PDCD1 biallelic knockout

Abstract

A composition, method and use thereof comprising an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within any of the sequences set forth in SEQ ID NOs: 1 to 6215.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 376,276 (filed September 19, 2022), the contents of which are incorporated herein by reference.

[0002] Throughout this application, various publications are referenced, including those within parentheses. The entire disclosures of all publications mentioned in this application are incorporated by reference into this application in their entireties to inform the reader of the technology that may be used in or to which the present invention pertains.

[0003] Sequence Listing Reference This application was created on September 18, 2023 on an IBM PC using an operating system compatible with MS-Windows®, and incorporates by reference the nucleotide sequence contained in an XML file having a size of 5,383 kilobytes and filed as part of this application on September 18, 2023, entitled "230918_92043-A-PCT_Sequence_Listing_AWG.xml." [Background technology]

[0004] Chimeric antigen receptors (CARs) offer a promising approach to immunotherapy. However, to increase the accessibility of such therapies (e.g., CAR-T cell therapy), the development of allogeneic adoptive transfer strategies that allow multiple patients to be treated with universal CAR cells derived from healthy donor cells is highly desirable. To achieve this, the immunogenicity and reactivity of CAR-T cells must be optimized to avoid adverse reactions, such as host rejection and graft-versus-host disease. Summary of the Invention

[0005] PDCD1 is an immunosuppressive receptor expressed on T cells and is involved in the regulation and inhibition of T cell function. This paper describes a method for knocking out the PDCD1 gene in cells used in immunotherapy, such as CAR-T therapy. Cells engineered to knock out PDCD1 improve their performance in allogeneic adoptive transfer therapy. These cells have improved activity, retention, and / or growth characteristics for use in adoptive cancer immunotherapy.

[0006] The disclosure also provides a method of inactivating an allele of a programmed cell death protein 1 (PDCD1) gene in a cell, the method comprising: a CRISPR nuclease or a polynucleotide molecule encoding said CRISPR nuclease; and An RNA molecule comprising a guide sequence portion having 17 to 50 nucleotides, or a polynucleotide molecule encoding the RNA molecule introducing into said cells a composition comprising The complex of the CRISPR nuclease and the RNA molecule makes a double-stranded break in the allele of the PDCD1 gene.

[0007] In some embodiments, the RNA molecule comprises a guide sequence portion that targets a sequence located in any of exons 1-5 of the PDCD1 gene, or a sequence within a genomic span selected from any of 2:241858724-241858876, 2:241852582-241853018, 2:241852159-241852391, 2:241851910-241852021, and 2:241851019-241851335. In some embodiments, the guide sequence portion of the RNA molecule comprises 17-50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1-6215.

[0008] In an embodiment of the present invention, there is provided an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 6215.

[0009] In an embodiment of the present invention, there is provided a composition comprising an RNA molecule including a guide sequence portion having 17 to 50 consecutive nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 6215, and a CRISPR nuclease.

[0010]

[0010] Embodiments of the present invention provide methods for inactivating a PDCD1 allele in a cell, the method comprising delivering to the cell a composition comprising an RNA molecule comprising a guide sequence portion having 17-50 contiguous nucleotides within any of SEQ ID NOS: 1-6215 and a CRISPR nuclease. In some aspects, the cell is a lymphocyte. In some aspects, the cell is a T cell. In some aspects, the cell is a regulatory T cell. In some aspects, the cell is a B cell. In some aspects, the cell is a natural killer (NK) cell. In some aspects, the cell is a macrophage. In some aspects, the cell is a stem cell. In some aspects, the cell is an iPSC. In some aspects, the cell is a fibroblast, blood cell, hepatocyte, keratinocyte, or other cell that can be reprogrammed into an induced pluripotent stem cell (iPSC). In some aspects, delivery to the cell is performed in vivo, ex vivo, or in vitro. In some aspects, the method is performed ex vivo, and the cells are provided / explanted from an individual patient. In some embodiments, the method further comprises introducing cells having an altered / knocked-out PDCD1 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 to be introduced.

[0011] Embodiments of the present invention provide methods for improving the viability, retention, and / or proliferation of cells for adoptive cell therapy, the methods comprising delivering to cells of a subject in need of adoptive cell therapy a composition containing an RNA molecule comprising a guide sequence portion having 17-50 contiguous nucleotides within any of SEQ ID NOS: 1-6215 and a CRISPR nuclease. In some aspects, the methods are for improving the persistence and / or engraftment of cells in a host subject, the methods comprising delivering to the cells a composition containing an RNA molecule comprising a guide sequence portion having 17-50 contiguous nucleotides within any of SEQ ID NOS: 1-6215 and a CRISPR nuclease; and introducing the cells into the host subject. In some aspects, the cells are further differentiated before introduction into the host subject. In some aspects, the cells are further modified to express a chimeric antigen receptor. In some aspects, the cells are stem cells, iPSCs, or progenitor cells, and are differentiated into T cells before introduction into the host subject. In some aspects, the cells are T cells. In some embodiments, the cells are further modified to inactivate and / or knock out additional genes to improve the use of the cells for adoptive transfer (e.g., knocking out additional genes to avoid graft-versus-host disease (GVHD) after introducing the cells into a host subject).

[0012] An embodiment of the present invention provides a use of a composition for inactivating a PDCD1 allele in a cell, comprising delivering to the cell an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 6215, and a CRISPR nuclease.

[0013] In an embodiment of the present invention, there is provided a pharmaceutical for use in inactivating a PDCD1 allele in a cell, comprising an RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 6215, and a CRISPR nuclease, wherein the pharmaceutical is administered by delivering to the cell a composition containing an RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 6215, and a CRISPR nuclease.

[0014] In an embodiment of the present invention, there is provided a use of a composition comprising an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within any of SEQ ID NOs: 1 to 6215 and a CRISPR nuclease to improve the activity, maintenance and / or proliferation of cells for adoptive cell therapy or to improve the persistence of cells upon engraftment, comprising delivering the composition to cells of a subject in need of adoptive cell therapy.

[0015] In embodiments of this invention, there is provided a method for treating a disease or disorder, said method comprising delivering to a subject any of the compositions or modified cells described herein, wherein said disease or disorder is preferably cancer.

[0016] In an embodiment of the present invention, a kit for inactivating a PDCD1 allele in a cell is provided, comprising an RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1 to 6215, a CRISPR nuclease and, optionally, a tracrRNA molecule, and instructions for delivering the RNA molecule, CRISPR nuclease, and optionally, tracrRNA to the cell. [Brief explanation of the drawings]

[0017] [Figure 1]PDCD1 editing in HeLa cells. OMNI-103 CRISPR nuclease was co-transfected with sgRNA expression plasmids and expressed in mammalian cell lines (HeLa cells). The mCherry signal was measured by flow cytometry to determine transfection efficiency (percentage of transfection). All experiments were repeated three times. 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). [Figure 2] PDCD1 editing in primary T cells. Donor-derived T cells were thawed and activated with beads for 72 hours. Cleavage activity was then analyzed using OMNI-103 CRISPR nuclease (113 pmol), sgRNA (226 pmol), and 2 x 106 cells per treatment. After 7 days, approximately 100,000 cells were sorted by FACS and subjected to next-generation sequencing (NGS). NGS samples were prepared by robotic PCR of DNA lysates from Quick Extract. *NT cells express PD1 at approximately 33.6%. After normalization to NT cells, the editing rate with PDCD1_S98 is 61% (=100-((13.1 / 33.6) x 100)). **Parental frequencies and geometric means are shown in the PDCD1-positive column. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention, representative methods and / or materials are described below. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0019] The term "a" or "an" is understood to refer to "one or more" of the listed components. It will be apparent to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Thus, the terms "a" and "at least one" have the same meaning in this application.

