Pharmaceutical composition for treating eye disease and method

Abstract

A pharmaceutical composition for treating an eye disease and a method. A nucleic acid construct, comprising a cone cell specific promoter and a coding sequence for a CRX protein. The pharmaceutical composition can delay the death of cone cells in retinitis pigmentosa, and prolong patients' daytime visual function, and can be used for treating retinal degenerative diseases.

Description

Pharmaceutical compositions and methods for treating ocular diseases

[0001] This application claims priority to Chinese application No. 202410470843.1, filed on April 18, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of biomolecules, in particular to pharmaceutical compositions and methods for treating ocular diseases. BACKGROUND

[0003] Retinitis pigmentosa (RP) is the most common type of inherited retinal degenerative disease, with approximately one patient in every 4,000 people worldwide. It is currently 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 are completely dead, causing the patient's visual field to gradually shrink and eventually leading to the gradual loss of day vision, color vision, and central vision. So far, only a few treatment methods targeting a few specific 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 where all rod cells are dead, 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 after all rod cells are dead. This greatly limits the scope of use and the applicable stage of disease for 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 groups and disease stages.

[0004] 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

[0005] 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.

[0006] 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.

[0007] In one or more embodiments, the nucleic acid construct further comprises a polyadenylation signal.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] In one or more embodiments, the cone-specific promoter is a RedO promoter, a GNAT2 promoter, or an ARR3 promoter.

[0013] In one or more embodiments, the RedO promoter is 2 kb or 1.7 kb in length.

[0014] In one or more embodiments, the RedO promoter is RO1.7.

[0015] In one or more embodiments, the GNAT2 promoter is SynP136, 2 kb in length, or SynPVI, 0.5 kb in length.

[0016] 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.

[0017] 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).

[0018] In one or more embodiments, the nucleic acid construct further comprises an ITR region, including a 5’ ITR and a 3’ ITR.

[0019] 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.

[0020] In one or more embodiments, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 3.

[0021] In one or more embodiments, the nucleic acid construct is a vector.

[0022] In one or more embodiments, the vector is a cloning vector, an integration vector, or an expression vector. In one or more embodiments, the nucleic acid construct is a vector.

[0023] In one or more embodiments, the vector is an AAV vector, a lentivirus vector, or an adenovirus vector, preferably an AAV vector.

[0024] 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).

[0025] The second aspect of the present application provides an AAV virus comprising the nucleic acid construct described in the first aspect herein.

[0026] In one or more embodiments, the AAV virus further comprises a nucleic acid construct having AAV virus required genes.

[0027] 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.

[0028] 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.

[0029] In one or more embodiments, the host cell is a 293T cell.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] In one or more embodiments, the method comprises the steps of:

[0035] (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;

[0036] (2) transfecting the plasmid in (1) with Helper plasmid, and any one of the capsid plasmid selected from AAV2, AAV8 or AAV7m8 into 293T cells to package into AAV2, AAV8 or AAV7m8 capsid; and

[0037] (3) collecting the virus from the cell culture.

[0038] In one or more embodiments, the cell culture comprises cells or culture supernatant thereof.

[0039] 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, AAV virus, host cell described in any of the embodiments herein.

[0040] In one or more embodiments, the degenerative death of cone cells is non-autonomous degenerative death of cone cells.

[0041] In one or more embodiments, the degenerative eye disease is a degenerative eye disease associated with degenerative death of cone cells.

[0042] In one or more embodiments, the degenerative eye disease is not a disease caused by mutation of CRX gene.

[0043] 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.

[0044] In one or more embodiments, the degenerative eye disease is retinitis pigmentosa, age-related macular degeneration, etc. caused by mutation of non-CRX gene.

[0045] 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, the AAV virus of the second aspect of the present application.

[0046] In one or more embodiments, the excipient comprises a viscosity-inducing agent.

[0047] In one or more embodiments, the pharmaceutical composition is suitable for intraocular administration.

[0048] In one or more embodiments, the pharmaceutical composition is suitable for intravitreal or subretinal, subtenon, subconjunctival, submuscular, periocular, retrobulbar, suprachoroidal, and / or intrascleral administration.

[0049] 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.

[0050] 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.

[0051] In one or more embodiments, the administering is intraocular administration.

[0052] In one or more embodiments, the intraocular administration is selected from intravitreal or subretinal, subtenon, subconjunctival, submuscular, periocular, retrobulbar, suprachoroidal, and / or intrascleral administration.

[0053] In one or more embodiments, the degenerative death of cone cells is non-autonomous degenerative death of cone cells.

