An engineered cell and its application

Abstract

The present invention discloses an engineered cell and its application, wherein the engineered cell lacks or reduces the expression of HLA-A protein and / or HLA-B protein compared with the wild type. The engineered cell disclosed in the present invention can significantly reduce the immune rejection reaction after human hematopoietic cell infusion, while significantly improving the implantation rate and long-term reconstruction capacity in vivo. The cell will significantly expand the use of existing cord blood and bone marrow donor banks, reduce the need to recruit a large number of donors to match recipients, increase the probability of HLA matching donors, and thus expand the infusion of clinical-grade allogeneic cells. As a source of universal cells, engineered cells can realize HLA-matched off-the-shelf cell therapy, which has important prospects for clinical transplantation treatment.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular, to an engineered cell. Background Art

[0002] Cell infusion can be used to treat tumors, autoimmune diseases, and congenital genetic diseases. However, allogeneic cell infusion requires strict matching. Cell therapy is limited by bottlenecks such as difficulty in finding donors and low matching success rate. Immune rejection caused by human leukocyte antigen (HLA) mismatch can lead to failure of cell infusion. Transplantation failure and graft-versus-host disease (GVHD) are the main obstacles to cell therapy, which seriously affect the treatment effect and quality of life of patients. At present, there is no effective solution to the difficulty of cell infusion matching.

[0003] The success of allogeneic cell transplantation depends on the degree of matching of major histocompatibility complex genes between donor and recipient. Haploid cell infusion can improve the selection of HLA-matched donors, however, the incidence of transplantation failure and GVHD remains an obstacle to its therapeutic success due to cellular and antibody immune responses mediated by HLA molecules. Compared with adult donor cells, umbilical cord blood (UCB)-derived cells require less stringent HLA matching for transplantation. However, hematopoiesis is not fully restored after allogeneic UCB cell infusion due to HLA-specific antibodies in the recipient, and the occurrence of complications after cell infusion is exacerbated by the degree of HLA mismatch between UCB donors and recipients.

[0004] Therefore, how to regulate the immunogenicity of infused cells, reduce the rejection reaction of cell infusion, increase the probability of HLA-matched donors, and thus expand the transplantation of clinical-grade allogeneic cells and improve the engraftment and long-term reconstruction of donor cells is an important issue that needs to be addressed urgently. Summary of the invention

[0005] Technical issues solved:

[0006] One aspect of the present invention is to provide an engineered cell to address the problem of cell infusion failure caused by the immunogenicity of infused allogeneic cells in the prior art.

[0007] Specifically, the present disclosure aims at the deficiencies of the prior art and actual needs, and provides a method for lacking or reducing the expression of HLA-A protein and / or HLA-B protein in human cells, thereby obtaining a variety of low immunogenic cells, thereby solving the above-mentioned problems.

[0008] Technical solutions provided:

[0009] An engineered cell lacks or reduces the expression of HLA-A protein and / or HLA-B protein compared with a wild-type cell, and the cell is a human hematopoietic stem cell (HSPC).

[0010] In some embodiments of the present disclosure, the lack or reduction is achieved by knocking out or knocking down the HLA-A gene and / or HLA-B gene in the engineered cells.

[0011] In the present disclosure, any suitable method in the prior art can be used to knock out the above genes. In some embodiments of the present disclosure, the engineered cells contain a gene editing system for editing the HLA-A gene and / or the HLA-B gene. The gene knockout is achieved by the gene editing system.

[0012] Further, in some embodiments of the present disclosure, the gene editing system comprises transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs) or Cas9 protease.

[0013] Further, in some embodiments of the present disclosure, the gene editing system comprises a Cas9 protease and a guide RNA, wherein the Cas9 protease is exogenously transferred or expressed in the cell.

[0014] Further, in some embodiments of the present disclosure, the guide RNA includes one or more selected from the nucleotide sequences shown in SEQ ID No.1-21. The sgRNA shown in SEQ ID No.1-21 is used to efficiently knock out or knock down the HLA-A gene. In some embodiments of the present disclosure, the guide RNA also includes a nucleotide sequence shown in SEQ ID No.22, and the nucleotide sequence shown in SEQ ID No.22 is used to efficiently knock out or knock down the HLA-B gene.

[0015] Further, in some embodiments of the present disclosure, the guide RNA is selected from one or more of the nucleotide sequences shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3.

[0016] In the present disclosure, any suitable method in the prior art can be used to knock down the above genes. In some embodiments of the present disclosure, the engineered cells contain inhibitory nucleic acids targeting genes or mRNAs of HLA-A and / or HLA-B.

