A method for constructing a myopia animal model by knocking out NEB

Abstract

The application provides a method for constructing a myopia animal model by knocking out NEB, and belongs to the technical field of biological medicine. The method is targeted editing of homologous gene exon 5 of zebrafish by a CRISPR-CAS9 system, and a homozygous deletion strain which can be stably inherited is obtained. The strain of zebrafish shows pathological characteristics such as shortening of body length, reduction of eye interval and reduction of eyeball size, and conforms to the clinical manifestations of pathological myopia, and is an animal model which can stably reproduce the myopia phenotype. The animal model provided by the application can be widely applied to the exploration of the pathogenic mechanism of genetic myopia, verification of gene therapy targets and evaluation of the drug efficacy of candidate compounds.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, and specifically to a method for constructing a myopia animal model by knocking out NEB. Background Technology

[0002] High myopia (HM) is a severe refractive error, typically defined as a refractive error ≤ -6.00D or an axial length ≥ 26.5mm. In recent years, the public health burden of high myopia has been increasing dramatically. Epidemiological projections indicate that the prevalence of high myopia in the Chinese population is expected to rise from 5.3% in 2020 to 17.6% in 2050, implying significant clinical and social pressures in the coming decades. While multiple factors contribute to the development of high myopia, genetic factors play a crucial role in its progression.

[0003] In disease management, accurately understanding the progression dynamics of high myopia is a crucial prerequisite for developing intervention strategies. Existing research generally indicates that the rate of myopia progression tends to slow with age, with younger children experiencing faster progression and older children showing a more gradual plateau. However, this evidence primarily comes from the general myopic population (whose refractive error is typically below -6.00D). Due to the lack of large-scale longitudinal studies specifically targeting children and adolescents with high myopia, the academic community still knows very little about the unique progression trajectory of this population—especially how the rate of progression changes with age and whether there are non-linear patterns different from those in ordinary myopia. Therefore, treating the progression of high myopia as a quantitative longitudinal trait, rather than a simple dichotomy or cross-sectional trait, holds promise for revealing the biological mechanisms underlying the accelerated disease and providing a more refined analysis of genotype-phenotype associations. Because clinical longitudinal studies are lengthy, involve many confounding factors, and are difficult to validate mechanisms in humans, relying solely on population data cannot provide a deep understanding of the biological basis of the interaction between genetics and age.

[0004] Therefore, there is an urgent need in this field to establish an animal model that can reflect the interaction between genetic background and age in the progression of high myopia, so as to provide a reliable tool platform for elucidating the age-dependent genetic regulatory mechanism of high myopia progression and screening drugs to delay the progression of high myopia. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for constructing a myopia animal model by knocking out NEB.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The first aspect of this invention provides a method for preparing a NEB loss-of-function zebrafish mutant, the method comprising the following steps: 1) Design and synthesize sgRNA targeting zebrafish NEB, wherein the sgRNA has the nucleotide sequence shown in SEQ ID NO:1; 2) Inject the sgRNA described in step 1) together with the Cas9 protein or the mRNA encoding the Cas9 protein into zebrafish fertilized eggs; 3) Culture the injected fertilized eggs to obtain NEB loss-of-function zebrafish mutants.

[0007] In some embodiments, the preparation method further includes the following steps: 4) The NEB loss-of-function zebrafish mutant obtained in step 3) is cultured to sexual maturity, mated with wild-type zebrafish, and after screening and purification, a stable genetic homozygous NEB loss-of-function zebrafish mutant strain is obtained.

[0008] In this invention, NEB refers to nebulin, a giant cytoskeletal protein located on the long arm of chromosome 2 (2q23.3) with gene ID 4703. In zebrafish, NEB is encoded by the neb gene, with gene ID 566928.

[0009] In this invention, NEB loss of function refers to a decrease or loss of expression level of the NEB gene or its homologous genes, or a decrease or loss of activity of NEB or its homologous proteins. NEB loss of function can be achieved by knockout or knockdown, or by administering NEB inhibitors.

