Design of synthetic nucleic acid construct of tumor-associated antigen and preparation of engineering T cell

Abstract

The invention relates to the field of immunotherapy, in particular to a nucleic acid construct containing a nucleotide sequence of a synthetic T cell antigen receptor (STAR) encoding a specific binding tumor-associated antigen, and an engineering T cell prepared from the nucleic acid construct. The invention also relates to application of the nucleic acid construct and the engineering T cell in tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of immunotherapy, and in particular, to a nucleic acid construct comprising a nucleotide sequence encoding a synthetic T cell antigen receptor (STAR) that specifically binds to a tumor-associated antigen, and an engineered T cell prepared by the nucleic acid construct. The present application also relates to the use of the nucleic acid construct and the engineered T cell in drug development, treatment of tumors and autoimmune diseases, etc. Background Art

[0002] Cellular immunotherapy, such as chimeric antigen receptor T-cell (CAR-T) immunotherapy, has completely changed the treatment landscape of cancer in the past decade. Many patients with hematological malignancies that were no longer treatable have had their tumors significantly controlled after receiving CAR-T cell therapy, and some patients have even fully recovered. CAR-T cell therapy has shown great application prospects in the treatment of various cancers.

[0003] In cell immunotherapy, the successful preparation of CAR-T cells is the most critical step in the application of immunotherapy. At present, the preclinical and clinical preparation of autologous or allogeneic CAR-T cells largely relies on the use of viral vectors (such as retroviruses and lentiviruses) to transduce CAR transgenes that recognize tumor-specific antigens into T cells. However, the CAR transgenes delivered to T cells by viral transduction will randomly integrate into the host DNA, which may cause phenomena such as rapid proliferation or transcriptional silencing of T cells, bringing unpredictable risks and variable transgene expression. In addition, if CAR is integrated into the carcinogenic site, the random integration may lead to oncogenic transformation of T cells, and the preparation of viral vectors is time-consuming, expensive, and faces biosafety challenges. One method to overcome the random integration of viral transduction is site-specific gene integration, which uses CRISPR / Cas9 gene editing combined with AAV vector delivery system to insert the CAR transgene that recognizes tumor-specific antigens into a specific gene site of T cells. For example, the most widely used method is to insert the CAR transgene into the T cell TRAC (T-cell receptor alphaconstant) gene site. Compared with CAR T cells produced by lentiviral or retroviral transduction, this method inserts CAR into the T cell TRAC specific site, improves the consistent expression of CAR in T cells, reduces graft-versus-host response (GvHD) and Tonic signaling, and improves in vivo anti-tumor activity. However, the delivery capacity of AAV vectors is limited, and only 5Kb of target sequence can be inserted at most. In addition, the preparation process of AAV is complex, the production cycle is long, and the preparation cost is higher than that of lentivirus. In addition, high-titer virus needs to be produced to achieve experimental expectations. There are also biosafety risks and toxic side effects in in vitro preparation and in vivo treatment. Therefore, this method is time-consuming, expensive, labor-intensive, and has certain safety issues. To overcome these obstacles, instead of using viral vectors, non-viral double-stranded DNA (dsDNA) is used as a homology-directed repair (HDR) template. This template is produced by conventional PCR amplification, which is cheaper and faster than using viral vector-based delivery. This approach has the potential to reduce the cost and time of preparing autologous CAR T cells for cancer patients. However, the existing dsDNA used as an HDR template to prepare CAR-T cells has always faced challenges in terms of insertion integration efficiency and the proportion of live cells.Single-stranded DNA (ssDNA) is used as an HDR template to insert the CAR transgene into the T cell-specific gene site. Compared with double-stranded DNA, single-stranded DNA has lower toxicity, and combined with small molecule inhibitors, the knock-in efficiency and the proportion of live cells are about 1-2 times that of dsDNA HDR on average. However, single-stranded DNA is not fully suitable for the insertion of larger target genes and large-scale industrial production due to its poor stability, limited length of target genes that can be carried, complex preparation process, low yield, and high preparation and synthesis costs. Importantly, CRISPR / Cas9 gene editing has high off-target efficiency and potential safety risks.

[0004] Therefore, it is necessary to provide a nucleic acid construct and method to solve the problems of safe, efficient, rapid and economical preparation of CAR T cells. Summary of the invention

[0005] After a large number of experiments and repeated explorations, the inventors of this application have optimized the structure of non-viral double-stranded DNA as an HDR template, and combined with the CRISPR / Cas12a Ultra gene editing system to achieve efficient site-specific editing of a variety of different target genes in human T cells, which can achieve site-specific insertion and integration of large fragments of target genes, and can be applied to a variety of different types of human and mouse cells to achieve site-specific gene editing of human and mouse cells. The present invention provides a method and application of efficient site-specific editing of human cells based on structurally optimized double-stranded DNA and CRISPR / Cas12a Ultra system, which can efficiently, cheaply and quickly prepare safer STAR-T cells and site-specific editing cells, and achieve efficient site-specific insertion and integration at multiple gene-specific sites in human cells, which can achieve site-specific editing of genes above 7kb.

[0006] Nucleic acid construct

[0007] Therefore, in a first aspect, the present application provides a nucleic acid construct comprising a nucleotide sequence encoding a synthetic T cell antigen receptor (STAR) that specifically binds to a tumor-associated antigen, and a first guide sequence and a second guide sequence on both sides of the nucleotide sequence of the STAR; wherein the first guide sequence and the second guide sequence comprise a PAM sequence that can be recognized by the Cas12a protein and a crRNA sequence that can bind to the Cas12a protein.

[0008] In certain embodiments, the PAM sequences in the first and second leader sequences are located near one end of the STAR.

[0009] In certain embodiments, the first guide sequence is located at the 3' end of the STAR, and the first guide sequence comprises the PAM sequence and the crRNA sequence in sequence from the 5' end to the 3' end.

[0010] In certain embodiments, the second guide sequence is located at the 5' end of the STAR, and the second guide sequence comprises the crRNA sequence and the PAM sequence in sequence from the 5' end to the 3' end.

[0011] In certain embodiments, the nucleic acid construct further comprises a first homology arm sequence between the STAR and the first guide sequence, and the nucleic acid construct further comprises a second homology arm sequence between the STAR and the second guide sequence.

[0012] In certain embodiments, the first homology arm sequence and the second homology arm sequence are identical to the sequences on both sides of the target sequence.

[0013] In certain embodiments, the first homology arm sequence is identical to a sequence downstream of the target sequence, and the second homology arm sequence is identical to a sequence upstream of the target sequence.

[0014] In certain embodiments, the target sequence comprises a sequence that is complementary to the crRNA sequence.

[0015] In certain embodiments, the target sequence is adjacent to a PAM sequence recognized by the Cas12a protein.

[0016] In certain embodiments, the target sequence is located 3' to the PAM sequence.

[0017] In certain embodiments, the target sequence is located in the genome of an immune cell.

[0018] In certain embodiments, the target sequence is located within the sequence of an endogenous gene of the T cell.

[0019] In certain embodiments, the endogenous gene is selected from B2M, TET2, PD1, BCOR, Regnase, Roquin-1 or TRAC.

[0020] In certain embodiments, the crRNA used to target the endogenous B2M gene of T cells has a sequence as shown in SEQ ID NO:2.

[0021] In certain embodiments, the crRNA used to target the endogenous TET2 gene of T cells has a sequence as shown in SEQ ID NO:3.

[0022] In certain embodiments, the crRNA used to target the endogenous PD1 gene of T cells has a sequence as shown in SEQ ID NO:4.

