Gene drug for treating retinal degenerative diseases

Abstract

Provided is a gene drug for treating retinal degenerative diseases. Provided is a gene expression cassette, which comprises a coding sequence of a photosensitive protein, and optionally, a promoter, an enhancer, and / or an intron. Optionally, the photosensitive protein comprises a microbial photosensitive protein or an animal photosensitive protein. The provided gene drug has demonstrated a good therapeutic effect in cells and mice, making the optogenetic treatment method targeting bipolar cells a potential treatment method with broad clinical application prospects.

Description

A gene therapy drug for treating retinal degeneration

[0001] Priority and related applications

[0002] This disclosure claims priority to PCT international application PCT / CN2024 / 130095, filed on November 6, 2024, entitled “A gene therapy for the treatment of retinal degenerative diseases,” the entire contents of which, including its appendices, are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of biotechnology, and more specifically to a gene therapy for the treatment of retinal degenerative diseases. Background Technology

[0004] Hereditary and age-related retinal degenerative diseases cause the gradual loss of function of retinal photoreceptor cells, eventually leading to blindness. Leber's congenital amaurosis (LCA) is a hereditary retinal dystrophy characterized by early loss of retinal function accompanied by degenerative changes in retinal cell structure. LCA is inherited in an autosomal recessive manner, and more than ten mutated genes have been identified. The most common pathogenic gene is RPE65, which causes LCA2. The RPE65 gene is highly expressed in retinal pigment epithelial cells. RPE65 protein is a retinol isomerase; a lack of RPE65 protein severely affects the function of retinal photoreceptor cells, thus causing serious damage to visual function. Retinitis pigmentosa (RP) is a retinal degenerative disease. The prevalence of RP is approximately 1 in 4000. Early symptoms are usually night blindness, followed by gradual loss of peripheral vision, and eventually loss of central vision. Retinopathy of prematurity (RP) is inherited in three modes: autosomal recessive (50-60%), autosomal dominant (30-40%), and X-linked recessive (5-15%). Most cases are caused by single-gene mutations, and more than seventy mutated genes have been identified. The most common causative gene is RHO, causing approximately 25% of dominant RP, about 20% of recessive RP by the USH2A gene, and about 70% of X-linked RP by the RPGR gene. Stargardt disease is an autosomal recessive genetic disorder originating in the retinal pigment epithelium, with a prevalence of approximately 1 / 8000-1 / 10000. Stargardt disease is caused by mutations in the ABCA4 gene. First identified by Karl Stargardt in 1909, Stargardt disease is characterized by oval-shaped atrophic areas of the macula and surrounding yellow spots on the retina, commonly presenting in adolescence and leading to permanent vision loss in later stages. Age-related macular degeneration (AMD) is a degenerative disease that occurs in the central area of ​​the retina and is one of the most common causes of blindness in the elderly. AMD can be divided into dry AMD and wet AMD. All AMD initially presents as dry AMD, while approximately 10% of patients develop wet AMD. Dry AMD causes thinning of the macular tissue, accumulation of waste products from photoreceptor cells forming drusen, and in later stages, it develops into geographic atrophy (GA). GA is characterized by loss of the retinal pigment epithelium (RPE) due to photoreceptor degeneration and retinal thinning; approximately 20% of GA patients develop legal blindness. Wet AMD develops from dry AMD and is characterized by neovascularization of the choroid in the macular region, detachment of the retinal pigment epithelium, macular edema, and hemorrhage. Wet AMD causes a rapid decline in vision.

[0005] Retinitis pigmentosa (RP), Stargardt disease, Leber congenital amaurosis, and age-related macular degeneration are among the leading causes of visual loss. Currently, there is no cure; existing methods and medications can only stabilize the condition or slow disease progression. Gene therapy can deliver missing genes to the retina via viral vectors, allowing retinal cells to potentially express the corresponding gene products normally, thereby restoring the corresponding biological functions. Adeno-associated virus (AAV) has advantages such as good safety, small size, and good persistence, making it the preferred vector for retinal gene delivery. Current research has shown that AAV virus has been used to achieve retinal gene delivery, restoring vision in LCA model mice.

[0006] Although gene therapy can achieve the re-expression of missing genes in the retina, the retinal photoreceptor cells of some patients with advanced retinal degenerative diseases have already progressed to the degenerative stage, and conventional gene therapy can no longer deliver the missing genes to the corresponding cells, resulting in reduced treatment efficacy. Therefore, there is an urgent need to find new solutions.

[0007] Optogenetic therapy is a novel gene therapy technique. It restores visual function by ectopically expressing photosensitizing proteins in nerve cells within the retinal tissue to reconstruct visual signals. Photosensitizing proteins, also known as light-sensitive proteins, are a major optogenetic tool. These proteins, acting as photoresponsive ion pumps or photoreceptors, are widely found in eukaryotes and prokaryotes. They are mainly divided into two categories: microbial photosensitizing proteins (primarily found in prokaryotes, algae, and fungi) and animal photosensitizing proteins (found only in higher eukaryotes, primarily responsible for visual function). Microbial photosensitizing proteins can capture light energy and actively transport ions out of the cell membrane, or passively pump ions out of the cell membrane by opening ion channels. As optogenetic tools, microbial photosensitizing proteins can provide high-speed neural activation or silencing without relying on chemical substances. ChRs are a common microbial photosensitizing protein; studies have shown that expressing ChR2 in retinal ganglion cells can restore photosensitivity after complete degeneration of retinal photoreceptor cells. Compared to microbial opsins, animal photosensitizing proteins can amplify light signals through G protein-coupled signal cascades, thus exhibiting higher photosensitivity. Studies have shown that rhodopsin, as an optogenetic tool, can induce neuronal excitability under low light. By delivering photosensitizing proteins to specific retinal neurons via viral vectors, light causes these proteins to generate electrical signals. These signals are then integrated by multiple levels of neurons and enter the brain, forming vision in the visual cortex. Optogenetics makes it possible to acquire photosensitivity in retinal cells, thus providing new methods for treating various hereditary retinal degenerative diseases and age-related retinal degeneration.

[0008] Although there are many light-sensitive proteins that can be used to restore vision through optogenetic methods, no light-sensitive protein has yet been found that can perfectly adapt to retinal nerve cells and restore visual function. Therefore, discovering or creating light-sensitive proteins that can adapt to retinal nerve cells is of paramount importance.

[0009] Besides screening for photosensitive proteins, the specificity of photosensitive protein expression is also crucial. Retinal neurons include various types such as photoreceptor cells, bipolar cells, retinal ganglion cells, horizontal cells, and non-secreting cells. Gene therapy for retinal diseases often achieves precision treatment by specifically targeting the cells containing the pathogenic genes. In retinal degenerative diseases, photoreceptor cells and retinal pigment epithelium are early targets for gene therapy. As the disease progresses, these cells gradually degenerate. 78%-88% of severe and moderate patients still retain nuclear layer cells, and bipolar cells become the lowest rank among the remaining retinal neurons. This makes bipolar cells an attractive target for treating patients with advanced retinal degeneration. By targeting the expression of photosensitive proteins in bipolar cells, their photosensitivity can be restored, helping to recover more advanced and realistic visual functions. Targeting bipolar cells has significant therapeutic application potential.

[0010] Bipolar cells are mainly divided into ON-type and OFF-type bipolar cells, which can respond to increases and decreases in light to form contrast vision. Optogenetic gene therapy studies have shown that in mouse models of retinal degeneration, specifically targeting the expression of photosensitive proteins in ON-type bipolar cells can help restore visual function at the retinal, cortical, and behavioral levels in blind mice.

[0011] However, although gene therapy targeting ON-type bipolar cells already exists, there is still a lack of effective targeting molecular tools in the human retina, as well as elements and combinations such as bipolar cell-specific promoters with high expression intensity and good specificity. Summary of the Invention

[0012] The problem the invention aims to solve

[0013] In view of the above-mentioned problems existing in the prior art, the purpose of this disclosure is to provide a gene therapy for treating retinal degenerative diseases.

[0014] Solution for solving the problem

[0015] [1]. A gene expression cassette comprising a coding sequence for a light-sensitive protein, and optionally, a promoter, an enhancer, and / or an intron;

[0016] Optionally, the photosensitizing protein includes microbial photosensitizing proteins or animal photosensitizing proteins;

[0017] Optionally, the microbial photosensitizing protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:13-33 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:13-33;

[0018] Optionally, the animal photosensitizing protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:34-44 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:34-44.

[0019] [2]. The gene expression cassette according to [1], wherein the microbial photosensitive protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:13, 15, 29, 30 and 33 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:13, 15, 29, 30 and 33;

[0020] Preferably, the microbial photosensitizing protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:15, 30 and 33 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:15, 30 and 33;

[0021] More preferably, the microbial photosensitive protein comprises a protein encoded by a sequence as shown in SEQ ID NO:15 or 30 or a sequence having at least 85% identity with the sequence shown in SEQ ID NO:15 or 30.

[0022] [3]. The gene expression cassette according to [1], wherein the animal photosensitive protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:34, 35, 37, 39, 43 and 44 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:34, 35, 37, 39, 43 and 44;

[0023] Preferably, the animal photosensitive protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:34, 35, 43 and 44 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:34, 35, 43 and 44.

[0024] [4]. According to any one of [1] to [3], the coding sequence of the light-sensitive protein includes any one of SEQ ID NO:47, 79-86, or a sequence having 85% identity with the sequence shown in any one of SEQ ID NO:47, 79-86.

[0025] [5]. The gene expression cassette according to any one of [1] to [4], wherein the promoter comprises a sequence as shown in any one of SEQ ID NO:6-10 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:6-10;

[0026] Preferably, the promoter comprises a sequence as shown in SEQ ID NO:7 or a sequence having at least 85% identity with the sequence shown in SEQ ID NO:7.

[0027] [6]. The gene expression cassette according to any one of [1] to [5], wherein the enhancer comprises a sequence as shown in any one of SEQ ID NO:1-5 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:1-5;

[0028] Preferably, the enhancer comprises a sequence as shown in SEQ ID NO:2 or a sequence having at least 85% identity with the sequence shown in SEQ ID NO:2.

[0029] [7]. A gene expression cassette according to any one of [1] to [6], wherein the intron comprises a sequence as shown in SEQ ID NO:11 or 12 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:11 or 12;

[0030] Preferably, the intron comprises a sequence as shown in SEQ ID NO:12 or a sequence having at least 85% identity with SEQ ID NO:12.

[0031] [8]. The gene expression cassette according to any one of [1] to [7], wherein the gene expression cassette further comprises a polyadenylation region;

[0032] Optionally, the polyadenylation region comprises a sequence as shown in any one of SEQ ID NO:48, 77 and 78 or a sequence having at least 85% identity with any one of SEQ ID NO:48, 77 and 78;

[0033] Preferably, the polyadenylation region is the human growth hormone polyadenylation region; the human growth hormone polyadenylation region comprises a sequence as shown in SEQ ID NO:48 or a sequence having at least 85% identity with it.

