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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

• the present invention relates to a method for producing hematopoietic stem cells in which the enhancer of the BCL11A gene has been partially or completely destroyed, and a kit for use in said method.
• BCL11A B-cell lymphoma 11A
• BCL11A B-cell lymphoma 11A
• BCL11A suppresses the expression of ⁇ -globin in adult erythroid progenitor cells and functions as a major transcription factor for the switch from ⁇ -globin to ⁇ -globin expression during the fetal-to-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 ⁇ -globins and two ⁇ -globins.
• Non-Patent Document 1 Elenoe C. Smith et al., Blood., 2016, Nov. 10; 128(19): p. 2338-2342 (Non-Patent Document 2)).
• Patent Document 1 (WO 2014/085593) describes a method for producing precursor cells in which expression of the BCL11A gene is suppressed, and as a method for suppressing the expression of the BCL11A gene, a method of introducing a mutation into hematopoietic stem cells within 60,716,189 to 60,728,612 on the same chromosome 2 as the BCL11A gene is mentioned.
• Patent Document 2 (WO 2019/113149) describes hematopoietic stem cells in which a mutation has been introduced into +58 DHS (DNase I hypersensitive site) in the enhancer region of the BCL11A gene. Methods for introducing the mutation include cleaving the site with a site-specific nuclease such as zinc finger nuclease, CRISPR-Cas9 system, or TALEN to introduce the mutation.
• a site-specific nuclease such as zinc finger nuclease, CRISPR-Cas9 system, or TALEN to introduce the mutation.
• the present inventors investigated the suppression of expression of the BCL11A gene, they found that, for example, even if a mutation is introduced into the enhancer region of the BCL11A gene in hematopoietic stem cells using the above-mentioned CRISPR-Cas9 system, the suppression of BCL11A gene expression in erythroid progenitor cells differentiated from these cells is not sufficient, and as a result, the suppression of ⁇ -globin expression by BCL11A is not completely released, and the amount of ⁇ -globin expression does not increase sufficiently.
• the CRISPR-Cas9 system has the problem that the target sequence of the guide RNA is short (usually, preferably about 20 bases), and therefore, there are problems such as insufficient specificity and off-target occurrence.
• the present invention was made in consideration of the problems with the above-mentioned conventional technology, and aims to provide a method for producing hematopoietic stem cells capable of differentiating into blood cell progenitor cells in which expression of the BCL11A gene is specifically and sufficiently suppressed with high efficiency, and a kit that can be suitably used for the production method.
• the present inventors have conducted extensive research to achieve the above object, and have attempted to disrupt the enhancer of the BCL11A gene in hematopoietic stem cells using the CRISPR-Cas3 system.
• the genome editing efficiency (deletion rate) at the target site (edited site) in the enhancer was lower than when the conventional CRISPR-Cas9 system was used, the expression level of ⁇ -globin was significantly higher in erythroid progenitor cells differentiated from hematopoietic stem cells in which the enhancer was destroyed by the CRISPR-Cas3 system.
• the inventors discovered that by using the CRISPR-Cas3 system particularly for disrupting the enhancer of the BCL11A gene, the enhancer of the BCL11A gene can be specifically and highly efficiently disrupted in hematopoietic stem cells, and as a result, expression of the BCL11A gene is sufficiently suppressed in blood cell progenitor cells differentiated from the hematopoietic stem cells, and therefore the CRISPR-Cas3 system is particularly effective for suppressing expression of the BCL11A gene, leading to the completion of the present invention.
• a method for producing hematopoietic stem cells in which a part or all of the enhancer of the BCL11A gene has been disrupted The following (A) to (C): (A) a Cas3 protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; (B) a Cascade protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; and (C) a CRISPR-Cas3 system comprising a crRNA targeting an enhancer of the BCL11A gene, a polynucleotide encoding the crRNA, or an expression vector comprising the polynucleotide, into a hematopoietic stem cell.
• [5] A method for preventing or treating hemoglobinopathy, comprising a step of administering to a subject hematopoietic stem cells obtained by the method according to any one of [1] to [3].
• [6] A kit for use in the method according to any one of [1] to [5], The following (A) to (C): (A) a Cas3 protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; (B) a cascade protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; and (C) a crRNA targeting an enhancer of the BCL11A gene, a polynucleotide encoding the crRNA, or an expression vector comprising the polynucleotide.
• the present invention makes it possible to provide a method for producing hematopoietic stem cells capable of differentiating into blood cell progenitor cells in which expression of the BCL11A gene is specifically and sufficiently suppressed with high efficiency, and a kit that can be used for the method.
• the present invention is useful for preventing or treating a group of diseases in which the amount of ⁇ -globin expression is involved. Furthermore, such a method using the CRISPR-Cas3 system is useful from the standpoint of safety as it can avoid off-targets.
• 1 is a graph showing the relationship between the amount of Cas3 protein/Cascade protein (Cas3 protein/(Cascade protein complex + crRNA)) or the amount of Cas9 protein introduced and the deletion rate (% Deletion) of the target site as a result of digital PCR in Test Example 1.
