Knock-in strategy at the APLP2 safe harbor site

Abstract

An RNA molecule, composition, method, and use thereof, comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown by any of SEQ ID NOs: 1 to 159641.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 478,586, filed on January 5, 2023, and 63 / 368,566, filed on July 15, 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 disclosure of all publications mentioned in this application is incorporated herein by reference to supplement the technology that can be used in the present invention and the technology related to the present invention.

[0003] Reference to Sequence Listing This application was created on July 13, 2023, in the form of an IBM-PC machine using an operating system compatible with MS-Windows (registered trademark), and is included in a compact disc sent by express on July 14, 2023, as part of this application. The XML file with a size of 141,616,317 bytes, named "230714_91781-A-PCT_Sequence_Listing_AWG.xml", incorporates the nucleotide sequences by reference. Attachment A is a delivery note for the compact disc containing the sequence listing and was sent by express together with the compact disc.

Background Art

[0004] The safe harbor site of amyloid-β precursor-like protein 2 (APLP2) can be targeted to introduce a desired sequence into that site without causing harmful disruptions to the APLP2 gene or affecting its expression. Such a targeting strategy may be utilized to enable the expression mediated by the APLP2 promoter of the introduced sequence.

Summary of the Invention

[0005] The present disclosure also provides a method for modifying at least one allele of the amyloid-β precursor-like protein 2 (APLP2) gene in a cell, the method comprising At least one CRISPR nuclease or a polynucleotide molecule encoding a CRISPR nuclease; and An RNA molecule comprising a guide sequence portion of 17 to 50 nucleotides or a nucleotide sequence encoding the same comprising introducing a composition comprising the same into a cell, wherein the complex of the CRISPR nuclease and the RNA molecule double-strand breaks at least one allele of the APLP2 gene.

[0006] In some embodiments, the composition further comprises a donor molecule, and the nucleotide sequence of the donor molecule is inserted or copied at or near the double-strand break site. Similarly, in some embodiments, the composition further comprises a donor molecule containing a nucleotide sequence introduced at the double-strand break site such that expression of the introduced sequence is mediated by the promoter of the APLP2 gene. Accordingly, this specification also discloses strategies that enable the expression of a gene or a portion thereof under the control of the APLP2 promoter. In some embodiments, expression of a gene or a portion thereof under the control of the APLP2 promoter is possible without knocking out the expression of the APLP2 gene.

[0007] According to an aspect of the present invention, there is provided a first RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 159641. In some embodiments, the composition further comprises a CRISPR nuclease. In some embodiments, the composition further comprises a donor molecule.

[0008] In some aspects of the present invention, cells are provided that have been modified to express and secrete a protein of interest in a desired tissue of the body. Such cells can be modified by inserting the sequence of a gene of interest under the control of a promoter of a selected gene (e.g., APLP2) that contains a safe harbor site. Non-limiting examples of methods for modifying cells to express a gene of interest include knock-in by utilizing a CRISPR nuclease system that causes double-strand breaks and a donor molecule encoding the sequence of the gene of interest. In some embodiments, the donor molecule is an ssODN, dsDNA, plasmid, AAV, lentivirus, or transposon. Further, knock-in may be mediated by a composition comprising i) a fusion protein comprising a nickase and a reverse transcriptase, and ii) an RNA donor molecule.

[0009] According to an aspect of the present invention, cells modified by any of the methods described in the specification are provided. 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 and progenitor cells (HSPCs), bone marrow 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 in the specification to the 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 an individual patient. In some embodiments, the method further comprises introducing the cells modified or edited for the APLP2 allele into an individual patient (e.g., autologous transplantation).

[0011] In some embodiments of the present invention, provided is the use of a composition for modifying or editing the APLP2 allele intracellularly, the composition comprising an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 159641 and a CRISPR nuclease, said use comprising delivering the composition to the cell. In some embodiments, the composition further comprises a donor molecule.

[0012] According to an embodiment of the present invention, provided is a medicament for use in modifying or editing the APLP2 allele intracellularly, the medicament comprising an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 159641 and a CRISPR nuclease, said medicament being administered by delivering the composition to the cell. In some embodiments, the medicament further comprises a donor molecule.

[0013] In some embodiments of the present invention, provided is a kit for modifying or editing the APLP2 allele intracellularly, the kit comprising an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 159641, a CRISPR nuclease and / or a tracrRNA molecule; and instructions for delivering the RNA molecule; the CRISPR nuclease and / or the tracrRNA to the cell. In some embodiments, the kit further comprises a donor molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

FIG. 1A-1B

FIG. 2

FIG. 3A-3E

FIG. 4A-4B

FIG. 5A-5B

Best Mode for Carrying Out the Invention

[0015] Detailed Description Unless otherwise defined, all technical and / or scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described in this specification can be used in the practice or testing of embodiments of the present invention, but representative methods and / or materials are described below. In case of conflict, this specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0016] The term "one" is understood to refer to "one or more" of the recited components. The use of the singular form is clearly understood by those skilled in the art to include the plural form unless specifically stated otherwise. Thus, the terms "one" and "at least one" have the same meaning in this application.

[0017] For the purpose of better understanding this disclosure and without in any way limiting the scope of the disclosure, unless otherwise indicated, all numbers and other numerical values used in this specification and the claims to represent amounts, percentages, or ratios are understood to be modified in all instances by the term "about". Thus, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximate values 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 reported number of significant digits and by applying ordinary rounding techniques.

[0018] Unless otherwise indicated, adjectives, such as "substantially" and "about" that modify a state or relationship indicating a feature of an aspect of the present invention, are understood to mean that the state or property is defined within an acceptable range that is acceptable for the operation of the aspect for its intended application. Unless otherwise indicated, the term "or" in this specification and the claims is to be regarded as an inclusive "or" rather than an exclusive "or", indicating at least one or any combination of the items it connects.

[0019] In this specification and the claims, the verbs "comprise", "comprising", "include" and "including" and their conjugations are used to indicate that the object of the verb is not necessarily an exhaustive listing of the components, elements or parts 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 a cell, for example during the repair of double-strand and single-strand breaks in DNA. HDR requires nucleotide sequence homology and uses a "nucleic acid template" (in this specification, nucleic acid template and donor template have the same meaning) to repair the sequence in which the double-strand or single-strand break occurred (e.g., a DNA target sequence). Thereby, for example, genetic information is transferred from the nucleic acid template to the DNA target sequence. HDR may result in a change (e.g., insertion, deletion, mutation) in the DNA target sequence if the sequence of the nucleic acid template is different from that of the DNA target and part or all of the nucleic acid template polynucleotide or oligonucleotide is incorporated into the DNA target sequence. In some aspects, 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 nucleotide sequences that are inserted into or copied into the genome. A nucleic acid template can be added to a target nucleic acid, used as a template for changes in the target nucleic acid, or used for modification of the target sequence, and includes, for example, a nucleotide sequence of 1 nucleotide or more. The length of the nucleic acid template sequence can be arbitrary, for example, it can be 2 to 10,000 nucleotides in length. The nucleic acid template can be a single-stranded nucleic acid or a double-stranded nucleic acid. In some embodiments, the nucleic acid template includes, for example, a nucleotide sequence of 1 nucleotide or more corresponding to the wild-type sequence of the target nucleic acid at the target position. In some embodiments, the nucleic acid template includes a nucleotide sequence of, for example, 1 ribonucleotide or more corresponding to the wild-type sequence of the target nucleic acid at the target position. In some embodiments, the nucleic acid template includes modified nucleotides.

[0022] Insertion of an exogenous sequence (also referred to as "donor sequence", "donor template", "donor molecule" or "donor") can also be performed. For example, the donor sequence can include non-homologous sequences flanked by two homologous regions, enabling efficient homologous recombination repair (HDR) at the desired position. Further, the donor sequence can include a vector molecule having a sequence that is not homologous to the region of interest in the cell chromatin. The donor molecule can include several discontinuous regions that are homologous to the cell chromatin. For example, for insertion targeting a sequence that is not normally present in the region of interest, the sequence can be included in the donor nucleic acid molecule and regions homologous to the sequence of the region of interest can be adjacent thereto. The length of the donor molecule can be arbitrary, for example, it can be from several bases (e.g., 10 to 20 bases) to several kilobases.

[0023] The donor polynucleotide may be DNA or RNA, single-stranded and / or double-stranded, and can be introduced into cells in linear or circular form. See, for example, U.S. Patent Application Publication Nos. 2010 / 0047805; 2011 / 0281361; 2011 / 0207221 and 2019 / 0330620. See also Anzalone et al. (2019). When introduced in linear form, the ends of the donor sequence can be protected by methods known to those skilled in the art (e.g., from exonuclease degradation). For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule, and / or self-complementary oligonucleotides are ligated to one or both ends. See, for example, 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 endogenous sequence alteration by a target. The oligonucleotide may be introduced into cells by a vector, by electroporation into cells, or by other methods known in the art. The donor polynucleotide can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as liposomes or poloxamers, 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 the complex of the RNA molecule and the CRISPR nuclease makes a double-stranded break as a result of hybridization with the target sequence, i.e., on-target hybridization. The term "modified cell" may further include a cell in which editing or modification including the introduction of an exogenous sequence has been performed after the double-stranded break.

[0026] The present invention provides modified cells obtained by use of any of the methods described in this specification. 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, iPS-derived monocytes or iPS-derived macrophages.

[0027] The present invention also provides a composition comprising these modified cells and a pharmaceutically acceptable carrier. Also provided is an in vitro or ex vivo method of preparing this composition, which comprises mixing 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, the targeting sequence has a nucleotide sequence that is at least partially complementary to the target sequence of interest. The targeting sequence or targeting molecule may be part of an RNA molecule that can form a complex with a CRISPR nuclease, alone or in combination with other RNA molecules, and the targeting sequence serves as the targeting moiety 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), is capable of directing the CRISPR nuclease to a specific target sequence. By way of non-limiting example, the guide sequence portion of a CRISPR RNA molecule or a single guide RNA molecule may serve as a targeting molecule. Each possibility is an individual embodiment. The targeting sequence can be custom designed to target a desired sequence.

[0029] As used herein, the term "targeting" refers to preferentially hybridizing a targeting sequence of a targeting molecule to a nucleic acid having a target nucleotide sequence. It is understood that the term "targeting" preferentially targets a nucleic acid having a target nucleotide sequence, but includes various hybridization efficiencies, such as the possibility of unintended off-target hybridization in addition to on-target hybridization. When an RNA molecule targets a sequence, it is understood that a complex of the RNA molecule and a CRISPR nuclease molecule targets that sequence for nuclease activity.

