WO2023080755A1 - Method for preparing stem cell-derived cardiomyopathy model cell line by using crispr-based base editing technology, and cardiomyopathy cell line prepared by same method - Google Patents

Abstract

The present invention relates to a method for preparing a cardiomyopathy model cell line on the basis of 4th generation base editing technology, and the like. The present invention can provide a cell line of an accurate genetic hypertrophic cardiomyopathy model by accurately inducing mutation of only 1 bp base, sgRNA of the present invention is applicable to various human-derived cells, and a cardiomyopathy model cell line prepared by means of generation in an intron that does not have off-target occurrence or does not affect cell phenotype can be used for research on cardiomyopathy, and a therapeutic agent and patient-specific treatment thereof.

Description

Method for manufacturing stem cell-derived cardiomyopathy model cell line using CRISPR-based base correction technology and cardiomyopathy cell line prepared by the method

The present invention relates to a method for preparing a cardiomyopathy model cell line based on 4th generation base editing technology, and the like.

Heart disease has a very high prevalence worldwide and ranks first among all causes of death from a single disease. Cardiomyopathy is a disease that occurs primarily in the muscle of the heart itself, and it is often impossible to determine the cause of the disease, so it is classified into hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Familial hypertrophic cardiomyopathy is an autosomal genetic disorder caused by mutations in genes that make up sarcomere proteins. Currently, about 50-60% of patients with hypertrophic cardiomyopathy have been identified as having mutations in the sarcomere protein gene.

Recently, genome-wide association studies (GWAS), next-generation sequencing (NGS), and big data studies have been actively conducted, which is expected to have a high correlation with the pathophysiology of cardiovascular disease. Variant unknown significance (VUS) or single nucleotide polymorphism (SNP) data are accumulating.

Hereditary hypertrophic cardiomyopathy (HCM) comprises a group of highly penetrant, monogenic, autosomal dominant cardiomyopathy, characterized by over 1,000 point mutations in any one of the proteins contributing to sarcomere, a functional unit of the myocardium. It can be caused by more than one, and generally about 1 in 500 people have left ventricular hypertrophy due to other causes, such as hypertension and valvular disease, and other hereditary disorders, such as lysosomal storage disorders, metabolic or invasive causes. Hypertrophic cardiomyopathy may occur.

In the case of new drug candidates related to cardiovascular diseases such as heart failure, arrhythmia, or cardiomyopathy in adults, the efficacy and toxicity of the drug must be determined using cells derived from cardiovascular disease patients, but it is difficult to secure various cardiovascular disease patients-derived cells having mutations. In addition, since there are various genetic mutations in cardiovascular diseases seen in humans, it is quite difficult to evaluate new drugs using patient-derived cells for each genetic mutation. Most cardiovascular diseases such as heart failure, arrhythmia, or cardiomyopathy induce disease by translating an incorrect amino acid into a single nucleotide mutation in a disease-inducing gene. Therefore, for cardiovascular disease modeling, a technique that can be precisely changed at the level of a single base is required.

With the development of CRISPR/Cas9 gene editing technology (gene scissors) with a simple structure and high efficiency, a system application method that can be easily applied to cell/animal and plant genome editing was first reported in 2012. First-generation zinc fingers (ZNFs) and second-generation tallens Gene editing technology is developing rapidly, up to the development of CRISPRs, a third-generation gene editing technology that has a simpler structure than TALENs, is easier to manufacture, has a wider target range, and has higher DNA editing efficiency. However, the main problem is that part of the gene is deleted because even CRISPR, a third-generation gene editing technology, cuts and repairs the target DNA. On the other hand, the recently reported fourth-generation base-editors-based gene editing technology can accurately edit only 1 bp of base without DNA cutting.

The present inventors intend to prepare a human pluripotent stem cell cell line of a genetic hypertrophic cardiomyopathy model using CRISPR/Cas9 gene editing technology and provide it as a model for cardiomyopathy research and drug development.

The purpose of the present invention is to improve the limitations of disease modeling due to the diversity of genetic mutations, TTN, the target gene of DCM, a cardiomyopathy, by using 4th generation Base-Editors gene editing technology capable of single base mutations. , Methods for constructing human pluripotent stem cells and fibroblast-derived cardiomyopathy human pluripotent stem cell lines corrected for MYH7, the target gene of LMNA, DES and HCM, and providing guide RNA (gRNA) for the production of the cell lines there is.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to solve the above problems, the present invention provides DCM target genes TTN, LMNA, DES and HCM target gene MYH7 correction guide RNA for preparing a cardiomyopathy model.

The guide RNA is applied to embryonic stem cells, induced pluripotent stem cells, and fibroblasts together with the CRISPR/Cas system to induce mutations in DES, LMNA, MYH7 or TTN genes, and the fibroblasts are dedifferentiated to produce induced pluripotent stem cells , and ultimately, pluripotent stem cells in which mutations in DES, LMNA, MYH7 or TTN genes are induced can differentiate into cardiomyocytes, and the guide RNA is used to prepare pluripotent stem cell-derived cardiomyopathy model cell lines can be provided.

