Genetic editing of pcsk9 or angptl3 and compositions and methods of using the same for treatment of disease

Abstract

To provide a new gene editing system capable of realizing in vivo editing.SOLUTION: An in vivo hybrid-guided gene editing system comprising: (a) a gene editor protein or a component thereof comprising a nucleic acid binding domain, or a nucleic acid encoding the gene editor protein or the component thereof; and (b) a spacer sequence comprising (i) deoxyribonucleotides and ribonucleotides comprising ribose, wherein: And (ii) a hybrid guide nucleic acid comprising a spacer sequence, wherein the 2 ' hydroxyl group of the ribose is covalently linked to a methyl group, and a binding scaffold for a gene editor protein or component thereof, wherein (a) and (b) are components comprising a pharmaceutical composition comprising a lipid nanoparticle (LNP) containing (a) or (b), wherein the LNP comprises an amino lipid, a phospholipid, a sterol, and a PEG lipid.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119 of Provisional Application No. 63 / 247,236, filed September 22, 2021, Provisional Application No. 63 / 255,333, filed October 13, 2021, and Provisional Application No. 63 / 389,679, filed July 15, 2022, the disclosures of which are incorporated by reference in their entireties herein. Summary of the Invention

[0002]

[0002] Described herein is an in vivo hybrid guide gene editing system comprising: (a) a gene editor protein or component thereof comprising a nucleic acid-binding domain, or a nucleic acid encoding the gene editor protein or component thereof; and (b) a hybrid guide nucleic acid comprising: (i) a spacer sequence comprising deoxyribonucleotides and ribonucleotides comprising ribose, wherein the 2' hydroxyl group of the ribose is covalently linked to a methyl group (2'-OMe); and (ii) a binding scaffold for the gene editor protein or component thereof, wherein (a) and (b) are components constituting a pharmaceutical composition comprising a lipid nanoparticle (LNP) containing (a) or (b), wherein the LNP comprises an amino lipid, a phospholipid, a sterol, and a PEG-lipid. In some embodiments, the spacer sequence corresponds to a protospacer on a target gene, and the target gene is ANGPTL3. In some embodiments, the gene editor protein or component thereof comprises a deaminase.

[0003]

[0003] Described herein is an in vivo hybrid guide gene editing system comprising: (a) a gene editor protein or component thereof comprising a nucleic acid-binding domain, or a nucleic acid encoding the gene editor protein or component thereof; and (b) a hybrid guide nucleic acid comprising: (i) a spacer sequence comprising deoxyribonucleotides and ribonucleotides, the spacer sequence corresponding to a protospacer on the ANGPTL3 gene; and (ii) a binding scaffold for the gene editor protein or component thereof, wherein (a) and (b) are components constituting a pharmaceutical composition comprising a lipid nanoparticle (LNP) containing (a) or (b), the LNP comprising an amino lipid, a phospholipid, a sterol, and a PEG-lipid. In some embodiments, the gene editor protein or component thereof comprises a deaminase. In some embodiments, the ribonucleotide comprises a ribose, and the ribose comprises a 2' hydroxyl group covalently linked to a methyl group (2'-OMe).

[0004]

[0004] Described herein is an in vivo hybrid guide gene editing system comprising: (a) a gene editor protein or component thereof comprising a nucleic acid binding domain and a deaminase, or a nucleic acid encoding the gene editor protein or component thereof; and (b) a hybrid guide nucleic acid comprising (i) a spacer sequence comprising deoxyribonucleotides and ribonucleotides, and (ii) a binding scaffold for the gene editor protein or component thereof, wherein (a) and (b) are components that comprise a pharmaceutical composition comprising a lipid nanoparticle (LNP) containing (a) or (b), and the LNP comprises an amino lipid, a phospholipid, a sterol, and a PEG lipid. In some embodiments, the spacer sequence corresponds to a protospacer on a target gene, and the target gene is ANGPTL3. In some embodiments, the ribo Nucleotides contain a ribose, which contains a 2' hydroxyl group covalently linked to a methyl group (2'-OMe).

[0005] In some embodiments, the spacer sequence comprises unmodified ribonucleotides. In some embodiments, the nucleic acid encoding the gene editor protein or component thereof is mRNA. In some embodiments, the gene editor protein or component thereof comprises a single fusion protein or two or more proteins. In some embodiments, the spacer sequence comprises a phosphorothioate backbone modification (PS). In some embodiments, the spacer sequence comprises a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA)(DNA) motif. In some embodiments, the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence. In some embodiments, the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 30 or 31. In some embodiments, the spacer sequence comprises a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA) motif. In some embodiments, the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence. In some embodiments, the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 28 or 29. In some embodiments, the spacer sequence comprises a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(DNA)(DNA)(DNA) motif. In some embodiments, the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(DNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence. In some embodiments, the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO: 5, 11, or 12.In some embodiments, a hybrid guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or similarity to a sequence selected from the group consisting of SEQ ID NOs: 110-113. In some embodiments, a hybrid guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to a sequence selected from the group consisting of SEQ ID NOs: 106-109. In some embodiments, a guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to a sequence selected from the group consisting of SEQ ID NOs: 79-82.

[0006] In some embodiments, the nucleic acid binding domain is capable of binding to DNA. In some embodiments, the nucleic acid binding domain is capable of binding to RNA. In some embodiments, the deoxyribonucleotides are located at positions 3, 4, 6, 7, or 8 from the 5' end of the spacer sequence. In some embodiments, the spacer sequence comprises deoxyribonucleotides at positions 3, 4, 6, and 7 from the 5' end of the spacer sequence. In some embodiments, the spacer sequence comprises deoxyribonucleotides at positions 3 and 4 from the 5' end of the spacer sequence. In some embodiments, the spacer sequence comprises deoxyribonucleotides at positions 6 and 7 from the 5' end of the spacer sequence. In some embodiments, the spacer sequence comprises deoxyribonucleotides at positions 3, 4, 6, 7, and 8 from the 5' end of the spacer sequence. In some embodiments, the spacer sequence comprises 1 to 10 deoxyribonucleotides. In some embodiments, the spacer sequence comprises 1 to 5 deoxyribonucleotides. In some embodiments, the DNA binding domain The domain comprises a CRISPR protein or a fragment thereof. In some embodiments, the DNA binding domain comprises a catalytically impaired nuclease. In some embodiments, the DNA binding domain comprises a prime editing protein or a fragment thereof.

[0007] In some embodiments, the gene editor protein or components thereof affect less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% editing of all off-target sites compared to a gene editing system comprising a corresponding gRNA that does not contain deoxyribonucleotides. In some embodiments, the gene editor protein or components thereof affect more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, or more than about 95% editing of target genes compared to a gene editing system comprising a corresponding gRNA that does not contain deoxyribonucleotides. In some embodiments, the gene editor protein or components thereof affect more than about 95%, more than about 96%, more than about 97%, more than about 98%, or more than about 99% editing of target genes compared to a gene editing system comprising a corresponding gRNA that does not contain deoxyribonucleotides. In some embodiments, the gene editor protein or component thereof affects about 95% to about 99% of editing of the target gene compared to a gene editing system comprising a corresponding gRNA that does not contain deoxyribonucleotides. In some embodiments, the LNP comprises an N-acetylgalactosamine (GalNAc) lipid acceptor targeting conjugate. In some embodiments, the target gene is expressed in the liver or cells or tissues of liver origin. In some embodiments, the target gene is expressed in a non-liver organ or cells or tissues of non-liver origin.

[0008]

[0008] Described herein are methods for treating or preventing atherosclerotic cardiovascular disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an in vivo hybrid guide gene editing system described herein. In some embodiments, the subject is a primate. In some embodiments, the primate is a human.

[0009]

[0009] Described herein is a gene editing system comprising: (a) a gene editor protein or component thereof comprising a nucleic acid binding domain, or a nucleic acid encoding the gene editor protein or component thereof; and (b) a hybrid guide nucleic acid comprising a spacer sequence, wherein the spacer sequence comprises deoxyribonucleotides and ribonucleotides comprising ribose, and the 2' hydroxyl group of the ribose is covalently linked to a methyl group (2'-OMe).

[0010]

[0010] Described herein is a gene editing system comprising: (a) a gene editor protein or component thereof comprising a nucleic acid binding domain, or a nucleic acid encoding the gene editor protein or component thereof; and (b) a hybrid guide nucleic acid comprising a spacer sequence, wherein the spacer sequence comprises deoxyribonucleotides and ribonucleotides, and the spacer sequence corresponds to a protospacer on the ANGPTL3 gene.

[0011]

[0011] Described herein is a gene editing system comprising: (a) a gene editor protein or component thereof comprising a nucleic acid binding domain and a deaminase, or a nucleic acid encoding the gene editor protein or component thereof; and (b) a hybrid guide nucleic acid comprising a spacer sequence, wherein the spacer sequence comprises deoxyribonucleotides and ribonucleotides.

[0012] A hybrid guide nucleic acid for a gene editing system, comprising: Described herein are hybrid guide nucleic acids comprising: (i) a spacer sequence comprising deoxyribonucleotides and ribonucleotides comprising ribose, wherein the 2' hydroxyl group of the ribose is covalently linked to a methyl group (2'-OMe); and (ii) a binding scaffold. In some embodiments, the spacer sequence comprises a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA)(DNA) motif. In some embodiments, the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence. In some embodiments, the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO: 30 or 31. In some embodiments, the spacer sequence comprises a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA) motif. In some embodiments, the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence. In some embodiments, the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 28 or 29. In some embodiments, the spacer sequence comprises a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(DNA)(DNA)(DNA) motif. In some embodiments, the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(DNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence. In some embodiments, the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO: 5, 11, or 12.In some embodiments, a hybrid guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or similarity to a sequence selected from the group consisting of SEQ ID NOs: 110-113. In some embodiments, a hybrid guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to a sequence selected from the group consisting of SEQ ID NOs: 106-109. In some embodiments, a guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to a sequence selected from the group consisting of SEQ ID NOs: 79-82.

[0013]

[0013] Described herein are compositions comprising a Cas9 nickase, wherein the Cas9 nickase comprises a mutation, the mutation selected from the group consisting of N692A, M694A, Q695A, H698A, K810A, K855A, K848A, K1003A, and R1060A, relative to the Cas9 nickase of SEQ ID NO: 695. In some embodiments, the Cas9 nickase comprises a sequence having at least 90% identity to SEQ ID NO: 695. In some embodiments, the Cas9 nickase comprises a sequence having at least 95% identity to SEQ ID NO: 695. In some embodiments, the Cas9 nickase comprises a sequence having at least 98% identity to SEQ ID NO: 695. In some embodiments, the Cas9 nickase comprises a sequence having at least 99% identity to SEQ ID NO: 695. In some embodiments, the Cas9 nickase comprises a sequence having at least 99.5% identity to SEQ ID NO: 695. In some embodiments, the mutations include N692A, M694A, Q695A, and H698A relative to the Cas9 nickase of SEQ ID NO: 695. In some embodiments, the mutations include K855A relative to the Cas9 nickase of SEQ ID NO: 695. In some embodiments, the mutations include K848A, K1003A, and R1060A relative to the Cas9 nickase of SEQ ID NO: 695. In some embodiments, the mutations include K810A, K1003A, R1060A relative to the Cas9 nickase of SEQ ID NO: 695.

[0014]

[0014] Described herein is a composition comprising a nucleic acid encoding a Cas9 nickase, wherein the nucleic acid encoding the Cas9 nickase comprises mutations selected from the group consisting of an AAC to GCC mutation at codon 692, an AUG to GCC mutation at codon 694, a CAG to GCC mutation at codon 695, a CAC to GCC mutation at codon 698, an AAG to GCC mutation at codon 855, an AAG to GCC mutation at codon 810, an AAG to GCC mutation at codon 848, an AAG to GCC mutation at codon 1003, and a CGG to GCC mutation at codon 1060, compared to the nucleic acid of SEQ ID NO: 694. In some embodiments, the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 90% identity to SEQ ID NO: 694. In some embodiments, the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 95% identity to SEQ ID NO: 694. In some embodiments, the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 98% identity to SEQ ID NO: 694. In some embodiments, the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 99% identity to SEQ ID NO: 694. In some embodiments, the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 99.5% identity to SEQ ID NO: 694. In some embodiments, the mutations comprise an AAC to GCC mutation at codon 692, an AUG to GCC mutation at codon 694, a CAG to GCC mutation at codon 695, and a CAC to GCC mutation at codon 698, relative to nucleic acid SEQ ID NO: 694. In some embodiments, the mutations comprise an AAG to GCC mutation at codon 855, relative to nucleic acid SEQ ID NO: 694. In some embodiments, the mutations include an AAG to GCC mutation at codon 878, an AAG to GCC mutation at codon 1003, and a CGG to GCC mutation at codon 1060, relative to nucleic acid sequence number 694.In some embodiments, the mutations include an AAG to GCC mutation at codon 810, an AAG to GCC mutation at codon 1003, and a CGG to GCC mutation at codon 1060, compared to nucleic acid sequence number 694.

[0015]

[0015] Described herein is a gene editing system comprising: (a) a Cas9 nickase; and (b) a guide RNA comprising a spacer corresponding to a protospacer sequence on a target gene and a binding scaffold for the Cas9 nickase, wherein the Cas9 nickase comprises a mutation, the mutation being selected from the group consisting of N692A, M694A, Q695A, H698A, K810A, K855A, K848A, K1003A, and R1060A compared to the Cas9 nickase of SEQ ID NO: 695. In some embodiments, the gene editing system comprises a deaminase. In some embodiments, the gene editing system comprises a polymerase. In some embodiments, the polymerase is a reverse transcriptase. In some embodiments, the target gene is PCSK9. In some embodiments, the target gene is ANGPTL3. In some embodiments, the Cas9 nickase comprises a sequence having at least 90% identity to SEQ ID NO: 695. In some embodiments, the Cas9 nickase comprises a sequence having at least 95% identity to SEQ ID NO: 695. In some embodiments, the Cas9 nickase comprises a sequence having at least 98% identity to SEQ ID NO: 695. In some embodiments, the Cas9 nickase comprises a sequence having at least 99% identity to SEQ ID NO: 695. In some embodiments, the Cas9 nickase comprises a sequence having at least 99.5% identity to SEQ ID NO: 695. In some embodiments, the mutations comprise N692A, M694A, Q695A, and H698A relative to the Cas9 nickase of SEQ ID NO: 695. In some embodiments, the mutations comprise K855A relative to the Cas9 nickase of SEQ ID NO: 695. In some embodiments, the mutations comprise K848A, K1003A, and R1060A relative to the Cas9 nickase of SEQ ID NO: 695. In some embodiments, the mutations are K810A, K1003, K2004, K3005, K4006, K5007, K6008, K7009, K810A, K9001, K9002, K9003, K9004, K9005, K9006, K9007, K9008, K9009, K9009, K9001 A, including R1060A.

[0016]

[0016] Described herein is a gene editing system comprising: (a) a nucleic acid encoding a Cas9 nickase; and (b) a guide RNA comprising a spacer corresponding to a protospacer sequence on a target gene and a binding scaffold for the Cas9 nickase, wherein the nucleic acid encoding the Cas9 nickase comprises mutations, relative to the nucleic acid of SEQ ID NO: 694, selected from the group consisting of: AAC at codon 692 to GCC, AUG at codon 694 to GCC, CAG at codon 695 to GCC, CAC at codon 698 to GCC, AAG at codon 855 to GCC, AAG at codon 810 to GCC, AAG at codon 848 to GCC, AAG at codon 1003 to GCC, and CGG at codon 1060 to GCC. In some embodiments, the gene editing system comprises a nucleic acid encoding a deaminase. In some embodiments, the gene editing system comprises a nucleic acid encoding a polymerase. In some embodiments, the polymerase is a reverse transcriptase. In some embodiments, the target gene is PCSK9. In some embodiments, the target gene is ANGPTL3. In some embodiments, the nucleic acid encoding a Cas9 nickase comprises a sequence having at least 90% identity to SEQ ID NO: 694. In some embodiments, the nucleic acid encoding a Cas9 nickase comprises a sequence having at least 95% identity to SEQ ID NO: 694. In some embodiments, the nucleic acid encoding a Cas9 nickase comprises a sequence having at least 98% identity to SEQ ID NO: 694. In some embodiments, the nucleic acid encoding a Cas9 nickase comprises a sequence having at least 99% identity to SEQ ID NO: 694. In some embodiments, the nucleic acid encoding a Cas9 nickase comprises a sequence having at least 99.5% identity to SEQ ID NO: 694. In some embodiments, the mutations include an AAC to GCC mutation at codon 692, an AUG to GCC mutation at codon 694, a CAG to GCC mutation at codon 695, and a CAC to GCC mutation at codon 698, relative to nucleic acid SEQ ID NO: 694.In some embodiments, the mutations comprise an AAG to GCC mutation at codon 855 relative to nucleic acid SEQ ID NO: 694. In some embodiments, the mutations comprise an AAG to GCC mutation at codon 878, an AAG to GCC mutation at codon 1003, and a CGG to GCC mutation at codon 1060 relative to nucleic acid SEQ ID NO: 694. In some embodiments, the mutations comprise an AAG to GCC mutation at codon 810, an AAG to GCC mutation at codon 1003, and a CGG to GCC mutation at codon 1060 relative to nucleic acid SEQ ID NO: 694.

[0017]

[0017] Nucleic acids comprising a sequence selected from the group consisting of SEQ ID NOs: 723, 725, 727 and 729 are described herein.

[0018]

[0018] Described herein are base editor proteins, wherein the base editor protein comprises a sequence selected from the group consisting of SEQ ID NOs: 724, 726, 728, and 730.

[0019]

[0019] Described herein is a pharmaceutical composition for a human subject, the pharmaceutical composition comprising: (a) an mRNA encoding a base editor protein, the base editor protein comprising a nucleic acid binding domain; (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) an LNP comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol, wherein the total amount of the guide RNA and mRNA is about 0.01 mg / kg to about 3 mg / kg. In some embodiments, the total amount of the guide RNA and mRNA is about 0.1 mg / kg to about 3 mg / kg. In some embodiments, the total amount of the guide RNA and mRNA is about 0.01 mg / kg to about 2 mg / kg. In some embodiments, the total amount of the guide RNA and mRNA is about 0. In some embodiments, the total amount of guide RNA and mRNA is about 0.01 mg / kg to about 1.5 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.1 mg / kg to about 1.5 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.01 mg / kg to about 1.25 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.1 mg / kg to about 1.25 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.01 mg / kg to about 1 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.1 mg / kg to about 1 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.1 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.2 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.3 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.4 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.5 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.6 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.7 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.8 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 0.9 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 1 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 1 mg / kg to about 3 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 1.1 mg / kg to about 3 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 1.2 mg / kg to about 3 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 1.3 mg / kg to about 3 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 1.4 mg / kg to about 3 mg / kg.In some embodiments, the total amount of guide RNA and mRNA is about 1.5 mg / kg to about 3 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 1.6 mg / kg to about 3 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 1.7 mg / kg to about 3 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 1.8 mg / kg to about 3 mg / kg. In some embodiments, the total amount of guide RNA and mRNA is about 1.9 mg / kg to about 3 mg / kg.

[0020]

[0020] Described herein is a pharmaceutical composition for a human subject, the pharmaceutical composition comprising: (a) an mRNA encoding a base editor protein, the base editor protein comprising a nucleic acid binding domain; (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (b) an LNP comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol; wherein administration of the gene editing system results in a plasma Cmax of the mRNA in the human subject of about 0.05 μg / mL to about 5 μg / mL. In some embodiments, the plasma Cmax of the mRNA in the human subject is about 0.1 μg / mL to about 5 μg / mL. In some embodiments, the plasma Cmax of the mRNA in the human subject is about 0.2 μg / mL to about 5 μg / mL. In some embodiments, the plasma Cmax of the mRNA in the human subject is about 0.5 μg / mL to about 5 μg / mL. In some embodiments, the plasma Cmax of the mRNA in a human subject is about 1 μg / mL to about 5 μg / mL. In some embodiments, the plasma Cmax of the mRNA in a human subject is about 2 μg / mL to about 5 μg / mL. In some embodiments, the plasma Cmax of the mRNA in a human subject is about 3 μg / mL to about 5 μg / mL. In some embodiments, the plasma Cmax of the mRNA in a human subject is about 4 μg / mL to about 5 μg / mL.

[0021]

[0021] Described herein is a pharmaceutical composition for a human subject, the pharmaceutical composition comprising: (a) an mRNA encoding a base editor protein, the base editor protein comprising a nucleic acid binding domain; (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) an LNP comprising an amino lipid, a PEG-lipid, a phospholipid, and a sterol; wherein administration of the gene editing system results in an AUC of the mRNA in the human subject that is between about 1 μg x h / mL and about 100 μg x h / mL. In some embodiments, the AUC of the mRNA in the human subject is between about 1 μg x h / mL and about 50 μg x h / mL. In some embodiments, the AUC of the mRNA in the human subject is between about 1 μg x h / mL and about 20 μg x h / mL. In some embodiments, the AUC of the mRNA in the human subject is between about 1 μg x h / mL and about 10 μg x h / mL. In some embodiments, the AUC of mRNA in a human subject is about 10 μg×h / mL to about 100 μg×h / mL. In some embodiments, the AUC of mRNA in a human subject is about 10 μg×h / mL to about 50 μg×h / mL. In some embodiments, the AUC of mRNA in a human subject is about 10 μg×h / mL to about 20 μg×h / mL. In some embodiments, the AUC of mRNA in a human subject is about 20 μg×h / mL to about 100 μg×h / mL. In some embodiments, the AUC of mRNA in a human subject is about 20 μg×h / mL to about 50 μg×h / mL. In some embodiments, the AUC of mRNA in a human subject is about 20 μg×h / mL to about 30 μg×h / mL. In some embodiments, the AUC of mRNA in a human subject is about 30 μg×h / mL to about 100 μg×h / mL. In some embodiments, the AUC of the mRNA in a human subject is about 30 μg×h / mL to about 50 μg×h / mL, hi some embodiments, the AUC of the mRNA in a human subject is about 50 μg×h / mL to about 100 μg×h / mL.

[0022]

[0022] Described herein is a pharmaceutical composition for a human subject, the pharmaceutical composition comprising: (a) an mRNA encoding a base editor protein, the base editor protein comprising a nucleic acid binding domain; (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) an LNP comprising an amino lipid, a PEG-lipid, a phospholipid, and a sterol; wherein administration of the gene editing system results in a plasma Cmax of the amino lipid in the human subject of about 1 μg / mL to about 100 μg / mL. In some embodiments, the plasma Cmax of the amino lipid in the human subject is about 1 μg / mL to about 50 μg / mL. In some embodiments, the plasma Cmax of the amino lipid in the human subject is about 1 μg / mL to about 30 μg / mL. In some embodiments, the plasma Cmax of the amino lipid in the human subject is about 1 μg / mL to about 20 μg / mL. In some embodiments, the plasma Cmax of the amino lipid in the human subject is about 1 μg / mL to about 10 μg / mL. In some embodiments, the plasma Cmax of the amino lipid in a human subject is about 10 μg / mL to about 100 μg / mL. In some embodiments, the plasma Cmax of the amino lipid in a human subject is about 10 μg / mL to about 50 μg / mL. In some embodiments, the plasma Cmax of the amino lipid in a human subject is about 10 μg / mL to about 30 μg / mL. In some embodiments, the plasma Cmax of the amino lipid in a human subject is about 20 μg / mL to about 100 μg / mL. In some embodiments, the plasma Cmax of the amino lipid in a human subject is about 20 μg / mL to about 50 μg / mL. In some embodiments, the plasma Cmax of the amino lipid in a human subject is about 50 μg / mL to about 100 μg / mL.

[0023]

[0023] A pharmaceutical composition for a human subject, comprising: (a) mRNA encoding a base editor protein, wherein the base editor protein comprises a nucleic acid binding domain; (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) an LNP comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol; wherein administration of the gene editing system is at a concentration of about 100 μg x h / mL to about 10,000 μg x h / mL. Described herein are pharmaceutical compositions that provide an AUC of an amino lipid in a human subject of about 100 μg×h / mL to about 5000 μg×h / mL. In some embodiments, the AUC of an amino lipid in a human subject is about 100 μg×h / mL to about 2000 μg×h / mL. In some embodiments, the AUC of an amino lipid in a human subject is about 100 μg×h / mL to about 1000 μg×h / mL. In some embodiments, the AUC of an amino lipid in a human subject is about 100 μg×h / mL to about 500 μg×h / mL. In some embodiments, the AUC of an amino lipid in a human subject is about 500 μg×h / mL to about 10000 μg×h / mL. In some embodiments, the AUC of an amino lipid in a human subject is about 500 μg×h / mL to about 5000 μg×h / mL. In some embodiments, the AUC of an amino lipid in a human subject is about 500 μg×h / mL to about 5000 μg×h / mL. In some embodiments, the AUC of the amino lipid in a human subject is about 500 μg×h / mL to about 2000 μg×h / mL. In some embodiments, the AUC of the amino lipid in a human subject is about 500 μg×h / mL to about 1000 μg×h / mL. In some embodiments, the AUC of the amino lipid in a human subject is about 1000 μg×h / mL to about 10,000 μg×h / mL. In some embodiments, the AUC of the amino lipid in a human subject is about 1000 μg×h / mL to about 5,000 μg×h / mL. In some embodiments, the AUC of the amino lipid in a human subject is about 5,000 μg×h / mL to about 10,000 μg×h / mL.

[0024]

[0024] Described herein is a pharmaceutical composition for a human subject, the pharmaceutical composition comprising: (a) an mRNA encoding a base editor protein, the base editor protein comprising a nucleic acid binding domain; (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) an LNP comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol; wherein administration of the gene editing system results in a plasma Cmax of the PEG lipid in the human subject of about 0.1 μg / mL to about 50 μg / mL. In some embodiments, the plasma Cmax of the PEG lipid in the human subject is about 0.1 μg / mL to about 25 μg / mL. In some embodiments, the plasma Cmax of the PEG lipid in the human subject is about 0.1 μg / mL to about 10 μg / mL. In some embodiments, the plasma Cmax of the PEG lipid in the human subject is about 0.1 μg / mL to about 5 μg / mL. In some embodiments, the plasma Cmax of the PEG lipid in a human subject is about 0.1 μg / mL to about 1 μg / mL. In some embodiments, the plasma Cmax of the PEG lipid in a human subject is about 1 μg / mL to about 50 μg / mL. In some embodiments, the plasma Cmax of the PEG lipid in a human subject is about 1 μg / mL to about 25 μg / mL. In some embodiments, the plasma Cmax of the PEG lipid in a human subject is about 1 μg / mL to about 10 μg / mL. In some embodiments, the plasma Cmax of the PEG lipid in a human subject is about 10 μg / mL to about 50 μg / mL. In some embodiments, the plasma Cmax of the PEG lipid in a human subject is about 10 μg / mL to about 25 μg / mL. In some embodiments, the plasma Cmax of the PEG lipid in a human subject is about 25 μg / mL to about 50 μg / mL.

[0025]

[0025] Described herein is a pharmaceutical composition for a human subject, the pharmaceutical composition comprising: (a) an mRNA encoding a base editor protein, wherein the base editor protein comprises a nucleic acid binding domain; (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) an LNP comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol, wherein administration of the gene editing system results in an AUC of the PEG lipid in the human subject that is about 10 μg x h / mL to about 5000 μg x h / mL. In some embodiments, the AUC of the PEG lipid in the human subject is about 10 μg x h / mL to about 2000 μg x h / mL. In some embodiments, the AUC of the PEG lipid in the human subject is about 10 μg x h / mL to about 1000 μg x h / mL. In some embodiments, the AUC of the PEG lipid in the human subject is about 10 μg x h / mL to about 2000 μg x h / mL. ×h / mL to about 500 μg ×h / mL. In some embodiments, the AUC of the PEG lipid in a human subject is about 10 μg ×h / mL to about 100 μg ×h / mL. In some embodiments, the AUC of the PEG lipid in a human subject is about 100 μg ×h / mL to about 5000 μg ×h / mL. In some embodiments, the AUC of the PEG lipid in a human subject is about 100 μg ×h / mL to about 2000 μg ×h / mL. In some embodiments, the AUC of the PEG lipid in a human subject is about 100 μg ×h / mL to about 1000 μg ×h / mL. In some embodiments, the AUC of the PEG lipid in a human subject is about 100 μg ×h / mL to about 500 μg ×h / mL. In some embodiments, the AUC of the PEG lipid in a human subject is about 500 μg ×h / mL to about 5000 μg ×h / mL. In some embodiments, the AUC of the PEG lipid in a human subject is about 500 μg×h / mL to about 2000 μg×h / mL. In some embodiments, the AUC of the PEG lipid in a human subject is about 500 μg×h / mL to about 1000 μg×h / mL. In some embodiments, the AUC of the PEG lipid in a human subject is about 1000 μg×h / mL to about 5000 μg×h / mL. In some embodiments, the AUC of the PEG lipid in a human subject is about 1000 μg×h / mL to about 2000 μg×h / mL. In some embodiments, the AUC of the PEG lipid in a human subject is about 2000 μg×h / mL to about 5000 μg×h / mL.

[0026] In some embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the sterol is cholesterol. In some embodiments, the LNP comprises an N-acetylgalactosamine (GalNAc) lipid receptor targeting conjugate.

[0027]

[0027] Described herein is a method for treating or preventing atherosclerotic cardiovascular disease in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described in any one of claims 116 to 219.