[0023] In certain embodiments, the crRNA used to target the endogenous Regnase gene of T cells has a sequence as shown in SEQ ID NO:5.

[0024] In certain embodiments, the crRNA used to target the endogenous Roquin-1 gene in T cells has a sequence as shown in SEQ ID NO:6.

[0025] In certain embodiments, the crRNA used to target the endogenous TRAC gene of T cells has a sequence as shown in SEQ ID NO:7.

[0026] In certain embodiments, the nucleic acid construct further comprises a nucleotide sequence encoding a regulatory element.

[0027] In certain embodiments, the regulatory element is selected from a promoter, a self-cleaving polypeptide, an internal ribosome entry site (IRES), a signal peptide, or any combination thereof.

[0028] In certain embodiments, the self-cleaving polypeptide is selected from P2A, T2A, E2A or F2A.

[0029] In certain embodiments, the promoter is selected from EF1a or an endogenous gene promoter.

[0030] In certain embodiments, the suicide gene is selected from CD20, HER2, or HSV-TK.

[0031] In certain embodiments, the nucleic acid construct further comprises a nucleotide sequence encoding a detectable label.

[0032] In certain embodiments, the detectable label is a fluorescent protein.

[0033] In certain embodiments, the various elements of the nucleic acid construct are optionally connected via a linker (eg, a flexible peptide linker) or not.

[0034] In certain embodiments, the nucleic acid construct comprises 1, 2, 3, 4 or 5 STARs. In certain embodiments, each STAR is connected with or without an IRES.

[0035] In certain embodiments, the nucleic acid construct comprises: a second guide sequence, a second homology arm, a STAR, a first homology arm, and a first guide sequence.

[0036] In certain embodiments, the nucleic acid construct comprises, from the 5' end to the 3' end, a second guide sequence, a second homology arm, a STAR, a first homology arm, and a first guide sequence.

[0037] In certain embodiments, the nucleic acid construct comprises: a second guide sequence, a second homology arm, a promoter, a STAR, a self-cleaving polypeptide, a first homology arm, and a first guide sequence.

[0038] In certain embodiments, the nucleic acid construct comprises, from the 5' end to the 3' end, in order: a second guide sequence, a second homology arm, a promoter, a STAR, a self-cleaving polypeptide, a first homology arm, and a first guide sequence.

[0039] In certain embodiments, the nucleic acid construct further comprises an IRES and / or a fluorescent protein at the 3' end of the STAR.

[0040] In certain embodiments, the nucleic acid construct has an amino acid sequence as shown in SEQ ID NO:37, 38, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.

[0041] In certain embodiments, the Cas12a protein is selected from a natural Cas12a protein, a mutant of the Cas12a protein, a homolog of the Cas12a protein, or any combination thereof.

[0042] In certain embodiments, the tumor-associated antigen is selected from the group consisting of: CD19, EGFR, CD20, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), mesothelin MSLN, epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigens, β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate enzyme-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGF1 receptor, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, A1 domain of tenascin-C (TnC A1), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF17), TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), FCRL5 (UNIPROT Q68SN8), or any combination thereof.

[0043] In certain embodiments, the length of the first homology arm is 100-3000bp; for example, 100-3000bp, 100-2000bp, 100-1000bp, 100-900bp, 100-800bp, 100-700bp, 100-600bp, 100-500bp, 100-400bp, 100-300bp, 100-200bp.

[0044] In certain embodiments, the length of the second homology arm is 100-3000bp; for example, 100-3000bp, 100-2000bp, 100-1000bp, 100-900bp, 100-800bp, 100-700bp, 100-600bp, 100-500bp, 100-400bp, 100-300bp, 100-200bp.

[0045] In certain embodiments, the length of the STAR is 100-6000 bp; for example, 100-6000 bp, 300-5000 bp, 500-4000 bp, 800-3000 bp, 1000-3000 bp, 1000-2000 bp.

[0046] In certain embodiments, the length of the nucleic acid construct is 100-10000 bp; for example, 100-10000 bp, 500-9000 bp, 1000-8000 bp, 2000-7000 bp, 3000-6000 bp, 4000-5000 bp.

[0047] In certain embodiments, the crRNA has a length of 5-50 bp; for example, 5-50 bp, 8-40 bp, 10-30 bp, 15-25 bp.

[0048] In certain embodiments, the Cas12a protein has the sequence shown in SEQ ID NO:1.

[0049] In certain embodiments, the PAM has a sequence as shown in any one of SEQ ID NOs: 9-15.

[0050] In certain embodiments, the crRNA has a sequence shown in any one of SEQ ID NOs: 2-8.

[0051] In certain embodiments, the EF1a has the sequence shown in SEQ ID NO:40.

[0052] In certain embodiments, the P2A has the sequence shown in SEQ ID NO:39.

[0053] In certain embodiments, the STAR has the sequence shown in SEQ ID NO:37.

[0054] In certain embodiments, the second homology arm has a sequence shown in any one of SEQ ID NOs: 23-29.

[0055] In certain embodiments, the first homology arm has a sequence shown in any one of SEQ ID NOs: 30-36.

[0056] Carrier

[0057] In a second aspect, the present application provides a vector comprising the nucleic acid construct as described in the first aspect.

[0058] In certain embodiments, the vector is a cloning vector or an expression vector.

[0059] Host cells

[0060] In a third aspect, the present application provides a host cell comprising the nucleic acid construct as described in the first aspect or the vector as described in the second aspect.

[0061] In certain embodiments, the cell is a mammalian cell.

[0062] Reagent test kit

[0063] In a fourth aspect, the present application provides a kit comprising at least one of the following (1) to (3) (e.g., 1, 2, 3):

[0064] (1) The nucleic acid construct according to the first aspect;

[0065] (2) The vector as described in the second aspect;

[0066] (3) The host cell as described in the third aspect.

[0067] In certain embodiments, the kit further comprises any one or more of the following (4) to (8) (e.g., 1, 2, 3, 4, 5):

[0068] (4) Cas12a protein;

[0069] (5) crRNA capable of binding to Cas12a protein;

[0070] (6) Complexes formed by Cas12a protein and crRNA (e.g., T cells, macrophages, DCs, NK);

[0071] (7) immune cells (e.g., T cells);

[0072] (8) Donor vector.

[0073] (9) one or more pharmaceutically active agents (eg, pharmaceutically active agents that enhance cell survival).

[0074] In certain embodiments, the donor vector is used to amplify the nucleic acid construct. In certain embodiments, the nucleic acid construct is constructed in the donor vector to amplify the nucleic acid construct.

[0075] In certain embodiments, the kit further comprises: a polymerase (e.g., a DNA polymerase), an RNase inhibitor, a reagent for extracting and / or separating T cells, a reagent for activating T cells (e.g., an anti-CD3 antibody, an anti-CD28 antibody), a reagent for electroporation, or any combination thereof.

[0076] In certain embodiments, the Cas12a protein is as defined in the first aspect.

[0077] In certain embodiments, the crRNA is as defined in the first aspect.

[0078] In certain embodiments, the pharmaceutically active agent is selected from Troglitazone, Inauhzin, H151, or any combination thereof.

[0079] Engineered T cells and preparation methods thereof

[0080] In a fifth aspect, the present application provides a method for preparing engineered T cells, the method comprising:

[0081] (1) Provide T cells;

[0082] (2) providing a nucleic acid construct as described in the first aspect;

[0083] (3) providing a Cas12a protein and a crRNA capable of binding to the Cas12a protein;

[0084] (4) contacting the nucleic acid construct, Cas12a protein, crRNA, and T cells under conditions that allow transformation;

[0085] Wherein, steps (1), (2) and (3) are performed in any order (for example, performed sequentially in any order, and / or, performed simultaneously).

