Compounds and methods for allele-specific editing of the ELANE gene

Abstract

The present invention relates to nucleic acid molecules for allele-specific editing of the ELANE gene, vectors containing the nucleic acid molecules, compositions containing the nucleic acid molecules or the vectors, methods for allele-specific editing of the ELANE gene in vitro in biological material containing genetic material encoding the ELANE gene, and methods for preventing, treating and / or testing diseases in organisms.

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid molecule for allele-specific editing of the ELANE gene, a vector containing the nucleic acid molecule, a composition containing the nucleic acid molecule or the vector, a method for allele-specific editing of the ELANE gene in a biomaterial containing genetic material encoding the ELANE gene in vitro, and a method for preventing, treating and / or testing for diseases in organisms.

[0002] This invention relates to the field of molecular medicine, and more specifically to the field of genetic engineering applications, and more preferably to knockdown targeting disease-related genes. [Background technology]

[0003] Genetic disorders are health problems caused by one or more abnormalities in the genome. Genetic disorders can be caused by mutations in a single gene (monogenic), mutations in multiple genes (polygenic), or chromosomal abnormalities.

[0004] Monogenic disorders are caused by a mutation in a single gene. Monogenic disorders are inherited in several ways by subsequent generations. Genomic imprinting and uniparental disomy can influence the inheritance pattern. Monogenic disorders can be classified into recessive or dominant inheritance patterns, and further into autosomal (i.e., non-sex chromosome linked) or X-linked patterns. They are generally referred to simply as autosomal recessive monogenic disorders or autosomal dominant monogenic disorders.

[0005] For a person to develop an autosomal recessive disorder, two copies of the gene (i.e., two alleles) must be mutated. Individuals with autosomal recessive disorders typically have parents who do not have the disorder, but each parent carries one copy of the mutated gene; these parents are called carriers. On the other hand, for a person to develop an autosomal dominant disorder, only one copy of the mutated gene (i.e., only one allele) is required. Individuals with autosomal dominant disorders typically have one parent who has the disorder. In some cases, a child with a sporadic mutation can be born to healthy, non-diseased parents. Also, one parent may be mosaic for a dominant mutation, and their child may be heterozygous for that mutation. Dominant genetic disorders involve a dominant gene in relation to a normal gene (a functional gene or a healthy gene). Therefore, in dominant genetic disorders, only one copy of the abnormal gene (i.e., only one allele) is required for the onset or contribution of the symptoms of a specific genetic disorder. One example of such mutations is gain-of-function mutation, in which the altered gene product acquires a new molecular function or a new gene expression pattern.

[0006] Neutropenia is a disease characterized by an abnormally low concentration of neutrophils in the blood and bone marrow. Neutrophils make up the majority of circulating white blood cells and play a major role in the defense against infection by destroying bacteria, their fragments, and viruses bound to immunoglobulins in the blood. Patients with neutropenia are susceptible to bacterial infections and, if not treated promptly, are at risk of developing a life-threatening condition (neutropenic sepsis). Neutropenia can be acute (transient) or chronic (long-term). The term "neutropenia" is sometimes used interchangeably with "leukopenia" ("decreased white blood cell count").

[0007] Neutropenia can be classified into acquired and congenital types. Congenital neutropenia mainly has two types: severe congenital neutropenia (CN or SCN) and cyclic neutropenia (CyN).

[0008] Cyclic neutropenia is characterized by fluctuations in neutrophil counts ranging from normal to zero. On the other hand, severe congenital neutropenia is characterized by a significant decrease in absolute neutrophil count (ANC) at birth (500 cells / ml), cessation of bone marrow hematopoietic maturation at the promyelocyte / myelocyte stage in the bone marrow, and the early onset of bacterial infections.

[0009] Severe congenital neutropenia can be diagnosed by extremely low absolute neutrophil counts in the blood and cessation of myeloid cell maturation in bone marrow aspiration. Severe congenital neutropenia is usually diagnosed shortly after birth, while cyclic neutropenia generally develops at various ages, with the main clinical symptom being recurrent acute oral disease. Bone marrow examination is often required to rule out malignant hematopoietic transformation, measure cell solidity, assess bone marrow maturation, and detect signs of the precise etiology. Currently, cytogenetic bone marrow examination is extremely important when severe congenital neutropenia (CN / SCN) is suspected. When evaluating severe congenital neutropenia (CN / SCN) and cyclic neutropenia (CyN), anti-neutrophil antibody assays, immunoglobulin assays (Ig GAM), lymphocyte immunophenotyping, pancreatic markers (serum trypsinogen and fecal elastase), and fat-soluble vitamin levels (vitamins A, E, and D) are also important.

