Variants of light-sensitive ion channel protein ChRmine and uses thereof

Abstract

The invention relates to variants of light-sensitive ion channel protein ChRmine and application of the variants in treatment of retina neurodegenerative diseases. Compared with ChRmine, the variant has improved stability, expression efficiency and light activation characteristics in vivo, and can significantly improve the effect of treating retina neurodegenerative diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of treatment of eye diseases. In particular, the present application relates to variants of the light-sensitive ion channel protein ChRmine and their use in the treatment of retinal neurodegenerative diseases. BACKGROUND

[0002] Retinal neurodegenerative diseases, including blinding eye diseases such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), are caused by degeneration and death of key retinal cells such as photoreceptors, leading to loss of visual function and even blindness. The pathogenesis of retinal neurodegenerative diseases seriously damages the vision of patients, and currently all the existing treatment means cannot avoid the irreversible loss of visual function. The pathogenesis of retinal neurodegenerative diseases often occurs due to the apoptosis response of photoreceptors caused by genetic mutations in photoreceptors or the imbalance of the retinal microenvironment (such as neovascularization, death of retinal pigment epithelial cells, etc.) which exacerbates the apoptosis of photoreceptors. According to the pathogenesis, the treatment strategies mainly focus on: 1) overexpression or gene editing to repair the relevant mutant genes to delay the degeneration of photoreceptors; 2) inhibition of neovascularization to reduce the damage to photoreceptors; and 3) expression of photopigments in other retinal neurons (such as bipolar cells or ganglion cells) to perform photoreceptor functions.

[0003] WO2007024391, WO2008022772 or WO2009127705 describe the use of protease genes from plants and microorganisms that encode light-sensitive ion channels and ion pumps such as the channelrhodopsin type 2 [ChR2], which are engineered to be expressed in mammalian neurons and can be implanted into specific neurons at the genetic level by viral vectors. When exposed to light with the appropriate wavelength, action potentials can be triggered in neurons expressing light-sensitive proteins, thus making these cells light-sensitive.

[0004] WO2017187272A1 discloses a light-sensitive ion channel protein ChrimsonR, after administration, patients can see the objects on the table with the help of special glasses. In particular, it is found that ChrimsonR fused with tdTomato (tdT) fluorescent protein or green fluorescent protein (GFP) can respond to light stimulation more effectively than ChrimsonR protein alone.

[0005] AU2017372351B2 discloses a light-sensitive ion channel protein MCO. After subretinal injection of adeno-associated virus carrying MCO, the MCO reporter protein is expressed effectively and stably in the mouse retina. It is also shown that expression of MCO in the retina of a mouse model of photoreceptor degeneration can achieve behavioral visual recovery.

[0006] Therefore, in the field of optogenetics, researchers are working to develop new light-sensitive ion channel proteins in order to achieve better therapeutic effects in the treatment of retinal neurodegenerative diseases. Specifically, by introducing light-sensitive ion channel proteins through optogenetic technology, the remaining functional retinal cells respond to light signals, thereby partially restoring vision.

[0007] However, natural light-sensitive ion channel proteins may face a series of problems in treatment, such as low light activation efficiency, unsatisfactory response spectrum, poor protein stability, etc. Therefore, through genetic engineering means to mutate and optimize the amino acid or nucleotide sequence is the key to improve the therapeutic effect of light-sensitive ion channel proteins. First, the gene of light-sensitive ion channel protein can be modified through site-directed mutagenesis or directed evolution technology to improve its light activation efficiency. For example, by changing the amino acid sequence of the light-sensitive region of the protein, it can be made more sensitive to light of a specific wavelength, thereby achieving more efficient light activation. Second, by mutation and screening, the spectral specificity of the light-sensitive protein can be adjusted to have a better response to light of a specific wavelength. In addition, the stability and expression efficiency of the protein are also the focus of optimization. Through in-depth study of the protein structure, more stable mutants can be designed to reduce the degradation of the protein in the body and improve its expression level in the target cells. For example, by introducing mutations that are beneficial to protein folding and stability, the lifespan of the light-sensitive ion channel protein in cells can be extended, improving its long-term therapeutic effect. Finally, the optimized light-sensitive ion channel protein needs to be verified for its function and safety through a series of in vitro and in vivo experiments. In vitro experiments can evaluate the light activation characteristics, stability, and expression efficiency of the protein, while in vivo experiments need to test its therapeutic effect and potential side effects in animal models.

[0008] Therefore, there is a need in the art for improved light-sensitive ion channel proteins for the treatment of various retinal neurodegenerative diseases. SUMMARY

[0009] ChRmine protein is derived from marine microorganisms and was first discovered in 2019. In recent years, it has been widely used in the brain as an optogenetic tool. The inventors have unexpectedly found that ChRmine protein has extremely high light sensitivity, generates a large photocurrent, and has an optimal absorption wavelength of 580 nm, which is a safe red-shifted wavelength with minimal phototoxicity, which is superior to all light-sensitive ion channel protein products currently on the market. This provides a new strategy for treating retinal neurodegenerative diseases, i.e., using other neurons in the retina (such as bipolar cells or ganglion cells) to express light-sensitive ion channel proteins with photosensitive function to restore vision in blind patients.

[0010] To this end, the present inventors screened a plurality of different variants by mutating and optimizing a light-sensitive ion channel protein Chrmine. Compared with the Chrmine protein, these variants not only have improved protein expression and stability, but more importantly, have improved light-sensitive ability, which can significantly improve the effect of treating retinal neurodegenerative diseases. This result not only helps to develop new vision recovery therapies, but also provides important theoretical and technical support for the application of optogenetic technology in other neurological diseases.

[0011] In one aspect, the present application provides a light-sensitive ion channel protein Chrmine variant, which has an amino acid sequence shown in SEQ ID NO: 3, 7, 11 or 15, or an amino acid sequence having at least 80% identity to SEQ ID NO: 3, 7, 11 or 15.

[0012] In one embodiment, the ChRmine variant consists of an amino acid sequence shown in SEQ ID NO: 3, 7, 11 or 15, or an amino acid sequence having at least 80% identity to SEQ ID NO: 3, 7, 11 or 15.

[0013] In another aspect, the present application provides a nucleic acid encoding the ChRmine variant. In one embodiment, the nucleic acid comprises a nucleotide sequence shown in SEQ ID NO: 4, 8, 12 or 16, or a nucleotide sequence having at least 70% identity to SEQ ID NO: 4, 8, 12 or 16.

[0014] In one embodiment, the nucleic acid consists of a nucleotide sequence shown in SEQ ID NO: 4, 8, 12 or 16, or a nucleotide sequence having at least 70% identity to SEQ ID NO: 4, 8, 12 or 16.

[0015] In another aspect, the present application provides a fusion protein comprising the ChRmine variant of the present application. In one embodiment, the fusion protein further comprises a hemagglutinin signal peptide, a bacterial rhodopsin-like insertion loop, a kv2.1 channel protein, a Golgi export signal and / or a rhodopsin trafficking sequence, or any combination thereof.

[0016] In a specific aspect, the present application provides a fusion protein having an amino acid sequence shown in SEQ ID NO: 5, 9, 13 or 17, or an amino acid sequence having at least 80% identity to SEQ ID NO: 5, 9, 13 or 17.

[0017] In one embodiment, the fusion protein consists of the amino acid sequence set forth in SEQ ID NO: 5, 9, 13, or 17, or an amino acid sequence having at least 80% identity to SEQ ID NO: 5, 9, 13, or 17.

[0018] In another aspect, the present application provides a nucleic acid encoding the fusion protein. In one embodiment, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 6, 10, 14, or 18, or a nucleotide sequence having at least 70% identity to SEQ ID NO: 6, 10, 14, or 18.

[0019] In one embodiment, the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 6, 10, 14, or 18, or a nucleotide sequence having at least 70% identity to SEQ ID NO: 6, 10, 14, or 18.

