Method for producing hematopoietic stem cells in which some or all of BCL11A gene enhancer has been destroyed, and kit for use in said method

Abstract

A method for producing hematopoietic stem cells in which a part or all of the enhancer of the BCL11A gene has been destroyed, the method comprising: a step for introducing a CRISPR-Cas3 system comprising the following (A)-(C) into hematopoietic stem cells: (A) a Cas3 protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; (B) a cascade reaction protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; and (C) a crRNA having an enhancer of the BCL11A gene as a target, a polynucleotide encoding the crRNA, or an expression vector comprising the polynucleotide.

Description

Technical Field

[0001] This invention relates to a method for manufacturing hematopoietic stem cells in which some or all of the enhancer of the BCL11A gene is destroyed, and a kit for using the method. Background Technology

[0002] BCL11A (B-cell lymphoma 11A) is a C2H2 type zinc finger protein involved in the expression control of various genes. In particular, BCL11A inhibits the expression of γ-globin in adult erythrocyte precursor cells, functioning as a key transcriptional regulator in the transition from γ-globin to β-globin expression during the fetal-adult transition. Hemoglobin (Hb) is a tetramer composed of four globin peptides. The main form of hemoglobin in the fetus is fetal hemoglobin (HbF), which is composed of two α-globins and two γ-globins. However, just before birth, the expression of γ-globin is suppressed through a control mechanism mainly controlled by BCL11A, and HbF is switched to adult hemoglobin (HbA), which is composed of two α-globins and two β-globins (Haydar Frangoul et al., N Engl J Med, 2021, 384, pp. 252-260 (Non-Patent Literature 1)). However, even after birth, HbF exists in whole hemoglobin at a proportion of less than 1%. It has been reported that due to the high oxygen transport capacity of HbF, increasing the expression of γ-globin, i.e. increasing HbF, can alleviate the clinical severity of abnormal hemoglobinopathy such as hemoglobinopathy, sickle cell disease (SCD), and β-thalassemia caused by mutations and reduced expression of β-globin (Non-Patent Literature 1; Elenoe C. Smith et al., Blood., 2016, Nov 10; 128(19): p. 2338-2342 (Non-Patent Literature 2)).

[0003] Therefore, for example, with the aim of treating the aforementioned abnormal hemoglobinemia, Patent Document 1 (International Publication No. 2014 / 085593) discloses a method for manufacturing precursor cells that inhibit the expression of the BCL11A gene. As a method for inhibiting the expression of the BCL11A gene, a method of introducing a mutation into hematopoietic stem cells at 60, 716, 189 to 60, 728, 612 on chromosome 2, which is the same as the BCL11A gene. Further, for example, Patent Document 2 (International Publication No. 2019 / 113149) discloses hematopoietic stem cells in which a mutation has been introduced into the +58 DHS (DNase I hypersensitive site) located within the enhancer region of the BCL11A gene. Additionally, as a method for introducing the mutation, methods of introducing the mutation by cleaving the site with site-specific nucleases such as zinc finger nucleases, the CRISPR-Cas9 system, and TALEN are listed.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: International Publication No. 2014 / 085593

[0007] Patent Document 2: International Publication No. 2019 / 113149

[0008] Non-patent literature

[0009] Non-patent literature 1: Haydar Frangoul et al., N Engl J Med, 2021, 384, pp. 252-260

[0010] Non-patent literature 2: Elenoe C. Smith et al., Blood., 2016, Nov 10; 128(19): pp. 2338-2342 Summary of the Invention

[0011] The problem that the invention aims to solve

[0012] However, the inventors studied the inhibition of BCL11A gene expression and found that, for example, even when a mutation was introduced into the enhancer region of the BCL11A gene in hematopoietic stem cells using the aforementioned CRISPR-Cas9 system, the inhibition of BCL11A gene expression in subsequently differentiated erythrocyte precursor cells was insufficient. This resulted in incomplete relief of the inhibition of γ-globin expression caused by BCL11A and an insufficient increase in γ-globin expression. Furthermore, in the CRISPR-Cas9 system, the target sequence of the guide RNA is short (typically around 20 bases), leading to insufficient specificity and off-target effects.

[0013] The present invention was made in view of the problems of the prior art described above, and aims to provide a method for manufacturing hematopoietic stem cells capable of differentiating into blood cell line progenitor cells with the expression of the BCL11A gene specifically and sufficiently suppressed, and a kit suitable for use in the manufacturing method.

[0014] Methods for solving problems

[0015] To achieve the aforementioned objectives, the inventors conducted repeated and in-depth research, attempting to disrupt the enhancer of the BCL11A gene in hematopoietic stem cells using the CRISPR-Cas3 system. Surprisingly, compared to using the existing CRISPR-Cas9 system, although the genome editing efficiency (defect rate) at the target site (editable site) within the enhancer was low, the expression level of γ-globin was significantly increased in erythrocyte precursor cells differentiated from hematopoietic stem cells whose enhancer had been disrupted using the CRISPR-Cas3 system. In other words, it was found that using the CRISPR-Cas3 system, compared to using the CRISPR-Cas9 system, the success rate of BCL11A gene expression inhibition was higher, specifically and efficiently disrupting the BCL11A gene enhancer more reliably. Furthermore, it was found that the above-mentioned technique using the CRISPR-Cas3 system resulted in virtually no off-target effects, and the target efficiency was relatively high relative to this low off-target frequency. Therefore, the inventors discovered that by specifically using the CRISPR-Cas3 system to disrupt the enhancer of the BCL11A gene, the enhancer of the BCL11A gene can be specifically and efficiently disrupted in hematopoietic stem cells. As a result, the expression of the BCL11A gene is sufficiently suppressed in hematopoietic cell line progenitor cells differentiated from the hematopoietic stem cells. Thus, the CRISPR-Cas3 system is particularly effective in inhibiting the expression of the BCL11A gene, thereby completing the present invention.

[0016] The solution of the present invention, based on the above understanding, is as follows.

[0017] [1]

[0018] Methods for manufacturing hematopoietic stem cells in which some or all of the enhancers of the BCL11A gene are destroyed. This method includes the step of introducing a CRISPR-Cas3 system comprising (A) to (C) the following into hematopoietic stem cells. (A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide. (B) cascade reaction proteins, polynucleotides encoding those proteins, or expression vectors containing those polynucleotides, and (C) crRNA targeting the enhancer of the BCL11A gene, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

[0019] [2]

[0020] According to the method described in [1], the nucleotide sequence of the crRNA comprises the nucleotide sequence described in sequence number 27.

[0021] [3]

[0022] According to the method described in [1] or [2], the hematopoietic stem cells produced can differentiate into erythrocyte precursor cells with increased expression of γ-globin.

[0023] [4]

[0024] A method for increasing the expression level of γ-globin in the erythrocyte precursor cells of a subject, comprising administering hematopoietic stem cells obtained by any one of the methods described in [1] to [3] to the subject.

[0025] [5]

[0026] Prevention or treatment of abnormal hemoglobinemia, including the procedure of administering hematopoietic stem cells obtained by any one of the methods described in [1] to [3] to a recipient.

