Compositions and methods for gene editing in T cells using CRISPR / CPF1

Abstract

To provide a composition and method for modifying primary T cells.SOLUTION: The present invention is a method of gene editing comprising administering stem-loop CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (st-crRNA) and Cpf1 enzyme to a cell.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 501,371, filed May 4, 2017, which is hereby incorporated by reference in its entirety.

[0002] Statement Regarding Federally Sponsored Research or Development This invention was made with government support under Grant No. 2R01CA120409 from the National Institutes of Health. The U.S. government has certain rights in this invention.

Background Art

[0003] Background of the Invention Initial clinical data on chimeric antigen receptor (CAR) T cells for cancer treatment have shown promising results. However, the development of CAR immunotherapy for cancer and infectious diseases is hampered by the lack of immediately available, highly potent, antigen - and patient - specific T lymphocytes. Treating patients with highly individualized CAR T - cell immunotherapy can be quite costly and time - consuming. Autologous T cells from patients with advanced disease may be dysfunctional and tolerant to the desired antigen, thus necessitating the modification of allogeneic donor - derived T cells. Furthermore, the endogenous αβ T - cell receptor on the injected allogeneic T cells can recognize major and minor histocompatibility antigens in the recipient, potentially leading to graft - versus - host disease (GVHD). As a result, most current clinical trials injecting autologous CAR T cells rely on immune tolerance to prevent the TCR - mediated harmful recognition of normal tissues after transplantation. Although this approach has achieved initial clinical success, the time and cost required to produce patient - specific T - cell products are limiting factors. Therefore, there is a need for a safer method of modifying T cells while minimizing the time and cost required to produce patient - specific T - cell products.

[0004] While several reports claim success in creating general-purpose T cells that evade GVHD by inhibiting TCR expression, allogeneic T cells can still be rejected by the host immune system through HLA-A molecule recognition. Due to the complexity of targeting strategies manipulating multiple genes, as well as the low efficiency of ZFNs and TALENs in T cells, no studies have yet achieved the goal of simultaneously preventing GVHD and host-versus-graft response. To create truly general-purpose CART cells, complete depletion of TCR α-chain, β-chain, and β-2 microglobin must be achieved.

[0005] T cell genomic manipulation is highly promising for cell therapies against cancer, HIV, primary immunodeficiency, and autoimmune diseases, but genetically manipulating human T cells remains a challenge. While CRISPR / Cas9 technology has facilitated genomic manipulation in many cell types, including T cells, the gene-editing capabilities of CRISPR / Cpf1 in human T cells remain largely hypothetical.

[0006] There is a demand for novel compositions and methods for therapeutic genome manipulation techniques in primary human T cells. This invention addresses this demand. [Overview of the project]

[0007] As described herein, the present invention relates to compositions and methods for gene editing.

[0008] One aspect of the present invention includes a gene editing method comprising the step of administering to cells an exogenous nucleic acid containing stem-loop CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (st-crRNA) and an exogenous nucleic acid encoding the Cpf1 enzyme.

[0009] Another aspect of the present invention includes a method for producing modified T cells by gene editing. This method includes administering exogenous nucleic acids, including st-crRNA and exogenous nucleic acids encoding the Cpf1 enzyme, to T cells.

[0010] Another aspect of the present invention includes genetically modified cells comprising exogenous nucleic acids encoding st-crRNA and exogenous nucleic acids encoding the Cpf1 enzyme.

[0011] Another aspect of the present invention includes a method of adoptive cell transfer therapy. This method includes administering a population of modified cells, including the modified cells of the present invention, to a subject in need thereof.

[0012] In another aspect, the present invention includes a method for treating a disease or condition in a subject. The method includes administering a population of modified cells, including the modified cells of the present invention, to a subject in need.

[0013] In various aspects of the above and any other aspects of the present invention described herein, the cell is a T cell. In one aspect, the T cell is a primary T cell.

[0014] In one embodiment, the administration step includes electroporating the exogenous nucleic acid into cells. In one embodiment, electroporation is performed multiple times.

[0015] In one embodiment, the Cpf1 enzyme includes Acidaminococcus Cpf1 (AsCpf1). In another embodiment, the Cpf1 enzyme includes Lachnospiraceae Cpf1 (LbCpf1).

[0016] In one embodiment, st-crRNA contains a stem-loop structure at the 3' end of crRNA. In one embodiment, st-crRNA contains a stem-loop structure at the 5' end of crRNA. In one embodiment, the stem-loop structure further contains three glycine residues added to the 5' end of the stem-loop. In one embodiment, the protospacer region of st-crRNA further contains partial phosphorothioate (PMS) modification.

[0017] In one embodiment, gene editing involves mutating a gene selected from the group consisting of: the TCR α-chain constant region (TRAC), the TCR β-chain constant region (TRBC), and β-2 microglobulin (B2m).

[0018] In one embodiment, the disease or condition is selected from the group consisting of infectious diseases, autoimmune diseases, and cancer. In one embodiment, the method of the present invention further includes a step of providing secondary treatment for the disease or condition.

[0019] In one embodiment, the subject is a human being. [Invention 1001] A gene editing method comprising the step of administering exogenous nucleic acids, including stem-loop CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (st-crRNA), and exogenous nucleic acids encoding the Cpf1 enzyme, to cells. [Invention 1002] The method of the present invention 1001, wherein the cell is a T cell. [Invention 1003] The method of the present invention 1002, wherein the T cells are primary T cells. [Invention 1004] The method of the present invention 1001, wherein the administration step includes electroporating the exogenous nucleic acid into cells. [Invention 1005] A method according to the present invention 1004, wherein electroporation is performed multiple times. [Invention 1006] The method of the present invention 1001, wherein the Cpf1 enzyme comprises Acidaminococcus Cpf1 (AsCpf1). [The present invention 1007] The method of the present invention 1001, wherein the Cpf1 enzyme comprises Lachnospiraceae Cpf1 (LbCpf1). [The present invention 1008] The method of the present invention 1001, wherein the st-crRNA comprises a stem-loop structure at the 3' end of the crRNA. [The present invention 1009] The method of the present invention 1001, wherein the st-crRNA comprises a stem-loop structure at the 5' end of the crRNA. [The present invention 1010] The method of the present invention 1009, wherein the stem-loop structure further comprises three glycine residues added to the 5' end of the stem-loop. [The present invention 1011] The method of the present invention 1010, wherein the protospacer region of the st-crRNA further comprises a partial phosphorothioate (PMS) modification. [The present invention 1012] The gene editing is TCR α constant region (TRAC), TCR β constant region (TRBC), and β-2 microglobulin (B2m) The method of the present invention 1001, which comprises mutating a gene selected from the group consisting of [The present invention 1013] A method for producing a modified T cell by gene editing, comprising administering to the T cell an exogenous nucleic acid comprising st-crRNA and an exogenous nucleic acid encoding a Cpf1 enzyme. [The present invention 1014] The method of the present invention 1013, wherein the T cell is a primary T cell. [The present invention 1015] The method of the present invention 1013, wherein the administering step comprises electroporating the nucleic acid into the T cell. [The present invention 1016] The method of the present invention 1015, wherein the electroporation is performed multiple times. [The present invention 1017] The method of the present invention 1013, wherein the Cpf1 enzyme contains Cpf1 (AsCpf1) of the genus Salvia. [Invention 1018] The method of the present invention 1013, wherein the Cpf1 enzyme contains Lachnospira family Cpf1 (LbCpf1). [Invention 1019] The method of the present invention 1013, wherein the st-crRNA contains a stem-loop structure at the 3' end of the crRNA. [Invention 1020] The method of the present invention 1013, wherein st-crRNA contains a stem-loop structure at the 5' end of crRNA. [Invention 1021] The method of the present invention 1020, wherein the stem-loop structure further comprises three glycine residues attached to the 5' end of the stem-loop. [Invention 1022] The method of the present invention 1021, further comprising partial phosphorothioate (PMS) modification of the stem-loop structure. [Invention 1023] Gene editing, TCR α-chain constant region (TRAC), TCR β-chain constant region (TRBC), and β-2 microglobulin (B2m) The method of the present invention 1013, comprising mutating a gene selected from the group consisting of the following. [Invention 1024] Genetically modified cells containing exogenous nucleic acids encoding st-crRNA and exogenous nucleic acids encoding the Cpf1 enzyme. [Invention 1025] A T cell, which is a genetically modified cell according to the present invention 1024. [Invention 1026] Genetically modified cells according to Invention 1025, in which the T cells are primary T cells. [Invention 1027] Genetically modified cells according to Invention 1024, wherein the Cpf1 enzyme contains Cpf1 (AsCpf1) of the genus Salvia. [Invention 1028] Genetically modified cells according to Invention 1024, wherein the Cpf1 enzyme contains Lacnospira family Cpf1 (LbCpf1). [Invention 1029] Genetically modified cells according to Invention 1024, wherein st-crRNA contains a stem-loop structure at the 5' end of the crRNA. [Invention 1030] Genetically modified cells according to Invention 1024, wherein st-crRNA contains a stem-loop structure at the 3' end of the crRNA. [Invention 1031] Genetically modified cells according to the present invention 1030, wherein the stem-loop structure further includes three glycine residues added to the 5' end of the stem-loop. [Invention 1032] Genetically modified cells according to the present invention 1031, wherein the protospacer region of st-crRNA further comprises partial phosphorothioate (PMS) modification. [Invention 1033] A method for adoptive cell transfer therapy, comprising the step of administering a population of modified cells, including any modified cells according to items 1024 to 1032 of the present invention, to a subject in need thereof. [Invention 1034] A method for treating a disease or condition in a subject, comprising the step of administering to a subject in need of such treatment a population of modified cells, including any of the modified cells described in items 1024 to 1032 of the present invention. [Invention 1035] The method of the present invention 1034, wherein the disease or condition is selected from the group consisting of infectious diseases, autoimmune diseases, and cancer. [Invention 1036] Any method according to invention 1033 to 1034, wherein the subject is a human. [Invention 1037] Any method of the present invention 1033 to 1034, further comprising the step of providing secondary treatment for a disease or condition. [Brief explanation of the drawing]

