Optogenetic gene therapy for treating blindness

Abstract

Provided herein are AAV2 vectors encoding SEQ ID NO:4 as optogenetic proteins and methods of use, for example, for retinitis pigmentosa.

Description

Related Applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 359,822, filed July 9, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on Jun. 26, 2023, is named BIS-001WO_SL.xml, and is 20,730 bytes in size. [Background technology]

[0003]

[0003] Age-related macular degeneration (AMD) and retinitis pigmentosa (RP) collectively affect 2 million people in the United States and 25 million people worldwide. In both retinal degenerative diseases, it is the retinal input that degenerates: in AMD, the cones degenerate, and in RP, the rods degenerate. More specifically, RP is a large group of inherited retinal disorders in which progressive degeneration of photoreceptor cells or the retinal pigment epithelium causes vision loss. Clinical symptoms in affected individuals initially manifest as night blindness, followed by reduced peripheral vision, and eventually loss of central vision.

[0004]

[0004] Current treatments for patients with retinal degenerative diseases are limited. Although there is some evidence suggesting that vitamin A and fish oil supplements may slow vision loss in some patients with early-stage disease, vitamin A and fish oil supplements cannot reverse the disease. In a subset of patients in whom retinal degeneration is caused by mutations in the RPE65 gene, targeted gene therapy is now possible and is currently being used to treat patients. However, because RP is a genetically heterogeneous disease with over 100 different genes or loci that lead to a common endpoint, for example, vision loss, gene therapy is not necessarily applicable to the majority of patients.

[0005] However, new gene therapies using optogenetics are opening up new treatment options for patients with RP. Optogenetics allows for the treatment of this disease in a manner that is independent of the underlying genetic defect, potentially benefiting a much broader range of patients. While conceptually highly promising, these types of approaches can have therapeutic limitations due, for example, to the need for extremely bright light to activate proteins and / or phototoxic effects due to short excitation wavelengths. Summary of the Invention

[0006]

[0006] Therefore, there is a significant unmet need for treatment modalities (e.g., for RP) that can improve vision in human patients, for example, giving patients an enhanced ability to detect light, shape, motion, and / or color.

[0007] The disclosure herein provides, at least in part, compositions and methods for treating retinal degenerative disorders, and provides, for example, an adeno-associated virus (AAV) 2 (AAV2) vector having a nucleic acid encoding a gene that expresses an optogenetic fusion protein (e.g., a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:2 or, e.g., a sequence having 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2) or a protein having 98% or 99% identity to the amino acid sequence of SEQ ID NO:3 (e.g., a sequence having 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1). AAV vectors contemplated as such may be administered, for example, intravitreally to a patient at an initial dose (e.g., administered to the retinal surface of the patient's eye).

[0008] For example, the disclosed method of delivering contemplated optogenetic proteins to the human eye provides substantially greater light sensitivity (over 100-fold) than would be expected from measurements of the light sensitivity produced by the protein in cultured neurons. Blind or near-blind patients treated with, for example, SEQ ID NO: 1 were able to detect light at much lower light levels, including daylight and television light levels, than would be expected from published reports using the protein.

[0009]

[0009] Approximately 1×10 11 ~Approx. 1×10 13 Also provided herein are methods comprising administering an effective amount of vector dose (e.g., a vector contemplated herein, e.g., SEQ ID NO: 9) of vector genome (vg) / eye. Surprisingly, for example, upon administration, approximately 1.2 x 10 12 A dose response of vg / eye or higher produces unexpected supralinear improvements in light sensitivity; for example, the amplitude of the electroretinogram (ERG) response in mice shows a roughly linear dose response at lower doses, but a supralinear increase in amplitude at these higher doses.

[0010]

[0010] Contemplated methods may further include administering to a patient a light delivery device that sends light pulses to optogenetic proteins in the neural cord of the retina, causing ganglion cells, the output cells of the retina, to fire in a pattern that mimics that of a normal retina. Surprisingly, administration of both the contemplated vector and the neurally encoded stimulation enables patients (e.g., treated patients) to move, detect direction of movement, and, in some patients, recognize objects.

[0011] For example, in patients with moderate blindness, administration of a contemplated vector, e.g., SEQ ID NO: 9, unexpectedly enhances the patient's ability to detect shapes, count fingers, and recognize objects, due to interaction between the vector and the patient's remaining retinal circuitry. Even more surprisingly, for example, in patients with moderate blindness, administration of a contemplated vector, as provided herein, enables color perception due to interaction with the patient's cone system.

[0012]

[0012] A DNA polynucleotide or its RNA equivalent encoding an optogenetic fusion protein, wherein the optogenetic fusion protein comprises a light-activated ion channel protein fused to a reporter protein, the light-activated ion channel protein having the amino acid sequence of SEQ ID NO: 5, the reporter protein being fused to the 3' end of the optogenetic protein, and the polynucleotide being operably linked to a CAG promoter having the nucleic acid sequence of SEQ ID NO: 6 and a WPRE enhancer having the nucleic acid sequence of SEQ ID NO: 7.

[0013]

[0013] The present disclosure is based, at least in part, on the surprising discovery that a vector encoding a light-sensitive protein (e.g., a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 4 or a nucleic acid encoding a protein having the amino acid sequence of SEQ ID NO: 5) was effective in humans over a wide range of vector doses and stimulation light intensities. Furthermore, the contemplated vectors are well tolerated when administered to human patients. In some embodiments, the present disclosure provides a combination therapy comprising optogenetic gene therapy (e.g., a vector encoding a light-sensitive protein (e.g., a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 4 or a nucleic acid encoding a protein having the amino acid sequence of SEQ ID NO: 5)) and neurally encoded stimulation. The combination therapy described herein has the surprising advantage of providing unprecedented visual restoration to patients with retinal degenerative disorders, such as those with advanced blindness due to retinitis pigmentosa. Thus, the compositions and methods described herein can be used to restore vision to patients with retinal degenerative diseases. [Brief explanation of the drawings]

[0014] [Figure 1A]1A-1B are a set of graphs depicting the robust electroretinogram (ERG) responses in Pde6brd1 (rd1) mice following administration of Compound A, an adeno-associated virus 2 (AAV2) whose genome contains the nucleic acid sequence of SEQ ID NO: 1 and whose expression cassette contains the nucleic acid sequence of SEQ ID NO: 9. Figure 1A shows raw ERG responses (mean ± SEM) from untreated eyes (top, red) and Compound A-treated eyes (bottom, blue) from the same animals. Horizontal black lines on each plot indicate the location of stimulus artifacts, e.g., small upward and downward electrical transients at stimulus onset and offset. The right side of the lower panel shows a set of traces from individual, non-overlapping epochs in the ERG, demonstrating consistency across the entire acquisition period. Figure 1B shows a set of graphs of ERG responses for all animals. For each eye of each animal, responses were quantified by the size of the photopic negative response (PhNR)-like wave (e.g., the ERG component corresponding to the ganglion cell response), measured as the difference between the potential at time zero and the mean potential during the second half of the stimulation period, just before the stimulus artifact. PhNR-like waves were observed in 6 of 7 animals tested. The wave size was significant at both the group level (p<0.001, unpaired t-test) and the individual animal level (p<0.004, paired t-test, for each animal, comparing the treated eye with its untreated counterpart, shown on the right). Some variability in responses is expected due to injection variability in the small target (mouse eye). Results were measured 10 weeks after vector injection. Light stimulation was 0.06 mW / mm², 505 nm. [Figure 1B] Same as description for Figure 1A. [Figure 2]

[0015] Figures 2A-2B are a set of graphs depicting the assessment of ERG responses in rd1 mice over a 20-fold dose range. Figure 2A is a graph depicting ERG responses to light stimulation from animals treated with Compound A. Five groups are shown: untreated eyes (n=7), eyes treated with a dose of 5x107 vg / eye (n=7), eyes treated with a dose of 1x108 vg / eye (n=3), eyes treated with a dose of 5x108 vg / eye (n=12), and eyes treated with a dose of 1x109 vg / eye (n=11). The mean response amplitude for each dose group was statistically significantly different from the control mean response amplitude (p<0.01), and, as expected, response amplitude increased with increasing dose. Figure 2B is a set of graphs depicting raw ERG responses (mean ± SEM) from each of the five groups. Responses for each eye were quantified by the size of the PhNR-like wave. All injections were performed 10–15 weeks prior to recording. Light stimulation was 0.06 mW / mm², 505 nm. [Figure 3]

[0001] Figure 1 is a graph depicting the assessment of ERG responses to lower light levels in Compound A-treated rd1 mice. In animals treated with the two highest doses, 1 x 109 vg / eye and 5 x 108 vg / eye, light levels were substantially reduced from the level used in Figures 2A-2B and 3 (0.06 mW / mm2) and were still able to produce ERG responses well above baseline (see untreated eyes in Figures 2A-2B) (p<0.01). All injections were performed 10-15 weeks prior to recording. [Figure 4]

[0016] Figure 4 is a graph depicting the absence of retinal ganglion cell loss in Compound A-treated retinas compared to untreated retinas. Mean density of Brn3a-positive cells from control, low-dose Compound A-treated, and high-dose Compound A-treated retinas. Data are plotted as the number of ganglion cells per linear mm of retina (mean ± SEM). No statistically significant differences in retinal ganglion cell numbers were observed between the low-dose and control groups (p>0.1, Student's t-test) or between the high-dose and control groups (p>0.5, Student's t-test). The low-dose group included 10 eyes, the high-dose group included 9 eyes, and the control group included 3 vehicle-treated and 2 untreated eyes. [Figure 5]

[0017] Figures 5A-5B are a set of graphs depicting the lack of loss of Compound A-expressing ganglion cells and photoreceptors in retinas treated with both Compound A and light. Figure 5A is a graph showing a comparison of the density (mean ± SEM) of Compound A-expressing cells in the Compound A alone group and the Compound A plus light treatment group; no statistically significant difference was observed between the two groups (p>0.7, Student's t-test). Figure 5B is a graph showing a comparison of the density (mean ± SEM) of cones in untreated retinas and Compound A plus light treatment retinas; no statistically significant difference was observed (p>0.4, Student's t-test). [Figure 6]

[0018] Figures 6A-6B are a set of graphs depicting no statistically significant reduction in ERG component amplitude between treated and untreated eyes of non-human primates. Figure 6A is a graph depicting representative ERG responses to a flash stimulus showing the three main ERG components: the a-wave, the b-wave, and the PhNR. Figure 6B is a set of graphs depicting the mean maximum velocity of rise (Vmax) values for the three ERG components in untreated and treated groups. No statistically significant reduction in Vmax was observed for any of the three components as a result of treatment (p>0.2, for all waves, comparing Vmax values in the treated group with Vmax values in the untreated group). ERGs in treated animals were performed 7 months after vector injection. [Figure 7]

[0019] Figures 7A-7B are a set of graphs depicting that the distributions of ganglion cell receptive field size and firing frequency from Compound A-expressing retinas were not statistically significantly different from those from untreated retinas. Figure 7A shows histograms of receptive field size from untreated retinas (top) and SEQ ID NO:1-treated retinas (bottom) using comparable retinal eccentricities (3-12 mm from the central retina) (p>0.2, Kolmogorov-Smirnov test). Figure 7B shows histograms of ganglion cell firing frequency from untreated retinas (top) and Compound A-expressing retinas (bottom); the two distributions were not statistically significantly different (p>0.2, Kolmogorov-Smirnov test). Firing frequency was measured in response to videos of natural scenes, including trees, landscapes, and walking people. All eyes were intravitreally injected with Compound A vector 3-6 months before eye removal for electrophysiological recording. [Figure 8]

[0020] 8 is a set of graphs depicting the results of an individual human patient in a two-alternative forced-choice task to test light sensitivity. The patient received an initial vector dose of at least 1 x 10 vg / eye of Compound A intravitreally and was subsequently tested in the paradigm. [Figure 9]

[0021] 9 is a set of graphs depicting the results of individual human patients taken over time (e.g., at 3, 6, 9, and / or 12 months) in a two-algebra forced-choice task to test light sensitivity. The patients were administered an initial vector dose of an effective amount of Compound A (e.g., at least about 1 x 10 vg / eye) intravitreally and subsequently tested in the paradigm. [Figure 10]

[0022] 10 is a set of graphs depicting the results of an individual human patient in four paradigms, each of which measures the ability to detect movement ("Movement"), the ability to detect the direction of movement ("Direction"), the ability to discriminate live movement (e.g., an arm moving up vs. down, or e.g., arm movement vs. hand movement; "Live"), and the ability to discriminate objects (e.g., apples vs. other fruits or vegetables, or e.g., playing card suits; "Objects"). The patient was administered an initial vector dose of an effective amount (e.g., at least about 1 x 10 vg / eye) of Compound A intravitreally, followed by paradigm testing. [Figure 11]

[0023] Figure 11 is a set of graphs depicting the results of an individual human patient as a function of time in four paradigms, including motion, orientation, live, and object, respectively, described in Figure 10. The patient was administered an initial vector dose of an effective amount of Compound A (e.g., at least about 1 x 10 vg / eye) intravitreally and subsequently tested in the paradigms. [Figure 12]

[0024] 12A-12B are a set of graphs depicting the results of an individual human patient with baseline visual acuity of <20 / 200 in the index eye ( FIG. 12A ) and an individual human patient in the motion / object recognition test ( FIG. 12B ), respectively. The patient received an initial vector dose of an effective amount (e.g., at least about 1×10 vg / eye) of Compound A intravitreally and subsequently underwent paradigm testing. [Figure 13]

