Engineered cells and use thereof

Abstract

Disclosed are engineered cells and use thereof. Compared with the wild type, the engineered cells lack or reduce the expression of the HLA-A protein and / or the HLA-B protein. The disclosed engineered cells can significantly reduce the immune rejection response after artificial blood cell infusion, and significantly improve the implantation rate and long-term reconstruction ability in vivo. The cells will significantly expand the use of existing cord blood and bone marrow donor libraries, reduce the need to recruit a large number of donors to match receptors, and increase the probability of HLA matching donors, thereby expanding the infusion of clinical-grade allogeneic cells. As a source of universal cells, the engineered cells can enable HLA-matching off-the-shelf cell therapy, showing great promise in the area of clinical transplantation treatment.

Description

An engineered cell and its applications

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202410772710.X, filed with the Chinese Patent Office no earlier than June 17, 2024, entitled "An Engineered Cell and Its Application", the entire contents of which are incorporated herein by reference.

[0003] This application includes a sequence list, which was submitted electronically in .XML format on June 17, 2024. The sequence list contained in the .XML file is part of the specification and is hereby incorporated herein by reference in its entirety. Technical Field

[0004] This disclosure relates to the field of biotechnology, and in particular, to an engineered cell. Background Technology

[0005] Cell infusion can be used to treat tumors, autoimmune diseases, and congenital genetic disorders. However, allogeneic cell infusion requires strict matching. Cell therapy is limited by bottlenecks such as difficulty in finding donors and low matching success rates. Immune rejection caused by human leukocyte antigen (HLA) mismatch can lead to cell infusion failure. Transplantation failure and graft-versus-host disease (GVHD) are major obstacles to cell therapy, seriously affecting patient treatment outcomes and quality of life. Currently, there is no effective solution to the difficulty of cell infusion matching.

[0006] The success of allogeneic cell transplantation depends on the degree of genetic matching of the major histocompatibility complex (MHC) between the donor and recipient. Haploidentical cell infusion can improve the selection of HLA-matched donors; however, due to HLA-mediated cellular and antibody immune responses, transplantation failure and the incidence of GVHD remain obstacles to its therapeutic success. Compared to adult donor cells, umbilical cord blood (UCB)-derived cells require less stringent HLA matching for transplantation. However, due to HLA-specific antibodies in the recipient, hematopoiesis may not be fully restored after allogeneic UCB cell infusion; and the occurrence of post-infusion complications is exacerbated by the degree of HLA incompatibility between the UCB donor and recipient.

[0007] Therefore, how to regulate the immunogenicity of infused cells, reduce cell infusion rejection, increase the probability of HLA-matched donors, thereby expanding the transplantation of clinical-grade allogeneic cells and improving the implantation and long-term reconstitution of donor cells, is an important problem that urgently needs to be solved. Summary of the Invention

[0008] Technical problems to be solved:

[0009] One aspect of the present invention addresses the problem of cell infusion failure caused by the immunogenicity of allogeneic cells in the prior art by providing an engineered cell.

[0010] Specifically, this disclosure addresses the shortcomings of existing technologies and practical needs by providing a method for reducing or eliminating the expression of HLA-A and / or HLA-B proteins in human cells, thereby obtaining a variety of low-immunogenic cells and solving the aforementioned problems.

[0011] Technical solutions provided:

[0012] An engineered cell, which, compared to wild-type cells, lacks or reduces the expression of HLA-A and / or HLA-B proteins, wherein the cell is a human hematopoietic stem cell (HSPC).

[0013] In some embodiments of this disclosure, the absence or reduction is achieved by knocking out or knocking down the HLA-A gene and / or HLA-B gene in the engineered cells.

[0014] In this disclosure, the aforementioned genes can be knocked out using any suitable method available in the prior art. In some embodiments of this disclosure, the engineered cells include a gene editing system for editing the HLA-A gene and / or the HLA-B gene. The gene knockout is achieved through this gene editing system.

[0015] Furthermore, in some embodiments of this disclosure, the gene editing system includes transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), or Cas9 protease.

[0016] Furthermore, in some embodiments of this disclosure, the gene editing system includes a Cas9 protease and a guide RNA, wherein the Cas9 protease is exogenously introduced or expressed in the cells.

