Construction method and application of ECHS1D disease animal model

Abstract

The invention discloses a construction method of an ECHS1D disease animal model and an application of the ECHS1D disease animal model, and discloses the construction method of the ECHS1D disease animal model. The invention discloses an application and a method of the animal model constructed by the construction method in the aspect of simulating animal ECHS1D disease and Like's syndrome pathological research and screening a pharmaceutical composition for treating ECHS1D disease and Like's syndrome, a new mouse Like's syndrome disease model is constructed aiming at a new Echs1 gene mutation mode for the first time, and the application and the method of the animal model in screening the pharmaceutical composition for treating ECHS1D disease and Like's syndrome are used for screening the pharmaceutical composition for treating ECHS1D disease and Like's syndrome. The method can be used for simulating a mitochondrial function damage mechanism caused by Echs1 gene homozygous point mutation.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a method for constructing an ECHS1D disease animal model and application thereof. Background Art

[0002] Single gene genetic disease refers to a genetic disease controlled by a pair of alleles. Although the incidence of each single gene genetic disease is low, the overall prevalence is not low due to the large number of types. Single gene genetic diseases have different pathogenesis according to different pathogenic genes. According to the inheritance mode, they can be divided into autosomal dominant genetic diseases, autosomal recessive genetic diseases, X-linked dominant genetic diseases, X-linked recessive genetic diseases, linked genetic diseases and genetic diseases caused by mitochondrial genome mutations. Common causes of disease include developmental disorders, metabolic defects, and abnormal host immune defense and immune regulation. In recent years, the rapid development and widespread application of sequencing technology have greatly accelerated the diagnosis of single gene genetic diseases and the identification of new pathogenic mutations. With the continuous discovery of new pathogenic genes and in-depth research on pathogenic mechanisms, the treatment of single gene genetic diseases has also entered a new era. The treatment methods used for different diseases in clinical practice are different, and different treatment methods also differ in the molecular mechanisms of targeted diseases.

[0003] Leigh Syndrome (also known as subacute necrotizing encephalopathy) is a severe neurodegenerative disease characterized by brain lesions represented by focal bilateral symmetrical lesions, especially in the basal ganglia, thalamus and brainstem, but with considerable clinical and genetic heterogeneity. Clinically, Leigh Syndrome is characterized by varying degrees of neurological abnormalities, ranging from severe neurological problems to almost no lesions. The most common is damage to the central nervous system, manifested as psychomotor retardation, seizures, nystagmus, ophthalmoparesis, optic atrophy, ataxia, dystonia or respiratory failure. Since its early discovery, Leigh Syndrome has been shown to be not only genetically heterogeneous, but also clinically heterogeneous, sometimes presenting with atypical but highly Leigh Syndrome-like neurological abnormalities, also known as Leigh-like syndrome. Leigh syndrome is associated with more than 75 genes. Defects in the respiratory chain (especially complex I, II, IV or V), coenzyme Q or pyruvate dehydrogenase complex can cause Leigh syndrome or Leigh-like syndrome. Mutations in the structural and assembly factors of complex I of the respiratory chain (OXPHOS) are the most common causes of the disease. Leigh syndrome and Leigh-like syndrome are the mitochondrial diseases with the most extensive genetic heterogeneity. In a few cases, non-mitochondrial diseases may also manifest as Leigh syndrome.

[0004] ECHS1 (short-chain enoyl-CoA α-hydratase), also known as crotonase, is a 290-amino acid protein located in the mitochondrial matrix that participates in multiple metabolic pathways such as fatty acid oxidation and degradation of essential amino acids such as valine (Val). ECHS1 deficiency (ECHS1D) is a rare mitochondrial disease caused by biallelic mutations that leads to reduced ECHS1 enzyme activity. It usually develops at birth or in early childhood, and some cases die within two days after birth. The clinical features of the disease are represented by Leigh-like syndrome, characterized by but not limited to severe developmental delay or regression, feeding difficulties, growth retardation, extreme irritability, central hypotonia, seizures, metabolic acidosis, visual impairment, and cardiomyopathy. Almost all reported cases of ECHS1D have cranial magnetic resonance imaging (MRI) findings consistent with Leigh syndrome, but there are still differences between the clinical manifestations of ECHS1D and the typical features of Leigh syndrome. Therefore, ECHS1D is classified as a unique form of Leigh-like syndrome associated with severe progressive encephalopathy, accompanied by bilateral brain damage and mitochondrial dysfunction. ECHS1D has a wide mutation spectrum, and more than sixty patients have been reported, including more than twenty missense mutations causing ECHS1D disease, and the only homozygous truncating mutation found in a pair of siblings caused the patient to die within 48 hours of birth. Although molecular diagnostic methods have been used in the diagnosis of ECHS1D disease, patients carrying the same gene mutation may have very different disease manifestations, so the symptoms of the disease are highly heterogeneous, making it difficult to diagnose it. Existing studies still cannot confirm the cause of ECHS1D, making it difficult to treat patients with ECHS1D.

[0005] There are five reported mouse models of Leigh syndrome and Leigh-like syndrome, which mainly focus on mutations in mitochondrial respiratory chain proteins.

[0006] 1. A mouse model of Leigh syndrome was constructed by knocking out the gene of Ndufs4, a component of mitochondrial complex I. Homozygous mutant mice with Ndufs4 knockout will develop encephalopathy and fatal respiratory disorders and die 50 days after birth. It is suitable for the development and degenerative lesions of the nervous system caused by Leigh syndrome and is currently the most commonly used Leigh syndrome model mouse in the field.

[0007] 2. A mouse model of Leigh syndrome constructed by knocking out the nuclear gene Surf1. Sfurf1 is a component of the mitochondrial translation regulatory assembly intermediate cytochrome c oxidase complex (MITRAC complex), and is involved in regulating the assembly of cytochrome c oxidase (mitochondrial complex IV). Surf1 homozygous knockout mice will develop mitochondrial diseases with cytochrome c oxidase deficiency, accompanied by high embryonic lethality and reproductive disorders. They also show early spontaneous death after birth, and their fertility is impaired even in adulthood.

