EA043449B1 - IDENTIFICATION OF CHANNEL-OPSIN-2 (Chop2) MUTATIONS AND METHODS OF APPLICATION - Google Patents

Description

Field of technology to which the invention relates

The invention relates mainly to the field of molecular biology. Mutations in the channel opsin-2 (Channelopsin-2) gene (Chop2) have been identified. Compositions comprising the mutant Chop2 gene are used in therapeutic methods for improving and restoring vision loss.

Prior Art

The retina consists of photoreceptors (or photoreceptor cells, rod and cone). Photoreceptors are highly specialized neurons that are responsible for photoconversion, or the conversion of light (in the form of electromagnetic radiation) into electrical and chemical signals that stimulate a cascade of events within the visual system, ultimately generating a representation of our world.

Loss or degeneration of photoreceptors significantly compromises, if not completely inhibits, phototransduction of visual information within the retina. Loss of photoreceptor cells and/or loss of photoreceptor cell function is the primary cause of decreased visual performance, decreased light sensitivity, and blindness. There is a need in the art to provide compositions and a method that restores the photosensitivity of the retina of a subject who has experienced vision loss.

The essence of the invention

The invention proposes a solution to the urgent need for a method for restoring and/or increasing the photosensitivity of photoreceptor cells by expressing preferred mutations and/or combinations thereof, the channel opsin-2 gene (Chop2), and then proposes methods of gene therapy based on the channel opsin gene. 2 (Chop2).

Gene therapy based on the channel opsin 2 (Chop2) gene offers an excellent strategy for restoring retinal photosensitivity after photoreceptor degeneration. The protein product of the Chop2 gene, when bound to light-isomerizing chromophores, all trans-retinal, forms a functional light-regulated channel called channel-rhodopsin-2 (ChR2). Native ChR2 exhibits low photosensitivity. Recently, two ChR2 mutants, L132C and T159C, were reported to significantly increase their photosensitivity (Kleinlogel 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, each of which is incorporated herein by reference in its entirety). The properties of these two ChR2 mutants (ie, L132C and T159C) were examined and compared with a number of double mutants at these two sites to identify suitable candidates for therapeutic modalities. Compositions containing one or more of these mutations are offered to a subject in need for the purpose of restoring vision. Specifically, targeted mutations in the Chop2 gene are introduced into a cell and/or integrated into the cell's genomic DNA to improve or restore vision. Targeted mutations in the Chop2 gene that are introduced into a cell to improve or restore vision can also remain episomal, not being integrated into genomic DNA.

Mutations at amino acid positions L132 or T159 of Chop2 (and thus the resulting ChR2) significantly reduce the threshold photosensitivity required to induce ChR2-mediated photocurrent. Double mutants at amino acid positions L132 and T159 further increase the photocurrent at low light intensities, exceeding the values corresponding to single mutations. Retinal ganglion cells expressing double mutants at positions L132 and T159 can respond to light intensities that fall within the range of natural daylight conditions but still maintain adequate and high temporal resolution that is suitable for restoring effective vision. Thus, the mutant Chop2 protein of the present invention, which produces mutant ChR2s having improved sensitivity, is used individually or in combination to restore vision.

Specifically, the invention provides a polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L). In some embodiments of 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 include or consist of SEQ ID NO: 13. When the amino acid at position 132 is alanine (A), the polypeptide molecule may include or consist of SEQ ID NO: 20 .

The invention provides a polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein the amino acid at position 159 of SEQ ID NO: 26 is not threonine (T). In some embodiments 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 (C), the polypeptide molecule may include or consist of SEQ ID NO: 14. When the amino acid at position 159 is serine (S), the polypeptide molecule may include or consist of SEQ ID NO: 17. When the amino acid at position 159 is alanine (A), the polypeptide molecule may include or consist of SEQ ID NO: 23.

The invention provides an isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L). In some embodiments of the isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein 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), the amino acid at position 132 is cysteine (C) and the amino acid at position 159 is cysteine (C). In a preferred embodiment of an isolated polypeptide molecule, the polypeptide molecule includes or consists of SEQ ID NO: 16. The invention provides an isolated nucleic acid molecule that encodes an isolated polypeptide comprising or consisting of SEQ ID NO: 16. Preferably, an isolated nucleic acid molecule that encodes an isolated polypeptide comprising or consisting of SEQ ID NO: 16 is a nucleic acid molecule that includes or consisting of SEQ ID NO: 15.

In some embodiments of the isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein 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), the amino acid at position 132 is cysteine (C) and the amino acid at position 159 is serine (S). An isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein 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 include or consist of SEQ ID NO: 19. Alternatively or additionally isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein 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), wherein the amino acid at position 132 is cysteine (C) and where the amino acid at position 159 is serine (S), may include or consist of SEQ ID NO: 19. The invention provides an isolated nucleic acid molecule that encodes an isolated polypeptide that includes or consists of SEQ ID NO: 19. Preferably, the nucleic acid molecule includes or consists of SEQ ID NO: 18.

In some embodiments of the isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein 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), the amino acid at position 132 is alanine (A) and the amino acid at position 159 is cysteine (C). An isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is threonine (T), may include or consist of SEQ ID NO: 22. An alternative or additionally isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein 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 ), wherein the amino acid at position 132 is alanine (A) and where the amino acid at position 159 is cysteine (C), may include or consist of SEQ ID NO: 22. The invention provides an isolated nucleic acid molecule that encodes an isolated polypeptide, which includes or consists of SEQ ID NO: 22. Preferably, the nucleic acid molecule includes or consists of SEQ ID NO: 21.

In some embodiments of the isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein 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), the amino acid at position 132 is cysteine (C) and the amino acid at position 159 is alanine (A). An isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein 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 include or consist of SEQ ID NO: 25. An alternative or additionally isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26, wherein 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), wherein the amino acid at position 132 is cysteine (C) and where the amino acid at position 159 is alanine (A), may include or consist of SEQ ID NO: 25. The invention provides an isolated nucleic acid molecule that encodes an isolated polypeptide that includes or consists of SEQ ID NO: 25. Preferably, the nucleic acid molecule includes or consists of SEQ ID NO: 24.

The invention provides any of the isolated polypeptide molecules described herein, wherein the polypeptide molecule encodes a mutant Chop2 protein that forms a mutant

- 2 043449

ChR2, which produces a current in response to a threshold light intensity that is lower than the wild-type ChR2 protein threshold. In addition, current conducts cations. Typical cations include, but are not limited to, H+, Na+, K+ and Ca2+ ions. The wild-type ChR2 and mutant proteins described herein conduct cations nonspecifically. Therefore, one or more of the following ions conduct current: H+, Na+, K+ and Ca2+.

The invention provides any of the isolated polypeptide molecules described herein, further comprising a pharmaceutically acceptable carrier. The invention also provides a composition comprising at least one isolated polypeptide molecule described herein. The composition may further include a pharmaceutically acceptable carrier.

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

The invention provides a cell, where the cell includes or contacts an isolated polypeptide molecule of the invention. The invention further provides a cell that includes or contacts an isolated nucleic acid molecule that encodes an isolated polypeptide molecule of the invention. The invention provides a composition comprising, essentially consisting of, or consisting of, a cell that includes an isolated polypeptide molecule of the invention or a nucleic acid molecule that encodes an isolated polypeptide molecule of the invention. The cell of the invention may be contacted with the isolated polypeptide in vitro, ex vivo, in vivo or in situ. In some embodiments of the invention, the cell is a photoreceptor; horizontal cell; bipolar cell; amacrine cell and especially AII-amacrine cell; or a retinal ganglion cell, including a photosensitive retinal ganglion cell. Preferably, the cell is a retinal ganglion cell, a photosensitive retinal ganglion cell, a bipolar cell, an ON-type bipolar cell, a rod bipolar cell, or an AII amacrine cell. In some 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 AII-amacrine cell.

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

The methods described herein may also be applied to those subjects who are healthy, blind (partially or completely), and/or to subjects with retinal degeneration (characterized by loss of rod and/or cone photoreceptor cells), but may be activity dependent photosensitive retinal ganglion cells to detect environmental light levels. For example, the methods described herein can be used to preserve, improve, or restore the activity of photosensitive retinal ganglion cells that mediate the transduction of light information to synchronize circadian rhythms with the 24-hour light/dark cycle, pupillary control and reflexes, and for light-regulated release melatonin.

In some embodiments of the methods of the invention, the subject may have normal vision or impaired vision. Alternatively or additionally, the subject may be at risk of developing an eye disease that results in visual impairment. For example, the subject may have a family history of eye diseases including macular degeneration and retinitis pigmentosa. The subject may be at risk of being exposed to eye damage that causes damage to photosensitive cells in the retina. The subject may have a genetic marker or a genetic/congenital disease that results in impaired vision, low vision, partial blindness, or total blindness. The subject may have a refractive error that results in myopia (nearsightedness) or hypertrophy (farsightedness).

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

Other features and advantages of the invention will be understood from and covered by the following detailed description and claims.

Brief description of drawings

In fig. Figure 1 shows representative recordings of light-evoked currents from wild-type (WT) ChR2, L132C, L132C/T159C, and L132C/159S mutants in HEK cells to compare their light sensitivity (A). Light stimuli (photons/cm ² /s at 460 nm) were generated using a xenon arc lamp and attenuated using a neutral gray filter: ND4.0 (2.8x10 ¹⁴ ), ND3.0 (1.4x10 ¹⁵ ),

ND2.5 (4.8x10 ¹⁵ ); ND2.0 (1.6x1016), ND1.0 ( ^1.3x1017 ), ND0 ( ^1.2x1018 ). (B) The same current traces are presented at different current scales. The traces indicated by the arrows are caused by the same light intensity (ND2.5).

In fig. Figure 2 shows representative recordings of light-evoked currents from (WT) ChR2 wild type, L132C, L132C/T159C and L132C/159S mutants at a 10 ms light pulse (1.2 x 10 ¹⁸ photons/cm ² /s at 460 nm) in cells NEK to compare the time dependence of their deactivation (decay time after turning off the light).

In fig. Figure 3 shows representative multichannel chip recordings of WT ChR2, L132C, L132C/T159C, and L132C/T159S-mediated spike activities from retinal ganglion cells in a whole-mount retina to compare their light sensitivity. Light stimuli (photons/cm ² /s) were generated using a 473 nm blue laser and attenuated using a neutral gray filter: ND0 (6.3x1016), ND1.0 (7.4x10 ¹⁵ ), ND1.5 (2 ,7x10 ¹⁵ ), ND2.0 (7.3x10 ¹⁴ ), ND2.5 (3.2x10 ¹⁴ ), ND3.0 (8.5x10 ¹³ ), ND3.5 (3.8x10 ¹³ ) and ND4.0 (9 ,5x10 ¹² ).

In fig. Figure 4 shows representative multichannel chip recordings of WT ChR2, L132C, L132C/T159C, and L132C/T159S-mediated spike activities from retinal ganglion cells in a whole retinal mount to compare their temporal dynamics. Each panel presents a two-dimensional point array of 10 consecutive light-evoked spikes originating from a single neuron (top) and a histogram of the average spike strength (bottom). Light pulses at different frequencies were generated using a 473 nm laser source in the blue region of the spectrum at intensities approximately one log unit above the threshold intensity of each mutant. The WT ChR2 and L132C recordings are presented in (A), and the L132C/T159C and L132C/T159S recordings are presented in (B).

Detailed description

Visual system.

The central nervous system mediates vision (also referred to herein as visual imagery) through specialized cells and unique signal transduction modes present in the visual system. The principal responsibility of the visual system is to convert light in the form of electromagnetic radiation into an image or image of the environment. In addition to the visual system's function, the visual system also regulates the pupillary light reflex (PLR), the adjustment of the circadian rhythm to periodic light/dark cycles, and the release of the hormone melatonin.

Retinal cells are the first cells in the visual or nervous system to encounter light (electromagnetic radiation of varying wavelengths and intensities). Photons pass through the cornea, pupil and lens before reaching the retina. The retina has a unique structure because the photoreceptor cells that directly absorb photons are located on the outer layer of the retina. Photons that travel to the lens first encounter an inner layer of retinal ganglion cells (a small portion of which are photosensitive through the expression of opsin, melanopsin) and an intermediate layer of bipolar cells before reaching the outer layer of photoreceptor cells (also known as rod and cone cells). Rod photoreceptors function in dim light conditions (scotopic vision), while photoreceptors function in bright light conditions (photopic vision) and are responsible for color vision. Cone photoreceptors directly synapse with ON- and OFF-type cone bipolar cells, which in turn directly synapse with ON- and OFF-type retinal ganglion cells. Rod photoreceptors synapse with rod bipolar cells (a unique type of cell that is ON-type), which synapse with Anamacrine cells. An-amacrine cells then transmit visual signals to ON-type cone bipolar cells via gap junctions and to OFF-type cone bipolar cells and OFF-type ganglion cells via inhibitory glycinergic synapses. Retinal ganglion cells are responsible for binding visual information to neurons in the brain.

Photoconversion.