[0020] For the purpose of better understanding the present teachings, and without in any way limiting the scope of the teachings, unless otherwise indicated, all numbers and other numerical values ​​expressing quantities, percentages or proportions used in the specification and claims are understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should be construed in light of the number of recited significant digits and by applying ordinary rounding techniques.

[0021] Unless otherwise stated, adjectives such as "substantially" and "about," modifying a condition or relationship characterizing an embodiment of this invention, are understood to mean that the condition or characteristic is defined to the extent acceptable in the operation of the embodiment for its intended application. Unless otherwise indicated, the term "or" in this specification and claims is considered to be an inclusive "or," indicating at least one or any combination of the items it connects, rather than an exclusive "or."

[0022] In this specification and claims, the verbs "comprise," "contain," and "have," and their conjugations, are used to indicate that the object of the verb is not necessarily an exhaustive list of components, elements, or moieties of the subject of the verb. Other terms in this specification are intended to be defined by their well-known meanings in the art.

[0023] In some embodiments of the present invention, DNA nucleases are used to cleave DNA at target sites and trigger 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) are ligated in a rapid but imprecise manner (i.e., frequently resulting in DNA mutations at the break site in the form of small insertions or deletions).

[0024] As used herein, the term "modified cell" refers to a cell in which a complex of an RNA molecule and a CRISPR nuclease makes a double-stranded break as a result of hybridization with a target sequence, i.e., on-target hybridization.

[0025] The invention provides modified cells obtained by use of any of the methods described herein. In some embodiments, these modified cells are capable of giving rise to progeny cells. In some embodiments, these modified cells are capable of giving rise to progeny cells after engraftment. By way of non-limiting example, the modified cells may be hematopoietic stem cells (HSCs) or cells suitable for allogeneic or autologous cell transplantation.

[0026] As used herein, the term "targeting sequence" or "targeting molecule" refers to a nucleotide sequence or molecule comprising a nucleotide sequence capable of hybridizing to a specific target sequence; for example, a targeting sequence has a nucleotide sequence along its length that is at least partially complementary to the sequence being targeted. A targeting sequence or targeting molecule may be a portion of an RNA molecule that can form a complex with a CRISPR nuclease, either alone or in combination with other RNA molecules, with the targeting sequence serving as the targeting portion of the CRISPR complex. When a molecule having a targeting sequence is present simultaneously with a CRISPR nuclease, the RNA molecule, alone or in combination with one or more other RNA molecules (e.g., a tracrRNA molecule), can target the CRISPR nuclease to a specific target sequence. As a non-limiting example, a CRISPR RNA molecule or the guide sequence portion of a single-guide RNA molecule may serve as a targeting molecule. Each possibility is a separate embodiment. A targeting sequence can be custom designed to target a desired sequence.

[0027] In this specification, the term "targeting" refers to the targeting sequence of targeting molecule being preferentially hybridized with the nucleic acid having the target nucleotide sequence.It is understood that the term "targeting" includes various hybridization capabilities, such as the nucleic acid having the target nucleotide sequence being preferentially targeted, but in addition to on-target hybridization, unintended off-target hybridization may also occur.It is understood that when an RNA molecule targets a sequence, the complex of the RNA molecule and the CRISPR nuclease molecule will target this sequence for nuclease activity.

[0028] The "guide sequence portion" of an RNA molecule refers to a nucleotide sequence that can hybridize to a specific target DNA sequence; for example, the guide sequence portion has a nucleotide sequence along its length that is partially or fully complementary to the targeted DNA sequence. In some embodiments, the length of 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, or 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, 17-44, 17-45, 17-46, 17-47, 17-48, 17-49, 17-49, 17-50, 17-51, 17-52, 17-53, 17-54, 17-55, 17-56, 17-57, 17-58, 17-5 ...60, 17-61, 7 to 39, 17 to 38, 17 to 37, 17 to 36, 17 to 35, 17 to 34, 17 to 33, 17 to 31, 17 to 30, 17 to 29, 17 to 28, 17 to 27, 17 to 26, 17 to 25, 17 to 24, 17 to 22, 17 to 21, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 22, 18 to 20, 20 to 21, 21 to 22, or 17 to 20. Preferably, the entire length of the guide sequence portion is completely complementary to the target DNA sequence along its length. The guide sequence portion may be a portion of an RNA molecule that can form a complex with a CRISPR nuclease, and 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 other RNA molecules (e.g., a tracrRNA molecule), the RNA molecule can target the CRISPR nuclease to a specific target DNA sequence. Thus, a CRISPR complex can be formed by directly binding a CRISPR nuclease to an RNA molecule having a guide sequence portion, or by binding a CRISPR nuclease to an RNA molecule having a guide sequence portion and one or more other RNA molecules. Each possibility is a separate embodiment. The guide sequence portion can be custom designed and directed to a desired sequence. Thus, a molecule containing a "guide sequence portion" is a type of targeting molecule.In some embodiments, the guide sequence portion has the same sequence as a guide sequence portion described herein (e.g., a guide sequence set forth in any of SEQ ID NOS: 1-6215), or a different sequence by no more than 1, 2, 3, 4, or 5 nucleotides. Each possibility is a separate embodiment. In some of these embodiments, the guide sequence portion has the same sequence as a sequence set forth in any of SEQ ID NOS: 1-6215. Throughout this application, the terms "guide molecule," "RNA guide molecule," "guide RNA molecule," and "gRNA molecule" are synonymous with a molecule that includes a guide sequence portion.

[0029] As used herein, the term "non-discriminatory" refers to the guide sequence portion of an RNA molecule that targets a specific DNA sequence that is common to all alleles of a gene.

[0030] In an embodiment of the present invention, the RNA molecule comprises a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence set forth in any one of SEQ ID NOs: 1 to 6215.

[0031] The RNA molecule and / or the guide sequence portion of the RNA molecule may contain modified nucleotides. Exemplary modifications to nucleotides / polynucleotides may be synthetic and may include polynucleotides with nucleotides containing bases other than the naturally occurring adenine, cytosine, thymine, uracil, or guanine. Modifications to polynucleotides include polynucleotides with synthetic, non-naturally occurring nucleosides, e.g., locked nucleic acids. Modifications to polynucleotides may be used to increase or decrease RNA stability. An example of a modified polynucleotide is mRNA with 1-methylpseudouridine. For examples of modified polynucleotides and their uses, see U.S. Pat. No. 8,278,036, WO 2015 / 006747, and Weissman and Kariko (2015), incorporated herein by reference.

[0032] As used herein, "consecutive nucleotides" as set forth in a SEQ ID NO: refers to the nucleotides of the sequence in the order set forth in the SEQ ID NO:, without any intervening nucleotides.

