Photoreceptor cell-specific bidirectional promoter and use thereof

Abstract

Provided are a photoreceptor cell-specific bidirectional promoter and the use thereof. Specifically, the present invention relates to a new photoreceptor cell-specific bidirectional promoter, an expression cassette, vector, virus particle, cell and pharmaceutical composition containing the bidirectional promoter, and the use thereof in the treatment of ocular diseases such as retinal diseases.

Description

Photoreceptor cell-specific bidirectional promoter and uses thereof TECHNICAL FIELD

[0001] The present application relates to the fields of bioengineering and medicine. In particular, the present application relates to a novel photoreceptor cell-specific bidirectional promoter, expression cassettes, vectors, viral particles, cells and pharmaceutical compositions comprising the bidirectional promoter, and uses thereof in treating eye diseases, such as retinal diseases. BACKGROUND

[0002] Inherited retinal diseases (IRDs) are one of the most common genetic diseases in humans, and are a group of heterogeneous genetic diseases affecting the retina, which cause visual impairment due to dysfunction and degeneration of photoreceptors, retinal pigment epithelium or choroid, or cause vision loss due to improper development, dysfunction or premature death of photoreceptor cells in the retina. Common forms of IRDs include retinitis pigmentosa (RP), cone / rod dystrophy (CD / CRD), Leber congenital amaurosis (LCA), macular dystrophy (MD), and achromatopsia (rod monochromacy), etc. IRDs are also one of the most genetically heterogeneous diseases in humans, and more than 270 related genes have been identified so far. It can be inherited by autosomal recessive (AR), autosomal dominant (AD) or X-linked (XL), in addition, mitochondrial and digenic inheritance patterns have also been reported. The pathogenesis of IRDs often occurs due to the apoptosis response of photoreceptor cells themselves due to gene mutations or the imbalance of the retinal microenvironment (such as the death of neovascularization, retinal pigment epithelial cells, etc.) exacerbating the apoptosis of photoreceptor cells.

[0003] The retina includes a variety of cells, among which the outer nuclear layer is mainly composed of photoreceptor cells. Photoreceptor cells, also known as photoreceptor cells, are a special type of neural epithelial cells with light signal conversion function located in the retina, which can sense light or color. In mammals, photoreceptor cells include rod cells, cone cells and intrinsic photosensitive retinal ganglion cells.

[0004] The functions of rod cells and cone cells are different. Rod cells are responsible for sensing the intensity of light and mainly dominate vision in dark environments or at night. Rod cells are extremely sensitive and can be triggered by a single photon. If the rod cells are damaged, the ability to adapt to darkness will be reduced and it will be difficult to see things in low light. Cone cells have high resolution ability for strong light and color. If the cone cells are damaged, color weakness will occur, which can affect photopic vision, and in severe cases, it can also affect scotopic vision with varying degrees of damage to rod cells.

[0005] According to the pathogenesis of inherited retinal diseases, current treatment strategies mainly focus on (1) overexpression or gene editing to repair the relevant mutant genes, delaying photoreceptor degeneration; (2) inhibiting neovascularization, reducing damage to photoreceptors; and (3) expressing photoreceptor proteins in retinal cells to perform photoreceptor functions.

[0006] The strategy of using retinal cells to express photoreceptor proteins to restore vision is currently the most popular. Photoreceptor proteins include endogenous photoreceptor proteins (such as opsin, rhodopsin, melanopsin, etc.) and exogenous photoreceptor proteins. Studies have found that expressing endogenous photoreceptor proteins or their respective fusions with metabotropic glutamate receptors mGLUR can restore partial vision in blind mice. Using exogenous photoreceptor proteins, such as light-sensitive ion channel proteins (such as chr2, chrimsonR, MCO, etc.), has also achieved vision restoration in mice, and related products have reached the clinic.

[0007] On the one hand, gene expression in retinal cells involves the use of promoters. A promoter is a cis-element that regulates gene expression, responsible for initiating the process of gene transcription, and can effectively regulate the initiation, shutdown, and abundance of downstream gene expression, thereby allowing gene expression to produce a desired level of protein. There have been reports of using constitutive promoters to express target genes in retinal cells. Constitutive promoters can provide strong but non-tissue-specific gene expression patterns in tissues. Constitutive eukaryotic promoters include promoters derived from chicken beta-actin (CBA) genes or phosphoglycerate kinase (PGK) or elongation factor 1 alpha (EF1 alpha). Other constitutive promoters of viral origin include promoters derived from cytomegalovirus (CMV) or synthetic promoter sequences such as CAG. However, it has been shown that the regulation of the CMV promoter depends on many cell signaling pathways that can change the expression of the transgene. In addition, since these promoters are not limited to a given cell type and cause expression in all cells into which they are delivered, such as retinal pigment epithelial (RPE), retina, and other eye tissues outside the retina, such as the ciliary body, iris, cornea, etc., this can reduce the efficiency of infection of target retinal cells and produce potential interference and risks.

[0008] To achieve specific expression of photoreceptor proteins in retinal cells, particularly photoreceptor cells, a specific promoter capable of driving high-efficiency expression of the target gene, i.e., a cell-specific promoter, is needed. Cell-specific promoters are important elements in gene regulation, with the ability to initiate gene expression in specific types of cells, thereby allowing precise regulation of gene function in specific cell types, and thus play a key role in maintaining cell specificity and tissue specificity in living organisms.

[0009] Cell-specific promoters can be an important resource for gene therapy applications. Using cell-specific promoters to design gene expression vectors can enable gene therapy and gene regulation in specific cell types. In addition, the specificity of the promoters can also be used in gene editing techniques to achieve precise editing and regulation of specific cell populations.

[0010] There are reports that tissue-specific promoters that cause expression in RPE or photoreceptor cells have been used to obtain gene expression restricted to retinal cells. Promoters such as those based on RPE65, VMD2, and OA1 cause gene expression in RPE cells, while promoters for human (RK) or bovine (RHO) rhodopsin kinase or the mouse opsin (mOP) cause expression restricted to photoreceptor cells.

[0011] On the other hand, delivery of expression systems to photoreceptor cells often relies on viral infection, with the most widely used being adeno-associated virus (AAV). AAV is highly regarded for its safety, but because its vector capacity is very small, it is prone to a significant drop in viral titer when carrying larger functional proteins (such as Cas9 protein) or simultaneously expressing two or more target genes or proteins due to length exceeding capacity. Obtaining a small length but retaining a promoter that can express downstream genes in specific tissues or cell types can to some extent solve this problem, such a promoter can not only ensure the specific expression of the target gene, but also reduce the capacity occupation of the AAV genome, thereby facilitating the loading of larger or more target genes.

[0012] Promoters include both unidirectional promoters and bidirectional promoters. Bidirectional promoters can drive the expression of downstream structural genes in both the positive and negative strands. Compared with traditional unidirectional promoters, bidirectional promoters not only can achieve the expression of a single exogenous gene, but also can achieve the simultaneous expression of two exogenous genes, and have more extensive applications in the fields of synthetic biology, genetic engineering, etc. Recent studies have shown that bidirectional promoters are widely present in eukaryotes and prokaryotes. Since bidirectional promoters can simultaneously drive the expression of two downstream genes in opposite directions, they are very suitable for use in AAV viral vectors with small loading capacity. In addition, the number of promoters currently available for biological engineering is limited, and using the same type of promoter or a promoter with a similar sequence when simultaneously transferring multiple genes into an organism can cause "co-suppression" of gene expression, leading to gene silencing. Bidirectional promoters can avoid this undesirable transgenic silencing because one promoter can simultaneously express two exogenous genes.

[0013] However, there are currently no reports of the use of bidirectional promoters, especially photoreceptor cell-specific bidirectional promoters, in expression delivery systems for photoreceptor cells.

[0014] Therefore, there is a strong need in the field of retinal disease treatment for a photoreceptor cell-specific bidirectional promoter that facilitates the construction of a high-efficiency expression system for delivery to photoreceptor cells. Screening and identifying a high-efficiency photoreceptor cell-specific bidirectional promoter is of great significance for understanding the mechanism of retinal cell fate determination, the occurrence and progression of related diseases, and especially for developing new means of treating retinal diseases. SUMMARY

[0015] In response to the need in the art for a photoreceptor cell-specific bidirectional promoter, the inventors of the present application have obtained a novel photoreceptor cell-specific bidirectional promoter by artificial recombination, and have identified the specificity and transcriptional activity of the promoter, thereby completing the present application. This novel photoreceptor cell-specific bidirectional promoter has important application value in the fields of biological engineering and medicine, especially in the field of retinal disease treatment.

[0016] In one aspect of the present application, a bidirectional promoter having photoreceptor cell-specific promoter activity is provided. In one embodiment, the bidirectional promoter is capable of bidirectionally driving the expression of positive and negative downstream genes at a high level in photoreceptor cells.

[0017] In one embodiment, the bidirectional promoter comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0018] In one embodiment, the bidirectional promoter comprises a nucleotide sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0019] In one embodiment, the bidirectional promoter comprises a nucleotide sequence as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0020] In one embodiment, the bidirectional promoter consists of a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0021] In one embodiment, the bidirectional promoter consists of a nucleotide sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0022] In one embodiment, the bidirectional promoter consists of a nucleotide sequence as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0023] In yet another aspect of the application, an expression cassette is provided comprising a bidirectional promoter according to the application.

[0024] In one embodiment, the expression cassette further comprises a nucleic acid of interest operably linked to a bidirectional promoter as described in the application.

[0025] In one embodiment, the nucleic acid of interest encodes a therapeutic protein, an optogenetic driver protein, or a reporter protein.

[0026] In one embodiment, the therapeutic protein can be selected from MT-ND4, MT-ND1, MT-ND6, MT-CYB, MT-CO3, MT-ND5, MT-ND2, MT-COI, MT-ATP6, MT-ND4L, OPA1, OPA3, OPA7, and ACO2, or can be a neurotrophic factor selected from GDNF, VEGF, CNTF, FGF2, BDNF, and EPO, an anti-apoptotic protein selected from BCL2 and BCL2L1, an anti-angiogenic factor selected from endostatin, angiostatin, and sFlt, an anti-inflammatory factor selected from IL10, IL1R1, TGFBI, and IL4, or a rod-derived cone viability factor (RdCVF).

