Knock-in strategies at B2M safe harbor sites

Abstract

An RNA molecule, composition, method and use thereof comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence set forth in any one of SEQ ID NOs: 1 to 28998.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 477,888 (filed December 30, 2022) and 63 / 373,990 (filed August 30, 2022), the contents of each 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 August 16, 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 the file name "230829_91780-A-PCT_Sequence_Listing_AWG.xml," 25,695 kilobytes in size, submitted as part of this application on August 29, 2023. [Background technology]

[0004] The beta-2 microglobulin (B2M) safe harbor site can be targeted to introduce desired sequences into the site without detrimentally disrupting the B2M gene or affecting its expression. Such a targeting strategy may be used to allow B2M promoter-mediated expression of the introduced sequence. Summary of the Invention

[0005] The present disclosure also provides a method of modifying at least one allele of a beta-2 microglobulin (B2M) gene in a cell, the method comprising: At least one CRISPR nuclease or a polynucleotide molecule encoding said CRISPR nuclease; and An RNA molecule containing a guide sequence portion of 17 to 50 nucleotides, or a nucleotide sequence encoding the same introducing into the cell a composition comprising The complex of the CRISPR nuclease and the RNA molecule makes a double-stranded break in at least one allele of the B2M gene.

[0006] In some embodiments, the composition also includes a donor molecule, and a nucleotide sequence of the donor molecule is inserted or copied at or near the site of the double-strand break. Similarly, in some embodiments, the composition further includes a donor molecule comprising a nucleotide sequence to be introduced at the site of the double-strand break, and expression of the introduced sequence is mediated by the promoter of the B2M gene. Thus, this specification also discloses strategies that enable expression of a gene or a portion thereof under the control of the B2M promoter. In some embodiments, expression of a gene or a portion thereof under the control of the B2M promoter is possible without knocking out expression of the B2M gene.

[0007] In some embodiments, the composition further comprises a CRISPR nuclease. In some embodiments, the composition further comprises a donor molecule.

[0008] Some aspects of the present invention provide cells engineered to express and secrete a protein of interest in desired tissues of the body. Such cells can be engineered by inserting the sequence of the gene of interest under the control of a promoter of a selected gene (e.g., B2M) containing a safe harbor site. Non-limiting examples of methods for engineering cells to express a gene of interest include knock-in using a CRISPR nuclease system that generates a double-strand break and a donor molecule encoding the sequence of the gene of interest. In some embodiments, the donor molecule is a ssODN, dsDNA, plasmid, AAV, lentivirus, or transposon. Furthermore, knock-in can be mediated by a composition comprising i) a fusion protein comprising a nickase and a reverse transcriptase, and ii) an RNA donor molecule.

[0009] Aspects of the invention provide cells modified by any of the methods described herein. In some embodiments, the cells are stem cells. In some embodiments, the cells are monocytes. In some embodiments, the cells are macrophages. In some embodiments, the cells are iPS-derived monocytes or macrophages. In some embodiments, the cells are hematopoietic stem cells (HSCs), hematopoietic stem / progenitor cells (HSPCs), myeloid progenitor cells, myeloblasts, lymphoblasts, erythroid progenitor cells, platelet cells, natural killer (NK) cells, B lymphocytes, T lymphocytes, eosinophils, neutrophils, or basophils. In some embodiments, the cells are iPS-derived cells.

[0010] In some embodiments, delivering any of the compositions described herein to cells is performed in vitro, ex vivo, or in vivo. In some embodiments, the method is performed ex vivo, and the cells are provided / explanted from the individual patient. In some embodiments, the method further comprises introducing the cells with the modified or edited B2M allele into the individual patient (e.g., autologous transplant).

[0011] In some embodiments, the present invention provides use of a composition comprising an RNA molecule and a CRISPR nuclease, the composition comprising a guide sequence portion having 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1 to 28998, in modifying or editing a B2M allele in a cell, the use comprising delivering the composition to the cell. In some embodiments, the composition further comprises a donor molecule.

[0012] An embodiment of the present invention provides a pharmaceutical composition for use in modifying or editing a B2M allele in a cell, the pharmaceutical composition 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 28998, and a CRISPR nuclease, wherein the pharmaceutical composition is administered to the cell by delivering to the cell a composition 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 28998, and a CRISPR nuclease. In some embodiments, the pharmaceutical composition further comprises a donor molecule.

[0013] In some embodiments, the present invention provides a kit for modifying or editing a B2M allele in a cell, the kit comprising an RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within any of SEQ ID NOS: 1 to 28998, a CRISPR nuclease and / or a tracrRNA molecule, and instructions for delivering the RNA molecule and the CRISPR nuclease and / or tracrRNA to the cell. In some embodiments, the kit further comprises a donor molecule. [Brief explanation of the drawings]

[0014] [Figure 1A] Figure 1A: Screening of guides to optimize OMNI-50 nuclease-mediated editing of intron 1 of B2M in HeLa cells. Cells were harvested 72 hours after DNA transfection, genomic DNA was extracted, and the region of mutation was analyzed by next-generation sequencing (NGS). [Figure 1B]The editing activity of selected guides was tested in HSCs using OMNI-50 or OMNI-50 V6172 CRISPR nuclease to identify the optimal composition for editing intron 1 of B2M. HSCs were electroporated with RNPs (CRISPR nuclease and RNA guides), and 72 hours later, genomic DNA was extracted and the region of mutation was analyzed by next-generation sequencing. The graph shows the percentage of editing ± standard deviation (SD) from two independent measurements. [Figure 2] Insertion of GFP into the B2M endogenous locus by HDR to generate a bicistronic transcript. Before editing, the native B2M locus is shown encoding the B2M protein, including its signal peptide (SP) at its N-terminus. The SP portion of B2M is encoded by exon 1. Also shown are the left and right homology arms (LHA and RHA, respectively), as well as the donor sequence carrying GFP-2A-SP flanked by splice acceptor and splice donor sites. Successful integration results in a novel middle exon and a novel chimeric polypeptide, SP-GFP-2A-SP-B2M. The chimeric polypeptide self-cleaves into two distinct proteins, including the SP. [Figures 3A-3D]Integration of GFP into the B2M locus of HSCs allows for the expression and secretion of GFP without altering B2M expression. Figure 3A: FACS analysis of HSCs treated with donor virus (GFP-2A) and RNP reveals cells expressing GFP. No GFP signal was detected in HSCs treated with donor virus alone or with unrelated RNP. Figure 3B: Examination of total B2M transcript levels normalized to GAPDH revealed no change in B2M transcript levels after HDR integration. Data from two biological and three technical measurements are shown (nested analysis). Figure 3C: Examination of B2M protein expression by FACS confirmed that most edited cells retained B2M protein expression. B2M expression in GFP+ edited cells after HDR (top) was compared with all live cells in the untreated and ISO-stained control groups. The dashed line indicates the expression cutoff determined by the ISO-stained control. Figure 3D: Analysis of GFP secretion by ELISA detects GFP in HSC culture medium after HDR at the B2M locus (GFP concentration (pg / ml) normalized to 106 cell numbers). Data from two biological and two technical measurements are shown (nested analysis). Graphs represent mean ± SD. [Figures 4A-4C] Integration of GFP into the B2M locus of HSCs and subsequent differentiation into macrophages allows for the preparation of GFP-secreting macrophages. Figure 4A: FACS analysis of macrophages to confirm the effectiveness of the differentiation protocol and verify GFP expression. Figure 4B: Examination of B2M protein expression by FACS confirmed that most edited cells retained B2M protein expression. B2M expression (top) in GFP+ edited cells after HDR was compared with all live cells in the untreated and ISO-stained control groups. Cells were divided into those with WT level expression (untreated population), reduced expression, and no expression (ISO-stained control). Figure 4C: GFP secretion measured by ELISA detects GFP in macrophage culture media after HDR at the B2M locus of HSCs. Data are shown for two biological and two technical measurements (nested analysis). Graphs represent mean ± SD. [Figure 5A-5B]Integration of GFP into the B2M locus of iPSCs allows for the expression and secretion of GFP. Figure 5A: FACS of iPSCs treated with donor virus (GFP-2A) and RNP reveals GFP expression. When combined with irrelevant RNP, GFP was not observed in cells treated with the donor virus. Figure 5B: GFP secretion measured by ELISA detects GFP in the cell medium after HDR at the B2M locus. Data are shown for two biological and two technical measurements (nested analysis). Graphs represent mean ± SD. DETAILED DESCRIPTION OF THE INVENTION

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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."

[0019] 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.

[0020] The term "homologous recombination repair" or "HDR" refers to a mechanism for repairing DNA damage in cells, e.g., repairing double- and single-strand breaks in DNA. HDR requires nucleotide sequence homology and uses a "nucleic acid template" (as used herein, nucleic acid template and donor template are synonymous) to repair a sequence (e.g., a DNA target sequence) where a double- or single-strand break has occurred. This results in, for example, the transfer of genetic information from the nucleic acid template to the DNA target sequence. HDR can result in a change in the sequence of the DNA target (e.g., an insertion, deletion, or mutation) if the sequence of the nucleic acid template differs from that of the DNA target and some or all of the nucleic acid template polynucleotide or oligonucleotide is incorporated into the DNA target sequence. In some embodiments, the entire nucleic acid template polynucleotide, a portion of the nucleic acid template polynucleotide, or a copy of the nucleic acid template is incorporated into the DNA target sequence.

[0021] The terms "nucleic acid template" and "donor" refer to a nucleotide sequence to be inserted or copied into a genome. A nucleic acid template comprises a nucleotide sequence, e.g., one or more nucleotides, that can be added to a target nucleic acid, template a change in a target nucleic acid, or used to modify a target sequence. The nucleic acid template sequence can be any length, e.g., from 2 to 10,000 nucleotides. A nucleic acid template can be a single-stranded or double-stranded nucleic acid. In some embodiments, a nucleic acid template comprises a nucleotide sequence, e.g., one or more nucleotides, that corresponds to the wild-type sequence of a target nucleic acid, e.g., at a target location. In some embodiments, a nucleic acid template comprises a nucleotide sequence, e.g., one or more ribonucleotides, that corresponds to the wild-type sequence of a target nucleic acid, e.g., at a target location. In some embodiments, a nucleic acid template comprises modified nucleotides.

[0022] Insertion of an exogenous sequence (also referred to as a "donor sequence," "donor template," "donor molecule," or "donor") can also be performed. For example, the donor sequence can contain a non-homologous sequence flanked by two regions of homology, enabling efficient homology-directed repair (HDR) at the desired location. Furthermore, the donor sequence can comprise a vector molecule having a sequence that is not homologous to the region of interest in cellular chromatin. The donor molecule can contain several discontinuous regions homologous to cellular chromatin. For example, to target and insert a sequence not normally present in the region of interest, the sequence can be included in the donor nucleic acid molecule and flanked by regions of homology to the sequence of the region of interest. The donor molecule can be any length, from a few bases (e.g., 10-20 bases) to several kilobases.