[0008] 3. Leigh syndrome mice constructed by knocking out mitochondrial superoxide dismutase (SOD). Mice lacking mitochondrial manganese superoxide dismutase accumulate reactive oxygen species and thus exhibit neurological symptoms similar to Leigh syndrome.

[0009] 4. A mouse model of Leigh-like syndrome disease constructed by knocking out the mitochondrial membrane protease PARL showed necrotizing encephalomyelopathy similar to Leigh-like syndrome, accompanied by changes in brain mitochondrial ultrastructure and impaired mitochondrial complex III activity, coenzyme Q (CoQ) biosynthesis, and mitochondrial calcium metabolism. Mutations in the PARL gene are more likely to manifest as Parkinson's disease, Leber hereditary optic neuropathy, and type 2 diabetes in patients, and no association with Leigh-like syndrome has been reported, so this model is still questionable in exploring Leigh-like syndrome.

[0010] 5. Knockout of the C subunit of the SDH protein, a component of mitochondrial complex II, caused mice to show Leigh-like syndrome. Inducible knockout mice showed hyperlactosis and other symptoms and died within four weeks of induced knockout. No tissue damage was found in autopsy. Clinically, the gene mutation of the A subunit of the SDH protein is associated with Leigh-like syndrome, and mutations in SDHC mostly show increased susceptibility to multiple tumors. This model still has some uncertainty when exploring the pathogenesis of Leigh-like syndrome. Summary of the invention

[0011] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0012] The present invention provides a method for constructing an ECHS1D disease animal model, wherein the animal model expresses a homozygous point mutation protein of an Echs1 gene, and the point mutation site is p.C225S.

[0013] Furthermore, the point mutation site is c.673T>C.

[0014] Furthermore, the construction method includes the step of using CRISPR / Cas9 technology and / or TALEN technology to point mutate the Echs1 gene in the animal model.

[0015] Furthermore, the construction method comprises the following steps: The recombinant vector is injected into a single-cell embryo or embryonic stem cell through gene editing technology and transplanted into a pseudo-pregnant mother to obtain an F0 generation chimeric individual. The F0 generation chimeric individual is hybridized with a wild-type individual to obtain an F1 generation positive heterozygous individual. The F1 generation individual is hybridized with a wild-type individual to obtain an F2 generation heterozygous individual. The F2 generation heterozygous individual is self-pollinated to obtain an F3 generation homozygous individual, i.e., the ECHS1D disease animal model.

[0016] Furthermore, the gene editing technology includes CRISPR / Cas9 technology and / or TALEN technology.

[0017] The term "single-cell embryo" used in the present invention refers to cells that have cell totipotency and can grow into a complete individual after differentiation, including fertilized eggs, embryonic cells, etc.

[0018] The term "embryonic stem cells" used in the present invention refers to stem cells that can differentiate into embryonic cells with different functions and in different parts and stages. Embryonic stem cells are a type of cells separated from early embryos or primitive gonads, with high proliferation ability and multidirectional differentiation potential, and can differentiate into primitive cells of all cell types in the three germ layers. According to the different sources of embryonic stem cells, they are divided into ES cells, which come from the blastocyst stage; EC cells, which are cells separated and screened from teratocarcinomas and have the ability to proliferate and self-renew; EG cells, which are separated and obtained from primitive germ cells.

[0019] Further, the construction method includes the following steps: designing, constructing, and in vitro transcribing sgRNA according to the Echs1 gene mutation, and connecting the sgRNA to the homologous recombination vector; the p.[Cys225Ser]; [Cys225Ser] point mutation is a mutation of the codon TGT at the 225th position to AGT. Fertilized eggs are collected and pseudo-pregnant female mice to be transplanted with fertilized eggs are prepared, and Cas9, sgRNA and homologous recombination vectors are introduced into the fertilized eggs of mice for homologous recombination. The injected fertilized eggs are transplanted into pseudo-pregnant female mice, and after the mice are born, the genotypes are identified by PCR amplification and sequencing to obtain F0 generation chimeric mice. F0 generation mice are mated with C57BL / 6J mice, and the genotypes are identified by PCR amplification and sequencing to obtain positive F1 generation heterozygous mice. F1 generation mice are mated with C57BL / 6J mice, and the genotypes are identified by PCR amplification and sequencing to obtain F2 generation heterozygous mice. The F2 generation heterozygous mice were self-fertilized, and their genotypes were identified by PCR amplification and sequencing to obtain F3 generation homozygous mice.

[0020] Furthermore, the ECHS1D disease animal model appears in the F3 generation, and the ECHS1D disease animal model is a homozygous point mutation Echs1 gene.

[0021] Furthermore, the amino acid sequence of the homozygous point mutation of the Echs1 gene is shown in SEQ ID NO:1.

[0022] The SEQ ID The sequence of NO:1 is as follows: MAALRALLPRACSSLLSSVRCPELRRFASGANFQYIITEKKGKNSSVGLIQLNRPKALNALCNGLIEELNQALETFEQDPAVGAIVLTGGDKAFAAGADIKEMQNRTFQDCYSSKFLSHWDHITRVKKPVIAAVNGYAL GGGCELAMMCDIIYAGEKAQFGQPEILLGTIPGAGGTQRLTRAVGKSLAMEMVLTGDRISAQDAKQAGLVSKIFPVEKLVEEAIQSAEKIASNSKIVVAMAKESVNAAFEMLTEGNKLEKRLFYSTFATDDRREGMTAFVEKRKANFKDH.

[0023] Furthermore, the homozygous point mutation of the Echs1 gene includes a nucleotide sequence formed by a mutation of the 673rd nucleotide in the protein coding region sequence of the wild-type Echs1 gene from T to C.

