Myopia treatment method

Abstract

The invention provides application of a reagent for improving CPNE1 protein expression or activity in prevention and treatment of myopia or related diseases thereof.

Description

Technical Field

[0001] This invention relates to the field of treatment for myopia and related diseases, particularly to the field of gene therapy for myopia and related diseases. Specifically, based on the original research finding that the CPNE1 gene can serve as a significant protective genetic factor against myopia, this application provides the application of reagents for increasing CPNE1 protein expression or its activity in the treatment of myopia and related diseases. Background Technology

[0002] Myopia has become the most prevalent vision impairment worldwide. Axial length is a key indicator determining the refractive state of the eye. Most vertebrates regulate axial length to ensure light focuses accurately on the retina; malfunction of this regulatory mechanism leads to refractive imbalance. In myopia, especially axial myopia, the excessive elongation of the eye axis results in excessive refractive error, causing light to focus in front of the retina rather than on it, thus causing myopia. Myopia is influenced by both genetic and environmental factors. In other words, increased refractive error is largely a direct consequence and outward manifestation of axial elongation. The occurrence of myopia is influenced by both genetic and environmental factors. Education level, reading habits, and electronic product use are all considered potential risk factors. Furthermore, population- and family-based studies indicate that myopia has a certain genetic predisposition.

[0003] Currently, the main clinical interventions for myopia include optical correction, refractive surgery, and medication (such as low-concentration atropine). However, these are all limited to symptom management of the disease phenotype and lack effective interventions targeting the fundamental mechanisms of disease development. Myopic individuals—especially those with high myopia—still have a significantly increased risk of developing secondary eye diseases such as retinal detachment, cataracts, and glaucoma. Furthermore, intraocular optical correction and medication treatments carry the risk of causing intraocular inflammation, while refractive surgery carries a certain risk of corneal ectasia and even keratoconus.

[0004] Therefore, developing highly safe drugs that can effectively prevent the occurrence or progression of myopia from the root cause has significant clinical importance and potential value. Summary of the Invention

[0005] Based on extensive and in-depth research, the inventors of this application, through large-scale post-GWAS analysis of the UK Biobank (UKB) and Fingen clinical databases, have for the first time discovered that the CPNE1 gene reaches a genome-wide significant association level (p<5×10⁻⁶). -8 The study has verified that it is a significant protective genetic factor against myopia and its application in the prevention and treatment of myopia.

[0006] Therefore, in a first aspect, the present invention provides the use of a reagent for increasing the expression of CPNE1 protein or its activity in the preparation of a medicament for the prevention and / or treatment of myopia and / or myopia-related diseases.

[0007] In some embodiments, the drug is administered in combination with other pharmaceutically active agents or therapies for the prevention and / or treatment of myopia or related diseases, such as simultaneously, separately, or sequentially.

[0008] In some embodiments, the drug is used in subjects to prevent and / or treat myopia and / or myopia-related diseases.

[0009] This application is not limited in the types of subjects to which it applies. In some embodiments, the subjects are selected from animals (e.g., mammals), preferably humans.

[0010] In some implementations, the subject contains an endogenous CPNE1 gene.

[0011] The reagents described herein for enhancing CPNE1 protein expression or its activity can be selected from any reagent capable of enhancing CPNE1 protein expression or its activity through optional pathways. Such reagents may affect processes including but not limited to gene coding, genome epigenetic modification, transcription, post-transcriptional modification, translation, and post-translational modification.

[0012] In some embodiments, the reagent comprises one or more of the following:

[0013] (i) Reagents used to increase the copy number of the CPNE1 gene;

[0014] (ii) Reagents for enhancing transcription of the CPNE1 gene;

[0015] (iii) Reagents for enhancing the translation of the mRNA product of the CPNE1 gene;

[0016] (iv) Reagents for enhancing the activity of CPNE1 protein;

[0017] (v) Reagents used to increase CPNE1 protein or mRNA levels.

[0018] Those skilled in the art will readily understand that the reagents provided in this application for increasing CPNE1 protein expression or its activity can be used for the prevention and / or treatment of myopia and / or myopia-related diseases, or for the preparation of related drugs, because they can cause an increase in the number of CPNE1 proteins and / or an enhancement of CPNE1 activity in subjects. Thus, for the purpose of reducing unnecessary immunogenicity of drugs and / or improving therapeutic effects, the reagents can be specifically designed according to the type of subjects to which they are intended to be administered.

