NZ727041B2 - Identification of channelrhodopsin-2 (chop2) mutations and methods of use - Google Patents

Abstract

Disclosed are expression vectors comprising a polynucleotide encoding a polypeptide molecule comprising SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is cysteine (C) or alanine (A), and the amino acid at position 159 is cysteine (C), serine (S), or alanine (A). Also disclosed is compositions comprising said vectors and use of said vectors in improving or restoring vision in a subject. d is compositions comprising said vectors and use of said vectors in improving or restoring vision in a subject.

Description

FICATION OF CHANNELOPSIN-2 (Chop2) MUTATIONS AND METHODS OF USE RELATED APPLICATIONS The t application is a divisional application of New Zealand Application No. , which is orated in its entirety herein by reference. [01a] This ation claims priority to and benefit of U.S. Provisional Application No. ,663 filed March 5, 2012, the contents of which are incorporated herein in its entirety.

GOVERNMENT SUPPORT This invention was made with U.S. Government support under the National Institutes of Health/National Eye Institute grant NIH EY 17130. The Government has certain rights in the invention.

FIELD OF THE INVENTION This invention relates lly to the field of molecular biology. Mutations in the Channelopsin-2 (Chop2) gene are identified. Compositions comprising a mutant Chop2 gene are used in therapeutic methods to improve and restore vision loss.

BACKGROUND OF THE INVENTION [03a] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

The retina is composed of photoreceptors (or photoreceptor cells, rods and .

Photoreceptors are highly specialized neurons that are responsible for phototransduction, or the conversion of light (in the form of electromagnetic radiation) into electrical and chemical s that propagate a cascade of events within the visual system, ultimately generating a representation of our world.

Photoreceptor loss or degeneration severely compromises, if not completely inhibits, phototransduction of visual information within the retina. Loss of eceptor cells and/or loss of a photoreceptor cell function are the primary causes of diminished visual acuity, shed light sensitivity, and blindness. There is a long-felt need in the art for compositions and method that restore photosensitivity of the retina of a subject experiencing vision loss. [05a] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be ued in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". [05b] It is an object of the present invention to overcome or ameliorate at least one of the antages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION [05c] According to a first aspect, the t invention provides an expression vector comprising a polynucleotide encoding a polypeptide molecule comprising SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is cysteine (C) or alanine (A), and the amino acid at position 159 is cysteine (C), serine (S) ), or alanine (A). [05d] According to a second aspect, the present invention provides a pharmaceutical composition comprising the expression vector of the invention. [05e] According to an third aspect, the present invention provides use of the expression vector of the invention or the pharmaceutical composition of the ion in the preparation of a medicament for improving or restoring vision in a subject.

The invention relates to the long-felt need for a method of restoring and/or increasing the light sensitivity of eceptor cells by sion of advantageous mutations, and/or combinations thereof, of the Channelopsin-2 (Chop2) gene, and subsequently providing methods for lopsin-2 (Chop2)- based gene therapy.

Channelopsin-2 (Chop2)-based gene therapy offers a superior strategy for restoring retinal photosensitivity after photoreceptor degeneration. The n product of the Chop2 gene, when bound to the light-isomerizable chromophore all-irans-retinal, forms a functional light-gated channel, called channelrhodopsin-2 (ChR2). Native ChR2 shows low light sensitivity. Recently, two mutant ChR2s, L132C and T159C, were reported to markedly se their light sensitivity logel et al. (2011) Nat Neurosci. 14:513-8; Berndt et al. (2011) Proc Natl Acad Sci USA. 108:7595-600; Prigge et al. (2012) J Biol Chem. 287(38)3104: 12; the contents of each of which are incorporated herein in their entireties). 2a followed by page 3 The properties of these two ChR2 mutants (i.e. , L132C and T159C) were examined and compared with a number of double mutants at these two sites to identify suitable ates for therapeutic methods.

Compositions sing one or more of these mutations are ed to a subject in need thereof for the purpose of restoring vision. Specifically, desired mutations in the Chop2 gene are uced to a cell and/or integrated into the genomic DNA of a cell to improve or restore vision. Desired mutations in the Chop2 gene that are introduced to a cell to improve or restore vision may also remain episomal, not having integrated into the genomic DNA.

Mutations at the L132 or T159 amino acid ons of Chop2 (and therefore, the ing ChR2) markedly lower the threshold light intensity that is required to elicit the ChR2-mediated photocurrent.

Double mutants at the amino acid positions LI 32 and T159 further increase the photocurrent at low light intensities, exceeding that of either of the corresponding single mutations. Retinal ganglion cells expressing the double mutants at the LI 32 and T159 positions can respond to light intensities that fall within the range of normal outdoor lighting conditions but should still in adequate, and high temporal resolution that are suitable for restoring useful vision. 'lhus, mutant Chop2 protein 01’ the t invention that form mutant ChR2s having improved light sensitivity are used alone or in combination to restore or improve Specifically, the invention provides an isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L). In certain embodiments 01' the isolated polypeptide molecule, the amino acid at position 132 is cysteine (C) or alanine (A).

When the amino acid at position 132 is cysteine (C), the polypeptide molecule may se or consist of SEQ 11) N0: 13. When the amino acid at position 132 is alanine (A). the polypeptide molecule may comprise or consist of SEQ 11) NO: 20.

The invention provides an isolated polypeptide molecule comprising or consisting of SEQ 11) NO: 26 in which the amino acid at position 159 of SEQ 11) NO: 26 is not a threonine (T). In certain ments of the isolated polypeptide molecule, the amino acid at position 159 is cysteine (C). serine (S), or alanine (A).

When the amino acid at position 159 is cysteine ((T). the polypeptide molecule may comprise or consist of SEQ 11) NO: 14. When the amino acid at position 159 is serine (S). the polypeptide molecule may comprise or consist of SEQ ID N0: 17. When the amino acid at position 159 is alanine (A), the polypeptide molecule may comprise or consist of SEQ ID NO: 23.

The invention provides isolated polypeptide molecule sing or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T). In certain embodiments of the isolated ptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (1.) and the amino acid at position 159 is not Lhreonine ('1'). the amino acid at position 132 is cysteine (C), and the atnino acid at position 159 is cysteine (C). In a preferred embodiment of this isolated polypeptide molecule, the polypeptide molecule comprises or consists of SEQ ID NO: 16. The invention provides an isolated c acid le that encodes for the isolated ptide comprising or consisting of SEQ ID NO: 16. Preferably, the ed c acid le that encodes for the isolated polypeptide comprising or consisting of SEQ ID NO: 16, is a nucleic acid le that comprises or consists of SEQ ID NO: IS.

In certain embodiments of the isolated polypeptide molecule sing or consisting of SEQ II) NO: 26 in which the amino acid at position 132 of SIZQ ll) NO: 26 is not leucine (L) and the amino acid at position 159 is not thrconine (T), the amino acid at position 132 is cysteine (C) and the amino acid at position 159 is serine(S).

The isolated polypeptide molecule sing or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ II) N0: 26 is not leucine (L) and the amino acid at position 159 is not threonine (1‘), may comprise or consist of SEQ ID NO: 19. atively, or in addition, the isolated polypeptide molecule comprising or consisting oI‘SIEQ [1) NO: 26 in which the amino acid at position 132 of SIEQ [1) NO: 26 is not leucine (L) and the amino acid at position 159 is not thrconine (T). wherein the amino acid at position I32 is ne (C) and wherein the amino acid at position 159 is serine(S) may comprise or consist of SEQ ID NO: 19. The invention provides an isolated c acid molecule that encodes for the isolated polypeptide that comprises or consists of SEQ ID N0: 19. Preferably, the nucleic acid molecule comprises or consists of SEQ ID NO: 18.

In certain embodiments of the isolated polypeptide molecule comprising or consisting of SIEQ ID NO: 26 in which the amino acid at position 132 of SIEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not thrconine (T), the amino acid at position 132 is alanine (A) and the amino acid at position 159 is cysteine (C).

The isolated polypeptide tnolecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T) may comprise or consist of SEQ ID NO: 22. Altemalively, or in addition, the ed polypeptide le comprising or consisting ol‘SlEQ ID NO: 26 in which the amino acid at position 132 of SEQ 11) NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T), wherein the amino acid at position I32 is alanine (A) and wherein the amino acid at position 159 is cysteine (C) may comprise or t of SEQ II) N0: 22. The invention provides an isolated nucleic acid molecule that s for the isolated polypeptide that comprises or consists of SEQ ID NO: 22. Preferably, this nucleic acid molecule ses or consists of SEQ ll) NO: 21.

In certain embodiments of the isolated polypeptide molecule sing or consisting of SEQ ID NO: 26 in which the amino acid at on 132 of SEQ ID NO: 26 is not leucine (1.) and the amino acid at position 159 is not threonine ('1‘), the amino acid at position 132 is cysteine ((7) and the amino acid at. on 159 is alanine (A).

The isolated polypeptide molecule comprising or consisting of SEQ [1) NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (1‘) may comprise or consist of SEQ ID NO: 25. Alternatively, or in addition, the isolated ptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position I32 of SEQ 11) NO: 26 is not leucine (1.) and the amino acid at position [59 is not threonine ('1‘), wherein the amino acid at position 132 is cysteine (C) and wherein the amino acid at position [59 is alanine (A) may comprise or consist of SEQ ID NO: 25. The invention es an isolated nucleic acid molecule that encodes for the isolated polypeptide that comprises or ts of SEQ 11) NO: 25. Preferably, this nucleic acid molecule comprises or consists of SEQ ID NO: 24.

The invention provides any one of the isolated polypeptide les described herein, wherein the polypeptide molecule encodes for a mutant Chop2 protein that forms a mutant ChR2, which elicits a current in response to a threshold intensity of light that is lower than the threshold of a wild type ChR2 protein.

Moreover. the current conducts cations. Exemplary cations include, but are not limited to, 11*. Na". K+, and Ca2+ ions. The (‘hR2 wild type and mutant proteins bed herein ecifically conduct s. Consequently, the current conducts one or more of the following: 11". Na+, K", and Ca" ions.

The invention provides any one of the isolated polypeptide molecules described herein further sing a pharmaceutically acceptable carrier. The invention also es a composition comprising at least one isolated polynucleotide molecule described herein. The composition may further se a pharmaceutically-acceptable carrier.

The invention provides an isolated nucleic acid molecule that encodes for any of the isolated polypeptides described herein. Moreover, the isolated nucleic acid molecule may further include a phannaceutically acceptable carrier. The invention also provides a composition comprising at least one isolated nucleic acid molecule described . The composition may further comprise a pharmaceutically- acceptable carrier.

The invention provides a cell. wherein the cell has been contacted with or comprises an isolated ptide molecule of the invention. Moreover. the invention provides a cell, wherein the cell has been contacted with or comprises an isolated nucleic acid molecule that encodes for an isolated polypeptide molecule of the invention. The invention provides, a composition comprising, consisting essentially of, or consisting of a cell that comprises an ed polypeptide molecule of the invention or a nucleic acid molecule that encodes for an isolated polypeptide molecule of the invention. Cells of the ion may be contacted with the isolated polypeptide or an isolated nucleic acid encoding the ptide in vitro, ex vii-'0, in rim, or in situ. In certain embodiments of the invention. the cell is a photoreceptor: a horizontal cell; a bipolar cell; an amacrine cell, and, ally, an All amacrine cell; or a retinal ganglion cell. including a photosensitive retinal on cell. Preferably, the cell is a retinal ganglion cell, a photosensitive retinal ganglion cell. a bipolar cell, an e bipolar cell, a rod bipolar cell, or an All amacrine cell. In certain aspects of the invention, the cell is a photoreceptor, a bipolar cell, a rod bipolar cell, an ON—type cone bipolar cell. a retinal ganglion cell. a photosensitive retinal ganglion cell, a horizontal cell, an amacrine cell, or an All amacrine cell.

The invention provides a method of improving or restoring vision, comprising administering to a subject any one of the compositions described herein. The invention further provides a lactic method of preserving vision, comprising administering to a subject any one of the compositions described herein.

The s described herein may also be d to those subjects who are y, blind (in part or in total), and/or those subjects with retinal degeneration (characterized by a loss of rod and/or cone photoreceptor cells), but may be dependent upon the activity of ensitive retinal ganglion cells for a detemiination of ambient light levels. For example, the methods described herein can be used to preserve. improve. or restore the activity of a photosensitive retinal ganglion cell that mediates the transduction of light ation for synchronizing circadian rhythms to the 24-hour light/dark cycle, pupillary control and reflexes, and photic regulation of melatonin release.

In certain embodiments of the methods of the invention, the t may have normal vision or impaired vision. Alternatively, or in addition. the subject may be at risk for developing an ocular disease that leads to impairment of . For example, the subject may have a family history of, ocular disease. including, r degeneration and tis pigmentosa. The subject may be at risk for incurring an eye injury that causes damage to photosensitive cells in the retina. The subject may have a genetic marker or genetic/congenital condition that results in impaired vision, low , legal blindness, partial blindness, or complete blindness. Subjects may have a refractive defect that results in myopia (near-sightedness) or hyperopia (far~ sightedness).

Compositions of methods of the invention may be administered to a subject either systemically or locally. A preferred route of local administration is intravitreal injection.

Other features and advantages of the invention will be apparent from and are encompassed by the following ed ption and claims.

BRIEF DESCRIPTION OF THE FIGURES Figure I shows representative recordings of the light-evoked currents from wild-type (WT) ChRZ, , ’l‘159C, and I.l32C/IS9S mutants in Him cells for comparison of their light sensitivity (A). The light stimuli (photons/011123 at 460 nm) were generated by a xenon arc lamp and attenuated by neutral density filters: Nl)4.0 (2.8x10"). ND3.() (1.4x no"). ND2.5 0'5); ND2.()(1.6xl()'6). NDLO (l.3xl0’7), NDO (1.2x10'8). (B) The same current traces are shown at a different current scale. The traces d by arrows are evoked by the same light intensity (Nl)2.5).

Figure 2 shows representative recordings of the light-evoked currents from wild-type (WT) ChRZ, 'l'159C, L132C, Ll32C/l‘lS9C, and Ll32C/l‘l598 mutants to a 10 ms light pulse (1.2x10l8 photons/cmZIS at 460 run) in IIEK cells for comparison of their deactivation time course (decay time course after light off).

