Non-electroporation method for preparing genetically modified cells

Abstract

The present invention relates to the field of biomedicine. Specifically, the present invention relates to a non-electroporation method for preparing genetically modified cells. The method is particularly suitable for preparing therapeutic immune cells, such as therapeutic T cells.

Description

Non-electroporation methods for preparing genetically modified cells

[0001] This application claims priority to Chinese patent application No. 202510125111.3, filed on January 26, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedicine. Specifically, it relates to a non-electroporation method for preparing genetically modified cells. This method is particularly suitable for preparing therapeutic immune cells, such as therapeutic T cells.

[0003] Background of the Invention

[0004] Chimeric antigen receptor T-cell (CAR-T) therapy has shown significant potential in cancer treatment. Currently, the preparation of CAR-T cells mainly relies on lentiviral vectors for gene transduction, but this technology carries the risk of random insertion of exogenous genes into the host genome, potentially leading to genotoxicity and carcinogenicity. Therefore, developing safe preparation technologies that enable targeted CAR gene insertion has become an important research direction in the field.

[0005] The CRISPR / Cas system (such as the commonly used Cas9 and Cas12a proteins) is a highly efficient gene editing tool that enables site-specific integration. Its mechanism of action is as follows: the ribonucleoprotein (RNP) complex formed by the guide RNA (gRNA) and the Cas protein can target and cleave specific sites in the genome, causing DNA double-strand breaks (DSBs). The cell then integrates exogenous donor DNA at the break site through mechanisms such as homology-directed repair (HDR), thereby achieving precise knock-in of the target gene.

[0006] Currently, the mainstream strategies for targeted CAR gene insertion based on the CRISPR / Cas system are typically "electroporation of RNP combined with adeno-associated virus (AAV) infection" or "electroporation of RNP combined with DNA donor." While these methods can achieve targeted editing, they generally suffer from the following significant drawbacks: 1) cumbersome procedures relying on expensive electroporation equipment; 2) severe damage to cell viability, proliferation capacity, and function during electroporation, leading to decreased cell survival and editing efficiency; 3) high production costs of AAV vectors and limitations in packaging size. Therefore, developing a simple, cost-effective (e.g., reducing AAV dosage) and cell-friendly non-electroporation gene editing technology is urgently needed and of great significance for promoting the industrialization of cell therapy products. The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and provide a simple, cost-controllable, and cell-viability-maximally-preserving non-electroporation method for preparing genetically modified therapeutic immune cells.

[0007] Invention Summary

[0008] Through in-depth research and creative work, the inventors have deeply integrated CRISPR / Cas gene editing technology with AAV vectors, developing a CRISPR / Cas gene editing method that utilizes the cell-penetrating peptide CPP to deliver AAV vector viruses. This method employs a non-electroporation process and subsequently incorporates the non-homologous end joining (NHEJ) inhibitor AZD7684. Gene editing efficiency can be improved by enhancing the delivery efficiency of the CRISPR system. This invention enables the one-step or step-by-step knock-in of large fragments and the simultaneous knockout of multiple genes via non-electroporation.

[0009] In addition to the construction of CAR T cells, this invention can also be used for the construction of TCR T cells and STAR T cells, as well as for the targeted knock-in and knock-out editing of cells such as natural killer cells, dendritic cells, macrophages, hematopoietic stem cells, and nerve cells.

[0010] Brief description of the attached diagram

[0011] Figure 1. TRAC-MND-STAR vector diagram.

[0012] Figure 2. Comparison of gene editing effects of Cas9 and Cas12a with and without AZD7648 in the presence of CPP.

[0013] Figure 3. Comparison of the preparation of general-purpose STAR-T by Cas12a and Cas9 using non-electro-spinning or electro-spinning methods.

[0014] Figure 4. The effect of different cell densities and different start times on editing results.

[0015] Figure 5. Knockout efficiency of general-purpose CD19-STAR and general-purpose BCMA-LILRB4-STAR.

[0016] Figure 6. In vitro killing of NALM6-LUC target cells by universal CD19-STAR.

[0017] Figure 7. In vitro killing of NCI-H929 target cells by the universal BCMA-LILRB4-STAR.

[0018] Figure 8. Results of multi-gene editing experiments.

[0019] Invention Details

[0020] definition

[0021] Unless otherwise indicated or defined, all terms used herein have their ordinary meaning as will be understood by those skilled in the art. References include, for example, standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), Volumes 1–3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York (1990); and Roitt et al., “Immunology” (2nd edition), Gower Medical Publishing, London, New York (1989), and general prior art cited herein; furthermore, unless otherwise stated, all methods, procedures, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as will be understood by those skilled in the art. Also refer to, for example, standard manuals, the aforementioned general prior art, and other references cited therein.

[0022] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0023] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, when describing a specific polypeptide amino acid sequence in this specification and claims, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still included, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0024] The terms “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” or “nucleic acid fragment” are used interchangeably and refer to single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides are designated by their single-letter names as follows: “A” for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), “C” for cytidine or deoxycytidine, “G” for guanosine or deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” for purine (A or G), “Y” for pyrimidine (C or T), “K” for G or T, “H” for A, C, or T, “I” for inosine, and “N” for any nucleotide. Although nucleotide sequences may be represented as DNA sequences (containing T) herein, when referring to RNA, those skilled in the art can readily determine the corresponding RNA sequence (i.e., replacing T with U).

[0025] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0026] A polypeptide or nucleic acid molecule is considered "isolated" when it has been separated from at least one other component (e.g., another protein / peptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or trace component) that is normally associated with it in that source or medium (culture medium), compared to its natural biological source and / or the reaction medium or culture medium from which the molecule is obtained. Specifically, a polypeptide or nucleic acid molecule is considered "isolated" when it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, and up to 1000-fold or more. "Isolated" polypeptide or nucleic acid molecules are preferably substantially homogeneous, as determined by suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).

[0027] As used in this article, “exogenous” means a protein or nucleic acid sequence that is derived from a foreign species, or, if derived from the same species, a protein or nucleic acid sequence whose composition and / or location have been significantly altered from its natural form through deliberate human intervention.

[0028] As used in this article, the synthetic T-cell receptor antigen receptor (STAR) refers to a modified TCR in which the variable region sequence of the TCR is replaced with an antibody variable region sequence or other receptor sequences, while the constant region sequence of the TCR can also be modified.

[0029] As used herein, an "antigen-binding region" (e.g., an antigen-binding region in STAR) means that it can specifically bind to a target antigen, either alone or in combination with another antigen-binding region. The antigen-binding region can be derived from an antibody that specifically binds to the target antigen, including any commercially available antibody. An antigen-binding region can also be derived from a receptor that binds to a specific target protein.

[0030] As used herein, “antibody” refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetic (e.g., recombinant), including any fragment that retains the binding specificity of the full-length immunoglobulin molecule, containing at least a portion of the variable region of the immunoglobulin molecule. Therefore, antibodies include any protein having a binding domain homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies include antibody fragments. As used herein, the term antibody includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, camel antibodies, single-domain antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0031] As used herein, a variable domain or variable region is a specific Ig domain of the antibody heavy or light chain, containing a variable amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has a variable region VL (also denoted as V). L ) and VH (or also represented as V) L Variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region contains a CDR and a frame region (FR), with the CDR being part of the antigen-binding site.

[0032] As used herein, “hypervariant region,” “HV,” “complementarity-determining region,” and “CDR” and “antibody CDR” are interchangeably used to refer to one of the multiple portions within each variable region that together form the antigen-binding site of an antibody. Each variable region contains three CDRs, named CDR1, CDR2, and CDR3. For example, for a conventional four-chain antibody, the light chain variable region contains three CDRs, named VL CDR1, VL CDR2, and VL CDR3 (or LCDR1, LCDR2, and LCDR3); the heavy chain variable region contains three CDRs, named VH CDR1, VH CDR2, and VH CDR3 (or HCDR1, HCDR2, and HCDR3). For camel antibodies or single-domain antibodies, since they have only one variable region, they contain only three CDRs, named CDR1, CDR2, and CDR3.

[0033] As used herein, "amino acid number reference SEQ ID NO:x" (SEQ ID NO:x being a specific sequence listed herein) refers to the position number of the described specific amino acid being the position number of the corresponding amino acid in SEQ ID NO:x. The correspondence between amino acids in different sequences can be determined using sequence alignment methods known in the art. For example, amino acid correspondence can be determined using the EMBL-EBI online alignment tool (https: / / www.ebi.ac.uk / Tools / psa / ), where two sequences can be aligned using the Needleman-Wunsch algorithm with default parameters. For example, if the alanine at position 46 from the N-terminus of a polypeptide is aligned with the 48th amino acid in SEQ ID NO:x in sequence alignment, then the alanine in that polypeptide can also be described herein as "the alanine at position 48 of the polypeptide, the position of which is referenced to SEQ ID NO:x".

[0034] The proteins / peptides mentioned in this invention may contain a signal peptide (or guide sequence) at their N-terminus. Those skilled in the art will understand that in cells, the signal peptide sequence can guide the protein / peptide to a specific location on the cell membrane, and it may itself be cleaved and not included in the final product. Exemplary signal peptides include, but are not limited to, IgE signal peptide, GM-CSF signal peptide, bovine prolactin pre-signal peptide, and natural signal peptides of the mentioned proteins / peptides such as IL-15Ra signal peptide, IL-15 signal peptide, etc. These signal peptide sequences are known in the art or can be readily identified by those skilled in the art based on existing knowledge in the art.

[0035] The "expression vector" of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or a translatable RNA (such as mRNA). In some preferred embodiments, the expression vector is a viral vector, such as an adenovirus-associated virus (AAV vector) or a lentiviral vector.

[0036] As used herein, the term "operably linked" refers to the linking of an expression regulatory element (e.g., but not limited to, promoter sequences, transcription termination sequences, etc.) to a nucleic acid sequence (e.g., coding sequences or open reading frames) such that transcription of the nucleotide sequence is controlled and regulated by the transcription regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art. The terms "regulatory sequence" and "regulatory element" are used interchangeably, referring to a nucleotide sequence located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding sequence and influencing transcription, RNA processing, or stability or translation of the relevant coding sequence. An expression regulatory element refers to a nucleotide sequence capable of controlling transcription, RNA processing, or stability or translation of a nucleotide sequence of interest. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, enhancers, and polyadenylation recognition sequences. Suitable promoters include, but are not limited to, the PGK promoter, hEF1a promoter, HTLV promoter, and MND promoter.

[0037] As used herein, "object" refers to an organism that suffers from or is susceptible to a disease (such as cancer) that can be treated by the antibodies, cells, methods, or pharmaceutical compositions of the present invention. Non-limiting examples include humans, cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals. In a preferred embodiment, the object is a human.

[0038] The term "pharmaceutically acceptable carrier" as used herein includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0039] As used herein, "therapeutic effective amount" or "therapeutic effective dose" or "effective amount" refers to an amount of substance, compound, material, or cell that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition. For example, an "effective amount" of the cellular or pharmaceutical composition of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of damage or disability caused by disease suffering. For example, in the treatment of tumors, an "effective amount" of the antibody, cell, expression vector, or pharmaceutical composition of the present invention preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system for predicting the efficacy against human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit tumor cell growth, which can be determined in vitro by assays known to those skilled in the art.

[0040] Combinations / kits for preparing genetically modified cells via non-electroporation methods

[0041] In one aspect, the present invention provides a combination for preparing genetically modified cells by a non-electrotransfer method, comprising:

[0042] i) An expression vector containing a foreign nucleotide sequence to be inserted into the genome of the cell;

[0043] ii) Gene editing systems that target one or more endogenous genes in cells; and

[0044] iii) Cell penetration reagents and / or non-homologous end joining (NHEJ) inhibitors.

[0045] The cells described herein can be any type of cell, such as immune cells (T cells), natural killer cells, dendritic cells, macrophages, hematopoietic stem cells, or nerve cells. Preferably, the cells are immune cells such as T cells. In some preferred embodiments, the genetically modified cells are therapeutic immune cells such as therapeutic T cells.

[0046] The cell penetration reagents described herein may include, but are not limited to, Ctr, R10, Pep-1, TAT, Bac7, Penetratin, SynB1, PreS2, MAP12, NF-κB, TAT-HA2, HA2, A5K, LAH2, Vectofusion-a, Polybrene, Protamine, PEI, PLL, and NLSMATS. In some preferred embodiments, the cell penetration reagent is a cell penetration peptide (CCP), such as TAT, TAT-HA2, HA2, etc., more preferably TAT-HA2.

[0047] The non-homologous end joining (NHEJ) inhibitors described herein may include, but are not limited to, SCR7, NU7441 (KU-57788), L189, Brefeldin A (BFA), L755507, RS-1, and AZD7648. In some preferred embodiments, the NHEJ inhibitor is AZD7648.

[0048] The structure of AZD7648 is shown in Equation I below.

[0049] In this invention, the genetic modification may refer to the knockout / knockdown of endogenous genes and / or the knock-in of exogenous nucleotide sequences.

[0050] In some embodiments, the gene editing system may be a CRISPR gene editing system, a TALEN gene editing system, or a ZFN gene editing system, etc. Preferably, the gene editing system is a CRISPR gene editing system. Preferably, the gene editing system can create double-strand breaks at or near a target sequence within the endogenous gene in the cell, thereby knocking out or knocking down the endogenous gene. Double-strand breaks within a specific endogenous gene can also promote the site-specific insertion (knock-in) of exogenous nucleotide sequences at that location.

[0051] In some embodiments, the one or more endogenous genes contain the locus where the exogenous nucleotide sequence is to be inserted.

[0052] In some embodiments, the gene editing system targets the endogenous TRAC gene. In some embodiments, the gene editing system targets the endogenous TRAC, HLA-A, and HLA-B genes. In some embodiments, the gene editing system targets the endogenous TRAC, HLA-A, HLA-B, and CIITA genes.