[0023] The donor polynucleotide may be DNA or RNA, single-stranded and / or double-stranded, and may be introduced into cells in linear or circular form. See, e.g., U.S. Patent Application Publication Nos. 2010 / 0047805; 2011 / 0281361; 2011 / 0207221; and 2019 / 0330620. See also Anzalone et al. (2019). If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonuclease degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues can be added to the 3' end of the linear molecule and / or self-complementary oligonucleotides can be ligated to one or both ends. See, e.g., Chang et al. (1987) and Nehls et al. (1996). Other methods of protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups, modified internucleotide linkages such as phosphorothioates, phosphoramidates, and the use of O-methyl ribose or deoxyribose residues.

[0024] The donor sequence may be an oligonucleotide and may be used for targeted alteration of an endogenous sequence. The oligonucleotide may be introduced into the cell using a vector, electroporated into the cell, or by other methods known in the art. The donor polynucleotide may be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or delivered by a virus (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)).

[0025] 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. The term "modified cell" may also encompass cells that have been edited or modified (including by introduction of an exogenous sequence) after the double-stranded break.

[0026] The invention provides modified cells obtained by use of any of the methods described herein. In certain embodiments, these modified cells are capable of giving rise to progeny cells. In certain embodiments, these modified cells are capable of giving rise to progeny cells after transplantation. By way of non-limiting example, the modified cells may be hematopoietic stem cells (HSCs) or cells suitable for allogeneic or autologous cell transplantation. By way of non-limiting example, the modified cells may be stem cells, monocytes, macrophages, or iPS-derived monocytes or macrophages.

[0027] The invention also provides compositions comprising these modified cells and a pharmaceutically acceptable carrier, as well as in vitro or ex vivo methods for preparing the compositions, which involve combining the cells with a pharmaceutically acceptable carrier.

[0028] 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 molecule, 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.

[0029] 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.

[0030] 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 comprises the same sequence as a guide sequence portion described herein (e.g., a guide sequence set forth in any of SEQ ID NOS: 1-28998), 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 comprises the same sequence as a sequence set forth in any of SEQ ID NOS: 1-28998. Throughout this application, the terms "guide molecule," "RNA guide molecule," "guide RNA molecule," and "gRNA molecule" are synonymous with a molecule comprising a guide sequence portion.

[0031] As used herein, the term "non-discriminatory" refers to a guide sequence portion of an RNA molecule that targets a specific DNA sequence that is common to a pair of alleles of a gene. For example, a non-discriminatory guide sequence portion can target a pair of alleles of a gene present in a cell.

[0032] In embodiments of the invention, the RNA molecule comprises a guide sequence portion having 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 28998. In some embodiments, the guide sequence portion comprises the same sequence as a sequence set forth in any of SEQ ID NOs: 1 to 28998, or a sequence that differs by 1, 2, or not more than 3 nucleotides.

[0033] The RNA molecule and / or the guide sequence portion of the RNA molecule may contain modified nucleotides. Exemplary modifications to nucleotides or polynucleotides may be synthetic and include polynucleotides with nucleotides containing bases other than the naturally occurring adenine, cytosine, thymine, uracil, or guanine bases. 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.

[0034] 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.

[0035] In embodiments of the invention, the guide sequence portion may be 25 nucleotides in length, or may contain 20 to 22 consecutive nucleotides within the sequence set forth in any of SEQ ID NOs: 1 to 28998. In embodiments of the invention, the guide sequence portion may be less than 22 nucleotides in length. For example, in embodiments of the 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 28998. For example, the guide sequence portion of the 17 consecutive nucleotide sequence set forth in SEQ ID NO: 28999 may be any of the following nucleotide sequences (nucleotides removed from the consecutive sequence are struck through):

[0036] [ka]

[0037] 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 includes 17 to 50 nucleotides having a sequence of 20, 21, or 22 consecutive nucleotides set forth in any of SEQ ID NOs: 1 to 28998, and nucleotides adjacent to the 3' end, 5' end, or both, of the target sequence, or nucleotides completely complementary to said sequences.

[0038] 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 comprising a DNA-targeting RNA portion that includes a guide sequence portion and a second RNA molecule comprising a CRISPR protein-binding RNA sequence interact by base pairing to form an RNA complex that targets the CRISPR nuclease to a DNA target site, or they are fused to each other to form an RNA molecule that complexes with a CRISPR nuclease and targets the CRISPR nuclease to a DNA target site.

[0039] 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 the guide sequence portion. In such embodiments, the tracrRNA may hybridize with the RNA molecule via base pairing, which may be advantageous in certain applications of the invention described herein.

[0040] 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.

[0041] 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.

[0042] "Eukaryotic" cells include, but are not limited to, fungal cells (eg, yeast), plant cells, animal cells, mammalian cells, and human cells.

[0043] 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 modified or synthetic proteins that retain phosphodiester bond cleavage activity. Genetic modifications can be achieved using nucleases (e.g., CRISPR nucleases).

[0044] Embodiments of the invention provide RNA molecules comprising a guide sequence portion (e.g., a targeting sequence) having a nucleotide sequence that is fully or partially complementary to a target located within or near an allele of the B2M gene. In some embodiments, the guide sequence portion of the RNA molecule consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more than 26 nucleotides. In some embodiments, the guide sequence portion is configured to target a CRISPR nuclease to the B2M target site and induce a double-stranded or single-stranded break within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides of the B2M target site. In some embodiments, the RNA molecule is a guide RNA molecule, such as a crRNA molecule or a single-guide RNA molecule. In some embodiments, the guide sequence portion is complementary to a target sequence located 30 base pairs upstream to 30 base pairs downstream of exon 3, exon 4, intron 1, intron 2, intron 3, or the 3' untranslated region (3'UTR) of the B2M gene. In some embodiments, the guide sequence portion is complementary to a target sequence located 50 base pairs upstream to 50 base pairs downstream of exon 3, exon 4, intron 1, intron 2, intron 3, or the 3' untranslated region (3'UTR) of the B2M gene. Each possibility is a separate embodiment. In some embodiments, the guide sequence portion is complementary to a target sequence located 7 base pairs upstream to 7 base pairs downstream of exon 3, exon 4, intron 1, intron 2, intron 3, or the 3' untranslated region (3'UTR) of the B2M gene.

[0045] As used herein, the term "HSC" refers to both hematopoietic stem cells and hematopoietic progenitor stem cells. Non-limiting examples of stem cells include myeloid cells, myeloid progenitor cells, multipotent progenitor cells, and lineage-restricted progenitor cells.

[0046] As used herein, "progenitor cell" refers to a lineage cell that is derived from a stem cell and retains mitotic capacity and multipotency (e.g., the ability to differentiate or develop into multiple, but not all, types of mature cell lineages). As used herein, "hematopoiesis" refers to the formation and development in the body (e.g., bone marrow) of various blood cells (e.g., erythrocytes, megakaryocytes, myeloid cells (e.g., monocytes, macrophages, and neutrophils), and lymphocytes) and other formed elements.

[0047] In some aspects of the present invention, there is provided a method for modifying an allele of a β2 microglobulin (B2M) gene in a cell, the method comprising: At least one CRISPR nuclease or a polynucleotide molecule encoding said CRISPR nuclease; and an RNA molecule comprising a guide sequence portion of 17 to 50 nucleotides, or a nucleotide 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 B2M gene.

[0048] In some embodiments, the RNA molecule is a crRNA molecule and the composition further comprises a tracrRNA molecule that forms a crRNA:tracrRNA molecule with the crRNA molecule. In some embodiments, the RNA molecule is an sgRNA molecule.

[0049] In some aspects, the composition also includes a donor molecule. In some aspects, the nucleotide sequence of the donor molecule is inserted or copied at or near the site of the double-strand break. In some aspects, the composition further includes a donor molecule that includes the nucleotide sequence to be introduced at the site of the double-strand break.

[0050] In some embodiments, the composition further comprises a donor molecule comprising a nucleotide sequence to be introduced at the site of the double-stranded break, wherein expression of the introduced sequence is mediated by the promoter of the B2M gene.

[0051] In some embodiments, the introduced sequence is selected from the group consisting of alpha 1-antitrypsin, glucose-6-phosphatase (G6PC), serpin family A members (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthase, arginase, argininosuccinase, carbamoyl phosphate synthase, and N-acetylglutamate synthase, alpha galactosidase A, clotting factor IX, clotting factor VII, lysosomal alpha glucosidase, fibrinogen, phenylalanine 4-hydroxylase, alkaline phosphatase, glucosylceramidase, beta-galactosidase, porphobilinogen deaminase, arylsulfatase B, beta-glucuronidase, alpha-N-acetylglucosaminidase, lysosomal alpha, alpha L-iduronidase, mannosidase, phosphatidylcholinesterol acyltransferase, N-sulfoglucosamine sulfohydrolase, coagulation factor X, N-acetylgalactosamine-6-sulfatase, sphingomyelin phosphodiesterase, iduronate-2-sulfatase, lysosomal alpha glucosidase, cyclin-dependent kinase-like 5, pro-low density lipoprotein receptor-related protein 1, phenylalanine ammonia-lyase, protein glutamine gamma glutamyltransferase K, or lysosomal protective protein.

[0052] In some embodiments, the introduced sequence is selected from the group consisting of acid α-glucosidase, α-L-iduronidase, α-galactosidase, iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, lysophosphatidylcholine metabolism-related protein, preferably phospholipase A2, T-REC or K-REC-related protein, β-glucosidase, β-glucocerebrosidase, arylsulfatase A, Factor VIII, insulin-like growth factor 1 (IGF-1), surfactant protein A, surfactant protein B, aspartyl-β-glucosaminidase, acetyl-CoA α-glucosaminide, acetyl-CoA-arylamine N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylglucosaminidase, acid ceramidase, aspartoacylase, lysosomal acid lipase, acid sphingomyelinase, arylsulfatase B, α-L-fucosidase, galactosylceramidase, galactocerebrosidase, β-galactosidase, protective protein / cathepsin A, β-glucuronidase, heparan N-sulfatase, β-hexosaminidase A, hyaluronidase-1, α-D-mannosidase, β-mannosidase, α-neuraminidase, β-hexosaminidase A, β-hexosaminidase B, palmitase and sequences derived from genes encoding thiazol-1, thiazol-1, thiazol-2, thiazol-3, thiazol-4, thiazol-5, thiazol-6, thiazol-7, thiazol-8, thiazol-9, thiazol-10, thiazol-11, thiazol-12, thiazol-13, thiazol-14, thiazol-15, thiazol-16, thiazol-17, thiazol-18, thiazol-19, thiazol-20, thiazol-19, thiazol-19, thiazol-21, thiazol-19, thiazol-19, thiazol-22, thiazol-19, thiazol-19, thiazol-23, thiazol-19, thiazol-19, thiazol-24, thiazol-19, thiazol-25, thiazol-19, thiazol-26, thiazol-19, thiazol-27, thiazol-19, thiazol-28, thiazol-29, thiazol-29, thiazol-30, thiazol-31, thiazol-32, thiazol-33, thiazol-34, thiazol-35, thiazol-36, thiazol-37, thiazol-38, thiazol-39, thiazol-40, thiazol-41, thiazol-39, thiazol-39, th

[0053] In some aspects, the donor molecule comprises a sequence derived from a gene that encodes a protein that is secreted by the cell.