[0027] In one embodiment, the optogenetic driver can be an optogenetic activator, which is preferably selected from the group consisting of a rhodopsin, a photopsin, a melanopsin, a pinealopsin, a parapinealopsin, a VA opsin, a peropsin, a neuroopsin, a cerebellum opsin, a retinal pigment, a RGR opsin, a microbial opsin with red-shifted spectral properties (e.g. ReaChR, Chrimson or ChrimsonR), a vertebrate opsin that recruits Gi / o signaling (e.g. a short-wavelength vertebrate opsin or a long-wavelength vertebrate opsin), a channelrhodopsin from Chlamydomonas microalgae (e.g. channelrhodopsin-1 and channelrhodopsin-2), and variants of the above proteins; or an optogenetic inhibitor, which is preferably selected from the group consisting of a halorhodopsin (e.g. NpHR, eNpHR2.0, eNpHR3.0 and Halo57), an archaeal rhodopsin (e.g. Arch and AR-3), a bacterial rhodopsin (e.g. eBR, Proteus rhodopsin and Xanthomonas rhodopsin), a Leptosphaeria discolor fungal opsin (Mac), a Megachasma pelagios cross-halorhodopsin, and variants of the above proteins.

[0028] In one embodiment, the reporter protein can be selected from the group consisting of a fluorescent protein, a calcium indicator, an alkaline phosphatase, a beta-galactosidase, a beta-lactamase, a horseradish peroxidase, and variants of the above proteins.

[0029] In one embodiment, the target nucleic acid encodes a Cas9 protein.

[0030] In one embodiment, the target nucleic acid encodes a nucleic acid selected from the group consisting of an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme and a deoxyribozyme.

[0031] In yet another aspect of the application, a vector comprising the bidirectional promoter according to the application or the expression cassette according to the application is provided.

[0032] In one embodiment, the vector is a viral vector.

[0033] In one embodiment, the viral vector is an adeno-associated virus (AAV) vector, a retroviral vector or a parvovirus vector.

[0034] In one embodiment, the viral vector is a Moloney murine leukemia virus (MoMLV), MSCV, SFFV, MPSV or SNV vector, a lentiviral vector (e.g., derived from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) or equine infectious anemia virus (EIAV)), an adenoviral (Ad) vector, an adeno-associated viral (AAV) vector, a simian virus 40 (SV-40) vector, a bovine papilloma virus vector, an Epstein-Barr virus vector, a herpes virus vector, a vaccinia virus vector, a Harvey murine sarcoma virus vector, a murine mammary tumor virus vector, an anellovirus vector or a Rous sarcoma virus vector.

[0035] In yet another aspect of the application, a viral particle comprising a vector according to the application is provided.

[0036] In one embodiment, the vector is an AAV vector.

[0037] In one embodiment, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74 and AAVdj. In a preferred embodiment, the AAV serotype is selected from the group consisting of AAV-2, AAV-5, AAV2-7m8, AAV-9 and AAV-8. In a more preferred embodiment, the AAV serotype is selected from the group consisting of AAV-2, AAV2-7m8 or AAV-8.

[0038] In yet another aspect of the application, a cell comprising a bidirectional promoter according to the application, an expression cassette according to the application, a vector according to the application or a viral particle according to the application is provided.

[0039] In one embodiment, the cell is a photoreceptor cell.

[0040] In one embodiment, the cell is a cone cell.

[0041] In one embodiment, the cell is a rod cell.

[0042] In one embodiment, the cell is a retinal pigment epithelial cell (RPE).

[0043] In one embodiment, the cell is a HEK293, HEK293T, BHK or CHO cell.

[0044] In yet another aspect of the application, a pharmaceutical composition comprising the bidirectional promoter according to the application, the expression cassette according to the application, the vector according to the application, the viral particle according to the application, or the cell according to the application, and a pharmaceutically acceptable excipient or carrier is provided. The pharmaceutical composition is for use in the treatment or prevention of an eye disease, preferably for use in the treatment or prevention of a retinal disease, more preferably for use in the treatment or prevention of a genetic retinal disease.

[0045] In one embodiment, the eye disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration, Stargardt disease, Leber's hereditary optic neuropathy, cone / rod dystrophy (CD / CRD), Leber congenital amaurosis (LCA), diabetic retinopathy, retinal detachment, Best's disease, choroideremia, tapetochoroidal dystrophy, achromatopsia (rod monochromacy), retinal pigmentary dystrophy, night blindness, X-linked retinoschisis, and Usher syndrome.

[0046] In yet another aspect of the application, a method for the treatment or prevention of an eye disease is provided, the method comprising administering to a subject in need thereof the bidirectional promoter according to the application, the expression cassette according to the application, the vector according to the application, the viral particle according to the application, the cell according to the application, or the pharmaceutical composition according to the application.

[0047] In one embodiment, the eye disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration, Stargardt disease, Leber's hereditary optic neuropathy, cone / rod dystrophy (CD / CRD), Leber congenital amaurosis (LCA), diabetic retinopathy, retinal detachment, Best's disease, choroideremia, tapetochoroidal dystrophy, achromatopsia (rod monochromacy), retinal pigmentary dystrophy, night blindness, X-linked retinoschisis, and Usher syndrome.

[0048] In one embodiment, the method comprises further administering to the subject one or more other therapies, such as one or more other therapeutic agents, gene therapy, immunotherapy, surgical procedures, etc.

[0049] In yet another aspect of the application, the use of the bidirectional promoter according to the application, the expression cassette according to the application, the vector according to the application, the viral particle according to the application, the cell according to the application, or the pharmaceutical composition according to the application for the manufacture of a medicament for the treatment or prevention of an eye disease is provided.

[0050] In one embodiment, the eye disease is a genetic retinal disease.

[0051] In one embodiment, the eye disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration, Stargardt disease, Leber's hereditary optic neuropathy, cone / rod dystrophy (CD / CRD), Leber congenital amaurosis (LCA), diabetic retinopathy, retinal detachment, Best's disease, choroideremia, tapetoretinal degeneration, achromatopsia (rod monochromacy), retinitis pigmentosa, night blindness, X-linked retinoschisis, and Usher syndrome.

[0052] In yet another aspect of the application, a method for expressing a polypeptide or nucleic acid of interest in a cell is provided, comprising introducing into said cell a bidirectional promoter according to the application, an expression cassette according to the application, a vector according to the application, or a viral particle according to the application.

[0053] In one embodiment, the cell is a photoreceptor cell.

[0054] In one embodiment, the cell is a cone cell.

[0055] In one embodiment, the cell is a rod cell.

[0056] In one embodiment, the cell is a retinal pigment epithelial cell (RPE). BRIEF DESCRIPTION OF DRAWINGS

[0057] The subject matter of the present disclosure has been generally described above, and the following drawings are part of the present specification, and are included to further illustrate certain aspects of the present application. The application can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0058] Figure 1 shows the plasmid map of vector VB211226-1096bhj, which was used to verify the activity of the CA10 promoter bidirectional driving downstream gene expression.

[0059] Figure 2 shows the results of immunofluorescence staining experiments performed on vectors pAAV[Exp]-{CA10}>NLS-EGFP:WPRE (containing CA10 promoter) and pAAV[Exp]-{re_CA10}>NLS-EGFP:WPRE (containing re_CA10 promoter) after subretinal injection in mice. Sections were imaged under three magnifications (40x / 100x / 200x) of the fluorescence microscope with corresponding exposure times (2000ms / 1000ms / 500ms), and three pictures were shown for each magnification from left to right. The first picture from left shows the expression of the green fluorescent protein driven by the promoter alone; the second picture from left is a merged image of the fluorescence (green) expressed by the cells and the immunofluorescence staining (red, indicating the cone cell surface protein in the photoreceptor layer) (exposure times for each channel were adjusted); the third picture from left is a merged image of the fluorescence (green) expressed by the cells, the immunofluorescence staining (red, indicating the cone cell surface protein in the photoreceptor layer), and the DAPI staining (blue, indicating the cell nuclei) (exposure times for each channel were adjusted). CA and re_CA represent the CA10 promoter and the re_CA10 promoter, respectively.

[0060] Figure 3 shows the results of immunofluorescence staining experiments performed on vectors pAAV-SV40 late pA-NLS_mCherry <ca10>Figure 4 shows the plasmid map of the vector pAAV-SV40 late pA-NLS_mCherry<re_CA10>NLS_EGFP-BGH pA, wherein the protein expression cassette of NLS_mCherry is inserted in reverse upstream of the re_CA10 promoter.

[0061] Figure 4 shows the plasmid map of the vector pAAV-SV40 late pA-NLS_mCherry<re_CA10>NLS_EGFP-BGH pA, wherein the protein expression cassette of NLS_mCherry is inserted in reverse upstream of the re_CA10 promoter.

[0062] Figure 5 shows the plasmid map of the vector pAAV-SV40 late pA-NLS_mCherry <ca10>Results of immunofluorescence staining experiments performed after subretinal injection of the NLS_EGFP-BGH pA and pAAV-SV40 late pA-NLS_mCherry <re_CA10> NLS_EGFP-BGH pA vector. Sections were photographed under two magnifications (100x / 200x) of the fluorescence microscope with corresponding exposure times (1000ms / 500ms). Three pictures are shown for each magnification from left to right. The first picture from left shows the expression of the promoter-driven nuclear-localized green fluorescent protein (NLS-EGFP) alone; the second picture from left shows the expression of the promoter-driven nuclear-localized red fluorescent protein (NLS_mCherry) alone; the third picture from left is a merged picture of the green fluorescent protein, the red fluorescent protein and DAPI staining (blue, indicating the nucleus) expressed by the cells (exposure times for each channel were adjusted). CA and re_CA stand for CA10 promoter and re_CA10 promoter, respectively.

