TET2 diallele knockout

Abstract

A composition comprising an RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides comprising the nucleotide of the sequence set forth in any one of SEQ ID NO: 1 to 37421, as well as methods and uses thereof.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 376,279, filed September 19, 2022, the contents of which are incorporated herein by reference.

[0002] Throughout this application, various publications are cited, including references in parentheses. The disclosures of all publications mentioned in this application are incorporated herein by reference in their entirety to provide additional information on the field to which the invention pertains and the features of the art to which the invention may be applied. References to sequence listings

[0003] This application incorporates by reference the nucleotide sequence in the file named "230918_92039-A-PCT_Sequence_Listing_AWG.xml", which is 32,380 kilobytes in size and was created on September 17, 2023 in IBM-PC machine format and is compatible with the MS-Windows operating system, which is included as part of this application in the XML file filed on September 18, 2023. Background of the Invention

[0004] Chimeric antigen receptors (CARs) provide a promising immunotherapy approach. However, in order to make such therapies (e.g., CAR-T cell therapies) more accessible, it is highly desirable to develop an allogeneic adoptive transfer strategy in which universal CAR cells derived from cells of healthy donors can be used to treat multiple patients. In order to implement this strategy, the immunogenicity and reactivity of CAR-T cells must be optimized to avoid adverse reactions, such as host rejection or graft-versus-host disease. Summary of the Invention

[0005] Tet proteins play a vital role in regulating gene expression levels by balancing DNA methylation during hematopoietic and immune cell activation and amplification. A method for knocking out the TET2 gene in cells for use in immunotherapy methods (such as CAR-T treatment) is disclosed. Cells modified with TET2 knockout improve the performance of the cells in allogeneic adoptive transfer therapy. Such cells have improved activity, retention and / or amplification properties for use in adoptive cancer immunotherapy.

[0006] The present disclosure also provides a method for inactivating an allele of the Tet methylcytosine dioxygenase 2 (TET2) gene in a cell, the method comprising introducing into the cell a composition comprising: A CRISPR nuclease, or a polynucleotide molecule encoding the CRISPR nuclease; and An RNA molecule comprising a guide sequence portion having 17-50 nucleotides, or a polynucleotide molecule encoding the RNA molecule, wherein the complex of the CRISPR nuclease and the RNA molecule effects a double-strand break in an allele of the TET2 gene.

[0007] In some embodiments, the RNA molecule comprises a guide sequence portion that targets a sequence located within any one of exons 3-11 of the TET2 gene, or a sequence located within the genome of any one of 4:105272525-105272956, 4:105275009-105276557, 4:105233904-105237389, 4:105241300-105241467, 4:105242795-105242965, 4:105243531-105243816, 4:105259580-105259807, 4:105261720-105261886, and 4:105269571-10526978. In some embodiments, the guide sequence portion of the RNA molecule comprises 17-50 consecutive nucleotides comprising nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-37421.

[0008] According to an embodiment of the present invention, an RNA molecule is provided, which comprises a guide sequence portion having 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421.

[0009] According to an embodiment of the present invention, a composition comprising an RNA molecule and a CRISPR nuclease is provided, wherein the RNA molecule comprises a guide sequence portion having 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421.

[0010] According to an embodiment of the present invention, there is provided a method for inactivating a TET2 allele in a cell, the method comprising delivering a composition comprising an RNA molecule and a CRISPR nuclease to the cell, the RNA molecule comprising a guide sequence portion having 17-50 consecutive nucleotides, the 17-50 consecutive nucleotides containing nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a T regulatory cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a macrophage. In some embodiments, the cell is a stem cell. In some embodiments, the cell is an iPSC. In some embodiments, the cell is a fibroblast, a blood cell, a hepatocyte, a keratinocyte, or any other cell type capable of being reprogrammed into an induced pluripotent stem cell (iPSC). In some embodiments, delivery to the cell is performed in vivo, ex vivo, or in vitro. In some embodiments, the method is performed ex vivo and the cells are provided / explanted from an individual patient. In some embodiments, the method further comprises the step of introducing the resulting cells having a modified / knocked-out TET2 allele into the individual patient. In some embodiments, the cells are derived from the individual patient to be treated. In some embodiments, the cells are derived from a donor. In some embodiments, the cells are allogeneic to the individual patient into which they are introduced.

[0011] According to an embodiment of the present invention, a method for improving the activity and / or retention and / or expansion of cells for adoptive cell therapy is provided, the method comprising delivering a composition comprising an RNA molecule and a CRISPR nuclease to cells of a subject in need of adoptive cell therapy, the RNA molecule comprising a guide sequence portion having 17-50 consecutive nucleotides, the 17-50 consecutive nucleotides containing nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421. In some embodiments, the method is used to increase the persistence and / or engraftment of cells in cells of a host subject, the method comprising delivering a composition comprising an RNA molecule and a CRISPR nuclease to the cells, the RNA molecule comprising a guide sequence portion having 17-50 consecutive nucleotides, the 17-50 consecutive nucleotides containing nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421; and introducing the cells into the host subject. In some embodiments, the cells are further differentiated before being introduced into the host subject. In some embodiments, the cells are further engineered to express a chimeric antigen receptor. In some embodiments, the cell is a stem cell, iPSC or progenitor cell and is differentiated into a T cell before the cell is introduced into the host subject. In some embodiments, the cell is a T cell. In some embodiments, the cell is further engineered to inactivate and / or knock out other genes to improve the use of the cell for adoptive transfer, such as knocking out other genes to avoid graft-versus-host disease (GVHD) after the cell is introduced into the host subject.

[0012] According to an embodiment of the present invention, there is provided a use of a composition comprising an RNA molecule and a CRISPR nuclease for inactivating a TET2 allele in a cell, wherein the RNA molecule comprises a guide sequence portion having 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421, and the use comprises delivering to the cell a composition comprising an RNA molecule and a CRISPR nuclease, wherein the RNA molecule comprises a guide sequence portion having 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421.

[0013] According to an embodiment of the present invention, a drug comprising an RNA molecule and a CRISPR nuclease for inactivating a TET2 allele in a cell is provided, wherein the RNA molecule comprises a guide sequence portion having 17-50 consecutive nucleotides, wherein the consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421, wherein the drug is administered by delivering a composition comprising an RNA molecule and a CRISPR nuclease to the cell, wherein the RNA molecule comprises a guide sequence portion having 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421.

[0014] According to an embodiment of the present invention, there is provided a use of a composition comprising an RNA molecule and a CRISPR nuclease for improving the activity and / or retention and / or expansion of cells used for adoptive cell therapy or increasing the persistence of the cells after transplantation, wherein the RNA molecule comprises a guide sequence portion having 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides in the sequence of any one of SEQ ID NOs: 1-37421, and the use comprises delivering a composition comprising an RNA molecule and a CRISPR nuclease to cells of a subject in need of adoptive cell therapy, wherein the RNA molecule comprises a guide sequence portion having 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides in the sequence of any one of SEQ ID NOs: 1-37421.

[0015] According to an embodiment of the present invention, there is provided a method of treating a disease or condition comprising delivering any of the compositions or modified cells described herein to a subject, preferably wherein the disease or condition is cancer.

[0016] According to an embodiment of the present invention, a kit for inactivating a TET2 allele in a cell is provided, comprising an RNA molecule, a CRISPR nuclease, and optionally a tracrRNA molecule, wherein the RNA molecule comprises a guide sequence portion having 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421; and instructions for delivering the RNA molecule, the CRISPR nuclease, and optionally the tracrRNA to the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Figure 2: TET2 editing in HeLa cells. OMNI-103 CRISPR nuclease was expressed in a mammalian cell system (HeLa cells) by DNA transfection along with an sgRNA expression plasmid. Transfection efficiency (% transfection) was determined by flow cytometric measurement of mCherry signal. All assays were performed in triplicate. Cells transfected with "OMNI nuclease only" (i.e., no guide) served as a negative control, and no editing was observed in these cells (data not shown).

[0018] Figure 2 : TET2 editing in primary T cells. T cells obtained from two donors were thawed and activated with microbeads for 72 hours. Then, OMNI-103 CRISPR nuclease (113 pmol) + sgRNA (226 pmol) and 2×10 6 Seven (7) days later, genomic DNA was isolated from approximately 100,000 cells, subjected to manual PCR, and analyzed by next generation sequencing (NGS). DETAILED DESCRIPTION

[0019] Unless otherwise specified, all technical and / or scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of embodiments of the present invention, exemplary methods and / or materials will be described below. In the event of conflict, the patent specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be necessarily limiting.

[0020] It should be understood that the terms "a" and "an" as used above and elsewhere herein refer to "one or more" of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms "a," "an," and "at least one" are used interchangeably in this application.

[0021] For a better understanding of the present teachings and in no way limiting the scope of the present teachings, all numbers and other numerical values ​​expressing quantities, percentages or ratios used in the specification and claims should be understood as being modified in all cases by the term "about," unless otherwise indicated. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0022] Unless otherwise indicated, adjectives such as "substantially" and "about" that modify a condition or relational characteristic of one or more features of an embodiment of the present invention should be understood to mean that the condition or characteristic is defined within an acceptable tolerance for the operation of the embodiment to which it is intended to be applied. Unless otherwise indicated, the word "or" in the specification and claims is considered to be an inclusive "or" rather than an exclusive or, and indicates at least one or any combination of the items to which it is associated.