[0086] In certain embodiments, the Cas12a protein and crRNA in step (3) exist in the form of a complex, or each exists separately.

[0087] In certain embodiments, the Cas12a protein is as defined in the first aspect.

[0088] In certain embodiments, the crRNA is as defined in the first aspect.

[0089] In certain embodiments, step (2) of the method is achieved by the following steps:

[0090] (a) selecting at least one target sequence (e.g., 1, 2, 3, 4, 5) in the T cell; wherein the target sequence comprises a sequence complementary to the crRNA sequence;

[0091] (b) designing the crRNA sequence, the first homology arm sequence and the second homology arm sequence according to the selected target site;

[0092] (c) selecting at least one STAR and constructing the nucleic acid construct as described in the first aspect.

[0093] In certain embodiments, after step (c), the nucleic acid construct is amplified.

[0094] In certain embodiments, the nucleic acid construct of the first aspect is amplified by constructing the nucleic acid construct in a donor vector.

[0095] In certain embodiments, the target sequence is adjacent to a PAM sequence recognized by the Cas12a protein.

[0096] In certain embodiments, the target sequence is located 3' to the PAM sequence.

[0097] In certain embodiments, the Cas12a protein and crRNA targeting the endogenous gene of the T cell are introduced into the T cell in the form of a complex or separately, thereby forming a double-strand break site in the endogenous gene. In certain embodiments, the nucleic acid construct described in the first aspect is introduced into the T cell, thereby homologously recombining the nucleic acid construct described in the first aspect to the double-strand break site of the endogenous gene. In certain embodiments, by inserting the nucleic acid construct described in the first aspect into the endogenous gene seat of the T cell, to prepare an engineered T cell.

[0098] In a sixth aspect, the present application provides an engineered T cell, which is prepared by the method described in the fifth aspect.

[0099] In certain embodiments, the engineered T cells express the STAR.

[0100] In certain embodiments, the genome of the engineered T cell comprises a nucleotide sequence encoding the STAR.

[0101] In certain embodiments, the engineered T cells are chimeric antigen receptor T cells (CAR-T).

[0102] In certain embodiments, the engineered T cells are T cell receptor gene-modified T cells (TCR-T).

[0103] Immune cell composition

[0104] In the seventh aspect, the present application provides an immune cell composition comprising the engineered T cells described in the eighth aspect.

[0105] In certain embodiments, the number of engineered T cells accounts for at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) of the total number of cells in the immune cell composition.

[0106] Methods and uses

[0107] In an eighth aspect, the present application provides a method for modifying a target sequence, comprising: contacting the nucleic acid construct described in the first aspect with the target sequence, or delivering the nucleic acid construct described in the first aspect into a cell containing the target sequence.

[0108] In certain embodiments, the target sequence is present in an immune cell (eg, a T cell).

[0109] In certain embodiments, the methods result in breaks in the target sequence, such as DNA double-strand breaks or RNA single-strand breaks.

[0110] In certain embodiments, the method further comprises inserting a STAR into the break.

[0111] In the ninth aspect, the present application provides the nucleic acid construct described in the first aspect, or the vector described in the second aspect, or the host cell described in the third aspect, or the kit described in the fourth aspect, for use in nucleic acid editing (e.g., gene or genome editing).

[0112] In certain embodiments, the gene or genome editing comprises modifying a gene, knocking out a gene, altering the expression of a gene product, repairing a mutation, and / or inserting a polynucleotide.

[0113] In the tenth aspect, the present application provides the use of the nucleic acid construct described in the first aspect, or the vector described in the second aspect, or the host cell described in the third aspect, or the kit described in the fourth aspect for preparing engineered T cells.

[0114] In the eleventh aspect, the present application provides the use of the nucleic acid construct described in the first aspect, or the vector described in the second aspect, or the host cell described in the third aspect, or the kit described in the fourth aspect, or the engineered T cell described in the sixth aspect, or the immune cell composition described in the seventh aspect for preparing a drug, wherein the drug is used to prevent and / or treat a disease (e.g., tumor, autoimmune disease, organ fibrosis) in a subject.

[0115] In certain embodiments, the nucleic acid construct specifically binds to an antigen expressed by the tumor.

[0116] In certain embodiments, the engineered T cells are derived from the subject.

[0117] In certain embodiments, the engineered T cells are derived from a donor other than the subject.

[0118] In certain embodiments, the tumor is selected from a solid tumor or a hematological tumor.

[0119] In certain embodiments, the tumor is selected from a malignancy of the B-cell lineage, such as a B-cell lymphoma or a B-cell leukemia, such as non-Hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia.

[0120] In certain embodiments, the subject is a mammal, such as a human.

[0121] Definition of terms

[0122] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0123] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0124] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0125] As used herein, the term "Cas12a protein" has a meaning commonly understood by those skilled in the art, and generally refers to a Class 2 Cas protein derived from Acidaminococcus sp. In certain exemplary embodiments, the Cas12a protein is an effector protein AsCas12aUltra contained in the CRISPR / ASCas12a Ultra system. In certain exemplary embodiments, the CRISPR / ASCas12a Ultra system also contains a scaffold sequence and a guide sequence of a guiding crRNA, and the guide sequence specifically binds to the target sequence. In certain embodiments, the effector protein AsCas12aUltra comprises an amino acid sequence as shown in SEQ ID NO: 1, or an ortholog thereof, a homolog, a variant, or a functional fragment thereof. In certain embodiments, the CRISPR / ASCas12a Ultra system or the effector protein contained therein can be purchased from IDT, Cat. No.: 10001273, and its product description is incorporated herein by reference.

[0126] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival period (if not receiving treatment), extending the survival period.

[0127] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) has a tumor, or is at risk of having a tumor.

[0128] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0129] The terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade neighboring tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies.

[0130] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0131] As used herein, the terms "guide RNA", "crRNA" are used interchangeably and have the meanings commonly understood by those skilled in the art. In general, guide RNA may include a direct repeat sequence and a guide sequence, or may be substantially composed of or composed of a direct repeat sequence and a guide sequence (also referred to as a spacer in the context of an endogenous CRISPR system). In some cases, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target sequence to hybridize with the target sequence and guide the specific binding of a CRISPR / Cas complex to the target sequence. In certain embodiments, when optimally aligned, the degree of complementarity between a guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. It is within the capabilities of those of ordinary skill in the art to determine the optimal alignment. For example, there are publicly available and commercially available alignment algorithms and programs, such as, but not limited to, ClustalW, Smith-Waterman algorithm in matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0132] As used herein, the term "target sequence" refers to a polynucleotide targeted by a guide sequence designed to have targeting in the case of forming a CRISPR / Cas complex, such as a sequence having complementarity with the guide sequence, wherein the hybridization between the target sequence and the guide sequence will promote the formation of a CRISPR / Cas complex. Complete complementarity is not required, as long as there is enough complementarity to cause hybridization and promote the formation of a CRISPR / Cas complex. The target sequence can include any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. A sequence or template that can be used to recombine into a target locus containing the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In certain embodiments, the editing template is an exogenous nucleic acid. In certain embodiments, the recombination is homologous recombination.

[0133] In the present invention, the expression "target sequence" or "target polynucleotide" can be any endogenous or exogenous polynucleotide for a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a useless DNA). In some cases, the target sequence should be related to a protospacer adjacent motif (PAM). The precise sequence and length requirements for PAM vary depending on the Cas effector enzyme used, but PAM is typically a 2-5 base pair sequence adjacent to the protospacer sequence (i.e., the target sequence). Those skilled in the art can identify the PAM sequence used with a given Cas effector protein. As used herein, the term "PAM sequence" refers to a specific motif sequence recognized by the Cas protein.