[0033] In embodiments of the present invention, the guide sequence portion may be 17 to 50 nucleotides in length, or may contain 20 to 22 consecutive nucleotides within the sequence set forth in any of SEQ ID NOs: 1 to 6215. In embodiments of the present invention, the guide sequence portion may be less than 22 nucleotides in length. For example, in embodiments of the present invention, the guide sequence portion may be 17, 18, 19, 20, or 21 nucleotides in length. In such embodiments, the guide sequence portion may consist of 17, 18, 19, 20, or 21 nucleotides, respectively, within the 17 to 22 consecutive nucleotide sequence set forth in any of SEQ ID NOs: 1 to 6215. For example, the guide sequence portion of the 17 consecutive nucleotide sequence set forth in SEQ ID NO: 6216 may be any of the following nucleotide sequences (nucleotides removed from the consecutive sequence are struck through):

[0034] [ka]

[0035] In embodiments of the present invention, the guide sequence portion may be greater than 20 nucleotides in length. For example, in embodiments of the present invention, the guide sequence portion may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In such embodiments, the guide sequence portion has 17 to 50 nucleotides comprising a sequence of 20, 21, or 22 consecutive nucleotides set forth in any of SEQ ID NOs: 1 to 6215, and nucleotides that are completely complementary to the nucleotides (sequences) adjacent to the 3' end, 5' end, or both, of the target sequence.

[0036] In an embodiment of the present invention, a CRISPR nuclease and an RNA molecule comprising a guide sequence portion bind to a target DNA sequence to form a CRISPR complex that cleaves the target DNA sequence. The CRISPR nuclease (e.g., Cpf1) may form a CRISPR complex comprising the CRISPR nuclease and the RNA molecule without an additional tracrRNA molecule. Alternatively, the CRISPR nuclease (e.g., Cas9) may form a CRISPR complex between the CRISPR nuclease, the RNA molecule, and the tracrRNA molecule. The guide sequence portion, which has a nucleotide sequence capable of hybridizing to a specific target DNA sequence, and the sequence portion involved in CRISPR nuclease binding (e.g., the tracrRNA sequence portion) can be present in the same RNA molecule. Alternatively, the guide sequence portion can be present in one RNA molecule, and the sequence portion involved in CRISPR nuclease binding (e.g., the tracrRNA portion) can be present in a separate RNA molecule. A single RNA molecule comprising a guide sequence portion (e.g., a DNA-targeting RNA sequence) and at least one CRISPR protein-binding RNA sequence portion (e.g., a tracrRNA sequence portion) can form a complex with a CRISPR nuclease and serve 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 another RNA molecule (e.g., a tracrRNA molecule) comprising a CRISPR protein-binding RNA sequence interact by base pairing to form an RNA complex (e.g., a crRNA:tracrRNA complex) that targets a CRISPR nuclease to a DNA target site, or they fuse to each other to form an RNA molecule (e.g., an sgRNA molecule) that complexes with a CRISPR nuclease and targets the CRISPR nuclease to a DNA target site.

[0037] In embodiments of the invention, the RNA molecule containing the guide sequence portion may further comprise the sequence of a tracrRNA molecule. Such embodiments may be designed as a synthetic fusion of the guide portion of the RNA molecule and a transactivating crRNA (tracrRNA) molecule (see Jinek et al., 2012). In such embodiments, the RNA molecule is a single-guide RNA (sgRNA) molecule. Some embodiments of the invention may also form CRISPR complexes utilizing individual tracrRNA molecules and individual RNA molecules containing guide sequence portions (e.g., crRNA molecules). In such embodiments, the tracrRNA may hybridize to the RNA molecule via base pairing, which may be advantageous in certain applications of the inventions described herein.

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

[0039] As used herein, a "gene" includes a DNA region that encodes a gene product and all DNA regions that control the production of that gene product, whether or not the regulatory sequence is contiguous with the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation control sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

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

[0041] As used herein, the term "nuclease" refers to an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of nucleic acids. Nucleases may be isolated from or derived from natural sources. Natural sources may be living organisms. Alternatively, nucleases may be engineered or synthetic proteins that retain phosphodiester bond cleavage activity. Genetic modification can be achieved using nucleases (e.g., CRISPR nucleases).

[0042] In an embodiment of the invention, there is provided a method of inactivating an allele of the Programmed Cell Death Protein 1 (PDCD1) gene in a cell, said method 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 same; introducing into said cells a composition comprising the complex of the CRISPR nuclease and the RNA molecule makes a double-stranded break in the allele of the PDCD1 gene; The guide sequence portion of the RNA molecule has 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 6215.

[0043] In some embodiments, the guide sequence portion comprises a nucleotide sequence set forth in any of SEQ ID NOs: 1-6215, or a portion of a sequence set forth in any of SEQ ID NOs: 1-6215, optionally including additional nucleotides added to the beginning or end of the nucleotide sequence. As a non-limiting example, a 17-nucleotide sequence within the sequence set forth in SEQ ID NO: 1 may form the guide sequence portion. The guide sequence portion may have a 17-nucleotide sequence within the sequence set forth in SEQ ID NO: 1, and may include additional nucleotides 5' or 3' of the 17-nucleotide sequence.

[0044] In some embodiments, the RNA molecule is a crRNA molecule and the composition further contains 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 contains another RNA molecule that includes a guide sequence portion having 17 to 50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 1-6215.

[0046] In some embodiments, the guide sequence portion of the RNA molecule has 17 to 50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 1-6215 that have been modified to contain up to five mismatches relative to the target sequence.

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

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

[0049] In some aspects, the cells are lymphocytes, T cells, regulatory T cells, B cells, natural killer (NK) cells, macrophages, stem cells, fibroblasts, blood cells, hepatocytes, keratinocytes, or other cells that can be reprogrammed into induced pluripotent stem cells (iPSCs).

[0050] In some aspects, the cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPS cell), an iPSc-derived cell, a natural killer (NK) cell, an iPS-induced NK cell (iNK), a T cell, an innate-like T cell (iT), a natural killer T cell (NKT), a gamma delta T cell, an iPSc-induced T cell, an invariant NKT cell (iNKT), an iPSc-induced 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 aspects, an allele of the PDCD1 gene in the cell undergoes an insertion or deletion mutation.

[0053] In some aspects, the insertion or deletion mutation results in a premature stop codon.

[0054] In some embodiments, inactivation results in a truncated protein encoded by the mutated allele, for example, an inactivation method mutates a PDCD1 allele such that the mutated allele encodes a truncated form of the PDCD1 protein.

[0055] In some embodiments, the composition to be introduced into the cell further contains a second RNA molecule comprising a guide sequence portion, and the guide sequence portion of the second RNA molecule preferably has 17 to 50 consecutive nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 6215, and the guide sequence portion of the second RNA molecule is different from the guide sequence portion of the first RNA molecule.

[0056] In an embodiment of the invention, there is provided a method of inactivating an allele of the Programmed Cell Death Protein 1 (PDCD1) gene in a cell, said method 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 introducing into said cells a composition comprising the complex of the CRISPR nuclease and the RNA molecule makes a double-stranded break in the allele of the PDCD1 gene; The guide sequence portion of the RNA molecule has 17 to 50 consecutive nucleotides within the sequence set forth in any one of SEQ ID NOs: 1 to 6215 that have been modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches with respect to a sequence that is completely complementary to its target sequence.

[0057] In some embodiments, the guide sequence portion of the RNA molecule contains 1, 2, 3, 4, or 5 nucleotide mismatches relative to a sequence that is perfectly complementary to its target sequence.

[0058] In some embodiments, the guide sequence portion provides increased targeting specificity to the complex of the CRISPR nuclease and the RNA molecule relative to a guide sequence portion that has a higher degree of complementarity to an allele of the PDCD1 gene.