[0054] In some aspects, the introduced sequence comprises a sequence encoding a polypeptide of interest that the cell will express.

[0055] In some aspects, the expressed polypeptide of interest is secreted by the cells.

[0056] In some aspects, the expressed polypeptide of interest further comprises a signal peptide.

[0057] In some embodiments, the signal peptide is encoded by an allele of the B2M gene.

[0058] In some embodiments, the introduced sequence comprises a sequence encoding a 2A self-cleaving peptide.

[0059] In some aspects, the introduced sequence includes a sequence encoding a signal peptide.

[0060] In some embodiments, the signal peptide is a B2M signal peptide.

[0061] In some aspects, the introduced sequences include a splice acceptor sequence and a splice donor sequence.

[0062] In some embodiments, the introduced sequences include a splice acceptor sequence, a sequence encoding a polypeptide of interest, a sequence encoding a 2A self-cleaving peptide, a signal peptide, and a splice donor sequence.

[0063] In some embodiments, the donor molecule comprises a first homology arm sequence having at least 90%, 90-95%, 95-100%, or preferably 100% sequence identity with the B2M sequence upstream of the double-stranded break, and a second homology arm sequence having at least 90%, 90-95%, 95-100%, or preferably 100% sequence identity with the B2M sequence downstream of the double-stranded break.

[0064] In some embodiments, the length of the first homology arm sequence and the second homology arm sequence is about 20 to 50, 50 to 100, 100 to 200, 200 to 500, 500 to 1000, or 1000 to 2000 nucleotides, respectively.

[0065] In some aspects, the polypeptide of interest is a soluble protein.

[0066] In some aspects, the length of the polypeptide of interest is about 20-50, 50-100, 100-200, 200-500, 500-1000, or 1000-2000 amino acids.

[0067] In some embodiments, the RNA molecule comprises a guide sequence portion having 17 to 50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 1-28998.

[0068] In some embodiments, the RNA molecule comprises a non-discriminatory guide sequence portion that targets both alleles of B2M.

[0069] In some embodiments, the RNA molecule comprises a non-discriminatory guide portion that targets exon 3, exon 4, intron 1, intron 2, intron 3, or the 3' untranslated region (3'UTR) of the B2M gene.

[0070] In some embodiments, the RNA molecule is selected from the group consisting of 15:44716329-44716356, 15:44717607-44717813, 15:44717825-44718145, 15:44716357-44717323, 15:44717327-44717606, 15:44711614-44712998, 15:44713001-44713065, 15:44714 and a non-discriminatory guide portion that targets a sequence within a genomic range selected from any of 15:44713067-44713182, 15:44713184-44713461, 15:44713463-44714419, 15:44714421-44714474, 15:44714476-44715422, and 15:44715702-44716328.

[0071] In some embodiments, the modified allele of the B2M gene expresses a B2M gene product.

[0072] In some aspects, the modified allele of the B2M gene expresses a B2M polypeptide and a polypeptide of interest.

[0073] In some aspects, the cell is a stem cell, a monocyte, a macrophage, an iPS-derived monocyte, an iPS-derived macrophage, a hematopoietic stem cell (HSC), a hematopoietic stem or progenitor cell (HSPC), a myeloid progenitor cell, a myeloblast, a lymphoblast, an erythroid progenitor cell, a platelet cell, a natural killer (NK) cell, a B lymphocyte, a T lymphocyte, an eosinophil, a neutrophil, an iPS-derived cell, or a basophil.

[0074] In some aspects, the cells are stem cells and the method further comprises differentiating the stem cells after modifying the stem cells.

[0075] Aspects of the invention provide modified cells obtainable by the method of any of the aspects set out herein.

[0076] In some aspects, the cell is a stem cell, a monocyte, a macrophage, an iPS-derived monocyte, an iPS-derived macrophage, a hematopoietic stem cell (HSC), a hematopoietic stem or progenitor cell (HSPC), a myeloid progenitor cell, a myeloblast, a lymphoblast, an erythroid progenitor cell, a platelet cell, a natural killer (NK) cell, a B lymphocyte, a T lymphocyte, an eosinophil, a neutrophil, an iPS-derived cell, or a basophil.

[0077] In an embodiment of the present invention, an RNA molecule is provided that includes a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 28998.

[0078] An embodiment of the invention provides a composition comprising an RNA molecule and at least one CRISPR nuclease.

[0079] In some embodiments, the composition further comprises a donor molecule.

[0080] In some embodiments, the donor molecule is selected from the group consisting of alpha 1-antitrypsin, glucose-6-phosphatase (G6PC), serpin family A members (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthase, arginase, argininosuccinase, carbamoyl phosphate synthase, and N-acetylglutamate synthase, alpha galactosidase A, clotting factor IX, clotting factor VII, lysosomal alpha glucosidase, fibrinogen, and phenylalanine. The present invention also includes sequences derived from genes encoding 4-hydroxylase, alkaline phosphatase, glucosylceramidase, beta-galactosidase, porphobilinogen deaminase, arylsulfatase B, beta-glucuronidase, alpha-N-acetylglucosaminidase, lysosomal alpha, alpha L-iduronidase, mannosidase, phosphatidylcholinesterol acyltransferase, N-sulfoglucosamine sulfohydrolase, coagulation factor X, N-acetylgalactosamine-6-sulfatase, sphingomyelin phosphodiesterase, iduronate-2-sulfatase, lysosomal alpha glucosidase, cyclin-dependent kinase-like 5, pro-low density lipoprotein receptor-related protein 1, phenylalanine ammonia-lyase, protein glutamine gamma glutamyltransferase K, or lysosomal protective protein.

[0081] In some embodiments, the donor molecule is selected from the group consisting of acid α-glucosidase, α-L-iduronidase, α-galactosidase, iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, lysophosphatidylcholine metabolism-related protein, preferably phospholipase A2, T-REC or K-REC-related protein, β-glucosidase, β-glucocerebrosidase, arylsulfatase A, Factor VIII, insulin-like growth factor 1 (IGF-1), surfactant protein A, surfactant protein B, aspartyl-β-glucosaminidase, acetyl-CoA α-glucosaminide, acetyl-CoA-arylamine N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylglucosaminidase, acid ceramidase, aspartoacylase, lysosomal acid lipase, acid sphingomyelinase, arylsulfatase B, α-L-fucosidase, galactosylceramidase, galactocerebrosidase, β-galactosidase, protective protein / cathepsin A, β-glucuronidase, heparan N-sulfatase, β-hexosaminidase A, hyaluronidase-1, α-D-mannosidase, β-mannosidase, α-neuraminidase, β-hexosaminidase A, β-hexosaminidase B, palmitase and sequences derived from genes encoding thiazol-1, thiazol-1, thiazol-2, thiazol-3, thiazol-4, thiazol-5, thiazol-6, thiazol-7, thiazol-8, thiazol-9, thiazol-10, thiazol-11, thiazol-12, thiazol-13, thiazol-14, thiazol-15, thiazol-16, thiazol-17, thiazol-18, thiazol-19, thiazol-20, thiazol-19, thiazol-19, thiazol-21, thiazol-19, thiazol-19, thiazol-22, thiazol-19, thiazol-19, thiazol-23, thiazol-19, thiazol-19, thiazol-24, thiazol-19, thiazol-25, thiazol-19, thiazol-26, thiazol-19, thiazol-27, thiazol-19, thiazol-28, thiazol-29, thiazol-29, thiazol-30, thiazol-31, thiazol-32, thiazol-33, thiazol-34, thiazol-35, thiazol-36, thiazol-37, thiazol-38, thiazol-39, thiazol-40, thiazol-41, thiazol-39, thiazol-39, th

[0082] In some aspects, the donor molecule comprises a sequence derived from a gene that encodes a protein that is secreted by the cell.

[0083] In some embodiments, the donor molecule comprises a sequence encoding a polypeptide of interest. A nucleotide sequence encoding a polypeptide desired to be expressed in and secreted by a target cell can be inserted into the B2M allele so that the modified B2M allele can express both the polypeptide encoded by the inserted sequence and the original B2M gene product.

[0084] In some embodiments, the donor molecule comprises a sequence encoding a 2A self-cleaving peptide.

[0085] In some embodiments, the donor molecule comprises a sequence encoding a signal peptide.

[0086] In some embodiments, the signal peptide is a B2M signal peptide.

[0087] In some aspects, the donor molecule comprises a splice acceptor sequence and a splice donor sequence.

[0088] In some embodiments, the donor molecule comprises a splice acceptor sequence, a sequence encoding a polypeptide of interest, a sequence encoding a 2A self-cleaving peptide, a signal peptide, and a splice donor sequence.

[0089] In some embodiments, the donor molecule comprises a first homology arm sequence having at least 90%, preferably 100%, sequence identity with a first sequence of the B2M gene, and a second homology arm sequence having at least 90%, preferably 100%, sequence identity with a second sequence of the B2M gene.

[0090] In some embodiments, the length of the first homology arm sequence and the second homology arm sequence is about 20 to 50, 50 to 100, 100 to 200, 200 to 500, 500 to 1000, or 1000 to 2000 nucleotides, respectively.

[0091] In some aspects, the polypeptide of interest is a soluble protein.

[0092] In some aspects, the length of the polypeptide of interest is at most 20-50, 50-100, 100-200, 200-500, 500-1000, or 1000-2000 amino acids.

[0093] In some embodiments, the composition further comprises a tracrRNA molecule.Embodiments of this invention provide a method of modifying or editing a B2M allele in a cell, the method comprising delivering to the cell a composition of any of the embodiments described herein.

[0094] In accordance with an aspect of the invention, there is provided a use of a composition as described herein in modifying or editing a B2M allele in a cell, said use comprising delivering to said cell a composition according to any of the aspects described herein.

[0095] In one aspect of the present invention, there is provided a pharmaceutical composition for use in modifying or editing a B2M allele in a cell, the pharmaceutical composition comprising any of the compositions of the aspects set forth in the specification, wherein the pharmaceutical composition is administered to the cell by delivering any of the compositions of the aspects set forth in the specification.

[0096]

[0009] Aspects of the invention provide kits for modifying or editing a B2M allele in a cell, the kits comprising an RNA molecule, a CRISPR nuclease, and / or a tracrRNA molecule according to any of the embodiments described herein; and instructions for delivering the RNA molecule, the CRISPR nuclease, and / or the tracrRNA to the cell. In some embodiments, the kits further comprise a donor molecule and instructions for delivering the donor molecule to the cell.

[0097] In some embodiments, the present invention provides a gene editing composition comprising an RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1 to 28998. 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.

[0098] In some embodiments, the RNA molecule further comprises a portion having a tracr mate sequence.

[0099] In some embodiments, the RNA molecule may further comprise one or more linker moieties.

[0100] 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 is a separate embodiment. In embodiments of the invention, the length of the RNA molecule may be from 17 to at most 300 nucleotides, from 100 to at most 300 nucleotides, from 150 to at most 300 nucleotides, from 100 to at most 500 nucleotides, from 100 to at most 400 nucleotides, from 200 to at most 300 nucleotides, from 100 to at most 200 nucleotides, or from 150 to at most 250 nucleotides. Each possibility is a separate aspect.

