Supplementation of liver enzyme expression

Abstract

Described herein are methods, compositions, and systems derived from uncultured microorganisms that can be used to supplement liver enzyme deficiency. Disclosed is an engineered nuclease system comprising: a) an endonuclease comprising an amino acid sequence; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene; and c) a donor template comprising a nucleic acid sequence encoding a Factor VIII (FVIII) gene or a functional fragment thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 345,526 filed on May 25, 2022, U.S. Provisional Patent Application No. 63 / 359,288 filed on July 8, 2022, and U.S. Provisional Patent Application No. 63 / 396,421 filed on August 9, 2022, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] The electronic sequence listing (MTG-017WO_SL.xml; size: 384,067 bytes; and creation date: May 17, 2023) is incorporated herein by reference in its entirety. Summary of the invention

[0005] Various diseases are caused by the lack of factors (e.g., liver enzymes) produced by the liver, which are themselves caused by genetic mutations. For example, hemophilia A and hemophilia B are hereditary diseases caused by mutations in genes encoding coagulation factors (e.g., factor VIII produced by the liver). Individuals suffering from hemophilia have lower levels of these coagulation factors, thereby causing their blood to be unable to coagulate normally. The gene therapy method for treating these diseases using the liver gene encoding functional liver enzymes (e.g., factor VIII) integrated into the genome in the patient allows functional liver enzymes to be continuously expressed. Therefore, methods, compositions and systems for supplementing liver enzymes by gene therapy methods are described herein.

[0006] In certain embodiments, disclosed herein is an engineered nuclease system comprising: a) an endonuclease comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene; and c) a donor template comprising a nucleic acid sequence encoding a factor VIII (FVIII) gene or a functional fragment thereof. In some embodiments, the target nucleic acid sequence within the albumin gene is located within intron 1 of the albumin gene. In some embodiments, the sequence encoding the factor VIII (FVIII) gene or a functional fragment thereof is linked to a splice acceptor sequence targeting exon 1 of the albumin gene. In some embodiments, the endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence having 100% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having 100% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-6.In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3-6. In some embodiments, the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 3-6. In some embodiments, the donor template further comprises a polyadenylation signal. In some embodiments, the donor template further comprises a nuclear targeting sequence. In some embodiments, the nuclear targeting sequence comprises a plurality of transcription factor binding sites. In some embodiments, the transcription factor is TCF1, HNF1, NFY, CEBP, OCT1, AP1, HNF1-α, HNF1-β, CEBPA, LEF-1, FOX D1, IRF1, HNF3, HNF4, HNF5, Tal1β / E47 or MyoD. In some embodiments, the nuclear targeting sequence is located at the 5' end and the 3' end of the donor template. In some embodiments, the donor template further comprises a recognition site sequence for the endonuclease at the 5' end or the 3' end. In some embodiments, when the donor template is flanked on the 5' end, the nuclear targeting sequence is located 5' of the recognition site sequence. In some embodiments, when the donor template is flanked on the 3' end, the nuclear targeting sequence is located 3' of the recognition site sequence. In some embodiments, the donor template comprises, from 5' to 3': NTS(1)-NRS(1)-SA-FVIII-NRS(2)-NTS(2), wherein NTS(1) represents a first nuclear targeting sequence; NTS(2) represents a second nuclear targeting sequence; NRS(1) represents a first nuclease recognition site sequence; NRS(2) represents a second nuclease recognition site sequence; SA represents the splice acceptor sequence targeting exon 1 of the albumin gene; and FVIII represents the factor VIII gene or a fragment thereof. In some embodiments, the 5' to 3' orientation of NRS (1) and NRS (2) is according to: (a) forward, forward; (b) reverse, reverse; (c) forward, reverse; (d) reverse, forward; wherein forward represents the same 5' to 3' orientation as the target nucleic acid sequence, and reverse represents the opposite 5' to 3' orientation as the target nucleic acid sequence. In some embodiments, the donor template comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.In some embodiments, the donor template comprises a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the FVIII gene or its functional fragment is codon-optimized to remove at least one cytosine-guanine (CG or CpG) motif. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having 100% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having at least 80% identity to SEQ ID NOs: 10. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having at least 90% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having 100% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or its functional fragment is modified to comprise a B domain comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 71-79 and 89. In some embodiments, the FVIII gene or its functional fragment is modified to comprise a B domain comprising a sequence having 100% identity to any one of SEQ ID NOs: 71-79 and 89. In some embodiments, the FVIII gene or its functional fragment comprising the modified B domain comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 86-87 and 90. In some embodiments, the FVIII gene or its functional fragment comprising the modified B domain comprises a sequence having 100% identity to any one of SEQ ID NOs: 86-87 and 90.

[0007] In certain embodiments, disclosed herein is a method for supplementing liver enzyme expression in a subject in need thereof, the method comprising administering to the subject: a) an endonuclease comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene; and c) a donor template comprising a nucleic acid sequence encoding a factor VIII (FVIII) gene or a functional fragment thereof, thereby supplementing liver enzyme expression in the subject. In some embodiments, the target nucleic acid sequence within the albumin gene is located within intron 1 of the albumin gene. In some embodiments, the sequence encoding the factor VIII (FVIII) gene or a functional fragment thereof is operably linked to a splice acceptor sequence targeting exon 1 of the albumin gene. In some embodiments, the endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence having 100% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having 100% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.In some embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-6. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3-6. In some embodiments, the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 3-6. In some embodiments, the donor template further comprises a polyadenylation signal. In some embodiments, the donor template further comprises a nuclear targeting sequence. In some embodiments, the nuclear targeting sequence comprises a plurality of transcription factor binding sites. In some embodiments, the transcription factor is TCF1, HNF1, NFY, CEBP, OCT1, AP1, HNF1-α, HNF1-β, CEBPA, LEF-1, FOX D1, IRF1, HNF3, HNF4, HNF5, Tal1β / E47 or MyoD. In some embodiments, the nuclear targeting sequence is located at the 5' end and the 3' end of the donor template. In some embodiments, the donor template further comprises a recognition site sequence for the endonuclease on the 5' end or the 3' end. In some embodiments, when the donor template is flanked on the 5' end, the nuclear targeting sequence is located at 5' of the recognition site sequence. In some embodiments, when the donor template is flanked on the 3' end, the nuclear targeting sequence is located at 3' of the recognition site sequence. In some embodiments, the donor template comprises from 5' to 3': NTS(1)-NRS(1)-SA-FVIII-NRS(2)-NTS(2), wherein NTS(1) represents a first nuclear targeting sequence; NTS(2) represents a second nuclear targeting sequence; NRS(1) represents a first nuclease recognition site sequence; NRS(2) represents a second nuclease recognition site sequence; SA represents the splice acceptor sequence targeting exon 1 of the albumin gene; and FVIII represents the factor VIII gene or a fragment thereof. In some embodiments, the 5' to 3' orientation of NRS (1) and NRS (2) is according to: (a) forward, forward; (b) reverse, reverse; (c) forward, reverse; (d) reverse, forward; wherein forward represents the same 5' to 3' orientation as the target nucleic acid sequence, and reverse represents the opposite 5' to 3' orientation as the target nucleic acid sequence. In some embodiments, the donor template comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.In some embodiments, the donor template comprises a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the FVIII gene or its functional fragment is codon-optimized to remove at least one cytosine-guanine (CG or CpG) motif. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having 100% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having at least 80% identity to SEQ ID NOs: 10. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having at least 90% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or its functional fragment comprises a sequence having 100% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or its functional fragment is modified to comprise a B domain comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 71-79 and 89. In some embodiments, the FVIII gene or its functional fragment is modified to comprise a B domain comprising a sequence having 100% identity to any one of SEQ ID NOs: 71-79 and 89. In some embodiments, the FVIII gene or its functional fragment comprising the modified B domain comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 86-87 and 90. In some embodiments, the FVIII gene or its functional fragment comprising the modified B domain comprises a sequence having 100% identity to any one of SEQ ID NOs: 86-87 and 90.

[0008] In certain embodiments, disclosed herein are cells comprising an engineered nuclease system disclosed herein. In some embodiments, the cell is a liver cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell or a derivative thereof. In some embodiments, the cell is an engineered cell. In some embodiments, the cell is a stable cell.

[0009] In certain embodiments, disclosed herein are lipid nanoparticles (LNPs) comprising components (a) and (b) or components (a), (b) and (c) of the engineered nuclease system disclosed herein. In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, cholesterol or a cholesterol analog, and a lipid connected to PEG. In some embodiments, the cationic lipid comprises C12-200 (1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)amino)bis(dodecane-2-ol)), the neutral lipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or the PEG-connected lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000).

[0010] In certain embodiments, disclosed herein are viral vectors comprising the engineered nuclease systems disclosed herein. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF , AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, or derivatives thereof. In some embodiments, the AAV is AAV6. In some embodiments, the AAV is AAV8.

[0011] In certain embodiments, disclosed herein is a method of supplementing liver enzyme expression in an individual in need thereof, the method comprising administering to the individual: (a) an endonuclease comprising a RuvC domain or a nucleic acid encoding the endonuclease, the endonuclease having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 30; (b) an engineered guide RNA (i) configured to form a complex with the endonuclease and (ii) comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene; and (c) a donor template comprising a nucleic acid sequence encoding a therapeutic gene encoding a functional liver enzyme (e.g., factor VIII), thereby supplementing the expression of a liver enzyme (e.g., factor VIII) in the individual. In some embodiments, the spacer sequence is configured to hybridize with intron 1 of the albumin gene. In some embodiments, the nucleic acid sequence encoding the therapeutic gene is linked to a splice acceptor sequence targeting exon 1 of the albumin gene.

[0012] In certain embodiments, disclosed herein is a method for supplementing liver enzyme expression in an individual in need thereof, the method comprising administering to the individual: (a) an endonuclease comprising a RuvC domain or a nucleic acid encoding the endonuclease, wherein the endonuclease has at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 31; (b) an engineered guide RNA, the engineered guide RNA (i) being configured to form a complex with the endonuclease and (ii) comprising a spacer sequence configured to hybridize with a target nucleic acid sequence within an albumin gene; and (c) a donor template comprising a nucleic acid sequence encoding a functional liver enzyme (e.g., Factor VIII) linked to a splice acceptor sequence targeting exon 1 of the albumin gene, thereby supplementing liver enzyme expression in the subject.

[0013] In some embodiments, the endonuclease induces single-strand breaks or double-strand breaks at or near the target nucleic acid sequence. In some embodiments, the endonuclease induces double-strand breaks at or near the target nucleic acid sequence. In some embodiments, the donor template is integrated into the target nucleic acid sequence at a double-strand break. In some embodiments, the donor template is integrated into the target nucleic acid sequence at a double-strand break by non-homologous end joining (NHEJ). In some embodiments, the donor template is integrated into the target nucleic acid sequence at a double-strand break by homology-directed repair (HDR).

[0014] In some embodiments, the engineered guide RNA is configured to hybridize to a sequence having at least 80% identity to any one of SEQ ID NOs: 3-6. In some embodiments, the engineered guide RNA comprises a sequence according to any one of SEQ ID NOs: 24-27.

[0015] In some embodiments, the donor template further comprises a polyadenylation signal. In some embodiments, the donor template is a linear duplex with closed ends. In some embodiments, the donor template further comprises a recognition site sequence for the endonuclease at the 5' end or the 3' end of the donor template. In some embodiments, the donor template further comprises a nuclear targeting sequence: (a) when the coding sequence is flanked on the 5' end of the donor template, the nuclear targeting sequence is located at 5' of the recognition site sequence, or (b) when the coding sequence is flanked on the 3' end of the donor template, the nuclear targeting sequence is located at 3' of the recognition site sequence. In some embodiments, the donor template comprises nuclear targeting sequences at both the 5' end and the 3' end of the donor template. In some embodiments, the donor template comprises, from 5' to 3', NTS(1)-NRS(1)-SA-TG-NRS(2)-NTS(2), wherein NTS(1) represents a first nuclear targeting sequence; NTS(2) represents a second nuclear targeting sequence; NRS(1) represents a first nuclease recognition site sequence; NRS(2) represents a second nuclease recognition site sequence; SA represents a splice acceptor sequence targeting exon 1 of an albumin gene; and TG represents a therapeutic gene. In some embodiments, the 5' to 3' orientation of NRS(1) and NRS(2) is according to: (a) forward, forward; (b) reverse, reverse; (c) forward, reverse; (d) reverse, forward; wherein forward represents the same 5' to 3' orientation as the target nucleic acid sequence, and reverse represents the opposite 5' to 3' orientation to the target nucleic acid sequence. In some embodiments, the first nuclear targeting sequence or the second nuclear targeting sequence comprises multiple binding sites for LEF / TCF1, HNF1, NFY, CEBP, OCT1, AP1, HNF1-A, HNF1-B, CEBPA, LEF-1, FOX D1, or IRF1. In some embodiments, the first nuclear targeting sequence or the second nuclear targeting sequence comprises a sequence having at least 80% identity to SEQ ID NO: 1 or SEQ ID NO: 2 appended to the 5' or 3' end of the nuclear targeting sequence.

[0016] In some embodiments, the donor template comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, and 32-33. In some embodiments, the donor template comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 16-23. In some embodiments, the donor template comprises a sequence having at least 75% identity to any one of SEQ ID NOs: 16-19. In some embodiments, the donor template comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 20-23.

[0017] In some embodiments, the therapeutic gene is a factor VIII (FVIII) gene or a functional fragment thereof. In some embodiments, the FVIII gene or its functional fragment is codon-optimized to remove at least one cytosine-guanine (CG or CpG) motif. In some embodiments, the codon-optimized FVIII gene or its functional fragment comprises a sequence having at least 80% identity to SEQ ID NO: 10.

[0018] In certain embodiments, disclosed herein is a vector comprising an endonuclease system disclosed herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the vector is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, cholesterol or a cholesterol analog or a lipid connected to PEG. In some embodiments, the cationic lipid comprises C12-200 (1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)amino)bis(dodecane-2-ol)), the neutral lipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or the lipid connected to PEG comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000).

[0019] In certain embodiments, disclosed herein are systems comprising: (a) an endonuclease capable of cleaving at least one strand of a target nucleic acid within a first nuclease recognition site sequence or a second nuclease recognition site sequence, and (b) a nucleic acid comprising, from 5' to 3': (i) a first nuclear targeting sequence; and (ii) a coding sequence of a therapeutic gene encoding a functional liver enzyme (e.g., Factor VIII), wherein the coding sequence is flanked on the 5' end by the first nuclease recognition site sequence and / or on the 3' end by the second nuclease recognition site sequence. In some embodiments, the nucleic acid is double-stranded DNA. In some embodiments, the double-stranded DNA is a linear duplex with closed ends. In some embodiments, the coding sequence of the coding sequence of the therapeutic gene is flanked on the 5' end by the first nuclease recognition sequence, and the first recognition site sequence is located at 3' of the first nuclear targeting sequence. In some embodiments, the coding sequence of the therapeutic gene is flanked on the 3' end by the second nuclease recognition site sequence. In some embodiments, the nucleic acid further comprises a second nuclear targeting sequence. In some embodiments, the nucleic acid comprises a first nuclear targeting sequence on the 5' end of the nucleic acid and a second nuclear targeting sequence on the 3' end. In some embodiments, the nucleic acid comprises, from 5' to 3': NTS(1)-NRS(1)-SA-TG-NRS(2)-NTS(2), wherein NTS(1) represents the first nuclear targeting sequence; NTS(2) represents the second nuclear targeting sequence; NRS(1) represents the first nuclease recognition site sequence; NRS(2) represents the second nuclease recognition site sequence; SA represents a splice acceptor sequence targeting exon 1 of the albumin gene; and TG represents a therapeutic gene. In some embodiments, the 5' to 3' orientation of the NRS(1) and NRS(2) is according to: (a) forward, forward; (b) reverse, reverse; (c) forward, reverse; (d) reverse, forward; wherein forward represents the same 5' to 3' orientation as the target nucleic acid sequence, and reverse represents the opposite 5' to 3' orientation as the target nucleic acid sequence. In some embodiments, the nucleic acid comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 12-13, 16-23, or 32-33. In some embodiments, the first nuclear targeting sequence or the second nuclear targeting sequence comprises a binding site for LEF / TCF1, HNF1, NFY, CEBP, OCT1, AP1, HNF1-A, HNF1-B, CEBPA, LEF-1, FOX D1, or IRF1. In some embodiments, the first nuclear targeting sequence or the second nuclear targeting sequence comprises a sequence having at least 80% identity to SEQ ID NO: 1 or SEQ ID NO: 1 and SEQ ID NO: 2 appended to the 5' or 3' end of the nuclear targeting sequence.In some embodiments, the therapeutic gene is a factor VIII (FVIII) gene or a functional fragment thereof. In some embodiments, the FVIII gene or a functional fragment thereof is codon-optimized to remove at least one cytosine-guanine (CG or CpG) motif. In some embodiments, the codon-optimized FVIII gene or a functional fragment thereof comprises a sequence having at least 80% identity to SEQ ID NO: 10.

[0020] In certain embodiments, disclosed herein is a viral vector comprising, from 5' to 3': (a) a nucleic acid comprising a nucleic acid sequence having at least 80% identity with the first intron 1 sequence of the albumin gene and comprising a KTTN (K=G or T, N=any base) or an AAANNN sequence (N=any base); (b) a splice acceptor sequence targeting exon 1 of the albumin gene; (c) a coding sequence of a therapeutic gene, the therapeutic gene encoding a functional liver enzyme (e.g., Factor VIII) linked to a polyadenylation signal and a splice acceptor sequence; and (d) a sequence comprising a nucleic acid sequence having at least 80% identity with the second intron 1 sequence of the albumin gene and comprising a KTTN (K=G or T, N=any base) or an AAANNN (N=any base), wherein the second intron 1 sequence is located 3' of the first intron 1 sequence of the albumin gene. In some embodiments, the nucleic acid further comprises a first nuclear targeting sequence (NTS), which is located 5' of the recognition site sequence when the coding sequence is flanked on the 5' end of the nucleic acid, or 3' of the recognition site sequence when the coding sequence is flanked on the 3' end of the nucleic acid. In some embodiments, the nucleic acid further comprises a second nuclear targeting sequence (NTS). In some embodiments, the nucleic acid comprises a first nuclear targeting sequence on the 5' end of the nucleic acid and a second nuclear targeting sequence on the 3' end of the nucleic acid. In some embodiments, the nucleic acid comprises, from 5' to 3': NTS(1)-NRS(1)-SA-TG-NRS(2)-NTS(2), wherein NTS(1) represents the first nuclear targeting sequence; NTS(2) represents the second nuclear targeting sequence; NRS(1) represents the first nuclease recognition site sequence; NRS(2) represents the second nuclease recognition site sequence; SA represents a splice acceptor sequence targeting exon 1 of the albumin gene; and TG represents the coding sequence of the therapeutic gene. In some embodiments, the 5' to 3' orientation of NRS (1) and NRS (2) is according to: (a) forward, forward; (b) reverse, reverse; (c) forward, reverse; (d) reverse, forward; wherein forward means the same 5' to 3' orientation as the target nucleic acid sequence, and reverse means the opposite 5' to 3' orientation as the target nucleic acid sequence. In some embodiments, the nucleic acid comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 12-13, 16-23, or 32-33. In some embodiments, the first nuclear targeting sequence or the second nuclear targeting sequence comprises a binding site for LEF / TCF1, HNF1, NFY, CEBP, OCT1, AP1, HNF1-A, HNF1-B, CEBPA, LEF-1, FOX D1, or IRF1.In some embodiments, the first nuclear targeting sequence or the second nuclear targeting sequence comprises a sequence having at least 80% identity to SEQ ID NO: 1 or SEQ ID NO: 1 and SEQ ID NO: 2 appended to the 5' or 3' end. In some embodiments, the therapeutic gene is a factor VIII (FVIII) gene or a functional fragment thereof. In some embodiments, the FVIII gene or its functional fragment is codon-optimized to remove at least one cytosine-guanine (CG or CpG) motif. In some embodiments, the codon-optimized FVIII gene or its functional fragment comprises a sequence having at least 80% identity to SEQ ID NO: 10.

[0021] Other aspects and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described in the detailed description. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details can be modified in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and description are to be regarded as illustrative rather than restrictive in nature. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description and accompanying drawings which set forth illustrative embodiments in which the principles of the present disclosure are utilized, and in which:

[0023] Figure 1 Depicted are the locations of the MG29-1 cleavage sites on the target and non-target strands as determined from in vitro editing assays. Figure 1 SEQ ID NOs 123-124, 126, 125 and 127-128 are disclosed respectively in order of appearance.

[0024] Figure 2 Depicted are editing efficiencies in whole livers of mice 5 days after intravenous injection of three doses of LNPs encapsulating MG29-1 mRNA and guide mAlb29-8-50 (mA29-8-50), spCas9 mRNA, and guide mAlbR2, or PBS buffer (control). Each circle represents an individual mouse, and the bars indicate the mean and standard deviation.

[0025] Figure 3Depicted are editing efficiencies in whole liver of mice 12 days after intravenous injection of 0.5 mg / kg dose of LNPs encapsulating MG29-1 mRNA and sgRNA mA29-12b-50, MG29-1 mRNA and sgRNA mA29-8b-50, or PBS buffer (control). Each circle represents an individual mouse, and the bars indicate the mean and standard deviation.

[0026] Figure 4 Depicted is a gel showing integration of the FVIII gene cassette in the forward orientation by In-Out PCR.

[0027] Figure 5 Depicted are editing by MG29-1 and five guides targeting albumin intron 1 in primary hepatocytes from macaques.

[0028] Figure 6 Depicted are editing in the liver of NHPs 8 days after administration of LNP-encapsulated MG29-1 mRNA and guide RNA.

[0029] Figure 7 Depicted are the frequencies of MG29-1 and Guide 12-mediated forward integration of the FVIII gene cassette into the albumin intronic site measured by dd-PCR in liver and normalized to cytochrome C1.

[0030] Figure 8 Depicted are the average frequencies of MG29-1 and Guide 12-mediated forward integration of the FVIII gene cassette into the albumin intronic site measured by dd-PCR in liver and normalized to cytochrome C1.

[0031] Fig. 9 Depicted are the frequencies of MG29-1 and Guide 12-mediated forward and reverse integration of the FVIII gene cassette into the albumin intronic site measured by dd-PCR in liver and normalized to cytochrome C1.

[0032] Fig.10 Depicted are the frequencies of MG29-1 and Guide 8 mediated forward and reverse integration of the FVIII gene cassette into the albumin intronic site measured by dd-PCR in liver and normalized to cytochrome C1.

[0033] Fig.11 Depicted are the average frequencies of MG29-1 and Guide 8 mediated forward integration of the FVIII gene cassette into the albumin intronic site measured by dd-PCR in liver and normalized to cytochrome C1.

[0034] Fig.12Depicted are the frequencies of MG29-1 and Guide 8 mediated forward and reverse integration of the FVIII gene cassette into the albumin intronic site measured by dd-PCR in liver and normalized to cytochrome C1.

[0035] Fig.13 The predicted mRNA sequence of the junction between mouse albumin exon 1 and human FVIII after correct splicing is depicted. Fig.13 SEQ ID NOs 129 and 111-113 are disclosed in order of appearance, respectively.

[0036] Fig.14 Depicted is the administration of 4 AAV8-FVIII donors (1x 10 13 Quantification of albumin-FVIII fusion mRNA in the liver of mice treated with either LNPs (mA29-8b-50, 0.5 mpk dose) or LNPs encapsulating MG29-1 mRNA and guide RNA8.

[0037] Fig.15 Depicted is the quantification of albumin-FVIII fusion mRNA in the liver of mice dosed with either of four AAV8-FVIII donors and LNPs encapsulating MG29-1 mRNA and guide RNA 12 (mA29-12b-50).

[0038] Fig.16Depicted are edits (Indels) in the liver of mice dosed with various AAV8-FVIII donors or PBS and LNPs encapsulating MG3-6 / 3-4 mRNA and either mA364-34-1 or mA364-59-1. From left to right: The first 4 columns are mice dosed with AAV8-pMG4012 to AAV8-pMG4015 at 0.5 mpk, followed by LNPs encapsulating MG3-6 / 3-4 mRNA and guide mA364-34-1. The next 4 columns are mice dosed with AAV8-pMG4012 to AAV8-pMG4015 at 0.7 mpk, followed by LNPs encapsulating MG3-6 / 3-4 mRNA and guide mA364-34-1. The next 4 columns are mice dosed with AAV8-pMG4012 to AAV8-pMG4015 at 0.5 mpk followed by LNPs encapsulating MG3-6 / 3-4 mRNA and guide mA364-59-1. The next 4 columns are mice dosed with AAV8-pMG4012 to AAV8-pMG4015 at 0.7 mpk followed by LNPs encapsulating MG3-6 / 3-4 mRNA and guide mA364-59-1. The next column is mice dosed with PBS only (control). The last 4 columns are mice dosed with AAV8-pMG4012 to AAV8-pMG4015 only.

[0039] Fig.17 Depicted are the frequencies of integration of the FVIII gene cassette in the forward orientation into albumin intron 1 mediated by MG3-6 / 3-4 and the guide mA364-34-1 (guide 34).

[0040] Fig.18 Depicted are the average frequencies of integration of the FVIII gene cassette into albumin intron 1 in the forward orientation mediated by MG3-6 / 3-4 and the guide mA364-34-1 (guide 34).

[0041] Fig.19 Depicted are the frequencies of integration of the FVIII gene cassette into albumin intron 1 in the forward and reverse orientations mediated by MG3-6 / 3-4 and the guide mA364-34-1 (guide 34).

[0042] Fig. 20 Depicted is the quantification of albumin-FVIII fusion mRNA in the liver of mice dosed with either of four AAV8-FVIII donors and LNPs encapsulating MG3-6 / 3-4 mRNA and guide RNA 34 (mA364-34-1).

[0043] Fig.21Depicted are mean albumin-FVIII fusion mRNA levels in the liver of mice dosed with either of the four AAV8-FVIII donors and LNPs encapsulating MG3-6 / 3-4 mRNA and guide RNA 34 (mA364-34-1).

[0044] Fig. 22 Depicted is the sequence of human FVIII around the B domain junction aligned with different designs of B domain replacement sequences. Fig. 22 SEQ ID NOs 130-143, 99, 160, 73-74 and 78 are disclosed respectively in order of appearance.

[0045] Fig.23 Depicted is the administration of AAV8-pMG4017, AAV8-pMG4018, AAV8-pMG4019, AAV8-pMG40120, AAV8-pMG4021 (1 x 10 13 vg / kg), followed by editing in the liver of mice encapsulated with MG29-1 mRNA and guide RNA mAlb28-8b-50 (0.5 mpk dose). The assay was performed at the end of the study.

[0046] Fig.24 Depicted is a 1x 10 13 Quantification of albumin-FVIII fusion mRNA in the liver of mice dosed with AAV8-pMG4017, AAV8-pMG4018, AAV8-pMG4019, AAV8-pMG4020, AAV8-pMG4021 vg / kg followed by LNP delivery of MG29-1 mRNA and guide RNA mA29-8b50 (0.5 mpk).

[0047] Fig.25 Depicted is a 1x 10 13 FVIII protein levels in the blood of mice dosed with AAV8-pMG4017, AAV8-pMG4018, AAV8-pMG4019, AAV8-pMG4020, AAV8-pMG4021 vg / kg followed by LNP delivery of MG29-1 mRNA and guide RNA mA29-8b50 (0.5 mpk).

[0048] Fig.26 Depicted are sequence elements present in the cyno FVIII donor cassette pMG4016 (not to scale). TS = target site of the designated guide.

[0049] Fig. 27 Depicted is the administration of 1x 10 13vg / kg of AAV6(sf)-pMG4016, AAV8(sf)-pMG4016, or AAV8(HK)-pMG4016 in the liver of mice.

[0050] Fig.28 Depicts the integration of the cyno-FVIII gene cassette into 1x 10 13 Quantification of albumin intron 1 in mice treated with vg / kg of AAV6(sf)-pMG4016, AAV8(sf)-pMG4016, or AAV8(HK)-pMG4016 followed by a mixture of LNP A and LNP B.

[0051] Fig.29 Depicted is the administration of 1x 10 13 Quantification of albumin-cynoFVIII fusion mRNA levels in the liver of mice given vg / kg of AAV6(sf)-pMG4016, AAV8(sf)-pMG4016, or AAV8(HK)-pMG4016, followed by a mixture of LNP A and LNP B.