[0134] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0135] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0136] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the desired expression level, etc. A vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including a protein, fusion protein, isolated nucleic acid molecule, etc. as described herein (e.g., a CRISPR transcript, such as a nucleic acid transcript, protein, or enzyme).

[0137] As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences), which are described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990). In some cases, regulatory elements include those sequences that direct the constitutive expression of a nucleotide sequence in many types of host cells and those sequences that direct the nucleotide sequence to be expressed only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). In some cases, regulatory elements can also direct expression in a timing-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I ((Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); SV40 enhancer; and intron sequences between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0138] As used herein, the term "promoter" has a meaning well known to those skilled in the art, and refers to a non-coding nucleotide sequence located upstream of a gene that can initiate expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of a gene product in a cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell essentially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of a gene product in the cell essentially only when the cell is a cell of the tissue type corresponding to the promoter.

[0139] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0140] Advantageous Effects of the Invention

[0141] Compared with the prior art, the nucleic acid construct provided by the present application has higher gene editing efficiency, lower off-target efficiency and higher safety. Specifically, the nucleic acid construct provided by the present application significantly improves the site-specific editing efficiency of donor fragments of different lengths (e.g., between 0.5kb and 7kb) (the efficiency is increased by 7-10 times), and especially achieves efficient site-specific editing of donor fragments of 7kb and above.

[0142] In addition, the nucleic acid constructs and engineered T cells provided in the present application have a strong killing effect on tumor cells, with a basic killing efficiency of more than 80%. In addition, the nucleic acid constructs and engineered T cells provided in the present application are particularly suitable for site-specific editing of double-stranded donor fragments, and the preparation cycle of double-stranded donor fragments is shorter and the preparation cost is lower than that of single-stranded donor fragments. Therefore, the nucleic acid constructs, engineered T cells and preparation methods provided in the present application have great application potential in large-scale production or medical treatment.

[0143] Embodiments of the present invention will be described in detail below by examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] Figure 1 A nucleic acid construct STAR dsDNA-HDR was designed with optimized structural sequences.

[0145] Figure 2A The efficiency of site-directed knock-in of P2A-FMC63-OX40STAR at the TRAC locus.

[0146] Figure 2B The efficiency of site-directed knock-in of EF1a-FMC63-OX40STAR-IRES-GFP at the TRAC site.

[0147] Figure 3 A in the figure is the editing expression efficiency of genes with different gene fragment lengths (3.3Kb, 4.5Kb, 5.3Kb, 5.7Kb, 6.9Kb) knocked into the TRAC site by flow cytometry analysis.

[0148] Figure 3 B in the figure represents the killing effect of the STAR positive cells constructed in the present application on the target cell Raji.

[0149] Figure 4 A and B in the figure are flow cytometry analyses of the editing expression efficiency of different genes knocked into multiple different gene loci.

[0150] Figure 5 Comparison of the integration efficiency of nucleic acid constructs containing and not containing specific crRNA-PAM.

[0151] Figure 6 A, B and C in the figure are the survival rate of cells and the rate of positive cells containing STAR after electroporation on different days.

[0152] Figure 7 The survival rate of T cells and the efficiency of STAR integration expression at different days after electroporation.

[0153] Figure 8 The results of in vitro and in vivo anti-tumor experiments of Lenti-STAR, AAV-STAR and dsDNA-STAR are shown in Figure 2. Figure 8 A in the figure is the efficiency of STAR expression on the membrane detected by flow cytometry; Figure 8 B in the figure is the result of in vitro anti-tumor experiment; Figure 8 C in Figure 8 to F in Figure 8 show the process and results of the in vivo anti-tumor experiment.

[0154] Fig. 9 The cell viability of electroporated cells was determined by adding small molecule drugs at different concentrations. The PBMC used in the experiment were frozen cells with an initial cell concentration of 1.5e 5 cells per well. Fig. 9 A-9 and D in the figure respectively represent drug concentrations of 0.5uM, 1uM, 5uM and 10uM, n=3. The electroporated dsDNA concentration is 1ug / 1e 6 cell.

[0155] Fig.10 Figure 2 is the fold change diagram of cell viability and number after dsDNA electroporation. The initial cell number is 1.5e 5 , dsDNA concentration is 1ug / 1e 6 Cells, PBMCs are frozen and thawed cells, n = 3. Fig.10 The drug concentrations added to C in A-10 were 1uM, 5uM, and 10uM respectively; Fig.10 F in D-10 in the figure respectively represents the cell viability after adding drugs i, b, and l; Fig.10 I in G-10 represents the fold change of cell number after adding drugs i, b, and l, respectively. P values ​​were calculated by two-way ANOVA, * indicates p<0.05, ** indicates p<0.01, and no * indicates no significant difference.

[0156] Sequence information

[0157] The information of the partial sequences involved in the present invention is provided in Table 1 below.

[0158] Table 1: Description of sequences

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181] DETAILED DESCRIPTION

[0182] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0183] Unless otherwise specified, the experiments and methods described in the embodiments are carried out basically according to the conventional methods well known in the art and described in various references. For example, the conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., ed., (1987)); METHODS IN ENZYMOLOGY series (Academic Publishing Company): PCR 2: A PRACTICAL METHOD. APPROACH) (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)), ANTIBODIES, A LABORATORY MANUAL, eds. Harlow and Lane, eds. (1988), and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

[0184] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0185] Example 1. Design and selection of Cas12a / crRNA with the highest efficiency in targeted gene knockout

[0186] 1.1 Download the target gene sequence: Taking TRAC as an example, first download the target genome sequence that needs to be site-specifically integrated and inserted from the UCSC website https: / / genome-asia.ucsc.edu / cgi-bin / hgGateway. Enter the website: click the species option and select Human; select Dec.2013 (GRCh38 / hg38) at Find Position-Human Assembly; enter the name of the gene to be targeted (TRAC) at Position / Search Term, click GO, and the webpage will jump to the next page; click the name of the gene to be targeted (TRAC is a transparent white font on a blue background); select GemomicSequence on the new page; check 5'UTR Exons, CDS Exons, 3'UTR Exons and Introns on the new jump page, and click submit to download the target gene sequence.

[0187] 1.2 Design and screen Cas12a / crRNA with high knockout efficiency: Taking the TRAC gene as an example, enter the downloaded TRAC gene sequence into the box of Step 1 on the http: / / crispor.tefor.net / page, select Homosapiens-Human-UCSC Feb.2009(GRCh37 / hg19)+SNPs:1000Genomes,ExaC in Step 2, and select TTT(A / C / G)-21bp-Cas12a(Cpf1)-21bp guides recommended by IDT in Step 3. After Submit, a series of guide RNAs are obtained. Select the guide RNA sequence with TTT(A / C / G) PAM sequence close to 5'UTR and located in the Exons region with a higher score. Generally, 3-5 guide RNA sequences are selected and sent to IDT for synthesis. At the same time, As.Cas12aUltra protein was ordered from IDT, and incubated with synthetic crRNA (the above-mentioned guide RNA) to prepare RNP, and activated Human T cells were electroporated. The knockout efficiency was analyzed by flow cytometry four days after electroporation, and the knockout efficiency of these crRNAs was identified to be above 70%, and the crRNA sequence with the highest knockout efficiency shown in SEQ ID NO: 7 (above 95%) was selected.