[0023] In the specification and claims of this application, each of the verbs "comprise," "include," and "have," and their conjugated forms, is used to indicate that the object or objects of the verb are not necessarily a complete list of components, elements, or parts of the subject or subjects of the verb. Other terms used herein are intended to be defined by their commonly understood meanings in the art.

[0024] In some embodiments of the present invention, DNA nucleases are used to affect DNA breakage at the target site to induce cellular repair mechanisms, such as, but not limited to, non-homologous end joining (NHEJ). In classical NHEJ, the two ends of a double-strand break (DSB) site are joined together in a rapid but inaccurate manner (i.e., often resulting in mutations in the DNA at the cut site in the form of small insertions or deletions).

[0025] As used herein, the term "modified cell" refers to a cell in which a double-strand break is effected by a complex of an RNA molecule and a CRISPR nuclease due to hybridization to a target sequence, i.e., on-target hybridization.

[0026] The present invention provides one or more modified cells obtained by using any method described herein. In one embodiment, these one or more modified cells can produce daughter cells (progeny cell). In one embodiment, these one or more modified cells can produce daughter cells after transplantation (engraftment). As a non-limiting example, the modified cell can be a hematopoietic stem cell (HSC), or any cell suitable for allogeneic cell transplantation or autologous cell transplantation.

[0027] As used herein, the term "targeting sequence" or "targeting molecule" refers to a nucleotide sequence or a molecule comprising a nucleotide sequence that can hybridize with a specific target sequence, for example, a targeting sequence having a nucleotide sequence that is at least partially complementary to the targeted sequence along the length of the targeting sequence. The targeting sequence or targeting molecule can be part of an RNA molecule that can form a complex with the CRISPR nuclease alone or in combination with other RNA molecules, wherein the targeting sequence serves as the targeting portion of the CRISPR complex. When a molecule with a targeting sequence is present simultaneously with the CRISPR nuclease, the RNA molecule, alone or in combination with one or more additional RNA molecules (e.g., tracrRNA molecules), is capable of targeting the CRISPR nuclease to a specific target sequence. As a non-limiting example, the guide sequence portion of a CRISPR RNA molecule or a single guide RNA molecule can serve as a targeting molecule. Each possibility represents a separate embodiment. The targeting sequence can be custom-designed to target any desired sequence.

[0028] As used herein, the term "targeting" refers to preferential hybridization of a targeting sequence of a targeting molecule to a nucleic acid having a target nucleotide sequence. It should be understood that the term "targeting" includes variable hybridization efficiencies such that nucleic acids having a target nucleotide sequence are preferentially targeted, but in addition to on-target hybridization, unintentional off-target hybridization may also occur. It should be understood that when an RNA molecule targets a sequence, the complex of the RNA molecule and the CRISPR nuclease molecule will target the sequence to achieve nuclease activity.

[0029] The "guide sequence portion" of an RNA molecule refers to a nucleotide sequence that is capable of hybridizing to a specific target DNA sequence, for example, a guide sequence portion has a nucleotide sequence that is partially or completely complementary to the targeted DNA sequence along the length of the guide sequence portion. In some embodiments, the guide sequence portion is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or is about 17-50, 17-49, 17-48, 17-47, 17-46, 17-45, 17-44, 17-43, 17-42, 17-41, 17-40, 17-41, 17-42, 17-43 Preferably, the full length of the guide sequence portion is fully complementary to the target DNA sequence along the length of the guide sequence portion. The guide sequence portion can be part of an RNA molecule that can form a complex with a CRISPR nuclease, wherein the guide sequence portion serves as the DNA targeting portion of the CRISPR complex. When an RNA molecule having a guide sequence portion is present simultaneously with a CRISPR molecule, alone or in combination with one or more additional RNA molecules (e.g., tracrRNA molecules), the RNA molecule is capable of targeting the CRISPR nuclease to a specific target DNA sequence. Thus, a CRISPR complex can be formed by direct binding of an RNA molecule having a guide sequence portion to a CRISPR nuclease or by binding of an RNA molecule having a guide sequence portion and one or more additional RNA molecules to a CRISPR nuclease. Each possibility represents a separate embodiment. The guide sequence portion can be custom-designed to target any desired sequence. Therefore, a molecule comprising a "guide sequence portion" is a class of targeting molecules. In some embodiments, the guide sequence portion comprises a sequence that is identical to or differs from a guide sequence portion described herein by no more than 1, 2, 3, 4, or 5 nucleotides, such as a guide sequence shown in any one of SEQ ID NOs: 1-37421. Each possibility represents a separate embodiment. In some such embodiments, the guide sequence portion comprises a sequence identical to the sequence set forth in any one of SEQ ID NOs: 1-37421.In this application, the terms "guide molecule," "RNA guide molecule," "guide RNA molecule," and "gRNA molecule" are synonymous with molecules that comprise a guide sequence portion.

[0030] As used herein, the term "indiscriminate" means that the guide sequence portion of the RNA molecule targets a specific DNA sequence that is common to all alleles of a gene.

[0031] In an embodiment of the present invention, the RNA molecule comprises a guide sequence portion having 17-50 consecutive nucleotides comprising nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-37421.

[0032] The guide sequence portion of the RNA molecule and / or RNA molecule may contain modified nucleotides. Exemplary modifications to nucleotides / polynucleotides may be synthetic and include polynucleotides containing nucleotides containing bases other than naturally occurring adenine, cytosine, thymine, uracil, or guanine bases. Modifications to polynucleotides include polynucleotides containing synthetic non-naturally occurring nucleosides such as locked nucleic acids. Modifications to polynucleotides may be used to increase or decrease the stability of RNA. An example of a modified polynucleotide is an mRNA containing 1-methyl pseudouridine. For examples of modified polynucleotides and their uses, see U.S. Patent No. 8,278,036, PCT International Publication No. WO / 2015 / 006747, and Weissman and Kariko (2015), each of which is incorporated herein by reference.

[0033] As used herein, "consecutive nucleotides" as shown in a SEQ ID NO refers to the nucleotides in a nucleotide sequence in the order shown in the SEQ ID NO without any intervening nucleotides.

[0034] In embodiments of the present invention, the guide sequence portion can be 17-50 nucleotides in length and contain 20-22 consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 1-37421. In embodiments of the present invention, the guide sequence portion can be less than 22 nucleotides in length. For example, in embodiments of the present invention, the guide sequence portion can be 17, 18, 19, 20, or 21 nucleotides in length. In such embodiments, the guide sequence portion can consist of 17, 18, 19, 20, or 21 nucleotides, respectively, of a sequence of 17-22 consecutive nucleotides set forth in any one of SEQ ID NOs: 1-37421. For example, a guide sequence portion of 17 nucleotides of a sequence of 17 consecutive nucleotides set forth in SEQ ID NO: 37422 can consist of any of the following nucleotide sequences (nucleotides excluded from the consecutive sequence are marked with a strikethrough): AAAAAAUGUACUUGGUUCC(SEQ ID NO:37422) 17 nucleotide guide sequence 1: 17 nucleotide guide sequence 2: 17 nucleotide guide sequence 3: 17 nucleotide guide sequence 4:

[0035] In embodiments of the present invention, the guide sequence portion can be greater than 20 nucleotides in length. For example, in embodiments of the present invention, the guide sequence portion can be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In such embodiments, the guide sequence portion comprises 17-50 nucleotides of a sequence comprising 20, 21, or 22 consecutive nucleotides as set forth in any one of SEQ ID NOs: 1-37421, and additional nucleotides that are fully complementary to the nucleotides or nucleotide sequence adjacent to the 3' end of the target sequence, the 5' end of the target sequence, or both.

[0036] In an embodiment of the present invention, a CRISPR nuclease and an RNA molecule comprising a guide sequence portion form a CRISPR complex that binds to a target DNA sequence to achieve cleavage of the target DNA sequence. A CRISPR nuclease, such as Cpf1, can form a CRISPR complex comprising a CRISPR nuclease and an RNA molecule without an additional tracrRNA molecule. Alternatively, a CRISPR nuclease, such as Cas9, can form a CRISPR complex between a CRISPR nuclease, an RNA molecule, and a tracrRNA molecule. The guide sequence portion, which comprises a nucleotide sequence capable of hybridizing with a specific target DNA sequence, and the sequence portion involved in CRISPR nuclease binding, such as a tracrRNA sequence portion, can be located on the same RNA molecule. Alternatively, the guide sequence portion can be located on one RNA molecule, and the sequence portion involved in CRISPR nuclease binding, such as a tracrRNA portion, can be located on a separate RNA molecule. A single RNA molecule comprising a guide sequence portion (e.g., a DNA-targeting RNA sequence) and an RNA sequence portion bound by at least one CRISPR protein (e.g., a tracrRNA sequence portion) can form a complex with the CRISPR nuclease and act as a DNA-targeting molecule. In some embodiments, a first RNA molecule (e.g., a crRNA molecule) comprising a DNA-targeting RNA portion that includes a guide sequence portion and a separate RNA molecule (e.g., a tracrRNA molecule) comprising a CRISPR protein-binding RNA sequence interact through base pairing to form an RNA complex (e.g., a crRNA:tracrRNA complex) that targets the CRISPR nuclease to the DNA target site, or, alternatively, the first RNA molecule and the separate RNA molecule are fused together to form an RNA molecule (e.g., an sgRNA molecule) that complexes with and targets the CRISPR nuclease to the DNA target site.

