US 20160361439A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0361439 A1 AGBANDUE-MCKENNA et al. (43) Pub. Date: Dec. 15, 2016

(54) CAPSD-MUTATED RAAV VECTORS AND Publication Classification METHODS OF USE (51) Int. Cl. (71) Applicant: University of Florida Research A6IR 48/00 (2006.01) Foundation, Inc., Gainesville, FL (US) CI2N 5/86 (2006.01) (52) U.S. Cl. (72) Inventors: Mavis AGBANDJE-MCKENNA, CPC ...... A61K 48/0058 (2013.01): CI2N 15/86 Gainesville, FL (US); William W. (2013.01); C12N 27.10/10043 (2013.01); C12N HAUSWIRTH, Gainesville, FL (US); 2830/008 (2013.01) Arun SRIVASTAVA, Gainesville, FL (US); Li ZHONG, Boxborough, MA (57) ABSTRACT (US) Disclosed are capsid-mutated ra AV vectors and methods for their use in gene therapy, and particularly for use in deliv (21) Appl. No.: 15/246,385 ering therapeutic transgenes to treat a variety of mammalian diseases and disorders, including dysfunctions and abnormal conditions of the . VP3 capsid compris (22) Filed: Aug. 24, 2016 ing a modification of one or more of the Surface-exposed tyrosine residues are disclosed, and in particular. VP3 capsid Related U.S. Application Data comprising tyrosine-to-phenylalanine mutations at positions corresponding to Y444F, Y500F, and Y730F of the (63) Continuation of application No. 14/298,553, filed on wild-type AAV2 sequence. Also provided are ra AV virions Jun. 6, 2014, which is a continuation-in-part of ap and viral particles that comprise Such a mutated AAV capsid plication No. 12/993,092, filed on Jun. 10, 2011, now protein and a nucleic acid molecule that expresses one or abandoned, filed as application No. PCT/US09/.44753 more selected therapeutic or reporter transgenes in one or on May 20, 2009. more mammalian cells of interest. Advantageously, the (60) Provisional application No. 61/054,571, filed on May capsid-mutated ra AV vectors and virions disclosed herein 20, 2008, provisional application No. 61/199,241, afford improved transduction efficiency in a variety of cells, filed on Nov. 14, 2008, provisional application No. tissues and organs of interest, when compared to their 61/200,430, filed on Nov. 26, 2008. unmodified (i.e., wild-type) ra AV vector counterparts.

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US 2016/0361439 A1 Dec. 15, 2016

CAPSD-MUTATED RAAV VECTORS AND facilitating or inhibiting the flow of positive or negative ions METHODS OF USE through cell membranes, the cell can be briefly depolarized, depolarized and maintained in that State, or hyperpolarized. CROSS-REFERENCE TO RELATED Thus, the control of the depolarization of cells can be APPLICATIONS beneficial for a number of different purposes, including 0001. The present application is a continuation of U.S. visual, muscular and sensory control. -sensitive protein patent application Ser. No. 14/298,533, filed Jun. 6, 2014 channels, pumps, and receptors can permit millisecond (Atty. Dkt. No. 3.6689.370; pending), which is a continua precision optical control of cells. Although light-sensitive tion-in-part of U.S. patent application Ser. No. 12/993,092, proteins in combination with light can be used to control the filed Jun. 10, 2011 (Atty. Dkt. No. 3.6689.358; now aban flow of ions through cell membranes, targeting and delivery doned), which was the U.S. national-stage filing of PCT Intl. remain to be addressed for specific diseases, disorders, and Pat. Appl. No. PCT/US2009/044753, filed May 20, 2009 circuits. (now abandoned); which claims priority to U.S. Prov. Pat. Appl. No. 61/054,571, filed May 20, 2008 (expired); U.S. BRIEF SUMMARY OF THE INVENTION Prov. Pat. Appl. No. 61/199,241, filed Nov. 14, 2008 (ex 0008. In one aspect, the invention provides a recombinant pired); and U.S. Prov. Pat. Appl. No. 61/200,430, filed Nov. nucleic acid comprising a nucleic acid encoding a light 26, 2008 (expired); the contents of each of which is spe sensitive protein operatively linked to a metabotropic glu cifically incorporated herein in its entirety by express ref tamate receptor 6 (mGluR6) regulatory sequence or frag erence thereto. ment thereof. In one embodiment, the light-sensitive protein can be selected from the group consisting of ChR1, ChR2, STATEMENT REGARDING FEDERALLY VChR1, ChR2C128A, ChR2 C128S, ChR2 C128T, ChR1 SPONSORED RESEARCH OR DEVELOPMENT ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, 0002 This invention was made with government support eNpHR, , and variants thereof. In another under Grant Nos. DK-058327 GM-082946, HL-076901, and embodiment the light-sensitive protein is ChR2 or a light HL-097088 awarded by the National Institutes of Health. sensitive protein that is at least about 70%, at least about The government has certain rights in the invention. 80%, at least about 90% or at least about 95% identical to ChR2. In another embodiment the mGluR6 regulatory NAMES OF THE PARTIES TO A JOINT sequence fragment comprises less than about 1000, less than RESEARCH AGREEMENT about 750, less than about 500, less than about 250, or less than about 100 base pairs. In a related embodiment, the 0003) Not Applicable. mGluR6 regulatory sequence or fragment thereof is a mGluR6 promoter or enhancer. In a specific related embodi BACKGROUND OF THE INVENTION ment, the nucleic acid further comprises a sequence that 0004 Field of the Invention encodes a green fluorescent protein. In another embodiment, 0005. The present disclosure relates to the fields of the nucleic acid is encapsidated within a recombinant adeno molecular biology, ophthalmology, and gene therapy. In associated virus (AAV). In certain embodiments, the recom particular embodiments, capsid-mutated ra AV vectors and binant AAV is of a combinatorial hybrid of 2, 3, 4, 5, 6, 7, methods for their use in gene therapy are disclosed. In 8, 9, 10, 11, 12, 13, 14, 15 or more serotypes or mutants exemplary embodiments, capsid proteins comprising a thereof. In certain embodiments, the recombinant adeno modification of one or more of the Surface-exposed tyrosine associated virus is of a serotype selected from the group residues are disclosed, including VP3 capsid proteins that consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, include tyrosine-to-phenylalanine mutations at positions AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids corresponding to Y444F,Y500F, and Y730F of the wild-type thereof. In a related embodiment, the nucleic acid is encapsi AAV2 sequence. Also provided are ra AV virions and viral dated within a recombinant virus selected from the group particles that comprise Such a mutated AAV capsid protein consisting of recombinant adeno-associated virus (AAV), and a nucleic acid molecule that expresses one or more recombinant retrovirus, recombinant lentivirus, and recom selected therapeutic or reporter transgenes in one or more binant poxvirus. mammalian cells of interest. 0009. In another aspect, the invention provides a vector 0006. Description of Related Art comprising a nucleic acid encoding a light-sensitive protein, 0007. A gene-delivery therapy to treat a disease or dis wherein the nucleic acid is operatively linked to a metabo order independent of treating an underlying mutation could tropic glutamate receptor 6 (mGluR6) regulatory sequence have potential value. Methods capable of controlling, regu or fragment thereof. In one embodiment, the light-sensitive lating, and/or driving specific neural circuits so as to mediate protein is selected from the group consisting of ChR1, naturalistic neural responses and high resolution perception ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2C128T, and control could also be of enormous potential therapeutic ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, value. Neurons are an example of a type of cell that uses the NpHR, eNpHR, melanopsin, and variants thereof. In a electrical currents created by depolarization to generate related embodiment the light-sensitive protein is ChR2 or a communication signals (e.g., nerve impulses). Other elec light-sensitive protein that is at least about 70%, at least trically excitable cells include skeletal muscle, cardiac about 80%, at least about 90% or at least about 95% identical muscle, and endocrine cells. Neurons use rapid depolariza to ChR2. In another embodiment, the mGluR6 regulatory tion to transmit signals throughout the body and for various sequence fragment is less than about 1000, less than about purposes, such as motor control (e.g., muscle contractions), 750, less than about 500, less than about 250, or less than sensory responses (e.g., touch, hearing, and other senses) about 100 base pairs. In a related embodiment, the mGluR6 and computational functions (e.g., functions). By regulatory sequence fragment is represented by the sequence US 2016/0361439 A1 Dec. 15, 2016

in FIG. 6. In another embodiment the vector comprises an bipolar cell (e.g., ON or OFF bipolar cells; rod and adeno-associated virus (AAV). In a related embodiment the cone bipolar cells) comprising introducing into a a vector comprises a recombinant virus selected from the vector comprising the exogenous nucleic operatively linked group consisting of a recombinant adeno-associated virus to a retinal bipolar (e.g., ON or OFF retinal bipolar cells; rod (ra AV), a recombinant retrovirus, a recombinant lentivirus, and cone bipolar cells) cell-specific regulatory sequence and a recombinant poxvirus. In a specific embodiment, the wherein the method results in at least about a 25-30% AAV is of a serotype selected from the group consisting of transduction efficiency. In other embodiment, Such method AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, results in at least about a 10% transduction efficiency. In one AAV9, AAV10, AAV11, AAV12, and hybrids thereof. In embodiment, the method results in at least about 20%, 30%, other specific embodiments, the recombinant AAV is of a 40%, 50%, 60%, 70%, 80%, or 90% transduction efficiency. combinatorial hybrid of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 0013. In a related embodiment, the transduction effi 14, 15 or more serotypes or mutants thereof. In a related ciency is measured by quantifying the total number of retinal embodiment, the AAV comprises mutated capsid protein. In bipolar cells (e.g., ON or OFF retinal bipolar cells; rod and one specific embodiment, the capsid protein comprises at cone bipolar cells) infected. In another embodiment, the least one mutated tyrosine residue. The mutated tyrosine exogenous nucleic acid comprises a light-sensitive protein. residue can be selected from the group consisting of Y252F, In a related embodiment, the light-sensitive protein is Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, selected from the group consisting of ChR1, ChR2. VChR1, Y281F, Y508F, Y576F, Y612G, Y673F, and Y720F. In a ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 specific embodiment, the mutated capsid protein comprises hybrids/chimeras, ChD, ChEF, ChF ChIEF, NpHR, eNpHR, one or more tyrosine residues, each mutated to a phenyl melanopsin, and variants thereof. In a specific embodiment, alanine residue. the light-sensitive protein is ChR2 or a light-sensitive pro 0010. In another aspect the present invention provides a tein that is at least about 70%, at least about 80%, at least method of treating a subject suffering from a disease or about 90% or at least about 95% identical to ChR2. disorder of the eye comprising introducing into an affected 0014. In another embodiment, the regulatory sequence eye a ra AV vector comprising a light-sensitive protein comprises a metabotropic glutamate receptor 6 regulatory operatively linked to a metabotropic glutamate receptor 6 sequence (mGluR6) or a fragment thereof. In a related regulatory sequence (mGluR6) or fragment thereof. In one embodiment, the mGluR6 regulatory sequence fragment is embodiment, the disease or disorder of the eye is caused by less than about 1000, less than about 750, less than about degeneration. In another embodiment, the 500, less than about 250, or less than about 100 base pairs. light-sensitive protein is selected from the group consisting In a specific embodiment, the mGluR6 regulatory sequence of ChR1, ChR2, VChR1, ChR2C128A, ChR2C128S, ChR2 fragment is represented by the sequence in FIG. 6. In another C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, embodiment, the exogenous nucleic acid is introduced using ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. In a recombinant adeno-associated viral vector (AAV). In a a related embodiment, the light-sensitive protein is ChR2 or related embodiment the AAV comprises a mutated capsid a light-sensitive protein that is at least about 70%, at least protein. In a specific embodiment, the capsid protein com about 80%, at least about 90% or at least about 95% identical prises a mutated tyrosine residue. In a related embodiment, to ChR2. In another embodiment, the mGluR6 promoter the mutated tyrosine residue is selected from the group fragment is less than about 1000, less than about 750, less consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, than about 500, less than about 250, or less than about 100 Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and base pairs. In a related embodiment, the mGluR6 promoter Y720F. In another embodiment the mutated capsid protein fragment is represented by the sequence in FIG. 6. comprises a tyrosine residue mutated to a phenylalanine. 0011. In another embodiment, the AAV is of a serotype 0015. In another embodiment, the exogenous nucleic acid selected from the group consisting of AAV1, AAV2, AAV3, is introduced using intravitreal injection, Subretinal injec AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, tion, and/or ILM peel. In another embodiment, the AAV is AAV11, AAV12, and hybrids thereof. In other embodiments, of a serotype is selected from the group consisting of AAV1, the recombinant AAV is of a combinatorial hybrid of 2, 3, 4, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more serotypes or AAV10, AAV11, AAV12, and hybrids thereof. In yet another mutants thereof. In a related embodiment, the AAV com embodiments, the recombinant AAV is of a combinatorial prises a mutated capsid protein. In another related embodi hybrid of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more ment, the capsid protein comprises a mutated tyrosine resi serotypes, or mutants thereof. due. In a specific embodiment, the mutated tyrosine residue 0016. In yet another aspect, the present invention pro is selected from the group consisting of Y252F, Y272F, vides a method of introducing an exogenous nucleic acid Y444F, Y500F, Y700F, Y704F, Y73OF, Y275F, Y281F, into the nucleus of a retinal cell comprising introducing a Y508F, Y576F, Y612G, Y673F and Y720F. In a related vector comprising an exogenous nucleic acid operatively embodiment, the mutated capsid protein comprises a tyro linked to a retinal cell-specific regulatory sequence into a sine residue mutated to a phenylalanine. In another embodi retinal cell, wherein the vector is specifically designed to ment, the AAV is introduced using intravitreal injection, avoid ubiquitin-mediated protein degradation. In one subretinal injection and/or ILM peel. In a specific embodi embodiment, the degradation is proteasome-mediated. In ment, the AAV is introduced into a retinal bipolar cell (e.g., another embodiment, the exogenous nucleic acid comprises ON or OFF retinal bipolar cells; rod and cone bipolar cells). a light-sensitive protein. In a related embodiment, the light In another embodiment, the method further comprises using sensitive protein is selected from the group consisting of a light-generating device external to the eye. ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 0012. In another aspect the present invention provides a C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, method of expressing an exogenous nucleic acid in a retinal ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. In US 2016/0361439 A1 Dec. 15, 2016

