Knock-in strategy in the AAVS1 safe harbor area

Abstract

RNA molecules containing a guide sequence portion having 17 to 50 consecutive nucleotides within any of the sequences shown in Sequence ID No. 1 to 24195, as well as compositions, methods, and uses thereof.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 491,660, filed Mar. 22, 2023, the contents of which are incorporated by reference herein. Throughout this application, various publications are referenced, including those within parentheses. The entire disclosure of all publications mentioned in this application is incorporated by reference herein to supplement the technology that can be used in this invention and the technology related to this invention.

[0002] Sequence listing reference This application was created on Mar. 22, 2023, in the form of an IBM-PC machine using an operating system compatible with MS-Windows (registered trademark), and incorporates by reference the nucleotide sequences in an XML file of 20,924 kilobytes, filed Mar. 22, 2024, with the file name "230322_92134-PRO_Sequence_Listing_AWG.xml".

Background Art

[0003] The safe harbor site of adeno-associated virus integration site 1 (AAVS1) can be targeted to introduce a desired sequence into the AAVS1 site without causing harmful disruption to the protein phosphatase 1 regulatory (inhibitory) subunit 12C (PPP1R12C) gene that constitutes the AAVS1 site, or without affecting the expression of PPP1R12C or the expression of other endogenous genes. Such a targeting strategy may be utilized to enable the expression of the introduced sequence.

Summary of the Invention

[0004] This specification discloses a strategy that enables the expression of a gene or a portion thereof under the control of an endogenous promoter located within or near the AAVS1 locus without interfering with the expression of endogenous genes or transcripts, including but not limited to PPP1R12C.

[0005] This specification also discloses strategies to enable the expression of an introduced gene or coding sequence, or a portion thereof, independently of an endogenous promoter located within or near the target AAVS1 locus.

[0006] This disclosure also provides a method for modifying at least one adeno-associated virus integration site 1 (AAVS1) site within a cell, the method being At least one CRISPR nuclease, or a sequence encoding a CRISPR nuclease; and An RNA molecule containing a guide sequence of 17-50 nucleotides, or a nucleotide sequence encoding it. The process includes introducing a composition containing into cells, Here, the complex of the CRISPR nuclease and the RNA molecule cleaves the double-strand at least one AAVS1 site.

[0007] In some embodiments, the composition also contains a donor molecule, the nucleotide sequence of the donor molecule being inserted into or near a double-strand break site. Similarly, in some embodiments, the composition further contains a donor molecule containing a nucleotide sequence to be introduced into a double-strand break site, the expression of the introduced sequence being mediated by an endogenous gene promoter, or more preferably by an exogenously introduced promoter. For example, the exogenous promoter may be provided by the donor molecule.

[0008] Aspects of this invention provide a first RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides in a sequence shown in any of Sequence IDs 1 to 24195. In some aspects, the composition further comprises a CRISPR nuclease. In some aspects, the composition further comprises a donor molecule. In some aspects, the donor molecule is a single-stranded or double-stranded DNA molecule, a plasmid, a PCR product, an adeno-associated virus (AVV), or an integrase-deficient lentivirus. In some aspects, the donor molecule is an RNA molecule.

[0009] In some embodiments, the donor consists of transcription elements that enable the independent expression of the inserted gene. Such elements may be, but are not limited to, enhancers, core promoter elements, 5'UTR, 3'UTR, and introns.

[0010] Aspects of this invention provide cells modified by the methods described in the specification. In some aspects, the cells are dividing cells. In some aspects, the cells are non-dividing cells. In some aspects, the cells are terminating cells. In some aspects, the cells are stem cells. In some aspects, the cells are hematopoietic stem cells (HSCs). In some aspects, the cells are induced pluripotent stem cells (iPSCs). In some aspects, the cells are hematopoietic stem progenitor cells (HSPCs). In some aspects, the cells are lymphocytes. In some aspects, the cells are hepatocytes. In some aspects, the cells are neurons.

[0011] In some embodiments, the delivery of the compositions described in the specification to cells is carried out in vitro, ex vivo, or in vivo. In some embodiments, the method is carried out ex vivo, and cells are provided / excised from individual patients. In some embodiments, the method further includes the step of introducing (e.g., autologous transplantation) the resulting cells having the modified or edited AAVS1 allele into individual patients.

[0012] Some aspects of this invention provide the use of a composition for modifying or editing an intracellular AAVS1 allele, comprising delivering the composition to a cell, which includes an RNA molecule containing a guide sequence portion having 17 to 50 consecutive nucleotides in a sequence shown in any of SEQ ID NOs: 1 to 24195, and a CRISPR nuclease. In some aspects, the composition further contains a donor molecule.

[0013] Aspects of this invention provide a pharmaceutical formulation containing an RNA molecule and a CRISPR nuclease, for use in modifying or editing an AAVS1 allele in a cell, the RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides in a sequence shown in any of SEQ ID NOs: 1 to 24195, wherein the pharmaceutical formulation is administered by delivering a composition containing the RNA molecule and the CRISPR nuclease to a cell. In some embodiments, the pharmaceutical formulation further contains a donor molecule.

[0014] Some aspects of this invention provide a kit for modifying or editing the AAVS1 allele in a cell, the kit comprising an RNA molecule containing a guide sequence portion having 17 to 50 consecutive nucleotides in a sequence shown in any of SEQ ID NOs: 1 to 24195, 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 aspects, the kit further comprises a donor molecule. [Brief explanation of the drawing]

[0015] Figure 1A-1F: Editing and excision in HeLa cells using DNA transfection. HeLa cells were transfected with plasmids encoding nucleases and guide sequences. Editing activity was screened for a total of 16 nucleases and 39 guide sequences. Cells were harvested 72 hours after DNA transfection, genomic DNA was extracted, the target guide regions were amplified, and analyzed by NGS. The graph shows the edit rate ± STDV in two independent experiments. [Figure 1A] Figure 1A shows the edit rates for AAVS_s29, AAVS_s30, AAVS_s31, AAVS_s37, and AAVS_s43 using OMNI-159. [Figure 1B] Figure 1B shows the editing rates for AAVS_s4~AAVS_s7 and AAVS_s10~AAVS_s15 using OMNI-103. [Figure 1C]Figure 1C shows the editing rates for AAVS_s33 to AAVS_s35 using OMNI-110. [Figure 1D] Figure 1D shows the editing rates for AAVS_s22~AAVS_s26 and AAVS_s39~AAVS_s41 using OMNI-274. [Figure 1E] Figure 1E shows the editing rates for AAVS_s17~AAVS_s19 using OMNI-308. [Figure 1F] Figure 1F shows the editing rates for AAVS_s3 using OMNI-50; AAVS_s39 and AAVS_s40 using OMNI-75; AAVS_s41 using OMNI-93; AAVS_s32 using OMNI-127; AAVS_s27 and AAVS_s28 using OMNI-231; AAVS_s43 using OMNI-269; AAVS_s20, AAVS_s21 and AAVS_s39-AAVS_s42 using OMNI-281; ​​AAVS_s37 using OMNI-286; AAVS_s36 and AAVS_s38 using OMNI-291; AAVS_s36-AAVS_s38 using OMNI-302; and AAVS_s16 using OMNI-366. [Modes for carrying out the invention]

[0016] Detailed explanation Unless otherwise defined, all technical and / or scientific terms used in this specification have the same meaning as those generally understood by those skilled in the art to the extent of this invention. Methods and materials similar or equivalent to those described in this specification may be used in carrying out or testing aspects of this invention, but representative methods and / or materials are described below. In case of any conflict, the specification, including definitions, shall prevail. In addition, the materials, methods and examples are illustrative only and are not necessarily intended to be limiting.

[0017] It is understood that the term "one" refers to "one or more" of the recited components. The use of the singular form is 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.

[0018] 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 the specification and 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 number of reported significant digits and by applying ordinary rounding techniques. <000​​​​​​​​​The term "homologous recombination repair" or "HDR" refers to a mechanism for repairing DNA damage in cells, such as when repairing double-stranded and single-stranded breaks of DNA. HDR requires nucleotide sequence homology and uses a "nucleic acid template" (in this specification, the nucleic acid template and the donor template have the same meaning) to repair the sequence where a double-stranded or single-stranded break has occurred (e.g., a DNA target sequence). Thereby, for example, genetic information is transmitted from the nucleic acid template to the DNA target sequence. If the sequence of the nucleic acid template is different from the sequence of the DNA target and part or all of the nucleic acid template polynucleotide or oligonucleotide is incorporated into the DNA target sequence, the sequence of the DNA target may change (e.g., insertion, deletion, mutation) by HDR. In some embodiments, the entire nucleic acid template polynucleotide, a portion of the nucleic acid template polynucleotide, or a copy of the nucleic acid template is incorporated into the DNA target sequence.

[0022] The terms "nucleic acid template" and "donor" refer to a nucleotide sequence that is inserted or copied into the genome. The nucleic acid template can be added to the 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 or more nucleotides. The length of the nucleic acid template sequence can be arbitrary, for example, it can be 2 to 10,000 nucleotides. 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 or more nucleotides corresponding to the wild-type sequence of the target nucleic acid at the target position. In some embodiments, the nucleic acid template includes, for example, a nucleotide sequence of 1 or more ribonucleotides 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.

[0023] Insertion of exogenous sequences (also called "donor sequences," "donor templates," "donor molecules," or "donors") can also be performed. For example, a donor sequence can include a non-homologous sequence flanked by two homologous regions, enabling efficient homologous recombination repair (HDR) at the target site. Furthermore, a donor sequence can include a vector molecule having a sequence that is not homologous to the target region in cellular chromatin. A donor molecule can include several discontinuous regions homologous to cellular chromatin. For example, when inserting a sequence that is not normally present in the target region, the sequence may be included in the donor nucleic acid molecule and flanked by regions homologous to the sequence in the target region. The length of the donor molecule is arbitrary, and may range from a few bases (e.g., 10-20 bases) to several kilobases.

[0024] 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 Publications 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 (e.g., from degradation by exonucleases) in a manner known to those skilled in the art. For example, by adding one or more dideoxynucleotide residues to the 3' end of the linear molecule and / or by ligating a self-complementary oligonucleotide to one or both ends. See, for example, Chang et al. (1987) and Nehls et al. (1996). Other methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups, modified nucleotide bonds such as phosphorothioates and phosphoramidates, and the use of O-methylribose or deoxyribose residues.

[0025] The donor sequence may be an oligonucleotide and may be used for modification of the endogenous sequence by the target. The oligonucleotide may be introduced into cells using a vector, by electroporation of cells, or by other methods known in the art. The donor polynucleotide can be introduced as a naked nucleic acid, as a nucleic acid complexed with a substance such as a liposome or poloxamer, or by a virus (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase-deficient lentivirus (IDLV)).

[0026] In this specification, the term “modified cell” refers to a cell in which the complex of an RNA molecule and a CRISPR nuclease undergoes a double-strand break as a result of hybridization with a target sequence, i.e., on-target hybridization. The term “modified cell” may further encompass cells that have been edited or modified (including the introduction of an exogenous sequence) after the double-strand break.