Inside the retina, photoreceptor cells absorb photon particles and transform the raw frequency and wavelength information of light into chemical and then electrical signals that transmit this raw information through the visual and nervous systems. Specifically, an opsin protein localized on the surface of the photoreceptor (rod, cone, and/or photosensitive retinal ganglion cell) absorbs the photon and initiates an intracellular signaling cascade that leads to hyperpolarization of the photoreceptor. In the dark, the opsin protein does not absorb photons, and the photoreceptors are depolarized. The visual signals from the photoreceptors are then transmitted through bipolar cells, amacrine cells and ganglion cells to the higher visual centers in the brain. Specifically, when rod and cone photoreceptors are depolarized (in the dark), they cause rod bipolar cells to depolarize ON-type cone bipolar cells, but cause hyperpolarization of OFF-type cone bipolar cells, which in turn cause AII-type depolarization. amacrine cells and increase the adhesions of ON-type retinal ganglion cells and decrease the adhesions of OFF-type retinal ganglion cells. The opposite effect is observed (for rod, ON and OFF bipolar cells, AII amacrine cells, and ON and OFF ganglion cells) when rod and cone photoreceptors are hyperpolarized (in response to light).

Light information is processed and refined in a significant way through the functioning of photoreceptors, bipolar cells, horizontal cells, amacrine cells and retinal ganglion cells. To add to the complexity of this system, photoreceptors are found in three main variations, including rods, cones (of which three types are particularly responsible for different wavelengths of light), and photosensitive retinal ganglion cells. Thus, the first layer of information processing occurs at the level of photoreceptors, which differentially respond to specific wavelengths and intensities of light. Bipolar cells in the retina receive information from both types of cells, photoreceptor and horizontal. Horizontal retinal cells receive information from multiple photoreceptor cells and thus integrate information between cell types and across distances in the retina. Bipolar cells further integrate information directly from photoreceptor cells and horizontal cells by receiving mostly step potentials to retinal ganglion cells, although some recent studies indicate that some bipolar cells can generate action potentials. Cone bipolar cells form connections by synapse with retinal ganglion cells and amacrine cells, while rod bipolar cells form connections by synapse with AII amacrine cells. Similar to horizontal cells, most amacrine cells integrate information laterally within the retina. Unlike horizontal cells, most amacrine cells are inhibitory (GABAergic) interneurons. Amacrine cells are also more specialized than horizontal cells, since each amacrine cell makes a specific connection through a synapse with a specific type of bipolar cell (one of ten types of bipolar cell). Specifically, the AII-amacrine cell is a critical interneuron in the scotopic signaling pathway (in scotopic vision where cone photoreceptors do not respond). An-amacrine cells receive synaptic input from rod bipolar cells and then transmit signals to the cone cell signaling pathway via ON- and OFF-cone bipolar cells to ON- and OFF-ganglion cells, as described above. Thus, expression of Chop2, and the resulting formation of ChR2, in rod bipolar cells or AII amacrine cells may generate both ON and OFF responses in retinal ganglion cells. In addition, retinal ganglion cells integrate information from bipolar cells and amacrine cells. Although retinal ganglion cells vary considerably in size, conductivity, and response to visual stimulation (eg, visual field), all retinal ganglion cells extend a long axon into the brain. With the exception of a minimal portion of the retinal ganglion cells, which convey non-visual information concerning the pupillary light reflex and the imposition of the circadian rhythm, the entire collection of axons extending from the retinal ganglion cells forms the second cranial nerve, the optic chiasm, and the optic tract of the central nervous system. Consequently, a significant amount of information processing occurs in the retina itself.

Photoreceptor cells express endogenous opsin proteins such as rhodopsin. The Chop2 mutant proteins of the invention can be expressed in any cell type and form functional ChR2 channels. Preferably the cell is a retinal cell. Typical cells include, but are not limited to, photoreceptor cells (eg, rods, cones, and photosensitive retinal ganglion cells), horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells.

Channel-opsin-2 (Chop2).

Channel opsin 2 (Chop2) was first isolated from the green algae Chlamydomonas reinhardtii. Channel-opisin-2 is a protein containing seven transmembrane domains that becomes light-regulated (photosensitive) when it binds to an all-transretinal chromophore. Chop2, when bound to a retinal molecule through a Schiff base bond, forms a light-gated, nonspecific, internally rectifying cation channel called channel-rhodopsin-2 (Chop2 retinalidene, abbreviated ChR2).

When referred to herein, channel-opsin-2 or Chop2 refers to the gene that encodes channel-opsin-2, which then forms channel-rhodopsin-2 (ChR2) associated with retinal. The gene constructs of the present invention relate primarily to channel opsin-2 (ie, without retinal), and all Chop2 variants disclosed herein form functional caalrhodopsin-2 variants. The methods disclosed herein may involve delivering Chop2 to cells without exogenous retinal. It is understood that when Chop2 is expressed in cells (ie, retinal neurons), endogenously available retinal binds to wild-type Chop2 or mutant Chop2 of the present invention to form functional light-gated channels, WT ChR2 or mutant ChR2.

As such, Chop2 proteins, when referred to herein, may also be synonymous with ChR2.

As used herein, channel-rhodopsin-2 or ChR2 refers to the retinal-associated functional light-sensitive channel. In one embodiment, bound retinal can be provided exogenously. In a preferred embodiment, bound retinal is provided at an endogenous level available in the cell. The present invention also covers functional channel-rhodopsin-2 channels formed by polypeptides and polynucleotides encoding the Chop2 mutants described herein.

When illuminated with a preferred dose of light radiation, ChR2 opens a channel pore through which H ⁺ , Na ⁺ , K ⁺ , and/or Ca ²⁺ ions flow into the cell from the extracellular space. Activation of the ChR2 channel typically causes depolarization of the cell expressing the channel. A depolarized cell produces step potentials and/or action potentials to carry information from the Chop2/ChR2-expressing cell to other cells in the retina or brain.

The high temporal resolution form of wild-type or mutant ChR2 is a central focus of neurobiological research. When expressed in a mammalian neuron, ChR2 mediates light-controlled depolarization in vitro or ex vivo cultures. Wild-type ChR2 or mutant ChR2s with high temporal resolution (the latter typically exhibiting low photosensitivity) present several challenges that must be resolved before they can be used for vision restoration. For the purpose of vision restoration, ChR2 with high light sensitivity is more appropriate than ChR2 with high temporal resolution.

Wild-type ChR2 proteins require high-intensity light in the blue region of the spectrum (ie, ^1018-1019 photons s ^-1 cm ^-2 at 480 nm) for full activation. Prolonged lighting of this type can destroy cells.

Wild-type ChR2 protein kinetics are suboptimal for maximum channel efficiency.

Efficiency can be increased by modifying one or more amino acids of the wild-type ChR2 protein, either to lengthen the duration of the channel's open state, or to increase channel conductance units, or both. The single-channel conductance of wild-type ChR2 is small. Thus, neural activation in vivo will require either high expression of the wild-type channel or very intense activation using the preferred blue wavelength. A simpler solution can be found by changing the conductivity of the channel or lengthening the duration of the channel opening time. Each of these mechanisms, and specifically a combination of these mechanisms, makes it possible to use lower and safer light intensities to achieve the same level of cellular depolarization.

For example, the ChR2 mutant proteins of the invention achieve higher photosensitivity by extending the duration of the channel open state. Therefore, each mutant ChR2 channel conducts a higher photocurrent than the wild-type ChR2 channel when activated at the same light intensities. Thus, the mutant channels are activated by light intensities that are lower than those required to activate wild-type ChR2 channels. Quantitatively, detectable spike activity from retinal ganglion cells expressing mutant ChR2 proteins can be evoked by light intensities that are 1.5 to 2 log units lower than the light intensity required to evoke spike activity from retinal ganglion cells expressing wild-type ChR2. Thus, the light intensities required to activate mutant ChR2 proteins are close to or within the normal daylight range.

The following sequences provide specific examples of mutant and wild-type Chop2 proteins and polynucleotides encoding the indicated WT and mutant Chop2 proteins of the invention and forming the WT and mutant ChR2 of the invention.

The wild-type Chop2 (WT) of the invention may be encoded by the following Chlamydomonas reinhardtii light-regulated chlamyopsin ion channel 4 (COP4) mRNA sequence (GenBank No. XM_001701673 and SEQ ID NO: 1.

- 6 043449 gcagcaccat acttgacatc tgtcgccaag caagcattaa acatggatta tggaggcgcc ctgagtgccg ttgggcgcga gctgctattt gtaacgaacc cagtagtcgt caatggctct