[0019] For example, when the subject is a person, the reagent is preferably a reagent for increasing the expression or activity of human CPNE1 protein, such as a reagent for increasing the copy number of the human CPNE1 protein encoding gene, a reagent for enhancing the transcription of the human CPNE1 gene (e.g., the subject's endogenous CPNE1 gene), a reagent for enhancing the translation of human CPNE1 protein mRNA (e.g., the subject's endogenous CPNE1 mRNA product), a reagent for enhancing the activity of human CPNE1 protein, or a reagent for increasing the level of human CPNE1 protein or its mRNA.

[0020] Based on the description of the above exemplary embodiments, the protocols covered by this application, including those applicable to any subject, have been fully disclosed in the content of this application and will not be repeated here.

[0021] Based on the disclosure of this application, those skilled in the art will readily understand that the increase in CPNE1 protein or mRNA levels can be achieved through a variety of pathways, including but not limited to, an increase in the copy number of the CPNE1 gene, enhanced transcription and / or translation of the subject's endogenous CPNE1 gene, and an increase in CPNE1 protein or mRNA levels through exogenous delivery, etc.

[0022] In some embodiments, the reagent may be an in vitro prepared CPNE1 protein or mRNA encoding the CPNE1 protein for administration to a subject. In some embodiments, the reagent further comprises a delivery vector (e.g., lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns, or viral vectors) for delivering the CPNE1 protein or mRNA.

[0023] In some implementations, the reagent includes a gene editing system.

[0024] In some embodiments, the reagent is capable of enhancing CPNE1 gene expression.

[0025] Reagents used to increase the copy number of the CPNE1 gene

[0026] In some embodiments, the reagent is used to increase the copy number of the CPNE1 gene. The gene copy number can be determined using conventional techniques in the art, such as PCR, first-generation sequencing (Sanger sequencing), and second-generation sequencing (NGS sequencing).

[0027] In some embodiments, the reagent is used to introduce an exogenous nucleotide sequence encoding the CPNE1 protein into the subject. In some embodiments, the nucleotide sequence encodes a CPNE1 protein homologous to the subject. For example, in some embodiments where the subject is human, the nucleotide sequence encodes a human CPNE1 protein.

[0028] In some embodiments, the reagent comprises a nucleic acid (e.g., a vector containing the nucleotide sequence) encoding a CPNE1 protein (e.g., a CPNE1 protein homologous to the subject, such as a human CPNE1 protein). In some embodiments, the nucleotide sequence encoding the CPNE1 protein may be naturally occurring or codon-optimized, for example, having a codon preference adapted to the subject. In some preferred embodiments, the nucleic acid further comprises a regulatory element (e.g., a promoter sequence) operatively linked to the nucleotide sequence encoding the CPNE1 protein.

[0029] In some embodiments, the nucleotide sequence encoding the CPNE1 protein can exist in a non-integrating form, such as in plasmid DNA, extrachromosomal DNA (ecDNA), or viral DNA. In other embodiments, the nucleotide sequence encoding the CPNE1 protein can be integrated into the genome of a subject. In these embodiments, the reagent further comprises a gene editing system capable of inserting the target sequence into the genome of a subject, said gene editing system being any site-specific (sequence-specific) genome editing system currently known. In some embodiments, the reagent further comprises: an effector enzyme of the gene editing tool (e.g., a nuclease (e.g., a Cas effector protein of the CRISPR system, such as Cas9; e.g., Fok I nuclease), a transposase), and / or, a nucleic acid comprising the nucleotide sequence encoding the effector enzyme of said gene editing tool (e.g., a nuclease (e.g., a Cas effector protein of the CRISPR system, such as Cas9; e.g., Fok I nuclease), a transposase). In some embodiments, the reagent further comprises: a guide molecule for a gene editing tool (e.g., guide RNA (gRNA) or guide protein (e.g., TALE protein, zinc finger protein)), and / or, a nucleic acid containing a nucleotide sequence encoding the guide RNA; wherein the guide molecule is capable of being bound by the effector enzyme. In some preferred embodiments, the guide molecule is a guide RNA of the CRISPR system, the guide RNA comprising a backbone sequence and a guide sequence, the guide sequence being capable of targeting endogenous sequences in the cellular genome, and the backbone sequence being capable of being bound by the effector enzyme.

[0030] Reagents for enhancing CPNE1 gene transcription

[0031] In some embodiments, the reagent is used to enhance the transcription of the CPNE1 gene (e.g., the subject's endogenous CPNE1 gene). The transcription level can be determined by a variety of methods known in the art, such as Northern blotting, PCR, RT-PCR, real-time RT-PCR, or RNA-seq.