[0034] [9]. The gene expression cassette according to any one of [1] to [8], wherein the structure of the gene expression cassette is as follows: [enhancer]-[promoter]-[coding sequence of light-sensitive protein]-[intron]-[polyadenylation region].

[0035]

[0010] . The gene expression cassette according to any one of [1] to [9], wherein the nucleotide sequence of the gene expression cassette comprises a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:49-57;

[0036] Preferably, the gene expression cassette contains a sequence as shown in any one of SEQ ID NO:49, 51-54 and 56 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:49, 51-54 and 56.

[0037]

[0011] . A gene delivery vector comprising the gene expression cassette described in any one of [1] to

[0010] .

[0038]

[0012] . According to the gene delivery vector described in

[0011] , wherein the gene delivery vector is a viral vector derived from a virus;

[0039] Preferably, the gene delivery vector is a recombinant adeno-associated virus.

[0040]

[0013] . According to the gene delivery vector of

[0012] , wherein the recombinant adeno-associated virus contains a capsid protein, and the gene expression cassette is capsidated within the capsid protein;

[0041] Optionally, the capsid protein is selected from any one of the adeno-associated virus serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 or a variant thereof.

[0042]

[0014] . According to the gene delivery vector described in

[0013] , wherein the capsid protein is AAV2 capsid protein or a variant thereof;

[0043] Preferably, the capsid protein is an AAV2 capsid protein variant;

[0044] More preferably, the AAV2 capsid protein variant comprises a sequence as shown in SEQ ID NO:65, or a sequence having at least 85% identity with SEQ ID NO:65.

[0045]

[0015] . A pharmaceutical composition comprising a gene expression cassette as described in any one of [1] to

[0010] or a gene delivery vector as described in any one of

[0011] to

[0014] .

[0046] And, optionally, pharmaceutically acceptable carriers.

[0047]

[0016] . Use of the gene expression cassette as described in any one of [1] to

[0010] or the gene delivery vector as described in any one of

[0011] to

[0014] in the preparation of a medicament for treating a disease;

[0048] Optionally, the disease is an eye disease;

[0049] Preferably, the eye disease includes retinal degenerative diseases;

[0050] More preferably, the eye disease includes hereditary and / or age-related retinal degenerative diseases.

[0051]

[0017] . A method of treating a disease, comprising administering to a subject a therapeutically effective amount of a gene expression cassette as described in any one of [1] to

[0010] or a gene delivery vector as described in any one of

[0011] to

[0014] or a pharmaceutical composition as described in

[0015] ;

[0052] Optionally, the disease is an eye disease;

[0053] Preferably, the eye disease includes retinal degenerative diseases;

[0054] More preferably, the eye disease includes hereditary and / or age-related retinal degenerative diseases.

[0055] The effects of the invention

[0056] In some embodiments of the present invention, combinations of promoters and other elements are screened and modified based on ON-type bipolar cell-specific expression genes, and their targeting specificity to bipolar cells is verified. The modified combinations of promoters and other elements can significantly improve the targeting efficiency and expression intensity for bipolar cells, helping to improve the accuracy and efficiency of retinal gene delivery.

[0057] In some embodiments of the present invention, multiple photosensitive proteins are screened and modified, and the expression level and photosensitivity specificity of the screened and modified photosensitive proteins are verified to screen out photosensitive proteins with high photosensitivity and capable of generating effective electrical signals.

[0058] In some embodiments of the present invention, this disclosure uses a self-designed and modified AAV2 viral vector that can be expressed in the retina, and the infection efficiency of the vector is far greater than that of wild-type AAV2 virus, enabling the drug to be stably expressed in the inner retina.

[0059] In some embodiments of the present invention, this disclosure has achieved good therapeutic effects in cells and mice, making optogenetic therapy targeting bipolar cells a promising treatment method with broad clinical application prospects. Attached Figure Description

[0060] Figure 1: Comparison of expression intensity of bipolar cell-specific promoters in SK-N-SH cells.

[0061] Figures 2A-2H: Comparison of in vivo expression levels of bipolar cell-specific promoters. Wherein:

[0062] Figure 2A: pIVB1082; Figure 2B: pIVB1083; Figure 2C: pIVB1084; Figure 2D: pIVB1085; Figure 2E: pIVB1087; Figure 2F: pIVB1088; Figure 2G: pIVB2025; Figure 2H: pIVB2026. The scale bar in Figures 2A-2H is 150 μm.

[0063] Figure 3: Validation of bipolar cell-specific capsid and promoter expression levels in vivo. GFP expression mediated by the bipolar cell promoter encapsulated by the IVT18 capsid in wild-type WT mice. Wild-type C57BL / 6J mice were injected intravitreal with a single dose of 5.0E+9 vg / eye of IVT18-2422 virus. Eye samples were collected 4 weeks later, frozen sections were prepared, and staining with anti-GFP antibody and anti-bipolar cell marker PKCα antibody to confirm the co-localization of virus-infected cells and bipolar cells. Scale bar: 150 μm.

[0064] Figures 4A-4D: Photoreactivity of candidate prokaryotic photosensitizing proteins detected by the MED64 microarray electrode system. Different candidate prokaryotic photosensitizing proteins were overexpressed in an in vitro HEK293T cell system, and cellular electrochemical signals were collected using the MED64 microarray electrode system. During the experiment, after detecting the background response for 30 seconds, cells were stimulated with different wavelengths (blue, green, red, and white light) at a frequency of 5 seconds on and 5 seconds off. Data analysis was performed using the MED64 accompanying software after the experiment.

[0065] in:

[0066] Figure 4A: Cellular electrical signal intensity of cells overexpressing candidate light-sensitive proteins after 150 s of blue light irradiation;

[0067] Figure 4B: Cellular electrical signal intensity of cells overexpressing candidate light-sensitive proteins after 150 s of green light irradiation;

[0068] Figure 4C: Cellular electrical signal intensity of cells overexpressing candidate light-sensitive proteins after 150 s of white light irradiation;

[0069] Figure 4D: Cellular electrical signal intensity of cells overexpressing candidate light-sensitive proteins after 150 s of red light irradiation.

[0070] Figures 5A-5K: Photoreactivity of candidate eukaryotic photosensitizing proteins detected by the MED64 microarray electrode system. Different candidate eukaryotic photosensitizing proteins were overexpressed in an in vitro HEK293T cell system, and cellular electrochemical signals were collected using the MED64 microarray electrode system. During the experiment, after detecting the background response for 30 seconds, cells were stimulated with different wavelengths (blue, green, red, and white light) at a frequency of 5 seconds on and 5 seconds off. After the experiment, data analysis was performed using the MED64 software. The figures show the changes in excitability of cells overexpressing different eukaryotic photosensitizing proteins under light stimulation. (Note:)

[0071] Figure 5A: pIVB2220; Figure 5B: pIVB2323; Figure 5C: pIVB2221; Figure 5D: pIVB2222; Figure 5E: pIVB2275; Figure 5F: pIVB2277; Figure 5G: pIVB2281; Figure 5H: pIVB2282; Figure 5I: pIVB2283; Figure 5J: pIVB2284; Figure 5K: pIVB2285;

[0072] Figures 5A-5K are standardized at the baseline level.

[0073] Figures 6A-6E: Behavioral testing results of rd1 and wt mice overexpressing prokaryotic photosensitizers in light and dark chambers. Different prokaryotic photosensitizers were overexpressed in the retinal bipolar cells of rd1 mice via a single intravitreal injection. Ten weeks after administration, behavioral testing was performed on the rd1 and wt mice in light and dark chambers. The mice were placed in a light chamber and allowed to move freely within the chamber for 5 minutes. The movement patterns of the mice were recorded and analyzed. The light source in the light chamber was white light. (The details are not provided in the original text.)

[0074] Figure 6A: Percentage of time mice spend in the dark box; Figure 6B: Ratio of time mice spend in the dark box to time mice spend in the light box; Figure 6C: Time required for mice to go from being placed in the light box to entering the dark box for the first time; Figure 6D: Average moving speed of mice; Figure 6E: Number of times mice shuttle between the light and dark boxes.

[0075] Figures 7A-7E: Behavioral testing results of rd1 and wt mice overexpressing eukaryotic photosensitizing proteins in light and dark boxes. Different eukaryotic photosensitizing proteins were overexpressed in the retinal bipolar cells of rd1 mice via a single intravitreal injection. Ten weeks after administration, behavioral testing was performed on the rd1 and wt mice in light and dark boxes. The mice were placed in a light box and allowed to move freely within the box for 5 minutes. The movement patterns of the mice were recorded and analyzed. The light source in the light box was white light. (The details are not provided in the original text.)

[0076] Figure 7A: Percentage of time mice spend in the dark box; Figure 7B: Ratio of time mice spend in the dark box to time mice spend in the light box; Figure 7C: Time required for a mouse to go from being placed in the light box to entering the dark box for the first time; Figure 7D: Average moving speed of mice; Figure 7E: Number of times mice shuttle between the light and dark boxes.

[0077] Figures 8A-8C: N1 amplitude levels in visual evoked potentials (VEPs) of rd1 mice overexpressing prokaryotic light-sensitive proteins. Different prokaryotic light-sensitive proteins were overexpressed in the retinal bipolar cells of rd1 mice via a single intravitreal injection. Ten weeks after administration, visual evoked potentials (VEPs) were measured in the mice, and higher-order visual electrical signals were detected in the cerebral cortex. Among them:

[0078] Figure 8A: N1 amplitude level in mouse cortex under different light intensities of white light stimulation; Figure 8B: N1 amplitude level in mouse cortex under different light intensities of blue light stimulation; Figure 8C: N1 amplitude level in mouse cortex under different light intensities of green light stimulation.

[0079] Figures 9A-9C: N1 amplitude levels in visual evoked potentials (VEPs) of rd1 mice overexpressing eukaryotic light-sensitive proteins. Different eukaryotic light-sensitive proteins were overexpressed in the retinal bipolar cells of rd1 mice via a single intravitreal injection. Ten weeks after administration, visual evoked potentials (VEPs) were measured in the mice, and higher-order visual electrical signals were detected in the cerebral cortex. Among them:

[0080] Figure 9A: N1 amplitude level in mouse cortex under different light intensities of white light stimulation; Figure 9B: N1 amplitude level in mouse cortex under different light intensities of blue light stimulation; Figure 9C: N1 amplitude level in mouse cortex under different light intensities of green light stimulation. Detailed Implementation

[0081] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0082] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0083] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0084] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0085] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0086] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0087] In this specification, the terms "substantially" or "truly" are used to indicate that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.

[0088] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0089] In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event occurs and the scenario in which the event does not occur.

[0090] In this specification, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0091] In this specification, the term "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0092] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0093] According to this disclosure, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein to refer to a polymeric form of amino acids of any length, including encoded and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a similar peptide backbone.