[0063] Figure 6 shows the plasmid map of the vector VB231203-1125phu, which comprises a lacZ gene, used to verify the activity of the CA10 promoter to drive expression of a large downstream gene.

[0064] Figure 7 shows the results of immunofluorescence staining experiments performed after subretinal injection of the pAAV[Exp]-CA10>LacZ_HA vector. Sections were photographed under two magnifications (100x / 200x) of the fluorescence microscope with corresponding exposure times (500ms / 200ms). Two pictures are shown for each magnification from left to right. The first picture from left shows the expression of the promoter-driven lacZ and HA proteins (red fluorescent protein) alone; the second picture from left is a merged picture of the red fluorescent protein and DAPI staining (blue, indicating the nucleus) expressed by the cells (exposure times for each channel were adjusted). CA stands for CA10 promoter.

[0065] Figure 8 shows the results of immunofluorescence staining experiments to verify the promoter activity of the variant promoters CA10V1, CA10V2, CA10V3 and CA10V4. The sections were photographed under two magnifications (100x / 200x) of the fluorescence microscope with corresponding exposure times (1000ms / 500ms), and three photos were shown for each magnification from left to right. The first photo from left shows the expression of the promoter-driven nuclear-localized green fluorescent protein (NLS-EGFP) alone; the second photo from left shows the expression of the promoter-driven nuclear-localized red fluorescent protein (NLS_mCherry) alone; the third photo from left is the merged image of the green fluorescent protein, the red fluorescent protein and DAPI staining (blue, indicating the nucleus) expressed by the cells (the exposure time for each channel was adjusted). CA10V1, CA10V2, CA10V3 and CA10V4 represent the respective variant promoters. DETAILED DESCRIPTION

[0066] The present inventors have obtained a novel photoreceptor cell-specific bidirectional promoter by artificial recombination, which has photoreceptor cell-specific promoter activity, for example, the promoter can specifically bidirectionally drive the expression of a downstream gene in photoreceptor cells. This novel photoreceptor cell-specific bidirectional promoter can be applied in the field of retinal disease treatment, for example, by simultaneously regulating the expression of two different therapeutic proteins and / or therapeutic nucleic acids in photoreceptor cells, thereby treating retinal diseases.

[0067] DEFINITIONS

[0068] In order to make the present application more easily understood, certain terms are first defined. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of the present application belong. In describing and claiming the embodiments of the present application, the following terms are used in preference, according to the definitions set out below.

[0069] In the present application, unless otherwise specifically indicated, the use of the singular includes the plural, the term "one" means "at least one", and the term "or" means "and / or".

[0070] The terms "comprising" and "including" are generally used herein, and are intended to specify the presence of stated features, integers, steps, or components as being included, but do not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. "Consisting essentially of" and "consisting of" are generally used herein to indicate the presence of stated features, integers, steps, or components as being consist of only those items specifically recited. The text of the specification, including the claims, is to be taken as written and is not to be construed against the patent applicant.

[0071] The term "about" as used herein refers to a variable amount that can be quantified, including but not limited to mass, volume, time, distance, and quantity, for example, the variation in the number of values that can occur through typical measurement techniques and equipment. The term "about" also encompasses these variations, which can be up to ±10%, but also ±9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, etc. For example, "about 100" includes all values within the range of 100 plus or minus 10%. Whether or not modified by the term "about", the claims include equivalents of the recited amounts.

[0072] The term "promoter" as used herein is a nucleotide sequence that permits binding of RNA polymerase and directs the transcription of a gene. A promoter can regulate both the rate and efficiency of transcription of a nucleic acid operably linked thereto. A promoter can also be operably linked to other regulatory elements that enhance ("enhancers") or repress ("repressors") promoter-dependent transcription of the nucleic acid.

[0073] The term "bidirectional promoter" as used herein refers to a stretch of DNA sequence located between two adjacent and transcriptionally opposite genes, which can simultaneously drive the transcription and expression of downstream structural genes of both the positive and negative strands (bidirectional transcriptional gene pairs, also known as "head-to-head" gene pairs), the distance between the transcription start sites of the two adjacent and transcriptionally opposite genes is usually no more than 1 kb.

[0074] The term "promoter activity" as used herein refers to the ability of a promoter to initiate transcription of a nucleic acid operably linked thereto. Promoter activity can be measured using procedures known in the art. For example, promoter activity can be measured by measuring the amount of mRNA transcribed using, for example, Northern blotting or the polymerase chain reaction (PCR). Alternatively, promoter activity can be measured, for example, by Western blotting, ELISA, colorimetric assays, and various activity assays, including reporter gene assays, as the amount of protein product translated.

[0075] The term "operably linked" as used herein refers to the association of nucleic acid sequences on a single nucleic acid molecule such that function of one is affected by the other and the association between the elements permits them to carry out their respective functions in their intended manner. For example, a promoter is operably linked to a coding sequence when the promoter is capable of influencing the expression of the coding sequence, i.e., the coding sequence is under the transcriptional control of the promoter.

[0076] The term "nucleic acid" or "polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, which can be either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising a plurality of purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as most commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a synthetic subunit, such as a polymer of amino phosphonates, and thus can be an oligodeoxynucleoside amino phosphonate (P-NH2) or a mixed amino phosphonate-phosphodiester oligomer. The nucleic acids of the present application can be prepared by any technique known to one skilled in the art, including chemical synthesis, recombination, and mutation. In a preferred embodiment, the nucleic acids of the present application are DNA molecules, which are preferably synthesized by recombinant methods well known to one skilled in the art.

[0077] The terms "polypeptide" and "protein" as used herein can be used interchangeably and refer to polymers of amino acid residues, and are not limited to a minimum length. The polymers of amino acid residues can contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. The terms also include post-translational modifications of the polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In addition, for purposes of the present application, "polypeptide" can refer to proteins that include modifications of the parent sequence, such as deletions, additions, and substitutions, so long as the protein maintains a desired activity. These modifications can be deliberately engineered, through site-directed mutagenesis, or can be accidental, such as by mutation of a host producing the protein or errors caused by PCR amplification.

[0078] The terms "variant" or "functional variant" used herein can be used interchangeably and refer to a nucleotide or amino acid sequence that differs from an original sequence but retains its essential properties and / or function. Generally, a variant is closely similar in sequence to the original nucleotide or amino acid sequence and is identical in many regions. The sequence of a variant can differ from the original sequence by one or more nucleotides or amino acids substitutions, deletions or insertions which do not impair the activity of the sequence. A variant can have the same length as the original sequence or can be shorter or longer.

[0079] The terms "sequence identity" or "identity" used herein refer to the number (%) of positions in which an alignment of two polynucleotide sequences results in a match (identical nucleic acid residue) at that position. Sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing gaps between the sequences. In particular, sequence identity can be determined using any of a variety of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm that aligns the sequences optimally over the entire length (e.g. the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), while sequences of significantly different length are preferably aligned using a local alignment algorithm (e.g. the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for determining the percentage of nucleic acid sequence identity can be achieved in various different ways within the skill in the art, for example using publicly available computer software available at websites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . A person skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required to obtain maximum alignment over the full length of the sequences being compared. For the purposes of the present application, the percentage value of nucleic acid sequence identity refers to the value generated using the pairwise sequence alignment program EMBOSS Needle, which generates the best global alignment of two sequences using the Needleman-Wunsch algorithm, with all search parameters set to default values, i.e. score matrix = BLOSUM62, gap opening penalty = 10, gap extension penalty = 0.5, end gap penalty = error, end gap opening penalty = 10, end gap extension penalty = 0.5.

[0080] For purposes of comparing sequence identity, the two polynucleotide sequences being aligned can contain any proportion of reverse complement sequence segments without affecting the calculation of the percent sequence identity. For example, the two polynucleotide sequences can contain less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of reverse complement sequence segments. The two polynucleotide sequences can contain 10%-90% of reverse complement sequence segments. The two polynucleotide sequences can contain 30%-70% of reverse complement sequence segments. The two polynucleotide sequences can contain 50%-60% of reverse complement sequence segments. The two polynucleotide sequences can contain 100% of reverse complement sequence segments, i.e., the two polynucleotide sequences are completely reverse complement over the entire sequence. The reverse complement sequence segments can be present anywhere in the two polynucleotide sequences being aligned, e.g., starting at the 1st, 2nd, 3rd, 4th, or n-th nucleotide and ending at the m-4th, m-3rd, m-2nd, m-1st, or m-th nucleotide, where m is the length of the nucleotide sequence of the two polynucleotide sequences being aligned and n < m. There can be one or more reverse complement sequence segments in the two polynucleotide sequences being aligned, e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 reverse complement sequence segments. In one embodiment, there are 1-100 reverse complement sequence segments in the two polynucleotide sequences being aligned. The number of reverse complement sequence segments is not limited in sequence identity alignment, and the present application is intended to include two aligned polynucleotide sequences containing any number of reverse complement sequence segments. The reverse complement sequence segments can be obtained by dot plot-based sequence alignment algorithms, e.g., publicly available computer software available at https: / / www.ebi.ac.uk / jdispatcher / emboss. Those skilled in the art will appreciate that the presence of reverse complement sequence segments in the two polynucleotide sequences being aligned does not affect the calculation of the percent sequence identity, which means that the percent sequence identity is 100% if the two polynucleotide sequences are completely reverse complement over the entire length, or if part of the sequence is reverse complement and the other sequence is identical.

[0081] The terms "subject" and "patient" used herein can be used interchangeably, and refer to an animal, preferably a mammal, such as including humans, monkeys, cows, horses, camels, pigs, goats, sheep, dogs, cats, rabbits, rats, mice, etc., more preferably a human, including adults, children, and humans at pre-natal stages, and most preferably a human suffering from a retinal disease, such as a genetic retinal disease.

[0082] The term "treatment" as used herein refers to any action aimed at improving the health status of a patient, such as the reduction, prevention, limitation, prevention or retardation of a disease and its symptoms, which includes prophylactic and / or therapeutic treatment. Treatment is prophylactic if it is administered prior to the clinical appearance of a condition (e.g. a disease or other unwanted symptoms in a subject) (i.e. it protects a subject from developing an unwanted disease); treatment is therapeutic if it is administered after the condition has manifested (i.e. it aims to reduce, ameliorate or stabilise an existing unwanted disease or its symptoms).