[0032] The reagent for enhancing CPNE1 gene transcription can increase the transcriptional level of the CPNE1 gene through any mechanism. The reagent may comprise a small molecule compound, peptide, nucleic acid molecule, or a combination thereof, which, for example, can activate the transcription of the CPNE1 gene by activating upstream signaling pathways of the CPNE1 gene, or directly act on the transcriptional regulatory elements of the CPNE1 gene to activate its transcription. In some embodiments, the reagent for enhancing CPNE1 gene transcription comprises a sequence-specific endogenous transcription activation system or a polynucleotide encoding such system. Such sequence-specific endogenous transcription activation systems include systems that act on the transcriptional regulatory regions of the CPNE1 gene in the genome to activate the transcription of the CPNE1 gene.

[0033] In some embodiments, the reagent is capable of enhancing the expression of the CPNE1 gene (e.g., the subject's endogenous CPNE1 gene).

[0034] In some embodiments, the reagent is capable of activating the transcription of the CPNE1 gene (e.g., the subject's endogenous CPNE1 gene).

[0035] In some embodiments, the reagent can specifically act on the regulatory sequence (e.g., promoter or enhancer sequence) of the CPNE1 gene to activate transcription of the CPNE1 gene.

[0036] In some embodiments, the reagent comprises:

[0037] (1) A polypeptide construct or a nucleotide sequence encoding the polypeptide construct, the polypeptide construct comprising a transcriptional activation domain; and,

[0038] (2) A guide nucleic acid molecule or guide protein that targets and recognizes and / or binds to the transcriptional regulatory sequence (e.g., promoter or enhancer sequence) of the CPNE1 gene, or a nucleotide sequence encoding the guide nucleic acid molecule or guide protein.

[0039] Based on the disclosure of this application, those skilled in the art will readily understand that, under the guidance of the guide nucleic acid molecule or guide protein, the transcriptional activation domain can directly or indirectly act on the transcriptional regulatory sequence of the CPNE1 gene (e.g., optionally in the synergy of a corresponding transcriptional activator (e.g., an endogenous corresponding transcriptional activator)) to activate the transcription initiation program, thereby activating the transcription of the CPNE1 gene and enhancing the expression of the CPNE1 gene.

[0040] In some embodiments, the guide nucleic acid molecule or the guide protein is linked to the polypeptide construct (e.g., covalently and / or non-covalently), or not linked. In some embodiments, the guide nucleic acid molecule or the guide protein may be conjugated to the polypeptide construct.

[0041] In some embodiments, the reagent possesses one or more of the following characteristics:

[0042] (a) The transcription activation domain has one or more activities selected from the following: transcription activation activity, histone modification activity, histone methyltransferase activity, DNA methyltransferase activity, histone demethyltransferase activity and / or DNA demethyltransferase activity; preferably, the transcription activation domain is selected from: VP16, VP48, VP64, p65, TET1, VPR, VPH, Rta, p300 and any combination thereof;

[0043] (b) The guide nucleic acid molecule is selected from guide RNA molecules of the CRISPR / Cas system;

[0044] (c) The guide protein is selected from programmable DNA-binding proteins, such as TALE protein and zinc finger protein;

[0045] (d) The transcriptional regulatory sequence of the CPNE1 gene includes the sequence shown in SEQ ID NO: 1;

[0046] (e) The polypeptide construct is a fusion protein;

[0047] (f) The polypeptide construct further includes one or more nuclear localization signals (NLS).

[0048] In some embodiments, the reagent comprises a guide RNA molecule of a CRISPR / Cas system that targets and recognizes and / or binds to transcriptional regulatory sequences (e.g., promoter or enhancer sequences) of the CPNE1 gene, or the nucleotide sequence encoded thereon.

[0049] In some embodiments, the polypeptide construct comprises the transcriptional activation domain and a Cas effector protein capable of binding the guide RNA molecule. In some embodiments, the polypeptide construct is a fusion protein comprising the transcriptional activation domain and a Cas effector protein capable of binding the guide RNA molecule. In some embodiments, the Cas effector protein does not have nucleic acid cleavage activity; preferably, the nuclease is nuclease-inactivated Cas9 (dCas9). More preferably, the nuclease is nuclease-inactivated Staphylococcus aureus Cas9 (dSaCas9).

[0050] In some embodiments, the transcriptional activation domain is attached to (e.g., optionally via a linker or not) the N-terminus and / or C-terminus of the Cas effector protein.