[0053] In some implementations, the gene editing system causes partial or complete inactivation (knockout or knockdown) of the targeted endogenous gene.

[0054] The CRISPR gene editing system can be the Cas12 system, preferably the Cas12a system.

[0055] In some implementations, the CRISPR gene editing system comprises:

[0056] i) CRISPR nucleases and / or expression constructs containing the encoding nucleotide sequences of CRISPR nucleases; and

[0057] ii) Guide RNA and / or an expression construct containing a coding nucleotide sequence of guide RNA, wherein the guide RNA is capable of targeting a target sequence in a cell.

[0058] In some preferred embodiments, the CRISPR gene editing system comprises:

[0059] i) CRISPR nuclease; and

[0060] ii) Guide RNA, wherein the guide RNA is capable of targeting the target sequence of the endogenous gene in the cell.

[0061] As used herein, the term "CRISPR nuclease" generally refers to a nuclease present in the naturally occurring CRISPR system, as well as its modified forms, variants, or catalytically active fragments thereof. CRISPR nucleases can recognize, bind to, and / or cleave target nucleic acid structures by interacting with guide RNA. The term encompasses any CRISPR-based nuclease or its functional variants capable of intracellular gene editing. In some embodiments, the functional variants retain their double-strand cleavage activity, i.e., the ability to form double-strand breaks (DSBs) in the target sequence.

[0062] The CRISPR nuclease used in this invention may be selected from, for example, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, GSU0054, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx11, Csx16, CsaX, Csx3, Csx1, and Csx15. Csf1, Csf2, Csf3, Csf4, C2c3, C2c8, C2c10, Cas12a (also known as Cpf1), Cas12a2, Cas12b (also known as C2c1), Cas12c, Cas12c1, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, Cas12f, Cas12k, Cas12m, Cas12n, Cas13a (also known as C2c2), Cas13b, Cas13c, Cas13d, Cas13m.3, Cas13m.6, Cas14, Casφ, Casλ, TnpB proteins, or functional variants of these nucleases. In some preferred embodiments, the CRISPR nuclease is a Cas12a nuclease or a variant thereof. Gene editing systems based on Cas12a nucleases are also called Cas12a gene editing systems, and so on for other gene editing systems.

[0063] As used herein, "gRNA" and "guide RNA" are used interchangeably and refer to RNA molecules capable of forming a complex with a CRISPR nuclease and, due to complementarity with the target sequence, directing the complex to the target sequence. For example, for the Cas9 nuclease, its gRNA typically consists of partially complementary crRNA and tracrRNA molecules forming a complex, wherein the crRNA contains a sequence sufficiently identical to the target sequence to hybridize with its complementary sequence and guide the CRISPR complex (Cas9 + crRNA + tracrRNA) to specifically bind to the target sequence. However, it is known in the art that single guide RNAs (sgRNAs) can be designed that simultaneously contain the characteristics of both crRNA and tracrRNA. In the Cas12a (also known as Cpf1) nuclease, the gRNA typically consists only of mature crRNA molecules. Designing suitable gRNAs based on the CRISPR nuclease used and the target sequence to be edited is within the capabilities of those skilled in the art.

[0064] As used herein, a “target sequence” is a sequence that is complementary to or identical (depending on the specific CRISPR nuclease) to a guide sequence of approximately 20 nucleotides contained in the guide RNA. The guide RNA targets the target sequence by base pairing with the target sequence or its complementary strand. In the Cas12a guide RNA, the target sequence is located at the 5' end of the scaffold sequence. In some embodiments, the sgRNA for Cas12a comprises the scaffold sequence shown in SEQ ID NO:32.

[0065] In some embodiments, the guide RNA targets or includes the target sequence shown in SEQ ID NO:33 of the endogenous TRAC gene.

[0066] In some embodiments, the guide RNA targets or includes the target sequence shown in SEQ ID NO:34 of the endogenous HLA-A gene.

[0067] In some embodiments, the guide RNA targets or includes the target sequence described in SEQ ID NO:35 of the endogenous HLA-B gene.

[0068] In some embodiments, the guide RNA targets or includes the target sequence shown in SEQ ID NO:36 of the endogenous CIITA gene.

[0069] In some embodiments, the guide RNA contains chemical modifications. For example, the first three bases at the 3' and 5' ends of the guide RNA contain thio and 2'-O-methyl modifications.

[0070] In some specific implementations, the gene editing system includes:

[0071] i) Ca12a nuclease; and

[0072] ii) guide RNA that targets or contains the target sequence shown in SEQ ID NO:33, guide RNA that targets or contains the target sequence shown in SEQ ID NO:34, guide RNA that targets or contains the target sequence shown in SEQ ID NO:35, and / or, guide RNA that targets or contains the target sequence shown in SEQ ID NO:36.

[0073] In some specific implementations, the gene editing system includes:

[0074] i) Ca12a nuclease; and

[0075] ii) Guide RNA that targets or contains the target sequence shown in SEQ ID NO:33.

[0076] In some specific implementations, the gene editing system includes:

[0077] i) Ca12a nuclease; and

[0078] ii) Guide RNA that targets or contains the target sequence shown in SEQ ID NO:33, and guide RNA that targets or contains the target sequence shown in SEQ ID NO:34.

[0079] In some specific implementations, the gene editing system includes:

[0080] i) Ca12a nuclease; and

[0081] ii) guide RNA that targets or contains the target sequence shown in SEQ ID NO:33, guide RNA that targets or contains the target sequence shown in SEQ ID NO:34, and guide RNA that targets or contains the target sequence shown in SEQ ID NO:35.

[0082] In some specific implementations, the gene editing system includes:

[0083] i) Ca12a nuclease; and

[0084] ii) guide RNA that targets or contains the target sequence shown in SEQ ID NO:33, guide RNA that targets or contains the target sequence shown in SEQ ID NO:34, guide RNA that targets or contains the target sequence shown in SEQ ID NO:35, and guide RNA that targets or contains the target sequence shown in SEQ ID NO:36.

[0085] In some embodiments, the exogenous nucleotide sequence to be inserted into the cell genome may include a 5' homologous arm (5'HA) at its 5' end and a 3' homologous arm (3'HA) at its 3' end, wherein the 5'HA and 3'HA contain nucleotide sequences homologous to sequences in the locus to which the exogenous nucleotide sequence is to be inserted. Through the 5'HA and 3'HA, the exogenous nucleotide sequence can be site-directedly integrated into the endogenous locus via homologous recombination. The length of the homologous arm can be 100 nt to 1000 nt, for example, 300 nt to 500 nt.

[0086] In some embodiments, a foreign nucleotide sequence is to be inserted into an endogenous TRAC gene. In some embodiments, the foreign nucleotide sequence includes a 5' homologous arm (5'HA) as shown in SEQ ID NO:18 at its 5' end and a 3' homologous arm (3'HA) as shown in SEQ ID NO:19 at its 3' end. Thus, the foreign nucleotide sequence can be inserted into a TRAC gene locus in a cell, for example, at the location defined by SEQ ID NO:18 and SEQ ID NO:19 within an endogenous TRAC gene locus.

[0087] In some embodiments, the expression vector is an AAV vector. Methods for preparing AAV vectors (viral particles) are known in the art.

[0088] In some embodiments, the exogenous nucleotide sequence is an expression cassette for CAR, TCR, or STAR. In some preferred embodiments, the exogenous nucleotide sequence is an expression cassette for CAR or STAR.

[0089] In some embodiments, the expression cassette of the CAR or STAR contains the coding nucleotide sequence of the CAR or STAR and an expression regulatory element, such as a promoter, operatively linked thereto.

[0090] Examples of promoters include the MND promoter. An exemplary nucleotide sequence of the MND promoter is shown in SEQ ID NO:20.

[0091] In some embodiments, the STAR expression cassette includes a coding nucleotide sequence of a fusion protein of the α chain and β chain of the STAR linked by a self-cleaving peptide, preferably the coding nucleotide sequence being operatively linked to an MND promoter.

[0092] As used herein, "self-cleaving peptide" refers to a peptide capable of self-cleaving within a cell. For example, the self-cleaving peptide may contain a protease recognition site, thereby being recognized and specifically cleaved by intracellular proteases. Alternatively, the self-cleaving peptide may be a 2A peptide. 2A peptides are a class of short peptides derived from viruses whose self-cleavage occurs during translation. When two different target proteins are expressed in the same reading frame using a 2A peptide, the two target proteins are generated in an almost 1:1 ratio. Commonly used 2A peptides include P2A from porcine techovirus-1, T2A from the β-tetrasomatic moth virus (Thosea asigna virus), E2A from equine rhinitis A virus, and F2A from foot-and-mouth disease virus. P2A has the highest cleavage efficiency and is therefore preferred. Various functional variants of these 2A peptides are also known in the art and can also be used in this invention. 2A peptides can also be combined with a Furin recognition sequence to remove additional introduced amino acid sequences.

[0093] In some embodiments, the self-cleaving peptide is a 2A peptide, such as a P2A peptide. In some embodiments, the self-cleaving peptide is a Furin-2A peptide, such as the Furin-P2A peptide shown in SEQ ID NO:17.

[0094] In some embodiments, the different portions of the fusion polypeptide can be arranged in different ways, as long as they are separated by self-cleaving peptides. For example, in some embodiments, the fusion polypeptide may include the β chain, a self-cleaving peptide such as Furin-P2A, and the α chain from the N-terminus to the C-terminus.

[0095] In some embodiments, the STAR comprises an α chain and a β chain, the α chain comprising a first constant region, the β chain comprising a second constant region, and wherein the α chain and / or the β chain further comprises an antigen-binding region that specifically binds to the target antigen.

[0096] In some embodiments, the first constant region is a natural TCRα chain constant region, such as a natural human TCRα chain constant region or a natural mouse TCRα chain constant region. An exemplary natural human TCRα chain constant region comprises the amino acid sequence shown in SEQ ID NO:1. An exemplary natural mouse TCRα chain constant region comprises the amino acid sequence shown in SEQ ID NO:2.

[0097] In some implementations, the first constant region is a modified TCRα chain constant region.

[0098] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein the amino acid at position 48, for example threonine T, is mutated to cysteine ​​C, relative to the wild-type mouse TCRα chain constant region.

[0099] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 112, such as serine S, is replaced with leucine L; the amino acid at position 114, such as methionine M, is replaced with isoleucine I; and the amino acid at position 115, such as glycine G, is replaced with valine V.

[0100] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, which, relative to the wild-type mouse TCRα chain constant region, has its 6th amino acid, such as E, replaced by D, its 13th K replaced by R, and its 15th-18th amino acids deleted.

[0101] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, which, relative to the wild-type mouse TCRα chain constant region, has the following modifications: the amino acid at position 48, such as threonine (T), is mutated to cysteine ​​(C); the amino acid at position 112, such as serine (S), is mutated to leucine (L); the amino acid at position 114, such as methionine (M), is mutated to isoleucine (I); and the amino acid at position 115, such as glycine (G), is mutated to valine (V).

[0102] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, which, relative to the wild-type mouse TCRα chain constant region, has the following modifications: amino acid at position 6, such as E, is replaced by D; amino acid at position 13, K, is replaced by R; amino acids at positions 15-18 are deleted; amino acid at position 48, such as threonine (T), is mutated to cysteine ​​(C); amino acid at position 112, such as serine (S), is replaced by leucine (L); amino acid at position 114, such as methionine (M), is replaced by isoleucine (I); and amino acid at position 115, such as glycine (G), is replaced by valine (V).

[0103] In some embodiments, the TCRα chain constant region is a non-intracellular region relative to the wild-type TCRα chain constant region, for example, the non-intracellular region of the constant region is missing amino acids 136-137.

[0104] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, which, relative to the wild-type mouse TCRα chain constant region, lacks the intracellular region of the constant region, for example, the deletion of amino acids 136-137.

[0105] In some specific embodiments, the modified TCRα chain constant region contains an amino acid sequence shown in one of SEQ ID NO:3-7.

[0106] In some embodiments, the second constant region is a natural TCRβ chain constant region, such as a natural human TCRβ chain constant region or a natural mouse TCRβ chain constant region. An exemplary natural human TCRβ chain constant region comprises the amino acid sequence shown in SEQ ID NO:8. An exemplary natural mouse TCRβ chain constant region comprises the amino acid sequence shown in SEQ ID NO:9.

[0107] In some implementations, the second constant region is a modified TCRβ chain constant region.

[0108] In some embodiments, the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, wherein the amino acid at position 56, for example serine S, is mutated to cysteine ​​C, relative to the wild-type mouse TCRβ chain constant region.

[0109] In some embodiments, the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, which, relative to the wild-type mouse TCRβ chain constant region, has the following modifications: the amino acid at position 3, such as R, is replaced by K; the amino acid at position 6, such as T, is replaced by F; the amino acid at position 9, such as K, is replaced by E; the amino acid at position 11, such as S, is replaced by A; the amino acid at position 12, such as L, is replaced by V; and the amino acids at positions 17 and 21-25 are deleted.

[0110] In some embodiments, the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, which, relative to the wild-type mouse TCRβ chain constant region, has the following modifications: the amino acid at position 56, such as serine (S), is mutated to cysteine ​​(C); the amino acid at position 3, such as R, is replaced by K; the amino acid at position 6, such as T, is replaced by F; the amino acid at position 9, such as K, is replaced by E; the amino acid at position 11, such as S, is replaced by A; the amino acid at position 12, such as L, is replaced by V; and the amino acids at positions 17 and 21-25 are deleted.

[0111] In some embodiments, the TCRβ chain constant region is a non-intracellular region relative to the wild-type TCRβ chain constant region, for example, the intracellular region of the constant region is missing amino acids 167-172.