[0027] This invention provides modified cells obtained by using the methods described in the specification. In some embodiments, these modified cells are capable of generating progeny cells. In some embodiments, these modified cells are capable of generating progeny cells after transplantation. For example, the modified cells may be hematopoietic stem cells (HSCs) or cells suitable for allogeneic or autologous cell transplantation. For example, the modified cells may be stem cells.

[0028] This invention also provides compositions comprising these modified cells and a pharmaceutically acceptable carrier. It also provides in vitro or ex vivo methods for preparing these compositions, comprising mixing the cells with the pharmaceutically acceptable carrier.

[0029] In this specification, the terms “targeting sequence” or “targeting molecule” refer to a nucleotide sequence or molecule containing a nucleotide sequence that can hybridize with a specific target sequence. For example, a targeting sequence has a nucleotide sequence that, along its length, is at least partially complementary to the target sequence. A targeting sequence or targeting molecule may be a portion of an RNA molecule that can form a complex with a CRISPR nuclease, either alone or in combination with other RNA molecules, with the targeting sequence acting as the targeting portion of the CRISPR complex. When a molecule containing a targeting sequence is present simultaneously with a CRISPR molecule, the RNA molecule, either alone or in combination with one or more other RNA molecules (e.g., a tracrRNA molecule), can direct the CRISPR nuclease to a specific target sequence. As a non-limiting example, the guide sequence portion of a CRISPR RNA molecule or a single guide RNA molecule may act as a targeting molecule. Each feasibility is in a separate manner. Targeting sequences can be custom designed and directed to desired sequences.

[0030] In this specification, the terms “targeting” and “directing” refer to preferentially hybridizing the targeted sequence of a targeted molecule with a nucleic acid containing the target nucleotide sequence. It is understood that the terms “targeting” and “directing” include various hybridization capabilities, such as preferentially targeting nucleic acids containing the target nucleotide sequence, but also the possibility of unintended off-target hybridization in addition to on-target hybridization. It is understood that when an RNA molecule targets a sequence, the complex of the RNA molecule and the CRISPR nuclease molecule targets that sequence for nuclease activity.

[0031] The "guide sequence portion" of an RNA molecule refers to a nucleotide sequence that can hybridize with a specific target DNA sequence. For example, the guide sequence portion has a nucleotide sequence that is partially or completely complementary to the target DNA sequence along its length. In some embodiments, the nucleotide lengths of the guide sequence portion are 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, 1 These are 7-39, 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 entire length of the guide sequence portion is perfectly complementary to the target DNA sequence along its length. The guide sequence portion may be a part of an RNA molecule capable of forming a complex with a CRISPR nuclease, and the guide sequence portion acts as the DNA targeting portion of the CRISPR complex. When an RNA molecule containing the guide sequence portion is present simultaneously with a CRISPR molecule, either alone or in combination with one or more other RNA molecules (e.g., a tracrRNA molecule), the RNA molecule can direct the CRISPR nuclease to a specific target DNA sequence. Therefore, a CRISPR complex can be formed by directly binding the RNA molecule containing the guide sequence portion to a CRISPR nuclease, or by binding the CRISPR nuclease to an RNA molecule containing both the guide sequence portion and one or more other RNA molecules. Each feasibility is in a different form. The guide sequence portion can be custom designed to direct it to a desired sequence. Therefore, a molecule containing a "guide sequence portion" is a type of targeting molecule.In some embodiments, the guide sequence portion includes the same sequence as the guide sequence portion described in the specification (e.g., the guide sequence shown in any of SEQ ID NOs: 1 to 24195), or a different sequence of 1, 2, 3, 4, or 5 nucleotides or less. Each feasibility is a separate embodiment. In some of these embodiments, the guide sequence portion includes the same sequence as the sequence shown in any of SEQ ID NOs: 1 to 24195. Throughout this application, the terms “guide molecule,” “RNA guide molecule,” “guide RNA molecule,” and “gRNA molecule” are synonymous with the molecule containing the guide sequence portion.

[0032] In this specification, the term "non-distinguishable" refers to a guide sequence portion of an RNA molecule that targets a specific DNA sequence common to a pair of gene alleles. For example, a non-distinguishable guide sequence portion can target a pair of gene alleles present in a cell.

[0033] In aspects of this invention, the RNA molecule includes a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 24195. In some aspects, the guide sequence portion includes the same sequence as the sequence shown in any of SEQ ID NOs: 1 to 24195, or a different sequence of 1, 2, or 3 nucleotides or less.

[0034] RNA molecules and / or guide sequences of RNA molecules may have modified nucleotides. Typical modifications to nucleotides or polynucleotides may be synthetic and may include polynucleotides having nucleotides containing bases other than naturally occurring adenine, cytosine, thymine, uracil, or guanine bases. Modifications to polynucleotides include synthetic polynucleotides having nucleosides that are not naturally occurring, such as locked nucleic acids. Modifications to polynucleotides may be used to increase or decrease the stability of RNA. An example of a modified polynucleotide is mRNA having 1-methylpsoiduridine. For examples of modified polynucleotides and their uses, see U.S. Patent No. 8,278,036, International Publication No. 2015 / 006747, and Weissman and Kariko (2015) (incorporated in the specification by reference).

[0035] In this specification, "consecutive nucleotides" as indicated by the sequence number refers to nucleotides in the order shown in the sequence number, without any nucleotide intervention.

[0036] In aspects of this invention, the guide sequence portion may be 25 nucleotides long and may contain 20 to 22 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 24195. In aspects of this invention, the guide sequence portion may be less than 22 nucleotides long. For example, in aspects of this invention, the guide sequence portion may be 17, 18, 19, 20, or 21 nucleotides long. In such aspects, the guide sequence portion may consist of 17, 18, 19, 20, or 21 nucleotides within the sequence of 17 to 22 consecutive nucleotides shown in any of SEQ ID NOs: 1 to 24195. For example, the guide sequence portion of the sequence of 17 consecutive nucleotides shown in SEQ ID NO: 24196 may be any of the following nucleotide sequences (nucleotides removed from the sequence are crossed out):

[0037] [ka]

[0038] In aspects of this invention, the nucleotide length of the guide sequence portion may exceed 20. For example, in aspects of this invention, the nucleotide length of the guide sequence portion may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In such aspects, the guide sequence portion comprises 17 to 50 nucleotides having a sequence of 20, 21, or 22 consecutive nucleotides as shown in any of SEQ ID NOs: 1 to 24195, and nucleotides adjacent to or perfectly complementary to the 3' end, 5' end, or both of the target sequence.

[0039] In aspects of this invention, a CRISPR nuclease and an RNA molecule containing a guide sequence portion bind to a target DNA sequence to form a CRISPR complex that cleaves the target DNA sequence. A CRISPR nuclease (e.g., Cpf1) may form a CRISPR complex containing the CRISPR nuclease and RNA molecule without an additional tracrRNA molecule. Alternatively, a 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 having a nucleotide sequence that can hybridize with a specific target DNA sequence and the sequence portion involved in CRISPR nuclease binding (e.g., the tracrRNA sequence portion) can reside in the same RNA molecule. Alternatively, the guide sequence portion may reside in one RNA molecule, and the sequence portion involved in CRISPR nuclease binding (e.g., the tracrRNA portion) may reside in another RNA molecule. A single RNA molecule containing a guide sequence portion (e.g., a DNA-targeting RNA sequence) and at least one CRISPR protein-binding RNA sequence portion (e.g., a tracrRNA sequence portion) can form a complex with a CRISPR nuclease and act as a DNA-targeting molecule. In some embodiments, a first RNA molecule containing a DNA-targeting RNA portion including a guide sequence portion and a second RNA molecule containing a CRISPR protein-binding RNA sequence interact by base pairing to form an RNA complex that directs the CRISPR nuclease to a DNA target site, or they fuse with each other to form an RNA molecule that complexes with a CRISPR nuclease and directs the CRISPR nuclease to a DNA target site.

[0040] In aspects of this invention, the RNA molecule containing the guide sequence portion may further contain the sequence of a tracrRNA molecule. Such aspects may be designed as a synthetic fusion of the guide portion of the RNA molecule and transactivated crRNA (tracrRNA) (see Jinek et al., 2012). In such aspects, the RNA molecule is a single guide RNA (sgRNA) molecule. Some aspects of this invention may also form a CRISPR complex utilizing individual tracrRNA molecules and individual RNA molecules containing the guide sequence portion. In such aspects, the tracrRNA may hybridize with the RNA molecule via base pairing, which may be advantageous in certain applications of the invention described in the specification.

[0041] The term "tracrmate sequence" refers to a sequence that is sufficiently complementary to the tracrRNA molecule so as to hybridize with tracrRNA via base pairing and promote the formation of the CRISPR complex (see U.S. Patent No. 8,906,616). In aspects of this invention, the RNA molecule may further contain a tracrmate sequence portion.

[0042] In this invention, "gene" includes the DNA region that codes for a gene product and all DNA regions that control the production of the gene product, and the sequence of the control region is not limited to whether it is adjacent to the coding sequence and / or transcription sequence. Therefore, a gene includes, but is not limited to, promoter sequences, terminators, translation control sequences, such as ribosome binding sites and intra-sequence ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and gene locus regulatory regions.

[0043] Eukaryotic cells include, but are not limited to, fungal cells (e.g., yeast), plant cells, animal cells, mammalian cells, and human cells.

[0044] In this specification, the term "nuclease" refers to an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of nucleic acids. Nucleases may be isolated from or derived from natural products. Natural products may be living organisms. Alternatively, nucleases may be modified or synthetic proteins that retain phosphodiester bond cleavage activity. Genetic modification can be achieved using nucleases (e.g., CRISPR nucleases).

[0045] Aspects of this invention provide an RNA molecule comprising a guide sequence portion (e.g., a targeting sequence) having a nucleotide sequence that is completely or partially complementary to a target located within or near at least one AAVS1 site. 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 AAVS1 target site and induce a double-strand or single-strand break within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides from the AAVS1 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 intron 1 of at least one STED5 allele containing the AAVS1 site. In some embodiments, the guide sequence portion is complementary to a target sequence located 50 base pairs upstream to 50 base pairs downstream of intron 1 of at least one STED5 allele containing the AAVS1 site. Each feasibility is a separate embodiment. In some embodiments, the guide sequence portion is complementary to a target sequence located 7 base pairs upstream to 7 base pairs downstream of intron 1 of at least one STED5 allele containing the AAVS1 site.

[0046] In this specification, the term "HSC" refers to both hematopoietic stem cells and hematopoietic progenitor stem cells. Examples of stem cells that are not limited to these include myeloid cells, myeloid progenitor cells, pluripotent progenitor cells, and lineage-limited progenitor cells.

[0047] In this specification, “progenitor cells” refers to lineage cells derived from stem cells that possess mitotic and pluripotency (e.g., the ability to differentiate or develop into multiple, though not all, types of mature cell lineages). In this specification, “hematopoiesis” refers to the formation and development of various blood cells (e.g., erythrocytes, megakaryocytes, myeloid cells (e.g., monocytes, macrophages, and neutrophils), and lymphocytes) and other forming elements within the body (e.g., bone marrow).