[0017] Furthermore, in some embodiments of this disclosure, the guide RNA comprises one or more nucleotide sequences selected from those 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 this disclosure, the guide RNA further comprises a nucleotide sequence shown in SEQ ID No. 22, which is used to efficiently knock out or knock down the HLA-B gene.

[0018] Furthermore, in some embodiments of this disclosure, the guide RNA is selected from one or more nucleotide sequences such as those shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

[0019] In this disclosure, the aforementioned genes can be knocked down using any suitable method available in the prior art. In some embodiments of this disclosure, the engineered cells contain repressive nucleic acids that target genes or mRNAs of HLA-A and / or HLA-B.

[0020] Furthermore, in some embodiments of this disclosure, the repressive nucleic acid includes antisense oligonucleotides targeting genes or proteins of HLA-A and / or HLA-B, small interfering RNA (siRNA), or dsRNA used in RNA interference (RNAi).

[0021] Another aspect of this disclosure is the use of the aforementioned engineered cells in the preparation of products for cell infusion.

[0022] Another aspect of this disclosure is to provide a pharmaceutical composition comprising the engineered cells, the gene editing system, or the inhibitory nucleic acid described above. Beneficial effects:

[0023] The engineered cells disclosed herein significantly reduce the activation of allogeneic T cells without activating NK cells, thereby reducing infusion-induced immune rejection. Furthermore, the engineered cells exhibit significantly improved in vivo engraftment rates and long-term hematopoietic reconstitution capabilities. These cells will significantly expand the use of existing umbilical cord blood and bone marrow donor banks, reducing the need to recruit large numbers of donors to match recipients and increasing the probability of HLA-matched donors, thus expanding the infusion of clinical-grade allogeneic cells. As a universal cell source, engineered cells enable off-the-shelf HLA-matched cell therapy, holding significant clinical therapeutic potential. Attached Figure Description

[0024] Figure 1 is a flow cytometry result of the knockout / knockdown efficiency of cells in the Ctrl group and after knocking out / knocking down HLA-A, HLA-B, HLA-A and B, and HLA-C respectively in the embodiments of this disclosure.

[0025] Figure 2 is a statistical result 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 this disclosure;

[0026] Figure 3 is a statistical result 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 this disclosure;

[0027] Figure 4 shows the flow cytometry results of the Ctrl group, HLA-A- group, HLA-B- group, HLA-C- group and HLA-AB- group evading the heterologous T cell immune response in the embodiments of this disclosure;

[0028] Figure 5 shows the statistical results of the Ctrl group, HLA-A- group, HLA-B- group, HLA-C- group and HLA-AB- group evading the heterologous T cell immune response in the embodiments of this disclosure;

[0029] Figure 6 is a statistical result of NK activation in the Ctrl group, HLA-AB- group, HLA-ABC- group and HLA-B2M- group in the embodiments of this disclosure;

[0030] Figure 7 shows the levels of human CD45 in the peripheral blood of mice after cell infusion into the Ctrl group, HLA-A- group, HLA-B- group, and HLA-AB- group in this embodiment of the present disclosure. + Statistical results of cell percentage;

[0031] Figure 8 shows the statistical results of the proportion of each cell lineage in the peripheral blood of mice after infusion of Ctrl group, HLA-A- group, HLA-B- group and HLA-AB- group cells in the embodiments of this disclosure;

[0032] Figure 9 shows the human CD45 concentration in the bone marrow of mice after cell infusion into the Ctrl group, HLA-A- group, HLA-A- group and HLA-AB- group in this embodiment of the present disclosure. + Statistical results of cell implantation rate;

[0033] Figure 10 shows the statistical results of the proportion of each cell lineage in the bone marrow of mice after infusion of Ctrl group, HLA-A- group, HLA-B- group and HLA-AB- group cells in the embodiments of this disclosure;

[0034] Figure 11 shows the human CD34 concentration in the bone marrow of mice after cell infusion into the Ctrl group, HLA-A- group, HLA-B- group and HLA-AB- group in this embodiment of the present disclosure. + CD38 - Statistical results of cell percentage;

[0035] Figure 12 shows the CD34 content in the bone marrow of mice after cell infusion into the Ctrl group, HLA-A- group, HLA-B- group and HLA-AB- group in this embodiment of the present disclosure. + CD38 + Statistical results of cell percentage;

[0036] Figure 13 shows the knockout effect of 21 sgRNAs screened by transient transfection in the 293T cell line in this embodiment of the present disclosure. The figure shows the proportion of HLA-A low expression cells after each sgRNA knockout. The HLA-A-ex2-g13 group with the best knockout effect can reach 60% of HLA-A knockout cells. 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.