[0112] In some embodiments, the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, which, relative to the wild-type mouse TCRβ chain constant region, lacks the intracellular region of the constant region, for example, the deletion of amino acids 167-172.

[0113] In some specific embodiments, the modified TCRβ chain constant region contains an amino acid sequence shown in one of SEQ ID NO:10-14.

[0114] In some embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:3, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:10. In some embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:6, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:10. In some embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:3, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:13. In some preferred embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:6, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:13.

[0115] In some embodiments, the α-chain and / or β-chain, preferably the α-chain and β-chain, have at least one exogenous intracellular functional domain attached to their C-terminus. In some embodiments, the exogenous intracellular functional domain is connected directly or via a linker to the α-chain and / or β-chain, preferably to the C-terminus of the constant region of the α-chain and β-chain. In some embodiments, the exogenous intracellular functional domain is connected via a linker to the α-chain and / or β-chain missing from the intracellular region, preferably to the C-terminus of the constant region of the α-chain and β-chain. In some embodiments, the linker is a (G4S)n linker, where n represents an integer from 1 to 10, preferably n is 3.

[0116] In some embodiments, the first constant region is a modified TCRα chain constant region derived from a mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 48, for example threonine (T), is mutated to cysteine ​​(C); the amino acid at position 112, for example serine (S), is mutated to leucine (L); the amino acid at position 114, for example methionine (M), is mutated to isoleucine (I); and the amino acid at position 115, for example glycine (G), is mutated to valine (V). The modified TCRα chain constant region, relative to the wild-type mouse TCRα chain constant region, lacks an intracellular region of the constant region, for example, the amino acids at positions 136-137. The α chain includes an intracellular domain of OX40 connected to the C-terminus of the constant region (e.g., via a linker, such as a (G4S)n linker, where n represents an integer from 1 to 10, preferably n is 3).

[0117] The second constant region is a modified TCRβ chain constant region derived from the mouse TCRβ chain constant region, wherein, relative to the wild-type mouse TCRβ chain constant region, the amino acid at position 56, for example serine S, is mutated to cysteine ​​C, and the modified TCRβ chain constant region, relative to the wild-type mouse TCRβ chain constant region, lacks the intracellular region of the constant region, for example, the amino acids at positions 167-172 are missing, and the β chain contains an intracellular domain of OX40 connected to the C-terminus of the constant region (e.g., via a linker, such as a (G4S)n linker, where n represents an integer from 1 to 10, preferably n is 3).

[0118] As used in this article, "exogenous intracellular functional domain" can be the intracellular domain of co-stimulatory molecules such as CD40, OX40, ICOS, CD28, 4-1BB, CD27, and CD137; it can also be the intracellular domain of co-inhibitory molecules, such as TIM3, PD1, CTLA4, and LAG3; it can also be the intracellular domain of cytokine receptors such as interleukin receptors (e.g., IL-2β, IL-7α, or IL-21 receptors), interferon receptors, tumor necrosis factor superfamily receptors, colony-stimulating factor receptors, chemokine receptors, growth factor receptors, or other membrane proteins; or the domain of intracellular proteins such as NIK.

[0119] In some preferred embodiments, the exogenous intracellular functional domain is an intracellular domain of a co-stimulatory molecule, preferably an intracellular domain of OX40. In some embodiments, the intracellular domain of OX40 comprises the amino acid sequence of SEQ ID NO:15.

[0120] In some implementations, the antigen-binding region is fused directly or indirectly (e.g., via a linker) to the N-terminus of the first and / or second constant region.

[0121] In some embodiments, the α chain includes a first antigen-binding region and a first constant region, and the β chain includes a second constant region. In this case, the β chain does not contain an antigen-binding region.

[0122] In some embodiments, the α chain includes a first constant region, and the β chain includes a second antigen-binding region and a second constant region. In this case, the α chain does not contain an antigen-binding region.

[0123] In some embodiments, the α chain includes a first antigen-binding region and a first constant region, and the β chain includes a second antigen-binding region and a second constant region.

[0124] In some embodiments, the first antigen-binding region and the second antigen-binding region each bind specifically to the target antigen independently or in combination. Those skilled in the art will understand that when the antigen-binding region contains a single-domain antibody or a single-chain antibody, it can bind to the target antigen alone. However, if the first antigen-binding region contains a conventional antibody heavy chain variable region and the second antigen-binding region contains a conventional antibody light chain variable region, then the first and second antigen-binding regions bind to the target antigen in combination, and vice versa.

[0125] The target antigen described in this invention can be a disease-associated antigen, preferably a cancer-associated antigen, such as those selected from the following disease-associated antigens: GPC3 (phosphatidylinositol proteoglycan 3), BCMA, mesothelin, LILRB4, CD16, CD64, CD78, CD96, CLL1, CD7, CD70, CD38, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), Claudin 18.2, and carcinoembryonic antigen (CEA). Epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD276, CD19, CD22, CD20, CD30, CD40, disialotetrahexosylganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alpha fetal globulin (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostaglandin, prostaglandin-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, liver glycoside B2, CD22, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, major histocompatibility complex (MHC) molecule presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor matrix antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, A1 domain (TnC) of tendin-C 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, endothelial factors, major histocompatibility complex (MHC) molecules, TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5.

[0126] In some embodiments, the target antigen is an antigen derived from a pathogen or a surface antigen of cells infected by a pathogen, such as RSVF (prevention of respiratory syncytial virus), PA (inhalation anthrax), CD4 (HIV infection), etc.

[0127] In some implementations, the target antigen is a molecule produced or secreted by disease-causing cells, such as CD3 (involved in transplant rejection), CD25 (involved in acute kidney transplant rejection), C5 (involved in paroxysmal nocturnal hemoglobinuria), IL-1β (involved in cold pyridine-associated periodic syndrome), RANKL (involved in cancer-related bone injury), von Willebrand factor (involved in adult acquired thrombotic thrombocytopenic purpura), plasma kallikrein (involved in angioedema), calcitonin gene-related peptide receptor (involved in adult migraine), FGF23 (involved in X-linked hypophosphatemia), etc.

[0128] The antigen-binding region may be derived from one or more known antibodies, including any commercially available antibody such as FMC63, rituximab, alemtuzumab, epratuzumab, trastuzumab, bivatuzumab, cetuximab, labetuzumab, palivizumab, sevirumab, and tuvirumab. Baliximab, daclizumab, infliximab, omalizumab, efalizumab, keliximab, siplizumab, natalizumab, clenoliximab, pemtumomab, edrecolomab, cantuzumab, etc.

[0129] In some embodiments, the first antigen-binding region comprises a heavy chain variable region of an antibody that specifically binds to the target antigen, and the second antigen-binding region comprises a light chain variable region of the antibody; or, the first antigen-binding region comprises a light chain variable region of an antibody that specifically binds to the target antigen, and the second antigen-binding region comprises a heavy chain variable region of the antibody.

[0130] In some embodiments, the first antigen-binding region contains a single-chain antibody (e.g., scFv) or a single-domain antibody that specifically binds to the target antigen; and / or the second antigen-binding region contains a single-chain antibody or a single-domain antibody that specifically binds to the target antigen.

[0131] In some embodiments, the single-chain antibody (e.g., scFv) comprises a heavy chain variable region and a light chain variable region connected by a linker, such as a flexible linker. Those skilled in the art can readily utilize existing knowledge and methods to construct single-chain antibodies from the heavy chain variable regions and light chain variable regions of conventional antibodies.

[0132] In some implementations, the first antigen-binding region and the second antigen-binding region bind the same target antigen.

[0133] In some implementations, the first antigen-binding region and the second antigen-binding region bind to different regions (e.g., different epitopes) of the same target antigen.

[0134] In some implementations, the first antigen-binding region and the second antigen-binding region bind different target antigens.

[0135] In some embodiments, the antigen-binding region includes the heavy chain variable region shown in SEQ ID NO:21 and / or the light chain variable region shown in SEQ ID NO:22, thereby the STAR targets CD19. In some embodiments, the first antigen-binding region includes the heavy chain variable region shown in SEQ ID NO:21, and the second antigen-binding region includes the light chain variable region shown in SEQ ID NO:22. In some embodiments, the first antigen-binding region includes the light chain variable region shown in SEQ ID NO:22, and the second antigen-binding region includes the heavy chain variable region shown in SEQ ID NO:21. In some embodiments, the first antigen-binding region includes both the heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:22.

[0136] In some embodiments, the α chain comprises the amino acid sequence shown in SEQ ID NO:30, and the β chain comprises the amino acid sequence shown in SEQ ID NO:31. (CD19-STAR)

[0137] In some embodiments, the antigen-binding region includes the heavy chain variable region shown in SEQ ID NO:29 and / or the light chain variable region shown in SEQ ID NO:28, thereby the STAR targets GPC3. In some embodiments, the first antigen-binding region includes the heavy chain variable region shown in SEQ ID NO:29, and the second antigen-binding region includes the light chain variable region shown in SEQ ID NO:28. In some embodiments, the first antigen-binding region includes the light chain variable region shown in SEQ ID NO:28, and the second antigen-binding region includes the heavy chain variable region shown in SEQ ID NO:29. In some embodiments, the first antigen-binding region includes both the heavy chain variable region shown in SEQ ID NO:29 and the light chain variable region shown in SEQ ID NO:28.

[0138] In some embodiments, the antigen-binding region comprises one or more amino acid sequences selected from SEQ ID NO:24-27.

[0139] In some embodiments, the antigen-binding region comprises an amino acid sequence selected from SEQ ID NO:24, thereby enabling the STAR to target mesothelin (MSLN).

[0140] In some embodiments, the antigen-binding region comprises an amino acid sequence selected from SEQ ID NO:25, thereby enabling the STAR to target LILRB4.

[0141] In some embodiments, the antigen-binding region comprises an amino acid sequence selected from SEQ ID NO:26, thereby enabling the STAR to target LILRB4.

[0142] In some embodiments, the antigen-binding region comprises an amino acid sequence selected from SEQ ID NO:27, thereby allowing the STAR to target BCMA.

[0143] In some embodiments, the chimeric antigen receptor (CAR) of the present invention includes at least one extracellular antigen-binding region. The antigen and antigen-binding region are as defined above.

[0144] In some embodiments, the CAR further includes a transmembrane domain, such as a CD8α transmembrane domain or a CD28 transmembrane domain, preferably a CD8α transmembrane domain.

[0145] In some embodiments, the CAR further includes a hinge region located between the extracellular antigen-binding region and the transmembrane domain, for example, the hinge region being the CD8α hinge region.

[0146] In some embodiments, the CAR further includes a signal transduction domain, such as a signal transduction domain that can be used for T cell activation, for example, a signal transduction domain selected from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d. In some preferred embodiments, the CAR includes a CD3ζ signal transduction domain.

[0147] In some embodiments, the CAR further includes one or more co-stimulatory domains, such as co-stimulatory domains selected from CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, and 4-1BBL.

[0148] In some embodiments, the CAR includes, from the N-terminus to the C-terminus, the extracellular antigen-binding region, the hinge region, the transmembrane domain, the co-stimulatory domain, and the signal transduction domain. In some embodiments, the hinge region is a CD8α hinge region, the transmembrane domain is a CD8α transmembrane domain, the signal transduction domain is a CD3ζ signal transduction domain, and the co-stimulatory domain is a 4-1BB co-stimulatory domain.

[0149] The components of the combination described in this invention, such as the expression vector, the nuclease in the gene editing system, the guide RNA, and cell penetration reagents such as CPP and NHEJ inhibitors, can exist independently or in any combination as a composition. This invention also covers kits comprising the combination.

[0150] Non-electroporation methods for preparing genetically modified cells

[0151] In another aspect, the present invention provides a non-electrotransfer method for preparing genetically modified cells, comprising:

[0152] Step 1) Provide the starting cells;

[0153] Step 2) Contact the starting cells with the combination of the present invention; and

[0154] Step 3) Harvest the cells obtained in Step 2).

[0155] In some implementations, the expression vector, gene editing system, cell penetration reagent, and NHEJ inhibitor in the combination can be contacted with the starting cells simultaneously or separately (sequentially).

[0156] The initiating cells described herein can be any type of cell, such as immune cells, T cells, natural killer cells, dendritic cells, macrophages, hematopoietic stem cells, or nerve cells. Preferably, the initiating cells are T cells.

[0157] In some embodiments, the method described in this invention is an in vitro method. In some embodiments, the starting cells are isolated or ex vivo cells.

[0158] In some embodiments, step 2) of "contacting the starting cells with the combination of the present invention" includes incubating the starting cells and the combination of the present invention in an incubation system for a period of time. In some embodiments, the method of the present invention, such as step 2), does not include the electroporation step.

[0159] In some implementations, the method is used to prepare therapeutic immune cells, such as therapeutic T cells.

[0160] In some embodiments, the therapeutic immune cells, such as therapeutic T cells, comprise chimeric antigen receptors (CARs), T cell receptors (TCRs), or synthetic T cell receptor antigen receptors (STARs), and one or more endogenous genes selected from TRAC, HLA-A, HLA-B, and CIITA in the therapeutic immune cells are partially or completely inactivated.

[0161] Therefore, in some embodiments, the starting cell is a starting immune cell. In some preferred embodiments, the starting cell is a T cell.

[0162] The initiating immune cells, such as T cells, of the present invention can be obtained from a variety of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors, using various non-limiting methods. In some embodiments, the cells can be derived from healthy donors or from patients diagnosed with cancer. In some embodiments, the cells can be part of a mixed population of cells exhibiting different phenotypic characteristics. For example, initiating immune cells, such as T cells, can be obtained by isolating peripheral blood mononuclear cells (PBMCs) and then activating and expanding them with specific antibodies.

[0163] In some embodiments, the initiating immune cells, such as T cells, described in this invention are isolated (ex vivo) immune cells, such as T cells. Therefore, the immune cells, such as T cells, obtained by this invention are isolated (ex vivo) immune cells, such as T cells.