[0020] In another aspect, the present application provides a vector comprising a nucleic acid encoding a ChRmine variant or fusion protein of the present application. In one embodiment, the vector is a viral vector. In one embodiment, the vector is a retroviral vector or a parvovirus vector. In one embodiment, the vector is a Moloney murine leukemia virus (MoMLV), MSCV, SFFV, MPSV, or SNV vector, a lentivirus vector (e.g., derived from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), or equine infectious anemia virus (EIAV)), an adenovirus (Ad) vector, an adeno-associated virus (AAV) vector, a simian virus 40 (SV-40) vector, a bovine papilloma virus vector, an Epstein-Barr virus vector, a herpes virus vector, a vaccinia virus vector, a Harvey murine sarcoma virus vector, a murine mammary tumor virus vector, or a Rous sarcoma virus vector. In one embodiment, the vector is an adeno-associated virus (AAV) vector and can comprise two ITRs flanking the nucleic acid of interest.

[0021] In another aspect, the present application provides a viral particle comprising a vector of the present application. In one embodiment, the vector is an AAV vector. In one embodiment, the viral particle further comprises an AAV-derived capsid. In one embodiment, the AAV serotype includes, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, and AAVdj. In one embodiment, the AAV serotype is preferably selected from the group consisting of AAV-2, AAV-5, AAV2-7m8, AAV-9, and AAV-8, more preferably AAV-2 or AAV2-7m8.

[0022] In another aspect, the present application provides a composition comprising a ChRmine variant or fusion protein of the present application, or a nucleic acid encoding the same.

[0023] In another aspect, the present application provides a pharmaceutical composition comprising a ChRmine variant or fusion protein of the present application, or a nucleic acid encoding the same, a viral vector or viral particle, and a pharmaceutically acceptable carrier.

[0024] In another aspect, the present application provides a cell transduced by, or comprising, a vector of the present application. In one embodiment, the vector is a viral vector. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a non-human cell. In one embodiment, the cell is a retinal cell. In one embodiment, the cell is a bipolar cell or a ganglion cell. In one embodiment, the cell is in vitro. In one embodiment, the cell is in vivo.

[0025] In another aspect, the present application provides a method of treating a retinal neurodegenerative disease, comprising administering to the eye of a subject in need thereof a therapeutically effective amount of a ChRmine variant or fusion protein, a nucleic acid, a viral vector, a viral particle, a pharmaceutical composition, or a cell of the present application. In one embodiment, the retinal neurodegenerative disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration (AMD), Stargardt disease, Leber’s hereditary optic neuropathy, cone-rod dystrophy, Leber’s congenital amaurosis, diabetic retinopathy, retinal detachment, Best’s disease, choroideremia, lamellar macular dystrophy, inherited optic neuropathies (Leber’s hereditary optic neuropathy, dominant optic neuropathy), compressive optic neuropathy (orbital pseudotumor, thyroid eye disease), autoimmune optic neuropathy (lupus), diabetic retinopathy, glaucomatous optic neuropathy (GOND), glaucoma, arterial ischemic optic neuropathy (giant cell arteritis), non-arteritic ischemic optic neuropathy, infiltrative optic neuropathy (sarcoidosis), infectious optic neuropathy (syphilis, Lyme disease, toxoplasmosis, shingles), optic neuritis from demyelinating disease, and post-radiation optic neuropathy. In one embodiment, the subject is a human.

[0026] In another aspect, the present application provides the use of a ChRmine variant or fusion protein, a nucleic acid, a viral vector, a viral particle, a pharmaceutical composition, or a cell of the present application in the manufacture of a medicament for treating a retinal neurodegenerative disease in a subject in need thereof. In one embodiment, the retinal neurodegenerative disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration (AMD), Stargardt disease, Leber’s hereditary optic neuropathy, cone-rod dystrophy, Leber’s congenital amaurosis, diabetic retinopathy, retinal detachment, Best’s disease, choroideremia, lamellar macular dystrophy, inherited optic neuropathies (Leber’s hereditary optic neuropathy, dominant optic neuropathy), compressive optic neuropathy (orbital pseudotumor, thyroid eye disease), autoimmune optic neuropathy (lupus), diabetic retinopathy, glaucomatous optic neuropathy (GOND), glaucoma, arterial ischemic optic neuropathy (giant cell arteritis), non-arteritic ischemic optic neuropathy, infiltrative optic neuropathy (sarcoidosis), infectious optic neuropathy (syphilis, Lyme disease, toxoplasmosis, shingles), optic neuritis from demyelinating disease, and post-radiation optic neuropathy. In one embodiment, the subject is a human.

[0027] In another aspect, the present application provides use of a viral vector described herein in the manufacture of a medicament for treating a retinal neurodegenerative disease in a subject in need thereof, wherein the viral vector is transducible to ocular retinal cells to cause the transduced ocular retinal cells to express a ChRmine variant or fusion protein of the present application. In one embodiment, the ocular retinal cells are bipolar cells or ganglion cells. In one embodiment, the retinal neurodegenerative disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration (AMD), Stargardt disease, Leber's hereditary optic neuropathy, cone-rod dystrophy, Leber congenital amaurosis, diabetic retinopathy, retinal detachment, Best disease, choroideremia, fundus BRIEF DESCRIPTION OF DRAWINGS

[0028] The present application will be better understood with reference to the accompanying drawings.

[0029] Figure 1 The photosensitivity of various ChRmine variants in HEK293T cells compared to ChRmine is shown.

[0030] Figure 2 Pharmacodynamic results of ChRmine variant M1 are shown. A: retinal ganglion cell imaging; B: optic nerve imaging; C: infection RGC efficiency results; D: light stimulation results at different wavelengths; E: current response results induced at different light intensities; F: relationship between light wave and voltage response; G: relationship between light intensity and current response; H: ERG signals (a wave + b wave) of normal C57 mice, blind C3H mice given control AAV, and blind C3H mice given AAV-M1.

[0031] Figure 3Retinal current graphs (ERG signals) are shown for various ChRmine variants in blind C3H mice compared to normal C57 mice and ChRmine. DETAILED DESCRIPTION

[0032] In this application, the use of the singular includes the plural, the word "one" means "at least one", and the word "or" means "and / or", unless specifically stated otherwise. Further, the words "comprise", "comprises" and "comprising" are to be construed in an open-ended fashion, i.e., as meaning "including, but not limited to", "in the case of a process, method, etc., comprising steps A, B, and C, the process, method, etc. can comprise only those steps A, B, and C, or it can include one or more additional steps".

[0033] The term "about", as used herein when used in connection with a percentage or other quantity, means all values within 10% of that percentage or other quantity. For example, "about 80%" includes all values within 8% of 80%.

[0034] All documents cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. To the extent that the definition of any term in one or more incorporated documents and this application contradict, the definition in this application controls.

[0035] The term "nucleic acid" or "polynucleotide", as used herein, refers to a polymeric form of nucleotides of any length, which can be either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, cDNA, a DNA-RNA hybrid, or a polymer comprising both purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugar and phosphate groups (as can typically be found in RNA or DNA) or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a synthetic subunit, such as a phosphoramidate, and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidatc-phosphodiester oligomer. The nucleic acids of the application can be prepared by any technique known to one skilled in the art, including chemical synthesis, recombination, and mutagenesis. In a preferred embodiment, the nucleic acids of the application are DNA molecules, which are preferably synthesized by recombinant methods well known to one skilled in the art.

[0036] The term "isolated nucleic acid" as used herein refers to a nucleic acid molecule that has been identified and separated from a component of its natural environment. In particular, this term refers to a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. For example, with respect to genomic DNA, the term "isolated" includes a nucleic acid molecule that is separated from the chromosome with which it is naturally associated. Preferably, an "isolated" nucleic acid molecule is free of sequences that naturally flank the nucleic acid molecule in the genome of the organism from which the nucleic acid molecule was derived.

[0037] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues, and are not limited to a minimum length. The polymers of amino acid residues can contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. The term also includes post-translational modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In addition, for purposes of the present application, "polypeptide" can refer to proteins that include modifications of the parent sequence, such as deletions from, additions to, and substitutions of, amino acid residues, as long as the proteins maintain the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as by mutation of the host that produces the protein or by errors caused by PCR amplification.

