Method of enhancing viral mediated gene delivery

Abstract

Viral constructs and methods of use in the treatment of diseases and disorders are provided. The viral constructs include a plurality of transgenes attached to each viral vector, allowing for an increase in protein expression without increasing viral load. The viral constructs may include a polynucleotide molecule including multiple nucleotide sequences, a promoter sequence, and a post-transcriptional regulatory element. The nucleotide sequences may be separated by a cleaving or non-cleaving peptide.

Description

METHOD OF ENHANCING VIRAL MEDIATED GENE DELIVERYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 614,406, entitled Optogenetic Gene Therapy, filed on December 22, 2023, and which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under contract EY04068 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing isMethod_of_enhancing_viral_mediated_gene_delivery_PCT.xml. The XML file is 200,800 bytes in size, was created on December 20, 2024, and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0004] The present disclosure relates generally to gene therapy. Specifically, the present disclosure relates to viral-mediated gene therapy to treat disease.BACKGROUND OF THE DISCLOSURE

[0005] Gene therapy is a technique that uses a gene to treat, prevent, or cure a disease or medical disorder by replacing a defective gene with a new one. The new gene is often delivered using viral transduction. Typically, a viral vector used in viral transduction has three components, a protein capsid and / or envelope that encapsidates the genetic payload and defines the vector’s tissue or cell tropism and antigen recognition; the transgene of interest; and elements that control expression of the transgene. Commonly used viruses for viral transduction include adeno- associated virus (AAV), retroviruses, lentiviruses, and herpes simplex viruses (HSV).

[0006] While viral-mediated gene therapy is promising, high therapeutic gene expression in targeted cells is needed to achieve the desired efficacy. Merely increasing the amount of virus administered may lead to viral-vector dose-dependent immunogenicity, decreasing theeffectiveness of the delivery mechanism. There is therefore a need to enhance transgene expression without increasing viral load.SUMMARY OF THE DISCLOSURE

[0007] Gene therapy uses gene(s) to treat, prevent, or cure a disease or medical disorder through the introduction of specific cell function-altering genetic material into the patient. The new genetic material is generally delivered via a vector including viral and non-viral vectors. Provided is a method of delivering multiple copies of the therapeutic gene including two, three, four, five, six, seven, eight, or more copies to the target in a single construct without increasing immunogenicity.

[0008] The viral constructs described herein include a viral vector with multiple copies of the transgene of interest. In some aspects, the viral construct may include a promoter or other regulatory element such as a post-transcriptional regulatory element. In some aspects, the copies of the transgene of interest may be separated by cleavage and non-cleavage peptides.

[0009] Exemplary viral vectors that can be used to deliver multiple copies of a transgene of interest include mutated or wild-type retroviral vectors, adenoviral vectors, adeno-associated vectors (AAV), herpes simplex virus (HSV)-based vectors, alphavirus-based vectors, such as semliki forest virus (SFV) and sindbis virus (SIN), lentiviral vectors, or any engineered or recombinant viral vector known in the art. Additional vectors may include fusion proteins and chemical conjugates.

[0010] Viral vectors may be selected based on their ability to support multiple copies of the transgene. For instance, the lentiviral payload capacity is about 9 kb, SIN payload capacity is about 6.5 kb, and AAV payload capacity is about 5 kb. Thus, the type of transgene, or the number of transgenes, as well as the size and types of regulatory elements may be varied depending on the viral vector being used. In some aspects, the viral vector may be selected based on its ability to transfect a particular cell type. In some aspects, the viral vector may be a recombinant adeno- associated viral (rAAV) vector including a capsid protein with a mutated tyrosine residue. The mutation in the rAAV vector enables the vector to have improved transduction efficiency of a target cell, e.g., retinal bipolar cells (e.g., ON or OFF retinal bipolar cells (ON or OFF BCs); rod or cone bipolar cells), retinal ganglion cells (RGCs), amacrine cells, or rod and cone photoreceptors.

[0011] Exemplary promoters included in the viral constructs include ubiquitous and cell-specific promoters. Exemplary ubiquitous promoters include cytomegalovirus immediate enhancer / p-actin (CAG), cytomegalovirus (CMV), synapsin, elongation factor-1 alpha (EF1 a), or thymocyte differentiation antigen 1 (Thy-1 ). Exemplary cell-specific promoters may include retinal promoters such as metabolic glutamate receptor 6 (mGluR6), neurokinin 3 (NK-3), Purkinje cell protein 2(Pcp2(L7)), gamma-synuclein gene (SNCG), HKamac, and rhodopsin (RHO). In some aspects, the promoters may include variants such as shortened promoters, for example, shortened mGluR6 promoters or combinations of promoters or portions of promoters.

[0012] Exemplary post-transcriptional regulatory elements included in the viral constructs include woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or mutated woodchuck hepatitis virus posttranscriptional regulatory element (mWPRE). In some aspects, exemplary post-transcriptional regulatory elements may include human growth hormone polyadenylation signal (hGHpA), bovine growth hormone polyadenylation signal (bGHpA), or SV40 early polyadenylation signal (SV40pA).

[0013] In some aspects, the viral constructs may include a polynucleotide molecule including a first nucleotide sequence, a second nucleotide sequence, a promoter sequence, and a posttranscriptional regulatory element. In some aspects, the polynucleotide molecule may include a third, fourth, fifth, sixth, seven, or more nucleotide sequences. In some aspects, the nucleotide sequences may be separated by cleaving or non-cleaving peptides. The sequences may be separated by the same or different cleaving or non-cleaving promoters and may be under the control of the same or different regulatory elements such as promoters and post-transcriptional regulatory elements. In some aspects, there may be no cleaving or non-cleaving peptides.

[0014] Exemplary viral constructs are provided using light-sensitive proteins including microbial, invertebrate, and vertebrate opsins. Exemplary microbial channel-based opsins include Chloromonas oogama channelrhodopsin (CoChR), channel rhodopsin 2 (ChR2), Volvox carteri channelrhodopsin (VChR), red activatable channelrhodopsin (ReaChR), Platymonas (Tetraselmis) subcordiformis channelrhodopsin (PsChR), Chronos, or Chrimson.

[0015] Photoreceptors are highly specialized neurons that are responsible for the conversion of light into signals that can be interpreted by the brain. Photoreceptor cells are vulnerable to retinal degenerative diseases including degenerative diseases caused by genetic mutations in photoreceptor cells or retinal pigment epithelial (RPE) cells, as well as geographic atrophy (or dry type age-related macular degeneration). Loss of photoreceptor cells and / or loss of photoreceptor cell function is the primary cause of diminished visual acuity, diminished light sensitivity, and blindness. Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Some of the drawings submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0017] FIG. 1. Imaging of expression of bAAV2.7m8(Y444F)-mGluR6-Co3M-GFP-hGHpA (viral vector #1) in the retina of triple knock-out (TKO) (Opn47Gnatl7’ Cnga3 ) blind mice showing selective targeting in ON bipolar cells (ON BCs).

[0018] FIG. 2. Image of expression of bAAV2.7m8(Y444F)-mGluR6(s)-Co3M-GFP-hGHpA (viral vector #2) in the retina of triple knock-out (TKO) (Opn47-Gnatl7’ Cnga37) blind mice showing selective targeting of ON BCs.

[0019] FIG. 3. Image of expression of bAAV2.7m8(Y444F)-mGluR6-Co3M-WPRE-hGHpA (viral vector #3) in the retinal of triple knock-out (TKO) (Opn47' Gnatl7’ Cnga37’) blind mice showing predominant targeting in ON BCs but with some off targeting in the ganglion cell layer (GCL) ( transfection of retinal ganglion cells shown at 302, 304, and 306).

[0020] FIG. 4. Image of expression of bAAV2.7m8(Y444F)-mGluR6(s)-Co3M-GFP-WPRE-hGHpA (viral vector #4) in the retinal of triple knock-out (TKO) (Opn47-Gnatl7’ Cnga37) blind mice showing predominant targeting in ON BCs but with some off targeting in the ganglion cell layer (GCL) (transfection of retinal ganglion cells shown at 402, 404, and 406).

[0021] FIG. 5. Image of expression of bAAV2.7m8(Y444F)-mGluR6-Co3M-P2A-Co3M-hGHpA (viral vector #5) in the retina of triple knock-out (TKO) (Opn47-Gnatl7-Cnga3- / ) blind mice showing selective targeting in ON BCs.

[0022] FIG. 6. Image of expression of bAAV2.7m8(Y444F)-mGluR6(s)-Co3M-P2A-Co3M-P2A- Co3M-hGHpA (viral vector #6) in the retina of triple knock-out (TKO) (Opn47Gnatl Cnga3 ' ) blind mice showing selective targeting in ON BCs.

[0023] FIG. 7. Image of expression of bAAV2.7m8(Y444F)-mGluR6(s)-Co3M-P2A-Co3M-WPRE- hGHpA (viral vector #7) in the retina of triple knock-out (TKO) (Opn4- / -Gnatl7-Cnga37-) blind mice. It shows selective targeting in ON BCs.

[0024] FIG. 8 Image of expression of bAAV2.7m8(Y444F)-mGluR6(s)-Co3M-pHK-Co3M- mWPRE-bGHpA (viral vector #8) in the retina of triple knock-out (TKO) (Opn47Gnatl7Cnga37) blind mice showing selective targeting in ON BCs.

[0025] FIG. 9. Illustration of an exemplary optomotor system 900 including a camera 902, a computer 904, a power supply 906, light stimulus 918, and a motor control 908 with a rotating platform 912 and a spinning drum 910.

[0026] FIG. 10. A graph showing the comparison of the restored light sensitivity in TKO blind mice treated with viral vectors #1 - #8.

[0027] FIG. 11. Illustration of an exemplary optomotor test system 1100 for use in testing visual acuity with a video camera 1102, and an LCD monitor 1104 or 1 106 on each wall of the arena frame 1108.

[0028] FIG. 12. A graph showing the comparison of the restored visual acuity in TKO blind mice treated with viral vectors #1 - #8.

[0029] FIG. 13 shows a construct map of viral vector #1 , AAV2-mGluR6-Co3M-GFP-hGHpA (SEQ ID NO: 1 ).

[0030] FIG. 14 shows a construct map of viral vector #2, AAV2-mGluR6(s)-Co3M-GFP-hGHpA (SEQ ID NO: 2).

[0031] FIG. 15 shows a construct map of viral vector #3, AAV2-mGluR6-Co3M-WPRE-hGHpA (SEQ ID NO: 3).

[0032] FIG. 16 shows a construct map of viral vector #4, AAV2-mGluR6(s)-Co3M-GFP-WPRE- hGHpA (SEQ ID NO: 4).

[0033] FIG. 17 shows a construct map of viral vector #5, bAAV2-mGluR6-Co3M-P2A-Co3M- hGHpA (SEQ ID NO: 5).

[0034] FIG. 18 shows a construct map of viral vector #6, bAAV2-mGluR6(s)-Co3M-P2A-Co3M- P2A-Co3M-hGHpA (SEQ ID NO: 6).

[0035] FIG. 19 shows a construct map of viral vector #7, bAAV2-mGluR6(s)-Co3M-P2A-Co3M- WPRE-hGHpA (SEQ ID NO: 7).

[0036] FIG. 20 shows a construct map of viral vector #8, bAAV2-mGluR6(s)-Co3M-[3HK-Co3M- mWPRE-bGHpA (SEQ ID NO: 8).DETAILED DESCRIPTION

[0037] Gene therapy uses gene(s) to treat, prevent, or cure a disease or medical disorder through the introduction of specific cell function-altering genetic material into the patient. The genes may be delivered to the cells using viral and non-viral vectors. Provided is a method of delivering multiple copies of the therapeutic gene including two, three, four, five, six, seven, eight, or more copies to the target without increasing immunogenicity.

[0038] The viral constructs described herein include a viral vector and multiple copies of the transgene of interest. In some aspects, the viral construct may include a promoter or other regulatory element such as a post-transcriptional regulatory element. In some aspects, the copies of the transgene of interest may be separated by cleavage and / or non-cleavage proteins.

[0039] Exemplary applications of the viral constructs were demonstrated using light-sensitive proteins including microbial, invertebrate, and vertebrate opsins. However, it should be understood by one of ordinary skill in the art that such examples are not intended to be limiting and the viral constructs as described may include a variety of transgenes suitable for delivery to a variety of cells for use in the treatment, amelioration, and prevention of one or more diseases and disorders.

[0040] The retina is a complex tissue in the back of the eye that contains photoreceptor cells (e.g., rods, cones), horizontal cells, retinal bipolar cells, amacrine cells, retinal ganglion cells, Muller glial cells, and retinal pigment epithelial cells. Each photoreceptor absorbs light according to its spectral sensitivity and converts the light into signals that can be decoded by the brain into a visual image.

[0041] The retina has multiple layers including an inner limiting membrane forming the boundary between the vitreous and the retina; the outer nuclear layer which contains the cell bodies of photoreceptor layers; the outer plexiform layer which contains synapses that connect photoreceptors to bipolar and horizontal cells; the inner nuclear layer (INL) which contains bipolar cells, horizontal cells, amacrine cells, interplexiform neurons, and Muller cells; the inner plexiform layer (IPL) which provides synaptic connections between the axons of bipolar cells and dendrites of ganglion cells; and the ganglion cell layer (GCL) which transmits signals to the brain via the optic nerve.

[0042] Retinal bipolar cells provide the link between photoreceptors and retinal ganglion cells which function as output neurons. Information regarding color, depth, shape, and motion is captured and parsed into different signals which are simultaneously sent to the brain for processing. Retinal parallel visual processing starts at a retinal bipolar cell. There are two types of retinal bipolar cells, ON-center bipolar cells, and OFF-center bipolar cells, both of which receive a glutamate neurotransmitter. Upon light stimulation, the ON-center bipolar cells depolarize, whereas the OFF-center bipolar cells hyperpolarize, providing information regarding light increments and decrements. This segregation of ON and OFF channels is a fundamental principle of retinal processing, and the processing remains separate as they are transmitted to retinal ganglion cells and higher brain visual centers.

[0043] Retinal ganglion cells are final output neurons in the retina that propagate visual stimuli to the brain. There are about 1 .2 to 1 .5 million retinal ganglion cells in the human retina with at least eighteen types of retinal ganglion cells (Kim US et al., Front Neurol. 2021 May 21 ;12:661938). Retinal ganglion cells also process additional features of visual processing, such as orientation and directional selectivity.

[0044] Channelrhodopsin (ChR)-based optogenetics is a particularly promising approach to restore vision lost due to photoreceptor degenerative diseases. A common strategy is to express a depolarizing ChR in retinal ganglion cells, driven by a ubiquitous promoter and delivered by AAV vectors through intravitreal injection. However, this strategy would convert all transduced cells to ON cells, changing the intrinsic visual processing features. A second treatment strategy is to target a depolarizing ChR in ON bipolar cells. AAV-mediated targeted expression in such cells can be achieved using mGluR6 promoters. Targeting ON bipolar cells can lead to the generation of downstream ON and OFF responses in retinal ganglion cells, comparable to intrinsic visual processing features. However, AAV-mediated transduction in bipolar cells is less efficient than in retinal ganglion cells.

[0045] The feasibility of optogenetic gene therapy has been demonstrated, especially through AAV-mediated expression of channelrhodopsins, in surviving inner retinal neurons (retinal bipolar cells and ganglion cells) in rodent models (see U.S. Patent Publication No. 20180214513 and U.S. Patent No. 9,730,981 ). In primates and humans, AAV-mediated transduction efficiency in inner retinal neurons through intravitreal delivery is further hampered by the inner limiting membrane (ILM). The transduction efficiency of channelrhodopsins in bipolar cells is further decreased due to the presence of additional retinal layers for viral vectors to reach bipolar cells. Thus, methods to increase transgene expression are desired to improve the outcome of optogenetic gene therapy.