[0024] Furthermore, the introduction method includes electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, and viral infection.

[0025] Furthermore, the vector includes retrovirus, adenovirus, adeno-associated virus, herpes virus, poxvirus, baculovirus, papillomavirus, papovavirus, phage, and plasmid.

[0026] The term "ECHS1" used in the present invention refers to wild-type ECHS1 (ENSMUSG00000025465), the website of which is: http: / / asia.ensembl.org / Mus_musculus / Gene / Summary?db=core;g=ENSMUSG00000025465;r=7:140105710-140116476;t=ENSMUST00000026538. Specifically in the present invention, the transcript targeted by the point mutation of the ECHS1 gene is Echs1-201 (ENSMUST00000026538.12), the targeted site is C225S, and the exon where the mutation site is located is exon 6.

[0027] The term "sgRNA" used in the present invention refers to guide RNA (gRNA), also known as small guide RNA (sgRNA), which is a post-transcriptional modification process called RNA editing in kinetoplastids. SgRNA is a small non-coding RNA that can pair with pre-mRNA and insert some uracil into it to produce mRNA with function. The RNA molecule edited by the guide RNA is about 60 to 80 nucleotides in length, transcribed from a separate gene, has a 3' oligo U tail, a sequence in the middle that is precisely complementary to the edited mRNA, and an anchor sequence at the 5' end that is complementary to the non-edited mRNA sequence.

[0028] In some embodiments, the homologous recombination vector refers to a composition that can promote transduction of a selected nucleic acid into a cell or expression of the nucleic acid in the cell, for example, a plasmid, a cosmid, a virus, a YAC, a bacterium, polylysine, a chromosomal integration vector, an episomal vector, etc.

[0029] In some embodiments, the animal model is a rodent, rabbit, pig, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques); preferably, a rodent (e.g., mice, rats, and the like); particularly preferably, a mouse.

[0030] Furthermore, the rodents include the family Cricetidae, the family Cricetidae, the family Muridae, the family Myrmecophaga, the family Myrmecophaga, the family Myrmecophaga, the family Myrmecophaga, and the family Myrmecophaga.

[0031] Further, the family Muridae includes black rats, brown rats, gerbils, New World rats, Old World rats, SD rats, Polynesian rats, tree rats, wood rats, stick rats, rice rats, kangaroo rats, and climbing rats.

[0032] In some embodiments, rodents of the present disclosure include, by way of non-limiting examples, mice, rats, and hamsters.

[0033] In some embodiments, rodents of the present disclosure include mice and rats as non-limiting examples.

[0034] In some embodiments, the rodent is selected from the superfamily Muroidea.

[0035] In some embodiments, the rodent of the present disclosure is from a family selected from the group consisting of Calomyscidae (e.g., Calomyscidae, Bart's hamster, Goodman's hamster, Golden hamster, Hall's hamster, Bearded hamster, Chu's hamster, Ulaty hamster), Cricetidae (e.g., Black-lined hamster, Vole, Crowned hamster), Muridae (Black rat, Brown rat, Gerbil, New World rat, Old World rat, Norway rat, Polynesian rat, Tree rat, Cotton rat, Wood rat, Stick rat, Rice rat, Kangaroo rat, Climbing rat), Nesomyidae (Long-tailed giant rat, South African pocket rat, African giant rat, Malvinas white-tailed rat), Platacanthomyidae (e.g., Spiny rat, Pig-tailed rat), and Spalacidae (e.g., Mole, Bamboo rat, and Zokrat).

[0036] In some embodiments, the rodent of the present disclosure is selected from the group consisting of mouse or rat (family Muridae), gerbil, spiny mouse, and crested mouse.

[0037] In some embodiments, the rats of the present disclosure are from a member of the family Muridae.

[0038] The term "vector" used in the present invention refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to cause the expression of the protein. The vector can be used to transform, transduce or transfect a host cell to express the genetic elements carried in the host cell.

[0039] The present invention provides the use of the animal model generated by the above-mentioned method in simulating ECHS1D disease in animals.

[0040] The present invention provides the application of the animal model generated by the above-mentioned method in the study of the pathology of Leigh-like syndrome.

[0041] The present invention provides the use of the animal model generated by the method described above in screening a pharmaceutical composition for treating ECHS1D disease and Leigh-like syndrome.

[0042] Furthermore, the pharmaceutical composition includes a pharmaceutically acceptable adjuvant.

[0043] Furthermore, the auxiliary agent includes an excipient, a glidant, a sweetener, a diluent, a preservative, a dye / colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersant, a suspending agent, a stabilizer, an isotonic agent, a pH adjusting and / or buffering agent, a solvent, a surfactant or an emulsifier.

[0044] Furthermore, the administration methods of the pharmaceutical composition include oral administration, suppository administration, perilesional administration, lesion surface administration, local application, corneal stroma injection, subconjunctival injection, intravenous administration, parenteral administration, intraperitoneal administration, muscle administration, intralesional administration, intrathecal administration, intranasal administration or subcutaneous administration.

[0045] The present invention provides a method for screening one or more candidate drugs using the animal model / animal model cells produced by the method described above, the method comprising the step of administering the one or more candidate drugs to the animal model / animal model cells.

[0046] Furthermore, the candidate drugs include protein analogs, antibodies, DNA, and RNA.

[0047] Furthermore, the drug candidate includes various pharmaceutically acceptable salt forms.

[0048] Furthermore, the DNA includes single-stranded DNA, closed-circle DNA, and linked DNA.

[0049] Furthermore, the RNA includes mRNA, tRNA, rRNA, snRNA, hRNA, antisense RNA, tCRNA, dsRNA, SCRNA, RNA with catalytic activity, and various viral RNAs.