[0010] A second aspect of the present invention provides a gene-editing reagent for targeting zebrafish NEB, said gene-editing reagent being selected from any of the following: i) sgRNA targeting zebrafish NEB, said sgRNA having a nucleotide sequence as shown in SEQ ID NO:1; ii) An expression vector containing the sgRNA described in i); iii) Contains the sgRNA described in i), and the Cas9 protein or mRNA encoding the Cas9 protein.

[0011] A third aspect of the present invention provides a composition comprising the gene editing reagent described in the second aspect, and a pharmaceutically acceptable carrier or delivery excipient.

[0012] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, to a reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that meet a reasonable benefit / risk ratio. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in pharmaceutical compositions may comprise fluids such as water, saline, glycerol, and ethanol. Such carriers may also contain excipients such as wetting agents or emulsifiers, pH buffers, etc.

[0013] The fourth aspect of the present invention provides in vitro tissue, cell or genomic DNA samples of NEB loss-of-function zebrafish mutants obtained by the preparation method described in the first aspect.

[0014] In some embodiments, the ex vivo tissue is selected from ocular tissue, retinal tissue, choroidal tissue, scleral tissue, or lens tissue.

[0015] In some embodiments, the cells are selected from retinal ganglion cells, photoreceptor cells, Müller cells, scleral fibroblasts, or lens epithelial cells.

[0016] In some embodiments, the RNA sample is selected from total RNA, mRNA, miRNA, or lncRNA.

[0017] The fifth aspect of the present invention provides the use of NEB or its encoded protein in screening or preparing medicaments for treating high myopia.

[0018] The sixth aspect of the present invention provides the use of the ex vivo tissue, cell, or genomic DNA samples described in the fourth aspect in screening drugs for the treatment of high myopia.

[0019] The seventh aspect of the present invention provides a method for screening drugs for the treatment of high myopia, the method comprising the following steps: administering a candidate drug to a NEB loss-of-function zebrafish mutant obtained by the preparation method described in the first aspect; The zebrafish mutant was tested for at least one of the following indicators: eye size, body length, optokinetic response, and average swimming speed under light and dark stimuli. The test results were compared with those of control zebrafish mutants that had not been given the candidate drug. If the candidate drug could improve at least one of the following phenotypes: reduced eye size, shortened body length, reduced optokinetic response, and reduced swimming speed, then the candidate drug was a potential treatment for high myopia.

[0020] In some embodiments, the improvement refers to an improvement of at least about 10%, at least about 30%, at least about 50%, at least about 80%, or more in the mutant’s symptoms (such as eye size, body length, optokinetic response, average swimming speed under light and dark stimuli) compared to before administration of the candidate drug.

[0021] In this invention, therapeutic candidates can be obtained from a variety of sources, including but not limited to synthetic, naturally occurring, or recombinant molecules, including small molecules, peptides, antibodies, or other polypeptides. For example, a variety of organic compounds and biomolecules can be synthesized randomly and directionally, or libraries of natural compounds in the form of bacterial, fungal, plant, or animal extracts, or natural or synthetic libraries and compounds modified by conventional chemical, physical, or biochemical means, or known pharmacological agents can be chemically modified in a directed or random manner, such as by acylation, alkylation, esterification, amidation, etc., to generate structural analogs.

[0022] Furthermore, the candidate drug may be a protein analog, antibody, nucleic acid, or other small molecule compound.

[0023] In some embodiments, the nucleic acid may be in the form of DNA, RNA, or a chemically modified analogue thereof.

[0024] In some embodiments, the DNA may be in the form of single-stranded DNA, double-stranded DNA, circular DNA, or linker DNA.

[0025] In some embodiments, the RNA may be in the form of mRNA, tRNA, rRNA, snRNA, hRNA, antisense RNA, tCRNA, dsRNA, SCRNA, catalytically active RNA, or various viral RNAs.