[0010] Severe congenital neutropenia (CN / SCN) and cyclic neutropenia (CyN) are generally autosomal dominant disorders, often caused by heterozygous mutations in the so-called "neutrophil elastase gene" (ELANE gene). However, there are also cases of autosomal recessive severe congenital neutropenia, such as those caused by HAX1 mutations. The ELANE gene encodes neutrophil elastase, a serine protease involved in the function of neutrophil extracellular traps (NETs) (fibrous networks that bind to pathogens). Several studies have suggested that the mutant ELANE gene product suppresses the maturation of bone marrow hematopoietic stem cells into neutrophils, leading to neutropenia. These studies have shown that mutations in neutrophil elastase trigger an endoplasmic reticulum stress response (UPR), resulting in neutrophil formation defects in the bone marrow. In addition to severe congenital neutropenia (CN / SCN) and cyclic neutropenia (CyN), other ELANE gene-related disorders such as pulmonary emphysema and emphysematous changes are also known in this field.

[0011] Currently, neutropenia is treated with granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor. G-CSF stimulates neutrophil production and delays apoptosis. Recombinant G-CSF factor preparations such as filgrastim may be effective in patients with various forms of neutropenia, including severe congenital neutropenia and cyclic neutropenia. The dose required to induce neutrophil production varies significantly depending on the individual patient's condition. Currently, the overall survival rate for patients with severe congenital neutropenia is estimated to be over 80%, but 10% of patients still die from severe bacterial infections or sepsis. While G-CSF treatment has been successful in preventing death from sepsis, it has been found that long-term G-CSF treatment in patients with severe congenital neutropenia increases the risk of developing myelodysplastic syndrome (MDS) and leukemia.

[0012] Hematopoietic stem cell transplantation (HSCT) is an alternative curative treatment used for patients who do not respond to G-CSF therapy or who have developed acute myeloid leukemia or myelodysplastic syndrome (MDS). However, patients with congenital neutropenia who undergo HSCT treatment are at high risk of developing infection complications, such as fungal infections, and graft-versus-host disease.

[0013] WO2019 / 217294(A1) discloses a method for knocking out the expression of the mutant ELANE gene using CRISPR / Cas9 technology. However, the method disclosed in the aforementioned document relies on the presence of single nucleotide polymorphisms (SNPs), and SNPs are not found in many mutant ELANE genes. Therefore, it has been found that this method cannot be used for knockdown targeting the mutant ELANE gene. [Overview of the project] [Problems that the invention aims to solve]

[0014] Under these circumstances, the present invention aims to provide a novel targeted method or compound capable of preventing, treating, and / or testing ELANE gene-related diseases. Furthermore, it provides a compound capable of treating mutated ELANE genes in an allele-specific manner. Specifically, it provides a compound capable of treating leukemia-related ELANE gene mutations or G-CSF nonresponsiveness-related ELANE gene mutations, which are frequently observed and constitute the majority of the population, rather than the rarely observed ELANE gene polymorphisms. [Means for solving the problem]

[0015] The aforementioned problem can be solved by providing a nucleic acid molecule for allele-specific editing of the ELANE gene, which contains a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 5. [Effects of the Invention]

[0016] The inventors of the present invention have found that the nucleic acid molecules of the present invention can be used to specifically edit the ELANE gene. For example, a nucleic acid molecule containing the nucleotide sequence shown in SEQ ID NO: 1 or a nucleic acid molecule consisting of the nucleotide sequence shown in SEQ ID NO: 1 is configured to specifically target the c.170C>T mutation in exon 2 of the human ELANE gene, and / or a nucleic acid molecule containing the nucleotide sequence shown in SEQ ID NO: 4 or a nucleic acid molecule consisting of the nucleotide sequence shown in SEQ ID NO: 4 is configured to specifically target the p.A57V mutation in the gene product of the mutant ELANE gene. For example, a nucleic acid molecule containing the nucleotide sequence shown in SEQ ID NO: 2 or a nucleic acid molecule consisting of the nucleotide sequence shown in SEQ ID NO: 2 is configured to specifically target the c.641G>T mutation in exon 5 of the human ELANE gene, and / or a nucleic acid molecule containing the nucleotide sequence shown in SEQ ID NO: 5 or a nucleic acid molecule consisting of the nucleotide sequence shown in SEQ ID NO: 5 is configured to specifically target the p.G214V mutation in the gene product of the mutant ELANE gene. According to the findings of the inventors of the present invention, both of these mutations are often found in autosomal dominant severe congenital neutropenia (CN / SCN). Since the nucleic acid molecules of the present invention have a specific configuration, they do not target the non-mutated ELANE gene. Therefore, due to this specific configuration of the nucleic acid molecules of the present invention, the ELANE gene can be specifically edited at the allele level.