[0054] In one or more embodiments, the degenerative ocular disease is a degenerative ocular disease associated with degenerative death of cone cells.

[0055] In one or more embodiments, the subject’s cone cells do not carry a CRX gene pathogenic mutation.

[0056] In one or more embodiments, the degenerative ocular disease is not a disease caused by a mutation in the CRX gene.

[0057] 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.

[0058] 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.

[0059] The present application also provides a method of delaying functional vision loss 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.

[0060] In one or more embodiments, the administering is intraocular administration.

[0061] 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.

[0062] In one or more embodiments, the degenerative death of cone cells is non-autonomous degenerative death of cone cells.

[0063] In one or more embodiments, the degenerative eye disease is a degenerative eye disease associated with degenerative death of cone cells.

[0064] In one or more embodiments, the subject’s cone cells do not carry a CRX gene pathogenic mutation.

[0065] In one or more embodiments, the degenerative eye disease is not a disease caused by a mutation in the CRX gene.

[0066] 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.

[0067] 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.

[0068] 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 functional vision loss 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.

[0069] In one or more embodiments, the degenerative death of cone cells is non-autonomous degenerative death of cone cells.

[0070] In one or more embodiments, the degenerative eye disease is a degenerative eye disease associated with degenerative death of cone cells.

[0071] In one or more embodiments, the degenerative eye disease is not a disease caused by a mutation in the CRX gene.

[0072] 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.

[0073] In one or more embodiments, the degenerative eye disease is retinitis pigmentosa, age-related macular degeneration, etc. caused by mutations in non-CRX genes.

[0074] Advantages of the present application:

[0075] (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.

[0076] (2) The present application has a significant protective effect on the survival of cone cells in 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

[0077] Figure 1: AAV-RO1.7-CRX schematic diagram.

[0078] Figure 2: AAV-RO1.7-CRX vector map.

[0079] Figure 3: High expression, high specificity cone promoter (RedO, GNAT2) fluorescent protein expression result graph. Highlight signal: fluorescent protein expressed by AAV-promoter; Cone cell: nucleus position outside the ONL layer (1-2 layers at the top of the picture), nucleus is large; Rod cell: nucleus position inside the ONL layer (about 10 layers inside at the bottom of the picture), nucleus is small.

[0080] Figure 4: Representative image of 50-day-old RD1 retina after AAV-RO1.7-CRX treatment.

[0081] Figure 5: Statistics of cone counting in the 1 / 2 radius of the retina of 50-day-old RD1 mice overexpressing CRX in cone cells by AAV-RO1.7-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.

[0082] Figure 6: Representative image of 20-day-old RD1 retina after AAV-RO1.7-CRX treatment.

[0083] Figure 7: Statistics of cone counts in the inner 1 / 2 radius of the retina of 20-day-old RD1 mice overexpressing CRX in cone cells with AAV-RO1.7-CRX. ROHG: control group injected with AAV8-RedO-H2BGFP only.

[0084] Figure 8: Statistics of cone counts in the inner 1 / 2 radius of the retina of 20-day-old, 50-day-old RD1 mice overexpressing CRX in cone cells with AAV-RO1.7-CRX. Data combined from Figures 5 and 7 for easy visual comparison of degenerative changes. Control: control group injected with AAV8-RedO-H2BGFP only, equivalent to ROHG group.

[0085] Figure 9: Representative images of 50-day-old RD1 retinas and statistics of cone counts in the inner 1 / 2 radius of the retina of 50-day-old RD1 mice after treatment with AAV-Arr3-CRX overexpressing CRX in cone cells.

[0086] Figure 10: Representative images of 20-day-old RD1 retinal sections after treatment and statistics of ONL thickness.

[0087] Figure 11: Representative images of 50-day-old RD1 retinas and statistics of cone counts in the inner 1 / 2 radius of the retina of 50-day-old RD1 mice after overexpression of CRX in rod cells with AAV-Rho-CRX.

[0088] Figure 12: Representative images of 130-day-old RD10 retinas and statistics of cone counts in the inner 1 / 2 radius of the retina of 130-day-old RD10 mice after treatment with AAV-RO1.7-CRX overexpressing CRX in cone cells.

[0089] Figure 13: Representative images of 150-day-old RHO- / - retinas and statistics of cone counts in the inner 1 / 2 radius of the retina of 150-day-old RHO- / - mice after treatment with AAV-RO1.7-CRX overexpressing CRX in cone cells.