[0051] In some embodiments, the reagent comprises (i) a first nucleotide sequence encoding the polypeptide construct and / or (ii) a second nucleotide sequence encoding the guide nucleic acid molecule or the guide protein.

[0052] In some implementations, the first nucleotide sequence and / or the second nucleotide sequence are present in the vector (e.g., in different vectors or in the same vector).

[0053] In some embodiments, the vectors are each independently selected from viral vectors. In some embodiments, the vectors are each independently selected from adeno-associated virus vectors (e.g., AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh10). In some embodiments, the vector is AAV2.

[0054] carrier

[0055] In some embodiments, the reagent comprises a carrier system containing one or more carriers;

[0056] The one or more vectors comprise: a first nucleotide sequence encoding a polypeptide construct as defined above; and a second nucleotide sequence encoding a guide nucleic acid molecule or guide protein as defined above.

[0057] In some embodiments, the one or more vectors comprise a viral vector. In some embodiments, the viral vector is an adeno-associated virus vector (e.g., AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh10). In some embodiments, the viral vector is AAV2.

[0058] Prevention and / or treatment of myopia and / or myopia-related diseases

[0059] In some embodiments, the myopia is selected from: axial myopia and refractive myopia. In some embodiments, the prevention or treatment includes at least inhibiting the development of refractive error and / or inhibiting axial growth.

[0060] As used herein, the terms "axial myopia" and "refractive myopia" have their common meanings in the art. Generally speaking, axial myopia refers to a refractive error caused by an excessively long axial length (the eyeball's anteroposterior diameter), resulting in parallel light rays focusing in front of the retina after refraction by the eye's refractive system. Its root cause lies in an abnormal anatomical structure of the eye, specifically a mismatch between axial length and refractive power. Refractive myopia, on the other hand, refers to a refractive error where the axial length is within the normal range, but the refractive power of the eye's refractive system (mainly the cornea and lens) is too strong, causing parallel light rays to focus prematurely and form an image in front of the retina. Its root cause lies in abnormal optical properties of the refractive media, rather than an abnormal anatomical structure of the eye.

[0061] In some embodiments, the myopia-related disease is a disease associated with excessive axial growth or its functional abnormalities. In some embodiments, the myopia-related disease is selected from: posterior staphyloma, myopic macular degeneration (e.g., macular atrophy, cracked lacquer, Fuchs' spot), myopic choroidal neovascularization, retinal schisis, macular hole, retinal detachment, glaucoma, cataract (e.g., nuclear cataract), vitreous diseases, peripapillary diseases (e.g., peripapillary atrophy, tilted optic disc), and anisometropic amblyopia.

[0062] In another aspect, the present invention provides a method for preventing and / or treating myopia and / or myopia-related diseases. The method includes administering to a subject in need an effective amount of the reagent described in the first aspect for enhancing CPNE1 protein expression or its activity.

[0063] In some embodiments, the method further includes administering to the subject additional pharmaceutically active agents or therapies for the prevention and / or treatment of myopia or related diseases, such as simultaneously, separately, or sequentially.

[0064] application

[0065] The reagents described herein can be used to form pharmaceutical compositions with pharmaceutically acceptable carriers and / or excipients. "Pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, which are well known in the art and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives.

[0066] The reagents described herein can be administered by any suitable method known in the art. Preferred routes of administration include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes. Parenteral administration refers to administration methods that are typically administered by injection rather than intravenous or local administration, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Alternatively, administration may be via non-parenteral routes, such as local, epidermal, or mucosal administration, for example, intranasal, oral, vaginal, rectal, sublingual, or local administration.

[0067] In some embodiments, the reagents described herein are applied to a specific organ or tissue or cell (e.g., eye, eyeball, scleral tissue, scleral fibroblasts) of a subject. For example, in some embodiments, the reagents described herein are administered via local targeted delivery, such as through subcapsular injection in Tenon's capsule and diffusion to the target tissue (e.g., sclera). In some embodiments, the reagents described herein are administered in an organ- or tissue- or cell-specific manner (e.g., eye tissue-specific, scleral-specific, scleral fibroblast-specific). For example, in some embodiments, the reagents described herein are endowed with targeting capabilities (e.g., targeting the eye, eyeball, scleral tissue, or scleral fibroblasts), for example, by being endowed with the ability to accumulate and / or exert activity at a target site in the subject (e.g., targeting the eye, eyeball, scleral tissue, or scleral fibroblasts).

[0068] The reagents described herein can be formulated into dosage forms compatible with their intended route of administration. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the reagents described herein into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use.