[0017] Further, in some embodiments of the present disclosure, the inhibitory nucleic acid includes antisense oligonucleotides, small interfering RNA (siRNA) or dsRNA used in RNA interference (RNAi) against genes or proteins of HLA-A and / or HLA-B.

[0018] Another aspect of the present disclosure is to provide use of the engineered cells described above in preparing products for cell infusion.

[0019] Another aspect of the present disclosure is to provide a pharmaceutical composition comprising the engineered cells, the gene editing system or the inhibitory nucleic acid.

[0020] Beneficial effects:

[0021] The engineered cells provided by the present disclosure can significantly reduce the activation of allogeneic T cells and will not activate NK cells, thereby reducing the infusion immune rejection reaction. In addition, the implantation rate and long-term hematopoietic reconstruction ability of the engineered cells in the body are significantly improved. The cells will significantly expand the use of existing cord blood and bone marrow donor banks, reduce the need to recruit a large number of donors to match recipients, and increase the probability of HLA matching donors, thereby expanding the infusion of clinical-grade allogeneic cells. As a source of universal cells, engineered cells can achieve HLA-matched off-the-shelf cell therapy, which has important clinical treatment prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow cytometry result diagram of the knockout / knockdown efficiency of the Ctrl group and each group of cells after knocking out / knockdown of HLA-A, HLA-B, HLA-A and B, and HLA-C in the disclosed embodiment;

[0023] Figure 2 It is a statistical result diagram of the editing efficiency of the Ctrl group, HLA-A group, HLA-B group, HLA-C group and HLA-AB group in the embodiments of the present disclosure;

[0024] Figure 3 It is a statistical result diagram of the knockout efficiency of the Ctrl group, HLA-A group, HLA-B group, HLA-C group and HLA-AB group in the embodiments of the present disclosure;

[0025] Figure 4 This is a flow cytometry result diagram of the Ctrl group, HLA-A group, HLA-B group, HLA-C group and HLA-AB group escaping from the heterologous T cell immune response in the embodiments of the present disclosure;

[0026] Figure 5It is a statistical result diagram of escaping heterologous T cell immune response of Ctrl group, HLA-A group, HLA-B group, HLA-C group and HLA-AB group in the embodiment of the present disclosure;

[0027] Figure 6 It is a statistical result diagram of NK activation of the Ctrl group, HLA-AB group, HLA-ABC group and HLA-B2M group in the embodiment of the present disclosure;

[0028] Figure 7 The human CD45 in the peripheral blood of mice after the cells of the Ctrl group, HLA-A group, HLA-B group and HLA-AB group were infused into the mice in the embodiments of the present disclosure. + Statistical results of cell proportion;

[0029] Figure 8 This is a statistical result diagram of the proportion of each lineage of cells in the peripheral blood of mice after the cells of the Ctrl group, HLA-A group, HLA-B group and HLA-AB group were infused in the embodiments of the present disclosure;

[0030] Fig. 9 The human CD45 in the bone marrow of the Ctrl group, HLA-A group, HLA-A group and HLA-AB group cells were infused into mice in the disclosed embodiments. + Statistical results of cell implantation rate;

[0031] Fig.10 This is a statistical result diagram of the proportion of each lineage of cells in the bone marrow of mice after the cells of the Ctrl group, HLA-A group, HLA-B group and HLA-AB group were infused in the embodiments of the present disclosure;

[0032] Fig.11 The human CD34 in the bone marrow of the Ctrl group, HLA-A group, HLA-B group and HLA-AB group cells were infused into mice in the disclosed embodiments. + CD38 - Statistical results of cell proportion;

[0033] Fig.12 The CD34 expression in the bone marrow of the Ctrl group, HLA-A group, HLA-B group and HLA-AB group cells after infusion into mice in the disclosed embodiments + CD38 + Statistical results of cell proportion;

[0034] Fig.13This is a knockout effect result diagram of 21 sgRNAs screened by transient transfection method on 293T cell line in the disclosed embodiment, showing the proportion of HLA-A low-expressing cells after each sgRNA knockout, and the proportion of HLA-A knockout cells in the HLA-A-ex2-g13 group with the best knockout effect can reach 60%. In addition, the HLA-A gene site targeted by each sgRNA is marked in the figure, where P is Promoter, Ex is exon, and Int is Intron;

[0035] Fig.14 This is a diagram showing the knockout site results of HLA-A-ex2-g13 (SEQ ID No. 1), HLA-A-ex3-g167 (SEQ ID No. 2), and HLA-A-ex2-g452 (SEQ ID No. 3) on the human HLA-A gene in the embodiments of the present disclosure;