[0030] The "guide sequence portion" of an RNA molecule refers to a nucleotide sequence capable of hybridizing with a specific target DNA sequence. For example, the guide sequence portion has a nucleotide sequence that is partially or completely complementary to the DNA sequence targeted along the guide sequence portion. In some embodiments, the length of the nucleotides in 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, or approximately 17 - 50, 17 - 49, 17 - 48, 17 - 47, 17 - 46, 17 - 45, 17 - 44, 17 - 43, 17 - 42, 17 - 41, 17 - 40, 17 - 39, 17 - 38, 17 - 37, 17 - 36, 17 - 35, 17 - 34, 17 - 33, 17 - 31, 17 - 30, 17 - 29, 17 - 28, 17 - 27, 17 - 26, 17 - 25, 17 - 24, 17 - 22, 17 - 21, 18 - 25, 18 - 24, 18 - 23, 18 - 22, 18 - 21, 19 - 25, 19 - 24, 19 - 23, 19 - 22, 19 - 21, 19 - 20, 20 - 22, 18 - 20, 20 - 21, 21 - 22 or 17 - 20. Preferably, the full length of the guide sequence portion is completely complementary to the DNA sequence targeted along the guide sequence portion. The guide sequence portion may be part of an RNA molecule capable of forming 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, either alone or in combination with one or more additional RNA molecules (e.g., tracrRNA molecule), the RNA molecule has the ability to target the CRISPR nuclease to a specific target DNA sequence. Thus, the CRISPR complex can be formed by direct binding of an RNA molecule having a guide sequence portion to a CRISPR nuclease, or by binding of an RNA molecule having a guide sequence portion and one or more additional RNA molecules to a CRISPR nuclease. Each possibility is an individual embodiment. The guide sequence portion can be custom-designed to target 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 the guide sequence portion described in this specification (e.g., the guide sequence shown in any of SEQ ID NOs: 1 to 159641), or a different sequence that is 1, 2, 3, 4, or 5 nucleotides or less. Each possibility is an individual embodiment. In some of these embodiments, the guide sequence portion comprises the same sequence as the sequence shown in any of SEQ ID NOs: 1 to 159641. Throughout this application, the terms "guide molecule", "RNA guide molecule", "guide RNA molecule", and "gRNA molecule" are synonymous with a molecule that comprises a guide sequence portion.

[0031] As used herein, the term "indiscriminate" refers to the guide sequence portion of an RNA molecule that targets a specific DNA sequence common to a pair of alleles of a gene. For example, an indiscriminate 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 the sequence shown in any of SEQ ID NOs: 1 to 159641. In some embodiments, the guide sequence portion comprises the same sequence as the sequence shown in any of SEQ ID NOs: 1 to 159641, or a different sequence that is 1, 2, or 3 nucleotides or less.

[0033] The RNA molecule and / or the guide sequence portion of the RNA molecule may have modified nucleotides. Representative modifications to nucleotides or polynucleotides may be synthetic and may include polynucleotides having nucleotides with bases other than the naturally occurring adenine, cytosine, thymine, uracil, or guanine bases. Modifications to polynucleotides may include synthetic, non-naturally occurring nucleosides, such as polynucleotides having locked nucleic acids. Modifications to polynucleotides may be utilized to increase or decrease the stability of the RNA. An example of a modified polynucleotide is mRNA having 1-methylpseudouridine. Examples of modified polynucleotides and their uses are described in U.S. Patent No. 8,278,036, International Publication No. 2015 / 006747, and Weissman and Kariko (2015), which are incorporated herein by reference.

[0034] As used herein, "consecutive nucleotides" indicated by a sequence number refers to the nucleotides of the sequence in the order indicated by the sequence number, without any intervening nucleotides.

[0035] In aspects of the invention, the guide sequence portion may be 25 nucleotides in length and may contain 20-22 consecutive nucleotides within the sequences shown in any of SEQ ID NOs: 1-159641. In aspects of the invention, the guide sequence portion may be less than 22 nucleotides in length. For example, in aspects of the invention, the guide sequence portion may be 17, 18, 19, 20, or 21 nucleotides in length. In such aspects, the guide sequence portion may consist of 17, 18, 19, 20, or 21 nucleotides, respectively, within the sequence of 17-22 consecutive nucleotides shown in any of SEQ ID NOs: 1-159641. For example, a guide sequence portion having the sequence of 17 consecutive nucleotides of the sequence shown in SEQ ID NO: 159642 may be any of the following nucleotide sequences (nucleotides removed from the consecutive sequence are struck through):

[0036]

Chemical formula

[0037] In an aspect of the present invention, the nucleotide length of the guide sequence portion may exceed 20. For example, in an aspect of the present invention, the nucleotide length of the guide sequence portion may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In such an aspect, the guide sequence portion contains 17 to 50 nucleotides containing a sequence of 20, 21, or 22 consecutive nucleotides shown in any of SEQ ID NOs: 1 to 159641, and nucleotides that are completely complementary to the nucleotides or their sequences adjacent to the 3'-end, 5'-end, or both of the target sequence.

[0038] In aspects of the 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 comprising 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) may be present in the same RNA molecule. Alternatively, the guide sequence portion may be present in one RNA molecule and the sequence portion involved in CRISPR nuclease binding (e.g., the tracrRNA portion) may be present in another 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 the CRISPR nuclease and serve as a DNA-targeting molecule. In some aspects, a first RNA molecule comprising a DNA-targeting RNA portion comprising the 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 fuse to form a complex with the CRISPR nuclease to construct an RNA molecule that targets the CRISPR nuclease to a DNA target site.

[0039] In aspects of the invention, an RNA molecule comprising a guide sequence portion may further comprise the sequence of a tracrRNA molecule. Such aspects may be designed as synthetic fusions of the guide portion of the RNA molecule and trans-activating crRNA (tracrRNA) (see Jinek et al., 2012). In such aspects, the RNA molecule is a single guide RNA (sgRNA) molecule. Some aspects of the invention may also form a CRISPR complex that utilizes an individual tracrRNA molecule and an individual RNA molecule comprising a guide sequence portion. In such aspects, the tracrRNA may hybridize to 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 that is sufficiently complementary to a tracrRNA molecule such that it hybridizes to the tracrRNA via base pairing and promotes the formation of a CRISPR complex (see U.S. Patent No. 8,906,616). In aspects of the invention, the RNA molecule may further comprise a portion having a tracr mate sequence.

[0041] In the present invention, a “gene” includes a DNA region encoding a gene product and all DNA regions that control the production of the gene product, and the sequence of the control region may or may not be adjacent to the coding sequence and / or the transcription sequence. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translational 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 (e.g., 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. The nuclease may be isolated from a natural product or may be derived from a natural product. The natural product may be a living organism. Alternatively, the nuclease may be a modified or synthetic protein that retains phosphodiester bond cleavage activity. Gene modification can be achieved using nucleases, such as CRISPR nucleases.

[0044] According to an aspect of the present invention, there is provided an RNA molecule comprising a guide sequence portion (e.g., a targeting sequence) that is fully or partially complementary to a target located within or near an allele of the APLP2 gene. In some aspects, the guide sequence portion of the RNA molecule consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 nucleotides, or more than 26 nucleotides. In some aspects, the guide sequence portion is configured to direct a CRISPR nuclease to the APLP2 target site and induce a double-strand break or a single-strand break within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides from the APLP2 target site. In some aspects, the RNA molecule is a guide RNA molecule, such as a crRNA molecule or a single-guide RNA molecule. In some aspects, the guide sequence portion is complementary to a target sequence located 30 base pairs upstream to 30 base pairs downstream of an intron 1 or exon 17 of the APLP2 gene. In some aspects, the guide sequence portion is complementary to a target sequence located 50 base pairs upstream to 50 base pairs downstream of an intron 1 or exon 17 of the APLP2 gene. Each possibility is an individual aspect. In some aspects, the guide sequence portion is complementary to a target sequence located 7 base pairs upstream to 7 base pairs downstream of an intron 1 or exon 17 of the APLP2 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 bone marrow cells, bone marrow progenitor cells, pluripotent progenitor cells, and lineage-restricted progenitor cells.

[0046] In this specification, a "progenitor cell" refers to a lineage cell derived from a stem cell and retaining mitotic ability and pluripotency (e.g., capable of differentiating or developing into a plurality of, but not all, types of a cell maturation lineage). In this specification, "hematopoiesis" refers to the formation and development in the body (e.g., bone marrow) of various blood cells (e.g., red blood cells, megakaryocytes, myeloid cells (e.g., monocytes, macrophages, and neutrophils) and lymphocytes) and other formed elements.

[0047] In some embodiments of the present invention, a method of modifying an allele of the amyloid β precursor-like protein 2 (APLP2) gene in a cell is provided, the method comprising at least one CRISPR nuclease or a polynucleotide molecule encoding a CRISPR nuclease; and an RNA molecule comprising a guide sequence portion of 17 to 50 nucleotides or a nucleotide molecule encoding the same introducing a composition comprising the same into the cell, wherein the complex of the CRISPR nuclease and the RNA molecule double-strand breaks the allele of the APLP2 gene.

[0048] In some embodiments, the composition also includes a donor molecule. In some embodiments, the nucleotide sequence of the donor molecule is inserted or copied at or near the double-strand break site. In some embodiments, the composition further includes a donor molecule comprising a nucleotide sequence to be introduced at the double-strand break site.

[0049] In some embodiments, the composition further includes a donor molecule comprising a nucleotide sequence to be introduced at the double-strand break site such that the expression of the introduced sequence is mediated by the promoter of the APLP2 gene.

[0050] In some embodiments, the introduced array comprises sequences derived from genes encoding α1 - antitrypsin, glucose - 6 - phosphatase (G6PC), serpin family A member (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthetase, arginase, argininosuccinase, carbamoyl phosphate synthetase and N - acetylglutamate synthetase, α - galactosidase A, coagulation factor IX, coagulation factor VII, lysosomal α - glucosidase, fibrinogen, phenylalanine 4 - hydroxylase, alkaline phosphatase, glucosylceramidase, β - galactosidase, porphobilinogen deaminase, arylsulfatase B, β - glucuronidase, α - N - acetylglucosaminidase, lysosomal α, α - L - iduronidase, mannosidase, phosphatidylcholine - cholesterol 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 lysosomal protective protein.

[0051] In some embodiments, the introduced array comprises sequences derived from genes encoding acid alpha-glucosidase, alpha-L-iduronidase, alpha-galactosidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, lysophosphatidylcholine metabolism-related protein, preferably phospholipase A2, T-REC or K-REC-related protein, beta-glucosidase, beta-glucocerebrosidase, arylsulfatase A, factor VIII, insulin-like growth factor 1 (IGF-1), surfactant protein A, surfactant protein B, aspartyl-beta-glucosaminidase, acetyl CoA alpha-glucosaminide, acetyl CoA-arylamine N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-1-phosphotransferase, alpha-N-acetylglucosaminidase, acid ceramidase, aspartoacylase, lysosomal acid lipase, acid sphingomyelinase, arylsulfatase B, alpha-L-fucosidase, galactosylceramidase, galactocerebrosidase, beta-galactosidase, protective protein / cathepsin A, beta-glucuronidase, heparan N-sulfatase, beta-hexosaminidase A, hyaluronidase-1, alpha-D-mannosidase, beta-mannosidase, alpha-neuraminidase, beta-hexosaminidase A, beta-hexosaminidase B, palmitoylated protein thioesterase, tripeptidyl peptidase I, battenin, ceroid lipofuscinosis neuronal protein 5 (CLN5), ceroid lipofuscinosis neuronal protein 6 (CLN6), ceroid lipofuscinosis neuronal protein 7 (CLN7), ceroid lipofuscinosis neuronal protein 8 (CLN8), (cathepsin D), cystinosin, cathepsin K, cystatin, lysosome-associated membrane protein 2 (LAMP2), human growth hormone, follicle-stimulating hormone, erythropoietin, cytokine, chemokine, IL-10, IGF1, TGFβ, IL-15, CXCR4, IL-4 or granulocyte colony-stimulating factor (G-CSF).

[0052] In some embodiments, the donor molecule comprises a sequence derived from a gene encoding a protein secreted by the cell.

[0053] In some embodiments, the introduced sequence comprises a sequence encoding a polypeptide of interest expressed by the cell.

[0054] In some embodiments, the polypeptide of interest expressed is secreted by the cell.

[0055] In some embodiments, the polypeptide of interest expressed further comprises a signal peptide.

[0056] In some embodiments, the signal peptide is encoded by an allele of the APLP2 gene.

[0057] In some embodiments, the introduced sequence comprises a sequence encoding a 2A self-cleaving peptide.

[0058] In some embodiments, the introduced sequence comprises a sequence encoding a signal peptide.