[0188] Example 2. Design of double-stranded DNA HDR with optimized structure and sequence

[0189] Select Cas12a / crRNA with the best knockout efficiency, select a suitable insertion site near the Cas12a cleavage site, select the left homology arm (LHA) shown in SEQ ID NO:23 and the right homology arm (RHA) shown in SEQ ID NO:30 300bp upstream and downstream of the insertion site, and between the left and right homology arms is the insertion sequence or gene of interest (Insert / GOI). Our optimization improvement is to add reverse complementary crRNA-PAM sequences or gRNA-PAM sequences (applicable to Cas9) on both sides of the left and right homology arms, respectively. This embodiment constructs a crRNA-PAM sequence as shown in SEQ ID NO:17. For example, the double-stranded DNA HDR of T cell receptor antigen receptor (STAR) is designed and synthesized and inserted into the TRAC site of Human T cells. The double-stranded STAR DNAHDR (STAR ​​dsDNA-HDR) with optimized structure sequence is as follows Figure 1 shown.

[0190] Example 3. CRISPR / Cas12a Ultra / dsDNA-HDR system targets TRAC loci in human T cells Effective integration of STAR

[0191] 3.1. Donor DNA amplification, recovery, purification and concentration

[0192] The designed structure-optimized STAR dsDNA-HDR (300 bp homology arm length, for site-specific insertion into the TRAC gene specific site) as shown in SEQ ID NO: 38 was cloned into the template vector pcDNA3.1 or AAV vector (Donorplasmid). PCR amplification primers were designed, and the structure-optimized STAR dsDNA-HDR target fragment was amplified using a high-fidelity DNA polymerase. The DNA size was confirmed and gel purified using 1% agarose gel electrophoresis, and the target sequence was concentrated and precipitated with isopropanol (concentrated to 1-5 ug / ul).

[0193] Isopropanol concentration and precipitation of target sequence

[0194] Components System (μl) Gel-recovered dsDNA-HDR target fragments 80 Isopropyl alcohol 100 NaCl(5M) 2 H2O upto ～200

[0195] Vortex mix and incubate at room temperature for 15 minutes, then centrifuge at 15,000g for 15 minutes at room temperature to precipitate DNA. Discard the supernatant, resuspend with 1ml ice-cold (-20℃) 80% (vol / vol) ethanol, wash twice, and dry at room temperature. Resuspend the DNA in 5-10μl of DNase / RNase free water, fully dissolve the precipitate in a 55℃ metal bath for 10 minutes, and adjust the concentration to 1-5ug / ul.

[0196] 3.2 Preparation of Cas12a / TRAC RNP

[0197] The TRAC-crRNA ordered from IDT was dissolved in DEPC water to a final concentration of 75 μM. As.Cas12a Ultra protein was ordered from IDT at a concentration of 10 μg / μL. 0.25 μL As.Cas12a Ultra protein and 0.5 μL crRNA were mixed and incubated at 37°C for 15 min.

[0198] 3.3 Isolation and culture of human T cells

[0199] Human peripheral blood cells were obtained, and CD4 and CD8 T cells were purified using a complete T cell magnetic bead separation kit. T cells were then stimulated and activated in culture dishes coated with anti-CD3 / CD28 (anti-CD3 5ug / ml, purchased from BioLegend, #317315; anti-CD28 1ug / ml, purchased from BD Biosciences, #555725) and human fibronectin (5ug / ml, purchased from BD Biosciences, #354008) for 24-36 hours, and the cells were collected, washed twice with Opti-MEM without RNAse, the residual liquid was aspirated, and the reagent in the LONZA P3 primary cell 4D nuclear transfection system X unit kit S was used to resuspend the cells so that the final concentration was 1.5E6 / 20μL.

[0200] 3.4 Electrotransfer of Cas12a-TRAC RNP / STAR dsDNA-HDR into activated human T cells

[0201] 0.5-1pm (1-2ug) of purified structure-optimized STAR dsDNA-HDR was added to the Cas12a-TRAC RNP tube after incubation at 37°C for 15min, and 20μL of cell suspension was immediately added. After gently blowing and mixing, all were transferred to the electroporation cup in the LONZA P3 primary cell 4D nuclear transfection system X unit kit S, placed in the electroporator of the LONZA X unit, and electroporated using the EO115 program provided by the instrument. After the electroporation, 100ul T cell complete medium (1640+20% Inactivated FBSGibco+GlutMAX+NEAA+P / S+SP (sodium pyruvate)+IL2-100U / ml) was immediately added and transferred to a 48-well flat-bottom plate. Three days later, the cells were transferred to a 12-well flat-bottom plate, and the efficiency of site-specific integration was detected by flow cytometry 6-7 days after electroporation.

[0202] 3.5 Streaming Analysis Integration Efficiency

[0203] For the TRAC site, this scheme used the TRAC endogenous promoter, used the P2A sequence in the knock-in template, and site-directed knock-in the FMC63-OX40 STAR sequence shown in SEQ ID NO:41; and the EF1a exogenous promoter, and site-directed knock-in the EF1a-FMC63-OX40STAR-IRES-GFP sequence shown in SEQ ID NO:42. The results are shown in Figure 2. The knockout efficiency of TCR reached more than 92%, and the efficiency of FMC63-OX40 STAR site-directed knock-in was 55.6% ( Figure 2A ). The efficiency of EF1a-FMC63-OX40STAR-IRES-GFP sequence FMC63-OX40STAR site-directed knock-in was 35.9%, the efficiency of GFP site-directed knock-in was 40.6%, and the FMC63-OX40STAR and GFP double-positive population cells reached 40.6%. Figure 2B Based on this, this example has obtained STAR-T cells with high integration efficiency through CRISPR / Cas12a Ultra and structure-optimized dsDNA-HDR, and the integration efficiency of the 6kb EF1a-FMC63-OX40STAR-IRES-GFP DNA fragment is as high as 36%.

[0204] Example 4. CRISPR / Cas12aUltra and dsDNA-HDR systems can efficiently integrate gene fragments of different lengths The segment was inserted into the TRAC site of human T cells, and the successfully edited cells had efficient tumor killing function.

[0205] 4.1. Insertion of gene fragments of different lengths into TRAC loci of human PBMC cells

[0206] Target gene sequences with different structures and fragment sizes were designed and constructed (such as P2A-STAR 3.3 kb as shown in SEQ ID NO: 43; P2A-STAR-IRES-RFP 4.5 kb as shown in SEQ ID NO: 44; P2A-STAR-IRES-RFP-P2A-eGFP 5.3 kb as shown in SEQ ID NO: 45; EF1a-STAR-IRES-GFP 5.7 kb as shown in SEQ ID NO: 46; P2A-STAR-IRES-RFP-P2A-HER2 6.9 kb as shown in SEQ ID NO: 47), and the site-directed knockout integration method established in this patent was used to knock out the TRAC gene in human T cells, and double-stranded DNA fragments of different lengths were site-directedly integrated at the TRAC knockout site. After 6 days of electroporation, the editing efficiency of the site-directed insertion of gene fragments of different lengths by the method was detected by flow cytometry. The results are as follows: Figure 3 As shown in A, constructs of different sizes ranging from 3.3 kb to 6.9 kb showed high integration and expression efficiencies, such as the integration and expression efficiencies of STAR, GFP, and RFP were close to 60%.

[0207] 4.2 STAR cells edited by this technical solution have strong tumor killing function

[0208] The STAR positive cell population successfully edited by this scheme was sorted, and incubated with Raji tumor cells at a MOI ratio of 1:1; 1:2 for 16 hours. The killing effect of STAR positive cells on target cells Raji was detected using an LDH kit. The results showed that the STAR cells constructed by this technical scheme had a very good killing effect on Raji, with a basic killing efficiency of more than 80%. The results are as follows Figure 3 As shown in B, this shows that the technical solution has good application prospects.