Figure 3 shows entative hannel array recordings of WT ChRZ, Ll32C, L132C/T159C, and I/T159S mediated spiking activities from l ganglion cells in retinal whole-mounts for comparison of their light sensitivity. Light stimuli (photons/cmzls) was generated by a 473 nm blue laser and attenuated by neutral density filters: NDO (6.3x10'"), Nl)l.0 (7.4x10'5). Nl)l.5 (2.7x10'5), ND2.() (7.3x10"), ND2.5 (3.2x10"), Nl)3.() (8.5x10"). ND3.5 (3.8x10"), and ND4.0 0'2).

Figure 4 shows representative multichannel array recordings of WT ChRZ, Ll32C, 7/1‘159C, and LI32C/’l‘159S mediated spiking activities from retinal ganglion cells in retinal whole-mounts for comparison of their temporal dynamics. In each panel, the raster plots of 10 consecutive light~elicited spikes originated from a single neuron (top) and the averaged spike rate histograms (bottom) are shown. Light pulses at different frequency was generated by a 473 nm blue laser with intensities about one log unit above the threshold intensity of each mutant. Recordings of WT ChR2 and Ll32(.‘ are shown in (A). and recordings of Ll32(.‘./1"159(.‘ and l.l32(.‘/'l‘l59S are shown in (B).

DETAILED DESCRIPTION Visual System The central nervous system mediates vision (also referred to herein as sight) through specialized cells and unique methods of signal transduction present in the visual system. The principle sibility of the visual system is to transfonn light. in the form of electromagnetic radiation. into a representation or image of the surrounding world. In addition to the "visual" function of this system. the visual system also regulates the pupillary light reflex (PLR), circadian photoentrainment to periodic light/dark , and release of the hormone melatonin.

The cells of the retina are the first cells of the visual or nervous system to encounter light romagnetic radiation of varying wavelengths and intensities). s travel through the cornea, pupil, and lens before reaching the . The retina has a unique structure because the eceptor cells that directly absorb photons are located in the outer layer of the retina. Photons that traverse the lens first encounter an inner layer of retinal ganglion cells (a minority of which are photosensitive through the expression of the opsin. melanopsin) and an intermediate layer of r cells before reaching the outer layer of photoreceptor cells (also known as rods and cones). Rod photoreceptors operate in dim nation condition (scotopic vision) while cone photoreceptors operate in bright illumination conditions pic vision) responsible for color vision. Cone photoreceptors synapse directly onto ()N— and OFF-type cone bipolar cells, which in turn, synapse directly onto ()N— and OFF-type retinal ganglion cells. Rod photoreceptors synapse to rod bipolar cells (a unique type of bipolar cells, which is ON-type), which synapse to All amacrine cells. ’lhe All ne cells then relay the visual signals to ()N-type cone bipolar cells through gap junction and to OFF-type cone bipolar cells as well as Old" ganglion cells through inhibitory glycinergic synapses. Retinal ganglion cells are responsible for relating visual information to neurons of the brain.

Phototrausduction Within the retina. photoreceptor cells absorb photon particles and transform the raw data of light frequency and wavelength into chemical and subsequently electrical signals that propagate this initial information throughout the visual and nervous systems. Specifically. an opsin protein located on the surface of a photoreceptor (rod, cone, and/or photosensitive retinal ganglion cell) absorbs a photon and initiates an intracellular signaling e, which results in the olan'zation of the photoreceptor. [n the dark. the opsin proteins absorb no photons, the photoreceptors are depolarized. The visual signals of eceptors then relay through bipolar cells, amacrine cells, and on cells to the high visual centers in the brain. Specifically, when rod and cone photoreceptors are depolarized (in the dark), they cause the depolarization of rod bipolar cells and ()N—type cone bipolar cells, but the hyperpolarization of OFF—type cone bipolar cells, which in turn cause the depolarization of All amacrine cells and the se of the spiking of pe retinal ganglion cells and the decrease of the spiking of OFF-type retinal ganglion cells. The opposite happens (to nod. ()N- and polar cells. All amacrine and ON- and OFF-ganglion cells), when rod and cone photoreceptors are hyperpolarized (in response to light).

Light infomiation is processed and refined icantly by the actions of photoreceptors. bipolar cells, horizontal cells, amacrine cells, and retinal ganglion cells. To add to the complexity of this system, eceptors are found in three main varieties, including rods. cones (of which three types respond most strongly to distinct wavelengths oflight), and photosensitive retinal ganglion cells. Thus. a first layer of infonnation processing occurs at the level of the photoreceptors which respond differentially to certain ngths and intensities of light. Bipolar cells of the retina receive infonnation from both photoreceptor cells and ntal cells. Horizontal cells of the retina receive information from multiple photoreceptor cells, and, therefore, ate information between cell types and across distances in the retina. r cells further integrate infonnation directly from photoreceptor cells and horizontal cells by producing mainly graded potentials to retinal on cells, although some recent studies indicate that some bipolar cells can generate action potentials. Cone bipolar cells synapse on retinal ganglion cells and amacrine cells while rod r cells synapse only to All amacrine cells. Similar to horizontal cells, most amacrine cells integrate information laterally within the retina. Unlike ntal cells, most amacrine cells are inhibitory (GABAergic) intemeurons. Amacrine cells are also more specialized than horizontal cells, because each amacrine cell specifically synapses on a particular type of bipolar cell (one of the ten varieties of bipolar cell). Particularly, the All amacrine cell is a critical relay neuron in the rod pathway (under scotopic vision when cone eceptors do not respond). ’lhe All amacinre cells receive synaptic inputs from rod bipolar cells and then piggy—back the signals to cone pathway through ON- and OFF-cone bipolar cells to ON— and OFF— ganglion cells as described above. ore, expression of (Thop’l. and the resulting ion of (ThRCZ, in rod bipolar cells or All amacrine cells can create both ON and OH7 responses in retinal on cells. Furthermore, retinal ganglion cells integrate information from bipolar cells and from amacrine cells. Although retinal ganglion cells vary significantly with respect to size, connectivity, and responses to visual stimulation fag. visual fields). all retinal ganglion cells extend a long axon into the brain. lixcept for a minute portion of the l ganglion cells that transduce non- visual information regarding the ary light reflex and circadian entrainment. the totality of axons ing from the retinal ganglion cells fortn the optic nerve, optic chiasm, and optic tract of the central nervous system. Consequently, a icant amount of infonnation processing occurs in the retina . l’hotoreceptor cells express nous opsin proteins, such as rhodopsin. The mutant ChopZ proteins of the invention may be expressed in any cell type, and form functional ChR2 channels. Preferably. the cell is a retinal cell. ary cells, include, but are not limited to, photoreceptor cells (e.g., rods, cones. and photosensitive retinal ganglion cells), horizontal cells, bipolar cells. antacrine cells. and retinal ganglion cells.

Channelopsin-Z (ChapZ) Channelopsin-Z (ChopZ) was first isolated from the green algae, Chlamydomonas rein/mrdlii. Channelopsin-2 is a seven transmembrane domain protein that becomes photo-switchable (light sensitive) when bound to the chromophore ans—rctinal. ChopZ. when linked to a retinal molecule via Schiff base linkage forms a light-gated, nonspecific, inwardly rectifying, cation channel, called elrhodopsin-2 (Chop2 rctinalidene, abbreviated ChR’l).

As referred to herein, "channelopsin-Z" or " refers to the gene that encodes channelopsin-Z. which then forms (Thannelrhodopsin—Z ((ThR2) once bound to retinal. Gene constructs of the present invention refer primarily to channelopsin-2 (i. e., without the retinal). and all ChopZ variants disclosed herein form functional channelrhodopsin-2 variants. The methods disclosed herein may include delivering Chopl to cells without exogenous retinal. It is understood that upon expression of Chopl in cells (Le, retinal neurons), endogenously ble retinal binds to the wild- type (‘hop’l or the (Thopl mutants of the present ion to form functional light— gated channels, WT (‘hRZ or mutant (ThRZ. As such, ChopZ proteins, as referred to , can also be synonymous with ChRZ.

As used herein. "channelrhodopsin-Z" or "(ThRT refers to the retinal—bound onal light-sensitive channel. In one embodiment, the bound retinal may be provided exogenously. In a preferred embodiment. the bound retinal is ed from endogenous levels available in the cell. The present invention also encompasses the functional channelrhodopsin—‘Z channels formed by the polypeptides and polynucleotides encoding the Chop'Z mutants described herein.

Upon illumination by the preferred dose of light radiation. ChR’l opens the pore of the channel, h which 11*, NF, K", and/or (Ta2+ ions flow into the cell from the extracellular space. Activation of the ChR’.’ channel typically causes a depolarization of the cell expressing the channel. Depolarized cells e graded potentials and or action potentials to carry information from the ChopZ/ChRZ- expressing cell to other cells of the retina or brain. 'lhe wild type l‘onn ofChRQ or mutant ChRZs with high temporal resolution have become a central focus of neuroscience research. When expressed in a ian neuron, ChR2 es controlled depolarization of in vitro or ex viva cultures. Wild type ChRQs or mutant ChRQs with high al resolution (the latter usually dispay low light sensitivity) presents several challenges that must be addressed to enable their use for the purpose of vision restoration. lior the purpose of vision restoration, the (‘hR2 with high light sensitivity rather than high temporal resolution is desired.

Wild type ChR’Z proteins require illumination from high blue light intensities for full activation (Le. 10'8-1019 photons s' ' cm'2 at a wavelength of 480nm).

Continuous illumination of this type can damage cells.

The cs of the wild type ChR'l protein is suboptimal for maximizing channel efficacy. Efficacy can be increased by modifying one or more amino acids of the wild type ChR2 protein either to prolong the open state of the channel or increase the unit tance of the channel. or both. The —channel conductance of wild- type (‘hR2 is small. Thus, al activation in vivo would either require high expression of the wild type channel or very intense activation with the preferred wavelength of blue~lighL A simpler solution may be found by altering the channel conductance or to prolong the channel open time. [Either one of these mechanisms and. in particular, the combination of these isms, enable lower and safer light intensities to be used to achieve the same level of cellular depolarization.

For example. mutant ChR2 proteins of the invention achieve greater light sensitivity through the prolongation of the channel open state. Consequently, each mutant ChR’Z channel conducts a greater photocurrent than a wild type ChR2 channel when ted by the same light intensities. 'lherefore, the mutant channels are activated by light intensities that are lower than those required for activation of the wild type ChR’Z ls. Quantitatively, detectable spiking activity of retinal ganglion cells expressing mutant ChR2 proteins can be elicited by a light ity that[S l .-25 log units lower than the light intensity required to elicit spiking activity from retinal ganglion cells expressing wild type ChRZ. Thus. the light intensities required to activate the mutant ChRZ proteins are close to or fall within the range of normal Outdoor lighting conditions. 1116 following ces provide non-limiting examples of wild type and mutant Chop2 proteins, and polynucleotides encoding said WT and mutant ChopC’. proteins of the invention, and forming WT and mutant ChRZs of the invention.

