Application of multiple promoters capable of efficiently targeting on ON-type bipolar cells in gene therapy technology

Abstract

The invention relates to the technical field of biology and the technical field of gene therapy. The invention relates to a retina light-donating bipolar cell specific promoter sequence and application thereof to regulation and control of expression of therapeutic genes in retina or retina organoid cells in a gene therapy technology so as to improve and / or recover vision. The retina bipolar cells are divided into light-supplying activated bipolar cells (ON type bipolar cells or depolarized bipolar cells) and light-removing activated bipolar cells (OFF type bipolar cells or hyperpolarized bipolar cells), and accurate and specific expression of transgenosis is realized by utilizing gene differential expression characteristics of different types of bipolar cells. The efficient and accurate treatment on the retina diseases is realized. The promoter has the advantage that the promoter of the transient receptor potential cation channel protein (TRPM1) gene with high-level and high-specificity expression can be realized in light-activated bipolar cells.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to the application of multiple enhancer or promoter elements capable of regulating the specific expression of genes in retinal bipolar cells in human ophthalmology retinal diseases, retinal macular region-related diseases, especially retinitis pigmentosa, to drive the expression of therapeutic transgenes in retinal bipolar cells to achieve the purpose of restoring and / or improving vision. Background Art

[0002] Inherited retinal diseases (IRDs) have an incidence of 1 in 5,000 to 1 in 3,000 individuals and are currently one of the most common causes of blindness (PMID: 36362249). Retinal diseases such as retinitis pigmentosa (RP), Stargardt's disease, and age-related macular degeneration (AMD) are characterized by progressive photoreceptor cell death, leading to partial or complete blindness (PMID: 33686777). Traditional treatments for RP, Stargardt's disease, and AMD have limited efficacy and do not alter the photoreceptor cell death and eventual blindness. Gene therapy involves the delivery of therapeutic endogenous or exogenous DNA or RNA to replace or silence defective genes, aiming to slow disease progression, alleviate symptoms, or restore lost function. In the retinas of patients with RP, Stargardt's disease, and AMD, despite photoreceptor cell death, the remaining retinal structures can maintain structural and functional integrity for a limited period of time. Optogenetics has been shown to effectively improve vision in RP model mice by expressing light-sensitive proteins in residual retinal cells, causing cells that are not sensitive to light to respond to light. Some published clinical trials have also shown that optogenetics can improve vision in RP patients. For example, an ongoing optogenetic therapy clinical trial (NCT04945772) uses multi-characteristic opsin (vMCO) to specifically express on retinal light-sensitive bipolar cells (On-BCs) to restore light sensitivity in the retina. Phase 2b results of this clinical trial indicate that this therapy can improve patient vision to a certain extent, revealing that the strategy of expressing light-sensitive proteins on the retina to improve vision in patients with retinitis pigmentosa is feasible under certain conditions in clinical practice.

[0003] Retinal bipolar cells are divided into light-activated bipolar cells (ON bipolar, ON-BC) and light-off bipolar cells (OFF bipolar, Off-BC). In the normal retina, upon exposure to light, the photoreceptor cells connected to the presynaptic terminals of ON-BCs reduce glutamate release, uninhibiting the mGluR6-TRPM1 channels previously inhibited by glutamate and allowing cation influx, resulting in a depolarizing response and activation of the ON-BCs. Meanwhile, Off-BCs are activated by light exposure due to the reduced glutamate signaling from photoreceptor cells, which closes the cation channels and produces a hyperpolarizing response. Therefore, gene therapy techniques that specifically target depolarizing light-sensitive proteins in light-activated bipolar cells hold great promise for improving visual acuity in patients with retinitis pigmentosa. The ON-BC promoter that has been frequently used in previous research reports is an approximately 200-bp enhancer sequence upstream of the mouse GRM6 gene. The artificial promoter composed of this sequence and the SV40 mini promoter can be specifically expressed in bipolar cells. However, this promoter was later shown to have reduced activity in the retina of rd1 model mice, affecting its clinical application in disease treatment.

[0004] Therefore, there is an urgent need to provide a bipolar cell-specific expression regulatory element that is different from the GRM6 gene promoter to exert its effect in treating inherited retinal diseases. Summary of the Invention

[0005] The purpose of this application is to improve the targeting specificity of existing ophthalmic gene therapy technologies, to provide a variety of novel hTRPM1 series promoters that have high specificity and high expression levels for On-type bipolar cells, and the use of said promoters in the preparation of therapeutic drugs for retinal degenerative diseases. The innovation of this application: Unlike most existing technologies that are based on the modification of promoters or regulatory sequences of the GRM6 gene, this application is based on a novel promoter or DNA regulatory sequence of the TRPM1 gene, which is a promoter composed of a completely new, unreported DNA sequence. The promoter or DNA regulatory sequence involved in this application has expression specificity and sufficient expression activity in retinal ON-type bipolar cells, can be efficiently expressed in human retinal organoids, and has the potential to be used to treat human ophthalmic retinal diseases, especially retinal macular diseases.

[0006] The purpose of this application is to provide a variety of new artificially synthesized ON-type bipolar cell-specific promoters.

[0007] To achieve the above objectives, this application adopts the following technical solutions:

[0008] Item 1. An enhancer or enhancer variant, wherein the enhancer comprises the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:6, SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:39, or is truncated from the 5'-end or 3'-end of the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:6, SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:39 to a length of 40-910 base pairs, preferably truncated to a length of 56-880 base pairs;

[0009] wherein the enhancer variant is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the enhancer.

[0010] Item 2. An enhancer or enhancer variant according to Item 1, wherein the enhancer comprises the nucleotide sequence shown in any one of SEQ ID NOs: 4-14 and 35-39, or a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0011] Item 3. A promoter or promoter variant, wherein the promoter comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, or is truncated from the 5'-end or 3'-end of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33 to a length of 500-800 base pairs, preferably truncated to a length of 550-750 base pairs;

[0012] wherein the promoter variant is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the promoter.

[0013] Item 4. A promoter or promoter variant according to Item 3, wherein the promoter comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1-3 and 33-34, or a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0014] Item 5. An isolated nucleic acid molecule comprising an enhancer and a promoter, wherein:

[0015] The enhancer is an enhancer as described in item 1 or 2; and / or

[0016] The promoter is the promoter described in item 3 or 4.

[0017] Item 6. An isolated nucleic acid molecule according to Item 5, wherein the isolated nucleic acid molecule consists of one or more of the enhancers or enhancer variants, one or more of the promoters or promoter variants, and optionally a spacer separating the enhancer or enhancer variant from the promoter or promoter variant.

[0018] Item 7. An isolated nucleic acid molecule according to Item 6, wherein the isolated nucleic acid molecule comprises or consists of a sequence selected from SEQ ID NOs: 15-31, 40-42, or comprises or consists of a sequence having at least 60% identity with a sequence selected from SEQ ID NOs: 15-31, 40-42.

