Epigenetic regulation-based method for preparing universal car-t cell

Abstract

Provided are a method for producing a universal CAR-T cell, and a universal CAR-T cell prepared by using the method.

Description

A method for preparing universal CAR-T cells based on epigenetic regulation

[0001] priority

[0002] This application claims the rights and priority of Chinese application No. 202510125743X, filed January 26, 2025, and Chinese application No. 2025101257548, filed January 26, 2025. The entire contents of both are incorporated herein by reference for all purposes. Technical Field

[0003] This disclosure belongs to the field of biotechnology, and in particular relates to a universal method for preparing CAR-T cells based on epigenetic regulation. Background Technology

[0004] Universal CAR-T cell therapy is an innovative immunotherapy strategy designed to overcome the individual variability inherent in traditional CAR-T therapy, which relies on the patient's own T cells. The core technology of universal CAR-T lies in editing the T-cell receptor (TCR). By deleting or modulating TCR function, the modified T cells no longer recognize the patient's own tissues, thus avoiding immune rejection. Universal CAR-T cell therapy solves the immune rejection problem in traditional CAR-T therapy through TCR editing technology and improves production efficiency and therapeutic universality through the use of allogeneic cells. In the preparation of universal CAR-T cells, CRISPR / Cas9 and adenosine deaminase editing (ABE) technologies are widely used for TCR gene editing. However, despite the powerful gene editing capabilities offered by these technologies, studies have shown that editing with Cas9 and ABE may induce chromosomal translocations, large-scale chromosomal deletions, potential carcinogenic risks, and other adverse effects, posing challenges to the safety and efficacy of universal CAR-T.

[0005] Host-graft resistance (HVG) refers to the immune rejection of foreign transplanted cells or tissues by the host's immune system, a phenomenon particularly important in universal CAR-T cell therapy. Since universal CAR-T cells are typically derived from allogeneic donors, the host's immune system may recognize and attack these foreign cells, leading to loss of CAR-T cell function or immune side effects. Therefore, reducing HVG response is crucial for improving the efficacy of universal CAR-T cell therapy. In this context, epigenetic regulation technology offers an important solution for optimizing the immune characteristics of CAR-T cells and reducing immune rejection. By precisely regulating the expression of genes related to immune rejection, especially HLA Class I and HLA Class II molecules and other immune-related molecules such as CD52, the risk of CAR-T cells being recognized and attacked by the host's immune system can be effectively reduced.

[0006] HLA Class I molecules consist of three parts: the α chain, β2-microglobulin (B2M), and the antigenic peptide. The antigenic peptide displayed by HLA Class I molecules is typically transported by the transmembrane proteins TAP1 and TAP2 in the endoplasmic reticulum. The TAP1 and TAP2 genes encode the TAP1 and TAP2 proteins, respectively, which are responsible for transporting short peptides (typically 8-10 amino acids) degraded by the proteasome from the cytoplasm to the endoplasmic reticulum, where they bind to the HLA Class I molecule to form an antigen-presenting complex. Key genes associated with HLA Class I molecules include: HLA-A, HLA-B, and HLA-C: These genes encode different alleles of the HLA Class I α chain, enabling the presentation of different antigenic peptides and enhancing the immune system's recognition and immune response to various pathogens. β2-microglobulin (B2M): This gene encodes β2-microglobulin, which binds to the HLA Class I α chain, stabilizing the overall molecular structure and ensuring its expression on the cell surface. TAP1 and TAP2: These two genes encode transmembrane proteins that are important components of the antigen presentation pathway, responsible for transporting antigenic peptides to the endoplasmic reticulum, binding to HLA Class I molecules, and ultimately displaying them on the cell surface.

[0007] HLA Class II molecules consist of two chains: an α chain and a β chain. Together, they form an antigen-binding groove that binds to exogenous antigenic peptides. After digestion, the exogenous antigenic peptide is processed via the endoplasmic reticulum, endocytosis, and lysosomes, ultimately binding to the HLA Class II molecule and being displayed on the cell surface for recognition by CD4+ T cells. This process is a crucial step in initiating the adaptive immune response. The expression and function of HLA Class II molecules are finely regulated by multiple transcription factors and regulatory factors, among which the complex composed of RFXAP, RFX5, CIITA, and RFXANK is the core regulatory factor controlling HLA Class II gene expression.

[0008] These factors work synergistically through a complex regulatory network to ensure the efficient expression of HLA Class II molecules, enabling antigen-presenting cells to effectively initiate an immune response from CD4+ T cells. CD52 is a glycoprotein on the surface of immune cells that plays a role in regulating immune tolerance. CD52 is expressed on T cells, B cells, and natural killer cells, and is one of the important immunomodulatory factors in the immune system. By regulating CD52 expression, especially using anti-CD52 monoclonal antibodies, the number and activity of immune cells can be effectively regulated, thereby affecting the immune rejection response. Therefore, CD52 also plays an important role in CAR-T cell immune escape.

[0009] However, CAR-T cell therapy still faces some challenges in clinical application. For example, a high incidence of tumor recurrence exists in the treatment of hematological malignancies, and CAR-T cells do not persist long enough in the patient's body. These issues may be caused by factors such as tumor antigen loss, CAR-T cell depletion, and host immune cell rejection. Therefore, improvements to existing CAR-T cell therapies are still needed to promote CAR-T cell proliferation in vivo and reduce host cell rejection. Summary of the Invention

[0010] On one hand, this disclosure provides a method for producing universal CAR-T cells, the method comprising:

[0011] The expression of target genes in CAR-T cells was downregulated by using gene expression regulatory molecules;

[0012] The gene expression regulatory molecule comprises i) an epigenetic modification domain, ii) a transcriptional regulatory domain, and iii) a DNA-binding domain, wherein the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to one end of the DNA-binding domain via a linker sequence, and the epigenetic modification domain is optionally linked to one end of the transcriptional regulatory domain via a linker sequence; or the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to both ends of the DNA-binding domain via a linker sequence.

[0013] The target genes are selected from CD3D, CD3G, CD3E, CD3Z, HLA I, HLA II, CD52, TCRα, or TCRβ.

[0014] In some specific embodiments, the method uses gene expression regulating molecules to downregulate the expression of the TCR-CD3 complex in the T cell receptor (TCR) of the CAR-T cells, preferably downregulating the expression of one or more of CD3D, CD3G, CD3E, CD3Z, TCRα, and TCRβ;

[0015] The gene expression regulatory molecule comprises i) an epigenetic modification domain, ii) a transcriptional regulatory domain, and iii) a DNA-binding domain, wherein the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to one end of the DNA-binding domain by a linker sequence, and the epigenetic modification domain is optionally linked to one end of the transcriptional regulatory domain by a linker sequence; or the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to both ends of the DNA-binding domain by a linker sequence.

[0016] In some specific embodiments, the method uses gene expression regulating molecules to downregulate the expression of CD3D / CD3E or CD3G / CD3E dimers.

[0017] In some specific embodiments, the epigenetic modification domain comprises DNA methyltransferases DNMT 3A and DNMT3L; the transcriptional regulatory domain is selected from at least one of ZIM3, KRAB, HP1a, and SETDB1; the DNA binding domain is selected from: TALE domain, zinc finger domain, tetR domain, a wide range of nucleases, Cas proteins such as inactivated Cas9 protein (dCas9) or inactivated Cas12 protein (dCas12), Argonaute (Ago) protein, and their homologues, modified forms, or variants.

[0018] In some specific embodiments, the gene expression regulatory molecule further comprises (iv) a first recruitment domain and a second recruitment domain, wherein the first recruitment domain and the second recruitment domain are respectively at one end or both ends of the DNA binding domain. Preferably, the first recruitment domain is selected from universal control non-derepressor protein 4 (GCN4), a GFP11 fragment derived from split green fluorescent protein (GFP), or a GVKESLV polypeptide; the second recruitment domain is selected from a single-chain antibody (scFv), a GFP1-10 fragment derived from split green fluorescent protein (GFP), or a PDZ protein domain; preferably, the first recruitment domain is selected from GCN4, and the second recruitment domain is selected from scFv.

[0019] In some specific embodiments, the order of the gene expression regulatory molecule from the N-terminus to the C-terminus includes:

[0020] 1) Epigenetic modification domains, DNA-binding domains, and transcriptional regulatory domains;

[0021] 2) DNA-binding domain, transcriptional regulatory domain, and epigenetic modification domain;

[0022] 3) The second recruitment domain, transcriptional regulatory domain, cleavage peptide, epigenetic modification domain, DNA binding domain, and first recruitment domain; the first recruitment domain and the second recruitment domain can interact with each other.

[0023] In some specific embodiments, the linker sequence is XTEN80, and the cleavage peptide is selected from P2A, T2A, E2A, and F2A.

[0024] In some specific embodiments, the DNA-binding domain is capable of binding to guide RNA, which is capable of specifically recognizing and hybridizing with the target sequence of the target locus.

[0025] In some specific embodiments, the target sequence of the target locus is selected from: SEQ ID NOs: 1621-1625.

[0026] In some specific embodiments, the guide RNA (sgRNA) targeting the CD3D gene comprises a sequence selected from SEQ ID NOs: 39-584; the guide RNA (sgRNA) targeting the CD3E gene comprises a sequence selected from SEQ ID NOs: 1075-1439; and the guide RNA (sgRNA) targeting the CD3G gene comprises a sequence selected from SEQ ID NOs: 172, 173, 185-188, 191-194, 196, 198, 219, 225, 228, 233-235, 237, 238, 247, 256-258, 260, 261, 266, 268, 269, 271, 272, 329, 331, 332, 336, 340, 346-351, 3 The sequences 56, 358-361, 373, 375, 381-385, 388, 390, 391, 393-395, 398, 409-411, 413, 418-420, 425-429, 438-440, 443, and 585-1074; and / or the guide RNA (sgRNA) targeting the CD3Z gene contains sequences selected from SEQ ID NOs: 1440-1459.

[0027] In some specific embodiments, the guide RNA (sgRNA) targeting the CD3D gene comprises a subset selected from SEQ ID NOs: 39-58, 172, 173, 185-188, 191-194, 196, 198, 219, 225, 228, 233-235, 237, 238, 247, 256-258, 260, 261, 266, 268, 269, 271, 272, 329, 331, 332, 336, 340. The sequences 346-351, 356, 358-361, 373, 375, 381-385, 388, 390, 391, 393-395, 398, 409-411, 413, 418-420, 425-429, 438-440, and 443; the guide RNA (sgRNA) targeting the CD3E gene contains sequences selected from SEQ ID NOs: 1075-1094; the guide RNA (sgRNA) targeting the CD3G gene contains sequences selected from SEQ ID NOs: 1075-1094. NOs: 172, 173, 185-188, 191-194, 196, 198, 219, 225, 228, 233-235, 237, 238, 24 7. 256-258, 260, 261, 266, 268, 269, 271, 272, 329, 331, 332, 336, 340, 346-351, 3 The sequences 56, 358-361, 373, 375, 381-385, 388, 390, 391, 393-395, 398, 409-411, 413, 418-420, 425-429, 438-440, 443, and 585-604; and / or the guide RNA (sgRNA) targeting the CD3Z gene contains sequences selected from SEQ ID NOs: 1440-1459.

[0028] In some specific embodiments, the sequence of DNMT 3A includes SEQ ID NO: 15, and the sequence of DNMT 3L includes SEQ ID NO: 16 or SEQ ID NO: 17. Preferably, the sequence of the epigenetic modification domain is selected from DNMT 3A-DNMT 3L, DNMT 3L-DNMT 3A, DNMT 3A-hDNMT 3L, and hDNMT 3L-DNMT 3A.

[0029] In some specific embodiments, the sequence of ZIM3 contains SEQ ID NO: 20, the sequence of KRAB contains SEQ ID NO: 19, the sequence of HP1a contains SEQ ID NO: 23, and the sequence of SETDB1 contains SEQ ID NO: 24.

[0030] In some specific embodiments, the DNA binding domain comprises SEQ ID NO: 18.

[0031] In some specific embodiments, the sequence of said XTEN80 includes SEQ ID NO: 25.

[0032] In some specific embodiments, the amino acid sequence of the gene expression regulatory molecule comprises any sequence selected from SEQ ID NO: 1-7.

[0033] In some specific embodiments, the nucleic acid sequence encoding the gene expression regulatory molecule comprises any sequence selected from SEQ ID NOs: 8-14.

[0034] On the other hand, this disclosure provides a universal CAR-T cell that is prepared by the method provided in this disclosure.

[0035] In some specific embodiments, the CAR-T cells are CARs that specifically bind to tumor antigens, preferably anti-CD19 CAR-T cells, anti-CD20 CAR-T cells, anti-CD22 CAR-T cells, anti-CD23 CAR-T cells, anti-CD28 CAR-T cells, or anti-CD137 CAR-T cells.

[0036] On one hand, this disclosure provides a method for producing universal CAR-T cells, the method comprising:

[0037] By using gene expression regulatory molecules, the expression of HLA class I genes, HLA class II genes, and / or CD52 genes in the CAR-T cells was downregulated;

[0038] The gene expression regulatory molecule comprises i) an epigenetic modification domain, ii) a transcriptional regulatory domain, and iii) a DNA-binding domain, wherein the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to one end of the DNA-binding domain by a linker sequence, and the epigenetic modification domain is optionally linked to one end of the transcriptional regulatory domain by a linker sequence; or the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to both ends of the DNA-binding domain by a linker sequence.

