Genetically modified megakaryocytes, modified platelets, and methods for producing them

Abstract

Provided is a method for producing a genetically modified megakaryocyte, the method comprising a step for introducing a CRISPR-associated (Cas) family protein and guide RNA (gRNA) into a megakaryocyte to modify a gene of interest, wherein the Cas family protein and the gRNA to be introduced into the megakaryocyte at said step are formed as a complex beforehand. The method is useful as a technique for producing a genetically modified megakaryocyte and a modified platelet.

Description

[Technical Field]

[0001] The present invention relates to genetically modified megakaryocytes, modified platelets, and methods for producing them. This application claims priority based on Japanese Patent Application No. 2020-034255, filed on February 28, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] Platelets are a type of cell produced from the cytoplasm of megakaryocytes in the bone marrow. Platelets play a central role in the formation of blood clots, and when a blood vessel wall is damaged, they gather together to seal the wound and stop bleeding.

[0003] Platelet transfusions are performed to stop or prevent bleeding by replenishing platelet components in cases where severe bleeding or bleeding is predicted due to a decrease in platelet count or abnormality in platelet function.

[0004] However, it is known that transplanted platelets are destroyed in patients with platelet refractoriness due to the abnormal production of antibodies against Human Leukocyte Antigen (HLA) class 1. In contrast, if HLA-deficient platelets can be produced, it is expected that they will be transplantable into patients with platelet refractoriness who have antibodies against HLA class 1.

[0005] In recent years, the CRISPR-Cas9 system, a type of prokaryotic adaptive immune system known as the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, has been widely used in genome editing techniques such as gene disruption (knockout). This system induces double-stranded DNA breaks (DSBs) at desired sites in genomic DNA, which then undergo deletion or insertion via the host cell's inherent repair mechanisms. It is also known that other Cas family proteins can be used for genome editing.

[0006] Since platelets do not have a genome, one possible method for producing HLA protein-deficient platelets is to disrupt the HLA gene in megakaryocytes (the precursor stage) by genome editing and obtain the platelets from the HLA protein-deficient megakaryocytes. However, as described in Non-Patent Document 1, for example, it has been known that it is difficult to express foreign genes in megakaryocytes using methods other than viral vectors. Megakaryocytes are giant cells with diameters of 35 to 160 μm, and produce platelets by stretching a portion of their cell membrane and tearing it off under intense shear stress caused by turbulent flow. In other words, the cell membrane of megakaryocytes is more flexible and tougher than that of other cells, and the unique nature of this membrane structure is thought to make exogenous gene transfer difficult.

[0007] Furthermore, Non-Patent Document 2 describes that genome editing could not be performed on megakaryocytes induced to differentiate from iPS cells. For this reason, Non-Patent Document 2 describes that megakaryocytes were reinitialized to return to iPS cells, and after genome editing, they were again induced to differentiate into megakaryocytes.

[0008] Furthermore, Non-Patent Document 3 describes genome editing of megakaryocytes by introducing Cas9 and gRNA into megakaryocytes using a lentiviral vector. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Isakari Y., et al., Efficient gene expression in megakaryocytic cell line using nucleofection, Int J Pharm., 338 (1-2), 157-64, 2007. [Non-patent document 2] Seo H., et al., A beta1-tubulin-based megakaryocyte maturation reporter system identifies novel drugs that promote platelet production, Blood Adv., 2 (17), 2262-2272, 2018. [Non-patent document 3] Kahr WH, et al., Loss of the Arp2 / 3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease, Nat Commun., 8:14816, 2017. Summary of the Invention [Problem to be solved by the invention]

[0010] However, the method of Non-Patent Document 2 is inefficient, requiring a great deal of time and effort for the reprogramming of iPS cells and subsequent differentiation into megakaryocytes. Furthermore, the method of Non-Patent Document 3 involves the risk that the transgene is inserted into the genomic DNA of megakaryocytes and cannot be removed, resulting in the risk of exogenous gene expression becoming a new antigen or adversely affecting endogenous gene function. More specifically, genome editing using lentiviral vectors involves the insertion of the Cas9 gene, sgRNA, etc. into the genome. Therefore, the resulting megakaryocytes are likely to be at risk of off-target mutations, antigenicity due to Cas9 protein expression, and effects on endogenous gene function (such as activation of oncogenes), making them unsuitable for clinical application. Furthermore, the use of lentiviruses poses problems such as limited facilities and high production costs, necessitating the need for a simpler method.

[0011] Against this background, an object of the present invention is to provide a technique for producing genetically modified megakaryocytes and genetically modified platelets. [Means for solving the problem]

[0012] The present invention includes the following aspects. [1] A method for producing genetically modified megakaryocytes, comprising the step of introducing a CRISPR-associated (Cas) family protein and a guide RNA (gRNA) into megakaryocytes to modify a target gene, wherein the Cas family protein and the gRNA introduced into the megakaryocytes in this step have already formed a complex. [2] The method according to [1], wherein the introduction of the Cas family protein and the gRNA is carried out by lipofection or electroporation, which is suitable for protein introduction. [3] The method according to [1] or [2], wherein the introduction of the Cas family protein and the gRNA is carried out by electroporation. [4] The method according to any one of [1] to [3], wherein the target gene is (i) a human leukocyte antigen (HLA)-A, HLA-B, or HLA-C gene, or (ii) a β2-microglobulin (B2M) gene, and the modification is disruption of the target gene. [5] The method according to any one of [1] to [4], wherein the megakaryocytes are obtained by differentiating pluripotent stem cells. [6] Genetically modified megakaryocytes in which (i) the HLA-A, HLA-B and HLA-C genes or (ii) the B2M gene have been disrupted. [7] A method for producing modified platelets, comprising the steps of introducing a Cas family protein and gRNA into megakaryocytes to modify a target gene and obtain genetically modified megakaryocytes, and producing platelets from the genetically modified megakaryocytes to obtain modified platelets, wherein in the step of obtaining genetically modified megakaryocytes, the Cas family protein and the gRNA introduced into the megakaryocytes have previously formed a complex. [8] The method according to [7], wherein the introduction of the Cas family protein and the gRNA is carried out by lipofection or electroporation, which is suitable for protein introduction. [9] The method according to [7] or [8], wherein the introduction of the Cas family protein and the gRNA is carried out by electroporation.

[10] The method according to any one of [7] to [9], wherein the megakaryocytes are obtained by differentiating pluripotent stem cells.