[0048] Some aspects of this invention provide a method for modifying at least one adeno-associated virus integration site 1 (AAVS1) site within a cell, the method being: At least one CRISPR nuclease, or a sequence encoding a CRISPR nuclease; and An RNA molecule containing a guide sequence of 17-50 nucleotides, or a nucleotide sequence encoding it. The step includes introducing a composition containing into the cells, Here, the CRISPR nuclease-RNA molecule complex cleaves at least one AAVS1 site in the double strand.

[0049] In some embodiments, the composition also contains a donor molecule. In some embodiments, the nucleotide sequence of the donor molecule is inserted or copied at or near a double-strand break site.

[0050] In some embodiments, the composition further comprises a donor molecule containing a nucleotide sequence to be introduced into the double-strand break site, the expression of the introduced sequence being mediated by an endogenous promoter. Alternatively, the donor molecule may contain a nucleotide sequence to be introduced into the double-strand break site, which includes a promoter that mediates the expression of the introduced sequence.

[0051] In some embodiments, the introduced sequences include α1-antitrypsin, glucose-6-phosphatase (G6PC), serpine family A member (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthase, arginase, argininosuccinate, carbamoyl phosphate synthase, and N-acetylglutamate synthase, α-galactosidase A, coagulation factor IX, coagulation factor VII, lysosomal α-glucosidase, fibrinogen, and phenylalanine. These are sequences derived from genes encoding 4-hydroxylase, alkaline phosphatase, glucosylceramidase, β-galactosidase, porphobilinogen deaminase, arylsulfatase B, β-glucuronidase, α-N-acetylglucosaminidase, lysosomal α, α-L-idulonidase, mannosidase, phosphatidylcholinesterol acyltransferase, N-sulfoglucosamine sulfohydrolase, coagulation factor X, N-acetylgalactosamine-6-sulfatase, sphingomyelin phosphodiesterase, iduronic acid-2-sulfatase, lysosomal α-glucosidase, cyclin-dependent kinase-like 5, pro-low-density lipoprotein receptor-related protein 1, phenylalanine ammonia lyase, protein glutamine γ-glutamyltransferase K, or lysosomal protective proteins.

[0052] In some embodiments, the introduced sequence may be acid α-glucosidase, α-L-iduronidase, α-galactosidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, lysophosphatidylcholine metabolism-related proteins, preferably phospholipase A2, T-REC or K-REC-related proteins, β-glucosidase, β-glucocerebrosidase, arylsulfatase A, factor VIII, insulin-like growth factor 1 (IGF-1), surfactant protein A, surfactant protein B, aspartyl-β-glucosaminidase, acetyl-CoA α-glucosaminide, or acetyl-CoA arylamine. N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylglucosaminidase, acid ceramidase, aspartoacylase, lysosomal acid lipase, acid sphingomyelinase, arylsulfatase B, α-L-fucosidase, galactosylceramidase, galactocerebrosidase, β-galactosidase, protective protein / cathepsin A, β-glucuronidase, heparan N-sulfatase, β-hexosaminidase A, hyaluronidase-1, α-D-mannosidase, β-mannosidase, α-neuraminidase, β-hexosaminidase These are sequences derived from genes encoding saminidase A, β-hexosaminidase B, palmitoylated protein thioesterase, tripeptidyl peptidase I, battenin, ceroid lipofuscinosis neuron protein 5 (CLN5), ceroid lipofuscinosis neuron protein 6 (CLN6), ceroid lipofuscinosis neuron protein 7 (CLN7), ceroid lipofuscinosis neuron protein 8 (CLN8), (cathepsin D), cystinosine, cathepsin K, sialin, lysosome-associated membrane protein 2 (LAMP2), human growth hormone, follicle-stimulating hormone, erythropoietin, or granulocyte colony-stimulating factor (G-CSF).

[0053] In some embodiments, the donor molecule includes a sequence derived from a gene that codes for a protein secreted by the cell.

[0054] In some embodiments, the RNA molecule includes a non-distinguishable guide region that targets a pair of AAVS1 alleles.

[0055] In some embodiments, the RNA molecule includes a non-distinguishable guide region that targets intron 1 of the PPP1R12C allele containing the AAVS1 site.

[0056] In some embodiments, the RNA molecule includes a non-distinguishable guide region that targets a sequence located within a genomic range selected from 19:55115657-55115880, 19:55115981-55117130, 19:55115881-55115980, 19:55115557-55115656, and 19:55112832-55115556.

[0057] Aspects of this invention provide modified cells obtained by the method of the aspect described in the specification.

[0058] According to some embodiments, the cells are stem cells, hematopoietic stem cells (HSCs), iPSCs, lymphocytes, hepatocytes, or neurons.

[0059] An aspect of this invention provides an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within a sequence shown in any of sequence numbers 1 to 24195.

[0060] One aspect of this invention provides a composition comprising an RNA molecule and at least one CRISPR nuclease.

[0061] According to some embodiments, the composition further contains a donor molecule.

[0062] In some embodiments, the donor molecules include α1-antitrypsin, glucose-6-phosphatase (G6PC), serpine family A member (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthase, arginase, argininosuccinate, carbamoyl phosphate synthase, and N-acetylglutamate synthase, α-galactosidase A, coagulation factor IX, coagulation factor VII, lysosomal α-glucosidase, fibrinogen, and phenylalanine. The sequences include those derived from genes encoding 4-hydroxylase, alkaline phosphatase, glucosylceramidase, β-galactosidase, porphobilinogen deaminase, arylsulfatase B, β-glucuronidase, α-N-acetylglucosaminidase, lysosomal α, α-L-idulonidase, mannosidase, phosphatidylcholinesterol acyltransferase, N-sulfoglucosamine sulfohydrolase, coagulation factor X, N-acetylgalactosamine-6-sulfatase, sphingomyelin phosphodiesterase, iduronic acid-2-sulfatase, lysosomal α-glucosidase, cyclin-dependent kinase-like 5, pro-low-density lipoprotein receptor-related protein 1, phenylalanine ammonia lyase, protein glutamine γ-glutamyltransferase K, or lysosomal protective proteins.

[0063] In some embodiments, the donor molecule may be acid α-glucosidase, α-L-iduronidase, α-galactosidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, lysophosphatidylcholine metabolism-related proteins, preferably phospholipase A2, T-REC or K-REC-related proteins, β-glucosidase, β-glucocerebrosidase, arylsulfatase A, factor VIII, insulin-like growth factor 1 (IGF-1), surfactant protein A, surfactant protein B, aspartyl-β-glucosaminidase, acetyl-CoA α-glucosaminide, or acetyl-CoA arylamine. N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylglucosaminidase, acid ceramidase, aspartoacylase, lysosomal acid lipase, acid sphingomyelinase, arylsulfatase B, α-L-fucosidase, galactosylceramidase, galactocerebrosidase, β-galactosidase, protective protein / cathepsin A, β-glucuronidase, heparan N-sulfatase, β-hexosaminidase A, hyaluronidase-1, α-D-mannosidase, β-mannosidase, α-neuraminidase, β-hexosaminidase The sequence contains genes that encode saminidase A, β-hexosaminidase B, palmitoylated protein thioesterase, tripeptidyl peptidase I, battenin, ceroid lipofuscinosis neuron protein 5 (CLN5), ceroid lipofuscinosis neuron protein 6 (CLN6), ceroid lipofuscinosis neuron protein 7 (CLN7), ceroid lipofuscinosis neuron protein 8 (CLN8), (cathepsin D), cystinosine, cathepsin K, sialin, lysosome-associated membrane protein 2 (LAMP2), human growth hormone, follicle-stimulating hormone, erythropoietin, or granulocyte colony-stimulating factor (G-CSF).

[0064] In some embodiments, the donor molecule includes a sequence derived from a gene that codes for a protein secreted by the cell.

[0065] According to some embodiments, the composition further contains a tracrRNA molecule. Embodiments of this invention provide a method for modifying or editing an AAVS1 allele in a cell, the method comprising delivering the cell a composition according to the embodiment described in the specification.

[0066] Aspects of this invention provide the use of the compositions described in the specification in the modification or editing of the AAVS1 allele within a cell, wherein such use includes delivering the compositions described in the specification to the cell.

[0067] Aspects of this invention provide a pharmaceutical product comprising a composition as described in the specification for use in modifying or editing the AAVS1 allele within a cell, wherein the pharmaceutical product is administered to the cell by delivering the composition as described in the specification.

[0068] Aspects of this invention provide a kit for modifying or editing the AAVS1 allele in a cell, the kit comprising an RNA molecule, a CRISPR nuclease and / or a tracrRNA molecule as described in the specification; and instructions for delivering the RNA molecule; the CRISPR nuclease and / or the tracrRNA to the cell. In some aspects, the kit further comprises a donor molecule and instructions for delivering the donor molecule to the cell.

[0069] Aspects of this invention provide a gene editing composition containing an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within a sequence shown in any of Sequence IDs 1 to 24195. 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.

[0070] In some embodiments, the RNA molecule further comprises a portion having a tracrmate sequence.

[0071] In some embodiments, the RNA molecule may further include one or more linker moieties.

[0072] In aspects of this invention, the length of an RNA molecule may be up to 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. The feasibility of each is a separate aspect. In aspects of this invention, the length of an RNA molecule may be up to 17 to 300 nucleotides, up to 100 to 300 nucleotides, up to 150 to 300 nucleotides, up to 100 to 500 nucleotides, up to 100 to 400 nucleotides, up to 200 to 300 nucleotides, up to 100 to 200 nucleotides, or up to 150 to 250 nucleotides. The feasibility of each is different in nature.

[0073] According to some aspects of this invention, the composition further contains a tracrRNA molecule.

[0074] Some aspects of this invention provide a method for modifying or editing the AAVS1 allele in a cell, the method comprising delivering to the cell a composition containing an RNA molecule having a guide sequence portion having 17 to 50 consecutive nucleotides in a sequence shown in any of SEQ ID NOs: 1 to 24195, and a CRISPR nuclease. In some aspects, the composition further contains a donor molecule.

[0075] Some aspects of this invention provide a method for treating a disorder or disease, the method comprising delivering to target cells having the disorder or disease a composition containing an RNA molecule having a guide sequence portion having 17 to 50 consecutive nucleotides in a sequence shown in any of SEQ ID NOs: 1 to 24195, and a CRISPR nuclease. In some aspects, the composition further contains a donor molecule.

[0076] Some aspects of this invention provide a method for treating a disorder or disease, the method comprising delivering a composition of the above aspect to cells of a subject having the disorder, or delivering modified cells of the above aspect to the subject.