[0020] The following detailed description of specific embodiments of the present invention will be better understood in conjunction with the accompanying drawings. Exemplary embodiments are shown in the drawings to illustrate the present invention. However, it should be understood that the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings. [Figure 1A] Figures 1A–1E are a series of plots and images illustrating how chemical modifications and stem-loop structures of crRNAs enable efficient gene targeting by Cpf1. Figure 1A illustrates the structures of crRNAs and sgRNAs, as well as schematic designs of stem-loop and chemical modification sites. Gray text: stem-loop; asterisk: modification. Figure 1B depicts the gene targeting efficiency of crRNAs with various stem-loop structures. No: no crRNA; Un: unmodified crRNA; Lst: left stem-loop; Mst: central stem-loop; Rst: right stem-loop. TRAC and TRBC disruption was determined by measuring CD3 expression on T cells by flow cytometry. B2m disruption was determined by measuring B2m or HLA-I expression on T cells by flow cytometry. Figure 1C is an explanatory diagram of chemically modified PMS-crRNA. MS-crRNA: 2'-O-methyl3'-phosphorothioate-modified crRNA; PMS-crRNA: phosphorothioate-modified MS-crRNA. Figure 1D shows the gene targeting efficiency of chemically modified crRNA after a single electroporation. FPMS-crRNA: fully phosphorothioate-modified PMS-crRNA. Figure 1E shows the gene targeting efficiency of chemically modified crRNA after multiple electroporations. [Figure 1B] See the explanation in Figure 1A. [Figure 1C] See the explanation in Figure 1A. [Figure 1D] See the explanation in Figure 1A. [Figure 1E] See the explanation in Figure 1A. [Figure 2A]Figures 2A-2B are a series of plots illustrating the stringent guide selectivity of Cpf1 in primary T cells. Figure 2A shows guides that work favorably for various RGENs. Three genes, TRAC, TRBC, and B2m, were edited in primary T cells using Cpf1 and wild-type Cas9 and high-fidelity Cas9 (eSpCas9). Ten different guide RNAs were examined for each gene. Gene disruption was measured based on protein expression by flow cytometry. Figure 2B shows the guide length requirements for various RGENs. TRAC, TRBC, and B2m were targeted with various RGENs that have truncated guide RNAs. Gene disruption was measured based on protein expression by flow cytometry. [Figure 2B] See the explanation in Figure 2A. [Figure 3A]Figures 3A–3C are a series of plots illustrating the enhancement of gene targeting specificity by Cpf1 in primary T cells. Gene ablation of TRAC, TRBC, and B2m was performed to measure off-target events for various RGENs. The frequency of mutagenesis was measured by the T7E1 assay. Gene targeting was performed using either a 20-mer guide RNA (Figure 3A) or an 18-mer guide RNA (Figure 3B), and the off-target effects of various RGENs were investigated using the mutated guide RNA. The frequency of mutagenesis at target and off-target sites was calculated by the T7E1 assay. Figure 3C shows the decrease in Cpf1's off-target capability, confirmed by gene targeting using mutated guide RNA. Nonspecific guiding of Cpf1 or Cas9 was investigated using guide RNA with single base pair mutations. A single mutation within 10 base pairs adjacent to the PAM site reduced Cas9 targeting efficiency, while a single mutation proximal to the PAM site did not significantly affect gene targeting efficiency. A single base pair mutation in the guide RNA greatly reduced Cpf1's gene disruption ability, indicating that Cpf1's target recognition is more specific than that of Cas9. [Figure 3B] See the explanation in Figure 3A. [Figure 3C] See the explanation in Figure 3A. [Figure 4] Figure 4 shows a series of plots illustrating the enhancement of gene disruption by stem-loop crRNA in primary T cells. Figure 4A shows a comparison of the gene disruption efficiency of Cas9, AsCpf1, and LbCpf1 in primary T cells using a multi-pass electroporation protocol. Figure 4B shows the enhancement of Cpf1 gene disruption by stem-loop crRNA. In the multi-pass electroporation protocol, nearly twice the gene disruption was observed when using stem-loop crRNA compared to wild-type crRNA. [Figure 5]Figure 5 is a plot illustrating the loss of function due to chemical modification of the crRNA handle structure. It shows TRAC disruption using handle "AA" chemically modified crRNA via single and multiple electroporation protocols. [Modes for carrying out the invention]

[0021] Detailed explanation definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in practice for testing the present invention, but preferred materials and methods are described herein. The following technical terms are used in describing and claiming the present invention:

[0022] Furthermore, it should be understood that the technical terms used in this specification are intended solely to describe specific embodiments and are not intended to be restrictive.

[0023] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “one element” means one or more elements.

[0024] When referring to measurable values ​​such as quantity or duration, the term "about" as used herein includes a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variation is reasonable for carrying out the disclosed method.

[0025] As used herein, the term "activation" refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" specifically refers to T cells undergoing cell division.

[0026] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be intact immunoglobulins derived from natural or recombinant sources, or they may be the immune reaction portion of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in this invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as single-chain antibodies (scFv), humanized antibodies and human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0027] The term "antibody fragment" refers to a portion of an intact antibody, specifically the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, ScFv antibodies, and multispecific antibodies formed from antibody fragments.

[0028] As used herein, "antibody heavy chain" refers to the longer of the two polypeptide chains present in all antibody molecules in their natural higher-order structure.

[0029] In this text, "antibody light chain" refers to the shorter of the two polypeptides present in all antibody molecules in their natural higher-order structure. α-light chain and β-light chain refer to the two main antibody light chain isotypes.

[0030] As used herein, "synthetic antibody" means an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage as described herein. This term should also be considered to mean an antibody produced by the synthesis of an antibody-encoding DNA molecule, wherein the DNA molecule expresses an antibody protein or an amino acid sequence that designates the antibody, and the DNA or amino acid sequence is obtained using DNA or amino acid sequence synthesis techniques that are available and well known in the art.

[0031] The terms “antigen” or “Ag,” as used herein, are defined as molecules that induce an immune response. This immune response may include either or both antibody production or activation of specific immune-qualified cells. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can act as an antigen. Furthermore, antigens may be derived from recombinant DNA or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response, therefore, encodes an “antigen” as the term is used herein. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded by the full-length nucleotide sequence of a gene. It is immediately apparent that the present invention non-limitingly involves the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences are arranged in various combinations that elicit a desired immune response. Moreover, those skilled in the art will understand that antigens do not necessarily have to be encoded by a “gene.” It is immediately apparent that antigens can be synthesized or obtained from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0032] As used herein, the term “self” refers to any material originating from the same individual that is later reintroduced into that individual.

[0033] "Same species" refers to any material derived from different animals of the same species.

[0034] "Different species" refers to any material derived from animals of different species.

[0035] The terms “chimeric antigen receptor” or “CAR,” as used herein, refer to an artificial T cell receptor that has been engineered to be expressed on immune effector cells and specifically bind to an antigen. CARs can be used as a therapeutic agent involving adoptive cell transfer. T cells are removed from a patient and modified to express a receptor that is specific to a particular form of antigen. In some embodiments, CARs have specificity to a selected target, such as a B cell surface receptor. CARs may also include an extracellular domain containing an intracellular activation domain, a transmembrane domain, and a tumor-associated antigen-binding region. In some aspects, CARs include an extracellular domain containing an anti-B cell-binding domain fused with a CD3-ζ transmembrane domain and an intracellular domain.

[0036] The term "cleavage" refers to the breaking of covalent bonds in the backbone of nucleic acid molecules, or the hydrolysis of peptide bonds. Cleavage can be initiated by a variety of methods, non-limitingly including enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand breaks are possible. Double-strand breaks can occur as a result of two different single-strand break events. DNA breaks can result in the formation of either blunt or adherent ends. In some embodiments, fusion polypeptides can be used to target cleaved double-stranded DNA.

[0037] "Disease" refers to a state of animal health in which the animal is unable to maintain homeostasis, and whose health will continue to deteriorate unless the disease is improved. In contrast, "disorder" in animals refers to a state of health in which the animal is able to maintain homeostasis, but whose health is worse than it would be in the absence of the disorder. Even if left untreated, a disorder does not necessarily lead to a further decline in the animal's health.

[0038] As used herein, the term "downregulation" refers to a reduction or loss of gene expression of one or more genes.

[0039] "Effective dose" or "therapeutic effective dose" is used interchangeably herein and refers to the amount of any compound, formulation, material or composition described herein that is effective in achieving a particular biological outcome or in providing a therapeutic or prophylactic benefit. Such outcomes may, but are not limited to, include antitumor activity as determined by any suitable means in the art.

[0040] "Code" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide such as a gene, cDNA, or mRNA, which has either a given nucleotide sequence (i.e., rRNA, tRNA, or mRNA) or a given amino acid sequence, and the resulting biological properties, and acts as a template for the synthesis of other polymers and macromolecules in biological processes. In other words, a gene codes for a protein when the transcription and translation of the mRNA corresponding to that gene produces that protein in a cell or other biological system. Both the coding strand, which has a nucleotide sequence identical to the mRNA sequence and is usually presented as a sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, can be said to code for a protein, or other products of that gene or cDNA.

[0041] As used herein, “endogenous” means any material that originates from or is produced within a living organism, cell, tissue, or system.

[0042] As used herein, the term “exogenous” means any material that is introduced into or produced outside of a living organism, cell, tissue, or system.

[0043] The term "increased," as used herein, refers to an increase in number, such as in the case of an increase in the number of T cells. In one aspect, the number of T cells increased ex vivo is greater than the number originally present in the culture. In another aspect, the number of T cells increased ex vivo is greater than the number of other cell types in the culture. The term "ex vivo," as used herein, refers to cells that have been removed from a living organism (e.g., a human) and grown outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0044] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence that is acted upon by its promoter.

[0045] An "expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence functionally linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus), incorporating recombinant polynucleotides.

[0046] As used herein, the term "immune response" is defined as a cellular response to an antigen in which lymphocytes recognize an antigen molecule as foreign and induce the formation of antibodies to eliminate the antigen, and / or activate the lymphocytes.

[0047] When an "immunologically effective dose," "autoimmune disease inhibitory dose," or "therapeutic dose" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician or researcher, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the patient's (subject's) condition.

[0048] As used herein, “explanatory materials” include publications, records, diagrams, or any other expressive medium that can be used to convey the usefulness of the compositions and methods of the present invention. The explanatory materials for the kit of the present invention may, for example, be attached to the container containing the nucleic acids, peptides, and / or compositions of the present invention, or may be shipped together with the container containing the nucleic acids, peptides, and / or compositions. Alternatively, the explanatory materials may be shipped separately from the container, with the intention that the explanatory materials and compounds be used collaboratively by the recipient.

