x 10⁶ 8 Beads / mL (Dynabeds M-450 Epoxy, ThermoFisher Scientific, Cat# 14011, Waltham, MA, USA) were vortexed to uniformly distribute the MB in the solution. Next, 1 mL of the stock MB solution was pipette-transferred to 50 mL of 0.02 M sodium hydroxide (NaOH, MW 39.997 g / mol) in Tris 10x buffer (MW 121.4 g / mol) and spun at room temperature for 24 hours to remove protective epoxy groups from the MB. A magnet was attached to the bottom of the tube to collect the magnetic MB. The tube was then oriented horizontally and spun at room temperature for an additional 4 hours. The supernatant of the MB solution was removed using a micropipette, while the magnetic MB was resuspended in 50 mL of Tris 10x buffer until uniformly resuspended. The MB was washed three times with 5 mL of ultrapure laboratory-grade water by vortexing for 2 minutes. The MB was collected by attaching a magnet to the bottom of the tube. The MB was washed three additionally with BSS in a laminar flow hood by aspiration using a micropipette. The MB was 2.4 x 10⁻⁶ 6 Beads / μL (high concentration) or 1.6 x 10⁶ 6 Resuspended in BSS to a final concentration of 3.6 x 10⁶ beads / μL (low concentration). 6 Beads / μL or 2.4 x 10⁶ 6Each bead was injected into the anterior chamber with a final volume of 1.5 μL. The MB was stored at 4°C until use. Mice received an anterior chamber injection of 1.5 μL of magnetic MB into the experimental model eye on Day 0. IOP was measured using the Icare TONOLAB tonometry method (Icare TONOVET, Vantaa, Finland). The average of three measurements per eye was used at each time point. The IOP was considered successfully elevated if the baseline IOP of the corresponding eye increased by more than 18 or more than double (whichever was lower) from the minimum at two separate time points after MB injection.

[0254] Test-motor reaction record (OKR)

[0255] Visual function was evaluated by measuring OKR using commercial software and instruments (OptoMetry; CerebralMechanics, Inc., Medicine Hat, AB, Canada). OKR was determined as the highest spatial frequency at which the mouse tracked a 100% contrast grid projected at different spatial frequencies. Briefly, mouse movements were captured by placing the mouse unconstrained on a platform at the center of four computer monitors equipped with video cameras. A rotating cylinder with a vertical sine wave grid was computed and projected onto the monitors. Consisting of black and white bars at 100% contrast and 12 degrees / second, the sine wave grid provided a virtual-reality environment to measure the spatial resolution of the left eye when rotating clockwise and the spatial resolution of the right eye when rotating counterclockwise. Thus, asymmetry in the ophthalmokinetic reflexes allowed for the examination of visual function for each eye separately. The examination was performed by a shielded investigator: OKR responses were recorded for both eyes, and the presence of any central opacity (corneal staining or visible cataracts) was checked. Two experiments were performed.

[0256] Experiment 1 used high concentrations of MB to induce a severe increase in IOP, but the MB caused significant corneal staining, thus limiting the ability to track visual function. Experiment 2 used two concentrations of MB and still caused a significant increase in IOP, but corneal staining decreased at lower MB concentrations.

[0257] In Experiment 1 (Figs. 7a to 7c), IOPs measured by shielded investigators showed a significant increase in IOPs across all treatment groups without significant differences. Unlike previous studies, visual acuity loss also did not show significant differences between treatment groups; however, the OKR response may have been reduced due to corneal staining induced by MB. Even after removing the eye with the most severe corneal staining resulting in no detected visual response, milder corneal staining in the other eye could explain the decline in visual acuity even in the SIRT1-treated group. The primary outcome of RGC survival showed a tendency for both AR0012 and AR0018 to reduce RGC loss when injected before or after disease induction.

[0258] IOP measurements (Figs. 8a to 8d) demonstrate that elevated IOP disease was successfully induced in mice injected with microbeads (MB). OKR scores show visual function in control mice, untreated MB mice, and MB mice treated with the indicated vector at the stated time. The number of RGCS and their axons is plotted from control mice, untreated MB mice, and MB mice treated with the indicated vector at the stated time.

