Melanopsin variants for vision restoration

Abstract

In this specification, there are provided, inter alia, melanopsin variants that exhibit greater amplitude / conductance and / or faster off-kinetics than wild-type human melanopsin. Also provided are related nucleic acids, virions, host cells, methods for producing recombinant virions, and pharmaceutical compositions. Further provided is a method for restoring or enhancing visual function in a subject using such melanopsin variants.

Description

【Technical Field】 【0001】 Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 349,970, filed on June 7, 2022, U.S. Provisional Application No. 63 / 411,523, filed on September 29, 2022, and U.S. Provisional Application No. 63 / 466,181, filed on May 12, 2023, the contents of each of which are hereby incorporated by reference in their entirety. 【0002】 Reference to Electronic Sequence Listing The contents of the electronic sequence listing (627002001440SEQLIST.xml, size: 162,974 bytes, and creation date: June 5, 2023) are hereby incorporated by reference in their entirety. 【0003】 This application relates to melanopsin variants and their use for restoring or enhancing visual function in a subject whose vision has been reduced due to photoreceptor loss. 【Background Art】 【0004】 Degeneration of photoreceptors in the human retina, such as rods and cones (e.g., due to disease, infection, or injury), typically results in severe visual impairment and, in some cases, legal blindness for millions of people worldwide (McClements et al. (2020) Front Neurosci. 14:57090). However, while the causes of retinal degeneration can be diverse, there are significant similarities in the physiological changes that occur in the retina. When patients suffer from the loss of photoreceptor cells but the remaining cell layers are maintained in the neural retina, there is a possibility of restoring vision by optogenetic therapy, i.e., providing light-sensitive molecules that enable light perception through the remaining neurons to the surviving cell types in the retina. Current optogenetic approaches are limited by low light sensitivity, slow kinetics, and / or narrow spectral responses. Furthermore, current approaches lack adaptation to changes in ambient light. Therefore, there is a need in the art for improved optogenetic approaches for the treatment of vision loss due to rod and cone degeneration. 【Prior Art Documents】 【Non-Patent Documents】 【0005】 【Non-Patent Document 1】 McClements et al. (2020) Front Neurosci. 14:57090 【Summary of the Invention】 【0006】 In some embodiments, a melanopsin variant comprising amino acids 1 to 425 or less of the wild-type human melanopsin set forth in SEQ ID NO: 1, wherein the amplitude / conductance and / or off-kinetics of the melanopsin variant are greater than the amplitude / conductance of the wild-type human melanopsin and / or faster than the off-kinetics of the wild-type human melanopsin, is provided. In some embodiments, the melanopsin variant comprises the sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 82, 83, and 84, or a variant thereof comprising one or more amino acid substitutions. In some embodiments, the melanopsin variant comprises one or more amino acid substitutions (s) at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390, the amino acid position(s) being relative to the wild-type human melanopsin set forth in SEQ ID NO: 1. In some embodiments, the one or more substitution mutations (s) are selected from the group consisting of P10F, T83L, T129S, Q135N, S183A, Y212F or Y212A, E215S, M226S or M226T, Y382E or Y382D, S384D, R386A, and R390A or R390D. In some embodiments, the melanopsin variant comprises a P10F substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 5. In some embodiments, the melanopsin variant comprises (e.g., further comprises) a T83L substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 6. In some embodiments, the melanopsin melanopsin variant comprises (e.g., further comprises) a T129S substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 7. In some embodiments, the melanopsin variant comprises (e.g., further comprises) a Q135N substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 8 or 9. In some embodiments, the melanopsin variant comprises (e.g., further comprises) an S183A substitution. In some embodiments, the melanopsin variant comprises any one of SEQ ID NOs: 10-12. In some embodiments, the melanopsin variant comprises (e.g., further comprises) a Y212F substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 13. In some embodiments, the melanopsin variant comprises (e.g., further comprises) an M226S or M226T substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 14 or 15. In some embodiments, the melanopsin variant comprises (e.g., further comprises) a Y382E or Y382D substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, the melanopsin variant comprises (e.g., further comprises) an S384D substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 17. In some embodiments, the melanopsin variant comprises (e.g., further comprises) an R386A substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 18 or 19. In some embodiments, the melanopsin variant comprises (e.g., further comprises) an R390A or R390D substitution.In some embodiments, the melanopsin variant comprises the sequence set forth in any one of SEQ ID NOs: 20-23. 【0007】 In some embodiments, a melanopsin variant comprising at least amino acids 1 to 377 of wild-type human melanopsin as set forth in SEQ ID NO: 1 fused to the C-terminal domain (CTD) of a heterologous G protein-coupled receptor (GPCR) or a CTD variant thereof, wherein the amplitude / conductance and / or off-kinetics of the melanopsin variant are greater than the amplitude / conductance of the wild-type human melanopsin and / or faster than the off-kinetics of the wild-type human melanopsin, is provided. In some embodiments, the CTD of the heterologous GPCR or its CTD variant is the CTD of a visual opsin or a variant thereof. In some embodiments, the CTD of the visual opsin or its variant is the CTD of (i) wild-type D. melanogaster rhodopsin 1, (ii) wild-type human rhodopsin, (iii) wild-type human short wavelength opsin (hOPN1SW), (iv) wild-type human middle wavelength opsin, or (v) wild-type human long wavelength opsin. In some embodiments, the CTD of the visual opsin or its variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 27-30 and 32-33. In some embodiments, the melanopsin variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 39-42, 44-45, and 63-65. In some embodiments, the melanopsin variant further comprises one or more amino acid substitution mutations (multiple possible) at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, R390, and the amino acid position(s) (multiple possible) are relative to the wild-type human melanopsin set forth in SEQ ID NO: 1. In some embodiments, the one or more substitution mutations (multiple possible) are selected from the group consisting of P10F, T83L, T129S, Q135N, S183A, Y212F or Y212A, E215S, M226S or M226T, Y382E or Y382D, S384D, R386A, and R390A or R390D. In some embodiments, the melanopsin variant further comprises one or more amino acid substitutions in the CTD of the heterologous GPCR or its CTD variant. 【0008】 In some embodiments, a melanopsin variant comprising one or more amino acid substitutions at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384D, R386, and R390, wherein the amino acid positions are relative to the wild-type human melanopsin set forth in SEQ ID NO: 1, and the amplitude / conductance and / or off-kinetics of the melanopsin variant are greater than the amplitude / conductance of wild-type human melanopsin and / or faster than the off-kinetics of wild-type human melanopsin, are provided. In some embodiments, the one or more substitution mutations (s) are selected from the group consisting of P10F, T83L, T129S, Q135N, S183A, Y212F or Y212A, E215S, M226S or M226T, Y382E or Y382D, S384D, R386A, R390A or R390D. 【0009】 In some embodiments, the melanopsin variant comprises any one of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% overall sequence homology or identity to the melanopsin variant set forth in any one of SEQ ID NOs: 2-4, 5-23, 39-42, 44-45, 63-65, and 82-84. In some embodiments, the melanopsin variant has at least about 90% overall sequence homology or identity to the melanopsin variant set forth in any one of SEQ ID NOs: 2-4, 5-23, 39-42, 44-45, 63-65, and 82-84. In some embodiments, the melanopsin variant has at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% overall sequence homology or identity to the melanopsin variant set forth in any one of SEQ ID NOs: 2-4, 5-23, 39-42, 44-45, 63-65, and 82-84. 【0010】 In some embodiments, the amplitude / conductance of the melanopsin variants provided herein is at least 1.25-fold greater than the amplitude / conductance of wild-type human melanopsin in HEK293T cells. In some embodiments, the off-kinetics of the melanopsin described herein is at least 1.10-fold faster than the off-kinetics of wild-type human melanopsin in HEK293T cells. 【0011】 Also provided herein are nucleic acids comprising a polynucleotide sequence encoding a melanopsin variant provided herein. In some embodiments, the nucleic acid is operably linked to a promoter. In some embodiments, the promoter is a retinal cell-specific promoter. In some embodiments, the retinal cell-specific promoter is selected from the group consisting of human synapsin (hSyn), SNCG, NEFH, NEFL, 4xgrm6, and grm6. In some embodiments, the nucleic acid further comprises one or more enhancer sequences, intron sequences, leader sequences, Kozak sequences, polyA sequences, stuffer sequences, and / or terminal inverted repeat (ITR) sequences. 【0012】 Also provided herein are recombinant virions comprising (a) a capsid protein and (b) a nucleic acid provided herein. In some embodiments, the capsid protein is selected from AAV2-7m8, AAV2, AAV2-4YF, AAV9, AAV9-7m8, R100, and LSV1. 【0013】 In some embodiments, host cells comprising the nucleic acids described herein are provided. In some embodiments, the host cell further comprises (i) a polynucleotide encoding a capsid protein, (ii) a polynucleotide encoding a rep protein, and (iii) one or more of the AAV helper functions. In some embodiments, provided is a method for generating recombinant virions, comprising (a) culturing the host cell of claim 47 under conditions to produce recombinant virions, and (b) recovering the recombinant virions produced by the host cell. In some embodiments, the method further comprises the step of purifying the recombinant virions. 【0014】 Also provided herein are pharmaceutical compositions comprising the recombinant virions described herein and a pharmaceutically acceptable excipient. In some embodiments, provided is a method of restoring or enhancing visual function in a subject, comprising administering the pharmaceutical composition provided herein to the eye of the subject. In some embodiments, administering comprises intravitreal injection, subretinal injection, suprachoroidal injection, or intravitreal injection. In some embodiments, the subject has an eye disease or disorder selected from the group consisting of retinitis pigmentosa, macular degeneration, retinal detachment, Leber congenital amaurosis, diabetic retinopathy, geographic atrophy, choroideremia, cone dystrophy, and cone-rod dystrophy. In some embodiments, the subject has experienced retinal detachment or photoreceptor loss due to an eye disease, an infectious disease, trauma, injury, head impact, acute light injury, UV light injury, laser injury, or chemical injury. In some embodiments, the subject is human. 【0015】 It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those of ordinary skill in the art. These and other embodiments of the present invention are further described by the following detailed description. 【0016】 All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. 【Brief Description of the Drawings】 【0017】 【Figure 1】 A schematic diagram of wild-type (“WT”) human melanopsin (SEQ ID NO: 1), a seven-transmembrane G protein-coupled receptor (GPCR) with a cytoplasmic tail, is provided. Putative protein kinase A (PKA) sites, putative protein kinase C (PKC) sites, and putative phosphorylation and / or G protein-coupled receptor kinase (GRK) / arrestin binding are shown. 【Figure 2】 Shows the results of experiments performed to determine the amplitude of the calcium light response from melanopsin truncation variants. 【Figure 3】 Shows the results of experiments performed to determine the Tau off of the calcium light response from melanopsin truncation variants. 【Figure 4】 Shows a comparison of the amplitude and Tau off of the truncation variants. Variants such as 419AA (black circles) have both a smaller Tau off and a larger amplitude than wild-type (white circles). 