[0038] As used in the present application, unless otherwise indicated, the terms "treatment" and "therapy" refer to an action that occurs when a subject is suffering from a disease (e.g., a retinal neurodegenerative disease), and thereby reduces the severity of one or more symptoms or the effects of the disease. As used in the present application, unless otherwise indicated, the term "prevention" refers to an action that occurs before a subject begins to suffer from a disease (e.g., a retinal neurodegenerative disease), which delays the onset of the disease, and / or inhibits or reduces the severity of the disease. It will be appreciated that treatment can be prophylactic treatment as well as treatment after diagnosis of a disease or condition. Treatment of the present application can reduce or eliminate symptoms or characteristics of a disorder, disease, or condition, or can eliminate the disorder, disease, or condition itself. It will be appreciated that methods of treatment of the present application can slow or eliminate the development of a disease, or disorder condition, and in some cases can lead to regression of the disease, disorder, or condition. In some embodiments of the present application, ChRmine variants of the present application can be expressed in a population of cells and used to treat a retinal neurodegenerative disease.

[0039] As used herein in the specification and claims, a "therapeutically effective amount" of a compound is an amount sufficient to provide any therapeutic benefit in the treatment of a disease, or an amount sufficient to delay or lessen one or more symptoms associated with a disease. A therapeutically effective amount of a compound means an amount of a compound, alone or in combination with one or more other therapies and therapeutic agents, that provides any therapeutic benefit in the treatment of a disease. The term "therapeutically effective amount" can include an amount that reduces retinal neurodegenerative disease, improves or reduces visual impairment, improves overall treatment, or enhances the efficacy of another therapeutic agent.

[0040] As used herein in the specification and claims, a "patient" or "subject" includes a mammal, such as a human and a non-human mammal, non-limiting examples of which include rodents, mice, rats, non-human primates, companion animals (such as dogs and cats), and livestock (such as sheep, cattle, horses, etc.), that is suffering from or susceptible to a disease as described herein.

[0041] As used herein, the term "vision" is defined as the ability of an organism to effectively detect light as a stimulus. "Vision" is intended to encompass the following aspects: (1) light detection or perception, i.e., the ability to discriminate whether light is present or not; (2) light projection, i.e., the ability to discriminate from which direction a light stimulus comes; (3) resolution, i.e., the ability to detect different levels of brightness (i.e., contrast) in a grating or letter target; and (4) recognition, i.e., the ability to recognize the shape of a visual target by reference to different contrasts within the target. Thus, "vision" includes the ability to simply detect whether light is present or not, the light preferably being red light, more preferably the light having a wavelength of between about 365 nm and about 700 nm, between about 530 nm and about 640 nm, and in some embodiments, peak activation can occur upon exposure to light having a wavelength of about 550 nm.

[0042] As used herein in the specification and claims, the term "ChRmine variant" refers to a protein formed by the introduction of one or more additions, deletions, or substitutions of amino acids relative to a parent ChRmine protein. In the context of the present application, the ChRmine variant has an improvement in at least one property, activity, or function as compared to the parent ChRmine protein. Such properties, activities, or functions include, but are not limited to, light activation properties of the protein, stability, expression efficiency, and therapeutic effectiveness. It is understood that such a definition does not mean that the ChRmine variant is superior to the parent ChRmine protein in all respects. For example, the ChRmine variant can be improved in one or more aspects as compared to the parent ChRmine protein, but remain the same or be worse in one or more other aspects.

[0043] The term "ChRmine variants" can also include conservative amino acid residue substitutions. As used in the present application, the term "conservative substitution" generally refers to amino acid substitutions that preserve the structural and functional properties of a protein or polypeptide. Such functionally equivalent (conservative substitution) peptide amino acid sequences include, but are not limited to, the addition or substitution of an amino acid residue in an amino acid sequence encoded by a nucleotide sequence that results in a silent change, thereby making a functionally equivalent gene product. Conservative amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the relevant residues. For example: non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0044] Conservative amino acid substitutions can also be made based on the hydrophilic index of the amino acids. According to the hydrophobic and charge characteristics of each amino acid, a hydrophilic index is assigned to it. These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The use of the hydrophilic amino acid index in imparting biological functions of interest to proteins is known in the art (Kyte and Doolittle, J. Mol. Biol., 157: 105-132, 1982). It is known that in certain instances, certain amino acids can be substituted for other amino acids having a similar hydrophilic index or score and still retain a similar biological activity. In making changes based on similar hydrophilic indices, in certain embodiments include amino acid substitutions within the range of +-2 of the hydrophilic index, in other embodiments include amino acid substitutions within the range of +-1 of the hydrophilic index, and in yet other embodiments include amino acid substitutions within the range of +-0.5 of the hydrophilic index.

[0045] Conservative amino acid substitutions can also be made on the basis of hydrophilicity, particularly where the resulting biologically functional protein or peptide is involved in immunological embodiments. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein. These amino acid residues are assigned hydrophilicity values as follows: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +1); glutamate (+3.0 +1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 +1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based on similar hydrophilicity values, in certain embodiments include amino acid substitutions that have a hydrophilicity value within +2 of the preferred substitution, in other embodiments include substitutions that have a hydrophilicity value within +1 of the preferred substitution, and in yet other embodiments include substitutions that have a hydrophilicity value within +0.5 of the preferred substitution.

[0046] Accordingly, ChRmine variants of the present application can have the amino acid sequence set forth in SEQ ID NO: 3, 7, 11, or 15, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, 7, 11, or 15.

[0047] Amino acid changes are accomplished by altering the codons of the corresponding nucleic acid sequence. Such protein variants are known to be obtained by replacing certain amino acids with others in the protein structure in order to modify or improve biological activity. For example, by replacing amino acids, small conformational changes can be imparted to a protein that results in increased activity. Alternatively, amino acid replacements in certain proteins can be used to provide residues that can then be linked to other molecules to provide peptide-molecule conjugates that retain sufficient properties of the starting polypeptide for other purposes.

[0048] In one embodiment, the ChRmine variants of the present application can be encoded by a nucleotide sequence set forth in SEQ ID NO: 4, 8, 12, or 16, or a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4, 8, 12, or 16. In one embodiment, the nucleotide sequence is substantially identical to SEQ ID NO: 4, 8, 12, or 16.

[0049] 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4, 8, 12, or 16. In one embodiment, the nucleotide sequence is substantially identical to SEQ ID NO: 4, 8, 12, or 16.

[0050] The ChRmine variants of the present application can also be provided in the form of fusion proteins. One or more functional domains can be linked to the ChRmine variants to provide an improvement in a certain biological activity. The functional domains include, but are not limited to, a hemagglutinin signal peptide, a bacterial rhodopsin-like insertion loop, a kv2.1 channel protein, a Golgi export signal, and / or a rhodopsin trafficking sequence, or any combination thereof. Such functional domains can be linked to the ChRmine variants directly, or indirectly through a linker, or both. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is composed of glycine, or composed of glycine and serine. The linker of the fusion proteins of the present disclosure can have various lengths, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids long. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of GG, GGG, GGGG, GGGGG, GGGGGG, (GGS)2, (GGS)3, (GGS)4, and (GGS)5, or any combination thereof.

[0051] The term "hemagglutinin (HA) signal peptide", as used herein, refers to a short peptide chain derived from influenza virus, comprising about 13-30 amino acid residues. It is usually located at the N-terminus of a protein, and can help the target protein to be correctly inserted and oriented to the membrane of a cell or an organelle. An exemplary hemagglutinin signal peptide has the amino acid sequence set forth in MKTIIALSYIFCLVFA (SEQ ID NO: 19).

[0052] The term "bacterial rhodopsin-like insertion loop (BRIL)", as used herein, refers to a membrane protein derived from a halophilic archaea, which is designed to improve the expression, stability of a membrane protein. An exemplary BRIL has the amino acid sequence set forth in

[0053] The amino acid sequence shown is ADLEDNWETLNDNLKVIEKADNAAQVKDALTKMRAAALDAQKATPPKLEDKSPDSP EMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAEQLKTTRNAYIQKYL (SEQ ID NO:20).

[0054] As used in this article, the term "kv2.1 channel protein" refers to a voltage-gated potassium channel that, through a specific localization mechanism, reduces its distribution in axons, facilitating protein localization to the cell body and dendrites. Exemplary kv2.1 channel proteins possess...