[0052] Brief Description of Sequence Listing

[0053] The sequence listing submitted herewith provides exemplary polynucleotide and polypeptide sequences for use in the methods, compositions and systems according to the present disclosure.Below is an exemplary description of the sequences therein.

[0054] SEQ ID NO: 1 shows the DNA sequence of the human albumin target site.

[0055] SEQ ID NO: 2 shows the DNA sequence of the transcription enhancer.

[0056] SEQ ID NOs: 3-6 show the DNA sequences of the mouse albumin target sites.

[0057] SEQ ID NO: 7 shows the nucleotide sequence of the 20 bp spacer.

[0058] SEQ ID NO: 8 shows the DNA sequence of the human albumin target site and transcription enhancer.

[0059] SEQ ID NO:9 shows the DNA sequence of the splice acceptor.

[0060] SEQ ID NO: 10 shows the DNA sequence of the human Factor VIII coding sequence.

[0061] SEQ ID NO: 11 shows the DNA sequence of the stop codon, spacer and polyadenylation signal.

[0062] SEQ ID NOs: 12-13, 16-23 and 32-33 show the DNA sequences of Factor VIII donor templates.

[0063] SEQ ID NOs: 14-15 and 24-25 show the nucleotide sequences of MG29-1 sgRNA targeting mouse albumin.

[0064] SEQ ID NOs: 26-27 show the nucleotide sequences of MG3-6 / 3-4 sgRNA targeting mouse albumin.

[0065] SEQ ID NOs: 28-29 show the nucleotide sequences of PCR primers.

[0066] SEQ ID NO: 30 shows the DNA sequence encoding MG3-6 / 3-4 mRNA.

[0067] SEQ ID NO: 31 shows the DNA sequence encoding MG29-1 mRNA.

[0068] SEQ ID NOs: 37, 39 and 41 show the nucleotide sequences of transcription factor binding sequences.

[0069] SEQ ID NOs:43-45 show the nucleotide sequences of MG29-1 sgRNA targeting mouse albumin.

[0070] SEQ ID NOs: 46-49 show the nucleotide sequences of PCR primers.

[0071] SEQ ID NO: 50 shows the nucleotide sequence of MG29-1 sgRNA targeting mouse albumin.

[0072] SEQ ID NO: 51 shows the DNA sequence encoding spCas9 mRNA.

[0073] SEQ ID NO:52 shows the spCas9 amino acid sequence.

[0074] SEQ ID NO: 53 shows the DNA sequence encoding MG29-1 mRNA.

[0075] SEQ ID NO:54 shows the MG29-1 amino acid sequence.

[0076] SEQ ID NO: 55 shows the nucleotide sequence of MG29-1 sgRNA targeting mouse albumin.

[0077] SEQ ID NOs:56-59 show the DNA sequences of Factor VIII donor templates.

[0078] SEQ ID NOs:60-63 show the nucleotide sequences of MG29-1 sgRNA targeting mouse albumin.

[0079] SEQ ID NOs:64-68 show the nucleotide sequences of MG29-1 sgRNA targeting human albumin intron 1.

[0080] SEQ ID NO: 69 shows the protein sequence of the protease recognition site.

[0081] SEQ ID NOs: 71-79 show protein sequences intended to replace the B domain of FVIII.

[0082] SEQ ID NO: 80 shows the protein sequence of the SQ linker.

[0083] SEQ ID NOs: 81-85 show the nucleotide sequences of the human FVIII donor cassettes.

[0084] SEQ ID NOs: 86-87 show the protein sequences of cynomolgus FVIII sequences in which the B domain has been replaced.

[0085] SEQ ID NO: 88 shows the nucleotide sequence of the cynomolgus FVIII donor cassette.

[0086] SEQ ID NO: 89 shows the protein sequence intended to replace the B domain of FVIII.

[0087] SEQ ID NO: 90 shows the protein sequence of the human FVIII sequence in which the B domain has been replaced.

[0088] SEQ ID NOs:91-94 show the nucleotide sequences of the human FVIII donor cassettes.

[0089] SEQ ID NO:95 shows the nucleotide sequence of the messenger RNA encoding the MG3-6 / 3-4 nuclease with nuclear localization signals added at both the N-terminus and the C-terminus.

[0090] SEQ ID NO:96 shows the protein sequence of MG3-6 / 3-4 nuclease with nuclear localization signals added at both the N-terminus and the C-terminus.

[0091] SEQ ID NOs:97-98 show the nucleotide sequences of MG3-6 / 3-4 sgRNA targeting mouse albumin intron 1. DETAILED DESCRIPTION

[0092] Although various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Various changes, modifications and substitutions may be envisioned by those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0093] Unless otherwise indicated, the practice of certain methods disclosed herein employs techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th edition (2012); the book series Current Protocols in Molecular Biology (FM Ausubel et al., eds.); the book series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th edition (RI Freshney, ed., (2010)).

[0094] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, where the terms "including," "include," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0095] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within one or more than one standard deviation as practiced in the art. Alternatively, "about" may mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0096] As used herein, the term "nucleotide" refers to a base-sugar-phosphate combination. Envisioned nucleotides include naturally occurring nucleotides and synthetic nucleotides. Nucleotide is a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphates, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or its derivatives. Such derivatives include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that impart nuclease resistance to nucleic acid molecules containing it. As used herein, the term nucleotide encompasses dideoxyribonucleoside triphosphates (ddNTP) and derivatives thereof. Illustrative examples of ddNTP include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP and ddTTP. Nucleotide can be unlabeled or detectably labeled, such as using a part or quantum dot comprising an optically detectable portion (e.g., fluorophore). Detectable labeling includes, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. The fluorescent labeling of nucleotides includes, but is not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N, N, N', N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxyl-X-rhodamine (ROX), 4-(4' dimethylaminophenylazo) benzoic acid (DABCYL), waterfall blue, Oregon green, Texas red, cyanine, and 5-(2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, available from Perkin Elmer, Foster City, Calif.; FluoroLink deoxyribonucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, available from Boehringer Mannheim, Indianapolis, Ind.; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2′-dATP, available from Molecular Probes, Eugene, Oreg.; and chromosome-labeled nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-FL-5-UTP, available from Molecular Probes, Eugene, Oreg. IPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP and Texas Red-12-dUTP. The term nucleotide encompasses chemically modified nucleotides. Exemplary chemically modified nucleotides are biotin-dNTPs.Non-limiting examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0097] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides or their analogs, in single-stranded, double-stranded or multi-stranded forms. The contemplated polynucleotides include genes or fragments thereof. Exemplary polynucleotides include, but are not limited to, coding or non-coding regions of DNA, RNA, genes or gene fragments, multiple loci defined according to connection analysis (locus), exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides, nucleic acid probes and primers comprising cell-free DNA (cfDNA) and cell-free RNA (cfRNA). In a polynucleotide, when T is mentioned, T means U (uracil) in RNA and T (thymine) in DNA. Polynucleotides can be exogenous or endogenous to cells and / or present in a cell-free environment. The term polynucleotide encompasses modified polynucleotides (e.g., altered backbones, sugars or nucleobases). If present, modification of the nucleotide structure is imparted before or after assembling the polymer. Non-limiting examples of modification include: 5-bromouracil, peptide nucleic acids, heterologous nucleic acids, morpholinos, locked nucleic acids, glycerol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, braided glycosides and wyoside. The nucleotide sequence can be interspersed with non-nucleotide components.

[0098] The term "transfection" or "transfected" refers to the introduction of a polynucleotide into a cell by a non-viral or viral-based method. The polynucleotide can be a gene sequence encoding a complete protein or a functional portion thereof. See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0099] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues connected by a peptide bond. This term does not indicate the specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally present. The term applies to naturally occurring amino acid polymers and amino acid polymers including at least one modified amino acid. In some cases, the polymer may be interspersed with non-amino acids. The term includes amino acid chains of any length, including full-length proteins and proteins with or without secondary or tertiary structures (e.g., domains). The term also encompasses amino acid polymers that have been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other operations, such as conjugation with a labeling component. As used herein, the term "amino acid (amino acid / amino acids)" refers to natural and non-natural amino acids, including but not limited to modified amino acids. Modified amino acids include natural amino acids that have been chemically modified to include groups or chemical moieties that are not naturally present on amino acids. The term "amino acid" includes both D-amino acids and L-amino acids.

[0100] As used herein, "non-natural" refers to non-natural nucleic acid or polypeptide sequences. Non-natural refers to non-natural nucleic acid or polypeptide sequences that contain modifications such as mutations, insertions or deletions. The term non-natural encompasses fusion nucleic acids or polypeptides that encode or show activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) of the nucleic acid or polypeptide sequence fused to the non-natural sequence. Non-natural nucleic acids or polypeptide sequences include those non-natural nucleic acids or polypeptide sequences that are connected to naturally occurring nucleic acids or polypeptide sequences (or variants thereof) by genetic engineering to produce chimeric nucleic acids or polypeptide sequences encoding chimeric nucleic acids or polypeptides.

[0101] As used herein, the term "promoter" refers to a regulatory DNA region that controls polynucleotide (e.g., gene) transcription or expression and can be located adjacent to or overlapping a nucleotide or nucleotide region that initiates RNA transcription. A promoter can contain a specific DNA sequence that binds a protein factor (commonly referred to as a transcription factor) that promotes the binding of RNA polymerase to DNA, thereby resulting in gene transcription. Eukaryotic basal promoters typically (although not necessarily) contain a TATA box and / or a CAAT box.

[0102] As used herein, the term "expression" refers to the process of transcribing a nucleic acid sequence or polynucleotide from a DNA template (e.g., transcribed into mRNA or other RNA transcripts) and / or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, the term expression includes splicing of mRNA in eukaryotic cells.

[0103] As used herein, "operably connected", "operably connected", "operably connected" or its grammatical equivalent refers to genetic elements, such as the arrangement of promoters, enhancers, polyadenylation sequences, etc., wherein the operation (for example, movement or activation) of the first genetic element has some influence on the second genetic element. The influence on the second genetic element can be but need not be the same type as the operation of the first genetic element. For example, if the movement of the first element causes the activation of the second element, the two genetic elements are operably connected. For example, if the regulatory element helps to start the transcription of the coding sequence, the regulatory element that can include promoters and / or enhancer sequences is operably connected to the coding region. As long as this functional relationship is maintained, there may be an insertion residue between the regulatory element and the coding region.

[0104] As used herein, "vector" refers to a macromolecule or an associate of macromolecules that contains or associates with a polynucleotide and mediates the delivery of the polynucleotide to a cell. Examples of vectors include nucleic acid-based vectors (e.g., plasmids and viral vectors) and liposomes. Exemplary nucleic acid-based vectors include genetic elements (e.g., regulatory elements) that are operably linked to a gene to promote expression of the gene in a target.

[0105] As used herein, "expression cassette" and "nucleic acid cassette" are used interchangeably to refer to components of a vector comprising a combination of nucleic acid sequences or elements (e.g., therapeutic genes, promoters, and terminators) expressed together or operably linked for expression. These terms encompass expression cassettes that include a combination of regulatory elements and one or more genes to which they are operably linked for expression.

[0106] A "functional fragment" of a DNA or protein sequence refers to a fragment that retains a biological activity (functional or structural) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence includes its ability to affect expression in a manner attributed to the full-length sequence.

[0107] The terms "engineered", "synthetic" and "artificial" are used interchangeably herein to refer to objects that are modified by human intervention. For example, these terms refer to non-naturally occurring polynucleotides or polypeptides. Engineered peptides have, but do not require, low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) with naturally occurring human proteins. For example, the VPR and VP64 domains are synthetic transactivation domains. Non-limiting examples include the following: nucleic acids are modified by changing their sequence to a sequence that does not exist in nature; nucleic acids are modified by connecting them to nucleic acids that are not associated with them in nature, so that the connection product has a function that does not exist in the original nucleic acid; engineered nucleic acids are synthesized in vitro with sequences that do not exist in nature; proteins are modified by changing the amino acid sequence of proteins to sequences that do not exist in nature; engineered proteins obtain new functions or properties. An "engineered" system includes at least one engineered component.

[0108] The term "tracrRNA" or "tracr sequence" means trans-activating CRISPR RNA. TracrRNA interacts with CRISPR (cr) RNA to form guide (g) RNA in type II and subtype VB CRISPR-Cas systems. If tracrRNA is engineered, it may have about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% sequence identity and / or sequence similarity with a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes (S. pyogenes), Staphylococcus aureus (S. aureus)). TracrRNA may refer to a modified form of tracrRNA, which may include nucleotide changes, such as deletions, insertions or substitutions, variants, mutations or chimeras. The term tracrRNA encompasses nucleic acids that are at least about 60% identical to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) over a stretch of at least 6 consecutive nucleotides. For example, the tracrRNA sequence is at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) over a stretch of at least 6 consecutive nucleotides. Type II tracrRNA sequences can be predicted on genomic sequences by identifying regions that are complementary to a portion of a repeat sequence in an adjacent CRISPR array.

[0109] As used herein, "guide nucleic acid" or "guide polynucleotide" refers to a nucleic acid that can hybridize with a target nucleic acid and thereby guide the relevant nuclease to the target nucleic acid. A guide nucleic acid is, but is not limited to, RNA (guide RNA or gRNA), DNA, or a mixture of RNA and DNA. Guide nucleic acids include crRNA or tracrRNA or a combination of the two. The term guide nucleic acid covers engineered guide nucleic acids and programmable guide nucleic acids to specifically bind to a target nucleic acid. A portion of a target nucleic acid may be complementary to a portion of a guide nucleic acid. The chain of a double-stranded target polynucleotide that is complementary to and hybridized with a guide nucleic acid is a complementary chain. The chain of a double-stranded target polynucleotide that is complementary to a complementary chain and is therefore not complementary to a guide nucleic acid is referred to as a non-complementary chain. A guide nucleic acid with a polynucleotide chain is a "single guide nucleic acid". A guide nucleic acid with two polynucleotide chains is a "double guide nucleic acid". If not otherwise specified, the term "guide nucleic acid" is inclusive and refers to both a single guide nucleic acid and a double guide nucleic acid. A guide nucleic acid may include a segment referred to as a "nucleic acid targeting segment" or a "nucleic acid targeting sequence" or a "spacer". The nucleic acid targeting segment may include sub-segments, which are referred to as "protein binding segments" or "protein binding sequences" or "Cas protein binding segments."

[0110] As used herein, the term "Cas12a" refers to a family of Cas endonucleases that belong to class 2, type VA Cas endonucleases and (a) use relatively small guide RNAs (approximately 42-44 nucleotides) that are processed by the nuclease itself after transcription from the CRISPR array, and (b) cleave DNA to leave staggered cleavage sites.

[0111] As used herein, the term "RuvC_III domain" refers to the third discontinuous segment of the RuvC nuclease domain (the RuvC nuclease domain comprises three discontinuous segments, RuvC_I, RuvC_II and RuvC_III). RuvC domains or their segments can generally be identified by comparing with recorded domain sequences, with the structural comparison of proteins with annotated domains, or by comparing with a Hidden Markov Model (HMM) constructed based on recorded domain sequences (e.g., Pfam HMM PF18541 of RuvC_III).

[0112] As used herein, the term "wedge-shaped" (WED) domain refers to a domain that primarily interacts with the repeat: anti-repeat duplex of the sgRNA and PAM duplex (e.g., present in Cas proteins). WED domains can generally be identified by alignment with recorded domain sequences, structural alignment with proteins with annotated domains, or by comparison with a hidden Markov model (HMM) constructed based on recorded domain sequences.

[0113] As used herein, the term "PAM interaction domain" or "PI domain" refers to a domain that interacts with a protospacer adjacent motif (PAM) outside of a seed sequence in a region targeted by a Cas protein. Examples of PAM interaction domains include, but are not limited to, topoisomerase homology (TOPO) domains and C-terminal domains (CTDs) present in Cas proteins. PAM interaction domains or segments thereof can typically be identified by alignment with recorded domain sequences, alignment with proteins with annotated domains, or by comparison with a hidden Markov model (HMM) constructed based on recorded domain sequences.

[0114] As used herein, the term "REC domain" refers to a domain (e.g., present in a Cas protein) comprising at least one of two segments (REC1 or REC2), which are alpha helical domains that are thought to contact guide RNAs. A REC domain or a segment thereof can generally be identified by comparison with a recorded domain sequence, with a structural alignment of a protein with annotated domains, or by comparison with a hidden Markov model (HMM) constructed based on a recorded domain sequence (e.g., Pfam PF19501 of domain REC1).

[0115] As used herein, the term "BH domain" refers to a domain that is a bridge helix between the NUC and REC lobes of a type II Cas enzyme (e.g., present in a Cas protein). A BH domain or a segment thereof can generally be identified by alignment with a recorded domain sequence, alignment with a protein having annotated domains, or by comparison with a hidden Markov model (HMM) constructed based on a recorded domain sequence (e.g., Pfam PF16593 for domain BH).

[0116] As used herein, the term "HNH domain" refers to an endonuclease domain having characteristic histidine and asparagine residues. HNH domains can generally be identified by comparison with recorded domain sequence alignments, with structural alignments of proteins with annotated domains, or by comparison with a hidden Markov model (HMM) constructed based on recorded domain sequences (e.g., Pfam HMM PF01844 for domain HNH).

[0117] "Factor VIII" or "FVIII" refers to the antihemophilic factor (i.e., blood coagulation or clotting protein) encoded by the FVIII gene. Factor VIII binds to von Willebrand factor in its inactive form. In response to injury, the two factors separate, and FVIII activates and interacts with FIX to start a chain of chemical reactions that lead to blood clots. Genetic defects in factor VIII lead to hemophilia A.

[0118] The term "donor template" refers to a polynucleotide comprising an exogenous polynucleotide sequence (e.g., a nucleic acid sequence of a therapeutic gene) and one or more polynucleotide sequences for mediating recombination, such as by non-homologous end joining (NHEJ) or homology directed repair (HDR).

[0119] As used herein, the term "complex" refers to the connection of at least two components. The two components can each retain the properties / activity they had before forming the complex or acquire properties as a result of forming the complex. Connection includes, but is not limited to, covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, van der Waals interactions, and hydrophobic bonds), use of linkers, fusion, or any other suitable method. The components of the contemplated complex include polynucleotides, polypeptides, or combinations thereof. For example, the complex comprises an endonuclease and a guide polynucleotide.

[0120] The terms "sequence identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, for example: BLASTP for polypeptide sequences longer than 30 residues using parameters of a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix with gap penalties set to existence of 11, extension of 1, and adjustment using a conditional composition scoring matrix; BLASTP for sequences less than 30 residues using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and a PAM30 scoring matrix with gap penalties set to gap open of 9 and extension of gap of 1 (these are the default parameters for BLASTP in the BLAST suite, available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW using the Smith-Waterman homology search algorithm parameters of matches of 2, mismatches of -1, and gaps of -1; MUSCLE using default parameters; MAFFT using parameters of retree of 2 and maximum iterations of 1000; Novafold using default parameters; HMMER hmmalign using default parameters.

[0121] The term "optimally aligned" in the context of two or more nucleic acid or polypeptide sequences refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that have been aligned for maximum correspondence of amino acid residues or nucleotides, e.g., as determined by the alignment that produces the highest or "optimizing" percent identity score.

[0122] The present disclosure includes variants of any enzyme in the enzymes described herein with one or more conservative amino acid substitutions. Such conservative substitutions can be carried out in the amino acid sequence of a polypeptide without destroying the three-dimensional structure or function of the polypeptide. Conservative substitutions can be completed by replacing each other with amino acids having similar hydrophobicity, polarity, and R chain length. Additionally or alternatively, by comparing the alignment sequences of homologous proteins from different species, conservative substitutions can be identified by locating the amino acid residues (e.g., non-conservative residues) that have mutated between species without changing the basic function of the encoded protein. Such conservatively substituted variants may include variants having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity with any of the endonuclease protein sequences described herein (e.g., MG3 or MG29 family endonucleases described herein, or any other family nuclease described herein). In some embodiments, such conservatively substituted variants are functional variants. Such functional variants may encompass sequences having substitutions such that the activity of one or more key active site residues or guide RNA binding residues of the endonuclease is not destroyed. In some embodiments, a functional variant of any of the proteins described herein lacks a substitution of at least one conserved or functional residue.

[0123] The present disclosure also encompasses variants of any of the enzymes described herein that replace one or more catalytic residues to reduce or eliminate the activity of the enzyme (e.g., variants with reduced activity). In some embodiments, variants with reduced activity of proteins described herein include disruptive substitutions of at least one, at least two, or all three catalytic residues.

[0124] Conservative substitution tables providing functionally similar amino acids are available from various references (see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd ed. (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another:

[0125] 1) Alanine (A), glycine (G);

[0126] 2) Aspartic acid (D), glutamic acid (E);

[0127] 3) Asparagine (N), glutamine (Q);

[0128] 4) Arginine (R), Lysine (K);

[0129] 5) Isoleucine (I), leucine (L), methionine (M), valine (V);

[0130] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0131] 7) serine (S), threonine (T); and

[0132] 8) Cysteine ​​(C), methionine (M).

[0133] Overview

[0134] Treatment of diseases and conditions caused by genetic defects can include using gene editing systems to correct the genetic defects. This can be done by integrating a copy of the gene into the genome at the appropriate site and in the appropriate cells or tissues so that the gene is expressed and a functional protein is produced.

[0135] Hemophilia A is caused by mutations in factor VIII (FVIII) genes, which reduce FVIII expression or inactivate the function of FVIII protein. Because many different mutations in FVIII cause hemophilia A, it is preferred that the gene therapy method is not specific for individual mutations. A promising method is to supplement the genome copy of defective FVIII with a functional transgenic copy of a gene. In one embodiment of this method, a carrier of a guide polynucleotide comprising a nuclease and a target nucleic acid at a targeting safe harbor locus (such as an albumin locus) is administered systemically (e.g., intravenously), and a functional copy of the FVIII gene is delivered to the hepatocytes of the liver. Then, the FVIII gene is integrated into the target nucleic acid at the break produced by the nuclease by non-homologous end joining (NHEJ) or homology directed repair (HDR), a combination of two DNA repair mechanisms, or by other DNA repair mechanisms.

[0136] CRISPR / Cas enzymes

[0137] The discovery of new Cas enzymes with unique functions and structures offers the possibility to further gene editing technologies with improved speed, specificity, functionality, and ease of use. Relative to the predicted prevalence of clustered regularly interspaced short palindromic repeat (CRISPR) systems in microorganisms and the sheer diversity of microbial species, relatively few functionally characterized CRISPR / Cas enzymes exist in the literature. This is in part because a large number of microbial species may not be easily cultured under laboratory conditions. Metagenomic sequencing of natural environmental niches containing a large number of microbial species may offer the potential to substantially increase the number of new CRISPR / Cas systems characterized and accelerate the discovery of new oligonucleotide editing functions. A recent example of the fruitfulness of this approach was demonstrated by the discovery of the CasX / CasY CRISPR system in 2016 through metagenomic analysis of natural microbial communities.

[0138] The CRISPR / Cas system is an RNA-guided nuclease complex that acts as an adaptive immune system in microorganisms. In its natural environment, the CRISPR / Cas system occurs in a CRISPR (clustered regularly interspaced short palindromic repeats) operator or locus, and the system consists of two parts: (i) a short repeat array (30-40bp) separated by a short spacer sequence, which encodes an RNA-based targeting element; and (ii) an ORF encoding a Cas nuclease. The efficient nuclease targeting of a specific target nucleic acid sequence generally requires the following two: (i) complementary hybridization between the first 6-8 nucleic acids of the target nucleic acid and the crRNA guide; and (ii) according to a specific Cas nuclease, a protospacer adjacent motif (PAM) sequence (PAM is generally a sequence not often represented in the host genome) exists in a certain vicinity of the target nucleic acid sequence. According to the exact function and organization of the system, the CRISPR-Cas system is generally divided into 2 categories, 5 types, and 16 subtypes based on shared functional properties and evolutionary similarities.

[0139] Class 1 CRISPR-Cas systems have large multi-subunit effector complexes and include type I, III, and IV Cas nucleases. Class 2 CRISPR-Cas systems typically have single-polypeptide multi-domain nuclease effectors and include type II, V, and VI Cas nucleases.

[0140] Type I CRISPR-Cas systems are considered to have medium complexity in terms of components. In type I CRISPR-Cas systems, the array of RNA targeting elements is transcribed into long precursor crRNA (pre-crRNA), which is processed at the repeat element to release short mature crRNA, when followed by a suitable short consensus sequence called a protospacer adjacent motif (PAM), the short mature crRNA guides the nuclease complex to the nucleic acid target. This treatment is carried out by the endoribonuclease subunit (Cas6) of the large endonuclease complex called cascade, and the endonuclease complex also includes the nuclease (Cas3) protein component of the nuclease complex guided by crRNA. Cas I nuclease mainly serves as DNA nuclease.

[0141] Type III CRISPR systems are characterized by the presence of a central nuclease called Cas10 and repeat-associated mysterious proteins (RAMPs) including Csm or Cmr protein subunits. As in type I systems, mature crRNAs are processed by pre-crRNA using a Cas6-like enzyme. Unlike type I and type II systems, type III systems appear to target and cleave DNA-RNA duplexes (such as DNA chains used as templates for RNA polymerases).

[0142] Type IV CRISPR-Cas systems have an effector complex consisting of a highly reduced large subunit nuclease (csf1), two genes for the Cas5 (csf3) and Cas7 (csf2) groups of RAMP proteins, and in some cases a gene for a predicted small subunit; such systems are often found on endogenous plasmids.

[0143] Class 2 CRISPR-Cas systems typically have single-polypeptide multi-domain nuclease effectors and include types II, V, and VI.

[0144] Type II CRISPR-Cas systems are considered to be the simplest in terms of components. In type II CRISPR-Cas systems, the CRISPR array is processed into mature crRNA without the presence of special endonuclease subunits, but requires a small trans-encoded crRNA (tracrRNA), whose region is complementary to the array repeat sequence; tracrRNA interacts with its corresponding effector nuclease (e.g., Cas9) and repeat sequence to form a precursor dsRNA structure, which is cut by endogenous RNase III, thereby generating a mature effector enzyme loaded with both tracrRNA and crRNA. Cas II nucleases are identified as DNA nucleases. Type 2 effectors generally show a structure including a RuvC-like endonuclease domain, which is folded using RNase H, wherein the folding of the RuvC-like nuclease domain is inserted with an unrelated HNH nuclease domain. The RuvC-like domain is responsible for the cutting of the target (e.g., crRNA complementary) DNA chain, and the HNH domain is responsible for the cutting of the replaced DNA chain.

[0145] The V-type CRISPR-Cas system is characterized by a nuclease effector (e.g., Cas12) structure similar to the structure of the type II effector including a RuvC-like domain. Similar to type II, most (but not all) V-type CRISPR systems use tracrRNA to process pre-crRNA into mature crRNA. However, unlike the type II system that requires RNase III to cut pre-crRNA into multiple crRNAs. The V-type system can use the effector nuclease itself to cut pre-crRNA. Like the type II CRISPR-Cas system, the V-type CRISPR-Cas system is again identified as a DNA nuclease. Unlike the type II CRISPR-Cas system, some V-type enzymes (e.g., Cas12a) appear to have a powerful single-stranded nonspecific deoxyribonuclease activity activated by the first crRNA-directed cutting of a double-stranded target sequence.

[0146] Type VI CRISPR-Cas systems have RNA-guided RNA endonucleases. The single polypeptide effector of a type VI system (e.g., Cas13) includes two HEPN ribonuclease domains instead of a RuvC-like domain. Unlike type II and type V systems, type VI systems also do not appear to require tracrRNA to process pre-crRNA into crRNA. However, similar to type V systems, some type VI systems (e.g., C2C2) appear to have powerful single-stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA-directed cutting of the target RNA.

[0147] Gene editing system

[0148] In certain embodiments, described herein is an engineered nuclease system comprising: a) an endonuclease; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene; and c) a donor template comprising a nucleic acid sequence encoding a Factor VIII (FVIII) gene or a functional fragment thereof.

[0149] MG endonuclease

[0150] Disclosed herein are systems and methods for supplementing liver enzymes. In some embodiments, the systems and methods comprise an endonuclease. In some embodiments, the endonuclease is functional in a prokaryotic or eukaryotic cell for in vitro, in vivo, or ex vivo applications. In some embodiments, the endonuclease is a nucleic acid-guided nuclease, a chimeric nuclease, or a fusion nuclease.