In the present invention, guide RNA for DES gene correction for preparing a cardiomyopathy model may include or consist of the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the guide RNA for LMNA gene correction for preparing a cardiomyopathy model may include or consist of the nucleotide sequence of SEQ ID NO: 2.

In the present invention, guide RNA for MYH7 gene correction for preparing a cardiomyopathy model may include or consist of the nucleotide sequence of SEQ ID NO: 3.

In the present invention, the guide RNA for TTN gene correction for preparing a cardiomyopathy model may include or consist of the nucleotide sequence of SEQ ID NO: 4.

The cardiomyopathy model cell line constructed using guide RNA for DES gene correction, guide RNA for LMNA gene correction, or guide RNA for TTN gene correction may be a dilated cardiomyopathy (DCM) model cell line.

In addition, the cardiomyopathy model cell line prepared using the guide RNA for correcting the MHY7 gene may be a hypertrophic cardiomyopathy (HCM) model cell line. In addition, the present invention relates to DES, LMNA, MYH7 or A vector for TTN gene correction is provided.

The vector may further include a gene encoding the Cas protein, and the guide RNA and/or the gene encoding the Cas protein are preferably operably linked to a promoter.

In addition, the present invention provides a DES, LMNA, MYH7 or TTN gene correction composition comprising the guide RNA or a vector for expressing the guide RNA.

The composition may further include a vector expressing the Cas protein, or the vector for expressing the guide RNA may further include a gene encoding Cas.

In addition, the present invention provides a method for preparing a cardiomyopathy model cell line comprising the step of transforming mammalian cells using the gene correction composition.

The mammalian cells may be pluripotent stem cells or somatic cells capable of dedifferentiation.

The pluripotent stem cells may be embryonic stem cells or induced pluripotent stem cells.

The method for preparing the cardiomyopathy model cell line may further include the step of isolating the cells into single cells after the transformation and culturing the cells to form clusters, and after isolating the cells from each cluster, gene sequences are analyzed. A step of determining whether editing has occurred may be further included.

As a result of determining whether gene editing has occurred, wild-type clusters in which gene editing has not occurred, heterotype clusters in which 50% gene editing has occurred, and homotype clusters in which 100% gene editing has occurred can be identified. The cardiomyopathy model cell line prepared through the method may be used for other purposes depending on the purpose of production. In general, patients with cardiomyopathy have a heterotype mutant gene, so the heterotype cluster can be used for patient-specific drug screening, the homotype cluster can be used for each mutation and mechanism study of cardiomyopathy, and the wild type can be used as a control.

On the other hand, if the transformed cells are embryonic stem cells or induced pluripotent stem cells in the manufacturing method, a step of differentiating the cells into cardiomyocytes may be further included after the step of determining whether gene editing has occurred. If the cells are somatic cells, a step of dedifferentiating into induced pluripotent stem cells through reprogramming and differentiating them into cardiomyocytes may be further included after the step of determining whether gene editing has occurred.

In addition, the present invention provides a cardiomyopathy model cell line produced by the above method.

The present invention can provide a cell line of an accurate genetic hypertrophic cardiomyopathy model by accurately inducing mutation of only 1 bp base using 4th generation gene editing technology. The sgRNA of the present invention can be applied to various human-derived cells, and the cell line of the cardiomyopathy model prepared by generating in an intron that does not generate off-targets or does not affect cell phenotype is suitable for research on cardiomyopathy and its therapeutic agents and patient-specific treatment. can be used

Figure 1 shows the nucleotide sequence of the sgRNA target region for DES, LMNA, MYH7 or TTN gene editing of the present invention. The target region is marked in red (underlined), and the PAM binding site is marked in blue.

FIG. 2 shows the result of Sanger sequencing of the cloning of each sgRNA encoding gene in the sgRNA expression vector for DES, LMNA, MYH7 or TTN gene correction of the present invention.

Figure 3 shows the results of performing gene editing in embryonic stem cells using sgRNA for DES gene correction and BE4max and performing targeted deep sequencing.

4 shows the results of gene editing and targeted deep sequencing in induced pluripotent stem cells using DES gene correction sgRNA and BE4max.

5 shows the results of performing gene editing in embryonic stem cells using sgRNA for DES gene correction and BE4max and performing Sanger sequencing.

6 shows the results of performing gene editing in fibroblasts using sgRNA for DES gene correction and BE4max and performing targeted deep sequencing.

7 shows regions other than the target where gene editing by the DES gene editing sgRNA is expected to be possible, and bases that are not complementary to the sgRNA are indicated in red (lowercase letters).

8 is a result of performing gene editing in human-derived embryonic stem cells using sgRNA for DES gene correction and BE4max, and confirming whether off-target cleaved sequences are mutated by a targeted deep sequencing method.

9 shows the results of gene editing and targeted deep sequencing in human-derived embryonic stem cells using LMNA gene correction sgRNA and BE4max.

10 shows the results of gene editing and targeted deep sequencing in human-derived embryonic stem cells using LMNA gene correction sgRNA and NG_BE4max.

11 shows the results of gene editing and targeted deep sequencing in human-derived induced pluripotent stem cells and human-derived fibroblasts using LMNA gene-editing sgRNA and BE4max.

12 shows regions other than the target where gene editing by the LMNA gene editing sgRNA is expected to be possible.