[0055] The amino acid sequence shown is QSQPILNTKEMAPQSKPPEELEMSSMPSPVAPLPARTEGVIDMRSMSSIDSFISCATDFP EATRF (SEQ ID NO:21).

[0056] As used herein, the term "Golgi output signal" refers to a signal sequence responsible for the transport of membrane proteins from the Golgi apparatus to the cell membrane or other parts. An exemplary Golgi output signal has the amino acid sequence shown in RSRFVKKDGHCNVQFINV (SEQ ID NO: 22).

[0057] As used herein, the term "rhodopsin transport sequence" refers to an important signaling sequence of rhodopsin responsible for its proper folding and transport in the endoplasmic reticulum. An exemplary rhodopsin transport sequence has the amino acid sequence shown in TETSQVAPA (SEQ ID NO:23).

[0058] In one embodiment, the fusion protein described herein includes, but is not limited to, the following structures:

[0059] HA signal peptide-ChRmine variant;

[0060] HA signal peptide-BRIL-ChRmine variant;

[0061] HA signal peptide-BRIL-ChRmine variant-kv2.1;

[0062] HA signal peptide-BRIL-ChRmine variant-kv2.1-Golgi output signal;

[0063] HA signal peptide-BRIL-ChRmine variant-kv2.1-Golgi output signal-rhodopsin transport sequence;

[0064] HA signal peptide - ChRmine variant - BRIL;

[0065] HA signal peptide - ChRmine variant - BRIL - kv2.1 ;

[0066] HA signal peptide - ChRmine variant - BRIL - kv2.1 - Golgi export signal;

[0067] HA signal peptide - ChRmine variant - BRIL - kv2.1 - Golgi export signal - rhodopsin trafficking sequence;

[0068] HA signal peptide - ChRmine variant - kv2.1 ;

[0069] HA signal peptide - ChRmine variant - Golgi export signal;

[0070] HA signal peptide - ChRmine variant - rhodopsin trafficking sequence;

[0071] HA signal peptide - kv2.1 - ChRmine variant - BRIL - Golgi export signal - rhodopsin trafficking sequence;

[0072] HA signal peptide - Golgi export signal - ChRmine variant - BRIL - kv2.1 - rhodopsin trafficking sequence;

[0073] HA signal peptide - rhodopsin trafficking sequence - ChRmine variant - BRIL - kv2.1 - Golgi export signal;

[0074] wherein each domain is directly connected, or indirectly connected via a linker.

[0075] It should be understood that the number and relative position of each domain in the fusion proteins described herein is not limited, so long as their respective corresponding functions are not significantly affected.

[0076] Accordingly, the fusion proteins of the present application can have an amino acid sequence set forth in SEQ ID NO: 5, 9, 13, or 17, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5, 9, 13, or 17.

[0077] In one embodiment, the ChRmine variants of the application can be encoded by the nucleotide sequence set forth in SEQ ID NO: 6, 10, 14, or 18, or a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6, 10, 14, or 18. In one embodiment, the nucleotide sequence is substantially identical to SEQ ID NO: 6, 10, 14, or 18.

[0078] In the context of two or more nucleic acid or polypeptide sequences, the term percent "identity" means the designated percentage of amino acid residues or nucleotides that are the same or have identical function when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0079] For sequence comparison, typically one sequence is compared to a reference sequence, and the test sequence is compared to the reference sequence. When using a sequence alignment algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated (if necessary), and program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence against the reference sequence based on the designated program parameters.

[0080] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith&Waterman (Adv. Appl. Math. 2:482 (1981)), by using the homology alignment algorithm of Needleman&Wunsch (J Mol. Biol., 48:443 (1970)), by searching the similarity method of Pearson&Lipman (Proc. Nat'l. Acad. Sci. USA 85:2444 (1988)), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement) (Ausubel)).

[0081] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, e.g., the parameters described in Altschul et al. (1997) supra.

[0082] For nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0) are used to compute the cumulative score. For amino acid sequences, a scoring matrix is used to compute the cumulative score. Extension of the word hits in each direction is terminated when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation value (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0083] Another indication that two nucleic acid or polypeptide sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. Thus, a first polypeptide is typically substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0084] The nucleic acids according to the present application can include additional sequences including, but not limited to, one or more signal sequences (e.g., enhancers, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites) and / or promoter sequences, or other coding segments, or combinations thereof. The promoter can be an inducible or constitutive general or cell-specific promoter. One example of a cell-specific promoter is the bipolar cell-specific mGlu6 promoter or the ganglion cell-specific SNCG promoter. Some embodiments are any of the disclosed methods, wherein the promoter is a constitutive promoter. Some embodiments are any of the disclosed methods, the constitutive promoter includes, but is not limited to, a CMV promoter or a CAG promoter (consists of the cytomegalovirus (CMV) early enhancer and chicken beta-actin promoter). The promoter, vector, enhancer, polyadenylation site are routine choices for one of skill in the art. These elements are well described in the literature and are commercially available.

[0085] The present invention relates in certain aspects to the expression of light- sensitive ion channel proteins in cells that can be activated by contact with one or more light pulses, resulting in strong depolarization of the cell. The light-sensitive ion channel proteins according to the present invention, also referred to as light-activated ion channels, can be expressed in specific cells, tissues and / or organisms and used to control the response of cells in vivo, ex vivo and in vitro to light pulses of appropriate wavelength. The light-sensitive ion channel proteins according to the present invention include, but are not limited to, ChRmine variants of the present invention and fusion proteins. In the present invention, the term "ion channel" refers to a transmembrane polypeptide that forms a pore, which opens upon activation, allowing ion conductance through the pore across the membrane.

[0086] The light-sensitive ion channel proteins according to the present invention are strongly activated by contact with red light, preferably light having a wavelength between about 365 nm and about 700 nm, and in some embodiments, peak activation occurs upon contact with light having a wavelength of about 550 nm.

[0087] Contacting excitable cells comprising a light-sensitive ion channel protein according to the present invention with light having a wavelength in the activation range will strongly depolarize the cell. Exemplary wavelengths of light that can be used to depolarize cells expressing a light-sensitive ion channel protein according to the present invention include wavelengths of at least about 365 nm, 385 nm, 405 nm, 425 nm, 445 nm, 465 nm, 485 nm, 505 nm, 525 nm, 545 nm, 565 nm, 585 nm, 590 nm, 605 nm, 625 nm, 645 nm, 665 nm, 685 nm and 700 nm, including all wavelengths between the aforementioned wavelengths. In some embodiments, the light-sensitive ion channel proteins according to the present invention have peak wavelength sensitivity at 550 nm.

[0088] The light-sensitive ion channel proteins according to the present invention can be used to depolarize excitable cells in which a light-sensitive ion channel protein according to the present invention is expressed. In some embodiments, the light-sensitive ion channel proteins according to the present invention can be expressed in a subpopulation of cells of a cell population that also includes one or more additional subpopulations of cells that express a light-sensitive ion channel protein that is not activated by the wavelength of light that activates the light-sensitive ion channel proteins according to the present invention.

[0089] While the peptide amino acid sequences described herein can be chemically synthesized, large polypeptide sequences can preferably be produced by recombinant DNA technology using techniques well known in the art for expressing nucleic acids containing nucleotide sequences encoding the desired peptide. Such methods can be used to construct expression vectors containing nucleotide sequences encoding the peptide and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination, which are well known in the art.

[0090] In the present application, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been operatively linked between different genetic environments.

[0091] A nucleic acid is "operably linked" when it is within a functional relationship with another nucleic acid sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous. However, enhancers do not need to be contiguous. Linking is accomplished by ligation at conventional restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0092] The term "vector" also refers to a virus or organism that is capable of transporting a nucleic acid molecule. One type of vector is an episome, i.e., a nucleic acid molecule capable of extra-chromosomal replication. Some useful vectors are those that are capable of autonomous replication and / or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". Expression vectors and their use are well known in the art. The present application provides non-limiting examples of suitable expression vectors and methods of using the same. In preferred embodiments, the vector is suitable for use in gene therapy, particularly for viral-mediated gene transfer. Viral vectors suitable for gene therapy include retroviruses, adenoviruses, adeno-associated viruses (AAV), lentiviruses, poxviruses (e.g., MVA), alphaviruses, herpesviruses, and the like. Examples of AAVs can be found in Davidson et al., PNAS (2000) 97:3428-3432. AAVs and lentiviruses can confer persistent expression, while adenoviruses can provide transient expression.