[0037] Figure 14 shows 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) in the human HLA-A gene in the embodiments of this disclosure;

[0038] Figure 15 shows the results of further testing the knockout effect of the three sgRNAs with the best HLA-A protein knockout effect selected from the 293T cell line in this embodiment of the present disclosure: 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 umbilical cord blood. The flow cytometry plot is CD34. + CD34 + CD90 + and CD34 + CD90 - Cellular gatening strategies;

[0039] Figures 16 and 17 show the knockout effects 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 umbilical cord blood in the embodiments of this disclosure. Figure 16 shows that all three sgRNAs can effectively knock out CD34. + CD34 + CD90 + and CD34 + CD90 - Cell surface HLA-A protein, 3 sgRNAs knocked out CD34 + CD90 + The cell proportions were all 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%. Figure 17 shows the statistical analysis, which shows that the proportion of HLA-A positive cells decreased significantly after knocking out 3 sgRNAs.

[0040] Figures 18 and 19 are specific results from embodiments of this disclosure. Figure 18 shows that, compared with the control group, knocking out HLA-A with three sgRNAs resulted in CD34... + CD90 + No significant difference was observed in HLA-ABC expression on the cell surface, indicating that the three sgRNAs targeting and knocking out HLA-A had significant specificity. Knocking out HLA-A with these three sgRNAs did not affect CD34. + CD34 + CD90 + and CD34 + CD90 - The expression of HLA-B and HLA-C molecules on the cell surface was analyzed. Figure 19 shows the statistical analysis. After knocking out the three sgRNAs, the proportion of HLA-ABC positive cells did not change significantly.

[0041] Figures 20 and 21 are specific results from embodiments of this disclosure. Figure 20 shows that, compared with the control group, knocking out HLA-A with three sgRNAs resulted in CD34... + CD90 + There was no significant difference in the expression of HLA-DR molecules on the cell surface. Figure 21 shows the statistical analysis. After knocking out the three sgRNAs, the proportion of HLA-DR positive cells did not change significantly, indicating that the three sgRNAs have significant specificity in targeting and knocking out HLA-A. Knocking out HLA-A with these three sgRNAs did not affect CD34. + CD34 + CD90 + and CD34 + CD90 - The expression of HLA-II molecules on the cell surface.

[0042] Sequence description.

[0043] sequence list Detailed Implementation

[0044] This invention discloses an engineered cell, which can be implemented by those skilled in the art by appropriately modifying the process parameters based on the content herein. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately alter and combine the content described herein without departing from the scope, spirit, and meaning of this invention to implement and apply the technology of this invention.

[0045] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., shall be understood to include the stated elements or components without excluding other elements or other components. The term "a," "an," and "the" includes plural indicators. The term "a plurality of" 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 this disclosure.

[0046] In this disclosure, when a range of values ​​is provided, it should be understood that, unless the context otherwise explicitly indicates otherwise, the range includes endpoints and each intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range and any value within a smaller range between specified values.

[0047] In this disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 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.

[0048] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common molecular biology terms can be found in Lewin's *GENES*, Twelfth Edition, by Jocelyn E. Krebs, Elliott S. Goldstein, and Stephen T. Kilpatrick, published by Jones & Bartlett Learning. Definitions of common biochemistry terms can be found in Lehninger's *Principles of Biochemistry*, Eighth Edition, by David L. Nelson and Michael M. Cox, published by WH Freeman. Definitions of common cell biology terms can be found in *Molecular Biology of the Cell*, Sixth Edition, by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, and Peter Walter, published by Garland Science. Definitions of common genetics terms can be found in *Genetics: Analysis of Genes and Genomes*, Eighth Edition, by Daniel L. Hartl and Maryellen Ruvolo, published by Jones & Bartlett Learning.

[0049] Unless otherwise specified, the experimental techniques used in this paper employ standard techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as *Molecular Cloning: A Laboratory Manual* and *Cell Biology: A Laboratory Handbook*.