[0090] Figure 14: Statistics of spatial frequency threshold results of the photopic optokinetic response of 60-day-old RD10 mice after treatment with AAV-RO1.7-CRX overexpressing CRX in cone cells. DETAILED DESCRIPTION

[0091] 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).

[0092] The term "disease and / or condition" refers to the physical state of a subject associated with a disease and / or condition described herein.

[0093] The term "subject" or "patient" can refer to a patient or other animal, especially a mammal, such as a human, dog, monkey, cow, horse, etc., who receives a pharmaceutical composition of the present application to treat, prevent, ameliorate, and / or alleviate a disease or condition of the present application.

[0094] 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 rod cell degeneration and death, but such mutations have less direct effect on cone cell function and survival, with cones only undergoing non-autonomous degeneration and death after the surrounding rods have died off, affecting day-time color and central vision. As noted above, the cause of RP cone non-autonomous degeneration and death has not been fully determined.

[0095] Prior to the present application, the art did not clearly disclose the relationship between the CRX gene and non-autonomous death of cone cells after rod cell death, and the art could not have predicted that specific overexpression of CRX protein in RP cone cells would delay non-autonomous death of cones at the post-rod death stage.

[0096] The present application first discovers that, in RP cone cells (non-CRX gene defect subtype), using a cone-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 CRX gene expression has an excellent protective effect on the cone cells of non-CRX gene defect subtype RP, can delay or even block the non-autonomous death of RP cone cells, prolong the patient's diurnal visual function, and therefore can be widely used in the treatment of various types of degenerative eye diseases that cause cone cell degeneration and death, thereby completing the present application.

[0097] Nucleic acid construct, virus and cell

[0098] The present application first provides a nucleic acid construct (such as an expression cassette or a vector) comprising, in order, a cone-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-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-specific promoter and the polyadenylation signal.

[0099] 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.

[0100] In this context, the cone-specific promoter can be any promoter known in the art that can specifically initiate protein expression in cones, such as the RedO promoter, the GNAT2 promoter, or the ARR3 promoter (https: / / pubmed.ncbi.nlm.nih.gov / 12135752 / ). The sequence of the RedO promoter is shown in SEQ ID NO: 8 (RedO-1.7kb, RO1.7) or SEQ ID NO: 9 (RedO-2kb). The sequence of the GNAT2 promoter is shown in SEQ ID NO: 10 (GNAT2-2kb, SynP136) or SEQ ID NO: 11 (GNAT2-0.5kb, SynPVI). The sequence of the ARR3 promoter is shown in SEQ ID NO: 12.

[0101] In this context, the sequence of the mouse CRX protein has SEQ ID NO: 6 or a variant having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity thereto; the sequence of the human CRX protein has SEQ ID NO: 7 or a variant having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0102] In this context, 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.

[0103] A variant of an amino acid sequence described herein 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 described herein has at least 95%, 96%, 97%, 98%, or 99% identity to the sequence from which it is derived, and the amino acid sequence encoded by the variant of the nucleic acid sequence is identical to the sequence encoded by the sequence from which it is derived or is different but retains the biological function of the 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 is different but has the function of a CRX protein). Sequence identity described herein can be measured using sequence analysis software, for example, the computer program BLAST, such as BLASTP or BLASTN, using default parameters.

[0104] The full-length nucleic acid sequence of the present application or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. Once the relevant sequence is obtained, recombination can be used to obtain the relevant sequence in large quantities. This is usually by cloning it into a vector, then into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules that exist in isolated form.

[0105] The DNA sequence can then be introduced into a variety of existing DNA molecules (or as a vector) and cells known in the art. Recombinant vectors can be constructed using methods well known to those of skill in the art, see, e.g., 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 a liposome for delivery to the target cell. Vectors containing the polynucleotides of the application can be 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).

[0106] As will be appreciated by those of skill in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be made that all encode the fusion polypeptides of the application. Thus, given a particular amino acid sequence, one of skill in the art can make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the encoded protein. Therefore, the present application also relates to polynucleotides that hybridize to the above polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application specifically relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under 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 polypeptides encoded by the hybridizable polynucleotides have the same biological function and activity as the mature polypeptides.

[0107] The nucleic acid constructs described herein can be expression cassettes, which can comprise a cone photoreceptor cell-specific promoter, a coding sequence for a 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.

[0108] 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 include a cloning vector, an integration vector, and can also be an expression vector. In addition to the nucleotide molecules of the present application, an expression vector typically contains other elements normally found in a vector, such as a multiple cloning site, a resistance gene, an origin of replication, etc. Vectors that are effective in expressing proteins in humans, particularly in cone cells, are preferred for use in the present application, including but not limited to AAV vectors, lentivirus vectors, adenovirus vectors, etc.