[0037] In an embodiment of the present invention, the RNA molecule comprising a guide sequence portion may further comprise the sequence of a tracrRNA molecule. These embodiments can be designed as a synthetic fusion of the guide portion of the RNA molecule and a transactivating crRNA (tracrRNA). (See Jinek et al., 2012). In such embodiments, the RNA molecule is a single guide RNA (sgRNA) molecule. The embodiments of the present invention can also form a CRISPR complex using a separate tracrRNA molecule and a separate RNA molecule (e.g., crRNA molecule) comprising a guide sequence portion. In such embodiments, the tracrRNA molecule can hybridize with the RNA molecule by base pairing, and may be advantageous in certain applications of the invention described herein.

[0038] The term "tracr pairing sequence" refers to a sequence that is sufficiently complementary to a tracrRNA molecule to hybridize with the tracrRNA via base pairing and promote formation of a CRISPR complex. (See U.S. Patent No. 8,906,616). In embodiments of the present invention, the RNA molecule may further comprise a portion having a tracr pairing sequence.

[0039] For the purposes of this disclosure, a "gene" includes a DNA region that encodes a gene product, as well as all DNA regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to the coding sequence and / or the transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0040] "Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells, and human cells.

[0041] As used herein, the term "nuclease" refers to an enzyme that is capable of cleaving the phosphodiester bond between the nucleotide subunits of a nucleic acid. Nucleases can be isolated or derived from natural sources. The natural source can be any living organism. Alternatively, the nuclease can be a modified or synthetic protein that retains phosphodiester bond cleavage activity. Genetic modification can be achieved using nucleases, such as CRISPR nucleases.

[0042] According to an embodiment of the present invention, there is provided a method for inactivating an allele of the Tet methylcytosine dioxygenase 2 (TET2) gene in a cell, the method comprising introducing into the cell a composition comprising: at least one CRISPR nuclease, or a polynucleotide molecule encoding the same; and an RNA molecule comprising a guide sequence portion, or a polynucleotide molecule encoding the RNA molecule, wherein the complex of the CRISPR nuclease and the RNA molecule affects a double-strand break in an allele of the TET2 gene, and The guide sequence portion of the RNA molecule comprises 17-50 consecutive nucleotides, and the 17-50 consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421.

[0043] In some embodiments, the guide sequence portion comprises a nucleotide sequence as set forth in SEQ ID NOs: 1-37421, or a portion of any one of SEQ ID NOs: 1-37421, and optionally comprises additional nucleotides before or after the beginning or end of the nucleotide sequence. As a non-limiting example, the 17-nucleotide sequence found in SEQ ID NO: 1 can form a guide sequence portion. Additionally, the guide sequence portion can comprise the 17-nucleotide sequence found in SEQ ID NO: 1 and further comprise additional nucleotides 5' or 3' to the 17-nucleotide sequence found in SEQ ID NO: 1.

[0044] In some embodiments, the RNA molecule is a crRNA molecule, and the composition further comprises a tracrRNA molecule that forms a crRNA:tracrRNA complex with the crRNA molecule. In some embodiments, the RNA molecule is an sgRNA molecule.

[0045] In some embodiments, the composition comprises an additional RNA molecule comprising a guide sequence portion comprising 17-50 consecutive nucleotides comprising nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-37421.

[0046] In some embodiments, the guide sequence portion of the RNA molecule comprises 17-50 consecutive nucleotides comprising nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-37421, modified to contain up to five mismatches relative to the target site.

[0047] In some embodiments, the method is a method of preparing a modified immune cell (eg, T cell) for use in immunotherapy. In some embodiments, the method is performed in vitro or ex vivo.

[0048] In some embodiments, the composition is introduced into cells of a subject or into cells in culture.

[0049] In some embodiments, the cell is a lymphocyte, a T cell, a T regulatory cell, a B cell, a natural killer (NK) cell, a macrophage, a stem cell or a fibroblast, a blood cell, a hepatocyte, a keratinocyte, or any other cell type capable of being reprogrammed into an induced pluripotent stem cell (iPSC).

[0050] In some embodiments, the cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPS cell), an iPSC-derived cell, a natural killer cell (NK), an iPS-derived NK cell (iNK), a T cell, an innate-like T cell (iT), a natural killer T cell (NKT), a γδ T cell, an iPSC-derived T cell, an invariant NKT cell (iNKT), an iPSC-derived NKT, a monocyte, or a macrophage.

[0051] In some embodiments, the CRISPR nuclease and the RNA molecule are introduced into the cell at substantially the same time or at different times.

[0052] In some embodiments, the allele of the TET2 gene in the cell undergoes an insertion or deletion mutation.

[0053] In some embodiments, the insertion or deletion mutation creates a premature stop codon.

[0054] In some embodiments, the inactivation results in a truncated protein encoded by the mutant allele. For example, the inactivation method mutates a TET2 allele such that the mutant allele encodes a truncated form of the TET2 protein.

[0055] In some embodiments, the composition introduced into the cell further comprises a second RNA molecule comprising a guide sequence portion, wherein the guide sequence portion of the second RNA molecule comprises 17-50 consecutive nucleotides comprising nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-37421, preferably wherein the guide sequence portion of the second RNA molecule is different from the guide sequence portion of the first RNA molecule.

[0056] According to an embodiment of the present invention, there is provided a method for inactivating an allele of the Tet methylcytosine dioxygenase 2 (TET2) gene in a cell, the method comprising introducing into the cell a composition comprising: at least one CRISPR nuclease, or a polynucleotide molecule encoding a CRISPR nuclease; and An RNA molecule comprising a guide sequence portion, or a polynucleotide molecule encoding the RNA molecule, wherein the complex of the CRISPR nuclease and the RNA molecule effects a double-strand break in an allele of the TET2 gene, The guide sequence portion of the RNA molecule comprises 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides comprise nucleotides in the sequence of any one of SEQ ID NOs: 1-37421, which are modified to contain 1, 2, 3, 4 or 5 nucleotide mismatches relative to the fully complementary target sequence of the guide sequence portion.

[0057] In some embodiments, the guide sequence portion of the RNA molecule comprises 1, 2, 3, 4, or 5 nucleotide mismatches relative to the fully complementary target sequence of the guide sequence portion.

[0058] In some embodiments, the guide sequence portion provides greater targeting specificity for the complex of the CRISPR nuclease and RNA molecule relative to a guide sequence portion that has greater complementarity to an allele of the TET2 gene.

[0059] In some embodiments, the composition introduced into the cell further comprises a second RNA molecule comprising a guide sequence portion, wherein the guide sequence portion of the second RNA molecule comprises 17-50 contiguous nucleotides comprising nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-37421, or any one of SEQ ID NOs: 1-37421 modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches relative to the fully complementary target sequence of the guide sequence portion, preferably wherein the guide sequence portion of the second RNA molecule is different from the guide sequence portion of the first RNA molecule.

[0060] According to an embodiment of the present invention, a composition comprising an RNA molecule is provided, wherein the RNA molecule comprises a guide sequence portion comprising 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides comprise nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421.

[0061] In some embodiments, the composition further comprises a second RNA molecule comprising a guide sequence portion, wherein the guide sequence portion comprises 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides comprise nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-37421, preferably wherein the guide sequence portion of the second RNA molecule is different from the guide sequence portion of the first RNA molecule.

[0062] According to an embodiment of the present invention, a composition is provided comprising an RNA molecule comprising a guide sequence portion comprising 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides comprise nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421, or any one of SEQ ID NOs: 1-37421 modified to contain 1, 2, 3, 4 or 5 nucleotide mismatches relative to the fully complementary target sequence of the guide sequence portion.

[0063] In some embodiments, the composition further comprises a second RNA molecule comprising a guide sequence portion comprising 17-50 contiguous nucleotides comprising nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-37421, or any one of SEQ ID NOs: 1-37421 modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches relative to the fully complementary target sequence of the guide sequence portion, preferably wherein the guide sequence portion of the second RNA molecule is different from the guide sequence portion of the first RNA molecule.

[0064] In some embodiments, any of the compositions described herein further comprises a CRISPR nuclease.

[0065] In some embodiments, any of the compositions described herein further comprises a tracrRNA molecule.

[0066] According to an embodiment of the present invention, cells modified by any of the methods described herein or using any of the compositions described herein are provided. The modified cells may also have other genes altered to improve their use for adoptive transfer, for example, to reduce or prevent graft-versus-host disease (GVHD).

[0067] Preferably, all alleles of the TET2 gene are inactivated such that the modified cell is unable to express a full-length, functional TET2 protein product.