[0151] In some embodiments, the endonuclease is MG29-1 (i.e., SEQ ID NO: 54). In some embodiments, the endonuclease is MG3-6 / 3-4 (i.e., SEQ ID NO: 96). MG29-1 is a type V CRISPR nuclease, and MG3-6 / 3-4 is a type II CRISPR nuclease, which is generated by exchanging the PAM interaction domain (PID) of MG3-6 with the PID of MG3-4 to change the PAM recognition specificity. In some embodiments, the PAM of MG29-1 is functionally defined as KTTN (K=G or T, N=any base) in mammalian cells. In some embodiments, the PAM of MG3-6 or MG3-4 is functionally defined as AAANN (N=any base) in mammalian cells.

[0152] In some embodiments, the endonuclease comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NO: 54 and 96. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to SEQ ID NO: 54 or SEQ ID NO: 96. In some embodiments, the endonuclease comprises a sequence that is 100% identical to SEQ ID NO:54 or SEQ ID NO:96.

[0153] In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least about 75% identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least about 80% identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least about 85% identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least about 90% identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least about 95% identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least about 96% identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least about 97% identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least about 98% identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having at least about 99% identity to any one of SEQ ID NOs: 30, 31, 53, and 95. In some embodiments, the endonuclease is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 30, 31, 53, and 95.

[0154] In some embodiments, the endonuclease comprises one or more fragments or domains of a nuclease, such as a nucleic acid-guided nuclease. In some embodiments, the endonuclease comprises one or more fragments or domains of a nuclease from an organism, genus, species, or other phylogenetic group as described herein. In some embodiments, the endonuclease comprises one or more fragments or domains of a nuclease from a nuclease ortholog from a different species.

[0155] In some embodiments, the endonuclease comprises one or more fragments or domains of a nuclease, such as a nucleic acid-guided nuclease. In some embodiments, the endonuclease comprises one or more fragments or domains of a nuclease from an organism, genus, species, or other phylogenetic group as described herein. In some embodiments, the endonuclease comprises one or more fragments or domains of a nuclease from a nuclease ortholog from a different species.

[0156] In some embodiments, the endonuclease comprises one or more fragments or domains from at least two different nucleases. In some embodiments, the endonuclease comprises one or more fragments or domains from at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different nucleases. In some embodiments, the endonuclease comprises one or more fragments or domains from at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleases from different species. In some embodiments, the endonuclease comprises 2 fragments or domains, each fragment or domain is from a different nuclease. In some embodiments, the endonuclease comprises 3 fragments or domains, each fragment or domain is from a different nuclease. In some embodiments, the endonuclease comprises 4 fragments or domains, each fragment or domain is from a different nuclease. In some embodiments, the endonuclease comprises 5 fragments or domains, each fragment or domain is from a different nuclease. In some embodiments, the endonuclease comprises 3 fragments or domains, at least one of which is from a different nuclease. In some embodiments, the endonuclease comprises 4 fragments or domains, wherein at least one fragment or domain is from a different nuclease. In some embodiments, the endonuclease comprises 5 fragments or domains, wherein at least one fragment or domain is from a different nuclease.

[0157] In some embodiments, the junctions between fragments or domains from different nucleases or species occur in multiple unstructured regions. Unstructured regions in polynucleotides include, for example, regions without predicted secondary structural elements (such as alpha helices or beta strands). Unstructured regions may include, for example, regions exposed within the protein structure, loop regions, or regions that are not conserved within various protein orthologs as predicted by sequence or structural comparisons.

[0158] In some embodiments, the endonuclease comprises one or more nuclear localization sequences (NLS) near the N-terminus or C-terminus of the endonuclease. In some embodiments, the NLS comprises a sequence of any one of SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence of any one of SEQ ID NOs: 144-159 or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any one of SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 80% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 85% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 90% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 91% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 92% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 93% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 94% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 95% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 96% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 97% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 98% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having at least about 99% identity to SEQ ID NOs: 144-159. In some embodiments, the NLS comprises a sequence having 100% identity to SEQ ID NOs: 144-159.

[0159] Table 1A: Example NLS sequences for use with Cas effectors according to the present disclosure.

[0160]

[0161] Guide polynucleotide

[0162] The systems and methods described herein for supplementing liver enzymes can include a guide polynucleotide, e.g., a guide RNA (gRNA), a single gRNA, or a dual guide RNA for supplementing liver enzymes. In a polynucleotide, when T is mentioned, T means U (uracil) in RNA and T (thymine) in DNA.

[0163] In some embodiments, the target gene or locus is albumin. In some embodiments, the guide polynucleotide targets or hybridizes to a target nucleic acid sequence in albumin. In some embodiments, the guide polynucleotide targets or hybridizes to a target nucleic acid sequence in intron 1 of albumin. In some embodiments, the guide polynucleotide targets or hybridizes to a target nucleic acid sequence in exon 1 of albumin.

[0164] In some embodiments, the target gene or locus is albumin. In some embodiments, the guide polynucleotide targeting albumin is encoded by any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides that is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98.In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98.

[0165] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene (e.g., SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98.In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that has 100% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98.

[0166] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene (e.g., SEQ ID NOs: 3-6). In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 3-6 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-6. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 3-6. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 3-6. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 3-6. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 3-6. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 3-6. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 3-6. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 3-6. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 3-6. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 3-6.

[0167] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene (e.g., SEQ ID NOs: 1, 2, and 8). In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 1, 2, and 8 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 1, 2, and 8. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 1, 2, and 8.

[0168] In some embodiments, the guide polynucleotide is configured to form a complex with an endonuclease. In some embodiments, the guide polynucleotide binds to the endonuclease to form a complex. In some embodiments, the guide polynucleotide binds to the endonuclease (e.g., non-covalently via electrostatic interactions or hydrogen bonds) to form a complex. In some embodiments, the guide polynucleotide is fused to the endonuclease to form a complex.

[0169] In some embodiments, the guide polynucleotide comprises a spacer sequence. In some embodiments, the spacer sequence is configured to hybridize to a target nucleic acid sequence. In some embodiments, the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence.

[0170] In some embodiments, the guide polynucleotide (e.g., gRNA) targets a gene or locus in a cell. In some embodiments, the guide polynucleotide targets a gene or locus in a mammalian cell. In some embodiments, the mammalian cell is a pig, cow, goat, sheep, rodent, rat, mouse, non-human primate, or human cell.

[0171] In some embodiments, the guide polynucleotide (e.g., guide RNA) includes various structural elements, including but not limited to: a spacer sequence, crRNA, and optional tracrRNA that binds to the original spacer sequence (target sequence). In some embodiments, the genome editing system includes a CRISPR guide RNA. In some embodiments, the guide RNA includes a crRNA that includes a spacer sequence. In some embodiments, the guide RNA additionally includes tracrRNA or modified tracrRNA.

[0172] In some embodiments, the systems provided herein comprise one or more guide polynucleotides. In some embodiments, the guide polynucleotide comprises a sense sequence. In some embodiments, the guide polynucleotide comprises an antisense sequence. In some embodiments, the guide polynucleotide comprises a nucleotide sequence other than a region that is complementary or substantially complementary to a region of a target sequence. For example, a crRNA is or is considered to be part of a guide polynucleotide, or is included in a guide polynucleotide, such as a crRNA:tracrRNA chimera.

[0173] In some embodiments, the guide polynucleotide comprises synthetic nucleotides or modified nucleotides. In some embodiments, the guide polynucleotide comprises one or more internucleoside linkers modified by natural phosphodiester. In some embodiments, all internucleoside linkers of the guide polynucleotide or a contiguous nucleotide sequence thereof are modified. For example, in some embodiments, the internucleoside bond comprises sulfur (S), such as a thiophosphate internucleoside bond. In some embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% of modified internucleoside linkers. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified internucleoside linkers (e.g., thiophosphate internucleoside bonds).

[0174] In some embodiments, the guide polynucleotide comprises a modification to a ribose sugar or a nucleobase. In some embodiments, the guide polynucleotide comprises one or more nucleosides comprising a modified sugar moiety, wherein a modified glucose moiety is a modification of the sugar moiety when compared to the ribose sugar moiety present in deoxyribonucleic acid (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring having a diradical bridge between the C2 carbon and the C4 carbon on the ribose ring (e.g., locked nucleic acid (LNA)), or an unconnected ribose ring (e.g., UNA) that typically lacks a bond between the C2 carbon and the C3 carbon. In some embodiments, sugar-modified nucleosides include bicyclic hexose nucleic acids or tricyclic nucleic acids. In some embodiments, modified nucleosides include nucleosides in which the sugar moiety is replaced by a non-sugar moiety, such as a peptide nucleic acid (PNA) or a morpholino nucleic acid.

[0175] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, sugar modifications include modifications by changing the substituents on the ribose ring to groups other than hydrogen or 2'-OH groups naturally present in DNA and RNA nucleosides. In some embodiments, the substituents are introduced at the 2', 3', 4', 5' positions or a combination thereof. In some embodiments, nucleosides having modified sugar moieties include 2' modified nucleosides, e.g., 2' substituted nucleosides. In some embodiments, 2' sugar modified nucleosides are nucleosides having substituents other than H or -OH at the 2' position (2' substituted nucleosides) or including 2' linked diradicals, and include 2' substituted nucleosides and LNA (2'-4' diradical bridged) nucleosides. Examples of 2'-substituted modified nucleosides include, but are not limited to, 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. In certain embodiments, the modification in the ribose group includes modification at the 2' position of the ribose group. In certain embodiments, the modification at the 2' position of the ribose group is selected from the group consisting of: 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxyethyl).

[0176] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, the guide polynucleotide comprises only modified sugars. In some embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a disaccharide. In some embodiments, the modified sugar comprises 2'-O-methyl. In some embodiments, the modified sugar comprises 2'-fluoro. In some embodiments, the modified sugar comprises 2'-O-methoxyethyl. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified sugars (e.g., comprising 2'-O-methyl or 2'-fluoro).

[0177] In some embodiments, the guide polynucleotide comprises both an internucleoside linker modification and a nucleoside modification. In some embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified internucleoside linkers and greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified internucleoside linkers (e.g., phosphorothioate internucleoside linkages) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified sugars (e.g., comprising 2'-O-methyl or 2'-fluoro).

[0178] In some embodiments, the guide polynucleotide comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a plant genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a human genomic polynucleotide sequence.

[0179] In some embodiments, the guide polynucleotide is 30-250 nucleotides in length. In some embodiments, the guide polynucleotide is more than 90 nucleotides in length. In some embodiments, the guide polynucleotide is less than 245 nucleotides in length. In some embodiments, the guide polynucleotide is 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, or more than 240 nucleotides in length. In some embodiments, the guide polynucleotide is about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 10 In some embodiments, the present invention relates to a polypeptide having at least one nucleotide sequence and at least one nucleotide sequence. In some embodiments, the polypeptide has at least one nucleotide sequence and at least one nucleotide sequence. In some embodiments, the polypeptide has at least one nucleotide sequence and at least one nucleotide sequence.

[0180] MG Gene Editing System

[0181] In certain embodiments, described herein is an engineered nuclease system comprising: a) an endonuclease; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene; and c) a donor template comprising a nucleic acid sequence encoding a Factor VIII (FVIII) gene or a functional fragment thereof.

[0182] In some embodiments, the endonuclease induces a single-strand break at or near the target nucleic acid sequence. In some embodiments, the endonuclease induces a double-strand break at or near the target nucleic acid sequence. In some embodiments, the donor template is integrated into the target nucleic acid sequence at a double-strand break. In some embodiments, the donor template is integrated into the target nucleic acid sequence at a double-strand break by non-homologous end joining (NHEJ). In some embodiments, the donor template is integrated into the target nucleic acid sequence at a double-strand break by homology-directed repair (HDR).

[0183] In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 70% identity to SEQ ID NO: 54 or SEQ ID NO: 96; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 75% identity to SEQ ID NO: 54 or SEQ ID NO: 96; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 80% identity to SEQ ID NO: 54 or SEQ ID NO: 96; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 85% identity to SEQ ID NO: 54 or SEQ ID NO: 96; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 90% identity to SEQ ID NO: 54 or SEQ ID NO: 96; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 95% identity to SEQ ID NO: 54 or SEQ ID NO: 96; b) an engineered guide polynucleotide; and c) a donor template comprising a sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 96% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 97% identity to SEQ ID NO: 54 or SEQ ID NO: 96; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 98% identity to SEQ ID NO: 54 or SEQ ID NO: 96; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 99% identity to SEQ ID NO: 54 or SEQ ID NO: 96; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising 100% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.

[0184] In some embodiments, the engineered guide polynucleotide is a single guide nucleic acid. In some embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In some embodiments, the engineered guide polynucleotide is RNA. In some embodiments, the endonuclease is complexed with the engineered guide polynucleotide. In some embodiments, the endonuclease is linked to the engineered guide polynucleotide.

[0185] In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 70% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs:14-15, 24-27, 43-45, 50, 55, 60-68 and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 75% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs:14-15, 24-27, 43-45, 50, 55, 60-68 and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 80% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs:14-15, 24-27, 43-45, 50, 55, 60-68 and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 85% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs:14-15, 24-27, 43-45, 50, 55, 60-68 and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 90% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs:14-15, 24-27, 43-45, 50, 55, 60-68 and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 95% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs:14-15, 24-27, 43-45, 50, 55, 60-68 and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 96% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs:14-15, 24-27, 43-45, 50, 55, 60-68 and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 97% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs:14-15, 24-27, 43-45, 50, 55, 60-68 and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 98% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs:14-15, 24-27, 43-45, 50, 55, 60-68 and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 99% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs:14-15, 24-27, 43-45, 50, 55, 60-68 and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising 100% identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs:14-15, 24-27, 43-45, 50, 55, 60-68 and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.

[0186] In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 75% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 80% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 85% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 90% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 95% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 96% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 97% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 98% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 99% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising 100% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.

[0187] In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide being encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 75% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide being encoded by a nucleic acid sequence having at least about 75% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 80% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide being encoded by a nucleic acid sequence having at least about 80% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 85% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide being encoded by a nucleic acid sequence having at least about 85% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 90% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide being encoded by a nucleic acid sequence having at least about 90% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 95% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide being encoded by a nucleic acid sequence having at least about 95% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 96% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide being encoded by a nucleic acid sequence having at least about 96% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 97% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide being encoded by a nucleic acid sequence having at least about 97% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 98% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide being encoded by a nucleic acid sequence having at least about 98% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.In some embodiments, the engineered nuclease system comprises: a) an endonuclease encoded by a nucleic acid sequence having at least about 99% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide being encoded by a nucleic acid sequence having at least about 99% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising 100% identity to any one of SEQ ID NOs: 30, 31, 53, and 95; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or within an intron of an albumin gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, and 97-98; and c) a donor template comprising a nucleic acid sequence encoding a FVIII gene or a functional fragment thereof.

[0188] In some embodiments, the donor template comprises a polyadenylation signal. In some embodiments, the polyadenylation signal is located at the C-terminus of a factor VIII gene or a fragment thereof. In some embodiments, the polyadenylation signal is connected to a factor VIII gene or a fragment thereof. In some embodiments, the polyadenylation signal is fused to a factor VIII gene or a fragment thereof.

[0189] In some embodiments, the donor template comprises a nuclear targeting sequence (NTS). In some embodiments, the nuclear targeting sequence comprises a plurality of transcription factor binding sites. In some embodiments, the transcription factor (TF) binding site is an SV40 enhancer region. In some embodiments, the transcription factor is TCF1, HNF1, NFY, CEBP, OCT1, AP1, HNF1-α, HNF1-β, CEBPA, LEF-1, FOX D1, IRF1, HNF3, HNF4, HNF5, Tal1β / E47 or MyoD. Table 1B includes examples of TFs found in promoters and enhancers of genes highly expressed in the liver. Common liver-specific transcription factors include: HNF3, HNF4, HNF5, C / EBP, HNF1α, LEF1, FOX, IRF and TCF.

[0190] Table 1B: Binding sites of TFs known to be highly expressed in the liver

[0191]

[0192] In some embodiments, a functional fragment of the albumin promoter is used with a donor template. In some embodiments, the functional fragment comprises a sequence of about 100 bp located 5' of the transcription start site and containing binding sites for several TFs, including HNF1, CEBP, LEF-1, FOX, IRF1, and LEF1.

[0193] In some embodiments, the transcription factor binding sequence comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 37, 39, and 41, or any sequence listed in Table 1B. In some embodiments, the transcription factor binding sequence comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 37, 39, and 41, or any sequence listed in Table 1B. In some embodiments, the transcription factor binding sequence comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 37, 39, and 41, or any sequence listed in Table 1B. In some embodiments, the transcription factor binding sequence comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 37, 39, and 41, or any sequence listed in Table 1B. In some embodiments, the transcription factor binding sequence comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 37, 39, and 41, or any sequence listed in Table 1B. In some embodiments, the transcription factor binding sequence comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 37, 39, and 41, or any sequence listed in Table 1B. In some embodiments, the transcription factor binding sequence comprises a sequence that is 100% identical to any one of SEQ ID NOs: 37, 39, and 41, or any of the sequences listed in Table IB.

[0194] In some embodiments, the nuclear targeting sequence is located on the 5' end of the donor template. In some embodiments, the nuclear targeting sequence is located on the 3' end of the donor template. In some embodiments, the nuclear targeting sequence is located on the 5' end and the 3' end of the donor template.

[0195] In some embodiments, the donor template further comprises a recognition site sequence for the endonuclease on the 5' end or the 3' end of the donor template. In some embodiments, when the donor template is flanked by the nuclear targeting sequence on the 5' end, the nuclear targeting sequence is located 5' of the recognition site sequence. In some embodiments, when the donor template is flanked by the nuclear targeting sequence on the 3' end, the nuclear targeting sequence is located 3' of the recognition site sequence.

[0196] In some embodiments, the donor template comprises a splice acceptor sequence. In some embodiments, the splice acceptor sequence targets an albumin gene. In some embodiments, the splice acceptor sequence targets exon 1 of an albumin gene. In some embodiments, the splice acceptor sequence targets intron 1 of an albumin gene. In some embodiments, the splice acceptor sequence is connected to a factor VIII gene or a fragment thereof. In some embodiments, the splice acceptor sequence is an intron sequence connected to an exon sequence of a factor VIII gene or a fragment thereof. In some embodiments, the splice acceptor sequence is connected to a factor VIII gene or a fragment thereof at the 5' end of the factor VIII gene or a fragment thereof. In some embodiments, the splice acceptor sequence is connected to a factor VIII gene or a fragment thereof at the 3' end of the factor VIII gene or a fragment thereof. In some embodiments, the splice acceptor sequence is connected to a factor VIII gene or a fragment thereof using a linker. In some embodiments, the linker comprises a sequence having at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, the linker comprises a sequence having at least 90% sequence identity to SEQ ID NO: 80. In some embodiments, the linker comprises a sequence having at least 95% sequence identity to SEQ ID NO: 80. In some embodiments, the linker comprises a sequence having at least 96% sequence identity to SEQ ID NO: 80. In some embodiments, the linker comprises a sequence having at least 97% sequence identity to SEQ ID NO: 80. In some embodiments, the linker comprises a sequence having at least 98% sequence identity to SEQ ID NO: 80. In some embodiments, the linker comprises a sequence having at least 99% sequence identity to SEQ ID NO: 80. In some embodiments, the linker comprises a sequence having 100% sequence identity to SEQ ID NO: 80. In some embodiments, the splice acceptor sequence is fused to a Factor VIII gene or a fragment thereof.

[0197] In some embodiments, the donor template comprises, from 5' to 3', NTS(1)-NRS(1)-SA-FVIII-NRS(2)-NTS(2), wherein NTS(1) represents a first nuclear targeting sequence; NTS(2) represents a second nuclear targeting sequence; NRS(1) represents a first nuclease recognition site sequence; NRS(2) represents a second nuclease recognition site sequence; SA represents the splice acceptor sequence targeting exon 1 of the albumin gene; and FVIII represents the Factor VIII gene or a fragment thereof. In some embodiments, the 5' to 3' orientation of NRS(1) and NRS(2) is according to: (a) forward, forward; (b) reverse, reverse; (c) forward, reverse; (d) reverse, forward; wherein forward represents the same 5' to 3' orientation as the target nucleic acid sequence, and reverse represents the opposite 5' to 3' orientation to the target nucleic acid sequence. In some embodiments, the donor template comprises a KTTN (K=G or T, N=any base) or AAANNN (N=any base) sequence.

[0198] In some embodiments, the donor template comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.In some embodiments, the donor template comprises a sequence that is at least about 99% identical to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94. In some embodiments, the donor template comprises a sequence that is 100% identical to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.

[0199] In some embodiments, the FVIII gene or a functional fragment thereof is codon-optimized to remove at least one cytosine-guanine (CG or CpG) motif.

[0200] In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs: 10, 71-79 and 89. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 10, 71-79 and 89. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 10, 71-79 and 89. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 10, 71-79, and 89. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having 100% identity to any one of SEQ ID NOs: 10, 71-79, and 89.

[0201] In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 70% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 75% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 80% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 85% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 90% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 95% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 96% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 97% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 98% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having at least about 99% identity to SEQ ID NO: 10. In some embodiments, the FVIII gene or a functional fragment thereof comprises a sequence having 100% identity to SEQ ID NO: 10.

[0202] In some embodiments, the FVIII gene or its functional fragment is modified to include a B domain comprising a sequence having at least about 90% identity with any one of SEQ ID NOs: 71-79 and 89. In some embodiments, the FVIII gene or its functional fragment is modified to include a B domain comprising a sequence having at least about 95% identity with any one of SEQ ID NOs: 71-79 and 89. In some embodiments, the FVIII gene or its functional fragment is modified to include a B domain comprising a sequence having at least about 96% identity with any one of SEQ ID NOs: 71-79 and 89. In some embodiments, the FVIII gene or its functional fragment is modified to include a B domain comprising a sequence having at least about 97% identity with any one of SEQ ID NOs: 71-79 and 89. In some embodiments, the FVIII gene or its functional fragment is modified to include a B domain comprising a sequence having at least about 98% identity with any one of SEQ ID NOs: 71-79 and 89. In some embodiments, the FVIII gene or a functional fragment thereof is modified to comprise a B domain comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 71-79 and 89. In some embodiments, the FVIII gene or a functional fragment thereof is modified to comprise a B domain comprising a sequence having 100% identity to any one of SEQ ID NOs: 71-79 and 89.

[0203] In some embodiments, the FVIII gene or functional fragment thereof comprising a modified B domain comprises a sequence having at least about 90% identity with any one of SEQ ID NOs: 86-87 and 90. In some embodiments, the FVIII gene or functional fragment thereof comprising a modified B domain comprises a sequence having at least about 95% identity with any one of SEQ ID NOs: 86-87 and 90. In some embodiments, the FVIII gene or functional fragment thereof comprising a modified B domain comprises a sequence having at least about 96% identity with any one of SEQ ID NOs: 86-87 and 90. In some embodiments, the FVIII gene or functional fragment thereof comprising a modified B domain comprises a sequence having at least about 97% identity with any one of SEQ ID NOs: 86-87 and 90. In some embodiments, the FVIII gene or functional fragment thereof comprising a modified B domain comprises a sequence having at least about 98% identity with any one of SEQ ID NOs: 86-87 and 90. In some embodiments, the FVIII gene or functional fragment thereof comprising a modified B domain comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 86-87 and 90. In some embodiments, the FVIII gene or functional fragment thereof comprising a modified B domain comprises a sequence having 100% identity to any one of SEQ ID NOs: 86-87 and 90.

[0204] How to use

[0205] Provided herein is a method for supplementing liver enzymes using an engineered nucleic acid enzyme system as described herein. The method for supplementing liver enzymes is included in the appropriate site and in appropriate cells or tissues by integrating liver genes into the genome so that genes are expressed and functional proteins (e.g., functional FVIII) are produced, and therefore deficiencies are supplemented.

[0206] In some embodiments, the engineered nuclease systems described herein are used to integrate the Factor VIII gene into the genome of an individual in need. In some embodiments, the engineered nuclease systems described herein are used to integrate the Factor VIII gene into the genome of an individual in need, thereby treating hemophilia A. In some embodiments, the engineered nuclease systems described herein are used to treat hemophilia A in an individual in need.

[0207] In some embodiments, the FVIII gene or a functional fragment thereof is delivered to hepatocytes of the liver by systemic (e.g., intravenous) administration of a vector comprising an endonuclease and a guide polynucleotide targeting a target nucleic acid sequence in a safe harbor locus (e.g., an albumin locus).

[0208] In certain embodiments, the integration site of FVIII gene is located in albumin gene. In certain embodiments, the integration site of FVIII gene is intron 1 of albumin gene. Since albumin gene is expressed at high level in hepatocyte, it is expected that the promoter of albumin gene drives the effective expression of integrated FVIII gene. This method of integration into intron has the advantage that the double-strand break subsequently repaired by error-prone NHEJ will not adversely affect the function of albumin gene. In certain embodiments, in order to capture transcription initiated from albumin promoter, the 5' end of donor template immediately before the N-terminal of FVIII protein coding sequence includes splice acceptor site. This splice acceptor can capture some splicing events from exon 1 of albumin, thereby generating mRNA of 5'UTR and exon 1 of albumin fused to the coding sequence of FVIII in frame.

[0209] Delivery and vectors

[0210] Disclosed herein, in some embodiments, are nucleic acid sequences encoding the engineered nuclease systems described herein, or components thereof (e.g., endonucleases, engineered guide polynucleotides, or donor templates).

[0211] In some embodiments, the nucleic acid encoding the engineered nuclease system described herein or its components is DNA, such as linear DNA, plasmid DNA or minicircle DNA. In some embodiments, the nucleic acid encoding the engineered nuclease system described herein or its components is RNA, such as mRNA.

[0212] In some embodiments, nucleic acids encoding engineered nuclease systems described herein or components thereof are delivered by nucleic acid-based vectors. In some embodiments, nucleic acid-based vectors are plasmids (e.g., circular DNA molecules that can replicate autonomously in cells), cosmids (e.g., pWE or sCos vectors), artificial chromosomes, human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), P1-derived artificial chromosomes (PACs), phagemids, phage derivatives, bacmids, or viruses. In some embodiments, the nucleic acid-based vector is selected from the list consisting of: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 vector, pEF1a-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP tag (m), pSF-CMV-PURO-NH2-CMYC, pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, pSF-Tac, pRI 101-AN DNA, pCambia2301, pTYB21, pKLAC2, pAc5.1 / V5-His A and pDEST8.

[0213] In some embodiments, the nucleic acid-based vector includes a promoter. In some embodiments, the promoter is selected from the group consisting of: a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof. In some embodiments, the promoter is selected from the group consisting of: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p19, p40, synaptic protein, CaMKII, GRK1, and derivatives thereof. In some embodiments, the promoter is a U6 promoter. In some embodiments, the promoter is a CAG promoter.

[0214] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a dengue virus, a lentivirus, a herpes virus, a poxvirus, anellovirus, a bocavirus, a vaccinia virus, or a retrovirus. In some embodiments, the virus is an alphavirus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In some embodiments, the virus is a baculovirus. In some embodiments, the virus is a dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpes virus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is an anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is a retrovirus.

[0215] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF , AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 or derivatives thereof. In some embodiments, the herpes virus is HSV type 1, HSV type 2, VZV, EBV, CMV, HHV-6, HHV-7 or HHV-8.

[0216] In some embodiments, the virus is AAV1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV10 or a derivative thereof. In some embodiments, the virus is AAV11 or a derivative thereof. In some embodiments, the virus is AAV12 or a derivative thereof. In some embodiments, the virus is AAV13 or a derivative thereof. In some embodiments, the virus is AAV14 or a derivative thereof. In some embodiments, the virus is AAV15 or a derivative thereof. In some embodiments, the virus is AAV16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rh10 or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-1 or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof. In some embodiments, the virus is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the virus is AAV-2tYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the virus is AAV-HSC8 or a derivative thereof. In some embodiments, the virus is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof.In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV-HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof. In some embodiments, the virus is AAV-DJ / 8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the virus is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.

[0217] In some embodiments, the virus is HSV-1 or a derivative thereof. In some embodiments, the virus is HSV-2 or a derivative thereof. In some embodiments, the virus is VZV or a derivative thereof. In some embodiments, the virus is EBV or a derivative thereof. In some embodiments, the virus is CMV or a derivative thereof. In some embodiments, the virus is HHV-6 or a derivative thereof. In some embodiments, the virus is HHV-7 or a derivative thereof. In some embodiments, the virus is HHV-8 or a derivative thereof.

