Black vision protein variants for vision recovery

Abstract

Provided herein, inter alia, are melanopsin variants that exhibit greater amplitude / conductance and / or faster off kinetics than the amplitude / conductance and / or off kinetics of wild-type human melanopsin. Related nucleic acids, virions, host cells, methods of producing recombinant virions, and pharmaceutical compositions are also provided. Also provided are methods of restoring or enhancing visual function in a subject using such melanopsin variants.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to 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. 63,466181 filed on May 12, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (627002001440SEQLIST.xml; size: 162,974 bytes; and creation date: June 5, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present application relates to melanopsin variants and their use for restoring or enhancing visual function in subjects whose vision is impaired due to loss of photoreceptors. Background Art

[0006] Degeneration of light-detecting rod and cone photoreceptors in the human retina (e.g., due to disease, infection, or injury) often leads to severe visual impairment and, in some cases, legal blindness in millions of people worldwide (McClements et al. (2020) Front Neurosci. 14:57090). However, although the causes of retinal degeneration may be different, there are considerable similarities in the physiological changes that occur in the retina. When patients suffer from photoreceptor cell loss but retain the remaining layers of cells in the neural retina, it is possible to restore vision through optogenetic therapy, i.e., providing light-sensitive molecules to the surviving cell types of the retina, which enable light perception by residual neurons. Current optogenetic methods are limited by low light sensitivity, slow kinetics, and / or narrow spectral responses. In addition, current methods lack adaptation to changes in ambient light. Therefore, there is a need in the art for improved optogenetic methods to treat vision loss caused by rod and cone degeneration. Summary of the invention

[0007] In some embodiments, a melanopsin variant is provided, comprising no more than amino acids 1-425 of the wild-type human melanopsin shown in SEQ ID NO: 1, wherein 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 the wild-type human melanopsin. In some embodiments, the melanopsin variant comprises a sequence as set forth in any one of SEQ ID NO: 2, 3, 4, 82, 83, and 84, or a variant thereof, containing one or more amino acid substitutions. In some embodiments, the melanopsin variant comprises one or more amino acid substitutions at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390, wherein the amino acid position is relative to the wild-type human melanopsin shown in SEQ ID NO: 1. In some embodiments, the one or more substitution mutations 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.

[0008] In some embodiments, the melanopsin variant comprises a P10F substitution. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO:5. In some embodiments, the melanopsin variant comprises (such as also comprising) a T83L substitution. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO:6. In some embodiments, the melanopsin variant comprises (such as also comprising) a T129S substitution. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO:7. In some embodiments, the melanopsin variant comprises (such as also comprising) a Q135N substitution. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO:8 or 9. In some embodiments, the melanopsin variant comprises (such as also comprising) an S183A substitution. In some embodiments, the melanopsin variant comprises any one of SEQ ID NO:10-12. In some embodiments, the melanopsin variant comprises (such as also comprising) a Y212F substitution. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO:13. In some embodiments, the melanopsin variant comprises (such as also comprises) an M226S or M226T substitution. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO: 14 or 15. In some embodiments, the melanopsin variant comprises (such as also comprises) a Y382E or Y382D substitution. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO: 16. In some embodiments, the melanopsin variant comprises (such as also comprises) an S384D substitution. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO: 17. In some embodiments, the melanopsin variant comprises (such as also comprises) an R386A substitution. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO: 18 or 19. In some embodiments, the melanopsin variant comprises (such as also comprises) an R390A or R390D substitution. In some embodiments, the melanopsin variant comprises the sequence shown in any one of SEQ ID NO: 20-23.

[0009] In some embodiments, a melanopsin variant is provided, comprising at least amino acids 1-377 of wild-type human melanopsin as shown 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 wild-type human melanopsin and / or faster than the off kinetics of wild-type human melanopsin. In some embodiments, the CTD of the heterologous GPCR or its CTD variant is the CTD of vision opsin or its variant. In some embodiments, the CTD of vision 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. In some embodiments, the CTD of the vision opsin or its variant comprises the amino acid sequence shown in any one of SEQ ID NOs: 27-30 and 32-33. In some embodiments, the black opsin variant comprises the amino acid sequence shown in any one of SEQ ID NOs: 39-42, 44-45 and 63-65. In some embodiments, the black opsin variant further comprises one or more substitution mutations at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, R390, wherein the amino acid position is relative to the wild-type human black opsin shown in SEQ ID NO: 1, wherein the amino acid position is relative to the wild-type human black opsin shown in SEQ ID NO: 1. In some embodiments, the one or more substitution mutations 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 a heterologous GPCR or a CTD variant thereof.

[0010] In some embodiments, a melanopsin variant is provided, comprising one or more amino acid substitutions at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and R390, wherein the amino acid positions are relative to the wild-type human melanopsin shown in SEQ ID NO: 1, and 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. In some embodiments, one or more substitution mutations 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.

[0011] In some embodiments, the melanopsin variant comprises any of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% overall sequence homology or identity with the melanopsin variant shown 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 with the melanopsin variant shown 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.

[0012] In some embodiments, the amplitude / conductance of the melanopsin variants provided herein is at least 1.25 times 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 times the off kinetics of wild-type human melanopsin in HEK293T cells.

[0013] Also provided herein is a nucleic acid 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 synaptic protein (hSyn), SNCG, NEFH, NEFL, 4xgrm6 and grm6. In some embodiments, the nucleic acid also comprises one or more enhancer sequences, intron sequences, leader sequences, Kozak sequences, polyA sequences, stuffing sequences and / or inverted terminal repeat (ITR) sequences.

[0014] Also provided herein is a recombinant virion comprising: (a) a capsid protein, and (b) a nucleic acid provided herein. In some embodiments, the capsid protein is selected from the group consisting of: AAV2-7m8, AAV2, AAV2-4YF, AAV9, AAV9-7m8, R100, and LSV1.

[0015] In some embodiments, a host cell is provided, comprising a nucleic acid as described herein. In some embodiments, the host cell further comprises one or more of the following: (i) a polynucleotide encoding a capsid protein; (ii) a polynucleotide encoding a rep protein; and (iii) an AAV helper function. In some embodiments, a method for producing a recombinant virion is provided, comprising: (a) culturing a host cell as described in claim 47 under conditions that produce a recombinant virion, and (b) recovering the recombinant virion produced by the host cell. In some embodiments, the method further comprises the step of purifying the recombinant virion.

[0016] Also provided herein is a pharmaceutical composition comprising a recombinant virion as described herein and a pharmaceutically acceptable excipient. In some embodiments, a method for restoring or enhancing the visual function of a subject is provided, comprising applying a pharmaceutical composition provided herein to the subject's eyes. In some embodiments, administration includes intraocular injection, subretinal injection, suprachoroidal injection or intravitreal injection. In some embodiments, the subject suffers from an ocular disease or disorder selected from the group consisting of: retinitis pigmentosa, macular degeneration, retinoschisis, Leber's congenital amaurosis (Leber's 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 ocular disease, infection, trauma, injury, head impact, acute light damage, UV light damage, laser damage or chemical damage. In some embodiments, the subject is a person.

[0017] It should be understood that one, some or all of the properties 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 become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description.

[0018] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety to the same extent as if each reference was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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 indicated.

[0020] Figure 2 Shown are the results of experiments performed to determine the amplitude of the calcium light response from truncation variants of melanopsin.

[0021] Figure 3 Shown are the results of experiments performed to determine TauOFF from calcium light responses of truncation variants of melanopsin.

[0022] Figure 4 A comparison of the amplitude of the truncated variants to TauOFF is shown. For example, the 419AA variant (black circles) has a smaller TauOFF and a larger amplitude than the wild type (white circles).

[0023] Figure 5 Shown are the results of experiments performed to determine the amplitude of calcium light responses from chimeric variants of melanopsin.

[0024] Figure 6 Shown are the results of experiments performed to measure TauOFF from calcium light responses of chimeric melanopsin variants.

[0025] Figure 7 Shown is the determination of Figure 6 Results of experiments performed with TauOFF of the calcium light response of the fastest chimeric melanopsin variant shown in . Error bars = SEM.

[0026] Figure 8 Shows Figure 7Amplitudes of the fastest chimeric melanopsin variants compared to TauOFF shown in. Variants are black circles and wild type are white circles.

[0027] Fig. 9 Shown are the results of experiments performed to determine the amplitude of calcium light responses from substituted full-length melanopsin variants.

[0028] Fig.10 Shown are the results of experiments performed to determine TauOFF from calcium light responses of substituted full-length melanopsin variants.

[0029] Fig.11 Shown is the determination of Fig.10 Results of experiments performed to determine the TauOFF calcium light response of the fastest substituted full-length melanopsin variant shown in . Error bars = SEM.

[0030] Fig.12 Shows Fig.10 Amplitudes of the fastest substituted full-length melanopsin variants shown in compared to TauOFF. Variants are black circles and wild type are white circles. Error bars = SEM.

[0031] Fig.13 Shown are the results of experiments performed to determine the amplitude of the calcium light response of melanopsin variants derived from substitutions in the truncated backbone (419AA, SEQ ID NO: 3).