another related embodiment, the light-sensitive protein is AAV is of a combinatorial hybrid of 2, 3, 4, 5, 6, 7, 8, 9, 10, ChR2 or a light-sensitive protein that is at least about 70%, 11, 12, 13, 14, 15 or more serotypes or mutants thereof. In at least about 80%, at least about 90% or at least about 95% a related embodiment, the AAV comprises a mutated capsid identical to ChR2. In another embodiment, the retinal cell is protein. In another embodiment, the capsid protein com a retinal bipolar cell (e.g., ON or OFF retinal bipolar cells; prises a mutated tyrosine residue. In another embodiment, rod and cone bipolar cells). the mutated tyrosine residue is selected from the group 0017. In a related embodiment, the regulatory sequence consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, comprises a metabotropic glutamate receptor 6 promoter Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and (mGluR6 promoter) or fragment thereof. In another embodi Y720F. In another embodiment, the mutated capsid protein ment, the mGluR6 fragment is less than 1000, 750, 500, 250, comprises a tyrosine residue mutated to a phenylalanine. In or 100 base pairs. In another embodiment, the mGluR6 another embodiment, the vector is introduced using intrav promoter fragment is represented by the sequence in FIG. 6. itreal injection, subretinal injection, and/or ILM peel. In another embodiment, the vector is selected from the group consisting of recombinant adeno-associated virus (AAV), INCORPORATION BY REFERENCE recombinant retrovirus, recombinant lentivirus, and recom 0021 All publications, patents, and patent applications binant poxvirus. In a related embodiment, the vector is a mentioned in this specification are herein incorporated by recombinant adeno-associated viral vector (AAV). In reference to the same extent as if each individual publica another embodiment, the AAV is of a serotype selected from tion, patent, or patent application was specifically and indi the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, vidually indicated to be incorporated by reference. AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof. In other embodiments, the recombinant BRIEF DESCRIPTION OF THE DRAWINGS AAV is of a combinatorial hybrid of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more serotypes, or mutants thereof. 0022. For promoting an understanding of the principles 0018. In another embodiment, the AAV comprises a of the invention, reference will now be made to the embodi mutated capsid protein. In another embodiment, the capsid ments, or examples, illustrated in the drawings and specific protein comprises a mutated tyrosine residue. In another language will be used to describe the same. It will, never embodiment, the mutated tyrosine residue is selected from theless be understood that no limitation of the scope of the the group consisting of Y252F, Y272F, Y444F, Y500F, invention is thereby intended. Any alterations and further Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, modifications in the described embodiments, and any further Y612G, Y673F and Y720F. In another embodiment, the applications of the principles of the invention as described mutated capsid protein comprises a tyrosine residue mutated herein are contemplated as would normally occur to one of to a phenylalanine. In another embodiment, the vector is ordinary skill in the art to which the invention relates. introduced using intravitreal injection, Subretinal injection, 0023 The novel features of the invention are set forth and/or ILM peel. with particularity in the appended claims. A better under 0019. In another aspect the present invention provides a standing of the features and advantages of the present method of transducing a retinal bipolar cell (e.g., ON or OFF invention will be obtained by reference to the following retinal bipolar cells; rod and cone bipolar cells) comprising detailed description that sets forth illustrative embodiments, introducing into a retina a vector comprising an exogenous in which the principles of the invention are utilized, and the nucleic acid operatively linked to a regulatory sequence. In accompanying drawings of which: one embodiment, the regulatory sequence is a non-cell type 0024 FIG. 1 depicts the ChR1 nucleic acid sequence specific promoter. In another embodiment, the regulatory (SEQ ID NO:2): sequence is a guanine nucleotide binding protein alpha 0025 FIG. 2 depicts the ChR2 nucleic acid sequence activating activity polypeptide O (GNAO1) promoter or a (SEQ ID NO:3); fusion of the cytomegalovirus (CMV) immediate early 0026 FIG. 3 depicts the NpHR nucleic acid sequence enhancer and the bovine B-actin promoter plus intronl-exon1 (SEQ ID NO:4); junction (CBA, SmCBA). In another embodiment, the exog 0027 FIG. 4 depicts the melanopsin nucleic acid enous nucleic acid comprises a light-sensitive protein. In a sequence (SEQ ID NO:5); related embodiment, the light-sensitive protein is selected 0028 FIG. 5 depicts the ChR2 nucleic acid sequence that from the group consisting of ChR1, ChR2, VChR1, ChR2 is mammalian codon-optimized and that encodes a ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/ fused with Green Fluorescent Protein (GFP) (SEQ ID chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, mel NO:6): anopsin, and variants thereof. In another related embodi (0029 FIG. 6 depicts a fragment of the GRM6 (metabo ment, the light-sensitive protein is ChR2 or a light-sensitive tropic glutamate receptor 6) regulatory nucleic acid protein that is at least about 70%, at least about 80%, at least sequence capable of regulating expression in a bipolar cell about 90% or at least about 95% identical to ChR2. specific manner (SEQ ID NO:7): 0020. In another embodiment, the vector is selected from 0030 FIG. 7 depicts a smCBA regulatory nucleic acid the group consisting of recombinant adeno-associated virus sequence (SEQ ID NO:8); (AAV), recombinant retrovirus, recombinant lentivirus, and 0031 FIG. 8A and FIG. 8B depict neurons expressing recombinant poxvirus. In a related embodiment, the vector ChR2 and firing. (FIG. 8A) Neurons expressing ChR2 with is a recombinant adeno-associated viral vector (AAV). In light stimulation: (FIG. 8B) Neurons firing in response to another embodiment, the AAV is of a serotype selected from fast trains of blue light pulses; the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, 0032 FIG.9A, FIG.9B, FIG.9C, FIG.9D, FIG.9E, FIG. AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and 9F, FIG. 9G, and FIG. 9H depict AAV delivery to retinal hybrids thereof. In certain embodiments, the recombinant bipolar cells. Column 1 shows GFP expression in retinal US 2016/0361439 A1 Dec. 15, 2016 bipolar cells after a subretinal injection with the AAV7 implementation to another. Moreover, it will be appreciated CBA-GFP vector after 8 weeks of age. Column 2 shows that such a development effort might be complex and PKCo. staining (an antibody that is specific to bipolar cells). time-consuming, but would be a routine undertaking for Column 3 shows DAPI staining for cell nuclei. Column 4 those of ordinary skill in the art having the benefit of this shows merged images of GFP expression, PKCC. and DAPI disclosure. stains. The first row is 20x magnification and the second row 0038 Light-Sensitive Proteins: is 40x magnification; 0039. The present invention provides recombinant 0033 FIG. 10A, FIG. 10B, FIG.10C, FIG.10D, and FIG. nucleic acids encoding light-sensitive proteins, viral and 10E depict expression of the ChR2-GFP fused protein in non-viral vectors for the delivery of recombinant nucleic rdl, rho-/-, and rd 16 in retinal bipolar cells. In each image, acids encoding light-sensitive proteins, and methods for the retinal pigment epithelium (RPE), bipolar cells or inner delivery of light-sensitive proteins. nuclear layer (INL), inner plexiform layer (IPL), and gan 0040 Light-sensitive proteins are proteins that belong to glion cell layer (GCL) are noted. The brighter white areas the family and include () and inver show GFP expression. There are ringlets of expression in the tebrate . The animal , rhodopsins, are bipolar cells of the INL (except for the AAV5 intravitreal G-protein coupled receptors (GPCRs) with seven transmem injection); brane helices that can regulate the activity of ion channels. 0034 FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. Invertebrate rhodopsins are usually not GPCRs, but instead 11E, and FIG. 11F depict analysis of EGFP expression in are light-sensitive, or light-activated, ion pumps or ion frozen retinal sections by immunohistochemistry at 1 month, channels. following subretinal injections with the Tyrosine-mutant 0041 An algal opsin such as (ChR2) AAV vectors. Example sections depicting spread and inten from Chlamydomonas reinhardtii allows blue light-induced sity of EGFP fluorescence throughout the retina after trans action potentials to be triggered with millisecond-precision duction with serotype 2 Y444 (FIG. 11A) or serotype 8Y733 in cells due to depolarizing cation flux through a light-gated (FIG. 11B). The images are oriented with the vitreous pore. An archaeal opsin, such as from Natron toward the bottom and the photoreceptor layer toward the Omonas pharaonis, allows for light-activated chloride top. EGFP fluorescence in photoreceptors, RPE and gan pumping; the pump can be hyperpolarized and inhibited glion cells from mouse eyes injected Subretinally with from firing action potentials when exposed to yellow light. serotype 2 Y444 (FIG. 11C) EGFP fluorescence in photo Use of Such light-sensitive opsins allows for temporal and receptors, RPE and Muller cells after serotype 8 Y733 spatial regulation of neuronal firing activity. delivery (FIG. 11D) Detection of Muller cells processes 0042. As referred to herein, a “light-sensitive' protein (red) by immunostaining with a glutamine-synthetase (GS) includes (ChR1, ChR2), antibody (FIG. 11E) Merged image showing colocalization (NpHR), , pineal opsins, , and of EGFP fluorescence (green) and GS staining (red) in variants thereof. A light-sensitive protein of this invention retinal sections from eyes treated with serotype 8 Y733 can occur naturally in plant, animal, archaebacterial, algal, (FIG. 11F) Calibration bar 100 gcl., ganglion cell layer; ipl. or bacterial cells, or can alternatively be created through inner plexiform layer; inl, inner nuclear layer, onl, outer laboratory techniques. nuclear, os, outer segment; rpe, retinal pigment epithelium; 0043 Channelrhodopsins ChR1 (GenBank accession 0035 FIG. 12A-1, FIG. 12A-2, FIG. 12B, and FIG. 12C number AB058890/AF385748; FIG. 1) (SEQID NO:2) and depict training mice on a water maze task. (FIG. 12A-1 and ChR2 (GenBank accession number AB058891/AF461397: FIG. 12A-2) A schematic (FIG. 12A-1) of the water maze FIG. 2) (SEQ ID NO:3) are two rhodopsins from the green (FIG. 12A-2) used to measure scotopic threshold (Hayes and alga, Chlamydomonas reinhardtii (Nagel, 2002; Nagel, Balkema, 1993). (FIG. 12B) Time it took each mouse group 2003). Both are light-sensitive channels that, when (retinal degenerated—treated, retinal degenerated—un expressed and activated in neural tissue, allow a cell to be treated, and wild type) to find the target (black platform-- depolarized when stimulated with light (Boyden, 2005). LED array) as a function of training sessions. (FIG. 12C) 0044. In some embodiments, hybrid or chimeric chan Time it took each mouse group (treated rdl., treated rd 16, nelrhodopsins can be created and used by combining dif treated rho, untreated retinal degenerated, and wild type) ferent portions of the ChR1 and ChR2 proteins. to find the target (black platform--LED array) as a function 0045. In one embodiment, a hybrid or chimeric channel of different light intensities; and can be created and used by replacing the N-ter 0036 FIG. 13 depicts a goggle-like device with an asso minal segments of ChR2 with the homologous counterparts ciated light generation/production element (LED array/laser of ChR1 (and vice-versa). In some embodiments, the hybrid system) that can trigger expression of light-sensitive pro channelrhodopsins result in a shift of sensitivity into a teins. different wavelength spectrum (for example, into the red wavelength spectrum) with negligible desensitization and DESCRIPTION OF ILLUSTRATIVE slowed turning-on and turning-off kinetics. EMBODIMENTS 0046. In another embodiment, a ChR1 (amino acids 0037 Illustrative embodiments of the invention are 1-345) and ChR2 (amino acids 1-315) hybrid/chimera can described below. In the interest of clarity, not all features of be created and used. an actual implementation are described in this specification. 0047. In yet another embodiment, ChR1-ChR2 hybrids/ It will of course be appreciated that in the development of chimeras retaining the N-terminal portion of ChR1 and any such actual embodiment, numerous implementation replacing the C-terminal portion with the corresponding specific decisions must be made to achieve the developers ChR2 segment can be created and used. In specific embodi specific goals, such as compliance with system-related and ments, hybrids/chimeras of ChR1 and ChR2 can be con business-related constraints, which will vary from one structed and utilized including mutant residues around the US 2016/0361439 A1 Dec. 15, 2016 retinal binding pockets of the chimeras. In exemplary created. In specific embodiments, single or multiple point embodiments, the following chimeras can be created and mutations in the melanopsin protein can result in melanopsin used: variants. 0048 (a). ChD: a hybrid/chimera of a ChR1 N-terminal 0059 Light-sensitive proteins may also include proteins portion and a ChR2 C-terminal portion where the crossover that are at least about 10%, at least about 20%, at least about site is at a point of homology at helixD of the two channel 25%, at least about 30%, at least about 35%, at least about rhodopsins; 40%, at least about 45%, at least about 50%, at least about 0049 (b). ChEF: a hybrid/chimera of a ChR1 N-terminal 55%, at least about 60%, at least about 70%, at least about portion and a ChR2 C-terminal portion where the crossover 80%, at least about 90%, at least about 95%, or at least about site is at the loop between helices E and F of the two 99% identical to the light-sensitive proteins ChR1, ChR2, channelrhodopsins; NpHR, and melanopsin. For example, the ChR2 protein may 0050 (c). ChIEF: a variant of the ChEF chimera with include proteins that are at least about 60%, at least about isoleucine 170 mutated to valine; or 70%, at least about 80%, at least about 90% or at least about 0051 (d). ChF: a hybrid/chimera of a ChR1 N-terminal 95% identical to ChR2. In addition, these proteins may portion and a ChR2 C-terminal portion where the crossover include ChR2 that is photosensitive and can be activated by site is at the end of helix F of the two channelrhodopsins. specific wavelengths of high intensity light. 0.052. In some embodiments, the chimeras retain the 0060. In some embodiments, light-sensitive proteins can reduced inactivation of ChR1 in the presence of persistent modulate signaling within neural circuits and bidirectionally light, but can allow the permeation of sodium and potassium control behavior of ionic conductance at the level of a single ions in addition to protons. In other embodiments, the neuron. In some embodiments, the neuron is a retinal chimeras can improve the kinetics of the channel by enhanc neuron, a retinal bipolar cell (e.g., ON or OFF retinal bipolar ing the rate of the channel closure after stimulation. cells; rod and cone bipolar cells), a retinal ganglion cell, a 0053. In some embodiments, other ChR1 and ChR2 photoreceptor cell, or a retinal amacrine cell. variants can be engineered. In specific embodiments, single 0061 Adeno-Associated Viral Vectors: or multiple point mutations to the ChR2 protein can result in 0062. The present invention provides viral vectors com ChR2 variants. In exemplary embodiments, mutations at the prising nucleic acids encoding a light-sensitive proteins and C128 location of ChR2 can result in altered channel prop methods of use, as described herein. erties. In related embodiments, C128A, C128S, and C128T 0063 Adeno-associated virus (AAV) is a small (25-mm), ChR2 mutations can result in greater overall mean open nonenveloped virus that packages a linear single-stranded times (Berndt, 2009). In other related embodiments, ChR2 DNA genome of 4.7 Kb. The small size of the AAV genome variants can result in altered kinetics. and concerns about potential effects of Rep on the expres 0054. In another embodiment, a VChR1 can be used sion of cellular genes led to the construction of AAV vectors (GenBank accession number EU622855). that do not encode Rep and that lack the cis-active IEE, 0055. In specific embodiments, a mammalian codon which is required for frequent site-specific integration. The optimized version of ChR2 is utilized (FIG. 5) (SEQ ID ITRS are kept because they are the cis signals required for NO:6). packaging. Thus, current recombinant AAV (r.AAV) vectors 0056 NpHR (Halorhodopsin) (GenBank accession num persist primarily as extrachromosomal elements. ber EF474018: FIG. 3) (SEQ ID NO:4) is from the haloal 0064. A variety of recombinant adeno-associated viral kaliphilic archaeon, Natronomonas pharaonis. In certain vectors (ra AV) may be used to deliver genes of interest to embodiments, variants of NpHR can be created. In specific a cell and to effect the expression of a gene of interest, e.g., embodiments single or multiple point mutations to the a gene encoding a light-sensitive protein. For example, NpHR protein can result in NpHR variants. In specific rAAV can be used to express light-sensitive proteins, e.g., embodiments, a mammalian codon optimized version of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 NpHR can be utilized. C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, 0057. In one embodiment, NpHR variants are utilized. In ChIEF, NpHR, eNpHR, melanopsin, and variants thereof or one specific embodiment, eNpHR (enhanced NpHR) is any light-sensitive protein described herein, in a target cell. utilized. Addition of the amino acids FCYENEV (SEQ ID At times herein, “transgene' is used to refer to a polynucle NO: 1) to the NpHR C-terminus along with the signal otide encoding a polypeptide of interest, wherein the poly peptide from the B-subunit of the nicotinic acetylcholine nucleotide is encapsidated in a viral vector (e.g., ra AV). receptor to the NpHR N-terminus results in the construction 0065 Adeno-associated viruses are small, single of eNpHR. stranded DNA viruses, which require helper virus to facili 0058 Melanopsin (GenBank accession number 6693702: tate efficient replication. The 4.7-kb genome of AAV is FIG. 4) (SEQ ID NO:5) is a photopigment found in spe characterized by two inverted terminal repeats (ITR) and cialized photosensitive ganglion cells of the retina that are two open reading frames, which encode the Rep proteins and involved in the regulation of circadian rhythms, pupillary Cap proteins, respectively. The Rep reading frame encodes light reflex, and other non-visual responses to light. In four proteins of molecular weight 78 kDa, 68 kDa, 52 kDa, structure, melanopsin is an opsin, a retinylidene protein and 40 kDa. These proteins function mainly in regulating variety of G-protein-coupled receptor. Melanopsin AAV replication, and rescue and integration of the AAV into resembles invertebrate opsins in many respects, including its a host cell's chromosomes. The Cap reading frame encodes sequence, and downstream signaling cascade. three structural proteins of molecular weight 85 kDa (VP1), Like invertebrate opsins, melanopsin appears to be a bi 72 kDa (VP2), and 61 kDa (VP3) (Berns), which form the stable photopigment, with intrinsic photoisomerase activity. virion capsid. More than 80% of total proteins in AAV virion In certain embodiments, variants of melanopsin can be comprise VP3. US 2016/0361439 A1 Dec. 15, 2016