[0039] In some specific embodiments, the gene expression regulatory molecule further includes (iv) a first recruitment domain and a second recruitment domain.

[0040] In some specific embodiments, the gene expression regulatory molecule comprises, from the N-terminus to the C-terminus, a second recruitment domain, a transcriptional regulatory domain, a splicing peptide, an epigenetic modification domain, a DNA-binding domain, and a first recruitment domain; the first recruitment domain and the second recruitment domain can interact with each other.

[0041] In some specific embodiments, the epigenetic modification domain comprises DNA methyltransferases DNMT 3A and DNMT3L; the transcriptional regulatory domain is selected from at least one of ZIM3, KRAB, HP1a, and SETDB1; the DNA binding domain is selected from: TALE domain, zinc finger domain, tetR domain, a wide range of nucleases, Cas proteins such as inactivated Cas9 protein (dCas9) or inactivated Cas12 protein (dCas12), Argonaute (Ago) protein, and their homologues, modified forms, or variants.

[0042] In some specific embodiments, the first recruitment domain is selected from universal control non-derepressor protein 4 (GCN4), a GFP11 fragment derived from split green fluorescent protein (GFP), or a GVKESLV polypeptide; the second recruitment domain is selected from a single-chain antibody (scFv), a GFP1-10 fragment derived from split green fluorescent protein (GFP), or a PDZ protein domain; more preferably, the first recruitment domain is selected from GCN4, and the second recruitment domain is selected from scFv.

[0043] In some specific embodiments, the HLA class I gene is selected from one or more of HLA-A, HLA-B, HLA-C, β2 microglobulin (B2M), TAP1, and TAP2.

[0044] In some specific implementations, the HLA class II gene is selected from one or more of RFXAP, RFX5, CIITA, and RFXANK.

[0045] In some specific embodiments, the linker sequence is XTEN80, and the cleavage peptide is selected from P2A, T2A, E2A, and F2A.

[0046] In some specific embodiments, the DNA-binding domain is capable of binding to guide RNA, which is capable of specifically recognizing and hybridizing with the target sequence of the target locus.

[0047] In some specific embodiments, the target sequence of the target locus is selected from SEQ ID NOs: 1626-SEQ ID NO: 1633.

[0048] In some specific embodiments, the guide RNA (sgRNA) targeting the B2M gene comprises a sequence selected from SEQ ID NOs: 1460-1478; the guide RNA (sgRNA) targeting the HLA-A gene comprises a sequence selected from SEQ ID NOs: 1479-1487; the guide RNA (sgRNA) targeting the HLA-B gene comprises a sequence selected from SEQ ID NOs: 1488-1501; the guide RNA (sgRNA) targeting the TAP1 gene comprises a sequence selected from SEQ ID NOs: 1502-1517; the guide RNA (sgRNA) targeting the TAP2 gene comprises a sequence selected from SEQ ID NOs: 1518-1529; the guide RNA (sgRNA) targeting the CIITA gene comprises a sequence selected from SEQ ID NOs: 1530-1549; and the guide RNA (sgRNA) targeting the RFX5 gene comprises a sequence selected from SEQ ID NOs: 1460-1478. The sequence NOs: 1550-1568; the guide RNA (sgRNA) targeting the RFXANK gene contains a sequence selected from SEQ ID NOs: 1569-1584; the guide RNA (sgRNA) targeting the RFXAP gene contains a sequence selected from SEQ ID NOs: 1585-1601; and / or the guide RNA (sgRNA) targeting the CD52 gene contains a sequence selected from SEQ ID NOs: 1602-1620.

[0049] In some specific embodiments, the sequence of DNMT 3A includes SEQ ID NO: 15, and the sequence of DNMT 3L includes SEQ ID NO: 16 or SEQ ID NO: 17. Preferably, the sequence of the epigenetic modification domain is selected from DNMT 3A-DNMT 3L, DNMT 3L-DNMT 3A, DNMT 3A-hDNMT 3L, and hDNMT 3L-DNMT 3A.

[0050] In some specific embodiments, the sequence of ZIM3 contains SEQ ID NO: 20, the sequence of KRAB contains SEQ ID NO: 19, the sequence of HP1a contains SEQ ID NO: 23, and the sequence of SETDB1 contains SEQ ID NO: 24.

[0051] In some specific embodiments, the sequence of the DNA-binding domain includes SEQ ID NO: 18.

[0052] In some specific embodiments, the sequence of said XTEN80 includes SEQ ID NO: 25.

[0053] In some specific embodiments, the sequence of the single-chain antibody (scFv) includes SEQ ID NO: 21, and the sequence of the GCN4 includes SEQ ID NO: 22.

[0054] In some specific embodiments, the amino acid sequence of the gene expression regulatory molecule comprises a sequence selected from that shown in SEQ ID NO: 4.

[0055] In some specific embodiments, the nucleic acid sequence encoding the gene expression regulatory molecule comprises a sequence selected from the sequence shown in SEQ ID NO: 11.

[0056] On the other hand, this disclosure provides a universal CAR-T cell that is prepared by the method provided in this disclosure.

[0057] In some specific embodiments, the CAR-T cells are CARs that specifically bind to tumor antigens, preferably anti-CD19 CAR-T cells, anti-CD20 CAR-T cells, anti-CD22 CAR-T cells, anti-CD23 CAR-T cells, anti-CD28 CAR-T cells, or anti-CD137 CAR-T cells. Attached Figure Description

[0058] This disclosure can be more fully understood with reference to the following figures.

[0059] Figure 1 shows (A) the whole-genome methylation level and RAN expression level of the CD3D gene, and the modification levels of various histones, including K3K4me1, H3K3me3, H3K36me3, H3K9me3, H3K27me3, and H3K27ac, using the CUT&Tag method; (B) the experimental workflow of CD3D gRNA plasmid library construction, viral transfection, cell sorting, and sequencing analysis; (C) the changes in TCR complex protein levels detected by flow cytometry in human primary T cells after transfection with the CD3D gRNA library, followed by epigenetic regulation using EPIREG-04; (D) the enrichment level of sgRNA readcounts at different positions of the target sequence (sequence number) in TCR-negative cells; (E) the inhibitory effect of 20 CD3D sgRNAs on TCR complex proteins; and (F) the results of long-term editing efficiency detection of 5 CD3D sgRNAs (numbered 9, 10, 11, 15, and 18).

[0060] Figure 2 shows (A) the whole-genome methylation level and RAN expression level of the CD3G gene, and the modification levels of various histones, including K3K4me1, H3K3me3, H3K36me3, H3K9me3, H3K27me3, and H3K27ac, using the CUT&Tag method; (B) the experimental workflow of CD3G gRNA plasmid library construction, viral transfection, cell sorting, and sequencing analysis; (C) the changes in TCR complex protein levels detected by flow cytometry after epigenetic regulation using EPIREG-04 in human primary T cells transfected with the CD3G gRNA library; (D) the enrichment level of sgRNA readcounts at different positions of the target sequence (sequence number) in TCR-negative cells; (E) the inhibitory effect of 20 CD3G sgRNAs on the protein expression level of the TCR complex; and (F) the results of long-term editing efficiency detection of 5 CD3G sgRNAs (numbered 1, 3, 10, 12, and 18).

[0061] Figure 3 shows the inhibitory effect of 77 sgRNAs on the protein expression level of the TCR complex.

[0062] Figure 4 shows (A) the whole-genome methylation level and RAN expression level of the CD3E gene, and the modification levels of various histones, including K3K4me1, H3K3me3, H3K36me3, H3K9me3, H3K27me3, and H3K27ac, using the CUT&Tag method; (B) the experimental workflow of CD3E gRNA plasmid library construction, viral transfection, cell sorting, and sequencing analysis; (C) the changes in TCR complex protein levels detected by flow cytometry after epigenetic regulation using EPIREG-04 in human primary T cells transfected with the CD3E gRNA library; (D) the enrichment level of sgRNA readcount at different positions of the target sequence (sequence number) in TCR-negative cells; (E) the inhibitory effect of 20 CD3E sgRNAs on TCR complex protein expression; and (F) the results of long-term editing efficiency detection of 5 CD3E sgRNAs (numbered 7, 8, 12, 17, and 18).

[0063] Figure 5 shows (A) the whole-genome methylation level and RAN expression level of the CD247 gene, and the modification levels of various histones, including K3K4me1, H3K3me3, H3K36me3, H3K9me3, H3K27me3, and H3K27ac, by the CUT&Tag method; (B) the inhibitory effect of 20 CD247 sgRNAs on the protein expression of the TCR complex; and (C) the results of long-term editing efficiency detection of 5 CD247 sgRNAs (numbered 7, 8, 12, 17, and 18).

[0064] Figure 6 shows the flow cytometry (Biolegend, 317318) staining analysis and comparison of the inhibitory effects of CD3 single subunit and multi-subunit combinations on CD3 gene expression.

[0065] Figure 7 shows the inhibitory effect of different versions of EPIREG on the protein expression of the TCR complex.

[0066] Figure 8 shows the structure of CAR.

[0067] Figure 9 shows (A) the preparation protocol of CAR T and the regulation of TCR epigenes; (B) the gene editing of sorted cells with human Burkitt lymphoma cells (Raji) added at a cell number ratio of 1:1; and (C) the gene editing of cells without human Burkitt lymphoma cells (Raji) added under normal culture.

[0068] Figure 10 shows the expression level and subset ratio of CAR (AC); (D) the expression of CAR T cells in the experimental and control groups after the addition of target cells Raji; (E) the cell number of Raji cells reflecting the killing effect of CAR T cells on Raji cells; and (F) the expansion of CAR-T cells when human Burkitt lymphoma cells (Raji) are added to the sorted cells at a cell number ratio of 1:1 every ten days to activate CAR-T cells.

[0069] Figure 11 shows the survival of mice after CAR T injection.

[0070] Figure 12 shows the inhibitory effect of B2M sgRNA on B2M protein.

[0071] Figure 13 shows the inhibitory effect of HLA-A sgRNA on HLA-A protein.

[0072] Figure 14 shows the inhibitory effect of HLA-B sgRNA on HLA-B protein.

[0073] Figure 15 shows the inhibitory effect of TAP-1sgRNA on HLA class I protein.

[0074] Figure 16 shows the inhibitory effect of TAP-2sgRNA on HLA class II protein.

[0075] Figure 17 shows the inhibitory effect of CIITA sgRNA on the HLA class II complex.

[0076] Figure 18 shows the inhibitory effect of RFX5 sgRNA on the HLA class II complex.

[0077] Figure 19 shows the inhibitory effect of RFXANK sgRNA on the HLA class II complex.

[0078] Figure 20 shows the inhibitory effect of RFXAP sgRNA on the HLA class II complex.

[0079] Figure 21 shows the inhibitory effect of CD52 sgRNA on CD52.

[0080] Figure 22 shows (A) the inhibitory effect of targeting MHC class I molecule HLA-A; (B) the inhibitory effect of targeting MHC class I molecule HLA-B; (C) the inhibition and short-term maintenance of the TCR complex by targeting CD3; (D) the long-term inhibitory stability test of targeting CD3; and (E) the inhibition and maintenance of MHC class II molecule (HLA-DR) by targeting CIITA.

[0081] Figure 23 shows the combined editing effect of (A) four targets (HLA-A, HLA-B, CD3, CIITA) and (B) the combined editing effect of alternative target combination (TAP1, RFX5, CD3). Detailed Implementation

[0082] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0083] Gene expression regulatory molecules

[0084] As used herein, the term "gene expression regulatory molecule" refers to a chimeric protein directly or indirectly generated through the covalent or non-covalent linkage of two or more genes, which originally encode individual proteins. In some embodiments, the translation of the gene expression regulatory molecule gene produces a single polypeptide having functional properties derived from each original protein. The gene expression regulatory molecule of this application can be widely used in gene editing, particularly for epigenetic modification of the TCR gene, to prepare UCART products with greater immunotherapeutic potential. It has significant technical advantages in cancer immunotherapy, especially in the application of CAR-T cells, helping to improve and enhance the efficacy of existing CAR-T therapies and providing important technical support for the development of next-generation T-cell therapies. Furthermore, the gene expression regulatory molecule of this application can broaden the scope of TCR gene epigenetic modification by using different types of epigenetic modification domains and transcriptional regulatory domains, thereby optimizing the immune response of T cells. For the preparation of UCART, this technology can introduce more site-specific modifications into TCR regulation, improving the specificity, persistence, and anti-tumor efficacy of T cells.

[0085] This application provides a gene expression regulatory molecule for gene editing and its uses, particularly for TCR editing and the preparation of UCART (universal CAR-T cells). This gene expression regulatory molecule comprises i) an epigenetic modification domain, ii) a transcriptional regulatory domain, and iii) a DNA-binding domain. By fusing the epigenetic modification domain and the transcriptional regulatory domain to the DNA-binding domain, the gene expression regulatory molecule of this application exhibits higher efficiency in the precise regulation of the TCR gene. Compared to existing fusions with the same or similar domains and regulatory elements, the gene expression regulatory molecule of this application can more effectively regulate TCR expression and avoid unnecessary genomic mutations during the editing process.