[0094] According to this disclosure, the terms “nucleic acid molecule,” “polynucleotide,” “polynucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or similar substances.

[0095] According to this disclosure, the terms "upstream" and "downstream" are relative terms that define the linear positions of at least two elements in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in a 5' to 3' direction.

[0096] According to this disclosure, the term "amino acid" can include natural amino acids, non-natural amino acids, amino acid analogs, and all their D and L stereoisomers. According to this disclosure, the three-letter and single-letter codes for amino acids are as described in J. Biol. Chem., 243, p3558 (1968). The amino acids and their abbreviations and English abbreviations in this disclosure are as follows: Histidine (His, H); Serine (Ser, S); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Threonine (Thr, T); Phenylalanine (Phe, F); Aspartic acid (Asp, D); Tyrosine (Tyr, Y); Leucine (Leu, L); Isoleucine (Ile, I); Arginine (Arg, R); Alanine (Ala, A); Valine (Val, V); Tryptophan (Trp, W); Methionine (Met, M); Asparagine (Asn, N); Cysteine ​​(Cys, C); Lysine (Lys, K); Proline (Pro, P).

[0097] According to this disclosure, "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, at optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0098] According to this disclosure, “application,” “giving,” and “treatment,” when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid. “Application,” “giving,” and “treatment” can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cell treatment includes contact between a reagent and a cell, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cell. “Application,” “giving,” and “treatment” also mean, by means of a reagent, diagnostic agent, conjugated composition, or by means of another cell in vitro and ex vivo treatment, such as of cells. “Treatment,” when applied to humans, veterinary, or research subjects, refers to therapeutic, preventative, or prophylactic measures, research, and diagnostic applications.

[0099] According to this disclosure, "treatment" means administering an oral or topical therapeutic agent, such as an antibody comprising any of the present disclosure, to a patient who has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of a disease, whether by inducing the regression of such symptoms or inhibiting their progression to any clinically measurable degree. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms.

[0100] According to this disclosure, the term "prevention" refers to preventive treatment for subjects who currently do not have or have not had any disease but are at risk of developing a disease, or who have had a disease in the past but are currently not at risk of disease recurrence. In some embodiments, subjects have a higher risk of developing a disease or a higher risk of disease recurrence compared to the average healthy member of the subject population.

[0101] According to this disclosure, an "effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0102] According to this disclosure, a "therapeuticly effective amount" is an amount sufficient to provide therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount refers to the amount of a therapeutic agent, alone or in combination with other therapies, that provides therapeutic benefit in the treatment of a condition. The term "therapeuticly effective amount" can include amounts that improve overall therapy; reduce or avoid symptoms, signs, or causes of the condition; and / or enhance the therapeutic efficacy of another therapeutic agent.

[0103] According to this disclosure, a "preventive effective amount" is an amount sufficient to prevent a disease or one or more symptoms associated with a disease, or to prevent its recurrence. A preventive effective amount refers to the amount of a therapeutic agent, alone or in combination with other agents, that provides preventive benefit in preventing a disease. The term "preventive effective amount" may also include amounts that improve overall prevention or enhance the preventive efficacy of another preventive agent.

[0104] According to this disclosure, the term "subject" refers to a human (i.e., a male or female of any age, such as a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young adult, middle-aged adult, or elderly person)) or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or rhesus monkey), a commercially relevant mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog) or a bird. The non-human animal can be male or female at any developmental stage. The non-human animal can be a transgenic animal or a genetically engineered animal.

[0105] According to this disclosure, a "vector" refers to a polymer or polymeric association, which includes or is associated with a polynucleotide and can be used to mediate the delivery of the polynucleotide to a cell. Illustrative vectors include, for example, plasmids, viral vectors (i.e., viruses, such as adeno-associated virus), liposomes, and other gene delivery agents.

[0106] According to this disclosure, "expression vector" encompasses vectors that include gene expression cassettes encoding the gene product of interest, such as plasmids, microcircles, viral vectors, liposomes, etc., and are used to deliver the gene expression cassettes to intended target cells.

[0107] According to this disclosure, the term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term covers all subtypes, as well as naturally occurring and recombinant forms, unless otherwise required. The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects bovine mammals, etc.

[0108] According to this disclosure, "AAV virus," "AAV virus particle," or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically composed of all capsid proteins of wild-type AAV) and capsidated polynucleotides. If the particle includes heteropolynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes to be delivered to mammalian cells), it is generally referred to as a recombinant AAV vector or rAAV. Typically, the heteropolynucleotides are flanked by an AAV inverted terminal repeat (ITR).

[0109] According to this disclosure, the terms "gene," "coding sequence," or "coding gene" refer to a nucleotide sequence that encodes a gene product in vitro or in vivo. The term "transgenic" refers to a coding sequence or gene delivered into cells via a vector. A coding sequence or gene may encode a peptide or polypeptide molecule.

[0110] According to this disclosure, the term "operably linked" refers to the juxtaposition of genetic elements (e.g., promoters, enhancers, termination signal sequences, polyadenylation sequences, etc.) in a relationship that allows them to operate in a intended manner. For example, if a promoter helps initiate transcription of a coding sequence, then the promoter is operably linked to the coding region. Intercalation residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

[0111] According to this disclosure, the term "heterologous" refers to an entity with a different genotype from the rest of the entity being compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species via genetic engineering is a heterologous polynucleotide. As another example, a promoter that has its natural coding sequence removed and operatively linked to a coding sequence not found to be linked naturally is a heterologous promoter. Thus, for example, an rAAV containing a heterologous nucleic acid encoding a heterologous gene product is an rAAV containing nucleic acids not normally contained in naturally occurring wild-type AAVs, and the encoded heterologous gene product is a gene product not normally encoded by naturally occurring wild-type AAVs.

[0112] According to this disclosure, the term "endogenous" in relation to nucleotide molecules or gene products refers to nucleic acid sequences (e.g., genes or genetic elements) or gene products (e.g., RNA, proteins) that are naturally present in or associated with host viruses or cells.

[0113] Invention Details

[0114] Gene Expression Kit

[0115] According to some embodiments of this disclosure, a gene expression cassette is provided, comprising a coding sequence for a light-sensitive protein, and optionally, one or more of a promoter, enhancer, and intron.

[0116] (Coding sequence of the photosensitive protein)

[0117] In some embodiments of this disclosure, the photosensitizing protein includes microbial photosensitizing proteins or animal photosensitizing proteins.

[0118] In some embodiments of this disclosure, the photosensitizing protein includes a microbial photosensitizing protein; the microbial photosensitizing protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:13-33 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:13-33.

[0119] In some preferred embodiments, the microbial photosensitive protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:13, 15, 29, 30 and 33 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:13, 15, 29, 30 and 33.

[0120] In some preferred embodiments, the microbial photosensitizing protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:15, 30 and 33 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:15, 30 and 33.

[0121] In some preferred embodiments, the microbial photosensitizing protein comprises a protein encoded by a sequence as shown in SEQ ID NO:15 or 30 or a sequence having at least 85% identity with the sequence shown in SEQ ID NO:15 or 30.

[0122] In some embodiments of this disclosure, the photosensitive protein includes an animal photosensitive protein; the animal photosensitive protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:34-44 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:34-44.

[0123] In some preferred embodiments, the animal photosensitizing protein comprises a protein encoded by a sequence such as any one of SEQ ID NO:34, 35, 37, 39, 43 and 44 or a sequence having at least 85% identity with any one of SEQ ID NO:34, 35, 37, 39, 43 and 44.

[0124] In some preferred embodiments, the animal photosensitizing protein comprises a protein encoded by a sequence such as any one of SEQ ID NO:34, 35, 43 and 44 or a sequence having at least 85% identity with a sequence such as any one of SEQ ID NO:34, 35, 43 and 44.

[0125] In some embodiments of this disclosure, the coding sequence of the photosensitive protein may be an uncodon-optimized or codon-optimized coding sequence for encoding the corresponding photosensitive protein.

[0126] According to this disclosure, the term "codon optimization" refers to a polynucleotide sequence modified from its natural form. Such modifications result in differences in one or more base pairs, with or without alterations in the corresponding amino acid sequence, potentially enhancing or suppressing gene expression and / or cellular responses to the modified polynucleotide sequence. The coding sequence is a portion of the mRNA sequence that encodes the amino acids used for translation. During translation, each of the 61 trinucleotide codons is translated into one of 20 amino acids, resulting in degeneracy or redundancy in the genetic code. However, different cell types and different animal species utilize tRNAs (each carrying an anticodon) that encode the same amino acids at different frequencies. When a gene sequence contains codons that are not frequently represented by the corresponding tRNA, the ribosomal translation mechanism may be slowed, thus hindering efficient translation. Expression can be improved through species-specific "codon optimization," where the coding sequence is altered to encode the same protein sequence while utilizing codons that are highly represented and / or utilized by highly represented human proteins (Cid-Arregui et al., 2003; Journal of Virology 77:4928).

[0127] For example, the plasmid sequences in Examples 7 and 8, and the coding sequences for the photosensitive protein in SEQ ID NO:49-57 (as shown in Table 7, the photosensitive protein coding sequences in SEQ ID NO:47, 79-86) are codon-optimized coding sequences for the human species.

[0128] Therefore, in some embodiments, the coding sequence of the photosensitive protein includes a sequence as shown in any one of SEQ ID NO:47, 79-86, or a sequence having 85% identity with the sequence shown in any one of SEQ ID NO:47, 79-86.

[0129] In some embodiments of this disclosure, in addition to the sequence encoding the protein product, the gene expression cassette also includes polynucleotide elements for controlling the expression of the protein product, such as promoters, enhancers, introns, polyadenylation signals, etc. Promoters and enhancers can be natural, artificial, or chimeric sequences, i.e., prokaryotic or eukaryotic sequences.

[0130] (Promoter)

[0131] According to this disclosure, a "promoter" encompasses a DNA sequence that guides the binding of RNA polymerase and thereby promotes RNA synthesis. The expression of the promoter and the corresponding protein or polypeptide can be ubiquitous (meaning highly active in a wide range of cells, tissues, and species) or cell-type specific, tissue-specific, or species-specific. A promoter can be "constitutive" (meaning persistently active) or "inducible" (meaning that the promoter can be activated or inactivated by the presence or absence of biological or abiotic factors).

[0132] In some alternative embodiments, the promoter comprises a sequence as shown in any one of SEQ ID NO:6-10 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:6-10.

[0133] In some preferred embodiments, the promoter comprises a sequence as shown in SEQ ID NO:7 or a sequence having at least 85% identity with the sequence shown in SEQ ID NO:7.

[0134] (Enhancer)

[0135] According to this disclosure, "enhancer" encompasses cis-acting elements that stimulate or repress transcription of adjacent genes. Enhancers that repress transcription are also referred to as "silencers." Enhancers can act in any orientation at a distance of several thousand base pairs (kb) downstream of the coding sequence and the transcribed region (i.e., they can be associated with the coding sequence).