13 is a result of performing gene editing in human-derived embryonic stem cells using LMNA gene editing sgRNA and BE4max, and confirming whether off-target cleavage sequences are mutated by a targeted deep sequencing method.

Figure 14 induces the differentiation of cells of Wild type, Hetero type, and Homo type colonies of human-derived embryonic stem cells subjected to gene editing using LMNA gene correction sgRNA and BE4max into cardiomyocytes, and cardiomyocyte markers (cTnT, cTnI, MLC2v, MLC2a, TBX18) and mature differentiation markers (Bin-1, JPH2, HCN4) were confirmed by Western blotting and immunofluorescence.

15 shows the expression of LaminA/C, H3k9me2, and Emerin in Wild type, Hetero type, and Homo type cells of human-derived embryonic stem cells subjected to gene editing using LMNA gene correction sgRNA and BE4max as organoids. This is the result confirmed by Western blot and immunofluorescence.

16 shows the results of gene editing and targeted deep sequencing in human-derived embryonic stem cells using sgRNA for MYH7 gene correction and BE4max.

17 shows the results of performing gene editing in human-derived embryonic stem cells using MYH7 gene editing sgRNA and BE4max and performing Sanger sequencing.

18 shows the results of performing gene editing and targeted deep sequencing in human-derived induced pluripotent stem cells using sgRNA for MYH7 gene correction and BE4max.

19 shows regions other than the target where gene editing by the MYH7 gene editing sgRNA is expected to be possible, and bases that are not complementary to the sgRNA are indicated in red (lowercase letters).

20 is a result of performing gene editing in human-derived embryonic stem cells using MYH7 gene editing sgRNA and BE4max, and confirming whether off-target cleavage sequences are mutated by a targeted deep sequencing method.

21 shows the results of performing gene editing and targeted deep sequencing in human-derived embryonic stem cells using sgRNA for TTN gene editing, and confirming the deletion efficiency in TTN exon 336 of 5 cell lines obtained.

22 is a brief summary of cell lines secured as a result of performing gene editing using the sgRNA of the present invention.

In the case of human cardiovascular disease, it is difficult to evaluate new drugs using patient-derived cells for each gene mutation because genetic mutations are diverse and it is difficult to secure the entire nucleotide sequence. Since the 4th generation base editor can accurately induce mutations in 1bp bases without cutting DNA, the present inventors use human pluripotent stem cells based on the 4th generation base editing technology to specifically target only the target sequence in a target gene. We tried to create a cardiomyopathy cell model that reflects genetic cardiomyopathy by selectively selecting it.

Cardiomyopathy is a disease that occurs in the muscle itself of the heart and causes damage to the structure and function of the muscle wall. Cardiomyopathy is divided into dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and restrictive cardiomyopathy. known to occur. Currently, about 50-60% of patients with hypertrophic cardiomyopathy have been found to be caused by mutations in the sarcomere protein gene, and in the case of patients with dilated cardiomyopathy, dominant mutations in the TTN (titin) gene have been known to be the cause.

The present inventors used the previously reported TTN gene mutation sequence of dilated cardiomyopathy as a positive control group and, in addition to the TTN gene mutation, conducted a GWAS study on three previously unreported genes (DES, LMNA, and MYH7 genes) to determine their relevance to cardiomyopathy. These known SNP sequences were selected as targets for cell line construction of the cardiomyopathy model.

Specifically, the TTN mutation designed sgRNA to delete the base at position exon336 and used the CRISPR/Cas9 system, and the LMNA mutation designed sgRNA to induce His222Try mutation with C to T base substitution and used a base editor. DES For the mutation, a base editor was used by designing sgRNA to induce Arg454Trp mutation with C to T base substitution, and for the MYH7 mutation, sgRNA was designed to induce Arg249Gln mutation with C to T base substitution and a base editor was used. The specific sequence of sgRNA for editing each gene is shown in Figure 1 below (see Example 1).

Subsequently, the present inventors constructed DES, LMNA, or MYH7 mutant cell lines using a cytocine base editor (CBE). In order to confirm the applicability of the sgRNA designed in various cell lines, human-derived embryonic stem cells (ESC), human-derived induced pluripotent stem cells (iPSC), and human-derived fibroblasts were transformed with sgRNA expression vectors, respectively, and gene editing was performed. The results were confirmed. Human-derived embryonic stem cells and human-derived induced pluripotent stem cells were separated into single cells after transformation, cultured into colonies, and cells were collected from each colony, and nucleotide substitution from C to T was confirmed by targeted deep sequencing analysis. After transformation, human-derived fibroblasts were separated into single cells, each was cultured, and base substitution from C to T was confirmed by targeted deep sequencing (see Example 1).

As a result of transformation using sgRNA for DES gene mutation induction, wild type without gene editing, heterotype with 50% substitution, and homotype with 100% substitution were identified in human embryonic stem cells. Human-derived fibroblasts were also identified as wild type without gene editing and homotyped cells with 100% substitution. sgRNA for DES gene editing did not induce gene editing in human induced pluripotent stem cells. On the other hand, in order to confirm whether the designed sgRNA induces the operation of CBE off-target, that is, to verify the safety of the DES mutagenized cell line in the manufactured cell line, off-target cleavage (off-target) by the sgRNA for editing the DES gene As a result of checking, it was confirmed that 1 region with the possibility of off-target cleavage was identified, but it was found that the region was located in an intron and did not affect the cell phenotype by off-target cleavage (see Example 2).