[0059] In some embodiments, the signal peptide is the APLP2 signal peptide.

[0060] In some embodiments, the introduced sequence comprises a splice acceptor sequence and a splice donor sequence.

[0061] In some embodiments, the introduced sequence 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.

[0062] In some embodiments, the donor molecule comprises a first homology arm sequence having at least 90%, preferably 100%, sequence identity with the APLP2 sequence upstream of the double-strand break, and a second homology arm sequence having at least 90%, preferably 100%, sequence identity with the APLP2 sequence downstream of the double-strand break.

[0063] In some embodiments, the lengths of the first homology arm sequence and the second homology arm sequence are each about 20-50, 50-100, 100-200, 200-500, 500-1000 or 1000-2000 nucleotides, respectively.

[0064] In some embodiments, the polypeptide of interest is a soluble protein.

[0065] In some embodiments, the length of the polypeptide of interest is about 20-50, 50-100, 100-200, 200-500, 500-1000 or 1000-2000 amino acids.

[0066] In some embodiments, the RNA molecule comprises a guide sequence portion having 17-50 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1-159641.

[0067] In some embodiments, the RNA molecule comprises an undifferentiated guide sequence portion that targets a pair of APLP2 alleles.

[0068] In some embodiments, the RNA molecule comprises an undifferentiated guide portion that targets either an intron 1 of APLP2 or an undifferentiated guide portion that targets the 3' untranslated region (3'UTR) of APLP2.

[0069] In some embodiments, the RNA molecule comprises an undifferentiated guide portion that targets a sequence within a genomic range selected from either 11:130142110-130144147 or 11:130070140-130109426.

[0070] In some embodiments, the modified allele of the APLP2 gene expresses the APLP2 gene product.

[0071] In some embodiments, the modified allele of the APLP2 gene expresses the APLP2 polypeptide and the polypeptide of interest.

[0072] In some embodiments, the cell is a stem cell, monocyte, macrophage, iPS-derived monocyte, iPS-derived macrophage, hematopoietic stem cell (HSC), hematopoietic stem and progenitor cell (HSPC), bone marrow progenitor cell, myeloblast, lymphoblast, erythroid progenitor cell, platelet cell, natural killer (NK) cell, B lymphocyte, T lymphocyte, eosinophil, neutrophil, iPS-derived cell, or basophil.

[0073] In some embodiments, the cell is a stem cell, and the method further includes differentiating the stem cell after modification of the stem cell.

[0074] According to an embodiment of the present invention, a modified cell obtained by the method described in the specification is provided.

[0075] In some embodiments, the cell is a stem cell, monocyte, macrophage, iPS-derived monocyte, iPS-derived macrophage, hematopoietic stem cell (HSC), hematopoietic stem and progenitor cell (HSPC), bone marrow progenitor cell, myeloblast, lymphoblast, erythroid progenitor cell, platelet cell, natural killer (NK) cell, B lymphocyte, T lymphocyte, eosinophil, neutrophil, iPS-derived cell, or basophil.

[0076] According to 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 of SEQ ID NOs: 1 to 159641.

[0077] According to an embodiment of the present invention, a composition is provided that includes the RNA molecule and at least one CRISPR nuclease.

[0078] In some embodiments, the composition further includes a donor molecule.

[0079] In some embodiments, the donor molecule comprises a sequence derived from a gene encoding α1-antitrypsin, glucose-6-phosphatase (G6PC), serpin family A member (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthetase, arginase, argininosuccinase, carbamoyl phosphate synthetase, and N-acetylglutamate synthetase, α-galactosidase A, coagulation factor IX, coagulation factor VII, lysosomal α-glucosidase, fibrinogen, phenylalanine 4-hydroxylase, alkaline phosphatase, glucosylceramidase, β-galactosidase, porphobilinogen deaminase, arylsulfatase B, β-glucuronidase, α-N-acetylglucosaminidase, lysosomal α, α-L-iduronidase, mannosidase, phosphatidylcholine cholesterol acyltransferase, N-sulfo-glucosamine 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 lysosomal protective protein.

[0080] In some embodiments, the donor molecule comprises a sequence derived from a gene encoding acidic α-glucosidase, α-L-iduronidase, α-galactosidase, iduronic acid-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, acidic 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, battenin, ceroid lipofuscinosis neuronal protein 5 (CLN5), ceroid lipofuscinosis neuronal protein 6 (CLN6), ceroid lipofuscinosis neuronal protein 7 (CLN7), ceroid lipofuscinosis neuronal protein 8 (CLN8), (cathepsin D), cystinosin, cathepsin K, cystatin, lysosome-associated membrane protein 2 (LAMP2), human growth hormone, follicle-stimulating hormone, erythropoietin, cytokine, chemokine, IL-10, IGF1, TGFβ, IL-15, CXCR4, IL-4 or granulocyte colony-stimulating factor (G-CSF).

[0081] In some embodiments, the donor molecule comprises a sequence derived from a gene encoding a protein secreted by a cell.

[0082] In some embodiments, the donor molecule comprises a sequence encoding a polypeptide of interest. A nucleotide sequence encoding a polypeptide that is desired to be expressed and secreted by the target cell may be inserted into the APLP2 allele such that the modified APLP2 allele can express both the polypeptide encoded by the inserted sequence and the original APLP2 gene product.

[0083] In some embodiments, the donor molecule comprises a sequence encoding a 2A self-cleaving peptide.

[0084] In some embodiments, the donor molecule comprises a sequence encoding a signal peptide.

[0085] In some embodiments, the signal peptide is the APLP2 signal peptide.

[0086] In some embodiments, the donor molecule comprises a splice acceptor sequence and a splice donor sequence.

[0087] 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.

[0088] 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 APLP2 gene, and a second homology arm sequence having at least 90%, preferably 100% sequence identity with a second sequence of the APLP2 gene.

[0089] In some embodiments, the lengths of the first homology arm sequence and the second homology arm sequence are each about 20-50, 50-100, 100-200, 200-500, 500-1000 or 1000-2000 nucleotides, respectively.

[0090] In some embodiments, the polypeptide of interest is a soluble protein.

[0091] In some embodiments, 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.

[0092] In some embodiments, the composition further comprises a tracrRNA molecule. According to an aspect of the invention, there is provided a method for modifying or editing the APLP2 allele in a cell, the method comprising delivering the composition as described in the specification to the cell.

[0093] According to an aspect of the invention, there is provided the use of the composition as described in the specification for modifying or editing the APLP2 allele in a cell, the use comprising delivering the composition as described in the specification to the cell.

[0094] According to an aspect of the invention, there is provided a medicament for use in modifying or editing the APLP2 allele in a cell, comprising the composition as described in the specification, the medicament being administered by delivering the composition as described in the specification to the cell.

[0095] According to an aspect of the invention, there is provided a kit for modifying or editing the APLP2 allele in a cell, the kit comprising the RNA molecule, CRISPR nuclease and / or tracrRNA molecule as described in the specification; and instructions for delivering the RNA molecule; CRISPR nuclease and / or tracrRNA to the cell. In some embodiments, the kit further comprises a donor molecule and instructions for delivering the donor molecule to the cell.

[0096] According to an aspect of the present invention, there is provided a gene editing composition comprising an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 159641. In some aspects, the RNA molecule further comprises a portion having a sequence that binds to a CRISPR nuclease. In some aspects, the sequence that binds to the CRISPR nuclease is a tracrRNA sequence. In some aspects, the RNA comprising the guide sequence portion is a crRNA molecule. In some aspects, the RNA molecule comprising the guide sequence portion is a single guide RNA (sgRNA) molecule.

[0097] In some aspects, the RNA molecule further comprises a portion having a tracr mate sequence.

[0098] In some aspects, the RNA molecule may further comprise one or more linker portions.

[0099] According to an aspect of the present 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 an individual aspect. In an aspect of the present invention, the length of the RNA molecule is 17 to at most 300 nucleotides, 100 to at most 300 nucleotides, 150 to at most 300 nucleotides, 100 to at most 500 nucleotides, 100 to at most 400 nucleotides, 200 to at most 300 nucleotides, 100 to 200 nucleotides or 150 to at most 250 nucleotides. Each possibility is an individual aspect.

[0100] In some aspects of the present invention, the composition further comprises a tracrRNA molecule.

[0101] In some aspects of the present invention, a method for modifying or editing the APLP2 allele within a cell is provided, the method comprising delivering to the cell a composition comprising an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 159641 and a CRISPR nuclease. In some aspects, the composition further comprises a donor molecule.

[0102] In some aspects of the present invention, a method for treating a disorder or disease is provided, the method comprising delivering to the cells of a subject having the disorder or disease a composition comprising an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 159641 and a CRISPR nuclease. In some aspects, the composition further comprises a donor molecule.

[0103] In some aspects of the present invention, a method for treating a disorder or disease is provided, the method comprising delivering to the cells of a subject having the disorder the composition of the above aspect, or delivering to the subject the modified cells of the above aspect.

[0104] In some embodiments, the disease or disorder is Pompe disease, mucopolysaccharidosis type I, Fabry disease, mucopolysaccharidosis type II, mucopolysaccharidosis IVA, mucopolysaccharidosis VI, adrenoleukodystrophy, severe combined immunodeficiency disease, 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, I-cell 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 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-Hartiala disease, Jansky-Bielschowsky 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, pycnodysostosis, Salla disease, Danon disease and / or α1-antitrypsin deficiency.

[0105] In some embodiments, the composition or modified cell is delivered to the tissue or tumor of a subject.

[0106] In some embodiments of the invention, there is provided a medicament for use in modifying the APLP2 allele intracellularly, comprising the composition of the above embodiments, wherein the medicament is administered by delivering the composition of the above embodiments to the cell.

[0107] In some aspects of the present invention, there is provided the use of the composition of the above aspect or the modified cell of the above aspect for the treatment, amelioration or prevention of a disorder or disease, said use comprising delivering the composition of the above aspect to the cells of a subject having or at risk of having a disorder, or delivering the modified cell of the above aspect to the subject.

[0108] In some aspects of the present invention, there is provided a medicament for use in treating, ameliorating or preventing a disorder or disease, comprising the composition of the above aspect or the modified cell of the above aspect, said medicament being administered by delivering the composition of the above aspect to the cells of a subject having or at risk of having a disorder, or delivering the modified cell of the above aspect to the subject.

[0109] In some aspects, the disorder or disease is Pompe disease, mucopolysaccharidosis type I, Fabry disease, mucopolysaccharidosis type II, mucopolysaccharidosis IVA, mucopolysaccharidosis 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, I-cell 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 IVB (MPS IVB) or galactosialidosis, Sly disease, mucopolysaccharidosis type III, late-onset Tay-Sachs disease, hyaluronidase 1 deficiency, α-mannosidosis, β-mannosidosis, sialidosis, Stanworth disease, Santavuori-Hartiala disease, Jansky-Bielschowsky 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, pycnodysostosis, Salla disease, Danon disease and / or α1-antitrypsin deficiency.

[0110] In some embodiments, the disorder or disease is a lysosomal storage disorder.

[0111] In some embodiments, the disorder or disease is a disorder or disease of the blood, lung, brain, liver, intestine, intestinal tract, bone, muscle or central nervous system or an inflammatory or autoinflammatory disease.

[0112] In some embodiments, the composition or modified cell of the above embodiment is a medicament for enzyme replacement therapy.

[0113] In some embodiments of the present invention, a method for treating a disease or disorder is provided, the method being an immunotherapy comprising delivering the modified cell of the above embodiment to a subject.

[0114] In some embodiments, the disease or disorder is cancer.

[0115] In some embodiments, the composition or modified cell of the above embodiment is used to treat, ameliorate or prevent a disorder or disease.