[0027] [6]

[0028] The kit used in any of the methods described in [1] to [5] The kit contains the following (A) to (C): (A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide. (B) cascade reaction protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide, and (C) crRNA targeting the enhancer of the BCL11A gene, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

[0029] The effects of the invention

[0030] According to the present invention, a method for manufacturing hematopoietic stem cells capable of differentiating into blood cell line progenitor cells with specific and sufficient inhibition of BCL11A gene expression can be provided, as well as a kit that can be used in the manufacturing method.

[0031] If hematopoietic stem cells with disrupted BCL11A gene enhancers differentiate into erythrocyte precursor cells, the expression of the BCL11A gene is suppressed in these precursor cells. This suppression caused by BCL11A is then relieved, thereby inducing γ-globin expression and increasing its level. Therefore, this invention is useful for the prevention or treatment of disease groups related to γ-globin expression levels. Furthermore, this method using a CRISPR-Cas3 system avoids off-target effects, making it useful from a safety perspective. Attached Figure Description

[0032] Figure 1 The results of digital PCR in Example 1 show the relationship between the amount of Cas3 protein / cascade reaction protein (Cas3 protein / (cascade reaction protein complex + crRNA)) introduced or the amount of Cas9 protein introduced and the defect rate (% defect) at the target site.

[0033] Figure 2 The graph shows the results of quantitative PCR as Experiment Example 1, displaying the relationship between the amount of Cas3 protein / cascade reaction protein (Cas3 protein / (cascade reaction protein complex + crRNA)) introduced or the amount of Cas9 protein introduced and the relative expression level of γ-globin (relative to β-globin). Detailed Implementation

[0034] The following describes specific examples of preferred embodiments of the present invention, but the present invention is not limited thereto.

[0035] <Methods for manufacturing hematopoietic stem cells with disrupted BCL11A gene enhancers>

[0036] This invention provides a method (sometimes referred to herein as "the manufacturing method of this invention") for manufacturing hematopoietic stem cells in which part or all of the enhancer of the BCL11A gene is destroyed, comprising the step of introducing a CRISPR-Cas3 system comprising (A) to (C) below into the hematopoietic stem cells. (A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide. (B) cascade reaction protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide, and (C) crRNA targeting the enhancer of the BCL11A gene, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

[0037] (BCL11A gene, BCL11A gene enhancer, hematopoietic stem cells with destroyed BCL11A gene enhancer)

[0038] The BCL11A gene is a C2H2 type zinc finger protein that encodes BCL11A (B-cell lymphoma 11A), a subunit of the BAF chromatin remodeling complex (human BCL11A gene reference number (Gene ID): 53335). BCL11A is also known as EVI9, CTIP1, DILOS, ZNF856, HBFQTL5, BCL11A-L, BCL11A-S, BCL11a-M, or BCL11A-XL. BCL11A is known to be highly expressed in hematopoietic cells and contributes to the switching of γ-globin to β-globin expression during the transition from fetal hemoglobin (HbF) to adult hemoglobin (HbA). γ-globin in humans is encoded by the HBG1 and HBG2 genes. Two γ-globin chains together with two α-globin chains constitute fetal hemoglobin (HbF), which is usually replaced by adult hemoglobin (HbA) after birth (Non-Patent Literature 1).

[0039] An enhancer of the BCL11A gene is a region bound by a transcription factor that promotes the transcription of the BCL11A gene. In human genomic DNA, various enhancers are known to exist within introns of the BCL11A gene, upstream of the BCL11A gene, or downstream of the BCL11A gene. However, as the enhancer of the BCL11A gene involved in this invention, an enhancer located within an intron of the BCL11A gene is preferred. More preferably, the region at 60, 489, 054–60, 501, 476 (12,423 bp) on human chromosome 2 is preferred. Furthermore, transcription factors that bind to enhancers of the BCL11A gene include GATA, STAT1, ELF1, KLF1, RREB1, and TAL1. For example, "GATA" is a protein belonging to the GATA transcription factor family, also known as GF1, GF-1, NFE1, XLTT, ERYF1, NF-E1, XLANP, XLTDA, and GATA-1. The regions in which GATA binds (GATA-binding regions) within the enhancer region of the BCL11A gene can be listed as DNase I hypersensitive sites (DHS), such as DHS+55, DHS+58, and DHS+62, which will be discussed later.

[0040] Hematopoietic stem cells (HSCs) are cells capable of self-replication and differentiation into precursor cells of hematopoietic cell lines (sometimes referred to as "hematopoietic cell line precursor cells" in this specification, depending on the context). Examples of hematopoietic cell lines include leukocytes, erythrocytes, platelets, mast cells, dendritic cells, eosinophils, neutrophils, monocytes, macrophages, granulocytes, T cells, B cells, and NK cells. Examples of hematopoietic cell line precursor cells include leukocyte precursor cells, erythrocyte precursor cells, megakaryocyte precursor cells, granulocyte precursor cells, precursor B cells, precursor T cells, and precursor NK cells. As for the hematopoietic stem cells used in the manufacturing method of the present invention, considering, for example, the increased expression of γ-globin as a result of the treatment method of the present invention described below, erythrocyte precursor cells differentiated into γ-globin-producing cells are preferred. Furthermore, in this invention, the term "differentiation into hematopoietic stem cell progenitor cells (preferably erythrocyte progenitor cells)" means that in addition to the hematopoietic stem cell itself differentiating into the progenitor cells, it also includes one or more hematopoietic stem cells generated from the self-replication of the hematopoietic stem cell differentiating into the progenitor cells, thereby producing one or more progenitor cells.

[0041] Hematopoietic stem cells exhibit a variety of known phenotypes, such as CD34+ and CD38-. The hematopoietic stem cells used in the manufacturing method of the present invention can be isolated from peripheral blood, bone marrow, umbilical cord, placenta, etc., by means of the aforementioned phenotypes or by methods according to the present invention.

[0042] Animals can be cited as sources of hematopoietic stem cells according to the present invention, preferably humans or mammals other than humans. Examples of mammals other than humans include, for instance, even-toed ungulates such as cattle, wild boars, pigs, sheep, and goats; odd-toed ungulates such as horses; rodents such as mice, rats, guinea pigs, hamsters, and squirrels; and rabbits, dogs, cats, and ferrets. When the hematopoietic stem cells manufactured by the method of the present invention are administered to the recipient, the animal that is the same as the recipient is preferably the hematopoietic stem cell used in the manufacturing method of the present invention. Furthermore, the hematopoietic stem cells used in the manufacturing method of the present invention can be, in addition to cells collected from the peripheral blood, bone marrow, umbilical cord, placenta, etc., of these animals, cultured cells derived from the cells constituting the animal, cells from organs / tissues removed from the animal (e.g., induced pluripotent stem cells, iPS cells, etc.).

