Mutant photo-induced ion channel ChR-2 protein and application thereof

Abstract

The invention relates to the technical field of biomedicine, discloses mutant photo-induced ion channel ChR-2 protein, and further discloses a nucleic acid construct, an expression vector, a cell, related application and a computer model. The light-sensitive channel ChR2 protein mutant obtained by the invention has stronger light current, and the light-sensitive capability of the light-sensitive channel ChR2 protein mutant is at least improved by 100 times; besides, the invention also obtains a nucleotide sequence for coding the light-sensitive channel ChR2 protein mutant, constructs a recombinant expression vector, and obtains the light-sensitive channel ChR2 protein with higher expression quantity and stronger light sensitivity, and the light-sensitive channel ChR2 protein is very suitable for expression in cells of mammals (especially human); according to the invention, the rhodopsin in different channels is systematically studied by modifying the position G224 in the helix 6 of seven transmembrane helix motifs, which proves that the mutation of the position G224 in the helix 6 in WT ChR2 accelerates the photosensitivity of the channels, and the considered position is homologous in the helix 6 of the rhodopsin in different channels.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the mutant photoinduced ion channel ChR-2 protein and its applications. Background Technology

[0002] Optogenetic cataract surgery is a novel treatment strategy for patients blinded by retinal degenerative diseases. Since the demonstration in 2006 that ChR2 optogenetics can restore vision, scientists have conducted research on restoring vision using different optogenetic proteins, including molecularly engineered ChR2s or newly discovered mutants in nature. ChR2 was first discovered in Chlamydomonas reinhardtii and has since been artificially mutated and modified to be suitable for mammalian cells, with stable expression and no cellular toxicity. When excited by blue light (with a maximum excitation peak around 470 nm), this protein induces the opening of cation channels, promoting neuronal depolarization, thereby inducing action potentials and activating neurons. The natural ChR2 protein is a light-controlled cation channel containing 737 amino acids. Endogenous retinal is sufficient to assist the truncated 309-amino acid ChR2 protein in achieving its function, without the need for the addition of exogenous chromophores. Channelopsin-2 (Chop2) is a seven-span membrane protein, first isolated from the green alga *Chlamydomonas reinhardtii*, which becomes photosensitive upon binding to the chromophore all-trans-retinal. Chop2 forms a light-gated, non-specific, internally rectified cation channel, called channelrhodopsin-2 (ChR2), upon binding to retinal molecules via Schiff base linkage. Channelopsin-2 forms channelrhodopsin-2 (ChR2) upon binding to retinal. It should be understood that after Chop2 is expressed in cells (i.e., retinal neurons), endogenously available retinal binds to Chop2 to form the functional light-gated channel ChR2. Therefore, as mentioned herein, ChR2 and Chop2 proteins can be synonymous.

[0003] Computational modeling of photosensitive proteins can characterize their photocurrents and calculate the photocurrents of their expression in different cells. Preliminary work in this field includes constructing classical theoretical derivations of three-, four-, and six-state Markov models of the ChR2 photocycle, exploring photo-peaking processes under continuous and pulsed illumination, photostimulation in subcellular compartments, and analyzing the sensitivity of activation thresholds to tissue media (scattering and absorption) and illumination devices. Mechanistic models have been widely used to study the biophysics of potential information processing in individual neurons and small networks. In contrast to phenomenological models used for recognition in the nervous system, these models attempt to preserve certain physical properties of the system under study to facilitate interpretation and causal understanding. For example, biophysical models can include detailed neuronal anatomy, ion channel types and distribution, and synaptic connections with other cells (the level of modeling can be adjusted as needed for all these attributes). To date, no sophisticated mechanistic models have been available to systematically study the photosensitive excitability of retinal neurons remaining after different optogenetic tools targeting different RP lesions and the visual signal transmission in the retinal network after optogenetic restoration of sight.

[0004] Therefore, there is still a need for evaluation methods of mutant photosensitive ion channels with higher photosensitivity and optogenetic methods for restoring sight. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a mutant photoinduced ion channel ChR-2 protein and its applications.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A mutant photoinduced ion channel ChR-2 protein, wherein the amino acid sequence of the mutant photoinduced ion channel ChR-2 protein is as follows: it has at least 84% similarity and / or at least 75% identity with the full-length amino acid sequence of the wild-type photoinduced ion channel ChR2 protein SEQ ID NO: 1 (ChR-2), and the only difference between the mutant photoinduced ion channel ChR2 protein and the wild-type photoinduced ion channel ChR2 protein is the substitution at the position of G224 in SEQ ID NO: 1;

[0008] When measured in whole-cell mode via patch clamp at a clamp potential of -70 mV, bath solution of 138 mM NaCl, 1 mM NaHCO3, 0.3 mM Na2HPO4, 5 mM KCl, 0.3 mM KH2PO4, 1.25 mM CaCl2, 0.5 mM MgSO4, 0.5 mM MgCl2, 5 mM HEPES, 22.2 mM glucose, pH 7.2, and pipette solution of 110 mM Cs-Methanesulfonate, 30 mM TEA-Cl, 2 mM MgCl2, 0.1 mM CaCl2, 10 mM EGTA, 10 mM HEPES, pH 7.25, the substitution in (b) enhances the photosensitivity of the mutant photoinduced ion channel ChR-2 protein compared to the parental channel.

[0009] Preferably, the mutant photoinduced ion channel ChR-2 protein has at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least 92%, more preferably at least 94%, more preferably at least 96%, more preferably at least 98%, and more preferably at least 99% similarity to the full-length sequence of SEQ ID NO: 1 (ChR-2); and / or the mutant photoinduced ion channel ChR-2 protein has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% identity to the full-length sequence of SEQ ID NO: 1 (ChR-2).

[0010] Preferably, the substitution is made with glycine at position 224.

[0011] Preferably, the mutant photoinduced ion channel ChR-2 protein is obtained by mutating glycine (G) at position 224 of the amino acid sequence of the wild-type photoinduced ion channel ChR2 protein to serine (S), and the amino acid sequence of the mutant photoinduced ion channel ChR-2 protein is shown in SEQ ID NO.2.

[0012] Preferably, the mutant photoinduced ion channel ChR-2 protein further comprises at least one of the following amino acid residues: aspartic acid at position 253 of SEQ ID NO: 1; lysine at position 257 of SEQ ID NO: 1; tryptophan at position 260 of SEQ ID NO: 1; glutamic acid at position 123 of SEQ ID NO: 1; histidine or arginine, preferably arginine, at position 134 of SEQ ID NO: 1; threonine, serine or alanine at position 128 of SEQ ID NO: 1; and / or alanine at position 156 of SEQ ID NO: 1.

[0013] Preferably, the mutant photoinduced ion channel ChR-2 protein is a membrane protein with at least 5 transmembrane helices and capable of binding all-trans retinaldehyde, preferably a membrane protein with 6 or 7 transmembrane helices; the mutant photoinduced ion channel ChR-2 protein, in addition to the transmembrane portion, also includes C-terminal and / or N-terminal sequences, wherein both the C-terminal and N-terminal sequences can extend into the lumen surrounded by the membrane or into the liposome, or be arranged on the outer surface of the membrane; the length of the C-terminal sequence of the mutant photoinduced ion channel ChR-2 protein is preferably 1-1000 amino acids not embedded in the membrane, more preferably 1-500 amino acids, particularly preferably 5-50 amino acids; and / or the length of the N-terminal sequence is preferably 1-500 amino acids not embedded in the membrane, particularly preferably 5-50 amino acids.