[0116] In some embodiments of the present invention, a method for modifying a DNA target site of a subject's monocytes or macrophages is provided, the method comprising inducing expression of a desired protein encoded by the modification in monocytes or macrophages of the DNA target site, the method comprising delivering to the subject's cells a composition comprising an RNA molecule having a guide sequence portion of 17 to 50 contiguous nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 159641 and a CRISPR nuclease. In some embodiments, the composition further comprises a donor molecule.

[0117] According to an embodiment of the present invention, at least one CRISPR nuclease and one or more RNA molecules are delivered to a subject and / or cell substantially simultaneously or at different times.

[0118] In some embodiments, the tracrRNA molecule is delivered to the subject and / or cell substantially simultaneously with, or at a different time point than, the CRISPR nuclease and one or more RNA molecules.

[0119] According to an embodiment of the invention, the RNA molecule targets a selective splicing signal sequence between an exon and an intron of the APLP2 allele.

[0120] According to an embodiment of the invention, the RNA molecule is non-discriminatory and targets sequences present in a pair of APLP2 alleles. In some embodiments, the sequences are present in a pair of APLP2 alleles. In some embodiments, the sequences are present in an intron of the APLP2 gene. In some embodiments, the intron is intron 1 following coding exon 1 of the APLP2 gene.

[0121] Any or a combination of the above strategies for modifying or editing the APLP2 allele may be used in connection with the present invention.

[0122] In some embodiments, the method comprises contacting at least one allele of a target gene with a non-discriminatory 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., Cas9 protein), wherein the non-discriminatory RNA molecule and the CRISPR nuclease bind to the nucleotide sequence of at least one allele of the target gene to modify or edit at least one allele. In particular, introduction of the non-discriminatory RNA molecule into the cell may result in cleavage of both alleles, but insertion of the nucleotide sequence at the cleavage site may occur in only one allele, not both alleles. Thus, by inducing cleavage of both alleles with a non-discriminatory RNA molecule targeting an intron of the APLP2 gene, expression of the endogenous APLP2 gene from one allele is maintained and a nucleotide sequence (e.g., a nucleotide sequence derived from a donor molecule) may be introduced into the other allele. Introduction of the nucleotide sequence into the APLP2 allele may or may not disrupt the expression of the gene product encoded by APLP2.

[0123] In some embodiments, the method comprises contacting an allele of a target gene with an RNA molecule and a CRISPR nuclease (e.g., Cas9 protein), wherein the RNA molecule and the CRISPR nuclease bind to a nucleotide sequence of the allele of the target gene that is at least one nucleotide different from the nucleotide sequence of the other allele of the target gene to modify or edit the target allele.

[0124] In some embodiments, the RNA molecule and the CRISPR nuclease are introduced into a cell encoding the target gene. In some embodiments, the cell encoding the target gene is a cell of a mammalian subject.

[0125] Embodiments of the compositions described herein include at least one CRISPR nuclease, an RNA molecule comprising a guide sequence portion, and a tracrRNA molecule, wherein the tracrRNA molecule may be separate from or linked to the RNA molecule comprising the guide sequence portion and is effective simultaneously in a subject or cell. The at least one CRISPR nuclease, the RNA molecule comprising the guide sequence portion, and the tracrRNA may be delivered substantially simultaneously, or may be delivered at different times but exert their effects simultaneously. For example, this includes delivering the CRISPR nuclease to a subject or cell before the RNA molecule comprising the guide sequence portion and / or the tracrRNA is substantially present in the subject or cell.

[0126] 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 and 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.

[0127] Genetic APLP2 Safe Harbor Knock-In for Treating Diseases and Disorders In some embodiments, the method of the present invention may be used to knock in a sequence into the safe harbor site of APLP2. In some embodiments, the expression mediated by APLP2 of the knocked-in sequence is involved in or related to the treatment of a disorder or disease.

[0128] For example, in some embodiments, the APLP2 DNA target site of a target cell (e.g., a monocyte, macrophage, hematopoietic stem cell (HSC), hematopoietic stem and progenitor cell (HSPC), bone marrow progenitor cell, myeloblast, lymphocyte, 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 the protein product encoded by the modification (e.g., an introduced or knocked-in protein coding sequence). These target cells may be used, for example, in the treatment of lysosomal storage diseases, or other disorders of the blood, lung, brain, liver, intestine, gut, bone, muscle or central nervous system, or inflammatory or autoinflammatory diseases. In some embodiments, these modified cells serve as an alternative to conventional enzyme replacement therapy. In some embodiments, these modified cells are used in immunotherapy such as cancer immunotherapy.

[0129] By way of non-limiting example, the expression of the knocked-in sequence may be involved in or related to the treatment of a disease or disorder of the blood, lung, brain, intestine, gut, bone, liver, muscle or central nervous system. By way of non-limiting example, the knocked-in sequence may be the sequence of A1AT, G6PC, SERPINA, TTR, ornithine transcarbamylase, argininosuccinate synthetase, arginase, argininosuccinase, carbamoyl phosphate synthetase or N-acetylglutamate synthetase or a portion thereof.

[0130] By way of non-limiting example, the expression of the knocked-in array may be involved in or associated with the treatment of inflammatory or autoinflammatory diseases or disorders such as inflammatory bowel disease (IBD). For example, insertion of IL-4 into nerve cells reduces the symptoms of multiple sclerosis mice. Furthermore, insertion of IL-10 into type 1 diabetes model mice reduces insulitis and decreases T cell activation. By way of non-limiting example, the knocked-in array 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, the secreted knocked-in cytokine or chemokine facilitates the manipulation of immune and inflammatory responses for the treatment of the disease or disorder.

[0131] By way of non-limiting example, the expression of the knocked-in array may be involved in or associated with the treatment of cancer. By way of non-limiting example, the knocked-in array may be a cytokine, chemokine, factor or protein that can activate the immune system or assist in mobilizing immune system cells to the tumor site to remove the tumor. For example, IL-15 may be knocked in and secreted to enhance the persistence of T cells or natural killer cells, and CXCR4 may be knocked in to increase the mobilization of T cells and natural killer cells to the tumor site.

[0132] By way of non-limiting example, target cells (e.g., monocytes or macrophages) may be modified to express cytokines or chemokines including but not limited to IL-10, IGF1, TGF-β, IL-15, CXCR4 and / or IL-4.

[0133] By way of non-limiting example, the expression of the knocked-in array may be involved in or related to the treatment of lysosomal storage diseases or other disorders. By way of non-limiting example, target cells (e.g., monocytes or macrophages) may be modified to express alpha-galactosidase A, coagulation factor IX, coagulation 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, phosphatidylcholine sterol acyltransferase, N-sulfo-glucosamine sulfohydrolase, coagulation factor X, N-acetylgalactosamine-6-sulfatase, sphingomyelin phosphodiesterase, alpha-1 antitrypsin, iduronic acid-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.

[0134] By way of non-limiting example, the expression of the knocked-in array is for the following diseases or disorders (each with the related gene or enzyme noted in parentheses): Pompe disease (acid α-glucosidase), mucopolysaccharidosis type I (α-L-iduronidase), Fabry disease (α-galactosidase), mucopolysaccharidosis type II (iduronate-2-sulfatase), mucopolysaccharidosis IVA (N-acetylgalactosamine-6-sulfatase), mucopolysaccharidosis VI (N-acetylgalactosamine-4-sulfatase), adrenoleukodystrophy (genes related to phospholipid metabolism, 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), I-cell disease (N-acetylglucosamine-1-phosphotransferase), Schindler disease (α-N-acetylglucosaminidase), 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 (MPSIVB) 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), Stanworth disease (β-hexosaminidase A and / or β-hexosaminidase B), Santavuori-Haltia disease (palmitoylated protein thioesterase), Jansky-Bielschowsky 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 cathepsin D deficiency (cathepsin D), cystinosis (cystinosin), pycnodysostosis (cathepsin K), salla disease (sialin) and / or Danon disease (lysosome-associated membrane protein 2 (LAMP2)), or may be involved in the treatment of any of these. By way of non-limiting example, target cells (e.g., monocytes or macrophages) may be modified to express a metabolic modulator. By way of non-limiting example, target cells may be modified 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-hexosaminidase.

[0135] In particular, monocytes and macrophages are present in target tissues including the lung and brain and can serve as expression vectors for long-term secretion of proteins in these target tissues. Expression of the transgene under the control of the APLP2 promoter is achieved by CRISPR-mediated knock-in at the safe harbor site targeted by the guide sequence portion described in the specification. The protein expressed by the transgene may be secreted. Thus, the ability to prepare blood and / or tissue containing modified target cells and continuously secrete the protein of interest therefrom serves as an alternative to enzyme replacement therapy (ERT). For example, modified monocytes may target the central nervous system (CNS) or the lung and may be useful for secreting the protein of interest in the target tissue.

[0136] Such an approach is also useful, for example, in the treatment of lysosomal storage diseases. Some of these diseases also exhibit phenotypes in the CNS. In particular, due to the blood-brain barrier (BBB), it is difficult to treat brain damage with enzyme replacement therapy. Monocytes may be utilized for delivery across the BBB such that the secreted protein of interest is secreted in the CNS.

[0137] For example, lysosomal storage diseases may be treated using modified monocytes or macrophages delivered to the brain; antitrypsin deficiency (A1AT), D-surfactant deficiency, or protein deposition diseases may be treated using modified monocytes or macrophages delivered to the lung; or A1AT or adenosine deaminase 2 (DADA2) deficiency may be treated using modified monocytes or macrophages in the blood.

[0138] APLP2 editing strategies may, in some cases, include strategies that enable the expression of a desired sequence under the control of the APLP2 promoter without knocking out the edited APLP2 allele. This can be achieved by strategy (1) knock-in into intron 1 of the APLP2 allele or intron 1 following coding exon 1 of the APLP2 allele, or (2) knock-in by replacement of the stop codon of the APLP2 allele.

[0139] In the case of the first strategy (i.e., mediating cleavage of both alleles using an RNA molecule containing a guide sequence portion targeting intron 1 of the APLP2 gene), since cleavage is mediated within a non-regulatory region, it is expected not to affect the expression of the APLP2 gene. When knocking in a desired sequence without knocking out the endogenous APLP2 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), such that the desired sequence is inserted 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. Furthermore, to not interfere with the expression of the endogenous APLP2 gene, the cassette contains a self-cleaving peptide (e.g., 2A self-cleaving peptides such as P2A, F2A, E2A, T2A or combinations thereof) and / or the signal peptide of APLP2 at the C-terminus of the inserted gene, which is cleaved within the endoplasmic reticulum to enable separation of the APLP2 gene product and the inserted gene product.

[0140] In the case of the second strategy (i.e., knock-in by replacing the stop codon of APLP2), cleavage of both alleles 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 cleavage position is expected not to affect the expression of the APLP2 gene. The desired sequence to be knocked in 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, enabling separation of the inserted gene product from the APLP2 gene product.

[0141] An APLP2 editing strategy may be utilized to edit T cells for use in chimeric antigen receptor T cell (CAR-T) therapy. For example, a target sequence (e.g., CD19 sequence) encoded by a donor molecule may be introduced into a safe harbor site of APLP2 targeted by an RNA molecule containing the guide sequence portion described in the specification. Such editing may be utilized to alter T cell receptor expression and / or receptor signal transduction by the cell.

[0142] 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 further does not include a tracrRNA. In non-limiting examples where the RNA-guided DNA nuclease is a CRISPR protein, at least one nucleotide that differs between APLP2 alleles may be present within and / or in the vicinity of the PAM site within a region designed such that the RNA molecule hybridizes. Those skilled in the art will understand that RNA molecules can be manipulated in methods generally known in the art to bind to selected targets in the genome.

