RU2787071C2 - Compositions and methods for improvement of visual function - Google Patents

Abstract

FIELD: medicine; ophthalmology.

SUBSTANCE: invention relates to medicine, namely to ophthalmology; it can be used for restoration or improvement of a visual function in an individual with retinal degeneration. The method includes administration to the individual of at least one recombinant expression vector containing one or several of: i) a nucleotide sequence encoding MW-opsin; ii) a nucleotide sequence encoding LW-opsin; and iii) a nucleotide sequence encoding SW-opsin, where the specified nucleotide sequence is functionally connected to a promotor other than an opsin promotor, and it is expressed in a retinal cell in the individual, which is a retinal ganglion cell, an amacrine cell, a horizontal cell, or a bipolar cell.

EFFECT: method provides restoration or improvement of a visual function in an individual due to expression of the specified nucleic acid in retinal cells of this individual.

22 cl, 22 dwg, 1 ex

Description

This application claims priority over U.S. Provisional Application No. 62/585,237 filed November 13, 2017, U.S. Provisional Application No. 62/589,476 filed November 21, 2017, and U.S. Provisional Application No. 62/641,783 filed November 12 March 2018, each of these applications is incorporated herein by reference in its entirety.

Federally funded research statement

The present invention was made with government support in accordance with EY018241 awarded by the National Institutes of Health. The government has certain rights to the present invention.

Introduction

Hereditary and age-related degenerative diseases of the retina cause a progressive loss of rod and cone photoreceptors, leading to total blindness. Despite the loss of light-sensitive cells essential for vision, downstream neurons of the inner retina survive in a functional state, providing a target for optogenetic therapy. To date, optogenetic approaches have faced certain limitations, including: a) the very low photosensitivity of bacterial opsins and chemically engineered mammalian receptors; b) very slow kinetics in retinal opsins; and c) the lack of adaptation mechanisms to provide natural vision with high sensitivity over a very wide range of ambient light levels.

There is a need in the art for improved optogenetics approaches to the treatment of visual impairment.

The essence of the invention

The present invention provides a method for restoring or improving visual function in an individual, the method comprising administering to the individual a nucleic acid comprising a nucleotide sequence encoding one or more medium wavelength conopsins (MW-opsin), long wavelength conopsins (LW-opsin), and short wavelength conopsins (SW-opsin). opsin). One or more of MW opsin, LW opsin and SW opsin is expressed in a retinal cell in an individual, thereby restoring or improving visual function.

Brief description of graphic materials

In FIG. 1A-1C demonstrate that light activation of MW-opsin or rhodopsin activates GIRK channels to generate internal photocurrent at negative initial potentials in high external potassium.

In FIG. 2A-2G demonstrate protein expression and light responses in retinas isolated from rd1 mice whose rods and cones have degenerated. Demonstrated expression of MW-opsin in retinal cells (Fig. 2A-2D), and demonstrated light-induced responses that are mediated by MW-opsin (Fig. 2E-2G).

In FIG. 3A-3G depict the light response time, size, and high sensitivity of MW-opsin or rhodopsin in retinal cells isolated from blind rd1 mice.

In FIG. 4A-4I show behavioral tests demonstrating the same MW-opsin and rhodopsin mediated light-to-dark ability in mouse rd1 retinal cells (FIGS. 4A-4C), but only MW-opsin allows animals to distinguish between flashing and steady light (Fig. 4D-4E), or various light spatial patterns (FIGS. 4F-4G). The MW opsin also supports line differentiation at the mass level between parallel lines at different intervals, which are displayed as still images or in motion (FIGS. 4H-4I).

In FIG. 5A-5F depict MW-opsin-mediated light adaptation in rd1 mice: in MEA recordings on exposed retina (FIGS. 5A-5C) and visually mediated behavior (FIGS. 5D-5F).

In FIG. 6 depicts behavioral tests demonstrating the restoration of the ability to distinguish between parallel lines at a distance of 1 vs 6 cm with MW opsin, LW opsin and SW opsin.

In FIG. 7 depicts the threshold response of optogenetic systems in blind retinas compared to normal vision.

In FIG. 8A-8E depict intensity dependent kinetic responses mediated by MW opsin.

In FIG. 9A-9G depict light avoidance behavior and learned pattern recognition behavior.

In FIG. 10A-10G depict light adaptation in retinal and behavioral mice.

In FIG. 11 is Table 1, which shows the statistical significance of light avoidance and learned visually mediated behavior.

In FIG. 12A-12B depict an amino acid sequence alignment of a human SW opsin (SEQ ID NO: 5) and a mouse SW opsin (SEQ ID NO: 6), and an exemplary amino acid sequence of a chimeric human/mouse SW opsin (SEQ ID NO: 7). ). The transmembrane (TM) domains of the human SW opsin and the chimeric human/mouse SW opsin are underlined; intracellular domains of mouse SW opsin and chimeric human/mouse SW opsin are double underlined.

In FIG. 13A-13L depict the restoration of visually mediated behavior with MW opsin.

In FIG. 14A-14B depict MW-opsin expression in the rd1 mouse retina.

In FIG. 15A-15B depict rhodopsin expression in the rd1 mouse retina.

In FIG. 16A-16D depict the transduction efficiency of MW-opsin in rd1 retina.

In FIG. 17A-17D depict the characterization of the slow component of the light response in rd1 retina expressing MW opsin.

In FIG. 18A-18C depict contrast detection in isolated mouse retina expressing mouse MW opsin rd1.

In FIG. 19A-19E depict the in vivo light response in a V1 mouse rd1 expressing MW opsin.

In FIG. 20A-20B depict in vivo contrast detection in rd1 mice expressing MW-opsin.

In FIG. 21A-21C depict the temporal properties of in vivo V1 responses in rd1 mice expressing MW opsin.

In FIG. 22A-22B depict location preferences in discrimination tasks.

Definitions

The term "retinal cell" may refer herein to any of the types of cells that make up the retina, such as retinal ganglion cells; amacrine cells; horizontal cells; bipolar cells; and photoreceptor cells, including rods and cones.

"Operably linked" refers to the matching of genetic elements, where the elements are in a relationship that allows them to function in the expected way. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. Intermediate residues may be present between the promoter and the coding region, as long as this functional relationship is maintained.

An "expression vector" is a vector containing a region that encodes a polypeptide of interest and is used to effect expression of a protein in the intended target cell. The expression vector also contains control elements operably linked to the coding region to facilitate protein expression in the target. The combination of control elements and the gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette", a large number of which are known and available in the art, or can be easily constructed from components available in the art.

The terms "treatment", "process of treatment" and the like, in the context of this document, refers to obtaining the desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, and/or may be therapeutic in terms of partially or completely curing a disease and/or an adverse effect associated with the disease. "Treatment", in the context of this document, covers any treatment of a disease in a mammal, in particular a human, and includes: (a) preventing the onset of the disease in a subject who may be predisposed to the disease or at risk of acquiring the disease, but has not yet been diagnosed as having a disease; (b) the suppression of the disease, that is, the cessation of its development; (c) alleviating the disease, ie causing regression of the disease; and (d) replacement of lost function as a result of the disease.

The terms "individual", "host", "subject" and "patient" are used interchangeably herein and refer to a mammal, including, but not limited to, humans and primates, including monkeys and humans; sports animals (eg horses, camels, etc.); farm animals (eg sheep, goats, cows, etc.); pets (dogs, cats, etc.); and rodents (eg mice, rats, etc.). In some cases, the individual is a human.

Before the present invention is further described, it should be understood that the present invention is not limited to the particular embodiments described, as they may, as such, be varied. It should also be understood that the terminology used herein is only intended to describe specific embodiments and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

When a range of values is provided, each intermediate value, to the nearest tenth of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range and any other specified or intermediate value within the specified range, is intended to be within the scope of the present invention. . The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also covered by the present invention, subject to any specifically excluded limit in the specified range. If the specified range includes one or both of the limits, ranges excluding one or both of these included limits are also included in the present invention.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications cited in this document are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It should be noted that the singular forms used herein and in the appended claims are also intended to include the plural, unless the context clearly dictates otherwise. Thus, for example, reference to "MW-opsin polypeptide" includes a plurality of such polypeptides, and reference to "retinal" includes reference to one or more retinal cells and their equivalents known to those skilled in the art, and so on. In addition, it should be noted that the claims may be drafted in such a way as to exclude any optional feature. As such, this statement is intended to serve as a foregoing basis for the use of such exclusive terminology as "only" and the like in connection with the enumeration of features of a claim or the use of a "negative" limitation.

It should be appreciated that certain features of the invention, which are described in the context of separate embodiments for clarity, may also be provided as a combination in one embodiment. Conversely, various features of the present invention, which for brevity are described in the context of one embodiment, may also be provided separately or in any suitable combination. All combinations of embodiments pertaining to the present invention are specifically covered by the present invention and are disclosed herein as if each combination were disclosed individually and expressly. In addition, all intermediate combinations of various embodiments and elements thereof are also specifically covered by the present invention and are disclosed herein as if each such combination were individually and expressly disclosed herein.

The publications discussed herein are solely for their disclosure prior to the filing date of this application. Nothing contained herein should be construed as an admission that the present invention is not entitled to precede such publication by virtue of prior invention. In addition, the dates of publication indicated may differ from the actual dates of publication, which may need to be confirmed independently.

Detailed Description of the Invention

The present invention provides a method for restoring or improving visual function in an individual, the method comprising administering to the individual a nucleic acid comprising a nucleotide sequence encoding one or more medium wavelength conopsins (MW-opsin), long wavelength conopsins (LW-opsin), and short wavelength conopsins (SW-opsin). opsin). One or more of MW opsin, LW opsin and SW opsin is expressed in a retinal cell in an individual, thereby restoring or improving visual function.

The MW-opsin polypeptide may contain an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the following amino acid. human MW opsin sequences:

maqqwslqrl agrhpqdsye dstqssifty tnsnstrgpf egpnyhiapr wvyhltsvwm ifvviasvft nglvlaatmk fkklrhplnw ilvnlavadl aetviastis vvnqvygyfv lghpmcvleg ytvslcgitg lwslaiiswe rwmvvckpfg nvrfdaklai vgiafswiwa avwtappifg wsrywphglk tscgpdvfsg ssypgvqsym ivlmvtccit plsiivlcyl qvwlairava kqqkesestq kaekevtrmv vvmvlafcfc wgpyaffacf aaanpgypfh plmaalpaff aksatiynpv iyvfmnrqfr ncilqlfgkk vddgselssa sktevssvss vspa (SEQ ID NO:1).

The MW-opsin polypeptide may contain an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the following amino acid. mouse MW opsin sequences:

maqrltgeqt ldhyedstha siftytnsns tkgpfegpny hiaprwvyhl tstwmilvvv asvftnglvl aatmrfkklr hplnwilvnl avadlaetii astisvvnqi ygyfvlghpl cviegyivsl cgitglwsla iiswerwlvv ckpfgnvrfd aklatvgivf swvwaaiwta ppifgwsryw pyglktscgp dvfsgtsypg vqsymmvlmv tccifplsii vlcylqvwla iravakqqke sestqkaeke vtrmvvvmvf ayclcwgpyt ffacfatahp gyafhplvas lpsyfaksat iynpiiyvfm nrqfrncilh lfgkkvddss elsstsktev ssvssvspa (SEQ ID NO:8)

The rhodopsin polypeptide may contain an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the following amino acid sequence of rhodopsin:

mngtegpnfy vpfsnatgvv rspfeypqyy laepwqfsml aaymfllivl gfpinfltly vtvqhkklrt plnyillnla vadlfmvlgg ftstlytslh gyfvfgptgc nlegffatlg geialwslvv laieryvvvc kpmsnfrfge nhaimgvaft wvmalacaap plagwsryip eglqcscgid yytlkpevnn esfviymfvv hftipmiiif fcygqlvftv keaaaqqqes attqkaekev trmviimvia flicwvpyas vafyifthqg snfgpifmti paffaksaai ynpviyimmn kqfrncmltt iccgknplgd deasatvskt etsqvapa (SEQ ID NO:2).

The channel rhodopsin polypeptide may contain an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the following amino acid sequence channel rhodopsin:

MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVLQ WLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQW LRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLG LCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVY GSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAG AVP (SEQ ID NO:3).

The LW opsin may contain an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the following amino acid sequence Human LW opsin:

maqqwslqrl agrhpqdsye dstqssifty tnsnstrgpf egpnyhiapr wvyhltsvwm ifvvtasvft nglvlaatmk fkklrhplnw ilvnlavadl aetviastis ivnqvsgyfv lghpmcvleg ytvslcgitg lwslaiiswe rwmvvckpfg nvrfdaklai vgiafswiwa avwtappifg wsrywphglk tscgpdvfsg ssypgvqsym ivlmvtccii plaiimlcyl qvwlairava kqqkesestq kaekevtrmv vvmifaycvc wgpytffacf aaanpgyafh plmaalpayf aksatiynpv iyvfmnrqfr ncilqlfgkk vddgselssa sktevssvss vspa (SEQ ID NO:4).

The LW opsin may contain an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the following amino acid sequence mouse LW opsin:

mngtegpnfy vpfsnvtgvv rspfeqpqyy laepwqfsml aaymfllivl gfpinfltly vtvqhkklrt plnyillnla vadlfmvfgg ftttlytslh gyfvfgptgc nlegffatlg geialwslvv laieryvvvc kpmsnfrfge nhaimgvvft wimalacaap plvgwsryip egmqcscgid yytlkpevnn esfviymfvv hftipmivif fcygqlvftv keaaaqqqes attqkaekev trmviimvif flicwlpyas vafyifthqg snfgpifmtl paffaksssi ynpviyimln kqfrncmltt lccgknplgd ddasataskt etsqvapa (SEQ ID NO:9).

The SW-opsin polypeptide may contain an amino acid sequence that is at least 85% identical, at least 87% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical. % or 100% of the following human SW-opsin amino acid sequence:

mrkmseeefy lfknissvgp wdgpqyhiap vwafylqaaf mgtvfligfp lnamvlvatl rykklrqpln yilvnvsfgg fllcifsvfp vfvascngyf vfgrhvcale gflgtvaglv tgwslaflaf eryivickpf gnfrfsskha ltvvlatwti gigvsippff gwsrfipegl qcscgpdwyt vgtkyrsesy twflfifcfi vplslicfsy tqllralkav aaqqqesatt qkaerevsrm vvvmvgsfcv cyvpyaafam ymvnnrnhgl dlrlvtipsf fsksaciynp iiycfmnkqf qacimkmvcg kamtdesdtc ssqktevstv sstqvgpn (SEQ ID NO:5).

In some cases, the SW opsin contains an amino acid sequence that is at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence. SW-opsin human (SEQ ID NO: 5), responsive to light in the visible range and exhibiting excitatory activity.

The SW-opsin polypeptide may contain an amino acid sequence that is at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the following amino acid sequence mouse sw-opsin:

msgeddfylf qnissvgpwd gpqyhlapvw afrlqaafmg fvffvgtpln aivlvatlhy kklrqplnyi lvnvslggfl fcifsvftvf iaschgyflf grhvcaleaf lgsvaglvtg wslaflafer yvvickpfgs irfnskhalm vvlatwiigi gvsippffgw srfipeglqc scgpdwytvg tkyrseyytw flfifcfiip lslicfsysq llrtlravaa qqqesattqk aerevshmvv vmvgsfclcy vpyaalamym vnnrnhgldl rlvtipaffs ksscvynpii ycfmnkqfra cilemvcrkp madesdvsgs qktevstvss skvgph (SEQ ID NO:6). In some cases, the SW opsin contains an amino acid sequence that is at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence. Mouse SW-opsin (SEQ ID NO: 6) responsive to ultraviolet light and exhibiting inhibitory activity.

In some cases, both human SW opsin and mouse SW opsin are produced in a retinal cell in an individual. In some cases, both human SW opsin and mouse SW opsin (but not LW opsin or MW opsin) are produced in a retinal cell in an individual. In some cases, both human SW opsin and mouse SW opsin, as well as LW opsin (but not MW opsin) are produced in a retinal cell in an individual. In some cases, both human SW opsin and mouse SW opsin, as well as MW opsin (but not LW opsin) are produced in a retinal cell in an individual. In some cases, both human SW opsin and mouse SW opsin, as well as LW opsin and MW opsin, are produced in a retinal cell in an individual. For example, in some cases, the first recombinant expression vector containing a nucleotide sequence encoding a SW-opsin polypeptide containing an amino acid sequence identical to at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the amino acid sequence of human SW-opsin (SEQ ID NO: 5); and a second recombinant expression vector containing a nucleotide sequence encoding a SW-opsin polypeptide containing an amino acid sequence that is at least 87%, at least 90%, at least 95%, at least 98% identical, according to at least 99% or 100% of the amino acid sequence of mouse SW-opsin (SEQ ID NO: 6) is introduced into the retinal cell.

In some cases, both MW opsin and LW opsin (but not SW opsin) are produced in a retinal cell in an individual. In some cases, both MW opsin and SW opsin (but not LW opsin) are produced in a retinal cell in an individual. In some cases, MW-opsin, LW-opsin and SW-opsin are expressed in a retinal cell in an individual.

In some cases, a suitable opsin is a chimeric opsin, for example an opsin that contains the amino acid sequence(s) from the first species and the amino acid sequence(s) from the second species. For example, in some cases, the chimeric opsin contains intracellular regions of mouse SW opsin and transmembrane regions of human SW opsin. An example of a suitable chimeric SW opsin is shown in FIGS. 12A-12B.

In some instances, a suitable chimeric SW opsin contains an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the chimeric SW opsin. shown in FIG. 12A-12B, with the chimeric SW opsin having the following amino acid sequence:

EDDFYLFQNISSVGPWDGPQYHLAPVWAFYLQAAFMGTVFLIGFPLNAMVLVATLHY KKLRQPLNYILVNVSFGGFLLCIFSVFPVFVASCNGYFVFGRHVCALEGFLGTVAGLVTGWSL AFLAFERYVVICKPFGSIRFNSKHALTVVLATWTIGIGVSIPPFFGWSRFIPEGLQCSCGPDWYT VGTKYRSESYTWFLFIFCFIVPLSLICFSYTQLLRALRAVAAQQQESATTQKAEREVSHMVVV MVGSFCVCYVPYAAFAMYMVNNRNHGLDLRLVTIPSFFSKSACIYNPIIYCFMNKQFRACILE MVCRKPMADESDVSGSQKTEVSTVSSSKVGPH (SEQ ID NO:7).