[0017] As an advantageous aspect of this method, when there are a mutated first allele and a non-mutated second allele of the ELANE gene, only the mutated first allele is targeted, for example, knockdown is performed, but the non-mutated second allele can express a functional ELANE gene product. Thereby, the expression of the mutated ELANE gene product having abnormal function is inhibited or blocked. Instead, only the functional ELANE gene product becomes dominant. This enables gene correction of dominant mutations without using a repair template.

[0018] In vitro, the inventors have demonstrated that by using the nucleic acid molecule of the present invention (for example, a nucleic acid molecule in the form of a single-guide RNA (sgRNA)) to knockdown the ELANE gene, it is possible to restore the reduced differentiation of hematopoietic stem cells and progenitor cells (HSPCs) derived from patients with congenital neutropenia into granulocytes. The repaired and differentiated HSPCs showed significant ROS production compared to HSPCs that did not undergo editing of the ELANE gene by the nucleic acid molecule of the present invention.

[0019] This finding of the inventors is surprising and unexpected.

Mode for Carrying Out the Invention

[0020] As used herein, "nucleic acid molecule" includes both single-stranded and double-stranded deoxyribonucleic acid (DNA) molecules and ribonucleic acid (RNA) molecules in which natural nucleotides and / or chemically modified nucleotides are linearly linked.

[0021] According to the present invention, the ELANE gene, that is, the "neutrophil elastase gene" (human variant: Gene ID: 1991, Ensembl: ENSG00000197561, Uni-ProtKB / Swiss-Prot: P08246), encodes a serine protease and is also called neutrophil elastase. Neutrophil elastase belongs to the same family as chymotrypsin and has a broad substrate specificity. Neutrophil elastase is secreted by neutrophils and macrophages in inflammation and destroys bacteria and host tissues.

[0022] According to the present invention, "gene editing," or genome editing, is a type of genetic engineering technique that involves the insertion, deletion, modification, or substitution of DNA in the genome of a living organism. In the context of the present invention, "gene editing" refers, for example, to the modification of the ELANE gene in a living cell. Therefore, according to the present invention, "gene editing" includes knockout, knockdown, or modification of the ELANE gene, preferably including knockout, knockdown, or modification of a mutated ELANE gene. According to the present invention, "allele-specific" means that gene editing is performed with a specific target of the mutated allele of the ELANE gene, but the non-mutated allele is not subjected to gene editing. In one embodiment of the present invention, this gene editing results in knockdown of the expression of the mutated allele of the ELANE gene and specific functional loss of the expression of the mutated allele of the ELANE gene. Therefore, the gene editing of the present invention includes inactivating a heterozygous mutated allele of the ELANE gene in a living cell, i.e., the living cell further contains at least one non-mutated allele of the ELANE gene.

[0023] The fundamental problem underlying this invention has been completely solved by this method.

[0024] In one embodiment of the present invention, the nucleic acid molecule is configured for allele-specific editing of an autosomal dominant mutation in the ELANE gene.

[0025] This method has the advantage of being able to specifically treat the majority of ELANE gene-related disorders. For example, mutations in the ELANE gene in patients with severe congenital neutropenia (CN / SCN) very often occur in an autosomal dominant manner. Mutation in one of the two alleles leads to the disease phenotype, even if the other allele is unmutated. The present invention can effectively treat such a genetic configuration.

[0026] In another embodiment of the present invention, the nucleic acid is a "single guide RNA (sgRNA)".

[0027] This means is a requirement for using the nucleic acid molecule according to the present invention using the CRISPR / Cas9 method, and specifically, this nucleic acid molecule contains or consists of the nucleotide sequence of Sequence ID No. 1 (for treating the c.170C>T mutation in exon 2 of the human ELANE gene or the p.A57V mutation in the corresponding gene product) and contains or consists of the nucleotide sequence of Sequence ID No. 2 (for treating the c.641G>T mutation in exon 5 of the human ELANE gene or the p.G214V mutation in the corresponding gene product).

[0028] According to the present invention, a "single guide RNA (sgRNA)," or guide RNA, is a component of the CRISPR complex and plays a role in guiding the CRISPR endonuclease to its target (e.g., the ELANE gene). sgRNA is a short non-coding ribonucleic acid (RNA) sequence that binds to a complementary target DNA sequence. After binding to the CRISPR endonuclease enzyme, sgRNA guides the CRISPR complex to a specific ELANE gene site on the DNA via pairing, where the CRISPR endonuclease cleaves the double strand.