[0068] In some embodiments, the cell is any of the following, wherein the cell is a lymphocyte, a T cell, a T regulatory cell, a B cell, a natural killer (NK) cell, a macrophage, a stem cell or a fibroblast, a blood cell, a hepatocyte, a keratinocyte, or any other cell type capable of being reprogrammed into an induced pluripotent stem cell (iPSC).

[0069] In some embodiments, the cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPS cell), an iPSC-derived cell, a natural killer cell (NK), an iPS-derived NK cell (iNK), a T cell, an innate-like T cell (iT), a natural killer T cell (NKT), a γδ T cell, an iPSC-derived T cell, an invariant NKT cell (iNKT), an iPSC-derived NKT, a monocyte, or a macrophage.

[0070] In some embodiments, the cell is a stem cell or any cell type capable of being reprogrammed into an induced pluripotent stem cell (iPSC).

[0071] In some embodiments, the stem cells are differentiated after they are modified.

[0072] In some embodiments, the stem cells differentiate into any of lymphocytes, T cells, T regulatory cells, B cells, natural killer (NK) cells, innate-like T cells (iT), natural killer T cells (NKT), γδ T cells, invariant NKT cells (iNKT), monocytes, or macrophages.

[0073] According to an embodiment of the present invention, there is provided a medicament comprising any one of the compositions described herein for inactivating a TET2 allele in a cell, wherein the medicament is administered by delivering the composition to the cell.

[0074] According to an embodiment of the present invention, there is provided a use of any of the compositions or modified cells described herein for adoptive immunotherapy, for example, for treating cancer.

[0075] According to an embodiment of the present invention, there is provided a medicament comprising any of the compositions or modified cells described herein for use in adoptive immunotherapy, for example, for treating cancer.

[0076] According to an embodiment of the present invention, there is provided a kit for inactivating a TET2 allele in a cell, comprising any one of the compositions described herein and instructions for delivering the composition to the cell.

[0077] In some embodiments, the composition is delivered to the cell ex vivo.

[0078] According to an embodiment of the present invention, a kit for administering adoptive immunotherapy to a subject is provided, comprising any of the compositions or modified cells described herein and instructions for delivering any of the compositions or modified cells to a subject in need of adoptive immunotherapy.

[0079] According to an embodiment of the present invention, there is provided any of the compositions or modified cells described herein for use in adoptive immunotherapy, comprising delivering any of the compositions or modified cells described herein to a subject in need of adoptive immunotherapy.

[0080] A method of treating a disease or condition, the method comprising delivering any of the compositions or modified cells described herein to a subject, preferably wherein the disease or condition is cancer.

[0081] According to an embodiment of the present invention, there is provided a composition, method, process, kit or use characterized by one or more elements disclosed herein.

[0082] According to an embodiment of the present invention, the immune cell (for example, T cell) of the modification obtained by the method is intended to be used as the medicine for treating the cancer, infection or immune disease of the experimenter in need.The immune cell of modification or its colony can be carried out in any convenient way known in the art to the application of the experimenter, including but not limited to aerosol inhalation, injection, intake, blood transfusion, implantation or transplantation.Injection or infusion can be subcutaneous, intradermal, intratumor, intranodule, intramedullary, intramuscular, by intravenous or intralymphatic injection or intraperitoneal.According to an embodiment of the present invention, there is provided a method for adoptive cell therapy or prevention, which includes applying the modified cell to the experimenter suffering from cancer or infection or determining to be in the risk of suffering from cancer or infection.

[0083] According to an embodiment of the present invention, there is provided a use of any of the compositions or modified cells described herein for adoptive immunotherapy, comprising delivering a composition of any of the compositions or modified cells described herein to a subject in need of adoptive immunotherapy.

[0084] According to an embodiment of the present invention, a medicament for adoptive immunotherapy comprising any of the compositions or modified cells described herein is provided, wherein the medicament is administered by delivering any of the compositions or modified cells described herein to a subject in need of adoptive immunotherapy.

[0085] According to an embodiment of the present invention, there is provided an RNA molecule for modifying cells (e.g., lymphocytes, T cells, CAR-T cells) that can be used for adoptive immunotherapy. The RNA molecule can be delivered to cells in vitro, in vitro, or in vivo.

[0086] According to an embodiment of the present invention, there is provided a kit for inactivating a TET2 allele in a cell, comprising any one of the compositions described herein and instructions for delivering the composition to the cell.

[0087] According to embodiments of the present invention, cells modified by the methods described herein or using the compositions described herein are provided.

[0088] According to an embodiment of the present invention, a gene editing composition is provided, comprising an RNA molecule, wherein the RNA molecule comprises a guide sequence portion having 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421. In some embodiments, the RNA molecule further comprises a portion having a sequence that binds to a CRISPR nuclease. In some embodiments, the sequence that binds to a CRISPR nuclease is a tracrRNA sequence. In some embodiments, the RNA comprising the guide sequence portion is a crRNA molecule. In some embodiments, the RNA molecule comprising the guide sequence portion is a single guide RNA (sgRNA) molecule.

[0089] In some embodiments, the RNA molecule further comprises a portion having a tracr mate sequence.

[0090] In some embodiments, the RNA molecule may further comprise one or more linker moieties.

[0091] According to embodiments of the present invention, the length of the RNA molecule can be at most 1000, 900, 800, 700, 600, 500, 450, 400, 350, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110 or 100 nucleotides. Each possibility represents a separate embodiment. In embodiments of the present invention, the RNA molecule may be 17 to a maximum of 300 nucleotides in length, 100 to a maximum of 300 nucleotides in length, 150 to a maximum of 300 nucleotides in length, 100 to a maximum of 500 nucleotides in length, 100 to a maximum of 400 nucleotides in length, 200 to a maximum of 300 nucleotides in length, 100 to 200 nucleotides in length, or 150 to a maximum of 250 nucleotides in length. Each possibility represents a separate embodiment.

[0092] According to some embodiments of the invention, the composition further comprises a tracrRNA molecule.

[0093] According to an embodiment of the present invention, a method for inactivating TET2 expression in a cell is provided, the method comprising delivering a composition comprising an RNA molecule and a CRISPR nuclease to the cell, the RNA molecule comprising a guide sequence portion having 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421.

[0094] According to embodiments of the present invention, at least one CRISPR nuclease and one or more RNA molecules are delivered to a subject and / or cell substantially simultaneously or at different times.

[0095] In some embodiments, the tracrRNA molecule is delivered to a subject and / or cell substantially simultaneously with the CRISPR nuclease and one or more RNA molecules, or at different times.

[0096] The compositions and methods of the present disclosure can be used for improved adoptive immunotherapy.

[0097] Any one or a combination of the strategies described herein for inactivating TET2 expression can be used in the context of the present invention.

[0098] In an embodiment of the present invention, a guide RNA molecule is used to direct the CRISPR nuclease to an exon or splice site of a TET2 allele to generate a double-strand break (DSB), resulting in the insertion or deletion of nucleotides and the formation of a frameshift mutation in the TET2 allele by inducing an error-prone non-homologous end joining (NHEJ) mechanism. Frameshift mutations can result in, for example, inactivation or knockout of the TET2 allele by generating an early stop codon in the TET2 allele, and the production of a truncated protein or nonsense-mediated mRNA decay of the allelic transcript. In a further embodiment, a single RNA molecule is used to direct the CRISPR nuclease to the promoter of the TET2 allele.

[0099] Embodiments of the compositions described herein include at least one CRISPR nuclease, one or more guide RNA molecules, and optionally one or more tracrRNA molecules, that are simultaneously effective in a subject or cell. The at least one CRISPR nuclease, one or more guide RNA molecules, and optionally one or more tracrRNA molecules can be delivered substantially simultaneously, or can be delivered at different times but have an effect simultaneously. For example, this includes delivering the CRISPR nuclease to a subject or cell before the guide RNA molecule and / or tracrRNA molecule is substantially present in the subject or cell.

[0100] In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a T regulatory cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a macrophage. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a fibroblast, a blood cell, a hepatocyte, a keratinocyte, or any other cell type that can be reprogrammed into an induced pluripotent stem cell (iPSC). TET2 editing strategy

[0101] The present invention provides methods for knocking out a TET2 allele in cells of a subject, thereby improving the performance of the cells or cells derived from the cells in adoptive transfer therapy.

[0102] TET2 editing strategies include, but are not limited to, biallelic knockout by targeting any one or a combination of exons 3–11, including within thirty nucleotides upstream and downstream of the exons flanking the splice donor and acceptor sites, as frameshifts in these exons result in nonfunctional, truncated TET2 proteins or nonsense-mediated decay of mutant TET2 transcripts. CRISPR nucleases and PAM recognition

[0103] In some embodiments, the sequence-specific nuclease is selected from CRISPR nuclease or a functional variant thereof. In some embodiments, the sequence-specific nuclease is an RNA-guided DNA nuclease. In such embodiments, the RNA sequence of the guide RNA-guided DNA nuclease (e.g., Cpf1) binds to all TET2 alleles in the cell and / or directs the RNA-guided DNA nuclease to all TET2 alleles in the cell. In some embodiments, the CRISPR complex does not further comprise tracrRNA. It will be understood by those skilled in the art that RNA molecules can be engineered to bind to a target selected in the genome by methods well known in the art.