[0046] The present disclosure generally relates to improved gene therapy compositions and methods of using the same to treat, prevent, or ameliorate disease. The present disclosure is based, in part, on the discovery that introducing multiple copies of a therapeutic transgene in a viral construct can increase delivery efficacy without increasing the viral load. While transgene delivery to retinal cells is provided as an exemplary system, this example is not intended to be limiting and multiple copies of transgenes may be delivered to other cells using the same or different transgenes and viral vectors.

[0047] Thus, the present disclosure provides methods for increased efficiency of delivery of transgenes. In some aspects, such delivery may be to the eye for treating an ocular disease or disorder, or for restoring or improving vision. Transgenes of particular interest for restoration of photosensitivity or vision include photosensitive proteins, such as microbial or mammalian opsin genes. As used herein “transgene” refers to a polynucleotide encoding a polypeptide of interest, wherein the polynucleotide is present in a nucleic acid expression vector suitable for gene therapy (e.g., a viral vector such as AAV).

[0048] Previous studies in non-human primates have shown that injection of a recombinant adeno- associated viral vector encoding a transgene, such as channelopsin-2 (Chop2), results in poor delivery of the vector and low expression of Chop2 in the inner retinal cells, especially bipolar cells. Also, AAV-mediated gene transfection was found to be more efficient in peripheral retina, parafovea, and along blood vessels, suggesting that the inner limiting membrane (ILM), which is the boundary between the retina and the vitreous space, is a major barrier (Ivanova et aL, Neuroscience. 2010 Jan 13;165(1 ):233-43).

[0049] The methods described herein provide enhanced delivery of therapeutic transgenes. The enhanced delivery of transgenes is demonstrated by increased transduction efficiency, increased expression of the therapeutic transgene (i.e., CoChR-3M), and increased efficacy of the therapeutic compound (i.e., increased light sensitivity or restored visual acuity).

[0050] By constructing a viral cassette that contains multiple copies of the same therapeutic transgene linked by 2A self-cleaving peptides (P2A, E2A, F2A, and T2A) or non-cleaving peptides (e.g., the transmembrane helix of the rat gastric H+ / K+ ATPase beta-subunit [pHK], or internal ribosome entry site (IRES)), the expression of proteins may be increased in the targeted cells without increasing the viral load. Constructs include a viral vector, a promoter, a plurality of transgenes, and optionally a post-transcriptional regulatory protein. In some aspects, the construct may include cleaving and / or non-cleaving peptides. Promoters may be ubiquitous or retinal specific promoters. Exemplary viral vectors include adeno-associated virus (AAV), lentivirus, and herpes simplex virus (HSV). Exemplary promoters include ubiquitous and cell specific promoters. Exemplary ubiquitous promoters include cytomegalovirus immediate enhancer / p-actin (CAG), cytomegalovirus (CMV), synapsin, elongation factor-1 alpha (EF1 a), or thymocyte differentiation antigen 1 (Thy-1 ) promoter. Exemplary cell-specific promoters may include retinal promoters such as metabolic glutamate receptor 6 (mGluR6), neurokinin 3 (NK-3), or Purkinje cell protein 2 (Pcp2(L7)), gamma-synuclein gene (SNOG), Hkamac, or rhodopsin (RHO). In some aspects, the promoters may include variants such as shortened promoters, for example, shortened mGluR6 as described in further detail below. Exemplary post-transcriptional regulatory proteins include Woodchuck Hepatitis Virus (WHP) Post-transcriptional Regulatory Element (WPRE), modified WPRE (mWPRE), human growth hormone polyadenylation signal (hGHpA), bovine growth hormone polyadenylation signal (bGHpA), and / or SV40 early polyadenylation signal (SV40pA). In some aspects, the viral cassette may include the transmembrane helix of the rat gastric H+ / K+ ATPase beta-subunit (PHK). While not wishing to be bound, it is currently thought that the use of [3HK assists in maintaining the transmembrane topology.

[0051] The viral constructs described herein may be administered concurrently or sequentially with a therapeutic agent. For example, for concurrent administration, a construct such as an AAV construct may be formulated with a therapeutic agent in a single composition suitable for delivery, for example, injection, by methods known in the art. Alternatively, the construct may be injected in separate compositions, simultaneously or sequentially with a therapeutic agent. In a preferred embodiment, the construct may be administered prior to administration of a therapeutic agent.

[0052] Such formulations include a pharmaceutically and / or physiologically acceptable vehicle, diluent, carrier, or excipient, such as buffered saline or other buffers, e.g., HEPES, to maintain physiologic pH. For a discussion of such components and their formulation, see, generally, Gennaro, A E., Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishers; 2003 or latest edition; see also, W02000015822. If the preparation is to be stored for long periods, it may be frozen, for example, in the presence of glycerol.

[0053] The gene of interest to be delivered by the methods described herein is any therapeutic transgene known in the art for treating, alleviating, reducing, or preventing a disease. In some aspects, the gene of interest (i.e., therapeutic transgene) is known in the art for treating, alleviating, reducing, or preventing a symptom of an ocular disease, an ocular disorder, or an ocular condition. For example, the viral vectors described herein may be used to treat diseases including macular degeneration, age-related macular degeneration (AMD), diabetic retinopathy (DR), retinitis pigmentosa (RP), glaucoma, and other inherited retinal degenerations, uveitis, retinal detachment, paraneoplastic retinopathies, and eye cancers (ocular melanoma and retinoblastoma) each of which can lead to visual loss or complete blindness.

[0054] Examples of nucleic acids suitable for use in the methods described herein include viral vectors encoding therapeutic transgenes (i.e., channelrhodopsins, or any genetically encoded photosensitive proteins), or RNA interference molecules (i.e., short hairpins, siRNA, or microRNAs). Particularly preferred viral vectors are rAAV vectors that encode a rhodopsin such as channelrhodopsins for expression in the retina to restore light sensitivity.

[0055] Aspects of the current disclosure are now described with additional details and options as follows: (I) Expression Vectors; (II) Exemplary Embodiments; (III) Ocular diseases; (IV) Restoration of Light Sensitivity; (V) Pharmaceutical compositions; (VI) Kits; (VII) Experimental Examples; and (VIII) Closing Paragraphs. These headings do not limit the interpretation of the disclosure and are provided for organizational purposes only.(I) Expression vectors

[0056] The term “vector” is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, e.g., replication defective retroviruses and lentiviruses.

[0057] The term “nucleic acid cassette” or “expression cassette” as used herein refers to genetic sequences within the vector that can express RNA, and subsequently a polypeptide. In one aspect, the nucleic acid cassette contains one or more gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest. In another aspect, the nucleic acid cassette contains one or more expression control sequences and one or more gene(s)-of-interest. Vectors may include one, two, three, four, five, or more nucleic acid cassettes. The nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo appropriate post-translational modifications required for activity in the transformed cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into extracellular compartments. Preferably, the cassette has its 3' and 5' ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end. In a preferred aspect of the invention, the nucleic acid cassette contains the sequence of a plurality of therapeutic genes used to treat, prevent, or ameliorate a genetic disorder, such as an ocular disorder. The cassette can be removed and inserted into a plasmid or viral vector as a single unit.Viral vectors

[0058] As will be evident to one of skill in the art, the term “viral vector” is widely used to refer either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate the transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s).

[0059] The term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. Theterm “adeno-associated viral vector” (AAV) refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof that are primarily derived from an adenovirus. The term “retroviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof that are primarily derived from a retrovirus. The term “lentiviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus. The term “hybrid” refers to a vector, LTR, or other nucleic acid containing both viral and non-viral viral sequences.

[0060] In particular aspects, the terms “viral vector” and “viral expression vector” may be used to refer to viral transfer plasmids and / or infectious viral particles. Where reference is made herein to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that the sequences of these elements are present in RNA form in the viral particles of the invention and are present in DNA form in the DNA plasmids of the invention. Nucleic acid expression vectors suitable for use in gene therapy are known in the art. For example, the nucleic acid expression vector is a viral vector. The viral vectors can be retroviral vectors, adenoviral vectors, adeno-associated vectors (AAV), herpes simplex virus (HSV)-based vectors, or alphavirus-based vectors, such as those made from semliki forest virus (SFV) and sindbis virus (SIN), or any engineered or recombinant viral vector known in the art. Additional vectors may include fusion proteins and chemical conjugates.

[0061] Retroviruses, such as C-type retroviruses and lentiviruses, may also be used. For example, retroviral vectors may be based on murine leukemia virus (MLV), as provided by Hu and Pathak, Pharmacol. Rev. 52:493511 , 2000 and Fong et al., Grit. Rev. Ther. Drug Carrier Syst. 17:1-60, 2000. MLV-based vectors may contain up to 8 kb of heterologous (therapeutic) DNA in place of the viral genes. Additional retroviral vectors may be used including replication-defective lentivirus- based vectors, including human immunodeficiency (HlV)-based vectors (Vigna and Naldini, J. Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Virol. 72:8150-8157, 1998). Lentiviral vectors may be derived from human and non-human (including SIV) lentiviruses. Examples of lentiviral vectors include nucleic acid sequences required for vector propagation as well as a tissue-specific promoter operably linked to an mCoChop gene. Nucleic acid sequences may include the viral LTRs, a primer binding site, a polypurine tract, att sites, and an encapsidation site.

[0062] A lentiviral vector may be packaged into any suitable lentiviral capsid. The substitution of one particle protein with another from a different virus is referred to as “pseudotyping”. The vector capsid may contain viral envelope proteins from other viruses, including murine leukemia virus(MLV) or vesicular stomatitis virus (VSV). The use of the VSV G-protein yields a high vector titer and results in greater stability of the vector virus particles.

[0063] Recombinant, replication-defective alphavirus vectors may be advantageous because they are capable of high-level heterologous (therapeutic) gene expression and can infect a wide target cell range. Alphavirus replicons may be targeted to specific cell types by displaying on their virion surface a functional heterologous ligand or binding domain that would allow selective binding to target cells expressing a cognate binding partner. Alphavirus replicons may establish latency, and therefore long-term heterologous nucleic acid expression in a target cell. The replicons may also exhibit transient heterologous nucleic acid expression in the target cell.

[0064] Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors may be used with the compositions and methods of the disclosure. The adenovirus vector may result in a shorter-term expression (e.g., less than about a month) than adeno-associated virus, in some aspects, and may exhibit much longer expression. Viral vectors may be selected based on their ability to support multiple copies of the transgene. For instance, the lentiviral vector payload capacity is about 9 kb, the retroviral payload capacity, that is, the size of the transgenes and regulatory elements it can carry is about 8 kb, and the adeno-associated virus (AAV) payload capacity is about 5 kb. Thus, the type of transgene, the number of transgenes, and the size and type of regulatory elements may be varied depending on the viral vector being used. In some aspects, the viral vector may be selected based on its ability to transfect a particular cell type.

[0065] AAV is a naturally replication-deficient virus that requires a helper virus for replication. This self-limiting infection coupled with the ability to stably infect dividing and non-dividing cells makes AAV a useful target for in vivo gene therapy. Further, AAV vectors have tissue-targeting abilities that can be enhanced with capsid re-engineering. Exemplary adeno-associated vectors, include AAV-1 (SEQ ID NO: 9), AAV-2 (SEQ ID NO: 10), AAV-3 (SEQ ID NO: 11 ), AAV-4 (SEQ ID NO: 12), AAV-5 (SEQ ID NO: 13), AAV-6 (SEQ ID NO: 14), AAV-7 (SEQ ID NO: 15), AAV-8 (SEQ ID NO:16), AAV-9 (SEQ ID NO: 17), AAV-10 (SEQ ID NO: 18), AAV-1 1 (SEQ ID NO: 19), AAV-12 (SEQ ID NO: 20) or any engineered or recombinant AAV known in the art. For example, D100 is a recently developed AAV vector (Kotterman et al., bioRxiv 2021.06.24.449775 doi.org / 10.1101 / 2021 .06.24.449775 (2021 )). In some aspects, the AAV vectors are produced using a baculovirus system with sf9 insect cell line. In some aspects, the vector is recombinant AAV-2 (rAAV2).

[0066] In some aspects, the rAAV vector includes a capsid having at least one mutation. For example, the AAV2.7m8 (AAV2588LALGETTRP) (SEQ ID NO:21 ) vector is an engineered capsid with a 9-amino acid insertion in the AAV surface variable region VIII (VR-VIII) resulting in thealteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration (Dalkara, D. et aL, Sci. TransL Med. 5(189):ra76 (2013)). In other aspects, it may be AAV2.7m8(Y444F) which is a capsid variant of AAV that can be used to deliver genes to retinal cells.

[0067] In some embodiments, a recombinant adeno-associated viral (rAAV) vector includes a capsid protein with a mutated tyrosine residue which enables the vector to have improved transduction efficiency of a target cell, e.g., a retinal bipolar cell (e.g., ON or OFF retinal bipolar cells; rod or cone bipolar cells) or retinal ganglion cells. In some cases, the rAAV further includes a promoter (e.g., mGluR6, or fragment thereof, or other ubiquitous or cell specific promoter) capable of driving the expression of a protein of interest in the target cell.

[0068] In one embodiment, a mutation may be made in any one or more of the tyrosine residues of the capsid protein of AAV 1 -12 or hybrid AAVs. In specific embodiments, these are surface- exposed tyrosine residues. In a related embodiment, the tyrosine residues are part of the VP1 , VP2, or VP3 capsid protein. In exemplary embodiments, the mutation may be made at one or more of the following amino acid residues of an AAV-VP3 capsid protein: Tyr252, Tyr272, Tyr444, Tyr500, Tyr700, Tyr704, Tyr730; Tyr275, Tyr281 , Tyr508, Tyr576, Tyr612, Tyr673 or Tyr720. Exemplary mutations are tyrosine-to-phenylalanine mutations including, but not limited to, Y252F, Y272F, Y444F, Y500F, Y700F Y704F, Y730F, Y275F, Y281 F, Y508F, Y576F, Y612G, Y673F and Y720F. In a specific embodiment, these mutations are made in the AAV2 serotype. In some cases, an AAV2 serotype includes a Y444F mutation, and / or an AAV8 serotype includes a Y733F mutation, wherein 444 and 733 indicate the location of a point tyrosine mutation of the viral capsid. In further embodiments, such mutated AAV2 and AAV8 serotypes encode a light-sensitive protein and also include a modified mGluR6 promoter to drive the expression of such light-sensitive protein. Such AAV vectors are described in, for example, Petrs-Silva et aL, Mol Ther., 201 1 19:293-301.

[0069] In some embodiments, the amino acid mutation is of one or more of the surface tyrosine residues (e.g., Y252, Y272, Y444, Y500, Y700, Y704, and Y730 of an AAV2 capsid protein), surface threonine residues (e.g., T251 , T329, T330, T454, T455, T503, T550, T592, T581 , T597, T491 , T671 , T659, T660, T701 , T713, and T716 of an AAV2 capsid protein), surface serine residues (e.g., S261 , S264, S267, S276, S384, S458, S468, S492, S498, S578, S658, S662, S668, S707, S721 of an AAV2 capsid protein), and / or surface lysine residues (e.g., 258, K321 , K459, K490, K507, K527, K572, K532, K544, K549, K556, K649, K655, K665, K706 of an AAV2 capsid protein). These residues are highly conserved between AAV1 -AAV12 capsids, thus embodiments utilizing AAV1 -AAV12 are encompassed herein and could be readily developed bythose of ordinary skill. Preferred capsid mutants of the invention increase transduction efficiency compared with wild-type capsid proteins.

[0070] In one aspect, the mutation is a tyrosine (Y) to phenylalanine (F) at one or more of Y252, Y272, Y444, Y500, Y700, Y704, and Y730 of an AAV2 capsid protein or an equivalent conserved residue of AAV1 or AAV3-12. In a preferred embodiment, the Y to F mutation is at amino acid position 444 and / or position 730 of an AAV2 capsid protein or an equivalent conserved residue of AAV1 or AAV3-12. In another preferred embodiment, the mutant is a quadruple mutant with Y to F mutations at Y272, Y444, Y500, and Y730 (Petrs-Silva, H. et al., Mol. Then, vol. 19(2): 293- 301 (2011 )).