[0043] The manufacturing method of the present invention produces hematopoietic stem cells in which the enhancer of the BCL11A gene is destroyed. Furthermore, in the present invention, the phrase "destruction of the enhancer" includes not only complete destruction of the enhancer but also partial destruction. The "hematopoietic stem cells in which part or all of the enhancer of the BCL11A gene is destroyed" obtained by the manufacturing method of the present invention refers to cells in which part or all of the enhancer of the BCL11A gene is missing or replaced (preferably missing), and its enhancer function is suppressed. The term "suppression of the enhancer function of the BCL11A gene" more specifically means, for example, that at least one, preferably at least one or more, GATA transcription factors are not bound.

[0044] The site of deletion or replacement in the enhancer of the BCL11A gene is preferably part or all of the region 60, 489, 054 to 60, 501, 476 in human chromosome 2 as described above. As a site of deletion or replacement (preferably deletion) in the enhancer of the BCL11A gene, it is particularly preferred to be part or all of the GATA-binding region, more specifically, preferably one of the following three regions: DHS+55 (60,498,289 to 60,498,552 (264 bp) in human chromosome 2), DHS+58 (60,495,103 to 60,495,330 (228 bp) in human chromosome 2), and DHS+62 (60,490,907 to 60,491,050 (144 bp) in human chromosome 2), more preferably part (e.g., more than 75%) or all of two of these regions, and further preferably all of all three regions.

[0045] Confirmation that part or all of the enhancer of the BCL11A gene is destroyed can be made using existing known methods, such as PCR (digital PCR, real-time PCR, endpoint PCR, etc.), sequence determination, DNA blotting, next-generation sequencing, etc.

[0046] (CRISPR-Cas3 system)

[0047] CRISPR-Cas systems are classified into two categories: Category 1, which cleaves the target region (the region to be cleaved) through a complex of multiple proteins, and Category 2, which cleaves through a single protein. To date, all CRISPR-Cas9, CRISPR-Cas12 (Cpf1), and CRISPR-Cas13 systems developed as genome editing tools have been classified into Category 2. However, the CRISPR-Cas3 system discussed in this invention belongs to Category 1, Type I. Regarding the type I system, it was previously classified into several subtypes, now classified as IA, IB, IC, ID, IE, and IF types, as well as IG type, which is a subtype of IB type (see, for example, [van der OostJ et al., Nature Reviews Microbiology, 2014, Vol. 12 (No. 7), pp. 479-492], [Jackson RN et al., Current Opinion in Structural Biology, 2014, Vol. 24, pp. 106-114], [Makarova et al., Nat Rev Microbiol., 2020, 18, pp. 67-83]).

[0048] The "CRISPR-Cas3 system" involved in this invention specifically includes the following (A) to (C): (A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide. (B) cascade reaction protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide, and (C) crRNA targeting the enhancer of the BCL11A gene, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

[0049] [Cas protein group]

[0050] The Cas3 proteins that make up the CRISPR-Cas3 system possess DNA cleavage and helicase activities. Cas3 proteins, through their interaction with cascade proteins and crRNA that constitute the CRISPR-Cas3 system, can cleave target DNA at multiple sites. Furthermore, in this specification, when referred to simply as "Cas3," it means "Cas3 protein." Additionally, in this specification, Cas3 proteins and cascade proteins are collectively referred to as the "Cas protein group," depending on the circumstances.

[0051] The CRISPR-Cas3 system involved in this invention comprises all seven isotypes of type I. The typical IE type of the type I CRISPR-Cas3 system, representing the Cas protein group, includes Cas3, Cas8 (Cse1), Cas11 (Cse2), Cas5, Cas6, and Cas7. This Cas protein group cleaves target DNA in cooperation with crRNA. In this case, the Cas protein group typically forms a complex (cascade reaction complex) consisting of one Cas3 molecule, one Cas8 molecule, two Cas11 molecules, six Cas7 molecules, one Cas5 molecule, and one Cas6 molecule.

[0052] In the IA type system, the Cas protein group includes Cas3-HD, Cas3-HEL, Cas5, Cas6, Cas7, Cas8, and Cas11; in the IB type system, the Cas protein group includes Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11; in the IC type system, the Cas protein group includes Cas3, Cas5, Cas7, Cas8, and Cas11; in the ID type system, the Cas protein group includes Cas3, Cas5, Cas6, Cas7, Cas10, and Cas11; in the IF type system, the Cas protein group includes Cas2-3, Cas5, Cas6, Cas7, and Cas8; and in the IG type system, the Cas protein group includes Csb2 (Cas6-like), Cas7, Cas8g, Cas3, and Cas11. However, even when Cas11 is excluded from the components of a type I system, the systems still exhibit DNA cleavage activity (for example, it is known that in type IB and type IC systems, DNA cleavage activity is observed even at a lower level compared to systems containing Cas11, and the same is true in type ID systems). Therefore, the CRISPR-Cas3 system according to this invention also includes systems that do not contain Cas11.

[0053] The source of the Cas protein group involved in this invention is not particularly limited, but from the viewpoint of suitability for genome editing in animal cells, it is preferably from *E. coli*. The amino acid sequences of each protein constituting the Cas protein group involved in this invention can be obtained from, for example, public databases (Genbank, etc.), but as one of the preferred configurations of the Cas protein group, the following are examples of sequences from a typical *E. coli*-derived IE type system: Cas3: A protein containing the amino acid sequence shown in sequence number 1. Cas8 (Cse1): A protein containing the amino acid sequence shown in sequence number 3. Cas11 (Cse2): A protein containing the amino acid sequence shown in sequence number 5. Cas5: A protein containing the amino acid sequence shown in sequence number 7. Cas6: A protein containing the amino acid sequence shown in sequence number 9. Cas7: A protein containing the amino acid sequence shown in sequence number 11.

[0054] Furthermore, the Cas protein group involved in this invention also includes mutants that are generated in nature or artificially modified. Therefore, as another preferred embodiment of the Cas protein group involved in this invention, each protein can be described as containing amino acid sequences that have high identity with the amino acid sequences of the typical Cas protein group described above, and Cas3 has DNA cleavage activity (preferably further, the Cas protein group has the ability to form cascade reaction complexes). High identity is, for example, an amino acid sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more), and even more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more). The amino acid sequence identity can be determined using, for example, BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) (e.g., using default, i.e., initially set parameters).

[0055] As another preferred embodiment of the Cas protein group involved in this invention, each protein may be a protein comprising an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in each amino acid sequence of the typical Cas protein group described above, and Cas3 has DNA cleavage activity (preferably further comprising the Cas protein group having the ability to form cascade reaction complexes). The term "multiple" typically refers to 50 amino acids or less, preferably 30 amino acids or less, more preferably 20 amino acids or less, and particularly preferably 10 amino acids or less (e.g., 5 amino acids or less, 3 amino acids or less, 2 amino acids or less, 1 amino acid or less).

[0056] Furthermore, the term "having DNA cleavage activity" indicates the ability to cleave a DNA strand at at least one site. The Cas3 possesses both DNA cleavage activity and the ability to form cascade reaction complexes, which can be confirmed, for example, by exhibiting DNA cleavage activity equal to or greater than that of typical Cas protein groups described above (e.g., 50% or more, 80% or more).