0066. The genome of ra AV is generally comprised of: (1) examples of suitable inducible promoters include inducible a 5' adeno-associated virus ITR, (2) a coding sequence (e.g., promoters sensitive to an antibiotic, e.g., tetracycline-re transgene) for the desired gene product (e.g., a light-sensi sponsive promoters such as “tet-on' and/or “tet-off pro tive protein) operatively linked to a sequence that regulates moters. Inducible promoters may also include promoters its expression in a cell (e.g., a promoter sequence Such as a sensitive to chemicals other than antibiotics. mGluR6 or fragment thereof), and (3) a 3' adeno-associated 0072 The rAAV vector may also contain additional virus inverted terminal repeat. In addition, the rAAV vector sequences, for example from an adenovirus, which assist in may preferably contain a polyadenylation sequence. effecting a desired function for the vector. Such sequences 0067 Generally, ra AV vectors have one copy of the AAV include, for example, those that assist in packaging the ITR at each end of the transgene or gene of interest, in order rAAV vector into virus particles. to allow replication, packaging, and efficient integration into 0073 Packaging cell lines suitable for producing adeno cell chromosomes. The ITR consists of nucleotides 1 to 145 associated viral vectors may be accomplished given avail at the 5'-end of the AAV DNA genome, and nucleotides 4681 able techniques (see e.g., U.S. Pat. No. 5,872,005). Methods to 4536 (i.e., the same sequence) at the 3'-end of the AAV for constructing and packaging ra A7I vectors are described DNA genome. The rAAV vector may also include at least 10 in, for example, PCT Intl. Pat. Appl. Publ. No. WO nucleotides following the end of the ITR (i.e., a portion of OO/54813. the “D region”). 0074 Flanking the rep and cap open reading frames at the 0068. The transgene sequence (e.g., the polynucleotide 5' and 3' ends are 145-bp inverted terminal repeats (ITRs), encoding a light-sensitive protein) can be of about 2- to 5-kb the first 125 bp of which are capable of forming Y- or in length (or alternatively, the transgene may additionally T-shaped duplex structures. The two ITRs are the only cis contain a “stuffer or “filler” sequence to bring the total size elements essential for AAV replication, rescue, packaging of the nucleic acid sequence between the two ITRs to and integration of the AAV genome. There are two confor between 2 and 5 kb). Alternatively, the transgene may be mations of AAV ITRs called “flip' and “flop.” These dif composed of repeated copies of the same or similar heter ferences in conformation originated from the replication ologous sequence several times (e.g., two nucleic acid model of adeno-associated virus, which uses the ITR to molecules which encode one or more light-sensitive proteins initiate and reinitiate the replication (R. O. Snyder et al., J. separated by a ribosome readthrough, or alternatively, by an Viral., 67:6096-6104; 1993; K. I. Berns, Microbiol. Rev., Internal Ribosome Entry Site or “IRES), or several differ 54:316-329; 1990). The entire rep and cap domains can be ent heterologous sequences (e.g., ChR2 and NpHR sepa excised and replaced with a therapeutic or reporter trans rated by a ribosome readthrough or an IRES; or any two or gene. more of the light-sensitive proteins described herein includ 0075. In some embodiments, self-complementary AAV ing, but not limited to, ChR1, ChR2, VChR1, ChR2C128A, vectors are used. Self-complementary vectors have been ChR2C128S, ChR2C128T, ChR1-ChR2 hybrids/chimeras, developed to circumvent rate-limiting second-strand synthe ChD, ChEF, ChF ChIEF, NpHR, eNpHR, melanopsin, and sis in single-stranded AAV vector genomes and to facilitate variants thereof). robust transgene expression at a minimal dose. In specific 0069. Recombinant AAV vectors of the present invention embodiments, a self-complementary AAV of any serotype or may be generated from a variety of adeno-associated hybrid serotype or mutant serotype, or mutant hybrid sero viruses, including for example, any of serotypes 1 through type increases expression of a light-sensitive protein Such as 12, as described herein. For example, ITRs from any AAV ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 serotype are expected to have similar structures and func C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, tions with regard to replication, integration, excision and ChIEF, NpHR, eNpHR, melanopsin, and variants thereof by transcriptional mechanisms. at least 10%, at least 15%, at least 20%, at least 25%, at least 0070. In some embodiments, a cell-type specific pro 30%, at least 35%, at least 40%, at least 45%, at least 50%, moter (or other regulatory sequence Such as an enhancer) is at least 55%, at least 60%, at least 65%, at least 70%, at least employed to drive expression of a gene of interest, e.g., a 75%, at least 80%, at least 85%, at least 90%, at least 95%, light-sensitive protein, ChR2, etc., in one or more specific at least 100%, at least 125%, at least 150%, at least 175%, cell types. In other cases, within certain embodiments of the at least 200%, or more than 200%, when compared to a invention, expression of the light-sensitive transgene may be non-self-complementary ra AV of the same serotype. accomplished by a separate promoter (e.g., a viral, eukary 0076 Adeno-Associated Viral Serotypes: otic, or other promoter that facilitates expression of an 0077. In one embodiment, the vector comprises a recom operatively linked sequence in a eukaryotic cell, particularly binant AAV of a particular serotype, either naturally occur a mammalian cell). Representative examples of Suitable ring or engineered. promoters in this regard include a mGluR6 promoter, a 0078 AAVs have been found in many animal species, GNA01 promoter, a CBA/smCBA (fusion of the CMV including primates, canine, fowl and human. immediate early enhancer and bovine beta actin promoter 0079 Viral serotypes are strains of microorganisms hav plus introl-exon 1 junction) promoter, CBA promoter ing a set of recognizable antigens in common. There are (chicken B-actin), CMV promoter, RSV promoter, SV40 several known serotypes of AAV, and the efficacy of trans promoter, MoMLV promoter, or derivatives, mutants and/or fection or transduction within the retina may vary as a fragments thereof. Promoters and other regulatory function of the specific serotype and the nature of the target sequences are further described herein. cells. ra AV, or a specific serotype of ra AV or AAV, may 0071. Other promoters that may similarly be utilized provide tissue-specific or cell-type specific tropism for gene within the context of the present invention include cell or delivery to retinal bipolar cells (e.g., ON or OFF retinal tissue specific promoters (e.g., a rod, cone, or ganglia bipolar cells; rod and cone bipolar cells). While ra AV and/or derived promoter), or inducible promoters. Representative AAV are relatively-safe vectors for delivering a transgene to US 2016/0361439 A1 Dec. 15, 2016