[0071] In another aspect, the mutation is a threonine (T) to valine (V) at one or more of T251 , T329, T330, T454, T455, T503, T550, T592, T581 , T597, T491 , T671 , T659, T660, T701 , T713, and T716 of an AAV2 capsid protein or an equivalent conserved residue of AAV1 or AAV3-12. In a preferred embodiment, the T to V mutation is at amino acid position 491 of an AAV2 capsid protein or an equivalent conserved residue of AAV1 or AAV3-12. In yet another embodiment, the mutant capsid includes Y to F mutations at Y272, Y444, Y500, and Y730, as well as a T to V mutation at amino acid position 491 of an AAV2 capsid protein or an equivalent conserved residue of AAV1 or AAV3-12 (Kay, C. N. et al., “PLoS One, vol. 8(4): e62097 (2013)).

[0072] In another embodiment, the capsid protein is engineered to include a 9-amino acid stretch of a conformationally variable region of the AAV8 capsid protein between positions 585 and 593. In some embodiments, the capsid protein includes SEQ ID NO: 22, PERTAMSLP. In preferred embodiments, SEQ ID NO: 22 is inserted between positions 585 and 593 of an AAV2 capsid protein or the equivalent conserved residues of AAV1 or AAV3-12. The capsid proteins including this sequence effectively transduce ocular cells, including bipolar and ganglion cells. In some embodiments, the capsid protein includes SEQ ID NO: 23, SFSRAVLCD. In some embodiments, SEQ ID NO: 23 is inserted between positions 585 and 593 of an AAV2 capsid protein or the equivalent conserved residues of AAV1 or AAV3-12. The capsid proteins including this sequence may transduce ocular cells, preferably ON bipolar cells (Cronin, T. et al., EMBO Mol. Med., vol. 6(9): 1 175-1 190 (2014)).

[0073] In some aspects, the mutation in the capsid protein is a tyrosine to phenylalanine at amino acid position 444. The ordinarily skilled artisan could readily design nucleic acid sequences that encode said mutated capsid protein. A nucleic acid sequence for such an exemplary capsid protein is provided in SEQ ID NO: 24.Promoters

[0074] The term “promoter” as used herein refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The term “enhancer” refers to a segment of DNA that contains sequences capable of providing enhanced transcription. In some instances, the enhancement can function independently of its orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and / or other enhancer elements. The term “promoter / enhancer” refers to a segment of DNA that contains sequences capable of providing both promoter and enhancer functions.

[0075] In some embodiments, the expression of the therapeutic transgene is driven by a ubiquitous promoter, i.e., cytomegalovirus immediate enhancer / p-actin (CAG), cytomegalovirus (CMV), synapsin, elongation factor-1 alpha (EF1 a), or Thymocyte differentiation antigen 1 (Thy-1) promoter or long terminal repeat (LTR). In some aspects, the promoter may be a cell type-specific promoter such as metabolic glutamate receptor 6 (mGluR6), neurokinin 3 (NK-3), or Purkinje cell protein 2 (Pcp2(L7)), gamma-synuclein gene (SNCG), HKamac, or rhodopsin (RHO).

[0076] In some aspects, the nucleic acid molecule or nucleic acid expression vector may include a mGluR6 enhancer or a variant thereof. As used herein, the term “modified mGluR6 promoter” refers to the combination of regulatory elements that include at least a 200 bp mGluR6 enhancer sequence and at least a fragment of the promoter region from mGluR6 gene (e.g., about a 1 kb or a 500 bp fragment). In some aspects, the modified mGluR6 promoter may be a shortened mGluR6 promoter(mGluR6(s)). In some aspects, the modified mGluR6 promoter includes at least a 200 bp mGluR6 enhancer of SEQ ID NO: 25, the human 198 bp mGluR6 enhancer (corresponding to the mouse 200 bp enhancer) is provided in SEQ ID NO: 26 (US Patent No. 1 1 ,583,595).

[0077] The modified mGluR6 promoter further includes at least a fragment of the mGluR6 promoter region. The length of the promoter and the sequence that includes control elements of mGluR6 can be adjusted by increasing or decreasing the fragment length. For example, a fragment of the promoter of about 1 kb in length can be used as a mGluR6 promoter sequence. Promoter analysis can be used to identify promoter functional fragments and derivatives (McGowen et al. Mol. Vision 1998, 4:2; Bookstein et al. PNAS 1990, 87 (19) 7762-66). The sequences provided herein are meant to be exemplary and in no way limit the scope of the invention. A person of ordinary skill in the art, given the instant disclosure, would be enabled to identify further suitable enhancer, promoter, transgene, and vector sequences.

[0078] For example, the mGluR6 promoter sequence used herein may be a 1095 bp fragment of the mGluR6 promoter region as shown in SEQ ID NO: 28. SEQ ID NO: 29 is a nucleic acidsequence of the human 1784 promoter (corresponding to the mouse mGluR6 1095 bp promoter) fragment of the human mGluR6 promoter region. In some aspects, the mGLUR6 promoter may include intron 3 and intron 4 such as mouse intron 4 (SEQ ID NO: 64), mouse intron 3 (SEQ ID NO: 65), human intron 4 (SEQ ID NO: 66) and human intron 3 (SEQ ID NO: 67). In some aspects, the mGluR6 promoter may be a shortened mGluR6 promoter in which intron 3 has been removed, for example the mGlurR6 promoter of SEQ ID NO: 61 . In other aspects, the mGluR6 promoter of SEQ ID NO: 62 may be used. In other aspects, the mGluR6 promoter sequence may be a 500 bp fragment of the mGluR6 promoter region (SEQ ID NO: 30). The 500 bp fragment described in SEQ ID NO: 30 is encompassed by the 1095 bp fragment described above (SEQ ID NO: 28). The 500 bp fragment is preferred because it results in higher expression than the 1095 bp fragment. In some aspects, a nucleic acid sequence of the 547 bp human mGluR6 promoter, encompassed by the 1784 bp fragment described above (SEQ ID NO: 29) and provided in SEQ ID NO: 31 may be used. In some aspects the mGluR6 promoter may include combinations of enhancers and promoters, for example, it may be a combination of mouse enhancer SEQ ID NO: 25 and mouse promoter SEQ ID NO: 69, or human enhancer SEQ ID NO: 68 and human promoter SEQ ID NO: 70. Intron 3 and / or intron 4 may also be included. In some aspects, the mGluR6 promoter may be a combination of the 200 bp mGluR6 enhancer (SEQ ID NO: 25) and the 500 bp mGluR6 promoter (SEQ ID NO: 30) resulting in SEQ ID NO: 71. Other promoters such as the SV40 promoter of SEQ ID NO: 72 or combinations of promoters such as the combination of the 200 bp mGluR6 enhancer (SEQ ID NO: 25) and the SV40 promoter (SEQ ID NO:72) as shown, for example, in SEQ ID NO: 73.

[0079] In various aspects, vectors may include modified 5' long terminal repeats (LTRs) and / or 3' LTRs. Long terminal repeats (LTRs) are domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R, and U5 regions. LTRs generally provide functions fundamental to the expression of viral genes (e.g., promotion, initiation, transcriptional control elements, replication, and polyadenylation of gene transcripts) and to viral replication. The U3 region contains the enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. Modifications of the 3' LTR are often made to improve the safety of the viral systems by rendering viruses replication defective. As used herein, the term “replication-defective” refers to virus that is not capable of complete, effective replication such that infective virions are not produced (e.g., replication-defective lentiviral progeny). The term “replication-competent” refers to wild-type virusor mutant virus that is capable of replication, such that viral replication of the virus is capable of producing infective virions (e.g., replication-competent lentiviral progeny).

[0080] Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site). As used herein, the term “packaging signal” or “packaging sequence” refers to sequences located within the viral genome that are required for insertion of the viral RNA into the viral capsid or particle (see e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101 -2109). As used herein, the terms “packaging sequence,” “packaging signal,” “psi” and the symbol ‘“PSI,” are used in reference to the non-coding sequence required for encapsidation of retroviral RNA strands during viral particle formation.

[0081] “ Self-inactivating” (SIN) vectors refer to replication-defective vectors, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion and / or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region during viral replication and, thus, the viral transcript cannot be made without the U3 enhancer-promoter. In a further aspect of the invention, the 3' LTR is modified such that the U5 region is replaced, for example, with a heterologous or synthetic poly(A) sequence, one or more insulator elements, and / or an inducible promoter. It should be noted that modifications to the LTRs such as modifications to the 3' LTR, the 5' LTR, or both 3' and 5' LTRs, are also included in the invention.

[0082] An additional safety enhancement is provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Examples of heterologous promoters which can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. Typical promoters are able to drive high levels of transcription in a TAT-independent manner. This replacement reduces the possibility of recombination to generate replication-competent virus because there is no complete U3 sequence in the virus production system. In certain aspects, the heterologous promoter may be inducible, such that transcription of all or part of the viral genome will occur only when one or more induction factors are present. Induction factors include one or more chemical compounds or physiological conditions, e.g., temperature or pH, in which the host cells are cultured.

[0083] In some aspects, viral vectors include a TAR element. The term “TAR” refers to the “transactivation response” genetic element located in the R region of lentiviral (e.g., HIV) LTRs. This element interacts with the viral trans-activator (tat) genetic element to enhance viral replication.However, this element is not required in aspects wherein the U3 region of the 5' LTR is replaced by a heterologous promoter.

[0084] As used herein, the term “FLAP element” refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, et aL, 2000, Cell, 101 :173. During HIV-1 reverse transcription, central initiation of the plus-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) lead to the formation of a three-stranded DNA structure: the HIV-1 central DNA flap. While not wishing to be bound by any theory, the DNA flap may act as a cis- active determinant of lentiviral genome nuclear import and / or may increase the titer of the virus. In particular aspects, the retroviral or lentiviral vector backbones include one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vectors. For example, in particular aspects a transfer plasmid includes a FLAP element. In one aspect, a vector of the invention includes a FLAP element isolated from HIV-1 .

[0085] In other embodiments, the promoter is an inducible or a cell-specific promoter. Cell typespecific promoters that enable transgene expression in specific subpopulations of cells, i.e., retinal neuron cells or degenerating cells, may be preferred. These cells may include a retinal ganglion cell, a photoreceptor cell, a bipolar cell, a rod bipolar cell, an ON-type cone bipolar cell, a photosensitive retinal ganglion cell, a horizontal cell, an amacrine cell, an All amacrine cell, or a retinal pigment epithelial cell. Cell type-specific promoters are well known in the art. Cell typespecific promoters include mGluR6, NK-3, Purkinje cell protein 2 (L7)(Pcp2(L7)), human gamma- synuclein gene (SNCG), HKamac, and Rhodopsin (Rho). Cell type-specific promoters may be modified using recombinant DNA techniques known in the art to increase efficiency of expression and selective targeting. For example, a modified mGluR6 promoter contains a combination of regulatory elements from the mGluR6 gene, as described in U.S. Patent Publication No. US 2017- 0021038 A1.

[0086] Generally, promoter sequences and / or any associated regulatory sequences may include about at least 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp or 10000 bp. Promoter sequences and any associated regulatory sequences may include about at most 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp or 10000 bp.

[0087] In any of the isolated nucleic acid molecules or nucleic acid expression vectors described herein, the mGluR6 promoter or other ubiquitous or cell specific promoter is upstream of a transgene to be expressed in the eye. Preferably, the transgene encodes a gene product thatincreases light sensitivity, increases light detection, increases photosensitivity, increases visual evoked potential, or restores vision in a retina. More preferably, the transgene is an opsin gene. Examples of opsin genes include channelrhodopsins (channelrhodopsin 1 or 2 (ChR1 or ChR2), CoChR, Volvox carteri channelrhodopsins 1 or 2 (vChR1 or vChR2), mVChRI , ex3mV1Co, red activatable channelrhodopsin (ReaChR), Chronos, Chrimson, Platymonias subcordiformus channelrhodopsin (PsChR), ChRmine, ChroME2s, melanopsin, pineal opsin, mammalian photopsins (rhodopsin, cone opsins, green cone opsin), halorhodopsin, bacteriorhodopsin, proteorhodopsin, Opto-mGluR6, Mela(CTmGluR6), or any functional variants or chimeras thereof. Suitable opsin variants may be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% 98%, or 99% identical to the opsin. Preferably, the opsin variant has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the functional activity of the opsin. Functional activity can be measured by any means known in the art, for example, by electroretinography.Transgenes

[0088] The gene of interest (i.e., therapeutic transgene) can be any transgene that may treat, prevent, or ameliorate a disease or disorder. Exemplary transgenes are light-sensitive opsins. A light-sensitive protein can occur naturally in plant, animal, archaebacterial, algal, or bacterial cells, or can alternatively be created through laboratory techniques. The opsin family of genes includes vertebrate (animal) and invertebrate opsins.

[0089] Channelopsins are seven transmembrane domain proteins that become photo-switchable (light sensitive) when bound to the chromophore all-trans-retinal. Animal opsins are G-protein coupled receptors (GPCRs) with 7-transmembrane helices which regulate the activity of ion channels. Invertebrate rhodopsins are usually not GPCRs but are light-sensitive or light-activated ion pumps or ion channels.

[0090] When linked to a retinal molecule via Schiff base linkage, channelopsins form a light-gated, nonspecific, inwardly rectifying, cation channel, called a channelrhodopsin. Examples of opsin genes or light-sensitive proteins include channelrhodopsins, such as channelrhodopsin 1 or 2 (ChR1 or ChR2), CoChR, Volvox carteri channelrhodopsins 1 or 2 (vChR1 or vChR2), mVChRI , ex3mV1 Co, red activatable channelrhodopsin (ReaChR), Chrimson, Platymonas subcordiformus channelrhodopsin (PsChR), ChRmine, ChroME2s), melanopsin, pineal opsin, mammalian photopsins (rhodopsin, cone opsins, green cone opsin), halorhodopsin, bacteriorhodopsin, proteorhodopsin, Opto-mGluR6, Mela(CTmGluR6), or any functional variants or chimeras thereof (see, e.g., U.S. Patent Publication Nos. 20220089660 and 20180214513).

[0091] The nucleotide sequence for a wild-type channelrhodopsin is shown in SEQ ID NO: 51 and the corresponding amino acid sequence is shown at SEQ ID NO: 52. These light-sensitive channels, when expressed and activated in neural tissue, allow for a cell to be depolarized when stimulated with light (Boyden, 2005). A Chop2 fragment (315 amino acids) (SEQ ID NO: 40) has been shown to efficiently increase photosensitivity and vision in mouse models of photoreceptor degeneration (Bi A, Cui J, Ma YP, Olshevskaya E, Pu M, Dizhoor AM, Pan ZH. Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration. Neuron. 2006 Apr 6;50(1 ):23-33, and U.S. Pat. No. 8,470,790). Chop2 mutants and variants as described in PCT Publication WO 2013 / 134295 may also be expressed using the promoters described herein. Any channelrhodopsins (ChRs), or microbial opsins, or other genetically encoded light sensors or switches, presently known or as yet undiscovered, are useful in generating the compositions and practicing the methods of the invention. Other suitable transgenes include Volvox carter! channelrhodopsins (e.g., vChR1 (SEQ ID NO: 45 (GenBank Accession No. EU285658.1 ))) and vChR2 (SEQ ID NO: 47 (GenBank Accession No. EU285660)) with corresponding amino acid sequences SEQ ID NO: 46 (vCHR1 ) and SEQ ID NO: 48 (vChR2). Additional suitable transgenes include the mutants disclosed in U.S. Patent No. 11 ,041 ,004, and U.S. Patent No. 10,307,492, such as mutant CoChR amino acid sequences L1 12C (SEQ ID NO: 53), C68S / V69I (SEQ ID NO: 54), T139C (SEQ ID NO: 55), T145A / S146A (SEQ ID NO: 56), C68T / V69L (SEQ ID NO: 57), L112C / T139C (SEQ ID NO: 58), L1 12C / H94E (SEQ ID NO: 59) and H94E / L112C / K264T (abbreviated as Co3M) (SEQ ID NO: 60).