[0057] Each protein constituting the Cas protein group may be further supplemented with functional molecules as needed. Examples of such functional molecules include, for instance, nuclear transfer signals that promote translocation into the nucleus of eukaryotic cells (e.g., described in Wu J et al., 2009, Biophysical Journal, Vol. 96 (Issue 9), pp. 3840-3849), tags that facilitate purification (e.g., HN tags, His tags, FLAG tags, glutathione S-transferase (GST) tags), and reporter proteins that facilitate detection (e.g., fluorescent proteins such as green fluorescent protein (GFP), chemiluminescent proteins such as luciferase). One or more of these may be used, but the combination is not limited to these. When adding such functional molecules, they may be added, for example, to the N-terminal and / or C-terminal sides of each protein.

[0058] The following explanation uses the IE-type CRISPR-Cas3 system as a representative example. However, for other types of CRISPR-Cas3 systems, simply replace the proteins that make up the cascade reaction of the system appropriately.

[0059] In the CRISPR-Cas3 system of this invention, each protein constituting the Cas protein group can be independently introduced into the hematopoietic stem cells in the form of a protein, a polynucleotide encoding the protein, or an expression vector containing the polynucleotide.

[0060] When the Cas protein group is introduced into cells in the form of proteins, the amount of each protein can be appropriately adjusted, which is preferred from the viewpoint of operation and from the viewpoint of not introducing DNA into the genome.

[0061] Each protein constituting such a Cas protein group can be prepared individually by known methods or by methods based on known methods. For example, the method described in International Publication No. 2022 / 186063 can be cited as a method for preparing Cas3 protein. In addition, cascade reaction proteins can be prepared, for example, by the method described in Kazuto Yoshimi et al., Nature Communications, 2022, 13:4917, https: / / doi.org / 10.1038 / s41467-022-32618-0 (hereinafter referred to as "Reference I").

[0062] When introducing the Cas protein group into cells in the form of proteins, from the viewpoint of intracellular cleavage efficiency, it is preferable to first form a complex composed of two or more of the proteins constituting the Cas protein group before introducing it into the cells. Examples of protein combinations in the complex introduced into the cells include, for example, a complex composed of 2 molecules of Cas11; a complex composed of 6 molecules of Cas7; a complex composed of 1 molecule of Cas8, 1 molecule of Cas5, and 1 molecule of Cas6; a complex composed of 1 molecule of Cas3, 1 molecule of Cas8, 2 molecules of Cas11, 6 molecules of Cas7, 1 molecule of Cas5, and 1 molecule of Cas6; and combinations of one or more of these complexes. Further, it is more preferable to first form a complex of the complex with the crRNA described below before introducing it into the cells. Such a complex can be prepared by known methods or methods based on known methods, for example, by the method described in the aforementioned document I.

[0063] When the Cas protein group is introduced into cells in the form of a polynucleotide, the polynucleotide can be composed of DNA alone, or it can be composed of RNA, GNA, LNA, BNA, PNA, TNA, or a mixture thereof. Furthermore, it can be modified with components other than nucleic acids, such as glycans. Such polynucleotides can be prepared by known methods or methods based on known methods, for example, they can be synthesized artificially.

[0064] When introducing the Cas protein group into cells via an expression vector, the preferred expression vector is one that does not integrate into the host genome and can stably express the encoded protein. Various commonly used vectors can be used as the base vector for such an expression vector, and the appropriate vector can be selected based on the cells to be introduced and the introduction method. Examples of such base vectors include, for instance, phage vectors, plasmid vectors, viral vectors, retroviral vectors, chromosome vectors, episome vectors, and virus-derived vectors (bacterial plasmids, bacteriophages, yeast episomes, etc.), yeast chromosome elements and viruses (baculoviruses, papillomaviruses, vaccinia viruses, adenoviruses, fowlpox viruses, pseudorabies viruses, herpesviruses, lentiviruses, retroviruses, etc.), and vectors derived from two or more of these (plasmids, phage particles, etc.).

[0065] As the expression vector, it is preferable to further include sites for transcription initiation and termination, and to include a ribosome-binding site in the transcription region. Additionally, as the expression vector, it is also preferable to include one or more of the following: a promoter sequence suitable for the cell type to which it is to be introduced; a sequence for enhancing transcription from DNA (e.g., an enhancer sequence); and a sequence for stabilizing the transcribed RNA (e.g., a poly-A addition sequence). Furthermore, in the expression vector, the polynucleotides encoding each protein constituting the Cas protein group can be independently and appropriately codon-optimized.

[0066] When introducing multiple proteins constituting the Cas protein group into cells via expression vectors, multiple polynucleotides encoding these proteins can be contained within the same expression vector. The number of polynucleotides is not particularly limited as long as the CRISPR-Cas3 system functions correctly within the host cell in which the expression vector has been introduced. For example, it is possible to carry all polynucleotides encoding each protein constituting the Cas protein group into one (same) expression vector. Furthermore, it is also possible to carry a portion or one type of polynucleotide encoding each protein constituting the Cas protein group into separate expression vectors. For example, it is possible to carry polynucleotides encoding cascade reaction proteins into one (same) expression vector and polynucleotides encoding Cas3 into different expression vectors. From the viewpoint of expression efficiency, it is preferable to carry polynucleotides encoding each protein constituting the Cas protein group into six different expression vectors. Furthermore, for purposes such as adjusting expression levels, multiple polynucleotides encoding the same protein can be carried within the same expression vector. For example, it is possible to place the polynucleotide encoding Cas3 in two locations within one (same) expression vector. In addition, when the expression vector contains multiple polynucleotides that encode multiple proteins that constitute the Cas protein group, nucleotide sequences encoding amino acid sequences (such as 2A peptides) that are cleaved by intracellular proteases can be inserted between these multiple polynucleotides.

[0067] Such expression vectors can be prepared by known methods or by methods based on known methods. For example, in addition to the methods described in the instruction manual accompanying the vector preparation kit, various guidelines can be used, such as the method described in "Joseph Sambrook & David W. Russell, Molecular cloning: a laboratory manual 3rd Ed., New York: Cold Spring Harbor Laboratory Press, 2001".

[0068] [crRNA]

[0069] The crRNA (CRISPR RNA) constituting the CRISPR-Cas3 system has a nucleotide sequence complementary to the target sequence on the target DNA. The crRNA involved in this invention targets the enhancer region of the BCL11A gene as the cleavage region (cutting target region). In humans, the target DNA is located on chromosome 2 where the enhancer of the BCL11A gene is present, and the cutting target region is within the enhancer region of the BCL11A gene (preferably at least one of DHS+55, DHS+58, and DHS+62). The target sequence can be selected based on the target region, and more specifically, preferably within the range of 60, 485, 907 to 60, 503, 552 in human chromosome 2, and preferably within 5,000 bp before and after the target region (the enhancer region of the BCL11A gene, preferably at least one of DHS+55, DHS+58, and DHS+62). For example, more preferably, it includes 60, 495, 486 to 60, 495, 517 in human chromosome 2. In addition, the length of the target sequence is generally preferably 32 to 37 bases (Ming Li et al., Nucleic Acids Res., 2017, May 5; 45(8): p. 4642-4654).