[0164] In some embodiments of various aspects of the invention, the initiating immune cells, such as T cells, are derived from the subject's own cells. As used herein, "autologous" means that the cells, cell lines, or cell populations used to treat the subject are derived from the subject. In some embodiments, the initiating immune cells, such as T cells, are derived from allogeneic cells.

[0165] In some alternatives, the T cells are allogeneic (from different donors of the same species) to the recipient cells or the cells to be received (e.g., the cells are in the form of a therapeutic composition); in other alternatives, the T cells are autologous (the donor and recipient are the same).

[0166] The immune cells used in this application can be autologous / autogenetic (“own”) or non-autologous (“non-own”, such as allogeneic, syngeneic, or allogeneic). In this application, the term “autologous” generally refers to cells from the same subject. “Allogeneic” generally refers to cells of the same species as the comparison but genetically different. “Syngeneic” generally refers to cells from a different subject that are genetically identical to the comparison cells. “Allogeneic” generally refers to cells of a different species than the comparison cells. In some embodiments, the cells used in this application are autologous or allogeneic.

[0167] In some implementations, the method includes:

[0168] Step 1) Provide initial immune cells such as T cells;

[0169] Step 2) Contact the starting cell with a combination comprising:

[0170] i) AAV vectors containing expression cassettes of CAR, TCR, or STAR.

[0171] ii) Target one or more of the Cas12a gene editing systems selected from TRAC, HLA-A, HLA-B and CIITA;

[0172] iii) Cell penetration reagents such as TAT-HA2, and / or NEHJ inhibitors such as AZD7648; and

[0173] Step 3) Harvest the cells obtained in Step 2).

[0174] In some embodiments, the expression cassette of the CAR, TCR, or STAR includes a 5' homologous arm (5'HA) as shown in SEQ ID NO:18 at its 5' end and a 3' homologous arm (3'HA) as shown in SEQ ID NO:19 at its 3' end, thereby allowing it to be inserted into the intracellular intracellular TRAC gene at the location defined by SEQ ID NO:18 and SEQ ID NO:19.

[0175] In some implementations, step 1) includes isolating and activating the immune cells, such as T cells. Methods known in the art for isolating and activating immune cells, such as T cells, may be employed, such as those exemplified in the embodiments herein.

[0176] In some implementations, step 1) is performed on day 0, or step 2 is performed on day 1, day 2, or day 3, preferably starting on day 2.

[0177] In some preferred embodiments, step 2) includes:

[0178] 2-1) Incubate the Cas12a nuclease with one or more guide RNAs targeting TRAC, HLA-A, HLA-B, and CIITA for the first time period to form a ribonucleoprotein complex (RNP);

[0179] 2-2) Incubate the RNP with the initiating immune cells, the AAV vector, and a cell penetration reagent such as TAT-HA2 for a second time period; and

[0180] 2-3) The product obtained in 2-2) is cultured in a cell culture medium, preferably, the cell culture medium contains an NHEJ inhibitor such as AZD7648.

[0181] In some embodiments, the first time period is approximately 10-30 minutes, preferably approximately 15 minutes. In some embodiments, the second time period is approximately 15-60 minutes, preferably approximately 30 minutes.

[0182] In some preferred embodiments, step 2) includes:

[0183] 2-1) Incubate the Cas12a nuclease with one or more guide RNAs targeting TRAC, HLA-A, HLA-B, and CIITA for the first time period to form a ribonucleoprotein complex (RNP);

[0184] 2-2) Incubate the RNP with the initiating immune cells and the AAV vector for a second time period;

[0185] 2-3) Incubate the product obtained in 2-2) with a cell penetration reagent such as TAT-HA2 for the third time period; and

[0186] 2-4) The product obtained in 2-3) is cultured in a cell culture medium, preferably, the cell culture medium contains an NHEJ inhibitor such as AZD7648.

[0187] In some embodiments, the first time period is approximately 10-30 minutes, preferably approximately 15 minutes. In some embodiments, the second time period is approximately 15-60 minutes, preferably approximately 30 minutes. In some embodiments, the third time period is approximately 15-60 minutes, preferably approximately 30 minutes.

[0188] The cell culture medium used in this article can be X-VIVO medium. The medium may also be supplemented with cytokines such as IL-2, and serum such as fetal bovine serum (FBS).

[0189] In the methods described in this invention, the working concentration of Cas12a can be approximately 0.25 μM to approximately 1 μM. The working concentration of cell penetration reagents such as TAT-HA2 can be approximately 10 μM to 100 μM, preferably approximately 60 μM. The working concentration of NEHJ inhibitors such as AZD7648 can be approximately 0.1 μM to 10 μM, preferably approximately 1 μM. The amount of the AAV carrier is approximately 1 x 10⁻⁶. 4 MOI - approximately 1x10 5 MOI, preferably approximately 2x10 4 MOI.

[0190] In some implementations, step 2) may include one or more sub-steps, each of which may target a different endogenous gene or combination of endogenous genes.

[0191] In some implementations, step 2) is performed separately on day 2 and day 4.

[0192] In some implementations, step 2) includes:

[0193] 2-1) On the second day, the Cas12a nuclease is incubated with the guide RNA targeting HLA-A and HLA-B for a first time period to form a ribonucleoprotein complex (RNP). The RNP is then incubated with the initiating immune cells and a cell penetration reagent such as TAT-HA2 for a second time period, and the resulting cells are cultured in cell culture medium.

[0194] 2-2) On the 4th day, the Cas12a nuclease is incubated with the guide RNA targeting CIITA and TRAC for the first time period to form a ribonucleoprotein complex (RNP). The RNP is then incubated with the cells obtained in 2-1), the AAV vector, and a cell penetration reagent such as TAT-HA2 for the second time period. The cells are then cultured in cell culture medium.

[0195] Preferably, the cell culture medium contains an NHEJ inhibitor such as AZD7648.

[0196] In some implementations, step 2) includes: 2-1) incubating the Cas12a nuclease with guide RNA targeting CIITA, HLA-A and HLA-B for a first time period on the second day to form a ribonucleoprotein complex (RNP), incubating the RNP with the initiating immune cells and a cell penetration reagent such as TAT-HA2 for a second time period, and culturing the resulting cells in a cell culture medium;

[0197] 2-2) On the 4th day, the Cas12a nuclease is incubated with the guide RNA targeting TRAC for the first time period to form a ribonucleoprotein complex (RNP). The RNP is then incubated with the cells obtained in 2-1), the AAV vector, and a cell penetration reagent such as TAT-HA2 for the second time period. The cells are then cultured in cell culture medium.

[0198] Preferably, the cell culture medium contains an NHEJ inhibitor such as AZD7648.

[0199] Pharmaceutical Compositions and Applications

[0200] In another aspect, the present invention provides therapeutic immune cells, such as T cells, that can be obtained or acquired through the combination of the present invention or the methods of the present invention.

[0201] In another aspect, the present invention provides a pharmaceutical composition comprising the therapeutic immune cells of the present invention and a pharmaceutically acceptable carrier.

[0202] In another aspect, the present invention provides the use of the therapeutic immune cells of the present invention and / or the pharmaceutical compositions of the present invention in the preparation of a medicament for treating a disease in a subject.

[0203] In another aspect, the present invention provides a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the therapeutic immune cells of the present invention and / or the pharmaceutical composition of the present invention.

[0204] In practical applications, the dosage levels of cells and / or expression vectors in the pharmaceutical compositions of this invention may be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and route of administration, while being non-toxic to the patient. The selected dosage level depends on a variety of pharmacokinetic factors, including the activity of the specific composition of this invention applied, the route of administration, the time of administration, the excretion rate of the specific compound applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition applied, the age, sex, weight, condition, general health status, and medical history of the patient receiving treatment, and similar factors known in the medical field.

[0205] The administration of the combination, therapeutic immune cell, or pharmaceutical composition or drug according to the present invention can be carried out in any convenient manner, including by injection, infusion, implantation, or transplantation. The administration of the combination, therapeutic immune cell, or pharmaceutical composition described herein can be by intravenous, intralymphatic, intradermal, intratumoral, intramedullary, intramuscular, or intraperitoneal administration. In one embodiment, the combination, therapeutic immune cell, or pharmaceutical composition of the present invention is preferably administered by intravenous injection.

[0206] In embodiments of various aspects of the present invention, the disease depends on the target antigen targeted by the CAR, TCR, or STAR.

[0207] In embodiments of various aspects of the present invention, the disease is, for example, cancer, and examples of such cancers include, but are not limited to, myeloma, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, endometrial tumors, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, etc. Adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of the aforementioned cancers.

[0208] In various embodiments of the present invention, the disease is, for example, a pathogen infection, and examples of the pathogens include, but are not limited to, respiratory syncytial virus, anthrax, and human immunodeficiency virus.

[0209] In various embodiments of the present invention, the disease is, for example, cardiovascular disease, diabetes, neurological disease, post-transplant rejection, or some other disease.

[0210] In various embodiments of the present invention, the disease may also be an autoimmune disease, including but not limited to systemic lupus erythematosus (SLE), myositis, scleroderma, Sjögren's syndrome, autoimmune hemolytic anemia, and rheumatoid arthritis.

[0211] In embodiments of various aspects of the present invention, the disease is a phosphatidylinositol proteoglycan-3 (GPC3)-related disease, such as a disease related to abnormal GPC3 expression, or GPC3-related cancer. The cancer is, for example, liver cancer such as hepatocellular carcinoma, lung cancer such as squamous cell carcinoma (SqCC), gastric cancer, ovarian cancer, melanoma, or pediatric embryonal tumors.

[0212] In embodiments of various aspects of the present invention, the disease is a CD19-related disease, such as a disease related to abnormal CD19 expression, such as CD19-related cancer. The cancer can be a B-cell malignancy, such as chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphocytic lymphoma, non-Hodgkin's lymphoma, and combinations of said cancers. Alternatively, the disease is an autoimmune disease, such as systemic lupus erythematosus.

[0213] In embodiments of various aspects of this invention, the disease is a B-cell maturation antigen (BCMA)-related disease, such as a disease related to abnormal BCMA expression, such as BCMA-related cancer. The cancer is, for example, myeloma, such as multiple myeloma (MM), particularly relapsed or refractory multiple myeloma (RRMM); or an autoimmune disease, including but not limited to systemic lupus erythematosus (SLE), myositis, scleroderma, Sjögren's syndrome, autoimmune hemolytic anemia, and rheumatoid arthritis.

[0214] In embodiments of various aspects of the present invention, the disease is a mesothelin (MSLN)-related disease, such as a disease related to abnormal MSLN expression, such as MSLN-related cancer. Examples of such cancers include ovarian cancer, breast cancer, colorectal cancer, and pancreatic cancer.

[0215] In embodiments of various aspects of the present invention, the disease is a leukocyte immunoglobulin-like receptor B4 (LILRB4) related disease, such as a disease related to abnormal LILRB4 expression, such as LILRB4-related cancer. The cancer is, for example, leukemia, particularly acute myeloid leukemia. Example

[0216] Example 1: Construction of STAR structure and carrier

[0217] 1.1 Synthetic T-cell receptor antibody receptor (STAR) structure

[0218] B cell secretory antibodies (Abs) or B cell receptors (BCRs) share significant similarities with T cell receptors (TCRs) in gene structure, protein structure, and spatial conformation. Both antibodies and TCRs consist of variable and constant regions. The variable region is responsible for antigen recognition and binding, while the constant region plays a role in structural interaction and signal transduction. By replacing the variable regions of the TCRα and β chains (or TCRγ and δ chains) with the heavy chain variable regions (VH) and light chain variable regions (VL) of the antibody, a synthetic chimeric molecule called a synthetic T-cell receptor and antibody receptor (STAR) can be constructed.

[0219] The STAR molecule has two chains. The first chain is formed by fusing an antigen recognition sequence (such as the variable region VH of the antibody heavy chain) with the constant region (Cα) of the T cell receptor α chain (TCRα). The second chain is formed by fusing an antigen recognition sequence (such as the variable region VL of the antibody light chain) with the constant region (Cβ) of the T cell receptor β chain (TCRβ). The antigen recognition domains (such as VH, VL, or scFv) and constant region domains (the constant regions of TCRα, β, γ, and δ) in this construct can be arranged and combined to form various constructs with different configurations but similar functions.

[0220] After the first and second chains of the STAR molecule are expressed in T cells, they bind with endogenous CD3εδ, CD3γε, and CD3ζζ chains in the endoplasmic reticulum to form an eight-subunit complex, which is then displayed on the cell membrane surface as a complex. The immunoreceptor tyrosine-based activation motif (ITAM) is a signal transduction motif in the TCR molecule, with a conserved sequence of YxxL / V. The intracellular regions of the CD3ε, δ, γ, and ε chains contain one ITAM sequence, and the intracellular region of the CD3ζ chain contains three ITAM sequences, so a complete STAR complex contains a total of 10 ITAM sequences. When the antigen recognition sequence of the STAR receptor binds to its specific antigen, the intracellular ITAM sequences are successively phosphorylated, thereby activating downstream signaling pathways, activating transcription factors such as NF-κB, NFAT, and AP-1, triggering T cell activation, and producing effector functions.

[0221] The inventors have previously modified the constant region of STAR to improve its performance. Specifically, this includes:

[0222] First, the constant region is modified to be mouse-derived: Since the constant region sequences of human, primate and mouse TCRα / β chains (mouse TCRAC / mouse TCRBC) are highly conserved in function and have the same key amino acid sequences, they can be substituted for each other. After substitution, the efficiency of correct pairing of STAR molecules is increased, the possibility of mismatches causing unknown specificity is reduced and the safety is increased.