[0029] As will be easily understood by those skilled in the art, the thymine (t) contained in the DNA molecule corresponding to the aforementioned position is replaced by uracil (u) in the RNA molecule or sgRNA molecule. When this is applied to SEQ ID NO: 1 and SEQ ID NO: 2, the sequences of the corresponding sgRNA molecules are 5'-UCAGGGUGACGCCGCAGAAG-3' (SEQ ID NO: 4) and 5'-GGACGAAGGAGGCAAUUACG-3' (SEQ ID NO: 5), respectively.

[0030] In another embodiment of the present invention, the nucleic acid molecule is a repair template.

[0031] This method has the advantage that, for example, a mutant allele of the ELANE gene can be knocked down using sgRNA containing the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and / or SEQ ID NO: 5, or sgRNA consisting of these nucleotide sequences. Furthermore, the mutant allele of the ELANE gene can be repaired using, for example, a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 3, or a nucleic acid molecule consisting of this sequence, or a single-stranded oligonucleotide DNA (ssODN) containing this sequence, or ssODN consisting of this sequence. The nucleotide sequence of SEQ ID NO: 3 is derived from exon 5 of an unmutated human ELANE gene. This method makes it possible to construct another copy of an unmutated, functional ELANE gene or allele, thereby increasing the expression of the gene product.

[0032] Therefore, another subject of the present invention is a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 5.

[0033] In another embodiment, the nucleic acid molecule of the present invention is configured for use in the prevention, treatment and / or diagnosis of a disease, the disease being preferably an ELANE gene-related disorder, more preferably congenital neutropenia, and even more preferably severe congenital neutropenia (CN / SCN) and / or cyclic neutropenia (CyN).

[0034] This method has the advantage that, by using the present invention as a causal therapy, it can not only treat ELANE gene-related disorders, particularly severe congenital neutropenia (CN / SCN) or cyclic neutropenia (CyN), but also treat pulmonary emphysema or emphysematous changes.

[0035] Another subject of the present invention relates to vectors containing nucleic acid molecules of the present invention.

[0036] According to the present invention, a "vector" includes any DNA molecule used as a medium to artificially deliver nucleic acid molecules, such as sgRNA, to another cell capable of replicating and / or expressing nucleic acid molecules. Viral vectors are particularly preferred because they are characterized by having a virus-specific molecular mechanism for efficiently transporting the genome into an infected cell. In a preferred embodiment of the present invention, the viral vector may be an adeno-associated virus vector (AAV) or any other viral vector.

[0037] The features, properties, advantages, and embodiments described for the nucleic acid molecules of the present invention also apply to the vectors of the present invention.

[0038] Another subject of the present invention relates to compositions comprising nucleic acid molecules and / or vectors of the present invention.

[0039] The features, properties, advantages, and embodiments described for the nucleic acid molecules and vectors of the present invention also apply to the compositions of the present invention.

[0040] In one embodiment of the present invention, the composition further comprises a vector encoding CRISPR-related protein 9 (Cas9), and more preferably further comprises a vector encoding CRISPR Cas9 (SaCas9) derived from Staphylococcus aureus.

[0041] This method establishes the requirements for using CRISPR / Cas9 technology. Cas9 (CRISPR-related protein 9; formerly known as Cas5, Csn1, or Csx12) is a 160-kilodalton protein that cleaves phosphodiester bonds within polynucleotide chains and is a component of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) complex. Therefore, Cas9 can specifically bind to sgRNAs that guide endonucleases to target nucleic acids. CRISPR Cas9 derived from Staphylococcus aureus (SaCas9) is a small Cas9 ortholog that overcomes the limitations of AAV packaging capacity and can be packaged in AAVs. Effective gene targeting by SaCas9 has been validated in adult rats. Recently, other small Cas9s derived from Staphylococcus auricularis, Campylobacter jejuni, or Neisseria meningitidis have also been discovered, and these small Cas9s are also suitable for the present invention.

[0042] In one embodiment of the composition of the present invention, the Cas9 is under the control of the CAG promoter.

[0043] Expression induced by the CAG promoter (CMV enhancer / chicken β-actin promoter) is superior to that of other promoters, and particularly superior to that of common synapsin promoters in the long term. This aspect is important in the treatment, prevention, or modeling of neurodegenerative diseases requiring long-term expression and monitoring. CAG is generally preferred as a promoter in recent studies using AAV.PHP.EB.

[0044] In another embodiment of the present invention, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0045] A "pharmaceutical composition" is a composition suitable for administration to animals and / or humans in a medical setting. The pharmaceutical composition is preferably sterilized and preferably manufactured in accordance with GMP guidelines.