[0104] As used herein, the term "PAM" refers to a nucleotide sequence of a target DNA that is located near a target DNA sequence and is recognized by a CRISPR nuclease complex. The PAM sequence may differ depending on the identity of the nuclease. In addition, there are CRISPR nucleases that can target almost all PAMs. In some embodiments of the present invention, the CRISPR system utilizes one or more RNA molecules having a guide sequence portion to direct the CRISPR nuclease to the target DNA site by Watson-Crick base pairing between the guide sequence portion and the pre-spacer of the target DNA site, which is adjacent to the pre-spacer adjacent motif (PAM), which is an additional requirement for target recognition. The CRISPR nuclease then mediates the cutting of the target DNA site to produce a double-strand break in the pre-spacer. In a non-limiting example, a type II CRISPR system utilizes a mature crRNA:tracrRNA complex that directs a CRISPR nuclease (e.g., Cas9) to the target DNA by Watson-Crick base pairing between the guide sequence portion of the crRNA and the pre-spacer on the target DNA adjacent to the pre-spacer adjacent motif (PAM).It will be understood by those skilled in the art that each of the engineered RNA molecules of the present invention is further designed to associate with a target genomic DNA sequence adjacent to a protospacer adjacent motif (PAM), such as a PAM that matches a sequence associated with the type of CRISPR nuclease being utilized, such as, for non-limiting examples, NGG or NAG for Streptococcus pyogenes Cas9 WT (SpCAS9), where "N" is any nucleobase; NNGRRT for Staphylococcus aureus (SaCas9); NNNVRYM for Campylobacter jejuni Cas9 WT; NGAN or NGNG for SpCas9-VQR variant; NGCG for SpCas9-VRER variant; NGAG for SpCas9-EQR variant; NRRH for SpCas9-NRRH variant, wherein N is any nucleobase, R is A or G and H is A, C or T; NRTH for SpCas9-NRTH variant, wherein N is any nucleobase, R is A or G and H is A, C or T; NRCH for SpCas9-NRCH variant, wherein N is any nucleobase, R is A or G and H is A, C or T; NG for SpCas9 SpG variant, wherein N is any nucleobase; NG or NA for SpCas9 SpCas9-NG variants, wherein N is any nucleobase; NR or NRN or NYN for SpRY variants of SpCas9, wherein N is any nucleobase, R is A or G and Y is C or T; NNG for Streptococcus canis Cas9 variant (ScCas9), wherein N is any nucleobase; NNNRRT for SaKKH-Cas9 variant of Staphylococcus aureus (SaCas9), wherein N is any nucleobase and R is A or G; NNNNGATT for Neisseria meningitidis (NmCas9), wherein N is any nucleobase; TTN for Alicyclobacillus acidocaldarius Cas12b (AacCas12b), wherein N is any nucleobase; or TTTV for Cpf1, wherein V is A, C or G. Each of the RNA molecules of the present invention is designed to bind to one or more different CRISPR nucleases to form a complex and is designed to target a polynucleotide sequence of interest using one or more different PAM sequences corresponding to the CRISPR nuclease utilized.

[0105] In some embodiments, RNA-guided DNA nucleases, such as CRISPR nucleases, can be used to cause DNA breaks at a desired location in the cell genome, whether double-stranded or single-stranded in nature. The most commonly used RNA-guided DNA nucleases are derived from the CRISPR system, however, other RNA-guided DNA nucleases are also contemplated for use in the genome editing compositions and methods described herein. For example, see U.S. Patent Publication No. 2015 / 0211023, which is incorporated herein by reference.

[0106] The CRISPR systems that can be used in the practice of the present invention vary widely. The CRISPR system can be a type I, type II, or type III system. Non-limiting examples of suitable CRISPR proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas11, Cas12, Cas13, Cas14, Cas15, Cas16, Cas17, Cas18, Cas19, Cas20, Cas21, Cas22, Cas23, Cas24, Cas25, Cas26, Cas27, Cas28, Cas29, Cas30, Cas31, Cas32, Cas33, Cas34, Cas35, Cas36, Cas37, Cas38, Cas39, Cas31, Cas3 Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966.

[0107] In some embodiments, the RNA-guided DNA nuclease is a CRISPR nuclease derived from a type II CRISPR system (e.g., Cas9). The CRISPR nuclease can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Neisseria meningitidis, Treponema denticola, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenogenum, selenitireducens), Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsonii watsoni), Pseudoalteromonas shaloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp.), Petrotoga mobilis, Thermosiphoafricanus, Acaryochloris marina, or any species encoding a CRISPR nuclease with a known PAM sequence. CRISPR nucleases encoded by uncultured bacteria can also be used in the context of the present invention. (See Burstein et al., Nature, 2017). CRISPR protein variants with known PAM sequences, such as the SpCas9 D1135E variant, the SpCas9 VQR variant, the SpCas9 EQR variant, or the SpCas9 VRER variant, can also be used in the context of the present invention.

[0108] Thus, RNA-guided DNA nucleases of CRISPR systems, such as Cas9 proteins or modified Cas9 or homologs or orthologs of Cas9, or other RNA-guided DNA nucleases belonging to other types of CRISPR systems, such as Cpf1 and its homologs and orthologs, can be used in the compositions of the present invention. Other CRISPR nucleases, such as those described in PCT International Application Publication Nos. WO2020 / 223514 and WO2020 / 223553, which are incorporated herein by reference, may also be used.

[0109] In certain embodiments, the CRISPR nuclease may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound having the same qualitative biological properties as a native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of native sequences and derivatives of native sequence polypeptides and fragments thereof, provided that they have the same biological activity as the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of a functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" includes amino acid sequence variants, covalent modifications, and fusions thereof of a polypeptide. Suitable derivatives of a Cas polypeptide or its fragments include, but are not limited to, mutants, fusions, and covalent modifications of a Cas protein or its fragments. Derivatives include, but are not limited to, CRISPR nickases, catalytically inactive or "dead" CRISPR nucleases, and fusions of CRISPR nucleases or their derivatives with other enzymes such as base editors or retrotransposons. See, for example, Anzalone et al. (2019) and PCT International Application No. PCT / US2020 / 037560.

[0110] In some embodiments, CRISPR nuclease or its derivatives can be fused with a protein having enzymatic activity. In some embodiments, enzymatic activity modifies target DNA. In some embodiments, enzymatic activity is nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase (photolyase) activity or glycosylase activity. In some cases, enzymatic activity is nuclease activity. In some cases, nuclease activity introduces double-strand breaks in target DNA. In some cases, enzymatic activity modifies the target polypeptide related to target DNA. In some cases, the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity. In some cases, the target polypeptide is a histone and the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity or deubiquitinating activity.

[0111] Cas proteins, including Cas proteins or fragments thereof, and derivatives of Cas proteins or fragments thereof, can be obtained from cells or chemically synthesized or obtained by a combination of these two methods. The cell can be a cell that naturally produces Cas proteins, or a cell that naturally produces Cas proteins and is genetically engineered to produce endogenous Cas proteins at higher expression levels or to produce Cas proteins from exogenously introduced nucleic acids, the nucleic acids encoding Cas proteins that are the same or different from the endogenous Cas. In some cases, the cell does not naturally produce Cas proteins and is genetically engineered to produce Cas proteins.

[0112] In some embodiments, the CRISPR nuclease is Cpf1. Cpf1 is a single RNA-guided endonuclease that utilizes a T-rich protospacer-adjacent motif. Cpf1 cleaves DNA through staggered double-strand breaks. Two Cpf1 enzymes from Acidaminococcus and Lachnospiraceae have been shown to perform efficient genome editing activity in human cells (see Zetsche et al., 2015).

[0113] Thus, RNA-guided DNA nucleases of a type II CRISPR system, such as Cas9 protein or modified Cas9 or homologs, orthologs or variants of Cas9, or other RNA-guided DNA nucleases belonging to other types of CRISPR systems, such as Cpf1 and homologs, orthologs or variants thereof, can be used in the present invention.

[0114] In some embodiments, the guide molecule comprises one or more chemical modifications that impart new or improved properties (e.g., improved degradation stability, improved hybridization energy characteristics, or improved binding properties to RNA-guided DNA nucleases). Suitable chemical modifications include, but are not limited to, modified bases, modified sugar moieties, or modified internucleoside linkages. Non-limiting examples of suitable chemical modifications include: 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methyluridine, "β, D-galactosylquinoside", 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, "2, 2-dimethylguanosine", 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, "β, D-mannosylqueuosine", 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methoxyguanosine, 2-methylthio-N6-isopentenyladenosine, N- ((9-β-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, N-((9-β-D-ribofuranosylpurin-6-yl)N-methylcarbamoyl)threonine, uridine-5-hydroxyacetic acid methyl ester, uridine-5-hydroxyacetic acid, wybutoxosine, queuosine, 2-thiacytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5- Methyluridine, N-((9-β-D-ribofuranosylpurin-6-yl)-carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine, "3-(3-amino-3-carboxypropyl)uridine, (acp3)u", 2'-O-methyl (M), 3'-thiophosphate (MS), 3'-thioPACE (MSP), pseudouridine or 1-methylpseudouridine. Each possibility represents a separate embodiment of the present invention.