[0209] Example 5. CRISPR / Cas12aUltra and dsDNA-HDR system can be used in B2M, TET2, PD1, BCOR, Regnase and other multiple different sites to efficiently integrate multiple genes

[0210] According to the experimental scheme and steps described in the above examples, we designed crRNAs targeting multiple different genes in human T cells (B2M, TET2, PD1, BCOR, Regnase,), and used the technical method of this patent to knock in different genes at the knockout site of the gene. The tools involved in the experiment and their sequences are specifically shown in Table 2 and as follows:

[0211] (1) Site-specific knock-in of HER2 or eGFP at the B2M locus (RefSeq NM_004048);

[0212] (2) site-specific knock-in of eGFP at the TET2 locus (RefSeq NM_001127208);

[0213] (3) site-specific knock-in of HER2-G4S3-HSVTK at the PD1 locus (RefSeq NM_005018);

[0214] (4) site-specific knock-in of eGFP at the BCOR locus;

[0215] (5) site-specific knock-in of RFP at the Regnase locus (RefSeq NM_025079);

[0216] (6) Site-specific knock-in of eGFP and RFP at the BCOR and Regnase sites; wherein, the scheme is to simultaneously electroporate BCOR-eGFP-HDR and Regnase-RFP-HDR double-stranded DNA, incubate BCOR-RNP and Regnase-RNP separately, and immediately add 1.5ug of BCOR-eGFP-HDR and Regnase-RFP-HDR double-stranded DNA at the end of incubation, and then add PBMC for electroporation.

[0217] Table 2. Components used and their sequence

[0218]

[0219] The integration efficiency was analyzed by flow cytometry 7 days after electroporation. Figure 4 As shown in A in Figure 1, the target gene was successfully integrated and expressed at multiple different gene loci using this technical solution for site-specific editing of human cells. Importantly, the same cell can achieve site-specific knock-in editing of multiple loci, such as knocking out BCOR and Regnase at the same time in human T cells, and successfully knocking in eGFP and RFP at the two knockout sites at the same time, with the proportion of double-positive cells being around 4%. Figure 4 As shown in B.

[0220] Example 6. Adding reverse complementary crRNA-PAM sequences on both sides of the dsDNA-HDR homology arms can The input efficiency is increased by 7-10 times

[0221] Taking the site-specific insertion of STAR at the TRAC knockout site as an example, the inverted repeat sequence of the specific crRNA-PAM (the left crRNA-PAM is shown in SEQ ID NO:54; the right crRNA-PAM is shown in SEQ ID NO:55) and the unrelated sequence that does not recognize the match are added to the homologous arms on the left and right sides of the STAR dsDNA, respectively, to obtain the STAR dsDNAHDR with specific crRNA-PAM and the STAR dsDNA HDR with nonsense crRNA-PAM. The electroporation results of these two different characteristics of STAR dsDNAHDR showed that the editing and integration efficiency of the inverted repeat sequence STAR dsDNA HDR with specific crRNA-PAM was 7-10 times that of the nonsense crRNA-PAM STAR dsDNA HDR (for example, Figure 5 40.8 and 3.96 in ; and Figure 5 5.04 and 37.0 in , and the electrotransfection efficiency of dsDNA was highest at 0.75-1pM. Figure 5 shown.

[0222] Example 7. Compared with single-stranded donor DNA fragments, the dsDNA-HDR system has superior integration efficiency and good Cell viability

[0223] For contrasting the single-stranded donor DNA fragments and double-stranded donor DNA fragments of the same structure under CRISPR / Cas12a systems, the effect (integration efficiency, cell viability, etc.) of the same site of site-specific integration;And under the same single-stranded donor DNA fragments and double-stranded donor DNA structure gene fragments, compared to the CRISPR / Cas9 system, the advantages and disadvantages of the same site of site-specific integration of CRISPR / Cas12a and dsDNA-HDR systems. We have compared the viability and target gene integration efficiency of cells in the same single-stranded donor DNA fragments and double-stranded donor DNA fragments in CRISPR / Cas12a and CRISPR / Cas9 systems respectively. Wherein, the sequence of the donor fragment, the insertion site selected and the sequence of crRNA, etc. are the same as in Example 3.

[0224] The results showed that the cell viability of Cas12a and Cas9 ssDNA was 45-47% on the third day of electroporation, while that of Cas12a and Cas9 dsDNA was 38% on the third day of electroporation. The double-stranded electroporation cell viability was lower than that of the single-stranded one, but on the fifth day of electroporation, the double-stranded electroporation cell viability exceeded 60%. When detected on the tenth day of electroporation, the efficiency of single-stranded and double-stranded cells was consistent, both exceeding 85%.

[0225] Importantly, if Figure 6 The results shown in B and C in 6 show that the site-specific integration efficiency of the CRISPR / Cas12a-dsDNA system on days 3, 5, and 10 is significantly higher than that of other systems (e.g., CRISPR / Cas12a-ssDNA system, CRISPR / Cas9-dsDNA system, and CRISPR / Cas9-ssDNA system). In particular, the positive cell rate containing STAR on the 10th day was 200% higher in the CRISPR / Cas12a-dsDNA (54.4%) system compared with the CRISPR / Cas9-dsDNA (23.1%) and CRISPR / Cas9-ssDNA (18.7%) systems! This shows that compared with the CRISPR / Cas9 dsDNA-ssDNA system and CRISPR / Cas12a-ssDNA, the CRISPR / Cas12a-dsDNA system has a superior integration efficiency and good cell viability.

[0226] Example 8. Compared with plasmid DNA, dsDNA-HDR has superior integration efficiency and cell viability

[0227] To compare the integration efficiency and cell viability of the same target gene in T cells, plasmid donor DNA (pcDNA3.1) and double-stranded donor DNA fragments were used as templates under the CRISPR / Cas12a system. Plasmid and dsDNA-HDR of the same molar amount (0.5pM) were electrotransferred to activated T cells (0.5pM plasmid+TRAC-RNP; 0.5pM dsDNA-HDR+TRAC-RNP). The sequence of the donor fragment, the selected insertion site, and the sequence of crRNA were the same as in Example 3.

[0228] The T cell viability and STAR integration expression efficiency were then tested. The results showed that on the 3rd, 5th and 8th days after electroporation, the viability of T cells electroporated with plasmid DNA was lower than that of cells electroporated with double-stranded DNA (dsDNA-HDR), and the integration efficiency of T cells electroporated with plasmid DNA was significantly lower than that of cells electroporated with double-stranded DNA (dsDNA-HDR). Figure 7 As shown, the results indicate that compared with electroporation integration using plasmid as a template, the CRISPR / Cas12a-dsDNA system has superior integration efficiency and cell viability.

[0229] Example 9. In vitro and in vivo anti-tumor experiments confirm that CRISPR / Cas12a-dsDNA edited engineered T cells have Has powerful tumor-killing function

[0230] To further verify the tumor killing function of engineered T cells prepared by CRISPR / Cas12a-dsDNA construction in vitro and in vivo, we compared the targeted killing function of Lenti-STAR, AAV-STAR and dsDNA-STAR in Raji tumors in vitro and in vivo (the specific preparation method is described in 3.1. In short, the STAR dsDNA-HDR shown in SEQ ID NO: 38 is cloned into the template vector Lenti vector (Phage, PMID: 33762437 DOI: 10.1126 / scitranslmed.abb5191), AAV vector (rAAV6, doi: 10.1038 / nature21405) or pcDNA3.1 vector).