【Figure 5】 Shows the results of experiments performed to determine the amplitude of the calcium light response from melanopsin chimeric variants. 【Figure 6】 Shows the results of experiments performed to determine the Tau off of the calcium light response from chimeric melanopsin variants. 【Figure 7】 Shows the results of experiments performed to determine the Tau off of the calcium light response from the fastest chimeric melanopsin variant shown in Figure 6. Error bars = SEM. 【Figure 8】Shows the comparison of the amplitude with Tau off for the fastest chimeric melanopsin variant shown in Fig. 7. The variant is a black circle and the wild type is a white circle. 【Figure 9】 Shows the results of experiments carried out to determine the amplitude of the calcium light response from the substituted full-length melanopsin variant. 【Figure 10】 Shows the results of experiments carried out to determine the Tau off of the calcium light response from the substituted full-length melanopsin variant. 【Figure 11】 Shows the results of experiments carried out to determine the Tau off of the calcium light response from the fastest substituted full-length melanopsin variant shown in Fig. 10. Error bars = SEM. 【Figure 12】 Shows the comparison of the amplitude with Tau off for the fastest substituted full-length melanopsin variant in Fig. 10. The variant is a black circle and the wild type is a white circle. Error bars = SEM. 【Figure 13】 Shows the results of experiments conducted to measure the amplitude of the calcium light response of the substituted melanopsin variant in the truncated backbone (419AA, SEQ ID NO: 3). 【Figure 14】 Shows the results of experiments conducted to measure the Tau off of the calcium light response of the substituted melanopsin variant in the truncated backbone (419AA, SEQ ID NO: 3). 【Figure 15】 Shows the results of experiments carried out to determine the Tau off of the calcium light response from the fastest substituted melanopsin variant in the truncated backbone shown in Fig. 14. 【Figure 16】 Shows the comparison of the amplitude with Tau off for the fastest substituted melanopsin variant in the truncated backbone shown in Fig. 14. The variant is a black circle and the wild type is a white circle. Error bars = SEM. 【Figure 17】 Shows a schematic diagram of the development strategy for designing melanopsin variants. 【Figure 18】Shows the results of experiments performed to determine the amplitude of the calcium light response over time for WT human melanopsin, melanopsin variants 405AA, 425AA, V370-R377Del, and K356-R377Del. 【Figure 19】 Shows the results of experiments performed to determine the Tau off of the calcium light response over time for WT human melanopsin, melanopsin variants 405AA, 425AA, V370-R377Del, and K356-R377Del. 【Figure 20】 Shows the results of experiments performed to determine the amplitude of the calcium light response of cells transduced with AAV2.7m8 carrying WT human melanopsin or a melanopsin variant containing SEQ ID NO: 19. 【Figure 21】 Shows the results of experiments performed to determine the amplitude of the calcium light response of HEK293T cells transduced with AAV2.7m8-CMV-SEQ ID NO: 19 at different MOIs. 【Mode for Carrying Out the Invention】 【0018】 Summary One goal of optogenetic therapies is to provide the expression of a light-sensitive protein, namely an opsin, in damaged or degenerated retinal cells. However, many opsins are excluded from practical use in the treatment of vision loss because they have low light sensitivity and slow response rates (seconds). Described herein are melanopsin variants that exhibit, for example, a larger amplitude / conductance (such as an amplitude / conductance light response) and / or faster off kinetics (e.g., a faster off light response) compared to wild-type human melanopsin. Such melanopsin variants are used, for example, in methods for restoring or enhancing visual function in subjects who have experienced photoreceptor loss or retinal detachment due to eye disease, infection, trauma, injury, head impact, acute light damage, UV light damage, laser damage, or chemical damage. 【0019】 Definitions The compositions and methods described herein may, unless otherwise indicated, use conventional techniques and descriptions within the skill of the art in molecular biology (including recombinant techniques), cell biology, biochemistry, immunochemistry, and ophthalmic techniques. Such conventional techniques include methods for observing and analyzing the retina or vision of a subject, cloning and propagating recombinant viruses, formulating pharmaceutical compositions, and performing biochemical purification and immunochemistry. By referring to the examples herein, specific examples of suitable techniques can be obtained. However, of course, equivalent conventional procedures can also be used.Such conventional techniques and descriptions are described in general experimental manuals such as Green, et al., Eds., Genome Analysis: A Laboratory Manual Series (Vols. I-IV) (1999), Weiner, et al., Eds., Genetic variation: A Laboratory Manual (2007), Dieffenbach, Dveksler, Eds., PCR Primer: A Laboratory Manual (2003), Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003), Mount, Bioinformatics: Sequence and Genome Analysis (2004), Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2006), and Sambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (all from Cold Spring Harbor Laboratory Press), Stryer, L., Biochemistry (4th Ed.) W.H. Freeman, N.Y. (1995), Gait, “Oligonucleotide Synthesis: A Practical Approch” IRL Press, London (1984), Nelson and Cox, Lehninger, Principles of Biochemistry, 3rd Ed., W.H. Freeman Pub., New York (2000), and Berg et al., Biochemistry, 5th Ed., W.H. Freeman Pub., New York (2002), all of which are hereby incorporated by reference in their entirety for all purposes. 【0020】 Before describing embodiments in detail herein, it is to be understood that the present disclosure is not limited to a particular composition or biological system and can, of course, vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the same. 【0021】 As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules. 【0022】 The term "about" as used herein refers to the normal error range of each value that would be readily understood by one of ordinary skill in the art. References to "about" values or parameters herein include (and describe) embodiments that are directed to that value or parameter itself. 【0023】 It is understood that aspects and embodiments of the present disclosure include aspects and embodiments "comprising," "consisting of," and "consisting essentially of." 【0024】 The terms "polypeptide," "protein," and "peptide" are used interchangeably herein and can refer to a polymer of two or more amino acids. 【0025】 The terms "treat", "treating", "treatment", "ameliorate", or "ameliorating" and other grammatical equivalents, as used herein, refer to reducing, weakening, or improving an eye disease or disorder, or a symptom thereof, preventing additional symptoms of an eye disease or disorder, ameliorating or preventing metabolic causes underlying the symptoms, inhibiting an eye disease or disorder, e.g., arresting the development of an eye disease or disorder, alleviating an eye disease or disorder, regressing an eye disease or disorder, or arresting the symptoms of an eye disease or disorder, and are intended to include prevention. The above terms further include achieving a therapeutic benefit and / or a prophylactic benefit. The term "therapeutic benefit" refers to the eradication or improvement of an eye disease or disorder being treated. A therapeutic benefit may, in some embodiments, be achieved by the eradication or improvement of one or more physiological symptoms associated with an eye disease or disorder such that improvement is observed in the subject even though the subject still suffers from the eye disease or disorder. For prophylactic benefit, a pharmaceutical composition is administered to a patient who has experienced vision loss, or who is at risk of vision loss (e.g., due to loss of photoreceptor cells), or who reports one or more of the physiological symptoms of vision loss (e.g., due to loss of photoreceptor cells). A patient in whom vision loss has developed asynchronously may receive a therapeutic benefit from treatment of the more advanced eye with vision loss and a prophylactic benefit from treatment of the less advanced eye with vision loss. 【0026】 The terms "administer", "administering", "administration", etc., as used herein, may refer to methods used to enable delivery of a therapeutic agent or pharmaceutical composition to a site where a biological effect is desired. Such methods include intravitreal injection into the eye, subretinal injection, intraocular injection, or suprachoroidal injection. Other suitable modes of administration are described elsewhere in this specification. 【0027】 As used herein, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not inhibit the biological activity or properties of the compounds disclosed herein and is relatively non-toxic (i.e., when the substance is administered to an individual, the substance not only does not produce undesirable biological effects, but also does not interact in a harmful manner with any of the components of the composition containing the substance). 【0028】 As used herein, the term "pharmaceutical composition" or simply "composition" may refer to a biologically active compound optionally mixed with at least one pharmaceutically acceptable chemical component, such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickening agent, excipient, etc. 【0029】 As used herein, the term "vector" or "viral vector" or "recombinant viral vector" refers to a viral vector (e.g., an adeno-associated vector or "AAV") or a recombinant viral vector (e.g., recombinant AAV or "rAAV") that contains a polynucleotide sequence of non-viral origin (e.g., a therapeutic transgene, such as a nucleic acid sequence encoding a melanopsin variant described herein, a polynucleotide heterologous to the virus) for transduction into a target cell or into a target tissue. In the case of rAAV, the heterologous polynucleotide is flanked by at least one, generally two, AAV inverted terminal repeat (ITR) sequences. The rAAV vector can be either single-stranded (ssAAV) or self-complementary (scAAV). The term "recombinant viral vector" (e.g., rAAV) encompasses both viral vector particles / virions and viral vector plasmids. 【0030】 The terms "virus", "virus particle", "virion", "recombinant vector particle", "recombinant particle", and "recombinant virion" are used interchangeably to refer to virus particles that contain at least one viral capsid protein and a polynucleotide vector. When such particles contain a heterologous polynucleotide (e.g., a polynucleotide other than the wild-type viral genome such as a transgene (e.g., a melanopsin variant) to be delivered to a target cell or target tissue), they are typically referred to as "recombinant vector particles", "recombinant vectors", or "recombinant virions". Thus, the production of recombinant virus particles (e.g., rAAV) necessarily involves the production of a recombinant polynucleotide vector, as such vectors are contained within the recombinant virus particles. 【0031】 The term "packaging", as used herein, may refer to a series of intracellular events that can result in the assembly and capsid formation of recombinant AAV particles. 【0032】 The AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and capsid-forming proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes". 【0033】 The term "polypeptide" may encompass proteins, peptides, fragments, mutants, derivatives, and analogs of both naturally occurring and non-naturally occurring entities (an example of a non-naturally occurring protein is a fusion protein). A polypeptide may be a monomer, dimer, trimer, or polymer. Further, a polypeptide may contain multiple distinct domains, each of which has one or more distinct activities. To avoid doubt, a "polypeptide" may be of any length greater than two amino acids. 【0034】 As used herein, "polypeptide variant" or simply "variant" refers to a polypeptide whose sequence contains amino acid modifications. In some embodiments, the modifications are insertions, duplications, deletions, rearrangements, or substitutions of one or more amino acids as compared to the amino acid sequence of a comparative protein or polypeptide, such as a native or wild-type protein. A variant may have one or more amino acid point substitutions (where a single amino acid at a position has been changed to another amino acid), one or more insertions and / or deletions (where one or more amino acids have been inserted or deleted, respectively, in the sequence of the comparative protein), and / or truncations of the amino acid sequence at either or both the amino or carboxy termini. A variant may have similar or different biological activities as compared to the comparative protein or unmodified protein. 