[0136] In some specific embodiments, the enhancer comprises a sequence as shown in any one of SEQ ID NO:1-5 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:1-5.

[0137] In some preferred embodiments, the enhancer comprises a sequence as shown in SEQ ID NO:2 or a sequence having at least 85% identity with the sequence shown in SEQ ID NO:2.

[0138] In some specific implementations, the enhancer is located upstream of the promoter.

[0139] (Intron)

[0140] According to this disclosure, "intron" includes splicing donor / recipient regions. Introns are DNA polynucleotides that are transcribed into RNA via intron splicing and removed during mRNA processing. Gene expression cassettes containing introns are generally expressed at higher rates than those without introns.

[0141] In some specific embodiments, the introns in the gene expression cassette described above contain sequences as shown in any one of SEQ ID NO:11 or 12, or sequences that have at least 85% identity with the sequences shown in SEQ ID NO:11 or 12.

[0142] In some preferred embodiments, the intron comprises a sequence as shown in SEQ ID NO:12 or a sequence having at least 85% identity with SEQ ID NO:12.

[0143] (Polyadecylation region)

[0144] In some optional embodiments of this disclosure, the gene expression cassette further includes a polyadenylation region (or polynucleotide tailing signal or polyadenylation signal).

[0145] As understood in the art, RNA polymerase II transcripts terminate by cleavage and addition of a polyadenylated region, also referred to as a polynucleotide tail signal, polyA signal, polyA region, or polyA tail. The polyA region contains multiple consecutive adenosine monophosphates, typically with repeats of the motif AAUAAA. Several effective polyadenylated sites have been identified, including those from SV40, bovine growth hormone (bGH), human growth hormone (hGH), and rabbit β-globin. The most effective polyA signal for transgene expression in mammalian cells can vary depending on cell type, species of interest, and the specific vector used. In some embodiments of this disclosure, the gene expression cassette includes a polyA region selected from the group consisting of SV40, bovine growth hormone (bGH), human growth hormone (hGH), and β-globin (β-globin).

[0146] In some specific implementations, the polyA region contains a sequence as shown in any one of SEQ ID NO:48, 77 and 78 or a sequence having at least 85% identity with any one of SEQ ID NO:48, 77 and 78.

[0147] In some preferred embodiments, the polyA region is the human growth hormone polyA region. In some specific embodiments, the human growth hormone polyA region comprises a sequence as shown in SEQ ID NO:48 or a sequence having at least 85% identity with it.

[0148] (Reverse end repetition)

[0149] In some embodiments of this disclosure, in addition to the sequence encoding the protein product, the gene expression cassette also includes a variety of regulatory elements to package the gene expression cassette into a virus.

[0150] According to this disclosure, the term "inverted terminal repeat (ITR)" includes any AAV viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as a cis element to mediate viral replication, packaging, and integration. The ITRs described herein include, but are not limited to, terminal repeat sequences from types 1-11 AAVs (avian AAV, bovine AAV, canine AAV, equine AAV, and sheep AAV). Furthermore, the AAV terminal repeat sequence need not have a naturally occurring terminal repeat sequence, as long as it is usable for viral replication, packaging, and integration.

[0151] In some exemplary embodiments, the ITR can be an upstream and downstream ITR from the AAV2 genome. Optionally, the upstream ITR contains a sequence as shown in SEQ ID NO:45 or a sequence having at least 85% identity with it; the introns of the downstream ITR contain a sequence as shown in SEQ ID NO:46 or a sequence having at least 85% identity with it.

[0152] In some specific implementations, the gene expression cassette includes, from the 5' end to the 3' end:

[0153] [Enhancer]-[Promoter]-[Coding sequence of light-sensitive protein]-[Intron]-[Polyadecylate region].

[0154] Furthermore, the gene expression cassette includes, from the 5' end to the 3' end:

[0155] [Upstream ITR]-[Enhancer]-[Promoter]-[Coding sequence of light-sensitive protein]-[Intron]-[Polyadecytylation region]-[Downstream ITR].

[0156] In some specific embodiments, the gene expression cassette contains a sequence as shown in any one of SEQ ID NO:49-57 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:49-57.

[0157] In some preferred embodiments, the gene expression cassette contains a sequence as shown in any one of SEQ ID NO:49, 51-54 and 56 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:49, 51-54 and 56.

[0158] <Gene Delivery Vector>

[0159] In some aspects of this disclosure, the gene expression cassette is used to deliver genes into animal cells, for example, to determine the effect of the gene on cell viability and / or function, to treat cellular diseases, etc. Therefore, in some aspects of this disclosure, a gene delivery vector is provided that comprises the gene expression cassette of this disclosure. In some preferred embodiments, the gene delivery vector is used to express transgenes in mammalian cells.

[0160] The gene delivery vectors disclosed herein encompass any convenient gene delivery vector for delivering polynucleotide sequences into mammalian cells. For example, the vector may comprise a single-stranded or double-stranded nucleic acid, such as single-stranded or double-stranded DNA. For example, the gene delivery vector may be DNA, such as naked DNA, such as plasmids or microcircles. The vector may comprise single-stranded or double-stranded RNA, containing a modified form of RNA. In another instance, the gene delivery vector may be RNA, such as mRNA or modified mRNA.

[0161] As another example, the gene delivery vector can be a viral vector derived from a virus, such as adenovirus, adeno-associated virus (AAV), lentivirus, herpesvirus, alpha virus, or retrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibberish leukemia virus (GaLV), feline leukemia virus (FLV), foam virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), or lentivirus. While embodiments covering the use of adeno-associated viruses are described in more detail below, it is expected that those skilled in the art will recognize that similar knowledge and skills in the art can also be applied to non-AAV gene delivery vectors.

[0162] In some embodiments, the gene delivery vector is a recombinant adeno-associated virus (rAAV). In this embodiment, the gene expression cassette has functional AAV inverted terminal repeat (ITR) sequences flanked at the 5' and 3' ends. A “functional AAV ITR sequence” refers to an ITR sequence intended for rescuing, replicating, and packaging AAV viral particles. Therefore, the AAV ITR used in the gene delivery vector of this disclosure does not need to have a wild-type nucleotide sequence and can be altered by nucleotide insertion, deletion, or substitution, or the AAV ITR can be derived from any of several AAV serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. Preferred AAV vectors have all or part of the wild-type Rep and Cap genes deleted, but retain the functional flanked ITR sequences. In certain embodiments, the AAV viral vector is an AAV variant. In some embodiments, the AAV variant is an AAV viral vector containing a variant AAV capsid (or, more specifically, an AAV capsid protein variant).

[0163] In some embodiments, the gene expression cassette is capsidated within an AAV capsid, which can be derived from any adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc., and any of these adeno-associated virus serotypes can serve as a gene delivery vector. For example, the AAV capsid can be a wild-type capsid or a natural capsid. However, similar to ITRs, the capsid does not need to have a wild-type nucleotide sequence, but can be altered relative to the wild-type sequence by inserting, deleting, or substituting nucleotides in the VP1, VP2, or VP3 sequences, as long as the capsid is capable of transducing mammalian cells. In other words, the AAV capsid can be a variant AAV capsid comprising one or more amino acid substitutions, deletions, or insertions relative to the derived parental capsid protein or AAV capsid protein.

[0164] The AAV capsid is an icosahedron composed of 60 VP capsid protein monomers, including 5 VP1 monomers, 5 VP2 monomers, and 50 VP3 monomers. VP1, VP2, and VP3 monomers are all transcribed and translated from the AAV cap gene. VP1 is the longest, containing approximately 735 amino acids. VP2 and VP3 are truncated versions of VP1, omitting the N-terminal amino acids of the VP1 protein. Conventionally, capsid protein modification sites are named according to the amino acid sequence of the VP1 protein. For example, inserting a targeting peptide after amino acid 587 of the AAV2 capsid / capsid protein means inserting the targeting peptide after amino acid 587 of the VP1 monomer (containing 735 amino acids) of AAV2, so that the corresponding positions in AAV2 VP1, VP2, and VP3 all contain this targeting peptide. The amino acid sequence of the AAV2 capsid / capsid protein is usually represented by the amino acid sequence of VP1.

[0165] In some preferred embodiments, AAV is AAV2.

[0166] In some implementations, AAV2 includes AAV2 VP1 or a variant thereof, AAV2 VP2 or a variant thereof, and / or AAV2 VP3 or a variant thereof.

[0167] For example, the amino acid sequence of AAV2 VP1 is shown below (SEQ ID NO:58):

[0168] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL*(Total 735 amino acids, the targeting peptide insertion site is between amino acids N587 and R588)

[0169] The amino acid sequence of AAV2 VP2 is shown below (SEQ ID NO:59):

[0170] *(A total of 598 amino acids, corresponding to amino acids 138-736 of AAV2 VP1. The target peptide insertion site is between amino acids N450 and R451, that is, between amino acids N587 and R588 of AAV2 VP1.)

[0171] The amino acid sequence of AAV2 VP3 is shown below (SEQ ID NO:60):

[0172] *(A total of 533 amino acids, corresponding to amino acids 203-736 of AAV2 VP1. The target peptide insertion site is between amino acids N385 and R386, that is, between amino acids N587 and R588 of AAV2 VP1.)

[0173] In some embodiments of this disclosure, the targeting peptide is inserted at the 587th amino acid position of AAV2 VP1 or a variant thereof. That is, the targeting peptide is located between the 587th and 588th amino acids of AAV2 VP1 or a variant thereof.

[0174] In some embodiments, the AAV capsid protein variant may be an AAV capsid protein variant containing a targeting peptide that targets a specific tissue. In some specific embodiments, the targeting peptide is a targeting peptide that has good targeting properties to the eye, and the target peptide may be as shown in SEQ ID NO:61.

[0175] In some more specific implementations, the AAV capsid protein variant is the AAV2 capsid protein variant.

[0176] In some preferred embodiments, the AAV2 capsid protein variant is the AAV2 capsid protein variant IVT18.

[0177] The aforementioned targeting peptide and IVT18 are described in PCT international application number PCT / CN2024 / 094226 (in PCT / CN2024 / 094226, they are described as targeting peptide 13, with the sequence SEQ ID NO:13, and the corresponding AAV capsid protein name is IVT13, with the sequence SEQ ID NO:27), which is incorporated herein by reference. The sequences of the targeting peptide and IVT18, as well as the specific construction method, are also described in Example 2 below.

[0178] In some preferred embodiments, the AAV2 capsid protein variant is AAV2 capsid protein variant IVT18, which is formed by inserting a targeting peptide (AAARGSLAA (SEQ ID NO:61)) with good ocular targeting properties at the 587th amino acid position of AAV2 VP1. That is, the targeting peptide is located between the 587th and 588th amino acids of AAV2 VP1, resulting in AAV2 capsid protein / AAV2 containing this capsid protein.