• 1 is a graph showing the relationship between the amount of Cas3 protein/Cascade protein (Cas3 protein/(Cascade protein complex + crRNA)) introduced or the amount of Cas9 protein introduced and the relative expression level of ⁇ -globin (Ratio to ⁇ -globin) as a result of quantitative PCR in Test Example 1.
• the present invention provides a method for producing hematopoietic stem cells in which a part or all of the enhancer of the BCL11A gene has been disrupted,
• BCL11A gene refers to a gene (human BCL11A gene reference number (Gene ID): 53335) that encodes BCL11A (B-cell lymphoma 11A), a C2H2-type zinc finger protein and a subunit of the BAF chromatin remodeling complex.
• BCL11A is also referred to as EVI9, CTIP1, DILOS, ZNF856, HBFQTL5, BCL11A-L, BCL11A-S, BCL11a-M, or BCL11A-XL.
• BCL11A is highly expressed in hematopoietic cells and is known to contribute to the switch in expression from ⁇ -globin to ⁇ -globin during the transition from fetal hemoglobin (HbF) to adult hemoglobin (HbA).
• ⁇ -globin is a protein encoded by the HBG1 and HBG2 genes in humans.
• the "BCL11A gene enhancer” is a region to which a transcription factor capable of promoting transcription of the BCL11A gene binds, and is known to exist in multiple locations in the introns of the BCL11A gene or upstream or downstream of the BCL11A gene in human genomic DNA.
• the BCL11A gene enhancer of the present invention is preferably an enhancer located within an intron of the BCL11A gene. More preferably, the BCL11A gene enhancer region of the present invention is the region from 60,489,054 to 60,501,476 (12,423 bp) on human chromosome 2.
• examples of the transcription factors that bind to the enhancer of the BCL11A gene include GATA, STAT1, ELF1, KLF1, RREB1, TAL1, etc.
• GATA is a protein belonging to the GATA transcription factor family, and is also called GF1, GF-1, NFE1, XLTT, ERYF1, NF-E1, XLANP, XLTDA, and GATA-1.
• regions to which GATA binds include DNase I hypersensitive sites (DHS), such as DHS+55, DHS+58, and DHS+62, which will be described later.
• DHS DNase I hypersensitive sites
• Hematopoietic stem cells are cells capable of self-renewal and differentiation into precursor cells of blood cells (sometimes referred to as "blood cell precursor cells” in this specification).
• blood cells include white blood cells, red blood cells, platelets, mast cells, dendritic cells, eosinophils, neutrophils, monocytes, macrophages, granulocytes, T cells, B cells, and NK cells
• the blood cell precursor cells include white blood cell precursor cells, red blood cell precursor cells, megakaryocyte precursor cells, granulocyte precursor cells, pro-B cells, pro-T cells, and pro-NK cells.
• the hematopoietic stem cells used in the production method of the present invention are differentiated into red blood cell precursor cells, which are cells that produce gamma-globin, from the viewpoint that the expression level of gamma-globin can be improved as a result of, for example, the treatment method of the present invention described below.
• “differentiation of hematopoietic stem cells into blood cell precursor cells (preferably erythroid precursor cells)" means not only that the hematopoietic stem cells themselves differentiate into the precursor cells, but also that one or more hematopoietic stem cells produced by the self-replication of the hematopoietic stem cells each differentiate into the precursor cells to produce one or more precursor cells.
• Hematopoietic stem cells exhibit many known phenotypes, such as CD34+ and CD38-.
• Hematopoietic stem cells used in the production method of the present invention can be isolated from peripheral blood, bone marrow, umbilical cord, placenta, etc., by conventionally known methods or methods similar thereto, using the above phenotypes as indicators.
• the hematopoietic stem cells according to the present invention can be derived from animals, preferably humans or non-human mammals.
• the non-human mammals include even-toed animals such as cows, wild boars, pigs, sheep, and goats; odd-toed animals such as horses; rodents such as mice, rats, guinea pigs, hamsters, and squirrels; and rabbits, dogs, cats, and ferrets.
• the hematopoietic stem cells produced by the method of the present invention are administered to a subject, it is preferable that the hematopoietic stem cells used in the method of the present invention are the same animal as the subject.
• the hematopoietic stem cells used in the method of the present invention may be cells collected from the peripheral blood, bone marrow, umbilical cord, placenta, etc. of these animals, as well as cultured cells derived from cells constituting an animal or cells constituting an organ or tissue extracted from an animal (e.g., stem cells such as induced pluripotent stem (iPS) cells).
• stem cells such as induced pluripotent stem (iPS) cells.
• the manufacturing method of the present invention produces hematopoietic stem cells in which the enhancer of the BCL11A gene has been destroyed.
• the term "destruction of the enhancer” refers not only to the destruction of the entire enhancer, but also to the destruction of a portion of the enhancer.