[0231] In vitro experiments co-cultured Lenti-STAR, AAV-STAR and dsDNA-STAR with Raji-luciferase tumor cells at different ratios for 16 hours, and then tested the killing of tumor cells by different preparation groups. The results showed that ( Figure 8 A in and B in 8), CRISPR / Cas12a-dsDNA-edited STAR T cells have a strong tumor-killing function, and the killing effect is basically consistent with the results of the Lenti-STAR and AAV-STAR groups.

[0232] In the in vivo experiment, NCG mice were first inoculated with Raji-luciferase tumor cells via the tail vein on day 0, with 0.5e6 tumor cells inoculated per mouse. Four days after inoculation, the mice were imaged and grouped according to the imaging results (5 mice per group for Lenti-GFP (which is a Lenti vector with only GFP), Lenti-STAR, AAV-STAR, and dsDNA-STAR). On the fifth day of tumor inoculation, the constructed T cells were infused back into the tail vein of each group of mice, with 1e6 control and STAR-positive cells infused back into each mouse ( Figure 8 C in the figure). Then, the mice were imaged every four days to detect tumor killing and clearance, and blood was collected to analyze the proliferation of cells in each group. The results showed ( Figure 8 D-8F), compared with the Lenti-GFP control group, dsDNA-STAR showed strong tumor killing function in vivo, and its killing ability was comparable to that of the Lenti-STAR and AAV-STAR groups. At the same time, the proliferation ability of T cells in vivo also showed strong proliferation potential. These results show that CRISPR / Cas12a-dsDNA edited STAR T cells have strong tumor killing function.

[0233] Example 10. Screening of small molecule drug library to enhance the viability of CRISPR / Cas12a-dsDNA electroporated T cells

[0234] CRISPR / Cas12a-dsDNA electroporation can efficiently target and break double-stranded DNA in eukaryotic cells. At the same time, the DNA molecules delivered into the cells can be used as repair templates for homologous directed repair to achieve site-specific integration and expression of target genes. However, high concentrations of double-stranded DNA can be highly toxic to primary cells, leading to increased cell mortality, which to some extent limits the practical application of this system.

[0235] By screening two small molecule drug libraries related to apoptosis and epigenetics, a total of 1524 drugs were screened out, and 16 drugs (a: co1003, b: Inauhzin, c: Dihydrocoumarin, d: UBCS039, e: NSC7458, i: troglitzone, f: NU7026, g: Trovafloxacin, h: llaprazole, j: Remodelin, i: Troglitazone, k: G140, l: H151, m: ZL0420, n: XL4B, o: CPTH2) were initially screened out, which can significantly improve the cell viability after double-stranded DNA electroporation. Further concentration gradient experiments revealed that 0.5uM and 1uM drugs can improve cell viability without causing strong toxicity to cells. At concentrations of 5uM and 10uM, most of the screened drugs can enhance cell viability, but a few drugs significantly inhibit cell survival at the same concentration ( Fig. 9 ). By continuously detecting the cell viability and number on the 2nd, 3rd, 5th and 7th days after electroporation, it was found that the effect of drugs in promoting cell repair and proliferation mainly appeared in the later stage of electroporation (5-7 days). Three drugs that can significantly improve the cell viability after dsDNA electroporation were screened out: i: Troglitazone, b: Inauhzin, l: H151. They did not affect cell proliferation at a concentration of 1uM. Flow cytometric analysis further showed that these drugs, while enhancing cell viability, did not interfere with the knockout process of the target gene, nor did they affect the upper membrane expression of the target gene ( Fig.10 ). This may provide a new drug strategy for the application of electroporation technology in gene editing and cell therapy.

[0236] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A nucleic acid construct comprising a nucleotide sequence encoding a synthetic T cell antigen receptor (STAR) that specifically binds to a tumor-associated antigen, and a first guide sequence and a second guide sequence on both sides of the nucleotide sequence of the STAR; wherein, The first guide sequence and the second guide sequence include a PAM sequence that can be recognized by the Cas12a protein and a crRNA sequence that can bind to the Cas12a protein.

2. The nucleic acid construct according to claim 1, wherein The PAM sequences in the first and second guide sequences are located at one end close to the STAR; Preferably, the first guide sequence is located at the 3' end of the STAR, and the first guide sequence comprises the PAM sequence and the crRNA sequence in sequence from the 5' end to the 3' end; Preferably, the second guide sequence is located at the 5' end of the STAR, and the second guide sequence comprises the crRNA sequence and the PAM sequence in sequence from the 5' end to the 3' end.

3. The nucleic acid construct according to claim 1 or 2, wherein The nucleic acid construct further comprises a first homology arm sequence between the STAR and the first guide sequence, and the nucleic acid construct further comprises a second homology arm sequence between the STAR and the second guide sequence; Preferably, the first homology arm sequence and the second homology arm sequence are identical to the sequences on both sides of the target sequence; Preferably, the first homology arm sequence is identical to a sequence downstream of the target sequence, and the second homology arm sequence is identical to a sequence upstream of the target sequence.

4. The nucleic acid construct according to claim 3, wherein The target sequence comprises a sequence complementary to the crRNA sequence; Preferably, the target sequence is adjacent to the PAM sequence recognized by the Cas12a protein; Preferably, the target sequence is located at the 3' end of the PAM sequence; Preferably, the target sequence is located in the genome of an immune cell; Preferably, the target sequence is located in the sequence of an endogenous gene of a T cell; Preferably, the endogenous gene is selected from B2M, TET2, PD1, BCOR, Regnase, Roquin-1 or TRAC.

5. The nucleic acid construct according to any one of claims 1 to 4, wherein The nucleic acid construct further comprises a nucleotide sequence encoding a regulatory element; Preferably, the regulatory element is selected from a promoter, a self-cleaving polypeptide, an internal ribosome entry site (IRES), a signal peptide, a suicide gene, or any combination thereof; Preferably, the self-cleaving polypeptide is selected from P2A, T2A, E2A or F2A; Preferably, the promoter is selected from EF1a or an endogenous gene promoter; Preferably, the suicide gene is selected from CD20, HER2, iCasp9 or HSV-TK; Preferably, the nucleic acid construct further comprises a nucleotide sequence encoding a detectable label; Preferably, the detectable label is a fluorescent protein.

6. The nucleic acid construct according to any one of claims 1 to 5, wherein The nucleic acid construct comprises: a second guide sequence, a second homology arm, a STAR, a first homology arm, and a first guide sequence; Preferably, the nucleic acid construct comprises, from the 5' end to the 3' end, in sequence: a second guide sequence, a second homology arm, a STAR, a first homology arm, and a first guide sequence.

7. The nucleic acid construct according to any one of claims 1 to 6, wherein The nucleic acid construct comprises: a second guide sequence, a second homology arm, a promoter, a STAR, a self-cleaving polypeptide, a first homology arm, and a first guide sequence; Preferably, the nucleic acid construct comprises, from the 5' end to the 3' end, in order: a second guide sequence, a second homology arm, a promoter, a STAR, a self-cleaving polypeptide, a first homology arm, and a first guide sequence; Optionally, the nucleic acid construct further comprises an IRES and / or a fluorescent protein at the 3' end of the STAR; Preferably, the nucleic acid construct has the amino acid sequence shown in SEQ ID NO:37, 38, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.

8. The nucleic acid construct according to any one of claims 1 to 7, wherein The Cas12a protein is selected from a natural Cas12a protein, a mutant of the Cas12a protein, a homolog of the Cas12a protein, or any combination thereof.