【0035】 In some embodiments, a variant may have, for example, any one of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% overall sequence identity with its corresponding comparative protein. In some embodiments, a variant may have at least about 90% overall sequence identity with a wild-type protein. In some embodiments, a variant exhibits at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% overall sequence identity. 【0036】 As used herein, "recombinant" refers to a biomolecule such as a gene or protein that (1) has been removed from its natural environment, (2) is not linked to all or part of the polynucleotide in which the gene naturally occurs, (3) is operably linked to a polynucleotide to which it is not linked in nature, or (4) is a biomolecule such as the gene or protein that does not occur in nature. The term "recombinant" can be used in connection with cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant if, for example, it is synthesized from an mRNA synthesized from a recombinant gene present in the cell. 【0037】 The term "expression vector" or "expression construct" or "cassette" or "plasmid" or simply "vector" includes any type of gene construct that is a gene product and contains a nucleic acid or polynucleotide that encodes a gene product (e.g., a melanopsin variant as described herein) where some or all of the nucleic acid encoding the sequence is transcribable and is suitable for gene therapy, including vectors. The transcript can be translated into a protein. In some embodiments, the transcript is partially translated or not translated. In certain aspects, expression includes both transcription of the gene and translation of the mRNA into the gene product (e.g., a melanopsin variant). In other aspects, expression includes only transcription of the nucleic acid encoding the gene of interest. An expression vector may also include regulatory components operably linked to the coding region to facilitate expression of the protein in the target cell. The combination of a regulatory component and the gene or genes to which the regulatory component is operably linked for expression may sometimes be referred to as an "expression cassette", many of which are known and available in the art or can be readily constructed from components available in the art. In some embodiments, the term "expression vector" refers to both a genetic construct and a viral particle containing the genetic construct. 【0038】 The term "heterologous" may refer to an entity that has a different genotype from the remainder of the entity that is the comparison control for that entity. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques may be a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to a coding sequence to which the promoter would not be linked in nature may be a heterologous promoter. 【0039】 As used herein, the terms "subject", "individual", and "patient" are used interchangeably and refer to vertebrates, such as mammals. Mammals include, but are not limited to, mice, monkeys, humans, non-human primates (e.g., cynomolgus monkeys, African green monkeys, macaques), livestock, sport animals, and pets. 【0040】 It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the invention. These and other aspects of the invention will be apparent to those of ordinary skill in the art. These and other embodiments of the invention are further described in the following detailed description. 【0041】 Melanopsin variant Truncated melanopsin variant In some embodiments, the present application provides a melanopsin variant comprising amino acids 1 to 425 or less of wild-type human melanopsin represented by SEQ ID NO: 1, wherein the amplitude / conductance (e.g., amplitude / conductance light response), and / or the off kinetics (e.g., off light response of the melanopsin variant) is greater than the amplitude / conductance of wild-type human melanopsin and / or faster than the off kinetics of wild-type human melanopsin. Such melanopsin variants are also referred to herein as "truncated melanopsin variants". FIG. 1 provides a schematic diagram of melanopsin incorporated into the cell membrane. MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISIRRRQESLGSESEVGWTHMEAAAVWGAAQQANGRSLYGQGLEDLEAKAPPRPQGHEAETPGKTKGLIPSQDPRM (SEQ ID NO: 1) 【0042】 In some embodiments, the melanopsin variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2-4. MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISIRRRQESLGSESEVGW (SEQ ID NO: 2) MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISIRRRQESLGSESEVG (SEQ ID NO: 3) MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISIRRRQESLG (SEQ ID NO: 4) MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNL (SEQ ID NO: 82) MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISI(SEQ ID NO: 83) MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISIRRRQESLGSESEVGWTHMEA(SEQ ID NO: 84) The sites where wild-type human melanopsin (SEQ ID NO: 1) was truncated to generate SEQ ID NOs: 2, 3, 4, 82, 83, and 84 are indicated by arrows in Figure 1. 【0043】 In some embodiments, the melanopsin variant is a variant of SEQ ID NO: 2, 3, 4, 82, 83, or 84 that includes one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2, 3, 4, 82, 83, or 84, respectively. In some embodiments, the variant of SEQ ID NO: 2, 3, 4, 82, 83, or 84 includes, for example, in any combination, one or more amino acid substitution(s) at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390, and the amino acid position(s) is / are relative to the reference melanopsin variant described in SEQ ID NO: 2, 3, 4, 82, 83, or 84, respectively. In some embodiments, the variant of SEQ ID NO: 2, 3, 4, 82, 83, or 84 includes, for example, in any combination, at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitution(s) at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390, and the amino acid position(s) is / are relative to the reference melanopsin variant described in SEQ ID NO: 2, 3, 4, 82, 83, or 84, respectively. Each of the amino acid positions that can be substituted is indicated by a black circle in FIG. 1. In some embodiments, the variant of SEQ ID NO: 2, 3, 4, 82, 83, or 84 includes one or more substitution(s) selected from the group consisting of P10F, T83L, T129S, Q135N, S183A, Y212F or Y212A, E215S, M226S or M226T, Y382E or Y382D, S384D, R386A, and R390A or R390D, or any combination thereof. In some embodiments, the variant of SEQ ID NO: 2, 3, 4, 82, 83, or 84 includes at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitution(s) selected from the group consisting of P10F, T83L, T129S, Q135N, S183A, Y212F or Y212A, E215S, M226S or M226T, Y382E or Y382D, S384D, R386A, and R390A or R390D, or any combination thereof. 【0044】 In some embodiments, the melanopsin variant includes a P10F substitution. In some embodiments, the melanopsin variant includes the sequence set forth in SEQ ID NO: 5. In some embodiments, the melanopsin variant includes (e.g., further includes) a T83L substitution. In some embodiments, the melanopsin variant includes the sequence set forth in SEQ ID NO: 6. In some embodiments, the melanopsin variant includes (e.g., further includes) a T129S substitution. In some embodiments, the melanopsin variant includes the sequence set forth in SEQ ID NO: 7. In some embodiments, the melanopsin variant includes (e.g., further includes) a Q135N substitution. In some embodiments, the melanopsin variant includes the sequence set forth in SEQ ID NO: 8. In some embodiments, the melanopsin variant includes T129S and Q135N substitutions. In some embodiments, the melanopsin variant includes the sequence set forth in SEQ ID NO: 9. In some embodiments, the melanopsin variant includes (e.g., further includes) an S183A substitution. In some embodiments, the melanopsin variant includes the sequence set forth in SEQ ID NO: 10. In some embodiments, the melanopsin variant includes Q135N and S183A substitutions. In some embodiments, the melanopsin variant includes the sequence set forth in SEQ ID NO: 11. In some embodiments, the melanopsin variant includes T129S, Q135N, and S183A substitutions. In some embodiments, the melanopsin variant includes the sequence set forth in SEQ ID NO: 12. In some embodiments, the melanopsin variant includes (e.g., further includes) a Y212F substitution. In some embodiments, the melanopsin variant includes the sequence set forth in SEQ ID NO: 13. In some embodiments, the melanopsin variant includes (e.g., further includes) an M226S or M226T substitution. In some embodiments, the melanopsin variant includes the sequence set forth in SEQ ID NO: 14. In some embodiments, the melanopsin variant includes Y212F and M226S substitutions. In some embodiments, the melanopsin variant includes the sequence set forth in SEQ ID NO: 15.In some embodiments, the melanopsin variant comprises (e.g., further comprises) a Y382E or Y382D substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, the melanopsin variant comprises (e.g., further comprises) an S384D substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 17. In some embodiments, the melanopsin variant comprises (e.g., further comprises) an R386A substitution. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 18. In some embodiments, the melanopsin variant comprises S183A, S384D, and R386A substitutions. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the melanopsin variant comprises (e.g., further comprises) an R390A or R390D substitution. In some embodiments, the melanopsin variant comprises any one of SEQ ID NO: 20. In some embodiments, the melanopsin variant comprises (e.g., further comprises) Y382D, R386A, and R390A or R390D substitutions. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 21. In some embodiments, the melanopsin variant comprises (e.g., further comprises) S183A, Y382D, R386A, and R390A or R390D substitutions. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 22. In some embodiments, the melanopsin variant comprises (e.g., further comprises) T129S, S183A, M226T, Y382D, R386A, and R390A or R390D substitutions. In some embodiments, the melanopsin variant comprises the sequence set forth in SEQ ID NO: 23. 【0045】 The amino acid sequences of SEQ ID NOs: 5-23 are provided below. The substitution positions in each of SEQ ID NOs: 5-23 are in bold text and underlined type. 【Chemical formula】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【0046】 In some embodiments, the truncated melanopsin variants comprising one or more substitutions are the substitution truncated variants listed in Table A. 【0047】 Chimeric melanopsin variants In some embodiments, the present application provides a melanopsin variant comprising at least amino acids 1 to 377 of the wild-type human melanopsin set forth in SEQ ID NO:1 fused (e.g., via a peptide bond) to a heterologous G protein-coupled receptor (GPCR) or a C-terminal domain (CTD) variant thereof, wherein the amplitude / conductance (e.g., amplitude / conductance light response) and / or off-kinetics (e.g., off-light response) is greater than the amplitude / conductance of wild-type human melanopsin and / or faster than the off-kinetics of wild-type human melanopsin. Such melanopsin variants are also referred to herein as "chimeric melanopsin variants". 【0048】 In some embodiments, the melanopsin variant comprises amino acids 1 to 377 of the wild-type human melanopsin set forth in SEQ ID NO:1. In some embodiments, the melanopsin variant comprises amino acids 1 to 380 of the wild-type human melanopsin set forth in SEQ ID NO:1. The chimeric fusion point (i.e., the point at which the N-terminus of the CTD of the GPCR or its CTD variant binds to the C-terminus of at least amino acids 1 to 377) is indicated by a dotted line in Figure 1. 【0049】 In some embodiments, the CTD of a heterologous GPCR or a CTD variant thereof is the CTD of a visual opsin or a variant thereof. In some embodiments, the CTD of a visual opsin or a variant thereof is the CTD of (i) wild-type D. melanogaster rhodopsin 1, (ii) wild-type human rhodopsin, (iii) wild-type human short-wavelength opsin (hOPN1SW), (iv) wild-type human middle-wavelength opsin, or (v) wild-type human long-wavelength opsin. In some embodiments, the CTD of a visual opsin comprises the amino acid sequence set forth in any one of SEQ ID NOs: 27-30 and 32-33 shown below. In some embodiments, the CTD of a visual opsin comprises a variant of any one of SEQ ID NOs: 27-30 and 32-33 that includes one or more amino acid substitutions, deletions, or insertions. TEVSTVSSTQVGPN (SEQ ID NO: 27) NKQFQACIMKMVCGKAMTDESDTCSSQKTEVSTVSSTQVGPN (SEQ ID NO: 28) AMTDESDTCSSQKTEVSTVSSTQVGPN (SEQ ID NO: 29) DAQSQATASEAESKA (SEQ ID NO: 30) TEVSSVSSVSPA (SEQ ID NO: 32) QFRNCILQLFGKKVDDGSELSSASKTEVSSVSSVSPA (SEQ ID NO: 33) 【0050】 In some embodiments, the N-terminus of the CTD of a heterologous GPCR or a CTD variant thereof is directly fused to the C-terminus of a melanopsin variant comprising at least amino acids 1-377 of wild-type human melanopsin set forth in SEQ ID NO: 1, for example, via a peptide bond. In some embodiments, the melanopsin variant (e.g., a chimeric melanopsin variant) comprises the amino acid sequence set forth in any one of SEQ ID NOs: 39-42 and 44-45 shown below. The CTD of the heterologous GPCR in each chimeric melanopsin variant is shown in bold text. 