As a result of transformation using sgRNA for inducing LMNA gene mutation, wild-type colonies without gene editing, heterotype with 50% substitution, and homotype with 100% substitution were confirmed in human-derived embryonic stem cells. In addition, in human-derived fibroblasts, wild type without gene editing and homotype cells with 100% substitution were identified. sgRNA for LMNA gene editing did not induce gene editing in human induced pluripotent stem cells. In addition, as a result of confirming the presence or absence of off-target cleavage for verification of the produced mutant cell line, it was confirmed that base substitution did not occur in all of the target sequences with promising editing potential (see Example 3).

As a result of transformation using sgRNA for inducing MYH7 gene mutation, wild-type colonies without gene editing and homotype colonies with 100% substitution were confirmed in human-derived induced pluripotent stem cells. Furthermore, as a result of verification of the prepared mutant cell line, off-target cleavage by the sgRNA for editing MYH7 did not occur (see Example 4).

In addition, it was confirmed that the sgRNA for inducing mutation of the TTN gene induces deletion of the TTN gene in embryonic stem cells, and it was possible to secure 5 cell lines usable as positive controls for cardiomyopathy (see Example 5).

On the other hand, in order to confirm the applicability of the mutant cell line prepared through base substitution as a cardiomyopathy model, differentiation of the LMNA mutant cell line among DES, LMNA, or MYH7 mutant cell lines into cardiomyocytes was induced and organoids were prepared. In the case of transformed fibroblasts, dedifferentiated stem cells were prepared through reprogramming and then differentiated into cardiomyocytes. As a result, the expression of cTnT, cTnI, MLC2v, MLC2a, and TBX18, which are cardiomyocyte markers, and Bin-1, JPH2, and HCN4, which are mature differentiation markers, were confirmed in each cell. Through this, it was found that it can be used as a cardiomyopathy model cell line (see Examples 3-4 and 3-5).

From the above, the present inventors confirm that a cardiomyopathy cell line can be constructed by inducing a mutation by correcting one nucleotide sequence in human pluripotent stem cells, and provide an sgRNA for preparing a cardiomyopathy model cell line.

In the present invention, "cardiomyopathy model cell line" means a cell line containing a DES, LMNA, MYH7, or TTN gene mutation associated with cardiomyopathy, and the present invention is a stem cell line inducing the gene mutation Since differentiation into cardiomyocytes was confirmed, stem cells prior to cardiomyocyte differentiation may be referred to as cardiomyopathy model cell lines. On the other hand, the gene mutation refers to a mutation that has a difference in phenotype from normal cells.

In the present invention, “gene editing” may be used in the same sense as gene editing or genome editing. Genetic editing refers to a mutation (substitution, insertion or deletion) that causes mutations in one or more bases at a target site in a target gene, but in the present invention, gene editing aims to induce mutations in only one base .

In one embodiment of the present invention, the mutation or gene correction that induces mutation with respect to the one base inactivates the target gene by generating a stop codon at the target site or generating a codon encoding an amino acid different from the wild type ( loss of function, knock-out). or inactivating a gene or correcting a genetic mutation by changing the initiation codon to another amino acid, inactivating a gene by frameshift by insertion or deletion, correcting a gene mutation, or not producing a protein It may be in various forms, such as introducing a mutation into a non-coding DNA sequence that does not cause a disease, or changing a DNA sequence different from the wild type that causes a disease to the same sequence as the wild type, but is not limited thereto.

In the present invention, “base sequence” refers to a nucleotide sequence including a corresponding base, and may be used in the same sense as a nucleotide sequence, a nucleic acid sequence, or a DNA sequence.

In the present invention, “target gene” refers to a gene to be subjected to gene correction, and the present invention provides DES, LMNA, MYH7, and TTN as target genes for preparing a cardiomyopathy model cell line.

In the present invention, "target site (target site)" refers to a site where gene editing or correction by a target-specific nuclease within a target gene occurs. When the target gene is TTN, the target site is located in the TTN exon. It is the 336th base, and if the target gene is DES, it is a site that causes mutation of Arg454 in the protein expressed by DES, and if the target gene is LMNA, it is a site that causes mutation of His222 in the protein encoded by LMNA, and the target When the gene is MYH7, it is a site where mutation occurs in Arg249 in the protein encoded by MYH7.

In the present invention, "guide RNA" refers to an RNA comprising a targeting sequence hybridizable to a specific nucleotide sequence (target sequence) within a target site in a target gene, in vitro or in vivo (or cell). ), binds to a nuclease protein such as Cas and guides it to a target gene (or target site), and is used interchangeably with the term sgRNA (single guide RNA) in the present specification.

The present invention provides a composition for gene correction for preparing a cardiomyopathy model cell line by inducing TTN, LMNA, DES, or MYH7 gene mutations, and the composition includes the guide RNA described above. The guide RNA may be used in the form of RNA (or included in the composition) or in the form of a vector containing DNA encoding the same (or included in the composition).

