Pharmaceutical composition and method for treating eye diseases

Abstract

The invention relates to a pharmaceutical composition for treating eye diseases and a method thereof, and particularly provides a nucleic acid construct which comprises a cone cell specific promoter and a coding sequence of CRX protein. The pharmaceutical composition can delay death of cone cells in retinitis pigmentosa and prolong the daytime visual function of a patient, and can be used for treating retinal degenerative diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomolecules, and in particular to pharmaceutical compositions and methods for treating ocular diseases. BACKGROUND

[0002] Retinitis pigmentosa (RP) is the most common type of inherited retinal degenerative disease, with a prevalence of approximately 1 in 4,000 people worldwide. Currently, it is known that mutations in about 100 genes (such as RHO, PDE6B) can cause RP. And any single gene mutation in these about 100 genes will cause RP. Directly affected by these RP gene mutations, rod cells first undergo autonomous degenerative death. Although cone cells are not directly affected by these pathogenic gene mutations (some RP pathogenic genes are not expressed in cone cells, such as RHO, PDE6B), for reasons that have not yet been fully determined (including but not limited to: lack of trophic factors, metabolic imbalance, increased oxidative stress, abnormal immune response, excessive activity of the vitamin A visual cycle, and other environmental changes), cone cells undergo a second wave of death, i.e., non-autonomous degenerative death, after the surrounding rod cells have died out, resulting in a gradual reduction in the patient's visual field and ultimately leading to the gradual loss of day vision, color vision, and central vision. To date, only a few treatment methods targeting a very small number of pathogenic genes have been developed, and their long-term effects are still being observed. The vast majority of RP cannot be effectively treated. In addition, when RP progresses to a certain stage, i.e., the stage at which all rod cells have died, even if a gene therapy vector is used to introduce a normal gene into retinal cells to replace the pathogenic gene or to edit the pathogenic gene to modify the pathogenic gene to normal at this stage, cone cells will not be able to benefit from these gene replacement or gene editing therapies to avoid non-autonomous degenerative death due to their inability to completely overcome or correct certain adverse environmental changes that occur after all rods have died. This greatly limits the scope of use and the applicable stage of disease of the above-mentioned traditional gene therapy methods targeting traditional target cells (rod cells). Based on this, it is particularly necessary to develop a universal gene therapy method that can target a wider range of patient populations and disease stages.

[0003] However, the non-autonomous degeneration death mechanism of RP cones is not completely clear, and no method with complete protection effect on RP cones has been detected in animal models. At present, the research on general gene therapy for RP mainly focuses on overexpression of NRF2, TGFB1, CX3CL1, CD47, TXNIP, RDCVF, NR2E3 and other genes in retinal cells (for example: cones) to improve the ability of cone cells to adapt to the environment after the death of all rods, and to partially protect the day vision. Only RDCVF and NR2E3 related gene therapy products have entered the clinical trial stage, but their effectiveness still needs to be detected. For the non-autonomous degeneration death of cone cells, the design of RP general gene therapy drugs provides a more effective treatment method, which will benefit more patients. SUMMARY

[0004] The present application finds that the increase of the expression amount of the CRX gene in the cone cells driven by the cone cell specific promoter has a protective effect on the non-autonomous death of the RP cone cells. Therefore, the present application provides a gene therapy product with a protective effect on the RP cone cells.

[0005] The first aspect of the present application provides a nucleic acid construct comprising a cone cell specific promoter and a coding sequence of a CRX protein.

[0006] In one or more embodiments, the nucleic acid construct further comprises a polyadenylation signal.

[0007] In one embodiment, the polyadenylation signal is selected from the group consisting of a human growth hormone polyadenylation signal, a bovine growth hormone polyadenylation signal and an SV40 polyadenylation signal.

[0008] In one or more embodiments, the nucleic acid construct comprises, in order, a cone cell specific promoter, a coding sequence of a CRX protein and a polyadenylation signal. The coding sequence of the CRX protein is operably linked to the cone specific promoter and the polyadenylation signal.

[0009] In one or more embodiments, the CRX protein has the sequence shown in SEQ ID NO: 6 or 7 or a sequence having at least 80%, at least 90%, at least 99% sequence identity to the sequence shown in SEQ ID NO: 6 or 7.

[0010] In one or more embodiments, the coding sequence of the CRX protein has the sequence shown in SEQ ID NO: 4 or 5 or a variant having at least 80%, at least 90%, at least 99% sequence identity to the sequence shown in SEQ ID NO: 4 or 5.

[0011] In one or more embodiments, the cone-specific promoter is a RedO promoter, a GNAT2 promoter, or an ARR3 promoter.

[0012] In one or more embodiments, the RedO promoter is 2 kb or 1.7 kb in length.

[0013] In one or more embodiments, the RedO promoter is RO1.7.

[0014] In one or more embodiments, the GNAT2 promoter is SynP136, 2 kb in length, or SynPVI, 0.5 kb in length.

[0015] In one or more embodiments, the sequence of the RedO promoter is set forth in SEQ ID NO: 8 or 9. In one or more embodiments, the sequence of the GNAT2 promoter is set forth in SEQ ID NO: 10 or 11. In one or more embodiments, the sequence of the ARR3 promoter is set forth in SEQ ID NO: 12.

[0016] In one or more embodiments, the nucleic acid construct further comprises a woodchuck hepatitis virus post-transcriptional regulatory element WPRE (set forth in SEQ ID NO: 13) or a variant thereof. In one or more embodiments, the WPRE variant is WPRE3 (set forth in SEQ ID NO: 14).

[0017] In one or more embodiments, the nucleic acid construct further comprises an ITR region, including a 5’ ITR and a 3’ ITR.

[0018] In one or more embodiments, the nucleic acid construct comprises, in 5’ to 3’ order, a 5’ ITR operably linked to, a cone-specific promoter as described in any embodiment herein, a coding sequence for a CRX protein as described in any embodiment herein, a woodchuck hepatitis virus post-transcriptional regulatory element as described in any embodiment herein, and a polyadenylation signal as described in any embodiment herein. In some embodiments, the nucleic acid construct comprises, in 5’ to 3’ order, a 5’ ITR operably linked to, a RedO promoter, a coding sequence for a CRX protein, WPRE3, and an SV40 polyadenylation signal.

[0019] In one or more embodiments, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 3.

[0020] In one or more embodiments, the nucleic acid construct is a vector.

[0021] In one or more embodiments, the vector is a cloning vector, an integration vector, or an expression vector.

[0022] In one or more embodiments, the vector is an AAV vector, a lentivirus vector, or an adenovirus vector, preferably an AAV vector.

[0023] The present application also provides a nucleic acid delivery reagent comprising the nucleic acid construct described in any one of the embodiments herein and a delivery vehicle, which includes a virus (e.g., AAV, lentivirus, or adenovirus), a virus-like particle (e.g., PEG10), a lipid nanoparticle (LNP).

[0024] The second aspect of the present application provides an AAV virus comprising the nucleic acid construct described in the first aspect herein.

[0025] In one or more embodiments, the AAV virus further comprises a nucleic acid construct having AAV virus required genes.

[0026] In one or more embodiments, the AAV virus required genes include one or more selected from the group consisting of Rep, Cap, E2A, E4, and VA genes.

[0027] The present application provides a host cell comprising the nucleic acid construct described in the first aspect herein or the AAV virus described in the second aspect.

[0028] In one or more embodiments, the host cell is a 293T cell.

[0029] In one or more embodiments, the CRX protein has the sequence set forth in SEQ ID NO: 6 or 7 or a sequence having at least 80%, at least 90%, at least 99% sequence identity to the sequence set forth in SEQ ID NO: 6 or 7.

[0030] In one or more embodiments, the coding sequence of the CRX protein has the sequence set forth in SEQ ID NO: 4 or 5 or a variant having at least 80%, at least 90%, at least 99% sequence identity to the sequence set forth in SEQ ID NO: 4 or 5.

[0031] The present application also provides a method of preparing the AAV virus described herein, the method comprising the steps of: (1) transfecting a cell with the nucleic acid construct described herein as an AAV vector and a helper plasmid, (2) incubating the cell to produce the AAV virus, and (3) collecting the virus.

[0032] In one or more embodiments, the helper plasmid comprises a rep gene and a cap gene. Preferably, the rep gene encodes Rep78, Rep68, Rep52, and / or Rep40; and the cap gene encodes VP1, VP2, and / or VP3.

[0033] In one or more embodiments, the method comprises the steps of:

[0034] (1) cloning the coding sequence of CRX protein (such as SEQ ID NO: 4 or 5) described in any of the embodiments herein into the empty vector of AAV-RO1.7 to obtain plasmid AAV-RO1.7-CRX;

[0035] (2) transfecting the plasmid in (1) with Helper plasmid and AAV8 capsid plasmid into 293T cells to package into AAV8 capsid; and

[0036] (3) collecting the virus from the cell culture.

[0037] In one or more embodiments, the cell culture comprises cells or culture supernatant thereof.