[0259] IOP (Fig. 8a) measurements show average IOP levels increased 1.5 to 2.0-fold in all lower concentration MB-injected treatment groups. Eyes were injected with a sham, the vector 14 days prior to MB injection, or the vector 10 days after MB injection (n = 10 eyes / group). OKR (Fig. 8b) shows significant visual impairment in MB-injected eyes without SIRT1 gene therapy (***p<0.001 for control eyes), including only eyes with documented IOP elevation and no severe corneal staining on the day of OKR measurement (n = 5-10 eyes / treatment). Treatment with AR0012 administered 14 days prior to MB injection, and treatment with AR0018 administered 14 days prior or 10 days after MB injection, respectively, significantly improved OKR response (*p<0.05 and **p<0.01 for MB-injected eyes treated with the control vector), and AR0012 administered 10 days after MB injection showed a similar strong trend toward improved OKR response. RGC / retina (Fig. 8c) showed significant RGC loss in MB-injected eyes treated with the control vector (#p<0.05 for control eyes), and treatment with both AR0012 and AR0018 before and after MB injection resulted in increased RGC survival in all groups when compared by ANOVA (n = 7-10 eyes / treatment; *p<0.05 and **p<0.01 for MB-injected eyes treated with the control vector). Data were shown from MB-injected eyes with elevated IOP. Cross-sections of the optic nerves of 4 randomly selected mice from each treatment group (n=4 nerves / treatment) were stained with toluidine blue, and the count of RGC axons by a shielded investigator showed a strong trend toward increased RGC axon density in treated versus control-treated MB-injected mice (Fig. 8d).

[0260] The degree of RGC neuroprotection in both EAE optic neuritis and MB glaucoma models was impressive, and all hSIRT1 gene therapy candidates prevented more than 80% of disease-induced RGC loss in EAE and more than 90% of disease-induced RGC loss in glaucoma (Figs. 8a to 8d). The inventors consider this impressive based on the magnitude of the effect, as well as the fact that this large effect was demonstrated using hSIRT1 transduction, which was detected in 65 to 75% of RGCs near the injection site and in only 40% of RGCs across the entire retina. Possible reasons for the observed effect being stronger than the detected transduction efficiency include 1) potential limitations using the inventors' detection of hSIRT1 (i.e., some RGCs have lower levels of hSIRT1 expression that can be transduced but are not detected in immunohistochemistry); and / or 2) there may be a bystander effect in which successfully transduced surviving RGCs secrete survival-promoting factors that can help support subsequent surrounding RGCs.

[0261] In EAE experiments, the inventors find that the RGC survival data is highly compelling. The failure of hSIRT1 gene therapy to consistently prevent the decline in OKR scores is not unexpected and is consistent with over 10 years of experience using various methods to promote SIRT1 as a means to reduce RGC loss in this model. The inventors' published work has consistently demonstrated that SIRT1 prevents RGC death by a mechanism independent of the suppression of optic nerve inflammation that induces RGC loss. This inflammation also impairs visual function, and thus OKR scores may continue to be suppressed in this EAE model that induces chronic inflammation. In human patients with optic neuritis, inflammation is transient, and studies show that only patients who develop significant RGC loss end up with permanent vision loss after the inflammation resolves. Therefore, the promotion of RGC survival shown in this model suggests strong potential for clinically relevant improved visual outcomes in patients.