[0092] Chopl and Chop2 are two rhodopsins from the green algae Chlamydomonas reinhardtii (Nagel G, Ollig D, Fuhrmann M, Kateriya S, Musti AM, Bamberg E, Hegemann P. Channelrhodopsin-1 : a light-gated proton channel in green algae. Science. 2002 Jun 28;296(5577):2395-8; Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, Ollig D, Hegemann P, Bamberg E. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13940-5.). A nucleic acid sequence encoding an exemplary Chopl of the disclosure includes GenBank accession number AB058890 (SEQ ID NO: 32). The corresponding amino acid sequence encoding an exemplary Chopl of the disclosure includes GenBank accession number BAB68566 (SEQ ID NO: 36). A nucleic acid sequence encoding an exemplary Chopl of the disclosure includes GenBank accession number AF385748 (SEQ ID NO: 33). The corresponding amino acid sequence encoding an exemplary Chopl of the disclosure includes GenBank accession number AAL08946 (SEQ ID NO: 37). A nucleic acid sequence encoding an exemplary Chopl of the disclosure includes GenBank accession number AB058891 (SEQ ID NO: 34). The correspondingamino acid sequence encoding an exemplary Chopl of the disclosure includes GenBank accession number BAB68567.1 (SEQ ID NO: 38). A nucleic acid sequence encoding an exemplary Chopl of the disclosure includes GenBank accession number AF461397 (SEQ ID NO: 35). The corresponding amino acid sequence encoding an exemplary Chopl of the disclosure includes GenBank accession number AAM15777 (SEQ ID NO: 39). A nucleic acid sequence encoding Chop2 (also known as ChR2) includes (GenBank accession number AB058891 (SEQ ID NO:34) / AF461397 (SEQ ID NO: 35)), In some aspects, the channelrhodopsin may be a Volvox carter! f. nagariensis channelrhodopsin (vChR) as shown for example in SEQ ID NOs: 46-49. Volvox carter! f. nagariensis channelrhodopsins (vChRs) such as VchR1 have a peak excitation wavelength around 589nm, which is red-shifted compared to the commonly used ChR2.

[0093] In some aspects, the opsin may include NpHR (Halorhodopsin) (GenBank accession number EF474018, SEQ ID NO: 41 and SEQ ID NO: 42), a photosensitive chloride pump from the haloalkallphilic archaeon Natronomaspharonis. In some aspects, single or multiple point mutations to the NpHR protein can result in NpHR variants. In some aspects, codon optimized or enhanced embodiments of NpHR may be used. The addition of the amino acids FCYENEV to the NpHR C-terminus along with the signal peptide from the p subunit of the nicotinic acetylcholine receptor to the NpHR N-terminus results in the construction of enhanced NpHR (eNpHR).

[0094] Melanopsin (GenBank accession number 6693702) (SEQ ID NO: 43) and (GenBank accession number AF147789_1 ) (SEQ ID NO: 44) is a photopigment found in specialized photosensitive ganglion cells of the retina that are involved in the regulation of circadian rhythms, pupillary light reflex, and other non-visual responses to light. In structure, melanopsin is a retinylidene protein variety of G-protein-coupled receptor. Melanopsin resembles invertebrate opsins in many respects, including its amino acid sequence and downstream signaling cascade. Like invertebrate opsins, melanopsin appears to be a bistable photopigment, with intrinsic photoisomerase activity. In certain embodiments variants of melanopsin can be created for example via single or multiple point mutations to the melanopsin protein. A nucleic acid sequence encoding an exemplary Melanopsin of the disclosure includes GenBank accession number 6693702 (SEQ ID: 43). The corresponding amino acid sequence encoding an exemplary Melanopsin of the disclosure includes SEQ ID NO: 44.

[0095] Light-sensitive proteins may also include proteins that are at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% identical to any ofthe light-sensitive proteins described herein (i.e., ChR1 , ChR2, vChR1 , vChR2, NpHR ReaChR, PsChR, Chronos, Chrimson, and melanopsin and its chimeras, such as Opto-mGluR6, Mela(CT mGluR6)). The light-sensitive proteins of the present invention may also include proteins that have at least one mutation. The mutation may be a point mutation.

[0096] In some embodiments, light-sensitive proteins can modulate signaling within neural circuits and bidirectionally control the behavior of ionic conductance at the level of a single neuron. In some embodiments, the neuron is a retinal neuron, a retinal bipolar cell (e.g., ON or OFF retinal bipolar cells; rod and cone bipolar cells), a retinal ganglion cell, a photoreceptor cell, or a retinal amacrine cell.

[0097] In some embodiments, a polyA tail may be inserted downstream of the transgene in an expression cassette or nucleic acid expression vector of the present invention. Suitable polyA tails are known in the art, and include, for example, human growth hormone poly A tail (hGHpA), bovine growth hormone polyA tail (bGHpA), bovine polyA, SV40 polyA, and AV40pA. An exemplary hGHpA is provided in SEQ ID NO: 50.

[0098] A polyA sequence may include a length of 1-10 bp, 10-20 bp, 20-50 bp, 50-100 bp, 100- 500 bp, 500 bp-1 kb, 1 Kb-2 kb, 2 Kb-3 kb, 3 kb-4 kb, 4 Kb-5 kb, 5 Kb-6 kb, 6 Kb-7 kb, 7 Kb-8 kb, 8 kb-9 kb, and 9 Kb-10 kb in length. A polyA sequence may include a length of at least 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, and 10 kb in length. A polyA sequence may include a length of at most 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb. and 10 kb in length.

[0099] In some cases, polyA sequences may be optimized for various parameters affecting protein expression, including the mRNA half-life of the transgene in the cell, stability of the mRNA of the transgene, or transcriptional regulation. For example, polyA sequences may be altered to increase mRNA transcript of the transgene, which may result in increased protein expression. In some cases, the polyA sequences may be altered to decrease the half-life of the mRNA transcript of the transgene, which may result in decreased protein expression.

[0100] Upon illumination by the preferred dose of light radiation, rhodopsin proteins open the pore of the channel through which H+, Na+, K+, and / or Ca2+ ions flow into the cell from the extracellular space. Activation of the rhodopsin channel typically causes a depolarization of the cell expressing the channel. Depolarized cells produce graded potentials and or action potentials to carry information from the rhodopsin-expressing cell to other cells of the retina or brain, toincrease light sensitivity or restore vision. Methods of improving vision or light sensitivity by administration of a vector encoding a channelopsin (or variant thereof) are described for example in U.S. Patent No. 8,470,790.

[0101] In some aspects, a dual rhodopsin system may be used to recapitulate the ON and OFF pathways integral to visual processing and acuity. Briefly, a CoChR protein of the present invention can be specifically targeted to ON type retinal neurons (i.e., ON type ganglion cells and / or ON type bipolar cells), while a hypopolarizing light sensor (i.e., halorhodopsin or other chloride or proton pump, or chloride or potassium-selective channelrhodopsins, known in the art) can be targeted to OFF type retinal neurons (i.e. OFF type ganglion cells and / or OFF type bipolar cells) to create ON and OFF pathways. The specific targeting to preferred cell subpopulations may be achieved through the use of different cell type-specific promoters. For example, CoChR expression may be driven by the mGluR6 promoter for targeted expression in ON-type retinal neurons (i.e., ON type ganglion cells and / or ON type bipolar cells) while a hypopolarizing channel, such as halorhodopsin, expression is driven by the NK-3 promoter or other promoters for targeted expression in OFF-type retinal neurons (i.e., OFF type ganglion cells and / or OFF type bipolar cells).

[0102] An alternative approach to restore ON and OFF pathways in the retina is achieved by expressing a depolarizing light sensor, such as CoChR, to rod bipolar cells or All amacrine cells. In this approach, the depolarization of rod bipolar cells or All amacrine cells can lead to the ON and OFF responses at the levels of cone bipolar cells and the downstream retinal ganglion cells. Thus, the ON and OFF pathways that are inherent in the retina are maintained.

[0103] In some aspects, a plurality of transgenes may be inserted with a single viral vector in an expression cassette. For example, there may be one, two, three, four, five, six, seven, or more copies of CoChR3M or other opsin included in a single expression cassette, as shown in FIGs. 5-8. The transgenes of the plurality of transgenes in the single viral vector may be the same or different. In some aspects, the transgenes are separated, for example, self-cleaving or noncleaving peptides such 2A peptides, for example P2A. Using the techniques described herein, the expression of transgene proteins can be multiplied in the targeted cells without increasing the viral load. However, there are limits to the number of copies that may be inserted in a single expression cassette as after a certain length, the expression cassette may become too large. For instance, the lentiviral vector payload capacity is about 9 kb, the retroviral payload capacity is about 8 kb, and the adeno-associated virus (AAV) payload capacity is about 5 kb. Thus, different viral vectors may be selected based on the size of the transgene or the number of copies of the transgene to be included in the expression cassette. In some aspects, modified or shortenedpromoters and post-transcriptional regulatory elements may be used to decrease the overall length of the expression cassette as seen for example in viral construct 8 (bAAV2-mGluR6(s)- Co3M-pHK-Co3M-mWPRE-bGHpA) (SEQ ID NO: 8).Post-transcriptional regulatory elements

[0104] In particular aspects, expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors. A variety of posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid at the protein, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) (Zufferey R, et aL, Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol. 1999 Apr;73(4):2886-92.); (Huang et aL, Mol. Cell. Biol., 5:3864); and the like or modified WPRE.

[0105] Elements directing the efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. The term “polyA site” or “polyA sequence” as used herein denotes a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. Illustrative examples of polyA signals that can be used in a vector of the invention include an ideal polyA sequence (e.g., AATAAA, ATT AAA AGTAAA), a human growth hormone polyadenylation signal (hGHpA), bovine growth hormone polyA sequence (BGHpA), a rabbit p-globin polyA sequence (r|3gpA) , or mouse growth hormone polyA sequence (mGHpA) or another suitable heterologous or endogenous polyA sequence known in the art.

[0106] In certain aspects, the viral vector further includes one or more insulator elements. Insulator elements may contribute to protecting lentivirus-expressed sequences, e.g., therapeutic polypeptides, from integration site effects, which may be mediated by cis-acting elements present in genomic DNA and lead to deregulated expression of transferred sequences (i.e., position effect; see, e.g., Burgess-Beusse et aL, 2002, Proc. Natl. Acad. ScL, USA, 99:16433; and Zhan et aL, 2001 , Hum. Genet., 109:471 ). In some aspects, transfer vectors include one or more insulator elements the 3' LTR, and upon integration of the provirus into the host genome, the provirus includes the one or more insulators at both the 5' LTR or 3' LTR, by virtue of duplicating the 3' LTR. Suitable insulators for use in the invention include the chicken [3-globin insulator (see, e.g., Chung et aL, A 5' element of the chicken beta-globin domain serves as an insulator in humanerythroid cells and protects against position effect in Drosophila. Cell. 1993 Aug 13;74(3):505-14.; Chung et al., Characterization of the chicken beta-globin insulator. Proc Natl Acad Sci U S A. 1997 Jan 21 ;94(2):575-80; and Bell et al., The Protein CTCF is Required for the Enhancer Blocking Activity of Vertebrate Insulators 1999. Cell issue 3 98:387, incorporated by reference herein). Examples of insulator elements include an insulator from an Pglobin locus, such as chicken HS4.(II) Exemplary Embodiments.

[0107] 1. A method of enhancing delivery of a gene of interest to an eye of a subject including administering a viral vector containing multiple copies of the gene of interest to the eye.

[0108] 2. The method of embodiment 1 , wherein the gene of interest is a light sensitive protein.

[0109] 3. The method of embodiment 2, wherein the light sensitive protein is a microbial opsin or a vertebrate opsin, or an invertebrate opsin.

[0110] 4. The method of embodiment 3, wherein the microbial opsin is a channelrhodopsin (ChR) or a pump-based opsin.

[0111] 5. The method of embodiment 4, wherein the ChR is Chloromonas oogama channelrhodopsin (CoChR), channel rhodopsin 2 (ChR2), Volvox carteri channelrhodopsin (vChR), red activatable channelrhodopsin (ReaChR), Chronos, Platymonas (Tetraselmis) subcordiformis channelrhodopsin (PsCHR), or Chrimson.

[0112] 6. The method of embodiment 5, wherein the CoChR is a mutant CoChR having the sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEO ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

[0113] 7. The method of embodiment 6, wherein the ChR is CoChR-H94E / L112C / K264T having a sequence of SEQ ID NO: 60.

[0114] 8. The method of embodiment 4, wherein the CHR has at least 90% sequence identity with SEQ ID NO: 60.

[0115] 9. The method of any of embodiments 1 to 8, wherein the delivery of the gene of interest increases light sensitivity in the subject.

[0116] 10. The method of any of embodiments 1 to 9, wherein the delivery of the gene of interest increases visual acuity in the subject.

[0117] 11. The method of any of embodiments 2 to 10, wherein the viral vector further includes a promoter arranged to promote expression from multiple copies of the gene of interest.

[0118] 12. The method of embodiment 11 , wherein the promoter is a ubiquitous promoter or a retinal cell-specific promoter.

[0119] 13. The method of embodiment 12, wherein the ubiquitous promoter is cytomegalovirus immediate enhancer / p-actin (CAG), cytomegalovirus (CMV), synapsin, elongation factor-1 alpha (EFI a), or thymocyte differentiation antigen 1 (Thy-1 ).

[0120] 14. The method of embodiment 12, wherein the retinal cell-specific promoter is metabolic glutamate receptor 6 (mGluR6), neurokinin 3 (NK-3), or purkinje cell protein 2 (Pcp2(L7)), gamma-synuclein gene (SNCG), HKamac, or rhodopsin (RHO).

[0121] 15. The method of embodiment 14, wherein the promoter is a mGluR6 promoter.

[0122] 16. The method of embodiment 15, wherein the mGluR6 promoter is a human promoter or a mouse promoter.

[0123] 17. The method of embodiment 16, wherein the mGluR6 promoter is a shortened mGluR6 having the sequence of SEQ ID NO: 62, SEQ ID NO: 71 , or SEQ ID NO: 73.

[0124] 18. The method of any of embodiments 1 to 17, wherein the viral vector includes an adeno-associated virus (AAV) vector.

[0125] 19. The method of embodiment 18, wherein the AAV vector includes AAV2.7m8 or AAV2.7m8(Y444F).

[0126] 20. The method of embodiment 16, wherein the viral vector further includes at least one post-transcriptional regulatory element.

[0127] 21. The method of embodiment 20, wherein the at least one post-transcriptional regulatory element is woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or mutated woodchuck hepatitis virus posttranscriptional regulatory element (mWPRE).

[0128] 22. The method of embodiment 20, wherein the at least one post-transcriptional regulatory element further includes human growth hormone polyadenylation signal (hGHpA), bovine growth hormone polyadenylation signal (bGHpA), or SV40 early polyadenylation signal (SV40pA).

[0129] 23. The method of any of embodiments 1 to 22, wherein the viral vector encodes two copies of the gene of interest.

[0130] 24. The method of any of embodiments 1 to 22, wherein the viral vector encodes at least three copies of the gene of interest.

[0131] 25. A method of increasing light sensitivity or improving visual acuity in a subject including administering a viral vector that encodes a plurality of opsins to vitreous of an eye.

[0132] 26. The method of embodiment 25, wherein the plurality of opsins are microbial opsins, vertebrate opsins, invertebrate opsins, or a combination thereof.

[0133] 27. The method of embodiment 26, wherein the microbial opsins are channelrhodopsins (ChR) or pump-based opsins.