[0014] To achieve the above objectives, the present invention also provides a nucleic acid construct comprising a nucleotide sequence encoding a mutant photoinduced ion channel ChR-2 protein.

[0015] To ensure optimal expression, the coding DNA can be appropriately modified by adding suitable regulatory and / or targeting sequences and / or by matching the coding DNA sequence with the host's preferred codon usage. The targeting sequence may encode a C-terminal extension that directs the light-sensitive ion channel to a specific site or compartment within the cell, such as targeting a synapse or postsynaptic site, axon-tumulus, or endoplasmic reticulum. The nucleic acid can be combined with other elements, such as promoters and transcription initiation and termination signals, as well as translation initiation and termination signals and polyadenylation signals, to provide expression of the mutant light-sensitive ion channel sequence of this disclosure. The promoter can be inducible or constitutive, general or cell-specific. An example of a cell-specific promoter is the mGlu6 promoter, which is specific to bipolar cells. The selection of promoters, vectors, and other elements is a routine design issue at the level of common skill in the art. Many such elements are described in the literature and are available from commercial suppliers.

[0016] Preferably, the nucleotide sequence is shown in SEQ ID NO.4.

[0017] To achieve the above objectives, the present invention also provides an expression vector comprising a nucleotide sequence or nucleic acid construct encoding a mutant photoinduced ion channel ChR-2 protein.

[0018] In a preferred embodiment, the vector is suitable for gene therapy, particularly where the vector is suitable for virus-mediated gene transfer. The term "suitable for virus-mediated gene transfer" in this invention means that the vector can be packaged in a virus and thus delivered to a target site or cell. Examples of viruses suitable for gene therapy are retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, poxviruses, alphaviruses, rabies virus, semliki forest virus, and herpesviruses. These viruses differ in the extent to which they transfer genes to cells they recognize and are able to infect, and whether they permanently or temporarily alter the cell's DNA. However, gene therapy also includes non-viral methods, such as the use of naked DNA, lipid complexes and polyplexes, and dendritic macromolecules.

[0019] As described above, the obtained nucleic acid sequence can be introduced into cells, for example, using a virus as a vector or through transfection (including, for example, via chemical transfectants (such as Lipofectamine, Fugene, etc.), electroporation, calcium phosphate co-precipitation, and direct diffusion of DNA). Methods for transfecting cells are detailed in the examples and can be adapted to their respective recipient cells. Transfection with DNA produces stable cells or cell lines if the transfected DNA integrates into the genome, or unstable (transient) cells or cell lines if the transfected DNA is present in an extrachromosomal form. Furthermore, stable cell lines can be obtained by using an adjoint replication plasmid, meaning that the inheritance of the extrachromosomal plasmid is controlled by control elements integrated into the cell's genome. Typically, the choice of a suitable vector or plasmid depends on the intended host cell.

[0020] To achieve the above objectives, the present invention also provides a cell comprising a nucleic acid construct or expression vector.

[0021] Preferably, the cell is a mammalian cell, and more preferably HEK293T cells.

[0022] Incorporating mutant light-sensitive ion channels into cell membranes that do not naturally express the corresponding channels can be achieved, for example, simply by using known recombinant DNA techniques, first integrating the DNA encoding the ion channel into a suitable expression vector (e.g., plasmid, granulosome, or virus), then transforming the target cell with this vector and expressing the protein in the host. Next, the cell is treated in a suitable manner (e.g., with retinal) to form a Schiffs base bond between the protein and retinal.

[0023] The expression of the photosensitive ion channel of the present invention can be advantageously achieved in certain mammalian cell systems. Therefore, in a preferred embodiment, the cells are mammalian cells. Expression is achieved using a free vector, preferably as transient expression in neuroblastoma cells (e.g., NG108-15- cells), melanoma cells (e.g., BLM cell line), COS cells (generated by infection with “African green monkey kidney CV1” cells), or HEK cells (“human embryonic kidney cells,” e.g., HEK293 cells), or BHK cells (“juvenile hamster kidney cells”), or in a stable expression form (by integration into the genome) in CHO cells (“Chinese hamster ovary cells”), myeloma cells, or MDCK cells (“Madine-Darby canine kidney cells”), or in baculovirus-infected Sf9 insect cells. Therefore, in a more preferred embodiment, the mammalian cell is HEK293 cell.

[0024] Photoreceptor cells are specialized neurons found in the retina that are capable of light transduction. Two classic photoreceptors are rods and cones, each providing information used by the visual system. Retinal ganglion cells are neurons located near the inner surface of the retina. These cells have dendrites and long axons that project into the suprachiasmatic nucleus and lateral geniculate body (thalamus) in the hypothalamus. A small fraction contribute little or nothing to vision, but they are themselves light-sensitive. Their axons form the retinohypothalamic tract and contribute to circadian rhythms and pupillary light reflexes, adjusting pupil size. They receive visual information from photoreceptors through two types of interneurons: bipolar cells and amacrine cells. Amacrine cells are interneurons in the retina responsible for 70% of the input to retinal ganglion cells. Bipolar cells are responsible for the remaining 30% of retinal ganglion input and are regulated by amacrine cells. As part of the retina, bipolar cells exist between photoreceptors (rods and cones) and ganglion cells. They transmit signals directly or indirectly from photoreceptors to ganglion cells.

[0025] Cells can be isolated (and genetically modified) and cultured under suitable temperature and gas mixtures (typically 37°C, 5% CO2) (optionally in a cell culture incubator known to those skilled in the art and as illustrated for certain cell lines or cell types). Culture conditions may differ for each cell type, and variations in conditions for a particular cell type can lead to different phenotypes. Besides temperature and gas mixtures, the most common variable factor in cell culture systems is the growth medium. The formulation of the growth medium can vary in pH, glucose concentration, the presence of growth factors, and other nutrients. Growth media are commercially available or can be prepared according to compositions obtained from the American Tissue Culture Collection (ATCC). Growth factors used to supplement the medium are typically derived from animal blood, such as calf serum. Additionally, antibiotics may be added to the growth medium. Common operations performed on cultured cells include medium replacement and cell passage.

[0026] To achieve the above objectives, the present invention also provides the application of mutant photoinduced ion channel ChR-2 protein in the preparation of products for high-throughput screening.

[0027] To achieve the above objectives, the present invention also provides the use of cells in the preparation of products for high-throughput screening.

[0028] To achieve the above objectives, the present invention also provides the application of mutant photoinduced ion channel ChR-2 protein in the preparation of non-therapeutic products for photostimulation of neuronal cells.

[0029] To achieve the above objectives, the present invention also provides the application of mutant photoinduced ion channel ChR-2 protein in the preparation of pharmaceutical products for treating retinal degeneration diseases.