[0179] Preferably, rAAV is replication-defective because the AAV vector cannot independently replicate and package its genome further. For example, when cone cells are transduced with rAAV viral particles, the gene is expressed in the transduced cone cells; however, rAAV cannot replicate because the transduced cone cells lack the AAV rep and cap genes, as well as the helper genes.

[0180] Gene delivery vectors (e.g., rAAV viral particles) that capsidate the gene expression cassettes of this disclosure can be generated using standard methods. For example, in the case of rAAV viral particles, the AAV expression vector according to this disclosure can be introduced into production cells, followed by the introduction of an AAV helper construct, wherein the helper construct contains an AAV coding region capable of expression in the production cells and said AAV coding region supplements AAV helper functions not present in the AAV vector. A helper virus and / or additional vectors are then introduced into the production cells, wherein the helper virus and / or additional vectors provide helper functions capable of supporting efficient rAAV viral production. The production cells are then cultured to produce rAAV. These steps are performed using standard methods. Replication-deficient AAV viral particles capsidating the recombinant AAV vectors of this disclosure are prepared using AAV packaging cells and packaging techniques known in the art.

[0181] Viral particles of any concentration suitable for effective transduction into mammalian cells can be prepared for contact with mammalian cells in vitro or in vivo. Similarly, any total quantity of viral particles suitable for providing appropriate cellular transduction to confer the desired effect or treat disease can be administered to mammals. Any suitable quantity of the vector can be administered to the eyes of mammals or primates.

[0182] A viral vector can be formulated into a pharmaceutical composition comprising any suitable unit dose of the vector, and the pharmaceutical composition can be administered to a subject to produce changes in the subject or to treat the subject's disease.

[0183] In some cases, the multiple of infection (MOI) can be used to measure the unit dose of a drug composition. MOI refers to the ratio or multiple of the vector or viral genome to the nucleic acid that can be delivered to cells.

[0184] In preparing the rAAV composition, any host cell used to produce rAAV viral particles can be used, including, but not limited to, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can also be a packaging cell or a production cell, in which the AAV rep and cap genes are stably maintained within the host cell, and the AAV vector genome is stably maintained and packaged in the production cell. Exemplary packaging and production cells are derived from SF-9, 293, A549, or HeLa cells. The AAV vector is purified and formulated using standard techniques known in the art.

[0185] <Pharmaceutical Composition>

[0186] As disclosed herein, in some aspects of this disclosure, a pharmaceutical composition is provided comprising the gene expression cassette or gene delivery vector provided herein, and optionally, a pharmaceutically acceptable vector.

[0187] In some embodiments, the pharmaceutical composition described herein contains the gene expression cassette or gene delivery vector described above as an active ingredient.

[0188] In some specific embodiments, the pharmaceutical composition described herein contains a gene delivery vector as an active ingredient, said gene delivery vector being a recombinant adeno-associated virus. In these embodiments, the pharmaceutical composition comprises about 1 × 10⁻⁶ 8 Up to approximately 1×10 15 Viral genome (vg), approximately 1×10 9 Up to approximately 1×10 14 vg, approximately 1×10 9 Up to approximately 1×10 13 vg, for example 2.0×109 vg, 1×10 10 vg.

[0189] The individual dose is generally not less than the amount required to produce a measurable effect in the subject and can be determined based on the pharmacokinetics and pharmacology of the absorption, distribution, metabolism, and excretion (“ADME”) of the pharmaceutical composition or its byproducts, and therefore on the disposal of the composition within the subject. This includes consideration of the route of administration and dosage. Effective doses and / or dosing regimens can be readily determined empirically based on preclinical assays, safety and escalation and dose range trials, individual clinician-patient relationships, and in vitro and in vivo assays.

[0190] As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents that are compatible with drug administration.

[0191] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, intravenous, intra-arterial, subcutaneous, intraperitoneal, intrathecal, intramuscular, or injection or infusion administration. Thus, delivery can be systemic or local. For example, for delivery to the retina, subretinal or intravitreal injection can be used (see, for example, Ochakovski et al., Front Neurosci. 2017; 11:174; Xue et al., Eye (Lond). 2017 Sep; 31(9):1308-1316).

[0192] In some specific embodiments, the pharmaceutical compositions of this disclosure are designed, engineered, or adapted for administration to primates (e.g., non-human primates and human subjects) via intravitreal or subretinal injection.

[0193] Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY).

[0194] <Uses and Methods>

[0195] In some aspects of this disclosure, this disclosure provides methods for treating or preventing a disease (e.g., an eye disease) in a subject in need, the methods comprising administering an effective amount of the gene expression cassette, gene delivery vector, or pharmaceutical composition of this disclosure to the subject in need.

[0196] In other aspects of this disclosure, the use of the gene expression cassette or gene delivery vector of this disclosure in the preparation of medicaments for treating diseases (e.g., eye diseases) is also provided.

[0197] In other aspects of this disclosure, the use of gene expression cassettes, gene delivery vectors, or pharmaceutical compositions disclosed herein in the preparation of reagents for optogenetic therapy is also discussed.

[0198] In some specific embodiments, the gene expression cassette, gene delivery vector, or pharmaceutical composition of this disclosure can at least partially improve retinal degenerative diseases. In some embodiments, the gene expression cassette, gene delivery vector, or pharmaceutical composition of this disclosure can be delivered to the eye of a human subject.

[0199] In some implementations, the eye disease includes retinal degenerative diseases.

[0200] In some specific implementations, the retinal degenerative diseases include hereditary and age-related retinal degenerative diseases.

[0201] In some specific implementations, the retinal degenerative diseases include Leber congenital amaurosis, retinitis pigmentosa, occult macular dystrophy, or age-related macular degeneration.

[0202] In some embodiments, the gene expression cassette, gene delivery vector, or pharmaceutical composition disclosed herein is used to treat retinal degenerative diseases via optogenetic therapy.

[0203] In some specific embodiments, gene expression cassettes, gene delivery vectors, or pharmaceutical compositions can be administered parenterally via intravenous injection or oral infusion. In some more specific embodiments, the gene expression cassettes, gene delivery vectors, or pharmaceutical compositions are administered to the eye, for example, the retina, lower retina, or vitreous humor, by injection. In other more specific embodiments, the gene expression cassettes, gene delivery vectors, or pharmaceutical compositions are administered via retinal injection, lower retinal injection, or intravitreal injection. In still more specific embodiments, the gene expression cassettes, gene delivery vectors, or pharmaceutical compositions are administered locally or directly to the tissue or organ of interest, for example, by injection into the liver. In some embodiments, the method includes a single administration; in other embodiments, multiple administrations may be performed over time as deemed appropriate by the attending clinician.

[0204] Subjects may be mammals, including human subjects who require treatment for specific diseases (e.g., retinal degeneration).

[0205] In some specific implementation schemes, the therapeutically effective dose for achieving changes or producing a therapeutic effect in a subject can be approximately 1 × 10⁻⁶ when the gene delivery vector (recombinant adeno-associated virus) is administered to the subject. 8 Viral genome (vg) or more, in some cases, approximately 1 × 10⁻⁶ 9 1×10 10 1×10 11 1×10 12 Or 1×10 13 Viral genome or more.

[0206] Example

[0207] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0208] Example 1: Construction of plasmid vector

[0209] The plasmid was synthesized by General Biosystems (Anhui) Co., Ltd.

[0210] The enhancers, promoters, and introns used in each plasmid in Examples 3-5 are shown in Tables 1-3. The sequence of the exemplary plasmid pIVB1088 (SEQ ID NO:73) in Example 3 is shown in Appendix Table 1; the sequence of the plasmid pIVB2422 (SEQ ID NO:76) in Example 5 is shown in Appendix Table 1.

[0211] The nucleotide sequences encoding the light-sensitive proteins in each plasmid in Example 6 are shown in Tables 4-5. The sequences of the exemplary prokaryotic light-sensitive protein plasmid pIVB919 (SEQ ID NO:74) and the exemplary eukaryotic light-sensitive protein plasmid pIVB2220 (SEQ ID NO:75) in Example 6 are shown in Appendix Table 1.

[0212] The sequences of upstream ITR, downstream ITR, and polyA are shown in Table 6.

[0213] The nucleotide sequences encoding light-sensitive proteins in each plasmid in Examples 7-8 are shown in Table 7.

[0214] The plasmid sequences (SEQ ID NO:49-57) of Examples 7-8 are shown in Appendix Table 1.

[0215] Example 2: Preparation and Detection of Recombinant AAV Virus

[0216] 1. Design and construction of AAV capsid protein expression plasmid encoding the capsid protein of interest

[0217] 1.1 Construction of the intermediate plasmid RC2_IVB-NotI

[0218] In this embodiment, a reverse P5 promoter sequence was added upstream of the Rep sequence on pAAV-RC2 (purchased from CellBiolabs, catalog number: VPK-410-SER2), and a NotI restriction endonuclease site was inserted at 1752 bp of the Cap2 sequence. The intermediate plasmid RC2_IVB-NotI was constructed by Anhui General Biotechnology, and the specific sequence is shown below:

[0219] RC2_IVB-NotI plasmid vector sequence (SEQ ID NO:62)

[0220] 1.2 Construction of AAV capsid protein expression plasmid encoding the capsid protein of interest

[0221] The AAV capsid protein expression plasmid encoding the capsid protein of interest was constructed using the Gibson assembly method (see Gibson for detailed steps). Chemical Transformation Protocol (E2611) uses Gibson assembly to assemble PCR fragments and fragments from the NotI-digested linearized intermediate plasmid RC2_IVB-NotI plasmid constructed in step 1, thereby obtaining different AAV capsid plasmids encoding the capsid protein of interest. In obtaining the PCR fragments, no template is required for PCR; primers pair naturally for PCR amplification. The primer sequences are as follows:

[0222] Experimental Results: In this embodiment, the plasmid DNA to be constructed was identified by enzyme digestion and Sanger sequencing, proving that the expression plasmid of the corresponding capsid protein was successfully constructed. The amino acid sequence of the AAV capsid protein expressed in the obtained AAV capsid protein expression plasmid is as follows:

[0223] 2. AAV virus production

[0224] Recombinant AAV virus was obtained by packaging using a three-plasmid co-transfection method (Xiao X, et al. J Virol. 1998; 72(3): 2224-2232.), and the AAV virus was separated, purified and packaged by iodixanol density gradient centrifugation.

[0225] Specifically,

[0226] Recombinant AAV virus was obtained by packaging using a three-plasmid co-transfection method. The three plasmids included any of the plasmid vectors constructed in Example 1, the RC plasmid (capsid & replication plasmid; containing the nucleotide sequence of the capsid Cap, i.e., the AAV capsid protein expression plasmid RC2_IVT18 constructed in step 1 of this example), and the Helper plasmid (or Ad Helper plasmid, which provides the auxiliary factors required for AAV generation).