[0109] Expression vectors typically contain sequences for plasmid maintenance and for cloning and expression of foreign nucleotide sequences. The sequences, collectively referred to in some 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.

[0110] 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 6 His) 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 the affinity matrix. The tag can optionally be subsequently removed from the purified protein by various means, such as using certain peptidases for cleavage.

[0111] 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.

[0112] 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.

[0113] An "AAV vector" as used herein 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.

[0114] 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 include the Helper plasmid set forth in SEQ ID NO: 16 and / or the AAV8 capsid plasmid set forth in SEQ ID NO: 17.

[0115] 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.

[0116] 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.

[0117] The method of producing an AAV virus herein specifically comprises: (1) cloning the CRX nucleotide sequence (such as 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).

[0118] The present disclosure also provides a method of introducing a target gene into a cell of a subject, comprising contacting the cell with an effective amount of any of the AAV virus particles described herein, wherein the particle contains any of the AAV vectors comprising a CRX coding sequence described herein.

[0119] The present disclosure also includes host cells that comprise, express, and / or secrete a CRX nucleotide or polypeptide described in any of the embodiments of the present disclosure, 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.

[0120] Pharmaceutical compositions

[0121] 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.

[0122] 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., a virus particle or a recombinant vector) with excipients or other auxiliary ingredients, dividing or packaging the product into dosage units. The virus 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.

[0123] 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 mimetic, 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.

[0124] 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, for example, a dose and / or a convenient ratio of such dose, for example, one-half or one-third of such a dose.

[0125] As used herein, the term "pharmaceutically acceptable excipient" encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, and emulsions, such as oil / water or water / oil emulsions, with various types of wetting agents, excipients, stabilizers, preservatives, viscosity inducers. See Martin (1975) Remington's Pharm. Sci. 15th Ed. (Mack Publ. Co., Easton) for examples of excipients.

[0126] 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.

[0127] 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.

[0128] Delivery vehicles

[0129] 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 BNT16262 and Moderna’s mRNA-1273.

[0130] Methods and uses

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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, the effective amount is the amount sufficient to result in partial or full restoration of function of a defective gene in the subject. In other some embodiments, the effective amount of the AAV virus is the amount sufficient to result in expression of the gene in the subject. The skilled artisan will be able to determine an appropriate amount depending on these and other factors.

[0138] 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.

[0139] 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.

[0140] Dose and administration

[0141] 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.

[0142] 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.

[0143] 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.

[0144] Kit

[0145] 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.

[0146] In some embodiments, the kit further comprises instructions for use. In particular, such a kit can include one or more of the reagents described herein, along with instructions describing the intended use and proper use of the reagents. In some embodiments, the kit 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.

[0147] 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 an active form), e.g., 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 storage solution (e.g., a cryopreservation solution). Non-limiting examples of storage solutions include DMSO, paraformaldehyde. In some embodiments, the storage solution contains an amount of metalloprotease inhibitor.

[0148] In some embodiments, the kit contains any one or more of the components described herein in one or more containers. Thus, in some embodiments, the kit can include a container holding a reagent described herein. The reagent can be in the form of a liquid, gel, or solid (powder). The reagent can be prepared aseptically, packaged in a syringe, 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 a syringe, vial, tube, or other container. The kit can have one or more or all of the components necessary to administer the reagent to a subject, such as a syringe, a topical application device, or an IV needle and bag.

[0149] Some specific embodiments

[0150] Item 1. A nucleic acid construct comprising a cone cell specific promoter and a coding sequence for a CRX protein,

[0151] Preferably, the nucleic acid construct further comprises a polyadenylation signal.

[0152] Item 2. The nucleic acid construct of item 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.

[0153] Preferably, the nucleic acid construct comprises, in order, a cone cell-specific promoter, a coding sequence of a CRX protein, and a polyadenylation signal,

[0154] More preferably, the coding sequence of the CRX protein is operably linked to the cone-specific promoter, the polyadenylation signal.

[0155] Item 3. The nucleic acid construct of item 2, wherein,

[0156] 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

[0157] 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.

[0158] Item 4. The nucleic acid construct of item 2, wherein the cone-specific promoter is a RedO promoter, a GNAT2 promoter, or an ARR3 promoter,

[0159] Preferably,

[0160] the sequence of the RedO promoter is as set forth in SEQ ID NO: 8 or 9,

[0161] the sequence of the GNAT2 promoter is as set forth in SEQ ID NO: 10 or 11,

[0162] the sequence of the ARR3 promoter is as set forth in SEQ ID NO: 12.