[0038] The present application also provides the use of an agent that specifically increases the expression or activity of CRX protein in cone cells in the manufacture of a medicament for treating a degenerative eye disease or a symptom thereof by inhibiting degenerative death of cone cells. The agent that specifically increases the expression or activity of CRX protein in cone cells is the nucleic acid construct, the AAV virus, the host cell described in any of the embodiments herein.

[0039] In one or more embodiments, the degenerative death of cone cells is non-autonomous degenerative death of cone cells.

[0040] In one or more embodiments, the degenerative eye disease is a degenerative eye disease associated with degenerative death of cone cells.

[0041] In one or more embodiments, the degenerative eye disease is not a disease caused by mutation of CRX gene.

[0042] In one or more embodiments, the degenerative eye disease is selected from the group consisting of retinitis pigmentosa (i.e. rod-cone dystrophy), age-related macular degeneration and cone-rod dystrophy.

[0043] In one or more embodiments, the degenerative eye disease is retinitis pigmentosa, age-related macular degeneration, etc. caused by mutation of non-CRX gene.

[0044] The present application also provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient, and one or more selected from the group consisting of the nucleic acid construct of the first aspect of the present application and the AAV virus of the second aspect of the present application.

[0045] In one or more embodiments, the excipient comprises a viscosity-inducing agent.

[0046] In one or more embodiments, the pharmaceutical composition is suitable for intraocular administration.

[0047] In one or more embodiments, the pharmaceutical composition is suitable for intravitreal or subretinal, subtenon, subconjunctival, intramuscular, periocular, retrobulbar, suprachoroidal, and / or intrascleral administration.

[0048] The present application also provides a kit comprising any one or more of the nucleic acid construct, AAV virus, host cell, and pharmaceutical composition described in any of the embodiments herein.

[0049] The present application also provides a method of treating or preventing a degenerative ocular disease or a symptom thereof in a subject by inhibiting degenerative death of cone cells, the method comprising administering to the subject a therapeutically effective amount of an agent that specifically increases expression or activity of CRX protein in cone cells. The method does not require increasing expression or activity of CRX in rod cells. The agent that specifically increases expression or activity of CRX protein in cone cells includes any one or more of the nucleic acid construct, AAV virus, or pharmaceutical composition of the present application.

[0050] In one or more embodiments, the administering is intraocular administration.

[0051] In one or more embodiments, the intraocular administration is selected from intravitreal or subretinal, subtenon, subconjunctival, intramuscular, periocular, retrobulbar, suprachoroidal, and / or intrascleral administration.

[0052] In one or more embodiments, the degenerative death of cone cells is non-autonomous degenerative death of cone cells.

[0053] In one or more embodiments, the degenerative ocular disease is a degenerative ocular disease associated with degenerative death of cone cells.

[0054] In one or more embodiments, the subject’s cone cells do not carry a CRX gene pathogenic mutation.

[0055] In one or more embodiments, the degenerative ocular disease is not a disease caused by a mutation in the CRX gene.

[0056] In one or more embodiments, the degenerative ocular disease is selected from the group consisting of: retinitis pigmentosa (i.e., rod-cone dystrophy), age-related macular degeneration, and cone-rod dystrophy.

[0057] In one or more embodiments, the degenerative ocular disease is retinitis pigmentosa or age-related macular degeneration, etc., caused by a mutation in a non-CRX gene.

[0058] The present application also provides a method of delaying loss of functional vision in a subject having a degenerative eye disease by inhibiting degenerative death of cone cells, the method comprising administering to the subject a therapeutically effective amount of any one or more of the nucleic acid construct, the AAV virus or the pharmaceutical composition of the present application.

[0059] In one or more embodiments, the administering is intraocular administration.

[0060] In one or more embodiments, the intraocular administration is selected from the group consisting of intravitreal or subretinal, subtenon, subconjunctival, subtenon, periocular, retrobulbar, suprachoroidal and / or intrascleral administration.

[0061] In one or more embodiments, the degenerative death of cone cells is non-autonomous degenerative death of cone cells.

[0062] In one or more embodiments, the degenerative eye disease is a degenerative eye disease associated with degenerative death of cone cells.

[0063] In one or more embodiments, the subject’s cone cells do not carry a CRX gene pathogenic mutation.

[0064] In one or more embodiments, the degenerative eye disease is not a disease caused by a mutation in the CRX gene.

[0065] In one or more embodiments, the degenerative eye disease is selected from the group consisting of: retinitis pigmentosa (i.e. rod-cone dystrophy), age-related macular degeneration and cone-rod dystrophy.

[0066] In one or more embodiments, the degenerative eye disease is retinitis pigmentosa, age-related macular degeneration, etc. caused by a mutation in a non-CRX gene.

[0067] The present application also provides use of the nucleic acid construct, the AAV virus, the host cell of any one of the embodiments herein in the manufacture of a medicament for delaying loss of functional vision in a subject having a degenerative eye disease by inhibiting degenerative death of cone cells, delaying death of cone cells in retinitis pigmentosa and / or prolonging a patient’s diurnal visual function.

[0068] In one or more embodiments, the degenerative death of cone cells is non-autonomous degenerative death of cone cells.

[0069] In one or more embodiments, the degenerative eye disease is a degenerative eye disease associated with degenerative death of cone cells.

[0070] In one or more embodiments, the degenerative eye disease is not a disease caused by a mutation in the CRX gene.

[0071] In one or more embodiments, the degenerative eye disease is selected from the group consisting of retinitis pigmentosa (i.e. rod-cone dystrophy), age-related macular degeneration and cone-rod dystrophy.

[0072] In one or more embodiments, the degenerative eye disease is retinitis pigmentosa, age-related macular degeneration, etc. caused by mutations in non-CRX genes.

[0073] Advantages of the present application:

[0074] (1) The present application can delay the death of cone cells in retinitis pigmentosa, prolong the day vision function of patients, and can be used for the treatment of other related retinal degenerative diseases, and has a protective effect on the survival of cone cells and the day vision of patients in other retinal / eye blinding degenerative diseases, such as age-related macular degeneration.

[0075] (2) The present application has a significant protective effect on the survival of cone cells of the disease model mice by specifically overexpressing the CRX gene in the cone cells of the RP animal model (RD1, i.e. PDE6B gene deletion mice). BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 : AAV-RO1.7-CRX schematic diagram.

[0077] Figure 2 : AAV-RO1.7-CRX vector diagram.

[0078] Figure 3 : High expression and high specificity cone promoter (RedO, GNAT2) fluorescent protein expression result diagram. Highlight signal: fluorescent protein expressed driven by AAV-promoter; Cone cell: the nucleus position is outside the ONL layer (1-2 layers above the picture), and the nucleus is large; Rod cell: the nucleus position is inside the ONL layer (about 10 layers inside the lower part of the picture), and the nucleus is small.

[0079] Figure 4 : Representative image of 50-day-old RD1 retina after treatment.

[0080] Figure 5 : Statistics of cone counting in the 1 / 2 radius of the retina of 50-day-old RD1 mice overexpressing CRX. Sample number n: represents the number of retinas measured, a total of from 4 litters of mice. ROHG: control group injected with AAV8-RedO-H2BGFP only.

[0081] Figure 6 : Representative image of 20-day-old RD1 retina after treatment.

[0082] Figure 7Statistical analysis of cone counts in the inner 1 / 2 radius of the retina of 20 day old RD1 mice overexpressing CRX. ROHG: control group injected with AAV8-RedO-H2BGFP only.

[0083] Figure 8 Statistical analysis of cone counts in the inner 1 / 2 radius of the retina of 20 day, 50 day old RD1 mice overexpressing CRX. Data combined from Figure 5 and Figure 7 for easy and intuitive comparison of degenerative changes.

[0084] Figure 9 Representative images of 20 day old RD1 retinal sections after treatment, and statistical analysis of ONL thickness. DETAILED DESCRIPTION

[0085] Unless otherwise defined, the practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987 and periodic updates thereto); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0086] The term "disease and / or condition" refers to the physical state of a subject associated with a disease and / or condition described herein.

[0087] The term "subject" or "patient" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., who receives a pharmaceutical composition of the present application for the treatment, prevention, amelioration, and / or alleviation of a disease or condition of the present application.

[0088] The CRX gene is located on a mammalian autosome. Studies have shown that in CRX- caused RP subtypes, mutations in the CRX gene directly cause the degeneration and death of rod cells, but such mutations have little direct effect on the function and survival of cone cells, and the cone cells only undergo non-autonomous degeneration and death after the death of the surrounding rod cells, thereby affecting diurnal color and central vision. As described above, the cause of the non-autonomous degeneration and death of RP cone cells has not been fully determined.

[0089] Prior to the present application, the relationship between the CRX gene and the non-autonomous death of cone cells after the death of rod cells was not clearly disclosed in the art, and it was not expected in the art that specific overexpression of the CRX protein in RP cone cells could delay the non-autonomous death of cone cells that occurs after the death of rod cells.