[0059] In some embodiments, the composition to be introduced into a cell further comprises a second RNA molecule comprising a guide sequence portion, wherein the guide sequence portion of the second RNA molecule comprises 17 to 50 consecutive nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 6215, or 17 to 50 consecutive nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 6215 that has been modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches with respect to a sequence that is completely complementary to its target sequence, and the guide sequence portion of the second RNA molecule is preferably different from the guide sequence portion of the first RNA molecule.

[0060] In an embodiment of the present invention, a composition is provided that contains an RNA molecule that includes a guide sequence portion having 17 to 50 consecutive nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 6215.

[0061] In some embodiments, the composition further comprises a second RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1 to 6215, and the guide sequence portion of the second RNA molecule is preferably different from the guide sequence portion of the first RNA molecule.

[0062] In an embodiment of the present invention, there is provided a composition comprising an RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 6215, or 17 to 50 contiguous nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 6215 that has been modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches with respect to a sequence that is fully complementary to the target sequence of the guide sequence portion.

[0063] In some embodiments, the composition further comprises a second RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1-6215, or 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1-6215 modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches relative to a sequence fully complementary to the target sequence of the guide sequence portion, wherein the guide sequence portion of the second RNA molecule is preferably different from the guide sequence portion of the first RNA molecule.

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

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

[0066] Embodiments of the invention provide cells modified by the methods described herein or using the compositions described herein. The modified cells may be further genetically modified to improve their use in adoptive transfer, for example, to reduce or prevent graft-versus-host disease (GVHD).

[0067] Preferably, all alleles of the PDCD1 gene are inactivated so that the modified cells are unable to express full-length functional PDCD1 protein.

[0068] In some aspects, the cells are lymphocytes, T cells, regulatory T cells, B cells, natural killer (NK) cells, macrophages, stem cells, fibroblasts, blood cells, hepatocytes, keratinocytes, or other cells that can be reprogrammed into induced pluripotent stem cells (iPSCs).

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

[0070] In some aspects, the cells are stem cells or cells that can be reprogrammed into induced pluripotent stem cells (iPSCs).

[0071] In some aspects, the stem cells differentiate after being modified.

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

[0073] In an embodiment of the invention, there is provided a medicament for use in inactivating a PDCD1 allele in a cell, comprising a composition as described herein, wherein the medicament is administered by delivering the composition to the cell.

[0074] Embodiments of the invention provide for the use of any of the compositions or modified cells described herein in adoptive immunotherapy (e.g., for the treatment of cancer).

[0075] In embodiments of the invention, there is provided a medicament for use in adoptive immunotherapy (e.g., treating cancer) comprising any of the compositions or modified cells described herein.

[0076] In an embodiment of the invention, a kit is provided for inactivating a PDCD1 allele in a cell, comprising a composition as 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] In embodiments of the invention, kits are provided for administering adoptive immunotherapy to a subject, comprising any of the compositions or modified cells described herein and instructions for delivering the composition or cells to a subject in need of adoptive immunotherapy.

[0079] Embodiments of the invention provide any of the compositions or modified cells described herein for use in adoptive immunotherapy, comprising delivering any of the compositions or modified cells to a subject in need thereof.

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

[0081] In embodiments of the present invention, there are provided compositions, methods, processes, kits or uses characterized by one or more elements disclosed herein.

[0082] In embodiments of the invention, modified immune cells (e.g., T cells) obtained by the methods described herein are intended to be used as a medicament to treat cancer, infectious diseases, or immune disorders in a subject in need thereof. The modified immune cells or populations thereof may be administered to a subject by any convenient method known in the art, including, but not limited to, aerosol inhalation, injection, ingestion, transfer, implantation, or transplantation. Injection or transfer may be subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous, or intralymphatic injection, or intraperitoneal. In embodiments of the invention, methods of adoptive cell therapy or prophylaxis are provided, comprising administering the modified cells to a subject identified as having, or at risk of having, cancer or an infectious disease.

[0083] In embodiments of the invention, there is provided the use of any of the compositions or modified cells described herein in adoptive immunotherapy, comprising delivering any of the compositions or cells to a subject in need of adoptive immunotherapy.

[0084] In embodiments of the invention, a medicament for use in adoptive immunotherapy is provided, comprising any of the compositions or modified cells described herein, wherein the medicament is administered to a subject in need of adoptive immunotherapy by delivering either the composition or the cells.

[0085] In embodiments of the present invention, RNA molecules are provided for use in modifying cells (e.g., lymphocytes, T cells, CAR-T cells) that may be used in adoptive immunotherapy. The RNA molecules may be delivered to cells ex vivo, in vitro, or in vivo.

[0086] In an embodiment of the invention, a kit is provided for inactivating a PDCD1 allele in a cell, comprising a composition as described herein and instructions for delivering the composition to the cell.

[0087] Embodiments of the invention provide cells modified by the methods described herein or using the compositions described herein.

[0088] In some embodiments of the present invention, a gene editing composition is provided, comprising an RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1 to 6215. In some aspects, the RNA molecule further comprises a portion having a sequence that binds to a CRISPR nuclease. In some aspects, the sequence that binds to a CRISPR nuclease is a tracrRNA sequence. In some aspects, the RNA comprising the guide sequence portion is a crRNA molecule. In some aspects, 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] In embodiments of the invention, the length of the RNA molecule may 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 invention, the length of the RNA molecule may be 17 to at most 300 nucleotides, 100 to at most 300 nucleotides, 150 to at most 300 nucleotides, 100 to at most 500 nucleotides, 100 to at most 400 nucleotides, 200 to at most 300 nucleotides, 100-200 nucleotides, or 150 to at most 250 nucleotides. Each possibility is a separate aspect.

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

[0093] In some embodiments, the present invention provides a method for inactivating expression of PDCD1 in a cell, the method comprising delivering to the cell a composition containing an RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1 to 6215 and a CRISPR nuclease.

[0094] In embodiments of the invention, at least one CRISPR nuclease and an RNA molecule are delivered to a subject and / or cell at substantially the same time or at different times.

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

[0096] The compositions and methods of this disclosure may be used to improve adoptive immunotherapy.

[0097] Any or a combination of the strategies for inactivating PDCD1 expression described herein may be used in the present invention.

[0098] In one embodiment of the invention, a guide RNA molecule is used to target a CRISPR nuclease to an exon or splice site of a PDCD1 allele to generate a double-strand break (DSB), triggering the error-prone mechanism of non-homologous end joining (NHEJ), resulting in the insertion or deletion of nucleotides and creating a frameshift mutation in the PDCD1 allele. A frameshift mutation can inactivate or knock out a PDCD1 allele, for example, by generating a premature stop codon in the PDCD1 allele and producing a truncated protein, or by nonsense-mediated mRNA decay of the allele's transcript. In another embodiment, a single RNA molecule is used to target a CRISPR nuclease to the promoter of a PDCD1 allele.

[0099] Embodiments of the compositions described herein include at least one CRISPR nuclease, a guide RNA molecule, and optionally a tracrRNA molecule, which are simultaneously effective in a subject or cell. The at least one CRISPR nuclease, the guide RNA molecule, and the tracrRNA molecule (optionally) may be delivered substantially simultaneously, or may be delivered at different times, but are effective simultaneously. For example, this includes delivering the CRISPR nuclease to a subject or cell before the guide RNA molecule and / or the tracrRNA molecule are 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 regulatory T 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, blood cell, hepatocyte, keratinocyte, or other cell that can be reprogrammed into an induced pluripotent stem cell (iPSC).