[0019] Item 8. A nucleic acid expression vector comprising the isolated nucleic acid molecule according to any one of Items 5 to 7.

[0020] Item 9. The nucleic acid expression vector according to Item 8, wherein the nucleic acid expression vector is a recombinant adeno-associated vector (AAV), in particular wherein the nucleic acid expression vector is a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12 vector or a derivative variant of the above recombinant vector.

[0021] Item 10. The nucleic acid expression vector according to any one of Items 8 to 9, further comprising:

[0022] a. A transgene, wherein the transgene is a light-sensitive protein gene, in particular a light-sensitive protein gene as shown in SEQ ID NO: 46 or 47 or comprises a light-sensitive protein gene as shown in SEQ ID NO: 46 or 47, and.

[0023] b. Sequence encoding capsid protein.

[0024] Item 11. An adeno-associated virus particle, or a virus-like particle, or a non-viral particle, comprising the isolated nucleic acid molecule according to any one of Items 5-7 or the nucleic acid expression vector according to any one of Items 8-10.

[0025] Item 12. An agent selected from the isolated nucleic acid molecule according to any one of Items 5 to 7 or the nucleic acid expression vector according to any one of Items 8 to 10 and the adeno-associated virus particle, or virus-like particle, or non-viral particle according to Item 11 and used as a drug.

[0026] Item 13. An agent selected from the isolated nucleic acid molecule according to any one of Items 5-7, the nucleic acid expression vector according to any one of Items 8-10 and the adeno-associated virus particle, or virus-like particle, or non-viral particle according to Item 11 and used to treat congenital stationary night blindness (CSBN1) or rod-cone and cone-rod dystrophy, more particularly for the treatment of retinitis pigmentosa, Stargardt and age-related macular degeneration, or other diseases that achieve therapeutic effects by expressing therapeutic genes in bipolar cells.

[0027] Item 14. A medicament, wherein the medicament is selected from the isolated nucleic acid molecule according to any one of Items 5 to 7 or the nucleic acid expression vector according to any one of Items 8 to 10 and the adeno-associated virus particle, virus-like particle, or non-viral particle according to Item 11, wherein the medicament is administered by:

[0028] a. Intravitreal injection,

[0029] b. Subretinal injection,

[0030] c. Choroidal injection, or

[0031] d. Administer eye drops. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the chromatin localization of enhancer and promoter elements of the human hTRPM1 gene selected for promoter design in Example 1. (A) PhastCons 100way is a conserved peak plot across 100 vertebrate genomes. FetalRetina-125D-DNase is a sequencing peak plot of the fetal retinal genome after treatment with DNase I at 125 days of development. Sites with high peaks represent high chromatin accessibility, i.e., strong genomic activity. The gray boxes show that four selected enhancer regions (En1, En2, En3, En4) and one core promoter region (P) have strong chromatin accessibility. All four enhancers are located in the first intron region of the hTRPM1 gene described in Example 1. (BF) Localization of the (B) En1, (C) En2, (D) En3, (E) En4, and (F) P core promoters and their respective truncated sequences in Figure A on human chromosome 15. The light gray boxes show ChIP-seq enriched regions for transcription factors. Dark grey boxes indicate the positions of truncated sequences, and numbers indicate chromatin position numbers.

[0033] Figure 2Diagram showing the design of promoter testing vector components. (A) Diagram showing the enhancer in the promoter vector linked 5' to the core promoter and the GFP-NanoLuc fusion reporter gene linked 3' to the core promoter. (B) Diagram showing the promoter testing combination scheme (see Example 2 for details), promoter ID, and fold change in promoter strength normalized to the NanoLuc expression intensity of the pAAV-P750(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA vector.

[0034] Figure 3 is a schematic diagram of the promoter test vectors used in Examples 1, 2, 3, and 4. The novel En4_400-P750(TRPM1) (A) and En2_460-En4_160-P750(TRPM1) (B) promoters are inserted 5' of GFP and NanoLuc fusion reporter genes. The Kozak sequence is a translation initiation regulatory sequence, while WPRE and hGHpA (i.e., hGH poly(A)) are transcriptional regulatory sequences. The 5' ITR and 3' ITR sequences mediate transgene packaging within the AAV capsid.

[0035] Figure 4 The following is an SDS-PAGE silver staining image of the promoter test virus in Examples 2, 3, and 4. The image shows obvious VP1, VP2, and VP3 capsid protein bands of the AAV virus, indicating high virus purity.

[0036] Figure 5 This is an immunofluorescence staining diagram of the mouse retina 3 weeks after the AAV virus containing different promoters in Example 3 was injected into the vitreous cavity. The figure shows the expression of green fluorescent protein mediated by P550 (hTRPM1), P750 (hTRPM1), P1061 (hTRPM1), En4_622-P550 (hTRPM1), En4_400-P750 (hTRPM1), En2_460-En4_160-P750 (hTRPM1), En4_622-P1061 (hTRPM1) and hGRM6 (1243) promoters in the inner nuclear layer (INL) of the retina. The green fluorescence displayed by the green fluorescence channel (GFP channel) indicates the expression of green fluorescent protein (GFP) mediated by the promoter, the red fluorescence displayed by the PKCα channel is the rod bipolar cells recognized by the PKCα antibody, the blue fluorescence displayed by the DAPI channel is the nucleus staining labeled by DAPI, and Merge is the overlap of the fluorescence images displayed by the above three channels. Bar=50μm.

[0037] Figure 6Fluorescence intensity of hTRPM1 promoters expressed in (A) the retina and (B) ON bipolar cells. (A) Fluorescence signal intensity after background correction. (B) Percentage of cells co-labeled with GFP and PKCα compared to PKCα-labeled cells, indicating the promoter's specificity in ON bipolar cells.

[0038] Figure 7 Immunofluorescence staining of bipolar cells in human retinal organoids infected with AAV containing the hTRPM1 promoter. The green fluorescence (GFP) channel indicates promoter-mediated GFP protein expression. The red fluorescence (PKCα) channel indicates rod bipolar cells recognized by the PKCα antibody. The blue fluorescence (DAPI) channel indicates DAPI-labeled nuclei. Merge represents the overlap of the fluorescence images displayed by the three channels. Bar = 50 μm.

[0039] Figure 8 Visual acuity analysis of rd10 mice after treatment with an AAV gene therapy containing a promoter-light-sensitive protein. The exemplary En4_622-P550(hTRPM1)-PsCatch2.0 and En4_400-P750(hTRPM1)-PsCatch2.0 significantly improved the visual acuity of rd10 mice. DETAILED DESCRIPTION

[0040] The following describes the specific embodiments of the present invention in detail, but it should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods in the following examples where specific experimental conditions are not specified are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0041] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0042] the term

[0043] In the context of this specification, the term "AAV capsid" refers to a synthetic capsid (cap) gene. The AAV capsid disclosed herein is a recombinant adeno-associated virus that can be used to package for gene therapy.