[0025] 13A-13B are a set of graphs depicting the results of a second individual human patient with baseline visual acuity of <20 / 200 in the index eye ( FIG. 13A ) and a second individual human patient in the motion / object recognition test ( FIG. 13B ). The patient received an initial vector dose of Compound A (e.g., at least about 1×10 vg / eye) intravitreally and subsequently underwent paradigm testing. [Figure 14]

[0026] FIG. 14 is a set of graphs depicting the results of two individual human patients each with baseline visual acuity of <20 / 400 on color testing. [Figure 15]

[0027] 15 is a graph showing the amplitude of the ERG response in mice administered different doses of Compound A. Doses, from left to right, include: untreated, 5×10 vg / eye, 1×10 vg / eye (equivalent to human "dose 1, 1×10 vg / eye"), 5×10 vg / eye (approximately equivalent to human "dose 3, 6×10 vg / eye"), and 1×10 vg / eye (equivalent to human "dose 4, 1×10 vg / eye"). [Figure 16-1]

[0028] FIG. 16 is a depiction of the nucleic acid sequence of Compound A (SEQ ID NO:1). [Figure 16-2] Figure 16 (continued 1). [Figure 16-3] Figure 16 (continued 2). [Figure 16-4] Figure 16 (continued 3). [Figure 16-5] Figure 16 (continued 4). [Figure 16-6] Figure 16 (continued 5). [Figure 16-7] Figure 16 (continued 6). [Figure 16-8] Figure 16 (continued 7). [Figure 16-9] Figure 16 (continued 8). [Figure 16-10] Figure 16 (continued 9). DETAILED DESCRIPTION OF THE INVENTION

[0015] Optogenetic Protein

[0029] The compounds of the present disclosure include genes that express optogenetic proteins. Optogenetic proteins are often light-gated ion channels or pumps that absorb light at specific wavelengths. When activated by light, these channels and pumps respond by opening or closing, allowing ions to flow into or out of the cells in which the protein is expressed, respectively. Provided herein are optogenetic proteins that include light-sensitive channels from, for example, Stigeoclonium helveticum, such as an optogenetic protein encoded by a nucleic acid having the nucleic acid sequence of SEQ ID NO: 4 below. ATGGAAACAGCCGCCACAATGACCCACGCCTTTATCTCAGCCGTGCCTAGCGCCGAAGCCACAATTAGAGGCCTGCTGAGCGCCGCAGCAGTGGTGACACCAGCAGCAGACGCTCACGGAGAAACCTCTAACGCCACAACAGCCGGAGCCGATCACGGTTGCTTCCCCCACATCAACCACGGAACCGAGCTGCAGCACAAGATCGCAGTGGGACTCCAGTGGTTCACCGTGATCGTGGCTATCGTGCAGCTCATCTTCTACGGTTGGCACAGCTTCAAGGCCACAACCGGCTGGGAGGAGGTCTACGTCTGCGTGATCGAGCTCGTCAAGTGCTTCATCGAGCTGTTCCACGAGGTCGACAGCCCAGCCACAGTGTACCAGACCAACGGAGGAGCCGTGATTTGGCTGCGGTACAGCATGTGGCTCCTGACTTGCCCCGTGATCCTGATCCACCTGAGCAACCTGACCGGACTGCACGAAGAGTACAGCAAGCGGACCATGACCATCCTGGTGACCGACATCGGCAACATCGTGTGGGGGATCACAGCCGCCTTTACAAAGGGCCCCCTGAAGATCCTGTTCTTCATGATCGGCCTGTTCTACGGCGTGACTTGCTTCTTCCAGATCGCCAAGGTGTATATCGAGAGCTACCACACCCTGCCCAAAGGCGTCTGCCGGAAGATTTGCAAGATCATGGCCTACGTCTTCTTCTGCTCTTGGCTGATGTTCCCCGTGATGTTCATCGCCGGACACGAGGGACTGGGCCTGATCACACCTTACACCAGCGGAATCGGCCACCTGATCCTGGATCTGATCAGCAAGAACACTTGGGGCTTCCTGGGCCACCACCTGAGAGTGAAGATCCACGAGCACATCCTGATCCACGGCGACATCCGGAAGACAACCACCATCAACGTGGCCGGCGAGAACATGGAGATCGAGACCTTCGTCGACGAGGAGGAGGAGGGAGGAGTG。

[0016]

[0030] In some embodiments, a contemplated optogenetic protein is a protein having the amino acid sequence of SEQ ID NO: 5 below. METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFPHINHGTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIELFHEVDSPATVYQTNGGAVIWLRYSMWLLTCPVILIHLSNLTGLHEEYS KRTMTILVTDIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKIMAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIHEHILIHGDIRKTTTINVAGENMEIETFVDEEEEGGV.

[0017]

[0031] In some embodiments, a contemplated optogenetic protein is fused to a reporter protein.

[0032] The reporter protein may include, for example, a fluorescent protein, luciferase, beta-galactosidase, alkaline phosphatase, beta-lactamase, a protein or enzyme that confers resistance to cytotoxic substances or to minimal media, a cytotoxic or pro-apoptotic protein, or a protein that alters the growth or morphology of the cells in which the protein is expressed. For example, in some embodiments, the reporter protein fused to the optogenetic protein is a fluorescent protein, e.g., luciferase, and / or the reporter protein may be fused to an optogenetic protein that includes one of beta-galactosidase, alkaline phosphatase, or beta-lactamase. In some embodiments, the reporter protein fused to the optogenetic protein is a protein or enzyme that confers resistance to cytotoxic substances or to minimal media, e.g., a cytotoxic or pro-apoptotic protein, and / or a protein that alters the growth or morphology of the cells in which the protein is expressed.

[0018]

[0033] In some embodiments, the reporter protein fused to the optogenetic protein is a fluorescent protein, for example, the reporter protein is fused to the 3' end of the optogenetic protein.

[0019]

[0034] The fluorescent protein of the present disclosure can be any suitable fluorescent protein, such as a green fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, or a red fluorescent protein. For example, the reporter protein is a green fluorescent protein.

[0020]

[0035] Exemplary fluorescent proteins include green fluorescent protein (GFP) (e.g., excitation maximum 395 / 475 nm, emission maximum 509 nm and relative brightness (e.g., 48% of EGFP)), as well as green fluorescent proteins such as EFTP, Emerald, superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, or T-Sapphire; blue fluorescent proteins such as EBFP, EBFP2, Azurite, mTagBFP; cyan fluorescent proteins such as ECFP, mECFP, cerulean, mTurqoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal); yellow fluorescent proteins such as EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, or mBanana; The fluorescent protein may be selected from orange fluorescent proteins such as Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), DsRed-Monomer, and mTangerine; or red fluorescent proteins such as mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, or AQ143.

[0021]

[0036] In some embodiments, the fluorescent protein is a green fluorescent protein. For example, in some embodiments, the green fluorescent protein is GFP. In some embodiments, the green fluorescent protein is EGFP. In some embodiments, the green fluorescent protein is Emerald. In some embodiments, the green fluorescent protein is superfolder GFP. In some embodiments, the green fluorescent protein is Azami Green. In some embodiments, the green fluorescent protein is mWasabi. In some embodiments, the green fluorescent protein is TagGFP. In some embodiments, the green fluorescent protein is TurboGFP. In some embodiments, the green fluorescent protein is AcGFP. In some embodiments, the green fluorescent protein is ZsGreen. In some embodiments, the green fluorescent protein is T-Sapphire.

[0022]

[0037] Contemplated optogenetic fusion proteins may be encoded by a nucleic acid sequence that is at least 94% identical to the nucleic acid molecule of SEQ ID NO: 2. In some embodiments, the optogenetic fusion protein is encoded by a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid molecule of SEQ ID NO: 2.

[0023]

[0038] For example, the optogenetic fusion protein may be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:2 below.

[0024]

[0039] In some embodiments, the optogenetic fusion protein may have the amino acid sequence of SEQ ID NO:3 below. .

[0025]

[0040] In some embodiments, a composition of the present disclosure comprises an AAV2 vector having a gene that expresses an optogenetic fusion protein (e.g., a nucleic acid encoding a protein having 98% or 99% identity to the amino acid sequence of SEQ ID NO: 3). For example, in some embodiments, a composition of the present disclosure comprises an AAV2 vector having a nucleic acid encoding a protein having 98% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, a composition of the present disclosure comprises an AAV2 vector having a nucleic acid encoding a protein having 99% identity to the amino acid sequence of SEQ ID NO: 3.

[0026]

[0041] Effective intracellular concentrations of the genes disclosed herein can be achieved through stable expression (e.g., by integration into the nuclear or mitochondrial genome of mammalian cells) of vectors encoding genes, such as genes expressing optogenetic proteins, as described herein. To introduce such genes into mammalian cells, the genes can be incorporated into vectors. Vectors can be introduced into cells by various methods, including transformation, transfection, direct uptake, gene guns, and encapsulation of vectors in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. The genes disclosed herein can also be introduced into mammalian cells by targeting a vector containing a polynucleotide encoding such a gene to cell membrane phospholipids. For example, the vector can be targeted to phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to the VSV-G protein, a viral protein with affinity for all cell membrane phospholipids. Stable expression of exogenous polynucleotides in mammalian cells can be achieved by integrating a polynucleotide containing a gene into the nuclear genome of the mammalian cell. The expression vectors used in the compositions and methods described herein contain polynucleotide sequences encoding genes and additional sequence elements, for example, used for expressing these genes and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used include plasmids containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' UTR regions, internal ribosome entry sites (IRES), and polyA to direct the efficient transcription of genes carried on the expression vector.Expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, nourseothricin, etc.

[0027]

[0042] The genes described herein can be incorporated into recombinant AAV (rAAV) vectors to facilitate their introduction into cells, such as target cells, and / or for administration.The rAAV vectors useful in conjunction with the compositions and methods described herein include recombinant nucleic acid constructs that contain (1) genes and (2) nucleic acids that facilitate the expression of heterologous genes.The viral nucleic acid may contain AAV sequences (e.g., functional ITRs) that are required in cis for DNA replication and packaging into virions.Such rAAV vectors may also contain marker or reporter genes.

[0028]

[0043] Useful rAAV vectors include vectors in which one or more naturally occurring AAV genes have been deleted, either in whole or in part, but which retain functional flanking ITR sequences. The AAV ITRs can be of any serotype suitable for a particular application (e.g., from serotype 2 or 5). In some embodiments, the AAV contains two ITRs, the first ITR (ITR1) and the second ITR (ITR2), where ITR1 is located 5' to a polynucleotide encoding an optogenetic fusion protein and ITR2 is located 3' to the polynucleotide, forming a cassette containing the structure ITR1-optogenetic fusion protein-ITR2, for example, the two ITRs are AAV serotype 2 ITRs.

[0029]

[0044] The genes described herein (e.g., genes encoding optogenetic proteins) can be incorporated into rAAV virions to facilitate the introduction of nucleic acids or vectors into cells. AAV capsid proteins constitute the external, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, which are required for virion assembly. rAAV virions useful in conjunction with the compositions and methods described herein include virions derived from various AAV serotypes, including AAV1, 2, 3, 4, 5, 6, 7, 8, and 9. Pseudotyped rAAV vectors are also useful in conjunction with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype that have been shoot-typed with capsid genes derived from a serotype other than the given serotype (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, among others). For example, a representative pseudotype vector is an AAV2 vector encoding a therapeutic protein pseudotyped with a capsid gene from AAV serotype 8 or AAV serotype 9. For example, in certain embodiments, AAV virions with mutations within the virion capsid may be used to infect specific cell types more efficiently than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations to facilitate targeting of AAV to specific cell types. Other rAAV virions that can be used in the methods of the invention include capsid hybrids generated by molecular breeding of viruses and by exon shuffling.

[0030]

[0045] Contemplated vectors may contain appropriate expression control sequences, including, but not limited to, transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing, and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if necessary, sequences that enhance protein processing and / or secretion. In eukaryotic cells, expression control sequences typically include a promoter, an enhancer, such as those derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, which may include splice donor and acceptor sites. The polyadenylation sequence is generally inserted following the transgene sequence and before the 3' ITR sequence. In one embodiment, bovine growth hormone polyA is used. Another regulatory component of a useful vector is an internal ribosome entry site (IRES). IRES sequences, or other suitable systems, may be used to produce more than one polypeptide from a single gene transcript. An IRES (or other suitable sequence) is used to produce proteins containing more than one polypeptide chain or to express two different proteins from or within the same cell. An example of an IRES is the poliovirus internal ribosome entry sequence, which supports transgene expression in retinal cells.

[0031]

[0046] The promoter used in the vector can be selected from a number of constitutive or inducible promoters capable of expressing a selected optogenetic protein in ocular cells. In one embodiment, the promoter is cell-specific. The term "cell-specific" means that the particular promoter selected for the recombinant vector can direct the expression of a selected optogenetic protein in a specific cell type. In one embodiment, the promoter is specific for the expression of an optogenetic protein in retinal ganglion cells. In one embodiment, the promoter is specific for the expression of an optogenetic protein in bipolar cells. For example, a light-sensitive protein can be expressed in retinal ganglion cells via a ganglion cell-specific gene promoter, such as Thy-I.