[0070] In the CRISPR-Cas3 system of this invention, crRNA can be introduced into the hematopoietic stem cells in the form of RNA, in the form of a polynucleotide encoding the RNA, or in the form of an expression vector containing the polynucleotide.

[0071] As a CRISPR-Cas3 system, when the crRNA and the Cas protein group are introduced separately into the hematopoietic stem cells, which are eukaryotic cells, in the form of RNA, or in the form of a polynucleotide encoding the crRNA or an expression vector containing the polynucleotide, the preferred crRNA is the precursor crRNA described in International Publication No. 2018 / 225858. In this invention, the term "precursor crRNA" refers to intracellular crRNA (referred to as "mature crRNA" depending on the case) with added repeat sequences, typically having a structure of "leader sequence-repeat sequence-spacer sequence-repeat sequence (LRSR structure)" or "repeat sequence-spacer sequence-repeat sequence (RSR structure)". Typical schemes for the leader sequence and repeat sequence are described in International Publication No. 2018 / 225858. The precursor crRNA becomes mature crRNA when cleaved by a cascade reaction protein (e.g., Cas6 in types IA, B, D-E, and Cas5 in type IC).

[0072] Mature crRNA typically has a nucleotide sequence complementary to the target sequence and a 3' end repeat sequence (each unit contains approximately 10–70 bases, preferably 30–50 bases, and at least one hairpin structure). This hairpin structure interacts with one or more proteins (typically Cas6) constituting the Cas protein group to form a complex. Therefore, crRNA recognizes the target sequence and binds to the target DNA, inducing the Cas protein group capable of forming a complex with the crRNA to the vicinity of the cleavage target region. The induced Cas protein group, through the helicase activity of Cas3 exposing the single-stranded DNA, simultaneously cleaves the cleavage target region through DNA cleavage activity.

[0073] In the enhancer (target region) of the BCL11A gene, cleavage occurs using the CRISPR-Cas3 system at a position determined by the complementarity of the base pairs formed between the nucleotide sequence of the crRNA and the target sequence, and by the PAM sequence present on the 5' side of the complementary strand of the target sequence.

[0074] The PAM sequence of the CRISPR-Cas3 system involved in this invention is an "AAG" or similar base sequence (e.g., "AGG", "GAG", "TAC", "ATG", "TAG", etc.) adjacent to the 5' side of the target sequence.

[0075] The sequence of the crRNA (including mature crRNA and precursor RNA) can be appropriately designed according to the target sequence. As one of the preferred embodiments of the crRNA involved in this invention, for example, sequences containing nucleotide sequences complementary to the target sequence (60, 495, 486-60, 495, 517 of human chromosome 2) can be listed, and more specifically, sequences containing the nucleotide sequence described in sequence number 27 can be listed.

[0076] When the crRNA is introduced into a cell in the form of RNA or in the form of a polynucleotide encoding the RNA, these polynucleotides can be prepared by known methods or by methods based on known methods, for example, they can be synthesized artificially.

[0077] In addition, when the crRNA is introduced into cells in the form of an expression vector, the expression vector can be prepared by a known method or by a method according to a known method, for example, including its preferred embodiment, the expression vector described above for the Cas protein group can be used as its expression vector.

[0078] (Importing Process)

[0079] In the manufacturing method of the present invention, the CRISPR-Cas3 system is introduced into the hematopoietic stem cells, and the DNA cleavage activity of Cas3 induced to the cleavage target region on the enhancer of the BCL11A gene is induced through the binding of the target region to crRNA, thereby cleaving the enhancer of the BCL11A gene (preferably multiple cleavages) and thus destroying part or all of the enhancer of the BCL11A gene.

[0080] The method for introducing the CRISPR-Cas3 system into the hematopoietic stem cells is as described above. It can be a method of introducing the CRISPR-Cas3 system as a protein or RNA, or a method of introducing polynucleotides encoding them, or an expression vector containing the polynucleotides, to express them intracellularly. Therefore, as the CRISPR-Cas3 system, each protein constituting the Cas protein group can be introduced into the cell independently as a protein, or as RNA or DNA (polynucleotide) encoding the protein, to express it intracellularly, or as a vector expressing the protein (expression vector), to express it intracellularly. Furthermore, independently, the crRNA can be introduced into the cell as RNA (polynucleotide), or as DNA (polynucleotide) encoding the RNA, to express it intracellularly, or as a vector expressing the RNA (expression vector), to express it intracellularly.

[0081] When the CRISPR-Cas3 system is introduced into cells in the form of expression vectors to express it in the cells, for example, the vectors expressing each protein constituting the Cas protein group and the vector expressing the crRNA can be introduced into the cells separately. Alternatively, two or more vectors expressing them in combination can be introduced into the cells.

[0082] The method for introducing the CRISPR-Cas3 system involved in this invention into cells is not particularly limited. Methods for introducing proteins, DNA fragments, RNA fragments, and vectors into cells can be appropriately employed depending on the cell type. Examples of such methods include electroporation, microinjection, gene gun methods, calcium phosphate methods, polyethyleneimine (PEI) methods, liposome methods (liposome transfection), DEAE-dextrose glycoside methods, cationic lipid-mediated transfection, viral methods (adenovirus, lentivirus, adeno-associated virus, baculovirus, etc.), Agrobacterium methods, lithium acetate methods, protoplast methods, and heat shock methods (calcium chloride method, rubidium chloride method), etc. Such methods are described in numerous standard laboratory manuals, such as "Daviset et al., Basic methods in molecular biology, New York: Elsevier, 1986".

[0083] If the CRISPR-Cas3 system is introduced into a cell (including expression within the cell), the CRISPR-Cas3 system contacts the enhancer (cutting target region) of the BCL11A gene through the binding of the target region to crRNA. By utilizing the DNA cutting activity of the CRISPR-Cas3 system to cut the cutting target region, part or all of the enhancer of the BCL11A gene is destroyed (preferably destroyed).

[0084] (Uses of hematopoietic stem cells with disrupted BCL11A gene enhancers)

[0085] The manufacturing method of the present invention can also be manifested as a method for manufacturing hematopoietic stem cells that can differentiate into erythrocyte precursor cells with suppressed BCL11A gene expression, or a method for manufacturing hematopoietic stem cells that can differentiate into erythrocyte precursor cells with promoted γ-globin expression. That is, when the hematopoietic stem cells obtained by the manufacturing method of the present invention differentiate into erythrocyte precursor cells, the expression of their BCL11A gene is suppressed by the disruption of enhancers, thereby promoting the expression of γ-globin, which is normally suppressed by BCL11A in adults. Furthermore, in the present invention, "expression" includes either transcriptional expression (the process of DNA being transcribed into mRNA) or translational expression (the process of mRNA being translated into peptides, polypeptides, or proteins), and also includes the step of mRNA splicing, but when referred to as "expression of the BCL11A gene," it mainly refers to transcriptional expression.