[0050] In some embodiments, pharmaceutically acceptable salt forms include acid / base addition salts, specifically acid addition salts including but not limited to hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, hexanoate, arginine, fumarate, nicotinate, phthalate or oxalate of FTY720, and base addition salts including but not limited to lithium, sodium, potassium, barium, calcium, magnesium, aluminum, iron, ferrous, copper, zinc salts of FTY720, or FTY720 with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

[0051] The term "pharmaceutically acceptable" as used in the present invention refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, within the scope of reasonable medical judgment, and in accordance with a reasonable benefit / risk ratio.

[0052] The term "antibody" as used in the present invention includes heterotetrameric glycoproteins of about 150,000 daltons with identical structural features, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH). This is followed by multiple constant regions. One end of each light chain has a variable region (VL) and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0053] The present invention provides a method for identifying a therapeutic agent for treating ECHS1D disease and Leigh-like syndrome for non-therapeutic purposes, the method comprising: using the agent in an animal model produced by the method described above, and detecting whether the animal model has changes in abnormal symptoms related to ECHS1D disease and Leigh-like syndrome; when the use of the agent has a therapeutic effect on ECHS1D disease and Leigh-like syndrome, the agent is identified as a therapeutic agent.

[0054] Advantages and beneficial effects of the present invention: ECHS1D is a genetic disease associated with impaired mitochondrial fatty acid oxidation. Although its disease manifestations are similar to those of Leigh-like syndrome, its clinical phenotype and pathogenesis show significant heterogeneity. Its specific pathogenesis remains in question. ECHS1 is involved in fatty acids and amino acids, and its deficiency is expected to lead to impaired mitochondrial fatty acid oxidation. However, since the membrane-bound enzyme machinery for long-chain fatty acids in ECHS1D patients remains intact, ketone body production should not be completely interrupted in ECHS1D patients, and it can be assumed that the resulting clinical phenotype should be a milder and relatively benign clinical phenotype similar to SCAD deficiency. However, ECHS1D patients show mild to moderate reduction in ketosis, but neither hypoketotic hypoglycemia and hypoglycemic encephalopathy occur, nor do they have acylcarnitines in the blood due to decreased levels of fatty acid β-oxidation. In addition, three of the ECHS1D patients showed abnormal palmitic acid metabolism, indicating that their short-chain fatty acid oxidation was impaired, rather than long-chain fatty acid metabolism involving the ECHS1 protein.

[0055] To complicate matters further, although the metabolic disorders of ECHS1D patients are expected to lead to relatively mild clinical phenotypes, they actually show severe clinical symptoms, such as Leigh-like syndrome, neonatal lactic acidosis, sensorineural deafness, hypotonia, cardiomyopathy, and a high incidence of respiratory failure. ECHS1D patients are often accompanied by neurological diseases, which are quite rare in diseases related to fat oxidation defects. In fact, the clinical manifestations of ECHS1D patients have some similarities with those of long-chain acyl-CoA α dehydrogenase (LCHAD) deficiency and mitochondrial trifunctional protein (MTP) deficiency, similar to primary OXPHOS deficiency. Due to the significant difference between the clinical manifestations of moderately impaired mitochondrial fatty acid oxidation and the severe clinical manifestations of ECHS1D patients, ECHS1D may not be caused by impaired mitochondrial fatty acid oxidation function, but there are other pathological mechanisms. In fact, short-chain enoyl-CoA α hydratase (ECHS1) is not only an essential enzyme for valine degradation in mitochondria, but also participates in the degradation of other branched-chain amino acids.

[0056] Secondary OXPHOS defects similar to Leigh syndrome have also been found in ECHS1D. These defects range from isolated defects in complex I, complex III, or complex IV to multiple defects in complex I / III / IV or complex I / IV / V. Although it is not clear what causes these secondary oxidative phosphorylation defects in ECHS1D, it is currently speculated that the accumulation of 2-methacrylyl-CoA caused by impaired valine metabolism may cause loss of mitochondrial complex OXPHOS activity, inhibiting mitochondrial respiration. However, it is difficult to predict the impact of ECHS1D on OXPHOS. Many ECHS1D patients do not show symptoms of OXPHOS, while patients with secondary OXPHOS defects usually show more severe Leigh-like symptoms and have more severe clinical manifestations. Therefore, the relationship between ECHS1D patients and Leigh-like syndrome is not completely equivalent, and the existing disease model of Leigh-like syndrome is not completely applicable to the exploration of the causes of ECHS1D patients.

[0057] The only disease model of ECHS1D mice is the heterozygous knockout mouse of the Echs1 gene. There is no report on the construction of Echs1 point mutation mice, and the homozygous knockout mouse of the Echs1 gene is lethal in early embryonic development. The disease of Echs1 heterozygous mice manifests as neurological diseases such as epilepsy, and there is still a lack of disease models for the metabolic characterization of ECHS1D. Moreover, since heterozygous knockout mice carry one normal copy of the Echs1 gene, their disease phenotype may be relatively mild or not obvious, which makes it possible for heterozygous knockout mice to not fully simulate the pathological process of the disease when studying diseases caused by ECHS1 deficiency. Therefore, constructing a mouse model of the mutation site of ECHS1D patients will help to more accurately study the pathogenesis and causes of ECHS1D, clarify the relationship between ECHS1D and OXPHOS dysfunction, and further studies will reveal whether ECHS1 deficiency will lead to impaired stability and / or biogenesis of the OXPHOS complex, promote the diagnosis and treatment of mitochondrial diseases, and based on a deeper understanding, more effective treatment strategies can be developed to improve the quality of life of patients.

[0058] Although a variety of mitochondrial disease models have been established, the complexity and heterogeneity of ECHS1D still require further study. Building a more accurate ECHS1D mouse model and further clarifying its pathogenesis will be important directions for future research.