[0036] Fig.15 The three sgRNAs with the best knockout effect on HLA-A protein in 293T cell lines were selected in the present embodiment: HLA-A-ex2-g13 (SEQ ID No. 1), HLA-A-ex3-g167 (SEQ ID No. 2), and HLA-A-ex2-g452 (SEQ ID No. 3). The knockout effect was further tested on hematopoietic cells derived from umbilical cord blood. The flow cytometry graph is CD34 + , CD34 + CD90 + and CD34 + CD90 - Cell gating strategy;

[0037] Fig.16 , Fig.17 The knockout effect of HLA-A-ex2-g13 (SEQ ID No. 1), HLA-A-ex3-g167 (SEQ ID No. 2), and HLA-A-ex2-g452 (SEQ ID No. 3) on hematopoietic cells derived from cord blood in the embodiments of the present disclosure, wherein: Fig.16 It showed that all three sgRNAs could effectively knock out CD34 + , CD34 + CD90 + and CD34 + CD90 - HLA-A protein on the cell surface, 3 sgRNAs knocked out CD34 + CD90 + The cell ratio was above 94%, and the HLA-A-ex2-g13 (SEQ ID No.1) group CD34 +CD90 + The proportion of HLA-A knockout cells can reach 97%. Fig.17 Statistical analysis showed that after knockout of the three sgRNAs, the proportion of HLA-A positive cell population decreased significantly;

[0038] Fig.18 , Fig.19 This is a specific result diagram in the embodiment of the present disclosure, wherein: Fig.18 Compared with the control group, CD34 + CD90 + There was no significant difference in the expression of HLA-ABC on the cell surface, indicating that the three sgRNAs targeting HLA-A knockout had significant specificity. The use of these three sgRNAs to knock out HLA-A did not affect CD34 + , CD34 + CD90 + and CD34 + CD90 - The expression of HLA-B and HLA-C molecules on the cell surface Fig.19 Statistical analysis showed that after knockout of the three sgRNAs, the proportion of HLA-ABC-positive cell populations did not change significantly;

[0039] Fig. 20 , Fig.21 This is a specific result diagram in the embodiment of the present disclosure, wherein: Fig. 20 Compared with the control group, CD34 + CD90 + There was no significant difference in the expression of HLA-DR molecules on the cell surface. Fig.21 Statistical analysis showed that after the three sgRNA knockout, the proportion of HLA-DR positive cell population did not change significantly, indicating that the three sgRNA targeted knockout of HLA-A had significant specificity, and the use of these three sgRNAs to knock out HLA-A did not affect CD34 + , CD34 + CD90 + and CD34 + CD90 - Expression of HLA-II class molecules on the cell surface.

[0040] Sequence Description

[0041] Sequence Listing

[0042] DETAILED DESCRIPTION

[0043] The present invention discloses an engineered cell, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention, and relevant personnel can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0044] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" etc. will be understood to include the elements or components stated, without excluding other elements or other components. The term "a", "an" and "the" include plural indicators. The term "multiple" refers to two or more. The terms "such as", "for example", etc. are intended to refer to exemplary embodiments, and are not intended to limit the scope of the present disclosure.

[0045] In the present disclosure, when a range of values ​​is provided, it should be understood that the endpoints are included in the range and each intervening value between the upper and lower limits of the range and any other specified value or intervening value in the specified range and any value in the smaller range between the specified values ​​are included unless the context clearly dictates otherwise.

[0046] In this disclosure, the term "about" generally refers to variations within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0047] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The definitions of common terms in molecular biology can be found in Lewin's GENES, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Publisher: Jones & Bartlett Learning. The definitions of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: WH Freeman. The definitions of common terms in cell biology can be found in Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Publisher: Garland Science. The definitions of common terms in genetics can be found in Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Publisher: Jones & Bartlett Learning.

[0048] Unless otherwise specified, the experimental techniques herein employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found in standard books such as the following: Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook, etc.

[0049] the term:

[0050] The term "hematopoietic stem and progenitor cells (HSPC)" used in the present disclosure refers to hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs). Hematopoietic stem cells are adult stem cells in the blood system, which have the ability of long-term self-renewal and the potential to differentiate into various types of mature blood cells. The various types of mature blood cells include red blood cells and white blood cells. Hematopoietic progenitor cells are progenitor cells of various types of blood cells that proliferate and differentiate into hematopoietic stem cells under the regulation of a certain microenvironment and certain factors. It is also a rather primitive cell with proliferation ability, but has lost its multidirectional differentiation ability and can only proliferate and differentiate toward one or several blood cell lines, so it is also called committed stem cells. In some embodiments of the present disclosure, the hematopoietic stem and progenitor cells can be derived from bone marrow, peripheral blood or umbilical cord blood.