[0049] "Isolated" means that it has been modified or removed from its natural state. For example, a nucleic acid or peptide that exists naturally in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the substance that coexists with it in its natural state is "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form, or they may exist in a non-native environment, such as a host cell.

[0050] As used herein, the term "knockdown" refers to a reduction in the gene expression of one or more genes.

[0051] As used herein, the term "knockout" refers to the loss of gene expression in one or more genes.

[0052] The term "modified" as used herein means an altered state or structure of the molecules or cells of the present invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0053] The term “modulate” means mediating a detectable increase or decrease in the level of response in a subject compared to the level of response in the subject in the absence of the treatment or compound, and / or compared to the level of response in an otherwise identical but untreated subject. This term includes, in a subject, preferably a human, disrupting and / or influencing native signals or responses, thereby mediating a beneficial therapeutic response.

[0054] In connection with the present invention, the following abbreviations are used for commonly existing nucleic acid bases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0055] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotides that are degenerate and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may include introns, insofar as protein-coding nucleotide sequences may contain introns in some types.

[0056] Parenteral administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion.

[0057] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art have general knowledge that nucleic acids are polynucleotides and that they can be hydrolyzed to monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR®, and synthetic means.

[0058] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, also commonly called peptides, oligopeptides, and oligomers in the art, and long chains, both commonly called proteins in the art, of which there are many types. Examples of “polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0059] The term "stimulation" refers to a primary response induced by a stimulating molecule (e.g., the TCR / CD3 complex) binding to its cognitive ligand, thereby mediating a signaling event, such as, but not limited to, signaling mediated by the TCR / CD3 complex. Stimulation can mediate alterations to the expression of certain molecules, such as the downregulation of TGF-β, and / or rearrangements of the cytoskeleton.

[0060] As used herein, "stimulating molecule" refers to a molecule on a T cell that specifically binds to a cognitive stimulating ligand present on an antigen-presenting cell.

[0061] As used herein, "stimulating ligand" means a ligand that, when present on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.), specifically binds to a cognitive-binding partner on T cells (referred to herein as "stimulating molecule"), thereby mediating a primary response by T cells, including but not limited to activation, initiation of an immune response, and proliferation. Stimulating ligands are well known in the art and, in particular, include peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0062] The term “subject” is intended to include living organisms (e.g., mammals) capable of eliciting an immune response. “Subject” or “patient” may, where used, be human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as sheep, bovines, pigs, canids, felines, and rodents. Preferably, the subject is human.

[0063] As used herein, “substantially purified” cells mean cells that essentially contain no other cell types. Substantially purified cells also mean cells isolated from other cell types that normally accompany them in their native state. In some cases, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to cells isolated from cells that normally accompany them in their native state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0064] A "target site" or "target sequence" refers to a genomic nucleic acid sequence that clearly defines the portion of the nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0065] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. The TCR is responsible for the recognition of antigens bound to major histocompatibility complex molecules. The TCR is composed of an alpha (a) and beta (β) chain heterodimer, although in some cells, the TCR consists of γ and δ (γ / δ) chains. The TCR may also exist in α / β and γ / δ forms, which are structurally similar but differ in anatomical location and function. Each chain consists of two extracellular domains, one variable domain, and one constant domain. In some embodiments, the TCR can be modified on any cell containing a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0066] As used herein, the term "therapeutic" means treatment and / or prevention. Therapeutic effects are obtained by suppression, remission, or eradication of a disease state.

[0067] The terms “transfected,” “transformed,” or “transduced,” as used herein, refer to the process by which an exogenous nucleic acid is introduced into a host cell. A “transfected,” “transformed,” or “transduced” cell is one that has been transfected, transformed, or transduced with an exogenous nucleic acid. This includes primary target cells and their offspring.

[0068] To “treat” a disease, as used herein, means to reduce the frequency or severity of at least one sign or symptom of the disease or disorder that the subject is suffering from.

[0069] A "vector" is a composition containing isolated nucleic acids that can be used to deliver those isolated nucleic acids into a cell. Numerous vectors, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses, are known in the art. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmidal and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

[0070] Scope: Throughout this disclosure, various aspects of the invention can be presented in range form. It should be understood that descriptions in range form are merely for convenience and brevity and should not be considered inflexible limitations on the scope of the invention. Therefore, a range description should be considered to specifically disclose individual numbers as well as all possible partial ranges within that range. For example, a range description such as 1 to 6 should be considered to specifically disclose individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6, as well as partial ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6. This applies regardless of the breadth of the range.

[0071] explanation The present invention provides compositions and methods for the creation and use of genetically modified cells. Genetically modified cells include exogenous nucleic acids encoding stem-loop CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (st-crRNA) and exogenous nucleic acids encoding the Cpf1 enzyme. One aspect of the present invention includes methods for gene editing in cells and methods for creating modified cells. It also includes methods and pharmaceutical compositions containing modified cells for adoptive therapy and for treating diseases or conditions.

[0072] The data disclosed herein demonstrate efficient genomic manipulation techniques in human T cells using Cpf1 and crRNA. CRISPR / Cpf1 gene editing resulted in the loss of the TCR α chain, an essential component of the TCR / CD3 complex. Cpf1 gene editing resulted in the loss of high levels of TCR surface expression in up to 80% of cells. These results confirm the potential of CRISPR / Cpf1 for use in a variety of experimental and therapeutic genomic manipulation applications in primary human T cells.

[0073] CRISPR / Cpf1 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CR1SPR-related (Cas) proteins, which provide adaptive immunity to heterologous nucleic acids in bacteria, are being used for another purpose: targeted genome editing in human cells and other cell types, and even in animals and plants. CRISPR / Cas9 technology originated from the Type II CRISPR / Cas system, which consists of one DNA endonuclease protein, Cas9, and two small RNAs: CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). These small RNAs, or chimeric single guide RNAs (sgRNAs), bind to Cas9, thereby forming an RNA-guided DNA endonuclease (RGEN) complex that cleaves specific DNA targets. The double-strand blunt-end breaks (DSBs) of chromosomes are subsequently repaired via homologous recombination (HR) or non-homologous end joining (NHEJ), resulting in genetic modification.

[0074] Cpf1 is another type of RGEN derived from the V-type CRISPR system. Cpf1 differs from Cas9 in several ways. Firstly, Cpf1 requires only crRNA, not a crRNA / tracrRNA pair, for its function. Secondly, Cas9 cleavage produces blunt double-spindle breaks (DSBs), while Cpf1 cleavage produces adherent ends. Thirdly, Cpf1 recognizes thymidine-rich DNA sequences such as protospacer-adjacent motifs (PAMs) (e.g., 5'-TTTN-3') at the 5' end of the target sequence. These characteristics of Cpf1 allow for a wider range of genomic sites editable by CRISPR-endonucleases than guanosine-rich sequences recognized by various Cas9 enzymes. Cpf1 requires only crRNA and does not utilize tracrRNA, and Cpf1 crRNA is significantly shorter than the nearly 100-nucleotide sgRNA required by Cas9, making guide RNA preparation cheaper and easier. This characteristic is particularly useful when using chemically modified guides. Recent studies have shown that chemically modified sgRNAs enhance gene targeting efficiency in primary human cells; however, prior to this study, the extent to which chemically modified crRNAs can assist Cpf1-mediated gene targeting had not been investigated.

[0075] Based on whole-genome off-target analysis, researchers have reported that Cpf1 exhibits better gene targeting specificity than Cas9 in certain cell types. The specificity of Cpf1 in T cells remains unclear.

[0076] Genome editing targeting T cells holds great potential for facilitating T cell-based cancer immunotherapy. Gene editing in primary T cells using Cas9-sgRNA has been widely studied. However, the gene-editing capabilities of Cpf1 in T cells have not yet been investigated. This study investigated the effects of structural and chemical modifications to crRNA, as well as the efficiency and specificity of gene editing using Cpf1-crRNA in T cells.

[0077] composition One aspect of the present invention includes genetically modified cells. In one aspect, the present invention includes genetically modified cells comprising an exogenous nucleic acid encoding st-crRNA and an exogenous nucleic acid encoding the Cpf1 enzyme. Genetically modified cells may be any type of cell, not limited to T lymphocytes, B lymphocytes, NK cells, monocytes, macrophages, neutrophils, epithelial cells, hematopoietic stem cells, and induced pluripotent stem cells (iPS).

[0078] In one aspect, the genetically modified cells are human cells. In another aspect, the cells, the genetically modified cells, are autologous cells.

[0079] In one embodiment, the genetically modified cell is a T cell. The T cell can be any type of T cell known in the art, including but not limited to CD3+ cells, CD4+ cells, CD8+ cells, regulatory T cells (Treg), helper T cells (Th1 and Th2), cytotoxic T cells (CTLs), natural killer T cells (NKT cells), γδT cells, effector T cells, memory T cells, and naive T cells. In one embodiment, the genetically modified T cell is a primary T cell. In another embodiment, the cell, the genetically modified cell, is an autologous T cell.

[0080] The genetically modified cells of the present invention contain an exogenous nucleic acid encoding the Cpf1 enzyme. The Cpf1 enzyme may originate from any genera of microorganisms, including, but not limited to, Parcubacteria, Lachnospiraceae, Butyrivibrio, Peregrinibacteria, Tinecoccus, Porphyromonas, Lachnospiraceae, Porphromonas, Prevotella, Moraxela, Smithella, Leptospira, Lachnospiraceae, Francisella, Candidatus, and Eubacterium. In one embodiment, Cpf1 is derived from a species of the genus Tinecoccus (AsCpf1). In another embodiment, Cpf1 is derived from a species of the genus Lachnospirae (LbCpf1).

[0081] The genetically modified cells of the present invention contain an exogenous nucleic acid encoding a CRISPR RNA (st-crRNA) to which at least one additional stem-loop structure is attached. Native crRNAs generally consist of approximately 42-44 nucleotides (a 19-nucleotide repeat sequence and a 23-15 nucleotide spacer sequence) and a single stem-loop structure, also known as a "handle" structure. The crRNAs of the present invention contain at least one additional stem-loop structure (in addition to the handle) and are referred to herein as stem-loop-crRNA (st-crRNA). The additional stem-loop structure may be attached to the 5' end and / or the 3' end of the crRNA. The st-RNA may contain one additional stem-loop structure or multiple additional stem-loop structures. In one embodiment, the st-crRNA contains a stem-loop structure at the 5' end of the crRNA adjacent to the handle. In another embodiment, the st-crRNA contains a stem-loop structure at the 3' end of the crRNA. The st-RNA may further contain modifications. In one embodiment, the st-RNA further comprises three glycine residues attached to the 5' end of the stem-loop. In another embodiment, the protospacer region of the st-crRNA further comprises partial phosphorothioate (PMS) modification. In yet another embodiment, the st-RNA further comprises three glycine residues attached to the 5' end of the stem-loop, and partial phosphorothioate (PMS) modification in the protospacer region.