121 gtacttgtgc ctgaggacca gtgttactgc gcgggctgga ttgagtcgcg tggcacaaac

181 ggtgcccaaa cggcgtcgaa cgtgctgcaa tggcttgctg ctggcttctc catcctactg

241 cttatgtttt acgcctacca aacatggaag tcaacctgcg gctgggagga gatctatgtg

301 tgcgctatcg agatggtcaa ggtgattctc gagttcttct tcgagtttaa gaacccgtcc

361 atgctgtatc tagccacagg ccaccgcgtc cagtggttgc gttacgccga gtggcttctc

421 acctgcccgg tcattctcat tcacctgtca aacctgacgg gcttgtccaa cgactacagc

481 aggcgcacca tgggtctgct tgtgtctgat attggcacaa ttgtgtgggg cgccacttcc

541 gccatggcca ccggatacgt caaggtcatc ttcttctgcc tgggtctgtg ttatggtgct

601 aacacgttct ttcacgctgc caaggcctac atcgagggtt accacaccgt gccgaagggc

661 cggtgtcgcc aggtggtgac tggcatggct tggctcttct tcgtatcatg gggtatgttc

721 cccatcctgt tcatcctcgg ccccgagggc ttcggcgtcc tgagcgtgta cggctccacc

781 gtcggccaca ccatcattga cctgatgtcg aagaactgct ggggtctgct cggccactac

841 ctgcgcgtgc tgatccacga gcatatcctc atccacggcg acattcgcaa gaccaccaaa

901 ttgaacattg gtggcactga gattgaggtc gagacgctgg tggaggacga ggccgaggct

961 ggcgcggtca acaagggcac cggcaagtac gcctcccgcg agtccttcct ggtcatgcgc

1021 gacaagatga aggagaaggg cattgacgtg cgcgcctctc tggacaacag caaggaggtg

1081 gagcaggagc aggccgccag ggctgccatg atgatgatga acggcaatgg catgggtatg

1141 ggaatgggaa tgaacggcat gaacggaatg ggcggtatga acgggatggc tggcggcgcc

1201 aagcccggcc tggagctcac tccgcagcta cagcccggcc gcgtcatcct ggcggtgccg

1261 gacatcagca tggttgactt cttccgcgag cagtttgctc agctatcggt gacgtacgag

1321 ctggtgccgg ccctgggcgc tgacaacaca ctggcgctgg ttacgcaggc gcagaacctg

1381 ggcggcgtgg actttgtgtt gattcacccc gagttcctgc gcgaccgctc tagcaccagc

1441 atcctgagcc gcctgcgcgg cgcgggccag cgtgtggctg cgttcggctg ggcgcagctg

1501 gggcccatgc gtgacctgat cgagtccgca aacctggacg gctggctgga gggcccctcg

1561 ttcggacagg gcatcctgcc ggcccacatc gttgccctgg tggccaagat gcagcagatg

1621 cgcaagatgc agcagatgca gcagattggc atgatgaccg gcggcatgaa cggcatgggc

1681 ggcggtatgg gcggcggcat gaacggcatg ggcggcggca acggcatgaa caacatgggc

1741 aacggcatgg gcggcggcat gggcaacggc atgggcggca atggcatgaa cggaatgggt

1801 ggcggcaacg gcatgaacaa catgggcggc aacggaatgg ccggcaacgg aatgggcggc

1861 ggcatgggcg gcaacggtat gggtggctcc atgaacggca tgagctccgg cgtggtggcc

1921 aacgtgacgc cctccgccgc cggcggcatg ggcggcatga tgaacggcgg catggctgcg

1981 ccccagtcgc ccggcatgaa cggcggccgc ctgggtacca acccgctctt caacgccgcg

2041 ccctcaccgc tcagctcgca gctcggtgcc gaggcaggca tgggcagcat gggaggcatg

2101 ggcggaatga gcggaatggg aggcatgggt ggaatggggg gcatgggcgg cgccggcgcc

2161 gccacgacgc aggctgcggg cggcaacgcg gaggcggaga tgctgcagaa tctcatgaac

2221 gagatcaatc gcctgaagcg cgagcttggc gagtaaaagg ctggaggccg gtactgcgat

2281 acctgcgagc tcgcgcgcct gactcgtcgt acacacggct caggagcacg cgcgcgtgga

2341 cttctcaacc tgtgtgcaac gtatctagag cggcctgtgc gcgaccgtcc gtgagcattc

2401 cggtgcgatc ttcccgcctt cgcaccgcaa gttcccttcc tggccctgct gcgcctgacg

2461 catcgtccga acggaagggc ggcttgatca gtaaagcatt gaagactgaa gtcgtgcgac

2521 cgtagtgcta tggctctgca cgtaagtggg cgctgccctg cttactacgc attgcccaag

2581 actgcttcct tttggtggcc gaggccctgg tcccacatca ttcatttgca taacgtactg

2641 tttagttaca tacgctttgc ttaacctcga caattgcaac atgggctgag agtccgtacg

2701 gcggctatgg acgaaggtgt tatcggatgt gattaggaat ctcggttgaa aggcttcgag

2761 aaagtgagct tcatctgtgg cttctgttgg ggtcatcaag aagaacgacg gtaaggcaaa

2821 cgaggtaaaa gtggcacgtc tttgtgcaca acgggcccgt ggagagtggg ggagtgcatg

2881 tgtgcggtcc taacacgcga gtgcaaagcg ggcttttctg gagctgggtt acggtctggc

2941 tcggcaactg ctctgtgttt taaccacagc ttcggaagtc tgggtatgtt ttgttggcag

3001 aaacatttgg gtaacttgag ggtgattcgt ctggagtcgg acaacatggc tgccgtccgt

3061 gtgcagggac ggtaatcaat gagctggagc tgtgatgctc accacacgtt gcatacccct

3121 gcttacaaaa acactttgat gtcgtggcca aactatgcgt gagcaaagag ttaaagaggc

3181 atgagtgcat ggttgcggac gtgcgcaaca attgcatcaa gtatttgacg ccttcaagcc

3241 aacaagtgcg cgcgcggcaa cttgattaac acgccggacg cagtggtggg ggcgtgtaca

3301 gtgtttatga gctgccattc tgcgatccgt agtgttaggt tgcgtgtgac gccgcgcggc

3361 tgtgggccct tacatggaga gttgggtgct tcaccacacg gttggcgccg ctgaagggtg

3421 tgctatgttt tggtaaagcc ggggccctga agaccgcaac cgtagaaccg tactgaaagg

3481 gtgtcagccc ggggtaactg gatgccctgg gacatagcta ttaatgttga agtgaagccg

3541 tcaagccgag tgccgtgcgc cgctgtatca ccaaggcccg tecta

The wild type ChR2 (WT) of the invention may be encoded by the following amino acid sequence of the light-gated ion channel chlamyopsin 4 (COP4) (GenBank No. XP 001701725 and SEQ ID NO: 2).

- 7 043449 mdyggalsav grellfvtnp vvvngsvlvp edqcycagwi esrgtngaqt asnvlqwlaa gfsilllmfy ayqtwkstcg weeiyvcaie mvkvilefff efknpsmlyl atghrvqwlr

121 yaewlltcpv ilihlsnltg Isndysrrtm gllvsdigti vwgatsamat gyvkviffcl

181 glcygantff haakayiegy htvpkgrcrq vvtgmawlff vswgmfpilf ilgpegfgvl

241 svygstvght iidlmskncw gllghylrvl ihehilihgd irkttklnig gteievetlv

301 edeaeagavn kgtgkyasre sflvmrdkmk ekgidvrasl dnskeveqeq aagaalshshp

361 gngmgmgmgm ngmngmggmn gmaggakpgl eltpqlqpgr vilavpdism vdffreqfaq

421 Isvtyelvpa Igadntlalv tqaqnlggvd fvlihpeflr drsstsilsr Irgagqrvaa

481 fgwaqlgpmr dliesanldg wlegpsfgqg ilpahivalv akmqqmrkmq qmqqigmmtg

541 gmngmgggmg ggmngmgggn gmnnmgngmg ggmgngmggn gmngmgggng mnnmggngma

601 gngmgggmgg ngmggsmngm ssgvvanvtp saaggmggrm nggmaapqsp gmnggrlgtn

661 plfnaapspl ssqlgaeagm gsmggmggms gmggmggmgg mggagaattq aaggnaeaem

721 Iqnlmneinr Ikrelge

The wild type Chop2 (WT) of the invention may be encoded by the following sequence of the Chlamydomonas reinhardtii retinal binding protein gene (cop4) (GenBank No. AF461397 and SEQ ID NO: 3).

gcatctgtcg ccaagcaagc attaaacatg gattatggag gcgccctgag tgccgttggg cgcgagctgc tatttgtaac gaacccagta gtcgtcaatg gctctgtact tgtgcctgag

121 gaccagtgtt actgcgcggg ctggattgag tcgcgtggca caaacggtgc ccaaacggcg

181 tcgaacgtgc tgcaatggct tgctgctggc ttctccatcc tactgcttat gttttacgcc

241 taccaaacat ggaagtcaac ctgcggctgg gaggagatct atgtgtgcgc tatcgagatg

301 gtcaaggtga ttctcgagtt cttcttcgag tttaagaacc cgtccatgct gtatctagcc

361 acaggccacc gcgtccagtg gttgcgttac gccgagtggc ttctcacctg cccggtcatt

421 ctcattcacc tgtcaaacct gacgggcttg tccaacgact acagcaggcg caccatgggt

481 ctgcttgtgt ctgatattgg cacaattgtg tggggcgcca cttccgccat ggccaccgga

541 tacgtcaagg tcatcttctt ctgcctgggt ctgtgttatg gtgctaacac gttctttcac

601 gctgccaagg cctacatcga gggttaccac accgtgccga agggccggtg tcgccaggtg

661 gtgactggca tggcttggct cttcttcgta tcatggggta tgttccccat cctgttcatc

721 ctcggccccg agggcttcgg cgtcctgagc gtgtacggct ccaccgtcgg ccacaccatc

781 attgacctga tgtcgaagaa ctgctggggt ctgctcggcc actacctgcg cgtgctgatc

841 cacgagcata tcctcatcca cggcgacatt cgcaagacca ccaaattgaa cattggtggc

901 actgagattg aggtcgagac gctggtggag gacgaggccg aggctggcgc ggtcaacaag

961 ggcaccggca agtacgcctc ccgcgagtcc ttcctggtca tgcgcgacaa gatgaaggag

1021 aagggcattg acgtgcgcgc ctctctggac aacagcaagg aggtggagca ggagcaggcc

1081 gccagggctg ccatgatgat gatgaacggc aatggcatgg gtatgggaat gggaatgaac

1141 ggcatgaacg gaatgggcgg tatgaacggg atggctggcg gcgccaagcc cggcctggag

1201 ctcactccgc agctacagcc cggccgcgtc atcctggcgg tgccggacat cagcatggtt

1261 gacttcttcc gcgagcagtt tgctcagcta tcggtgacgt acgagctggt gccggccctg

1321 ggcgctgaca acacactggc gctggttacg caggcgcaga acctgggcgg cgtggacttt

1381 gtgttgattc accccgagtt cctgcgcgac cgctctagca ccagcatcct gagccgcctg

1441 cgcggcgcgg gccagcgtgt ggctgcgttc ggctgggcgc agctggggcc catgcgtgac

1501 ctgatcgagt ccgcaaacct ggacggctgg ctggagggcc cctcgttcgg acagggcatc

1561 ctgccggccc acatcgttgc cctggtggcc aagatgcagc agatgcgcaa gatgcagcag

1621 atgcagcaga ttggcatgat gaccggcggc atgaacggca tgggcggcgg tatgggcggc

1681 ggcatgaacg gcatgggcgg cggcaacggc atgaacaaca tgggcaacgg catgggcggc

1741 ggcatgggca acggcatggg cggcaatggc atgaacggaa tgggtggcgg caacggcatg

1801 aacaacatgg gcggcaacgg aatggccggc aacggaatgg gcggcggcat gggcggcaac

1861 ggtatgggtg gctccatgaa cggcatgagc tccggcgtgg tggccaacgt gacgccctcc

1921 gccgccggcg gcatgggcgg catgatgaac ggcggcatgg ctgcgcccca gtcgcccggc

1981 atgaacggcg gccgcctggg taccaacccg ctcttcaacg ccgcgccctc accgctcagc

2041 tcgcagctcg gtgccgaggc aggcatgggc agcatgggag gcatgggcgg aatgagcgga

2101 atgggaggca tgggtggaat ggggggcatg ggcggcgccg gcgccgccac gacgcaggct

2161 gcgggcggca acgcggaggc ggagatgctg cagaatctca tgaacgagat caatcgcctg

2221 aagcgcgagc ttggcgagta a

The wild-type Chop2 of the invention may be encoded by the following amino acid sequence of Chlamydomonas reinhardtii retinal binding protein (cop4) (GenBank No. AAM15777 and SEQ ID NO: 4).

mdyggalsav grellfvtnp vvvngsvlvp edqcycagwi esrgtngaqt asnvlqwlaa gfsilllmfy ayqtwkstcg weeiyvcaie mvkvilefff efknpsmlyl atghrvqwlr

121 yaewlltcpv ilihlsnltg Isndysrrtm 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 gmaggakpgl eltpqlqpgr vilavpdism vdffreqfaq

421 Isvtyelvpa Igadntlalv tqaqnlggvd fvlihpeflr drsstsilsr Irgagqrvaa

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 mggagaattq aaggnaeaem

721 Iqnlmneinr Ikrelge

The wild type Chop2 of the invention may be encoded by the following mRNA sequence

- 8043449 Chlamydomonas reinhardtii sensor opsin B (CSOB) (GenBank No. AF508966 and SEQ ID NO: 5).

ttgacatctg tcgccaagca agcattaaac atggattatg gaggcgccct gagtgccgtt gggcgcgagc tgctatttgt aacgaaccca gtagtcgtca atggctctgt acttgtgcct