a target tissue, the efficacy of delivery, and possibly the 40%, 50%, 60%, 70%, 80%, or 90% of cells of a particular safety of delivery, may depend on the coat proteins of the cell type, e.g., retinal bipolar cells (e.g., ON or OFF retinal AAV. The protein coat, or capsid, determines which cells can bipolar cells; rod and cone bipolar cells), or of cells within take up the viral payload. Different AAV serotypes, i.e., a particular tissue, e.g., retinal tissue. viruses that differ in their protein coats or capsids, may differ I0083. There is a need in the art for AAV serotypes that in their tissue tropism, and/or their ability to transduce can effectively transduce retinal bipolar cells (e.g., ON or targeted cells. Transgenes can be packaged within AAV OFF retinal bipolar cells; rod and cone bipolar cells), particles with many functionally different coat proteins, or particularly when such transduction enables the delivery and capsids. These different capsids are what define the serotype expression of a light-sensitive protein, e.g., ChR2. An and may contribute (either entirely or in part) to its ability to important therapy to treat eye disorders or diseases (e.g., transduce particular cell types. The entry of the viral vector visual impairment, blindness), may involve using a particu begins with the interaction of the capsid and the target cell lar AAV serotype to express light-sensitive proteins such as surface proteins. Without wishing to be bound by theory, it ChR2 in retinal bipolar cells (e.g., ON or OFF retinal bipolar is at this point in the transduction pathway that different cells; rod and cone bipolar cells). For example, in a preferred serotypes may significantly influence the efficiency of trans embodiment, AAV5 or AAV7 serotypes are used to target gene delivery. expression of a gene of interest (e.g., a light-sensitive 0080. In certain embodiments, the AAV vector is of a protein, ChR2, etc.) in retinal cells, e.g., retinal bipolar cells serotype or variant/mutant thereof including, but not limited (e.g., ON or OFF retinal bipolar cells; rod and cone bipolar to: AAV1 (GenBank accession number AY724675), AAV2 cells). In some cases, AAV5 and/or AAV7 may more effi (GenBank accession number AF043303), AAV3, AAV4, ciently transduce retinal cells, e.g., retinal bipolar cells (e.g., AAV5 (GenBank accession number M61166), AAV6, AAV7 ON or OFF retinal bipolar cells; rod and cone bipolar cells), (GenBank accession number AF513851), AAV8 (GenBank than other AAV serotypes. For example, in some embodi accession number AF513852), AAV9 (GenBank accession ments, the AAV5 and/or AAV7 serotypes, but not AAV1 number AX753250), AAV10, AAV11 (GenBank accession serotype, are used to transduce retinal bipolar cells (e.g., ON number AY 631966), or AAV12 (GenBank accession number or OFF retinal bipolar cells; rod and cone bipolar cells). In DQ813647), or mutants, hybrids, or fragments thereof. In other cases, AAV2 and/or AAV8 serotypes are used to certain embodiments the AAV vector comprises one or transduce retinal bipolar cells (e.g., ON or OFF retinal more, two or more, three or more, four or more, or five or bipolar cells; rod and cone bipolar cells). In some cases, a more of the following serotypes: AAV1 (GenBank accession specific serotype, e.g., AAV2, AAV5, AAV7 or AAV8, may number AY724675), AAV2 (GenBank accession number be generally applied to a tissue, e.g., retinal tissue, but then AF043303), AAV3, AAV4, AAV5 (GenBank accession preferentially transduces a specific cell-type over another number M61166), AAV6, AAV7 (GenBank accession num cell type. ber AF513851), AAV8 (GenBank accession number I0084. In some cases, an AAV, e.g., AAV2, AAV5, AAV7, AF513852), AAV9 (GenBank accession number or AAV8 that is introduced to the retina may preferentially AX753250), AAV10, AAV11 (GenBank accession number transduce retinal bipolar cells (e.g., ON or OFF retinal AY 631966), or AAV12 (GenBank accession number bipolar cells; rod and cone bipolar cells) so that the trans DQ813647), or mutants, hybrids, or fragments thereof. In gene is expressed more highly in retinal bipolar cells com other embodiments, the AAV vector is of a natural serotype pared to other retinal cells. In some cases, the particular or variant/mutant thereof, heretofore yet undiscovered and serotype e.g., AAV2, AAV5, AAV7 or AAV8, of the AAV uncharacterized. may be the cause or contribute to the cause of such prefer 0081. In certain embodiments, the recombinant AAV is of ential transduction. In some cases, only a small Subset of the a combinatorial hybrid of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, bipolar cells is transduced. 14, 15 or more serotypes or mutants thereof. I0085. In some embodiments, a specific serotype of AAV. 0082 In some embodiments, the AAV vector may be e.g., AAV5 and/or AAV7 (or any other AAV serotype or used to specifically transduce a specific cell type, e.g., retinal mutant described herein) comprising a non-cell-type-spe cells or retinal bipolar cells (e.g., ON or OFF retinal bipolar cific promoter is used to drive expression of a light-sensitive cells; rod and cone bipolar cells). In some cases, a specific protein in a particular cell type. In some cases, a specific serotype, e.g., AAV2, AAV5, AAV7 or AAV8, may be better serotype of AAV that has been demonstrated to preferen than other serotypes at transducing a particular cell type tially transduce a particular cell type is used along with a e.g., retinal bipolar cells (e.g., ON or OFF retinal bipolar cell-type specific promoter to drive expression of a protein cells; rod and cone bipolar cells), neurons or tissue. For of interest, e.g., a light-sensitive protein, in a specific cell example, a specific AAV serotype such as AAV2, AAV5, type. AAV7 or AAV8 may transduce a specific cell type, e.g., I0086. The AAV ITR sequences and other AAV sequences retinal bipolar cells (e.g., ON or OFF retinal bipolar cells; employed in generating the minigenes, vectors, and capsids, rod and cone bipolar cells), with an increased transduction and other constructs used in certain embodiments may be efficiency of at least 10%, at least 15%, at least 20%, at least obtained from a variety of sources. For example, the 25%, at least 30%, at least 35%, at least 40%, at least 45%, sequences may be provided by presently-identified human at least 50%, at least 55%, at least 60%, at least 65%, at least AAV types or by AAV serotypes as-yet-to-be-identified. 70%, at least 75%, at least 80%, at least 85%, at least 90%, Similarly, AAVs known to infect other may also at least 95%, at least 100%, at least 125%, at least 150%, at provide the ITRs employed in the molecules or constructs of least 175%, at least 200%, or more than 200%, when this invention. Similarly, the capsids from a variety of compared to a different AAV serotype. In some cases, a serotypes of AAV may be “mixed-and-matched with the specific serotype e.g., AAV2, AAV5, AAV7 or AAV8, may other vector components (see, e.g., PCT Intl. Pat. Appl. Publ. permit transduction of at least 2%. 5%, 10%, 20%, 30%, No. WO2001/83692; published Nov. 8, 2001, and incorpo US 2016/0361439 A1 Dec. 15, 2016 rated herein by reference). A variety of these viral serotypes As described herein, codon-optimization of the polynucle and strains are available from the American TypeCulture otide encoding the light-sensitive protein may also improve Collection (Manassas, Va., USA), or are available from a transduction efficiency. variety of academic or commercial sources. Alternatively, it 0092. The ubiquitin-proteasome pathway plays a role in may be desirable to synthesize sequences used in preparing AAV-intracellular trafficking. Substitution of surface the vectors and viruses of the invention using known tech exposed tyrosine residues on, for example, AAV2 or AAV8 niques, which may utilize AAV sequences that are published capsids permits the vectors to either have limited ubiquitina and/or available from a variety of databases. tion or to escape ubiquitination altogether. The reduction in, 0087 Adeno-Associated Viruses & Mutations of Sur or absence of ubiquitination may help prevent the capsid face-Exposed Residues from undergoing proteasome-mediated degradation. AAV or 0088 Recombinant adeno-associated virus (ra AV) vec rAAV capsids can be phosphorylated at tyrosine residues by tors are in use in several clinical trials, but relatively large EGFR-PTK in an in vitro phosphorylation assay, and the vector doses are needed to achieve therapeutic benefits. phosphorylated AAV capsids retain their structural integrity. Large vector doses may also trigger an immune response as Although phosphorylated AAV vectors may enter cells as a significant fraction of the vectors may fail to traffic efficiently as their unphosphorylated counterparts, their efficiently to the nucleus and may be targeted for degrada transduction efficiency may be significantly impaired. tion by the host cell proteasome machinery. It has been 0093. In some cases, a recombinant adeno-associated reported that epidermal growth factor receptor protein tyro viral (ra AV) vector comprises a capsid protein with a sine kinase (EGFR-PTK) signaling negatively affects trans mutated tyrosine residue which enables to the vector to have duction by AAV serotype 2 vectors by impairing nuclear improved transduction efficiency of a target cell, e.g., a transport of the vectors (Zhong, 2007). Tyrosine-phospho retinal bipolar cell (e.g., ON or OFF retinal bipolar cells; rod rylated AAV2 vectors enter, but do not transduce effectively, and cone bipolar cells). In some cases, the rAAV vector in part because of the ubiquitination of AAV capsids fol further comprises a promoter (e.g., mGluR6, or a fragment lowed by proteasome-mediated degradation. Point muta thereof) capable of driving the expression of a protein of tions in tyrosine residues in AAV2 lead to high-efficiency interest in the target cell. transduction at lower virus titers (Zhong, 2008). 0094. In some cases, expression in a specific cell type is 0089. In one embodiment, tyrosine-mutated AAVs (e.g., further achieved by including a cell-type specific promoter AAV2 or AAV8) are used in order to improve the efficiency described herein within the rAAV vector. of transduction of retinal cells, e.g., retinal bipolar cells 0.095. In one embodiment, a recombinant adeno-associ (FIG. 9A through FIG. 12C). In one embodiment, mutations ated viral (ra AV) vector comprises at least a first capsid of the surface-exposed tyrosine residues of the rAAV capsid protein comprising at least a first phosphorylated tyrosine allow the vectors to evade phosphorylation and Subsequent amino acid residue, and at least a first nucleic acid segment ubiquitination and, thus, prevent proteasome-mediated deg that encodes a light-sensitive protein operably linked to a radation, leading to greater transduction and Subsequent promoter capable of expressing the segment in a host cell. gene expression of light-sensitive proteins. 0096. In one embodiment, a mutation may be made in 0090. In a related embodiment, any one or more surface any one or more of tyrosine residues of the capsid protein of exposed residues other than tyrosine may be mutated to AAV1 to AAV12, or in one or more hybrid AAVs. In specific improve the transduction efficiency, tissue/cell-type tropism, embodiments, these are surface-exposed tyrosine residues. expression characteristics, and titers needed for effective In a related embodiment, the tyrosine residues are part of the infection. VP1,VP2, or VP3 capsid protein. In exemplary embodi 0091. As described herein, modification and changes to ments, the mutation may be made at one or more of the the structure of the polynucleotides and polypeptides of following amino acid residues of an AAV-VP3 capsid pro wild-type ra AV vectors may result in improved ra AV tein: Tyr252, Tyr272, Tyr444, TyrS00, Tyr700, Tyr704, virions possessing desirable characteristics. For example, Tyr730; Tyr275, Tyr281, TyrS08, TyrS76, Tyró12, Tyró73 or mutated raAV vectors may improve delivery of light-sen Tyr720. Exemplary mutations are tyrosine-to-phenylalanine sitive gene constructs to selected mammalian cell, tissues, mutations including, but not limited to, Y252F, Y272F, and organs for the treatment, prevention, and prophylaxis of Y444F, Y500F, Y700F, Y704F, Y73OF, Y275F, Y281F, various diseases and disorders. Such approach may also Y508F, Y576F, Y612G, Y673F and Y720F. In a specific provide a means for the amelioration of symptoms of Such embodiment, these mutations are made in the AAV2 sero diseases, and to facilitate the expression of exogenous type. In some cases, an AAV2 serotype comprises a Y444F therapeutic and/or prophylactic polypeptides of interest via mutation and/or an AAV8 serotype comprises a Y733F rAAV vector-mediated gene therapy. The mutated ra AV mutation, wherein 444 and 733 indicate the location of a vectors may encode one or more proteins, e.g., the light point tyrosine mutation of the viral capsid. In further sensitive proteins, e.g., ChR2, described herein. The creation embodiments, such mutated AAV2 and AAV8 serotypes (or insertion) of one or more mutations into specific poly encode a light-sensitive protein, e.g., ChR2, and may also nucleotide sequences that encode one or more of the light comprise a regulatory sequence (e.g., mGluR6) to drive sensitive proteins encoded by the disclosed ra AV constructs expression of Such light-sensitive protein. are provided herein. In certain circumstances, the resulting 0097. In a related embodiment, 1, 2, 3, 4, 5, 6, or 7 light-sensitive polypeptide sequence is altered by these mutations are made to the tyrosine residue on an AAV1, 2, mutations, or in other cases, the sequence of the polypeptide 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or hybrid serotype. In one is unchanged by one or more mutations in the encoding exemplary embodiment, 3 tyrosines are mutated to create an polynucleotide to produce modified vectors with improved AAV serotype with a triple-mutation consisting of: Y444F, properties for effecting gene therapy in mammalian systems. Y500F, and Y730F. US 2016/0361439 A1 Dec. 15, 2016

0098. The rAAV vectors of the present invention may be ChEF, ChF ChIEF, NpHR, eNpHR, melanopsin, and vari comprised within an adeno-associated viral particle or infec ants thereof or any light-sensitive protein in a cell of the eye, tious ra AV virion, including for example, virions selected particularly within a retinal cell, more particularly within a from the group consisting of an AAV serotype 1, an AAV non-photoreceptor cell e.g., amacrine cells, retinal ganglion serotype 2, an AAV serotype 3, an AAV serotype 4, an AAV cells, retinal bipolar cells, (ON or OFF retinal bipolar cells; serotype 5 and an AAV serotype 6, an AAV serotype 7, an rod and cone bipolar cells), are within the scope of the AAV serotype 8, an AAV serotype 9, an AAV serotype 10, present invention. Gene delivery vectors can be viral (e.g., an AAV serotype 11, an AAV serotype 12, or a hybrid AAV derived from or containing sequences of viral DNA or RNA, serotype. preferably packaged within a viral particle), or non-viral 0099. The rAAV vectors of the present invention may (e.g., not packaged within a viral particle, including "naked' also be comprised within an isolated mammalian host cell, polynucleotides, nucleic acid associated with a carrier par including for example, human, primate, murine, feline, ticle Such as a liposome or targeting molecule, and the like). canine, porcine, ovine, bovine, equine, epine, caprine, and Other exemplary gene delivery vectors are described below. lupine host cells. The rAAV vectors may be comprised 0104 Recombinant Adenoviral Vectors (AD): within an isolated mammalian host cell Such as a human 0105. In other embodiments, the gene delivery vector is endothelial, epithelial, vascular, liver, lung, heart, pancreas, a recombinant adenoviral vector. U.S. Pat. No. 6,245.330 intestinal, kidney, muscle, bone, neural, blood, or brain cell. describes recombinant adenoviruses, which may be suitable 0100. In certain embodiments, the transduction efficiency for use in the invention. Ad vectors do not integrate into the of an AAV comprising a mutated capsid protein (e.g., a host cell genome, particularly preferred when short-term mutation of a tyrosine residue described herein) expressing gene is required, typically about 14 days. Thus, use of Ad a light-sensitive protein such as ChR1, ChR2. VChR1, ChR2 vectors can require repeated intraocular injections to treat a C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/ retinal disease, which continues over decades in the average chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, mel patient. anopsin, and variants thereof is increased by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 0106 The viral tropism of Ad and AAV in the retina is 35%, at least 40%, at least 45%, at least 50%, at least 55%, can be different. The subset of cells that are transduced by at least 60%, at least 65%, at least 70%, at least 75%, at least the vector is usually a receptor-mediated event. Ad vectors 80%, at least 85%, at least 90%, at least 95%, at least 100%, have been shown to primarily transduce retinal Muller cells at least 125%, at least 150%, at least 175%, at least 200%, and Retinal pigment epithelial cells following injection. or more than 200%, when compared to a wild-type AAV AAV vectors are very efficient at transferring their genetic expressing a light-sensitive protein. This disclosure also payload to retinal photoreceptor and non-photoreceptor cells provides mutated ra AV vectors (e.g., the AAV2 Y444F when injected into the eye. vector or the AAV8 Y733F vector) capable of transducing at 01.07 Retroviral Gene Delivery Vectors: least 2%. 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 0108. The gene delivery vectors of the invention can be or 90% of (ON or OFF retinal bipolar cells; rod and cone a retroviral gene delivery vector adapted to express a bipolar cells) bipolar cells. The improvement in transduction selected gene (S) or sequence (S) of interest (e.g., ChR1, created by the mutated capsid may permit transduction of ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2C128T, bipolar cells by intravitreal injection. For example, in some ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, embodiments, a mutated ra AV vector or a ra AV combina NpHR, eNpHR, melanopsin, and variants thereof). Retro torial serotype hybrid vector or a mutated combinatorial viral gene delivery vectors of the present invention may be serotype hybridra AV vector may be capable of transducing readily constructed from a wide variety of retroviruses, at least 2%. 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, including for example, B, C, and D type retroviruses as well 80%, or 90% of retinal bipolar cells (e.g., ON or OFF retinal as spumaviruses and lentiviruses. For example, in some bipolar cells; rod and cone bipolar cells) is introduced to the cases, a retrovirus, e.g., a lentivirus, is pseudotyped with an retina by intravitreal injection. In a specific embodiment, envelope protein or other viral protein to facilitate entry into only a subset of the retinal bipolar cells is transduced. In target cells. In some cases, a lentivirus is pseudotyped with another specific embodiment, only the highly sensitive bipo vesicular-stomatitis virus . (see, e.g., RNA Tumor lar cells are transduced. Viruses, Second Edition, Cold Spring Harbor Laboratory 0101. In certain embodiments the ubiquitin or protea Press, Cold Spring Harbor, N.Y., USA; 1985). Such retro some-mediated degradation of an AAV comprising a capsid viruses may be readily obtained from depositories or col protein with a mutation expressing a light-sensitive protein, lections such as the American TypeCulture Collection such as ChR1, ChR2, VChR1, ChR2C128A, ChR2 C128S, (“ATCC), or isolated from known provided herein, and ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, standard recombinant techniques (e.g., Sambrook et al., ChF. ChIEF, NpHR, eNpHR, melanopsin, and variants Molecular Cloning: A Laboratory Manual 2" ed., Cold thereof, is decreased by at least 10%, at least 15%, at least Spring Harbor Laboratory Press, 1989; Kunkel, Proc. Natl. 20%, at least 25%, at least 30%, at least 35%, at least 40%, Acad. Sci. USA, 82(2):488-492: 1985). at least 45%, at least 50%, at least 55%, at least 60%, at least 0109. In addition, within certain embodiments of the 65%, at least 70%, at least 75%, at least 80%, at least 85%, invention, portions of the retroviral gene delivery vectors or at least 90%, when compared to a wild-type AAV may be derived from different retroviruses. For example, expressing a light-sensitive protein. within one embodiment of the invention, retrovirus LTRs 0102). Other Gene Delivery Vectors: may be derived from a Murine Sarcoma Virus, a tRNA 0103) Any of a variety of other vectors adapted for binding site from a Rous Sarcoma Virus, a packaging signal expression of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 from a Murine Leukemia Virus, and an origin of second C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, Strand synthesis from an Avian Leukosis Virus. US 2016/0361439 A1 Dec. 15, 2016

