Base editing of angptl3 and methods of using the same for treatment of disease

Abstract

To provide specific compositions and methods capable of safely and effectively editing gene targets expressed in a liver, to durably lower LDL-C, thereby treating a leading cause of a cardiovascular disease.SOLUTION: A composition for editing a gene target comprises (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same, and (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on an ANGPTL3 gene.SELECTED DRAWING: Figure 49

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of Provisional Application No. 63 / 007,803, filed April 9, 2020. This application claims the benefit under 35 U.S.C. § 119 of Provisional Application No. 63 / 007,797, filed April 9, 200, Provisional Application No. 63 / 136,087, filed January 11, 2021, Provisional Application No. 63 / 045,032, filed June 26, 2020, and Provisional Application No. 63 / 045,033, filed June 26, 2020, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing

[0002] This application has been submitted electronically in ASCII format and is hereby incorporated by reference. Contains a Sequence Listing, which is incorporated herein in its entirety. Said ASCII copy was created on April 8, 2021, is named 53989_711_601_SL.txt and is 1,105,762 bytes in size.

[0003] Field of Disclosure

[0003] Compositions for modifying or editing genes and methods for treating cardiovascular diseases and conditions Methods of using the compositions are provided herein that can treat or prevent certain conditions, such as diseases associated therewith, such as diabetes. [Background technology]

[0004] All publications, patents, and patent applications mentioned herein are the property of their respective owners. Each publication, patent, or patent application is incorporated by reference herein to the same extent as if it were specifically and individually indicated to be incorporated by reference. No admission is made that any publication or information specifically or implicitly referenced herein is prior art or necessarily relevant to the claimed subject matter. To the extent that a publication or patent or patent application incorporated by reference is inconsistent or inconsistent with the disclosure contained herein, such cited or incorporated references should be considered in support of the present disclosure, with the understanding that the present specification is intended to replace and / or supersede any conflicting, inconsistent, or contradictory material. Summary of the Invention

[0005] The subject matter of the invention disclosed in this application is a method for editing a polynucleotide or a target gene. The present invention is directed to compositions and methods of using these compositions that can be used to treat a variety of conditions, including, but not limited to, cancer, cancers, and cancers. The compositions and their components, individually and in combination, are considered separate aspects of the inventive subject matter, which may be defined and claimed by their respective physical and / or functional attributes described herein in any combination, without limitation. The breadth, specificity, and variability of the inventive subject matter are further illustrated by the specific embodiments summarized herein.

[0006]

[0006] One prominent aspect of the subject matter described herein is the treatment of cardiovascular disease (CVD). Treatment targets: CVD is the leading cause of death worldwide, accounting for nearly one-third of deaths according to the World Health Organization. CVD is also the leading cause of reduced life expectancy and is one of the most costly health conditions to consider. According to the Centers for Disease Control and Prevention (CDC), CVD is a significant economic burden, costing the U.S. healthcare system more than $320 billion per year in annual expenses and lost productivity. CVD collectively refers to heart and vascular disease, which are specifically diagnosed as atherosclerotic cardiovascular disease (ASCVD) or cardiomyopathies. ASCVD is a large subset of CVD. Cholesterol promotes the development of atherosclerotic plaques, a mixture of cholesterol, cells and cell debris in the vessel walls that leads to arteriosclerosis. The current reliance in treating and preventing ASCVD is to lower cumulative exposure to blood lipids with the goal of maintaining low-density lipoprotein cholesterol (LDL-C, commonly known as "bad" cholesterol) and / or triglycerides as low as possible for as long as possible. For example, there is significant evidence demonstrating that individuals who maintain low enough levels of LDL-C for a long enough period of time are substantially less likely to develop ASCVD. The relationship between lowering LDL-C and reducing ASCVD is the best understood of all relationships in medicine. It has been shown that a 5-year reduction of 39 mg / dL of LDL-C in patients with established ASCVD reduces the risk of ASCVD by 21%, while the same 39 mg / dL reduction in LDL-C over a lifetime reduces first ASCVD events by 88%. Current standard treatments are chronic treatment models that typically require multiple pills and / or intermittent injections every day, and often do not adequately control cumulative exposure to LDL-C. Despite the availability of such chronic treatments, cumulative exposure to LDL-C is often not adequately controlled in many patients with ASCVD, and a large percentage of individuals with established ASCVD have LDL-C levels above recommended targets. High cumulative LDL-C exposure accelerates the buildup of cholesterol plaques in the heart or carotid arteries, the rupture of which can lead to heart attack, cardiac death, stroke, and the need for invasive medical procedures such as intracoronary stent placement and coronary artery bypass surgery.

[0007]

[0007] One aspect disclosed herein is a method for safely and effectively targeting genes expressed in the liver. The present invention provides compositions and methods that can selectively edit genes to sustainably lower LDL-C and / or triglycerides, thereby treating CVD, such as ASCVD.

[0008] Another aspect disclosed herein is a single course or dose (once and done) The efficacy and safety of the gene editing compositions described herein when administered as a combination gene editing therapy dose, in repeated or sequential doses, as a first-and-done therapy, and as a combination gene editing therapy dose. The efficacy and safety of the compositions described herein are shown in the in vitro and in vivo studies described herein, including various cell and animal studies, including mouse and non-human primate studies, and the results or functions of the compositions presented in these studies and disclosed herein each constitute an aspect of the invention.

[0009] Another aspect of the compositions and methods disclosed herein is a method for detecting nucleotides, e.g., at splice junctions. The present invention relates to sites for gene editing, including compositions and methods directed to gene editing in

[0010] Another aspect of the compositions and methods disclosed herein are component guide RNA (gRNA) and base editors that comprise compositions capable of precisely editing genes at a single base pair without imparting a double-stranded break in the target gene. Compositions and methods of using these gRNA and base editors, including nucleotide or mRNA sequences that express and encode the base editors, constitute yet another aspect.

[0011]

[0011] Another aspect disclosed herein is the lipid nanoparticle (LNP) formulation encapsulating the gRNA and base editor drug substance, the selection of the LNP, and the relative ratios between the various components of the drug substance composition alone and as part of the LNP.

[0012]

[0012] Another aspect disclosed herein is directed to the dosing of gene editing compositions as described herein, and the effects of dosing and repeated dosing on indicators of efficacy and safety profile.

[0013]

[0013] Another aspect disclosed herein is that the compositions and methods of use include implementations designed to target or edit specific genes, such as PCSK9, ANGPTL3, APOC3, and / or Lp(a), and / or have an effect on the protein levels of proteins expressed by these genes.

[0014]

[0014] In one aspect, provided herein is a composition for editing a gene target, comprising (i) a base editor fusion protein, or an mRNA encoding the same, comprising a programmable DNA binding domain and a deaminase, (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence corresponding to a protospacer on the ANGPTL3 gene, wherein the guide RNA, when administered to a mammalian subject, directs the base editor fusion protein to effect a nucleic acid base change in the ANGPTL3 gene in vivo, and when the guide RNA and mRNA are administered in a total amount of at least 0.5 mg / kg, the base change occurs in at least 35% of the total hepatocytes of the mammalian subject, as measured by next generation sequencing or Sanger sequencing. In some embodiments, the mammalian subject is a cynomolgus monkey, and when the guide RNA and mRNA are administered in a total amount of about 1 mg / kg, the base change occurs in at least 35% of the total hepatocytes of the cynomolgus monkey, as measured by next generation sequencing or Sanger sequencing. In some embodiments, the mammalian subject is a cynomolgus monkey, and when the guide RNA and mRNA are administered at a total dose of about 3 mg / kg, the base modification occurs in at least 50% of the total liver cells of the cynomolgus monkey as measured by next generation sequencing or Sanger sequencing. In some embodiments, the nucleobase modification results in at least a 20% reduction in blood triglyceride levels in the cynomolgus monkey compared to before administration. In some embodiments, the nucleobase modification results in at least a 50% reduction in blood triglyceride levels in the cynomolgus monkey compared to before administration. In some embodiments, the protospacer is located at a splice site. In some embodiments, the protospacer complementary sequence is in the antisense strand of the ANGPTL3 gene. In some embodiments, the protospacer complementary sequence is in the sense strand of the ANGPTL3 gene.In some embodiments, the base change occurs outside the protospacer (off-target site) on the ANGPTL3 gene, and the editing percentage of the off-target site described in Table 14 is equal to or less than the editing percentage described in Table 14, respectively. 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 Cas9 nickase. 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, referenced in SEQ ID NO:7. 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, a premature stop codon, an insertion or deletion in the transcript encoded by the ANGPTL3 gene. In some embodiments, the nucleobase alteration results in an aberrant transcript encoded by the ANGPTL3 gene. In some embodiments, the guide RNA is chemically modified. In some embodiments, the tracr sequence of the guide RNA is chemically modified according to the scheme shown in FIG. 7. In some embodiments, the spacer sequence comprises an ANGPTL3 ABE guide RNA spacer sequence as described in Table 1. In some embodiments, the guide RNA comprises an ANGPTL3 ABE guide RNA spacer sequence as described in Table 1: GA067, GA091, GA098, GA099, GA100, GA101, GA102, GA103, GA347, GA441, GA442, GA472, GA473, GA474, GA475, GA476, GA517, or GA547. In some embodiments, the protospacer sequence comprises the ANGPTL3 ABE protospacer sequence set forth in Table 1. In some embodiments, the protospacer comprises the sequence 5'-AAGATACCTGAATAACTCTC-3' (SEQ ID NO: 14), 5'-AAGATACCTGAATAACCCTC-3' (SEQ ID NO: 15), 5'-GATACCTGAATAACTCTC-3' (SEQ ID NO: 1606), 5'-AGATACCTGAATAACCCTC-3' (SEQ ID NO: 248), or 5'-GATACCTGAATAACCCTC-3' (SEQ ID NO: 249). In some embodiments, the base editor fusion protein comprises the amino acid sequence of SEQ ID NO: 2137. In some embodiments, the GC% content of the mRNA sequence is greater than 50%. In some embodiments, the GC% content of the mRNA sequence is greater than 56%. In some embodiments, the GC% content of the mRNA sequence is 63% or greater. In some embodiments, the mRNA comprises an adenine tTNA deaminase (TadA) region, a Cas9 region, and a nuclear localization sequence (NLS) region. In some embodiments, the mRNA further comprises a first linker region linking the TadA region and the Cas9 region and a second linker region linking the Cas9 region and the NLS region. In some embodiments, the GC% content of the TadA region is greater than 60%. In some embodiments, the GC% content of the TadA region is greater than 70% or greater. In some embodiments, the GC% content of the Cas9 region is greater than 56%. In some embodiments, the GC% content of the Cas9 region is greater than 62% or greater. In some embodiments, the GC% content of the NLS region is greater than 54%. In some embodiments, the GC% content of the NLS region is greater than 63% or greater. In some embodiments, the GC% content of the first linker region is greater than 65%. In some embodiments, the GC% content of the first linker region is greater than 79% or greater. In some embodiments, the GC% content of the second linker region is greater than 67%. In some embodiments, the GC% content of the second linker region is 83% or more. In some embodiments, the GC% content of the TadA region is greater than 60%, the GC% content of the Cas9 region is greater than 56%, the GC% content of the NLS region is greater than 54%, the GC% content of the first linker region is greater than 65%, and the GC% content of the second linker region is greater than 67%.In some embodiments, the mRNA comprises an mRNA sequence selected from Table 23. In some embodiments, the mRNA comprises an mRNA sequence of SEQ ID NO: 2136. In some embodiments, the mRNA comprises a poly-A tail. In some embodiments, the composition further comprises a lipid nanoparticle (LNP) encapsulating (i). In some embodiments, the LNP further encapsulates (ii). In some embodiments, the composition further comprises a second LNP encapsulating (ii). In some embodiments, the ratio of guide RNA to mRNA encoding the base editor fusion protein is about 1:10 to about 10:1 by weight. In some embodiments, the ratio of guide RNA to mRNA encoding the base editor fusion protein is about 1:1, 1.5:1, 2:1, 3:1, 4:1, 1:1.5, 1:2, 1:3, or 1:4 by weight. In some embodiments, the ratio of guide RNA to mRNA encoding the base editor fusion protein is about 1:1 by weight.

[0015]

[0015] In another aspect, provided herein is a pharmaceutical composition comprising a composition provided herein and a pharma- ceutically acceptable carrier or excipient.

[0016] In another aspect, provided herein is a method of treating or preventing a condition in a subject in need thereof, comprising administering a therapeutically effective amount of a composition provided herein to the subject. In some embodiments, the administration is via intravenous infusion. In some embodiments, the method comprises sequential administration of LNPs encapsulating (i) and LNPs encapsulating (ii). In some embodiments, the method comprises simultaneous administration of LNPs encapsulating (i) and LNPs encapsulating (ii). In some embodiments, the method comprises administering a single dose of LNPs encapsulating (ii) followed by staggered doses of LNPs encapsulating (i) at one-day intervals. In some embodiments, the method comprises administering a single dose of LNPs encapsulating (ii) followed by staggered doses of LNPs encapsulating (i) at two-day intervals. In some embodiments, the method comprises administering a single dose of LNPs encapsulating (ii) followed by staggered doses of LNPs encapsulating (i) at three-day intervals. In some embodiments, the method includes administering a single dose of LNP encapsulating (ii) followed by staggered doses of LNP encapsulating (i) at 4 day intervals. In some embodiments, the method includes administering a single dose of LNP encapsulating (ii) followed by staggered doses of LNP encapsulating (i) at 5 day intervals. In some embodiments, the method includes administering a single dose of LNP encapsulating (ii) followed by staggered doses of LNP encapsulating (i) at 6 day intervals. In some embodiments, the method includes administering a single dose of LNP encapsulating (ii) followed by staggered doses of LNP encapsulating (i) at 7 day intervals. In some embodiments, the method includes administering a single dose of LNP encapsulating (i) and (ii). In some embodiments, the single dose of LNP is about 0.3 to about 3 mg / kg. In some embodiments, the method includes administering to the subject a treatment course of one or more treatments, each one of the one or more treatments comprising one or more single doses of LNP. In some embodiments, the method includes administering a treatment course of 2 to 10 treatments. In some embodiments, the method includes administering a treatment course of 2 to 5 treatments. In some embodiments, the method includes administering a treatment course of 2 treatments. In some embodiments, the method includes administering a treatment course of 3 treatments. In some embodiments, the method includes administering a treatment course of 4 treatments. In some embodiments, the method includes administering a treatment course of 5 treatments. In some embodiments, the condition is atherosclerotic cardiovascular disease. In some embodiments, the condition is atherosclerotic vascular disease. In some embodiments, the subject is a human.