121 gaggaccagt gttactgcgc gggctggatt gagtcgcgtg gcacaaacgg tgcccaaacg

181 gcgtcgaacg tgctgcaatg gcttgctgct ggcttctcca tcctactgct tatgttttac

241 gcctaccaaa catggaagtc aacctgcggc tgggaggaga tctatgtgtg cgctatcgag

301 atggtcaagg tgattctcga gttcttcttc gagtttaaga acccgtccat gctgtatcta

361 gccacaggcc accgcgtcca gtggttgcgt tacgccgagt ggcttctcac ctgcccggtc

421 attctcattc acctgtcaaa cctgacgggc ttgtccaacg actacagcag gcgcaccatg

481 ggtctgcttg tgtctgatat tggcacaatt gtgtggggcg ccacttccgc catggccacc

541 ggatacgtca aggtcatctt cttctgcctg ggtctgtgtt atggtgctaa cacgttcttt

601 cacgctgcca aggcctacat cgaggttac cacaccgtgc cgaagggccg gtgtcgccag

661 gtggtgactg gcatggcttg gctcttcttc gtatcatggg gtatgttccc catcctgttc

721 atcctcggcc ccgagggctt cggcgtcctg agcgtgtacg gctccaccgt cggccacacc

781 atcattgacc tgatgtcgaa gaactgctgg ggtctgctcg gccactacct gcgcgtgctg

841 atccacgagc atatcctcat ccacggcgac attcgcaaga ccaccaaatt gaacattggt

901 ggcactgaga ttgaggtcga gacgctggtg gaggacgagg ccgaggctgg cgcggtcaac

961 aagggcaccg gcaagtacgc ctcccgcgag tccttcctgg tcatgcgcga caagatgaag

1021 gagaagggca ttgacgtgcg cgcctctctg gacaacagca aggaggtgga gcaggagcag

1081 gccgccaggg ctgccatgat gatgatgaac ggcaatggca tgggtatggg aatgggaatg

1141 aacggcatga acggaatggg cggtatgaac gggatggctg gcggcgccaa gcccggcctg

1201 gagctcactc cgcagctaca gcccggccgc gtcatcctgg cggtgccgga catcagcatg

1261 gttgacttct tccgcgagca gtttgctcag ctatcggtga cgtacgagct ggtgccggcc

1321 ctgggcgctg acaacacact ggcgctggtt acgcaggcgc agaacctggg cggcgtggac

1381 tttgtgttga ttcaccccga gttcctgcgc gaccgctcta gcaccagcat cctgagccgc

1441 ctgcgcggcg cgggccagcg tgtggctgcg ttcggctggg cgcagctggg gcccatgcgt

1501 gacctgatcg agtccgcaaa cctggacggc tggctggagg gcccctcgtt cggacagggc

1561 atcctgccgg cccacatcgt tgccctggtg gccaagatgc agcagatgcg caagatgcag

1621 cagatgcagc agattggcat gatgaccggc ggcatgaacg gcatgggcgg cggtatgggc

1681 ggcggcatga acggcatggg cggcggcaac ggcatgaaca acatgggcaa cggcatgggc

1741 ggcggcatgg gcaacggcat gggcggcaat ggcatgaacg gaatgggtgg cggcaacggc

1801 atgaacaaca tgggcggcaa cggaatggcc ggcaacggaa tgggcggcgg catgggcggc

1861 aacggtatgg gtggctccat gaacggcatg agctccggcg tggtggccaa cgtgacgccc

1921 tccgccgccg gcggcatggg cggcatgatg aacggcggca tggctgcgcc ccagtcgccc

1981 ggcatgaacg gcggccgcct gggtaccaac ccgctcttca acgccgcgcc ctcaccgctc

2041 agctcgcagc tcggtgccga ggcaggcatg ggcagcatgg gaggcatggg cggaatgagc

2101 ggaatgggag gcatgggtgg aatggggggc atgggcggcg ccggcgccgc cacgacgcag

2161 gctgcgggcg gcaacgcgga ggcggagatg ctgcagaatc tcatgaacga gatcaatcgc

2221 ctgaagcgcg agcttggcga gtaaaaggct ggaggccggt actgcgatac ctgcgagctc

2281 gcgcgcctga ctcgtcgtac acacggctca ggagcacgcg cgcgtggact tctcaacctg

2341 tgtgcaacgt atctagagcg gcctgtgcgc gaccgtccgt gagcattccg gtgcgatctt

2401 cccgccttcg caccgcaagt tcccttcctg gccctgctgc gcctgacgca tcgtccgaac

2461 ggaagggcgg cttgatcagt aaagcattga agactgaagt cgtgcgaccg tagtgctatg

2521 gctctgcacg taagtgggcg ctgccctgct tactacgcat tgcccaagac tgcttccttt

2581 tggtggccga ggccctggtc ccacatcatt catttgcata acgtactgtt tagttacata

2641 cgctttgctt aacctcgaca attgcaacat gggctgagag tccgtacggc ggctatggac

2701 gaaggtgtta tcggatgtga ttaggaatct cggttgaaag gcttcgagaa agtgagcttc

2761 ttctgtggct tctgttgggg tcatcaagaa gaacgacggt aaggcaaacg aggtaaaagt

2821 ggcacgtctt tgtgcacaac gggcccgtgg agagtggggg agtgcatgtg tgcggtccta

2881 acacgcgagt gcaaagcggg cttttctgga gctgggttac ggtctggctc ggcaactgct

2941 ctgtgtttta accacagctt cggaagtctg ggtatgtttt gttggcagaa acatttgggt

3001 aacttgagg tgattcgtct ggagtcggac aacatggctg ccgtccgtgt gcagggacgg

3061 taatcaatga agctgaagct gtgatgctca ccacacgttg catacccctg cttacaaaaa

3121 cactttgatg tcgtggccaa actatgcgtg agcaaagagt taaagaggca tgagtgcatg

3181 gttgcggacg tgcgcaacaa ttgcatcaag tatttgacgc cttcaagcca acaagtgcgc

3241 gcgcggcaac ttgattaaca cgccggacgc agtggtgggg gcgtgtacag tgtttatgag

3301 ctgccattct gcgatccgta gtgttaggtt gcgtgtgacg ccgcgcggct gtgggccctt

3361 acatggagag ttgggtgctt caccacacgg ttggcgccgc tgaagggtgt gctatgtttt

3421 ggtaaagccg gggccctgaa gaccgcaacc gtagaaccgt actgaaaggg tgtcagcccg

3481 gggtaactgg atgccctggg acatagctat taatgttgaa gtgaagccgt caagccgagt

3541 gccgtgcgcc gctgtatcac caaggcccgt ccaaaaaaaa aaaaaaaaaa aaaaaaaaa

The wild-type Chop2 of the invention may be encoded by the following amino acid sequence of Chlamydomonas reinhardtii sensor opsin B (CSOB) (GenBank. No. AAM44040 and SEQ ID NO: 6).

mdyggalsav grellfvtnp vvvngsvlvp edqcycagwi esrgtngaqt asnvlqwlaa gfsilllmfy ayqtwkstcg weeiyvcaie mvkvilefff efknpsmlyl atghrvqwlr

121 yaewlltcpv ilihlsnltg Isndysrrtm gllvsdigti vwgatsamat gyvkviffcl

181 glcygantff haakayiegy htvpkgrcrq vvtgmawlff vswgmfpilf ilgpegfgvl

- 9 043449

241 svygstvght iidlmskncw gllghylrvl ihehilihgd irkttklnig gteievetlv

301 edeaeagavn kgtgkyasre sflvmrdkmk ekgidvrasl dnskeveqeq aaraammmmn

361 gngmgmgmgm ngmngmggmn gmaggakpgl eltpqlqpgr vilavpdism vdffreqfaq

421 Isvtyelvpa Igadntlalv tqaqnlggvd fvlihpeflr drsstsilsr Irgagqrvaa

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 mggagaattq aaggnaeaem

721 Iqnlmneinr Ikrelge

The wild-type Chop2 of the invention may be encoded by the following mRNA sequence of the protozoan opsin-2 Chlamydomonas reinhardtii acop2 (GenBank No. AB058891 and SEQ ID NO: 7).

catctgtcgc caagcaagca ttaaacatgg attatggagg cgccctgagt gccgttgggc gcgagctgct atttgtaacg aacccagtag tcgtcaatgg ctctgtactt gtgcctgagg

121 accagtgtta ctgcgcgggc tggattgagt cgcgtggcac aaacggtgcc caaacggcgt

181 cgaacgtgct gcaatggctt gctgctggct tctccatcct actgcttatg ttttacgcct

241 accaaacatg gaagtcaacc tgcggctggg aggagatcta tgtgtgcgct atcgagatgg

301 tcaaggtgat tctcgagttc ttcttcgagt ttaagaaccc gtccatgctg tatctagcca

361 caggccaccg cgtccagtgg ttgcgttacg ccgagtggct tctcacctgc ccggtcattc

421 tcattcacct gtcaaacctg acgggcttgt ccaacgacta cagcaggcgc accatgggtc

481 tgcttgtgtc tgatattggc acaattgtgt ggggcgccac ttccgccatg gccaccggat

541 acgtcaaggt catcttcttc tgcctgggtc tgtgttatgg tgctaacacg ttctttcacg

601 ctgccaaggc ctacatcgag ggttaccaca ccgtgccgaa gggccggtgt cgccaggtgg

661 tgactggcat ggcttggctc ttcttcgtat catggggtat gttccccatc ctgttcatcc

721 tcggccccga gggcttcggc gtcctgagcg tgtacggctc caccgtcggc cacaccatca

781 ttgacctgat gtcgaagaac tgctggggtc tgctcggcca ctacctgcgc gtgctgatcc

841 acgagcatat cctcatccac ggcgacattc gcaagaccac caaattgaac attggtggca

901 ctgagattga ggtcgagacg ctggtggagg acgaggccga ggctggcgcg gtcaacaagg

961 gcaccggcaa gtacgcctcc cgcgagtcct tcctggtcat gcgcgacaag atgaaggaga

1021 agggcattga cgtgcgcgcc tctctggaca acagcaagga ggtggagcag gagcaggccg

1081 ccagggctgc catgatgatg atgaacggca atggcatggg tatgggaatg ggaatgaacg

1141 gcatgaacgg aatgggcggt atgaacggga tggctggcgg cgccaagccc ggcctggagc

1201 tcactccgca gctacagccc ggccgcgtca tcctggcggt gccggacatc agcatggttg

1261 acttcttccg cgagcagttt gctcagctat cggtgacgta cgagctggtg ccggccctgg

1321 gcgctgacaa cacactggcg ctggttacgc aggcgcagaa cctgggcggc gtggactttg

1381 tgttgattca ccccgagttc ctgcgcgacc gctctagcac cagcatcctg agccgcctgc

1441 gcggcgcggg ccagcgtgtg gctgcgttcg gctgggcgca gctggggccc atgcgtgacc

1501 tgatcgagtc cgcaaacctg gacggctggc tggagggccc ctcgttcgga cagggcatcc

1561 tgccggccca catcgttgcc ctggtggcca agatgcagca gatgcgcaag atgcagcaga

1621 tgcagcagat tggcatgatg accggcggca tgaacggcat gggcggcggt atgggcggcg

1681 gcatgaacgg catgggcggc ggcaacggca tgaacaacat gggcaacggc atgggcggcg

1741 gcatgggcaa cggcatgggc ggcaatggca tgaacggaat gggtggcggc aacggcatga

1801 acaacatggg cggcaacgga atggccggca acggaatggg cggcggcatg ggcggcaacg

1861 gtatgggtgg ctccatgaac ggcatgagct ccggcgtggt ggccaacgtg acgccctccg

1921 ccgccggcgg catgggcggc atgatgaacg gcggcatggc tgcgccccag tcgcccggca

1981 tgaacggcgg ccgcctgggt accaacccgc tcttcaacgc cgcgccctca ccgctcagct

2041 cgcagctcgg tgccgaggca ggcatgggca gcatgggagg catgggcgga atgagcggaa

2101 tgggaggcat gggtggaatg gggggcatgg gcggcgccgg cgccgccacg acgcaggctg

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 gcaagttccc ttcctggccc tgctgcgcct gacgcatc

The wild-type Chop2 of the invention may be encoded by the following Chlamydomonas reinhardtii acop2 mRNA sequence encoding the amino acid sequence of prototype opsin-2 (GenBank No. BAB68567 and SEQ ID NO: 8).

mdyggalsav grellfvtnp vvvngsvlvp edqcycagwi esrgtngaqt asnvlqwlaa gfsilllmfy ayqtwkstcg weeiyvcaie mvkvilefff efknpsmlyl atghrvqwlr

121 yaewlltcpv ilihlsnltg Isndysrrtm 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 gmaggakpgl eltpqlqpgr vilavpdism vdffreqfaq

421 Isvtyelvpa Igadntlalv tqaqnlggvd fvlihpeflr drsstsilsr Irgagqrvaa

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 mggagaattq aaggnaeaem

721 Iqnlmneinr Ikrelge

ChR2 mutants.

The present invention provides Chop2 mutants, wherein one or more amino acids under

- 10 043449 mutations have been turned down. In some embodiments, Chop2 is a full-length polypeptide, such as SEQ ID NO: 2, 4, 6 and 8, containing at least one amino acid mutation. In some embodiments, the mutation is located at amino acid 132 and/or amino acid 159. In some preferred embodiments, the amino acid at position 132 is mutated from leucine to cysteine or alanine. In some preferred embodiments, the amino acid at position 159 is mutated from threonine to alanine, cysteine, or serine. In all embodiments, Chop2 mutants form a functional ChR2 channel.

The present invention also covers Chop2 proteins and nucleic acids that encode a biologically active fragment or conservative amino acid substitution or other mutant variant of Chop2. Particular examples of useful fragments include polypeptides encoding amino acids 1-315 of wild type Chop2, i.e. SEQ ID NO: 26, wherein at least one amino acid is subject to mutation or conservative substitution, for example, at amino acid positions 132 and/or 159. Shorter fragments of wild-type Chop2, wherein at least one amino acid is subject to mutation or conservative substitution (i.e. e. at amino acid positions 132 and/or 159) can also be used in the present invention. Accordingly, the Chop2 polypeptides and nucleic acids of the present invention further include, in particular, biologically active fragments encoding amino acids 1-315, 1-310, 1-300, 1-275, 1-250, 1-225, 1-200, 1- 175, or 1-160 wild-type Chop2, wherein at least one amino acid is subject to mutation or conservative substitution, for example at amino acid positions 132 and/or 159. In other embodiments, the Chop2 polypeptides and nucleic acids of the present invention can be about or up to 315 amino acids, 310 amino acids, 300 amino acids, 275 amino acids, 250 amino acids, 225 amino acids, 200 amino acids, 175 amino acids or 160 amino acids.

The Chop2 single mutant of the invention can be encoded by the following synthetic gene sequence construct hVChR1-mKate-betahChR2 (L132C) (GenBank No. JN836746 and SEQ ID NO: 9) containing the following annotations, the GFP sequence is indicated in bold, the Chop2 L132C sequence is underlined.

atggattacc ctgtggcccg gtccctgatt gtaagatacc ccaccgatct gggcaatgga accgtgtgca tgcccagagg acaatgctac tgcgaggggt ggctgaggag ccggggcact

121 agtatcgaaa aaaccatcgc tatcaccctc cagtgggtag tgttcgctct gtccgtagcc

181 tgtctcggct ggtatgcata ccaagcctgg agggctacct gtgggtggga ggaagtatac

241 gtggccctga tcgagatgat gaagtccatc atcgaggctt tccatgagtt cgactcccca

301 gccacactct ggctcagcag tgggaatggc gtagtgtgga tgagatatgg agagtggctg

361 ctgacctgtc ccgtcctgct cattcatctg tccaatctga ccgggctgaa agatgactac

421 tccaagagaa caatgggact gctggtgagt gacgtggggt gtattgtgtg gggagccacc

481 tccgccatgt gcactggatg gaccaagatc ctctttttcc tgatttccct ctcctatggg

541 atgtatacat acttccacgc cgctaaggtg tatattgagg ccttccacac tgtacctaaa

601 ggcatctgta gggagctcgt gcgggtgatg gcatggacct tctttgtggc ctgggggatg

661 ttccccgtgc tgttcctcct cggcactgag ggatttggcc acattagtcc ttacgggtcc

721 gcaattggac actccatcct ggatctgatt gccaagaata tgtggggggt gctgggaaat

781 tatctgcggg taaagatcca cgagcatatc ctgctgtatg gcgatatcag aaagaagcag

841 aaaatcacca ttgctggaca ggaaatggag gtggagacac tggtagcaga ggaggaggac

901 gggaccgcgg tcgccaccat ggtgtctaag ggcgaagagc tgattaagga gaacatgcac

961 atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc

1021 gaaggcaagc cctacgagg cacccagacc atgagaatca aggtggtcga gggcggccct

1081 ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcaa aaccttcatc

1141 aaccacaccc agggcatccc cgacttcttt aagcagtcct tccctgaggg cttcacatgg

1201 gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc

1261 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc

1321 cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagatgct gtaccccgct

1381 gacggcggcc tggaaggcag agccgacatg gccctgaagc tcgtgggcgg gggccacctg

1441 atctgcaact tgaagaccac atacagatcc aagaaacccg ctaagaacct caagatgccc

1501 ggcgtctact atgtggacag aagactggaa agaatcaagg aggccgacaa agagacctac

1561 gtcgagcagc acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac

1621 aaacttaatt gcctgcagga gaagaagtca tgcagccagc gcatggccga attccggcaa

1681 tactgttgga acccggacac tgggcagatg ctgggccgca ccccagcccg gtgggtgtgg

1741 atcagcctgt actatgcagc tttctacgtg gtcatgactg ggctctttgc cttgtgcatc

1801 tatgtgctga tgcagaccat tgatccctac acccccgact accaggacca gttaaagtca

1861 ccgggggtaa ccttgagacc ggatgtgtat ggggaaagag ggctgcagat ttcctacaac

1921 atctctgaaa acagctctag acaggcccag atcaccggac gtccggagac tgagacattg

1981 s s a s cggtgg actacggggg ggccctgagc gctgtgggca gagaactcct gttcgtgaca

2041 aatccagtcg tggtgaacgg ctccgtactc gtacccgagg 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 ctgcggtatg ccgaatggct gctgacttgc ccagtgattt gcatccacct gtccaacctg