A wild type (WT) Chop2 of the ion may be encoded by the following Chlamydomonas reinhardtii chlamyopsin 4 light-gated ion channel (COP4) mRNA sequence (Ganank Accession No. XM_001701673. and SEQ ID NO: 1): 1 gcagcaccat a0 I" rt \Q I'D () [ut: :gtcgccaag caagcattaa acatggatta tggaggcgcc 61 ctgagtgccg r" rt 10 \Q n ()0U»)01 gctgctattt gtaa:gaacc cagtagtcgt caatggctct 121 gtacttgtgc 0 0"quth H n» min gtgttactgc gcgggctgga ttgagtcgcg tggcacaaac 11 Ill .4 m P 0 ‘38 ggtgcccaaa 0 0 W 00 0(II1 1Q (lrt Mn (to W :ggcttgctg tctc catcctactg .pgsmw’v Mme.» .1. cttatgttt: m0'11"!)10091000 0 1110 (1110thWIQWm _ Isaacctgcg gctgggagga gatctatgtg HHHHHHH tgcgctatcg m H n m0 mm Dru 10w '0 inn Q\Qa: a: Ll (D!) :aattctc ttct - tcgagtttaa gaacccgtcc atgctgtatc er 0 IQ cgtc cagtggttgc gttacgccga gtggcttctc acctgcccgg mtg n 0 M fl 0 9'0nr9 \QH 0 BI acctgacgg gcttgtccaa cgactacagc m aggcgcacca r1 (11011) DithQf' SD 11'00 n IO (1 (1 1‘1 (t {I (1 0 rt 1Q HI r1 DI (1 r' acaa ttgtgtgggg cgccacttcc VOIONUW molb gccatggcca () m0 r0 11 rt "'9 rt O(1 tgggtctgcg ttatggtgct ttct r1 n10 0 r1 tQWtQtQDth(3 0 D: D) KQll}0 )i 0‘ O m(0 '0 DirItQ ()thO H r1 accacaccgt gccgaagggc cggtgtcgcc fl) nut} 1" (Q{Q I" 10 0 fl (QQ () I (1 rt () rt "in (1 tcgtatcatg gggtatgttc [\\ 1.4 cccatcctgt r1 r1 0 0 (1 0 it) 00 (ll0 Oman NW! 11 IQ LQO n tgagcgtgta cggctccacc 0.19 .bm Ht»! gtcggccaca ceaceaataa cctg)l I l ) I I r0010 DJ fl! 1001900 0: HI (l 1’le QLQlflrtrftQtQD ggggtctgct cggccactac ctgcgcgtgc tgatccacga gcata 1» r9 0 0 It! 0 In n acattcgcaa gaccaccaaa m o H :tgaacattg gtggcactga oattgaggtc aWluOI n zon \ tQLQrtnrt tggaggacga ggccgaggct 961 ggcgcggtca acaagggcac cggcaagtac gcctcccgcg agtccttcct ggtcatgcgc 1021 gacaagatga aggagaaggg :attgacgtg :gcgcctctc tggacaacao caaggaggtg 1061 gagc aggccgccag ggctgccatg atgatgatga atgg catgggtatg 1141 ggaatgggaa tgaacggcat gaasggaatg ggcggtatga tccc tggcggcgcc 1201 aagcccggcc tggagctcac tzcgcagcta cagcccggcc gcgtcatcct ggcggtgccg 1261 gacatcagca tggttgact: :ttccgcgag cagtttgctc cggt gacgtacgag 1321 ccgg :cctgggcgc tgacaacaca ctggcgctgg ttacgcagg: gcagaacctg 1381 ggcggcgtgg actttgtgt: gattcacccc gagttcctgc gcgaccgct: tagcaccagc 1441 agc: gcgg cgcggcccag :gtgtggctg :gttcggctg ggcgcagctgl l 1501 gggcccatgc gtgacctgat cgca aacctggacg gctggctgga gggcccctcg 1561 ttcggacagg gcatcctgcc ggcccacatc gttgccctgg tggccaagat gcagcagatg 1621 cgcaagatgc agcagatgca gcaga :ggc atgatgaccg gcggcatgaa cggcatgggc 1651 atgg gcggcggca: gaacggcatg ggcggcggca acggcatgaa caacatgggc 174 aacggcatgg gcggcggcat cggc ggca atggcatgaa cggaatgggt 1501 ggcggcaacg gcatgaacaa catgggcggc aacggaatgg ccggcaacgg aatgggcggc 1861 ggcatgggcg gcaacggtat ctc: atgaacggca ccgg cgtggtggcc 1921 aacgtgacgc cctccgccgc cggcggcatg acga tgaacggcgg catggctgcg 1951 :cccagtcgc ccggcatgaa cggcggccgc ctgggtacca acccgctct: caacgccgcg 2041 ccctcaccgc :cagctcgca gctcggt_cc gaggcaggca tgggcagcat gggaggcatg 2101 azga QCQQEat??? aggcatggg: 3953199??? gcacgggcgg C§CC 2161 gccacgacgc aggctgcggg cggcaa:gcq gaggcggaga tgctgcagaa tctcatgaac 2221 aatc gc-ctgaagcg cgagcttggc gagtaaaagg :tggaqgccg gtactgcga: 2281 acctgcgagc tcgcgcgcct gactcgtcgt acacacggct caggagcacg cgcgcgtgga 2341 cttctcaacc tgtgtgcaac agag cggcctgtgc gcgaccgtcc gtgagcattc 2401 cggtgcgatc ttcccgcctt cgcaccgcaa gttcccttcc tgct gcgcctgacg 2461 ccga acggaagggc ggcttgatca gtaaagcatt gaagactgaa gtcgtgcgac 2521 cgtagtgcta tggctctgca cgtaagtggg cgctgccctg cttactacgc attgcccaag 2581 tcc: tttggtggcc gaggccctgg atca :catttgca 2641 :ttagttaca tacgctttgc taacctcga caattgcaac atgggctgag 2701 9599323599 acgaaggtgt tatcggatgc gattaggaat ctcggttgaa 2761 aaagtgagct tcatctgtgg cttctgttgg ggtcatcaag aagaacgacg gtaaggcaaa 2821 cgaggtaaaa gzgqcacgtc tttgtgcaca acgggcccgt ggagagtggg ggagtgcatg 2381 :gtgcggtcc taacacgcga gtgcaaagcg ggcttttctg gagctgggtt acggtctggc 2941 tcggcaactg ctctgtgttt taaccacagc ttcggaagtc tgggtatgtt ttgttggcag 3001 aaacatttgg gtaacttgag ggtgattcgt tcgg tggc tgccgtccgt 3061 gtgcagggac ggtaatcaat gagctggagc gctc accacacgtt gcatacccc: 3121 gcttacaaaa acactttgat gtcgtggcca aactatgcgt gagcaaagag :taaagaggc 3181 atgagtgcat gqttgcggac gtgcgcaaca attgcatcaa gtatttgacg ccttcaagcc 3241 tgcg cgcgcggcaa cttgattaac acgccggacg cagtggtggg taca 3301 gtgtttatga attc tgcgatccgt agtgttaggt tgcgtgtgac cggc 3361 :gtgggccct tacatggaga gttgggtgc: tcaccacacg gttggcgccg ctgaagggtg 3421 tgctatgtt: tggtaaagcc ggggccctga agaccgcaac :gtagaaccg tactgaaagg 3481 gtgtcagccc ggggtaactg gatgccctgg gacatagcta ttaatgttga agtgaagccg 3541 :caagccgag tgccgtgcgc cgctgtatca ccaaggcccg tccta A wild type (WT) ChR2 of the invention may be encoded by the following Chlamydomonas reinhardtii chlamyopsin 4 light-gated ion channel (COP4) amino acid sequence (GenBank Accession No. XP_001701725. and SEQ ID NO: 2): 1 mdygga1sav gre11fvtnp vvvngsvlvp edqcycagwi esrgtngaqt asnvlqwlaa El gfsilllmfy aythkstcg weeiyvcaie mvkviiefff efknpsm1y1 qwl: 121 yaewiltcpv ilihlsnltg rrtm gllvsdigti vwgatsamat gyvkvificl 181 icygantff haakayiegy htvpkgrcrq vvtgmawlff pilf fgvi 241 svygstvght kncw gllghylrvl ihenilihgd irkttklnig gteievetlv 301 edeaeagavn kgtgkyasre sflvmrdkmk ekgidvrasl dnskeveqeq aaraammmmn 361 gngmgmgmgm ggmn gmaggakpgl eltpqlqur vilavpdism vdffreqiaq 421 isvtyelvpa 1gadnt1a1v tqaqnlggvd fvlihpeflr drsstsilsr rvaa 451 fgwaqlgpmr dliesanldg wlegpsfgqg ilpahivalv akmqqukmq qmqqigmmtg 541 ggmg ggmngmgggn gmnnmgngmg ggmgngmggn gmngmgggng mnnmggngma 501 gngmgggmgg ngmggsmngm ssgvvanvtp saaggmggmm nggmaapqsp gmnggrlgtn 651 plfnaapspl eagm smggmggms gmgg mggagaattq aaggnaeaem 721 lqnlmneinr 1krelge A wild type (WT) Chop2 of the invention may be encoded by the following Chlamydomonas reinhardtii retinal binding protein (cop4) gene sequence (GenBank Accession No. AF461397, and SEQ ID NO: 3): H gcatctgtcg ccaagcaagc attaaacatg gattazggag gcgccctgag tgccqttggg «memObflJK‘O‘. FIJIJIJIJIJIJ cgcgagctgc taac gaacccagta gtcgtcaatg gctctgtact tgtgcctgag Hbfl gaccagtgtn actgcgcggg ctggattgag tcgcgtggca caaacggtgc ccaaacggcg tcgaacgtgc tgcaatggct tgctgctggc atcc tactgcttat cgcc nhnhhbulk‘ :accaaaca: ggaagtcaac ctgcggctgg gaggagatct atgtgtgcgc :atcgagatg gtcaaggtga ttctcgagtt cttcttcgag tttaagaac: cgtccatgct gtatctagcc acaggccac: gcgtccagtg gttgcgttac tggc ttctcacctg cccggtcatt ctcattcacc tgtcaaacct gacgggcttg tccaacgact acagcaggcg caccatgggt H ctgcttgtgt ctgatattgg cacaatt :g tggggcgcca cttccgccat ggccaccgga ("11' o PH aagg tcatcttctt ctgcctgggt c:gtgttatg gtgctaaca: gttctttcacgctgccaagg :ctacatcga gggttaccac accgtgccga agggccggtg :cgccaggtg (1" m ,4. gtgactggca tggcttggct cgta tcatggggta tgttccccat cctgttcatc 721 ctcggccccg agggcttcgg :gtcctgagc gtgtacggct :caccgtcgg cacc 781 attgac:tga tgtcgaagaa :tgctggggt ctgctcggcc actacctgcg cgtgctgatc 841 ownm tcctcatcca :ggcgacatt cgcaagacca ccaaattgaa 901 monm aggtcgagac gctggtggag gacgaggccg §5§5 961 Wk" n-mo m n n agtacgcctc ccgcgagtcc ttcctggtca :"cgcgacaa gatgaaggag 1021 m\Q0 Wu)HtQ I10 as» acgtgcgcgc ~gac aagg aggtggagca ggagcaggcc 1081 ccatgatgat gatgaacgg: aatggcatgg gtatgggaat gggaatgaac 1141 0ou ruaruumruun 061000000 tflfltfldm \Qfi nad0nQam rtIQ m 0: 9‘0 L0 gcgg cggg ' gcgccaagcc cggcctggag 1201 n agctacagcc cggccgcgtc tgccggacat cagcatggtt 1261 r' gcgagcagtt tgctcagcta acgagctggt gccggccctg 1321 0 tggc gctggttacg acctgggcgg cgtggacttt 1351 OLQKQIQ 10‘" tan £00 accccgagtt cctgcgcgac :cagcatcc: gagccgcctg 1441 0 gccagcgtgt ggctgcgttc agctggggcc catgcgtgac 1501 D 00! f. 0QDIQN ccgcaaacct ggacggctgg :ctcgttcgg acagggcatc .1" fiU" m H ctgccggccc acatcgttgc cctggtggcc aagatgcagc agatgcgcaa gatgcagcag ‘AOIDU'JPU H atgcagcaga tgat gaccggcggc atgaacggca Igggcggng tatgggcggc Pl (h! Ho4»-Aier)r-w aacg gcatgggcgg cggcaacggc atgaacaaca tgggcaacgg catgggcgqc v.4 ggcatgggca acggcatggg cggcaatggc atgaacggaa caacggcatg Iggqtggcgg aacaacatgg gcggcaacgg aacggaatgg gggcqgcaac ._. l-‘OlOKDWuJ‘J aatggccggc gcggcggcat ggtatgggtg gctccatgaa cggcatgagc tccggcgcgg acgt gacgccctcc 0‘ ggcg gcatgggcgg gaac ggcggcatgg ctgcgcccca gtcgcccggc l-J atgaacggcg gccgcctggg taccaacccg ctcttcaacg :cgcgccct: acc :tcagc |\\ :cgcagctcg gtgccgaggc aggcatgggc agcatgggag gcatgggcgg aatgagcgga l\\ atgggaggca tgggtggaat ggggggcatg ggcggcgccg gcgccgccac gacgcaggc: \NJ NH Mm HP chggcggca acgcggaggc ggagatgctg Cagaatctca tgaacgagat caatcgcctg aagcgcgagc ttggcgagta a A wild type (WT) Chop’l of the invention may be d by the following Chlamydomonas neinhardtii l g n (cop4) amino acid ce (GenBank Accession No. AAM15777. and SEQ ID NO: 4): 1 mdyggalsav grellfvtnp vvvngsvlvp edqcycaqwi esrgtngaqt asnvlqwlaa 61 gfsilllmfy aythkstcg weeiyvcaie mvkvilefff efknpsmlyl atghrvqwlr 121 yaewlltcpv nltg lsndysrrtm gllvsdigti vwgatsamat gyvkviffcl 181 glcygantff haakayiegy htvpkgrcrq vvtgmawlff vswgmfpilf ilgpegfgvl 241 svygstvght iidlmskncw gllghylrvl ihehilihgd irkttklnig gteievetlv 301 edeaeagavn kgtgkyasre sflvmrdkmk ekgidvrasl dnskeveqeq aaraammmmn 361 gngmgmgmgm ngmngmggmn kpgl eltpqlqur vilavpdism vdffreqfaq 421 lsvtyelvpa 1gadntlalv t qaqn lggvd fvlihpeflr drsstsilsr lrgagqrvaa 481 fgwaqlgpmr dliesanldg fgqg ilpahivalv akmqqmrkmq qmqqigmmtg 541 gmngmgggmg ggmngmgqgn gmnnmgngmg ggmgngmggn gmngmgggng mnnmggngma 601 gngmgggmgg ngmggsmngm ssgvvanvtp saaggmggmm nggmaapqsp gmnggrlgtn 661 plfnaapspl eagm gsmggmggms gmggmggmgg mggagaattq aaggnaeaem 721 lqnlmneinr 1krelge A wild type (WI‘) Chop’2 of the invention may be encoded by the following Chlamydomonas reinhardtii sensory opsin B (CSOB) mRNA ce (GenBank Accession No. 1413508966, and SEQ ID NO: 5): 1 ttgacatctg tcgccaagca agcattaaac atggattatg gaggcgccct gagtgccgtt 61 gggcgcgagc tgctatttgt aacgaaccca gtagtcgtca atggctctgt acttgtgcct 121 gaggaccagt gttactgcgc gggctggatt gagtcgcgtg gcacaaacgg tgcccaaacg 181 gcgtcgaacg tgctgcaatg gcttgctgct ggcttctcca tcctactgct tatgttttac 241 gcctaccaaa catggaagtc aacctgcggc tgggaqgaga tqtg cgctatcgag 301 atggtcaagg tgattctcga cttc gagtttaaga acccgtccat tcta 361 gccacaggcc accgcgtcca gtggttcht tacgccgagt ggcttctcac ctgcccggtc 421 attctcattc acctgtcaaa cctgacgggc ttgtccaacg actacagcag catg 481 ggtctgcttg tgtctgatat tggcacaatt gtgtggggcq ccacttccgc cacc 541 ggatacgtca aggtcatctt cttctgcctq ggtctgtgtt atggtgctaa cacgttcttt 601 cacgctgcca acat cgagggttac cacaccgtgc cgaagggccg gtgtcgccag 661 qtggtgactq gcatggcttg gctcttcttc gtatcatgqg gtatgttccc catcctgttc 721 atcctcggcc ccgagggctt cggcgtcctg agcgtgtacg gctccaccgt cggccacacc 781 atcattgacc tgatgthaa gaactgctgg ggtctgctcg gccactacct gctg 841 atccacgagc tcat ccacggcgac aaga ccaccaaatt gaacattggt 901 ggcactgaga ttgaggtcga qa¢gctqqtg gaggacgagg ccgaggctgg cgcggtcaac 961 aagggcaccg gcaagtacgc ctcccgcgag tccttcctgq tcatgcgcga gaag 1021 gagaagggca ttgacgtgcg cgcctctctg gacaacagca aggaggtgga gcaggagcag 1081 gccgccaggg ctgccatgat gatgatgaac ggcaatggca tgggtatggg aatgggaatg 1141 aacggcatga acggaatggg gaac gggatggctg ccaa gcccggcctg 1201 gagctcactc cgcagctaca gcccggccgc gtcatcctgq cggtgccgga catcagcatg 1261 gttgacttct tccgcgagca tcaq ctatcggtga cgtacgagct ggtgccggcc 1321 ctgggcgctg acaacacact ggcgctggtt acgcaggcgc agaacctggg cggcgtggac 1381 tttgtgttga ttcaccccga gttcctgcgc tcta gcaccagcat cctgagccgc 1441 ctgcgcggcg cgggccagcg tqtggctgcg ttcggctggg tggg gcccatgcgt 1501 gacctgatcg agtccgcaaa cctggacggc gagg gcccctcgtt cqgacagggc 1561 atcctgccgg cccacatcgt tgccctggtg gccaagatgc agcagatgcg caagatgcag 1621 cagatgcagc agattggcat gatgaccggc ggcatgaacg gcatgggcgg cggtatgggc 1681 ggcggcatga acggcatggg cggcggcaac ggcatgaaca acatgggcaa cggcatgggc 1741 ggcggcatgg gcat caat aacg gaatgggtgg cggcaacggc 1801 atgaacaaca tgggcggcaa cggaatggcc ggcaacggaa tgggcggcgg catgggcggc 1861 aacggtatgg gtggctccat gaacggcatg agctccggcg tggtggccaa cgtgacgccc 1921 tccgccgccg gcggcatggg gatg aacggcggca tggctgcgcc gccc 1981 ggcatgaacg gcct gggtaccaac ccgctcttca acgccgcgcc ctcaccgctc 2041 agctcgcaqc tcggtgccga ggcaggcatg ggcagcatgg tggg cggaatgagc 2101 ggaatgggag gcatgggtgg aatggqqggc atgggcggcg ccggcgccgc cacgacgcag 2161 9¢t9¢999¢9 gcaacgcgga ggcggagatg ctgcagaatc tcatgaacga gatcaatcgc 2221 ctgaagcgcg agcttggcga gtaaaaggct ggaggccgqt atac ctgcgagctc 2281 gcgcgcctga ctcgtcgtac acacggctca ggagcacgcg cgcgtggact tctcaacctg 2341 tgtgcaacgt atctagagcg gcctgtgcgc gaccgtccgt gagcattccg gtgcgatctt 2401 cccgccttcg caccgcaagt tcccttcctg gccctgctgc gcctgacgca tcgtccgaac 2461 ggaagggcgg cttqatcagt aaagcattga agactgaagt cgtgcgaccg tagtgctatg 2521 gctctgcacg taagtgggcg ctgccctgct gcat tgcccaagac tgcttccttt 2581 tggtggccga ggccctggtc ccacatcatt catttgcata acgtactgtt taqttacata 2641 cgctttgctt aacctcgaca attgcaacat gggctgagag tccgtacggc ggctatggac 2701 gaaggtgtta tcggatgtga atct cggttgaaag gcttcgagaa agtgagcttc 2761 ttctgtggct tctgttgggg tcatcaagaa gaacgacggt aaggcaaacg aggtaaaagt 2821 ggcacgtctt tgtgcacaac QQQCCCQtQQ agagtggggg agtgcatgtg tgcggtccta 2881 acacgcgagt gcaaagcggg cttttctgga gctgggttac ggtctggctc ggcaactgct 2941 ctgtgtttta accacagctt cggaagtctg ggtatgtttt gttggcagaa acatttgggt 3001 aacttgaggg tgattcgtct ggagtcggac gctg gtgt acgg 3061 taatcaatga agctgaagct ctca ccacacgttg catacccctg cttacaaaaa 3121 cactttgatg ccaa actatgcgtg agcaaagagt taaagaggca tgagtgcatg 3181 gacg tgcgcaacaa ttgcatcaag tatttgacgc cttcaagcca acaagtgcgc 3241 gcgcggcaac ttgattaaca cgccggacgc aqtggtggqg gcgtgtacag tgag 3301 ctgccattct gcgatccgta gtgttaggtt gcgtgtgacg ccgcgcggct gtgggccctt 3361 acatggagag ttgggtgctt acgg ttggcgccgc tgaagggtgt gctatgtttt 3421 ggtaaagccg gggccctgaa aacc gtagaaccgt actgaaaggq tgtcagcccg 3481 gggtaactgg atgccctggg acatagctat taatgttgaa gtgaagccgt caagccgagt 3541 gccgtgcgcc gctgtatcac caaggcccgt ccaaaaaaaa aaaaaaaaaa aaaaaaaaa A wild type (WT) Chop2 of the ion may be encoded by the following domonas reinhardtii sensory opsin B (CSOB) amino acid sequence (GenBank Accession No. AAM44040, and SEQ ID NO: 6): 1 mdyggalsav grellfvtnp vvvngsvlvp agwi esrgtngaqt asnvlqwlaa 61 gfsilllmfy aythkstcg weeiyvcaie mvkvilefff efknpsmlyl atghrvqwlr 121 yaewlltcpv ilihlsnltg lsndysrrtm gllvsdigti vwgatsamat gyvkviffcl 181 glcygantff haakayiegy htvpkgrcrq vvtgmawlff vswgmfpilf ilgpegfgvl 241 vght kncw gllghylrvl ihehilihgd irkttklnig gteievetlv 301 edeaeagavn kgtgkyasre sflvmrdkmk ekgidvrasl dnskeveqeq aaraammmmn 361 gngmgmgmgm ngmngmggmn gmagqakpql eltpqlqur Vilavpdism vdffreqfaq 421 lsvtyelvpa 1gadnt1alv tqaqnlggvd fvlihpeflr drsstsilsr lrgagqrvaa 481 fgwaqlgpmr dliesanldg wlegpsfgqg ilpahivalv akmqqmrkmq qmqqigmmtg 541 gmngmgggmg ggmngmgggn gmnnmgngmg ggmgngmggn gmngmgggng mnnmggngma 601 gngmgggmgg ngmggsmngm ssgvvanvtp saaggmggmm nggmaapqsp gmnggrlgtn 661 plfnaapspl ssqlgaeagm gsmggmggms gmggmggmgg attq aaggnaeaem 721 lqnlmneinr lkrelge A wild type (W'l‘) Chop?! of the invention may be encoded by the following Chlamydomonas neinhardtii ac0p2 111RNA for archacal-typc opsin 2 nucleic acid sequence (GenBank Accession No. ABOS8891, and SEQ 1]) NO: 7): 1 tcgc caagcaagca ttaaacatgg attatggagg cgccctgagt gccgttgggc 61 gcgagctgct atttgtaacg aacccagtag tcgtcaatgg actt 9t9CCt9a99 121 accagtgtta ctgcgcgggc tggattgagt cgcgtggcac tgcc caaacggcgt 181 cgaacgtgct gctt gctgctggct tctccatcct actgcttatg ttttacgcct 241 catg gaagtcaacc tgcggctggg aggagatcta tgtgtgcgct atcgagatgg 301 tgat tctcgagttc ttcttcgagt ttaagaaccc gtccatgctg tatctagcca 361 caggccaccg cgtccagtgg ttgcgttacg ccgagtggct tctcacctgc ccggtcattc 421 tcattcacct gtcaaacctg acqggcttgt ccaacgacta cagcaggcgc accatgggtc 481 tgcttgtgtc tgatattggc acaattgtgt ccac ttccgccatg gccaccggat 541 acgtcaaggt catcttcttc tgcctgggtc tgtgttatgg tgctaacacg cacg 601 ctgccaaggc ctacatcgag ggttaccaca ccgtgccgaa ggqccgqtgt cgccaggtgg 661 tgactggcat ggcttggctc ttcttcgtat catggggtat gttccccatc ctgttcatcc 721 ccga gggcttcggc agcg tgtacggctc caccgtcggc cacaccatca 781 tgat gtcgaagaac ggtc tgctcggcca ctacctgcgc gtgctgatcc 841 acgagcatat cctcatccac ggcgacattc gcaagaccac caaattgaac attggtgqca 901 ctqagattga gqtcgagacg ctggtggagg acgaqgccga ggctggcgcg gtcaacaagg 961 gcaa gtacgcctcc cgcgagtcct tcat gcgcgacaag atgaaggaga 1021 agggcattga cgtgcgcgcc tctctggaca acagcaagga ggtggagcag gagcaggccg 1081 ccagggctgc catgatgatg atgaacggca atqgcatqqq tatgggaatg ggaatgaacg 1141 gcatgaacgg aatgggcggt atgaacggga tggctggcgg cgccaagccc gagc 1201 tcactccgca gctacagccc ggccgcgtca cggt gccggacatc agcatggttg 1261 acttcttccg cgagcagttt gctcagctat cggtgacgta cgagctggtg ctgg 1321 gcgctgacaa cacactggcg ctqgttacgc aggcgcagaa cctgggcggc gtggactttg 1381 ttca ccccgagttc ctgcgcgacc gctctagcac cagcatcctg agccgcctgc 1441 gcggcgcggg ccagcgtgtg gctgcgtth gctgggcgca gccc atchtgacc 1501 tgatcgagtc cgcaaacctg gacggctggc tggagggccc ctcgttcgga cagggcatcc 1561 tgccggccca catcgttgcc ctggtggcca agatgcagca gatgcgcaag atgcagcaga 1621 tgcagcagat tggcatgatg accggcggca tgaacggcat gggcggcggt atqggcggcg 1681 gcatgaacgg cggc ggcaacggca tgaacaacat gggcaacggc atgggcggcg 1741 gcatggqcaa cggcatqggc ggcaatggca tgaacggaat gggtggcggc atga 1801 acaacatggg cggcaacgga atggccggca acqgaatggg cggcgqcatg ggcggcaacg 1861 gtatgggtgg ctccatgaac ggcatgagct ccggcgtggt ggccaacgtg acgccctccg 1921 CCgCngng catgggcggc atgatgaacg chgcatggc tgcgccccag tcgcccggca 1981 9099 ccgcctgggt accaacccgc tcttcaacgc cgcgccctca ccgctcagct 2041 cgcagctcgg tgccgaggca ggcatgggca gcatqggagg catgggcgga atgagcggaa 2101 gcat qqqtggaatg atgq gcggcgccgg cgccgccacg gctg 2161 cgggcggcaa cgcggaggcg gagatgctgc agaatctcat gaacgagatc aatcgcctga 2221 agcgcgagct tggcgagtaa aaggctggag gccggtactg cgatacctgc gagctcgcgc 2281 gcctgactcg tcgtacacac ggctcaggag cacgcgcgcg tggacttctc aacctgtgtg 2341 caacgtatct agagcggcct gtgcgcgacc gtccgtgagc attccggtgc gatcttcccg 2401 ccttcgcacc tccc ttcctggccc tgctgcgcct gacqcatc A wild type (W'I‘) ChopZ of the ion may be d by the ing Chiamydomonas reinhardtii acop2 mRNA for archaeal-type opsin 2 amino acid sequence (GenBank Accession No. BAB68567, and 8150 II) NO: 8): 1 mdyggalsav grellfvtnp vvvngsvlvp edqcycagwi esrgtngaqt asnvlqwlaa 61 gfsilllmfy aythkstcg weeiyvcaie mvkvilefff efknpsmlyl atghrvqwlr 121 yaewlltcpv ilihlsnltg 15ndysrrtm gllvsdigti vwgatsamat gyvkviffcl 181 glcygantff haakayiegy htvpkgrcrq vvtgmawlff vswgmfpilf ilgpegfgvl 241 svygstvght iidlmskncw gllghylrvl ihehilihgd irkttklnig gteievetlv 301 gavn kgtgkyasre sflvmrdkmk ekgidvrasl dnskeveqeq aaraammmmn 361 gngmgmgmgm ngmngmggmn gmaqgakpgl eltpqlqur vilavpdism vdffreqfaq 421 lsvtyelvpa lalv tqaqnlggvd fvlihpeflr ilsr lrgagqrvaa 481 fgwaqlgpmr dliesanldg wlegpsfgqq ilpahivalv akmqqmrkmq mmtg 541 gmngmgggmg ggmngmgggn gmnnmgngmg ggmgngmggn gmngmgggng mnnmggngma 601 gngmgggmgg ngmggsmngm ssgvvanvtp saaggmggmm nggmaapqsp lgtn 661 pspl ssqlgaeagm gsmqgmggms gmggmggmgg mggagaattq aaggnaeaem 721 lqnlmneinr lkrelge ChRZ s The present invention provides Chop2 mutants wherein one or more amino acids are mutated. In some embodiments, the (‘hopZ is the full-length ptide, such as SEQ 1]) N05: 2, 4. 6, and 8, with at least one amino acid mutation. In some ments, the mutation is at amino acid 132 and/or amino acid 159. In some preferred embodiments. the amino acid at position 132 is mutated from a e to a cysteine or an alanine. In some preferred embodiments, the amino acid at position 159 is mutated from a threonine to an alanine. a cysteine, or a serine. In all eembodiments. the Chop2 mutants fonn a functional ChRZ channel.