[0083] For example, in certain embodiments, the term refers to the amelioration or eradication of a disease or symptoms associated with a disease. In other embodiments, the term refers to minimization of the spread or worsening of a disease resulting from the administration of one or more therapeutic agents to a subject having a disease. In particular, the term "treatment of an eye disease" can refer to a treatment that provides improved vision, prevents the disease from progressing to complete blindness, prevents the spread of damage to non-damaged eye cells, improves damage in damaged eye cells, prevents the occurrence of retinal damage, or rescues eyes with mild or late-stage disease. In certain embodiments, the term refers to a treatment that prevents, reduces or halts retinal disease by providing a therapeutic protein that corrects a genetic deficiency in a patient. In certain other embodiments, the term refers to a treatment that restores the retina or restores vision using optogenetics.

[0084] The term "prevention" as used herein refers to the prevention of the occurrence of a disease, disorder or condition. When used in relation to a medical condition, such as an eye disease, for example a retinal disease, it refers to a reduction in the frequency of occurrence of symptoms of the medical condition (e.g. an eye disease or a retinal disease, especially a genetic retinal disease) in a subject, or a delay in the onset of its symptoms, or a complete elimination thereof, relative to a subject who has not received the administration of the promoter, expression cassette, vector, viral particle, cell and / or composition of the application, when administered to a subject in need thereof.

[0085] Photoreceptor cell-specific bidirectional promoter

[0086] In one aspect of the application, the present application provides a photoreceptor cell-specific bidirectional promoter having photoreceptor cell-specific promoter activity. For example, the photoreceptor cell-specific bidirectional promoter is capable of bidirectionally driving the expression of a positive strand and a negative strand downstream gene in a photoreceptor cell at a high level. Alternatively, the photoreceptor cell-specific bidirectional promoter can also be used to unidirectionally drive the expression of a downstream gene in a photoreceptor cell at a high level. The term "photoreceptor cell" as used herein refers to a special type of neural epithelial cell with the function of light signal conversion located in the retina, typically including rod cells, cone cells, intrinsic photosensitive retinal ganglion cells, and also refers to a photoreceptor cell precursor cell or progenitor cell capable of differentiating into a photoreceptor cell after being transplanted into the subretinal space.

[0087] The photoreceptor cell-specific bidirectional promoters of the application are capable of driving expression of a gene of interest primarily in photoreceptor cells (e.g., cone and rod cells), preferably, the photoreceptor cell-specific bidirectional promoters are capable of driving expression of a gene of interest only in photoreceptor cells. The term "photoreceptor cell-specific" as used herein in reference to a promoter is to be interpreted as being active primarily in photoreceptor cells. It is to be understood that residual expression activity in other tissues or cells than photoreceptor cells cannot be completely excluded. Preferably, the activity of a photoreceptor cell-specific bidirectional promoter in non-photoreceptor cells is less than 90%, e.g., less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the activity in photoreceptor cells. In one embodiment, the activity of a photoreceptor cell-specific bidirectional promoter in non-photoreceptor cells is between 1% and 90%, 1% and 80%, 1% and 70%, 1% and 60%, 1% and 50%, 1% and 40%, 1% and 30%, 1% and 20%, 1% and 10%, 1% and 9%, 1% and 8%, 1% and 7%, 1% and 6%, 1% and 5%, 1% and 4%, 1% and 3%, or 1% and 2% of the activity in photoreceptor cells. In a preferred embodiment, a photoreceptor cell-specific bidirectional promoter is essentially inactive in non-photoreceptor cells. In a more preferred embodiment, a photoreceptor cell-specific bidirectional promoter is inactive in non-photoreceptor cells. In a preferred embodiment, the photoreceptor cell-specific promoters of the application are inactive in ganglion cells, bipolar cells, amacrine cells, horizontal cells, Muller cells, and / or glial cells.

[0088] In one embodiment, the photoreceptor cell-specific bidirectional promoters of the application comprise a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0089] In one embodiment, the photoreceptor cell-specific bidirectional promoters of the application comprise a nucleotide sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0090] In one embodiment, the photoreceptor cell-specific bidirectional promoter of the application comprises a functional variant of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the functional variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or more substitutions, deletions, and / or insertions of nucleotides compared to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0091] In one embodiment, the functional variant is capable of hybridizing to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or its complementary strand under low, medium, or high stringency conditions.

[0092] The term "low stringency conditions" as used herein means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms / mL sheared and denatured salmon sperm DNA, and 25% formamide, followed by washing in 2X SSC with 0.2% SDS at 50°C, for 15 min. three times, followed by standard Southern blotting procedures.

[0093] The term "medium stringency conditions" as used herein means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms / mL sheared and denatured salmon sperm DNA, and 35% formamide, followed by washing in 2X SSC with 0.2% SDS at 55°C, for 15 min. three times, followed by standard Southern blotting procedures.

[0094] The term "high stringency conditions" as used herein means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms / mL sheared and denatured salmon sperm DNA, and 50% formamide, followed by washing in 2X SSC with 0.2% SDS at 65°C, for 15 min. three times, followed by standard Southern blotting procedures.

[0095] In one embodiment, the photoreceptor cell-specific bidirectional promoter of the application comprises a nucleotide sequence as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0096] In one embodiment, the photoreceptor cell-specific bidirectional promoter of the application consists of a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0097] In one embodiment, the photoreceptor cell-specific bidirectional promoter of the application consists of a nucleotide sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0098] In one embodiment, the photoreceptor cell-specific bidirectional promoter of the application consists of a functional variant of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the functional variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more nucleotide substitutions, deletions and / or insertions relative to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the functional variant is capable of hybridizing to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or the complement thereof under low, medium or high stringency conditions.

[0099] In one embodiment, the photoreceptor cell-specific bidirectional promoter of the application consists of a nucleotide sequence as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0100] Expression cassette

[0101] In yet another aspect of the application, an expression cassette comprising the photoreceptor cell-specific bidirectional promoter of the application is provided. The term "expression cassette" as used herein refers to a nucleic acid construct comprising a coding sequence and one or more control sequences required for expression of the coding sequence. In particular, one of these control sequences is the promoter of the application. Typically, the expression cassette comprises the coding sequence required for expression of a selected gene product and regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. Thus, an expression cassette typically comprises a promoter sequence, a coding sequence, and a 3' non-translated region which usually contains a polyadenylation site and / or a transcription termination signal. The expression cassette can also comprise other regulatory elements, such as enhancer sequences, a multiple cloning site sequence to facilitate insertion of the DNA fragment within a vector, and / or a splicing signal sequence. The expression cassette is usually comprised within a vector to facilitate cloning and transformation.

[0102] In one embodiment, the expression cassette further comprises a nucleic acid of interest operably linked to the photoreceptor cell-specific bidirectional promoter of the application. In one embodiment, the nucleic acid of interest is a nucleic acid encoding a polypeptide of interest. The polypeptide of interest can be any polypeptide that needs to be expressed in photoreceptor cells. For example, the polypeptide of interest can be a therapeutic protein, an optogenetic driver protein, or a reporter protein. Preferably, the photoreceptor cell-specific bidirectional promoter of the application is operably linked to two different nucleic acids of interest, thereby driving the specific expression of the two different nucleic acids of interest in photoreceptor cells simultaneously.

[0103] In one embodiment, the nucleic acid of interest is a therapeutic gene, i.e. a gene encoding a therapeutic protein. The term "therapeutic gene" as used herein refers to a gene encoding a therapeutic protein useful in the treatment of a pathological condition. The therapeutic gene, when expressed, provides a beneficial effect to the cell or tissue in which it is present or to the patient in which the gene is expressed in vivo. Examples of beneficial effects include improvement or alleviation of a sign or symptom of a disease, disorder, or condition, prevention or inhibition of a disease, disorder, or condition, or provision of a desirable characteristic. Therapeutic genes include genes that partially or completely correct a genetic deficiency in a patient. In particular, the therapeutic gene can be, but is not limited to, a nucleic acid sequence encoding a protein useful in gene therapy to alleviate a deficiency caused by the absence, deficiency, or non-optimal level of the protein in the cells or tissues of the subject. The therapeutic polypeptide may, for example, provide a polypeptide and / or enzyme activity that is absent, deficient, or present at a non-optimal level in photoreceptor cells, a polypeptide and / or enzyme activity that indirectly counteracts an imbalance in photoreceptor cells. The therapeutic polypeptide can also be used to decrease the activity of a polypeptide by, for example, acting as a dominant negative polypeptide. Preferably, the therapeutic polypeptide provides a polypeptide and / or enzyme activity that is absent, deficient, or present at a non-optimal level in photoreceptor cells, more preferably a polypeptide and / or enzyme activity that is absent or deficient in photoreceptor cells.

[0104] Examples of therapeutic genes include, but are not limited to, nucleic acids for replacing missing or mutated genes known to cause retinal disease, such as MT-ND4, MT-ND1, MT-ND6, MT-CYB, MT-CO3, MT-ND5, MT-ND2, MT-COI, MT-ATP6, MT-ND4L, OPAl, OPA3, OPA7, and ACO2. The therapeutic genes can also encode neurotrophic factors such as GDNF, CNTF, FGF2, BDNF, and EPO, anti-apoptotic genes such as BCL2 and BCL2L1, anti-angiogenic factors such as endostatin, angiostatin, and sFlt, anti-inflammatory factors such as IL10, IL1R1, TGFBI, and IL4, or rod-derived cone viability factor (RdCVF). Preferably, the photoreceptor cell-specific bidirectional promoter of the application drives expression of one, two, or more of the above therapeutic genes in photoreceptor cells simultaneously.

[0105] Additional signal peptides can be added to the therapeutic proteins, particularly in order to import them into certain organelles (e.g. mitochondria), to secrete them out of the cell, or to insert them into the cell membrane.