• "Hematopoietic stem cells in which a part or all of the enhancer of the BCL11A gene has been destroyed" obtained by the manufacturing method of the present invention means that the enhancer function of the BCL11A gene is suppressed by the deletion or replacement (preferably deletion) of a part or all of the enhancer of the BCL11A gene. More specifically, “suppression of the enhancer function of the BCL11A gene” refers to, for example, the absence of binding of at least one of the transcription factors, preferably at least one or more GATAs.
• the site to be deleted or replaced in the enhancer of the BCL11A gene is preferably at least part or all of the region from 60,489,054 to 60,501,476 in human chromosome 2 as described above.
• the site to be deleted or replaced (preferably deleted) in the enhancer of the BCL11A gene is preferably a part or all of the GATA binding region, more specifically, at least one of the following three regions: DHS+55 (60,498,289-60,498,552 (264 bp) in human chromosome 2), DHS+58 (60,495,103-60,495,330 (228 bp) in human chromosome 2), and DHS+62 (60,490,907-60,491,050 (144 bp) in human chromosome 2), more preferably a part (e.g., 75% or more) or all of two or even all of these three regions, and even more preferably all of them.
• the destruction of part or all of the enhancer of the BCL11A gene can be confirmed based on conventionally known methods, for example, by PCR (digital PCR, real-time PCR, end-point PCR, etc.), sequencing, Southern blotting, next-generation sequencing, etc.
• PCR digital PCR, real-time PCR, end-point PCR, etc.
• sequencing Southern blotting, next-generation sequencing, etc.
• CRISPR-Cas3 system CRISPR-Cas3 system
• CRISPR-Cas systems are divided into class 1, in which a target region (region to be cleaved) is cleaved by a complex of multiple proteins, and class 2, in which a target region is cleaved by a single protein.
• the CRISPR-Cas9 system, CRISPR-Cas12 (Cpf1) system, CRISPR-Cas13 system, and the like that have been developed as genome editing tools so far are all classified into class 2, but the CRISPR-Cas3 system according to the present invention belongs to class 1, type I.
• the "CRISPR-Cas3 system” includes the following (A) to (C): (A) a Cas3 protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; (B) a cascade protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; and (C) a crRNA targeting an enhancer of the BCL11A gene, a polynucleotide encoding the crRNA, or an expression vector comprising the polynucleotide.
• the Cas3 protein constituting the CRISPR-Cas3 system has DNA cleavage activity and helicase activity.
• the Cas3 protein can cleave the target DNA at multiple sites by cooperating with the cascade protein and crRNA constituting the CRISPR-Cas3 system.
• Cas3 when simply described as “Cas3", it means “Cas3 protein”.
• the Cas3 protein and the cascade protein are sometimes collectively referred to as "Cas protein group”.
• the CRISPR-Cas3 system of the present invention includes all seven subtypes of Type I.
• Type I-E systems which are common among Type I CRISPR-Cas3 systems, include Cas3, Cas8 (Cse1), Cas11 (Cse2), Cas5, Cas6, and Cas7 as the Cas protein group, and the Cas protein group cooperates with crRNA to cleave the target DNA.
• the Cas protein group typically forms a complex (cascade complex) of one molecule of Cas3, one molecule of Cas8, two molecules of Cas11, six molecules of Cas7, one molecule of Cas5, and one molecule of Cas6.
• the Cas protein group includes Cas3-HD, Cas3-HEL, Cas5, Cas6, Cas7, Cas8, and Cas11; in the type I-B system, the Cas protein group includes Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11; in the type I-C system, the Cas protein group includes Cas3, Cas5, Cas7, Cas8, and Cas11.
• the Cas protein group includes Cas3, Cas5, Cas6, Cas7, Cas10, and Cas11; in the type I-F system, the Cas protein group includes Cas2-3, Cas5, Cas6, Cas7, and Cas8; in the type I-G system, the Cas protein group includes Csb2 (Cas6-like), Cas7, Cas8g, Cas3, and Cas11.
• the CRISPR-Cas3 system of the present invention also includes a system that does not contain Cas11.
• the origin of the Cas protein group according to the present invention is not particularly limited, but is preferably derived from E. coli from the viewpoint of suitability for genome editing in animal cells.
• the amino acid sequence of each protein constituting the Cas protein group according to the present invention can be obtained, for example, from a public database (Genbank, etc.), and one preferred embodiment of the configuration of the Cas protein group is the following configuration as a typical sequence of the E. coli-derived type I-E system:
• Cas3 a protein comprising the amino acid sequence shown in SEQ ID NO: 1.
• Cas8 (Cse1) a protein comprising the amino acid sequence shown in SEQ ID NO: 3.
• Cas11 (Cse2) a protein comprising the amino acid sequence shown in SEQ ID NO: 5.
• Cas5 a protein comprising the amino acid sequence shown in SEQ ID NO: 7.
• Cas6 a protein comprising the amino acid sequence shown in SEQ ID NO: 9.
• Cas7 a protein comprising the amino acid sequence shown in
• the Cas protein group of the present invention also includes mutants that have occurred in nature or have been artificially modified. Therefore, another preferred embodiment of the configuration of the Cas protein group of the present invention is a configuration in which each protein is a protein containing an amino acid sequence that has high identity with each amino acid sequence of the typical Cas protein group, and Cas3 has DNA cleavage activity (preferably, the Cas protein group also has the ability to form a cascade complex).