[11] The method according to any one of [7] to

[10] , wherein the target gene is (i) an HLA-A, HLA-B, or HLA-C gene, or (ii) a B2M gene, and the modification is a disruption of the target gene.

[12] HLA-deficient platelets, which lack HLA-A, HLA-B, and HLA-C proteins.

[13] The HLA-deficient platelets according to

[12] , produced by the production method according to

[11] .

[14] A method for producing genetically modified megakaryocytes, comprising the step of introducing a Cas family protein and gRNA into megakaryocytes to modify a target gene, wherein the introduction of the Cas family protein and the gRNA is carried out by electroporation.

[15] The method according to

[14] , wherein the megakaryocytes are obtained by differentiating pluripotent stem cells.

[16] A manufacturing method according to

[14] or

[15] , in which electroporation is performed using 0.1 to 1 pmol of a Cas family protein and 0.1 to 1 pmol of gRNA per 1,000 megakaryocyte cells.

[17] The method according to any one of

[14] to

[16] , wherein the target gene is (i) a human leukocyte antigen (HLA)-A, HLA-B, or HLA-C gene, or (ii) a β2-microglobulin (B2M) gene, and the modification is disruption of the target gene.

[18] A genetically modified megakaryocyte produced by the method described in

[17] , in which (i) the HLA-A, HLA-B and HLA-C genes, or (ii) the B2M gene, have been disrupted.

[19] A method for producing modified platelets, comprising: a step of introducing a Cas family protein and gRNA into megakaryocytes using electroporation to modify a target gene and obtain genetically modified megakaryocytes; and a step of producing platelets from the genetically modified megakaryocytes to obtain the modified platelets.

[0013] The present invention can also be said to include the following aspects. [P1] A method for producing genetically modified megakaryocytes, comprising the step of introducing a CRISPR-associated (Cas) family protein and a guide RNA (gRNA) into megakaryocytes to modify a target gene. [P2] The method for producing genetically modified megakaryocytes according to [P1], wherein the introduction of the Cas family protein and gRNA is carried out by electroporation. [P3] The method for producing genetically modified megakaryocytes according to [P1] or [P2], wherein the target gene is (i) human leukocyte antigen (HLA)-A, HLA-B, and HLA-C genes, or (ii) β2-microglobulin (B2M) gene, and the modification is the disruption of the target gene. [P4] The method for producing genetically modified megakaryocytes according to any one of [P1] to [P3], wherein the megakaryocytes are obtained by differentiating pluripotent stem cells. [P5] Genetically modified megakaryocytes in which (i) the HLA-A, HLA-B and HLA-C genes or (ii) the B2M gene have been disrupted. [P6] A method for producing modified platelets, comprising the steps of: introducing a Cas family protein and gRNA into megakaryocytes to modify a target gene and obtain genetically modified megakaryocytes; and producing platelets from the genetically modified megakaryocytes to obtain modified platelets. [P7] The method for producing modified platelets according to [P6], wherein the introduction of the Cas family protein and gRNA is carried out by electroporation. [P8] A method for producing modified platelets according to [P6] or [P7], wherein the target gene is (i) HLA-A, HLA-B, and HLA-C genes, or (ii) B2M gene, and the modification is disruption of the target gene. [P9] The method for producing modified platelets according to any one of [P6] to [P8], wherein the megakaryocytes are obtained by differentiating pluripotent stem cells. [P10] HLA-deficient platelets lacking HLA-A protein, HLA-B protein, and HLA-C protein. [Effects of the Invention]

[0014] According to the present invention, a technique for producing genetically modified megakaryocytes and genetically modified platelets can be provided. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a graph showing the results of flow cytometry analysis in Experimental Example 1. [Figure 2] 1 is a graph showing the results of flow cytometry analysis in Experimental Example 2. [Figure 3] 1(a) is a diagram showing the gating settings for flow cytometry analysis in Experimental Example 3. FIG. 1(b) is a graph showing the results of flow cytometry analysis in Experimental Example 3. [Figure 4] 6(a) and 6(b) are graphs showing the results of flow cytometry analysis in Experimental Example 4. [Figure 5] 10(a) and 10(b) are graphs showing the results of flow cytometry analysis in Experimental Example 5. [Figure 6] 10(a) and 10(b) are graphs showing the results of flow cytometry analysis in Experimental Example 5. [Figure 7] 10(a) and 10(b) are graphs showing the results of flow cytometry analysis in Experimental Example 5. [Figure 8] 10(a) and 10(b) are graphs showing the results of flow cytometry analysis in Experimental Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0016] [Method for producing genetically modified megakaryocytes] In one embodiment, the present invention provides a method for producing genetically modified megakaryocytes, comprising the step of introducing a Cas family protein and gRNA into megakaryocytes to modify a gene of interest. In this production method, the Cas family protein and gRNA to be introduced into megakaryocytes have previously formed a complex.

[0017] Generally, the amount of intracellular protein is higher when an expression vector is introduced into a cell and the protein is expressed in the cell than when the protein is directly introduced into the cell. Therefore, in order to perform genome editing with high efficiency, it is common knowledge among those skilled in the art to introduce a Cas family protein and gRNA into a cell in the form of an expression vector. In contrast, as described below in the Examples, the inventors have demonstrated that target genes can be efficiently modified by introducing a complex of a Cas family protein and gRNA into megakaryocytes and performing genome editing.

[0018] Furthermore, as described later in the Examples, gene modification can be performed quickly and efficiently by introducing a complex of a Cas family protein and gRNA, compared to genome editing performed by introducing plasmid DNA.

[0019] (megakaryocyte) In the production method of this embodiment, the megakaryocytes may be derived from humans or non-human animals, and may be appropriately selected depending on the purpose. They may be megakaryocytes collected from humans or non-human animals, or megakaryocytes obtained by inducing differentiation of hematopoietic stem cells collected from humans or non-human animals, or megakaryocytes obtained by inducing differentiation of pluripotent stem cells derived from humans or non-human animals. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).

[0020] Megakaryocytes may be genetically engineered, such as immortalized megakaryocytes immortalized by forced expression of the BMI1 gene, c-MYC gene, and BCL-xL gene in the presence of doxycycline. Such megakaryocytes can be expanded in the presence of doxycycline, and platelets can be produced by removing doxycycline.

[0021] (Cas family proteins) Examples of Cas family proteins include Cas9, Cpf1 (also known as Cas12a), C2C1 (also known as Cas12b), C2C2 (also known as Cas13a), CasX, CasY, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, and Cas10. CRISPR-Cas systems are classified into class 1 and class 2. Traditionally, class 2 systems, centered around Cas9, have been commonly used, but class 1 systems are now also available.