[0090] The present application first discovered that, in RP cone cells (non-CRX gene defect subtypes), the use of a cone cell-specific promoter with high expression intensity and good specificity to express the CRX gene can protect RP cone cells from non-autonomous death. The increase in the expression of the CRX gene has an excellent protective effect on the cone cells of non-CRX gene defect subtypes of RP, can delay or even block the non-autonomous death of RP cone cells, and prolong the diurnal visual function of patients, and thus can be widely used in the treatment of various types of degenerative eye diseases that cause the degeneration and death of cone cells, thereby completing the present application.

[0091] Nucleic acid construct, virus, and cell

[0092] The present application first provides a nucleic acid construct (e.g., an expression cassette or a vector) comprising, in order, a cone cell-specific promoter and a CRX nucleotide sequence. The nucleic acid construct can include a polyadenylation signal. Thus, one nucleic acid construct herein comprises, in order, a cone cell-specific promoter, a coding sequence for a CRX protein, and a polyadenylation signal, the coding sequence for the CRX protein being operably linked to the cone cell-specific promoter and the polyadenylation signal.

[0093] In this context, the polyadenylation signal can be a human growth hormone polyadenylation signal, a bovine growth hormone polyadenylation signal, or an SV40 polyadenylation signal.

[0094] Herein, the cone-specific promoter can be any promoter known in the art that is capable of specifically initiating protein expression in cones, for example the RedO promoter, the GNAT2 promoter or the ARR3 promoter (https: / / pubmed.ncbi.nlm.nih.gov / 12135752 / ). The sequence of the RedO promoter is set forth in SEQ ID NO: 8 (RedO-1.7kb, RO1.7) or SEQ ID NO: 9 (RedO-2kb). The sequence of the GNAT2 promoter is set forth in SEQ ID NO: 10 (GNAT2-2kb, SynP136) or SEQ ID NO: 11 (GNAT2-0.5kb, SynPVI). The sequence of the ARR3 promoter is set forth in SEQ ID NO: 12.

[0095] Herein, the mouse CRX protein sequence has the sequence set forth in SEQ ID NO: 6 or a variant thereof having at least 80%, at least 90%, at least 95% or at least 99% sequence identity thereto; the human CRX protein sequence has the sequence set forth in SEQ ID NO: 7 or a variant thereof having at least 80%, at least 90%, at least 95% or at least 99% sequence identity thereto.

[0096] Herein, the mouse CRX nucleotide sequence has the sequence set forth in SEQ ID NO: 4 or a variant thereof having at least 80%, at least 90%, at least 95% or at least 99% sequence identity thereto; the human CRX nucleotide sequence has the sequence set forth in SEQ ID NO: 5 or a variant thereof having at least 80%, at least 90%, at least 95% or at least 99% sequence identity thereto.

[0097] A variant of an amino acid sequence according to the present application has at least 95%, 96%, 97%, 98% or 99% identity to the sequence from which it is derived and retains the biological function of the sequence from which it is derived (e.g. a variant of a CRX protein has the function of a CRX protein). A variant of a nucleic acid sequence according to the present application has at least 95%, 96%, 97%, 98% or 99% identity to the sequence from which it is derived and, in addition, the amino acid sequence encoded by the variant of the nucleic acid sequence is identical to the amino acid sequence encoded by the sequence from which it is derived or, although different, retains the biological function of the amino acid sequence encoded by the sequence from which it is derived (e.g. a variant of a nucleic acid sequence of a CRX protein encodes a protein that is identical to or, although different, has the function of a CRX protein). Sequence identity according to the present application can be measured using sequence analysis software, for example the computer program BLAST, e.g. BLASTP or BLASTN, using default parameters.

[0098] The full-length sequence of the nucleic acid of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually to clone it into a vector, then into cells, and then to separate the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules in isolated form.

[0099] The DNA sequence can then be introduced into a variety of existing DNA molecules (or as vectors) and cells known in the art. Recombinant vectors can be constructed using methods well known to those skilled in the art, see for example Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory), Ausubel et al. (1989, Short Protocols in Molecular Biology, Wiley) or techniques described in other standard textbooks. Alternatively, the polynucleotide and vector can be reconstituted into liposomes for delivery to target cells. Vectors containing the polynucleotides of the present application can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts, see Sambrook et al. (supra).

[0100] As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be made which all encode the fusion polypeptides of the present application. Thus, given the identification of a particular amino acid sequence, one skilled in the art can make any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. Therefore, the present application also relates to polynucleotides which hybridize to the above polynucleotide sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides which hybridize to the polynucleotides described herein under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95% or more. And the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0101] The nucleic acid construct described herein can be an expression cassette, which can consist of a cone cell-specific promoter, a coding sequence for CRX protein, and a polyadenylation signal, depending on the expression. In some embodiments, the expression cassette further comprises a 5' ITR and a 3' ITR.

[0102] In some embodiments, the nucleic acid construct is a vector. The vector can be a plasmid, a cosmid, a virus, a viral vector. The vector can be a cloning vector, an integration vector, and can also be an expression vector. In addition to the nucleotide molecule described in the present application, the expression vector usually contains other elements commonly contained in vectors, such as a multiple cloning site, a resistance gene, a replication initiation site, etc. Vectors that can effectively express proteins in the human body, especially in cone cells, are preferred for use in the present application, including but not limited to AAV vectors, lentiviral vectors, adenoviral vectors, etc.

[0103] The expression vector usually contains sequences for plasmid maintenance and for cloning and expression of foreign nucleotide sequences. The sequences, collectively referred to in certain embodiments as "flanking sequences", typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of nucleic acids encoding proteins to be expressed, and optional marker elements.

[0104] The vector can optionally contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the fusion polypeptide; the oligonucleotide sequence encodes a polyhistidine (such as 6His) or another "tag", such as FLAG, HA, or myc. This tag is typically fused to the polypeptide when the polypeptide is expressed, and can serve as a means for affinity purification or detection of the protein from host cells. Affinity purification can be accomplished, for example, by column chromatography using an antibody against the tag as an affinity matrix. The tag can optionally be subsequently removed from the purified protein by various means, such as using certain peptidases for cleavage.

[0105] The flanking sequences can be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or native. Likewise, the source of the flanking sequences can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences function in and are activatable by the host cell machinery.

[0106] In some embodiments, the fusion polypeptides of the present application are expressed by recombinant AAV (rAAV) vector systems. The term "adeno-associated virus" or "AAV" as used herein includes members of the virus class associated with that name, which belongs to the genus Dependoparvovirus, family Parvoviridae. Adeno-associated virus is a single-stranded DNA virus that grows only in cells in which certain functions are provided by a co- infecting helper virus. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228. Various serotypes of the virus are known to be suitable for gene delivery, and the various serotypes are closely related in structure and function. All AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins. AAV serotypes are known in the art, for example, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV7m8, AAVPHP.B, or AAVrh74. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of these serotypes, for example, AAV2, AAV5, AAV8, AAV9. The AAV viruses provided in the present application comprise the CRX nucleotide sequence or nucleic acid construct described in any of the embodiments herein.

[0107] As used herein, "AAV vector" refers to a vector comprising, consisting essentially of, or consisting of one or more coding sequences for the fusion polypeptides herein and one or more AAV inverted terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that provides the functional rep and cap gene products; for example, by transfecting a host cell. In one or more embodiments, the AAV vector contains a promoter, at least one nucleic acid encoding CRX, and / or an enhancer and / or terminator packaged into the flanking ITRs of an infectious AAV particle. A plasmid containing the AAV vector can also contain elements for use in making, for example, antibiotic resistance genes, etc. In one or more embodiments, the AAV virus comprises the sequence set forth in SEQ ID NO: 3.

[0108] As used herein, the term "helper" or "helper plasmid" with respect to a virus or plasmid refers to a virus or plasmid used to provide additional components necessary for replication and packaging of any of the AAV vectors disclosed herein. The components encoded by the helper virus can include any genes required for virion assembly, encapsidation, genome replication, and / or packaging. For example, the helper virus or plasmid can encode enzymes necessary for viral genome replication. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELPer (plasmid), adenovirus (virus), or herpesvirus (virus). In some embodiments, the one or more helper plasmids include a first helper plasmid comprising a rep gene and a cap gene and a second helper plasmid comprising an Ela gene, an Elb gene, an E4 gene, an E2a gene, and a VA gene. In some embodiments, the rep gene is a rep gene derived from AAV2 and the cap gene is derived from AAV44.9. Helper plasmids and methods of making such plasmids are known in the art and are commercially available. Exemplary helper plasmids are the Helper plasmid set forth in SEQ ID NO: 16 and / or the AAV8 capsid plasmid set forth in SEQ ID NO: 17.

[0109] As used herein, the term "viral capsid" or "capsid" refers to the protein shell or coat of a viral particle. The function of the capsid is to encapsidate, protect, transport, and release the viral genome into a host cell. The capsid is typically composed of oligomeric structural subunits of protein ("capsid proteins"). As used herein, the term "encapsidate" refers to being contained within a viral capsid. The viral capsid of AAV comprises, consists essentially of, or consists of the following three major viral proteins: VP1, VP2, and VP3.