[0069] The reagents described herein can be formulated in dosage units for ease of administration. Dosage unit form refers to physically discrete units suitable for use as a single dose in the subject of treatment; each unit contains a predetermined amount of the active ingredient, calculated to be combined with the desired drug carrier to produce the desired therapeutic effect.

[0070] The reagents described herein are administered to subjects who require them. In some embodiments, the subject is a mammal, such as a guinea pig or a human. In some embodiments, the subject is a human.

[0071] Terminology Definition

[0072] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0073] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0074] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0075] As used herein, the term "CPNE1" or "CPNE1 protein" refers to Copine 1, also known as Chromobindin 17, CPN1, or COPN1, and has the meaning commonly understood by those skilled in the art. CPNE1 as used herein may be human or a homologous gene encoding CPNE1 from other species (e.g., non-human mammals, fish, reptiles, or birds, such as rodents like mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cattle, sheep, pigs, goats, primates, etc.). The term "CPNE1 gene" or "Cpne1 gene" refers to any nucleotide sequence encoding the CPNE1 protein. Those skilled in the art can obtain the amino acid sequence of the CPNE1 protein, the nucleotide sequence of the CPNE1 gene, and its location in the genome from public databases (e.g., NCBI). For example, the exemplary nucleotide coding sequence and its location of the human CPNE1 gene can be obtained from NCBI using gene ID 8904, and the exemplary nucleotide sequence of the human CPNE1 protein can be found in UniProtKB:Q99829; the exemplary nucleotide coding sequence and its location of the guinea pig Cpne1 gene can be obtained from NCBI using gene ID 100732374, and the nucleotide sequence of the guinea pig CPNE1 protein can be found in UniProtKB:H0WB17.

[0076] As used herein, the terms “transcriptional regulatory element,” “transcriptional regulatory sequence,” “regulatory sequence,” or “regulatory element” refer to nucleic acid sequences that regulate the initiation, efficiency, and timing of transcription of a target gene. These elements typically do not encode proteins but precisely control gene expression spatially and temporally through interactions with transcription factors and other protein complexes. Transcriptional regulatory elements include, but are not limited to, promoters, enhancers, silencers, insulators, and response elements. Their functions can be constitutive, tissue-specific, developmental stage-specific, or inducible.

[0077] In this document, the term "promoter" is used in its broadest sense, generally referring to a core transcriptional regulatory element located upstream of the transcription start site of a gene. This element provides specific binding sites for RNA polymerase and its associated transcription factors, thereby mediating and determining the basic location and level of transcription initiation. The promoters used in this document include constitutive promoters, tissue-specific promoters, and inducible promoters. Furthermore, depending on the host species, the promoters used in this application may also include prokaryotic and eukaryotic promoters. In particular, based on their sensitivity to α-amanitin, the promoters used in this document include type I, type II, and type III promoters.

[0078] The term "enhancer" as used in this article refers to a transcriptional regulatory element that can significantly increase promoter activity. It typically functions independently of its relative position and orientation to the target gene, and can be located upstream, downstream, or within introns of the gene. It recruits coactivators by binding to specific transcription factors, thereby altering chromatin structure or promoting the assembly of the transcription initiation complex, thus enhancing the gene's transcriptional level.

[0079] In this document, the term "peptide construct" is used in its broadest sense. Generally, "peptide construct" is used to refer to a construct comprising one or more peptide or protein components, wherein the one or more peptide or protein components may each have different origins or different biological activities or functions, and are linked by covalent and / or non-covalent means (e.g., covalently linked by covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds, and / or non-covalently linked by hydrogen bonds). The peptide constructs of the present invention are not limited in the number of their molecular chains (e.g., peptide chains). For example, the peptide constructs of the present invention may contain only one molecular chain (e.g., a peptide chain), or two or more molecular chains (e.g., peptide chains) linked covalently and / or non-covalently (e.g., covalently linked by covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds, and / or non-covalently linked by hydrogen bonds). Similarly, those skilled in the art will readily understand that, in embodiments comprising multiple polypeptide or protein components, the multiple polypeptide or protein components contained in the polypeptide construct of the present invention may be located entirely or partially in the same molecular chain (e.g., peptide chain), or may each be located in different molecular chains (e.g., peptide chains).

[0080] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received).