[0143] In this specification, the term "PAM" refers to the nucleotide sequence of the target DNA that is located near the target DNA sequence and is recognized by the CRISPR nuclease complex. The PAM sequence may vary depending on the type of nuclease. In addition, there are CRISPR nucleases that can target almost all PAMs. In some embodiments of the present invention, the CRISPR system forms Watson-Crick base pairs between the guide sequence portion and the protospacer of the target DNA site adjacent to the protospacer adjacent motif (PAM), which is an additional requirement for target recognition, and utilizes one or more RNA molecules having a guide sequence portion that directs the CRISPR nuclease to the target DNA site. Subsequently, the CRISPR nuclease cleaves the target DNA site, creating a double-strand break within the protospacer. In a non-limiting example, the type II CRISPR system utilizes a mature crRNA:tracrRNA complex that forms Watson-Crick base pairs between the guide sequence portion of the crRNA and the protospacer on the target DNA adjacent to the protospacer adjacent motif (PAM) to direct the CRISPR nuclease (e.g., Cas9) to the target DNA. Those skilled in the art will understand that the engineered RNA molecules of the present invention are further designed to bind to the target genomic DNA sequence of interest adjacent to a protospacer adjacent motif (PAM), e.g., a PAM corresponding to the sequence associated with the CRISPR nuclease utilized.The PAM is, 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 variant; NGCG for SpCas9-VRER variant; NGAG for SpCas9-EQR variant; NRRH (where N is any nucleobase, R is A or G, and H is A, C, or T) for SpCas9-NRRH variant; NRTH (where N is any nucleobase, R is A or G, and H is A, C, or T) for SpCas9-NRTH variant; NRCH (where N is any nucleobase, R is A or G, and H is A, C, or T) for SpCas9-NRCH variant; NG (where N is any nucleobase) for SpG variant of SpCas9; NG or NA (where N is any nucleobase) for SpCas9-NG variant of SpCas9; NR or NRN or NYN (where N is any nucleobase, R is A or G, and Y is C or T) for SpRY variant of SpCas9; NNG (where N is any nucleobase) for Streptococcus canis Cas9 variant (ScCas9); NNNRRT (where N is any nucleobase and R is A or G) for SaKKH-Cas9 variant (SaCas9) of Staphylococcus aureus; NNNNGATT (where N is any nucleobase) for Neisseria meningitidis (NmCas9); TTN (where N is any nucleobase) for Alicyclobacillus acidiphilus Cas12b (AacCas12b); or TTTV (where V is A, C, or G) for Cpfl. The RNA molecules of the present invention are each designed to form a complex with one or more different CRISPR nucleases and are designed to target a polynucleotide sequence of interest using one or more different PAM sequences corresponding to the CRISPR nucleases.

[0144] In some embodiments, an RNA-guided DNA nuclease (e.g., a CRISPR nuclease) may be used to introduce a double-stranded or single-stranded DNA break at a desired location in the genome of a cell. The most commonly used RNA-guided DNA nucleases are derived from the CRISPR system, although other RNA-guided DNA nucleases are also contemplated for use in the genome editing compositions and methods described herein. See, for example, U.S. Patent Application Publication No. 2015 / 0211023, which is incorporated herein by reference.

[0145] The CRISPR systems that can be used in the practice of the present invention are diverse. 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, Casl0, Casl 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.

[0146] In some embodiments, the RNA-guided DNA nuclease is a CRISPR nuclease (e.g., Cas9) derived from a type II CRISPR system. CRISPR nucleases are from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Neisseria meningitidis, Treponema denticola, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp.) Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.) may also be derived from Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, or any species encoding a CRISPR nuclease having a known PAM sequence. CRISPR nucleases encoded by unculturable bacteria may also be used in the present invention. (See Burstein et al. Nature, 2017). Variants of CRISPR proteins having a known PAM sequence, for example, the SpCas9 D1135E variant, the SpCas9 VQR variant, the SpCas9 EQR variant, or the SpCas9 VRER variant, may also be used in the present invention.

[0147] Therefore, an RNA-guided DNA nuclease of the CRISPR system, such as a Cas9 protein or a modified Cas9, or a homolog or ortholog of Cas9, or another RNA-guided DNA nuclease belonging to another CRISPR system, such as Cpf1 and its homologs and orthologs, may be used in the compositions of the present invention. Other CRISPR nucleases, for example, the nucleases described in International Publication Nos. 2020 / 223514 and 2020 / 223553 (incorporated herein by reference), may also be used.

[0148] In certain embodiments, the CRISPR nuclease may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound having biological properties substantially common to the native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of the native sequence and derivatives of the native sequence polypeptide and fragments thereof that share biological activity with the corresponding native sequence polypeptide. The biological activity contemplated herein 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 both its covalent modifications and fusions. Suitable derivatives of the Cas polypeptide or fragment thereof include, but are not limited to, variants, fusions, and covalent modifications of the Cas protein or fragment thereof. Derivatives include CRISPR nickases, catalytically inactive CRISPR nucleases or "dead" CRISPR nucleases, and fusions of the CRISPR nuclease or derivative thereof with other enzymes such as base editors or retrotransposons. See, e.g., Anzalone et al. (2019) and PCT International Patent Application No. PCT / US2020 / 037560.

[0149] The Cas protein, including the Cas protein or fragment thereof, and in addition derivatives of the Cas protein or fragment thereof, may be obtainable from cells, or may be chemically synthesized, or may be obtained by a combination of these methods. The cells may be cells that naturally produce the Cas protein, or may be genetically engineered cells that naturally produce the Cas protein and produce the endogenous Cas protein at a higher expression level, or produce the Cas protein from an introduced exogenous nucleic acid encoding the same or a different Cas as the endogenous Cas. In some cases, the cells are genetically engineered to produce the Cas protein when they do not naturally produce it.

[0150] In some embodiments, the CRISPR nuclease is Cpf1. Cpf1 is a single RNA-guided endonuclease that utilizes a T-rich protospacer adjacent motif. Cpf1 cleaves the double-stranded DNA in a twisted form. Two Cpf1 enzymes derived from Acidaminococcus and Lachnospiraceae have been shown to efficiently perform genome editing in human cells. (See Zetsche et al., 2015).

[0151] Thus, an RNA-guided DNA nuclease of a type II CRISPR system such as a Cas9 protein or a modified Cas9, or a homologue, orthologue or variant of Cas9, or another RNA-guided DNA nuclease belonging to another CRISPR system such as Cpf1 and its homologues, orthologues or variants may be used in the present invention.

[0152] In some embodiments, the guide molecule comprises one or more chemical modifications that confer new or improved properties (e.g., stability to degradation, hybridization energy, or binding to an RNA-guided DNA nuclease). 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-galactosylqueuosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, β,D-mannosylqueuosine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-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, methyl ester of uridine-5-oxyacetic acid, uridine-5-oxyacetic acid, wybutoxosine, queuosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, N-((9-β-D-ribofuranosylpurin-6-yl)-carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine, 3-(3-amino-3-carboxy-propyl)uridine, (acp3)u, 2'-O-methyl(M), 3'-phosphorothioate(MS), 3'-thioPACE(MSP), pseudouridine or 1-methylpseudouridine.The respective realizabilities of this invention are individual aspects.

[0153] Guide Sequences Targeting the APLP2 Allele A predetermined RNA molecule containing a guide sequence portion used to target a DNA site may result in degradation of the RNA molecule, limitation of activity, lack of activity, or off-target effects. Therefore, an appropriate guide sequence portion is necessary to target a predetermined DNA site in a gene.

[0154] According to the present invention, a novel set of guide sequence portions that target at least one APLP2 allele and introduce a nucleotide sequence expressed under the control of the APLP2 promoter into at least one allele has been identified. Such a gene editing approach may be used to treat a disorder or disease, or to modify the behavior of cells. Preferably, a non-discriminating RNA molecule capable of targeting a pair of APLP2 alleles is used for targeting.

[0155] In some aspects of the present invention, an RNA molecule is used to target a site in the APLP2 gene and introduce or knock in an exogenous nucleotide sequence into the APLP2 gene. In some aspects, the position of the site is in the vicinity of the intended knock-in site, preferably in the vicinity of the start codon or the stop codon, preferably within 150 nucleotides from the start codon or the stop codon. In some aspects, the site is within intron 1 following coding exon 1 of the targeted APLP2 allele. In some aspects, the site is within intron 1 or exon 17 of the targeted APLP2 allele.

[0156] Delivery to Cells The compositions described in this specification may be delivered to target cells by suitable means. The compositions of the present invention may target cells containing and / or expressing the APLP2 allele, such as mammalian cells, preferably monocytes or macrophages. For example, in one embodiment, an RNA molecule that specifically targets the APLP2 allele is delivered to monocytes or macrophages, which are the target cells. Delivery to cells may be performed in vitro, ex vivo, or in vivo. Furthermore, the nucleic acid compositions described in this specification 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.

[0157] In some embodiments, in vivo delivery of the compositions described in this specification includes delivery by lentivirus, adeno-associated virus (AAV), or nanoparticles. In some embodiments, in vivo delivery of the compositions described in this specification includes delivery by lentivirus, adeno-associated virus (AAV), or nanoparticles. The composition may be in the form of an RNP composition. Thus, delivery can be performed in vivo on the monocytes or macrophages of the subject.

[0158] In some embodiments, the compositions described in this specification 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 and progenitor cells (HSPCs), bone marrow progenitor cells, myeloblasts, lymphoblasts, erythroid progenitor cells, platelet cells, natural killer (NK) cells, B lymphocytes, T lymphocytes, eosinophils, neutrophils, or basophils. The composition may be delivered to the cells by known ex vivo delivery, and the methods include, but are not limited to, electroporation, viral transduction, nanoparticle delivery, liposomes, and the like. The composition may be in the form of an RNP composition. Details of the delivery method are described throughout this section.

[0159] In some embodiments, the RNA molecules of the compositions described herein include 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'-thioPACE (MSP), pseudouridine, and 1-methylpseudouridine. Each possibility of this invention is an individual embodiment.

[0160] A suitable viral vector system may be used to deliver a nucleic acid composition (e.g., a composition of the RNA molecules of the present invention). Conventional viral and non-viral based gene transfer methods can be used to introduce the nucleic acid and the target tissue. In certain embodiments, the nucleic acid is administered for in vivo or ex vivo gene therapy. Non-viral vector delivery systems include naked nucleic acids and nucleic acids complexed with delivery vehicles (e.g., liposomes or poloxamers). Reviews of gene therapy include 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).

[0161] Methods of non-viral delivery of nucleic acids and / or proteins include electroporation, lipofection, microinjection, biolistic particle delivery, particle gun acceleration, virosomes, liposomes, immunoliposomes, lipid nanoparticles (LNP), polycation or lipid:nucleic acid conjugates, artificial virions, and uptake of nucleic acids by facilitators, or nucleic acids and / or proteins can be delivered into 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). For example, sonoporation using a Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Delivery of proteins and / or nucleic acids by cationic lipids is also contemplated for in vivo, ex vivo, or in vitro delivery. (See Zuris et al. (2015); Coelho et al. (2013); Judge et al. (2006) and Basha et al. (2011)).

[0162] Non-viral vectors such as transposon-based systems (e.g., recombinant Sleeping Beauty transposon system or recombinant PiggyBac transposon system) may be delivered to target cells and may be utilized for translocation of the polynucleotide sequence of the molecule of the composition or the polynucleotide sequence encoding the molecule of the composition in the target cells.

[0163] Other representative nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, e.g., U.S. Patent No. 6,008,336). Lipofection is described, for example, in U.S. Patent 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 International Publication Nos. 91 / 17424 and 91 / 16024. Delivery to cells (ex vivo administration) or target tissues (in vivo administration) is possible.