The MW opsin and/or the LW opsin and/or the SW opsin may be used in combination with a heterologous opsin, for example a non-mammalian opsin such as an algal opsin, a protozoan opsin, or a variant thereof. Suitable heterologous opsins include depolarizing opsins, for example ChR2 channelorrhodopsin (Zhang et al. (2007) Nature 446:633; GenBank ABO64386.1) isolated from Chlamydomonas reinhardtii; step-functional opsin (SFO) (Berndt et al. (2009) Nat. Neurosci. 12:229) or stabilized step-functional opsin (SSFO) (WO 2010/056970; Yizhar et al. (2011) Nature 477:171) ; cation transfer channel opsin isolated from Volvox carteri (VChR1 - NCBI Gene ID: 9619570; U.S. Patent No. 9,249,200); C1V1 chimeric protein, a derivative of Volvox carteri VChR1 protein and ChR1 protein isolated from Chlamydomonas reinhardtii (U.S. Patent No. 9,175,095); C1C2 chimeric protein derived from ChR1 protein and ChR2 protein isolated from Chlamydomonas reinhardtii (Lin et al. (2009) Biophys. J. 96:1803); a redshifted depolarizing photosensitive polypeptide isolated from Chlamydomonas reinhardtii, where such photosensitive polypeptides are referred to as "ReaChR" (Lin et al. (2013) Nat. Neurosci. 16:1499); CnChR2 (Genbank Accession No.: AHH02139) isolated from Chlamydomonas noctigama; CsChrimson chimeric protein isolated from CsChR (Genbank Accession No.: AHH02144; Klapoetke et al. (2014) Nature Methods 11:338) Chloromonas subdivisa protein; ShChR1 (Genbank Accession No.: AHH02106) isolated from Stigeoclonium helveticum; opsin “ChETA” Gunaydin et al. (2010 Nat. Neurosci. 13:387) “SwiChR” protein (WO 2015/148974); bReaChes protein (WO/2017/048808); etc. Other suitable depolarizing opsins are known in the art; see, for example, Zhang et al. (2011) Cell 147:1446; Deisseroth (2015) Nature Neurosci. 18:1213; Berndt and Deisseroth (2015) Science 349:590; etc.

Expression of the MW opsin polypeptide and/or the LW opsin polypeptide and/or the SW opsin polypeptide in a retinal cell of an individual provides for pattern vision and image recognition by the individual. The image to be recognized may be a static image and/or a moving image.

Expression of the MW opsin polypeptide and/or the LW opsin polypeptide and/or the SW opsin polypeptide in a retinal cell of an individual provides image recognition at a light intensity of about 10 ^-4 W/cm ² to about 10 W/cm ² . For example, in some instances, expression of an MW opsin polypeptide and/or an LW opsin polypeptide and/or an SW opsin polypeptide in a retinal cell in an individual provides image recognition at a light intensity of about 10 ^-2 W/cm ² to about 10 ^{- 4} W/cm ² , from about 10 ^-4 W/cm ² to about 1 W/cm ² , from about 10 ^-4 W/cm ² to about 10 ^-1 W/cm ² or from about 10 ^-4 W/cm ² to about 5×10 ^-1 W/cm ² . In some instances, expression of an MW opsin polypeptide and/or an LW opsin polypeptide and/or an SW opsin polypeptide in a retinal cell of an individual provides image recognition at a light intensity of about 10 ^-4 W/cm ² to about 10 ^-3 W /cm ² , from about 10 ^-3 W/cm ² to about 10 ^-2 W/cm ² , from about 10 ^-2 W/cm ² to about 10 ^-1 W/cm ² or from about 10 ^-1 W/cm ² to about 1 W/cm ² . In some instances, expression of an MW opsin polypeptide and/or an LW opsin polypeptide and/or an SW opsin polypeptide in a retinal cell of an individual provides image recognition at light intensities up to 2 W/cm ² , up to 3 W/cm ² , up to ⁴ W/cm2, up to ⁵ W/cm2 or up to 10 W/cm2 ^. Expression of the MW-opsin polypeptide and/or the LW-opsin polypeptide and/or the SW-opsin polypeptide in a retinal cell of an individual provides image recognition at a light intensity of less than 5 W/cm ² , less than 4 W/cm ² , less than 3 W/cm ² or less than 2 W/cm ² .

Expression of the MW opsin polypeptide and/or the LW opsin polypeptide and/or the SW opsin polypeptide in a retinal cell of an individual allows the individual to recognize an image at a light intensity that is at least 10 times lower than the light intensity needed to provide image recognition by an individual expressing a channel rhodopsin polypeptide (eg, a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3) in a retinal cell. For example, expression of an MW opsin polypeptide and/or an LW opsin polypeptide and/or an SW opsin polypeptide in a retinal cell of an individual allows the individual to recognize an image at a light intensity that is at least 10 times lower, at least 25 times lower, at least 50 times lower, at least 100 times lower, at least 150 times lower, at least 200 times lower, at least 300 times lower, at least 400 times lower, or at least 500 times lower than the light intensity required to provide image recognition by an individual expressing the channel rhodopsin polypeptide in a retinal cell.

Expression of an MW opsin polypeptide and/or an LW opsin polypeptide and/or an SW opsin polypeptide in a retinal cell provides a kinetic that is at least 2-fold faster than that provided in a retinal cell by rhodopsin (e.g., a polypeptide, containing the amino acid sequence shown in SEQ ID NO: 2). For example, expression of an MW opsin polypeptide and/or an LW opsin polypeptide and/or an SW opsin polypeptide in a retinal cell provides kinetics that is at least 2-fold, at least 5-fold, at least 10-fold , at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 50 times, at least 100 times, or more than 100 times faster than the kinetics provided in the retinal cell by the polypeptide rhodopsin.

The method of the present invention includes delivering to the eye of an individual a nucleic acid containing a nucleotide sequence encoding an MW-opsin polypeptide and/or an LW-opsin and/or SW-opsin polypeptide. In some cases, the nucleotide sequence is operably linked to a transcriptional control element that functions in a mammalian cell. In some instances, the nucleotide sequence is operably linked to a transcriptional control element that allows for expression in a retinal cell, for example that allows for selective expression in a retinal cell. In some cases, the nucleotide sequence is operably linked to a promoter that allows for expression in a retinal cell, for example that allows for selective expression in a retinal cell. In some instances, the nucleotide sequence is operably linked to a promoter that typically allows expression in a eukaryotic or mammalian cell.

Suitable promoters include, but are not limited to, the CAG promoter (Miyazaki et al. (1989) Gene 79:269); cytomegalovirus (CMV) promoter; metabotropic glutamate receptor 6 (grm6) promoter (Cronin et al. (2014) EMBO Mol. Med. 6:1175); Pleiades promoter (Portales-Casamar et al. (2010) Proc. Natl. Acad. Sci. USA 107:16589); choline acetyltransferase (ChAT) promoter (Misawa et al. (1992) J. Biol. Chem. 267:20392); vesicular glutamate transporter (V-glut) promoter (Zhang et al. (2011) Brain Res. 1377:1); glutamate decarboxylase (GAD) promoter (Rasmussen et al. (2007) Brain Res. 1144:19; Ritter et al. (2016) J. Gene Med. 18:27); cholecystokinin (CCK) promoter (Ritter et al. (2016) J. Gene Med. 18:27); parvalbumin (PV) promoter; somatostatin promoter (SST); neuropeptide Y (NPY) promoter; and a vasoactive intestinal peptide (VIP) promoter. Suitable promoters include, but are not limited to, the red light sensitive conopsin promoter, the rhodopsin promoter, the rhodopsin kinase promoter, and the GluR promoter (eg, the GluR6 promoter; also referred to as grm6). Suitable promoters include, but are not limited to, the vitelliform macular degeneration 2 (VMD2) gene promoter or the interphotoreceptor retinol-binding protein (IRBP) gene promoter. Also suitable for use is the L7 promoter (Oberdick et al. (1990) Science 248:223), the thy-1 promoter, the recoverin promoter (Wiechmann and Howard (2003) Curr. Eye Res. 26:25); calbindin promoter; and a beta actin promoter. Suitable promoters include synthetic (non-naturally occurring) promoter/enhancer combinations.

In some cases, a nucleic acid containing a nucleotide sequence encoding a MW-opsin polypeptide and/or an LW-opsin polypeptide and/or a SW-opsin polypeptide is present in a recombinant expression vector. Suitable expression vectors include, but are not limited to, a lentiviral vector, a herpes simplex virus (HSV) vector, an adenovirus vector, a retroviral vector, an adeno-associated virus (AAV) vector, and the like. Thus, in some cases, the nucleic acid containing the nucleotide sequence encoding the MW-opsin polypeptide and/or the LW-opsin polypeptide and/or the SW-opsin polypeptide is a recombinant lentiviral vector, a recombinant HSV vector, a recombinant adenovirus vector, a recombinant retroviral vector or recombinant AAV vector.

In some cases, a nucleic acid (eg, a recombinant expression vector) containing a nucleotide sequence encoding an MW-opsin polypeptide and/or an LW-opsin polypeptide and/or a SW-opsin polypeptide is complexed with the nanoparticle.

In some cases, the nucleic acid containing the nucleotide sequence encoding the MW-opsin polypeptide and/or LW-opsin and/or SW-opsin polypeptide is a recombinant AAV vector (rAAV). In some instances, the rAAV vector contains a nucleotide sequence encoding a variant AAV capsid protein, wherein the variant AAV capsid protein provides infectivity to retinal cells and/or ability to cross the inner limiting membrane (ILM) in the eye. See, e.g., Day et al. (2014) Adv. Exp. Med. Biol. 801:687; Boye et al. (2016) J. Virol. 90:4215; Vandenberghe and Auricchio (2012) Gene Therapy 19:162; Klimczak et al. (2009) PLoS One 4:e7467; US Patent Publication No. 2012/0164106; and US Patent Publication No. 2016/0017295.

Retinal cells include retinal ganglion cells; amacrine cells; horizontal cells; bipolar cells; and photoreceptor cells, including rods and cones.

In some instances, the method of the present invention comprises administering to an individual in need thereof a pharmaceutical composition comprising: a) a nucleic acid (e.g., a recombinant expression vector) comprising a nucleotide sequence encoding a MW-opsin and/or LW-opsin polypeptide, and/or SW-opsin polypeptide; and b) a pharmaceutically acceptable excipient.

A pharmaceutical composition containing a nucleic acid (for example, a recombinant expression vector) containing a nucleotide sequence encoding an MW-opsin and/or LW-opsin and/or SW-opsin polypeptide can be administered to a patient alone or in combination with other additional active substances. . Pharmaceutical compositions can be made using any of a variety of methods, including, without limitation, conventional mixing, dissolving, granulating, panning, triturating, emulsifying, encapsulating, trapping, and lyophilizing. The pharmaceutical composition may take any of a variety of forms, including, without limitation, a sterile solution, suspension, emulsion, lyophilisate, tablet, pill, pellet, capsule, powder, syrup, elixir, or any other dosage form suitable for administration.

A pharmaceutical composition containing a nucleic acid (e.g., a recombinant expression vector) containing a nucleotide sequence encoding an MW-opsin polypeptide and/or an LW-opsin polypeptide and/or a SW-opsin polypeptide may optionally include a pharmaceutically acceptable carrier(s), which facilitates processing of the active ingredient into pharmaceutically acceptable compositions. The term "pharmacologically acceptable carrier", as used herein, refers to any carrier that has little or no lasting or permanent adverse effect when administered, and encompasses terms such as "pharmacologically acceptable carrier, stabilizer, diluent, excipient, or excipient". Such a carrier is usually admixed with the active compound (eg, the nucleic acid of the present invention) or diluted with or enclosed in the active compound, and such carrier may be a solid, semi-solid, or liquid agent. It is understood that the active ingredients may be soluble or may be delivered as a suspension in the desired carrier or diluent. Any of a variety of pharmaceutically acceptable carriers may be used, including, without limitation, an aqueous medium such as, for example, distilled, deionized water, saline; solvents; dispersion media; coatings; antibacterial and antifungal agents; isotonic and absorption delaying agents; or any other excipient. The choice of pharmacologically acceptable carrier may depend on the route of administration. Unless any pharmacologically acceptable carrier is incompatible with the active ingredient, it is intended to be used in pharmaceutically acceptable compositions. Non-limiting examples of the specific use of such pharmaceutical carriers can be found in "Pharmaceutical Dosage Forms and Drug Delivery Systems" (Howard C. Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7th ^ed . 1999); “Remington: The Science and Practice of Pharmacy” (Alfonso R. Gennaro ed., Lippincott, Williams & ^Wilkins , 20th 2000); “Goodman &Gilman's The Pharmacological Basis of Therapeutics” Joel G. Hardman et al., eds., McGraw-Hill Professional, 10.sup.th ed. 2001); and “Handbook of Pharmaceutical Excipients” (Raymond C. Rowe et al., ^APhA Publications, 4th edition 2003).

The pharmaceutical composition may optionally contain, without limitation, other pharmaceutically acceptable ingredients, including, but not limited to, buffers, preservatives, tonicity agents, salts, antioxidants, saline solutions, pharmacological agents, excipients, emulsifying agents, wetting agents, sweeteners or flavorings, and the like. Various buffer solutions and pH adjusters can be used to prepare a pharmaceutical composition, provided that the resulting preparation is pharmaceutically acceptable. Such buffers include, without limitation, acetate buffers, citrate buffers, phosphate buffers, neutral saline buffer, phosphate buffered saline, and borate buffer. It is understood that acids or bases can be used to adjust the pH of the composition as needed. Pharmaceutically acceptable antioxidants include, without limitation, sodium metabisulphite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene. Suitable preservatives include, without limitation, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, and a stabilized oxychloro complex such as PURITE™. Tonicity agents suitable for inclusion in the pharmaceutical composition include, without limitation, salts such as, for example, sodium chloride, potassium chloride, mannitol or glycerin, and other pharmaceutically acceptable tonicity agents. It is clear that these and other substances known in the field of pharmacology, can be included in the pharmaceutical composition.

Some examples of substances that can act as pharmaceutically acceptable carriers include (1) sugars such as lactose, glucose and sucrose; (2) starch such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) pH-adjusting buffer solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical compositions.

In some cases, a nucleic acid (eg, a recombinant expression vector) containing a nucleotide sequence encoding an MW-opsin polypeptide and/or an LW-opsin polypeptide and/or a SW-opsin polypeptide is formulated with one or more biocompatible polymers. Suitable biocompatible polymers include, but are not limited to, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl ethers, polyvinyl halides, poly(vinylpyrrolidone), polyglycolides, polysiloxanes, polyurethanes and their copolymers, alkylcelluloses, hydroxyalkylcelluloses , cellulose ethers, cellulose esters, nitrocelluloses, polymers of acrylic and methacrylic ethers, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, sodium cellulose sulfate, polymethyl methacrylate ), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate\poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacryl m), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), polyvinyl alcohols, polyvinyl acetate, polyvinyl chloride polystyrene, poly(vinylpyrrolidone), polyhyaluronic acids, casein, gelatin , glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate) poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate) , poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), and combinations of any of the foregoing.

In some instances, a nucleic acid (eg, a recombinant expression vector) containing a nucleotide sequence encoding an MW-opsin polypeptide and/or an LW-opsin polypeptide and/or a SW-opsin polypeptide is formulated into a liposome. See, for example, US Patent Publication No. 2017/0119666. In some cases, a nucleic acid (eg, a recombinant expression vector) containing a nucleotide sequence encoding an MW-opsin polypeptide and/or an LW-opsin polypeptide and/or a SW-opsin polypeptide is formulated into a nanoparticle. The nanoparticles include, for example, polyalkylcyanoacrylate nanoparticles, poly(lactic acid)-containing nanoparticles, nanoparticles including lactic acid-glycolic acid (PLGA) nanoparticles, and the like. In some instances, a nucleic acid (eg, a recombinant expression vector) containing a nucleotide sequence encoding an MW-opsin polypeptide and/or an LW-opsin polypeptide and/or a SW-opsin polypeptide is formulated into a liposome. Suitable hydrogel components include, but are not limited to, silk (see, for example, US Patent Publication No. 2017/0173161), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), lactide-glycolide copolymer (PLGA ), polyesters, poly(orthoester), poly(phosphazine), poly(phosphate ester), polycaprolactone, gelatin, collagen, cellulose, hyaluronan, poly(ethylene glycol) (PEG), triblock copolymers, polylysine, poly(ethylene oxide), poly (vinylpyrrolidone), hyaluronic acid, acrylated hyaluronic acid, poly(N-isopropylacrylamide), and the like.

In some cases, a composition containing a nucleic acid (eg, a recombinant expression vector) containing a nucleotide sequence encoding an MW-opsin polypeptide and/or an LW-opsin polypeptide and/or a SW-opsin polypeptide is in a saline buffer solution. In some cases, a composition containing a recombinant virus-based expression vector containing a nucleotide sequence encoding a MW-opsin and/or LW-opsin and/or SW-opsin polypeptide is in a saline buffer solution in an amount of from about 10 ⁸ to about 10 ¹⁵ viral genomes (vg) in volumes from about 50 µl to about 1000 µl. For example, in some cases, a composition containing a recombinant virus-based expression vector containing a nucleotide sequence encoding a MW-opsin and/or LW-opsin and/or SW-opsin polypeptide is in a saline buffer solution in an amount of from about 10 ⁸ wg up to about 10 ⁹ hr, from about 10 ⁹ hr to about 10 ¹⁰ hr, from about 10 ¹⁰ hr to about 10 ¹¹ hr, from about 10 ¹¹ hr to about 10 ¹² hr, from about 10 ¹² hr to about 10 ¹³ hr, from about 10 ¹³ vg to about 10 ¹⁴ vg, or from about 10 ¹⁴ vg to about 10 ¹⁵ vg, in a volume of about 50 µl to about 75 µl, from about 75 µl to about 100 µl, from about 100 µl to about 150 µl, from about 150 µl to about 200 µl, from about 200 µl to about 300 µl, from about 300 µl to about 400 µl, from about 400 µl to about 500 µl, from about 500 µl to about 600 µl, from about 600 µl to about 700 µl, from about 800 to 900 µl, or from about 900 to about 1000 µl. In some cases, a composition containing a nucleic acid (for example, a recombinant expression vector) containing a nucleotide sequence encoding a MW-opsin and/or LW-opsin and/or SW-opsin polypeptide is in a saline buffer solution containing a non-ionic detergent at a concentration about 0.001%. Suitable non-ionic detergents include, for example, Pluronic F68®. In some cases, a composition containing a recombinant viral expression vector containing a nucleotide sequence encoding a polypeptide MW-opsin and/or LW-opsin and/or SW-opsin is in a saline buffer solution in an amount of from about 10 ⁸ to about 10 ⁹ vg, from about 10 ⁹ vg to about 10 ¹⁰ vg, from about 10 ¹⁰ vg to about 10 ¹¹ vg, from about 10 ¹¹ vg to about 10 ¹² vg, from about 10 ¹² vg to about 10 ¹³ vg, from about 10 ¹³ vg to about 10 ¹⁴ vg, or from about 10 ¹⁴ vg to about 10 ¹⁵ vg, in a volume of about 50 µl to about 75 µl, from about 75 µl to about 100 µl, from about 100 µl to about 150 µl, from about 150 µl to about 200 µl, about 200 µl to about 300 µl, about 300 µl to about 400 µl, about 400 µl to about 500 µl, about 500 µl to about 600 µl, about 600 µl to about 700 µl, from about 800 to about 900 µl, or from about 900 to about 1000 µl, and buffered saline solution contains a non-ionic detergent at a concentration of about 0.001%. The saline solution may contain 0.9% NaCl.