[0115] In addition to targeting TET2 alleles by RNA-guided CRISPR nucleases, other means of inhibiting TET2 expression in target cells include, but are not limited to, the use of gapmers, shRNAs, siRNAs, custom TALENs, large-range nucleases or zinc finger nucleases, small molecule inhibitors, and any other methods known in the art for reducing or eliminating gene expression in target cells. See, for example, U.S. Patent Nos. 6,506,559; 7,560,438; 8,420,391; 8,552,171; 7,056,704; 7,078,196; 8,362,231; 8,372,968; 9,045,754; and PCT International Publication Nos. WO / 2004 / 067736; WO / 2006 / 097853 ; WO / 2003 / 087341; WO / 2000 / 0415661; WO / 2003 / 080809; WO / 2010 / 079430; WO / 2010 / 079430; WO / 2011 / 072246; WO / 2018 / 057989; and WO / 2017 / 164230, the entire contents of each of which are incorporated herein by reference.

[0116] Advantageously, when complexed with CRISPR nucleases in cells, the guide RNA molecules provided herein provide improved TET2 knockout efficiency relative to other guide RNA molecules. These specifically designed sequences can also be used to identify TET2 target sites for other nucleotide-targeted gene editing or gene silencing methods, such as siRNA, TALEN, meganucleases, or zinc finger nucleases. Delivery to cells

[0117] Any one of the compositions described herein can be delivered to the target cell by any suitable means. The RNA molecule composition of the present invention can target any cell containing and / or expressing TET2 alleles, such as mammalian lymphocytes or stem cells. For example, in one embodiment, the RNA molecule specifically targets the TET2 alleles in the target cell, and the target cell is a lymphocyte, T cell, T regulatory cell, B cell, natural killer (NK) cell, macrophage, stem cell or fibroblast, hemocyte, hepatocyte, keratinocyte or any other cell type that can be reprogrammed to induced pluripotent stem cell (iPSC). Delivery to cells can be carried out in vivo, in vitro or in vitro. In addition, nucleic acid compositions as described herein can be delivered to cells as one or more of DNA molecules, RNA molecules, ribonucleoprotein (RNP), nucleic acid vectors or any combination thereof.

[0118] In some embodiments, the RNA molecule comprises a chemical modification. Non-limiting examples of suitable chemical modifications include 2'-O-methyl (M), 2'-O-methyl, 3' phosphorothioate (MS) or 2'-O-methyl, 3' thioPACE (MSP), pseudouridine and 1-methylpseudouridine. Each possibility represents a separate embodiment of the present invention.

[0119] In some embodiments, any of the compositions described herein are delivered to cells in vivo. Compositions can be delivered to cells by any known in vivo delivery method, including but not limited to viral transduction, e.g., using lentivirus or adeno-associated virus (AAV), nanoparticle delivery, etc. Additional detailed delivery methods are described in this section.

[0120] In some embodiments, any of the compositions described herein is delivered to cells ex vivo. Compositions can be delivered to cells by any known ex vivo delivery method, including but not limited to nuclear transfection, electroporation, viral transduction, such as using lentivirus or adeno-associated virus (AAV), nanoparticle delivery, liposomes, etc. This section describes additional detailed delivery methods.

[0121] Any suitable viral vector system can be used to deliver nucleic acid compositions, such as the RNA molecule compositions of the present invention. Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids and target tissues. In certain embodiments, nucleic acids are administered for in vivo or ex vivo gene therapy purposes. Non-viral vector delivery systems include naked nucleic acids and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers. For a review of gene therapy methods, see Anderson (1992); Nabel & Felgner (1993); Mitani & Caskey (1993); Dillon (1993); Miller (1992); Van Brunt (1988); Vigne (1995); Kremer & Perricaudet (1995); Haddada et al. (1995); and Yu et al. (1994).

[0122] Non-viral delivery methods for nucleic acids and / or proteins include electroporation, lipofection, microinjection, gene guns, particle gun acceleration, virosomes, liposomes, immunoliposomes, lipid nanoparticles (LNPs), polycationic or lipid:nucleic acid conjugates, artificial virions, and agent-enhanced nucleic acid uptake, or can be delivered to plant cells via bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizobium meliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus, and cassava vein mosaic virus). (See, e.g., Chung et al., 2006) Sonoporation using, for example, the Sonitron 2000 system (Rich-Mar) can also be used to deliver nucleic acids. Cationic lipid-mediated delivery of proteins and / or nucleic acids is also contemplated as an in vivo, ex vivo, or in vitro delivery method. (See Zuri et al. (2015); see also Coelho et al. (2013); Judge et al. (2006); and Basha et al. (2011)).

[0123] Non-viral vectors, such as transposon-based systems, such as the recombinant Sleeping Beauty transposon system or the recombinant PiggyBac transposon system, can also be delivered to target cells and used to transpose the polynucleotide sequence of the composition molecule or the polynucleotide sequence encoding the composition molecule in the target cell.

[0124] Other exemplary nucleic acid delivery systems include those provided by Amaxa.RTM. Biosystems (Cologne, Germany), Maxyte Corporation (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics (see, e.g., U.S. Patent No. 6,008,336). Lipofection is described in, e.g., U.S. Patent No. 5,049,386, U.S. Patent No. 4,946,787; and U.S. Patent No. 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam.TM., Lipofectin.TM., and Lipofectamine.TM. RNAiMAX). Cationic and neutral lipids suitable for efficient receptor recognition polynucleotide lipofection include those disclosed in PCT International Publication Nos. WO / 1991 / 017424 and WO / 1991 / 016024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).

[0125] Preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunoliposomes, is well known to those skilled in the art (see, e.g., Crystal, Science (1995); Blaese et al., (1995); Behr et al., (1994); Remy et al., (1994); Gao and Huang (1995); Ahmad and Allen (1992); U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).

[0126] Other delivery methods include using the nucleic acid to be delivered packaged into EnGeneIC delivery vectors (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies, where one arm of the antibody is specific for the target tissue and the other arm is specific for the EDV. The antibody brings the EDV to the surface of the target cell and then brings the EDV into the cell through endocytosis. Once inside the cell, the contents are released (see MacDiarmid et al., 2009).

[0127] Delivery vehicles include, but are not limited to, bacteria (preferably non-pathogenic), vectors, nanoparticles, exosomes, microvesicles, gene gun delivery (e.g., by attaching the composition to gold particles that are fired into cells using a "gene gun"), viral vehicles (including but not limited to lentivirus, AAV and retrovirus), virus-like particles (VLP), large VLP (LVLP), lentivirus-like particles, transposons, viral vectors, naked vectors, DNA or RNA, and other delivery vehicles known in the art.

[0128] Delivery of CRISPR nucleases and / or polynucleotides encoding CRISPR nucleases and optional additional nucleotide molecules and / or additional proteins or peptides can be carried out using a single delivery vehicle or method or a combination of different delivery vehicles or methods. For example, CRISPR nucleases can be delivered to cells using LNPs, and crRNA molecules and tracrRNA molecules can be delivered to cells using AAVs. Alternatively, CRISPR nucleases can be delivered to cells using AAV particles, and crRNA molecules and tracrRNA molecules can be delivered to cells using separate AAV particles, which may be advantageous due to size limitations.

[0129] The use of RNA or DNA virus-based systems for virus-mediated nucleic acid delivery utilizes a highly evolved process for targeting viruses to specific cells in vivo and transporting viral payloads to the nucleus. Viral vectors can be directly applied to patients (in vivo), or they can be used for in vitro treatment of cells, and modified cells are applied to patients (ex vivo). Conventional virus-based systems for delivering nucleic acids include, but are not limited to, retroviruses, slow viruses, adenoviruses, adeno-associated viruses, vaccinia and herpes simplex virus vectors for gene transfer. RNA viruses can be used to deliver compositions as described herein. In addition, high transduction efficiencies have been observed in many different cell types and target tissues. The nucleic acids of the present invention can be delivered by non-integrating slow viruses. Optionally, slow viruses are used to deliver RNA. Optionally, slow viruses include mRNA and guide RNA molecules of nucleases. Optionally, slow viruses include nuclease proteins and guide RNA molecules. Optionally, slow viruses include mRNA, guide RNA molecules and tracrRNA molecules of nucleases. Optionally, slow viruses include nuclease proteins, guide RNA molecules and tracrRNA molecules.

[0130] Retroviral tropism can be altered by incorporating exogenous envelope proteins and expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically produce high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors are composed of cis-acting long terminal repeats with the ability to package up to 6-10 kb of exogenous sequence. The minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate the therapeutic gene into target cells to provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchschacher et al. (1992); Johann et al. (1992); Sommerfelt et al. (1990); Wilson et al. (1989); Miller et al. (1991); PCT International Publication No. WO / 1994 / 026877A1).

[0131] There are currently at least six viral vector approaches available for gene transfer in clinical trials that utilize methods that involve complementing defective vectors by inserting genes into helper cell lines to produce the transducing agent.