[0016]

[0017] In another aspect, provided herein is a composition for editing a gene target comprising: (i) a base editor fusion protein comprising a programmable DNA-binding domain and a deaminase, or an mRNA encoding same; (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on the ANGPTL3 gene, wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the ANGPTL3 gene in vivo when administered to a mammalian subject, and wherein the guide RNA comprises an ANGPTL3 ABE guide RNA sequence as set forth in Table 1. In another aspect, provided herein is a composition for editing a gene target comprising: (i) a base editor fusion protein comprising a programmable DNA-binding domain and a deaminase, or an mRNA encoding the same; (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on the ANGPTL3 gene, wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the ANGPTL3 gene in vivo when administered to a mammalian subject, and the mRNA comprises a sequence selected from Table 23.

[0017]

[0018] In another aspect, a method of treating or preventing atherosclerotic cardiovascular disease in a subject in need thereof comprises administering a therapeutically effective amount of (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or an mRNA encoding the same; (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on the PCSK9 gene, wherein the guide RNA, when administered to a mammalian subject, directs the base editor fusion protein to effect a nucleobase alteration in the PCSK9 gene in vivo, and wherein when the guide RNA and mRNA are administered in a total amount of at least 0.5 mg / kg, the base alteration occurs in at least 35% of total hepatocytes of the mammalian subject as measured by next generation sequencing or Sanger sequencing; and Provided herein are methods comprising administering to a subject a second composition comprising (i) a nucleic acid sequence encoding the nucleic acid sequence of the ANGPTL3 gene, (ii) a guide RNA comprising a tracr sequence that functions as a binding scaffold for a base editor fusion protein, and a spacer sequence that corresponds to a protospacer on the ANGPTL3 gene, wherein the guide RNA, when administered to a mammalian subject, directs the base editor fusion protein to effect a nucleobase alteration in the ANGPTL3 gene in vivo, and wherein, when the guide RNA and mRNA are administered in a total amount of at least 1 mg / kg, the base alteration occurs in at least 35% of total hepatocytes of the mammalian subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the method comprises sequential administration of the first composition and the second composition. In some embodiments, the method comprises administering one or more doses of the first composition followed by one or more doses of the second composition. In some embodiments, the method comprises administering one or more doses of the second composition followed by one or more doses of the first composition. In some embodiments, the method comprises simultaneous administration of the first composition and the second composition. In some embodiments, the method includes one or more doses of the first composition and the second composition.

[0018]

[0019] In another aspect, provided herein is a composition for editing a gene target, comprising: (i) a base editor fusion protein, or an mRNA encoding the same, comprising a programmable DNA binding domain and a deaminase; (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence corresponding to a protospacer on the ANGPTL3 gene, wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the ANGPTL3 gene in vitro, and wherein the base alteration occurs in at least 35% of total hepatocytes of a mammalian subject, as measured by next generation sequencing or Sanger sequencing, when the guide RNA and mRNA are administered in a total amount of at least 0.5 mg / kg. In some embodiments, the base alteration occurs in at least 40% of total hepatocytes of a mammalian subject, as measured by next generation sequencing or Sanger sequencing, when the guide RNA and mRNA are administered in a total amount of at least 1 mg / kg. In some embodiments, when the guide RNA and mRNA are administered in a total amount of at least 1.5 mg / kg, the base modification occurs in at least 45% of the total hepatocytes of the mammalian subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, when the guide RNA and mRNA are administered in a total amount of at least 2 mg / kg, the base modification occurs in at least 50% of the total hepatocytes of the mammalian subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, when the guide RNA and mRNA are administered in a total amount of at least 2.5 mg / kg, the base modification occurs in at least 55% of the total hepatocytes of the mammalian subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, when the guide RNA and mRNA are administered in a total amount of at least 3 mg / kg, the base modification occurs in at least 60% of the total hepatocytes of the mammalian subject as measured by next generation sequencing or Sanger sequencing.

[0019]

[0020] In another aspect, provided herein is a composition for editing the ANGPTL3 gene, comprising: (a) an mRNA encoding an adenine base editor protein having an editing window; and (b) a guide RNA comprising a tracr sequence that functions as a binding scaffold for the base editor protein, and a spacer sequence that functions to guide the base editor protein to a protospacer sequence on the ANGPTL3 gene, wherein the spacer sequence is at least partially complementary to a splice site or exon region of the ANGPTL3 gene. In some embodiments, the editing window is determined to be an ANGPTL3 splice site or exon region when the base editor protein is operably bound to the guide RNA and the guide RNA hybridizes to a strand complementary to the protospacer sequence on the ANGPTL3 gene. The editing window encompasses a region of an intron of the L3 gene. In some embodiments, when the base editor protein is operably bound to the guide RNA and the guide RNA is hybridized to a strand complementary to a protospacer sequence on the ANGPTL3 gene, the editing window encompasses a region of intron 1, intron 3, or intron 4 of the ANGPTL3 gene when the base editor protein is operably bound to the guide RNA and the guide RNA is hybridized to a strand complementary to a protospacer sequence on the ANGPTL3 gene. In some embodiments, when the base editor protein is operably bound to the guide RNA and the guide RNA is hybridized to a strand complementary to a protospacer sequence on the ANGPTL3 gene, the editing window encompasses a region of intron 1 of the ANGPTL3 gene when the base editor protein is operably bound to the guide RNA and the guide RNA is hybridized to a strand complementary to a protospacer sequence on the ANGPTL3 gene. In some embodiments, the spacer sequence has 80-100% nucleotide sequence identity with a spacer sequence selected from the group of guide RNA sequences identified as GA067, GA100, and GA574. In some embodiments, the tracr sequence has 80-100% nucleotide sequence identity to a tracr sequence selected from the group of guide RNA sequences identified as GA067, GA091, GA098, GA099, GA100, GA101, GA102, GA103, GA347, GA441, GA442, GA472, GA473, GA474, GA475, GA476, GA517, and GA547. In some embodiments, the mRNA has 80-100% sequence identity to an mRNA sequence identified as MA002, MA004, MA040, MA0041, or MA045. In some embodiments, the mRNA has 80-100% sequence identity to an mRNA sequence identified as the following table:

[0020] [Table 1]

[0021] The nucleotide sequence of the present invention may have one or more of the GC nucleotide region percentages set forth in In some embodiments, the mRNA is selected from the group consisting of the following:

[0022] [Table 2]

[0023] The nucleotide sequence of the present invention may have one or more of the GC nucleotide region percentages set forth in In some embodiments, the mRNA and gRNA are encapsulated in lipid nanoparticles. ... iLipid 40~65% DSPC 2~20% PEG 1-5% The remaining mole % balance is cholesterol; LNP particle size: Z-average hydrodynamic diameter 55-120 nm, and Polydispersity index less than 0.2 as determined by dynamic light scattering The compound is encapsulated in lipid nanoparticles having the formula:

[0024]

[0023] In some embodiments, the mRNA and gRNA are encapsulated in lipid nanoparticles with a LNP particle size of 50-70 nm Z-average hydrodynamic diameter. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can induce adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkey with a mean editing percentage of greater than 40 percent. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can induce adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkey with a mean editing percentage of greater than 50 percent. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can induce adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkey with an average editing percentage of greater than 30 percent. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can induce adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkey with an average editing percentage of greater than 40 percent. In some embodiments, the composition, when administered to groups of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, is capable of inducing adenine base editing at ANGPTL3 target splice sites in the livers of cynomolgus monkeys with an average editing percentage of greater than 50 percent.In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can induce adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkey with an average editing percentage of greater than 60 percent. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can induce adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkey with an average editing percentage of greater than 70 percent. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can induce adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkey with an average editing percentage of greater than 40 percent. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can induce adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkey with an average editing percentage of greater than 50 percent. In some embodiments, the composition, when administered to groups of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, induces an average editing percentage of greater than 60 percent at the ANGPTL3 target splice site in the liver of cynomolgus monkeys. In some embodiments, the composition can induce adenine base editing at the ANGPTL3 target splice site in the liver of cynomolgus monkeys with an average editing percentage of greater than 70 percent when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight. In some embodiments, the composition can induce adenine base editing at the ANGPTL3 target splice site in the liver of cynomolgus monkeys with an average editing percentage of greater than 80 percent when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can induce adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkey with an average editing percentage of greater than 40 percent. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can induce adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkey with an average editing percentage of greater than 50 percent. In some embodiments, the composition, when administered to groups of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, is capable of inducing adenine base editing at ANGPTL3 target splice sites in the livers of cynomolgus monkeys with an average editing percentage of greater than 60 percent.In some embodiments, the composition can induce adenine base editing at the ANGPTL3 target splice site in the liver of cynomolgus monkeys with an average editing percentage of greater than 70 percent when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight. In some embodiments, the composition can induce adenine base editing at the ANGPTL3 target splice site in the liver of cynomolgus monkeys with an average editing percentage of greater than 80 percent when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight. In some embodiments, the editing percentage is determined by analysis of the liver of the administered cynomolgus monkeys via liver biopsy or autopsy of the monkeys 15 days after administration. In some embodiments, the editing percentage is determined to be persistently maintained by periodic liver biopsy testing of the administered cynomolgus monkeys for a period of at least 168 days after dosing. In some embodiments, the editing percentage is determined to be persistently maintained by periodic liver biopsy testing of the administered cynomolgus monkeys for a period of at least 300 days after dosing. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 35 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 40 percent on average compared to baseline.In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of monkey body weight, can reduce ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys by at least 50 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the dosed monkeys by at least 50 percent on average compared to baseline. In some embodiments, the composition can reduce ANGPTL3 protein by at least 60 percent on average compared to baseline when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, in the plasma of the administered cynomolgus monkeys by at least 70 percent on average compared to baseline. In some embodiments, the composition can reduce ANGPTL3 protein by at least 80 percent on average compared to baseline when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, in the plasma of the administered cynomolgus monkeys by at least 80 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 35 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 40 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 50 percent on average compared to baseline. In some embodiments, the composition, when administered to groups of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of dosed cynomolgus monkeys by an average of at least 60 percent compared to baseline.In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 70 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 80 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 80 percent on average compared to baseline. The ANGPTL3 protein in the plasma of the administered cynomolgus monkeys can be reduced by at least 35 percent on average compared to baseline. In some embodiments, the composition can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 40 percent on average compared to baseline when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight. In some embodiments, the composition can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 50 percent on average compared to baseline when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight. In some embodiments, the composition, when administered to groups of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of dosed cynomolgus monkeys by an average of at least 60 percent compared to baseline. The composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 70 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 80 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 35 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 40 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 50 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 60 percent on average compared to baseline.In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 70 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce ANGPTL3 protein in the plasma of the administered cynomolgus monkeys by at least 80 percent on average compared to baseline. In some embodiments, the reduction in plasma protein is determined by blood sampling and analysis of the administered cynomolgus monkeys 15 days after dosing. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 20 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 25 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 30 percent on average compared to baseline.In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the dosed cynomolgus monkeys by an average of at least 35 percent compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the dosed cynomolgus monkeys by at least 35 percent compared to baseline. When administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the combined total weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, the composition can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by an average of at least 40 percent compared to baseline. In some embodiments, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the combined total weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, the composition can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by an average of at least 45 percent compared to baseline. In some embodiments, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the combined total weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, the composition can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by an average of at least 20 percent compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 25 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 30 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 35 percent on average compared to baseline.In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 40 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 45 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 50 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 55 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 60 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 20 percent on average compared to baseline.In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, is capable of reducing triglyceride levels in the plasma of the dosed cynomolgus monkeys by an average of at least 25 percent compared to baseline. When administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, the composition can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 30 percent on average compared to baseline. In some embodiments, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, the composition can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 35 percent on average compared to baseline. In some embodiments, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, the composition can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 40 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, reduces plasma In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 45 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 50 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 55 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 60 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 65 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 20 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, is capable of reducing triglyceride levels in the plasma of dosed cynomolgus monkeys by an average of at least 25 percent compared to baseline.In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 30 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 35 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 35 percent on average compared to baseline. The composition can reduce triglyceride levels in the plasma of treated cynomolgus monkeys by at least 40 percent on average compared to baseline. In some embodiments, the composition can reduce triglyceride levels in the plasma of treated cynomolgus monkeys by at least 45 percent on average compared to baseline when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight. In some embodiments, the composition can reduce triglyceride levels in the plasma of treated cynomolgus monkeys by at least 50 percent on average compared to baseline when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 55 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 60 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce triglyceride levels in the plasma of the administered cynomolgus monkeys by at least 65 percent on average compared to baseline. In some embodiments, reduction in triglyceride levels is determined 15 days after dosing by blood sampling and analysis of dosed cynomolgus monkeys.In some embodiments, the reduction in triglyceride levels is determined to be sustained over a period of at least 168 days by periodic blood sampling and analysis of the dosed cynomolgus monkeys. In some embodiments, the reduction in triglyceride levels is determined to be sustained over a period of at least 300 days by periodic blood sampling and analysis of the dosed cynomolgus monkeys. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce lipoprotein(a) in the plasma of the dosed cynomolgus monkeys by an average of at least 10 percent compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce lipoprotein(a) in the plasma of the dosed cynomolgus monkeys by an average of at least 15 percent compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce lipoprotein(a) levels in the plasma of the administered cynomolgus monkeys by at least 20 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce lipoprotein(a) levels in the plasma of the administered cynomolgus monkeys by at least 25 percent on average compared to baseline. In some embodiments, the composition, when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total combined weight of guide RNA and mRNA per kg of cynomolgus monkey body weight, can reduce lipoprotein(a) levels in the plasma of the administered cynomolgus monkeys by at least 30 percent on average compared to baseline.In some embodiments, the composition is dosed when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg total weight of combined guide RNA and mRNA per kg of cynomolgus monkey body weight. The lipoprotein(a) levels in the plasma of the cynomolgus monkeys can be reduced by an average of approximately 35 percent compared to baseline. In some embodiments, the reduction in lipoprotein(a) is determined 15 days after dosing by blood sampling and analysis of the dosed cynomolgus monkeys. In some embodiments, the reduction in lipoprotein(a) is determined to be persistently maintained over a period of at least 224 days by periodic blood sampling and analysis of the dosed cynomolgus monkeys. In some embodiments, the reduction in lipoprotein(a) is determined to be persistently maintained over a period of at least 300 days by periodic blood sampling and analysis of the dosed cynomolgus monkeys. In some embodiments, to the extent that dosing of the cynomolgus monkeys results in elevation of AST, ALT, or cytokines, the elevations resulting from dosing of the composition are transient and return to approximately baseline levels within 3-15 days after dosing. In some embodiments, the percent editing of ANGPTL3 is negligible outside of liver tissue, spleen tissue, and adrenal tissue as shown in FIG. 27. In some embodiments, the results of repeated dosing are additive with respect to the editing percentage of ANGPTL3 editing percentage. In some embodiments, repeated dosing does not induce cytokine activation or immune response. In some embodiments, the spacer sequence has at least 80% nucleotide correlation with the nucleotide sequence of the targeted protospacer on the ANGPTL3 gene, and the RNA nucleotides are correlated with the DNA nucleotides if the RNA nucleotides on the spacer sequence have the same nucleotides as the DNA nucleotides of the protospacer in the same order, where uracil and thymine bases are considered to be the same nucleotides for the purpose of determining correlation. In some embodiments, the spacer sequence has at least 85% nucleotide correlation with the nucleotide sequence of the targeted protospacer on the ANGPTL3 gene. In some embodiments, the spacer sequence has at least 90% nucleotide correlation with the nucleotide sequence of the targeted protospacer on the ANGPTL3 gene.In some embodiments, the spacer sequence has at least 95% nucleotide correlation with the nucleotide sequence of the targeted protospacer on the ANGPTL3 gene. In some embodiments, the spacer sequence has at least 99% nucleotide correlation with the nucleotide sequence of the targeted protospacer on the ANGPTL3 gene. In some embodiments, the spacer sequence has at least 100% nucleotide correlation with the nucleotide sequence of the targeted protospacer on the ANGPTL3 gene.