[0086] As used herein, the term "gene expression regulatory molecule" can downregulate gene expression in cells, for example, downregulating the expression of the TCR-CD3 complex in the T cell receptor (TCR) of CAR-T cells, preferably downregulating the expression of one or more of CD3D, CD3E, CD3G, CD3Z, TCRA, and TCRβ; more preferably downregulating the expression of the CD3D / CD3E or CD3G / CD3E dimer. The gene expression regulatory molecule comprises i) an epigenetic modification domain, ii) a transcriptional regulatory domain, and iii) a DNA-binding domain, wherein the epigenetic modification domain and the transcriptional regulatory domain are selectively linked to one end of the DNA-binding domain by a linker sequence, and the epigenetic modification domain is selectively linked to one end of the transcriptional regulatory domain by a linker sequence; or the epigenetic modification domain and the transcriptional regulatory domain are selectively linked to both ends of the DNA-binding domain by a linker sequence. In some embodiments, the gene expression regulatory molecule further comprises (iv) a first recruitment domain and a second recruitment domain, wherein the first recruitment domain and the second recruitment domain are respectively at one end or both ends of the DNA binding domain. Preferably, the first recruitment domain is selected from universal control non-derepressor protein 4 (GCN4), a GFP11 fragment derived from split green fluorescent protein (GFP), or a GVKESLV polypeptide; the second recruitment domain is selected from a single-chain antibody (scFv), a GFP1-10 fragment derived from split green fluorescent protein (GFP), or a PDZ protein domain. Preferably, the first recruitment domain is selected from GCN4, and the second recruitment domain is selected from scFv.

[0087] In some specific embodiments, the gene expression regulatory molecule, from the N-terminus to the C-terminus, comprises: 1) an epigenetic modification domain, a DNA-binding domain, and a transcriptional regulatory domain; 2) a DNA-binding domain, a transcriptional regulatory domain, and an epigenetic modification domain; and 3) a second recruitment domain, a transcriptional regulatory domain, a cleavage peptide, an epigenetic modification domain, a DNA-binding domain, and a first recruitment domain; the first recruitment domain and the second recruitment domain are capable of interacting. The linker sequence is XTEN80, and the cleavage peptide is selected from P2A, T2A, E2A, and F2A.

[0088] In some specific embodiments, the amino acid sequence of the gene expression regulatory molecule comprises any sequence selected from SEQ ID NOs: 1-7; the nucleic acid sequence encoding the gene expression regulatory molecule comprises any sequence selected from SEQ ID NOs: 8-14.

[0089] As used herein, the term "gene expression regulatory molecule" refers to a molecule that downregulates gene expression in cells, such as downregulating the expression of HLA class I genes, HLA class II genes, and / or the CD52 gene in CAR-T cells; wherein the HLA class I genes are selected from one or more of HLA-A, HLA-B, HLA-C, β2-microglobulin (B2M), TAP1, and TAP2, and the HLA class II genes are selected from one or more of RFXAP, RFX5, CIITA, and RFXANK. The gene expression regulatory molecule comprises i) an epigenetic modification domain, ii) a transcriptional regulatory domain, and iii) a DNA-binding domain, wherein the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to one end of the DNA-binding domain by a linker sequence, and the epigenetic modification domain is optionally linked to one end of the transcriptional regulatory domain by a linker sequence; or the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to both ends of the DNA-binding domain by a linker sequence.

[0090] In some embodiments, the gene expression regulatory molecule further comprises (iv) a first recruitment domain and a second recruitment domain, wherein the first recruitment domain and the second recruitment domain are respectively at one end or both ends of the DNA binding domain. Preferably, the first recruitment domain is selected from universal control non-derepressor protein 4 (GCN4), a GFP11 fragment derived from split green fluorescent protein (GFP), or a GVKESLV polypeptide; the second recruitment domain is selected from a single-chain antibody (scFv), a GFP1-10 fragment derived from split green fluorescent protein (GFP), or a PDZ protein domain. Preferably, the first recruitment domain is selected from GCN4, and the second recruitment domain is selected from scFv.

[0091] In some specific embodiments, the gene expression regulatory molecule comprises, from the N-terminus to the C-terminus, a second recruitment domain, a transcriptional regulatory domain, a splicing peptide, an epigenetic modification domain, a DNA-binding domain, and a first recruitment domain; the first recruitment domain and the second recruitment domain can interact with each other.

[0092] In some specific embodiments, the amino acid sequence of the gene expression regulatory molecule comprises a sequence selected from the sequence shown in SEQ ID NO: 4; and the nucleic acid sequence encoding the gene expression regulatory molecule comprises a sequence selected from the sequence shown in SEQ ID NO: 11.

[0093] DNA-binding domain

[0094] In certain embodiments of the methods and compositions according to this disclosure as defined herein, the DNA-binding domain refers to, for example, a DNA targeter comprising a (DNA) nuclease, such as a nuclease that can target DNA in a sequence-specific manner or can be directed or indicated to target DNA in a sequence-specific manner, such as the CRISPR-Cas system, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or a wide range of nucleases. In some embodiments, the DNA-binding protein is a DNA nuclease derived from the CRISPR-Cas system. In some specific embodiments, the DNA-binding domain is selected from the TALE domain, zinc finger domain, tetR domain, wide range of nucleases, Cas proteins such as inactivated Cas9 protein (dCas9) or inactivated Cas12 protein (dCas12), Argonaute (Ago) protein, and their homologues, modified forms, or variants. In some specific embodiments, the DNA-binding domain comprises SEQ ID NO: 18.

[0095] In some implementations, cutting efficiency can be modulated by introducing mismatches, such as one or more mismatches, for example, one or two mismatches between the spacer sequence and the target sequence (including the location of the mismatch along the spacer / target region). For example, the closer a double mismatch is to the center (i.e., not at 3′ or 5′), the greater its impact on cutting efficiency. Therefore, cutting efficiency can be adjusted by selecting the location of the mismatch along the spacer region. For example, if a cut of the target of less than 100% is desired (e.g., in a cell population), one or more, preferably two, mismatches can be introduced between the spacer region and the target sequence in the spacer sequence. The closer the mismatch location is to the center along the spacer region, the lower the cutting percentage.

[0096] In some embodiments, the DNA-binding domain and single guide RNA sequence are derived from a CRISPR-Cas system. This disclosure provides a CRISPR / Cas9-based engineered system for genome editing and the treatment of genetic diseases. The CRISPR / Cas9-based engineered system can be designed to target any gene (e.g., HLA class I genes, HLA class II genes, and / or the CD52 gene in CAR-T cells). This disclosure provides a CRISPR-Cas system comprising a genetically engineered Cas protein and / or guide RNA having desired specificity and activity (e.g., reduced or eliminated expression of HLA class I genes, HLA class II genes, and / or the CD52 gene product in CAR-T cells). The Cas9 includes, for example, an inactivated Cas9 protein (dCas9) or an inactivated Cas12 protein (dCas12), an Argonaute protein (Ago), and its homologues, modified forms, or variants.

[0097] Typically, the guide sequence (or spacer sequence) can be a polynucleotide sequence with sufficient complementarity to the HLA class I, HLA class II, and / or CD52 gene sequences to hybridize with the HLA class I, HLA class II, and / or CD52 gene sequences and guide the CRISPR complex to specifically bind to the polynucleotide sequences of the HLA class I, HLA class II, and / or CD52 genes. In some embodiments, when optimal alignment is performed using a suitable alignment algorithm, the complementarity between the guide sequence and its corresponding target sequence is equal to or greater than about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher.

[0098] In some embodiments, the DNA-binding domain and single guide RNA sequence are derived from a CRISPR-Cas system. This disclosure provides a CRISPR / Cas9-engineered system for genome editing and the treatment of genetic diseases. The CRISPR / Cas9-engineered system can be designed to target any gene (e.g., the TCR-CD3 complex in the T cell receptor (TCR)). This disclosure provides a CRISPR-Cas system comprising a genetically engineered Cas protein and / or guide RNA having desired specificity and activity (e.g., reduced or eliminated expression of CD3D, CD3E, CD3G, CD3Z, TCRα, and / or TCRβ gene products). The Cas9 includes, for example, an inactivated Cas9 protein (dCas9) or an inactivated Cas12 protein (dCas12), an Argonaute protein (Ago), and its homologues, modified forms, or variants.

[0099] Typically, the guide sequence (or spacer sequence) can be sufficiently complementary to the CD3D, CD3E, CD3G, CD3Z, TCRα, and / or TCRβ polynucleotide sequences to hybridize with said CD3D, CD3E, CD3G, CD3Z, TCRα, and / or TCRβ sequences and guide the CRISPR complex to specifically bind to said CD3D, CD3E, CD3G, CD3Z, TCRα, and / or TCRβ polynucleotide sequences. In some embodiments, when optimal alignment is performed using a suitable alignment algorithm, the complementarity between the guide sequence and its corresponding target sequence is equal to or greater than about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher.

[0100] Epigenetic modification domains

[0101] "Epigenetic modification domains" refer to agents that target gene expression through epigenetic modifications (e.g., through histone acetylation or methylation, or DNA methylation at regulatory elements of a target gene, such as promoters, enhancers, or transcription start sites). Chromatin remodeling and DNA methylation are two major mechanisms regulating gene transcription. Specific epigenetic markers (e.g., DNA methylation) structurally or biochemically direct gene transcription or gene silencing / repression. For example, DNA methylation in regions that regulate transcriptional activity alters gene expression without changing the underlying DNA sequence. Transcriptional regulation using epigenetic modifications (e.g., DNA methylation) allows for targeted regulation of gene expression without affecting the expression of other gene products. In some specific embodiments, the epigenetic modification regulatory domain is DNMT3A, DNMT3L, a DNMT3A-DNMT3L fusion peptide, a DNMT3L-DNMT3A fusion peptide, or a combination thereof. In some specific embodiments, the sequence of DNMT 3A includes SEQ ID NO: 15, and the sequence of DNMT 3L includes SEQ ID NO: 16 or SEQ ID NO: 17. Preferably, the sequence of the epigenetic modification domain is selected from DNMT 3A-DNMT 3L, DNMT 3L-DNMT 3A, DNMT 3A-hDNMT 3L, and hDNMT 3L-DNMT 3A.

[0102] Transcriptional regulatory domains

[0103] As used herein, the term "transcriptional regulatory domain" includes repressors of gene expression. These repressors can be any known gene expression repressor, such as repressors selected from the Kruppe1-associated box (KRAB) domain, the mSin3-interacting domain (SID), the MAX-interacting protein 1 (MXI1), the chromosome shadow domain, the EAR repressor domain (SRDX), eukaryotic releasing factor 1 (ERF1), eukaryotic releasing factor 3 (ERF3), tetracycline repressors, 1ad repressors, periwinkle G-box binding factors 1 and 2, Drosophila Groucho, Tripartite motif-containing 28 (TRTM28), nuclear receptor co-repressor 1, nuclear receptor co-repressor 2, or fragments or fusions thereof. In some specific implementations, the transcriptome repressor is selected from one or more of the following domains: KRAB, ZIM3, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF35 0, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZF P82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP 1. ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, C BX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPL IRF-2BP1_2N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9,ZFP28,ZN334,ZN568,ZN37A,ZN181,ZN510,ZN862,ZN140,ZN208,ZN248,ZN571,ZN699,ZN726,ZIK1,ZNF2,Z705F,ZNF14,ZN471,ZN624,ZNF84,ZNF7,ZN891,ZN337,Z705G,ZN529,ZN729,ZN419,Z705A,ZN302,ZN486,ZN621,ZN688,ZN33A,ZN554,ZN878,ZN772,ZN224,ZN184,ZN544,ZNF57,ZN283,ZN549,ZN211,ZN615,ZN253,ZN226,ZN730,Z585A,ZN732,ZN681,ZN667,ZN649,ZN470,ZN484,ZN431,ZN382,ZN254,ZN124,ZN607,ZN317,ZN620,ZN141,ZN584,ZN540,ZN75D,ZN555,ZN658,ZN684,RBAK,ZN829,ZN582,ZN112,ZN716,HKR1,ZN350,ZN480,ZN416,ZNF92,ZN100,ZN736,ZNF74,ZN443,ZN195,ZN530,ZN782,ZN791,ZN331,Z354C,ZN157,ZN727,ZN550,ZN793,ZN235,ZN724,ZN573,ZN577,ZN789,ZN718,ZN300,ZN383,ZN429,ZN677,ZN850,ZN454,ZN257,ZN264,ZN485,ZN737,ZNF44,ZN596,ZN565,ZN543,ZFP69,SUMO1,ZNF12,ZN169,ZN433,ZN175,ZN347,ZNF25,ZN519,Z585B,ZN517,ZN846,ZN230,ZNF66,ZN713,ZN816,ZN426,ZN674,ZN627,ZNF20,Z587B,ZN316,ZN233,ZN611,ZN556,ZN234,ZN560,ZNF77,ZN682,ZN614,ZN785,ZN445,ZFP30,ZN225,ZN551,ZN610,ZN528,ZN284,ZN418,ZN490,ZN805,Z780B,ZN763,ZN285,ZNF85,ZN223,ZNF90,ZN557,ZN425,ZN229,ZN606,ZN155,ZN222,ZN442,ZNF91,ZN135,ZN778,ZN534,ZN586,ZN567,ZN440,ZN583,ZN441,ZNF43,ZN589,ZN563,ZN561,ZN136,ZN630,ZN527,ZN333,Z324B,ZN786,ZN709,ZN792,ZN599,ZN613,ZF69B,ZN799,ZN569,ZN564,ZN546,ZFP92,ZN723,ZN439,ZFP57,ZNF19,ZN404,ZN274,CBX3,ZN250,ZN570,ZN675,ZN695,ZN548,ZN132,ZN738,ZN420,ZN626,ZN559,ZN460,ZN268,ZN304,ZN605,ZN844,SUMO5,ZN101,ZN783,ZN417,ZN182,ZN823,ZN177,ZN197,ZN717,ZN669,ZN256,ZN251,CBX4,CDY2,CDYL2,ZN562,ZN461,Z324A,ZN766,ID2,ZN214,CBX7,ID1,CREM,SCX,ASCL1,ZN764,SCML2,TWST1,CREB1,TERF1,ID3,CBX8,GSX1,NKX22,ATF1,TWST2,ZNF17,TOX3,TOX4,ZMYM3,I2BP1,RHXF1,SSX2,I2BPL,ZN680,TRI68,HXA13,PHC3,TCF24,HXB13,HEY1,PHC2,ZNF81,FIGLA,SAM11,KMT2B,HEY2,JDP2,HXC13,ASCL4,HHEX,GSX2,ETV7,ASCL3,PHC1,OTP,I2BP2,VGLL2,HXA11,PDLI4,ASCL2,CDX4,ZN860,LMBL4,PDIP3,NKX25,CEBPB,ISL1,CDX2,PROP1,SIN3B,SMBT1,HXC11,HXC10,PRS6A,VSX1,NKX23,MTG16,HMX3,HMX1,KIF22,CSTF2,CEBPE,DLX2,PPARG,PRIC1,UNC4,BARX2,ALX3,TCF15,TERA,VSX2,HXD12,CDX1,TCF23,ALX1,HXA10,RX,CXXC5,SCML1,NFIL3,DLX6,MTG8,CEBPD,SEC13,FIP1,ALX4,LHX3,PRIC2,MAGI3,NELL1,PRRX1,MTG8R,RAX2,DLX3, DLX1, NKX26, NAB1, SAMD7, PITX3, WDR5, MEOX2, NAB2, DHX8, CBX6, EMX2, CPSF6, HXC12, KDM4B, LMBL3, PHX2A, EMX1, NC2B, DLX4, SRY, ZN777, ZN398, GATA3, BSH, SF3B4, TEAD1, TEAD3, RGAP1, PHF1, GATA2, FOXO3, ZN212, IRX4, ZBED6, LHX4, SIN3A, RBBP7 The transcriptional repressor comprises, and preferably, NKX61, R51A1, MB3L1, DLX5, NOTC1, TERF2, ZN282, RGS12, ZN840, SP12B, PAX7, NKX62, ASXL2, FOXO1, GATA1, ZMYM5, LRP1, MIXL1, SGT1, LMCD1, CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and their functionally active fragments. Preferably, the transcriptional repressor is selected from KRAB, ZIM3, HP1a, and SETDB1.