A nucleic acid (e.g., a recombinant expression vector, such as a recombinant viral vector) containing a nucleotide sequence encoding an MW-opsin and/or LW-opsin and/or SW-opsin polypeptide may be administered to an individual in need according to any one of a variety of methods of administration. Suitable routes of administration include, for example, periocular, intraocular, intravitreal, subconjunctival, retrobulbar, intrascleral, and intercameral. In some cases, a nucleic acid (eg, a recombinant expression vector) containing a nucleotide sequence encoding a MW-opsin and/or LW-opsin and/or SW-opsin polypeptide is delivered by intravitreal injection. In some instances, a nucleic acid (eg, a recombinant expression vector) containing a nucleotide sequence encoding an MW-opsin and/or LW-opsin and/or SW-opsin polypeptide is delivered intraocularly. In some cases, a nucleic acid (eg, a recombinant expression vector) containing a nucleotide sequence encoding a MW-opsin and/or LW-opsin and/or SW-opsin polypeptide is delivered via subretinal injection.

In some cases, the method of the present invention comprises a single administration to an individual in need (per eye) of a composition containing a nucleic acid (e.g., a recombinant expression vector, such as a recombinant viral vector) containing a nucleotide sequence encoding a MW-opsin and/or LW polypeptide. -opsin, and/or SW-opsin.

In some cases, an individual is administered multiple doses of a nucleic acid (eg, a recombinant expression vector) containing a nucleotide sequence encoding an MW opsin and/or an LW opsin. For example, in some cases, a nucleic acid (for example, a recombinant expression vector) containing a nucleotide sequence encoding a MW-opsin and/or LW-opsin and/or SW-opsin polypeptide is administered once a month, twice a month, three once a month, every second week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

A nucleic acid (e.g., a recombinant expression vector) containing a nucleotide sequence encoding a MW-opsin and/or LW-opsin and/or SW-opsin polypeptide may be administered to an individual over a period of time from about 1 day to about 1 year, or over 1 year. For example, a nucleic acid (e.g., a recombinant expression vector) containing a nucleotide sequence encoding a MW-opsin and/or LW-opsin and/or SW-opsin polypeptide can be administered to an individual over a period of 1 week to 2 weeks, 2 weeks to 1 month, 1 month to 4 months, 4 months to 6 months, 6 months to 1 year, or more than 1 year.

When the nucleic acid containing the nucleotide sequence is a recombinant viral expression vector containing a nucleotide sequence encoding an MW opsin and/or LW opsin and/or SW opsin polypeptide, the recombinant viral vector may be introduced in an amount of about 10 ⁸ bg to about 10 ⁹ bg, about 10 ⁹ bg to about 10 ¹⁰ bg, from about 10 ¹⁰ bg to about 10 ¹¹ bg, from about 10 ¹¹ to about 10 ¹² bg, from about 10 ¹² bg to about 10 ¹³ vg, from about 10 ¹³ vg to about 10 ¹⁴ vg, or from about 10 ¹⁴ vg to about 10 ¹⁵ vg per dose.

A nucleic acid (e.g., a recombinant expression vector) containing a nucleotide sequence encoding an MW-opsin and/or LW-opsin and/or SW-opsin polypeptide is administered in an amount effective to improve visual function in an individual by at least 10%. , at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 2 times, at least 5-fold, at least 10-fold, or more than 10-fold compared to visual function prior to nucleic acid administration. Visual function tests are known in the art and any test can be used to evaluate visual function.

Individuals suitable for treatment with the method of the present invention include individuals having reduced visual function due to loss of rod and cone photoreceptors. In some cases, the individual has an inherited degenerative retinal disease such as retinitis pigmentosa, retinoschisis, or Leber's congenital amaurosis. In some cases, the individual has an eye disease (eg, an inherited eye disease) selected from retinitis pigmentosa, macular degeneration, age-related macular degeneration, retinoschisis and Leber's congenital amaurosis, and diabetic retinopathy. Individuals suitable for treatment with the method of the present invention include individuals having a retinal degeneration condition in which sensitivity to natural light is reduced and therefore vision is impaired, but where neurons are at the end of the retinal circuit (e.g., bipolar cells or amacrine interneurons, or ganglion cells that are excreted in the brain) are protected and can be made directly light sensitive by administration of conopsin(s).

Individuals suitable for treatment with the method of the present invention include individuals having retinal lesions that are traumatic or acute, without a genetic or hereditary basis. For example, in some cases, an individual has experienced retinal detachment due to blunt trauma, such as a blast injury (eg, in a military battle), or as a result of a blow to the head, such as during a car accident or other accident resulting in a blow to the head. head. In some cases, photoreceptors die due to traumatic retinal detachment from the underlying RPE, but intrinsic retinal neurons are intact. Subjects suitable for treatment with the method of the present invention include those having loss of a photoreceptor due to acute light damage, laser exposure, or chemical toxicity.

Compositions

The present invention provides a composition containing one or more recombinant nucleic acid vectors containing one or more nucleotide sequences encoding one or more conopsins. In the event that the composition is administered to an individual in need thereof, one or more nucleotide sequences encoding one or more conopsins are expressed in the eye of the subject in need such that one or more conopsins are produced in the subject's eye, resulting in one or more positive clinical effects. results. For example, when the composition is administered to the eye of a subject in need, one or more nucleotide sequences encoding one or more conopsins are expressed in the eye of the subject in need such that one or more conopsins are produced in the subject's eye, resulting in one or more positive clinical results. When one or more nucleotide sequences encoding one or more conopsins are expressed in the eye of a subject in need such that one or more conopsins are produced in the subject's eye, one or more positive clinical results result. Positive clinical results include: 1) the subject can distinguish between an image containing a vertical line and an image containing a horizontal line in a spatial pattern recognition assay; 2) the subject can distinguish between an image containing a static line and an image containing a moving line in a spatial pattern recognition analysis; 3) the subject can distinguish between a flashing light and a constant light in the analysis of a temporary light pattern; 4) a subject can recognize an image at a light intensity of about 10 ^-4 W/cm ² to about 10 W/cm ² in an image recognition assay; and 5) the subject can distinguish between an area with white light and an area without white light in a light avoidance assay.

Whether the composition provides one or more of the above positive clinical results can be determined using tests known in the art. See, for example, Leinonen and Tanila (2017) Behavioral Brain Research pii: S0166-4328(17)30870-7; Caporale et al. (2011). Molecular Therapy 19, 1212-9; Gaub et al. (2014) Proc. Natl. Acad. sci. USA 111, E5574-83; Gaub et al. (2015) Molecular Therapy 23:1562; and Berry et al. (2017) Nat. commun. 8:1862.

Provided herein is a composition comprising one or more recombinant nucleic acid vectors comprising one or more nucleotide sequences encoding one or more conopsins, wherein: i) the composition is administered to an individual in need thereof; or ii) the composition is administered to the eye of a subject in need such that one or more nucleotide sequences are expressed in the eye of the subject in need (so that one or more opsins are produced in the subject's eye), the subject can distinguish between an image containing a vertical line, and an image containing a horizontal line in a spatial pattern recognition analysis. A composition of the present invention containing one or more recombinant vectors of a nucleic acid containing one or more nucleotide sequences encoding one or more conopsins, and when one or more nucleotide sequences are expressed in the eye of a subject in need, (so that one or more opsins are produced in the subject's eye), the subject can distinguish between an image containing a vertical line and an image containing a horizontal line in a spatial pattern recognition assay.

A composition of the present invention comprising one or more recombinant nucleic acid vectors containing one or more nucleotide sequences encoding one or more conopsins, wherein: i) the composition is administered to an individual in need thereof; or ii) the composition is administered to the eye of a subject in need such that one or more nucleotide sequences are expressed in the eye of the subject in need (so that one or more opsins are produced in the subject's eye), the subject can distinguish between an image containing a static line, and an image containing a moving line in a spatial pattern recognition analysis. The present invention relates to a composition containing one or more recombinant nucleic acid vectors containing one or more nucleotide sequences encoding one or more conopsins, and when these one or more nucleotide sequences are expressed in the eye of a subject in need (so that one or more more opsins are produced in the subject's eye), the subject can distinguish between an image containing a static line and an image containing a moving line in a spatial pattern recognition assay.

The composition of the present invention, containing one or more recombinant nucleic acid vectors containing one or more nucleotide sequences encoding one or more conopsins, and when: i) the composition is administered to an individual in need, ; or ii) the composition is administered to the eye of a subject in need such that one or more nucleotide sequences are expressed in the eye of the subject in need (so that one or more opsins are produced in the subject's eye), the subject can distinguish between a flashing light and a steady light in the analysis of the temporal light pattern. The present invention relates to a composition containing one or more recombinant nucleic acid vectors containing one or more nucleotide sequences encoding one or more conopsins, and when these one or more nucleotide sequences are expressed in the eye of a subject in need (so that one or more opsins are produced in the subject's eye), the subject can distinguish between a flashing light and a constant light in the analysis of a temporal light pattern.

A composition of the present invention comprising one or more recombinant nucleic acid vectors containing one or more nucleotide sequences encoding one or more conopsins, wherein: i) the composition is administered to an individual in need thereof; or ii) the composition is administered to the eye of a subject in need such that one or more nucleotide sequences are expressed in the eye of the subject in need (such that one or more opsins are produced in the subject's eye), the subject can recognize an image with a light intensity of about 10 ^-4 W/cm ² to about 10 W/cm ² in image recognition analysis. The present invention relates to a composition containing one or more recombinant nucleic acid vectors containing one or more nucleotide sequences encoding one or more conopsins, and when these one or more nucleotide sequences are expressed in the eye of a subject in need (so that one or more opsins are formed in the subject's eye), the subject can recognize an image at light intensity from about 10 ^-4 W/cm ² to about 10 W/cm ² (for example, light intensity from about 10 ^-4 W/cm ² to about 10 ^-3 W/cm ² , from about 10 ^-3 W/cm ² to about 10 ^-2 W/cm ² , from about 10 ^-2 W/cm ² to about 10 ^-1 W/cm ² or from about 10 ^-1 W/cm 2 cm ² to about 1 W/cm ^2. In some instances, expression of an MW opsin polypeptide and/or an LW opsin polypeptide and/or a SW opsin polypeptide in a retinal cell in an individual provides image recognition at light intensity up to 2 W/cm ² to ³ W/cm2, up to ⁴ W/cm2, up to 5 W/cm2 or up to ¹ 0 W/cm ² ) in image recognition analysis.

A composition of the present invention comprising one or more recombinant nucleic acid vectors comprising one or more nucleotide sequences encoding one or more conopsins, wherein: i) the composition is administered to an individual in need thereof; or ii) the composition is administered to the eye of a subject in need such that one or more nucleotide sequences are expressed in the eye of the subject in need (so that one or more opsins are produced in the subject's eye), the subject can distinguish between a white light area and a without white light in the light avoidance assay. The present invention relates to a composition containing one or more recombinant nucleic acid vectors containing one or more nucleotide sequences encoding one or more conopsins, and when these one or more nucleotide sequences are expressed in the eye of a subject in need (so that one or more opsins are produced in the subject's eye), the subject can distinguish between an area with white light and an area without white light in a light avoidance assay.

Conopsins that can be encoded by one or more recombinant nucleic acids present in the composition of the present invention are described above and include, for example, MW-opsin, LW-opsin, SW-opsin, chimeric opsin, and the like.

In some instances, the one or more conopsins encoded by the one or more recombinant nucleic acids present in the composition of the present invention are selected from the group consisting of:

a) an MW opsin containing an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid the sequence shown in SEQ ID NO: 1;

b) an LW opsin containing an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid the sequence shown in SEQ ID NO: 4;

c) an SW opsin containing an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid the sequence shown in SEQ ID NO: 5; and

d) a chimeric SW-opsin comprising: i) intracellular regions of a mouse SW-opsin containing an amino acid sequence that is at least 85%, at least 87%, at least 90%, at least 95% identical , at least 98%, at least 99% or 100% of the amino acid sequence shown in SEQ ID NO: 6; and ii) transmembrane regions of human SW-opsin containing an amino acid sequence that is at least 85%, at least 87%, at least 90%, at least 95%, at least 98% identical, at least 99% or 100% of the amino acid sequence shown in SEQ ID NO: 5.

In some cases, the composition of the present invention contains a recombinant nucleic acid containing a nucleotide sequence that encodes one conopsin. In some cases, one or more recombinant nucleic acid vectors present in the composition of the present invention contain one or more nucleotide sequences encoding 2 different conopsins. For example, in some cases, one or more recombinant nucleic acid vectors present in the composition of the present invention contain one or more nucleotide sequences encoding MW-opsin and LW-opsin. As another example, in some cases, one or more recombinant nucleic acid vectors present in the composition of the present invention contain one or more nucleotide sequences encoding MW-opsin and SW-opsin. As another example, in some cases, one or more recombinant nucleic acid vectors present in the composition of the present invention contain one or more nucleotide sequences encoding LW-opsin and SW-opsin. 2 different conopsins can be encoded in one recombinant nucleic acid. 2 different conopsins can be encoded in 2 separate recombinant nucleic acids.

In some cases, one or more recombinant nucleic acid vectors present in the composition of the present invention contain one or more nucleotide sequences encoding 3 different conopsins. For example, in some cases, one or more recombinant nucleic acid vectors present in the composition of the present invention contain one or more nucleotide sequences encoding MW-opsin, SW-opsin and LW-opsin. 3 different conopsins can be encoded on a single recombinant nucleic acid. 3 different conopsins can be encoded in 3 separate recombinant nucleic acids.

Suitable recombinant nucleic acid vectors include an adeno-associated virus vector, a recombinant lentiviral vector, a recombinant herpes simplex vector, and a recombinant retroviral vector. In some instances, one or more recombinant nucleic acid vectors are/are recombinant adeno-associated virus vectors. In some instances, the recombinant AAV vector contains a nucleotide sequence encoding a variant capsid polypeptide that confers increased infectivity to retinal cells and/or confers increased ability to cross the inner limiting membrane compared to the wild-type AAV capsid.

The nucleotide sequence encoding conopsin may be operably linked to one or more transcriptional control elements. For example, the nucleotide sequence encoding conopsin may be operably linked to a promoter. Examples of suitable promoters include, but are not limited to, synapsin promoter, CAG promoter, CMV promoter, grm6 promoter, Pleiades promoter, ChAT promoter, V-glut promoter, GAD promoter, PV promoter, somatostatin (SST) promoter, neuropeptide Y (NPY) promoter ), the VIP promoter, the red light-sensitive conopsin promoter, the rhodopsin promoter, the rhodopsin kinase promoter, the vitelliform macular degeneration 2 (VMD2) gene promoter, and the interphotoreceptor retinol-binding protein (IRBP) gene promoter.

The composition of the present invention is administered to an individual in need thereof. In some instances, the composition is administered directly to the eye of an individual, such as an eye with impaired visual function. In some cases, the subject has an eye disease selected from retinitis pigmentosa, macular degeneration, retinoschisis and Leber's congenital amaurosis, as well as diabetic retinopathy. In some cases, the subject has experienced retinal detachment or loss of a photoreceptor due to head trauma or injury. In some cases, the subject has a retinal degeneration condition in which sensitivity to natural light is reduced and therefore vision is impaired, but neurons located at the end of the retina (eg, bipolar cells or amacrine interneurons, or ganglion cells leading to the brain) , remain intact and, by administration of the conopsin(s), may become sensitive to light.

The composition of the present invention may contain, in addition to one or more recombinant nucleic acids, a pharmaceutically acceptable excipient. Suitable pharmaceutically acceptable excipients are known in the art and are described elsewhere herein. In some cases, the composition of the present invention is suitable for administration to a human; for example, in some cases, the composition is sterile and free of pyrogens, contaminants, and the like.

Recombinant viral vector

The present invention relates to a recombinant viral vector containing a nucleotide sequence encoding a polypeptide MW-opsin and/or LW-opsin and/or SW-opsin. Suitable virus-based expression vectors include, but are not limited to, a lentiviral vector, a herpes simplex virus (HSV) vector, an adenovirus vector, a retrovirus vector, an adeno-associated virus (AAV) vector, and the like. Thus, in some cases, a recombinant viral vector containing a nucleotide sequence encoding an MW-opsin polypeptide and/or an LW-opsin polypeptide and/or a SW-opsin polypeptide is a recombinant lentiviral vector, a recombinant HSV vector, a recombinant adenovirus vector, a recombinant a retrovirus vector; or a recombinant AAV vector.

In some instances, the nucleotide sequence encoding the MW opsin and/or the LW opsin and/or the SW opsin is operably linked to a promoter that allows expression in a retinal cell. In some cases, the nucleotide sequence is operably linked to a promoter, which typically allows expression in a eukaryotic or mammalian cell.

Suitable promoters include, but are not limited to, the CAG promoter (Miyazaki et al. (1989) Gene 79:269); cytomegalovirus (CMV) promoter; metabotropic glutamate receptor 6 (grm6) promoter (Cronin et al. (2014) EMBO Mol. Med. 6:1175); Pleiades promoter (Portales-Casamar et al. (2010) Proc. Natl. Acad. Sci. USA 107:16589); choline acetyltransferase (ChAT) promoter (Misawa et al. (1992) J. Biol. Chem. 267:20392); vesicular glutamate transporter (V-glut) promoter (Zhang et al. (2011) Brain Res. 1377:1); glutamate decarboxylase (GAD) promoter (Rasmussen et al. (2007) Brain Res. 1144:19; Ritter et al. (2016) J. Gene Med. 18:27); cholecystokinin (CCK) promoter (Ritter et al. (2016) J. Gene Med. 18:27); parvalbumin (PV) promoter; somatostatin promoter (SST); neuropeptide Y (NPY) promoter; and a vasoactive intestinal peptide (VIP) promoter. Suitable promoters include, but are not limited to, the red light sensitive conopsin promoter, the rhodopsin promoter, the rhodopsin kinase promoter, and the GluR promoter (eg, the GluR6 promoter). Suitable promoters include, but are not limited to, the vitelliform macular degeneration 2 (VMD2) gene promoter or the interphotoreceptor retinol-binding protein (IRBP) gene promoter. Also suitable for use is the L7 promoter (Oberdick et al. (1990) Science 248:223), the thy-1 promoter, the recoverin promoter (Wiechmann and Howard (2003) Curr. Eye Res. 26:25); calbindin promoter; and a beta actin promoter.