[0082] The st-crRNA of the present invention can be designed to target any gene of interest. For example, in one embodiment, the crRNA can be designed to target the TCR α chain constant region (TRAC) and / or the TCR β constant region (TRBC) and / or the β-2 microglobulin (B2m) gene.

[0083] method In one aspect, the present invention includes a method for gene editing. The method includes administering exogenous nucleic acids, including stem-loop CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (st-crRNA) and exogenous nucleic acids, including the Cpf1 enzyme, to cells. In one embodiment, the cells to be edited are T cells. In another embodiment, the cells are primary T cells.

[0084] Another aspect of the present invention includes a method for producing modified T cells. This method includes the step of administering exogenous nucleic acids, including st-crRNA and Cpf1 enzyme, to cells.

[0085] In one embodiment of the present invention, an exogenous nucleic acid is administered by electroporation into a cell. In one embodiment, the cell undergoes multiple electroporations. For example, the cell may be electroporated once with a first st-crRNA, followed by a second st-crRNA.

[0086] The Cpf1 enzyme can originate from microorganisms of any genera, including, but not limited to, Parcobacteria, Lachnospiraceae, Butylivibrio, Peregrinibacteria, Asidaminococcus, Porphynomonas, Lachnospiraceae, Porflomonas, Prevotella, Moraxella, Sumicella, Leptospiraceae, Francisella, Candidatus, and Eubacterium. In one embodiment, Cpf1 originates from the genus Asidaminococcus (AsCpf1). In another embodiment, Cpf1 originates from the family Lachnospiraceae (LbCpf1).

[0087] One aspect of the present invention includes a stem-loop-bound CRISPR-RNA (crRNA) (st-crCRNA). The stem-loop structure may be bound to the 3' end and / or the 5' end of the crRNA. The stem-loop may further include three glycine residues attached to the 5' end of an additional stem-loop. The protospacer region of the st-crRNA may further include partial phosphorothioate (PMS) modification. In one embodiment, the st-RNA further includes three glycine residues attached to the 5' end of the stem-loop and partial phosphorothioate (PMS) modification in the protospacer region.

[0088] The gene editing method of the present invention can be used to mutate any gene of interest. For example, this method can mutate the TCR α chain constant region (TRAC) and / or the TCR β constant region (TRBC) and / or β-2 microglobulin (B2m). This method can be used in conjunction with other CRISPR systems, such as type I or type II CRISPR systems. In one non-restrictive example, the Cpf1 / st-crRNA gene editing system can be used in conjunction with the CRISPR / Cas 9 system. It is believed that using the two systems in a multi-faceted manner will enable broader screening and target selectivity.

[0089] Nucleic acid introduction Methods for introducing nucleic acids into cells include physical, biological, and chemical methods. Physical methods for introducing polynucleotides such as RNA into host cells include calcium phosphate precipitation, lipofection, particulate guns, microinjection, and electroporation. RNA can be introduced into target cells using commercially available methods including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Cologne.), Multiporator (Eppendort, Hamburg, Germany)). RNA can also be introduced into cells using commercially available methods including cationic liposome-mediated transfection using lipofection, polymer capsule encapsulation, peptide-mediated transfection, or bioristic particle delivery systems such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0090] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA vectors and RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0091] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as polymer complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. One exemplary colloidal system for use as a delivery medium in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0092] Suitable lipids can be obtained from suppliers. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; diacetyl phosphatidyl ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid storage solutions in chloroform or chloroform / methanol can be stored at approximately -20°C. Since chloroform evaporates more readily than methanol, it is preferable to use it as the sole solvent. "Liposomes" is a general term encompassing a range of monolayers and multilayers of lipid media formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure by a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-rearrangement, followed by the formation of a closed structure that encapsulates water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions with structures different from the usual vesicle structure in solution are also envisioned. For example, lipids may take the form of micellar structures, or they may simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also envisioned.

[0093] Regardless of the method used to introduce exogenous nucleic acids into host cells, various assays can be used to confirm the presence of nucleic acids in host cells. Such assays include, for example, “molecular biological” assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; and “biochemical” assays, such as immunological means (ELSA and Western blotting) or the assays described herein for identifying active substances within the scope of the present invention to detect the presence or absence of specific peptides.

[0094] Furthermore, nucleic acids may be introduced by any means such as cell transduction, cell transfection, and cell electroporation. One type of nucleic acid may be introduced by one method, and another type of nucleic acid may be introduced by a different method.

[0095] RNA In one embodiment, RNA is introduced into target cells. In another embodiment, the RNA is mRNA, including in vitro transcribed RNA or synthetic RNA. The RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable source of DNA. The desired template for in vitro transcription is a chimeric membrane protein. For example, the template encodes an antibody, a fragment of an antibody, or a portion of an antibody. For another example, the template includes an extracellular domain containing a single-chain variable domain of an antibody, such as anti-CD3, and an intracellular domain of a costimulatory molecule. In one embodiment, a template for an RNA chimeric membrane protein encodes a chimeric membrane protein comprising an extracellular domain containing an antigen-binding domain derived from an antibody against a costimulatory molecule, and an intracellular domain derived from a portion of the intracellular domains of CD28 and 4-1BB.

[0096] PCR can be used to create templates for in vitro transcription of mRNA, which are then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have a region substantially complementary to the region of DNA to be used as a template for PCR. "Substantially complementary," as used herein, means a sequence of nucleotides in which most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched. A substantially complementary sequence can anneal or hybridize with the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, primers can be designed to amplify a portion of a gene (open reading frame) that is normally transcribed in cells, including the 5' UTR and 3' UTR. Alternatively, primers can be designed to amplify a portion of a gene that codes for a specific domain of interest. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or part of the 5' UTR and 3' UTR. Primers useful for PCR are prepared by synthetic methods well known in the art. A “forward primer” is a primer that contains a region of nucleotides substantially complementary to the nucleotides on the DNA template, located upstream of the DNA sequence to be amplified. “Upstream” is used herein to mean the location on the 5' side of the DNA sequence to be amplified, with respect to the coding strand. A “reverse primer” is a primer that contains a region of nucleotides substantially complementary to the double-stranded DNA template, located downstream of the DNA sequence to be amplified. “Downstream” is used herein to mean the location on the 3' side of the DNA sequence to be amplified, with respect to the coding strand.

[0097] Chemical structures that enhance the stability and / or translation efficiency of RNA can also be used. The RNA preferably has a 5' UTR and a 3' UTR. In one embodiment, the 5' UTR is 0 to 3000 nucleotides long. The lengths of the 5' UTR and 3' UTR sequences appended to the coding region can be varied in various ways, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, those skilled in the art can modify the lengths of the 5' UTR and 3' UTR to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0098] The 5' UTR and 3' UTR may be the native endogenous 5' UTR and 3' UTR of the gene of interest. Alternatively, a non-endogenous UTR sequence for the gene of interest may be added by incorporating the UTR sequence into forward and reverse primers, or by any other modification of the template. The use of a non-endogenous UTR sequence for the gene of interest may be useful for modifying RNA stability and / or translation efficiency. For example, AU-rich elements in the 3' UTR sequence are known to reduce mRNA stability. Therefore, based on UTR properties well known in the art, the 3' UTR can be selected or designed to enhance the stability of the transcribed RNA.

[0099] In one embodiment, the 5' UTR may contain the Kozak sequence of an endogenous gene. Alternatively, if a 5' UTR that is not endogenous for the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. While Kozak sequences can improve the translation efficiency of some RNA transcripts, they do not appear to be necessary to enable efficient translation for all RNAs. The need for Kozak sequences for many mRNAs is well known in the art. In another embodiment, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in the cell. In yet another embodiment, various nucleotide analogs can be used for the 3' UTR or 5' UTR to prevent exonuclease degradation of mRNA.

[0100] To enable RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter must be linked to the DNA template upstream of the sequence to be transcribed. By adding a sequence that functions as a promoter for RNA polymerase to the 5' end of a forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, as described elsewhere herein, the promoter is the T7 polymerase promoter. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0101] In one embodiment, mRNA possesses both a 5' cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and intracellular mRNA stability. On circular DNA templates, such as plasmid DNA, RNA polymerase produces long concatemer products unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end yields normal-sized mRNA that, even if polyadenylated post-transcriptionally, is not effective for eukaryotic transfection.

[0102] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0103] The conventional method for incorporating polyA / T linkages into DNA templates is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. This makes the cloning procedure not only cumbersome and time-consuming, but also often unreliable. This is why a method that allows for the construction of DNA templates with polyA / T 3' linkages without cloning is highly desirable.

[0104] The poly(A) tail of the transcription DNA template can be generated during PCR by using a reverse primer containing a poly(T) tail, e.g., a 100T tail (size can be 50–5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. The poly(A) tail also provides stability to the RNA and reduces its degradation. Generally, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail consists of 100–5000 adenosine molecules.

[0105] The poly(A) tail of RNA can be further elongated after in vitro transcription using poly(A) polymerase, such as E. coli poly(A) polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides approximately doubles the RNA translation efficiency. Furthermore, mRNA stability can be enhanced by attaching various chemical groups to the 3' end. Such appendages may include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further enhance RNA stability.

[0106] A 5' cap also provides stability to the RNA molecule. In one preferred embodiment, RNA produced by the method disclosed herein includes a 5' cap. The 5' cap is conferred using a method known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0107] RNA produced by the methods disclosed herein may also include an intra-sequence ribosome entry site (IRES) sequence. The IRES sequence may be any viral sequence, chromosomal sequence, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and facilitates translation initiation. It may also include any solute suitable for cell electroporation, which may include factors that enhance cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0108] In some embodiments, RNA, such as in vitro transcribed RNA, is electroporated into the cell.

[0109] The disclosed methods can be applied to the modulation of T cell activity in basic research and therapeutics, including the evaluation of the ability of genetically modified T cells to kill target cancer cells in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases.