[0227] HEK 293T cells were seeded in 100 mm TC-treated culture dishes and grown to 70%–80% confluence for plasmid transfection. For AAV packaging, 5 μg of any of the plasmid vectors constructed in Example 1, 5 μg of RC2_IVT18, and 10 μg of Hepler plasmid (purchased from Cell Biolabs, catalog number: VPK-410-SER2) were co-transfected into HEK 293T cells with polyethyleneimine (PEI). Cells and supernatant were harvested 72 hours after transfection. Cells were precipitated by low-speed centrifugation, and then lysed by adding 0.5% sodium deoxycholate (w / v). Free DNA molecules were digested by adding 50 U / ml Benzonase and 2 mM MgCl2 and incubating at 37°C for 2 hours. Simultaneously, the supernatant was precipitated on ice with a 1:5 volume solution of 40% PEG8000 and 2.5M NaCl. The cell lysate and supernatant were mixed and centrifuged. The supernatant was purified by ultracentrifugation with iodixanol. The purified AAV titer was determined by RT-qPCR and stored at -80°C.

[0228] 3. The genomic titer of the prepared AAV virus was determined using ddPCR.

[0229] The specific process is as follows:

[0230] Primers ITR-F and ITR-R, and probe ITR-P were designed for ITR. The 5' end of the probe was labeled with 6-carboxyfluorescein (6-FAM), and the 3' end was labeled with tetramethylrhodamine (TAMRA).

[0231] ITR-F: 5'-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO: 66)

[0232] ITR-R: 5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO: 67)

[0233] ITR-P: 5'-CACTCCCTCTCTGCGCGCTCG-3' (SEQ ID NO: 68)

[0234] A 62 bp ITR fragment was specifically amplified using ITR-F and ITR-R primers. The viral genome titer was detected using the ddPCR probe method with 2×ddPCR Mix reagent (Sinap, Suzhou, China) and a fluorescence ddPCR instrument (model: DQ24, Sinap). The procedure was performed according to the 2×ddPCR Mix reagent instructions. The viral titers obtained are shown below. Note: E represents 10 to the power of 10. For example, "E+12" means 10 to the power of 12, which is 10^12. 12 .

[0235] Example 3: Screening for in vitro expression of bipolar cell-specific elements

[0236] First, SK-N-SH cells (Wuhan Pronosai Life Science Technology Co., Ltd.) were used at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of / wells in 24-well cell culture plates and cultured overnight at 37°C with 5% CO2. The cells were then transfected with plasmids pIVB002, pIVB1082, pIVB1083, pIVB1084, pIVB1085, pIVB1087, pIVB1088, pIVB2025, and pIVB2026 in sequence. 1 μg of plasmid DNA was transfected into each well, and cells were harvested 72 h after transfection.

[0237] Total RNA was extracted from the samples using the TRIzol method, and the RNA concentration was determined. The culture medium was discarded, and the samples were rinsed once with pre-warmed D-PBS. Then, 1 mL of TRIzol lysis buffer was added, and the cells were gently lysed using a 1 mL pipette. The lysate was then transferred to new EP tubes and incubated at room temperature for 5 min. The samples were then transferred to a fume hood, and 200 μL of chloroform was added to each tube. The mixture was vigorously vortexed and incubated for 3 min, followed by centrifugation at 12000 g for 15 min at 4 °C. After centrifugation, the supernatant was collected and transferred to a new centrifuge tube. 500 μL of isopropanol was added, and the tubes were incubated at -20 °C for 30 min, followed by centrifugation at 12000 g for 10 min at 4 °C. After centrifugation, the supernatant was discarded, and the precipitate was retained. 1000 μL of 75% ethanol was added, and the precipitate was briefly vortexed to wash it. The precipitate was then centrifuged at 7500 g for 5 min at 4 °C. After centrifugation, discard the ethanol and place the precipitate in a fume hood to air dry for 2–5 minutes. When the precipitate is almost dry, add 30 μL of enzyme-free water and gently pipette to completely dissolve the RNA. Then, take 2 μL of the RNA sample and measure the RNA concentration using a micro-spectrophotometer. For each sample, take 500 ng for reverse transcription. The reverse transcription system is 20 μL: 500 ng RNA, 1 μL dT18 Primer, 10 μL 2×Mix, 1 μL gDNA Remover, and bring the enzyme-free water to 20 μL. The PCR program is 37℃ for 15 min; 75℃ for 5 s. Finally, qPCR is used to detect the expression level of the target gene when promoters and other elements are used in each group of samples. The specific procedure is as follows:

[0238] Design primers for mCherry and GAPDH:

[0239] mCherry-F: 5'-CCTGAGGGGCTTCAAGTGGGA-3' (SEQ ID NO: 69)

[0240] mCherry-R: 5'-TCACAGGGCCATCAGAGGGA-3' (SEQ ID NO:70)

[0241] GAPDH-F: 5'-AATGTGTCCGTCGTGGATCT-3' (SEQ ID NO:71)

[0242] GAPDH-R: 5'-AGACAACCTGGTCCTCAGTG-3' (SEQ ID NO:72)

[0243] The qPCR system consisted of 10 μL 2×qPCR Mix, 0.4 μL forward primer, 0.4 μL reverse primer, 2 μL cDNA, and 7.2 μL water. The qPCR program was as follows: 94℃ for 30 s (1×), 94℃ for 5 s, 60℃ for 15 s, 72℃ for 10 s (40×), and 95℃ for 15 s (1×). The data were processed using the 2^-(ΔΔCt) method to obtain the final results.

[0244] Experimental results: SK-N-SH cells were cultured at a density of 1.5 × 10⁻⁶. 5 The cells were seeded at a density of 1 μg / well in 24-well cell culture plates. The plasmid constructs pIVB002 (control), pIVB1082, pIVB1083, pIVB1084, pIVB1085, pIVB1087, pIVB1088, pIVB2025, and pIVB2026 obtained in Example 1 were transfected into SK-N-SH cells, with 1 μg of plasmid DNA transfected into each well. Cell samples were collected 72 h after transfection. Total RNA was extracted from the cells using the TRIzol assay, and the expression levels of the target gene were detected using qPCR when each promoter and other elements were in use.

[0245] As shown in Figure 1, the combination of promoter and other elements in the SK-N-SH cell samples transfected with plasmid pIVB1088 resulted in the highest expression level of the target gene, followed by pIVB1085.

[0246] Example 4: Determination of bipolar cell-specific promoter expression location and intensity in C57BL / 6J mice via intravitreal injection of recombinant virus.

[0247] Twenty-nine male C57BL / 6J mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into nine groups of three mice each. As described in Example 2, the bipolar cell promoter was used to package the virus, and the titer was measured to obtain eight viruses: AAV2.7m8-pIVB1082, AAV2.7m8-pIVB1083, AAV2.7m8-pIVB1084, AAV2.7m8-pIVB1085, AAV2.7m8-pIVB1087, AAV2.7m8-pIVB1088, AAV2.7m8-pIVB2025, and AAV2.7m8-pIVB2026. Six weeks after birth, mice were injected intravitreally with recombinant virus at a dose of 1.9E+10 vg / eye. Four weeks after administration, C57BL / 6J mice were anesthetized and euthanized by cervical dislocation. The eyeballs were removed and placed in 2 mL of 4% paraformaldehyde, fixed overnight on a shaker at 4°C. The eyeballs were then removed and dehydrated in 30% sucrose for at least 12 hours. The dehydrated eyeballs were washed once with OCT and placed in an embedding cassette filled with OCT, then flash-frozen in liquid nitrogen. The embedded tissue samples were sectioned using a cryostat, with 12 sections taken every 80 μm until 80% of the tissue sample was covered.

[0248] Experimental Results: Eight recombinant viruses were obtained through packaging as described in Example 2. The recombinant AAV virus was injected intravitreally into both eyes of C57BL / 6J mice at a dose of 1.9E+10 vg / eye (vg represents viral genome). Eye samples were collected from the C57BL / 6J mice 28 days post-injection, and frozen sections were used to detect the main expression locations and intensities of red fluorescence in the retina. The results are shown in Figures 2A-2H. The expression levels of the promoters pIVB1083, pIVB1085, and pIVB1088 were high, with pIVB1088 showing the highest expression level. Figure 2A: pIVB1082; Figure 2B: pIVB1083; Figure 2C: pIVB1084; Figure 2D: pIVB1085; Figure 2E: pIVB1087; Figure 2F: pIVB1088; Figure 2G: pIVB2025; Figure 2H: pIVB2026.

[0249] Example 5: Expression specificity was confirmed in C57BL / 6J mice via intravitreal injection of a novel recombinant viral capsid bound to a bipolar cell-specific promoter.

[0250] As described in Example 2, the virus was packaged with the IVT18 shell and plasmid pIVB2422, and the titer was detected to obtain AAV.IVT18-pIVB2422 virus. The recombinant virus was injected intravitreally into the eyes of 6-week-old C57BL / 6J mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) at a single intravitreal injection dose of 5.0E+9 vg / eye. Four weeks after administration, the C57BL / 6J mice were anesthetized and euthanized by cervical dislocation. The eyeballs were removed and placed in 2 mL of 4% paraformaldehyde, fixed overnight on a shaker at 4°C. The eyeballs were then removed and dehydrated in 30% sucrose for at least 12 hours. After washing once with OCT, the dehydrated eyeballs were placed in an embedding cassette filled with OCT and flash-frozen in liquid nitrogen. The embedded tissue samples were sectioned using a cryostat, with 12 sections collected every 80 μm until 80% of the tissue sample was covered. Immunofluorescence staining was performed on frozen sections using antibodies including anti-GFP (Chicken anti-GFP, Invitrogen), anti-bipolar cell marker PKCα (Rabbit anti-PKCα, Abcam), goat anti-chicken secondary antibody (AF488 Goat anti-Chicken, Invitrogen), goat anti-rabbit secondary antibody (AF594 Goat anti-Rabbit, Invitrogen), and DAPI (Invitrogen) to detect colocalization of virus-infected cells and bipolar cells.

[0251] Experimental Results: The recombinant AAV.IVT18-pIVB2422 virus, packaged as in Example 2, was injected intravitreally into both eyes of C57BL / 6J mice at a dose of 5.0E+9 vg / eye (viral genome, vg). Four weeks post-injection, eye samples were collected from the C57BL / 6J mice, and frozen sections were analyzed to detect the main expression locations of green fluorescence in the retina and its co-localization with bipolar cells. The results are shown in Figure 3. Green GFP represents virus-infected cells, and red fluorescent staining represents the bipolar cell-specific marker PKCα. The virus-infected cells showed excellent co-localization with the bipolar cell marker, indicating that the combination of the novel viral capsid IVT18 and the bipolar cell-specific promoter in plasmid pIVB2422 can specifically infect bipolar cells.