In some cases, the recombinant viral vector of the present invention contains nucleotide sequences encoding MW-opsin, LW-opsin and SW-opsin. In some cases, the nucleotide sequences encoding MW-opsin, LW-opsin and SW-opsin are operably linked to the same promoter. In some cases, the recombinant viral vector contains an internal ribosome entry site (IRES) between the nucleotide sequences encoding the MW-opsin and the LW-opsin, and between the nucleotide sequences encoding the LW-opsin and the SW-opsin.

In some instances, the recombinant viral vector containing the nucleotide sequence encoding the MW opsin and/or LW opsin and/or SW opsin polypeptide is a recombinant AAV vector (rAAV). In some cases, the rAAV vector contains: i) a nucleotide sequence encoding a MW-opsin and/or LW-opsin and/or SW-opsin polypeptide; and ii) a nucleotide sequence encoding a variant AAV capsid protein, wherein the variant AAV capsid protein provides infectivity to retinal cells and/or increased ability to cross the inner limiting membrane (ILM) in the eye compared to the original wild-type AAV. See, e.g., Day et al. (2014) Adv. Exp. Med. Biol. 801:687; Boye et al. (2016) J. Virol. 90:4215; Vandenberghe and Auricchio (2012) Gene Therapy 19:162; Klimczak et al. (2009) PLoS One 4:e7467; US Patent Publication No. 2012/0164106; and US Patent Publication No. 2016/0017295.

For example, a variant AAV capsid protein may contain an amino acid substitution at amino acid 451 of the AAV6 capsid or at the corresponding position in another AAV serotype. In some cases, the amino acid substitution at amino acid 451 of the AAV6 capsid, or the corresponding position in another AAV serotype, is a substitution of asparagine for aspartic acid. As another example, a variant AAV capsid protein may contain an amino acid substitution at amino acid 532 of the AAV6 capsid or the corresponding position in another AAV serotype. In some cases, the amino acid substitution at amino acid 532 of the AAV6 capsid, or the corresponding position in another AAV serotype, is a substitution of aspartic acid for asparagine.

QSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKT KTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDK

FFPMSGVMIFGKESAGASNTA LDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQ GPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVS VEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL (SEQ ID NO:10), где Asn-451 и Asp-532 выделены жирным шрифтом и подчеркнуты.Капсид AAV6 может иметь следующую аминокислотную последовательность: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVP DPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTST RTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNW GFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPA DVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSL DRLMNPLIDQYLYYLNRTQ

QSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKT KTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDK

FFPMSGVMIFGKESAGASNTA LDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQ GPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVS VEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL (SEQ ID NO:10), где Asn-451 и Asp-532 выделены жирным шрифтом и подчеркнуты.

В некоторых случаях белок капсида AAV содержит следующую аминокислотную последовательность: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVP DPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTST RTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNW GFRPKRLNFKLFNVQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPA DVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSL DRLMNPLIDQYLYYLNRTQ D QSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKT KTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDK N KFFPMSGVMIFGKESAGASNT ALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYL QGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQV SVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL (SEQ ID NO:11).

The present invention relates to a method for delivering MW opsin and/or LW opsin and/or SW opsin to a retinal cell, comprising administering a recombinant viral vector of the present invention (or a viral particle containing a recombinant viral vector) into the eye of an individual. Upon administration of the recombinant viral vector (or a viral particle containing the recombinant viral vector), MW-opsin is produced in the retinal cell.

The present disclosure provides a method for improving or restoring visual function in an individual, the method comprising administering a recombinant viral vector of the present invention (or a viral particle containing the recombinant viral vector) to the eye of the individual. Upon administration of a recombinant viral vector (or a viral particle containing a recombinant viral vector), MW opsin and/or LW opsin and/or SW opsin are produced in the retinal cell. The production of MW opsin and/or LW opsin and/or SW opsin in a retinal cell provides enhanced or restored visual function in an individual.

The present disclosure provides a pharmaceutical composition comprising: a) a recombinant viral vector of the present invention or a viral particle containing a recombinant viral vector; and b) a pharmaceutically acceptable excipient. Suitable pharmaceutically acceptable substances are described above.

The present disclosure provides a method for delivering MW opsin and/or LW opsin and/or SW opsin to a retinal cell, comprising administering a pharmaceutical composition containing a recombinant viral vector (or a viral particle containing a recombinant viral vector) to the eye of an individual. Upon administration of a recombinant viral vector (or a viral particle containing a recombinant viral vector), MW opsin and/or LW opsin and/or SW opsin are produced in the retinal cell.

The present disclosure provides a method for improving or restoring visual function in an individual, the method comprising administering a pharmaceutical composition containing a recombinant viral vector of the present disclosure (or a viral particle containing a recombinant viral vector) to the eye of the individual. Upon administration of a recombinant viral vector (or a viral particle containing a recombinant viral vector), MW opsin and/or LW opsin and/or SW opsin are produced in the retinal cell. The production of MW opsin and/or LW opsin and/or SW opsin in a retinal cell provides improvement or restoration of visual function in an individual.

The recombinant expression vector of the present invention (or a viral particle containing a recombinant viral vector) can be administered to an individual in need thereof by any of a variety of administration routes. Suitable routes of administration include, for example, periocular, intraocular, intravitreal, subconjunctival, retrobulbar, intrascleral, and intercameral. In some cases, a recombinant expression vector of the present invention (or a viral particle containing a recombinant viral vector) is delivered by intravitreal injection. In some cases, the recombinant expression vector of the present invention (or a viral particle containing a recombinant viral vector) is delivered intraocularly. In some cases, a recombinant expression vector of the present invention (or a viral particle containing a recombinant viral vector) is delivered via subretinal injection.

Individuals suitable for treatment with the method of the present invention include individuals having reduced visual function due to loss of rod and cone photoreceptors. In some cases, the individual has an inherited degenerative retinal disease. In some cases, the individual has an ocular disease selected from retinitis pigmentosa, macular degeneration, retinoschisis, and Leber's congenital amaurosis, as well as diabetic retinopathy. In some cases, the individual has age-related degenerative retinal disease. In some cases, an individual has age-related macular degeneration.

Examples of non-limiting aspects of the present invention

Aspects, including embodiments, of the subject matter of the present invention described above may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the above description, some non-limiting aspects of the present invention, numbered 1-54, are presented below. As will be appreciated by those skilled in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to the combinations of aspects that are expressly provided below:

Aspect 1. A method of restoring or improving visual function in an individual comprising administering to the individual a nucleic acid comprising a nucleotide sequence encoding a medium wavelength opsin (MW opsin) and/or a long wavelength opsin (LW opsin) and/or a short wavelength opsin (SW opsin). ), and the specified introduction provides the expression of MW-opsin and/or LW-opsin and/or SW-opsin in the cell of the retina in the individual and the restoration or improvement of visual function.

Aspect 2. The method of Aspect 1, wherein the MW opsin comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% or 100% of the amino acid sequence shown in SEQ ID NO: 1; wherein the LW-opsin contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence, presented in SEQ ID NO: 4, and the SW-opsin contains an amino acid sequence identical to at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% of the amino acid sequence shown in SEQ ID NO: 5.

Aspect 3. The method of Aspect 1 wherein the SW opsin comprises an amino acid sequence that is at least 87% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% or 100% of the human SW opsin amino acid sequence set forth in SEQ ID NO: 5, or at least 87% identical to the mouse SW opsin amino acid sequence set forth in SEQ ID NO: 6.

Aspect 4. The method according to aspect 1, comprising administering to the individual: i) a first nucleic acid containing a nucleotide sequence encoding a SW-opsin containing an amino acid sequence identical to at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the human SW-opsin amino acid sequence shown in SEQ ID NO: 5; and ii) a second nucleic acid containing a nucleotide sequence encoding a SW-opsin containing an amino acid sequence that is at least 87%, at least 90%, at least 95%, at least 98% identical, according to at least 99% or 100% of the mouse SW-opsin amino acid sequence shown in SEQ ID NO: 6.

Aspect 5. The method of Aspect 1 wherein the SW opsin is a chimeric SW opsin comprising intracellular domains of mouse SW opsin and transmembrane domains of human SW opsin.

Aspect 6. The method of Aspect 5 wherein the chimeric SW opsin comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical the amino acid sequence of the chimeric SW-opsin shown in SEQ ID NO: 7.

Aspect 7. The method of any one of aspects 1-6, wherein expression of an MW opsin and/or an LW opsin and/or a SW opsin in a retinal cell provides for pattern vision and image recognition by an individual.

Aspect 8. The method of aspect 7, wherein the image recognition is a static image or pattern recognition.

Aspect 9. The method of aspect 7 wherein the image recognition is a moving image or pattern recognition.

Aspect 10. The method of any one of aspects 1-9, wherein expression of MW opsin and/or LW opsin and/or SW opsin in a retinal cell provides image recognition at a light intensity of about 10 ^-4 W/cm ² up to about 1 W/cm ² .

Aspect 11. The method of any one of aspects 1-9, wherein expression of MW opsin and/or LW opsin and/or SW opsin in a retinal cell provides image recognition at a light intensity that is at least 10 times lower than the light intensity required for image recognition by an individual expressing the channel rhodopsin polypeptide in a retinal cell.

Aspect 12. The method of any one of aspects 1-9, wherein expression of MW opsin and/or LW opsin and/or SW opsin in a retinal cell provides kinetics that is at least 2-fold faster than kinetics in retinal cell caused by the polypeptide rhodopsin.

Aspect 13. The method of any one of aspects 1-12, wherein the nucleic acid is a recombinant expression vector.

Aspect 14. The method of aspect 13 wherein the recombinant expression vector is a recombinant viral vector.

Aspect 15. The method of aspect 14 wherein the recombinant viral vector is an adeno-associated virus vector, a lentiviral vector, a herpes simplex vector, or a retrovirus vector.

Aspect 16. The method of any one of aspects 1-15, wherein the nucleotide sequence is operably linked to a transcription control element that functions in a retinal cell.

Aspect 17 The method of aspect 16 wherein the transcriptional control element is a retinal cell-specific promoter.

Aspect 18. The method of aspect 17 wherein the promoter is a synapsin promoter, CAG promoter, CMV promoter, grm6 promoter, Pleiades promoter, ChAT promoter, V-glut promoter, GAD promoter, PV promoter, somatostatin (SST) promoter, neuropeptide Y (NPY) promoter, VIP promoter, red light sensitive conopsin promoter, rhodopsin promoter, rhodopsin kinase promoter, vitelliform macular degeneration 2 (VMD2) gene promoter, or interphotoreceptor retinol binding protein (IRBP) gene promoter.

Aspect 19. The method of any one of aspects 1-18, wherein said administration is by intraocular injection.

Aspect 20. The method of any one of aspects 1-18, wherein said administration is by intravitreal injection.

Aspect 21. The method of any one of aspects 1-18, wherein said administration is by subretinal injection.

Aspect 22 The method of any one of aspects 1-21 wherein the subject has an ocular disease selected from retinitis pigmentosa, macular degeneration, retinoschisis and Leber's congenital amaurosis, and diabetic retinopathy.

Aspect 23. The method of any one of aspects 1-21, wherein the subject has experienced retinal detachment or loss of a photoreceptor due to trauma or injury to the head.

Aspect 24. The method of any one of aspects 1-23, wherein the nucleic acid is complexed with the nanoparticle.

Aspect 25. A recombinant viral vector containing a nucleotide sequence encoding a medium wavelength opsin (MW opsin) and/or a long wavelength opsin (LW opsin) and/or a short wavelength opsin (SW opsin).

Aspect 26 The recombinant viral vector of aspect 25, wherein the viral vector is an adeno-associated virus vector.

Aspect 27. The recombinant viral vector according to aspect 25 or aspect 26, wherein the MW-opsin contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98% identical, at least 99% or 100% of the amino acid sequence shown in SEQ ID NO: 1; wherein the LW-opsin contains an amino acid sequence identical to the amino acid sequence by at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% the amino acid sequence shown in SEQ ID NO: 4, and wherein the SW-opsin contains an amino acid sequence that is at least 87%, at least 90%, at least 95%, at least 98% identical, at least 99% or 100% of the amino acid sequence shown in one of SEQ ID NOs: 5-7.

Aspect 28. A recombinant viral vector according to any one of aspects 25-27, wherein the nucleotide sequence is operably linked to a promoter.

Aspect 29. The recombinant viral vector of aspect 28 wherein the promoter is synapsin promoter, CAG promoter, CMV promoter, grm6 promoter, Pleiades promoter, ChAT promoter, V-glut promoter, GAD promoter, PV promoter, somatostatin (SST) promoter, neuropeptide Y (NPY) promoter, VIP promoter, red light sensitive conopsin promoter, rhodopsin promoter, rhodopsin kinase promoter, vitelliform macular degeneration 2 (VMD2) gene promoter, or interphotoreceptor retinol-binding protein (IRBP) gene promoter.

Aspect 30. The recombinant viral vector of any one of aspects 25-29, wherein the recombinant viral vector is a recombinant AAV vector, wherein the recombinant AAV vector contains a nucleotide sequence encoding a variant capsid polypeptide that confers increased infectivity to retinal cells and/or confers increased ability to cross the inner limiting membrane compared to the wild-type AAV capsid.

Aspect 31. A pharmaceutical composition comprising:

a) a recombinant viral vector according to any one of aspects 25-30; and

b) a pharmaceutically acceptable excipient.

Aspect 32. A method of restoring or improving visual function in an individual, the method comprising administering to the individual a recombinant viral vector according to any one of aspects 25-30 or a pharmaceutical composition according to aspect 31, said administration providing expression of an MW opsin and/or an LW opsin , and/or SW-opsin in a retinal cell in an individual and restoration or improvement of visual function.

Aspect 33 A chimeric SW opsin comprising intracellular regions of a mouse SW opsin containing an amino acid sequence identity of at least 87%, at least 90%, at least 95%, at least 98%, at least 99% or 100% of the amino acid sequence shown in SEQ ID NO: 6 and containing transmembrane regions of a human SW-opsin containing an amino acid sequence that is at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the amino acid sequence shown in SEQ ID NO: 5.

Aspect 34 The chimeric SW opsin of aspect 33 comprising an amino acid sequence at least 90% identical to the amino acid sequence shown in SEQ ID NO: 7.

Aspect 35. A recombinant expression vector containing a nucleotide sequence encoding a chimeric SW opsin of Aspect 33 or Aspect 34.

Aspect 36. A composition comprising:

a) a recombinant expression vector according to aspect 35; and

b) a pharmaceutically acceptable excipient.

Aspect 37. A composition comprising one or more recombinant nucleic acid vectors comprising one or more nucleotide sequences encoding one or more conopsins, wherein when said one or more nucleotide sequences are expressed in the eye of a subject in need, said subject can distinguish between an image , containing a vertical line, and an image containing a horizontal line, in the spatial pattern recognition analysis.

Aspect 38. A composition comprising one or more recombinant nucleic acid vectors comprising one or more nucleotide sequences encoding one or more conopsins, where said one or more nucleotide sequences are expressed in the eye of a subject in need thereof, said subject can distinguish between an image containing a static line and an image containing a moving line in a spatial pattern recognition analysis.

Aspect 39. A composition comprising one or more recombinant nucleic acid vectors comprising one or more nucleotide sequences encoding one or more conopsins, where said one or more nucleotide sequences are expressed in the eye of a subject in need thereof, said subject can distinguish between flashing light and constant light in the analysis of a temporary light pattern.

Aspect 40. A composition comprising one or more recombinant nucleic acid vectors comprising one or more nucleotide sequences encoding one or more conopsins, wherein if said one or more nucleotide sequences are expressed in the eye of a subject in need, said subject can recognize image at a light intensity of about 10 ^-4 W/cm ² to about 10 W/cm ² in an image recognition assay.

Aspect 41. A composition comprising one or more recombinant nucleic acid vectors comprising one or more nucleotide sequences encoding one or more conopsins, where said one or more nucleotide sequences are expressed in the eye of a subject in need thereof, said subject can distinguish between an area with white light and an area without white light in the light avoidance analysis.

Aspect 42 The composition of any one of aspects 37-41 wherein the one or more conopsins are selected from the group consisting of: a) an MW opsin containing an amino acid sequence at least 85% identical to the amino acid sequence shown in SEQ ID NO : 1; b) an LW-opsin containing an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 4; c) SW-opsin containing an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 5; and d) a chimeric SW opsin comprising: i) intracellular regions of a mouse SW opsin comprising an amino acid sequence at least 87% identical to the amino acid sequence shown in SEQ ID NO: 6; and ii) transmembrane regions of a human SW opsin comprising an amino acid sequence at least 87% identical to the amino acid sequence shown in SEQ ID NO: 5.

Aspect 43 The composition of any one of aspects 37-41, wherein the one or more recombinant nucleic acid vectors comprise one or more nucleotide sequences encoding 2 different conopsins.

Aspect 44 The composition of any one of aspects 37-41, wherein the one or more recombinant nucleic acid vectors comprise one or more nucleotide sequences encoding 3 different conopsins.

Aspect 45 The composition of any one of aspects 37-44, wherein the one or more recombinant nucleic acid vectors is an adeno-associated viral vector, a lentiviral vector, a herpes simplex vector, or a retroviral vector.

Aspect 46 The composition of any one of aspects 37-44, wherein the one or more recombinant nucleic acid vectors are recombinant adeno-associated viral vectors.

Aspect 47 The composition of aspect 46, wherein the recombinant AAV vector comprises a nucleotide sequence encoding a variant capsid polypeptide that confers increased infectivity to retinal cells and/or confers increased ability to cross the inner limiting membrane compared to the wild-type AAV capsid.

Aspect 48 A composition according to any one of aspects 37-47 wherein one or more nucleotide sequences are operably linked to a promoter.

Aspect 49 The composition of aspect 48 wherein the promoter is a synapsin promoter, CAG promoter, CMV promoter, grm6 promoter, Pleiades promoter, ChAT promoter, V-glut promoter, GAD promoter, PV promoter, somatostatin (SST) promoter, neuropeptide Y (NPY) promoter, VIP promoter, red-light sensitive conopsin promoter, rhodopsin promoter, rhodopsin kinase promoter, vitelliform macular degeneration 2 (VMD2) gene promoter, or interphotoreceptor retinol-binding protein (IRBP) gene promoter.