[0051] The term "wild type" used in the present disclosure is a concept relative to mutant type, and generally refers to individuals obtained from nature in research without artificial mutagenesis. Such individuals carry a wild type genome. In some embodiments of the present disclosure, wild type refers to unengineered.

[0052] The term "reduction" used in the present disclosure means that the expression level (expression amount) of the target protein in the cell is less than the expression level (expression amount) of the target protein in the wild-type cell. The reduction can be reduced to about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50% or less, but not 0%. The method for determining the expression level (expression amount) of the target protein may include, for example, Western Blot, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, flow cytometry, etc.

[0053] The term "knockout" used in this disclosure is used to describe the process of completely or partially inactivating a specific gene in an organism through gene editing technology. Knockout technology generally involves the use of gene editing tools, such as the CRISPR-Cas9 system, to accurately locate and cut the DNA sequence of the target gene. After the DNA sequence is cut, the cell usually attempts to repair the broken DNA through mechanisms such as non-homologous end joining (NHEJ) or homologous recombination (HDR). However, these repair processes may result in the insertion, deletion or replacement of the gene sequence, causing the target gene to lose its original function or expression level.

[0054] The term "knockdown" as used in this disclosure refers to reducing the expression level of a gene in an organism by specific technical means, rather than completely eliminating the function of the gene, such as through antisense RNA, ribonuclease or CRISPR-Cas system interference (CRISPRi) and other methods. The main difference between knockdown and knockout is the degree: knockout is the complete or partial elimination of the function of a gene, while knockdown only reduces its expression level. Therefore, knockdown allows researchers to study the effects of reduced gene expression rather than complete absence.

[0055] The term "HLA-A" used in the present disclosure is an abbreviation for human leukocyte antigen A, also known as MHC class I antigen. It is a glycoprotein expressed on the surface of almost all cells in the human body and is part of the major histocompatibility complex (MHC). The heavy chain of the HLA-A molecule is about 45 kDa, and its gene contains 8 exons. Exon 1 encodes the leader peptide, exons 2 and 3 encode the α1 and α2 domains (both of which bind peptides), exon 4 encodes the α3 domain, exon 5 encodes the transmembrane region, and exons 6 and 7 encode the cytoplasmic tail. In particular, polymorphisms within exon 2 and exon 3 are related to the peptide binding specificity of each HLA-A molecule, and typing of these polymorphisms is often used for matching of bone marrow and kidney transplants. In some embodiments of the present disclosure, the lack or reduction of the expression of HLA-A protein also includes the lack or reduction of the expression of the allele corresponding to the HLA-A protein, including HLA-A1, HLA-A2, HLA-A3, HLA-A11 or HLA-A24.

[0056] CRISPR-Cas system:

[0057] The CRISPR-Cas system is widely present on the chromosomes of bacteria and archaea, and is related to their immunity, used to defend against foreign genetic material and acquire the ability to resist bacteriophages. The CRISPR-Cas system is the third generation of gene editing technology after the introduction of gene editing technologies such as ZFN and TALENs. The CRISPR-Cas system can be divided into three main types: Type I, Type II, and Type III. The CRISPR Type I system is the earliest discovered and studied CRISPR system, which is mainly present in most bacteria and archaea. The system consists of multiple Cas proteins and multiple CRISPR RNAs (crRNAs) to form a complex Cas protein complex. This complex recognizes and cuts the target fragment through complementary pairing of crRNA and exogenous DNA. The Type I system is characterized by having multiple Cas proteins, among which the Cas3 protein has RNA-dependent DNA nuclease activity and is responsible for cutting the target DNA. The CRISPR Type II system, also known as the CRISPR / Cas9 system, is currently the most widely used gene editing tool. The CRISPR / Cas9 system only requires Cas9 protein and crRNA to form a complex with trans-activating CRISPR-derived RNA (tracrRNA) to identify and shear the target DNA fragment. Cas9 protein has two nuclease activities, HNH and RuvC, which can shear the two chains of the target DNA respectively. The structure and function of the CRISPR Type III system are similar to those of the Type I system, but it has more Cas proteins and more complex mechanisms. Similar to the Type I system, the Type III system also requires multiple Cas proteins and crRNA to form a complex, and recognizes and shears the target fragment through complementary pairing with exogenous DNA. However, the shearing mechanism of the Type III system is different from that of the Type I system, and it relies on the RNase activity of the Cas protein to cut the target DNA. In the embodiments of the present disclosure, the sgRNA is used in the Type II CRISPR-Cas system.