[0025] In another aspect, a method for treating or preventing atherosclerotic cardiovascular disease in a subject in need thereof comprises administering a therapeutically effective amount of (a) an mRNA encoding an adenine base editor protein having an editing window; (b) a first guide RNA comprising a tracr sequence that functions as a binding scaffold for the base editor protein and a spacer sequence that functions to guide the base editor protein to a protospacer sequence on the PCSK9 gene, wherein the spacer sequence is at least partially complementary to a splice site or exon region of the PCSK9 gene; and Disclosed herein is a method comprising administering to a subject a second guide RNA comprising a tracr sequence that functions as a binding scaffold for a base editor protein and a spacer sequence that functions to guide the base editor protein to a protospacer sequence on the ANGPTL3 gene, the spacer sequence being at least partially complementary to a splice site or exon region of the ANGPTL3 gene. In some embodiments, the method further comprises a first LNP encapsulating (a). In some embodiments, the first LNP encapsulates (b) and (c). In some embodiments, the first LNP is administered repeatedly. In some embodiments, the first LNP is administered repeatedly at intervals of 1-60 days. In some embodiments, the first LNP is administered repeatedly at intervals of 7 days. In some embodiments, the first LNP further encapsulates (b). In some embodiments, the method further comprises a second LNP encapsulating (a) and (c). In some embodiments, the first LNP and the second LNP are administered sequentially. In some embodiments, the first LNP and the second LNP are administered sequentially at an interval between 1 day and 12 months. In some embodiments, the interval is 1 day. In some embodiments, the interval is 5 days. In some embodiments, the interval is 10 days. In some embodiments, the interval is 15 days. In some embodiments, the interval is 20 days. In some embodiments, the interval is 25 days. In some embodiments, the interval is 1 month. In some embodiments, the interval is 2 months. In some embodiments, the interval is 3 months. In some embodiments, the interval is 5 months. In some embodiments, the interval is 8 months. In some embodiments, the interval is 10 months. In some embodiments, the interval is 12 months.

[0026]

[25] Features, principles, advantages, and illustrative embodiments, implementations, analyses, and examples of the subject matter of the present application are described herein, including in the appended claims, aspects of which are illustrated in the accompanying drawings. [Brief description of the drawings]

[0027] [Figure 1A]

[0026] Figures 1A-1C illustrate the modes of operation of Cas9, a cytidine base editor (CBE), and an adenine base editor (ABE), respectively, along with associated nomenclature used in this application, including "protospacer," "PAM," and "spacer" (Mali, P. et al. 2013 Nat Methods 10, 957-963; Anzalone, AV. 2020 Nat Biotechnol 38, 824-844). [Figure 1B]

[0026] Figures 1A-1C illustrate the modes of operation of Cas9, a cytidine base editor (CBE), and an adenine base editor (ABE), respectively, along with associated nomenclature used in this application, including "protospacer," "PAM," and "spacer" (Mali, P. et al. 2013 Nat Methods 10, 957-963; Anzalone, AV. 2020 Nat Biotechnol 38, 824-844). [Figure 1C]

[0026] Figures 1A-1C illustrate the modes of operation of Cas9, a cytidine base editor (CBE), and an adenine base editor (ABE), respectively, along with associated nomenclature used in this application, including "protospacer," "PAM," and "spacer" (Mali, P. et al. 2013 Nat Methods 10, 957-963; Anzalone, AV. 2020 Nat Biotechnol 38, 824-844). [Figure 1D]FIG. 1D is a schematic representation of how the mcPCSK9 guide RNA (gRNA)-Tracr disclosed herein was designed. Shown is the ribonucleoprotein (RNP)-single guide RNA (sgRNA) alignment at the stem-loop guide intramolecular interaction (WC base pairing in the stem). The tracrRNA sequence of the gRNA serves as a binding scaffold for the cas protein. When designing the gRNA, the loop nucleotides can be aligned with the protein, and the interactions of the base (H-bond), 2'-hydroxyl (2'-OH), 4'-oxygen (ring oxygen) of the sugar moiety, and the phosphate linkage of each nucleotide to the amino acid side chain of the protein can be considered in the design of the gRNA-Tracr. The spatial organization of the nucleotides within the RNP steric interactions, room to accept bulky substitutions such as 2'-O-methyl (2'-OMe) and phosphorothioate (PS) can also be taken into consideration. FIG. 1D discloses SEQ ID NOs: 70-71, respectively, in order of appearance. [Diagram 2]

[0027] Figure 2 shows two tables that describe how the SpCas9 crystal structure was selected in conjunction with the design of the gRNA disclosed herein based on the structure. The top panel is a table summarizing the different components of the CRISPR / Cas system with their Protein Data Bank (PDB) IDs and states. The bottom panel shows the root mean square deviation (RMSD) values ​​after alignment between the protein chains of each structure. The RNP state (4ZT0) and the precatalytic ternary complex (5F9R) are the most relevant to determine the contacts for RNP formation and the contacts for catalysis. Some rearrangements of the protein occur between these two states. Large-scale rearrangements can occur in the protein between the apo state and the RNP. [Diagram 3]

[0028] Figure 3 depicts the gRNA secondary structure of the sgRNA based on the 4ZT0 and 5F9R crystal structures, where the sgRNA is bound to SpCas9 either as an RNP alone or as part of a ternary complex with DNA. The secondary structure relationships are shown in the Leontis and Westhof nomenclature (RNA, 2001, 7, pp. 499-512) (SEQ ID NO: 72). [Figure 4]

[0029] FIG. 4 depicts the gRNA secondary structure with the PCSK9 spacer and shows predicted contacts based on RNP PDB 4ZT0 (sgRNA+SpCas9 RNP). Positions 1-10 and 83-100 were not modeled in this crystal structure due to lack of clear electron density (SEQ ID NO: 73). Black circles with open letter labels: protein contacts, light grey with black letters: steric clashes or distal contacts when 2'-O-Me is incorporated, dark grey with black letters: RNA contacts. As one of skill in the art would understand, the term "clash" as used herein refers to atomic volumes in the structure that are unlikely to physically overlap. Incorporation of 2'O-Me modifications in these situations would potentially result in structural rearrangements that could be detrimental to RNP function. As one of skill in the art would understand, the term "contact" as used herein means a stable, non-covalent interaction between two functional groups, e.g., hydrogen bonds. [Diagram 5]

[0030] Figure 5 depicts the gRNA secondary structure with the PCSK9 spacer and shows predicted contacts based on PDB 5F9R (pre-catalytic ternary complex) (SEQ ID NO: 74). Black circles with open letter labels: protein contacts, light grey with black letters: steric clashes or distal contacts when 2'-O-Me is incorporated, dark grey with black letters: RNA contacts. [Figure 6]

[0031] Figure 6 depicts the reasonable positions of 2'-OMe substitutions determined by structure-based design (SEQ ID NO: 73). Filled circles with open label: 2'-OMe and phosphorothioate substitutions, light grey circles with closed label: 2'-OMe substitutions only, open circles with closed label: unmodified nucleotides. [Figure 7A-1]

[0032] Figure 7A depicts three patterns of 2'-OMe positions identified in the tracr region of single guide RNAs from structure-guided incorporation of 2'-O-methyl ribosugar modifications that generated robust edits in vivo: (1) mouse: Figure 16, GA054 (tracr1) and GA055 (tracr2) and (2) NHP: Figure 28, GA096 (tracr1), GA097 (tracr2) and GA346 (tracr3). Filled circles with open letter labels: 2'-OMe and phosphorothioate substitutions, light grey circles with black letter labels: 2'-OMe substitutions only, open circles with black letter labels: unmodified nucleotides. (SEQ ID NO: 73) [Figure 7A-2]

[0032] Figure 7A depicts three patterns of 2'-OMe positions identified in the tracr region of single guide RNAs from structure-guided incorporation of 2'-O-methyl ribosugar modifications that generated robust edits in vivo: (1) Mouse: Figure 16, GA054 (tracr1) and GA055 (tracr2) and (2) NHP: Figure 28, GA096 (tracr1), GA097 (tracr2) and GA346 (tracr3). Black circles with open letter labels: 2'-OMe and phosphorothioate substitutions, light grey circles with black letter labels: 2'-OMe substitutions only, open circles with black letter labels: unmodified nucleotides. (SEQ ID NO: 73) [Figure 7A-3]

[0032] Figure 7A depicts three patterns of 2'-OMe positions identified in the tracr region of single guide RNAs from structure-guided incorporation of 2'-O-methyl ribosugar modifications that generated robust edits in vivo: (1) Mouse: Figure 16, GA054 (tracr1) and GA055 (tracr2) and (2) NHP: Figure 28, GA096 (tracr1), GA097 (tracr2) and GA346 (tracr3). Black circles with open letter labels: 2'-OMe and phosphorothioate substitutions, light grey circles with black letter labels: 2'-OMe substitutions only, open circles with black letter labels: unmodified nucleotides. (SEQ ID NO: 73) [Figure 7B]Figure 7B depicts a sequence alignment of unmodified SEQ ID NO:61, the reference tracrRNA sequence ("Lit Tracr"), tracr1, tracr2, and tracr3. (SEQ ID NO:73) [Figure 8]

[0033] Figure 8 shows the target splice site base editing by the adenosine base editor system in modifying PCSK9 in primary human hepatocytes. The dark line represents the percentage of splice site editing obtained using gRNA identified in GA066. [Figure 9]

[0034] Figure 9 depicts Sanger sequencing chromatograms demonstrating the editing of adenine bases in the antisense strand at the splice donor at the end of PCSK9 exon 1 (PCR amplification from genomic DNA of cells transfected with a dose of 2500ng / mL) and how splice site interference results in an in-frame stop codon. Heterozygosity of naturally occurring single nucleotide polymorphisms (SNPs) is evident downstream of the editing site. The scheme shows A to G base editing by adenosine base editor system to knock out PCSK9 in primary human hepatocytes. Figure 9 discloses SEQ ID NO: 75. [Figure 10]

[0035] Figure 10 depicts a Sanger sequencing chromatogram demonstrating the editing of an adenine base in the antisense strand at the splice donor at the end of ANGPTL3 exon 6 (PCR amplification from genomic DNA of cells transfected with a dose of 2500ng / mL) and illustrating how splice site interference results in an in-frame stop codon. A to G base editing provided by the adenosine base editor system in the modification of ANGPTL3 in primary human hepatocytes. Figure 10 discloses SEQ ID NO: 76. [Figure 11-1]

[0036] FIG. 11 shows three Sanger sequencing chromatograms from PCR-amplified genomic DNA isolated from 1) untreated primary hepatocytes (top panel); 2) primary hepatocytes treated with SpCas9 mRNA / PCSK9 gRNA GA097 (middle panel); 3) primary hepatocytes treated with ABE8.8 mRNA / PCSK9 gRNA (bottom panel). The PCSK9-gRNA GA097 protospacer sequence is highlighted in grey. The arrow points to position 6 of the protospacer targeted for A to G base editing by the ABE8.8 editor. The scissors depict the general site of double-strand break that occurs upon cleavage by SpCas9 nuclease at this target site. SpCas9 mRNA and GA097 delivery in cells resulted in significant gene editing at the site of the double-strand break (as represented by the scissors), which was not seen in ABE8.8 and GA097 delivery in cells. Instead, the same gRNA GA097 in combination with ABE8.8 mRNA resulted in robust A to G base editing. FIG. 11 discloses SEQ ID NOs: 77-79, respectively, in order of appearance. [Figure 11-2]