[0044] In the context of this specification, the term "transgenic" refers to a gene or genetic material that has been transferred from one organism to another. In the context of this article, the term can also refer to the transfer of a natural or physiologically intact variant of a gene sequence into a patient tissue in which it is absent. It can also refer to the transfer of a native coding sequence whose expression is driven by a promoter that is absent or silent in the target tissue. The term transgenic as used herein refers to a polynucleotide encoding a polypeptide of interest that, when expressed in a damaged or diseased retina, can be used to improve or restore vision. Transgenics of particular interest for restoring light sensitivity or vision include light-sensitive proteins, such as opsin genes, i.e., channelrhodopsins, vertebrate opsins, and variants thereof.

[0045] In the context of this specification, the term "intravitreal administration" or "vitreous injection" refers to a route of administration of a pharmaceutical agent (e.g., a virus) by which the agent is delivered to the vitreous body of the eye. Intravitreal administration (or intravitreal injection) is a procedure in which a drug is placed directly into the space at the back of the eye, called the vitreous cavity, which is filled with a jelly-like fluid called vitreous hydrogel.

[0046] In the context of this specification, the term "subretinal administration" or "subretinal injection" relates to a route of administering an agent, particularly a virus in the context of this specification, into the space between bipolar cells, photoreceptors and retinal pigment epithelial cells.

[0047] In the context of this specification, a broad "promoter" is a nucleic acid sequence that can regulate the transcription level of a gene. The term promoter in this context includes the core promoter sequence adjacent to the gene translation start site and also includes enhancer sequences.

[0048] In the context of this specification, an "enhancer" is a nucleic acid, a type of non-coding DNA cis-acting element, which can be located upstream, downstream or intronic regions of a gene and has the function of regulating gene transcription levels.

[0049] In a first aspect, the present application provides an enhancer or enhancer variant, wherein the enhancer comprises the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:6, SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:39, or is truncated from the 5'-end or 3'-end of the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:6, SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:39 to a length of 40-910 base pairs, such as 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 910 base pairs, etc., preferably truncated to 56-880 base pairs in length, such as 56, 57, 58, 59, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820, 840, 860, 880 base pairs, or any base pair length between 56-880 base pairs.

[0050] In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO: 4 or is truncated from the 5'-end or 3'-end of the nucleotide sequence of SEQ ID NO: 4 to a length of 40-910 base pairs, for example, 40, 50, 52, 54, 56, 58, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820, 840, 850, 860, 870, 880, 900, 910 base pairs. 80, 860, 880 base pairs, or any base pair length between 56 and 880 base pairs.

[0051] In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO: 35 or is truncated from the 5'-end or 3'-end of the nucleotide sequence of SEQ ID NO: 35 to a length of 40-910 base pairs, for example, 40, 50, 52, 54, 56, 58, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820, 840, 850, 860, 870, 880, 900, 910 bases. 80, 860, 880 base pairs, or any base pair length between 56 and 880 base pairs.

[0052] In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO: 6 or is truncated from the 5'-end or 3'-end of the nucleotide sequence of SEQ ID NO: 6 to a length of 40-910 base pairs, for example, 40, 50, 52, 54, 56, 58, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820, 840, 850, 860, 870, 880, 900, 910 base pairs. 80, 860, 880 base pairs, or any base pair length between 56 and 880 base pairs.

[0053] In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO: 37 or is truncated from the 5'-end or 3'-end of the nucleotide sequence of SEQ ID NO: 37 to a length of 40-910 base pairs, for example, 40, 50, 52, 54, 56, 58, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820, 840, 850, 860, 870, 880, 900, 910 bases. 80, 860, 880 base pairs, or any base pair length between 56 and 880 base pairs.

[0054] In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO: 9 or is truncated from the 5'-end or 3'-end of the nucleotide sequence of SEQ ID NO: 9 to a length of 40-910 base pairs, for example, 40, 50, 52, 54, 56, 58, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820, 840, 850, 860, 870, 880, 900, 910 base pairs. 80, 860, 880 base pairs, or any base pair length between 56 and 880 base pairs.

[0055] In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO: 10 or is truncated from the 5'-end or 3'-end of the nucleotide sequence of SEQ ID NO: 10 to a length of 40-910 base pairs, for example, 40, 50, 52, 54, 56, 58, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820, 840, 850, 860, 870, 880, 900, 910 bases. 80, 860, 880 base pairs, or any base pair length between 56 and 880 base pairs.

[0056] In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO: 39 or is truncated from the 5'-end or 3'-end of the nucleotide sequence of SEQ ID NO: 39 to a length of 40-910 base pairs, for example, 40, 50, 52, 54, 56, 58, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 820, 840, 850, 860, 870, 880, 900, 910 bases. 80, 860, 880 base pairs, or any base pair length between 56 and 880 base pairs.

[0057] In some embodiments, enhancer variants and their reverse complements are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the enhancer, and preferably 99% identical.

[0058] In some embodiments, the truncated enhancer variants and their reverse complements are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the truncated enhancer, and preferably 99% identical.

[0059] In some embodiments, the enhancer comprises the nucleotide sequence of any one of SEQ ID NOs: 4-14 and 35-39, or its reverse complement, or a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto, preferably a nucleotide sequence having at least 99% sequence identity thereto.

[0060] A second aspect of the present application provides a promoter or promoter variant, wherein the promoter comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, or is truncated from the 5'-end or 3'-end of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33 to a length of 500-800 base pairs, such as 500, 550, 600, 650, 700, 750, 800, preferably truncated to a length of 550-750 base pairs, such as 550, 600, 650, 700, 750, or any base pair length between 550-750 base pairs;

[0061] In some embodiments, the promoter comprises the nucleotide sequence shown in SEQ ID NO: 1 or is truncated from the 5'-end or 3'-end of the nucleotide sequence shown in SEQ ID NO: 1 to a length of 500-800 base pairs, for example, 500, 550, 600, 650, 700, 750, 800, preferably truncated to a length of 550-750 base pairs, for example, 550, 600, 650, 700, 750, or any base pair length between 550-750 base pairs.

[0062] In some embodiments, the promoter comprises the nucleotide sequence shown in SEQ ID NO:33 or is truncated to a length of 500-800 base pairs, for example, 500, 550, 600, 650, 700, 750, 800, from the 5'-end or 3'-end of the nucleotide sequence shown in SEQ ID NO:33, preferably truncated to a length of 550-750 base pairs, for example, 550, 600, 650, 700, 750, or any base pair length between 550-750 base pairs.