[0163] Item 5. The nucleic acid construct of item 4, wherein the nucleic acid construct further comprises a woodchuck hepatitis virus post-transcriptional regulatory element WPRE or a variant WPRE3 thereof,

[0164] 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,

[0165] More preferably, the nucleic acid construct further comprises an ITR region comprising a 5’ ITR and a 3’ ITR.

[0166] Item 6. The nucleic acid construct of any one of items 1-5, wherein the nucleic acid construct comprises a sequence as set forth in SEQ ID NO: 3,

[0167] Preferably, the nucleic acid construct is a vector,

[0168] More preferably, the vector is a cloning vector, an integration vector or an expression vector,

[0169] Further preferably, the vector is an AAV vector, a lentivirus vector or an adenovirus vector, preferably an AAV vector.

[0170] Item 7. A nucleic acid delivery reagent comprising the nucleic acid construct of any one of items 1-6 and a delivery vehicle, preferably the delivery vehicle comprises a virus, a virus-like particle, a lipid nanoparticle.

[0171] Item 8. An AAV virus comprising the nucleic acid construct of any one of items 1-6,

[0172] Item 9. A host cell comprising the nucleic acid construct of any one of items 1-6 or the AAV virus of item 8.

[0173] Item 10. 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 ocular disease or a symptom thereof by inhibiting degenerative death of cone cells,

[0174] 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 items 1-6, the AAV virus of item 8, the host cell of item 9,

[0175] Preferably, the degenerative ocular disease is a degenerative ocular disease associated with degenerative death of cone cells; more preferably, the degenerative ocular disease is selected from the group consisting of retinitis pigmentosa, age-related macular degeneration and cone-rod dystrophy.

[0176] Item 11. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more of the following: the nucleic acid construct of any one of items 1-6, the AAV virus of item 8, the host cell of item 9.

[0177] Item 12. A kit comprising any one or more of the nucleic acid construct of any one of items 1-6, the AAV virus of item 8, the host cell of item 9 and the pharmaceutical composition of item 11.

[0178] Item 13. Use of the nucleic acid construct of any one of items 1-6, the AAV virus of item 8, or the host cell of item 9 in the manufacture of a medicament for delaying loss of functional vision in a subject having a degenerative ocular disease by inhibiting degenerative death of cone cells, delaying death of cone cells in retinitis pigmentosa, or prolonging diurnal visual function in a patient.

[0179] The application will be illustrated hereinafter in the manner of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the application. The methods and materials used in the examples are conventional in the art unless otherwise stated.

[0180] Examples

[0181] Example 1: AAV vector construction

[0182] The CRX mouse full-length cDNA was purchased from Shenguo Bioengineering (Shanghai) Co., Ltd. The CRX cDNA (SEQ ID NO: 4) was obtained by PCR reaction using forward primer F (SEQ ID NO: 1): 5’-ggacagggctttccatagcAATGGCTCAGTGGTTAAGAA; reverse primer R (SEQ ID NO: 2): 5’-gattatctcgagcggGAATTCTACAAGATCTGAAACTTCC, and cloned into the empty vector of AAV-RO1.7 (RedO 1.7 kb) (SEQ ID NO: 15) with SpeI and EcoRI enzyme sites by Gibson Assembly Master Mix (New England BioLabs). The reaction temperature was 50 degrees Celsius, and the reaction time was 20 minutes. The obtained plasmid AAV-RO1.7-CRX is shown in SEQ ID NO: 3. The expression frame and vector map were confirmed by Sanger sequencing, marker enzyme digestion, and whole plasmid sequencing, as shown in FIG. 1 and FIG. 2, respectively.

[0183] Example 2: Selection of promoters

[0184] Using published AAV fluorescent protein vectors containing RedO (2 kb, 1.7 kb), GNAT2 (2 kb, 0.5 kb), RHO, GRK1 promoters (https: / / pubmed.ncbi.nlm.nih.gov / 30833387 / , https: / / pubmed.ncbi.nlm.nih.gov / 33847261 / ), packaged into AAV8 capsid, injected into the retinas of P0 WT mice, and their eyes were collected at P20-P30 to make frozen sections, and confocal imaging was used to detect the spatial location of fluorescent protein expression in the sample to identify the cell expression specificity and expression intensity of different promoters. The results of the in vivo expression verification experiment of the cone-specific promoter are shown in Figure 3. The results show that the expression of RedO and GNAT2 promoters in cone cells is high, and the cone specificity is strong; the expression of RHO promoter in rod cells is high, and the rod specificity is strong; the expression of GRK1 promoter in cone cells is low, and it shows certain rod specificity, and the expression in rod cells is lower than that of RHO promoter.