[0110] Methods of producing and using AAV viruses are known in the art. Methods of producing AAV viruses and heterologous nucleic acids are also known in the art and are commercially available (see, e.g., US 2007 / 0015238 and US 2012 / 0322861, which are incorporated by reference herein in their entireties). For example, a plasmid comprising a CRX nucleic acid sequence can be combined with one or more helper plasmids (e.g., comprising rep genes (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and cap genes (encoding VP1, VP2, and / or VP3) and transfected or permanently integrated into a producer cell line such that the AAV virus can be packaged and subsequently purified. In some embodiments, the cell line is a mammalian cell line, e.g., a human embryonic kidney (HEK) 293 cell line. Provided herein are cells comprising any of the AAV vectors and / or AAV viruses disclosed herein.

[0111] The method of producing an AAV virus herein specifically comprises: (1) cloning the CRX nucleotide sequence (e.g., SEQ ID NO: 4 or 5) or nucleic acid construct described herein into the empty vector of AAV-RO1.7 to obtain plasmid AAV-RO1.7-CRX; (2) co-transfecting the plasmid in (1) and Helper plasmid, AAV8 capsid plasmid into 293T cells to package into AAV8 capsid; (3) collecting the virus from the cell culture (e.g., using iodixanol gradient to collect the virus after 72 h).

[0112] The present disclosure also provides a method of introducing a gene of interest into a cell of a subject, comprising contacting the cell with an effective amount of any of the AAV viral particles described herein, wherein the particle contains any of the AAV vectors comprising a CRX-encoding sequence described herein.

[0113] The present disclosure also includes host cells that comprise, express, and / or secrete a CRX nucleotide or polypeptide according to any of the embodiments described herein, or that comprise a nucleic acid construct described herein or an AAV virus described herein. The nucleic acid construct or AAV virus is transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts, see Sambrook et al. (supra). Suitable host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), and the like.

[0114] Pharmaceutical compositions

[0115] The present disclosure also provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient, and one or more of: a nucleic acid construct described herein, an AAV virus described herein, a host cell. Typically, the pharmaceutical composition is administered for therapy.

[0116] As described herein, the pharmaceutical composition can be formulated by any method known or developed in the field of pharmacology, including but not limited to contacting the active ingredient (e.g., viral particle or recombinant vector) with excipients or other auxiliary ingredients, dividing or packaging the product into dosage units. The viral particles herein can be formulated to have desirable characteristics, such as increased stability, increased transfection of cells, sustained or delayed release, biodistribution or tropism, modulation or enhanced translation of encoded proteins in vivo, and release profile of encoded proteins in vivo.

[0117] Accordingly, the pharmaceutical composition can further comprise saline, a lipidoid, a liposome, a lipid nanoparticle, a polymer, a lipoplex, a core-shell nanoparticle, a peptide, a protein, a cell transfected with a viral vector (e.g., for implantation into a subject), a nanoparticle mimic, or a combination thereof. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle. In some embodiments, the nanoparticle is a self-assembling nucleic acid nanoparticle.

[0118] The pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as single unit doses, and / or as multiunit doses. The amount of active ingredient can be determined by providing dosages and / or multiples of dosages, for example, half or third of a dosage.

[0119] As used herein, the term "pharmaceutically acceptable excipient" encompasses any standard pharmaceutical carrier, such as sterile phosphate buffered saline solution, water, and emulsions, such as oil / water or water / oil emulsions, with or without various types of wetting agents, excipients, stabilizers, preservatives, viscosity-inducing agents. Examples of excipients are found in Martin (1975) Remington's Pharm. Sci. 15th Ed. (Mack Publ. Co., Easton).

[0120] The pharmaceutical compositions of the present disclosure can include one or more excipients, each excipient in an amount that together increases the stability of the viral vector, increases cell transfection or viral vector transduction, increases expression of the protein encoded by the viral vector, and / or alters the release profile of the protein encoded by the viral vector. Non-limiting examples of excipients include solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, or a combination thereof.

[0121] In some embodiments, the pharmaceutical composition comprises a cryoprotectant. The term "cryoprotectant" refers to an agent that is capable of reducing or eliminating damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and / or mannitol.

[0122] Delivery vehicles

[0123] The present disclosure also provides a nucleic acid delivery reagent comprising a nucleic acid construct (e.g., an expression cassette or a vector) described herein and a delivery vehicle, which includes a virus (e.g., AAV, lentivirus, or adenovirus), a virus-like particle (e.g., PEG10), a lipid nanoparticle (LNP). A lipid nanoparticle is a spherical vesicle composed of a phospholipid bilayer, typically consisting of four components: a cationic lipid, a helper lipid, cholesterol, and a PEG lipid. Exemplary lipid nanoparticles include, e.g., BioNTech’s BNT162b2 and Moderna’s mRNA-1273.

[0124] Methods and uses

[0125] The present disclosure provides a method of preventing or treating a disease, comprising administering to a subject a therapeutically effective amount of a nucleic acid construct, an AAV virus, or a pharmaceutical composition described herein. The method does not require increasing the expression or activity of CRX in rod cells.

[0126] The therapeutic method herein achieves treatment of degenerative eye diseases by increasing the expression or activity of CRX protein in cone cells, thereby inhibiting degenerative death of cone cells. In one aspect, the method can treat degenerative eye diseases even when rod cells are damaged or dead, or even all dead. In another aspect, the present disclosure can be widely applied to the treatment of various types of degenerative eye diseases in which degenerative death of cone cells occurs, and is not limited to eye diseases caused by autonomous death of rod cells. Therefore, the method of the present disclosure can be used to treat: degenerative eye diseases in which degenerative death of rod cells occurs and degenerative death of cone cells begins; degenerative eye diseases in which degenerative death of rod cells does not occur but degenerative death of cone cells begins; wherein the degenerative death of rod cells is autonomous degenerative death of rod cells; wherein the degenerative death of cone cells is non-autonomous degenerative death of cone cells.

[0127] In addition, the method can be used for degenerative eye diseases caused by mutations in the CRX gene, and can also be used for degenerative eye diseases in which the CRX gene is normal (e.g., RP subtypes that are not CRX gene defects). That is, the subject’s cone cells can not carry pathogenic mutations in the CRX gene, and the degenerative eye disease can not be a disease caused by mutations in the CRX gene.

[0128] In one or more embodiments, the degenerative eye disease is a degenerative eye disease associated with degenerative death of cone cells. In one embodiment, the degenerative eye disease is retinitis pigmentosa (i.e., rod-cone dystrophy), age-related macular degeneration, and cone-rod dystrophy, in particular, retinitis pigmentosa, age-related macular degeneration, and the like caused by mutations in other genes than the CRX gene.

[0129] As used herein, "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and can include even a small reduction in one or more measurable markers of the disease or condition in treatment (e.g., degenerative ocular disease). Treatment can optionally include a reduction or alleviation of symptoms of a disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. As used herein, "treatment" of a disease in a subject refers to (1) preventing the disease from occurring in a subject that is predisposed or does not yet exhibit symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or symptoms of the disease. As understood in the art, "treatment" is a method for obtaining beneficial or desired results, including clinical results. Beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, whether detectable or undetectable, reduction in extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether partia l or total.

[0130] A "subject" for diagnosis or treatment is a cell or an animal, such as a mammal or a human. The subject is not limited to a particular species, including non-human animals that receive diagnosis or treatment and those animals that receive infection or animal models, including but not limited to simian, murine, rat, canine, or rabbit species, as well as other livestock, sport, or pet animals. In some embodiments, the subject is a human.

[0131] As used herein, the term "effective amount" is intended to mean the amount that is sufficient to achieve the desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition in question and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, in some embodiments, an effective amount is an amount sufficient to result in partial or full restoration of function of a defective gene in a subject. In other some embodiments, an effective amount of an AAV virus is an amount sufficient to result in expression of a gene in a subject. The skilled artisan will be able to determine an appropriate amount based on these and other factors.

[0132] In some embodiments, the effective amount will depend on the size and nature of the application in question. It also depends on the nature and sensitivity of the target subject and the method used. The person skilled in the art will be able to determine the effective amount based on these and other considerations. According to the embodiments, the effective amount can comprise, consist essentially of, or consist of one or more administrations of the composition.

[0133] As used herein, the term "administering" is intended to mean delivering a substance to a subject, such as an animal or human. Administration can be carried out in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective dosage and regimen are known to those of skill in the art and will vary depending on the composition used for therapy, the purpose of the therapy, and the age, health, or gender of the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, or in the case of pets and other animals, by the treating veterinarian. Herein, the administration is intraocular administration. In one or more embodiments, the intraocular administration is selected from the group consisting of intravitreal or subretinal, subtenon, subconjunctival, intramuscular, periocular, retrobulbar, suprachoroidal, and / or intrascleral administration.