[0223] Secondly, point mutations were introduced into disulfide bonds using cysteine: a threonine T mutation at position 48 was replaced with a cysteine ​​C mutation in the constant region of the murine TCR α chain, and a serine S mutation at position 56 was replaced with a cysteine ​​C mutation in the constant region of the murine TCR β chain. These two newly added cysteine ​​residues form disulfide bonds between the two STAR chains, reducing mismatches between the two STAR chains and the endogenous TCR chain, and helping the STAR molecule form a more stable complex. The obtained α chain constant region was named TRAC(Cys), and the obtained β chain constant region was named TRBC(Cys).

[0224] The STAR transmembrane region was designed with hydrophobic amino acid substitutions: Mutations were made at three amino acid sites within the transmembrane region of the TCR α-chain constant region, from amino acid positions 111 to 119. Serine (S) at position 112 was replaced with leucine (L), methionine (M) at position 114 with isoleucine (I), and glycine (G) at position 115 with valine (V). The overall amino acid sequence of this region changed from LSVGMLRIL to LLVIVLRIL. This design increased the hydrophobicity of the transmembrane region, counteracting the instability caused by the positive charge carried by the TCR transmembrane region, allowing the STAR molecule to exist more stably on the cell membrane and thus acquire better function. The α-chain constant region obtained by combining cysteine ​​and hydrophobic region mutations was named TRAC(Cys-TM), and the corresponding β-chain constant region was named TRBC(Cys-TM), where TRBC(Cys-TM) is identical to TRBC(Cys).

[0225] To further optimize the design of the STAR molecule, a specific rearrangement was performed on the N-terminus of the STAR molecule's constant region, based on murine derivatization of the constant region, cysteine ​​point mutations, and hydrophobic amino acid mutations in the α-chain constant region, to achieve better results. Rearrangement involves partial sequence deletion and humanization mutation of other sequences. The significance of humanization mutation lies in minimizing non-human sequences in the STAR molecule while maintaining its function, thereby minimizing the possibility of receptor rejection of STAR-T cells in clinical applications. Therefore, the N-terminus of the TCR α-chain constant region was further modified, including replacing amino acid E at position 6 with D, replacing K at position 13 with R, and deleting amino acids 15-18. The resulting α-chain constant region was named TRAC (Nrec-Cys-TM). Further modifications were made to the N-terminal 25 amino acids of the TCR β-chain constant region, including replacing the 3rd amino acid (R) with K, the 6th amino acid (T) with F, the 9th amino acid (K) with E, the 11th amino acid (S) with A, and the 12th amino acid (L) with V, and the 17th and 21-25th amino acids were deleted. The resulting β-chain constant region was named TRBC (Nrec-Cys-TM).

[0226] Furthermore, STAR function can be further enhanced by linking co-stimulatory molecules, such as the OX40 cytoplasmic region, to the C-terminus of the α-chain constant region and / or the β-chain constant region. These co-stimulatory molecules can be linked to the C-terminus of the α-chain constant region and / or the β-chain constant region via adapters, such as (G4S)3 adapters. In addition to the modifications described above, the constant regions linked to the co-stimulatory molecules can also lack the native intracellular region relative to the wild-type constant region, which further improves STAR function. For example, the α-chain constant region may lack amino acids 136-137; and / or, the β-chain constant region may lack amino acids 167-172.

[0227] 1.2 STAR-AAV vector design and viral packaging for CRISPR knockin templates

[0228] 1.2.1 STAR-AAV Carrier Design for CRISPR Knockin Template

[0229] The sequence used for CRISPR knock-in contains the following elements: a 5' homologous arm (5'HA), an MND promoter sequence, the gene to be inserted (STAR ​​coding sequence), a PolyA sequence, and a 3' homologous arm (3'HA).

[0230] In this experiment, a STAR structure was inserted at the TRAC site. The 5'HA for homologous recombination contained the nucleotide sequence shown in SEQ ID NO:18, and the 3'HA contained the nucleotide sequence shown in SEQ ID NO:19. The MND promoter contained the nucleotide sequence shown in SEQ ID NO:20.

[0231] The above knock-in sequence was integrated between the 5' and 3' LTR sequences of the AAV6 vector to obtain the vector TRAC-MND-STAR as shown in Figure 1. It was then packaged into AAV6 virus according to the instructions provided by the supplier (Guangzhou Paizhen Biotechnology Co., Ltd.).

[0232] The STAR structure used in this experiment includes the constant region TRAC (Cys-TM) of the TCRα chain and the constant region TRBC (Cys-TM) of the TCRβ chain, as well as the co-stimulatory factor OX40. The expressed STAR molecule contains two polypeptide chains: a first polypeptide chain formed by the TCRβ chain and OX40, and a second polypeptide chain formed by the TCRα chain and OX40. The coding sequences of the two STAR polypeptide chains are linked by the coding sequences of furin and the P2A protease cleavage site. The coding sequences of the two polypeptide chains are transcribed and translated together into a fusion polypeptide, which is then cleaved by the proteases corresponding to furin and P2A into two independent protein subunits. These two subunits are covalently bound by disulfide bonds and form a complex with the endogenous CD3 subunits (ε, δ, γ, ζ) of T cells.

[0233] The amino acid sequence of the TCRα chain constant region mutant used in this experiment is SEQ ID NO:3; the amino acid sequence of the TCRβ chain constant region mutant is SEQ ID NO:10; the amino acid sequence of OX40 used in this experiment is SEQ ID NO:15, which is directly linked to the constant region in the α chain or β chain, and there are no deletions in the intracellular region of the constant region.

[0234] The antibodies used in this experiment include CD19 antibody FMC63 single-chain Fv (scFv), LILRB4 nanobodies NLB4 and NLB14, MSLN nanobodies NM5 and NM24, BCMA nanobodies NBC11, and GPC3 antibody GC33. The specific description is as follows: VH and VL of CD19-specific mouse monoclonal antibody (clone number FMC63) are used, the amino acid sequence of FMC63-VH is SEQ ID NO:21, the amino acid sequence of FMC63-VL is SEQ ID NO:22; the amino acid sequence of LILRB4 nanobody NLB4 is SEQ ID NO:25; the amino acid sequence of NLB14 is SEQ ID NO:26; the amino acid sequence of MSLN nanobody NM5 is SEQ ID NO:24; the amino acid sequence of BCMA nanobody NBC11 is SEQ ID NO:27; the VH amino acid sequence of GPC3 antibody GC33 is SEQ ID NO:29, and the VL amino acid sequence is SEQ ID NO:28.

[0235] The amino acid sequence of the CD19-STARα chain used in this experiment is shown in SEQ ID NO:30, and the amino acid sequence of the β chain is shown in SEQ ID NO:31. The universal CD19-STAR nucleic acid sequence prepared is SEQ ID NO:38. The amino acid sequence of the BCMA-LILRB4-STARα chain used in this experiment is shown in SEQ ID NO:39, and the amino acid sequence of the β chain is shown in SEQ ID NO:40. The universal BCMA-LILRB4-STAR nucleic acid sequence prepared is SEQ ID NO:41.

[0236] 1.2.2 STAR-AAV virus packaging for CRISPR knockin template

[0237] Taking T75flask as an example, the cell number was 9M-10M after passage the previous afternoon.

[0238] The reagents used included: 7.5 μL vector plasmid (1.0 μg / μL); 7.5 μL packaging plasmid (1.0 μg / μL); and 7.5 μL helper plasmid (1.0 μg / μL).

[0239] OptiMEM 1.5ml; TanslT-VirusGen: 45ul.

[0240] AAV virus collection: Virus particles are present in both packaging cells and culture supernatant.

[0241] AAV virus concentration and purification.

[0242] Example 2: Electroporation-free preparation method using the CRISPR-Cas system

[0243] 2.1 Design of crRNA

[0244] This invention relates to the design of CrRNAs for TRAC, HLA-A, HLA-B, and CIITA genes using the CHOPCHOP online tool. Specifically, the method includes the following steps: (1) Target gene region selection and sequence acquisition: The coding regions or key functional domains of TRAC (T cell receptor α constant region), HLA-A, HLA-B (major histocompatibility complex class I gene), and CIITA (MHC class II transactivator) genes are identified as editing targets to disrupt gene function and reduce the immunogenicity of transplanted cells. Reference mRNA or genomic DNA sequences of each gene are obtained from the NCBI or Ensembl database. (2) CrRNA design and screening: Access the CHOPCHOP online platform (https: / / chopchop.cbu.uib.no / ) and select "Cas12a (Cpfl)" as the nuclease type. Set parameters: The genome version is consistent with the reference sequence; the prototype spacer adjacent motif (PAM) is selected as "TTTN" (applicable to Cas12a); the CrRNA length is set to 20-24 nt. crRNAs with the following characteristics are preferred: a) high specificity score to minimize off-target effects; b) targeting the anterior exon near the 5' end of the gene to improve frameshift mutation or gene knockout efficiency; c) low predicted off-target activity, especially without highly homologous sites in other gene coding or regulatory regions; d) moderate GC content (e.g., 30%-70%) to ensure stability and binding efficiency. The crRNA sequences of TRAC, HLA-A, HLA-B, and CIITA genes are shown in Table 1.

[0245] Table 1. crRNA target sequences used in the examples

[0246] 2.2 Non-electric operation procedure of Crispr-Cas system

[0247] Step 1: Resuscitate PBMCs and isolate and activate T cells.

[0248] PBMC thawing: Remove frozen PBMCs from liquid nitrogen and place them in a 37°C water bath until they are 70% thawed. Transfer the cells to a 50mL centrifuge tube containing X-VIVO medium and centrifuge (500g, 7 minutes) to remove the supernatant.

[0249] T cell isolation, activation, and culture: PBMC cells were resuspended in MACS Buffer (60 μL for 1E7 cells). CD4 microbeads (20 μL for 1E7 cells) and CD8 microbeads (20 μL for 1E7 cells) were added to the cell suspension, and the mixture was thoroughly incubated at 2–8 °C for 15 minutes. After incubation, cells were collected by centrifugation (300 g, 10 minutes) with MACS Buffer (1–2 ml for 1E7 cells). The cells were then resuspended in MACS Buffer (500 μL for 10E8 cells) to obtain a cell suspension. An LS column was prepared in advance after rinsing with MACS Buffer (3 ml). The cell suspension was added to the LS column (which was placed on a magnet), and the LS column was rinsed three times with MACS Buffer (3 ml each time). The LS column was removed from the magnet, and MACS Buffer (5 ml) was added. The cell suspension was quickly displaced using a rubber stopper, and the cell suspension was centrifuged. Remove the supernatant from the cell suspension, resuspend the cells in X-VIVO + IL-2 + FBS medium (X-VIVO medium with added IL-2 and FBS) for counting, and add TransAct to the cell suspension (cell suspension: TransAct = 100:1 volume ratio (TransAct: manufacturer: Miltenyi, catalog number 130-111-160)) and mix well. Incubate the obtained T cells in an incubator for 1-4 days. Ready for further application.

[0250] In this example, the time for PBMC resuscitation to isolate and activate T cells is recorded as day 0, and the subsequent T cell culture is recorded as day 1, and so on; in this example, T cell culture is recorded from day 0 to day 3.

[0251] On day 1, T cells can be cultured or the subsequent "Step 2: RNP Preparation" experiment can be carried out; on day 2, T cells can be cultured or the subsequent "Step 2: RNP Preparation" experiment can be carried out; on day 3, T cells can be cultured or the subsequent "Step 2: RNP Preparation" experiment can be carried out; other operations can be deduced in the same way.

[0252] The subsequent steps 2 through 6 involve cell editing.

[0253] Step 2: Preparation of RNP

[0254] In this embodiment, the specific reagent dosage will be adjusted and administered according to subsequent embodiments. This embodiment begins non-electroplated CRISPR gene editing on day 3.

[0255] First, RNPs were formed by incubating Cas12a / Cas9 protein with TRAC-crRNA: Take an appropriate amount of TRAC-crRNA and place it on the sidewall of a 1.5ml EP tube (for multi-gene experiments, take appropriate amounts of HLA-A-crRNA, HLA-B-crRNA, and CIITA-crRNA and place them at different positions on the sidewall of the same 1.5ml EP tube, and refer to the TRAC-RNP experiment to obtain their respective RNP cell systems). Add an appropriate amount of Cas12a / Cas9 to the above TRAC-crRNA droplet, mix well by pipetting, and incubate at 37°C for 15 minutes to obtain the RNP cell system.

[0256] Step 3: Preparation of RNP-AAV system

[0257] Secondly, the RNP system was mixed with AAV (customized - Paizhen Biotechnology) to form the RNP-AAV system: Take out the EP tube from the incubator, add an appropriate amount of AAV-STAR to the above TRAC-RNP-Cas12a / Cas9 droplet system and mix by pipetting (if the RNPs of HLA-A, HLA-B, and CIITA in step 2 above are not treated with AAV-STAR, incubate at 37℃ for 15 minutes to obtain the four-gene RNP-AAV system.

[0258] Step 4: Preparation of RNP-AAV-T system

[0259] The above-mentioned 4-gene RNP-AAV system was added to T cells for incubation to form the RNP-AAV-T system: After the T cells cultured in step 1 were removed, sampled, counted, and centrifuged (500g, 7 minutes). The supernatant was removed, and the cells were resuspended in Opti-MEM (Gibco catalog number 31985062) for counting (2E7 / ml) to obtain a T cell suspension. After RNP-AAV incubation, 50 μL of the above T cell suspension was added to an EP tube of the RNP-AAV system, mixed thoroughly by pipetting, and incubated at 37°C for 30 minutes to obtain the RNP-AAV-T cell system.

[0260] Step 5: Non-electro-electric system

[0261] Next, CPP (custom-made - Jier Biochemical) was added to the RNP-AAV-T cell system for incubation: After the RNP-AAV-T cell incubation was completed, an appropriate amount of CPP (peptide TAT-HA2: RRRQRRKKRGGDIMGEWGNEIFGAIAGFLG) was added to the incubation system. The CPP was added and immediately mixed by pipetting. The cells were then incubated at 37°C for 30-45 minutes.