2401 actgggctgt ctaacgatta cagtaggaga acaatgggac tgctcgtatc cgacatcggc

2461 actatcgtat ggggcgcaac tagtgccatg gccactggat acgtgaaagt gatcttcttc

- 11 043449

2521 tgcctgggac tctgctacgg agcaaacaca ttttttcatg ccgcaaaagc atatatcgag

2581 gggtatcata ccgtcccaaa gggccggtgt agacaagtgg tgactggcat ggcttggctg

2641 ttcttcgtgt cctgggggat gtttcccatc ctctttatcc tgggcccaga aggcttcggg

2701 gtgctgagtg tgtatggcag taccgtagga cacactatca ttgacctgat gagcaaaaac

761 tgctgggggc tgctcggcca ctacctgaga gtactcatcc acgagcatat cctgattcat

2821 ggcgatatcc ggaaaactac caagctcaat atcggggggca ccgagattga agtggagaca

2881 ctcgtggagg acgaggccga ggccggagca gtgaacaaag gcactggcaa gtatgcctcc

2941 agagaatcct ttctggtgat gcgggacaaa atgaaggaga aaggcattga tgtacggtgc

3001 agtaatgcca aagccgtcga gactgatgtg tag

The ChR2 single mutant of the invention can be encoded by the following synthetic amino acid sequence construct hVChR1-mKate-betahChR2 (L132C) (GenBank No. AER29839 and SEQ ID NO: 10) containing the following annotations, the GFP sequence is indicated in bold, the Chop2 L132C sequence is underlined.

mdypvarsli vryptdlgng tvcmprgqcy cegwlrsrgt siektiaitl qwvvfalsva clgwyayqaw ratcgweevy valiemmksi ieafhefdsp atlwlssgng vvwmrygewl

121 Itcpvllihl snltglkddy skrtmgllvs dvgcivwgat samctgwtki Ifflislsyg

181 mytyfhaakv yieafhtvpk gicrelvrvm awtffvawgm fpvlfllgte gfghispygs

241 aighsildli aknmwgvlgn ylrvkihehi llygdirkkq kitiagqeme vetlvaeeed

301 gtavatmvsk geelikenmh mklymegtvn nhhfkctseg egkpyegtqt mrikweggp

361 Ipfafdilat sfmygsktfi nhtqgipdff kqsfpegftw ervttyedgg vltatqdtsl

421 qdgcliynvk irgvnfpsng pvmqkktlgw eastemlypa dgglegradm alklvggghl

481 icnlkttyrs kkpaknlkmp gvyyvdrrle rikeadkety veqhevavar ycdlpsklgh

541 klnclqekks csqrmaefrq ycwnpdtgqm Igrtparwvw islyyaafyv vmtglfalci

601 yvlmqtidpy tpdyqdqlks pgvtlrpdvy gerglqisyn isenssrqaq itgrpetetl

661 ppvdyggals avgrellfvt npvvvngsvl vpedqcycag wiesrgtnga qtasnvlqwl

721 aagfsilllm fyayqtwkst cgweeiyvca iemvkvilef ffefknpsml ylatghrvqw

781 Iryaewlltc pvicihlsnl tglsndysrr tmgllvsdig tivwgatsam atgyvkviff

841 clglcygant ffhaakayie gyhtvpkgrc rqvvtgmawl ffvswgmfpi Ifilgpegfg

901 vlsvygstvg htiidlmskn cwgllghylr vlihehilih gdirkttkln iggteievet

961 Ivedeaeaga vnkgtgkyas resflvmrdk mkekgidvrc snakavetdv

The Chop2 single mutant of the invention may be encoded by the following gene sequence of the synthetic construct hVChR1-mKate-betahChR2 (L132C) (GenBank. No. JN836745 and SEQ ID NO: 11) containing the following annotations, the GFP sequence is indicated in bold, the Chop2 L132C sequence is underlined.

atggattacc ctgtggcccg gtccctgatt gtaagatacc ccaccgatct gggcaatgga accgtgtgca tgcccagagg acaatgctac tgcgaggggt ggctgaggag ccggggcact

121 agtatcgaaa aaaccatcgc tatcaccctc cagtgggtag tgttcgctct gtccgtagcc

181 tgtctcggct ggtatgcata ccaagcctgg agggctacct gtgggtggga ggaagtatac

241 gtggccctga tcgagatgat gaagtccatc atcgaggctt tccatgagtt cgactcccca

301 gccacactct ggctcagcag tgggaatggc gtagtgtgga tgagatatgg agagtggctg

361 ctgacctgtc ccgtcctgct cattcatctg tccaatctga ccgggctgaa agatgactac

421 tccaagagaa caatgggact gctggtgagt gacgtggggt gtattgtgtg gggagccacc

481 tccgccatgt gcactggatg gaccaagatc ctctttttcc tgatttccct ctcctatggg

541 atgtatacat acttccacgc cgctaaggtg tatattgagg ccttccacac tgtacctaaa

601 ggcatctgta gggagctcgt gcgggtgatg gcatggacct tctttgtggc ctgggggatg

661 ttccccgtgc tgttcctcct cggcactgag ggatttggcc acattagtcc ttacgggtcc

721 gcaattggac actccatcct ggatctgatt gccaagaata tgtggggggt gctgggaaat

781 tatctgcggg taaagatcca cgagcatatc ctgctgtatg gcgatatcag aaagaagcag

841 aaaatcacca ttgctggaca ggaaatggag gtggagacac tggtagcaga ggaggaggac

901 gggaccgcgg tcgccaccat ggtgtctaag ggcgaagagc tgattaagga gaacatgcac

- 12 043449

961 atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc

1021 gaaggcaagc cctacgagg cacccagacc atgagaatca aggtggtcga gggcggccct

1081 ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcaa aaccttcatc

1141 aaccacaccc agggcatccc cgacttcttt aagcagtcct tccctgaggg cttcacatgg

1201 gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc

1261 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc

1321 cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagatgct gtaccccgct

1381 gacggcggcc tggaaggcag agccgacatg gccctgaagc tcgtgggcgg gggccacctg

1441 atctgcaact tgaagaccac atacagatcc aagaaacccg ctaagaacct caagatgccc

1501 ggcgtctact atgtggacag aagactggaa agaatcaagg aggccgacaa agagacctac

1561 gtcgagcagc acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac

1621 aaacttaatt gcctgcagga gaagaagtca tgcagccagc gcatggccga attccggcaa

1681 tactgttgga acccggacac tgggcagatg ctgggccgca ccccagcccg gtgggtgtgg

1741 atcagcctgt actatgcagc tttctacgtg gtcatgactg ggctctttgc cttgtgcatc

1801 tatgtgctga tgcagaccat tgatccctac acccccgact accaggacca gttaaagtca

1861 ccgggggtaa ccttgagacc ggatgtgtat ggggaaagag ggctgcagat ttcctacaac

1921 atctctgaaa acagctctag acaggcccag atcaccggac gtccggagac tgagacattg

1981 ccaccggtgg actacgggggg ggccctgagc gctgtgggca gagaactcct gttcgtgaca

2041 aatccagtcg tggtgaacgg ctccgtactc gtacccgagg 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 ctgcggtatg ccgaatggct gctgacttgc ccagtgattc tgatccacct gtccaacctg

2401 actgggctgt ctaacgatta cagtaggaga acaatgggac tgctcgtatc cgacatcggc

2461 actatcgtat ggggcgcaac tagtgccatg gccactggat acgtgaaagt gatcttcttc

2521 tgcctqggac tctqctacqq aqcaaacaca ttttttcatg ccgcaaaagc atatatcgag

2581 gggtatcata ccgtcccaaa gggccggtgt agacaagtgg tgactggcat ggcttggctg

2641 ttcttcgtgt cctgggggat gtttcccatc ctctttatcc tgggcccaga aggcttcggg

2701 gtgctgagtg tgtatggcag taccgtagga cacactatca ttgacctgat gagcaaaaac

2761 tgctggggggc tgctcggcca ctacctgaga gtactcatcc acgagcatat cctgattcat

2821 ggcgatatcc ggaaaactac caagctcaat atcggggggca ccgagattga agtggagaca

2881 ctcgtggagg acgagqccqa gqccqgagca gtgaacaaag gcactqgcaa gtatgcctcc

2941 agagaatcct ttctggtgat gcgggacaaa atgaaggaga aaggcattga tgtacggtgc

3001 agtaatgcca aagccgtcga gactgatgtg tag

The Chop2 single mutant of the invention can be encoded by the following amino acid sequence of the synthetic construct hVChR1-mKate-betahChR2 (L132C) (GenBank No. AER29838 and SEQ ID NO: 12) containing the following annotations, the GFP sequence is indicated in bold, the Chop2 L132C sequence is underlined.

mdypvarsli vryptdlgng tvcmprgqcy cegwlrsrgt siektiaitl qwvvfalsva clgwyayqaw ratcgweevy valiemmksi ieafhefdsp atlwlssgng vvwmrygewl

121 Itcpvllihl snltglkddy skrtmgllvs dvgcivwgat samctgwtki Ifflislsyg

181 mytyfhaakv yieafhtvpk gicrelvrvm awtffvawgm fpvlfllgte gfghispygs

241 aighsildli aknmwgvlgn ylrvkihehi llygdirkkq kitiagqeme vetlvaeeed

301 gtavatmvsk geelikenmh mklymegtvn nhhfkctseg egkpyegtqt mrikweggp

361 Ipfafdilat sfmygsktfi nhtqgipdff kqsfpegftw ervttyedgg vltatqdtsl

421 qdgcliynvk irgvnfpsng pvmqkktlgw eastemlypa dgglegradm alklvggghl

481 icnlkttyrs kkpaknlkmp gvyyvdrrle rikeadkety veqhevavar ycdlpsklgh

541 klnclqekks csqrmaefrq ycwnpdtgqm Igrtparwvw islyyaafyv vmtglfalci

601 yvlmqtidpy tpdyqdqlks pgvtlrpdvy gerglqisyn isenssrqaq itgrpetetl

661 ppvdyggals avgrellfvt npvvvngsvl vpedqcycag wiesrgtnga qtasnvlqwl

721 aagfsilllm fyayqtwkst cgweeiyvca iemvkvilef ffefknpsml ylatghrvqw

781 Iryaewlltc pvilihlsnl tglsndysrr tmgllvsdig tivwgatsam atgyvkviff

841 clglcygant ffhaakayie gyhtvpkgrc rqvvtgmawl ffvswgmfpi Ifilgpegfg

901 vlsvygstvg htiidlmskn cwgllghylr vlihehilih gdirkttkln iggteievet

961 Ivedeaeaga vnkgtgkyas resflvmrdk mkekgidvrc snakavetdv

The L132C Chop2 single mutant of the invention may be encoded by the following amino acid sequence (position 132 is underlined and in bold, SEQ ID NO: 13).

MDYGGALSAV GRELLFVTNP VWNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA

GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR

121 YAEWLLTCPV ICIHLSNLTG LSNDYSRRTM GLLVSDIGTI VWGATSAMAT GYVKVIFFCL

181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ WTGMAWLFF VSWGMFPILF ILGPEGFGVL

241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV

301 EDEAEAGAVN KGTGK

- 13 043449

The T159C Chop2 single mutant of the invention may be encoded by the following amino acid sequence (position 159 is underlined and in bold, SEQ ID NO: 14).