[0028] Incorporation by Reference

[0028] All publications, patents, and patent applications mentioned in this application are incorporated by reference in their entirety into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0029] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0030] [Figure 1]

[0030] Figure 1 shows data from an in vitro efficacy study using an LNP PCSK9 base editor formulation containing a payload of mRNA MA004 and guide RNA GA346 at a 1:1 mRNA:gRNA weight ratio and titrated at different concentrations against multiple lots of primary human hepatocyte (PHH) cells and primary cynomolgus monkey hepatocyte (PCH) cells, demonstrating the rate of PCSK9 editing in PHH compared to primary non-human primate hepatocytes (e.g., PCH). As shown in Figure 1, the administered PCSK9 base editor formulation appears to be more potent in human than non-human primate hepatocytes. [Figure 2]

[0031] Figure 1 shows a study protocol using an LNP PCSK9 base editor formulation containing a payload of mRNA MA004 and guide RNA GA346 at a 1:1 mRNA:gRNA weight ratio, with a first group of 4 NHPs intravenously infused with a single dose of 0.75 mg / kg, a second group of 22 NHPs intravenously infused with a single dose of 1.5 mg / kg, and a third group of 10 NHPs intravenously infused with a single dose of the same LNP delivery vehicle without the gRNA and mRNA drug substrate payload ("vehicle control"). Dosage is measured by the total weight of the mRNA and gRNA relative to the subject's body weight. [Figure 3]

[0032] Percent change in PCSK9 editing from baseline by liver biopsy assessing total liver DNA editing at day 15 post-infusion and PCSK9 protein reduction at day 14 post-infusion, data from the study described in Figure 2. [Figure 4] 3 is data from the study described in FIG. 2 showing PCSK9 protein reduction over an extended post-infusion period. [Figure 5] LDL-C reduction over an extended post-infusion period, data from the study described in FIG. [Figure 6] ALT enzyme levels over an extended post-infusion period, data from the study described in FIG. [Figure 7] 3 is data from the study described in FIG. 2 showing fasting glucose levels over an extended post-infusion period. [Figure 8]

[0033] Mouse test protocol shown. Each dot in the graphs shown in Figures 8-9 represents a mouse. [Figure 9] Figures 9-10 show data generated from studies supporting the durability of liver editing using the PCSK9 base editor LNP formulations described herein in regenerated PCSK9-edited liver cells after intravenous infusion. Each dot in the graphs shown in Figures 8-9 represents a mouse. [Figure 10]Data generated from studies supporting the durability of liver editing using the PCSK9 base editor LNP formulations described herein in regenerated PCSK9-edited liver cells following intravenous infusion are shown (Figures 9-10). [Figure 11]

[0034] 10 shows data from an NHP study showing no evidence of editing in sperm from sexually mature male NHPs following intravenous infusion of a PCSK9 base editor LNP formulation described herein. [Figure 12]

[0035]

[0023] Figure 1 shows the study protocol and data generated from evaluating potential off-target candidate sites for the PCSK9 base editor formulations described herein.

[0024] Figure 2 shows a schematic diagram of the experimental method used to evaluate the presence of actual off-target editing by the PCSK9 base editor formulations described herein. [Figure 13] 1 shows the study protocol and data generated from evaluating potential off-target candidate sites of the PCSK9 base editor formulations described herein, demonstrating the high sensitivity of the validation assay used. [Figure 14]

[0023] Figure 1 shows a test protocol and data generated from evaluating potential off-target candidate sites for PCSK9 base editor formulations described herein. A jitter plot generated from the off-target protocol shows that the validation assay used demonstrated no detectable off-target editing in primary human liver and spleen cells among the top 244 off-target candidate sites identified. [Figure 15]

[0023] Figure 1 shows a test protocol and data generated from evaluating potential off-target candidate sites for PCSK9 base editor formulations described herein. A jitter plot generated from the off-target protocol shows that the validation assay used demonstrated no detectable off-target editing in primary human liver and spleen cells among the top 244 off-target candidate sites identified. [Figure 16]

[0036] This figure shows data from a biodistribution study of NHPs administered a single 1 mg / kg dose of LNPs constructed with ABE mRNA MA004 and guide RNA GA346 intravenously. NHPs were euthanized 15 days after injection, and tissue samples were collected and analyzed for adenine editing in the PCSK9 gene. PCSK9 editing was observed to be distributed primarily to the liver. [Figure 17]

[0037] Figure 1 shows the efficacy of the disclosed gRNA variants directed against ANGPTL3 in human primary hepatocytes, and their on-target editing percentage of ANGPTL3 at position 6. It should be understood that "position 6," as used in the data reference position 6, refers to the sixth position counting from the 5' end of the protospacer sequence (i.e., the end opposite from the PAM). The gRNA variants were transfected into human primary hepatocytes using the methods described herein along with the ABE mRNA MA004 at the concentrations indicated therein. [Figure 18]

[0038] The potency of truncated guide RNA GA441 (n-1: GA475, n-2: GA576, n-3: GA477) and the percentage of on-target editing of ANGPTL3 at the desired editing location in human primary hepatocytes were compared with the observed corresponding reduction in potential off-target editing at selected off-target candidate sites (OT#1, OT#2, OT#3, OT#4). The gRNA truncation variants were transfected into human primary hepatocytes along with mRNA MA004 as described herein to demonstrate how such truncated variants of mRNA GA441 affect potential off-target sites more than on-target editing. [Figure 19]

[0039] Figure 1 shows the percentage of on-target editing of ANGPTL3 at position 6 in primary human hepatocytes (PHH) transfected with gRNA GA441, along with the identified ABE variant at the indicated concentration, using the methods described herein. Each sample was normalized to the respective MA004 ABE mRNA sample per concentration. Each set of columns in the graph shown corresponds to MA004, MA040, MA041, MA045, MA064, MA065, MA066, and MA067, in order read from left to right, as further indicated by the hash marks in the legend. [Figure 20A]

[0040] Figure 1 shows data generated using various ANGPTL3 gRNAs and the ABE mRNA, mRNA MA004 and its variants MA040, MA041, and MA045, demonstrating the relative efficacy of on-target editing for each ANGPTL3 gRNA and mRNA combination in transfected human primary hepatocytes at the indicated concentrations. Results are from Experiment 1, using MA004 encoding ABE8.8, MA040 encoding ABE8.8 D1135E, and MA041 encoding ABE8.8 R691A / D1135E. ANGPTL3 is targeted by gRNAs GA441, GA442, GA472, GA473, GA474, GA475, GA476, and GA477, as specified in the gRNA legend, shown in order from left to right on the graph. [Figure 20B]Figure 1 shows data generated using various ANGPTL3 gRNAs and the ABE mRNA, mRNA MA004 and its variants MA040, MA041, and MA045, demonstrating the relative efficacy of on-target editing for each ANGPTL3 gRNA and mRNA combination in transfected human primary hepatocytes at the indicated concentrations. Results are from Experiment 1, using MA004 encoding ABE8.8, MA040 encoding ABE8.8 D1135E, and MA041 encoding ABE8.8 R691A / D1135E. ANGPTL3 is targeted by gRNAs GA441, GA442, GA472, GA473, GA474, GA475, GA476, and GA477, as specified in the gRNA legend, shown in order from left to right on the graph. [Figure 20C] Figure 1 shows data generated using various ANGPTL3 gRNAs and the ABE mRNA, mRNA MA004 and its variants MA040, MA041, and MA045, demonstrating the relative efficacy of on-target editing for each ANGPTL3 gRNA and mRNA combination in transfected human primary hepatocytes at the indicated concentrations. Results are from Experiment 1, using MA004 encoding ABE8.8, MA040 encoding ABE8.8 D1135E, and MA041 encoding ABE8.8 R691A / D1135E. ANGPTL3 is targeted by gRNAs GA441, GA442, GA472, GA473, GA474, GA475, GA476, and GA477, as specified in the gRNA legend, shown in order from left to right on the graph. [Figure 20D] Figure 1 shows data generated using various ANGPTL3 gRNAs and ABE mRNAs, mRNA MA004 and its variants MA040, MA041, and MA045, demonstrating the relative efficacy of on-target editing for each combination of ANGPTL3 gRNA and mRNA in transfected human primary hepatocytes at the indicated concentrations. Results are from Experiment 2, using MA004, encoding ABE8.8, and MA045, encoding ABE8.8 R691A. [Figure 20E] Figure 1 shows data generated using various ANGPTL3 gRNAs and ABE mRNAs, mRNA MA004 and its variants MA040, MA041, and MA045, demonstrating the relative efficacy of on-target editing for each combination of ANGPTL3 gRNA and mRNA in transfected human primary hepatocytes at the indicated concentrations. Results are from Experiment 2, using MA004, encoding ABE8.8, and MA045, encoding ABE8.8 R691A. [Figure 21]

[0041] 1 is a comparative graph showing the on-target editing rate of PCSK9 in human primary hepatocytes transfected with a reference gRNA variant and ABE mRNA MA004 at the four indicated concentrations. The graph shows the relative efficacy of different gRNAs in human primary hepatocytes. From left to right on the graph, the gRNA variants at the four concentrations are GA346, GA376, GA377, GA380, GA381, GA382, GA383, GA384, GA385, GA386, GA387, GA388, GA389, and GA391, respectively. [Figure 22]

[0042] 1 is a comparative graph showing the on-target editing rate of PCSK9 in cynomolgus monkey primary hepatocytes transfected with a reference gRNA variant and ABE mRNA MA004 at the four indicated concentrations. The graph shows the relative efficacy in primary hepatocytes between the spacer of gRNA GA346 embodied in the reference gRNA variant and tracr chemical variants. From left to right on the graph, the gRNA variants at the four concentrations are GA346, GA376, GA377, GA380, GA381, GA382, GA383, GA384, GA385, GA386, GA387, GA388, GA389, and GA391, respectively. [Figure 23]

[0043] This is a comparative graph showing the on-target editing rate of PCSK9 in primary hepatocytes transfected with ABE mRNA M004 in combination with gRNAs GA376, GA377, GA380-GA389, GA391, and GA066 (left to right) at guide concentrations ranging from 312.5 ng / TA / mL to 2500 ng / TA / mL. Four different lots of GA066 were used in this study. GA376, GA377, GA380, GA383, GA385, and GA386 showed a dose response comparable to that of GA066. This graph shows the relative potency among gRNAs in human primary hepatocytes. [Figure 24]

[0044] 1 is a table showing predicted exposure of LNPs composed of ABE mRNA MA004 and guide RNA GA346 in humans. Pharmacokinetic ("PK") modeling using the NHP data disclosed herein was used to predict exposure correlations and potential safety margins in human subjects. Abbreviations: AUC = area under the plasma concentration-time curve; plasma C = maximum plasma concentration; NHP = non-human primate. The following assumptions were used for modeling: (a) Gradual infusion: The infusion rate (in terms of volume) for the first 15 minutes is 1 mL / min. For the remaining infusion time, the infusion rate is 3 mL / min. (b) Predicted exposure for a 75 kg person. (c) For NHPs, 2 mg / kg (single dose) plasma C and AUC were mean values. [Figure 25]

[0045] 1 is a table showing percent PCSK9 reduction in human subjects at different doses of ABE mRNA MA004 and guide RNA 346, formulated into LNPs described herein based on predictive PK-PD modeling and disrupted with equivalent and 3-fold greater efficacy in humans than in NHPs, depending on the body weight of the human subject. % PCSK9 reduction = 100% - % PCSK9 remaining relative to baseline. This is only one way to predict human dosing and other factors may affect the particular mRNA used and assay performed. [Figure 26]

[0046] Figure 1 shows the relative editing rate of the potential off-target site B2 in primary human hepatocytes transfected with reference ABE mRNA and the indicated ANGPTL3 guide RNAs GA441, GA475, and GA476. The relative editing rate for each sample was normalized to the GA441 / MA004 sample at site B2. [Figure 27]

[0047] Figure 1 shows the relative editing rate of the potential off-target site B6 in primary human hepatocytes transfected with reference ABE mRNA and the indicated ANGPTL3 guide RNAs GA441, GA475, and GA476. The relative editing rate for each sample was normalized to the GA441 / MA004 sample at site B6. [Figure 28A]

[0048] 1 shows an exemplary scheme of a gene editing system including a guide nucleic acid. The guide nucleic acid includes a spacer sequence having a first exemplary motif. The gRNAs GA837, GA693, and GA749 disclosed herein are embodiments of this motif. [Figure 28B]

[0049]

[0023] Figure 1 shows another exemplary scheme of a gene editing system comprising a guide nucleic acid. The guide nucleic acid comprises a spacer sequence having a second exemplary motif. The gRNAs GA745, GA692, and GA748 disclosed herein are embodiments of this motif. [Figure 28C]

[0050]

[0023] Figure 1 shows another exemplary scheme of a gene editing system comprising a guide nucleic acid. The guide nucleic acid comprises a spacer sequence having a third exemplary motif. The gRNAs GA682, GA723, and GA746 disclosed herein are embodiments of this motif. [Figure 28D]

[0051]

[0023] Figure 1 shows another exemplary scheme of a gene editing system comprising a guide nucleic acid. The guide nucleic acid comprises a spacer sequence having a fourth exemplary motif. The gRNAs GA691, GA724, and GA747 disclosed herein are embodiments of this motif. [Figure 29]

[0052] This figure shows the target editing rate of the target gene ANGPTL3 resulting from an initial on-target in vitro screening of guide nucleic acids containing spacer sequences (guide nucleic acids GA675-GA684) under MessengerMax transfection conditions. GA441 was used as a control. Primary human hepatocyte (PHH) cells (lot STL) were used for this study. Each guide was screened at concentrations of 5,000 ng / mL, 2,500 ng / mL, 1,250 ng / mL, and 625 ng / mL, and the results are represented in columns in descending order of concentration from left to right for each guide in the graph. [Figure 30]

[0053] This figure shows the target editing rate of the target gene ANGPTL3 resulting from an initial on-target screening of guide nucleic acids containing spacer sequences (guide nucleic acids GA685-GA694) and control guide RNA GA441 under MessengerMax transfection conditions in PHH cells (lot STL). Each guide was screened at concentrations of 5,000 ng / mL, 2,500 ng / mL, 1,250 ng / mL, and 625 ng / mL, and the results are represented in columns in descending order of concentration from left to right for each guide in the graph. [Figure 31]

[0054] This shows the results of in vitro off-target (OT1) editing based on the ANGPTL3 guide nucleic acid containing the spacer sequence GA675-GA684 and the control guide RNA GA441 under MessengerMax transfection conditions in PHH cells (lot STL). [Figure 32]

[0055] This shows the results of in vitro off-target (OT1) editing based on the ANGPTL3 guide nucleic acid containing the spacer sequence GA685-GA694 and the control guide RNA GA441 under MessengerMax transfection conditions in PHH cells (lot STL). [Figure 33]

[0056] 1 shows results from an initial in vitro on-target screen of ANGPTL3 guide nucleic acids containing the additional spacer sequence of GA695-GA715 and control guide RNA GA441 under MessengerMax transfection conditions in PHH cells (lot STL). [Figure 34]

[0057] This figure shows in vitro on-target and selected off-target (OT1 and OT3 sites) editing results from a follow-up experiment using guide nucleic acids containing selected spacer sequences (GA675, GA677, GA678, GA680, GA682, GA683, GA685-GA695) and the ANGPTL3 control guide RNA GA441 via MessengerMax transfection in PHH cells (lot IRZ). [Figure 35A]

[0058] Figure 1 shows the results of SureSelect in vitro testing of on-target and off-target (OT1, OT2, OT3, and other OT) editing using 50 ng DNA input of 16 ANGPTL3 guide nucleic acids containing selected spacer sequences and the ANGPTL3 control guide RNA GA441 under MessengerMax transfection conditions in PHH cells (lot IRZ). [Figure 35B]

[0059] Figure 1 shows the results of SureSelect in vitro testing of on-target and off-target (OT1, OT2, OT3, and other OT) editing using 150 ng DNA input of 10 ANGPTL3 guide nucleic acids containing selected spacer sequences and ANGPTL3 control guide RNA GA441 under MessengerMax transfection conditions in PHH cells (lot IRZ). [Figure 36A]

[0060] Figure 1 shows results from an LNP transfection experiment using various ANGPTL3 guides. Figure 2 shows on-target in vitro editing results from the use of LNP for transfection in PHH cells (lot IRZ) at two different doses of LNP. [Figure 36B]Results from LNP transfection experiments using various ANGPTL3 guides are shown. Except for the control, off-target editing from the same LNP transfection experiment using only one dose is shown. The LNPs used in these studies are listed in Table 5. [Figure 37]

[0061]

[0039] Figure 5 shows the results of SureSelect in vitro studies of ANGPTL3 on-target and off-target (OT1, OT2, OT3, and other OT) editing obtained from LNP transfection experiments in PHH cells (lot IRZ). The LNPs used in these studies are listed in Table 5. [Figure 38A]

[0062] A subset of SureSelect in vitro on-target and off-target (OT1, OT2, OT3, and other OT) editing results from LNP transfection experiments in PHH cells (lot IRZ) are shown. The LNPs used in these studies are listed in Table 5. [Figure 38B]

[0063] Targeted amplicon in vitro on-target and OT1 editing results from the same subset of data from the LNP transfection experiment are shown. [Figure 39A]

[0064] Shown is a subset of SureSelect in vitro on-target and off-target (OT1, OT2, OT3 and other OT) editing results from a MessengerMax transfection experiment in PHH cells (lot IRZ). [Figure 39B]

[0065] Targeted amplicon in vitro on-target and off-target OT1 and OT3 editing results from the same subset of data from the MessengerMax transfection experiment are shown. [Figure 40]

[0066]

[0049] Figure 1 shows the results of an in vitro on-target dose-response study of ANGPTL3 editing produced by different LNP formulations containing spacer-modified ANGPTL3 guide nucleic acids in PHH cells (Lot IRZ) evaluated using targeted amplicon sequencing. The three digits after the decimal point following the formulation, gRNA, and mRNA ID indicate the lot or batch number of each formulation, gRNA, and mRNA. The LNPs used in these studies are listed in Table 5. [Figure 41]

[0067] Figure 41A shows a comparison of off-target editing percentage data generated by different LNP formulations containing ANGPTL3 guide nucleic acids with various modified spacer sequences as specified and described herein. Figure 41A shows in vitro off-target editing results using OT1 primers. The LNPs used in these studies are listed in Table 5. Figure 41B shows off-target editing results using OT3 primers. The three digits after the decimal point following the formulation, gRNA, and mRNA IDs indicate the lot or batch number of the respective formulation, gRNA, and mRNA. The LNPs used in these studies are listed in Table 5. [Figure 42]

[0068]

[0023] Figure 5 shows the results of an in vitro on-target dose-response study of ANGPTL3 editing using LNPs containing GA837 and MA079 in PHH cells (lots NFX, GNA, IRZ, and LFQ) from four different donors assessed using targeted amplicon sequencing. The LNP (VF1542) is listed in Table 5. [Figure 43A]

[0069] Figure 1 shows in vitro on-target ANGPTL3 editing results (as net editing of treated samples minus untreated samples) for LNPs containing GA837 and MA079 transfected at a single dose in PHH cells (lot IRZ) for two biological replicates. The average % net editing shown above each bar is derived from two technical replicates. LNP (VF1542) is listed in Table 5. [Figure 43B]

[0070] Figure 43A shows an OGM (optical genome mapping) analysis of genomic DNA from one replicate (replicate 1) of the same subset of ANGPTL3 editing data. Circos plots show all SV (structural variation) categories (insertion, duplication, deletion, inversion, and translocation) for individual untreated (left) and ABE-treated (i.e., LNP(VF1542)-treated) (center) samples. The Bionano Dual analysis pipeline was used to identify SVs unique to the treatment condition. No particularly unique SVs were identified, as shown by the Circos plot of the distinct ABE-untreated control (right). The gRNA, mRNA, and LNP formulations are the same as in Figure 43A. [Figure 44A]

[0071] 1 shows hepatic ANGPTL3 on-target editing by selected (cynomolgus monkey equivalent) spacer-modified guide nucleic acids GA347, GA663, GA666, GA668, GA665, GA720, and mRNA MA004 containing the same spacer sequence in LNP-treated NHPs. [Figure 44B] The corresponding ANGPTL3 protein reduction at day 15 after a single IV dose of LNPs is shown. The LNPs used in this study are listed in Table 6. [Figure 45]

[0072] The in vitro on-target editing rates of selected (cynomolgus monkey equivalent) spacer-modified guide nucleic acids GA347, GA663, GA666, GA668, GA665, and GA720 in LNPs in PCH are shown. The corresponding human gRNA IDs with equivalent spacer sequences are listed below. The LNPs used in this study are listed in Table 6. [Figure 46]

[0073] This figure shows on-target liver ANGPTL3 editing in NHPs after a single IV injection of LNPs constructed with selected spacer-modified guide nucleic acids GA347, GA668, GA748, and GA749, which contain the same spacer sequence, and mRNA MA079. Editing was assayed 15 days after injection in cynomolgus monkeys, achieving greater than 54% liver ANGPTL3 editing. The LNPs used in this study are listed in Table 6. [Figure 47]

[0074] This shows that spacer-modified guide nucleic acids containing the same spacer sequence achieved a significant reduction in plasma ANGPTL3 protein 15 days after NHP administration. All spacer-modified guide nucleic acids resulted in a greater than 90% reduction in plasma ANGPTL3 protein at all dose levels evaluated. The LNPs used in this study are listed in Table 6. DETAILED DESCRIPTION OF THE INVENTION

[0031]

[0075] Certain details of the present description are set forth to provide a more thorough understanding of various aspects and embodiments. However, those skilled in the art will understand that the present disclosure may be practiced without certain details disclosed herein. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0032]

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in practicing or testing the present disclosure, suitable methods and materials are described below. Furthermore, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, the compositions of the present disclosure can be used to achieve the methods of the present disclosure.

[0033] definition

[0077] To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0034]

[0078] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0035]

[0079] Also, note that the term "or" is generally used in its sense, including "and / or," unless the context clearly dictates otherwise. The terms "and / or" and "any combination thereof," and their grammatical equivalents used herein, may be used interchangeably. These terms may convey that any combination is specifically contemplated. For illustrative purposes only, the phrase "A, B, and / or C" or "A, B, C, or any combination thereof" below may mean "individually A, individually B, individually C, A and B, B and C, A and C, and A, B, and C." The term "or" may be used conjunctively or disjunctively, except where the context specifically dictates disjunctive use.

[0036]

[0080] The term "about" or "approximately" can mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, in accordance with practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or more preferably within 2-fold of a value. Where specific values ​​are described in this application and claims, unless otherwise specified, the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0037]

[0081] As used in this specification and claim(s), the words "comprising" (and any form of "comprise" such as "comprise" and "comprises"), "having" (and any form of "having" such as "have" and "has"), "including" (and any form of "includes" and "include") or "containing" (and any form of "containing" such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0038]

[0082] As used herein, a reference to "some embodiments," "an embodiment," "one embodiment," "multiple embodiments," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0039]

[0083] The term "nucleic acid," as used herein, refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form, including DNA and RNA, hybrids of DNA and RNA, and combinations thereof. The term "nucleic acid," as used herein, also refers to a polymer containing at least two chemically modified nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form, including DNA and RNA, hybrids of DNA and RNA, and combinations thereof.

[0040]

[0084] The term "nucleotide" refers to a molecule containing the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group. "Base" includes purines and pyrimidines, and further includes the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogs, as well as synthetic derivatives of purines and pyrimidines, including modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0041]

[0085] Nucleic acids include any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides commonly referred to as oligonucleotides, and longer fragments referred to as polynucleotides. Deoxyribo-oligonucleotides consist of a five-carbon sugar called deoxyribose, covalently linked to phosphate at the 5' and 3' carbons of this sugar to form alternating, unbranched polymers. Ribo-oligonucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose. Thus, the terms "polynucleotide" and "oligonucleotide" can refer to polymers or oligomers of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. In addition, nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, non-standard, and / or non-naturally occurring and have similar binding properties to the reference nucleic acid. Nucleic acids can be modified at the base moiety (e.g., one or more atoms typically available to form hydrogen bonds with a complementary nucleotide, and / or one or more atoms typically incapable of forming hydrogen bonds with a complementary nucleotide), sugar moiety, or phosphate backbone. Backbone modifications can include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and phosphorodiamidate linkages. Phosphorothioate linkages replace sulfur atoms in the phosphate backbone, retarding nuclease degradation of oligonucleotides. Phosphorodiamidate linkages (N3'→P5') prevent nuclease recognition and degradation. Backbone modifications can also include peptide bonds (e.g., N-(2-aminoethyl)-glycine units linked by peptide bonds in peptide nucleic acids) in place of phosphorus in the backbone structure, or linking groups including carbamate, amide, and linear and cyclic hydrocarbon groups. Oligonucleotides with modified backbones are described in Micklefield, Backbone modification of nucleic acids: synthesis, structure, and structure. and therapeutic applications, Curr. Med. Chem., 8(10):1157-79, 2001 and Lyer et al., Modified oligonucleotides - synthesis, properties and applications, Curr. Opin. Mol. Ther., 1(3):344-358, 1999.

[0042]

[0086] The nucleic acid molecules described herein contain sugar moieties including ribose or deoxyribose, as found in naturally occurring nucleotides, or modified sugar moieties or sugar analogs. Modified sugar moieties may include, but are not limited to, 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-aminoethyl, 2'-fluoro, N3'→P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic-modified sugars. 2'-O-methyl or 2'-O-methoxyethyl modifications promote A-form or RNA-like conformations in oligonucleotides, increasing binding affinity to RNA and enhancing nuclease resistance. Modified sugar moieties may also include those with extra bridges (e.g., a methylene bridge connecting the 2'-O and 4'-C atoms of ribose in locked nucleic acids) or sugar analogs such as morpholine rings (e.g., phosphorodiamidate morpholinos).Examples of such analogs and / or modified residues include diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl Examples of suitable nucleotides include, but are not limited to, thyl-2-thiouracil, beta-D-mannosylqueosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, methylphosphonate, chiral-methylphosphonate, 2'-O-methylribonucleotide, peptide nucleic acid (PNA), etc. In some cases, the nucleotide may contain a modification in its phosphate moiety, including a modification to the triphosphate moiety. Non-limiting examples of such modifications include modifications with longer phosphate chains (e.g., phosphate chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphate). Such modified or substituted oligonucleotides are often preferred over native forms due to properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.Thus, the terms "polynucleotide" and "oligonucleotide" can also include polymers or oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly.

[0043]

[0087] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., 1999). al., Nucleic Acid Res., 19:5081 (1991), Ohtsuka et al., J. Biol. Chem., 260: 2605-2608 (1985), Rossolini et al., Mol. Cell. Probes, 8: 91-98 (1994)).

[0044]

[0088] The term "nucleoside" refers to a compound consisting of a base combined with deoxyribose or ribose. Nucleosides include, but are not limited to, ribonucleosides and deoxyribonucleosides. Nucleosides can be phosphorylated to yield nucleotides. Ribonucleosides include adenosine (A), guanosine (G), 5-methyl- ... Thiouridine (m 5Deoxyribonucleosides include deoxyadenosine (dA), deoxyguanosine (dG), deoxythymidine (dT), deoxyuridine (dU), and deoxycytidine (dC). The deoxyribose or ribose (i.e., the sugar moiety) can be modified. Examples of modified sugar moieties include, but are not limited to, 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-aminoethyl, 2'-fluoro, N3'→P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic-modified sugars. 2'-O-methyl or 2'-O-methoxyethyl modifications promote an A-form or RNA-like conformation in oligonucleotides, increasing binding affinity to RNA and enhancing nuclease resistance. Modified sugar moieties can also include those with extra bridges (e.g., a methylene bridge connecting the 2'-O and 4'-C atoms of ribose in locked nucleic acids) or sugar analogs such as morpholine rings (e.g., phosphorodiamidate morpholino).Examples of such analogs and / or modified residues include diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl These include, but are not limited to, chiral-2-thiouracil, beta-D-mannosylqueosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, methyl phosphonate, chiral-methyl phosphonate, 2'-O-methyl ribonucleotide, peptide nucleic acid (PNA), and the like.

[0045]

[0089] The terms "deoxyribonucleotide" and "2'-deoxyribonucleotide" are used interchangeably and refer to both unmodified and chemically modified deoxyribonucleotides, unless otherwise specified.

[0046]

[0090] The term "motif," as used herein, refers to a particular pattern or arrangement of nucleotides; for example, a motif may refer to (i) one or more 2'-deoxyribonucleotides within a spacer, (ii) a combination of one or more 2'-deoxyribonucleotides and one or more ribonucleotides within a spacer, (iii) a combination of one or more 2'-deoxyribonucleotides, one or more ribonucleotides, and one or more 2'-OMe ribonucleotides within a spacer (wherein the 2'-OMe ribonucleotides can be at the 5' or 3' end of the 2'-deoxyribonucleotides), (iv) a combination of one or more 2'-deoxyribonucleotides and one or more 2'-OMe ribonucleotides within a spacer (wherein the 2'-OMe ribonucleotides can be positioned / located at the 5' or 3' end of the 2'-deoxyribonucleotides), or (v) any of the motifs (i-iv) disclosed above further comprising a phosphonothioate backbone.

[0047]

[0091] The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some instances, vectors are expression vectors capable of directing the expression of nucleic acids to which they are operatively linked. The term "operably linked," as used herein, means that the nucleotide sequence of interest is linked to a regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. The term "regulatory sequence," as used herein, includes, but is not limited to, promoters, enhancers, and other expression control elements. Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Examples of expression vectors include, but are not limited to, plasmid vectors, vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, viral vectors based on retroviruses (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors.

[0048]

[0092] As used herein, the terms "off-targeting," "off-site targeting," or "off-target effect" refer to when a spacer of a guide nucleic acid binds to a sequence in a genome other than the target sequence to which the spacer is designed to specifically bind. The resulting unintended binding will cause unintended editing of a gene other than the target gene.

[0049]

[0093] As used herein, the terms "protein," "polypeptide," and "peptide" are used interchangeably and refer to a polymer of amino acid residues linked via peptide bonds and may be composed of two or more polypeptide chains. The terms "polypeptide," "protein," and "peptide" refer to a polymer of at least two amino acid monomers connected together via amide bonds. The amino acids may be the L-optical isomer or the D-optical isomer. More specifically, the terms "polypeptide," "protein," and "peptide" refer to a molecule composed of two or more amino acids in a specific order (e.g., the order determined by the base sequence of nucleotides in a gene or RNA encoding the protein). Proteins are essential for the structure, function, and regulation of cells, tissues, and organs of the body, and each protein has a unique function. Examples include hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein may be a portion of a protein, such as a protein domain, subdomain, or motif. In some cases, a protein may be a variant (or mutation) of a protein, in which one or more amino acid residues are inserted into, deleted from, and / or substituted into the naturally occurring (or at least known) amino acid sequence of the protein. The protein or its variant may be naturally occurring or recombinant. Methods for detecting and / or measuring polypeptides in biological materials are well known in the art and include, but are not limited to, Western blotting, flow cytometry, ELISA, RIA, and various proteomics techniques. An exemplary method for measuring or detecting polypeptides is an immunoassay such as an ELISA. This type of protein quantification can be based on an antibody capable of capturing a specific antigen and a second antibody capable of detecting the captured antigen. An exemplary assay for detecting and / or measuring polypeptides is described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

[0050]

[0094] As used herein, the term "sequence identity" refers to the amount of exact nucleotide or amino acid matches between two different sequences. When comparing RNA and DNA sequences, uracil and thymine bases are considered to be the same base. Gaps are No count is given, and measurements are typically relative to the shorter of the two sequences. For example, for a nucleotide sequence: A:AAGGCTT B:AAGGC C:AAGGCAT where identity(A,B)=100% (5 identical nucleotides / min(length(A), length(B))). Identity(B,C)=100%, but identity(A,C)=85% (6 identical nucleotides / 7). Therefore, 100% identity does not mean that the two sequences are the same. For amino acid sequences, A:Ala Ala Gly Gly Gln His His B: Ala Ala Gly Gly Gln C: Ala Ala Gly Gly Gln Ala His where identity(A,B)=100% (5 identical amino acids / min(length(A), length(B))). Identity(B,C)=100% but identity(A,C)=85% (6 identical amino acids / 7).