[0262] Finally, the RNP-AAV-T-CPP cell system was transferred to culture medium: After the RNP-AAV-T-CPP incubation was completed, it was transferred to X-VIVO culture medium supplemented with IL-2 (manufacturer: Beijing Sihuan, catalog number S10970016), AZD7648, and FBS (brand: Lonza, catalog number 04-418Q) and placed in an incubator for culture.

[0263] The chemical formula of AZD7648 (purchased from Selleck, product number S8843) is C 18 H 20 N8O2 has the following structural formula, as shown in Formula I:

[0264] Step 6: Cell Expansion and Editing Efficiency Detection

[0265] Days 4-9: Cell observation under a microscope. Gently agitate the cells and collect a cell suspension for counting. Add fluid. Return to the incubator for further culture. Day 10: Flow cytometry analysis. Cell observation under a microscope. Gently agitate the cells and collect a cell suspension for counting. Analyze the cell editing efficiency using flow cytometry, and calculate the viability and amplification value.

[0266] All reagents used in this patent are from Table 2. Details are as follows:

[0267] Table 2. Related Reagents

[0268] Example 3: Comparison of non-electroporation experiments of different proteins using CRISPR-Cas

[0269] Following the single-gene non-electrotransfer procedure in Example 2.2, a site-directed knock-in experiment of the TRAC gene locus was conducted. Cas12a was replaced with Cas9 in the experiment, and the effects of the two proteins, Cas12a and Cas9, on the experiment were compared. At the same time, the X-vivo medium containing AZD7648 was replaced with X-vivo medium without AZD7648 to evaluate the effect of AZD7648 on the experiment.

[0270] Experimental conditions: Cas12a or Cas9 were used at working concentrations of 0.25 μM, 0.5 μM, and 1 μM. The molar ratio of Cas12a, Cas9, and CrRNA / gRNA was 1:1.2. Two experimental groups were designed: one using X-vivo medium with AZD7648 and the other using X-vivo medium without AZD7648. The amount of AZD7648 used was 1 μM at a dilution of 1:1000. Specific amounts of other materials are shown in Tables 3 and 4. At the end of the experiment, TRAC knockout efficiency, STAR knock-in efficiency, cell viability, amplification, and editing efficiency were measured.

[0271] Table 3. Working concentrations of Cas12a reagent and corresponding cell dosages

[0272] Table 4. Working concentrations of Cas9 reagent and corresponding cell dosages

[0273] The experimental results are shown in Figure 2. In the TRAC single-knockout non-electroporation system, under the same incubation system, the TRAC knockout and STAR knock-in efficiencies of Cas12a and Cas9 proteins were: Cas12a > Cas9, with the difference being more pronounced at lower concentrations. Under different incubation systems, for the same working concentration of CRISPR protein, the higher the STAR positivity rate and the better the TRAC knockout efficiency, with the 1 μM system showing the highest efficiency compared to the 0.5 μM system, which in turn showed the highest efficiency compared to the 0.25 μM system.

[0274] The experimental results are shown in Figure 2. In the STAR-positive T cell knock-in efficiency, the addition of AZD7648 significantly improved the editing efficiency of Cas12a and Cas9. Specifically, the addition of CPP (peptide TAT-HA2) and AZD7648 significantly improved the editing efficiency of Cas12a and Cas9. Adding AZD7648 can simultaneously improve both knockout and knock-in efficiencies; moreover, the STAR positivity rate of Cas12a protein can reach over 60% even without the addition of AZD7648.

[0275] Example 4: Electrospinning and Non-electrospinning Experiments of Cas12a and Cas9

[0276] Referring to the experimental protocol for site-specific knock-in of the TRAC gene locus and knockout of HLA-A, HLA-B and CIITA genes in Example 2.2 of this specification, an experiment was conducted to prepare universal CD19-STAR-T cells using the Cas9 / Cas12a non-electroporation method; wherein the CD19 antibody used was the FMC63 antibody.

[0277] The following is an experiment to prepare universal STAR-T cells by electroporation of Cas9 / Cas12a: (1) First, PBMCs were resuscitated, then T cells were isolated and activated, and placed in T-VIVO medium for culture. (2) Electroporation buffer and RNP were prepared, mixed thoroughly, and incubated at 37°C for 20 minutes. The activated PBMC cells were centrifuged at 90g for 10 minutes, resuspended in PBS for counting, centrifuged according to the required number of cells, resuspended in electroporation buffer, and then electroporation was started. (3) After electroporation, 100ul of preheated medium was added to each electroporation cup, and the cells were transferred to a 6-well plate or a 48-well plate. After about 10 minutes, the virus AAV (CD19-STAR AAV virus, see section 1.2 of Example 1) was added and placed in an incubator for culture. (4) On day 8 of cell culture, an appropriate amount of cells were taken for flow cytometry to detect STAR positivity rate, TRAC knockout (hTCR expression), cell viability, gene knockout efficiency, etc.

[0278] The experimental results (Figure 3 and Table 5) show that: (1) Under electroporation conditions, the STAR gene knock-in efficiency mediated by Cas12a protein is 20% higher than that Cas9 protein, while the TRAC gene knockout efficiency mediated by both is basically the same; (2) Under non-electroplation conditions, the STAR gene knock-in efficiency and TRAC gene knockout efficiency mediated by Cas12a protein are both about 10% higher than those Cas9 protein. In summary, compared with Cas9 protein, Cas12a protein has better gene knockout and knock-in efficiencies under both electroporation and non-electroplation conditions; in addition, the STAR gene knock-in efficiency of Cas12a protein under non-electroplation conditions is 15% higher than that under electroporation conditions, while the TRAC gene knockout efficiency is 3.5% lower than that under electroporation conditions.

[0279] Table 5. Editing efficiency of electronic and non-electronic editing

[0280] Example 5: Experiments with different material ratios and editing times

[0281] This embodiment establishes an efficient CRISPR-Cas12a non-electro-transfer and gene editing protocol by systematically optimizing key parameters such as cell dosage, cell-penetrating peptide (CPP, sequence TAT-HA2) dosage, and initial editing time.

[0282] Following the single-gene non-electrotransfer procedure in Example 2.2, site-specific knock-in experiments were conducted on the TRAC gene locus in T cells. The editing program was initiated on either Day 2 (after cell culture) or Day 3 (after cell culture). First, Cas12a protein and TRAC crRNA were incubated at room temperature for 15 minutes to form a ribonucleoprotein complex (RNP). Then, a specified number of T cells, recombinant cathode ray tube virus (AAV), and CPP were added to the RNP complex system, and incubation continued at 37°C for 60 minutes. The specific amounts of Cas12a, crRNA, CPP, and cells in each reaction system (25 μL, 50 μL, 100 μL, and 200 μL) are detailed in Table 6. This experiment aimed to comprehensively evaluate the effects of editing initiation timing and cell density on TRAC gene knockout efficiency and STAR gene knock-in efficiency.

[0283] The experimental results are shown in Figure 4 and Table 7. Significant progress was made in optimizing key parameters: cell density: at 5 × 10⁻⁶ cells / year... 6 / mL to 4×10 7 Within the test range of / mL, cell density did not significantly affect the final editing efficiency. Editing start time: Compared to editing started on Day 3, editing started on Day 2 achieved both higher STAR insertion efficiency and TRAC knockout efficiency, with a difference of approximately 15%. Regarding CPP addition amount, when the CPP amount was 3µl, 1×10⁻⁶... 6 At a speed of / mL, STAR has the highest insertion efficiency, while Day2 editing has the highest knockout efficiency.

[0284] In summary, this embodiment successfully determined a set of preferred operating parameters for achieving high-efficiency CRISPR-Cas12a gene editing under non-electrotransfer conditions, providing a stable and reliable technical solution for applications in related fields.

[0285] Table 6. Reagent Working Concentrations and Corresponding Cell Doses

[0286] Table 7. Editing efficiency at different editing times and cell densities

[0287] Example 6: Editing time and in vitro functional verification in a single-gene system

[0288] 6.1 Optimization of Cas12a protein editing time and steps

[0289] Referring to the single-gene non-electrotransfer procedure in Example 2.2, a TRAC gene locus knock-in experiment was conducted to compare the effects of incubation steps and times of various reagents on editing efficiency in the TRAC single knock-in system.

[0290] The specific amounts of Cas12a, crRNA, and CPP used in the 50 μL incubation system are shown in Table 8, and the specific incubation steps and other experimental conditions are shown in Table 9. The final TRAC knockout efficiency, as well as the CD19-STAR (AAV-TRAC-MND-FMC-STAR-abOX40) and BCMA-LILRB4-STAR (AAV-TRAC-MND-NBC11-NLB14-STAR-mbIL15) knock-in efficiency were detected.

[0291] Table 8. Reagent Dosage

[0292] Table 9. Specific incubation steps for each scheme

[0293] Figure 5 and Table 10 show that optimized schemes 1-5 are no weaker than or even better than the original incubation step (scheme 6) in terms of KO / KI, D9 proliferation (compared to when CRISPR gene editing started on Day 3), and killing function. Scheme 1 has the shortest operation time, requiring only 45 minutes to complete all operations.

[0294] Table 10. Editing efficiency of CD19-STAR / BCMA-LILRB4-STAR under different incubation conditions

[0295] 6.2 In vitro functional validation of the universal STAR-T

[0296] Luciferase is a common substance used in cell function research. Enzyme activity is determined by adding a luciferase substrate to a system, and luciferase activity is closely related to the expression and binding strength of the target gene, as well as the number of cells. In this invention, a target cell line stably expressing luciferase is established, and the amount of luciferase is used to indicate the number of target cells, thereby indicating the cytotoxic function of functional cells.

[0297] Experiment 1: Constructing NALM6-LUC target cells to verify the killing level of CD19-STAR-T cells against NALM6-LUC target cells. NALM6-LUC target cells were seeded in 24-well plates at a density of 4E5 / well. The universal CD19-STAR-T cells prepared in section 6.1 were added to the target cells at STAR-positive T cell to target cell ratios of 0.3:1, 0.1:1, and 0.03:1, with a co-culture volume of 1 mL. After 24 hours of co-culture, the co-cultured cell suspension was collected, and the LUC luminescence value was detected using a luciferase reporter gene assay kit to calculate the killing efficiency of STAR-T cells against target cells. As shown in Figure 6, it can be seen that the universal CD19-STAR-T cells prepared by each scheme can effectively kill the target cells NALM6-LUC. When the E:T ratio is 0.3:1, the universal CD19-STAR-T cells prepared by each scheme can effectively kill the target cells with basically the same efficiency. When the E:T ratio is 0.1:1 and 0.03:1, the universal CD19-STAR-T cells prepared by schemes 4 and 5 have better killing efficiency, which is comparable to that of scheme 6.

[0298] Experiment 2: Constructing NCI-H929 target cells to verify the killing level of BCMA-LILRB4-STAR-T cells against NCI-H929 target cells. NCI-H929 target cells were seeded in 24-well plates at a density of 4E5 / well. The universal BCMA-LILRB4-STAR-T cells prepared in step 6.1 were added to the target cells at STAR-positive T cell to target cell ratios of 1:1 and 0.3:1, with a co-culture volume of 1 mL. After 24 hours of co-culture, the co-cultured cell suspension was collected, and the LUC (luciferase-reporter gene) luminescence value was detected using a luciferase reporter gene assay kit to calculate the killing efficiency of STAR-T cells against target cells. As shown in Figure 7, it can be seen that the universal BCMA-LILRB4-STAR-T cells prepared by each scheme can effectively kill the target cells NALM6-LUC. When the E:T ratio is 1:1, the universal BCMA-LILRB4-STAR-T cells prepared by each scheme can effectively kill the target cells with basically the same efficiency. When the E:T ratio is 0.3:1, the universal BCMA-LILRB4-STAR-T cells prepared by scheme 5 have the best killing efficiency, which is comparable to that of scheme 6.

[0299] Example 7: TRAC Fixed-Point Insertion Multiple Knockout System Validation

[0300] 7.1. One-step editing validation of multi-gene editing

[0301] Following the multi-gene non-electroporation procedure described in Example 2.2, knock-in and knockout experiments were conducted targeting four gene loci: TRAC, HLA-A, HLA-B, and CIITA. The specific amounts of Cas12a protein, CrRNA at each target site, and cell-penetrating peptide CPP in a 50 μL reaction system are shown in Table 11. To assess the impact of editing initiation time, Cas12a non-electroporation CRISPR gene editing was initiated on day 2 or day 3 after T cell culture. After editing, the following indicators were detected and analyzed: TRAC knockout efficiency, HLA-A knockout efficiency, HLA-B knockout efficiency, CIITA knockout efficiency, STAR knock-in efficiency, cell viability, and differences in cell proliferation capacity after editing. The effects of the editing process on cell activity, cell size, and cell activation status were also assessed.

[0302] Indirect indicators were used to evaluate the efficiency of CIITA gene knockout. Since CIITA is a key transcriptional activator regulating the expression of HLA class II molecules (including HLA-DP, HLA-DQ, and HLA-DR), its knockout leads to a downregulation of HLA class II molecule expression levels on the cell surface. Based on this principle, the extent of HLA-DP / DQ / DR expression downregulation was detected by flow cytometry, which can indirectly and effectively reflect the efficiency of CIITA gene knockout. Furthermore, the knockout efficiency can also be directly verified by sequencing analysis of the insertion / deletion mutation rate at the CIITA gene locus.