[0059] The present invention proposes a method for constructing a mouse model of homozygous point mutation of amino acid at position 225 of Echs1 gene and its application. The construction method adopts CRISPR / Cas9 technology to construct homozygous point mutation of Echs1 gene p.C225S, perform homologous recombination on fertilized eggs, and then transplant them into pseudo-pregnant female mice to wait for pregnancy and littering, and screen them in F0 generation mice; the mouse models are all Echs1 C225S The mice obtained by gene unit point mutation, rather than complete knockout of Echs1 gene sequence, showed impaired mitochondrial respiratory capacity and body metabolic defects, simulating the type and severity of metabolic impairment caused by Echs1 gene 225 site mutation carried by clinical patients. The present invention for the first time targets a new Echs1 gene mutation mode: c.[673T>C];[673T>C], p.[Cys225Arg];[Cys225Arg] point mutation, and constructs a new mouse Leigh-like syndrome disease model p.[Cys225Ser]; [Cys225Ser], which can be used to simulate the mechanism of mitochondrial dysfunction caused by homozygous point mutation of Echs1 gene, and is helpful for the mechanism research and treatment-related research of rare Leigh-like syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1The Echs1 gene is a heterozygous knockout mouse (Echs1 + / - ) Statistical results of genotypes of offspring from paired breeding.

[0061] Figure 2 It is the plasmid map of the homologous recombination vector, where Amp represents the ampicillin resistance screening gene, 5'arm represents the 5' homologous arm, 3'arm represents the 3' homologous arm, and HR represents the homologous recombination sequence.

[0062] Figure 3 Echs1 gene locus C225S Schematic diagram of single-site mutation. In double-stranded DNA, the target sequence of sgRNA (guide RNA) is shown in blue, the PAM sequence is shown in red, and the red arrow indicates the cleavage site of Cas9 protein. In the homologous recombination plasmid, the replaced nucleotide is shown in red (point mutation).

[0063] Figure 4 This is a schematic diagram of the F0 generation mouse identification strategy, in which the PCR sequence is shown in brown, the homology arm sequence is shown in black, the scissors indicate the cutting site of the Cas9 protein, and the red and purple asterisks are the mutation sites.

[0064] Figure 5 This is the result of genotyping of offspring mice. C225S Results of sequencing of PCR products from mutant mice (below).

[0065] Figure 6 Figure 1 shows the results of mitochondrial stress and glycolytic stress tests in mouse adult fibroblasts (MAFs). AB: wild type (WT) and Echs1 C225S Mitochondrial function of mouse MAF cells under various drug stimulations (mean ± standard error, unpaired t test; WT cells in 4 groups, Echs1 C225S Cell group 5). CE: wild type (WT) and Echs1 C225S Glycolytic function of mouse MAF cells under various drug stimulations (mean ± standard error, unpaired t test; WT cells and Echs1 C225S 5 groups of cells each).

[0066] Figure 7 The results of the assay of the enzyme activity of the respiratory chain complex in mouse liver are shown in Figure AE: wild type (WT) and Echs1 C225S Mouse liver 20 mg, enzyme activity was determined according to the method recommended by the kit manufacturer (mean ± standard error, using unpaired Welch test; A, C, D, 5 WT mice, Echs1 C225S 6 mice; B, E, 14 WT mice, Echs1C225S 16 mice).

[0067] Figure 8 The results of mouse liver metabolism mass spectrometry. A: Wild type (WT) and Echs1 C225S Metabolite differential analysis in mouse liver (9 WT mice, Echs1 C225S BE: wild type (WT) and Echs1 C225S The contents of intermediates of palmitic acid metabolic pathway in mouse liver (mean ± standard error, unpaired t test, 9 WT mice, Echs1 C225S 12 mice).

[0068] Fig. 9 The results of mouse liver metabolism mass spectrometry. AD: wild type (WT) and Echs1 C225S Metabolite contents in mouse liver (mean ± standard error, unpaired Welch test, 9 WT mice, Echs1 C225S 12 mice).

[0069] Fig.10 It is Echs1 C225S Figure 3. Results of blood glucose and glucose tolerance in wild-type mice after starvation and refeeding. A: Wild-type (WT) and Echs1 C225S Blood glucose levels of mice at different time points after fasting for 17 hours, and after refeeding for 2 hours (mean ± standard error, one-way ANOVA combined with Bonferroni test, number of mice in each group n = 13-14). BD: At ZT10 (B) and ZT22 (C), wild type and Echs1 C225S Mutant mice underwent intraperitoneal glucose tolerance test (IPGTT), and the results were normalized to baseline fasting blood glucose concentration, and the area under the curve (AUC) (D) was calculated (mean ± SD, one-way ANOVA combined with Bonferroni test, n = 6-7 mice per group). DETAILED DESCRIPTION

[0070] The present invention will be described in further detail below. It should be understood that the terms are intended for description purposes only and are not intended to limit the present invention.

[0071] Example 1

[0072] The present invention uses heterozygous knockout mice of the Echs1 gene (Echs1 + / - ) to pair up and breed offspring, and identify and count the genotypes of the offspring.

[0073] Over a two-year period, six cages of mice gave birth 70 times, including 169 wild-type mice and 220 Echs1 + / - mice, no homozygous knockout mice (Echs1 - / - According to genetic principles, when two heterozygous (Echs1 + / - ) are paired for breeding, the genotype of their offspring should be wild type (Echs1 + / + ), heterozygous (Echs1 + / - ) and homozygous knockout (Echs1 - / - The experimental results show that the number of wild-type and heterozygous mice roughly conforms to the above ratio (although there are random fluctuations), but the number of homozygous knockout mice is zero, as shown in the statistical chart. Figure 1 This indicates that during embryonic development, mice with homozygous knockout of Echs1 cannot survive to birth. Since no homozygous knockout mice were observed, it can be inferred that the loss of the Echs1 gene has a serious impact on embryonic development, resulting in the inability of homozygous knockout mice to be born.