[0063] In some embodiments, the promoter variants and their reverse complements are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the promoter, preferably 99% identical.

[0064] In some embodiments, the truncated promoter variants and their reverse complements are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the truncated promoter, preferably 99% identical.

[0065] In some embodiments, the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 1-3 and 33-34, or a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto, preferably a nucleotide sequence having at least 99% sequence identity thereto.

[0066] In some embodiments, the enhancer is located upstream, downstream, or anywhere else on the vector relative to the promoter.

[0067] The third aspect of the present application provides an isolated nucleic acid molecule comprising an enhancer and a promoter, wherein the enhancer is the enhancer described above; and / or the promoter is the promoter described above.

[0068] In some embodiments, the isolated nucleic acid molecule consists of one or more enhancers or enhancer variants, one or more promoters or promoter variants, and optionally a spacer separating the enhancer or enhancer variant from the promoter or promoter variant.

[0069] In some embodiments, the isolated nucleic acid molecule further comprises a spacer sequence having a length of 1 to 1000 base pairs, particularly 1 to 394 base pairs. In some embodiments, the spacer is located between the enhancer and the promoter. In some embodiments, the isolated nucleic acid molecule further comprises a spacer sequence having a length of 1 to 1000 base pairs, particularly 1 to 394 base pairs, and the spacer is located between the enhancer and the promoter.

[0070] In some embodiments, the isolated nucleic acid molecule comprises or consists of a sequence selected from SEQ ID NOs: 15-31, 38-40. In some embodiments, the isolated nucleic acid molecule comprises or consists of a sequence that is at least 60%, e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to a sequence selected from SEQ ID NOs: 15-31, 38-40.

[0071] The fourth aspect of the present application provides a nucleic acid expression vector comprising the isolated nucleic acid molecule described above.

[0072] In some embodiments, the nucleic acid expression vector is a recombinant adeno-associated vector (AAV).

[0073] In some embodiments, the nucleic acid expression vector is a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12 vector or a derivative variant thereof. Preferably, the vector is a recombinant AAV2 vector.

[0074] In some embodiments, the nucleic acid expression vector further comprises:

[0075] a transgenic, wherein the transgenic is a light-sensitive protein gene, in particular a light-sensitive protein gene as shown in SEQ ID NO: 46 or 47 or comprises a light-sensitive protein gene as shown in SEQ ID NO: 46 or 47, and

[0076] b. Sequence encoding capsid protein.

[0077] From the 5' end to the 3' end, the isolated nucleic acid molecule comprises: first, the enhancer, followed by an optional spacer, and then the promoter. The transgene is located 3' to the promoter. In some embodiments, the transgene is preceded by an optimized Kozak sequence.

[0078] In some embodiments, the transgene is a photosensitive protein gene or an opsin gene that restores light detection or vision.

[0079] In some embodiments, the light-sensitive protein or opsin is selected from the group consisting of channelrhodopsin, melanopsin, rhodopsin, cone opsin, pinealopsin, photopsin, halorhodopsin, bacteriorhodopsin, proteorhodopsin, aequorin, jumping spider opsin, or any functional variant or fragment thereof.

[0080] In some embodiments, the opsin is a chimeric protein between opsin and the metabotropic glutamate receptor mGluR6 of the retinal OBC. Preferably, the chimeric protein is Opto-mGluR6.

[0081] In some embodiments, the nucleic acid expression vector further comprises a WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) regulatory sequence. WPRE is a DNA sequence that generates a tertiary structure that enhances expression when transcribed. In some embodiments, the nucleic acid expression vector further comprises a polyadenylic acid tail inserted downstream of the transgene. The polyadenylic acid tail promotes translation of the transgene.

[0082] The fifth aspect of the present application provides an adeno-associated virus particle, or a virus-like particle, or a non-viral particle, comprising the isolated nucleic acid molecule according to the third aspect or the nucleic acid expression vector according to the fourth aspect.

[0083] The sixth aspect of the present application provides an agent selected from the isolated nucleic acid molecule according to the third aspect or the nucleic acid expression vector according to the fourth aspect and the adeno-associated virus particle, or virus-like particle, or non-viral particle according to the fifth aspect and used as a drug.

[0084] Another aspect relates to an agent selected from the group consisting of an isolated nucleic acid molecule according to the third aspect, a nucleic acid expression vector according to the fourth aspect, and an adeno-associated virus particle, or a virus-like particle, or a non-viral particle according to the fifth aspect and for use in treating congenital stationary night blindness or rod-cone and cone-rod dystrophy, in particular retinitis pigmentosa, Stargardt, or macular degeneration.

[0085] Another aspect relates to an agent selected from the group consisting of the isolated nucleic acid molecule according to the third aspect, the nucleic acid expression vector according to the fourth aspect, and the adeno-associated virus particle, or virus-like particle, or non-viral particle according to the fifth aspect, wherein the agent is administered by:

[0086] a. Intravitreal administration, in particular intravitreal injection,

[0087] b. Subretinal administration, especially subretinal injection,

[0088] c. Choroidal injection, or

[0089] d. Administer eye drops.

[0090] Another aspect relates to a method of treatment comprising administering the agents of the present application to a patient in need thereof.

[0091] Example

[0092] 1. Materials and Methods

[0093] 1.1 Intravitreal injection

[0094] Mice were anesthetized with an isoflurane gas anesthesia system (Ruiwode Life Science Co., Ltd.), and then intravitreal injection was performed. After sufficient anesthesia, the ocular surface and the skin around the orbit were disinfected with 0.5% active iodine. In order to reduce the discomfort caused by intravitreal injection to mice, the mouse eyeballs were anesthetized with proparacaine hydrochloride eye drops (Alcaine). The mice were fixed and the eyeballs were exposed. A microsyringe was used with a 32G insulin needle to draw 1.5 μL of rAAV virus (titer of 1E+13 vg / mL), and the needle was inserted 0.5 mm below the corneoscleral limbus on the nasal side of the mouse to complete the intravitreal injection. The intravitreal injection of the other eye was completed in the same way, and a control group with only the injection preparation was set up. After the injection, levofloxacin hydrochloride eye gel (Jeqi) was applied to the mouse eyeball to prevent infection.

[0095] 1.2 NanoLuc luciferase activity assay

[0096] One month after intravitreal injection, mice were killed by cervical dislocation, and the eyeballs were removed and the retinas were dissected using ophthalmic scissors in pre-cooled PBS. As described in the Luciferase Assay kit (Promega, N1110), the retina was transferred to a lysis buffer and ultrasonicated. After sonication for 1 minute, the reaction was incubated at room temperature for 10 minutes and then centrifuged to remove the precipitate. The reaction substrate was prepared according to the instructions and mixed with the retinal lysis buffer. After incubation at room temperature for 5 minutes, luciferase activity was measured using a multi-function microplate reader (Tecan Spark).