[0086] Therefore, for example, if hematopoietic stem cells obtained by the manufacturing method of the present invention are administered to a subject, and the hematopoietic stem cells differentiate into erythrocyte precursor cells in the subject's organism, the expression level of γ-globin in the subject's erythrocyte precursor cells can be increased. The increase in the expression level of γ-globin, and the resulting increase in fetal hemoglobin (HbF), contributes to the prevention and treatment of abnormal hemoglobinopathies such as hemoglobinopathy, sickle cell disease (SCD), and β-thalassemia (Non-Patent Literature 1, Non-Patent Literature 2).

[0087] Therefore, the present invention also provides a method for increasing the expression level of γ-globin in the erythrocyte precursor cells of the subject, including the step of administering hematopoietic stem cells obtained by the manufacturing method of the present invention to the subject, and a method for preventing or treating abnormal hemoglobinemia, including the step of administering hematopoietic stem cells obtained by the manufacturing method of the present invention to the subject (in this specification, they are sometimes collectively referred to as "the treatment method of the present invention" as appropriate).

[0088] The objects described above can be animals, with humans being a preferred candidate. Methods of administration can include, for example, injection or transplantation.

[0089] <Reagent Kit>

[0090] The present invention provides a kit comprising the following (A) to (C) as a kit for use in the manufacturing method and treatment method of the present invention described above. (A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide. (B) cascade reaction protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide, and (C) crRNA targeting the enhancer of the BCL11A gene, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

[0091] The above-described (A) to (C) also include their preferred embodiments, as described in the CRISPR-Cas3 system of the manufacturing method of the present invention. They can each independently be in the form of a protein or RNA, a polynucleotide encoding the protein or RNA, or an expression vector containing the polynucleotide expressing the protein or RNA. These methods also include their preferred embodiments, as described above.

[0092] Furthermore, in the case where (C) is an expression vector, in order for the user to design crRNA based on the target sequence, the expression vector can be an expression vector containing (C') an insertion site of a polynucleotide encoding crRNA targeting an enhancer of the BCL11A gene. In this case, the insertion site can be a configuration where only a nucleotide sequence complementary to the target sequence is inserted into the polynucleotide encoding the crRNA, and the other polynucleotide encoding the crRNA is pre-included in the expression vector. Moreover, in the case where (C) is a polynucleotide encoding crRNA or an expression vector, the aforementioned precursor crRNA is preferred as the crRNA.

[0093] In the kit of the present invention, the combination of (A) to (C) is not particularly limited. It can be a composition that individually contains (A) to (C), or a composition that pre-mixes two or more of (A) to (C). It can also be a composition containing other components (solvents, etc.). For example, (B) can be a different type of each of the aforementioned cascade reaction proteins, or a complex or composition containing two or more cascade reaction proteins, a polynucleotide encoding two or more cascade reaction proteins, or an expression vector containing such a polynucleotide. Furthermore, (A) and (B) can be a complex or composition containing multiple proteins constituting the Cas protein group, a polynucleotide encoding two or more proteins, or an expression vector containing such a polynucleotide. Further, for example, when (A) and (B) are introduced into cells as proteins and (C) is introduced into cells as RNA, it can be a pre-formed complex of two or more of them; or when (A) to (C) are introduced into cells as expression vectors, it can be an expression vector containing two or more polynucleotides.

[0094] As a kit of the present invention, it may further include one or more additional reagents. Examples of such additional reagents include, but are not limited to, reagents for isolating hematopoietic stem cells, culture media for culturing hematopoietic stem cells, dilution buffers, recombination solutions, washing buffers, nucleic acid delivery reagents, protein delivery reagents, and control reagents. Furthermore, the kit may further include instructions for use for carrying out the manufacturing method and treatment method of the present invention.

[0095] Furthermore, as a kit for use in the treatment method of the present invention, it may further include, for example, pharmaceutical additives and apparatus for administering the manufactured hematopoietic stem cells as the target of the pharmaceutical composition. The pharmaceutical composition can be prepared by conventional methods. More specifically, hematopoietic stem cells manufactured by the manufacturing method of the present invention described above can be prepared, for example, by mixing with pharmaceutical additives according to the administration method. Examples of pharmaceutical additives include excipients, binders, disintegrants, lubricants, flow agents (anti-caking agents), colorants, plasticizers, solvents, co-solvents, emulsifiers, suspending agents (adhesives), thickeners, pH adjusters (acidifying agents, alkalinizing agents, buffers), humectants (compressants), antibacterial preservatives, chelating agents, medical water, stabilizers, preservatives, etc., and may be one or more of these.

[0096] The components of the kit can be stored either individually in a single container or in the same container. Each component can be stored in a single-use quantity or in multiple-use quantities within a single container. Components can be stored in the container either dry or dissolved in a suitable solvent (containing buffers, stabilizers, preservatives, etc.).

[0097] Example

[0098] The present invention will now be described in more detail with reference to the embodiments, but the present invention is not limited to the embodiments described below.

[0099] <Experimental Example 1> Disruption of the enhancer of the BCL11A gene in artificial hematopoietic stem cells using the CRISPR-Cas3 system

[0100] (1) Enhancers of the BCL11A gene

[0101] In this experimental case, the target region for cleavage within the enhancer of the BCL11A gene, which was the target of the disruption, is around chromosome 2 (chr2) of the human genome DNA at points 60, 495, and 269.

[0102] (2) CRISPR-Cas system

[0103] (CRISPR-Cas3 System (Example))

[0104] The CRISPR-Cas3 system used in this experiment was constructed. The amino acid sequence of the Cas3 protein and the nucleotide sequence encoding it are shown in Serial No. 1 and Serial No. 2, respectively. The Cas3 protein was prepared and purified according to the method described in International Publication No. 2022 / 186063.

[0105] The cascade reaction protein is composed of Cas8(Cse1)-Cas11(Cse2)-Cas7-Cas5-Cas6, and is a cascade reaction protein complex consisting of 1 Cas8 molecule, 2 Cas11 molecules, 6 Cas7 molecules, 1 Cas5 molecule, and 1 Cas6 molecule. The amino acid sequences of the Cas8, Cas11, Cas5, Cas6, and Cas7 proteins and the nucleotide sequences encoding them are shown in sequence numbers 3–12. Each cascade reaction protein and the cascade reaction protein complex were prepared and purified using the method described in reference I.

[0106] The crRNA targeting the enhancer of the BCL11A gene was synthesized using sequences 60, 495, 486–60, 495, 517 of chromosome 2 (chr2) of the human genomic DNA as target sequences. Table 1 below shows the target sequences (underlined) and PAM sequences (bold) of the crRNA. The crRNA was prepared and purified as mature crRNA using the method described in Reference I. Additionally, the Cas3 protein, the cascade reaction protein complex, and the crRNA were used to form a protein-RNA complex, which was then used as the CRISPR-Cas3 system.

[0107] (CRISPR-Cas9 system (comparative example))

[0108] As a comparison, a CRISPR-Cas9 system was constructed. The Cas9 protein was obtained from Integrated DNA Technologies. Furthermore, the gRNA was synthesized using sequences 60, 495, 264–60, 495, 283 of chromosome 2 (chr2) of the human genome as target sequences. Table 1 below shows the target sequences (underlined) and PAM sequences (bold) of the gRNA.