[0030] To achieve the above objectives, the present invention also provides a computer model for evaluating the ability of optogenetics to treat retinal degenerative diseases using mutant light-induced ion channel ChR-2 protein.

[0031] The beneficial effects of this invention are as follows: Compared with the wild-type ChR2 protein, the photosensitive channel ChR2 protein mutant obtained by this invention has a stronger photocurrent, and its photosensitivity is improved by at least 100 times; In addition, this invention has also carried out targeted optimization design of the coding sequence of the wild-type photosensitive channel ChR2 protein, obtained the nucleotide sequence encoding the photosensitive channel ChR2 protein mutant, constructed a recombinant expression vector of the photosensitive channel ChR2 protein mutant, and transferred the optimized coding sequence of the photosensitive channel ChR2 protein mutant into host cells, resulting in a photosensitive channel ChR2 protein with higher photosensitivity, which is highly suitable for expression in mammalian (especially human) cells; This invention has also conducted a systematic study on different channel rhodopsin by modifying position G224 in helix 6 of the seven transmembrane helical motifs, demonstrating that the mutation at position G224 in helix 6 of WT ChR2 accelerates the photosensitivity of the channel, and the considered position is homologous in helix 6 of different channel rhodopsin. Attached Figure Description

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0033] Figure 1 The ChR2(WT) and ChR2(G224S) in Example 3 were tested at 1000 ms and 1.026-12.164 mW / mm. 2 Normalized photocurrent recording results under 470nm blue light stimulation;

[0034] Figure 2 The ChR2(WT) and ChR2(G224S) in Example 3 were tested at 1000 ms and 1.026-12.164 mW / mm. 2 The results of the normalized peak photocurrent (Ip), the static current (Iss) after deactivation (top column), and the time-current integration (bottom column) under 470nm blue light stimulation.

[0035] Figure 3 The ChR2s four-state model and simulation results in Example 4;

[0036] Figure 4 This is a diagram of the lightweight retinal neural network model in Example 4;

[0037] Figure 5 The simulation results of the retinal lightweight neural network for ON-type CBC are represented by ChR2(WT), ChR2(G224S), and ChR2(H134R) in Example 5. Detailed Implementation

[0038] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0039] Example 1: Explanation

[0040] To facilitate a clearer understanding of this embodiment, certain terms are first defined. As used herein, unless otherwise expressly specified, each of the following terms shall have the meaning given below. Other definitions are set forth throughout this application.

[0041] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0042] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0043] Sequence identity (or homology) is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of a reference nucleotide sequence or protein) and determining the number of positions where identical residues occur. This is typically expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0044] As used herein, the terms “subject” and “required subject” refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cattle, horses, dogs, cats, pigs, sheep, goats, giraffes, deer, camels, ibex, hares, and rabbits.

[0045] As used in this article, “light-sensitive channel”, “light-sensitive channel protein”, “ChR2”, “light-sensitive channel ChR2 protein”, “channel rhodopsin 2” or “peptide” can be used interchangeably.

[0046] As used in this embodiment, "wild-type light-sensitive channel ChR2 protein" refers to a naturally occurring, unmodified light-sensitive channel ChR2 protein. Its nucleotides can be obtained through genetic engineering techniques, such as genome sequencing and polymerase chain reaction (PCR), and its amino acid sequence can be deduced from the nucleotide sequence. The wild-type light-sensitive channel ChR2 protein is derived from the green algae *Chlamydomonas reinhardtii*.

[0047] The amino acid sequence of the wild-type light-sensitive channel ChR2 protein (wild-type light-induced ion channel ChR2 protein) mentioned in this embodiment is shown in SEQ ID NO.1, and is as follows:

[0048] MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDI GTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAVNKGTGK

[0049] As used in this embodiment, the terms "mutant protein", "mutant protein of the present invention", "ChR2 mutant protein of the present invention", "mutant photosensitive channel ChR2 protein of the present invention", "photosensitive channel ChR2 protein mutant", "mutant ChR2", "mutant ChR2 protein", "ChR2 mutant", and "mutant photoinduced ion channel ChR-2 protein" are used interchangeably and all refer to non-naturally occurring mutant photosensitive channel ChR2 protein, and the mutant photosensitive channel ChR2 protein is a protein artificially modified based on the protein shown in SEQ ID NO. 1 above.

[0050] It should be understood that the amino acid numbering in the mutated light-sensitive channel ChR2 protein of this embodiment is based on the wild-type light-sensitive channel ChR2 protein (preferably, SEQ ID NO. 1). When a specific mutated protein has 80% or more homology with the sequence shown in SEQ ID NO. 2, the amino acid numbering of the mutated protein may be misaligned relative to the amino acid numbering in SEQ ID NO. 1, such as misalignment 1-5 positions towards the N-terminus or C-terminus of the amino acid. Using conventional sequence alignment techniques in the art, those skilled in the art can generally understand that such misalignment is within a reasonable range, and that mutated proteins with 80% (e.g., 90%, 95%, 98%) homology and the same or similar ChR2 protein activity should not be excluded from the scope of the mutated proteins of this invention due to amino acid numbering misalignment.

[0051] The mutant proteins of this invention are synthetic or recombinant proteins, meaning they can be chemically synthesized products or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production protocol, the mutant proteins of this invention can be glycosylated or non-glycosylated. The mutant proteins of this invention may also include or exclude an initial methionine residue.

[0052] The present invention also includes fragments, derivatives, and analogs of the mutant protein. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to proteins that substantially retain the same biological function or activity as the mutant protein.

[0053] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells generated by genetic engineering using the vectors or protein-coding sequences of the present invention, and methods for expressing the ChR2 protein mutant of the light-sensitive channel of the present invention using said host cells via recombinant technology.

[0054] Host cells expressing the ChR2 protein mutant of the light-sensitive channel of the present invention can be obtained using conventional recombinant DNA technology and the polynucleotide sequence of the present invention.

[0055] The host cell can be a prokaryotic cell, a lower eukaryotic cell, or a higher eukaryotic cell, such as mammalian cells (including human and non-human mammals). Representative examples include animal cells such as CHO, NSO, COS7, or 293 cells. In a preferred embodiment of the invention, HEK cells, photoreceptor cells (including cone cells and / or rod cells), other visual cells (such as biganglionic cells), and nerve cells are selected as host cells. In another preferred embodiment, the host cell is selected from the group consisting of rod cells, cone cells, light-exposed bipolar cells, light-deactivated bipolar cells, horizontal cells, ganglion cells, cells without long processes, or combinations thereof.

[0056] In a preferred embodiment of the present invention, the amino acid sequence (SEQ ID NO.2) of the wild-type photosensitive channel ChR2 mutant G224S protein (mutant photoinduced ion channel ChR-2 protein) is as follows:

[0057] MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSHI GSIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAVNKGTGK

[0058] This embodiment also provides an expression vector containing the coding sequence of a photosensitive channel ChR2 protein mutant. With the provided sequence information, skilled technicians can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transduction into cells.