0110. Within one aspect of the present invention, retro containing liposomes (e.g., PCT Intl. Pat. Appl. Publ. Nos. viral vector constructs are provided comprising a 5'-LTR, a WO95/24929 and WO95/12387), and certain eukaryotic tRNA binding site, a packaging signal, one or more heter cells. ologous sequences, an origin of second strand DNA synthe 0119 Regulatory Sequences: sis and a 3'-LTR, wherein the vector construct lacks gag, pol, 0120 In some embodiments, regulatory sequences or or env coding sequences. elements are utilized to allow for cell-type or tissue-type 0111. Other retroviral gene delivery vectors may likewise specific targeting of ChR1, ChR2, VChR1, ChR2 C128A, be utilized within the context of the present invention, and ChR2C128S, ChR2C128T, ChR1-ChR2 hybrids/chimeras, are well known in the art. Packaging cell lines suitable for ChD, ChEF, ChF ChIEF, NpHR, eNpHR, melanopsin, and use with the above-described retroviral vector constructs can variants thereof. In related embodiments, regulatory ele be readily prepared according to methods well known in the ments are used to specifically target retinal neurons, or art, and utilized to create producer cell lines for the produc retinal bipolar cells (e.g., ON or OFF retinal bipolar cells; tion of recombinant vector particles. rod and cone bipolar cells), or retinal ganglion cells, or 0112 Alphavirus Delivery Vectors: photoreceptor cells, or amacrine cells. Examples of regula 0113 Gene delivery vectors suitable for use in the inven tory sequences or elements include, but are not limited to tion can also be based upon alphavirus vectors. For example, promoter, silencer, enhancer, and insulator sequences. the Sindbis virus is the prototype member of the alphavirus 0121. In some embodiments, regulatory sequences such genus of the togavirus family. The unsegmented genomic as promoters Suitable for use in the present invention include RNA (49S RNA) of Sindbis virus is approximately 11,703 constitutive promoters, strong promoters (e.g., CMV pro nucleotides in length, contains a 5' cap and a 3' poly moters), inducible promoters, and tissue-specific or cell adenylated tail, and displays positive polarity. Infectious specific promoters (e.g., promoters that preferentially facili enveloped Sindbis virus is produced by assembly of the viral tate expression in a limited number of tissues or cell types nucleocapsid proteins onto the viral genomic RNA in the (e.g., eye tissues, retina, retinal cells, photoreceptor cells, cytoplasm and budding through the cell membrane embed and the like). ded with viral encoded glycoproteins. Entry of virus into 0.122 Any of a variety of regulatory sequences can be cells is by endocytosis through clatharin-coated pits, fusion used in the gene delivery vectors of the invention to provide of the viral membrane with the endosome, release of the for a suitable level or pattern of expression of the light nucleocapsid, and uncoating of the viral genome. During sensitive protein of interest. The regulatory sequences are viral replication, the genomic 49S RNA serves as template generally derived from eukaryotic regulatory sequences. for synthesis of the complementary negative strand. This I0123. In some embodiments, non-cell specific regulatory negative strand in turn serves as template for genomic RNA elements are used. In one embodiment, the promoter com and an internally initiated 26S subgenomic RNA. prises (from 5'-to-3') a viral enhancer (a CMV immediate 0114. The Sindbis viral nonstructural proteins are trans early enhancer), and a B-actin promoter (a bovine or chicken lated from the genomic RNA while structural proteins are B-actin promoter-exon 1-intron 1 element). In a specific translated from the subgenomic 26S RNA. All viral genes embodiment, the promoter comprises (from 5'-to-3') CMV are expressed as a polyprotein and processed into individual immediate early enhancer (381 bp)/bovine or chicken B-ac proteins by post-translational proteolytic cleavage. The tin (CBA) promoter-exon 1-intron 1 (1352 bp) element, packaging sequence resides within the nonstructural coding which together are termed herein the "CBA promoter” (FIG. region, therefore only the genomic 49S RNA is packaged 7). In some embodiments a nucleic acid encoding a light sensitive protein is delivered to a cell using a viral vector into virions. Such as AAV carrying a selected light-sensitive transgene 0115 Several different Sindbis vector systems may be encoding DNA regulated by a non-cell-specific promoter constructed and utilized within the present invention. Rep and/or other regulatory sequences that expresses the product resentative examples of Such systems include those of the DNA. In some related embodiments, the non-cell described within U.S. Pat. Nos. 5,091,309 and 5,217,879, specific promoter is general promoter Such as a ubiquitin and PCT Intl. Pat. Appl. Publ. No. WO95/07994. based promoter, for e.g., a ubiquitin C promoter. 0116. Other viral gene delivery vectors. In addition to 0.124. In other embodiments, a light-sensitive protein is retroviral vectors and alphavirus vectors, numerous other delivered to a cell type or tissue type of interest using a viral viral vectors systems may also be utilized as a gene delivery vector Such as AAV carrying a selected light-sensitive trans vector. Representative examples of Such gene delivery vec gene-encoding DNA regulated by a promoter and/or other tors include viruses such as pox viruses, such as canary pox regulatory sequences that expresses the product of the DNA virus or vaccinia virus. in selected retinal cells of a subject. In specific embodi 0117 Non-Viral Gene Delivery Vectors: ments, expression is targeted to particular types of cells 0118. In addition to the above viral-based vectors, numer within the retina through the use of a specific promoter ous non-viral gene delivery vectors may likewise be utilized nucleotide sequence and/or other regulatory regions such as within the context of the present invention. Representative silencer, enhancer, or insulator sequences which are engi examples of such gene delivery vectors include direct deliv neered into the vector. In some embodiments, different ery of nucleic acid expression vectors, naked DNA (e.g., regulatory sequences are used to drive expression of differ DNA not contained in a viral vector) (PCT Intl. Pat. Appl. ent engineered genes in different populations of cells. Publ. No. WO 90/11092), polycation condensed DNA linked 0.125. In other embodiments, retinal bipolar cell-specific or unlined to killed adenovirus (Curiel et al., Hum. Gene regulatory sequences such as promoter, enhancer, silencer, Ther, 3:147-154; 1992), DNA ligand linked to a ligand with and insulator sequences are used. In specific embodiments, or without one of the high affinity pairs described above (Wu the ON bipolar cells are targeted. In other embodiments, the et al., J. Biol. Chem., 264:16985-16987: 1989), nucleic acid OFF bipolar cells are targeted. In other embodiments, the US 2016/0361439 A1 Dec. 15, 2016

rode bipolar cells are targeted. In other embodiments, the system of amacrine cells (Bi, 2006; Greenberg, 2007; cone bipolar cells are targeted. Tomita, 2007). These groups have reported very few behav 0126. In one embodiment, specific expression of a light ioral changes. sensitive proteins in ON bipolar cells is targeted using a 0.132. The majority of retinal cells are either ON-center light-sensitive protein such as ChR1, ChR2. VChR1, ChR2 (increased firing rate as a result of a step increase in contrast C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/ within the center of the receptive field) or OFF center type chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, mel (increased firing rate as a result of a step decrease in contrast anopsin, and variants thereof operatively linked to a GRM6 in the center of the receptive field), working in a push-pull (metabotropic glutamate receptor 6, mGluR6) regulatory inhibitory fashion (Wassle, 2004). In order to maintain this sequence or a fragment thereof. In one embodiment, the relationship between the two pathways, these two pathways full-length mCluR6 regulatory sequence is utilized. can be driven independently. ON and OFF channels of 0127. In another embodiment, specific expression of a information traveling from bipolar to ganglion cells are light sensitive proteins in ON bipolar cells is targeted using partially modulated through a network of inhibitory ama a light-sensitive protein such as ChR1, ChR2. VChR1, ChR2 crine cells within the inner nuclear layer (Roska, 2001). This C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/ bipolar-amacrine network produces temporally-distinct par chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, mel allel channels of information: Sustained-activity neurons, for anopsin, and variants thereof operatively linked to a example, maintain activity throughout the light step, mGluR6 regulatory sequence fragment. In a specific whereas transient-activity neurons have activity only at the embodiment, the mGluR6 regulatory sequence fragment is onset or offset. These distinct patterns of response code for the sequence presented in FIG. 6. In a related embodiment, visual information luminance, shape, edges, and motion the mGluR6 regulatory sequence fragment is substantially (Wassle, 2004). In some embodiments, cells that are pre the same as the sequence presented in FIG. 6, or is about synaptic to the retinal ganglion cells are genetically targeted 60% identical, or is about 70% identical, or is about 80% to maintain the naturalism of these pathways and elicit identical, or is about 90% identical, or is about 95% identical naturalistic ganglion cell spiking. to the sequence presented in FIG. 6. I0133. In some embodiments, retinal bipolar cells (e.g., 0128 Many known promoters are too large to fit into the ON or OFF retinal bipolar cells; rod and cone bipolar cells) genome of the AAV. Indeed, the original cell-specific regu are genetically targeted. Targeting retinal bipolar cells may latory sequence for mGluR6 (Dhingra, 2008) was far too allow the retina to respond to external light and, more large (approximately 10.5 Kb) to be used in AAV. In a importantly, convey meaningful image information to the preferred embodiment of the current invention, a mGluR6 brain even in the absence of natural photoreceptors. regulatory sequence fragment is used, one that is Small 0.134 Conditions Amenable to Treatment enough to be used in AAV-mediated delivery. In related I0135) In some embodiments, the present invention pro embodiments the mGluR6 regulatory sequence fragment is vides methods of treating a subject Suffering from a disease less than about 2000 base pairs, less than about 1000 base or disorder. The compositions and methods described herein pairs, less than about 750 base pairs, less than about 500 can be utilized to treat central and peripheral nervous system base pairs, less than about 250 base pairs, or less than about diseases and disorders. 100 base pairs in length. In another related embodiment, the 0.136. In one aspect, the compositions and methods of this mGluR6 regulatory sequence fragment is a variant of the invention are utilized to treat photoreceptor diseases. Pho sequence presented in FIG. 6. toreceptor diseases such as retinitis pigmentosa (RP) and 0129. In another embodiment, specific expression of a age-related macular degeneration (ARMD) cause blindness light sensitive proteins in ON bipolar cells is targeted using (Congdon, 2004) in 15 million people worldwide (Chader, a light-sensitive protein such as ChR1, ChR2. VChR1, ChR2 2002), a number that is increasing with the age of the C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/ population. There have been attempts to restore basic visual chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, mel function through gene replacement therapy or cellular trans anopsin, and variants thereof operatively linked to a GNA01 plantation (Acland, 2001, Acland, 2005, Batten, 2005, (guanine nucleotide binding protein (G protein), alpha acti Pawlyk, 2005, Aguirre, 2007, MacLaren, 2006). Vating activity polypeptide 0) regulatory sequence. In a 0.137 However, current approaches are fundamentally limited in scope and extent of potential impact, as they related embodiment the regulatory sequence is substantially attempt to correct mechanistically distinct genetic pathways the same as GNA01 sequence, or is about 60% identical, or on a one-at-a-time basis (Punzo, 2007). Photoreceptor dis is about 70% identical, or is about 80% identical, or is about eases are genetically diverse, with over 160 different muta 90% identical, or is about 95% identical to the GNA01 tions leading to degeneration (Punzo, 2007). There have also Sequence. been efforts in utilizing electrical stimulation with implanted 0130 Retinal Bipolar Cells and Targeting acute, semi-acute, and long-term retinal prostheses in human 0131 Although only a fraction of the human visual subjects (de Balthasar, 2008; Horsager, 2009). They have system, the retina is a complex system that filters, amplifies, shown elementary progress, but are gene-nonspecific; elec and modulates the visual signal before it is sent to the rest trical stimulation offers only gross specificity and indis of the visual system (Wassle, 2004). The vast majority of this criminately drives visual information channels mediated by processing happens within the inner plexiform layer (IPL) unique cell types. Activating retinal neurons requires large where a system of bipolar and amacrine cells refine the disc electrodes (at least 20 times the diameter of a retinal visual signal into its primary components (e.g., motion, ganglion cell), leading to stimulation of broad areas of retina contrast, resolution) (Mills, 1999; Roska, 2001). Some in a nonselective fashion, greatly limiting the achievable groups are currently targeting ChR2 to the ganglion cell visual resolution (Winter, 2007). In this aspect, the compo layer, which bypasses the processing power of the IPL and sitions and methods of this invention consist of introducing US 2016/0361439 A1 Dec. 15, 2016 a gene encoding a light-sensitive protein (e.g., ChR1, ChR2, necessarily limited to, Bardet-Biedl syndrome (autosomal VChR1, ChR2C128A, ChR2 C128S, ChR2 C128T, ChR1 recessive); congenital amaurosis (autosomal recessive); ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, cone or cone-rod dystrophy (autosomal dominant and eNpHR, melanopsin, and variants thereof) to induce light X-linked forms); congenital stationary night blindness (au sensitivity in 2" order neurons (e.g., bipolar cells) delivered tosomal dominant, autosomal recessive and X-linked using a viral vector Such as an AAV8 with a single tyrosine forms); macular degeneration (autosomal dominant and to phenylalanine mutation, under the control of a regulatory autosomal recessive forms); optic atrophy (autosomal domi element (e.g., GRM6). The activation of these light-sensitive nant and X-linked forms); retinitis pigmentosa (autosomal proteins could be controlled by ambient light or through a dominant, autosomal recessive and X-linked forms); syn light-delivery device Such as the goggles described in FIG. dromic or systemic retinopathy (autosomal dominant, auto 13. Somal recessive and X-linked forms); and Usher syndrome 0138. The methods of the invention can be used to treat (autosomal recessive). (e.g., prior to or after the onset of symptoms) in a susceptible 0144. In another aspect, the compositions and methods of Subject or Subject diagnosed with a variety of eye diseases. this invention are utilized to treat peripheral injury, nocice The eye disease may be a result of the environment (e.g., ption, or chronic pain. Nociception (pain) for prolonged chemical insult, thermal insult, and the like), mechanical periods of time can give rise to chronic pain and may arise insult (e.g., injury due to accident or Surgery), or genetic from injury or disease to visceral, Somatic and neural factors. The Subject having the condition may have one or structures in the body. Although the range of pharmacologi both eyes affected, and therapy may be administered accord cal treatments for neuropathic pain has improved over the ing to the invention to the affected eye or to an eye at risk past decade, many patients do not get effective analgesia, of photoreceptor degeneration due to the presence of Such a and even effective medications often produce undesirable condition in the subject's other, affected eye. side effects. Substance P (SP) is involved in nociception, 0.139. The present invention provides methods which transmitting information about tissue damage from periph generally comprise the step of intraocularly administering eral receptors to the central nervous system to be converted (e.g., by Subretinal injection or by intravitreal injection) a to the sensation of pain. It has been theorized that it plays a gene delivery vector which directs the expression of a part in fibromyalgia A role of Substance P in nociception is light-sensitive protein to the eye to treat, prevent, or inhibit Suggested by the reduction in response thresholds to noxious the onset or progression of an eye disease. As utilized herein, stimuli by central administration of NK1 and NK2 agonists. it should be understood that the terms “treated, prevented, or, Pain behaviors induced by mechanical, thermal and chemi inhibited’ refers to the alteration of a disease onset, course, cal stimulation of Somatic and visceral tissues were reduced or progress in a statistically significant manner. in the mutant mice lacking SP/NKA. In one embodiment, 0140. Another condition amenable to treatment accord light-sensitive proteins can silence the activity of over-active ing to the invention is Age-related Macular Degeneration neurons (i.e., Substance P expressing peripheral neurons) (AMD). The macula is a structure near the center of the due to peripheral injury or chronic pain using NpHR or retina that contains the fovea. This specialized portion of the eNpHR. NpHR/eNpHR can be genetically targeted to sub retina is responsible for the high-resolution vision that stance P expressing cells using the Substance P promoter permits activities such as reading. The loss of central vision sequence. In another embodiment, light-sensitive proteins in AMD is devastating. Degenerative changes to the macula enhance the activity of neurons that are inactive due to (maculopathy) can occur at almost any time in life but are peripheral injury or chronic pain. much more prevalent with advancing age. Conventional 0145. In another aspect, the compositions and methods of treatments are short-lived, due to recurrent choroidal neo this invention are utilized to treat spinal cord injury and/or vascularization. AMD has two primary pathologic pro motor neuron diseases. Spinal cord injury can cause cesses, choroidal neovascularization (CNV) and macular myelopathy or damage to white matter and myelinated fiber photoreceptor cell death. tracts that carry sensation and motor signals to and from the 01.41 Exemplary conditions of particular interest which brain. It can also damage gray matter in the central part of are amenable to treatment according to the methods of the the spine, causing segmental losses of interneurons and invention include, but are not necessarily limited to, retinitis motor neurons. Spinal cord injury can occur from many pigmentosa (RP), diabetic retinopathy, and glaucoma, causes, including but not limited to trauma, tumors, isch including open-angle glaucoma (e.g., primary open-angle emia, abnormal development, neurodevelopmental, neuro glaucoma), angle-closure glaucoma, and secondary glauco degenerative disorders or vascular malformations. In one mas (e.g., pigmentary glaucoma, pseudoexfoliative glau embodiment, light-sensitive proteins activate damaged neu coma, and glaucomas resulting from trauma and inflamma ral circuits to restore motor or sensory function. In one tory diseases). specific embodiment, the elements act to allow control of 0142 Further exemplary conditions amenable to treat autonomic and visceral functions. In other embodiments, the ment according to the invention include, but are not neces elements act to allow control of Somatic skeletal function. sarily limited to, retinal detachment, age-related or other The neural control of storage and Voiding of urine is maculopathies, photic retinopathies, Surgery-induced retin complex and dysfunction can be difficult to treat. One opathies, toxic retinopathies, retinopathy of prematurity, treatment for people with refractory symptoms is continuous retinopathies due to trauma or penetrating lesions of the eye, electrical nerve stimulation of the sacral nerve roots using inherited retinal degenerations, Surgery-induced retinopa implanted electrodes and an implanted pulse generator. thies, toxic retinopathies, retinopathies due to trauma or However, stimulation of this nerve root can result in a penetrating lesions of the eye. number of different complications or side effects. Being able 0143 Specific exemplary inherited conditions of interest to directly control the Sacral nerve through genetically for treatment according to the invention include, but are not targeted tools would be highly beneficial. In one embodi US 2016/0361439 A1 Dec. 15, 2016