[0164] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of skill in the art. (See, for example, 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.)

[0165] 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 has specificity for the EDV. The antibody transports the EDV to the surface of the target cell and the EDV is carried intracellularly by endocytosis. Once inside the cell, the contents are released. (See MacDiarmid et al., 2009).

[0166] The use of RNA or DNA virus-based systems for the delivery of nucleic acids by viruses exploits the highly evolved ways in which viruses target specific cells in the body and transport the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo), or used to treat cells in vitro and the modified cells administered to patients (ex vivo). Conventional virus-based systems for delivering nucleic acids include, but are not limited to, vectors of retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vaccinia virus and herpes simplex virus for gene transfer.

[0167] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding the population of targetable cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and usually have high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors are composed of cis-acting terminal repeat sequences and have the ability to package foreign sequences up to 6 - 10 kb in size. The minimal cis-acting LTR is sufficient for vector replication and packaging and is used to integrate therapeutic genes into target cells and permanently 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, for example, Buchschacher et al. (1992); Johann et al. (1992); Sommerfelt et al. (1990); Wilson et al. (1989); Miller et al. (1991); WO 94 / 26877).

[0168] For gene transfer in clinical trials, at least six viral vectors are currently available, and they utilize an approach that involves complementation of defective vectors by genes inserted into helper cell lines to prepare transducing agents.

[0169] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (see Dunbar et al., 1995; Kohn et al., 1995; Malech et al., 1997). PA317 / pLASN was the first therapeutic vector used in gene therapy trials (Blaese et al., 1995). The transduction efficiency of the MFG-S packaging vector was greater than 50% (Ellem et al., (1997); Dranoff et al., 1997).

[0170] Packaging cells are used to form virus particles capable of infecting host cells. Such cells include adenovirus, 293 cells that package AAV, and Psi-2 cells or PA317 cells that package retrovirus. Virus vectors used in gene therapy are usually generated by producer cell lines that package nucleic acid vectors into virus particles. The vector usually contains the minimal viral sequences required for packaging and (where applicable) subsequent integration into the host, and other viral sequences are replaced with an expression cassette encoding the protein to be expressed. The defective viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy usually have only the inverted terminal repeats (ITRs) derived from the AAV genome required for packaging and integration into the host genome. The viral DNA is packaged in the cell line, which lacks other AAV genes, i.e., the 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 facilitates the replication of the AAV vector and the expression of the AAV genes from the helper plasmid. Due to the lack of ITR sequences, the helper plasmid is not packaged in large amounts. Contamination with adenovirus can be reduced, for example, by heat treatment in which adenovirus is more sensitive than AAV. Furthermore, AAV can be produced at the clinical scale using a baculovirus system (see U.S. Patent No. 7,479,554).

[0171] In many gene therapies, it is desirable for the gene therapy vector to be delivered with a high degree of specificity to a particular tissue. Thus, viral vectors can be modified to have specificity for a given cell 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 an affinity for a receptor known to be present on the target cell. For example, Han et al. (1995) reported that Moloney murine leukemia virus could be modified to express human heregulin fused to gp70, and that the recombinant virus infected specific human breast cancer cells that express the human epidermal growth factor receptor. This principle can be extended to other virus-target cell combinations, in which 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 cell receptor. This explanation applies mainly to viral vectors, but the same principle can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences that preferentially allow uptake by specific target cells.

[0172] As described below, gene therapy vectors can typically be delivered in vivo by administration to an individual patient, either by systemic administration (e.g., intravitreal, intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial injection) or by local application. Alternatively, the vector can be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells, and subsequently the cells can be re-transplanted into the patient after selection of the cells incorporating the vector. A representative ex vivo approach, without limitation, may involve removing tissue (e.g., peripheral blood, bone marrow and spleen) from a patient for culture, introducing nucleic acid into cultured cells (e.g., hematopoietic stem cells), and then transplanting the cells into the target tissue (e.g., bone marrow and spleen) of the patient. In some embodiments, the stem cells or hematopoietic stem cells may be further treated with a survival enhancer.

[0173] Ex vivo cell transfection for diagnosis, research, or gene therapy (e.g., by reinjection of transfected cells into a host) is well known to those skilled in the art. In a preferred embodiment, cells are isolated from a subject, transfected with a nucleic acid composition, and reinjected into the subject (e.g., patient). A variety of cells suitable for ex vivo transfection are well known to those skilled in the art (see, e.g., Freshney, “Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications (6th edition, 2010) and the references cited therein for methods of isolating and culturing cells from patients).

[0174] 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), in addition, 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. Further, primary cells may be isolated and used ex vivo for return to a subject after treatment with an inducible nuclease system (e.g., CRISPR / Cas). Suitable primary cells include peripheral blood mononuclear cells (PBMC) and other blood cell subsets, such as, but not limited to, CD4+ T cells or CD8+ T cells. Also included as examples of suitable cells are stem cells such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neural stem cells, and mesenchymal stem cells.

[0175] In certain embodiments, stem cells are treated ex vivo for cell transfection and gene therapy. The advantages of using stem cells are that they can differentiate into other cells in vitro or can be introduced into a mammal (such as the cell donor) and transplanted into the bone marrow. Methods for differentiating CD34+ cells into clinically important immune cells in vitro using cytokines such as GM-CSF, IFN-γ, and TNF-α are known (see, for example, Inaba et al., 1992, for non-limiting examples).

[0176] Stem cells are isolated for transduction and differentiation by known methods. For example, stem cells can be 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 example, Inaba et al., 1992 as a non-limiting example). Modified stem cells may also be used in some embodiments.

[0177] Vectors having therapeutic nucleic acid compositions (e.g., retroviruses, liposomes) can also be administered directly to an organism for in vivo transduction of cells. Administration can be by any route commonly used to introduce molecules into 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 skilled in the art, and multiple routes can be used to administer a particular composition, although often a particular route can provide a more rapid and effective response than another route. In some embodiments, the composition is delivered by IV injection.

[0178] Vectors suitable for introduction of a transgene into immune cells (e.g., T cells) include non-integrating lentiviral vectors. See, for example, U.S. Patent Application Publication No. 2009 / 0117617.

[0179] The pharmaceutically acceptable carrier is determined in part by the composition being administered and in addition by the method used to administer the composition. Thus, there are a wide variety of suitable formulations of pharmaceutical compositions available, as described, for example, in Remington's Pharmaceutical Sciences, 17th ed., 1989.

[0180] Also, the compositions and methods of the present invention may be used in the manufacture of a medicament for treating a disease or disorder in a patient.

[0181] Mechanism of Action of the APLP2 Safe Harbor Knock-In Method Although not bound by any theory or mechanism, the present invention may be utilized to apply CRISPR nuclease to process the APLP2 allele by introducing a sequence into the safe harbor site of the APLP2 allele and controlling the expression of the sequence into which the promoter of the APLP2 allele is introduced. Specific guide sequences 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).

[0182] The APLP2 gene is located on chromosome 11 and encodes an amyloid beta precursor-like protein 2 protein. A donor molecule may be used to knock-in a desired nucleotide sequence into the APLP2 safe harbor site.

[0183] One strategy is to knock-in a nucleotide sequence into intron 1 of the APLP2 gene or intron 1 following coding exon 1. This strategy targets intron 1 of the APLP2 gene with CRISPR nuclease, thereby utilizing an RNA molecule to make a double-strand break. Since the cleavage occurs in a non-coding region, it is not expected to affect gene expression. In the knock-in of a sequence without knockout of APLP2, 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 the expression of the APLP2 gene, the donor cassette includes a 2A self-cleaving peptide and / or the signal peptide of APLP2 at the C-terminus of the inserted gene, which is cleaved within the endoplasmic reticulum and enables separation of the inserted sequence protein expression product from the APLP2 protein.

[0184] Another strategy is to replace the stop codon and knock in a nucleotide sequence into the APLP2 gene. In this case, both alleles are cleaved 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 cleavage positions in these regions do not affect the expression of APLP2. The knocked-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 enables the separation of the protein expression product by the inserted sequence from the APLP2 protein.

[0185] Examples of RNA Guide Sequences Specifically Targeting Alleles of the APLP2 Gene To target the APLP2 allele, numerous guide sequences can be designed, but the nucleotide sequences shown in Table 1 specified by SEQ ID NOs: 1 to 159641 were specially selected to effectively execute the method described in this specification.

[0186] Table 1 shows guide sequences designed for use in the manner described above to associate specific sequences within the APLP2 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, but not limited to, 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), SaCas9WT (PAM sequence: NNGRRT), SpRY (PAM sequence: NRN or NYN), NmCas9WT (PAM sequence: NNNNGATT), Cpf1 (PAM sequence: TTTV), or JeCas9WT (PAM sequence: NNNVRYM). The RNA molecules of the present invention are each designed to form a complex with one or more different CRISPR nucleases and to utilize the CRISPR nucleases used and their respective one or more different PAM sequences to target a polynucleotide sequence of interest.

[0187]

Table 1

[0188] Examples are shown below to facilitate a more complete understanding of the present invention. The following examples illustrate representative ways of constructing and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only.

Examples

[0189] Details of the Experiment Example 1 Analysis of APLP2 On-Target Activity In HeLa cells, using CRISPR nuclease, screen for guide sequences containing 17 - 50 consecutive nucleotides within the sequences shown in any of SEQ ID NOs: 1 - 159641 with high on - target activity. The on - target activity is determined by DNA capillary electrophoresis analysis.

[0190] Example 2 Insertion of the Sequence of Interest into the APLP2 Safe Harbor Site 1) APLP2 is highly expressed in the target cells; 2) Exon 1 of APLP2 encodes a signal peptide (SP) that can be utilized to enable the secretion of the introduced gene of interest; and 3) Since there is no risk of APLP2 haploinsufficiency, APLP2 was selected as a genomic safe - harbor site for macrophages. To take advantage of the SP of APLP2, a strategy was designed to insert donor DNA into intron 1 of APLP2 downstream of the SP. This insertion, which depends on homologous recombination repair (HDR), is made possible by combining the donor sequence encoding the gene of interest with the DNA damage induced by CRISPR. This strategy aims to enable the expression and secretion of the gene of interest while maintaining the expression and function of the safe - harbor site - containing gene.

[0191] The CRISPR - based ribonucleoprotein (RNP) composition enables editing in intron 1 of APLP2 Guides were screened in HeLa cells, and a composition that could be a CRISPR - based RNP enabling editing in intron 1 of APLP2 was identified. Briefly, using the jetOPTIMUS reagent (Polyplus), a plasmid encoding OMNI - 50 nuclease (64 ng) and a plasmid expressing the guide (20 ng) were co - transfected in a 96 - well plate. After 72 hours, the cells were harvested, genomic DNA was extracted, and then the DNA was analyzed by next - generation sequencing (NGS). The percentage of editing measured by the abundance of indels varied depending on the guide used and reached a maximum of 50% when using OMNI - 50 nuclease (Figure 1A).

[0192] Next, hematopoietic stem cells (HSCs) were electroporated with RNP and the top guides were tested. A composition of single guide RNA (sgRNA) (120 pmole) containing OMNI-50 or OMNI-50 V6172 nuclease (105 pmole) was electroporated into the cells using Lonza P3 cell 4D-nucleofecuor X Kiu S (CA-137 program, PBC2-00675, Lonza). The HSCs were collected 72 hours later and the genomic DNA was analyzed by NGS. As a result, the editing level was found to be comparable to the level observed in HeLa cells (Figure 1B). The APLP2 guide s30 showed the highest editing efficiency of approximately 70% when either OMNI-50 or OMNI-50 V6172 was used, and OMNI-50 V6172 was selected as the composition for future tests. When the protein level of APLP2 was tested by Western blot, no change in the expression of APLP2 was observed (see Figures 3B and 3C).