Aspect 50 The composition of any one of aspects 37-49 wherein the subject has an ocular disorder selected from retinitis pigmentosa, macular degeneration, retinoschisis, Leber's congenital amaurosis, and diabetic retinopathy.

Aspect 51 The composition of any one of aspects 37-49, wherein the subject has had retinal detachment or photoreceptor loss due to trauma or injury to the head.

Aspect 52 The composition of any one of aspects 37-51, wherein the composition contains a pharmaceutically acceptable excipient.

Aspect 53 The composition of aspect 52 wherein the pharmaceutically acceptable excipient comprises saline.

Aspect 54 The composition of any one of aspects 37-53, wherein the composition is sterile.

Examples

The following examples are provided to provide those skilled in the art with a full disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors consider their invention, and the experiments below are not all or the only ones performed. experiments. Efforts have been made to ensure accuracy with respect to the numbers used (eg amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, eg b.p., base pair(s); kb, thousand base pairs; pl, picoliter(s); s, second(s); min, minute(s); h, hour(s); aa, amino acid(s); kb, thousand base pairs; b.p., pair(s) of bases; nt, nucleotide(s); i / m intramuscularly; in / b intraperitoneally; s / c subcutaneously; etc.

Example 1 Influence of MW-opsin expression in retinal cells in vitro and in vivo

Hereditary and age-related degenerative diseases of the retina cause a progressive loss of rod and cone photoreceptors, leading to total blindness. Despite the loss of light-sensitive cells essential for vision, downstream neurons of the inner retina survive in a functional state, providing a target for optogenetic therapy. To date, optogenetic approaches have faced two major limitations: the very low light sensitivity of microbial opsins and the very slow kinetics of retinal opsins. The data presented below show that vertebrate wavelength media conopsin (MW-opsin) overcomes these limitations and maintains structured vision in low light. Expression of MW-opsin in rd1 blind mouse retinal ganglion cells (RGCs) induces an ON transient light response that is highly sensitive to light and has fast kinetics. MW-opsin restores photo-avoidance at the same photosensitivity as in wild animals, three orders of magnitude below the level required for microbial opsins and at the level of rhodopsin. The MW opsin allows rd1 animals to distinguish between both different temporal light patterns and different spatial light patterns, even when the spatial patterns are in motion. In contrast, rd1 animals with rhodopsin in the RGC are not capable of either temporal or spatial discrimination. Strikingly, the light response of the MW opsin adapts to ambient light in the ~1000-fold range. Thus, the MW opsin combines speed, sensitivity, and adaptability to restore steerable patterned vision at realistic light intensity. LW opsin and SW opsin also support vision discrimination between static visual patterns, suggesting that conopsins can individually restore natural monochromatic vision and that, when combined, they can restore color vision.

Materials and methods

Animals and AAV

Experiments on mice were performed with the consent of the Committee for the Care and Use of Animals at the University of California. Wt mice (C57BL/6J) and rd1 mice (C3H) were purchased from the Jackson laboratory and placed on a 12 hour light/dark cycle with food and water ad libitum. cDNA encoding vertebrate medium wavelength conopsin or rhodopsin was linked to the yellow fluorescent protein (YFP) gene at the C-terminus and inserted into an established viral cassette under the control of the human synapsin promoter (hsyn-1). The gene and promoter were flanked by inverted terminal repeat domains, stabilized by a polyadenylation signal sequence (polyA) with the woodchuck hepatitis post-transcriptional regulatory element (WPRE), and packaged in an AAV 2/2-4YF capsid. The AAV titer was determined by qPCR against the inverted repeat domain standard and was reported to contain 10 ¹⁰ -10 ¹² viral genomes. AAV was prepared as previously described ¹⁵ . The vector was delivered at a volume of 2 μl to the vitreous of the rd1 mouse eye by microinjection using a blunt 32 gauge Hamilton syringe, although the incision was made behind the ora serrata with a sharp 30 gauge needle. rAAV injections were performed at p30–p60, and in vivo and in vitro experiments at p90–p160. Mice were anesthetized ip with ketamine (72 mg/kg) and xylazine (64 mg/kg). The eyes were anesthetized with proparacaine (0.5%) and the pupils were dilated with phenylephrine (2.5%) and tropicamide (1%).

Electrophysiology and light stimulation

Registration of HEK cells was performed as previously described ^15,51,52 . Briefly, cells were fixed in whole cell mode in high external potassium solution (50 mM) and kept at V _H = -80 mV to provide internal driving force for potassium. 5-10 s light pulses were applied at low intensity (1 mW/cm ^-2 ) at 535 nm (for MW-opsin) or 500 nm (for rhodopsin).

MEA recordings were performed on wt mice (C57BL/6J), and in untreated and treated rd1 mice at >p90, 6-10 weeks after AAV injection, the experimental retina was excised from the eye under dim red light, mounted on 4 µm cell membranes, and placed in incubator (35°C) for 30 min and perfused with exogenous 9-cis retinal chromophore. The retinal tissue was placed ganglion cell side down ⁵³ into the recording chamber (pMEA 100/30iR-Tpr; multi-channel systems) of a 60-channel MEA system with constant perfusion of Ames recorder (32° C.). The weight of the Multi Channel Systems (Scientific Instruments - Slice grids) was placed on the retina to prevent movement, and a vacuum was applied to the retina with a pump (MEA1060 perforated system with CVP; Multi Channel Systems), improving electrode-tissue contact and providing a consistent relationship signal/noise on the retina. In addition, a dry crystal of the exogenous 9-cis-retinal chromophore was dissolved in µl of 100% ethanol in the dark. Dissolved retinal was then added to 100 µl of Matrigel on ice and added to the top of the mesh just above the retina to ensure a continuous supply of the chromophore. In addition, 9-cis-retinal was dissolved in the recording solution and continuously perfused into the recording chamber. More detailed information about the methods of MEA previously described in detail in Gaub. et al. (2015) ¹⁵ . In vitro illumination was provided by a 300 W mercury arc lamp (DG-4; Sutter Instruments) with a 535/50 nm bandpass filter for MW-opsin or a 510/89 nm bandpass filter for rhodopsin. Light intensity was controlled by changing the duty cycle of the light source or using neutral density filters and ranged from 0.038 μW cm ^-2 to 38.2 mW/cm ² . Relative comparisons with natural light intensity were obtained in various environments using direct light measurement with a power meter (Thorlabs). The spectral component of white light was measured using a CCD spectrometer with Fourier transform optical spectrum analysis software (Thorlabs).

MEA Data collection and analysis

Retinal activity on MEA was sampled at 25 kHz, filtered between 100 and 2000 Hz and recorded using MC_rack software (multichannel systems). The voltage traces were converted to spike sequences offline and the peaks recorded at each electrode were sorted into individual units, which are defined as "cells", through principal component analysis using an offline sorter (Plexon-64bit), where each electrode is usually identifies 1-3 cells. Single spike clusters were exported to MATLAB (MathWorks) and analyzed and graphically represented using special software. All ripple frequencies were extracted from traces averaged over 3-10 cycles of light response, unless otherwise noted, the details of which are indicated in the legend in the figures. The responses between cells and the retina were normalized using the light response index (LRI) taken from Tochitsky, I., et al. (2014) ³⁷ and Gaub et al. (2014) ⁴⁷ (LRI =(peak ripple frequency in light - average response speed in dark)/peak ripple frequency in light + average ripple frequency in dark). In experiments that varied retinal conditions (light sensitivity, light and dark sensitivity, and response versus flash duration), responses were normalized to the peak with the highest response from baseline, and channels were tracked across all recording parameters. All curve fitting and kinetic analysis were performed in Clampfit 10.6 (Molecular Devices). Cells were defined as "responding" if the LRI satisfied the condition LRI> 0.1 or LRI <-0.1. The half-peak response width from baseline was determined using custom MATLAB analysis software (MathWorks), which interpolated individual cell traces and applied smoothing until FWHM was determined. The intensity-response relationships corresponded to a single Boltzmann distribution and were normalized to fit between 0 and 1.

Comparison of photosensitivity of optogenetic probes

The natural sensitivity of human vision spans a wide range, from the ^scotopic (dim 10 ⁴ - 10 ¹¹ photons ⁻² s ⁻¹ ) to the photopic band (bright 10 ¹⁰ – 10 ¹⁷ photons cm ⁻² s . In order to relate the sensitivity of MW-opsin to that of other optogenetic systems and normal vision, experiments were performed that measured the intensity of natural light under various external and internal conditions using a portable power meter (Thorlabs), comparing them with experimental paradigms and light sensitivity thresholds ( lowest measurable light responses) described in the literature using monochromatic light wavelengths at or near maximum functional maxima for retinal explants derived from rd1 mice delivered with an optogenetic probe via AAV transfection (Fig. 7).

Electrophysiology and light stimulation for in vivo brain recording

In vivo recordings were performed as previously described by Veit et. al. (2017) ⁵⁸ . Mice were anesthetized with isoflurane (vapor concentration 2.5%). The scalp was removed, the fascia retracted, and the skull lightly etched with a 27-gauge needle. After applying Vetbond to the surface of the skull, a special stainless steel plate was attached to the skull using dental cement (Metabond). The mice were allowed to recover from the operation for at least 2 days. Then, the mice were accustomed for 2–5 days to fix their heads on a freely rotating circular treadmill. On the day of recording, mice were briefly anesthetized with isoflurane (2%), the skull over V1 was thinned, and a small (<250 µm) craniotomy was opened over the primary visual cortex with a fine needle. A 16-channel linear electrode array at 25 micron intervals (NeuroNexus, A1x16-5mm-25-177-A16) was introduced into the brain using a micromanipulator (Sensapex) and a stereo microscope (Leica). Electrical activity was amplified and digitized at 30 kHz (Spike Gadgets) and stored on a computer hard drive. The cortical depth of each electrical contact was determined by zeroing out the lower contact with the brain surface. The electrode was inserted close to perpendicular to the surface of the brain.

Visual stimuli were generated using Psychophysics Toolbox ⁵⁹ running on an Apple Mac Mini and presented on a 23-inch Eizo FORIS FS2333 LCD with gamma correction and a 60 Hz refresh rate. The monitor was positioned 15 cm from the mouse's right eye, covering approximately 108 by 61 degrees of viewing angle. Mice were dark adapted 7–10 min before each recording block. Two different stimulation paradigms were used: 1) Contrast: Mice were shown full screen flashes of 500ms at 0.5Hz at four different brightness levels (15%, 25%, 50% and 100% of maximum brightness) which were repeated 10 times in different random orders. 2) Flicker: Mice were shown 20,500 ms of full-screen flashes (maximum brightness 116 μW cm ^-2 ) at 0.0167 Hz (one per minute) or 100 flashes at 0.5, 1, 2 and 4 Hz in separate blocks.

In vivo cortical data recording, collection and analysis

Peak activity was extracted by filtering the raw signal between 800 and 7000 Hz. Splash detection was performed using the UltraMega Sort ⁶⁰ package. The detected signal peaks were sorted using the MClust package (http://redishlab.neuroscience.umn.edu/MClust/MClust.html). Waveforms were first grouped automatically using KlustaKwik and then manually adjusted to meet criteria for further analysis. Units with more than 2% of their individual waveforms violating the 2 ms refractory period were classified as multi-units. The depth of each block was assigned based on the computed electrode depth on the array, which showed its largest sorted signal amplitude. Pulsing frequencies were calculated by counting the peaks in a two second window starting 250 ms after the onset of the visual stimulus for FIG. 20b and in window 5c starting immediately after the appearance of the stimulus for FIG. 21c. Values for histograms of peristimulus time (PSTH) were generated by splitting the burst response into either 20 ms (FIG. 19), 50 ms (FIG. 20), or 25 ms (FIG. 21), and smoothing the resulting values with using a moving average filter. For FIG. 21, the modulation amplitude was calculated from the average probe peak responses, which were grouped at 25 ms into a window of 2, 1, 0.5, or 0.25 s for 0.5, 1, 2, and 4 Hz flicker, respectively. The corresponding base modulation amplitudes were obtained from a test averaging the same number of windows of the same length prior to the onset of the first visual stimulus (during the dark adaptation period). The local field potentials were extracted by low pass filtering the raw signal sampled at 30 kHz below 200 Hz and downsampled down to 1 kHz.

Statistics

To evaluate the statistical significance of the MEA records, non-parametric two-tailed Mann-Whitney U-tests were used. For learned dark avoidance behavior and learned pattern recognition behavior, significance was determined in two ways. (1) The significance of behavioral characteristics was calculated using two-tailed unpaired Student's t-tests with Bonferroni's correction when applicable. The value was also determined by calculating the percentage of successful performances (2). Success was defined as greater than the sum of the mean of the control group and one S.D, and failure was any value that did not meet this criterion. Success rates were then calculated for each condition. To determine the significance of differences between conditions, a pairwise contingency table was constructed and Pearson's two-tailed chi-square test was initially performed. To correct for the small n conditions, Fisher's exact one-tailed test was also performed. Fig. 11 (table 1).

Tissue preparation and immunohistochemistry

Mice >4-6 weeks after treatment with AAV2/2-hsyn-MW-conopsin-YFP were sacrificed, eyes fixed in 4% paraformaldehyde (Ted Pella) (30 min), retinas removed and thoroughly washed with PBS and mounted on a flat surface on slides using Vectashield medium (Vector Laboratories) impregnated with DAPI (staining of cell nuclei - blue). For retinal sections, whole mounts were embedded in agarose (Sigma) and cut transversely using a vibratome (Leica Microsystems) with medium speed, maximum vibration, and a thickness of 180 µm. Retinal tissues used for immunohistochemistry in retinal cryosection or total preparations were processed and examined using confocal microscopy (Leica TCS SP5; Leica Microsystems).

Passive avoidance - open field test

The open field test was performed as previously described ^16,47 . Briefly, the double (light and dark) shuttle box (Colbourn Instruments) allows the mouse to move freely through a small opening connecting the two compartments. The bright compartment was illuminated by an LCD panel above the compartment. White light, as well as wavelengths of 535 nm and 460 nm, were used at light intensities of ~100 μW cm ^-2 (white light) and 0.5-25 μW cm ^-2 (blue and green light), which were evenly distributed over the floor. Day 1 - Mice were transferred to the test box and allowed to acclimate to the new environment with their littermates for 45 minutes. The mice were then returned to their home cage and then tested individually. Day 2 - Mice were placed in the light compartment and allowed a maximum of 3 min to discover that there was a second compartment. The 15-minute test began when they entered the dark compartment, and the time spent in the light was recorded. Mice that crossed the hole only once and remained in the dark compartment the entire time were disqualified. For adaptation experiments, mice were pre-exposed to white light for 1 hour (1 mW/cm ^-2 ) or dark conditions, and then immediately tested in an open field. Animal movements were tracked using IR sensors on the shuttle box. Time spent on both sides was recorded and analyzed using the Graphic State and RT Graphic State programs (Colbourn Instruments).

Visually Tuned Fear Formation Paradigm

Fear shaping experiments were conducted using Colbourn impact chambers - a Colbourn Habitest chamber with test cage (Coulbourn Instruments, PA) with an LED screen attached to the ceiling. On Day 1, animals were brought to the home cage test room and then individually acclimatized for 30 minutes to clean the Colbourn shock chambers. On days 2-3, the animals were subjected to mild paired or unpaired fear formation, consisting of 5 min habituation to the chamber (darkness) followed by three shock tests at 0.7 mA for 15 min. For pair tests, the transition from a static or flashing (2 Hz) light on the LCD coincided with a short shock to the leg with a 2 s (within 10 s) interval between surges. For unpaired trials, animals also received shocks but were not affected by stimulus transition. These short, weak shocks provided minimal unpleasant stimuli to create a fear memory associated with patterned light. On day 4, the flooring of the box was replaced with plastic instead of a shock grate. Mice were accustomed to the chamber for 5 min and subjected to the same light stimulation protocol as on days 2 and 3, but without electric shock. Freezing behavior in anticipation of shock was recorded by the Colbourn FreezeFrame software and normalized to the movement behavior obtained before stimulation. The results were then compared between paired and unpaired cohorts to determine if the fear response was driven by stimulus transition.

Modified active avoidance protocol

Avoidance protocols were performed using the Colbourn Shuttle Box (H10-11M-SC) Colbourn Habitest Isolation Cubicle (H10-24) and Graphic State and Graphic State RT software (Colbourn Instruments, PA). On day 1, the animals acclimatized in a darkened treatment room for 30 min in their home cages, followed by individual adaptation to the darkened shuttle box until the animals were no longer afraid and spent approximately the same amount of time on each side of the shuttle box (exploratory behavior). ). On days 2-3, iPad screens were installed in the cell shuttle wall and displayed two different images of the same shape, size, and light intensity. Each run began when the animal moved to the other side of the shuttle box from where it was placed. Each run lasted 15 min. The aversive side of the image was paired with a 0.7 mA leg shock at 5 second intervals until the animal returned to the "safe" side. Any animal remaining on the aversive side for more than 60 seconds was removed from the cage and the move was discarded. On day 4, the light patterns were reversed (thus the averse screen was where the "safe" side used to be) to avoid shifting the location rather than the pattern. In addition, the cage flooring has been replaced with plastic instead of a shock grid. This ensures that the only link to the training day is the light stimuli. Again, the animals ran for 15 minutes and the time spent on each side was recorded. In the light adaptation experiments, the same protocol was used, except that on day 4 the recall phase was preceded by either a light (65 W white light bulb) or dark adaptation phase for one hour. The optical angle of visual discrimination calculations were performed using the behavioral shuttle cell parameters (15.24 × 36 cm), distance from the decision point (divider), central position of the LCD panel (18.85 cm), and stimulus pattern parameters (distance between parallel lines 1 - 6 cm) using the equation of the optical (physical) angle. Viewing angle = V = 2tan ^-1 ((D/2)/(L)) = 18 deg = 0.33 - 0.49 rad. Cycles per degree = 1/V ~0.056 c/deg. This is approximately 9 times lower than visually intact mice wt (~0.3-0.5 c/deg) as reported in other studies ^39,54,55,56 .