【Chemical formula】 【Chemical formula】 【Chemical formula】 【Chem.】 【Chem.】 【Chem.】 【0051】 In some embodiments, the chimeric melanopsin variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 63, 64, and 65. See Table A. 【0052】 In some embodiments, the N-terminus of the CTD of a heterologous GPCR or a CTD variant thereof is fused via one or more linker(s) to the C-terminus of a melanopsin variant comprising at least amino acids 1-377 of wild-type human melanopsin set forth in SEQ ID NO: 1. In some embodiments, the one or more linker(s) comprise one or more peptide linkers, including, for example, but not limited to, G(4)S (SEQ ID NO: 161), G(4)S)2 (SEQ ID NO: 162), and G(4)S)3 (SEQ ID NO: 163), GGGGS (SEQ ID NO: 161), GGGGSGGGGS (SEQ ID NO: 164), or GGGGSGGGGSGGGGS (SEQ ID NO: 165). In some embodiments, the N-terminus of the CTD of a heterologous GPCR or a CTD variant thereof is fused via one or more linker(s) and one or more spacer(s) to the C-terminus of a melanopsin variant comprising at least amino acids 1-377 of wild-type human melanopsin set forth in SEQ ID NO: 1. See, for example, Klein et al. (2014) Protein Eng Sel. 27(10):325-330. 【0053】 In some embodiments, a melanopsin variant (e.g., a chimeric melanopsin variant described herein) further comprises one or more amino acid substitutions. In some embodiments, a melanopsin variant (e.g., a chimeric melanopsin variant described herein) further comprises, for example, in any combination, one or more substitutions (s) at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390, and the amino acid position(s) is / are relative to the wild-type human melanopsin set forth in SEQ ID NO: 1. In some embodiments, a melanopsin variant (e.g., a chimeric melanopsin variant described herein) further comprises, for example, in any combination, at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions (s) at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390, and the amino acid position(s) is / are relative to the wild-type human melanopsin set forth in SEQ ID NO: 1. As described above, each of the amino acid positions that can be substituted is indicated by a black circle in FIG. 1. In some embodiments, a melanopsin variant (e.g., a chimeric melanopsin variant described herein) further comprises one or more substitutions (s) selected from the group consisting of P10F, T83L, T129S, Q135N, S183A, Y212F or Y212A, E215S, M226S or M226T, Y382E or Y382D, S384D, R386A, and R390A or R390D, or any combination thereof. In some embodiments, a melanopsin variant (e.g., a chimeric melanopsin variant described herein) further comprises at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions (s) selected from the group consisting of P10F, T83L, T129S, Q135N, S183A, Y212F or Y212A, E215S, M226S or M226T, Y382E or Y382D, S384D, R386A, and R390A or R390D, or any combination thereof.Additionally or alternatively, in some embodiments, the melanopsin variant (e.g., the chimeric melanopsin variant described herein) further comprises one or more amino acid substitutions (e.g., at least any 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) in the CTD of a heterologous GPCR or variant thereof. 【0054】 Substituted full-length melanopsin variants In some embodiments, the present application provides a melanopsin variant that includes one or more amino acid substitutions at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and R390, or any combination thereof, wherein the amino acid positions are relative to the wild-type human melanopsin set forth in SEQ ID NO: 1, and the amplitude / conductance (e.g., amplitude / conductance light response) and / or off kinetics (e.g., off light response) of the melanopsin variant are greater than the amplitude / conductance of wild-type human melanopsin and / or faster than the off kinetics of wild-type human melanopsin. Such melanopsin variants are referred to herein as "substituted full-length melanopsin variants." In some embodiments, the melanopsin variant includes, for example, in any combination, at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions (s) at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390, and the amino acid position(s) are relative to the wild-type human melanopsin set forth in SEQ ID NO: 1. In some embodiments, one or more substitutions (e.g., at least any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions) are selected from the group consisting of P10F, T83L, T129S, Q135N, S183A, Y212F or Y212A, E215S, M226S or M226T, Y382E or Y382D, S384D, R386A, R390A or R390D. In some embodiments, the melanopsin variant further includes one or more amino acid substitutions at positions 391-478, and the amino acid position(s) are relative to the wild-type human melanopsin set forth in SEQ ID NO: 1. In some embodiments, the full-length melanopsin variant comprising one or more substitutions is the substitution variant listed in Table A. 【0055】 In some embodiments, the melanopsin variant comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% overall sequence homology or identity to any one of, for example, SEQ ID NOs: 2-4, 5-23, 39-42, 44-45, 63-65, and 82-84. In some embodiments, the melanopsin variant has at least about 90% overall sequence homology or identity to any one of SEQ ID NOs: 2-4, 5-23, 39-42, 44-45, 63-65, and 82-84. In some embodiments, the melanopsin variant has at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% overall sequence homology or identity to any one of SEQ ID NOs: 2-4, 5-23, 39-42, 44-45, 63-65, and 82-84. 【0056】 Functional characteristics of melanopsin variants The amplitude / conductance (e.g., amplitude / conductance light response) of the melanopsin variants described herein is greater than that of wild-type human melanopsin (e.g., wild-type human melanopsin as set forth in SEQ ID NO: 1). In some embodiments, the amplitude / conductance of the melanopsin variants described herein is at least about 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, and 5 times greater than the amplitude / conductance of wild-type human melanopsin. In some embodiments, the amplitude / conductance of the melanopsin variants described herein is at least about 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, and 5 times greater than the amplitude / conductance of wild-type human melanopsin, for example, as measured in HEK293T cells via a calcium release assay as described in the examples herein. 【0057】 Additionally or alternatively, in some embodiments, the off-off kinetics (e.g., off light response) of the melanopsin variants described herein are faster than the off kinetics of wild-type human melanopsin (e.g., wild-type human melanopsin as set forth in SEQ ID NO: 1). In some embodiments, the off kinetics of the melanopsin variants described herein are at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10, 10.25, 10.5, 10.75, 11, 11.25, 11.5, 11.75, 12, 12.25, 12.5, 12.75, 13, 13.25, 13.5, 13.75, 14, 14.25, 14.5, 14.75, or 15 times faster than the off kinetics of wild-type human melanopsin. In some embodiments, the off kinetics of the melanopsin variants described herein are at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or 5 times faster than the off kinetics of wild-type human melanopsin, e.g., as measured in HEK293T cells via a calcium release assay as described in the examples herein. 【0058】 Nucleic Acids, Expression Vectors, Virus Particles, and Host Cells Also provided herein are nucleic acids comprising a nucleotide sequence encoding a melanopsin variant described herein. In some embodiments, the nucleotide sequence encoding the melanopsin variant is operably linked to a transcriptional control element (e.g., a promoter) that provides for the expression (e.g., selective expression) of the nucleic acid in retinal cells (e.g., retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, or photoreceptor cells, such as rod cells or cone cells, Müller cells, and retinal pigment epithelial cells). In some embodiments, the nucleic acid encoding the melanopsin variant is operably linked to a transcriptional control element (e.g., a promoter) that provides for expression in eukaryotic cells (e.g., mammalian cells). Exemplary transcriptional control elements and promoters include ubiquitous or tissue-specific promoters, such as the CAG promoter (Miyasaki et al. (1989) Gene 79:269), the cytomegalovirus (CMV) promoter, the human synapsin (hSyn) promoter, the metabotropic glutamate receptor-6 (grm6) promoter (also referred to as the GluR or GluR6 promoter, see Cronin et al. (2014) EMBO Mol. Med. 6:1175), the 4xgrm6 promoter (i.e., a concatemer of 4 copies of the minimal version of the grm6 promoter, see Lagali, et al. (2008) Nat Neurosci 11:667-675 and Masu, et al. (1995) Cell. 80:757-765), the NEFL promoter (Simpson et al. (2019) Human Gene Therapy. 30(3);257-272), the SNCG (gamma-synuclein) promoter (Chaffiol et al. (2017) Mol Ther. 25:2546-60), the NEFH promoter (Millington-Ward et al. (2020) Sci Rep. 10:16515), the 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 (SST) promoter, neuropeptide Y (NPY) promoter, and vasoactive intestinal peptide (VIP) promoter, red cone opsin promoter, rhodopsin promoter, rhodopsin kinase promoter, vitreoretinal macular dystrophy 2 (VMD2) gene promoter, and interphotoreceptor / retinoid-binding protein (IRBP) gene promoter, L7 promoter (Oberdick et al. (1990) Science 248:223), thy-1 promoter, recoverin promoter (Wiechmann and Howard (2003) Curr. Eye Res. 26:25), calbindin promoter, and beta-actin promoter, including but not limited to these. In some embodiments, the promoter is a synthetic (non-natural) promoter / enhancer combination or includes it. In some embodiments, the promoter includes the human cytomegalovirus (CMV) immediate early enhancer and promoter, followed by the adenovirus tripartite leader sequence (TPL), an enhancer element (eMLP) from the major late promoter, a synthetic intron, and a Kozak sequence. Such a promoter, also known as "C11", is described in Grishanin et al. (2019) Mol Ther.It is described in 27(1):118-129. Further, in addition to the above, in some embodiments, a nucleic acid comprising a sequence encoding a melanopsin variant described herein may include additional sequences such as enhancer(s), intron(s), leader sequence(s), Kozak sequence(s), polyA sequence(s), stuffer sequence(s), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence(s), and / or one or more terminal inverted repeat (ITR) sequences, such as, but not limited to, AAV ITR sequences, etc. 【0059】 In some embodiments, the nucleic acids provided herein are within recombinant gene constructs, such as expression vectors. Suitable expression vectors include, but are not limited to, lentiviral vectors, herpes simplex virus (HSV) vectors, adenoviral vectors, retroviral vectors, adeno-associated virus (AAV) vectors, which can be natural or engineered serotypes (such as, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAV11, or AAV12 vectors). In some embodiments, the AAV vector genome is single-stranded (ssAAV). In some embodiments, the viral vector genome is self-complementary (scAAV). In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a melanopsin variant described herein is within a recombinant lentiviral vector, recombinant HSV, recombinant adenoviral vector, recombinant retroviral vector, or recombinant AAV ("rAAV") vector (such as, for example, rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAVrh.8, rAAVrh.10, rAAV11, or rAAV12 vector). 【0060】 In some embodiments, the rAAV vector is AAV2.5T.LSV1 (also known as “LSV1”), which is described in detail in WO2020 / 180951 and US2020 / 0297869, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the rAAV vector is AAV2-4YF, which is described in Petrs-Silva et al. (2009) Mol. Ther. 17, 463-471 and Petrs-Silva et al. (2011) Mol. Ther. 19, 293-301, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the rAAV vector is AAV2.7m8, which is described in Dalkara et al. (2013) Sci Transl Med. 5, 189ra76, WO2012 / 145601, and US2014 / 0364338, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the rAAV vector is AAV9.7m8, which is described, for example, in Khabou et al. (2016) Biotechnol Bioeng. (12):2712-2724 and Khabou et al. (2018) JCI Insight. 