[0036] Figure 11 shows three Sanger sequencing chromatograms from PCR-amplified genomic DNA isolated from 1) untreated primary hepatocytes (top panel); 2) primary hepatocytes treated with SpCas9 mRNA / PCSK9 gRNA GA097 (middle panel); 3) primary hepatocytes treated with ABE8.8 mRNA / PCSK9 gRNA (bottom panel). The PCSK9-gRNA GA097 protospacer sequence is highlighted in grey. The arrow indicates the 6th position of the protospacer targeted for A to G base editing by the ABE8.8 editor. The scissors depict the general site of double-strand break that occurs upon cleavage by SpCas9 nuclease at this target site. SpCas9 mRNA and GA097 delivery in cells resulted in significant gene editing at the site of the double-strand break (as represented by the scissors), which was not seen in ABE8.8 and GA097 delivery in cells. Instead, the same gRNA GA097 in combination with ABE8.8 mRNA resulted in robust A to G base editing. FIG. 11 discloses SEQ ID NOs: 77-79, respectively, in order of appearance. [Figure 12]

[0037] Figure 12 depicts a schematic showing potential splicing outcomes with disruption of splice donor or splice acceptor sequences. Other outcomes are possible, such as inclusion of a portion of intron 1 in the splicing product. Alterations of splice donor or splice acceptor sites are shown (top panel). An alternative splice donor site within PCSK9 intron 1 resulting from editing of the PCSK9 exon 1 splice donor adenine base in primary human hepatocytes is shown in the bottom panel. *Four different primer pairs (Table 9) were used for RT-PCR of control / treated samples. [Figure 13]

[0038] Figure 13 shows the absence of guide RNA-dependent DNA off-target editing in primary human hepatocytes. Human primary hepatocytes were incubated with gRNA (GA097 or GA346) and ABE8.8 mRNA as described in Example 4. Off-target readings were calculated as the net adenine editing (the proportion of sequencing reads with one or more adenine base changes in LNP-treated cells compared to untreated cells) at the on-target PCSK9 site and more than 50 candidate off-target PCSK9 sites in primary human hepatocytes from four individual donors. [Figure 14]

[0039] Figure 14 depicts guide RNA-independent RNA editing assessed in SpCas9-treated or ABE8.8-treated hepatocytes after 2 days (n=4 biological replicates). Each replicate was compared to each of the four non-treated hepatocyte samples to eliminate any positions with editing common to both conditions. Jitter plot depicts transcriptome loci with editing in treated samples (numbers indicate total number of edited loci identified in treated samples, box plots indicate median ± interquartile range of percentage of edited reads across all edited loci in samples). gRNA GA097, SpCas9 mRNA MS010, and ABE8.8 mRNA MA004 were used in this study as described in Example 4. [Figure 15]

[0040] Figure 15 shows PCSK9 and ANGPTL3 base editing in primary human hepatocytes and primary non-human primate (NHP) hepatocytes using lipid nanoparticles (LNPs) formulated with one embodiment of the base editor system, ABE mRNA and guide RNA. Human-specific ANGPTL3 guide RNA showed low editing efficiency in NHP hepatocytes within the concentrations evaluated, while PCSK9 guide RNA cross-reactive to both human and NHP showed high editing efficiency in both cell lines. [Figure 16]

[0041] FIG. 16 shows gene editing of Pcsk9 in mice (n=2-5 mice) via LNPs containing SpCas9 mRNA MS002 (TriLink Biotechnologies) and mouse Pcsk9-targeting gRNA with different tracr designs (GA052, GA053, GA054, and GA055) at a 1:1 weight ratio. Wild-type C57BL / 6 mice were dosed with 2 mg / kg of LNP test article. The mg / kg dose was calculated based on total RNA, which is the quantified sum of mRNA and gRNA present in the LNP after formulation. Seven days after dosing, mice were euthanized and genomic DNA was harvested from mouse liver and then assessed for base editing of the target site using next-generation sequencing. All four guide RNAs evaluated have the same spacer and the same pattern of chemical modifications within the first 20 nucleotides from the 5' end, but all four differ in the chemical modification pattern within the tracr between nucleotides 21 and 100 of the 100-mer guide RNA. [Figure 17]

[0042] Figure 17 shows base editing of PCSK9 in mice (n=5) via LNP using an embodiment of the base editor system, ABE mRNA MA004 and PCSK9 guide RNA GA256 (targeting mouse intron 1 splice donor). Wild-type C57BL / 6 mice were dosed with 2 mg / kg of total RNA LNP test article. Seven days after dosing, the mice were euthanized and genomic DNA was collected from the mouse liver and then evaluated for base editing at the target site using next-generation sequencing. [Figure 18]

[0043] FIG. 18 depicts Pcsk9 exon 1 splice donor adenine base editing in wild-type mouse liver assessed after 1 week of treatment with different doses of the same LNP formulation containing ABE8.8 mRNA MA004 and Pcsk9 gRNA GA256 (n=4-5 mice / dosing group, bars indicate average editing in group). [Figure 19]

[0044] Figure 19 depicts the editing of Pcsk9 exon 1 splice donor adenine base in wild-type mouse liver after administration of 0.05 mg / kg total RNA dose of LNP containing different ratios of gRNA GA256 and mRNA MA002. Additional guides GA255 and GA257 with different chemical modifications were also evaluated for base editing efficiency at a 1:1 weight ratio of mRNA to gRNA. [Figure 20]

[0045] FIG. 20 shows results from dosing mice with LNPs containing ABE8.8 mRNA and mouse Angptl3-targeting gRNAs (GA258, GA259, GA260, GA349, GA353) in a 1:1 weight ratio at a total RNA dose of 0.05 mg / kg. GA258, GA259, and GA260 contain three different construct-guided tracr designs. GA349 and GA353 tracr designs were from published literature (Cell Reports, 2018 22, pp. 2227-2235). Mice were later euthanized and genomic DNA was harvested from mouse liver and then assessed for base editing of the targeted splice site using next-generation sequencing. [Figure 21]

[0046] FIG. 21 is a schematic showing a general dosing strategy for introducing adenine base editing of target genes in NHPs via LNPs formulated with one embodiment of the base editor system, ABE mRNA and guide RNA, and subsequent analysis after 2 weeks. [Figure 22]

[0047] FIG. 22 shows that administration of LNPs formulated with an embodiment of the base editor system, ABE mRNA MA002 and PCSK9 guide RNA GA066, at total RNA doses of 1 mg / kg and 3 mg / kg to cynomolgus monkeys via intravenous infusion induced adenine base editing at the PCSK9 target splice site in the liver of cynomolgus monkeys. [Figure 23]

[0048] FIG. 23 shows that administration of LNPs formulated with an embodiment of the base editor system, ABE mRNA MA004 and PCSK9 guide RNA GA066, to cynomolgus monkeys via intravenous infusion resulted in a reduction in blood PCSK9 protein levels compared to pre-dosing levels two weeks after dosing. [Figure 24]

[0049] FIG. 24 shows that administration of 3 mg / kg of LNP formulated with an embodiment of the base editor system, ABE mRNA MA002 and PCSK9 guide RNA GA066, to cynomolgus monkeys via intravenous infusion resulted in a reduction in blood low-density lipoprotein cholesterol (LDL-C) levels 1 and 2 weeks after dosing compared to pre-dosing levels. [Figure 25A]

[0050] Figures 25A-25C show short-term adenine base editing of PCSK9 in non-human primates. Figure 25A depicts PCSK9 exon 1 splice donor adenine base editing in the liver of cynomolgus monkeys given an intravenous infusion of a 1 mg / kg dose of LNP formulation containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA097 with necropsy either 2 weeks (3 animals) or 24 hours (2 animals) after treatment. For each animal, editing was assessed in samples collected from sites distributed throughout the liver (n=8 samples; bars indicate average editing in animals). Reductions in blood PCSK9 protein levels (Figure 25B) or blood LDL-C levels (Figure 25C) in three animals that underwent necropsy after 2 weeks of treatment are shown comparing the levels at 2 weeks to baseline pre-treatment levels (n=1 blood sample per animal). [Figure 25B]

[0050] Figures 25A-25C show short-term adenine base editing of PCSK9 in non-human primates. Figure 25A depicts PCSK9 exon 1 splice donor adenine base editing in the liver of cynomolgus monkeys given an intravenous infusion of a 1 mg / kg dose of LNP formulation containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA097 with necropsy either 2 weeks (3 animals) or 24 hours (2 animals) after treatment. For each animal, editing was assessed in samples collected from sites distributed throughout the liver (n=8 samples; bars indicate average editing in animals). Reductions in blood PCSK9 protein levels (Figure 25B) or blood LDL-C levels (Figure 25C) in three animals that underwent necropsy after 2 weeks of treatment are shown comparing the levels at 2 weeks to baseline pre-treatment levels (n=1 blood sample per animal). [Figure 25C]

[0050] Figures 25A-25C show short-term adenine base editing of PCSK9 in non-human primates. Figure 25A depicts PCSK9 exon 1 splice donor adenine base editing in the liver of cynomolgus monkeys given an intravenous infusion of a 1 mg / kg dose of LNP formulation containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA097 with necropsy either 2 weeks (3 animals) or 24 hours (2 animals) after treatment. For each animal, editing was assessed in samples collected from sites distributed throughout the liver (n=8 samples; bars indicate average editing in animals). Reductions in blood PCSK9 protein levels (Figure 25B) or blood LDL-C levels (Figure 25C) in three animals that underwent necropsy after 2 weeks of treatment are shown comparing the levels at 2 weeks to baseline pre-treatment levels (n=1 blood sample per animal). [Figure 26]

[0051] Figures 26A-26C show adenine base editing of PCSK9 in non-human primates. Figure 26A depicts PCSK9 exon 1 splice donor adenine base editing in the liver of cynomolgus monkeys given an intravenous infusion of 0.5, 1.0, or 1.5 mg / kg doses of LNP formulations containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA346. Reduction in blood PCSK9 protein levels (Figure 26B) or blood LDL-C levels (Figure 26C) for the animals is shown. [Figure 27]

[0052] Figure 27 depicts the tissue distribution of PCSK9 exon 1 splice donor adenine base editing in three animals that underwent necropsy after two weeks of treatment (n=1 sample per animal for each indicated organ except liver; liver data represents the average shown in the calculation from eight liver samples each). LNPs constructed with ABE mRNA MA004 and guide RNA GA346 were used in this study, and the administered dose was 0.5 mg / kg. [Figure 28]

[0053] Figure 28 illustrates the editing of PCSK9 exon 1 splice donor adenine base editing in the liver of cynomolgus monkeys after intravenous injection of individual lipid nanoparticles (LNPs) constructed with ABE mRNA MA004 and different guide RNAs with the same spacer but different tracer modifications. The guide RNAs used in this study were GA066, GA096, GA097 and GA346. gRNA GA097 from two different sources was used in the same study, and the sources are identified as (1) and (2). For the tracr comparison study, LNPs were dosed at a total RNA dose of 1 mg / kg, and guide RNA and mRNA were mixed at a weight ratio of 1:1. GA066, which has a published tracr design (Cell Reports, 2018 22, pp. 2227-2235), produced lower base edits in monkeys under the same experimental conditions compared to all other tracr designs (Figure 7) (GA095, GA097, and GA346). [Figure 29]

[0054] Figure 29 shows SpCas9 nuclease for adenine base editing of PCSK9 in non-human primates. LNPs containing either SpCas9 mRNA and PCSK9 gRNA (MS010 / GA097) or ABE8.8 mRNA and PCSK9 gRNA (MA004 / GA097) were intravenously injected into cynomolgus monkeys. MS010 / GA097 LNPs were dosed at 0.75 and 1.5 mg / kg, and MA004 / GA097 LNPs were dosed at 1 mg / kg total RNA dose. MS010 / GA097 LNP test article at 1.5 mg / kg produced low single digit gene edits in NHPs, while ABE / GA097 test article produced approximately 40% adenine base edits at 1 mg / kg, demonstrating the robustness of the ABE base editor over the SpCas9 system. All LNPs used in this study were prepared using the same excipients and composition. [Diagram 30]

[0055] Figure 30 shows adenine base editing of PCSK9 in non-human primates. The graph depicts PCSK9 exon 1 splice donor adenine base editing in the liver of cynomolgus monkeys given intravenous infusions of LNP#1 at 0.5, 1.0, 1.5, or 3 mg / kg total RNA doses (each bar is an individual animal and editing was recorded for multiple sampling plots). LNP#2 at 3 mg / kg total RNA was dosed as a benchmark from a previous study. Both LNP formulations contain ABE8.8 mRNA MA004 and PCSK9 gRNA GA346. [Diagram 31]

[0056] FIG. 31 shows the reduction in PCSK9 protein levels from basal on day 15 from the same experiment as described in FIG. [Diagram 32]

[0057] Figure 32 depicts PCSK9 exon 1 splice donor adenine base editing in the liver of four cynomolgus monkeys given an intravenous infusion of a 3 mg / kg total RNA dose of an LNP formulation containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA066. For each animal, editing was assessed in liver biopsy samples after 2 weeks of treatment (n=1 sample per animal). [Diagram 33]

[0058] Figure 33 depicts the reduction in blood PCSK9 protein levels in four animals from Figure 32 (animals given a 3 mg / kg total RNA dose of LNP formulations containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA066) and two control animals given phosphate buffered saline over the same period, comparing levels at various time points after treatment to baseline pre-treatment levels (mean ± standard deviation for each group at each time point, n=4 or n=2). The dotted lines indicate 100% and 10% of baseline levels, respectively. [Figure 34-1]

[0059] Figure 34 depicts the reduction of blood LDL-C levels in four animals from Figure 32 (animals given a total RNA dose of 3 mg / kg of LNP formulations containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA066) and two control animals given phosphate-buffered saline for the same period, comparing levels at various time points after treatment to baseline pre-treatment levels (mean ± standard deviation for each group at each time point, n=4 or n=2). Dotted lines indicate 100% and 40% of baseline levels, respectively (upper panel). Absolute values ​​for individual animals are shown in the lower panel. [Figure 34-2]

[0059] Figure 34 depicts the reduction of blood LDL-C levels in four animals from Figure 32 (animals given a total RNA dose of 3 mg / kg of LNP formulations containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA066) and two control animals given phosphate-buffered saline for the same period, comparing levels at various time points after treatment to baseline pre-treatment levels (mean ± standard deviation for each group at each time point, n=4 or n=2). Dotted lines indicate 100% and 40% of baseline levels, respectively (upper panel). Absolute values ​​for individual animals are shown in the lower panel. [Diagram 35]