[0058] Cas proteins:

[0059] In the embodiments of the present disclosure, the Cas protein used in the CRISPR-Cas system is mainly Cas9. Cas9 is a nuclease that can accurately recognize and cut DNA sequences under the guidance of sgRNA (small guide RNA). In some embodiments of the present disclosure, the Cas9 protein can be isolated or loaded on a vector for expression in cells. In addition to Cas9, other auxiliary proteins may also be included in other embodiments of the present disclosure, including but not limited to Cas1, Cas2, etc. They may play an auxiliary role in the DNA cutting process, or participate in other links of the CRISPR system, such as DNA acquisition and processing.

[0060] In an embodiment of the present disclosure, the Cas protein may be modified. The modification may include mutation, insertion or deletion of amino acid residues, optimization of codons, and other chemical modifications. The purpose may be to change the activity or specificity of the Cas protein without changing its main function. For example, U.S. Patent US20180100148A1 describes mutations in Streptococcus pyogenes Cas9 nucleic acids and proteins to reduce the off-target effects of the system.

[0061] Inhibitory nucleic acids:

[0062] In the present disclosure, the term "inhibitory nucleic acid" (INA) is a class of nucleic acid molecules that can inhibit the expression of specific genes or protein functions. Through specific sequence design and modification, they can bind to the complementary sequence of target genes or proteins, thereby interfering with their normal functions or expression levels. Common examples of inhibitory nucleic acids include antisense oligonucleotides, small interfering RNA (siRNA), RNA interference (RNAi), etc.

[0063] Guide RNA and its combination:

[0064] In the embodiments of the present disclosure, the nucleotide sequences of the sgRNAs shown in SEQ ID No. 1-21 can efficiently knock down and / or knock out the human HLA-A gene. The nucleotide sequence shown in SEQ ID No. 22 is used to efficiently knock out or knock down the HLA-B gene. After knowing the nucleotide sequence, it can be obtained by a suitable method in the prior art, including but not limited to chemical synthesis, polymerase chain reaction (PCR), etc.

[0065] In the embodiments of the present disclosure, the above-mentioned sgRNA is used to recognize and knock down and / or knock out the human HLA-A gene in cells through the CRISPR gene editing system, and its efficiency in the 293T cell line can reach more than 20%, and the highest efficiency in cord blood cells can reach 97%, which is much better than the methods in the prior art. Among them, the efficiency of the three sgRNAs HLA-A-ex2-g13, HLA-A-ex3-g167, and HLA-A-ex2-g452 is the best. Therefore, as a preference, in some embodiments of the present disclosure, the nucleotide sequence of the sgRNA is shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3.

[0066] In the process of gene editing, the above sgRNA can be used alone, and in some embodiments of the present disclosure, it can achieve an editing efficiency of about 20%, about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%; it can also be used in combination, or other sgRNAs can be added, and in other embodiments of the present disclosure, it can also achieve an editing efficiency of about 20%, about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%. In some embodiments of the present disclosure, sgRNA can be loaded in the same container or loaded in different containers and transferred into cells.

[0067] The nucleotide sequence of the above-mentioned sgRNA can be modified. The modification includes, but is not limited to, substitution, insertion, deletion or exchange or replacement region of one or more nucleotides for the purpose of improvement, and modification of the nucleotide sequence using other groups. In some embodiments of the present disclosure, the mutated sgRNA nucleotide sequence can have at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, at least 80%, at least 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity compared to SEQ No.1-21.

[0068] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0069] Example:

[0070] Example 1: Construction of cells with knockout / knockdown of HLA-A, HLA-B, HLA-C and HLA-AB genes

[0071] 1. CD34 derived from cord blood, bone marrow or peripheral blood + The cells were divided into 5 groups, namely, Ctrl group, knockout / knockdown HLA-A, HLA-B, HLA-C and HLA-AB groups. Each group of cells was resuspended in electroporation buffer, and RNPs without sgRNA were added to the Ctrl group, RNPs containing HLA-A sgRNA were added to the knockout HLA-A group, RNPs containing HLA-BsgRNA were added to the knockout HLA-B group, RNPs containing HLA-C sgRNA were added to the knockout HLA-C group, and RNPs containing HLA-AsgRNA and HLA-B sgRNA were added to the HLA-AB group.