[0032] Fig.14 Shown are the results of experiments performed to determine the TauOFF of the calcium light response of melanopsin variants derived from substitutions in the truncated backbone (419AA, SEQ ID NO: 3).

[0033] Fig.15 Shown is the determination of Fig.14 Results of TauOFF experiments performed on the calcium light response of the fastest substituted melanopsin variant in the truncated backbone are shown.

[0034] Fig.16 Shows Fig.14 Amplitudes of the fastest substituted melanopsin variants in the truncated backbone shown in , compared to TauOFF. Variants are black circles and wild type are white circles. Error bars = SEM.

[0035] Fig.17 Schematic showing the development strategy used to design melanopsin variants

[0036] Fig.18Shown are the results of experiments performed to determine the amplitude of the calcium light response over time for WT human melanopsin and melanopsin variants 405AA, 425AA, V370-R377Del, and K356-R377Del.

[0037] Fig.19 Shown are the results of experiments performed to determine TauOFF of the calcium light response of WT human melanopsin and melanopsin variants 405AA, 425AA, V370-R377Del, and K356-R377Del over time.

[0038] Fig. 20 Shown are the results of experiments performed to determine the amplitude of calcium light responses from cells transduced with AAV2.7m8 carrying WT human melanopsin or a melanopsin variant comprising SEQ ID NO:19.

[0039] Fig.21 Shown are 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-CMV-SEQ ID NO: 19. DETAILED DESCRIPTION

[0040] Overview

[0041] One of the purposes of optogenetic therapy is to provide expression of light-sensitive proteins, i.e., opsins, in damaged or degenerated retinal cells. However, the low photosensitivity and slow (seconds) kinetics of many opsins make them impractical for treating vision loss. Melanopsin variants are described herein, which exhibit, for example, greater amplitude / conductance (such as amplitude / conductance photoresponse) and / or faster OFF kinetics (such as faster OFF photoresponse) than wild-type human melanopsin. Such melanopsin variants can be used in methods for restoring or enhancing the visual function of a subject, who experiences photoreceptor loss or retinal detachment due to, for example, eye disease, infection, trauma, injury, head impact, acute light damage, UV light damage, laser damage, or chemical damage.

[0042] definition

[0043] Unless otherwise indicated, the compositions and methods described herein may use conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, immunochemistry, and ophthalmology techniques, which are within the skill of those skilled in the art. Such conventional techniques include methods for observing and analyzing the retina or vision of a subject, cloning and propagating recombinant viruses, preparing pharmaceutical compositions, and biochemical purification and immunochemistry. The specific description of suitable techniques may refer to the embodiments herein. However, of course, equivalent conventional methods may also be used. Such routine techniques and descriptions can be found in standard laboratory manuals, such as Green et al., ed., Genome Analysis: A Laboratory Manual Series (Volumes I-IV) (1999); Weiner et al., ed., Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler, ed., 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 Edition) WH Freeman, NY (1995); Gait, "Oligonucleotide Synthesis: A Practical Approach" IRL Press, London (1984); Nelson and Cox, Lehninger, Principles of Biochemistry, 3rd edition, WH Freeman Pub., New York (2000); and Berg et al., Biochemistry, 5th edition, WH Freeman Pub., New York (2002), all of which are incorporated herein by reference in their entirety for all purposes.

[0044] Before describing the embodiments herein in detail, it is to be understood that the present disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0045] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0046] As used herein, the term "about" refers to the usual error range of the corresponding value that is readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments for that value or parameter itself.

[0047] It should be understood that aspects and embodiments of the present disclosure include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0048] The terms "polypeptide," "protein," and "peptide" are used interchangeably herein and may refer to a polymer of two or more amino acids.

[0049] As used herein, the terms "treat / treating / treatment" or "ameliorate / ameliorating" and other grammatical equivalents refer to alleviating, reducing or ameliorating an ocular disease or condition or a symptom of an ocular disease or condition, preventing other symptoms of an ocular disease or condition, ameliorating or preventing a potential metabolic cause of a symptom, inhibiting an ocular disease or condition, such as arresting the development of an ocular disease or condition, alleviating an ocular disease or condition, causing regression of an ocular disease or condition, or stopping symptoms of an ocular disease or condition, and are intended to include prevention. These terms also include achieving a therapeutic benefit and / or a preventive benefit. The term "therapeutic benefit" refers to eradication or amelioration of the ocular disease or condition being treated. In addition, a therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with an ocular disease or condition, such that an improvement is observed in the patient, although in some embodiments, the patient still suffers from the ocular disease or condition. For preventive benefit, the pharmaceutical composition is administered to a patient who is experiencing or is at risk of vision loss, for example, due to photoreceptor cell loss, or to a patient who reports one or more physiological symptoms of vision loss, for example, due to photoreceptor cell loss. Patients whose vision loss progresses asynchronously may derive therapeutic benefit from treatment of the eye with more advanced vision loss and preventive benefit from treatment of the eye with less advanced vision loss.

[0050] As used herein, the term "administer / administering / administration, etc." may refer to methods for delivering a therapeutic agent or pharmaceutical composition to a desired biological site of action. These methods include intravitreal, subretinal, intraocular or suprachoroidal injection into the eye. Other suitable modes of administration are described elsewhere herein.

[0051] As used herein, the term "pharmaceutically acceptable" may refer to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compounds disclosed herein and is relatively non-toxic (i.e., it does not cause undesirable biological effects when the substance is administered to a subject, nor does it interact in a deleterious manner with any component of the composition in which it is contained).

[0052] 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, thickener, excipient, etc.

[0053] As used herein, "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") for transduction into target cells or target tissues, which contains a polynucleotide sequence of non-viral origin (e.g., a polynucleotide heterologous to the virus, such as a nucleic acid sequence encoding a therapeutic transgene (e.g., a melanopsin variant described herein)). In the case of rAAV, the heterologous polynucleotide is flanked by at least one and typically two AAV inverted terminal repeats (ITRs). rAAV vectors can be single-stranded (ssAAV) or self-complementary (scAAV). The term "recombinant viral vector" (e.g., rAAV) encompasses viral vector particles / virions and viral vector plasmids.

[0054] The terms "virus", "viral particle", "virion", "recombinant vector particle", "recombinant particle" and "recombinant virion" refer interchangeably to a viral particle comprising at least one viral capsid protein and a polynucleotide vector. If the particle comprises a heterologous polynucleotide (e.g., a polynucleotide different from the wild-type viral genome, such as a transgene to be delivered to a target cell or target tissue (e.g., a melanopsin variant)), it is typically referred to as a "recombinant vector particle", "recombinant vector" or "recombinant virion". Therefore, the production of a recombinant viral particle (e.g., rAAV) necessarily includes the production of a recombinant polynucleotide vector, such as a vector contained within a recombinant viral particle.

[0055] As used herein, the term "packaging" may refer to the series of intracellular events that may lead to the assembly and encapsidation of recombinant AAV particles.

[0056] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes".

[0057] The term "polypeptide" may encompass naturally occurring and non-naturally occurring proteins (e.g., fusion proteins), peptides, fragments, mutants, derivatives and analogs thereof. A polypeptide may be a monomer, dimer, trimer or multimer. In addition, a polypeptide may comprise a number of different domains, each of which has one or more different activities. For the avoidance of doubt, a "polypeptide" may be any length greater than two amino acids.

[0058] As used herein, "polypeptide variant" or simply "variant" refers to a polypeptide whose sequence contains amino acid modifications. In some embodiments, the modification is an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a reference protein or polypeptide such as a native or wild-type protein. The variant may have one or more amino acid point substitutions (wherein a single amino acid at one position has been changed to another amino acid), one or more insertions and / or deletions (wherein one or more amino acids are inserted or deleted, respectively, in the sequence of the reference protein), and / or truncations of the amino acid sequence at one or both of the amino and carboxyl termini. Compared to a reference protein or unmodified protein, a variant may have the same or different biological activity.

[0059] In some embodiments, the variant may have, for example, any of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% overall sequence homology with its corresponding reference protein. In some embodiments, the variant may have at least about 90% overall sequence homology with the wild-type protein. In some embodiments, the 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.

[0060] As used herein, "recombinant" can refer to a biological molecule, such as a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide to which the gene is found in nature, (3) is operably linked to a polynucleotide to which it is not linked in nature, or (4) does not occur in nature. The term "recombinant" can be used to refer to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNA encoded by such nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant if it is synthesized by mRNA synthesized by a recombinant gene present in a cell.

[0061] The term "expression vector" or "expression construct" or "box" or "plasmid" or simply "vector" may include any type of genetic construct, including vectors containing nucleic acids or polynucleotides encoding gene products (e.g., black opsin variants described herein), wherein part or all of the nucleic acid coding sequence can be transcribed and is suitable for gene therapy. The transcript can be translated into protein. In some embodiments, the transcript is partially translated or not translated. In certain aspects, expression includes transcription of the gene and translation of mRNA into a gene product (e.g., black opsin variant). In other aspects, expression includes only transcription of the nucleic acid encoding the target gene. The expression vector may also include control elements operably linked to the coding region to promote expression of the protein in the target cell. The combination of control elements and one or more genes to which they are operably linked for expression may sometimes be referred to as an "expression cassette," a large number of which are known and available in the art, or can be easily constructed from components available in the art. In some embodiments, the term "expression vector" refers to a genetic construct as well as a viral particle containing a genetic construct.