[0110] This method also offers the ability to control expression levels over a wide range, for example, by changing the promoter or the amount of input RNA, thus allowing for individual adjustment of expression levels. Furthermore, PCR-based mRNA generation methods greatly facilitate the design of mRNAs with various structures and combinations of their domains.

[0111] One advantage of the RNA transfection method of the present invention is that RNA transfection is inherently transient and does not require a vector. The RNA transgene can be delivered to lymphocytes and, after a short period of in vitro cell activation, expressed as a minimal expression cassette that does not require any other viral sequence. Under these conditions, the likelihood of the transgene being incorporated into the host cell genome is low. Due to the high efficiency of RNA transfection and its ability to homogeneously modify an entire lymphocyte population, cell cloning is not required.

[0112] Genetic modification of T cells using in vitro transcribed RNA (IVT-RNA) employs two strategies that have been continuously studied in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. To achieve long-term expression of the transferred IVT-RNA, it is desirable to stabilize the IVT-RNA using various modifications.

[0113] Several IVT vectors are known in the literature that are used in a standardized manner as templates for in vitro transcription and are genetically modified to produce stabilized RNA transcripts. Currently, protocols used in the art are based on plasmid vectors having the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest flanked by untranslated regions (UTRs) on the 3' and / or 5' sides, and a 3' polyadenylic cassette containing 50–70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylic cassette by a type II restriction enzyme (recognition sequences corresponding to cleavage sites). Thus, the polyadenylic cassette corresponds to the later poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzymatic cleavage sites after linearization, either elongating or masking the poly(A) sequence at the 3' end. It is unclear whether this non-physiological overhang affects the amount of protein produced intracellularly from such constructs.

[0114] RNA offers several advantages over more conventional plasmid or viral approaches. Gene expression from RNA sources does not require transcription, and protein products are rapidly produced after transfection. Furthermore, RNA only needs to reach the cytoplasm, not the nucleus, thus resulting in extremely high transfection rates with typical transfection methods. In addition, plasmid-based approaches require the promoter that activates the expression of the gene of interest to be active in the cells under test.

[0115] In another aspect, RNA constructs can be delivered into cells by electroporation. See, for example, formulations and methods for electroporation of nucleic acid constructs into mammalian cells, as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, and US 2004 / 0092907A1. Various parameters, including the electric field strength required for electroporation of any known cell type, are generally known in the relevant research literature and numerous patents and applications in the art. See, for example, US Patents 6,678,556, 7,171,264, and 7,173,116. Devices for the therapeutic application of electroporation are commercially available, including, for example, the MedPulser® DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif), which is described in U.S. patents such as U.S. Patent No. 6,567,694; U.S. Patents No. 6,516,223, U.S. Patent No. 5,993,434, U.S. Patent No. 6,181,964, U.S. Patent No. 6,241,701 and U.S. Patent No. 6,233,482; and electroporation can also be used for in vitro cell transfection, as described, for example, in U.S. Patent No. 20070128708A1. Electroporation can also be used to deliver nucleic acids to cells in vitro. Thus, electroporation-mediated administration of nucleic acids containing expression constructs to cells using any of the many available devices and electroporation systems known to those skilled in the art represents a remarkable new means of delivering RNA of interest to target cells.

[0116] treatment The modified cells described herein may be included in a composition for therapeutic purposes. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition containing the modified cells may be administered.

[0117] In one aspect, the present invention includes a method for adoptive cell transfer therapy, comprising the step of administering the modified cells of the present invention to a subject in need thereof. In another aspect, the present invention includes a method for treating a disease or condition in a subject, comprising the step of administering a population of modified cells to a subject in need thereof.

[0118] In one aspect, the modified cells are T cells. The T cells may be primary T cells. Modified T cells prepared as described herein have T cell function.

[0119] Modified cells can be administered to mammals, preferably humans, to suppress immune responses common to autoimmune diseases such as diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, and GVHD, as well as to enhanced alloimmune tolerance induction and transplant rejection. In addition, the cells of the present invention can also be used to treat any condition in which attenuation or other inhibition of an immune response, particularly a cell-mediated immune response, is desired to treat or alleviate a disease. In one aspect, the present invention includes a treatment of a condition in a subject, such as an autoimmune disease, which comprises administering a therapeutically effective amount of a pharmaceutical composition containing a population of modified cells to the subject. Examples of autoimmune diseases include acquired immunodeficiency syndrome (AIDS, a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac plusprüffe syndrome; chronic fatigue immune deficiency syndrome (CFIDS); chronic inflammatory demyelinating polyneuropathy (CIPD); bullous pemphigoid scarring, cold agglutinin disease, CRESTO syndrome, Crohn's disease, Degos disease, juvenile dermatomyositis, discoid lupus erythematosus, essential mixed cryoglobulinemia, fibromyalgia / fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, and idiopathic lung disease. Fibrosis, Idiopathic Thrombocytopenic Purpura (ITP), IgA Nephropathy, Insulin-Dependent Diabetes Membraneum, Juvenile Chronic Arthritis (Stil's Disease), Juvenile Rheumatoid Arthritis, Meniere's Disease, Mixed Connective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis, Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, Syndrome of Polyglandular Disease, Polymyalgia Rheumatica, Polymyositis / Dermatomyositis, Primary Gammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, This category includes, but is not limited to, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjögren's syndrome, generalized rigidity syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0120] Furthermore, modified cells produced as described herein can be enlarged and used to treat inflammatory disorders. Examples of inflammatory disorders include, but are not limited to, chronic and acute inflammatory disorders. Examples of inflammatory disorders include, but are not limited to, Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemia, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy, and ventilator-induced lung injury.

[0121] In another embodiment, the cells described herein may be used for the manufacture of pharmaceuticals for treating immune responses in subjects requiring such treatment. In yet another embodiment, the present invention includes modified cells described herein for use in a method for treating immune responses in subjects requiring such treatment.

[0122] The cells of the present invention can be administered to animals, preferably mammals, and more preferably humans, for the treatment of cancer. In addition, where it is desirable to treat or alleviate a disease, the cells of the present invention can also be used for the treatment of any cancer-related condition, particularly for cell-mediated immune responses against tumor cells. Examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain malignancies, lymphoma, leukemia, lung cancer, and thyroid cancer.

[0123] In one embodiment, the subject is offered secondary treatment. Secondary treatment includes, but is not limited to, chemotherapy, radiation therapy, surgery, and drug therapy.

[0124] The cells of the present invention can be administered in doses, routes, and frequencies determined by appropriate preclinical and clinical experiments and trials. The cell composition can be administered multiple times in doses within these ranges. The administration of the cells of the present invention may be combined with other methods useful for treating a desired disease or condition, as determined by those skilled in the art.

[0125] The cells of the present invention administered to the patient may be autologous, allogeneic, or heterogeneous to the patient receiving the treatment.

[0126] The administration of the cells of the present invention can be carried out in any convenient manner known to those skilled in the art. The cells of the present invention can be administered to a subject by aerosol inhalation, injection, oral ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodulely, intramedullarily, intramuscularly, intravenously (iv) or intraperitoneally. In other cases, the cells of the present invention may be injected directly into the site of inflammation, local disease site, lymph nodes, organs, tumors, etc., in the subject.

[0127] Furthermore, the cells described herein can also be administered using several matrices. The present invention utilizes such matrices in novel contexts, typically acting as artificial lymphoid organs to support, maintain, or modulate the immune system through T cell modulation. Therefore, the present invention can utilize such matrix compositions and formulations that have demonstrated utility in tissue engineering. Thus, the types of matrices that can be used in the compositions, apparatus, and methods of the present invention are virtually limiting and may include both biological and synthetic matrices. One specific example utilizes compositions and apparatuses described in U.S. Patents 5,980,889; 5,913,998; 5,902,745; 5,843,069; 5,787,900; or 5,626,561, and these patents are therefore incorporated herein by reference in their entirety. The matrices include features generally associated with biocompatibility when administered to a mammalian host. The matrices may be formed from natural and / or synthetic materials. The matrix may be non-biodegradable if it is desirable to leave a permanent or removable structure in the animal's body, such as an implant; or it may be biodegradable. The matrix may take the form of a sponge, implant, tube, telfa pad, fiber, hollow fiber, freeze-dried component, gel, powder, porous composition, or nanoparticles. In addition, the matrix may be designed to allow for the sustained release of seeded cells or produced cytokines or other active substances. In some embodiments, the matrix of the present invention may also be described as a flexible and stretchable semi-solid scaffold that is permeable to substances such as inorganic salts, aqueous solutions, and dissolved gaseous substances containing oxygen.

[0128] In this specification, "matrix" is used as an example of a biocompatible material. However, since the present invention is not limited to matrices, wherever the term "matrix" appears, it should be interpreted to include devices or other materials that enable the retention or passage of cells, are biocompatible, and are themselves semipermeable membranes, enabling the direct passage of polymers through the material or the passage of polymers when used in conjunction with certain semipermeable materials.

[0129] T cell source In one embodiment, the source of T cells is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. Preferably, the subject is human. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord blood, and tumors. In one embodiment, any variety of T cell lines available in the art can be used. In one embodiment, T cells can be obtained from blood units collected from a subject using any variety of techniques known to those skilled in the art, such as Ficoll isolation. In one preferred embodiment, cells derived from the blood of an individual are obtained by apheresis or leukocyte apheresis. Apheresis preparations typically contain T cells, monocytes, granulocytes, lymphocytes including B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected by apheresis can be washed to remove the plasma fraction and then placed in a suitable buffer or culture medium, such as phosphate-buffered saline (PBS), or in a washing solution that is calcium-free, magnesium-free, or free of many but not all divalent cations, for subsequent processing steps. After washing, the cells can be resuspended in various biocompatible buffers, such as Ca-free and Mg2-free PBS. Alternatively, undesirable components of the apheresis sample may be removed, and the cells may be resuspended directly in culture medium.

[0130] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation on a PERCOLL® gradient. Alternatively, T cells can be isolated from umbilical cord blood. In either case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0131] From umbilical cord blood mononuclear cells isolated in this manner, cells expressing specific antigens, including CD34, CD8, CD14, CD19, and CD56 in a non-limiting manner, can be depleted. This depletion of cells can be achieved using isolated antibodies, antibodies bound to a physical support such as an antibody-containing biological sample (e.g., ascites fluid), and antibodies bound to cells.