[0060] FIG. 35 depicts the reduction in lipoprotein(a) in four animals from FIG. 32 and two control animals from the same period that received phosphate-buffered saline, comparing levels at various time points after treatment to baseline pre-treatment levels (mean±standard deviation for each group at each time point, n=4 or n=2). [Figure 36-1]

[0061] Figure 36 shows the long-term phenotypic effects of hepatic PCSK9 base editing in non-human primates. Absolute values ​​of aspartate aminotransferase (AST) (upper panel) and alanine aminotransferase (ALT) (lower panel) in individual animals depicted in Figure 32 at various time points after treatment (n=4 animals treated with LNP formulations containing ABE8.8 mRNA and PCSK9-gRNA at a dose of 3 mg / kg, and n=2 animals treated with phosphate buffered saline). [Figure 36-2]

[0061] Figure 36 shows the long-term phenotypic effects of hepatic PCSK9 base editing in non-human primates. Absolute values ​​of aspartate aminotransferase (AST) (upper panel) and alanine aminotransferase (ALT) (lower panel) in individual animals depicted in Figure 32 (n = 4 animals treated with a 3 mg / kg dose of LNP formulation containing ABE8.8 mRNA and PCSK9-gRNA, and n = 2 animals treated with phosphate buffered saline) at various time points after treatment. [Figure 37A-1]

[0062] Figures 37A-37G show individual animal liver function markers: AST (Figure 37A, Figure 37B), ALT (Figure 37A, Figure 37C), Alkaline phosphate (Figure 37A, Figure 37D), gamma-glutamyltransferase (Figure 37A, Figure 37E), total bilirubin (Figure 37A, Figure 37F), and albumin (Figure 37A, Figure 37G) from cynomolgus monkeys given intravenous infusions of LNP formulations containing ABE8.8 mRNA and PCSK9 gRNA at doses of 0.5, 1.0, or 1.5 mg / kg up to 15 days post-dosing. [Figure 37A-2]

[0062] Figures 37A-37G show individual animal liver function markers. AST (Figure 37A, Figure 37B), ALT (Figure 37A, Figure 37C), alkaline phosphate (Figure 37A, Figure 37D), gamma-glutamyltransferase (Figure 37A, Figure 37E), total bilirubin (Figure 37A, Figure 37F), and albumin (Figure 37A, Figure 37G) from cynomolgus monkeys given intravenous infusions of LNP formulations containing ABE8.8 mRNA and PCSK9 gRNA at doses of 0.5, 1.0, or 1.5 mg / kg up to 15 days post-dosing. [Figure 37B]

[0062] Figures 37A-37G show individual animal liver function markers. AST (Figure 37A, Figure 37B), ALT (Figure 37A, Figure 37C), alkaline phosphate (Figure 37A, Figure 37D), gamma-glutamyltransferase (Figure 37A, Figure 37E), total bilirubin (Figure 37A, Figure 37F), and albumin (Figure 37A, Figure 37G) from cynomolgus monkeys given intravenous infusions of LNP formulations containing ABE8.8 mRNA and PCSK9 gRNA at doses of 0.5, 1.0, or 1.5 mg / kg up to 15 days post-dosing. [Figure 37C]

[0062] Figures 37A-37G show individual animal liver function markers. AST (Figure 37A, Figure 37B), ALT (Figure 37A, Figure 37C), alkaline phosphate (Figure 37A, Figure 37D), gamma-glutamyltransferase (Figure 37A, Figure 37E), total bilirubin (Figure 37A, Figure 37F), and albumin (Figure 37A, Figure 37G) from cynomolgus monkeys given intravenous infusions of LNP formulations containing ABE8.8 mRNA and PCSK9 gRNA at doses of 0.5, 1.0, or 1.5 mg / kg up to 15 days post-dosing. [Figure 37D]

[0062] Figures 37A-37G show individual animal liver function markers. AST (Figure 37A, Figure 37B), ALT (Figure 37A, Figure 37C), alkaline phosphate (Figure 37A, Figure 37D), gamma-glutamyltransferase (Figure 37A, Figure 37E), total bilirubin (Figure 37A, Figure 37F), and albumin (Figure 37A, Figure 37G) from cynomolgus monkeys given intravenous infusions of LNP formulations containing ABE8.8 mRNA and PCSK9 gRNA at doses of 0.5, 1.0, or 1.5 mg / kg up to 15 days post-dosing. [Figure 37E]

[0062] Figures 37A-37G show individual animal liver function markers. AST (Figure 37A, Figure 37B), ALT (Figure 37A, Figure 37C), alkaline phosphate (Figure 37A, Figure 37D), gamma-glutamyltransferase (Figure 37A, Figure 37E), total bilirubin (Figure 37A, Figure 37F), and albumin (Figure 37A, Figure 37G) from cynomolgus monkeys given intravenous infusions of LNP formulations containing ABE8.8 mRNA and PCSK9 gRNA at doses of 0.5, 1.0, or 1.5 mg / kg up to 15 days post-dosing. [Figure 37F]

[0062] Figures 37A-37G show individual animal liver function markers. AST (Figure 37A, Figure 37B), ALT (Figure 37A, Figure 37C), alkaline phosphate (Figure 37A, Figure 37D), gamma-glutamyltransferase (Figure 37A, Figure 37E), total bilirubin (Figure 37A, Figure 37F), and albumin (Figure 37A, Figure 37G) from cynomolgus monkeys given intravenous infusions of LNP formulations containing ABE8.8 mRNA and PCSK9 gRNA at doses of 0.5, 1.0, or 1.5 mg / kg up to 15 days post-dosing. [Figure 37G]

[0062] Figures 37A-37G show individual animal liver function markers. AST (Figure 37A, Figure 37B), ALT (Figure 37A, Figure 37C), alkaline phosphate (Figure 37A, Figure 37D), gamma-glutamyltransferase (Figure 37A, Figure 37E), total bilirubin (Figure 37A, Figure 37F), and albumin (Figure 37A, Figure 37G) from cynomolgus monkeys given intravenous infusions of LNP formulations containing ABE8.8 mRNA and PCSK9 gRNA at doses of 0.5, 1.0, or 1.5 mg / kg up to 15 days post-dosing. [Figure 38]

[0063] Figure 38 is a schematic diagram of representative candidate ONE-seq sites using a unique library designed against the cynomolgus genome. The PCSK9 protospacer is depicted at the top with a ONE-seq score of 1.00. All sites listed are ranked by decreasing ONE-seq score with mismatches to the identified protospacer sequence. Figure 38 discloses SEQ ID NOs: 80, 2193-2196, 566, 2197-2205, 675, 2206-2220, 564, 2221-224, 638, 644, 506, 621, 2225-2226, 681, and 2227-2230, respectively, in order of appearance. [Figure 39-1]

[0064] Figure 39 shows gRNA-dependent DNA off-target analysis of the sites identified in Figure 38. Samples from primary cynomolgus monkey hepatocytes (upper panel) or cynomolgus monkey liver (lower panel) were assessed for net A>G base editing (n=3 treated samples, n=3 untreated samples). [Figure 39-2]

[0064] Figure 39 shows gRNA-dependent DNA off-target analysis of the sites identified in Figure 38. Samples from primary cynomolgus monkey hepatocytes (upper panel) or cynomolgus monkey liver (lower panel) were assessed for net A>G base editing (n=3 treated samples, n=3 untreated samples). [Diagram 40]

[0065] FIG. 40 shows the % net A>G base editing at one off-target site (C5), identified in FIG. 39, from the livers of NHPs given either 0.5, 1.0, or 1.5 mg / kg of LNP. [Diagram 41]

[0066] 41 shows base editing of ANGPTL3 in NHPs via LNPs formulated with an embodiment of the base editor system, ABE mRNA MA004 and ANGPTL3 guide RNA GA067. Liver editing (left panel), ANGPTL3 protein levels (middle panel), and triglyceride levels (right panel) are shown for three NHPs. [Diagram 42]

[0067] FIG. 42 shows simultaneous ANGPTL3 and PCSK9 base editing using lipid nanoparticles (LNPs) formulated with an embodiment of the base editor system, ABE mRNA MA002 and dual guide RNAs GA095 (hcPCSK9) and GA098 (hANGPTL3), in human primary hepatocytes at concentrations ranging from 0-2500 ng / test article / mL. [Figure 43-1]

[0068] Figure 43 shows adenosine base editing results of liver biopsies on days 15 and 44. NHPs were dosed with LNPs formulated with ABE8.8 mRNA MA004 and either gRNA targeting PCSK9 (GA346) or gRNA targeting ANGPTL3 (GA347) via intravenous infusion at total RNA doses ranging from 0.5 to 2 mg / kg. Two weeks after administration of the test article, biopsies were taken to assess base editing. Thirty days after the start of the study, the opposite LNP was administered. After a second biopsy two additional weeks later, gDNA was extracted and base editing was assessed using next-generation sequencing. Results are shown for PCSK9 base editing (upper panel) and ANGPTL3 base editing (lower panel). A total RNA dose of 2 mg / kg of LNPs encapsulating ABE8.8 mRNA, PCSK9 gRNA GA346 and ANGPTL3 gRNA GA347 at a weight ratio of 1:0.5:0.5 produced robust synchronized PCSK9 and ANGPTL3 gene editing (Example 10). [Figure 43-2]

[0068] Figure 43 shows adenosine base editing results of liver biopsies on days 15 and 44. NHPs were dosed with LNPs formulated with ABE8.8 mRNA MA004 and either gRNA targeting PCSK9 (GA346) or gRNA targeting ANGPTL3 (GA347) via intravenous infusion at total RNA doses ranging from 0.5 to 2 mg / kg. Two weeks after administration of the test article, biopsies were taken to evaluate base editing. Thirty days after the start of the study, the opposite LNP was administered. After a second biopsy two additional weeks later, gDNA was extracted and base editing was evaluated using next-generation sequencing. Results are shown for PCSK9 base editing (upper panel) and ANGPTL3 base editing (lower panel). A total RNA dose of 2 mg / kg of LNPs encapsulating ABE8.8 mRNA, PCSK9 gRNA GA346 and ANGPTL3 gRNA GA347 at a weight ratio of 1:0.5:0.5 produced robust synchronized PCSK9 and ANGPTL3 gene editing (Example 10). [Figure 44-1]

[0069] FIG. 44 illustrates the corresponding % changes in PCSK9 (upper panel) and ANGPTL3 (lower panel) protein levels from the NHPs described in FIG. [Figure 44-2] FIG. 44 illustrates the corresponding % changes in PCSK9 (upper panel) and ANGPTL3 (lower panel) protein levels from the NHPs described in FIG. [Diagram 45]

[0070] Figure 45 illustrates that repeated LNP dosing in NHPs causes additive adenosine base editing in the liver in liver biopsies after 14, 46, and 75 days. NHPs were dosed with either LNP#1 or LNP#2 formulated with ABE8.8 mRNA MA004 and gRNA targeting PCSK9 (GA097) via intravenous infusion at a total RNA dose of 0.5 mg / kg (see Example 10 for details regarding dosing intervals and related details). [Figure 46]

[0071] Figure 46 illustrates that repeated LNP dosing in NHPs causes additive base editing in the liver over 90 days, translating into dose-dependent additive reductions in plasma PCSK9 protein levels. NHPs were repeatedly dosed with LNPs formulated with ABE8.8 mRNA MA004 and gRNA targeting PCSK9 (GA097) as described in Figure 45. NHPs were dosed via intravenous infusion at a total RNA dose of 0.5 mg / kg on days 0, 30, and 60 (arrows are illustrated on the graph to depict dosing). See the detailed methods section for a description of the analysis of PCSK9 protein levels. [Figure 47-1]

[0072] Figure 47 illustrates that repeated LNP dosing in NHPs causes additive base editing in the liver with only transient increases in liver markers that correlate well with the day of administration of each dose. Data shows liver marker levels of ALT, AST, total bilirubin, and creatine kinase 71 days after the first dose (see Example 10, Figure 45 for details). [Figure 47-2]

[0072] Figure 47 illustrates that repeated LNP dosing in NHPs causes additive base editing in the liver with only transient increases in liver markers that correlate well with the day of administration of each dose. Data show liver marker levels of ALT, AST, total bilirubin, and creatine kinase 71 days after the first dose (see Example 10, Figure 45 for details). [Figure 47-3]

[0072] Figure 47 illustrates that repeated LNP dosing in NHPs causes additive base editing in the liver with only transient increases in liver markers that correlate well with the day of administration of each dose. Data show liver marker levels of ALT, AST, total bilirubin, and creatine kinase 71 days after the first dose (see Example 10, Figure 45 for details). [Figure 47-4]

[0072] Figure 47 illustrates that repeated LNP dosing in NHPs causes additive base editing in the liver with only transient increases in liver markers that correlate well with the day of administration of each dose. Data show liver marker levels of ALT, AST, total bilirubin, and creatine kinase 71 days after the first dose (see Example 10, Figure 45 for details). [Figure 48-1]

[0073] FIG. 48 illustrates that repeated LNP dosing in NHPs causes additive base editing in the liver, with only transient increases in liver markers showing liver enzyme levels of LDH, GLDH, GGT, and ALP up to 71 days post-dosing in NHPs (see Example 10, FIG. 45 for details). [Figure 48-2]

[0073] Figure 48 illustrates that repeated LNP dosing in NHPs causes additive base editing in the liver, with only transient increases in liver markers showing liver enzyme levels of LDH, GLDH, GGT, and ALP up to 71 days after dosing in NHPs (see Example 10, Figure 45 for details). [Figure 48-3]

[0073] Figure 48 illustrates that repeated LNP dosing in NHPs causes additive base editing in the liver, with only transient increases in liver markers showing liver enzyme levels of LDH, GLDH, GGT, and ALP up to 71 days after dosing in NHPs (see Example 10, Figure 45 for details). [Figure 48-4]

[0073] Figure 48 illustrates that repeated LNP dosing in NHPs causes additive base editing in the liver, with only transient increases in liver markers showing liver enzyme levels of LDH, GLDH, GGT, and ALP up to 71 days after dosing in NHPs (see Example 10, Figure 45 for details). [Figure 49]