[0062] The term "heterologous" may refer to an entity that is genotypically different from the rest of the entity to which it is being compared. 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 it is not naturally found linked may be a heterologous promoter.

[0063] As used herein, the terms "subject", "individual" and "patient" are used interchangeably to refer to vertebrates, such as mammals. Mammals include, but are not limited to, rodents, apes, humans, non-human primates (e.g., cynomolgus monkeys, African green monkeys, macaques, farm animals, sports animals and pets).

[0064] It should be understood that one, some or all of the properties 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 become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description.

[0065] Melanopsin variants

[0066] Truncated melanopsin variants

[0067] In some embodiments, the present application provides a melanopsin variant comprising no more than amino acids 1-425 of the wild-type human melanopsin shown in SEQ ID NO: 1, wherein the amplitude / conductance (e.g., amplitude / conductance photoresponse) and / or off kinetics (e.g., OFF photoresponse) of the melanopsin variant is greater than the amplitude / conductance of the wild-type human melanopsin and / or faster than the off kinetics of the wild-type human melanopsin. Such melanopsin variants are also referred to herein as "truncated melanopsin variants". Figure 1 A schematic diagram of melanopsin integrated into the cell membrane is provided.

[0068] MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMS FTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCC FVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAI AQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISIRRRQESLGSESEVGWTHMEAAAVWGAAQQANGRSLYGQGLEDLEAKAPPRPQGHEAETPGKTKGLIPSQDPRM(SEQ ID NO:1)

[0069] In some embodiments, the melanopsin variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2-4.

[0070] MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQW LFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISIRRRQESLGSESEVGW(SEQ ID NO:2)

[0071] MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISIRRRQESLGSESEVG(SEQ ID NO:3)

[0072] MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISIRRRQESLG(SEQ ID NO:4)

[0073] MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNL TVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNL(SEQ ID NO:82)

[0074] MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISI(SEQ ID NO:83)

[0075] MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISIRRRQESLGSESEVGWTHMEA(SEQ ID NO:84)

[0076] The sites at which the truncated wild-type human melanopsin (SEQ ID NO:1) was truncated to generate SEQ ID NO:2, 3, 4, 82, 83 and 84 are indicated by arrows in Figure 1 the figure

[0077] In some embodiments, the melanopsin variant is a variant of SEQ ID NO: 2, 3, 4, 82, 83, or 84, comprising 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 comprises one or more amino acid substitutions at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390 (e.g., in any combination), wherein the amino acid positions are relative to the reference melanopsin variant set forth in SEQ ID NO: 2, 3, 4, 82, 83, or 84, respectively. In some embodiments, a variant of SEQ ID NO: 2, 3, 4, 82, 83, or 84 comprises at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390 (e.g., in any combination), wherein the amino acid positions are relative to the reference melanopsin variant shown in SEQ ID NO: 2, 3, 4, 82, 83, or 84, respectively. Each amino acid position that can be substituted is Figure 1 Indicated by black circles. In some embodiments, a variant of SEQ ID NO: 2, 3, 4, 82, 83, or 84 comprises 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, or any combination thereof. In some embodiments, the variant of SEQ ID NO: 2, 3, 4, 82, 83 or 84 comprises at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 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, or any combination thereof.

[0078] In some embodiments, the melanopsin variant comprises a P10F substitution. In some embodiments, the melanopsin variant comprises the sequence shown 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 shown in SEQ ID NO:6. In some embodiments, the melanopsin variant comprises (e.g., further comprises) a T129S substitution. In some embodiments, the melanopsin variant comprises the sequence shown 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 shown in SEQ ID NO:8. In some embodiments, the melanopsin variant comprises T129S and Q135N substitutions. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO:9. In some embodiments, the melanopsin variant comprises (e.g., further comprises) an S183A substitution. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO:10. In some embodiments, the melanopsin variant comprises Q135N and S183A substitutions. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO: 11. In some embodiments, the melanopsin variant comprises T129S, Q135N and S183A substitutions. In some embodiments, the melanopsin variant comprises the sequence shown in SEQ ID NO: 12. In some embodiments, the melanopsin variant comprises (e.g., further comprises) a Y212F substitution. In some embodiments, the melanopsin variant comprises the sequence shown 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 shown in SEQ ID NO: 14. In some embodiments, the melanopsin variant comprises Y212F and M226S substitutions. In some embodiments, the melanopsin variant comprises the sequence shown 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 19. In some embodiments, the melanopsin variant comprises (e.g., further comprises) R390A or R390D substitutions. 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 shown 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 shown 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 shown in SEQ ID NO:23.

[0079] The amino acid sequences of SEQ ID NOs: 5-23 are provided below. The substituted positions in each of SEQ ID NOs: 5-23 are in bold, underlined type.

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] In some embodiments, the truncated melanopsin variant comprising one or more substitutions is a substituted truncated variant listed in Table A.

[0088] Chimeric melanopsin variants

[0089] In some embodiments, the present application provides a melanopsin variant comprising at least amino acids 1-377 of the wild-type human melanopsin shown in SEQ ID NO: 1 fused (e.g., by a peptide bond) to the C-terminal domain (CTD) of a heterologous G protein-coupled receptor (GPCR) or a CTD variant thereof, wherein the amplitude / conductance (e.g., amplitude / conductance light response) and / or off kinetics (e.g., OFF light response) of the melanopsin variant is greater than the amplitude / conductance of the wild-type human melanopsin and / or faster than the off kinetics of the wild-type human melanopsin. Such melanopsin variants are also referred to herein as "chimeric melanopsin variants".

[0090] In some embodiments, the melanopsin variant comprises amino acids 1-377 of wild-type human melanopsin as shown in SEQ ID NO: 1. In some embodiments, the melanopsin variant comprises amino acids 1-380 of wild-type human melanopsin as shown 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 is linked to the C-terminus of at least amino acids 1-377 of melanopsin) is Figure 1 Indicated by dotted lines.

[0091] In some embodiments, the CTD of the heterologous GPCR or its CTD variant is a CDT of a vision opsin or its variant. In some embodiments, the CTD of a vision 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. In some embodiments, the CTD of the vision opsin comprises the amino acid sequence shown in any one of SEQ ID NOs: 27-30 and 32-33 shown below. In some embodiments, the CTD of the vision opsin comprises a variant of any one of SEQ ID NOs: 27-30 and 32-33, which comprises one or more amino acid substitutions, deletions or insertions.

[0092] TEVSTVSSTQVGPN (SEQ ID NO: 27)

[0093] NKQFQACIMKMVCGKAMTDESDTCSSQKTEVSTVSSTQVGPN(SEQ ID NO:28)

[0094] AMTDESDTCSSQKTEVSTVSSTQVGPN(SEQ ID NO:29)

[0095] DAQSQATASEAESKA (SEQ ID NO:30)

[0096] TEVSSVSSVSPA (SEQ ID NO: 32)

[0097] QFRNCILQLFGKKVDDGSELSSASKTEVSSVSSVSPA(SEQ ID NO:33)

[0098] In some embodiments, the N-terminus of the CTD of the heterologous GPCR or its CTD variant is fused directly to the C-terminus of a melanopsin variant comprising at least amino acids 1-377 of wild-type human melanopsin as shown in SEQ ID NO: 1, for example, by a peptide bond. In some embodiments, the melanopsin variant (e.g., a chimeric melanopsin variant) comprises the amino acid sequence shown 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 indicated in bold type.

[0099]

[0100]

[0101]

[0102] 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.

[0103] In some embodiments, the N-terminus of the CTD or CTD variant thereof of the heterologous GPCR is fused to the C-terminus of the melanopsin variant comprising at least amino acids 1-377 of the wild-type human melanopsin shown in SEQ ID NO: 1 via one or more linkers. In some embodiments, the one or more linkers comprise one or more peptide linkers, including but not limited to, for example, 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 or CTD variant thereof of the heterologous GPCR is fused to the C-terminus of the melanopsin variant comprising at least amino acids 1-377 of the wild-type human melanopsin shown in SEQ ID NO: 1 via one or more linkers and one or more spacers. See, e.g., Klein et al. (2014) Protein Eng Des Sel. 27(10):325–330.