3(2):e96029, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the rAAV vector is R100 described in Kotterman, et al. (2021) “Directed Evolution of AAV Targeting Primate Retina by Intravitreal Injection Identifies R100, a Variant Demonstrating Robust Gene Delivery and Therapeutic Efficacy in Non-Human Primates.” bioRxiv.In some embodiments, the vector comprises a nucleic acid comprising a nucleotide sequence encoding a melanopsin variant described herein and a nucleotide sequence encoding a variant AAV capsid protein, wherein the variant AAV capsid protein confers infectivity to retinal cells and / or confers the ability to cross the inner limiting membrane (ILM) in the eye (e.g., the eye of a mammal such as a mouse, human, or non-human primate). In some embodiments, the AAV capsid protein is AAV.ShH10, AAV.GL, or AAV.NN.Such capsid proteins, and other proteins, are described in detail, for example, in Day et al. (2014) Adv. Exp. Med. Biol. 801:687, Boye et al. (2016) J. Viral. 90:4215, Vandenberghe and Auricchio (2012) Gene Therapy 19:162, Klimczak et al. (2009) PLoS One 4:e7467, Byrne et al. (2020) “In vivo-directed evolution of adeno-associated virus in the primate retina.” JCI Insight. 2020;5(10):e135112, Pavlou et al. (2021) “Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders.” EMBO Mol Med. 13(4):e13392, Miyadera et al. (2022) “Targeting ON-bipolar cells by AAV gene therapy stably reverses LRIT3-congenital stationary night blindness.” Proc Natl Acad Sci USA. 119(13):e2117038119, Öztuerk et al. (2021) “scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution.” Elife. 10:e64175, US2012 / 0164106, and US2016 / 0017295. In some embodiments, the retinal cells are retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, or photoreceptor cells, such as rod cells or cone cells, Müller cells, or retinal pigment epithelial cells. 【0061】 In some embodiments, virions (i.e., virus particles) are provided that include (a) a capsid protein and (b) a nucleic acid described herein or an expression vector described herein. In some embodiments, the capsid protein is a lentiviral capsid protein, an HSV capsid protein, an adenoviral capsid protein, a retroviral capsid protein, an AAV capsid protein (e.g., an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAV11, or AAV12 capsid protein), and the like. In some embodiments, the capsid protein is a recombinant capsid protein such as, for example, a recombinant lentiviral capsid protein, a recombinant HSV capsid protein, a recombinant adenoviral capsid protein, a recombinant retroviral capsid protein, a recombinant AAV capsid protein (e.g., an rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAVrh.8, rAAVrh.10, rAAV11, or rAAV12 capsid protein). 【0062】 In some embodiments, the virion comprises a variant capsid protein that includes a peptide inserted into variable loop 8 of the capsid protein relative to the corresponding parental envelope protein, the insertion comprising an amino acid sequence selected from LALGETTRPA (SEQ ID NO: 46), LANETITRPA (SEQ ID NO: 47), LAKAGQANNA (SEQ ID NO: 48), LAKDPKTTNA (SEQ ID NO: 49), KDTDTTR (SEQ ID NO: 50), RAGGSVG (SEQ ID NO: 51), AVDTTKF (SEQ ID NO: 52), STGKVPN (SEQ ID NO: 53), LAKDTDTTRA (SEQ ID NO: 54), LARAGGSVGA (SEQ ID NO: 55), LAAVDTTKFA (SEQ ID NO: 56), and LASTGKVPNA (SEQ ID NO: 57). In some embodiments, the virion comprises a variant capsid protein that includes the insertion of a peptide set forth in any one of SEQ ID NOs: 46 - 57 at the following amino acid positions to generate a variant capsid: between amino acids 587 and 588 of the AAV2 capsid protein, between amino acids 590 and 591 of the AAV1 capsid protein, between amino acids 575 and 576 of the AAV5 capsid protein, between amino acids 590 and 591 of the AAV6 capsid protein, between amino acids 589 and 590 of the AAV7 capsid protein, between amino acids 590 and 591 of the AAV8 capsid protein, between amino acids 588 and 589, or between 589 and 590, of the AAV9 capsid protein, or between amino acids 589 and 590 of the AAV10 capsid protein. In some embodiments, the virion comprises the variant capsid protein AAV2.7m8 (see, e.g., Dalkara et al. (2013) Sci Transl Med. 5, 189ra76, WO2012 / 145601, and US2014 / 0364338, the contents of which are hereby incorporated by reference in their entirety).In some embodiments, the virion comprises the variant capsid protein AAV9.7m8 (see Khabou et al. (2016) Biotechnol Bioeng. (12):2712-2724 and Khabou et al. (2018) JCI Insight. 3(2):e96029, the contents of which are hereby incorporated by reference in their entirety). AAV2.7m8 and AAV9.7m8 can transduce the retina when delivered intravitreally. 【0063】 In some embodiments, the virion comprises a variant capsid protein, the variant capsid protein comprising a modified sequence that includes one or more amino acid substitutions within amino acid residues 570-579 relative to the parental AAV capsid protein, the modified sequence including HKFKSGD (SEQ ID NO: D58), the numbering of the amino acid residues corresponding to the AAV5 VP1 capsid protein. In some embodiments, the parental AAV capsid protein is the AAV5 capsid protein or an AAV5 and AAV2 hybrid capsid protein. In some embodiments, the parental AAV capsid protein is the AAV2.5T capsid protein. In some embodiments, "AAV2.5T capsid protein" or "AAV2.5T variant" refers to a hybrid capsid protein comprising regions from AAV2 and AAV5 as described in U.S. Patent No. 9,441,244, the disclosure of which is incorporated in its entirety. AAV2.5T can transduce the retina when delivered subretinally, but not when injected intravitreally. Transduction by AAV2.5T can be blocked by the inner limiting membrane (ILM) rich in heparan sulfate proteoglycan (HSPG). The domain exposed on the surface of AAV2.5T is identical to the domain of AAV5 except for a single substitution from A to T at aa582 (aa581 of AAV5) of AAV2.5T, and this mutation appears to increase infectivity in mammalian cells without affecting typical sialic acid receptor binding of AAV5. AAV5 and AAV2.5T have very little binding to heparan sulfate, while AAV2 has a high affinity for heparan sulfate. In some embodiments, the parental AAV capsid protein is the AAV2.5T VP1 capsid protein. In some embodiments, the modified sequence includes LAHKFKSGDA (SEQ ID NO: 59). In some embodiments, the rAAV is AAV2.5T.LSV1. In some embodiments, "AAV2.5 "T.LSV1" or "AAV2.5T.LSV1 variant" refers to an rAAV variant comprising a variant capsid protein, the variant capsid protein comprising a loop substitution variant, the loop substitution variant comprising the amino acid loop sequence LAHKFKSGDA (SEQ ID NO:60) at amino acid residues 570 - 579 relative to AAV2.5T, which is the parental AAV capsid protein. In some embodiments, the virion comprises the AAV2 - 4YF capsid protein (see Petrs - Silva et al. (2009) Mol. Ther. 17, 463 - 471 and Petrs - Silva et al. (2011) Mol. Ther. 19, 293 - 301, the contents of which are incorporated herein by reference in their entirety), or the R100 capsid protein (see Kotterman, et al. (2021) “Directed Evolution of AAV Targeting Primate Retina by Intravitreal Injection Identifies R100, a Variant Demonstrating Robust Gene Delivery and Therapeutic Efficacy in Non - Human Primates.” BioRxiv, the contents of which are incorporated herein by reference in their entirety). 【0064】 In some embodiments, the virion comprises a variant capsid protein, the variant capsid protein comprising an insertion of a peptide within variable loop 8 of the capsid protein relative to the corresponding parental capsid protein, the insertion being LAHQDTTKNS (SEQ ID NO: 85), LALGETTRAA (SEQ ID NO: 86), LAHQDTTRPA (SEQ ID NO: 87), LARQDTTKNA (SEQ ID NO: 88), LAHQDSTKNA (SEQ ID NO: 89), LAHQDATKNA (SEQ ID NO: 90), LAHQDTTKPA (SEQ ID NO: 91), IALSETTRPA (SEQ ID NO: 92), LAHQDTTKKC (SEQ ID NO: 93), LALGEATRPA (SEQ ID NO: 94), LALGETTRTA (SEQ ID NO: 95), LALSEATRPA (SEQ ID NO: 96), LAKDETKNSA (SEQ ID NO: 97), LALGETTKPA (SEQ ID NO: 98), LAHQATTKNA (SEQ ID NO: 99), LQRGNRQTTTADVNTQ (SEQ ID NO: 100), LQRGNRQATTADVNTL (SEQ ID NO: 101), LQRGNRQATTEDVNTQ (SEQ ID NO: 102), LQRGNRQAATEDVNTQ (SEQ ID NO: 103), LQRGNRQAATADVNSL (SEQ ID NO: 104), LQRGNRQAATADVNKL (SEQ ID NO: 105), LQRGVRVPSVLEVNGQ (SEQ ID NO: 106), LQRGNRQAATADVNIL (SEQ ID NO: 107), LQRGKRQATTADVNTQ (SEQ ID NO: 108), LHRGNRQAATADVNTL (SEQ ID NO: 109), SRTNTPSGTTTQPTLQFSQ (SEQ ID NO: 110), SKTDTPSGTTTQSRLQFSQ (SEQ ID NO: 111), SRTDTPSETTTQSRLQFSQ (SEQ ID NO: 112), SRTNSPSGTTTQSSLQFSQ (SEQ ID NO: 113), SRTDIPSGTTTQSRLQFSQ (SEQ ID NO: 114), HQDTTKN (SEQ ID NO: 115), LGETTRA (SEQ ID NO: 116), HQDTTRP (SEQ ID NO: 117), RQDTTKN (SEQ ID NO: 118), HQDSTKN (SEQ ID NO: 119), HQDATKN (SEQ ID NO: 120), HQDTTKP (SEQ ID NO: 121), LSETTRP (SEQ ID NO: 122), HQDTTKK (SEQ ID NO: 123), LGEATRP (SEQ ID NO: 124), LGETTRT (SEQ ID NO: 125), LSEATRP (SEQ ID NO: 126),It comprises an amino acid sequence selected from KDETKNS (SEQ ID NO: 127), LGETTKP (SEQ ID NO: 128), and HQATTKN (SEQ ID NO: 129). For example, in some embodiments, the insertion site is between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, between amino acids 588 and 589 of AAV10, or between amino acids 585 and 586 of AAV4. Additional details regarding such variant capsid proteins and virus particles (e.g., recombinant virus particles) comprising such variant capsid proteins are provided in WO2021 / 243085 and US2021 / 0371879, the contents of which are hereby incorporated by reference in their entirety. 【0065】 In some embodiments, the virion comprises a variant capsid protein, the variant capsid protein comprising a peptide insertion within the GH-loop relative to the corresponding parental capsid protein, the insertion being selected from the group consisting of ISDQTKH (SEQ ID NO: 130), QADTTKN (SEQ ID NO: 131), ASDSTKA (SEQ ID NO: 132), NQDYTKT (SEQ ID NO: 133), HDITKNI (SEQ ID NO: 134), HPDTTKN (SEQ ID NO: 135), HQDTTKN (SEQ ID NO: 136), NKITNKD (SEQ ID NO: 137), ISNENEH (SEQ ID NO: 138), QANANEN (SEQ ID NO: 139), GKSKVID (SEQ ID NO: 140), TNRTSPD (SEQ ID NO: 141), PNSTHGS (SEQ ID NO: 142), KDRAPST (SEQ ID NO: 143), LAQAD1TKNA (SEQ ID NO: 144), LAISDQTKHA (SEQ ID NO: 145), LGISDQTKHA (SEQ ID NO: 146), LAASDSTKAA (SEQ ID NO: 147), LANQDYTKTA (SEQ ID NO: 148), LAHDITKNIA (SEQ ID NO: 149), LAHPDTTKNA (SEQ ID NO: 150), LAHQDTTKNA (SEQ ID NO: 151), LANKTTNKDA (SEQ ID NO: 152), LPISNENEHA (SEQ ID NO: 153), LPQANANENA (SEQ ID NO: 154), LAGKSKVIDA (SEQ ID NO: 155), LATNRTSPDA (SEQ ID NO: 156), LAPNSTHGSA (SEQ ID NO: 157), and LAKDRAPSTA (SEQ ID NO: 158). For example, in some embodiments, the insertion site is between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, between amino acids 588 and 589 of AAV10, or between amino acids 585 and 586 of AAV4. Additional details regarding such variant capsid proteins and virus particles (e.g., recombinant virus particles) comprising such variant capsid proteins are provided in WO2017 / 197355, the contents of which are incorporated herein by reference in their entirety. 【0066】 Similarly, provided herein are host cells for producing the virions described herein. The host cells contain the nucleic acids provided herein. For example, suitable host cells include human-derived cell lines such as HeLa, A549, or 293 cells, or insect-derived cell lines such as SF-9. In some embodiments, the host cells further contain a polynucleotide encoding a capsid protein (e.g., any of the capsid proteins described herein), a polynucleotide encoding a rep protein, and AAV helper functions. In some embodiments, the nucleic acids described herein further contain a polynucleotide encoding a capsid protein and a polynucleotide encoding a rep protein. In some embodiments, the nucleic acids described herein do not further contain a polynucleotide encoding a capsid protein and a polynucleotide encoding a rep protein. 【0067】 Production of Recombinant Virus Particles In the art, many methods for producing rAAV vectors are known, including transfection, production of stable cell lines, and infectious hybrid virus production systems including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, et al. (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids. All rAAV production cultures for the production of rAAV virus particles require, to support rAAV production, 1) a suitable host cell, such as a human-derived cell line such as HeLa, A549, or 293 cells, or an insect-derived cell line such as Sf9, e.g., a baculovirus-free Sf9 cell (in the case of a baculovirus production system), 2) a suitable helper virus function provided by a wild-type or mutant adenovirus (such as a temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct that provides helper functions, 3) AAV rep and cap genes and gene products, 4) a transgene (such as a therapeutic transgene) flanked by at least one AAV ITR sequence (more typically two ITR sequences), and 5) a suitable medium and medium components. In some embodiments, the AAV rep and cap gene products can be derived from any AAV serotype. Although not essential, generally, the AAV rep gene product is of the same serotype as the ITR of the rAAV vector genome, as long as the rep gene product can function to replicate and package the rAAV genome. Suitable media known in the art can be used for the production of rAAV vectors. These media include, but are not limited to, modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), custom formulations such as those described in U.S. Patent No. 6,566,118, and media manufactured by Hyclone Laboratories and JRH, including the Sf-900 II SFM medium described in U.S. Patent No. 6,723,551, each of which is hereby incorporated by reference in its entirety, particularly with respect to custom medium formulations for use in the production of recombinant AAV vectors.In some embodiments, the AAV helper function is provided by adenovirus or HSV. In some embodiments, the AAV helper function is provided by baculovirus and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cells). 