[0104] In some specific embodiments, the sequence of ZIM3 contains SEQ ID NO: 20, the sequence of KRAB contains SEQ ID NO: 19, the sequence of HP1a contains SEQ ID NO: 23, and the sequence of SETDB1 contains SEQ ID NO: 24.

[0105] Recruitment domain

[0106] The gene expression regulatory molecule described in this application further includes a recruitment domain. The transcriptional regulatory domain and the epigenetic modification domain may coexist or not have a recruitment domain. When both recruitment domains are present, the interaction between the recruitment domains they contain forms an aggregated effector domain. Therefore, this application provides non-limiting examples of combinations of a first recruitment domain and a second recruitment domain: (1) one of the first recruitment domain and the second recruitment domain is GCN4, and the other domain is scFv; or (2) one of the first recruitment domain and the second recruitment domain is a GFP11 fragment, and the other domain is GFP1-10; or (3) one of the first recruitment domain and the second recruitment domain is GVKESLV, and the other domain is a PDZ protein domain. Similarly, the situation where GFP11 and GFP1-10 are derived from splitting GFP to form the first recruitment domain and the second recruitment domain, respectively, can also be applied to other types of fluorescent proteins, such as mCherry (GenBank: QSL83322.1), eYFP (GenBank: AAO48597.1), eCFP (GenBank: AHJ09746.1), etc. Different sets of the first recruitment domain and the second recruitment domain can be obtained by splitting mCherry, splitting eYFP, or splitting eCFP, respectively, and used in the complex provided in this application.

[0107] In some embodiments, the first recruitment domain and the second recruitment domain are respectively located at one end or both ends of the DNA binding domain. Preferably, the first recruitment domain is selected from Universal Control Non-Derepressor Protein 4 (GCN4), a GFP11 fragment derived from split green fluorescent protein (GFP), or a GVKESLV polypeptide; the second recruitment domain is selected from a single-chain antibody (scFv), a GFP1-10 fragment derived from split green fluorescent protein (GFP), or a PDZ protein domain. Preferably, the first recruitment domain is selected from GCN4, and the second recruitment domain is selected from scFv.

[0108] The epigenetic modification domain and the transcriptional regulatory domain described herein may be selectively connected to one end of the DNA-binding domain via a linker sequence, and the epigenetic modification domain may be selectively connected to one end of the transcriptional regulatory domain via a linker sequence; or the epigenetic modification domain and the transcriptional regulatory domain may be selectively connected to both ends of the DNA-binding domain via a linker sequence.

[0109] In some embodiments, the linker sequence is an XTEN linker. Such linkers may comprise a portion of an XTEN sequence (Schellenberger et al., Nat Biotechnol (2009) 27(1): 1186-90), which is a nonstructured hydrophilic polypeptide consisting only of residues G, S, P, T, and E, and the term “XTEN” as used herein refers to a recombinant peptide or polypeptide lacking hydrophobic amino acid residues. XTEN linkers are typically unstructured and contain a limited set of native amino acids. Fusion of XTEN to a protein alters its hydrodynamic properties and reduces the clearance and degradation rate of the fusion protein. These XTEN fusion proteins are produced using recombinant technologies without chemical modification and degrade naturally. The length of the XTEN linker can be, for example, 5, 10, 16, 20, 26, or 80 amino acids. In some embodiments, the length of the XTEN linker is 16 amino acids. In some embodiments, the length of the XTEN linker is 80 amino acids. In some embodiments, the XTEN linker can be XTEN10, XTEN16, XTEN20, or XTEN80.

[0110] Universal CAR-T cells

[0111] Chimeric antigen receptors (CARs) are engineered membrane protein receptor molecules that endow immune effector cells with the specificity required, such as the ability to bind specific tumor antigens. CARs typically consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some cases, the antigen-binding domain is the scFv sequence responsible for recognizing and binding a specific antigen. The intracellular signaling domain typically includes an immune receptor tyrosine activation motif (ITAM), such as the signaling domain derived from the CD3z molecule, which is responsible for activating immune effector cells and producing cytotoxic effects. Furthermore, CARs may also contain a signal peptide responsible for the intracellular localization of the nascent protein at its N-terminus, as well as a hinge region between the antigen-binding domain and the transmembrane domain. In addition to the signaling domain, the intracellular signaling domain may also contain a co-stimulatory domain derived from molecules such as 4-1BB or CD28.

[0112] In this document, "CAR cell" refers to a cell that expresses a CAR molecule on its cell surface. In most cases, these cells are immune cells, such as T cells or NK cells. Therefore, CAR-expressing T cells are referred to herein as "CAR-T" or "CAR-T cells." Furthermore, when CAR-T cells are mentioned herein, unless otherwise stated, it refers not only to cells directly modified with CAR, but also to daughter cells generated after these cells proliferate in vitro or in vivo. In some specific embodiments, the CAR-T cells are CARs that specifically bind to tumor antigens, preferably anti-CD19 CAR-T cells, anti-CD20 CAR-T cells, anti-CD22 CAR-T cells, anti-CD23 CAR-T cells, anti-CD28 CAR-T cells, or anti-CD137 CAR-T cells.

[0113] In this article, "universal CAR-T cells (UCAR-T)" refers to cells that are not limited to CAR-T cells infused into a specific patient. In existing technologies, to prevent GvHD and host rejection of the graft, cells (e.g., T cells) are typically collected from the patient, modified with CAR, and then reinfused into the patient. This method is not only time-consuming and expensive, but in some cases, it is impossible to obtain a sufficient number of patient T cells for CAR modification. In contrast, universal CAR-T cells here refer to cells suitable for allogeneic transplantation; the same batch of CAR-T cells can be used for different patients, and these universal CAR-T cells are usually not derived from these patients.

[0114] The term “B2M” used in this article refers to β2-microglobulin (Gene ID: 627).

[0115] The terms “HLA-A”, “HLA-B”, and “HLA-C” used in this article refer to Human Leukocyte Antigen A (Gene ID: 3104), Human Leukocyte Antigen B (Gene ID: 3105), and Human Leukocyte Antigen C (Gene ID: 3106), respectively.

[0116] As used in this article, the terms “TAP1” and “TAP2” refer to antigen presentation-associated transporter 1 (ATP-binding cassette sub-family B member 2, Gene ID: 6882) and antigen presentation-associated transporter 2 (ATP-binding cassette sub-family B member 3, Gene ID: 6883).

[0117] The terms “RFXAP”, “RFX5” and “RFXANK” used in this article refer to Regulatory Factor X Associated Protein (Gene ID: 5992), Regulatory Factor X 5 (Gene ID: 5994), and Regulatory Factor X Associated Nuclear Factor (Gene ID: 5993).

[0118] The term “CIITA” used in this article refers to Class II Transactivator (Gene ID: 5711).

[0119] The term "CD52" as used in this article refers to the CD52 antigen (CD52 molecule, Gene ID: 975).

[0120] As used herein, the terms "coding sequence" or "coding nucleic acid" refer to a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence encoding a protein. The coding sequence may further include start and stop signals operatively linked to regulatory elements, including promoters and polyadenylation signals capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon-optimized.

[0121] Example

[0122] To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0123] Example 1: Epigenetic Editing Tool (EPIREG)

[0124] The following are some typical appearance editing tool (EPIREG) structures used in this invention:

[0125] EPIREG-01: DNMT3A-DNMT3L-XTEN80-dCas9-KRAB;

[0126] EPIREG-02: DNMT3A-hDNMT3L-XTEN80-ZIM3-XTEN80-dCas9

[0127] EPIREG-03: dCas9-XTEN80-ZIM3-XTEN80-hDNMT3L-DNMT3A;

[0128] EPIREG-04: scFV-ZIM3-2a-DNMT3A-hDNMT3L-dCas9-GCN4, hDNMT3L is of human origin;

[0129] EPIREG-05: DNMT3A-DNMT3L-XTEN80-dCas9-HPIa;

[0130] EPIREG-06: DNMT3A-DNMT3L-XTEN80-dCas9-SETDB1;

[0131] EPIREG-07: DNMT3A-DNMT3L-XTEN80-dCas9-ZIM3.

[0132] The amino acid sequences of the epigenetic editing tool used in this invention are shown in SEQ ID NOs: 1-7, and the corresponding mRNA sequences are shown in SEQ ID NOs: 8-14. The domain sequences of the epigenetic editing tool (EPIREG) are shown in SEQ ID NOs: 15-26, and the corresponding mRNA sequences are shown in SEQ ID NOs: 27-38.

[0133] Table 1. Appearance Editing Tools (EPIREG)

[0134] Table 2. Domain sequence information of the appearance editing tool (EPIREG)

[0135] Example 2: Construction and screening of sgRNA libraries targeting the CD3D gene

[0136] (1) Prediction of adjustable regions in CD3D based on apparent AI large model

[0137] This invention first performed a multi-omics analysis on human primary T cells. Whole-genome methylation sequencing (WGBS) and RNA-seq were performed. Furthermore, the CUT & Tag method was used to sequence and analyze various histone modifications, including K3K4me1, H3K3me3, H3K36me3, H3K9me3, H3K27me3, and H3K27ac. After analysis using a large AI model (analysis method referenced: Yang, Q. et al. EpiCas-DL: Predicting sgRNA activity for CRISPR-mediated epigenome editing by deep learning. Comput Struct Biotechnol J 21, 202-211(2023). https: / / doi.org / 10.1016 / j.csbj.2022.11.034), it was found that the 2000bp upstream and downstream of the CD3D transcription start site (Chr11: 118340710-118344696, SEQ ID NO: 1621) are regulatory regions (Figure 1A).

[0138] (2) CD3D gRNA design and library construction

[0139] Based on the analysis results of the large epigenetic AI model, we screened gRNA libraries for CD3D (Gene ID: 915). The genomic range covered by the gRNAs was Chr11: 118340710-118344696.

[0140] The CD3D gRNA sequence designed in this embodiment is shown in Table 3.

[0141] Table 3. CD3D gRNA Sequence

[0142] (3) Construction and screening of gRNA plasmid libraries

[0143] Figure 1B shows the experimental workflow for gRNA plasmid library construction, viral transfection, cell sorting, and sequencing analysis.

[0144] gRNA plasmid library construction: The sgRNA library fragment was amplified by PCR, digested with enzymes to obtain the template vector, and the sg-library fragment was digested with the same enzyme. Ligation was then performed using T4 ligase. The ligation product was transformed into DH5α competent cells, and the transformation product was plated onto culture plates with appropriate antibiotics and incubated upside down at 37°C for 12–16 hours. Single clones were picked, cultured, and sent for assays. After confirmation of correctness, plasmid extraction was performed. The extracted plasmid was used to extract the fragment containing the sgRNA library using nested PCR, and then sent for NGS sequencing verification. After verification, lentivirus packaging was performed.