[0262] Example 5: Discussion

[0263] This study was undertaken to address the need to better understand the properties that enhance AAV-mediated recombinant therapeutic transplant gene expression in RGCs, a key target for gene therapy in optic neuropathy. Previous studies have shown that the utilization of the AAV2 serotype facilitates gene delivery to neuronal and some non-neuronal cells at various anatomical sites of the central nervous system, including the retina. 46 The inclusion of the CMV promoter helped provide rapid and stronger recombinant gene expression, but the latter tended to weaken over time. The decline in transplanted gene expression was thought to result from the silencing of the CMV promoter by hypermethylation, the loss of transduced cells, and / or the loss of the AAV vector in the absence of genomic integration. 46, 47AAV vectors containing CMV early enhancers / CBA promoters were shown to provide organ and robust transplant gene expression. 37 However, the universality of this promoter hindered the targeting of therapeutic gene expression in RGCs with high efficacy, as it excluded them from other retinal cells. A major challenge in improving the efficiency and selectivity of AAV vectors was to combine a minimal RGC-selective promoter with other optimal regulatory sequences to provide potent, RGC-selective, long-lasting, and widespread expression of therapeutic genes upon intravitreal injection of recombinant AAV vectors. The selection of regulatory elements is always limited by the packaging capacity of AAV vectors, which cannot exceed approximately 4.7 kb. 45 .

[0264] To overcome these challenges, the inventors designed a series of constructs in which an RGC-selective promoter and a transplanted gene are lateralized by various regulatory sequences. RGC selectivity was achieved using a human promoter containing a 0.953 kb regulatory region of the previously described SNCG gene to allow for strong expression in ganglion cells across species. 34 The inventors' data show that, unlike the CBA-CMV promoter which induced the eGFP reporter gene in cells of various retinal layers, the hSNCG promoter allowed the targeted expression of the eGFP reporter in RGCs in GCL. Even though a small number of cells in GCL and INL expressed the hSNCG promoter-driven eGFP reporter, since one fraction of RGCs (<10%) does not express Brn3a, these cells may be translocated RGCs or Brn3a-negative RGCs. 48-50 Previous studies examined the suitability of mouse phosphoglycerate kinase and other promoters, such as human synapsin, for direct gene transport to RGCs. 34However, the hSNCG promoter possesses a higher degree of RGC specificity and transduction efficiency than, for example, the synapsin promoter, which yielded only 30% expression efficacy across the retina. Interestingly, a study by Wang et al. showed that the activity of the 1.4-kb mouse SNCG promoter surpassed that of the human SNCG promoter in RGCs from various sources, and that the truncation of the mouse SNCG promoter to 1.03, 0.66, or 0.27-kb led to a progressively significant decrease in RGC specificity. 51 However, the functionality and efficacy of mouse SNCG promoters in the context of human and non-human primate retinas remain areas that need to be addressed. In contrast, the 0.963-kb hSNCG promoter tested in the inventors' study provided extra packaging capacity and strong RGC selectivity that would enable larger therapeutic gene packaging. It is also noteworthy that the RGC selectivity of the hSNCG promoter allows for application in clinical settings where mouse SNCG promoters are not applicable.

[0265] When inserted at the 3' end of the reporter gene, the WPRE sequence induced a notable trend toward improved transgenic expression intensity and distribution across the retina. The inventors' fundus imaging of the retina suggested that the inclusion of post-transcriptional WPRE elements in the expression cassette increased the functionality of all vectors containing the hSNCG promoter. Consistent with this, eGFP transduction efficiency detected on retinal flat mounts after intravitreal injection consistently tended to be higher for all AAV vectors designed with the WPRE sequence compared to those without the sequence. The positive effect associated with the insertion of WPRE into the 3' UTR is consistent with prior studies optimizing gene expression cassette designs. 52-55Functionally, the WPRE location in the gene cassette can be important as it can affect the proper folding of mRNA. Thus, the inventors' results suggest that the introduction of WPRE into the 3' UTR is useful for improving RGC-selective transplant gene expression.

[0266] Other factors that positively influenced the transduction efficiency of eGFP expression were identified when the hSNCG promoter was lateralized with a CMV enhancer and an SV40 intron. Although the inventors did not quantify the fluorescence intensity of eGFP in fundus images of living mice after transduction with recombinant vectors, qualitative evaluation, as shown in representative images, suggests that the eGFP reporter was intensely and extensively expressed in the retinas of mice transduced with the V6.G vector containing these regulatory sequences, and is more likely to be so than with vectors containing other sequences, such as the SV40 enhancer or the Arc promoter. The use of the CMV initial enhancer demonstrated the utility of this enhancer in conferring efficient and sustained cell type-specific gene expression when inserted upstream of a cell type-specific promoter, as demonstrated by Gruh et al. 56 It has been previously introduced by. Although CMV enhancers do not have cell type- or species-specific preference itself, two In vitro and In vivo Enhances transcription. It contains several binding sites recognized by various nuclear factors that additionally interact with the basic transcriptional machinery assembled in the promoter to increase transcription. 57 Despite these characteristics, the inventors observed that the CMV enhancer can maintain the specificity of the cell-specific promoter used, making it suitable for the AAV-mediated RGC-selective expression of therapeutic genes.