ment, both ChR2 and NpHR could be expressed in this nerve known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine to control storage and Voiding of the bladder. spongiform encephalopathy (BSE), chronic pain, Canavan 0146 In another aspect, the compositions and methods of disease, Cockayne syndrome, corticobasal degeneration, this invention are utilized to treat Parkinson's disease. Creutzfeldt-Jakob disease, Huntington's disease, HIV-asso Parkinson's disease belongs to a group of conditions called ciated dementia, Kennedy's disease, Krabbe's disease, movement disorders. They are characterized by muscle Lewy body dementia, Machado-Joseph disease (Spinocer rigidity, tremor, a slowing of physical movement (bradyki ebellar ataxia type 3), multiple Sclerosis, multiple system nesia) and, in extreme cases, a loss of physical movement atrophy, narcolepsy, neuroborreliosis, Parkinson's disease, (akinesia). The primary symptoms are the results of Pelizaeus-Merzbacher Disease, Pick's disease, primary lat decreased stimulation of the motor cortex by the basal eral Sclerosis, prion diseases, Refsum’s disease, Sandhoffs ganglia, normally caused by the insufficient formation and disease, Schilder's disease, Subacute combined degeneration action of dopamine, which is produced in the dopaminergic of spinal cord secondary to pernicious anemia, Schizophre neurons of the brain. Parkinson's disease is both chronic and nia, Spielmeyer-Vogt-Sjogren-Batten disease (also known as progressive. Deep brain stimulation (DBS) is an effective Batten disease), spinocerebellar ataxia (multiple types with surgical treatment for advanced Parkinson's disease (PD), varying characteristics), spinal muscular atrophy, Steele with significant advantages in morbidity-mortality and qual Richardson-Olszewski disease, and Tables dorsalis. ity of life when compared to lesion techniques such as 0150. In another aspect the compositions and methods of thalamotomy and/or pallidotomy. The procedure is indicated this invention are utilized to treat a neurodevelopmental in patients with severe resting tremor, unresponsive to disease selected from but not limited to attention deficit conventional medical treatment or with motor complica hyperactivity disorder (ADHD), attention deficit disorder tions. The most commonly reported complications in the (ADD), schizophrenia, obsessive-compulsive disorder intra- and post-Surgical period are aborted procedure, mis (OCD), mental retardation, autistic spectrum disorders placed leads, intracranial hemorrhage, seizures and hard (ASD), cerebral palsy, Fragile-X Syndrome, Downs Syn ware complications, whereas in the long-term period, symp drome, Retts Syndrome, Asperger's syndrome, Williams toms may include high level cognitive dysfunction, Beuren Syndrome, childhood disintegrative disorder, articu psychiatric, and Subtle language problems. Indeed, this lation disorder, learning disabilities (i.e., reading or method of therapy would be improved by being able to arithmetic), dyslexia, expressive language disorder and target specific cell types within a given region to avoid these mixed receptive-expressive language disorder, Verbal or side effects. In one embodiment, light-sensitive proteins performance aptitude. Diseases that can result from aberrant specifically activate dopaminergic circuits. neurodevelopmental processes can also include, but are not 0147 In another specific aspect, the compositions and limited to bi-polar disorders, anorexia, general depression, methods of this invention are utilized to treat epilepsy and seizures, obsessive compulsive disorder (OCD), anxiety, seizures. Epilepsy is a neurological disorder that is often bruixism, Angleman’s syndrome, aggression, explosive out characterized by seizures. These seizures are transient signs burst, self-injury, post-traumatic stress, conduct disorders, and/or symptoms due to abnormal, excessive or asynchro Tourette's disorder, stereotypic movement disorder, mood nous neuronal activity in the brain. Over 30% of people with disorder, sleep apnea, restless legs syndrome, dysomnias, epilepsy do not have seizure control even with the best paranoid personality disorder, Schizoid personality disorder, available medications. Epilepsy is not a single disorder, but Schizotypal personality disorder, antisocial personality dis rather as a group of syndromes with vastly divergent symp order, borderline personality disorder, histrionic personality toms but all involving episodic abnormal electrical activity disorder, narcissistic personality disorder, avoidant person in the brain. Acute deep brain stimulation (DBS) in various ality disorder, dependent personality disorder, reactive thalamic nuclei and medial temporal lobe structures has attachment disorder, separation anxiety disorder, opposi recently been shown to be efficacious in small pilot studies. tional defiant disorder, dyspareunia, pyromania, kleptoma There is little evidence-based information on rational targets nia, trichotillomania, gambling, pica, neurotic disorders, and stimulation parameters. Amygdalohippocampal DBS alcohol-related disorders, amphetamine-related disorders, has yielded a significant decrease of seizure counts and cocaine-related disorders, marijuana abuse, opioid-related interictal EEG abnormalities during long-term follow-up. disorders, phencyclidine abuse, tobacco use disorder, buli Data from pilot studies suggest that chronic DBS for epi mia nervosa, delusional disorder, sexual disorders, phobias, lepsy may be a feasible, effective, and safe procedure. Again, Somatization disorder, enuresis, encopresis, disorder of writ being able to genetically-target activation to specific Subsets ten expression, expressive language disorder, mental retar of cells would improve the quality of the therapy as well as dation, mathematics disorder, transient tic disorder, Stutter minimize overall side effects. In specific embodiments, the ing, selective mutism, Crohn's disease, ulcerative colitis, light-sensitive proteins are utilized to alter the asynchronous bacterial overgrowth syndrome, carbohydrate intolerance, electrical activity leading to seizures in these deep brain celiac sprue, infection and infestation, intestinal lymphangi aaS. ectasia, short bowel syndrome, tropical sprue. Whipple's 0148. In another aspect, the compositions and methods of disease, Alzheimer's disease, Parkinson's Disease, ALS, this invention are utilized to effect the light-stimulated spinal muscular atrophies, and Huntington's Disease. Fur release of implanted drug or vaccine stores for the preven ther examples, discussion, and information on neurodevel tion, treatment, and amelioration of diseases. opmental disorders can be found, for example, through the 0149. In another aspect, the compositions and methods of Neurodevelopmental Disorders Branch of the National Insti this invention are utilized to treat neurodegenerative disease tute of Mental Health. Additional information on neurode selected from but not limited to alcoholism, Alexander's velopmental disorders can also be found, for example, in disease, Alper's disease, Alzheimer's disease, Amyotrophic Developmental Disabilities in Infancy and Childhood: Neu lateral Sclerosis, Ataxia telangiectasia, Batten disease (also rodevelopmental Diagnosis and Treatment, Capute and US 2016/0361439 A1 Dec. 15, 2016