[0109]

[0110] (3) Cell Culture

[0111] Human hematopoietic stem cells (hHSCs) were used, derived from umbilical cord blood of healthy individuals obtained from StemExpress, Folsom CA. The hHSCs were first cultured in a medium supplemented with StemExpress... (TM)CD34+ Expansion Supplement (10×) was cultured in StemSpan SFEM (Veritas Corporation). Then, on day 4 from the start of culture, the CRISPR-Cas system constructed in (2) above was introduced by electroporation and cultured under the same conditions until day 7 from the start of culture (3 days after the introduction of the CRISPR-Cas system).

[0112] Next, on day 7 of culture, to induce hHSC differentiation into erythrocyte precursor cells, the culture medium was first replaced with the following composition: StemSpan SFEM (Veritas Corporation), 50 ng / mL SCF, 20 ng / mL EPO, 10 ng / mL IL-3, 10 ng / mL IGF-1, and 1 μM dexamethasone, and cultured for 5 days (day 12 from the start of culture). Then, on day 12 from the start of culture, the culture medium was replaced with the following composition: StemSpan SFEM (Veritas Corporation), 20 ng / mL EPO, 10 ng / mL IGF-1, 0.5 mg / mL human transferrin, and 2% BSA for differentiation induction, and cultured for another 6 days (day 18 from the start of culture) to obtain erythrocyte precursor cells.

[0113] (4) Electroporation

[0114] The electroporation in (3) above was performed on a cell population of 1 million hHSCs on day 4 from the start of culture using the electroporation method (Maxcyte-Atx, program: HSC-5). The CRISPR-Cas systems constructed in (2) above were configured as follows: (CRISPR-Cas3 system) Protein-RNA complex: Cas3 protein / cascade protein: Cas3 protein / (cascade protein complex + crRNA) (Cas3 protein : (cascade protein complex + crRNA) = 1:1 (molar ratio)): 2.5 μM, 5 μM, or 10 μM (CRISPR-Cas9 system) Cas9 protein: 1.5μM, 3μM, or 6μM gRNA: 6μM The cell population was introduced in this manner.

[0115] (5) Digital PCR

[0116] In (3) above, genomic DNA was recovered from cells on day 3 after introduction of each CRISPR-Cas system (day 7 from the start of culture), and the proportion of the target site deletion within the enhancer of the BCL11A gene was determined by digital PCR. In this experiment, region A (60, 495, 229–60, 495, 399: 171 bp on human chromosome 2, which is part of the GATA-binding region DHS+58) was used as the site (target site) for determining the presence or absence of genome editing. If region A was not amplified by PCR, it was determined that "the target site within the enhancer of the BCL11A gene is deleted". Genome extraction was performed using the DNeasy Blood & Tissue Kit (QIAGEN), and digital PCR was performed using QIAcuity (QIAGEN). In addition, as a comparison, unedited cells (NoEP) cultured under the same conditions without being introduced into any of the CRISPR-Cas systems constructed in (2) above were used. In addition, each defect rate was calculated using the region of genomic DNA not cleaved by the CRISPR-Cas system near the BCL11A gene as an endogenous control, and the defect rate (% defect) was calculated using the following formula: (1 - (amount of amplified product of region A introduced into cells by the CRISPR-Cas system / amount of amplified product of the endogenous control introduced into cells by the CRISPR-Cas system) / (amount of amplified product of region A of NoEP / amount of amplified product of the endogenous control of NoEP)) × 100. Table 2 below shows the nucleotide sequences of each primer and probe used in digital PCR. In Table 2, HEX / BHQ1 and FAM / BHQ1 represent the combination of reporter pigment and quencher pigment, respectively, and the amplified product amount is the fluorescence intensity derived from the reporter pigment.

[0117]

[0118] (6) Quantitative PCR (qPCR)

[0119] Following (3) above, mRNA was recovered from the cells on day 18 from the start of culture. After reverse transcription, the expression levels (mRNA levels) of γ-globin and β-globin were calculated by quantitative PCR. Furthermore, as a comparison, unedited cells (NoEP) cultured under the same conditions without any CRISPR-Cas system constructed in (2) above were used. Additionally, the expression levels were calculated using the ACTB gene as an endogenous control (normalized), and the relative expression level (ratio to β-globin) of β-globin relative to the expression level of γ-globin was determined. Table 3 below shows the nucleotide sequences of the primers used in the quantitative PCR.

[0120]

[0121] As a result of the digital PCR described in (5) above, the relationship between the amount of Cas3 protein / cascade reaction protein (Cas3 protein / (cascade reaction protein complex + crRNA)) introduced or the amount of Cas9 protein introduced and the defect rate (% defect) at the target site is shown in the figure. Figure 1 Furthermore, as a result of the quantitative PCR described in (6) above, the relationship between the amount of Cas3 protein / cascade reaction protein (Cas3 protein / (cascade reaction protein complex + crRNA)) introduced or the amount of Cas9 protein introduced and the relative expression level of γ-globin (relative to β-globin) is shown in the figure. Figure 2 The relationship between the defect rate and the relative expression level of γ-globin when the introduction amount of Cas3 protein / cascade reaction protein (Cas3 protein / (cascade reaction protein complex + crRNA)) is 10 μM and the introduction amount of Cas9 protein is 6 μM is shown in Table 4 below.

[0122]

[0123] like Figure 1 As shown, the proportion of target site deletions within the enhancer of the BCL11A gene using the CRISPR-Cas3 system was 16% (10 μM), compared to 35% (6 μM) using the CRISPR-Cas9 system. The CRISPR-Cas9 system demonstrated a higher target site deletion rate, indicating higher genome editing efficiency. On the other hand, as... Figure 2 As shown in Table 4, γ-globin expression increased 3.6-fold (10 μM) in unedited cells using the CRISPR-Cas3 system, compared to a 2.9-fold (6 μM) increase using the CRISPR-Cas9 system, demonstrating a significant increase in γ-globin expression when using the CRISPR-Cas3 system. Furthermore, as shown in Table 4, the relative expression level of γ-globin relative to the defect rate of the target site within the enhancer of the BCL11A gene ((b) / (a)) increased approximately 3-fold when using the CRISPR-Cas3 system.

[0124] Thus, although the genome editing efficiency (the proportion of the target site missing) is high when using the CRISPR-Cas9 system, the increase in γ-globin expression is far greater when using the CRISPR-Cas3 system, confirming that γ-globin expression can be induced with high efficiency. This confirms that the method using the CRISPR-Cas3 system of the present invention can specifically and efficiently disrupt the enhancer of the BCL11A gene in hematopoietic stem cells, resulting in sufficient suppression of BCL11A gene expression in differentiated hematopoietic cell line precursor cells.