[0267] Similarly, the introduction of an SV40 intron sequence upstream of the reporter gene produced high transfection efficiency.

[0268] To further verify the inventors' findings and investigate the effects on the expression of the transplant gene of interest, the inventors evaluated the expression of hSIRT1. The upregulation of SIRT1 activity by pharmacological, genetic, cell-based, and gene therapy approaches consistently demonstrated that this deacetylase can mediate RGC neuroprotection in various optic neuropathy. 26-28, 60-63 Some of these studies are RGC-selective hSIRT1 gene therapy 26, 27, 61 Although the therapeutic potential of [the subject] has already been demonstrated, these studies did not optimize the AAV vector, such as the hSNCG promoter and the included regulatory elements. Here, following the analysis of variable regulatory elements, the eGFP reporter gene was replaced by hSIRT1, which is codon-optimized from the AAV2 cassette. Upon intravitreal injection, the inventors found that hSIRT1 expression was prevalent, reaching nearly 40% of RGCs counted in a standardized area across the entire retina, similar to the expression of eGFP delivered in the corresponding vector construct. Importantly, hSIRT1 expression was detected across 70% of RGCS in the central retinal region with the highest observed transduction efficiency, suggesting that some RGC populations may be more receptive to the AAV vector, or that proximity to the injection site may influence the percentage of transduced RGCs. The vector tested herein demonstrated excellent transduction efficiency. An AAV vector containing the hSIRT1 gene lateralized by a WPRE at the 3' end and an eCMV-hSNCG-iSV40 enhancer-promoter-intron combination at the 5' end showed a tendency toward the highest local expression levels of hSIRT1. The ability to replace eGFP with a therapeutic gene of interest, such as hSIRT1, while maintaining strong transduction efficiency suggests that this vector design may be useful for introducing other potential therapeutic genes into RGCs in the future.

[0269] In summary, these studies demonstrate the benefits of the hSNCG promoter when combined with other regulatory sequences in maximizing transduction efficiency and increasing maximal specificity for RGCs. The inventors conclude that the vectors examined herein, e.g., eCMV-hSNCG-iSV40 enhancer-promoter-intron, combined with WPRE sequences, are generally advantageous for conveying recombinant AAV vector selectivity, distribution in the retina, and intensity of recombinant gene expression. The specific combination of highly efficient enhancers, intronic, poly(A), and translational elements with the most selective minimal RGC-specific promoter improves vector fitness while maximizing the remaining space for therapeutic transplant gene coding sequences, such as those of SIRT1. Overall, the identification of highly efficient AAV vectors provides proof of concept for their utility in clinical applications for AAV-based gene therapy to provide neuroprotection in neurodegenerative diseases.

[0270] Example 6: Therapeutic evaluation of combination therapy using an optimized SIRT1 AAV construct co-administered with complement-modulating gene therapy in experimental optic neuritis.

[0271] SIRT1 upregulation can reduce RGC loss in several preclinical optic neuropathy models by promoting mitochondrial biosynthesis within injured neurons and reducing oxidative stress. Complement regulation also reduces neuronal loss in several models, including RGC loss in EAE and glaucoma models, by reducing inflammatory responses that likely induce neuronal damage. By targeting distinct mechanisms of RGC loss, the inventors hypothesize that these two therapies can provide synergistic effects leading to improved efficacy.