[0050] the term:

[0051] As used in this disclosure, the term "hematopoietic stem-progenitor cells (HSPCs)" refers to hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs). Hematopoietic stem cells are adult stem cells in the blood system, possessing the ability to self-renew long-term and the potential to differentiate into various types of mature blood cells. These mature blood cells include erythrocytes and leukocytes. Hematopoietic progenitor cells are progenitor cells that proliferate and differentiate into various types of blood cells under the regulation of a specific microenvironment and certain factors. They are also a relatively primitive type of cell with proliferative capacity, but have lost their multi-lineage differentiation ability and can only proliferate and differentiate into one or a few blood cell lines in a directed manner; hence, they are also called committed stem cells. In some embodiments of this disclosure, the hematopoietic stem-progenitor cells may be derived from bone marrow, peripheral blood, or umbilical cord blood.

[0052] As used in this disclosure, the term "wild type" is a concept relative to "mutant" and generally refers to an individual obtained from nature without artificial mutation in a study. This individual carries a wild-type genome. In some embodiments of this disclosure, wild type refers to an unengineered organism.

[0053] As used in this disclosure, the term "reduction" means that the expression level (expression amount) of the target protein in cells is lower than that in wild-type cells. The reduction can be 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%. Methods for determining the expression level (expression amount) of the target protein may include, for example, Western blotting, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, flow cytometry, etc.

[0054] As used in this disclosure, the term "knockout" describes the process of completely or partially inactivating a specific gene in an organism using gene editing techniques. Knockout techniques typically involve using gene editing tools, such as the CRISPR-Cas9 system, to precisely locate and cut the DNA sequence of a target gene. After the DNA sequence is cut, cells usually attempt 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 substitution of gene sequences, causing the target gene to lose its original function or expression level.

[0055] As used in this disclosure, the term "knockdown" refers to reducing the expression level of a gene in an organism through specific technical means, rather than completely eliminating the gene's function. This can be achieved through methods such as interference with antisense RNA, ribonucleases, or the CRISPR-Cas system (CRISPRi). The main difference between knockdown and knockout lies in the degree: knockout completely or partially eliminates gene function, while knockdown only reduces its expression level. Therefore, knockdown allows researchers to study the effects of reduced gene expression, rather than the effects of complete gene deletion.

[0056] As used in this disclosure, the term "HLA-A" is an abbreviation for Human Leukocyte Antigen A, also known as an 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 approximately 45 kDa, and its gene contains eight exons. Exon 1 encodes a leader peptide, exons 2 and 3 encode α1 and α2 domains (both binding peptides), exon 4 encodes an α3 domain, exon 5 encodes a transmembrane region, and exons 6 and 7 encode a cytoplasmic tail. In particular, polymorphisms within exons 2 and 3 are associated with peptide-binding specificity for each HLA-A molecule, and typing of these polymorphisms is commonly used for matching in bone marrow and kidney transplants. In some embodiments of this disclosure, the absence or reduction of HLA-A protein expression also includes the absence or reduction of expression of the alleles corresponding to HLA-A protein, including HLA-A1, HLA-A2, HLA-A3, HLA-A11, or HLA-A24.

[0057] CRISPR-Cas system:

[0058] The CRISPR-Cas system is widely found on the chromosomes of bacteria and archaea, and is related to their immunity, used to defend against foreign genetic material and acquire resistance to bacteriophages. The CRISPR-Cas system is the third generation of gene editing technology following ZFN and TALENs. The CRISPR-Cas system can be divided into three main types: Type I, Type II, and Type III. CRISPR Type I was the first CRISPR system discovered and studied, and is mainly found in most bacteria and archaea. This system consists of multiple Cas proteins and multiple CRISPR RNAs (crRNAs), forming a complex Cas protein complex. This complex recognizes and cuts the target fragment through complementary pairing of crRNA with foreign DNA. A key characteristic of the Type I system is the presence of 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 requires only the Cas9 protein and crRNA to form a complex with trans-activating CRISPR-derived RNA (tracrRNA) to recognize and cleave target DNA fragments. The Cas9 protein possesses two nuclease activities, HNH and RuvC, which can cleave the two strands of the target DNA, respectively. The CRISPR Type III system has a similar structure and function to the Type I system, but with more Cas proteins and a more complex mechanism. Similar to the Type I system, the Type III system also requires multiple Cas proteins and crRNA to form a complex, recognizing and cleaving target fragments through complementary pairing with exogenous DNA. However, the cleavage mechanism of the Type III system differs from that of the Type I system; it relies on the RNase activity of the Cas proteins to cut the target DNA. In embodiments of this disclosure, the sgRNA is used in the Type II CRISPR-Cas system.