[0252] Example 6: Detection of the light response of candidate photosensitive proteins using MED64 microarray electrodes

[0253] Candidate photosensitizing proteins were overexpressed in HEK293T cells. The electrochemical signals of cells before and after light exposure were detected using the MED64 microarray electrode system (Alpha MED Scientific) to reflect cellular excitability and analyze the degree of response of different photosensitizing proteins to light stimulation. Cells were cultured in DMEM complete medium containing 10% fetal bovine serum and P / S antibiotics. After passage, cells were seeded onto MED electrode plates, and different photosensitizing protein constructs were overexpressed in HEK293T cells using Lipo3000 transfection reagent. Twenty-four hours after transfection, the medium was replaced with complete medium containing 5 μM 9-cis-retinal and 5 μM all-trans-retinal and cultured for another 24 hours. Subsequently, the cell firing frequency was detected under blue, green, red, and white light using a MED instrument to analyze the effect of light on cellular excitability.

[0254] Experimental results: HEK293T cells were cultured at a density of 2.5 × 10⁻⁶. 4The cells were seeded at a density of / wells in MED64 cell culture plates. The candidate prokaryotic photosensitive protein particle constructs pIVB920, pIVB921, pIVB922, pIVB2270, pIVB2274, pIVB2318, pIVB2269, pIVB2319, pIVB2315, pIVB2316, pIVB2271, pIVB2330, pIVB2317, pIVB2332, pIVB2333, pIVB2357, pIVB2331, pIVB2273, pIVB923, pIVB924, and pIVB919 obtained from Example 1 were transfected into HEK293T cells. 500 ng of plasmid DNA was transfected into each well. 24 hours after transfection, the culture medium was replaced with a solution containing 5 μM... Cells were cultured in complete medium containing 9-cis-retinal and 5 μM all-trans-retinal. Forty-eight hours after transfection, cellular electrochemical signals were collected using a MED64 microarray electrode system. After 30 seconds of background response, cells were stimulated with different wavelengths (blue, green, red, and white light) at 5-second on-off intervals. Data analysis was performed using the MED64 software after the experiment. Figures 4A-4D show the excitability of cells overexpressing different prokaryotic photosensitizers under light stimulation. Prokaryotic photosensitizers open ion channels under light, typically leading to a gradual increase in cell excitability. Among them, pIVB921, pIVB2330, pIVB2331, and pIVB2357 showed the strongest photosensitivity under blue and white light irradiation, pIVB2330, pIVB2357, and pIVB2331 showed the strongest photosensitivity under green light irradiation, and pIVB919 showed the strongest photosensitivity under red light irradiation. Figure 4A: Cellular electrochemical signal intensity of cells overexpressing candidate photosensitizing proteins after 150 s of blue light irradiation; Figure 4B: Cellular electrochemical signal intensity of cells overexpressing candidate photosensitizing proteins after 150 s of green light irradiation; Figure 4C: Cellular electrochemical signal intensity of cells overexpressing candidate photosensitizing proteins after 150 s of white light irradiation; Figure 4D: Cellular electrochemical signal intensity of cells overexpressing candidate photosensitizing proteins after 150 s of red light irradiation.

[0255] Experimental results: HEK293T cells were cultured at a density of 2.5 × 10⁻⁶. 4The wells were laid at a density that allowed up to the desired density in a MED64 cell culture plate. The candidate eukaryotic photosensitive protein plasmid constructs pIVB2220, pIVB2323, pIVB2221, pIVB2222, pIVB2275, pIVB2277, pIVB2281, pIVB2282, pIVB2283, pIVB2284, and pIVB2285 obtained in Example 1 were transfected into HEK293T cells, with 500 ng of plasmid DNA transfected into each well. Cells transfected with pIVB2220, pIVB2221, pIVB2222, and pIVB2323 were also co-transfected with GIRK expression plasmids pIVB2256 and pIVB2257 (purchased from Miaoling Biotechnology, P27805 and P18701) to assist in the detection of cell reactivity, with 500 ng of pIVB2256 and pIVB2257 plasmids transfected into each well. Twenty-four hours after transfection, the culture medium was replaced with complete medium containing 5 μM 9-cis-retinal and 5 μM all-trans-retinal for further culture. Forty-eight hours after transfection, cellular electrochemical signals were collected using the MED64 microarray electrode system. During the experiment, after detecting the background response for 30 seconds, cells were stimulated with light of different wavelengths (blue, green, red, and white light) at a frequency of 5 seconds on and 5 seconds off. After the experiment, data analysis was performed using the MED64 software. Eukaryotic photosensitizing proteins are GPCRs, which activate downstream signaling pathways through G proteins. Activation of most eukaryotic photosensitizing proteins leads to the closure of cellular ion channels, resulting in a decrease in cellular excitability. Figures 5A-5K show the changes in cellular excitability of cells overexpressing different eukaryotic photosensitizing proteins under light stimulation. Figure 5A: pIVB2220; Figure 5B: pIVB2323; Figure 5C: pIVB2221; Figure 5D: pIVB2222; Figure 5E: pIVB2275; Figure 5F: pIVB2277; Figure 5G: pIVB2281; Figure 5H: pIVB2282; Figure 5I: pIVB2283; Figure 5J: pIVB2284; Figure 5K: pIVB2285. As the results showed, the excitability of cells expressing pIVB2220, pIVB2221, pIVB2222, pIVB2275, pIVB2277, pIVB2281, pIVB2282, pIVB2283, pIVB2284, and pIVB2285 gradually decreased after light exposure. Among them, pIVB2221, pIVB2281, and pIVB2285 showed a more significant downregulation of cell excitability even under weak light stimulation.

[0256] Example 7: Light-dark box behavioral testing of wt mice and rd1 mice after intravitreal injection of the drug.

[0257] Based on the results of Examples 3, 4, and 5, the bipolar cell-specific promoter of pIVB1088 was selected to drive gene expression. Based on the results of Example 6, the prokaryotic photosensitizing protein genes pIVB2331, pIVB2357, and pIVB921 were selected, and the eukaryotic photosensitizing protein genes pIVB2220, pIVB2221, pIVB2323, pIVB2281, pIVB2285, and pIVB2275 were selected. The plasmids were then reconstructed to obtain the final vectors pIVB2364, pIVB2365, pIVB2366, pIVB2367, pIVB2368, pIVB2369, pIVB2370, pIVB2371, and pIVB2373.

[0258] As described in Example 2, nine AAV viruses, AAV.IVT18-pIVB2364, AAV.IVT18-pIVB2365, AAV.IVT18-pIVB2366, AAV.IVT18-pIVB2367, AAV.IVT18-pIVB2368, AAV.IVT18-pIVB2369, AAV.IVT18-pIVB2370, AAV.IVT18-pIVB2371, and AAV.IVT18-pIVB2373, were packaged and their titers were detected.

[0259] Different photosensitizing proteins were overexpressed in the retinal bipolar cells of rd1 mice via a single intravitreal injection. Male rd1 mice, purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd., were used in the experiment. The mice were 6-7 weeks old at the time of administration, and the dosage was 2.0E+9vg / eye. Ten weeks after administration, rd1 mice and age-matched male wt mice underwent light-dark box behavioral tests to assess their photosensitivity. Three days prior to the light-dark box behavioral tests, mice were placed in a 12h / 12h light cycle with light at night and no light during the day, with free access to water and food. On the day of the experiment, mice were placed in the behavioral testing environment for 30 minutes to acclimatize. Then, each mouse underwent a light-dark box test, with the light box illuminated and the dark box dark. Mice were gently placed in the light box, and their movement was recorded for 5 minutes. Data such as the time spent in the light / dark box, movement speed, and number of times the mice moved between the light and dark boxes were analyzed.

[0260] Experimental Result 1: Three recombinant AAV viruses, AAV.IVT18-pIVB2364, AAV.IVT18-pIVB2365, and AAV.IVT18-pIVB2366, were packaged as described in Example 2. The recombinant AAV viruses were injected intravitreally into both eyes of rd1 mice at a dose of 2.0E+9 vg / eye (viral genome, vg). Ten weeks post-injection, light-dark box behavioral tests were performed on the administered rd1 mice and age-matched wt mice. The mice were placed in a light box and allowed to move freely for 5 minutes. The movement of the mice was recorded and analyzed. The light source in the light box was white light. Figures 6A-6E show the light-dark box behavioral test results of rd1 mice and wt mice overexpressing prokaryotic photosensitizers. Mice injected with AAV.IVT18-pIVB2364 and AAV.IVT18-pIVB2366 showed better photosensitivity recovery. Figure 6A: Percentage of time mice spend in the dark box; Figure 6B: Ratio of time mice spend in the dark box to time mice spend in the light box; Figure 6C: Time required for mice to go from being placed in the light box to entering the dark box for the first time; Figure 6D: Average moving speed of mice; Figure 6E: Number of times mice shuttle between the light and dark boxes.

[0261] Experimental Result 2: Six recombinant AAV viruses, AAV.IVT18-pIVB2367, AAV.IVT18-pIVB2368, AAV.IVT18-pIVB2369, AAV.IVT18-pIVB2370, AAV.IVT18-pIVB2371, and AAV.IVT18-pIVB2373, were obtained through packaging in Example 2. The recombinant AAV viruses were injected intravitreally into both eyes of rd1 mice at a dose of 2.0E+9 vg / eye (viral genome, vg). Ten weeks after injection, the rd1 mice and age-matched weighted mice underwent light-dark box behavioral testing. The mice were placed in a light box and allowed to move freely within the box for 5 minutes. The movement trajectories of the mice were recorded and analyzed. The light source in the light box was white light. Figures 7A-7E show the behavioral results of rd1 mice and wt mice overexpressing eukaryotic photosensitizing proteins in light and dark chambers. All six photosensitizing proteins showed certain therapeutic effects. Among them, mice injected with AAV.IVT18-pIVB2367, AAV.IVT18-pIVB2368, AAV.IVT18-pIVB2369, and AAV.IVT18-pIVB2371 showed better photosensitivity recovery. Figure 7A: Percentage of time spent in the dark chamber; Figure 7B: Ratio of time spent in the dark chamber to time spent in the light chamber; Figure 7C: Time required for a mouse to enter the dark chamber for the first time after being placed in the light chamber; Figure 7D: Average movement speed of the mouse; Figure 7E: Number of times the mouse shuttled between the light and dark chambers.