[0051]

[0095] As used herein, the term "sequence similarity" can be described as an optimal matching problem that finds the minimum number of editing operations (insertions, deletions, and substitutions) to transform one sequence into an exact copy (edit distance) of another sequence to be aligned. Using this, the sequence similarity percentages for the example above are sim(A,B) = 60%, sim(B,C) = 60%, and sim(A,C) = 86% (semi-global, sim = 1 - (edit distance / unaligned length of the shorter sequence)).

[0052]

[0096] A "subject" in need thereof refers to an individual who has a disease, a symptom of a disease, or a predisposition toward a disease, and for whom the goal is to cure, heal, alleviate, relieve, alter, treat, improve, ameliorate, or affect the disease, symptom of a disease, or predisposition toward a disease. In some embodiments, the subject has hypercholesterolemia. In some embodiments, the subject has atherosclerotic vascular disease. In some embodiments, the subject has hypertriglyceridemia. In some embodiments, the subject has diabetes. The term "subject" or "patient" encompasses mammals. Examples of mammals include, but are not limited to, any member of the mammalian genus, non-human primates such as humans, chimpanzees, and other ape and monkey species; farm animals such as cows, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as rats, mice, and guinea pigs.

[0053]

[0097] The term "condition" as used herein includes disease, disorder, and susceptibility. In some embodiments, the condition is atherosclerotic vascular disease. In some embodiments, the condition is hypertriglyceridemia. In some embodiments, the condition is diabetes.

[0054]

[0098] The term "atherosclerosis" or "atherosclerotic vascular disease," as used herein, refers to a narrowing of the inside of an artery due to plaque buildup, which in some cases can lead to coronary artery disease, stroke, peripheral artery disease, or kidney problems.

[0055]

[0099] The term "hypertriglyceridemia" as used herein refers to the condition of fatty acids, which are the most abundant fat molecules in most organisms. Hypertriglyceridemia refers to high blood levels of triglycerides (hyper-emia). Elevated triglyceride levels, even in the absence of hypercholesterolemia (high cholesterol levels), may be associated with atherosclerosis and may predispose to cardiovascular disease. Extremely high triglyceride levels may increase the risk of acute pancreatitis. Hypertriglyceridemia may be associated with overeating, obesity, diabetes and insulin resistance, excessive alcohol consumption, renal failure, nephrotic syndrome, genetic predisposition (e.g., familial combined hyperlipidemia, i.e., type II hyperlipidemia), lipoprotein lipase deficiency, lysosomal acid lipase deficiency, cholesteryl ester storage disease, certain medications (e.g., isotretinoin, hydrochlorothiazide diuretics, beta-blockers, protease inhibitors), hypothyroidism (thyroid dysfunction), systemic lupus erythematosus and related autoimmune responses, glycogen storage disease type 1, propofol, or HIV medications.

[0056]

[0100] The term "diabetes" as used herein refers to a group of metabolic disorders characterized by prolonged high blood sugar levels. Diabetes may be type 1 diabetes, which results from the failure of the pancreas to produce sufficient insulin due to the loss of beta cells. Diabetes may be type 2 diabetes, which is characterized by insulin resistance, a condition in which cells do not respond properly to insulin. Diabetes may be gestational diabetes, which occurs when pregnant women with no history of diabetes develop high blood sugar levels.

[0057]

[0101] The term "low-density lipoprotein (LDL)," as used herein, refers to a microscopic mass composed of an outer rim of lipoprotein and a cholesterol core. LDL may have a highly hydrophobic core composed of a polyunsaturated fatty acid known as linoleate and hundreds to thousands of esterified and unesterified cholesterol molecules. The core of LDL may also carry triglycerides and other fats and may be surrounded by a shell of phospholipids and unesterified cholesterol.

[0058]

[0102] The term "high-density lipoprotein (HDL)" as used herein refers to the smallest lipoprotein particle. The plasma enzyme lecithin-cholesterol acyltransferase (LCAT) converts free cholesterol into cholesterol, which is then trapped in the core of the lipoprotein particle, ultimately giving the newly synthesized HDL its spherical shape. HDL particles can increase in size as they circulate in the bloodstream and incorporate more cholesterol and phospholipid molecules from cells and other lipoproteins.

[0059]

[0103] The term "cholesterol," as used herein, refers to a lipid with a unique structure composed of four linked hydrocarbon rings that form a bulky steroid structure. The term "triglyceride," as used herein, refers to a triester composed of glycerol bound to three fatty acid molecules. In some embodiments, the fatty acids are saturated or unsaturated fatty acids.

[0060]

[0104] The terms "treat," "treating," or "treatment," and their grammatical equivalents as used herein, can include alleviating, relieving, or ameliorating at least one symptom of a disease or condition; preventing additional symptoms; inhibiting a disease or condition, e.g., delaying, lowering, suppressing, attenuating, reducing, inhibiting, or stabilizing the onset or progression of a disease or condition; relieving a disease or condition; causing regression of a disease or condition; alleviating symptoms caused by a disease or condition; reducing the severity of the disease; or arresting symptoms of a disease or condition, either prophylactically and / or therapeutically. "Treating" also includes reducing the frequency of occurrence or recurrence or severity of any symptoms or other adverse effects associated with a disease or condition and / or side effects related to a disease or condition. "Treating" does not necessarily require a curative result. It is understood, although not excluded, that treating a disorder or condition also does not require that the associated disorder, condition, or symptoms be completely eliminated. The term "treating" encompasses the concept of "managing," which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, for example, extending the period of remission in a patient who has been suffering from the disease. "Treating" can refer to the application or administration of a composition to a subject after the onset or suspected onset of a disease or condition.

[0061]

[0105] The term "treating" further encompasses the concepts of "prevent," "preventing," and "prevention." The terms "prevent," "preventing," and "prevention," as used herein, refer to a reduction in the occurrence of pathology of a condition in a subject who does not have the disease or condition but who is at risk of or susceptible to developing the disease or condition. Prevention may be complete, e.g., the complete absence of pathology of the condition in the subject. Prevention may also be partial, such that the occurrence of the target condition of the condition in the subject is less than would have occurred without the present disclosure.

[0062]

[0106] "Treating or preventing a condition" can involve, for example, alleviating the symptoms of a disorder compared to an equivalent untreated control, by at least a 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% reduction or prevention as measured by any standard technique. In some embodiments, alleviating symptoms of a disorder may involve at least a 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000-fold degree of reduction or prevention compared to an equivalent untreated control.

[0063]

[0107] As used herein, "delaying" the onset of a disease means postponing, inhibiting, slowing, decelerating, stabilizing, and / or postponing the onset of the disease. This delay can be of varying lengths of time, depending on the disease being treated and / or the individual's medical history. A method that "delays" or alleviates the onset of a disease or delays the occurrence of a disease is a method that reduces the likelihood of developing one or more symptoms of the disease within a given time frame and / or reduces the severity of symptoms within a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to provide statistically significant results.

[0064]

[0108] "Onset" or "progression" of a disease refers to the initial onset and / or subsequent progression of the disease. Disease onset is detectable and assessable using standard clinical techniques well known in the art. However, onset also refers to progression, which may not be detectable. For purposes of this disclosure, onset or progression refers to the biological course of symptoms. "Onset" includes occurrence, recurrence, and onset.

[0065]

[0109] As used herein, the "onset" or "occurrence" of a disease includes initial onset and / or recurrence.

[0066]

[0110] As used herein, "administering" and its grammatical equivalents may refer to providing a pharmaceutical composition described herein to a subject or patient. Conventional methods known to those skilled in the medical arts can be used to administer the composition to a subject, depending on the type of disease or site of disease being treated. For example, the composition can be administered, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, via an implanted reservoir, or by injection. One or more such routes can be used.

[0067]

[0111] The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intradermal, intraarterial, intrasynovial, intrasternal, intrathecal, intravascular, intralesional, and intracranial injection or infusion techniques. In addition, the drug may be administered to a subject via an injectable depot route of administration, such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

[0068]

[0112] "Co-administering" means administering one or more additional therapeutic regimens or agents or treatments, or a treatment and composition of the present disclosure, close enough in time to enhance the effect of one or more additional therapeutic agents, or vice versa. In this regard, the compositions of the present disclosure described herein can be administered simultaneously with one or more additional therapeutic regimens or agents or treatments, at different times, or on entirely different treatment schedules (e.g., a first treatment can be administered daily, while an additional treatment is weekly). For example, in some embodiments, a secondary therapeutic regimen or agent or treatment is administered simultaneously with, before, or after a composition of the present disclosure.

[0069]

[0113] The term "pharmaceutical composition" and its grammatical equivalents as used herein may refer to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients, carriers, and / or therapeutic agents that are administered to a subject, e.g., a human in need thereof.

[0070]

[0114] As used herein, the term "pharmaceutically acceptable" and its grammatical equivalents can refer to the attributes of a material useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and not biologically undesirable, and that is acceptable for both veterinary and human pharmaceutical use. "Pharmaceutically acceptable" can refer to a material, such as a carrier or diluent, that does not abolish the biological activity or properties of the compound and is relatively non-toxic, i.e., that may be administered to a subject without causing undesirable biological effects or interacting in a deleterious way with any of the components of the pharmaceutical composition in which it is contained.

[0071]

[0115] A "pharmaceutically acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject together with a drug, does not destroy its pharmacological activity, and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0072]

[0116] A "pharmaceutically acceptable salt" can be any acid or base salt generally considered in the art to be suitable for use in contact with the tissues of humans or animals without excessive toxicity, irritation, allergic response, or other problem or complication. Such salts include inorganic and organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Particular pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids, such as acetic acid, HOOC-(CH)-COOH, where n is 0 to 4. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize from this disclosure and the knowledge of those skilled in the art that additional pharmaceutically acceptable salts include those listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985). Generally, pharmaceutically acceptable acid or base salts can be prepared using any conventional acid or base salt. The salts can be synthesized from parent compounds that contain a basic or acidic moiety by chemical methods such as those described below. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0073]

[0117] The term "therapeutic agent" may refer to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. A therapeutic agent may also be referred to as an "active substance" or "active agent." Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0074]

[0118] As used herein, a "therapeutically effective amount" refers to the amount of each composition of the present disclosure required to provide a therapeutic effect to a subject, either alone or in combination with one or more other therapeutic agents. Thus, as used herein, the term "therapeutically effective amount" refers to the amount of delivered agent (e.g., nucleic acid, composition, therapeutic agent, prophylactic agent, etc.) that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. In the context of treatment, a "therapeutically effective amount" is an amount sufficient to alleviate, ameliorate, stabilize, reverse, or slow the progression of a disease or condition, such as atherosclerotic vascular disease, hypertriglyceridemia, or diabetes. As will be recognized by those skilled in the art, a "therapeutically effective amount" will vary depending on the particular condition being treated, the severity of the condition, individual subject parameters including age, physical condition, size, sex, and weight, duration of treatment, the nature of concurrent therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of a medical practitioner. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. Generally, it is preferred to use the maximum dose of each component or combination thereof, i.e., the highest safe dose according to sound medical judgment. However, those skilled in the art will understand that a subject may insist on a lower or tolerated dose for medical, psychological, or virtually any other reason. In addition, other medications a patient may be taking will affect the determination of the therapeutically effective amount of the therapeutic agent to be administered. Empirical considerations such as half-life will generally contribute to determining the dosage. A "therapeutically effective amount" can be any therapeutically effective amount of the composition of the present disclosure used alone or in combination with one or more drugs used to treat a condition. A therapeutically effective amount can be administered in one or more doses.

[0075]

[0119] An effective initial method for determining a "therapeutically effective amount" can be by performing cell culture assays (e.g., using neuronal cells) or by using animal models (e.g., mice, rats, rabbits, dogs, or pigs). A dose may be formulated in an animal model to achieve a concentration range that includes the IC50 (i.e., the concentration of the composition that achieves half-maximal inhibition of symptoms), as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. In addition to determining the appropriate concentration range for the compositions of the present disclosure to be therapeutically effective, animal models can also yield other relevant information, such as preferred routes of administration for maximal efficacy. Based on the above and other methods, it is well within the capabilities of one of ordinary skill in the art to adjust doses to achieve maximal efficacy in humans.

[0076]

[0120] Ranges provided herein are understood to be shorthand for all values ​​within that range. For example, a range of 1 to 50 consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. It is understood to include any number, combination of numbers, or subrange from the group, as well as all intervening decimal values ​​between the recited integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "subranges within" are specifically contemplated that extend from either end of the range. For example, subranges within the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0077]

[0121] As used herein, the terms "protospacer" or "target sequence," and their grammatical equivalents, may refer to the DNA sequence of a target gene. In its natural state, the protospacer is adjacent to a PAM (protospacer adjacent motif). The cleavage site for the RNA-guided nuclease is located within the protospacer sequence.

[0078]

[0122] The term "spacer" or "spacer sequence" refers to a nucleic acid sequence that is complementary to and binds to the complementary strand of a target gene. The spacer can be within a guide nucleic acid (e.g., gRNA, gDRNA).

[0079]

[0123] As used herein, the terms "base editing," "gene editing," "genome editing," or "genetic modification," and their grammatical equivalents, may refer to genetic manipulation in which one or more nucleotides are inserted, replaced, or removed from a genome. Gene editing can be performed using a nuclease (e.g., a naturally occurring nuclease or an artificially engineered nuclease). The genetic modification can include the introduction of a double-strand break, a nonsense mutation, a frameshift mutation, a splice site change, or an inversion of a polynucleotide sequence, e.g., a target polynucleotide sequence.

[0080]

[0124] The term "base editor" as used herein may refer to an agent that binds to a polynucleotide and has nucleobase-modifying activity. A base editor may include a nucleobase-modifying polypeptide (e.g., a deaminase) and a nucleic acid-programmable nucleotide-binding domain, or a nucleic acid encoding a programmable nucleotide-binding domain and a deaminase, in conjunction with a guide polynucleotide (e.g., a guide RNA). An agent may be a biomolecular complex including a protein domain with base-editing activity, i.e., a domain capable of modifying bases (e.g., A, T, C, G, or U) in a nucleic acid molecule (e.g., DNA), or a nucleic acid encoding the same. A polynucleotide-programmable DNA-binding domain may be fused or linked to a deaminase domain, resulting in a base editor fusion protein. A base editor may include a nucleic acid encoding a base editor fusion protein, e.g., an mRNA encoding a base editor fusion protein. A base editor fusion protein may include one or more linkers, e.g., peptide linkers. An agent may be a fusion protein including a domain with base-editing activity. The protein domain with base editing activity can be linked to a guide RNA (e.g., via an RNA-binding motif on the guide RNA and the RNA-binding domain fused to a deaminase). In some cases, the domain with base editing activity can deaminate a base in a nucleic acid molecule. In some cases, the base editor can deaminate one or more bases in a DNA molecule. In some cases, the base editor can deaminate an adenosine (A) in DNA. The base editor can be an adenosine base editor (ABE). In some cases, the base editor can deaminate a cytosine (C) in DNA. The base editor can be a cytosine base editor (CBE).

[0081]

[0125] The term "base editor system" refers to a gene editing system for editing a single nucleic acid base of a target nucleotide sequence. In various embodiments, the base editor system comprises: (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9); (2) a deaminase domain (e.g., adenosine deaminase or cytidine deaminase) for deaminating the nucleobases described above, and (3) one or more guide polynucleotides (e.g., guide RNAs). In some embodiments, the base editor system comprises a base editor fusion protein comprising (1) and (2). In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a polynucleotide-programmable DNA-binding domain. In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is a cytidine base editor (CBE).

[0082]

[0126] As used herein, the term "prime editing" refers to a form of precise genome editing that allows direct, irreversible, targeted small insertions, deletions, and base swapping without the need for double-stranded DNA breaks (DSBs) or donor DNA templates. DNA prime editors contain a catalytically inactive Cas9 nuclease fused to a reverse transcriptase. Prime editing involves a prime editing guide RNA (pegRNA), which is substantially larger than the standard sgRNA used in CRISPR Cas9 editing. The pegRNA contains a primer binding sequence (PBS) and a template containing the desired RNA sequence attached to its 3' end.

[0083]

[0127] The term "CRISPR RNA" (crRNA) herein refers to an RNA sequence that can form a complex with one or more Cas proteins (e.g., Cas9) and provide DNA-binding specificity to the complex. The crRNA provides DNA-binding specificity because it contains a "spacer sequence" that is complementary to a strand of the DNA target sequence. The crRNA also contains a "repeat sequence" ("tracrRNA mate sequence") encoded by the repeat region of the CRISPR locus from which the crRNA is derived. The repeat sequence of the crRNA can anneal to a sequence at the 5' end of the tracrRNA. In natural CRISPR systems, the crRNA is derived from a "pre-crRNA" transcribed from the CRISPR locus. The pre-crRNA contains a spacer region and a repeat region, and the spacer region contains a unique sequence complementary to the DNA target site sequence. In natural systems, the pre-crRNA is processed into multiple different crRNAs, each of which has a guide sequence along with a portion of the repeat sequence. CRISPR systems utilize crRNAs, for example, for DNA-targeting specificity.

[0084]

[0128] The term "trans-activating CRISPR RNA" (tracrRNA) herein refers to a non-coding RNA used in type II CRISPR systems, which contains, in the 5'-3' direction, (i) a sequence that anneals to the repeat region of CRISPR type II crRNA, and (ii) a stem-loop-containing portion (Deltcheva et al., Nature 471:602-607). Modified tracrRNA refers to a tracrRNA with modified ribonucleotides (e.g., 2-OMe modified RNA).

[0085]

[0129] As used herein, the term "guide nucleic acid" refers to a polynucleotide sequence that can form a complex with a Cas endonuclease and enable the Cas endonuclease to recognize and optionally cleave a DNA target site. A guide nucleic acid can be a single molecule or a double molecule. A guide nucleic acid sequence can be DNA only (gDNA). The DNA can be modified or unmodified. A guide nucleic acid sequence can be RNA only (gRNA). The RNA can be modified or unmodified. A guide nucleic acid sequence can be a combination of DNA and RNA. Optionally, a guide nucleic acid contains at least one nucleotide, phosphodiester bond, or bond modification, such as, but not limited to, locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, phosphorothioate bond, bond to a cholesterol molecule, bond to a polyethylene glycol molecule, spacer 1 The guide nucleic acid may include a linkage to an 8 (hexaethylene glycol) molecule, or a 5'-3' covalent bond that results in cyclization. A guide nucleic acid that contains only ribonucleic acid is referred to as a "guide RN." A guide nucleic acid that contains both RNA and DNA is referred to as a "guide RDNA."

[0086]

[0130] "Partial hepatectomy," as used herein, refers to surgery to remove a portion of the liver. In some embodiments, partial hepatectomy is used to model liver regeneration in vivo.

[0087]

[0131] As used herein, a spacer sequence corresponding to a protospacer can make modifications to bases in the complementary strand of a target protospacer nucleic acid sequence. A spacer sequence corresponding to a protospacer sequence can be identical or substantially identical to the protospacer sequence.

[0088]

[0132] As used herein, "corresponding" refers to a region to which a different sequence or constituent iodine can react or bind. For example, the corresponding region of a Cas9 nickase to a scaffold component of a guide RNA refers to a region of the Cas9 nickase that can react and bind to the scaffold component of a guide RNA. In another example, the spacer sequence corresponds to a protospacer sequence, and refers to the fact that the spacer sequence binds to the protospacer sequence. The binding may have one or more mismatches.

[0089] Gene editing

[0133] The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for gene editing, revolutionizing the fields of biotechnology and life sciences. Despite being a powerful gene editing tool, CRISPR has its own drawbacks. One of the main challenges is off-target mutations caused by the system (Yin et al., Nature Chemical Biology 14, 311-316 (2018)). It has been shown that partial replacement of RNA nucleotides (ribonucleosides) from guide RNAs with DNA nucleotides (deoxyribonucleosides) can reduce off-target activity compared to unmodified guide RNAs. Such DNA-RNA chimeric guides are also known as CRISPR hybrid DNA-RNA (chRDNA). However, while chRDNA can reduce off-target effects, this is often at the expense of on-target editing efficiency. Therefore, there is an urgent need to find optimized guide nucleic acids for CRISPR systems that maintain strong on-target editing efficiency with low off-target effects, especially in vivo.

[0090]

[0134] The present disclosure provides a novel gene editing system that offers many of the advantages of using CRISPR systems for gene editing, i.e., highly efficient programmable gene editing, while overcoming the limitations of off-target editing. The novel gene editing system described herein achieves efficient gene editing results with reduced off-target effects by using novel guide nucleic acids to direct gene editor proteins to affect changes to target genes. As disclosed herein, the novel guide nucleic acids are engineered to contain a mixture of deoxyribonucleotides and ribonucleotides within a spacer sequence, including at least one deoxyribonucleotide and at least one ribonucleotide within the spacer sequence, while maintaining the ability to hybridize to the target gene. The number, position, and specific modifications of deoxyribonucleotides and / or ribonucleotides within the guide nucleic acid are optimized to enable a gene editing system comprising the guide nucleic acid to perform gene editing with high on-target efficiency but low off-target effects. In some embodiments, the 2' hydroxyl group on the ribose of a ribonucleotide can be covalently modified to be linked to a methyl group. Figures 28A-28D show exemplary spacer sequences disclosed herein. An exemplary gene editing system is provided having a guide nucleic acid comprising a sequence. As shown in Figure 28A, the exemplary gene editing system (100) includes a target gene (110) and a gene editor protein (150) containing a nucleic acid binding domain capable of binding to the guide nucleic acid (120). The exemplary guide nucleic acid (120) includes a spacer sequence (130) composed of both ribonucleotides (RNA) and deoxyribonucleotides (DNA). The spacer sequence (130) is complementary to a protospacer (160) from the target gene (110). Replacing ribonucleotides with deoxyribonucleotides at several specific positions in the spacer sequence (130) with 2'-OMe and phosphorothioate backbone chemical modifications results in a unique DNA-RNA hybrid sequence motif at the 5' end relative to the spacer sequence (130), which can guide the gene editor protein (150) to the target sequence (160) and induce site-specific on-target genetic alterations with reduced off-target effects. Here, an exemplary motif within the spacer sequence (130) is (2'-OMeRNA) (2'-OMeRNA) (DNA) (DNA) (RNA) (DNA) (DNA) (DNA). Note that the specific phosphorothioate backbone modification is not shown. The guide nucleic acid (120) further comprises a tracrRNA (140) or its equivalent at the 3' end of the spacer sequence (130). The tracrRNA (140) or its equivalent recruits a gene editor protein (150) to the target sequence (160) to affect changes in the target gene (110). The tracrRNA (140) can be modified. For example, the tracrRNA (140) can comprise a 2'-OMeRNA (not shown). The gene editor protein (150) can comprise a deaminase that can be used for base editing. Figures 1B-1D show other exemplary gene editing systems with guide nucleic acids comprising other exemplary spacer sequences. For example, Figure 28B shows a spacer sequence with the motif (2'-OMeRNA)(2'-OMeRNA)(DNA)(DNA)(RNA)(DNA)(DNA).Figure 28C shows another spacer sequence with another motif of (2'-OMeRNA)(2'-OMeRNA)(DNA)(DNA)(DNA)(DNA)(DNA). Figure 28D shows another spacer sequence with another motif of (2'-OMeRNA)(2'-OMeRNA)(2'-OMeRNA)(DNA)(DNA)(DNA)(DNA)(RNA)(DNA)(DNA)(RNA)(DNA)(DNA). Note that the specific phosphorothioate backbone modification is not shown. Note that the figures are for schematic purposes only.

[0091]

[0135] The present disclosure further provides novel gene editing systems for base editing that have high on-target efficiency and low off-target editing. The novel gene editing systems for base editing described herein achieve these benefits by using novel guide nucleic acids described herein to direct gene editor proteins, including deaminases, to modify nucleobases in target genes.

[0092]

[0136] The present disclosure further provides novel gene editing systems for editing the ANGPTL3 gene that have high on-target efficiency and low off-target editing. The novel gene editing systems for editing the ANGPTL3 gene described herein achieve these benefits by using novel guide nucleic acids to direct gene editor proteins to affect changes to the ANGPTL3 gene.

[0093] CRISPR / Cas system

[0137] Several different CRISPR / Cas systems exist, and their nomenclature and classification will change as the systems are further characterized. CRISPR / Cas systems have advantages over other genome editing methods, including endonucleases, meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs), which may require the engineering of new proteins for each new target locus.

[0094]

[0138] Based on the genes encoding the effector modules (i.e., proteins involved in the interference step), CRISPR / Cas systems can be classified as either Class 1 or Class 2. Class 1 systems have a multi-subunit crRNA effector complex, while Class 2 systems have a single protein, e.g., Cas9, Cpfl, C2cl, C2c2, C2c3, or crRNA effector complex. Class 1 systems include Type I, Type III, and Type IV systems. Class 2 systems include Type II and Type V systems (Makarova et al., Nature Review Microbiology 13:1-15 (2015)).

[0095]

[0139] All type I systems possess a Cas3 protein with helicase and cleavage activities. Type I systems are divided into seven subtypes (IA-IF and IU). Type III systems possess the Cas10 gene, which encodes a multidomain protein containing a Palm domain (a variant of an RNA recognition motif (RRM)) that is homologous to the core domains of many nucleic acid polymerases and cyclases and is the largest subunit of type III crRNA-effector complexes. All III loci also encode a small subunit protein, one Cas5 protein, and typically several Cas7 proteins. Type III can be divided into four subtypes, III-A to III-D. Subtype III-A possesses the csm2 gene encoding the small subunit and also possesses the casl, cas2, and cas6 genes. Subtype III-B possesses the cmr5 gene encoding the small subunit and typically lacks the casl, cas2, and cas6 genes. Subtype III-C has a Cas10 protein with an inactive cyclase-like domain and lacks the casl and cas2 genes. Subtype III-D has a Cas10 protein lacking the HD domain, lacks the casl and cas2 genes, and has a cas5-like gene known as csx10. Type IV systems encode a minimal multisubunit crRNA-effector complex containing a partially resolved large subunit, Csfl, Cas5, Cas7, and in some cases, a putative small subunit. Type IV systems lack the casl and cas2 genes. Type IV systems do not have subtypes, but exist in two distinct variants. One variant contains a DinG family helicase, while the other lacks the DinG family helicase but contains a gene encoding a small α-helical protein.

[0096]

[0140] Type II systems contain the casl, cas2, and cas9 genes. cas9 encodes a multidomain protein that couples the function of the crRNA-effector complex with targeted DNA cleavage. Type II systems also encode a tracrRNA. Type II systems are divided into three subtypes: subtype II-A, II-B, and II-C. Subtype II-A contains an additional gene, csn2. Subtype II-B lacks csn2 but contains cas4. Subtype II-C is the most common type II system found in bacteria and contains only three proteins: Casl, Cas2, and Cas9. Type V systems contain the cpf1 gene and the casl and cas2 genes. The cpf1 gene encodes the protein Cpfl, which contains RuvC-like nuclease domains homologous to the respective domains of Cas9 but lacks the HNH nuclease domain present in the Cas9 protein.

[0097]

[0141] In Class 1 systems, the expression and interference steps involve a multisubunit CRISPR RNA (crRNA)-effector complex. In these systems, the pre-crRNA binds to the multisubunit crRNA-effector complex and is processed into mature crRNA. In Type I and Type III systems, this involves an RNA endonuclease, e.g., Cas6.

[0098]

[0142] In class 1 systems, crRNA associates with a crRNA-effector complex and achieves interference by combining nuclease activity via the RNA-binding domain with base pairing between the crRNA and the target nucleic acid. In type I systems, target binding of the crRNA and crRNA-effector complex involves Cas7, Cas5, and Cas8 fused to small subunit proteins. Target nucleic acid cleavage in type I systems involves an HD nuclease domain, which is either fused to the superfamily 2 helicase Cas3' or encoded by a separate gene, cas3". In type III systems, target binding of the crRNA and crRNA-effector complex involves Cas7, Cas5, Cas10, and a small subunit protein. Target nucleic acid cleavage in type III systems involves the combined action of Cas7 and Cas10 proteins, with a separate HD nuclease domain fused to Cas10, which is thought to cleave single-stranded DNA during interference.

[0099]

[0143] In Class 2 systems, the expression and interference steps involve a single large protein, e.g., Cas9, Cpfl, C2C1, C2C2, or C2C3. In most Class 2 Type II systems, the pre-crRNA binds to Cas9 and is processed into mature crRNA in a step involving RNase III and tracrRNA.

[0100]

[0144] In class 2 systems, crRNA associates with a single protein, achieving interference by combining nuclease activity via the RNA-binding domain with base pairing between the crRNA and the target nucleic acid. In type II systems, crRNA and target binding, as well as target nucleic acid cleavage, involve Cas9. In type II systems, the RuvC-like nuclease (RNase H-fold) domain and HNH (McrA-like) nuclease domain of Cas9 each cleave one strand of the target nucleic acid. Cas9 cleavage activity in type II systems also requires hybridization of crRNA to tracrRNA, forming a duplex that facilitates crRNA and target binding by Cas9. In type V systems, crRNA and target binding, as well as target nucleic acid cleavage, involve Cpf1. In type V systems, the RuvC-like nuclease domain of Cpf1 cleaves both strands of the target nucleic acid in a staggered configuration, generating 5' overhangs, as opposed to the blunt ends generated by Cas9 cleavage. These 5' overhangs can facilitate the insertion of DNA by the non-homologous end joining (NHEJ) method.