[0059] A nucleic acid sequence encoding a mutant of the light-sensitive channel ChR2 protein is used as a vector, preferably an expression vector, which can serve as a gene therapy vector suitable for transduction and expression in target retinal cells. The vector can be viral or non-viral (e.g., plasmid). Viral vectors include those derived from: adenovirus, including mutant forms of adeno-associated virus (AAV), retrovirus, lentivirus, herpesvirus, vaccinia virus, poliovirus, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, poxvirus, and SV40. Preferably, the viral vector is replication-defective, although it is envisioned to be replication-deficient, capable of replication, or conditionally replicating. Viral vectors generally maintain an extrachromosomal state without integrating into the genome of the target retinal cells. A preferred viral vector for introducing a nucleic acid sequence encoding a mutant of the light-sensitive channel ChR2 protein into retinal target cells is an AAV vector, such as a self-complementary adeno-associated virus (scAAV). Selective targeting can be achieved using specific AAV serotypes (AAV serotypes 2 to 12) or modified versions of any of these serotypes (including AAV 4YF and AAV 7m8 vectors).

[0060] Viral vectors can be modified to delete any non-essential sequences. For example, in AAV, the virus can be modified to delete all or part of the IX gene, Ela, and / or Elb gene. For wild-type AAV, the absence of helper viruses such as adenovirus makes replication very inefficient. For recombinant adeno-associated viruses, preferably, the replication gene and capsid gene are provided in trans form (in the pRep / Cap plasmid), and only the 2ITRs of the AAV genome are preserved and packaged into the virion, while the required adenovirus genes are provided by adenovirus or another plasmid. Similar modifications can be made to lentiviral vectors.

[0061] Viral vectors have the ability to enter cells. However, non-viral vectors, such as plasmids, can be conjugated with agents to facilitate the uptake of viral vectors by target cells. Such agents include polycationic agents. Alternatively, delivery systems such as liposome-based delivery systems may be used.

[0062] The carrier used in this invention is preferably suitable for use in vivo or in vitro, and is preferably suitable for use in humans.

[0063] The vector will preferably contain one or more regulatory sequences to guide the expression of the nucleic acid sequence in target retinal cells. The regulatory sequences may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operatively linked to the nucleic acid sequence. The vector may also include selective markers, for example, to determine the expression of the vector in a growth system (e.g., bacterial cells) or in target retinal cells.

[0064] "Operationally linked" means that nucleic acid sequences are functionally related to their operationally linked sequences such that they are linked in a way that causes them to affect each other's expression or function. For example, a nucleic acid sequence operationally linked to a promoter will have an expression pattern influenced by the promoter.

[0065] A promoter mediates the expression of a nucleic acid sequence linked to it. Promoters can be constitutive or inducible. Promoters can direct ubiquitous expression in inner retinal cells or neuron-specific expression. In the latter case, promoters can direct cell-type-specific expression, such as for photosensitive or desensitive bipolar cells. Suitable promoters will be known to those skilled in the art. For example, suitable promoters can be selected from the group consisting of: L7, thy-1, recovery protein, calcium-binding protein, human CMV, GAD-67, chicken β-actin, hSyn, Grm6, and the Grm6 enhancer SV40 fusion protein. Cell-specific promoters can be used to achieve targeting, such as Grm6-SV40 for selective targeting of photosensitive bipolar cells. The Grm6 promoter is a fusion of the 200-base-pair enhancer sequence of the Grm6 gene and the SV40 eukaryotic promoter. The Grm6 gene encodes the photosensitive bipolar cell-specific metabolite glutamate receptor mGluR6. Preferred sources of the Grm6 gene are mice and humans. Ubiquitous expression can be achieved using pan-neuronal promoters, examples of which are known and available in the art. One such example is CAG. The CAG promoter is a fusion of the early CMV enhancer and the chicken β-actin promoter.

[0066] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered as part of the invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operatively linked to the inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0067] Many expression vectors can be used to express mutants of the light-sensitive channel ChR2 protein in mammalian cells (preferably human, more preferably human visual cells or photoreceptor cells). This invention preferably uses adeno-associated virus as the expression vector.

[0068] The present invention also provides a host cell for expressing a mutant of the light-sensitive channel ChR2 protein. Preferably, the host cell is a mammalian cell (preferably human, more preferably human visual cells or photoreceptor cells).

[0069] The present invention provides a formulation or composition comprising (1) the carrier described in the third aspect of the present invention or the adeno-associated virus carrier described in the fourth aspect of the present invention, and (2) a pharmaceutically acceptable carrier.

[0070] In a preferred embodiment, the pharmaceutical preparation is used to treat an eye disease caused by the degeneration of photoreceptor cells. The pharmaceutical preparation is used to expand or restore the photoreceptor function of the retina, restore the subject's vision, and / or treat retinal degenerative diseases.

[0071] The "active ingredient" in the pharmaceutical composition described in this invention refers to the vector described in this invention, such as a viral vector (including adeno-associated virus vectors). The "active ingredient," formulation, and / or composition described in this invention can be used to treat eye diseases.

[0072] In this invention, the carrier can be administered to the eye via subretinal or intravitreal application. In either administration mode, preferably, the carrier is provided as an injectable liquid. Preferably, the injectable liquid is provided as a capsule or syringe.

[0073] The compositions of the present invention can be provided for use in methods of providing photoreceptor function to cells. In one embodiment, the compositions of the present invention can be provided for use in methods of restoring photoreceptor function to the retina. In one embodiment, the compositions of the present invention can be provided for use in methods of restoring vision to a subject. In one embodiment, the compositions of the present invention can be provided for use in methods of treating conditions of retinal degeneration, such as retinal dystrophy, including rod dystrophy, rod-cone dystrophy, cone-rod dystrophy, cone dystrophy, and macular dystrophy; other forms of retinal or macular degeneration, ischemic conditions, uveitis, and any other disease resulting from loss of photoreceptor capacity, such as retinitis pigmentosa.

[0074] This invention provides a method for delivering photoreceptor function to cells, comprising introducing a vector containing a nucleic acid encoding a mutant of the photosensitive channel ChR2 protein into the eye; comprising administering the nucleic acid vector subretinally or intravitreal to the inner retinal cells of the eye. This invention provides a nucleic acid vector for use in methods of treating retinal degeneration by delivering photoreceptor function to cells, the nucleic acid vector containing a nucleic acid sequence encoding a mutant of the photosensitive channel ChR2 protein. The compositions of this invention can be administered alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

[0075] This invention also provides a method for enhancing photoreceptor function in the retina, particularly after degeneration of rods and / or cones, comprising introducing a nucleic acid carrier into the vitreous cavity of the eye, the nucleic acid carrier containing a nucleic acid sequence encoding a mutant of the light-sensitive channel ChR2 protein. The method may include administering the nucleic acid carrier to inner retinal cells, subretinal, or vitreous cells of the eye. This invention provides a nucleic acid carrier for use in the treatment of retinal degeneration by enhancing photoreceptor function in the retina, the nucleic acid carrier containing a nucleic acid sequence encoding a mutant of the light-sensitive channel ChR2 protein.