[0185] Example 3: Animal experiment for protecting cone cells from non-autonomous death

[0186] 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 from Vintory Biotechnology, carrying PDE6B gene deletion mutation). First, the left and right eyes of the same litter animals were grouped, for example: the experimental group injected 10 left eyes, and the control group injected 10 right eyes, mixed AAV and PBS, 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 nuclei of cone cells. The control group was only injected with the same dose of AAV8-RedO-H2BGFP virus to specifically label the nuclei of cone cells.

[0187] At P50 or P20, the retinas were collected, flat-mounted and the cone cells within 1 / 2 radius were counted using a published MATLAB script (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 tabulated. The results are shown in Figures 4-8. The left panel of Figure 4 is a representative P50 retinal flatmount image from the control group injected with AAV-RedO-H2BGFP only, and 4185 is the corresponding cone count within 1 / 2 radius. The right panel is a representative retinal flatmount image from the group co-injected with AAV-RO1.7-CRX and AAV-RedO-H2BGFP, and 14019 is the corresponding cone count within 1 / 2 radius. Figure 5 shows the overall distribution of cone cell counts within 1 / 2 radius of P50 RD1 retinas from the control and experimental groups. Each data point in the figure is from a single retinal count, and the statistical analysis found that 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). 4185 and 14019 in Figure 4 correspond to one data point in each of the two groups in Figure 5.

[0188] The left panel of Figure 6 is a representative P20 retinal flatmount image from the control group injected with AAV-RedO-H2BGFP only, and 12602 is the corresponding cone count within 1 / 2 radius. The right panel is a representative retinal flatmount image from the group co-injected with AAV-RO1.7-CRX and AAV-RedO-H2BGFP, and 12854 is the corresponding cone count within 1 / 2 radius. Figure 7 shows the overall distribution of cone cell counts within 1 / 2 radius of P20 RD1 retinas from the control and experimental groups. Each data point in the figure is from a single retinal count, and the statistical analysis found that the cone survival rate in the experimental group injected with AAV-RO1.7-CRX (n = 9 retinas) was not significantly different from that in the control group (ROHG, n = 5 retinas). 12602 and 12854 in Figure 6 correspond to one data point in each of the two groups in Figure 7.

[0189] Figure 8 is a plot of the data in Figures 5 and 7 plotted on the same graph with age as the horizontal axis. The results show that the AAV-RO1.7-CRX group RD1 retinas maintained the mid- view cone cell count at 1 / 2 radius from P20 to P50, while the control group mid-view cone cell count decreased substantially during the same period, suggesting that AAV-RO1.7-CRX blocked the RP cone cell autonomous death.

[0190] As shown in Figure 9, using the method described above, the successfully constructed plasmid AAV-ARR3-CRX was packaged into AAV8 capsid and purified. The ARR3 promoter sequence is SEQ ID NO: 12 and the mCRX sequence is SEQ ID NO: 4. The injection experiment was performed at P0 in RD1 mice and the retinas were collected at P50 and the mid-view cone cell count was performed. The left panel is a representative P50 retina raw image selected from the control group injected with AAV-RedO-H2BGFP and the 4971 is the mid-view cone cell count corresponding to this image. The middle panel is a representative retina raw image selected from the group co-injected with AAV-ARR3-CRX and AAV-RedO-H2BGFP and the 8043 is the mid-view cone cell count corresponding to this image. The right panel shows the overall distribution of the P50 RD1 retina mid-view cone cell count at 1 / 2 radius in the control and experimental groups. Each data point in the graph is from a single retina count result. It was found by statistics that the survival rate of the mid-view cone cell in the experimental group injected with AAV-ARR3-CRX (n = 9 retinas) was 66% higher than that in the control group (Control, n = 9 retinas). 4971 and 8043 in the left and middle panels correspond to one data point in the two groups of data in the right panel, respectively. The results suggest that overexpression of CRX in the mid-view cone cell using different promoters can protect RP mid-view cone cells from death. The degree of protection seems to depend on the strength of the promoter driving the expression of the gene in the mid-view cone cell. It is known that the ARR3 promoter drives the expression of fluorescent proteins in the mid-view cone cell significantly weaker than the RO1.7 promoter (https: / / pubmed.ncbi.nlm.nih.gov / 30833387 / ), so a higher expression of CRX in the mid-view cone cell seems to be more conducive to protecting RP mid-view cone cells from autonomous death.