[0101]

[0145] The Cpf1 cleavage activity of the V-type system also does not require hybridization of the crRNA to the tracrRNA to form a duplex. Rather, the crRNA in the V-type system uses a single crRNA with a stem-loop structure that forms an internal duplex. Cpf1 binds to the crRNA in a sequence- and structure-specific manner, recognizing the stem-loop and sequences adjacent to the stem-loop, most notably the 5' nucleotide of the spacer sequence that hybridizes to the target nucleic acid. This stem-loop structure typically ranges from 15 to 19 nucleotides in length. Substitutions that disrupt this stem-loop duplex abolish cleavage activity, while other substitutions that do not disrupt the stem-loop duplex do not. In the V-type system, the crRNA forms a stem-loop structure at the 5' end, and the sequence at the 3' end is complementary to a sequence in the target nucleic acid.

[0102]

[0146] Other proteins involved in V-type crRNA and target binding and cleavage include class 2 candidate 1 (C2c1) and class 2 candidate 3 (C2c3). C2c1 and C2c3 proteins are similar in length to Cas9 and Cpf1, ranging from approximately 1,100 amino acids to approximately 1,500 amino acids. C2c1 and C2c3 proteins also contain a RuvC-like nuclease domain and have a similar architecture to Cpf1. C2c1 protein is similar to Cas9 protein in that it requires crRNA and tracrRNA for target binding and cleavage, but has an optimal cleavage temperature of 50°C. The C2c1 protein, like Cpf1, targets AT-rich PAMs 5' to the target sequence (Shmakov et al., Molecular Cell 60(3):385-397(2015)). Class 2 candidate 2 (C2c2) shares no sequence similarity with other CRISPR effector proteins and may therefore be included in the putative type VI system. The C2c2 protein has two HEPN domains and is predicted to have RNase activity, thus allowing it to target and cleave mRNA. The C2c2 protein appears similar to the Cpfl protein in that it does not require the tracrRNA but does require the crRNA for target binding and cleavage. Also, like Cpfl, the crRNA in the case of the C2c2 protein forms a stable hairpin or stem-loop structure, which may aid in its association with the C2c2 protein.

[0103]

[0147] The gene editing systems provided herein may include components of Class 1 or Class 2 systems, including ribonucleoprotein complexes. The Class 2 Cas nuclease family of proteins are enzymes with DNA endonuclease activity that can be directed to cleave desired nucleic acid targets by designing appropriate guide RNAs, as further described herein. Components of Class 2 CRISPR / Cas systems may be derived from Type II, Type IIA, Type IIB, Type IIC, Type V, or Type VI systems. Class 2 Cas nucleases include, for example, Cas9 (also known as Csn1 or Csx12), Csn2, Cas4, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas13a (C2c2), Cas13b, Cas13c, and Cas13d proteins. In some embodiments, the Cas protein is from a type II CRISPR / Cas system, i.e., a Cas9 protein from a CRISPR / Cas9 system, or from a type V CRISPR / Cas system, e.g., a Cas12 protein. In some embodiments, the Cas protein is from a class 2 CRISPR / Cas system, i.e., a single protein Cas nuclease such as a Cas9 protein or a Cas12a protein. Other non-limiting examples of Cas proteins may include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, Cas9HiFi, homologs thereof, or modified versions thereof.

[0104] Base editing / base editing system

[0148] Class 2 CRISPR / Cas systems, particularly the CRISPR / Cas9 system, have been used for precise genome editing. By using a guide RNA (gRNA) with a sequence homologous to a sequence of DNA (known as a protospacer) in the target genome adjacent to a specific protospacer adjacent motif (PAM) containing the sequence NGG (where N is any standard base) in the DNA, Cas9 can be used to create a double-strand break (DSB) at the target sequence. NHEJ at the DSB can be used to create an indel at the locus and knock out the gene. Similarly, homology-directed repair (HDR) can be used with the introduced template DNA to insert a gene or modify the target sequence.

[0105]

[0149] In some aspects, provided herein are base editor systems capable of nucleic acid base modification. In some embodiments, the base editor system comprises: (i) a guide polynucleotide, or a nucleic acid encoding the same; and (ii) a base editor fusion protein comprising a programmable DNA binding domain (e.g., Cas9 or dCas9) and a deaminase, or a nucleic acid encoding the same. In some embodiments, the base editor system comprises: In some embodiments, the base editor system comprises a nucleic acid encoding a guide polynucleotide. In some embodiments, the base editor system comprises a base editor fusion protein comprising a programmable DNA-binding domain and a deaminase. In some embodiments, the base editor system comprises a nucleic acid encoding a base editor fusion protein comprising a programmable DNA-binding domain and a deaminase.

[0106]

[0150] In some embodiments, the guide polynucleotide, when administered to a subject, directs the base editor system to effect nucleobase changes in a PCSK9 or ANGPTL3 gene in vivo.

[0107]

[0151] In some embodiments, the base change occurs in at least 35% of all liver cells in the subject as measured by next generation sequencing or Sanger sequencing.

[0108]

[0152] In some embodiments, the base change occurs in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of all liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base changes occur in at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of all liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base change is present in between 1% and 99.9%, 2% and 99.9%, 3% and 99.9%, 4% and 99.9%, 5% and 99.9%, 6% and 99.9%, 7% and 99.9%, 8% and 99.9%, 9% and 99.9%, 10% and 99.9%, 15% and 99.9%, 20% and Occurs in 99.9%, 25% to 99.9%, 30% to 99.9%, 35% to 99.9%, 40% to 99.9%, 45% to 99.9%, 50% to 99.9%, 55% to 99.9%, 60% to 99.9%, 65% to 99.9%, 70% to 99.9%, 75% to 99.9%, 80% to 99.9%, 85% to 99.9%, 90% to 99.9%, or 95 to 99.9%.In some embodiments, the base change occurs in between 1% and 99.5%, between 1% and 99%, between 1% and 98%, between 1% and 97%, between 1% and 96%, between 1% and 95%, between 1% and 90%, between 1% and 85%, between 1% and 80%, between 1% and 75%, between 1% and 70%, between 1% and 65%, between 1% and 60%, between 1% and 55%, between 1% and 50%, between 1% and 45%, between 1% and 40%, between 1% and 35%, between 1% and 30%, between 1% and 25%, between 1% and 20%, between 1% and 15%, between 1% and 10%, between 1% and 9%, between 1% and 8%, between 1% and 7%, between 1% and 6%, between 1% and 5%, between 1% and 4%, between 1% and 3%, or between 1% and 2% of all liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base change occurs in 1% to 90%, 5% to 85%, 10% to 80%, 15% to 75%, 20% to 70%, 25% to 65%, 30% to 60%, 35% to 55%, or 40% to 50% of all liver cells in the subject, as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base change occurs in 100% of all liver cells in the subject, as measured by next generation sequencing or Sanger sequencing.

[0109]

[0153] In some embodiments, the base change occurs in hepatocytes of the subject. In some embodiments, the base change occurs in at least 30% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, The base change occurs in hepatocytes of the subject. In some embodiments, the base change occurs in at least % of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base change occurs in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base changes occur in at most 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base change is present in 1% to 99.9%, 2% to 99.9%, 3% to 99.9%, 4% to 99.9%, 5% to 99.9%, 6% to 99.9%, 7% to 99.9%, 8% to 99.9%, 9% to 99.9%, 10% to 99.9%, 15% to 99.9%, 20% to 99.9%, 21% to 99.9%, 22% to 99.9%, 23% to 99.9%, 24% to 99.9%, 25% to 99.9%, 26% to 99.9%, 27% to 99.9%, 28% to 99.9%, 30% to 99.9%, 31% to 99.9%, 32% to 99.9%, 33% to 99.9%, 34% to 99.9%, 35% to 99.9%, 36% to 99.9%, 37% to 99.9%, 38% to 99.9%, 39% to 40% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. Occurs in 9.9%, 25% to 99.9%, 30% to 99.9%, 35% to 99.9%, 40% to 99.9%, 45% to 99.9%, 50% to 99.9%, 55% to 99.9%, 60% to 99.9%, 65% to 99.9%, 70% to 99.9%, 75% to 99.9%, 80% to 99.9%, 85% to 99.9%, 90% to 99.9%, or 95 to 99.9%.In some embodiments, the base changes occur in between 1% and 99.5%, between 1% and 99%, between 1% and 98%, between 1% and 97%, between 1% and 96%, between 1% and 95%, between 1% and 90%, between 1% and 85%, between 1% and 80%, between 1% and 75%, between 1% and 70%, between 1% and 65%, between 1% and 60%, between 1% and 55%, between 1% and 50%, between 1% and 45%, between 1% and 40%, between 1% and 35%, between 1% and 30%, between 1% and 25%, between 1% and 20%, between 1% and 15%, between 1% and 10%, between 1% and 9%, between 1% and 8%, between 1% and 7%, between 1% and 6%, between 1% and 5%, between 1% and 4%, between 1% and 3%, or between 1% and 2% of liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base change occurs in 1% to 90%, 5% to 85%, 10% to 80%, 15% to 75%, 20% to 70%, 25% to 65%, 30% to 60%, 35% to 55%, or 40% to 50% of the hepatocytes in the subject, as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base change occurs in 100% of the hepatocytes in the subject, as measured by next generation sequencing or Sanger sequencing.

[0110]

[0154] In some embodiments, the base change occurs in the whole liver cells of a subject measured by next-generation sequencing. In some embodiments, the base change occurring in the whole liver cells of a subject is measured by Sanger sequencing. In some embodiments, the base change occurring in the liver cells of a subject is measured by next-generation sequencing. In some embodiments, the base change occurring in the liver cells of a subject is measured by Sanger sequencing.

[0111]

[0155] In some embodiments, the nucleobase alteration results in at least a 35% decrease in blood PCSK9 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. In some embodiments, the nucleobase alteration results in at least a 35% decrease in blood ANGPTL3 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS.

[0112]

[0156] In some embodiments, the nucleobase change is a decrease in blood P in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. In some embodiments, the nucleobase changes result in at least a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% decrease in blood PCSK9 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. resulting in a 6%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% reduction. In some embodiments, the nucleobase change is a decrease in blood PCSK9 protein levels in the subject by up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, %, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% reduction.In some embodiments, the nucleobase change is between 1% and 99.9%, 2% and 99.9%, 3% and 99.9%, 4% and 99.9%, 5% and 99.9%, 6% and 99.9%, 7% and 99.9%, 8% and 99.9%, 9% and 99.9%, 10% and 99.9%, 15% and 99.9%, 20% and 99.9%, 25% and 99.9%, 30% and 99.9% of the blood PCSK9 protein level in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. , resulting in a reduction of 31% to 99.9%, 32% to 99.9%, 33% to 99.9%, 34% to 99.9%, 35% to 99.9%, 36% to 99.9%, 37% to 99.9%, 38% to 99.9%, 39% to 99.9%, 40% to 99.9%, 45% to 99.9%, 50% to 99.9%, 55% to 99.9%, 60% to 99.9%, 65% to 99.9%, 70% to 99.9%, 75% to 99.9%, 80% to 99.9%, 85% to 99.9%, 90% to 99.9%, or 95% to 99.9%. In some embodiments, the nucleobase change is between 1% and 99.5%, 1% and 99%, 1% and 98%, 1% and 97%, 1% and 96%, 1% and 95%, 1% and 90%, 1% and 85%, 1% and 80%, 1% and 79%, 1% and 78%, 1% and 77%, 1% and 76%, 1% and 75%, 1% and 74%, 1% and 73%, 1% and 72%, 1% and 1% of blood PCSK9 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. % to 71%, 1% to 70%, 1% to 65%, 1% to 60%, 1% to 55%, 1% to 50%, 1% to 45%, 1% to 40%, 1% to 39%, 1% to 38%, 1% to 37%, 1% to 36%, 1% to 35%, 1% to 34%, 1% to 33%, 1% to 32%, 1% to 31%, 1% to 30%, 1% to 25%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, 1% to 3%, or 1% to 2% reduction.In some embodiments, the nucleobase alterations result in a 1% to 99.9%, 5% to 99.5%, 10% to 99%, 15% to 97%, 20% to 95%, 25% to 90%, 30% to 85%, 31% to 80%, 32% to 79%, 33% to 78%, 34% to 77%, 35% to 76%, 36% to 76%, 37% to 75%, 38% to 74%, 39% to 73%, 40% to 72%, 45% to 71%, 50% to 70%, or 55% to 65% decrease in blood PCSK9 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. In some embodiments, the nucleobase alterations result in a 100% decrease in blood PCSK9 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS.

[0113]

[0157] In some embodiments, the nucleobase change is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% decrease in blood PCSK9 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. In some embodiments, the nucleobase alterations result in at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or greater than 10-fold decrease in blood PCSK9 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS.

[0114]

[0158] In some embodiments, the blood PCSK9 protein level or decrease in blood PCSK9 protein level in the subject compared to before administration is measured by ELISA (enzyme-linked immunosorbent assay). In some embodiments, the blood PCSK9 protein level or decrease in blood PCSK9 protein level in the subject compared to before administration is measured by Western blot analysis. In some embodiments, the blood PCSK9 protein level or decrease in blood PCSK9 protein level in the subject compared to before administration is measured by LC-MS / MS (liquid chromatography-tandem mass spectrometry).

[0115]

[0159] In some embodiments, the nucleobase alteration results in at least a 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% decrease in blood ANGPTL3 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. In some embodiments, the nucleobase alteration results in at least a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, or 40% decrease in blood ANGPTL3 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. resulting in a 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% reduction. In some embodiments, the nucleobase change is at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134 resulting in a 6%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% reduction. In some embodiments, the nucleobase change is a decrease in blood ANGPTL3 protein levels in the subject by 1% to 99.9%, 2% to 99.9%, 3% to 99.9%, 4% to 99.9%, 5% to 99.9%, 6% to 99.9%, 7% to 99.9%, 8% to 99.9%, 9% to 99.9%, or 10% to 99.9%, as compared to before administration, as measured by ELISA, Western blot, or LC-MS / MS.%, 10%~99.9%, 15%~99.9%, 20%~99.9%, 25%~99.9%, 30%~99.9%, 31%~99.9%, 32%~99.9%, 33%~99.9%, 34%~99.9%, 35%~99.9%, 36%~99.9%, 37%~99.9%, 38%~99.9%, 39% A reduction of up to 99.9%, 40% to 99.9%, 45% to 99.9%, 50% to 99.9%, 55% to 99.9%, 60% to 99.9%, 65% to 99.9%, 70% to 99.9%, 75% to 99.9%, 80% to 99.9%, 85% to 99.9%, 90% to 99.9%, or 95 to 99.9%. In some embodiments, the nucleobase change is between 1% and 99.5%, 1% and 99%, 1% and 98%, 1% and 97%, 1% and 96%, 1% and 95%, 1% and 90%, 1% and 85%, 1% and 80%, 1% and 79%, 1% and 78%, 1% and 77%, 1% and 76%, 1% and 75%, 1% and 74%, 1% and 73%, 1% and 72%, 1% and 1% of blood ANGPTL3 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. A decrease of 1% to 71%, 1% to 70%, 1% to 65%, 1% to 60%, 1% to 55%, 1% to 50%, 1% to 45%, 1% to 40%, 1% to 39%, 1% to 38%, 1% to 37%, 1% to 36%, 1% to 35%, 1% to 34%, 1% to 33%, 1% to 32%, 1% to 31%, 1% to 30%, 1% to 25%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, 1% to 3%, or 1% to 2% results. In some embodiments, the nucleobase alterations result in a 1% to 99.9%, 5% to 99.5%, 10% to 99%, 15% to 97%, 20% to 95%, 25% to 90%, 30% to 85%, 31% to 80%, 32% to 79%, 33% to 78%, 34% to 77%, 35% to 76%, 36% to 76%, 37% to 75%, 38% to 74%, 39% to 73%, 40% to 72%, 45% to 71%, 50% to 70%, or 55% to 65% decrease in blood ANGPTL3 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS.In some embodiments, the nucleobase alteration results in a 100% decrease in blood ANGPTL3 protein levels in the subject compared to pre-administration as measured by ELISA, Western blot, or LC-MS / MS.

[0116]

[0160] In some embodiments, the nucleobase change is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% of the blood ANGPTL3 protein level in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. In some embodiments, the nucleobase alterations result in at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or greater than a 10-fold decrease in blood ANGPTL3 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS.

[0117]

[0161] In some embodiments, the blood ANGPTL3 protein level or the decrease in blood ANGPTL3 protein level in the subject compared to before administration is measured by ELISA (enzyme-linked immunosorbent assay). In some embodiments, the blood ANGPTL3 protein level or the decrease in blood ANGPTL3 protein level in the subject compared to before administration is measured by Western blot analysis. In some embodiments, the blood ANGPTL3 protein level or the decrease in blood ANGPTL3 protein level in the subject compared to before administration is measured by LC-MS / MS (liquid chromatography-tandem mass spectrometry). It is measured by NMR mass spectrometry.

[0118]

[0162] In some embodiments, the nucleobase alterations result in at least a 35% decrease in blood or low-density lipoprotein cholesterol (LDL-C) levels in the subject compared to before administration. In some embodiments, the nucleobase alterations result in at least a 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% decrease in blood low-density lipoprotein cholesterol (LDL-C) levels in the subject compared to before administration. In some embodiments, the nucleobase alterations result in at least a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% decrease in blood low-density lipoprotein cholesterol (LDL-C) levels in the subject compared to before administration. In some embodiments, the nucleobase alterations result in up to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% decrease in blood low-density lipoprotein cholesterol (LDL-C) levels in the subject compared to before administration.In some embodiments, the nucleobase change increases blood low-density lipoprotein cholesterol (LDL-C) levels in the subject by 1% to 99.9%, 2% to 99.9%, 3% to 99.9%, 4% to 99.9%, 5% to 99.9%, 6% to 99.9%, 7% to 99.9%, 8% to 99.9%, 9% to 99.9%, 10% to 99.9%, 15% to 99.9%, 20% to 99.9%, 30% to 99.9%, 31% to 99.9%, 32% to 99.9%, 33% to 99.9%, 34% to 99.9%, 35% to 99.9%, 36% to 99.9%, 37% to 99.9%, 38% to 99.9%, 39% to 40% A reduction of 0% to 99.9%, 25% to 99.9%, 30% to 99.9%, 35% to 99.9%, 40% to 99.9%, 45% to 99.9%, 50% to 99.9%, 55% to 99.9%, 60% to 99.9%, 65% to 99.9%, 70% to 99.9%, 75% to 99.9%, 80% to 99.9%, 85% to 99.9%, 90% to 99.9%, or 95% to 99.9% results. In some embodiments, the nucleobase alterations result in a 1% to 99.5%, 1% to 99%, 1% to 98%, 1% to 97%, 1% to 96%, 1% to 95%, 1% to 90%, 1% to 85%, 1% to 80%, 1% to 75%, 1% to 70%, 1% to 65%, 1% to 60%, 1% to 55%, 1% to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1% to 25%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, 1% to 3%, or 1% to 2% decrease in blood low density lipoprotein cholesterol (LDL-C) levels in the subject compared to before administration. In some embodiments, the nucleobase alterations result in a 1% to 99.9%, 5% to 99.5%, 10% to 99%, 15% to 97%, 20% to 95%, 25% to 90%, 30% to 85%, 35% to 80%, 40% to 75%, 45% to 70%, 50% to 65%, or 55% to 60% reduction in blood low-density lipoprotein cholesterol (LDL-C) levels in the subject compared to before administration. In some embodiments, the nucleobase alterations result in a 100% reduction in blood low-density lipoprotein cholesterol (LDL-C) levels in the subject compared to before administration.In some embodiments, the nucleobase change increases blood low-density lipoprotein cholesterol (LDL-C) levels in the subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280% compared to before administration. 290%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% reduction. In some embodiments, the nucleobase alterations result in at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or greater than 10-fold reduction in blood low-density lipoprotein cholesterol (LDL-C) levels in the subject compared to before administration.

[0119]

[0163] In some embodiments, the nucleobase alteration results in at least a 35% decrease in blood triglyceride levels in the subject compared to before administration, hi some embodiments, the nucleobase alteration results in at least a 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% decrease in blood triglyceride levels in the subject compared to before administration. In some embodiments, the nucleobase alterations result in at least a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% decrease in blood triglyceride levels in the subject compared to before administration. In some embodiments, the nucleobase alterations result in up to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% decrease in blood triglyceride levels in the subject compared to before administration.In some embodiments, the nucleobase change is between 1% and 99.9%, between 2% and 99.9%, between 3% and 99.9%, between 4% and 99.9%, between 5% and 99.9%, between 6% and 99.9%, between 7% and 99.9%, between 8% and 99.9%, between 9% and 99.9%, between 10% and 99.9%, between 15% and 99.9%, between 20% and 99.9%, between 25% and 25% of blood triglyceride levels in the subject compared to before administration. A reduction of 25% to 99.9%, 30% to 99.9%, 35% to 99.9%, 40% to 99.9%, 45% to 99.9%, 50% to 99.9%, 55% to 99.9%, 60% to 99.9%, 65% to 99.9%, 70% to 99.9%, 75% to 99.9%, 80% to 99.9%, 85% to 99.9%, 90% to 99.9%, or 95% to 99.9% results. In some embodiments, the nucleobase alteration results in a 1% to 99.5%, 1% to 99%, 1% to 98%, 1% to 97%, 1% to 96%, 1% to 95%, 1% to 90%, 1% to 85%, 1% to 80%, 1% to 75%, 1% to 70%, 1% to 65%, 1% to 60%, 1% to 55%, 1% to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1% to 25%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, 1% to 3%, or 1% to 2% decrease in blood triglyceride levels in the subject compared to before administration. In some embodiments, the nucleobase alteration results in a 1% to 99.9%, 5% to 99.5%, 10% to 99%, 15% to 97%, 20% to 95%, 25% to 90%, 30% to 85%, 35% to 80%, 40% to 75%, 45% to 70%, 50% to 65%, or 55% to 60% decrease in blood triglyceride levels in the subject compared to before administration, hi some embodiments, the nucleobase alteration results in a 100% decrease in blood triglyceride levels in the subject compared to before administration. In some embodiments, the nucleobase change increases blood triglyceride levels in the subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930 90%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% reduction. In some embodiments, the nucleobase alterations result in at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or greater than 10-fold reduction in blood triglyceride levels in the subject compared to before administration.

[0120]

[0164] In some embodiments, the blood triglyceride level or reduction in blood triglyceride level in the subject compared to before administration is measured by any standard technique. In some embodiments, the blood low-density lipoprotein cholesterol (LDL-C) level or reduction in blood low-density lipoprotein cholesterol (LDL-C) level in the subject compared to before administration is measured by any standard technique. For example, a clinical analyzer may be used to measure a "lipid panel" in a serum sample, which involves direct enzymatic measurement of cholesterol (total C), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C). Reagent kits specific to each analyte include buffers, calibrators, blanks, and controls. As used in this disclosure, cholesterol, triglycerides, and HDL-C may be quantified using absorbance measurements of specific enzyme reaction products. LDL-C may also be determined indirectly. In some cases, the majority of circulating cholesterol can be found in three major lipoprotein fractions: very low-density lipoprotein (VLDL), LDL, and HDL. In some embodiments, total circulating cholesterol can be estimated using the formula [total C] = [VLDL-C] + [LDL-C] + [HDL-C]. Therefore, LDL-C can be calculated from the measured values ​​of total cholesterol, triglycerides, and HDL-C according to the following relationship: [LDL-C] = [total C] - [HDL-C] - [TG] / 5, where [TG] / 5 is an estimate of VLDL-cholesterol. A triglyceride-specific reagent kit containing buffers, calibrators, blanks, and controls may be used. As used herein, serum samples from studies may be analyzed, and triglycerides may be measured using a series of coupled enzymatic reactions. In some embodiments, HO may be used to quantify the analyte as the final analyte end product, whose absorbance at 500 nm and color intensity are proportional to triglyceride concentration.

[0121]

[0165] In some embodiments, the guide polynucleotide is a guide RNA, and the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of a PCSK9 gene having 0, 1, or 2 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of a PCSK9 gene having no mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of a PCSK9 gene having one mismatch. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of a PCSK9 gene having two mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of a PCSK9 gene having three mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of a PCSK9 gene having four mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of a PCSK9 gene having five mismatches.

[0122]

[0166] In some embodiments, the guide polynucleotide is a guide RNA, and the guide RNA comprises a spacer sequence that binds to the complementary strand of the protospacer sequence of the ANGPTL3 gene with 0, 1, or 2 mismatches. The guide RNA comprises a spacer sequence that binds to the complementary strand of the protospacer sequence of the ANGPTL3 gene with no mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of the protospacer sequence of the ANGPTL3 gene with one mismatch. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of the protospacer sequence of the ANGPTL3 gene with two mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of the protospacer sequence of the ANGPTL3 gene with three mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of the protospacer sequence of the ANGPTL3 gene with four mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of the protospacer sequence of the ANGPTL3 gene with five mismatches.

[0123]

[0167] In some embodiments, the nucleobase changes are located outside the protospacer sequence in less than 1% of the subject's total liver cells, as measured by net nucleobase editing. In some embodiments, the nucleobase changes are located outside the protospacer sequence in less than 1% of the subject's liver cells, as measured by net nucleobase editing. In some embodiments, the nucleobase changes are located only inside the protospacer sequence, as measured by net nucleobase editing.

[0124]

[0168] In some embodiments, the nucleobase changes are outside the protospacer sequence in less than 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 65%, 80%, 85%, 90% of total liver cells of the subject, as measured by net nucleobase editing. In some embodiments, the nucleobase changes are outside the protospacer sequence in less than 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 65%, 80%, 85%, 90% of the subject's hepatocytes, as measured by net nucleobase editing. In some embodiments, the nucleobase changes are outside the protospacer sequence in less than 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 65%, 80%, 85%, 90% of the subject's cells, as measured by net nucleobase editing.

[0125]

[0169] In some embodiments, the deaminase is an adenine deaminase. In some embodiments, the nucleobase change is an A·T to G·C change. In some embodiments, the deaminase is an adenine deaminase and the nucleobase change is an A·T to G·C change. In some embodiments, the programmable DNA binding domain comprises a nuclease-inactive Cas9 or a Cas9 nickase. In some embodiments, the programmable DNA binding domain comprises Cas9.

[0126]

[0170] In some embodiments, the nucleobase variation is at a splice site of the PCSK9 gene. In some embodiments, the nucleobase change is at a splice donor site of the PCSK9 gene. In some embodiments, the splice donor site is at the 5' end of PCSK9 intron 1, as referenced in SEQ ID NO: 5. In some embodiments, the nucleobase change is at a splice acceptor site of the PCSK9 gene. In some embodiments, the nucleobase change results in a frameshift, premature stop codon, insertion, or deletion in a transcript encoded by the PCSK9 gene. In some embodiments, the nucleobase change results in an aberrant transcript encoded by the PCSK9 gene. In some embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the guide RNA comprises a tracrRNA sequence. In some embodiments, the guide RNA comprises a chemical modification. Exemplary guide RNAs comprising chemical modifications can be found in PCT Application Publication No. WO2021 / 207712, which is incorporated herein by reference in its entirety.

[0127]

[0171] In some embodiments, the nucleobase change is at a splice site of the ANGPTL3 gene. In some embodiments, the nucleobase change is at a splice donor site of the ANGPTL3 gene. In some embodiments, the splice donor site is at the 5' end of ANGPTL3 intron 6, as referenced in SEQ ID NO: 1. In some embodiments, the nucleobase change is at a splice acceptor site of the ANGPTL3 gene. In some embodiments, the nucleobase change results in a frameshift, premature stop codon, insertion, or deletion in a transcript encoded by the ANGPTL3 gene. In some embodiments, the nucleobase change results in an aberrant transcript encoded by the ANGPTL3 gene. In some embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the guide RNA comprises a tracrRNA sequence. In some embodiments, the guide RNA comprises a chemical modification. Exemplary guide RNAs comprising chemical modifications can be found in PCT Application Publication No. WO 2021 / 207712, which is incorporated herein by reference in its entirety.

[0128]

[0172] In some embodiments, the guide RNA comprises a guide RNA sequence. Additional exemplary guide RNA sequences can be found in PCT Application Publication No. WO2021 / 207712, which is incorporated by reference in its entirety.

[0129]

[0173] In some embodiments, the protospacer sequence comprises the sequence 5'-CCCGCACCTTGGCGCAGCGG-3' (SEQ ID NO: 731), AAGATACCTGAATAACTCTC-3' (SEQ ID NO: 732), and 5'-AAGATACCTGAATAACCCTC-3' (SEQ ID NO: 733).

[0130]

[0174] In some embodiments, the base editor fusion protein comprises the sequence of SEQ ID NO: 734. In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 734, or to any of the adenosine deaminases provided herein. It is understood that the adenosine deaminases provided herein can include one or more mutations (e.g., any of the mutations provided herein). The present disclosure provides any deaminase domain having a certain percent identity, as well as any of the mutations described herein or a combination thereof. In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 734, or to any of the adenosine deaminases provided herein. The adenosine deaminases include amino acid sequences having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to the amino acid sequence of any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues compared to any one of the amino acid sequences set forth in SEQ ID NO: 734, or any of the adenosine deaminases provided herein.

[0131]

[0175] In some embodiments, the nucleic acid encoding the base editor protein is an mRNA. The mRNA may comprise a modification, for example, a modification at the 3' or 5' end of the mRNA. In some embodiments, the mRNA comprises a cap analog.

[0132]

[0176] In some embodiments, the mRNA comprises at least one, two, or three nucleotides at the 5' end comprising a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least one, two, or three nucleotides at the 5' end comprising a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least one nucleotide at the 5' end comprising a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least two nucleotides at the 5' end comprising a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least three nucleotides at the 5' end comprising a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least four nucleotides at the 5' end comprising a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least five nucleotides at the 5' end comprising a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least six nucleotides at the 5' end comprising a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least seven nucleotides at the 5' end comprising a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least eight nucleotides at the 5' end comprising a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof.In some embodiments, the mRNA comprises at least 9 nucleotides at the 5' end that comprise a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least 10 nucleotides at the 5' end that comprise a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof.

[0133]

[0177] In some embodiments, the mRNA comprises a poly-A tail, which may be at the 3' end of the mRNA.

[0134]

[0178] In some embodiments, the GC% content of the mRNA sequence is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC% content of the mRNA sequence is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

[0135]

[0179] In some embodiments, the mRNA sequence comprises an adenine tTNA deaminase (TadA) region. In some embodiments, the GC% of the TadA region is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC% content of the TadA region is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

[0136]

[0180] In some embodiments, the mRNA sequence comprises a Cas9 region. In some embodiments, the GC% of the Cas9 region is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%. In some embodiments, the GC% content of the Cas9 region is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%.