[0077] In some aspects, the disorder or disease is Pompe disease, mucopolysaccharidosis type 1, Fabry disease, mucopolysaccharidosis type 2, mucopolysaccharidosis type IVA, mucopolysaccharidosis type VI, adrenoleukodystrophy, severe combined immunodeficiency, Gaucher disease, metachromatic leukodystrophy (MLD), primary immunodeficiency, hemophilia A, hemophilia B, IGF1 deficiency, surfactant deficiency, aspartylglucosamineuria, Sanfilippo syndrome, mucopolysaccharidosis type 3, Sanfilippo syndrome type 3d, Iesel's disease, Schindler's disease, Faber's disease (FD), spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), Canavan disease, lysosomal acid lipase deficiency, Niemann-Pick disease, mucopolysaccharidosis type 6, fucosidosis, Krabbe disease, GM1 gangliosidosis, mucopolysaccharidosis type IVB (MPS) IVB), or galactosialidosis, Sleigh's disease, mucopolysaccharidosis type III, late-onset Tay-Sachs disease, hyaluronidase 1 deficiency, α-mannosidosis, β-mannosidosis, sialidosis, Stanhoff disease, Santavori-Hartia disease, Jansky-Beersawski disease, Batten disease, neurogenic ceroid lipofuscinosis type 5, neurogenic ceroid lipofuscinosis type 6, neurogenic ceroid lipofuscinosis type 7, neurogenic ceroid lipofuscinosis type 8, congenital cathepsin D deficiency, cystinosis, concentrated dysostosis, Salla disease, Danon disease, and / or α1-antitrypsin deficiency.

[0078] Some aspects of this invention provide a pharmaceutical comprising the composition of the above aspect for use in modifying AAVS1 in cells, the pharmaceutical being administered to the cells by delivering the composition of the above aspect.

[0079] Some aspects of this invention provide the use of the composition or modified cells of the above aspects for the treatment, improvement or prevention of a disorder or disease, wherein such use involves delivering the composition or modified cells of the above aspects to cells of a subject having or at risk of having the above aspects.

[0080] Some aspects of this invention provide a pharmaceutical product for use in the treatment, improvement, or prevention of a disorder or disease, comprising a composition of the above-described aspect or modified cells of the above-described aspect, wherein the pharmaceutical product delivers the composition of the above-described aspect or modified cells of the above-described aspect to cells of a subject having or at risk of having the above-described aspect.

[0081] In some aspects, the disorder or disease is Pompe disease, mucopolysaccharidosis type 1, Fabry disease, mucopolysaccharidosis type 2, mucopolysaccharidosis type IVA, mucopolysaccharidosis type VI, adrenoleukodystrophy, severe combined immunodeficiency, Gaucher disease, metachromatic leukodystrophy (MLD), primary immunodeficiency, hemophilia A, hemophilia B, IGF1 deficiency, surfactant deficiency, aspartylglucosamineuria, Sanfilippo syndrome, mucopolysaccharidosis type 3, Sanfilippo syndrome type 3d, Iesel's disease, Schindler's disease, Faber's disease (FD), spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), Canavan disease, lysosomal acid lipase deficiency, Niemann-Pick disease, mucopolysaccharidosis type 6, fucosidosis, Krabbe disease, GM1 gangliosidosis, mucopolysaccharidosis type IVB (MPS) IVB), or galactosialidosis, Sleigh's disease, mucopolysaccharidosis type III, late-onset Tay-Sachs disease, hyaluronidase 1 deficiency, α-mannosidosis, β-mannosidosis, sialidosis, Stanhof's disease, Santavori-Hartia disease, Jansky-Beersawski disease, Batten disease, neurogenic ceroid lipofuscinosis type 5, neurogenic ceroid lipofuscinosis type 6, neurogenic ceroid lipofuscinosis type 7, neurogenic ceroid lipofuscinosis type 8, congenital cathepsin D deficiency, cystinosis, concentrated dysostosis, Salla disease, Danon disease, and / or α1-antitrypsin deficiency.

[0082] In some manifestations, the disorder or disease is a lysosomal storage disorder.

[0083] In some embodiments, the disorder or disease is a disorder or disease of the blood, brain, lungs, or central nervous system.

[0084] In some embodiments, the composition of the above embodiment or the modified cells of the above embodiment are pharmaceuticals for enzyme replacement therapy.

[0085] Some aspects of this invention provide a method for treating a disease or disorder, the method comprising an immunotherapy that delivers modified cells according to the above aspects to a subject.

[0086] In some cases, the disease or disorder is cancer.

[0087] In some embodiments, the composition of the above embodiment or the modified cells of the above embodiment are used for the treatment, improvement, or prevention of a disorder or disease.

[0088] Some aspects of this invention provide a method for modifying a DNA target site in a target cell, wherein the modification of the DNA target site induces the cell to express a desired protein encoded by the modification, and the method comprises delivering to the target a composition comprising an RNA molecule containing a guide sequence portion having 17 to 50 consecutive nucleotides in a sequence shown in any of SEQ ID NOs: 1 to 24195, and a CRISPR nuclease. In some aspects, the composition further contains a donor molecule.

[0089] According to aspects of this invention, at least one CRISPR nuclease and an RNA molecule, or an RNA molecule, are delivered to a target and / or cell substantially simultaneously or at different time points.

[0090] In some embodiments, the tracrRNA molecule is delivered to the target and / or cell substantially simultaneously or at different time points with the CRISPR nuclease and the RNA molecule, or with the RNA molecule.

[0091] According to an aspect of this invention, the RNA molecule targets an alternative splicing signal sequence between the exon and intron of the PPP1R12C allele containing the AAVS1 site.

[0092] According to aspects of this invention, the RNA molecule is non-distinguishable and targets a sequence present in a pair of PPP1R12C alleles. In some aspects, the target sequence is present in a pair of PPP1R12C alleles. In some aspects, the sequence is present in an intron of the PPP1R12C gene.

[0093] Any or a combination of the above strategies for modifying or editing the AAVS1 region may be used in connection with this invention.

[0094] In some embodiments, the method involves contacting at least one allele of a target gene with a non-distinguishable RNA molecule (e.g., an RNA molecule containing a guide sequence region capable of targeting a pair of alleles of a gene) and a CRISPR nuclease (e.g., the Cas9 protein), the non-distinguishable RNA molecule and the CRISPR nuclease binding to the nucleotide sequence of at least one allele of the target gene to modify or edit at least one allele. In particular, while the introduction of a non-distinguishable RNA molecule into a cell may result in bi-allele cleavage, the insertion of nucleotide sequences at the cleavage site may occur in only one allele, not both. Therefore, induction of bi-allele cleavage by a non-distinguishable RNA molecule targeting the intron of the PPP1R12C allele containing the AAVS1 site may result in the maintenance of endogenous PPP1R12C gene expression from one allele, while the other PPP1R12C allele may be subjected to the introduction of a nucleotide sequence, such as one derived from a donor molecule. Introducing a nucleotide sequence into the AAVS1 site may or may not inhibit the expression of the PPP1R12C coding gene product containing the AAVS1 site.

[0095] In some embodiments, the method involves contacting an allele of a target gene with an RNA molecule and a CRISPR nuclease (e.g., Cas9 protein), wherein the RNA molecule and CRISPR nuclease modify or edit the target allele by binding to a nucleotide sequence of the target gene allele that is at least one nucleotide different from the nucleotide sequence of the other allele of the target gene.

[0096] In some embodiments, RNA molecules and CRISPR nucleases are introduced into cells encoding the gene of interest. In some embodiments, the cells encoding the gene of interest are cells of the target mammal.

[0097] The embodiments of the compositions described in this specification comprise at least one CRISPR nuclease, an RNA molecule containing a guide sequence, and a tracrRNA molecule, the tracrRNA molecule may be separated from or ligated to the RNA molecule containing the guide sequence, and will be simultaneously effective in the subject or cell. The at least one CRISPR nuclease, the RNA molecule containing the guide sequence, and the tracrRNA may be delivered substantially simultaneously, or at different time points, but will be effective at the same time. For example, this may involve delivering the CRISPR nuclease to the subject or cell before the RNA molecule containing the guide sequence and / or the tracrRNA are substantially present in the subject or cell.

[0098] In some embodiments, the cell is a dividing cell. In some embodiments, the cell is a non-dividing cell. In some embodiments, the cell is a terminated cell. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a hematopoietic stem cell (HSC). In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, the cell is a hematopoietic stem progenitor cell (HSPC). In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a neuron.

[0099] Genetic AAVS1 safe harbor knock-in for treating diseases and disorders In some embodiments, the method of this invention may be used to knock in a sequence into the safe harbor region of AAVS1. In some embodiments, the AAVS1-mediated expression of the knocked-in sequence is involved in or associated with the treatment of a disorder or disease.

[0100] For example, in some embodiments, the AAVS1 DNA target site in target cells is modified so that the target cells express and secrete a protein product encoded by the modification (e.g., an introduced or knocked-in protein coding sequence). These target cells may be used, for example, to treat lysosomal storage disorders or other disorders of the blood, lungs, brain, or central nervous system. In some embodiments, these modified cells serve as an alternative to conventional enzyme replacement therapy. In some embodiments, these modified cells are used in immunotherapies such as cancer immunotherapy.

[0101] Examples, though not limited to those mentioned, include the expression of a knock-in sequence that may be involved in or associated with the treatment of diseases or disorders of the blood, lungs, brain, or central nervous system. Examples, though not limited to those mentioned, include the sequences or portions thereof of A1AT, G6PC, SERPINA, TTR, ornithine transcarbamylase, argininosuccinate synthase, arginase, argininosuccinate, carbamoyl phosphate synthase, or N-acetylglutamate synthase.

[0102] Examples, though not limited to those mentioned, include the potential involvement or association of knock-in sequence expression with the treatment of lysosomal storage disorders or other disorders. Examples, though not limited to those mentioned, include the modification of target cells (e.g., monocytes or macrophages) to express α-galactosidase A, coagulation factor IX, coagulation factor VII, lysosomal α-glucosidase, fibrinogen, and phenylalanine. 4-hydroxylase, alkaline phosphatase, glucosylceramidase, β-galactosidase, porphobilinogen deaminase, arylsulfatase B, β-glucuronidase, α-N-acetylglucosaminidase, lysosomal α, α-L-idulonidase, mannosidase, phosphatidylcholinesterol acyltransferase, N-sulfoglucosamine sulfohydrolase, coagulation factor X, N-acetylgalactosamine-6-sulfatase, sphingomyelin phosphodiesterase, α-1 antitrypsin, 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, lysosomal protective proteins, or a portion thereof may be expressed.