For exploratory behavioral analysis, two objects were placed in a 50 cm × 50 cm box. Animals were placed in an empty box and allowed to explore it freely for 10 min. The next day, two new objects were placed in the box, and the animals were again placed along the wall of the box and allowed to freely explore it for 10 minutes while the arena was filmed continuously. Using Noldus Ethosvision XT v13.5 technology, video was analyzed for delay to reach and explore each object, travel speed (cm/s) and distance traveled (cm).

results

Characterization of MW-opsin in HEK293 cells

Recent studies have shown that vertebrate rhodopsin, found in the outer segments of the rods, can be ectopically used to control Gi/o signaling in cultured cells at low light intensity ²⁵ but decreases with repeated stimulation and has a slow deactivation ^24,26 . Slow kinetics are also observed when rhodopsin is expressed in ON-bipolar cells of the blind retina ^{14,15 raising} concerns that it may not support vision of natural scenes due to the movement of the observer and surrounding objects. The question was raised whether another vertebrate opsin would have high rhodopsin sensitivity but have faster kinetics. Melanopsin and extraretinal opsins have been measured outside of photoreceptor cells in various cell types, including non-photoreceptor cells in the retina, and all have been found to be slow or slower than rhodopsin ^14-16,27-29 . Opsins from mammalian cone photoreceptor cells were chosen because they are deactivated faster in cones than rhodopsin in rods ³⁰ . Among the cone opsins, the MW opsin was chosen, which acts in the center of the visible spectrum. MW-opsin or rhodopsin was expressed in HEK293T cells with GIRK1 (F137S), a version of the potassium channel associated with the internal G rectifier that forms a homotetramer that is activated by Gβγ upon activation of the Gαi-coupled receptor ³¹ (Fig. 1). In the presence of 9-cis-retinal (a functionally stable analog of the natural chromophore 11-cis-retinal), cells expressing MW-opsin and rhodopsin exhibited large internal photocurrents at negative holding potentials at high external potassium levels, which were triggered by low-intensity flashes of light ( 1 mW cm ^-2 ) at 535 nm (for MW-opsin) or 500 nm (for rhodopsin) (Fig. 1a,b). After illumination, the MW-opsin-induced current decayed ∼8 times faster than the rhodopsin-induced current and recovered more completely, providing reproducible responses upon repeated flashes of illumination (Fig. 1a,c), as shown previously in heterologous cells for conopsins. short and long wave ²⁴ . This suggests that the higher rate may be inherent in MW opsin signaling rather than cone specialization. MW-opsin was then tested in non-photoreceptor retinal neurons.

Fig. 1a-1c. Activation by MW-opsin and rhodopsin of GIRK channels in HEK293 cells. (a,b). Representative traces of activation of channels of the GIRK homotetramer (F137S) by photostimulation of rhodopsin (a) or MW-opsin (b), measured in a layer of whole cells at 50 mM [K ⁺ ] _ext at V _H = -80 mV in response to low-intensity light pulses (1 mW cm ^-2 ) at 535 nm (for MW-opsin) or 500 nm (for rhodopsin). (c) Photoresponse decay (Tau OFF) for rhodopsin (blue) and MW opsin (green). Values are average + SOS; n = 6 (Rho), 8 (MW-opsin) cells.

MW opsin recovers quickly and elicits sensitive light responses in the blind retina

MW-opsin was tested in the rd1 mouse retina, which has a mutation in the PDE-6-β gene that results in progressive loss of rod and cone photoreceptor cells. MW-opsin under the control of the human synapsin promoter (hSyn-1) with a yellow fluorescent (YFP) C-terminal tag for expression tracking was packaged in AAV2/2 (4YF) and injected intravitreally at postnatal day 45 (Fig. 2a,b). The retinas were isolated after 4-8 weeks. Expression was found to be panretinal with a transfection rate of 45% + 19% (SD), restricted to the RGC layer and localized in the soma and dendrites of both ON- and OFF-RGC (Fig. 2c, d and 14), a well-defined profile expression is consistent with the previously shown targeting ³² and is similar to the expression of rhodopsin under identical parameters (Fig. 15). The retinas were mounted on a multi-electrode array (MEA) with an RGC layer in contact with the electrodes to test for light-induced activity. Due to complete photoreceptor degeneration in >3 months ³³ animals, no light-induced response was detected in the retinas of control rd1 littermates (Fig. 2e), with the exception of a few RGCs that exhibited slow responses characteristic of intrinsically photosensitive RGCs ⁵⁷ . In contrast, retinas from animals injected with AAV-encoding MW-opsin exhibited a strong light-induced increase in potential exposure, consisting of a large fast transient component and a small (~30% size) slow component (Figs. 2f, g, 3a, 16 and 17). Light responses decreased with repeated attacks of light stimulation, as would be expected after removal of the retinal pigment epithelium, the source of 11-cis. The contraction was reduced by adding 9-cis-retinal (a stable analogue of 11-cis-retinal) to the recording solution (Fig. 2g).

Fig. 2a-2g. Expression and function of MW-opsin in the RGC of the rd1 mouse retina. (a). Viral DNA expression cassette. MW-opsin with YFP (green) under the control of the hSyn-1 promoter, flanked by inverted terminal repeat (ITR) domains, stabilized by a polyadenylation signal sequence (polyA) and a woodchuck hepatitis post-transcriptional regulatory element (WPRE). (b) Schematic of the degenerated rd1 mouse retina with targeted RGCs highlighted (green). ONL: outer nuclear layer. IPL: inner plexiform layer. Photoreceptor degeneration is indicated in light gray. (c,d) En face image of (c) flat mount and (d) transverse section of confocal images of mouse rd1 retinal MW-opsin expression 4 weeks after intravitreal administration of AAV2/2-hSyn-MW-opsin-YFP. Images of YFP fused to the C-terminus of MW opsin (green) show pan-retinal distribution (c) in the RGC layer in relation to nuclear DAPI staining (d, blue). Scales 60 µm (c) and 20 µm (d). (e,f) MEA recordings from uninjected control (e) and MW-opsin expressing (f) mouse retinal rd1. (Top) Raster plot with spikes for each RGC (e: n = 75 cells; g: n = 84 units). (Bottom) Histogram of peristimulus time (PSTH). Light stimulation protocol: 4 light pulses of 1 s duration (λ = 535 nm, green bars) separated by dark intervals of 30 s. (g) Normalized light response index (LRI) for rd1 retina without (grey) and with MW-opsin expression (green) (grey: N=3 retinas, n=190 cells; green: N=6 retinas, n=273 cells ). ^LRI for 1st and 5th flash of light without (light green; N=3 retinas, n=106 cells) and with (dark green; N=3 retinas, n=167 cells) 9-cis retinal. Light intensity 3.38 mW cm ^-2 . Wavelength: λ = 535 nm (MW-O), values are average + SOS. All cells belong to sorted units. Statistical significance was assessed using the Mann-Whitney U-test (*p<0.001).

Fig. 14a-14b. MW opsin is expressed in the rd1 mouse retina. Flat mount (a) and section (b) confocal images of MW-opsin-YFP in RGC rd1 mouse retina 4-6 weeks after intravitreal administration of AAV2/2-hSyn-MW-opsin-YFP. YFP fluorescence (green) and DAPI nuclear staining (blue). INL = inner nuclear layer, RGC = retinal ganglion cell layer. Scale 40 µm (a) and 20 µm (b).

Fig. 15a-15b. Rhodopsin is expressed in the rd1 mouse retina. Flat mount (a) and section (b) confocal images of rhodopsin-YFP in RGC rd1 mouse retina 4-6 weeks after intravitreal administration of AAV2/2-hSyn-rhodopsin-YFP. YFP fluorescence (green) and DAPI nuclear staining (blue). INL = inner nuclear layer, RGC = retinal ganglion cell layer. Scale 40 µm (a) and 20 µm (b).

Fig. 16a-16d. Transduction efficiency of MW-opsin in rd1 retina. (a) Mean percentage of channels identified in the retina of rd1 mice expressing MW-opsin that show light sensitivity in MEA recordings. Light sensitivity, defined as Light Sensitivity Index (LRI) > 0.1, parameters set by Tochitsky et al. (2014) ³⁷ , Gaub. et al (2014) ⁴⁷ and Berry et al. (2017) ⁶² . (LRI = peak dark ripple - average dark ripple/peak dark ripple + average dark ripple). (b) Mean normalized retinal response). (a,b) N = 8 rd1 retinas, N = 5 wt retinas. (c) Mean cross-correlation values in MW-opsin rd1 (n=370 cells, N=3 retinas) and wild-type (n=237 cells, N=3 retinas). Cross-correlation of all photosensitive units over a period of 1 s to 2 s after the light pulse. (d) Distribution of correlation values for photosensitive units in the same retina for MW-opsin (n=370 cells, N=3 retinas) and wt retina (n=237 cells, N=3 retinas).

Fig. 17a-17d. Characterization of the slow component of the light response in the rd1 retina expressing MW-opsin. (a) Representative RGC population mean response curve for rd1 expressing MW-opsin (λ = 510 nm; pulse width 1 second; 5 flashes averaged at 30 second intervals) showing a fast transient component and a slow light response component (SOS in gray color). (b) LRI comparison of the fast transient (green) and slow (steel grey) components of the light response and their (black) fast/slow ratio (n = 122 channels, N = 3 retinas). (c) Kinetics of the slow component: time to peak (T. - peak) and decay time constant (tau decay). (d) LRI of fast (green) and slow (steel grey) components in dark (left) and light (right) conditions.

One potential advantage of retinal opsins is their high sensitivity to light in their normal cellular environments. Rhodopsin and melanopsin remain highly sensitive to ectopic expression ²⁵ , including in the retina, as shown for rhodopsin in ON-BC and RGC and melanopsin in RGC ^14-17 . Consistent with this, the MW-opsin in the RGC in the rd1 mouse retina was highly sensitive to light to a degree indistinguishable from rhodopsin (Fig. 3b,c). This sensitivity is about 1000 times higher than channel rhodopsin ^8,13 or halorodopsin ^11,12 and is in the range of normal indoor light intensities. Fig. 7. Despite the similarity in sensitivity to rhodopsin, MW-opsin showed kinetics of ~3.5-fold acceleration of rise and ~4-fold acceleration to peak at the beginning of the light pulse and ~7-fold acceleration of decay after the end of the light pulse (Fig. 3d,e), leading to an impulse response with a duration of 270 ± 21 ms at half amplitude, ~10 times shorter than the duration of 2.61 ± 0.21 s at half amplitude of the impulse response of rhodopsin. Tau ON, time to peak, and tau OFF changed little with decreasing light intensity, retaining a speed advantage over rhodopsin (Fig. 8a-c) ^14,15 . The rise and fall kinetics of the response in the MW-opsin-expressing rd1 retinal RGC was similar to that of the RGC transient ON response observed in the wt retina, except that the former had a longer latency (Figs. 3e and 8d, e). The fast response kinetics and sensitivity of the MW opsin suggested that it would respond to short bursts of light. Indeed, illumination pulses as short as 25 ms still elicited responses that reached ∼60% of the maximum peak response (Fig. 3f,g), similar to that observed in retinal wt ³⁴ .

Contrast sensitivity in the distant retina and in the primary visual cortex was examined in vivo by measuring the response to full halftone steps. In excised rd1 retinas expressing MW-opsin, RGC activity changed in response to changes in brightness by only 25% (Fig. 18a,b), approaching, but not equivalent to, the contrast sensitivity of the wild-type retina (Fig. 18c). In additional in vivo experiments on rd1 animals expressing MW-opsin in the RGC, the experiments included measuring unit responses and visually evoked potentials across layers of the primary visual cortex in awake, free-running animals (Fig. 19) and observing similar contrast sensitivity. using a standard computer monitor (FIG. 20). Cortical responses followed the flash frequency up to at least 4 Hz (Fig. 21). The sensitivity and kinetics of light responses mediated by MW opsin in the RGC suggest that it may support visually mediated behavior.

Fig. 3a-3g. Light response in the isolated retina of an rd1 mouse with MW-opsin in the RGC. (a) (Top) Average response to a 1 second flash of light of a population of RGCs expressing MW-opsin (green) or rhodopsin (blue) in the rd1 mouse retinal RGC. (Bottom) Raster mean RGC response plot of mouse rd1 retina to 5 1 s flashes of light at 535 nm for MW-opsin (n=88 cells) and 510 nm for rhodopsin (n=54 cells) expressed in RGC. (b, c) Light sensitivity for MW-opsin (N=2 retinas, n=127 cells) and rhodopsin (N=2 retinas, n=95 cells) in rd1 mouse retinal RGC. The peak pulsation frequency normalizes to a maximum at maximum intensity. (d, e) Response time to light. Population averages are traced over time from light appearance to maximum excitation (time to peak: 355 + 21 ms), exponential fits for excitation phase (Tau ON: 112 + 25 ms) and decay (Tau OFF: 260 + 31 ms) and full width at half maximum (FWHM: 183 + 85 ms) (e) for MW-opsin (d; e, green; N = 3 retinas, n = 95 cells) and rhodopsin (e, blue; N = 2 retinas, n = 67 cells). (f, g) Dependence of the light response of the MW opsin on the flash duration. (f) Representative light response of the retina (n = 117 cells): mean pulsation frequency across the population (top) and unit response raster plot (bottom). (h) Normalized peak responses for various durations of stimulation (N=2 retinas, n=183 cells). Light intensity 3.82×10 ^-1 mW cm ^-2 , unless otherwise indicated, wavelength: λ = 535 nm (MW-opsin) or 510 nm (rhodopsin). N = number of retinas, n = number of cells/units. All cells belong to sorted units. Values mean +SOS. Statistical significance was assessed using the Mann-Whitney U-test (*p<0.001).

Fig. 7. Threshold response of optogenetic systems in blind retinas compared to normal vision. Comparison of the threshold sensitivity of natural vision (top) with the sensitivity of various optogenetic systems introduced into a specific set of surviving neurons in the retina after photoreceptor cell degeneration (bottom). Mammalian MW-opsin (green), rhodopsin (blue), and melanopsin (cyan) are ~1000 times more sensitive than microbial channel rhodopsin2 and halorhodopsin. Threshold for alternative optogenetic approaches from Bi et al. (2006)⁸, Zhang et al (2009)¹², Sengupta et al. (2016)¹³, Doroudchi et al. (2011)⁴⁵, Gaub et al. (2014)⁴⁷, Cronin et al (2014)⁶⁵, Berry et al. (2017)⁶⁶ De Silva et al. (2017)¹⁷. (8) Bi et al. (2006) Canalorhodopsin expressed with AAV2-CAG promoter stimulated at 460 nm in rd1 mouse with a detection limit in RGC = 2.2 × 10¹⁵photons cm⁻² With⁻¹ ~1 mW cm^-2. (12) Zhang et al (2009) Halorhodopsin expressed with the AAV2-CMV promoter stimulated at 555 – 575 nm with an rd1 bandwidth in an rd1 mouse with a detection limit in RGC = 5.8 × 10^sixteen photons cm⁻² With⁻¹ ~20 mW cm^-2. (13) Sengupta et al. (2016) - Redshifted channelorhodopsin expressed with AAV2-hSyn promoter stimulated at 595nm in mouse rd1 with sensitivity limit in RGC = 2.5 × 10¹⁵photons cm⁻² With⁻¹~1 mW cm^-2. (45) Doroudchi et al. (2011) Channel rhodopsin expressed with the AAV8-Y733F & SV-40 promoter was stimulated at 450–490 nm with a bandwidth in the rd10 mouse with a sensitivity limit in ON-BC = 4 × 10^sixteenphotons cm⁻² With⁻¹~ 17 mW cm^-2. (47) Gaub et al. (2014) LiGluR-MAG₄₆₀ expressed with promoter AAV2-hSyn and AAV2-4xGrm6 stimulated at 445/20 nm in rd1 mice with a detection limit in RGC and ON-BC = 7.1 x 10^fourteenphotons cm⁻² With⁻¹ ~0.3 mW cm^-2. (65) Cronin et al (2014) Canalorhodopsin expressed with the AAV2/8BP2 promoter and 4xGRM6 was stimulated in rd1 mice with a sensitivity limit in ON-BC = 1 × 10^sixteenphotons cm⁻² With⁻¹ ~5 mW cm^-2. (66) Berry et al. (2017) SNAG-MGluR2 expressed with the AAV2-hSyn promoter was stimulated at 445/50 nm in rd1 mice with a detection limit in RGC = 1 × 10^fourteenphotons cm⁻² With⁻¹ ~0.5 mW cm^-2.

(17) De Silva et al. (2017) expressed with AAV2/8 (Y733F) and CMV enhancer CMV/CBA promoter stimulated at 480/20 nm in an rd1 mouse with a melanopsin sensitivity limit of RGC = 1.20 × 10 ¹² photons cm– ² s– ¹ ~0.5 μW cm ^-2 .

Fig. 8a-8e. Intensity-dependent kinetics of the light reaction of MW-opsin. (a-c) Dependence of light intensity (in seconds) on Tau ON (a), Tau OFF (b) and time to peak (T-peak) (c) in rd1 retina expressing MW-opsin, n = 111 cells, N = 3 retinas. All cells belong to sorted units. Values are average, errors are SOS. Wavelength: λ = 535 nm. (d, e) Average population response of RGCs with COS in gray (top) and averaged raster plot over 5 1 second flashes of light (bottom) for rd1 expressing MW-opsin (d; λ = 510 nm; n = 117 cells ) and wt (e, white light; n = 50 cells).

Fig. 18a-18c. Contrast detection in isolated rd1 mouse retina expressing MW-opsin. MEA registration of responses to steps from a dark to full tone scale of varying intensity on the example of an isolated retina from an MW-opsin-expressing mouse rd1 (on average, 14 channels) (a). (b, c) Normalized change in RGC pulsation rate in response to contrast changes for rd1 mice expressing MW-opsin (N = 2 retinas) (b) and wt mice (N = 2 retinas) (c) 100% light = 25 μW cm ^-2 .

Fig. 19a-19e. Light responses in vivo in V1 mouse rd1 expressing MW-opsin. (a) Schematic representation of a mouse with a fixed head on a running wheel. Stimuli are displayed on a standard computer monitor located within the field of view of one eye. (b) Response to a 500 ms light pulse. Top, representative visually evoked potential from V1 layer 4 (mean 20 responses, shaded area represents mean ± SOS). Below, a heat map of individual unit responses from all 16 electrodes of the linear electrode array across the entire depth of the visual cortex (mean 20 responses). (c) Scatterplot of light-induced dependence on baseline pulsation frequency for 39 units in 3 rd1 mice expressing MW-opsin. (d) PSTH (50 ms interval) of a typical device in response to a flash of light. (e) Plot of percentage change in neuronal firing after stimulation at 39 units in 3 mice.

Fig. 20a-20d. In vivo contrast detection in MW-opsin expressing mouse rd1. (a) Illustration of visual cortex recordings from an awake free-running rd1 mouse expressing MW-opsin. Randomized contrast changes are presented on a computer monitor placed in the field of view of the animals. (b) Average pulsation frequency in response to full contrast steps (500 ms duration) recorded in layer 4 of V1. The inset shows PSTH emitted from 20 ms. 100% light = 115 µW cm ^-2 .