• 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).
• Amino acid sequence identity can be determined using, for example, BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., using default, i.e., default parameters).
• each protein contains an amino acid sequence in which one or more amino acids are substituted, deleted, added, and/or inserted in each amino acid sequence of the above typical Cas protein group, and Cas3 has DNA cleavage activity (and preferably the Cas protein group also has the ability to form a cascade complex).
• “multiple” usually means 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).
• the phrase "having DNA cleavage activity" refers to the ability to cleave a DNA strand at at least one site.
• the fact that the Cas3 has DNA cleavage activity and the ability to form a cascade complex can be confirmed, for example, by showing DNA cleavage activity equal to or greater than that of the typical Cas protein group described above (e.g., 50% or more, 80% or more).
• Functional molecules may be added to each protein constituting the Cas protein group as necessary.
• the functional molecules include nuclear transport signals for promoting transport into the nucleus of eukaryotic cells (e.g., those described in Wu J et al., 2009, Biophysical journal, Vol. 96 (Issue 9), p.
• tags for facilitating purification e.g., HN tag, His tag, FLAG tag, glutathione-S-transferase (GST) tag
• reporter proteins for facilitating detection e.g., fluorescent proteins such as green fluorescent protein (GFP), chemiluminescent proteins such as luciferase
• GFP green fluorescent protein
• chemiluminescent proteins such as luciferase
• the amount of each protein can be adjusted appropriately, which is preferable from the standpoint of handling and the fact that DNA is not introduced into the genome.
• Each protein constituting such a Cas protein group can be prepared by a known method or a method similar thereto.
• a method for preparing a Cas3 protein can be described in International Publication No. 2022/186063.
• the cascade protein can be prepared by, for example, a 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 "Document I").
• the Cas protein group When the Cas protein group is introduced into a cell in the form of a protein, it is preferable to form a complex consisting of two or more of the proteins constituting the Cas protein group in advance and introduce it into a cell, from the viewpoint of cleavage efficiency in the cell.
• the polynucleotide may be composed of only DNA, or may be composed of RNA, GNA, LNA, BNA, PNA, TNA, etc., or a mixture of these. It may also be modified with a component other than nucleic acid, such as a sugar chain.
• Such polynucleotides can be prepared by known methods or methods similar thereto, and can be artificially synthesized, for example.
• the expression vector is capable of stably expressing the encoded protein without being incorporated into the host genome.
• the base vector for such an expression vector various commonly used vectors can be used, and can be appropriately selected depending on the cell to be introduced and the introduction method.
• base vectors examples include phage vectors, plasmid vectors, virus vectors, retrovirus vectors, chromosomal vectors, episomal vectors and virus-derived vectors (bacterial plasmids, bacteriophages, yeast episomes, etc.), yeast chromosomal elements and viruses (baculoviruses, papovaviruses, vaccinia viruses, adenoviruses, avian poxviruses, pseudorabies viruses, herpes viruses, lentiviruses, retroviruses, etc.), and vectors derived from a combination of two or more of these (cosmids, phagemids, etc.).
• phage vectors examples include phage vectors, plasmid vectors, virus vectors, retrovirus vectors, chromosomal vectors, episomal vectors and virus-derived vectors (bacterial plasmids, bacteriophages, yeast episomes, etc.), yeast chromosomal elements and viruses (bacul
• the expression vector preferably further includes sites for transcription initiation and termination, and includes a ribosome binding site in the transcription region.
• the expression vector also preferably includes one or more of the following: a promoter sequence according to the type of cell 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 polyA addition sequence).
• a promoter sequence according to the type of cell to be introduced
• a sequence for enhancing transcription from DNA e.g., an enhancer sequence
• a sequence for stabilizing the transcribed RNA e.g., a polyA addition sequence
• the polynucleotides encoding each protein constituting the Cas protein group may each be independently and appropriately codon-optimized.
• the same expression vector may contain multiple polynucleotides encoding each of the multiple proteins, and the number of polynucleotides is not particularly limited as long as the function of the CRISPR-Cas3 system can be exerted in the host cell into which the expression vector is introduced.
• the number of polynucleotides is not particularly limited as long as the function of the CRISPR-Cas3 system can be exerted in the host cell into which the expression vector is introduced.
• a polynucleotide encoding a cascade protein it is possible to design a polynucleotide encoding a cascade protein to be mounted on one type of (same) expression vector, and a polynucleotide encoding Cas3 to be mounted on another expression vector. From the viewpoint of expression efficiency, etc., a method in which polynucleotides encoding each protein constituting the Cas protein group are mounted on six different expression vectors is preferable. In addition, for the purpose of regulating the expression amount, etc., multiple polynucleotides encoding the same protein may be mounted on the same expression vector. For example, it is possible to design a polynucleotide encoding Cas3 to be placed at two locations within one (same) type of expression vector.