[0022] The Cas family protein may be a modified version of these proteins. For example, it may be a nickase-modified nuclease in which one of the two wild-type nuclease domains is modified to be inactive, or dCas9 in which both nuclease domains are modified to be inactive. Alternatively, it may be Cas9-HF, HiFi-Cas9, eCas9, or the like, which have improved target specificity. Furthermore, it may be a Cas9 fused with another protein (enzyme, etc.).

[0023] Examples of Cas9 proteins include those derived from Streptococcus pyogenes, Staphylococcus aureus, Streptococcus thermophilus, Geobacillus stearothermophilus, etc. Examples of Cpf1 proteins include those derived from Acidaminococcus, Lachnospira, Chlamydomonas, Francisella novicida, etc.

[0024] (gRNA) The gRNA used corresponds to the Cas family protein used, and determines the location in the genomic DNA where the Cas family protein will induce double-stranded DNA breaks, i.e., the target gene to be modified.

[0025] As used herein, the target base sequence of a gRNA refers to the base sequence on a single-stranded DNA that forms a complementary strand with the DNA to which the spacer base sequence of the gRNA hybridizes. The spacer base sequence of the gRNA binds complementarily to the antisense strand of the target sequence. Therefore, the spacer base sequence of the gRNA and the base sequence of the sense strand of the target sequence have high sequence identity, and the spacer base sequence of the gRNA and the base sequence of the antisense strand of the target sequence are generally complementary.

[0026] The gRNA may be a complex of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), or a single gRNA (sgRNA) that combines tracrRNA and crRNA.

[0027] Methods for preparing gRNA in the form of RNA include synthesizing it by in vitro transcription reaction using a construct in which a promoter such as T7 is added upstream of a nucleic acid fragment encoding the gRNA, and chemical synthesis. When chemically synthesizing gRNA, chemically modified RNA may also be used.

[0028] For example, if the base sequence obtained by removing the PAM sequence from the target base sequence is "5'-NNNNNNNNNNNNNNNNNNNN-3'" (SEQ ID NO: 1), the base sequence of the sgRNA that specifically cleaves the target base sequence can be "5'-NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU-3'" (SEQ ID NO: 2).

[0029] As long as the sgRNA functions as an sgRNA, the nucleotide sequence may have a mutation relative to the nucleotide sequence set forth in SEQ ID NO: 2. More specifically, the nucleotide sequence may be a nucleotide sequence in which one or several nucleotides have been deleted, substituted, or added in the nucleotide sequence set forth in SEQ ID NO: 2. Here, one or several nucleotides may be, for example, 1 to 10 nucleotides, for example, 1 to 5 nucleotides, or for example, 1 to 3 nucleotides.

[0030] When the gRNA is a complex of crRNA and tracrRNA, the base sequences of the crRNA and tracrRNA can be as follows:

[0031] First, the base sequence obtained by removing the PAM sequence from the target base sequence is designated as the spacer base sequence. Next, a base sequence is designed in which a scaffold sequence is linked to the 3' end of the spacer base sequence, and this is designated as the base sequence of the crRNA. For example, if the base sequence obtained by removing the PAM sequence from the target base sequence (spacer base sequence) is "5'-NNNNNNNNNNNNNNNNNNNN-3'" (SEQ ID NO: 1), the base sequence of the crRNA can be "5'-NNNNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUG-3'" (SEQ ID NO: 3). Furthermore, the base sequence of the tracrRNA can be, for example, "5'-CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'" (SEQ ID NO: 4).

[0032] The base sequence of the crRNA may have a mutation relative to the base sequence set forth in SEQ ID NO: 3, as long as it functions as a crRNA. More specifically, the base sequence may be a base sequence in which one or several bases are deleted, substituted, or added in the base sequence set forth in SEQ ID NO: 3. Here, one or several bases may be, for example, 1 to 10 bases, for example, 1 to 5 bases, or for example, 1 to 3 bases.

[0033] Furthermore, as long as the tracrRNA functions as a tracrRNA, the nucleotide sequence may be a nucleotide sequence in which one or several nucleotides are deleted, substituted, or added in the nucleotide sequence set forth in SEQ ID NO: 4. Here, one or several nucleotides may be, for example, 1 to 10 nucleotides, for example, 1 to 5 nucleotides, or for example, 1 to 3 nucleotides.

[0034] (Introduction of Cas family proteins and gRNA) The method for introducing Cas family proteins and gRNA into megakaryocytes (transfection method) is not particularly limited, and examples include lipofection, electroporation, and microinjection. As used herein, "transfection" refers to the introduction of nucleic acids or proteins into cells by a method other than viral infection. Commercially available transfection reagents can be used as appropriate for transfection.

[0035] As a transfection reagent, a reagent capable of introducing a protein into cells is preferred, and for example, Lipofectamine CRISPRMAX (Thermo Fisher Scientific), Pro-DeliverIN CRISPR Transfection Reagent (OZ Biosciences), Avalanche®-CRISPR Transfection Reagent (EZ Biosynthetics), etc. can be used.

[0036] Electroporation can also be performed using equipment such as NEPA21 (Nepgene Co., Ltd.), Neon (Thermo Fisher Scientific), 4D-Nucleofector (Lonza), and MaxCyte system (MaxCyte).

[0037] The Cas family protein and gRNA are preferably introduced into megakaryocytes in the form of a complex. The Cas family protein and gRNA can be mixed to form a complex. Before being introduced into megakaryocytes, the Cas family protein and gRNA may be mixed and incubated for several minutes (e.g., 1 to 10 minutes, 1 to 7 minutes, 1 to 5 minutes, 3 to 5 minutes, or around 5 minutes), for example, for about 5 minutes, to promote complex formation.

[0038] (Target gene) In the manufacturing method of this embodiment, a base sequence on a target gene is set as a target base sequence of a gRNA. In the manufacturing method of this embodiment, the target gene may be (i) a combination of an HLA-A gene, an HLA-B gene, and an HLA-C gene. Alternatively, the target gene may be (ii) a B2M gene. Furthermore, "modifying a target gene" may mean destroying the target gene.

[0039] Here, disruption of a target gene means that a deletion or insertion is introduced at the cleavage site of the target gene by the complex of a Cas family protein and gRNA, resulting in a frameshift, introduction of a stop codon, or large-scale deletion, and thus preventing the target gene from expressing a functional protein.