[0093] However, gene therapy further includes non-viral methods, such as the use of naked DNA or nucleic acids bound to liposomes. Vectors suitable for use in some methods according to the present application can genetically insert the light-sensitive ion channel protein into dividing and non-dividing cells, and can insert the light-sensitive ion channel protein into cells in vivo, in vitro, or ex vivo.

[0094] In some preferred embodiments, the nucleic acid expression vector comprising the gene of the light-sensitive ion channel protein according to the application is selected from the group consisting of AAV viral vectors. The term "serotype" refers to an AAV, e.g. a primate AAV, distinguished from other AAVs by a defined antiserum and based on the capsid protein reactivity with said defined antiserum, e.g. there are many known serotypes of AAV (e.g. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV-Rh74 and AAV Rh10 and modified capsids of these serotypes).

[0095] "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein, preferably all capsid proteins of a wild type AAV, and a polynucleotide encapsidated by the capsid. According to preferred embodiments, the AAV viral vectors used in the present application are of the serotypes AAV-2, AAV-5, AAV2-7m8, AAV-9 and AAV-8, more preferably of the serotypes AAV-2 or AAV2-7m8.

[0096] Some embodiments are methods of treating or preventing a retinal neurodegenerative disease, the method comprising: (a) delivering to a target cell a nucleic acid expression vector encoding a light-sensitive ion channel protein according to the application, said light-sensitive ion channel protein being capable of being expressed in said target cell, said vector comprising an open reading frame encoding a light-sensitive ion channel protein according to the application, operably linked to a promoter sequence and optionally to a transcriptional regulatory sequence; (b) expressing said vector in said target cell, wherein the expressed light-sensitive ion channel protein activates said target cell upon exposure to light.

[0097] The light-sensitive ion channel protein according to the application has been found to be suitable for expression and use in mammalian cells under normal cellular environmental conditions and ion concentrations, without any kind of chemical supplementation. The light-sensitive ion channel protein according to the application has been found to be activated in the range of 365 nm to 700 nm of light wavelength, preferably at a wavelength of 530 nm to 640 nm, with a peak activation wavelength of 550 nm.

[0098] An effective amount of a light-sensitive ion channel protein or a nucleic acid expression vector thereof is an amount that elevates the level of the light-sensitive ion channel protein in a cell, tissue, or subject to a level that is beneficial to the subject. An effective amount can also be determined by assessing the physiological effects on a cell or subject after administration, e.g., a reduction in symptoms. Other assays are known to those of ordinary skill in the art and can be used to determine the level of response to treatment. The amount of treatment can be varied, e.g., by increasing or decreasing the amount of light-sensitive ion channel protein or nucleic acid expression vector administered, varying the therapeutic composition containing the administered light-sensitive ion channel protein or nucleic acid expression vector, varying the route of administration, varying the timing of administration, and the like. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors that those of ordinary skill in the art will recognize. These factors are well known to those of ordinary skill in the art and are readily adapted to the particular situation by routine experimentation. Those of ordinary skill in the art will appreciate that a subject can insist on a lower dose or tolerate a dose for medical, psychological, or almost any other reason.

[0099] A light-sensitive ion channel protein or a nucleic acid expression vector thereof according to the present application can be administered using methods known in the art. In certain embodiments, a nucleic acid encoding a light-sensitive ion channel protein according to the present application is administered to a subject. In certain embodiments, a light-sensitive ion channel protein is administered to a subject. The manner and dosage of administration can be adjusted by a physician as is particularly indicated, especially in the event of any complications. The absolute amounts administered will depend on a variety of factors including the material selected for administration, whether single or multiple doses are administered, and the personal parameters of the subject, including age, weight, health, and the extent of the disease. These factors are well known to those of ordinary skill in the art and are readily adapted to the particular situation by routine experimentation.

[0100] A pharmaceutical composition comprising a light-sensitive ion channel protein or a nucleic acid expression vector thereof according to the present application can be administered alone and / or in combination with other pharmaceutical therapies or other treatment regimens administered to a subject. Various modes of administration are known to those of ordinary skill in the art. Suitable modes of administration can be topical administration, intravenous administration, oral administration, intracavitary administration, intrathecal administration, intrasynovial administration, buccal administration, sublingual administration, intranasal administration, transdermal administration, subretinal administration, subtenon administration, subcutaneous administration, intramuscular administration, and intradermal administration. The present application is not limited by the particular mode of administration disclosed herein.

[0101] The present disclosure includes, in certain aspects, preparing a nucleic acid comprising a nucleotide sequence; expressing a polypeptide encoded by the prepared nucleic acid and nucleotide sequence in a cell and / or membrane; irradiating the cell and / or membrane with appropriate light, and verifying the rapid depolarization of the cell and / or change in transmembrane conductance in response to the light, and the rapid escape from depolarization after light decay. The ability to controllably change the transmembrane voltage and cell depolarization by light has been verified. The present invention enables light control of cell function in vivo, ex vivo, and in vitro, and the light-sensitive ion channel proteins according to the present invention have a wide range of applications in drug screening, therapy, and research applications, some of which are described in the present invention.

[0102] The following examples section provides further detailed information regarding various specific embodiments. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques and / or compositions found to be useful by the inventors. However, it is understood that those skilled in the art will be able to prepare many variations of the disclosed embodiments without the use of undue experimentation, in light of the disclosure herein. These examples are merely for illustrative purposes and are not meant to be limiting on the scope of the application.

[0103] Example 1: Construction of Chrmine variants

[0104] Based on the wild-type Chrmine protein (SEQ ID NO: 1), mutation sites were introduced by primer design, followed by PCR amplification of the mutated gene fragment, which was then cloned into an expression vector. The mutation sites were verified by DNA sequencing.

[0105] Through preliminary screening of the light-activated properties, stability, and expression efficiency of the protein, the present inventors obtained Chrmine variants M1 (L228A / I232A), M2 (Y125G), M3 (L118A), and M4 (M122A), and constructed their respective fusion proteins M1-A, M2-A, M3-A, and M4-A.

[0106] Example 2: In vitro light stimulation to record the photosensitive ability of Chrmine variants

[0107] Genes encoding Chrmine variants of light-sensitive ion channel proteins are transiently transfected into HEK293T cells to ensure their efficient expression. Then, these cells expressing light-sensitive ion channel proteins are placed in an electrophysiological recording system. In current clamp or voltage clamp mode, cells are stimulated with light pulses of different wavelengths and intensities to activate the light-sensitive ion channel proteins. By recording changes in cell membrane potential or current responses, the response of the protein to light stimulation is evaluated. By adjusting the parameters of light (such as wavelength and intensity) and applying specific ion channel inhibitors, the functional characteristics of the light-sensitive ion channel proteins are further analyzed and verified. Statistical analysis shows that the light sensitivity of variants M3 and M3-A is significantly stronger than that of Chrmine itself, and the remaining variants are slightly better. See Figure 1 .

[0108] The specific steps are as follows:

[0109] 1. Cell culture and transfection

[0110] Cell culture: HEK293T cells are cultured in DMEM medium containing 10% FBS, and cultured at 37°C in a 5% CO2 environment until the cells reach 70-80% confluence.

[0111] Plasmid transfection: Use Lipofectamine 3000 to transfect plasmid DNA encoding light-sensitive proteins into HEK293T cells. Follow the reagent instructions for transfection, usually replace fresh medium after 6-8 hours.

[0112] 2. Protein expression and cell preparation

[0113] Expression induction: No special induction is required. 24-48 hours after transfection, protein expression reaches a peak.

[0114] Cell treatment: Before recording, wash the cells with PBS and replace them with buffer suitable for recording, such as HBSS.

[0115] 3. Light stimulation experiment

[0116] Microscope preparation: Use a fluorescence microscope or confocal microscope. Install a light source of specific wavelength (such as LED or laser) to stimulate light-sensitive proteins.

[0117] Light stimulation: Set different light intensities and durations according to the experimental design. Ensure that the recording equipment (such as an electrophysiological recorder or a fluorescence imaging system) is calibrated and ready to record signals.