The vector may encode Cas 9, and Cas 9 may be provided as a vector separate from a vector for expressing guide RNA.

In the present invention, "Cas9 (CRISPR associated protein 9)" is a protein that plays an important role in the immunological defense of certain bacteria against DNA viruses and is widely used in genetic engineering applications. Since the main function of the protein is to cleave DNA, It can be applied to modify the genome of a cell. Specifically, CRISPR/Cas9 recognizes, cuts, and edits a specific nucleotide sequence to be used as a third-generation gene scissors, and inserts a specific gene into the target site of the genome or stops the activity of a specific gene simply, quickly, and efficiently. useful for carrying out Cas9 protein or gene information may be obtained from a known database such as GenBank of National Center for Biotechnology Information (NCBI), but is not limited thereto. In addition, a person skilled in the art can appropriately connect additional domains to the Cas9 protein depending on its purpose. In the present invention, the Cas9 protein may include not only wild-type Cas9, but also variants of Cas9 as long as they have a function of a nuclease for gene editing. In addition, in the present invention, Cas9 may be mutated to lose endonuclease activity for cleaving DNA double strands.

The origin of the Cas9 protein or variant thereof is not limited, and as non-limiting examples, Streptococcus pyogenes, Francisella novicida, Streptococcus thermophilus, Legionella pneumophila It may be from Legionella pneumophila, Listeria innocua, or Streptococcus mutans.

The Cas9 protein or variant thereof may be isolated from a microorganism or produced artificially or non-naturally, such as by a recombinant or synthetic method. The Cas9 may be used in the form of pre-transcribed mRNA or pre-produced protein in vitro, or in the form included in a recombinant vector or virus such as AAV or lenti for expression in a target cell or in vivo. In one embodiment, the Cas9 may be a recombinant protein made by recombinant DNA (rDNA). Recombinant DNA refers to DNA molecules artificially created by genetic recombination methods such as molecular cloning to contain heterologous or homologous genetic material obtained from various organisms.

In the present invention, the “base editor (BE)” is not limited as long as it can induce substitution or deletion of the base of the target site described above, and is not limited to cytosine base editor (CBE) and adenine base editor (adenine base editor). editor, ABE), or a guanine base editor (GBE).

In the present invention, "pluripotent stem cell (PSC)" means a stem cell capable of induced differentiation into any type of cell constituting the human body, and includes embryonic stem cells (ESCs) and It is used to mean including induced pluripotent stem cells (iPSCs, dedifferentiated stem cells). Specifically, embryonic stem cells are derived from the inner cell mass of a blastocyst in the pre-implantation stage. Induced. Induced cells are maintained in a specific environment and are capable of unlimited culture and pluripotent differentiation. On the other hand, induced pluripotent stem cells may refer to pluripotent differentiated cells made by de-differentiation from body somatic cells, and cells Pluripotent stem cells are formed by making somatic cells into a state very similar to embryonic stem cells through a process called reprogramming, such as fusion, nuclear transfer, and overexpression of pluripotency regulators. It is not limited to cells, and may include both cells having differentiation pluripotency and self-renewal ability.However, pluripotent stem cells are preferably mammalian cells, more preferably human-derived pluripotent stem cells. there is.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes can be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents or substitutes to the embodiments are included within the scope of rights.

Terms used in the examples are used only for descriptive purposes and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

In addition, in the description with reference to the accompanying drawings, the same reference numerals are given to the same components regardless of reference numerals, and overlapping descriptions thereof will be omitted. In describing the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description will be omitted.

[Example]

Example 1. Mutagenesis using sgRNA design and base editor

1-1. sgRNA design

Cardiomyopathy target genes were designed to construct a cardiomyopathy human pluripotent stem cell line using 4th-generation base-correction factor-based gene editing technology. NCBI ClineVar database was used to find DCM and HCM-induced genetic mutations, which are cardiomyopathy.

The DCM target gene (i) TTN was designed using Cas9, a Cas9 gene editing technique, to induce gene deletion mutations in exon336.

DCM target gene (ii) LMNA gene was designed to induce His222Try mutation through cytosine nucleotide correction technique with C to T base substitution.

DCM target gene (iii) DES gene was designed to induce Arg454Trp mutation through cytosine base correction technique by replacing C to T base.

(iv) MYH7 gene, an HCM target gene, was designed to induce Arg249Gln mutation through cytosine nucleotide correction technique by substituting C to T nucleotides.

All targets were designed so that the C to T base conversion site was located between the 4th and 8th positions in the 5' direction among the 20 target bases, which are the most active editing windows (FIG. 1).

The sgRNA sequence targeting the target gene region is as follows.