Fig. 21a-21c. Temporal properties of V1 responses in vivo in rd1 mice expressing MW opsin. (a-c) Changes in neuronal firing in the visual cortex evoked by flickering full-field stimuli at 1, 2, and 4 Hz for 100 cycles.

MW opsin restores innate light avoidance

Observing that MW opsin elicits large light responses at low intensity in the highlighted rd1 retina, an intact animal was used to assess the ability of MW opsin to maintain visual behavior. Sighted mice innately avoid illuminated areas, a survival mechanism related to capture evasion ³⁵ . This behavior is lost after photoreceptor degeneration in the rd1 mouse model ^9,16 . To determine if this behavior could be restored, rd1 mice with MW-opsin in RGC were tested in a behavioral box consisting of adjacent light and dark compartments (Fig. 4a). The fraction of time spent in each compartment was recorded and compared with untreated rd1 mice and wt mice (Fig. 4, Fig. 9a-c, Fig. 11), as was the proportion of successful avoidance for each cohort (Fig. 9a-c and Fig.11, table 1). The light compartment was illuminated with low-intensity white light, which is equivalent to indoor lighting (100 μW/cm ^-2 ). While untreated rd1 animals cannot visually distinguish between compartments, they spend about 40-50% of their time in the dark compartment, consistent with the established bias in favor of the light compartment due to familiarity with the release site (see Methods ) (Fig. 4b). In contrast, rd1 mice expressing either rhodopsin or MW-opsin showed a strong preference for the dark compartment, similar to animals with normal vision (Fig. 4b and Fig. 9a). Then, using the same behavioral paradigm, white light was replaced with blue (460 + 22 nm) or green (534 + 25 nm) light, and the intensity was lowered to the lower threshold of the highlighted retina intensity-response curves for MW-opsin and rhodopsin ( 1 μW cm ^-2 Fig. 3b). Under green light conditions, animals expressing MW-opsin and rhodopsin exhibited light avoidance (Fig. 4c, left and Fig. 9b), however, under blue light, only animals expressing rhodopsin exhibited light avoidance (Fig. 4c, right and Fig. 9c). ). This manifestation of monochromatic sensitivity is consistent with the action spectrum of MW opsin ³⁶ .

Fig. 9a-9g. Light avoidance and learned pattern recognition behavior. (ac) Light compartment avoidance success rate (avoidance trial success rate) for rd1 expressing rhodopsin in RGC (blue; n=6 mice) or MW-opsin (green; n=5) and wt mice (white; n=5 mice) under illumination with either (a) white light (100 μW cm ^-2 ), (b) green light 25 μW cm ^-2 (535 nm) or (c) blue light 25 μW cm ^-2 (470 nm). Statistical significance was assessed using Pearson's two-tailed chi-square test and Fisher's exact one-tailed test when applicable (FIG. 11). Success is defined as greater than average avoidance + SD control untreated rd1 mice (see Methods). (d) Schematic of the pattern recognition experiment. Mice were adapted on day 1 and subjected to an electric shock in association with a particular type of light (stimulus A/B) randomly paired with any chamber on days 2 and 3 and tested (time spent in each chamber ) on the 4th day in the absence of electric shock with a change in the structure of the light to avoid location shift. (eg) Proportion of successful discrimination of the model in combination with electric shock (proportion of successful avoidance trials) compared to the untreated control group rd1. (e) Horizontal vs vertical parallel lines. Discrimination of parallel static (f) or moving (g) squares at a distance of 1 vs. 6 cm. Respectively for e, f and g: rd1 rhodopsin (blue; n = 8 (e), 6 (f) mice), rd1 MW-opsin (n = 17 (e), 11 (f), 6 (g) mice) and wt (n = 5 (e), 6 (f), 9 (g) mice). (25 μW cm ^-2 ). Statistical significance was assessed using Pearson's two-tailed chi-square test and Fisher's exact one-tailed test when applicable (FIG. 11). Success is defined as greater than average avoidance + SD compared to control untreated rd1 mice.

MW opsin supports detection of temporal light patterns

Noting that MW-opsin elicits faster light responses than rhodopsin in the isolated rd1 retina, the question was raised as to whether this would result in a superior ability to distinguish between different temporal patterns of light. A visually tuned fear formation paradigm was used to test the animals' ability to distinguish between flash and steady light. Wt mice or rd1 mice expressing either MW-opsin or rhodopsin in the RGC were placed in a single-compartment behavioral apparatus in which a low-intensity LCD screen (100 μW cm ^-2 ) switched between constant light and flashing light (2 Hz). In each animal, constant light or flashing light was constantly combined with a light shock to the foot. This was done for 1 trial per day for 2 days (Fig. 4d) and animals were tested on day 3 with light cues presented in the absence of foot shock, with freeze time used to measure fear association, as done previously ^{15 ,27,37} . The freezing time was measured within 10 seconds after switching the time light pattern. Measurements in animals in which the visual cue and the stomp were paired (paired group) were compared with measurements in a cohort of animals in which the training shocks were randomized (i.e. not matched sequentially with one of the stimuli) (unpaired group). Freezing time in untreated rd1 mice did not differ between paired and unpaired conditions, consistent with the animals' expected inability to see visual cues (Fig. 4e, grey). In contrast, rd1 mice expressing MW-opsin in the RGC showed greater freezing under paired conditions, as observed in animals (Fig. 4e, green and white). Strikingly, rd1 mice expressing rhodopsin in the RGC did not differ between paired and unpaired conditions (Fig. 4e, blue). This suggests that, unlike MW-conopsin-expressing blind mice, rhodopsin-expressing mice cannot distinguish between flashes of light at 2 Hz from constant light, consistent with slow light response kinetics observed in MEA (Fig. 3e) .

MW opsin restores spatial pattern recognition

It was then determined whether the MW opsin in RGC rd1 mice would allow detection of spatial light patterns. A behavioral chamber with two adjacent compartments (Fig. 4f), each with a low intensity LCD tablet (iPad) mounted on the wall, was used. Each tablet displayed a pair of parallel lines. In one case, the lines were oriented vertically (||), and in the other, horizontally (=). For MW-opsin, the wavelength was centered at 535 nm (520-560) and for rhodopsin at 497 nm (480-520) (Fig. 9d). First, the mice were given time to get used to the compartments with the visual displays turned off (day 1). During the two-day training period, foot shocks were combined with vertical or horizontal lines. The negative signal was randomly assigned and maintained consistently for this animal. On day 4, stimulus locations were switched to avoid location bias and conditional evasion was tested (Fig. 9d). It was found that rd1 animals with MW-opsin in the RGC showed avoidance of an unpleasant visual signal that was significantly higher than in untreated rd1 control animals and similar to that of wt mice (Fig. 4g and Fig. 9e). In contrast, rd1 animals with rhodopsin in the RGC did not differ from untreated rd1 control animals and showed a slight preference for an unfavorable stimulus, a prime example of the location bias seen in blind animals. These observations indicate that MW-opsin regains the ability to recognize spatial light patterns, while rhodopsin does not.

Noting that MW-opsin supports spatial pattern recognition, the question was asked whether mice could distinguish differences between lines of the same orientation but different distances, a visual task adopted from human and animal visual acuity tests ^38,39 . Parallel vertical lines were separated by 1 or 6 cm. As above, foot shock was randomly associated with one of the stimuli during the training period on days 2 and 3, and recall was tested on day 4 as an avoidance measure. MW-opsin-expressing rd1 mice were found to be able to distinguish between the two patterns with a performance preference for non-aversive stimuli that was similar to that observed in wt mice, while rhodopsin-expressing animals were similar to untreated rd1 mice (Fig. 4h). , Fig. 9f and Fig. 22). The MW opsin also maintained lineage differentiation when the parallel lines were in motion (1 cm/s) (FIGS. 4i and 9g).

The question has been asked whether the rate of "refresh" of the MW opsin is fast enough to support line pattern recognition when the pattern is in motion. To test this, the same problem of differentiating close lines was performed, but now with parallel lines moving at a speed of 5 cm per second. MW-opsin-expressing rd1 mice were found to show a preference for a moving pattern that was paired with an irreversible stimulus and worked in the same way as in wt mice (Fig. 4i and Fig. 9g). These results show that the MW opsin in the RGC supports the recognition of both static and moving spatial patterns at low LCD internal illumination intensity.

Fig. 4a-4i. Light avoidance and learned visually mediated behavior in rd1 mice expressing MW-opsin or rhodopsin in the RGC. (a) Light/dark box layout for the light avoidance test. (b, c) Proportion of time spent in the dark compartment (avoidance fraction) to control rd1 (grey; n = 4 mice), rd1 expressing rhodopsin in RGC (blue; n = 6 mice) or MW-opsin (green; n = 5) and wt mice (white; n = 5 mice) under illumination with either (b) white light (100 μW cm ^-2 ), (c) 25 μW cm ^-2 blue light (470 nm) (right) or green light (535 nm) (left). (d) Scheme of the experiment on the formation of fear of the freezing reaction. (e) Quantification of the fear response to stimulus discrimination in a temporal pattern. Freeze times above baseline are shown for cases where illumination transitions from static stimulation to 2 Hz (100 μW/cm ^-2 ) were paired or uncoupled with electrical current for rd1 control mice, mice expressing rhodopsin, MW-opsin, and wt ( n = 4, 6, 12, 10 paired, n = 7, 8, 7, 12 unpaired). (f) Schematic of the pattern recognition experiment. The mice adapted during day 1 and then were given an electric shock in connection with a specific pattern of light projected onto the ipad and randomly combined in any chamber (adaptation days 2 and 3). On day 4, recall tests (time spent in each chamber) were performed in the absence of an electric shock with a change in light pattern to avoid location bias (see Fig. 8d). (gi) Learned pattern recognition. The time spent avoiding the pattern in combination with the electric shock. (g) Horizontal and vertical parallel stripes. (h) Discrimination of parallel static (h) or moving (i) bands at a distance of 1 vs. 6 cm. For g, h, and i, respectively: control rd1 (n = 8,5,16 mice), rd1 rhodopsin (blue; n = 8 mice), rd1 MW-opsin (n = 17.11.6 mice) and wt (n = 5.6.9 mice). (25 μW cm ^-2 ). (Note the success rates for these experiments are shown in Figures 9a-c). Light intensity = 25-100 μW cm ^-2 ; Wavelength: = 535 nm (MW-opsin), 510 nm (rhodopsin) or white light (MW-opsin). n = number of mice. Statistical significance was assessed using two-tailed Student's t-test with Bonferroni correction: *p <0.01.

Fig. 22a-22b. Location of preference in the discrimination problem. (a) Illustration of an arena used in recognition tasks, showing a virtual divider dividing each compartment in half to determine the ratio of time spent near the central divider between the two cameras to the screen. (b) The ratio of time spent in the area closest to the center divider to the area closest to the screen for negative and non-avversive sides indicates the preference for the area closest to the center divider. Values are averages; errors are SOS.

MW opsin undergoes adaptation to light

A fundamental characteristic of vision is the ability to distinguish objects in a wide range of ambient light intensities. This adaptation is mediated by several different mechanisms in photoreceptor cells. The question has been raised as to whether any aspect of adaptation will pass into the RGC with the MW opsin. Retinas expressing MW-opsin in the RGC were excised, mounted on MEA, and perfused with 9cis-retinal. The retina was first kept in complete darkness for 15 min (dark-adapted); then a series of short (1 s) flashes of green light (535 + 25 nm) was carried out at large intervals (60 s) and in a range of intensities. The light was then adapted for 10 minutes to a moderate level of illumination in the room (adapted to light; white light at 100 μW/cm ^-2 ) and the series of light pulses was repeated.

The kinetics of light responses was investigated. The light response decayed rapidly, as shown above, for both the light and dark-adapted retinas, except for one case: the response to a bright flash in the dark-adapted retina decayed about 12 times slower (3.14 + 0.63 s , n = 171, N = 3) than in the same retina under light adaptation conditions (Fig. 5a and Fig. 10a, b). Light responses elicited by intact photoreceptors show a similar behavior and this is believed to play a role in desensitization to subsequent stimulation ^40,41 and underlies the "afterimage" in humans after a bright flash that temporarily obscures vision ⁴² . Due to its unusual properties, this response of the dark-adapted retina to the brightest flash of light was excluded from the analysis of the dependence of the response on intensity.

The intensity response curve showed that dark-adapted retinas were highly light-sensitive, responding at ~0.5 μW/cm ^-2 , while light-adapted retinas were much less sensitive, requiring ~100 μW/cm ^-2 for response ( Fig. 5b, c and 10c, d). This adaptation shifted the intensity curve by ~3 orders of magnitude (780 + 82), N = 3) (Fig. 5b,c). A striking feature of adaptation was that the maximum light response was similar in dark-adapted and light-adapted retinas (Fig. 5c), as shown previously in native cone photoreceptors ^43,44 .

By observing that the light response mediated by MW-opsin in the RGC undergoes light adaptation in the highlighted retina, it was asked whether this could lead to visually beneficial light adaptation in a behavioral animal. The MW-opsin-mediated light response in RGC has been investigated in the context of light avoidance behavior. Prior to testing, rd1 mice expressing MW-opsin in the RGC were kept for 1 hour either in total darkness (dark adaptation) or under indoor lighting (white light, 1 mW cm ^-2 /535 nm, light component, 50 μW cm ^-2 ) (Fig. 5d). Animals were then immediately tested in a two-chamber light-dark box for light avoidance behavior when the box was illuminated with green light (535 nm) at 1 μW cm ^-2 (internal light) or 100 μW cm ^-2 (external light). Light-adapted MW-opsin-expressing rd1 mice exhibited strong light avoidance when the test light was brighter (535 nm, 100 μW cm ⁻² ) than the light level to which they were light adapted (FIG. 5e). Light avoidance was reduced when the test light was dimmer (535 nm, 1 μW cm ^-2 ) than the light level to which the mice were adapted (Fig. 5e and Fig. 10e). In contrast, a dim test light of 1 μW/cm ^-2 produces a high level of light avoidance in dark-adapted animals, indicating behavioral adaptation to light.

The question was asked whether the light adaptation effect associated with light avoidance behavior would also operate for pattern recognition in a visual image recognition task. As indicated above (Fig. 4), mice were adapted for a 3-day training period by combining light foot shock with one of two parallel line displays at different intervals presented on LCD screens in two adjacent chambers (Figs. 4h and 9d ). Once again, mice were tested with visual stimulus only on day 4, but this time, immediately prior to testing, they were subjected to 1 hour of either dark adaptation (no light) or light adaptation (white light) for 4 or 8 hours. The intensity of the line patterns during recall was either 0.25 µW cm ^-2 or 10 µW cm ^-2 after light adaptation. It was found that rd1 mice expressing MW-opsin in the RGC that were dark adapted were able to distinguish between line patterns and avoid an unpleasant signal, whether presented at low (0.25 μW cm ^-2 ) or moderate (10 μW cm ^-2 ) light intensity in the room (Fig. 5f and Fig. 10f). In contrast, light-adapted animals only succeeded with the brighter samples of the test lines and were identical in performance in groups that were light-adapted for 1, 4, and 8 hours (FIGS. 5f and 10g). The results show that MW-opsin-mediated spatial pattern recognition is adaptive over a range of natural light intensities.

MW-opsin restores a new object of study

The above experiments show that MW-opsin provides pattern recognition over a wide range of light intensities using illuminated displays. The question was how would it work in a natural environment where ambient random light illuminates 3D objects. To overcome this problem, the experiments used the open field arena, which is commonly used to test novel object recognition and exploratory behavior ^62,63 . Mice naturally avoid open spaces and maintain closeness to the walls of their environment. Exploratory excursions from these safe places can be motivated by new incentives. Although mice use several sensory modalities during research, vision has been shown to be critical for spatial navigation ⁶⁴ . The arena consisted of a cube containing two different new objects. The mouse was positioned against the wall of the arena, far enough away from objects that were themselves far enough apart that there was little chance of an accidental collision, whether the animal was walking along the wall or exploring another object. Raw rd1, rd1 sham intraocular injection, rd1 expressing rhodopsin or rd1 mice expressing MW-opsin, and wt animals were videotaped. Their movements were tracked for 10 minutes the first time they were placed in the arena (FIGS. 13a-d). Animals were found to travel 1.57 times farther and move at 1.55 times faster environmental speeds than blind rd1 animals, consistent with the known visual component of exploratory behavior. Strikingly, like wt animals, MW-opsin-expressing rd1 animals traveled further (1.39-fold) and faster (1.37-fold) than their untreated rd1 littermates (Fig. 13e, f). assuming that the MW-opsin keeps the new object of study normal. To analyze this further, the experiments focused on aspects of exploratory behavior most likely to depend on distance vision; namely, the delay in exploring new objects, and the speed and distance traveled on excursions to objects. Mice given sham intraocular injection and rd1 mice expressing rhodopsin behaved similarly to untreated rd1 animals, but mice expressing MW-opsin reached the first and second targets 3.88 and 3.62 times faster, respectively (Fig. 13g, h), moving at speeds that were 2.1 times and 1.83 times higher towards the first and second objects, respectively (Fig. 13i, j), and took shorter paths that were at 0, 69 times and 0.64 times respectively shorter to the first and second objects (Fig. 13k, l) compared to untreated rd1 mice. In each of these measures, mice expressing MW-opsin rd1 reached levels that were similar to those in wt animals (Fig. 13e-g). These results indicate that the MW opsin in the RGC provides previously blind animals with naturalistic vision of objects in ambient light.

Fig. 5a-5f. Light adaptation in RGC activity and MW-opsin-mediated visual-mediated behavior. (ac) MEA recordings in isolated retina of the MW-opsin-mediated RGC light response in the rd1 mouse retinal RGC show a difference in sensitivity with dark and light adapted retinas. (a) Light response attenuation (Tau OFF) as a function of flash intensity under dark and light adaptation conditions (N = 3 retinas, n = 171 cells). (b) An example intensity response curve for a representative retina first dark-adapted (filled symbols) and then light-adapted (open symbols) (n = 57 cells). adaptation to white light. The minimum ChR2 value from Bi et al. (2006) ⁸ & Sengupta et al. (2016) ¹³ . (c) Mean (error bars - SOS) normalized light response index (LRI) at 3 flash intensities in the same retina first dark adapted and then light adapted (N = 3 retinas, n = 171 cells). (df) Behavior shows light adaptation in visually mediated tasks. (d) Schematic of adaptation to darkness or light prior to testing of innate avoidance behavior or learned pattern recognition behavior. (e) Proportion of time spent in the dark compartment (avoidance fraction) under outdoor light (100 μW cm ^-2 ) or indoor light (1 μW cm ^-2 ) during 1 hour of dark adaptation (n = 11 mice) or adaptation to light (white light; spectral component 1 mW cm ^-2 /535 nm; 50 μW cm ^-2 ; n = 12.13 mice). (f) Learned pattern recognition of parallel stripes spaced 1 vs 6 cm displayed at low (0.25 µW cm ^-2 ) or indoor (10 µW cm ^-2 ) light levels for 1 hour of dark adaptation (n = 8.8 mice) or light (white light; spectral component 1 mW cm ^-2 /535 nm; 50 μW cm ^-2 ; n = 7.7 mice). The dotted line represents the average performance of untreated rd1 control mice. Light intensity 3.82×10 ^-1 mW cm ^-2 , wavelength: λ = 535 nm. All cells belong to sorted units. Values mean +SOS. Statistical significance was assessed using the Mann-Whitney U-test (*p<0.01). Two-tailed Student's t-test with Bonferroni correction: * p<0.05.