[0132] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (see Dunbar et al., 1995; Kohn et al., 1995; Malech et al., 1997). PA317 / pLASN was the first therapeutic vector used in gene therapy trials (Blaese et al., 1995). Transduction efficiencies of 50% or greater have been observed with MFG-S-packaged vectors (Ellem et al., 1997; Dranoff et al., 1997).

[0133] Packaging cells are used to form viral particles capable of infecting host cells. These cells include 293 cells, which package adenoviruses, AAVs, and Psi-2 cells or PA317 cells, which package retroviruses. Viral vectors used for gene therapy are typically produced by production cell lines, which package nucleic acid vectors into viral particles. The vector typically contains the minimum viral sequences required for packaging and subsequent integration into the host (if applicable), with other viral sequences replaced by expression cassettes encoding proteins to be expressed. The missing viral functions are provided in trans by the packaging cell line. For example, AAV vectors used for gene therapy typically only have inverted terminal repeat (ITR) sequences from the AAV genome, which are necessary for packaging and integration into the host genome. The viral DNA is packaged in a cell line that contains a helper plasmid that encodes other AAV genes, namely rep and cap, but lacks ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes the replication of the AAV vector and the expression of the AAV genes in the helper plasmid. Due to the lack of ITR sequences, the helper plasmid cannot be packaged in large quantities. Adenoviral contamination can be reduced by, for example, heat treatment, to which adenovirus is more sensitive than AAV. Additionally, AAV can be produced on a clinical scale using a baculovirus system (see US Pat. No. 7,479,554).

[0134] In many gene therapy applications, it is desirable that gene therapy vectors be delivered to specific tissue types with a high degree of specificity. Therefore, viral vectors can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with the viral coat protein on the outer surface of the virus. Ligands with affinity for receptors known to be present on the target cell type are selected. For example, Han et al. (1995) reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and that this recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand of the cell surface receptor. For example, filamentous phages can be engineered to display antibody fragments (e.g., FAB or Fv) with specific binding affinity for almost any selected cell receptor. Although the above description primarily applies to viral vectors, the same principles can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences that are beneficial for uptake by specific target cells.

[0135] Gene therapy vectors can be delivered in vivo by administration to an individual patient, for example, by systemic administration (eg, intravitreal, intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or local administration.

[0136] Alternatively, the vector can be delivered ex vivo to cells, such as cells transplanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells, and then optionally after selecting cells that have incorporated the vector, the cells are reimplanted into the patient. Non-limiting exemplary ex vivo methods may include removing tissue (e.g., peripheral blood, bone marrow, and spleen) from the patient for culture, transferring the nucleic acid to the cultured cells (e.g., hematopoietic stem cells), and then transplanting the cells into the patient's target tissue (e.g., bone marrow and spleen). In some embodiments, the stem cells or hematopoietic stem cells may be further treated with a vitality enhancer.

[0137] In vitro cell transfection for diagnosis, research, or for gene therapy (e.g., by reinfusing the transfected cells into a host organism) is well known to those skilled in the art. In a preferred embodiment, cells are isolated from a subject organism, transfected with a nucleic acid composition, and reinfused back into the subject organism (e.g., a patient). Various cell types suitable for in vitro transfection are well known to those skilled in the art (see, e.g., Freshney, "Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications" (6th ed., 2010) and references cited therein for discussions of how to isolate and culture cells from patients).

[0138] The carrier (such as retrovirus, liposome etc.) containing therapeutic nucleic acid compositions can also be directly administered to organism for in vivo cell transduction.Use by being generally used for any approach that molecule is introduced into and finally contacts with blood or tissue cells, including but not limited to injection, infusion, topical application (such as eye drops and eye cream) and electroporation.The suitable method of using this type of nucleic acid is obtainable and well known to those skilled in the art, and although more than one approach can be used to use specific composition, specific approach can provide more direct and more effective reaction than another approach usually.According to some embodiments, compositions is delivered by IV injection.

[0139] Vectors suitable for introducing transgenes into immune cells (eg, T cells) include non-integrating lentiviral vectors. See, for example, U.S. Patent Publication No. 2009 / 0117617.

[0140] As described above, the compositions described herein may be administered using a non-integrating lentiviral particle approach, e.g. The system is delivered to the target cell. This method can be used to deliver mRNA or other types of RNA to the target cell, so that the delivery of RNA to the target cell causes the composition described herein to assemble within the target cell. See also PCT International Publication Nos. WO / 2013 / 014537, WO / 2014 / 016690, WO / 2016 / 185125, WO / 2017 / 194902, and WO / 2017 / 194903.

[0141] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as the particular method used to administer the composition. Thus, as described below, a variety of suitable pharmaceutical composition formulations are available (see, e.g., Remington's Pharmaceutical Sciences, 17th edition, 1989). Examples of RNA guide sequences that specifically target an allele of the TET2 gene

[0142] While a large number of guide sequences can be designed to target the TET2 gene, the nucleotide sequences described in Table 1 and identified by SEQ ID NOs: 1-37421 were specifically selected to effectively perform the methods described herein.

[0143] Table 1 lists guide sequences designed for use in the above embodiments to associate with specific sequences within the TET2 allele. Each engineered guide molecule is further designed to associate with a target genomic DNA sequence of interest that is located next to a pre-spacer adjacent motif (PAM), such as a PAM that matches the sequence NGG or NAG, where "N" is any nucleobase. The guide sequence is designed to work in concert with one or more different CRISPR nucleases, including but not limited to, for example, SpCas9WT (PAM SEQ: NGG), SpCas9.VQR.1 (PAM SEQ: NGAN), SpCas9.VQR.2 (PAM SEQ: NGNG), SpCas9.EQR (PAM SEQ: NGAG), SpCas9.VRER (PAM SEQ: NGCG), SaCas9WT (PAM SEQ: NNGRRT), SpRY (PAM SEQ: NRN or NYN), NmCas9WT (PAM SEQ: NNNNGATT), Cpf1 (PAM SEQ: TTTV), JeCas9WT (PAM SEQ: NNNVRYM), OMNI-50 (PAM SEQ: NGG), OMNI-79 (PAM SEQ: NGG), OMNI-103 (PAM SEQ: NNRACT), OMNI-159 (NNNNCMAN), or OMNI-124 (PAM SEQ: NNGNRMNN).

[0144] OMNI is provided in PCT International Application Publication No. WO 2023 / 019269 A2, PCT International Application Publication No. WO 2022 / 170199 A2 and WO 2023 / 107946 A2, U.S. Patent No. 11,666,641 B2 and PCT International Application Publication No. WO 2020 / 223514 A2, WO 2022 / 098693 A1 and WO 2023 / 019263 A1, U.S. Application Publication No. 2023 / 0122086 A1 and PCT International Application Publication No. WO 2021 / 248016 A2 and WO 2023 / 102407 A2, PCT International Application Publication No. WO 2022 / 087135 A1 and PCT International Application Publication No. WO 2022 / 226215 A1 Additional descriptions of CRISPR nucleases are provided, the contents of each of which are incorporated herein by reference.

[0145] Each of the RNA molecules of the invention is designed to bind to one or more different CRISPR nucleases to form a complex, and is designed to target a polynucleotide sequence of interest using one or more different PAM sequences associated with the CRISPR nuclease being used.

[0146] As used herein, the following nucleotide identifiers are used to indicate the referenced nucleotide bases: Table 1: Guide sequence segments designed to associate with specific TET2 gene targets The positions listed in column 1 of Table 1 are based on the gnomAD v3 database and the UCSC Genome Browser assembly ID: hg38, sequencing / assembly provider ID: Reference Genome Consortium Human GRCh38.p12 (GCA_000001405.27). Assembly date: initial release December 2013; patch release 12 December 2017.

[0147] Provide examples below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes for making and implementing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are intended to illustrate the present invention. Experimental details Example 1: TET2 modification analysis

[0148] CRISPR nucleases in T cells were used to screen guide sequences containing 17-50 consecutive nucleotides containing nucleotides in the sequence of any one of SEQ ID NOs: 1-37421 for high on-target activity. On-target activity was determined by DNA capillary electrophoresis analysis. Example 2: TET2 editing in HeLa cells and primary T cells

[0149] TET2 edited in HeLa cells and primary T cells using a subset of publicly available TET2 targeting guide sequence segments is shown in Figure 1 and Figure 2 middle.