DES-rs267607490-sgRNA Gx20 (+): 5'-ACACGGGAUGGGGAGGUAAG-3' (SEQ ID NO: 1)

LMNA-rs28928901-sgRNA Gx20 (+):5'-CCGUCAUGAGACCCGACUGG-3 (SEQ ID NO: 2)

MYH7-rs3218713-sgRNA Gx20 (-): 5'-GAAUUCGAAUGAAUUUCCCC-3' (SEQ ID NO: 3)

TTN sgRNA-2, gx20 (+): 5'-UGAAACUACAGAGCCAGUGA-3' (SEQ ID NO: 4)

1-2. gene editing

Cytosine nucleotide correction (CBE) was used for C to T base conversion, and Cas9 expression vector was purchased from Addgene. After that, it was cloned into a Cas9 expression vector so that the green fluorescent protein was co-expressed. To construct a vector for expressing target guide RNA, a backbone vector having a U6 promoter was purchased from Addgene. In order to design guide RNAs in target genes, target guide RNAs were designed using the RGEN tools program. A target guide RNA expression sequence was cloned into the vector so that it could be expressed under the U6 promoter. Cloning of the guide RNA expression sequence in the vector was confirmed by performing Sanger sequencing (FIG. 2).

In order to confirm the efficiency of gene editing using the designed sgRNA, three cell lines were used: human embryonic stem cells, human induced pluripotent stem cells, and human fibroblasts. In addition, the following two methods were used to construct myocardial hypertrophy cell lines of induced pluripotent stem cells. (1) Cell lines were constructed using base correction factors in induced pluripotent stem cell lines, and (2) cell lines were constructed through cell conversion to induced pluripotent stem cells after substitution with base correction factors in fibroblast cell lines.

Example 2. DES mutant cell line construction

The DES gene is involved in the Desmosome complex among the target genes of hereditary myocardial hypertrophy. gRNA designed for DES gene editing in Example 1 was applied to human-derived embryonic stem cells, human-derived pluripotent stem cells, and human-derived fibroblasts to obtain a DES knock-out cardiomyopathy cell line in each cell It was confirmed that production was possible.

2-1. Establishment of DES gene mutant cell lines from human-derived embryonic stem cells and induced pluripotent stem cells

DES gene mutations were transfected with BE4max and sgRNA plasmids in human embryonic stem cells and induced pluripotent stem cells using Lipofectamine.

In the case of embryonic stem cells, it was confirmed that green fluorescence was expressed through a fluorescence microscope after transfection, and separated into single cells using Accutase.

In the case of human-derived pluripotent stem cells, it was confirmed that green fluorescence was expressed through a fluorescence microscope after transfection, and only cells expressing green fluorescence were isolated using flow cytometry.

After isolation, the replacement efficiency of colonies grown from single cells was confirmed through targeted deep sequencing. As a result, Wild type with no substitution, Hetero type with 50% substitution, and Homo type with 100% substitution were confirmed, and each cell line was selected.

2-2. Confirmation of DES gene mutation induction in human-derived embryonic stem cells

Embryonic stem cell lines were transfected with BE4max, a base editing method, and sgRNA using Lipofectamine, and the efficiency of base substitution from C to T was confirmed by targeted deep sequencing of colonies grown from single cells.

As a result of confirming the replacement efficiency in embryonic stem cell lines, colonies of the Wild type with no replacement, the Hetero type with 50% replacement, and the Homo type with 100% replacement were identified. In addition, it was possible to additionally screen mutation colony of mosaic pattern with 60% mutation rate. Genotypes were confirmed again through Sanger sequencing of the WT, hetero, and homozygote cell lines constructed in this experiment (FIG. 5).

2-3. Confirmation of DES gene mutation induction in human-derived induced pluripotent stem cells and fibroblasts

In induced pluripotent stem cells and fibroblasts, BE4max and sgRNA, which are base correction methods, are base-corrected using lipofectamine 3000 and lipofectamine stem, and then base substitution from C to T is performed on colonies grown from single cells by targeted deep sequencing. Efficiency was confirmed.

In human-derived fibroblasts, Wild type and Homo type with 100% substitution efficiency were obtained (Fig. 6). In addition, it was confirmed through Sanger sequencing that off-target cleavage did not appear in the acquired Homo type.

2-4. Verification of DES mutant cell lines: confirmation of off-target effects

To verify the safety of the DES target, we wanted to confirm the presence or absence of off-target cleavage at the cellular level. Targets that were judged to have off-target cleavage potential for the DES target were allowed up to 3 base mismatches through the RGEN tools program to select possible sequences. In addition, an NGS library was constructed using a library primer designed for the DES target through a cell line into which the desired genetic mutation was introduced, and the selected off-target cleavage sequences were analyzed by targeted deep sequencing analysis.

Sequences with potential off-target cleavage for the DES target were selected through the RGEN tools program by allowing up to 3 base mismatches, and 22 possible sequences were identified (FIG. 6). Mutations in off-target cleavage sequences were confirmed by targeted deep sequencing. Through the analysis results, it was confirmed that in the case of off-target cleavage target 18 (OT-18), editing was induced with high efficiency in the cell line into which the DES mutation was introduced. However, since OT-18 is a mutation present in the intron of the CLMN gene, it is judged that it will not affect the cell phenotype (FIG. 7 and FIG. 8).

Example 3. LMNA mutant cell line construction

The LMNA gene is involved in mechano signaling.

The gRNA designed for LMNA gene editing in Example 1 was applied to embryonic stem cells, induced pluripotent stem cells, and fibroblasts to confirm whether LMNA knock-out cardiomyopathy cell lines could be prepared in each cell.