[0101] In some embodiments of the invention, the composition further comprises a tracrRNA molecule.

[0102] In some embodiments, the present invention provides a method for modifying or editing a B2M allele in a cell, the method comprising delivering to the cell a composition comprising a CRISPR nuclease and 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 28998. In some embodiments, the composition further comprises a donor molecule.

[0103] In some embodiments, the present invention provides a method for treating a disorder or disease, the method comprising delivering to a cell of a subject having the disorder or disease a composition comprising an RNA molecule including a guide sequence portion having 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 28998 and a CRISPR nuclease. In some embodiments, the composition further comprises a donor molecule.

[0104] In some aspects of this invention, there is provided a method of treating a disorder or disease, the method comprising delivering to cells of a subject having the disorder a composition of any of the above aspects, or delivering to the subject a modified cell of any of the above aspects.

[0105] In some aspects, the disorder or disease is Pompe disease, mucopolysaccharidosis type I, Fabry disease, mucopolysaccharidosis type II, mucopolysaccharidosis type IVA, mucopolysaccharidosis type VI, adrenoleukodystrophy, severe combined immunodeficiency, Gaucher disease, metachromatic leukodystrophy (MLD), primary immunodeficiency, hemophilia A, hemophilia B, IGF1 deficiency, surfactant deficiency, aspartylglucosaminuria, Sanfilippo syndrome, mucopolysaccharidosis type III, Sanfilippo syndrome type IIId, Eissel disease, Schindler disease, Farber disease (FD), spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), Canavan disease, lysosomal acid lipase deficiency, Niemann-Pick disease, mucopolysaccharidosis type 6, fucosidosis, Krabbe disease, GM1 gangliosidosis, mucopolysaccharidosis type IVB (MPS IVB), or galactosialidosis, Sly disease, mucopolysaccharidosis type III, late-onset Tay-Sachs disease, hyaluronidase 1 deficiency, α-mannosidosis, β-mannosidosis, sialidosis, Stanhoff disease, Santavuori-Hartier disease, Jansky-Bielschowski disease, Batten disease, neuronal ceroid lipofuscinosis type 5, neuronal ceroid lipofuscinosis type 6, neuronal ceroid lipofuscinosis type 7, neuronal ceroid lipofuscinosis type 8, congenital cathepsin D deficiency, cystinosis, pyknodysostosis, Salla disease, Danon disease, and / or α1-antitrypsin deficiency.

[0106] In some aspects, the composition or modified cells are delivered to a tissue or tumor of a subject.

[0107] In some aspects of the present invention, there is provided a pharmaceutical agent for use in modifying a B2M allele in a cell, the pharmaceutical agent comprising the composition of any of the above aspects, wherein the pharmaceutical agent is administered by delivering the composition of any of the above aspects to the cell.

[0108] Some aspects of this invention provide a use of a composition of any of the above aspects or a modified cell of any of the above aspects for the treatment, amelioration, or prevention of a disorder or disease, said use comprising delivering a composition of any of the above aspects to cells of a subject having or at risk of having said disorder, or delivering a modified cell of any of the above aspects to said subject.

[0109] In some aspects of the invention, there is provided a medicament for use in treating, ameliorating, or preventing a disorder or disease, comprising a composition of any of the above aspects or a modified cell of any of the above aspects, wherein the medicament delivers the composition of any of the above aspects to cells of a subject having or at risk of having the disorder, or delivers the modified cell of any of the above aspects to the subject.

[0110] In some aspects, the disorder or disease is Pompe disease, mucopolysaccharidosis type I, Fabry disease, mucopolysaccharidosis type II, mucopolysaccharidosis type IVA, mucopolysaccharidosis type VI, adrenoleukodystrophy, severe combined immunodeficiency, Gaucher disease, metachromatic leukodystrophy (MLD), primary immunodeficiency, hemophilia A, hemophilia B, IGF1 deficiency, surfactant deficiency, aspartylglucosaminuria, Sanfilippo syndrome, mucopolysaccharidosis type III, Sanfilippo syndrome type IIId, Eissel disease, Schindler disease, Farber disease (FD), spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), Canavan disease, lysosomal acid lipase deficiency, Niemann-Pick disease, mucopolysaccharidosis type 6, fucosidosis, Krabbe disease, GM1 gangliosidosis, mucopolysaccharidosis type IVB (MPS IVB), or galactosialidosis, Sly disease, mucopolysaccharidosis type III, late-onset Tay-Sachs disease, hyaluronidase 1 deficiency, alpha-mannosidosis, beta-mannosidosis, sialidosis, Stanhof disease, Santavuori-Hartier disease, Jansky-Bielschowski disease, Batten disease, neuronal ceroid lipofuscinosis type 5, neuronal ceroid lipofuscinosis type 6, neuronal ceroid lipofuscinosis type 7, neuronal ceroid lipofuscinosis type 8, congenital cathepsin D deficiency, cystinosis, pyknodysostosis, Salla disease, Danon disease, and / or alpha-1-antitrypsin deficiency.

[0111] In some aspects, the disorder or disease is a lysosomal storage disorder.

[0112] In some aspects, the disorder or disease is a disorder or disease of the blood, lung, brain, liver, gut, intestinal tract, bone, muscle, or central nervous system, or an inflammatory or autoinflammatory disease.

[0113] In some aspects, the composition of any of the above aspects or the modified cell of any of the above aspects is an enzyme replacement therapy medicament.

[0114] In some aspects of this invention, there is provided a method of treating a disease or disorder, the method being immunotherapy comprising delivering to a subject the modified cells of any of the above aspects.

[0115] In some aspects, the disease or disorder is cancer.

[0116] In some aspects, the composition of any of the above aspects or the modified cells of any of the above aspects are used to treat, ameliorate, or prevent a disorder or disease.

[0117] In some embodiments, the present invention provides a method for modifying a DNA target site in monocytes or macrophages of a subject, wherein the modification of the DNA target site induces the monocytes or macrophages to express a desired protein encoded by the modification, the method comprising delivering to the subject a composition comprising a CRISPR nuclease and 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 28998. In some embodiments, the composition further comprises a donor molecule.

[0118] In embodiments of the invention, at least one CRISPR nuclease and an RNA molecule, or the RNA molecules, are delivered to a subject and / or cell at substantially the same time or at different times.

[0119] In some embodiments, the tracrRNA molecule is delivered to a subject and / or cell at substantially the same time or at different times as the CRISPR nuclease and the RNA molecule, or the RNA molecule.

[0120] In an embodiment of this invention, the RNA molecule targets the alternative splicing signal sequence between the exon and intron of the B2M allele.

[0121] In some embodiments, the RNA molecule is non-discriminatory and targets a sequence present in a pair of B2M alleles. In some embodiments, the sequence is present in a pair of B2M alleles. In some embodiments, the sequence is present in an intron of the B2M gene. In some embodiments, the intron is intron 1 following coding exon 1 of the B2M gene.

[0122] Any or a combination of the above strategies for modifying or editing the B2M allele may be used in connection with the present invention.

[0123] In some embodiments, the methods include contacting at least one allele of a gene of interest with a non-discriminating RNA molecule (e.g., an RNA molecule comprising a guide sequence portion capable of targeting a pair of alleles of the gene) and a CRISPR nuclease (e.g., a Cas9 protein), where the non-discriminating RNA molecule and the CRISPR nuclease bind to a nucleotide sequence in at least one allele of the gene of interest to modify or edit at least one allele. Notably, introduction of the non-discriminating RNA molecule into a cell can result in biallelic cleavage, but insertion of a nucleotide sequence at the cleavage site can occur in only one allele, but not both alleles. Thus, inducing biallelic cleavage with a non-discriminating RNA molecule targeted to an intron of the B2M gene can maintain expression of the endogenous B2M gene from one allele and introduce a nucleotide sequence (e.g., a nucleotide sequence derived from a donor molecule) into the other allele. Introduction of a nucleotide sequence into a B2M allele may or may not perturb expression of the B2M-encoded gene product in the B2M allele.

[0124] In some embodiments, the methods include contacting an allele of a gene of interest with an RNA molecule and a CRISPR nuclease (e.g., a Cas9 protein), where the RNA molecule and the CRISPR nuclease bind to a nucleotide sequence of the allele of the gene of interest that differs by at least one nucleotide from the nucleotide sequence of the other allele of the gene of interest, thereby modifying or editing the targeted allele.

[0125] In some embodiments, the RNA molecule and CRISPR nuclease are introduced into a cell that encodes a gene of interest. In some embodiments, the cell that encodes the gene of interest is a cell of a mammalian subject.

[0126] Embodiments of the compositions described herein include at least one CRISPR nuclease, an RNA molecule comprising a guide sequence portion, and a tracrRNA molecule, which may be separate from or linked to the RNA molecule comprising a guide sequence portion, and are effective simultaneously in a subject or cell. The at least one CRISPR nuclease, the RNA molecule comprising a guide sequence portion, and the tracrRNA 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 RNA molecule comprising a guide sequence portion and / or the tracrRNA are substantially present in the subject or cell.

[0127] In some embodiments, the cell is a stem cell. In some embodiments, the cell is a monocyte. In some embodiments, the cell is a macrophage. In some embodiments, the cell is an iPS-derived monocyte. In some embodiments, the cell is an iPS-derived macrophage. In some embodiments, the cell is a hematopoietic stem cell (HSC), a hematopoietic stem / progenitor cell (HSPC), a myeloid progenitor cell, a myeloblast, a lymphoblast, an erythroid progenitor cell, a platelet cell, a natural killer (NK) cell, a B lymphocyte, a T lymphocyte, an eosinophil, a neutrophil, an iPS-derived cell, or a basophil.

[0128] Genetic B2M safe harbor knock-ins to treat diseases and disorders In some embodiments, the methods of the invention may be used to knock in a sequence into a safe harbor site of B2M, hi some embodiments, B2M-mediated expression of the knocked-in sequence is involved in or relevant to the treatment of a disorder or disease.

[0129] For example, in some embodiments, the B2M DNA target site in a target cell (e.g., a monocyte, macrophage, hematopoietic stem cell (HSC), hematopoietic stem / progenitor cell (HSPC), myeloid progenitor cell, myeloblast, lymphoblast, erythroid progenitor cell, platelet cell, natural killer (NK) cell, B lymphocyte, T lymphocyte, eosinophil, neutrophil, basophil, or iPS cell) is modified such that the target cell expresses and secretes a protein product encoded by the modification (e.g., an introduced or knocked-in protein-coding sequence). These target cells may be used, for example, to treat lysosomal storage diseases or other disorders of the blood, lung, brain, liver, intestine, intestinal tract, bone, muscle, or central nervous system, or inflammatory or autoinflammatory diseases. In some embodiments, these modified cells serve as an alternative to traditional enzyme replacement therapy. In some embodiments, these modified cells are used in immunotherapies, such as cancer immunotherapy.