[0097] 1.3 Immunofluorescence staining

[0098] Mouse eyeballs or retinal organoids were removed and fixed in 4% paraformaldehyde at room temperature for 40 minutes. Retinal tissue was placed in a 30% sucrose solution and dehydrated overnight in a 4°C refrigerator. After embedding in OCT gel, the retinas were sectioned into 14 μm thick sections using a frozen tissue slicer (Leica, Germany, model CM 1950) for immunofluorescence staining. The cut retinas were blocked with 4% BSAT solution for 2 hours at room temperature, incubated with primary antibodies overnight in a 4°C refrigerator, and incubated with secondary antibodies of the corresponding species at room temperature for 2 hours. DAPI (Sevier Biotechnology, Cat. No. G1012) was incubated at room temperature for 5 minutes, followed by anti-fluorescence quenching mounting medium (Sevier Biotechnology, Cat. No. G1401). Primary antibodies against GFP (chicken, diluted 1:2000 in retinal sections, Abacm, USA, Catalog No. ab13970) and PKCα (a rod bipolar cell marker, goat, diluted 1:500, Santa Cruz Biotechnology, Catalog No. SC-208-G) were used. Secondary antibodies included Alexa 594 (diluted 1:500, Jackson ImmunoResearch, USA, Catalog No. 705-585-147) and Alexa 488 (diluted 1:500, Jackson ImmunoResearch, USA, Catalog No. 715-545-150). Whole-mount images of the retinas were captured using a Leica Thunder slide scanner and a Zeiss laser confocal microscope (LSM880). Cell counts were analyzed using Cellpose 2.0 and Cellprofiler software.

[0099] 1.4 AAV infection of human retinal organoids

[0100] 300 μl of (DMEM+N2+FBS) medium was added to a 24-well low-adhesion cell culture plate, and the virus was added at a titer of 1E+11 vg / organoid. After mixing, human retinal organoids differentiated for 200 days were added. The organoid and virus mixture was incubated in a 37°C cell culture incubator for 2 hours. Each dish was then supplemented with 700 μl of (DMEM+N2+FBS) medium and mixed, and co-cultured in a 37°C cell culture incubator. After 2 days of co-culture, the cells were washed twice with culture medium and the medium was completely replaced to remove all excess viral particles. The transduced organoids were cultured in (DMEM+N2+FBS) medium until 10 days after transduction, when samples were collected for observation. Retinal organoids were induced from the H9 human embryonic stem cell line (WiCell, USA).

[0101] Example 1 Design of hTRPM1 promoter

[0102] The applicant analyzed the single-cell RNA sequencing data published in the literature (PMID: 37388908; PMID: 27565351; PMID: 32555229) to check the expression pattern of TRPM1. TRPM1 is expressed at a high abundance in ON-BCs of the human retina, and no significant expression is seen in other retinal cells such as photoreceptor cells, ganglion cells, horizontal sacral cells, and vertical cells, indicating that the TRPM1 promoter has great potential for mining ON-BC-specific promoters. According to the literature information (PMID: 32555229), the hTRPM1 transcript with Ensembl gene ID ENST00000542188.5 (NCBI gene ID NM_001252020.2) is enriched in retinal cells. Given that this promoter is primarily intended for the treatment of human ophthalmic diseases, to ensure its specific and robust activity in the retina of retinitis pigmentosa, we reviewed the previously published TRPM1 expression pattern in the retina of the rd1 mouse model (PMID: 33665228). These data showed that TRPM1 gene expression was not downregulated by disease progression, indicating that TRPM1 remains active or even more active in this model, making it suitable for gene therapy of retinitis pigmentosa-related ophthalmic diseases. Human retinal DNase I sequencing data (PMID: 29233477) were viewed using the IGV genome browser to analyze cell-type-specific chromatin access within TRPM1 gene regulatory sequences. PhastCons 100-way (genomic conservation data for 100 vertebrate species) was downloaded from the UCSC website (https: / / hgdownload.cse.ucsc.edu / goldenpath / hg38 / phastCons100way / ) to examine conservation across 100 vertebrate genomes, including humans and mice. Four enhancer regions (En) with both chromatin openness and genomic conservation and one core promoter region (P) were obtained. The GTRD website was used to view the transcription factor ChIP sequencing peaks of these four enhancer regions (named En1 to En4 according to the order in which the enhancers appear on the chromatin) and the core promoter region. The transcription factor enrichment regions are as follows: Figure 1 As shown. Considering that the AAV capsid can only accommodate a core plasmid of about 4.5kb in length, the length of the promoter sequence should be shortened as much as possible. Based on the transcription factor ChIP-seq enrichment sites, we made a series of truncation of enhancers and promoters. The specific sequence is shown as follows Figure 1 The specific description is as follows:

[0103] 1) Core promoter sequence: There are three versions of the core promoter sequence, namely P1061 (hTRPM1), P750 (hTRPM1), and P550 (hTRPM1). Figure 1 As shown in Figures A and F, the longest promoter sequence, P1061, is located at -1 to -1061 bp upstream of the 5' end of the translation start site (TLSS) of the TRPM1 (ENST00000542188.5) transcript. Based on the sequence length, it is abbreviated as P1061 (SEQ ID NO: 1). This version was truncated at the 5' end to obtain sequences of 750 bp and 550 bp in length, respectively, abbreviated as P750 (SEQ ID NO: 2) and P550 (SEQ ID NO: 3). The mouse homologous sequence of P1061 (hTRPM1) is mP1488 (mTRPM1) (SEQ ID NO: 33); the mouse homologous sequence of P550 (hTRPM1) is mP528 (mTRPM1) (SEQ ID NO: 34).

[0104] 2) The first enhancer sequence: Figure 1 As shown in Figures A and B, there are two versions of the first enhancer region: En1_802 (hTRPM1) and En1_480 (hTRPM1), with sequence lengths of 802 bp and 480 bp, respectively (corresponding to SEQ ID NO: 4 and SEQ ID NO: 5, respectively). The mouse homologous sequence of En1_802 (hTRPM1) is mEn1_984 (mTRPM1) (SEQ ID NO: 35). The mouse homologous sequence of En1_480 (hTRPM1) is mEn1_664 (mTRPM1) (SEQ ID NO: 36).

[0105] 3) The second enhancer sequence: Figure 1As shown in A and C of , the second enhancer region has 3 versions, namely En2_1251 (hTRPM1), En2_880 (hTRPM1), and En2_460 (hTRPM1). The sequence lengths are 1251bp, 880bp, and 460bp, respectively (corresponding to SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively). Among them, En2_880 (hTRPM1) is derived from the original 1251bp version by removing redundant sequences at both ends and removing sequences in which some central transcription factors are relatively not enriched. En2_460 (hTRPM1) is further truncated on the basis of En2_880 (hTRPM1), retaining retinal-specific transcription factor OTX2 and NEUROD1 binding sites and removing epigenetic factor-related binding sites. The mouse homologous sequence of En2_1251 (hTRPM1) is mEn2_1187 (mTRPM1) (SEQ ID NO: 37). The mouse homologous sequence of En2_460 is mEn2_542 (mTRPM1) (SEQ ID NO: 38).