[0132] The enrichment of T cell populations by negative selection can be achieved using a combination of antibodies targeting surface markers specific to negatively selected cells. One preferred method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, a monoclonal antibody cocktail for enriching CD4+ cells by negative selection typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0133] To isolate a desired cell population by positive or negative selection, the concentrations of cells and surfaces (e.g., particles such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In another embodiment, a concentration of 1 billion cells / ml is used. In yet another embodiment, a concentration higher than 100 million cells / ml is used. In yet another embodiment, cell concentrations of 10 million / ml, 15 million / ml, 20 million / ml, 25 million / ml, 30 million / ml, 35 million / ml, 40 million / ml, 45 million / ml, or 50 million / ml are used. In yet another embodiment, cell concentrations of 75 million / ml, 80 million / ml, 85 million / ml, 90 million / ml, 95 million / ml, or 100 million / ml are used. In yet another embodiment, concentrations of 125 million / ml or 150 million / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell growth.

[0134] Furthermore, T cells can be frozen after the washing step without requiring a monocyte removal step. While we do not wish to be bound by theory, the freezing step and the subsequent thawing step provide a more homogeneous formulation by removing granulocytes and some monocytes from the cell population. After the washing step to remove plasma and platelets, these cells may be suspended in a freezing medium. Many freezing media and parameters are known in the art and are considered useful in this situation, but one method involves the use of PBS containing 20% ​​DMSO and 8% human serum albumin, or other suitable cell freezing medium. These cells are then frozen to -80°C at a rate of 1°C per minute and stored in the gas phase in a liquid nitrogen storage tank. In addition to other controlled freezing methods, immediate -20°C or uncontrolled freezing in liquid nitrogen may be used.

[0135] In one embodiment, a population of T cells is contained within cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified T cell populations, and T cell lines. In another embodiment, peripheral blood mononuclear cells contain a population of T cells. In yet another embodiment, purified T cells contain a population of T cells.

[0136] T cell growth In one embodiment, T cells can be enlarged. This enlargement can increase the T cell population by approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 100,000, 1,000,000, 10,000,000, or more, as well as multiples of any and all integers in between. In one embodiment, T cells are enlarged in the range of approximately 20 to 50 times.

[0137] After culture, the T cells are incubated in cell medium within a culture device for a period of time, or until the cells reach a dense or high cell density for optimal subculturing, and then subculturised into another culture device. The culture device may be any culture device commonly used for culturing cells in vitro. Preferably, the density level before subculturing the cells into another culture device is 70% or higher. More preferably, the density level is 90% or higher. The period may be any time suitable for culturing cells in vitro. The T cell medium may be changed at any point during T cell culture. Preferably, the T cell medium is changed approximately every 2-3 days. The T cells are then collected from the culture device and can be used immediately or cryopreserved for later use. In one embodiment, the present invention includes cryopreserving enlarged T cells. The cryopreserved T cells are thawed before nucleic acids are introduced into the T cells.

[0138] In another embodiment, the method comprises isolating T cells and amplifying T cells. In yet another embodiment, the present invention further comprises cryopreserving T cells before amplification. In yet another embodiment, the cryopreserved T cells are thawed for electroporation using RNA encoding chimeric membrane proteins.

[0139] Another procedure for ex vivo cell augmentation is described in U.S. Patent No. 5,199,942 (which is incorporated herein by reference). Augmentation as described in U.S. Patent No. 5,199,942 may be an alternative option or may be added to other augmentation methods described herein. Briefly, ex vivo culture and augmentation of T cells involves the addition of cell growth factors, such as those described in U.S. Patent No. 5,199,942, or other factors, such as flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, augmenting T cells involves culturing T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0140] The culture stage described herein (contact with the active substance as described herein) may be very short, for example, less than 24 hours, for example, less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culture stage described herein (contact with the active substance as described herein) may be longer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or longer.

[0141] Various terms are used to describe cells in culture. A cell culture generally refers to cells taken from a living organism and grown under controlled conditions. A primary cell culture is a culture of cells, tissues, or organs taken directly from a living organism before the first passage. Cells grow in culture when placed in a growth medium under conditions that promote cell proliferation and / or division, resulting in a larger cell population. When cells grow in culture, the rate of cell proliferation is typically measured by the time it takes for the number of cells to double, also known as the doubling time.

[0142] Each passage is referred to as a passage. When cells are passaged, they are referred to as having been passaged. A particular population of cells or cell lines may sometimes be referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged 10 times may be referred to as a P10 culture. A primary culture, i.e., the first culture after isolation of cells from tissue, is designated as P0. After the first passage, the cells are described as a secondary culture (P1 or passage 1). After the second passage, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those skilled in the art that many doublings of the population can occur during the passage period. Therefore, the number of doublings of a culture population is greater than the number of passages. The growth of cells during the interpassage period (i.e., the number of population doublings) depends on many factors, including seeding density, substrate, medium, and interpassage time, not limited to these.

[0143] In one embodiment, cells can be cultured for several hours (approximately 3 hours) to approximately 14 days, or any integer unit of time in between. Suitable conditions for culturing T cells include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) that may contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-β and TNF-α, or other additives for cell proliferation known to those skilled in the art. Other additives for cell proliferation include, but are not limited to, surfactants, plasmamenates, and reducing agents, such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may contain serum-free RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or a certain amount of cytokines sufficient for T cell proliferation and enlargement. Antibiotics such as penicillin and streptomycin are included only in experimental cultures and not in the cultures of cells intended for injection into the target. Target cells are maintained under conditions necessary to support proliferation, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0144] The culture medium used to cultivate T cells may contain agents that can co-stimulate T cells. For example, an agent that can stimulate CD3 is an antibody against CD3, and an agent that can stimulate CD28 is an antibody against CD28. This is precisely why, as demonstrated by the data disclosed herein, cells isolated by the methods disclosed herein can increase in size by approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 100,000, 1,000,000, 10,000,000 or more. In one embodiment, T cells can be increased in size by approximately 20 to 50 times or more by culturing a population that has undergone electroporation.

[0145] In one embodiment, a method for augmenting T cells may further include a step of isolating the augmented T cells for further use. In yet another embodiment, a method for augmenting may further include subsequent electroporation and subsequent culture of the augmented T cells. The subsequent electroporation may include introducing a nucleic acid encoding a certain active agent into a population of augmented T cells, for example, transduction of the augmented T cells using nucleic acid, transfecting the augmented T cells, or electroporating the augmented T cells, wherein the active agent further stimulates the T cells. The active agent can stimulate the T cells by stimulating further augmentation, effector function, or another T cell function.

[0146] Pharmaceutical composition The pharmaceutical compositions of the present invention may contain modified cells as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or additives. Such compositions may include buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0147] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The quantity and frequency of administration are determined by factors such as the patient's condition, as well as the type and severity of the patient's disease, although the appropriate dosage may be determined by clinical trials.

[0148] The cells of the present invention administered to the patient may be autologous, allogeneic, or heterogeneous to the patient receiving the treatment.

[0149] The cells of the present invention can be administered in doses, routes, and frequencies determined by appropriate preclinical and clinical experiments and trials. The cell composition can be administered multiple times in doses within these ranges. The administration of the cells of the present invention may be combined with other methods useful for treating a desired disease or condition, as determined by those skilled in the art.

[0150] The pharmaceutical composition containing the modified cells described herein is cell 10 4 ~10 9 cells / kg body weight, or in some cases, 10 cells 5 ~10 6The cells can be administered in doses of cells / kg body weight, including all integer values ​​within this range. The cell composition can also be administered multiple times at these doses. Cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by those skilled in the art by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0151] The modified cells of the present invention can be administered in any convenient manner known to those skilled in the art. The cells of the present invention can be administered in any convenient manner, including aerosol inhalation, injection, oral ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodulely, intramedullarily, intramuscularly, intravenously (iv) or intraperitoneally. In other cases, the cells of the present invention may be injected directly into the site of inflammation, local disease site, lymph nodes, organs, tumors, etc.

[0152] It should be understood that the methods and compositions considered useful in the present invention are not limited to the specific formulations shown in the examples. The following examples are provided for the purpose of providing a thorough disclosure and explanation to those skilled in the art of how the cells, growth and culture methods and therapeutics of the present invention are prepared and used, and are not intended to limit the scope of what the inventors consider to be their invention.

[0153] Unless otherwise indicated, the implementation of the present invention involves conventional methods of molecular biology (including recombinant methods), microbiology, cell biology, biochemistry, and immunology that are within the scope of the skills in the art. Such techniques are well described in literature such as "Molecular Cloning: A Laboratory Manual", fourth edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting", (Babar, 2011); and "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the preparation of polynucleotides and polypeptides of the present invention and can therefore be considered in the preparation and implementation of the present invention. The following sections will discuss methods that are particularly useful for specific applications. [Examples]

[0154] Experimental example The present invention will now be described with reference to the following experimental examples. These examples are provided for illustrative purposes only, and the present invention is not limited to these examples, but rather includes all variations that become apparent as a result of the teachings provided herein.

[0155] The materials and methods used in these experiments are described below.

[0156] Primary human lymphocytes Primary human CD4 and CD8 T cells were isolated from healthy volunteer donors after leukocyte apheresis by negative selection using the RosetteSep kit (Stem Cell Technologies, Vancouver BC, Canada). As previously described (Barrett et al. (2011) Human Gene Therapy 22(12): 1575-1586), primary lymphocytes were stimulated with microbeads coated with CD3 and CD28 stimulating antibodies (Life Technologies, Grand Island, NY, Catalog). On day 10, T cells in a solution of 90% fetal bovine serum and 10% dimethyl sulfoxide (DMSO) were isolated at 1 × 10⁶ cells. 8 They were frozen and stored individually in vials.

[0157] Proliferation of primary T cells Primary human T cells were cultured in RPMI 1640 supplemented with 10% FCS, 100-U / ml penicillin, 100-g / ml streptomycin sulfate, and 10-mM Hepes, and stimulated with magnetic beads coated with anti-CD3 / anti-CD28 at a cell-to-bead ratio of 1:3. Cell counting and nutrient supplementation were performed every two days. When the T cells appeared to have entered a quiescent state based on both decreased proliferation and cell size, they were used for functional assays or cryopreserved.

[0158] Design and construction of CRISPR CrRNAs were selected using NTTTN20, which contains the NTTT PAM site. The constant regions of TCR α, β, and β-2 microglobin, targeted by Cpf1 and crRNA, were transcribed in vitro. CrRNAs were designed to target either a sequence in exon 1 of the TCR α constant region, or a consensus sequence common to both TCR β constant regions 1 and 2, as well as exon 1 of B2m. mRNAs were stored at -80°C in single-use, nuclease-free vials.