[0103] Without limiting examples, the expression of knock-in sequences may be involved in or related to the treatment of the following diseases or disorders (each with the associated gene or enzyme listed in parentheses): Pompe disease (acid α-glucosidase), mucopolysaccharidosis type I (α-L-iduronidase), Fabry disease (α-galactosidase), mucopolysaccharidosis type II (iduronic acid-2-sulfatase), mucopolysaccharidosis type IVA (N-acetylgalactosamine-6-sulfatase), mucopolysaccharidosis type VI (N-acetylgalactosamine- 4-sulfatase), adrenoleukodystrophy (lysophosphatidylcholine metabolism-related genes, e.g., phospholipase A2), severe combined immunodeficiency (genes related to T-REC or K-REC), Gaucher disease (β-glucosidase or β-glucocerebrosidase), metachromatic leukodystrophy (MLD) (arylsulfatase A), primary immunodeficiency, hemophilia A and B (factor VIII), IGF1 deficiency (IGF-1), surfactant deficiency (surfactant protein A (SP-A) and / or This includes surfactant protein B (SP-B), aspartylglucosamiuria (aspartyl-β-glucosaminidase), Sanfilippo syndrome (acetyl-CoAα-glucosaminidase), mucopolysaccharidosis type III (acetyl-CoA-arylamine N-acetyltransferase), Sanfilippo syndrome type IIId (N-acetylglucosamine-6-sulfatase), Iesel's disease (N-acetylglucosamine-1-phosphotransferase), and Schindler's disease (α-N-acetylglucosaminidase). ), Faber's 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 (aryl sulfatase B), fucosidosis (α-L-fucosidase), Krabbe disease (galactosylceramidase, galactocerebrosidase), GM1 gangliosidosis, mucopolysaccharidosis type IVB (MPS)IVB) or galactosialidosis (galactosidase-β-1 or β-galactosidase (GLB1) or protective protein / cathepsin A), Sleigh's disease (β-glucuronidase), mucopolysaccharidosis type III (heparan N-sulfatase), late-onset Tay-Sachs disease (β-hexosaminidase A), hyaluronidase 1 deficiency (hyaluronidase-1), α-mannosidosis (α-D-mannosidase), β-mannosidosis (β-mannosidase), sialidosis (α-neuraminidase), Stanhof's disease (β-hexosaminidase A and / or β-hexosaminidase B), Santavori-Hartia disease ( (Palmitoylated protein thioesterase), Jansky-Beersawski disease (tripeptidyl peptidase I), Batten disease (Battenin), neuronal ceroid lipofuscinosis type 5 (ceroid lipofuscinosis neuron 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 (cystinosine), concentrated dysostosis (cathepsin K), Salah disease (sialin) and / or Danon disease (lysosome-associated membrane protein 2 (LAMP2)). As an example, though not limited to these, target cells (e.g., monocytes or macrophages) may be modified to express metabolic modulators. For example, the 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, granulorulocyte colony-stimulating factor (G-CSF), galactosamine-6-sulfatase and / or β-hexosamine enzyme.

[0104] In particular, the cells are present in target tissues, including the lungs and brain, and can serve as expression vectors to induce long-term protein secretion in these target tissues. Expression of the transgene under the control of the AAVS1 promoter is achieved by CRISPR-mediated knock-in at a safe harbor site targeted by the guide sequence portion described in the specification. The protein expressed by the transgene may be secreted. Therefore, the ability to prepare blood and / or tissues containing modified target cells and to induce continuous secretion of the desired protein serves as an alternative to enzyme replacement therapy (ERT). For example, modified monocytes may be useful for targeting the central nervous system (CNS) or the lungs and inducing the secretion of the desired protein in the target tissue.

[0105] Such approaches are also useful in the treatment of lysosomal storage disorders, for example. Some of these diseases exhibit phenotypes in the central nervous system (CNS). In particular, treating brain damage with enzyme replacement therapy is difficult due to the blood-brain barrier (BBB). Monocytes may be used for delivery across the BBB so that the target secreted protein is secreted in the CNS.

[0106] For example, modified cells delivered to the brain may be used to treat lysosomal storage disorders; modified cells delivered to the lungs may be used to treat antitrypsin deficiency (A1AT), D-surfactant deficiency, or protein deposition disorders; or modified cells in the blood may be used to treat A1AT or adenosine deaminase 2 (DADA2) deficiency.

[0107] CRISPR nuclease 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-induced DNA nuclease. In some embodiments, the CRISPR complex further does not contain tracrRNA. In examples where the RNA-induced DNA nuclease is not limited to a CRISPR protein, at least one nucleotide different between the AAVS1 alleles may be located within and / or near the PAM site in the region where the RNA molecule is designed to hybridize. Those skilled in the art will understand that the RNA molecule can be manipulated in ways commonly known in the art to bind to a selected target in the genome.

[0108] In this specification, the term "PAM" refers to a nucleotide sequence of target DNA located near the target DNA sequence and recognized by the CRISPR nuclease complex. The PAM sequence may differ depending on the type of nuclease. In addition, there are CRISPR nucleases that can target almost all PAMs. In some aspects of this invention, the CRISPR system utilizes one or more RNA molecules having a guide sequence portion that guides the CRISPR nuclease to the target DNA site by forming a Watson-Crick base pair between the protospacer and the guide sequence portion of the target DNA site adjacent to the protospacer-adjacent motif (PAM), which is an additional requirement for target recognition. The CRISPR nuclease then cleaves the target DNA site, resulting in a double-strand break within the protospacer. In a non-limiting example, a type II CRISPR system utilizes a mature crRNA:tracrRNA complex that guides a CRISPR nuclease (e.g., Cas9) to target DNA by forming a Watson-Crick base pair between a protospacer on target DNA adjacent to a protospacer-adjacent motif (PAM) and a guide sequence portion of the crRNA. Those skilled in the art will understand that the manipulated RNA molecule of this invention may be further designed to bind to a target genomic DNA sequence of interest adjacent to a protospacer-adjacent motif (PAM), for example, a PAM corresponding to a sequence associated with the CRISPR nuclease being utilized.PAM is, for example, not limited to, NGG or NAG (where N is any nucleic acid base) in Streptococcus pyogenes Cas9 WT (SpCAS9); NNGRRT (where N is any nucleic acid base) in Staphylococcus aureus (SaCas9); NNNVRYM (where Jejuni Cas9 WT); NGAN or NGNG (where SpCas9-VQR variant); NGCG (where SpCas9-VRER variant); NGAG (where SpCas9-EQR variant); NRRH (where N is any nucleic acid base, R is A or G, H is A, C or T) in SpCas9-NRTH variant; NRTH (where N is any nucleic acid base, R is A or G, H is A, C or T) in SpCas9-NRCH variant In Ant, NRCH (where N is any nucleic acid base, R is A or G, and H is A, C, or T); in the SpG variant of SpCas9, NG (where N is any nucleic acid base); in the SpCas9-NG variant of SpCas9, NG or NA (where N is any nucleic acid base); in the SpRY variant of SpCas9, NR, NRN, or NYN (where N is any nucleic acid base, R is A or G, and Y is C or T); Streptococcus canis In the canis)Cas9 variant (ScCas9), the format is NNG (where N is any nucleic acid base); in the Staphylococcus aureus SaKKH-Cas9 variant (SaCas9), it is NNNRRT (where N is any nucleic acid base and R is A or G); in Neisseria meningitidis (NmCas9), it is NNNNGATT (where N is any nucleic acid base); in Alicyclobacillus acidiphilus Cas12b (AacCas12b), it is TTN (where N is any nucleic acid base); or in Cpf1, it is TTTV (where V is A, C, or G). Each RNA molecule of this invention is designed to form a complex with one or more different CRISPR nucleases and is designed to target a desired polynucleotide sequence using one or more different PAM sequences corresponding to the CRISPR nucleases.

[0109] In some embodiments, RNA-inducible DNA nucleases (e.g., CRISPR nucleases) may be used to induce breaks in double-stranded or single-stranded DNA at desired locations in the cell's genome. While the most commonly used RNA-inducible DNA nucleases are derived from the CRISPR system, other RNA-inducible DNA nucleases are also intended for use in the genome editing compositions and genome editing methods described in this specification. See, for example, U.S. Patent Application Publication 2015 / 0211023, incorporated by reference in this specification.

[0110] A wide range of CRISPR systems can be used in carrying out this invention. The CRISPR system may be a type I, type II, or type III system. Examples of suitable CRISPR proteins, though not limited to them, include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, Casl This includes 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, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966.

[0111] In some embodiments, the RNA-induced DNA nuclease is a CRISPR nuclease derived from the type II CRISPR system (e.g., Cas9). CRISPR nucleases are effective against Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Neisseria meningitidis, Treponema denticola, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, and Streptosporangium roseum. roseum), Streptosporandium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas species (Polaromonas Crocosphaera watsonii, Cyanothece sp.), Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum Thermopropionicum), Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.The CRISPR nuclease may 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 non-cultured bacteria may also be used in this invention (see Burstein et al. Nature, 2017). Variants of CRISPR proteins having a known PAM sequence, such as the SpCas9 D1135E variant, SpCas9 VQR variant, SpCas9 EQR variant, or SpCas9 VRER variant, may also be used in this invention.

[0112] Therefore, CRISPR-based RNA-inducible DNA nucleases such as Cas9 protein or modified Cas9, or homologs or orthologues of Cas9, or other RNA-inducible DNA nucleases belonging to other CRISPR systems such as Cpf1 and its homologs and orthologues, may also be used in the composition of this invention. Other CRISPR nucleases, such as those described in International Publication Nos. 2020 / 2235I4, 2020 / 223553, 2022 / 087135, 2022 / 170199, 2022 / 170216, 2022 / 2262I5, 2023 / 091987, and 2023 / 019269 (each incorporated into the specification by reference), may also be used.

[0113] In certain embodiments, CRISPR nucleases may be “functional derivatives” of naturally occurring Cas proteins. “Functional derivatives” of natural sequence polypeptides are compounds that have biological properties qualitatively common to the natural sequence polypeptide. “Functional derivatives” include, but are not limited to, fragments of natural sequence polypeptides and derivatives of natural sequence polypeptides and their fragments that share biological activity with the corresponding natural sequence polypeptide. The biological activity referred to here is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term “derivative” encompasses both amino acid sequence variants of polypeptides and their covalent modifications and fusions. Suitable derivatives of Cas polypeptides or their fragments include, but are not limited to, variants, fusions, and covalent modifications of Cas proteins or their fragments. Derivatives include, but are not limited to, CRISPR nickase, catalytically inactive CRISPR nucleases or “dead” CRISPR nucleases, and fusions of CRISPR nucleases or their derivatives with other enzymes such as base editors or retrotransposons. See, for example, Anzalone et al. (2019) and PCT International Patent Application No. PCT / US2020 / 037560.

[0114] Cas proteins, including Cas proteins or their fragments, as well as derivatives of Cas proteins or their fragments, may be obtained from cells, chemically synthesized, or by a combination of these methods. The cells may be naturally occurring Cas protein-producing cells, or genetically engineered cells that naturally produce Cas proteins and produce endogenous Cas proteins at higher expression levels, or produce Cas proteins from introduced exogenous nucleic acids encoding the same or different Cas as endogenous Cas. In some cases, cells that do not naturally produce Cas proteins are genetically engineered to produce them.

[0115] In some embodiments, the CRISPR nuclease is Cpf1. Cpf1 is a single RNA-inducible endonuclease that utilizes T-rich protospacer-adjacent motifs. Cpf1 cleaves DNA double strands in a staggered manner. Two Cpf1 enzymes from the genera Acidaminococcus and the family Lachnospiraceae have been shown to efficiently perform genome editing in human cells (see Zetsche et al., 2015).

[0116] Therefore, the present invention may also use type II CRISPR RNA-induced DNA nucleases such as Cas9 protein or modified Cas9, or homologs, orthologues, or variants of Cas9, or other RNA-induced DNA nucleases belonging to other CRISPR systems, such as Cpf1 and its homologs, orthologues, or variants.