[0218] In some embodiments, the nucleic acid encoding the engineered nuclease system described herein or its components is delivered by a delivery system (e.g., non-viral delivery system) based on non-nucleic acids. In some embodiments, the non-viral delivery system is a liposome. In some embodiments, the nucleic acid is associated with a lipid. In some embodiments, the nucleic acid associated with a lipid can be encapsulated in the aqueous interior of the liposome, spread in the lipid bilayer of the liposome, connected to the liposome via a connecting molecule associated with the liposome and the nucleic acid, captured in the liposome, compounded with the liposome, dispersed in a solution containing lipids, mixed with lipids, merged with lipids, contained as a suspension in the lipid, containing micelles or compounded with micelles, or otherwise associated with lipids. In some embodiments, the nucleic acid is included in a lipid nanoparticle (LNP).

[0219] In some embodiments, the engineered nuclease system described herein or its components are stably or transiently introduced into the cell in any suitable manner. In some embodiments, the engineered nuclease system described herein or its components are transfected into the cell. In some embodiments, the cell is transduced or transfected with a nucleic acid construct encoding the engineered nuclease system described herein or its components. For example, a nucleic acid encoding the engineered nuclease system described herein or its components or a nucleic acid transduced (e.g., with a virus encoding the engineered nuclease system described herein or its components) or transfected (e.g., with a plasmid encoding the engineered nuclease system described herein or its components) cells. In some embodiments, transduction is a stable or transient transduction. In some embodiments, for example, when the engineered nuclease system described herein or its components include a CRISPR nuclease, one or more gRNA molecules are transduced or transfected to express the engineered nuclease system described herein or its components or cells containing the engineered nuclease system described herein or its components. In some embodiments, the plasmid expressing the engineered nuclease system described herein or its components is introduced into the cell by electroporation, transient (e.g., lipofection) and stable genomic integration (e.g., piggybac) and viral transduction (e.g., lentivirus or AAV) or other methods known to those skilled in the art. In some embodiments, the gene editing system is introduced into the cell as one or more polypeptides. In some embodiments, delivery is achieved by using RNP complexes. The delivery methods of polypeptides and / or RNPs to cells are known in the art, such as by electroporation or by cell extrusion.

[0220] Exemplary delivery methods for nucleic acids include lipofection, nucleofection, electroporation, stable genomic integration (e.g., piggybac), microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycationic or lipid nucleic acid conjugates, naked DNA, artificial virus particles, and agent-enhanced DNA uptake. Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam TM 、Lipofectin TM and SF cell lines 4D-Nucleofectin X Kit TM(Lonza). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include cationic lipids and neutral lipids in WO 91 / 17424 and WO 91 / 16024. In some embodiments, delivery to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). In some embodiments, the nucleic acid is included in a liposome or nanoparticle that specifically targets a host cell.

[0221] Additional methods of delivering nucleic acids to cells are known to those skilled in the art. See, for example, US 2003 / 0087817.

[0222] In some embodiments, the disclosure provides a cell comprising a vector or nucleic acid described herein. In some embodiments, the cell expresses a gene editing system or a portion thereof. In some embodiments, the cell is a human cell. In some embodiments, the cell is an ex vivo edited genome. In some embodiments, the cell is an in vivo edited genome.

[0223] Lipid Nanoparticles

[0224] In certain embodiments, disclosed herein are lipid nanoparticles comprising the engineered nuclease system of the present disclosure for delivering the engineered nuclease system into a cell.

[0225] In some embodiments, the lipid nanoparticle comprises an engineered nuclease system or a nucleic acid encoding an engineered nuclease system. In some embodiments, the lipid nanoparticle comprises one or more components of an engineered nuclease system. In some embodiments, the lipid nanoparticle comprises an endonuclease or a nucleic acid encoding an endonuclease. In some embodiments, the lipid nanoparticle comprises an engineered guide polynucleotide. In some embodiments, the lipid nanoparticle comprises a donor template.

[0226] In some embodiments, the lipid nanoparticle is tethered to an engineered nuclease system.

[0227] Lipid nanoparticles as described herein can be 4-component lipid nanoparticles. Such nanoparticles can be configured to deliver RNA or other nucleic acids (e.g., synthetic RNA, mRNA or in vitro synthesized mRNA), and can be generally formulated as described in WO2012135805A2. Such nanoparticles can generally include: (a) cationic lipids; (b) neutral lipids (e.g., DSPC or DOPE); (c) sterols (e.g., cholesterol or cholesterol analogs); or (d) PEG-modified lipids (e.g., PEG-DMG).

[0228] The cationic lipid referred to herein as "C12-200" is disclosed by Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869 and Liu and Huang, Molecular Therapy. 2010 669-670. In addition to polynucleotides, primary constructs or RNA (e.g., mRNA), cationic lipid formulations can include particles comprising 3 or 4 or more components. As an example, formulations containing certain cationic lipids include, but are not limited to, 98N12-5, and can contain 42% lipid, 48% cholesterol, and 10% PEG (alkyl chain length of C14 or greater). As another example, formulations with certain lipids include, but are not limited to, C12-200, and can contain 50% cationic lipids, 10% distearoylphosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG.

[0229] In some embodiments, cationic lipid nanoparticles include cationic lipids, PEG-modified lipids, sterols and non-cationic lipids. In some embodiments, the molar ratio of cationic lipid nanoparticles is about 20-60% of cationic lipids: about 5-25% of non-cationic lipids: about 25-55% of sterols; and about 0.5-15% of PEG-modified lipids. In some embodiments, cationic lipid nanoparticles include about 50% of cationic lipids, about 1.5% of PEG-modified lipids, about 38.5% of cholesterol and about 10% of non-cationic lipids. In some embodiments, cationic lipid nanoparticles include about 55% of cationic lipids, about 2.5% of PEG-modified lipids, about 32.5% of cholesterol and about 10% of non-cationic lipids. In some embodiments, cationic lipids are ionizable cationic lipids, non-cationic lipids are neutral lipids, and sterols are cholesterol. In some embodiments, the cationic lipid nanoparticles have a molar ratio of 50:38.5:10:1.5 cationic lipid: cholesterol: PEG2000-DMG: DSPC or DMG: DOPE. In some embodiments, lipid nanoparticles as described herein may include cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)amino)bis(dodecan-2-ol) (C12-200) and DMG-PEG-2000 in a molar ratio of 47.5:16:35:1.5.

[0230] cell

[0231] In certain embodiments, described herein is a cell comprising an engineered nuclease system described herein.

[0232] In some embodiments, the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungal cell), a mammalian cell (Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK) cell, human embryonic kidney (HEK) cell, mouse myeloma (NS0) cell, or human retinal cell), an immortalized cell (e.g., HeLa cell, COS cell, HEK-293T cell, MDCK cell, 3T3 cell, PC12 cell, Huh7 cell, HepG2 cell, K562 cell, , N2a cells or SY5Y cells), insect cells (e.g., Spodoptera frugiperda cells, Trichoplusia ni cells, Drosophila melanogaster cells, S2 cells or Heliothis virescens cells), yeast cells (e.g., Saccharomyces cerevisiae cells, Cryptococcus cells or Candida cells), plant cells (e.g., parenchyma cells, collenchyma cells or sclerenchyma cells), fungal cells (e.g., Saccharomyces cerevisiae) cells, Cryptococcus cells or Candida cells); or prokaryotic cells (e.g., Escherichia coli cells, Streptococcus cells, Streptomyces soil bacteria cells or Archaea cells). In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immortalized cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are yeast cells. In some embodiments, the cells are plant cells. In some embodiments, the cells are fungal cells. In some embodiments, the cells are prokaryotic cells.

[0233] In some embodiments, the cell is A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof.

[0234] In some embodiments, the cell is a liver cell.

[0235] Reagent test kit

[0236] In some embodiments, the present disclosure provides kits comprising one or more nucleic acid constructs encoding various components of the engineered nuclease system described herein. In some embodiments, the nucleotide sequence comprises a heterologous promoter driving expression of the engineered nuclease system component.

[0237] In some embodiments, the engineered nuclease system disclosed herein or its components are assembled into a pharmaceutical kit, a diagnostic kit, or a research kit to facilitate its use in therapeutic, diagnostic, or research applications. The kit may include one or more containers containing any vector disclosed herein and instructions for use.

[0238] Kit can be designed to help researchers use the methods described herein, and can take many forms. Each composition of the kit, where applicable, can be provided in liquid form (e.g., in solution form) or in solid form (e.g., dry powder). In some cases, some compositions can be configurable or otherwise processable (e.g., to form an active form), for example, by adding a suitable solvent or other species (e.g., water or cell culture medium), the composition can be provided together with the kit, or it may not be provided together with the kit. As used herein, "instructions" can define the composition of instructions and / or promotion, and are generally related to written instructions on or associated with the packaging of the present disclosure. Instructions can also include any oral or electronic instructions provided in any way, so that the user will clearly recognize that the instructions will be related to the kit, for example, audio-visual (e.g., videotapes, DVDs, etc.), the Internet and / or network-based communications, etc. In certain embodiments, the written instructions are in the form of regulations of government agencies regulating the manufacture, use or sale of drugs or biological products, and the instructions can also reflect the permission of the agency for animal administration in terms of manufacture, use or sale.

[0239] Examples

[0240] Example 1 - Design of a DNA donor template optimized for non-viral delivery for the purpose of integrating FVIII into the genome and producing functional FVIII protein

[0241] An 88 bp sequence from the human albumin promoter covering several transcription factor (TF) binding sites was selected as part of the donor template. This 88 bp sequence spans from 111 bp at 5' of the transcription start site to only 8 bp at 3' of the transcription start site and contains binding sites for transcription factors LEF / TCF1, HNF1, NFY and CEBP. This sequence is designated as the human albumin nuclear targeting sequence (hANTS) and comprises the following sequence: 5'-TGAATTTTGTAATCGGTTGGCAGCCAATGAAA TACAAAGATGAGTCTAGTTAATAATCTACAATTATTGGTTAAAGAAGTAT ATTAGT-3' (SEQ ID NO: 1). In addition, the 72 bp SV40 enhancer: ATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCAC (SEQ ID NO: 2) is optionally included in the template.

[0242] The donor DNA template to be used for non-viral delivery is designed to have the following general structure: 5' closed end (CE)-nuclear targeting DNA sequence (NTDS)-spacer-CRISPR nuclease or other sequence-specific nuclease target site-therapeutic gene (TG)-polyA signal-spacer-CRISPR nuclease or other sequence-specific nuclease target site-spacer-nucleus-targeting DNA sequence (NTDS)-closed end (CE). The target site of the CRISPR nuclease or other sequence-specific nuclease in the donor DNA is the same as the target site selected as the integration site in the genome in the genomic locus. The closed end (CE) indicates a synthetic DNA sequence so that the 5' and 3' ends of the duplex are covalently linked, which increases the stability against nuclease degradation.

[0243] Since the specific sequence of NTDS may potentially affect the integration efficiency of the donor template, donor DNAs with different NTDS are designed. In one example, the NTDS includes a single copy of hANTS (SEQ ID NO: 1) present at one or both ends of the DNA donor. In another example, the NTDS includes a single copy of SV40e (SEQ ID NO: 2) present at one or both ends of the DNA donor. In another example, the NTDS includes a single copy of hANTS (SEQ ID NO: 1) present at one or both ends of the DNA donor and a single copy of SV40e (SEQ ID NO: 2).

[0244] In the case of the factor VIII gene, where the target locus in the genome is the albumin locus (particularly intron 1 of the albumin gene), the donor DNA template is designed to have the following components in order: 5' closed end (CE) - nuclear targeting sequence (NTS) - 20 bp spacer - target sites for guides 8 and 12 of nuclease MG29-1 in mouse albumin intron 1 - spacer (37 bp) - splice acceptor - FVIIICDS - polyA - spacer (36 nt) - target sites for guides 8 and 12 of CRISPR nuclease MG29-1 in mouse albumin intron 1 - 20 bp spacer - nuclear targeting sequence (NTS) - closed end (CE) - 3'. A total of 12 possible combinations of guide polynucleotide target site orientations and NTDS sequences were designed, including each of the 4 guide orientations combined with hANTS alone, SV40e alone, or a combination of nANTS and SV40e at both ends of the donor. The individual sequence components are listed in SEQ ID NOs: 3 to 11.

[0245] Since the relative orientation of the cleavage site in the donor relative to the genomic site is considered to be a factor affecting integration efficiency, examples with all combinations of cleavage site orientations in the donor DNA are designed. Four possible orientations are envisioned: (1) forward-forward, (2) reverse-reverse, (3) forward-reverse and (4) reverse-forward, wherein forward means that the target site is located in the same orientation as in the genome, and reverse means that the target site is the reverse complement of the sequence in the genome. Two examples of the full sequence of the donor template of FVIII are FF orientation (pMG4010, SEQ ID NO: 12) with a guide cleavage site comprising both hANTS and CMVe's NTDS and RR orientation (pMG4011, SEQ ID NO: 13) with a guide cleavage site comprising both hANTS and CMVe's NTDS. The other 2 possible orientations of the guide cleavage site are created by reversing the orientation of the cleavage site at the appropriate end to keep the FR (pMG4022, SEQ ID NO: 32) and RF (pMG4023, SEQ ID NO: 33) variants unchanged from all other sequence elements. The length of pMG4010 and pMG4011 is 4931bp. In one embodiment, the FVIII coding sequence is codon optimized to improve the expression of the FVIII gene after the FVIII gene is integrated into the target locus in the genome. The innate immune system can recognize and eliminate DNA by recognizing unmethylated CG dinucleotides (CpG motifs). Codon optimization generally involves selecting the most commonly used codons for each amino acid. In the case of the FVIII coding sequence in SEQ ID NO: 10, all CpG motifs are eliminated by carefully selecting alternative codons after codon optimization. In addition, a spacer sequence without CpG residues is designed. There are a total of 6 CpG residues in each of pMG4010 and pMG4011.

[0246] Example 2 - In vivo testing of non-viral delivery of a DNA donor template containing a FVIII gene cassette flanked by NTDS and guide RNA target sites (predictive)

[0247] In order to evaluate whether the DNA donor templates designed in Example 1 (e.g., SEQ ID NO: 12, 13, 32, and 33) can work in vivo after non-viral delivery, donor DNA templates were synthesized with blocked ends. This DNA was encapsulated in lipid nanoparticles. The lipids were dissolved in ethanol. The donor DNA was prepared in water and then diluted in 100 mM sodium acetate (pH 4.0) to prepare a DNA working stock solution. The four lipid components were combined in ethanol at a desired ratio to prepare a lipid working stock solution. A typical lipid mixture included cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)aminoidene)bis(dodecan-2-ol) (C12-200), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) in a molar ratio of 47.5:16:35:1.5. The lipid working stock and DNA working stock were combined in a microfluidic mixing device (Precision Nanosystems) at a flow rate of 12 ml / min and a ratio of 1 volume of lipid working stock to 3 volumes of DNA working stock. The mass ratio of C12-200 to DNA in the formulation was between 5 to 1 and 20 to 1. The formulated LNPs were diluted 1:1 with 1x PBS and then dialyzed twice in 1x PBS for 1 hour each, followed by concentration in an Amicon spin concentrator. The final LNPs were formulated into 1x PBS buffer, filter sterilized through a 0.2 μM filter, and stored at 4°C. The concentration of DNA inside and outside the LNPs was measured. The average diameter and polydispersity of the LNPs were measured in the final concentrated LNPs by dynamic light scattering. The expected size of the LNPs is in the range of 80 to 100 nanometers, with a PDI <0.15 and a DNA encapsulation efficiency greater than 90%.

[0248] Individual LNPs were formulated with mRNA encoding MG29-1 nuclease and guide RNA 8 or guide RNA 12, both target sites within mouse albumin intron 1. The guide RNAs contained a specific set of chemical modifications that have been optimized to improve stability and in vivo efficacy (SEQ ID NOs: 14 and 15). The guide RNA and mRNA were packaged separately. The lipids were dissolved in ethanol. The mRNA or guide RNA was prepared in water and then diluted in 100 mM sodium acetate (pH 4.0) to prepare the RNA working stock solution. The four lipid components were combined in ethanol at the desired ratio to prepare the lipid working stock solution. A typical lipid mixture contained cholesterol, DOPE, C12-200, and DMG-PEG-2000 in a molar ratio of 47.5:16:35:1.5. The lipid working stock solution and the RNA working stock solution were combined in a microfluidic mixing device at a flow rate of 12 ml / min and a ratio of 1 volume of lipid working stock solution to 3 volumes of RNA working stock solution. The mass ratio of C12-200 to RNA in the formulation is 10 to 1. The formulated LNPs were diluted 1:1 with 1x PBS, then dialyzed twice in 1x PBS for 1 hour each time, and then concentrated in a rotary concentrator. The final LNPs were formulated into 1x PBS buffer, sterilized by filtration through a 0.2 μM filter, and stored at 4°C. The concentration of RNA inside and outside the LNPs was measured. The average diameter and polydispersity of the LNPs were measured in the final concentrated LNPs by dynamic light scattering. Typically, the size of the LNPs is in the range of 80 to 100 nanometers, with PDI<0.15 and an RNA encapsulation efficiency greater than 90%. The LNPs encapsulating the guide RNA mAlb29-8-50 or mAlb29-12-50 were mixed with the LNPs encapsulating the MG29-1 mRNA at an RNA mass ratio of 1:1 (guide RNA: mRNA).

[0249] To initiate gene therapy, wild-type C57B16 mice were injected intravenously (0.1 mL, via the tail vein) with LNP-encapsulated donor DNA pMG4010 or pMG4011 (or other variants of the donor as described in Example 1) at a dose of 0.1 mg / kg to 2 mg / kg. The timing of the administration of LNPs encapsulating MG29-1 mRNA and guide RNA relative to the DNA donor LNP was evaluated. In one dosing regimen, the DNA donor-LNP and MG29-1 mRNA / guide RNA LNP were premixed and administered in a single injection. In another dosing regimen, the DNA donor-LNP was administered to mice between 1 hour and 48 hours before the administration of MG29-1 mRNA / guide RNA LNP. In still another dosing regimen, the MG29-1 mRNA / guide RNA LNP was administered to mice between 1 hour and 48 hours before the administration of DNA donor-LNP. The dose of DNA-donor LNP was in the range of 0.1mg / kg to 2mg / kg DNA in 0.1mL total volume per mouse, and the dose of MG29-1mRNA / guide RNA LNP was initially set to 0.25mg / kg, with the goal of achieving editing (cutting) of the target site in the range of 20% to 40%. Plasma was collected from mice 7 days and 14 days after administration, and human FVIII levels were analyzed using a capture CoA test assay that detects the activity of human FVIII in the mouse FVIII background. Mice were killed at 14 days, and the whole liver was quickly frozen and stored at -80°C. The entire left lateral lobe of the liver was homogenized using 0.4mL of buffer / 100mg tissue weight in a bead mill. Genomic DNA was purified from aliquots of the homogenate. The integration of genomic DNA at the predicted target site in albumin intron 1 was analyzed by internal-external PCR using a primer complementary to a sequence in the genome near the target site and a primer in the DNA donor template. The frequency of integration in the correct or reverse orientation was measured using quantitative real-time PCR or droplet digital PCR versions of the inside-out PCR assay. The percentage of cells expressing integrated human FVIII mRNA was measured on liver sections by in situ hybridization with a fluorescent probe designed to detect hybridized mRNA transcripts at the junction between albumin mRNA and FVIII mRNA.

[0250] Example 3 - Design of a DNA Donor Template Optimized for Viral Delivery by AAV for the Purpose of Integrating a Functional FVIII Gene into a Genome Producing a Functional FVII Protein (Predictive)

[0251] The donor DNA template box is designed to have the following sequence elements in the order of 5' to 3': target site of CRISPR nuclease-spacer-splicing acceptor-therapeutic gene-polyA signal-spacer-target site of CRISPR nuclease. This donor template box is flanked by AAV inverted terminal repeats (ITRs) to enable packaging into any AAV virus serotype of interest. In the case of studies in mice, AAV8 serotype or AAV6 serotype is selected. In this example, the target site of CRISPR nuclease or other sequence-specific nuclease is selected from the target site of nuclease MG29-1 or MG3-6 / 3-4. The specific guide RNA target site of MG29-1 or MG3-6 / 3-4 is selected from the guide target site at the genomic target locus, which is identified by screening for active guides that promote effective DSB formation. In this specific example, the target locus in the genome is albumin, and the specific region of albumin targeted is intron 1. It is envisioned that other genomic target sites can be selected to integrate the FVIII gene at different genomic loci. Active guide screens against mouse and human albumin intron 1 of both MG29-1 and MG3-6 / 3-4 identified a number of highly active guide RNAs.

[0252] The orientation of the guide RNA target site in the donor template can be designed to be the same as the orientation of the target site in albumin intron 1 or another target site (forward orientation F) or the reverse complement of the target site in albumin intron 1 or another target site (reverse orientation R). Therefore, there are 4 possible combinations of guide target site orientations designated as FF, RR, FR, RF. The FVIII donor cassette can be integrated into albumin intron 1 in any orientation, and only the "forward" orientation in which the splice acceptor in the donor is located near the albumin promoter is expected to produce functional FVIII protein. The orientation of the guide RNA target site in the donor template may affect the efficiency of the donor template cassette to integrate into albumin intron 1 in the forward orientation, which may cause the production of functional FVIII protein. MG29-1 and MG3-6 / 3-4 nucleases have not been tested in vivo in this context for integration of DNA donor templates in hepatocytes. MG29-1 is a type V CRISPR nuclease, and it performs staggered cuts at the target site, which is in contrast to the blunt-end cuts produced by the Cas9 nuclease and by MG3-6 / 3-4. In the absence of donor DNA, the final result of DSB repair produced by MG29-1 and MG3-6 / 3-4 in mammalian cells is mainly the deletion of alleles with very few inserted bases. In the case of MG29-1, deletions in the size range of 1 to 15 bases are the most common, while MG3-6 / 3-4 tends to produce smaller deletions on average in the range of 1 to 6 bases. The profile of insertions and deletions (INDELs) produced by DSBs (INDEL profiles) reflects the DNA repair process used by cells to repair DSBs. Larger deletions usually indicate alternative NHEJ repair pathways, while short deletions indicate typical NHEJ repair pathways. MG29-1 produces staggered cuts in the genome and leaves a 5' overhang of approximately 18 to 22 bases 3' to the PAM end in the target site and the DNA donor template, as Figure 1 Based on Figure 1 In vitro assays shown, the size of the single-stranded 5' overhang is expected to be 3 to 6 nucleotides. Due to base pairing interactions, the single-stranded overhang can be more effectively connected to the complementary single-stranded overhang in the same way that the "sticky end" connection in traditional cloning methods is more effective than the blunt end connection. Therefore, including a guide target site on each side of the donor box can not only produce a linear double-stranded template in the endosome of the hepatocyte, but also the resulting product can have a single-stranded overhang compatible with the overhang at the double-strand break in the genome. In some cases, one of the 4 possible orientations of the guide RNA target site in the donor will cause a superior integration frequency in the forward orientation, so the frequency can be determined empirically.

[0253] It is important to note that in cases where the two guide target sites in the donor DNA template are in the same orientation as the same target site in the genome and the staggered cleavages produced by MG29-1 are linked to the donor through complementarity of the single-stranded 5' overhangs, the guide target sites can be fully reconstructed at both junctions and the DNA can be re-cleaved, thereby releasing the donor DNA (Tables 2 and 3).

[0254] Table 2: Predicted complementarity and ligation results for DNA donors with different guide target site orientations containing the donor's MG29-1 guide 8 target site and assuming creation of a 6 base 5' overhang at each site

[0255]

[0256] Table 3: Effect of different guide target site orientations on integration outcomes for MG29-1 guide 8 target sites containing donors, assuming creation of 6 base 5' overhangs at each site, which are then joined by annealing of complementary single stranded overhangs, without any sequence deletions at either end

[0257]

[0258]

[0259] *If the ends that do not exhibit sequence complementarity at the two overhangs are joined by a mechanism where the ends are resected, the target site will not be recreated because the position of the PAM is incompatible

[0260] In the case where 5' overhangs in the donor DNA and target cleave and promote integration, the effect on integration can be hypothesized as follows. For a FF-oriented donor with a guide target site, integration is expected to occur only in the forward orientation (not in the reverse orientation) by cleavage of complementary ends, however this is expected to recreate the guide target site, which can then be re-cut, thereby excising the donor template. Alignment of the cleaved donor template in the reverse orientation with the cleaved genome will not result in complementary 5' overhangs at either junction.

[0261] For RR-oriented donors with guide target sites, integration in the reverse orientation can occur by bonding of complementary ends, while integration in the forward orientation is disadvantageous due to the lack of bonding of complementary ends. For FR-oriented donors with guide target sites, integration in both orientations can occur only by bonding of complementary ends at the 5' junction. For RF-oriented donors with guide target sites, integration in both orientations can occur only by bonding of complementary ends at the 3' junction. For each junction formed by complete bonding of complementary overhangs, a guide target site can be recreated, which will then be re-cut, thereby excising the donor template, which may be detrimental to integration efficiency. However, since the NHEJ repair process is error-prone and insertions and deletions are introduced at the site of the DSB during the repair process, these integration events may not simply occur without insertions or deletions of DNA as described above. This is clearly seen in the case of MG29-1 and MG3-6 / 3-4 by the observed INDEL curves.

[0262] One possible mechanism for donor DNA integration is a repetitive process of end joining and re-cleavage by nucleases until an insertion / deletion (indel) occurs at the junction that prevents the nuclease from cleaving. If complementary 5' overhangs at the ends of the donor template and DSBs in the genome promote bonding as an integral part of the NHEJ-driven repair process, the junction formed by bonding of complementary ends can become fixed and stable once enough indels are introduced at the junction to block re-cleavage. Given the complexity of the DNA repair process, it may be necessary to empirically determine the optimal design of the donor template, especially in the case of novel nucleases.

[0263] For MG29-1, two guides called mAlb29-8-50b (SEQ ID NO: 24) and mAlb29-12-50b (SEQ ID NO: 25) were selected based on editing activity in the mouse liver cell line Hepa1-6 and in vivo editing activity in mouse liver. These guides contain 20 nucleotide spacers and various chemical modifications and additional stem loops at the 5' end, which together significantly improve the in vivo stability and efficacy of the guide RNA. The target sites of both mAlb29-8-50b and mAlb29-12-50b are present in the albumin intron 1 of mice in a forward orientation (defined as the PAM sequence located at the 5' side of the guide target site, or closest to exon 1).

[0264] For MG3-6 / 3-4, two guides, called mAlb3634-34 (SEQ ID NO: 26) and mAlb3634-59 (SEQ ID NO: 27), were selected based on editing activity in the mouse liver cell line Hepa1-6 and in vivo editing activity in mouse liver. These guides contain chemical modifications that improve the in vivo stability and efficacy of the guides. The target site of guide mAlb3634-34 is present in the albumin intron 1 of the mouse in the reverse orientation (defined as the PAM sequence is located at the 3' side of the guide target site, or closest to exon 2). The target site of guide mAlb3634-59 is present in the albumin intron 1 of the mouse in the forward orientation (defined as the PAM sequence is located at the 5' side of the guide target site, or closest to exon 1). Four FVIII donor DNA template boxes containing 4 guide RNA target site orientations were designed for each of the nucleases MG29-1 and MG3-6 / 3-4. For MG29-1, donor DNA templates with FF, RR, FR, and RF orientations were designated pMG4006 (SEQ ID NO: 16), pMG4007 (SEQ ID NO: 17), pMG4008 (SEQ ID NO: 18), and pMG4009 (SEQ ID NO: 19), respectively. The four MG29-1 donors contained target sites for both guides mAlb29-8 and mAlb29-12 on both sides of the donor cassette.

[0265] For use with the MG3-6 / 3-4 nuclease, donor DNA templates with FF, RR, FR, and RF orientations of the guide target site were designated pMG4012 (SEQ ID NO: 20), pMG4013 (SEQ ID NO: 21), pMG4014 (SEQ ID NO: 22), and pMG4015 (SEQ ID NO: 23), respectively. The four MG3-6 / 3-4 donors contained target sites for both guides mAlb3634-34 and mAlb3634-59 on both sides of the donor cassette. Since the "F" designation means the same orientation as the target site in the genome, the F orientation of mAlb3634-34 means the PAM site on the 3' side of the target site, and the F orientation of mAlb3634-59 means the PAM site on the 5' side of the target site.