[0159] Flow cytometry The following monoclonal antibodies and reagents with the specified specificities were used, along with appropriate isotype controls: from BD Biosciences (San Jose, CA): APC-conjugated anti-CD3 (555335), PE-anti-β-2 microglobin (551337), and FITC-anti-HLA (555552). Data were acquired using CellQuest version 3.3 (BD ​​Biosciences, San Jose, CA) via FACS Accuri (BD Biosciences, San Jose, CA) and analyzed using FCS Express version 3.00 (De Novo Software, Los Angeles, CA) or FlowJo version 7.6.1 (Tree Star, Inc. Ashland, OR).

[0160] CRISPR-mediated gene disruption Prior to electroporation, T cells were stimulated with CD3 / CD28 DynaBeads for 3 days. 10 million primary T cells were debeaded, and then 20 μg of Cpf1 (Cas9 or eSpCas9) and 10 μg of crRNA (or sgRNA) were electrotransferred into the cells using a BTX830 at 360 V and 1 ms, followed by a second electrotransfer of 5 μg of crRNA (or sgRNA). Alternatively, 20 μg of Cpf1 mRNA and 10 μg of chemically modified crRNA were simultaneously electroporated into the T cells. After electroporation, the cells were immediately placed in 2 mL of preheated medium and cultured for 1 day at 37°C, 5% CO2 or 32°C, 5% CO2, followed by a return to 37°C, 5% CO2. TIFF0007884292000001.tif43147

[0161] CRISPR gene editing Cpf1 and Cas9 mRNA were transcribed in vitro using the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies, AMI 345, Carlsbad, CA). SgRNA and crRNA were transcribed in vitro using the MEGAscript T7 transcription kit. Chemically modified crRNA was purchased from Integrated DNA Technologies. T cells were stimulated with CD3 / CD28 DynaBeads. On day 3, Cas9 or Cpf1 mRNA was electroporated into the T cells. Briefly, the T cells were washed three times with OPTI-MEM and then rehydrated in OPTI-MEM (Invitrogen) at a final concentration of 1-3 × 10⁶. 8 The cells were resuspended at a concentration of cells / ml. Subsequently, 0.1 ml of the cell suspension was mixed with IVT RNA and electroporated in a 2 mm cuvette. Next, 20 μg of Cas9 or Cpf1 mRNA and 5 μg of guide RNA were electroporated into the cells using a BTX830 system (Harvard Apparatus BTX) at 360 V and 1 ms. After electroporation, the cells were immediately placed in 2 ml of preheated medium and cultured at 37°C and 5% CO2 in the presence of IL-2 (100 IU / ml). On day 4, an additional 5 μg of guide RNA was electroporated into the T cells.

[0162] Measurement of allele modification frequency using the T7E1 assay The levels of genomic disruption of TRAC, TRBC, and B2m in T cells were determined using the T7E1 Nuclease Assay (NEB). Target disruption rates were quantified by concentration measurements.

[0163] The results of the experiment are explained below.

[0164] Example 1: Enhancement of targeted mutagenesis using stem-loop crRNA in primary T cells Previously, Cpf1 proteins derived from various genera have been tested for their genome editing efficiency in mammalian cells (Zetsche et al. Cell. 2015; 163(3): 759-771). Of the eight Cpf1 proteins tested, only two proteins, AsCpf1 and LbCpf1, derived from the genera *As* and *Lb* respectively, were found to induce detectable mutations in mammalian cells. Therefore, in this specification, the gene targeting efficiency of AsCpf1 and LbCpf1 was evaluated in T cells. The efficiency of CRISPR-mediated genome editing in primary T cells was compared among AsCpf1, LbCpf1, and Cas9 using a protocol previously developed for efficient T cell gene disruption (Ren et al. (2016) Clinical Cancer Research (2016): clincanres-1300). Cas9 or Cpf1 was delivered to primary T cells via a single electroporation along with guide RNA. Targeted mutagenesis was measured at three endogenous target sites: the TCR α-chain constant region (TRAC), the TCR β-chain constant region (TRBC), and β-2 microglobulin (B2m). With Cas9, 10%–20% targeted mutagenesis was observed, but no detectable mutagenesis was observed with either AsCpf1 or LbCpf1. When multiple guide RNA deliveries were used, Cas9 achieved 80–90% gene disruption at all three gene loci. With AsCpf1, gene disruption exceeded 50%, and with LbCpf1, approximately 30% was achieved (Figure 4). The improved effectiveness of gene disruption was associated with delaying the second delivery of guide RNA after Cas9 and Cpf1 electroporation. This finding indicates that small guide RNAs, including sgRNA and crRNA, are far more susceptible to degradation than Cas9 and Cpf1 mRNA. AsCpf1 showed substantially higher gene disruption efficiency than LbCpf1, so subsequent experiments focused on AsCpf1.

[0165] Gene targeting with Cpf1-crRNA achieved substantial gene disruption in T cells, but its efficiency was lower than that of Cas9-sgRNA. Structural differences between sgRNA and crRNA may be the reason for the difference in stability. Interestingly, sgRNA differs from crRNA in the various stem-loop structures that interact with Cas9, and therefore it was found to protect sgRNA from RNases. Stem-loops have been reported to enhance RNA stability by reducing the activity of RNases. AU flips and stem-loop extensions have been shown to increase sgRNA stability. Furthermore, it has been shown that the efficiency of gene disruption can be enhanced by optimizing the structure of sgRNA. In this specification, we investigated whether gene disruption efficiency can be increased by adding a stem-loop to crRNA to create stem-loop-crRNA (st-crRNA) (Figure 1A). In all three targeted genes, approximately twofold increase was observed after introducing various stem-loop structures of sgRNA to either the 5' (Lst) or 3' (Rst) end of the crRNA. The 5' end stem-loop structure-3 (Lst-3) resulted in the greatest increase in gene disruption, reaching 85% (83.6% ± 7.3, n=5) for TRAC, 83% (81.7 ± 5.7, n=4) for TRBC, and 81% (78.7 ± 8.4, n=4) for B2m (Figure 1B). However, extension of the original handle stem-loop in crRNA (Mst-crRNA) resulted in loss of function, and adding stem-loops to both the 5' and 3' ends reduced targeting efficiency, revealing structural requirements and length constraints of crRNA for Cpf1 binding (Figure 1B).

[0166] Example 2: Enhancement of targeted mutagenesis by chemically modified stem-loop-crRNA Chemical modifications to enhance sgRNA stability have been shown to improve gene disruption efficiency. To further enhance crRNA stability, chemically modified crRNAs targeting TRAC were constructed herein. We also investigated whether efficient gene disruption could be achieved by single electroporation, and therefore whether the toxicity caused by a second electroporation could be reduced. To confirm that chemical modifications do not cause loss of crRNA function, modified crRNAs were examined using two electroporation protocols. Modification of the tail "AA" nucleotide in the crRNA handle caused loss of function, indicating that "AA" is essential for proper handle structure formation (Figure 5). To avoid this effect, three "G" residues were added to the handle tail, followed by 2'-O-methyl3'-phosphorothioate (MS) of these three "G" residues and three tail protospacer nucleotides (Figure 1C). In this case, the modification did not cause loss of crRNA function, and gene disruption was still not observed after single electroporation of Cpf1 and MS-crRNA. Using partially phosphorothioated protospacer MS-crRNA (PMS-crRNA), 9.1% (9.7±1.7, n=3) of TRAC gene disruption was achieved. However, complete phosphorothioation of crRNA (FPMS-crRNA) resulted in loss of function, indicating that crRNA is vulnerable to modification of its handle structure (Figure 1D).

[0167] To investigate the function of the stem-loop structure on crRNA, PMS-modified Lst-3 crRNA targeting TRAC was constructed. No gene disruption was observed after single electroporation of Cpf1 and unmodified Lst-3 crRNA; however, PMS modification of Lst-3 crRNA resulted in over 61% (58.3±6.5, n=3) TRAC disruption, which was nearly seven times greater than the disruption obtained with PMS-crRNA without the stem-loop structure (Figure 1D).

[0168] The presence of both PMS modification and stem-loop significantly enhanced the efficiency of Cpf1-crRNA gene targeting in the single-pass electroporation protocol; however, this did not further improve the efficiency of stem-loop-crRNA gene disruption in the double-pass electroporation protocol. This suggests that the stem-loop structure provides sufficient protection for crRNA in experiments requiring only transient crRNA exposure (Figure 1E).

[0169] Example 3: Stringent guide selectivity of Cpf1 in primary T cells To investigate the characteristics of Cpf1 in T cells, gene disruption was performed in primary T cells using Cpf1 and st-crRNA. Cpf1 differs from Cas9 not only in its use of crRNA instead of sgRNA, but also in its recognition of the more stringent PAM sequence of TTTN, while Cas9 recognizes the PAM sequence of NGG. Compared to Cas9, Cpf1 disrupts target genes in a more stringent manner, but with comparable efficiency. The performance of Cpf1, Cas9, and the high-fidelity Cas9 eSpCas9 in T cells was investigated using 10 different crRNAs or sgRNAs targeting the TRAC, TRBC, and B2m gene loci. In the majority of cases, sgRNAs mediated efficient gene disruption by Cas9, while Cpf1 achieved comparable gene disruption with only 1-2 of the 10 crRNAs. These results indicate that crRNA selection by Cpf1 is stringenter than selection by Cas9. Interestingly, this characteristic of Cpf1 was similar to that of eSpCas9, a high-fidelity Cas9 that exhibited comparable gene disruption and also showed more stringent sgRNA selection (Figure 2A). Enhanced specificity through the use of shorter truncated sgRNAs has been reported. Therefore, we investigated whether a similar strategy could be used with Cpf1-crRNA to enhance gene targeting specificity. In contrast to Cas9, where the gene disruption efficiency was only slightly reduced with sgRNAs as short as 16 bp, even crRNAs shortened to 17 bp completely eliminated the function of Cpf1, a result similar to that observed with eSpCas9 (Figure 2B). Thus, Cpf1 behaves similarly to eSpCas9 in terms of both guide selectivity and truncated RNA, demonstrating that Cpf1 can be used as a high-fidelity gene editing tool in place of Cas9.