The present invention also encompases Chop’l proteins and nucleic acids that encode a biologically active fragment or a conservative amino acid substitution or other mutation variant of Chop'l. Non-limiting examples of useful fragments include polypeptides encoding amino acids l-315 of the wild-type ChopZ. i. e., SEQ [1) NO: 26, wherein at least one amino acid is mutated or conservatively substituted, for example at amino acid positions 132 and/or 159. Smaller fragments of wild-type Chop2. wherein at least one amino acid is mutated or conservatively tuted (i.e., at amino acid positions 132 and/or 159) may also be useful in the present invention.

Accordingly, Chop2 polypeptides and nucleic acids of the t invention further include, but are not limited to, biologically active fragments encoding amino acids 1- 315, [-310, 1-300, 1-275, 1-250, 1—225, 1-200, 1-175, or 1—160 of the wild-type Ch0p2, wherein at least one amino acid is mutated or conservatively substituted, for example at amino acid ons 132 and/or 159. In other embodiments, the Chopi! polypeptides and nucleic acids of the present invention can be up to, or abuot, 315 anfinoackblong.310anfinoackklong,300anunoackblong,275anfinoackblong, 250anfinoackblong,225anunoackblong,200anunoackmlong.l75annnoackm long,orl60zunhu)ackblong.