[0104] In some embodiments, the melanopsin variant (e.g., a chimeric melanopsin variant described herein) further comprises one or more amino acid substitutions. In some embodiments, the melanopsin variant (e.g., a chimeric melanopsin variant described herein) further comprises one or more substitutions at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390 (e.g., in any combination), wherein the amino acid position is relative to the wild-type human melanopsin shown in SEQ ID NO: 1. In some embodiments, the melanopsin variant (e.g., a chimeric melanopsin variant described herein) further has at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390 (e.g., in any combination), wherein the amino acid position is relative to the wild-type human melanopsin shown in SEQ ID NO: 1. As described above, each amino acid position that can be substituted is Figure 1Indicated by black circles in. In some embodiments, the melanopsin variant (e.g., a chimeric melanopsin variant described herein) further comprises 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, or any combination thereof. In some embodiments, the 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 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., a chimeric melanopsin variant described herein) further comprises one or more amino acid substitutions (e.g., at least any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) in the CTD of a heterologous GPCR or variant thereof.

[0105] Substituted full-length melanopsin variants

[0106] In some embodiments, the present application provides a melanopsin variant comprising 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 position is relative to the wild-type human melanopsin shown in SEQ ID NO: 1, and wherein the amplitude / conductance (e.g., amplitude / conductance photoresponse) and / or off kinetics (e.g., OFF photoresponse) of the melanopsin variant is greater than the amplitude / conductance of the wild-type human melanopsin and / or faster than the off kinetics of the wild-type human melanopsin. Such melanopsin variants are referred to herein as "substituted full-length melanopsin variants". In some embodiments, the melanopsin variant comprises at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, and / or R390 (e.g., in any combination), wherein the amino acid positions are relative to the wild-type human melanopsin shown 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 comprises one or more amino acid substitutions at positions 391-478, wherein the amino acid positions 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 a substituted variant listed in Table A.

[0107] In some embodiments, the melanopsin variant comprises any of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% overall sequence homology or identity to any 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 any 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.

[0108] Functional properties of melanopsin variants

[0109] The amplitude / conductance (e.g., amplitude / conductance light response) of the melanopsin variants described herein is greater than the amplitude / conductance of wild-type human melanopsin (e.g., the wild-type human melanopsin shown in SEQ ID NO: 1). In some embodiments, the amplitude / conductance of the melanopsin variants described herein is at least about any of 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 the amplitude / conductance of wild-type human melanopsin. In some embodiments, the amplitude / conductance of the melanopsin variants described herein is at least about any of 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 that of wild-type human melanopsin as measured by a calcium release assay in HEK293T cells (e.g., as described in the Examples herein).

[0110] Additionally or alternatively, in some embodiments, the 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., the wild-type human melanopsin 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. 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 greater than the OFF kinetics of wild-type human melanopsin as measured by a calcium release assay in HEK293T cells (e.g., as described in the Examples herein).

[0111] Nucleic acids, expression vectors, viral particles and host cells

[0112] Also provided herein are nucleic acids comprising nucleotide sequences encoding black opsin variants as described herein. In some embodiments, the nucleotide sequences encoding black opsin variants are operably linked to transcriptional control elements (e.g., promoters) that provide expression (e.g., selective expression) of nucleic acids in retinal cells (e.g., retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, or photoreceptor cells, such as rods or cones, Muller cells, and retinal pigment epithelial cells). In some embodiments, nucleic acids encoding black opsin variants are operably linked to transcriptional control elements (e.g., promoters) that provide expression in eukaryotic cells (e.g., mammalian cells).Exemplary transcriptional control elements and promoters include, but are not limited to, ubiquitous or tissue-specific promoters, such as the CAG promoter (Miyazaki et al. (1989) Gene 79:269); the cytomegalovirus (CMV) promoter; the human synapsin (hSyn) promoter; the glutamate metabotropic receptor-6 (grm6) promoter (also known as the GluR or GluR6 promoter, see Cronin et al. (2014) EMBO Mol. Med. 6:1175); the 4xgrm6 promoter (i.e., a four-copy concatemer of a 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); SNCG (γ-synuclein) promoter (Chaffiol et al. (2017) Mol Ther. 25:2546-60); NEFH promoter (Millington-Ward et al. (2020) Sci Rep. 10:16515); 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 polypeptide (VIP) promoter; red cone opsin promoter; rhodopsin promoter; rhodopsin kinase promoter; vitelliform 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); the calcium binding protein promoter; and the β-actin promoter.In some embodiments, the promoter is or comprises a synthetic (non-naturally occurring) promoter / enhancer combination. In some embodiments, the promoter comprises a human cytomegalovirus (CMV) immediate early enhancer and promoter, an adenovirus tripartite leader sequence (TPL), followed by an enhancer element from a major late promoter (eMLP), a synthetic intron, and a Kozak sequence. Such promoters (also referred to as "C11") are described in Grishanin et al. (2019) Mol Ther. 27 (1): 118-129. As further described above, in some embodiments, a nucleic acid comprising a sequence encoding a melanopsin variant as described herein comprises (such as also comprising) additional sequences, including but not limited to, for example, enhancers, introns, leader sequences, Kozak sequences, polyA sequences, stuffer sequences, woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE) sequences, and / or one or more inverted terminal repeat (ITR) sequences, such as AAV ITR sequences, etc.

[0113] In some embodiments, the nucleic acid provided herein is in a recombinant gene construct (e.g., expression vector). 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 may be natural or engineered serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAV11 or AAV12 vectors), etc. In some embodiments, the AAV vector genome is single-stranded (ssAAV). In some embodiments, the AAV vector genome is self-complementary (scAAV). In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a melanopsin variant described herein is in a recombinant lentiviral vector, a recombinant HSV, a recombinant adenoviral vector, a recombinant retroviral vector, or a recombinant AAV ("rAAV") vector (e.g., rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAVrh.8, rAAVrh.10, rAAV11, or rAAV12 vector).

[0114] In some embodiments, the rAAV vector is AAV2.5T.LSV1 (also referred to as "LSV1"), which is described in detail in WO2020 / 180951 and US 2020 / 0297869, the contents of which are incorporated herein 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 incorporated herein 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, WO 2012 / 145601 and US 2014 / 0364338, the contents of which are incorporated herein 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 incorporated herein by reference in their entirety. In some embodiments, the rAAV vector is R100, which is 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 of retinal cells in the eye (e.g., the eye of a mammal such as a mouse, a human, or a non-human primate) and / or the ability to cross the inner limiting membrane (ILM). In some embodiments, the AAV capsid protein is AAV.ShH10\AAV.GL or AAV.NN.Such capsid proteins and others 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 primateretina.” 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 AcadSci US A.119(13):e2117038119;. 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 cell is a retinal ganglion cell, an amacrine cell, a horizontal cell, a bipolar cell, or a photoreceptor cell, such as a rod cell or a cone cell, a Muller cell, or a retinal pigment epithelial cell.

[0115] In some embodiments, a virion (i.e., a viral particle) is provided, comprising (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., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAV11 or AAV12 capsid protein), etc. In some embodiments, the capsid protein is a recombinant capsid protein, such as 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., rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAVrh.8, rAAVrh.10, rAAV11 or rAAV12 capsid protein), etc.

[0116] In some embodiments, the virion comprises a variant capsid protein, wherein the variant capsid protein comprises a peptide insertion in the capsid protein variable loop 8 relative to the corresponding parent capsid protein, wherein the insertion comprises 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, wherein the variant capsid protein comprises an insertion of a peptide shown in any one of SEQ ID NOs:46-57 at the following amino acid positions to produce a variant capsid: between positions 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 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, WO 2012 / 145601, and US2014 / 0364338, the contents of which are incorporated herein 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 incorporated herein by reference in their entirety). AAV2.7m8 and AAV9.7m8 are capable of transducing the retina when delivered intravitreally.

[0117] In some embodiments, the virion comprises a variant capsid protein, wherein the variant capsid protein comprises a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 58), and wherein the amino acid residue numbering corresponds to the AAV5 VP1 capsid protein. In some embodiments, the parent AAV capsid protein is an AAV5 capsid protein or an AAV5 and AAV2 hybrid capsid protein. In some embodiments, the parent AAV capsid protein is an AAV2.5T capsid protein. In some embodiments, "AAV2.5T capsid protein" or "AAV2.5T variant" refers to a hybrid capsid protein containing regions from AAV2 and AAV5, which is described in U.S. Pat. No. 9,441,244, the disclosure of which is incorporated in its entirety. AAV2.5T is capable of transducing the retina when delivered subretinaly, but not when injected intravitreally. AAV2.5T transduction can be blocked by the inner limiting membrane (ILM) rich in heparin sulfate proteoglycans (HSPGs). The surface exposed domains of AAV2.5T are identical to those of AAV5, except for a single substitution of A to T in aa582 of AAV2.5T (aa581 of AAV5), a mutation that appears to increase infectivity in mammalian cells without affecting the typical sialic acid receptor binding of AAV5. AAV5 and AAV2.5T have negligible heparin sulfate binding, while AAV2 has a high affinity for heparin sulfate. In some embodiments, the parent AAV capsid protein is the AAV2.5TVP1 capsid protein. In some embodiments, the modified sequence comprises LAHKFKSGDA (SEQ ID NO: 59). In some embodiments, the rAAV is AAV2.5T.LSV1. In some embodiments, "AAV2.5T.LSV1" or "AAV2.5T.LSV1 variant" refers to an rAAV variant comprising a variant capsid protein, wherein the variant capsid protein comprises a loop substitution variant, wherein the loop substitution variant comprises the amino acid loop sequence LAHKFKSGDA (SEQ ID NO:60) at amino acid residues 570-579 relative to the AAV2.5T parent AAV capsid protein.In some embodiments, the virion comprises 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 entireties), or 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 entireties).