Fig. 10a-10g. Adaptation to light upon removal of the retina and visual behavior of the mouse. (ad) Representative trace of the average response of the RGC population in the retina expressing MW-opsin (n = 57). Response to light flashes 500 ms 3.82×10 ¹ mW/cm ^-2 (a, b) or 3.82×10 ^-3 mW cm ^-2 (c, d) after adaptation to darkness (a, c) or light (b, d). (e) Light compartment avoidance (proportion of successful avoidance trials) under indoor light (1 μW cm ^-2 ) during 1 hour of dark adaptation (n = 11 mice) compared to the proportion of mice with successful avoidance under indoor light ( 1 μW cm ^-2 ; n = 12 mice) and outdoor illumination (100 μW cm ^-2 ; n = 13 mice) for 1 hour of light adaptation (white light; spectral component 1 mW cm ^-2 /535 nm; 50 μW cm ^-2 ). (f) Recognition success rate of 1 vs 6 cm parallel bands displayed at low (0.25 µW cm ^-2 ) or indoor (10 µW cm ^-2 ) light levels after 1 hour, dark adaptation (n = 8 mice at 0.25 μW cm ^-2 , 8 mice at 10 μW cm ^-2 ) or light (n = 7 mice at 0.25 μW cm ^-2 , 7 mice at 10 μW cm ^-2 ). (g) Scientific recognition of patterns of parallel stripes spaced 1 vs. 6 cm displayed under indoor light (10 μW cm ^-2 ) after 1, 4, or 8 hours of light adaptation (n = 7,4,4) vs. with non-adapted rd1 controls.

Fig. 11 (table 1). Statistical significance of light avoidance and learned visually mediated behavior. Success rates were calculated for behavioral avoidance (FIGS. 4, 5 and FIGS. 9, 10). To determine the significance of differences between conditions, a pairwise contingency table was constructed and Pearson's two-tailed chi-square test was initially performed. To correct for the small n conditions, Fisher's exact one-tailed test was also performed.

Links

1. Tuo, J., Bojanowski, C.M. & Chan, C.-C. Genetic factors of age-related macular degeneration. Progress in retinal and eye research 23, 229-249 (2004).

2. Leveillard, T. & Sahel, J.A. Rod-derived cone viability factor for treating blinding diseases: from clinic to redox signaling. Sci Transl Med 2, 26ps16 (2010).

3. Bennett, J. Taking Stock of Retinal Gene Therapy: Looking Back and Moving Forward. Molecular Therapy 25, 1076-1094.

4. Daiger, S.P., Bowne, S.J. & Sullivan, L.S. Perspective on genes and mutations causing retinitis pigmentosa. Arch Ophthalmol 125, 151-158 (2007).

5. Ferrari, S., et al. Retinitis pigmentosa: genes and disease mechanisms. Curr Genomics 12, 238-249 (2011).

6. Mazzoni, F., Novelli, E. & Strettoi, E. Retinal ganglion cells survive and maintain normal dendritic morphology in a mouse model of inherited photoreceptor degeneration. J Neurosci 28, 14282-14292 (2008).

7. Haverkamp, S., et al. Synaptic plasticity in CNGA3(-/-) mice: cone bipolar cells react on the missing cone input and form ectopic synapses with rods. J Neurosci 26, 5248-5255 (2006).

8 Bi, A., et al. Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration. Neuron 50, 23-33 (2006).

9. Busskamp, V., et al. Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa. Science 329, 413-417 (2010).

10. Lagali, P.S., et al. Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Nat Neurosci 11, 667-675 (2008).

11. Thyagarajan, S., et al. Visual function in mice with photoreceptor degeneration and transgenic expression of channelrhodopsin 2 in ganglion cells. J Neurosci 30, 8745-8758 (2010).

12. Zhang, Y., Ivanova, E., Bi, A. & Pan, Z.H. Ectopic expression of multiple microbial rhodopsins restores ON and OFF light responses in retinas with photoreceptor degeneration. J Neurosci 29, 9186-9196 (2009).

13. Sengupta, A., et al. Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina. EMBO Molecular Medicine 8, 1248-1264 (2016).

14. Cehajic-Kapetanovic, J., et al. Restoration of Vision with Ectopic Expression of Human Rod Opsin. Current biology : CB 25, 2111-2122 (2015).

15. Gaub, B.M., Berry, M.H., Holt, A.E., Isacoff, E.Y. & Flannery, J.G. Optogenetic Vision Restoration Using Rhodopsin for Enhanced Sensitivity. Mol Ther 23, 1562-1571 (2015).

16. Lin, B., Koizumi, A., Tanaka, N., Panda, S. & Masland, R.H. Restoration of visual function in retinal degeneration mice by ectopic expression of melanopsin. Proceedings of the National Academy of Sciences of the United States of America 105, 16009-16014 (2008).

17. De Silva, S.R., et al. Long-term restoration of visual function in end-stage retinal degeneration using subretinal human melanopsin gene therapy. Proc Natl Acad Sci U S A (2017).

18. Cepko, C. Neuroscience. Seeing the light of day. Science 329, 403-404 (2010).

19. Klapper, S.D., Swiersy, A., Bamberg, E. & Busskamp, V. Biophysical Properties of Optogenetic Tools and Their Application for Vision Restoration Approaches. Front Syst Neurosci 10, 74 (2016).

20. The Lasker, I.I.f.I.i.V.S. Restoring Vision to the Blind: The Lasker/IRRF Initiative for Innovation in Vision Science. Transl Vis Sci Technol 3, 1 (2014).

21. Vinores, S.A., et al. Blood-retinal barrier breakdown in retinitis pigmentosa: light and electron microscopic immunolocalization. Histol Histopathol 10, 913-923 (1995).

22. Kawamura, S. & Tachibanaki, S. Rod and cone photoreceptors: molecular basis of the difference in their physiology. Comp Biochem Physiol A Mol Integr Physiol 150, 369-377 (2008).

23. Korenbrot, J.I. Speed, sensitivity, and stability of the light response in rod and cone photoreceptors: facts and models. Prog Retin Eye Res 31, 442-466 (2012).

24 Masseck, O.A., et al. Vertebrate cone opsins enable sustained and highly sensitive rapid control of Gi/o signaling in anxiety circuitry. Neuron 81, 1263-1273 (2014).

25. Gutierrez, D.V., et al. Optogenetic control of motor coordination by Gi/o protein-coupled vertebrate rhodopsin in cerebellar Purkinje cells. J Biol Chem 286, 25848-25858 (2011).

26. Levitz, J., et al. Optical control of metabotropic glutamate receptors. Nat Neurosci 16, 507-516 (2013).

27. Friedmann, D., Hoagland, A., Berlin, S. & Isacoff, E.Y. A spinal opsin controls early neural activity and drives a behavioral light response. Current biology: CB 25, 69-74 (2015).

28. Fischer, R.M., et al. Co-expression of VAL- and TMT-opsins uncovers ancient photosensory interneurons and motorneurons in the vertebrate brain. PLoS Biol 11, e1001585 (2013).

29 Liegertova, M., et al. Corrigendum: Cubozoan genome illuminates functional diversification of opsins and photoreceptor evolution. Sci Rep 5, 14396 (2015).

30. Chen, M.H., Kuemmel, C., Birge, R.R. & Knox, B.E. Rapid release of retinal from a cone visual pigment following photoactivation. Biochemistry 51, 4117-4125 (2012).

31. Mark, M.D. & Herlitze, S. G-protein mediated gating of inward-rectifier K+ channels. Eur J Biochem 267, 5830-5836 (2000).

32. Shevtsova, Z., Malik, J.M., Michel, U., Bahr, M. & Kugler, S. Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo . Exp Physiol 90, 53-59 (2005).

33. Sancho-Pelluz, J., et al. Photoreceptor cell death mechanisms in inherited retinal degeneration. Molecular neurobiology 38, 253-269 (2008).

34 Schwartz, E.A. Responses of bipolar cells in the retina of the turtle. The Journal of Physiology 236, 211-224 (1974).

35. Bourin, M. & Hascoet, M. The mouse light/dark box test. European journal of pharmacology 463, 55-65 (2003).

36 Merbs, S.L. & Nathans, J. Photobleaching difference absorption spectra of human cone pigments: quantitative analysis and comparison to other methods. Photochem Photobiol 56, 869-881 (1992).

37. Tochitsky, I., et al. Restoring visual function to blind mice with a photoswitch that exploits electrophysiological remodeling of retinal ganglion cells. Neuron 81, 800-813 (2014).

38. Bittner, A.K., Jeter, P. & Dagnelie, G. Grating Acuity and Contrast Tests for Clinical Trials of Severe Vision Loss. Optometry and vision science : official publication of the American Academy of Optometry 88, 1153-1163 (2011).

39. Wong, A.A. & Brown, R.E. Visual detection, pattern discrimination and visual acuity in 14 strains of mice. Genes, Brain and Behavior 5, 389-403 (2006).

40. Pepperberg, D.R. Bleaching desensitization: background and current challenges. Vision Res 43, 3011-3019 (2003).

41. Kang Derwent, J.J., Qtaishat, N.M. & Pepperberg, D.R. Excitation and desensitization of mouse rod photoreceptors in vivo following bright adapting light. The Journal of Physiology 541, 201-218 (2002).

42. Di Lollo, V., Clark, C.D. & Hogben, J.H. Separating visible persistence from retinal afterimages. Percept Psychophys 44, 363-368 (1988).

43. Hurley, J.B. Shedding light on adaptation. J Gen Physiol 119, 125-128 (2002).

44 Normann, R.A. & Werblin, F.S. Control of retinal sensitivity. I. Light and dark adaptation of vertebrate rods and cones. J Gen Physiol 63, 37-61 (1974).

45. Doroudchi, M.M., et al. Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness. Mol Ther 19, 1220-1229 (2011).

46. Caporale, N., et al. LiGluR restores visual responses in rodent models of inherited blindness. Mol Ther 19, 1212-1219 (2011).

47 Gaub, B.M., et al. Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells. Proc Natl Acad Sci U S A 111, E5574-5583 (2014).

48. Sommer, M.E., Hofmann, K.P. & Heck, M. Distinct loops in arrestin differentially regulate ligand binding within the GPCR opsin. Nat Commun 3, 995 (2012).

49. Polosukhina, A., et al. Photochemical restoration of visual responses in blind mice. Neuron 75, 271-282 (2012).

50 Holladay, J.T. Proper method for calculating average visual acuity. Journal of refractive surgery (Thorofare, N.J.: 1995) 13, 388-391 (1997).

51. Broichhagen, J., et al. Orthogonal Optical Control of a G Protein-Coupled Receptor with a SNAP-Tethered Photochromic Ligand. ACS Cent Sci 1, 383-393 (2015).

52. Levitz, J., Broichhagen, J., Leippe, P., Konrad, D., Trauner, D. and Isacoff, E.Y. Dual optical control and mechanistic insights into photoswitchable group II and III metabotropic glutamate receptors. Proc Natl Acad Sci U S A (2017).

53. Roska, B., Molnar, A. & Werblin, F.S. Parallel processing in retinal ganglion cells: how integration of space-time patterns of excitation and inhibition form the spiking output. Journal of neurophysiology 95, 3810-3822 (2006).

54. Leinonen H, Tanila H. Vision in laboratory rodents-Tools to measure it and implications for behavioral research. Behav Brain Res, (2017).

55. Prusky GT, West PW, Douglas RM. Behavioral assessment of visual acuity in mice and rats. Vision Res 40, 2201-2209 (2000).

56 Shi C, et al. Optimization of Optomotor Response-based Visual Function Assessment in Mice. Sci Rep 8, 9708 (2018).

57. Berson DM, Dunn FA, Takao M. Phototransduction by retinal ganglion cells that set the circadian clock. Science 295, 1070-1073 (2002).

58. Veit J, Hakim R, Jadi MP, Sejnowski TJ, Adesnik H. Cortical gamma band synchronization through somatostatin interneurons. Nat Neurosci 20, 951-959 (2017).

59. Brainard DH. The psychophysics toolbox. Spatial Vision 10, 433-436 (1997).

60. Hill DN, Mehta SB, Kleinfeld D. Quality Metrics to Accompany Spike Sorting of Extracellular Signals. Journal of Neuroscience 31, 8699-8705 (2011).

61 Berry M, et al. Restoration of Patterned Vision with an Engineered Photo-Activatable G Protein-Coupled Receptor. Nat Commun, (2017).

62. Antunes M, Biala G. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cognitive Processing 13, 93-110 (2012).

63. Christmas AJ, Maxwell DR. A comparison of the effects of some benzodiazepines and other drugs on aggressive and exploratory behavior in mice and rats. Neuropharmacology 9, 17-29 (1970).

64. Buhot MC, Dubayle D, Malleret G, Javerzat S, Segu L. Exploration, anxiety, and spatial memory in transgenic anophthalmic mice. Behavioral neuroscience 115, 455-467 (2001).

65 Cronin T, et al. Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter. EMBO Molecular Medicine 6, 1175-1190 (2014).

66 Berry MH, et al. Restoration of patterned vision with an engineered photoactivatable G protein-coupled receptor. Nat Commun 8, 1862 (2017).

While the present invention has been described with reference to specific embodiments thereof, those skilled in the art will appreciate that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, method, method step or steps to the purpose, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims (24)

1. A method of restoring or improving visual function in an individual having retinal degeneration, the method comprising administering to the individual at least one recombinant expression vector comprising one or more of: i) a nucleotide sequence encoding a medium wavelength opsin (MW-opsin); ii) a nucleotide sequence encoding a long wavelength opsin (LW-opsin); and iii) a nucleotide sequence encoding a short wavelength opsin (SW opsin), wherein the nucleotide sequence encoding the SW opsin, the nucleotide sequence encoding the MW opsin, or the nucleotide sequence encoding the LW opsin is operably linked to a promoter other than the opsin promoter. , and is expressed in a retinal cell of an individual, wherein said administration provides restoration or improvement of visual function in an individual by expressing said nucleic acid in retinal cells of that individual, where the retinal cell is a retinal ganglion cell, amacrine cell, horizontal cell, or bipolar cell. 2. The method according to p. 1, in which the MW-opsin contains an amino acid sequence identical to at least 85% of the amino acid sequence shown in SEQ ID NO: 1; wherein the LW opsin contains an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 4, and where the SW opsin contains an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: : five. 3. The method of claim 1, wherein the recombinant expression vector contains a nucleotide sequence encoding a SW opsin, wherein the SW opsin contains an amino acid sequence that is at least 87% identical to the human SW opsin amino acid sequence shown in SEQ ID NO : 5, or at least 87% identical to the amino acid sequence of the mouse SW opsin shown in SEQ ID NO: 6. 4. The method according to any one of paragraphs. 1-3, wherein expression of MW opsin and/or LW opsin and/or SW opsin in a retinal cell provides structured vision and image recognition to an individual. 5. The method according to claim 4, wherein the image to be recognized is: a) a static image or pattern, or b) a moving image or pattern. 6. The method according to any one of paragraphs. 1-5, in which the expression of MW-opsin and/or LW-opsin and/or SW-opsin in the retinal cell provides image recognition at light intensity from 10 ^-4 W/cm ² to 1 W/cm ² . 7. The method according to any one of paragraphs. 1-6, in which the expression of MW-opsin and/or LW-opsin and/or SW-opsin in the retinal cell provides image recognition at a light intensity that is at least 10 times lower than the light intensity required to provide image recognition by an individual expressing the channel rhodopsin polypeptide in a retinal cell. 8. The method according to any one of paragraphs. 1-7, in which the expression of MW-opsin and/or LW-opsin and/or SW-opsin in the retinal cell provides kinetics that is at least 2 times faster than the kinetics in the retinal cell caused by the rhodopsin polypeptide. 9. The method according to any one of paragraphs. 1-8, wherein the recombinant expression vector is a recombinant viral vector. 10. The method of claim 9, wherein said recombinant viral vector is an adeno-associated viral vector, a lentiviral vector, a herpes simplex vector, or a retroviral vector. 11. The method according to any one of paragraphs. 1-10, wherein the promoter is synapsin promoter, CAG promoter, CMV promoter, grm6 promoter, Pleiades promoter, ChAT promoter, V-glut promoter, GAD promoter, PV promoter, somatostatin (SST) promoter, neuropeptide Y (NPY) promoter , a VIP promoter, a rhodopsin kinase promoter, a vitelliform macular degeneration 2 (VMD2) gene promoter, or an interphotoreceptor retinol-binding protein (IRBP) gene promoter. 12. The method according to any one of paragraphs. 1-11, wherein said administration is by intraocular injection, intravitreal injection, subretinal injection, or by periocular, intraocular, intravitreal, subconjunctival, retrobulbar, scleral, subretinal, or intercameral route of administration. 13. The method according to any one of paragraphs. 1-12, wherein the subject has an ocular disease selected from retinitis pigmentosa, macular degeneration, retinoschisis, and Leber's congenital amaurosis, and diabetic retinopathy, or wherein the subject has had retinal detachment or photoreceptor loss due to trauma or injury to the head. 14. The method according to any one of paragraphs. 1-13, in which at least one recombinant expression vector forms a complex with a nanoparticle. 15. The method of claim 1, wherein the retinal cell is a retinal ganglion cell. 16. The method of claim. 1, characterized in that at least one recombinant expression vector is a recombinant adeno-associated viral vector. 17. The method of claim. 1, characterized in that the individual has retinitis pigmentosa. 18. The method of claim 16 wherein the adeno-associated viral vector is AAV2. 19. The method according to claim 1, characterized in that said administration is carried out by the periocular, intraocular, intravitreal, subconjunctival, retrobulbar, scleral, subretinal or intercameral route of administration. 20. The method of claim 1, wherein the promoter is a synapsin promoter. 21. The method of claim 1 wherein the promoter is a CAG promoter. 22. The method of claim 1 wherein the promoter is the V-glut promoter.

Images