[0134] Dosage and administration

[0135] Methods of determining the most effective dosage and regimen are known to those of skill in the art and will vary depending on the composition used for therapy, the purpose of the therapy, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. The dosage can be affected by the route of administration. Suitable dosage formulations and methods of administering the agents are known in the art. Non-limiting examples of such suitable dosages can be as low as 10E9 vector genomes per administration to as much as 10E17 vector genomes per administration.

[0136] Suitable for administration of the nucleic acid delivery agent, AAV virus, or pharmaceutical composition of the present application include intraocular administration, such as intravitreal or subretinal, subtenon, subconjunctival, intramuscular, periocular, retrobulbar, suprachoroidal, and / or intrascleral administration.

[0137] Administration of the AAV virus or pharmaceutical composition of the present application can be carried out in one dose, continuously or intermittently throughout the course of treatment. In some embodiments, the AAV virus or pharmaceutical composition of the present application is administered intraocularly by injection. The AAV viruses and compositions of the present application can be administered in combination with other known treatments for the disorder being treated.

[0138] Kits

[0139] In some embodiments, the agents, vectors, or compositions described herein can be assembled into a pharmaceutical or diagnostic or research kit to facilitate their use in therapeutic, diagnostic, or research applications. In some embodiments, the kits of the present application include any of the nucleic acid constructs, AAV viruses, host cells, or pharmaceutical compositions described herein.

[0140] In some embodiments, the kits further include instructions for use. In particular, such kits can include one or more of the reagents described herein, along with instructions describing the intended use and correct use of the reagents. In some embodiments, the kits can include instructions for mixing one or more components of the kit and / or isolating and mixing a sample and administering to a subject. In some embodiments, the reagents in the kit are in a pharmaceutical formulation and dosage appropriate for the particular application and method of administration of the reagents. Kits for research purposes can contain components in appropriate concentrations or amounts for conducting various experiments.

[0141] Kits can be designed to facilitate use of the methods described herein, and can take many forms. Each composition of a kit can be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder), as applicable. In certain instances, some compositions can be constitutable or otherwise processable (e.g., into active form), for example, by addition of a suitable solvent or other substance (e.g., water or cell culture media), which can or can not be provided with the kit. In some embodiments, compositions can be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde. In some embodiments, the preservation solution contains an amount of metalloprotease inhibitor.

[0142] In some embodiments, the kits contain any one or more of the components described herein in one or more containers. Thus, in some embodiments, the kits can include containers holding the reagents described herein. The reagents can be in the form of a liquid, gel, or solid (powder). The reagents can be prepared aseptically, packaged in syringes, and shipped frozen. Alternatively, they can be contained in vials or other containers for storage. A second container can have other reagents prepared aseptically. Alternatively, the kit can include pre-mixed active agents and shipped in syringes, vials, tubes, or other containers. The kits can have one or more or all of the components needed to administer the reagents to a subject, such as syringes, topical application devices, or IV needles and bags.

[0143] The present application will be illustrated hereinafter in the context of specific examples. It will be understood that these examples are merely illustrative and are not intended to limit the scope of the present application. The methods and materials used in the examples are those that are conventional in the art, unless otherwise specified.

[0144] Example

[0145] Example 1: AAV vector construction

[0146] CRX mouse full-length cDNA was purchased from Shenguo Bioengineering (Shanghai) Co., Ltd. The obtained CRX cDNA (SEQ ID NO: 4) was cloned into the empty vector of AAV-RO1.7 (RedO1.7 kb) (SEQ ID NO: 15) with SpeI and EcoRI enzyme sites by Gibson Assembly Master Mix (New England BioLabs) through forward primer F (SEQ ID NO: 1): 5'-ggacagggctttccatagcAATGGCTCAGTGGTTAAGAA; reverse primer R (SEQ ID NO: 2): 5'-gattatctcgagcggGAATTCTACAAGATCTGAAACTTCC, which was derived from the published article: Xue Y, Wang SK, Rana P, West ER, Hong CM, Feng H, Wu DM, Cepko CL. AAV-Txnip prolongs cone survival and vision in mouse models of retinitis pigmentosa. Elife. 2021 Apr 13;10:e66240. The reaction temperature was 50 degrees Celsius, and the reaction time was 20 minutes. The obtained plasmid AAV-RO1.7-CRX was confirmed by Sanger sequencing, marker enzyme digestion, and whole plasmid sequencing. The expression frame and vector map thereof are shown in FIGS. 1 and 2, respectively. Figure 1 and Figure 2

[0147] Example 2: Selection of Promoters

[0148] The published AAV fluorescent protein vectors containing RedO (2 kb, 1.7 kb), GNAT2 (2 kb, 0.5 kb), RHO, and GRK1 promoters (https: / / pubmed.ncbi.nlm.nih.gov / 30833387 / , https: / / pubmed.ncbi.nlm.nih.gov / 33847261 / ) were used. The vectors were packaged into AAV8 capsids, injected into the retinas of P0 WT mice, and the eyes were collected at P20-P30 for cryosectioning. Confocal imaging was used to detect the spatial position of the expression of fluorescent proteins in the samples, and the cell expression specificity and expression intensity of different promoters were identified. The results of the in vivo expression verification experiment of cone-specific promoters are shown in FIGS. 3-6. Figure 3 ​The results show that the RedO, GNAT2 promoter has high expression in cone cells and strong cone specificity; the RHO promoter has high expression in rod cells and strong rod specificity; the GRK1 promoter has low expression in cone cells and shows certain rod specificity, and the expression in rod cells is lower than that of the RHO promoter.

[0149] Example 3: Animal experiment for protecting cone cell from autonomous death

[0150] First, the successfully constructed plasmid AAV-RO1.7-CRX was co-transfected with 293T cells and Helper plasmid (SEQ ID NO: 16), AAV8 capsid plasmid (SEQ ID NO: 17) to package into AAV8 capsid, and after 72 h, the virus was purified using iodixanol gradient. The experiment was performed at P0 (day 0 of birth) in RD1 mice (FVB strain carrying PDE6B gene deletion mutation, purchased from Vintorbio). First, the left and right eyes of the same litter were grouped, for example: 10 left eyes were injected in the experimental group, and 10 right eyes were injected in the control group. AAV and PBS were mixed, and 1x10 9 vg / eye was injected subretinally, and AAV8-RedO (2 kb)-H2BGFP virus (H2BGFP sequence as shown in SEQ ID NO: 18) was co-injected to specifically label the cone cell nucleus. The control group was only injected with the same dose of AAV8-RedO-H2BGFP virus to specifically label the cone cell nucleus.

[0151] When the mice were P50 or P20, the retinas were collected, flattened, and the cone cells within 1 / 2 radius were counted using a published MATLAB script program (Xue Y, Wang SK, Rana P, West ER, Hong CM, Feng H, Wu DM, Cepko CL. AAV-Txnip prolongs cone survival and vision in mouse models of retinitis pigmentosa. Elife. 2021 Apr 13;10:e66240.) and the results were counted. The results are shown in Figure 4-8 . Figure 4 The left graph is a representative P50 retinal original image selected from the control group injected with only AAV-RedO-H2BGFP, and 4185 is the counting result of the cones in 1 / 2 radius corresponding to the graph; the right graph is a representative retinal original image selected from the group co-injected with AAV-RO1.7-CRX and AAV-RedO-H2BGFP, and 14019 is the counting result of the cones in 1 / 2 radius corresponding to the graph. Figure 5The overall distribution of cone counts in the P50 RD1 retinal half radius in the control and experimental groups is shown. Each data point in the figure is the result of a single retinal count. Statistically, the cone survival rate in the experimental group injected with AAV-RO1.7-CRX (n = 28 retinas) was 227% higher than that in the control group (ROHG, n = 19 retinas). Figure 4 4185 and 14019 correspond to Figure 5 A data point in two sets of data.

[0152] Figure 6 The left figure is a representative P20 retinal original image selected from the control group injected with AAV-RedO-H2BGFP only, and 12602 is the cone counting result in the 1 / 2 radius corresponding to the image; the right figure is a representative retinal original image selected from the group co-injected with AAV-RO1.7-CRX and AAV-RedO-H2BGFP, and 12854 is the cone counting result in the 1 / 2 radius corresponding to the image. Figure 7 The overall distribution of cone counts in the 1 / 2 radius of the P20RD1 retina in the control and experimental groups is shown. Each data point in the figure is the result of the count of a single retina. Statistically, no significant difference in cone survival was found in the experimental group injected with AAV-RO1.7-CRX (n = 9 retinas) compared with the control group (ROHG, n = 5 retinas). Figure 6 12602 and 12854 correspond to Figure 7 A data point in two sets of data.

[0153] Figure 8 It will Figure 5 and Figure 7 The data are plotted in the same figure with age as the horizontal axis. The results showed that the cone count in the radial half of the RD1 retina remained unchanged from P20 to P50 in the AAV-RO1.7-CRX group, while the cone count in the control group decreased significantly during the same period, suggesting that AAV-RO1.7-CRX blocked the non-autonomous death of RP cones.