[0032]

[0047] The structure of the ganglion cell layer (GCL) of the primate retina may also allow for targeting of specific cell types, for example, using mechanical means. Ganglion cell bodies are located within the GCL. Near the fovea, the GCL is at its thickest and contains several layers of cell bodies. The somata of different retinal ganglion cell types are located in different locations (e.g., ON-type ganglion cells are more vitreous when observed by multielectrode recording), which may allow ganglion cells to be preferentially targeted (e.g., by intravitreal administration of a viral vector (e.g., AAV expressing an optogenetic protein)). Selective targeting of ON-type cells may also be achieved using a contemplated vector containing a non-specific promoter (e.g., CAG) because the cells are closer to the retinal surface (i.e., in the vitreous), and AAV does not penetrate the retina well when delivered by intravitreal injection.

[0033]

[0048] For example, significant visual recovery can be achieved by administering neurally encoded stimuli (e.g., via a neural coding device) and a contemplated optogenetic vector (e.g., an AAV expressing an optogenetic protein) that can preferentially target, for example, ON-type ganglion cells. The neural coding device can deliver optogenetic stimuli (i.e., light stimuli that activate optogenetic proteins) that are specific to different ganglion cell types (e.g., stimuli following an ON-type cell neural code or stimuli following an OFF-type cell neural code). When ON-type neurally encoded stimuli are applied to ON-type ganglion cells expressing optogenetic proteins, the ON-type cells can send normal ON-type visual signals to the brain because the ON-type code causes the cells to substantially mimic the normal response of ON-type cells.

[0034]

[0049] Examples of constitutive promoters that can be included in the vectors contemplated herein include, but are not limited to, the CAG promoter, the CMV immediate-early enhancer / chicken actin (CA) promoter-exon 1-intron 1 element, the RSV LTR promoter / enhancer, the SV40 promoter, the CMV promoter, the 381 bp CMV immediate-early gene enhancer, the dihydrofolate reductase promoter, the phosphoglycerate kinase (PGK) promoter, and the 578 bp CBA promoter-exon 1-intron 1. For example, a contemplated promoter is the CAG promoter. In some embodiments, the CAG promoter has the nucleic acid sequence of SEQ ID NO: 6. TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGCGCGCCAGGCGGGGC GGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG.

[0035]

[0050] In some embodiments, the vectors contemplated herein include an enhancer, such as, for example, the WPRE enhancer. In some embodiments, the WPRE enhancer has the nucleic acid sequence of SEQ ID NO: 7 below. .

[0036]

[0051] In some embodiments, vectors contemplated herein include a polyadenylation (poly(A)) element, such as, for example, SV40 poly(A). In some embodiments, the SV40 poly(A) has the nucleic acid sequence of SEQ ID NO: 8: TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTT.

[0037]

[0052] A vector contemplated herein can include, for example, an expression cassette having the nucleic acid sequence of SEQ ID NO:9 below.

[0038]

[0053] Vectors contemplated herein include AAV2, which comprises a DNA polynucleotide encoding an optogenetic fusion protein or its RNA equivalent. In some embodiments, the optogenetic protein is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:4 or is a protein having the amino acid sequence of SEQ ID NO:5 fused to a reporter protein. In some embodiments, the reporter protein is GFP. For example, compounds of the present disclosure are exemplified by AAV vectors having a nucleic acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:1. For example, in some embodiments, the AAV vector has a nucleic acid sequence at least 91% identical to the nucleic acid molecule of SEQ ID NO:1. In some embodiments, the AAV vector has a nucleic acid sequence at least 92% identical to the nucleic acid molecule of SEQ ID NO:1. In some embodiments, the AAV vector has a nucleic acid sequence at least 93% identical to the nucleic acid molecule of SEQ ID NO:1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 94% identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 95% identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 96% identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 97% identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 98% identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 99% identical to the nucleic acid molecule of SEQ ID NO: 1.

[0039]

[0054] In some embodiments, the AAV vectors described herein comprise the nucleic acid sequence of SEQ ID NO:1 below.

[0040] Pharmaceutical Compositions and Routes of Administration

[0055] Any one of the compositions described herein, such as a vector carrying a gene encoding an optogenetic protein (SEQ ID NO: 5), can be formulated into a pharmaceutical composition for administration to a mammalian (e.g., human) subject in a biologically compatible form suitable for in vivo administration. The compositions disclosed herein may be formulated in any suitable vehicle for delivery to a subject (e.g., human). For example, the composition may be formulated into a pharmaceutically acceptable suspension, dispersion, solution, or emulsion. Suitable vehicles include saline and liposomal formulations. Pharmaceutically acceptable carriers may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Recombinant human albumin (rAlbumin Human NF RECOMBUMIN® Prime) may also be used as a stabilizer with AAV vectors (Albumedix, Nottingham, UK). Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases and the like. Colloidal dispersion systems may also be used for targeted gene delivery. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The compositions described herein may be used in the form of a free base, a salt, a solvate, and as a prodrug. All forms are within the scope of the methods described herein.

[0041]

[0056] The optogenetic proteins and cell-specific promoters for use in the target ocular cells described above can be evaluated for contamination by conventional methods and then formulated into pharmaceutical compositions for retinal injection. Such formulations include the use of pharmaceutically and / or physiologically acceptable solvents or carriers, particularly those suitable for intravitreal, retinal, or subretinal injection, such as buffered saline or other buffers, e.g., HEPES, to maintain the pH at an appropriate physiological level. Also, the presence of glycerol.

[0042]

[0057] According to the method of the present invention for treating an ocular disorder characterized by retinal degeneration, the pharmaceutical composition described above is administered to a subject with such a blinding disease by intravitreal, retinal, or subretinal injection.

[0043] dosage

[0058] For example, a vector described herein, such as an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 9, can be expressed in an amount of approximately 1 x 10 11 ~1×10 13 (For example, about 2 × 10 11 vg / eye ~ approx. 1×10 13 vg / eye, 3×10 11 vg / eye ~ approx. 1×10 13 vg / eye, 4x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 5x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 6x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 7x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 8x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 9x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 1x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 2x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 3x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 4x1012 vg / eye ~ approx. 1x10 13 vg / eye, 5x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 6x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 7x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 8x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 9x10 12 vg / eye ~ approx. 1x10 13 Alternatively, an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 9 may be administered to a human patient at an initial vector dose of about 1 x 10 12 vg / eye ~ approx. 1×10 13 vg / eye or more. Such a dose (e.g., 1 x 10 12 vg / eye) can, for example, produce a supra-linear improvement in photosensitivity (e.g., improvement / dose increase is not linear).

[0044]

[0059] In some embodiments, the amount of initial vector (e.g., an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 9) is about 1 x 10 12 ~Approx. 1×10 14 vg / mL (e.g., approximately 2 × 10 12 vg / mL ~ approx. 1×10 14 vg / mL, 3 × 10 12 vg / mL ~ approx. 1×10 14 vg / mL, 4x10 12 vg / mL ~ approx. 1x10 14 vg / mL, 5x10 12 vg / mL ~ approx. 1x10 14 vg / mL, 6x10 12 vg / mL ~ approx. 1x10 14 vg / mL, 7x10 12 vg / mL ~ approx. 1x10 14 vg / mL, 8x10 12 vg / mL ~ approx. 1x10 14 vg / mL, 9x10 12 vg / mL ~ approx. 1x10 14 vg / mL, 1x10 13vg / mL ~ approx. 1x10 14 vg / mL, 2x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 3x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 4x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 5x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 6x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 7x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 8x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 9 × 10 13 At a concentration of 0.05 mg / mL, the volume is about 70 μL to about 130 μL (e.g., about 80 μL to about 120 μL, about 90 μL to about 110 μL, or about 100 μL).

[0045]

[0060] In some embodiments, the AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 1 is about 1 x 10 11 ~1×10 13 (For example, about 2 × 10 11 vg / eye ~ approx. 1×10 13 vg / eye, 3×10 11 vg / eye ~ approx. 1×10 13 vg / eye, 4x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 5x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 6x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 7x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 8x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 9x10 11 vg / eye ~ approx. 1x10 13 vg / eye, 1x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 2x10 12 vg / eye ~ approx. 1x1013 vg / eye, 3x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 4x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 5x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 6x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 7x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 8x10 12 vg / eye ~ approx. 1x10 13 vg / eye, 9x10 12 vg / eye ~ approx. 1x10 13 Alternatively, an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 1 may be administered to a human patient at an initial vector dose of about 1 x 10 12 vg / eye ~ approx. 1×10 13 vg / eye or more. Such a dose (e.g., 1 x 10 12 vg / eye) can result in, for example, a supra-linear improvement in photosensitivity (e.g., improvement / dose increase is not linear).

[0046]

[0061] In some embodiments, the amount of initial vector (e.g., an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 1) is about 1 x 10 12 ~Approx. 1×10 14 vg / mL (e.g., approximately 2 × 10 12 vg / mL ~ approx. 1×10 14 vg / mL, 3 × 10 12 vg / mL ~ approx. 1×10 14 vg / mL, 4x10 12 vg / mL ~ approx. 1x10 14 vg / mL, 5x10 12 vg / mL ~ approx. 1x10 14 vg / mL, 6x10 12 vg / mL ~ approx. 1x10 14 vg / mL, 7x10 12 vg / mL ~ approx. 1x10 14 vg / mL, 8x10 12 vg / mL ~ approx. 1x10 14vg / mL, 9x10 12 vg / mL ~ approx. 1x10 14 vg / mL, 1x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 2x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 3x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 4x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 5x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 6x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 7x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 8x10 13 vg / mL ~ approx. 1x10 14 vg / mL, 9 × 10 13 At a concentration of 0.05 mg / mL, the volume is about 70 μL to about 130 μL (e.g., about 80 μL to about 120 μL, about 90 to about 110 μL, or about 100 μL).

[0047]

[0062] In some cases, booster doses may be desirable.The amount and necessity of such booster doses can be monitored by the attending physician using, for example, the retinal and visual function tests and visual behavior tests described herein.Other similar tests may also be used to determine the status of the treated subject over time.The selection of appropriate tests can be made by the attending physician.Alternatively, the method of the present invention may also include injecting a larger volume of virus-containing solution in a single or multiple injections, so as to enable a level of visual function close to that found in normal retina.

[0048]

[0063] Also contemplated is a combination therapy that includes administering light stimulation to a patient after initial administration of an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 9. For example, the light stimulation can be ambient light (e.g., about 1×10 -7 mW / mm 2 ~Approx. 1×10 -2 mW / mm2 at a level of 1 × 10 -7 mW / mm 2 ~about 0.1mW / mm 2 may be given using (at the level of

[0049]

[0064] Alternatively, or in addition, the combination therapy may include administering to the patient an initial dose of Compound A (an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 9) followed by neurally encoded stimulation. In some embodiments, the neurally encoded light stimulation is administered at a dose of about 5×10 -2 mW / mm 2 ~about 0.1mW / mm 2 , about 1×10 -2 mW / mm 2 ~about 0.1mW / mm 2 , about 1×10 -3 mW / mm 2 ~about 0.1mW / mm 2 , about 1×10 -4 mW / mm 2 ~about 0.1mW / mm 2 , or approximately 1 × 10 -5 mW / mm 2 ~about 0.1mW / mm 2 It is administered at .

[0050]

[0065] In some embodiments, the neural-encoded stimulation includes using a light delivery device to send light pulses at the neural cord of the retina. Such stimulation may cause ganglion cell firing to mimic normal retinal firing. For example, in some embodiments, the neural-encoded stimulation includes using a light delivery device to send light pulses at the neural cord of the retina to cause ganglion cell firing to mimic normal retinal firing.

[0051]

[0066] Stimulation may be by any suitable mechanism, including optogenetic stimulators or other light-delivering stimulators, as described in U.S. Pat. No. 9,220,634, which is incorporated herein by reference in its entirety.

[0052]

[0067] The stimulus output interface may be a digital light processing (DLP) device. This DLP device would output a pulse of light. The light pulse would then drive optogenetic proteins in the ganglion cells, causing the ganglion cells to fire as an encoder instruction. In this example, the output interface functions as follows: the encoder output is sent from the processing unit to the output interface (e.g., DLP). The output interface then converts binary data representing action potential duration into a light pulse using a digital micromirror device (DMD) coupled with a light-emitting diode (LED). The DMD may be a grid of mirrors whose position can be switched with high temporal and spatial resolution. When the encoder instructs a ganglion cell at location (x, y) to fire an action potential, the mirror at location (x, y) on the device is switched to the "on" position for a short period of time (e.g., on the millisecond timescale) and then switched back to the "off" position. This reflects light from the LED onto the retina for a short period of time, triggering a light pulse at location (x, y). This light pulse stimulates the retinal ganglion cell at location (x, y) to fire.