[0118] 4. Data recording and analysis

[0119] Signal recording: Use the corresponding recording equipment to record the response of light-sensitive proteins.

[0120] Data analysis: analyze the recorded data, draw the response curve, and evaluate the photosensitive properties of the light-sensitive protein, such as response speed, sensitivity, dynamic range, etc.

[0121] Example 3: In vivo pharmacodynamic verification of Chrmine variants

[0122] The AAV2 / 7m8 serotype used in this study was produced by Genewiz Biotechnology (Shanghai) Co., Ltd. C3H mice (12 weeks old, male) with photoreceptor degeneration were selected for subretinal injection, and the dosage was 1.5 x 10 9 VG (vector genome) / eye (1.5 ul, 1 x 10 12 VG / ml).

[0123] The steps are as follows: 1% sodium pentobarbital was injected intraperitoneally to anesthetize the mouse (10 ul / g), the mouse whiskers were cut, and a drop of hydrochloric acid deoxyepinephrine eye drops (2.5%) was added to the mouse eye. The mouse was placed on an electric blanket, and after the pupil of the mouse was dilated, it was placed under a body microscope, and some absorbent paper was placed on the stage, and a thermostat was also placed to prevent the mouse from dying due to sudden drop in body temperature. The head of the mouse was raised to the level of the corneal limbus, and a drop of 0.5% hydrochloric acid propamocaine eye drops was added to the mouse eye for short-term surface anesthesia. A 31 gauge sharp needle was used for corneal puncture to release some aqueous humor to reduce intraocular pressure, and a 5 ul Hamilton microsyringe connected to a 33 gauge sharp needle was used to inject 1.5 ul of AAV virus particles vertically 1 mm behind the corneal limbus (avoiding large blood vessels), then the needle was tilted at 45°, and the microsyringe was slowly pushed to inject the virus particles into the subretinal space (about 1 min to push). After pushing, the needle was left for 1 min, and finally the needle was slowly pulled out to complete the subretinal injection.

[0124] Brief description of retinal stromal imaging: After perfusion with physiological saline (0.9% NaCl) and 4% PFA, mouse eyes were taken and fixed overnight in 4% PFA solution at 4°C. The eyes were washed three times with PBS for 5 minutes each time. Under a microscope, the cornea was cut off along the limbus using ophthalmic surgical scissors, while the lens was removed. The cornea was then dehydrated by placing it in 5% sucrose solution for 5 minutes, 15% sucrose solution for 1 hour, and 30% sucrose solution for 2 hours. In 30% sucrose solution, under a microscope, a small longitudinal incision was made along the limbus between the retina and sclera using ophthalmic surgical scissors. The sclera was held with forceps, and the sclera was carefully cut off piece by piece through this small incision, revealing the translucent retinal tissue. Four radial incisions were made in the retina, centered on the optic papilla. A square groove (50mm × 50mm) was constructed on a glass slide using a sealing film drawn into thin filaments. Place the retina in the center of the square slot with the opening facing upwards (i.e., the retinal ganglion cell layer facing upwards). Use a pipette and absorbent paper to remove any remaining sucrose solution. Place the retina in a petal shape against the slide, and simultaneously add a drop of sealing medium. Quickly cover with a coverslip and seal with nail polish. Use a Zeiss LSM880 confocal microscope or a Nikon turntable (CSU W1Sora) confocal microscope for microscopic imaging.

[0125] Brief description of optic nerve imaging: After perfusion with physiological saline (0.9% NaCl), mice were euthanized by cervical dislocation. Surgical scissors were used to remove as much muscle as possible around the eye socket, and the eye socket was cut open to allow the eyeball to protrude as much as possible. The eyeball and optic nerve were removed together with curved forceps and fixed in 4% PFA solution at room temperature for 1 hour. The eyes were washed three times with PBS for 5 minutes each time. At the same time, the optic nerve was cut off along the end of the optic papilla under a microscope. A square groove (50mm × 50mm) was constructed on a glass slide using a sealing film drawn into a thin filament. The optic nerve was placed in the center of the square groove and straightened as much as possible. A drop of sealing medium was added at the same time, and a coverslip was quickly placed on top. The slide was then sealed with nail polish. Microscopic imaging was performed using a Zeiss LSM880 confocal microscope and a Nikon turntable (CSU W1 Sora) confocal microscope.

[0126] Retinal patch and optic nerve imaging of C3H mice 7 weeks after drug administration revealed that variant M1 was highly expressed in retinal ganglion cells. Figure 2 A), and the axons converge at the optic nerve ( Figure 2 B), the infection efficiency of RGCs is approximately 20%. Figure 2 C).

[0127] The treated retina was prepared as a whole-mount and placed in an electrophysiological recording system. In whole-cell current-clamp mode, these retinal cells expressing photosensitive proteins were stimulated using light pulses of different wavelengths. Figure 2D shows that M1 variant can respond to different wavelength of light stimuli (305nm-635nm). Cells were placed in voltage clamp, which can precisely control the membrane potential of cells, and record the current response of M1 under different light intensity Figure 2 E). Figure 2 F is the relationship between light wave and voltage response. Figure 2 G is the relationship between light intensity and current response. Using 0.1 cd*s / m2light intensity for EGR data collection (a wave + b wave), it was found that C3H mice expressing AAV-M1 could record ERG signals compared with AAV drug control, indicating that the vision of blind mice was partially restored Figure 2 H).

[0128] Example 4: ERG efficacy summary of Chrmine variants

[0129] C3H mice after 7 weeks of administration were given 12h dark adaptation in advance, and the mice were anesthetized by intraperitoneal injection of Zoletil (25mg / kg) and Xylazine (2.5mg / kg). A drop of hydrochloric acid deoxyepinephrine eye drops (2.5%) was dropped on the mouse eye, and after the pupil of the mouse was enlarged, the corneal electrode was tightly attached to the cornea of the eye, and the reference electrode was fixed near the mouse tail and the mouse eyelid. Using 0.1 cd*s / m2and 2cd*s / m2light intensity, respectively, EGR data collection (retinal current map) was performed, and it was found that the experimental group expressing ChRmine could record ERG signals compared with the control under two light intensities, and the light intensity was enhanced, and the ERG signal was also enhanced, which conforms to the normal physiological phenomenon. As shown in Figure 3 M1, M1-A, M2, M2-A, M3, M3-A, M4 and M4-A, among which M3 and M3-A are the most effective.

[0130] Sequence information

[0131] Amino acid sequence of Chrmine, SEQ ID NO: 1

[0132] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLSAAAKSRITSEGEYIPLDQIDINV

[0133] Nucleotide sequence of Chrmine, SEQ ID NO: 2

[0134] ATGGCCCACGCCCCTGGCACCGACCAGATGTTCTACGTGGGAACAATGGACGGCTGGTATCTGGATACCAAGCTGAACAGCGTGGCCATTGGAGCCCACTGGTCCTGTTTTATCGTTCTGACCATCACCACCTTCTACCTGGGCTATGAGAGCTGGACCTCCCGGGGCCCCAGCAAGCGGACCAGCTTCTACGCCGGCTACCAGGAGGAACAGAACCTGGCCCTGTTCGTGAACTTCTTCGCCATGCTGAGCTACTTCGGGAAGATCGTGGCCGACACCCTGGGACACAACTTCGGCGATGTGGGCCCTTTCATCATAGGATTTGGAAATTACAGGTACGCCGACTACATGCTGACATGCCCTATGCTGGTCTACGATCTGCTGTACCAGCTGAGAGCTCCTTACCGGGTGTCTTGTAGCGCCATCATCTTCGCTATTCTGATGAGCGGCGTGCTGGCAGAGTTCTACGCCGAGGGCGACCCCAGACTGCGAAACGGCGCCTACGCCTGGTACGGCTTTGGCTGCTTCTGGTTTATCTTCGCCTATTCTATCGTGATGAGCATCGTGGCCAAGCAGTACTCCAGACTGGCTCAGCTGGCTCAGGACACAGGCGCCGAGCACAGCCTGCACGTGCTGAAGTTCGCCGTGTTCACCTTCAGCATGCTGTGGATCCTGTTCCCCCTGGTGTGGGCCATCTGCCCAAGAGGCTTCGGCTGGATCGACGACAATTGGACAGAGGTGGCTCATTGTGTGTGCGACATCGTCGCCAAGAGCTGCTACGGCTTCGCCCTGGCTAGATTCAGAAAGACATACGACGAGGAGCTGTTCCGGCTGCTCGAGCAACTGGGCCACGACGAGGACGAATTTCAGAAACTGGAACTGGACATGAGACTCAGCAGCAACGGCGAGAGACTGCGCAGACTGTCTGCCGCGGCTAAGAGCAGAATCACAAGTGAAGGCGAATACATCCCCCTGGATCAAATCGACATCAACGTG