MDYGGALSAV GRELLFVTNP VWNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA

GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR

121 YAEWLLTCPV ILIHLSNLTG LSNDYSRRTM GLLVSDIGCI VWGATSAMAT GYVKVIFFCL

181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ WTGMAWLFF VSWGMFPILF ILGPEGFGVL

241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEEAAGAVN KGTGK

The L132C/T159C Chop2 double mutant of the invention may be encoded by the following nucleotide sequence (SEQ ID NO: 15).

atggactacg ggggggctct gtctgctgtc gggagggaac tgctgtttgt gactaaccct 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 gtggctgaga

361 tatgcagaat ggctgctgac atgccccgtc atctgcattc acctgtccaa cctgacaggc

421 ctgagcaatg actactccag gagaactatg ggactgctgg tgtccgacat cggctgcatt

481 gtctggggag caacttctgc tatggcaacc ggatacgtga aggtcatctt tttctgcctg

541 gggctgtgct atggcgcaaa tacctttttc cacgcagcca aggcctacat tgaggggtat

601 cataccgtgc caaaaggccg gtgccgacag gtggtcacag gaatggcttg gctgtttttc

661 gtctcttggg gaatgtttcc 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 ctgaggctgg ggctgtgaac aaggggactg gcaaa

The L132C/T159C Chop2 double mutant of the invention can be encoded by the following amino acid sequence (positions 132 and 159 are underlined and in bold, SEQ ID NO: 16).

MDYGGALSAV GRELLFVTNP VWNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA

GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR

121 YAEWLLTCPV ICIHLSNLTG LSNDYSRRTM GLLVSDIGCI VWGATSAMAT GYVKVIFFCL

181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ WTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK

The T159C Chop2 single mutant of the invention may be encoded by the following amino acid sequence (position 159 is underlined and in bold, SEQ ID NO: 17).

MDYGGALSAV GRELLFVTNP VWNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA

GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR

121 YAEWLLTCPV ILIHLSNLTG LSNDYSRRTM GLLVSDIGSI VWGATSAMAT GYVKVIFFCL

181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ WTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK

The L132C/T159S Chop2 double mutant of the invention may be encoded by the following nucleotide sequence (SEQ ID NO: 18).

atggactacg ggggggctct gtctgctgtc gggagggaac tgctgtttgt gactaaccct 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 gtggctgaga

361 tatgcagaat ggctgctgac atgccccgtc atctgcattc acctgtccaa cctgacaggc

421 ctgagcaatg actactccag gagaactatg ggactgctgg tgtccgacat cggcagcatt

481 gtctggggag caacttctgc tatggcaacc ggatacgtga aggtcatctt tttctgcctg

541 gggctgtgct atggcgcaaa tacctttttc cacgcagcca aggcctacat tgaggggtat

601 cataccgtgc caaaaggccg gtgccgacag gtggtcacag gaatggcttg gctgtttttc

661 gtctcttggg gaatgtttcc 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 ctgaggctgg ggctgtgaac aaggggactg gcaaa

The L132C/T159S Chop2 double mutant of the invention can be encoded by the following amino acid sequence (positions 132 and 159 are underlined and in bold, SEQ ID NO: 19).

MDYGGALSAV GRELLFVTNP VWNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA

GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR

121 YAEWLLTCPV ICIHLSNLTG LSNDYSRRTM GLLVSDIGSI VWGATSAMAT GYVKVIFFCL

181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ WTGMAWLFF VSWGMFPILF ILGPEGFGVL

241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL· IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEEAAGAVN KGTGK

- 14 043449

The L132A Chop2 single mutant of the invention may be encoded by the following amino acid sequence (position 132 is underlined and in bold, SEQ ID NO: 20).

MDYGGALSAV GRELLFVTNP VWNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA

GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR

121 YAEWLLTCPV IAIHLSNLTG LSNDYSRRTM GLLVSDIGTI VWGATSAMAT GYVKVIFFCL

181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ WTGMAWLFF VSWGMFPILF ILGPEGFGVL

241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL· IHEHILIHGD IRKTTKLNIG GTEIEVETLV

301 EDEAEAGAVN KGTGK

The L132A/T159C Chop2 double mutant of the invention may be encoded by the following nucleotide sequence (SEQ ID NO: 21).

ATGGACTACG GGGGGGCTCT GTCTGCTGTC GGGAGGGAAC TGCTGTTTGT GACTAACCCT

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 GTGGCTGAGA

361 TATGCAGAAT GGCTGCTGAC ATGCCCCGTC ATCGCCATTC ACCTGTCCAA CCTGACAGGC

421 CTGAGCAATG ACTACTCCAG GAGAACTATG GGACTGCTGG TGTCCGACAT CGGCTGCATT

481 GTCTGGGGAG CAACTTCTGC TATGGCAACC GGATACGTGA AGGTCATCTT TTTCTGCCTG

541 GGGCTGTGCT ATGGCGCAAA TACCTTTTTC CACGCAGCCA AGGCCTACAT TGAGGGGTAT

601 CATACCGTGC CAAAAGGCCG GTGCCGACAG GTGGTCACAG GAATGGCTTG GCTGTTTTTC

661 GTCTCTTGGG GAATGTTTCC 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 CTGAGGCTGG GGCTGTGAAC AAGGGGACTG GCAAA

The L132A/T159C Chop2 double mutant of the invention can be encoded by the following amino acid sequence (positions 132 and 159 are underlined and in bold, SEQ ID NO: 22).

MDYGGALSAV GRELLFVTNP VWNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA

GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR

121 YAEWLLTCPV IAIHLSNLTG LSNDYSRRTM GLLVSDIGCI VWGATSAMAT GYVKVIFFCL

181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ WTGMAWLFF VSWGMFPILF ILGPEGFGVL 241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEAEAGAVN KGTGK

The T159A Chop2 single mutant of the invention may be encoded by the following amino acid sequence (position 159 is underlined and in bold, SEQ ID NO: 23).

MDYGGALSAV GRELLFVTNP VWNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA

GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR

121 YAEWLLTCPV ILIHLSNLTG LSNDYSRRTM GLLVSDIGAI VWGATSAMAT GYVKVIFFCL

181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ WTGMAWLFF VSWGMFPILF ILGPEGFGVL

241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV 301 EDEEAAGAVN KGTGK

The L132C/T159A Chop2 double mutant of the invention may be encoded by the following nucleotide sequence (SEQ ID NO: 24).

atggactacg ggggggctct gtctgctgtc gggagggaac tgctgtttgt gactaaccct 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 gtggctgaga

361 tatgcagaat ggctgctgac atgccccgtc atctgcattc acctgtccaa cctgacaggc

421 ctgagcaatg actactccag gagaactatg ggactgctgg tgtccgacat cggcgccatt

481 gtctggggag caacttctgc tatggcaacc ggatacgtga aggtcatctt tttctgcctg

541 gggctgtgct atggcgcaaa tacctttttc cacgcagcca aggcctacat tgaggggtat

601 cataccgtgc caaaaggccg gtgccgacag gtggtcacag gaatggcttg gctgtttttc

661 gtctcttggg gaatgtttcc 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 ctgaggctgg ggctgtgaac aaggggactg gcaaa

The L132C/T159A Chop2 double mutant of the invention can be encoded by the following amino acid sequence (positions 132 and 159 are underlined and in bold, SEQ ID NO: 25).

MDYGGALSAV GRELLFVTNP VWNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA

GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR

121 YAEWLLTCPV ICIHLSNLTG LSNDYSRRTM GLLVSDIGAI VWGATSAMAT GYVKVIFFCL

181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ WTGMAWLFF VSWGMFPILF ILGPEGFGVL

241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV

301 EDEAEAGAVN KGTGK

- 15 043449

The wild type Chop2 of the invention may be encoded by the following amino acid sequence (SEQ ID NO: 26).

MDYGGALSAV GRELLFVTNP VWNGSVLVP EDQCYCAGWI ESRGTNGAQT ASNVLQWLAA

GFSILLLMFY AYQTWKSTCG WEEIYVCAIE MVKVILEFFF EFKNPSMLYL ATGHRVQWLR

121 YAEWLLTCPV ILIHLSNLTG LSNDYSRRTM GLLVSDIGTI VWGATSAMAT GYVKVIFFCL

181 GLCYGANTFF HAAKAYIEGY HTVPKGRCRQ WTGMAWLFF VSWGMFPILF ILGPEGFGVL

241 SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV

301 EDEAEAGAVN KGTGK

The ChR2 mutant proteins of the invention also exhibit slower channel kinetics. Higher photosensitivity was found to correlate with channels with slower kinetics, revealing a relationship between photosensitivity and channel kinetics. The Chop2 proteins that form the ChR2 proteins of the present invention may also include additional mutations or modifications that may improve channel kinetics, or increase the degree of ChR2 deactivation. Particularly favored ChR2 mutants balance the photosensitivity threshold with channel kinetics.

Compositions and sets of reagents.

Compositions and reagent kits of the invention include at least one nucleic acid molecule or polypeptide molecule that encodes a mutant Chop2 protein, and the resulting ChR2, of the invention. The at least one nucleic acid molecule or polypeptide molecule that encodes the Chop2 mutant protein of the invention may further comprise a pharmaceutically acceptable carrier. The reagent kits of the invention further include instructions for administering a composition of the invention to the subject.

Therapeutic applications.

Mutations were performed on the codon-optimized Chop2-GFP cross-linked protein to create single and double mutations at sites L132 (Leucine 132) and T159 (threonine 159). The functional properties of each ChR2 mutant or combination thereof were first examined in HEK cells. ΛΛV2 viral vectors carrying mutant Chop2-GFP constructs driven by the CLG promoter were obtained and injected intravitreally into the eyes of an adult mouse. Mutant Chop2-mediated response to light was examined using multielectrode chip recordings from whole-mount retina.

ChR2 single mutant, i.e. L132 and T159C, significantly reduced the threshold photosensitivity required to induce ChR2-mediated photocurrent. In addition, several ChR2 double mutant variants, including L132C/T159C, L132L/T159C, and L132C/T159S, were found to further increase photocurrent above the results of any ChR2 single mutant at low light intensities. The double mutants exhibited slower turn-off, which likely contributes to the increased photocurrent at low light intensities. Spike activity of retinal ganglion cells mediated by the L132C/T159C double mutant was observed at a light intensity of 10 ¹³ photons/cm ² /s and a wavelength of 473 nm. This light level is approximately 1.5 to 2 log units lower than the light level required to elicit spike activity by wild-type ChR2. Spike firing in retinal ganglion cells expressing L132C/T159C can follow light flicker frequencies of up to 15 Hz. Ongoing studies are evaluating the long-term expression and safety of the ChR2 mutants of the invention in retinal neurons.

In addition, it was found that expression of mutant Chop2 proteins and the resulting ChR2 proteins of the present invention does not cause neurotoxicity up to two months after viral infection of mice, demonstrating the safety of the present invention for therapeutic use.

Vectors for use in the present invention may include various viral vectors such as plasmids and recombinant viruses, i.e. recombinant adeno-associated virus (rALV), recombinant adenoviruses, recombinant retroviruses, recombinant lentiviruses and other viruses known in the art.

In some embodiments, the expression of the Chop2 proteins of the present invention is driven by a constitutive promoter, i.e. CLG promoter, CMV promoter, LTR. In other embodiments, the promoter is inducible or cell specific. Cell-specific promoters that enable the expression of the Chop2 protein in specific subpopulations of cells, i.e. in retinal neuronal cells or in degrading cells may be preferable. These cells may include, but are not limited to, retinal ganglion cells, photoreceptor cells, bipolar cells, rod bipolar cells, ON-type cone bipolar cells, photosensitive retinal ganglion cells, horizontal cells, amacrine cells, or L11 amacrine cells. Cell-specific promoters are known in the art. Particularly preferred cell-specific promoters include, but are not limited to, mGluR6, NK-3, and Pcp2 (L7).

In some embodiments, the use of various opsin genes in addition to the Chop2 mutant proteins of the present invention and targeted gene expression can further enhance photosensitivity or improve vision. Visual information is processed through the retina in two pathways: the ON pathway, which transmits the light-on signal ON, and the OFF-pathway, which transmits the light-off signal OFF. The existence of an ON and OFF pathway is important for enhancing contrast sensitivity. The visual signal in the ON pathway is a switch from ON cone bipolar cells to ON ganglion cells. Both ON cone bipolar cells and ON ganglion cells depolarize 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 OFF cone bipolar cells and OFF ganglion cells hyperpolarize in response to light. Rod bipolar cells, which are responsible for the ability to see in low light (scotopic vision), are ON bipolar cells (depolarized in response to light). Rod bipolar cells transmit visual signals through AII amarcrine cells (ON-type retinal cells) to ON and OFF cone bipolar cells.

Accordingly, the dualistic rhodopsin system can be used to summarize the ON and OFF pathways in general with visual processing and visual acuity. Briefly, the Chop2 protein of the present invention can be specifically targeted to ON-type retinal neurons (i.e., ON-type ganglion cells and/or ON-type bipolar cells), while the light hypopolarization sensor (i.e., halorhodopsin or another chloride pump known in the art) can be directed to OFF-type retinal neurons (ie, OFF-type ganglion cells and/or OFF-type bipolar cells) to create the ON and OFF pathway. Specific targeting to preferred subpopulations can be achieved through the use of various cell-specific promoters. For example, expression of Chop2 may be driven by the mGluR6 promoter to target expression in ON-type retinal neurons (i.e., ON-type ganglion cells and/or ON-type bipolar cells), while expression of a hyperpolarization channel such as halogenorhodopsin driven by the NK-3 promoter for targeted expression in OFF-type retinal neurons (ie, OFF-type ganglion cells and/or OFF-type bipolar cells).