[0154] Example 4: Unveiling the mechanism of preventing non-autonomous cone cell death

[0155] During the experiment described in Example 3, 3 eyes of RD1 mice were taken at P20 (one eye of each mouse was injected with control group and the other eye was injected with experimental group). After the eyes were taken, they were fixed in 4% PFA fixation solution at room temperature for 2 hours, and then the retinas were dissected out and embedded in 1:1 ratio of OCT-30% sucrose solution, and then placed in a -80 degree Celsius freezer. After overnight freezing, frozen sections were made in a -20 degree Celsius environment, and glass slides were attached. After washing and staining with PBS solution containing DAPI, a few drops of Fluoromount-G mounting medium (SouthernBiotech) were added, and a cover glass was used for mounting. Then a confocal fluorescence microscope was used for imaging. Figure 9 The left image shows a representative image of the control group retinal section, Figure 9 The middle image shows a representative image of the AAV-RO1.7-CRX experimental group. Both images show that the ONL regressed to only one layer of nuclei, i.e. the nuclei of the cone cells, which is consistent with the reported photoreceptor degeneration in P20 RD1 mice (https: / / pubmed.ncbi.nlm.nih.gov / 19060896 / ). The ONL thickness of the experimental and control group retinal sections was measured using ImageJ software on a computer, respectively. This thickness reflects the P20 RD1 rod death, and the statistics are shown in Figure 9 The right image. The results show that there is no difference in the ONL thickness between the AAV-RO1.7-CRX treatment group and the control group, indicating that AAV-RO1.7-CRX has no effect on the spontaneous degeneration and death of RD1 rods, supporting Figure 4 、 Figure 5 and Figure 8 the protective effect of AAV-RO1.7-CRX on RP cones in

[0156] Example 5: Other RP mouse experiments

[0157] RD10: Mice carrying a point mutation in the Pde6b gene

[0158] AAV and PBS were mixed at P0 of RD10 mice, and subretinal injection was performed at a dose of 1 x 10 9 vg / eye, and AAV8-RedO-H2BGFP was co-injected to specifically label the nuclei of cone cells. During P30-P60, mouse optokinetic reflex behavioral experiments were performed to detect the diurnal vision of mice. When the mice reached P130, the retinas were collected and flattened, and the 1 / 2 radius cone cells were counted using the MATLAB script described above, and the results are shown in the table below. The results show that the number of cone cells in the AAV-RO1.7-CRX treatment group is higher than that in the control group.

[0159] RHO- / -: RHO knockout mice

[0160] AAV and PBS were mixed at P0 of RHO- / - mice at 1 x 10 9 vg / eye, and co-injected with AAV8-Redo-H2BGFP to specifically label the nuclei of cone cells. At around P140, mice were subjected to optokinetic behavioral assay to test the mice's diurnal vision. At P150, retinas were collected and flat-mounted, and 1 / 2 radius of the retinas were counted for cone cells using the MATLAB script described above. The results showed that the AAV-RO1.7-CRX treated group had more cone cells than the control group.

[0161] Example 6: Animal vision test

[0162] Under background light of about 70 cd / m 2 The mice's photopic optokinetic responses (two parameters tested: spatial frequency response threshold and contrast response threshold) were measured using OptoDrum system (Striatech). Spatial frequency response threshold test: the grating's contrast was set at 100%, the temporal frequency was 1.5 Hz, and the tester tested the mice's spatial frequency threshold without knowing the control and experimental groups. During each test, the direction of the grating's movement (clockwise or counterclockwise) was random, and the spatial frequency of each test segment was determined by a software algorithm until the software algorithm automatically determined the spatial frequency response threshold of the tested mice. Contrast threshold test: the spatial frequency was set at 0.128 c / d, the temporal frequency was 1.5 Hz, and the tester tested the mice's contrast threshold without knowing the control and experimental groups. During each test, the direction of the grating's movement (clockwise or counterclockwise) was random, and the spatial frequency of each test segment was determined by a software algorithm until the software algorithm automatically determined the contrast response threshold of the tested mice. The RD10 and RHO- / - mice were subjected to optokinetic test, but the RD1 line was not tested because the line had lost vision at a very young age and could not perform any meaningful vision test. The results showed that the AAV-RO1.7-CRX treated group had better optokinetic response than the control group (i.e., higher spatial frequency response threshold and lower contrast response threshold).

[0163] Example 7: Light-dark discrimination test

[0164] The diurnal vision of mice can be evaluated using their innate light-avoidance behavior. A 28 cm (length) x 28 cm (width) x 21 cm (height) plastic chamber is divided into two equal-sized compartments: a dark compartment and a bright, illuminated compartment (about 900 lux). The temperature of the two compartments differs by no more than 1 °C. A small opening connects the two compartments, allowing the test mice to move freely throughout the space. At the beginning of each test, the mice are placed in the bright room, and the system begins recording their activity for 9 minutes after the mice cross the narrow door into the dark room. The position of each mouse is determined by the bottom pressure sensor and analyzed using the device's accompanying software, and the percentage of time spent in the dark compartment is calculated. This light-avoidance behavior test is performed on RD1 mice older than P30, and the results show that the RD1 mice in the AAV-RO1.7-CRX treatment group spend more than 50% of their time in the dark compartment, significantly higher than the control group results (about 50%, indicating that blind mice have no light perception in the retina and cannot distinguish between dark and light rooms).

[0165] Part of the sequence in this paper

[0166] SEQ ID NO: 4 Mouse CRX nucleotide sequence

[0167] atggctcagtggttaagaacactgactgctctttcaaaggtgccctcatctgggccacagtctctgaagatcatgatggcatatatgaacccggggcctcactattcagtcaatgccttgg

[0168] ctctgagtggccccaatgtggacctgatgcaccaggctgtcccatactcaagtgcccctaggaagcagcggcgggagcggaccacattcaccaggagccagctggaggagctggaggccctgtttg

[0169] ccaagacccagtacccggatgtgtatgcacgtgaggaggttgctcttaagatcaatctgcctgagtccagggtccaggtctggttcaagaatcgtagggcgaaatgcagacagcagcgacagcagc

[0170] agaaacagcaacagcagcccccgggggcacagaccaaggctcgtcctgcgaagaggaaggcagggacatccccgagaccctctacagatgtttgtacagatcctttgggcatctcagattcttac

[0171] agcccatctctgcctggcccctcaggttcacctaccacagcagtggccaccgtgtccatttggagtccagcctcagaggctcctttgcctgaggcccagagagccggcctagtggcttctgggccctct

[0172] ctcacctcagccccttatgccatgacctatgccccggcttctgctttctgttcttccccttcagcttatgcatctccaagttcctatttcagtgggttggatccctacctttcgcccatggtgccccaacttgg

[0173] gggtccggctctcagccccctctcaggcccctctgtggggccatccctggcccagtcccccacctccttgtcaggccagagctatagcacctacagtcctgtggacagcctggaattcaaggacccca

[0174] caggcacctggaaatttacctacaatcccatggaccctctggactacaaagatcagagtgcctggaagtttcagatcttgtag

[0175] SEQ ID NO:5 human CRX nucleotide sequence:

[0176] atgatggcgtatatgaacccggggccccactattctgtcaacgccttggccctaagtggccccagtgtggatctgatgcaccaggctgtgccctacccaagcgcccccaggaagcagcg

[0177] gcgggagcgcaccaccttcacccggagccaactggaggagctggaggcactgtttgccaagacccagtacccagacgtctatgcccgtgaggaggtggctctgaagatcaatctgcctgagtccag

[0178] ggttcaggtttggttcaagaaccggagggctaaatgcaggcagcagcgacagcagcagaaacagcagcagcagcccccagggggccaggccaaggcccggcctgccaagaggaaggcgggca

[0179] cgtccccaagaccctccacagatgtgtgtccagaccctctgggcatctcagattcctacagtccccctctgcccggcccctcaggctccccaaccacggcagtggccactgtgtccatctggagccca

[0180] gcctcagagtcccctttgcctgaggcgcagcgggctgggctggtggcctcagggccgtctctgacctccgccccctatgccatgacctacgccccggcctccgctttctgctcttccccctccgcctatg

[0181] ggtctccgagctcctatttcagcggcctagacccctacctttctcccatggtgccccagctagggggcccggctcttagccccctctctggcccctccgtgggaccttccctggcccagtcccccacctc

[0182] cctatcaggccagagctatggcgcctacagccccgtggatagcttggaattcaaggaccccacgggcacctggaaattcacctacaatcccatggaccctctggactacaaggatcagagtgcctg