[0106] In one embodiment, the polypeptide of interest is a tool enzyme used in gene editing methods. In one embodiment, the polypeptide of interest is a tool enzyme in the CRISPR / Cas system, e.g. a Cas9 protein.

[0107] In one embodiment, the polypeptide of interest is a optogenetic kinesin. The term "optogenetic kinesin" as used herein refers to a photochemically reactive polypeptide that uses vitamin A or isoforms thereof as a chromophore. Optogenetic kinesins are light-gated ion pumps or channels that absorb light and are activated by light. The optogenetic kinesin can be from a prokaryote or a eukaryote. In particular, it can be a microbial opsin or a vertebrate opsin. The optogenetic kinesin can be an optogenetic activator or an optogenetic inhibitor.

[0108] Optogenetic activators cause cells to depolarize after exposure to light. When a cell depolarizes, the interior negative charge of the cell becomes positive for a short period of time. The shift from negative to positive in the interior environment of the cell allows for the transmission of an electrical pulse both within the cell and optionally between cells. Examples of optogenetic activators include, but are not limited to, rhodopsin, photopsin, melanopsin, pinealopsin, parapinealopsin, VA opsin, peropsin, neuroopsin, retiochrome, RGR opsin, microbial opsin with red-shifted spectral properties (e.g., ReaChR, Chrimson, or ChrimsonR), vertebrate opsin that recruits Gi / o signaling (e.g., short-wavelength vertebrate opsin or long-wavelength vertebrate opsin), channelrhodopsins from Chlamydomonas microalgae (e.g., channelrhodopsin-1 and channelrhodopsin-2 from Chlamydomonas reinhardtii), and variants of the above proteins. Numerous variants of channelrhodopsins (e.g., codon-optimized variants, mutants, chimeras) are continually being generated to improve certain characteristics of these proteins. Examples of these variants include, but are not limited to, hChR2(L132C), ChR2(H134R), ChETA(E123T), C1V1(E122T), C1V1(E162T), C1V1(E122 / 162T), hChR2(C128A), hChR2(C128S), hChR2(C128T), hChR2(C128A / H134R), hCatch(T159S), hChief, hChR2(C128S / D156A), hChR2(T159C), hChR2(E123T / T159C), hChR2c(C128T), ChR2c(C128T), ChR2e(Q117C), and SwitChR (for review see Prakash et al., Nat Methods. 2012 Dec;9(12):1171-9).

[0109] Optogenetic inhibitors cause cell hyperpolarization upon exposure to light. When a cell hyperpolarizes, the internal negative charge of the cell becomes more negative for a short period of time. The shift to more negative inhibits action potentials by increasing the stimulus needed to shift the membrane potential toward the action potential threshold. In particular embodiments, the optogenetic inhibitor is a light-gated ion pump that transports chloride ions inwards and / or cations outwards upon absorption of a photon. Any suitable light-gated, retinal-dependent ion pump that transports chloride ions inwards or cations outwards upon absorption of a photon can be used as an optogenetic inhibitor. Examples of optogenetic inhibitors include, but are not limited to, halorhodopsins such as halorhodopsin (NpHR), enhanced halorhodopsin (eNpHR2.0 and eNpHR3.0), and red-shifted halorhodopsin Halo57, archaeorhodopsin-3 (AR-3), archaerhodopsin (Arch), bacterial rhodopsins such as enhanced bacterial rhodopsin (eBR), proteorhodopsin, xanthorhodopsin, Aspergillus variecolor fungal opsin (Mac), great white shark cross-halorhodopsin, and variants of the above proteins.

[0110] In a particularly preferred embodiment, the optogenetic driver is an optogenetic activator, preferably selected from the group consisting of channelrhodopsin, ChrimsonR and variants thereof.

[0111] Preferably, the photoreceptor cell-specific bidirectional promoter of the application drives the expression of one, two or more of the above optogenetic drivers in photoreceptor cells simultaneously.

[0112] In an embodiment, the polypeptide of interest is a reporter protein. Preferably, the reporter protein is detectable in living photoreceptor cells. Expression of a reporter protein under the control of the promoter of the application allows for the specific detection or recognition of photoreceptor cells. The reporter protein can be a fluorescent protein (such as green fluorescent protein GFP including EGFP, red fluorescent protein RFP, yellow fluorescent protein YFP, blue fluorescent protein BFP, cyan fluorescent protein CFP, etc.), a calcium indicator (such as GCamP), luciferase, alkaline phosphatase, beta-galactosidase, beta-lactamase, horseradish peroxidase and variants thereof. In particular embodiments, the reporter protein is selected from the group consisting of a fluorescent protein, a calcium indicator, alkaline phosphatase, beta-galactosidase, beta-lactamase, horseradish peroxidase and variants thereof. Preferably, the photoreceptor cell-specific bidirectional promoter of the application drives the expression of one, two or more of the above reporter proteins in photoreceptor cells simultaneously.

[0113] In one embodiment, the nucleic acid of interest encodes any nucleic acid that needs to be expressed, for example any nucleic acid of interest that needs to be expressed in photoreceptor cells. In particular, the nucleic acid that needs to be expressed can be a therapeutic nucleic acid. In one embodiment, the therapeutic nucleic acid is selected from the group consisting of siRNA, shRNA, RNAi, miRNA, antisense RNA, sgRNA, ribozyme and DNAzyme. In a particular embodiment, the nucleic acid of interest, when transcribed by the promoter of the application, can treat or prevent a disease associated with an abnormal or excessive protein when translated or transcribed by interfering with the translation or transcription of said abnormal and / or excessive protein. For example, the RNA encoded by the nucleic acid of interest can highly specifically eliminate or reduce the expression level of the mRNA encoding the abnormal and / or excessive protein, thereby achieving the purpose of treating the eye disease. Preferably, the photoreceptor cell-specific bidirectional promoter of the application simultaneously drives the expression of one, two or more therapeutic nucleic acids described above in photoreceptor cells.

[0114] Vector

[0115] In yet another aspect of the application, a vector comprising the photoreceptor cell-specific bidirectional promoter of the application or the expression cassette of the application is provided. The term "vector" as used herein refers to a nucleic acid molecule that functions as a vehicle to transfer genetic material, in particular to deliver a nucleic acid into a host cell, either in vitro or in vivo. Vectors include, but are not limited to, plasmids, phagemids, cosmids, transposable elements, viruses and artificial chromosomes (e.g. YACs). Preferably, the vector of the application is a vector suitable for gene or cell therapy, in particular suitable for targeting the eye, for example photoreceptor cells.

[0116] In one embodiment, the vector is a viral vector. The term "viral vector" as used herein refers to a virus-based composition that is capable of acting as a vehicle to deliver a nucleic acid molecule of interest, such as a heterologous nucleic acid, into a cell. A heterologous nucleic acid can be delivered to a recipient in vivo with or without insertion into the recipient's genomic nucleic acid by means of a viral vector. In one embodiment, a viral vector is a virus in which the viral genome has been manipulated to accommodate a nucleic acid sequence that is non-native with respect to the viral genome. A viral vector can be generated by introducing one or more mutations into the viral genome of a virus to accommodate the insertion of a non-native nucleic acid sequence into the virus. Typically, the genome of a virus can be modified by deleting sequences that are non-essential therein and containing only the minimum components necessary to assemble a functional recombination virus or viral particle, thereby providing an efficient payload for a nucleic acid molecule of interest to be delivered. A viral vector includes any element necessary to establish expression of a polypeptide of interest in a host cell, such as a promoter, ITR, ribosome binding element, terminator, enhancer, selection marker, intron, polyA signal and / or origin of replication.

[0117] In one embodiment, the viral vector is, for example, derived from a vector of Moloney murine leukemia virus (MoMLV), MSCV, SFFV, MPSV or SNV, a lentiviral vector (e.g., derived from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) or equine infectious anemia virus (EIAV)), an adenoviral (Ad) vector, an adeno-associated viral (AAV) vector, a simian virus 40 (SV-40) vector, a bovine papilloma virus vector, an Epstein-Barr virus vector, a herpes virus vector, a vaccinia virus vector, a Harvey murine sarcoma virus vector, a murine mammary tumor virus vector, an anellovirus vector or a Rous sarcoma virus vector. In particular embodiments, the vector is a retroviral vector, preferably a lentiviral vector, or a non-pathogenic parvovirus vector. As is known in the art, depending on the particular viral vector contemplated for use, appropriate sequences should be introduced into the vector of the application to obtain a functional viral vector, such as AAV inverted terminal repeat sequences (ITRs) for AAV vectors or long terminal repeat sequences (LTRs) for lentiviral vectors.

[0118] In a preferred embodiment, the vector is an adeno-associated viral (AAV) vector. The human parvovirus adeno-associated virus (AAV) is a dependovirus that is naturally defective for replication, capable of integrating into the genome of the infected cell to establish a latent infection. This last property appears to be unique among mammalian viruses, as the integration occurs in the human genome at a specific site on chromosome 19, called AAV S1 (19ql3.3-qter). Thus, AAV has raised considerable interest as a potential vector for human gene therapy. Favorable properties of the virus include its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and the broad range of cell lines derived from different tissues that it can infect.

[0119] The term "AAV vector" as used herein refers to a polynucleotide vector comprising one or more heterologous sequences (i.e. nucleic acid sequences not of AAV origin) flanked by at least one AAV inverted terminal repeat sequence (ITR), preferably two ITRs. Such AAV vectors, when present in a host cell infected with a suitable helper virus (or a virus expressing suitable helper virus functions) and expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins), can be replicated and packaged in infectious viral particles. The "inverted terminal repeat" or "ITR" sequence is a term well known in the art and refers to a relatively short sequence present at the ends of the viral genome in opposite orientation. The "AAV inverted terminal repeat (ITR)" sequence is a sequence of about 145 nucleotides present at both ends of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, giving rise to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter regions of self-complementarity (referred to as A, A', B, B', C, C' and D regions), allowing intrastrand base pairing to occur within this portion of the ITR. The AAV ITR used in the vectors of the present application can have the wild-type nucleotide sequence, or can be altered by insertion, deletion or substitution. The terminal inverted repeat (ITR) of the AAV vector can be selected from any known human or non-human AAV serotype. The promoter or expression cassette of the present application can be introduced into the vector by any method known to the skilled person.