[0053]

[0068] In one embodiment, the stimulation output interface is a digital light processing (DLP) device as described above. The standard light source on the DLP device may be replaced with a high-brightness LED sufficient to activate an optogenetic protein such as SEQ ID NO:5. As described above, the DLP may include a digital micromirror device (DMD) (DLP1 3010LC, Texas Instruments, Dallas, TX), which consists of a grid of mirrors, each of which can be switched to reflect light from an LED onto the retina when a retinal ganglion cell at that location fires. Data is sent from the encoding device using the retinal neural code to the output interface over a high-definition multimedia interface (HDMI®, 22 MB / sec). The position of each mirror on the DMD is controlled with high temporal resolution (e.g., when an encoder signals a ganglion cell to fire an action potential, the mirror at the corresponding location is switched to the "on" position for a short period of time (e.g., 1.4 ms)). The mirror switching state causes the device to output a pulse of light to the corresponding location, which prompts the targeted retinal ganglion cell to fire an action potential. The mirror switching time may be shorter or longer, e.g., from 0.1 ms to 10 ms, depending on the amount of light required to activate the cell. In this embodiment, the mirror array on the DMD may be 480 x 320 mirrors, thus allowing over 150,000 locations (e.g., cells) to be independently targeted. It is contemplated that DLPs may also have more mirrors, e.g., 1024 x 768 mirrors, as in the case of the DLP5500A (Texas Instruments, Dallas, TX), thus allowing even more locations to be independently stimulated. Data movement between the encoding device and the interface follows standard specifications, as assigned in Texas Instruments Application Report DLPA021 - January 2010 - "Using the DLP Pico 2.0 Kit for Structured Light Applications."

[0054]

[0069] DLP is an example of an electric potential output interface. It is considered that the output interface can be implemented using any device that can activate optogenetic proteins. Examples include but are not limited to digital micromirror devices; LED arrays; spatial light modulators; fiber optics; lasers; xenon lamps; scanning mirrors; liquid crystal displays (LCDs); and combinations thereof (Golan L et al., 2009; Grossman N et al., 2010).

[0055]

[0070] Alternatively, or in addition, the combination therapy may include administering a corticosteroid (e.g., prednisone, prednisolone, cortisone, methylprednisolone, dexamethasone, betamethasone, or hydrocortisone), e.g., administered orally. In some embodiments, the corticosteroid is prednisone / prednisolone.

[0056]

[0071] The corticosteroid may be administered to the patient 1 to 3 (e.g., 2) days prior to intravitreal administration of an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 1. For example, in some embodiments, the patient is administered oral corticosteroids 1 to 3 days prior to intravitreal administration of SEQ ID NO: 1.

[0057]

[0072] In some embodiments, oral prednisone / prednisolone is administered at 1 mg / kg. In some embodiments, oral prednisone / prednisolone is administered at this dose for 7 days post-injection, including the day of injection (e.g., the day SEQ ID NO: 1 is administered), for a total of 10 days. For example, in some embodiments, the corticosteroid is oral prednisone / prednisolone, administered at 1 mg / kg prednisone / prednisolone for 1-3 (e.g., 2) days prior to injection or on the day of injection, and optionally continued at this dose for 7 days post-injection, including the day of injection, for a total of 10 days.

[0058]

[0073] Alternatively, or in addition, the combination therapy may include administering valacyclovir or acyclovir. Valacyclovir may be administered orally at 1000 mg / day or acyclovir at 400 mg twice daily, starting 3-7 days prior to intravitreal administration. For example, in some embodiments, valacyclovir may be administered orally at 1000 mg / day, starting 3-7 days prior to intravitreal administration. In some embodiments, acyclovir may be administered orally at 400 mg twice daily, starting 3-7 days prior to intravitreal administration.

[0059] Treatment method

[0074] Provided herein are methods for treating a retinal degenerative disorder in a human patient in need thereof, comprising administering an effective vector dose of an AAV2 vector carrying a gene expressing an optogenetic protein into the patient's eye, e.g., the vector comprises a sequence having, e.g., 95% to 100% identity over the length of SEQ ID NO: 1. Retinal degenerative diseases contemplated include retinitis pigmentosa (RP), age-related macular degeneration, Usher syndrome, Stargardt macular dystrophy, Leber congenital amaurosis, and Bardet-Biedl syndrome. Retinal disorders, including retinal detachment and retinal vascular occlusion, are also contemplated.

[0060]

[0075] Retinitis pigmentosa includes autosomal recessive, autosomal dominant, and X-linked recessive forms of retinitis pigmentosa. The most common type of retinitis pigmentosa is the autosomal recessive type, which accounts for approximately 35% of cases. The second most common type is the autosomal dominant type, which accounts for 10% of cases. The least common type is the X-linked (X-linked recessive) type, which accounts for approximately 5% of cases.

[0061]

[0076] Diseases in which retinal degeneration occurs as a complication are also contemplated by this disclosure. Such diseases include snowflake vitreoretinal degeneration; choroidal neovascularization caused by adult-onset foveomacular dystrophy; Bietti crystalline corneoretinal dystrophy; and diabetic retinopathy. A partial list of diseases in which retinal degeneration occurs as a symptom is aceruloplasminemia; adrenoleukodystrophy; Alström disease; Alström syndrome; respiratory insufficiency thoracic dysplasia; Bonnemann-Meinecke-Reich syndrome; Bonnemann-Meinecke-Reich syndrome; syndrome); CDG syndrome type IA; chorioretinopathy dominant-microcephaly; total choroidal atrophy-hypopituitarism; congenital glycosylation disorder type IA; congenital glycosylation disorder type Ia; cystinosis; hypotrichosis, syndactyly, and retinal degeneration; Jeune syndrome; mucolipidosis type IV; mucolipidosis type 4; mucopolysaccharidosis; muscle-eye-brain syndrome; neonatal ALD; olivopontocerebellar atrophy type 3; osteopetrosis, autosomal recessive type 4; pigmentary retinopathy; pseudoadrenoleukodystrophy; retinoschisis, X-linked; retinoschisis, X-linked, juvenile type; Santavoli disease; spastic paraplegia, autosomal recessive; and Werner syndrome. The present method can be used to treat any mammalian subject with RP. Any of the compositions described herein can be used in treatment methods, such as improving light sensitivity in a subject in need thereof. Such treatment may have a desired therapeutic effect, for example, in partially or completely curing the disease and / or adverse effects resulting from the disease.

[0062]

[0077] The human patient may have progressive blindness due to RP or mild, moderate, or severe visual impairment. For example, a contemplated patient may have poor light perception (BLP) or no light perception (NLP) at baseline (i.e., before treatment).

[0063]

[0078] Contemplated patients may have early-stage disease and / or have baseline visual acuity of only 20 / 200 or about 20 / 4200, e.g., the patient may have baseline visual acuity of about 20 / 200. Contemplated patients may have baseline visual acuity of no more than finger counting ability. For example, in some embodiments, the patient has baseline visual acuity of no more than 20 / 200 or less than finger counting ability. Contemplated patients may have baseline visual acuity of no more than 20 / 60 (or, e.g., about 20 / 60 to about 20 / 200 or more, and / or have a baseline visual field test showing, e.g., mild, moderate, or severe visual field impairment (e.g., tunnel vision)).

[0064]

[0079] The beneficial treatment effects of the compositions and methods described herein, such as the ability of Compound A described herein to cause ganglion cell firing to mimic normal retinal firing, may manifest clinically in a variety of ways. For example, in some embodiments, following initial administration of Compound A, patients have improved light sensitivity, evidence of which includes an increase in the amplitude of the electroretinogram (ERG) response and / or visual evoked potential (VEP) compared to baseline. In some embodiments, patients have improved light sensitivity manifested as an increase in the amplitude of the ERG or VEP response compared to baseline 4 months or more (e.g., 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more) after initial administration of Compound A.

[0065]

[0080] In some embodiments, the patient's light perception threshold is decreased as measured by a staircase or dichotomous search procedure following the initial administration of Compound A. In some embodiments, the patient's light perception threshold is decreased as measured by a staircase or dichotomous search procedure compared to baseline at 4 months or more (e.g., 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more) after the first dose of Compound A.

[0066]

[0081] In some embodiments, following the first dose of Compound A, the patient's ability to detect movement and / or direction of movement is increased as measured by a standard two-arm forced-choice paradigm. In some embodiments, the patient's ability to detect movement and / or direction of movement is increased as measured by a standard two-arm forced-choice paradigm at 3 months or more (e.g., 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more) after the first dose of Compound A compared to baseline.

[0067]

[0082] In some embodiments, following the first dose of Compound A:1, the patient's shape detection ability, as measured by a standard two-arm forced-choice paradigm, is increased. In some embodiments, the patient's shape detection ability, as measured by a standard two-arm forced-choice paradigm, is increased relative to baseline at 4 months or more (e.g., 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more) after the first dose of Compound A.

[0068]

[0083] In some embodiments, following the first dose of Compound A, the patient has an increased ability to detect and / or distinguish colors. For example, in some embodiments, following the first dose of Compound A, the patient has an increased ability to detect colors. In some embodiments, following the first dose of Compound A, the patient has an increased ability to distinguish colors. In some embodiments, the patient has an increased ability to detect colors 4 months or more (e.g., 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more) after the first dose of Compound A compared to baseline. In some embodiments, the patient has an increased ability to distinguish colors 4 months or more (e.g., 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more) after the first dose of Compound A compared to baseline.

[0069]

[0084] Other methods for measuring clinical efficacy include flash visual evoked responses (VEPs), pupillary light reflexes (PLRs), ERGs (including bilateral full-field ERGs), and nystagmus testing. Analysis may follow the International Standard Guidelines for Clinical Visual Electrophysiology. Pupillary responses may be recorded simultaneously in both eyes. Nystagmus may be characterized qualitatively and quantitatively by analyzing the motion path of videos taken at baseline and at various desired time points after treatment. Interpupillary distance may be measured directly from video frames. Subjective measures include, but are not limited to, standard tests of visual acuity (VA), dynamic visual fields, and mobility tests to assess the subject's ability to navigate an obstacle course. Mobility tests may use a different maze each time the test is administered, and the number of obstacles avoided or encountered, the number of landmarks identified, and the time spent in the maze can then be assessed.

[0070]

[0085] The compositions and methods described herein may provide beneficial clinical effects that may be long-lasting. For example, after administration of an initial dose of an AAV2 vector carrying a gene that expresses an optogenetic protein and comprising the nucleic acid sequence of SEQ ID NO: 1, a patient with RP may exhibit a therapeutic effect compared to baseline at 3 months or more (e.g., 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more) after administration. For example, in some embodiments, after administration of an initial dose of such a vector carrying a gene that expresses an optogenetic protein, a patient with RP may exhibit a therapeutic effect compared to baseline at 4, 5, 6, or 7 months or more after administration.

[0071] definition

[0086] The features and other details of the present disclosure will now be described in more detail. Certain terms used in the specification, examples, and appended claims are collected here. These definitions should be read and understood in light of the remainder of the disclosure as by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0072]

[0087] As used herein, "adeno-associated virus" (AAV) refers to any virus, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHPB, AAV. AAV vector refers to a vector derived from an adeno-associated virus serotype, including AV.PHP.EB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV-TT, AAV-DJ8, or AAV.HSC 16. An AAV vector can lack, in whole or in part, one or more of the AAV wild-type genes, such as the rep and / or cap genes, but retain functional flanking inverted terminal repeat (ITR) sequences. A functional ITR sequence promotes the rescue, replication, and packaging of AAV virions. Therefore, an AAV vector is defined herein as comprising at least a sequence (e.g., a functional ITR) required in cis for viral replication and packaging. The ITR does not need to be a wild-type polynucleotide sequence, and may be altered, for example, by the insertion, deletion, or substitution of nucleotides, as long as the sequence provides functional rescue, replication, and packaging. An AAV expression vector is constructed using known techniques to provide at least the following control elements in the direction of transcription, including a transcription initiation region, a DNA of interest (e.g., a vector carrying a gene encoding the optogenetic protein of the present disclosure (e.g., SEQ ID NO: 5)), and a transcription termination region, as operably linked components.The terms "adeno-associated virus inverted terminal repeat" and "AAV ITR" refer to art-recognized regions adjacent to each end of the AAV genome that function together in cis as an origin of DNA replication and as a packaging signal for the virus. The AAV ITR, together with the AAV rep coding region, provides efficient excision and integration of a polynucleotide sequence inserted between the two adjacent ITRs in a mammalian genome. The polynucleotide sequence of the AAV ITR region is known. As used herein, "AAV ITR" does not necessarily include the wild-type polynucleotide sequence, which may be altered, for example, by the insertion, deletion, or substitution of nucleotides. Moreover, AAV ITRs include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.EB, AAV.PHP.A, AAV.PHP.B, AAV.PHP.EB, AAV.PHP.A, AAV.PHP.B, AAV.PHP.C, AAV.PHP.D, AAV.PHP.E, AAV.PHP.F, AAV.PHP.H ... It may be derived from any of several AAV serotypes, including AV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV-TT, AAV-DJ8, or AAV.HSC16. Furthermore, the 5' and 3' ITRs flanking a selected polynucleotide sequence in an AAV vector need not be identical to or derived from the same AAV serotype or isolate, so long as these ITRs function as intended, for example, to allow excision and rescue of the sequence of interest from the host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome if the AAV Rep genome product is present in the cell.Additionally, AAV ITRs include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.EB, AAV.PHP.A ... It may be derived from any of several AAV serotypes, including AV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV-TT, AAV-DJ8, or AAV.HSC16.