[0046] "Pharmacologically acceptable carriers" or excipients are well known in the art and include, for example, nanocarriers; nanovectors; aqueous solutions such as water or buffered saline; or other solvents or vehicles such as glycols, glycerols, oils (e.g., olive oil), or organic esters for injection. In preferred embodiments, if the pharmaceutical composition is for administration to humans, for example, for parenteral administration, the aqueous solution is pyrogen-free or substantially pyrogen-free. For example, the excipient can be an excipient that can delay the release of the drug, or an excipient that can selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in dosage forms such as injections, tablets, capsules (including sprinkle capsules and gelatin capsules), granules, powders, syrups, or suppositories. The pharmaceutical composition may also be in the form of a solvent suitable for topical administration. Alternatively, the pharmaceutical composition may be in the form of an aerosol that can be administered by inhalation.

[0047] "Pharmacologically acceptable" means a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues without causing excessive toxicity, inflammation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit-risk ratio, within the bounds of reasonable medical judgment. Suitable pharmaceutical carriers or excipients and pharmaceutical additives used in pharmaceutical formulations are listed in the well-known reference books in the art, Remington—The Science and Practice of Pharmacy, 23rd edition, 2020 and the United States Pharmacopeia and the National Formulary (USP / NF). Other sources of information are also available to those skilled in the art.

[0048] The pharmaceutical compositions and methods of the present invention, described below, may be used to treat organisms in need of treatment. In certain embodiments, the organism is a mammal such as a human, or another non-human mammal.

[0049] In one embodiment of the composition of the present invention, the composition is configured for the prevention, treatment, and / or testing of a disease, wherein the disease is preferably an ELANE gene-related disorder, more preferably congenital neutropenia, and even more preferably severe congenital neutropenia (CN / SCN) and / or cyclic neutropenia (CyN).

[0050] Another subject of the present invention is a method for allele-specific editing of the ELANE gene in a biomaterial containing genetic material encoding the ELANE gene in vitro, comprising the step of introducing a nucleic acid molecule, vector and / or composition of the present invention into the biomaterial, preferably a method in which the editing is carried out using CRISPR / Cas9 technology.

[0051] According to the present invention, "biomolecules containing genetic material encoding the ELANE gene" include living cells, tissues, and parts of organisms.

[0052] In one embodiment of the present invention, the biomaterial includes hematopoietic stem cell / progenitor cells (HSPCs).

[0053] This method has the advantage of being able to edit the ELANE gene in biomaterials in which mutations are pathologically expressed. For example, in HSPCs of patients with severe congenital neutropenia (CN / SCN), differentiation into granulocytes is reduced, but according to our findings, the nucleic acid molecule of the present invention can restore this reduced differentiation into granulocytes.

[0054] Another subject of the present invention is a method for preventing, treating and / or testing for disease in an organism (e.g., an animal or a human), comprising the step of allele-specific editing of the ELANE gene in the organism by introducing a nucleic acid molecule, vector and / or composition of the present invention into the organism, preferably a method by which the gene editing is carried out using CRISPR / Cas9 technology.

[0055] The present invention is a method for inactivating a mutant allele of the neutrophil elastase gene (ELANE gene) in cells, Preferably, the ELANE gene has a mutation associated with congenital neutropenia, severe congenital neutropenia (CN / SCN), The ELANE gene in the cell is mutated at one or more nucleotide positions selected from c.170C>T (exon 2) and c.641G>T (exon 5), and / or the ELANE gene product of the cell is mutated at one or more amino acid positions selected from p.A57V and p.G214V. The method described above is The process involves introducing a composition comprising a CRISPR nuclease or a sequence encoding the CRISPR nuclease and a first RNA molecule containing the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 into the cells, thereby causing the complex of the CRISPR nuclease and the first RNA molecule to double-strand break the mutant allele of the ELANE gene. This provides a method that includes this.

[0056] In one embodiment of the method of the present invention, single-stranded oligonucleotide DNA (ssODN) containing the nucleotide sequence of SEQ ID NO: 3 is further introduced into the cells.

[0057] The present invention provides recombinant cells obtained by the method described above.

[0058] The present invention relates to a method for preparing a composition comprising recombinant cells in vitro or ex vivo, a) A step of isolating or providing HSPCs from cells obtained from an organism (preferably human) that has an ELANE gene mutation associated with severe congenital neutropenia (CN / SCN) or cyclic neutropenia (CyN) and / or is suffering from severe congenital neutropenia or cyclic neutropenia. b) A step to obtain recombinant cells by introducing a composition comprising a CRISPR nuclease or a sequence encoding the CRISPR nuclease and a first RNA molecule containing the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 into the cells of step (a), thereby inactivating the mutant allele of the ELANE gene by double-strand cleavage of the mutant allele of the ELANE gene in one or more cells. Includes, The ELANE gene within the cell is mutated at one or more nucleotide positions selected from c.170C>T (exon 2) and c.641G>T (exon 5), and / or the ELANE gene product of the organism is mutated at one or more amino acid positions selected from p.A57V and p.G214V. The method described above is c) Step of growing and culturing the recombinant cells from step (b) It may include, The recombinant cells are capable of engraftment and can produce progeny cells after engraftment. Provide a method.