[0118] In some embodiments, the virion comprises a variant capsid protein, wherein the variant capsid protein comprises a peptide insertion in variable loop 8 of the capsid protein relative to the corresponding parent capsid protein, wherein the insertion comprises an amino acid sequence selected from the group consisting of 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), 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 IDNO: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), 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 viral particles (e.g., recombinant viral particles) comprising such variant capsid proteins are provided in WO 2021 / 243085 and US 2021 / 0371879, the contents of which are incorporated herein by reference in their entireties.

[0119] In some embodiments, the virion comprises a variant capsid protein, wherein the variant capsid protein comprises a peptide insertion in the GH loop relative to the corresponding parent capsid protein, wherein the insertion is 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 TD 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), LAQD2TKNA (SEQ ID NO: 145), LAQD3TKNA (SEQ ID NO: 146), LAQD4TKNA (SEQ ID NO: 147), LAQD5TKNA (SEQ ID NO: 148), LAQD6TKNA (SEQ ID NO: 149), LAQD7TKNA (SEQ ID NO: 150), LAQD8TKNA (SEQ ID NO: 151), LAQD9TKNA (SEQ ID NO: 152), LAQD10TKNA (SEQ ID NO: 153), LAQD11TKNA (SEQ ID NO: 154), LAQD 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 1D 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 about such variant capsid proteins and viral particles (e.g., recombinant viral particles) comprising such variant capsid proteins are provided in WO 2017 / 197355, the contents of which are incorporated herein by reference in their entirety.

[0120] Also provided herein is a host cell for producing virions as described herein. The host cell comprises a nucleic acid as provided herein. Suitable host cells include, for example, cell lines of human origin, such as HeLa, A549 or 293 cells, or cell lines of insect origin, such as SF-9. In some embodiments, the host cell also comprises a polynucleotide encoding a capsid protein (e.g., any capsid protein as described herein), a polynucleotide encoding a rep protein, and an AAV helper function. In some embodiments, the nucleic acid as described herein also comprises a polynucleotide encoding a capsid protein and a polynucleotide encoding a rep protein. In some embodiments, the nucleic acid as described herein does not comprise a polynucleotide encoding a capsid protein and a polynucleotide encoding a rep protein.

[0121] Production of recombinant viral particles

[0122] Many methods for producing rAAV vectors are known in the art, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpes virus-AAV hybrids (Conway et al. (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids. The rAAV production culture for producing rAAV viral particles requires all: 1) suitable host cells, including, for example, cell lines of human origin, such as HeLa, A549 or 293 cells, or cell lines of insect origin, such as Sf9, for example, baculovirus-free Sf9 cells, in the case of baculovirus production systems; 2) suitable helper virus functions, which are provided by wild-type or mutant adenoviruses (such as temperature-sensitive adenoviruses), herpes viruses, baculoviruses, or plasmid constructs that provide helper functions; 3) AAV rep and cap genes and gene products; 4) transgenes (such as therapeutic transgenes) flanked by at least one AAV ITR sequence (more usually two ITR sequences); and 5) suitable culture media and culture media components that support rAAV production. In some embodiments, the AAV rep and cap gene products can be from any AAV serotype. Generally, but not necessarily, the AAV rep gene product has 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 culture media known in the art can be used to produce rAAV vectors. These media include, but are not limited to, media produced by Hyclone Laboratories and JRH, including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), custom formulations such as described in U.S. Pat. No. 6,566,118, and Sf-900 II SFM medium such as described in U.S. Pat. No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for producing recombinant AAV vectors. In some embodiments, AAV helper functions are provided by adenovirus or HSV. In some embodiments, AAV helper functions are provided by baculovirus, and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cell).

[0123] Suitable rAAV production medium of the present invention may be supplemented with serum or serum-derived recombinant proteins at a level of 0.5%-20% (v / v or w / v). Alternatively, as known in the art, rAAV vectors may be produced under serum-free conditions, which may also be referred to as a medium that does not contain animal-derived products. It will be appreciated by those of ordinary skill in the art that commercial or custom culture media designed to support rAAV vector production may also be supplemented with one or more cell culture components known in the art, including but not limited to glucose, vitamins, amino acids and / or growth factors, in order to increase the titer of rAAV in the production culture.

[0124] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtering through a series of depth filters, including, for example, DOHC grade Millipore Millistak + HC Pod filter, A1HC grade Millipore Millistak + HC Pod filter and 0.2 μm Filter Opticap XL Millipore Express SHC Hydrophilic Membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtering through any cellulose acetate filter of 0.2 μm or larger pore size known in the art. In some embodiments, the rAAV production culture harvest is further processed to digest any high molecular weight DNA present in the production culture.

[0125] 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) for concentrating 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; and rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in various combinations, or in different orders. In some embodiments, the method includes all steps of purifying rAAV particles in the order described below, see, for example, Xiao et al., (1998) Journal of Virology 72: 2224-2232; US Patent Nos. 6,989,264 and 8,137,948 and WO 2010 / 148143. Methods for purifying adenoviral particles are described in, for example, Bo, H et al., (2014) Eur. J. Pharm. Sci. 67C: 119-125. Methods for purifying lentiviral particles are described in, for example, Segura et al., (2013) Expert Opin Biol Ther. 13(7): 987-1011. Methods for purifying HSY particles are described in, for example, Goins, WF et al., (2014) Herpes Simplex Virus Methods in Molecular Biology 1144: 63-79.

[0126] Pharmaceutical compositions and kits

[0127] In some embodiments, a pharmaceutical composition is provided, comprising: a) a nucleic acid comprising a nucleotide sequence encoding a melanopsin variant 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 "pharmacologically acceptable excipient or carrier" refers to any excipient, carrier, diluent, stabilizer, etc. that has substantially no long-term or permanent adverse effects when administered to a subject (e.g., a mammal such as a mouse, a human, or a non-human primate). Typically, such excipients are mixed with active compounds (e.g., nucleic acids disclosed herein, expression vectors disclosed herein, or viral particles disclosed herein), or are allowed to dilute or encapsulate active compounds, and may be solid, semisolid, or liquid reagents. It should be understood that the active ingredient may be soluble or may be delivered as a suspension in a desired excipient or diluent. Any of a variety of pharmaceutically acceptable excipients may be used, including but not limited to aqueous media such as, for example, distilled water, deionized water, saline; solvents; dispersion media; coatings; antibacterial and antifungal agents; isotonic and absorption delaying agents; or any other inactive ingredients. The choice of a pharmacologically acceptable excipient may depend on the mode of administration. Unless any pharmacologically acceptable excipient is incompatible with the active ingredient, its use in a pharmaceutically acceptable composition is contemplated. Some examples of materials that can be used 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 carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) cocoa butter and other waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) 1) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffer solutions; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other nontoxic compatible substances used in pharmaceutical formulations.Other non-limiting examples of specific uses of such pharmaceutical carriers can be found in "Pharmaceutical Dosage Forms and Drug Delivery Systems" (Howard C. Ansel et al., ed., Lippincott Williams & Wilkins Publishers, 7th edition, 1999); "Remington: The Science and Practice of Pharmacy" (Alfonso R. Gennaro, ed., Lippincott, Williams & Wilkins, 20th edition, 2000); "Goodman & Gilman's The Pharmacological Basis of Therapeutics 13. th ed.” Brunton et al., McGraw-Hill Professional, 2017); and “Handbook of Pharmaceutical Excipients” (Sheskey et al., APhA Publications, 9th edition, 2020).

[0128] In some embodiments, the pharmaceutical composition further comprises one or more additional pharmaceutically acceptable components, such as buffers, preservatives, tension regulators, salts, antioxidants, physiological substances, pharmacological substances, fillers, emulsifiers, wetting agents, etc. Various buffers and means for adjusting pH can be used to prepare pharmaceutical compositions, provided that the resulting preparation is pharmaceutically acceptable. Such buffers include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers, neutral buffered saline, phosphate buffered saline, and borate buffers. It should be understood that, as needed, an acid or base is used to adjust the pH of the composition. Pharmaceutically acceptable antioxidants include, but are not limited to, sodium pyrosulfite, 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 stable oxychlorine compositions, such as PURITE TM Suitable tonicity modifiers for inclusion in the pharmaceutical composition include, but are not limited to, salts such as, for example, sodium chloride, potassium chloride, mannitol or glycerol and other pharmaceutically acceptable tonicity modifiers. It should be understood that these and other substances known in the art of pharmacology may be included in the pharmaceutical composition.