[0101] PDCD1 editing strategy The invention provides methods for knocking out a PDCD1 allele in cells of a subject, thereby improving the performance of those cells, or cells derived by the methods of the present application, in adoptive transfer therapy.

[0102] PDCD1 editing strategies include, but are not limited to, biallelic knockouts targeting any one or combination of exons 1-5, including 30 nucleotide stretches upstream and downstream of the exons flanking the splice donor and splice acceptor sites; frameshifts in these exons lead to nonsense-mediated degradation of the mutated PDCD1 transcript or to a non-functional, truncated PDCD1 protein.

[0103] CRISPR nucleases and PAM recognition In some embodiments, the sequence-specific nuclease is selected from a CRISPR nuclease or a functional variant thereof. In some embodiments, the sequence-specific nuclease is an RNA-guided DNA nuclease. In such embodiments, an RNA sequence that guides the RNA-guided DNA nuclease (e.g., Cpf1) binds to and / or directs the RNA-guided DNA nuclease to all PDCD1 alleles in the cell. In some embodiments, the CRISPR complex does not further comprise a tracrRNA. One of skill in the art will appreciate that RNA molecules can be engineered to bind to selected targets in the genome using methods commonly known in the art.

[0104] As used herein, the term "PAM" refers to a nucleotide sequence in a target DNA that is located adjacent to the target DNA sequence and recognized by a CRISPR nuclease complex. PAM sequences may vary depending on the type of nuclease. In addition, there are CRISPR nucleases that can target almost any PAM. In some embodiments of this invention, the CRISPR system utilizes one or more RNA molecules containing a guide sequence portion that guides the CRISPR nuclease to the target DNA site by forming Watson-Crick base pairs between the guide sequence portion and the protospacer of the target DNA site adjacent to the protospacer adjacent motif (PAM), an additional requirement for target recognition. The CRISPR nuclease then cleaves the target DNA site, creating a double-stranded break within the protospacer. In a non-limiting example, a Type II CRISPR system utilizes a mature crRNA:tracrRNA complex that guides a CRISPR nuclease (e.g., Cas9) to a target DNA by forming Watson-Crick base pairs between the guide sequence portion of the crRNA and a protospacer on the target DNA adjacent to a protospacer adjacent motif (PAM). One of skill in the art will understand that the engineered RNA molecules of the present invention are further designed to bind to a target genomic DNA sequence of interest adjacent to a protospacer adjacent motif (PAM), e.g., a PAM corresponding to a sequence associated with the CRISPR nuclease being utilized.The PAM may be, for example, but not limited to, NGG or NAG (where N is any nucleobase) for Streptococcus pyogenes Cas9 WT (SpCAS9); NNGRRT for Staphylococcus aureus (SaCas9); NNNVRYM for Jejuni Cas9 WT; NGAN or NGNG for SpCas9-VQR variants; NGCG for SpCas9-VRER variants; NGAG for SpCas9-EQR variants; NRRH for SpCas9-NRRH variants (where N is any nucleobase, R is A or G, and H is A, C, or T); NRTH for SpCas9-NRTH variants (where N is any nucleobase, R is A or G, and H is A, C, or T); or SpCas9-NRCH variants (where N is any nucleobase, R is A or G, and H is A, C, or T). NRCH (where N is any nucleobase, R is A or G, and H is A, C, or T) for the SpG variant of SpCas9; NG (where N is any nucleobase); NG or NA (where N is any nucleobase) for the SpCas9-NG variant of SpCas9; NR, NRN, or NYN (where N is any nucleobase, R is A or G, and Y is C or T) for the SpRY variant of SpCas9; and Streptococcus canis (Streptococcus canis Cas9 variant (ScCas9), NNG (where N is any nucleobase); NNNRRT (where N is any nucleobase and R is A or G) for Staphylococcus aureus SaKKH-Cas9 variant (SaCas9); NNNNGATT (where N is any nucleobase) for Neisseria meningitidis (NmCas9); TTN (where N is any nucleobase) for Alicyclobacillus acidophilus Cas12b (AacCas12b); or TTTV (where V is A, C, or G) for Cpfl. The RNA molecules of the invention are each designed to form a complex with one or more different CRISPR nucleases and to target a polynucleotide sequence of interest using one or more different PAM sequences corresponding to the CRISPR nucleases.

[0105] In some embodiments, RNA-guided DNA nucleases (e.g., CRISPR nucleases) may be used to create double- or single-stranded DNA breaks at desired locations in a cell's genome. While the most commonly used RNA-guided DNA nucleases are derived from CRISPR systems, other RNA-guided DNA nucleases are also contemplated for use in the genome editing compositions and methods described herein. See, e.g., U.S. Patent Application Publication No. 2015 / 0211023, which is incorporated herein by reference.

[0106] There are a variety of CRISPR systems that can be used to practice the present invention. The CRISPR system may 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, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, Casl These include Od, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl0, 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). CRISPR nucleases have been shown to inhibit the expression of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Neisseria meningitidis, Treponema denticola, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, and Streptosporangium roseum. roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas species sp.), Crocosphaera watsonii, Cyanothece sp.), Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.CRISPR nucleases may be derived from Bacillus subtilis, Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, or any species encoding a CRISPR nuclease with a known PAM sequence. CRISPR nucleases encoded by uncultured bacteria may also be used in the present invention (see Burstein et al., Nature, 2017). Variants of CRISPR proteins with known PAM sequences, such as the SpCas9 D1135E variant, SpCas9 VQR variant, SpCas9 EQR variant, or SpCas9 VRER variant, may also be used in the present invention.

[0108] Therefore, RNA-guided DNA nucleases of the CRISPR system, such as Cas9 protein or modified Cas9, or homologs or orthologs of Cas9, or other RNA-guided DNA nucleases belonging to other CRISPR systems, such as Cpf1 and its homologs and orthologs, may be used in the compositions of the invention. Other CRISPR nucleases, such as those described in WO2020 / 223514 and WO2020 / 223553 (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 that shares qualitative biological properties with the native sequence polypeptide. "Functional derivatives" include, but are not limited to, native sequence fragments and derivatives of native sequence polypeptides and their fragments that share biological activity with the corresponding native sequence polypeptide. The contemplated biological activity is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses both amino acid sequence variants of the polypeptide and covalently modified and fusion forms thereof. Suitable derivatives of a Cas polypeptide or a fragment thereof include, but are not limited to, mutants, fusions, and covalently modified forms of the Cas protein or a fragment thereof. Derivatives include, but are not limited to, CRISPR nickases, catalytically inactive or "dead" CRISPR nucleases, and fusions of CRISPR nucleases or derivatives thereof with other enzymes, such as base editors or retrotransposons. See, for example, Anzalone et al. (2019) and PCT International Patent Application No. PCT / US2020 / 037560.

[0110] In some embodiments, the CRISPR nuclease or its derivative may be fused to a protein having enzymatic activity. In some embodiments, the enzymatic activity modifies target DNA. In some embodiments, the 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 activity, or glycosylase activity. In some embodiments, the enzymatic activity is nuclease activity. In some embodiments, the enzymatic activity introduces double-strand breaks in target DNA. In some embodiments, the enzymatic activity modifies a target polypeptide associated with target DNA. In some cases, the enzymatic activity is a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitinating activity, an adenylating activity, a deadenylating activity, a sumoylating activity, a desumoylating activity, a ribosylation activity, a deribosylation activity, a myristoylating activity, or a demyristoylating activity. In some cases, the target polypeptide is a histone and the enzymatic activity is a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, or a deubiquitinating activity.