[0134] 28. The method of embodiment 27, wherein the ChR are Chloromonas oogama channelrhodopsin (CoChR), channel rhodopsin 2 (ChR2), Volvox carteri channelrhodopsin (vChR), red activatable channelrhodopsin (ReaChR), Platymonas (Tetraselmis) subcordiformis channelrhodopsin (PsChR), Chronos, or Chrimson.

[0135] 29. The method of embodiment 28, wherein the CoChRs are mutant CoChR having the sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

[0136] 30. The method of embodiment 29, wherein the ChR is CoChR-H94E / L112C / K264T having a sequence of SEQ ID NO: 60.

[0137] 31. The method of embodiment 27, wherein the ChR has at least 90% sequence identity with SEQ ID NO: 60.

[0138] 32. The method of any of embodiments 25 to 31 , wherein the viral vector further includes a promoter arranged to promote expression of the plurality of opsins.

[0139] 33. The method of embodiment 32, wherein the promoter is a ubiquitous promoter or a retinal cell-specific promoter.

[0140] 34. The method of embodiment 33, wherein the ubiquitous promoter is cytomegalovirus immediate enhancer / -actin (CAG), cytomegalovirus (CMV), synapsin, elongation factor-1 alpha (EFI a), or thymocyte differentiation antigen 1 (Thy-1 ).

[0141] 35. The method of embodiment 33, wherein the retinal cell-specific promoter is metabolic glutamate receptor 6 (mGluR6), neurokinin 3 (NK-3), or purkinje cell protein 2 (Pcp2(L7)), gamma-synuclein gene (SNCG), HKamac, or rhodopsin (RHO).

[0142] 36. The method of embodiment 35, wherein the promoter is mGluR6.

[0143] 37. The method of embodiment 35, wherein the mGluR6 promoter is a human promoter or a mouse promoter.

[0144] 38. The method of embodiment 35, wherein the promoter is a shortened mGluR6 promoter.

[0145] 39. The method of embodiment 38, wherein the mGluR6 promoter is a shortened mGluR6 having a sequence of SEQ ID NO: 62, SEQ ID NO: 71 , or SEQ ID NO: 73.

[0146] 40. The method of embodiment 25, wherein the viral vector includes an adeno- associated viral vector (AAV).

[0147] 41. The method of embodiment 40, wherein the AAV is AAV2.7m8 or AAV2.7m8(Y444F).

[0148] 42. The method of any of embodiments 25 to 41 , wherein the viral vector further includes at least one post-transcriptional regulatory element.

[0149] 43. The method of embodiment 42, wherein the at least one post-transcriptional regulatory element is woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or mutated woodchuck hepatitis virus posttranscriptional regulatory element (mWPRE).

[0150] 44. The method of embodiment 42, wherein the at least one post-transcriptional regulatory element further includes human growth hormone polyadenylation signal (hGHpA), bovine growth hormone polyadenylation signal (bGHpA), or SV40 early polyadenylation signal (SV40pA).

[0151] 45. The method of any of embodiments 25 to 45, wherein the plurality of opsins is two opsins.

[0152] 46. The method of any of embodiments 25 to 45, wherein the plurality of opsins is at least three opsins.

[0153] 47. An isolated nucleic acid molecule including: a polynucleotide molecule including a nucleic acid sequence encoding a channelrhodopsin for differential expression in subcellular regions of a retinal neuron, the nucleic acid sequence including: a first nucleotide sequence encoding a first Chloromonas oogama channelrhodopsin (CoChR); a second nucleotide sequence encoding a second CoChR, which second nucleotide sequence is linked to the first nucleotide sequence; a promoter sequence arranged to promote expression of the first and second channelrhodopsin; and a post-transcriptional regulatory element.

[0154] 48. The isolated nucleic acid molecule of embodiment 47, wherein the first nucleotide sequence encodes an amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEO ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

[0155] 49. The isolated nucleic acid molecule of embodiment 47 or 48, wherein the second nucleotide sequence encodes an amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

[0156] 50. The isolated nucleic acid molecule of embodiment 49, wherein the first nucleotide sequence encodes CoChR-H94E / L112C / K264T having a sequence of SEQ ID NO: 60.

[0157] 51. The isolated nucleic acid molecule of any of embodiments 47 to 51 , wherein the first channelrhodopsin and the second channelrhodopsin are alike.

[0158] 52. The isolated nucleic acid molecule of any of embodiments 47 to 52, wherein the promoter sequence is cytomegalovirus immediate enhancer / p-actin (CAG), cytomegalovirus (CMV), synapsin, elongation factor-1 alpha (EF1a), thymocyte differentiation antigen 1 (Thy-1 ),metabolic glutamate receptor 6 (mGluR6), neurokinin 3 (NK-3), or purkinje cell protein 2 (Pcp2(L7)), gamma-synuclein gene (SNCG), HKamac, or rhodopsin (RHO).

[0159] 53. The isolated nucleic acid molecule of embodiment 52, wherein the promoter sequence is shortened mGluR6 of SEQ ID NO: 62, SEQ ID NO: 71 , or SEQ ID NO: 73.

[0160] 54. The isolated nucleic acid molecule of any of embodiments 47 to 53, wherein the post-transcriptional regulatory element is woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or mutated woodchuck hepatitis virus posttranscriptional regulatory element (mWPRE).

[0161] 55. The isolated nucleic acid molecule of any of embodiments 47 to 54, wherein the post-transcriptional regulatory element further includes human growth hormone polyadenylation signal (hGHpA), bovine growth hormone polyadenylation signal (bGHpA), or SV40 early polyadenylation signal (SV40pA).

[0162] 56. The isolated nucleic acid molecule of any of embodiments 47 to 55, wherein the first nucleotide sequence and the second nucleotide sequence are separated by a sequence encoding a cleavage protein.

[0163] 57. The isolated nucleic acid molecule of embodiment 56, wherein the cleavage protein is P2A.

[0164] 58. The isolated nucleic acid molecule of any of embodiments 47 to 58, wherein the first nucleotide sequence and the second nucleotide sequence are separated by a sequence encoding a non-cleaving peptide, wherein the non-cleaving peptide is IRES or HK.

[0165] Provided are constructs with multiple copies of the transgene of interest. FIGs. 1 and 2 show the expression of viral vector #1 , bAAV2.7m8(Y444F)-mGluR6-Co3M-GFP-hGHpA (SEQ ID NO: 1 ), a control viral vector including the full mGluR6 promoter to drive CoChR-3M fused with GFP (FIG. 1 ) and the expression of viral vector #2, bAAV2.7m8(Y444F)-mGluR6(s)-Co3M-GFP- hGHpA, (SEQ ID NO: 2) a control viral vector including a shortened mGluR6 promoter in which intron 3 has been removed, named mGluR6(s) (SEQ ID NO: 61 ). While there is specific targeting of ON bipolar cells (ON BCs) of viral vector #1 and #2 shown in FIG. 1 and 2, the functional efficacy is relatively low in comparison to viral vectors #3-#8 (see FIG. 10 and 12).

[0166] FIG. 3 shows the expression of viral vector #3, bAAV2.7m8(Y444F)-mGluR6-Co3M- WPRE-hGHpA (SEQ ID NO: 3). In this construct shown in FIG. 3, the full mGluR6 promoter was used to drive CoChR-3M without GFP marker as there was insufficient space to include the GFP marker. In addition, WPRE was added to improve transgene expression. This transgene shows targeting to ON BCs but with some off targeting to retinal ganglion cells at 302, 304, and 306 inFIG. 3, but the functional efficacy is markedly improved compared to the viral vector #1 and #2 as shown in FIG. 10 and FIG. 12.

[0167] FIG. 4 shows the expression of viral vector #4, bAAV2.7m8(Y444F)-mGluR6(s)-Co3M- GFP-WPRE-hGHpA (SEQ ID NO: 4) in which the shortened mGluR6 promoter was used to drive CochR-3M fused with GFP and WPRE. This construct demonstrate that the shortened mGluR6 promoter was effective as the full length mGluR6 promoter. The addition of WPRE also increased the functional efficacy (FIG. 10 and 12) but targeting selectivity for ON BCs was somewhat compromised as shown at 402, 404, and 406 in FIG. 4 (also see Miyadera et al., Proc Natl Acad Sci USA. 2022; 119:e21 17038119.).

[0168] The inventors determined that a viral cassette with multiple copies of the same therapeutic transgene linked by 2A self-cleaving peptides or non-cleaving peptides increases the expression of transgene proteins in the targeted cells without increasing the viral load. As there are limits to the size of the constructs that will achieve delivery of the transgenes, the use of viral vectors with different payload capabilities or shorter or shortened versions of promoters allows for the inclusion of more copies of the transgenes in a single viral construct. Specific results for each construct are shown in Tables 2 and 3.

[0169] FIG. 5 shows the expression of viral vector #5, bAAV2.7m8(Y444F)-mGluR6-Co3M-P2A- Co3M-hGHpA (SEQ ID NO: 5), a construct in which the full mGluR6 promoter was used to drive two CoChR-3Ms linked by P2A self-cleaving peptide. The expression of this viral vector shows specific targeting of ON BCs with also markedly improved functional efficacy compared to controls (viral vector #1 and #2) (FIGs. 10 and 12).

[0170] FIG. 6 shows the expression of viral vector #6, bAAV2.7m8(Y444F)-mGluR6(s)-Co3M- P2A-Co3M-P2A-Co3M-hGHpA (SEQ ID NO: 6), a viral construct with multiple transgenes and cleavage peptides. In this construct, the shortened mGluR6 promoter (mGluR6(s)) was used to drive three CoChR-3Ms linked by two P2A self-cleaving peptides. As shown in FIG. 6, there was specific targeting of ON BCs (FIG.6) and the functional efficacy of the construct was also markedly improved in comparison to controls (viral vector #1 and #2) (FIG. 10 and 12).

[0171] As shown in the expression of bAAV2.7m8(Y444F)-mGluR6(s)-Co3M-P2A-Co3M-WPRE- hGHpA (SEQ ID NO: 7) (FIG. 7), the use of WPRE increased the efficacy of the double transgene. In this construct, the shortened mGluR6 promoter was used to drive two CoChR-3Ms linked by two P2A self-cleaving peptides and WPRE. As shown in FIG. 7, there was specific targeting of ON BCs and the functional efficacy of the construct was also markedly improved compared to controls (viral vector #1 and #2) (FIGs. 10 and 12).

[0172] FIG. 8 shows the expression of bAAV2.7m8(Y444F)-mGluR6(s)-Co3M-pHK-Oo3M- mWPRE-bGHpA (SEQ ID NO: 8). In this construct, a shortened mGluR6 promoter was used to drive two CoCHR-3Ms linked by a transmembrane fusion peptide (PHK) peptide and including a modified WPRE. As shown in FIG. 8, there was specific targeting of ON BCs and the functional efficacy of the construct was also markedly improved compared to controls (viral vector #1 and #2) (FIGs. 10 and 12).

[0173] As shown in FIG. 10, the light sensitivity between TKO mice treated with constructs #1 and #2 or among TKO mice treated with constructs #3 - #8 is not statistically significant. But the light sensitivity of TKO mice treated with constructs #3 - #8 is significantly higher than those of TKO mice treated with constructs #1 and #2.

[0174] Similarly, as shown in FIG. 12, the visual acuity between constructs #1 and #2 treated TKO mice or among TKO mice treated with constructs #3 - #8 are not statistically significant. But the visual acuity achieved by TKO mice treated with constructs #3 - #8 is significantly higher than those achieved by TKO mice treated with constructs #1 and #2.

[0175] Mice injected with the novel viral vectors #5: bAAV2.7m8(Y444F)-mGluR6-Co3M-P2A- Co3M-hGHpA, #6: bAAV2.7m8-mGluR6(s)-Co3M-P2A-Co3M-P2A-Co3M-hGHpA, #7: bAAV2.7m8(Y444F)-mGluR6(s)-Co3M-P2A-Co3M-WPRE-hGHpA, and #8: bAAV2.7m8(Y444F)- mGluR6(s)-Co3M-pHK-Co3M-mWPRE-bGHpA demonstrated increased visual acuity and light sensitivity in comparison to constructs #1 and #2 which were used as controls the expression of which is shown in FIGs. 1 and 2.(Ill) Ocular Disorders

[0176] The viral-mediated gene therapy of the present invention may be useful for the treatment, prevention, and amelioration of one or more diseases and conditions. In exemplary embodiments, viral-mediated gene therapy using light-sensitive protein may be useful for the treatment and / or restoration of at least partial vision to subjects that have lost vision due to ocular disorders. Ocular disorders for which the methods and compositions of the present invention are intended and may be used to improve one or more parameters of vision include developmental abnormalities that affect both anterior and posterior segments of the eye. Anterior segment disorders include glaucoma, cataracts, corneal dystrophy, and keratoconus; and posterior segment disorders including blinding disorders caused by photoreceptor malfunction and / or death caused by retinal dystrophies and degenerations. Other ocular diseases that may benefit from the methods described herein include retinoblastoma, ocular melanoma, diabetic retinopathy, hypertensive retinopathy, any inflammation of the ocular tissues (i.e., chorioretinal inflammation, scleritis,keratitis, uveitis, etc.), or infection (i.e., bacterial or viral). In particular, for optogenetic gene therapy, ocular disorders additionally include retinal disorders such as congenital stationary night blindness, age-related macular degeneration, congenital cone dystrophies, and a large group of retinitis-pigmentosa (RP)-related disorders. These disorders include genetically pre-disposed death of photoreceptor cells, rods, and cones in the retina, occurring at various ages. Among those are severe retinopathies, such as subtypes of RP itself that progress with age and cause blindness in childhood and early adulthood, and RP-associated diseases, such as genetic subtypes of LCA, which frequently results in loss of vision during childhood, as early as the first year of life. The latter disorders are generally characterized by severe reduction, and often complete loss of photoreceptor cells, rods, and cones.(IV) Restoration of Light Sensitivity

[0177] These methods and compositions described herein may be used in subjects of normal and / or impaired vision. The enhanced expression of transgene proteins, as described herein, may preserve, improve, or restore vision. The term “vision” as used herein is defined as the ability of an organism to usefully detect light as a stimulus for differentiation or action. Vision is intended to encompass the following:1 . Light detection or perception — the ability to discern whether or not light is present;2. Light projection — the ability to discern the direction from which a light stimulus is coming;3. Resolution — the ability to detect differing brightness levels (i.e., contrast) in a grating or letter target; and4. Recognition— the ability to recognize the shape of a visual target by reference to the differing contrast levels within the target.

[0178] Thus, “vision” includes the ability to simply detect the presence of light. The compositions and methods of the present invention can be used to improve or restore vision, wherein the improvement or restoration in vision includes, for example, increases in light detection or perception, increase in light sensitivity or photosensitivity in response to a light stimulus, increase in the ability to discern the direction from which a light stimulus is coming, increase in the ability to detect differing brightness levels, increase in the ability to recognize the shape of a visual target, and increases in visual evoked potential or transmission from the retina to the cortex. As such, improvement or restoration of vision may or may not include full restoration of sight, i.e., wherein the vision of the patient treated with the present invention is restored to the degree of the vision of a non-affected individual. The visual recovery described in the animal studies described below may, in human terms, place the person on the low end of vision function by increasing one aspectof vision (i.e., light sensitivity, or visual evoked potential) without restoring full sight. Nevertheless, placement at such a level would be a significant benefit because these individuals could be trained in mobility and potentially in low-order resolution tasks which would provide them with a greatly improved level of visual independence compared to total blindness. Even basic light perception can be used by visually impaired individuals whose vision is improved using the present compositions and methods, to accomplish specific daily tasks and improve general mobility, capability, and quality of life.