【0068】 In a suitable rAAV production medium of the present invention, serum or a serum-derived recombinant protein can be added at a level of 0.5% - 20% (v / v or w / v). Alternatively, as is well known in the art, rAAV vectors can be produced under serum-free conditions, which can also be referred to as media that do not contain animal-derived products. Those skilled in the art will understand that commercially available or custom media designed to support the production of rAAV vectors may also have added thereto, but are not limited to, one or more cell culture components known in the art, such as glucose, vitamins, amino acids, and / or growth factors, to increase the titer of rAAV in the production culture. 【0069】 In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the harvest of the production culture is clarified by filtration through a series of depth filters including, for example, a Grade DOHC Millipore Millistak+HC Pod filter, a Grade A1HC Millipore Millistak+HC Pod filter, and a 0.2 μm filter Opticap XL Millipore Express SHC hydrophilic membrane filter. Clarification can also be achieved by various other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 μm or greater known in the art. In some embodiments, the rAAV production culture is further processed to digest any high molecular weight DNA present in the production culture. 【0070】 rAAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation, flow-through anion exchange filtration, tangential flow filtration (TFF) to concentrate rAAV particles, rAAV capture by apatite chromatography, heat inactivation of helper virus, rAAV capture by hydrophobic interaction chromatography, buffer exchange by size exclusion chromatography (SEC), nanofiltration, anion exchange chromatography, cation exchange chromatography, or rAAV capture by affinity chromatography. These steps can be used alone, in various combinations, or in different orders. In some embodiments, the method includes all steps in the order described below to purify rAAV particles, and this step is found, for example, in Xiao et al., (1998) Journal of Virology 72:2224-2232, U.S. Pat. Nos. 6,989,264 and 8,137,948, and WO2010 / 148143. Methods for purifying adenovirus particles are found, for example, in Bo, H et al., (2014) Eur. J. Pharm. Sci. 67C:119-125. Methods for purifying lentivirus particles are found, for example, in Segura et al., (2013) Expert Opin Biol Ther. 13(7):987-1011. Methods for purifying HSY particles are found, for example, in Goins, WF et al., (2014) Herpes Simplex Virus Methods in Molecular Biology 1144:63-79. 【0071】 Pharmaceutical Compositions and Kits In some embodiments, there is provided a pharmaceutical composition comprising: a) a nucleic acid comprising a nucleotide sequence encoding a melanopsin variant described herein, an expression vector described herein, or a virion comprising a nucleic acid or expression vector described herein; and b) a pharmaceutically acceptable excipient or carrier. In some embodiments, a “pharmaceutically acceptable excipient or carrier” refers to any excipient, carrier, diluent, stabilizer, etc. that, when administered to a subject (e.g., a mammal such as a mouse, human, or non-human primate), does not have substantially long-term or permanent adverse effects. Typically, such excipients can be mixed with the active compound (e.g., a nucleic acid disclosed herein, an expression vector disclosed herein, or a viral particle disclosed herein), or can dilute or encapsulate the active compound, and can be solid, semi-solid, or liquid agents. It is understood that the active ingredient can be soluble or delivered as a suspension in a desired excipient or diluent. Without limitation, for example, any of a variety of pharmaceutically acceptable excipients can be used, including aqueous media such as distilled deionized water, physiological saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, or any other inert ingredient. The choice of pharmaceutically acceptable excipient can depend on the mode of administration. Pharmaceutically acceptable excipients are contemplated for use in pharmaceutically acceptable compositions, provided they are not incompatible with the active ingredient.Examples of substances that can function as pharmaceutically acceptable excipients include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, 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) cocoa butter and other waxes; (9) oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, 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; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic and compatible substances used in pharmaceutical formulations. Other non-limiting examples of the specific use of such pharmaceutical carriers are described 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 13. thIt can be found in “Brunton et al., eds., McGraw-Hill Professional, 2017)” and “Handbook of Pharmaceutical Excipients” (Sheskey et al., APhA Publications, 9th edition 2020). 【0072】 In some embodiments, the pharmaceutical composition further comprises one or more additional pharmaceutically acceptable components, such as, for example, buffers, preservatives, isotonic agents, salts, antioxidants, physiological substances, pharmacological substances, fillers, emulsifiers, wetting agents, etc. Assuming that the resulting formulation is pharmaceutically acceptable, the pharmaceutical composition can be prepared using various buffers and methods for adjusting pH. Non-limiting examples of such buffers include acetate buffer, citrate buffer, phosphate buffer, neutral buffered saline, phosphate buffered saline, and borate buffer. It is understood that the pH of the composition can be adjusted using an acid or a base as needed. Pharmaceutically acceptable antioxidants include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene. Useful preservatives include, but are not limited to, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate and stabilized oxychloride compositions, such as PURITE™. Suitable tonicity modifiers for inclusion in the pharmaceutical composition include, for example, salts such as sodium chloride, potassium chloride, mannitol or glycerin, and other pharmaceutically acceptable tonicity modifiers, but are not limited thereto. It is understood that these and other substances known in the art of pharmacology can be included in the pharmaceutical composition. 【0073】 In some embodiments, the nucleic acids, expression vectors, or virions described herein are formulated with one or more biocompatible polymers. In some embodiments, the nucleic acids, expression vectors, or virions described herein are formulated within liposomes. See, for example, US2017 / 0119666. In some embodiments, the nucleic acids, expression vectors or virions described herein are formulated within nanoparticles. Examples of nanoparticles include, but are not limited to, polyalkylcyanoacrylate nanoparticles, nanoparticles containing poly(lactic acid), nanoparticles containing poly(lactic-co-glycolic acid) (PLGA) nanoparticles, and the like. In some embodiments, the nucleic acids, expression vectors or virions described herein are formulated within hydrogels. 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), poly(lactide-co-glycolide) (PLGA), polyester, hyaluronic acid, and the like. In some embodiments, the nucleic acids, expression vectors, or virions described herein are present in buffered saline. In some embodiments, the buffered saline is from about 50 μL to 1000 μL by volume (including any range between these values). In some embodiments, a volume of 50 μL to 1000 μL contains a unit dose of the nucleic acids, expression vectors, or virions described herein. 【0074】 Method for restoring or enhancing visual function In some embodiments, a method of restoring or enhancing visual function in a subject, the method comprising administering to the subject's eye a nucleic acid described herein, an expression vector described herein, a virion described herein, or a pharmaceutical composition described herein. In some embodiments, the subject is a mammal, such as a mouse, a human, or a non-human primate (e.g., a macaque or a cynomolgus monkey). In some embodiments, the nucleic acid, expression vector, virion, or pharmaceutical composition described herein is administered via intravitreal (IVT) injection, subretinal (SR) injection, intraocular injection, or suprachoroidal injection. Other suitable modes of administration include, for example, periocular injection, subconjunctival injection, retrobulbar injection, intrascleral injection, and intracameral injection. In some embodiments, the nucleic acid, expression vector, virion, or pharmaceutical composition described herein is administered to the subject over a period ranging from about 1 day to about 1 year (including any range between these values, e.g., about 1 week to about 2 weeks, about 2 weeks to about 1 month, about 1 month to about 6 months, about 6 months to about 1 year). In some embodiments, the nucleic acid, expression vector, virion, or pharmaceutical composition described herein is administered to the subject over a period exceeding 1 year. After administration of the nucleic acid, expression vector, virion, or pharmaceutical composition, the melanopsin variant described herein is produced in retinal cells (e.g., retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, or photoreceptor cells, e.g., rod cells or cone cells, Müller cells, or retinal pigment epithelial cells), and expression of the melanopsin variant in retinal cells results in enhancement or restoration of the subject's visual function. Tests for visual function are known in the art, and any known test can be applied to evaluate the visual function of a subject administered the nucleic acid, expression vector, virion, or pharmaceutical composition described herein. 【0075】 In some embodiments, the subject is a human subject. In some embodiments, the human subject has a degenerative disease or disorder that affects the retina. In some embodiments, the human subject has reduced sensitivity to light. In some embodiments, neurons in the subject's retinal circuitry (e.g., bipolar cells, amacrine interneurons, and / or ganglion cells that output to the brain) are made directly light-sensitive by expressing the melanopsin variants described herein within the subject's retina. In some embodiments, the human subject has reduced visual function due to loss of rod and cone photoreceptors. In some embodiments, the subject has a hereditary retinal degenerative disease (IRD), such as Leber congenital amaurosis (LCA), retinitis pigmentosa, Usher syndrome, Stargardt disease, cone-rod dystrophy (CRD), color blindness, choroideremia, retinal detachment, or Bardet-Biedl syndrome. In some embodiments, the human subject has an eye disease. In some embodiments, the human subject has an eye disease including, but not limited to, macular degeneration (such as age-related macular degeneration), diabetic retinopathy, geographic atrophy, and cone dystrophy. In some embodiments, the human individual has retinal damage or retinal detachment due to injury (e.g., blunt trauma, blast injury, head impact, acute light injury, UV light injury, laser injury, chemical injury, etc.). In some embodiments, the human individual has retinal damage or retinal detachment due to an infectious disease. 【0076】 Kits and products In some embodiments, provided is a kit or product comprising one or more nucleic acids, expression vectors, virions, or pharmaceutical compositions disclosed herein for use according to a method of restoring or enhancing the visual functions described herein. In some embodiments, the kit comprises a pharmaceutical composition in lyophilized form and a solution for reconstituting the pharmaceutical composition prior to administration to a subject. In some embodiments, the kit further comprises instructions for administering one or more nucleic acids, expression vectors, virions, or pharmaceutical compositions herein to the eye of a subject (e.g., a human subject) via intravitreal injection, subretinal injection, intraocular injection, suprachoroidal injection, or other administration routes described herein. 