3-1. Establishment of LMNA gene mutant cell line in embryonic stem cells

Introduction of LMNA gene mutation in embryonic stem cells induced C to T substitution using NG_BE4max as a target for the LMNA gene involved in mechano-signaling. NG_BE4max is a base correction method capable of substituting T from C, but if a target sequence is recognized as an NGG sequence among existing target sequences, NG_BE4max recognizes the NG sequence and edits the target sequence. Therefore, it has the advantage of being able to target a wider range of bases C. Human embryonic stem cell lines were transfected with NG_BE4max and sgRNA using Lipofectamine.

After transfection, it was confirmed that green fluorescence was expressed through a fluorescence microscope, and separated into single cells using accutease.

After isolation, the replacement efficiency of colonies grown from single cells was confirmed through targeted deep sequencing.

As a result of confirmation through the targeted deep sequencing method, 3 cell lines were identified as normal WT type without substitution, and 4 cell lines were identified as heterotype with 50% substitution. Although substitution did not occur at 100%, 10 cell lines of the Homo type were identified. In addition, each genotype was confirmed again by Sanger sequencing (FIG. 10).

3-2. Construction of LMNA gene mutant cell line in human-derived fibroblasts

Human-derived pluripotent stem cells and human-derived fibroblasts were transfected with NG_BE4max and sgRNA using Lipofectamine.

After transfection, it was confirmed that green fluorescence was expressed through a fluorescence microscope, and only cells expressing green fluorescence were isolated using flow cytometry.

After isolation, the replacement efficiency of the colony proliferated from a single cell was confirmed through targeted deep sequencing. As a result, 3 cell lines of the Homo type in which 100% substitution occurred in fibroblasts were obtained. (FIG. 11).

3-3. Verification of LMNA mutant cell lines: confirmation of off-target effects

To verify the safety of the LMNA target, we wanted to confirm the presence or absence of off-target cleavage, which is off-target cleavage at the cellular level. Targets judged to have off-target cleavage potential for the LMNA target were allowed up to 3 base mismatches through the RGEN tools program to select possible sequences. In addition, an NGS library was constructed using a library primer designed for the LMNA target through a cell line into which the desired genetic mutation was introduced, and the selected off-target cleavage sequences were analyzed by targeted deep sequencing analysis.

Sequences with potential off-target cleavage for the LMNA target were selected by allowing up to 3 base mismatches through the RGEN tools program, and 5 possible sequences were identified. It was confirmed by a targeted deep sequencing method that mutations were not induced in all five off-target cleavage target sequences (FIG. 12 and FIG. 13).

3-4. Characterization of human pluripotent stem cell line in LMNA gene mutation cardiomyopathy model

After securing target gene mutant cell lines, differentiation into cardiomyocytes was induced to identify the characteristics of each cell line, and the degree of cardiomyocyte maturation and differentiation was compared and evaluated using qualitative and quantitative differences in cardiomyocyte expression genes for characteristic evaluation. .

As a result of evaluating the degree of cardiomyocyte differentiation using the His222Try mutation-induced LMNA gene cell line, it was confirmed that differentiation into cardiomyocytes was induced in all three cell lines, wild type, hetero type, and homo type. As a result of confirming the expression of cardiomyocyte markers (cTnT, cTnI, MLC2v, MLC2a, TBX18) and mature differentiation markers (Bin-1, JPH2, HCN4) through polymerase chain reaction and Western blot after 15 days of induction of differentiation, all cardiomyocytes It was confirmed that the expression of cTnT, a marker, was increased in Homo type cell lines through polymerase chain reaction. As a result of comparing the expression of the cardiomyocyte marker (cTnT) and the mature cardiomyocyte marker (cTnI) through fluorescence staining, the expression of all cardiomyocyte markers was strong in the Homo type, but in the case of the mature cardiomyocyte marker, the expression of the Wild type and Hetero type was high. It was confirmed that it was strong and it was confirmed that it was consistent with the results of Western blot (FIG. 14).

3-5. LMNA gene mutation = characterization of cardiomyopathy organoids derived from cardiomyopathy cell lines

After securing the target gene mutant cell lines, differentiation into cardiomyocytes was induced in 3D (Organoid) for wild type and Homo type inducing His222Try mutation in order to identify the characteristics of each cell line. Gene expression levels of wild type organoid and homo type organoid were compared and evaluated using LMNA gene-related markers. Samples were taken 20 days after induction of differentiation of each cell line into organoids, and the expression of LADs (Laminassociated Domains) markers (LaminA/C, H3k9me2, Emerin) was measured by western blotting and immunofluorescence staining in each sample. and confirmed (FIG. 15).

Example 4. MYH7 mutant cell line construction

The MYH7 gene is involved in Sarcomere organization among the target genes of genetic myocardial hypertrophy. MYH7 knock-out cardiomyopathy cell line in each cell by applying the gRNA designed for MYH7 gene editing in Example 1 to human-derived embryonic stem cells, human-derived pluripotent stem cells, and human-derived fibroblasts It was confirmed that production was possible.