[0074] Example 2

[0075] 1. Experimental Materials PrimeStar GXL (from TaKaRa, Catalog No. R050A), 2 × Rapid Taq Master Mix (from Nanjing Novozymes, Catalog No. P222), succinate dehydrogenase detection kit (from sigma, Catalog No. MAK184-1KT), Complex I Enzyme Activity Assay Kit (Colorimetric) (from Abcam, Catalog No. ab109721), CheKine™ Micro Mitochondrial complex V Activity Assay Kit (from Abbkine, Catalog No. KTB1890), CheKine™ Micro Mitochondrial complex IV Activity Assay Kit (from Abbkine, Catalog No. KTB1880), Yuyue blood glucose meter (from Yuyue, model 590), 10% glucose injection (from Chuangxin, veterinary drug number 140091535).

[0076] 2. Experimental methods and results According to the Echs1 gene mutation, sgRNA is designed, constructed, and in vitro transcribed, and the sgRNA is connected to the homologous recombination vector; the p.[Cys225Ser]; [Cys225Ser] point mutation is a mutation of the codon TGT at the 225th position to AGT. Fertilized eggs are collected and pseudo-pregnant female mice to be transplanted with fertilized eggs are prepared. Cas9, sgRNA and homologous recombination vectors are injected into the fertilized eggs of mice by microinjection for homologous recombination. The injected fertilized eggs are transplanted into pseudo-pregnant female mice. After the mice are born, their genotypes are identified by PCR amplification and sequencing to obtain F0 generation chimeric mice. F0 generation mice are mated with C57BL / 6J mice, and their genotypes are identified by PCR amplification and sequencing to obtain positive F1 generation heterozygous mice. F1 generation mice are mated with C57BL / 6J mice, and their genotypes are identified by PCR amplification and sequencing to obtain F2 generation heterozygous mice. The F2 generation heterozygous mice were self-fertilized, and their genotypes were identified by PCR amplification and sequencing to obtain F3 generation homozygous mice.

[0077] Name of the target gene for mutation (Ensembl number): Echs1 (ENSMUSG00000025465).

[0078] Ensembl website link of the mutant target gene: http: / / asia.ensembl.org / Mus_musculus / Gene / Summary?db=core;g=ENSMUSG00000025465;r=7:140105710-140116476;t=ENSMUST00000026538.

[0079] Transcript targeted by mutation (Ensembl number): Echs1-201 (ENSMUST00000026538.12).

[0080] The site targeted by the mutation: C225S.

[0081] The exon where the mutation site is located: exon 6.

[0082] 2.1 Design of sgRNA and construction of homologous recombination vector 2.1.1. The reference Echs1 gene transcript is: Echs1-201 (ENSMUST00000026538.12), and the editing was performed on exon 6. The DNA sequence of the Echs1 gene is shown below.

[0083] The uppercase bases are the exon6 sequence, and the bases at positions 126, 287, and 572 are the bases that need to be mutated, among which the bases at positions 126 and 572 are the mutated bases made to prevent secondary cutting by gRNA (SEQ ID NO: 2).

[0084] The following is the genome sequence information after mutation.

[0085] , including a target mutated base (position 287) and two base mutations (positions 126 and 572) to prevent secondary cutting by gRNA (SEQ ID NO: 3).

[0086] 2.1.2 gRNA sequence information: Three gRNAs were designed based on the sequence and mutation site information and transcribed in vitro. The sequence information is as follows.

[0087] gRNA1: AGGACTGTGGTCACTGGCAAGGG (SEQ ID NO: 4).

[0088] gRNA2: GAGGACTGTGGTCACTGGCAAGG (SEQ ID NO: 5).

[0089] gRNA3: CAATGCAGTATCAGGCACAGAGG (SEQ ID NO: 6).

[0090] gRNA4: AATGCAGTATCAGGCACAGAGGG (SEQ ID NO:7).

[0091] 2.1.3 Construction of homologous recombination donor plasmid: Shanghai Nanmo Biotechnology was commissioned to construct the homologous recombination plasmid ( Figure 2 ).

[0092] , where the mutated base is at position 270, and the mutated bases to prevent secondary cutting by gRNA are at positions 1 and the last (SEQ ID NO: 8).

[0093] 2.1.4 Homology arm sequence.

[0094]

[0095]

[0096] 2.2 Construction of point mutation mice: Cas9 mRNA, homologous recombination plasmid and gRNA targeting Echs1 gene were mixed, and then an appropriate amount of the mixture was introduced into the fertilized eggs of mice (C57BL / 6J strain) by microinjection, and then transplanted into pseudopregnant female mice (C57BL / 6J strain) by embryo transfer, and the genotype of the offspring was identified to obtain homozygous point mutation mice of F0 generation. In this process, the preparation of superovulated female mice, pseudopregnant female mice, superovulation, fertilization, preparation and collection of fertilized eggs, and microinjection were completed by Shanghai South Model Organisms Company ( Figure 3 ). The injected fertilized eggs are transplanted into pseudo-pregnant mother mice. The mice born around 20 days later are F0 generation mice. Since the cleavage rate of the fertilized eggs is very fast in the early stage, the F0 generation mice obtained are chimeras and may not have the ability of stable inheritance. They need to be subcultured to obtain F1 generation mice with stable inheritance.

[0097] 2.3 Identification of point mutation mice: 2.3.1. Mouse paw DNA extraction: Cut the toes of one-week-old mice to collect DNA. The position of the toes is used as the mouse number. The cut toes are placed in a numbered 1.5ml centrifuge tube, and 29µL of mouse paw digestion solution and 1µL of proteinase K are added. Digest at 55℃ overnight. The digested mouse paw is placed in a boiling water bath for 10 minutes, centrifuged at 12000rpm for 10 minutes, and the supernatant is the mouse paw identification sample.

[0098] 2.3.2 PCR identification of genotype: Mouse paw identification samples were taken and PCR was performed with identification primers to identify the mouse genotype.

[0099] m-Echs1-Forward: 5'-CAAGGATTCAGTAGTTATTCAGTC-3' (SEQ ID NO: 11).