[0130] By way of non-limiting example, expression of the knocked-in sequence may be involved in or associated with the treatment of a disease or disorder of the blood, lung, brain, gut, intestinal tract, bone, liver, muscle, or central nervous system. By way of non-limiting example, the knocked-in sequence may be an A1AT, G6PC, SERPINA, TTR, ornithine transcarbamylase, argininosuccinate synthase, arginase, argininosuccinase, carbamoyl phosphate synthase, or N-acetylglutamate synthase sequence, or a portion thereof.

[0131] By way of non-limiting example, expression of the knocked-in sequence may be involved in or relevant to the treatment of inflammatory or autoinflammatory diseases or disorders, such as inflammatory bowel disease (IBD). For example, insertion of IL-4 into neural cells reduces symptoms in mice with multiple sclerosis. Furthermore, insertion of IL-10 into a mouse model of type 1 diabetes reduces insulitis and T cell activation. By way of non-limiting example, the knocked-in sequence may be a cytokine or chemokine, such as an anti-inflammatory cytokine or chemokine (e.g., IL-10, IGF1, TGF-β, and IL-4). In such embodiments, secretion of the knocked-in cytokine or chemokine facilitates manipulation of immune and inflammatory responses for the treatment of the disease or disorder.

[0132] By way of non-limiting example, expression of the knocked-in sequence may be involved in or associated with cancer treatment. By way of non-limiting example, the knocked-in sequence may be a cytokine, chemokine, factor, or protein that can activate the immune system or recruit immune system cells to the tumor site to assist in tumor elimination. For example, IL-15 may be knocked in and secreted to enhance the persistence of T cells or natural killer cells, or CXCR4 may be knocked in to increase the recruitment of T cells and natural killer cells to the tumor site.

[0133] By way of non-limiting example, target cells (e.g., monocytes or macrophages) may be engineered to express cytokines or chemokines, including, but not limited to, IL-10, IGF1, TGF-β, IL-15, CXCR4, and / or IL-4.

[0134] By way of non-limiting example, expression of the knocked-in sequence may be involved in or associated with the treatment of lysosomal storage diseases or other disorders. By way of non-limiting example, target cells (e.g., monocytes or macrophages) may express or express alpha-galactosidase A, clotting factor IX, clotting factor VII, lysosomal alpha-glucosidase, fibrinogen, phenylalanine, or the like. The cells may be modified to express 4-hydroxylase, alkaline phosphatase, glucosylceramidase, beta-galactosidase, porphobilinogen deaminase, arylsulfatase B, beta-glucuronidase, alpha-N-acetylglucosaminidase, lysosomal alpha, alpha L-iduronidase, mannosidase, phosphatidylcholinesterol acyltransferase, N-sulfoglucosamine sulfohydrolase, coagulation factor X, N-acetylgalactosamine-6-sulfatase, sphingomyelin phosphodiesterase, alpha-1 antitrypsin, iduronate-2-sulfatase, lysosomal alpha glucosidase, cyclin-dependent kinase-like 5, pro-low density lipoprotein receptor-related protein 1, phenylalanine ammonia-lyase, protein glutamine gamma glutamyltransferase K, lysosomal protective protein, or a portion thereof.

[0135] By way of non-limiting example, expression of the knocked-in sequence may be involved in or relevant to the treatment of the following diseases or disorders (each with the associated gene or enzyme listed in parentheses below): Pompe disease (acid α-glucosidase), mucopolysaccharidosis type I (α-L-iduronidase), Fabry disease (α-galactosidase), mucopolysaccharidosis type II (iduronate-2-sulfatase), mucopolysaccharidosis type IVA (N-acetylgalactosamine-6-sulfatase), mucopolysaccharidosis type VI (N-acetylgalactosamine- 4-sulfatase), adrenoleukodystrophy (lysophosphatidylcholine metabolism-related genes, e.g., phospholipase A2), severe combined immunodeficiency (genes related to T-REC or K-REC), Gaucher disease (β-glucosidase or β-glucocerebrosidase), metachromatic leukodystrophy (MLD) (arylsulfatase A), primary immunodeficiency, hemophilia A and B (factor VIII), IGF1 deficiency (IGF-1), surfactant deficiency (surfactant protein A (SP-A) and / or surfactant protein B (SP-B), aspartylglucosaminuria (aspartyl-β-glucosaminidase), Sanfilippo syndrome (acetyl-CoA α-glucosaminide), mucopolysaccharidosis type III (acetyl-CoA-arylamine N-acetyltransferase), Sanfilippo syndrome type IIId (N-acetylglucosamine-6-sulfatase), Eisel disease (N-acetylglucosamine-1-phosphotransferase), Schindler disease (α-N-acetylglucosaminidase) enzyme), Farber disease (FD) or spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) (acid ceramidase), Canavan disease (aspartoacylase), lysosomal acid lipase deficiency (lysosomal acid lipase), Niemann-Pick disease (acid sphingomyelinase), mucopolysaccharidosis type 6 (arylsulfatase B), fucosidosis (α-L-fucosidase), Krabbe disease (galactosylceramidase, galactocerebrosidase), GM1 gangliosidosis, mucopolysaccharidosis type IVB (MPS)IVB) or galactosialidosis (galactosidase-β-1 or β-galactosidase (GLB1) or protective protein / cathepsin A), Sly disease (β-glucuronidase), mucopolysaccharidosis type III (heparan N-sulfatase), late-onset Tay-Sachs disease (β-hexosaminidase A), hyaluronidase 1 deficiency (hyaluronidase-1), α-mannosidosis (α-D-mannosidase), β-mannosidosis (β-mannosidase), sialidosis (α-neuraminidase), Stanhof disease (β-hexosaminidase A and / or β-hexosaminidase B), Santavuori-Hartia disease (palmitoylated protein Protein thioesterase), Jansky-Bielschowski disease (tripeptidyl peptidase I), Batten disease (Battenin), neuronal ceroid lipofuscinosis type 5 (ceroid lipofuscinosis neuronal protein 5 (CLN5)), neuronal ceroid lipofuscinosis type 6 (CLN6), neuronal ceroid lipofuscinosis type 7 (CLN7), neuronal ceroid lipofuscinosis type 8 (CLN8), congenital Examples of target cells include those with cathepsin D deficiency (cathepsin D), cystinosis (cystinosin), pyknodysostosis (cathepsin K), Salla disease (sialin), and / or Danon disease (lysosomal-associated membrane protein 2 (LAMP2)). By way of non-limiting example, target cells (e.g., monocytes or macrophages) may be engineered to express metabolic modulators. By way of non-limiting example, target cells may be engineered to express human growth hormone, insulin-like growth factor 1 (IGF-1), factor VIII (hemophilia A and B), follicle-stimulating hormone, erythropoietin, cytokines, chemokines, IL-10, IGF1, TGF-β, IL-15, CXCR4, IL-4, granulocyte colony-stimulating factor (G-CSF), galactosamine-6-sulfatase, and / or β-hexosamine enzymes.

[0136] In particular, monocytes and macrophages reside in target tissues, including the lung and brain, and can serve as expression vectors for the long-term secretion of proteins in these target tissues. Expression of a transgene under the control of the B2M promoter is achieved by CRISPR-mediated knock-in at safe harbor sites targeted by the guide sequence moieties described herein. The transgene-expressed protein may be secreted. Thus, the ability to prepare blood and / or tissues containing modified target cells and have them continuously secrete a protein of interest serves as an alternative to enzyme replacement therapy (ERT). For example, modified monocytes may be useful for targeting the central nervous system (CNS) or lung to secrete a protein of interest in target tissues.

[0137] Such an approach would also be useful for treating, for example, lysosomal storage diseases. Some of these diseases manifest in the CNS. In particular, the blood-brain barrier (BBB) ​​makes it difficult to treat brain damage with enzyme replacement therapy. Monocytes may be used to deliver secreted proteins of interest across the BBB so that they are secreted in the CNS.

[0138] For example, modified monocytes or macrophages delivered to the brain may be used to treat lysosomal storage diseases; modified monocytes or macrophages delivered to the lung may be used to treat antitrypsin deficiency (A1AT), D-surfactant deficiency, or proteinosis; or modified monocytes or macrophages in the blood may be used to treat A1AT or adenosine deaminase 2 (DADA2) deficiency.

[0139] B2M editing strategies include those that allow expression of a desired sequence under the control of the B2M promoter, in some cases without knocking out the edited B2M allele, which can be achieved by strategies (1) knocking in the B2M allele into intron 1 or into the intron 1 following the coding exon 1 of the B2M allele, or (2) knocking in the B2M allele by replacing the stop codon.

[0140] In the first strategy (i.e., mediating biallelic cleavage using an RNA molecule containing a guide sequence portion targeting intron 1 of the B2M gene), cleavage is mediated within a non-regulatory region and is therefore not expected to affect B2M gene expression. To knock in a desired sequence without knocking out the endogenous B2M gene, splicing acceptor (SA), branch site, and splicing donor (SD) elements are added to the knock-in cassette (e.g., as part of the donor molecule), resulting in the insertion of the desired sequence as a novel exon (e.g., novel exon 2). In this way, an mRNA containing two coding regions (referred to as a "bicistronic transcript") is prepared. Additionally, to avoid interfering with the expression of the endogenous B2M gene, the cassette may contain a self-cleaving peptide (e.g., a 2A self-cleaving peptide such as P2A, F2A, E2A, T2A, or a combination thereof) and / or a B2M signal peptide at the C-terminus of the inserted gene, which is cleaved in the endoplasmic reticulum to allow separation of the B2M gene product from the inserted gene product.

[0141] In the second strategy (i.e., knock-in by replacing the B2M stop codon), biallelic truncation is mediated in the 3'UTR region up to 150 nucleotides downstream from the stop codon or upstream of the stop codon but within the intron region. The location of the truncation is not expected to affect B2M gene expression. The desired knock-in sequence is inserted in place of the stop codon, and the knock-in cassette contains a 2A self-cleaving peptide at the N-terminus of the inserted sequence, allowing for separation of the inserted gene product from the B2M gene product.

[0142] B2M editing strategies may be used to edit T cells for use in chimeric antigen receptor T cell (CAR-T) therapy. For example, a sequence of interest (e.g., a CD19 sequence) encoded by a donor molecule may be introduced into a B2M safe harbor site targeted by an RNA molecule containing a guide sequence portion as described herein. Such editing may be used to alter T cell receptor expression and / or receptor signaling by the cell.

[0143] 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 some embodiments, the CRISPR complex does not further comprise a tracrRNA. In a non-limiting example where the RNA-guided DNA nuclease is a CRISPR protein, the at least one nucleotide that differs between the B2M alleles may be located within and / or near a PAM site within the region to which the RNA molecule is designed to hybridize. Those skilled 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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 (each of which is incorporated herein by reference), may also be used.

[0149] 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.

[0150] 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 a double-strand break in the target DNA. In some embodiments, the enzymatic activity modifies a target polypeptide associated with the 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.

[0151] 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.

[0152] In some embodiments, the CRISPR nuclease is Cpf1. Cpf1 is a single RNA-guided endonuclease that utilizes a T-rich protospacer adjacent motif. Cpf1 produces 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).

[0153] 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 Cpf1 and its homologs, orthologs or variants, may be used in the present invention.