[0040] The most important role in distinguishing between self and non-self cells is played by cell surface proteins called HLA (Human Leukocyte Antigen) or Major Histocompatibility Complex (MHC).

[0041] HLA is classified into class 1 and class 2. Class 1 HLA proteins are expressed on most types of cells in the body. Class 1 HLA proteins form heterodimers with β2-microglobulin (B2M) and are expressed on the cell surface, where they function to present peptides to CD8-positive cytotoxic T cells and induce their activation. The antigen peptides they present are endogenous and are often 8 to 10 amino acids long.

[0042] HLA class 1 is classified into six main genes: HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In addition, numerous pseudogenes (HLA-H, HLA-J, HLA-K, HLA-L, HLA-P, HLA-T, HLA-U, HLA-V, HLA-W, HLA-X, HLA-Y, etc.) are also known. Of these, the three genes with the greatest inter-individual sequence diversity are HLA-A, HLA-B, and HLA-C, which play a major role in distinguishing self from non-self in transplantation immunity.

[0043] Class 2 HLA proteins are primarily expressed in immune cells such as macrophages, dendritic cells, activated T cells, and B cells. Class 2 HLA proteins form heterodimers of α and β chains and function to present peptides to CD4 helper T cells and induce their activation. The antigen peptides they present are exogenous and often 15 to 24 amino acids in length.

[0044] HLA class 2 includes HLA-DR (α chain: HLA-DRA, β chain: HLA-DRB), HLA-DQ (α chain: HLA-DQA1, β chain: HLA-DQB1), and HLA-DP (α chain: HLA-DPA1 or HLA-DPA2, β chain: HLA-DPB1 or HLA-DPB2). Many other pseudogenes (HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB) are also known to exist.

[0045] HLA-A, HLA-B, and HLA-C proteins are highly associated with rejection during cell transplantation. Therefore, by producing genetically modified megakaryocytes in which the HLA-A, HLA-B, and HLA-C genes of megakaryocytes have been disrupted and then producing platelets, it is possible to eliminate the expression of HLA proteins on the platelet surface. This is expected to reduce antigenicity during platelet transplantation. Furthermore, transplantable platelet preparations can be provided for patients with antibodies against HLA class 1.

[0046] The target base sequence of the gRNA for disrupting the HLA-A, HLA-B, and HLA-C genes of megakaryocytes can be designed based on the base sequences of the determined HLA-A, HLA-B, and HLA-C genes after determining the HLA alleles of the megakaryocytes.

[0047] HLA alleles can be determined by conventional methods such as PCR-rSSO (PCR-reverse sequence specific oligonucletide), PCR-SSP (sequence specific primer), PCR-SBT (sequence based typing), and next-generation sequencing, and can be determined using commercially available HLA typing kits, etc. Known software for HLA typing from sequence data obtained by next-generation sequencers such as whole genome sequencing (WGS), exome sequencing (WES), and RNA-seq includes HLAreporter, HLA-PRG, hla-genotyper, PHLAT, Optitype, neXtype, Athlates, HLAforest, SOAP-HLA, HLAminer, seq2HLA, and GATK HLA Caller, and these software may also be used.

[0048] Furthermore, megakaryocytes can be deficient in HLA-A, HLA-B, and HLA-C proteins by deleting the B2M gene, which is required for cell surface presentation of class 1 HLA proteins.

[0049] Furthermore, "modifying a target gene" is not limited to disrupting the target gene, but may also mean editing the base sequence of the target gene, resulting in the expression of a modified protein from the modified target gene.

[0050] Specifically, by specifically cleaving a target gene in the presence of donor DNA to induce double-stranded DNA breaks, homologous recombination can be induced during the repair process of the double-stranded DNA break, thereby modifying the target gene.

[0051] The donor DNA used should have sequence identity with the region surrounding the double-stranded DNA break in the target gene and have the desired base sequence. The donor DNA may be single-stranded or double-stranded. The donor DNA may also be DNA with a single base sequence or a mixture of DNAs with multiple base sequences.

[0052] Here, having sequence identity means that 90% or more of the base sequence between the donor DNA and the region containing the double-strand break in the target gene of interest matches. Preferably, 95% or more of the base sequence of the donor DNA matches, and more preferably, 99% or more of the base sequence matches, with the region containing the double-strand break in the genomic DNA.

[0053] The donor DNA may be a single-stranded DNA of about 50 to 5,000 base pairs, or a double-stranded DNA of about 50 to 5,000 base pairs. When the donor DNA is a single-stranded DNA, the donor DNA may have sequence identity with either strand of the double strand of the genomic DNA.

[0054] Here, the sequence identity of a query base sequence to a reference base sequence can be determined, for example, as follows: First, the reference base sequence and the query base sequence are aligned. Here, gaps may be included in each base sequence to maximize sequence identity. Next, the number of matching bases in the reference base sequence and the query base sequence is calculated, and the sequence identity can be determined according to the following formula (F1): Sequence identity (%) = number of matched bases / total number of bases in query sequence × 100 (F1)

[0055] [Genetically modified megakaryocytes] In one embodiment, the invention provides genetically modified megakaryocytes in which (i) the HLA-A, HLA-B and HLA-C genes, or (ii) the B2M gene, have been disrupted.

[0056] The genetically modified megakaryocytes of this embodiment lack expression of HLA proteins. Furthermore, HLA-deficient platelets can be produced from the megakaryocytes of this embodiment. Therefore, even in allotransplantation, the genetically modified megakaryocytes of this embodiment can provide megakaryocytes or platelets with reduced HLA antigen-mediated immune rejection. Since platelet preparations produced from donated blood are inactivated within a few days, cells that can be expanded inexhaustibly while maintaining platelet-producing capacity, such as iPS cell-derived megakaryocytes, are highly useful in medical and industrial applications.

[0057] Conventionally, megakaryocytes and platelets could only be produced from iPS cells derived from donors with matching HLA types. In contrast, the method of this embodiment makes it possible to transplant a single HLA-deficient platelet preparation into multiple recipients. As a result, the cost of platelet preparations can be dramatically reduced compared to autologous transplantation.

[0058] [Method of manufacturing modified platelets] In one embodiment, the present invention provides a method for producing modified platelets, comprising the steps of: introducing a Cas family protein and gRNA into megakaryocytes to modify a target gene and obtain genetically modified megakaryocytes; and producing platelets from the genetically modified megakaryocytes to obtain modified platelets.