[0120] The vector can also comprise one or more of the following nucleic acid sequences encoding a selectable marker such as an auxotrophic marker (e.g. LEU2, URA3, TRP 1 or HIS3), a detectable tag such as a fluorescent or luminescent protein (e.g. GFP, eGFP, DsRed, CFP) or a protein providing resistance to a chemical / toxic compound (e.g. the MGMT gene providing resistance to temozolomide). These markers can be used to select or detect host cells comprising the vector and can be easily selected by the skilled person depending on the host cell.

[0121] Viral particle

[0122] In yet another aspect of the present application, a viral particle is provided, comprising the vector of the present application.

[0123] In one embodiment, the vector is an AAV vector and is packaged in an AAV-derived capsid to generate an adeno-associated viral particle or AAV particle. Thus, the term "adeno-associated viral particle / AAV particle" as used herein refers to a viral particle composed of at least one AAV capsid protein and an encapsidated AAV vector genome. AAV viruses are generally classified and referred to according to their serotype. A "serotype" corresponds to a variant subspecies of AAV that has a unique reactivity useful for distinguishing it from other variant subspecies by virtue of the expression profile of its capsid surface antigens. AAV serotypes include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, and AAVdj, among others. In addition, non-natural engineered variants and chimeric AAVs can also be useful. In particular, the capsid proteins can be variants comprising one or more amino acid substitutions that enhance transduction efficiency. In one embodiment, the capsid is preferably selected from the group consisting of AAV-2, AAV-5, AAV2-7m8, AAV-9, and AAV-8 serotype capsids, more preferably an AAV-2 derived capsid such as an AAV-2 or AAV2-7m8 capsid.

[0124] Different AAV serotypes are used to optimize transduction of specific target cells or to target specific cell types within a particular target tissue (e.g., photoreceptor cells). The AAV particles can comprise viral proteins and viral nucleic acids of the same serotype or any natural or artificial sequence variant of AAV. For example, the AAV particles can comprise AAV2 capsid proteins and at least one, preferably two, AAV2 ITRs. Any combination of AAV serotypes for producing AAV particles is provided herein. AAV viruses can be engineered using conventional molecular biology techniques such that these particles can be optimized for cell-specific delivery of nucleic acid sequences, for minimizing immunogenicity, for modulating stability and particle longevity, for efficient degradation, for accurate delivery to the nucleus.

[0125] In the art, a large number of methods for producing viral particles, in particular AAV particles, are known, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpes virus-AAV hybrids (Conway, JE et al., (1997) Virology 71(11):8780-8789), and baculovirus-AAV hybrids. AAV production cultures for producing AAV viral particles require: 1) a suitable host cell, including for example a cell line of human origin such as HeLa, A549, or 293T cells, or in the case of baculovirus production systems a cell line of insect origin such as SF-9; 2) a suitable helper virus function, provided by a wild-type or mutant adenovirus (e.g. temperature sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper virus functions; 3) AAV rep and cap genes and gene products; 4) a nucleic acid of interest flanked by at least one AAV ITR sequence, such as a vector of the application; and 5) suitable media and media components known in the art that support AAV production.

[0126] In the present application, host cells for producing AAV particles include mammalian cells, insect cells, plant cells, microorganisms, and yeast. The host cell can be a packaging cell in which AAV rep and cap genes are stably maintained in the host cell or a producer cell in which AAV vector genomes are stably maintained. Exemplary packaging and producer cells are derived from 293T, A549, or HeLa cells. AAV particles are then purified and formulated using standard techniques known in the art.

[0127] Cell

[0128] In yet another aspect of the present application, a cell comprising or having been transformed with a photoreceptor cell-specific bi-directional promoter, expression cassette, vector, or viral particle of the present application is provided. The cell can be any cell, including an animal cell, a plant cell, a bacterial cell, or a yeast cell. Preferably, the cell is a mammalian cell or an insect cell. More preferably, the cell is a mammalian cell, such as a cell of a human, monkey, cow, horse, camel, pig, goat, sheep, dog, cat, rabbit, rat, or mouse. Most preferably, the cell is a human cell.

[0129] In one embodiment, the cell is a photoreceptor cell. In one embodiment, the cell is a cone cell. In one embodiment, the cell is a rod cell. In one embodiment, the cell is a retinal pigment epithelial cell (RPE). In one embodiment, the cell is a HEK293, HEK293T, BHK, or CHO cell.

[0130] The promoters, expression cassettes, vectors, or viral particles of the present application can be transferred into a cell using any technique known in the art, for example, including but not limited to calcium phosphate-DNA precipitation, DEAE-Dextran transfection, electroporation, microinjection, biolistics, lipofection, or viral infection, and the promoters, expression cassettes, vectors, or viral particles of the present application can be maintained in the host cell in an episomal form or can be integrated into the genome. In a preferred embodiment, the promoters, expression cassettes, vectors, or viral particles of the present application are delivered into a host cell by viral infection, preferably using the viral particles of the present application, more preferably using the AAV particles of the present application.

[0131] Pharmaceutical composition

[0132] In yet another aspect of the present application, a pharmaceutical composition comprising the photoreceptor cell-specific bidirectional promoter, expression cassette, vector, viral particle, or cell of the present application, and a pharmaceutically acceptable excipient or carrier is provided. The pharmaceutical composition is used for treating or preventing an eye disease, preferably for treating or preventing a retinal disease, more preferably for treating or preventing a genetic retinal disease.

[0133] The term "pharmaceutical composition" as used herein refers to a preparation which contains an active compound or ingredient, i.e., the promoter, expression cassette, vector, viral particle, or cell of the present application, which is intended to prevent, maintain, or treat a tissue state or disease in a subject.

[0134] The term "pharmaceutically acceptable" as used herein means that the drug or agent is approved or recognized by a national regulatory agency (e.g., the National Medical Products Administration (NMPA) or the U.S. Food and Drug Administration (FDA)) or listed in a pharmacopoeia (e.g., the People's Republic of China Pharmacopoeia or the United States Pharmacopoeia) for use in animals, e.g., mammals, particularly humans. The term "excipient" refers to a natural or synthetic substance that is attached together with a pharmaceutically active ingredient in a drug and enhances the action of the pharmaceutically active ingredient, which is harmless to a subject at a concentration consistent with the effective activity of the pharmaceutically active ingredient, which includes, for example, buffers, disintegrants, binders, lubricants, fillers, plasticizers, surfactants, humectants, film-forming agents, and coating materials, as well as colorants, and the like. The term "carrier" herein refers to a substance that can deliver a pharmaceutically active ingredient to a desired compartment, e.g., a specific cell type (e.g., retinal cells, preferably photoreceptor cells), in a subject, and can be used to provide and control the release of the drug after administration by a selected route and regimen. "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" can also be used interchangeably in the present application.

[0135] In general, pharmaceutically acceptable excipients / carriers are relatively inert substances that facilitate the administration of the pharmaceutical active and can be provided as liquid solutions or suspensions, emulsions, or solid forms suitable for dissolution or suspension in a liquid prior to use. For example, the excipient / carrier can provide bulk or consistency, or act as a diluent. Suitable excipients / carriers include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for varying osmotic pressure, encapsulating agents, pH buffering substances, and buffers. Suitable excipients / carriers include any agent suitable for direct delivery to the eye, which can be administered without undue toxicity. Pharmaceutically acceptable excipients / carriers include, but are not limited to, water, petroleum, animal or vegetable oils, mineral oil, synthetic oil, sorbitol, Tween compounds, glycerol, saline solution, magnesium chloride solution, dextrose or other sugar solution, various alcohols such as glycols, e.g., ethylene glycol, propylene glycol, or polyethylene glycol, ethanol. Pharmaceutically acceptable excipients / carriers can also include pharmaceutically acceptable salts, such as mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, or the like, and salts of organic acids such as acetates, propionates, malonates, benzoates, or the like. A thorough discussion of pharmaceutically acceptable excipients / carriers is available, for example, in Remington's Pharmaceutical Sciences, 15thEdition. The selection and precise nature of the pharmaceutically acceptable excipients / carriers can be determined by one skilled in the art based on the particular route of administration.

[0136] Preferably, the pharmaceutical composition of the present application is formulated for administration to the eye, e.g., intraocular, intraocular, intravitreal, or subretinal, in particular by direct intraretinal, subretinal space, and / or intravitreal injection. For ocular delivery, the pharmaceutical composition is preferably in the form of an aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. A person skilled in the relevant art is able to prepare a suitable aqueous solution using, for example, isotonic carriers such as sodium chloride solution, Ringer's solution, lactated Ringer's solution, Hartmann's solution, and the like. If desired, the pharmaceutical composition of the present application can also contain a preservative, a stabilizer, a buffer, an antioxidant, and / or other additives. The pharmaceutical active ingredient can also be formulated in a preparation for slow release to achieve delayed release, or contained in microcapsules formed from biocompatible polymers or in a liposome carrier system.

[0137] The pharmaceutical composition of the present application can be packaged in unit dose or multiple dose forms, or in the form of a kit. The specific dose and dosage regimen of the pharmaceutical composition of the present application to be administered can be adjusted and determined by a physician depending on the purpose of use, the type and severity of the disease to be treated, the age, sex, body weight, health status, tolerance, and administration method of the subject, and the like.

[0138] In one embodiment, the pharmaceutical composition of the application comprises cells of the application, preferably human cells, i.e. cells transformed or transfected with an expression cassette, a vector or a viral particle of the application, preferably with an AAV particle. Optionally, the composition comprising cells can be stored frozen at any temperature suitable for storing cells. For example, the cells can be frozen at about -20°C, -80°C or any other suitable temperature. Cryofrozen cells can be stored in suitable containers and prepared for storage to reduce the risk of cell damage and maximize the likelihood of cell survival upon thawing. Alternatively, the cells can also be maintained at refrigerated room temperature, e.g. about 4°C.