[0154] 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, or 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-methyl-2-methyl- ... 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.

[0155] Guide sequence targeting the B2M allele A given RNA molecule containing a guide sequence portion used to target a DNA site may result in degradation of the RNA molecule, limited activity, lack of activity, or off-target effects. Therefore, an appropriate guide sequence portion is required to target a given DNA site in a gene.

[0156] The present invention identifies a novel set of guide sequence segments that target at least one B2M allele and introduce a nucleotide sequence into at least one allele that is expressed under the control of a B2M promoter. Such gene editing approaches can be used to treat disorders or diseases or modify cell behavior. Preferably, non-discriminatory RNA molecules that can target a pair of B2M alleles are used for targeting.

[0157] In some embodiments of this invention, RNA molecules are used to target sites in the B2M gene and introduce or knock-in exogenous nucleotide sequences into the B2M gene. In some embodiments, the site is located near the intended knock-in site, preferably near the start or stop codon, preferably within 150 nucleotides of the start or stop codon. In some embodiments, the site is within intron 1 following coding exon 1 of the targeted B2M allele. In some embodiments, the site is within exon 3, exon 4, intron 1, intron 2, intron 3, or the 3' untranslated region (3'UTR) of the targeted B2M allele.

[0158] Cellular delivery The compositions described herein may be delivered to target cells by any suitable means. The compositions of the present invention may be targeted to cells containing and / or expressing the B2M allele, such as mammalian cells, preferably monocytes or macrophages. For example, in one embodiment, an RNA molecule specifically targeting the B2M allele is delivered to the target cell, a monocyte or macrophage. Delivery to the cell may be performed in vitro, ex vivo, or in vivo. Furthermore, the nucleic acid compositions described herein may be delivered as one or more of a DNA molecule, an RNA molecule, a ribonucleoprotein (RNP), a nucleic acid vector, or a combination thereof.

[0159] In some embodiments, in vivo delivery of the compositions described herein includes delivery via lentivirus, adeno-associated virus (AAV), or nanoparticles. In some embodiments, in vivo delivery of the compositions described herein includes delivery via lentivirus, adeno-associated virus (AAV), or nanoparticles. The compositions may be in the form of an RNP composition. Thus, delivery can be in vivo to monocytes or macrophages in a subject.

[0160] In some embodiments, the compositions described herein are delivered to cells ex vivo. In some embodiments, the cells are stem cells. In some embodiments, the cells are monocytes. In some embodiments, the cells are macrophages. In some embodiments, the cells are iPS-derived monocytes or macrophages. In some embodiments, the cells are hematopoietic stem cells (HSCs), hematopoietic stem / progenitor cells (HSPCs), myeloid progenitor cells, myeloblasts, lymphoblasts, erythroid progenitor cells, platelet cells, natural killer (NK) cells, B lymphocytes, T lymphocytes, eosinophils, neutrophils, or basophils. The compositions may be delivered to cells by known ex vivo delivery methods, including, but not limited to, electroporation, viral transduction, nanoparticle delivery, liposomes, and the like. The compositions may be in the form of RNP compositions. Details of delivery methods are provided throughout this section.

[0161] In some embodiments, the RNA molecules of the compositions described herein comprise chemical modifications. 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.

[0162] 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 based 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 or 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).

[0163] 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, and cassava vein mosaic virus). (See, e.g., Chung et al., 2006). 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)).

[0164] Non-viral vectors, such as transposon-based systems (e.g., recombinant Sleeping Beauty transposon systems or recombinant PiggyBac transposon systems), 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.

[0165] 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.

[0166] 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).

[0167] 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).

[0168] 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.

[0169] 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.

[0170] 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 RNA 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. In some cases, the lentivirus contains a nuclease mRNA, a guide RNA, and a donor template. In some cases, the lentivirus comprises a nuclease protein and a guide RNA. In some cases, the lentivirus comprises a nuclease protein, a guide RNA, and / or a donor template for gene editing, e.g., by homology-directed repair. In some cases, the lentivirus comprises a nuclease mRNA, a DNA-targeting RNA, and a tracrRNA. In some cases, the lentivirus comprises a nuclease mRNA, a DNA-targeting RNA, a tracrRNA, and a donor template. In some cases, the lentivirus comprises a nuclease protein, a DNA-targeting RNA, and a tracrRNA. In some cases, the lentivirus comprises a nuclease protein, a DNA-targeting RNA, a tracrRNA, and a donor template for gene editing, e.g., by homology-directed repair.

[0171] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding the target cell population. 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 foreign sequences up to 6-10 kb. 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).

[0172] 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.

[0173] 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). The transduction efficiency of the MFG-S packaging vector was greater than 50% (Ellem et al., (1997); Dranoff et al., 1997).

[0174] 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 prepared 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. Patent No. 7,479,554.)

[0175] In many gene therapies, it is desirable for gene therapy vectors to be delivered with high specificity to particular 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 or 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.

[0176] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravitreal, intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection) or local application, as described below. Alternatively, vectors can be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirate, tissue biopsy) or universal donor hematopoietic stem cells, followed by re-implantation into the patient after selection of cells incorporating the vector. Non-limiting exemplary ex vivo approaches may include removing tissues (e.g., peripheral blood, bone marrow, and 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 and spleen). In some embodiments, the stem cells or hematopoietic stem cells may be further treated with a survival-enhancing agent.

[0177] Ex vivo cell transfection for diagnostics, research, or 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).

[0178] Suitable cells include, but are not limited to, eukaryotic cells and / or cell lines. Non-limiting examples of such cells, or cell lines prepared from such cells, include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6 cells, any plant cell (differentiated or undifferentiated), as well as insect cells, such as Spodoptera fugiperda (Sf), or fungal cells, such as Saccharomyces, Pichia, and Schizosaccharomyces. In certain embodiments, the cell line is a CHO-K1, MDCK, or HEK293 cell line. Additionally, primary cells may be isolated and used ex vivo for treatment with an inducible nuclease system (e.g., CRISPR / Cas) followed by reintroduction into the subject. Suitable primary cells include peripheral blood mononuclear cells (PBMCs) and other blood cell subsets, such as, but not limited to, CD4+ T cells or CD8+ T cells. Suitable cells also include stem cells, such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neural stem cells, and mesenchymal stem cells.

[0179] In some embodiments, stem cells are processed ex vivo for cell transfection and gene therapy. An advantage of using stem cells is that they can be differentiated into other cells in vitro or introduced into a mammal (such as the cell donor) and engrafted into the bone marrow. Methods for differentiating CD34+ cells in vitro into clinically important immune cells using cytokines such as GM-CSF, IFN-γ, and TNF-α are known (see, for non-limiting examples, Inaba et al., 1992).

[0180] Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies that bind to unwanted cells such as CD4+ and CD8+ (T cells), CD45+ (pan B cells), GR-1 (granulocytes), and Iad (differentiated antigen-presenting cells) (see, for non-limiting examples, Inaba et al., 1992). Modified stem cells may also be used in some embodiments.

[0181] Vectors (e.g., retroviruses, liposomes) carrying therapeutic nucleic acid compositions can also be administered directly to an organism to transduce 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 and creams), and electroporation. Suitable methods for administering such nucleic acids are available and well known to those of skill 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.

[0182] 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.

[0183] As previously mentioned, the compositions described herein can 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 within the target cell. See also WO 2013 / 014537, WO 2014 / 016690, WO 2016 / 185125, WO 2017 / 194902, and WO 2017 / 194903.

[0184] 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.

[0185] The compositions and methods of the present invention may also be used in the manufacture of a medicament for treating a disease or disorder in a patient.

[0186] Mechanism of action of the B2M safe harbor knock-in method Without being bound by any theory or mechanism, the present invention may be used to apply a CRISPR nuclease to process the B2M allele so as to introduce a sequence into the safe harbor site of the B2M allele and control expression of the introduced sequence with the promoter of the B2M allele. A particular guide sequence may be selected from Table 1 based on the target location and the type of CRISPR nuclease used (e.g., according to the required PAM sequence).

[0187] The B2M gene is located on chromosome 15 and encodes the β2 microglobulin protein. A donor molecule may be used to knock in a desired nucleotide sequence into the B2M safe harbor site.

[0188] One strategy is to knock-in a nucleotide sequence into intron 1 of the B2M gene, or into intron 1 following coding exon 1. This strategy utilizes an RNA molecule to target a CRISPR nuclease into intron 1 of the B2M gene, thereby creating a double-stranded break. Because the break occurs in a non-regulatory region, it is not expected to affect gene expression. In knock-in of a sequence without a knockout in B2M, the sequence is inserted as a new exon (i.e., exon 2) by adding splicing acceptor (SA) and splicing donor (SD) elements to the knock-in donor cassette. Additionally, to prevent disruption of B2M gene expression, the donor cassette contains a 2A self-cleaving peptide and / or a B2M signal peptide at the C-terminus of the inserted gene, which is cleaved in the endoplasmic reticulum and allows the inserted sequence protein expression product to be separated from the B2M protein.

[0189] Another strategy is to knock-in a nucleotide sequence into the B2M gene, replacing the stop codon. In this case, both alleles are truncated in the 3'UTR region up to 150 nucleotides downstream of the stop codon or in the intron region upstream of the stop codon. The location of the truncation in these regions does not affect B2M expression. The knock-in nucleotide sequence is inserted in place of the stop codon, and the knock-in donor cassette contains a 2A self-cleaving peptide at the N-terminus of the inserted nucleotide sequence, which allows separation of the B2M protein from the protein expression product of the inserted sequence.

[0190] Example of an RNA guide sequence that specifically targets an allele of the B2M gene While numerous guide sequences can be designed to target the B2M allele, the nucleotide sequences set forth in Table 1, identified below as SEQ ID NOS: 1-28998, were specifically selected to effectively carry out the methods set forth herein.

[0191] Table 1 shows guide sequences designed for use in the previously described embodiments to associate specific sequences within the B2M allele. 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), SpCas9 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).

[0192] OMNI CRISPR 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 each of which are incorporated herein by reference.

[0193] 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.

[0194] [Table 1]

[0195] To facilitate a more complete understanding of the present invention, the following examples are provided. 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]

[0196] Experiment details Example 1 Analysis of B2M on-target activity Guide sequences containing 17 to 50 consecutive nucleotides within any of SEQ ID NOs: 1 to 28998 are screened for high on-target activity in target cells using CRISPR nucleases. On-target activity is determined by DNA capillary electrophoresis analysis.

[0197] Example 2 Insertion of a Sequence of Interest into a B2M Safe Harbor Site B2M is highly expressed in target cells (e.g., macrophages) and contains a signal peptide (SP) in exon 1. These characteristics make the B2M locus an excellent candidate for the integration and secretion of a gene of interest. To take advantage of the SP of B2M, we designed a strategy to insert donor DNA into intron 1 of B2M, downstream of the SP. This homology-directed repair (HDR)-dependent insertion is possible by combining a donor sequence encoding the gene of interest with CRISPR-induced DNA damage. This strategy aims to enable the expression and secretion of the gene of interest while maintaining the expression and function of genes with safe harbor sites.