[0081] As used herein, the term "effective amount" is at least the minimum concentration required to achieve measurable improvement or prevention of a particular condition. Effective amounts as used herein can vary with factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit the desired response in the individual. An effective amount is also the amount at which the beneficial effect of treatment outweighs any toxic or adverse effects of treatment. For prophylactic use, beneficial or desired outcomes include results such as elimination or reduction of risk, mitigation of severity, or delay of disease onset, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during disease development. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reduction of one or more symptoms arising from the disease, improvement of the quality of life of those suffering from the disease, reduction of the dosage of other medications required to treat the disease, enhancement of the effect of another medication (e.g., via targeted therapy), delay of disease progression, and / or prolongation of survival. Effective amounts may be administered in one or more doses.

[0082] Beneficial effects of the invention

[0083] The inventors of this application have discovered for the first time that the CPEN1 gene is a protective genetic factor against myopia, and experimental verification has shown that increasing the expression level of the CPEN1 gene can improve refractive error, reduce the axial length growth rate, and inhibit the development of myopia.

[0084] Furthermore, this application provides gene therapy methods for myopia treatment, such as treating myopia by AAV-mediated overexpression of the CPNE1 gene. This method has high treatment safety (e.g., it does not cause intraocular inflammation, retinal structural abnormalities, or retinal barrier disruption), and can achieve long-term treatment with relatively few side effects, thus having significant clinical value.

[0085] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0086] Figure 1 Schematic diagram of the GV369 backbone plasmid structure.

[0087] Figure 2 The expression level of CPNE1 mRNA was shown, which represented the results of qPCR detection of the relative expression level of CPNE1 mRNA in scleral tissue of each group; the LIM group was normalized to 1, and the differences between groups were assessed by one-way ANOVA, with FDR as the post-hoc test; #, p < 0.05.

[0088] Figure 3 Line graph showing the refractive error change trend of different treatment groups during the experimental period; data are expressed as mean ± SEM, and one-way ANOVA was used to assess differences between groups, with FDR as a post-hoc test; #, p < 0.05 indicates a significant difference between the LIM+AAV-NC and LIM+AAV-CPNE1 groups; ##, p < 0.01.

[0089] Figure 4 Line graph showing axial length changes in different treatment groups during the experimental period; data are expressed as mean ± SEM, and one-way ANOVA was used to assess differences between groups, with FDR as a post-hoc test; #, p < 0.05 indicates a significant difference between the LIM+AAV-NC and LIM+AAV-CPNE1 groups.

[0090] Sequence information

[0091] The description of the sequences involved in this application is provided in the table below.

[0092] Table 1: Sequence Information Detailed Implementation

[0093] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it). Those skilled in the art will appreciate that the embodiments are described by way of example and are not intended to limit the scope of protection claimed by the invention.

[0094] Example 1: The CPNE1 gene is associated with myopia in the population.

[0095] The inventors, targeting the myopia phenotype, utilized genotype, gene expression, and disease GWAS data from the UK Biobank (UKB) (OpenGWAS ID: ukb-b-6353) and Finngen (finngen_pheno: H7_MYOPIA) clinical databases. Using SMR software (SMR | Yang Lab, https: / / yanglab.westlake.edu.cn / software / smr / #Overview), they conducted a large-scale GWAS-eQTL integration analysis, finding that the CPNE1 gene reached a genome-wide significant association level (p<5×10⁻⁶). -8 It was identified as a significant protective genetic factor against myopia (detailed results are shown in Table 2).

[0096] Table 2: SMR Analysis Results (CPNE1 Only)

[0097] Example 2: Overexpression of CPNE1 in a myopia model guinea pig inhibits myopia progression

[0098] 2.1 Preparation process of recombinant adeno-associated virus (AAV) with endogenous activation and overexpression of CPNE1

[0099] (1) Construction of recombinant expression vector

[0100] The CRISPR activation system (CRISPRa) was used to target the CPNE1 promoter region, achieving upregulation of endogenous CPNE1 gene expression. The guinea pig Cpne1 gene sequence (ID: 100732374) was obtained from the NCBI website. The GV639 vector (AAV-EFS-NLS-dSaCas9-NLS-VP64-bGHpA-U6-sgRNA) (purchased from GCI Gene) was selected as the expression backbone. The backbone vector sequence is as follows: Figure 1 As shown. The guide sequence of the sgRNA was designed to target the sequence 5'-TTTAGGCTTAGGCTGGATGGT-3' (SEQ ID NO: 1), which is located in the promoter regulatory element within 200 bp upstream of the CPNE1 transcription start site. Single-stranded primers (SEQ ID NO: 2-3) were synthesized based on the sgRNA target sequence using Oligo sequences, and annealed to form double-stranded DNA. After digestion of the vector with Bsal restriction endonuclease, the annealed product was ligated to the vector. All recombinant plasmids were verified by bidirectional Sanger sequencing to ensure the integrity of the gene sequence and the correctness of the reading frame.