[0266] A summary of the relative orientation of the different guide target sites in the donors and the genomic target relative to the position of the PAM is shown in Tables 4 and 5 below.

[0267] Table 4: Relative orientation of different guide target sites and genomic targets in MG29-1 based DNA donors relative to the position of the PAM

[0268]

[0269] Table 5: Relative orientation of different guide target sites and genomic targets relative to the position of the PAM in the MG3-6 / 3-4 based DNA donor

[0270]

[0271] Example 4 - Testing the integration of FVIII donor template delivered by AAV into albumin intron 1 in mice (predictive)

[0272] Human FVIII donor templates pMG4006 to pMG4009 (MG29-1 targeted) and pMG4012 to pMG4015 (MG3-6 / 3-4 targeted) were packaged into AAV8 viral capsids using molecular biology techniques. AAV viruses were purified using CsCl gradients and analyzed for purity by protein gel electrophoresis to visualize viral capsid proteins. The titer of each virus was determined by quantitative PCR with primers for inverted terminal repeats (ITRs) and expressed as vector genome copies per mL (vg / mL).

[0273] The messenger RNA encoding MG29-1 nuclease and MG3-6 / 3-4 nuclease was produced by in vitro transcription of a linearized plasmid template using a mixture of T7 RNA polymerase, ribonucleotides rATP, rCTP and rGTP, N1-methyl pseudouridine and a capping agent. The SV40-derived nuclear localization sequence (PKKKRKVGGGGS (SEQ ID NO: 103)) followed by a short linker was included at the N-terminus of the coding sequence of both MG3-6 / 3-4 and MG29-1. For both MG3-6 / 3-4 and MG29-1, the nuclear localization signal of the nucleoplasmic protein before the short linker (SGGKRPAATKKAGQAKKKK (SEQ ID NO: 104)) was added to the C-terminus of the coding sequence. Therefore, the same nuclear localization signal was used for both MG29-1 and MG3-6 / 3-4. The plasmid also encodes about 100nt polyA tail at the 3' end of both MG3-6 / 3-4 and MG29-1 coding sequences, which produces a polyA tail in the mRNA. The coding sequences of both MG3-6 / 3-4 and MG29-1 are codon optimized. The DNA sequence encoding MG3-6 / 3-4 mRNA is shown in SEQ ID NO:30, and the DNA sequence encoding MG29-1 mRNA is shown in SEQ ID NO:31. The mRNA is purified on a spin column, the concentration is determined by the absorbance at 260nM, and the purity is determined, and the purity of both MG3-6 / 3-4 mRNA and MG29-1 mRNA is equivalent. For delivery in mice, MG3-6 / 3-4 mRNA or MG29-1 mRNA and its corresponding guide RNA are packaged in lipid nanoparticles (LNPs) respectively. Guide RNA and mRNA are packaged for both MG3-6 / 3-4 and MG29-1 respectively. The lipid is dissolved in ethanol. The mRNA or guide RNA was prepared in water and then diluted in 100 mM sodium acetate (pH 4.0) to prepare an RNA working stock solution. The four lipid components were combined in ethanol at a desired ratio to prepare a lipid working stock solution. A typical lipid mixture contains cholesterol, DOPE, C12-200, and DMG-PEG-2000 in a molar ratio of 47.5:16:35:1.5. The lipid working stock solution and the RNA working stock solution were combined in a microfluidic mixing device at a flow rate of 12 ml / min and a ratio of 1 volume of lipid working stock solution to 3 volumes of RNA working stock solution. The mass ratio of C12-200 to RNA in the formulation was 10 to 1. The formulated LNPs were diluted 1:1 with 1x PBS, then dialyzed twice in 1x PBS for 1 hour each, and then concentrated in a rotary concentrator. The final LNPs were formulated into 1x PBS buffer, sterilized by filtration through a 0.2 μM filter, and stored at 4°C. Measure the concentration of RNA inside and outside the LNP.The average diameter and polydispersity of the LNPs were measured in the final concentrated LNPs by dynamic light scattering. Typically, the size of the LNPs ranged from 80 to 100 nm with a PDI < 0.15 and an RNA encapsulation efficiency greater than 90%.

[0274] LNPs encapsulating guide RNA mAlb29-8-50b (SEQ ID NO: 24) or mAlb29-12-50b (SEQ ID NO: 25) and MG29-1 mRNA were mixed at a 1:1 RNA mass ratio (guide RNA: mRNA). LNPs encapsulating guide RNA mAlb3634-34 (SEQ ID NO: 26) or mAlb3634-59 (SEQ ID NO: 27) and MG3-6 / 3-4 mRNA were mixed at a 1:1 RNA mass ratio (guide RNA: mRNA). A mixture of guide RNA LNPs and matched mRNA LNPs was injected intravenously into wild-type C57Bl / 6 mice via the tail vein at a total RNA dose of 1 mg / kg to 0.25 mg / kg of RNA in a total volume of 0.1 mL per mouse (N = 5 mice / LNP dose).

[0275] The optimal order and timing of administration of AAV-encapsulated donor templates and LNP-encapsulated nuclease mRNA and guide RNA are determined empirically. After AAV transduction of mammalian cells, the single-stranded DNA packaged in AAV (referred to as the AAV genome) is converted into double-stranded DNA in the nucleus of mammalian cells by bonding of complementary (positive and negative single strands) packaged in equal proportions in bulk AAV preparations or by synthesizing new complementary strands from scratch or by a combination of these two mechanisms. Once converted to double-stranded DNA, the AAV genome undergoes concatenation, thereby connecting multiple copies of the AAV genome to form circular concatemers, which can persist in non-dividing cells in vivo for several years. Double-stranded DNA is a substrate for NHEJ-mediated integration at DSB, while single-stranded DNA is not a substrate for this integration mechanism. Given that it takes time for single-stranded AAV genomes to be converted into double-stranded DNA, and that double-stranded AAV genomes persist in vivo for months to years, it is logical to administer AAV viruses before editing nucleases and guide RNAs. This is particularly important when the editing nuclease is encoded in mRNA and delivered with the guide RNA in LNPs, as this results in transient expression of the nuclease by translating a limited amount of mRNA into nuclease protein. Nuclease proteins have a limited lifespan within cells. Therefore, it may be beneficial to first administer the AAV-encapsulated donor template, followed by administration of LNPs encapsulating the nuclease mRNA and guide RNA at a later time. The optimal time between AAV administration and LNP administration is determined empirically and can vary between mammalian species. In the case of mice, LNPs are administered between 24 hours and 21 days after the AAV is evaluated. In one potential study design, 5 x 10 11 vg / kg to 1x 10 13The dosage of vg / kg is to wild-type C57B16 mice or hemophilia A mice (mouse FVIII deficiency) administration encapsulates the AAV of different FVIII donor boxes. After AAV administration 1 day, 7 days, 14 days or 21 days, suitable LNPs are injected to the same mice, and the LNPs encapsulate the nuclease mRNA and guide RNA that match the guide target site in the donor template previously applied. After LNP administration, plasma is collected from mice on the 7th and 14th day, and human FVIII protein or human FVIII activity is measured using a suitable assay. In order to detect human FVIII protein, human FVIII specific ELISA can be used to measure. In order to detect human FVIII activity, capture-Coatest can be used to measure, wherein first the human FVIII in the mouse plasma sample is captured on the surface of 96 well plates by the human FVIII specific antibody that is not combined with mouse FVIII. After washing away the uncombined mouse FVIII in plasma, the human FVIII combined can be quantitatively measured using human FVIII activity. In each assay run, the purified human FVIII protein diluted into the original mouse plasma is used to create a human FVIII standard curve so that the level of mouse plasma is the same as the level in the sample during the capture step. Alternatively, if a hemophilia A mouse strain lacking mouse FVIII is used, the FVIII activity in plasma can be directly measured. Mice were killed 14 days after LNP administration, and the whole liver was quickly frozen and stored at -80 ° C. The whole lobe of the liver was homogenized in genomic digestion buffer using 0.4 mL of buffer / 100 mg tissue weight in a bead mill. Genomic DNA was purified from aliquots of the homogenate. To measure the total editing efficiency at the target site in albumin intron 1, the albumin intron 1 region was PCR amplified from 50ng of genomic DNA in a reaction solution containing 0.5 micromolar primers mAlb90F (SEQ ID NO: 28, CTCCTCTTCGTCTCCGGC) and mAlb1073R (SEQ ID NO: 29, CTGCCACATTGCTCAGCAC) and 1x high-fidelity PCR master mix. The resulting 984bp PCR product spanning the entire intron 1 of mouse albumin was purified using a column-based purification kit. The PCR products were sequenced by next-generation sequencing (NGS). When nucleases generate double-strand breaks (DSBs) in DNA within living cells, DSBs can be repaired by cellular DNA repair mechanisms. In actively dividing cells in culture, such as transformed mammalian cells, and in the absence of a repair template, this repair can occur through the NHEJ pathway. The NHEJ pathway can be an error-prone process that introduces insertions or deletions of bases at double-strand break sites. These insertions and deletions (INDELs) are therefore markers of double-strand breaks that have occurred and will be subsequently repaired and are widely used as readouts of the editing or cleavage efficiency of nucleases.Sequencing reads were analyzed, each sequence read was aligned to the wild-type target sequence (in this case, albumin intron 1), and the number of reads containing at least one INDEL within a window spanning 10 base pairs on either side of the predicted target cleavage site of the nuclease was calculated, regardless of INDEL size. Integration of the same liver genomic DNA at the predicted target site in albumin intron 1 was analyzed by inside-out PCR using one primer complementary to a sequence in the genome near the target site and one primer in the DNA donor template. Integration frequencies in the correct orientation or in the reverse orientation were measured using quantitative real-time PCR or droplet digital PCR versions of the inside-out PCR assay. The percentage of cells expressing integrated human FVIII mRNA was measured on liver sections by in situ hybridization with a fluorescent probe designed to detect hybridized mRNA transcripts at the junction between albumin mRNA and FVIII mRNA.

[0276] Example 5-Comparison of in vivo editing efficiency between MG29-1 and spCas9

[0277] In order to compare the in vivo editing efficiency of MG29-1 nuclease and spCas9, a dose response was performed in wild-type C57B16 mice. Albumin intron 1 was selected as the genomic target locus for both spCas9 and MG29-1. A total of 39 potential guides were identified by searching for spCas9 guide target sites in mouse intron 1 in computer using the Chop-Chop algorithm, and these guides were ranked according to their efficiency scores and off-target predictions. In addition, guide target sites within 50bp of exon 1 or exon 2 were excluded. The first three guides from this sorting were named mAlbR1 (SEQ ID NO: 43), mAlbR2 (SEQ ID NO: 44) and mAlbR3 (SEQ ID NO: 45), and chemically synthesized with chemical modifications at both 5' and 3' ends, the chemical modifications comprising methylated bases (represented by nomenclature mA, mC, mG and mU) and phosphorothioate backbone bonds (represented by nomenclature A*, C*, G* and U*). The editing efficiency of these three guides was evaluated in the mouse liver cell line Hepa1-6 by nucleofection of ribonucleoprotein complexes formed by mixing guide RNA and spCas9 protein at a molar ratio of 1:2.5 (protein for guide RNA). 20 moles of spCas9 protein were mixed with 50 moles of guide RNA and then nucleofected into 2×10 5 Transfer the nucleofected cells individually to wells of a 48-well plate in fresh growth medium and incubate at 5% CO 2 / 37 ℃ humidified incubator for 48 hours. Genomic DNA was purified from cells, and primers mAlb90F and mAlb1073R (SEQ ID NO: 46 and 47) and a high-fidelity PCR enzyme mixture were used to analyze the editing at the target site in albumin intron 1 by PCR amplification of the target locus. Primers mAlb282F or mAlb460F (SEQ ID NO: 48 and 49) were used to perform Sanger sequencing on the PCR product. Insertions and deletions ("indels") of the Sanger sequencing chromatogram were analyzed. The presence of indels at the target site is the result of the generation of double-strand breaks in DNA, which are then repaired by the error-prone cell repair mechanism that introduces insertions and deletions. The results of the TIDE analysis are shown in Table 6. All three guides produce indel frequencies greater than 90%, thereby proving that all three guides are highly active.

[0278] Table 6: INDEL frequency in Hepa1-6 cells nucleofected with guide RNAs for spCas9 targeting mouse albumin intron 1 and spCas9 as RNP

[0279] Sample ID Wizard INDEL% R2 1 mAlbR1 92 0.95 2 mAlbR2 91 0.91 3 3A 96 0.96

[0280] The guide mALBR2 was synthesized using extensive chemical modifications. The chemical modifications included modification of 3 bases at the 5' end and 3 bases at the 3' end with 2'-O-methyl bases, as well as phosphorothioate bonds between the 3 bases at the 5' end and the 3 bases at the 3' end. In addition, 33 of the internal bases were modified with 2'-O-methyl (SEQ ID NO: 50). It is reported that these chemical modifications of the guide RNA of spCas9 enable efficient editing in vivo in mouse liver after delivery of the mRNA and guide RNA of spCas9 in lipid nanoparticles.

[0281] A guide screen was performed for guides that target the MG29-1 nuclease to mouse albumin intron 1 and promote cleavage and indel formation. The two guides with the highest editing activity in Hepa1-6 cells were mAlb29-8 and mAlb29-12 when the nuclease was delivered as mRNA. The guide mAlb29-8 was selected for comparison with the spCas9 guide mAlbR2 in mice. Chemical and structural modifications to the guide RNA for MG29-1 were optimized by evaluating the effects of different chemical modifications including 2'O-methyl and 2'-fluoro modified bases, phosphorothioate bonds, and additional stem-loops on the stability and editing activity of the guides.

[0282] Experiments on guide chemistry optimization showed that guide chemistry #50 was the most active guide chemistry among those tested. When delivered to mice in vivo using LNPs encapsulating MG29-1 mRNA and the same guide RNA sequence targeting mouse albumin intron 1, but with two different guide chemistries (#37 and #50), at a dose of 0.5 mg / kg, the potency of chemical #50 was about 4 times that of chemical #37. Therefore, MG29-1 guide chemical #50 was selected for in vivo testing in comparison with spCas9 and its cognate guide mALBR2 (SEQ ID NO: 50).

[0283] The messenger RNA encoding MG29-1 nuclease or spCas9 nuclease is produced by in vitro transcription of a linearized plasmid template using a mixture of T7 RNA polymerase and ribonucleotides rATP, rCTP and rGTP, N1-methyl pseudouridine and CleanCAP capping reagents. SV40-derived nuclear localization sequence (PKKKRKVGGGGS (SEQ ID NO: 103)), followed by a short linker, is included at the N-terminus of the coding sequence of both spCas9 and MG29-1. For both spCas9 and MG29-1, the nuclear localization signal of the nucleoplasmic protein before the short linker (SGGKRPAATKKAGQAKKKK (SEQ ID NO: 104)) is added to the C-terminus of the coding sequence. Therefore, the same nuclear localization signal is used for both MG29-1 and spCas9. The plasmid also encodes approximately 100nt polyA tails at the 3' end of both spCas9 and MG29-1 coding sequences, which produce polyA tails in mRNA. The coding sequences of both spCas9 and MG29-1 were codon optimized using the same algorithm. The DNA sequence encoding spCas9 mRNA is in SEQ ID NO: 51, and the amino acid sequence encoded by spCas9 mRNA is in SEQ ID NO: 52. The mRNA was purified on a commercial spin column, the concentration was determined by the absorbance at 260nM, and the purity was determined, and it was found that the purity of both spCas9 mRNA and MG29-1 mRNA was equivalent. In order to deliver to mice, the spCas9 mRNA / mAlbR2 guide or the MG29-1mRNA / mAlb29-8-50 guide was packaged in lipid nanoparticles (LNP). Guide RNA and mRNA were packaged for both spCas9 and MG29-1, respectively. Lipids were dissolved in ethanol. The mRNA or guide RNA was prepared in water and then diluted in 100mM sodium acetate (pH 4.0) to prepare RNA working stock solution. The four lipid components were combined in ethanol at a specified ratio to prepare a lipid working stock solution. An exemplary lipid mixture comprises cholesterol, a neutral lipid such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), a cationic lipid such as 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)aminoidene)bis(dodecan-2-ol) (C12-200), and a PEG-linked lipid such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) in a molar ratio of 47.5:16:35:1.5.The lipid working stock solution and RNA working stock solution were combined in a microfluidic mixing device at a flow rate of 12 ml / min and a ratio of 1 volume of lipid working stock solution to 3 volumes of RNA working stock solution. The mass ratio of C12-200 to RNA in the formulation was 10 to 1. The formulated LNP was diluted with 1x PBS at 1:1, then dialyzed twice in 1x PBS for 1 hour each, and then concentrated in a rotary concentrator. The resulting LNP was formulated into 1x PBS buffer, sterilized by filtration through a 0.2 μM filter, and stored at 4°C. The concentration of RNA inside and outside the LNP was measured. The average diameter and polydispersity of LNP were measured in the resulting concentrated LNP by dynamic light scattering. The size of the representative LNP is in the range of 80 to 100 nanometers, wherein PDI<0.15 and the RNA encapsulation efficiency is greater than 90%. The average diameter, polydispersity and RNA encapsulation efficiency are shown in Table 7 below.

[0284] Table 7: Overview of LNP characteristics

[0285]

[0286] LNPs encapsulating guide RNAmAlb29-8-50 and MG29-1 mRNA were mixed at an RNA mass ratio of 1:1. LNPs encapsulating guide RNAmAlbR1 and spCas9 mRNA were mixed at an RNA mass ratio of 1:1. The two LNP mixtures were injected intravenously into wild-type C57Bl / 6 mice via the tail vein, with a total RNA dose of 1 mg / kg, 0.5 mg / kg, or 0.25 mg / kg of RNA in a total volume of 0.1 mL per mouse (N=5 mice / LNP dose). Mice were sacrificed 5 days after dosing, and the whole liver was quickly frozen and stored at -80°C. The entire left lateral lobe of the liver was homogenized using 0.4 mL of buffer / 100 mg of tissue weight in a bead mill. Genomic DNA was purified from aliquots of the homogenate. The albumin intron 1 region was PCR amplified from 50 ng of genomic DNA in a reaction containing 0.5 micromolar of each of the primers mAlb90F (SEQ ID NO: 46, CTCCTCTTCGTCTCCGGC) and mAlb1073R (SEQ ID NO: 47, CTGCCACATTGCTCAGCAC) and 1x high-fidelity PCR master mix. The resulting 984 bp PCR product spanning the entire intron 1 of mouse albumin was purified using a column-based purification kit. The PCR products were sequenced by next-generation sequencing (NGS) and analyzed for the generation of indels in the target sequence, which were used as indicators of double-strand breaks generated by Cas enzymes and the joining of NHEJ pathways.

[0287] Sequencing reads were analyzed by aligning each sequence read to the wild-type target sequence (in this case, albumin intron 1) and counting the number of reads containing at least one indel within a window spanning 10 base pairs on either side of the predicted target cleavage site for the nuclease, regardless of indel size. Editing efficiency (indel frequency) for each of the five mice per group, as well as the group mean and standard deviation, are shown in Table 2. Figure 2 Summary. No editing was detected in control mice injected with PBS buffer. Both spCas9 mRNA / mAlbR2 LNP and MG29-1 mRNA / mAlb29-8-50LNP resulted in dose-dependent editing. At all 3 doses, the editing efficiency of MG29-1 mRNA / mAlb29-8-50LNP was higher than that of spCas9mRNA / mAlbR2 LNP. The average editing efficiency of the 3 doses is summarized in Table 8. At a dose of 1 mg / kg (0.5 mg / kg mRNA and 0.5 mg / kg guide RNA), MG29-1 was slightly more potent than spCas9, resulting in more than about 15% indel. At a dose of 0.5 mg / kg (0.25 mg / kg mRNA and 0.25 mg / kg guide RNA), MG29-1 resulted in more than about 50% indel. At a dose of 0.25 mg / kg (0.125 mg / kg mRNA and 0.125 mg / kg guide RNA), MG29-1 resulted in more than 100% indels. These data demonstrate that the MG29-1 nuclease in combination with appropriately optimized guide RNA is more potent than the spCas9 nuclease and appropriately modified guide RNA using the same LNP for delivery and the same method for producing mRNA. The superior in vivo editing efficiency of MG29-1 is particularly evident at the lowest dose tested, where MG29-1 is 2 times more potent than spCas9 at the same dose. These results suggest that MG29-1 nuclease and appropriately modified guide RNA exemplified by chemical #50 may have advantages for in vivo gene editing delivered using LNPs.

[0288] Table 8. Average editing efficiency in whole liver of mice 5 days after intravenous injection of LNPs encapsulating MG29-1 mRNA and the guide mAlb29-8-50 (mA29-8-50) or spCas9 mRNA and three doses of the guide mAlbR2 or PBS buffer (control).

[0289]

[0290] Example 6 - Sequence-specific double-stranded DNA cleavage by MG29-1 RNA-guided nuclease mediates integration of the FVIII gene cassette at albumin intron 1 in the liver of mice

[0291] To evaluate whether the human FVIII gene can integrate into albumin intron 1 in the liver of mice and produce human FVIII protein in the blood of mice, a two-vector approach was utilized, in which the FVIII cassette was delivered in an AAV8 virus, and lipid nanoparticles were used to deliver mRNA encoding the MG29-1 nuclease and sgRNA targeting albumin intron 1. The human FVIII gene cassette was designed as described in Example 3.

[0292] The FVIII gene cassettes pMG4006 (SEQ ID NO: 56), pMG4007 (SEQ ID NO: 57), pMG4008 (SEQ ID NO: 58) and pMG4009 (SEQ ID NO: 59) were packaged into adeno-associated virus serotype 8 (AAV8) using molecular biology techniques. The viruses were titrated by measuring the encapsulated DNA by quantitative PCR and expressed as genome copies per mL. Synthetic mRNA encoding the MG29-1 nuclease flanked by a nuclear localization signal (NLS) was generated as described in Example 1 above. Using the methods described in Example 5 above, MG29-1 mRNA (SEQ ID NO: 53) encoding the amino acid sequence of SEQ ID NO: 54 and sgRNA mA29-8b-50 (SEQ ID NO: 60) or mA29-12b-50 (SEQ ID NO: 61) were co-formulated into LNPs at a 1: 1 RNA mass ratio (mRNA: sgRNA). After systemic administration of AAV8 virus by intravenous injection, it is mainly taken up by the liver, but also by other tissues. After AAV transduction of hepatocytes, the single-stranded AAV genome is converted to a double-stranded form, and this is then concatenated to form circular head-to-tail and head-to-head polymers, which are believed to represent the main free form of the AAV genome that persists in the nucleus for a long time. The process of cell transduction and conversion to a stable concatemer AAV genome occurs over a period of days to weeks, and can at least partially explain why the expression of the transgene encoded in the AAV genome may take several weeks to reach maximum levels. In contrast, LNP delivery of mRNA is a rapid process in which maximum gene expression occurs within 24 hours of systemic IV administration of mice, as demonstrated by mRNA encoding reporter protein luciferase. Therefore, AAV8-FVIII virus was injected 21 days before the administration of LNP-encapsulated MG29-1 mRNA / sgRNA, so as to allow time for effective AAV transduction of hepatocytes in the liver and conversion of the single-stranded AAV genome into a stable double-stranded form. 1x 10 13Each of the four AAV8 viruses encapsulating the FVIII gene cassettes pMG4006, pMG4007, pMG4008, and pMG4009 was injected intravenously (IV) to five wild-type C57Bl / 6 mice in each group through the tail vein at a dose of 0.5 mg total RNA / kg body weight (prepared at a 1:1 mass ratio of mRNA:sgRNA) IV injection of LNPs encapsulating MG29-1 mRNA and sgRNA mA29-12b-50 or MG29-1 mRNA and sgRNA mA29-8b-50. On the 7th day after LNP administration, plasma was collected from each mouse to analyze human FVIII levels. Mice were sacrificed on the 12th day after LNP administration, and plasma was collected from all mice by cardiac puncture, and liver samples were collected for genomic DNA extraction.

[0293] Liver samples were homogenized using 0.4 mL buffer / 100 mg tissue weight in a bead mill. Genomic DNA was purified from aliquots of the homogenate. The albumin intron 1 region was PCR amplified from 50 ng of genomic DNA in a reaction solution containing 0.5 micromolar primers mAlb90F (SEQ ID NO:46, CTCCTCTTCGTCTCCGGC) and mAlb1073R (SEQ ID NO:47, CTGCCACATTGCTCAGCAC) and 1x high-fidelity PCR master mix. The resulting 984 bp PCR product spanning the entire intron 1 of mouse albumin was purified using a column-based purification kit. The PCR product was sequenced by next generation sequencing (NGS).

[0294] Sequencing reads were analyzed using a custom Python script that aligned each sequence read to the wild-type target sequence (in this case, albumin intron 1) and counted the number of reads containing at least one INDEL within a window spanning 10 base pairs on either side of the predicted target cleavage site for the nuclease, regardless of INDEL size. Figure 3 The data are summarized, which plots the editing efficiency (INDEL frequency) in each of the 5 mice in each group and the mean and standard deviation of the group. The editing data are also summarized in Table 9 below. The average INDEL frequency in the 8 groups treated with LNPs encapsulating MG29-1 mRNA and albumin targeting sgRNA ranged from 43% to 53%. There was no significant difference in INDEL frequency between the individual groups or between Groups 1 to 4 edited with guide mA29-12b-50 and Groups 5 to 8 edited with guide mA29-8b-50.

[0295] Table 9: Editing efficiency in whole liver of mice 12 days after intravenous injection of a 0.5 mg / kg dose of LNPs encapsulating MG29-1 mRNA and sgRNA mA29-12b-50 or MG29-1 mRNA and sgRNA mA29-8b-50 or PBS buffer (control).

[0296]

[0297] Human FVIII in mouse plasma in Groups 1 to 8 was measured using a human FVIII-specific ELISA kit (for the record, this kit does not have cross-reactivity to mouse FVIII). Plasma from control untreated mice (Group 9) was included in each assay and the values ​​were subtracted from the experimental samples. Background signals from untreated mouse plasma were low. In the range of 10mIU / mL to 200mIU / mL, a standard curve consisting of plasma-derived full-length human FVIII drug products diluted in 25% of the original C57BL / 6 mouse plasma matrix was run in duplicate on each assay plate. Plasma samples from treated mice were also diluted to 25% plasma and then added to the wells of the ELISA plate in duplicate. After binding to the capture antibody and washing with PBS / 0.05% Tween, the bound human FVIII was detected using the biotin-labeled detection antibody in the kit, followed by the addition of streptavidin-HRP and TMB substrates, and the absorbance at 450nM was measured in a plate reader. The concentration of human FVIII in the plasma samples of treated mice (mIU FVIII per mL of plasma) was interpolated according to the standard curve. On the 7th day after LNP administration, the human FVIII levels in the treated mice were in the range of 13mIU / mL to 46mIU / mL, representing 1.3% to 4.6% of the normal FVIII levels in humans (Tables 10 and 11). On the 12th day after LNP administration, the human FVIII levels in the treated mice were in the range of 39mIU / mL to 120mIU / mL, representing 3.9% to 12% of the normal FVIII levels (Tables 10 and 11).

[0298] Table 10: Human FVIII antigen levels in plasma from mice on day 7 and day 12 after LNP. Normal human plasma was assayed in parallel as a positive control

[0299]

[0300] Table 11: Human FVIII antigen levels in plasma from mice on day 7 and day 12 after LNP. Normal human plasma was assayed in parallel as a positive control

[0301]

[0302] The 7-day plasma samples from mice in groups 5 to 8 (treated with LNPs containing guide mA29-8b-50) were re-assayed using the same human FVIII-specific ELISA kit, but using a recombinant human B-domain deleted FVIII drug product (Xyntha). The average human FVIII levels measured using Xyntha as a standard were 67±36mIU / mL, 33±18mIU / mL, 104±62mIU / mL, and 78±53mIU / mL in groups 5, 6, 7, and 8, respectively. The FVIII levels measured using Xyntha as a standard were an average of 2.8 times the FVIII levels measured when Octanate was used as a standard. The FVIII gene delivered to the mice encodes a B-domain deleted FVIII protein and is the same protein sequence in all 4 viruses (pMG4006, pMG4007, pMG4008, pMG4009). The B domain of FVIII contains 908 amino acids (38% of the full-length FVIII protein) and therefore constitutes a substantial proportion of the FVIII protein in Octanate. Taken together, these data demonstrate that delivery of a human FVIII donor cassette to mice, followed by delivery of LNPs encapsulating MG29-1 mRNA and an sgRNA targeting albumin intron 1, achieves measurable expression of human FVIII protein detectable in mouse blood on days 7 and 12 after LNP administration.