[0303] The experimental results are shown in Figure 8 and Table 12. Multi-gene CRISPR editing initiated on day 2 after T cell culture showed a higher overall editing efficiency than the group initiated on day 3, with an efficiency improvement of approximately 10%. In the multi-gene knockout system, both day 2 and day 3 initiation of editing achieved high editing efficiencies. Specifically, at day 2 initiation, the STAR gene knock-in positivity rate was approximately 68.4%, the TRAC gene knockout efficiency was 80.6%, the HLA-A gene knockout efficiency was 72.5%, the HLA-B gene knockout efficiency was 74.8%, and the CIITA knockout efficiency, indirectly assessed by downregulation of HLA-DP / DQ / DR expression, was 77.0%. These data indicate that this Cas12a non-electroplated multi-gene editing system can achieve efficient gene knock-in and high-efficiency simultaneous multi-gene knockout. Furthermore, according to the data in Table 12, the editing initiation time (day 2 and day 3) had minimal impact on key cell state monitoring indicators such as cell viability, particle size, and activation status, indicating that this editing system has good cell compatibility.

[0304] Table 11. Reagent working concentrations and corresponding cell dosages

[0305] Table 12. Effects of editing time on monitoring cell viability, particle size, and activation status.

[0306] 7.2. Validation of Multi-Gene Knock-In Stepwise Editing

[0307] Following the multi-gene non-electroporation procedure in Example 2.2, knock-in and knockout experiments were conducted at four loci: TRAC, HLA-A, HLA-B, and CIITA. The specific amounts of Cas12a, CrRNA, and CPP in a 50 μL system are shown in Table 13, and the specific CRISPR editing time and order are shown in Table 14. Two-step CRISPR gene editing with non-electroporation of Cas12a was initiated on day 2, 3, 4, or 5 to verify the effect of starting CRISPR gene editing at different time points on editing efficiency. Differences in TRAC knockout efficiency, HLA-A knockout efficiency, HLA-B knockout efficiency, CIITA knockout efficiency, STAR knock-in efficiency, cell viability, amplification, and editing efficiency were detected. Cell activity, particle size, and activation status were also monitored.

[0308] Table 13. Reagent Working Concentrations and Corresponding Cell Doses

[0309] Table 14. Specific locus editing times in the experiment

[0310] The experimental results are shown in Table 15:

[0311] (1) In terms of editing time: the combination of Day 2 first gene editing and Day 4 second gene editing has the best knock-in efficiency and knock-out efficiency. The combination of Day 1 first gene editing and Day 3 second gene editing has an extremely low knock-out efficiency of KO% but an ideal knock-in efficiency of KI%. The combination of Day 3 first gene editing and Day 5 second gene editing has an acceptable knock-out efficiency of KO% and an acceptable knock-in efficiency of KI%.

[0312] (2) In terms of RNP usage: Under the same editing system, RNP usage increases KO% but decreases KI%.

[0313] (3) In terms of gene combination: the knock-in efficiency KI% of the combination of Day 2 first gene editing and Day 2 second gene editing has an impact, while the knock-out efficiency KO% of the combination of Day 3 first gene editing and Day 5 second gene editing has a smaller impact.

[0314] (4) Optimal system: The combination of Day 2 first gene editing (HLA-A, HLA-B) and Day 4 second gene editing (CIITA, TRAC) has a relatively ideal gene knockout efficiency at an RNP dosage of 1.6; the combination of Day 2 first gene editing (HLA-A, HLA-B, CIITA) and Day 4 second gene editing (TRAC) has a relatively ideal gene knock-in efficiency at an RNP dosage of 1.6.

[0315] Table 15. Editing Efficiency Results

[0316] Related sequence information

[0317] SEQ ID NO:1 Human T cell receptor α chain constant region

[0318] SEQ ID NO:2 Mouse T cell receptor α chain constant region

[0319] SEQ ID NO:3 Mouse T cell receptor α chain constant region containing cysteine ​​substitution and hydrophobic region modification (TRAC-Cys-TM)

[0320] SEQ ID NO:4 Mouse T cell receptor α chain constant region (TRAC-Nrec-Cys-TM) containing N-terminal modification, cysteine ​​substitution and hydrophobic modification of the transmembrane region.

[0321] SEQ ID NO:5 Mouse T cell receptor α chain homeostasis region lacking intracellular region

[0322] SEQ ID NO:6 Mouse T cell receptor α chain constant region lacking intracellular region and containing cysteine ​​substitutions and hydrophobic regions.

[0323] SEQ ID NO:7 Mouse T cell receptor α chain constant region lacking intracellular region, containing N-terminal modification, cysteine ​​substitution, and hydrophobic modification of the transmembrane region.

[0324] SEQ ID NO:8 Human T cell receptor β chain constant region

[0325] SEQ ID NO:9 Mouse T cell receptor β chain constant region

[0326] SEQ ID NO:10 Contains cysteine-substituted mouse T cell receptor β-chain constant region (TRBC-Cys-TM)

[0327] SEQ ID NO:11 Mouse T cell receptor β chain constant region containing N-terminal modification and cysteine ​​substitution (TRBC-Nrecc-Cys-TM)

[0328] SEQ ID NO:12 Mouse T cell receptor β chain homeostasis region lacking intracellular region

[0329] SEQ ID NO:13 Deletion of intracellular region, containing cysteine-substituted mouse T cell receptor β chain constant region

[0330] SEQ ID NO:14 Mouse T cell receptor β chain constant region lacking intracellular region, containing N-terminal modification and cysteine ​​substitution.

[0331] SEQ ID NO:15 OX40 intracellular domain

[0332] SEQ ID NO:16 (G4S)3 connector

[0333] SEQ ID NO:17 Furin-P2A

[0334] SEQ ID NO:18 5'HA

[0335] SEQ ID NO:19 3'HA

[0336] SEQ ID NO:20 MND promoter

[0337] SEQ ID NO:21 Anti-CD19 FMC63 VH

[0338] SEQ ID NO:22 Anti-CD19 FMC63 VL

[0339] SEQ ID NO:23 CD19 antibody FMC63 ScFv amino acid sequence

[0340] SEQ ID NO:24 Anti-MSLN NM5 VHH

[0341] SEQ ID NO:25 Anti-LILRB4 NLB4 VHH

[0342] SEQ ID NO:26 Anti-LILRB4 NLB14 VHH

[0343] SEQ ID NO:27 Anti-BCMA NBC11 VHH

[0344] SEQ ID NO:28 GC33-VL

[0345] SEQ ID NO:29 GC33-VH

[0346] SEQ ID NO:30 CD19-STARα chain

[0347] SEQ ID NO:31 CD19-STARβ chain

[0348] SEQ ID NO:32 3'Cas binding fragment (sgRNA scaffold sequence)

[0349] SEQ ID NO:33 Guide TRAC

[0350] SEQ ID NO:34 Guide HLA-A

[0351] SEQ ID NO:35 Guide HLA-B

[0352] SEQ ID NO:36 Guide CIITA

[0353] SEQ ID NO:37 CPP polypeptide TAT-HA2

[0354] SEQ ID NO:38 CD19-STAR nucleic acid sequence

[0355] SEQ ID NO:39 BCMA-LILRB4-STARβ chain

[0356] SEQ ID NO:40 BCMA-LILRB4-STARα chain

[0357] SEQ ID NO:41 BCMA-LILRB4-STAR nucleotide sequence.

Claims

1. A combination for preparing genetically modified cells by a non-electroporation method, comprising: i) An expression vector containing a foreign nucleotide sequence to be inserted into the genome of the cell; ii) Gene editing systems that target one or more endogenous genes in cells; and iii) Cell penetration reagents and / or non-homologous end joining (NHEJ) inhibitors.

2. The combination of claim 1, wherein the cell penetration reagent is selected from Ctr, R10, Pep-1, TAT, Bac7, Penetratin, SynB1, PreS2, MAP12, NF-κB, TAT-HA2, HA2, A5K, LAH2, Vectofusion-a, Polybrene, Protamine, PEI, PLL, NLSMATS, preferably, the cell penetration reagent is a cell penetration peptide (CCP), such as TAT, TAT-HA2, HA2, etc., more preferably TAT-HA2.

3. A combination of claim 1 or 2, wherein the non-homologous end joining (NHEJ) inhibitor is selected from SCR7, NU7441 (KU-57788), L189, Brefeldin A (BFA), L755507, RS-1, AZD7648, preferably, the NHEJ inhibitor is AZD7648.

4. A combination of any one of claims 1-3, wherein the gene editing system is a CRISPR gene editing system.

5. The combination of claim 4, wherein the CRISPR gene editing system is a Cas12 gene editing system, preferably a Cas12a gene editing system.

6. The combination of claim 5, wherein the Cas12a gene editing system comprises: i) Cas12 nucleases such as Cas12a nucleases; and ii) Guide RNA, wherein the guide RNA is capable of targeting the target sequence of the endogenous gene in the cell.

7. A combination of any one of claims 1-6, wherein the gene editing system targets one or more of the endogenous TRAC, HLA-A, HLA-B and CIITA genes.

8. The combination of claim 7, wherein The guide RNA targets or contains the target sequence shown in SEQ ID NO:33 of the endogenous TRAC gene; The guide RNA targets or contains the target sequence shown in SEQ ID NO:34 of the endogenous HLA-A gene; The guide RNA targets or contains the target sequence described in SEQ ID NO:35 of the endogenous HLA-B gene; and / or The guide RNA targets or contains the target sequence shown in SEQ ID NO:36 of the endogenous CIITA gene.

9. A combination of any one of claims 1-8, wherein the expression vector is an AAV vector.

10. A combination of any one of claims 1-9, wherein the foreign nucleotide sequence to be inserted into the cell genome may include a 5' homologous arm (5'HA) at its 5' end and a 3' homologous arm (3'HA) at its 3' end, wherein the 5'HA and 3'HA contain nucleotide sequences homologous to sequences in the locus to which the foreign nucleotide sequence is to be inserted.

11. The combination of claim 10, wherein the exogenous nucleotide sequence comprises a 5' homologous arm (5'HA) shown in SEQ ID NO:18 at its 5' end and a 3' homologous arm (3'HA) shown in SEQ ID NO:19 at its 3' end.

12. A combination of any one of claims 1-11, wherein the exogenous nucleotide sequence is an expression cassette of a CAR, TCR, or STAR, for example, the expression cassette of the CAR, TCR, or STAR comprises a coding nucleotide sequence of the CAR, TCR, or STAR, and an expression regulatory element, such as a promoter, operatively linked thereto.

13. The combination of claim 12, wherein an example of a promoter includes the MND promoter. An exemplary nucleotide sequence of the MND promoter is shown in SEQ ID NO:

20.

14. A combination of claim 12 or 13, wherein the STAR expression cassette comprises a coding nucleotide sequence of a fusion protein of the α chain and the β chain of the STAR linked by a self-cleaving peptide, preferably wherein the coding nucleotide sequence is operatively linked to an MND promoter.

15. The combination of claim 14, wherein the self-cleaving peptide is a 2A polypeptide, such as the Furin-2A polypeptide, for example, the Furin-P2A polypeptide shown in SEQ ID NO:

17.

16. A combination of any one of claims 12-15, wherein the STAR comprises an α chain and a β chain, the α chain comprising a first constant region, the β chain comprising a second constant region, and wherein the α chain and / or the β chain further comprises an antigen-binding region that specifically binds to the target antigen.

17. The combination of claim 16, wherein the first constant region is a natural TCRα chain constant region, for example, a natural human TCRα chain constant region or a natural mouse TCRα chain constant region; or, the first constant region is a modified TCRα chain constant region.

18. The combination of claim 17, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 48, for example threonine T, is mutated to cysteine ​​C.

19. The combination according to claim 17 or 18, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 112, such as serine S, is replaced with leucine L, the amino acid at position 114, such as methionine M, is replaced with isoleucine I, and the amino acid at position 115, such as glycine G, is replaced with valine V.

20. The combination according to any one of claims 17-19, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 6, such as E, is replaced by D, the amino acid at position 13, K, is replaced by R, and the amino acids at positions 15-18 are deleted.

21. The combination according to any one of claims 17-20, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 48, such as threonine (T), is mutated to cysteine ​​(C), the amino acid at position 112, such as serine (S), is mutated to leucine (L), the amino acid at position 114, such as methionine (M), is mutated to isoleucine (I), and the amino acid at position 115, such as glycine (G), is mutated to valine (V).

22. The combination according to any one of claims 17-21, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 6, such as E, is replaced by D; the amino acid at position 13, K, is replaced by R; the amino acids at positions 15-18 are deleted; the amino acid at position 48, such as threonine T, is mutated to cysteine ​​C; the amino acid at position 112, such as serine S, is replaced by leucine L; the amino acid at position 114, such as methionine M, is replaced by isoleucine I; and the amino acid at position 115, such as glycine G, is replaced by valine V.

23. The combination according to any one of claims 17-22, wherein the TCRα chain constant region is missing an intracellular region relative to the wild-type TCRα chain constant region, for example, the 136th-137th amino acid is missing.

24. The combination according to any one of claims 17-23, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, and is derived from the wild-type mouse TCRα chain constant region by lacking the intracellular region of the constant region, for example, by lacking amino acids 136-137.

25. The combination according to any one of claims 17-24, wherein the modified TCRα chain constant region comprises an amino acid sequence shown in one of SEQ ID NO: 3-7.

26. The combination according to any one of claims 16-25, wherein the second constant region is a natural TCRβ chain constant region, for example, a natural human TCRβ chain constant region or a natural mouse TCRβ chain constant region; or, the second constant region is a modified TCRβ chain constant region.

27. The combination of claim 26, wherein the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, wherein, relative to the wild-type mouse TCRβ chain constant region, the amino acid at position 56, for example serine S, is mutated to cysteine ​​C.

28. The combination according to claim 26 or 27, wherein the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, wherein, relative to the wild-type mouse TCRβ chain constant region, the amino acid at position 3, such as R, is replaced by K; the amino acid at position 6, such as T, is replaced by F; the amino acid at position 9, such as K, is replaced by E; the amino acid at position 11, such as S, is replaced by A; the amino acid at position 12, such as L, is replaced by V; and the amino acids at positions 17 and 21-25 are deleted.