[0193] Integration of the HDR editing template into intron 1 of APLP2 AAV particles carrying a donor construct encoding GFP fused to the self-cleaving P2A-T2A element (2A) and SP were prepared to test the possibility of integrating an HDR editing template into intron 1 of APLP2. As shown in Figure 2, the donor of the present invention targets intron 1 of APLP2 downstream of the SP of APLP2. The donor construct of the present invention contains a GFP-2A-SP sequence sandwiched between a splice acceptor and a splice donor and flanked by 800 base pair homology arms corresponding to the APLP2 s30 cleavage site (see the details of the donor construct sequence for each element shown in SEQ ID NO: 159654 and the following table). CRISPR-mediated DNA cleavage promotes integration of the donor sequence into intron 1 by HDR. Thereby, an open reading frame encoding SP-GFP-2A-SP-APLP2 is obtained, and after 2A self-cleavage, two polypeptides, SP-GFP and SP-APLP2, are obtained. Importantly, since the donor contains neither a promoter nor other mRNA stabilizing elements, GFP expression is not possible unless integration into the desired locus is successful.

[0194]

Table A

[0195] Integration of GFP into the APLP2 locus of HSCs The above approach was first tested in primary HSCs. Cells were electroporated with an RNP composed of OMNI-50 V6172 nuclease and APLP2 guide s30, and 10 5AAV6 particles carrying GFP donor molecules were infected at a multiplicity of infection (MOI) of 6 . Three days after the treatment, GFP expression by HSCs was analyzed by FACS (Figure 3A). By combining RNP containing APLP2 guide s30 with donor AAV, 53% GFP-expressing cells were obtained (average of two times). To confirm that GFP expression was due to successful integration into the APLP2 locus, two control groups were also tested: a group infected with donor AAV only (without RNP) and a group infected with donor AAV combined with a non-related RNP (NR RNP). In these groups, no significant GFP expression was observed, similar to the untreated HSC control group. Next, to confirm whether APLP2 expression was maintained after integration by HDR, the expression of APLP2 protein and transcript levels were tested. To evaluate the protein level, cell lysates were analyzed by Western blot (Figure 3B and Figure 3C), and no change in APLP2 protein expression was observed between the untreated group and the HDR group, suggesting that APLP2 protein was expressed normally. To evaluate the total APLP2 transcript level, qRT-PCR was performed using a primer set that discriminates between the natural transcript and the transcript integrated with GFT, which is downstream of the editing site and targets exon 4 of APLP2 (Figure 3D). The levels of transcripts in the HDR group and the untreated control group were equivalent, further confirming that APLP2 expression was not changed by the integration of GFP into the APLP2 locus. Finally, to test whether GFP was secreted smoothly from HSCs after integration into the APLP2 locus, cell culture medium was sampled using a GFP ELISA kit (Abcam, ab171581). HSCs in the HDR group secreted approximately 1.3 ng of GFP per cell, while GFP was not detected in the control group (Figure 3E - note that this value is the absolute GFP level detected after considering the volume of the medium).

[0196] Differentiation of edited HSCs into macrophages maintains GFP expression from the APLP2 locus The edited HSCs can differentiate into multiple hematopoietic cell lineages with various therapeutic capabilities. Therefore, after differentiating into macrophages, we test whether the edited HSCs retain GFP expression and secretion. To this end, according to previous descriptions (Gomez-Ospina et al., Nature Communications, 2019), the edited HSCs were differentiated into macrophages. Specifically, the cells were seeded in a 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), penicillin / streptomycin (10 U / mL)) for 48 hours. The adherent cells were maintained in a 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 the differentiation, the cells were scraped and analyzed with a FACS panel: CD14, CD16 and HLA-DR. As shown in Figure 4A, by examining the staining panels of the edited and unedited controls, it can be understood that most of the cells tested differentiated successfully into macrophages and all macrophage markers tested were expressed. The proportion of macrophages in the population did not change with editing and was equivalent between the edited and unedited controls. Furthermore, from the FITC channel of the FACS analysis, it can be understood that approximately 20% of the cells still expressed GFP after differentiation. To test for GFP secretion, the cell medium was assayed by ELISA (Figure 4B). Similar to the original HSC population, GFP secretion was also observed in the edited population, confirming that the insertion of GFP into the APLP2 locus enables both the expression and secretion of the gene of interest.

[0197] Integration of GFP into the APLP2 locus of iPSCs To confirm that this system is related to other pluripotent cells, these experiments were repeated with iPSCs. Similar to HSCs, RNPs were delivered by electroporation and donor DNA was delivered using AAV. AAV-DJ was used to increase the infection rate of iPSCs. Testing for GFP expression in iPSCs 3 days after treatment showed that GFP was successfully expressed in iPSCs after integration by HDR (Figure 5A). Consistent with the results shown in HSCs, the control group without CRISPR activity did not express GFP, indicating that the integration of the GFP cassette is essential for GFP expression. The secretion of GFP from the modified iPSCs was evaluated by ELISA (Figure 5B). Secretion of GFP was detected only in the HDR group, and it was confirmed that integration of GFP into intron 1 of APLP2 enables the expression of a polypeptide with an SP, which leads to subsequent secretion.

[0198] FACS analysis of iPSCs

Table 2

[0199]

Table 3

[0200] References 1. Ahmad and Allen (1992) “Antibody-mediated Specific Binging and Cytotoxicity of Lipsome-entrapped Doxorubicin to Lung Cancer Cells in Vitro”, Cancer Research 52:4817-20. 2. Anderson (1992) “Human gene therapy”, Science 256:808-13. 3. Anzalone et al. (2019) “Search-and-replace genomme editing without double-strand brakes or donor DNA”, Nature 576, 149-157. 4. Basha et al. (2011) “Influence of Cationic Lipid Composition on Gene Silencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting Cells”, Mol. Ther. 19(12):2186-200. 5. Behr (1994) Gene transfer with synthetic cationic amphiphiles: Prospects for gene therapy”, Bioconjuage Chem 5:382-89. 6. Blaese (1995) “Vectors in cancer therapy: how will they deliver”, Cancer Gene Ther. 2:291-97. 7. Blaese et al. (1995) “T lympocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years”, Science 270(5235):475-80. 8. Buchschacher and Panganiban (1992) “Human immunodeficiency virus vectors for inducible expression of foreign genes”, J. Virol. 66:2731-39. 9. Burstein et al. (2017) “New CRISPR-Cas systems from uncultivated microbes”, Nature 542:237-41. 10. Chang and Wilson (1987) “Modification of DNA ends can decrease end-joining relative to homologous recombination in mammalian cells”, Proc. Natl. Acad. Sci. USA 84:4959-4963. 11. Chung et al. (2006) “Agrobacterium is not alone: gene transfer to plants by viruses and other bacteria”, Trends Plant Sci. 11(1):1-4. 12. Coelho et al. (2013) “Safety and efficacy of RNAi therapy for transthyretin amyloidosis” N. Engl. J. Med. 369, 819-829. 13. Crystal (1995) “Transfer of genes to humans: early lessons and obstacles to success”, Science 270(5235):404-10. 14. Dillon (1993) “Regulation gene expression in gene therapy” Trends in Biotechnology 11(5):167-173. 15. Dranoff et al. (1997) “A phase I study of vaccination with autologous, irradiated melanoma cells engineered to secrete human granulocyte macrophage colony stimulating factor”, Hum. Gene Ther. 8(1):111-23. 16. Dunbar et al. (1995) “Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation”, Blood 85:3048-57. 17. Ellem et al. (1997) “A case report: immune responses and clinical course of the first human use of ganulocyte / macrophage-colony-stimulating-factor-tranduced autologous melanoma cells for immunotherapy”, Cancer Immunol Immunother 44:10-20. 18. Gao and Huang (1995) “Cationic liposome-mediated gene transfer” Gene Ther. 2(10):710-22. 19. Haddada et al. (1995) “Gene Therapy Using Adenovirus Vectors”, in: The Molecular Repertoire of Adenoviruses III: Biology and Pathogenesis, ed. Doerfler and Boehm, pp. 297-306. 20. Han et al. (1995) “Ligand-directed retro-viral targeting of human breast cancer cells”, Proc Natl Acad Sci U.S.A. 92(21):9747-51. 21. Inaba et al. (1992) “Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte / macrophage colony-stimulating factor”, J Exp Med. 176(6):1693-702. 22. Jinek et al. (2012) “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science 337(6096):816-21. 23. Johan et al. (1992) “GLVR1, a receptor for gibbon ape leukemia virus, is homologous to a phosphate permease of Neurospora crassa and is expressed at high levels in the brain and thymus”, J Virol 66(3):1635-40. 24. Judge et al. (2006) “Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo”, Mol Ther. 13(3):494-505. 25. Kohn et al. (1995) “Engraftment of gene-modified umbilical cord blood cells in neonates with adnosine deaminase deficiency”, Nature Medicine 1:1017-23. 26. Kremer and Perricaudet (1995) “Adenovirus and adeno-associated virus mediated gene transfer”, Br. Med. Bull. 51(1):31-44. 27. Macdiarmid et al. (2009) “Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug”, Nat Biotehcnol. 27(7):643-51. 28. Malech et al. (1997) “Prolonged production of NADPH oxidase-corrected granulocyes after gene therapy of chronic granulomatous disease”, PNAS 94(22):12133-38. 29. Miller et al. (1991) “Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus”, J Virol. 65(5):2220-24. 30. Miller (1992) “Human gene therapy comes of age”, Nature 357:455-60. 31. Mitani and Caskey (1993) “Delivering therapeutic genes - matching approach and application”, Trends in Biotechnology 11(5):162-66. 32. Nabel and Felgner (1993) “Direct gene transfer for immunotherapy and immunization”, Trends in Biotechnology 11(5):211-15. 33. Remy et al. (1994) “Gene Transfer with a Series of Lipphilic DNA-Binding Molecules”, Bioconjugate Chem. 5(6):647-54. 34. Sentmanat et al. (2018) “A Survey of Validation Strategies for CRISPR-Cas9 Editing”, Scientific Reports 8:888, doi:10.1038 / s41598-018-19441-8. 35. Sommerfelt et al. (1990) “Localization of the receptor gene for type D simian retroviruses on human chromosome 19”, J. Virol. 64(12):6214-20. 36. Van Brunt (1988) “Molecular framing: transgenic animals as bioactors” Biotechnology 6:1149-54. 37. Vigne et al. (1995) “Third-generation adenovectors for gene therapy”, Restorative Neurology and Neuroscience 8(1,2): 35-36. 38. Weissman and Kariko (2015) “mRNA: Fulfilling the promise of gene therapy”, Molecular Therapy (9):1416-7. 39. Wilson et al. (1989) “Formation of infectious hybrid virion with gibbon ape leukemia virus and human T-cell leukemia virus retroviral envelope glycoproteins and the gag and pol proteins of Moloney murine leukemia virus”, J. Virol. 63:2374-78. 40. Yu et al. (1994) “Progress towards gene therapy for HIV infection”, Gene Ther. 1(1):13-26. 41. Zetsche et al. (2015) “Cpf1 is a single RNA-guided endonuclease of a class 2 CRIPSR-Cas system” Cell 163(3):759-71. 42. Zuris et al. (2015) “Cationic lipid-mediated delivery of proteins enables efficient protein based genome editing in vitro and in vivo” Nat Biotechnol. 33(1):73-80.