[0129] In some embodiments, nucleic acid, expression vector or virosome as described herein are formulated with one or more biocompatible polymers. In some embodiments, nucleic acid, expression vector or virosome as described herein are formulated in liposomes. See, for example, US 2017 / 0119666. In some embodiments, nucleic acid, expression vector or virosome as described herein are formulated in nanoparticles. Nanoparticles include, for example, polyalkyl cyanoacrylate nanoparticles, nanoparticles comprising poly (lactic acid), nanoparticles comprising poly (lactic acid-co-glycolic acid) (PLGA) nanoparticles, etc. In some embodiments, nucleic acid, expression vector or virosome as described herein are formulated in hydrogel. Suitable hydrogel components include, but are not limited to, silk (see, for example, U.S. Patent Publication No. 2017 / 0173161), poly (lactic acid) (PLA), poly (glycolic acid) (PGA), poly (lactide-co-glycolide) (PLGA), polyester, hyaluronic acid, etc. In some embodiments, nucleic acid, expression vector or virosome as described herein are present in a buffered saline solution. In some embodiments, the volume of the buffered saline solution is between about 50 μL and 1000 μL, including any ranges between these values. In some embodiments, a volume of 50 μL to 1000 μL contains a unit dose of a nucleic acid, expression vector, or virion described herein.

[0130] Methods of restoring or enhancing visual function

[0131] In some embodiments, a method for restoring or enhancing the visual function of a subject is provided, comprising administering a nucleic acid as described herein, an expression vector as described herein, a virion as described herein, or a pharmaceutical composition as described herein to the subject's eye. 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 as described herein is administered by 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 as described herein is administered to a subject within a time period of about 1 day to about 1 year, including any range between these values ​​(e.g., between about one week to about two weeks, between about two weeks to about 1 month, between about one month to about 6 months, between about 6 months to about 1 year). In some embodiments, the nucleic acid, expression vector, virion, or pharmaceutical composition as described herein is administered to a subject within a time period greater than one year. After administration of nucleic acid, expression vector, virion or pharmaceutical composition, the melanopsin variants described herein are produced in retinal cells (e.g., retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells or photoreceptor cells, such as rods or cones, Muller cells or retinal pigment epithelial cells), and the expression of melanopsin variants in retinal cells provides enhanced or restored visual function in the subject. Visual function tests are known in the art, and any known test can be applied to assess the visual function of a subject to which a nucleic acid, expression vector, virion or pharmaceutical composition as described herein is administered.

[0132] In some embodiments, the subject is a human subject. In some embodiments, the human subject suffers from a degenerative disease or disorder affecting the retina. In some embodiments, the sensitivity of the human subject to light is reduced. In some embodiments, the neurons in the retinal circuit of the subject (e.g., bipolar cells, amacrine interneurons and / or ganglion cells output to the brain) are directly sensitive to light by the expression of the black opsin variants described herein in the retinal cells of the subject. In some embodiments, the human subject has reduced visual function due to the loss of rods and cone photoreceptors. In some embodiments, the subject suffers from hereditary retinal degenerative diseases (IRD), such as Leber's congenital amaurosis (LCA), retinitis pigmentosa, Usher syndrome (Usher syndrome), Stargardt disease (Stargardtdisease), cone-rod dystrophy (CRD), achromatopsia, choroideremia, retinoschisis or Bardet-Beidl syndrome (Bardet-Beidl syndrome). In some embodiments, the human subject suffers from an ocular disease. In some embodiments, the human subject suffers from an eye disease, including, but not limited to, for example, macular degeneration (such as age-related macular degeneration), diabetic retinopathy, geographic atrophy, and cone dystrophy. In some embodiments, the human subject has retinal damage or retinal detachment due to injury (e.g., blunt trauma, impact injury, head impact, acute light damage, UV light damage, laser damage, chemical damage, etc.). In some embodiments, the human subject has retinal damage or retinal detachment due to infection.

[0133] Kits and Products

[0134] In some embodiments, a kit or article is provided, which comprises one or more nucleic acids disclosed herein, expression vectors, virions or pharmaceutical compositions, for use according to the methods described herein or for restoring or enhancing visual function. In some embodiments, the kit comprises a lyophilized form of the pharmaceutical composition and a solution for reconstructing the pharmaceutical composition before administration to the subject. In some embodiments, the kit also comprises instructions for administering one or more nucleic acids, expression vectors, virions or pharmaceutical compositions described herein to the eyes of a subject (e.g., a human subject) by intravitreal injection, subretinal injection, intraocular injection, suprachoroidal injection or other administration routes described herein.

[0135] In some embodiments, the kit includes pharmaceutically acceptable excipients, buffers, solutions, etc. for administering the pharmaceutical composition. In some embodiments, the kit also includes instructions for suitable operating parameters in the form of a label or a separate insert. For example, the kit may have a standard instruction for informing a physician or laboratory technician to prepare a therapeutically effective dose of nucleic acid, expression vector, virion, or pharmaceutical composition and / or reconstruct a lyophilized composition. In some embodiments, the kit also includes a device for administration, such as a syringe, a filter needle, an extension tube, a cannula, or other apparatus that facilitates the injection of the pharmaceutical composition into the subject's eye. Exemplary injection routes are described elsewhere herein. In some embodiments, the kit includes a pharmaceutical composition in the form of a suspension or a frozen suspension, and a syringe and / or buffer for dilution. In some embodiments, the kit includes a prefilled syringe containing a suspension or a frozen suspension.

[0136] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0137] The foregoing description is presented to enable one of ordinary skill in the art to make and use various embodiments. The descriptions of specific compositions, techniques, and applications are provided as examples only. Various modifications to the embodiments described above will be apparent to one of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the examples described and shown herein, but to conform to a scope consistent with the claims.

[0138] Example

[0139] The present disclosure will be more fully understood by reference to the following examples. However, the examples should not be construed as limiting the scope of the present disclosure. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes according to the examples and embodiments will be proposed to those skilled in the art, and the various modifications or changes are included in the spirit and scope of the present application and within the scope of the appended claims.

[0140] Example 1A: Characterization of the Amplitude / Conductance Photoresponse and OFF Photoresponse of Melanopsin Variants

[0141] Table A

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] The following experiments were performed to measure the amplitude / conductance photoresponse and OFF photoresponse of the melanopsin variants shown in Table A above.

[0161] Briefly, HEK293T cells were transfected with: (a) nucleic acid encoding one of the melanopsin variants from Table A and (b) nucleic acid encoding GCaMP6 (an exemplary fluorescent calcium sensor is described in Chen et al. (2013) Nature. 499(7458):295–300) or (c) nucleic acid R-GECO1 (an exemplary fluorescent calcium sensor is described in Zhao et al. (2011) Science. 333(6051):1888–1891). Parallel transfection groups were performed using: (a) nucleic acid encoding wild-type melanopsin and (b) nucleic acid encoding GCaMP6 or (c) nucleic acid R-GECO1. After 72 hours, cells were imaged in an ImageXpress micro-confocal system (Molecular Devices) to record 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 enables recording from GCaMP6 calcium-induced fluorescence while activating melanopsin or melanopsin variants. To measure the OFF light response, cells co-transfected with R-GECO1 were briefly illuminated with 480 nm light to activate melanopsin or melanopsin variants and then recorded under the TexasRed channel to record from 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 reached. The average lifetime of the decay from the OFF assay, TauOFF, was calculated from the inverse of the rate constant K, which was determined by the first-order rate law from the decay of the measured fluorescence over time.

[0162] In the ON assay, light-induced intracellular calcium flux by melanopsin or melanopsin variants results in fluorescence from GCaMP6. The light-induced fluorescence increases logarithmically over time. The amplitude of the calcium-induced fluorescence correlates with the photosensitivity of WT human melanopsin or melanopsin variants. Melanopsin variants with greater amplitude / light sensitivity were identified.

[0163] The OFF assay measures the cessation of the light response of WT human melanopsin or melanopsin variants, where depletion of intracellular calcium exponentially reduces R-GECO1 calcium-induced fluorescence over time. Melanopsin variants that switch off faster (ie, have smaller TauOFF) than WT melanopsin were identified.

[0164] Figure 2 Tables A and B show the results of experiments performed to determine the amplitude of calcium light responses ("ON" light responses) from melanopsin truncation variants. In the amplitude assay using GCaMP6 ("ON" assay), the difference between the baseline and the peak light-induced fluorescence was used to determine the amplitude of the light response. Several truncation variants (including 419aa and 425aa) had larger amplitudes than the wild type. Error bars = standard error of the mean (SEM).

[0165] Table B. Amplitudes of calcium light responses from melanopsin truncation variants

[0166]

[0167] Figure 3 Tables A and C show the results of experiments performed to determine TauOFF of calcium light responses from melanopsin truncation variants. The rate constant k for the decay of the induced fluorescence response in the OFF assay using RGECO-1 was used to find the average decay time TauOFF. Several truncation variants (including 413AA, 419AA, and 420AA) had lower TauOFF than the wild type. Error bars = SEM

[0168] Table C. TauOFF of calcium light responses from melanopsin truncated variants

[0169]

[0170] Figure 4 Shows Figure 2 and Figure 3 Comparison of amplitude and TauOFF of truncated variants shown in . Variants such as 419AA (black circles) have smaller TauOFF and larger amplitude than wild type (white circles). Error bars = SEM.