[0072] 2. Each group of cells with RNP added was electroporated under an electroporator. The gene editing method is as follows:

[0073] (1) Place the protein and sgRNA (1:2) in the Lonza electroporation kit into a 1.5 ml EP tube and react at room temperature for 10-15 minutes.

[0074] (2) Count the cells, aspirate the cell suspension and place it in a 50 ml centrifuge tube. Centrifuge at 500g for 5 minutes.

[0075] (3) Discard the supernatant, resuspend the cells in PBS and wash each tube once, then centrifuge at 500g for 5 min.

[0076] (4) Discard the supernatant, resuspend the cells in buffer, add the RNP prepared in step (1) (final volume is 20ul system), mix well by pipetting with the tip of a pipette, and place in the electroporation well plate.

[0077] (5) After electroporation on the Lonza electroporator, pipette the cell suspension from the electroporation plate into a 15 ml centrifuge tube. Centrifuge at 500 g for 5 min and discard the supernatant.

[0078] (6) Resuspend each tube in 2 ml of culture medium and transfer to a culture dish.

[0079] 3. After electroporation, the cells were quickly resuspended in culture medium and cultured in an incubator for 24 hours.

[0080] 4. Sequence the gene fragments to detect the efficiency of knockout, and use flow cytometry to detect the editing efficiency of each gene.

[0081] The results are as follows Figure 1 , 2As shown in Figures 3 and 4, the gene editing efficiency percentages of the HLA-A, HLA-B, HLA-C and HLA-AB groups were 86.25±3.30, 66±3.36, 74.75±4.42, and 66.75±4.78, respectively; the gene knockout efficiency percentages were 25.99±1.67, 29.81±4.15, 22.23±0.79, and 1.71±0.13, respectively.

[0082] Example 2: Obtaining three engineered cells with reduced immunogenicity by knocking out / knockdown of HLA-A, HLA-B and HLA-AB genes

[0083] 1. The cells in the Ctrl group, HLA-A, HLA-B, HLA-C, and HLA-AB knockout / knockdown groups were pretreated with 50 ng / mL IFN-γ for 48 hours, and sgRNA and RNP were added to each group for electroporation (RNP without sgRNA was added to the Ctrl group).

[0084] 2. Isolate human peripheral blood cells as follows:

[0085] (1) Place freshly obtained healthy human peripheral blood (PBMC) in a sterile 50 mL centrifuge tube, add red blood cell lysis buffer, mix thoroughly, and let stand at room temperature for at least 6 min to lyse red blood cells;

[0086] (2) Centrifuge at 1500 rpm for 5 min at 4°C;

[0087] (3) Discard the supernatant, resuspend the cells in 20 mL PBE per tube, mix thoroughly, take 10 μL for counting, and centrifuge at 1500 rpm for 5 min at 4°C;

[0088] (4) Discard the supernatant and resuspend the collected human peripheral cells in IMDM-FBS for later use.

[0089] 3. Add PBMC to 500 μL PBS and mix well to make a single cell suspension for sorting T cells. Preparation of RPMI 1640 complete medium: RPMI 1640 basal medium + 10% FBS + 1% Glutamax + 1% penicillin-streptomycin solution; all subsequent steps are performed under light protection. Preparation of CFSE working solution: 1 μL CFSE + 500 μL PBS in each portion, mix thoroughly by pipetting.

[0090] 4. Add one portion of CFSE working solution to each resuspended single cell suspension, mix thoroughly by pipetting, and let stand at room temperature for 10 minutes.

[0091] 5. Quickly add 1 ml of pre-cooled 50% FBS and mix by pipetting to terminate staining. Let stand on ice for 2 min and centrifuge at 300 g for 10 min.

[0092] 6. Add 2 ml of 1640 complete medium to each tube, mix thoroughly by pipetting, and centrifuge at 300 g for 10 min.

[0093] 7. Repeat step 6.

[0094] 8. The engineered cells pretreated for 48 h on day 1 were mixed with T cells at a 1:1 ratio in 96-well U-bottom plates in RPMI-1640 containing glutamine, 10% FBS, and 20 U / mL IL-2 for 3-5 days.

[0095] 9. Flow cytometry to detect proliferative CD8 + The percentage of T cells (T cells that have become negative for CFSE labeling).

[0096] The results are as follows Figure 4 , 5 The results showed that after co-culture of gene-edited cells with T cells, knockout / knockdown of HLA-A, HLA-B and HLA-AB could significantly reduce their activation of allogeneic T cells compared with the Ctrl group, but knockout of HLA-C still significantly activated T cells.