[0059] The megakaryocytes, Cas family proteins, gRNA, target genes, etc. are the same as those described above. Megakaryocytes may be obtained by differentiating pluripotent stem cells. Modified platelets can be manufactured by producing platelets from genetically modified megakaryocytes. Here, modified platelets refer to platelets in which the expression of the protein encoded by the target gene has been changed compared to wild-type platelets. The method for producing platelets from megakaryocytes is not particularly limited, and any commonly used method can be used as appropriate.

[0060] In the production method of this embodiment, the introduction of the Cas family protein and gRNA into megakaryocytes is preferably performed by electroporation. As described later in the Examples, the inventors have demonstrated that target genes can be modified more efficiently by introducing a complex of the Cas family protein and gRNA into megakaryocytes and performing genome editing.

[0061] In the production method of this embodiment, the target gene may be (i) the HLA-A, HLA-B, and HLA-C genes, or (ii) the B2M gene, and the modification may be disruption of the target gene. The HLA-A gene, HLA-B gene, HLA-C gene, and B2M gene are the same as those described above.

[0062] By disrupting these genes, HLA-deficient megakaryocytes can be produced, and HLA-deficient platelets can be produced from HLA-deficient megakaryocytes. In other words, in this case, the modified platelets are HLA-deficient platelets.

[0063] In one embodiment, the present invention provides a method for producing low-antigenic platelets, comprising the following steps: (a) inducing differentiation of human-derived pluripotent stem cells into immortalized megakaryocytes; (b) using CRISPER-Cas gene modification technology to introduce a complex of a Cas family protein and gRNA into the immortalized megakaryocytes, thereby modifying or deleting two or more HLA class 1 proteins; (c) expanding the immortalized megakaryocytes in which two or more HLA class 1 proteins have been modified or deleted; and (d) producing platelets from the expanded megakaryocytes.

[0064] [HLA-deficient platelets] In one embodiment, the present invention provides an HLA-deficient platelet that is deficient in two or more HLA class 1 proteins, preferably two or more proteins selected from the group consisting of HLA-A protein, HLA-B protein, and HLA-C protein, more preferably HLA-A protein, HLA-B protein, and HLA-C protein.

[0065] In one embodiment, the present invention provides HLA-deficient platelets that are deficient in HLA-A protein, HLA-B protein, and HLA-C protein. Alternatively, the present invention can also provide the HLA-deficient platelets that are deficient in HLA-A protein, HLA-B protein, and / or HLA-C protein. As described above, the HLA-deficient platelets of this embodiment are deficient in HLA proteins. Therefore, even in allogeneic transplantation, immune rejection mediated by HLA antigens can be reduced upon transplantation. Furthermore, the HLA-deficient platelets of this embodiment can be mass-produced by the method described above.

[0066] [Other embodiments] In one embodiment, the present invention provides a method for treating platelet-related diseases, comprising the steps of: introducing a complex of a Cas family protein and gRNA into megakaryocytes to modify (i) HLA-A, HLA-B, and HLA-C genes or (ii) the B2M gene to obtain HLA-deficient megakaryocytes; producing platelets from the HLA-deficient megakaryocytes to obtain HLA-deficient platelets; and administering an effective amount of the HLA-deficient platelets to a patient in need of treatment. The megakaryocytes, Cas family protein, gRNA, target gene, etc. are the same as those described above.

[0067] In the treatment method of this embodiment, the platelet-related disease refers to a pathological condition in which severe bleeding or bleeding is predicted due to a decrease in platelet count or abnormality in platelet function. The patient may also have platelet refractoriness. The HLA-deficient platelets are preferably administered to the patient by intravenous drip infusion. [Example]

[0068] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0069] [Experimental Example 1] (HLA-A / B / C gene knockout in megakaryocytes) Cas9 protein and sgRNA were introduced into megakaryocytes to knock out the HLA-A gene, HLA-B gene, and HLA-C gene (hereinafter sometimes referred to as "HLA-A / B / C genes").

[0070] We used an immortalized human megakaryocyte cell line (imMKCL) that overexpresses the BMI1, c-MYC, and BCL-xL genes in the presence of doxycycline (Clontech) (see Nakamura S., et al., Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells, Cell Stem Cell, 14 (4), 535-548, 2014). The cells can be expanded in the presence of doxycycline, and platelets can be produced by removing doxycycline.

[0071] The Cas9 protein was a commercially available product (product name: TrueCut TM Cas9 Protein v2, catalog number A36498, Thermo Fisher Scientific) was used.

[0072] Three types of sgRNAs were used: a synthetic sgRNA targeting the HLA-A gene ("HLA-A-ex3g1", the target base sequence of which is shown in SEQ ID NO: 5), a synthetic sgRNA targeting the HLA-B gene ("HLA-B-ex2g1", the target base sequence of which is shown in SEQ ID NO: 6), and a synthetic sgRNA targeting the HLA-C gene ("HLA-C12:02-ex3g1", the target base sequence of which is shown in SEQ ID NO: 7).

[0073] 15 μg of Cas9 protein, 1.25 μg of HLA-A-ex3g1, 1.25 μg of HLA-B-ex2g1, and 1.25 μg of HLA-C12:02-ex3g1 were mixed and incubated, and then 3 × 10 5 The cells were transfected into megakaryocytes. Electroporation was performed using 4D-Nucleofector. TM (Lonza) and P4 Primary Cell 4D-Nucleofector TMX KitS (catalog number "V4XP-4032", Lonza) was used.

[0074] The electroporated megakaryocytes were added to a medium and cultured at 37°C and 5% CO2. The composition of the medium is shown in Table 1 below.

[0075] [Table 1]

[0076] Megakaryocytes were collected 3 and 6 days after the start of culture and stained with FITC-labeled anti-HLA-A / B / C antibody (catalog number 555552, BD Biosciences). Flow cytometry analysis was then performed using a FACS Verse (BD Biosciences) to determine the percentage of cells in which all HLA-A / B / C genes were knocked out.

[0077] Figure 1 is a graph showing the results of flow cytometry analysis. In Figure 1, "Wild type" indicates the results for wild-type megakaryocytes, "HLA-A / B / C KO" indicates the results for megakaryocytes that were subjected to knockout treatment of the HLA-A gene, HLA-B gene, and HLA-C gene. "Stained" indicates the results stained with FITC-labeled anti-HLA-A / B / C antibody, and "unstained" indicates the results without staining. "Day 3" indicates the results measured 3 days after the start of culture, and "Day 6" indicates the results measured 6 days after the start of culture.