[0303] The FVIII gene cassette packaged in AAV lacks a promoter to drive the expression of the FVIII gene in the free-type AAV genome. However, because AAV ITR has a weak promoter activity, the RNA encoding the FVIII coding sequence is likely to be transcribed from the free-type genome. However, for two reasons, the free-body-derived RNA is unlikely to be translated into the FVIII protein secreted into the blood. The first, the FVIII gene cassette does not contain an in-frame translation initiation codon (ATG), and also lacks a translation initiation consensus sequence (KOZAK sequence) at the 5' end of the cassette. Therefore, any FVIII protein is unlikely to be translated from any presumed RNA produced by the free-type AAV genome. Second, any FVIII protein produced by the free-type AAV genome will not include a signal peptide at the N-terminal. Due to the need for a signal peptide to guide the secretion of protein from cells, any FVIII protein that may be expressed from the free-type AAV genome will not be secreted into the blood. The FVIII gene cassette is designed to express the FVIII protein after being integrated into albumin intron 1. After integration into albumin intron 1 in the forward orientation, transcription from the endogenous albumin promoter will produce a pre-mRNA encoding albumin exon 1, followed by intron 1 and a portion of the human FVIII coding sequence. Splicing of the pre-mRNA from the albumin exon 1 splice donor to a splice acceptor included in the FVIII gene cassette will produce a hybrid mRNA transcript in which the albumin exon is fused in frame to the mature B domain-deleted human FVIII coding sequence. Since albumin exon 1 encodes the signal peptide of albumin, this will provide the signal peptide required for FVIII secretion into the blood.

[0304] The frequency of FVIII gene integration in albumin intron 1 in the liver of mice is measured using a quantitative assay designed to measure forward integration and reverse integration. These assays use digital droplet PCR (dd-PCR) technology, in which sample genomic DNA is encapsulated in a separate droplet so that the droplet contains a single DNA molecule or does not contain DNA. Droplets containing DNA that is positive for the integration junction (as detected by PCR-based amplification by incorporating fluorescent probes) are scored as positive, and droplets lacking the integration junction are scored as negative. The dd-PCR instrument counts the number of positive droplets and uses an algorithm to determine the absolute number of copies of the target amplicon (in this case, the integration junction) in the original genomic DNA sample. PCR primers and probes are optimized to ensure that the assay is specific and quantitative. The copies of the genomic DNA in each sample are normalized using an internal control assay for the cytochrome C1 gene. The results are expressed as integration percentages, calculated as the number of copies of the FVIII gene integration junction per 100 cytochrome C1 copies. Two assays for FVIII integration were used, one detecting the 5' junction of the forward integration product and the other detecting the 5' junction of the reverse integration product. The sequences of the primers and probes used to quantify the forward integration product were KAS_401_F1-forward: 5'GCACAGATATAAACACTTAACGGGT3' (SEQ ID NO: 105); KAS_401_F1-reverse: 5'GGAGGAAATCTAGCATCCACAG3' (SEQ ID NO: 106); KAS_401_F1-probe: 5'+C+CACCAGAAGA+TAT+T+ACCTG3'6-FAM / 3'IBFQ (SEQ ID NO: 107). The sequences of the primers and probes used to quantify the reverse integration products were: KAS_501_R2-forward: 5'GCACAGATATAAACACTTAACGGG 3' (SEQ ID NO: 108); KAS_501_R2-reverse: 5'TGCTCTGAGAATGGAAGTGC 3' (SEQ ID NO: 109); KAS_501_R2-probe: 5'+C+GATCAGT+AGAGGTCCTGAGC 3'6-FAM / 3'IBFQ (SEQ ID NO: 110) (+ indicates locked nucleic acid base). These results for individual mice are summarized in Table 12.

[0305] Table 12: Frequency of integration of the FVIII cassette in the forward orientation in the liver of individual mice and quantification of albumin-FVIII fusion mRNA

[0306]

[0307]

[0308]

[0309] Figure 7 The frequency of forward integration in individual mice from each group is shown. Animals m1, m4, and m5 were control mice injected with PBS buffer only, and no integration was detected, indicating that this assay did not have background signal. All mice that received AAV8-pMG4006, AAV8-pMG4007, AAV8-pMG4008, or AAV8-pMG4009, followed by LNPs encapsulating MG29-1 mRNA and guide RNA12 (mA29-12b-50) had measurable integration in the forward direction, ranging from 0.25% to 2% (0.25 to 2 copies per 100 cytochrome C1 copies). Due to technical issues with the dd-PCR assay on this sample, data for mouse m21 are not reported. The average forward integration frequency for each group is shown in Figure 8 There were no significant differences between the four groups receiving four AAV donors (AAV8-pMG4006, AAV8-pMG4007, AAV8-pMG4008, AAV8-pMG4009) that differed in the orientation of the guide RNA target sites flanking the donor, but pMG4008 showed the most consistent frequency in mice and the highest average forward integration of 2%. The frequency of integration of the FVIII cassette into albumin intron 1 in the reverse orientation was measured and plotted together with the forward integration frequency in Fig. 9 The frequency of reverse integration ranged from 1% to 2% and was not statistically significantly different between the groups receiving the four AAV donors (AAV8-pMG4006, AAV8-pMG4007, AAV8-pMG4008, AAV8-pMG4009). Overall, the frequency of reverse integration in each mouse was similar to the frequency of forward integration, indicating that there was no preferential integration of the FVIII cassette in the forward or reverse orientation.

[0310] Fig.10 The forward integration frequency of individual mice from each group that received AAV8-pMG4006, AAV8-pMG4007, AAV8-pMG4008, or AAV8-pMG4009 followed by LNPs encapsulating MG29-1 mRNA and guide RNA 8 (mA29-8b-50) is shown. All mice had measurable integration in the forward orientation, ranging from 0.25% to 2% (0.25 to 2 copies per 100 cytochrome C1 copies). The average forward integration frequency for each group is shown in Fig.11There were no significant differences between the four groups that received four AAV donors (AAV8-pMG4006, AAV8-pMG4007, AAV8-pMG4008, AAV8-pMG4009) that differed in the orientation of the guide RNA target sites flanking the donor. The frequency of integration of the FVIII cassette into albumin intron 1 in the reverse orientation was measured and plotted together with the forward integration frequency in Fig.12 The frequency of reverse integration ranged from 0.5% to 3% and was not statistically significantly different between the groups receiving the four AAV donors (AAV8-pMG4006, AAV8-pMG4007, AAV8-pMG4008, AAV8-pMG4009). Overall, the frequency of reverse integration in each mouse was similar to the frequency of forward integration, indicating that there was no preferential integration of the FVIII cassette in the forward or reverse orientation.

[0311] Expression of the expected FVIII-encoding mRNA from the integrated FVIII cassette in the liver of the same mice was quantified using a dd-PCR assay. Integration of the FVIII cassette in the forward orientation (defined as the 5' end of the FVIII cassette adjacent to albumin exon 1) at the double-strand break generated by the MG29-1 nuclease and guide RNA was predicted to produce a hybrid mRNA generated by RNA splicing between the albumin exon 1 splice donor and the splice acceptor at the 5' end of the FVIII cassette. Therefore, this hybrid mRNA will contain a novel sequence junction between albumin exon 1 and the 5' end of the coding sequence for mature FVIII, such as Fig.13 A dd-PCR assay was designed in which the forward primer was complementary to a sequence within albumin exon 1, the reverse primer was complementary to a sequence within the 5' end of human FVIII, and the probe spanned the predicted junction between albumin exon 1 and the 5' end of human FVIII after correct splicing. The sequences of the primers and probe were: MG101-set1-forward: 5'TAACCTTTCTCCTCCTCCTCTT3' (SEQ ID NO: 111); MG101-set1-reverse: 5'TCCACAGCTCCCAGGTAATA3' (SEQ ID NO: 112); MG101-set1-probe: 5'TCTTCTGGTGGCCAGTGCTTCTC3'FAM, ZEN / 3'IBFQ (SEQ ID NO: 113).

[0312] Total RNA was purified from the left lateral lobe of the liver of each mouse. After DNA enzyme digestion to eliminate any remaining genomic DNA, cDNA was prepared from 500ng of total RNA. The albumin-FVIII fusion mRNA and cytochrome C1 mRNA of cDNA were determined. The level of cytochrome C1 mRNA is used to normalize the quality and quantity of the measured mRNA by dividing the absolute copy number of albumin-FVIII hybrid mRNA by the absolute copy number of cytochrome C1 mRNA and expressing it as a percentage. The results of mice receiving any of 4 AAV8-FVIII donors (AAV8-pMG4006, AAV8-pMG4007, AAV8-pMG4008, AAV8-pMG4009) and LNPs encapsulating MG29-1 mRNA and guide RNAmA29-8b-50 (guide 8) are shown in Fig.14 No signal was detected in control mice injected with PBS, indicating the specificity of this assay. The levels of albumin-FVIII fusion mRNA ranged from 20% relative to cytochrome C1 to 30% relative to cytochrome C1 in the four groups. Although the differences between the groups were not statistically significant, the levels of albumin-FVIII fusion mRNA tended to be higher in mice receiving AAV8-pMG4008 or AAV8-pMG4009.

[0313] Results for mice that received any of the four AAV8-FVIII donors (AAV8-pMG4006, AAV8-pMG4007, AAV8-pMG4008, AAV8-pMG4009) and LNPs encapsulating MG29-1 mRNA and guide RNA mA29-12b-50 (guide 12) are shown in Fig.15No signal was detected in control mice injected with PBS, indicating that this assay is specific. In the four groups, the levels of albumin-FVIII fusion mRNA ranged from 5% relative to cytochrome C1 to 10% relative to cytochrome C1. Although the differences between the groups were not statistically significant, the levels of albumin-FVIII fusion mRNA in mice receiving AAV8-pMG4007, AAV8-pMG4008, or AAV8-pMG4009 tended to be higher. Overall, the levels of albumin-FVIII fusion mRNA were lower in the group of mice receiving Guide 12 (mA29-12b-50) compared to the group of mice receiving Guide 8 (mA29-8b-50). The higher levels of albumin-FVIII fusion mRNA observed in mice in which the FVIII gene was integrated at the Guide 8 target site were not related to a higher frequency of FVIII gene integration in the forward orientation. The highest frequency of FVIII integration in the forward orientation was observed in mice receiving pMG4008 and guide 12, with an albumin-FVIII fusion mRNA frequency of 10%, which was lower than the 30% measured in mice receiving pMG4008 and pMG4008 and guide 8.

[0314] Example 7 - Demonstration of Integration of the Human FVIII Gene Cassette into Albumin Intron 1 of Mouse

[0315] Cohorts of 15 adult C57BL / 6 mice (8 weeks old) were injected IV with 5 × 10 12 vg / kg of pMG4006 packaged in AAV8 (Groups 2, 3, and 4). One group of 5 mice was not injected with AAV (Group 1). 21 days later, 5 mice in Group 3 were injected IV with LNPs encapsulating MG29-1 mRNA and sgRNAmA29-8-37 (SEQ ID NO: 62), and 5 mice in Group 4 were injected IV with LNPs encapsulating MG29-1 mRNA and sgRNAmA29-12-37 (SEQ ID NO: 63). sgRNAmA29-12-37 targets the same genomic sequence in albumin intron 1 as mA29-12b-50 (SEQ ID NO: 61), and sgRNA mA29-8-37 targets the same genomic sequence in albumin intron 1 as mA29-8b-50 (SEQ ID NO: 60). PBS buffer was injected IV as a control to mice in Group 1 and Group 2. On day 12 after LNP administration, plasma was collected from all mice by cardiac puncture, and liver tissues were collected for genomic DNA analysis.

[0316] As described in the previous examples, the editing frequency at the target site in the albumin intron in genomic DNA purified from mouse liver was measured by NGS (Table 13).

[0317] Table 13: INDEL frequency in liver and FVIII protein level in blood of mice

[0318]

[0319]

[0320] Groups 1 and 2, which did not receive LNPs, had background editing levels as expected. The INDEL frequencies of mice in Group 3 injected with LNPs encapsulating MG29-1 mRNA and sgRNA mA29-8-37 ranged from 43% to 51%. Two mice (mouse No. 16 and No. 19) in Group 4 injected with LNPs encapsulating MG29-1 mRNA and sgRNA mA29-12-37 had high INDEL frequencies of 45% and 49%, similar to those observed in Group 3. One mouse in Group 4 (mouse No. 18) had a moderate INDEL frequency of 19%. Two mice in Group 4 (mouse No. 17 and No. 20) had lower or undetectable INDEL frequencies similar to those of uninjected mice, indicating that the administration of LNPs may not be successful in these 2 mice.

[0321] The human FVIII level in the plasma sample was measured using a capture chromogenic activity assay, which measures FVIII activity after the human FVIII in the plasma was captured on the surface of the plate using a human FVIII-specific antibody that does not bind to mouse FVIII. A 96-well plate was first coated with a mixture of two human-specific anti-FVIII antibodies. After the plate surface was blocked with a PBS buffer containing milk powder and washed with PBS plus 0.05% Tween, the appropriately diluted mouse plasma samples and standards were added to the wells and incubated at 37°C for 2 hours. Standards were prepared by diluting the European Pharmacopoeia (EP) reference standard derived from human plasma in the original C57BL / 6 mouse plasma. The wells were washed 3 times with PBS containing 0.05% Tween, and the FVIII activity bound to each well was determined using a commercial FVIII chromogenic assay kit according to the manufacturer's protocol. The results were back-calculated as the percentage of the FVIII level in normal human plasma present in mouse plasma. The results (Table 13) show that human FVIII activity was not detected in the plasma of the first group of mice that did not receive AAV and LNP. The 2nd group of mice that received AAV encoding human FVIII gene cassettes but did not receive LNPs (and therefore did not edit at the albumin locus) also did not have detectable human FVIII activity in their blood. This indicates that the AAV virus alone that delivers the FVIII gene cassette and is expected to produce free-type AAV genomes cannot produce active human FVIII protein. Three of the five mice in the 3rd group that received both AAV and LNPs (with guide mA29-8-37) had detectable human FVIII activity in their blood, which was about 4% of normal human levels. Two of the five mice in the 4th group that received both AAV and LNPs (with guide mA29-12-37) had detectable human FVIII activity in their blood, which was 2.6% and 13% of normal human levels. The two mice without INDELs in the 4th group did not have detectable FVIII. Taken together, the data from Groups 3 and 4 demonstrated that 5 of 8 mice edited at the albumin locus expressed detectable human FVIII activity in their blood on day 12 after LNP administration.

[0322] In order to confirm that the expression of human FVIII in the blood of these mice is associated with the integration of the FVIII gene cassette into the sgRNA target site in albumin intron 1, an inside-out PCR assay was performed. A pair of PCR primers complementary to a DNA sequence flanking the predicted junction between albumin intron 1 and the 5' end of the human FVIII gene cassette packaged in the AAV virus was designed. The primer that binds to albumin intron 1 is located 5' of the target site of the sgRNA. In the case of integrating the FVIII gene cassette at the target site of the sgRNA in albumin intron 1, genomic DNA purified from the liver of mice was used as a template to generate a PCR product of 286bp for Group 3 (integrated into the mA29-8-37 target site) and a PCR product of 221bp for Group 4 (integrated into the mA29-12-37 target site). The products of the PCR reactions were fractionated on an agarose gel and imaged by staining. As Figure 4 As shown, the endo-external PCR analysis of the genomic DNA of the mice in the 1st group (lanes 1 to 3) does not have bands, indicating that there is no background PCR amplification. Four mice (mice 6,7,9,10) in the five mice in the 2nd group injected with AAV (without LNP) also failed to produce PCR products, and the mouse 8 in the 2nd group gave the incorrect fuzzy bands of size for the FVIII gene integrated. This shows that in the mice injected with the AAV carrying the FVIII gene cassette, the FVIII gene cassette is not integrated at the guide RNA target site, but is not edited by the LNP delivery of MG29-1 and homologous sgRNA. The liver genomic DNA of all five mice (mouse numbering 11 to numbering 15) in the 3rd group produces PCR products, and its size matches the predicted size 286bp of the FVIII cassette integrated in the forward (expression ability) orientation at mA29-8-37 target site. This PCR determination is not quantitative, so the PCR band intensity does not represent the relative integration frequency between different mice. Liver genomic DNA from 3 mice (mouse No. 16, No. 18, No. 19) out of 5 mice in Group 4 produced PCR products that matched the expected 221bp size of the FVIII cassette integrated in the forward (expression capacity) orientation at the mA29-12-37 target site. No PCR products were generated for mice No. 17 and No. 20 from Group 4, indicating that in these two mice, the integration of the FVIII cassette integrated in the forward (expression capacity) orientation could not be detected using this assay. This is consistent with the fact that there are no INDELs at the guide target site in albumin intron 1 for mice No. 17 and No. 20 (possibly due to unsuccessful LNP administration), and provides further evidence that integration depends on editing by MG29-1.

[0323] Taken together, these data demonstrate that the MG29-1 nuclease, in combination with an appropriately designed guide RNA, can induce integration of an appropriately designed human FVIII gene cassette into the genome in the liver of mice at albumin intron 1. Furthermore, this integration results in the expression of functional human FVIII protein in the blood.

[0324] Example 8 - Efficient editing by MG29-1 nuclease in the liver of non-human primates following systemic administration of MRG29-1 mRNA and appropriate guide RNA packaged in lipid nanoparticles

[0325] Based on testing 23 guides in the liver cell line Hep3B, five guide RNAs targeting MG29-1 intron 1 of human albumin were selected. These five guide RNAs, designated chA29-74B-50 (SEQ ID NO: 64), cA29-78B-50 (SEQ ID NO: 65), chA29-83B-50 (SEQ ID NO: 66), cA29-84B-50 (SEQ ID NO: 67), and cA29-87B-50 (SEQ ID NO: 68), were synthesized using specific chemical modifications to the RNA to improve the in vitro stability and in vivo efficacy of gene editing of MG29-1 (Chemistry 50). The nomenclature for the modifications to the RNA is as follows; m indicates that the base has a 2'-O-methyl group (e.g., mG), f indicates that the base has a 2'-fluoro group (e.g., fG), and * indicates that the backbone contains a phosphorothioate bond (e.g., G*G).

[0326] The messenger RNA encoding the MG29-1 nuclease is produced by in vitro transcription of a linearized plasmid template using a mixture of T7 RNA polymerase and ribonucleotides rATP, rCTP and rGTP, N1-methyl pseudouridine and CleanCAP end-capping reagents. The SV40-derived nuclear localization sequence (PKKKRKVGGGGS (SEQ ID NO: 103)) followed by a short linker is included at the N-terminus of the coding sequence. The nuclear localization signal of the nucleoplasmic protein before the short linker (SGGKRPAATKKAGQAKKKK (SEQ ID NO: 104)) is added to the C-terminus of the coding sequence. The plasmid also encodes approximately 100nt polyA tail at the 3' end, which produces a polyA tail in the mRNA. The coding sequence of MG29-1 is codon optimized. The DNA sequence encoding MG29-1 mRNA is as in SEQ ID NO: 53, and the amino acid sequence encoded by MG29-1 mRNA is as in SEQ ID NO: 54. The mRNA was column purified, the concentration was determined by absorbance at 260 nm, and the purity was determined.

[0327] The relative efficacy of five leading guide RNAs was evaluated in primary hepatocytes from macaques (PCH). Cryopreserved PCH cells were plated on 24-well plates according to the supplier's instructions. Each guide in the MG29-1 mRNA and guide was mixed at a 1:20 mRNA:guide molar ratio, mixed with a transfection reagent according to the manufacturer's protocol, and then applied to PCH. After transfection, the culture medium on the cells was replaced every 24 hours, and genomic DNA was purified from the cells after 48 hours. The target region in albumin intron 1 was PCR amplified using primer pairs and a high-fidelity PCR master mix. The purified PCR product of the correct size was sequenced by next-generation sequencing. The sequence reads were aligned with the reference sequence of the albumin gene from macaques, and the number of reads containing insertions or deletions (indels) at the target site of the specific guide RNA was counted using a custom script. The editing frequency is defined as the percentage of the total number of sequence reads containing indels. Results ( Figure 5 ) showed that all 5 guides edited at the intended target site in a dose-dependent manner. The ranking of guide potency from most to least effective was cA29-87B-50>chA29-74B-50>cA29-78B-50>cA29-84B-50>chA29-83B-50. For guides chA29-74B-50, cA29-78B-50, chA29-83B-50, cA29-84B-50, and cA29-87B-50, the editing efficiencies of these guides and MG29-1 mRNA in primary human hepatocytes at a single dose were 38%, 43%, 55%, 40%, and 40.5%, respectively. Therefore, in primary human hepatocytes, the guide chA29-83B-50 appears to be the most effective. Based on these data from PCH and PHH, two guides, cA29-87B-50 and chA29-83B-50, were selected for testing in non-human primates.

[0328] MG29-1 mRNA and guide chA29-83B-50 (SEQ ID NO: 66) or guide cA29-87B-50 (SEQ ID NO: 68) were co-formulated in two lipid nanoparticle formulations referred to as L1 and L2 at a mass ratio of 1: 1 (mRNA: guide RNA). The average diameter of the lipid nanoparticles (LNPs) was less than 100 nm, the polydispersity index was less than 0.12, as measured by dynamic light scattering, and the encapsulation efficiency was greater than 85%, as measured by ribogreen assay. After formulation, the LNPs were buffer exchanged into phosphate-buffered saline containing sucrose, frozen, and stored at -80°C for several weeks before administration.

[0329] Prior to dosing, intentionally bred naive macaques (Macaca fascicularis) with an average body weight of 1.8 kg ± 0.16 kg were adapted for 2 weeks. The LNP formulations were thawed and diluted to 0.3 mg total RNA / mL by dilution with sterile 0.9% sodium chloride. Each of the four LNP formulations was administered to a group of three monkeys by infusion into the tail vein (5 mL / kg body weight) over a period of 60 minutes. All animals were monitored by veterinary staff and no serious adverse events were observed. The RNA concentration of the retentate of the dosing solution was determined using the ribogreen assay, in which the standard contained guides and mRNA for the production of LNPs. Based on this analysis, the actual doses of RNA given to each group were 1.4, 1.3, 1.25, and 1.25 mg / kg, respectively, for Groups 1, 2, 3, and 4, as shown in Table 14.

[0330] Table 14: NHP Study Groups and Doses

[0331]

[0332] Eight days after dosing, all groups underwent autopsy and collected samples of different tissues for analysis. Samples from each of the five lobes of the liver were collected individually from each animal and stored frozen before extraction and purification of genomic DNA. The target region in albumin intron 1 was PCR amplified from the purified genomic DNA from each of the five liver lobes of each animal using primer pairs and high-fidelity PCR master mix. The purified PCR products of the correct size were sequenced by next-generation sequencing on an Illumina MiSeq instrument. The sequence reads were aligned with the reference sequence of the albumin gene from macaques and the number of reads containing insertions or deletions (indels) at the target site of the specific guide RNA was counted using a custom script. The editing frequency is defined as the percentage of the total number of sequence reads containing indels. Editing at the expected target site of each guide was detected in the livers of all 12 animals ( Figure 6 ). Table 15 lists the average editing percentages for the five liver lobes per animal and the average editing values ​​for each group. The average editing in each group ranged from 29% to 50%. Group 4 (treated with LNP L2 encapsulating MG29-1 mRNA and guide cA29-87B-50) exhibited the highest average editing (50%) and the most consistent editing among the three animals in each group. Animal 2502 exhibited scaly skin and elevated cytokines at the injection site on days 1 to 3 post-dose, indicating that this animal experienced an acute but self-resolving inflammatory response to the test article. This suggests that the low editing level (3%) in this animal was caused by an inflammatory response that prevented effective delivery to the liver. No animals received anti-inflammatory medications before or during the course of the study.

[0333] Collectively, these data demonstrate that MG29-1 nuclease, when combined with an appropriate guide RNA, can mediate efficient editing in the liver of nonhuman primates when delivered systemically as RNA packaged in LNPs.

[0334] Table 15: Editing percentage in macaque liver 8 days after systemic administration of LNPs encapsulating MG29-1 mRNA and 2 different guide RNAs (average of 5 liver lobes per animal)

[0335]

[0336] Example 9 - Sequence-specific double-stranded DNA cleavage by MG3-6 / 3-4 RNA-guided nuclease mediates integration of the FVIII gene cassette at albumin intron 1 in the liver of mice

[0337] To evaluate whether the type II CRISPR system MG3-6 / 3-4 can mediate the integration of the human FVIII gene into albumin intron 1 in the liver of mice and produce human FVIII protein in the blood of mice, a dual vector approach was used. The FVIII cassette was delivered in the AAV8 virus, and lipid nanoparticles were used to deliver mRNA encoding the MG3-6 / 3-4 nuclease and the albumin intron 1 targeting sgRNA. The human FVIII gene cassette contained the same human FVIII coding sequence as in pMG4006 (SEQ ID NO: 16), pMG4007 (SEQ ID NO: 17), pMG4008 (SEQ ID NO: 18), and pMG4009 (SEQ ID NO: 19), but the flanking sequences were modified to contain target sites for two MG3-6 / 3-4 guide RNAs (called mA364-34 and mA364-59) targeting intron 1 of mouse albumin. These guide RNAs were selected based on the editing (INDEL) efficiency of a guide screen against mG3-6 / 3-4 in the mouse liver cell line Hepa1-6. The FVIII cassettes in pMG4012 (SEQ ID NO: 20), pMG4013 (SEQ ID NO: 21), pMG4014 (SEQ ID NO: 22), and pMG4015 (SEQ ID NO: 23) differ in the orientation of the flanking guide RNA target site: pMG4012 (forward-forward orientation), pMG4013 (reverse-reverse orientation), pMG4014 (forward-reverse orientation), and pMG4015 (reverse-forward orientation).

[0338] The FVIII cassettes in pMG4012 (SEQ ID NO: 20), pMG4013 (SEQ ID NO: 21), pMG4014 (SEQ ID NO: 22), and pMG4015 (SEQ ID NO: 23) were packaged into adeno-associated virus serotype 8 (AAV8) using standard methods. The viruses were titrated by measuring the encapsulated DNA by quantitative PCR and expressed as genome copies per mL. Synthetic mRNA encoding the MG3-6 / 3-4 nuclease flanked by a nuclear localization signal (NLS) was generated as described in Example 1 above. Using the methods described in Example 5 above, MG3-6 / 3-4 mRNA (SEQ ID NO: 95) encoding the amino acid sequence of SEQ ID NO: 96 and sgRNA mA364-34-1 (SEQ ID NO: 97) or mA364-59-1 (SEQ ID NO: 98) were co-formulated into LNPs at a 1: 1 RNA mass ratio (mRNA: sgRNA).

[0339] 1x 10 13 Each of the four AAV8 viruses encapsulating the FVIII gene cassettes pMG4012, pMG4013, pMG4014, and pMG4015 was injected intravenously (iv) into groups of five immunodeficient mice via the tail vein at a dose of 0.5 mg or 0.7 mg total RNA / kg body weight (formulated at a 1:1 mass ratio of mRNA:sgRNA) iv to the same mice encapsulating MG3-6 / 3-4 mRNA and sgRNA mA364-34-1 or MG3-6 / 3-4 mRNA and sgRNA mA364-59-1. Additional mice were administered the four AAV viruses but no LNPs were given.

[0340] At the end of the study, when the mice were sacrificed (approximately 5 months after LNP administration), genomic DNA was purified from the liver tissue of each mouse and analyzed by NGS for editing of the guide RNA at the target site. The results are shown in Fig.16In the whole liver of mice dosed with LNPs encapsulating MG3-6 / 3-4 mRNA and guide RNA mA364-34-1 at 0.5 or 0.7 mg / kg body weight, INDELs ranged between 63% and 70%. Since hepatocytes account for approximately 60%-70% of the cells in the liver and the LNPs used are primarily delivered to hepatocytes, 70% of Indels in the whole liver represent saturated editing of hepatocytes. In contrast, INDELs ranged between 0% and 35% in the whole liver of mice dosed with LNPs encapsulating MG3-6 / 3-4 mRNA and guide RNA mA364-59-1 at 0.5 or 0.7 mg / kg body weight. The two groups that received AAV8-pMG4014 and AAV8-pMG4015 and LNPs encapsulating MG3-6 / 3-4 mRNA and guide RNA mA364-59-1 at 0.5 mpk showed almost no editing, which was attributed to dosing failure. In general, the editing efficiency of guide mA364-34-1 was at least 2-fold higher than that of guide mA364-59-1.