[0059] In one embodiment of the method of the present invention, single-stranded oligonucleotide DNA (ssODN) containing the nucleotide sequence of SEQ ID NO: 3 is further introduced into the cells.

[0060] In another embodiment of the method of the present invention, the method further comprises the step of administering the cells of step (b) or step (c) to the organism in order to treat the subject's severe congenital neutropenia or cyclic neutropenia.

[0061] Accordingly, the present invention provides a method for treating an organism (preferably a human) suffering from severe congenital neutropenia or cyclic neutropenia, the method comprising the step of administering a therapeutically effective amount of the recombinant cells.

[0062] The features, properties, advantages, and embodiments described for the nucleic acid molecules, vectors, and compositions of the present invention also apply to the methods of the present invention described above.

[0063] The features described above and those described later are not limited to the combinations shown in each embodiment, but can also be used in other combinations or individually without departing from the scope of the present invention.

[0064] The present invention will be described in more detail by reference to the following embodiments. These embodiments describe further features, characteristics, and advantages of the present invention. Furthermore, the following embodiments are for illustrative purposes only and do not limit the spirit or scope of the present invention. Features described in specific embodiments are general features of the present invention and can be applied not only to specific embodiments but also individually and to all embodiments of the present invention. [Brief explanation of the drawing]

[0065] The present invention will be described and explained in more detail with reference to the following embodiments and drawings, but the present invention is not limited to these embodiments and drawings. [Figure 1]This study demonstrates that allele-specific knockout can rescue granulocyte generation in patients with congenital neutropenia (CN) who have the p.A57V mutant ELANE gene. (A) The experimental scheme illustrating the processes of allele-specific gene knockout and allele-specific gene modification is shown. HSPCs derived from CN patients with the mutant ELANE gene were grown in vitro, and allele-specific Hifi Cas9 RNPs were electroporated. For allele-specific gene modification, a single-strand DNA repair template (ssODN) was further used. After 3 days, the cells were seeded in liquid medium and differentiated into neutrophils. On day 14, differentiation into neutrophils was evaluated by flow cytometry and morphological analysis. (B) On day 14 of differentiation induction by liquid culture, the efficiency of gene modification by V57-specific RNPs was analyzed in CN patients with the mutant ELANE gene and healthy controls. (C) Differentiation into granulocytes of ELANE V57 knockout CD34+ cells was evaluated by flow cytometry investigating the expression of surface markers. (D) Representative images of cells smeared with cytospin and stained with Wright-Giemsa stain 14 days after differentiation induction are shown. (E) Results of colony-forming unit (CFU) assay of HSPCs from CN patients with mutant ELANE genes that were nucleofected with non-targeted RNPs or mutation-specific RNPs are shown. Colonies were counted 14 days after CFU culture. (F) CFU-G colonies were picked from mutation-specific KO cells and analyzed by Sanger sequencing. (G) Neutrophils differentiated by liquid culture were treated with fMLP to investigate ROS production capacity. [Figure 2]This study demonstrates that allele-specific gene modification of the ELANE gene in CN patients with the p.G214V mutation improved granulocyte differentiation compared to unedited control cells. (A) HSPCs derived from CN patients with the ELANE gene with the p.G214V mutation were nucleofected with either a non-targeted RNP alone or with a V214-specific RNP and an ssODN modification template, and then differentiated in liquid culture. Surface marker expression was investigated after 14 days. (B) Representative Wright-Giemsa stained images of cytospin smears of cells 14 days after differentiation induction are shown. (C) The allele-specific gene editing efficiency of the p.G214V mutation was evaluated using Sanger sequencing and the Sequence Trace Degradation Algorithm (TIDE), revealing that indels occurred in 64% of cases. The homologous recombination repair rate (HDR) was approximately 36%. R2 was calculated to evaluate the goodness of fit of the ICE algorithm, and R2 > 0.9 was considered a reliable predictor. [Examples]