[0078] As a result, it was revealed that in megakaryocytes that had undergone HLA-A / B / C gene knockout treatment, 24.0% (after 3 days) and 19.7% (after 6 days) of the entire cell population became negative for HLA-A protein, HLA-B protein, and HLA-C protein (hereinafter sometimes referred to as "HLA-A / B / C protein"). This result demonstrates that genome editing in megakaryocytes was efficiently performed using the method of this experimental example.

[0079] [Experimental Example 2] (B2M gene knockout in megakaryocytes) Cas9 protein and sgRNA were introduced into megakaryocytes to knock out the B2M gene. For comparison, HLA-A / B / C genes were also knocked out. The megakaryocytes used were the same immortalized human megakaryocyte line (imMKCL) as in Experimental Example 1.

[0080] The Cas9 protein was a commercially available product (product name: TrueCut TM Cas9 Protein v2, catalog number A36498, Thermo Fisher Scientific) was used.

[0081] The sgRNAs used were a synthetic sgRNA targeting the B2M gene ("B2M-ex1g2", the target base sequence is shown in SEQ ID NO: 8), a synthetic sgRNA targeting the HLA-A gene ("HLA-A-ex3g1", the target base sequence is shown in SEQ ID NO: 5), a synthetic sgRNA targeting the HLA-B gene ("HLA-B-ex2g1", the target base sequence is shown in SEQ ID NO: 6), and a synthetic sgRNA targeting the HLA-C gene ("HLA-C12:02-ex3g1", the target base sequence is shown in SEQ ID NO: 7).

[0082] For knockout of the B2M gene, 5 μg of Cas9 protein and 1.25 μg of B2M-ex1g2 were mixed and electroporated to 3 × 10 5 For knockout of HLA-A / B / C genes, 15 μg of Cas9 protein, 1.25 μg of HLA-A-ex3g1, 1.25 μg of HLA-B-ex2g1, and 1.25 μg of HLA-C12:02-ex3g1 were mixed and transfected into 3 × 10 megakaryocytes by electroporation. 5 The cells were transfected into megakaryocytes using MaxCyte STX (MaxCyte).

[0083] The electroporated megakaryocytes were added to the medium with the composition shown in Table 1 above and cultured at 37°C and 5% CO2. Six days after the start of culture, the megakaryocytes were collected and stained with FITC-labeled anti-HLA-A / B / C antibody (catalog number "555552", BD Biosciences). Next, flow cytometry analysis was performed using a FACS Verse (BD Biosciences) to measure the percentage of the cell population in which all HLA-A / B / C genes were knocked out.

[0084] Figure 2 is a graph showing the results of flow cytometry analysis. In Figure 2, "Wild type" indicates the results for wild-type megakaryocytes, "B2M KO" indicates the results for megakaryocytes subjected to B2M gene knockout treatment, and "HLA-A / B / C KO" indicates the results for megakaryocytes subjected to HLA-A, HLA-B, and HLA-C gene knockout treatment. "Unstained" indicates the results without staining with FITC-labeled anti-HLA-A / B / C antibody.

[0085] As a result, it was revealed that in megakaryocytes where the B2M gene was knocked out, 48.9% of the entire cell population became HLA-A / B / C protein negative. Furthermore, it was revealed that in megakaryocytes where the HLA-A / B / C gene was knocked out, 59.5% of the entire cell population became HLA-A / B / C protein negative. These results further support the idea that genome editing can be efficiently performed in megakaryocytes using the method of this experimental example.

[0086] [Experimental Example 3] (Subcloning of HLA-A / B / C gene knockout megakaryocytes) Subcloning by colony formation was performed to purify megakaryocytes that had undergone HLA-A / B / C gene knockout treatment in Experimental Example 1. The medium used for subcloning had the composition shown in Table 2 below.

[0087] [Table 2]

[0088] Megakaryocytes whose HLA-A / B / C genes had been knocked out in Experimental Example 1 were added to the medium whose composition is shown in Table 2 above, stirred, and seeded on a 100 mm dish. The number of cells seeded was 5 × 10 4 The cells were then cultured at 37°C in 5% CO2 for 8 to 12 days.

[0089] Next, single colonies were collected under a microscope and seeded into each well of a 96-well plate in which 200 μL of the medium shown in Table 1 above had been dispensed into each well, and further cultured for 3 to 7 days at 37°C and 5% CO2.

[0090] The proliferated megakaryocytes were then collected and stained with FITC-labeled anti-HLA-A / B / C antibody (catalog number 555552, BD Biosciences), followed by flow cytometry analysis using a FACS Verse (BD Biosciences).

[0091] First, as shown in Figure 3(a), wild-type megakaryocytes were analyzed by flow cytometry for unstained and stained cells with anti-HLA-A / B / C antibodies, and gating was performed to define the HLA-A / B / C protein-negative cell population. In Figure 3(a), "stained" indicates the results of staining with FITC-labeled anti-HLA-A / B / C antibodies, and "unstained" indicates the results of unstained cells. The oval in the graph indicates the gating for defining the HLA-A / B / C protein-negative cell population.

[0092] Next, using the gating settings described above, the expression of HLA-A / B / C proteins in the megakaryocytes cloned by subcloning was evaluated. Figure 3(b) shows the results of flow cytometry analysis of each megakaryocyte clone. The graph enclosed in a bold line in Figure 3(b) shows the megakaryocyte clones negative for HLA-A / B / C proteins.

[0093] As a result, 13 of the 71 clones were found to be HLA-A / B / C protein-negative, as shown in Figure 3(b). These results demonstrate that it is possible to generate megakaryocyte lines necessary for producing HLA-A / B / C protein-deficient platelet products.

[0094] [Experimental Example 4] (Platelet production from HLA null megakaryocytes) As in Experimental Example 1, the HLA-A / B / C genes of an immortalized human megakaryocyte cell line (imMKCL) were knocked out, and HLA-A / B / C protein-negative megakaryocytes (hereinafter sometimes referred to as "HLA null megakaryocytes") were sorted and collected using a FACS Aria (BD Biosciences).

[0095] Subsequently, the collected megakaryocytes were washed and cultured in a doxycycline-free medium to release the forced expression of the BMI1 gene, c-MYC gene, and BCL-xL gene. Specifically, wild-type megakaryocytes and HLA-null megakaryocytes were cultured in an E125 flask (catalog number "431143", Corning) at 1 x 10 in the medium whose composition is shown in Table 3 below. 5 The cells were seeded at a cell density of 100 cells / mL in 25 mL / flask and cultured at 37°C in 5% CO2 with shaking at 100 rpm. As a result, platelet production was induced from megakaryocytes.