9. The nucleic acid construct according to any one of claims 1 to 8, wherein The tumor-associated antigen is selected from the group consisting of CD19, EGFR, CD20, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), mesothelin MSLN, epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigens, β-human chorionic gonadotropin, alpha fetoprotein (AFP), exogenous lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate enzyme-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGF1 receptor, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, A1 domain of tenascin-C (TnC A1), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF17), TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), FCRL5 (UNIPROT Q68SN8), or any combination thereof.

10. The nucleic acid construct according to any one of claims 1 to 9, wherein The nucleic acid construct has one or more characteristics selected from the following: (1) The length of the first homology arm is 100-3000 bp; for example, 100-3000 bp, 100-2000 bp, 100-1000 bp, 100-900 bp, 100-800 bp, 100-700 bp, 100-600 bp, 100-500 bp, 100-400 bp, 100-300 bp, 100-200 bp; (2) the length of the second homology arm is 100-3000 bp; for example, 100-3000 bp, 100-2000 bp, 100-1000 bp, 100-900 bp, 100-800 bp, 100-700 bp, 100-600 bp, 100-500 bp, 100-400 bp, 100-300 bp, 100-200 bp; (3) The length of the STAR is 100-6000 bp; for example, 100-6000 bp, 300-5000 bp, 500-4000 bp, 800-3000 bp, 1000-3000 bp, 1000-2000 bp; (4) The length of the nucleic acid construct is 100-10000 bp; for example, 100-10000 bp, 500-9000 bp, 1000-8000 bp, 2000-7000 bp, 3000-6000 bp, 4000-5000 bp; (5) The length of the crRNA is 5-50 bp; for example, 5-50 bp, 8-40 bp, 10-30 bp, 15-25 bp.

11. The nucleic acid construct according to any one of claims 1 to 10, wherein The nucleic acid construct has one or more characteristics selected from the following: (1) The Cas12a protein has the sequence shown in SEQ ID NO: 1; (2) the PAM has a sequence shown in any one of SEQ ID NOs: 9-15; (3) The crRNA has a sequence shown in any one of SEQ ID NOs: 2-8; (4) The STAR has the sequence shown in SEQ ID NO:

37.

12. The nucleic acid construct according to any one of claims 1 to 11, wherein The nucleic acid construct has one or more characteristics selected from the following: (1) The EF1a has the sequence shown in SEQ ID NO:40; (2) the P2A has the sequence shown in SEQ ID NO:39; (3) the second homology arm has a sequence shown in any one of SEQ ID NOs: 23-29; (4) The first homology arm has a sequence shown in any one of SEQ ID NOs: 30-36.

13. A vector comprising the nucleic acid construct according to any one of claims 1 to 12; preferably, the vector is a cloning vector or an expression vector.

14. A host cell comprising the nucleic acid construct according to any one of claims 1 to 12 or the vector according to claim 13; Preferably, the cell is a mammalian cell.

15. A kit comprising at least one (e.g., one, two, or three) of the following (1) to (3): (1) The nucleic acid construct according to any one of claims 1 to 12; (2) The vector according to claim 13; (3) The host cell according to claim 14.

16. The kit of claim 15, further comprising any one or more (e.g., 1, 2, 3, 4, 5) of the following (4) to (8): (4) Cas12a protein; (5) crRNA capable of binding to Cas12a protein; (6) Complex formed by Cas12a protein and crRNA; (7) immune cells (e.g., T cells, macrophages, DCs, NK cells); (8) donor vector; (9) one or more pharmaceutically active agents (e.g., pharmaceutically active agents that enhance cell survival); Preferably, the donor vector is used to amplify the nucleic acid construct; preferably, the nucleic acid construct is amplified by constructing the nucleic acid construct into the donor vector; Optionally, the kit further comprises: a polymerase (e.g., DNA polymerase), an RNase inhibitor, a reagent for extracting and / or separating T cells, a reagent for activating T cells (e.g., anti-CD3 antibody, CD28 antibody), a reagent for electroporation, or any combination thereof; Preferably, the Cas12a protein is as defined in any one of claims 1 to 12; Preferably, the crRNA is as defined in any one of claims 1-12.

17. A method for preparing engineered T cells, the method comprising: (1) Provide T cells; (2) providing the nucleic acid construct according to any one of claims 1 to 12; (3) providing a Cas12a protein and a crRNA capable of binding to the Cas12a protein; (4) contacting the nucleic acid construct, Cas12a protein, crRNA, and T cells under conditions that allow transformation; Wherein, steps (1), (2) and (3) are performed in any order (for example, performed sequentially in any order, and / or, performed simultaneously); Optionally, the Cas12a protein and crRNA in step (3) exist in the form of a complex, or each exists separately; Preferably, the Cas12a protein is as defined in any one of claims 1 to 12; Preferably, the crRNA is as defined in any one of claims 1-12.

18. The method of claim 17, wherein: Step (2) of the method is achieved by the following steps: (a) selecting at least one target sequence (e.g., 1, 2, 3, 4, 5) in the T cell; wherein the target sequence comprises a sequence complementary to the crRNA sequence; (b) designing the crRNA sequence, the first homology arm sequence and the second homology arm sequence according to the selected target site; (c) selecting at least one STAR to construct the nucleic acid construct according to any one of claims 1 to 12; Optionally, after step (c), amplifying the nucleic acid construct; Preferably, the nucleic acid construct according to any one of claims 1 to 12 is constructed in a donor vector to amplify the nucleic acid construct; Preferably, the target sequence is adjacent to the PAM sequence recognized by the Cas12a protein; Preferably, the target sequence is located at the 3' end of the PAM sequence.

19. An engineered T cell prepared by the method of claim 17 or 18.

20. The engineered T cell of claim 19, which expresses the STAR; Preferably, the genome of the engineered T cell comprises a nucleotide sequence encoding the STAR; Preferably, the engineered T cells are chimeric antigen receptor T cells (CAR-T); Preferably, the engineered T cells are T cell receptor gene-modified T cells (TCR-T).

21. An immune cell composition comprising the engineered T cells according to claim 19 or 20.

22. A method for modifying a target sequence, comprising: Contacting the nucleic acid construct of any one of claims 1 to 12 with the target sequence, or delivering the nucleic acid construct of any one of claims 1 to 12 into a cell comprising the target sequence; Preferably, the target sequence is present in an immune cell (e.g., a T cell); Preferably, the method results in target sequence breaks, such as DNA double-strand breaks or RNA single-strand breaks; Preferably, the method further comprises inserting a STAR into the break.

23. Use of the nucleic acid construct of any one of claims 1 to 12, or the vector of claim 13, or the host cell of claim 14, or the kit of claim 15 or 16 for nucleic acid editing (e.g., gene or genome editing); For example, the gene or genome editing includes modifying a gene, knocking out a gene, altering the expression of a gene product, repairing a mutation, and / or inserting a polynucleotide.

24. Use of the nucleic acid construct according to any one of claims 1 to 12, or the vector according to claim 13, or the host cell according to claim 14, or the kit according to claim 15 or 16 for preparing engineered T cells.

25. Use of the nucleic acid construct of any one of claims 1 to 12, or the vector of claim 13, or the host cell of claim 14, or the kit of claim 15 or 16, or the engineered T cell of claim 19 or 20, or the immune cell composition of claim 21 for preparing a medicament for preventing and / or treating a disease (e.g., tumor, autoimmune disease, organ fibrosis) in a subject; Preferably, the nucleic acid construct specifically binds to an antigen expressed by the tumor; Preferably, the engineered T cells are derived from the subject; Preferably, the engineered T cells are derived from a donor other than the subject; Preferably, the tumor is selected from a solid tumor or a blood tumor; Preferably, the subject is a mammal, such as a human.

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