[0066] 1. Allele-specific design method and validation of sgRNA targeting the mutant ELANE gene. We created allele-specific single guide RNAs (sgRNAs) that selectively target mutations in the ELANE gene (Table 1). Using the CCTop website, we designed a p.A57V mutation-specific sgRNA (cleavage site: chr19[+852,969:-852,969], NM_001972.3 exon 2, 161bp; NP_001963.1 p.F54) to knock out the mutant allele of the ELANE gene, and commissioned Integrated DNA Technologies (IDT) to synthesize it as a chemically modified sgRNA. Furthermore, using the CCTop website, we designed a p.G214V mutation-specific sgRNA (cleavage site: chr19[+856,001:-856,001], NM_001972.3 exon 5, 641 bp; NP_001963.1 p.V214) that selectively targeted the mutant allele of the ELANE gene, and commissioned Integrated DNA Technologies (IDT) to synthesize it as a chemically modified sgRNA.

[0067] CRISPR / Cas9-gRNA RNP complexes were constructed by incubating V57 sgRNA with recombinant HiFi-Cas9 protein. V57 sgRNA targeting the mutated exon 2 of the ELANE gene was selected, and a stop codon mutation was introduced using this V57 sgRNA. This induced nonsense mutation-dependent mRNA degradation (NMD) in only the mutated ELANE mRNA during the first translation of ELANE mRNA transcribed from the mutated allele, specifically due to the stop codon mutation or frameshift mutation. It is hypothesized that such stop codon mutation introduction and NMD can reverse granulocyte production impairment in patients with congenital neutropenia (CN) and severe congenital neutropenia (SCN) (Figure 1A). To evaluate the allele selectivity of V57 sgRNA, RNP complexes consisting of HiFi Cas9-V57 sgRNA were electroporated into HSPCs derived from CN / SCN patients with the p.A57V mutation or healthy control HSPCs. Sanger sequencing performed 96 hours after electroporation revealed that V57 sgRNA exhibits highly selective on-target activity against the mutant allele. In contrast, no double-strand breaks (DSBs) were induced by V57 mutant-specific sgRNA in healthy control HSPCs (Figure 1B), further demonstrating the allele specificity of V57 sgRNA.

[0068] [Table 1]

[0069] To further evaluate whether this allele-specific design method based on CRISPR / Cas9 is applicable to a wide range of ELANE mutations, we designed an sgRNA targeting the p.G214V mutation in exon 5 of the ELANE gene, named V214 sgRNA (cleavage site: chr19[+856,002:-856,002]). The HiFi Cas9-V214 sgRNA complex and single-stranded oligonucleotide DNA (ssODN) repair template (Table 1) were electroporated into HSPCs derived from CN / SCN patients with the p.G214V mutation. Sanger sequencing analysis showed that R214 sgRNA had high selectivity for the mutant allele. On the other hand, no introduction of double-strand breaks (DSBs) by the R214 mutant-specific sgRNA was observed in healthy control HSPCs (Figure 2A).

[0070] These results confirmed that gene editing using allele-specific CRISPR / Cas9 can be applied to congenital neutropenia associated with the ELANE gene.

[0071] 2. Restoration of decreased granulocyte differentiation by ELANE knockout in HSPCs derived from patients with congenital neutropenia (CN) and severe congenital neutropenia (SCN) who have mutant ELANE genes. To further evaluate the clinical applicability of allele-specific knockout of ELANE for the treatment of CN / SCN related to the ELANE gene, primary bone marrow CD34 + HSPCs from CN / SCN patients with the ELANE gene carrying the p.A57V mutation were gene-edited with CRISPR / Cas9 RNPs using V57 sgRNA and differentiated into neutrophils in vitro. The complex constructed from V57 sgRNA and HiFi-Cas9 protein was electroporated into human CD34 + HSPCs to knockout the allele-specific ELANE gene in CD34 + HSPCs. The percentages of CD15 + CD11b + CD45 + cells, the percentages of CD16 + CD11b + CD45 + cells and the percentages of CD15 + CD16+ CD45 + The cell proportion (Figure 1C) was evaluated, and morphological examination (Figure 1D) of mature granulocytes formed on day 14 after in vitro differentiation induction of granulocytes by liquid culture was performed using cytospin smears. It was found that allele-specific ELANE gene knockout increased granulocyte differentiation. Furthermore, CD34 cells derived from CN / SCN patients who underwent allele-specific knockout were found to be affected. + A colony-forming unit (CFU) assay of cells revealed that CD34 was obtained from CD34 differentiated from HSPCs derived from CN / SC patients treated with non-targeted RNP MOCK by electroporation of a complex constructed from sgRNA and HiFi-Cas9 that does not have a target in the human genome. + Compared to cells, the number of CFU-G colonies increased, while the number of CFU-M colonies decreased (Figure 1E). These data suggest that allele-specific ELANE gene knockout restores granulocyte differentiation in CN / SCN cells. CFU-G colonies were picked from the CFU assay, and Sanger sequencing of exon 2 of the ELANE gene was performed to genotyping of the gene editing results. 66.67% of HSPCs with allele-specific ELANE gene knockout formed CFU-G, while only 13.33% of spontaneously repaired HSPCs formed CFU-G, with 20% remaining unedited (Figure 1F).