[0100] m-Echs1-Reverse: 5'-AGTTGGCCTTCCTTTTCTCTA-3' (SEQ ID NO: 12).

[0101] 2.3.3 The reaction system is shown in Table 1.

[0102] Table 1

[0103] 2.3.4 The reaction procedure is shown in Table 2.

[0104] Table 2

[0105] 2.3.5 Identification: Gel electrophoresis was performed and the sample group with a 7.8 kb band was selected and sent to Tianyi Huiyuan Company for sequencing. The sequencing results were compared to determine the F0 generation positive chimeric mice carrying the point mutation gene ( Figure 5 ).

[0106] 2.4 Identification of mouse metabolic phenotype: Determination of animal metabolic levels indicates Echs1 C225S The mice had impaired metabolic behavior.

[0107] 2.4.1. Mitochondrial function of mouse adult fibroblasts 2.4.1.1 Cell separation: Take a one-month-old male mouse, inject 2mL of anesthetic to anesthetize the mouse, apply depilatory cream around the ear, remove the ear hair, and then cut the outer ear (without cartilage) into an EP tube. In the clean bench, transfer the ear in the EP tube to a 15mL centrifuge tube, add 75% ethanol to wash for 5min, transfer the ear to a new 15mL centrifuge tube, add PBS solution containing three antibodies, and wash the tissue twice. Transfer the ear to an EP tube, add 50-100μL HBSS collagenase solution, and cut the tissue into small pieces with autoclaved scissors until all the pieces are uniform in size. Make up the volume to 1mL and incubate in a 37℃ incubator for 30min. Wrap it in a 50mL centrifuge tube with PE gloves and centrifuge at 1000rpm for 5min, discard the supernatant (do not discard all the supernatant), and add 1mL HBSS containing three antibodies to wash once (flick the cells to wash). Add 500 μL of 0.05% trypsin to the precipitate for 20 min, centrifuge at 1000 rpm for 5 min, remove the supernatant, add 500 μL of complete nutrient medium, blow the cells evenly with a 200 μL gun, plate them into a 6-well plate, add 2 mL of complete nutrient medium, and culture them in a 37°C, 5% CO2 incubator. After 1-2 days of cell attachment, replace the complete nutrient medium and perform normal cell culture.

[0108] 2.4.1.2 Determination of cell mitochondrial pressure and glycolysis pressure: Cell plating: Digest the cells with trypsin, centrifuge, resuspend the cells with 10% DMEM medium, and then count the cells. Ensure that the total number of cells in each well of the cells loaded on the machine at the same time is consistent, dilute according to the total number of cells, count the total number of cells again, and the number of cells in each well should be 1×10 4 Up to 8×10 4 Add 100 μL of diluted cell suspension (2×10 4 pcs / hole = 2×10 5After the cells are plated, place them at room temperature for 1 hour. After 1 hour, observe whether the cells have successfully adhered to the wall. After adhesion, add 150 μL of culture medium. If not, place them until they adhere, and then add culture medium. The adhered cells are transferred to the incubator for overnight culture. The cells are treated and the probe plate is assembled according to the instructions provided by the manufacturer. The cell plate is assembled according to the instructions and the cell metabolism indicators are measured.

[0109] 2.4.1.3 Mitochondrial stress test: After measuring the basal oxygen consumption (OCR) of the cells three times, oligomycin A was added to the cells to inhibit the function of mitochondrial complex V, and the oxygen consumption was measured three times; carbon cyano-p-trifluoromethoxyphenylhydrazone (FCCP) was added to permeabilize the mitochondrial inner membrane, and the oxygen consumption was measured three times; rotenone / antimycin A (Rot / AA) was added to the cells to inhibit the activity of mitochondrial complex I and complex III, and the oxygen consumption was measured three times.

[0110] 2.4.1.4 Glycolysis stress test: Culture the cells in a sugar-free medium, measure the cell pH three times, add glucose to make the cells enter glycolysis, and measure the cell pH three times; add oligomycin A to inhibit ATP synthase to maximize the glycolysis level of the cells, and measure the cell pH three times; finally, add 2-deoxy-D-glucose (2-DG) to inhibit hexokinase by a competitive mechanism, completely inhibit glycolysis, and measure the cell pH three times.

[0111] 2.4.1.5 Results: Echs1 C225S There are obvious differences between the mitochondrial respiratory function of adult mouse fibroblasts and wild-type cells, especially in Echs1 C225S The maximum respiratory capacity retained by the mitochondria of the cells is significantly different from that of wild-type cells ( Figure 6 A, B), may be due to impaired mitochondrial function. Mitochondrial function can be maintained under normal activities, but the respiratory capacity retained is less, which may be one of the reasons for the patient's muscle development disorder. C225S The glycolytic capacity of cells was improved, and Echs1 C225S The glycolytic retention capacity of the cells is low ( Figure 6 CE), indicating that the glycolysis level of cells is higher than that of wild type cells under normal conditions, which may be due to Echs1 C225S This results in a decrease in mitochondrial respiratory function, and the cell energy supply is more dependent on glycolysis than the wild type. This result proves that Echs1 C225S The mice have impaired mitochondrial respiratory function and altered cellular metabolism, which can be used to explore the relationship between Leigh-like syndrome and cellular metabolism.

[0112] 2.4.2 Mitochondrial enzyme activity in mouse liver.

[0113] Liver collection: After anesthetizing the mice, PBS buffer was perfused into the heart, and the largest lobe of the liver was taken out and placed in an EP tube and frozen in liquid nitrogen.

[0114] Enzyme activity assay: 20 mg of liver was cut from each mouse for enzyme activity assay, and the activities of various mitochondrial enzymes were assayed according to the manufacturer's instructions.