[0074] Figures 49A and 49B illustrate that base editing of ANGPTL3 results in long-term reduction of ANGPTL3 protein and triglyceride levels after a single dose of LNP constructed with ABE8.8 mRNA MA004 and ANGPTL3 gRNA GA067. Results illustrate the effect of long-term adenine base editing of ANGPTL3 on ANGPTL3 protein (Figure 49A) and triglyceride (Figure 49B) in non-human primates over a 6-month period. ANGPTL3 protein (96% reduction) and triglyceride levels are substantially reduced with a single dose of LNP and remain stably reduced for more than 170 days (see Example 10 for details). [Figure 50A]

[0075] Figures 50A-50E illustrate the effect of LNP dosing in NHPs on cytokine activation and immune response. Cynomolgus monkeys were given intravenous infusions of LNP formulations containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA346 at a dose of 0.5 mg / kg at the specified time points (Figures 50A and 50B). Blood was collected at the specified time points and graphs and analyzed for IP-10 and MCP-1. In an additional study, IL-6, MCP-1, and SC5b-9 (Figures 50C, 50D, and 50E, respectively) were analyzed at different time points from blood collected from NHPs given intravenous infusions of a 1.0 mg / kg total RNA dose of LNPs formulated with MA004 and PCSK9 gRNA GA346. [Figure 50B]

[0075] Figures 50A-50E illustrate the effect of LNP dosing in NHPs on cytokine activation and immune response. Cynomolgus monkeys were given intravenous infusions of LNP formulations containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA346 at a dose of 0.5 mg / kg at the specified time points (Figures 50A and 50B). Blood was collected at the specified time points and graphs and analyzed for IP-10 and MCP-1. In an additional study, IL-6, MCP-1, and SC5b-9 (Figures 50C, 50D, and 50E, respectively) were analyzed at different time points from blood collected from NHPs given intravenous infusions of 1.0 mg / kg total RNA doses of LNPs formulated with MA004 and PCSK9 gRNA GA346. [Figure 50C]

[0075] Figures 50A-50E illustrate the effect of LNP dosing in NHPs on cytokine activation and immune response. Cynomolgus monkeys were given intravenous infusions of LNP formulations containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA346 at a dose of 0.5 mg / kg at the specified time points (Figures 50A and 50B). Blood was collected at the specified time points and graphs and analyzed for IP-10 and MCP-1. In an additional study, IL-6, MCP-1, and SC5b-9 (Figures 50C, 50D, and 50E, respectively) were analyzed at different time points from blood collected from NHPs given intravenous infusions of 1.0 mg / kg total RNA doses of LNPs formulated with MA004 and PCSK9 gRNA GA346. [Figure 50D]

[0075] Figures 50A-50E illustrate the effect of LNP dosing in NHPs on cytokine activation and immune response. Cynomolgus monkeys were given intravenous infusions of LNP formulations containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA346 at a dose of 0.5 mg / kg at the specified time points (Figures 50A and 50B). Blood was collected at the specified time points and graphs and analyzed for IP-10 and MCP-1. In an additional study, IL-6, MCP-1, and SC5b-9 (Figures 50C, 50D, and 50E, respectively) were analyzed at different time points from blood collected from NHPs given intravenous infusions of 1.0 mg / kg total RNA doses of LNPs formulated with MA004 and PCSK9 gRNA GA346. [Figure 50E]

[0075] Figures 50A-50E illustrate the effect of LNP dosing in NHPs on cytokine activation and immune response. Cynomolgus monkeys were given intravenous infusions of LNP formulations containing ABE8.8 mRNA MA004 and PCSK9 gRNA GA346 at a dose of 0.5 mg / kg at the specified time points (Figures 50A and 50B). Blood was collected at the specified time points and graphs and analyzed for IP-10 and MCP-1. In an additional study, IL-6, MCP-1, and SC5b-9 (Figures 50C, 50D, and 50E, respectively) were analyzed at different time points from blood collected from NHPs given intravenous infusions of 1.0 mg / kg total RNA doses of LNPs formulated with MA004 and PCSK9 gRNA GA346. [Figure 51]

[0076] Figure 51 illustrates liver editing in NHPs 15 days after treatment with LNPs containing SpCas9 mRNA / gRNA. Figure 51 shows results from gene editing of ANGPTL3 or PCSK9 in non-human primates. Cynomolgus monkeys were given an intravenous infusion of a dose of 1.5 mg / kg of LNP formulations containing SpCas9 mRNA MS004 and one gRNA targeting either ANGPTL3 (GA261-GA263) or PCSK9 (GA266-GA271). At necropsy two weeks later, two pieces were isolated from each liver lobe (total of eight pieces) and gDNA was extracted. Samples were processed as described in the detailed methods section. Indel % was analyzed for each separate piece and is graphed as individual points. High editing efficiency was observed in most NHP livers. [Figure 52]

[0077] Figure 52 illustrates LDL-C levels in NHPs 15 days after treatment with SpCas9 / gRNA. Figure 52 shows reduction in LDL-C from gene editing of ANGPTL3 or PCSK9 in non-human primates. Cynomolgus monkeys were given an intravenous infusion of a dose of 1.5 mg / kg of LNP formulation containing SpCas9 mRNA MS004 and one gRNA targeting either ANGPTL3 (GA261-GA263) or PCSK9 (GA266-GA271). Samples were processed as described in the detailed methods section. All NHPs given LNPs containing SpCas9 mRNA / PCSK9 gRNA had at least a 35% reduction in circulating LDL-C levels. Although more modest, LNPs containing SpCas9 mRNA / ANGPTL3 gRNA had a 10-25% reduction in circulating LDL-C levels. [Diagram 53]

[0078] Figure 53 illustrates triglyceride levels in NHPs 15 days after treatment with SpCas9 / gRNA. Figure 53 shows triglyceride levels from gene editing of ANGPTL3 or PCSK9 in non-human primates. Cynomolgus monkeys were given an intravenous infusion of a dose of 1.5 mg / kg of LNP formulation containing SpCas9 mRNA MS004 and one gRNA targeting either ANGPTL3 (GA261-GA263) or PCSK9 (GA266-GA271). Samples were processed as described in the detailed methods section. NHPs given LNPs containing SpCas9 mRNA / ANGPTL3 gRNA had approximately 10-50% reduction in triglyceride levels. NHPs given LNPs containing SpCas9 mRNA / PCSK9 gRNA did not show a significant reduction in triglyceride levels. [Figure 54]

[0079] Figure 54A and Figure 54B illustrate that PACE modifications to gRNAs reduce off-target editing efficiency. Human primary hepatocytes were transfected with SpCas9 mRNA (purchased commercially from Trilink) at 2500, 1250, 500, and 250 ng / test article / mL and gRNAs targeting PCSK9 with modifications to tracr. Genomic DNA was processed, sequenced, and analyzed as described in the detailed method section. GA156 was transfected to serve as a positive control. GA248 and GA249 contain PACE modifications to gRNAs that have been previously demonstrated to reduce off-target editing efficiency. Indeed, GA248 and GA249 had lower on-target editing compared to the unmodified gRNA, GA156 (Figure 54A), but GA248 and GA249 showed reduced off-target editing at the identified off-target sites (Figure 54B). [Figure 55A-1]

[0080] Figure 55A illustrates a GC comparison of the ABE-encoding nucleotides MA004, MA019, MA020, MA021, and ABE8.8m (Table 23), as well as a more detailed look at MA004 (Figure 55A, bottom panel). Figure 55B illustrates the % editing obtained using the ABE-encoding nucleotides MA004, MA019, MA020, and MA021 (Table 23). [Figure 55A-2]

[0080] Figure 55A illustrates a GC comparison of the ABE-encoding nucleotides MA004, MA019, MA020, MA021, and ABE8.8m (Table 23), as well as a more detailed look at MA004 (Figure 55A, bottom panel). Figure 55B illustrates the % editing obtained using the ABE-encoding nucleotides MA004, MA019, MA020, and MA021 (Table 23). [Figure 55A-3]

[0080] Figure 55A illustrates a GC comparison of the ABE-encoding nucleotides MA004, MA019, MA020, MA021, and ABE8.8m (Table 23), as well as a more detailed look at MA004 (Figure 55A, bottom panel). Figure 55B illustrates the % editing obtained using the ABE-encoding nucleotides MA004, MA019, MA020, and MA021 (Table 23). [Figure 55B]

[0080] Figure 55A illustrates a GC comparison of the ABE-encoding nucleotides MA004, MA019, MA020, MA021, and ABE8.8m (Table 23), as well as a more detailed look at MA004 (Figure 55A, bottom panel). Figure 55B illustrates the % editing obtained using the ABE-encoding nucleotides MA004, MA019, MA020, and MA021 (Table 23). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028]

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

[0029]

[82] 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 or materials similar or equivalent to those described herein can be used in the practice or testing of this 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 intended that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0030] definition

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

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

[0031]

[85] It should also be noted that the term "or" is generally employed in its sense including "and / or" unless the context clearly dictates otherwise. As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably. These terms may convey that any combination is specifically intended. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C." The term "or" may be used conjunctively or disjunctively unless the context specifically dictates a disjunctive use.

[0032]

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

[0033]

[87] As used in this specification and the claims, the words "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "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.

[0034]

[88] As used herein, “some embodiments,” “embodiment,” “one embodiment,” “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.

[0035]

[89] As used herein, the term "nucleic acid" 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. A "nucleotide" includes the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other through the phosphate group. "Base" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that introduce novel reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. Nucleic acids include any oligonucleotide or polynucleotide, and may contain up to 60 nucleotides. Fragments containing the ribonucleotide are generally named oligonucleotides, while longer fragments are named polynucleotides. Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose covalently joined to phosphate at the 5' and 3' carbons of the sugar, forming an unbranched alternating polymer. The DNA may be in the form of, for example, an antisense molecule, a plasmid DNA, a precondensed DNA, a PCR product, a vector, an expression cassette, a chimeric sequence, a chromosomal DNA, or derivatives and combinations of these groups. Ribooligonucleotides consist of a similar repeating structure, where the five-carbon sugar is a ribose. Thus, the terms "polynucleotide" and "oligonucleotide" can refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. Additionally, nucleic acids include synthetic, nonstandard, and / or non-naturally occurring nucleic acids that contain known nucleotide analogs or modified backbone residues or linkages that have similar binding properties to the reference nucleic acid. Nucleic acids can be modified in the base moiety (e.g., typically at one or more atoms available to form hydrogen bonds with complementary nucleotides, and / or typically at one or more atoms not capable of forming hydrogen bonds with complementary nucleotides), in the sugar moiety, or in the phosphate backbone. Backbone modifications include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, phosphoroamidate, and phosphorodiamidate linkages. Phosphorothioate linkages replace non-bridging oxygen in the phosphate backbone with sulfur atoms to retard nuclease degradation of oligonucleotides. Phosphorodiamidate linkages (N3'→P5') allow for nuclease recognition and prevention of degradation. Backbone modifications can also include having peptide bonds in place of phosphorus in the backbone structure (e.g., N-(2-aminoethyl)glycine units linked by peptide bonds in peptide nucleic acids), or linking groups including carbamates, amides, and linear and cyclic hydrocarbon groups.Oligonucleotides with modified backbones are described by Micklefield et al., "Backbone modification of nucleic acids: synthesis, structure and. "Modified oligonucleotides - synthesis, properties and 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. The nucleic acid molecules described herein can contain sugar moieties that include ribose or deoxyribose, as occurs in naturally occurring nucleotides, or modified sugar moieties or sugar analogs. 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, increase binding affinity to RNA, and have enhanced nuclease resistance. Modified sugar moieties can also include those having additional bridges (e.g., a methylene bridge joining the 2'-O and 4'-C atoms of ribose in immobilized nucleic acids) or sugar analogs such as morpholine rings (e.g., as in phosphorodiamidate morpholinos). Examples of such analogs and / or modified residues include, but are not limited to, 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-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queuosine, 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 phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. In some cases, nucleotides may include modifications in their phosphate moieties, including modifications to triphosphate moieties. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphate). Such modified or substituted oligonucleotides are often preferred over natural forms due to properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases. That is, the terms "polynucleotide" and "oligonucleotide" may also include polymers or oligomers that include non-naturally occurring monomers or portions thereof that function similarly.

[0036]

[90] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, and also encompasses the explicitly indicated sequence. 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., 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)).

[0037]

[91] The present disclosure encompasses isolated or substantially purified nucleic acid molecules and compositions comprising these molecules. As used herein, an "isolated" or "purified" DNA or RNA molecule is a DNA or RNA molecule that exists away from its natural environment. An isolated DNA or RNA molecule may exist in purified form or may exist in a non-native environment, such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or a biologically active portion thereof is substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In one embodiment, an "isolated" nucleic acid does not include sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid). For example, in some embodiments, an isolated nucleic acid molecule can include less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived.

[0038]

[92] The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some instances, a vector is an expression vector capable of directing the expression of a nucleic acid to which it is operably linked. The term "operably linked" as used herein means that a nucleotide sequence of interest is linked to a control sequence in a manner that allows for the expression of the nucleotide sequence. The term "control sequence" as used herein includes, but is not limited to, promoters, enhancers, and other expression control elements. Such control 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, retroviruses (e.g., viral vectors based on murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentiviruses, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors.

[0039]

[93] As used herein, the terms "protein", "polypeptide", and "peptide" are used interchangeably and refer to a polymer of amino acid residues linked through peptide bonds, which may consist of two or more polypeptide chains. The terms "polypeptide", "protein", and "peptide" refer to a polymer of at least two amino acid monomers joined together through 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 consisting of two or more amino acids in a specific order, e.g., an order determined by the base sequence of nucleotides in a gene or RNA that encodes the protein. Proteins are important for the structure, function, and control of cells, tissues, and organs of living organisms, and each protein has a unique function. Examples include hormones, enzymes, antibodies, and any fragments thereof. In some examples, a protein may be a portion of a protein, e.g., a domain, subdomain, or motif of a protein. In some examples, the 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 for the amino acid sequence of a naturally occurring (or at least known) protein. The protein or 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 blot, flow cytometry, ELISA, RIA, and various proteomic techniques. Exemplary methods for measuring or detecting polypeptides include immunoassays, such as ELISA. This type of protein quantification method may be based on an antibody capable of capturing a specific antigen and a second antibody capable of detecting the captured antigen. Exemplary assays for detecting and / or measuring polypeptides are described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor. Published in Laboratory Press.