【0077】 In some embodiments, the kit comprises pharmaceutically acceptable excipients, buffers, solutions, etc. for administering the pharmaceutical composition. In some embodiments, the kit further comprises instructions regarding appropriate operating parameters in the form of a label or separate insert. For example, the kit may have standard instructions notifying a physician or laboratory technician of preparing a therapeutically effective dose of a nucleic acid, expression vector, virion, or pharmaceutical composition and / or reconstituting a lyophilized composition. In some embodiments, the kit further comprises an administration device such as a syringe, filter needle, extension tube, cannula, or other instrument to facilitate injection of the pharmaceutical composition into the eye of the subject. Exemplary injection routes are described elsewhere herein. In some embodiments, the kit comprises a pharmaceutical composition in the form of a suspension or refrigerated suspension, as well as a syringe and / or a buffer for dilution. In some embodiments, the kit comprises a prefilled syringe containing a suspension or refrigerated suspension. 【0078】 All publications and patent applications mentioned herein are hereby incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. 【0079】 The foregoing description is presented to enable a person skilled in the art to make and use various embodiments. The description of specific compositions, techniques, and applications is provided by way of example only. Various modifications to the embodiments described above will be readily apparent to those skilled in the art, and the general principles defined herein can be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not intended to be limited to the examples described and shown herein, but rather should be accorded a scope consistent with the claims. 【Examples】 【0080】 A more complete understanding of the present disclosure may be obtained by referring to the following examples. However, these examples should not be construed as limiting the scope of the present disclosure. Of course, the examples and embodiments described herein are for illustrative purposes only, and in view of them, various modifications or changes will be shown to those skilled in the art, but they are included in the spirit and scope of the present application as well as the appended claims. Example 1A: Characterization of the Amplitude / Conductance Photoresponse and Off-Photon Response of Melanopsin Variants 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 【Table 1-7】 【Table 1-8】 【Table 1-9】 【Table 1-10】 【Table 1-11】 【Table 1-12】 【Table 1-13】 【Table 1-14】 【Table 1-15】 【Table 1-16】 【Table 1-17】 【Table 1-18】 【Table 1-19】 【Table 1-20】 【Table 1-21】 【Table 1-22】 【Table 1-23】 【0081】 To measure the amplitude / conductance light response and off-light response of the melanopsin variants shown in Table A above, the following experiments were conducted. 【0082】 Briefly, HEK293T cells were transfected with either (a) a nucleic acid encoding one of the melanopsin variants of Table A, and (b) a nucleic acid encoding GCaMP6s (an exemplary fluorescent calcium sensor described in Chen et al. (2013) Nature. 499(7458):295 - 300) or (c) a nucleic acid R - GECO1 (an exemplary fluorescent calcium sensor described in Zhao et al. (2011) Science. 333(6051):1888 - 1891). Parallel sets of transfections were performed using either (a) a nucleic acid encoding wild - type melanopsin, and (b) a nucleic acid encoding GCaMP6s or (c) a nucleic acid R - GECO1. After 72 hours, the cells were imaged with an ImageXpress Micro confocal system (Molecular Devices) to record the changes in fluorescence over time. To measure the amplitude / conductance light - on response, cells co - transfected with GCaMP6 were imaged under the FITC channel, which enabled recording from the calcium - induced fluorescence of GCaMP6s while the melanopsin or melanopsin variant was activated. To measure the off - light response, cells co - transfected with R - GECO1 were irradiated briefly with 480 nm light to activate the melanopsin or melanopsin variant and then recorded under the TexasRed channel to record from the R - GECO1 calcium - induced fluorescence. The amplitude of the light response from the on - assay was calculated from the starting baseline recording point to the maximum fluorescence achieved. The average decay lifetime, Tau off, from the off - assay was calculated from the inverse of the rate constant K, which was determined by the first - order rate law from the decay of fluorescence measured over time. 【0083】 In the on-assay, the light-induced intracellular calcium flux of melanopsin or melanopsin variants resulted in fluorescence from GCaMP6s. The light-induced fluorescence increased logarithmically over time. The amplitude of the calcium-induced fluorescence correlated with the photosensitivity of WT human melanopsin or melanopsin variants. Melanopsin variants with larger amplitude / photosensitivity were identified. 【0084】 The off-assay measures the termination of the light response of WT human melanopsin or melanopsin variants, and the fluorescence induced by R-GECO1 calcium decreases exponentially over time due to the depletion of intracellular calcium. Melanopsin variants that turned off faster than WT melanopsin, i.e., had a smaller Tau off than WT melanopsin, were identified. 【0085】 Figures 2 and Table B show the results of experiments performed to determine the amplitude of the calcium light response ("on" light response) of melanopsin truncation variants. In the amplitude assay ("on" assay) using GCaMP6s, the amplitude of the light response was determined using the difference between the baseline and the peak of the light-induced fluorescence. Some truncation variants containing 419aa and 425aa had a larger amplitude than the wild type. Error bar = standard error of the mean (SEM). [Table 2] 【0086】 Figures 3 and Table C show the results of experiments performed to determine the Tau off of the calcium light response of melanopsin truncation variants. In the off-assay using RGECO-1, the average decay time Tau off was determined using the rate constant k for the decay of the induced fluorescence response. Some truncation variants containing 413AA, 419AA, and 420AA had a lower Tau off than the wild type. Error bar = SEM [Table 3] 【0087】 Figure 4 shows a comparison of the amplitudes for Tau off in the truncation variants shown in Figures 2 and 3. Variants such as 419AA (black circles) have both a smaller Tau off and a larger amplitude than the wild type (white circles). Error bars = SEM. 【0088】 Figures 5 and Table D show the results of experiments carried out to determine the amplitude of the calcium light response of the chimeric melanopsin variants. In the on assay using GCaMP6s, the amplitude of the light response was determined using the difference between the baseline and the peak of the light-induced fluorescence. Error bars = SEM. 【Table 4】 【0089】 Figures 6 and Table E show the results of experiments carried out to determine the Tau off of the calcium light response of the chimeric melanopsin variants. In the off assay using RGECO-1, the average decay time Tau off was determined using the rate constant k for the decay of the induced fluorescence response. Error bars = SEM. 【Table 5】 【0090】 Figures 7 and Table F show the results of experiments carried out to determine the Tau off of the calcium light response of the fastest chimeric melanopsin variant shown in Figure 6. In the off assay using RGECO-1, the average decay time Tau off was determined using the rate constant k for the decay of the induced fluorescence response. Error bars = SEM. Figure 8 shows a comparison of the amplitude of the on response to Tau off in the fastest chimeric melanopsin variant shown in Figures 7 and Table F. The variants are black circles and the wild type is white circles. 【Table 6】 【0091】 Figures 9 and Table G show the results of experiments performed to determine the amplitude of the calcium light response of substituted full-length melanopsin variants. In the on-assay using GCaMP6s, the amplitude of the light response was determined using the difference between the baseline and the peak of the light-induced fluorescence. Error bars = SEM. 【Table 7】 【0092】 Figures 10 and Table H show the results of experiments performed to determine the Tau off of the calcium light response of substituted full-length melanopsin variants. In the off-assay using RGECO-1, the average decay time Tau off was determined using the rate constant k for the decay of the induced fluorescence response. Error bars = SEM. 【Table 8】 【0093】 Figures 11 and Table I show the results of experiments performed to determine the Tau off of the calcium light response of the fastest substituted full-length melanopsin variant shown in Figure 10. In the off-assay using RGECO-1, the average decay time Tau off was determined using the rate constant k for the decay of the induced fluorescence response. Error bars = SEM. Figure 12 shows a comparison of the amplitude of the on-response to Tau off in the fastest substituted full-length melanopsin variant of Figure 11. The variants are black circles and the wild type is white circles. Error bars = SEM 【Table 9】 【0094】 Figures 13 and Table J show the amplitude of the calcium light response of substituted melanopsin variants in a truncated backbone (419AA, SEQ ID NO: 3). In the assay using GCaMP6s, the amplitude of the light response was determined using the difference between the baseline and the peak of the light-induced fluorescence. Error bars = SEM. 【Table 10】 【0095】 Figures 14 and Table K show the results of experiments carried out to determine the Tau off of the calcium photoreaction of substituted melanopsin variants in a truncated backbone (419AA, SEQ ID NO: 3). In the off assay using RGECO-1, the average decay time Tau off was determined using the rate constant k for the decay of the induced fluorescence response. Error bars = SEM. [Table 11] 【0096】 Figures 15 and Table L show the results of experiments carried out to determine the Tau off of the calcium photoreaction of the fastest substituted melanopsin variant in the truncated backbone shown in Figure 14. In the off assay using RGECO-1, the average decay time Tau off was determined using the rate constant k for the decay of the induced fluorescence response. Error bars = SEM. Figure 16 shows a comparison of the amplitude of the on response to the Tau off in the fastest substituted full-length melanopsin variant of Figure 14. The variants are black circles and the wild type is white circles. Error bars = SEM. [Table 12] 【0097】 Example 1B: Evaluation of the electrophysiological properties of melanopsin variants Patch-clamp experiments were performed to measure the ionic activity of HEK293T cells expressing the melanopsin variants shown in Table A. Parallel experiments were performed using HEK293T cells expressing wild-type melanopsin. Briefly, HEK293T cells were transfected with (a) a nucleic acid encoding wild-type melanopsin or a melanopsin variant, and (b) the G protein-gated inwardly rectifying K + channel 1 F137S (GIRK1 F137STransfect nucleic acids encoding ( 2 ). Place the transfected cells in an external recording solution of 110 mM NaCl, 30 mM KCl, 2 mM CaCl2, 10 mM HEPES, 5 mM glucose (pH 7.4), and record using a glass pipette with a high potassium solution containing 120 mM KCl, 1.75 mM MgCl2, 5.735 mM CaCl2, 10 mM EGTA, 5 mM Na2ATP, and 10 mM HEPES (pH 7.2). Cells are whole-cell patch-clamped using a HEKA amplifier. 10 mM acetylcholine (as a GIRK activator) and 5 mM BaCl2 (full block) are applied using a gravity-driven perfusion system and controlled using a DG-4 system (Sutter) in combination with an excitation filter for irradiation. Irradiate the cells with light of different wavelengths to find the wavelength at which WT human melanopsin and melanopsin variants are most sensitive (i.e., the "sensitive wavelength"). While irradiating at the "sensitive wavelength", expose WT human melanopsin and melanopsin variants to a light intensity range (1E+10 ph / cm 2 / s to 10E+14 ph / cm 【0098】 / s) and identify the intensity at which WT human melanopsin and melanopsin variants are most sensitive. The activities of WT human melanopsin and melanopsin variants in Table A are characterized in a mouse model of retinal degeneration. In the C3H / HeJ (Rd1) mouse strain, mice experience retinal degeneration by weaning age due to a mutation in PDE beta that encodes the beta subunit of rod photoreceptor cGMP phosphodiesterase. This degeneration is consistent with retinitis pigmentosa experienced by human individuals with mutations in human PDE beta. Package WT human melanopsin or melanopsin variants under the ubiquitous CMV promoter into AAV2.7m8 and intravitreally inject 40-day-old Rd1 mice at approximately 1E+10 vg / eye. After 49 - 50 days, measure visual acuity by optokinetic response test (OKR), determine retinal light responses by full-field electroretinogram (ffERG), and record brain light responses by visual evoked potential (VEP). 