[0026] In some embodiments, the chemically modified analogue may be a short DNA or RNA fragment that has been artificially designed and chemically modified, such as an antisense oligonucleotide or a morpholino oligonucleotide.

[0027] In some embodiments, the candidate drug comprises various pharmaceutically acceptable salt forms.

[0028] In some embodiments, the candidate drug includes a drug used alone or a drug composition.

[0029] In some embodiments, the pharmaceutical composition refers to a combination of a candidate drug and a pharmaceutically acceptable carrier.

[0030] In some embodiments, the small molecule compounds are sourced from: newly synthesized or existing databases; wherein existing databases include, but are not limited to, general natural product databases, plant natural product databases, traditional Chinese medicine natural product databases, microbial natural product databases, marine natural product databases, natural product databases from different countries and regions, food natural product databases, toxic natural product databases, natural product industrial catalogs, databases deduplicated using MS data, databases deduplicated using NMR data, etc.

[0031] The eighth aspect of this invention provides a kit for constructing NEB loss-of-function zebrafish mutants, the kit comprising: a) the gene editing reagents described in the second aspect; and / or, b) The composition described in the third aspect; c) Microinjection buffer; d) User manual.

[0032] The ninth aspect of the present invention provides reagents for knocking down or inhibiting NEB or its homologous genes, and applications of the ex vivo tissue, cell, or genomic DNA samples described in the fourth aspect, said applications including any one of the following: 1) Application in constructing NEB loss-of-function animal models or high myopia animal models; 2) Application in the preparation of research tools for studying the pathogenesis of high myopia or assessing visual function.

[0033] Advantages and beneficial effects of this invention: This invention discloses for the first time the correlation between NEB and hereditary high myopia, and provides a method for constructing an animal model of myopia by knocking out NEB. Through experimental exploration, this invention has determined that homozygous deletion strains of zebrafish can be stably inherited by targeting exon 5 of the homologous gene using the CRISPR-CAS9 system. These zebrafish strains exhibit pathological characteristics such as shortened body length, reduced interocular distance, and reduced eye size, consistent with the clinical manifestations of pathological myopia, and represent an animal model that can stably reproduce the myopia phenotype. The animal model provided by this invention can be widely used for exploring the pathogenesis of hereditary myopia, validating gene therapy targets, and evaluating the efficacy of candidate compounds. Attached Figure Description

[0034] Figure 1 Manhattan plot showing the association between SNP allele frequency and the progression of high myopia.

[0035] Figure 2Figure 1 shows the effects of neb gene knockout on eye development and swimming speed in zebrafish. A represents the targeting efficiency of the neb gene as assessed by Sanger sequencing. Red nucleotides indicate the target sequence, black nucleotides or "-" indicate CRISPR / Cas9-mediated insertions or deletions, and gray nucleotides correspond to non-target bases in the original sequence. BD shows the body length, interocular distance, and eye area statistics for the negative control group (neb-NC) and the knockdown group (neb-KD). E shows the eye movement frequency in the neb NC and neb KD groups. F shows the difference in average swimming speed per minute between the NC and KD groups during alternating light and dark stimuli (5 minutes each time). Orange areas represent light stimulation, and blue areas represent dark environments. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates no significant difference. Detailed Implementation

[0036] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] Example I. Materials and Methods 1. Research Subjects This study included Chinese children aged 6 to 18 years at baseline, from the population-based, nationwide, million-scale Child and Adolescent Myopia Survey (CAMS) conducted in Wenzhou starting in September 2019, with a 4-year follow-up period ending in June 2023. Automated refraction without cycloplegia was performed using a Goaleye RM-9000 (Shenzhen Aster Industrial Co., Ltd., China; formerly known as GoldEyeRM-9000) autorefractor. Right eye spherical power (RSP), left eye spherical power (LSP), right eye cylindrical power (RLP), and left eye cylindrical power (LLP) were measured for each individual. The equivalent spherical power (SE) was calculated as the spherical power plus half the cylindrical power. The worse-eye SE was defined based on baseline SE measurements, and the average progression over 5 years was calculated for subsequent analysis. This study was approved by the Ethics Committee of the Eye Hospital of Wenzhou Medical University and conducted in accordance with the principles of the Declaration of Helsinki. All participants signed written informed consent forms.