[0341] The dd-PCR assay was used to measure the integration of the FVIII gene cassette into the target site in albumin intron 1 in the forward or reverse orientation. Genomic DNA purified from the liver of mice was digested with EcoRI. For quantification in the forward orientation, the primers used to amplify the 5' forward junction were KAS_401_F1-Forward: 5'GCACAGATATAAACACTTAACGGGT3' (SEQ ID NO: 105); KAS_401_F1-Reverse: 5'GGAGGAAATCTAGCATCCACAG3' (SEQ ID NO: 106); KAS_401_F1-Probe: 5'+C+CACCAGAAGA+TAT+T+ACCTG3'6-FAM / 3'IBFQ (SEQ ID NO: 107). For quantification in the reverse direction, the primers used to amplify the 5' reverse junction were KAS_501_R2-Forward: 5'GCACAGATATAAACACTTAACGGG 3' (SEQ ID NO: 108); KAS_501_R2-Reverse: 5'TGCTCTGAGAATGGAAGTGC 3' (SEQ ID NO: 109); KAS_501_R2-Probe: 5'+C+GATCAGT+AGAGGTCCTGAGC 3'6-FAM / 3'IBFQ (SEQ ID NO: 110) (+ indicates locked nucleic acid base).

[0342] The copies of genomic DNA in each sample were corrected using a dd-PCR assay for cytochrome C1. The results were calculated as the percentage of forward integration (the number of copies of the forward integration junction per 100 copies of cytochrome C1). The integration of the FVIII gene cassette of mice administered with any of the four AAV8 FVIII donors (pMG4012, pMG4013, pMG4014, or pMG4015), followed by LNPs encapsulating MG3-6 / 3-4 mRNA and sgRNA mA364-34-1, was analyzed at the end of the study. Due to the low level of editing achieved, the integration of mice from the group treated with AAV, followed by LNPs encapsulating MG3-6 / 3-4 mRNA and sgRNA mA364-59-1 was not determined. The results showed that the frequency of forward integration was between 0.2% and 1.3% in the 24 mice analyzed ( Fig.17 The average forward integration frequency in each group was approximately 0.5% ( Fig.18 ), and there were no significant differences between the groups. In the 24 mice analyzed, the frequency of reverse integration ranged from 0.05% to 0.8% ( Fig.19 In each of the individual mice that received pMG4013 (10 mice in total, 5 of which received 0.7 mpk LNP and 5 of which received 0.5 mpk LNP) and pMG4014 (5 mice in total), the frequency of reverse integration was lower than the frequency of forward integration ( Fig.19 ). In contrast, the forward integration frequency of each individual mouse in the groups receiving AAV8-pMG4012 and AAV8-pMG4015 was similar to the reverse integration frequency. In AAV8-pMG4012 and AAV8-pMG4015, the guide 34 (mA364-34-1) target site flanking the FVIII cassette was in the forward-forward and reverse-forward orientations, respectively. In AAV8-pMG4013 and AAV8-pMG4014, which caused preferential integration in the desired forward orientation, the guide 34 (mA364-34-1) target site flanking the FVIII cassette was in the reverse-reverse and forward-reverse orientations, respectively. These data indicate that the guide target site of MG3-6 / 3-4, including the FVIII cassette flanked in the reverse-reverse or forward-reverse orientation, causes a favorable preferential integration of the FVIII cassette in the forward orientation in albumin intron 1. The forward orientation of integration is advantageous as it enables expression from the albumin promoter to produce mRNA encoding FVIII.

[0343] To assess the expression of the FVIII transgene driven by the endogenous albumin promoter, a dd-PCR assay was used. Integration of the FVIII cassette in the forward orientation (defined as the 5' end of the FVIII cassette adjacent to albumin exon 1) at the double-strand break generated by the MG3-6 / 3-4 nuclease and guide RNA was predicted to produce a hybrid mRNA resulting from RNA splicing between the albumin exon 1 splice donor and the splice acceptor at the 5' end of the FVIII cassette. Thus, this hybrid mRNA would contain a novel sequence junction between albumin exon 1 and the 5' end of the coding sequence for mature FVIII, such as Fig.13 A dd-PCR assay was designed in which the forward primer was complementary to a sequence within albumin exon 1, the reverse primer was complementary to a sequence within the 5' end of human FVIII, and the probe spanned the predicted junction between albumin exon 1 and the 5' end of human FVIII after correct splicing. The sequences of the primers and probe were: MG101-set1-forward: 5'TAACCTTTCTCCTCCTCCTCTT3' (SEQ ID NO: 111); MG101-set1-reverse: 5'TCCACAGCTCCCAGGTAATA3' (SEQ ID NO: 112); MG101-set1-probe: 5'TCTTCTGGTGGCCAGTGCTTCTC3'FAM, ZEN / 3'IBFQ (SEQ ID NO: 113).

[0344] Total RNA was purified from the left lateral lobe of the liver of each mouse using the QIAGEN RNeasy Plus Mini Kit with a genomic DNA Eliminator column. cDNA was prepared from 500 ng of total RNA after ezDNase digestion to eliminate any remaining genomic DNA. The cDNA was assayed for albumin-FVIII fusion mRNA and cytochrome C1 mRNA. The level of cytochrome C1 mRNA was used to normalize the quality and quantity of the mRNA assayed by dividing the absolute copy number of albumin-FVIII hybrid mRNA by the absolute copy number of cytochrome C1 mRNA and expressing it as a percentage. In mice that received one of the AAV8-FVIII donor viruses (AAV8-pMG4012, AAV8-pMG4013, AAV8-pMG4014, or AV8-pMG4015) and LNPs encapsulating MG3-6 / 3-4 mRNA and guide RNA mA364-34-1, albumin-FVIII fusion mRNA levels ranged from 1% to 20% of endogenous cytochrome C1 mRNA levels ( Fig. 20Two mice had very low levels of albumin-FVIII fusion mRNA, while the remaining mice exhibited between 3% and 20% of endogenous cytochrome C1 mRNA levels. The average albumin-FVIII fusion mRNA levels were between 7% and 11%, and there were no significant differences between the groups ( Fig.21 ).

[0345] Example 10 - Evaluation of FVIII-B domain alternative sequences incorporating varying numbers of N-linked glycosylation sites and furin cleavage sites

[0346] Untreated full-length wild-type FVIII protein contains 6 domains, referred to as (in order from N-terminus to C-terminus): A1-A2-B-A3-C1-C2. During post-translational processing, the full-length FVIII protein is cleaved by furin, which results in the removal of most of the B domain and produces a mature 2-chain form of FVIII, in which the heavy chain and light chain are held together by a metal bridge. The B domain of FVIII contains most of the N-linked glycosylation sites in FVIII, but is not essential for the biological activity of the protein, and is generally not present in the recombinantly produced FVIII (commonly referred to as B domain-deleted FVIII) sold as a drug for the treatment of hemophilia A patients. In these B domain-deleted FVIII proteins, the B domain is generally referred to as a short linker sequence replacement of "SQ linker", which is derived from the N-terminus and C-terminus of the B domain, and retains the natural furin cleavage site (RHQR, SEQ ID NO: 69), thereby ensuring that the protein is processed into a natural 2-chain FVIII protein during expression.

[0347] The consensus sequence for N-linked glycosylation sites is the triplex amino acid sequence NXS / T, where X represents any amino acid, N is asparagine, and S / T represents a serine or threonine residue in the 3rd position. The glycan chain is linked to the asparagine. Not all NXS / T sequences in a protein will be glycosylated, and it is not possible to accurately predict which specific sequences will be glycosylated based on the surrounding sequences. The N6 amino acid sequence (SFSQNATNVSNNSNTSNDSNVSPPVLKRHQR, SEQ ID NO: 70) has been shown to work in mice.

[0348] like Fig. 22As shown, nine possible designs of B domain replacement sequences that meet these criteria were created. These designs, referred to as VAR1 to VAR9 (SEQ ID NOs: 71-79), contain 1 to 3 N-linked glycosylation sites and 0 to 1 amino acid changes of wild-type human FVIII. Four of these designs, VAR2 (ENRSFSQNPPVLKRHQR; SEQ ID NO: 72), VAR3 (EPRSFSQNCSQNPPVLKRHQR; SEQ ID NO: 73), VAR4 (EPRNFSQNCSQNPPVLKRHQR; SEQ ID NO: 74) and VAR8 (ENRSNFSQNCSQNPPVLKRHQR; SEQ ID NO: 78) were selected for experimental evaluation. VAR 2 and VAR 3 contain 1 N-linked glycan site and 1 amino acid difference relative to wild-type FVIII. VAR 4 contains 2 N-linked glycan sites and 1 amino acid difference relative to wild-type FVIII. VAR8 contains 3 N-linked glycan sites and 3 amino acid differences relative to wild-type FVIII. All 4 B domain replacement designs were inserted in place of the B domain in the human FVIII coding sequence along with an SQ linker (EPRSFSQNPPVLKRHQR; SEQ ID NO: 80) and an N6 glycan B domain replacement comprising the sequence ENRSFSQNATNVSNNSNTSNASNVSPPVLKRHQR (SEQ ID NO: 99) to create constructs referred to as pMG4017 (SQ linker; SEQ ID NO: 81), pMG4018 (VAR2; SEQ ID NO: 82), pMG4019 (VAR3; SEQ ID NO: 83), pMG4020 (VAR4; SEQ ID NO: 84), and pMG4021 (VAR8; SEQ ID NO: 85). It should be noted that for each of these B domain replacements (SEQ ID NOs: 71-79), only the sequence between SFSQN (SEQ ID NO: 114) and PPVLKRHQR (SEQ ID NO: 115) is part of the previously defined linker for the B domain in FVIII replacing the B domain deletion. The sequence listing includes the 3 or 4 residues before "SFSQN (SEQ ID NO: 114)" because some of the new variants contain amino acid changes or additions within this sequence before "SFSQN (SEQ ID NO: 114)". Since each of these B domain replacements contains a native furin cleavage site (RHQR (SEQ ID NO: 69)), it is expected that the FVIII proteins expressed from all of these constructs will be cleaved by furin to produce 2-chain FVIII.Aside from the differences in the B domain replacement sequence, the human FVIII coding sequence in these constructs was identical and consisted of a co1 codon-optimized DNA sequence as described below.

[0349] The human FVIII coding sequence used here lacks a signal peptide and is codon optimized, which is designed to improve expression by selecting more commonly used codons and eliminating hidden splice sites and other undesirable sequence features. When applied to the FVIII coding sequence for human cells, this sequence optimization increases the number of CpG dinucleotides in the FVIII coding sequence in the original human FVIII from 53 to 210. Since CpG dinucleotides are recognized by innate immune responses, all CpG dinucleotides are eliminated by changing any one of the adjacent codons to the sub-frequent codons that remove CpG dinucleotides. This codon optimization is designated as copt1. The overall G / C content of the FVIII sequence with the B domain deletion of the copt1 codon-optimized SQ joint is 51%, which is similar to the original human FVIII. The DNA sequence identity between the FVIII-BDD after copt1 codon optimization and the original human FVIII is about 80%. Since most DNA base changes are at the last position of a codon, 80% identity represents about 3 x 20% = 60% change of codons in the original FVIII.

[0350] Each of the constructs pMG4017, pMG4018, pMG4019, pMG4020, and pMG4021 contains the same sequence flanking the FVIII gene cassette. At the 5' end of the FVIII coding sequence, target sites for MG29-1 guide RNA 8 (mAlb29-8-50) and MG29-1 guide RNA 12 (mAlb29-12b-50) in the forward orientation are added, followed by a spacer sequence, a splice acceptor sequence, and a dinucleotide TG (required to maintain the correct reading frame after RNA splicing between albumin exon 1 and the splice acceptor site). At the 3' end of the FVIII coding sequence, after the stop codon, a polyadenylation signal, a short spacer sequence, a target site for MG29-1 guide RNA 12 (mAlb29-12b-50) in the reverse orientation are added, followed by a target site for MG29-1 guide RNA 8 (mAlb29-8-50).

[0351] AAV8 viruses were generated from constructs pMG4017, pMG4018, pMG4019, pMG4020, and pMG4021 using standard methods, and viral genome copy number was measured by PCR. 13Wild-type adult C57BL / 6 mice were IV injected with each of the AAV8 viruses (AAV8-pMG4017, AAV8-pMG4018, AAV8-pMG4019, AAV8-pMG4020, AAV8-pMG4021) at a dose of 0.5 mg / kg. Three weeks later, all mice were IV injected with hepatotrophic LNPs encapsulating mRNA of MG29-1 and guide RNA mA29-8b-50 at a dose of 0.5 mg / kg (total RNA dose per kg body weight). LNPs were prepared as described in the previous example. All mice were sacrificed 16 days after LNP administration, plasma was collected by cardiac puncture, and liver tissue was collected for purification of genomic DNA or total RNA. In the 5 groups, the editing of the whole liver ranged from 40% to 50% ( Fig.23 ), where no editing was detected in control mice injected with PBS.

[0352] Total RNA was purified from the left lateral lobe of the liver of each mouse using a QIAGEN RNeasy Plus mini kit with a genomic DNA eliminator column. cDNA was prepared from 500 ng of total RNA after ezDNase digestion to eliminate any remaining genomic DNA. The cDNA of albumin-FVIII fusion mRNA and cytochrome C1 mRNA were determined using primers MG101-set1-forward (5'TAACCTTTCTCTCCTCCTCTT3' (SEQ ID NO: 111)) and MG101-set1-reverse (5'TCCACAGCTCCCAGGTAATA3' (SEQ ID NO: 112)) and MG101-set1-probe: (5'TCTTCTGGTGGCCAGTGCTTCTC3'FAM, ZEN / 3'IBFQ (SEQ ID NO: 113)). The level of cytochrome C1 mRNA was used to normalize the quality and quantity of the measured mRNA by dividing the absolute copy number of albumin-FVIII hybrid mRNA by the absolute copy number of cytochrome C1 mRNA and expressing it as a percentage. Fig.24) showed that mice dosed with AAV8-pMG4020 had the highest levels of albumin-FVIII fusion mRNA expressed from the integrated FVIII gene cassette. All four constructs containing B domain replacements containing 1 or more N-linked glycosylation sites had higher levels of albumin-FVIII fusion mRNA than AAV8-pMG4017, in which the B domain was replaced by an SQ linker lacking an N-linked glycosylation site. These data suggest that the inclusion of 1, 2, or 3 N-linked glycosylation sites in place of the B domain of FVIII improves the expression level of mRNA. Although AAV8-pMG4020 (which contains 2 N-linked glycan sites) has a higher mRNA expression level than AAV8-pMG4018 and AAV8-pMG4019 (both of which contain 1 N-linked glycan), AAV8-pMG4021 (which contains 3 N-linked glycans) has a similar mRNA expression level to constructs with a single N-linked glycan. Therefore, more N-linked glycans do not necessarily correlate with higher mRNA expression. Based on these results, the B domain var4 replacement design present in pMG4020, which contains 2 N-linked glycans and only 1 amino acid change compared to wild-type FVIII, produced the highest levels of albumin-FVIII fusion mRNA encoding the integrated FVIII protein.

[0353] The human FVIII protein in plasma collected from the same mice 16 days after LNP administration was determined using a human FVIII-specific ELISA assay. Recombinant human FVIII (Ren Jie) spiked into the same percentage of original mouse plasma was used for the standard curve. FVIII above background in control mice injected with PBS was detected in mice administered AAV8-pMG4018, AAV8-pMG4019, AAV8-pMG4020, and AAV8-pMG4021, but not in plasma from mice administered AAV8-pMG4017 (which contains an SQ linker instead of the B domain). Therefore, the inclusion of the B domain replacement sequences var2, var3, var4, and var8 (which contain 1, 2, or 3 N-linked glycosylation sites) enables the expression of detectable levels of human FVIII ( Fig.25 The highest levels of FVIII were observed in mice receiving AAV8-pMG4020(var4), which correlated with higher levels of albumin-FVIII fusion mRNA ( Fig.24 ). AAV8-pMG4020(var4) contains a B domain replacement with 2 N-glycan sites.

[0354] Example 11 - Design of a macaque FVIII gene donor cassette and evaluation in mice

[0355] Macaque (cyno) is a recognized preclinical model, which more closely predicts the behavior of drugs in humans. Due to the difference in amino acid sequence between human and macaque FVIII protein, neutralizing antibodies are produced within the first 1 to 2 months after administration, and human FVIII protein is immunogenic when expressed or applied to non-human primates (such as macaques). The genome editing method for treating hemophilia A envisioned herein integrates the FVIII gene into the genome, with the aim of providing a lasting curative therapy from a single administration. In order to assess the persistence of this method in NHP, the FVIII gene encoding macaque FVIII protein is used. By comparing with human FVIII BDD-SQ, a B domain deletion form of long-tailed macaque FVIII protein sequence is produced, wherein the B domain is replaced by a so-called "SQ" joint, and the joint includes a sequence "SFSQNPPVLKRHQR (SEQ ID NO: 116)". Macaque FVIII has the same sequence as human FVIII around the B domain junction, so that the deletion of the B domain from macaque FVIII produces a junction identical to the SQ linker. After comparing the B domain deleted versions of human and macaque FVIII (both of which have SQ linkers), it is apparent that the macaque FVIII protein sequence has 28 amino acid differences from human FVIII. In order to be able to detect macaque FVIII proteins from integrated transgenes, a single amino acid change F2196K (lysine is indicated in bold underlined in the sequence: ASSY K TNM (SEQ ID NO: 117)) was introduced into the B domain deleted (SQ linker) macaque FVIII protein sequence (SEQ ID NO: 86). Including lysine instead of phenylalanine at residue 2196 (numbered according to full-length human FVIII) blocked the binding of the neutralizing anti-FVIII monoclonal antibody BO2C11. This enabled the activity of macaque FVIII-F2196K protein to be measured in plasma after neutralization of endogenous macaque FVIII protein with BO2C11 antibody. To generate a DNA sequence encoding macaque FVIII-F2196K, the DNA sequence encoding human B domain deleted FVIII with the var 4 B domain replacement identified in Example 10 (present in pMG4020) was modified by selecting the most frequent codons encoding macaque FVIII amino acids but not creating CG dinucleotides in the DNA sequence to change the codons of 28 amino acids that are different from macaque FVIII. In 3 cases, the previous codon was changed to avoid the creation of a CG dinucleotide (CpG). In addition, residue F2196 was changed to lysine, selecting the most frequent codon for lysine, thereby generating protein SEQ ID NO: 87. The sequence changes made to create protein SEQ ID NO: 87 are shown in Table 16.

[0356] Table 16. DNA sequence changes made to the FVIII coding sequence in pMG4020 to convert it to encode cynomolgus FVIII using F2196K.

[0357]

[0358]

[0359] Numbering: Numbering (positions) is based on mature (no signal peptide) human FVIII-BDD numbering, but after position 728, the numbering is shifted by 4 amino acids due to the inclusion of the var4 B domain substitution

[0360] For integration and expression of FVIII from albumin intron 1 in macaques, cA29-87b (guide 87) was selected for nuclease MG29-1, and ch364-58 (guide 58) was selected for nuclease MG3-6 / 3-4. The target sites for both guides were included in the donor DNA sequence flanking the FVIII donor cassette. When describing the orientation of the guide target site in the FVIII donor cassette, the orientation is relative to the orientation of the target site in the genome, so the forward orientation means the same orientation as the guide's target site in the genome, in this case, within albumin intron 1. For MG29-1, based on the studies described in Example 6, the orientation of the flanking guide had no significant effect on integration efficiency, and the forward-FVIII gene-reverse orientation was selected. For the MG29-1 guide cA29-87b, the orientation of the two guide target sites in the donor was forward (PAM to 5' side) at the 5' end of the FVIII cassette and reverse (PAM to 3' side) at the 3' end of the FVIII cassette.

[0361] In the case of the MG3-6 / 3-4 nuclease, inclusion of a guide cleavage site flanking the FVIII donor in either the reverse-FVII gene-reverse or forward-FVIII gene-reverse orientation resulted in a higher frequency of integration of the FVIII gene cassette into albumin 1 in the preferred forward orientation (see Example 9, Fig.19 Based on this data, the reverse-FVIII gene-reverse orientation of the MG3-6 / 3-4 guide was selected for use in the macaque FVIII construct. For the MG3-6 / 3-4 guide ch364-58, the orientation of the two guide target sites in the donor was reversed (PAM to 3' side) at the 5' end of the FVIII cassette and reversed (PAM to 3' side) at the 3' end of the FVIII cassette.

[0362] In addition to the guide RNA cleavage site, after splicing from albumin exon 1, the same splice acceptor sequence used in pMG4008 is inserted at the 5' end of the FVIII coding sequence, followed by the dinucleotide TG, which maintains the correct reading frame in the mRNA. The same polyadenylation signal is inserted at the 3' of the stop codon in the FVIII coding sequence. In addition, a short spacer sequence is inserted between the guide target site (TS) and the splice acceptor and between the polyA signal and the guide target site. These spacer sequences are designed to isolate the FVIII gene cassette from a small deletion at the guide cleavage site that may occur as a part of the repair process driven by NHEJ before integration.

[0363] The DNA sequence elements present in the complete macaque FVIII donor cassette designated as pMG4016 (SEQ ID NO: 88) are as follows: 5' TS of cA29-87b - TS of chA364-58 - spacer (16 bp) - splice acceptor - TG - mature macaque FVIII with var4 B domain replacement - F2196K coding sequence - stop codon - polyadenylation signal - spacer (10 bp) - TS of chA364-58 - TS of cA29-87b - 3' ( Fig.26 ).

[0364] To evaluate the macaque FVIII donor cassette in mice, pMG4016 was packaged into AAV6 or AAV8 viruses using a HEK293 (HK)-based packaging system or the sf9 insect cell packaging system (sf) using standard methods. Wild-type C57Bl / 6 mice were injected IV with 1x 10 13 vg / kg of AAV6(sf)-pMG4016, AAV8(sf)-pMG4016 or AAV8(HK)-pMG4016. 21 days later, the same mice were IV injected with 0.5 mg / kg of LNP A and 0.5 mg / kg of LN B. LNP A contains MG29-1 mRNA and mA29-8b-50 guide RNA co-formulated in a 1:1 mass ratio. LNP B contains MG29-1 mRNA and cAlb29-87b-50 guide RNA co-formulated in a 1:1 mass ratio. The guide RNA in LNP A targets mouse albumin intron 1 in the genome of the mouse. The guide RNA in LNP B targets the MG29-1 guide RNA target site flanking the FVIII gene cassette in pMG4016. Two weeks later, the mice were sacrificed and liver tissues were collected for genomic DNA purification and total RNA purification.

[0365] Editing of the albumin intron 1 target site in the liver of mice (targeted by the guide mA29-8b-50) ranged from 45% to 50% throughout the liver, indicating efficient delivery of the MG29-1 editing system and intended editing of genomic targets ( Fig. 27 ).

[0366] Integration of the macaque_FVIII gene cassette into the target site in mouse albumin intron 1 was quantified using a dd-PCR assay. Genomic DNA purified from mouse liver was digested with EcoRI. To quantify integration in the forward orientation, a primer and probe set called MG401_set3 that detects the 5' integration junction was used. The sequences of the primers and probes were: MG401-set3-forward: 5'TCTTGAGTTTGAATGCACAGAT3' (SEQ ID NO: 118); MG401-set3-reverse: 5'TAGTCCCAGCTCAGTTCCA3' (SEQ ID NO: 119); MG401-set3-probe: 5'TGGCCACCAGAAGATATTACCTGGGA3'FAM, ZEN / 3'IBFQ (SEQ ID NO: 120).

[0367] The dd-PCR assay for cytochrome C1 was used to correct the copies of genomic DNA in each sample. The results were calculated as the percentage of forward integration (the number of copies of the forward integration junction per 100 copies of cytochrome C1). Integration of the macaque FVIII gene in the forward orientation was detected in all mice, although the levels varied, ranging from about 0.1% to about 2% ( Fig.28 ). Comparing the average forward integration of 5 mice in each group receiving 3 different AAV viruses, the group receiving AAV8(HK)-pMG4016 (mice number 22 to number 25) had the highest forward integration, with an average integration rate of 1% ( Fig.28 The average integration frequency of AAV8(sf)-pMG4016 and AAV6(sf)-pMG4016 was about 0.5%.

[0368] Expression of predicted macaque FVIII encoding mRNA from the integrated macaque FVIII cassette in the liver of the same mice was quantified using a dd-PCR assay. Integration of the macaque FVIII cassette in the forward orientation (defined as the 5' end of the macaque FVIII cassette adjacent to albumin exon 1) at the double-strand break generated by the MG29-1 nuclease and guide RNA was predicted to produce a hybrid mRNA generated by RNA splicing between the albumin exon 1 splice donor and the splice acceptor at the 5' end of the macaque FVIII cassette. Therefore, this hybrid mRNA will contain a novel sequence junction between albumin exon 1 and the 5' end of the coding sequence for mature FVIII, such as Fig.13 A dd-PCR assay was designed in which the forward primer was complementary to a sequence within albumin exon 1, the reverse primer was complementary to a sequence within the 5' end of macaque FVIII, and the probe spanned the predicted junction between albumin exon 1 and the 5' end of macaque FVIII after correct splicing. The sequences of the primers and probe were: MG113-set1: MG113-set1-forward: 5'CTCTTCGTCTCCGGCTCT3' (SEQ ID NO: 121); MG113-set1-reverse: 5'TCCACAGCTCCCAGGTAATA3' (SEQ ID NO: 112); MG113-set1-probe: 5'TCTTCTGGTGGCCAGTGCTTCTC3'FAM, ZEN / 3'IBFQ (SEQ ID NO: 113).

[0369] Total RNA was purified from the left lateral lobe of the liver of each mouse. cDNA was prepared from 500ng of total RNA after DNase digestion to eliminate any remaining genomic DNA. The cDNA was assayed for albumin-FVIII fusion mRNA and cytochrome C1 mRNA. The level of cytochrome C1 mRNA was used to normalize the quality and quantity of the measured mRNA by dividing the absolute copy number of albumin-FVIII hybrid mRNA by the absolute copy number of cytochrome C1 mRNA and expressing it as a percentage. Albumin-cynomolgus FVIII fusion mRNA was detected in all mice, and the levels ranged from 2% to 40% (95% confidence interval [CI], 2.0) relative to cytochrome C1. Fig.29). The average albumin-cynomolgus FVIII fusion mRNA levels in each group of 5 mice dosed with 3 different AAV viruses were 5% for AAV6(sf)-pMG4016, 10% for AAV8(sf)-pMG4016, and 18% for AAV8(HK)-pMG4016. These mRNA levels correlated with the forward integration frequency measured in the liver of the same mice. The observation that AAV8-pMG4016 packaged in the HEK293 production system caused a higher forward integration frequency and higher levels of albumin-cynomolgus FVIII fusion mRNA suggests that differences in AAV virus quality are an important factor in optimizing integration using this gene editing approach. Overall, these results validate the pMG4016 cynomolgus FVIII cassette design.

[0370] Example 12 - Design of an additional FVIII donor sequence consisting of a human FVIII coding sequence encoding a single-chain FVIII protein

[0371] The native FVIII protein in circulation is composed of two separate protein chains, called heavy and light chains, which are generated from a single FVIII protein by post-translational cleavage by a protease called furin. To generate a single-chain version of human FVIII containing a B-domain deletion containing the var4 B-domain replacement, the furin cleavage site at the C-terminus of the B-domain replacement sequence was inactivated. The sequence of the var4 B-domain replacement sequence containing 2 N-linked glycosylation sites is: NFSQNCSQNPPVLK QUR (SEQ ID NO:100), wherein the furin cleavage site is underlined.Three of the four amino acids constituting the furin cleavage site are deleted, resulting in a sequence known as var4sc (NFSQNCSQNPPVLKR, SEQ ID NO:89).When incorporated into the FVIII of the human B domain deletion, this can create a protein with SEQ ID NO:90, wherein, relative to natural FVIII, there is 1aa substitution (S to N, creating one of 2 N polysaccharide sites), 4 amino acid whose insertion (SQNC (SEQ ID NO:122)), this is the normal sequence in the B domain of FVIII, and the disappearance (RHQ) of 3 residues.The partial reason for selecting this sequence is that it minimizes the sequence difference of the FVIII protein with natural human FVIII or the recombinant B domain deletion.