[0139] In one embodiment, the pharmaceutical composition of the application comprises viral particles of the application and each unit dose comprises 10 8 to 10 13 viral particles, preferably 10 9 to 10 12 viral particles, more preferably 10 10 to 10 11 viral particles.

[0140] In one embodiment, the pharmaceutical composition of the application can further comprise one or several other active compounds such as corticosteroids, antibiotics, analgesics, immunosuppressants, trophic factors or any combination thereof.

[0141] The pharmaceutical composition of the application can also be used in combination with one or more other therapies for the treatment or prevention of an eye disease, preferably a retinal disease, more preferably a genetic retinal disease. In one embodiment, the eye disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration, Stargardt disease, Leber's hereditary optic neuropathy, cone / rod dystrophy (CD / CRD), Leber congenital amaurosis (LCA), diabetic retinopathy, retinal detachment, Best disease, choroideremia, tapetochoroidal dystrophy, achromatopsia (rod monochromacy), retinitis pigmentosa, night blindness, X-linked retinoschisis and Usher syndrome.

[0142] Method of treatment

[0143] In yet another aspect of the application, a method for the treatment or prevention of an eye disease is provided, the method comprising administering a photoreceptor cell-specific bi-directional promoter, an expression cassette, a vector, a viral particle, a cell or a pharmaceutical composition of the application to a subject in need thereof, preferably to the eye of a subject. In one embodiment, a photoreceptor cell-specific bi-directional promoter, an expression cassette, a vector, a viral particle, a cell or a pharmaceutical composition of the application is administered in a therapeutically or prophylactically effective amount to a subject in need thereof, e.g. in the eye.

[0144] As used herein, "therapeutically or prophylactically effective amount" means an amount of a promoter, expression cassette, vector, viral particle, cell or pharmaceutical composition of the application, which when administered to a subject, is capable of achieving the desired therapeutic or prophylactic effect, such as reducing, preventing, halting, slowing or eliminating the progression of a physical change associated with an eye disease (e.g. a retinal disease), or a symptom resulting therefrom. The "therapeutically or prophylactically effective amount" will generally vary depending on the gender, age, and general condition of the subject, the mode of administration, and the like, and can be determined in a routine manner by a person skilled in the art. In one embodiment, the subject is a mammal, preferably a human.

[0145] In one embodiment, the eye disease is a retinal disease. In one embodiment, the eye disease is an inherited retinal disease (IRD). In one embodiment, the eye disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration, Stargardt disease, Leber's hereditary optic neuropathy, cone / rod dystrophy (CD / CRD), Leber congenital amaurosis (LCA), diabetic retinopathy, retinal detachment, Best disease, choroideremia, tapetochoroidal dystrophy, achromatopsia (rod monochromacy), retinal pigmentary dystrophy, night blindness, X-linked retinoschisis, and Usher syndrome.

[0146] In one embodiment, the method of treatment of the application comprises administering a promoter, expression cassette, vector, viral particle, cell or pharmaceutical composition of the application to the eye of a subject, e.g. intraocularly, intraocularly, intravitreally or subretinally.

[0147] In one embodiment, the method of treatment of the application further comprises administering to the subject one or more other therapies, including administering one or more other therapeutic agents, gene therapy, immunotherapy, surgical procedures, and the like. For example, the therapeutic agent can be selected from the group consisting of a corticosteroid, an antibiotic, an analgesic, an immunosuppressant or a trophic factor, or any combination thereof.

[0148] In one embodiment, the promoter, expression cassette, vector, viral particle, cell or pharmaceutical composition of the application can be administered before or after the eye disease manifests symptoms, e.g. before or after partial or complete photoreceptor degeneration, and / or before or after partial or complete loss of vision.

[0149] Pharmaceutical uses

[0150] In yet another aspect of the application, there is provided the use of a photoreceptor cell-specific bidirectional promoter, expression cassette, vector, viral particle, cell or pharmaceutical composition of the application for the manufacture of a medicament for the treatment or prevention of an eye disease.

[0151] In one embodiment, the eye disease is a retinal disease. In one embodiment, the eye disease is an inherited retinal disease (IRD). In one embodiment, the eye disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration, Stargardt disease, Leber's hereditary optic neuropathy, cone / rod dystrophy (CD / CRD), Leber congenital amaurosis (LCA), diabetic retinopathy, retinal detachment, Best disease, choroideremia, tapetochoroidal dystrophy, achromatopsia (rod monochromacy), retinal pigmentary dystrophy, night blindness, X-linked retinoschisis, and Usher syndrome.

[0152] Expression methods

[0153] In yet another aspect of the application, a method for expressing a polypeptide or nucleic acid of interest in a cell is provided, comprising introducing the photoreceptor cell-specific bidirectional promoter, expression cassette, vector or viral particle of the application into a cell of interest. In one embodiment, the cell is a photoreceptor cell. In one embodiment, the cell is a cone cell. In one embodiment, the cell is a rod cell. In one embodiment, the cell is a retinal pigment epithelial cell (RPE).

[0154] The claimed technical solution of the application can be implemented using the specific description and sequences of the present application, in combination with techniques known to the person skilled in the art. Such techniques include, for example, routine cloning techniques in molecular biology and various experimental techniques commonly used in biochemistry, such as those described in textbooks, as well as any suitable method.

[0155] The following non-limiting examples further describe the application.

[0156] Example

[0157] Example 1 : Expression vector construction and promoter activity verification (mouse)

[0158] A photoreceptor cell-specific bidirectional promoter sequence was obtained by artificial recombination, and the promoter was named CA10 promoter (SEQ ID NO: 1). In order to verify that the CA10 promoter is a promoter that can bidirectionally drive the expression of downstream genes, two expression vectors were constructed based on the VB211226-1096bhj vector (internally constructed, see https: / / en.vectorbuilder.com / vector / VB211226-1096bhj.html, the plasmid map is shown in Figure 1). The CAG promoter in the VB211226-1096bhj vector was replaced with the CA10 promoter by conventional enzyme digestion and ligation method, and the expression vector pAAV[Exp]-{CA10}>NLS-EGFP:WPRE with CA10 promoter upstream and nuclear green fluorescent protein (NLS-EGFP) gene downstream was constructed. And the CAG promoter in the VB211226-1096bhj vector was replaced with the reverse complementary sequence of the CA10 promoter (re_CA10, SEQ ID NO: 6), and the expression vector pAAV[Exp]-{re_CA10}>NLS-EGFP:WPRE with re_CA10 promoter upstream and nuclear green fluorescent protein (NLS-EGFP) gene downstream was constructed.

[0159] According to the conventional virus packaging method, the above-mentioned expression vectors containing CA10 and re_CA10 promoters were mixed with the remaining two auxiliary vector plasmids (carrying Rep\Capsid gene and E2\E4\VA gene respectively) for AAV packaging, and then transfected into 293T cells for virus packaging. After harvesting the virus, cesium chloride purification was performed, and finally the virus particles (AAV8 type) for animal in vivo verification experiment were obtained.

[0160] The mice (C57BL / 6J, 6-8 weeks old, purchased from Guangdong Medical Laboratory Animal Center) were injected with a virus injection dose of 1E+10 GC / eye in the subretinal space, and the samples were taken 2 weeks after injection for sectioning and immunofluorescence staining: The primary antibody used in the immunofluorescence process is rabbit anti-mouse polyclonal anti-Arrestin C antibody (purchased from Sigma-Aldrich, catalog number AB15282), which can specifically bind to the surface protein of cone cells in the photoreceptor (PR) layer, thereby effectively distinguishing cone cells and rod cells. The secondary antibody used is goat anti-rabbit IgG with red fluorescence (purchased from Thermofisher, catalog number A-11012). DAPI was also used to stain the nuclei of cells in each layer, and DAPI imaging was blue.

[0161] The sections were photographed, and finally the expression section maps of CA10 and re_CA10 promoters were obtained, as shown in Figure 2.

[0162] As can be seen from FIG. 2, green fluorescence only appears in the photoreceptor cells, proving that the CA10 promoter, either in forward or reverse, placed upstream of the open reading frame of the fluorescent protein can drive the expression of the downstream protein, and the expression intensity is very strong. From the overall expression, the CA10 promoter only expresses in the photoreceptor cells, and no expression in other cells, with very high photoreceptor cell expression specificity.

[0163] Example 2: Verification of bidirectional expression activity of the promoter (mouse)

[0164] Based on the expression vectors pAAV[Exp]-{CA10}>NLS-EGFP:WPRE and pAAV[Exp]-{re_CA10}>NLS-EGFP:WPRE described in Example 1, an NLS_mCherry protein expression frame is reversely inserted upstream of the CA10 and re_CA10 promoters in the two vectors respectively by a conventional enzyme digestion and ligation method, to construct the corresponding expression vectors pAAV-SV40 late pA-NLS_mCherry <ca10>NLS_EGFP-BGH pA and expression vector pAAV-SV40 late pA-NLS_mCherry <re_CA10> NLS_EGFP-BGH pA, for observing the expression of CA10 and re_CA10 promoters simultaneously driving the expression of the two flanking fluorescent expression cassettes. Vector pAAV-SV40 late pA-NLS_mCherry <ca10>The vector framework of NLS_EGFP-BGH pA and the vector pAAV-SV40 late pA-NLS_mCherry<re_CA10>NLS_EGFP-BGH pA is shown in FIGS. 3 and 4, respectively.

[0165] Using the same method as in Example 1, virus packaging was performed to obtain high-purity virus particles (AAV8 type).

[0166] Mice (C57BL / 6J, 6-8 weeks old, purchased from Guangdong Medical Laboratory Animal Center) were injected subretinally at a virus injection dose of 1E+9 GC / eye, and samples were taken for sectioning one month after injection. The expression of red fluorescent protein and green fluorescent protein was observed using different channels of a fluorescence microscope, and the nuclei of cells in each layer were stained using DAPI, which was imaged as blue.

[0167] The sections were photographed, and the final CA10 and re_CA10 promoter bidirectional expression section images are shown in FIG. 5.