[0137]

[0181] In some embodiments, the mRNA sequence comprises an NLS region, in which the GC% of the NLS region is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%. In some embodiments, the GC% content of the NLS region is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

[0138]

[0182] In some embodiments, the mRNA sequence comprises a first linker region connecting the TadA region and the Cas9 region. In some embodiments, the GC% of the first linker region is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. The GC % content of the linker region is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

[0139]

[0183] In some embodiments, the mRNA sequence comprises a second linker region connecting the Cas9 region and the NLS region. In some embodiments, the GC% of the second linker region is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC % content of the second linker region is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

[0140]

[0184] In some embodiments, the base editor systems provided herein further comprise a lipid nanoparticle (LNP) (i) surrounding a guide polynucleotide or a nucleic acid encoding the guide polynucleotide. In some embodiments, the LNP further surrounds a base editor fusion protein (ii) comprising a programmable DNA-binding domain and a deaminase, or nucleic acids encoding them. In some embodiments, the base editor systems further comprise a second LNP surrounding a base editor fusion protein (ii) comprising a programmable DNA-binding domain and a deaminase, or nucleic acids encoding them.

[0141]

[0185] The base editor systems provided herein may comprise one or more LNPs. For example, a base editor system may comprise an LNP surrounding both a guide polynucleotide and a nucleic acid encoding a base editor fusion protein, e.g., an mRNA encoding the base editor fusion protein. In another example, a base editor system may comprise an LNP surrounding a guide polynucleotide (e.g., a guide RNA) and an LNP surrounding a nucleic acid (e.g., an mRNA) encoding a base editor fusion protein. LNPs separately surrounding a guide polynucleotide and a base editor fusion protein or an mRNA encoding a base editor fusion protein may allow for flexible dosing and administration of the base editor system. For example, an LNP surrounding a guide RNA may be administered first, followed by an LNP surrounding an mRNA encoding a base editor fusion protein. In some embodiments, an LNP surrounding a guide RNA and a second LNP surrounding an mRNA encoding a base editor fusion protein are administered to a subject simultaneously. In some embodiments, an LNP surrounding a guide RNA and an LNP surrounding an mRNA encoding a base editor fusion protein are administered sequentially to a subject. In some embodiments, an LNP surrounding an mRNA encoding a base editor fusion protein is administered to a subject, followed by multiple administrations or doses of a second LNP surrounding a guide RNA 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more later. The multiple doses of the second LNP may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days apart or longer.

[0142]

[0186] In some embodiments, the ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 1:10 to about 101 by weight. In some embodiments, the ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 1:1, 1.5:1, 2:1, 3:1, 4:1, 1:1.5, 1:2, 1:3, 1:4, or any ratio between 4:1 or 1:4 ... The base editor fusion protein can be determined by titration of the nucleic acid encoding the base editor fusion protein with a polynucleotide.

[0143]

[0187] In some embodiments, the molar ratio of guide polynucleotide to nucleic acid encoding the base editor fusion protein is about 500:1 to about 1:500.

[0144]

[0188] In some embodiments, the ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 1000:1 to about 1:1000 by weight. In some embodiments, the ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40: 1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1. In some embodiments, the ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000.In some embodiments, the ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is at least about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1. In some embodiments, the ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is at least about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:15. 0, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000. In some embodiments, the ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is at most about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 4 0:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1.In some embodiments, the ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is at most about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1 4, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000.

[0145]

[0189] In some embodiments, the molar ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 10,000:1 to about 1:10,000. In some embodiments, the molar ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 10,000:1, 9,500:1, 9,000:1, 8,500:1, 8,000:1, 7,500:1, 7,000:1, 6,500:1, 6,000:1, 5,500:1, 5,000:1, 4,500:1, 4,000:1, 3,500:1, 3,000:1, 2,500:1, 2,000:1, 1,500:1, 1,000:1, 9,500:1 , 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 6 7:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29: 1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is at least about 1:10000, 1:9500, 1: 9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:5 00, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:62 :60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1.In some embodiments, the molar ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is at least about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 9000:1, 1 ... 50:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68: 1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 2 9:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1.In some embodiments, the molar ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:5 ...500, 1:500, 1:500, 1:500, 1:650, 1:600, 1:500, 1:500, 1:500, 1:500, 1:500, 1:650, 1:600, 1: :350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66 , 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1. 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1.In some embodiments, the molar ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is at most about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 9500:1, 10 ... 0:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1 , 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29 :1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1.In some embodiments, the molar ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is at most about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1: 950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68 ,1:67,1:66,1:65,1:64,1:63,1:62,1:61,1:60,1:59,1:58,1:57,1:56,1:55,1:54,1:53,1:52,1:51,1:50,1:49,1:48,1:47,1:46,1:45,1:44,1:43,1:42,1:41,1:40,1:39,1:38,1:37,1:36,1:35,1:34,1:33,1:32,1:31,1:30,1: 29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1.

[0146]

[0190] In some embodiments, the ratio of nucleic acid encoding the guide polynucleotide to nucleic acid encoding the base editor fusion protein is about 10:1 to about 1:10 by weight. In some embodiments, the ratio of nucleic acid encoding the guide polynucleotide to nucleic acid encoding the base editor fusion protein is about 4:1, 3:1, 2:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73: The molar ratio of nucleic acid encoding the guide polynucleotide to nucleic acid encoding the base editor fusion protein is 1:1, 1:1.5, 1:2, 1:3, or 1:4. In some embodiments, the molar ratio of nucleic acid encoding the guide polynucleotide to nucleic acid encoding the base editor fusion protein is about 500:1 to about 1:500.

[0147]

[0191] In some embodiments, the ratio of nucleic acid encoding a guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 1000:1 to about 1:1000 by weight. In some embodiments, the ratio of nucleic acid encoding a guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1 by weight. , 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1. In some embodiments, the ratio of nucleic acid encoding a guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000.In some embodiments, the ratio of nucleic acids encoding guide polynucleotides to nucleic acids encoding base editor fusion proteins is at least about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 4 5:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1. In some embodiments, the ratio of nucleic acid encoding a guide polynucleotide to nucleic acid encoding a base editor fusion protein is at least about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:. 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000. In some embodiments, the ratio of nucleic acids encoding guide polynucleotides to nucleic acids encoding base editor fusion proteins is at most about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45 ... :1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1. In some embodiments, the ratio of nucleic acid encoding a guide polynucleotide to nucleic acid encoding a base editor fusion protein is at most about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13 by weight , 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000.

[0148]

[0192] In some embodiments, the molar ratio of nucleic acid encoding a guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 10,000:1 to about 1:10,000. In some embodiments, the molar ratio of nucleic acid encoding a guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 10,000:1, 9,500:1, 9,000:1, 8,500:1, 8,000:1, 7,500:1, 7,000:1, 6,500:1, 6,000:1, 5,500:1, 5,000:1, 4,500:1, 4,000:1, 3,500:1, 3,000:1, 2,500:1, 2,000:1, 1,500:1, 1,000:1 , 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68 :1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 2 9:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of nucleic acid encoding a guide polynucleotide to nucleic acid encoding a base editor fusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:80 00, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:4 00, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59 , 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25 , 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1.In some embodiments, the molar ratio of nucleic acids encoding guide polynucleotides to nucleic acids encoding base editor fusion proteins is at least about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 10 00:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1 , 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1 , 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1.In some embodiments, the molar ratio of nucleic acids encoding guide polynucleotides to nucleic acids encoding base editor fusion proteins is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:4 00, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1: 66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1. 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1.In some embodiments, the molar ratio of nucleic acids encoding guide polynucleotides to nucleic acids encoding base editor fusion proteins is at most about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 100 0:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1.In some embodiments, the molar ratio of nucleic acid encoding a guide polynucleotide to nucleic acid encoding a base editor fusion protein is at most about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1 000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1.

[0149]

[0193] Precision genome editing is an area of ​​growing industrial, agricultural, and biomedical applications. One of the leading genome editing systems available today is clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated 9 (Cas9). By using a guide RNA (gRNA) with a sequence homologous to a sequence of DNA (known as a protospacer) in the target genome adjacent to a specific protospacer adjacent motif (PAM), including the sequence NGG (where N is any standard base), Cas9 can be used to create a double-strand break (DSB) at the targeted sequence. Non-homologous end joining (NHEJ) at the DSB can be used to create an indel at the locus and knock out a gene. Similarly, homology-directed repair (HDR) can be used with introduced template DNA to insert a gene or modify the targeted sequence. Various Cas9-based tools have been developed in recent years, including tools that methylate DNA, recognize a wider sequence space, or create single-strand nicks. In 2016, Komor et al. described the use of CRISPR-Cas9 to convert cytosine bases to thymine bases without introducing a template DNA strand and without the need for a DSB (Komor AC, Kim YB, Packer MS, et al. Programmable editing of a target DNA strand). base in genomic DNA without double-stranded DNA cleavage. Nature, 2016, 533:420-4, incorporated herein by reference in its entirety. After fusing the cytidine deaminase domain of rat APOBEC1 to the N-terminus of catalytic death Cas9 (dCas9) using the linker XTEN (resulting in a fusion protein called base editor 1, or BE1), cytosine-to-uracil conversion was observed between positions 4 and 8 within the 20-nt protospacer region of DNA (or, to express it differently, 13–17 nucleotides upstream of the PAM). Notably, any cytosine base within this “window” was editable, resulting in various outcomes depending on how many cytosines and which cytosines were edited. After DNA replication or repair, each uracil was replaced with a thymine, completing the C-to-T base editing. The next version of the base editor (BE2) incorporated a uracil glycosylase inhibitor fused to the C-terminus of dCas9, helping to inhibit base excision repair of the uracil base resulting from cytidine deaminase activity (which would otherwise act to restore the original cytosine base). This improved the efficiency of C to T base editing. The final version, BE3, used the Cas9 nickase instead of dCas9. The nickase cleaves the unedited strand opposite the edited C to T base, stimulating removal of the opposing guanidine via eukaryotic mismatch repair. BE2 and BE3 base editing was observed in both human and mouse cell lines. The specificity of base editing was further improved by adding mutations to the Cas9 nickase. In a similar manner, Cas9 has been mutated to narrow the width of its editing window from approximately 5 nucleotides to just 1–2 nucleotides (Rees HA, Komor et al., 2014). AC,Yeh WH,et al.Improving the DNA specificity and applicability of base editing through protein engineering and protein deliveryNat Commun,2017,8:15790,Kim YB,Komor AC,Levy JM,et al.Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol, 2017, 35:371-6, each of which is incorporated herein by reference in its entirety).

[0150]

[0194] An alternative cytosine base editing platform was demonstrated by linking the activation-induced cytosine deaminase domain PmCDA1 to dCas9 (Target-AID), which enabled targeted C to T base editing in yeast. Furthermore, an alternative C to T base editing strategy was also demonstrated without fusing the deaminase domain to Cas9. Instead, an SH3 (Src3 homology) domain was added to the C-terminus of dCas9, and an SHL (SH3-interacting ligand) was added to PmCDA1.6. The optimal efficiency was achieved. This was achieved by using Cas9 nickase rather than dCas9. Furthermore, a uracil DNA glycosylase inhibitor was added to enhance base editing in a mammalian CHO cell line. The resulting platform was able to consistently edit bases within 3–5 bases of the 18th nucleotide upstream of the PAM sequence (Nishida K et al., 2014). al.,Science,2016,353:aaf8729).

[0151]

[0195] A separate cytosine base editing platform used Cpf1 (also known as Cas12a) instead of Cas9 as the RNA-guided endonuclease. Catalytically inactive Cpf1 was fused to APOBEC1 (dLbCpf1-BE0), resulting in C to T conversion in human cell lines. While the Cas9 base editor variants BE3 and target-AID recognize the PAM sequence NGG, dLbCpf1-BE0 recognizes the T-rich PAM sequence TTTV. Base editing was observed between positions 8 and 13 of the protospacer sequence with dLbCpf1-BE0, but the introduction of additional mutations into Cpf1 could reduce the window to positions 10–12. However, narrowing the base editing window correlated with decreased editing efficiency (Li X et al., Nat. 2014). Biotechnol,2018,36:324-7).

[0152]

[0196] Cytosine base editing is not completely predictable. Indels can occur at target sites, albeit at a lower frequency than observed for C-to-T editing editors. Furthermore, cytosine editing editors can occasionally cause C-to-A or C-to-G edits rather than the expected C-to-T edits. A cytosine base editor, designated "BE4," was improved by adding a linker length of 16 to 32 amino acids between the Cas9 nickase and the rat APOBEC1 cytosine deaminase domain, a linker of 4 to 9 amino acids between the Cas9 nickase and a uracil glycosylase inhibitor, and a second uracil glycosylase inhibitor attached to the C-terminus of the new cytosine base editor using another 9-amino acid linker (Komor AC et al., Sci Adv, 2017, 3:eaao4774).

[0153]

[0197] Fusing the Escherichia coli adenine tTNA deaminase TadA (ecTadA) to dCas9 and mutagenesis of the ecTadA domain combined with selection for editing activity revealed that the A106V and D108N mutations yielded a base editor, designated ABE7.10, that allowed adenine to guanine editing in DNA (Gaudelli NM et al., Nature, 2017, 551:464-71). Koblan et al. improved the efficiency of ABE7.10 by modifying the nuclear localization signal and codon optimization to obtain a version called ABEmax. A similar approach improved the efficiency of the cytosine base editor BE4.10. Huang et al. further developed both adenine and cytosine base editors using alternative PAMs to extend their editing windows, thereby increasing their target range (Koblan LW et al., Nat Biotechnol, 2018, 36:843-6). Comparison of Cas9, cytosine base editors, and adenine base editors using the same gRNA has shown different off-target profiles, but this is also true for base editors (12-14).

[0154]

[0198] Comparison of Cas9, cytosine base editors, and adenine base editors, which use the same gRNA, shows different off-target profiles, but this is also found to be true for base editors.

[0155]

[0199] Various studies have demonstrated gRNA-independent off-regulation, triggered by the deaminase domain acting alone (without requiring DNA engagement by the Cas9-gRNA complex). This raises concerns about targeted base editing. Further studies have shown that the gRNA-independent off-target effects of base editors are not limited to DNA. RNA sequencing of cells treated with either a cytosine or adenine base editor revealed transcriptome-wide off-target editing of RNA, and the introduction of amino acid substitutions in the deaminase domain of this adenine base editor (e.g., R106W) reduced off-target editing of RNA without substantially reducing on-target DNA base editing efficiency. Zuo E, Sun Y, Wei W, et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos.Science,2019,364:289-92, Jin S,Zong Y,Gao Q,et al.Cytosine,but not adenine,base editors induce genome-wide off-target mutations in rice.Science,2019,364:292-5,Grunewald J,Zhou R,Garcia SP,et al.Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.Nature,2019,569:433-7, Grunewald J,Zhou R,Iyer S,et al.CRISPR DNA base editors with reduced RNA off-target and self-editing activities.Nat Biotechnol,2019,37:1041-8, Zhou C,Sun Y,Yan R,et al.Off-target RNA mutation induced by DNA Base editing and its elimination by mutagenesis. Nature, 2019, 571:275-8; Rees HA, Wilson C, Doman JL, et al. Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv, 2019, 5:eaax5717 (each of which is incorporated by reference in its entirety).

[0156]

[0200] Correction of disease-causing mutations through precision editing using standard Cas9 genome editing primarily requires HDR. Because HDR is limited to cells in the S or G2 phase of mitosis, precision editing of non-mitotic cells is challenging. However, base editors do not rely on HDR. Editing of postmitotic cochlear cells in mice is feasible using the cytosine base editor BE3. By injecting BE3 and gRNA in the form of a preassembled ribonucleoprotein via cationic liposomes, serine-33 in beta-catenin was edited to phenylalanine (TCT codon edited to TTT), enabling transdifferentiation of supporting cells to hair cells. Base editing in cochlear tissue was confirmed by sequencing, showing editing rates between 0.7% and 3.0%, depending on the cochlear region. In contrast, standard Cas9 editing via HDR showed negligible signs of efficacy in cochlear cells. A SaBE3 variant delivered to the liver via an adeno-associated virus (AAV) vector has been reported to directly correct a pathogenic T-to-C mutation in the Pah gene with a high editing rate of 29%, thereby treating the disease phenylketonuria in adult mice. Adenine base editing has also been shown to generate mutations in mice. Yeh WH, Chiang H, Rees HA, et al. In vivo base editing of post-mitotic sensory cells. Nat Commun, 2018, 9:2184; Villiger L, Grisch-Chan HM, Lindsay H, et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.Nat Med,2018,24:1519-25, Ryu SM, Koo T, Kim K, et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat Biotechnol, 2018, 36:536-9; Song CQ, Jiang T, Richter M, et al. Adenine base editing in an adult mouse model of tyrosinemia. Nat Biomed Eng, 2020, 4:125-30; Pisciotta L, Favari E, Magnolo L, et al. Characterization of three kindreds with familial combined hypolipidemia caused by loss-of-function mutations of ANGPTL3. Circ Cardiovasc Genet, 2012, 5:42-50 (each of which is incorporated by reference in its entirety).

[0157]

[0201] Provided herein are nucleobase editor system compositions that include nucleobase editor proteins, complexes, or compounds that can effect modifications or conversions to nucleobases (e.g., A, T, C, G, or U) within a target nucleotide sequence.

[0158]

[0202] Nucleic acid base editor or base editor (BE) refers to an agent comprising a polypeptide that can make modifications to bases (e.g., A, T, C, G, or U) in a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, a base editor can deaminate a base in a nucleic acid. In some embodiments, a base editor can deaminate a base in a DNA molecule. In some embodiments, a base editor can deaminate adenine (A) in DNA.

[0159]

[0203] In some embodiments, the base editor comprises a fusion protein comprising a programmable DNA-binding protein fused to an adenosine deaminase. In some embodiments, the base editor comprises a fusion protein comprising a Cas9 protein and an adenosine deaminase. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to an adenosine deaminase. In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, the base editor comprises a fusion protein comprising a programmable DNA-binding protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a fusion protein comprising a Cas9 protein and a cytidine deaminase. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a cytidine deaminase. In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to adenosine deaminase. In some embodiments, the base editor further comprises an inhibitor of base excision repair, e.g., a UGI domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair, e.g., a UGI or dISN domain. In some embodiments, the dCas9 domain of the fusion protein comprises D10A and H840A mutations, as numbered in the wild-type SpCas9 amino acid sequence. In some embodiments, UGI comprises the following amino acid sequence:

[0160]

[0204] >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SD APE YKPW ALVIQDS NGENKIKML (SEQ ID NO: 735).

[0161]

[0205] In some embodiments, the base editor systems provided herein comprise a base editor fusion protein. For example, the base editor fusion protein can comprise a programmable DNA-binding protein and a deaminase, such as an adenosine deaminase. In some embodiments, any of the fusion proteins provided herein is a base editor. In some embodiments, the programmable DNA-binding protein is a Cas9 domain, a Cpf1 domain, a CasX domain, a CasY domain, a Cas12b domain, a C2c2 domain, an aC2c3 domain, or an Argonaute domain. In some embodiments, the programmable DNA-binding protein is a Cas9 domain. The Cas9 domain can be any of the Cas9 domains or Cas9 proteins provided herein (e.g., a nuclease-inactive Cas9 or Cas9 nickase, or a Cas9 variant from any species). In some embodiments, any of the Cas9 domains or Cas9 proteins provided herein can be fused to any of the deaminases provided herein. In some embodiments, the base editor comprises a deaminase (e.g., adenosine deaminase) and a programmable DNA-binding protein (e.g., a Cas9 domain) connected via a linker. In some embodiments, the base editor comprises a fusion protein comprising a deaminase (e.g., adenosine deaminase) and a programmable DNA-binding protein (e.g., a Cas9 domain) connected via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is present between the deaminase domain and the Cas9 domain. In some embodiments, the deaminase and the programmable DNA-binding domain are fused via any of the peptide linkers provided herein. For example, the adenosine deaminase and the Cas9 domain may be fused via a linker comprising between 1 and 200 amino acids.In some embodiments, the adenosine deaminase and the programmable DNA binding protein are 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 60, 5 to 80, 5 to 100, 5 to 150, 5 to 200, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10-60, 10-80, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-80, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-80, 30-100, 30-150, 30-200, 40-50, 40-60, 40-80, 40-100, 40-150, 40-200, 50-60 The adenosine deaminase and the programmable DNA binding protein are fused via a linker comprising 50 to 80, 50 to 100, 50 to 150, 50 to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200, or 150 to 200 amino acids in length. In some embodiments, the adenosine deaminase and the programmable DNA binding protein are fused via a linker comprising 4, 16, 32, or 104 amino acids in length. In some embodiments, the adenosine deaminase and the programmable DNA binding protein are fused via a linker comprising the amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 736), SGGS (SEQ ID NO: 737), SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 738), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 739), or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 740). In some embodiments, the adenosine deaminase and the programmable DNA binding protein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 736), which may also be referred to as an XTEN linker. In some embodiments, the linker is 24 amino acids in length.In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 741). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 742). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 743). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 744).

[0162]

[0206] In some embodiments, the base editor systems provided herein comprise a base editor comprising a fusion protein comprising an inhibitor of base repair. In some embodiments, the base editor comprises a fusion protein comprising a cytidine deaminase and a programmable DNA binding domain (e.g., a Cas9 domain). In some embodiments, the base editor comprises a fusion protein comprising an adenosine deaminase and a programmable DNA binding domain (e.g., a Cas9 domain). In some embodiments, the base editor or fusion protein further comprises an inhibitor of base repair (IBR). In some embodiments, the IBR comprises an inhibitor of inosine base repair. In some embodiments, the IBR is an inhibitor of inosine base excision repair. In some embodiments, the inhibitor of inosine base excision repair is a catalytically inactive inosine-specific nuclease (dISN). In some embodiments, the dISN can inhibit inosine-removing enzymes from removing inosine residues from DNA (e.g., by steric hindrance). For example, catalytically dead inosine glycosylases (e.g., alkyladenine glycosylase [AAG]) bind to inosine but do not create an abasic site or remove the inosine, thereby sterically shielding the newly formed inosine moiety from potential DNA damage / repair mechanisms. Thus, the present disclosure contemplates fusion proteins comprising a programmable DNA-binding protein and an adenosine deaminase further fused to a dISN. The present disclosure also contemplates fusion proteins comprising any Cas9 domain, such as a Cas9 nickase (nCas9) domain, a catalytically inactive Cas9 (dCas9) domain, a high-fidelity Cas9 domain, or a Cas9 domain with reduced PAM exclusivity. It should be understood that the use of a dISN can increase the editing efficiency of adenosine deaminases capable of catalyzing the A to I modification. For example, a fusion protein comprising a dISN domain may be more efficient at deaminating A residues.

[0163]

[0207] In some embodiments, base editors provided herein comprise fusion proteins further comprising one or more nuclear targeting sequences, e.g., a nuclear localization sequence (NLS). In some embodiments, the fusion protein comprises multiple NLSs. In some embodiments, the fusion protein comprises an NLS at the N-terminus and C-terminus of the fusion protein. In some embodiments, the NLS comprises an amino acid sequence that facilitates transport of the protein comprising the NLS to a cell nucleus. In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of a programmable DNA binding protein (e.g., Cas9). In some embodiments, the NLS is fused to the C-terminus of a programmable DNA binding protein. In some embodiments, the NLS is fused to the N-terminus of an adenosine deaminase. In some embodiments, the NLS is fused to the C-terminus of an adenosine deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS has the amino acid sequence of any one of the NLS sequences provided or referenced herein. In some embodiments, the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 745) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 746). Additional nuclear localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are described in Plank et al., PCT / EP2000 / 011690, the contents of which are incorporated herein by reference for disclosure of exemplary nuclear localization sequences.

[0164]

[0208] In some embodiments, the fusion proteins provided herein do not comprise a linker. In some embodiments, a linker is present between one or more of the domains or proteins (e.g., adenosine deaminase, napDNAbp, NLS, and / or IBR). In some embodiments, the "-" used in the general architecture above indicates the presence of an optional linker.

[0165]

[0209] Some aspects of the present disclosure provide base editors or fusion proteins comprising a programmable DNA-binding protein and at least two adenosine deaminase domains. Without wishing to be bound by any particular theory, dimerization of adenosine deaminases (e.g., in cis or trans) may improve the ability (e.g., efficiency) of the fusion protein to modify a nucleobase, for example, the ability (e.g., efficiency) to deaminate adenine. In some embodiments, any of the fusion proteins may comprise two, three, four, or five adenosine deaminase domains. In some embodiments, any of the fusion proteins provided herein comprises two adenosine deaminases. In some embodiments, any of the fusion proteins provided herein contains only two adenosine deaminases. In some embodiments, the adenosine deaminases are the same. In some embodiments, the adenosine deaminase is any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminases are different. In some embodiments, the first adenosine deaminase is any of the adenosine deaminase provided herein, and the second adenosine deaminase is any of the adenosine deaminase provided herein, but is not identical to the first adenosine deaminase. In some embodiments, the first adenosine deaminase, when numbered in SEQ ID NO: 747, comprises any one of the mutations provided herein. In some embodiments, the second adenosine deaminase, when numbered in SEQ ID NO: 747, comprises any one of the mutations provided herein. In some embodiments, the first adenosine deaminase, when numbered in SEQ ID NO: 747, comprises any one of the mutations provided herein, and the second adenosine deaminase comprises a wild-type adenosine deaminase sequence. In some embodiments, the second adenosine deaminase comprises any one of the mutations provided herein, and the first adenosine deaminase comprises a wild-type adenosine deaminase sequence, as numbered in SEQ ID NO: 747.As one example, a fusion protein can comprise a first adenosine deaminase and a second adenosine deaminase, both of which comprise the A106V, D108N, D147Y, and E155V mutations from ecTadA (SEQ ID NO: 747). As another example, a fusion protein can comprise a first adenosine deaminase domain that comprises the A106V, D108N, D147Y, and E155V mutations from ecTadA (SEQ ID NO: 747) and a second adenosine deaminase that comprises the L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F mutations from ecTadA (SEQ ID NO: 747).

[0166]

[0210] In some embodiments, the adenosine deaminase comprises the following amino acid sequence: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHEIMALRQGGLVMQNYRLIDATL YVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 747).

[0167]

[0211] In some embodiments, the fusion protein comprises two adenosine deaminase (e.g., a first adenosine deaminase and a second adenosine deaminase). In some embodiments, the first adenosine deaminase is N-terminal to the second adenosine deaminase in the fusion protein. In some embodiments, the first adenosine deaminase is C-terminal to the second adenosine deaminase in the fusion protein. In some embodiments, the first adenosine deaminase and the second deaminase are fused directly or via a linker. In some embodiments, the linker is any of the linkers provided herein, e.g., any of the linkers described in the "Linkers" section. In some embodiments, the first adenosine deaminase is the same as the second adenosine deaminase. In some embodiments, the first adenosine deaminase and the second adenosine deaminase are any of the adenosine deaminase described herein. In some embodiments, the first adenosine deaminase and the second adenosine deaminase are different. In some embodiments, the first adenosine deaminase is any of the adenosine deaminase provided herein. In some embodiments, the second adenosine deaminase is any of the adenosine deaminase provided herein, but is not identical to the first adenosine deaminase. In some embodiments, the first adenosine deaminase is ecTadA adenosine deaminase. In some embodiments, the first adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 747, or to any of the adenosine deaminases provided herein.In some embodiments, the second adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO:747, or to any of the adenosine deaminases provided herein. In some embodiments, the second adenosine deaminase comprises the amino acid sequence of SEQ ID NO:747.

[0168]

[0212] In some embodiments, the fusion proteins provided herein do not include a linker, ie, a linker is present between one or more of the domains or proteins (e.g., the first adenosine deaminase, the second adenosine deaminase, the programmable DNA-binding protein, and / or the NLS).

[0169]

[0213] It should be understood that the fusion proteins of the present disclosure may include one or more additional features. For example, in some embodiments, the fusion protein may include an export sequence, such as a cytoplasmic localization sequence, a nuclear export sequence, or other localization sequence, as well as a sequence tag useful for solubilizing, purifying, or detecting the fusion protein. Suitable protein tags provided herein include biotin carboxylase carrier protein (BCCP) tag, myc tag, calmodulin tag, FLAG tag, hemagglutinin (HA) tag, polyhistidine tag, also known as histidine tag or His tag, maltose binding protein (MBP) tag, nus tag, glutathione-S-transferase (GST) tag, green fluorescent protein (GFP) tag, thioredoxin tag, S tag, Softag (e.g., Softag1, Softag3), strep tag, biotin ligase tag, FlA tag, and the like. Examples of suitable sequences include, but are not limited to, sH tags, V5 tags, and SBP tags. Additional suitable sequences will be apparent to those skilled in the art. In some embodiments, the fusion protein comprises one or more His tags.

[0170]

[0214] In some embodiments, the fusion protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the amino acid sequences listed in Table 4. In some embodiments, the fusion protein comprises any one of the amino acid sequences listed in Table 4. In some embodiments, the sequence of the fusion protein is any one of the amino acid sequences listed in Table 4. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the amino acid sequences in SEQ ID NOs: 688, 691, 695, 702, 705, and 707. In some embodiments, the sequence of the fusion protein is any one of the amino acid sequences in SEQ ID NOs: 688 and 702.

[0171]

[0215] In some embodiments, the fusion protein is encoded by a polynucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences listed in Table 4. In some embodiments, the fusion protein is encoded by any one of the polynucleotide sequences listed in Table 23. In some embodiments, the fusion protein is expressed by a polynucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences listed in Table 4. In some embodiments, the fusion protein is expressed by any one of the polynucleotide sequences listed in Table 4. In some embodiments, the fusion protein is encoded by a polynucleotide sequence comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences in SEQ ID NOs: 687, 690, 694, 701, 704, and 706. In some embodiments, the fusion protein is encoded by a polynucleotide sequence comprising any one of the polynucleotide sequences in SEQ ID NOs: 687, 690, 694, 701, 704, and 706. In some embodiments, the fusion protein is encoded by any one of the polynucleotide sequences in SEQ ID NOs: 687 and 701.In some embodiments, the fusion protein is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least identical to any one of the polynucleotide sequences of SEQ ID NOs: 687, 690, 694, 701, 704, and 706. In some embodiments, the fusion protein is expressed by a polynucleotide sequence comprising at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to the polynucleotide sequence in SEQ ID NOs: 687, 690, 694, 701, 704, and 706, or a combination thereof. In some embodiments, the fusion protein is encoded by a polynucleotide sequence comprising any one of the polynucleotide sequences in SEQ ID NOs: 687, 690, 694, 701, 704, and 706, or a combination thereof. In some embodiments, the fusion protein is expressed by any one of the polynucleotide sequences in SEQ ID NOs: 687, 690, 694, 701, 704, and 706. In some embodiments, the polynucleotide sequence quality further comprises one that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences of SEQ ID NOs: 687 and 701.