[0106] 4) The third enhancer sequence: Figure 1 As shown in A and D, the third enhancer sequence has one version, En3_277 (hTRPM1) (SEQ ID NO: 9);

[0107] 5) The fourth enhancer sequence: Figure 1 As shown in A and E, the fourth enhancer sequence has five versions: En4_622 (hTRPM1), En4_400 (hTRPM1), En4_284 (hTRPM1), En4_160 (hTRPM1), and En4_56 (hTRPM1). The corresponding sequence lengths are 622 bp, 400 bp, 284 bp, 160 bp, and 56 bp, respectively. The details are as follows: The longest enhancer sequence is En4_622 (SEQ ID NO: 10). By removing the less accessible chromatin regions at its 5' and 3' ends, the resulting truncation scheme is En4_400 (hTRPM1) (SEQ ID NO: 11). Further removal of the relatively closed chromatin regions, retention of evolutionarily conserved regions, and combination of different schemes with reference to transcription factor binding sites yielded the sequences En_284 (hTRPM1) (SEQ ID NO: 12), En4_160 (hTRPM1) (SEQ ID NO: 13), and En4_56 (hTRPM1) (SEQ ID NO: 14). The mouse homologous sequence of En4_622 (hTRPM1) is mEn4_876 (mTRPM1) (SEQ ID NO: 39).

[0108] Example 2: Packaging of promoter test vector and virus

[0109] In order to evaluate the performance of the promoter in this application, the promoter and enhancer sequences designed in Example 1 were combined and fused with the GFP-NanoLuc gene to construct a promoter test vector. The GFP on the reporter vector can be used to indicate the promoter expression position to determine the promoter specificity, and the NanoLuc gene on the reporter vector is used to indicate the promoter expression strength ( Figure 2 A and Figure 3). The specific combinations of the tested plasmids were divided into 4 categories ( Figure 2 B), as described below (the corresponding relationship between promoter number and promoter combination is described below 1 and Figure 2 ):

[0110] 1) Test vectors containing human and mouse TRPM1 core promoters, a total of 5 vectors, as shown below:

[0111] pAAV-P1061(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0112] pAAV-P750(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0113] pAAV-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0114] pAAV-mP1488(mTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0115] pAAV-mP528(mTRPM1)-GFP-NanoLuc-WPRE-hGHpA.

[0116] 2) Test vectors containing the human TRPM1 enhancer + basic version of the P550 core promoter and the mouse TRPM1 enhancer + basic version of the mP528 core promoter, a total of 14 vectors (corresponding promoter sequence information is shown in SEQ ID NOs: 15-25, 40-42), as shown below:

[0117] En1 enhancer:

[0118] pAAV-En1_802-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0119] pAAV-En1_480-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0120] En2 enhancer:

[0121] pAAV-En2_1251-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0122] pAAV-En2_880-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0123] pAAV-En2_460-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0124] En3 enhancer:

[0125] pAAV-En3_277-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0126] En4 enhancer:

[0127] pAAV-En4_622-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0128] pAAV-En4_400-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0129] pAAV-En4_284-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0130] pAAV-En4_160-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0131] pAAV-En4_56-P550(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0132] Mouse homologous sequences of some of the above enhancers:

[0133] pAAV-mEn1_664-mP528(mTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0134] pAAV-mEn2_542-mP528(mTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0135] pAAV-mEn4_876-mP528(mTRPM1)-GFP-NanoLuc-WPRE-hGHpA.

[0136] 3) Enhancer + core promoter combination, a total of 6 vectors (corresponding promoter sequence information is shown in SEQ ID NO: 26-31), as shown below:

[0137] pAAV-En4_284*2-P750(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0138] pAAV-En4_400-P750(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0139] pAAV-En4_160-P750(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0140] pAAV-En1_480-En4_160-P750(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0141] pAAV-En2_460-En4_160-P750(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA,

[0142] pAAV-En4_622-P1061(hTRPM1)-GFP-NanoLuc-WPRE-hGHpA.

[0143] The prior art (PMID: 32258214) was used as a positive control: pAAV-hGRM6(1243)-GFP-NanoLuc-WPRE-hGHpA.

[0144] The above core expression plasmid vector was packaged into rAAV vector, and the AAV2-NN mutant serotype (PMID: 33616280) with a higher efficiency of infecting retinal cells was used for intravitreal injection. The specific operation is as follows: the genomic DNA of HEK 293 cells or mouse tissues was extracted as a template, and the promoter and enhancer gene fragments of the human or mouse hTRPM1 gene were amplified from the human or mouse genome. The promoter sequence was connected to the linearized pAAV core plasmid vector backbone using recombinase (Quanshijin, product number: CU201), and the connection product was transformed into StbI3 chemical competent cells. The colonies with recombinant plasmids were screened, and the positive colonies of the colony PCR were expanded and cultured, and the plasmids were extracted for sequencing verification. The control promoter expression vector pAAV-hGRM6 (1243) -GFP-NanoLuc-WPRE-hGHpA was commissioned to Shanghai Bioengineering to construct. The recombinant adeno-associated virus vector AAV was obtained by packaging and purification using the three-plasmid transfection method of HEK293 cells, such as Figure 4 shown.

[0145] Example 3 Intravitreal injection of rAAV2-NN to detect the expression intensity of hTRPM1 promoter in mouse retina

[0146] C57BL / 6J WT mice were injected intravitreally at 4 weeks of age and observed for 1 month after administration. The expression strength of the promoter was confirmed by measuring the activity of NanoLuc luciferase. Figure 2 The specific results are analyzed as follows:

[0147] 3.1 Core promoter test results

[0148] The shortest P550(hTRPM1) promoter showed almost no activity, while the slightly longer P750(hTRPM1) promoter exhibited basal expression activity. We used the expression intensity of this promoter as a parameter to measure the expression intensity multiples of other promoters relative to the P750(hTRPM1) promoter. The longest P1061(hTRPM1) promoter exhibited 1.85 times the expression intensity of the P750(hTRPM1) promoter. This indicates that the P750(hTRPM1) promoter possesses both an appropriate sequence length and basal expression intensity, making it suitable for use as a core promoter in subsequent testing and clinical trials.