AkanmmmCmmhflmemmenmwbemmmwbymemmwmg Synthetic construct hVCth -mKate-betahChR2(Ll32C) gene sequence (GenBank Accession No.1N836746, and SliQ ID NO: 9) with the following annotations, Gl’l’ sequence is in bold, 1.132C ChopZ sequence is underlined:: 1 atggattacc cccg gtccctgatt tacc ccaccgatct tqga 61 accgtgtgca tgcccagaqg acaatgctac tgcgaqggqt ggctgagqag ccggggcact 121 agtatcgaaa aaaccatcgc tatcaccctc cagtgggtag tqttcgctct gtccgtagcc 181 tgtctcggct ggtatgcata ctgq agggctacct gtggqtqgga ggaagtatac 241 gtggccctga tcgagatgat gaagtccatc atcgaggctt tccatgagtt cgactcccca 301 gccacactct gcag tgggaatggc gtagtqtgqa tgagatatgg agagtggctg 361 ctgacctgtc ccgtcctgct cattcatctg tccaatctga ccgggctgaa agatgactac 421 tccaagagaa caatgggact gctgqtqaqt qacgtqggqt gtattgtgtg gggagccacc 481 tccgccatgt gcactggatg gaccaagatc ctctttttcc tgatttccct tggg 541 atgtatacat acttccacgc cgctaaggtg tatattgagg ccttccacac tgtacctaaa 601 ggcatctgta gggagctcgt gcqggtqatq gcatggacct tctttgtggc ctgggggatg 661 ttccccgtgc tqttcctcct cggcactgag ggatttggcc acattagtcc ttacgggtcc 721 ggac actccatcct ggatctgatt gccaagaata tgtngggqt gctgggaaat 781 tatctgcggg taaagatcca cgagcatatc ctgctgtatg gcgatatcag aaagaagcag 841 aaaatcacca ttgctggaca gqaaatqgaq gtggagacac tggtagcaga ggaggagqac 901 gggaccgcgg ccat ggtgtctaag ggcgaagagc tgattaagga guacatgcac 961 atgaagctgt aggg caccgtgaac aaccaccact tcaagtgcnc atccgagggc 1021 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct 1081 ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcaa aaccttcatc 1141 aaccacaccc agggcatccc cgacttcttt aagcagtcct tccctgaggg atgg 1201 gagagagtca ccacatacga SQQC gtgctgaccg ctacccagga caccagcctc 1261 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc 1321 cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgngatgct gtaccccgct 1381 gacggcggcc tggaaggcag agccgacatg aagc t¢9t999599 gggccacctg 1441 atctgcaact tgaagaccac atacagatcc aagaaacccg ctaagaacct caagatgccc 1501 ggcgtctaat atgtggacag angactggaa agaatcaagg aggccgacaa agagacctac 1561 cagc acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac 1621 aaacttaatt gcctgcagga gaagaagtca tgcagccagc gcatggccga attccggcaa 1681 tactgttgga acccggacac tqqgcaqatq ctgggccgca cccg gtgg 1741 atcagcctgt actatgcagc tttctacgtg gtcatgactg ggctctttgc catc 1801 ctga tgcagaccat tgatccctac acccccgact accaggacca gttaaagtca 1861 ccgggggtaa gacc ggatgtgtat ggggaaagag ggctgcagat ttcctacaac 1921 atctctgaaa acagctctag acaggcccag atcaccggac agac tgagacattg 1981 ccaccggtgg actacggggg ggccctgagc gctgtgggca gagaactcct gaca 2041 gtcg tggtgaacgg ctccgtactc gagg atcagtgcta ttgcgcagga 2101 tggatcgaga gcagaggcac aaacggcgca cagactgcat ccaacgtgct ccagtggttg 2161 gccgcaggct tttccattct cctgctcatg ttttacgcct accagacttg gaagtccaca 2221 tgtggctggg aggaaatcta cgtgtgtgca atcgaaatgg tgaaggtgat cctggagttt 2281 ttcttcgaat ttaaaaaccc aagcatgctg tacctggcta ctggccacag agtgcagtgg 2341 tatg ggct gctgacttgc ccagtgattt gcatccacct gtccaacctg 2401 ctgt ctaacgatta cagtaggaga acaatgggac tactcgtatc cgacatcggc 2461 actatcgtat gaggcgcaac tagtgccatg gccactggat acgtgaaagt gatcttcttc 2521 tgcctgggac tctgctacgg agcaaacaca ttttttcatg ccgcaaaagc atatatcgag 2581 gggtatcata ccgtcccaaa 333ccggtgt agacaagtgg tgactggcat ggcttggctg 2641 ttcttcgtgt cctgggggat gtttcccatc ctctttatcc taggcccaga aggcttcggg 2701 gtgctgagtg tgtatggcag taccgtagga cacactatca ttgacctgat gagcaaaaac 2761 tgctgggggc tgctcggcca ctacctgaga atcc acgagcatat cctgattcat 2821 ggcgatatcc ctac caagctcaat atcgggggca ttga agtggagaca 2881 ctcgtggagg acgaggccga ggccggagca gtgaacaaag gcactggcaa gtatgcctcc 2941 agagaatcct tgat gcgggacaaa atgaaggaga aaggcattga tgtacggtgc 3001 agtaatgcca aagccgtcga gactgatgtg tag A single mutant ChR2 of the invention may be encoded by the following Synthetic construct hVChRl—mKate-betahChR2(Ll32C) amino acid sequence (GenBank Accession No. Al3R29839, and SEQ ID NO: l0) with the following annotations, GFP sequence is in bold, L132C Chop2 sequence is underlined: 1 mdypvarsli vryptdlgng tvemprgqcy cegwlrsrgt siektiaitl quvfalsva 61 clgwyayqaw ratcgweevy valiemmksi ieafhefdsp atlwlssgng vvwmrygewl 121 ltcpvllihl snltglkddy skrtmgllvs dvgcivwgat samctgwtki lfflislsyg 181 mytyfhaakv yieafhtvpk gicrelvrvm awtffvawgm fpvlfllgte gfqhismgs 241 aighsildli aknmwgvlgn ylrvkihehi rkkq kitiagqeme vetlvaeeed 301 gtavatmvs: geelikenmh mklymegtvn nhhfkctseg egkpyegtqt mrikvveggp 361 1pfafdi1at sfmygsktfi nhtqgipdff kqsfpegftw ervttyedgg vltatthsl 421 ngcliynvk irgvnfpsng pvmqkktlgw eastemlypa dgglegradm alklvggghl 481 icnlkttyrs kkpnknlkmp gvyyvdxrle rikeadkety avar ycdlpsklgh 541 klnclqekks csqrmaefrq ycwnpdtgqm lgrtparwvw islyyaafyv alci 601 yvlmqtidpy tpdyqdqlks pgvtlrpdvy qerglqisyn isenssrqaq itgrpetetl 661 ppvdxggals lfvt nEvvvngsvl veedgcycag wiesrgtnga lgwl 721 aagfsilllm wkst cgweeixvca iemvkvilef ffefkngsml ylatghrvgg 781 lrxaewlltc Evicihlsnl tglsndxsrr tmgllvsdig tivwgatsam atgyvkviff 841 clglcxgant ffhaakaxie gxhtvgkgrc rgvvtgmawl ffvswggfgi lfilggegfg 901 Vlsvxgstvg htiidlmskn cwgllghxlr vlihehilih tkln igggeievet 961 lvedeaeaga vnkgtgkyas resflvmrdk mkekgidvrc snakavetdv A single mutant ChopiZ ol' the invention may be encoded by the following Synthetic construct hVCth-ntl(ate—betahChR2(L132C) gene sequence (GenBank Accession No.1N836745, and SEQ ID NO: I l) with the following annotations, GFP sequence is in bold. Ll32C (Thop2 sequence is underlined: 1 atggattacc ctgtggcccg gtccctgatt gtaagatacc ccaccgatct gggcaatgga 61 tgca tgcccagagg acaatgctac tchaqgggt 99a9 ccggggcact 121 agtatcgaaa aaaccatcgc tatcaccctc cagtgggtag ctct gtccgtagcc 181 tgtctcggct cata ccaagcctgg agggctacct gtgggtggga ggaagtatac 241 gtggccctga tcgagatgat gaagtccatc atcgaggctt tccatgagtt cgactcccca 301 gccacactct ggctcagcag tgggaatggc gtagtgtgga atgg agaqtqgctg 361 ctgacctgtc ccgtcctgct cattcatctg tccaatctga ccgggctgaa aqatgactac 421 tccaagagaa caatgggact gctggtgaqt gggt gtattgtgtg gggagccacc 481 tccgccatgt gcactggatg gaccaagatc ctctttttcc tgatttccct ctcctatggg 541 atgtatacat acttccacgc cgctaaggtg tatattgagq ccttccacac taaa 601 ggcatctgta QQQaQCtCQt chggtqatg gcatggacct tctttgtggc ctqggggatg 661 ttccccgtgc tgttcctcct cggcactgag ggcc acattagtcc ttacgggtcc 721 gcaattggac tcct gatt gccaagaata tgtggggggt gctgggaaat 781 tatctgcggg taaagatcca tatc tatg gcgatatcag aaagaagcag 841 aaaatcacca ttgctggaca ggag gtggagacac tggtagcaga ggaggaggac 901 gggaccgcgg tcgccaccat ggtgtctaag gagc tgattaagga gancatgcac 961 atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc 1021 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga ccct 1081 ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcaa aaccttcatc 1141 accc agggcatccc cgacttcttt tcct tccctgaggg atgg 1201 gagagagtca ccacatacga €98=§9999¢ gtgctgaccg agga caccagcctc 1261 caggacggct gcctcatcta caag atc‘Q‘QQQQ tgaacttccc atccaacggc 1321 cctgtgatgc agaagaaaac actcggctgg tcca tgct gtaccccgct 1381 gacggcggcc tggaaggcag agccgacatg gccctgaagc tcgtgggcgg gggccacctg 1441 atctgcaact tgaagaccac atacagatcc aaganacccg ctaagaacct cangatgccc 1501 ggcgtctact atgtggacag aagactggaa agaatcaagg aggccgacaa agagacctac 1561 gtcgagcagc tggc tgtggccaga tactgcgacc tccctagcaa actggggcac 1621 aaacttaatt gcctgcagga gtca tgcagccaqc gcatggccga attccggcaa 1681 tactgttgga acccggacac tgggcagatg ctgggccgca ccccagcccg gtgggtgtgg 1741 atcagcctgt actatgcagc tttctacgtg gtcatgactg ggctctttgc cttgtgcatc 1801 ctga tgcagaccat tqatccctac acccccgact accaggacca gttaaagtca 1861 ccgggggtaa ccttgagacc gtat 9999535959 ggctgcagat ttcctacaac 1921 atctctqaaa acagctctag acaggcccag atcaccggac gtccggagac tgagacattg 1981 ccaccggtgg actacggggg ggccctgagc actgtgggca gagaactcct gaca 2041 aatccagtcg tggtgaacgg ctccgtactc gtacccgagg gcta ttgcgcagga 2101 tggatcgaga gcagaggcac aaacggcgca cagactgcat ccaacgtgct ccagtggttg 2161 gccgcaggct tttccattct cctgctcatg ttttacgcct accagacttg gaagtccaca 2221 tgtggctggg aggaaatcta cgtgtgtgca atgg tgat cctggagttt 2281 ttcttcgaat ttaaaaaccc aagcatgctg tacctggcta ctggccacag agtgcagtgg 2341 ctgcggtatg ccgaatggct gctgacttgc ccagtgattc tgatccacct gtccaacctg 2401 actgggctgt ctaacgatta gaga acaatgggac tgctcgtatc cggc 2461 actatcgtat gaggcgcaac tagtgccatg gccactggat acgtgaaagt gatcttcttc 2521 tgcctgggac tctgctacgg agcaaacaca ttttttcatg ccgcaaaagc atatatcgag 2581 gggtatcata caaa gagecggtgt agacaagtgg tgactggcat ggcttggctg 2641 ttcttcgtgt cctgggggat gtttcccatc ctctttatcc taggcccaga aggcttcggg 2701 gtgctgagtg tgtatggcag taccgtagga cacactatca ttgacctgat gagcaaaaac 2761 tgctgggggc tgctcggcca ctacctgaga gtactcatcc acgagcatat cctgattcat 2821 ggcgatatcc ggaaaactac caagctcaat atcgggggca ccgagattga agtggagaca 2881 ctcgtggagg acgaggccga ggccggagca gtgaacaaag gcactggcaa gtatgcctcc 2941 aqagaatcct ttctggtgat gcgggacaaa atgaaggaga aaggcattga tgtacggtgc 3001 agtaatgcca aagccgtcga gactgatgtg tag A single mutant Chop2 of the invention may be encoded by the following Synthetic construct hVCth ~mKatc-betahChR2(Ll32C) amino acid sequence (Ganank Accession No. 38. and SEQ ID NO: 12) with the following annotations. GFP sequence is in bold, 1.132C ChopZ ce is underlined: 1 mdypvarsli vryptdlgng tvemprgqcy cegwlrsrgt siektiaitl quvfalsva 61 clgwyayqaw eevy mksi ieafhefdsp atlwlssgng vvwmrygewl 121 ltcpvllihl snltglkddy skrtmgllvs dvgcivwgat samctgwtki 1fflislsyg 181 mytyfhaakv yieafhtvpk gicrelvrvm awtffvawgm fpvlfllgte gfghism/gs 241 aighsildli aknmwgvlgn ylrvkihehi llygdirkkq kitiagqeme eeed 301 gtavatmvsk geelikenmh mklymegtvn nhhfkctseg egkpyegtqt mrikvveggp 361 lpfafdilat sfmygsktfi nhtqgipdff kqsfpegftw ervttyedgg vltatthsl 421 ngcliynvk irgvnfpsng tlgv eastemlypa dgglegradm alklvggghl 481 icnlkttyrs lkmp gvyyvdrrle rikeadkety veqhevavar ycdlpsklgh 541 klnclqekks csqrmaefrq ycwnpdtgqm lgrtparwvw islyyaafyv vmtglfalci 601 yvlmqtidpy tpdyqdqlks pgvtlrpdvy gerglqisyn isenssrqaq itgrpetetl 661 ppvdyggals avgrellfvt nEvvvngsvl yEedgcxcag wiesrgtnga gtasnvlgwl 721 aagfsilllm fxaygtwkst cgyeeizvca iemvkvilef ffefkngsml rvgfi 781 lrvaewlltc Bvilihlsnl tglsndysrr tmgllvsdig tivwgatsam atngkviff 841 clglcxgant ffhaakayie gvhtvgkgrc rgvvtgmawl mfgi 1filggegfg 901 vlsvxgstvg htiidlmskn cwgllghzlr vlihehilih tkln iggteievet 961 lvedeaeaga vnkgtgkyas resflvmrdk mkekgidvrc snakavetdv A L 132C single mutant Chop2 of the ion may be encoded by the following amino acid sequence (positions 132 underlined and bolded, SEQ ID NO: 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG CAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV IEIHLSNLTG LSNDYSRRTM GLLVSDIGTI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF HAAKAYIEGY RCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK A ’l‘ I 59C single mutant Chop2 of the invention may be encoded by the following amino acid sequence (positions 159 underlined and . SEQ ID NO: l MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY STCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV ILIHLSNLTG LSNDYSRRTM GLLVSDIGEI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD LNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK A LlBZC/T159C double mutant Chop’l of the invention may be encoded by the following nucleotide sequence (SEQ ID NO: 15): 1 atggactacg ggggggctct gtctgctgtc gaac tgctgtttgt qactaaccct 61 gtcgtcgtga acgggagtgt gctggtccct gaggaccagt gctactgtgc cggctggatc 121 gaatcacgcg gaaccaacgg ggcccagaca gctagcaatg tgctgcagtg cgct 181 gggtttagta tcctgctgct gatgttctac gcctatcaga cttggaagtc aacctgcggc 241 gaaa tctacgtgtg tgag atggtgaaag tgatcctgga gttcttcttc 301 gagttcaaga acccaagcat gctgtacctg gctactggac tgca gtggctgaga 361 gaat tgac atgccccgtc atctgcattc acctgtccaa cctgacaggc 421 ctgagcaatg actactccag gagaactatg ggactgctgg tgtccgacat cggctgcatt 481 gtctggggag caacttctgc tatggcaacc ggatacgtga aggtcatctt tttctgcctg 541 gggctgtgct atggcgcaaa tacctttttc gcca aggcctacat tgaggggtat 601 cataccgtgc caaaaggccg gtgccgacag acag gaatggcttg gctgtttttc 661 gtctcttggg gaatgtttcc catcctgttc attctggggc ctgaagggtt cggcgtgctg 21 tctgtctacg gaagtacagt ggggcatact atcattgacc tgatqtccaa aaactgttgg 781 ggcctgctgg gacactatct gagagtgctg atccacgagc atatcctgat tcatggcgat 841 attcggaaga ccacaaaact gaatatcggc ggaaccgaga ttgaagtgga aacactggtq 901 gaagacgagg ctgaggctgg gaac aaggggactg gcaaa A Ll32Cl'1‘159C double mutant Chop2 of the invention may be encoded by the following amino acid sequence (positions 132 and 159 underlined and bolded.