[0135] Amino acid sequence of Chrmine variant M1 (L228A / I232A), SEQ ID NO: 3

[0136] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPAVWAACPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLSAAAKSRITSEGEYIPLDQIDINV

[0137] Nucleotide sequence of Chrmine variant M1 (L228A / I232A), SEQ ID NO: 4

[0138] ATGGCCCACGCCCCTGGCACTGATCAGATGTTCTACGTGGGAACAATGGACGGCTGGTATCTGGATACAAAGCTGAACAGCGTGGCCATCGGCGCCCACTGGTCCTGCTTCATCGTGCTGACAATCACAACCTTCTACCTGGGCTACGAGAGCTGGACAAGCAGAGGCCCTAGCAAGCGGACCAGCTTCTACGCCGGCTACCAGGAGGAACAAAATCTGGCCCTGTTTGTGAACTTCTTCGCCATGCTGAGCTACTTCGGCAAGATCGTCGCCGACACCCTGGGCCACAACTTCGGCGATGTGGGCCCCTTCATCATTGGCTTCGGCAACTACAGATACGCCGATTACATGCTGACCTGCCCCATGCTTGTGTACGACCTGCTGTACCAACTGAGAGCCCCTTACCGGGTGTCCTGTTCTGCCATCATCTTCGCCATTCTGATGTCCGGCGTGCTCGCCGAGTTCTACGCCGAAGGCGACCCTAGGCTGCGGAACGGCGCTTATGCCTGGTACGGCTTTGGTTGTTTTTGGTTCATCTTTGCCTACAGCATCGTGATGTCTATCGTTGCGAAACAGTACTCCCGGCTGGCCCAGCTGGCTCAGGACACCGGCGCCGAGCACAGCCTGCACGTGCTGAAGTTCGCCGTGTTCACCTTCAGCATGCTGTGGATCCTGTTCCCTGCTGTGTGGGCCGCCTGCCCAAGAGGATTTGGATGGATCGACGACAATTGGACCGAGGTGGCCCATTGTGTGTGCGACATCGTGGCCAAGAGCTGCTACGGCTTCGCTCTGGCCAGATTCAGAAAGACCTACGACGAGGAACTGTTCCGGCTGCTGGAACAGCTGGGACACGACGAGGACGAATTTCAGAAGCTGGAACTGGATATGAGACTCAGCAGCAACGGCGAGAGACTCCGCAGACTGAGCGCCGCTGCTAAGTCTAGAATCACCAGCGAGGGAGAGTACATCCCCCTGGACCAGATCGATATCAACGTG

[0139] Amino acid sequence of Chrmine variant M1 -A (hemagglutinin signal peptide - Chrmine (L228A / I232A)), SEQ ID NO: 5

[0140] MKTIIALSYIFCLVFAMAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPAVWAACPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLSAAAKSRITSEGEYIPLDQIDINV

[0141] Nucleotide sequence of Chrmine variant M1 -A (hemagglutinin signal peptide - Chrmine (L228A / I232A)), SEQ ID NO: 6

[0142]

[0143] Amino acid sequence of Chrmine variant M2 (Y125G), SEQ ID NO: 7

[0144] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVGDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLSAAAKSRITSEGEYIPLDQIDINV

[0145] Nucleotide sequence of Chrmine variant M2 (Y125G), SEQ ID NO: 8

[0146] ATGGCCCATGCCCCTGGCACCGACCAGATGTTCTACGTGGGCACAATGGACGGCTGGTACCTGGACACAAAGCTGAACAGCGTGGCCATCGGCGCCCACTGGTCCTGTTTTATCGTGCTGACCATCACAACCTTCTACCTGGGATACGAGAGCTGGACCTCTAGAGGCCCCAGCAAGAGAACCAGCTTCTACGCAGGCTACCAGGAGGAACAGAACCTGGCCCTGTTTGTGAACTTCTTCGCCATGCTGTCCTACTTCGGCAAGATCGTGGCTGATACCCTGGGCCACAACTTCGGCGATGTGGGACCATTCATTATCGGCTTTGGCAATTACAGATACGCCGACTACATGCTCACATGTCCTATGCTGGTCGGCGATCTGCTGTACCAGCTGAGAGCCCCTTACAGAGTGTCCTGCAGCGCCATCATCTTCGCAATCCTGATGAGCGGCGTGCTGGCCGAGTTCTACGCTGAAGGCGACCCTAGACTGCGGAACGGAGCTTATGCCTGGTATGGATTCGGATGTTTTTGGTTCATCTTTGCCTACTCTATCGTGATGAGCATCGTGGCCAAACAGTACAGCAGACTGGCCCAGCTGGCCCAAGACACCGGAGCCGAGCACAGTCTGCACGTGCTGAAGTTCGCCGTGTTCACCTTCAGCATGCTGTGGATCCTGTTCCCTCTGGTGTGGGCCATCTGCCCCCGGGGCTTTGGCTGGATCGATGATAATTGGACCGAGGTGGCCCACTGCGTGTGCGACATCGTCGCCAAGTCTTGCTACGGCTTCGCCCTGGCTAGATTCAGAAAGACATACGACGAGGAACTGTTCCGGCTGCTCGAGCAACTGGGCCACGACGAGGACGAGTTCCAGAAGCTGGAACTGGACATGAGGCTGTCTAGCAACGGCGAGAGACTTCGCAGACTGAGCGCTGCTGCCAAGAGCCGGATCACCAGCGAGGGCGAATACATCCCCCTGGATCAGATCGACATCAACGTT

[0147] Amino acid sequence of Chrmine variant M2-A (hemagglutinin signal peptide-BRIL-Chrmine(Y125G)-kv2.1-Golgi export signal- rhodopsin transport sequence), SEQ ID NO: 9

[0148] MKTIIALSYIFCLVFAADLEDNWETLNDNLKVIEKADNAAQVKDALTKMRAAALDAQKATPPKLEDKSPDSPEMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAEQLKTTRNAYIQKYLMAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVGDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLSAAAKSRITSEGEYIPLDQIDINVQSQPILNTKEMAPQSKPPEELEMSSMPSPVAPLPARTEGVIDMRSMSSIDSFISCATDFPEATRFRSRFVKKDGHCNVQFINVTETSQVAPA

[0149] Nucleotide sequence of Chrmine variant M2-A (hemagglutinin signal peptide-BRIL-Chrmine(Y125G)-kv2.1-Golgi export signal- rhodopsin transport sequence), SEQ ID NO: 10

[0150]

[0151] Amino acid sequence of Chrmine variant M3 (L118A), SEQ ID NO: 11

[0152] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMATCPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLSAAAKSRITSEGEYIPLDQIDINV