[0038] 2. Whole exome sequencing and quality control All participants underwent whole-exome sequencing. Individuals with only one refractive measurement were excluded, as were those with a mean detection rate <0.9. Variant filtering criteria: genotype detection rates <0.9 or deviations from Hardy-Weinberg equilibrium (p<1×10⁻⁶) were excluded. -6 Variants of the variants were included. Only common variants (minor allele frequencies (MAF) > 0.01) were retained for subsequent analysis. Minor allele frequencies (MAFs) of variants in global, European, Asian, and African populations were obtained from the gnomAD database.

[0039] 3. Zebrafish Experiment Homologous genes from zebrafish were retrieved from the ENSEMBL database. Single guide RNAs (sgRNAs) (neb-Target1F: TTAATACGACTCACTATA) were designed using CRISPR / Cas9 technology via chopchop. GTTGCCTTCAGTCCC TCCATA GTTTTAGAGCTAGAAATAG (SEQ ID NO:1). During the single-cell stage, Cas9 protein was combined with targeted or non-targeted sgRNAs and microinjected (using PICOSPRITZER® Ⅲ (Parker, USA)) to construct zebrafish knockdown and control models. Sanger sequencing was used to assess target site cleavage efficiency. Eight to twenty zebrafish larvae aged 5 days post-fertilization (dpf) were selected for behavioral evaluation. Phenotypic observation focused primarily on eye development, including eye size, interocular distance, and eye area, quantified using ImageJ software. Optokinetic response (OKR) software (ViewPoint OKR 2.0, ViewPoint, France) was used to record eye movement responses over one minute. Furthermore, the difference in average swimming speed of zebrafish under alternating light and dark stimuli was measured using a behavioral assessment system (ZebraLab 3.22.3.31, Viewpoint, France) to evaluate their swimming behavior.

[0040] 4. Functional Analysis Amino acid sequence changes were assessed using ENSEMBL. RNA tertiary structures were generated using 3dRNA and AlphaFold2 and visualized in PyMOL. Structural similarity between RNA pairs was quantified using RNAalign, where the TM-score ranged from 0 to 1, with 1 indicating a perfect match and ≥0.45 indicating significant structural similarity.

[0041] 5. Statistical Analysis Using the 5-year mean progression value as a quantitative trait, genome-wide association analysis (GWAS) was performed using SNPTEST v2.5.2 to identify potential susceptible SNPs. Loci with a log-Bayes factor >3 (log(BF) > 3) were considered potential loci associated with high myopia. The Kruskal-Wallis test and Wilcoxon rank-sum test were used to assess differences in progression values ​​between different genotypes. The Wilcoxon rank-sum test was used for pairwise comparisons between groups. Genotype effects under a codominant model were assessed using SNPstats software. Generalized additive models (GAMs) were used to assess the interaction between genotype and age for susceptible SNPs, after adjusting for age and equivalent spherical lenses. Curve parameters, effective degrees of freedom (edf), and p-values ​​for the smoothing term were obtained. edf = 1 corresponds to a linear relationship, and edf > 1 reflects a non-linear effect. The significance level was defined as p < 0.05. All analyses were performed using R software (version 4.0.2).

[0042] II. Experimental Results 1. Characteristics of the research population This study included 7,635 school-aged adolescents aged 6 to 18 years at enrollment, all from the CAMS survey and with at least one follow-up record. The baseline mean age was 14.60 ± 2.04 years, with males comprising 52.35% (N = 3,997). The overall mean annual progression of high myopia (HM) was -0.53 ± 0.46 diopters / year (D / y) (see Table 1).