[0168] As can be seen from FIG. 5, the CA10 promoter can efficiently drive the expression of fluorescent proteins downstream of the positive strand and the negative strand at the same time, and the expression of fluorescent proteins is limited to photoreceptor cells, showing a high degree of photoreceptor cell specificity. Under the same experimental comparison conditions, the expression intensity of the CA10 promoter driving the downstream genes on both sides is basically the same.

[0169] Example 3: Verification of promoter activity (large gene)

[0170] Because the expression frame of the fluorescent protein is relatively short, there may be some differences with the expression of large genes, so lacZ gene is used as a substitute gene for large genes for experimental verification. Based on the VB231203-1125phu vector (internally constructed, see https: / / en.vectorbuilder.com / vector / VB231203-1125phu.html, as shown in FIG. 6), the CBh promoter was replaced with the CA10 promoter by conventional enzyme digestion and ligation method, and the expression vector pAAV[Exp]-CA10>LacZ_HA with the CA10 promoter upstream and the lacZ gene with a tag protein HA downstream was constructed.

[0171] Using the same method as in Example 1, virus packaging was performed to obtain high-purity virus particles (AAV8 type).

[0172] The mice (C57BL / 6J, 6-8 weeks old, purchased from Guangdong Medical Experimental Animal Center) were injected with a virus injection dose of 8E+9 GC / eye in the subretinal space, and the samples were taken for sectioning and immunofluorescence staining one month after injection: The primary antibody used in the immunofluorescence process is anti-HA tag antibody (purchased from thermofisher, catalog number 14-6756-81), which can specifically bind to the HA tag protein and can side verify whether the lacZ gene is normally transcribed and translated. The secondary antibody used is a red fluorescent goat anti-rabbit IgG (purchased from Thermofisher, catalog number A-11012). DAPI was also used to stain the nuclei of cells in each layer, and DAPI imaging is blue.

[0173] The slices were photographed, and finally the slice map of the CA10 promoter expressing large genes was obtained, as shown in FIG. 7.

[0174] As can be seen from FIG. 7, the CA10 promoter can normally drive the expression of lacZ gene and HA tag protein in photoreceptor cells, proving that the CA10 promoter has the ability to express large genes. Therefore, the promoter can be used to specifically express functional therapeutic proteins in photoreceptor cells.

[0175] Example 4: Construction of functional variants of CA10 promoter and verification of their activity

[0176] After confirming the bidirectional expression specificity of the CA10 promoter, a series of CA10 variant promoters were constructed by random mutation and deletion substitution of the CA10 promoter, including CA10V1 (SEQ ID NO: 2), CA10V2 (SEQ ID NO: 3), CA10V3 (SEQ ID NO: 4) and CA10V4 (SEQ ID NO: 5) promoters. The sequence identity between the sequences of the variant promoters and the sequence of the CA10 promoter is shown in the following table:

[0177] According to the method described in Example 2, a series of expression vectors with the variant promoters CA10V1, CA10V2, CA10V3 and CA10V4 in the middle, the nuclear red fluorescent protein (NLS-mCherry) gene upstream and the nuclear green fluorescent protein (NLS-EGFP) gene downstream were constructed. The promoter activity was verified in mice by virus packaging and purification, and finally the expression slice map of the promoter was obtained, as shown in FIG. 8.

[0178] As can be seen from FIG. 8, all the variant promoters CA10V1, CA10V2, CA10V3 and CA10V4 can bidirectionally drive the expression of positive and negative downstream genes in photoreceptor cells at high levels, with slightly different expression intensities, while maintaining the characteristics of photoreceptor cell-specific expression. Therefore, a series of photoreceptor cell-specific bidirectional promoters with different expression intensities are obtained. The reverse complementary sequences of the variant promoters CA10V1, CA10V2, CA10V3 and CA10V4 are shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0179] While the foregoing examples set forth preferred aspects of the application, each example is presented by way of explanation of the application and is not intended to limit the application. Based on the discussion and examples provided, one of ordinary skill in the art will be able to determine the essential characteristics of the aspects of the disclosure and, without departing from the spirit and scope of the disclosure, will be able to make various changes and modifications to the aspects to adapt them to various uses and conditions. Thus, from the foregoing description, one skilled in the art can effect various changes and modifications without departing from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited to the specific examples described and shown, but rather, the scope of the disclosure is to be determined entirely by the appended claims.

[0180] The sequence information related to the present application is as follows:

[0181] CA10 promoter sequence (347bp): SEQ ID NO: 1

[0182] CA10V1 promoter sequence (347bp): SEQ ID NO: 2

[0183] CA10V2 promoter sequence (347bp): SEQ ID NO: 3

[0184] CA10V3 promoter sequence (335bp): SEQ ID NO: 4

[0185] CA10V4 promoter sequence (333bp): SEQ ID NO: 5

[0186] re_CA10 promoter sequence (347bp): SEQ ID NO: 6

[0187] re_CA10V1 promoter sequence (347bp): SEQ ID NO: 7

[0188] re_CA10V2 promoter sequence (347bp): SEQ ID NO: 8

[0189] re_CA10V3 promoter sequence (335bp): SEQ ID NO: 9

[0190] re_CA10V4 promoter sequence (333bp): SEQ ID NO: 10

Claims

1. A bidirectional promoter having photoreceptor cell-specific promoter activity.

2. The bidirectional promoter of claim 1, wherein the bidirectional promoter comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably, the bidirectional promoter comprises a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably, the bidirectional promoter comprises a nucleotide sequence as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

3. The bidirectional promoter of claim 1, wherein the bidirectional promoter consists of a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably, the bidirectional promoter consists of a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably, the bidirectional promoter consists of a nucleotide sequence as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

4. An expression cassette, wherein the expression cassette comprises the bidirectional promoter of any one of claims 1 to 3.

5. The expression cassette of claim 4, wherein the expression cassette further comprises a nucleic acid of interest operably linked to the bidirectional promoter; wherein the nucleic acid of interest encodes a therapeutic protein, an optogenetic driver protein, or a reporter protein, or the nucleic acid of interest encodes a nucleic acid selected from the group consisting of siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, and deoxyribozyme.

6. A vector, wherein the vector comprises the bidirectional promoter of any one of claims 1 to 3 or the expression cassette of claim 4 or 5.

7. The vector of claim 6, which is a viral vector.

8. The vector of claim 7, wherein the viral vector is an adeno-associated virus (AAV) vector, a retroviral vector, or a parvovirus vector; optionally, the viral vector is a MoMLV vector, a MSCV vector, a SFFV vector, a MPSV vector, a SNV vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a monkey virus 40 vector, a bovine papilloma virus vector, an Epstein-Barr virus vector, a herpes virus vector, a vaccinia virus vector, a Harvey murine sarcoma virus vector, a murine mammary tumor virus vector, an anellovirus vector, or a Rous sarcoma virus vector.

9. A viral particle, wherein the viral particle comprises the vector of any one of claims 6 to 8; optionally, the vector is an AAV vector, preferably, wherein the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, and AAVdj, more preferably selected from the group consisting of AAV-2, AAV-5, AAV2-7m8, AAV-9, and AAV-8, more preferably AAV-2, AAV2-7m8, or AAV-8.

10. A cell, wherein the cell comprises the bidirectional promoter of any one of claims 1 to 3, the expression cassette of claim 4 or 5, the vector of any one of claims 6 to 8, or the viral particle of claim 9.

11. The cell of claim 10, wherein the cell is a photoreceptor cell, a cone cell, a rod cell, a retinal pigment epithelial cell (RPE), a HEK293, a HEK293T, a BHK, or a CHO cell.

12. A pharmaceutical composition, wherein the pharmaceutical composition comprises the bidirectional promoter of any one of claims 1 to 3, the expression cassette of claim 4 or 5, the vector of any one of claims 6 to 8, the viral particle of claim 9, or the cell of claim 10 or 11, and a pharmaceutically acceptable excipient or carrier.

13. Use of the bidirectional promoter of any one of claims 1 to 3, the expression cassette of claim 4 or 5, the vector of any one of claims 6 to 8, the viral particle of claim 9, the cell of claim 10 or 11, or the pharmaceutical composition of claim 12, in the manufacture of a medicament for the treatment or prevention of an eye disease.

14. The use of claim 13, wherein the eye disease is a hereditary retinal disease.

15. The use of claim 13, wherein the eye disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration, Stargardt’s disease, Leber’s hereditary optic neuropathy, cone / rod dystrophy (CD / CRD), Leber congenital amaurosis (LCA), diabetic retinopathy, retinal detachment, Best’s disease, choroideremia, tapetochoroidal dystrophy, achromatopsia (rod monochromacy), retinal pigmentary dystrophy, night blindness, X-linked retinoschisis, and Usher syndrome.

16. A method for expressing a polypeptide or nucleic acid of interest in a cell, wherein the method comprises introducing into the cell the bidirectional promoter of any one of claims 1 to 3, the expression cassette of claim 4 or 5, the vector of any one of claims 6 to 8, or the viral particle of claim 9.

17. The method of claim 16, wherein the cell is selected from a photoreceptor cell, a cone cell, a rod cell, or a retinal pigment epithelial cell (RPE).

18. A method for treating or preventing an eye disease, the method comprising administering to a subject in need thereof, preferably to the eye of the subject, the bidirectional promoter of any one of claims 1 to 3, the expression cassette of claim 4 or 5, the vector of any one of claims 6 to 8, the viral particle of claim 9, the cell of claim 10 or 11, or the pharmaceutical composition of claim 12.

19. The method of claim 18, wherein the eye disease is a genetic retinal disease.

20. The method of claim 18, wherein the eye disease is selected from retinitis pigmentosa (RP), age-related macular degeneration, Stargardt disease, Leber’s hereditary optic neuropathy, cone / rod dystrophy (CD / CRD), Leber congenital amaurosis (LCA), diabetic retinopathy, retinal detachment, Best’s disease, choroideremia, tapetochoroidal dystrophy, achromatopsia (rod monochromacy), retinal pigmentary changes, night blindness, X-linked retinoschisis, and Usher syndrome.

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