[0096] [Table 3]

[0097] After 6 days of culture, a portion of the cell suspension was taken and stained for 30 minutes with APC-labeled anti-CD41 antibody (catalog number 303710, BioLegend), PE-labeled anti-CD42b antibody (catalog number 303906, BioLegend), and FITC-labeled anti-HLA-A / B / C antibody (catalog number 555552, BD Biosciences).

[0098] Next, flow cytometry analysis was performed using FACS Verse (BD Biosciences) to examine the expression of HLA-A / B / C proteins on platelets (CD41 / CD42b positive cells).

[0099] Figures 4(a) and (b) are graphs showing the results of flow cytometry analysis. As shown in Figure 4(a), first, the small cell population was gated based on forward scatter signal (FSC) / side scatter signal (SSC), and then the CD41-positive fraction was gated.

[0100] Next, as shown in Figure 4(b), the CD42b-positive fraction of the gated fraction was defined as platelets, and the expression of HLA-A / B / C proteins was examined. In Figure 4(b), "Wild type" indicates the results for platelets derived from wild-type megakaryocytes, and "HLA-A / B / C KO" indicates the results for platelets derived from HLA-null megakaryocytes. "Stained" indicates the results stained with FITC-labeled anti-HLA-A / B / C antibodies, and "unstained" indicates the results without staining.

[0101] As a result, it was revealed that platelets derived from wild-type megakaryocytes were positive for HLA-A / B / C protein expression, whereas platelets derived from HLA null megakaryocytes were negative for HLA-A / B / C protein expression.

[0102] [Experimental Example 5] (Study of genome editing efficiency in megakaryocytes) We investigated the genome editing efficiency by introducing Cas9 protein and gRNA into megakaryocytes using various methods to knock out the B2M gene. The immortalized human megakaryocyte cell line (imMKCL) used in Experimental Example 1 was used as the megakaryocyte.

[0103] Table 4 below shows the conditions for introducing the Cas9 protein and gRNA examined. The Cas9 protein was used in the form of a Cas9 expression plasmid or the Cas9 protein itself. The Cas9 expression plasmid used was pHL-EF1a-SphcCas9-iC-A. pHL-EF1a-SphcCas9-iC-A was generated by cloning the SphcCas9 cDNA of pHL-EF1a-SphcCas9-iP-A described in Li HL, et al., "Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and CRISPR-Cas9," Stem Cell Reports, 4, 143-154, 2015, into the pHL-EF1a-GW-iC-A vector.

[0104] The Cas9 protein was a commercially available product (product name: TrueCut TM Cas9 Protein v2, catalog number A36498, Thermo Fisher Scientific) was used.

[0105] The gRNA was used in the form of an expression plasmid for gRNA targeting the B2M gene or in the form of sgRNA. The expression plasmid for gRNA targeting the B2M gene was pHL-H1-B2M-sgRNA-mEF1a-RiH (see Suzuki D., et al., iPSC-Derived Platelets Depleted of HLA Class I Are Inert to Anti-HLA Class I and Natural Killer Cell Immunity, Stem Cell Reports, 14, 49-59, 2020).

[0106] The sgRNA used was a synthetic sgRNA targeting the B2M gene ("B2M-ex1g2", the target base sequence is shown in SEQ ID NO: 8).

[0107] We also investigated the effects of incubating the Cas9 protein and gRNA for 5 minutes before introducing them into cells to form a complex, and the effects of not allowing the complex to form.

[0108] Lipofection and electroporation were investigated as methods for introducing Cas9 protein and gRNA. For lipofection, Lipofectamine 2000 (Thermo Fisher Scientific) and Lipofectamine CRISPRMAX (Thermo Fisher Scientific), which is suitable for protein introduction, were used. For electroporation, 4D-Nucleofector TM (Lonza) and Amaxa TM P4 Primary Cell 4D-Nucleofector TM X KitS (Lonza) was used.

[0109] [Table 4]

[0110] Megakaryocytes after introduction of the Cas9 protein and gRNA were cultured in a medium having the composition shown in Table 1 above under culture conditions of 37°C and 5% CO2.

[0111] Four and seven days after transfection with the Cas9 protein and gRNA, each megakaryocyte was stained with FITC-conjugated anti-HLA-A / B / C antibody (catalog number 555552, BD Biosciences). The same cells were also stained with propidium iodide (PI). Flow cytometry analysis was then performed using a FACS Verse (BD Biosciences) to assess the percentage of megakaryocytes in which the B2M gene had been knocked out. Cell death was also assessed by analyzing the percentage of PI-positive cells.

[0112] Figures 5(a) and (b) and Figures 6(a) and (b) are graphs showing the analysis results 4 days after introduction of the Cas9 protein and gRNA.

[0113] Figure 5(a) shows the results for the control group in which neither Cas9 protein nor gRNA was introduced. In Figure 5(a), "stained" indicates the results of staining with FITC-labeled anti-HLA-A / B / C antibody, and "unstained" indicates the results without staining.

[0114] Figure 5(b) shows the results for the group in which the Cas9 protein and gRNA were introduced using Lipofectamine 2000. In Figure 5(b), "Plasmid vector" indicates the result of introducing the Cas9 protein and gRNA in the form of an expression plasmid, "RNP separate" indicates the result of not allowing a complex of the Cas9 protein and gRNA to form, and "RNP pre-mix" indicates the result of mixing the Cas9 protein and gRNA and then incubating to allow the complex to form.

[0115] In Figures 5(a) and (b), "SSC" indicates the side-scattered light signal, and "Anti-HLA-ABC" indicates the staining intensity with FITC-labeled anti-HLA-A / B / C antibodies.

[0116] Figure 6(a) shows the results for the group in which the Cas9 protein and gRNA were introduced using Lipofectamine CRISPRMAX, and Figure 6(b) shows the results for the group in which the Cas9 protein and gRNA were introduced by electroporation.

[0117] 6(a) and (b), "Plasmid vector" indicates the result of introducing the Cas9 protein and gRNA in the form of an expression plasmid. "RNP separate" indicates the result of not allowing the Cas9 protein and gRNA to form a complex. "RNP premix" indicates the result of mixing the Cas9 protein and gRNA and then incubating to allow the complex to form. "SSC" indicates the side scatter signal. "Anti-HLA-ABC" indicates the staining intensity with FITC-labeled anti-HLA-A / B / C antibody.

[0118] Figures 7(a) and (b) and Figures 8(a) and (b) are graphs showing the analysis results 7 days after introduction of the Cas9 protein and gRNA.