Accardo, eds. 1996, Paul H Brookes Pub Co.; Hagerman, 0157. The gene delivery vector can be modified to Neurodevelopmental Disorders. Diagnosis and Treatment, enhance penetration of the blood-retinal barrier. Such modi 1999, Oxford Univ Press; Handbook of Neurodevelopmental fications may include increasing the lipophilicity of the and Genetic Disorders in Children, Goldstein and Reynolds, pharmaceutical formulation in which the gene delivery eds., 1999, Guilford Press; Handbook of Neurodevelopmen vector is provided. tal and Genetic Disorders in Adults, Reynolds and Gold 0158. The gene delivery vector can be delivered alone or stein, eds., 2005, Guilford Press; and Neurodevelopmental in combination, and may be delivered along with a phar Disorders, Tager-Flusberg, ed., 1999, MIT Press. maceutically acceptable vehicle. Ideally, such a vehicle 0151 Assessment of Therapy would enhance the stability and/or delivery properties. The 0152 The effects of therapy according to the invention as invention also provides for pharmaceutical compositions described herein can be assessed in a variety of ways, using containing the active factor, or fragment, or derivative methods known in the art. For example, the subjects vision thereof, which can be administered using a suitable vehicle can be tested according to conventional methods. Such Such as liposomes, microparticles or microcapsules. In vari conventional methods include, but are not necessarily lim ous embodiments of the invention, it may be useful to use ited to, electroretinogram (ERG), focal ERG, tests for visual Such compositions to achieve Sustained release of the active fields, tests for visual acuity, ocular coherence tomography component. (OCT), Fundus photography, Visual Evoked Potentials 0159. The amount of gene delivery vector (e.g., the (VEP) and Pupillometry. In other embodiments, the subject number of viral particles), and the amount of light-sensitive can be assessed behaviorally. In general, the invention protein expressed, effective in the treatment of a particular provides for maintenance of a Subject's vision (e.g., preven disorder or condition may depend of the nature of the tion or inhibition of vision loss of further vision loss due to disorder or condition and a variety of patient-specific fac photoreceptor degeneration), slows onset or progression of tors, and can be determined by standard clinical techniques. vision loss, or in Some embodiments, provides for improved 0160. In one embodiment, the gene delivery vectors are vision relative to the subjects vision prior to therapy. administered to the eye, intraocularly to a variety of loca 0153 Methods of Administration tions within the eye depending on the type of disease to be 0154 The gene delivery vectors of the present invention treated, prevented, or, inhibited, and the extent of disease. can be delivered to the eye through a variety of routes. They Examples of Suitable locations include the retina (e.g., for may be delivered intraocularly, by topical application to the retinal diseases), the vitreous, or other locations in or eye or by intraocular injection into, for example the vitreous adjacent the retina or in or adjacent the eye. (intravitreal injection) or subretinal (subretinal injection) 0.161 The human retina is organized in a fairly exact inter-photoreceptor space. Alternatively, they may be deliv mosaic. In the fovea, the mosaic is a hexagonal packing of ered locally by insertion or injection into the tissue Sur cones. Outside the fovea, the rods break up the close rounding the eye. They may be delivered systemically hexagonal packing of the cones but still allow an organized through an oral route or by Subcutaneous, intravenous or architecture with cones rather evenly spaced surrounded by intramuscular injection. Alternatively, they may be delivered rings of rods. Thus in terms of densities of the different by means of a catheter or by means of an implant, wherein photoreceptor populations in the human retina, it is clear that Such an implant is made of a porous, non-porous or gelati the cone density is highest in the foveal pit and falls rapidly nous material, including membranes such as Silastic mem outside the fovea to a fairly even density into the peripheral branes or fibers, biodegradable polymers, or proteinaceous retina (see Osterberg, G., “Topography of the layer of rods material. The gene delivery vector can be administered prior and cones in the human retina.’ Acta Ophthal., (Suppl.) to the onset of the condition, to prevent its occurrence, for 6:1-103, 1935; Curcio, C. A., Sloan, K. R., Packer, O., example, during Surgery on the eye, or immediately after the Hendrickson, A. E. and Kalina, R. E., “Distribution of cones onset of the pathological condition or during the occurrence in human and monkey retina: individual variability and of an acute or protracted condition. radial asymmetry,” Science, 236:579-582: 1987). 0155. In another embodiment the inner limiting mem 0162 Access to desired portions of the retina, or to other brane (ILM) is broken down to effect delivery. The ILM is parts of the eye may be readily accomplished by one of skill the boundary between the retina and the vitreous body, in the art (see, generally Medical and Surgical Retina: formed by astrocytes and the end feet of Muller cells. In both Advances, Controversies, and Management, Hillel Lewis, nonhuman primates and humans, the ILM is thick and Stephen J. Ryan, Eds., medical-illustrator, Timothy C. provides a Substantial barrier to the retina. Indeed, using Hengst, St. Louis, Mosby, 1994. xix., 534; see also Retina, intravitreal injections, most viral particles are incapable of Stephen J. Ryan, editor in chief, 2nd ed., St. Louis, Mo.: transducing retinal cells. In one embodiment, to improve Mosby, 1994. 3 v. (xxix. 2559). transduction efficiency, an ILM peel is conducted compris 0163. In one embodiment, the amount of the specific viral ing carrying out a Surgical procedure that comprises peeling vector applied to the retina is uniformly quite Small as the off a small part of the ILM. In another embodiment, to eye is a relatively contained structure and the agent is improve transduction efficiency, the ILM barrier can be injected directly into it. The amount of vector that needs to partially, or wholly, broken down comprising using enzy be injected is determined by the intraocular location of the matic techniques and one or more enzymes. chosen cells targeted for treatment. The cell type to be 0156. In one embodiment the ILM is maintained to limit transduced may be determined by the particular disease the therapeutic effect of the light-sensitive protein to the entity that is to be treated. macula. In another embodiment the ILM peel procedure 0164. For example, a single 20-microliter volume (e.g., and/or the ILM enzymatic digestion procedure, both containing about 10" physical particle titer/mL rAAV) may described herein is used to achieve a broader distribution of be used in a Subretinal injection to treat the macula and fovea the light-sensitive protein. of a human eye. A larger injection of 50 to 100 microliters US 2016/0361439 A1 Dec. 15, 2016

may be used to deliver the rAAV to a substantial fraction of 0173 Examples of non-aqueous solvents are propylene the retinal area, perhaps to the entire retina depending upon glycol, polyethylene glycol, hyaluronic acid, vegetable oils the extent of lateral spread of the particles. Such as olive oil, and injectable organic esters such as ethyl 0.165. A 100-microliter injection may provide several oleate. million active rAAV particles into the subretinal space. This 0.174 Aqueous carriers include water, alcoholic/aqueous calculation is based upon a titer of 10' physical particles per Solutions, emulsions or Suspensions, including saline and milliliter. Of this titer, it is estimated that 1/1000 to 1/10,000 buffered media. Parenteral vehicles include sodium chloride of the AAV particles are infectious. The retinal anatomy Solution, Ringer's dextrose, dextrose and Sodium chloride, constrains the injection Volume possible in the Subretinal lactated Ringer's or fixed oils. Intravenous vehicles can space (SRS). Assuming an injection maximum of 100 include fluid and nutrient replenishers, electrolyte replen microliters, this could provide an infectious titer of 10 to ishers (such as those based on Ringer's dextrose), and the 10 raAV in the SRS. This would have the potential of like. infecting all of the approximately 150x10° photoreceptors in 0.175. A composition of gene delivery vector of the the entire human retina with a single injection. invention may also be lyophilized using means well known in the art, for Subsequent reconstitution and use according to 016.6 Smaller injection volumes focally applied to the the invention. Where the vector is to be delivered without fovea or macula may adequately transfect the entire region being encapsulated in a viral particle (e.g., as “naked' affected by the disease in the case of macular degeneration polynucleotide), formulations for liposomal delivery, and or other regional retinopathies. formulations comprising microencapsulated polynucle (0167 Depending on the application at least 10, 10, 10. otides, may also be of interest. 10, 107, 10, 10, 10, 10'', 10°, 10, 10', or more 0176 Compositions comprising excipients are formu particles can be delivered into the tissue of interest. lated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, Chapter 0168 Gene delivery vectors can alternately be delivered 43, 14" Ed., Mack Publishing Co., Easton, Pa., USA). to the eye by intraocular injection into the vitreous, e.g., to 0177. In general, the pharmaceutical compositions can be treat glaucomatous loss of retinal ganglion cells through prepared in various forms, preferably a form compatible apoptosis. In the treatment of glaucoma, the primary target with intraocular administration. Stabilizing agents, wetting cells to be transduced are the retinal ganglion cells, the and emulsifying agents, salts for varying the osmotic pres retinal cells primarily affected. In such an embodiment, the Sure or buffers for securing an adequate pH value may also injection volume of the gene delivery vector could be optionally be present in the pharmaceutical composition. Substantially larger, as the Volume is not constrained by the 0.178 The amount of gene delivery vector in the phar anatomy of the Subretinal space. Acceptable dosages in this maceutical formulations can vary widely, i.e., from less than instance can range from about 25 microliters to 1000 micro about 0.1%, usually at or at least about 2% to as much as liters. 20% to 50% or more by weight, and may be selected (0169 Pharmaceutical Compositions: primarily by fluid volumes, viscosities, etc., in accordance 0170 Gene delivery vectors can be prepared as a phar with the particular mode of administration selected. maceutically acceptable composition Suitable for adminis 0179 The pharmaceutical composition can comprise tration. In general. Such pharmaceutical compositions com other agents Suitable for administration, which agents may prise an amount of a gene delivery vector Suitable for have similar to additional pharmacological activities to the delivery of light-sensitive protein-encoding polynucleotide light-sensitive protein to be delivered (e.g., ChR1, ChR2, to a cell of the eye for expression of a therapeutically VChR1, ChR2C128A, ChR2 C128S, ChR2 C128T, ChR1 effective amount of the light-sensitive protein, combined ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, with a pharmaceutically acceptable carrier or excipient. eNpHR, melanopsin, and variants thereof). Preferably, the pharmaceutically acceptable carrier is suit 0180 Kits: able for intraocular administration. Exemplary pharmaceu 0181. The invention also provides kits comprising vari tically acceptable carriers include, but are not necessarily ous materials for carrying out the methods of the invention. limited to, Saline or a buffered saline solution (e.g., phos In one embodiment, the kit comprises a vector encoding a phate-buffered saline). light-sensitive protein polypeptide (e.g., ChR1, ChR2, VChR1, ChR2C128A, ChR2 C128S, ChR2 C128T, ChR1 0171 Various pharmaceutically acceptable excipients are ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, well known in the art. As used herein, “pharmaceutically eNpHR, melanopsin, and variants thereof), which vector is acceptable excipient' includes any material, which, when adapted for delivery to a subject, particularly an eye of the combined with an active ingredient of a composition, allows Subject, and adapted to provide for expression of the light the ingredient to retain biological activity, preferably with sensitive polypeptide in a cell of an eye, particularly a out causing disruptive reactions with the Subject’s immune mammalian cell. The kit can comprise the vector in a sterile system or adversely affecting the tissues Surrounding the site vial, which may be labeled for use. The vector can be of administration (e.g., within the eye). provided in a pharmaceutical composition. In one embodi 0172 Exemplary pharmaceutically carriers include ster ment, the vector is packaged in a virus. The kit can further ile aqueous of non-aqueous Solutions, Suspensions, and comprise a needle and/or syringe Suitable for use with the emulsions. Examples include, but are not limited to, any of vial or, alternatively, containing the vector, which needle the standard pharmaceutical excipients such as a saline, and/or syringe are preferably sterile. In another embodiment, buffered saline (e.g., phosphate buffered saline), water, the kit comprises a catheter suitable for delivery of a vector emulsions such as oil/water emulsion, and various types of to the eye, which catheter may be optionally attached to a Wetting agents. syringe for delivery of the vector. The kits can further US 2016/0361439 A1 Dec. 15, 2016 comprise instructions for use, e.g., instructions regarding view are enhanced (e.g., moving cars, doorways) and less route of administration, dose, dosage regimen, site of admin important objects (e.g., clouds), are filtered out. The LED istration, and the like. and/or laser lighting array system could contain a high 0182 Devices: density LED array or a scanning laser system that consists 0183. The data in FIG. 12C demonstrate that the delivery of either one (1) or more lasers. The position of the of light-sensitive proteins can work in the range of normal could be either fixed or could move. For example, the vision. In some embodiments, for greater efficacy, an inter orientation of the lights relative to the eye could move as a nal or external device may be used. In one embodiment, an function of eye movements, using an eye movement track external device. Such as a goggle, can be used for generation ing device as an input. This is depicted in FIG. 13. and/or amplification of light. In embodiments where a 0188 In another related exemplary embodiment, an Subject having partial vision is being treated, i.e. a treatment image intensifying device. Such as those provided by of a subject whose photoreceptors are only partially dam Telesensory, may be combined with a retinal scanning aged and a light-sensitive protein such as ChR1, ChR2, device (RSD) as developed by Microvision, to provide a VChR1, ChR2C128A, ChR2 C128S, ChR2 C128T, ChR1 head-worn apparatus capable of delivering a bright, inten ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, sified image directly to the retina of a patient with impaired eNpHR, melanopsin, and variants thereof is being delivered, vision. Briefly, a RSD projects images onto the retina such the stimulation may be adjusted so that the Surviving and/or that an individual can view a large, full-motion image healthy photoreceptors are not overdriven by the light gen without the need for additional screens or monitors. Thus, by eration/amplification device. In various embodiments, lim projecting an intensified image directly onto the retina of an iting overdriving by the light generation/amplification individual with impaired vision, it may be possible to device can be achieved by i) stimulating evenly, but shield improve vision in those considered to be blind or near-blind. ing the Surviving or healthy photoreceptor cells from bright 0189 While some embodiments of the present invention light through an implanted or external contact-lens type have been shown and described herein, it will be obvious to partial Sunglass (tinting over photoreceptors, clear over those skilled in the art that such embodiments are provided light-sensitive protein transduction area); ii) adjustment of by way of example only. Numerous variations, changes, and the stimulation intensity to match the cell types being substitutions will now occur to those skilled in the art stimulated; or iii) adjustment of the stimulation to the be without departing from the invention. It should be under near the top of the visual dynamic range. stood that various alternatives to the embodiments of the 0184. In one embodiment, an internal light-generating invention described herein may be employed in practicing device is implanted. the invention. 0185. In another embodiment, a protective optic, or a contact lens-type barrier is implanted either in conjunction EXAMPLES with or independent of the device. In a specific embodiment, Such an optic or contact lens protects photoreceptors from 0190. The following examples are included to demon light stimulation. In a specific embodiment, the lens com strate illustrative embodiments of the invention. It should be prises tinting over photoreceptors, and clear over light appreciated by those of ordinary skill in the art that the sensitive protein transduction area. techniques disclosed in these examples represent techniques 0186. In some embodiments, a head-mounted, external discovered to function well in the practice of the invention, device or eyewear is utilized. In certain embodiments where and thus can be considered to constitute preferred modes for the light-sensitive element is not triggered to the extent its practice. However, those of ordinary skill in the art desired by natural or ambient light, an additional light should, in light of the present disclosure, appreciate that production or generation source Such as a LED array/laser many changes can be made in the specific embodiments that system is provided. In certain embodiments, the external are disclosed, and still obtain a like or similar result without eyewear can additionally include a camera and an image departing from the spirit and Scope of the invention. processing unit for the filtering, enhancement, processing, and resolution of the presented images. FIG. 13 depicts a Example 1 goggle-like device with an associated light production ele ment (LED array/laser system) that may trigger expression Injection Methods of light-sensitive proteins. 0187. In one embodiment, an exemplary camera system 0191 All procedures in animals were handled according would comprise at least three main components: 1) A small to the statement for the use of animals in Ophthalmic and camera built into the glasses, 2) an imaging processing unit, Vision Research of the Association of Research in Vision and 3) a light delivery system that includes either or both and Ophthalmology and the guidelines of the Institutional LEDs or a laser system. The camera could either be a single Animal Care and Use Committee at the University of lens camera or a dual camera system that could potentially Florida. provide binocular imaging and depth information. The cam 0.192 For intrvitreal injections, mice were anesthetized era could capture either visual light or infrared light. The with ketamine (72 mg/kg)/xylazine (4 mg/kg) by intraperi camera could either be adaptive to various lighting condi toneal injection. Following anesthetization, a Hamilton tions or could be fixed. The image processing unit (IPU) syringe fitted with a 33-gauge beveled needle was used. The could provide any number of signal transformations includ needle was passed through the Sclera, at the equator, next to ing amplification, increased or decreased contrast, structure the limbus, into the vitreous cavity. Injection occurred with from motion, edge enhancement, or temporal filtering (i.e., direct observation of the needle in the center of the vitreous integration). Additionally, Saliency algorithms could be cavity. The total volume delivered was 1.5 containing dif employed such that only certain objects within the field of ferent concentrations of the AAV vectors tested. US 2016/0361439 A1 Dec. 15, 2016