[0198] CRISPR-based ribonucleoprotein (RNP) composition enables editing in intron 1 of B2M We screened guides in HeLa cells to identify potential CRISPR-based RNP compositions that enabled editing in intron 1 of B2M. Briefly, OMNI-50 nuclease-encoding plasmids (64 ng) and guide-expressing plasmids (20 ng) were co-transfected in 96-well plates using jetOPTIMUS reagent (Polyplus). 72 hours later, cells were harvested, genomic DNA was extracted, and the DNA was analyzed by next-generation sequencing (NGS). The percentage of editing, as measured by indel abundance, varied depending on the guide used, reaching a maximum of 61% when OMNI-50 nuclease was used (Figure 1A).

[0199] Next, we electroporated the RNPs into hematopoietic stem cells (HSCs) and tested the best-performing guide. Single-guide RNA (sgRNA) (120 pmoles) containing either OMNI-50 or OMNI-50 V6172 nuclease (105 pmoles) was electroporated into cells using a Lonza P3 cell 4D-nucleofecuor X Kiu S (CA-137 program, PBC2-00675, Lonza). After 72 hours, HSCs were harvested and genomic DNA analyzed by next-generation sequencing. Analysis revealed that OMNI-50 V6172 nuclease was highly efficient, with editing levels of approximately 80%, while OMNI-50 nuclease was less efficient (Figure 1B). As a result, the composition chosen for HDR editing in intron 1 of B2M was the combination of the B2M_g57 guide molecule and the OMNI-50 V6172 nuclease.

[0200] Integration of HDR editing template into intron 1 of B2M We prepared AAV particles carrying a donor construct encoding GFP fused to a self-cleaving P2A-T2A element (2A) and SP to test the feasibility of integrating an HDR-edited template into intron 1 of B2M. As shown in Figure 2, the donor targets intron 1 of B2M, downstream of SP. The donor construct contains the GFP-2A-SP sequence flanked by a splice acceptor and splice donor and further flanked by 800 base pair homology arms matching the B2M_g57 cleavage site (see SEQ ID NO: 29012 and Table 4). CRISPR-mediated DNA cleavage promotes HDR-mediated integration of the donor sequence into intron 1 of B2M. This results in a bicistronic transcript with a single open reading frame encoding SP-GFP-2A-SP-B2M, which, after self-cleavage of 2A, yields two polypeptides: SP-GFP and SP-B2M. Importantly, the donor does not contain a promoter or other mRNA stabilizing elements, so no GFP expression is possible unless it is successfully integrated into the desired locus.

[0201] Integration of GFP into the B2M locus of HSCs We first tested this approach in primary HSCs. Cells were electroporated with RNPs composed of OMNI-50 V6172 nuclease and B2M_g57 guide molecules, and 10 5 The cells were infected with AAV6 particles carrying GFP donor molecules at a multiplicity of infection (MOI) of 1000.

[0202] Three days after treatment, GFP expression by HSCs was analyzed by FACS (Figure 3A). Combining RNP (B2M_g57 guide) with donor AAV resulted in 46% GFP-expressing cells (average of two replicates).

[0203] To confirm that GFP expression was due to successful integration into the B2M locus, we examined GFP expression in a group infected with donor AAV combined with an irrelevant RNP (NR RNP). Similar to the untreated HSC control group, no significant GFP expression was observed in this group. Next, to confirm whether B2M expression was maintained after HDR integration, we examined B2M transcript and protein levels at day 7.

[0204] To confirm that GFP integration into the B2M locus maintained transcription of the safe harbor gene, we assessed total B2M transcript levels by qRT-PCR using a primer set targeting exon 2 of B2M, which is downstream of the editing site and therefore recognizes both the native and GFP-integrated transcripts (Figure 3B). Transcript levels in the HDR group were slightly higher than in the untreated control group, and no decrease in B2M transcript levels was detected.

[0205] To assess the protein levels, cells were analyzed by FACS (Figure 3C). To better understand the impact of HDR on B2M expression, we focused our analysis on GFP+ cells in the GFP-P2A-B2M + RNP group compared with the "No HDR" and "ISO-stained" groups (not targeted but labeled with an antibody). Based on the latter group, we determined the background staining (no B2M expression). While the majority of GFP+ cells (approximately 86%) expressed B2M protein, a second peak of lower intensity was detected, accounting for approximately 37% of GFP+ cells and retaining moderate B2M expression. This population may be explained by reduced translation of genes located downstream of the 2A peptide. We also observed that approximately 14% of cells showed very low or no expression, corresponding to the signal in the ISO-stained group.

[0206] Finally, to test whether GFP was successfully secreted from HSCs after integration into the B2M locus, cell culture medium was sampled using a GFP ELISA kit (Abcam, ab171581). HSCs in the HDR group were cultured for 10 min at 4°C.6 Approximately 1.3 ng of GFP was secreted per cell, whereas no GFP was detected in the control group (Figure 3D - note that this value is the absolute level of GFP detected after accounting for the volume of medium).

[0207] Differentiation of edited HSCs into macrophages maintains GFP expression from the B2M locus Edited HSCs can differentiate into multiple hematopoietic cell lineages with diverse therapeutic potential. Therefore, we tested whether the edited HSCs retained GFP expression and secretion after differentiation into macrophages. To do so, we differentiated the edited HSCs into macrophages as previously described (Gomez-Ospina et al., Nature Communications, 2019). Briefly, cells were seeded in differentiation medium (SFEM II supplemented with SCF (200 ng / ml), IL-3 (10 ng / ml), IL-6 (10 ng / ml), FLT3-L (50 ng / ml), M-CSF (10 ng / ml), GM-CSF (10 ng / ml), and penicillin / streptomycin (10 U / ml)) for 48 hours. Adherent cells were maintained in maintenance medium (RPMI supplemented with FBS (10% v / v), M-CSF (10 ng / ml), GM-CSF (10 ng / ml), and penicillin / streptomycin (10 U / ml)) for 19 days. To verify differentiation, cells were scraped and analyzed using a FACS panel: CD14, CD16, and HLA-DR. As shown in Figure 4A, examination of the staining panel for edited and unedited controls reveals that most cells tested successfully differentiated into macrophages and expressed all macrophage markers tested. The proportion of macrophages in the population was unchanged by editing and was comparable between edited and unedited controls. Furthermore, the FITC channel of the FACS analysis reveals that approximately 20% of cells expressed GFP after differentiation.

[0208] We also measured B2M expression signals by FACS to confirm that the B2M gene remained expressed after differentiation and HDR (Figure 4B). While B2M expression decreased in the original HSCs at day 7, in this experiment, restoration of B2M expression was detected, as nearly all GFP-expressing cells were B2M+, with over 90% of cells showing WT-level signals. To test for GFP secretion, cell culture media was assayed by ELISA (Figure 4C), and GFP secretion was observed in the edited population. This confirmed that insertion of GFP into the B2M locus enabled both expression and secretion of the gene of interest.

[0209] Integration of GFP into the B2M locus of iPSCs To confirm the relevance of this system to other pluripotent cells, we repeated these experiments with iPSCs. As with HSCs, we delivered RNPs via electroporation and donor DNA molecules using AAV. To increase the infection rate of iPSCs, we used AAV-DJ. Testing GFP expression in iPSCs 3 days after treatment demonstrated that GFP was successfully expressed in iPSCs after HDR integration (Figure 5A). Consistent with the results shown with HSCs, the control group lacking CRISPR activity did not express GFP, demonstrating that integration of the GFP cassette is essential for GFP expression. GFP secretion from the engineered iPSCs was assessed by ELISA (Figure 5B). GFP secretion was detected only in the HDR group, confirming that integration of GFP into intron 1 of B2M enables the expression of a polypeptide containing the SP, leading to its subsequent secretion.

[0210] FACS analysis of iPSCs [Table 2]

[0211] [Table 3]

[0212] [Table 4]

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Claims

1. An ex vivo or in vitro method for modifying at least one allele of the β2 microglobulin (B2M) gene within a cell, At least one CRISPR nuclease, or a polynucleotide molecule encoding the CRISPR nuclease; and, An RNA molecule containing a guide sequence of 17 to 50 nucleotides, or a nucleotide sequence encoding it. The step includes introducing a composition containing the above into the cells, A method comprising a complex of the CRISPR nuclease and the RNA molecule causing a double-strand break in at least one allele of the B2M gene.

2. The composition further comprises a donor molecule containing a nucleotide sequence to be introduced into the double-strand break site, and optionally the introduced sequence is (i) α1-antitrypsin, glucose-6-phosphatase (G6PC), serpine family A member (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthase, arginase, argininosuccinate, carbamoyl phosphate synthase, N-acetylglutamate synthase, α-galactosidase A, coagulation factor IX, coagulation factor VII, lysosomal α-glucosidase, fibrinogen, phenylalanine (ii) A sequence derived from a gene encoding 4-hydroxylase, alkaline phosphatase, glucosylceramidase, β-galactosidase, porphobilinogen deaminase, arylsulfatase B, β-glucuronidase, α-N-acetylglucosaminidase, lysosomal α, α-L-idulonidase, mannosidase, phosphatidylcholinesterol acyltransferase, N-sulfoglucosamine sulfohydrolase, coagulation factor X, N-acetylgalactosamine-6-sulfatase, sphingomyelin phosphodiesterase, iduronic acid-2-sulfatase, lysosomal α-glucosidase, cyclin-dependent kinase-like 5, pro-low-density lipoprotein receptor-related protein 1, phenylalanine ammonia lyase, protein glutamine γ-glutamyltransferase K, or a lysosomal protective protein. Acid α-glucosidase, α-L-iduronidase, α-galactosidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, lysophosphatidylcholine metabolism-related proteins, preferably phospholipase A2, T-REC or K-REC-related proteins, β-glucosidase, β-glucocerebrosidase, arylsulfatase A, factor VIII, insulin-like growth factor 1 (IGF-1), surfactant protein A, surfactant protein B, aspartyl-β-glucosaminidase, acetyl-CoA α-glucosaminide, acetyl-CoA-arylamineN-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylglucosaminidase, acid ceramidase, aspartoacylase, lysosomal acid lipase, acid sphingomyelinase, arylsulfatase B, α-L-fucosidase, galactosylceramidase, galactocerebrosidase, β-galactosidase, protective protein / cathepsin A, β-glucuronidase, heparan N-sulfatase, β-hexosaminidase A, hyaluronidase-1, α-D-mannosidase, β-mannosidase, α-neuraminidase, β-hexosaminidase A, β-hexosaminidase B, palmitoylated protein The method according to claim 1, comprising a sequence derived from a gene encoding thioesterase, tripeptidyl peptidase I, battenin, ceroid lipofuscinosis neuron protein 5 (CLN5), ceroid lipofuscinosis neuron protein 6 (CLN6), ceroid lipofuscinosis neuron protein 7 (CLN7), ceroid lipofuscinosis neuron protein 8 (CLN8), (cathepsin D), cystinosine, cathepsin K, sialin, lysosome-associated membrane protein 2 (LAMP2), human growth hormone, follicle-stimulating hormone, erythropoietin, CD19, cytokines, chemokines, IL-10, IGF1, TGFβ, IL-15, CXCR4, IL-4, or granulocyte colony-stimulating factor (G-CSF).