[0040]

[94] The term "sequence identity" as used herein means the amount of exact nucleotide matches between two different sequences. When comparing RNA and DNA sequences, uracil and thymine bases are considered identical bases. Gaps are not counted, and measurements typically relate to the shorter of the two sequences. for example: A: AAGGCTT B: AAGGC C: AAGGCAT Here 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 identical.

[0041]

[95] The term "sequence similarity", as used herein, can be described as an optimal matching problem that finds the minimum number of editing operations (insertions, deletions, and substitutions) (edit distance) to convert one sequence into an exact copy of the other sequence that aligns with it. Using this, the sequence similarity percentages for the above example 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)).

[0042]

[96] A "subject" in need thereof means an individual having a disease, a symptom of a disease, or a predisposition to a disease, for the purpose of curing, treating, mitigating, alleviating, altering, relieving, ameliorating, improving, or affecting the disease, symptom of a disease, or predisposition to 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 class of mammals, i.e., humans; non-human primates, such as chimpanzees and other ape and monkey species; farm animals, such as cows, horses, sheep, goats, pigs, and the like; farm animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs, and the like.

[0043]

[97] 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.

[0044]

[98] The term "atherosclerosis" or "atherosclerotic vascular disease" as used herein means a disease in which the buildup of plaque narrows the inside of the arteries. In some embodiments, this can lead to coronary artery disease, stroke, peripheral artery disease, or kidney problems.

[0045]

[99] The term "hypertriglyceridemia," as used herein, refers to hyper-emia of triglycerides, the most abundant lipid molecules in most living organisms. In some embodiments, elevated levels of triglycerides are associated with atherosclerosis and increased susceptibility to cardiovascular disease even in the absence of hypercholesterolemia (high cholesterol levels). In some embodiments, extremely high triglyceride levels increase the risk of acute pancreatitis. In some embodiments, hypertriglyceridemia is associated with overeating, obesity, diabetes and insulin resistance, excessive alcohol consumption, renal failure, nephrotic syndrome, genetic predisposition (e.g., familial combined hyperlipidemia, or Type II hyperlipidemia), lipoprotein lipase deficiency, lysosomal acid lipase deficiency, cholesteryl ester deposition disease, certain medications (e.g., isotretinoin, hydrochlorothiazide diuretics, beta blockers, protease inhibitors), hypothyroidism (underactive thyroid), systemic lupus erythematosus and related autoimmune responses, type 1 glycogen deposition disease, propofol, or HIV medications.

[0046]

[0100] The term "diabetes" as used herein refers to a condition characterized by prolonged high blood sugar levels. In some embodiments, diabetes refers to a group of metabolic disorders characterized by the inability of the pancreas to produce enough insulin due to loss of beta cells. In some embodiments, diabetes is type 1 diabetes, which is caused by the inability of the pancreas to produce enough insulin due to loss of beta cells. In some embodiments, diabetes is type 2 diabetes, which is characterized by insulin resistance, a condition in which cells cannot respond properly to insulin. In some embodiments, diabetes is gestational diabetes, which occurs when pregnant women with no history of diabetes develop high blood sugar levels.

[0047]

[0101] The term "low density lipoprotein (LDL)" as used herein refers to lipoproteins. It refers to a microscopic sphere consisting of a rim of protein and a core of cholesterol. In some embodiments, LDL has a highly hydrophobic core consisting of polyunsaturated fatty acids known as linoleic acid esters and hundreds to thousands of esterified and unesterified cholesterol molecules. In some embodiments, the core of LDL also contains triglycerides and other fats, and is surrounded by a shell of phospholipids and unesterified cholesterol.

[0048]

[0102] The term "high density lipoprotein (HDL)" as used herein refers to the minimum In embodiments, the plasma enzyme lecithin-cholesterol acyltransferase (LCAT) converts free cholesterol to cholesteryl, which is then sequestered in the core of the lipoprotein particle, ultimately giving rise to newly synthesized HDL, which assumes a spherical shape. In embodiments, HDL particles circulate through the bloodstream, increasing in size as they take up more cholesterol and phospholipid molecules from cells and other lipoproteins.

[0049]

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

[0050]

[0104] The terms "treat," "treating," or "treatment" and their grammatical equivalents As used herein, may include alleviating, attenuating, or ameliorating at least one symptom of a disease or condition, preventing further symptoms, arresting a disease or condition, for example, delaying, reducing, inhibiting, attenuating, eradicating, arresting, or stabilizing the progression or development of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, relieving a condition caused by a disease or condition, reducing disease severity, or prophylactically and / or therapeutically arresting a symptom of a disease or condition. "Treatment" also includes reducing the onset or recurrence of any symptoms or other adverse effects associated with a disease or condition, or the severity and / or frequency of side effects associated with a disease or condition. "Treatment" does not necessarily require a curative outcome. It is recognized that treating a disorder or condition does not prevent, but does not require, the complete elimination of the disorder, condition, or symptoms associated therewith. The term "treating" encompasses the concept of "management," which means reducing the severity of or delaying the recurrence of a particular disease or disorder in a patient, for example, lengthening the period of remission in a patient suffering from a disease. "Treating" can mean applying or administering a composition to a subject after the onset or suspected onset of a disease or condition.

[0051]

[0105] The term "treating" is intended to encompass the terms "preventing," "preventing," and "prevention." The term "prevent", "preventing" and "prevention" as used herein means to reduce the occurrence of symptoms of a condition in a subject who does not have a disease or condition but is at risk or susceptible to developing it. Prevention can be complete, such as the complete absence of symptoms of a condition in a subject. Prevention can be partial, such that the occurrence of symptoms of a condition in a subject is less than would occur in the absence of the present disclosure.

[0052]

[0106] For example, by "treating or preventing a condition" compared to an equivalent untreated control. Therefore, the reduction in symptoms of the disorder is at least 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 100%, 105%, 107%, 109%, 110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120 In some embodiments, alleviating symptoms of a disorder may include a degree of reduction or prevention of at least 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 reduction or prevention as compared to an equivalent untreated control.

[0053]

[0107] As used herein, "slowing" the progression of a disease refers to slowing the progression of the disease. It means to prolong, hinder, slow, hinder, stabilize and / or postpone. This delay can vary in duration depending on the history of the disease and / or the individual being treated. A method of "delaying" or mitigating the progression of a disease or delaying the onset of a disease is a method that reduces the probability of one or more symptoms of the disease progressing in a given time frame and / or reduces the severity of symptoms in a given time frame when compared with not using the method. Such comparisons are typically based on clinical trials that use a sufficient number of subjects to give statistically significant results.

[0054]

[0108] The "progression" or "evolution" of a disease refers to the progression of early signs of the disease and / or the progression of the disease. "Progression" refers to ensuring progression. Disease progression is detectable and can be assessed using standard clinical techniques well known in the art. However, progression also refers to progression that may not be detectable. For purposes of this disclosure, progression or progression refers to the biological course of a condition. "Progression" includes onset, recurrence, and onset.

[0055]

[0109] As used herein, the "onset" or "development" of a disease refers to the initial onset and and / or recurrence.

[0110] As used herein, the term "administer" and its grammatical equivalents are intended to mean the same thing as the term "administer" or "administered" in this specification. It may mean providing a pharmaceutical composition described in to a subject or patient. The composition can be administered to a subject using conventional methods known to those skilled in the art of medicine, depending on the type of disease or the site of the disease to be 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 adopted.

[0056]

[0111] The term "parenteral" as used herein includes subcutaneous, intradermal, This includes intravenous, intramuscular, intraperitoneal, intradermal, intraarterial, intrasynovial, intrapedicle, intrathecal, intravascular, intralesional, and intracranial injection or infusion techniques. Additionally, it can be administered to a subject via an injectable depot administration route, for example, using 1, 3, or 6 month depot injectable or biodegradable materials and methods.

[0057]

[0112] "Co-administration" refers to the use of one or more additional therapeutic agents to enhance the effect of the agent(s). "Administered" refers to administering a composition of the present disclosure sufficiently close in time to one or more additional therapeutic regimens or agents or treatments, or vice versa. In this regard, the compositions of the present disclosure described herein may be administered simultaneously with one or more additional therapeutic regimens or agents or treatments, at different times, or on completely different treatment schedules (e.g., a first treatment is administered daily while an additional treatment is administered weekly). For example, in embodiments, a second therapeutic regimen or agent or treatment is administered simultaneously with, prior to, or subsequent to the composition of the present disclosure.

[0058]

[0113] The term "pharmaceutical composition" and its grammatical equivalents as used herein refer to The mixture or solution may be administered to a subject, e.g., a human in need thereof, comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharma- ceutical acceptable excipients, carriers, and / or therapeutic agents. The term "medicament for preparation of a pharmaceutical composition" may refer to a mixture or solution that includes a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharma- ceutically acceptable excipients, carriers, and / or therapeutic agents.

[0059]

[0114] The term "pharmaceutically acceptable" and its grammatical equivalents are used herein. In some cases, "pharmaceutical acceptable" may refer to the attributes of a material useful in preparing a pharmaceutical composition that is generally safe, non-toxic, not biologically or otherwise harmful, and acceptable for veterinary as well as human pharmaceutical use. "Pharmaceutically acceptable" may refer to a material, such as a relatively non-toxic carrier or diluent, that does not abolish the biological activity or properties of a compound. That is, the material may be administered to a subject without eliciting undesirable biological effects or interacting in a deleterious manner with any of the components of the pharmaceutical composition in which it is contained.

[0060]

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

[0061]

[0116] A "pharmaceutical acceptable salt" is a salt that does not cause excessive toxicity, irritation, allergic reaction, or other adverse reactions. The salt may be of an acid or base generally considered in the art to be suitable for use in contact with human or animal tissues without other problems or complications. Such salts include mineral or organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids. Salts of specific pharmaceutical agents include, but are not limited to, alkanoic 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, acetic acid, and the like; HOOC-(CH 2 )n-COOH, and the like. Similarly, pharma- ceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. One skilled in the art will recognize from this disclosure and knowledge in the art that additional pharma- ceutically acceptable salts include those listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985). In general, pharma- ceutically acceptable acid or base salts can be synthesized from parent compounds that contain a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0062]

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

[0063]

[0118] A "therapeutically effective amount," as used herein, refers to a single or multiple The term "therapeutically effective amount" refers to the amount of each composition of the present disclosure required to confer a therapeutic effect on a subject in combination with other therapeutic agents. Thus, as used herein, the term "therapeutically effective amount" refers to the amount of agent (e.g., nucleic acid, composition, therapeutic agent, prophylactic agent, etc.) to be delivered that is sufficient to treat, ameliorate symptoms, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. In the context of treatment, "therapeutically effective amount" refers to the amount of agent (e.g., nucleic acid, composition, therapeutic agent, prophylactic agent, etc.) to be delivered that is sufficient to treat, ameliorate symptoms, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. A "therapeutically effective amount" is an amount sufficient to palliate, ameliorate, stabilize, reverse, or slow the progression of a disease or condition, such as atherosclerotic vascular disease, hypertriglyceridemia, or diabetes. A "therapeutically effective amount," as recognized by those skilled in the art, will vary depending on the particular condition being treated, the severity of the condition, the parameters of the individual subject, including age, physical condition, size, sex, and weight, the duration of treatment, the nature of concomitant therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner. These factors are well known to those skilled in the art and can be addressed without more than routine experimentation. It is generally preferred that maximum doses of individual components or compositions thereof are used, i.e., the maximum safe dose according to sound medical judgment. However, those skilled in the art will understand that a subject may adhere to a lower or tolerated dose for medical reasons, psychological reasons, or virtually any other reason. In addition, other medications that a patient may be receiving 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 of any of the disclosed compositions used alone or in combination with one or more agents used to treat a condition. A therapeutically effective amount can be administered in one or more administrations.

[0064]

[0119] An effective initial method for determining a "therapeutically effective amount" is to perform a cell culture assay. and (e.g., using neuronal cells) or by using animal models (e.g., mice, rats, rabbits, dogs, or pigs). The dose may be formulated in the 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) 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 disclosed compositions to be therapeutically effective, the animal model can also provide other relevant information, such as preferred routes of administration to produce maximum efficacy. It is well within the capabilities of one of ordinary skill in the art to adjust the dose to achieve maximum efficacy in humans based on the above and other methods.

[0065]

[0120] It is understood that ranges provided herein are shorthand for all values ​​within the range. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting 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, as well as all intervening decimal values ​​between the above integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, nested subranges of 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.

[0066]

[0121] The terms "protospacer" or "target sequence," and their grammatical equivalents, , as used herein, may refer to the DNA sequence of a target gene. In nature, the protospacer is adjacent to a PAM (protospacer adjacent motif). The term "spacer" may be an RNA version of the protospacer that binds to the complementary strand of the protospacer. The spacer may be in a guide RNA (gRNA). The site of RNA-guided nuclease cleavage is in the protospacer sequence. See FIG. 1A for illustration.

[0067]

[0122] The terms "base editing," "gene editing," or "genetic modification," and their grammatical meanings Equivalents, as used herein, can refer to genetic manipulation in which one or more nucleotides are inserted, replaced, or removed from a genome. The genetic modification may be performed using a nuclease (e.g., a naturally occurring or artificially engineered nuclease). The genetic modification may include the introduction of a double-strand break, a nonsense mutation, a frameshift mutation, a splice site change, or an inversion in a polynucleotide sequence, e.g., a target polynucleotide sequence.

[0068]

[0123] The term "base editor (BE)" or "nucleobase editor (NBE)" refers to As used herein, it may refer to an agent that binds to a polynucleotide and has an activity of modifying a nucleobase. In various embodiments, the base editor comprises a nucleic acid programmable nucleotide binding domain linked to a polypeptide (e.g., a deaminase) that modifies a nucleobase and a guide nucleotide (e.g., a guide RNA), or a nucleic acid that encodes a programmable nucleotide binding domain and a deaminase. In various embodiments, the agent is a biomolecular complex that comprises a protein domain with base editing activity, i.e., a domain that can modify a base (e.g., A, T, C, G, or U) in a nucleic acid molecule (e.g., DNA), or a nucleic acid that encodes the same. In some embodiments, the polynucleotide programmable DNA binding domain is fused or linked to a deaminase domain, resulting in a base editor fusion protein. In some embodiments, the base editor comprises a nucleic acid that encodes a base editor fusion protein, e.g., an mRNA that encodes a base editor fusion protein. The base editor fusion protein may include one or more linkers, e.g., a peptide linker. In one embodiment, the agent is a fusion protein that comprises a domain with base editing activity. In another embodiment, the protein domain with base editing activity is linked to a guide RNA (e.g., via an RNA binding motif on the guide RNA and an RNA binding domain fused to a deaminase). In some embodiments, the domain with base editing activity is capable of deaminating a base in a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases in a DNA molecule. In some embodiments, the base editor is capable of deaminating an adenosine (A) in DNA. In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is capable of deaminating a cytosine (C) in DNA. In some embodiments, the base editor is a cytosine base editor (CBE).