An alternative way to restore ON and OFF pathways in the retina is achieved by expressing a light depolarization sensor such as ChR2 in rod bipolar cells or AII amacrine cells. In this method, depolarization of rod bipolar cells or AII amacrine cells can lead to ON and OFF responses at the level of cone bipolar cells and downstream retinal ganglion cell signaling pathways. Thus, ON and OFF pathways that are characteristic of the retina are supported.

The present invention may relate to a pharmaceutical composition or a drug suitable for administration to a subject or patient. Suitable routes of administration include, for example, intravitreal, intraocular or subretinal injection.

Such formulations include a pharmaceutically and/or physiologically acceptable vehicle, diluent, carrier or excipient, such as a buffer salt or other buffers, for example, HEPES, to maintain physiological pH. To discuss such components and their composition; see mainly Gennaro, A.E., Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishers; 2003 or latest edition). See also WO 00/15822. If the drug must be stored for a long time, it can be frozen, for example, in the presence of glycerol.

The pharmaceutical composition described above is administered to a subject having visual disease or blindness through a suitable route, preferably intravitreal or subretinal injection depending on the retinal layer that is targeted.

The description by Bennett and co-workers (cited herein) concerns targeting the retinal pigment epithelium, the most distal layer from the vitreal space. According to the present invention, the Chop2 construct or polypeptide acts specifically on retinal cells, i.e. retinal ganglion cells or bipolar cells. Such cells are known to be quite readily available for intravitreal injection, as disclosed herein. Intravitreal and/or subretinal injection may provide the necessary access to bipolar cells, especially in circumstances in which the photoreceptor cell layer is absent due to degeneration, as occurs in some forms of degeneration that the present invention is intended to overcome.

To test the ability of a vector to express Chop2 mutants of the present invention specifically in mammalian retinal neurons using AAV-mediated delivery, a combination of a preferred promoter sequence linked to a reporter gene such as LacZ or GFP linked to an SV40 poly A sequence can be inserted into a plasmid and packaged into rAAV viral particles, concentrated, tested for adenoviral contamination, and the rAAV titer determined using an infection assay. The right eyes of a number of test subjects, preferably inbred mice, can be injected subretinal with about 1 μl of rAAV preparation (eg, higher than about 10 ¹⁰ infectious units per ml). Two weeks later, the right (test) and left (control) eyes of half the animals can be removed, fixed and stained with a suitable substrate or antibody or other substance to detect the presence of

- 17 043449 action of the reporter gene. Most tested retinas in injected eyes show a focal staining area, such as blue for LacZ/Xgal, or green for GFP, consistent with the subretinal cavity 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 laterally sacrificed mice was detected for at least 10 weeks after injection, suggesting stable expression of the reporter transgene.

In one embodiment, Chop2 constructs are packaged into adenoviral vectors for transgene delivery. The effective number of rAAV virions carrying the Chop2 DNA coding sequence under the control of a selected promoter, preferably a constitutive CMV promoter or a cell-specific promoter such as mGluR6, preferably ranges from about 10 ¹⁰ to about 10 ¹³ rAAV infectious units in a volume of about from 150 to approximately 800 µl per injection. rAAV infectious units can be measured according to McLaughlin, SK et al., 1988, J. Virol., 62:1963. More preferably, the effective amount is from about 10 ¹⁰ to about 10 ¹² rAAV infectious units, and the injection volume is preferably from about 250 to about 500 μl. Other dosages and volumes, preferably within these ranges but possibly outside them, may be selected by the practitioner, taking into account the physical condition of the subject (preferably the person) being treated, including age, weight, health status and the nature and severity of the particular visual disorder.

It may also be advisable to administer additional doses (boosters) of nucleic acid or rAAV compositions. For example, depending on the duration of transgene expression in the target ocular cell, a second course of treatment may be administered after 6 months or annually and may be repeated in a similar manner. Neutralizing antibodies to AAV are not expected to be generated due to the doses and routes of administration used, thus allowing repeated rounds of treatment.

The need for such additional doses can be monitored by practitioners using, for example, well-known electrophysiological and other tests of retinal function and vision and tests of visual behavior. The medical professional will be able to select appropriate tests using standard skills known in the art. It may be advisable to inject a larger volume of the composition, either in a single dose or in multiple doses, to further improve the respective endpoints.

Eye diseases.

The ocular disorders targeted by the present Chop2 proteins and the resulting ChR2 proteins that may be used to improve one or more parameters of vision include, but are not limited to, developmental abnormalities that affect both early and late segments of the eye. Anterior segment disorders include glaucoma, cataracts, and corneal dystrophy, keratoconus. Posterior segment disorders include blinding disorders caused by abnormal functioning and/or death of photoreceptors caused by retinal degeneration and degradation. Retinal disorders include congenital permanent night blindness, age-related macular degeneration, congenital conical dystrophies, and a large group of disorders associated with retinitis pigmentosa (RP). These disorders include genetically predisposed death of photoreceptor cells, rods and cones in the retina, observed at different ages. These include severe retinopathy, such as the RP subtypes, which progress with age and cause blindness in childhood and early adulthood, and RP-associated diseases, such as the LCA genetic subtypes, which often result in vision loss in childhood as early as the first year of life. year of life. Later disorders are usually characterized by significant reduction and often complete loss of photoreceptor cells, rods and cones (Trabulsi, E.I., ed., Genetic Diseases of the Eye, Oxford University Press, NY, 1998).

Specifically, the Chop2 and ChR2 proteins of the present invention are used to treat and/or restore at least partial vision in subjects who have lost vision due to ocular disorders, such as RPE-associated retinopathy, which are characterized by long-term preservation of ocular tissue structure despite to loss of function, and the association between functional loss and a defect or absence of a normal gene in the subject's eye cells. Many such ocular disorders are known, such as childhood onchocerciasis, retinitis pigmentosa, macular degeneration and diabetic retinopathy, as well as blinding eye diseases known in the art. It is understood that these and other disorders, as well as disorders with blindness of a currently unknown cause, which are later characterized using the same description as above, can also be successfully treated with the Chop2 and ChR2 proteins of the present invention. Thus, the particular ocular disorder treated by the present invention may include the above-mentioned disorders and a number of diseases that have not yet been properly characterized.

Optogenetics.

The promising field of optogenetics involves a combination of genetic and optical techniques

- 18 043449 for monitoring specific events in target cells of living tissue. Optogenetics can be used in wild mammals and other animals. In addition, the temporal accuracy (time scale - milliseconds) of optogenetic methods is sufficient to function in intact biological systems.

The present invention provides Chop2 gene therapy for preserving retinal tissue by introducing into retinal cells a nucleic acid or polypeptide encoding at least one mutant form of Chop2. The Chop2/ChR2 mutant proteins of the invention are specifically adapted to be activated by light at lower light intensities than the corresponding wild-type proteins. Accordingly, the Chop2/ChR2 mutant proteins of the invention can be used to activate retinal and visual system cells using less disruptive light sources. Mutant Chop2/ChR2 proteins also conduct more photocurrent when activated, resulting in a more robust or efficient response from cells expressing mutant Chop2/ChR2.

For example, the Chop2 mutant proteins of the invention are administered to a subject by local intravitreal or subretinal injection of a nucleic acid molecule encoding a Chop2 mutant polypeptide molecule, a Chop2 mutant, or a cell expressing Chop2/ChR2. The subject's retinal cells express mutant Chop2 proteins in the plasma membrane. When transfected or transformed retinal cells encounter light radiation, the transfected or transformed retinal cells transmit an improved or restored signal.

These methods can be used in subjects with normal and/or impaired vision. The Chop2/ChR2 mutants of the invention can maintain, improve or restore vision. In addition, the Chop2/ChR2 mutants of the invention are used to preserve, improve or restore the transmission 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 effectively detect light as a stimulus for differentiation or function. Vision is understood to cover the following.

1. Light detection or perception - the ability to notice the presence or absence of light.

2. Light projection - the ability to notice the direction from which a light stimulus comes.

3. Resolution - the ability to detect different levels of brightness (i.e. contrast) on a diffraction or letter target.

4. Recognition - the ability to recognize the shape of a visual target by referring to different levels of contrast on the target.

Thus, vision involves the ability to facilitate the detection of the presence or absence of light. The polypeptides and polynucleotides encoding the mutant Chop2 of the present invention can be used to improve or restore vision, where the improvement or restoration of vision includes, for example, increased detection and perception of light, increased photosensitivity or photosensitivity in response to a light stimulus, increased ability to notice direction, from which light source comes from, increasing the ability to detect different levels of brightness, increasing the ability to recognize the shape of a visual target, and increasing the induced visual potential or transmission from the retina to the cerebral cortex. As such, improvement or restoration of vision may or may not include complete restoration of vision, i.e. wherein the vision of a patient treated with the present invention is restored to that of a healthy individual. Visual restoration, described in the animal studies below, may, in relation to humans, place an individual at the lower limit of visual function by increasing one aspect of vision (ie, light sensitivity or visual induced potential) without restoring full vision. However, placement at such a level would have significant benefits, as these individuals could be trained to move and could potentially solve low-order problems that would provide them with a much higher level of visual independence compared to complete blindness. Even general light sensing can be used by individuals with visual impairments that are improved using the compositions and methods of the present invention to perform specific daily tasks and improve overall mobility, capabilities, and quality of life.

The degree of vision recovery can be determined by measuring vision before and preferably after administration of a vector including, for example, DNA encoding Chop2. Vision can be measured using any of a number of methods well known in the art and methods that have not yet been developed. Vision improved or restored by the present invention can be measured by any of the following visual responses.

1. A response in the form of detection of light by an object after exposure to a light stimulus, in which the reliable response data sought is the appearance or movement in the general direction of light by the object when the light is turned on.

2. A response in the form of the projection of light by an object after exposure to a light stimulus, where the data sought is a reliable response of the individual manifesting or moving in a particular direction of light when the light is turned on.

- 19 043449

3. An object’s resolution of a light versus a dark visual stimulus, which is measured by the object’s ability to resolve a light versus a dark visual stimulus, which is determined using:

a) the presence of demonstrated reliable optokinetically derived nystagmoid gaze movements and/or associated head or body movements that demonstrate target tracking (see above); and/or

b) the presence of a reliable ability to discriminate a visual stimulus and to express such discrimination in verbal or non-verbal ways, including, for example, pointing or pressing a pen or button.

4. Electrical recording of the response of the visual cortex to a flash of light stimulus or to a reference visual stimulus, this response represents the end point of electrical transmission from the restored retina to the visual cortex, also referred to as visual induced potential (VEP). The measurement may be represented by electrical recording at the surface of the scalp at a site in the visual cortex, at the cortical surface, and/or recording within cells of the visual cortex.

Thus, improvement or restoration of vision according to the present invention may include, but is not limited to, increasing the amplitude or kinetics of photocurrents or electrical response to light stimuli in retinal cells, increasing photosensitivity (i.e., reducing the threshold of light intensity required to initiate a photocurrent or electrical response to a light stimulus, thus requiring less or weaker light to induce a photocurrent) of retinal cells, an increase in the number or amplitude of a light-evoked spike or spike excitations, an increase in the response to light of the visual cortex, which involves an increase in the induced visual potential transmitted from the retina or retinal cells into the visual cortex or brain.

Both in vitro and in vivo studies can be used to evaluate various parameters of the present invention, including known animal models of human eye disorders with blindness. Numerous animal models of human retinopathy, such as childhood blindness, are in use. The examples provided herein will allow one of ordinary skill in the art to readily recognize that this technique may similarly be used in the treatment of a number of retinal disorders.

While previous others' studies have demonstrated that retinal degeneration can be slowed using gene therapy techniques, the present invention demonstrates certain physiological restoration of function that is expected to generate or improve various parameters of vision, including behavioral parameters.

Behavioral measurements can be obtained using known animal models and tests, such as the water maze, where an object that is maintained or restored to varying degrees of vision swims into the light (Hayes, J.M. et al., 1993, Behav. Genet. , 23:395-403).

In models in which blindness is induced in adulthood, or hereditary blindness develops slowly enough that the individual has seen before vision loss, subject training can be performed on various tests. In this case, when these tests are reintroduced after vision loss to test the effectiveness of the present compositions and methods for their vision-restoring effects, the animals do not need to learn the tasks de novo as in the blind condition. Other behavioral tests require no training and rely on the instinctive nature of certain behavioral traits. An example is the optokinetic nystagmus test (Balkema G.W. et al., 1984, Invest. Ophthalmol. Vis. Sci., 25:795-800; Mitchiner J.C. et al., 1976, Vision Res., 16:1169-71).

The present invention may also be used in combination with other forms of vision therapy known in the art to improve or restore vision.