[0076] The present invention also provides a method for restoring vision in a subject, the method comprising introducing a vector containing a nucleic acid sequence encoding a mutant of the light-sensitive channel ChR2 protein into the eye. The method may include administering the nucleic acid vector subretinally or intravitreal to the inner retinal cells of the eye. The present invention provides a nucleic acid vector for use in restoring vision in a subject, the nucleic acid vector containing a nucleic acid sequence encoding a mutant of the light-sensitive channel ChR2 protein.

[0077] This invention also provides a method for treating retinal diseases in a subject, comprising introducing a vector containing a nucleic acid sequence encoding a mutant of the light-sensitive channel ChR2 protein into the eye. The method may include administering the nucleic acid vector subretinally or intravitreal to the inner retinal cells of the eye. Diseases may be retinal dystrophys, including rod dystrophy, rod-cone dystrophy, cone-rod dystrophy, cone dystrophy, and macular dystrophy; other forms of retinal or macular degeneration, ischemic conditions, uveitis, and any other disease resulting from loss of photoreceptor capacity.

[0078] As used in this embodiment, providing photoreceptor function to cells means that cells that previously lacked photoreceptor capacity or whose photoreceptor capacity had completely or partially degenerated become photosensitive after expressing a foreign nucleic acid sequence encoding a mutant of the light-sensitive channel ChR2 protein. Such cells may be referred to as transformed cells in this embodiment because they contain non-natural nucleic acids. Preferably, the transformed retinal cells exhibit some or all of the photoreceptor capacity of natural photoreceptor cells. Preferably, the transformed cells exhibit at least the same or substantially the same photosensitivity as natural retinal photoreceptor cells. Preferably, the transformed cells exhibit higher photosensitivity than diseased or degenerating natural retinal photoreceptor cells. Therefore, transformed cells will preferably have increased photoreceptors compared to untreated degenerated or diseased cells from the same source, maintained under the same conditions. Transformed cells can be distinguished from natural cells by the presence of foreign nucleic acids therein.

[0079] As used in this embodiment, enhancing photoreceptor function means increasing the photoreceptor function of the retina by increasing the function of photoreceptor cells such as rods or cones and / or by providing photoreceptor function to the cells. Therefore, the retina, compared to a retina not treated with the methods described in this embodiment, will have an increased ability to receive and transmit light signals, and this increase can be of any amount.

[0080] As used in this embodiment, restored vision in a subject means that the subject shows improved vision compared to before treatment, for example, using a vision test as described in this embodiment. Restoration includes any degree of improvement, including complete restoration of vision to perfect or near-perfect vision.

[0081] As used in this embodiment, treating a disease means applying a nucleic acid or vector as described in this embodiment to improve or alleviate one or more symptoms of a disease selected from the group consisting of: retinal dystrophy, including rod dystrophy, rod-cone dystrophy, cone-rod dystrophy, cone dystrophy, and macular dystrophy; another form of retinal or macular degeneration, ischemic condition, uveitis, and any other disease resulting from loss of photoreceptor capacity. Improvement or alleviation may result in improvement of peripheral or central vision, and / or daytime or nighttime vision.

[0082] The method of the present invention includes introducing a nucleic acid sequence encoding a mutant of the light-sensitive channel ChR2 protein into the vitreous cavity of the eye. Preferably, the method includes contacting cells with a vector (preferably a virus, more preferably adeno-associated virus) containing the nucleic acid sequence encoding the mutant of the light-sensitive channel ChR2 protein. Preferably, the cells are retinal cells, preferably photobipolar cells, dephotobipolar cells, horizontal cells, ganglion cells, and / or cells without long processes.

[0083] When nucleic acid sequences and one or more enzymes are provided in multiple (two or more) doses, these doses can be spaced out at appropriate time intervals, such as from 30 seconds to several hours or one day or more.

[0084] Each dose may contain an effective amount of nucleic acid sequence or viral vector. The effective dose of nucleic acid sequence or viral vector can be 1 × 10⁻⁶ per treatment regimen. 9 -1×10 13 The scope of the virus.

[0085] This invention is based on targeting nucleic acid sequences encoding mutant ChR2 proteins of the light-sensitive channel to retinal cells to compensate for the degeneration of photoreceptor cells in the retina. The cells to which the nucleic acid sequence is targeted are retinal cells that are alive and capable of expressing the foreign nucleic acid sequence. Retinal cells are cells of the retina that are nerve or neuronal cells capable of becoming excited and transmitting electrical signals. Preferably, the target retinal cells will be able to generate electrical signals and initiate signal cascades, resulting in signal transmission to the optic nerve. Preferably, the target retinal cells are cells of the inner retina. Target cells can be rod or cone cells, and / or can be non-photoreceptor cells (i.e., retinal cells that are not light-responsive in their natural form). Target retinal cells can include one or more cell types selected from the group consisting of: rod cells, cone cells, light-emitting bipolar cells, light-removing bipolar cells, horizontal cells, ganglion cells, Miller cells, and / or cells without long processes.

[0086] Therefore, when the target retinal cells are light-emitting bipolar cells, light-reducing bipolar cells, horizontal cells, ganglion cells, and / or cells without long processes targeting the retina, the expression of nucleic acid encoding a mutant of the photosensitive channel ChR2 protein can be termed ectopic expression. Therefore, this invention, within its scope, includes a method for ectopically expressing a nucleic acid sequence encoding a mutant of the photosensitive channel ChR2 protein in non-photoreceptor cells. Such ectopic expression, through the expression of the heterologous photosensitive protein therein, has the function of providing photoreceptor function to the cell. This is used to increase the photosensitivity observed in degenerating retina.

[0087] Horizontal cells are inner retinal cells that participate in signal processing and feedback to photoreceptor cells; bipolar cells are inner retinal cells and communicate between rod / cone cells and amacrine and / or ganglion cells; amacrine cells are found in the inner retina and allow communication between photoreceptor pathways and ganglion cells; ganglion cells are the innermost retinal cells that transmit signals from photoreceptor cells to the optic nerve.

[0088] References to cells herein include cell progeny. Preferably, the modification of cells according to the invention also occurs in progeny cells following the transformation of the host cell. Progeny cells may not be identical to the original target cells, but preferably will also exhibit expression of a non-natural light-sensitive channel ChR2 protein mutant.

[0089] Example 2: Optimized Sequence

[0090] In this embodiment, based on the amino acid sequence of the wild-type ChR2 protein (SEQ ID NO.1), the inventors mutated and optimized the amino acid sequence and the nucleic acid coding sequence. First, the glycine (G) at position 224 of the amino acid sequence of the wild-type ChR2 protein was mutated to a serine (S), resulting in the ChR2 protein mutant sequence G224S, the amino acid sequence of which is shown in SEQ ID NO.2.