[0179] The degree of restoration of vision can be determined through the measurement of vision before, and preferably after, administering a vector including, for example, DNA encoding a therapeutic transgene such as CoChR-3M (SEQ ID NO: 63). Vision can be measured using any of a number of methods well-known in the art or methods not yet established. Vision, as improved or restored by the present invention, can be measured by any of the following visual responses:1. a light detection response by the subject after exposure to a light stimulus — in which evidence is sought for a reliable response of an indication or movement in the general direction of the light by the subject individual when the light it is turned on;2. a light projection response by the subject after exposure to a light stimulus in which evidence is sought for a reliable response of indication or movement in the specific direction of the light by the individual when the light is turned on;3. light resolution by the subject of a light vs. dark patterned visual stimulus, which measures the subject's capability of resolving light vs dark patterned visual stimuli as evidenced by: a. the presence of demonstrable reliable optokinetically produced nystagmoid eye movements and / or related head or body movements that demonstrate tracking of the target (see above) and / or b. the presence of a reliable ability to discriminate a pattern visual stimulus and to indicate such discrimination by verbal or non-verbal means, including, for example pointing, or pressing a bar or a button; or4. electrical recording of a visual cortex response to a light flash stimulus or a pattern visual stimulus, which is an endpoint of electrical transmission from a restored retina to the visual cortex, also referred to as the visual evoked potential (VEP). Measurement may be by electrical recording on the scalp surface at the region of the visual cortex, on the cortical surface, and / or recording within cells of the visual cortex (see U.S. Patent No. 9,730,981).

[0180] Thus, improvement or restoration of vision, according to the present invention, can include, but is not limited to: increases in amplitude or kinetics of photocurrents or electrical response inresponse to light stimulus in the retinal cells, increases in light sensitivity (i.e., lowering the threshold light intensity required for initiating a photocurrent or electrical response in response to light stimulus, thereby requiring less or lower light to evoke a photocurrent) of the retinal cells, increases in number or amplitude of light-evoked spiking or spike firings, increases in light responses to the visual cortex, which includes increasing in visual evoked potential transmitted from the retina or retinal cells to the visual cortex or the brain.

[0181] Both in vitro and in vivo studies to assess the various parameters of the present invention may be used, including recognized animal models of blinding human ocular disorders. Large animal models of human retinopathy, e.g., childhood blindness, are useful. The examples provided herein allow one of skill in the art to readily anticipate that this method may be similarly used in treating a range of retinal diseases.

[0182] While earlier studies by others have demonstrated that retinal degeneration can be retarded by gene therapy techniques, the present invention demonstrates a definite physiological recovery of function, which is expected to generate or improve various parameters of vision, including behavioral parameters through the use of multiple copies of transgenes.

[0183] Behavioral measures can be obtained using known animal models and tests, for example performance in a water maze, wherein a subject in whom vision has been preserved or restored to varying extents will swim toward light (Hayes et aL, 1993; Behav Genet 23:395-403; Lu et al., IOVS, 2018; Ganjawala et al., 2019; U.S. Patent No. 11 ,583,595).

[0184] In models in which blindness is induced during adult life or congenital blindness develops slowly enough that the individual experiences vision before losing it, training of the subject in various tests may be done. In this way, when these tests are re-administered after visual loss to test the efficacy of the present compositions and methods for their vision-restorative effects, animals do not have to learn the tasks de novo while in a blind state. Other behavioral tests, such as the optokinetic nystagmus test, do not require learning and rely on the instinctiveness of certain behaviors (Balkema G W et al., 1984; Invest Ophthalmol Vis Sci. 25:795-800; Mitchiner J C et aL, 1976, Vision Res. 16:1169-71 ; Lu et al., IOVS, 2018; Ganjawala et aL, 2019).

[0185] The present invention may also be used in combination with other forms of vision therapy known in the art to improve or restore vision. For example, the use of visual prostheses, which include retinal implants, cortical implants, lateral geniculate nucleus implants, or optic nerve implants. Thus, in addition to genetic modification of surviving retinal neurons using the present methods, the subject being treated may be provided with a visual prosthesis before, at the same time as, or after the molecular method is employed. The effectiveness of visual prosthetics can be improved with training of the individual, thus enhancing the potential impact of the CoChR-3Mtransformation of patient cells as contemplated herein. Training methods, such as habituation training characterized by training the subject to recognize (i) varying levels of light and / or pattern stimulation, and / or (ii) environmental stimulation from a common light source or object as would be understood by one skilled in the art; and orientation and mobility training characterized by training the subject to detect visually local objects and move among said objects more effectively than without the training. In fact, any visual stimulation techniques that are typically used in the field of low vision rehabilitation are applicable here (see U.S. Patent Publication No. 20180214513).

[0186] In some embodiments, administration of the plurality of transgenes in a single expression cassette disclosed herein improves, prevents, delays, or ameliorates vision loss in a treated patient compared to an untreated patient or the same patient before treatment. In some embodiments, administration of the plurality of transgenes in a single expression cassette disclosed herein improves, prevents, delays, or ameliorates vision loss in a treated patient between day 1 and year 10. In some embodiments, administration of administration of the mutant the plurality of transgenes in a single expression cassette disclosed herein improves, prevents, delays, or ameliorates vision loss at about day 1 , about day 2, about day 3, about day 4, about day 5, about day 6, about week 1 , about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1 , about year 2, or about year 3 compared with vision loss in an untreated patient or the same patient before treatment. In some embodiments, administration of the plurality of transgenes in a single expression cassette compositions disclosed herein improves, prevents, delays, or ameliorates vision loss for about 1 day, about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more compared with vision loss in an untreated patient or the same patient before treatment.

[0187] In some embodiments, vision loss is decreased by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared with controls or patients treated with other compositions. In some embodiments, administration of the plurality of transgenes in a single expression cassette improves, prevents, ameliorates, or delays vision loss by about 1 %, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% at about day 1 , about day 2, about day 3, about day 4, about day 5, about day 6, about week 1 , about week 2, about week 3, about week 4, about week 5, about week 6, aboutweek 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1 , about year 2, or about year 3 compared with controls or patients treated with other compositions. In some embodiments, administration of the plurality of transgenes in a single expression cassette compositions disclosed herein improves, prevents, ameliorates, or delays vision loss by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 600%, about 70%, about 80%, about 90%, or about 100% for about 1 , about 2 days, about3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or more compared with controls or patients treated with other methods.

[0188] In some embodiments, administration of the plurality of transgenes in a single expression cassette disclosed herein increases light sensitivity in a treated patient compared to an untreated patient or the same patient before treatment. In some embodiments, administration of the plurality of transgenes in a single expression cassette disclosed herein increases light sensitivity in a treated patient between day 1 and year 10. In some embodiments, administration of administration of the plurality of transgenes in a single expression cassette disclosed herein increases light sensitivity at about day 1 , about day 2, about day 3, about day 4, about day 5, about day 6, about week 1 , about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1 , about year 2, or about year 3 compared with light sensitivity an untreated patient or the same patient before treatment. In some embodiments, administration of the plurality of transgenes in a single expression cassette disclosed herein increases light sensitivity for about 1 day, about 1 week, about 1 month, about 2 months, about 3 months, about4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more compared with light sensitivity in an untreated patient or the same patient before treatment.

[0189] In some embodiments, light sensitivity is increased by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared with controls or patients treated with other compositions. In some embodiments, administration of the plurality of transgenes in a single expression cassette increases light sensitivity by about 1 %, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% at about day 1 , aboutday 2, about day 3, about day 4, about day 5, about day 6, about week 1 , about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1 , about year 2, or about year 3 compared with controls or patients treated with other compositions. In some embodiments, administration of the plurality of transgenes in a single expression cassette disclosed herein increases light sensitivity by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% for about 1 , about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or more compared with controls or patients treated with other methods.

[0190] In some embodiments, administration of the plurality of transgenes in a single expression cassette disclosed herein decreases the light intensity required to elicit a photocurrent in a treated patient compared to an untreated patient or the same patient before treatment. In some embodiments, administration of the plurality of transgenes in a single expression cassette disclosed herein decreases the light intensity required to elicit a photocurrent in a treated patient between day 1 and year 10. In some embodiments, administration of administration of the plurality of transgenes in a single expression cassette disclosed herein decreases the light intensity required to elicit a photocurrent at about day 1 , about day 2, about day 3, about day 4, about day 5, about day 6, about week 1 , about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1 , about year 2, or about year 3 compared with the light intensity required to elicit a photocurrent in an untreated patient or the same patient before treatment. In some embodiments, administration of the plurality of transgenes in a single expression cassette disclosed herein decreases the light intensity required to elicit a photocurrent for about 1 day, about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more compared with the light intensity required to elicit a photocurrent in an untreated patient or the same patient before treatment.

[0191] In some embodiments, the light intensity required to elicit a photocurrent is decreased by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared with controls or patients treatedwith other compositions. In some embodiments, administration of the plurality of transgenes in a single expression cassette decreases the light intensity required to elicit a photocurrent by about 1%, about 5%, about 100%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% at about day 1 , about day 2, about day 3, about day 4, about day 5, about day 6, about week 1 , about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1 , about year 2, or about year 3 compared with controls or patients treated with other compositions. In some embodiments, administration the plurality of transgenes in a single expression cassette disclosed herein decreases the light intensity required to elicit a photocurrent by about 1 %6, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% for about 1 , about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or more compared with controls or patients treated with other methods.(V) Pharmaceutical compositions

[0192] The vector or recombinant viruses (virions) can be incorporated into pharmaceutical compositions for administration to mammalian patients, particularly humans. The vector or virions can be formulated in nontoxic, inert, pharmaceutically acceptable aqueous carriers, preferably at a pH ranging from 3 to 8, more preferably ranging from 6 to 8, most preferably ranging from 6.8 to 7.2. Such sterile compositions will include the vector or virion containing the nucleic acid encoding the therapeutic molecule dissolved in an aqueous buffer having an acceptable upon reconstitution.

[0193] Pharmaceutical compositions suitable for internal use with the compositions described herein include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It 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 (for example, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The 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 dispersion and by the use of surfactants such as polysorbates (Tween™), sodium dodecyl sulfate (sodium lauryl sulfate), lauryl dimethyl amine oxide, cetyltrimethylammonium bromide (CTAB), polvethoxylated alcohols, polyoxyethylene sorbitan, octoxvnol (Triton X100™), NN- dimethvIdodecylamine-N-oxide, hexadecyltrimethylammonium bromide (1 H4TAB), polyoxyl 10 lauryl ether, Brij 721 ™, bile salts (sodium deoxycholate, sodium cholate), pluronic acids (F-68, F- 127), polyoxyl castor oil (Cremophor™) nonylphenol ethoxylate (Tergitol™), cyclodextrins and, ethylbenzethonium chloride (Hy amine™). Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0194] Sterile solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze- drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof.

[0195] In some aspects, the pharmaceutical compositions provided herein include a therapeutically effective amount of a vector or virion in admixture with a pharmaceutical acceptable carrier and / or excipient, for example saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugar, buffers, preservatives, and other proteins. Exemplary amino acids, polymers and sugars and the like are octylphenoxy polvethoxy ethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene and glycol. Preferably, this formulation is stable for at least 14 months at -60° C.

[0196] Such formulations include a pharmaceutically and / or physiologically acceptable vehicle, diluent, carrier, or excipient, such as buffered saline or other buffers, e.g., HEPES, to maintain physiologic pH. For a discussion of such components and their formulation, see, generally, Gennaro, A. E., Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishers; 2003 or latest edition; see also, WOOO / 15822. If the preparation is to be stored for long periods, it may be frozen, for example, in the presence of glycerol.

[0197] In some aspects, the pharmaceutical composition provided herein includes a buffer, such as phosphate buffered saline (PBS) or sodium phosphate / sodium sulfate, tris buffer, glycine buffer, sterile water, and other buffers known to the ordinarily skilled artisan such as those described by Good et al. (1966) Biochemistry 5:467. In some aspects, pharmaceutical composition contains sodium phosphate, sodium chloride and orbital. In some aspects, the pharmaceutical composition contains 10 mM sodium phosphate, 350 mM sodium chloride, and 5% (v / v) sorbitol. The pH of the buffer in which the pharmaceutical composition including the mCoChop contained in the adenoviral vector delivery system, may be in the range of 6.5 to 7.75, 6.5 to 7.5, 6.8 to 7.4, or 6.8 to 7.2. In some aspects, the pharmaceutical composition provided herein includes substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran, in the amount of about 1 -10 percent, such as 1 , 2, 3, 4, 5, 6, 7,8, 9, or 10 percent (v / v). Preferably the sorbitol is about 3-6% (v / v), most preferably the sorbitol is about 5%. (v / v).

[0198] Before administration the pharmaceutical composition is free of components used during the production, e.g., culture components, host cell protein, host cell DNA, and plasmid DNA and substantially free of mycoplasma, endotoxin, and microbial contamination. Preferably, the pharmaceutical composition has less than 10, 5, 3, 2, or I CFU / swab. Most preferably composition has 0 CFU / swab. The endotoxin level in the pharmaceutical composition is less than 20 EU / mL, less than 10 EU / mL, or less than 5 EU / mL. The pharmaceutical composition must have sufficiently full capsid prior to administration. The pharmaceutical composition has at least 50%, at least 60%, at least 70%, at least 80% or greater full capsids.

[0199] Suitable routes of administration include, for example, intravitreal, intraocular, or subretinal injection. Preferably, the route of administration is by intravitreal injection. All retinal neurons, including retinal ganglion cells, bipolar cells, horizontal cells, amacrine cells, and photoreceptor cells are known to be reasonably well-accessible to intravitreal injection as disclosed herein. Intravitreal and / or subretinal injection may provide the necessary access to the bipolar cells, especially in circumstances in which the photoreceptor cell layer is absent due to degeneration.

[0200] In one embodiment, the constructs or nucleic acid expression vectors described herein are packaged in adenoviral vectors for transgene delivery. An effective amount of rAAV virions carrying a transgene under the control of the modified mGluR6 promoter is preferably in the range of between about 1010to about 1013rAAV infectious units in a volume of between about 150 and about 800 pl per injection. The rAAV infectious units can be measured according to McLaughlin, S K et al., 1988, J. Viral. 62:1963. More preferably, the effective amount is between about 1010and about 1012rAAV infectious units and the injection volume is preferably between about 250 and about 500 pl. Other dosages and volumes, preferably within these ranges but possibly outside them, may be selected by the treating professional, taking into account the physical state of the subject (preferably a human), who is being treated, including, age, weight, general health, and the nature and severity of the particular ocular disorder.

[0201] In some cases, the viral vector of the disclosure may be measured as pfu (plaque forming units). In some cases, the pfu of recombinant virus, or viral vector of the compositions and methods of the disclosure may be about 108to about 5x101° pfu. In some cases, recombinant viruses of this disclosure are at least about 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x101°, 2x101°, 3x101°, 4x 101°, and 5x 101° pfu. In some cases, recombinant viruses of this disclosure are at most about 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x1010, 2x1010, 3x1010, 4x1010, and 5x1010pfu.

[0202] In some cases, the viral vector of the disclosure may be measured as vector genomes. In some cases, recombinant viruses of this disclosure are 1 x 1010to 3x 1012vector genomes. In some cases, recombinant viruses of this disclosure are 1 x109to 3x1013vector genomes. In some cases, recombinant viruses of this disclosure are 1x108to 3x1014vector genomes. In some cases, recombinant viruses of the disclosure are at least about 1 x 101, 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x107, 1 x108, 1 x109, 1 x1O10, 1 x1011, 1x1012, 1x1013, 1x1014, 1x1015, 1x1016, 1x1017, and 1x1018vector genomes.

[0203] In some cases, the viral vector of the disclosure may be measured using a multiplicity of infection (MOI). In some cases, MOI may refer to the ratio, or multiple of vector or viral genomes to the cells to which the nucleic may be delivered. In some cases, the MOI may be 1 x106. In some cases, the MOI may be 1 x105-1x107. In some cases, the MOI may be 1 x104-1x108. In some cases, recombinant viruses of the disclosure are at least about 1 x 101, 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x106, 1 x107, 1 x108, 1 x109, 1 x1O10, 1 x1011, 1x1012, 1x1013, 1x1014, 1x1015, 1x1016, 1x1017, and 1 x1018MOI. In some cases, recombinant viruses of this disclosure are 1 x108to 3x1014MOI.