For example, the use of visual prostheses, which include retinal implants, cortical implants, lateral geniculate nucleus implants, or optic nerve implants. Thus, in addition to genetically modifying living retinal neurons using the present methods, the subject being treated may be provided with a visual prosthesis before, during, or after application of the molecular method. The effectiveness of visual prosthetics can be improved by training the individual, thereby enhancing the potential contribution of Chop2 transformation of the patient's cells, as suggested herein. Training methods, such as training training, characterized by training an object to recognize (i) various levels of light and/or reference stimulation, and/or (ii) environmental stimuli from a conventional light source or object, as understood by one skilled in the art; and orientation and mobility training, characterized by training an object to visually detect local subjects and move more efficiently among said subjects than without training. In fact, any visual stimulation methods commonly used in the field of low vision rehabilitation are applicable here.

- 20 043449

Examples

Example 1: Generation of tagged mutant Chop2 constructs.

Mutations were performed on the codon-optimized Chop2-GFP cross-linked protein to create single and double mutations at sites L132 (Leucine 132) and T159 (Threonine 159). Several mutants generated, for example, single mutants such as L132A, L132C, T159A, T159C and T159S, and double mutants such as L132C/T159C, L132C/T159S, L132A/T159C and L132C/T159A. Chop2-GFP transgenes were cloned into the rAAV vector under the control of the CAG promoter using methods known in the art.

Example 2: In vitro analysis of Chop2 mutant constructs.

The functional properties of each Chop2 mutant or combination thereof were first examined in HEK cells. Chop2 constructs were delivered to HEK cells using, for example, adenoviral infection. Expression of WT or mutant Chop2 produces functional WT and mutant ChR2 channels. Light sensitivity measurements and other properties of ChR2 channels were assessed as described herein. Light stimuli (photons/ ^cm2 /s at 460 nm) were generated using a xenon arc lamp and attenuated using a neutral gray filter: ND4.0 ( ^2.8x1014 ), ND3.0 ( ^1.4x1015 ), ND2. 5 (4.8χ 10 ¹⁵ ), ND2.0 (1.6x1016), ND1.0 (1.3x 10 ¹⁷ ), ND0 (1.2x10 ¹⁸ ). Light-evoked current was measured from wild-type ChR2, T159C, L132C, L132C/T159C, and L132C/T159S. Patch clamp registration was carried out using methods known in the art.

Representative recordings from this experiment comparing photosensitivity between Chop2 constructs showed that mutations in L132 alone or in combination with the mutation in T159 exhibited increased photocurrent compared with WT (Fig. 1A and 1B). In fig. Figure 1B displays the same current traces at different scales to more clearly illustrate the difference in photocurrent amplitude between WT ChR2 and mutant ChR2. Fig. 1B specifically compares current traces resulting from light stimulation using a neutral gray filter (ND 2.5), which is equivalent to 4.8 x 10 ¹⁵ photons/cm ² /s; the tracks were indicated by arrows. The photocurrent amplitude of the L132C mutant is higher than that of the WT; the photocurrent amplitude of the double mutant L132C/T159C is higher than L132C; and the photocurrent amplitude of the L132C/T159S mutant is higher than that of L132/T159C. Current traces of ChR2 mutants, especially the double mutants L132C/T159C and L132C/T159S, also show slower deactivation compared to WT and L132C.

In fig. Figure 2 shows representative recordings of light-evoked currents from WT ChR2, L132C, L132C/T159C, and L132C/T159S after stimulation with a 10 ms light pulse (1.2 x ^{10 18} photons/cm ² /s at 460 nm) to compare the time course of deactivation after turning off the lights. The ChR2 mutant exhibits deactivation dynamics, with the double mutant L132C/T159S having the longest deactivation. Higher photosensitivity, as demonstrated by L132C/T159C and L132C/T159S, may correlate with a channel with slower kinetics.

Example 3: In vivo ocular administration and analysis of Chop2 mutant constructs.

AAV2 viral vectors carrying mutant Chop2-GFP constructs driven by the CAG promoter were prepared and injected intravitreally into the eyes of an adult C57BL/6J mouse. An adult mouse was anesthetized using an IP injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Under a dissecting loupe, an incision was made using scissors through the eyelid to expose the sclera. A small hole was made in the area of the sclera behind the pupil using a needle and a viral vector suspension of 0.8-1.5 μl at a concentration of approximately 10 ¹¹ genomic particles/ml was injected into the intravitreal space through the hole using a Hamilton syringe with a blunt-tipped 32-gauge needle. For each animal, typically only one eye was injected using a viral vector carrying the Chop2 construct, and the other eye was not injected or was injected using control viral vectors carrying only GFP. Expression of WT or mutant Chop2 of the present invention produces functional WT or mutant ChR2 channels using endogenous retinal, and the properties of these ChR2 proteins were assessed as described herein.

ChR2-mediated responses to light were examined using retinal whole-mount multielectrode chip recordings. Light stimuli (photons/cm ² /s) were generated using a laser in the blue light region of 473 nm and attenuated using neutral gray filters: NDO (6.3x10 ¹⁶ ), ND1.0 (7.4x10 ¹⁵ ), ND1.5 ( 2.7x10 ¹⁵ ), ND2.0 (7.3x10 ¹⁴ ), ND2.5 (3.2x10 ¹⁴ ), ND3.0 (8.5x10 ¹³ ), ND3.5 (3.8x10 ¹³ ) and ND4.0 ( 9.5x10 ¹² ).

Multielectrode chip recordings are based on procedures published by Tian and Copenhagen (2003). Briefly, the retina was dissected and placed photoreceptor side down on nitrocellulose filter paper (Millipore Corp., Bedford, MA). The fixed retina was placed in the recording chamber of a MEA-60 multielectrode chip with a chamber diameter of 30 μm, with electrodes separated by 200 μm (Multi Channel System MCS GmbH, Reutlingen, Germany), together with a layer of ganglion cells facing the recording electrodes. The retina was continuously perfused in an oxygenated extracellular solution at 34°C during all experiments. Extracellular

- 21 043449 solution contained (in mM): NaCl, 124; KCl, 2.5; CaCl ₂ , 2; MgCl ₂ , 2; _NaH2PO4 , _1.25 ; NaHCO ₃ , 26; and glucose, (pH 7.35 with 95% O ₂ and 5% CO ₂ ). Recordings typically began 60 min after the retina was placed in the recording chamber. The interval between the onset of each light stimulus was 10–15 s. Signals were filtered at 200 Hz (low threshold) and 20 kHz (high threshold). Responses from individual neurons were analyzed using the Offline Sorter software package (Plexon, Inc., Dallas, TX).

Chop2/ChR2 single mutants, i.e. L132 and T159C significantly reduced the threshold light intensity than required to induce ChR2-mediated photocurrent. In addition, several double mutants, including L132C/T159C, L132A/T159C, and L132C/T159S, were found to further increase photocurrent at low light intensities. Various neutral gray filters were used to attenuate light stimuli to differentiate light-evoked responses of Chop2 constructs under low light conditions. Retinal ganglion cell spiking activity mediated by the mutants of the present invention was observed at light intensities approximately 1.5 to 2 log units lower than the light level required to induce spiking activity by wild-type ChR2 (FIG. 3). . Specifically, WT ChR2 did not show any spike activity in response to light stimuli with a 2.5 neutral gray filter (3.2 x ^{10 14} photons/cm ² /s), while ChR2 mutants (L132C, L132C/T159C and L132C/ T159S) exhibit spike activity. In fact, ChR2 mutants still showed spiking activity in response to light with neutral gray filters 3.0 and 3.5. Thus, the ChR2 mutants of the present invention have higher light sensitivity and thus require significantly lower light intensity to induce ChR2-mediated photocurrent. In addition, ChR2 double mutants have higher photosensitivity than single mutants, i.e. L132C. In addition, spike firing of retinal ganglion cells expressing L132C/T159C and L132/T159S can follow light flicker frequencies of up to 15 and 5 Hz, respectively ( Fig. 4 ).

The L132C/T159A mutant exhibits high light sensitivity, probably the most photosensitive of these mutants, but it also exhibits exceptionally slow shutdown (the channel remains open for many seconds after the light is turned off). Interestingly, it can be turned off faster using longer wavelength light, such as yellow light. The L132C/T159A mutant (encoded by SEQ ID NOs: 24 and 25) shows significant potential.

Considering the tradeoff between photosensitivity and channel kinetics, Chop2/ChR2 mutants that exhibit a balance between photosensitivity and channel kinetics, such as L132C/T159C or L132C/T159S, may be suitable for applications in vision restoration.

Example 4: Analysis of Chop2 mutant constructs in mouse models of disease.

Mouse models of degenerative eye diseases are known in the art. For example, homozygous rd1 (rd1/rd1) mice are widely used as a model of photoreceptor degeneration. Rd1 mice carry a null mutation in the cyclic GMP phosphodiesterase, PDE6, similar to some forms of retinitis pigmentosa in humans. Other well-studied mouse models of ocular disease that may be of particular interest for demonstrating the safety and efficacy of the ChR2 mutant include rds (also known as Prph ^Rd2 ), rd3, rd4, rd5, rd6, rd7, rd8, rd9, Pde6b ^rd10 , or cpf11.

The Chop2-GFP constructs of the present invention can be injected intravitreally into the eyes of neonatal (P1) or adult mice aged 2-12 months. The GFP signal can be observed in Chop2-6EP-injected retinas to determine the level of ChR2 expression or expression in a specific cell population, such as retinal ganglion cells. The expression of mutant Chop2-GFP can be monitored over time, i.e. 3-6 months or 1 year after viral infection. Patch clamp and multichannel chip recordings can be performed using methods known in the art and described herein for measuring light-evoked responses of mutant Chop2-6EP-expressing cells in vivo.

Additional methods and tests for testing the restoration of visual light sensitivity have been well studied in the field. Induced visual potentials from Chop2-GFP-expressing cells or visual cortex can be examined as described in PCT publication WO 2007/131180. Other tests include behavioral assessment of visual acuity in mice, e.g. virtual optomotor test and visual water maze.

Example 5. Analysis of long-term expression and safety of introduction of mutant Chop2 constructs into retinal neurons.

Neurotoxicity was assessed in C57BL/6J adult mice injected with the Chop2 constructs of the present invention. The safety of expression of Chop2 mutants in the retina was assessed by immunostaining and cell counting after exposure to hard ultraviolet light for two weeks. It was found that none of the mice showed symptoms of neurotoxicity for up to two months after the injection.

Additional ongoing studies are evaluating the long-term expression and safety of the Chop2/ChR2 mutants of the invention in retinal neurons.

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Other incarnations

Although the invention has been given in conjunction with a detailed description, the foregoing description is intended to be illustrative and does not limit the scope of the invention as defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the claims below.

The patent and scientific literature referenced herein represents knowledge available to those skilled in the art. All US patents and published and unpublished US patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All published references, documents, manuscripts and scientific literature cited in this document are incorporated herein by reference.

Although the present invention has been specifically shown and described with reference to its preferred embodiments, those skilled in the art will appreciate that various changes in form and details may be made without departing from the scope of the invention as defined by the appended claims.

Claims (16)

1. An expression vector containing a polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 26, in which the amino acid at position 132 of SEQ ID NO: 26 is cysteine (C) and the amino acid at position 159 of SEQ ID NO: 26 is cysteine (C) or serine (S). 2. The expression vector of claim 1, wherein the expression vector is an adeno-associated virus (AAV) based vector. 3. The expression vector of claim 2, wherein the AAV vector is an AAV2 vector. 4. An expression vector according to claim 1, 2 or 3, wherein the polypeptide contains cysteine (C) at positions 132 and 159 of SEQ ID NO: 26. 5. The expression vector of claim 4, wherein the polypeptide contains the amino acid sequence of SEQ ID NO: 16 and the polynucleotide contains the nucleic acid sequence of SEQ ID NO: 15. 6. An expression vector according to claim 1, 2 or 3, wherein the polypeptide contains a cysteine (C) at position 132 and a serine (S) at position 159 SEQ ID NO: 26. 7. The expression vector of claim 6, wherein the polypeptide contains the amino acid sequence of SEQ ID NO: 19 and the polynucleotide contains the nucleic acid sequence of SEQ ID NO: 18. 8. A method for improving or restoring vision in a subject, comprising administering to the subject an expression vector according to any one of claims 1 to 7. 9. The method of claim 8, wherein the subject has normal vision. 10. The method according to claim 8, wherein the subject suffers from an eye disease. 11. The method according to claim 10, wherein the eye disease is macular degeneration or retinitis pigmentosa. 12. The method according to any one of claims 8 to 11, wherein said improvement or restoration of vision includes any of the following: increasing photosensitivity, decreasing the threshold of light intensity required to induce photocurrent, and increasing induced visual potential in the visual cortex. 13. An expression vector containing a polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 26, in which the amino acid at position 132 of SEQ ID NO: 26 is alanine (A) and the amino acid at position 159 of SEQ ID NO: 26 is cysteine (WITH). 14. The expression vector of claim 13, wherein the expression vector is an adeno-associated virus (AAV) based vector. 15. The expression vector of claim 14, wherein the AAV vector is an AAV2 vector. 16. An expression vector according to claims 13, 14 or 15, wherein the polypeptide contains the amino acid sequence of SEQ ID NO: 22 and where the polynucleotide contains the nucleic acid sequence of SEQ ID NO: 21.