[0183] gaagtttcagatcttgtag

[0184] SEQ ID NO:6 Amino acid sequence of mouse CRX

[0185] MAQWLRTLTALSKVPSSGPQSLKIMMAYMNPGPHYSVNALALSGPNVDLMHQAVPYSSAPRKQRRERTTFTRSQLEELEALFAKTQYPDVYAR

[0186] EEVALKINLPESRVQVWFKNRRAKCRQQRQQQKQQQQPPGAQTKARPAKRKAGTSPRPSTDVCTDPLGISDSYSPSLPGPSGSPTTAVATVSIWSPASEAP

[0187] LPEAQRAGLVASGPSLTSAPYAMTYAPASAFCSSPSAYASPSSYFSGLDPYLSPMVPQLGGPALSPLSGPSVGPSLAQSPTSLSGQSYSTYSPVDSLEFKDPTGT

[0188] WKFTYNPMDPLDYKDQSAWKFQIL

[0189] SEQ ID NO:7 human CRX amino acid sequence

[0190] MMAYMNPGPHYSVNALALSGPSVDLMHQAVPYPSAPRKQRRERTTFTRSQLEELEALFAKTQYPDVYAREEVALKINLPESRVQVWFKNRRAK

[0191] CRQQRQQQKQQQQPPGGQAKARPAKRKAGTSPRPSTDVCPDPLGISDSYSPPLPGPSGSPTTAVATVSIWSPASESPLPEAQRAGLVASGPSLTSAPYAMT

[0192] YAPASAFCSSPSAYGSPSSYFSGLDPYLSPMVPQLGGPALSPLSGPSVGPSLAQSPTSLSGQSYGAYSPVDSLEFKDPTGTWKFTYNPMDPLDYKDQSAWKF

[0193] QIL

[0194] SEQ ID NO:8 RedO-1.7kb (RO1.7) promoter sequence

[0195] aatagaagggtgggtgcaggaggctgaggggtggggaaagggcatgggtgtttcatgaggacagagcttccgtttcatgcaatgaaaagagtttggagacggatggtggtgactgga

[0196] ctatacacttacacacggtagcgatggtacactttgtattatgtatattttaccacgatctttttaaagtgtcaaaggcaaatggccaaatggttccttgtcctatagctgtagcagccatcggctgttag

[0197] tgacaaagcccctgagtcaagatgacagcagcccccataactcctaatcggctctcccgcgtggagtcatttaggagtagtcgcattagagacaagtccaacatctaatcttccaccctggccaggg

[0198] ccccagctggcagcgagggtgggagactccgggcagagcagagggcgctgacattggggcccggcctggcttgggtccctctggcctttccccaggggccctctttccttggggctttcttgggccgc

[0199] cactgctcccgctcctctccccccatcccaccccctcaccccctcgttcttcatatccttctctagtgctccctccactttcatccacccttctgcaagagtgtgggaccacaaatgagttttcacctggcct

[0200] ggggacacacgtgcccccacaggtgctgagtgactttctaggacagtaatctgctttaggctaaaatgggacttgatcttctgttagccctaatcatcaattagcagagccggtgaaggtgcagaacc

[0201] TACCgcctttccaggcctcctcccacctctgccacctccactctccttcctgggatgtgggggctggcacacgtgtggcccagggcattggtgggattgcactgagctgggtcattagcgtaatcctgg

[0202] ACAAGGGCAGACAGGGCGAGCGGAGGGCCAGCTCCGGGGCTCAGGCAAGGCTGGGGGCTTCCCCCAGACACCCCAC TCCTCCTCTGCTGGACCCCCACTTCATAGGGC ACTTCGTGTTCTCAAAGG

[0203] GCTTCCAAATAGCATGGTGGCCTTGGATGCCCAGGGAAGCCTCAGAGTTGCTTATCTCCCTCTAGACAGAAGGGGAATCTCGGTCAAGAGGGAGAGGTCGCCCTGTTCAAGGCCACCCAGCCAGCTC

[0204] ATGGCGGTAA TGGGACAAGGCTGGCCAGCCATCCCAC CCTCAGAAGGGACCCG GTGGGGCAGGTGATCTCAGAGGAGGCTC ACTTCTGGGTCTCACATTCTTGGATCCGGTTCCAGGCCTCGGCCCT

[0205] AAATAGTCTCCCTGGGCTTTCAAGAGAACACATGAGAAAGGAGGATT CGGGCTCTGAGCAGTTTCACCACCCACCCCCCAGTCTGCAATCCTGACCCGTGGGTCCACCTGCCCAAAGGCGGAC

[0206] GCAGGACAGTAGAAGGGAACAGAGAACACATAAACACAGAGAGGGCCACAGCGGCTCCCACAGTCACCGCCACCTTCCTGGCGGGGATGGGTGGGGCGTCTGAGTTTGGTTCCCAGCAAATCCC

[0207] TCTGAGCCGCCCTTGC GGGCTCGCCTCAGGAGCAGGGGAGCAAGAGGTGGGAGGAGGAGGTCTAAGTCCCAGGCCCAATTAAGAGATCAGGTA GTGTAGGGTTTGGGAGCTTTTAAGGTGAAGAG

[0208] GCCC GGGCTGATCCCACAGGCCAGTATAAAGCGCCGTGACCCTCAGGTGATGC GCCAGGGCCGGCTGCCGTCGGGGACAGGGCTTTCCATAGC

[0209] SEQ ID NO: 9 RedO-2kb promoter sequence

[0210] CCTACAGCAGCCAGG GTGAGATTATGAGGCTGAGCTGAGAATATCAAGACTGTACC GAGTAGGGGGCCTTGGCAAGTGTGGAGAGCCC GGCAGCTGGGGCAGAGGGCGGAGTACGG

[0211] TGTGC GTTTACGGACCTCTTCAAACGAGG TAGGAAGGTCAGAAGTCAAAAAGGGAACAAATGATGTTTAACCACACAAAAATGAAAATCCAATGGTTGGATATCCATTCCAAATACAAAA GGCA

[0212] ACGGATAAGTGATCCGGGCCAGGCACAGAAGGCCATGCACCCG TAGGATTGC ACTCAGAGCTCCCAAATG CATAGGAATAGAAGGGTGGGTGCAGGAGGCTGAGGGGTGGGGAAAGGGCATGG

[0213] GTGTTT CATGAGGACAGAGCTTCCGTTTCATGCAATGAAAAGAGTTTGGAGACGGATGGTG GTGACTGGACTATACACTTACACACGGTAGCGATG GTACACTTTGTATTATGTATATT TTACCACGAT

[0214] tttttaaagtgtcaaaggcaaatggccaaatggttccttgtcctatagctgtagcagccatcggctgttagtgacaaagcccctgagtcaagatgacagcagcccccataactcctaatcggctctc

[0215] ccgcgtggagtcatttaggagtagtcgcattagagacaagtccaacatctaatcttccaccctggccagggccccagctggcagcgagggtgggagactccgggcagagcagagggcgctgacat

[0216] tggggcccggcctggcttgggtccctctggcctttccccaggggccctctttccttggggctttcttgggccgccactgctcccgctcctctccccccatcccaccccctcaccccctcgttcttcatatcct

[0217] tctctagtgctccctccactttcatccacccttctgcaagagtgtgggaccacaaatgagttttcacctggcctggggacacacgtgcccccacaggtgctgagtgactttctaggacagtaatctgctt

[0218] taggctaaaatgggacttgatcttctgttagccctaatcatcaattagcagagccggtgaaggtgcagaacctaccgcctttccaggcctcctcccacctctgccacctccactctccttcctgggatgt

[0219] gggggctggcacacgtgtggcccagggcattggtgggattgcactgagctgggtcattagcgtaatcctggacaagggcagacagggcgagcggagggccagctccggggctcaggcaaggctg

[0220] ggggcttcccccagacaccccactcctcctctgctggacccccacttcatagggcacttcgtgttctcaaagggcttccaaatagcatggtggccttggatgcccagggaagcctcagagttgcttatc

[0221] tccctctagacagaaggggaatctcggtcaagagggagaggtcgccctgttcaaggccacccagccagctcatggcggtaatgggacaaggctggccagccatcccaccctcagaagggacccgg

[0222] tggggcaggtgatctcagaggaggctcacttctgggtctcacattcttggatccggttccaggcctcggccctaaatagtctccctgggctttcaagagaaccacatgagaaaggaggattcgggctc

[0223] tgagcagtttcaccacccaccccccagtctgcaaatcctgacccgtgggtccacctgccccaaaggcggacgcaggacagtagaagggaacagagaacacataaacacagagagggccacagc

[0224] ggctcccacagtcaccgccaccttcctggcggggatgggtggggcgtctgagtttggttcccagcaaatccctctgagccgcccttgcgggctcgcctcaggagcaggggagcaagaggtgggagg

[0225] aggaggtctaagtcccaggcccaattaagagatcaggtagtgtagggtttgggagcttttaaggtgaagaggcccgggctgatcccacaggccagtataaagcgccgtgaccctcaggtgatgcgc

[0226] cagggccggctgccgtcggggacagggctttccatagc

[0227] SEQ ID NO: 10 SynP136 (GNAT2-2kb) sequence

[0228] acgcgtagaggcaggccgagtttgaggccagcctggtctacacaggcgttctaggagaacctgtctcatatgcacatgggcctgggattacacatacaactgcaatggcagcacttgg