[0073]

[0088] As used herein, "combination therapy," "administered in combination," or "further administering" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen defines the dose and periodicity of each agent so that the effects of the separate agents or treatments on the subject overlap. In some embodiments, the delivery of two or more agents or treatments is simultaneous or concomitant, and the agents may be co-formulated. In other embodiments, the two or more agents are not co-formulated but are administered in a sequential manner as part of a prescribed regimen. In some embodiments, the administration of two or more agents or treatments in combination aims to achieve a reduction in symptoms or other parameters related to the disorder that is greater than would be observed with one agent or treatment delivered alone or in the absence of the other agent or treatment. The effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each therapeutic agent can be carried out by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination can be administered by intravenous injection, and the second therapeutic treatment of the combination therapy can be photostimulation. In some embodiments, the combination therapy includes photostimulation.

[0074]

[0089] Throughout the specification and claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated word or group of words but not the exclusion of any other word or group of words.

[0075]

[0090] As used herein, "Compound A" refers to a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:1.

[0091] As used herein, the term " dose " refers to the amount of therapeutic agent, such as the viral vector described herein, that is administered to a subject at a specific time for the treatment of a disorder, such as for treating or ameliorating one or more symptoms of retinitis pigmentosa.The therapeutic agent described herein can be administered in a single dose or in multiple doses over a treatment period.In each case, the therapeutic agent can be administered using one or more unit dosage forms of the therapeutic agent, which is a term that refers to one or more separate compositions that contain the therapeutic agent and that together constitute a single dose of the drug.

[0076]

[0092] As used herein, the terms "effective amount," "therapeutically effective amount," and the like, when used to refer to a composition described herein, such as a vector having a gene encoding an optogenetic protein (SEQ ID NO: 5), refer to an amount sufficient to produce a beneficial or desired result (e.g., expression of an optogenetic protein), which may include a clinical result, when administered to a subject, including a mammal (e.g., a human). For example, an effective amount of one or more compositions described herein (e.g., a vector having a gene encoding an optogenetic protein (SEQ ID NO: 5)) may achieve expression of a protein of interest compared to expression of said protein without administration of the composition of interest. An "effective amount," "therapeutically effective amount," and the like of a composition, such as a vector having a gene encoding an optogenetic protein (SEQ ID NO: 5), also includes an amount that produces a beneficial or desired result in a subject compared to a control.

[0077]

[0093] The term "pharmaceutically acceptable" means safe for administration to mammals, such as humans. In some embodiments, a pharmaceutically acceptable composition is approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or any other generally recognized pharmacopoeia for use in animals (e.g., humans). As used herein, the term "pharmaceutically acceptable" refers to compounds, anions, cations, substances, compositions, carriers, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0078]

[0094] "Operably linked" refers to a juxtaposition in which the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. As used herein, the terms "heterologous promoter" and "heterologous control region" refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature. For example, a "transcriptional control region that is heterologous to a coding region (e.g., a transgene)" is a transcriptional control region that is not normally associated with that coding region in nature.

[0079]

[0095] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refer interchangeably to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may also contain other active compounds that provide supplementary, additional, or enhanced therapeutic functions together with one or more pharmaceutically acceptable excipients.

[0080]

[0096] It should be understood that the present disclosure also provides pharmaceutical compositions comprising any of the compounds described herein in combination with at least one pharmaceutically acceptable excipient or carrier.

[0081]

[0097] As used herein, the term "pharmaceutical composition" refers to a formulation containing a compound of the present disclosure in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or unit dosage form. The amount of active ingredient in a unit dose of the composition is an effective amount and varies depending on the specific treatment involved. Those skilled in the art will recognize that routine variations in dosage may be necessary depending on the age and condition of the subject. The dosage also depends on the route of administration. Various routes are contemplated, including intravitreal, oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. In some embodiments, the composition is administered intravitreally. In one embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives, buffers, or propellants.

[0082]

[0098] As used herein, the term "pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and not biologically or otherwise undesirable and is useful in preparing pharmaceutical compositions, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable excipient" includes both one such excipient and more than one such excipient.

[0083]

[0099] Pharmaceutical compositions containing the active compounds of the present disclosure can be prepared in a generally known manner, for example, by conventional mixing, dissolving, granulating, dragee-making, gelling, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers, including excipients and / or adjuvants that facilitate the processing of the active compounds into pharmaceutical preparations. Of course, the appropriate formulation depends on the selected route of administration.

[0084]

[0100] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, polyethylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Additionally, the composition may contain isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0085]

[0101] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the required ingredients listed above, followed by filtration sterilization.Generally, dispersions are prepared by incorporating the active compound into a sterile solvent containing a basic dispersion medium and the required other ingredients from the ingredients listed above.In the case of sterile powders for preparing sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient plus any additional ingredients from a previously sterile-filtered solution of the desired ingredient.

[0086]

[0102] The active compounds can be prepared using pharmaceutically acceptable carriers that protect the compound against rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations are apparent to those skilled in the art. Materials can also be purchased from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (containing liposomes targeted to infected cells with monoclonal antibodies directed against viral antigens) can also be used as pharmaceutically acceptable carriers. These carriers can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0087]

[0103] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives the transcription of the transgene. Exemplary promoters suitable for use with the compositions and methods described herein, such as the CAG promoter, are described herein. Furthermore, the term "promoter" can also refer to synthetic promoters, such as regulatory DNA sequences that do not naturally occur in biological systems. Synthetic promoters include portions of naturally occurring promoters combined with polynucleotide sequences that do not occur in nature and can be optimized to express recombinant DNA.

[0088]

[0104] As used herein, the term "optogenetic protein" refers to proteins, such as opsins, that are photogated ion channels or pumps that absorb light at specific wavelengths. When activated by light, these channels and pumps respond by opening or closing, which directs the flow of ions into or out of cells. Different optogenetic proteins respond to different wavelengths of light, such as blue or yellow light. Furthermore, in addition to naturally occurring optogenetic proteins, the disclosure herein includes engineered optogenetic proteins, such as optogenetic proteins with point mutations that alter their absorption spectrum and / or add signals to direct intracellular trafficking, such that the engineered optogenetic protein localizes to a specific location within the cell (e.g., the cell membrane). Optogenetic proteins may include, but are not limited to, SEQ ID NO: 5. Any suitable optogenetic protein, including any currently known or later discovered / engineered optogenetic protein, may be used in the methods described herein.

[0089]

[0105] As used herein, the term "retinal degenerative disease" refers to any disease caused by degeneration of the retina, examples of which include, for example, retinitis pigmentosa (RP), age-related macular degeneration, Usher syndrome, Stargardt macular dystrophy, Leber congenital amaurosis and Bardet-Biedl syndrome, retinal detachment, and retinal vascular occlusion.

[0090]

[0106] As used herein, "retinitis pigmentosa" refers to a genetic disease of the retina in which the photoreceptor cells and pigment epithelial cells of the retina are extensively degenerated. Retinitis pigmentosa typically presents with three symptoms: night blindness (difficulty seeing in the dark), narrowing of the visual field (tunnel visual field), and reduced visual acuity. Degeneration of only rod cells among photoreceptor cells is called rod dystrophy, while degeneration of both rod and cone cells among photoreceptor cells is called rod-cone dystrophy. As used herein, "retinitis pigmentosa" refers to autosomal recessive retinitis pigmentosa, autosomal dominant retinitis pigmentosa, and X-linked recessive retinitis pigmentosa.

[0091]

[0107] A "subject" can include any animal, including mammals, mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or non-human primates, and most preferably humans. The compositions of the invention can be administered to mammals, such as humans, but also other mammals, such as animals requiring veterinary treatment, for example, domestic animals (e.g., dogs, cats, and the like), livestock (e.g., cows, sheep, pigs, horses, and the like), and laboratory animals (e.g., rats, mice, guinea pigs, non-human primates, and the like).

[0092]

[0108] As used herein, the terms "transduction" and "transducing" refer to the process of introducing a viral vector construct or a portion thereof into a cell, resulting in the subsequent expression of a transgene encoded by the vector construct or portion thereof in the cell.

[0093]

[0109] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, diethylaminoethyl (DEAE) dextran transfection, NUCLEOFECTION™, squeezeporation, sonoporation, phototransfection, MAGNETOFECTION™, impalefection, and the like.

[0094]

[0110] The terms "treat," "treatment," "treating," and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic in that it partially or completely cures the disease and / or adverse effects caused by the disease. The term "treatment," as used herein, encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) suppressing the disease, e.g., preventing the disease from increasing in severity or extent; (b) relieving the disease, e.g., causing partial or complete recovery of the disease; or (c) preventing the recurrence of the disease, e.g., preventing the disease from returning to an active state following successful treatment of previous disease symptoms or successful treatment of the disease.

[0095]

[0111] As used herein, the term "vector" includes nucleic acid vectors, e.g., DNA vectors such as plasmids, RNA vectors, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for the delivery of exogenous polynucleotides or polynucleotides encoding proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994 / 011026, which is incorporated herein by reference as it relates to vectors suitable for expressing genes of interest. Expression vectors suitable for use with the compositions and methods described herein contain polynucleotide sequences and additional sequence elements used for the expression of heterologous nucleic acid material (e.g., a vector carrying a gene encoding an optogenetic protein (SEQ ID NO: 5)) in, for example, mammalian cells. Certain vectors that can be used for the expression of the genes described herein include plasmids containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors for the expression of the genetic elements disclosed herein contain polynucleotide sequences that enhance the translation rate of these polynucleotides or improve the stability or nuclear export of RNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, IRES, and polyA to direct efficient transcription of the gene carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin. [Example]

[0096]

[0112] The present disclosure is further illustrated by the following examples, which are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present disclosure in any way. [Example]

[0097] Example 1: Safety and Efficacy of Compound A (SEQ ID NO: 1) for Optogenetic Gene Therapy

[0113] Preclinical efficacy and safety data were obtained using a vector carrying a gene expressing an optogenetic fusion protein (e.g., an AAV2 vector containing a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:2 or a nucleic acid encoding a protein having the amino acid sequence of SEQ ID NO:3 as the optogenetic fusion protein). Efficacy was assessed in a dose-dependent manner in mice using electroretinograms (ERGs). Safety was assessed in rats, non-human primates, and mice using several tests, including immunohistochemistry and cell counts (rats), electroretinograms (non-human primates), and ocular toxicity assays (mice). Results indicated that the SEQ ID NO:3-expressing vector was effective across a wide range of vector doses, stimulating light intensities, and well tolerated; no test article-related findings were observed in the anatomical and electrophysiological assays performed.

[0098] material and method Vector injection

[0114] All vectors were prepared in balanced salt solution (BSS) with 0.014% Tween 20 and delivered to the eye via intravitreal injection. In rodents, animals were anesthetized with intraperitoneal ketamine / xylazine (72 mg / kg ketamine and 4 mg / kg xylazine in mice, and 80 mg / kg ketamine and 10 mg / kg xylazine in rats), and the pupils were dilated with atropine sulfate ophthalmic solution (1%). Using a Hamilton syringe under an operating microscope, the needle was passed through the sclera into the vitreous cavity. The injection volume was 1 μL in mice and 4 μL in rats. In nonhuman primates, animals were anesthetized with a ketamine / dexmedetomidine mixture (5–10 mg / kg ketamine and 0.01–0.02 mg / kg dexmedetomidine) and then maintained with an inhaled isoflurane / oxygen mixture. The pupils were dilated with topical administration of 1% atropine sulfate and 2.5% phenylephrine hydrochloride. The vector was injected intravitreally using a 3 / 10cc U-100 insulin syringe with a 30-gauge needle. The injection volume was 80–100 μL. All animal experiments and procedures were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC).

[0099] Electroretinography (ERG)

[0115] In mice, animals were anesthetized with intraperitoneal ketamine / xylazine (72 mg / kg ketamine and 4 mg / kg xylazine), and pupils were dilated with 1% atropine sulfate and 2.5% phenylephrine hydrochloride. To perform ERG, a tungsten wire electrode was placed on the corneal surface of the eye being recorded, referenced to an electrode in the mouth. Visual stimuli were delivered by an LED stimulator using a 505 nm peak wavelength. The stimulator was placed 1.7 cm from the cornea, for a visual angle of approximately 100 degrees, with a peak intensity of 0.06 mW / mm. 2 The stimulus was delivered as a pulsed light with a frequency of 10 Hz and a pulse width of 11.2 ms. Data collection was performed using an Espion E 3 Electroretinography was performed on a testing console (Diagnosys LLC, Lowell, MA).

[0100]

[0116] For non-human primates, animals were anesthetized with a mixture of ketamine (5 mg / kg–10 mg / kg) and dexmedetomidine (0.01 mg / kg–0.02 mg / kg). Pupils were dilated with topical agents (1% atropine sulfate, 2.5% phenylephrine hydrochloride). To conduct ERG, a tungsten wire electrode was placed on the corneal surface of the eye being examined and referenced to a needle electrode placed on the scalp. Visual stimuli were delivered using a mini-Ganzfeld stimulator placed near the eye being recorded. In a Naka-Rushton fit, photopic ERG stimuli were delivered at a constant 7 cd / m (Joshi, Ly, & Viswanathan, 2017): 2 On blue background, 0.00625~1.6cd.s / m 2 A 5ms red light Ganzfeld flash was used.