[0101] (2) AAV virus packaging and purification

[0102] An optimized three-plasmid co-transfection system (containing a recombinant expression vector, pHelper helper plasmid, and pRepCap packaging plasmid) was used to transfect 293T cells using transfection reagents (purchased from GKG). Cell culture supernatant was collected 72 hours after transfection, and the virus particles were initially purified using an optimized iodixanol density gradient ultracentrifugation method (28,000 rpm, 4°C, 2 hours). The virus was then concentrated using 100 kDa ultrafiltration centrifuge tubes and filtered through a 0.22 μm sterile membrane for sterilization. Finally, the AAV2 serotype viral genomic titer (4.94E+12 vg / mL) was accurately determined using TaqMan probe-based quantitative PCR. All viral preparations were stored in PBS buffer containing 0.001% Pluronic F-68 at -80°C for long-term storage.

[0103] 2.2 Grouping and Intervention Protocols of Experimental Animals

[0104] Three-week-old healthy calico guinea pigs were selected. After baseline screening to exclude individuals with spontaneous myopia and ocular inflammation, they were randomly divided into 4 groups (n=5 / group):

[0105] • Blank control group: Routine feeding, no intervention

[0106] •LIM model group: Right eye myopia induced by optical defocus, without viral injection.

[0107] • LIM+AAV-NC group: right eye myopia was induced by light defocusing, and Tenon's subcapsular injection of empty AAV vector (40 μL, 1×10¹² vg / mL) was performed.

[0108] • LIM+ AAV-CPNE1 group: right eye myopia induced by defocusing, and Tenon's subcapsular injection of AAV-CPNE1 (40μL, 1×10¹² vg / mL) was performed.

[0109] The treatment included: viral injection: at 3 weeks of age, a single injection of 40 μL AAV (titer 1×10¹² vg / mL) was administered into the subcapsular space of Tenon's capsule. Myopia induction: at 3 weeks of age, defocused myopia was induced using a -10D lens for 4 weeks.

[0110] The experiment employed a double-blind design, with participants unaware of their group assignments. A standardized environment was maintained, employing a 12-hour light / 12-hour dark cycle at a room temperature of 26±1℃. This experiment was ethically compliant, adhering to the Association for Research in Vision and Ophthalmology (ARVO) statement regarding animal use.

[0111] 2.3 Expression efficiency detection

[0112] Four weeks later, scleral tissue was collected from animals to assess the CPNE1 transfection efficiency. Specifically, qPCR was used to detect the expression level of CPNE1 mRNA in scleral tissue, with β-actin as an internal reference gene, and the 2-ΔΔCt method was used to analyze transfection efficiency. The results showed that compared with the LIM+AAV-NC group, the qPCR results of the LIM+AAV-CPNE1 group showed an increased CPNE1 mRNA level, indicating successful transfection. Figure 2 ).

[0113] 2.4 Overexpression of CPNE1 inhibits the development of myopia refractive error.

[0114] Refractive error was measured using a small animal refractometer at fixed weekly times, with three measurements taken per eye and the average value recorded. Results showed that, four weeks after optical defocusing-induced myopia, the myopia progression was inhibited in the LIM+AAV-CPNE1 group compared to the LIM+AAV-NC group, suggesting that CPNE1 transfection inhibits myopia progression. Figure 3 ).

[0115] 2.5 Overexpression of CPNE1 inhibits axial elongation in myopic eyes.

[0116] Axial length was measured using an A-scan biometer at fixed weekly times, with three measurements taken per eye and the average value recorded. Results showed that, four weeks after photodefocus-induced myopia, the axial elongation process was inhibited in the LIM+AAV-NC group, suggesting that CPNE1 transfection inhibits myopic axial length development. Figure 4 ).

[0117] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of a reagent for enhancing CPNE1 protein expression or its activity in the preparation of a medicament for the prevention and / or treatment of myopia and / or myopia-related diseases.

2. The use as described in claim 1, wherein, The reagent comprises one or more of the following: (i) Reagents used to increase the copy number of the CPNE1 gene; (ii) Reagents for enhancing transcription of the CPNE1 gene; (iii) Reagents for enhancing the translation of the mRNA product of the CPNE1 gene; (iv) Reagents for enhancing the activity of CPNE1 protein; (v) Reagents used to increase CPNE1 protein or mRNA levels.