SIEQ ID NO: 16): 1 MDYGGALSAV GRELLFVTNP VLVP EDQCYCAGWI GAQT WLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF MLYL ATGHRVQWLR 121 TCPV IEIHLSNLTG LSNDYSRRTM IGSI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK A T1598 single mutant Chop2 of the invention may be encoded by the following amino acid sequence (positions 159 underlined and bolded, SEQ ID NO: 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP AGWI ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL QWLR 121 YAEWLLTCPV ILIHLSNLTG LSNDYSRRTM GLLVSDIGEI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKITKLNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK ] A Ll32CI'11598 double mutant Chop2 of the invention may be encoded by the following nucleotide sequence (SEQ ID NO: 18): 1 atgqactacg ggggggctct gtctgctgtc gggagggaac tgctgtttgt gactaaccct 61 gtcgtcgtga acgggagtgt ccct gaggaccagt gctactgtgc cggctggatc 121 gaatcacgcg gaaccaacgg ggcccagaca aatg agtg cgct 181 gggtttagta tcctgctgct gatgttctac gcctatcaga cttggaagtc aacctgcggc 241 tgggaggaaa tctacgtgtg cgctattgag atggtgaaag tgatcctgga gttcttcttc 301 aaga acccaagcat gctgtacctg gctactggac accgagtgca gtggctgaga 361 tatgcagaat ggctgctgac atgccccgtc atctgcattc acctgtccaa cctgacaggc 421 ctgagcaatg ccag gagaactatg ggactgctqg tgtccgacat cggcagcatt 481 gtctggggag caacttctgc aacc ggatacgtga aggtcatctt tttctgcctg 541 ngctgtget atggcgcaaa tttc cacgcagcca acat tgaggggtat 601 cataccgtgc caaaaggccg gtgccgacag gtggtcacag cttg gctgtttttc 661 gtctcttggg ttcc catcctgttc attctggggc ctgaagggtt cggcgtgctg 721 tctgtctacg gaagtacagt ggggcatact atcattgacc tgatgtccaa aaactgttgg 781 ggcctgctgg gacactatct gagagtgctg atccacgagc atatcctgat tcatggcgat 841 attcggaaga ccacaaaact gaatatcggc ggaaccgaga ttgaagtgga aacactggtg 901 gaagacgagg ctgg ggctgtgaac aaggggactg gcaaa A Ll32C/T159S double mutant Chop2 of the invention may be encoded by the following amino acid sequence (positions 132 and 159 underlined and bolded.

SEQ ID NO: 19): l MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI ESRGTNGAQT WLAA 61 LMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV IEIHLSNLTG LSNDYSRRTM GLLVSDIGEI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF IEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW LRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK A L132A single mutant (‘hop2 of the invention may be encoded by the following amino acid sequence (position 132 underlined and bolded, SEQ ID NO: l MDYGGALSAV GRELLFVINP VVVNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE EFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV IAIHLSNLTG LSNDYSRRTM GLLVSDIGTI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKITKLNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK A L132A/I‘159C double mutant (ThopZ of the invention may be encoded by the following nucleotide ce (SEQ ID NO: 21): 1 ATGGACTACG GGGGGGCTCT GTCTGCTGTC GGGAGGGAAC TGCTGTTTGT GACTAACCCT 61 GTCGTCGTGA GTGT GCTGGTCCCI GAGGACCAGT GCTACTGTGC CGGCTGGAIC 121 GAATCACGCG GAACCAACGG GGCCCAGACA GCTAGCAATG AGTG GCTGGCCGCT 181 GGGTTTAGTA TGCT GATGTTCTAC GCCTATCAGA CTTGGAAGTC AACCTGCGGC 241 TGGGAGGAAA ICTACGTGTG CGCTATTGAG ATGGTGAAAG TGATCCTGGA GTTCTTCTTC 301 GAGTTCAAGA ACCCAAGCAT GCTGTACCIG GCTACTGGAC ACCGAGTGCA GTGGCTGAGA 361 TATGCAGAAT GGCTGCTGAC ATGCCCCGTC ATCGCCATTC ACCTGTCCAA CCTGACAGGC 421 CTGAGCAATG ACTACTCCAG GAGAACTATG GGACTGCTGG TGTCCGACAT CGGCTGCATT 481 GTCTGGGGAG CAACTTCTGC TATGGCAACC GGATACGTGA AGGTCATCTT TTTCTGCCTG 541 TGCT CAAA TTTC CACGCAGCCA AGGCCTACAT TGAGGGGTAT 601 CATACCGTGC CAAAAGGCCG GTGCCGACAG ACAG GAATGGCTTG GCTGTTTTTC 661 GTCTCTTGGG GAATGTTTCC CATCCTGTTC ATTCTGGGGC CTGAAGGGTT CGGCGTGCTG 721 TACG GAAGTACAGT GGGGCATACT ATCATTGACC TGATGTCCAA AAACTGTTGG 781 CTGG GACACTATCT GCTG GAGC ATATCCTGAT TCATGGCGAT 841 ATTCGGAAGA CCACAAAACT GAATATCGGC GGAACCGAGA TTGAAGTGGA AACACTGGTG 901 GAAGACGAGG CTGAGGCTGG GGCTGTGAAC ACTG GCAAA A Ll32A/T159C double mutant Chop2 of the invention may be encoded by the following amino acid sequence (positions 132 and 159 underlined and bolded, SEQ ID NO: 22): 1 MDYGGALSAV GRELLFVTNP VLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEBIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV IAIHLSNLTG RRTM GLLVSDIGEI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK A 'l‘159A single mutant Chop2 of the invention may be encoded by the following amino acid sequence (position 159 ined and bolded, SEQ ID NO: l MDYGGALSAV GRELLFVINP VVVNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF MLYL ATGHRVQWLR 121 TCPV ILIHLSNLTG LSNDYSRRTM IGAI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD LNIG GTEIEVETLV 301 BDEAEAGAVN KGTGK A L 132(7/1" 1 59A double mutant Chop’l of the invention may be encoded by the following nucleotide sequence (SEQ ID NO: 24): 1 tacg ggggggctct gtctgctgtc gggagggaac tgctgtttgt gactaaccct 61 gtcgtcgtga acgggagtgt gctggtccct gaggaccagt gctactgtgc cggctggatc 121 gaatcacgcg gaaccaacgg ggcccagaca gctagcaatg tgctgcagtg gctggccgct 181 gggtttagta tcctgctgct gatgttctac gcctatcaga cttggaagtc aacctgcggc 241 tgggaggaaa tctacgtgtg cgctattgag atggtgaaag tgatcctgga gttcttcttc 301 gagttcaaga acccaagcat gctgtacctg gctactggac accgagtgca gaga 361 tatgcagaat ggctgctgac atgccccgtc atctgcattc acctgtccaa aggc 421 ctgagcaatg actactccag gagaactatg ggactgctgg tgtccgacat cggcgccatt 481 gtctggggag caacttctgc tatggcaacc ggatacgtga aggtcatctt tttctgcctg 541 gggctgtgct atggcgcaaa tacctttttc gcca aggcctacat tgaggggtat 601 cataccgtgc caaaaggccg gtgccgacag gtggtcacag gaatggcttg gctgtttttc 661 gtctcttggg gaatgtttcc catcctgttc attctggggc ctgaagggtt cggcgtgctg 721 tctgtctacg gaagtacagt gqggcatact gacc ccaa aaactgttgg 781 ggcctgctgg gacactatct gctg atccacgagc atatcctgat tcatggcgat 841 attcggaaga ccacaaaact gaatatcggc ggaaccgaga ttgaagtgga aacactggtg 901 gaagacgaqg ctgg ggctgtgaac aaggggactg gcaaa A Ll32C/l‘159A double mutant Ch0p2 of the invention may be encoded by the following amino acid sequence (positions 132 and 159 underlined and bolded, SEQ ID NO: 25): 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA 61 LMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL QWLR 121 YAEWLLTCPV IEIHLSNLTG LSNDYSRRTM GLLVSDIGAI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF IEGY RCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK A wild type (W'l‘) ChopZ of the invention may be encoded by the following amino acid sequence (SEQ ID NO: 26): 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF MLYL ATGHRVQWLR 121 YAEWLLTCPV ILIHLSNLTG LSNDYSRRTM GLLVSDIGTI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ WLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK Mutant ChRZ proteins of the invention also demonstrate slower channel cs. Higher light sensitivity was found to correlate with slower channel kinetics. indicating a off between light sensitivity and channel kinetics. Chop2 proteins that fomi the ChR2 proteins of the present invention may also comprise onal mutations or modifications that may e l kinetics, or increase the deactivation rate, of the ChR2. Particularly preferred ChR2 mutants balance the threshold of light sensitivity with channel kinetics.

Compositions and kits Compositions and kits of the invention comprise at least one nucleic acid molecule or polypeptide le that s a mutant Chop2 protein, and the resulting ChRZ, of the invention. The at least one nucleic acid le or polypeptide molecule that encodes a mutant ChopZ protein of the invention may further include a phannaceutically-acceptible carrier. Kits of the invention further include instructions for administering a composition of the invention to a subject.

Therapeutic uses Mutations were made on a codon optimized Chop2-Gl'il’ fusion protein to create single and double mutations at the 1.132 (Leucine 132) and '1‘159 (thrconine [59) sites. The functional ties of each mutant ChRQ, or a combination thereof, were first examined in llEK cells. AAV2 virus vectors carrying mutant ChopZ—GFP constructs driven by CAG promoter were made and injected intravitreally into the eyes of adult mice. Mutant (‘hopZ-mediatcd light responses were ed by using multi-electrode array recordings from whole-mount retinas.

Single mutant (7th, Le, L132 and T159C, markedly lower the threshold light intensity that is required to evoke a ChRZ-mediated photocurrent. Moreover, several double mutant ChR2 variants, including Ll32(‘./T159C, Ll32AfI‘159C, and l.l32C/'l"159S. were found to further increase the photocurrent above the results of any single mutant (ThRIZ at low light intensities. The double mutants exhibited a slower off-rate, which is likely to contribute to the increased photocurrent at the low light intensities. Spiking activity of retinal ganglion cells mediated by the Li 32(T/Tl 59C double mutant was ed at the light intensity of 1013 photon/cmZ/s and at the wavelength of 473 nm. This light level is about 1.5 to 2 log units lower than the light level that is required to elicit the spiking activity with ype ChRZ. The spike firing of retinal ganglion cells expressing Ll32C/l‘lS9C could follow a light flicker frequency of up to IS 112. Ongoing studies are evaluating the long-term expression and safety of mutant ChR2s of the invention in retinal neurons.