3. The method according to claim 2, wherein the donor molecule comprises a sequence derived from a gene encoding a protein secreted by a cell, and / or the sequence to be introduced comprises a sequence encoding a polypeptide of interest expressed by the cell, wherein optionally the polypeptide of interest expressed is secreted by the cell, wherein optionally the polypeptide of interest expressed further comprises a signal peptide, wherein optionally the signal peptide is encoded by an allele of the B2M gene.

4. The introduced sequence includes a sequence encoding a 2A self-cleaving peptide, and / or the introduced sequence includes a sequence encoding a signal peptide, the signal peptide being a B2M signal peptide if necessary, and / or the introduced sequence includes a splice acceptor sequence and a splice donor sequence, and / or the introduced sequence includes a splice acceptor sequence, a sequence encoding the target polypeptide, a sequence encoding a 2A self-cleaving peptide, a signal peptide and a splice donor sequence, and / or the do The method according to claim 2 or 3, wherein the NER molecule comprises a first homology arm sequence having at least 90%, preferably 100%, sequence identity with the B2M sequence upstream of the double-strand break, and a second homology arm sequence having at least 90%, preferably 100%, sequence identity with the B2M sequence downstream of the double-strand break, wherein the lengths of the first homology arm sequence and the second homology arm sequence are, optionally, about 20 to 50, 50 to 100, 100 to 200, 200 to 500, 500 to 1000, or 1000 to 2000 nucleotides, respectively.

5. The method according to claim 3 or 4, wherein the target polypeptide is a soluble protein, and / or the length of the target polypeptide is about 20 to 50, 50 to 100, 100 to 200, 200 to 500, 500 to 1000 or 1000 to 2000 amino acids, and / or the modified allele of the B2M gene expresses the B2M polypeptide and the target polypeptide.

6. The RNA molecule includes a guide sequence portion having 17 to 50 consecutive nucleotides in any of the sequences shown in SEQ ID NOs: 1 to 28998, and / or the RNA molecule includes a non-distinguishable guide portion targeting exon 3, exon 4, intron 1, intron 2, intron 3, or 3' untranslated region (3'UTR) of the B2M gene, and / or the RNA molecule includes 15:44716329 to 44716 356, 15:44717607~44717813, 15:44717825~44718145, 15:44716357~44717323, 15:44717327~44717606, 15:44711614~44712998, 15:44713001~44713065, 15:44713067~44713182, 15:44713184~44713461, 15:44713463~4471441 9, comprising a non-distinguishable guide region that targets a sequence located within a genomic range selected from any of 15:44714421–44714474, 15:44714476–44715422 and 15:44715702–44716328, and / or the modified allele of the B2M gene expresses the B2M gene product, and / or the cells include stem cells, monocytes, macrophages, iPS cell-derived monocytes, and iPS cell-derived macrophages. The method according to any one of claims 1 to 5, wherein the cells are clophages, hematopoietic stem cells (HSCs), hematopoietic stem / progenitor cells (HSPCs), myeloprogenitor cells, myeloblasts, lymphoblasts, erythrocyte progenitor cells, platelet cells, natural killer (NK) cells, B lymphocytes, T lymphocytes, eosinophils, neutrophils, iPS-derived cells, or basophils, and / or the cells are stem cells, and the method further comprises differentiating the stem cells after modification.

7. A modified cell obtained by the method described in any one of claims 1 to 6, wherein the modified cell is, as necessary, a stem cell, a monocyte, a macrophage, an iPS cell-derived monocyte, an iPS cell-derived macrophage, a hematopoietic stem cell (HSC), a hematopoietic stem / progenitor cell (HSPC), a myeloprogenitor cell, a myeloblast, a lymphoblast, a erythrocyte progenitor cell, a platelet cell, a natural killer (NK) cell, a B lymphocyte, a T lymphocyte, an eosinophil, a neutrophil, an iPS cell-derived cell, or a basophil.

8. A composition comprising an RNA molecule containing a guide sequence portion having 17 to 50 consecutive nucleotides in any of the sequences shown in Sequence ID No. 1 to 28998, and optionally further comprising at least one CRISPR nuclease.

9. The mixture further contains donor molecules, which may optionally include: (i) α1-antitrypsin, glucose-6-phosphatase (G6PC), serpine family A member (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthase, arginase, argininosuccinate, carbamoyl phosphate synthase, N-acetylglutamate synthase, α-galactosidase A, coagulation factor IX, coagulation factor VII, lysosomal α-glucosidase, fibrinogen, and phenylalanine. (ii) A sequence derived from a gene encoding 4-hydroxylase, alkaline phosphatase, glucosylceramidase, β-galactosidase, porphobilinogen deaminase, arylsulfatase B, β-glucuronidase, α-N-acetylglucosaminidase, lysosomal α, α-L-idulonidase, mannosidase, phosphatidylcholinesterol acyltransferase, N-sulfoglucosamine sulfohydrolase, coagulation factor X, N-acetylgalactosamine-6-sulfatase, sphingomyelin phosphodiesterase, iduronic acid-2-sulfatase, lysosomal α-glucosidase, cyclin-dependent kinase-like 5, pro-low-density lipoprotein receptor-related protein 1, phenylalanine ammonia lyase, protein glutamine γ-glutamyltransferase K, or a lysosomal protective protein. Acid α-glucosidase, α-L-iduronidase, α-galactosidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, lysophosphatidylcholine metabolism-related proteins, preferably phospholipase A2, T-REC or K-REC-related proteins, β-glucosidase, β-glucocerebrosidase, arylsulfatase A, factor VIII, insulin-like growth factor 1 (IGF-1), surfactant protein A, surfactant protein B, aspartyl-β-glucosaminidase, acetyl-CoA α-glucosaminide, acetyl-CoA-arylamineN-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylglucosaminidase, acid ceramidase, aspartoacylase, lysosomal acid lipase, acid sphingomyelinase, arylsulfatase B, α-L-fucosidase, galactosylceramidase, galactocerebrosidase, β-galactosidase, protective protein / cathepsin A, β-glucuronidase, heparan N-sulfatase, β-hexosaminidase A, hyaluronidase-1, α-D-mannosidase, β-mannosidase, α-neuraminidase, β-hexosaminidase A, β-hexosaminidase B, palmitoylated protein thioesterase, tripeptidyl peptidase I, The composition according to claim 8, comprising a sequence derived from a gene encoding battenin, ceroid lipofuscinosis neuron protein 5 (CLN5), ceroid lipofuscinosis neuron protein 6 (CLN6), ceroid lipofuscinosis neuron protein 7 (CLN7), ceroid lipofuscinosis neuron protein 8 (CLN8), (cathepsin D), cystinosine, cathepsin K, sialin, lysosome-associated membrane protein 2 (LAMP2), human growth hormone, follicle-stimulating hormone, erythropoietin, CD19, cytokines, chemokines, IL-10, IGF1, TGFβ, IL-15, CXCR4, IL-4, or granulocyte colony-stimulating factor (G-CSF), and / or the donor molecule comprising a sequence derived from a gene encoding a protein secreted by a cell.

10. The donor molecule comprises a sequence encoding the target polypeptide, and / or the donor molecule comprises a sequence encoding a 2A self-cleaving peptide, and / or the donor molecule comprises a sequence encoding a signal peptide, the signal peptide being optionally a B2M signal peptide, and / or the donor molecule comprises a splice acceptor sequence and a splice donor sequence, and / or the donor molecule comprises a splice acceptor sequence, a sequence encoding the target polypeptide, a sequence encoding a 2A self-cleaving peptide, a signal peptide, and a splice donor. The composition according to claim 9, comprising a sequence and / or the donor molecule comprising a first homology arm sequence having at least 90%, preferably 100%, sequence identity with a first sequence of the B2M gene, and a second homology arm sequence having at least 90%, preferably 100%, sequence identity with a second sequence of the B2M gene, wherein the lengths of the first homology arm sequence and the second homology arm sequence are, optionally, about 20 to 50, 50 to 100, 100 to 200, 200 to 500, 500 to 1000, or 1000 to 2000 nucleotides, respectively.

11. The composition according to claim 10, wherein the target polypeptide is a soluble protein, and / or the length of the target polypeptide is at most 20 to 50, 50 to 100, 100 to 200, 200 to 500, 500 to 1000, or 1000 to 2000 amino acids.

12. The composition according to any one of claims 8 to 11, further comprising a tracrRNA molecule.

13. An ex vivo or in vitro method for modifying a B2M allele within a cell, comprising delivering a composition according to any one of claims 8 to 12 to the cell.

14. A composition or pharmaceutical for use in a method of treating, improving or preventing a disorder or disease, comprising: delivering the composition according to any one of claims 8 to 12 or the modified cells according to claim 7 to cells of a subject having the disorder or disease or at risk of having the disorder or disease, or delivering the pharmaceutical containing the composition according to any one of claims 8 to 12 or the modified cells according to claim 7 to the subject, wherein, optionally, (i) the pharmaceutical is an enzyme replacement therapy, (ii) the method is an immunotherapy, and / or the composition or the modified cells are delivered to the tissue or tumor of the subject.

15. (i) The said disorder or disease is a lysosomal storage disorder, (ii) The said disorder or disease is Pompe disease, mucopolysaccharidosis type I, Fabry disease, mucopolysaccharidosis type II, mucopolysaccharidosis type IVA, mucopolysaccharidosis type VI, adrenoleukodystrophy, severe combined immunodeficiency, Gaucher disease, metachromatic leukodystrophy (MLD), primary immunodeficiency, hemophilia A, hemophilia B, IGF1 deficiency, surfactant deficiency, aspartylglucosamineuria, Sanfils Po syndrome, mucopolysaccharidosis type III, Sanfilippo syndrome type IIId, Iesel's disease, Schindler's disease, Faber's disease (FD), spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), Canavan disease, lysosomal acid lipase deficiency, Niemann-Pick disease, mucopolysaccharidosis type 6, fucosidosis, Krabbe disease, GM1 gangliosidosis, mucopolysaccharidosis type IVB (MPS) (iv) The disorder or disease is galactosialidosis, Sleigh's disease, mucopolysaccharidosis type III, late-onset Tey-Sachs disease, hyaluronidase 1 deficiency, α-mannosidosis, β-mannosidosis, sialidosis, Stanhof's disease, Santavori-Hartia disease, Jansky-Birsawski disease, Batten disease, neurogenic ceroid lipofuscinosis type 5, neurogenic ceroid lipofuscinosis type 6, neurogenic ceroid lipofuscinosis type 7, neurogenic ceroid lipofuscinosis type 8, congenital cathepsin D deficiency, cystinosis, concentrated dysostosis, Salla disease, Danon disease and / or α1-antitrypsin deficiency, (iii) The disorder or disease is a disorder or disease of the blood, lungs, brain, liver, intestines, intestinal tract, bones, muscles or the central nervous system, or an inflammatory disease or autoinflammatory disease, or (iv) The disease or disorder is cancer. The composition or pharmaceutical product according to claim 14.

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