[0171] Figure 5 Tables A and D show the results of experiments performed to determine the amplitude of calcium light responses from chimeric melanopsin variants. In the ON assay using GCaMP6, the difference between the baseline and the light-induced fluorescence peak was used to determine the amplitude of the light response. Error bars = SEM.

[0172] Table D. Amplitudes of calcium light responses from chimeric melanopsin variants

[0173]

[0174] Figure 6 Tables A and E show the results of TauOFF experiments performed to determine calcium light responses from chimeric melanopsin variants. The rate constant k for the decay of the induced fluorescence response in the OFF assay using RGECO-1 was used to find the mean decay time TauOFF. Error bars = SEM.

[0175] Table E. TauOFF of calcium light responses from chimeric variants of melanopsin

[0176]

[0177] Figure 7 and Table F shows the Figure 6Results of experiments performed to determine the TauOFF of the calcium light response of the fastest chimeric melanopsin variant shown in . The rate constant k for the decay of the induced fluorescence response in the OFF assay using RGECO-1 was used to find the mean decay time TauOFF. Error bars = SEM. Figure 8 Shows Figure 7 Amplitude of ON responses of the fastest chimeric melanopsin variants compared to TauOFF shown in Table F. Variants are black circles and wild type are white circles.

[0178] Table F. TauOFF of calcium light responses of the fastest melanopsin chimeric variants from Table E.

[0179]

[0180] Fig. 9 Tables A and 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 GCaMP6, the difference between the baseline and the light-induced fluorescence peak was used to determine the amplitude of the light response. Error bars = SEM.

[0181] Table G. Amplitudes of calcium light responses from substituted full-length melanopsin variants.

[0182]

[0183] Fig.10 Tables A and H show the results of experiments performed to determine the TauOFF of calcium light responses of substituted full-length melanopsin variants. The rate constant k for the decay of the induced fluorescence response in the OFF assay using RGECO-1 was used to find the mean decay time TauOFF. Error bars = SEM.

[0184] Table H. TauOFF of calcium light responses from substituted full-length melanopsin variants

[0185]

[0186] Fig.11 and Table I shows the Fig.10 Results of experiments performed to measure the calcium light response of the fastest substituted full-length melanopsin variants shown in TauOFF. The rate constant k for the decay of the induced fluorescence response in the OFF assay using RGECO-1 was used to find the mean decay time TauOFF. Error bars = SEM. Fig.12 Shows Fig.11 Amplitude of the ON response of the fastest substituted full-length melanopsin variant in comparison with TauOFF. Variants are black circles and wild type is white circles. Error bars = SEM

[0187] Table I. TauOFF of calcium light responses of the fastest substituted full-length melanopsin variants from Table H

[0188]

[0189] Fig.13 Tables A and J show the amplitude of the calcium light response of melanopsin variants from substitutions in the truncated backbone (419AA, SEQ ID NO: 3). In the assay using GCaMP6, the difference between the baseline and the light-induced fluorescence peak was used to determine the amplitude of the light response. Error bars = SEM.

[0190] Table J. Amplitude of calcium light responses from melanopsin variants with substitutions in the truncated backbone

[0191]

[0192] Fig.14 Tables 3 and K show the results of experiments performed to determine the TauOFF of calcium light response from substituted melanopsin variants in a truncated backbone (419AA, SEQ ID NO: 3). The rate constant k for the decay of the induced fluorescence response in the OFF assay using RGECO-1 was used to find the average decay time TauOFF. Error bars = SEM.

[0193] Table K. TauOFF of calcium light responses from melanopsin variants with substitutions in the truncated backbone.

[0194]

[0195] Fig.15 and Table L shows the determination of Fig.14 Results of experiments performed to measure the calcium light response TauOFF of the fastest substituted melanopsin variants in the truncated backbone shown in . The rate constant k for the decay of the induced fluorescence response in the OFF assay using RGECO-1 was used to find the mean decay time TauOFF. Error bars = SEM. Fig.16 Shows Fig.14 Amplitude of ON response of the fastest substituted full-length melanopsin variants in Table L compared to TauOFF. Variants are black circles and wild type is white circles. Error bars = SEM. Table L. TauOFF of calcium light response of the fastest substituted melanopsin variants in truncated backbones from Table K

[0196]

[0197] Example 1B: Electrophysiological Characterization of Melanopsin Variants

[0198] 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) nucleic acid encoding wild-type melanopsin or a melanopsin variant and (b) a nucleic acid encoding a G protein-gated inwardly rectifier K + Channel 1F137S (GIRK1 F137S ) nucleic acid. The transfected cells were placed 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 recorded using a glass pipette using 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). The cells were patch clamped whole-cell using a HEKA amplifier. A gravity-driven perfusion system was used to apply 10 mM acetylcholine (as a GIRK activator) and 5 mM BaCl2 (full block), and illumination was controlled using a DG-4 system (Sutter) in conjunction with an excitation filter. The cells were illuminated with light of different wavelengths to find those wavelengths to which WT human melanopsin and melanopsin variants are most sensitive (i.e., "sensitive wavelengths"). When irradiated at the “sensitive wavelength”, WT human melanopsin and melanopsin variants were exposed to a range of light intensities (1E+10 ph / cm 2 / s-10E+14ph / cm 2 / s) to identify the most sensitive intensity of WT human melanopsin and melanopsin variants

[0199] Example 2: In vivo characterization of melanopsin variants

[0200] The activity of WT human melanopsin and melanopsin variants in Table A was characterized in a mouse model of retinal degeneration. In the C3H / HeJ (Rd1) mouse line, mice experience retinal degeneration at weaning due to a PDEbeta mutation encoding the β subunit of the cGMP phosphodiesterase of rod photoreceptors. This degeneration is consistent with retinitis pigmentosa experienced by individuals with human PDEbeta mutations. WT human melanopsin or melanopsin variants under the ubiquitous CMV promoter were packaged into AAV2.7m8 and injected into 40-day-old Rd1 mice at approximately 1E+10 vg / eye intravitreal. After 49-50 days, visual acuity was measured by optokinetic reflex testing (OKR), retinal light response was determined by full-field electrical response (ffERG), and brain light response was recorded by visual evoked potential (VEP).

[0201] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

[0202] Example 3: Further Characterization of Amplitude / Conductance Photoresponse and OFF Photoresponse of Melanopsin Variants

[0203] Melanopsin has many of the best features of its class, but the wild-type protein (sequence diagram as shown in Figure 1 ) have a slow inactivation, thereby preventing rapid and continuous reactivation. Table M describes the mutation strategy for making calcium-mediated rapid opsin. Such strategies are also discussed elsewhere herein, including in Example 1A and Example 1B and in the specific embodiments.

[0204] Fig.17 A schematic diagram of such a melanopsin variant development strategy is shown. Approximately 70 melanopsin variants (see, e.g., Example 1A and Example 1B and Table A and Table M), including truncated variants, point mutation variants, and chimeras, were screened in the first round of screening by fluorescent calcium imaging assays. The second round of screening identified approximately 40 melanopsin variants that were truncated or chimeras with synergistic point mutations. Melanopsin variants that showed increased amplitude and / or decreased TauOFF compared to WT human melanopsin (e.g., according to assays described in Example 1A and Example 1B and elsewhere herein) were selected for AAV production.

[0205] Fig.18 Shown are 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. Fig.19 Results of experiments performed to determine TauOFF of the calcium light response over time for wild-type human melanopsin, exemplary melanopsin variants 405AA, 425AA, V370-R377Del, and K356-R377Del are shown. In the amplitude assay ("ON" assay) using GCaMP6, the difference between the baseline and the light-induced fluorescence peak is used to determine the amplitude of the light response. In the OFF assay using RGECO-1, the rate constant k for the decay of the induced fluorescence response is used to find the average decay time TauOFF.

[0206] The selection of such mutations is Figure 1As indicated in the schematic in the Figure 2. Some N-terminal melanopsin mutations increased speed and light sensitivity. Only one mutation in the transmembrane domain was successful. The transmembrane domain of GPCRs is the most conserved. Reduction in alcohol consistently improved amplitude and TauOFF. The CTD chimera required the first 377 amino acids of melanopsin to be successful. Many of the successful mutations in putative phosphorylation and / or GRK / arrestin binding regions were phosphorylations of amino acids normally phosphorylated during inactivation, as well as mutations that reduced positive charge.

[0207] As mentioned above, Fig.12 An exemplary comparison of the amplitude of the ON response and TauOFF for a subset of melanopsin variants shown in Table A is provided. Error bars = standard error of the mean (SEM). The second round of melanopsin variants included truncated variants and chimeric variants containing synergistic point mutations. Melanopsin variants that showed increased amplitude and reduced TauOFF, such as 419AA_S183A_S384D_R386A (SEQ ID NO: 19), were selected for AAV production.

[0208] Fig. 20 Shown are the results of experiments performed to determine the amplitude of calcium light responses from cells transduced with AAV2.7m8 carrying WT human melanopsin or a melanopsin variant comprising SEQ ID NO 19.