[0111] Cas proteins, including Cas proteins or fragments thereof, as well as derivatives of Cas proteins or fragments thereof, may be obtainable from cells, may be chemically synthesized, or may be obtained by a combination of these methods. The cells may be cells that naturally produce Cas proteins, or may be cells that naturally produce Cas proteins and have been genetically engineered to produce higher expression levels of endogenous Cas proteins or to produce Cas proteins from introduced exogenous nucleic acids encoding the same or different Cas proteins as the endogenous Cas. In some cases, the cells do not naturally produce Cas proteins but are 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 makes staggered DNA double-strand breaks. Two Cpf1 enzymes from Acidaminococcus and Lachnospiraceae have been shown to efficiently edit genomes in human cells (see Zetsche et al., 2015).

[0113] Thus, RNA-guided DNA nucleases of Type II CRISPR systems, such as the Cas9 protein or modified Cas9, or homologs, orthologs or variants of Cas9, or other RNA-guided DNA nucleases belonging to other CRISPR systems, such as Cpfl and its homologs, orthologs or variants, may be used in the present invention.

[0114] In some embodiments, the guide molecule comprises one or more chemical modifications that confer new or improved properties (e.g., stability against degradation, hybridization energy, binding to RNA-guided DNA nucleases). Suitable chemical modifications include, but are not limited to, modified bases, modified sugars, 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-methylpseudouridine, β,D-galactosylqueosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methyladenosine, 1-methylguano ... Chirpseudouridine, 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-mannosylqueusine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine , 5-methoxyuridine, 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-oxyacetic acid methyl ester, uridine-5-oxyacetic acid, wybutoxocin, queusine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine Examples of uridine include uridine, 4-thiouridine, 5-methyluridine, N-((9-β-D-ribofuranosylpurin-6-yl)-carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosin, 3-(3-amino-3-carboxypropyl)uridine, (acp3)u, 2'-O-methyl (M), 3'-phosphorothioate (MS), 3'-thioPACE (MSP), pseudouridine, or 1-methylpseudouridine.Each realization of the invention is a separate embodiment.

[0115] In addition to targeting PDCD1 alleles with RNA-guided CRISPR nucleases, other means of blocking PDCD1 expression in target cells include, but are not limited to, the use of gapmers, shRNAs, siRNAs, customized TALENs, meganucleases, zinc finger nucleases, small molecule inhibitors, and other methods known in the art to reduce or eliminate gene expression in target cells. See, e.g., 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; WO 2004 / 067736; WO 2006 / 097853; WO 2003 / 08734 See No. 1; No. 2000 / 041566; No. 2003 / 080809; No. 2010 / 079430; No. 2010 / 079430; No. 2011 / 072246; No. 2018 / 057989 and No. 2017 / 164230 (the entire contents of which are incorporated herein by reference).

[0116] Advantageously, the guide RNA molecules provided herein, when complexed with CRISPR nucleases in cells, improve PDCD1 knockout efficiency compared to other guide RNA molecules. These specially designed sequences may also be useful for identifying target sites in PDCD1 for other nucleotide-targeting-based gene editing or gene silencing methods (e.g., siRNA, TALEN, meganucleases, or zinc finger nucleases).

[0117] Cellular delivery The compositions described herein may be delivered to target cells by any suitable means. The RNA molecule compositions of the present invention may be directed to cells containing and / or expressing a PDCD1 allele, such as mammalian lymphocytes or stem cells. For example, in certain embodiments, the RNA molecule specifically targets a PDCD1 allele in target cells, which may be lymphocytes, T cells, regulatory T cells, B cells, natural killer (NK) cells, macrophages, stem cells, fibroblasts, blood cells, hepatocytes, keratinocytes, or other cells that can be reprogrammed into induced pluripotent stem cells (iPSCs). Delivery to cells may be in vivo, ex vivo, or in vitro. Furthermore, the nucleic acid compositions described herein may be delivered to cells as one or more of a DNA molecule, an RNA molecule, a ribonucleoprotein (RNP), a nucleic acid vector, or a 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' thio PACE (MSP), pseudouridine, and 1-methylpseudouridine. Each realization of this invention is a separate embodiment.

[0119] In some embodiments, the compositions described herein are delivered to cells in vivo. The compositions may be delivered to cells by known in vivo delivery methods, including, but not limited to, viral transduction using, for example, lentivirus or adeno-associated virus (AAV), nanoparticle delivery, etc. Details of delivery methods are provided throughout this section.

[0120] In some embodiments, the compositions described herein are delivered to cells ex vivo. The compositions may be delivered to cells by known ex vivo delivery methods, including, but not limited to, nucleofection, electroporation, viral transduction using, for example, lentivirus or adeno-associated virus (AAV), nanoparticle delivery, liposomes, etc. Details of delivery methods are provided throughout this section.

[0121] Nucleic acid compositions (e.g., compositions of RNA molecules of the invention) may be delivered using an appropriate viral vector system. Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into target tissues. In certain embodiments, nucleic acids are administered for in vivo or ex vivo gene therapy. Non-viral vector delivery systems include naked nucleic acid and nucleic acid complexed with a delivery vehicle (e.g., liposomes, poloxamers). For reviews of gene therapy, 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 methods of nucleic acid and / or protein delivery include electroporation, lipofection, microinjection, biolistic bombardment, particle gun acceleration, uptake of nucleic acids by virosomes, liposomes, immunoliposomes, lipid nanoparticles (LNPs), polycation or lipid:nucleic acid conjugates, artificial virions, and facilitators, or nucleic acids and / or proteins can be delivered to plant cells by bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizobium meliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus, cassava vein mosaic virus) (see, e.g., Chung et al., J. Immunol. 2004, 103:111-114). (See Zuris et al. (2015); Coelho et al. (2013); Judge et al. (2006); and Basha et al. (2011). Sonoporation, for example using the Sonitron 2000 system (Rich-Mar), can also be used to deliver nucleic acids. Cationic lipid delivery of proteins and / or nucleic acids is also contemplated for in vivo, ex vivo, or in vitro delivery (see Zuris et al. (2015); Coelho et al. (2013); see also Judge et al. (2006); and Basha et al. (2011)).

[0123] Non-viral vectors, such as transposon-based systems (e.g., recombinant Sleeping Beauty transposon system, recombinant PiggyBac transposon system), may be delivered to target cells and used to transpose the polynucleotide sequences of or encoding the molecules of the composition in the target cells.

[0124] Other representative nucleic acid delivery systems include those offered by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, e.g., U.S. Pat. No. 6,008,336). Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam®, Lipofectin®, and Lipofectamine® RNAiMAX). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in WO 91 / 17424 and WO 91 / 16024. Delivery to cells (ex vivo administration) or target tissues (in vivo administration) is possible.

[0125] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, 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. Patent 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] Another method of delivery involves packaging the nucleic acid to be delivered into an EnGeneIC delivery vehicle (EDV). The EDV is specifically delivered to the target tissue using a bispecific antibody, where one arm of the antibody has specificity for the target tissue and the other arm has specificity for the EDV. The antibody carries the EDV to the surface of the target cell, where it is transported into the cell by endocytosis. After entering the cell, the contents are released (see MacDiarmid et al., 2009).