Furthennore, sion of the mutant Chop’l proteins, and the resulting ChR2 proteins, of the present ion was not. found to cause neurotoxicity of up to two months after viral ion in mice, demonstrating the safety of the present ion for therapeutic use. [7S] s for use in the present invention can include various viral vectors, such as plasmids and recombinant viruses, i.e.. recombinant adeno-associated virus (rAAV). recombinant adenoviruses, recombinant retroviruses, recombinant lentiviruses, and other viruses known in the art.

In some embodiments, the expression of the Chopl ns of the present invention is driven by a constitutive promoter, i.e., CAG promoter, CMV promoter, LTR. In other embodiments. the promoter is an inducible or a cell-specific er.

Cell type-specific promoters that enable Chop2 protein expression in specific subpopulations of cells, i. e., retinal neuron cells or degenerating cells, may be preferred. These cells may include, but are not limited to, a retinal ganglion cell, a photoreceptor cell. a bipolar cell, a rod bipolar cell. an ()N—type cone bipolar cell, a retinal ganglion cell. a ensitive retinal ganglion cell, a ntal cell. an amacn'ne cell, or an All amacrine cell. Cell type—specific promoters are well known in the art. Particularly preferred cell type-specific promoters e, but are not limited to mGluR6, NK-3, and Pep2( L7).

In some embodiments, use of different opsin genes in addition to the mutant ChopZ ns of the present invention and targeted gene expression may further increase light sensitivity or improve vision. Visual ation is processed through the retina through two pathways: an ()N pathway which s the light ON, and an OH" pathway which signals the light OH". The existence of the ON and OW" pathway is important for the enhancement of st sensitivity. The visual signal in the ON y is relay from e bipolar cells to ON ganglion cells. Both ()N— cone bipolar cells and ON-ganglion cells are depolarized in response to light. On the other hand, the visual signal in the OFF pathway is carried from OFF—cone bipolar cells to OFF ganglion cells. Both Ol'TF-cone bipolar cells and OFF-ganglion cells are hypopolarized in response to light. Rod bipolar cells, which are responsible for the ability to see in dim light (scotopic vision), are ON bipolar cells (depolarized in response to light). Rod bipolar cells relay the vision signal through All amacrine cells (an ON type retinal cells) to ON an Olil? cone bipolar cells.

Accordingly. a dual rhodopsin system can be used to recapitulate the ON and Old" pathways integral to visual sing and acuity. Briefly, a ChopZ protein of the present invention can be specifically targeted to ON type retinal neurons (i. e., ON type ganglion cells and/or ()N type bipolar , while a hypopolarizing light sensor (i. e.. halorhodopsin or other chloride pump known in the art) can be targeted to OFF type retinal neurons (Le. OFF type ganglion cells and/0r OFF type bipolar cells) to create ON and OH" pathways. The specific targeting to preferred cell subpopulations can be ed through the use of different cell type-specific promoters. For e, (‘hop2 expression may be driven by the mGluR6 promoter for targeted sion in ON-type retinal neurons (129., ON type ganglion cells and/or ON type bipolar cells) while a hypopolarizing channel, such as halorhodopsin, expression is driven by the NK-3 promoter for targeted expression in OFF-type retinal neurons (i.e., OFF type ganglion cells and/or OFF type bipolar cells).

An alternative approach to restore ON and OFF ys in the retina is achieved by. expressing a depolarizing light sensor, such as ChR2, to rod bipolar cells or All amacrine. In this approach, the depolarization of rod bipolar cells or All amacrine cells can lead to the ON and OFF responses at the levels of cone bipolar cells and the downstream retinal ganglion cells. Thus. the ON and OH" pathways that are inherent in the retina are maintained.

The t invention can be fonnulated to a phannaceutical composition or medicament suitable for administration into a subject or t. Suitable routes of administration include, for example, intravitreal. intraocular, or subretinal injection.

Such formulations comprise a ceutically and/or physiologically acceptable e, diluent, carrier or ent. such as buffered saline or other buffers, e.g., lIEPES. to maintain physiologic pl 1. For a discussion of such components and their formulation, see, generally, (iennaro, AE., Remington: The Science and Practice ofPharmacy, Lippincott Williams & Wilkins Publishers; 2003 or latest edition). See also, WOOD/15822. If the preparation is to be stored for long periods, it may be frozen. for example, in the presence of glycerol.

The pharmaceutical composition described above is administered to a subject having a visual or blinding disease by any riate route, preferably by intravitreal or subretinal injection. ing on the retinal layer being targeted.

Disclosures from t and colleagues (cited herein) concern targeting of retinal pigment epithelium — the most distal layer from the vitreal space. According to the present invention. the ChopZ construct or polypeptide is targeted to retinal cells. i.(’.., retinal ganglion cells or bipolar cells. Such cells are known to be reasonably well- accessible to intravitreal injection as disclosed . [ntravitreal and/or inal injection can provide the necessary access to the bipolar cells. especially in circumstances in which the photoreceptor cell layer is absent due to ration — which is the case in certain forms of degeneration that the present invention is intended to overcome.

To test for the vector's ability to express the Chop2 mutants of the t invention, specifically in mammalian retinal neurons, by AAV-medialed delivery, a combination of a preferred promoter sequence linked to a reporter gene such as LacZ or Gl-il’ linked to a SV40 poly A sequence can be inserted into a plasmid and packaged into rAAV virus particles. concentrated. tested for inating adenovirus and titered for rAAV using an infectious center assay. The right eyes of a number of test subjects, preferably inbred mice, can be injected sub—retinally with about In] of the rAAV preparation (e.g.. greater than about IO'0 infectious units ml).

Two weeks later, the right (test) and left (control) eyes of half the animals may be removed, fixed and stained with an appropriate substrate or antibody or other substance to reveal the presence of the reporter gene. A majority of the test retinas in injected eyes will exhibited a focal d , e.g., blue for Xgal, or green for GFP consistent with a subretinal bleb of the injected virus creating a localized retinal detachment. All control eyes may be negative for the reporter gene product.

Reporter gene expression examined in mice sacrificed at later periods is ed for at least IO weeks post-injection, which suggests persistent expression of the reporter transgene.

In one embodiment, the Chop2 constructs are packaged in adenoviral vectors for transgene ry. An effective amount of rAAV virions carrying a nucleic acid sequence encoding the (‘hop2 DNA under the l of the promoter of choice. ably a constitutive C‘MV promoter or a cell-specific promoter such as mGluRé, is preferably in the range of between about 10'0 to about If)" rAAV infectious units in a volume of between about [50 and about 800 pl per injection. The rAAV ious units can be measured according to McLaughlin, SK er (1]., I988, J Viral 6211963. More preferably, the ive amount is between about 10'" and about [0'2 rAAV infectious units and the injection volume is preferably n about 250 and about 500 pl. ()ther dosages and volumes, preferably within these ranges but possibly outside them, may be selected by the treating professional, taking into account the al state of the t (preferably a human), who is being treated, including, age, weight. general health. and the nature and severity of the particular ocular It may also be desirable to administer additional doses ("boosters") of the present nucleic acid(s) or rAAV compositions. For example, depending upon the duration of the ene expression within the ocular target cell, a second treatment may be administered after 6 months or yearly, and may be rly repeated.

Neutralizing antibodies to AAV are not expected to be generated in view of the routes and doses used, thereby permitting repeat treatment rounds.

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

Ocular Disorders The ocular disorders for which the present (‘hopZ proteins, and the ing ChR2 proteins. are intended and may be used to improve one or more parameters of vision include, but are not limited to, developmental abnormalities that affect both anterior and posterior segments of the eye. Anterior segment ers include glaucoma, cataracts, corneal dystrophy, keratoconus. Posterior segment disorders include blinding disorders caused by photoreceptor malfunction and/or death caused by retinal dystrophies and degenerations. Retinal disorders include congenital stationary night blindness, age-related macular degeneration. congenital cone dystrophies, and a large group of retinitis-pigmentosa (RM-related disorders. ’lhese disorders e genetically pre-disposed death of photoreceptor cells, rods and cones in the , occurring at various ages. Among those are severe retinopathics. such as subtypes of RP itself that progresses with age and causes blindness in childhood and early adulthood and RP-associated diseases. such as genetic subtypes of LCA, which frequently results in loss of vision during childhood, as early as the first year of life.

The latter disorders are generally characterized by severe ion. and often complete loss of photoreceptor cells, rods and cones. ('l‘rabulsi. liL ed., Genetic Diseases oft/1e Eye. Oxford sity Press, NY, 1998).

In particular, the Chop2 and ChR2 proteins of the present invention useful for the treatment and/or restoration of at least l vision to subjects that have lost vision due to ocular disorders, such as sociated retinopathies, which are characterized by a long-term preservation of ocular tissue structure despite loss of function and by the association between function loss and the defect or absence of a normal gene in the ocular cells of the subject. A y of such ocular disorders are known. such as childhood onset blinding diseases, retinitis pigmentosa, macular degeneration, and diabetic retinopathy. as well as ocular blinding diseases known in the art. It is anticipated that these other disorders, as well as blinding disorders of presently unknown ion which later are characterized by the same description as above. may also be successfully treated by the Chop2 and ChRIZ proteins of the present invention. Thus, the particular ocular disorder treated by the t invention may include the above-mentioned disorders and a number of diseases which have yet to be so terized.

Optagenetics The emerging field of optogenetics involves the combination of genetic and optical methods to control specific events in targeted cells of a living tissue. ()ptogeneics may be used within freely moving mamtnals and other animals. er. the temporal precision (millisecond-timescale) of optogeneic methods are sufficient to function within intact biological systems. ] ’lhe instant invention provides ChopZ-gene therapy to l tissues of the eye, by introducing into retinal cells a nucleic acid or polypeptide encoding for at least one mutant form ofChopZ. Mutant Chop2/ChR'.’ ns of the invention are specifically adapted to be light-activated at lower thresholds of light intensities than their wild type counterparts. ingly. the mutant (‘hopZ/ChRZ proteins of the invention can be used to activate cells of the retina and visual system using less damaging sources of illumination. The mutant ChopZ/ChRZ proteins also conduct larger photocurrents upon activation, resulting in a more robust or efficacious response from the mutant ChopZ/ChRZ-expressing cells.

For example, mutant Chop2 proteins of the ion are stered to a subject through local, intravitreous or subretinal, injection of a nucleic acid molecule encoding a mutant Chop2. a mutant (‘hop2 polypeptide molecule, or a cell expressing a mutant ChopZ/ChRZ. Retinal cells of the subject express the mutant Chop2 proteins within the plasma membrane. When the transfected or transformed retinal cells encounter light ion, the transfected or ormed retinal cells uce an improved or restored signal.

These methods may be used in subjects of normal and/or impaired vision.

ChopCZ/ChRZZ mutants of the invention may ve, improve, or restore vision.

Moreover. Ch0p2/ChR2 mutants of the invention are used to ve, improve. or restore the transduction of non—visual information from photosensitive retinal ganglion cells to the brain.

The term "vision" as used herein is defined as the ability of an organism to usefully detect light as a stimulus for differentiation or action. Vision is intended to encompass the following: I. Light detection or tion — the ability to discern whether or not light is present; l») Light projection - the ability to n the direction from which a light stimulus is coming; 3. Resolution — the ability to detect differing brightness levels (1.9.. contrast) in a grating or letter target; and 4. Recognition - the ability to recognize the shape of a visual target by reference to the differing contrast levels within the target. 'l‘hus, "vision" includes the ability to simply detect the presence of light. The polypeptides and polynucleotides encoding mutant (Ihop2 of the present invention can be used to improve or restore , wherein the ement or ation in vision includes, for example, increases in light detection or perception, increase in light sensitivity or photosensitivity in response to a light stimulus, increase in the ability to discern the direction from which a light stimulus is coming, increase in the ability to detect differing brightness levels, increase in the ability to recognize the shape of a visual target, and increases in visual evoked ial or transmission from the retina to the cortex. As such, improvement or restoration of vision may or may not include full restoration of sight, i.e., wherein the vision of the patient treated with the present invention is restored to the degree to the vision of a non-affected individual. "llie visual recovery described in the animal studies described below may, in human terms, place the person on the low end of vision function by increasing one aspect of vision (Le, light sensitivity, or visual evoked potential) without ing full sight.

Nevertheless, ent at such a level would be a significant benefit because these individuals could be trained in ty and potentially in low order resolution tasks which would provide them with a greatly improved level of visual independence compared to total blindness. liven basic light perception can be used by visually impaired individuals, whose vision is ed using the present compositions and methods, to accomplish specific daily tasks and improve general ty. capability. and quality of life.

The degree of restoration of vision can be determined through the measurement of vision before, and ably after, administering a vector comprising, for example, DNA encoding (Thop2. Vision can be measured using any of a number of methods well-known in the art or methods not yet established. Vision, as ed or restored by the present ion, can be measured by any of the following visual responses: I. a light detection response by the subject after exposure to a light stimulus — in which evidence is sought for a reliable response of an indication or movement in the general direction of the light by the subject individual when the light it is turned on; to a light projection response by the t after exposure to a light stimulus in which evidence is sought for a reliable response of indication or movement in the specific direction of the light by the individual when the light is turned on; 3. light tion by the t of a light vs. dark patterned visual stimulus. which es the subject’s capability of resolving light vs dark ned visual stimuli as evidenced by: a. the presence of demonstrable reliable optoki netically produced nystagmoid eye movements and/or related head or body movements that trate tracking of the target (see above) and/or b. the presence of a reliable ability to discriminate a pattern visual us and to indicate such discrimination by verbal or non-verbal means, including, for example pointing, or pressing a bar or a button; 4. electrical recording of a visual cortex response to a light flash stimulus or a pattern visual stimulus, which is an endpoint of electrical transmission from a restored retina to the visual cortex, also referred to as the visual evoked potential (VEP). ement may be by electrical recording on the scalp surface at the region of the visual cortex, on the cortical surface, and/or recording within cells of the visual cortex.

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

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

While r studies by others have demonstrated that retinal degeneration can be retarded by gene therapy ques, the present invention demonstrates a definite physiological recovery of function, which is expected to generate or improve various parameters of vision, including oral parameters.

Behavioral measures can be obtained using known animal models and tests, for example performance in a water maze, wherein a subject in whom vision has been preserved or restored to varying extents will swim toward light es, JM et ((1., 1993, Behav Genet 23:395-403).