[0209] MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVA A KRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYP D Y A STHRSTLTSHTSNLSWISIRRRQESLGSESEVG(SEQ ID NO:19)

[0210] SEQ ID NO: 19 comprises 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).

[0211] AAVMAX suspension cells, AVB columns, CsCl ultracentrifugation and buffer exchange were used to generate AAV2.7m8-CMV-WT human melanopsin and AAV2.7m8-CMV-SEQ ID NO: 19. The resulting rAAVs were analyzed by ddPCR titer, Western blot, silver staining and alkaline gel.

[0212] AAV2.7m8-CMV-SEQ ID NO: 19 or AAV2.7m8-CMV-WT human melanopsin and AAV2.7m8-CMV-GCAMP6 were used at an MOI of 5x10 4 HEK293T cells were transduced with vg / AAV and imaged at 480 nm in an ImageXpress Micro (Molecular Devices). AAV2.7m8 packaged with CMV-WT human melanopsin or CMV-SEQ ID NO: 19 successfully generated photoresponses in HEK293T. The photoresponse of SEQ ID NO: 19 was greater and faster than that of WT human melanopsin.

[0213] Fig.21 Shown are 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. Less AAV transduction resulted in lower maximum amplification. The amplitude of the light response varied with the dose of AAV2.7m8-SEQ ID NO: 19 applied.

[0214] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

Claims

1. A melanopsin variant comprising no more than amino acids 1-425 of wild-type human melanopsin as shown in SEQ ID NO: 1, wherein 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 the wild-type human melanopsin.

2. The melanopsin variant of claim 1, comprising a sequence shown in any one of SEQ ID NOs: 2, 3, 4, 82, 83 and 84 or a variant thereof containing one or more amino acid substitutions.

3. The melanopsin variant of claim 2, wherein the variant comprises one or more amino acid substitutions at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386 and / or R390, wherein the amino acid positions are relative to the wild-type human melanopsin shown in SEQ ID NO:

1.

4. The melanopsin variant of claim 3, wherein the one or more substitution mutations 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.

5. The melanopsin variant of claim 3 or 4, wherein the melanopsin variant comprises a P10F substitution.

6. The melanopsin variant of claim 5, wherein the melanopsin variant comprises SEQ ID NO:

5.

7. The melanopsin variant of claim 3 or 4, wherein the melanopsin variant comprises a T83L substitution.

8. The melanopsin variant of claim 5, wherein the melanopsin variant comprises SEQ ID NO:

6.

9. The melanopsin variant of claim 3 or 4, wherein the melanopsin variant comprises a T129S substitution.

10. The melanopsin variant of claim 9, wherein the melanopsin variant comprises SEQ ID NO:

7.

11. The melanopsin variant of any one of claims 3 to 4 and 9, comprising a Q135N substitution.

12. The melanopsin variant of claim 11, wherein the melanopsin variant comprises SEQ ID NO: 8 or 9.

13. The melanopsin variant of any one of claims 3 to 4, 9 and 11, comprising an S183A substitution.

14. The melanopsin variant of claim 13, wherein the melanopsin variant comprises any one of SEQ ID NOs: 10-12.

15. The melanopsin variant of claim 3 or 4, comprising a Y212F substitution.

16. The melanopsin variant of claim 15, wherein the melanopsin variant comprises SEQ ID NO:

13.

17. The melanopsin variant of any one of claims 3 to 4 and 15, comprising a M226S or M226T substitution.

18. The melanopsin variant of claim 17, wherein the melanopsin variant comprises SEQ ID NO: 14 or 15.

19. The melanopsin variant of claim 3 or 4, wherein the melanopsin variant comprises a Y382E or Y382D substitution.

20. The melanopsin variant of claim 19, wherein the melanopsin variant comprises SEQ ID NO:

16.

21. The melanopsin variant of claim 3 or 4, wherein the melanopsin variant comprises a S384D substitution.

22. The melanopsin variant of claim 21, wherein the melanopsin variant comprises SEQ ID NO:

17.

23. The melanopsin variant of any one of claims 3 to 4, 13 and 21, wherein the melanopsin variant comprises a R386A substitution.

24. The melanopsin variant of claim 23, wherein the melanopsin variant comprises SEQ ID NO: 18 or SEQ ID NO:

19.

25. The melanopsin variant of any one of claims 3 to 4, 13, 17, 20 and 23, wherein the melanopsin variant comprises a R390A or R390D substitution.

26. The melanopsin variant of claim 25, wherein the melanopsin variant comprises any one of SEQ ID NOs: 20-23.

27. A melanopsin variant comprising at least amino acids 1-377 of the wild-type human melanopsin shown 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 and / or faster than the off kinetics of the wild-type human melanopsin.

28. The melanopsin variant of claim 27, wherein the CTD or CTD variant thereof of the heterologous GPCR is the CTD of aphthous opsin or a variant thereof.

29. The melanopsin variant of claim 27 or 28, wherein the CTD of the vision opsin or a variant thereof is the CTD of: (i) wild-type Drosophila 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.

30. The melanopsin variant of any one of claims 27 to 29, wherein the CTD of the melanopsin or variant thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 27-30 and 32-33.

31. The melanopsin variant of any one of claims 27 to 30, comprising the amino acid sequence shown in any one of SEQ ID NOs: 39-42 and 44-45.

32. The melanopsin variant of any one of claims 17 to 29, further comprising one or more substitution mutations at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386, R390, wherein the amino acid positions are relative to the wild-type human melanopsin shown in SEQ ID NO: 1, wherein the amino acid positions are relative to the wild-type human melanopsin shown in SEQ ID NO:

1.

33. The melanopsin variant of claim 32, wherein the one or more substitution mutations 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.

34. The melanopsin variant of any one of claims 17 to 33, further comprising an amino acid substitution in the CTD of the heterologous GPCR or the CTD variant thereof.

35. A melanopsin variant comprising one or more amino acid substitutions at P10, T83, T129, Q135, S183, Y212, E215, M226, Y382, S384, R386 and R390, wherein the amino acid positions are relative to the wild-type human melanopsin shown in SEQ ID NO: 1, and wherein 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 the wild-type human melanopsin.

36. The melanopsin variant of claim 35, wherein the one or more substitution mutations 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.

37. The melanopsin variant of any one of claims 1 to 36, wherein the amplitude / conductance of the melanopsin variant is at least 1.25 times the amplitude / conductance of wild-type human melanopsin in HEK293T cells.

38. The melanopsin variant of any one of claims 1 to 37, wherein the off kinetics of the melanopsin variant are at least 1.10 times the off kinetics of the wild-type human melanopsin in HEK293T cells.

39. A nucleic acid comprising a polynucleotide sequence encoding the melanopsin variant of any one of claims 1 to 38.

40. The nucleic acid of claim 39, operably linked to a promoter.

41. The nucleic acid of claim 40, wherein the promoter is a retinal cell-specific promoter.

42. The nucleic acid of claim 41, wherein the retinal cell-specific promoter is selected from the group consisting of human synapsin (hSyn), SNCG, NEFH, NEFL, 4xgrm6, and grm6.

43. The nucleic acid of any one of claims 40 to 42, further comprising one or more enhancer sequences, intron sequences, leader sequences, Kozak sequences, polyA sequences, stuffer sequences and / or inverted terminal repeat (ITR) sequences.

44. A recombinant virion comprising: (a) capsid protein and (b) The nucleic acid of any one of claims 39 to 43.

45. The recombinant virion of claim 44, wherein the capsid protein is selected from the group consisting of: AAV2-7m8, AAV2, AAV2-4YF, AAV9, AAV9-7m8, R100, and LSV1.

46. ​​A host cell comprising the nucleic acid of any one of claims 39 to 43.

47. The host cell of claim 46, further comprising one or more of: (i) a polynucleotide encoding a capsid protein; (ii) a polynucleotide encoding a rep protein; and (iii) AAV helper function.

48. A method for producing a recombinant virion, comprising: (a) culturing the host cell of claim 47 under conditions that produce the recombinant virion, and (b) recovering the recombinant virion produced by the host cell.

49. The method of claim 48, further comprising purifying the recombinant virion.

50. A pharmaceutical composition comprising the recombinant virion according to any one of claims 41 to 45 and a pharmaceutically acceptable excipient.

51. A method for restoring or enhancing visual function in a subject, comprising administering the pharmaceutical composition of claim 50 to the eye of the subject.

52. The method of claim 51, wherein the administering comprises intraocular injection, subretinal injection, suprachoroidal injection, or intravitreal injection.

53. The method of claim 51 or 52, wherein the subject has an ocular disease or condition selected from the group consisting of retinitis pigmentosa, macular degeneration, retinoschisis, Leber's congenital amaurosis, diabetic retinopathy, geographic atrophy, choroideremia, cone dystrophy, and cone-rod dystrophy.

54. The method of claim 51 or 52, wherein the subject has experienced retinal detachment or photoreceptor loss due to ocular disease, infection, trauma, injury, head impact, acute light damage, UV light damage, laser damage, or chemical damage.

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