[0117] In some embodiments, the guide molecule includes one or more chemical modifications that confer new or improved properties (e.g., stability against degradation, hybridization energy, or binding to RNA-induced DNA nucleases). Suitable chemical modifications include, but are not limited to, modified bases, modified sugars, or modified nucleoside bonds. Examples of suitable chemical modifications that are not limited to these include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methylpsoiduridine, β,D-galactosyl quosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methyl Chilpsoiduridine, 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-mannosylquosine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine 5-Methoxyuridine, 2-Methylthio-N6-isopentenyladenosine, N-((9-β-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine, N-((9-β-D-ribofuranosylpurine-6-yl)N-methylcarbamoyl)threonine, Uridine-5-oxyacetate methyl ester, Uridine-5-oxyacetate, Weibtoxosin, Quosin, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine This includes 4-thiouridine, 5-methyluridine, N-((9-β-D-ribofuranosylpurine-6-yl)-carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, weibtosine, 3-(3-amino-3-carboxypropyl)uridine, (acp3)u, 2'-O-methyl (M), 3'-phosphorothioate (MS), 3'-thioPACE (MSP), pseudouridine, or 1-methylpsoiduridine.Each aspect of this invention is a distinct possibility.

[0118] Guide sequence targeting the AAVS1 allele A given RNA molecule containing a guide sequence portion used to target a DNA site may result in RNA molecule degradation, limited activity, lack of activity, or off-target effects. Therefore, an appropriate guide sequence portion is necessary to target a given DNA site in a gene.

[0119] This invention identifies a novel set of guide sequence regions that target at least one AAVS1 allele and introduce a nucleotide sequence expressed under the control of the AAVS1 promoter into at least one allele. Such gene editing approaches may be used to treat disorders or diseases or to modify cellular behavior. Preferably, non-distinguishable RNA molecules capable of targeting a pair of AAVS1 alleles are used for targeting.

[0120] In some embodiments of this invention, an RNA molecule is used to target the AAVS1 site and introduce or knock in an exogenous nucleotide sequence to the AAVS1 site. In some embodiments, the location of the target site is near the intended knock-in site, preferably near a start codon or stop codon, and preferably within 150 nucleotides from the start codon or stop codon.

[0121] Delivery to cells The compositions described in this specification may be delivered to target cells by appropriate means. The compositions of this invention may target cells containing and / or expressing the AAVS1 allele, such as mammalian cells. For example, in one embodiment, an RNA molecule that specifically targets the AAVS1 allele is delivered to target cells, which are stem cells, hematopoietic stem cells (HSCs), iPSCs, lymphocytes, hepatocytes, or neurons. Delivery to cells may be carried out in vitro, ex vivo, or in vivo. Furthermore, the nucleic acid compositions described in this specification may be delivered as one or more DNA molecules, RNA molecules, ribonucleoproteins (RNPs), nucleic acid vectors, or combinations thereof.

[0122] In some embodiments, in vivo delivery of the compositions described in the specification includes delivery by lentivirus, adeno-associated virus (AAV), or nanoparticles. The compositions may be in the form of RNP compositions. Therefore, delivery can be performed in vivo to target cells.

[0123] In some embodiments, the composition described in the specification is delivered to cells ex vivo. In some embodiments, the cells are dividing cells. In some embodiments, the cells are non-dividing cells. In some embodiments, the cells are terminated cells. In some embodiments, the cells are stem cells. In some embodiments, the cells are hematopoietic stem cells (HSCs). In some embodiments, the cells are induced pluripotent stem cells (iPSCs). In some embodiments, the cells are hematopoietic stem progenitor cells (HSPCs). In some embodiments, the cells are lymphocytes. In some embodiments, the cells are hepatocytes. In some embodiments, the cells are neurons. The composition may also be delivered to cells by known ex vivo delivery methods, including but not limited to electroporation, viral transduction, nanoparticle delivery, and liposomes. The composition may be in the form of an RNP composition. Details of the delivery methods are described throughout this section.

[0124] In some embodiments, the RNA molecules of the compositions described in the specification include chemical modifications. Examples of appropriate chemical modifications, though not limited to them, include 2'-O-methyl (M), 2'-O-methyl 3'-phosphorothioate (MS), or 2'-O-methyl, 3'-thioPACE (MSP), pseudouridine, and 1-methylpsoiduridine. Each of the feasibility of this invention is a separate embodiment.

[0125] Nucleic acid compositions (e.g., the RNA molecular compositions of this invention) may be delivered using appropriate viral vector systems. Nucleic acids and target tissues can be introduced using conventional viral and nonviral gene transfer methods. In certain embodiments, nucleic acids are administered for in vivo or ex vivo gene therapy. Nonviral vector delivery systems include bare nucleic acids and nucleic acids complexed with a delivery medium (e.g., liposomes or poloxamers). For reviews of gene therapy, see Anderson (1992); Nabel & Felgner (1993); Mitani & Caskey (1993); Dillon (1993); Miller (1992); Van Brunt (1988); Vigne (1995); Kremer & Perricaudet (1995); Haddada et al. (1995) and Yu et al. (1994).

[0126] Nonviral delivery methods for nucleic acids and / or proteins include electroporation, lipofection, microinjection, particle guns, particle gun acceleration, virosomes, liposomes, immunoliposomes, lipid nanoparticles (LNPs), polycations or lipids: nucleic acid conjugates, artificial virions, and nucleic acid uptake by accelerators, or nucleic acids and / or proteins can be delivered to plant cells by bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizobium meliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus, and cassava vein mosaic virus) (e.g., Chung See et al., 2006). For example, sonoporation using the Sonitron 2000 system (Rich-Mar) can also be used for nucleic acid delivery. Delivery of proteins and / or nucleic acids by cationic lipids is also planned 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)).

[0127] Nonviral 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 used in the target cells to transpose the polynucleotide sequence of the molecule of the composition or the polynucleotide sequence encoding the molecule of the composition.

[0128] Other representative nucleic acid delivery systems include those offered by Amaxa® Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, for example, U.S. Patent No. 6,008,336). Lipofection is described, for example, in U.S. Patents No. 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) or target tissues (in vivo) is possible.

[0129] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those skilled 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. Patents No. 4,186,183; No. 4,217,344; No. 4,235,871; No. 4,261,975; No. 4,485,054; No. 4,501,728; No. 4,774,085; No. 4,837,028 and No. 4,946,787).

[0130] Another delivery method involves packaging the nucleic acid to be delivered into an EnGeneIC delivery medium (EDV). EDVs are specifically delivered to target tissues using bispecific antibodies, where one arm of the antibody is specific to the target tissue and the other arm is specific to the EDV itself. The antibody carries the EDV to the surface of target cells, where it is transported into the cells via endocytosis. After entering the cell, the contents are released (see MacDiarmid et al., 2009).

[0131] The use of RNA or DNA viral systems for nucleic acid delivery leverages highly evolved methods by 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 process cells in vitro, and modified cells can be administered to patients (ex vivo). Conventional viral systems for nucleic acid delivery include, but are not limited to, retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, and herpes simplex virus vectors for gene transfer.

[0132] Retroviral tropism can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically have high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-terminal repeat sequences and have the ability to package foreign sequences up to 6–10 kb. A minimum cis-terminal repeat sequence (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 mouse leukemia virus (MuLV), gibbon 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); and International Publication No. 94 / 26877).

[0133] For gene transfer in clinical trials, at least six viral vectors are currently available, and they prepare transduction agents using approaches that include complementarity of deletion vectors with genes inserted into helper cell lines.

[0134] 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 over 50% (Ellem et al., (1997); Dranoff et al., 1997).

[0135] Packaging cells are used to form viral particles capable of infecting host cells. Such cells include adenoviruses, 293 cells for packaging AAV, and Psi-2 or PA317 cells for packaging retroviruses. Viral vectors used in gene therapy are typically prepared by a production cell line that packages nucleic acid vectors into viral particles. The vector usually contains the minimum viral sequence required for packaging and (where applicable) subsequent integration into the host, with other viral sequences replaced by expression cassettes encoding the proteins to be expressed. Missing viral function is supplied trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the terminal inversion (ITR) sequence derived from the AAV genome required for packaging and integration into the host genome. The viral DNA is packaged in a cell line that contains helper plasmids encoding other AAV genes, namely rep and cap, but lacks the ITR sequence. The cell line is also infected with adenovirus as a helper. Helper viruses promote the replication of AAV vectors and the expression of AAV genes from helper plasmids. Due to the deletion of the ITR sequence, helper plasmids are not packaged in large quantities. Adenovirus contamination can be mitigated, for example, by heat treatment, which makes adenoviruses more susceptible than AAV. Furthermore, AAV can be produced on a clinical scale using baculovirus systems (see U.S. Patent No. 7,479,554).

[0136] In many gene therapies, it is desirable that gene therapy vectors be delivered with a high degree of specificity to specific tissues. Therefore, viral vectors can be modified to exhibit specificity to a given cell by expressing a ligand as a fusion protein with the viral coating protein on the outer surface of the virus. The ligand is selected to have affinity for a receptor known to be present on the target cell. For example, Han et al. (1995) reported that Moloney mouse leukemia virus could be modified to express human heregurin fused with gp70, and that this recombinant virus infected specific human breast cancer cells expressing the human epidermal growth factor receptor. This principle can be extended to other virus-target cell combinations where the target cell expresses the receptor and the virus expresses a fusion protein containing the ligand for the cell surface receptor. For example, filamentous phages can be engineered to present antibody fragments (e.g., FAB or Fv) with specific binding affinity to substantially any chosen cell receptor. While this explanation primarily applies to viral vectors, the same principle can be applied to non-viral vectors. Such vectors can be manipulated to include specific uptake sequences that are preferred for uptake by particular target cells.

[0137] Gene therapy vectors can be delivered in vivo by individual patient administration, typically by systemic administration (e.g., intravitreous, intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection) or topical application, as described below. Alternatively, the vector can be delivered ex vivo to cells, such as cells extracted from individual patients (e.g., lymphocytes, bone marrow aspiration, tissue biopsy) or universal donor hematopoietic stem cells, and subsequently re-implanted into the patient after selection of cells incorporating the vector. Representative ex vivo approaches, though not limited to these, may include extracting tissue from the patient for culture (e.g., peripheral blood, bone marrow, and spleen), transferring nucleic acids into cultured cells (e.g., hematopoietic stem cells), and then transplanting the cells into the patient's target tissue (e.g., bone marrow and spleen). In some embodiments, the stem cells or hematopoietic stem cells may be further treated with viability enhancers.

[0138] Ex vivo cell transfection for diagnostic, 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., a patient). Various cells suitable for ex vivo transfection are well known to those skilled in the art (see, for example, Freshney, Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications (6th edition, 2010) and the literature cited in the discussion of methods for isolating and culturing cells from a patient).