[0115] Result: Echs1 C225S There are differences between the mitochondrial enzyme activities of mice and wild-type mice. The enzyme activities of mitochondrial complexes I and II (succinate dehydrogenase, SDH) are significantly decreased compared with wild-type mice ( Figure 7 A, B, while the enzyme activities of complexes IV and V increased significantly ( Figure 7 C, D), indicating Echs1 C225S The OXPHOS deficiency in mice may be a complex defect, that is, a common defect in respiratory chain complexes I and II. C225S The content of succinate, a substrate of complex II, in mice was significantly decreased ( Figure 7 E), indicating that the mitochondrial respiratory chain substrate supply is insufficient. This result proves that Echs1 C225S Mice can be used as a mouse model of Leyte disease to study the relationship between ECHS1D and respiratory chain defects and for mechanistic studies of Leyte disease.

[0116] 2.4.3 Metabolite levels in mouse liver.

[0117] Liver collection: After anesthetizing the mice, PBS buffer was perfused into the heart, and the largest lobe of the liver was taken out and placed in an EP tube and frozen in liquid nitrogen.

[0118] Metabolic mass spectrometry: 100 mg of liver was cut from each mouse for metabolic mass spectrometry, and Biomax was commissioned to perform non-targeted metabolomics.

[0119] Result: Echs1 C225S Changes in the contents of multiple intermediates in the palmitic acid metabolic pathway in which ECHS1 is involved in mouse liver ( Figure 8 ), indicating that ECHS1 enzyme activity was impaired. At the same time, the levels of citric acid, an intermediate product in the tricarboxylic acid cycle, citramalic acid, an intermediate product in glucose metabolism, and the important cofactor flavin mononucleotide were all changed compared to wild-type mice ( Fig. 9 ), indicating Echs1 C225S Affects multiple metabolic pathways in mice.

[0120] 2.4.4 The mice were starved and then fed with blood glucose.

[0121] Starvation treatment: The mice were starved 17 h in advance, at ZT21 and ZT3. Blood was collected from the tail of the mice and the starvation blood glucose level was measured using a Yuyue 590 blood glucose meter.

[0122] Refeeding: The mice that had been starved for 17 hours were fed with mouse chow. After 2 hours of free diet, blood was taken from the tail of the mice and the refeeding blood glucose level was measured using a Yuyue 590 blood glucose meter.

[0123] Result: Echs1 C225S After mice were starved for 17 hours, the starvation blood glucose level at ZT8 was different from that of wild-type mice. Moreover, after feeding for another two hours, Echs1 C225S The differential loss of the height of blood glucose rise between ZT8 and ZT20 in mice indicates that Echs1 C225S The diurnal difference in insulin secretion in mice was impaired to some extent ( Fig.10 A).

[0124] 2.4.5 Mouse glucose tolerance test.

[0125] Starvation treatment: The mice were starved 24 h in advance, at the time point ZT10.

[0126] Glucose preparation: physiological saline injection containing 10% glucose, injected at 20 μL / g per mouse.

[0127] Blood glucose measurement: weigh the mice, and inhale glucose with a syringe according to the proportion. Take blood from the tail of the mice and use a Yuyue 590 blood glucose meter to measure the basal blood glucose value. Intraperitoneally inject glucose, take blood from the tail of the mice and use blood glucose test strips to measure the blood glucose value 15min, 30min, 45min, 60min, and 120min after injection.

[0128] Result: Echs1 C225S After glucose injection, the blood glucose of mice decreased at the same rate during the day and at night, and the rate of blood glucose decrease during the day was significantly slower than that of wild-type mice, indicating that Echs1 C225S Glucose transport and energy storage are decreased in mice. Echs1 at night C225S There was no difference in the blood glucose tolerance level between mice and daytime, but there was a significant difference in the wild-type group, indicating that Echs1 C225S The loss of the difference in glucose metabolism between day and night in mice may be due to impaired cyclical insulin secretion or cyclical loss of insulin sensitivity ( Fig.10 BD).

Claims

1. A method for constructing an ECHS1D disease animal model, wherein the animal model expresses a homozygous point mutation protein of the Echs1 gene, and the point mutation site is p.C225S.

2. The construction method according to claim 1, wherein the point mutation site is c.673T>C.

3. The construction method according to claim 1, comprising the step of using CRISPR / Cas9 technology and / or TALEN technology to point mutate the Echs1 gene in the animal model.

4. The construction method according to claim 1, comprising the following steps: The recombinant vector is injected into the fertilized egg through gene editing technology and transplanted into the pseudo-pregnant mother to obtain the F0 generation chimeric individual. The F0 generation chimeric individual is hybridized with the wild-type individual to obtain the F1 generation positive heterozygous individual. The F1 generation individual is hybridized with the wild-type individual to obtain the F2 generation heterozygous individual. The F2 generation heterozygous individual is self-pollinated to obtain the F3 generation homozygous individual, which is the ECHS1D disease animal model.

5. The construction method according to claim 1, wherein the amino acid sequence of the homozygous point mutation of the Echs1 gene is shown in SEQ ID NO:

1.

6. Use of the animal model produced according to any one of claims 1 to 5 in simulating ECHS1D disease in animals.

7. Use of the animal model produced according to any one of claims 1 to 5 in the study of the pathology of Leigh-like syndrome.

8. Use of the animal model produced by any one of claims 1 to 5 in screening pharmaceutical compositions for treating ECHS1D disease and Leigh-like syndrome.

9. A method for screening one or more candidate drugs using the animal model / animal model cells produced according to any one of claims 1 to 5, the method comprising the step of administering the one or more candidate drugs to the animal model / animal model cells.

10. A method for identifying a therapeutic agent for treating ECHS1D disease or Leigh-like syndrome for non-therapeutic purposes, the method comprising: Using the agent in the animal model produced by any one of claims 1 to 5 to detect whether the animal model has changes in abnormal symptoms related to ECHS1D disease and Leigh-like syndrome; when the agent has a therapeutic effect on ECHS1D disease and Leigh-like syndrome, the agent is identified as a therapeutic agent.

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