【0099】 Although the present disclosure has been described in some detail by way of illustration and example for purposes of clear understanding, these descriptions and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patents and scientific documents cited herein are hereby expressly incorporated by reference in their entirety. 【0100】 Example 3: Further Characterization of the Amplitude / Conductance Photoresponse and Off-Light Response of Melanopsin Variants Melanopsin has many features of the highest class as a light-generating protein, but the wild-type protein (sequence schematic shown in FIG. 1) has slow deactivation and rapid continuous reactivation is hindered. Table M describes the mutagenesis strategy carried out to generate a calcium-mediated rapid opsin. Such strategies have been discussed elsewhere in this specification, including Examples 1A and 1B and the forms for carrying out the invention. 【0101】 FIG. 17 shows a schematic of such a melanopsin variant development strategy. Approximately 70 melanopsin variants, including truncation variants, point mutation variants, and chimeras (see, for example, Examples 1A and 1B and Tables A and M), were screened by a fluorescence calcium imaging assay in the first round of screening. In the second round of screening, approximately 40 melanopsin variants that were chimeras with truncations or synergistic point mutations were identified. For example, according to the assays described in Examples 1A and 1B and elsewhere in this specification, melanopsin variants showing an increase in amplitude and / or a decrease in Tau off compared to WT human melanopsin were selected for AAV production. 【0102】 Figure 18 shows the results of experiments performed to determine the amplitude of the calcium light response over time for WT human melanopsin and exemplary melanopsin variants 405AA, 425AA, V370-R377Del, and K356-R377Del. Figure 19 shows the results of experiments performed to determine the Tau off of the calcium light response over time for WT human melanopsin, exemplary melanopsin variants 405AA, 425AA, V370-R377Del, and K356-R377Del. In the amplitude assay using GCaMP6s (the "on" assay), the amplitude of the light response was determined using the difference between the baseline and the peak of the light-induced fluorescence. In the off assay using RGECO-1, the average decay time Tau off was determined using the rate constant k for the decay of the induced fluorescence response. 【0103】 The selection of such mutations is shown in the schematic diagram of Figure 1. Some N-terminal melanopsin mutations increase speed and light sensitivity. Only one mutation in the transmembrane domain was successful. The transmembrane domain of GPCRs is the most conserved. Loss of alcohol consistently improved amplitude and Tau off. The first 377 amino acids of melanopsin were required for the CTD chimera to be successful. Many of the successful mutations in the putative phosphorylation and / or GRK / arrestin binding regions were phosphomimics of amino acids that are normally phosphorylated during inactivation and mutations that decrease the positive charge. 【0104】 As described above, Figure 12 provides an exemplary comparison of the amplitude of the on response and the amplitude of the Tau off response for a subset of the melanopsin variants shown in Table A. Error bars = standard error of the mean (SEM). The second round of melanopsin variants included truncation variants and chimeric variants containing synergistic point mutations. Melanopsin variants that showed both an increase in amplitude and a decrease in Tau off, such as 419AA_S183A_S384D_R386A (SEQ ID NO: 19), were selected for AAV production. 【0105】 Figure 20 shows the results of an experiment performed to determine the amplitude of the calcium light response of cells transduced with AAV2.7m8 carrying WT human melanopsin or a melanopsin variant containing SEQ ID NO: 19. 【Chemical formula】 【0106】 SEQ ID NO: 19 contains amino acids 1-419 of WT human melanopsin (SEQ ID NO: 3) and has been engineered to contain S183A, S384D, and R386A substitutions (see Table A). 【0107】 AAV2.7m8-CMV-WT human melanopsin and AAV2.7m8-CMV-SEQ ID NO: 19 were generated using AAVMAX suspension cells, an AVB column, CsCl ultracentrifugation, and buffer exchange. The resulting rAAV was analyzed by ddPCR titer, Western blot, silver staining, and alkaline gel. 【0108】 HEK293T cells were transduced with AAV2.7m8-CMV-SEQ ID NO: 19 or AAV2.7m8-CMV-WT human melanopsin and AAV2.7m8-CMV-GCaMP6 at an MOI of 5 × 10 4 vg per AAV and imaged at 480 nm using an ImageXpress Micro (Molecular Devices). AAV2.7m8 packaged with CMV-WT human melanopsin or CMV-SEQ ID NO: 19 successfully generated a light response in HEK293T. The light response of SEQ ID NO: 19 was larger and faster than that of WT human melanopsin. 【0109】 Figure 21 shows the results of an experiment performed to determine the amplitude of the calcium light response of HEK293T cells transduced with different MOIs of AAV2.7m8-SEQ ID NO: 19. With fewer transduced AAVs, the maximum amplification is lower. The amplitude of the light response varied with the dose of AAV2.7m8-SEQ ID NO: 19 applied. 【0110】 The present disclosure has been described in some detail by way of illustration and example for the purpose of a clear understanding, but these descriptions and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety.

Claims

[Claim 1] A melanopsin variant comprising an amino acid sequence corresponding to amino acids 1 to 425 or less of wild-type human melanopsin described in Sequence ID No. 1, wherein the amplitude / conductance and / or off-kinetics of the melanopsin variant are greater than the amplitude / conductance of wild-type human melanopsin and / or faster than the off-kinetics of wild-type human melanopsin. [Claim 2] A melanopsin variant according to claim 1, comprising the sequence described in any one of SEQ ID NOs: 2, 3, 4, 82, 83, and 84, or a variant thereof comprising one or more amino acid substitutions. [Claim 3] The melanopsin variant according to claim 2, wherein the variant comprises an amino acid sequence having substitutions at one or more amino acid positions in P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and R390, the amino acid positions being those of the wild-type human melanopsin described in SEQ ID NO:

1. [Claim 4] A melanopsin variant according to claim 3, comprising one or more substitutions selected from the group consisting of P10F, T83L, T129S, Q135N, S183A, Y212F or Y212A, E215S, M226S or M226T, Y382E or Y382D, S384D, R386A, and R390A or R390D. [Claim 5] The melanopsin variant according to claim 1, wherein the melanopsin variant comprises any sequence of sequence numbers 5 to 23. [Claim 6] A melanopsin variant comprising an amino acid sequence corresponding to at least amino acids 1 to 377 of wild-type human melanopsin described in Sequence ID No. 1, fused with a heterologous G protein coupling receptor (GPCR) or its CTD variant, wherein the amplitude / conductance and / or ofkinetics of the melanopsin variant are greater than the amplitude / conductance and / or faster than the ofkinetics of wild-type human melanopsin. [Claim 7] (a) The CTD of the heterogeneous GPCR or its CTD variant is the CTD of the visual opsin or its variant, (b) The melanopsin variant according to claim 6, wherein the CTD of the visual opsin or its variant is the CTD of (i) wild-type D. melanogaster rhodopsin 1, (ii) wild-type human rhodopsin, (iii) wild-type human short-wavelength opsin (hOPN1SW), (iv) wild-type human medium-wavelength opsin, or (v) wild-type human long-wavelength opsin. [Claim 8] The melanopsin variant according to claim 7, wherein the CTD of the visual opsin or its variant comprises the amino acid sequence described in any one of SEQ ID NOs: 27-30 and 32-33. [Claim 9] A melanopsin variant according to claim 6, comprising the amino acid sequence described in any one of sequence numbers 39-42 and 44-45. [Claim 10] The melanopsin variant according to claim 6, wherein the variant comprises an amino acid sequence having substitutions at one or more amino acid positions in P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and R390, the amino acid positions being relative to wild-type human melanopsin as described in SEQ ID NO:

1. [Claim 11] A melanopsin variant according to claim 10, comprising one or more substitutions selected from the group consisting of P10F, T83L, T129S, Q135N, S183A, Y212F or Y212A, E215S, M226S or M226T, Y382E or Y382D, S384D, R386A, and R390A or R390D. [Claim 12] The melanopsin variant according to claim 6, wherein the CTD variant comprises one or more amino acid substitutions relative to wild-type CTD. [Claim 13] A melanopsin variant comprising an amino acid sequence having substitutions at one or more amino acid positions in P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and R390, wherein the amino acid positions are relative to wild-type human melanopsin described in Sequence ID No. 1, and the amplitude / conductance and / or off-kinetics of the melanopsin variant are greater than the amplitude / conductance and / or faster than the off-kinetics of wild-type human melanopsin. [Claim 14] A melanopsin variant according to claim 13, comprising one or more substitutions selected from the group consisting of P10F, T83L, T129S, Q135N, S183A, Y212F or Y212A, E215S, M226S or M226T, Y382E or Y382D, S384D, R386A, R390A or R390D. [Claim 15] (a) The amplitude / conductance of the melanopsin variant is at least 1.25 times greater than the amplitude / conductance of wild-type human melanopsin in HEK293T cells, or (b) The melanopsin variant according to claim 1, wherein the of-kinetics of the melanopsin variant are at least 1.10 times faster than the of-kinetics of wild-type human melanopsin in HEK293T cells. [Claim 16] A nucleic acid comprising a polynucleotide sequence encoding the melanopsin variant according to any one of claims 1 to 15. [Claim 17] The nucleic acid according to claim 16, which is operably linked to a promoter. [Claim 18] The nucleic acid according to claim 17, wherein the promoter is a retinal cell-specific promoter. [Claim 19] The nucleic acid according to claim 18, wherein the retinal cell-specific promoter is selected from the group consisting of human synapsin (hSyn), SNCG, NEFH, NEFL, 4xgrm6, and grm6. [Claim 20] The nucleic acid according to claim 17, further comprising one or more enhancer sequences, intron sequences, leader sequences, Kozak sequences, polyA sequences, stuffer sequences, and / or terminal inversion repeat (ITR) sequences. [Claim 21] Recombinant virions, (a) Capsid protein and (b) The recombinant virion comprising the nucleic acid according to claim 16. [Claim 22] The recombinant virion according to claim 21, wherein the capsid protein is selected from the group consisting of AAV2-7m8, AAV2, AAV2-4YF, AAV9, AAV9-7m8, R100, and LSV1. [Claim 23] A host cell comprising the nucleic acid described in claim 16. [Claim 24] The host cell according to claim 23, further comprising one or more of the following: (i) Polynucleotides encoding capsid proteins, (ii) Polynucleotides encoding the rep protein, and (iii) AAV Helper function. [Claim 25] A method for generating recombinant virions, (a) Culturing the host cells described in claim 24 under conditions that produce the recombinant virions, (b) The method comprising recovering the recombinant virions produced by the host cells. [Claim 26] The method according to claim 25, further comprising purifying the recombinant virion. [Claim 27] A pharmaceutical composition comprising a recombinant virion according to claim 21 and a pharmaceutically acceptable excipient. [Claim 28] A pharmaceutical composition according to claim 27 for restoring or enhancing the visual function of a target. [Claim 29] The pharmaceutical composition according to claim 28, for use by intraocular injection, subretinal injection, suprachoroidal injection, or intravitreal injection. [Claim 30] (a) Subjects having an eye disease or disorder selected from the group consisting of retinitis pigmentosa, macular degeneration, retinoschisis, Leber congenital amaurosis, diabetic retinopathy, geographic atrophy, choroidemia, cone dystrophy, and cone-rod dystrophy, or (b) Subjects who have experienced retinal detachment or photoreceptor loss due to eye disease, infection, trauma, injury, head impact, acute photoinjury, UV photoinjury, laser injury, or chemical injury. The pharmaceutical composition according to claim 28, for use in administration to a person.

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