[0097] Example 3: Effect of HLA-C knockout / knockdown cells on NK cell activity

[0098] The cells with HLA-B2M knocked out / knocked down were set as the positive control group, and HLA-A and HLA-B were knocked out / knocked down simultaneously using the method of Example 1 to obtain HLA-AB group cells, and HLA-A, HLA-B and HLA-C were knocked out / knocked down simultaneously to obtain HLA-ABC group cells.

[0099] 2. RNP without sgRNA was added to the Ctrl group, and the rest was the same as the experimental group. Each group of cells co-electroporated with RNP was pretreated with 50 ng / mL IFN-γ for 48 hours.

[0100] 3. Add PBMC to 500 μL PBS and mix well to make a single cell suspension, and use CD3 and CD56 antibodies to sort NK cells.

[0101] 4. Preparation of RPMI1640 complete medium: RPMI1640 basal medium + 10% FBS + 1% Glutamax + 1% Penicillin-Streptomycin (PS) solution.

[0102] 5. The cells in the Ctrl group, HLA-AB group, HLA-ABC group, and HLA-B2M group were co-cultured with NK cells in a U-bottom 96-well plate for 5 hours. Flow cytometry was used to detect the percentage of CD107a-positive cells in NK cells.

[0103] The statistical results are as follows Figure 6 The results showed that compared with the Ctrl group, knockout / knockdown of HLA-AB did not activate NK cells, but knockout / knockdown of HLA-C significantly activated NK cells.

[0104] Example 4: Engineered cells have significantly increased ability to engraft and reconstitute in vivo

[0105] The three engineered cells with low immunogenicity HSPC (human CD34+ cells with knockout / knockdown of HLA-A, HLA-B and HLA-AB) prepared in Examples 1 and 2 were transplanted into immunodeficient mice, respectively, and the cell engraftment rate and hematopoietic reconstruction in each group were dynamically detected as follows:

[0106] 1. Female NOG mice aged 6-8 weeks and weighing 18-22 g were randomly divided into groups and irradiated with 2 Gy of X-rays at a dose rate of 1.2 Gy / min before transplantation.

[0107] 2. The knockout / knockdown cells of human HLA-A, HLA-B and HLA-AB were obtained by the method of Example 1 and transplanted through the tail vein, respectively, at a rate of 1×10 5 The cells were transplanted into NOG mice, and the human CD45 in the peripheral blood of mice was continuously detected 4-20 weeks after transplantation. + The proportion of cells.

[0108] 3. 20 weeks after transplantation, mice were killed by cervical dislocation, and bone marrow cells were collected, washed with staining buffer and resuspended, antibodies were added, and incubated at 4°C in the dark for 30 min.

[0109] 4. Wash once with 2 mL of staining buffer and use flow cytometry to detect human cell engraftment and the composition of each lineage in the mouse bone marrow.

[0110] The results showed that compared with the control group, human engineered cells had a significantly higher expression of CD45 in the peripheral blood of mice. + The percentage of cells increased significantly, and the main cell composition was CD33 + Myeloid cells and CD19 + The results of B cells Figure 7 , 8 As shown. In the mouse bone marrow, the implantation rate of human engineered cells was also significantly higher than that of the control group, and the main cell composition was CD33 + Myeloid cells and CD19 +The results of B cells Fig. 9 , 10 As shown in the figure, it shows that after HLA gene modification, the reconstruction of various lineages of engineered cells in vivo is not affected. In addition, in the mouse bone marrow, the engineered cells CD34 + CD38 - and CD34 + CD38 + The proportion of cells was significantly higher than that of the control group (the results are shown in Fig.11 , 12 as shown).

[0111] Example 5: Testing the knockout effect of sgRNA in 293T cell line using transient transfection method

[0112] 1) 293T cells were plated in 24-well plates 24 hours in advance, with 2×10 cells per well. 5 cell.

[0113] 2) Replace DMEM complete medium with 0.4 ml DMEM medium (containing 10% FBS, without penicillin / streptomycin) and place in the incubator.

[0114] 3) Prepare the transfection system: first add 50ul Opti medium into a 1.5ml EP tube, then add 1ug / well of the target plasmid (0.5ug / well each of sg plasmid and Cas9 plasmid, sg plasmid is prepared by conventional methods), invert to mix, do not vortex.

[0115] 4) Take another 1.5ml EP tube, add 50ul optim medium first, then add 3ug / well PEI, invert to mix, do not vortex.

[0116] 5) Leave at room temperature for 5 minutes.

[0117] 6) Mix 3) and 4) in one tube, invert to mix well, do not vortex, and let stand for 20 minutes.