• an expression vector contains multiple polynucleotides that respectively encode multiple proteins that constitute the Cas protein group
• a nucleotide sequence encoding an amino acid sequence (such as 2A peptide) that is cleaved by a protease in the cell may be inserted between the multiple polynucleotides.
• Such expression vectors can be prepared by known methods or methods similar thereto.
• the method described in various manuals for example, the method described in "Joseph Sambrook & David W. Russell, Molecular cloning: a laboratory manual 3rd Ed., New York: Cold Spring Harbor Laboratory Press, 2001” can be adopted.
• the crRNA (CRISPR RNA) constituting the CRISPR-Cas3 system has a nucleotide sequence complementary to a target sequence on a target DNA.
• the crRNA according to the present invention targets the enhancer region of the BCL11A gene as a target region for cleavage (a region to be cleaved), and in humans, the target DNA is chromosome 2 where the enhancer of the BCL11A gene is present, and the region to be cleaved is within the enhancer region of the BCL11A gene (preferably within at least one of the regions DHS+55, DHS+58, and DHS+62).
• the target sequence can be selected according to the region to be cleaved, and more specifically, in human chromosome 2, it is preferably within the range of 60,485,907 to 60,503,552, and preferably within 5,000 bp before and after the region to be cleaved (the enhancer region of the BCL11A gene, preferably at least one of DHS+55, DHS+58, and DHS+62), and more preferably includes 60,495,486 to 60,495,517 in human chromosome 2.
• 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).
• the crRNA When the crRNA is introduced into the hematopoietic stem cell, which is a eukaryotic cell, separately from the Cas protein group in the form of RNA, or in the form of a polynucleotide encoding the crRNA or an expression vector containing the same, as the CRISPR-Cas3 system, the crRNA is preferably pre-crRNA described in WO 2018/225858.
• the "pre-crRNA” has a structure in which a repeat sequence or the like is added to the crRNA (sometimes referred to as “mature crRNA") that functions as a component of the CRISPR-Cas3 system in the cell, and typically has a structure of "leader sequence-repeat sequence-spacer sequence-repeat sequence (LRSR structure)” or “repeat sequence-spacer sequence-repeat sequence (RSR structure)". Typical aspects of the leader sequence and repeat sequence are as described in WO 2018/225858.
• Pre-crRNA becomes mature crRNA when cleaved by Cascade proteins (e.g., Cas6 in types I-A, B, D-E, and Cas5 in type I-C).
• CrRNA typically has a nucleotide sequence complementary to the target sequence and a repeating sequence at the 3' end (each unit is about 10-70 bases, preferably 30-50 bases, and includes at least one hairpin structure), and this hairpin structure interacts with one or more proteins (typically Cas6) that make up the Cas protein group to form a complex. Therefore, crRNA recognizes the target sequence and binds to the target DNA, and guides the Cas protein group that forms a complex with the crRNA to the vicinity of the region to be cleaved, and the induced Cas protein group exposes single-stranded DNA by the helicase activity of Cas3 and cleaves the region to be cleaved by its DNA cleavage activity.
• Cas6 proteins
• cuts by the CRISPR-Cas3 system occur at a position determined by both the complementarity of base pairing between the nucleotide sequence of the crRNA and the target sequence, and the PAM sequence present on the 5' side of the complementary strand of the target sequence.
• the PAM sequence for the CRISPR-Cas3 system of the present invention is "AAG” or a base sequence similar thereto (e.g., "AGG,” “GAG,” “TAC,” “ATG,” “TAG,” etc.) adjacent to the 5' side of the target sequence.
• the sequence of the crRNA may be appropriately designed depending on the target sequence.
• One preferred embodiment of the crRNA according to the present invention is, for example, a sequence that includes a nucleotide sequence complementary to the target sequence (60,495,486 to 60,495,517 of human chromosome 2), and more specifically, a sequence that includes the nucleotide sequence set forth in SEQ ID NO:27.
• these polynucleotides can be prepared by known methods or methods equivalent thereto, for example, they can be artificially synthesized.
• the expression vector can be prepared by a known method or a method equivalent thereto, and for example, the expression vectors described in the above Cas protein group, including their preferred embodiments, can be appropriately adopted.
• the CRISPR-Cas3 system is introduced into the hematopoietic stem cells, and the DNA cleavage activity of Cas3 induced near the cleavage target region on the enhancer of the BCL11A gene via binding between the target region and crRNA cleaves the enhancer of the BCL11A gene (preferably cleaves at multiple sites), thereby destroying part or all of the enhancer of the BCL11A gene.
• the method of introducing the CRISPR-Cas3 system into the hematopoietic stem cells may be, as described above, a method of introducing the CRISPR-Cas3 system in the form of a protein or RNA, or a method of introducing a polynucleotide encoding the CRISPR-Cas3 system or an expression vector containing the polynucleotide to express the system in the cells.
• each protein constituting the Cas protein group may be independently introduced into a cell in the form of a protein, or in the form of an RNA or DNA (polynucleotide) encoding the protein to express the protein in the cells, or in the form of a vector (expression vector) expressing the protein to express the protein in the cells.