[0262] Example 8: Visual Evoked Potential (VEP) Detection in wt Mice and rd1 Mice After Intravitreal Injection

[0263] Using the nine AAV viruses described in Example 7, different photosensitive proteins were overexpressed in the retinal bipolar cells of rd1 mice via a single intravitreal injection. The rd1 mice used were male and purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd. The mice were administered the drug at 6-7 weeks of age, with a dosage of 2.0E+9 vg / eye. Ten weeks after administration, visual evoked potentials (VEPs) were detected to assess higher-level visual signal transduction in the mouse cortex. During the test, mice were anesthetized via intraperitoneal injection of tribromoethanol anesthetic according to their body weight. Subsequently, mydriatic fluid and topical anesthetic were instilled into the eyes. After the mice were stably anesthetized, they were placed on a brain localization device, their heads were fixed, the hair on their heads was carefully trimmed, the scalp was disinfected, and the posterior fontanelle was opened along the midline using a craniotomy drill. The mice were then placed flat on the Diagnosys electrophysiology instrument, back up, allowing their bodies to stretch freely. Two electrodes were inserted into the tail and nose, respectively. Gel was applied to the stimulator area of ​​the instrument, and the electrodes were brought close to the mice's eyes, adjusted to ensure full contact. The brain filler electrode was then inserted into the posterior fontanelle, and the programmed sequence was executed to detect the VEP signal in the visual cortex at the lambdoid suture. After the test, the mouse scalp was sutured shut. Following the experiment, the VEP waveform and N1 amplitude were analyzed and statistically analyzed using software.

[0264] Experimental Result 1: Three recombinant AAV viruses, AAV.IVT18-pIVB2364, AAV.IVT18-pIVB2365, and AAV.IVT18-pIVB2366, were packaged as described in Example 2. The recombinant AAV viruses were injected intravitreally into both eyes of rd1 mice at a dose of 2.0E+9 vg / eye (viral genome, vg). Ten weeks post-injection, visual evoked potentials (VEPs) were detected in the mice, and higher visual electrical signals were detected in the cerebral cortex. Figures 8A-8C show the statistical results of the N1 amplitude of VEP waves detected in the mouse cortex under light stimulation of different wavelengths and intensities. The mice injected with AAV.IVT18-AAV.pIVB2364 and AAV.IVT18-pIVB2366 showed more significant recovery in N1 amplitude. Figure 8A: N1 amplitude level in mouse cortex under different light intensities of white light stimulation; Figure 8B: N1 amplitude level in mouse cortex under different light intensities of blue light stimulation; Figure 8C: N1 amplitude level in mouse cortex under different light intensities of green light stimulation.

[0265] Experimental Result 2: Six recombinant AAV viruses, AAV.IVT18-pIVB2367, AAV.IVT18-pIVB2368, AAV.IVT18-pIVB2369, AAV.IVT18-pIVB2370, AAV.IVT18-pIVB2371, and AAV.IVT18-pIVB2373, were packaged in Example 2. The recombinant AAV viruses were injected intravitreally into both eyes of rd1 mice at a dose of 2.0E+9 vg / eye (viral genome, vg). Ten weeks after administration, visual evoked potentials (VEPs) were detected in the mice, and higher visual electrical signals were detected in the cerebral cortex. Figures 9-9C show the statistical results of the N1 amplitude of VEP waves detected in the mouse cortex under light stimulation of different wavelengths and intensities. Among them, the mice injected with IVT18-pIVB2368 showed a more significant recovery in N1 amplitude. Figure 9A: N1 amplitude level in mouse cortex under different light intensities of white light stimulation; Figure 9B: N1 amplitude level in mouse cortex under different light intensities of blue light stimulation; Figure 9C: N1 amplitude level in mouse cortex under different light intensities of green light stimulation.

[0266] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0267] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

[0268] The following nucleotide or amino acid sequences are involved in this disclosure:

[0269] Table 1:

[0270] Table 2:

[0271] Table 3:

[0272] Table 4:

[0273] Table 5:

[0274] Table 6:

[0275] Table 7:

[0276] Appendix 1: Plasmid Sequences

[0277] In the following SEQ ID NO:49-57: double underlined portions are enhancers, single underlined portions are promoters, wavy lines represent photosensitive proteins, dotted underlined portions are introns, italicized portions represent ITRs, and lowercase letters represent polyA.

[0278] pIVB2364 (SEQ ID NO:49)

[0279] pIVB2365 (SEQ ID NO:50)

[0280] pIVB2366 (SEQ ID NO:51)

[0281] pIVB2367 (SEQ ID NO:52)

[0282] pIVB2368 (SEQ ID NO:53)

[0283] pIVB2369 (SEQ ID NO:54)

[0284] pIVB2370 (SEQ ID NO:55)

[0285] pIVB2371 (SEQ ID NO:56)

[0286] pIVB2373 (SEQ ID NO:57)

[0287] pIVB1088 (SEQ ID NO:73)

[0288] In this text, double underlined parts represent enhancers, single underlined parts represent promoters, wavy lines represent photosensitive proteins, dotted underlined parts represent introns, italic parts represent ITRs, lowercase letters represent polyA, and gray backgrounds represent mcherry.

[0289] pIVB919 (SEQ ID NO:74)

[0290] In this text, the single underlined part represents the promoter, the wavy line part represents the photosensitive protein, the dotted underlined part represents the intron, the italic part represents the ITR, the lowercase letter represents polyA, and the gray background represents mcherry.

[0291] pIVB2220:(SEQ ID NO:75)

[0292] In this diagram, the single underlined part represents the promoter, the wavy part represents the photosensitive protein, the italic part represents the ITR, the lowercase letter represents polyA, the gray background represents mcherry, the dotted underline represents Furin, and the lowercase letter + single underline represents T2A.

[0293] pIVB2422 (SEQ ID NO:76)

[0294] In this diagram, double underlines represent enhancers, single underlines represent promoters, wavy lines represent green fluorescent proteins, dotted underlines represent introns, italics represent ITRs, and lowercase letters represent polyA.

Claims

1. A gene expression cassette comprising a coding sequence for a light-sensitive protein, and optionally, a promoter, an enhancer, and / or an intron; Optionally, the photosensitizing protein includes microbial photosensitizing proteins or animal photosensitizing proteins; Optionally, the microbial photosensitizing protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:13-33 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:13-33; Optionally, the animal photosensitizing protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:34-44 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:34-44.

2. The gene expression cassette according to claim 1, wherein, The microbial photosensitive protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:13, 15, 29, 30 and 33 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:13, 15, 29, 30 and 33; Preferably, the microbial photosensitizing protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:15, 30 and 33 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:15, 30 and 33; More preferably, the microbial photosensitive protein comprises a protein encoded by a sequence as shown in SEQ ID NO:15 or 30 or a sequence having at least 85% identity with the sequence shown in SEQ ID NO:15 or 30.

3. The gene expression cassette according to claim 1, wherein, The animal photosensitive protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:34, 35, 37, 39, 43 and 44 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:34, 35, 37, 39, 43 and 44; Preferably, the animal photosensitive protein comprises a protein encoded by a sequence as shown in any one of SEQ ID NO:34, 35, 43 and 44 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:34, 35, 43 and 44.

4. The gene expression cassette according to any one of claims 1 to 3, wherein the coding sequence of the photosensitive protein comprises a sequence as shown in any one of SEQ ID NO:47, 79-86, or a sequence having 85% identity with the sequence shown in any one of SEQ ID NO:47, 79-86.

5. The gene expression cassette according to any one of claims 1 to 4, wherein, The promoter comprises a sequence as shown in any one of SEQ ID NO:6-10 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:6-10; Preferably, the promoter comprises a sequence as shown in SEQ ID NO:7 or a sequence having at least 85% identity with the sequence shown in SEQ ID NO:

7.

6. The gene expression cassette according to any one of claims 1 to 5, wherein, The enhancer comprises a sequence as shown in any one of SEQ ID NO:1-5 or a sequence having at least 85% identity with a sequence shown in any one of SEQ ID NO:1-5; Preferably, the enhancer comprises a sequence as shown in SEQ ID NO:2 or a sequence having at least 85% identity with the sequence shown in SEQ ID NO:

2.

7. The gene expression cassette according to any one of claims 1 to 6, wherein, The intron comprises a sequence as shown in SEQ ID NO:11 or 12 or a sequence having at least 85% identity with the sequence shown in either SEQ ID NO:11 or 12; Preferably, the intron comprises a sequence as shown in SEQ ID NO:12 or a sequence having at least 85% identity with SEQ ID NO:

12.

8. The gene expression cassette according to any one of claims 1 to 7, wherein, The gene expression cassette also includes a polyadenylation region; Optionally, the polyadenylation region comprises a sequence as shown in any one of SEQ ID NO:48, 77 and 78 or a sequence having at least 85% identity with any one of SEQ ID NO:48, 77 and 78; Preferably, the polyadenylation region is the human growth hormone polyadenylation region; the human growth hormone polyadenylation region comprises a sequence as shown in SEQ ID NO:48 or a sequence having at least 85% identity with it.

9. The gene expression cassette according to any one of claims 1 to 8, wherein, The structure of the gene expression cassette is as follows: [enhancer]-[promoter]-[coding sequence of light-sensitive protein]-[intron]-[polyadenylation region].

10. The gene expression cassette according to any one of claims 1 to 9, wherein, The nucleotide sequence of the gene expression cassette contains a sequence that has at least 85% identity with the sequence shown in any one of SEQ ID NO:49-57; Preferably, the gene expression cassette contains a sequence as shown in any one of SEQ ID NO:49, 51-54 and 56 or a sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:49, 51-54 and 56.

11. A gene delivery vector comprising a gene expression cassette according to any one of claims 1 to 10.

12. The gene delivery vector according to claim 11, wherein, The gene delivery vector is a viral vector derived from a virus; Preferably, the gene delivery vector is a recombinant adeno-associated virus.

13. The gene delivery vector of claim 12, wherein the recombinant adeno-associated virus comprises a capsid protein, and the gene expression cassette is capsidated within the capsid protein; Optionally, the capsid protein is selected from any one of the adeno-associated virus serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 or a variant thereof.

14. The gene delivery vector according to claim 13, wherein, The capsid protein is AAV2 capsid protein or a variant thereof; Preferably, the capsid protein is an AAV2 capsid protein variant; More preferably, the AAV2 capsid protein variant comprises a sequence as shown in SEQ ID NO:65, or a sequence having at least 85% identity with SEQ ID NO:

65.

15. A pharmaceutical composition comprising a gene expression cassette as described in any one of claims 1 to 10 or a gene delivery vector as described in any one of claims 11 to 14. And, optionally, pharmaceutically acceptable carriers.

16. Use of the gene expression cassette as described in any one of claims 1 to 10 or the gene delivery vector as described in any one of claims 11 to 14 in the preparation of a medicament for treating a disease; Optionally, the disease is an eye disease; Preferably, the eye disease includes retinal degenerative diseases; More preferably, the eye disease includes hereditary and / or age-related retinal degenerative diseases.

17. A method of treating a disease, comprising administering to a subject a therapeutically effective amount of a gene expression cassette as described in any one of claims 1 to 10, a gene delivery vector as described in any one of claims 11 to 14, or a pharmaceutical composition as described in claim 15; Optionally, the disease is an eye disease; Preferably, the eye disease includes retinal degenerative diseases; More preferably, the eye disease includes hereditary and / or age-related retinal degenerative diseases.

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