[0372] Example 13 - Design of a DNA sequence encoding a single-chain FVIII with two N-linked glycans instead of a B domain consisting of alternate codon optimization

[0373] To evaluate the function of a single-chain B-domain deleted human FVIII containing a var4sc B-domain replacement (SEQ ID NO:90), two DNA sequences encoding this protein were generated with the goal of optimizing expression. Due to redundancy in the genetic code, a given protein amino acid sequence can be encoded by a large number of unique DNA sequences, where there are several codons encoding each amino acid. On average there are about 4 codons per amino acid, with some amino acids being encoded by 2 codons and some being encoded by 5 or 6 codons, while one amino acid (methionine) is encoded by a single codon. The number of DNA sequences that can encode a given protein sequence can be calculated by multiplying the number of codons for each amino acid. For example, a protein with only 10 amino acids, where each amino acid can be encoded by 4 possible codons, can be encoded by 4 10 The number of possible DNA molecules that can encode even a short protein consisting of 100 amino acids is very large, about 2 x 10 60 (4 100 Large proteins, such as B-domain deleted FVIII (1438 amino acids), can consist of approximately 4 1438 Therefore, there are a large number of possible DNA sequences that can be used to encode the FVIII protein.

[0374] The frequency with which each codon is used in naturally produced proteins in living organisms tends to correlate with the cellular abundance of the transfer RNA (tRNA) for that codon, and it is generally accepted that codons that occur at lower frequencies can limit translation efficiency in mammalian cells due to limited concentrations of the corresponding tRNA. It is known that the frequency of codon usage between prokaryotes and eukaryotes varies significantly, and adjusting codon usage to match the codon usage of the organism in which the heterologous gene is being expressed can increase the level of protein produced, especially when genes identified in prokaryotes are expressed in eukaryotic systems, and vice versa. There are also reports that the codon usage of genes highly expressed in specific tissues (such as the liver) is different compared to the average codon usage of a large group of genes in the same organism, again indicating that codon usage was selected during evolution to enable high levels of expression. Codons that are utilized at particularly low frequencies in expressed human genes are defined herein as codons with a codon usage frequency of less than 10, based on published human codon usage tables. In this case, the codon frequency is defined as the number of times a codon is used per 1,000 amino acids.

[0375] This list of rare codons consists of the following 12 codons, where the amino acid and frequency are shown in brackets after each codon: GCG (A, 7.6), TGT (C, 9.6), CAT (H, 9.9), ATA (I, 5.5), CTA (L, 5.9), TTA (L, 5.7), CCG (P, 6.2), CGA (R, 6.1), CGT (R, 4.5), TCG (S, 4.9), ACG (T, 6.4), and GTA (V, 6.9). Alternatively, the rarest codon for each amino acid can be considered, which adds another 8 codons: GAA (E, 27.5), TTT (F, 17.1), GGT (G, 11 / .), AAA (K, 24.8), CAA (Q, 10.4), TAT (Y, 13.1), GAT (D, 22), and AAT (N, 17.5).

[0376] The native human FVIII DNA sequence in the human genome (where the B domain is artificially removed) contains 18 of these rare codons. Comparison of the codon usage frequency within the native human FVIII DNA sequence with the codon usage frequency of human genes expressed in the liver (Table 17) revealed codons that are used more or less frequently in FVIII than in genes expressed in the liver.

[0377] Table 17: Rare codons in the native human FVIII DNA sequence, where the B domain was removed resulting in a sequence encoding a 1443 amino acid protein.

[0378]

[0379]

[0380] The frequency in native FVIII is calculated as the number of occurrences of the codon in native FVIII divided by the total amino acid size of FVIII (1443 residues). #The frequency of codon usage is the frequency of occurrence per 1000 amino acids. *: The number is the number of times the codon occurs in native FVIII without the B domain. ^The frequency is the percentage of codons used for amino acids within native human FVIII without the B domain. Codon usage in FVIII that is 50% or less than the codon usage in the liver-expressed human gene is shown in bold, while those above 150% are shown in italics.

[0381] Specifically, GCT (A), GCG (A), CCG (P), CGG (R), TCG (S) and ACG (T) are used at a frequency less than 50% of their average frequency used in liver-expressed genes. In contrast, GAT (D), TTT (F), CAT (H), ATT (I), ACT (T) and GTA (V) are used in FVIII at a frequency greater than 150% of their average frequency used in liver-expressed genes. This indicates that natural FVIII contains a large number of codons that exist at frequencies different from those in the average gene expressed in the liver. This codon usage difference may have a negative impact on the ability of liver cells to express human FVIII. This provides a reason for modifying the codon usage of the artificially generated FVIII DNA sequence to improve expression without changing the encoded amino acid sequence.

[0382] The mature B domain deleted FVIII amino acid sequence containing the var4 B domain replacement was codon optimized and then all CG dinucleotides were removed using a custom designed algorithm that selects the less frequent codons for any of the codons that may span a given CG sequence. The rationale for eliminating CG dinucleotides is that CpGs can be recognized by the innate immune system as part of the cellular response to foreign viral and bacterial DNA, and therefore may have a negative impact on DNA delivery or potential expression in vivo. This algorithm removed a total of 210 CG sequences and changed approximately 210 codons in the process while retaining the same encoded amino acid sequence. The resulting DNA sequence was designed as codon optimized 1 (copt 1) and included in construct pMG4026 (SEQ ID NO: 91). Analysis of codon usage in the copt1 DNA sequence revealed that the copt1 sequence changed the frequency of specific codons compared to the native human FVIII DNA sequence. Among the 12 codons defined as rare in the human gene expressed in the liver (<10 codons / 1000 amino acids), compared with natural FVIII, the frequency of six codons in copt1 is reduced, and in natural FVIII, the CAT codon frequency is reduced by 50%, the TGT codon frequency is unchanged, and the frequency of 4 codons is already present at low frequencies (occurring 1 or 2 times in the whole sequence). The codons (i.e., frequency>10 / 1000 amino acids) that are not rare in the gene expressed in the liver with the lowest frequency for this amino acid are all reduced in the frequency in copt1 compared with natural FVIII, but only reduce by about 50% on average. These differences are summarized in Table 18.

[0383] Table 18: Comparison of codon usage of the native DNA sequence of human B-domain deleted FVIII and B-domain deleted FVIII after codon optimization 1 (copt1)

[0384]

[0385] As shown in Table 19, the copt1 sequence still contains many rare codons, which account for 27% to 48% of the codons encoding the specific amino acids. This is unlikely to be optimal for protein expression from this FVIII DNA sequence. Therefore, further reduction or elimination of these rare codons was included in a second codon optimization design called copt4.

[0386] Table 19: Rare codons present in the copt1 DNA sequence of B domain deleted FVIII

[0387]

[0388] Removing all CpG dinucleotides from sequences that have been codon-optimized using the most frequent codons particularly affects the distribution of arginine codons, because 4 of the 6 Arg codons contain CG sequences, and 2 of these are the most frequently used Arg codons. There are 70 arginine codons in the mature FVIII-BDD sequence containing the var 4B domain replacement. CpG can be recognized by the innate immune system as part of the cellular response to foreign viral and bacterial DNA. However, CpG also occurs naturally in mammalian genomic DNA, although the distribution is different from foreign DNA and the methylation pattern is different (for example, bacterial CpG is not methylated, and in eukaryotes, CpG tends to be methylated). Therefore, it may not be necessary to remove all CpG to minimize the innate immune response. Sequence elements or high-density CpGs known as CpG islands (defined as 200-bp regions of DNA with a G+C content greater than 50%) in mammalian genomes are more likely to be problematic.

[0389] The usage of arginine codons in cop1 is summarized in Table 20.

[0390] Table 20: Arginine codon usage in copt-optimized FVIII-BDD

[0391]

[0392]

[0393] The analysis of the arginine (R) codon in copt1 reveals the almost exclusive use of codon AGA (66 occurrences), wherein other codons are only used 4 times (AGG). This is contrary to the situation of natural FVIII, wherein all 6 codons are used with a bias to AGA and AGG codons. In view of the codon usage frequency of arginine in the gene expressed by the liver, which only changes in 3 times of range (4.55 to 11.11), wherein 4 of 6 codons have similar frequencies, the more even distribution of R codons in 4 most frequently used codons (AGA, AGG, CGC, CGG) may provide improved expression. Therefore, the use of adjusting the R codon is included in the second codon optimization design referred to as copt4, and the second codon optimization design necessarily increases the CG dinucleotide content. Although CpG can be immunogenic, it is also naturally present in mammalian genomic DNA, although distribution is different from foreign DNA and methylation patterns are different (for example, bacterial CpG is not methylated, and in eukaryotes, CpG tends to methylate). Therefore, it may not be necessary to remove all CpGs to minimize the innate immune response. Sequence elements or high density CpGs in mammalian genomes known as CpG islands (defined as 200-bp regions of DNA with a G+C content greater than 50%) are more likely to be problematic and are avoided. Other considerations when codon optimization of coding sequences is that some codon pairs are more frequently used in the coding region of a gene, while other codon pairs are avoided. In addition, out-of-frame TAA or TAG stop codons should be avoided if possible.

[0394] A random design approach was used to modify the copt1 sequence to ultimately create a codon-optimized copt 4 encoding a B-domain deleted FVIII containing a var4 B-domain replacement through several iterative steps. After each step, the impact on CpG content and codon usage was evaluated. The resulting DNA sequence encoding a B-domain deleted FVIII containing a var4 B-domain replacement was designated copt4 and included in construct pMG4029 (SEQ ID NO: 92). A comparison of the codon usage in copt4 with the codon usage in the native B-domain deleted FVIII is shown in Table 21.

[0395] Table 21: Comparison of codon usage in copt4 and native FVIII.

[0396]

[0397]

[0398]

[0399] Highlighted codons are rare codons (present in <10 / 1,000 amino acids in liver expressed genes). Codons in bold italics are the least frequently used codons but used in >10 / 1,000 codons in liver expressed genes.

[0400] All 12 rare codons were reduced in frequency compared to native FVIII, with only TGT and CAT remaining and the other 10 completely eliminated. Of the 6 least frequent but otherwise not rare codons for amino acids (i.e., present at >10 / 1,000 codons in the liver-expressed gene), all codons were significantly reduced in occurrence (2- to 3-fold) compared to native FVIII.

[0401] Codon usage for arginine is similar to that of native FVIII, except that the rare codon CGT, which is present in 7 of the 70 R codons in native FVIII, is eliminated. The total CG dinucleotide content of copt4 is 22, compared to 53 in native B-domain deleted FVIII. Thus, a mixture of arginine codons that is more representative of its usage in genes expressed in the liver is included, eliminating this rare arginine codon, and keeping the CpG content lower than native FVIII.

[0402] Another significant change in the copt4 sequence compared to the copt1 sequence is in the valine codon. In copt1, 78 of the 88 valine amino acids are encoded by GTG codons, which are not the most frequently used codons for valine. The most frequently used codon for valine in genes expressed in the liver (the number of codons per 1,000 amino acids is listed in brackets) is GTC (29.9), followed by GTG (15), GTT (11.3) and GTA (6.8). In copt4, 44 of the valine residues are encoded by GTC and 44 are encoded by GTG. There are 6 codons for serine, of which 3 codons (AGC, TCC, TCT) are the most frequently used in genes expressed in the liver. Although copt1 only utilized these 3 most frequent codons (compared to native FVIII which contains 23 AGT codons and 26 TCA codons), the distribution of these codons was heavily skewed towards 2 codons: AGC, which occurred 61 times, and TCT, which occurred 43 times. This was adjusted in copt 4 to be distributed between the codons AGC, TCC, and TCT, which occurred 61, 28, and 29 times, respectively.

[0403] The two DNA sequences encoding the same B domain deleted human FVIII protein containing the var4sc B domain replacement were synthesized using appropriate flanking sequences to enable expression after integration into albumin intron 1 by RNA splicing from albumin exon 1. Specifically, a splice acceptor site followed by the dinucleotide TG was included at the 5' position of the N-terminus of the FVIII coding sequence. After splicing from albumin exon one to the splice acceptor, the TG dinucleotide is required to maintain the correct reading frame. In addition, the guide target sites of MG29-1 guide 8 (mAb29-8) and MG3-6 / 3-4 guide 12 (mA364-12) and appropriate spacer sequences were placed at the 5' position of the splice acceptor. A stop codon and polyadenylation signal were added at the C-terminus of the FVIII coding sequence, followed by a spacer and the target sites of MG29-1 guide 8 (mAb29-8) and MG3-6 / 3-4 guide 12 (mA364-12). The resulting constructs were designated pMG4026 (SEQ ID NO:91) and pMG4029 (SEQ ID NO:92), wherein the codon optimization of the FVIII coding sequence used was copt1 or copt4, respectively (Table 22).

[0404] Table 22: Construct ID and description of single chain B domain deleted human FVIII design with var4 B domain replacement

[0405]

[0406] AAV8 viruses encapsulating the DNA sequences of pMG4026 and pMG4029m were produced in HEK293 cells using standard protocols. These AAV viruses can be tested in mice for integration efficiency and levels of circulating albumin-FVIII fusion protein and VIII protein using the methods described herein.

[0407] Example 14 - Design of an additional FVIII donor sequence consisting of a human FVIII coding sequence encoding a single-chain FVIII protein containing a B domain replacement with two N-linked glycosylation sites and the amino acid change F309S

[0408] Known FVIII protein is the limiting factor of FVIII protein secretion through the passage of ER-Golgi apparatus. Two DNA sequences containing var4sc B domain replacements encoding single-chain FVIII protein are modified to include serine instead of phenylalanine at position 309 (amino acid numbering is according to full-length FVIII). The resulting DNA sequence is designated as pMG4027_co1_F309S and pMG4029_co4_F309S, the former utilizing alternative codon optimization (SEQ ID NO:93) referred to as copt2, and the latter utilizing copt4 codon optimization (SEQ ID NO:94). Two sequences encode identical amino acid sequences. Both pMG4027_co2_F309S and pMG4029_co4_F309S include the same sequence of the FVIII coding sequence present in pMG4026 and pMG4029, which are necessary for splicing from albumin exon 1 after being integrated at the target site for guide.

[0409] Table 23 - List of additional protein and nucleic acid sequences mentioned herein that are not included in the sequence listing

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469] [...

Claims

1. An engineered nuclease system comprising: a) an endonuclease comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene; and c) a donor template, wherein the donor template comprises a nucleic acid sequence encoding a factor VIII (FVIII) gene or a functional fragment thereof.

2. The engineered nuclease system of claim 1, wherein the target nucleic acid sequence within the albumin gene is located within intron 1 of the albumin gene.

3. The engineered nuclease system according to any one of claims 1 to 2, wherein the sequence encoding the Factor VIII (FVIII) gene or a functional fragment thereof is linked to a splice acceptor sequence targeting exon 1 of the albumin gene.

4. The engineered nuclease system of any one of claims 1 to 3, wherein the endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO:54 or SEQ ID NO:

96.

5. The engineered nuclease system of any one of claims 1 to 3, wherein the endonuclease comprises a sequence having 100% sequence identity to SEQ ID NO:54 or SEQ ID NO:

96.

6. The engineered nuclease system according to any one of claims 1 to 5, wherein the endonuclease is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 30, 31, 53 and 95.

7. The engineered nuclease system according to any one of claims 1 to 5, wherein the endonuclease is encoded by a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 30, 31, 53 and 95.

8. The engineered nuclease system according to any one of claims 1 to 5, wherein the endonuclease is encoded by a nucleic acid sequence having 100% sequence identity to any one of SEQ ID NOs: 30, 31, 53 and 95.

9. The engineered nuclease system of any one of claims 1 to 8, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98.

10. The engineered nuclease system of any one of claims 1 to 8, wherein the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98. 11 . The engineered nuclease system of any one of claims 1 to 10 , wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.

12. The engineered nuclease system of any one of claims 1 to 10, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.

13. The engineered nuclease system according to any one of claims 1 to 10, wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1, 2 and 8.

14. The engineered nuclease system of any one of claims 1 to 10, wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-6.

15. The engineered nuclease system of any one of claims 1 to 10, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3-6.

16. The engineered nuclease system of any one of claims 1 to 10, wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 3-6.

17. The engineered nuclease system of any one of claims 1 to 16, wherein the donor template further comprises a polyadenylation signal.

18. The engineered nuclease system of any one of claims 1 to 17, wherein the donor template further comprises a nuclear targeting sequence.

19. The engineered nuclease system of claim 18, wherein the nuclear targeting sequence comprises a plurality of transcription factor binding sites.

20. The engineered nuclease system of claim 19, wherein the transcription factor is TCF1, HNF1, NFY, CEBP, OCT1, AP1, HNF1-α, HNF1-β, CEBPA, LEF-1, FOX D1, IRF1, HNF3, HNF4, HNF5, Tal1β / E47, or MyoD.

21. The engineered nuclease system of any one of claims 1 to 20, wherein the nuclear targeting sequence is located on the 5' end and the 3' end of the donor template.

22. The engineered nuclease system according to any one of claims 1 to 21, wherein the donor template further comprises a recognition site sequence for the endonuclease on the 5' end or the 3' end.

23. The engineered nuclease system of claim 22, wherein the nuclear targeting sequence is located 5' to the recognition site sequence when the donor template is flanked on the 5' end.

24. The engineered nuclease system of claim 22, wherein the nuclear targeting sequence is located 3' to the recognition site sequence when the donor template is flanked on the 3' end.

25. The engineered nuclease system of any one of claims 1 to 24, wherein the donor template comprises from 5' to 3': NTS(1)-NRS(1)-SA-FVIII-NRS(2)-NTS(2), where NTS(1) indicates the first nuclear targeting sequence; NTS(2) indicates the second nuclear targeting sequence; NRS(1) indicates the first nuclease recognition site sequence; NRS(2) indicates the second nuclease recognition site sequence; SA represents the splice acceptor sequence targeting exon 1 of the albumin gene; and FVIII represents the Factor VIII gene or a fragment thereof.

26. The engineered nuclease system of claim 25, wherein the 5' to 3' orientation of NRS (1) and NRS (2) is according to: (a) Positive, positive; (b) reverse, reverse; (c) forward, reverse; (d) reverse, forward; Therein, forward indicates the same 5' to 3' orientation as the target nucleic acid sequence, and reverse indicates the opposite 5' to 3' orientation as the target nucleic acid sequence.

27. The engineered nuclease system of any one of claims 1 to 26, wherein the donor template comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.

28. The engineered nuclease system of any one of claims 1 to 26, wherein the donor template comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.

29. The engineered nuclease system of any one of claims 1 to 26, wherein the donor template comprises a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.

30. The engineered nuclease system according to any one of claims 1 to 29, wherein the FVIII gene or a functional fragment thereof is codon optimized to remove at least one cytosine-guanine (CG or CpG) motif.

31. The engineered nuclease system according to any one of claims 1 to 30, wherein the FVIII gene or a functional fragment thereof comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 10, 71-79 and 89.

32. The engineered nuclease system according to any one of claims 1 to 30, wherein the FVIII gene or a functional fragment thereof comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 10, 71-79 and 89.

33. The engineered nuclease system according to any one of claims 1 to 30, wherein the FVIII gene or a functional fragment thereof comprises a sequence having 100% identity to any one of SEQ ID NOs: 10, 71-79 and 89.

34. The engineered nuclease system according to any one of claims 1 to 30, wherein the FVIII gene or a functional fragment thereof comprises a sequence having at least 80% identity to SEQ ID NO:

10.

35. The engineered nuclease system according to any one of claims 1 to 30, wherein the FVIII gene or a functional fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO:

10.

36. The engineered nuclease system according to any one of claims 1 to 30, wherein the FVIII gene or a functional fragment thereof comprises a sequence having 100% identity to SEQ ID NO:

10.

37. The engineered nuclease system according to any one of claims 1 to 30, wherein the FVIII gene or a functional fragment thereof is modified to comprise a B domain comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 71-79 and 89.

38. The engineered nuclease system according to any one of claims 1 to 30, wherein the FVIII gene or a functional fragment thereof is modified to comprise a B domain comprising a sequence having 100% identity to any one of SEQ ID NOs: 71-79 and 89.

39. The engineered nuclease system according to any one of claims 37 to 38, wherein the FVIII gene or a functional fragment thereof comprising a modified B domain comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 86-87 and 90.

40. The engineered nuclease system according to any one of claims 37 to 38, wherein the FVIII gene or a functional fragment thereof comprising a modified B domain comprises a sequence having 100% identity to any one of SEQ ID NOs: 86-87 and 90.

41. A method of supplementing liver enzyme expression in a subject in need thereof, the method comprising administering to the subject: a) an endonuclease comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:54 or SEQ ID NO:96; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene; and c) a donor template comprising a nucleic acid sequence encoding a Factor VIII (FVIII) gene or a functional fragment thereof, thereby supplementing liver enzyme expression in the subject.

42. The method of claim 41, wherein the target nucleic acid sequence within the albumin gene is located within intron 1 of the albumin gene.

43. The method according to any one of claims 41 to 42, wherein the sequence encoding the Factor VIII (FVIII) gene or a functional fragment thereof is operably linked to a splice acceptor sequence targeting exon 1 of the albumin gene.

44. The method of any one of claims 41 to 43, wherein the endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO:54 or SEQ ID NO:

96.

45. The method of any one of claims 41 to 43, wherein the endonuclease comprises a sequence having 100% sequence identity to SEQ ID NO:54 or SEQ ID NO:

96.

46. ​​The method according to any one of claims 41 to 45, wherein the endonuclease is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 30, 31, 53 and 95.

47. The method according to any one of claims 41 to 46, wherein the endonuclease is encoded by a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 30, 31, 53 and 95.

48. The method according to any one of claims 41 to 46, wherein the endonuclease is encoded by a nucleic acid sequence having 100% sequence identity to any one of SEQ ID NOs: 30, 31, 53 and 95.

49. The method of any one of claims 41 to 48, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98.

50. The method of any one of claims 41 to 48, wherein the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98.

51. The method of any one of claims 41 to 50, wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.

52. The method of any one of claims 41 to 50, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.

53. The method of any one of claims 41 to 50, wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.

54. The method of any one of claims 41 to 50, wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-6.

55. The method of any one of claims 41 to 50, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3-6.

56. The method of any one of claims 41 to 50, wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 3-6.

57. The method of any one of claims 41 to 56, wherein the donor template further comprises a polyadenylation signal.

58. The method of any one of claims 41 to 57, wherein the donor template further comprises a nuclear targeting sequence.

59. The method of claim 58, wherein the nuclear targeting sequence comprises a plurality of transcription factor binding sites.

60. The engineered nuclease system of claim 59, wherein the transcription factor is TCF1, HNF1, NFY, CEBP, OCT1, AP1, HNF1-α, HNF1-β, CEBPA, LEF-1, FOX D1, IRF1, HNF3, HNF4, HNF5, Tal1β / E47, or MyoD.

61. The method of any one of claims 41 to 60, wherein the nuclear targeting sequence is located on the 5' end and the 3' end of the donor template.

62. The method according to any one of claims 41 to 61, wherein the donor template further comprises a recognition site sequence for the endonuclease on the 5' end or the 3' end.

63. The method of claim 62, wherein the nuclear targeting sequence is located 5' to the recognition site sequence when the donor template is flanked on the 5' end.

64. The method of claim 62, wherein the nuclear targeting sequence is located 3' to the recognition site sequence when the donor template is flanked on the 3' end.

65. The method of any one of claims 41 to 64, wherein the donor template comprises from 5' to 3': NTS(1)-NRS(1)-SA-FVIII-NRS(2)-NTS(2), where NTS(1) indicates the first nuclear targeting sequence; NTS(2) indicates the second nuclear targeting sequence; NRS(1) indicates the first nuclease recognition site sequence; NRS(2) indicates the second nuclease recognition site sequence; SA represents the splice acceptor sequence targeting exon 1 of the albumin gene; and FVIII represents the Factor VIII gene or a fragment thereof.

66. The method of claim 65, wherein the 5' to 3' orientation of NRS (1) and NRS (2) is according to: (a) Positive, positive; (b) reverse, reverse; (c) forward, reverse; (d) reverse, forward; Therein, forward indicates the same 5' to 3' orientation as the target nucleic acid sequence, and reverse indicates the opposite 5' to 3' orientation as the target nucleic acid sequence.

67. The method of any one of claims 41 to 66, wherein the donor template comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.

68. The method of any one of claims 41 to 66, wherein the donor template comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.

69. The method of any one of claims 41 to 66, wherein the donor template comprises a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.

70. The method according to any one of claims 41 to 69, wherein the FVIII gene or a functional fragment thereof is codon-optimized to remove at least one cytosine-guanine (CG or CpG) motif.

71. The method according to any one of claims 41 to 70, wherein the FVIII gene or a functional fragment thereof comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 10, 71-79 and 89.

72. The method according to any one of claims 41 to 70, wherein the FVIII gene or a functional fragment thereof comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 10, 71-79 and 89.

73. The method according to any one of claims 41 to 70, wherein the FVIII gene or a functional fragment thereof comprises a sequence having 100% identity to any one of SEQ ID NOs: 10, 71-79 and 89.

74. The method according to any one of claims 41 to 70, wherein the FVIII gene or a functional fragment thereof comprises a sequence having at least 80% identity to SEQ ID NO:

10.

75. The method according to any one of claims 41 to 70, wherein the FVIII gene or a functional fragment thereof comprises a sequence that is at least 90% identical to SEQ ID NO:

10.

76. The method according to any one of claims 41 to 70, wherein the FVIII gene or a functional fragment thereof comprises a sequence having 100% identity to SEQ ID NO:

10.

77. The method according to any one of claims 41 to 70, wherein the FVIII gene or a functional fragment thereof is modified to comprise a B domain comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 71-79 and 89.

78. The method according to any one of claims 41 to 70, wherein the FVIII gene or a functional fragment thereof is modified to comprise a B domain comprising a sequence having 100% identity to any one of SEQ ID NOs: 71-79 and 89.

79. The method of any one of claims 77 to 78, wherein the FVIII gene or functional fragment thereof comprising a modified B domain comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 86-87 and 90.

80. The method according to any one of claims 77 to 78, wherein the FVIII gene or a functional fragment thereof comprising a modified B domain comprises a sequence having 100% identity to any one of SEQ ID NOs: 86-87 and 90.

81. A cell comprising the engineered nuclease system of any one of claims 1 to 40.

82. The cell of claim 81, wherein the cell is a liver cell.

83. The cell of claim 81, wherein the cell is a eukaryotic cell.

84. The cell of claim 81, wherein the cell is a mammalian cell.

85. The cell of claim 81, wherein the cell is an immortalized cell.

86. The cell of claim 81, wherein the cell is an insect cell.

87. The cell of claim 81, wherein the cell is a yeast cell.

88. The cell of claim 81, wherein the cell is a plant cell.

89. The cell of claim 81, wherein the cell is a fungal cell.

90. The cell of claim 81, wherein the cell is a prokaryotic cell.

91. The cell of claim 81, wherein the cell is A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, a primary cell, or a derivative thereof.

92. The cell of claim 81, wherein the cell is an engineered cell.

93. The cell of claim 81, wherein the cell is a stable cell.

94. A lipid nanoparticle (LNP) comprising components (a) and (b) or components (a), (b) and (c) of the engineered nuclease system according to any one of claims 1 to 40.

95. The lipid nanoparticle of claim 94, wherein the LNP comprises a cationic lipid, a neutral lipid, cholesterol or a cholesterol analog, and a lipid connected with PEG.

96. The lipid nanoparticle of claim 95, wherein the cationic lipid comprises C12-200 (1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)aminoidene)bis(dodecane-2-ol)), the neutral lipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or the PEG-attached lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000).

97. A viral vector comprising the engineered nuclease system of any one of claims 1 to 40.

98. The viral vector of claim 97, wherein the viral vector is an adeno-associated virus (AAV) vector.

99. The viral vector of claim 98, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV- AV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-H SC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 or their derivatives.

100. The viral vector of claim 98, wherein the AAV is AAV6.

101. The viral vector of claim 98, wherein the AAV is AAV8.

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