[0059] Cas protein:

[0060] In embodiments of this disclosure, the Cas protein used in the CRISPR-Cas system is primarily Cas9. Cas9 is a nuclease that, guided by sgRNA (small guide RNA), can precisely recognize and cleave DNA sequences. In some embodiments of this disclosure, the Cas9 protein can be isolated or loaded onto a vector for intracellular expression. In addition to Cas9, other accessory proteins may be included in other embodiments of this disclosure, including but not limited to Cas1, Cas2, etc. They may play an auxiliary role in the DNA cleavage process or participate in other aspects of the CRISPR system, such as DNA acquisition and processing.

[0061] In embodiments of this disclosure, the Cas protein may be modified. Such modifications may include mutations, insertions, or deletions of amino acid residues, codon optimization, and other chemical modifications. The purpose may be to alter the activity or specificity of the Cas protein without changing its primary function. For example, US Patent 20180100148A1 describes mutations in the nucleic acid and protein of Streptococcus pyogenes Cas9 to reduce off-target effects of the system.

[0062] Inhibitory nucleic acids:

[0063] In this disclosure, the term "inhibitory nucleic acid" (INA) refers to a class of nucleic acid molecules capable of inhibiting the expression of specific genes or the function of proteins. Through specific sequence design and modification, they can bind to complementary sequences of target genes or proteins, thereby interfering with their normal function or expression level. Common examples of inhibitory nucleic acids include antisense oligonucleotides, small interfering RNA (siRNA), RNA interference (RNAi), etc.

[0064] Guide RNA and its combinations:

[0065] In embodiments of this disclosure, the nucleotide sequences of the sgRNA shown in SEQ ID No. 1-21 are capable of efficiently knocking down and / or eliminating the human HLA-A gene. The nucleotide sequence shown in SEQ ID No. 22 is used to efficiently knock down or eliminate the HLA-B gene. Once the nucleotide sequences are known, they can be obtained by suitable methods in the prior art, including but not limited to chemical synthesis, polymerase chain reaction (PCR), etc.

[0066] In the embodiments of this disclosure, the human HLA-A gene is recognized, knocked down, and / or eliminated in cells using the aforementioned sgRNA via a CRISPR gene editing system. The efficiency achieved is over 20% in the 293T cell line and reaches a maximum of 97% in umbilical cord blood cells, significantly superior to existing methods. Among these, the sgRNAs HLA-A-ex2-g13, HLA-A-ex3-g167, and HLA-A-ex2-g452 exhibit the highest efficiency. Therefore, preferably, in some embodiments of this disclosure, the nucleotide sequence of the sgRNA is as shown in SEQ ID No. 1, SEQ ID No. 2, or SEQ ID No. 3.

[0067] In gene editing, the aforementioned sgRNAs can be used alone, achieving editing efficiencies of approximately 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99% in some embodiments of this disclosure. They can also be used in combination or with the addition of other sgRNAs, achieving editing efficiencies of approximately 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99% in other embodiments of this disclosure. In some embodiments of this disclosure, the sgRNAs can be loaded into the same container or into different containers before being transfected into cells.

[0068] The nucleotide sequence of the above-mentioned sgRNA can be modified. Such modifications include, but are not limited to, substitution, insertion, deletion, or exchange / replacement of one or more nucleotides for improvement purposes, as well as modifications to the nucleotide sequence using other groups. In some embodiments of this disclosure, the mutated sgRNA nucleotide sequence may 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 with SEQ No. 1-21.

[0069] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0070] Example:

[0071] Example 1: Constructing cells with knockout / knockdown of HLA-A, HLA-B, HLA-C, and HLA-AB genes

[0072] 1. CD34 derived from umbilical cord blood, bone marrow, or peripheral blood + Cells were divided into 5 groups: Ctrl group, HLA-A knockout / knockdown group, HLA-B group, HLA-C group, and HLA-AB group. Each group of cells was resuspended in electroporation buffer. RNPs without sgRNA were added to the Ctrl group, RNPs containing HLA-A sgRNA were added to the HLA-A knockout group, RNPs containing HLA-B sgRNA were added to the HLA-B knockout group, RNPs containing HLA-C sgRNA were added to the HLA-C knockout group, and RNPs containing both HLA-A and HLA-B sgRNAs were added to the HLA-AB group.