[0091] The mutant photoinduced ion channel ChR-2 protein (ChR2 protein mutant) obtained in this embodiment is a membrane protein with at least 5 transmembrane helices capable of binding all-trans retinaldehyde, with transmembrane proteins having 6 or 7 transmembrane helices being preferred. Furthermore, transmembrane proteins with more than 7 helices are also included, such as 8, 9, or 10 transmembrane helices. In addition, this embodiment covers transmembrane proteins that, in addition to the transmembrane portion, include C-terminal and / or N-terminal sequences, wherein the C-terminal sequence can extend into the lumen surrounded by the membrane (e.g., the cytoplasm of the cell) or into liposomes, or can also be arranged on the outer surface of the membrane. This also applies to optionally present N-terminal sequences, which can also be arranged within the lumen or on the outer surface of the membrane. The length of the C-terminal and / or N-terminal sequences is not limited in principle; however, photosensitive ion channels with C-terminal sequences not embedded in the membrane (having 1-1000 amino acids, preferably 1-500 amino acids, particularly preferably 5-50 amino acids) are preferred. Independent of the length of the C-terminal sequence, the N-terminal positioning sequence not embedded in the membrane preferably contains 1-500 amino acids, particularly preferably 5-50 amino acids. The concept of transmembrane helices is well known to those skilled in the art. These are typically α-helical protein structures, which usually contain 20 to 25 amino acids. However, depending on the nature of the membrane, which can be a natural membrane, such as a cell membrane or plasma membrane, or a synthetic membrane, the transmembrane segment can be shorter or longer. For example, transmembrane segments in artificial membranes can contain up to 30 amino acids, but on the other hand, they can contain only a few amino acids, such as 12 to 16 amino acids.

[0092] Furthermore, glycine should not be substituted at space-required locations, and proline should not be introduced into protein motifs with α-helical or β-sheet structures.

[0093] Typically, retinaldehyde or retinaldehyde derivatives, essential for the function of photosensitive ion channels, are produced by cells transfected with the ion channel to be used. Depending on its conformation, retinaldehyde can be all-trans-retinal, 11-cis-retinal, 1-cis-retinal, or 9-cis-retinal. However, it is also anticipated that the mutant photosensitive ion channel of this embodiment can be incorporated into vesicles, liposomes, or other artificial cell membranes. Therefore, by applying the mutant photosensitive ion channel of this embodiment, channel rhodopsin comprising the photosensitive ion channel of this embodiment and retinaldehyde or retinaldehyde derivatives can be obtained. The retinaldehyde derivatives are selected from 3,4-dehydroretinaldehyde, 1-ethylretinaldehyde, 9-dm-retinaldehyde, hydroxyretinaldehyde, 4-hydroxyretinaldehyde, naphthylretinaldehyde; 3,7,11-trimethyl-dodecyl-2,4,6,8,10-pentaenal; 3,7-dimethyl-decyl-2,4,6,8-tetraenal; 3,7-dimethyl-octyl-2,4,6-trienal; and 6-7-rotation-hindered retinaldehyde, 8-9-rotation-hindered retinaldehyde, and 10-11-rotation-hindered retinaldehyde.

[0094] The inventors further optimized sequence fragments that affect gene expression, including but not limited to: codon usage bias, elimination of secondary structures unfavorable to expression (such as hairpin structures), alteration of GC content, CpG dinucleotide content, mRNA secondary structure, hidden splicing sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning. Ultimately, they obtained the nucleotide sequence (SEQ ID NO) encoding the ChR2 protein mutant sequence G224S.4), as follows: atggactacggcggcgccctgagcgccgtgggcagagagctgctgttcgtgaccaaccccgtggtggtgaacggcagcgtgctggtgcccgaggaccagtgctactgcgccggctggatcgagagcagaggcaccaacggcgcccagaccgccagcaacgtgctgcagtggctggccgccggcttcagcatcctgctgctgatgttctacgcctaccagacctggaagagcacctgcggctgggaggagatctacgtgtgcgccatcgagatggtgaaggtgatcctggagttcttcttcgagttcaagaaccccagcatgctgtacctggccaccggccacagagtgcagtggctgagatacgccgagtggctgctgacctgccccgtgatcctgatccacctgagcaacctgaccggcctgagcaacgactacagcagaagaaccatgggcctgctggtgagcgacatcggcaccatcgtgtggggcgccaccagcgccatggccaccggctacgtgaaggtgatcttcttctgcctgggcctgtgctacggcgccaacaccttcttccacgccgccaaggcctacatcgagggctaccacaccgtgcccaagggcagatgcagacaggtggtgaccggcatggcctggctgttcttcgtgagctggagcatgttccccatcctgttcatcctgggccccgagggcttcggcgtgctgagcgtgtacggcagcaccgtgggccacaccatcatcgacctgatgagcaagaactgctggggcctgctgggccactacctgagagtgctgatccacgagcacatcctgatccacggcgacatcagaaagaccaccaagctgaacatcggcggcaccgagatcgaggtggagaccctggtggaggacgaggccgaggccggcgccgtgaacaagggcaccggcaag.

[0095] The nucleotide sequence encoding the wild-type ChR2 protein is shown in SEQ ID NO.3, as follows: atggactacggcggcgccctgagcgccgtgggcagagagctgctgttcgtgaccaaccccgtggtggtgaacggcagcgtgctggtgcccgaggaccagtgctactgcgccggctggatcgagagcagaggcaccaacggcgcccagaccgccagcaacgtgctgcagtggctggccgccggcttcagcatcctgctgctgatgttctacgcctaccagacctggaagagcacctgcggctgggaggagatctacgtgtgcgccatcgagatggtgaaggtgatcctggagttcttcttcgagttcaagaaccccagcatgctgtacctggccaccggccacagagtgcagtggctgagatacgccgagtggctgctgacctgccccgtgatcctgatccacctgagcaacctgaccggcctgagcaacgactacagcagaagaaccatgggcctgctggtgagcgacatcggcaccatcgtgtggggcgccaccagcgccatggccaccggctacgtgaaggtgatcttcttctgcctgggcctgtgctacggcgccaacaccttcttccacgccgccaaggcctacatcgagggctaccacaccgtgcccaagggcagatgcagacaggtggtgaccggcatggcctggctgttcttcgtgagctggggcatgttccccatcctgttcatcctgggccccgagggcttcggcgtgctgagcgtgtacggcagcaccgtgggccacaccatcatcgacctgatgagcaagaactgctggggcctgctgggccactacctgagagtgctgatccacgagcacatcctgatccacggcgacatcagaaagaccaccaagctgaacatcggcggcaccgagatcgaggtggagaccctggtggaggacgaggccgaggccggcgccgtgaacaagggcaccggcaag

[0096] Example 3: Packaging, purification, and transfection of ChR2 plasmids

[0097] The whole genome was synthesized into two plasmids: one carrying the sequence of the wild-type light-sensitive channel ChR2 protein (SEQ ID NO. 3) and YFP, and the other carrying the optimized sequence of the mutant light-sensitive channel ChR2 protein (SEQ ID NO. 4) and YFP. Except for the coding sequence of the ChR2 protein, the other sequences in the plasmids were identical to the 1DNA plasmid. The 1DNA or 2DNA plasmids were transfected into HEK293 cells according to the following steps:

[0098] HEK-293T cells were cultured in Advanced Dulbecco's Minimum Essential Medium (Bio-Channel, China) supplemented with 5% fetal bovine serum (Bio-Channel, China), 100 units / ml penicillin G, and 100 μg / ml streptomycin, and incubated at 37°C in a humidified 5% CO2 incubator. HEK 293T cells were cultured at a growth rate of 15,000–20,000 cells / cm². 2 Cells were seeded at the specified density in 12-well plates containing 16 mm diameter spreaders (Menzel Glaser, Braunschweig, Germany) and cultured at 37°C with 5% CO2 for 24 h before DNA transfection. WT-ChR2 or ChR2s mutants were transfected using lipofectamine 2000 (Life Technologies) at a ratio of 1 μl:1 μg (cationic liposome:recombinant plasmid). Six h after adding the plasmid-liposome mixture, all-trans retinaldehyde (1 μM) was added to the culture medium. Patch-clamp experiments were performed 48–72 h post-transfection.