[0101] Stimulation for photosafety

[0117] Light stimulation was administered over a 6- to 8-week period in 12 2- to 2.5-hour sessions. The light intensity was 0.1 mW / mm 2 The intensity was 100 Hz (peak wavelength 505 nm). The light was delivered in 5-ms pulses, as is the case when neurally encoded stimulation is used (Nirenberg and Pandarinath 2012; Yan et al. 2016). Stimuli covering a visual angle of approximately 60 degrees covered a large area of the central retina, 4 mm in diameter (Yan et al. 2016). For each session, animals were anesthetized with isoflurane (99.9%) to a depth that minimized eye movements. Each animal was placed on its left side, and the stimulus was delivered to its right eye. The pupils were dilated with atropine sulfate eye drops (1%), and the eyes were kept moist with periodic application of artificial tears (every 7 minutes). The left eye was left untreated. Between sessions, animals were exposed to normal room light with a standard day / night cycle, as is standard in rodent housing facilities. Two to four weeks after the sessions were completed, the animals were euthanized and the retinas were removed for testing.

[0102] Histological analysis

[0118] For whole-mount retina preparations, eyes were excised and fixed in 4% paraformaldehyde in PBS for 30 minutes. Retinas were dissected and fixed overnight in 4% paraformaldehyde. Autofluorescence was quenched with 1% sodium borohydride in PBS for 5 minutes. Retinas were permeabilized and blocked for 1 hour with 5% normal donkey serum (NDS), 1% bovine serum albumin (BSA) in PBS with 0.3% Triton X-100. Retinas were then labeled overnight with rabbit anti-GFP Alexa Fluor 555 1:200 (Invitrogen-Molecular Probes, Life Technologies, Carlsbad, CA) in 5% NDS, 1% BSA in PBS or with fluorescein peanut agglutinin (FITC PNA) 1:500 in 2% BSA in PBS (Vector Laboratories, Burlington, CA) for 15 minutes. The retinas were then washed five times in PBS and incubated with Alexa Fluor-647 donkey anti-goat IgG 1:100 (Invitrogen-Molecular Probes, Life Technologies, Carlsbad, CA) for 1.5 hours at room temperature. The retinas were thoroughly washed in PBS and mounted.

[0103]

[0119] Once sectioned, the eyes were fixed in 4% paraformaldehyde. After 1 hour, the cornea and lens were removed without disturbing the retina. The retinas were further fixed for an additional 2–3 hours at room temperature. The eyecups were rinsed in PBS and cryoprotected in 30% sucrose / PBS for 4 hours at room temperature, then embedded in cryostat compound (Tissue TEK OCT, Sakura Finetek USA, Inc., Torrance, CA) and frozen at -80°C. The retinas were sectioned vertically from dorsal to ventral at a thickness of 12 μm. For immunohistochemistry, retinal slices were rinsed in PBS, incubated in 0.3% Triton X-100 in PBS for 15 minutes, and then blocked in 5% BSA in PBS for 1 hour at room temperature. Next, the sections were incubated overnight at room temperature with anti-Brn3a (1:500, Santa Cruz, sc-31984) and anti-GFP (1:200 dilution, Life Technologies, A11122). The sections were washed three times with PBS and subsequently incubated with Alexa-594 and Alexa-488 tagged IgG secondary antibodies (1:500 dilution, Molecular Probes, Eugene, OR) for 2 hours at room temperature, followed by PBS washes. The sections were mounted with Vectashield mounting medium for fluorescence (Vector Lab, H-10400, Burlingame, CA) and coverslipped.

[0104] Multielectrode array recording

[0120] Electrophysiological recordings were obtained in vitro from isolated retinas. Briefly, the eyeball and anterior portion of the vitreous were excised immediately after removal of the eyeball, and the eyecup was placed in Ringer's solution and stored in the dark for at least 20 minutes before dissection. Under dim red light illumination, 1.5-3 mm diameter fragments were cut from the central region and placed on the multielectrode array for recording. Ringer's solution was bubbled with 95% O2 and 5% CO2, maintained at pH 7.4, and at 35-36°C. Stimulation and recording of retinal ganglion cells were performed as described by Nirenberg and Pandarinath (2012). Spike waveforms were recorded using a Plexon Instruments multichannel neuron acquisition processor (Dallas, TX). Standard spike sorting methods were used to identify individual cells as described in reference (Nirenberg and Pandarinath 2012).

[0105] Ocular toxicity assay

[0121] Before injection, an ophthalmological examination was performed. If ocular findings were present, the animal was excluded from the study. Ophthalmological examinations were also performed by a licensed veterinary ophthalmologist at 2, 4, 8, and 12 weeks after injection. A slit lamp was used to evaluate the anterior segment of the eye, including the cornea, iris, and lens. An indirect ophthalmoscope was used to evaluate the posterior segment, including the vitreous cavity and retina. All observations were performed in a masked state. There were two sacrifice time points: 4 and 12 weeks. At scheduled necropsies, the animals' eyes were collected in 4% paraformaldehyde. Following sufficient time for fixation, tissues were trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin, and the slides were examined. Immunohistochemical staining for green fluorescent protein was also performed to confirm transgene expression.

[0106] result Compound A produced reliable ERG responses in blind mice

[0122] To test for validity, photopic ERGs were performed in adult rd1 mice, a widely used model for retinal degenerative disease (Farber et al., 1994; Grimm et al., 2004; Hackam et al., 2004; Lagali et al., 2008; Thyragaraian et al., 2010), which have earlier onset and more severe retinal degeneration than rd10 (Pennesi et al., 2012). Briefly, the photopic ERG response in normal animals contains three components: the a-wave, which corresponds to photoreceptor signaling; the b-wave, which corresponds to bipolar cell signaling; and the photopic negative response (PhNR), which corresponds to ganglion cell signaling (Viswanathan et al., 1999). In rd1 animals, which lose photoreceptor outer segments by 8 weeks of age (Grimm et al., 2004; Nirenberg and Pandarinath, 2012), none of these components are present (Nishiguchi et al., 2015). When these animals are engineered to express optogenetic proteins in their ganglion cells, the expectation is that PhNR-like waves will appear, reflecting newly created optogenetic activity in the ganglion cells. ERGs were performed on Compound A-treated eyes to examine for the presence of newly created PhNR-like waves.

[0107]

[0123] Seven rd1 mice were treated with a human dose of 5 × 10 11 vg / eye, equivalent to a dose of 5 × 10 8One eye received Compound A via intravitreal injection at 10 weeks post-injection, at which time Compound A expression was expected to peak. The untreated eye of each animal served as a control. Results showed that six of seven animals produced reliable light-evoked ERG responses (PhNR-like waves) in their treated eyes; this contrasted with the ERGs produced by the untreated eyes of the same rd1 animals, which exhibited a flat ERG response (Figures 1A-1B). Results were significant at both the group level (p<0.001, unpaired t-test, comparing the magnitude of the ERG signal from the treated eye to that from the untreated eye) and the individual level (p<0.004, paired t-test, comparing the treated eye to its untreated counterpart for each animal).

[0108] The magnitude of the ERG response was dose-dependent for both virus dose and light dose

[0124] Following the experiment shown in Figures 1A-1B, 7 vg / eye)~2x or more (1×10 9 The efficacy of Compound A was investigated across a wider dose range, up to 100 mg / eye (vg / eye). Figures 2A-2B show the results across this 20-fold range, with all dose levels statistically significantly different from the control group (p<0.01, Student's t-test, comparing each dose group with the untreated group), and the amplitude of the response increasing with increasing vector dose.

[0109]

[0125] Given the robust PhNR-like responses at even higher vector doses, the intensity of the light stimulus was also tested to see if it could be reduced and still produce a light response. Specifically, using the two highest doses, we examined whether the light level could be lowered while still maintaining a PhNR-like amplitude significantly above control levels. Results showed that at 1 × 10 9 showed that the light level required for a vector dose of 1000 vg / eye could be reduced approximately 10-fold (Figure 3). 8At vg / eye, light levels could be reduced by six-fold (p<0.01, Student's t-test). The effect of even lower vector doses was not tested because there was little room for correction. These results may help narrow the range of light levels required for testing in clinical trials and reduce the burden on patients. Controls in this study (Figures 2A-2B) were blind, untreated eyes, but always received the maximum light intensity (0.06 mW / mm 2 ). Thus, without vector treatment, blind eyes showed no response to even the brightest light in this regimen, whereas with Compound A treatment, eyes responded with robust ERG signals even when light levels were substantially reduced. Light levels may be further reduced when sensory studies are conducted with human subjects, since it is well known that ERG responses are not as sensitive as sensory responses.

[0110] Evaluating safety

[0126] ERG responses in rd1 mice indicated that Compound A was effective in producing a light response in blind animals. To assess the safety of the vector, studies were conducted in rats, non-human primates, and mice using several tests, including immunohistochemical analysis and cell counts (rats), electroretinograms (non-human primates), and ocular tolerance / toxicology assays (mice).

[0111] To evaluate safety using immunohistochemistry and cell counts in rats

[0127] Similar to the rd1 mouse strain and human patients with RP, S334ter-3 rats have an inherited retinal degenerative disease that results in severe loss of photoreceptors (Martinez-Navarrete et al., 2011). Two sets of experiments were performed: the first evaluated the safety of the Compound A vector, and the second evaluated the safety of the Compound A vector in combination with the light stimulation required to activate it.

[0112]

[0128] In the first set, three groups of animals: 6.7 x 10 mice; 7 vg / eye equivalent to 6.8 × 10 8vg / eye in the low-dose group ( Onodera et al., 2015 ), and 2.6 × 10 in mice. 8 vg / eye corresponds to 2.7 × 10 9 A high-dose group receiving 1000 mg / eye and a control group containing both vehicle-treated and untreated eyes were used. Note that there is a wide range of vitreous volumes reported for rat eyes (13–54 μL) (Onodera et al., 2015). The maximum volume of rat vitreous (54 μL) was assumed when converting to a mouse-equivalent dose (5.3 μL for mouse vitreous). Therefore, the doses used here may be significantly higher (as much as four-fold higher) and therefore provide a much stronger assessment of Compound A's toxicity to ganglion cells. The assay performed was to count the number of cells in the ganglion cell layer. If treatment with Compound A is harmful to target cells, cell loss in the ganglion cell layer of treated retinas compared to controls would be expected. Ganglion cells were labeled with two markers: a marker that generally labels ganglion cells (Brn3) (Nadal-Nicolas et al., 2009) and a marker that specifically labels Compound A-expressing cells (antibody against GFP). Cell counts were performed 5 months after Compound A injection to account for cell death, if any, as well as removal of cellular debris (Elmore, 2007). Figure 4 shows that there was no statistically significant difference in retinal ganglion cell numbers between the low-dose and control groups (p>0.1, Student's t-test) or between the high-dose and control groups (p>0.5, Student's t-test) after 5 months of treatment.

[0113]

[0129] The second set of experiments evaluated the safety of vector plus light stimulation. These were divided into two parts. The first focused on testing for the presence of Compound A-expressing cells. These cells were made photosensitive by expressing Compound A within them, so light stimulation could potentially damage the cells and limit the value of treatments that required light stimulation. To evaluate the safety of photoactivating Compound A-expressing cells, two groups of four animals were used, both containing 2.7 x 10 9vg / eye with Compound A. One group of animals received a light stimulus (0.1 mW / mm ) similar to the exposure expected in clinical trials with optogenetic vectors. 2 One group of animals received 12 2-hour sessions over an 8-week period at 1000 Watts (at 12 2-hour sessions) while the other group did not receive light stimulation. Retinas were processed 5 months after light stimulation. Results showed no difference in the number of Compound A-expressing cells between the two groups, indicating no loss of Compound A-expressing cells as a result of light exposure (Figure 5A) (p>0.7, Student's t-test).

[0114]

[0130] The second part of the study focused on photoreceptors, assessing whether the light stimulus required to drive Compound A in ganglion cells would damage photoreceptors, the retina's naturally light-absorbing cells. In these experiments, wild-type (WT) rats were used, rather than S334ter-3, because adult S334ter-3 rats no longer have a photoreceptor layer as a result of their retinal degenerative disease (Martinez-Navarrete et al., 2011; McGill et al., 2012). Six WT animals were evaluated. In each WT animal, one eye received Compound A plus light treatment (0.1 mW / mm 2 (12 2.5-hour sessions over a 6-week period at 8.4 × 10 9 vg / eye, which is 8.2 × 10 8 vg / eye. Retinas were excised 6 months after injection. Results (Figure 5B) showed no loss of photoreceptors (e.g., cones) in Compound A plus light-treated retinas compared to untreated retinas (p>0.4, Student's t-test). These results are consistent with previous results on light stimulation for optogenetic therapy reported by Yan et al. (2016), who reported no loss of the photoreceptor layer, including both rods and cones, as measured by retinal thickness (outer nuclear layer thickness), using the same wavelength and light level.

[0115] Evaluating the safety of Compound A in non-human primates (NHPs) using electroretinograms

[0131] The rat study assessed tissue integrity using immunohistochemistry and cell count assays. To examine electrophysiological safety in a species similar to humans, ERGs were performed in cynomolgus monkeys. Three animals (six eyes total) were evaluated 7 months after vector administration via intravitreal injection. One animal (both eyes) received Compound A, and two animals (both eyes) received a variant using the same transgene (SEQ ID NO: 2) but packaged using a different AAV2 capsid (AAV2tYF capsid) (Petrs-Silva et al., 2009). Doses fell within the range used in efficacy studies in Figures 2A-2B; doses were 1 × 10 in mice. 8 vg / eye and 3.2 × 10 8 vg / eye corresponds to 3.7 × 10 10 vg / eye and 1.2 × 10 11 vg / eye (Onodera et al., 2015). Four animals (8 eyes in total) were untreated and served as controls.