[0149] 3.2 Enhancer test results

[0150] According to Example 1 and Figure 2 As described in

[15] , to test the enhancer's own activity, we fused the enhancer with P550 (hTRPM1) which has almost no expression intensity. The results are as follows:

[0151] (1) Regarding the En1-related enhancer, the expression intensity of En1_802-P550 (hTRPM1) was 3.78 times that of the basic version of P750, and the expression intensity of the truncated En1_480-P550 (hTRPM1) was 3.05 times that of the basic version of P750. Since the P550 (hTRPM1) promoter alone has almost no expression activity, it can be inferred that En1_802 (hTRPM1) and En1_480 (hTRPM1) have enhancer activity. The mouse homologous sequence of En1_480 (hTRPM1) also has enhancer activity, and the expression intensity of mEn1_664-mP528 (mTRPM1) relative to P750 is 2.71 times. Since the sequence of En1_480 (hTRPM1) is shorter, the activity loss is not much compared with En1_802 (hTRPM1), and it has greater potential for clinical application.

[0152] (2) All three En2-related enhancers have enhancer activity. The expression intensity of the P500 (hTRPM1) promoter relative to the P750 (hTRPM1) promoter after the long to short combinations is 3.72, 3.56, and 2.87 times, respectively. Among them, the En2_460 (hTRPM1) promoter has the characteristics of high expression activity and a relatively short sequence, and has great potential for clinical application. Its mouse homologous sequence mEn2_542 (mTRPM1) also has similar expression activity, indicating that conserved homologous sequences between species have similar evolutionarily conserved functions.

[0153] (3) The enhancer activity of En3_277 (hTRPM1) was weak and was not tested further.

[0154] (4) Five truncated sequences of different lengths related to En4 all had strong enhancer activity. The expression intensity of En4_622 (hTRPM1), En4_400 (hTRPM1), En4_284 (hTRPM1), En4_160 (hTRPM1), and En4_56 (hTRPM1) combined with the P500 (hTRPM1) promoter from long to short was 5.79, 5.04, 4.46, 3.77, and 2.19 times that of P750 (hTRPM1), respectively. All of them have the characteristics of short sequence and strong activity. Among them, the mouse homologous sequence of En4_622 (hTRPM1), mEn4_876 (mTRPM1), had similar expression activity (relative expression fold 4.73 times).

[0155] 3.3 Enhancer + core promoter combination test results

[0156] For the combined sequences: En4_284*2-P750(hTRPM1), En4_400-P750(hTRPM1), En4_160-P750(hTRPM1), En1_480-En4_160-P750(hTRPM1), En2_460-En4_160-P750(hTRPM1), and En4_622-P1061(hTRPM1), the test results showed that the above promoters all had high promoter activity, and the expression intensity multiples relative to P750 were 6.77, 6.28, 4.98, 5.61, 6.96, and 8.54 times, respectively.

[0157] Example 4 Intravitreal injection of rAAV2-NN to detect the expression specificity of the hTRPM1 promoter in mouse retinal bipolar cells

[0158] To verify the expression specificity of the hTRPM1 series promoters in the retina and ON-type bipolar cells, we injected the hTRPM1 series promoter-related AAV viruses described in Examples 2 and 3 into the vitreous cavity of mice. One month after the injection, the retinas were harvested and immunofluorescence staining was performed to detect the fluorescence expression intensity of the promoters in the retina.

[0159] like Figure 5 As shown, the promoter activities of the hTRPM1 series of novel promoters (such as P750 (hTRPM1), P1061 (hTRPM1), En4_622-P550 (hTRPM1), En4_400-P750 (hTRPM1), En2_460-En4_160-P750 (hTRPM1), En4_622-P1061 (hTRPM1)) and the prior art hGRM6 (1243) are mainly distributed in the inner nuclear layer (INL) and inner plexiform layer (IPL), indicating that they are mainly located in bipolar cells. The average fluorescence intensity (sum of fluorescence intensity / area) was analyzed using ImageJ, as shown in FIG. Figure 6As shown in Figure A, the expression activity of the exemplary P550 is almost invisible, while P750 (hTRPM1) and P1061 (hTRPM1) can be expressed in a small number of cells, with average GFP fluorescence intensities of 0.00AU (±0.01AU), 11.90AU (±1.85AU), and 12.33AU (±0.58AU), respectively. In addition, En4_622-P550 (hTRPM1), En4_400-P750 (hTRPM1), En2_460-En4_160-P750 (hTRPM1), En4_622-P1061 (hTRPM1) and hGRM6 (1243) were expressed at a high abundance in the retina, which was consistent with the results measured in the NanoLuc experiment. The GFP fluorescence intensities were 17.67AU (±1.53AU), 19.00AU (±2.00AU), 18.33AU (±3.215AU), 20.17AU (±2.26AU) and 19.13AU (±6.03AU), respectively.

[0160] The expression specificity of the promoter in ON-type bipolar cells was detected by co-labeling GFP antibody and PKCα antibody. PKCα antibody can mark retinal bipolar cells, which are a type of ON-type bipolar cells. Figure 6As shown in B, GFP(+)PKCα(+) / PKCα(+) represents the expression ratio of the promoter-mediated GFP reporter gene in rod bipolar cells. The hTRPM1 series of novel promoters involved in the present application (such as P550 (hTRPM1), P750 (hTRPM1), P1061 (hTRPM1), En4_622-P550 (hTRPM1), En4_400-P750 (hTRPM1), En2_460-En4_160-P750 (hTRPM1), En4_622-P1061 (hTRPM1)) have expression activity in 0.003% (±0.01%), 36.00% (±9.839%), 43.49% (±18.03%), 57.33% (±6.81%), 60.00% (±10.00%), 62.33% (±9.29%), and 67.18% (±23.96%) of rod bipolar cells (labeled with PKCα antibody), respectively. Almost no activity was observed in the photoreceptor layer and ganglion cell layer. The hGRM6 (1243) staining results were consistent with those reported in the literature (PMID: 32258214), and the co-labeled cells accounted for 78.23% (± 9.69%) of the rod bipolar cells. The hTRPM1 series of novel promoters involved in this application (such as En4_622-P550 (hTRPM1), En4_400-P750 (hTRPM1), En2_460-En4_160-P750 (hTRPM1), En4_622-P1061 (hTRPM1) promoters) can efficiently initiate gene expression in ON bipolar cells and have potential application value in the treatment of retinal diseases.