[0191] Example 4: Mechanism of preventing mid-view cone cell autonomous death

[0192] During the experiment described in Example 3, 3 eyes of RD1 mice were taken at P20 (one eye 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 solution at room temperature for 2 hours, and then the retinas were dissected out and embedded in 1:1 OCT-30% sucrose solution, and then placed in a -80 degree Celsius refrigerator for freezing. After overnight freezing, frozen sections were prepared 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. The left panel of Figure 10 shows a representative image of the control group retinal section, and the middle panel of Figure 10 shows a representative image of the AAV-RO1.7-CRX experimental group. In both images, the ONL regressed to only one layer of nuclei, i.e. the nuclei of the cone cells, which is consistent with the previously reported photoreceptor degeneration in P20 RD1 mice (https: / / pubmed.ncbi.nlm.nih.gov / 19060896 / ). Using ImageJ software on a computer, the ONL thickness of the experimental and control group retinal sections was measured, respectively, and this thickness reflects the rod death in P20 RD1 mice. The statistics are shown in the right panel of Figure 10. 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 rod cells, supporting the fact that the protective effect of AAV-RO1.7-CRX on RP cones in Figures 4, 5 and 8 is due to blocking the non-autonomous death of RP cones. This mechanism is also significantly different from the mechanism of gene replacement therapy disclosed in US2022175961.

[0193] As shown in FIG. 11, using the method as described above, the successfully constructed plasmid AAV-RHO-CRX was packaged into AAV8 capsid and purified. The RHO promoter sequence is SEQ ID NO: 19, and the mCRX sequence is SEQ ID NO: 4. The injection experiment was performed at P0 in RD1 mice, and the mice were waited until P50 to collect the retinas and count the cone cells within 1 / 2 radius. The left panel is a representative original image of P50 retina selected from the control group injected with AAV-RedO-H2BGFP, and 4894 is the counting result of the cones within 1 / 2 radius corresponding to the image; the middle panel is a representative original image of retina selected from the group co-injected with AAV-RHO-CRX and AAV-RedO-H2BGFP, and 4621 is the counting result of the cones within 1 / 2 radius corresponding to the image. The right panel shows the overall distribution of the counting results of the cone cells within 1 / 2 radius of P50 RD1 retina in the control group and the experimental group. Each data point in the figure comes from the counting result of a single retina, and it is found through statistics that the survival rate of the cones in the experimental group injected with AAV-RHO-CRX (n=9 retinas) has no significant difference compared with the control group (Control, n=9 retinas) (p>0.05). 4894 and 4621 in the left panel and the middle panel respectively correspond to one data point in the two groups of data in the right panel. The results suggest that overexpression of CRX in rod cells using a rod cell-specific promoter is ineffective in protecting RD1 cone cells from non-autonomous death, which supports the mechanism that the protection of RP cones by AAV-RO1.7-CRX in FIG. 4, FIG. 5 and FIG. 8, and AAV-ARR3-CRX in FIG. 9 is derived from blocking the non-autonomous death of RP cones. This mechanism is obviously different from the mechanism of gene replacement therapy disclosed in US2022175961.

[0194] Example 5: Other RP mouse experiments

[0195] RD10: a mouse carrying a point mutation in Pde6b gene

[0196] AAV and PBS were mixed at P0 in RD10 mice, and subretinal injection was performed at 1×10 9 vg / eye, and AAV8-RedO-H2BGFP was co-injected to specifically label the nuclei of cone cells. During P30-P60, the mouse optokinetic reflex behavior experiment was performed to detect the diurnal vision of the mice. When the mice reached P130, the retinas were collected and flattened, and the cone cells within 1 / 2 radius were counted using the MATLAB script program described above, and the counting results were statistically analyzed. As shown in FIG. 12, the counting results of the cone cells in the retina of the AAV-RO1.7-CRX treatment group were higher than those of the control group. The results suggest that the protection of RP cone cells by AAV-RO1.7-CRX is not limited to specific genetic mutations.

[0197] RHO- / -: RHO gene knockout mice

[0198] AAV and PBS were mixed at P0 of RHO- / - mice, and subretinal injection was performed 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, the optokinetic reflex behavior experiment was performed to detect the diurnal vision of mice. When the mice reached P150, the retinas were collected, flattened, and counted for 1 / 2 radius cone cells using the MATLAB script program described above, and the results were counted. The results are shown in Figure 13, and the cone cell count of the AAV-RO1.7-CRX treatment group was higher than that of the control group. This result suggests that the protection of AAV-RO1.7-CRX for RP cone cells is not limited to specific gene mutations.