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

[0128] Delivery of the CRISPR nuclease and / or polynucleotide encoding the CRISPR nuclease, and optionally additional nucleotide molecules and / or additional proteins or peptides, may be achieved using a single delivery vehicle or method, or a combination of different delivery vehicles or methods. For example, the CRISPR nuclease may be delivered to cells using LNPs, and the crRNA and tracrRNA molecules may be delivered to cells using AAV. Alternatively, the CRISPR nuclease may be delivered to cells using AAV particles, and the crRNA and tracrRNA molecules may be delivered to cells using separate AAV particles, which may be advantageous due to size limitations.

[0129] The use of RNA or DNA viral systems for viral nucleic acid delivery takes advantage of the highly evolved methods by which viruses target specific cells in the body and transport their viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or can be used to engineer cells in vitro, and the engineered cells are then administered to patients (ex vivo). Conventional viral systems for nucleic acid delivery include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated viral, vaccinia virus, and herpes simplex viral vectors for gene transfer. RNA viruses may also be used to deliver the compositions described herein. High transduction efficiencies have also been observed in a variety of cells and target tissues. The nucleic acids of the present invention may also be delivered by non-integrating lentiviruses. Optionally, lentiviral RNA delivery is utilized. In some cases, the lentivirus contains a nuclease mRNA and a guide RNA molecule. In some cases, the lentivirus contains a nuclease protein and a guide RNA molecule. In some cases, the lentivirus comprises a nuclease mRNA, a guide RNA molecule, and a tracrRNA molecule. In some cases, the lentivirus comprises a nuclease protein, a guide RNA molecule, and a tracrRNA molecule.

[0130] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding the potential population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats and are capable of packaging up to 6-10 kb of foreign sequence. A minimal set of cis-acting long terminal repeats is sufficient for vector replication and packaging, allowing therapeutic genes to be integrated into target cells and persistently express the transgene. 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); WO 94 / 26877).

[0131] At least six viral vectors are currently available for gene transfer in clinical trials, which utilize an approach involving complementation of a defective vector with a gene inserted into a helper cell line to prepare 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 a gene therapy trial (Blaese et al., 1995). Transduction efficiencies of MFG-S packaging vectors were greater than 50% (Ellem et al., (1997); Dranoff et al., 1997).

[0133] Packaging cells are used to form viral particles capable of infecting host cells. Such cells include 293 cells, which package adenovirus and AAV, and Psi-2 or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are typically generated by producer cell lines that package nucleic acid vectors into viral particles. The vectors typically contain minimal viral sequences required for packaging and (if applicable) subsequent integration into the host, with other viral sequences replaced by expression cassettes encoding the proteins to be expressed. Missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the inverted terminal repeats (ITRs) from the AAV genome required for packaging and integration into the host genome. Viral DNA is packaged in a cell line that lacks other AAV genes, i.e., ITR sequences, but contains a helper plasmid encoding rep and cap. The cell line is also infected with adenovirus as a helper. The helper virus promotes AAV vector replication and AAV gene expression from the helper plasmid. Due to the lack of ITR sequences, the helper plasmid is not packaged in large quantities. Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Furthermore, AAV can be produced on a clinical scale using the baculovirus system (see U.S. Pat. No. 7,479,554).

[0134] In many gene therapies, it is desirable for gene therapy vectors to be delivered with high specificity to specific tissues. Therefore, viral vectors can be engineered to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is selected to have affinity for a receptor known to be present in the target cell. For example, Han et al. (1995) reported that Moloney murine leukemia virus can be engineered to express human heregulin fused to gp70 and that the recombinant virus infects specific human breast cancer cells expressing the human epidermal growth factor receptor. This principle can be extended to other virus-target cell combinations, where the target cell expresses a receptor and the virus expresses a fusion protein containing a ligand for the cell surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB, Fv) with specific binding affinity for virtually any selected cellular receptor. While this discussion applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences that favor 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, intracranial injection) or by local application.

[0136] Alternatively, vectors can be delivered ex vivo to cells, e.g., cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirate, tissue biopsy) or universal donor hematopoietic stem cells, followed by re-implantation of the cells into the patient after selection of cells that have incorporated the vector. Non-limiting exemplary ex vivo methods may include removing tissue (e.g., peripheral blood, bone marrow, spleen) from the patient for culture, transferring the nucleic acid into the cultured cells (e.g., hematopoietic stem cells), and then transplanting the cells into the patient's target tissue (e.g., bone marrow, spleen). In some embodiments, the stem cells or hematopoietic stem cells may be further treated with a survival-enhancing agent.

[0137] Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., by re-infusion of the transfected cells into a host) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from a subject, transfected with a nucleic acid composition, and re-infused into the subject (e.g., patient). A variety of cells suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney, "Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications (6th edition, 2010) and references cited therein in the discussion of methods for isolating and culturing cells from patients).

[0138] Alternatively, vectors (e.g., retroviruses, liposomes) carrying therapeutic nucleic acid compositions can be administered directly to an organism for transduction of cells in vivo. Administration can be by any route typically used to introduce molecules into ultimate contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application (e.g., eye drops, creams), and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and while multiple routes of administration for a particular composition can be used, certain routes often provide a more rapid and effective response than others. In some embodiments, the composition is delivered by IV injection.

[0139] Suitable vectors for introducing transgenes into immune cells (e.g., T cells) include non-integrating lentiviral vectors, see, e.g., U.S. Patent Application Publication No. 2009 / 0117617.

[0140] As previously discussed, the compositions described herein may be delivered to target cells using non-integrating lentiviral particle methods (e.g., the LentiFlash® system). Such methods may be used to deliver mRNA or other RNA to target cells, such that delivery of the RNA to the target cell results in assembly of the compositions described herein inside the target cell. See also 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 composition being administered, as well as by the method used to administer the composition. Thus, there is a wide variety of suitable formulations of pharmaceutical compositions available, for example, as described in Remington's Pharmaceutical Sciences, 17th ed., 1989.

[0142] Example of an RNA guide sequence that specifically targets an allele of the PDCD1 gene While numerous guide sequences can be designed to target the PDCD1 gene, the nucleotide sequences shown in Table 1 and identified by SEQ ID NOs: 1-6215 were specifically selected to effectively carry out the methods described herein.

[0143] Guide sequences designed for use in the previously described embodiments to associate specific sequences within PDCD1 alleles are listed in Table 1. Each engineered guide molecule is further designed to bind to a target genomic DNA sequence of interest adjacent to a protospacer adjacent motif (PAM) (e.g., a PAM corresponding to the sequence NGG or NAG (where N is any nucleobase)). The guide sequences are designed to work with one or more different CRISPR nucleases, including, for example, SpCas9WT (PAM sequence: NGG), SpCas9.VQR.1 (PAM sequence: NGAN), SpCas9.VQR.2 (PAM sequence: NGNG), SpCas9.EQR (PAM sequence: NGAG), SpCas9.VRER (PAM sequence: NGCG), SpCas9WT (PAM sequence: NNGRRT), SpCas9.VQR.3 (PAM sequence: NGGG), SpCas9.VQR.4 (PAM sequence: NGGG), SpCas9.VQR.5 (PAM sequence: NGAG), SpCas9.VRER (PAM sequence: NGCG), SpCas9WT (PAM sequence: NNGRRT), SpCas9.VQR.6 (PAM sequence: NGGG), SpCas9.VQR.7 (PAM sequence: NGGG), SpCas9.VQR.8 (PAM sequence: NGGG), SpCas9.VQR.9 (PAM sequence: NGGG), SpCas9.VQR.10 (PAM sequence: NGAN), SpCas9.VQR.11 (PAM sequence: NGAN), SpCas9.VQR.12 (PAM sequence: NGNG), SpCas9.EQR (PAM sequence: NGAG), SpCas9.VRER (PAM sequence: NGCG), SpCas9WT (PAM sequence: NNGRRT), SpCas9.VQR.13 (PAM sequence: NGAG), SpCas9.VRER (PAM sequence: NGCG), Sp Examples of PAM sequences include, but are not limited to, NmCas9WT (PAM sequence: NNNNGATT), Cpf1 (PAM sequence: TTTV), JeCas9WT (PAM sequence: NNNVRYM), OMNI-50 (PAM sequence: NGG), OMNI-79 (PAM sequence: NGG), OMNI-103 (PAM sequence: NNRACT), OMNI-159 (NNNNCMAN), or OMNI-124 (PAM sequence: NNGNRMNN).