• the crRNA may be introduced into a cell in the form of an RNA (polynucleotide), or in the form of a DNA (polynucleotide) encoding the RNA to express the RNA in the cells, or in the form of a vector (expression vector) expressing the RNA to express the RNA in the cells.
• a vector expressing each protein constituting the Cas protein group and a vector expressing the crRNA may be introduced into the cell separately, or a vector expressing a combination of two or more of these may be introduced into the cell.
• the method of introducing the CRISPR-Cas3 system of the present invention into cells is not particularly limited, and known methods for introducing proteins, DNA fragments, RNA fragments, and vectors into cells can be appropriately adopted depending on the type of cells, etc.
• Such methods include, for example, electroporation, microinjection, particle gun, calcium phosphate, polyethyleneimine (PEI), liposome, DEAE-dextran, cationic lipid-mediated transfection, viruses (adenovirus, lentivirus, adeno-associated virus, baculovirus, etc.), Agrobacterium, lithium acetate, spheroplast, heat shock (calcium chloride, rubidium chloride), etc.
• PKI polyethyleneimine
• cationic lipid-mediated transfection viruses
• viruses adenovirus, lentivirus, adeno-associated virus, baculovirus, etc.
• Agrobacterium lithium acetate, spheroplast, heat shock (calcium chloride,
• the CRISPR-Cas3 system When the CRISPR-Cas3 system is introduced into a cell (including expression within the cell), the CRISPR-Cas3 system comes into contact with the enhancer (region to be cut) of the BCL11A gene via the binding between the target region and crRNA, and the DNA cutting activity of the CRISPR-Cas3 system cuts the region to be cut, destroying (preferably deleting) part or all of the enhancer of the BCL11A gene.
• the production method of the present invention can also be expressed as a method for producing hematopoietic stem cells capable of differentiating into erythroid progenitor cells in which the expression of the BCL11A gene is suppressed, or a method for producing hematopoietic stem cells capable of differentiating into erythroid progenitor cells in which the expression of ⁇ -globin is promoted.
• expression includes both expression at the transcription level (the process in which DNA is transcribed into mRNA) and expression at the translation level (the process in which mRNA is translated into peptides, polypeptides, or proteins), and also includes the step of splicing mRNA, but when referring to "expression of the BCL11A gene", it mainly means expression at the transcription level.
• the hematopoietic stem cells differentiate into erythroid progenitor cells in the subject's living body, and the expression level of ⁇ -globin in the erythroid progenitor cells of the subject can be increased.
• the increase in the expression level of ⁇ -globin and the resulting increase in fetal hemoglobin (HbF) contribute to the prevention and treatment of abnormal hemoglobinopathies such as ⁇ -hemoglobinopathy, sickle cell disease (SCD), and ⁇ -thalassemia (Non-Patent Document 1, Non-Patent Document 2).
• the present invention therefore also provides a method for improving the expression level of ⁇ -globin in erythroid progenitor cells of a subject, which comprises the step of administering to the subject hematopoietic stem cells obtained by the above-mentioned production method of the present invention, and a method for preventing or treating hemoglobinopathy, which comprises the step of administering to the subject hematopoietic stem cells obtained by the above-mentioned production method of the present invention (in this specification, these are sometimes collectively referred to as the "treatment method of the present invention").
• the subject may be any of the animals listed above, preferably humans.
• the administration method may be, for example, injection or transplantation.
• the present invention provides a kit for use in the above-mentioned production method of the present invention and the treatment method of the present invention, The following (A) to (C): (A) a Cas3 protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; (B) a cascade protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; and (C) a kit comprising a crRNA targeting an enhancer of the BCL11A gene, a polynucleotide encoding the crRNA, or an expression vector comprising the polynucleotide.
• the above (A) to (C), including their preferred embodiments, are as described above in the CRISPR-Cas3 system of the manufacturing method of the present invention.
• Each of these may be independently in the form of a protein or RNA, in the form of a polynucleotide that encodes the protein or RNA, or in the form of an expression vector that contains the polynucleotide and expresses the protein or RNA.
• Each of these forms, including their preferred embodiments, are as described above.
• the combination of the forms of (A) to (C) is not particularly limited, and may be a combination containing each of (A) to (C) individually, or may be a mixture of two or more of (A) to (C) in advance, or may be a composition containing other components (solvent, etc.).
• (B) may be in a different form for each cascade protein, or in the form of a complex or composition containing two or more cascade proteins, or in the form of a polynucleotide encoding each of two or more cascade proteins, or in the form of an expression vector containing the polynucleotide.
• (A) and (B) may be in the form of a complex or composition containing multiple proteins constituting the Cas protein group, or in the form of a polynucleotide encoding each of two or more proteins, or in the form of an expression vector containing the polynucleotide.
• (A) and (B) when introduced into a cell in the form of proteins and (C) is introduced into a cell in the form of RNA, they may be in the form of a complex of two or more of these in advance, or when (A) to (C) are introduced into a cell in the form of an expression vector, they may be in the form of an expression vector containing two or more polynucleotides.