Claims

1. A method for modifying at least one allele of the amyloid beta precursor-like protein 2 (APLP2) gene in a cell, 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 sequence encoding the same introducing a composition comprising the same into the cell, wherein a complex of the CRISPR nuclease and the RNA molecule double-strand breaks at least one allele of the APLP2 gene.

2. The method according to claim 1, wherein the composition further comprises a donor molecule comprising a nucleotide sequence to be introduced at the double-strand break site.

3. The introduced sequence is derived from a gene encoding alpha1-antitrypsin, glucose-6-phosphatase (G6PC), serpin family A member (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthetase, arginase, argininosuccinase, carbamoyl phosphate synthetase, and N-acetylglutamate synthetase, alpha-galactosidase A, coagulation factor IX, coagulation 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, phosphatidylcholine sterol acyltransferase, N-sulfo-glucosamine 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. The method according to claim 2.

4. The introduced array contains a sequence derived from a gene encoding acidic α-glucosidase, α-L-iduronidase, α-galactosidase, iduronic acid-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, acidic 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, battenin, ceroid lipofuscinosis neuronal protein 5 (CLN5), ceroid lipofuscinosis neuronal protein 6 (CLN6), ceroid lipofuscinosis neuronal protein 7 (CLN7), ceroid lipofuscinosis neuronal protein 8 (CLN8), (cathepsin D), cystinosin, cathepsin K, cystatin, lysosome-associated membrane protein 2 (LAMP2), human growth hormone, follicle-stimulating hormone, erythropoietin, CD19, cytokine, chemokine, IL-10, IGF1, TGFβ, IL-15, CXCR4, IL-4, or granulocyte colony-stimulating factor (G-CSF). The method according to claim 2.

5. The method according to any one of claims 2 to 4, wherein the donor molecule comprises a sequence derived from a gene encoding a protein secreted by a cell.

6. The method according to any one of claims 2 to 5, wherein the introduced sequence comprises a sequence encoding a polypeptide of interest expressed by the cell.

7. The method according to claim 6, wherein the polypeptide of interest to be expressed is secreted by the cell.

8. The method according to claim 6 or 7, wherein the polypeptide of interest to be expressed further comprises a signal peptide.

9. The method according to claim 8, wherein the signal peptide is encoded by an allele of the APLP2 gene.

10. The method according to any one of claims 2 to 9, wherein the introduced sequence comprises a sequence encoding a 2A self-cleaving peptide.

11. The method according to any one of claims 2 to 10, wherein the introduced sequence comprises a sequence encoding a signal peptide.

12. The method according to claim 11, wherein the signal peptide is an APLP2 signal peptide.

13. The method according to any one of claims 2 to 12, wherein the introduced sequence comprises a splice acceptor sequence and a splice donor sequence.

14. The method according to any one of claims 2 to 13, wherein the introduced sequence 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.

15. The method according to any one of claims 2 to 14, wherein the donor molecule comprises a first homology arm sequence having at least 90%, preferably 100% sequence identity with the APLP2 sequence upstream of the double-strand break, and a second homology arm sequence having at least 90%, preferably 100% sequence identity with the APLP2 sequence downstream of the double-strand break.

16. The method according to claim 15, wherein the lengths of the first homology arm sequence and the second homology arm sequence are each about 20 - 50, 50 - 100, 100 - 200, 200 - 500, 500 - 1000 or 1000 - 2000 nucleotides.

17. The method according to any one of claims 6 to 16, wherein the polypeptide of interest is a soluble protein.

18. The method according to any one of claims 6 to 17, wherein the length of the polypeptide of interest is about 20 to 50, 50 to 100, 100 to 200, 200 to 500, 500 to 1000 or 1000 to 2000 amino acids.

19. The method according to any one of claims 1 to 18, wherein the RNA molecule comprises a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 159641.

20. The method according to any one of claims 1 to 19, wherein the RNA molecule comprises a non-discriminatory guide portion targeting intron 1 of the APLP2 gene or the 3'untranslated region (3'UTR) of the APLP2 gene.

21. The method according to any one of claims 1 to 20, wherein the RNA molecule comprises a non-discriminatory guide portion targeting a sequence within a genomic range selected from any of 11:130142110 to 130144147 and 11:130070140 to 130109426.

22. The method according to any one of claims 1 to 21, wherein the modified allele of the APLP2 gene expresses an APLP2 gene product.

23. The method according to any one of claims 6 to 22, wherein the modified allele of the APLP2 gene expresses an APLP2 polypeptide and the polypeptide of interest.

24. The method according to any one of claims 1 to 23, wherein the cell is a stem cell, monocyte, macrophage, iPS-derived monocyte, iPS-derived macrophage, hematopoietic stem cell (HSC), hematopoietic stem and progenitor cell (HSPC), bone marrow progenitor cell, myeloblast, lymphoblast, erythroid progenitor cell, platelet cell, natural killer (NK) cell, B lymphocyte, T lymphocyte, eosinophil, neutrophil, iPS-derived cell or basophil.

25. The method according to any one of claims 1 to 24, wherein the cell is a stem cell, and the method further comprises differentiating the stem cell after modification of the stem cell.

26. A modified cell obtained by the method according to any one of claims 1 to 25.

27. The modified cells according to claim 26, wherein the modified cells are stem cells, monocytes, macrophages, iPS-derived monocytes, iPS-derived macrophages, hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), bone marrow progenitor cells, myeloblasts, lymphoblasts, erythroid progenitor cells, platelet cells, natural killer (NK) cells, B lymphocytes, T lymphocytes, eosinophils, neutrophils, iPS-derived cells or basophils.

28. A composition comprising an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 159641.

29. The composition according to claim 28, further comprising at least one CRISPR nuclease.

30. The composition according to claim 28 or 29, further comprising a donor molecule.

31. The donor molecule according to claim 30, wherein the donor molecule comprises a sequence derived from a gene encoding α1-antitrypsin, glucose-6-phosphatase (G6PC), serpin family A member (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthetase, arginase, argininosuccinase, carbamoyl phosphate synthetase, and N-acetylglutamate synthetase, α-galactosidase A, coagulation factor IX, coagulation factor VII, lysosomal α-glucosidase, fibrinogen, phenylalanine 4-hydroxylase, alkaline phosphatase, glucosylceramidase, β-galactosidase, porphobilinogen deaminase, arylsulfatase B, β-glucuronidase, α-N-acetylglucosaminidase, lysosomal α, αL-iduronidase, mannosidase, phosphatidylcholine cholesterol acyltransferase, N-sulfo-glucosamine 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 lysosomal protective protein.

32. The donor molecule according to claim 30, comprising a sequence derived from a gene encoding acidic α-glucosidase, α-L-iduronidase, α-galactosidase, iduronic acid-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, acidic 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, battenin, ceroid lipofuscinosis neuronal protein 5 (CLN5), ceroid lipofuscinosis neuronal protein 6 (CLN6), ceroid lipofuscinosis neuronal protein 7 (CLN7), ceroid lipofuscinosis neuronal protein 8 (CLN8), (cathepsin D), cystinosin, cathepsin K, cystatin, lysosome-associated membrane protein 2 (LAMP2), human growth hormone, follicle-stimulating hormone, erythropoietin, CD19, cytokine, chemokine, IL-10, IGF1, TGFβ, IL-15, CXCR4, IL-4, or granulocyte colony-stimulating factor (G-CSF). [

33. ] The composition according to any one of claims 30 to 32, wherein the donor molecule comprises a sequence derived from a gene encoding a protein secreted by a cell.

34. The composition according to any one of claims 30 to 33, wherein the donor molecule comprises a sequence encoding a polypeptide of interest.

35. The composition according to any one of claims 30 to 34, wherein the donor molecule comprises a sequence encoding a 2A self-cleaving peptide.

36. The composition according to any one of claims 30 to 35, wherein the donor molecule comprises a sequence encoding a signal peptide.

37. The composition according to claim 36, wherein the signal peptide is an APLP2 signal peptide.

38. The composition according to any one of claims 30 to 37, wherein the donor molecule comprises a splice acceptor sequence and a splice donor sequence.

39. The composition according to any one of claims 30 to 38, wherein 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.

40. The composition according to any one of claims 30 to 39, wherein the donor molecule comprises a first homology arm sequence having at least 90%, preferably 100%, sequence identity with a first sequence of the APLP2 gene, and a second homology arm sequence having at least 90%, preferably 100%, sequence identity with a second sequence of the APLP2 gene.

41. The composition according to claim 40, wherein the lengths of the first homology arm sequence and the second homology arm sequence are each about 20 to 50, 50 to 100, 100 to 200, 200 to 500, 500 to 1000, or 1000 to 2000 nucleotides.

42. The composition according to any one of claims 34 to 41, wherein the polypeptide of interest is a soluble protein.

43. The composition according to any one of claims 34 to 42, wherein the length of the polypeptide of interest is at most 20 to 50, 50 to 100, 100 to 200, 200 to 500, 500 to 1000, or 1000 to 2000 amino acids.

44. The composition according to any one of claims 28 to 43, further comprising a tracrRNA molecule.

45. A method for modifying the APLP2 allele intracellularly, the method comprising delivering to the cell the composition according to any one of claims 28 to 44.

46. A method for treating a disorder or disease, the method comprising delivering to the cells of a subject having the disorder or disease the composition according to any one of claims 28 to 44, or delivering to the subject the modified cell according to claim 26 or 27.

47. 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, I-cell 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-Haltia disease, Jansky-Bielschowsky 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, pycnodysostosis, Salla disease, Danon disease and / or α1-antitrypsin deficiency, the method according to claim 46.

48. The method according to claim 46 or 47, wherein the composition or the modified cell is delivered to the tissue or tumor of the subject.

49. A medicament for use in modifying the APLP2 allele intracellularly, the medicament comprising the composition according to any one of claims 28 to 44, the medicament being administered by delivering to the cell the composition according to any one of claims 28 to 44.

50. Use of a composition according to any one of claims 28 to 44 or a modified cell according to claim 26 or 27 for the treatment, amelioration or prevention of a disorder or disease, comprising delivering a composition according to any one of claims 28 to 44 to the cells of a subject having or at risk of having said disorder or disease, or delivering a modified cell according to claim 26 or 27 to said subject.

51. A medicament for use in treating, ameliorating or preventing a disorder or disease, comprising a composition according to any one of claims 28 to 44 or a modified cell according to claim 26 or 27, which is administered by delivering a composition according to any one of claims 28 to 44 to the cells of a subject having or at risk of having said disorder or disease, or delivering a modified cell according to claim 26 or 27 to said subject.

52. The method according to any one of claims 46 to 48, the use according to claim 50 or the medicament according to claim 51, wherein the disorder or disease is a lysosomal storage disorder.

53. 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, I-cell 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, Stanworth disease, Santavuori-Haltia disease, Jansky-Bielschowsky 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, pycnodysostosis, Salla disease, Danon disease and / or α1-antitrypsin deficiency, the method according to any one of claims 46 to 48, the use according to claim 50, or the medicament according to claim 51.

54. The disorder or disease is a disorder or disease of the blood, lung, brain, liver, intestine, intestinal tract, bone, muscle or central nervous system, or an inflammatory disease or an autoinflammatory disease, the method according to any one of claims 46 to 48, the use according to claim 50, or the medicament according to claim 51.

55. The medicament is enzyme replacement therapy, the medicament according to claim 51.

56. A method for treating a disease or disorder in a subject, which is an immunotherapy comprising delivering the modified cell according to claim 26 or 27 to the subject.

57. The disease or disorder is cancer, the method according to claim 56.

58. The composition according to any one of claims 28 to 44 or the modified cell according to claim 26 or 27 for use in treating, ameliorating or preventing a disorder or disease.

59. A composition, method, process, kit or use characterized by one or more elements disclosed in the specification.

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