[0144] OMNI nucleases are further described in WO 2023 / 019269, WO 2022 / 170199, WO 2023 / 107946, U.S. Pat. No. 11,666,641, WO 2020 / 223514, WO 2022 / 098693, WO 2023 / 019263, U.S. Patent Application Publication No. 2023 / 0122086, WO 2021 / 248016, WO 2023 / 102407, WO 2022 / 087135, and WO 2022 / 226215, the contents of which are incorporated herein by reference.

[0145] The RNA molecules of the invention are each designed to form a complex with one or more different CRISPR nucleases and to target a polynucleotide sequence of interest using one or more different PAM sequences corresponding to the CRISPR nucleases.

[0146] In this specification, the following nucleotide identifiers are used to represent nucleotide bases:

[0147] [Table A]

[0148] [Table 1]

[0149] Examples are presented to facilitate a more complete understanding of the present invention. The following examples illustrate representative modes of making and practicing the invention. However, the scope of the invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only. [Example]

[0150] Experiment details Example 1 Analysis of PDCD1 alterations Guide sequences containing 17-50 consecutive nucleotides within any of SEQ ID NOs: 1-6215 are screened for high on-target activity in T cells using CRISPR nucleases. On-target activity is determined by DNA capillary electrophoresis.

[0151] Example 2 Editing of PDCD1 in HeLa cells and primary T cells Editing of PDCD1 in HeLa cells and primary T cells using portions of the disclosed PDCD1 targeting guide sequence portions is shown in Figures 1 and 2.

[0152] A table summarizing data on PDCD1 editing is shown below. [Table B]

[0153] Sequences related to OMNI-103 are shown in the table below. [Table C]

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Claims

1. 1. A method for inactivating an allele of the Programmed Cell Death Protein 1 (PDCD1) gene in a cell, 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 said RNA molecule; introducing into said cells a composition comprising a complex of the CRISPR nuclease and the RNA molecule that makes a double-stranded break in the allele of the PDCD1 gene; The method, wherein the guide sequence portion of the RNA molecule comprises 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 6215.

2. The method of claim 1 , wherein the composition is introduced into a cell of a subject or into a cell in culture.

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

4. 3. The method of claim 1 or 2, wherein the cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPS cell), an iPSc-derived cell, a natural killer (NK) cell, an iPS-derived NK cell (iNK), a T cell, an innate immune-like T cell (iT), a natural killer T cell (NKT), a γδ T cell, an iPSc-induced T cell, an invariant NKT cell (iNKT), an iPSc-induced NKT, a monocyte, or a macrophage.

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

6. The method of any one of claims 1 to 5, wherein an allele of the PDCD1 gene in the cell is subjected to an insertion or deletion mutation.

7. The method of claim 6, wherein the insertion or deletion mutation results in a premature stop codon.

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

9. 9. The method of 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, the guide sequence portion of the second RNA molecule having 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 6215, and preferably the guide sequence portion of the second RNA molecule is different from the guide sequence portion of the first RNA molecule.

10. 1. A method for inactivating an allele of the Programmed Cell Death Protein 1 (PDCD1) gene in a cell, 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 said RNA molecule; introducing into said cells a composition comprising a complex of the CRISPR nuclease and the RNA molecule that makes a double-stranded break in the allele of the PDCD1 gene; The method of any one of claims 1 to 6215, wherein the guide sequence portion of the RNA molecule has 17 to 50 contiguous nucleotides within the sequence set forth in any one of SEQ ID NOs: 1 to 6215 that have been modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches with respect to a sequence that is completely complementary to the target sequence of the guide sequence portion.

11. 11. The method of claim 10, wherein the guide sequence portion of the RNA molecule contains 1, 2, 3, 4, or 5 nucleotide mismatches relative to a sequence perfectly complementary to its target sequence.

12. 12. The method of claim 10 or 11, wherein the guide sequence portion provides higher targeting specificity to the complex of the CRISPR nuclease and the RNA molecule compared to a guide sequence portion that has higher complementarity to an allele of the PDCD1 gene.

13. 13. The method of 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, the guide sequence portion of the RNA molecule comprising 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 6215, or 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 6215 modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches relative to a sequence fully complementary to its target sequence, and preferably 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 having 17 to 50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 1 to 6215.

15. 15. The composition of claim 14, further comprising a second RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 6215, wherein preferably 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 having 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1-6215, or 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1-6215 modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches relative to a sequence that is fully complementary to the target sequence of the guide sequence portion.

17. 17. The composition of claim 16, further comprising a second RNA molecule comprising a guide sequence portion having 17-50 contiguous nucleotides within any of SEQ ID NOs: 1-6215, or 17-50 contiguous nucleotides within any of SEQ ID NOs: 1-6215 modified to contain 1, 2, 3, 4, or 5 nucleotide mismatches relative to a sequence fully complementary to the target sequence of the guide sequence portion, wherein preferably the guide sequence portion of the second RNA molecule is different from the guide sequence portion of the first RNA molecule.

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

19. The composition of 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. 21. The modified cell of claim 20, wherein the cell is a lymphocyte, T cell, regulatory T cell, B cell, natural killer (NK) cell, macrophage, stem cell, fibroblast, blood cell, hepatocyte, keratinocyte, or other cell that can be reprogrammed into an induced pluripotent stem cell (iPSC).

22. 21. The modified cell of 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 (NK) cell, an iPS-derived NK cell (iNK), a T cell, an innate immune-like T cell (iT), a natural killer T cell (NKT), a γδ T cell, an iPSc-induced T cell, an invariant NKT cell (iNKT), an iPSc-derived NKT, a monocyte, or a macrophage.

23. 21. The modified cell of claim 20, wherein the cell is a stem cell or a cell that can be reprogrammed into an induced pluripotent stem cell (iPSC).

24. 24. The modified cell of claim 23, wherein the stem cell differentiates after being modified.

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

26. 20. A medicament for use in inactivating a PDCD1 allele in a cell, comprising the composition of any one of claims 14 to 19, wherein the medicament is administered by delivering the composition of any one of claims 14 to 19 to the cell.

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

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

29. 20. A kit for inactivating a PDCD1 allele in a cell, comprising the composition of any one of claims 14 to 19 and instructions for delivering said composition to said cell.

30. 30. The kit of claim 29, wherein the composition is delivered to the cells ex vivo.

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

32. 26. The composition of any one of claims 14 to 19 or the modified cells of any one of claims 20 to 25 for use in adoptive immunotherapy, comprising delivering the composition of any one of claims 14 to 19 or the modified cells of any one of claims 20 to 25 to a subject in need thereof.

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

34. A composition, method, process, kit or use characterized by one or more elements disclosed herein.

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