4-1. Establishment of MYH7 gene mutant cell line in human-derived embryonic stem cells

4-1-1. Embryonic stem cell lines were transfected using BE4max and sgRNA, which are base editing methods, and the efficiency of base substitution from C to T was confirmed by targeted deep sequencing for colonies grown from single cells. As a result, it was confirmed that about 25% of substitution occurred in MYH7.

4-1-2. Substitution from C to T was performed using a vector in which a selection marker was added to BE4max in an embryonic stem cell line. Green fluorescence was used as a selection marker using a vector expressing green fluorescence behind BE4max. Therefore, BE4max and sgRNA expressing green fluorescence were transfected through lipofection.

Afterwards, it was isolated and cultured as single cells, and targeted deep sequencing was performed on the colonies proliferated from single cells. 1 cell line was confirmed as the heterotype in which this occurred, and 4 cell lines of the Homo type in which 100% substitution occurred were confirmed. Each genotype was confirmed again by Sanger sequencing (FIG. 17).

4-2. Establishment of MYH7 gene mutant cell line in human-derived induced pluripotent stem cells

In induced pluripotent stem cells, BE4max and sgRNA targeting MYH7 were substituted from C to T through lipofection. After observing the green fluorescence expressed by BE4max through a fluorescence microscope, it was separated into single cells through flow cytometry. The isolated single cells were cultured to obtain colonies, and targeted deep sequencing was performed on the obtained colonies. As a result, it was possible to acquire 2 cell lines of Homo type with 100% substitution.

4-3. Verification of MYH7 mutant cell line: confirmation of off-target cleavage

To verify the safety of the MYH7 target, we tried to confirm the presence or absence of off-target cleavage at the cellular level. Sequences with potential off-target cleavage for the MYH7 target were selected by allowing up to 3 base mismatches through the RGEN tools program, and 6 possible sequences were identified (FIG. 19 and FIG. 20). As a result of analysis by the targeted deep sequencing method, it was confirmed that mutations were not induced in all 6 off-target sequences.

Example 5. TTN mutant cell line construction

Human-derived embryonic stem cells were induced to mutate the TTN gene by using the sgRNA designed in Example 1 to cause deletion. Deletion was confirmed in the transformed cells, and 5 cell lines usable as positive controls for cardiomyopathy were secured (FIG. 21).

The present inventors screened DCM and HCM-induced gene mutations, which are human cardiomyopathy, using human-derived embryonic stem cell lines, induced pluripotent stem cells, and fibroblasts. In addition, a cell line in a non-differentiated state was secured by analysis of additional mutation-induced cell lines in TTN, DES, LMNA, and MYH7 genes, which are targets of human cardiomyopathy-induced gene mutations (FIG. 22).

As described above, although the embodiments have been described with limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, the described techniques may be performed in an order different from the method described, and/or components of the described system, structure, device, circuit, etc. may be combined or combined in a different form than the method described, or other components may be used. Or even if it is replaced or substituted by equivalents, appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents of the claims are within the scope of the following claims.

Claims (12)

1. As a guide RNA for preparing a cardiomyopathy model cell line derived from human-derived pluripotent stem cells,

   The cardiomyopathy model cell line is one in which TTN, LMNA, DES, or MYH7 genes are knocked out (Knock-out),

   The guide RNA has a length of 20 to 30 bp,

   The guide RNA is a guide RNA comprising one base sequence selected from the group consisting of SEQ ID NOs: 1 to 4.
2. A vector for constructing a cardiomyopathy model cell line comprising a gene encoding the guide RNA of claim 1.
3. According to claim 2,

   The vector further comprises a gene encoding a Cas protein, a vector for constructing a cardiomyopathy model cell line.
4. According to claim 2,

   A vector for constructing a cardiomyopathy model cell line, wherein the gene encoding the guide RNA is operably linked to the U6 promoter.
5. A composition for preparing a cardiomyopathy model cell line comprising the guide RNA of claim 1 or a vector expressing the guide RNA.
6. According to claim 5,

   The composition further comprises a vector expressing the Cas protein, a composition for preparing a cardiomyopathy model cell line.
7. A method for preparing a cardiomyopathy model cell line comprising transforming embryonic stem cells, induced pluripotent stem cells, or fibroblasts using the composition of claim 5.
8. According to claim 7,

   The method further comprises the step of separating the cells into single cells after the transformation step and culturing to form a colony.
9. According to claim 8,

   The method further comprises the step of obtaining cells from each colony after the culturing step, isolating DNA from the obtained cells, and performing sequencing, a cardiomyopathy model cell line production method.
10. According to claim 9,

    When the transformed cells are embryonic stem cells or induced pluripotent stem cells,

    A method for preparing a cardiomyopathy model cell line, further comprising differentiating cells of a population in which correction of TTN, LMNA, DES, or MYH7 genes has been confirmed through sequencing analysis into cardiomyocytes.
11. According to claim 9,

    When the transformed cells are fibroblasts,

    induced pluripotent stem cells were produced by dedifferentiation of cells in the population in which TTN, LMNA, DES or MYH7 gene correction was confirmed through sequencing analysis,

    A method for producing a cardiomyopathy model cell line, further comprising differentiating the induced pluripotent stem cells into cardiomyocytes.
12. A cardiomyopathy model cell line produced by the method of any one of claims 7 to 11.

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