[0272] C57BL / 6 mice were transduced 2 weeks prior to EAE induction by a single intravitreal injection of the SIRT1 AAV2 vectors AR0012, AAV2.CR1, or AAV2.FHL1 alone at a dose of 3E9 vg / eye, or by a single injection of the two vectors: AR0012 + AAV2.CR1 or AR0012 + AAV2.FHL1, respectively, at a dose of 3E9 vg / eye. An eGFP-expressing AAV vector was injected as a sham control. EAE was induced in 8-week-old C57BL / 6 mice by immunization with MOG peptides, and disease severity was monitored using the EAE ascending paralysis score. Efficacy was evaluated based on RGC survival 8 weeks after EAE induction. Secondary outcomes were assessed for visual function using weekly OKRs, and optic nerve inflammation was scored on a relative 4-point scale using H&E-stained optic nerve longitudinal sections. In addition, vitreous fluid samples and retinas from four eyes in each treatment group were sent to a gyroscope for molecular research.

[0273] EAE scores showed that typical moderate ascending paralysis occurred in all treatment groups (Figs. 9a to 9d). Visual loss showed no significant difference between treatment groups, similar to the initial study of SIRT1 gene therapy alone. The primary outcome of RGC survival showed that only AR0012 significantly reduced RGC loss, and no improvement in optic nerve inflammation was found in any of the treatments, either alone or in combination.

[0274] All publications cited herein are incorporated herein by reference. Similarly, sequence numbers appearing in the appended sequence list referenced herein are incorporated by reference. U.S. provisional patent application no. 63 / 593,435 filed October 26, 2023 is incorporated herein by reference in its entirety. Although the invention has been described with reference to specific embodiments, it will be understood that modifications may be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

[0275]

[0276]

[0277]

[0278]

[0279]

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Claims

Claim 1 A recombinant adeno-associated virus (rAAV), wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, and the vector genome comprises (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) a CMV enhancer; (c) a human SNCG promoter; (d) an SV40 intron; (e) a coding sequence encoding human SIRT1; (f) a WPRE; (g) a bGH poly-A; and (h) an AAV 3' ITR sequence. Claim 2 In claim 1, (a) the AAV 5' inversion terminal repeat (ITR) sequence is SEQ ID NO: 4 or a sequence having at least 90% sequence identity therewith; (b) the CMV enhancer sequence is SEQ ID NO: 5 or 29 or a sequence having at least 90% sequence identity therewith; (c) the human SNCG promoter sequence is SEQ ID NO: 6 or a sequence having at least 90% sequence identity therewith; (d) the SV40 intron sequence is SEQ ID NO: 7 or a sequence having at least 90% sequence identity therewith; (e) the coding sequence encoding the human SIRT1 sequence is SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith; (f) the WPRE sequence is SEQ ID NO: 8 or a sequence having at least 90% sequence identity therewith; (g) the bGH poly A sequence is SEQ ID NO: 9 or 30 or a sequence having at least 90% sequence identity therewith; and (h) rAAV in which the above AAV 3' ITR sequence is one or more of SEQ ID NO: 10 or 31 or a sequence having at least 90% sequence identity therewith. Claim 3 rAAV according to claim 1 or 2, wherein the coding sequence encoding the human SIRT1 sequence is SEQ ID NO:

1. Claim 4 In claim 1, the vector genome comprises: (a) an AAV 5' ITR sequence preferably having SEQ ID NO: 4; (b) a CMV enhancer sequence having SEQ ID NO: 5 or 29; (c) a human SNCG promoter sequence having SEQ ID NO: 6; (d) an SV40 intron sequence having SEQ ID NO: 7; (e) a coding sequence encoding a human SIRT1 sequence having SEQ ID NO: 1; (f) a WPRE sequence having SEQ ID NO: 8; (g) a bGH poly-A sequence having SEQ ID NO: 9 or 30; and (h) an AAV 3' ITR sequence preferably having SEQ ID NO: 10 or 31, comprising an rAAV. Claim 5 rAAV according to any one of claims 1, 2, or 4, wherein the vector genome comprises SEQ ID NO: 11 or a sequence sharing at least 90% identity with it. Claim 6 A recombinant adeno-associated virus (rAAV), wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) an SV40 enhancer; (c) a human SNCG promoter; (d) an SV40 intron; (e) a coding sequence encoding human SIRT1; (f) a WPRE; (g) a bGH poly-A; and (h) an AAV 3' ITR. Claim 7 In claim 6, (a) the AAV 5' inversion terminal repeat (ITR) sequence is SEQ ID NO: 4 or a sequence having at least 90% sequence identity therewith; (b) the SV40 enhancer sequence is SEQ ID NO: 12 or a sequence having at least 90% sequence identity therewith; (c) the human SNCG promoter sequence is SEQ ID NO: 6 or a sequence having at least 90% sequence identity therewith; (d) the SV40 intron sequence is SEQ ID NO: 7 or a sequence having at least 90% sequence identity therewith; (e) the coding sequence encoding the human SIRT1 sequence is SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith; (f) the WPRE sequence is SEQ ID NO: 8 or a sequence having at least 90% sequence identity therewith; (g) the bGH poly A sequence is SEQ ID NO: 9 or 30 or a sequence having at least 90% sequence identity therewith; and (h) rAAV in which the above AAV 3' ITR sequence is one or more of SEQ ID NO: 10 or 31 or a sequence having at least 90% sequence identity therewith. Claim 8 In claim 6 or 7, rAAV, wherein the coding sequence encoding the human SIRT1 sequence is SEQ ID NO:

1. Claim 9 In claim 6, the vector genome comprises: (a) an AAV 5' ITR sequence preferably having SEQ ID NO: 4; (b) an SV40 enhancer sequence having SEQ ID NO: 12; (c) a human SNCG promoter sequence having SEQ ID NO: 6; (d) an SV40 intron sequence having SEQ ID NO: 7; (e) a coding sequence encoding a human SIRT1 sequence having SEQ ID NO: 1; (f) a WPRE sequence having SEQ ID NO: 8; (g) a bGH poly-A sequence having SEQ ID NO: 9 or 30; and (h) an AAV 3' ITR sequence preferably having SEQ ID NO: 10 or 31, comprising an rAAV. Claim 10 rAAV according to any one of claims 6, 7, or 9, wherein the vector genome comprises SEQ ID NO: 13 or a sequence sharing at least 90% identity with it. Claim 11 A recombinant adeno-associated virus (rAAV), wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, and the vector genome comprises (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) a CAG promoter; (c) a coding sequence encoding human SIRT1; (d) a WPRE; (e) a bGH poly-A; and (f) an AAV 3' ITR. Claim 12 rAAV according to claim 11, wherein (a) the AAV 5' inversion terminal repeat (ITR) sequence is SEQ ID NO: 4 or a sequence having at least 90% sequence identity therewith; (b) the CAG promoter is a sequence having SEQ ID NO: 28, 33, or 35 or a sequence having at least 90% sequence identity with SEQ ID NO: 28, 33, or 35; (c) the coding sequence encoding the human SIRT1 sequence is SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith; (d) the WPRE sequence is SEQ ID NO: 8 or a sequence having at least 90% sequence identity therewith; (e) the bGH poly A sequence is SEQ ID NO: 9 or 30 or a sequence having at least 90% sequence identity therewith; and (f) the AAV 3' ITR sequence is SEQ ID NO: 10 or 31 or a sequence having at least 90% sequence identity therewith, one or more of these. Claim 13 In claim 11 or 12, rAAV, wherein the coding sequence encoding the human SIRT1 sequence is SEQ ID NO:

1. Claim 14 In claim 11, the vector genome comprises (a) an AAV 5' ITR sequence, preferably having SEQ ID NO: 4; (b) a CAG promoter sequence, having SEQ ID NO: 28, 33, or 35; (c) a coding sequence encoding a human SIRT1 sequence, having SEQ ID NO: 1; (c) a WPRE sequence, having SEQ ID NO: 8; (e) a bGH poly-A sequence, having SEQ ID NO: 9 or 30; and (f) an AAV 3' ITR sequence, preferably having SEQ ID NO: 10 or 31, rAAV. Claim 15 rAAV according to any one of claims 11, 12, or 14, wherein the vector genome comprises SEQ ID NO: 18, or a sequence sharing at least 90% identity with it. Claim 16 A recombinant adeno-associated virus (rAAV), wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, said vector genome comprising (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) a CAG promoter; (c) a coding sequence encoding human SIRT1; (d) a bGH poly-A; and (e) an AAV 3' ITR. Claim 17 rAAV according to claim 16, wherein at least one of: (a) the AAV 5' inversion terminal repeat (ITR) sequence is SEQ ID NO: 4 or a sequence having at least 90% sequence identity therewith; (b) the CAG promoter sequence is SEQ ID NO: 37 or a sequence having at least 90% sequence identity therewith; (c) the coding sequence encoding the human SIRT1 sequence is SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith; (d) the bGH poly A sequence is SEQ ID NO: 9 or 30 or a sequence having at least 90% sequence identity therewith; and (e) the AAV 3' ITR sequence is SEQ ID NO: 10 or 31 or a sequence having at least 90% sequence identity therewith. Claim 18 In claim 16 or 17, rAAV, wherein the coding sequence encoding the human SIRT1 sequence is SEQ ID NO:

1. Claim 19 In claim 16, the vector genome comprises (a) an AAV 5' ITR sequence preferably having SEQ ID NO: 4; (b) a CAG promoter sequence having SEQ ID NO: 37; (c) a coding sequence encoding a human SIRT1 sequence having SEQ ID NO: 1; (d) a bGH poly A sequence having SEQ ID NO: 9 or 30; and (e) an AAV 3' ITR sequence preferably having SEQ ID NO: 10 or 31, rAAV. Claim 20 rAAV according to any one of claims 1 to 19, wherein the rAAV capsid is an AAV2 capsid or an AAV8 capsid. Claim 21 A cell transfected with an AAV vector of any one of claims 1 to 20. Claim 22 A pharmaceutical composition comprising rAAV according to any one of claims 1 to 20 or cells according to claim 21; and a pharmaceutically acceptable carrier or excipient. Claim 23 An aqueous suspension suitable for administration to a subject, wherein the suspension comprises an aqueous suspension and a recombinant adeno-associated virus (rAAV), wherein the rAAV has an AAV capsid and has (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) a CMV enhancer; (c) a human SNCG promoter; (d) an SV40 intron; (e) a coding sequence encoding human SIRT1; (f) a WPRE; (g) a bGH poly-A; and (h) a vector genome packaged therein comprising an AAV 3' ITR. Claim 24 In claim 23, (a) the AAV 5' inversion terminal repeat (ITR) sequence is SEQ ID NO: 4 or a sequence having at least 90% sequence identity therewith; (b) the CMV enhancer sequence is SEQ ID NO: 5 or 29 or a sequence having at least 90% sequence identity therewith; (c) the human SNCG promoter sequence is SEQ ID NO: 6 or a sequence having at least 90% sequence identity therewith; (d) the SV40 intron sequence is SEQ ID NO: 7 or a sequence having at least 90% sequence identity therewith; (e) the coding sequence encoding the human SIRT1 sequence is SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith; (f) the WPRE sequence is SEQ ID NO: 8 or a sequence having at least 90% sequence identity therewith; (g) the bGH poly A sequence is SEQ ID NO: 9 or 30 or a sequence having at least 90% sequence identity therewith; and (h) an aqueous suspension in which the AAV 3' ITR sequence is one or more of SEQ ID NO: 10 or 31 or a sequence having at least 90% sequence identity therewith. Claim 25 An aqueous suspension suitable for administration to a subject, wherein the suspension comprises an aqueous suspension and a recombinant adeno-associated virus (rAAV), wherein the rAAV has an AAV capsid and has a vector genome packaged therein comprising (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) an SV40 enhancer; (c) a human SNCG promoter; (d) an SV40 intron; (e) a coding sequence encoding human SIRT1; (f) a WPRE; (g) a bGH poly-A; and (h) an AAV 3' ITR. Claim 26 In claim 25, (a) the AAV 5' inversion terminal repeat (ITR) sequence is SEQ ID NO: 4 or a sequence having at least 90% sequence identity therewith; (b) the SV40 enhancer sequence is SEQ ID NO: 12 or a sequence having at least 90% sequence identity therewith; (c) the human SNCG promoter sequence is SEQ ID NO: 6 or a sequence having at least 90% sequence identity therewith; (d) the SV40 intron sequence is SEQ ID NO: 7 or a sequence having at least 90% sequence identity therewith; (e) the coding sequence encoding the human SIRT1 sequence is SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith; (f) the WPRE sequence is SEQ ID NO: 8 or a sequence having at least 90% sequence identity therewith; (g) the bGH poly A sequence is SEQ ID NO: 9 or 30 or a sequence having at least 90% sequence identity therewith; and (h) rAAV in which the above AAV 3' ITR sequence is one or more of SEQ ID NO: 10 or 31 or a sequence having at least 90% sequence identity therewith. Claim 27 An aqueous suspension suitable for administration to a subject, wherein the suspension comprises an aqueous suspension and a recombinant adeno-associated virus (rAAV), wherein the rAAV has an AAV capsid and has (a) an AAV 5' inversion terminal repeat (ITR) sequence; (b) a CAG promoter; (c) a coding sequence encoding human SIRT1; (d) a WPRE; (e) a bGH poly-A; and (f) a vector genome packaged therein comprising an AAV 3' ITR. Claim 28 An aqueous suspension according to claim 27, wherein (a) the AAV 5' inversion terminal repeat (ITR) sequence is SEQ ID NO: 4 or a sequence having at least 90% sequence identity therewith; (b) the CAG promoter sequence is a sequence having at least 90% sequence identity with SEQ ID NO: 28, 33, or 35 or SEQ ID NO: 28, 33, or 35; (c) the coding sequence encoding the human SIRT1 sequence is SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith; (d) the WPRE sequence is SEQ ID NO: 8 or a sequence having at least 90% sequence identity therewith; (e) the bGH poly A sequence is SEQ ID NO: 9 or 30 or a sequence having at least 90% sequence identity therewith; and (f) the AAV 3' ITR sequence is SEQ ID NO: 10 or 31 or a sequence having at least 90% sequence identity therewith, one or more of which. Claim 29 A suspension according to any one of claims 23 to 28, wherein the suspension further comprises a surfactant, a preservative, and / or a buffer dissolved in the aqueous suspension. Claim 30 In any one of paragraphs 22 to 29, the composition is a pharmaceutical composition or an aqueous suspension suitable for delivery to the eye. Claim 31 A method for treating or preventing eye disorders in a subject requiring treatment or prevention of eye disorders, comprising administering rAAV, cells, or a pharmaceutical composition according to any one of claims 1 to 30 to said subject. Claim 32 In paragraph 31, the method in which the above disorder is optic neuropathy. Claim 33 In paragraph 32, the above disorder is optic neuritis or glaucoma, preferably glaucoma. Claim 34 A method according to any one of claims 31 to 33, wherein the rAAV, cell, or pharmaceutical composition is administered into the eye. Claim 35 A method according to claim 34, wherein the rAAV, cell, or pharmaceutical composition is administered by subretinal, supracorbital, or intravitreal injection. Claim 36 In any one of paragraphs 31 to 35, the rAAV is approximately 1 x 10 9 to about 1 x 10 13 A method for delivering a vector genome / eye (vg / eye) in doses. Claim 37 In paragraph 36, the rAAV is 1 x 10⁻¹⁰ in a volume comprising about or at least 100 microliters. 9 Up to 1 x 10 13 A method of administering a dosage of rAAV, thereby preventing vision loss in the subject. Claim 38 A method according to any one of claims 31 to 37, wherein the rAAV is administered in a volume comprising about 100 to 250 microliters. Claim 39 A method according to any one of claims 31 to 37, wherein the rAAV is administered in a volume of 150 to 800 microliters. Claim 40 A method according to any one of claims 31 to 37, wherein the rAAV is administered in a volume comprising 250 to 500 microliters. Claim 41 A method according to any one of claims 31 to 37, wherein the rAAV is administered in a volume comprising about 500 microliters. Claim 42 A method according to any one of paragraphs 31 to 41, wherein the subject is a human.

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