[0153] Nucleotide sequence of Chrmine variant M3 (L118A), SEQ ID NO: 12

[0154] ATGGCTCATGCCCCTGGCACCGACCAGATGTTCTACGTGGGCACAATGGACGGCTGGTATCTGGATACAAAGCTGAACAGCGTGGCCATCGGCGCCCACTGGAGCTGTTTTATCGTGCTGACCATCACCACATTCTACCTGGGCTACGAGTCCTGGACAAGCAGAGGACCTAGCAAGCGGACCTCTTTTTACGCCGGCTATCAGGAGGAACAGAATCTGGCCCTGTTTGTGAACTTCTTCGCCATGCTGAGCTACTTCGGCAAGATCGTGGCCGACACACTGGGCCACAACTTTGGCGACGTGGGACCATTCATCATCGGGTTCGGCAATTACAGATACGCCGATTACATGGCCACCTGCCCCATGCTGGTGTACGACCTGCTGTACCAACTGAGAGCCCCTTACAGAGTGTCTTGTAGCGCCATCATTTTCGCCATTCTGATGAGCGGCGTGCTGGCCGAGTTCTACGCTGAAGGCGATCCCAGACTGAGAAACGGCGCTTATGCCTGGTACGGCTTCGGCTGCTTCTGGTTCATCTTTGCCTACAGCATCGTGATGTCCATCGTGGCCAAGCAGTACAGCCGGCTCGCCCAACTGGCCCAGGATACCGGAGCCGAGCACTCTCTGCACGTGCTAAAGTTCGCCGTGTTCACCTTCAGCATGCTGTGGATCCTGTTCCCCCTGGTCTGGGCCATCTGCCCTCGCGGCTTTGGCTGGATCGATGACAACTGGACCGAGGTGGCCCACTGTGTGTGCGACATCGTTGCTAAGAGCTGCTACGGCTTCGCCCTGGCTAGATTCCGGAAGACCTACGATGAGGAACTGTTCAGGCTGCTCGAGCAGCTGGGACACGACGAGGACGAGTTCCAGAAACTGGAACTGGACATGCGGCTGAGTAGCAACGGCGAGAGACTGCGGAGACTGTCTGCTGCCGCCAAGTCTAGAATCACAAGCGAGGGAGAATACATCCCTCTGGACCAGATCGACATCAACGTG

[0155] Amino acid sequence of Chrmine variant M3-A (hemagglutinin signal peptide-BRIL-Chrmine(L118A)-kv2.1-Golgi export signal- rhodopsin transport sequence), SEQ ID NO: 13

[0156] Amino acid sequence of Chrmine variant M3-A (hemagglutinin signal peptide-BRIL-Chrmine(L118A)-kv2.1-Golgi export signal- rhodopsin transport sequence), SEQ ID NO: 13

[0157] Nucleotide sequence of Chrmine variant M3-A (hemagglutinin signal peptide-BRIL-Chrmine(L118A)-kv2.1-Golgi export signal- rhodopsin transport sequence), SEQ ID NO: 14

[0158]

[0159] Amino acid sequence of Chrmine variant M4 (M122A), SEQ ID NO: 15

[0160] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPALVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLSAAAKSRITSEGEYIPLDQIDINV

[0161] Nucleotide sequence of Chrmine variant M4 (M122A), SEQ ID NO: 16

[0162] ATGGCCCACGCCCCTGGAACCGACCAGATGTTCTACGTGGGCACCATGGACGGCTGGTATCTGGACACCAAGCTGAACAGCGTGGCCATTGGAGCCCACTGGTCCTGCTTCATCGTGCTGACCATCACCACCTTCTACCTGGGCTACGAGAGCTGGACCAGCAGAGGCCCATCTAAAAGAACAAGCTTCTACGCCGGATATCAGGAGGAACAGAACCTGGCTCTGTTCGTGAACTTCTTCGCCATGCTGAGCTACTTCGGCAAGATCGTGGCTGATACCCTGGGCCACAATTTCGGCGATGTGGGCCCCTTCATCATCGGCTTCGGCAACTACAGATACGCCGACTACATGCTGACATGCCCCGCCCTGGTGTACGACCTGCTGTACCAGCTGAGAGCCCCTTACCGGGTGTCCTGTAGCGCCATCATCTTTGCTATCCTGATGAGCGGCGTGCTGGCCGAGTTCTACGCCGAGGGCGATCCTCGGCTCCGGAACGGCGCCTACGCCTGGTACGGATTTGGTTGTTTTTGGTTCATCTTCGCTTACAGCATCGTGATGTCTATCGTCGCCAAGCAGTACAGCCGGCTGGCCCAACTGGCCCAGGATACAGGCGCCGAACATTCTCTGCACGTGCTGAAGTTCGCCGTGTTCACCTTCAGCATGCTGTGGATCCTGTTCCCTCTGGTTTGGGCCATCTGCCCTAGAGGCTTTGGCTGGATCGACGACAACTGGACAGAGGTGGCCCACTGCGTGTGCGACATCGTGGCAAAGAGCTGTTACGGCTTCGCCTTGGCTAGATTCCGGAAGACCTACGATGAGGAACTGTTTAGACTCCTGGAACAACTGGGACACGACGAGGACGAGTTCCAGAAGCTGGAACTGGACATGAGGCTGTCTTCTAATGGCGAGAGACTGCGCAGACTGAGCGCCGCTGCCAAGTCCAGAATCACCAGCGAGGGCGAATACATCCCCCTGGATCAGATCGACATCAACGTG

[0163] Amino acid sequence of Chrmine variant M4-A (hemagglutinin signal peptide - Chrmine (M122A)), SEQ ID NO: 17

[0164] MKTIIALSYIFCLVFAMAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPALVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLSAAAKSRITSEGEYIPLDQIDINV

[0165] Nucleotide sequence of Chrmine variant M4-A (hemagglutinin signal peptide - Chrmine (M122A)), SEQ ID NO: 18

[0166]

Claims

1. A ChRmine variant, wherein the ChRmine variant comprises an amino acid sequence set forth in SEQ ID NO: 3, 7, 11, or 15, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 3, 7, 11, or 15; or wherein the ChRmine variant consists of an amino acid sequence set forth in SEQ ID NO: 3, 7, 11, or 15, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 3, 7, 11, or 15.

2. A fusion protein, wherein the fusion protein comprises the ChRmine variant of claim 1.

3. The fusion protein of claim 2, wherein the fusion protein further comprises a hemagglutinin signal peptide, a bacterial rhodopsin-like insertion loop, a kv2.1 channel protein, a Golgi export signal, and / or a rhodopsin trafficking sequence, or any combination thereof.

4. The fusion protein of claim 2 or 3, wherein: the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 5, 9, 13, or 17, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 5, 9, 13, or 17; or the fusion protein consists of an amino acid sequence set forth in SEQ ID NO: 5, 9, 13, or 17, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 5, 9, 13, or 17.

5. An isolated nucleic acid, wherein: the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18, or a nucleotide sequence that is at least 70% identical to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18; or the nucleic acid consists of a nucleotide sequence set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18, or a nucleotide sequence that is at least 70% identical to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18; or the nucleic acid encodes the Chrmine variant of claim 1 or the fusion protein of claim 2, 3, or 4.

6. A vector, wherein the vector comprises the nucleic acid of claim 5.

7. A viral particle, wherein the viral particle comprises the vector of claim 6, optionally wherein the vector is an AAV vector.

8. A pharmaceutical composition, wherein the pharmaceutical composition comprises the ChRmine variant of claim 1, the fusion protein of claim 2, 3, or 4, the nucleic acid of claim 5, the vector of claim 6, or the viral particle of claim 7, and a pharmaceutically acceptable carrier.

9. A cell, wherein the cell is transduced by or comprises the vector of claim 6.

10. Use of a ChRmine variant according to claim 1, a fusion protein according to claim 2, 3 or 4, a nucleic acid according to claim 5, a vector according to claim 6, a viral particle according to claim 7, a pharmaceutical composition according to claim 8 or a cell according to claim 9 in the manufacture of a medicament for the treatment of a retinal neurodegenerative disease in a subject in need thereof, optionally wherein the retinal neurodegenerative disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration (AMD), Batten disease, Leber’s hereditary optic neuropathy, cone-rod dystrophy, Leber’s congenital amaurosis, diabetic retinopathy, retinal detachment, Best’s disease, choroideremia, fundus albipunctatus, inherited optic neuropathies (Leber’s hereditary optic neuropathy, dominant optic neuropathy), compressive optic neuropathy (orbital pseudotumor, thyroid eye disease), autoimmune optic neuropathy (lupus), diabetic retinopathy, glaucomatous optic neuropathy (GOND), glaucoma, arterial ischemic optic neuropathy (giant cell arteritis), non-arteritic ischemic optic neuropathy, infiltrative optic neuropathy (sarcoidosis), infectious optic neuropathy (syphilis, Lyme disease, toxoplasmosis, shingles), optic neuritis from demyelinating disease, and post-radiation optic neuropathy.

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