[0116]

[0132] Whether ERG responses were adversely affected in treated vs. untreated eyes was examined using three standard photopic ERG components: the a-wave and b-wave, which reflect photoreceptor and bipolar cell responses, respectively, and the PhNR-wave, which reflects ganglion cell responses. Seven months after vector injection, intensity / response data were analyzed according to reference (Joshi, Ly, & Viswanathan, 2017), with the saturated amplitude V of each wave. max As shown in Figures 6A-6B, V for all three ERG components as a result of treatment was fitted with a generalized Naka-Rushton function to derive V. max There was no statistically significant decrease in V (p>0.2, for all waves) in the treatment group. max values and V of the untreated group max compare values).

[0117]

[0133] ERG experiments assess physiological function on a macroscale, e.g., whole retinal electrophysiology. Multielectrode array (MEA) recordings from excised retinas of cynomolgus monkeys previously injected with SEQ ID NO:3 expression vectors were evaluated for receptive field size and mean firing frequency. Retinas from six eyes (three animals) were treated with arrays of SEQ ID NO:3 expression vectors. In these experiments, the capsid was the AAV2 variant AAV2tYF, the promoters were CAMKII, hCACNA1G, and mNefL1.6, and the dose range was 2.2 x 10 11 ~7.6×10 12 vg / eye (5.8 × 10 in mice) 8 vg / eye~2×10 10 vg / eye). The results showed that the distribution of receptive field size and firing rate from the SEQ ID NO:3-treated group was not statistically significantly different from that from the untreated group (Figures 7A-7B), (p>0.2, Kolmogorov-Smirnov test). Retinal ganglion cells harvested from eyes intravitreally injected with SEQ ID NO:3 expression vector exhibited receptive field size and firing rate highly similar to those of retinal ganglion cells from untreated retinas using stimuli used to assess firing rate elicited from natural scenes (e.g., trees, landscapes, walking people).

[0118] Assessing local tolerance in blind mice

[0134] The safety of Compound A in terms of local tolerance was evaluated. A total of 120 rd1 mice were divided into three groups (40 animals per group): a 10-fold range of dose levels (4.25 × 10 8 vg / eye and 4.25 × 10 9 The animals were divided into two dose groups (20 vg / eye) and a vehicle-only group. Injections were administered intravitreally to the right eye, while the left eye was left untreated. Each group had two sacrifice time points: 4 weeks and 12 weeks (20 animals at each time point).

[0119]

[0135] SEQ ID NO:3 expression in injected eyes was verified and was measured by GFP immunolabeling (magenta) and was detected in the nerve fiber layer, inner plexiform layer, optic nerve head and elongating axons in all animals.

[0120]

[0136] Ophthalmic examinations were performed at 2, 4, 8, and 12 weeks postinjection, and the findings are summarized in Table 1 below. A slit lamp was used to evaluate the anterior segment, including the cornea, iris, and lens. An indirect ophthalmoscope was used to evaluate the posterior segment, including the vitreous cavity and retina. Previous phenotypic characterization of rd1 mice has shown that their eyes have attenuated blood vessels and pigmented patches at an early age (Hawes et al., 1999; Chang et al., 2002). Consistent with this, these phenotypes were present at similar frequencies in both Compound A- and vehicle-injected eyes in our study, suggesting that these phenotypes were not caused by the vector.

[0121]

[0137] In hematoxylin and eosin-stained retinal sections, Compound A-associated microscopic findings included minimal to slight mononuclear cell infiltration in the vitreous (Timmers et al., 2020), with minimal being the lowest level in a five-level classification (Table 2 below). By terminal sacrifice (12 weeks), only minimal (minimal) mononuclear cell infiltration was present.

[0122] [Table 1]

[0123] [Table 2] [Example]

[0124] Example 2: Single-Center, Phase 1 / 2, Safety and Efficacy Study of a Recombinant Adeno-Associated Viral Vector Carrying Compound A, SEQ ID NO: 1, in Patients with Retinitis Pigmentosa Study design

[0138] A non-randomized, open-label, Phase 1 / 2 dose-escalation study of the safety and efficacy of Compound A is being conducted by administering Compound A, utilizing SEQ ID NO: 3, as an optogenetic fusion protein by intravitreal injection in one eye of individuals with retinitis pigmentosa (RP). Secondary study results and efficacy are described below.

[0125]

[0139] Each participant will receive Compound A via a single intravitreal injection in one eye only, as outlined in Table 3 below.

[0126] [Table 3]

[0127]

[0140] Participants in all groups will be at least 18 years old and will receive the vector at a low dose (Group 1), low-intermediate dose (Group 2), high-intermediate dose (Group 3), or high dose (Group 4). Participants in Group 5 will be at least 18 years old and will receive the vector at the maximum tolerated dose (MTD) determined for Groups 1, 2, 3, and 4.

[0128]

[0141] If safety assessment confirms the absence of dose-limiting toxicity in Group 1, subjects will be enrolled in Group 2. If safety assessment confirms the absence of dose-limiting toxicity in Groups 1 and 2, participants will be enrolled in Group 3. The MTD will be the dose below which dose-limiting toxicity (DLT) is observed by the principal investigator. If the absence of DLT is confirmed, the MTD will be the highest dose tested. After review of safety data from Groups 1, 2, 3, and 4 by the DSMC, participants may be enrolled in the dose escalation group, Group 5, at the MTD confirmed in the previous group. If the DSMC determines from the Day 14 safety data of any group that the MTD is met, no further dose escalation will occur and all remaining subjects will be assigned to the MTD group.

[0129]

[0142] Within Arms 1, 2, 3, and 4, participant enrollment will be staggered by at least 2 weeks to allow sufficient time for review of safety information by the investigator and sponsor.

[0130]

[0143] Safety will be monitored by assessment of visible and invisible adverse events / adverse experiences (AEs), as well as hematology and clinical chemistry parameters. Other data collected will be the immune response to SEQ ID NO: 3 and the presence of vector DNA in blood, saliva, and tears.

[0131]

[0144] Efficacy is measured by evaluation of light threshold and motion and shape / object detection (see details in the Efficacy Parameters section below).

[0145] Other data collected may include electrophysiological measurements (ERG and / or VEP), fundus photography, immune response to AAV, and quality of life questionnaires.

[0132]

[0146] Approximately 40 participants will be enrolled in this study. Enrollment in this study is expected to take approximately 30 months. Enrolled participants will have frequent follow-up visits during the first year after receiving study medication.

[0133]

[0147] Participants will be followed annually for an additional 4 years after the 12-month visit to monitor for delayed AEs and to assess the duration of any changes in visual function or structure that occur. Identification of efficacy parameters

[0148] The following lists efficacy parameters. The procedures for obtaining these parameters are provided below in the Visual Function Measurement section below.

[0149] The change in light detection (detection threshold) in the treated eye is measured using a staircase or binary search method.

[0150] Changes in motion detection are measured using a standard two-alternate forced choice paradigm.

[0151] Changes in shape detection are measured using a standard two-alternate forced-choice paradigm.

[0152] Changes in the amplitude of the visual evoked potential (VEP) or ERG signal are measured.

[0134] Visual function measurement Psychophysical (perceptual) measurements

[0153] Psychophysical measurements in the study eyes draw on light detection, motion detection, and shape / object detection from a standard battery of tests used in subjects with very low vision ( Bach et al., 2010 ; Wilke et al., 2007 ).

[0135]

[0154] Primary efficacy endpoint: The primary efficacy endpoint is light detection (e.g., change in detection threshold). This is assessed using a stimulator (Diagnosys LLC) that delivers flashes of light at different intensities according to a staircase or dichotomous search method to determine the subject's threshold response. Subjects are tested at baseline and safety assessment visits at approximately 3, 6, 9, and 12 months, as well as at each long-term follow-up visit. This test evaluates whether the optogenetic protein in Compound A is expressed at a level sufficient to produce a sensory response in RP subjects. This test also evaluates the minimum light level required to activate the protein for subsequent tests evaluating light, motion, and shape detection.

[0136]

[0155] Secondary efficacy endpoints: Secondary endpoints will be assessed using a hierarchy of tests based on difficulty. Secondary endpoints include motion detection, motion direction detection, and shape / object recognition. Secondary endpoints will be performed using either the PSIII pattern stimulator (Diagnosys LLC) or the easier-to-use pattern stimulator (Optecks), a portable testing device that provides a wider field of view. Test results for the two stimulators will be assessed and described.

[0137]

[0156] Testing is conducted using a standard forced-choice paradigm, with subjects providing their responses at their own pace using a keypad or button box. If a subject's performance on a given test is at chance with respect to progressing through the hierarchy, the researcher or assessor can choose not to progress to the next level of difficulty so as not to unnecessarily burden the subject with tests they are unable to perform (e.g., if a subject is unable to detect movement versus no movement within approximately 10 presentations, tests based on this, such as detecting leftward versus rightward movement, may be eliminated from the test series).

[0138]

[0157] Motion detection (e.g., direction of motion): Stimuli are presented in a two-alternate forced-choice paradigm. Briefly, on each trial, the subject is presented with a moving bar in one of two directions in the visual field, chosen randomly with equal probability, and the subject must point to the direction of the movement. Performance is measured as the number of correct fractions across all trials. A correct fraction of 0.5 is chance, and values above this are assessed using standard statistics. If the subject is able to perform well, a four-alternate choice test may be administered.

[0139]

[0158] Shape / Object Detection: Stimuli are presented in a two-alternate forced-choice paradigm. Briefly, on each trial, subjects are presented with one of two shapes (e.g., target or non-target), selected at random with equal probability, and must indicate whether the presented stimulus is the target shape or not. Performance is measured as the number of correct fractions across all trials. A correct fraction of 0.5 is chance, and values above this are assessed using standard statistics. If subjects are able to perform well, a four-alternate choice test may be administered.

[0140]

[0159] Additional non-invasive measures of visual function with or without a stimulator, such as object detection (e.g., household items, items in the refrigerator) or light localization, may be performed at the discretion of the researcher or assessor.

[0141]

[0160] Training visits will also be conducted. Between baseline and Day 0, familiarization visits will be conducted with subjects to orient them to the stimulation device and testing procedures and record baseline testing values. Additionally, training sessions will be scheduled following study drug administration, generally in conjunction with the 3-6 month visit, to optimize stimulation to the subject's foveal ring and familiarize the subject with the new input, particularly to familiarize the subject with the connection between optogenetic activation of their retinal ganglion cells and visual perception. The number of training sessions and duration of testing time will be at the investigator's discretion; sessions are expected to last approximately 2 hours.

[0142] result

[0161] In the two-choice task, on each trial, patients pressed a bar to trigger a stimulus: light (3 flashes) or no light (for a duration equal to 3 flashes). An automated voice then prompted the patient to make a choice, and the patient was expected to indicate their answer by pressing a button. Stimuli were delivered automatically and randomly interleaved. Figure 8 shows that all patients demonstrated an increase in light sensitivity. Note that the y-axis is on a logarithmic scale, indicating that the increase in light sensitivity was substantial. These data provide evidence that Compound A was taken up and expressed SEQ ID NO:3 sufficiently to produce a light response. Figure 9 shows that this improvement in light sensitivity was stable or increased throughout the study period (up to 12 months) for all patients. These initial durability data suggest that the benefits of Compound A are sustained.

[0143]

[0162] Using the motion detection and shape / object paradigm described above, four endpoints were assessed (ranked on a hierarchy of difficulty), including the ability to detect motion, the ability to detect direction of motion, the ability to discriminate live motion (e.g., arm moving up vs. down, or e.g., arm movement vs. hand movement), and the ability to discriminate objects (e.g., apples vs. other fruits or vegetables, or e.g., playing card suits). Patients were observed to show improvement in several secondary endpoints. Notably, at baseline, performance on all tasks was at or relatively low to chance (chance is 50% for two-limb tasks). Figure 10 shows that after treatment, all patients gained the ability to detect motion, and several patients also gained the ability to detect direction and live motion. None of the patients on lower vector doses were able to recognize objects. Figure 11 shows that improvement remained stable or actually increased over the study period (up to 12 months).

[0144]

[0163] Patients were tested during the index task (Figures 12A and 13A), which required motion detection, motion direction detection, and object recognition; and motion / object set recognition testing (Figures 12B and 13B) at baseline or after treatment. The two depicted patients had baseline visual acuity: <20 / 200, 6.0 × 10 11 vg / eye of Compound A (Cohort 3). The exponential valve test was performed at various distances from the patient. At each distance, blocks of 8–10 trials were presented, randomly interleaved, and the percentage correct at each distance was tabulated. All other tests were performed as previously described. Baseline testing was a two-choice task. Post-treatment testing was a two- or, more challenging, four-choice task. Both exponential valve and motion / object recognition were observed to improve in both patients (Figures 12A–B and 13A–13B). Note that at baseline, this patient was able to detect motion but not the direction of motion or the objects on the object recognition task. After treatment, this patient was able to detect the direction of motion and easily perform the object recognition task, often naming the objects as she saw them (fruits, vegetables, playing card suits).