[0145] Viral transfection: Six hours after resuscitating PBMCs (peripheral blood mononuclear cells), cells were activated with anti-CD3 / 28 magnetic beads (GIBCO, 11161D). Lentiviral virus was added on the second day. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 was transfected into PBMCs by electroporation (specific electroporation method follows the kit instructions: Lonza P3 kit, V4XP-3024).

[0146] Cell sorting: On day 21 after electroporation, flow cytometry was used to detect changes in the protein level of the TCR complex. After flow cytometry sorting, TCR-negative and TCR-positive cells were lysed and subjected to nested PCR. The PCR products were then sent for NGS sequencing.

[0147] The components of the TCR complex work closely together to ensure that T cells can correctly recognize antigens and initiate an immune response. The TCR itself, composed of the TCRα and TCRβ chains, is responsible for antigen recognition; the CD3 complex, composed of the CD3ε, CD3δ, CD3γ, and ζ chains, is responsible for signal transduction and initiating T cell activation. The genetic coding of each subunit is crucial to the stability and function of the TCR complex.

[0148] However, the structure and function of the TCR complex depend on the normal expression and interaction of its various subunits. Knocking out any subunit of the TCR complex disrupts its overall structure, leading to dissociation and severely impacting its function. In particular, the loss of any subunit or TCR chain in the CD3 complex prevents the TCR complex from forming stably or transmitting signals normally. Whether it's the TCRα chain, TCRβ chain, or the CD3ε, CD3δ, CD3γ chains, or even the ζ chain in the CD3 complex, they are all crucial for maintaining the stability and normal function of the complex. The loss of any subunit prevents T cells from recognizing antigens or initiating an immune response, ultimately resulting in complete loss of T cell function. Therefore, the loss of each subunit in the TCR complex directly leads to dissociation and loss of function, severely interfering with the immune response of T cells.

[0149] Figure 1C shows primary human T cells transfected with a gRNA library. After epigenetic regulation using EPIREG-04, changes in the protein level of the TCR complex were detected by flow cytometry. TCR-negative and TCR-positive cells were sorted by flow cytometry, and gRNA sequencing analysis was performed to screen for gRNAs with regulatory functions.

[0150] (4) Analysis of sgRNA library screening results

[0151] This invention performed NGS sequencing on sgRNA libraries and then analyzed the regions enriched by sgRNAs to define the regulatory regions of CD3D. We calculated the read counts of the TCR-negative sgRNA enrichment results and normalized the sgRNA read counts using the median normalization default in Mageck (Model-based Analysis of Genome-wide CRISPR-CRISPR Knockout), ultimately obtaining the normalized count for each sgRNA. After matching the relative positions with the transcription start site (TSS), we found that a large number of sgRNAs capable of effectively repressing CD3D were successfully enriched within a 2000 bp range upstream and downstream of the TSS (Figure 1D).

[0152] (5) Validation experiment on the effectiveness of library screening for candidate sgRNAs

[0153] Based on the results of the above library screening and considering factors such as conservation, 20 CD3D sgRNAs, as shown in the table below, were selected and transfected into PBMC (Miaoshun (Shanghai) Biotechnology Co., Ltd.) for epigenetic editing to verify the inhibitory effect of the selected candidate sgRNAs on CD3D gene expression. The 20 sgRNA sequences used for verification in this example are as follows:

[0154] Table 4. Candidate CD3D gRNA Sequences

[0155] Specific experimental protocol: Six hours after resuscitating PBMCs, cells were activated with anti-CD3 / 28 magnetic beads (Thermofisher, Dynabeads). TM On day four, magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On day five, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs via electroporation (specific electroporation methods are described in the kit instructions: Lonza P3 kit, V4XP-3024). Samples were collected on days 3 and 7 post-transfection, and flow cytometry (Biolegend, 317318) staining was used to analyze the inhibitory effect of these 20 sgRNAs on the TCR complex protein. The results are shown in Figure 1E, indicating that the inhibition rate was over 80%. From these 20 sgRNAs, the 5 with the best editing performance (gRNA-CD3D-9, gRNA-CD3D-10, gRNA-CD3D-11, gRNA-CD3D-15, and gRNA-CD3D-18) were selected for further long-term editing efficiency testing. The statistical results are shown in Figure 1F.

[0156] Example 3: Construction and screening of sgRNA libraries targeting the CD3G gene

[0157] (1) Prediction of CD3G adjustable region based on apparent AI large model

[0158] This invention first performed a multi-omics analysis on human primary T cells. Whole-genome methylation sequencing (WGBS) and RNA-seq were performed. Furthermore, the CUT & Tag method was used to sequence and analyze various histone modifications, including K3K4me1, H3K3me3, H3K36me3, H3K9me3, H3K27me3, and H3K27ac. After analysis using a large AI model (analysis method referenced: Yang, Q. et al. EpiCas-DL: Predicting sgRNA activity for CRISPR-mediated epigenome editing by deep learning. Comput Struct Biotechnol J 21, 202-211(2023). https: / / doi.org / 10.1016 / j.csbj.2022.11.034), it was found that the 2000bp upstream and downstream of the CD3G transcription start site (Chr11: 118342401-118346166, SEQ ID NO: 1622) are regulatory regions (Figure 2A).

[0159] (2) CD3G gRNA design and library construction

[0160] Based on the predictions of the epigenetic AI model, this invention screens gRNA libraries for CD3G (Gene ID: 917), and the genomic range covered by the gRNAs is Chr11: 118342401-118346166.

[0161] The CD3G gRNA sequence designed in this embodiment is shown in Table 5.

[0162] Table 5. CD3G gRNA Sequence

[0163] Figure 2B shows the experimental workflow for gRNA plasmid library construction, viral transfection, cell sorting, and sequencing analysis. Specific steps are described in Example 2(3).

[0164] Figure 2C shows the changes in TCR complex protein levels in primary human T cells transfected with a gRNA library. After epigenetic regulation using EPIREG-04, the TCR complex protein levels were detected by flow cytometry. TCR-negative and TCR-positive cells were sorted by flow cytometry and then subjected to gRNA sequencing analysis to screen for gRNAs with regulatory functions.

[0165] (3) Analysis of sgRNA library screening results

[0166] This invention performs NGS sequencing on sgRNA libraries and then analyzes the regions enriched by sgRNAs to define the regulatory regions of CD3G. We calculated the read counts of the TCR-negative sgRNA enrichment results and normalized the sgRNA read counts using Mageck's default median normalization, ultimately obtaining the normalized count for each sgRNA. After matching the relative positions with the TSS, we found that a large number of sgRNAs capable of effectively inhibiting the epigenetic function of CD3G were successfully enriched within a 2000 bp range upstream and downstream of the TSS (Figure 2D).

[0167] (4) Validation experiment on the effectiveness of library screening for candidate sgRNAs

[0168] Based on the results of the above library screening and considering factors such as conservation, 20 CD3G sgRNAs, as shown in the table below, were selected and transfected into PBMCs for epigenetic editing to verify the inhibitory effect of the selected candidate sgRNAs on CD3G gene expression. The 20 sgRNA sequences used for verification in this example are gRNA-CD3G-1 to gRNA-CD3G-20 (Table 6).

[0169] Table 6. Candidate CD3G gRNA Sequences

[0170] Specific experimental protocol: Six hours after PBMC resuscitation, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs via electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). Samples were collected on days 3 and 7 post-transfection, and flow cytometry (Biolegend, 317318) staining was used to analyze the inhibitory effect of these 20 sgRNAs on the protein expression level of the TCR complex. The results are shown in Figure 2E, indicating that the inhibition rate was over 80%. From these 20 sgRNAs, the 5 with the best editing performance (gRNA-CD3G-1, gRNA-CD3G-3, gRNA-CD3G-10, gRNA-CD3G-12, and gRNA-CD3G-18) were selected for further long-term editing efficiency testing. The statistical results are shown in Figure 2F.

[0171] Example 4: Validation of sgRNA targeting the overlapping regulatory regions of CD3D and CD3G genes

[0172] Based on the AI ​​large-scale model analysis data of the epigenetic regulatory regions of CD3D and CD3G genes in this invention, we found that there is an overlap in the regulatory regions of CD3D and CD3G genes (Chr11: 118342488-118344696, SEQ ID NO: 1623). We anticipate that epigenetic modification of this region may better inhibit the protein expression of the TCR complex, and selected 77 sgRNAs to be transfected into PBMCs for epigenetic editing, thereby verifying the inhibitory effect of the selected candidate sgRNAs on TCR protein expression. The 77 sgRNA sequences used for verification in this embodiment are shown in Table 7:

[0173] Table 7. Candidate gRNA Sequences

[0174] Specific experimental protocol: Six hours after PBMC resuscitation, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs via electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On the seventh day after transfection, samples were collected, and flow cytometry (Biolegend, 317318) staining was used to analyze the inhibitory effect of these 77 sgRNAs on the protein expression level of the TCR complex. The results are shown in Figure 3. Epigenetic modification of overlapping regions showed that these sgRNAs could efficiently regulate the protein level of the TCR complex, with regulatory capabilities reaching or even exceeding the levels achieved by epigenetic modification of CD3D and CD3G alone.

[0175] Example 5: Construction and screening of sgRNA libraries targeting the CD3E gene

[0176] (1) Prediction of CD3E adjustable region based on apparent AI large model

[0177] This invention first performed a multi-omics analysis on human primary T cells. Whole-genome methylation sequencing (WGBS) and RNA-seq were performed. Furthermore, the CUT & Tag method was used to sequence and analyze various histone modifications, including K3K4me1, H3K3me3, H3K36me3, H3K9me3, H3K27me3, and H3K27ac. After analysis using a large AI model (analysis method referenced: Yang, Q. et al. EpiCas-DL: Predicting sgRNA activity for CRISPR-mediated epigenome editing by deeplearning. Comput Struct Biotechnol J 21, 202-211(2023). https: / / doi.org / 10.1016 / j.csbj.2022.11.034), it was found that the 2000bp upstream and downstream of the CD3E transcription start site (Chr11: 118302762-118306717, SEQ ID NO: 1624) are regulatory regions (Figure 4A).

[0178] (2) CD3E gRNA design and library construction

[0179] Based on the prediction results of the epigenetic AI large model, this invention performs gRNA library screening for CD3E (Gene ID: 916), and the genomic range covered by the gRNA is Chr11: 118302762-118306717.

[0180] The CD3E gRNA sequence designed in this embodiment is shown in Table 8:

[0181] Table 8. CD3E gRNA Sequence

[0182] Figure 4B shows the experimental workflow for gRNA plasmid library construction, viral transfection, cell sorting, and sequencing analysis. Specific steps are described in Example 2(3).

[0183] Figure 4C shows the changes in TCR complex protein levels in primary human T cells transfected with a gRNA library. After epigenetic regulation using EPIREG-04, the TCR complex protein levels were detected by flow cytometry. TCR-negative and TCR-positive cells were sorted by flow cytometry and then subjected to gRNA sequencing analysis to screen for gRNAs with regulatory functions.

[0184] (3) Analysis of sgRNA library screening results

[0185] This invention performs NGS sequencing on sgRNA libraries and then analyzes the regions enriched by sgRNAs to define the regulatory regions of CD3E. We calculated read counts for the TCR-negative sgRNA enrichment results and normalized the sgRNA read counts using Mageck's default median normalization, ultimately obtaining the normalized count for each sgRNA. After matching the relative positions with the TSS, we found that a large number of sgRNAs capable of effectively inhibiting the epigenetic function of CD3E were successfully enriched within a 2000 bp range upstream and downstream of the TSS (Figure 4D).

[0186] (4) Validation experiment on the effectiveness of library screening for candidate sgRNAs

[0187] Based on the results of the above library screening and considering factors such as conservation, 20 CD3E sgRNAs, as shown in the table below, were selected and transfected into PBMCs for epigenetic editing to verify the inhibitory effect of the selected candidate sgRNAs on CD3E gene expression. The 20 sgRNA sequences used for verification in this example are as follows:

[0188] Table 9. Candidate CD3E gRNA Sequences

[0189] Specific experimental protocol: Six hours after PBMC resuscitation, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs via electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On the 3rd and 7th days post-transfection, samples were collected for flow cytometry staining (Biolegend, 317318) to analyze the inhibitory effect of these 20 sgRNAs on the protein expression of the TCR complex. The results are shown in Figure 4E, indicating an inhibition rate ranging from 15 / 20 to over 80%. From these 20 sgRNAs, the 5 with the best editing performance (gRNA-CD3E-7, gRNA-CD3E-8, ​​gRNA-CD3E-12, gRNA-CD3E-17, and gRNA-CD3E-18) were selected for further long-term editing efficiency testing. The statistical results are shown in Figure 4F.

[0190] Example 6: Construction and screening of sgRNA libraries targeting the CD247 (CD3Z) gene

[0191] (1) Prediction of CD247 adjustable region based on apparent AI large model

[0192] This invention first performed a multi-omics analysis on human primary T cells. Whole-genome methylation sequencing (WGBS) and RNA-seq were performed. Furthermore, the CUT & Tag method was used to sequence and analyze various histone modifications, including K3K4me1, H3K3me3, H3K36me3, H3K9me3, H3K27me3, and H3K27ac. After analysis using a large AI model (analysis method referenced: Yang, Q. et al. EpiCas-DL: Predicting sgRNA activity for CRISPR-mediated epigenome editing by deep learning. Comput Struct Biotechnol J 21, 202-211(2023). https: / / doi.org / 10.1016 / j.csbj.2022.11.034), a 901bp region near the CD247 transcription start site (Chr1: 167518091-167518992, SEQ ID NO: 1625) was identified as the regulatory region (Figure 5A).