[0118] 7) Add the plasmid system into the dish and shake well.

[0119] 8) After 12-16 hours, replace the culture medium with DMEM complete medium.

[0120] 9) 48 hours after transfection, add 2ug / ml puromycin to screen.

[0121] 10) After 48 hours of puromycin screening, the HLA-A knockout effect of each sgRNA was detected.

[0122] 11) Flow cytometry results showed that among the 21 sgRNAs, HLA-A-ex2-g13 (SEQ ID No.1), HLA-A-ex3-g167 (SEQ ID No.2), and HLA-A-ex2-g452 (SEQ ID No.3) had the best knockout effects, and the proportion of HLA-A knockout cells in the HLA-A-ex2-g13 group could reach 60%. (Results are shown in Fig.13 ).

[0123] 12) The knockout sites of HLA-A-ex2-g13 (SEQ ID No. 1), HLA-A-ex3-g167 (SEQ ID No. 2), and HLA-A-ex2-g452 (SEQ ID No. 3) on the human HLA-A gene are as follows Fig.14 shown.

[0124] Example 6: Detection of the knockout effect of sgRNA on hematopoietic cells derived from cord blood

[0125] 1. Enrichment of cord blood CD34 + Cells were cultured overnight in hematopoietic cell basal medium. Hematopoietic cell basal medium was SFEMII medium supplemented with the following cytokines (Table 1):

[0126] Table 1

[0127]

[0128] 2. Select three sgRNAs with good knockout effects on 293T cells: HLA-A-ex2-g13 (SEQ ID No.1), HLA-A-ex3-g167 (SEQ ID No.2), and HLA-A-ex2-g452 (SEQ ID No.3), and knock out CD34 by electroporation RNP method according to the electroporation method of Lonza P3Primary Cell 4D-Nucleofector™ X Kit S + Cell surface HLA-A protein.

[0129] 1) CD34 + Cell count: 2 × 10 cells were collected from each sample. 5 cell.

[0130] 2) Prepare the electroporation system: first add buffer to each tube, then add 75pmol spCas9 protein and 150pmol sgRNA, a total of 5ul. React at room temperature for 20-30min.

[0131] 3) Resuspend the cells in 15ul of buffer, add RNP (to make a 20ul system), and pipette the system >10 times. Add liquid and do not add bubbles.

[0132] 4) Follow the instructions of the 4D electroporation instrument.

[0133] 5) After electroporation, transfer the cell suspension in the electroporation tank to a 1.5 ml centrifuge tube. Centrifuge at 500g for 5 minutes and discard the supernatant.

[0134] 3. Transfer cells to 24-well plates for culture and observation. After 72 hours, flow cytometry was used to detect CD34 + 、CD34 + CD90 + and CD34 + CD90 - Expression of HLA-A, HLA-ABC and HLA-DR on cell surface. Antibodies labeled by flow cytometry are shown in Table 2.

[0135] Table 2

[0136]

[0137] 4. Flow cytometry results showed that all three sgRNAs could significantly knock out CD34 + 、CD34 + CD90 + and CD34 + CD90 - HLA-A protein on the cell surface, 3 sgRNAs knocked out CD34 + The cell ratio was above 94%, and the HLA-A-ex2-g13 group CD34 + CD90 + The proportion of HLA-A knockout cells on cells can reach 97% ( Fig.15 It is a flow gate strategy, and the results are shown in Fig.16 , Fig.17 In addition, three sgRNA groups CD34 + 、CD34 + CD90 + and CD34 + CD90 - There was no significant change in the proportion of HLA-ABC and HLA-DR positive groups on the cells. After statistical analysis, the three sgRNAs knocked out HLA-A and had an effect on HLA-B and HLA-C in the HLA-I molecules of the three groups of cells (see the results). Fig.18 , Fig.19 ) and HLA-II class molecule expression had no significant effect (see Fig. 20 , Fig.21), that is, the sgRNA we used is highly targeted.

[0138] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An engineered cell, characterized in that: The engineered cells reduce the expression of HLA-A protein compared to wild-type cells, and the cells are human hematopoietic stem cells (HSPCs); The engineered cells contain a gene editing system for editing the HLA-A gene; The gene editing system includes Cas9 protease and guide RNA; The nucleotide sequence of the guide RNA is shown in SEQ ID No.

1.

2. The engineered cell according to claim 1, characterized in that: The reduction is achieved by knocking out or knocking down the HLA-A gene in the engineered cells.

3. Use of the engineered cells as claimed in claim 1 or 2 in the preparation of a product for cell infusion.

Images