[0172]

[0216] In some embodiments, the nucleobase editor ABE8.8 comprises a fusion protein comprising the sequence provided below. LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 734).

[0173]

[0217] Amino acid and nucleotide changes in MA040, MA041, and MA045 compared to MA004: MA004 has a codon CGG at amino acid position 691 of nCas9 and a codon GAC at amino acid position 1135. MA040 has a nCas9 amino acid mutation of D1135E. Codon 1135 is changed to GAG. MA041 has nCas9 amino acid mutations of R691A and D1135E. Codon 691 is changed to GCC and codon 1135 is changed to GAG. MA045 has a nCas9 amino acid mutation of R691A. Codon 691 is changed to GCC.

[0174]

[0218] Additional adenosine deaminase mutations and variants are described in patent application WO2018 / 119354, which is incorporated herein by reference in its entirety.

[0175]

[0219] Additional adenosine deaminases useful in the present application will be apparent to those skilled in the art and are within the scope of the present disclosure. For example, the adenosine deaminase may be a homolog of AD AT. Exemplary AD AT homologs include, but are not limited to:

[0176]

[0220] Staphylococcus aureus TadA: MGSHMTNDIYFMTLAIEEAKKAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAH AEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCS GS LMNLLQQS NFNHRAIVDKG VLKE AC S TLLTTFFKNLRANKKS TN(District 748)

[0177]

[0221] Bacillus subtilis TadA: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEML VIDE AC COLD WRLEG ATLY VTLEPCPMC AG AV VLS R VEK V VFG AFDPKGGC S GTLMN LLQEERFNHQ AE V VS G VLEEEC GGMLS AFFRELRKKKK A ARKNLS E(Office 749)

[0178]

[0222] Salmonella typhimurium(S.typhimurium)TadA: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEG WNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIG RVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIK ALKKADRAEGAGPAV (Knowledge Base 750)

[0179]

[0223] Shewanella putrefaciens (S. putrefaciens)TadA: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAF NHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE (SEQ ID NO: 751)

[0180]

[0224] Haemophilus influenzae F3031(H.influenzae)TadA: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAΑΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK (SEQ ID NO: 752)

[0181]

[0225] Caulobacter crescentus (C.crescentus)TadA: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI (SEQ ID NO: 753)

[0182]

[0226] Geobacter sulfurreducens(G.sulfurreducens)TadA: MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALF IDERKVPPEP (SEQ ID NO: 754)

[0183]

[0227] In other aspects, the present disclosure provides base editing systems and methods for editing polynucleotides. For example, genome / base editing systems and methods for editing polynucleotides encoding ANGPTL3 and variants thereof are provided herein. To edit a target gene, a target gene polynucleotide may be contacted with a system disclosed herein comprising an sgRDNA and an adenosine base editor protein, wherein the sgRDNA comprises a spacer sequence disclosed herein and a tracrRNA sequence, wherein the spacer sequence hybridizes to a target polynucleotide sequence in the ANGPTL3 gene, and the tracrRNA sequence binds to the adenosine base editor protein (e.g., the Cas9 component of the adenosine base editor). Thus, the sgRDNA can guide the base editor protein to the target polynucleotide sequence, resulting in an a to G modification in the target gene. The target gene or target polynucleotide may be a gene encoding ANGPTL3. The target polynucleotide sequence may be within the ANGPTL3 gene. The modification can reduce or abolish expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene in a cell. The introduction can be via lipid nanoparticles containing the composition.

[0184]

[0228] For example, an sgRDNA and an adenosine base editor protein may be expressed in a cell (e.g., a liver cell) in which targeted gene editing is required, thereby allowing the target gene to be contacted with a composition (e.g., an sgRNA and an adenosine base editor protein) disclosed herein. In some embodiments, binding of the adenosine base editing protein to its target polynucleotide sequence in the target gene is directed by a single guide RDNA disclosed herein, for example, a single guide RDNA comprising (i) a spacer sequence disclosed herein and (ii) a tracrRNA sequence. As shown, the spacer sequence hybridizes with the target polynucleotide sequence in the target gene. Thus, by designing the guide RDNA sequence, the adenosine base editor protein can be directed to edit any target polynucleotide sequence in a target gene (e.g., a target gene encoding ANGPTL3). The guide RDNA sequence can be co-expressed with the adenosine base editor protein in a cell in which editing is desired. To edit a gene encoding an ANGPTL3 protein, the gene is contacted with the system described herein. The target polynucleotide sequence in the target gene can be contacted with a single guide RDNA disclosed herein and an adenosine base editor protein or a nucleic acid sequence encoding an adenosine base editor protein, where the single guide RDNA directs the adenosine base editor protein to effect a modification in the target gene (e.g., a target gene encoding ANGPTL3). The target polynucleotide sequence can be a locus in the genomic DNA of a cell. The cell can be a cultured cell. The cell can be in vivo, in vitro, or ex vivo.

[0185]

[0229] The base editor system provided herein may include a programmable DNA-binding protein and a deaminase. As used herein, deaminase may refer to an enzyme that catalyzes the removal of an amine group from a molecule, or deamination, for example, by hydrolysis. In some embodiments, the deaminase is a cytidine deaminase, which catalyzes the deamination of cytidine (C) to uridine (U), deoxycytidine (dC) to deoxyuridine (dU), or 5-methylcytidine to thymidine (T, 5-methyl-U), respectively. Subsequent DNA repair mechanisms ensure that dU is replaced by T, as described in Komor et al., Nature, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, 533, 420-424 (2016), the entire contents of which are incorporated herein by reference. In some embodiments, the deaminase is a cytosine deaminase, which catalyzes and promotes the conversion of cytosine to uracil (e.g., in RNA) or thymine (e.g., in DNA). In some embodiments, the deaminase is an adenosine deaminase, which catalyzes and promotes the conversion of adenine to guanine. In some embodiments, the deaminase is a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is a variant of a naturally occurring deaminase from an organism, wherein the variant does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring deaminase from an organism.

[0186]

[0230] Cytidine deaminase (or cytosine deaminase) includes enzymes that catalyze the chemical reactions "cytosine + H20 → uracil + NH3" or "5-methyl-cytosine + H20 → thymine + NH3." In the context of a gene, such a nucleotide change or mutation can in turn cause a change in the amino acid of a protein, which can affect the function of the protein, for example, loss of function or gain of function. Subsequent DNA repair mechanisms ensure that the uracil base in DNA is replaced by T, as described in Komor et al. (Nature, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, 533, 420-424 (2016)), the entire contents of which are incorporated herein by reference.

[0187]

[0231] One exemplary and suitable class of cytosine deaminases is the apolipoprotein B mRNA editing complex (APOBEC) family of cytosine deaminases, which encompasses 11 proteins that serve to initiate mutagenesis in a controlled and beneficial manner. Apolipoprotein B editing complex 3 (APOBEC3) enzymes provide protection for human cells against certain HIV-1 strains through the deamination of cytosines in reverse-transcribed viral ssDNA. All of these cytosine deaminases require a Zn-coordination motif (His-X-Glu-X23_26-Pro-Cys-X2_4-Cys (SEQ ID NO: 29)) and a bound water molecule for catalytic activity. A glutamic acid residue acts to activate the water molecule to zinc hydroxide for nucleophilic attack in the deamination reaction. Each family member preferentially deaminates at its own specific "hot spot," e.g., WRC (W is A or T, and R is A or G) in the case of hAID, or TTC in the case of hAPOBEC3F. The recent crystal structure of the catalytic domain of APOBEC3G revealed a secondary structure containing a five-stranded β-sheet core flanked by six α-helices, which is believed to be conserved throughout the family. The active central loop has been shown to be involved in both ssDNA binding and determining "hot spot" identity. Overexpression of these enzymes has been associated with genomic instability and cancer, highlighting the importance of sequence-specific targeting. Another suitable cytosine deaminase is activation-induced cytidine deaminase (AID), which is involved in antibody maturation by converting cytosine in ssDNA to uracil in a transcription-dependent, strand-biased manner.

[0188]

[0232] Adenosine deaminases (or adenine deaminases) include enzymes that catalyze the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, adenosine deaminases catalyze the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be from any organism, such as bacteria. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism. In some embodiments, the deaminase or deaminase domain is not naturally occurring. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring deaminase. In some embodiments, the adenosine deaminase is from a bacterium, such as E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the adenosine deaminase is E. coli TadA deaminase. In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, a truncated ecTadA may lack one or more N-terminal amino acids relative to full-length ecTadA, hi some embodiments, a truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length ecTadA.In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to full-length ecTadA. In some embodiments, the ecTadA deaminase does not include an N-terminal methionine.

[0189]

[0233] In some embodiments, the adenosine deaminase is set forth in SEQ ID NO:747. The present disclosure provides any deaminase domain having a particular percent identity, as well as any of the mutations described herein, or a combination thereof. In some embodiments, the adenosine deaminase comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to the amino acid sequence set forth in SEQ ID NO: 747, or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues compared to any one of the amino acid sequences set forth in SEQ ID NO: 747 or any of the adenosine deaminases provided herein. Additional exemplary adenosine deaminases can be found in PCT Application Publication No. WO 2021 / 207712, which is incorporated herein by reference in its entirety.

[0190]

[0234] In some embodiments, an adenine base editor comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the amino acid sequences listed in Table 2 or Table 4. In some embodiments, an adenine base editor comprises any one of the amino acid sequences listed in Table 2 or Table 4. In some embodiments, the sequence of the adenine base editor is any one of the amino acid sequences listed in Table 2 or Table 4. In some embodiments, an adenine base editor comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the amino acid sequences in SEQ ID NOs: 688, 691, 695, 702, 705, 707, 718, 720, 722, 724, 726, 728, and 730. In some embodiments, an adenine base editor comprises any one of the amino acid sequences in SEQ ID NOs: 688, 691, 695, 702, 705, 707, 718, 720, 722, 724, 726, 728, and 730. In some embodiments, the sequence of the adenine base editor is any one of the amino acid sequences in SEQ ID NOs: 688, 702, 718, 720, 722, 724, 726, 728, and 730.

[0191]

[0235] In some embodiments, an adenine base editor has a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least 149%, at least 150%, at least 151%, at least 152%, at least 153%, at least 154%, at least 155%, at least 156%, at least 157%, at least 158%, at least 159%, at least In some embodiments, the adenine base editor is encoded by a polynucleotide sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences listed in Table 2 or Table 4. In some embodiments, the adenine base editor is encoded by a polynucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences listed in Table 2 or Table 4. In some embodiments, the adenine base editor is expressed by any one of the polynucleotide sequences listed in Table 2 or Table 4. In some embodiments, the adenine base editor is encoded by a polynucleotide sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences in SEQ ID NOs: 687, 690, 694, 701, 704, 706, 717, 719, 721, 723, 725, 727, and 729. In some embodiments, the adenine base editor is encoded by a polynucleotide sequence comprising any one of the polynucleotide sequences in SEQ ID NOs: 687, 690, 694, 701, 704, 706, 717, 719, 721, 723, 725, 727, and 729. In some embodiments, the adenine base editor is encoded by any one of the polynucleotide sequences in SEQ ID NOs: 687, 701, 717, 719, 721, 723, 725, 727, and 729.

[0192] Prime Edit

[0236] The disclosed gene editing system may include a prime-editing CRISPR system or its components. Prime editing is a variation of the CRISPR system that expands the role of the guide RNA to serve two purposes: to guide Cas9 to the targeted genomic location and to serve as an RNA template for copying new sequences into the DNA genome (Anzalone AV et al., Nature volume 576, pages 149-157 (2019)). Like CRISPR, prime editing requires the presence of a catalytically modified Cas endonuclease and a single guide RNA. The Cas9 endonuclease is catalytically modified to become a Cas9 nickase that nicks DNA rather than generating a double-strand break. The Cas9 nickase is fused to a reverse transcriptase. The prime-editing guide RNA (pegRNA) is significantly larger than a standard sgRNA. The pegRNA is an sgRNA with a primer binding sequence (PBS) and a template containing the desired RNA sequence added to the 3' end. Further information regarding prime editing can be found in published PCT application WO2020 / 191245A1, which is incorporated herein in its entirety.

[0193]

[0237] The hybrid guide gene editing system disclosed herein may include a polymerase. The polymerase functions to catalyze the polymerization of a nucleic acid strand using an existing nucleic acid as a template. Examples of useful polymerases include DNA polymerases and RNA polymerases. The polymerase may function with a nucleic acid-programmable nucleotide-binding domain or a nucleic acid-programmable DNA-binding protein (e.g., in the form of a fusion protein or bound or associated in trans with the hybrid guide nucleic acid sequence). The polymerase may be an RNA-dependent DNA polymerase (e.g., reverse transcriptase) or a DNA-dependent DNA polymerase (e.g., a prokaryotic polymerase including Pol I, Pol II, or Pol III, or a prokaryotic polymerase including Pol a, Pol b, Pol g, or Pol β). The polymerase may be a eukaryotic polymerase, including Pol d, Pol e, or Pol z.

[0194] Editing Efficiency

[0238] The gene editing system disclosed herein can achieve high efficiency in gene editing with low off-target editing effects. For example, a gene editor protein in a gene editing system having a guide nucleic acid containing a deoxyribonucleotide-containing motif(s) in a spacer disclosed herein can affect less than 10% of editing at all off-target sites compared to a gene editing system having a guide nucleic acid sequence that does not contain deoxyribonucleotides. For example, a gene editor protein in a gene editing system having a guide nucleic acid sequence containing any of the motifs in a spacer sequence disclosed herein can affect less than 7% of editing at all off-target sites compared to a gene editing system having a guide nucleic acid sequence that does not contain deoxyribonucleotides. A gene editor protein in a gene editing system having a guide nucleic acid sequence containing any of the motifs in a spacer sequence disclosed herein can affect less than 5% of editing at all off-target sites compared to a gene editing system having a guide nucleic acid sequence that does not contain deoxyribonucleotides. A gene editor protein in a gene editing system having a guide nucleic acid sequence comprising any of the motifs in its spacer sequence disclosed herein may affect less than 2% editing at all off-target sites compared to a gene editing system having a guide nucleic acid sequence without deoxyribonucleotides. A gene editor protein in a gene editing system having a guide nucleic acid sequence comprising any of the motifs in its spacer sequence disclosed herein may affect less than 1% editing at all off-target sites compared to a gene editing system having a guide nucleic acid sequence without deoxyribonucleotides. A gene editor protein in a gene editing system having a guide nucleic acid sequence comprising any of the motifs in its spacer sequence disclosed herein may affect less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% editing at all off-target sites compared to a gene editing system having a guide nucleic acid sequence without deoxyribonucleotides.

[0195]

[0239] A gene editor protein in a gene editing system having a guide nucleic acid sequence comprising any of the motifs in its spacer sequence disclosed herein can affect at least 50% editing in a target gene compared to a gene editing system having a guide nucleic acid sequence that does not contain deoxyribonucleotides. A gene editor protein in a gene editing system having a guide nucleic acid sequence comprising any of the motifs in its spacer sequence disclosed herein can affect at least 70% editing in a target gene compared to a gene editing system having a guide nucleic acid sequence that does not contain deoxyribonucleotides. A gene editor protein in a gene editing system having a guide nucleic acid sequence comprising any of the motifs in its spacer sequence disclosed herein can affect at least 90% editing in a target gene compared to a gene editing system having a guide nucleic acid sequence that does not contain deoxyribonucleotides. A gene editor protein in a gene editing system having a guide nucleic acid sequence comprising any of the motifs in its spacer sequence disclosed herein can affect more than 50% editing in a target gene compared to a gene editing system having a guide nucleic acid sequence that does not contain deoxyribonucleotides. Gene editor proteins in gene editing systems having a guide nucleic acid sequence comprising any of the motifs in its spacer sequence disclosed herein can affect greater than 70% editing in a target gene compared to a gene editing system having a guide nucleic acid sequence that does not comprise deoxyribonucleotides. Gene editor proteins in gene editing systems having a guide nucleic acid sequence comprising any of the motifs in its spacer sequence disclosed herein can affect greater than 90% editing in a target gene compared to a gene editing system having a guide nucleic acid sequence that does not comprise deoxyribonucleotides. Gene editor proteins in gene editing systems having a guide nucleic acid sequence comprising any of the motifs in its spacer sequence disclosed herein can affect greater than 90% editing in a target gene compared to a gene editing system having a guide nucleic acid sequence that does not comprise deoxyribonucleotides. The quality may affect at least 50%, 60%, 70%, 80%, or 90% editing in the target gene compared to a gene editing system with a guide nucleic acid sequence that does not contain deoxyribonucleotides. A gene editor protein in a gene editing system with a guide nucleic acid sequence that includes any of the motifs in a spacer sequence disclosed herein may affect more than 50%, 60%, 70%, 80%, or 90% editing in the target gene compared to a gene editing system with a guide nucleic acid sequence that does not contain deoxyribonucleotides.

[0196] Gene Editor Proteins

[0240] Provided herein are gene editor proteins comprising a nucleic acid binding domain. The nucleic acid binding domain may be capable of binding to DNA. The nucleic acid binding domain may be capable of binding to RNA. The nucleic acid binding domain may be a Cas9 domain. The term "Cas9" refers to an RNA-guided nuclease, including a Cas9 protein or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9). Cas9 nucleases are also referred to as Casnl nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nucleases. Cas9 may refer to a polypeptide having at least or at least about 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9 can refer to a polypeptide having at most, or at most, about 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 can refer to wild-type or modified forms of the Cas9 protein, which can include amino acid alterations such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0197]

[0241] Cas9 nuclease sequences and structures of variant Cas9 orthologs have been described in various species. Exemplary species from which the Cas9 protein or other components may be derived include, but are not limited to, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogenes, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polar omonas naphthalenivorans, Polar omonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulum osiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionium, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillator ia Examples of suitable Cas9 proteins include: Bacillus sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, or Acaryochloris marina. In some embodiments, the Cas9 protein is derived from Streptococcus pyogenes. In some embodiments, the Cas9 protein may be derived from Streptococcus thermophilus. In some embodiments, the Cas9 protein is derived from Staphylococcus aureus.

[0198]

[0242] Based on the present disclosure, additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art, including Cas9 sequences from the organisms and loci disclosed in Chylinski et al., (2013) RNA Biology 10:5, 726-737, which is incorporated herein by reference.

[0199]

[0243] The gene editing systems provided herein may include gene editor proteins, such as Cas nucleases, with reduced or disabled nuclease activity. For example, a Cas9 protein may be nuclease-inactive or may be a Cas9 nickase. Methods for generating a Cas9 protein (or a fragment thereof) with an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28;152(5):1173-83 (2013)). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: an HNH nuclease subdomain and a RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, and the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al, Cell. 28;152(5):1173-83(2013)). Cas9 nickases suitable for use according to the present disclosure have an active HNH domain and an inactive RuvC domain and can cleave only the strand of target DNA bound by the sgRNA (which is the strand opposite to the strand being edited via cytidine deamination). The Cas9 nickases of the present disclosure can include a mutation that inactivates the RuvC domain, e.g., a D10A mutation. It is understood that any mutation that inactivates the RuvC domain can be included in the Cas9 nickase, e.g., an insertion, deletion, or single or multiple amino acid substitution in the RuvC domain. In the Cas9 nickases described herein, the RuvC domain The RuvC domain is inactivated, but the HNH domain remains activated. Thus, the Cas9 nickase may contain mutations other than those that inactivate the RuvC domain (e.g., D10A), but these mutations do not affect the activity of the HNH domain. In a non-limiting example of a Cas9 nickase, the histidine at position 840 remains unchanged.

[0200]

[0244] Additional suitable mutations that inactivate Cas9 will be apparent to those skilled in the art based on this disclosure and knowledge in the art and are within the scope of this disclosure. Exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D839A and / or N863A (see, e.g., Prashant et al., Nature Biotechnology. 2013;31(9):833-838, incorporated herein by reference), or K603R (see, e.g., Chavez et al., Nature Methods 12,326-328, 2015, incorporated herein by reference). Cas9, dCas9, or Cas9 variants also encompass Cas9, dCas9, or Cas9 variants derived from any organism. It is also understood that dCas9, Cas9 nickase, or other suitable Cas9 variants derived from any organism may be used in accordance with the present disclosure.

[0201]

[0245] The Cas9 can be a Cas9 from the Archaea (e.g., Nanoarchaea), which comprise the domain and kingdom of unicellular prokaryotic microorganisms.

[0202]

[0246] Gene editor proteins may include CasX or CasY, or variants thereof, as described, for example, in Burstein et al., Cell Res. 2017 Feb 21. doi:10.1038 / cr.2017.21.

[0203]

[0247] The gene editor protein may comprise a high-fidelity Cas9 domain. A high-fidelity Cas9 domain is an engineered Cas9 domain that contains one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA compared to the corresponding wild-type Cas9 domain. Without wishing to be bound by any particular theory, a high-fidelity Cas9 domain with reduced electrostatic interaction with the sugar-phosphate backbone of DNA may have fewer off-target effects. The Cas9 domain may contain one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA. The Cas9 domain may contain one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or more. Any of the Cas9s provided herein may include one or more of the following mutations: N497X, R661X, Q695X, and / or Q926X, where X is any amino acid, as numbered in the wild-type Cas9 amino acid sequence or another Cas9. The Cas9 may include one or more of the following mutations: N497A, R661A, Q695A, and / or Q926A of the amino acid sequence provided in the wild-type Cas9 sequence, or the corresponding mutations as numbered in the corresponding amino acids in the wild-type Cas9 amino acid sequence or another Cas9. Cas9 domains with high fidelity are described in Kleinstiver, BP, et al., Nature 529, 490-495 (2016) and Slaymaker, IM, et al., Science 351, 84-88 (2015), the entire contents of each of which are incorporated herein by reference.

[0204]

[0248] Any of the gene editing systems provided herein may comprise any of the Ca2+ gene editing systems described herein. It should be understood that the s9 domain can be converted into a high-fidelity gene editing system by modifying it to generate a high-fidelity gene editor protein that includes a high-fidelity Cas9 domain, which can be a nuclease-inactive Cas9 domain or a Cas9 nickase domain.

[0205]

[0249] Gene editor proteins may comprise one or more nuclease domains. Gene editor proteins of the present disclosure may comprise an HNH or HNH-like nuclease domain, a RuvC or RuvC-like nuclease domain, and / or a HEPN superfamily-like nuclease. The HNH or HNH-like domain may comprise an McrA-like fold. The HNH or HNH-like domain may comprise two antiparallel β-strands and an a-helix. The HNH or HNH-like domain may comprise a metal binding site (e.g., a divalent cation binding site). The HNH or HNH-like domain can cleave one strand of a target nucleic acid (e.g., the complementary strand of the crRNA targeting strand). Proteins comprising an HNH or HNH-like domain may include endonucleases, colicins, restriction endonucleases, transposases, and DNA packaging factors.

[0206]

[0250] The gene editor protein may be a Cas9 protein, a Cpfl protein, a C2cl protein, a C2c2 protein, a C2c3 protein, Cas3, Cas5, Cas7, Cas8, Cas10, or a complex thereof, depending on the particular CRISPR system used. The gene editor protein may be a Cas9 protein or a Cpfl protein. The gene editor protein may have reduced nuclease activity. In some cases, the gene editor protein may be a nickase, i.e., modified to cleave one strand of a target nucleic acid duplex. The gene editor protein may be modified to have no nuclease activity, i.e., not to cleave any strand of a target nucleic acid duplex or any single strand of the target nucleic acid. Examples of gene editor proteins with reduced or no nuclease activity include, but are not limited to, Cas9 with modifications to the HNH nuclease domain and / or RuvC nuclease domain, and Cpfl with modifications to the RuvC nuclease domain. Non-limiting examples of such modifications include F. novicida These include changes to the RuvC nuclease domain of Cpfl, such as D917A, E1006A, and D1225A, and residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 of S. pyogenes Cas9, as well as their corresponding amino acid residues in other Cpfl and Cas9 proteins.

[0207]

[0251] Any variant of Cas9 known in the art may also be a gene editor protein. In some cases, the Cas9 variant is a catalytically inactive Cas9 (dCas9). dCas9 may have mutations in its two nuclease domains and thus lack nuclease activity. dCas9 can function as a programmable sequence-specific DNA-binding protein. dCas9 can be used to physically block the transcription process, turn off specific genes, or shuttle other proteins to specific sites in the genome. As used herein, "dCas" and "dCas protein" are used interchangeably and refer to catalytically inactive CRISPR-associated proteins. In some cases, the dCas protein contains one or more mutations in the DNA cleavage domain. The dCas protein may contain one or more mutations in the RuvC domain or the HN'H domain. The dCas protein may contain one or more mutations in both the RuvC domain and the HN'H domain. dCas can be a fragment of a wild-type Cas molecule. The dCas may comprise a functional domain derived from a wild-type Cas molecule, the functional domain being selected from a Reel domain, a bridge helix domain, or a PAM-interacting domain. The nuclease activity of the dCas molecule may be reduced or eliminated compared to the nuclease activity of the corresponding wild-type Cas. It can be reduced by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0208]

[0252] A suitable dCas can be derived from a wild-type Cas. Cas can be derived from a type I, type II, or type III CRISPR-Cas system. In some cases, a suitable dCas protein is derived from a Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, or Cas10 molecule. dCas can be derived from a Cas9 molecule. A dCas9 molecule can be obtained, for example, by introducing point mutations (e.g., substitutions, deletions, or additions) into a DNA cleavage domain, e.g., a nuclease domain, e.g., the RuvC domain and / or the HNH domain, in a Cas9 molecule. See, e.g., Jinek et al., Science (2012) 337:816-21, the entire contents of which are incorporated herein by reference. For example, introducing two point mutations into the RuvC domain and the HNH domain reduces Cas9 nuclease activity while retaining Cas9 sgRNA and DNA binding activity. In some cases, the two point mutations in the RuvC and HNH active sites are the DIOA and H840A mutations of an S. pyogenes Cas9 molecule. Alternatively, D10 and H840 of an S. pyogenes Cas9 molecule can be deleted to abolish Cas9 nuclease activity while maintaining its sgRNA and DNA binding activity. In some cases, the two point mutations in the RuvC and HNH active sites are the DIOA and N580A mutations of an S. aureus Cas9 molecule. The dCas can be an S. aureus dCas9 molecule containing mutations at D10 and / or N580. The dCas can be an S. aureus dCas9 molecule containing the DIOA and / or N580A mutations. The dCas can be an S. aureus dCas9 molecule, any variant or mutant, or any fragment thereof.

[0209]

[0253] Similar mutations can be applied to any other naturally occurring Cas9 (e.g., Cas9 from other species) or engineered Cas9 molecules, including Streptococcus pyogenes dCas9 molecules, Staphylococcus aureus dCas9 molecules, Campylobacter jejuni dCas9 molecules, Corynebacterium diphtheria dCas9 molecules, Eubacterium ventriosum dCas9 molecules, Streptococcus pasteurianus dCas9 molecules, Lactobacillus farciminis dCas9 molecule, Sphaerochaeta globus The dCas9 molecule may include a dCas9 molecule, an Azospirillum (strain B510) dCas9 molecule, a Gluconacetobacter diazotrophicus dCas9 molecule, a Neisseria cinerea dCas9 molecule, a Roseburia intestinalis dCas9 molecule, a Parvibaculum lavamentivorans dCas9 molecule, a Nitratifractor salsuginis (strain DSM 16511) dCas9 molecule, a Campylobacter lari (strain CF89-12) dCas9 molecule, a Streptococcus thermophilus (strain LMD-9) dCas9 molecule, or a fragment thereof.

[0210]

[0254] The gene editor proteins disclosed herein can be modified. Such modifications can include incorporating or fusing a domain from another polypeptide into the gene editor protein, or replacing a domain of the gene editor protein with a domain of another polypeptide. For example, a modified gene editor protein can contain a first domain from a Cas9 or Cpfl protein and a second domain from a protein other than Cas9 or Cpfl. Modifying a modified gene editor protein to include such domains can confer additional activities to the modified gene editor protein. Such activities can include modifying polypeptides that associate with target nucleic acids (e.g., histones). The activity can include a nuclease activity, a methyltransferase activity, a demethylase activity, a DNA repair activity, a DNA damage activity, a deamination activity, a dismutase activity, an alkylation activity, a depurination activity, an oxidation activity, a pyrimidine dimer formation activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, a photolyase activity, a glycosylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitination activity, an adenylation activity, a deadenylation activity, a sumoylation activity, a desumoylation activity, a ribosylation activity, a deribosylation activity, a myristoylation activity, or a demyristoylation activity).

[0211]

[0255] Gene editor proteins can introduce double-strand or single-strand breaks in nucleic acid sequences (e.g., genomic DNA). The nucleic acid sequence can be a target nucleic acid. Gene editor proteins can introduce blunt-end cleavage sites or generate sticky ends, i.e., cleavage sites with 5' or 3' overhangs. For example, Cpfl can introduce staggered DNA double-strand breaks with approximately 4 or 5 nucleotide (nt) 5' overhangs. The double-strand breaks can stimulate the cell's endogenous DNA repair pathways (e.g., NHEJ or alternative non-homologous end joining (A-NHEJ)). NHEJ can repair the cleaved target nucleic acid without the need for a homologous template, which can result in deletion of the target nucleic acid. Homologous recombination (HR) can be performed using a homologous template. The homologous template can include sequences homologous to sequences adjacent to the target nucleic acid cleavage site. After the target nucleic acid is cleaved by the gene editor protein, the cleavage site can be destroyed (e.g., the site may not be accessible for another round of cleavage by the nucleic acid-targeting polynucleotide and site-directed polypeptide).

[0212]

[0256] The gene editor protein can comprise a nucleic acid binding domain and thus can bind to a nucleic acid. The nucleic acid can be a DNA sequence. The DNA sequence can be a target DNA sequence. The nucleic acid binding domain can be a DNA binding domain.