[0170] Example 4: Enhancement of Cpf1 gene targeting specificity compared to Cas9 in primary T cells. To further investigate the safety of Cpf1, off-target events were measured using the three crRNAs with the highest gene disruption efficiency for targeting TRAC, TRBC, and B2m. Ten off-target sites were measured for each gene. Consistent with stringent guide selectivity, both Cpf1 and eSpCas9 showed significantly lower levels and fewer off-target events compared to Cas9, regardless of whether wild-type 20 bp or truncated 18 bp guide RNA was used (Figure 3A).

[0171] To investigate how faithful Cpf1 and Cas9 are to their guide RNAs, we mutated each nucleotide of sgRNA or crRNA targeting TRAC and TRBC, respectively. A single point mutation adjacent to the PAM site of Cas9 sgRNA had a significant impact on its function, while mutations 10 base pairs away from the PAM site had little to no effect. However, even a single mutation in crRNA, regardless of the location of the mutation, completely eliminated Cpf1 function. Interestingly, mutations in sgRNA targeting TRBC eliminated eSpCas9 function, while mutations in sgRNA targeting TRAC resulted in the retention of most of eSpCas9 function (Figure 3B). This finding suggests that Cpf1 may be far more faithful to its guide RNA than eSpCas9.

[0172] All of this data indicates that the target DNA recognition mechanisms of Cpf1 and Cas9 are different. In addition, in contrast to NGGs, which are mainly distributed in coding sequences within exons, many TTTN sequences are located within untranslated regions, which therefore leads to Cpf1's off-target functional effects being less significant than those of Cas9.

[0173] Example 5: Discussion One of the most intriguing applications of the CRISPR system is highly efficient gene editing, which holds great promise for advancing adoptive immunotherapy using T cells. However, the characteristics of Cpf1, a Cas9 homolog, and its guide crRNA remain largely unknown. To date, the potential for targeted genome editing in T cells using Cpf1 has not been explored. This study demonstrates that efficient and specific gene disruption can be achieved using Cpf1-crRNA.

[0174] Gene disruption exceeding 40-50% was achieved through multiple crRNA delivery, but this high efficiency also caused toxicity to T cells. Structural and chemical modifications were performed on crRNA to minimize toxicity associated with multiple electroporation cycles. Gene disruption exceeding 80% was achieved through the use of multiple stem-loop crRNA delivery cycles. However, no substantial gene disruption was observed after a single electroporation cycle. While enhancement of gene disruption through the use of 2'-O-methyl3'-phosphorothioate (MS) chemically modified sgRNA has been reported, MS modification of crRNA did not result in detectable gene disruption after a single electroporation cycle, as described herein. Gene editing with partial phosphorothioate of crRNA resulted in only 9% gene disruption, but incorporating a protective stem-loop into the crRNA achieved a nearly sevenfold increase in gene targeting, reaching over 60%.

[0175] CrRNA was found to be extremely sensitive to modifications of its handle structure, as its function was lost upon modification of either the tail AA or internal nucleotides. This finding suggests that the structure of crRNA is critically important for its formation, and therefore modifications of the handle structure can disrupt its interaction with Cpf1. The loss of function due to the extension of the crRNA handle stem loop points to the spatial constraints of the Cpf1 binding pocket, which have been previously elucidated in the crystal structure of Cpf1 in complex with crRNA.

[0176] Gene editing with Cpf1 resulted in fewer off-target mutagenesis for all three endogenous genes tested, compared to Cas9. Fidelity to guide RNA, assessed based on tolerance to mutations in crRNA and sgRNA, also demonstrated that Cpf1 was more rigorous than Cas9 in T cells. Both Cas9 and Cpf1 produced double-strand breaks (DSBs), but Cas9 used its RuvC-like and HNH-like domains to produce staggered cuts within the seed sequence, while Cpf1 used its RuvC-like domain to produce staggered cuts outside the seed sequence. As indicated by the crystal structure, targeted cuts require dissociation of double-stranded target DNA, triggered by the interaction between Cas9 and the PAM sequence. This result suggests that for Cas9, the energy required to dissociate double-stranded DNA through cuts within the seed region may be less than for Cpf1 when cutting outside the seed region. This characteristic can consequently result in Cas9 having a higher off-target potential than Cpf1.

[0177] In summary, this specification demonstrates that the CRISPR / Cpf1 system is an efficient and high-fidelity gene editing tool in T cells.

[0178] Other embodiments In this specification, descriptions of a list of elements in the definition of a variable include the definition of that variable as a single element or as a combination (or partial combination) of the listed elements. In this specification, descriptions of an aspect include that aspect as a single aspect or as a combination with other aspects or parts thereof.

[0179] Each and every disclosure of the patents, patent applications, and publications cited herein is incorporated herein by reference in their entirety. While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the present invention can be conceived by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to encompass all such embodiments and equivalent variations.

[0180] Sequence information SEQUENCE LISTING <110> The Trustees of the University of Pennsylvania <120> Compositions and Methods for Gene Editing in T cells using CRISPR / Cpf1 <150> US 62 / 501,371 <151> 2017-05-04 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sgRNA TRAC <400> 1 agagtctctc agctggtaca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sgRNA TRBC <400> 2 tgggagatct ctgcttctga 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sgRNA B2m <400> 3 cgcgagcaca gctaaggcca 20 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> crRNA TRAC <400> 4 gagtctctca gctggtacac ggc 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> crRNA TRBC <400> 5 agccatcaga agcagagatc tcc 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> crRNA B2m <400> 6 atccatccga cattgaagtt gac 23

Claims

1. (a) A protospacer region comprising a sequence that hybridizes to a target nucleic acid sequence, (b) A nucleic acid sequence that forms a handle stem loop structure at the 5' end of the protospacer region, (c) A nucleic acid sequence that forms an additional stem loop structure at the 5' end of the handle stem loop structure or at the 3' end of the protospacer region This includes manipulated CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA).

2. The modified crRNA according to claim 1, which is more resistant to degradation by RNases compared to wild-type crRNA without the additional stem-loop structure.

3. The manipulated crRNA according to claim 1 or 2, wherein the additional stem-loop structure is ligated to the 5' end of the handle-stem-loop structure.

4. The manipulated crRNA according to claim 1 or 2, wherein the additional stem-loop structure is ligated to the 3' end of the protospacer region.

5. The manipulated crRNA according to any one of claims 1 to 4, wherein the handle stem loop structure comprises the nucleic acid sequence AAUUUCUACUCUUGUAGAU in the 5' to 3' direction.

6. The manipulated crRNA according to any one of claims 1 to 3 and 5, wherein the additional stem-loop structure comprises the nucleic acid sequence UGAAAAAGUUCA in the 5' to 3' direction.

7. The manipulated crRNA according to any one of claims 1 to 6, which exhibits at least twice the gene editing efficiency when compared to wild-type crRNA without the additional stem-loop structure.

8. The manipulated crRNA according to any one of claims 1 to 7, wherein the 5' end of the manipulated crRNA contains three guanosine residues.

9. The manipulated crRNA according to claim 8, wherein the three guanosine residues are modified by phosphorothioate.

10. The manipulated crRNA according to any one of claims 1 to 9, wherein one or more nucleic acid residues in the protospacer region are modified by phosphorothioate.

11. The manipulated crRNA according to any one of claims 1 to 10, wherein the 3' end of the manipulated crRNA comprises three or more nucleic acid residues modified by phosphorothioate.

12. The manipulated crRNA according to any one of claims 1 to 11, wherein the nucleic acid of the handle stem loop structure is not modified by phosphorothioate.

13. The manipulated crRNA according to any one of claims 9 to 12, wherein the phosphorothioate modification is 2'-O-methyl3'-phosphorothioate.

14. The modified crRNA according to any one of claims 1 to 13, wherein the protospacer region hybridizes to a gene locus selected from the group consisting of the TCRα chain (TRAC) gene, the TCRβ chain (TRBC) gene, and the β-2 microglobulin (B2m) gene.

15. The protospacer region comprises a nucleic acid sequence that hybridizes to the TRAC gene and has SEQ ID NO:

4. The protospacer region hybridizes to the TRBC gene and contains the nucleic acid sequence of SEQ ID NO:5, or The protospacer region hybridizes to the B2m gene and contains the nucleic acid sequence of SEQ ID NO:

6. The manipulated crRNA according to claim 14.

16. The manipulated crRNA according to claim 1, comprising the nucleic acid sequence SEQ ID NO:

9.

17. A vector comprising the manipulated crRNA according to any one of claims 1 to 16, and a nucleic acid encoding a type V CRISPR DNA endonuclease.

18. The vector according to claim 17, comprising a nucleic acid sequence encoding a Cpf1 endonuclease.

19. The vector according to claim 18, wherein the Cpf1 endonuclease is an Acidaminococcus Cpf1 (AsCpf1) endonuclease or a Lachnospiraceae Cpf1 (LbCpf1) endonuclease.

20. comprising the manipulated crRNA according to any one of claims 1 to 16, and a type V CRISPR DNA endonuclease, or Including the vector according to any one of claims 17 to 19, Human T cells.

21. The human T cell according to claim 20, wherein the type V CRISPR DNA endonuclease is Cpf1 endonuclease.

22. The human T cell according to claim 21, wherein the Cpf1 endonuclease is Acidaminococcus Cpf1 (AsCpf1) endonuclease or Lachnospiraceae Cpf1 (LbCpf1) endonuclease.

23. A nucleic acid encoding a chimeric antigen receptor (CAR) having affinity for tumor-associated antigens. Human T cells according to any one of claims 20 to 22, further comprising:

24. A pharmaceutical agent for treating cancer, comprising a population of human T cells as described in Claim 23.

25. An ex vivo method for gene editing, comprising the step of administering the manipulated crRNA and type V CRISPR DNA endonuclease described in any one of claims 1 to 16 to human T cells.

26. The ex vivo method according to claim 25, wherein the administration step comprises electroporating the manipulated crRNA and the type V CRISPR DNA endonuclease into the human T cells.

27. ​​The method according to claim 26, further comprising electroporating an additional amount of the manipulated crRNA into the human T cells a second time.

28. The ex vivo method according to any one of claims 25 to 27, wherein the type V CRISPR DNA endonuclease is a Cpf1 endonuclease.

29. The ex vivo method according to claim 28, wherein the Cpf1 endonuclease is an Acidaminococcus Cpf1 (AsCpf1) endonuclease or a Lachnospiraceae Cpf1 (LbCpf1) endonuclease.

Images