0193 For subretinal injections, one hour before the anes (0197) The images depicted in FIG. 10A through FIG. 10E thesia, eyes of mice were dilated with eye drops of 1% show the overall expression of GFP (the reporter gene). This atropine, followed by topical administration of 2.5% phe expression is shown as white in the black and white images. nylephrine. Mice were then anesthetized with ketamine (72 This is indicative of the overall expression of ChR2 as the mg/kg)/xylazine (4 mg/kg) by intraperitoneal injection An ChR2-GFP complex is a fused protein. Note the ringlets of aperture within the pupil was made through the cornea with GFP expression in the INL, showing expression of the a 301/2-gauge disposable needle and a 33-gauge unbeveled ChR2-GFP protein complex is membrane bound. These data blunt needle in a Hamilton Syringe was introduced through show that delivery of the construct with an adeno-associated the corneal opening into the Subretinal space and 1.5 LL of virus leads to robust expression of ChR2-GFP in all 3 mouse AAV was delivered. models of blindness (rdl, rd 16, and rho-/-). This is con (0194 Typical titers of the AAV vectors were between ducted with 3 different serotypes using a subretinal injection 1.3x10° and 3.0x10'. (column 1). When using a tyrosine to phenylalanine mutant serotype, it is possible to get good expression in bipolar cells Example 2 (INL) using either a subretinal or intravitreal injection. However, wild type serotypes require a Subretinal injection Screening for AAV Serotypes 1, 2, 5, 7, 8, and 9 to get reasonable transduction of bipolar cells; intravitreal for Transduction of Retinal Bipolar Cells injections using wild type serotypes do not effectively 0.195 Screening of known and characterized viral vectors transduce bipolar cells (column 2). for optimal transduction of retina bipolar cells was carried out. AAV serotypes 1, 2, 5, 7, 8 and 9 carrying green Example 4 fluorescent protein (GFP) were individually subretinally injected in 4-week old rd 1 mice. Rd1 homozygous mice Creation of AAV7-GRM6-ChR2 to Establish Light carry ard1 mutation and rod photoreceptor degeneration in Sensitivity in Retinal ON-Bipolar Cells these mice begins around postnatal day (P)10 and is almost 0198 mGluR6 is a G-protein coupled metabotropic glu completed by P21. GFP was placed under control of the tamate receptor that is, in the retina, specifically expressed strong, non-cell type specific promoter CBA (fusion of the in ON bipolar cells (Tian, 2006). The adeno-associated CMV immediate early enhancer and the bovine B-actin virus, serotype 7 (AAV7), an mGluR6 regulatory sequence promoter plus intronll-exon 1 junction). 1 month later, mice fragment gene sequence (presented in FIG. 6), GRM6, and were tested for expression of GFP. Double labeling with the channelrhodopsin-2, ChR2 was constructed to form the PKCCantibody of mice injected with AAV7 demonstrated AAV7-GRM6-ChR2 construct. The cDNA encoding ChR2 that the transduced cells were most likely residual (no outer with eCFP was cloned downstream (i.e., 3') of the mGluR6 segment) photoreceptors rather than bipolar cells. Subretinal regulatory sequence fragment-SV40 minimal promoter. No injections with AAV7 were then performed in 8-week old IRES was used; ChR2 and GFP were fused. This viral vector mice, where there is less of a chance of residual photore construct once delivered using a viral delivery mechanism, ceptors. It was found that AAV7 was highly effective at and expressed, can establish photosensitivity in retinal ON transducing retinal bipolar cells, leading to GFP expression bipolar cells with high-spatial and temporal resolution. This in at least 75% of all bipolar cells after a single injection method can restore retinal responsiveness to optical infor (depicted in FIG. 9A through FIG. 9H). These images were mation, using the ChR2 class of light-activated molecules to obtained 16 weeks after injection, which additionally show directly sensitize spared retinal neurons to light. that GFP expression using an AAV7 delivery mechanism is stable for at least 4 months. AA7 is a serotype that can be Example 5 utilized to transduce bipolar cells in an effective and stable a. AAV8 Mutant Y733F-GRM6-ChR2 and AAV8 Mutant Y446F-CBA-ChR2 to Establish Light Example 3 Sensitivity in Retinal ON-Bipolar Cells 0199. Using a tyrosine-mutated version of AAV8 (at the Transduction of Retinal Bipolar Cells with 733 location), under the control of bipolar cell specific Serotypes AAV5, AAV2 Y444F Mutant, and AAV8 promoter GRM6, in the self-complementary configuration, Y733F Mutant it was possible to restore visual behavioral efficacy in rd1 (0196. As depicted in FIG. 10A-FIG. 10E, mice were mice, as depicted in FIG. 11A through FIG. 11F and FIG. subretinally (right eye) and intravitreally (left eye) injected 12A-1 through FIG. 12C. For example, FIG. 11A through with 1.5 L of adeno-associated viruses (AAV) of different FIG. 11F depict the analysis of EGFP expression in frozen serotypes. The serotypes tested included AAV2, AAV5, and retinal sections by immunohistochemistry at 1 month fol AAV8, all of which are traditional wild type serotypes. lowing subretinal injections with the tyrosine-mutated AAV Additionally, single tyrosine to phenylalanine mutated sero vectors. Exemplary sections depicting spread and intensity types AAV2 Y444F mutant and AAV8Y733F mutant, where of EGFP fluorescence throughout the retina after transduc 444 and 733 indicate the location of the point tyrosine tion with serotype 2 Y444 or serotype 8 Y733 are shown in mutation of the viral capsid, respectively. The virus con FIG. 11A and FIG. 11B, respectively. The images are tained the self-complementary DNA construct GRM6 oriented with the vitreous toward the bottom and the pho ChR2-GFP, where GRM6 is the metabotropic glutamate toreceptor layer toward the top. EGFP fluorescence in pho receptor 6 regulatory sequence driving cell-specific expres toreceptors, RPE and ganglion cells from mouse eyes sion in the ON bipolar cells (including rod bipolar), ChR2 is injected subretinally with serotype 2 Y444 (FIG. 11C) EGFP the therapeutic, light-sensitive protein gene, and GFP is the fluorescence in photoreceptors, RPE and Muller cells after reporter gene. serotype 8 Y733 delivery (FIG. 11D) Detection of Muller US 2016/0361439 A1 Dec. 15, 2016

cells processes (red) by immunostaining with a glutamine 0203 The animals performance on the task was then synthetase (GS) antibody (FIG. 11E) Merged image show evaluated at different light levels. FIG. 12C shows the ing colocalization of EGFP fluorescence (green) and GS average time it took for the rdl., rd 16, and rho-/- treated, staining (red) in retinal sections from eyes treated with sham injected (sham injected mice represent an average of serotype 8 Y733 (FIG. 11F) Calibration bar 100 uM. gcl. rd1, rd16, and rho-/- untreated), and wild type mice to find ganglion cell layer; ipl, inner plexiform layer, inl, inner the target, as a function of the light intensity. These data nuclear layer, onl, outer nuclear; os, outer segment; rpe, show that the treated mice can perform the task at multiple retinal pigment epithelium. light levels and their performance is dependent on the 0200. Using a tyrosine-mutated version of AAV8 (at the amount of light presented. 446 location), under the control of the non-cell specific 0204. It should be understood that the examples and promoter CBA (fusion of the CMV immediate early embodiments described herein are for illustrative purposes enhancer and the bovine B-actin promoter plus intronl-exon1 only and that various modifications or changes in light junction, and ChR2), in the self-complementary configura thereof will be suggested to persons skilled in the art and are tion, most or all bipolar cells can be targeted and visual to be included within the spirit and purview of this appli function is restored as depicted in FIG. 12A, FIG. 12B, and cation and the scope of the appended claims. FIG. 12C 0205 All references, including publications, patent appli cations and patents, cited herein are hereby incorporated by Example 6 reference to the same extent as if each reference was individually and specifically indicated to be incorporated by AAV5-CBA-ChR2 to Establish Light Sensitivity in reference and was set forth in its entirety herein. Retinal on Bipolar Cells 0206 Recitation of ranges of values herein are merely 0201 Using the AAV5, non-cell type specific promoter intended to serve as a shorthand method of referring indi CBA (fusion of the CMV immediate early enhancer and the vidually to each separate value falling within the range, bovine B-actin promoter plus intronl-exon 1 junction, and unless otherwise indicated herein, and each separate value is ChR2, in the self-complementary configuration, all bipolar incorporated into the specification as if it were individually cells can be targeted and visual function and behavior is recited herein. restored (FIG. 12A, FIG. 12B, and FIG. 12C). Treated mice 0207. The description herein of any aspect or embodi were subretinally (right eye) and intravitreally (left eye) ment of the invention using terms such as "comprising. injected with 1.5 uL of adeno-associated viruses (AAV) of “having”, “including,” or “containing,” with reference to an different serotypes. The serotypes tested included AAV2. element or elements is intended to provide Support for a AAV5, and AAV7, all of which are traditional wild type similar aspect or embodiment of the invention that “consists serotypes. Additionally, the single tyrosine to phenylalanine of.” “consists essentially of or “substantially comprises.” mutated serotypes AAV2 Y444F mutant and AAV8 Y733F that particular element or elements, unless otherwise stated mutant, where 444 and 733 indicate the location of the point or clearly contradicted by context (e.g., a composition tyrosine mutation of the viral capsid, respectively. The virus described herein as comprising a particular element should contained the self-complementary DNA construct GRM6 be understood as also describing a composition that contains ChR2-GFP, where GRM6 is the regulatory sequence driving and/or that includes that particular element, unless otherwise cell-specific expression in the ON bipolar cells (including explicated Stated, or clearly contradicted by context). rod bipolar), ChR2 is the therapeutic, light-sensitive protein 0208 All of the compositions and methods disclosed and gene, and GFP is the reporter gene. claimed herein can be made and executed without undue 0202 These mice were then trained on a water maze task experimentation in light of the present disclosure. While the FIG. 12A for 14 days (7 days for the wild type mice) and the compositions and methods of this invention have been time to find the target (a black platform with a 4x6 LED light described in terms of preferred embodiments, it will be source) was recorded. FIG. 12B shows the average time it apparent to those of skill in the art that variations may be took for the treated, untreated, and wild type mice to find the applied to the compositions and methods and in the steps or target, as a function of the training session. Both the in the sequence of steps of the method described herein untreated and treated groups contained samples from the without departing from the concept, spirit and scope of the rd1, rd 16, and rho-/-(different mouse models of blindness invention. More specifically, it will be apparent that certain that have different types of gene mutations that lead to agents that are chemically- and/or physiologically-related photoreceptor disease) groups. These data demonstrate that may be substituted for the agents described herein while the mice treated with ChR2 are able to learn a behavior task by same or similar results would be achieved. All such similar using visual information, Suggesting that a light sensitive Substitutes and modifications apparent to those skilled in the protein such as ChR2 has the ability to restore at least some art are deemed to be within the spirit, scope, and concept of visual function. the invention as defined by the appended claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 8

<21 Os SEQ ID NO 1 &211s LENGTH: 7 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence

US 2016/0361439 A1 Dec. 15, 2016 24

- Continued c cct coccac ccc caattitt g tatttattt atttitttaat tattttgttgc agcigatgggg 48O gcgggggggggggggggg.cg C9C9C Caggc gggg.cggggc gggg.cgaggg gC9gggcggg 54 O gcgaggcgga gaggtgcggc ggcagccalat cagagcggcg cgctic.cgaaa gttt CCttitt 6OO atgg.cgaggc gg.cggcgg.cg gC9gc cct at aaaaag.cgala gC9C9C9gcg gg.cgggagtic 660 gctg.cgacgc tigc ctitcgcc ccgtgcc.ccg. Ct.ccgc.cgcc gccticgc.gcc gcc.cgc.ccc.g 72 O gctctgactg accgcgttac toccacaggit gagcgggcgg gacggcc ctt Ctcct Coggg 78O

Ctgtaattag cqcttggttt aatgacggct ttitt Cttitt Ctgtggctgc gtgaaagcct 84 O tgaggggotic cqggagctag agcct ctdct aac catgttc atgcc ttctt Ctttitt.ccta 9 OO Cagctic ctgg gcaacgtgct ggittattgttg Ctgtct catc attittggcaa ag 952

What is claimed is: 7. A composition comprising: 1. A recombinant adeno-associated virus (rAAV) particle, (a) a recombinant adeno-associated virus (ra AV) particle comprising: that comprises: (i) an AAV2 VP3 capsid protein comprising tyrosine (a) an AAV2 VP3 capsid protein comprising tyrosine-to to-phenylalanine mutations at positions Y444F, phenylalanine mutations at positions Y444F, Y500F. Y500F, and Y73OF; and and Y73OF; and (ii) a ra AV nucleic acid vector comprising a 5' inverted (b) a ra AV nucleic acid vector comprising a 5' inverted terminal repeat (ITR), a transgene, and a 3" ITR; and terminal repeat (ITR), a transgene, and a 3" ITR. (b) a carrier or an excipient. 8. The composition of claim 7, formulated for injection or 2. The rAAV particle of claim 1, wherein the transgene is topical application to a mammalian eye. operatively linked to a sequence that regulates expression of 9. A method of transducing a cell, the method comprising the transgene in a cell. administering an effective amount of the rAAV particle of 3. The rAAV particle of claim 2, wherein the sequence claim 1, or the composition of claim 7, to the cell. that regulates expression of the transgene in a cell is a 10. The method of claim 9, wherein the cell is a mam promoter. malian eye cell. 11. The method of claim 10, wherein the mammalian eye 4. The rAAV particle of any one of claims 1 to 3, wherein cell is human. the transgene is about 2- to 5-kb in length. 12. The method of claim 10, wherein the mammalian eye 5. The rAAV particle of claim 1, wherein the nucleic acid cell is selected from the group consisting of an ON retinal vector is a self-complementary (sc) vector. bipolar cell, an OFF retinal bipolar cell, a rod bipolar cell, 6. The rAAV particle of claim 1, wherein the transgene is and a cone bipolar cell. a reporter transgene. k k k k k