[0213]

[0257] Any DNA-binding domain can be used in the system disclosed herein. The DNA-binding domain can include a zinc finger protein. The zinc finger protein can be non-naturally occurring in that it is engineered to bind to a selected target site. Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416. The engineered zinc finger binding domain can have novel binding specificity compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design, for example, involves using a database containing triplex (or quadruplex) nucleotide sequences and individual zinc finger amino acid sequences, where each triplex or quadruplex nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a particular triplex or quadruplex sequence. See, for example, co-owned U.S. Patent Nos. 6,453,242 and 6,534,261, which are incorporated herein by reference in their entireties.

[0214]

[0258] Exemplary selection methods, including phage display and two-hybrid systems, are described in U.S. Patent Nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,410,248, 6,140,466, 6,200,759, and 6,242,568, and WO 98 / 3718. 6, WO 98 / 53057, WO 00 / 27878, WO 01 / 88197, and GB 2,338,237. Additionally, enhanced binding specificity for zinc finger binding domains is described, for example, in co-owned WO 02 / 077227.

[0215]

[0259] The systems described herein can use meganuclease (homing endonuclease) DNA-binding domains to bind to target nucleic acids. Naturally occurring meganucleases recognize 15-40 base pair cleavage sites and are generally grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family, and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII. Their recognition sequences are known. See also U.S. Pat. No. 5,420,032, U.S. Pat. No. 6,833,252, Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388, Dujon et al. (1989) Gene 82:115-118, Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127, Jasin (1996) Trends Genet. 12:224-228, Gimble et al. (1996) J. Mol. Biol. 263:163-180, Argast et al. (1998) J. Mol. Biol. 280:345-353, and the New England Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites (see, e.g., Chevalier et al. (2002) Molec. Cell 10:895-905, Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962, Ashworth et al. (2006) Nature 441:656-659, Paques et al. (2007) Current Gene Therapy 7:49-66, U.S. Patent Publication No. 2007 / 0117128).The DNA binding domain of homing endonucleases and meganucleases may vary relative to the overall nuclease (ie, such that the nuclease contains a cognate cleavage domain) or may be fused to a heterologous cleavage domain.

[0216]

[0260] One or more DNA-binding domains of the nucleases used in the methods and compositions described herein comprise naturally occurring or engineered (non-naturally occurring) TAL effector DNA-binding domains. See, e.g., U.S. Patent No. 8,586,526, which is incorporated herein by reference in its entirety.

[0217] Deaminase domain

[0261] The gene editing system disclosed herein may include a deaminase. As used herein, "deaminase" may refer to an enzyme that catalyzes the removal of an amine group from a molecule, or deamination, for example, by hydrolysis. The deaminase may be a cytidine deaminase, which catalyzes the deamination of cytidine (C) to uridine (U), the deamination of deoxycytidine (dC) to deoxyuridine (dU), or the deamination of 5-methylcytidine to thymidine (T, 5-methyl-U), respectively. The subsequent DNA repair mechanism is described in Komor et al., Nature, Programmable editing of a target base in genomic DNA without double-stranded DNA As described in [Claim 1], cleavage, 533, 420-424 (2016), ensure that dU is replaced by T. The deaminase may be a cytosine deaminase, which converts cytosine to uracil (e.g., in RNA) or to thymine (e.g., in RNA). The deaminase catalyzes and promotes the conversion of adenine to guanine (e.g., in DNA). The deaminase may be an adenosine deaminase, which catalyzes and promotes the conversion of adenine to guanine. The deaminase may be a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. The deaminase may be a variant of a naturally occurring deaminase from an organism, where the variant does not occur in nature. For example, the deaminase or deaminase domain can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring deaminase from an organism.

[0218]

[0262] Cytidine deaminase (or cytosine deaminase) includes enzymes that catalyze the chemical reactions "cytosine + H20 → uracil + NH3" or "5-methyl-cytosine + H20 → thymine + NH3." In the context of a gene, such a nucleotide change, or mutation, can in turn cause a change in the amino acid of a protein, which can affect the function of the protein, for example, loss of function or gain of function. Subsequent DNA repair mechanisms ensure that the uracil base in DNA is replaced by T, as described in Komor et al. (Nature, 533, 420-424 (2016)), which is incorporated herein by reference in its entirety.

[0219]

[0263] One exemplary and suitable class of cytosine deaminases is the apolipoprotein B mRNA editing complex (APOBEC) family of cytosine deaminases, which encompasses 11 proteins that serve to initiate mutagenesis in a controlled and beneficial manner. Apolipoprotein B editing complex 3 (APOBEC3) enzymes provide protection for human cells against certain HIV-1 strains through the deamination of cytosines in reverse-transcribed viral ssDNA. All of these cytosine deaminases require a Zn-coordination motif (His-X-Glu-X23_26-Pro-Cys-X2_4-Cys) and a bound water molecule for catalytic activity. A glutamic acid residue acts to activate the water molecule to zinc hydroxide for nucleophilic attack in the deamination reaction. Each family member preferentially deaminates at its own specific "hot spot," e.g., WRC (W is A or T, and R is A or G) in the case of hAID, or TTC in the case of hAPOBEC3F. The recent crystal structure of the catalytic domain of APOBEC3G revealed a secondary structure containing a five-stranded β-sheet core flanked by six α-helices, which is believed to be conserved throughout the family. The active central loop has been shown to be involved in both ssDNA binding and determining "hot spot" identity. Overexpression of these enzymes has been associated with genomic instability and cancer, highlighting the importance of sequence-specific targeting. Another suitable cytosine deaminase is activation-induced cytidine deaminase (AID), which is involved in antibody maturation by converting cytosine in ssDNA to uracil in a transcription-dependent, strand-biased manner.

[0220]

[0264] Adenosine deaminases (or adenine deaminases) include enzymes that catalyze the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. Adenosine deaminases can catalyze the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be derived from any organism, such as bacteria. The deaminase or deaminase domain can be a variant of a naturally occurring deaminase from an organism. The deaminase or deaminase domain can be non-naturally occurring. For example, the deaminase or deaminase domain can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, or at least 90% similar to a naturally occurring deaminase. The adenosine deaminase may be 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical. The adenosine deaminase may be derived from a bacterium such as E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. The adenosine deaminase may be TadA deaminase. The TadA deaminase may be E. coli TadA deaminase. The TadA deaminase may be a truncated E. coli TadA deaminase. For example, the truncated ecTadA may lack one or more N-terminal amino acids relative to full-length ecTadA. The truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length ecTadA. The truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to full-length ecTadA. The ecTadA deaminase may not include an N-terminal methionine.

[0221]

[0265] It should be understood that the adenosine deaminase provided herein can comprise one or more mutations.Exemplary adenosine deaminase mutations and variants are described in patent application WO2018 / 119354, which is incorporated herein by reference in its entirety.Additional adenosine deaminase useful in this application will be apparent to those skilled in the art and fall within the scope of the present disclosure.

[0222] Guide nucleic acid

[0266] The guide nucleotide sequence may exist as a single nucleotide molecule and include two domains: (1) a domain that shares homology with the target nucleic acid and directs binding of the guide nucleotide sequence gene editor protein to the targeted gene sequence, and (2) a domain that binds to the guide nucleotide sequence gene editor protein. Domain (1) may include a spacer sequence. Domain (2) may be referred to as the tracrRNA sequence or an equivalent, such as a modified tracrRNA. Domain (2) may include a stem-loop structure. For example, domain (2) may be identical to or homologous to the tracrRNA provided in Jinek et al., Science 337:816-821 (2012), which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain (2)) can be found in U.S. Patent Application Publication Nos. US2016 / 0208288 and US2016 / 0200779, each of which is incorporated herein by reference in its entirety.

[0223]

[0267] Methods of using guide nucleotide sequence gene editor proteins such as Cas9 for site-specific cleavage (e.g., to modify a genome) are known in the art (see, e.g., Cong, L. et al., Science 339, 819-823 (2013); Mali, P. et al., Science 339, 823-826 (2013); Hwang, WY et al., Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., eLife 2, e00471 (2013); Dicarlo, JE et al., Nucleic acids research (2013); Jiang, W. et al., Nature biotechnology 31, 233-239 (2013), each of which is incorporated herein by reference).

[0224]

[0268] The guide nucleic acid of the gene editing system disclosed herein may comprise a DNA-RNA chimeric guide (i.e., chRDNA). The chRDNA may be a single guide RDNA. As used herein, the term "RDNA" refers to a ribonucleotide It refers to nucleic acids that contain a mixture of ribonucleotides (RNA) and deoxynucleotides (DNA), i.e., RDNA contains at least one ribonucleotide and at least one deoxynucleotide.

[0225]

[0269] As used herein, the terms "sgchRDNA," "sgRDNA," "single guide chRDNA," and "single guide RDNA" are used interchangeably and refer to a polynucleotide comprising a spacer sequence, where the spacer sequence comprises a mixture of DNA and RNA nucleotides complementary to a sequence within a target nucleic acid. The spacer sequence comprises at least one deoxyribonucleotide and at least one ribonucleotide. The ribose of the ribonucleotide of the spacer sequence may be modified. For example, the ribose may comprise a 2' hydroxyl group covalently linked to a methyl group (2'-O-methyl).

[0226]

[0270] As used herein, the terms "sgRNA," "sgRNA," "single guide RNA," and "single guide RNA" are used interchangeably and refer to a polynucleotide comprising a spacer sequence, where the spacer sequence comprises only RNA complementary to a sequence within a target nucleic acid. The spacer sequence comprises only ribonucleotides. The ribose of the ribonucleotides of the spacer sequence may be modified. For example, the ribose may comprise a 2' hydroxyl group covalently linked to a methyl group (2'-O-methyl).

[0227]

[0271] The guide nucleic acid disclosed herein may, for example, comprise a deoxyribonucleotide-deoxyribonucleotide-ribonucleotide-deoxyribonucleotide-deoxyribonucleotide (dN-dN-N-dN-dN) motif. In another example, the guide nucleic acid disclosed herein may comprise a deoxyribonucleotide-deoxyribonucleotide-ribonucleotide-deoxyribonucleotide-deoxyribonucleotide-deoxyribonucleotide (dN-dN-N-dN-dN-dN) motif. The guide nucleic acid comprises a spacer sequence. The spacer sequence comprises at least one deoxyribonucleotide. The spacer sequence may comprise 1 to 10 deoxyribonucleotides. The spacer sequence may comprise 1 to 9 deoxyribonucleotides. The spacer sequence may comprise 1 to 8 deoxyribonucleotides. The spacer sequence may comprise 1 to 7 deoxyribonucleotides. The spacer sequence may comprise 1 to 6 deoxyribonucleotides. The spacer sequence may comprise 1 to 5 deoxyribonucleotides. The spacer sequence may comprise 1 to 4 deoxyribonucleotides. The spacer sequence may comprise 1 to 3 deoxyribonucleotides. The spacer sequence may comprise 1 deoxyribonucleotide. The spacer sequence may comprise 2 deoxyribonucleotides. The spacer sequence may comprise 3 deoxyribonucleotides. The spacer sequence may comprise 4 deoxyribonucleotides. The spacer sequence may comprise 5 deoxyribonucleotides. The spacer sequence may comprise 6 deoxyribonucleotides. The spacer sequence may comprise 7 deoxyribonucleotides. The spacer sequence may comprise 8 deoxyribonucleotides. The spacer sequence may comprise 9 deoxyribonucleotides. The spacer sequence may comprise 10 deoxyribonucleotides.

[0228]

[0272] Positions where ribonucleotides can be replaced with deoxyribonucleotides can include, from the 5' end of the spacer sequence, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, or position 20. In some cases, deoxyribonucleotides are located at positions 3, 4, 6, 7, and / or 8 from the 5' end of the spacer sequence. The spacer sequence can include deoxyribonucleotides at positions 3, 4, 6, and 7 from the 5' end of the spacer sequence. The spacer sequence can include deoxyribonucleotides at positions 3 and 4 from the 5' end of the spacer sequence. The spacer sequence can include deoxyribonucleotides at positions 3 and 4 from the 5' end of the spacer sequence. The spacer sequence may comprise deoxyribonucleotides at positions 6 and 7 from the 5' end of the sequence. The spacer sequence may comprise deoxyribonucleotides at positions 3, 4, 6, 7, and 8 from the 5' end of the spacer sequence. The spacer sequence may comprise deoxyribonucleotides at positions 4, 5, 6, 7, 9, 10, 13, and 14 from the 5' end of the spacer sequence. The spacer sequence may comprise deoxyribonucleotides at positions 3, 4, 5, 6, and 7 from the 5' end of the spacer sequence. The spacer sequence may comprise deoxyribonucleotides at positions 3, 4, 6, and 7 from the 5' end of the spacer sequence. The spacer sequence may comprise deoxyribonucleotides at positions 3, 4, 6, and 7 from the 5' end of the spacer sequence. The spacer sequence may comprise deoxyribonucleotides at positions 3, 4, 6, 7, 8, and 9 from the 5' end of the spacer sequence.

[0229]

[0273] The spacer sequences disclosed herein may contain at least one modified ribonucleotide. For example, the ribonucleotide may be modified so that the 2' hydroxyl group is covalently linked to a methyl group (i.e., 2'-O-methyl). The spacer sequence may contain 1 to 3 modified ribonucleotides. The spacer sequence may contain 1 to 2 modified ribonucleotides. The spacer sequence may contain 1 modified ribonucleotide. The spacer sequence may contain 2 modified ribonucleotides. The spacer sequence may contain 3 modified ribonucleotides. In some cases, the spacer sequence does not have any modified ribonucleotides. The spacer sequence may contain only unmodified ribonucleotides. The spacer sequence may contain only unmodified ribonucleotides and 2'-deoxyribonucleotides. The modified ribonucleotide may be located at the 5' end of the spacer sequence. The modified ribonucleotide may be located at positions 1, 2, and 3 from the 5' end of the spacer sequence. The modified ribonucleotides may be located at positions 1, 3, and 4 from the 5' end of the spacer sequence. The modified ribonucleotides may be located at positions 1 and 2 from the 5' end of the spacer sequence. The modified ribonucleotides may be located at positions 1 and 3 from the 5' end of the spacer sequence. The modified ribonucleotides may be located at positions 2 and 3 from the 5' end of the spacer sequence. The modified ribonucleotides may be located at positions 3 and 4 from the 5' end of the spacer sequence. The modified ribonucleotides may be located at position 1 from the 5' end of the spacer sequence.

[0230]

[0274] In some cases, the spacer sequence has three modified ribonucleotides located at positions 1, 2, and 3 from the 5' end of the spacer sequence. The spacer sequence may have three modified ribonucleotides located at positions 1, 3, and 4 from the 5' end of the spacer sequence. The spacer sequence may have two modified ribonucleotides located at positions 1 and 2 from the 5' end of the spacer sequence. The spacer sequence may have two modified ribonucleotides located at positions 1 and 3 from the 5' end of the spacer sequence. The spacer sequence may have two modified ribonucleotides located at positions 2 and 3 from the 5' end of the spacer sequence. The spacer sequence may have two modified ribonucleotides located at positions 3 and 4 from the 5' end of the spacer sequence. The spacer sequence may have one modified ribonucleotide located at position 1 from the 5' end of the spacer sequence.

[0231]

[0275] In some cases, the spacer sequence comprises five deoxyribonucleotides and two 2'-OMe ribonucleotides. The spacer sequence may comprise five deoxyribonucleotides located at positions 3, 4, 5, 6, and 7 from the 5' end of the spacer sequence and two 2'-OMe ribonucleotides located at positions 1 and 2 from the 5' end of the spacer sequence. The spacer may comprise eight deoxyribonucleotides located at positions 4, 5, 6, 7, 9, 10, 13, and 14 from the 5' end of the spacer sequence and three 2'-OMe ribonucleotides located at positions 1, 2, and 3 from the 5' end of the spacer sequence. The spacer may comprise four deoxyribonucleotides located at positions 3, 4, 6, and 7 from the 5' end of the spacer sequence and two 2'-OMe ribonucleotides located at positions 1 and 2 from the 5' end of the spacer sequence. The spacer contains five deoxyribonucleotides located at positions 3, 4, 6, 7, and 8 from the 5' end of the spacer sequence and two 2'- OMe ribonucleotides.

[0232]

[0276] The spacer sequence may further comprise a phosphorothioate backbone modification (PS). The spacer sequence may comprise at least one phosphorothioate backbone modification. The spacer sequence may comprise at least two phosphorothioate backbone modifications. The spacer sequence may comprise at least three phosphorothioate backbone modifications. The spacer sequence may comprise two or three phosphorothioate backbone modifications. The 5'-terminal nucleotide residue of the spacer may comprise a phosphorothioate backbone modification. The phosphorothioate backbone modification may be between positions 1 and 2 from the 5' end of the spacer sequence. The phosphorothioate backbone modification may be between positions 2 and 3 from the 5' end of the spacer sequence. The phosphorothioate backbone modification may be between positions 3 and 4 from the 5' end of the spacer sequence. The spacer sequence may comprise three phosphorothioate backbone modifications, located from the 5' end of the spacer sequence between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4. The spacer sequence may comprise two phosphorothioate backbone modifications, located from the 5' end of the spacer sequence between positions 1 and 2 and between positions 2 and 3.

[0233]

[0277] The spacer sequence may comprise a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA)(DNA) motif. The spacer sequence may comprise a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA) motif. The spacer sequence may comprise a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(DNA)(DNA)(DNA) motif. The spacer sequence may comprise a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA)(DNA)(DNA) motif. As used herein, "2-OMe" refers to modified RNA in which the 2' hydroxyl group of the ribose of the RNA is covalently linked to a methyl group, "RNA" refers to unmodified RNA, and "DNA" refers to unmodified DNA.

[0234]

[0278] The spacer sequence may comprise or consist of SEQ ID NO: 30 or 31. The spacer sequence may comprise or consist of a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO: 30 or 31. The spacer sequence may comprise or consist of SEQ ID NO: 28 or 29. The spacer sequence may comprise or consist of a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO: 28 or 29. The spacer sequence may comprise or consist of SEQ ID NO: 11 or 12. The spacer sequence may comprise or consist of a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO: 11 or 12. The spacer sequence may comprise or consist of SEQ ID NO: 26 or 27. The spacer sequence may comprise or consist of a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO: 26 or 27. The spacer sequence may comprise or consist of SEQ ID NO: 41. The spacer sequence may comprise or consist of a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO:41.

[0235]

[0279] A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of SEQ ID NO: 110, 111, 112, or 113. A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 110, 111, 112, or 113. A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of SEQ ID NO: 106, 107, 108, or 109. A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO: 106, 107, 108, or 109. A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of SEQ ID NO: 79, 80, 81, or 82. A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO: 79, 80, 81, or 82. A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of SEQ ID NO: 102, 103, 104, or 105. A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 102, 103, 104, or 105. A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of SEQ ID NO: 79, 80, or 82.A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO: 79, 80, or 82. A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of SEQ ID NO: 132. A guide nucleic acid comprising a spacer sequence disclosed herein may comprise or consist of a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or similarity to SEQ ID NO: 132.

[0236]

[0280] The guide nucleic acid disclosed herein can be a single guide nucleic acid containing a spacer sequence at the 5' end and a tracrRNA at the 3' end. The tracrRNA can be modified. For example, some of the ribonucleotides of the tracrRNA can have one or more 2'-OMe modifications. The tracr nucleic acid (e.g., tracrRNA) can have a length of about 50 nucleotides to about 150 nucleotides.

[0237]

[0281] The tracrRNA sequence may contain more than one double-stranded region (e.g., hairpin, hybridized region). The tracrRNA sequence may contain two double-stranded regions. The tracrRNA may contain a secondary structure. The tracrRNA may contain more than one secondary structure. The tracrRNA sequence may contain a first secondary structure and a second secondary structure, where the first secondary structure contains more nucleotides than the second secondary structure. The tracrRNA may contain a first secondary structure, a second secondary structure, and a third secondary structure, where the first secondary structure contains fewer nucleotides than the second secondary structure and the second secondary structure contains more nucleotides than the third secondary structure. The number of secondary structures and the corresponding nucleotide length are not particularly limited.

[0238]

[0282] Unlike type II CRISPR systems, naturally occurring type V CRISPR systems do not require a tracrRNA for crRNA maturation and target nucleic acid cleavage. The tracrRNA may be modified (e.g., 2-OMe modified). The tracrRNA may have the sequence: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu. The tracrRNA may have the sequence: gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu.

[0239]

[0283] The target nucleic acid may comprise DNA, RNA, or a combination thereof, and may be a double-stranded or single-stranded nucleic acid. The spacer sequence can hybridize to the target nucleic acid located 5' or 3' of a protospacer adjacent motif (PAM), depending on the particular gene editor protein used. The PAM can vary depending on the gene editor protein used. For example, when using Cas9 from S. pyogenes, the PAM can be a sequence within the target nucleic acid comprising the sequence 5'-NRR-3', where R can be either A or G, and N is any nucleotide, and N is immediately 3' of the target nucleic acid sequence targeted by the targeting region sequence. The gene editor protein can be modified such that the PAM can be different compared to the PAM for the unmodified gene editor protein. For example, when using Cas9 from S. pyogenes, the Cas9 may be modified so that the PAM no longer comprises the sequence 5'-NRR-3' but instead comprises the sequence 5'-NNR-3', where R can be either A or G, and N is any nucleotide, and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence. Other gene editor proteins may recognize other PAMs, and one of skill in the art can determine the PAM for any particular gene editor protein. For example, Cpfl from Francisella novicida was identified as having a 5'-TTN-3' PAM (Zetsche et al. (Cell; 163(3):759-71 (2015))), but this failed to support site-specific cleavage of the target nucleic acid in vivo. Given the similarity of guide sequences between Francisella novicida and other Cpfl proteins, such as Cpfl from Acidaminocccus sp. BV3L6, which utilize the 5'-TTTN-3' PAM, it is more likely that the Francisella novicida Cpfl protein recognizes and cleaves a site on the target nucleic acid adjacent to the 5'-TTTN-3' PAM with greater specificity and activity than the site on the target nucleic acid adjacent to the cleavage 5'-TTTN-3' PAM incorrectly identified by Zetsche et al.The polynucleotides and CRISPR systems described in this application can be used with a Cpfl protein (e.g., from Francisella novicida) that is directed to a site on a target nucleic acid adjacent to a 5'-TTTN-3' PAM.

[0240]

[0284] The target nucleic acid sequence may be 20 nucleotides. The target nucleic acid may be less than 20 nucleotides. The target nucleic acid may contain at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. The target nucleotides may range from about 5 to 30 nucleotides, and therein. The selection of a particular PAM is within the knowledge of one of ordinary skill in the art based on the particular gene editor protein used in a given case.

[0241]

[0285] The spacer sequence of the present disclosure, which comprises DNA and RNA on the same strand, can be chemically synthesized.The chemical synthesis of polynucleotides is well understood by those skilled in the art.The chemical synthesis of polynucleotides of the present disclosure can be carried out in solution or on a solid support.Solid-phase synthesis is the preferred method for producing guide RNA for early evaluation.

[0242]

[0286] The guide nucleic acid containing DNA can provide the advantage of increasing the specificity of target nucleic acid such as DNA.Without being bound by any particular theory, the spacer sequence comprising DNA in a specific region as discussed herein presents the advantage of reducing off-target binding due to the local structural disturbance in the spacer, thereby reducing off-target site binding.

[0243]

[0287] The spacer sequence of the present disclosure may also include modifications that increase the stability of the polynucleotide, for example. Such modifications may include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates with normal 3'-5' linkages, 2-5' linkage analogs, and those with reversed polarity, where one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages. Suitable nucleic acid targeting polynucleotides with inverted polarity may contain a single 3'-3' linkage at the 3'-most internucleotide linkage (i.e., a single inverted nucleoside residue in which the nucleobase is missing or has a hydroxyl group instead). Various salts (e.g., potassium chloride or sodium chloride), mixed salts, and free acid forms may also be included.

[0244]

[0288] The deoxyribose or ribose (i.e., sugar moiety) on the deoxynucleotide or nucleotide of the spacer sequence can be modified. Examples of modified sugar moieties include, but are not limited to, 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-aminoethyl, 2'-fluoro, N3'→P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic-modified sugars. 2'-O-methyl or 2'-O-methoxyethyl modifications promote an A-form or RNA-like conformation in oligonucleotides, increasing binding affinity to RNA and enhancing nuclease resistance. Modified sugar moieties can also include those with an extra bridge (e.g., a methylene bridge connecting the 2'-O and 4'-C atoms of ribose in locked nucleic acids) or a sugar analog such as a morpholine ring (e.g., phosphorodiamidate morpholino).Examples of such analogs and / or modified residues include diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl These include, but are not limited to, chiral-2-thiouracil, beta-D-mannosylqueosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, methyl phosphonate, chiral-methyl phosphonate, 2'-O-methyl ribonucleotide, peptide nucleic acid (PNA), and the like.

[0245]

[0289] Spacer sequences of the present disclosure may also contain other nucleic acids or nucleic acid analogs, one example of which is peptide nucleic acid (PNA).

[0246] Linker

[0290] The gene editing systems provided herein may include a linker connecting one or more components of the gene editing system. Linkers may be used to link any of the proteins or protein domains described herein. A linker may be as simple as a covalent bond or may be a polymeric linker many atoms in length. A linker may be a polypeptide or may be based on an amino acid. A linker may not be peptide-like. A linker may be a carbon bond, a disulfide bond, a carbon-heteroatom bond, etc. A linker may be a carbon-nitrogen bond of an amide bond. A linker may be a cyclic or acyclic, substituted or unsubstituted, branched or unbranched, aliphatic or heteroaliphatic linker. A linker may be a polymer (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). A linker may include a monomer, dimer, or polymer of aminoalkanoic acid. The linker may comprise an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). The linker may comprise a monomer, dimer, or polymer of aminohexanoic acid (Ahx). The linker may be based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). The linker may comprise a polyethylene glycol moiety (PEG). The linker may comprise an amino acid. The linker may comprise a peptide. The linker may comprise an aryl or heteroaryl moiety. The linker may be based on a phenyl ring. The linker may comprise a functionalized moiety to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, alkyl halides, aryl halides, acyl halides, an...

Claims

1. 1. An in vivo hybrid guide gene editing system, comprising: (a) a gene editor protein or component thereof comprising a nucleic acid binding domain, or a nucleic acid encoding said gene editor protein or component thereof; (b) (i) a spacer sequence comprising deoxyribonucleotides and ribonucleotides comprising a ribose, wherein the 2′ hydroxyl group of the ribose is covalently linked to a methyl group (2′-OMe), and (ii) a hybrid guide nucleic acid comprising a binding scaffold for the gene editor protein or component thereof; An in vivo hybrid guide gene editing system, wherein (a) and (b) are components constituting a pharmaceutical composition comprising a lipid nanoparticle (LNP) containing (a) or (b), and the LNP comprises an amino lipid, a phospholipid, a sterol, and a PEG lipid.

2. 2. The in vivo hybrid guide gene editing system of Claim 1, wherein the spacer sequence corresponds to a protospacer on a target gene, and the target gene is ANGPTL3.

3. 2. The in vivo hybrid guide gene editing system of Claim 1, wherein the gene editor protein or component thereof comprises a deaminase.

4. 1. An in vivo hybrid guide gene editing system, comprising: (a) a gene editor protein or component thereof comprising a nucleic acid binding domain, or a nucleic acid encoding said gene editor protein or component thereof; (b) (i) a spacer sequence comprising deoxyribonucleotides and ribonucleotides, wherein the spacer sequence corresponds to a protospacer on the ANGPTL3 gene, and (ii) a hybrid guide nucleic acid comprising a binding scaffold for the gene editor protein or component thereof; An in vivo hybrid guide gene editing system, wherein (a) and (b) are components constituting a pharmaceutical composition comprising a lipid nanoparticle (LNP) containing (a) or (b), and the LNP comprises an amino lipid, a phospholipid, a sterol, and a PEG lipid.

5. 5. The in vivo hybrid guide gene editing system of Claim 4, wherein the gene editor protein or component thereof comprises a deaminase.

6. 5. The in vivo hybrid guide gene editing system of Claim 4, wherein the ribonucleotide comprises a ribose, the ribose comprising a 2' hydroxyl group covalently linked to a methyl group (2'-OMe).

7. 1. An in vivo hybrid guide gene editing system, comprising: (a) a gene editor protein or component thereof comprising a nucleic acid binding domain and a deaminase, or a nucleic acid encoding said gene editor protein or component thereof; (b) a hybrid guide nucleic acid comprising (i) a spacer sequence comprising deoxyribonucleotides and ribonucleotides, and (ii) a binding scaffold for said gene editor protein or component thereof; An in vivo hybrid guide gene editing system, wherein (a) and (b) are components constituting a pharmaceutical composition comprising a lipid nanoparticle (LNP) containing (a) or (b), and the LNP comprises an amino lipid, a phospholipid, a sterol, and a PEG lipid.

8. The spacer sequence corresponds to a protospacer on a target gene, and the target gene is 8. The in vivo hybrid guide gene editing system of claim 7, wherein the gene is ANGPTL3.

9. 8. The in vivo hybrid guide gene editing system of Claim 7, wherein the ribonucleotide comprises a ribose, the ribose comprising a 2' hydroxyl group covalently linked to a methyl group (2'-OMe).

10. 10. The in vivo hybrid guide gene editing system of any one of claims 1 to 9, wherein the spacer sequence comprises unmodified ribonucleotides.

11. 11. The in vivo hybrid guide gene editing system of any one of claims 1 to 10, wherein the nucleic acid encoding the gene editor protein or component thereof is mRNA.

12. 12. The in vivo hybrid guide gene editing system of any one of claims 1 to 11, wherein the gene editor protein or said components thereof comprise a single fusion protein or two or more proteins.

13. 13. The in vivo hybrid guide gene editing system of any one of claims 1 to 12, wherein the spacer sequence comprises a phosphorothioate backbone modification (PS).

14. 14. The in vivo hybrid guide gene editing system of any one of claims 1 to 13, wherein the spacer sequence comprises a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA)(DNA) motif.

15. 15. The in vivo hybrid guide gene editing system of Claim 14, wherein the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence.

16. 14. The in vivo hybrid guide gene editing system of any one of Claims 1-13, wherein the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 30 or 31.

17. 14. The in vivo hybrid guide gene editing system of any one of claims 1 to 13, wherein the spacer sequence comprises a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA) motif.