3. The use as described in claim 1 or 2, wherein, The reagents include a gene editing system.

4. The use according to any one of claims 1-3, wherein, The reagent can enhance CPNE1 gene expression.

5. The use according to any one of claims 1-4, wherein, The reagent can activate the transcription of the CPNE1 gene.

6. The use according to any one of claims 1-5, wherein, The reagents include: (1) A polypeptide construct or a nucleotide sequence encoding the polypeptide construct, the polypeptide construct comprising a transcriptional activation domain; and, (2) A guide nucleic acid molecule or guide protein that targets and / or binds to the transcriptional regulatory sequence (e.g., promoter or enhancer sequence) of the CPNE1 gene, or a nucleotide sequence encoding the guide nucleic acid molecule or guide protein; Preferably, the guide nucleic acid molecule or the guide protein forms a link (e.g., covalent and / or non-covalent link) with the polypeptide construct, or, no link is formed; Preferably, the guide nucleic acid molecule or the guide protein may be conjugated to the polypeptide construct.

7. The use as described in claim 6, wherein the use comprises one or more features selected from the following: (a) The transcription activation domain has one or more activities selected from the following: transcription activation activity, histone modification activity, histone methyltransferase activity, DNA methyltransferase activity, histone demethyltransferase activity and / or DNA demethyltransferase activity; preferably, the transcription activation domain is selected from: VP16, VP48, VP64, p65, TET1, VPR, VPH, Rta, p300 and any combination thereof; (b) The guide nucleic acid molecule is selected from guide RNA molecules of the CRISPR / Cas system; (c) The guide protein is selected from programmable DNA-binding proteins, such as TALE protein and zinc finger protein; (d) The polypeptide construct is a fusion protein; (e) The polypeptide construct further includes one or more nuclear localization signals (NLS).

8. The use as described in claim 6 or 7, wherein, The reagent contains a guide RNA molecule of the CRISPR / Cas system that targets and / or binds to transcriptional regulatory sequences (e.g., promoter or enhancer sequences) of the CPNE1 gene, or the nucleotide sequence it encodes. Preferably, the polypeptide construct comprises the transcriptional activation domain and a Cas effector protein capable of binding the guide RNA molecule; for example, the polypeptide construct is a fusion protein comprising the transcriptional activation domain and a Cas effector protein capable of binding the guide RNA molecule. Preferably, the Cas effector protein does not have nucleic acid cleavage activity; preferably, the nuclease is nuclease-inactivated Cas9 (dCas9); more preferably, the nuclease is nuclease-inactivated Staphylococcus aureus Cas9 (dSaCas9). Preferably, the transcriptional activation domain is attached to (e.g., optionally via a linker or not) the N-terminus and / or C-terminus of the Cas effector protein.

9. The use according to any one of claims 6-8, wherein, The reagent comprises (i) a first nucleotide sequence encoding the polypeptide construct and / or (ii) a second nucleotide sequence encoding the guide nucleic acid molecule or the guide protein; Preferably, the first nucleotide sequence and / or the second nucleotide sequence are present in the vector (e.g., in different vectors or in the same vector). Preferably, each of the vectors is independently selected from viral vectors; preferably, each of the vectors is independently selected from adeno-associated virus vectors (e.g., AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh10).

10. The use according to any one of claims 1-5, wherein, The reagent comprises a carrier system, which includes one or more carriers; The one or more vectors comprise: a first nucleotide sequence encoding a polypeptide construct as defined in any one of claims 6-9; and a second nucleotide sequence encoding a guide nucleic acid molecule or guide protein as defined in any one of claims 6-9; Preferably, the one or more vectors comprise a viral vector; preferably, the viral vector is an adeno-associated virus vector (e.g., AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh10).

11. The use according to any one of claims 1-10, wherein, The myopia referred to is selected from: axial myopia and refractive myopia.

12. The use according to any one of claims 1-11, wherein, The myopia-related diseases mentioned are those associated with excessive axial growth or abnormal function of the eye. Preferably, the myopia-related diseases are selected from: posterior staphyloma, myopic macular degeneration (e.g., macular atrophy, crack lacquer, Fuchs' spot), myopic choroidal neovascularization, retinal schisis, macular hole, retinal detachment, glaucoma, cataract (e.g., nuclear cataract), vitreous diseases, peripapillary diseases (e.g., peripapillary atrophy, tilted optic disc), and anisometropic amblyopia.

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