• the kit of the present invention may further include one or more additional reagents.
• additional reagents include, but are not limited to, a reagent for isolating hematopoietic stem cells, a medium for culturing hematopoietic stem cells, a dilution buffer, a reconstitution solution, a washing buffer, a nucleic acid introduction reagent, a protein introduction reagent, and a control reagent.
• the kit may further include instructions for carrying out the manufacturing method of the present invention and the treatment method of the present invention.
• kit of the present invention When the kit of the present invention is used in the treatment method of the present invention, it may further include, for example, pharmaceutical additives and equipment for administering the produced hematopoietic stem cells to a subject as a pharmaceutical composition.
• the pharmaceutical composition can be prepared by a conventional method. More specifically, the hematopoietic stem cells produced by the production method of the present invention can be prepared, for example, by mixing with pharmaceutical additives depending on the administration method.
• Examples of the pharmaceutical additives include excipients, binders, disintegrants, lubricants, flow agents (anti-solid agents), colorants, plasticizers, solvents, solubilizers, emulsifiers, suspending agents (adhesives), thickeners, pH adjusters (acidifiers, alkalizers, buffers), wetting agents (solubilizers), antibacterial preservatives, chelating agents, medical water, stabilizers, preservatives, etc., and one or more of these may be used.
• Each component included in the kit may be contained in a separate container, or may be contained in the same container. Each component may be contained in a container in an amount sufficient for one use, or multiple doses may be contained in one container. Each component may be contained in a container in a dry form, or in a form dissolved in an appropriate solvent (a solvent containing a buffer solution, a stabilizer, a preservative, an antiseptic, etc.).
• an appropriate solvent a solvent containing a buffer solution, a stabilizer, a preservative, an antiseptic, etc.
• ⁇ Test Example 1> Disruption of enhancer of BCL11A gene in human hematopoietic stem cells using the CRISPR-Cas3 system (1) Enhancer of BCL11A gene The region to be cleaved within the enhancer of the BCL11A gene to be disrupted in this test example was around 60,495,269 on chromosome 2 (chr2) of human genomic DNA.
• CRISPR-Cas3 system (Example)
• the CRISPR-Cas3 system used in this test example was constructed.
• the amino acid sequence of the Cas3 protein and the nucleotide sequence encoding it are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
• the Cas3 protein was prepared and purified by the method described in WO 2022/186063.
• the cascade protein complex was composed of Cas8 (Cse1)-Cas11 (Cse2)-Cas7-Cas5-Cas6, with one molecule of Cas8, two molecules of Cas11, six molecules of Cas7, one molecule of Cas5, and one molecule of Cas6.
• the amino acid sequences of the Cas8 protein, Cas11 protein, Cas5 protein, Cas6 protein, and Cas7 protein, and the nucleotide sequences encoding them, are shown in sequence numbers 3 to 12, respectively.
• Each cascade protein and cascade protein complex was prepared and purified by the method described in reference I.
• CrRNA targeting the enhancer of the BCL11A gene was synthesized using 60,495,486-60,495,517 of chromosome 2 (chr2) of human genomic DNA as the target sequence.
• the target sequence (underlined) and PAM sequence (bold) of the crRNA are shown in Table 1 below.
• the crRNA was prepared and purified as mature crRNA by the method described in Reference I.
• the Cas3 protein, Cascade protein complex, and crRNA were made into a protein-RNA complex by the method described in Reference I, and this was used as the CRISPR-Cas3 system.
• CRISPR-Cas9 system comparative example
• Cas9 protein was obtained from Integrated DNA Technologies.
• gRNA was synthesized with 60,495,264 to 60,495,283 of chromosome 2 (chr2) of human genomic DNA as the target sequence.
• Table 1 below shows the target sequence (underlined) and PAM sequence (bold) of gRNA.
• hHSCs Cell Culture Human hematopoietic stem cells
• StemExpress StemExpress
• Folsom CA derived from umbilical cord blood of healthy individuals.
• hHSCs were first cultured in a medium (StemSpan SFEM, Veritas, Inc., supplemented with StemSpan (TM) CD34+ Expansion Supplement (10x)).
• TM StemSpan
• CD34+ Expansion Supplement (10x) StemSpan
• the CRISPR-Cas system constructed in (2) above was introduced by electroporation, and the cells were cultured under the same conditions until the seventh day after the start of culture (three days after the introduction of the CRISPR-Cas system).
• the medium was changed to a medium with the following composition: StemSpan SFEM (Veritas), 50 ng/mL SCF, 20 ng/mL EPO, 10 ng/mL IL-3, 10 ng/mL IGF-1, 1 ⁇ M dexamethasone, and cultured for 5 days (until the 12th day from the start of culture).
• the medium was changed to a medium with the following composition: StemSpan SFEM (Veritas), 20 ng/mL EPO, 10 ng/mL IGF-1, 0.5 mg/mL human transferrin, 2% BSA, and differentiation was induced, and cultured for an additional 6 days (until the 18th day from the start of culture) to obtain erythroid progenitor cells.