[0193] (2) CD247 gRNA design and library construction

[0194] (3) Validation experiment of candidate sgRNA effect

[0195] Based on the prediction results of the comprehensive epigenetic AI model and considering factors such as conservation, 20 CD274 sgRNAs, as shown in the table below, were selected and transfected into PBMCs for epigenetic editing. This was to verify the inhibitory effect of the selected candidate sgRNAs on CD274 gene expression. The 20 sgRNA sequences used for verification in this example are as follows:

[0196] Table 10. Candidate CD247 gRNA sequences

[0197] Specific experimental protocol: Six hours after PBMC resuscitation, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 (and each of the above-mentioned sgRNAs) was electroporated (specific electroporation methods are as per the kit instructions: Lonza P3) The 20 sgRNAs (kit, V4XP-3024) were transfected into PBMCs. On days 3 and 7 post-transfection, samples were collected and analyzed using flow cytometry (Biolegend, 317318) to assess the inhibitory effect of these 20 sgRNAs on the vertical expression of the TCR complex protein. The results are shown in Figure 5B, indicating an inhibition rate of over 80% for 14 / 20 sgRNAs. Five sgRNAs with the best editing performance (gRNA-CD247-3, gRNA-CD247-10, gRNA-CD247-11, gRNA-CD247-13, and gRNA-CD247-17) were selected for further long-term editing efficiency testing. The statistical results are shown in Figure 5C.

[0198] Example 7: Combination Strategy of CD3 Single Subunit and Multi-Subunit

[0199] Based on the screening results of all the above embodiments, CD3D 11 (D11), CD3G 10 (G10), CD3E 17 (E17), and CD3Z 17 (Z17, or CD247) sgRNAs are located in conserved regions of the genome and exhibit the best inhibitory effect. This embodiment aims to verify and compare the inhibitory effect of CD3 single subunit and multi-subunit combinations on CD3 gene expression. Therefore, the experimental groups designed according to sgRNAs are as follows: Non-target control (NTC), D11, G10, E17, Z17, D11+G10, D11+G10+E17, D11+G10+E17+Z17. The sgRNAs and the mRNA encoding EPIREG-04 grouped above were transfected into PBMCs by electroporation (specific electroporation method according to the following kit instructions: Lonza P3 kit, V4XP-3024). Samples were collected on days 7, 14, 21, and 35 after electroporation, and CD3 was analyzed by flow cytometry antibody (Biolegend, 317318) staining to compare the inhibitory effects of single-subunit and multi-subunit combinations on CD3 gene expression. The results are shown in Figure 6. The dual-subunit combination can basically completely inhibit CD3 gene expression. Therefore, the dual-subunit gRNA combination CD3D-11+CD3G-10 will be used for subsequent experiments.

[0200] Example 8: TCR Editing Based on Different Versions of dCas's EPIREG

[0201] Six hours after PBMC resuscitation, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, mRNAs encoding different EPIRGs—EPIREG-01, EPIRG-02, EPIRG-03, EPIRG-04, EPIRG-05, EPIRG-06, EPIRG-07, and gRNA-CD3D-11+gRNA-CD3G-10—were transfected into PBMCs via electroporation (specific electroporation method follows the kit instructions: Lonza P3 kit, V4XP-3024). On days 1 and 5 post-transfection, samples were collected for flow cytometry staining (Biolegend, 317318) to analyze the inhibitory effect of different EPIRG versions on the protein expression of the TCR complex. The results are shown in Figure 7, indicating that the inhibition rate was above 90%.

[0202] Example 9: Epigenetic regulation of TCR in CAR-T cells

[0203] (1) Structure of CD19 CAR

[0204] The CAR structure used in this paper is not limited to any particular type of CAR. The CD19 CAR-T is used as an example for verification, as shown in Figure 9.

[0205] (2) CAR-T cell preparation and epigenetic regulation of TCR complex

[0206] The preparation protocol of CAR T cells and the regulation of TCR epigenes are shown in Figure 9A. Six hours after PBMC resuscitation, cells were activated with anti-CD3 / 28 magnetic beads. On the second day, CD19 CAR lentivirus was added. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA and sgRNA encoding EPIREG-04 were electroporated into the cells (specific electroporation method according to the following kit instructions: Lonza P3 kit, V4XP-3024). The sgRNA in the control group was NTC sgRNA, and the sgRNA in the experimental group was gRNA-CD3D-11, gRNA-CD3G-10, and gRNA-CD3D-11+gRNA-CD3G-10. On the sixth day after electroporation, a portion of the cells was stained with CD3 using flow cytometry (Biolegend, 317318). The positive cell population from the control group and the negative cell population from the experimental group were then sorted using a BD Airal flow cytometer. The sorted cells were further cultured in a 37°C CO2 incubator. After 4 days of culture, human Burkitt lymphoma cells (Raji) were added to the sorted cells at a 1:1 cell ratio. Raji cells were then added every 7 days at a 1:1 cell ratio. Cell proliferation and gene editing were continuously monitored throughout this process, and the statistical results are shown in Figure 9B. Before Raji activation, the cells proliferated at an average rate of twice the size per day. After Raji activation, the proliferation rate increased significantly until day 46, with the editing efficiency consistently remaining above 90%. The editing efficiency of unsorted and unactivated Raji cells is shown in Figure 9C, and the long-term editing efficiency also remained above 90%.

[0207] Example 10: UCAR-T in vitro functional analysis

[0208] Based on the above analysis, the dual-subunit combination (gRNA-CD3D-11+gRNA-CD3G-10) showed better CD3 inhibition than the single-subunit combination, and it could basically achieve complete inhibition. Therefore, the subsequent UCAR-T functional analysis selected the control group NTC and the experimental group gRNA-CD3D-1I+gRNA-CD3G-10 for related experiments.

[0209] On day 12 post-CAR T cell editing, CAR expression and subset proportions were detected by flow cytometry. The antibody used to detect CAR expression was a high-affinity anti-HA antibody (Roche, 11867423001). Since this antibody was not fluorescently labeled, a donkey anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (thermo, A78947) was added for additional fluorescent labeling. T cells were divided into two major subsets, CD4+ and CD8+, based on their cell surface differentiation antigens (CD). These were detected using PE anti-human CD4 (Biolegend, 317410) and PE / Cyanine7 anti-human CD8 (Biolegend, 344712) antibodies, respectively. T cells can be divided into four subsets according to their activation stage: naive T cells, central memory cells (TCM), effector memory cells (TEM), and effector cells (TE). The antibodies used to detect the proportion of these four subsets were APC anti-human CD62L (Biolegend, 304810) and PE anti-human CD45RA (Biolegend, 304108) (analysis method referred to www.biocompare.com / Product-Reviews / 348423-Classification-of-Naive-effector-and-memory-T-cells / ). The statistical results are shown in Figures 10A-C. There were no significant differences in CAR expression or subset proportions between the experimental group and the control group.

[0210] On day 17 after CAR T cells were edited with EPIREG-04 and gRNA-CD3D-11+gRNA-CD3G-10, most of the CAR T cells were cryopreserved in liquid nitrogen. On day 18 after editing, cells were harvested for in vitro antitumor function assays of CAR T cells, including cytokine and cytotoxicity assays. Raji cells were used as the target cells in this experiment. The cytokine assay method was as follows: CAR T cells and Raji cells were mixed and incubated at a 1:1 ratio. 1×Brefeldin A Solution (Biolegend, 420601) was added to the cells to fix the Golgi complex and endoplasmic reticulum. After 6 hours and 24 hours of incubation, portions of cells were harvested and processed according to BD Cytofix / Cytoperm... TMFixation / Permeabilization Kit (BD, 554714) was used for fixation and permeabilization, followed by staining with corresponding flow cytometry antibodies for cytokines. The cytokine antibodies used included PerCP / Cyanine 5.5 anti-human IFN-γ (Biolegend, 502526) and Alexa. 647anti-human / mouse Granzyme B (Biolegend, 515406), Brilliant Violet 421 TM anti-human IL-2 (Biolegend, 500328), Brilliant Violet 605 TM Anti-human TNF-α Antibody (Biolegend, 502936). The detection and analysis results are shown in Figure 10D. The expression of cytokines in the experimental group and the control group after the addition of target cells Raji was basically the same.

[0211] The in vitro killing function detection method is as follows: Effector cells CAR T and target cells Raji were mixed and incubated at a ratio of 1:1 / 1:5 / 1:10 (the number of Raji cells was kept consistent). Raji cells without CAR T (the same number as the experimental group) were used as a negative control. After 24 hours of incubation, a portion of the cells were taken and the target cells Raji were stained and counted by flow cytometry using PE anti-human CD19 Antibody (Biolegend, 302254). The number of Raji cells reflects the killing effect of CAR T on Raji. The statistical results are shown in Figure 10E. In vitro, CD3-edited CAR T and unedited CAR T have the same killing function. The 1:1 ratio of target cells basically achieved 100% killing, the 1:5 ratio of target cells achieved more than 60% killing, and the 1:10 ratio of target cells achieved more than 20% killing. When human Burkitt lymphoma cells (Raji) were added to the sorted cells at a cell ratio of 1:1 every ten days to activate CAR-T cells, the expansion of CAR-T cells is shown in Figure 10F.

[0212] Example 11: In vivo functional analysis of UCAR-T

[0213] In vivo functional analysis primarily examines the anti-tumor function of CAR T cells in vivo. This requires constructing a Rai hematologic malignancy model in mice, injecting CAR T cells into these mice, and then monitoring tumor growth after CAR injection to reflect its anti-tumor function. The mice used in the experiment were immunodeficient mice, B-NDG (Beijing Biocytogen Biosciences Co., Ltd., NOD.CB17-Prkdcscid Il2rgtm1Bcgen / Bcgen). This strain of mice exhibits NOD-scid-Il2rg null background characteristics, displaying a severe immunodeficiency phenotype, lacking mature T cells, B cells, and functional NK cells, and exhibiting impaired cytokine signaling. The specific experimental protocol is as follows: One million Rai cells carrying Luciferase were injected into B-NDG mice via tail vein injection to construct the hematologic malignancy mouse model. On day 7 post-injection, in vivo imaging was performed using the PerkinElmer IVIS Lumina III system. The number of photons emitted by Luciferase-expressing cells was quantified using Living Image software to confirm the successful establishment of the hematologic malignancy model. The frozen CAR T cells were revived, and 2 million CAR T cells were injected into each mouse via tail vein injection 6 hours later. In vivo imaging of the mice was performed every 7 days thereafter, and the survival status of the mice was recorded for three weeks. The statistical results are shown in Figure 11. The results indicate that both the CD3-inhibited CAR T cells in the experimental group and the unedited CAR T cells in the control group had good in vivo anti-tumor function, with no significant difference between them.

[0214] Example 12: Design and screening of sgRNAs targeting the B2M gene, which is involved in the HLA class I complex.

[0215] We designed gRNAs for B2M in the HLA class I complex, covering the region Chr15: 44711399-44711754 (SEQ ID NO: 1626), and selected 19 gRNAs for experimental validation. The B2M gRNA sequences designed in this embodiment are shown in Table 11:

[0216] Table 11. B2M gRNA

[0217] Specific experimental protocol: Six hours after resuscitation of PBMCs (Miaoshun (Shanghai) Biotechnology Co., Ltd.), anti-CD3 / 28 magnetic beads (Thermofisher, Dynabeads) were used. TMHuman T activators CD3 / CD28 (11132D) were used to activate cells. On the fourth day, magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the above-mentioned sgRNAs were transfected into PBMCs by electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On the 7th day after transfection, samples were collected, and flow cytometry was used to analyze the inhibitory effect of these 19 sgRNAs on B2M protein. The results are shown in Figure 12.

[0218] Example 13: Design and screening of sgRNAs targeting the HLA-A gene, which is involved in the HLA class I complex.

[0219] We designed gRNAs for HLA-A in the HLA class I complex, covering the region hg38 chr6: 29,940,554-29,944,554, and selected nine gRNAs for experimental validation. The designed HLA-A gRNA sequences in this embodiment are shown in Table 12:

[0220] Table 12. HLA-A gRNA

[0221] Specific experimental protocol: Six hours after reviving PBMCs, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs by electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On the 10th day after transfection, samples were collected, and flow cytometry was used to analyze the inhibitory effect of these nine sgRNAs on HLA-A protein. The results are shown in Figure 13.

[0222] Example 14: Design and screening of sgRNAs targeting HLA-B genes involved in the HLA class I complex

[0223] We designed gRNAs for HLA-B in the HLA class I complex, covering the region hg38 chr6:31, 355, 158-31, 359, 158, and selected 14 gRNAs for experimental validation. The designed HLA-B gRNA sequences in this embodiment are shown in Table 13:

[0224] Table 13. HLA-B gRNA

[0225] Specific experimental protocol: Six hours after reviving PBMCs, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the above-mentioned sgRNAs were transfected into PBMCs by electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On the 10th day after transfection, samples were collected, and HLA-B was analyzed by flow cytometry antibody staining to determine the inhibitory effect of these 14 sgRNAs on HLA-B protein. The results are shown in Figure 14.

[0226] Example 15: Design and screening of sgRNAs targeting the TAP1 gene, which is involved in the HLA class I complex.

[0227] We designed gRNAs for TAP1, a peptide transporter in the HLA class I complex. The gRNA coverage region is hg38 chr6: 32,853,236-32,854,217 (SEQ ID NO: 1627), and 16 gRNAs were selected for experimental validation. The designed TAP1 gRNA sequences in this embodiment are shown in Table 14.