[0229] aaagcggaagcaggagaatcggaatttcacactcatccttcattatagaagtccaaacctgtgctaagctacttggacatcgctaaaaaacagcaaaaatctttctaggaattgccaatgtatacac

[0230] accaagttgtatttttgtagggcagacttttccaacttgccagatgataataatcatttatattagctacatttcaggctccagaattaacactgttactgaaatgtataggttctaaaacataccatttc

[0231] caaatattttaaaggattaacatttttgaaaagcatgtgatcttccaaccatattttagggaacagacataagaagtaggcaagtttgggaagaaacagttcctgagggcatttcttcccaaccttga

[0232] atgccctgtgggttaactgaggtcttggtaaaatgtagattatttactggactttttgaactgagagtgcatttctaacaaaactccagggtatgtggtccttgcattacatatggggtagcaacattct

[0233] aaagcagtgtttttcaacctgtggatcaagacccacagaagtcacatagcagatatcctgcctattagatatttacattaagattcagagcagtagcagaataggagtcatcccaacctgaggaact

[0234] gcatcagagtcccagcatcaggaagggtgagagccactgagctaaagtcctctaggtgagggggctgccccggaaagactcatttaaatgaaaccactgacacagagagctgacagatgaggtg

[0235] ggttccgtgtctgtgaggctctgctggtcgtctcctcactcccatggaagaaccaccgagatgagggcgaggggcagagctaacccagcctctaagtaggcagagtctagtgtccagctgcccaagg

[0236] aagaagtttgctgtgtgaggtggccctgatgtccgcacacacaaaatgccagtgaagtctacttgaccaagtgaagctggtgtggaatgggaagaagcacacacagtcagtctctctgcacacact

[0237] ctgtccttcacttcttcacttaccagagatttgatgagaacctactagcagatcagatttgatccctgagtggaaaaaacgtacagtgggagataaaagaggaaaacaacggatctgagtttgaggt

[0238] tagcctagtctgagcaagccggatacacagtaagaccctgtctcacatacatccactcgcacgcgcgcgcacacaaacacacacacacacacacacacacacacacacacacacacacatcgtgc

[0239] taaggacaagataggcatcctgagagatgagccaggacaaagaaccagtaatagctcctggagcagcacatctgttttgccaggattatcccttggatctcttaaaaccgagaccttgtaatctgaa

[0240] gactcaacttgggctctacccttaaccttcagctctatgatgcaagtgagtccacaggaccggaggctttgagatgagcttttcagaagggagggttggccgcttgctcccagagctccagcacctg

[0241] cattcttctggctatgtcagaagccagatcatttccctcgttaaaaaacaaaaacaaaaaaaacaaacaaaaaaaatgttagtctttgccctttatctgcctggcaaagcttttaattggcttgatctgtca

[0242] ttccgctagacataaggggacaatccccggattaggaaggagctctccagctcgggtaaggagtctcaaggcaaggtaggcaagcaccaccggtccgcactctcgcccagctttacgggaagaa

[0243] agagaatgttactctatcctaacatattttcctttcctctatctcacagataggaaaaatttaagagccagagggaacgtcccttctcagaggagacagcagaag

[0244] SEQ ID NO:11SynPVI(GNAT2-0.5kb) sequence

[0245] catcctgagagatgagccaggacaaagaaccagtatagctcctggagcagcacatctgttttgccaggattatcccttggatctcttaaaaaccgagaccttgtaatctgaagaactcaa

[0246] cttgggctctacccttaaccttcagctctatgatgcaagtgagtccacaggaccggaggctttgagatgagcttttcagaagggagggttggccgcttgctcccagagctccagcacctgcattcttc

[0247] tggctatgtcagaagccagatcatttccctcgttaaaaacaaaaacaaaaaaacaaacaaacaaaatgttagtctttgccctttatctgcctggcaaagcttttaattggcttgatctgtcattccgct

[0248] agacataaaggggacaatccccggattaggaaggagctctccagctcgggtaaggagtctcaaggcaaggtaggcaagcaccaccggtccgcactctcgcccagcttttacgggaagaagaga

[0249] SEQ ID NO: 12 Mouse ARR3 (cone arrestin) promoter sequence

[0250] ggttcttcccattttggctacatggtctttttttttacctttttggttcctttggccttttggcttttggcttccagggcttctggatcccccccaacccctcccatacacatacacatgtgcactc

[0251] gtgcactcaacccagcacaggataatgttcattcttgacctttccacatacatctggctatgttctctctcttatctacaataaatctcctccactatacttaggagcagttatgttcttcttctttctttcttt

[0252] tttttttttttcattcagtaacatcatcagaatcccctagctctggcctacctcctcagtaacaatcagctgatccctggccactaatctgtactcactaatctgttttccaaactcttggcccctgagctaa

[0253] ttatagcagtgcttcatgccacccaccccaaccctattcttgttctctgactcccactaatctacacattcagaggattgtggatataagaggctgggaggccagcttagcaaccagagctggagg

Claims

1. A nucleic acid construct comprising a cone cell-specific promoter and a coding sequence of a CRX protein, Preferably, the nucleic acid construct further comprises a polyadenylation signal.

2. The nucleic acid construct of claim 1, wherein, The polyadenylation signal is selected from the group consisting of a human growth hormone polyadenylation signal, a bovine growth hormone polyadenylation signal and an SV40 polyadenylation signal, Preferably, the nucleic acid construct comprises, in order, a cone cell-specific promoter, a coding sequence of a CRX protein and a polyadenylation signal, More preferably, the coding sequence of the CRX protein is operably linked to the cone-specific promoter and the polyadenylation signal. 3.The nucleic acid construct of claim 2, wherein, The CRX protein has a sequence as set forth in SEQ ID NO: 6 or 7 or a sequence having at least 80% sequence identity to the sequence as set forth in SEQ ID NO: 6 or 7, and / or The coding sequence of the CRX protein has a sequence as set forth in SEQ ID NO: 4 or 5 or a variant having at least 80% sequence identity to the sequence as set forth in SEQ ID NO: 4 or 5.

4. The nucleic acid construct of claim 2, wherein The cone-specific promoter is a RedO promoter, a GNAT2 promoter or an ARR3 promoter, Preferably, The sequence of the RedO promoter is as set forth in SEQ ID NO: 8 or 9, The sequence of the GNAT2 promoter is as set forth in SEQ ID NO: 10 or 11, The sequence of the ARR3 promoter is as set forth in SEQ ID NO:

12.

5. The nucleic acid construct of claim 4, wherein The nucleic acid construct further comprises a woodchuck hepatitis virus post-transcriptional regulatory element WPRE or a variant WPRE3 thereof, Preferably, the sequence of the woodchuck hepatitis virus post-transcriptional regulatory element WPRE is as set forth in SEQ ID NO: 13 and the sequence of WPRE3 is as set forth in SEQ ID NO: 14, More preferably, the nucleic acid construct further comprises an ITR region comprising a 5’ ITR and a 3’ ITR.

6. The nucleic acid construct of any of claims 1-5, wherein The nucleic acid construct comprises a sequence as set forth in SEQ ID NO: 3, Preferably, the nucleic acid construct is a vector, More preferably, the vector is a cloning vector, an integration vector or an expression vector, Further preferably, the vector is an AAV vector, a lentivirus vector or an adenovirus vector, preferably an AAV vector. 7.A nucleic acid delivery reagent comprising the nucleic acid construct of any one of claims 1-6 and a delivery vehicle, preferably, the delivery vehicle comprises a virus, a virus-like particle, a lipid nanoparticle. 8.An AAV virus comprising the nucleic acid construct of any one of claims 1-6. 9.A host cell comprising the nucleic acid construct of any one of claims 1-6 or the AAV virus of claim 8. 10.Use of an agent specifically improving the expression or activity of a CRX protein in cone cells in the preparation of a medicament for treating a degenerative ocular disease or a symptom thereof by inhibiting degenerative death of cone cells, Preferably, the agent that specifically increases the expression or activity of CRX protein in cone cells is selected from one or more of the following: the nucleic acid construct of any one of claims 1-6, the AAV virus of claim 8, the host cell of claim 9, Preferably, the degenerative eye disease is a degenerative eye disease associated with degeneration and death of cone cells; more preferably, the degenerative eye disease is selected from the group consisting of retinitis pigmentosa, age-related macular degeneration, and cone-rod dystrophy.

11. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more of the following: the nucleic acid construct of any one of claims 1-6, the AAV virus of claim 8, the host cell of claim 9.

12. A kit comprising any one or more of the nucleic acid construct of any one of claims 1-6, the AAV virus of claim 8, the host cell of claim 9, and the pharmaceutical composition of claim 11.

13. Use of the nucleic acid construct of any one of claims 1-6, the AAV virus of claim 8, the host cell of claim 9 in the manufacture of a medicament for delaying loss of functional vision in a subject having a degenerative eye disease by inhibiting degeneration and death of cone cells, for delaying death of cone cells in retinitis pigmentosa, or for prolonging diurnal visual function in a patient.

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