18. 18. The in vivo hybrid guide gene editing system of Claim 17, wherein the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence.

19. 14. The in vivo hybrid guide gene editing system of any one of Claims 1-13, wherein the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 28 or 29.

20. 14. The in vivo hybrid guide gene editing system of any one of claims 1 to 13, wherein the spacer sequence comprises a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(DNA)(DNA)(DNA) motif.

21. 21. The in vivo hybrid guide gene editing system of Claim 20, wherein the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(DNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence.

22. 14. The in vivo hybrid guide gene editing system of any one of Claims 1-13, wherein the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 5, 11 or 12.

23. 14. The in vivo hybrid guide gene editing system of any one of Claims 1-13, wherein the hybrid guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to a sequence selected from the group consisting of SEQ ID NOs: 110-113.

24. 14. The in vivo hybrid guide gene editing system of any one of Claims 1-13, wherein the hybrid guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to a sequence selected from the group consisting of SEQ ID NOs: 106-109.

25. 14. The in vivo hybrid guide gene editing system of any one of Claims 1-13, wherein the guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to a sequence selected from the group consisting of SEQ ID NOs:79-82.

26. 26. The in vivo hybrid guide gene editing system of any one of claims 1 to 25, wherein the nucleic acid binding domain is capable of binding to DNA.

27. 26. The in vivo hybrid guide gene editing system of any one of claims 1 to 25, wherein the nucleic acid binding domain is capable of binding to RNA.

28. 14. The in vivo hybrid guide gene editing system of any one of claims 1 to 13, wherein the deoxyribonucleotide is located at position 3, 4, 6, 7 or 8 from the 5' end of the spacer sequence.

29. 14. The in vivo hybrid guide gene editing system of any one of claims 1 to 13, wherein the spacer sequence comprises deoxyribonucleotides at positions 3, 4, 6 and 7 from the 5' end of the spacer sequence.

30. 14. The in vivo hybrid guide gene editing system of any one of claims 1 to 13, wherein the spacer sequence comprises deoxyribonucleotides at positions 3 and 4 from the 5' end of the spacer sequence.

31. 14. The in vivo hybrid guide gene editing system of any one of claims 1 to 13, wherein the spacer sequence comprises deoxyribonucleotides at positions 6 and 7 from the 5' end of the spacer sequence.

32. 14. The in vivo hybrid guide gene editing system of any one of claims 1 to 13, wherein the spacer sequence comprises deoxyribonucleotides at positions 3, 4, 6, 7 and 8 from the 5' end of the spacer sequence.

33. 33. The in vivo hybrid guide gene editing system of any one of claims 1 to 32, wherein the spacer sequence comprises 1 to 10 deoxyribonucleotides.

34. 33. The in vivo hybrid guide gene editing system of any one of claims 1 to 32, wherein the spacer sequence comprises 1 to 5 deoxyribonucleotides.

35. 35. The in vivo hybrid guided gene editing system of any one of claims 1 to 34, wherein the DNA binding domain comprises a CRISPR protein or a fragment thereof.

36. 35. The in vivo hybrid guide gene editing system of any one of claims 1 to 34, wherein the DNA binding domain comprises a catalytically impaired nuclease.

37. 35. The in vivo hybrid guide gene editing system of any one of claims 1 to 34, wherein the DNA binding domain comprises a prime editing protein or a fragment thereof.

38. 35. The in vivo hybrid guide gene editing system of any one of Claims 1-34, wherein the gene editor protein or component thereof affects less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% editing at all off-target sites compared to a gene editing system comprising a corresponding gRNA that does not contain said deoxyribonucleotides.

39. 39. The in vivo hybrid guide gene editing system of any one of Claims 1-38, wherein the gene editor protein or component thereof affects more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, or more than about 95% of editing of the target gene compared to a gene editing system comprising a corresponding gRNA that does not contain deoxyribonucleotides.

40. 39. The in vivo hybrid guide gene editing system of any one of Claims 1-38, wherein the gene editor protein or component thereof affects more than about 95%, more than about 96%, more than about 97%, more than about 98%, or more than about 99% of editing of the target gene compared to a gene editing system comprising a corresponding gRNA that does not contain deoxyribonucleotides.

41. 39. The in vivo hybrid guide gene editing system of any one of Claims 1-38, wherein the gene editor protein or component thereof affects about 95% to about 99% editing of the target gene compared to a gene editing system comprising a corresponding gRNA that does not contain said deoxyribonucleotides.

42. 42. The in vivo hybrid guide gene editing system of any one of claims 1 to 41, wherein the LNP comprises an N-acetylgalactosamine (GalNAc) lipid acceptor targeting conjugate.

43. 43. The in vivo hybrid guide gene editing system of any one of claims 1 to 42, wherein the target gene is expressed in the liver or in cells or tissues of liver origin.

44. 44. The in vivo hybrid guide gene editing system of any one of claims 1 to 43, wherein the target gene is expressed in a non-liver organ or cell or tissue of non-liver origin.

45. 45. A method for treating or preventing atherosclerotic cardiovascular disease in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a compound according to any one of claims 1 to 44.

2. A method comprising administering the in vivo hybrid guide gene editing system of any one of claims 1 to 4.

46. 46. ​​The method of claim 45, wherein the subject is a primate.

47. 47. The method of claim 46, wherein the primate is a human.

48. A gene editing system, (a) a gene editor protein or component thereof comprising a nucleic acid binding domain, or a nucleic acid encoding said gene editor protein or component thereof; (b) a hybrid guide nucleic acid comprising a spacer sequence, the spacer sequence comprising deoxyribonucleotides and ribonucleotides comprising ribose, wherein the 2' hydroxyl group of the ribose is covalently linked to a methyl group (2'-OMe).

49. A gene editing system, (a) a gene editor protein or component thereof comprising a nucleic acid binding domain, or a nucleic acid encoding said gene editor protein or component thereof; (b) a hybrid guide nucleic acid comprising a spacer sequence, wherein the spacer sequence comprises deoxyribonucleotides and ribonucleotides, and the spacer sequence corresponds to a protospacer on the ANGPTL3 gene.

50. A gene editing system, (a) a gene editor protein or component thereof comprising a nucleic acid binding domain and a deaminase, or a nucleic acid encoding said gene editor protein or component thereof; (b) a hybrid guide nucleic acid comprising a spacer sequence, wherein the spacer sequence comprises deoxyribonucleotides and ribonucleotides.

51. 1. A hybrid guide nucleic acid for a gene editing system, the hybrid guide comprising: (i) a spacer sequence comprising deoxyribonucleotides and ribonucleotides comprising a ribose, wherein the 2' hydroxyl group of the ribose is covalently linked to a methyl group (2'-OMe); and (ii) a binding scaffold.

52. 52. The hybrid guide nucleic acid of Claim 51, wherein the spacer sequence comprises a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA)(DNA) motif.

53. 53. The hybrid guide nucleic acid of Claim 52, wherein the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence.

54. 52. The hybrid guide nucleic acid of Claim 51, wherein the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 30 or 31.

55. The spacer sequence is (2'-OMe)PS(2'-OMe)PS(DNA)PS( 52. The hybrid guide nucleic acid of Claim 51, comprising a (DNA)(RNA)(DNA)(DNA) motif.

56. 56. The hybrid guide nucleic acid of Claim 55, wherein the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(RNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence.

57. 52. The hybrid guide nucleic acid of Claim 51, wherein the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 28 or 29.

58. 52. The hybrid guide nucleic acid of Claim 51, wherein the spacer sequence comprises a (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(DNA)(DNA)(DNA) motif.

59. 59. The hybrid guide nucleic acid of Claim 58, wherein the (2'-OMe)PS(2'-OMe)PS(DNA)PS(DNA)(DNA)(DNA)(DNA) motif is located at the 5' end of the spacer sequence.

60. 52. The hybrid guide nucleic acid of Claim 51, wherein the spacer sequence comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 5, 11, or 12.

61. 52. The hybrid guide nucleic acid of Claim 51, wherein the hybrid guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to a sequence selected from the group consisting of SEQ ID NOs:110-113.

62. 52. The hybrid guide nucleic acid of Claim 51, wherein the hybrid guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to a sequence selected from the group consisting of SEQ ID NOs:106-109.

63. 52. The hybrid guide nucleic acid of Claim 51, wherein the guide nucleic acid comprises a sequence having at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% sequence identity or sequence similarity to a sequence selected from the group consisting of SEQ ID NOs:79-82.

64. 1. A composition comprising a Cas9 nickase, wherein the Cas9 nickase comprises a mutation selected from the group consisting of N692A, M694A, Q695A, H698A, K810A, K855A, K848A, K1003A, and R1060A compared to the Cas9 nickase of SEQ ID NO:

695.

65. 65. The composition of Claim 64, wherein the Cas9 nickase comprises a sequence having at least 90% identity to SEQ ID NO:

695.

66. 65. The composition of Claim 64, wherein the Cas9 nickase comprises a sequence having at least 95% identity to SEQ ID NO:

695.

67. The Cas9 nickase has at least 98% identity to SEQ ID NO:

695.

65. The composition of claim 64, comprising a sequence

68. 65. The composition of Claim 64, wherein the Cas9 nickase comprises a sequence having at least 99% identity to SEQ ID NO:

695.

69. 65. The composition of Claim 64, wherein the Cas9 nickase comprises a sequence having at least 99.5% identity to SEQ ID NO:

695.

70. 70. The composition of any one of claims 64 to 69, wherein the mutations include N692A, M694A, Q695A, and H698A compared to the Cas9 nickase of SEQ ID NO:

695.

71. 70. The composition of any one of claims 64 to 69, wherein the mutation comprises K855A compared to the Cas9 nickase of SEQ ID NO:

695.

72. 70. The composition of any one of claims 64 to 69, wherein the mutations include K848A, K1003A, and R1060A compared to the Cas9 nickase of SEQ ID NO:

695.

73. The composition of any one of claims 64 to 69, wherein the mutations include K810A, K1003A, and R1060A compared to the Cas9 nickase of SEQ ID NO:

695.

74. 1. A composition comprising a nucleic acid encoding a Cas9 nickase, wherein the nucleic acid encoding the Cas9 nickase comprises mutations selected from the group consisting of: an AAC to GCC mutation at codon 692, an AUG to GCC mutation at codon 694, a CAG to GCC mutation at codon 695, a CAC to GCC mutation at codon 698, an AAG to GCC mutation at codon 855, an AAG to GCC mutation at codon 810, an AAG to GCC mutation at codon 848, an AAG to GCC mutation at codon 1003, and a CGG to GCC mutation at codon 1060, relative to the nucleic acid of SEQ ID NO:

694.

75. 75. The composition of Claim 74, wherein the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 90% identity to SEQ ID NO:

694.

76. 75. The composition of Claim 74, wherein the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 95% identity to SEQ ID NO:

694.

77. 75. The composition of Claim 74, wherein the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 98% identity to SEQ ID NO:

694.

78. 75. The composition of Claim 74, wherein the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 99% identity to SEQ ID NO:

694.

79. 75. The composition of Claim 74, wherein the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 99.5% identity to SEQ ID NO:

694.

80. 80. The composition of any one of claims 74-79, wherein the mutations comprise an AAC to GCC mutation at codon 692, an AUG to GCC mutation at codon 694, a CAG to GCC mutation at codon 695, and a CAC to GCC mutation at codon 698, relative to nucleic acid SEQ ID NO:

694.

81. 80. The composition of any one of claims 74 to 79, wherein the mutation comprises a mutation of AAG to GCC at codon 855 compared to nucleic acid SEQ ID NO:

694.

82. 80. The composition of any one of claims 74 to 79, wherein the mutations comprise an AAG to GCC mutation at codon 878, an AAG to GCC mutation at codon 1003, and a CGG to GCC mutation at codon 1060, compared to nucleic acid SEQ ID NO:

694.

83. 80. The composition of any one of claims 74-79, wherein the mutations comprise an AAG to GCC mutation at codon 810, an AAG to GCC mutation at codon 1003, and a CGG to GCC mutation at codon 1060, compared to nucleic acid SEQ ID NO:

694.

84. A gene editing system, (a) a Cas9 nickase; and (b) a guide RNA comprising a spacer corresponding to a protospacer sequence on the target gene and a binding scaffold for the Cas9 nickase; The Cas9 nickase comprises a mutation, wherein the mutation is selected from the group consisting of N692A, M694A, Q695A, H698A, K810A, K855A, K848A, K1003A, R1060A compared to the Cas9 nickase of SEQ ID NO:

695. A gene editing system.

85. 85. The gene editing system of Claim 84, wherein the gene editing system comprises a deaminase.

86. 85. The gene editing system of Claim 84, wherein the gene editing system comprises a polymerase.

87. 87. The gene editing system of Claim 86, wherein the polymerase is a reverse transcriptase.

88. 88. The gene editing system of any one of claims 84 to 87, wherein the target gene is PCSK9.

89. The gene editing system of any one of claims 84 to 87, wherein the target gene is ANGPTL3.

90. The gene editing system of any one of claims 84-89, wherein the Cas9 nickase comprises a sequence having at least 90% identity to SEQ ID NO:

695.

91. The gene editing system of any one of claims 84-89, wherein the Cas9 nickase comprises a sequence with at least 95% identity to SEQ ID NO:

695.

92. The gene editing system of any one of claims 84-89, wherein the Cas9 nickase comprises a sequence with at least 98% identity to SEQ ID NO:

695.

93. The gene editing system according to any one of claims 84 to 89, wherein the Cas9 nickase comprises a sequence having at least 99% identity to SEQ ID NO:

695.

94. The gene editing system according to any one of claims 84 to 89, wherein the Cas9 nickase comprises a sequence having at least 99.5% identity to SEQ ID NO:

695.

95. The gene editing system according to any one of claims 84 to 94, wherein the mutation comprises N692A, M694A, Q695A, and H698A as compared to the Cas9 nickase of SEQ ID NO:

695.

96. The gene editing system according to any one of claims 84 to 94, wherein the mutation comprises K855A as compared to the Cas9 nickase of SEQ ID NO:

695.

97. The gene editing system according to any one of claims 84 to 94, wherein the mutation comprises K848A, K1003A, and R1060A as compared to the Cas9 nickase of SEQ ID NO:

695.

98. The gene editing system according to any one of claims 84 to 9`4, wherein the mutation comprises K810A, K1003A, and R1060A as compared to the Cas9 nickase of SEQ ID NO:

695.

99. A gene editing system comprising: (a) a nucleic acid encoding a Cas9 nickase; and (b) a guide RNA comprising a spacer corresponding to a protospacer sequence on a target gene and a binding scaffold for the Cas9 nickase, wherein the nucleic acid encoding the Cas9 nickase comprises a mutation, and the mutation is selected from the group consisting of the AAC at codon 692 mutating to GCC, the AUG at codon 694 mutating to GCC, the CAG at codon 695 mutating to GCC, the CAC at codon 698 mutating to GCC, the AAG at codon 855 mutating to GCC, the AAG at codon 810 mutating to GCC, the AAG at codon 848 mutating to GCC, the AAG at codon 1003 mutating to GCC, and the CGG at codon 1060 mutating to GCC as compared to the nucleic acid of SEQ ID NO:

694.

100. The gene editing system according to claim 99, wherein the gene editing system comprises a nucleic acid encoding a deaminase.

101. The gene editing system according to claim 99, wherein the gene editing system comprises a nucleic acid encoding a polymerase.

102. 102. The gene editing system of Claim 101, wherein the polymerase is a reverse transcriptase.

103. The gene editing system of any one of claims 99 to 102, wherein the target gene is PCSK9.

104. The gene editing system of any one of claims 99 to 102, wherein the target gene is ANGPTL3.

105. 105. The gene editing system of any one of Claims 99-104, wherein the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 90% identity to SEQ ID NO:

694.

106. 105. The gene editing system of any one of Claims 99-104, wherein the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 95% identity to SEQ ID NO:

694.

107. 105. The gene editing system of any one of Claims 99-104, wherein the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 98% identity to SEQ ID NO:

694.

108. 105. The gene editing system of any one of Claims 99-104, wherein the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 99% identity to SEQ ID NO:

694.

109. 105. The gene editing system of any one of Claims 99-104, wherein the nucleic acid encoding the Cas9 nickase comprises a sequence having at least 99.5% identity to SEQ ID NO:

694.

110. 110. The gene editing system of any one of claims 99-109, wherein the mutations comprise a mutation of AAC at codon 692 to GCC, a mutation of AUG at codon 694 to GCC, a mutation of CAG at codon 695 to GCC, and a mutation of CAC at codon 698 to GCC compared to nucleic acid SEQ ID NO:

694.

111. 110. The gene editing system of any one of Claims 99-109, wherein the mutation comprises a mutation of AAG to GCC at codon 855 compared to nucleic acid SEQ ID NO:

694.

112. 110. The gene editing system of any one of claims 99-109, wherein the mutations comprise an AAG to GCC mutation at codon 878, an AAG to GCC mutation at codon 1003, and a CGG to GCC mutation at codon 1060 compared to nucleic acid SEQ ID NO:

694.

113. 110. The gene editing system of any one of claims 99-109, wherein the mutations comprise an AAG to GCC mutation at codon 810, an AAG to GCC mutation at codon 1003, and a CGG to GCC mutation at codon 1060 compared to nucleic acid SEQ ID NO:

694.

114. A nucleic acid encoding a base editor protein, wherein said nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs: 723, 725, 727 and 729.

115. A base editor protein, wherein the base editor protein comprises a sequence selected from the group consisting of SEQ ID NOs: 724, 726, 728 and 730.

116. 1. A pharmaceutical composition for a human subject, comprising: (a) an mRNA encoding a base editor protein, wherein the base editor protein comprises a nucleic acid binding domain; and (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) LNPs comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol; A pharmaceutical composition, wherein the total amount of the guide RNA and the mRNA is from about 0.01 mg / kg to about 3 mg / kg.

117. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 0.1 mg / kg to about 3 mg / kg.

118. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 0.01 mg / kg to about 2 mg / kg.

119. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 0.1 mg / kg to about 2 mg / kg.

120. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 0.01 mg / kg to about 1.5 mg / kg.

121. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 0.1 mg / kg to about 1.5 mg / kg.

122. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 0.01 mg / kg to about 1.25 mg / kg.

123. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 0.1 mg / kg to about 1.25 mg / kg.

124. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 0.01 mg / kg to about 1 mg / kg.

125. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 0.1 mg / kg to about 1 mg / kg.

126. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is about 0.1 mg / kg.

127. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is about 0.2 mg / kg.

128. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is about 0.3 mg / kg.

129. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is about 0.4 mg / kg.

130. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is about 0.5 mg / kg.

131. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is about 0.6 mg / kg.

132. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is about 0.7 mg / kg.

133. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is about 0.8 mg / kg.

134. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is about 0.9 mg / kg.

135. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is about 1 mg / kg.

136. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 1 mg / kg to about 3 mg / kg.

137. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 1.1 mg / kg to about 3 mg / kg.

138. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 1.2 mg / kg to about 3 mg / kg.

139. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 1.3 mg / kg to about 3 mg / kg.

140. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 1.4 mg / kg to about 3 mg / kg.

141. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 1.5 mg / kg to about 3 mg / kg.

142. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 1.6 mg / kg to about 3 mg / kg.

143. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 1.7 mg / kg to about 3 mg / kg.

144. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 1.8 mg / kg to about 3 mg / kg.

145. 117. The pharmaceutical composition of claim 116, wherein the total amount of the guide RNA and the mRNA is from about 1.9 mg / kg to about 3 mg / kg.

146. 1. A pharmaceutical composition for a human subject, comprising: (a) an mRNA encoding a base editor protein, wherein the base editor protein comprises a nucleic acid binding domain; and (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) LNPs comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol; a pharmaceutical composition, wherein administration of the gene editing system results in a plasma Cmax of the mRNA in the human subject that is from about 0.05 μg / mL to about 5 μg / mL.

147. 147. The pharmaceutical composition of claim 146, wherein the plasma Cmax of the mRNA in the human subject is from about 0.1 μg / mL to about 5 μg / mL.

148. 147. The pharmaceutical composition of claim 146, wherein the plasma Cmax of the mRNA in the human subject is from about 0.2 μg / mL to about 5 μg / mL.

149. 147. The pharmaceutical composition of claim 146, wherein the plasma Cmax of the mRNA in the human subject is from about 0.5 μg / mL to about 5 μg / mL.

150. 147. The pharmaceutical composition of claim 146, wherein the plasma Cmax of the mRNA in the human subject is from about 1 μg / mL to about 5 μg / mL.

151. the plasma Cmax of the mRNA in the human subject is from about 2 μg / mL to about 5 μg 147. The pharmaceutical composition of claim 146, wherein the .mu.m.sup.2 is 0.1 mg / mL.

152. 147. The pharmaceutical composition of claim 146, wherein the plasma Cmax of the mRNA in the human subject is from about 3 μg / mL to about 5 μg / mL.

153. 147. The pharmaceutical composition of claim 146, wherein the plasma Cmax of the mRNA in the human subject is from about 4 μg / mL to about 5 μg / mL.

154. 1. A pharmaceutical composition for a human subject, comprising: (a) an mRNA encoding a base editor protein, wherein the base editor protein comprises a nucleic acid binding domain; and (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) LNPs comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol; wherein administration of the gene editing system results in an AUC of the mRNA in the human subject that is between about 1 μg×h / mL and about 100 μg×h / mL.

155. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 1 μg×h / mL to about 50 μg×h / mL.

156. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 1 μg×h / mL to about 20 μg×h / mL.

157. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 1 μg×h / mL to about 10 μg×h / mL.

158. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 10 μg×h / mL to about 100 μg×h / mL.

159. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 10 μg×h / mL to about 50 μg×h / mL.

160. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 10 μg×h / mL to about 20 μg×h / mL.

161. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 20 μg×h / mL to about 100 μg×h / mL.

162. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 20 μg×h / mL to about 50 μg×h / mL.

163. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 20 μg×h / mL to about 30 μg×h / mL.

164. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 30 μg×h / mL to about 100 μg×h / mL.

165. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 30 μg×h / mL to about 50 μg×h / mL.

166. 155. The pharmaceutical composition of claim 154, wherein the AUC of the mRNA in the human subject is from about 50 μg×h / mL to about 100 μg×h / mL.

167. 1. A pharmaceutical composition for a human subject, comprising: (a) an mRNA encoding a base editor protein, wherein the base editor protein comprises a nucleic acid binding domain; and (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) LNPs comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol; the pharmaceutical composition, wherein administration of the gene editing system results in a plasma Cmax of the amino lipid in the human subject that is from about 1 μg / mL to about 100 μg / mL.

168. 168. The pharmaceutical composition of claim 167, wherein the plasma Cmax of the amino lipid in the human subject is from about 1 μg / mL to about 50 μg / mL.

169. 168. The pharmaceutical composition of claim 167, wherein the plasma Cmax of the amino lipid in the human subject is from about 1 μg / mL to about 30 μg / mL.

170. 168. The pharmaceutical composition of claim 167, wherein the plasma Cmax of the amino lipid in the human subject is from about 1 μg / mL to about 20 μg / mL.

171. 168. The pharmaceutical composition of claim 167, wherein the plasma Cmax of the amino lipid in the human subject is from about 1 μg / mL to about 10 μg / mL.

172. 168. The pharmaceutical composition of claim 167, wherein the plasma Cmax of the amino lipid in the human subject is from about 10 μg / mL to about 100 μg / mL.

173. 168. The pharmaceutical composition of claim 167, wherein the plasma Cmax of the amino lipid in the human subject is from about 10 μg / mL to about 50 μg / mL.

174. 168. The pharmaceutical composition of claim 167, wherein the plasma Cmax of the amino lipid in the human subject is from about 10 μg / mL to about 30 μg / mL.

175. 168. The pharmaceutical composition of claim 167, wherein the plasma Cmax of the amino lipid in the human subject is from about 20 μg / mL to about 100 μg / mL.

176. 168. The pharmaceutical composition of claim 167, wherein the plasma Cmax of the amino lipid in the human subject is from about 20 μg / mL to about 50 μg / mL.

177. 168. The pharmaceutical composition of claim 167, wherein the plasma Cmax of the amino lipid in the human subject is from about 50 μg / mL to about 100 μg / mL.

178. 1. A pharmaceutical composition for a human subject, comprising: (a) an mRNA encoding a base editor protein, wherein the base editor protein comprises a nucleic acid binding domain; and (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) LNPs comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol; wherein administration of the gene editing system results in an AUC of the amino lipid in the human subject that is between about 100 μg×h / mL and about 10,000 μg×h / mL.

179. 179. The pharmaceutical composition of claim 178, wherein the AUC of the amino lipid in the human subject is from about 100 μg×h / mL to about 5000 μg×h / mL.

180. 179. The pharmaceutical composition of claim 178, wherein the AUC of the amino lipid in the human subject is from about 100 μg×h / mL to about 2000 μg×h / mL.

181. 179. The pharmaceutical composition of claim 178, wherein the AUC of the amino lipid in the human subject is from about 100 μg×h / mL to about 1000 μg×h / mL.

182. 179. The pharmaceutical composition of claim 178, wherein the AUC of the amino lipid in the human subject is from about 100 μg×h / mL to about 500 μg×h / mL.

183. 179. The pharmaceutical composition of claim 178, wherein the AUC of the amino lipid in the human subject is from about 500 μg×h / mL to about 10,000 μg×h / mL.

184. 179. The pharmaceutical composition of claim 178, wherein the AUC of the amino lipid in the human subject is from about 500 μg×h / mL to about 5000 μg×h / mL.

185. 179. The pharmaceutical composition of claim 178, wherein the AUC of the amino lipid in the human subject is from about 500 μg×h / mL to about 2000 μg×h / mL.

186. 179. The pharmaceutical composition of claim 178, wherein the AUC of the amino lipid in the human subject is from about 500 μg×h / mL to about 1000 μg×h / mL.

187. 179. The pharmaceutical composition of claim 178, wherein the AUC of the amino lipid in the human subject is from about 1000 μg×h / mL to about 10000 μg×h / mL.

188. 179. The pharmaceutical composition of claim 178, wherein the AUC of the amino lipid in the human subject is from about 1000 μg×h / mL to about 5000 μg×h / mL.

189. 179. The pharmaceutical composition of claim 178, wherein the AUC of the amino lipid in the human subject is from about 5000 μg×h / mL to about 10000 μg×h / mL.

190. 1. A pharmaceutical composition for a human subject, comprising: (a) an mRNA encoding a base editor protein, wherein the base editor protein comprises a nucleic acid binding domain; and (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) LNPs comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol; a pharmaceutical composition, wherein administration of the gene editing system results in a plasma Cmax of the PEG lipid in the human subject that is from about 0.1 μg / mL to about 50 μg / mL.

191. The pharmaceutical composition of claim 190, wherein the plasma Cmax of the PEG lipid in the human subject is from about 0.1 μg / mL to about 25 μg / mL.

192. The pharmaceutical composition of claim 190, wherein the plasma Cmax of the PEG lipid in the human subject is from about 0.1 μg / mL to about 10 μg / mL.

193. the plasma Cmax of the PEG-lipid in the human subject is from about 0.1 μg / mL to about 191. The pharmaceutical composition of claim 190, wherein the concentration is 5 μg / mL.

194. The pharmaceutical composition of claim 190, wherein the plasma Cmax of the PEG lipid in the human subject is from about 0.1 μg / mL to about 1 μg / mL.

195. The pharmaceutical composition of claim 190, wherein the plasma Cmax of the PEG lipid in the human subject is from about 1 μg / mL to about 50 μg / mL.

196. The pharmaceutical composition of claim 190, wherein the plasma Cmax of the PEG lipid in the human subject is from about 1 μg / mL to about 25 μg / mL.

197. The pharmaceutical composition of claim 190, wherein the plasma Cmax of the PEG lipid in the human subject is from about 1 μg / mL to about 10 μg / mL.

198. The pharmaceutical composition of claim 190, wherein the plasma Cmax of the PEG lipid in the human subject is from about 10 μg / mL to about 50 μg / mL.

199. The pharmaceutical composition of claim 190, wherein the plasma Cmax of the PEG lipid in the human subject is from about 10 μg / mL to about 25 μg / mL.

200. The pharmaceutical composition of claim 190, wherein the plasma Cmax of the PEG lipid in the human subject is from about 25 μg / mL to about 50 μg / mL.

201. 1. A pharmaceutical composition for a human subject, comprising: (a) an mRNA encoding a base editor protein, wherein the base editor protein comprises a nucleic acid binding domain; and (b) a guide RNA that serves to guide the nucleic acid binding domain to a protospacer sequence on a target gene; and (c) LNPs comprising an amino lipid, a PEG lipid, a phospholipid, and a sterol; wherein administration of the gene editing system results in an AUC of the PEG-lipid in the human subject that is between about 10 μg×h / mL and about 5000 μg×h / mL.

202. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 10 μg×h / mL to about 2000 μg×h / mL.

203. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 10 μg×h / mL to about 1000 μg×h / mL.

204. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 10 μg×h / mL to about 500 μg×h / mL.

205. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 10 μg×h / mL to about 100 μg×h / mL.

206. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 100 μg×h / mL to about 5000 μg×h / mL.

207. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 100 μg×h / mL to about 2000 μg×h / mL.

208. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 100 μg×h / mL to about 1000 μg×h / mL.

209. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 100 μg×h / mL to about 500 μg×h / mL.

210. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 500 μg×h / mL to about 5000 μg×h / mL.

211. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 500 μg×h / mL to about 2000 μg×h / mL.

212. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 500 μg×h / mL to about 1000 μg×h / mL.

213. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 1000 μg×h / mL to about 5000 μg×h / mL.

214. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 1000 μg×h / mL to about 2000 μg×h / mL.

215. The pharmaceutical composition of claim 201, wherein the AUC of the PEG-lipid in the human subject is from about 2000 μg×h / mL to about 5000 μg×h / mL.

216. 216. The pharmaceutical composition of any one of claims 116 to 215, wherein the phospholipid is distearoylphosphatidylcholine (DSPC).

217. 217. The pharmaceutical composition of any one of claims 116 to 216, wherein the sterol is cholesterol.

218. The pharmaceutical composition of any one of claims 116 to 217, wherein the LNP comprises an N-acetylgalactosamine (GalNAc) lipid receptor targeting conjugate.

219. 219. A method for treating or preventing atherosclerotic cardiovascular disease in a human subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 116 to 218.

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