[0073] 2. Each group of cells with added RNP was electroporated using an electroporator. The gene editing method is as follows:

[0074] (1) Take the protein and sgRNA (1:2) from the Lonza electroporation kit and put them into a 1.5ml EP tube. React at room temperature for 10-15min.

[0075] (2) Count the cells, aspirate the cell suspension and place it in a 50ml centrifuge tube. Centrifuge at 500g for 5min.

[0076] (3) Discard the supernatant, resuspend the cells in PBS and wash once, then centrifuge at 500g for 5min.

[0077] (4) Discard the supernatant, resuspend the cells in buffer, add the RNP prepared in step (1) (final volume of 20ul system), mix well by pipetting with the tip of a pipette, and place in the well plate for electroporation.

[0078] (5) After electroporation in a Lonza electroporator, aspirate the cell suspension from the electroporation plate into a 15ml centrifuge tube. Centrifuge at 500g for 5 minutes, then discard the supernatant.

[0079] (6) Resuspend each tube in 2 ml of culture medium and then transfer it to a petri dish.

[0080] 3. After electroporation, the cells were quickly resuspended in culture medium and incubated in an incubator for 24 hours.

[0081] 4. Sequencing gene fragments to detect the knockout efficiency, and using flow cytometry to detect the editing efficiency of each gene.

[0082] The results are shown in Figures 1, 2, and 3. The gene editing efficiency percentages for 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.

[0083] Example 2: Obtaining three types of engineered cells with reduced immunogenicity by knocking out / downgrading HLA-A, HLA-B, and HLA-AB genes.

[0084] 1. Cells in the Ctrl group, HLA-A knockout / knockdown group, HLA-B knockout group, and HLA-AB group were pretreated with 50 ng / mL IFN-γ for 48 hours. sgRNA and RNP were added to each group for electroporation (RNP without sgRNA was added to the Ctrl group).

[0085] 2. The method for separating human peripheral blood cells is as follows:

[0086] (1) Place freshly obtained peripheral blood (PBMC) from healthy individuals into a sterile 50mL centrifuge tube, add red blood cell lysis buffer, mix thoroughly, and let stand at room temperature for at least 6 minutes to lyse the red blood cells.

[0087] (2) Centrifuge at 1500 rpm for 5 min at 4℃;

[0088] (3) Discard the supernatant, resuspend the cells in 20 mL of PBE in each tube, mix thoroughly, take 10 μL for counting, and centrifuge at 1500 rpm for 5 min at 4℃.

[0089] (4) Discard the supernatant and resuspend the collected human peripheral cells in IMDM-FBS for later use.

[0090] 3. Add PBMC to 500 μL PBS and mix well to prepare a single-cell suspension for T cell sorting. Preparation of RPMI 1640 complete culture medium: RPMI 1640 basal medium + 10% FBS + 1% Glutamax + 1% penicillin-streptomycin solution; subsequent steps should be performed in the dark. Preparation of CFSE working solution: Add 1 μL CFSE to 500 μL PBS in each aliquot and mix thoroughly by pipetting.

[0091] 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.

[0092] 5. Quickly add 1 ml of pre-cooled 50% FBS and mix by pipetting to stop staining. Let stand on ice for 2 min, then centrifuge at 300g for 10 min.

[0093] 6. Add 2 ml of 1640 complete culture medium to each container, mix well by pipetting, and centrifuge at 300 g for 10 min.

[0094] 7. Repeat step 6.

[0095] 8. The engineered cells pretreated for 48 hours on day 1 were mixed with T cells at a 1:1 ratio in RPMI-1640 containing glutamine, 10% FBS and 20 U / mL IL-2 in a 96-well U-shaped plate for 3-5 days.

[0096] 9. Flow cytometry detection of proliferative CD8 + The percentage of T cells (T cells whose CFSE markers have become negative).

[0097] The results are shown in Figures 4 and 5. The results indicate that, compared to the Ctrl group, knockout / downdating of HLA-A, HLA-B, and HLA-AB significantly reduced the activation of heterologous T cells after co-culturing gene-edited cells with T cells. However, knockout of HLA-C still significantly activated T cells.