[0139] Suitable cells include, but are not limited to, eukaryotic cells and / or cell lines. Examples of such cells, or cell lines prepared from such cells, include, but are not limited to, 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 cells (differentiated or undifferentiated), and insect cells (e.g., fall armyworm (Spodoptera)). This includes fugiperda (Sf) or fungal cells (e.g., Saccharomyces, Pichia, and Schizosaccharomyces). In certain embodiments, the cell line is the CHO-K1, MDCK, or HEK293 cell line. Furthermore, primary cells may be isolated and used ex vivo for reintroduction to the therapeutic target after treatment with an inducible nuclease system (e.g., CRISPR / Cas). Suitable primary cells include peripheral blood mononuclear cells (PBMCs) and other hematopoietic cell subsets, such as, but not limited to, CD4+ T cells or CD8+ T cells. Also suitable cells include, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neural stem cells, and mesenchymal stem cells.

[0140] In some embodiments, stem cells are processed ex vivo for cell transfection and gene therapy. The advantage of using stem cells is that they can be differentiated into other cells in vitro or introduced into mammals (such as cell donors) and transplanted into the bone marrow. Methods for differentiating CD34+ cells into clinically important immune cells in vitro using cytokines (e.g., GM-CSF, IFN-γ, and TNF-α) are known (see Inaba et al., 1992, for example, but not limited to these).

[0141] Stem cells are isolated for transduction and differentiation by known methods. For example, stem cells are isolated from bone marrow cells by panning them with antibodies that bind to unwanted cells such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and Iad (differentiated antigen-presenting cells) (see Inaba et al., 1992 for examples, but not limited to these). In some embodiments, modified stem cells may be used.

[0142] Vectors containing therapeutic nucleic acid compositions (e.g., retroviruses, liposomes) can also be administered directly to organisms for transduction into cells in vivo. Administration includes, but is not limited to, injection, infusion, topical application (e.g., eye drops and creams) and electroporation, any route commonly used to introduce the molecule into final contact with blood or tissue cells. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and multiple routes for administering a particular composition may be available, although a particular route often provides a more rapid and effective response than another. According to some embodiments, the composition is delivered by IV injection.

[0143] Vectors suitable for introducing transgenes into immune cells (e.g., T cells) include non-integrated lentiviral vectors. See, for example, U.S. Patent Application Publication 2009 / 0117617.

[0144] The pharmaceutically acceptable carrier is determined, in part, by the composition being administered, and further by the method of administration of the composition. Therefore, there is a wide range of suitable formulations of available pharmaceutical compositions, as described, for example, in Remington's Pharmaceutical Sciences, 17th ed., 1989.

[0145] Furthermore, the disclosed compositions and methods may be used in the manufacture of pharmaceuticals for treating diseases or disorders in patients.

[0146] AAVS1 Safe Harbor Knock-In Method While not bound by any theory or mechanism, this invention may be used to apply a CRISPR nuclease to the safe harbor site of the AAVS1 site (also known as the AAVS1 locus) to process the AAVS1 site in order to introduce a sequence into the safe harbor site of the AAVS1 site. A specific guide sequence may be selected from Table 1 based on the location of the target and the type of CRISPR nuclease used (e.g., according to the required PAM sequence).

[0147] The objective of the presented strategy is to knock in an expression cassette into the AAVS1 safe harbor site following CRISPR-mediated DNA cleavage. The coding sequence introduced into the AAVS1 safe harbor site may be expressed under the control of an endogenous promoter. More preferably, the introduced coding sequence may be expressed via a transcription element supplied by an exogenous donor molecule. For example, the HDR cassette may contain a target sequence and a promoter that controls the expression of the target sequence, so that both the target sequence and the promoter are introduced into the AAVS1 safe harbor site after CRISPR-mediated DNA cleavage.

[0148] For example, strategies for inducing breaks and knocking in expression cassettes by homologous recombination repair (HDR) or homology-independent targeted insertion (HITI) include, but are not limited to, (1) targeting the PPP1R12C allele containing the AAVS1 site using a nuclease and one RNA guide molecule to mediate a double-strand break, and (2) targeting the PPP1R12C allele containing the AAVS1 site using two single-strand nickases and two guide RNA molecules (one guide molecule for each nickase) to induce the nickases to mediate a nick on the complementary DNA strand of the target, thereby forming a double-strand break.

[0149] Furthermore, a donor molecule may be used to knock in a desired nucleotide sequence into the AAVS1 safe harbor site. The donor molecule containing the template for the insertion sequence may be, for example, single-stranded DNA, a plasmid, a PCR product, AVV, or an integrase-deficient lentivirus. Alternatively, the insertion sequence may be introduced as an RNA template in a CRISPR-based system that includes, but is not limited to, a reverse transcriptase editor induced by at least one guide RNA molecule.

[0150] This includes, but is not limited to, hematopoietic stem cells, iPSCs, lymphocytes, hepatocytes, and neurons; it encompasses all dividing and non-dividing cells.

[0151] Examples of RNA guide sequences that specifically target the AAVS1 site While numerous guide sequences can be designed to target the AAVS1 allele, the nucleotide sequences listed in Table 1, identified by SEQ ID NOs: 1–24195, were specifically selected to effectively perform the methods described in the specification and to effectively distinguish the alleles.

[0152] Table 1 shows guide sequences designed for use in the embodiments described above to associate specific sequences within the AAVS1 allele. Each manipulated guide molecule is further designed to bind to the target genomic DNA sequence of interest adjacent to a protospacer fringe motif (PAM) (e.g., a PAM corresponding to the sequence NGG or NAG (where N is any nucleic acid base)). The guide sequence is 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), and JeCas9WT (PAM sequence: NNNVRYM). Each RNA molecule of this invention is designed to form a complex with one or more different CRISPR nucleases and is designed to target a desired polynucleotide sequence using one or more different PAM sequences corresponding to the CRISPR nucleases.

[0153] [Table 1]

[0154] Examples are provided below to facilitate a more complete understanding of this invention. The following examples illustrate typical modes of constructing and carrying out this invention. However, the scope of this invention is not limited to the specific embodiments disclosed in these examples, and these embodiments are for illustrative purposes only. [Examples]

[0155] Experiment details Example 1: Analysis of AAVS1 on-target activity Guide sequences containing 17–50 consecutive nucleotides within any of the sequences shown in Sequence IDs 1–24195 are screened for high on-target activity in HeLa cells using CRISPR nucleases (e.g., OMNI-50, OMNI-79, OMNI-103). On-target activity is determined by DNA capillary electrophoresis. The OMNI-50, OMNI-79, and OMNI-103 nucleases are described in International Publication Nos. 2020 / 223514, 2021 / 248016, and 2022 / 170199, respectively, the contents of which are incorporated into this specification by reference.

[0156] Example 2: Analysis of AAVS1 on-target activity The editing activity of a total of 16 nucleases and 39 guide sequences was screened. HeLa cells were transfected with plasmids encoding nucleases and guide sequences, as shown in Table 2. Cells were harvested 72 hours after DNA transfection, genomic DNA was extracted, the target guide regions were amplified, and analyzed by NGS. Table 2 shows the mean editing rates and standard deviations in two independent experiments.

[0157] [Table 2-1]

[0158] [Table 2-2]

[0159] [Table 2-3]

[0160] Figure 1A-1I shows the data from Table 2 in a graph, illustrating the editing rates for various OMNI nucleases by guide sequence.

[0161] Figure 1A shows the editing rates for AAVS_s29, AAVS_s30, AAVS_s31, AAVS_s37, and AAVS_s43 using OMNI-159. Figure 1B shows the editing rates for AAVS_s4~AAVS_s7 and AAVS_s10~AAVS_s15 using OMNI-103. Figure 1C shows the editing rates for AAVS_s33~AAVS_s35 using OMNI-110. Figure 1D shows the editing rates for AAVS_s22~AAVS_s26 and AAVS_s39~AAVS_s41 using OMNI-274. Figure 1E shows the editing rates for AAVS_s17~AAVS_s19 using OMNI-308. Figure 1F shows the edit rates for AAVS_s3 using OMNI-50, AAVS_s39 and AAVS_s40 using OMNI-75, AAVS_s41 using OMNI-93, AAVS_s32 using OMNI-127, AAVS_s27 and AAVS_s28 using OMNI-231, AAVS_s43 using OMNI-269, AAVS_s20, AAVS_s21 and AAVS_s39-AAVS_s42 using OMNI-281, AAVS_s37 using OMNI-286, AAVS_s36 and AAVS_s38 using OMNI-291, AAVS_s36-AAVS_s38 using OMNI-302, and AAVS_s16 using OMNI-366.

[0162] The OMNI nuclease of Example 2 is described in International Publication No. 2020 / 223514 (OMNI-50); International Publication No. 2022 / 087135 (OMNI-75); International Publication No. 2022 / 170199 (OMNI-103); International Publication No. 2022 / 170216 (OMNI-93, OMNI-110); International Publication No. 2022 / 226215 (OMNI-231) This is described in International Publication No. 2023 / 091987 (OMNI-269, OMNI-274, OMNI-281, OMNI-286, OMNI-291, OMNI-302, OMNI-308, OMNI-366) and International Publication No. 2023 / 019269 (OMNI-127, OMNI-159), the contents of which are incorporated into the specification by reference.

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Claims

1. A method for modifying at least one adeno-associated virus integration site 1 (AAVS1) site within a cell, At least one CRISPR nuclease, or a sequence encoding a CRISPR nuclease; and An RNA molecule containing a guide sequence of 17 to 50 nucleotides, or a nucleotide sequence encoding it. The step includes introducing a composition containing into the cells, A method comprising a complex of the CRISPR nuclease and the RNA molecule cleaving the double-strand of at least one AAVS1 site.

2. The method according to claim 1, wherein the composition further comprises a donor molecule containing a nucleotide sequence to be introduced into the double-strand break site, and the expression of the introduced sequence is mediated by an endogenous gene promoter, or more preferably by an exogenous promoter.

3. The sequences to be introduced include α1-antitrypsin, glucose-6-phosphatase (G6PC), serpine family A member (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthase, arginase, argininosuccinate, carbamoyl phosphate synthase, and N-acetylglutamate synthase, α-galactosidase A, coagulation factor IX, coagulation factor VII, lysosomal α-glucosidase, fibrinogen, and phenylalanine. The method according to claim 2, wherein the sequence is derived from a gene encoding 4-hydroxylase, alkaline phosphatase, glucosylceramidase, β-galactosidase, porphobilinogen deaminase, arylsulfatase B, β-glucuronidase, α-N-acetylglucosaminidase, lysosomal α, α-L-idulonidase, mannosidase, phosphatidylcholinesterol acyltransferase, N-sulfoglucosamine sulfohydrolase, coagulation factor X, N-acetylgalactosamine-6-sulfatase, sphingomyelin phosphodiesterase, iduronic acid-2-sulfatase, lysosomal α-glucosidase, cyclin-dependent kinase-like 5, pro-low-density lipoprotein receptor-related protein 1, phenylalanine ammonia lyase, protein glutamine γ-glutamyltransferase K, or a lysosomal protective protein.