[0125] <Experimental Example 2> Analysis of Genome Defect Maps Using the CRISPR-Cas System

[0126] (1) Analysis of genomic defect maps using the CRISPR-Cas9 system

[0127] Using a sample from the genomic DNA recovered on day 3 after the CRISPR-Cas9 system was introduced into the cells in <Example 1> (day 7 from the start of culture), which was determined by digital PCR in <Example 1> (5) to have a "deletion of the target site within the enhancer of the BCL11A gene", as a template, the region B (60, 494, 994–60, 495, 500: 507 bp) containing the DHS+58 region, which is the GATA binding region, was amplified by PCR. Then, the amplified fragment was sequenced using sequencing primers (60, 495, 481–60, 495, 500: 20 bp) of human chromosome 2. The obtained Senggelin sequencing waveform data (.ab1) was analyzed using SYNTEGO ICE Analysis (Synthego) to obtain a genomic defect map. In addition, as a comparison, genomic DNA recovered from unedited cells (NoEP) that were cultured under the same conditions without any CRISPR-Cas system was analyzed in the same manner.

[0128] (2) Analysis of genomic defect maps using the CRISPR-Cas3 system

[0129] (2-1) Preparation of NGS libraries

[0130] An Oligo Panel (manufactured by Twist Bioscience) was designed to concentrate the region containing DHS+58, which is the GATA-binding region, for region C (60, 493, 189–60, 543, 189 bp of human chromosome 2). From genomic DNA recovered on day 3 after the CRISPR-Cas3 system was introduced into cells in <Experiment 1> (day 7 from the start of culture), samples that were determined by digital PCR in <Experiment 1> (5) to have a "deletion of the target site within the enhancer of the BCL11A gene" were used. Region C was concentrated using the Twist Library Prep EF Kit 2.0, Twist UMI Adapter System TruSeqCompatible, Twist Std Hyb and Wash Kit, Twist Dry Down Beads, Twist UniversalBlockers, and the aforementioned Oligo Panel (manufactured by Twist BioScience). A library for analyzing the defect map of the enhancer of the BCL11A gene was constructed. The constructed NGS libraries were analyzed using Nova-seq (manufactured by Illumina, Inc.). Furthermore, as a comparison, libraries were prepared using genomic DNA recovered from unedited cells (NoEP) cultured under the same conditions without any CRISPR-Cas system.

[0131] (2-2) Analysis of genomic defect maps using the CRISPR-Cas3 system

[0132] The raw NGS data obtained in (2-1) above underwent quality checks and genome mapping to extract split reads (referring to structural variations). The extracted split reads were then analyzed using a CRISPR-Cas3 system to determine the length of genomic defects in the region containing DHS+58, which is a GATA-binding region.

[0133] The results of the analyses in (1) and (2) above show that only a few bases (1-20 bases) of deletion were identified in the genome defect map using the CRISPR-Cas9 system, while a wide range of deletions (49-20, 699 bases) spanning several k bases were identified in the genome defect map using the CRISPR-Cas3 system. That is, there are multiple GATA-binding regions (DHS+55, DHS+58, DHS+62) in the enhancer region of the BCL11A gene, but the range that can be deleted by a single system in the CRISPR-Cas9 system is limited to a part of a single GATA-binding region. In contrast, according to the method using the CRISPR-Cas3 system of the present invention, multiple GATA-binding regions can be deleted by a single system, which can more reliably disrupt the enhancer of the BCL11A gene.

[0134] <Experimental Example 3> Off-target analysis generated by the CRISPR-Cas3 system

[0135] (1) Extraction of off-target candidates for crRNA

[0136] Bioinformatics analysis was conducted to extract gene sequences and proto-oncogene sequences with high homology to the base sequences of crRNAs on chromosome 2 (chr2) of the human genome DNA prepared in <Experimental Example 1> (2) above, after removing the repeats from the top 50 loci, and then using these sequences as off-target candidate loci.

[0137] (2) Preparation of NGS libraries

[0138] Oligo Panels were designed for the 92 off-target candidate loci extracted in (1) above, and were concentrated in the same manner as in <Experimental Example 2> (2-1). NGS libraries for off-target analysis were constructed from samples of genomic DNA recovered on day 3 after the CRISPR-Cas3 system was introduced into cells in <Experimental Example 1> (day 7 from the start of culture) that were identified by digital PCR in <Experimental Example 1> (5) as having a "deficit in the target site within the enhancer of the BCL11A gene". Furthermore, during library construction, a 1kb deletion fragment of the human endogenous genome was added as a control for genome cleavage detection at a ratio of 1% relative to the amount of genomic DNA in each sample. The constructed NGS libraries were analyzed using a Nova-seq 6000 (manufactured by Illumina, Inc.).

[0139] (3) Off-target bioinformatics analysis generated by the CRISPR-Cas3 system

[0140] The raw NGS data obtained in (2) above were analyzed using Quality Check and genome mapping to extract split reads from off-target candidate loci. As a comparison, genomic DNA recovered from unedited cells (NoEP) cultured under the same conditions without any CRISPR-Cas system was also analyzed, and split reads were extracted and signal counts were compared. The number of reads detected for the missing human endogenous genomic regions added to each sample was used as an indicator of 1% genome editing efficiency to evaluate the cleavage of each off-target candidate locus.

[0141] The results of the above analysis show that no cleavage was detected at any off-target candidate loci, confirming that the induction of off-target cleavage was adequately suppressed by the disruption of the enhancer of the BCL11A gene using the CRISPR-Cas3 system of the present invention.

[0142] Industry availability

[0143] As described above, according to the present invention, a method for manufacturing hematopoietic stem cells capable of efficiently differentiating into hematopoietic progenitor cells whose expression of the BCL11A gene is specifically and sufficiently suppressed can be provided; and a kit suitable for use in the manufacturing method.

[0144] If hematopoietic stem cells with disrupted BCL11A gene enhancers are differentiated into erythrocyte precursor cells, then in these erythrocyte precursor cells, BCL11A gene expression is suppressed, the expression inhibition caused by BCL11A is lifted, and γ-globin expression is induced, thus increasing the expression level of γ-globin. Therefore, this invention is useful for the prevention or treatment of disease groups related to γ-globin expression levels. Furthermore, this method using a CRISPR-Cas3 system avoids off-target effects, and is therefore also useful from a safety point of view.

Claims

1. A method for manufacturing hematopoietic stem cells in which some or all of the enhancer of the BCL11A gene is destroyed. This method includes the step of introducing a CRISPR-Cas3 system comprising (A) to (C) the following into hematopoietic stem cells. (A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide. (B) cascade reaction proteins, polynucleotides encoding those proteins, or expression vectors containing those polynucleotides, and (C) crRNA targeting the enhancer of the BCL11A gene, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

2. The method according to claim 1, wherein the nucleotide sequence of the crRNA comprises the nucleotide sequence described in sequence number 27.

3. According to the method of claim 1, the hematopoietic stem cells produced can differentiate into erythrocyte precursor cells with increased expression of γ-globin.

4. A method for increasing the expression level of γ-globin in the erythrocyte precursor cells of a subject, comprising administering hematopoietic stem cells obtained by the method of claim 1 to the subject.

5. A method for the prevention or treatment of abnormal hemoglobinemia, comprising the step of administering hematopoietic stem cells obtained by the method of claim 1 to a subject.

6. A kit for use in the method according to any one of claims 1 to 5. The kit contains the following (A) to (C): (A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide. (B) cascade reaction protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide, and (C) crRNA targeting the enhancer of the BCL11A gene, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

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