[0043] Table 1. Demographic characteristics of high myopia cohorts grouped by 5-year average progression

[0044] 2. Genetic association between genotype and HM progression After adjusting for age, sex, and 3 PCAs, NEB rs149510427 (log10 (Bayes factor) = 3.03) showed a possible association with the 5-year mean progression of HM. For NEB rs149510427, 85.72% (N=6545) of participants carried only the wild-type allele, 13.58% (N=1037) carried the risk allele, and 0.68% (N=52) carried two risk alleles. Compared to GG, NEB rs149510427 showed a dose-dependent effect on GT and TT (-0.06D, -0.14D, ...). Figure 1 ).

[0045] 3. Potential regulation of risky SNPs and gene induction in computer analysis Based on the ENSEMBL annotation, rs149510427 is located in exon 145 of NEB, and rs149510427 in NEB does not cause any amino acid changes.

[0046] 4. Functional effects of NEB on zebrafish eye development Comparative analysis showed that NEB is the only gene in zebrafish with evolutionarily conserved homologs. Using the CRISPR-Cas9 system, we generated a knockdown group targeting exon 5 (neb KD) and a negative control group with a non-target site (neb NC). Sanger sequencing confirmed that the knockdown efficiency at the target site was 88.89%. Figure 2 (A) Regarding eye development, compared to the negative control group (neb-NC), knockdown of neb (neb KD) resulted in shorter body length, reduced interpupillary distance, and smaller eye size (pbody length = 0.010, resulting interpupillary distance = 0.002, eye size = 0.011). Figure 2 Regarding optomotor response (OKR), the neb KD group showed relatively fewer ocular motor responses, although the difference did not reach statistical significance. Figure 2 (E in the text). Finally, under light and dark stimuli, the average swimming speed of zebrafish in the neb-KD group was significantly lower than that in the neb-NC group (p=1.72×10). -4 () Figure 2 (F in the middle).

[0047] Based on the above results, this invention is the first to discover and identify the key SNPNEB rs149510427 in high myopia in children and adolescents. Functional studies in zebrafish have confirmed that knocking out neb leads to delayed eye development and impaired visual function, exhibiting pathological features such as shortened body length, reduced interocular distance, and reduced eye size, consistent with the clinical manifestations of pathological myopia. This is an animal model that can stably reproduce the myopic phenotype.

[0048] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a NEB loss-of-function zebrafish mutant, wherein the zebrafish mutant is used as a model of highly myopic zebrafish, characterized in that, The preparation method includes the following steps: 1) Design and synthesize sgRNA targeting zebrafish NEB, the nucleotide sequence of which is shown in SEQ ID NO:1; 2) Inject the sgRNA described in step 1) together with the Cas9 protein or the mRNA encoding the Cas9 protein into zebrafish fertilized eggs; 3) Culture the injected fertilized eggs to obtain NEB loss-of-function zebrafish mutants.

2. The preparation method according to claim 1, characterized in that, The preparation method further includes the following steps: 4) The NEB loss-of-function zebrafish mutant obtained in step 3) is cultured to sexual maturity, mated with wild-type zebrafish, and after screening and purification, a stable genetic homozygous NEB loss-of-function zebrafish mutant strain is obtained.

3. A gene-editing reagent for targeting zebrafish NEB, characterized in that, The gene editing reagent is selected from any of the following: i) sgRNA targeting zebrafish NEB, the nucleotide sequence of which is shown in SEQ ID NO:1; ii) An expression vector containing the sgRNA described in i); iii) Contains the sgRNA described in i), and the Cas9 protein or mRNA encoding the Cas9 protein.

4. A composition, characterized in that, The composition comprises the gene editing reagent of claim 3, and a pharmaceutically acceptable carrier or delivery excipient.

5. An in vitro tissue, cell, or genomic DNA sample of a NEB loss-of-function zebrafish mutant obtained by the preparation method according to any one of claims 1-2.

6. A kit for constructing NEB loss-of-function zebrafish mutants, characterized in that, The kit includes: a) The gene editing reagent according to claim 3; and / or, b) The composition according to claim 4; c) Microinjection buffer; d) User manual.

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