[0228] Table 14. TAP1 gRNA

[0229] Specific experimental protocol: Six hours after reviving PBMCs, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs via electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On the 14th day after transfection, samples were collected, and flow cytometry staining was performed to analyze the inhibitory effect of these 16 sgRNAs on HLA class I complex proteins. The results are shown in Figure 15.

[0230] Example 16: Design and screening of sgRNAs targeting the TAP2 gene, which is involved in the HLA class I complex.

[0231] We designed gRNAs for TAP2, a peptide transporter in the HLA class I complex. The gRNA coverage region is hg38 chr6: 32,838,356-32,838,754 (SEQ ID NO: 1628), and 12 gRNAs were selected for experimental validation. The designed TAP2 gRNA sequences in this embodiment are shown in Table 15:

[0232] Table 15. TAP2 gRNA

[0233] Specific experimental protocol: Six hours after reviving PBMCs, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs via electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On the 14th day after transfection, samples were collected, and flow cytometry staining was performed to analyze the inhibitory effect of these 12 sgRNAs on HLA class I complex proteins. The results are shown in Figure 16.

[0234] Example 17: Design and screening of sgRNAs targeting the CIITA gene, which is involved in regulating HLA class II complex transcription.

[0235] We designed gRNAs for CIITA, which are involved in the transcriptional regulation of the HLA class II complex. The gRNA coverage region is hg38 chr16: 10,876,812-10,877,580 (SEQ ID NO: 1629), and 20 gRNAs were selected for experimental validation. The CIITA gRNA sequences designed in this embodiment are shown in Table 16:

[0236] Table 16. CIITA gRNA

[0237] Specific experimental protocol: Six hours after PBMC resuscitation, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs via electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On the 14th day after transfection, samples were collected, and flow cytometry staining was performed to analyze the inhibitory effect of these 20 sgRNAs on HLA class II complex proteins. The results are shown in Figure 17.

[0238] Example 18: Design and screening of sgRNAs targeting the RFX5 gene, which is involved in regulating HLA class II complex transcription.

[0239] We designed gRNAs for RFX5, which are involved in the transcriptional regulation of the HLA class II complex. The gRNA coverage region is hg38 chr1: 151,346,853-151,347,430 (SEQ ID NO: 1630), and 19 gRNAs were selected for experimental validation. The RFX5 gRNA sequences designed in this embodiment are shown in Table 17:

[0240] Table 17. RFX5 gRNA

[0241] Specific experimental protocol: Six hours after reviving PBMCs, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs via electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On the 14th day after transfection, samples were collected, and flow cytometry staining was performed to analyze the inhibitory effect of these 19 sgRNAs on HLA class II complex proteins. The results are shown in Figure 18.

[0242] Example 19: Design and screening of sgRNAs targeting the RFXANK gene, which is involved in regulating HLA class II complex transcription.

[0243] We designed gRNAs for RFXANK, which are involved in the transcriptional regulation of the HLA class II complex. The gRNA coverage region is hg38 chr19:19,192,356-19,193,141 (SEQ ID NO: 1631), and 16 gRNAs were selected for experimental validation. The RFXANK gRNA sequences designed in this embodiment are shown in Table 18:

[0244] Table 18.RFXANK gRNA

[0245] Specific experimental protocol: Six hours after PBMC resuscitation, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs via electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On the 14th day after transfection, samples were collected, and flow cytometry staining was performed to analyze the inhibitory effect of these 16 sgRNAs on HLA class II complex proteins. The results are shown in Figure 19.

[0246] Example 20: Design and screening of sgRNAs targeting the RFXAP gene, which is involved in the formation of the HLA class II complex.

[0247] We designed gRNAs for RFXAP in the HLA class II complex, covering the region hg38 chr13: 36,819,211-36,819,616 (SEQ ID NO: 1632), and selected 17 gRNAs for experimental validation. The designed RFXAP gRNA sequences in this embodiment are shown in Table 19:

[0248] Table 19.RFXAP gRNA

[0249] Specific experimental protocol: Six hours after PBMC resuscitation, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs via electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On day 35 post-transfection, samples were collected, and flow cytometry staining was performed to analyze the inhibitory effect of these 17 sgRNAs on the HLA class II complex. The results are shown in Figure 20.

[0250] Example 21: Design and screening of sgRNAs targeting the CD52 gene

[0251] We designed CD52 gRNAs covering the chr1 region: 26,317,726-26,318,321 (SEQ ID NO: 1633), and selected 19 gRNAs for experimental validation. The designed CD52 gRNA sequences in this embodiment are shown in Table 20:

[0252] Table 20. CD52 gRNA

[0253] Specific experimental protocol: Six hours after reviving PBMCs, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA encoding EPIREG-04 and each of the aforementioned sgRNAs were transfected into PBMCs via electroporation (specific electroporation methods are as per the kit instructions: Lonza P3 kit, V4XP-3024). On the fifth day after transfection, samples were collected, and flow cytometry was used to analyze the inhibitory effect of these 19 sgRNAs on CD52. The results are shown in Figure 21.

[0254] Example 22: Epigenetic editing of different targets on T cells using different epigenetic editing tools

[0255] Six hours after reviving PBMCs, cells were activated with anti-CD3 / 28 magnetic beads. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack, and the cells were passaged. On the fifth day, the mRNA and gRNA encoding the epigenetic editing tools EPIREG-02 and EPIREG-04 were transfected into PBMCs by electroporation (the specific electroporation method is as follows: Lonza P3 kit, V4XP-3024).

[0256] For the HLA-A targeting example, the gRNA used was gRNA-HLAA-2 (SEQ ID NO: 1480), and for the HLA-B targeting example, the gRNA used was gRNA-HLAB-12 (SEQ ID NO: 1499). Both HLA-A and HLA-B belong to MHC class I molecules and are used by the immune system to recognize autologous and allogeneic cells. Inhibiting the expression of HLA-A and HLA-B can be used to prepare universal CAR-T therapy, thereby reducing treatment costs and accelerating the treatment cycle.

[0257] Samples were collected on day 5 after electroporation, and flow cytometry staining was performed on the samples (antibody used for HLA-A: BD Biosciences, catalog number 568024; antibody used for HLA-B: BD Biosciences, catalog number 569644) to analyze the inhibitory effects of different versions of epigenetic editing tools on the protein expression levels of HLA-A and HLA-B. By day 5 after electroporation, the mRNA and protein of the epigenetic editing tools had largely degraded. At this point, the proportion of target protein-positive cells was used to characterize the inhibitory effect of different tools; a lower proportion of positive cells indicated a stronger inhibitory effect. The results are shown in Figures 22A-B. Compared with the control, both epigenetic editing tools showed significant inhibitory effects on HLA-A and HLA-B.

[0258] For the example targeting CIITA, the gRNA used is gRNA-CIITA-4 (SEQ ID NO: 1533). CIITA is an essential transcription factor for the expression of MHC class II molecules. Inhibiting CIITA can inhibit the appearance of MHC class II molecules (including HLA-DR) on the surface of CAR-T cells, thereby inhibiting the immune system from recognizing autologous and allogeneic cells. Therefore, inhibiting the expression of MHC class II molecules can also be used for the preparation of universal CAR-T.

[0259] Samples were collected and analyzed on days 5 and 14 after electroporation. Flow cytometry staining was performed on the samples (HLA-DR antibody used: Biolegend, catalog number 327020; the inhibition of HLA-DR indirectly indicates the inhibitory effect of CIITA). The inhibitory effect of different versions of epigenetic editing tools on HLA-DR protein expression levels was analyzed. At the detection time points, the mRNA and protein of the epigenetic editing tools had been largely degraded. The proportion of HLA-DR-positive cells was then used to characterize the inhibitory effect of different tools; a lower proportion of positive cells indicated a stronger inhibitory effect of CIITA. The results are shown in Figure 22C. Compared with the control, both epigenetic editing tools produced significant inhibitory effects on CIITA, and the effects were similar on days 14 and 5, indicating that the inhibitory effects produced by these epigenetic editing tools are sustainable.

[0260] For the CD3-targeting embodiment, the gRNA used was a mixed gRNA of gRNA-CD3D-11 (SEQ ID NO: 49) and gRNA-CD3G-10 (SEQ ID NO: 594) mixed in equal proportions. Inhibiting CD3 expression prevents the endogenous TCR of T cells from transmitting signals, effectively suppressing TCR function. This allows CAR-T cells to transmit cellular signals solely through CAR, achieving universal CAR-T while delaying CAR-T cell exhaustion and improving therapeutic efficacy.

[0261] Samples were collected and analyzed on days 5 and 7 after electroporation. Flow cytometry staining (CD3 antibody used: Biolegend, catalog number 317318) was performed on the samples to analyze the inhibitory effect of different versions of epigenetic editing tools on CD3 protein expression. At the detection time points, the mRNA and protein of the epigenetic editing tools had largely degraded. The proportion of target protein-positive cells was used to characterize the inhibitory effect of different tools; a lower proportion of positive cells indicated a stronger inhibitory effect. The results are shown in Figure 22D. Compared with the control, both epigenetic editing tools showed significant inhibitory effects on CD3, and the effects were similar on days 5 and 7, indicating that the inhibitory effects of these epigenetic editing tools are sustainable.

[0262] For both appearance editing tools, we conducted longer-term inhibition tests on the CD3 target. Samples were collected and analyzed on days 14, 21, and 28 after electroporation. The results are shown in Figure 22E. Compared with the control, both appearance editing tools produced significant inhibition, and the effects were very stable on days 14, 21, and 28 after electroporation, indicating that the inhibition produced by these appearance editing tools can be maintained long-term.

[0263] Example 23: Simultaneous multi-target editing of TCR, HLA-A, HLA-B and CIITA in CAR-T cells

[0264] Six hours after reviving PBMCs, cells were activated with anti-CD3 / 28 magnetic beads. On the second day, CD19 CAR lentivirus was added. On the fourth day, the magnetic beads were removed from the cells using a magnetic rack and the cells were passaged. On the fifth day, the mRNA and corresponding gRNA encoding the epigenetic editing tool EPIREG-02 were transfected into PBMCs by electroporation (the specific electroporation method is as follows: Lonza P3 kit, V4XP-3024).

[0265] For the multi-target simultaneous targeting example, the first group simultaneously targeted HLA-A, HLA-B, CD3, and CIITA (detecting HLA-DR). Multi-target joint editing synergistically enhances the novelty of universal CAR-T. The gRNA combination used was gRNA-HLAA-2 (SEQ ID NO: 1480) + gRNA-HLAB-12 (SEQ ID NO: 1499) + gRNA-CD3D-11 (SEQ ID NO: 49) + gRNA-CD3G-10 (SEQ ID NO: 594) + gRNA-CIITA-4 (SEQ ID NO: 1533). All gRNAs were mixed in equal mass ratios and named gMix1. Samples were collected and analyzed on day 21 after electroporation. Flow cytometry staining was performed on the samples (using the same antibody as in Example 22) to analyze the inhibitory effect of the epigenetic editing tool EPIREG-02 on the expression of proteins corresponding to these targets. The control group consisted of mRNA electroporated with only the epigenetic editing tool EPIREG-02 added. At the detection time point, the mRNA and protein of the epigenetic editing tool had been largely degraded. The proportion of positive cells at different target sites in the control group was standardized to 100%, and the inhibitory effect of the epigenetic editing tool EPIREG-02 was tested. The results are shown in Figure 23A. On day 21 after electroporation, compared with the control, the epigenetic editing tool EPIREG-02 produced significant inhibitory effects at all tested target sites, indicating that multi-target epigenetic editing is entirely feasible and efficient, and the editing effect can be maintained long-term.

[0266] For the example of simultaneous multi-target targeting, the second group simultaneously targets TAP1, RFX5, and CD3. TAP1 and RFX5 are another type of inhibitory targets for MHC class I and MHC class II molecules, respectively. TAP1 is a key transport protein in the antigen presentation pathway of MHC class I molecules, and RFX5 is a core component of the MHC class II gene transcription complex. Joint inhibition of TAP1, RFX5, and CD3 can also achieve the preparation of universal CAR-T. The gRNA combination used is gRNA-TAP1-7 (SEQ ID NO: 1508) + gRNA-RFX5-5 (SEQ ID NO: 1554) + gRNA-CD3D-11 (SEQ ID NO: 49) + gRNA-CD3G-10 (SEQ ID NO: 594). All gRNAs are mixed in equal mass ratios and named gMix2.

[0267] Samples were collected and analyzed on day 21 after electroporation. Flow cytometry was used to stain samples (antibodies used to detect TAP1 inhibition: Biolegend, catalog number 311426; RFX5 inhibition: Biolegend, catalog number 327020; CD3 inhibition: Biolegend, catalog number 317318) to analyze the inhibitory effect of the epigenetic editing tool EPIREG-02 on the expression of proteins corresponding to these targets. The control group consisted of electroporated mRNA treated only with EPIREG-02. At the detection time point, the mRNA and protein of the epigenetic editing tool were largely degraded. The proportion of positive cells for different targets in the control group was normalized to 100% to test the inhibitory effect of EPIREG-02. As shown in Figure 23B, on day 21 after electroporation, compared with the control, the epigenetic editing tool EPIREG-02 produced a significant inhibitory effect on all tested targets, indicating that multi-target epigenetic editing is completely feasible and efficient, and the editing effect can be maintained for a long time.