• StemSpan SFEM Veritas
• Electroporation The electroporation in (3) above was performed by electroporation (Maxcyte-Atx (MaxCyte), program: HSC-5) on a group of 1 million cells including hHSC on the 4th day after the start of culture.
• region A (60,495,229 to 60,495,399: 171 bp of human chromosome 2) containing a part of DHS+58, which is a GATA binding region, was used as a point (target site) for determining the presence or absence of genome editing, and if this region A was not amplified by PCR, it was determined that "the target site in the enhancer of the BCL11A gene was deleted.”
• DNeasy Blood & Tissue Kit Qiagen
• QIAcuity Qiagen
• the deletion rate was calculated by the following formula: (1-(amount of amplification product in region A of cells introduced with the CRISPR-Cas system/amount of amplification product of endogenous control of cells introduced with the CRISPR-Cas system)/(amount of amplification product in region A of NoEP/amount of amplification product of endogenous control of NoEP)) x 100, with the region in which genomic DNA is not cleaved by the CRISPR-Cas system near the BCL11A gene being used as an endogenous control.
• Table 2 shows the nucleotide sequences of each primer and probe used in digital PCR.
• HEX/BHQ1 and FAM/BHQ1 each represent a combination of a reporter dye and a quencher dye, and the amount of each amplification product was the fluorescence intensity derived from the reporter dye.
• the rate at which the target site in the enhancer of the BCL11A gene was deleted by the CRISPR-Cas3 system was 16% (10 ⁇ M)
• the rate at which the target site in the enhancer of the BCL11A gene was deleted by the CRISPR-Cas9 system was 35% (6 ⁇ M)
• the deletion rate of the target site i.e., the genome editing efficiency, was higher when the CRISPR-Cas9 system was used.
• ⁇ Test Example 2> Analysis of genome deletion patterns by CRISPR-Cas system
• Sanger sequencing was performed on the amplified fragments using a sequence primer (60,495,481-60,495,500:20 bp of human chromosome 2).
• the obtained Sanger sequence waveform data (.ab1) was analyzed using SYNTEGO ICE Analysis (Synthego) to obtain a genome deletion pattern.
• genomic DNA similarly recovered from unedited cells (NoEP) cultured under the same conditions without introducing any of the CRISPR-Cas systems was used for the same analysis.
• Genomic DNA was collected on the third day (seventh day from the start of culture) after the introduction of the CRISPR-Cas3 system into the cells in the above ⁇ Test Example 1>, and samples determined to have "a target site in the enhancer of the BCL11A gene deleted" by the digital PCR in the above ⁇ Test Example 1> (5) were subjected to PCR using Twist Library Prep EF Kit 2.0, Twist UMI Adapter System TruSeq Compatible, Twist Std Hyb and Wash Kit, Twist Dry Down Beads, Twist Universal Blockers and the Oligo panel (all manufactured by TwistBioScience) were used to enrich region C, and a library for analyzing the deletion pattern of enhancers of the BCL11A gene was constructed.
• the constructed NGS library was analyzed by Nova-seq (manufactured by Illumina). As a comparison, a library was similarly prepared using genomic DNA similarly recovered from unedited cells (NoEP) cultured under the same conditions without introducing any of the CRISPR-Cas systems.
• GATA binding regions there are multiple regions (GATA binding regions) to which GATA binds in the enhancer region of the BCL11A gene (DHS+55, DHS+58, DHS+62), but while the range that can be deleted by a single CRISPR-Cas9 system is limited to a portion of a single GATA binding region, it was confirmed that the method using the CRISPR-Cas3 system of the present invention allows multiple GATA binding regions to be deleted by a single system, and thus the enhancer of the BCL11A gene can be more reliably destroyed.
• ⁇ Test Example 3> Off-target analysis using the CRISPR-Cas3 system (1) Extraction of off-target candidates of crRNA Gene sequences and proto-oncogene sequences with high homology to the base sequence of the crRNA targeted at 60,495,486 to 60,495,517 of chromosome 2 (chr2) of human genomic DNA, prepared in ⁇ Test Example 1> (2) above, were extracted from the human genome and human proto-oncogenes by in silico analysis, and 92 loci obtained by removing duplicates from the top 50 loci for each were determined as off-target candidate loci.
• a missing (1 kb missing) fragment of the human endogenous genomic region was added as a control for genome cleavage detection at a ratio of 1% to the amount of genomic DNA of each sample.
• the constructed NGS library was analyzed using Nova-seq 6000 (manufactured by Illumina).
• the present invention makes it possible to provide a method for producing hematopoietic stem cells capable of differentiating into blood cell progenitor cells in which expression of the BCL11A gene is specifically and sufficiently suppressed with high efficiency, and a kit that can be suitably used for the method.
• the present invention is useful for preventing or treating a group of diseases in which the expression level of ⁇ -globin is involved. Furthermore, such a method using the CRISPR-Cas3 system is useful from the standpoint of safety, as it can avoid off-targets.

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