4. The sequences to be introduced include acid α-glucosidase, α-L-iduronidase, α-galactosidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, lysophosphatidylcholine metabolism-related proteins, preferably phospholipase A2, T-REC or K-REC-related proteins, β-glucosidase, β-glucocerebrosidase, arylsulfatase A, factor VIII, insulin-like growth factor 1 (IGF-1), surfactant protein A, surfactant protein B, aspartyl-β-glucosaminidase, acetyl-CoA α-glucosaminide, and acetyl-CoA-arylamine. N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylglucosaminidase, acid ceramidase, aspartoacylase, lysosomal acid lipase, acid sphingomyelinase, arylsulfatase B, α-L-fucosidase, galactosylceramidase, galactocerebrosidase, β-galactosidase, protective protein / cathepsin A, β-glucuronidase, heparan N-sulfatase, β-hexosaminidase A, hyaluronidase-1, α-D-mannosidase, β-mannosidase, α-neuraminidase, β-hexosaminidase A, The method according to claim 2, wherein the sequence is derived from a gene encoding β-hexosaminidase B, palmitoylated protein thioesterase, tripeptidyl peptidase I, battenin, ceroid lipofuscinosis neuron protein 5 (CLN5), ceroid lipofuscinosis neuron protein 6 (CLN6), ceroid lipofuscinosis neuron protein 7 (CLN7), ceroid lipofuscinosis neuron protein 8 (CLN8), (cathepsin D), cystinosine, cathepsin K, sialin, lysosome-associated membrane protein 2 (LAMP2), human growth hormone, follicle-stimulating hormone, erythropoietin, CD19, or granulocyte colony-stimulating factor (G-CSF).

5. The method according to claim 2, wherein the donor molecule includes a sequence derived from a gene encoding a protein secreted by the cell.

6. The method according to any one of claims 1 to 5, wherein the RNA molecule includes a non-distinguishable guide portion that targets the PPP1R12C allele containing the AAVS1 site.

7. The method according to any one of claims 1 to 5, wherein the RNA molecule includes a non-distinguishable guide portion that targets a sequence located within a genomic range selected from any one of 19:55115657-55115880, 19:55115981-55117130, 19:55115881-55115980, 19:55115557-55115656 and 19:55112832-55115556.

8. The method according to any one of claims 1 to 7, wherein the PPP1R12C allele containing the modified AAVS1 site expresses the PPP1R12C gene product.

9. Modified cells obtained by the method described in any one of claims 1 to 8.

10. The modified cell according to claim 9, wherein the modified cell is a stem cell, hematopoietic stem cell (HSC), iPSC, lymphocyte, hepatocyte, or neuron.

11. A composition containing an RNA molecule that includes a guide sequence portion having 17 to 50 consecutive nucleotides within a sequence shown in any of Sequence IDs 1 to 24195.

12. The composition according to claim 11, further comprising at least one CRISPR nuclease.

13. The composition according to claim 11 or 12, further comprising a donor molecule.

14. The donor molecules include α1-antitrypsin, glucose-6-phosphatase (G6PC), serpine family A member (SERPINA), transthyretin (TTR), ornithine transcarbamylase, argininosuccinate synthase, arginase, argininosuccinate, carbamoyl phosphate synthase, and N-acetylglutamate synthase, α-galactosidase A, coagulation factor IX, coagulation factor VII, lysosomal α-glucosidase, fibrinogen, and phenylalanine. The composition according to claim 13, comprising a sequence derived from a gene encoding 4-hydroxylase, alkaline phosphatase, glucosylceramidase, β-galactosidase, porphobilinogen deaminase, arylsulfatase B, β-glucuronidase, α-N-acetylglucosaminidase, lysosomal α, α-L-idulonidase, mannosidase, phosphatidylcholinesterol acyltransferase, N-sulfoglucosamine sulfohydrolase, coagulation factor X, N-acetylgalactosamine-6-sulfatase, sphingomyelin phosphodiesterase, iduronic acid-2-sulfatase, lysosomal α-glucosidase, cyclin-dependent kinase-like 5, pro-low-density lipoprotein receptor-related protein 1, phenylalanine ammonia lyase, protein glutamine γ-glutamyltransferase K, or a lysosomal protective protein.

15. The donor molecules include acid α-glucosidase, α-L-iduronidase, α-galactosidase, iduronic acid-2-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, lysophosphatidylcholine metabolism-related proteins, preferably phospholipase A2, T-REC or K-REC-related proteins, β-glucosidase, β-glucocerebrosidase, arylsulfatase A, factor VIII, insulin-like growth factor 1 (IGF-1), surfactant protein A, surfactant protein B, aspartyl-β-glucosaminidase, acetyl-CoA α-glucosaminide, and acetyl-CoA arylamine. N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylglucosaminidase, acid ceramidase, aspartoacylase, lysosomal acid lipase, acid sphingomyelinase, arylsulfatase B, α-L-fucosidase, galactosylceramidase, galactocerebrosidase, β-galactosidase, protective protein / cathepsin A, β-glucuronidase, heparan N-sulfatase, β-hexosaminidase A, hyaluronidase-1, α-D-mannosidase, β-mannosidase, α-neuraminidase, β-hexosaminidase A, β -The composition according to claim 13, comprising a sequence derived from a gene encoding hexosaminidase B, palmitoylated protein thioesterase, tripeptidyl peptidase I, battenin, ceroid lipofuscinosis neuron protein 5 (CLN5), ceroid lipofuscinosis neuron protein 6 (CLN6), ceroid lipofuscinosis neuron protein 7 (CLN7), ceroid lipofuscinosis neuron protein 8 (CLN8), (cathepsin D), cystinosine, cathepsin K, sialin, lysosome-associated membrane protein 2 (LAMP2), human growth hormone, follicle-stimulating hormone, erythropoietin, CD19, or granulocyte colony-stimulating factor (G-CSF).

16. The composition according to claim 13, wherein the donor molecule comprises a sequence derived from a gene encoding a protein secreted by a cell.

17. The composition according to any one of claims 11 to 16, further comprising a tracrRNA molecule.

18. A method for modifying the AAVS1 allele within a cell, comprising delivering the cell to the cell a composition according to any one of claims 11 to 17.

19. A method for treating a disorder or disease, comprising delivering a composition according to any one of claims 11 to 17 to cells of a subject having the disorder or disease, or delivering modified cells according to claim 9 or 10 to the subject.

20. The aforementioned diseases or disorders include Pompe disease, mucopolysaccharidosis type 1, Fabry disease, mucopolysaccharidosis type 2, mucopolysaccharidosis type IV, mucopolysaccharidosis type VI, adrenoleukodystrophy, severe combined immunodeficiency, Gaucher disease, metachromatic leukodystrophy (MLD), primary immunodeficiency, hemophilia A, hemophilia B, IGF-1 deficiency, surfactant deficiency, aspartylglucosamineuria, and Sanfilippo. Syndrome, Mucopolysaccharidosis type III, Sanfilippo syndrome type IIId, Iysell's disease, Schindler's disease, Faber's disease (FD), Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), Canavan disease, Lysosomal acid lipase deficiency, Niemann-Pick disease, Mucopolysaccharidosis type 6, Fucosidosis, Krabbe disease, GM1 gangliosidosis, Mucopolysaccharidosis type IVB (MPS) The method according to claim 19, wherein the condition is IVB), or galactosialidosis, Sleigh's disease, mucopolysaccharidosis type III, late-onset Tay-Sachs disease, hyaluronidase 1 deficiency, α-mannosidosis, β-mannosidosis, sialidosis, Stanhoff disease, Santavori-Hartia disease, Jansky-Beersawski disease, Batten disease, neurogenic ceroid lipofuscinosis type 5, neurogenic ceroid lipofuscinosis type 6, neurogenic ceroid lipofuscinosis type 7, neurogenic ceroid lipofuscinosis type 8, congenital cathepsin D deficiency, cystinosis, concentrated dysostosis, Salla disease, Danon disease, and / or α1-antitrypsin deficiency.

21. A pharmaceutical for use in modifying an AAVS1 allele in a cell, comprising the composition according to any one of claims 11 to 17 or the modified cell according to claim 9 or 10, the pharmaceutical being administered by delivering the composition according to any one of claims 11 to 15 to the cell.

22. Use of the composition according to any one of claims 11 to 17 or the modified cells according to claim 9 or 10 for the treatment, improvement or prevention of a disorder or disease, comprising delivering the composition according to any one of claims 11 to 15 to cells of a subject having or at risk of having the disorder or disease, or delivering the modified cells according to claim 9 or 10 to the subject.

23. A pharmaceutical product for use in the treatment, improvement or prevention of a disorder or disease, comprising the composition according to any one of claims 11 to 17 or the modified cells according to claim 9 or 10, which is administered by delivering the composition according to any one of claims 11 to 17 to cells of a subject having the disorder or at risk of having the disorder, or by delivering the modified cells according to claim 9 or 10 to the subject.

24. The method according to claim 19 or 20, the use according to claim 22, or the pharmaceutical product according to claim 21, wherein the disorder or disease is a lysosomal storage disorder.

25. The aforementioned diseases or disorders include Pompe disease, mucopolysaccharidosis type 1, Fabry disease, mucopolysaccharidosis type 2, mucopolysaccharidosis type IV, mucopolysaccharidosis type VI, adrenoleukodystrophy, severe combined immunodeficiency, Gaucher disease, metachromatic leukodystrophy (MLD), primary immunodeficiency, hemophilia A, hemophilia B, IGF-1 deficiency, surfactant deficiency, aspartylglucosamineuria, and Sanfilippo. Syndrome, Mucopolysaccharidosis type III, Sanfilippo syndrome type IIId, Iysell's disease, Schindler's disease, Faber's disease (FD), Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), Canavan disease, Lysosomal acid lipase deficiency, Niemann-Pick disease, Mucopolysaccharidosis type 6, Fucosidosis, Krabbe disease, GM1 gangliosidosis, Mucopolysaccharidosis type IVB (MPS) The method according to claim 19 or 20, the use according to claim 22, or the pharmaceutical product according to claim 21, wherein the condition is IVB), or galactosialidosis, Sleigh's disease, mucopolysaccharidosis type III, late-onset Tay-Sachs disease, hyaluronidase 1 deficiency, α-mannosidosis, β-mannosidosis, sialidosis, Stanhof's disease, Santavori-Hartia disease, Jansky-Beersawski disease, Batten disease, neurogenic ceroid lipofuscinosis type 5, neurogenic ceroid lipofuscinosis type 6, neurogenic ceroid lipofuscinosis type 7, neurogenic ceroid lipofuscinosis type 8, congenital cathepsin D deficiency, cystinosis, concentrated dysostosis, Salla disease, Danon disease and / or α1-antitrypsin deficiency.

26. The method according to claim 19 or 20, the use according to claim 22, or the pharmaceutical product according to claim 21, wherein the disorder or disease is a disorder or disease of the blood, brain, lungs, or central nervous system.

27. The pharmaceutical product according to claim 23, wherein the pharmaceutical product is enzyme replacement therapy.

28. A method for treating a disease or disorder in a subject, comprising immunotherapy, which includes delivering the modified cells described in claim 9 or 10 to the subject.

29. The method according to claim 28, wherein the disease or disorder is cancer.

30. A composition according to any one of claims 11 to 17 or a modified cell according to claim 9 or 10, for use in the treatment, improvement, or prevention of a disorder or disease.

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