[0268] By incorporating via reference

[0269] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0270] Equivalence

[0271] This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. A method for producing universal CAR-T cells, the method comprising: The expression of target genes in CAR-T cells was downregulated by using gene expression regulatory molecules; The gene expression regulatory molecule comprises i) an epigenetic modification domain, ii) a transcriptional regulatory domain, and iii) a DNA-binding domain, wherein the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to one end of the DNA-binding domain via a linker sequence, and the epigenetic modification domain is optionally linked to one end of the transcriptional regulatory domain via a linker sequence; or the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to both ends of the DNA-binding domain via a linker sequence. The target genes are selected from CD3D, CD3G, CD3E, CD3Z, HLA I, HLA II, CD52, TCRα, or TCRβ.

2. The method of claim 1, comprising: By using gene expression regulatory molecules, the expression of the TCR-CD3 complex in the T cell receptor (TCR) of the CAR-T cells is downregulated, preferably by downregulating the expression of one or more of CD3D, CD3G, CD3E, CD3Z, TCRα, and TCRβ. The gene expression regulatory molecule comprises i) an epigenetic modification domain, ii) a transcriptional regulatory domain, and iii) a DNA-binding domain, wherein the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to one end of the DNA-binding domain by a linker sequence, and the epigenetic modification domain is optionally linked to one end of the transcriptional regulatory domain by a linker sequence; or the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to both ends of the DNA-binding domain by a linker sequence.

3. The method of claim 2, wherein the expression of CD3D / CD3E or CD3G / CD3E dimers is downregulated by using gene expression regulating molecules.

4. The method of claim 2, wherein the epigenetic modification domain comprises DNA methyltransferases DNMT 3A and DNMT 3L; The transcriptional regulatory domain is selected from at least one of ZIM3, KRAB, HP1a, and SETDB1; The DNA-binding domain is selected from: TALE domain, zinc finger domain, tetR domain, a wide range of nucleases, Cas proteins such as inactivated Cas9 protein (dCas9) or inactivated Cas12 protein (dCas12), Argonaute (Ago) protein, and their homologues, modified forms or variants.

5. The method of any one of claims 2 to 4, wherein the gene expression regulatory molecule further comprises (iv) a first recruitment domain and a second recruitment domain, wherein the first recruitment domain and the second recruitment domain are respectively at one end or both ends of the DNA binding domain; preferably, the first recruitment domain is selected from universal control non-derepressor protein 4 (GCN4), a GFP11 fragment derived from splitting green fluorescent protein (GFP), or a GVKESLV polypeptide; the second recruitment domain is selected from a single-chain antibody (scFv), a GFP1-10 fragment derived from splitting green fluorescent protein (GFP), or a PDZ protein domain; preferably, the first recruitment domain is selected from GCN4, and the second recruitment domain is selected from scFv.

6. The method of any one of claims 2 to 5, wherein the gene expression regulatory molecule comprises, from the N-terminus to the C-terminus, the following sequence: 1) Epigenetic modification domains, DNA-binding domains, and transcriptional regulatory domains; 2) DNA-binding domain, transcriptional regulatory domain, and epigenetic modification domain; 3) The second recruitment domain, transcriptional regulatory domain, cleavage peptide, epigenetic modification domain, DNA binding domain, and first recruitment domain; the first recruitment domain and the second recruitment domain can interact with each other.

7. The method of any one of claims 2 to 6, wherein the linker sequence is XTEN80 and the cleavage peptide is selected from P2A, T2A, E2A and F2A.

8. The method of any one of claims 2 to 7, wherein the DNA-binding domain is capable of binding to guide RNA, the guide RNA being capable of specifically recognizing and hybridizing with the target sequence of the target locus, preferably, the target sequence of the target locus is selected from SEQ ID NOs: 1621-1625.

9. The method of claim 8, wherein the guide RNA (sgRNA) targeting the CD3D gene comprises a sequence selected from SEQ ID NOs: 39-584, preferably selected from SEQ ID NOs: 39-584. NOs: 39-58, 172, 173, 185-188, 191-194, 196, 198, 219, 225, 228, 233-235, 237, 238, 247, 256-258, 260, 261, 266, 268, 269, 271, 272, 329, 331, 332, 336, 340, 346-351, 356, 358-361, 373, 375, 381-385, 388, 390, 391, 393-395, 398, 409-411, 413, 418-420, 425-429, 438-440, and 443; Guide RNA (sgRNA) targeting the CD3E gene contains ingredients selected from SEQ ID. The sequence NOs: 1075-1439, preferably selected from SEQ ID NOs: 1075-1094; the guide RNA (sgRNA) targeting the CD3G gene contains the sequence selected from SEQ ID NOs: 1075-1094. NOs: sequences of 172, 173, 185-188, 191-194, 196, 198, 219, 225, 228, 233-235, 237, 238, 247, 256-258, 260, 261, 266, 268, 269, 271, 272, 329, 331, 332, 336, 340, 346-351, 356, 358-361, 373, 375, 381-385, 388, 390, 391, 393-395, 398, 409-411, 413, 418-420, 425-429, 438-440, 443, and 585-1074, preferably selected from SEQ ID. NOs: 172, 173, 185-188, 191-194, 196, 198, 219, 225, 228, 233-235, 237, 238, 2 47, 256-258, 260, 261, 266, 268, 269, 271, 272, 329, 331, 332, 336, 340, 346-351 356, 358-361, 373, 375, 381-385, 388, 390, 391, 393-395, 398, 409-411, 413, 418-420, 425-429, 438-440, 443 and 585-604; and / or the guide RNA (sgRNA) targeting the CD3Z gene contains sequences selected from SEQ ID NOs: 1440-1459.

10. The method according to any one of claims 2 to 9, wherein the sequence of DNMT 3A comprises SEQ ID NO: 15, and the sequence of DNMT 3L comprises SEQ ID NO: 16 or SEQ ID NO: 17, preferably, the sequence of the epigenetic modification domain is selected from DNMT 3A-DNMT 3L, DNMT 3L-DNMT 3A, DNMT 3A-hDNMT 3L, and hDNMT 3L-DNMT 3A.

11. The method of any one of claims 2 to 10, wherein the sequence of ZIM3 comprises SEQ ID NO: 20, the sequence of KRAB comprises SEQ ID NO: 19, the sequence of HP1a comprises SEQ ID NO: 23, and the sequence of SETDB1 comprises SEQ ID NO:

24.

12. The method of any one of claims 2 to 11, wherein the DNA binding domain comprises SEQ ID NO:

18.

13. The method of any one of claims 2 to 12, wherein the sequence of said XTEN80 comprises SEQ ID NO:

25.

14. The method of any one of claims 2 to 13, wherein the amino acid sequence of the gene expression regulatory molecule comprises any sequence selected from SEQ ID NOs: 1-7.

15. The method of any one of claims 2 to 14, wherein the nucleic acid sequence encoding the gene expression regulatory molecule comprises any sequence selected from SEQ ID NOs: 8-14.

16. A universal CAR-T cell, prepared by the method according to any one of claims 2 to 15.

17. The universal CAR-T cell of claim 16, wherein the CAR-T cell is a CAR that specifically binds to tumor antigens, preferably anti-CD19 CAR-T cells, anti-CD20 CAR-T cells, anti-CD22 CAR-T cells, anti-CD23 CAR-T cells, anti-CD28 CAR-T cells, or anti-CD137 CAR-T cells.

18. The method of claim 1, wherein the method comprises: By using gene expression regulatory molecules, the expression of HLA class I genes, HLA class II genes, and / or CD52 genes in the CAR-T cells was downregulated; The gene expression regulatory molecule comprises i) an epigenetic modification domain, ii) a transcriptional regulatory domain, and iii) a DNA-binding domain, wherein the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to one end of the DNA-binding domain by a linker sequence, and the epigenetic modification domain is optionally linked to one end of the transcriptional regulatory domain by a linker sequence; or the epigenetic modification domain and the transcriptional regulatory domain are optionally linked to both ends of the DNA-binding domain by a linker sequence.

19. The method of claim 18, wherein the gene expression regulatory molecule further comprises iv) a first recruitment domain and a second recruitment domain.

20. The method of claim 19, wherein the gene expression regulatory molecule comprises, from N-terminus to C-terminus, a second recruitment domain, a transcriptional regulatory domain, a splicing peptide, an epigenetic modification domain, a DNA-binding domain, and a first recruitment domain; the first recruitment domain and the second recruitment domain are capable of interacting.

21. The method of any one of claims 18 to 20, wherein the epigenetic modification domain comprises DNA methyltransferases DNMT 3A and DNMT 3L; The transcriptional regulatory domain is selected from at least one of ZIM3, KRAB, HP1a, and SETDB1; The DNA-binding domain is selected from: TALE domain, zinc finger domain, tetR domain, a wide range of nucleases, Cas proteins such as inactivated Cas9 protein (dCas9) or inactivated Cas12 protein (dCas12), Argonaute (Ago) protein, and their homologues, modified forms or variants.

22. The method of any one of claims 18 to 21, wherein the first recruitment domain is selected from universal control non-derepressor protein 4 (GCN4), a GFP11 fragment derived from split green fluorescent protein (GFP), or a GVKESLV polypeptide; the second recruitment domain is selected from a single-chain antibody (scFv), a GFP1-10 fragment derived from split green fluorescent protein (GFP), or a PDZ protein domain; more preferably, the first recruitment domain is selected from GCN4, and the second recruitment domain is selected from scFv.

23. The method of claim 18, wherein the HLA class I gene is selected from one or more of HLA-A, HLA-B, HLA-C, β2 microglobulin (B2M), TAP1, and TAP2.

24. The method of claim 18, wherein the HLA class II gene is selected from one or more of RFXAP, RFX5, CIITA, and RFXANK.

25. The method of any one of claims 18 to 24, wherein the linker sequence is XTEN80 and the cleavage peptide is selected from P2A, T2A, E2A, and F2A.

26. The method of any one of claims 18 to 25, wherein the DNA-binding domain is capable of binding to guide RNA, the guide RNA being capable of specifically recognizing and hybridizing with the target sequence of the target locus, preferably, the target sequence of the target locus is selected from SEQ ID NOs: 1626-1633.

27. The method of any one of claims 18 to 26, wherein the guide RNA (sgRNA) targeting the B2M gene comprises a sequence selected from SEQ ID NOs: 1460-1478; the guide RNA (sgRNA) targeting the HLA-A gene comprises a sequence selected from SEQ ID NOs: 1479-1487; the guide RNA (sgRNA) targeting the HLA-B gene comprises a sequence selected from SEQ ID NOs: 1488-1501; the guide RNA (sgRNA) targeting the TAP1 gene comprises a sequence selected from SEQ ID NOs: 1502-1517; the guide RNA (sgRNA) targeting the TAP2 gene comprises a sequence selected from SEQ ID NOs: 1518-1529; the guide RNA (sgRNA) targeting the CIITA gene comprises a sequence selected from SEQ ID NOs: 1530-1549; and the guide RNA (sgRNA) targeting the RFX5 gene comprises a sequence selected from SEQ ID NOs: 1460-1478. The sequence NOs: 1550-1568; the guide RNA (sgRNA) targeting the RFXANK gene contains a sequence selected from SEQ ID NOs: 1569-1584; the guide RNA (sgRNA) targeting the RFXAP gene contains a sequence selected from SEQ ID NOs: 1585-1601; and / or the guide RNA (sgRNA) targeting the CD52 gene contains a sequence selected from SEQ ID NOs: 1602-1620.

28. The method of any one of claims 18 to 27, wherein the sequence of DNMT 3A comprises SEQ ID NO: 15, and the sequence of DNMT 3L comprises SEQ ID NO: 16 or SEQ ID NO: 17, preferably, the sequence of the epigenetic modification domain is selected from DNMT 3A-DNMT 3L, DNMT 3L-DNMT 3A, DNMT 3A-hDNMT 3L, and hDNMT 3L-DNMT 3A.

29. The method of any one of claims 18 to 28, wherein the sequence of ZIM3 comprises SEQ ID NO: 20, the sequence of KRAB comprises SEQ ID NO: 19, the sequence of HP1a comprises SEQ ID NO: 23, and the sequence of SETDB1 comprises SEQ ID NO:

24.

30. The method of any one of claims 18 to 29, wherein the sequence of the DNA binding domain comprises SEQ ID NO:

18.

31. The method of any one of claims 18 to 30, wherein the sequence of XTEN80 comprises SEQ ID NO:

25.

32. The method of any one of claims 18 to 31, wherein the sequence of the single-chain antibody (scFv) comprises SEQ ID NO: 21, and the sequence of the GCN4 comprises SEQ ID NO:

22.

33. The method of any one of claims 18 to 32, wherein the amino acid sequence of the gene expression regulatory molecule comprises a sequence selected from the sequence shown in SEQ ID NO:

4.

34. The method of any one of claims 18 to 33, wherein the nucleic acid sequence encoding the gene expression regulatory molecule comprises a sequence selected from the sequence shown in SEQ ID NO:

11.

35. A universal CAR-T cell, prepared by the method according to any one of claims 18 to 34.

36. The universal CAR-T cell of claim 35, wherein the CAR-T cell is a CAR that specifically binds to tumor antigens, preferably anti-CD19 CAR-T cells, anti-CD20 CAR-T cells, anti-CD22 CAR-T cells, anti-CD23 CAR-T cells, anti-CD28 CAR-T cells, or anti-CD137 CAR-T cells.

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