[0199] Example 6: Animal vision detection

[0200] Experimental / injection method:

[0201] The plasmid AAV-RO1.7-CRX was co-transfected with 293T cells and Helper plasmid (SEQ ID NO: 16), AAV7m8 capsid plasmid (amino acid sequence encoded by cap gene of capsid SEQ ID NO: 20, nucleic acid sequence of whole plasmid SEQ ID NO: 21) to package into AAV7m8 capsid, and after 72 h, the virus was purified using iodixanol gradient.

[0202] The virus was injected into the eye by intravitreal injection at P30 (30 days after birth) of RD10 mice, and the photopic optokinetic response experiment was performed at P60 to detect the spatial frequency response threshold (Acuity) of the animals.

[0203] The background light was about 70 cd / m 2Next, the OptoDrum system (Striatech) was used to measure the photopic optokinetic response (two parameters measured: spatial frequency response threshold and contrast response threshold) of the mice. Spatial frequency response threshold measurement: the grating contrast was set to 100%, the temporal frequency was 1.5 Hz, and the tester measured the spatial frequency threshold of the mice without knowing the control and experimental groups. During each test, the moving direction of the grating (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 measured mice. Contrast threshold measurement: the spatial frequency was set to 0.128 c / d, the temporal frequency was 1.5 Hz, and the tester measured the contrast threshold of the mice without knowing the control and experimental groups. During each test, the moving direction of the grating (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 measured mice. RD10 and RHO- / - mice were tested for optokinetic response, but the RD1 line was not tested because it had lost vision at a very young age and could not perform any meaningful vision test. The results showed that the retinal optokinetic response of the AAV-RO1.7-CRX treatment group was better than that of the control group (i.e., higher spatial frequency response threshold and lower contrast response threshold).

[0204] Example 7: Light / dark discrimination experiment

[0205] 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 was divided into two equal-sized compartments: a dark compartment and a bright, illuminated compartment (about 900 lux). The temperature difference between the two compartments was less than 1°C. A small opening connected the two compartments, allowing the test mice to move freely throughout the space. At the beginning of each test, the mice were placed in the bright room, and the system began recording their activity for 9 minutes after they crossed the narrow door into the dark room. The position of each mouse was determined by a bottom pressure sensor and analyzed using the device's accompanying software to calculate the percentage of time spent in the dark room. RD1 mice older than P30 were tested for this light-avoidance behavior, and the results showed that the AAV-RO1.7-CRX treatment group of RD1 mice spent more than 50% of their time in the dark compartment, significantly higher than the control group (about 50%, indicating that blind mice have no light perception and cannot distinguish between dark and light rooms).

[0206] Some sequences herein

[0207] SEQ ID NO: 4 Mouse CRX nucleotide sequence

[0208] SEQ ID NO: 5 Human CRX nucleotide sequence:

[0209] SEQ ID NO: 6 Mouse CRX amino acid sequence

[0210] SEQ ID NO: 7 Human CRX amino acid sequence

[0211] SEQ ID NO: 8 RedO-1.7kb (RO 1.7) promoter sequence

[0212] SEQ ID NO: 9 RedO-2kb promoter sequence

[0213] SEQ ID NO: 10 SynP136 (GNAT2-2kb) sequence

[0214] SEQ ID NO: 11 SynPVI (GNAT2-0.5kb) sequence

[0215] SEQ ID NO: 12 Mouse ARR3 (cone arrestin) promoter sequence

[0216] SEQ ID NO: 19 Human Rho (rhodopsin) promoter sequence

[0217] SEQ ID NO: 20 AAV-7m8 capsid cap gene encoded amino acid sequence

[0218] SEQ ID NO: 21 AAV-7m8 capsid full plasmid sequence

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. The cone-specific promoter is a RedO promoter, a GNAT2 promoter or an ARR3 promoter, Preferably, 4. The nucleic acid construct of claim 2, wherein 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. 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, 5. The nucleic acid construct of claim 4, wherein More preferably, the nucleic acid construct further comprises an ITR region comprising a 5’ ITR and a 3’ ITR. The nucleic acid construct comprises a sequence as set forth in SEQ ID NO: 3, Preferably, the nucleic acid construct is a vector, 6. The nucleic acid construct of any of claims 1-5, wherein 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 that specifically increases 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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