[0099] Single-cell patch-clamp recording was performed using standard procedures at room temperature (20-25°C) using an epatch (elements, Italy) or EPC-10USB single amplifier and Patchmaster software (Heka Electronik, Lambrecht / Pfalz, Germany) in whole-cell recording mode to obtain macro-photocurrent recordings of ChR2s. The recording electrode was ensured to have a resistance of 5-8 MΩ in the bath solution after the addition of electrode contents. The series resistance was maintained between 8 and 20 MΩ. Cell membrane capacitance was measured online using an epatch (Membrane TEST) or EPC-10USB single (Lock-In Extension) amplifier. Electrode and cell membrane capacitances were compensated online using the acquisition software. Recordings were performed in Hank's balanced salt solution containing (mM): 138 mM NaCl, 1 mM NaHCO3, 0.3 mM Na2HPO4, 5 mM KCl, 0.3 mM KH2PO4, 1.25 mM CaCl2, 0.5 mM MgSO4, 0.5 mM MgCl2, 5 mM HEPES, and 22.2 mM glucose; the pH was adjusted to 7.2 with 0.3 N NaOH. All-trans retinaldehyde (1 μM) was added to the recording solution. The solution inside the recording electrode contained (mM): 110 Cs-Methanesulfonate, 30 mM TEA-Cl, 2 mM MgCl2, 0.1 mM CaCl2, 10 mM EGTA, and 10 mM HEPES; the pH was adjusted to 7.25 with 1 M CsOH. The liquid junction potential was calibrated online using the acquisition software.

[0100] HEK293T cells expressing the heterologous phases ChR2-YFP and ChR2-YFP G224S were investigated in whole-cell mode using patch-clamp measurements at -70 mV. The response was 1000 ms light pulses (intensity 1–12 mW / mm²). 2 Photocurrent was measured at a wavelength of 470 nm. The photocurrent was normalized using membrane capacitance measured with patch clamp. Intensity-dependent parameters were obtained through a fitting program, thereby determining the photosensitivity. (See attached image.) Figure 1 For ChR2(WT) and ChR2(G224S), at 1000ms, 1.026-12.164mW / mm 2 Normalized photocurrent records were obtained under 470nm blue light stimulation. Normalization was performed by dividing the total cell photocurrent by the total cell membrane capacitance. The results suggest that G224S has a larger photocurrent and higher photosensitivity than WT ChR2. (See appendix) Figure 2The results confirmed that G224S has higher photosensitivity than WT ChR2. In addition, G224S has a larger photocurrent (Ip, Iss), and the photoresponse is linearly correlated with the light intensity (Ip, Iss and current integral).

[0101] Example 4: Construction of computer models of ChR2 and its mutants

[0102] Based on the data obtained in Example 3, parameters of the computer model of ChR2 and its mutants were extracted, and a four-state Markov model (as shown in the attached figure) was used. Figure 3 Simulations were performed. This model is used for a lightweight neural network model after RP lesions; a lightweight neural network model of the retina is attached. Figure 4 The left figure shows the convergence pattern of 16 ON-type CBCs projecting onto 1 ON-type RGC, and the right figure shows the ribbon synapse distribution pattern of 1 ON-type CBC on the dendrite of 1 ON-type RGC.

[0103] Igor Pro was used to extract the peak current Ip of multiple photostimulations, and the relationship between peak current and stimulation interval was plotted. The fitting function of recovery rate Gr is shown in Equation (1). Igor Pro was used to extract the steady-state current Iss of whole-cell voltage step photocurrent (extraction interval is 800-900ms after 1s photostimulation), and the relationship between steady-state current and voltage was plotted. Equation (1) was used for fitting to obtain model parameters. Igor Pro was used to divide the ChR2 (G224X) photocurrent into two parts I. light (Light stimulation part) and I off (After the light stimulation ends); extract I light Part of the Iss is constrained by formula (2), as shown in formula (5). Then, formula (2) is used to fit I. off In this part, the parameters of the four-state Markov model are obtained. These parameters are then incorporated into the complete four-state Markov model, and the parameters are fixed. Finally, the least squares algorithm is used to fit the experimental data to further optimize the model parameters.

[0104] I p =a+b*exp(-Gr*t) (1)

[0105] In the formula, I p t is the peak current; a and b are fitting parameters; Gr is the parameter of the classical photocyclic four-state Markov model; t is the stimulation interval.

[0106] I off =I slow *exp(A1*t)+I fast *exp(A2*t) (2)

[0107] In the formula, Ioff It is the part after the photocurrent is removed; I slow It represents the slowly decaying current after the light is removed; A1 is the fitting parameter; I fast It is the rapidly decaying current after the light is removed; A2 and A1 are as shown in formulas (3) and (4).

[0108] A1 + A2 = G d1 +G d2 +G f0 +G b0 (3)

[0109] In the formula, G d1 G d2 G f0 G b0 It is the dark state transition rate parameter of a four-state Markov.

[0110] A1*A2=G d1 *G d2 +G d1 *G b0 +G d2 *G f0 (4)

[0111] I ss =I slow +I fast (5)

[0112] All modeling was completed on a Windows 10 system. The .mod file (NMODEL language) for ChR2s was written using a text editor (PSPad editor). After writing, it was compiled into .c and .o text files using the NEURON built-in compiler mknrndll, and integrated into the nrnmech.dll file (NEURON native mechanism library). In the same folder, a HEK293T cell model expressing ChR2s was written using a .hoc file. The .hoc file was then run, and the photostimulation parameters were adjusted using the NEURON built-in SectionParameters GUI window. A whole-cell patch-clamp recording mode (clamp voltage -70mV) was simulated using the NEURON built-in VClamp simulator, and a photocurrent graph was plotted using the current axis. The simulation process was controlled using the NEURON built-in RunControl GUI window (sampling rate 40kHz). After the simulation, the current axis plotting results were saved as a .dat file (ASCII) using Print & FileWindow Manager. Then, the data was imported and analyzed using Igor por.