[0072] 3. ROS production after in vitro activation by neutrophils formed from ELANE KO HSPC remains unchanged. Neutrophils formed from HSPCs in which the ELANE gene was allele-specifically knocked out were cultured in liquid for 14 days to further evaluate their in vitro activation. Neutrophils in which the ELANE gene was allele-specific were activated with fMLP, and H2O2 levels (ROS) were evaluated. Significant ROS production was detected in allele-specific knocked-out neutrophils after activation with fMLP, but non-targeted RNP-MOCK neutrophils did not show a significant response even after activation with fMLP (Figure 1G).

[0073] 4. Restoration of reduced granulocyte differentiation by ELANE modification in HSPCs derived from patients with congenital neutropenia (CN) and severe congenital neutropenia (SCN) carrying the p.G214V mutation in the ELANE gene. To evaluate allele-specific ELANE modification, primary bone marrow CD34 from CN / SCN patients with the ELANE gene carrying the p.G214V mutation was used. + HSPCs were genetically modified using CRISPR / Cas9 RNP with V214 sgRNA and differentiated into neutrophils in vitro. The complex constructed from V214 sgRNA and HiFi-Cas9 protein was used together with ssODN as a repair template with human CD34. + By electroporating HSPC, CD34 + Allele-specific ELANE gene modification was performed in HSPCs. In contrast, no double-strand breaks (DSBs) were observed in healthy control HSPCs due to V214 mutation-specific sgRNA (Figure 2A). CD45 + CD15 + CD11b + cell percentage, CD45 + CD11b + CD16 + Cell percentage and CD45 + CD15 + CD16 + Evaluation of the cell proportions (Figure 2B) revealed that allele-specific modification of the ELANE gene increased granulocyte differentiation. Furthermore, morphological examination of cytospin smears of mature granulocytes formed on day 14 after in vitro granulocyte differentiation induction showed that cells with modified ELANE genes had an increased neutrophil count compared to control cells (Figure 2C). These data demonstrate that allele-specific modification of the ELANE gene restores granulocyte differentiation in CN / SCN cells. In addition, Sanger sequencing was performed to evaluate the results of allele-specific mutation modification, and trace analysis using the ICE algorithm revealed an indel efficiency of 64% and a homologous recombination repair rate (HDR) of 36% (Figure 2D).

[0074] 5. Conclusion Surprisingly, we were able to demonstrate that the ELANE gene can be edited in an allele-specific manner by using the nucleic acid molecule of the present invention, specifically a nucleic acid molecule containing the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and / or SEQ ID NO: 5. Such allele-specific editing of the ELANE gene can be performed, for example, by using CRISPR / Cas9 technology by constructing the nucleic acid molecule of the present invention as an sgRNA.

Claims

1. A composition for allele-specific editing of the c.641G>T mutation in exon 5 of the human ELANE gene, Includes a nucleic acid molecule or a vector containing said nucleic acid molecule, The nucleic acid molecule (a) a single guide RNA (sgRNA) containing the nucleotide sequence shown in SEQ ID NO: 2; and (b) Repair template containing the nucleotide sequence shown in Sequence ID No. 3 A composition containing the following:

2. The composition according to claim 1, for the prevention, treatment, and / or examination of a disease.

3. The composition according to claim 2, wherein the disease is congenital neutropenia.

4. The composition according to claim 3, wherein the congenital neutropenia is severe congenital neutropenia (CN / SCN) and / or cyclic neutropenia (CyN).

5. The composition according to claim 1, further comprising a vector encoding CRISPR-related protein 9 (Cas9).

6. The composition according to claim 1, further comprising a vector encoding CRISPR Cas9 (SaCas9) derived from Staphylococcus aureus.

7. The composition according to claim 5, characterized in that the Cas9 is under the control of the CAG promoter.

8. The composition according to any one of claims 1 to 7, which is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

9. A method for allele-specific editing of the ELANE gene in a biomaterial containing genetic material encoding the ELANE gene in vitro, comprising the step of introducing a composition according to any one of claims 1 to 7 into the biomaterial.

10. The method according to claim 9, characterized in that the editing is performed using CRISPR / Cas9 technology.

11. The method according to claim 9, wherein the biomaterial includes hematopoietic stem cells / progenitor cells (HSPCs).

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