[0150] A summary table of TET2 editing data is shown below:

[0151] OMNI-103 related sequences are provided in the table below: References 1.Ahmad and Allen(1992)“Antibody-mediated Specific Binging andCytotoxicity of Lipsome-entrapped Doxorubicin to Lung Cancer Cells in Vitro”,Cancer Research 52:4817-20. 2.Anderson(1992)“Human gene therapy”,Science 256:808-13. 3.Anzalone et al.(2019)“Search-and-replace genomme editing withoutdouble-strand brakes or donor DNA”,Nature 576,149-157. 4.Basha et al.(2011)“Influence of Cationic Lipid Composition on GeneSilencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting Cells”,Mol.Ther.19(12):2186-200. 5.Behr(1994)Gene transfer with synthetic cationic amphiphiles:Prospects for gene therapy”,Bioconjuage Chem 5:382-89. 6.Blaese(1995)“Vectors in cancer therapy:how will they deliver”,Cancer Gene Ther.2:291-97. 7.Blaese et al.(1995)“T lympocyte-directed gene therapy for ADA-SCID:initial trial results after 4years”,Science 270(5235):475-80. 8.Buchschacher and Panganiban(1992)“Human 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Claims

1. A method for inactivating an allele of the Tet methylcytosine dioxygenase 2 (TET2) gene in a cell, the method comprising introducing into the cell a composition comprising: at least one CRISPR nuclease, or a polynucleotide molecule encoding a CRISPR nuclease; and an RNA molecule comprising a guide sequence portion, or a polynucleotide molecule encoding the RNA molecule, wherein the complex of the CRISPR nuclease and the RNA molecule affects a double-strand break in an allele of the TET2 gene, and The guide sequence portion of the RNA molecule comprises 17-50 consecutive nucleotides, and the 17-50 consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421.

2. The method according to claim 1, wherein the composition is introduced into cells of a subject or into cultured cells.

3. The method according to any one of claims 1 to 2, wherein the cell is a lymphocyte, a T cell, a T regulatory cell, a B cell, a natural killer (NK) cell, a macrophage, a stem cell or a fibroblast, a blood cell, a hepatocyte, a keratinocyte or any other cell type that can be reprogrammed into an induced pluripotent stem cell (iPSC).

4. The method according to any one of claims 1-2, wherein the cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPS cell), an iPSC-derived cell, a natural killer cell (NK), an iPS-derived NK cell (iNK), a T cell, an innate-like T cell (iT), a natural killer T cell (NKT), a γδT cell, an iPSC-derived T cell, an invariant NKT cell (iNKT), an iPSC-derived NKT, a monocyte or a macrophage.

5. The method according to any one of claims 1 to 4, wherein the CRISPR nuclease and the RNA molecule are introduced into the cell at substantially the same time or at different times.

6. The method according to any one of claims 1 to 5, wherein the allele of the TET2 gene in the cell undergoes an insertion or deletion mutation.

7. The method according to claim 6, wherein the insertion or deletion mutation generates a premature stop codon.

8. The method according to any one of claims 1 to 7, wherein the inactivation results in a truncated protein encoded by the mutant allele.

9. The method according to any one of claims 1 to 8, wherein the composition introduced into the cell further comprises a second RNA molecule comprising a guide sequence portion, wherein the guide sequence portion of the second RNA molecule comprises 17-50 consecutive nucleotides, and the 17-50 consecutive nucleotides comprise nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421, preferably wherein the guide sequence portion of the second RNA molecule is different from the guide sequence portion of the first RNA molecule.

10. A method for inactivating an allele of the Tet methylcytosine dioxygenase 2 (TET2) gene in a cell, the method comprising introducing into the cell a composition comprising: at least one CRISPR nuclease, or a polynucleotide molecule encoding a CRISPR nuclease; and an RNA molecule comprising a guide sequence portion, or a polynucleotide molecule encoding the RNA molecule, wherein the complex of the CRISPR nuclease and the RNA molecule affects a double-strand break in an allele of the TET2 gene, and The guide sequence portion of the RNA molecule comprises 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides contain nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421, which are modified to contain 1, 2, 3, 4 or 5 nucleotide mismatches relative to the fully complementary target sequence of the guide sequence portion.

11. The method according to claim 10, wherein the guide sequence portion of the RNA molecule comprises 1, 2, 3, 4 or 5 nucleotide mismatches relative to the fully complementary target sequence of the guide sequence portion.

12. The method according to any one of claims 10 or 11, wherein the guide sequence portion provides higher targeting specificity for the complex of the CRISPR nuclease and RNA molecule relative to the guide sequence portion having higher complementarity to an allele of the TET2 gene.

13. The method according to any one of claims 10 to 12, wherein the composition introduced into the cell further comprises a second RNA molecule comprising a guide sequence portion, wherein the guide sequence portion of the RNA molecule comprises 17 to 50 consecutive nucleotides comprising nucleotides in the sequence of any one of SEQ ID NOs: 1 to 37421; or any one of SEQ ID NOs: 1 to 37421 modified to contain 1, 2, 3, 4 or 5 nucleotide mismatches relative to the fully complementary target sequence of the guide sequence portion, preferably wherein the guide sequence portion of the second RNA molecule is different from the guide sequence portion of the first RNA molecule.

14. A composition comprising an RNA molecule comprising a guide sequence portion comprising 17-50 consecutive nucleotides, wherein the 17-50 consecutive nucleotides comprise nucleotides in the sequence shown in any one of SEQ ID NOs: 1-37421.

15. The composition according to claim 14, further comprising a second RNA molecule comprising a guide sequence portion, wherein the guide sequence portion comprises 17-50 consecutive nucleotides, said 17-50 consecutive nucleotides comprising nucleotides in the sequence of any one of SEQ ID NOs: 1-37421, preferably wherein the guide sequence portion of the second RNA molecule is different from the guide sequence portion of the first RNA molecule.

16. A composition comprising an RNA molecule comprising a guide sequence portion comprising 17-50 contiguous nucleotides comprising nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-37421, or any one of SEQ ID NOs: 1-37421 modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches relative to a fully complementary target sequence of the guide sequence portion.

17. The composition according to claim 16, further comprising a second RNA molecule comprising a guide sequence portion comprising 17-50 consecutive nucleotides, said 17-50 consecutive nucleotides comprising nucleotides in the sequence of any one of SEQ ID NOs: 1-37421, or any one of SEQ ID NOs: 1-37421 modified to contain 1, 2, 3, 4 or 5 nucleotide mismatches relative to the fully complementary target sequence of said guide sequence portion, preferably wherein the guide sequence portion of the second RNA molecule is different from the guide sequence portion of the first RNA molecule.

18. The composition according to any one of claims 14-17, further comprising a CRISPR nuclease.

19. The composition according to any one of claims 14 to 18, further comprising a tracrRNA molecule.

20. A cell modified by the method of any one of claims 1 to 13 or using the composition of any one of claims 14 to 19.

21. The modified cell according to claim 20, wherein the cell is any of the following, wherein the cell is a lymphocyte, a T cell, a T regulatory cell, a B cell, a natural killer (NK) cell, a macrophage, a stem cell or a fibroblast, a blood cell, a hepatocyte, a keratinocyte or any other cell type capable of being reprogrammed into an induced pluripotent stem cell (iPSC).

22. The modified cell according to claim 20, wherein the cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPS cell), an iPSC-derived cell, a natural killer cell (NK), an iPS-derived NK cell (iNK), a T cell, an innate-like T cell (iT), a natural killer T cell (NKT), a γδ T cell, an iPSC-derived T cell, an invariant NKT cell (iNKT), an iPSC-derived NKT, a monocyte or a macrophage.

23. The modified cell according to claim 20, wherein the cell is a stem cell or any cell type capable of being reprogrammed into an induced pluripotent stem cell (iPSC).

24. The modified cell according to claim 23, wherein the stem cell differentiates after modification.

25. The modified cell according to claim 24, wherein the stem cell differentiates into any one of a lymphocyte, a T cell, a T regulatory cell, a B cell, a natural killer (NK) cell, an innate-like T cell (iT), a natural killer T cell (NKT), a γδ T cell, an invariant NKT cell (iNKT), a monocyte or a macrophage.

26. A medicament comprising the composition of any one of claims 14 to 19 for use in inactivating a CTLA4 allele in a cell, wherein the medicament is administered by delivering the composition of any one of claims 14 to 19 to the cell.

27. Use of the composition of any one of claims 14 to 19 or the modified cell of any one of claims 20 to 25 for adoptive immunotherapy, comprising delivering the composition of any one of claims 14 to 19 or the modified cell of any one of claims 20 to 25 to a subject in need of adoptive immunotherapy.

28. A medicament comprising the composition of any one of claims 14-19 or the modified cell of any one of claims 20-25 for use in adoptive immunotherapy, wherein the medicament is administered by delivering the composition of any one of claims 14-19 or the modified cell of any one of claims 20-25 to a subject in need of adoptive immunotherapy.

29. A kit for inactivating a TET2 allele in a cell, comprising the composition of any one of claims 14-19 and instructions for delivering the composition to the cell.

30. The kit according to claim 29, wherein the composition is delivered to the cell ex vivo.

31. A kit for administering adoptive immunotherapy to a subject, comprising the composition of any one of claims 14-19 or the modified cells of any one of claims 20-25, and instructions for delivering the composition or modified cells to a subject in need of adoptive immunotherapy.

32. Use of the composition of any one of claims 14-19 or the modified cell of any one of claims 20-25 for adoptive immunotherapy, comprising delivering the composition of any one of claims 14-19 or the modified cell of any one of claims 20-25 to a subject in need of adoptive immunotherapy.

33. A method of treating a disease or condition comprising delivering the composition of any one of claims 14-19 or the modified cell of any one of claims 20-25 to the subject, preferably wherein the disease or condition is cancer.

34. A composition, method, process, kit or use, characterized in that One or more elements disclosed herein.

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