[0161] Example 5 Detection of expression activity of hTRPM1 series promoters in human retinal cells

[0162] To verify the expression activity and specificity of the hTRPM1 promoter in human cells, we used AAV virus to infect human retinal organoids cultured in vitro. Human retinal organoids are highly similar to human retina in terms of cell morphology, function, and developmental status, and can therefore be used to assist in determining the expression characteristics of the promoter in the human retina. Figure 7As shown, human retinal organoids differentiated for 200 days were mixed and co-cultured with the detection virus of the hTRPM1 series promoter at a titer of 1E+11 vg / organoid. The expression of the promoter in human retinal organoids was detected by immunofluorescence staining 10 days after transduction. Human retinal organoid cells were labeled with GFP antibody, PKCα antibody (optic stalk bipolar cells), and DAPI nuclear dye to determine the activity of the promoter in human ON-type bipolar cells. GFP mediated by the hTRPM1 series promoter (such as the En400-P750 (hTRPM1) promoter) can be effectively expressed in ON-type bipolar cells of human retinal organoids and has a high co-labeling with the optic stalk bipolar cells of human retinal organoids. The hTRPM1 series promoter (such as the En400-P750 (hTRPM1) promoter) has similar activity to the prior art hGRM6 (1243) promoter in human retinal organoids, and both have the potential for application in human retinal cells and tissues.

[0163] Example 6 Restoring vision in rd10 mice using hTRPM1 series promoters combined with light-sensitive proteins

[0164] In order to verify the application of hTRPM1 series promoter in optogenetic gene therapy technology, it was fused with PsCatch2.0 photosensitive protein (PMID: 35430811) gene and constructed expression vector, packaged into AAV2-NN virus, and injected into 8-week-old wild-type rd10 mice through the vitreous cavity. This example uses the optokinetic response to evaluate the therapeutic effect of retinitis pigmentosa mice. The results are shown in Figure 2. Figure 8As shown, the average visual acuity of rd10 mice treated with hTRPM1 series promoter-fused photosensitive proteins (such as pAAV-En4_622-P550(hTRPM1)-PsCatch2.0-WPRE-hGHpA, pAAV-En4_400-P750(hTRPM1)-PsCatch2.0-WPRE-hGHpA, and pAAV-En4_622-P1061(hTRPM1)-PsCatch2.0-WPRE-hGHpA) was significantly improved compared with untreated rd10 mice. Specifically, the average maximum visual acuity of the two combinations of examples were 0.18c / d (±0.06c / d), 0.19c / d (±0.07c / d), and 0.2c / d (±0.08c / d), respectively. The average maximum visual acuity of untreated rd10 mice was 0.07c / d (±0.07c / d, n=5), and the average maximum visual acuity of the WT group was 0.46c / d (±0.04c / d). Although the visual acuity of rd10 mice after treatment did not recover to the level of WT mice, it was still significantly improved compared with untreated rd10 mice, indicating that the hTRPM1 series promoters (such as En4_622-P550 (hTRPM1), En4_400-P750 (hTRPM1), En4_622-P1061 (hTRPM1)) have extremely high application value in the treatment of retinal diseases characterized by retinal photoreceptor degeneration or apoptosis (such as retinitis pigmentosa, Stargardt, retinal macular degeneration, etc.), or other diseases that achieve therapeutic effects by expressing therapeutic genes in bipolar cells.

[0165] Table 1: Sequence Listing

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173] The above description is merely a preferred embodiment of the present application and does not constitute any limitation on the present application. Any person skilled in the art may utilize the technical content disclosed above to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments based on the technical essence of the present application that do not depart from the technical solution of the present application shall still fall within the scope of protection of the technical solution of the present application.

Claims

1. An enhancer or enhancer variant, wherein the enhancer comprises the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:6, SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:39, or is truncated from the 5'-end or 3'-end of the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:6, SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:39 to a length of 40-910 base pairs, preferably truncated to a length of 56-880 base pairs; wherein the enhancer variant is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the enhancer.

2. The enhancer or enhancer variant of claim 1 , wherein the enhancer comprises the nucleotide sequence of any one of SEQ ID NOs: 4-14 and 35-39, or a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

3. A promoter or promoter variant, wherein the promoter comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, or is truncated from the 5'-end or 3'-end of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33 to a length of 500-800 base pairs, preferably truncated to a length of 550-750 base pairs; wherein the promoter variant is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the promoter.

4. The promoter or promoter variant according to claim 3, wherein the promoter comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1-3 and 33-34, or a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

5. An isolated nucleic acid molecule comprising an enhancer and a promoter, wherein: The enhancer is the enhancer as claimed in claim 1 or 2; and / or The promoter is the promoter as claimed in claim 3 or 4.

6. An isolated nucleic acid molecule according to claim 5, wherein the isolated nucleic acid molecule consists of one or more of the enhancers or enhancer variants, one or more of the promoters or promoter variants, and optionally a spacer separating the enhancer or enhancer variant from the promoter or promoter variant.

7. The isolated nucleic acid molecule according to claim 6, wherein the isolated nucleic acid molecule comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 15-31, 40-42, or comprises or consists of a sequence that is at least 60% identical to a sequence selected from the group consisting of SEQ ID NOs: 15-31, 40-42.

8. A nucleic acid expression vector comprising the isolated nucleic acid molecule according to any one of claims 5 to 7.

9. The nucleic acid expression vector according to claim 8, wherein the nucleic acid expression vector is a recombinant adeno-associated virus vector (AAV), in particular wherein the nucleic acid expression vector is a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12 vector or a derivative variant of the above recombinant vector.

10. The nucleic acid expression vector according to any one of claims 8 to 9, further comprising: a transgenic, wherein the transgenic is a light-sensitive protein gene, in particular a light-sensitive protein gene as shown in SEQ ID NO: 46 or 47 or comprises a light-sensitive protein gene as shown in SEQ ID NO: 46 or 47, and b. Sequence encoding capsid protein.

11. An adeno-associated virus particle, virus-like particle, or non-viral particle, comprising the isolated nucleic acid molecule according to any one of claims 5 to 7 or the nucleic acid expression vector according to any one of claims 8 to 10.

12. A pharmaceutical agent selected from the group consisting of the isolated nucleic acid molecule according to any one of claims 5 to 7 or the nucleic acid expression vector according to any one of claims 8 to 10 and the adeno-associated virus particle, virus-like particle, or non-viral particle according to claim 11 and for use as a medicament.

13. A medicament selected from the group consisting of the isolated nucleic acid molecule according to any one of claims 5 to 7, the nucleic acid expression vector according to any one of claims 8 to 10, and the adeno-associated virus particle, virus-like particle, or non-viral particle according to claim 11, and for treating congenital stationary night blindness (CSBN1) or rod-cone and cone-rod dystrophy, more particularly retinitis pigmentosa and macular degeneration, or other diseases that achieve therapeutic effects by expressing therapeutic genes in bipolar cells.

14. A medicament selected from the group consisting of the isolated nucleic acid molecule according to any one of claims 5 to 7, the nucleic acid expression vector according to any one of claims 8 to 10, and the adeno-associated virus particle, virus-like particle, or non-viral particle according to claim 11, wherein the medicament is administered by: a. Intravitreal injection, b. Subretinal injection, c. Choroidal injection, or d. Administer eye drops.

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