[0098] Example 3: Effects of HLA-C knockout / knockdown cells on NK cell activity

[0099] 1. Cells with HLA-B2M knockout / knockdown were set as the positive control group. HLA-AB group cells were obtained by simultaneously knocking out / knocking down HLA-A and HLA-B using the method in Example 1. HLA-ABC group cells were obtained by simultaneously knocking out / knocking down HLA-A, HLA-B and HLA-C.

[0100] 2. The Ctrl group was supplemented with RNP without sgRNA, otherwise the same as the experimental group. Cells in each group co-electroporated with RNP were pretreated with 50 ng / mL IFN-γ for 48 hours.

[0101] 3. Add PBMC to 500 μL PBS and mix well to prepare a single-cell suspension. Use CD3 and CD56 antibodies to sort NK cells.

[0102] 4. Preparation of RPMI1640 complete medium: RPMI1640 basal medium + 10% FBS + 1% Glutamax + 1% penicillin-streptomycin (PS) solution.

[0103] 5. Cells from the Ctrl group, HLA-AB group, HLA-ABC group, and HLA-B2M group were co-cultured with NK cells in U-bottom 96-well plates for 5 hours. The percentage of CD107a-positive NK cells was detected by flow cytometry.

[0104] The statistical results are shown in Figure 6. The results indicate that, compared with the Ctrl group, knocking out / downgrading HLA-AB did not activate NK cells, but knocking out / downgrading HLA-C significantly activated NK cells.

[0105] Example 4: The ability of engineered cells to be implanted and reconstituted in vivo is significantly increased.

[0106] The three types of engineered low-immunogenic HSPCs (human CD34+ cells with HLA-A knockout / HLA-B knockdown and HLA-AB knockout) prepared in Examples 1 and 2 were transplanted into immunodeficient mice, and the cell engraftment rate and hematopoietic reconstitution in each group were dynamically detected. The methods are as follows:

[0107] 1. Female NOG mice aged 6-8 weeks and weighing 18-22g were randomly divided into groups. Before transplantation, the mice were irradiated with 2Gy X-rays at a dose rate of 1.2Gy / min.

[0108] 2. Using the method described in Example 1, human HLA-A, HLA-B, and HLA-AB cells were obtained and transplanted via tail vein at a rate of 1 × 10⁻⁶ cells per cell. 5 Human CD45 cells were transplanted into NOG mice, and the levels of human CD45 in the peripheral blood of the mice were continuously measured 4-20 weeks post-transplantation. + The proportion of cells.

[0109] 3. Twenty weeks after transplantation, mice were euthanized by cervical dislocation, and bone marrow cells were collected. The cells were washed with staining buffer, resuspended, and incubated with antibody at 4°C in the dark for 30 minutes.

[0110] 4. Wash once with 2 mL of staining buffer, and use flow cytometry to detect the implantation of human cells and the composition of each cell line in mouse bone marrow.

[0111] The results showed that, compared with the control group, human-engineered cells had higher CD45 levels in mouse peripheral blood. + The percentage of cells increased significantly, with CD33 being the main cell type. + Myeloid cells and CD19 + B cells were observed, and the results are shown in Figures 7 and 8. In mouse bone marrow, the engraftment rate of human-engineered cells was also significantly higher than that of the control group, with CD33 cells being the main cellular component. + Myeloid cells and CD19 + The results of the HLA gene modification of B cells, as shown in Figures 9 and 10, indicate that the regeneration of various cell lines in vivo by engineered cells was unaffected. Furthermore, in mouse bone marrow, engineered cells with CD34... + CD38 - and CD34 + CD38 +The proportion of cells was significantly higher in the control group than in the control group (the results are shown in Figures 11 and 12).

[0112] Example 5: Detection of sgRNA knockout effect in 293T cell line using transient transfection method.

[0113] 1) 293T cells were seeded in 24-well plates 2 × 10⁶ cells per well 24 hours in advance. 5 cell.

[0114] 2) Replace the DMEM complete medium with 0.4 ml of DMEM medium (containing 10% FBS, without penicillin / streptomycin) and place it in an incubator.

[0115] 3) Prepare the transfection system: Add 50ul of Optimum medium to a 1.5ml EP tube, then add 1ug / well of plasmid (0.5ug / well each of sg plasmid and Cas9 plasmid, sg plasmid is prepared using conventional methods), invert and mix well, do not vortex.

[0116] 4) Take another 1.5ml EP tube, add 50ul of Optimum medium, then add 3ug / well of PEI, invert to mix, do not vortex.