[0204] It may also be desirable to administer additional doses (“boosters”) of the present nucleic acid(s) or rAAV compositions. For example, depending upon the duration of the transgene expression within the ocular target cell, a second treatment may be administered after 6 months or yearly and may be similarly repeated. Neutralizing antibodies to AAV are not expected to be generated in view of the routes and doses used, thereby permitting repeat treatment rounds.

[0205] The need for such additional doses can be monitored by the treating professional using, for example, well-known electrophysiological and other retinal and visual function tests and visual behavior tests. The treating professional will be able to select the appropriate tests by applying routine skill in the art. It may be desirable to inject larger volumes of the composition in either single or multiple doses to further improve the relevant outcome parameters.(VI) Kits

[0206] Compositions and reagents useful for the present disclosure may be packaged in kits to facilitate application of the present disclosure. In some aspects, the present method provides for a kit including a recombinant nucleic acid of the disclosure. In some aspects, the present method provides for a kit including a recombinant virus of the disclosure. The instructions could be in any desired form, including but not limited to, printed on a kit insert, printed on one or more containers, as well as electronically stored instructions provided on an electronic storage medium, such as a computer readable storage medium. Also optionally included is a software package on a computer readable storage medium that permits the user to integrate the information and calculate a control dose. In another aspect, the present disclosure provides a kit including the pharmaceutical compositions provided herein. In yet another aspect, the disclosure provides kits for the treatment of diseases.

[0207] In one aspect, a kit includes: (a) a recombinant virus provided herein, and (b) instructions to administer to cells or an individual a therapeutically effective amount of the recombinant virus. In some aspects, the kit may include pharmaceutically acceptable salts or solutions for administering the recombinant virus. Optionally, the kit can further include instructions for suitable operational parameters in the form of a label or a separate insert. For example, the kit may have standard instructions informing a physician or laboratory technician to prepare a dose of recombinant virus.

[0208] Optionally, the kit may further include a standard or control information so that a patient sample can be compared with the control information standard to determine if the test amount of recombinant virus is a therapeutic amount Optionally, the kit could further include devices for administration, such as a syringe, filter needle, extension tubing, cannula, and subretinal injector.

[0209] Recombinant viruses may be generated by any suitable means. The methods and compositions of the disclosure provide for generation of recombinant viruses through various means, including the use of transgenic cells, which may include mammalian cells, insect cells, animal cells, or fungal cells.

[0210] For example, in some aspects, recombinant viruses may be generated through transfection of insect cells via recombinant baculovirus. In some cases, recombinant baculovirus may be generated as an intermediate, whereby the baculovirus may contain sequences necessary for the generation of other viruses such as AAV or rAAV2 viruses. In some cases, one or more baculoviruses may be used in the generation of recombinant viruses used for the composition and methods of treatment of this disclosure. In some cases, insect cells such as Sf9, High-Five, or Sf21 cell lines may be used. In some cases, cell lines may be generated using transient methods (i.e., infection with not stably integrated transgenes). In other cases, cell lines may be generated through the generation of stable cell lines (i.e., infection with transgenes stably integrated into the host cell genome.) In other aspects, the pharmaceutical composition provided herein is manufactured using adherent human embryonic kidney 293 (HEK293) cells. In an alternative aspect, the pharmaceutical composition provided herein is manufactured using suspension adapted HEK293 cells. In another aspect, the pharmaceutical composition provided herein is manufactured using the baculovirus expression system (BYES) in insect cells. In some aspects, the vector is produced using herpes-helper virus. In some aspects, the vector is produced using producer-clone methods. In some aspects, the vector is produced using Ad-AAV.

[0211] Generally, any suitable method may be used in the biochemical purification of recombinant viruses for use in a pharmaceutical composition as described herein. Recombinant viruses may be harvested directly from cells, or from the culture media surrounding host cells. Viruses may be purified using various biochemical means, such as gel filtration, filtration, chromatography, affinity purification, gradient ultracentrifugation, or size exclusion methods. Recombinant virus may be tested for content (i.e., identity), purity, or potency (i.e., activity) using any suitable means, before formulation into a pharmaceutical composition. Method may include immunoassays, ELISA, SDS- PAGE, western blot, Northern blot, Southern blot or PGR, HUVEC assays, and the like.(VII) Experimental ExamplesExample 1 : Generation of Optimized mGluR6 Promoter Constructs

[0212] A series of AAV2 expression cassettes were constructed with sequences of the mGluR6 promoter (Intron 4 + Intron 3 + 200 bp Enhancer + 500 bp Promoter) or the shortened mGluR6 promoter (Intron 4 + 200 bp Enhancer + 500 bp Promoter) (mGluR6(s)) where mouse intron 3 isSEQ ID NO: 65, mouse intron 4 is SEQ ID NO: 64, human intron 3 is SEQ ID NO: 67 and human intron 4 is SEQ ID NO: 66. The human 200bp enhancer is SEQ ID NIO: 58 and the mouse 200bp enhancer is SEQ ID NO: 25 . The mouse 500 bp promoter is SEQ ID NO: 69 and the human 500 bp promoter is SEQ ID NO: 70. The transgenes were either GFP-fused CoChR-3M (Co3M), or Co3M alone. The self-cleaving element was P2A. The post-transcriptional regulatory elements were WPRE, modified WPRE, bGHpA, and / or hGHPA. Exemplary constructs are shown in FIGs. 13-20 and Table 1.Table 1 : ConstructsExample 2: Viral Vector Injection

[0213] A triple knock-out (TKO), Opn4_ / _Gnat1_ / _Cnga3_ / _, blind mouse line was used (Lu et aL, Invest Ophthalmol Vis Sci. 2018 Mar 1 ;59(3):1288-1294). All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of Wayne State University and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

[0214] Animals aged at least 1 month were anesthetized with an intraperitoneal injection of a mixture of 100 mg / kg-1 ketamine and 12 mg / kg-1 xylazine. 1.5 pl at a titer of 2 x 1013vg / ml of the virus vectors as shown in Table 1 were injected intravitreally into both eyes of each animal except for construct #7 which was injected at the titer of 1 x 1013vg / ml. All experiments were performed at least one month after virus injection.Example 3: Light sensitivity detection

[0215] The homemade optomotor system used is shown in FIG. 9. The light stimulus 918 was generated by blue LEDs (SuperLightingLED) with a wavelength range of 465-475 nm mounted on the inner face of a central platform 914 (40 cm diameter x 51 cm height). The light intensity of the LEDs, controlled by voltage using power supply 906, reached a maximum of 1 x 1016photons / cm2s when measured at the center of the platform. A series of exchangeable drums 910 (30 cm diameter x 30 cm height) were made by marking stripes on the wall of acrylic cylinders at a spatial frequency of 0.042 cycles per degree. The drums 910 were covered with light-diffuser film.

[0216] Rotation of the drum was controlled by a digital motor 908 and set at four rotations per minute. During testing, unrestrained animals were placed on a central platform 914 (6.5 cm diameter) positioned 11.5 cm above the floor 912. A video camera 902 was mounted above the apparatus for animal behavior monitoring and video capture.

[0217] To determine the threshold light intensity at each grating frequency, the ability to elicit head tracking in each animal in response to drum rotation was assessed starting at a relatively high light intensity. Head tracking was tested in both clockwise and counterclockwise directions. Once tracking was confirmed, the light intensity was systematically decreased to determine the lowest light intensity that elicited tracking behavior. All data are expressed as the mean ± SD, with n indicating the number of animals unless otherwise specified and all animals were tested at least one month after injection with the respective viral vectors.

[0218] The lowest light intensity that can evoke OMR is defined as light sensitivity. As shown in FIG. 10 and Table 2, vectors 1 and 2 had decreased light sensitivity in comparison to vectors 3- 8.Table 2: Light SensitivityExample 4: Visual Acuity

[0219] Visual acuity was determined by using a commercially purchased virtual optomotor system (OptoMotry; CerebralMechanics, Lethbridge, Alberta, Canada) shown in FIG. 11. The visual optomotor system 1 100 includes a video camera 1102 over an area framel 108 with LCD panels 1 104 and 1106. The mouse is placed on a platform within the area frame as described in Prusky et al. (Prusky GT, Alam NM, Beekman S, Douglas RM. Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system. Invest Ophthalmol Vis Sci. 2004). As shown in FIG. 12 and Table 3, vectors 3-8 increased the visual acuity in comparison to vector 1 (control).Table 3: Visual acuityExample 5: Immunostaining

[0220] The eyes were enucleated at the end of the behavioral testing, approximately two months after viral injection. Enucleated eyes were fixed with 4% paraformaldehyde in phosphate buffer (PB) at room temperature for 20 minutes. Immunofluorescence staining was examined in retinal vertical sections. A mouse anti-GFP (1 :1000; Neuromab, UC Davis, Davis, CA, USA) was used to assess the expression of CoChR-3M for viral vectors containing CoChR-3M-GFP fusion transgene. A custom made anti-CoChR-3M antibody (SinoBiological) was used to assess the expression of CoChR-3M for viral vectors without carrying GFP marker. The secondary antibodies were conjugated to Alexa 488 (1 :600), and Alexa 555 (1 :1000) (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence images were obtained using a ZEISS APOTOME2 Optical Photomicroscope (Apotome; Carl Zeiss Microscopy GmbH, Jena, Germany) and are shown in FIGs. 1 -8.(VIII). Closing Paragraphs

[0221] The term “operably linked”, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In one aspect, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and / or enhancer or other expression control sequence) and a second polynucleotidesequence, e.g., a polynucleotide-of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0222] As used herein, the term “constitutive expression control sequence” refers to a promoter, enhancer, or promoter / enhancer that continually or continuously allows for transcription of an operably linked sequence. A constitutive expression control sequence may be a “ubiquitous” promoter, enhancer, or promoter / enhancer that allows expression in a wide variety of cell and tissue types or a “cell specific,” “cell type specific,” “cell lineage specific,” or “tissue specific” promoter, enhancer, or promoter / enhancer that allows expression in a restricted variety of cell and tissue types, respectively. Illustrative ubiquitous expression control sequences include a cytomegalovirus (CMV) immediate early promoter; a viral simian virus 40 (SV40) (e.g., early or late); a Moloney murine leukemia virus (MoMLV) LTR promoter; a Rous sarcoma virus (RSV) LTR; a herpes simplex virus (HSV) (thymidine kinase) promoter; H5, P7.5, and P11 promoters from vaccinia virus; an elongation factor 1 -alpha (EF1 a) promoter; early growth response 1 (EGR1 ); ferritin H (FerH); ferritin L (FerL); Glyceraldehyde 3-phosphate dehydrogenase (GAPDH); eukaryotic translation initiation factor 4A1 (EIF4A1 ); heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1 ), heat shock protein 70 kDa (HSP70); p-kinesin (p-KIN), the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)); a Ubiquitin C promoter (UBC); a phosphoglycerate kinase-1 (PGK) promoter; a cytomegalovirus enhancer / chicken p-actin (GAG) promoter; synapsin promoter; Thy-1 promoter; CaMKIla promoter; NK-3; and Pcp2(L7); gamma-synuclein gene (SNCG); HKamac (derived from the interphotoreceptor retinoid binding protein enhancer and the human GNAT2 promoter), and Rho (PPR); and a p-actin promoter.

[0223] As used herein, “conditional expression” may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological, or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression. Certain aspects of the invention provide conditional expression of a polynucleotide-of-interest, e.g., expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide-of-interest.

[0224] Illustrative examples of inducible promoters / systems include steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the “GeneSwitch” mifepristone-regulatable system (Sirin et aL, 2003, Gene, 323:67), the cumate inducible gene switch (WO 2002 / 088346), tetracycline-dependent regulatory systems, etc.

[0225] Conditional expression can also be achieved by using a site specific DNA recombinase. According to certain aspects of the invention, the vector includes at least one (typically two) site(s) for recombination mediated by a site specific recombinase. As used herein, the terms “recombinase” or “site specific recombinase” include excisive or integrative proteins, enzymes, co-factors, or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in particular aspects of the present invention include: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, PHI.C31 , Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, SpCCEl , and ParA.

[0226] The vectors may include one or more recombination sites for any of a wide variety of site specific recombinases. It is to be understood that the target site for a site specific recombinase is in addition to any site(s) required for integration of a vector. As used herein, the terms “recombination sequence,” “recombination site,” or “site specific recombination site” refer to a particular nucleic acid sequence to which a recombinase recognizes and binds.

[0227] For example, one recombination site for Cre recombinase is loxP which is a 34 base pair sequence including two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see FIG. 1 of Sauer, B., Current Opinion in Biotechnology 5:521 -527 (1994)) Other exemplary loxP sites may include: Iox51 1 , (Hoess et aL, Nucleic Acids Res. 14: 2287-2300, 1996; Bethke and Sauer, Nucleic Acids Res; 25: 2828-2834, 1997); Iox5171 , (Lee and Saito, Gene. 216: 55-65, 1998); Iox2272 (Lee and Saito, Gene. 216: 55-65, 1998); m2 (Langer et aL, Nucleic Acids Res. 30: 3067-3077, 2002), Iox71 (Albert et aL, Plant J.; 7: 649-659, 1995), and Iox66, (Albert et aL, Plant J.; 7: 649-659, 1995). Suitable recognition sites for the FLP recombinase include: FRT (McLeod M, Craft S, Broach JR. Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle. Mol Cell Biol. 1986 Oct;6(10):3357-67), F1 , F2, F3 (Schlake T, Bode J. Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry. 1994 Nov 1 ;33(43):12746-51), F4, F5 (Schlake and Bode, Biochemistry. 1994 Nov 1 ;33(43):12746-51 ), FRT(LE) (Senecoff JF,et aL, DNA recognition by the FLP recombinaseof the yeast 2 mu plasmid. A mutational analysis of the FLP binding site. J Mol Biol. 1988 May 20;201 (2):405-21 .) and FRT(RE), (Senecoff et aL, 1J Mol Biol. 1988 May 20;201 (2):405-2 1988).

[0228] As used herein, an “internal ribosome entry site” (IRES) refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene (See, e.g., Jackson et aL, 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995 Dec. RNA 1 (10):985- 1000). In particular aspects, the vectors contemplated by the invention, include one or more polynucleotides-of-interest that encode one or more polypeptides. In particular, aspects, to achieve efficient translation of each of the plurality of polypeptides, the polynucleotide sequences can be separated by one or more IRES sequences or polynucleotide sequences encoding selfcleaving polypeptides.

[0229] As used herein, the term “Kozak sequence” refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 45), where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48). In particular aspects, the vectors contemplated by the invention include polynucleotides that have a consensus Kozak sequence and that encode a desired polypeptide.

[0230] In certain aspects, vectors include a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, hygromycin, methotrexate, Zeocin, Blastocidin, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et a , 1977. Cell 1 1 :223-232) and adenine phosphoribosyltransferase (Lowy et a!., 1990. Cell 22:817-823) genes which can be employed in tk- or aprt-cells, respectively.

[0231] In various aspects, vectors of the invention are used to increase, establish, and / or maintain the expression of one or more polypeptides. The terms “polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogs of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0232] Particular aspects of the invention also include polypeptide “variants.” The recitation polypeptide “variant” refers to polypeptides that are distinguished from a reference polypeptide by the addition, deletion, truncations, and / or substitution of at least one amino acid residue, and that retains a biological activity. In certain aspects, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or nonconservative, as known in the art.

[0233] In certain aspects, a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity to a corresponding sequence of a reference polypeptide. In certain aspects, amino acid additions or deletions occur at the C-terminal end and / or the N-terminal end of the reference polypeptide.