[0113] Table 1. Parameters of the Four-State Model

[0114]

[0115] The parameters of the four-state model are shown in Table 1, and also in the appendix. Figure 3 Among them, (a) the four-state model of ChR2s assumes that ChR2(WT) and ChR2(G224S) have two closed states C1 and C2, and two open states O1 and O2; (b) simulation results of the classical photocyclic four-state Markov model of ChR2s; and (c) analysis of simulated photocurrent parameters. This model describes the conformational transition rate and photocurrent characteristics of ChR2s proteins. The characteristics of photocurrent reflect the conformational transition time of intermediate states of ChR2s proteins, and the photocurrent characteristics of ChR2s can also be predicted by the transition time between conformations. The results show that G224S (No. 45) has higher photosensitivity.

[0116] Example 5: Neural Network Computer Simulation to Evaluate Optogenetic Vision Restoration Capacity

[0117] Information on mouse retinal bipolar cells was retrieved from the morphological database Neuromorpho.org. Two ON-CBCs were selected based on the above criteria, with IDs NMO_63671 and NMO_63678 (as shown in Table 2). Based on the morphological data, two multi-compartment models of ON-CBCs (228 and 336 compartments) were constructed, including dendrites, cell bodies, axons, and axons.

[0118] Table 2

[0119]

[0120]

[0121] Based on existing research, this invention divides bipolar cells into six main subsets: distal dendrites (DD), proximal dendrites (PD), soma (S), proximal axon (PA), distal axon (DA), and axon terminals (AT) to describe the ion channel distribution and passive properties of bipolar cells. The availability and distribution of voltage- or ligand-gated ion channels determine neuronal responses. Here, ion channels are modeled as additional electrical elements on the cell membrane, whose conductance depends on time-varying parameters (such as membrane potential and intracellular calcium concentration). In addition to the equations governing channel dynamics, their location within the cell must also be determined. The ion channel distribution of the ON-BC in this embodiment is described in the table.

[0122] To assess the optogenetic rejuvenation ability of the mutants, we used a lightweight neural network model following RP lesions. We simulated the generation of action potentials in downstream retinal ganglion cells by retinal bipolar cells expressing heterologous phases ChR2-YFP, ChR2-YFP G224S, and ChR2-YFP H134R.

[0123] Optogenetic rejuvenation ability was determined by analyzing the frequency of action potentials; the results are attached. Figure 5 The optogenetic photosensitivity of ChR2(WT), ChR2(No.45), and ChR2(H134R) was comprehensively evaluated. It was found that ChR2(No.45) exhibits relatively high photosensitivity, capable of restoring sight within the range of 0.00001-0.1 μW / mm². 2 It generates photocurrent in the low light intensity range. Compared with ChR2(WT) and ChR2(H134R), ChR2(No.45) generates photocurrent in the low light intensity range (0.001-100 μW / mm²). 2 Its response frequency can reach 20-50Hz, which initially meets the requirements for visual frequency recovery. Among them, the 16 ON-type CBCs characterizing ChR2 (G224S) can induce high-frequency action potentials (above 50Hz) in ON-type RGCs at lower light intensities compared to ChR2 (WT) and ChR2 (H134R). Combining the results of this embodiment with the results of Example 3, it can be seen that G224S improves the photosensitivity calculated by retinal ganglion cell action potential firing in the retinal model by hundreds of times.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A mutant photoinduced ion channel ChR-2 protein, characterized in that, The amino acid sequence of the mutant photoinduced ion channel ChR-2 protein is as follows: it has at least 84% similarity and / or at least 75% identity with the full-length amino acid sequence of the wild-type photoinduced ion channel ChR2 protein SEQ ID NO: 1 (ChR-2), and the only difference from the wild-type photoinduced ion channel ChR2 protein is the substitution at the position of G224 in SEQ ID NO:

1.

2. The mutant photoinduced ion channel ChR-2 protein according to claim 1, characterized in that, The mutant photoinduced ion channel ChR-2 protein has at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least 92%, more preferably at least 94%, more preferably at least 96%, more preferably at least 98%, and more preferably at least 99% similarity to the full-length sequence of SEQ ID NO: 1 (ChR-2); and / or the mutant photoinduced ion channel ChR-2 protein has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% identity to the full-length sequence of SEQ ID NO: 1 (ChR-2).

3. The mutant photoinduced ion channel ChR-2 protein according to any one of claims 1 and 2, characterized in that, The substitution is made by replacing glycine at position 224. Preferably, glycine (G) at position 224 in the amino acid sequence of the wild-type photoinduced ion channel ChR2 protein is mutated to serine (S) to obtain the mutant photoinduced ion channel ChR-2 protein. The amino acid sequence of the mutant photoinduced ion channel ChR-2 protein is shown in SEQ ID NO.

2.

4. The mutant photoinduced ion channel ChR-2 protein according to any one of claims 1 and 2, characterized in that, The mutant photoinduced ion channel ChR-2 protein further comprises at least one of the following amino acid residues: aspartic acid at position 253 of SEQ ID NO: 1; lysine at position 257 of SEQ ID NO: 1; tryptophan at position 260 of SEQ ID NO: 1; glutamic acid at position 123 of SEQ ID NO: 1; histidine or arginine, preferably arginine, at position 134 of SEQ ID NO: 1; threonine, serine or alanine at position 128 of SEQ ID NO: 1; and / or alanine at position 156 of SEQ ID NO:

1.

5. The mutant photoinduced ion channel ChR-2 protein according to any one of claims 1 and 2, characterized in that, The mutant photoinduced ion channel ChR-2 protein is a membrane protein with at least 5 transmembrane helices and capable of binding all-trans retinaldehyde, preferably a membrane protein with 6 or 7 transmembrane helices. In addition to the transmembrane portion, the mutant photoinduced ion channel ChR-2 protein also includes C-terminal and / or N-terminal sequences, wherein both the C-terminal and N-terminal sequences can extend into the lumen surrounded by the membrane or into the liposome, or be arranged on the outer surface of the membrane. The length of the C-terminal sequence of the mutant photoinduced ion channel ChR-2 protein is preferably 1-1000 amino acids not embedded in the membrane, more preferably 1-500 amino acids, and particularly preferably 5-50 amino acids; and / or the length of the N-terminal sequence is preferably 1-500 amino acids not embedded in the membrane, particularly preferably 5-50 amino acids.

6. A nucleic acid construct, characterized in that, It comprises a nucleotide sequence encoding the mutant photoinduced ion channel ChR-2 protein as described in any one of claims 1-5, with the preferred nucleotide sequence shown in SEQ ID NO.

4.

7. An expression carrier, characterized in that, It comprises a nucleotide sequence encoding the mutant photoinduced ion channel ChR-2 protein of any one of claims 1-5 or a nucleic acid construct of any one of claims 6.

8. A cell, characterized in that, It comprises the nucleic acid construct of claim 6 or the expression vector of claim 7, wherein the cell is a mammalian cell, preferably HEK293T cell.

9. The use of the mutant photoinduced ion channel ChR-2 protein according to any one of claims 1-5 in the preparation of products for high-throughput screening, or in the preparation of non-therapeutic products for photostimulation of neuronal cells, or in the preparation of pharmaceutical products for the treatment of retinal degeneration diseases.

10. A computer model, characterized in that, The computer model is used to evaluate the ability of optogenetics to treat retinal degenerative diseases using the mutant light-induced ion channel ChR-2 protein of any one of claims 1-5.

Images