29. The combination according to any one of claims 26-28, wherein the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, wherein, relative to the wild-type mouse TCRβ chain constant region, the amino acid at position 56, such as serine S, is mutated to cysteine ​​C; the amino acid at position 3, such as R, is substituted with K; the amino acid at position 6, such as T, is substituted with F; the amino acid at position 9, K, is substituted with E; the amino acid at position 11, S, is substituted with A; the amino acid at position 12, L, is substituted with V; and the amino acids at positions 17 and 21-25 are deleted.

30. The combination according to any one of claims 26-29, wherein the intracellular region of the TCRβ chain constant region is missing relative to the wild-type TCRβ chain constant region, for example, the intracellular region of the constant region is missing amino acids 167-172.

31. The combination according to any one of claims 26-30, wherein the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, which, relative to the wild-type mouse TCRβ chain constant region, lacks the intracellular region of the constant region, for example, lacking amino acids 167-172.

32. The combination according to any one of claims 26-31, wherein the modified TCRβ chain constant region comprises the amino acid sequence shown in one of SEQ ID NO:10-14.

33. The combination according to any one of claims 16-32, wherein the α chain and / or β chain, preferably the α chain and β chain, are connected at their C-termini to at least one exogenous intracellular functional domain, such as the intracellular domain of a co-stimulatory molecule, preferably the intracellular domain of OX40, and more preferably, the intracellular domain of OX40 comprises the amino acid sequence of SEQ ID NO:

15.

34. The combination according to claim 33, wherein the exogenous intracellular functional domain is directly or via a linker connected to the α chain and / or β chain, preferably the C-terminus of the constant region of the α chain and β chain.

35. The combination of any one of claims 16-34, wherein the target antigen is a disease-associated antigen, for example... The target antigen is a cancer-associated antigen, such as those selected from the following cancer-associated antigens: GPC3 (phosphatidylinositol proteoglycan 3), BCMA, mesothelin, LILRB4, CD16, CD64, CD78, CD96, CLL1, CD7, CD70, CD38, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), Claudin 18.2, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule. Epidermal growth factor receptor (EpCAM), EGFR variant III (EGFRvIII), CD19, CD276, CD19, CD22, CD20, CD30, CD40, disialotetrahexosylganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alpha fetal globulin (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostaglandin, prostaglandin-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, liver glycoside B2, CD22, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, major histocompatibility complex (MHC) molecule presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor matrix antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, A1 domain (TnC) of tendin-C 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, endothelial factors, major histocompatibility complex (MHC) molecules, TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5; or The target antigen is an antigen derived from a pathogen or a surface antigen of cells infected by the pathogen, such as RSVF (prevention of respiratory syncytial virus), PA (inhalation anthrax), CD4 (HIV infection); or The target antigens are antigens associated with other diseases, such as CD3 (involved in transplant rejection), CD25 (involved in acute kidney transplant rejection), C5 (involved in paroxysmal nocturnal hemoglobinuria), IL-1β (involved in cold pyridine-associated periodic syndrome), RANKL (involved in cancer-related bone injury), von Willebrand factor (involved in adult acquired thrombotic thrombocytopenic purpura), plasma kallikrein (involved in angioedema), calcitonin gene-related peptide receptor (involved in adult migraine), and FGF23 (involved in X-linked hypophosphatemia).

36. The combination according to any one of claims 16-35, wherein i) The α chain comprises a first antigen-binding region and a first constant region, and the β chain comprises a second constant region; ii) The α chain contains a first constant region, and the β chain contains a second antigen-binding region and a second constant region; or iii) The α chain comprises a first antigen-binding region and a first constant region, and the β chain comprises a second antigen-binding region and a second constant region.

37. The combination according to claim 36, wherein the first antigen-binding region and the second antigen-binding region each specifically bind to the target antigen independently or in combination.

38. The combination of claim 36 or 37, wherein the first antigen-binding region comprises a heavy chain variable region of an antibody that specifically binds to the target antigen, and the second antigen-binding region comprises a light chain variable region of the antibody; or, the first antigen-binding region comprises a light chain variable region of an antibody that specifically binds to the target antigen, and the second antigen-binding region comprises a heavy chain variable region of the antibody.

39. The combination of claim 36 or 37, wherein the first antigen-binding region comprises a single-chain antibody (e.g., scFv) or a single-domain antibody that specifically binds to the target antigen; and / or the second antigen-binding region comprises a single-chain antibody or a single-domain antibody that specifically binds to the target antigen.

40. The combination of claim 39, wherein the first antigen-binding region and the second antigen-binding region bind the same target antigen.

41. The combination according to claim 40, wherein the first antigen-binding region and the second antigen-binding region bind different regions (e.g., different epitopes) of the same target antigen; or the first antigen-binding region and the second antigen-binding region bind different target antigens.

42. The combination according to any one of claims 16-41, wherein the antigen-binding region comprises the heavy chain variable region shown in SEQ ID NO:21 and / or the light chain variable region shown in SEQ ID NO:22; or The antigen-binding region includes the heavy chain variable region shown in SEQ ID NO:29 and / or the light chain variable region shown in SEQ ID NO:28; or The antigen-binding region contains one or more amino acid sequences selected from SEQ ID NO:24-27.

43. A non-electrotransfer method for preparing genetically modified cells, comprising: Step 1) Provide the starting cells; Step 2) Contact the starting cell with any combination of claims 1-42; and Step 3) Harvest the cells obtained in Step 2).

44. The method of claim 43, wherein the expression vector, gene editing system, cell penetration reagent, and NHEJ inhibitor in the combination are contacted with the starting cells simultaneously or separately (sequentially).

45. The method of claim 43 or 44, wherein the initiating cell may be an immune cell such as a T cell, natural killer cell, dendritic cell, macrophage, or hematopoietic stem cell or nerve cell.

46. ​​The method of any one of claims 43-45, wherein step 2) of "contacting the starting cells with the combination of the invention" comprises incubating the starting cells and the combination in an incubation system for a period of time.

47. The method of any one of claims 43-46, wherein the method, as in step 2), does not include the electroporation step.

48. The method of any one of claims 43-47, wherein the method is used to prepare therapeutic immune cells, such as therapeutic T cells.

49. The method of claim 48, wherein the therapeutic immune cell, such as a therapeutic T cell, comprises a synthetic T cell receptor antigen receptor (STAR), and one or more endogenous genes selected from TRAC, HLA-A, HLA-B, and CIITA in the therapeutic immune cell are partially or completely inactivated.

50. The method of claim 49, wherein the method comprises: Step 1) Provide initial immune cells such as T cells; Step 2) Contact the starting cell with a combination comprising: i) AAV vectors containing expression cassettes of CAR, TCR, or STAR. ii) Target one or more of the Cas12a gene editing systems selected from TRAC, HLA-A, HLA-B and CIITA; iii) Cell penetration agents such as TAT-HA2, and / or NEHJ inhibitors such as AZD7648; and Step 3) Harvest the cells obtained in Step 2).

51. The method of claim 50, wherein the expression cassette of the CAR, TCR or STAR includes a 5' homologous arm (5'HA) as shown in SEQ ID NO:18 at its 5' end and a 3' homologous arm (3'HA) as shown in SEQ ID NO:19 at its 3' end, thereby allowing it to be inserted into the intracellular TRAC gene at the location defined by SEQ ID NO:18 and SEQ ID NO:

19.

52. The method of claim 50 or 51, wherein step 1) comprises isolating and activating the immune cells, such as T cells.

53. The method of any one of claims 50-52, wherein step 1) is performed on day 0, or on day 1, day 2, or day 3, preferably starting on day 2.

54. The method of any one of claims 50-53, wherein step 2) comprises: 2-1) Incubate the Cas12a nuclease with one or more guide RNAs targeting TRAC, HLA-A, HLA-B, and CIITA for the first time period to form a ribonucleoprotein complex (RNP); 2-2) Incubate the RNP with the initiating immune cells, the AAV vector, and a cell penetration reagent such as TAT-HA2 for a second time period; and 2-3) The product obtained in 2-2) is cultured in a cell culture medium, preferably, the cell culture medium contains an NHEJ inhibitor such as AZD7648.

55. The method of claim 54, wherein the first time period is approximately 10-30 minutes, preferably approximately 15 minutes. In some embodiments, the second time period is approximately 15-60 minutes, preferably approximately 30 minutes.

56. The method of any one of claims 50-53, wherein step 2) comprises: 2-1) Incubate the Cas12a nuclease with one or more guide RNAs targeting TRAC, HLA-A, HLA-B, and CIITA for the first time period to form a ribonucleoprotein complex (RNP); 2-2) Incubate the RNP with the initiating immune cells and the AAV vector for a second time period; 2-3) Incubate the product obtained in 2-2) with a cell penetration reagent such as TAT-HA2 for the third time period; and 2-4) The product obtained in 2-3) is cultured in a cell culture medium, preferably, the cell culture medium contains an NHEJ inhibitor such as AZD7648.

57. The method of claim 56, wherein the first time period is about 10-30 minutes, preferably about 15 minutes; the second time period is about 15-60 minutes, preferably about 30 minutes; and the third time period is about 15-60 minutes, preferably about 30 minutes.

58. The method of any one of claims 50-53, wherein step 2) is performed separately on the second day and the fourth day.

59. The method of claim 58, step 2) comprises: 2-1) On the second day, the Cas12a nuclease is incubated with the guide RNA targeting HLA-A and HLA-B for a first time period to form a ribonucleoprotein complex (RNP). The RNP is then incubated with the initiating immune cells and a cell penetration reagent such as TAT-HA2 for a second time period, and the resulting cells are cultured in cell culture medium. 2-2) On day 4, the Cas12a nuclease is incubated with guide RNA targeting CIITA and TRAC for the first time period to form a ribonucleoprotein complex (RNP). The RNP is then incubated with the cells obtained in 2-1), the AAV vector, and a cell penetration reagent such as TAT-HA2 for the second time period. The resulting cells are then cultured in cell culture medium. Preferably, the cell culture medium contains an NHEJ inhibitor such as AZD7648.

60. The method of claim 58, step 2) comprises: 2-1) On the second day, the Cas12a nuclease is incubated with guide RNA targeting CIITA, HLA-A and HLA-B for a first time period to form a ribonucleoprotein complex (RNP). The RNP is then incubated with the initiating immune cells and cell penetration reagent such as TAT-HA2 for a second time period, and the resulting cells are cultured in cell culture medium. 2-2) On day 4, the Cas12a nuclease is incubated with the guide RNA targeting TRAC for the first time period to form a ribonucleoprotein complex (RNP). The RNP is then incubated with the cells obtained in 2-1), the AAV vector, and a cell penetration reagent such as TAT-HA2 for the second time period. The resulting cells are then cultured in cell culture medium. Preferably, the cell culture medium contains an NHEJ inhibitor such as AZD7648.

61. Therapeutic immune cells, such as T cells, that can be obtained or acquired by any combination of claims 1-42 or by any method of claims 43-60.

62. A pharmaceutical composition comprising the therapeutic immune cells of claim 61 and a pharmaceutically acceptable carrier.

63. Use of the therapeutic immune cells of claim 61 and / or the pharmaceutical composition of claim 62 in the preparation of a medicament for treating a disease in a subject.

64. A method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the therapeutic immune cells of claim 61 and / or the pharmaceutical composition of claim 62.

65. The use of claim 63 or the method of claim 64, wherein the disease is, for example, cancer, and examples of such cancers include, but are not limited to, myeloma, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, uterine fibroids, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer. Adenocarcinoma, adrenal carcinoma, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of the aforementioned cancers; or The disease is, for example, an infection caused by a pathogen, including but not limited to respiratory syncytial virus, anthrax, and human immunodeficiency virus; or The diseases mentioned include, for example, cardiovascular diseases, diabetes, neurological diseases, post-transplant rejection, or other diseases; or The disease can also be an autoimmune disease, including but not limited to systemic lupus erythematosus (SLE), myositis, scleroderma, Sjögren's syndrome, autoimmune hemolytic anemia, and rheumatoid arthritis.

66. The use of claim 63 or the method of claim 64, The disease is a phosphatidylinositol proteoglycan-3 (GPC3)-related disease, such as a disease related to abnormal GPC3 expression, or a GPC3-related cancer. Examples of such cancers include liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., squamous cell carcinoma of the lung, SqCC), gastric cancer, ovarian cancer, melanoma, or pediatric embryonal tumors; or The disease is a CD19-related disease, such as a disease related to abnormal CD19 expression, such as CD19-related cancer. The cancer can be a B-cell malignancy, such as chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphocytic lymphoma, non-Hodgkin's lymphoma, and combinations of the aforementioned cancers. Alternatively, the disease is an autoimmune disease, such as systemic lupus erythematosus; or The disease is a B-cell maturation antigen (BCMA)-related disease, such as a disease related to abnormal BCMA expression, such as BCMA-related cancer. The cancer is, for example, myeloma, such as multiple myeloma (MM), especially relapsed or refractory multiple myeloma (RRMM); or an autoimmune disease, including but not limited to systemic lupus erythematosus (SLE), myositis, scleroderma, Sjögren's syndrome, autoimmune hemolytic anemia, rheumatoid arthritis; or The disease is a mesothelin (MSLN)-related disease, such as a disease related to abnormal MSLN expression, or MSLN-related cancers. Examples of such cancers include ovarian cancer, breast cancer, colorectal cancer, and pancreatic cancer; or The disease in question is a leukocyte immunoglobulin-like receptor B4 (LILRB4) related disease, such as a disease related to abnormal LILRB4 expression, or LILRB4-related cancer. The cancer, for example, is leukemia, particularly acute myeloid leukemia.

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