[0234] A variant may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequences described herein. The term “% identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example, % identity is relative to the entire length of the coding regions of the sequences being compared.

[0235] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Percent identity is determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al., 1990, J. Mol. Biol. 215:3, 403- 410; Altschul et al., 1997, Nucleic Acids Res. 25:17, 3389-402).

[0236] As noted above, polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well-known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, J. D. et al., (Molecular Biology of the Gene, Fourth Edition,Benjamin / Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect the biological activity of the protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

[0237] A “host cell” includes cells transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or a polynucleotide of the invention. Host cells may include packaging cells, producer cells, and cells infected with viral vectors. In particular aspects, host cells infected with a viral vector of the invention are administered to a subject in need of therapy. In certain aspects, the term “target cell” is used interchangeably with the host cell and refers to transfected, infected, or transduced cells of a desired cell type.

[0238] Large scale viral particle production is often necessary to achieve a reasonable viral titer. Viral particles are produced by transfecting a transfer vector into a packaging cell line that includes viral structural and / or accessory genes, e.g., gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes.

[0239] As used herein, the term “packaging vector” refers to an expression vector or viral vector that lacks a packaging signal and includes a polynucleotide encoding one, two, three, four, or more viral structural and / or accessory genes. Typically, the packaging vectors are included in a packaging cell and are introduced into the cell via transfection, transduction, or infection. Methods for transfection, transduction or infection are well known by those of skill in the art. A retroviral / lentiviral transfer vector of the present invention can be introduced into a packaging cell line, via transfection, transduction, or infection, to generate a producer cell or cell line. The packaging vectors of the present invention can be introduced into human cells or cell lines by standard methods including, e.g., calcium phosphate transfection, lipofection, or electroporation. In some aspects, the packaging vectors are introduced into the cells together with a dominant selectable marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. A selectable marker gene can be linked physically to genes encoded by the packaging vector, e.g., by IRES or self-cleaving viral peptides.

[0240] Viral envelope proteins (env) determine the range of host cells that can ultimately be infected and transformed by recombinant retroviruses generated from the cell lines. In the case of lentiviruses, such as human immunodeficiency viruses HIV-1 , HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and equine immunodeficiency virus (EIV), the env proteins include gp41 and gp120. Preferably, the viral env proteins expressed by packagingcells of the invention are encoded on a separate vector from the viral gag and pol genes, as has been previously described.

[0241] As used herein, the term “packaging cell lines” is used in reference to cell lines that do not contain a packaging signal but do stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol, and env) which are necessary for the correct packaging of viral particles. Any suitable cell line can be employed to prepare the packaging cells of the invention. Generally, the cells are mammalian cells. In a particular aspect, the cells used to produce the packaging cell line are human cells. Suitable cell lines which can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRCS cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells. In preferred aspects, the packaging cells are 293 cells, 293T cells, or A549 cells. In another preferred aspect, the cells are A549 cells.

[0242] As used herein, the term “producer cell line” refers to a cell line that is capable of producing recombinant retroviral particles, including a packaging cell line and a transfer vector construct including a packaging signal. The production of infectious viral particles and viral stock solutions may be carried out using conventional techniques. Methods of preparing viral stock solutions are known in the art and are illustrated by, e.g., Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628- 633, and N. R. Landau et al. (1992) J. Virol. 66:5110-5113. Infectious virus particles may be collected from the packaging cells using conventional techniques. For example, the infectious particles can be collected by cell lysis, or collection of the supernatant of the cell culture, as is known in the art. Optionally, the collected virus particles may be purified if desired. Suitable purification techniques are well known to those skilled in the art. By “enhance” or “promote,” or “increase” or “expand” refers generally to the ability of the compositions and / or methods of the invention to elicit, cause, or produce higher numbers of transduced cells compared to the number of cells transduced by either vehicle or a control molecule / composition. In one embodiment, a hematopoietic stem cell transduced with compositions and methods of the present invention includes an increase in the number of transduced cells compared to existing transduction compositions and methods. Increases in cell transduction can be ascertained using methods known in the art, such as reporter assays, RT-PCR, and cell surface protein expression, among others. An “increased” or “enhanced” amount of transduction is typically a “statistically significant” amount and may include an increase that is 1.1 , 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above1 , e.g., 1 .5, 1 .6, 1 .7. 1 .8, etc.) the number of cells transduced by vehicle, a control composition, or another transduction method.

[0243] By “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refers generally to compositions or methods that result in comparably fewer transduced cells compared to cells transduced with compositions and / or methods according to the present invention. A “decrease” or “reduced” amount of transduced cells is typically a “statistically significant” amount, and may include an decrease that is 1.1 , 1 .2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1 , e.g., 1 .5, 1 .6, 1.7. 1.8, etc.) the number of transduced cells (reference response) produced by compositions and / or methods according to the present invention.

[0244] By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to a physiological response that is comparable to a response caused by either vehicle, a control molecule / composition, or the response in a particular cell lineage. A comparable response is one that is not significantly different or measurably different from the reference response.

[0245] As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Preferably, the subject is a human. A “subject in need thereof” is a subject suffering from or at risk of developing or suffering from an ocular disease or disorder. A subject at risk of developing or suffering from an ocular disease or disorder can be diagnosed by a physician or ocular specialist using routine methods in the art.

[0246] As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0247] As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also includes reducing the intensity, effect,symptoms, and / or burden of a disease or condition prior to onset or recurrence of the disease or condition.

[0248] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of a virus or transduced therapeutic cell to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

[0249] A “prophylactically effective amount” refers to an amount of a virus or transduced therapeutic cell effective to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0250] A “therapeutically effective amount” of a virus or transduced therapeutic cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient).

[0251] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant improvement in light sensitivity and / or visual acuity.

[0252] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary roundingtechniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0253] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0254] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0255] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0256] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0257] Furthermore, numerous references have been made to patents, printed publications, journal articles, other written text, and website content throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching(s), as of the filing date of the first application in the priority chain in which the specific reference was included. For instance, with regard to chemical compounds, nucleic acid, and amino acids sequences referenced herein that are available in a public database, the information in the database entry is incorporated herein by reference as of the date of an application in the priority chain in which the database identifier for that compound or sequence was first included in the text.

[0258] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0259] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0260] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Green and Sambrook,Molecular Cloning: A Laboratory Manual, 4nd Edition (2012); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (2003); the series Methods In Enzymology (Academic Press, Inc.); Behlke, et al., Polymerase Chain Reaction: Theory and Technology (2019); Greenfield, ed. Antibodies, A Laboratory Manual, Second Edition (2014); and Capes-Davis and R. I. Freshney, eds. Freshney's Culture of Animal Cells 8th Edition (2021 ).

[0261] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the example(s) or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 11 th Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006), and / or A Dictionary of Chemistry, 8’hEdition (Ed. J. Law & R. Rennie, Oxford University Press, 2020).

Claims

LISTING OF CLAIMSWhat is claimed is:

1. A method of enhancing delivery of a gene of interest to an eye of a subject comprising administering a viral vector containing multiple copies of the gene of interest to the eye.

2. The method of claim 1 , wherein the gene of interest is a light sensitive protein.

3. The method of claim 2, wherein the light sensitive protein is a microbial opsin or a vertebrate opsin, or an invertebrate opsin.

4. The method of claim 3, wherein the microbial opsin is a channelrhodopsin (ChR) or a pump-based opsin.

5. The method of claim 4, wherein the ChR is Chloromonas oogama channelrhodopsin CoChR), channelrhodopsin 2 (ChR2), Volvox carteri channelrhodopsin (vChR), red activatable channelrhodopsin (ReaChR), Chronos, Platymonas (Tetraselmis) subcordiformis channelrhodopsin (PsCHR), or Chrimson.

6. The method of claim 5, wherein the CoChR is a mutant CoChR comprising the sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

7. The method of claim 6, wherein the ChR is CoChR-H94E / L112C / K264T having a sequence of SEQ ID NO: 60.

8. The method of claim 4, wherein the CHR has at least 90% sequence identity with SEQ ID NO: 60.

9. The method of claim 6, wherein the delivery of the gene of interest increases light sensitivity in the subject.

10. The method of claim 6, wherein the delivery of the gene of interest increases visual acuity in the subject.11 . The method of claim 2, wherein the viral vector further comprises a promoter arranged to promote expression from multiple copies of the gene of interest.

12. The method of claim 1 1 , wherein the promoter is a ubiquitous promoter or a retinal cellspecific promoter.

13. The method of claim 12, wherein the ubiquitous promoter is cytomegalovirus immediate enhancer / p-actin (CAG), cytomegalovirus (CMV), synapsin, elongation factor-1 alpha (EF1 a), or thymocyte differentiation antigen 1 (Thy-1).

14. The method of claim 12, wherein the retinal cell-specific promoter is metabolic glutamate receptor 6 (mGluR6), neurokinin 3 (NK-3), or purkinje cell protein 2 (Pcp2(L7)), gamrna-synuctein gene (SNCG), HKamac, or rhodopsin (RHO).

15. The method of claim 14, wherein the promoter is a mGluR6 promoter.

16. The method of claim 15, wherein the mGluR6 promoter is a human promoter or a mouse promoter.

17. The method of claim 16, wherein the mGluR6 promoter is a shortened mGluR6 having the sequence of SEQ ID NO: 62, SEQ ID NO: 71 , or SEQ ID NO: 73.

18. The method of claim 1 , wherein the viral vector comprises an adeno-associated virus (AAV) vector.

19. The method of claim 18, wherein the AAV vector comprises AAV2.7m8 or AAV2.7m8(Y444F).

20. The method of claim 16, wherein the viral vector further comprises at least one post- transcriptional regulatory element.21 . The method of claim 20, wherein the at least one post-transcriptional regulatory element is woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or mutated woodchuck hepatitis virus posttranscriptional regulatory element (mWPRE).

22. The method of claim 20, wherein the at least one post-transcriptional regulatory element further comprises human growth hormone polyadenylation signal (hGHpA), bovine growth hormone polyadenylation signal (bGHpA), or SV40 early polyadenylation signal (SV40pA).

23. The method of claim 1 , wherein the viral vector encodes two copies of the gene of interest.

24. The method of claim 1 , wherein the viral vector encodes at least three copies of the gene of interest.

25. A method of increasing light sensitivity or improving visual acuity in a subject comprising administering a viral vector that encodes a plurality of opsins to vitreous of an eye.

26. The method of claim 25, wherein the plurality of opsins are microbial opsins, vertebrate opsins, invertebrate opsins, or a combination thereof.

27. The method of claim 26, wherein the microbial opsins are channelrhodopsins (ChR) or pump-based opsins.

28. The method of claim 27, wherein the ChR are Chloromonas oogama channelrhodopsin (CoChR), channel rhodopsin 2 (ChR2), V / i / ox carter / channelrhodopsin (vChR), red activatable channelrhodopsin (ReaChR), Platymonas (Tetraselmis) subcordiformis channelrhodopsin (PsChR), Chronos, or Chrimson.

29. The method of claim 28, wherein the CoChRs are mutant CoChR having the sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

30. The method of claim 29, wherein the ChR is CoChR-H94E / L112C / K264T having a sequence of SEQ ID NO: 60.31 . The method of claim 27, wherein the ChR has at least 90% sequence identity with SEQ ID NO: 60.

32. The method of claim 25, wherein the viral vector further comprises a promoter arranged to promote expression of the plurality of opsins.

33. The method of claim 32, wherein the promoter is a ubiquitous promoter or a retinal cellspecific promoter.

34. The method of claim 33, wherein the ubiquitous promoter is cytomegalovirus immediate enhancer / p-actin (GAG), cytomegalovirus (CMV), synapsin, elongation factor-1 alpha (EF1 a), or thymocyte differentiation antigen 1 (Thy-1 ).

35. The method of claim 33, wherein the retinal cell-specific promoter is metabolic glutamate receptor 6 (mGluR6), neurokinin 3 (NK-3), or purkinje cell protein 2 (Pcp2(L7)), gamma-synuclein gene (SNCG), HKamac, or rhodopsin (RHO).

36. The method of claim 35, wherein the promoter is mGluR6.

37. The method of claim 35, wherein the mGluR6 promoter is a human promoter or a mouse promoter.

38. The method of claim 35, wherein the promoter is a shortened mGluR6 promoter.

39. The method of claim 38, wherein the mGluR6 promoter is a shortened mGluR6 having a sequence of SEQ ID NO: 62, SEQ ID NO: 71 , or SEQ ID NO: 73.

40. The method of claim 25, wherein the viral vector comprises an adeno-associated viral vector (AAV).41 . The method of claim 40, wherein the AAV is AAV2.7m8 or AAV2.7m8(Y444F).

42. The method of claim 32, wherein the viral vector further comprises at least one post- transcriptional regulatory element.

43. The method of claim 42, wherein the at least one post-transcriptional regulatory element is woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or mutated woodchuck hepatitis virus posttranscriptional regulatory element (mWPRE).

44. The method of claim 42, wherein the at least one post-transcriptional regulatory element further comprises human growth hormone polyadenylation signal (hGHpA), bovine growth hormone polyadenylation signal (bGHpA), or SV40 early polyadenylation signal (SV40pA).

45. The method of claim 25, wherein the plurality of opsins is two opsins.

46. The method of claim 25, wherein the plurality of opsins is at least three opsins.

47. An isolated nucleic acid molecule comprising: a polynucleotide molecule comprising a nucleic acid sequence encoding a channelrhodopsin for differential expression in subcellular regions of a retinal neuron, the nucleic acid sequence comprising: a first nucleotide sequence encoding a first Chloromonas oogama channelrhodopsin (CoChR); a second nucleotide sequence encoding a second CoChR, which second nucleotide sequence is linked to the first nucleotide sequence; a promoter sequence arranged to promote expression of the first and second channelrhodopsin; and a post-transcriptional regulatory element.

48. The isolated nucleic acid molecule of claim 47, wherein the first nucleotide sequence encodes an amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

49. The isolated nucleic acid molecule of claim 47, wherein the second nucleotide sequence encodes an amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

50. The isolated nucleic acid molecule of claim 49, wherein the first nucleotide sequence encodes CoChR-H94E / L1 12C / K264T having a sequence of SEQ ID NO: 60.51 . The isolated nucleic acid molecule of claim 47, wherein the first channelrhodopsin and the second channelrhodopsin are alike.

52. The isolated nucleic acid molecule of claim 47, wherein the promoter sequence is cytomegalovirus immediate enhancer / p-actin (CAG), cytomegalovirus (CMV), synapsin, elongation factor- 1 alpha (EF1 a), thymocyte differentiation antigen 1 (Thy-1), metabolic glutamate receptor 6 (mGluR6), neurokinin 3 (NK-3), or purkinje cell protein 2 (Pcp2(L7)), gamma-synuclein gene (SNCG), HKamac, or rhodopsin (RHO).

53. The isolated nucleic acid molecule of claim 52, wherein the promoter sequence is shortened mGluR6 of SEQ ID NO: 62, SEQ ID NO: 71 , or SEQ ID NO: 73.

54. The isolated nucleic acid molecule of claim 47, wherein the post-transcriptional regulatory element is woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or mutated woodchuck hepatitis virus posttranscriptional regulatory element (mWPRE).

55. The isolated nucleic acid molecule of claim 54, wherein the post-transcriptional regulatory element further comprises human growth hormone polyadenylation signal (hGHpA), bovine growth hormone polyadenylation signal (bGHpA), or SV40 early polyadenylation signal (SV40pA).

56. The isolated nucleic acid molecule of claim 47, wherein the first nucleotide sequence and the second nucleotide sequence are separated by a sequence encoding a cleavage protein.

57. The isolated nucleic acid molecule of claim 56, wherein the cleavage protein is P2A.

58. The isolated nucleic acid molecule of claim 47, wherein the first nucleotide sequence and the second nucleotide sequence are separated by a sequence encoding a non-cleaving peptide, wherein the non-cleaving peptide is IRES or pHK.

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