In models in which blindness is induced during adult life or congenital blindness develops slowly enough that the individual experiences vision before losing it, training of the subject in various tests may be done. In this way, when these tests are re—administered after visual loss to test the efficacy of the present compositions and methods for their -restorative effects, animals do not have to learn the tasks de now while in a blind state. Other behavioral tests do not require ng and rely on the instinctiveness of certain behaviors. An example is the optokinetic nystagmus test (Balkema GW e1 ((1., 1984, Invest ()phtlmlmol Vis Sci. 25:795-800; Mitchiner JC et al., l976. Vision Res. 16:] l69-7l).

The present invention may also be used in combination with other forms of vision therapy known in the art to improve or restore vision. For example, the use of visual prostheses, which include retinal implants, cortical implants, lateral geniculate nucleus implants, or optic nerve ts. Thus, in addition to c modification of surviving retinal neurons using the present methods, the subject being treated may be provided with a visual prosthesis before, at the same time as, or after the molecular method is employed. The iveness of visual etics can be improved with training of the individual. thus enhancing the potential impact of the Chop2 transformation of patient cells as contemplated herein. Training methods, such as habituation ng terized by training the subject to recognize ize (i) varying levels of light and/or pattern stimulation, and/or (ii) environmental stimulation from a common light source or object as would be understood by one skilled in the art: and orientation and mobility training characterized by training the subject to detect visually local objects and move among said objects more effectively than without the training. In fact, any visual stimulation techniques that are typically used in the field of low vision rehabilitation are applicable here.

EXAMPLES Exam le 1: Generation of labeled mutant Cho 2 constructs.

Mutations were made on a codon optimized ChopZ-Gli‘l’ fusion protein to create single and double mutations at the 1-132 (Leucine 132) and '1‘159 ('l‘hreonine l59) sites. Several mutants were generated. for example, single mutants such as L132A, L132C, T159A, T159C, and T 1598, and double mutants such as l.l32C/'l‘159C, 1.132C/T159S, 1.132A/T159C, and 1.132C/l‘159A. Chop2-GH’ transgenes were cloned into a rAAV vector under the control of a (TAG promoter using methods known in the art.

Exam 1e 2: In vitro anal sis of mutant Cho 2 constructs.

The functional properties of each mutant Chopl, or a combination f. were first examined in 1115K cells. Chop2 constructs were delivered to 1115K cells by adenoviral infection, for e. Upon expression of the WT or mutant ChopZ, functional WT and mutant (‘hRZ channels were formed. ements of the light sensitivity and other properties of the Cth channels were assessed as described herein. The light stimuli ns/c1135 at 460 nm) were generated by a xenon arc lamp and attenuated by l density filters: ND4.0 (2.8x10'4), N1)3.0 0'5), ND2.5 (4.8x10'5); ND2.0 (_1.6x10"’), NDl.0 (1.3x10'7), NDO (‘1.2x1018). Light evoked currents were measured from wild-type ChR’l, C. L 132C. 14132C/T159C, and 3/1‘1598. Patch clamp recordings were performed using methods known in the art.

Representative recordings from this experiment comparing light ivity between the ChopZ constructs demonstrated that mutations at 1.132 alone or in combination with mutation at T159 show increased photocurrent in comparison to WI‘ e 1A and 113). Figure 1B shows the same current traces at a different scale to illustrate the difference in amplitude of the photocurrents between W'l‘ ChR2 and (7th mutants more clearly. Figure [B specifically compares the current traces resulting from light stimulation using the neutral density filter (NI) 2.5), equivalent to 4.8 x l()'5 photos/cmzls; the traces are designated by the arrows. The amplitude of the photocurrent of the 1.132(‘ mutant is larger than that of WT; the amplitude of the photocurrcnt of double mutant ./F 159C is larger than that of L132C; and the amplitude of the photocurrent of the 1.132(7/l‘159S mutant larger than 1.132/I'IS9C.

The t traces of the ChRZ mutants, particularly double mutants /F 1 59C and l.l32C/’l‘l 598. also show slower deactivation kinetics when compared to WT and 1.132C.

Figure 2 shows the representative recordings of light—evoked currents from WT ChRCZ, 1-132C. 1.132C/1'159C, and [.132Cfl‘159S after stimulation by a 10 ms light pulse (1.2 x [018 photons/cmz/s at 460 nm wavelength) to compare the deactivation time course, or decay time course after the light is off. Mutant ChR2 Show longer deactivation time courses, with the double mutant Ll32C/T159S having the longest. Higher light sensitivity, as demonstrated by 1.132C/l‘lS9C and L132C/T159S. may be correlated with slower channel kinetics.

Exam 1e 3: In vivo ocular administration and anal sis of mutant (he 2 constructs.

AAVZ virus s carrying mutant ChopQ-GFP constructs driven by CAG promoter were made and injected intravitreally into the eyes of (157BL/6J adult mice.

Adult mice were anesthetized by [P injection of ne (100 tug/kg) and xylazine (10 mg/kg). Under a dissecting microscope. an incision was made by scissors through the eyelid to expose the sclera. A small perforation was made in the sclera region ior to the lens with a needle and viral vector suspension of 0.8—1.5 N at the tration of approximately 10' ' genomic particles/1111 was injected into intravitreal space through the hole with a Hamilton syringe with a 32-gauge blunt- ended needle. For each animal. usually only one eye was injected with viral vectors carrying a Chop2 construct, and the other eye was uninjected or injected with control viral vectors carrying GFP alone. Upon expression of the WT or mutant ChopZ of the present ion, onal WT or mutant ChR2 channels were formed utilizing endogenous retina], and the ties of these ChR2 proteins were assessed as described .

ChR2-mediated light responses were examined by using multi-electrode array recordings from whole-mount retinas. Light stimuli (photons/cmz/s) was generated by a 473 nm blue laser and attenuated by neutral y filters: NDO (6.3xl()'°), NDl .0 (7.4xto'5), NDl.5 (2.7x10‘5), Nl)2.() (_7.3x10"). ND2.5 0"), ND3.() (8.5xt0"), ND3.5 (3.8x10'3), and ND4.0 (9.5x10'2).

The multielectrode array recordings were based on the procedures reported by Tim and Copenhagen (2003). y, the retina was dissected and placed photoreceptor side down on a nitrocellulose filter paper strip (Millipore Corp, Bedford, MA). The d retina was placed in the MBA-60 multielectrode array recording chamber of 30 um diameter electrodes spaced 200 um apart (Multi Channel System MCS Gmbll. Reutlingen, Germany), with the ganglion cell layer facing the recording electrodes. The retina was continuously ed in oxygenated extracellular solution at 34°C during all experiments. The extracellular solution contained (in mM): NaCl. I24; KC], 2.5; CaClz, 2; MgClz, 2; Nallgl’04, 1.25; Nall(‘()3, 26; and glucose, 22 (pH 7.35 with 95% ()2 and 5% (702). Recordings were usually started 60 min after the retina was positioned in the recording r. The interval between onsets of each light stimulus was 10—15 s. The signals were filtered between 200 Hz. (low cut ofl) and 20 kHz (high cutoff). The responses from individual neurons were analyzed using Offline Sorter software (Plexon, Inc.. Dallas, Single mutant /(ThR2 mutants. i.e., L132 and TlS9C. markedly lower the threshold light intensity that is required to evoke a (‘hR2~mediated photocurrent.

Moreover, several double mutants, including 1.132C/l‘159C. Ll 159C, and Ll32C/T159S. were found to further increase the photocurrent at low light intensities. ent l y filters were used to attenuate the light stimuli to differentiate the light—evoked responses of the (Thop2 constructs in low light. Spiking activity of retinal ganglion cells mediated by the mutants of the present invention was observed at the light intensities about l.5 to 2 log units lower than the light level that is required to elicit the spiking activity with wild-type ChR2 (Figure 3). Specifically, W'l‘ ChR2 exhibited did not exhibit any g activity in response to light stimuli with neutral density filter 2.5 (3.2 x 10" s/cmzls) while (‘hRZ mutants (L132C.

Ll32C/TIS9C, and l.l32C/T159S) demonstrate spiking ty. In fact, the ChR2 mutants still exhibited spiking activity in se to light with neutral density filters 3.0 and 3.5. 'lherel‘ore, ChR2 mutants of the present invention possess higher light sensitivity and. thus. a markedly lower threshold light ity that is required to elicit a ChRQ-mediated photocurrent. er, ChR2 double mutants possess a higher light sensitivity than single mutants, i.e. 1,132C. In addition, the spike firing of retinal ganglion cells expressing Ll32C/'l'lS9C and 1.132/1‘1598 could follow a light flicker frequency of up to l5 H7. and 5 Hz, respectively (Figure 4).

The Ll32(‘/'F159A mutant shows high light sensitivity. probably the most light sensitive among these mutants, but it also shows extremely slow off-rate (the l continue open for many many sends after light oil). Interestingly. it can be turned off more quickly using a light with long-wavelenghts, such as yellow light.

The Ll32C/T159A mutant (encoded by SEQ ID NOS: 24 and 25) demonstrates significant potential.

Given the trade—off between light sensitivity and channel kinetics, Chop2/ChR2 mutants that demonstrate a balance between light sensitivity and channel kinetics, such as Ll32(‘./l'159C or Ll32C/T159S, may be suitable for the application of vision restoration.

Exam le 4: Anal sis of mutant Cho 2 constructs in mouse models of disease.

Mouse models of degenerative ocular diseases are known in the art. For example, homozygous rd] (WU/WU) mice are a commonly used eceptor degeneration model. Rd} mice carry a null mutation in a cyclic GMP phosphodiesterase, PDE6, similar to some forms of retinitis pigmentosa in humans.

Other well-established mouse models of ocular disease that may be of particular interest to demonstrate ChR2 mutant safety and efficacy include rds (also known as Pip/1M2). r113, r114. rt15. 1716. NW. 1118, 11/9, Pde6b'w". or (73/71 mice.

The ChopZ-(lFP ucts of the present invention can be injected intravitreally into the eyes of newborn (Pl) or adult mice at 2-12 months of age. Glil’ signal can be observed in the ChopZ-Glillinjected retinas, to determine the levels of ChR2 sion or expression in particular populations of cells, such as the retinal ganglion cells. Mutant -Gl"l’ expression can be monitored for a predetermined amount of time, i.e. 3-6 months. or 1 year after viral injection. Patch-clamp and multichannel array recordings can be perfonned using the methods known in the art and described herein to measure the light—evoked ses of mutant ChopZ-(iFP- expressing cells in viva.

Additional techniques and tests are well-established in the art to test for the restoration of light sentivity or . Visual evoked potentials from the ChopZ-Glt‘l’ expressing cells or visual cortex can be examined, as described in PCT publication Wt) 2007/131180. Other tests include behavorial assessments of the visual acuity in the mice, i.e., virtual tor test and visual water maze.

Exam le 5: Anal sis of Ion -term ex ression and sal’et of administration of mutant Chopfl constructs to retinal neurons.

Neurotoxicity was assessed in 6J adult mice injected with ChopZ constructs of the t invention. The expression safety of Chop2 mutants in the retina was assessed by immunostaining and cell counting after exposure to strong blue light for two weeks. None of the mice were found to exhibit symptoms of neurotoxicity for up to two months after injection.

Additional ongoing studies are evaluating the long—term expression and safety of (‘hopZ/ChRZ mutants of the invention in retinal neurons.

OTHER EMBODIMENTS While the ion has been bed in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. which is defined by the scope of the appended claims.

Other aspects. advantages. and modifications are within the scope of the following claims.

The patent and ific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references. documents ripts and scientific literature cited herein are hereby orated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various s in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims (3)

1. An expression vector comprising a polynucleotide encoding a polypeptide molecule comprising SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is cysteine (C) or alanine (A), and the amino acid at position 159 is cysteine (C), serine (S), or alanine (A).

2. The expression vector of claim 1, wherein the vector is an adeno-associated virus (AAV) vector. 3. The expression vector of claim 2, wherein the AAV vector is AAV2. 4. The expression vector of any one of claims 1 to 3, wherein the amino acid at position 159 is cysteine (C), and wherein the amino acid at on 132 is ne (C). 5. The expression vector of claim 4, wherein the polypeptide molecule comprises SEQ ID NO: 16. 6. The expression vector of claim 4, wherein the polynucleotide comprises the cleotide sequence of SEQ ID NO: 15. 7. The expression vector of any one of claims 1 to 3, wherein the amino acid at position 159 is serine (S), and wherein the amino acid at position 132 is ne (C). 8. The expression vector of claim 7, wherein the ptide molecule comprises SEQ ID NO: 19. 9. The expression vector of claim 7, wherein the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 18. 10. The expression vector of any one of claims 1 to 3, wherein the polypeptide ses an alanine (A) residue at amino acid position 132 and a cysteine (C) residue at amino acid position 11. The sion vector of claim 10, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 22. 12. The expression vector of claim 10, wherein the cleotide comprises the polynucleotide sequence of SEQ ID NO: 21. 13. The expression vector of any one of claims 1 to 3, wherein the polypeptide comprises a cysteine (C) residue at amino acid position 132 and an e (A) residue at amino acid position 14. The expression vector of claim 13, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 25. 15. The expression vector of claim 13, wherein the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 24. 16. The expression vector of any one of claims 1 to 3, wherein the polypeptide comprises an alanine (A) residue at amino acid position 132 and a serine (S) e at amino acid position 159. 17. The expression vector of any one of claims 1-3, wherein the polypeptide comprises an alanine (A) residue at amino acid position 132 and alanine (A) e at amino acid position 159. 18. A pharmaceutical composition sing the expression vector of any one of claims 1 to 19. The pharmaceutical composition of claim 18, wherein the composition is a recombinant adeno-associated virus particle composition. 20. Use of the expression vector of any one of claims 1 to 17 or the pharmaceutical composition of claim 18 or 19 in the preparation of a medicament for improving or restoring vision in a subject. 21. The use of claim 20 wherein the subject has normal vision. 22. The use of claim 20 wherein the subject has impaired . 23. The use of claim 20 wherein the subject is suffering from an ocular disease. 24. 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