[0117] 5) Leave at room temperature for 5 minutes.

[0118] 6) Mix 3) and 4) in one tube, invert to mix well, do not vortex, and let stand for 20 minutes.

[0119] 7) Add the plasmid system to the dish and shake well.

[0120] 8) After 12-16 hours, switch to DMEM complete culture medium.

[0121] 9) Add 2ug / ml puromycin vial sieve 48 hours after transfection.

[0122] 10) The HLA-A knockout effect of each sgRNA was detected 48 hours after puromycin screening.

[0123] 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 better knockout effects, with the HLA-A-ex2-g13 group achieving a HLA-A knockout rate of up to 60% (see Figure 13).

[0124] 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 shown in Figure 14.

[0125] Example 6: Detection of sgRNA knockout effect on umbilical cord blood-derived hematopoietic cells

[0126] 1. Enrichment of umbilical cord blood CD34 + Cells were incubated overnight in hematopoietic cell basal medium. The hematopoietic cell basal medium was SFEMII medium supplemented with the following cytokines (Table 1):

[0127] Table 1

[0128] 2. Three sgRNAs with good knockout effects in 293T cells were selected: 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). CD34 was knocked out using the electroporation RNP method according to the Lonza P3 Primary Cell 4D-NucleofectorTMX Kit S. + HLA-A protein on the cell surface.

[0129] 1) CD34 + Cell counting, 2 × 10⁶ cells per sample 5 cell.

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

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

[0132] 4) Operate according to the instructions of the 4D electro-rotator.

[0133] 5) After electroporation, transfer the cell suspension from the electroporation tank to a 1.5ml 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, CD34 levels were detected by flow cytometry. + CD34 + CD90+ and CD34 + CD90 - Expression of HLA-A, HLA-ABC, and HLA-DR on the cell surface. The antibodies labeled by flow cytometry are shown in Table 2 below.

[0135] Table 2

[0136] 4. Flow cytometry results showed that all three sgRNAs could significantly knock out CD34. + CD34 + CD90 + and CD34 + CD90 - HLA-A protein on cell surface, 3 sgRNAs knocked out CD34 + The cell proportions were all above 94%, and the HLA-A-ex2-g13 group had CD34. + CD90 + The proportion of HLA-A knockout cells reached 97% (Figure 15 shows the flow cytometry gating strategy; results are shown in Figures 16 and 17). Furthermore, the three sgRNAs group CD34... + CD34 + CD90 + and CD34 + CD90 - The proportions of HLA-ABC and HLA-DR positive groups on cells did not change significantly. After statistical analysis, knocking out HLA-A with the three sgRNAs had no significant effect on the expression of HLA-B and HLA-C in HLA-I molecules in the three cell populations (see Figures 18 and 19) or HLA-II molecules (see Figures 20 and 21). In other words, the sgRNAs we used have high targeting specificity.

[0137] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An engineered cell characterized by, The engineered cells, compared to wild-type cells, lack or reduce the expression of HLA-A and / or HLA-B proteins, and the cells are artificial hematopoietic stem cells (HSPCs).

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

3. The engineered cell according to claim 1 or 2, characterized in that, The engineered cells contain a gene-editing system that edits the HLA-A gene and / or the HLA-B gene.

4. The engineered cell according to claim 1 or 2, characterized in that, The engineered cells contain repressive nucleic acids that target genes or proteins of HLA-A and / or HLA-B.

5. The engineered cell according to claim 3, characterized in that, The gene editing system includes transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), or Cas9 protease.

6. The engineered cell according to claim 5, characterized in that, The gene editing system includes a Cas9 protease and a guide RNA, wherein the Cas9 protease is either exogenously introduced or expressed in the cells.

7. The engineered cell according to claim 6, characterized in that, The guide RNA comprises one or more nucleotide sequences selected from those shown in SEQ ID No. 1-21.

8. The engineered cell according to claim 7, characterized in that, The guide RNA is selected from one or more nucleotide sequences such as those shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No.

3.

9. The engineered cell according to claim 4, characterized in that, The inhibitory nucleic acids include antisense oligonucleotides targeting genes or mRNAs of HLA-A and / or HLA-B, small interfering RNA (siRNA), or dsRNA used in RNA interference (RNAi).

10. Use in a product for cell infusion using the engineered cell preparation as described in any one of claims 1 to 9.

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