[0145]

[0164] The patients were further tested with a color discrimination task. The two depicted patients had baseline visual acuity of <20 / 200 and a visual acuity of 6.0 × 10 11 vg / eye of Compound A (Cohort 3). In each trial, patients were presented with red, green, or blue shapes. The percentage of correct answers for each color was tabulated. These tests were administered at two sets of post-treatment visits. Patient 108 showed moderate improvement in distinguishing between red, green, and blue colors. Patient 109 showed significant improvement in her ability to see red, moderate improvement in her ability to see green, and maintained her ability to see blue (Figure 14). These results are consistent with Subject 109's observations regarding colors at home: she suddenly realized that her couch was red, not black as she previously thought, and her newly developed ability to distinguish colored pills (red vs. blue) improved her quality of life.

[0146]

[0165] The above results provide clinical proof-of-concept. Notably, patients who started out blind or near-blind can now see light, with sensitivity increasing over time, and some able to see light in daylight or at television levels. All of these patients can now also detect motion, including three who can also detect the direction of motion, both in computer-generated images and live motion. Early data from patients with higher levels of residual retinal function suggest the potential for greater functional recovery.

[0147]

[0166] Additional studies addressed whether incremental benefit could be achieved using increasing doses of Compound A. These studies were based on in vivo data from mice demonstrating that a nonlinear dose response was observed in preclinical species (Figure 15). [Example]

[0148] Example 3: Design of a vector containing a gene expressing an optogenetic protein

[0167] A gene encoding an optogenetic fusion protein (e.g., a protein having the amino acid sequence of SEQ ID NO: 3) can be prepared, for example, by procedures known in the art. Techniques for solid-phase synthesis of polynucleotides are known in the art and are described, for example, in U.S. Pat. No. 5,541,307, the disclosure of which pertains to solid-phase polynucleotide synthesis and purification and is incorporated herein by reference. Furthermore, the prepared gene can be amplified, for example, using polymerase chain reaction (PCR)-based techniques known in the art and / or by transformation of Escherichia coli (E. coli) with a plasmid containing the optogenetic fusion protein. The bacteria can then be cultured to amplify the DNA therein, and the gene encoding the optogenetic fusion protein can be isolated by plasmid purification techniques known in the art, followed by restriction digestion and / or sequencing of the plasmid to confirm the identity of the optogenetic fusion protein.

[0149]

[0168] A DNA polynucleotide encoding an optogenetic fusion protein, or its RNA equivalent, may exhibit at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 2. In another example, a DNA polynucleotide encoding an optogenetic fusion protein may have a nucleic acid sequence that is identical to the nucleic acid sequence of SEQ ID NO: 2. An optogenetic fusion protein may have the amino acid sequence of SEQ ID NO: 3.

[0150]

[0169] The DNA polynucleotide encoding the optogenetic fusion protein, or its RNA equivalent, can then be incorporated into a plasmid, such as a viral vector. For example, an adeno-associated virus (AAV) vector, such as AAV2, can be created that incorporates the optogenetic fusion protein (SEQ ID NO: 3) between the 5' and 3' inverted terminal repeats of the vector, and the DNA nucleotides can be operably linked to a constitutive promoter (e.g., a CAG promoter). Such a vector can exhibit at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 1. In another example, an AAV encoding the optogenetic fusion protein can have a nucleic acid sequence identical to the nucleic acid sequence of SEQ ID NO: 1 (Figure 16).

[0151]

[0170] Those skilled in the art can monitor the expression of optogenetic fusion proteins by various methods. For example, those skilled in the art can transfect a viral vector containing an optogenetic fusion protein into cultured cells. The expression of the encoded fusion protein can then be monitored using expression assays such as qPCR, RNASeq, ELISA, or immunoblotting. [Example]

[0152] Example 4: Treatment of retinitis pigmentosa by administration of a vector containing a gene expressing an optogenetic protein

[0171] Using conventional molecular biology techniques known in the art, a gene encoding an optogenetic protein, such as SEQ ID NO: 4, can be incorporated into a vector, such as a viral vector, and administered to a patient suffering from retinitis pigmentosa. For example, a patient suffering from retinitis pigmentosa can be administered a viral vector containing a gene expressing SEQ ID NO: 4 under the control of transcriptional regulatory elements that promote the expression of SEQ ID NO: 4 in retinal ganglion cells. For example, an AAV vector, such as an AAV2 vector, can be created that incorporates SEQ ID NO: 4 (e.g., the vector may consist of or include the nucleic acid sequence of SEQ ID NO: 1) between the 5' and 3' inverted terminal repeats of the vector, and the gene may be placed under the control of transcriptional regulatory elements. The AAV vector can be, for example, at least about 1 x 10 11 An initial dose of vg / eye can be administered to a subject by a variety of routes of administration, including intravitreally.

[0153]

[0172] Following administration of the vector to a patient, those skilled in the art can monitor the improvement of the patient's response to treatment by various methods. For example, a physician can monitor the patient's improvement in light sensitivity, ability to detect movement, ability to detect the direction of movement, ability to distinguish live movements (e.g., arm moving up vs. down, or arm movement vs. hand movement), ability to distinguish objects (e.g., apples vs. other fruits or vegetables, or, for example, playing card suits), ability to count fingers, and / or ability to distinguish colors. Finding that the patient's function has improved in one of the above exemplary tests following administration of treatment may indicate that the patient is responding favorably to treatment. Subsequent doses can be determined and administered as needed.

[0154] Incorporation by Reference

[0173] The entire disclosure of each patent document and scientific article cited herein is incorporated by reference for all purposes.

[0155] equivalent

[0174] The present disclosure may be embodied in other specific forms without departing from its essential characteristics. Therefore, the foregoing embodiments should be considered illustrative rather than limiting of the disclosure set forth herein. The scope of the present disclosure is indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. A method for treating a retinal degenerative disorder in a human patient in need thereof, comprising the step of administering an effective initial vector dose of an AAV2 vector carrying a gene that expresses an optogenetic protein into the vitreous of the patient's eye, wherein the vector comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:

9.

2. The method of claim 1 , wherein the vector comprises the nucleic acid sequence of SEQ ID NO:

9.

3. 3. The method of claim 2, wherein the vector comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:

1.

4. The method of claim 3 , wherein the vector comprises the nucleic acid sequence of SEQ ID NO:

1.

5. 10. The method of claim 1, further comprising exposing the patient to light after administering the initial dose, wherein the light is ambient light or light stimulation via a device.

6. 10. The method of claim 1, further comprising administering neural-encoded optical stimulation after administering the initial dose.

7. The neurally encoded optical stimulation is about 5×10 －２ mW / mm ２ ~Approx. 0.1mW / mm ２ , about 1×10 －２ mW / mm ２ ~Approx. 0.1mW / mm ２ , about 1×10 －３ mW / mm ２ ~Approx. 0.1mW / mm ２ , about 1×10 －４ mW / mm ２ ~Approx. 0.1mW / mm ２ , or about 1 × 10 －５ mW / mm ２ ~Approx. 0.1mW / mm ２ 7. The method of claim 6, wherein the method is carried out by

8. The method of claim 7, wherein the neural-encoded optical stimulation is administered using a neural-coding device that delivers optogenetic stimulation substantially specific to ON-type ganglion cells.

9. The method of any one of claims 1 to 8, wherein the patient has advanced blindness due to a retinal degenerative disease.

10. The method of any one of claims 1 to 8, wherein the patient has mild, moderate or severe visual impairment.

11. 9. The method of any one of claims 1 to 8, wherein the patient has poor light perception (BLP) or no light perception (NLP) at baseline.

12. 12. The method of claim 11, wherein the patient has finger counting vision or less than finger counting vision at baseline.

13. 13. The method of any one of claims 1 to 12, wherein the patient has baseline visual acuity of no more than or about 20 / 200.

14. 13. The method of any one of claims 1 to 12, wherein the patient has early stage disease with baseline visual acuity of only or about 20 / 60.

15. 7. The method of claim 6, wherein the neurally encoded stimulation comprises using a light delivery device that delivers light pulses to the neural cord of the retina, causing ganglion cell firing to mimic normal retinal firing.

16. The effective amount is about 1×10 １１ ~1 x 10 １３ 16. The method of any one of claims 1 to 15, wherein the initial vector dose is vector genome (vg) / eye.

17. The effective amount is at least about 1.2×10 １２ 16. The method of any one of claims 1 to 15, wherein the initial vector dose is 0.05 vg / eye.

18. The initial vector amount is about 1 x 10 １２ ~Approx. 1×10 １４ 16. The method of any one of claims 1 to 15, wherein the volume is from about 70 μL to about 130 μL at a concentration of vg / mL.

19. The initial vector amount is about 1 x 10 １１ vg / eye, approximately 3.0×10 １１ vg / eye, approximately 6.0×10 １１ vg / eye, approximately 1.2×10 １２ 16. The method of any one of claims 1 to 15, wherein the amount is one of: vg / eye or higher.

20. A method for improving light sensitivity in a human patient suffering from a retinal degenerative disorder, comprising administering at least about 1 x 10 of an AAV2 vector carrying a gene that expresses an optogenetic protein. １１ 2. A method comprising administering an initial dose of 100 mg / eye of the vector intravitreally to the patient's eye, wherein the vector comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:

9.

21. 21. The method of claim 20, wherein the vector comprises the nucleic acid sequence of SEQ ID NO:

9.

22. 22. The method of claim 21, wherein the vector comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:

1.

23. 23. The method of claim 22, wherein the vector comprises the nucleic acid sequence of SEQ ID NO:

1.

24. 24. The method of claim 23, wherein at or after four months after the first dose, the patient has a supralinear improvement in photosensitivity compared to improvement at lower doses.

25. 25. The method of claim 24, wherein the visual improvement is measured as one or more of the amplitude of the electroretinogram (ERG) response or the amplitude of the VEP response.

26. 26. The method of any one of claims 20 to 25, wherein after administering the initial dose, the patient is further subjected to a light stimulus.

27. 26. The method of any one of claims 20 to 25, wherein after administering the initial dose, a neurally encoded stimulus is further administered.

28. 28. The method of any one of claims 1 to 27, wherein the patient has a decreased light valve threshold as measured by a staircase or dichotomous search method four months or more after the first administration.

29. 28. The method of any one of claims 1 to 27, wherein three months or more after the first administration, the patient has an increased ability to detect movement and / or direction of movement as measured by a standard two-arm forced choice paradigm.

30. 28. The method of any one of claims 1 to 27, wherein the patient has increased shape detection ability as measured by a standard two-arm forced choice paradigm three months or more after the first administration.

31. 28. The method of any one of claims 1 to 27, wherein three months or more after the first administration, the patient has an increased ability to detect and / or distinguish colors as measured by visual acuity testing or a standard two-choice forced choice paradigm.

32. 32. The method of any one of claims 1 to 31, wherein the retinal degenerative disorder is one or more of retinitis pigmentosa and macular degeneration.

33. 33. The method of any one of claims 1 to 32, further comprising administering to the patient an oral corticosteroid 1 to 3 days prior to the intravitreal administration.

34. 34. The method of claim 33, wherein the corticosteroid is oral prednisone / prednisolone administered at 1 mg / kg prednisone / prednisolone for 1-3 days prior to injection or on the day of injection, and optionally continuing at this dose for 7 days after injection, including the day of injection, for a total of 10 days.

35. 35. The method of any one of claims 1 to 34, further comprising the step of administering valacyclovir at 1000 mg / day or acyclovir at 400 mg twice daily orally starting 3 to 7 days prior to the intravitreal administration.

36. a nucleic acid sequence encoding an optogenetic fusion protein operably linked to a CAG promoter; the optogenetic fusion protein comprises a light-activated ion channel protein fused to the N-terminus of a reporter protein; the nucleic acid sequence encoding the light-activated ion channel protein comprises the nucleic acid sequence of SEQ ID NO: 4; Polynucleotide.

37. 37. The polynucleotide of claim 36, wherein the polynucleotide comprises DNA.

38. 37. The polynucleotide of claim 36, wherein the polynucleotide comprises RNA.

39. 37. The polynucleotide of claim 36, wherein the reporter protein is GFP.

40. 40. The polynucleotide of claim 39, wherein the optogenetic fusion protein is encoded by a nucleic acid molecule having a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:

2.

41. The polynucleotide of claim 40, wherein the optogenetic fusion protein is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:

2.

42. 42. A vector, optionally a plasmid, a DNA vector, an RNA vector, a virion, or a viral vector, comprising the polynucleotide of any one of claims 1 to 41.

43. 43. The vector of claim 42, wherein the vector is a viral vector.

44. 44. The vector of claim 43, wherein the viral vector is selected from the group comprising adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and synthetic virus.

45. 45. The vector of claim 44, wherein the viral vector is AAV2.

46. The AAV of claim 44 or 45, wherein the AAV further comprises two inverted terminal repeats (ITRs), the two ITRs comprising a first ITR (ITR1) and a second ITR (ITR2), wherein ITR1 is located on the 5' side of the polynucleotide and ITR2 is located on the 3' side of the polynucleotide to form a cassette comprising the structure ITR1-optogenetic fusion protein-ITR2.

47. 47. The AAV of claim 46, wherein the two ITRs are AAV serotype 2 ITRs.

48. 48. The vector of any one of claims 42 to 47, wherein the vector comprises a nucleic acid molecule that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:

1.

49. 49. The vector of claim 48, wherein the vector has the nucleic acid sequence of SEQ ID NO:

1.

50. A plasmid encoding the viral vector according to any one of claims 42 to 49.

51. 51. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 41, a vector according to any one of claims 42 to 49, or a plasmid according to claim 50, and a pharmaceutically acceptable carrier, diluent, or excipient.

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