[0119] Figure 7(a) shows the results for the control group in which neither Cas9 protein nor gRNA was introduced. In Figure 7(a), "stained" indicates the results of staining with FITC-labeled anti-HLA-A / B / C antibody, and "unstained" indicates the results without staining.

[0120] Figure 7(b) shows the results for the group in which the Cas9 protein and gRNA were introduced using Lipofectamine 2000. In Figure 7(b), "Plasmid vector" indicates the result of introducing the Cas9 protein and gRNA in the form of an expression plasmid, "RNP separate" indicates the result of not allowing a complex of the Cas9 protein and gRNA to form, and "RNP pre-mix" indicates the result of mixing the Cas9 protein and gRNA and incubating them to allow the complex to form.

[0121] In Figures 7(a) and (b), "SSC" indicates the side-scattered light signal, and "Anti-HLA-ABC" indicates the staining intensity with FITC-labeled anti-HLA-A / B / C antibodies.

[0122] Figure 8(a) shows the results for the group in which the Cas9 protein and gRNA were introduced using Lipofectamine CRISPRMAX, and Figure 8(b) shows the results for the group in which the Cas9 protein and gRNA were introduced by electroporation.

[0123] 8(a) and (b), "Plasmid vector" indicates the result of introducing the Cas9 protein and gRNA in the form of an expression plasmid, "RNP separate" indicates the result of not allowing the Cas9 protein and gRNA to form a complex, and "RNP pre-mix" indicates the result of mixing the Cas9 protein and gRNA and then incubating to allow the complex to form. Furthermore, "SSC" indicates the side scatter signal, and "Anti-HLA-ABC" indicates the staining intensity with FITC-labeled anti-HLA-A / B / C antibody.

[0124] As a result, first, in an analysis using Cas9 and gRNA expression plasmid vectors, almost no genome editing was observed with the lipofection method (0.0% or 0.1%, Figures 5(b), 6(a), 7(b), and 8(a) "Plasmid vector"), while only slight genome editing was observed with the electroporation method (3.0% (Figure 6(b)) and 4.6% (Figure 8(b)), "Plasmid vector"). This reaffirmed the difficulty of introducing genes (plasmid DNA) into megakaryocytes.

[0125] Next, in an analysis using Cas9 protein and gRNA, genome editing efficiency was very low when Lipofectamine 2000 was used, regardless of whether incubation was performed or not (both were below 0.5%, Figure 5(b) and Figure 7(b) for "RNP separate" and "RNP pre-mix"). This result was thought to be due to the fact that Lipofectamine 2000 was not a suitable reagent for introducing Cas9 protein or sgRNA.

[0126] In contrast, when a lipofection reagent for protein introduction (Lipofectamine CRISPRMAX) was used, the genome editing efficiency was 0.1% (after 4 days) and 0.0% (after 7 days) without incubation, but increased to 2.1% (after 4 days) and 3.5% (after 7 days) with incubation (Figures 6(a) and 8(a)). Furthermore, when the protein was introduced by electroporation, the genome editing efficiency increased significantly, from 2.8% (without incubation) to 45.3% (with incubation) after 4 days (Figure 6(b)), and from 2.4% (without incubation) to 51.9% (with incubation) after 7 days (Figure 8(b)).

[0127] Therefore, when genome editing is performed by introducing Cas family proteins and gRNA into megakaryocytes, whether using lipofection or electroporation, which are suitable methods for protein introduction, it has been revealed that the genome editing efficiency increases significantly (by an order of magnitude) when a complex between Cas family proteins and gRNA is formed before introduction. In particular, the genome editing efficiency obtained when the complex is introduced using electroporation is extremely high, and no examples of genome editing with such high efficiency in megakaryocytes have been reported.

[0128] Furthermore, the results of PI staining analysis showed that the percentage of cells stained with PI was similarly low whether the Cas9 protein and gRNA were introduced by lipofection or electroporation, regardless of whether the complex was formed or not (less than 4.3% after 4 days, and less than 1.5% after 7 days). Therefore, it was revealed that the method of genome editing by introducing a complex of Cas family proteins and gRNA into megakaryocytes also causes sufficiently low damage (cytotoxicity) to megakaryocytes. [Industrial Applicability]

[0129] According to the present invention, a technique for producing genetically modified megakaryocytes and genetically modified platelets can be provided.

Claims

1. A method for producing genetically modified megakaryocytes, comprising: The method includes the step of introducing a CRISPR-associated (Cas) family protein and a guide RNA (gRNA) into megakaryocytes to modify a gene of interest, The production method, wherein the Cas family protein and the gRNA to be introduced into the megakaryocyte in the step form a complex in advance.

2. The method of claim 1, wherein the introduction of the Cas family protein and the gRNA is carried out by lipofection or electroporation, which is suitable for protein introduction.

3. The production method according to claim 1 or 2, wherein the introduction of the Cas family protein and the gRNA is carried out by electroporation.

4. The production method according to any one of claims 1 to 3, wherein the target gene is (i) Human Leukocyte Antigen (HLA)-A, HLA-B, and HLA-C genes, or (ii) β2-Microglobulin (B2M) gene, and the modification is disruption of the target gene.

5. The method according to any one of claims 1 to 4, wherein the megakaryocytes are obtained by differentiating pluripotent stem cells.

6. 1. A method for producing modified platelets, comprising: A step of introducing a Cas family protein and gRNA into megakaryocytes to modify a target gene and obtain a gene-modified megakaryocyte; producing platelets from the genetically modified megakaryocytes to obtain modified platelets; The production method, wherein in the step of obtaining the genetically modified megakaryocyte, the Cas family protein and the gRNA to be introduced into the megakaryocyte form a complex in advance.

7. The production method according to claim 6, wherein the introduction of the Cas family protein and the gRNA is carried out by lipofection or electroporation, which is suitable for protein introduction.

8. The production method according to claim 6 or 7, wherein the introduction of the Cas family protein and the gRNA is carried out by electroporation.

9. The method according to any one of claims 6 to 8, wherein the target gene is (i) an HLA-A, HLA-B, or HLA-C gene, or (ii) a B2M gene, and the modification is a disruption of the target gene.

10. The method according to any one of claims 6 to 9, wherein the megakaryocytes are obtained by differentiating pluripotent stem cells.

11. A method for producing genetically modified megakaryocytes, comprising: The method includes the step of introducing a Cas family protein and gRNA into megakaryocytes to modify a target gene, In the step, the introduction of the Cas family protein and the gRNA is carried out by electroporation.

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