[0069]

[0124] The term "base editor system" refers to a system that edits nucleic acid bases of a target nucleotide sequence. In some embodiments, the base editor system comprises a base editor fusion protein comprising (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9); (2) a deaminase domain for deaminating said nucleobase (e.g., adenosine deaminase or cytosine deaminase); and (3) one or more guide polynucleotides (e.g., guide RNA). 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 cytosine base editor (CBE).

[0070] Nucleic acid base editor system

[0125] In some aspects, a base editor system capable of modifying a nucleobase is described herein. 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 and a deaminase, or a nucleic acid encoding the same. In some embodiments, the base editor system comprises a guide polynucleotide. In some embodiments, the base editor system comprises a nucleic acid encoding a guide polynucleotide. In some embodiments, the base editor system comprises a programmable D 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.

[0071]

[0126] In some embodiments, the guide polynucleotide, when administered to a subject, The base editor system is directed to effect nucleobase alterations in the PCSK9 or ANGPTL3 gene in vivo.

[0072]

[0127] In some embodiments, the base changes are identified using next generation sequencing or Sanger sequencing. Occurs in at least 35% of total liver cells in a subject, as determined by sequencing.

[0128] In some embodiments, the base changes are identified using next generation sequencing or Sanger sequencing. 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 the subject's total hepatocytes as measured by sequencing. In some embodiments, the base alteration occurs 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 of the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration 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 25% of all hepatocytes of the subject as measured by next generation sequencing or Sanger sequencing. Occurs in 99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9%. In some embodiments, the base modification 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 total liver cells of the subject as measured by next generation sequencing or Sanger sequencing.In some embodiments, the base alteration occurs in 1%-90%, 5%-85%, 10%-80%, 15%-75%, 20%-70%, 25%-65%, 30%-60%, 35%-55%, or 40%-50% of the total hepatocytes of the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 100% of the total hepatocytes of the subject as measured by next generation sequencing or Sanger sequencing.

[0073]

[0129] In some embodiments, the base alteration occurs in a liver cell of the subject. In some embodiments, the base alteration occurs in at least 30% of the subject's hepatocytes as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in the subject's hepatocytes. In some embodiments, the base alteration occurs in at least % of the subject's hepatocytes as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 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%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 7 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of the hepatocytes of the subject. In some embodiments, the base change occurs 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 the hepatocytes of the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration 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 25% of the subject's hepatocytes as measured by next generation sequencing or Sanger sequencing. Occurs in 99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9%. In some embodiments, the base modification 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 the subject's hepatocytes as measured by next generation sequencing or Sanger sequencing.In some embodiments, the base alteration occurs in 1%-90%, 5%-85%, 10%-80%, 15%-75%, 20%-70%, 25%-65%, 30%-60%, 35%-55%, or 40%-50% of the subject's hepatocytes as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 100% of the subject's hepatocytes as measured by next generation sequencing or Sanger sequencing.

[0074]

[0130] In some embodiments, the base alterations occurring in whole liver cells of the subject are identified by next generation sequencing. In some embodiments, the base changes occurring in the whole hepatocytes of the subject are measured by Sanger sequencing. In some embodiments, the base changes occurring in the hepatocytes of the subject are measured by next generation sequencing. In some embodiments, the base changes occurring in the hepatocytes of the subject are measured by Sanger sequencing.

[0075]

[0131] In some embodiments, the nucleobase alterations can be detected by ELISA, Western blot, or or LC-MS / MS. In some embodiments, the nucleobase alteration results in at least a 35% reduction in blood PCSK9 protein levels in the subject compared to pre-administration, as measured by ELISA, Western blot, or LC-MS / MS. In some embodiments, the nucleobase alteration results in at least a 35% reduction in blood ANGPTL3 protein levels in the subject compared to pre-administration, as measured by ELISA, Western blot, or LC-MS / MS.

[0076]

[0132] In some embodiments, the nucleobase alterations can be detected by ELISA, Western blot, or 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%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% reduction in blood PCSK9 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. %, 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 blood PCSK9 protein levels in the subject compared to pre-administration as measured by ELISA, Western blot, or LC-MS / MS. resulting in a reduction of 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%. In some embodiments, the nucleobase alteration increases or decreases 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%, 25% to 99.9%, 30% to 99.9%, 31% to 31%, or 32% of blood PCSK9 protein levels in the subject compared to before administration as measured by ELISA, Western blot, or LC-MS / MS. % 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% reduction.In some embodiments, the nucleobase alteration reduces the expression of PCSK9 in the blood of the subject by 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 79%, 1% to 78%, 1% to 77%, 1% to 76%, 1% to 75%, 1% to 74%, 1% to 73%, 1% to 72%, 1% to 74% or 1% to 75% 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 71%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-39%, 1%-38%, 1%-37%, 1%-36%, 1%-35%, 1%-34%, 1%-33%, 1%-32%, 1%-31%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2%. 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%, 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% reduction in blood PCSK9 protein levels in the subject compared to pre-administration as measured by ELISA, Western blot, or LC-MS / MS. In some embodiments, the nucleobase alteration results in a 100% reduction in blood PCSK9 protein levels in the subject compared to pre-administration as measured by ELISA, Western blot, or LC-MS / MS.

[0077]

[0133] In some embodiments, the nucleobase alterations can be detected by ELISA, Western blot, or or LC-MS / MS, compared to pre-dose, 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%, 500%, 600%, 700%, 800%, 900%, 1000% lower blood PCSK9 protein levels in the subject. In some embodiments, the nucleobase alteration results in 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 less, or less than 10 fold lower blood PCSK9 protein levels in the subject compared to pre-administration, as measured by ELISA, Western blot, or LC-MS / MS.

[0078]

[0134] In some embodiments, the blood PCSK9 protein in the subject compared to before administration. The reduction in blood PCSK9 protein level or blood PCSK9 level is measured by ELISA (enzyme-linked immunosorbent assay). In some embodiments, the reduction in blood PCSK9 protein level or blood PCSK9 level in a subject compared to before administration is measured by Western blot analysis. In some embodiments, the reduction in blood PCSK9 protein level or blood PCSK9 level in a subject compared to before administration is measured by LC-MS / MS (liquid chromatography-tandem mass spectrometry).

[0079]

[0135] In some embodiments, the nucleobase alterations can be detected by ELISA, Western blot, or 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%, 36%, or 37% reduction 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 reduction of 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%. In some embodiments, the nucleobase alteration increases blood ANGPTL3 protein levels in the subject by at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36% compared to pre-administration as measured by ELISA, Western blot, or LC-MS / MS. , resulting in a reduction of 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%.In some embodiments, the nucleobase alteration increases or decreases 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%, 25% to 99.9%, 30% to 99.9%, 35% to 99.9%, 40% to 99.9%, 45% to 99.9%, 50% to 99.9%, 60% to 99.9%, 70% to 99.9%, 80% to 99.9%, 90% to 99.9%, 100% to 99.9%, 110% to 99.9%, 120% to 99.9%, 130% to 99.9%, 140% to 99.9%, 150% to 99.9%, 160% to 99.9%, 170% to 99.9%, 180% to 99.9%, 190% to 100%%, 200% to 99.9%, 210% to 200%%, 220% to 230%%, 230% to 240%%, 240% to 250%%, 250% to 260%%, 260% to 270%%, 270% to 280%%, 280% to 290%%, 300% to 300%%, 310% to 320%%, 330% to 340% resulting in a reduction of 1%-99.9%, 32%-99.9%, 33%-99.9%, 34%-99.9%, 35%-99.9%, 36%-99.9%, 37%-99.9%, 38%-99.9%, 39%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9%. In some embodiments, the nucleobase alteration reduces blood ANGPTL3 protein levels in the subject by 1% to 99.5%, 1% to 9% compared to pre-administration, as measured by ELISA, Western blot, or LC-MS / MS. 9%, 1%~98%, 1%~97%, 1%~96%, 1%~95%, 1%~90%, 1%~85%, 1%~80%, 1%~79%, 1%~78%, 1%~77%, 1%~76%, 1%~75%, 1%~74%, 1%~73%, 1%~72%, 1%~71%, 1%~70%, 1%~65%, 1%~60%, 1%~55%, 1%~50%, 1%~45%, 1%~ resulting in a reduction of 40%, 1%-39%, 1%-38%, 1%-37%, 1%-36%, 1%-35%, 1%-34%, 1%-33%, 1%-32%, 1%-31%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2%. In some embodiments, the nucleobase alterations result in a 1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%, 31%-80%, 32%-79%, 33%-78%, 34%-77%, 35%-76%, 36%-76%, 37%-75%, 38%-74%, 39%-73%, 40%-72%, 45%-71%, 50%-70%, or 55%-65% reduction in blood ANGPTL3 protein levels in the subject compared to pre-administration as measured by ELISA, Western blot, or LC-MS / MS. In some embodiments, the nucleobase alteration results in a 100% reduction in blood ANGPTL3 protein levels in the subject compared to pre-administration, as measured by ELISA, Western blot, or LC-MS / MS.

[0080]

[0136] In some embodiments, the nucleobase alterations can be detected by ELISA, Western blot, or or LC-MS / MS, compared to pre-dose, 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%, 400%, 410%, %, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% lower blood ANGPTL3 protein levels in the subject. In some embodiments, the nucleobase alteration results in blood ANGPTL3 protein levels in the subject that are 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 less, or less than 10 fold lower, compared to before administration, as measured by ELISA, Western blot, or LC-MS / MS.

[0081]

[0137] In some embodiments, the blood ANGPTL3 protein level in the subject compared to before administration is The reduction in blood ANGPTL3 protein level or blood ANGPTL3 protein level is measured by ELISA (enzyme-linked immunosorbent assay). In some embodiments, the reduction in blood ANGPTL3 protein level or blood ANGPTL3 protein level in the subject compared to before administration is measured by Western blot analysis. In some embodiments, the reduction in blood ANGPTL3 protein level or blood ANGPTL3 protein level in the subject compared to before administration is measured by LC-MS / MS (liquid chromatography-tandem mass spectrometry).

[0082]

[0138] In some embodiments, the alteration of the nucleobase is in the blood or In some embodiments, the nucleobase alteration results in a reduction of blood low density lipoprotein cholesterol (LDL-C) levels in the subject by at least 35%, 40%, 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%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ...9%, 102%, 103%, 104%, 1 resulting in a reduction of 4%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. 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%, 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% reduction in blood low density lipoprotein cholesterol (LDL-C) levels in the subject compared to before administration. In some embodiments, the nucleobase alteration results in a reduction in blood low density lipoprotein cholesterol (LDL-C) levels in the subject by 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% compared to before administration. In some embodiments, the nucleobase alteration reduces or decreases 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 29% compared to before administration. resulting in a reduction of ~99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95%-99.9%.In some embodiments, the nucleobase alteration results in a 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 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%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% reduction in blood low density lipoprotein cholesterol (LDL-C) 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% reduction in blood low density lipoprotein cholesterol (LDL-C) levels in the subject compared to before administration. In some embodiments, the nucleobase alteration results 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 alteration 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%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 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%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 92 , resulting in 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% lower blood low density lipoprotein cholesterol (LDL-C) levels in the subject.In some embodiments, the nucleobase alteration results in blood low density lipoprotein cholesterol (LDL-C) levels in the subject that are 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 less, or less than 10 fold lower than before administration.

[0083]

[0139] In some embodiments, the alteration of the nucleobase reduces blood circulating fluid in the subject compared to before administration. In 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% reduction in blood triglyceride levels in the subject compared to before administration. 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%, 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% reduction in blood triglyceride levels in the subject compared to before administration. In some embodiments, the nucleobase alteration results in a reduction in blood triglyceride levels in the subject of 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% compared to before administration.In some embodiments, the alteration of the nucleobase reduces or decreases blood triglyceride 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%, 25% to 99.9%, 30% to 99.9%, 35% to 99.9%, 36% to 99.9%, 37% to 99.9%, 38% to 99.9%, 39% to 40%%, 40% to 40%%, 41% to 42%, 42% to 43%, 43% to 44%, 44% to 45%, 45% to 46%, 46% to 47%, 47% to 48%, 48% to 49%, 49% to 50%, 50% to 51%, 51% to 52%, 52% to 53%, 53% to 54%, 54% to 55%, 55% to 56%, 56% to 57%, 57% to 58%, 58% to 59%, 59% to 60%, 60% to 61%, 61% to 61%, 62% to 62%, 63% to 63%, 64% to 64%, 65% to 65%, 66% to 67%, 67% to 68%, 68% to 69%, 70% to 71%, 72% to 73%, 74% to resulting in a reduction of 5%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95%-99.9%. In some embodiments, the nucleobase alteration results in a 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 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%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% reduction 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% reduction in blood triglyceride levels in the subject compared to before administration. In some embodiments, the nucleobase alteration results in a 100% reduction in blood triglyceride levels in the subject compared to before administration.In some embodiments, the nucleobase alteration 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%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 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%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 93 %, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% lower blood triglyceride levels in the subject. In some embodiments, the nucleobase alteration is 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 less, or less than 10 fold lower, blood triglycylces in the subject compared to before administration. Brings ceride levels.

[0084]

[0140] In some embodiments, blood triglyceride levels in the subject compared to before administration. or reduction in blood triglyceride levels is measured by any standard technique. In some embodiments, blood low-density lipoprotein cholesterol (LDL-C) levels or reduction in blood low-density lipoprotein cholesterol (LDL-C) levels in a subject compared to before administration is measured by any standard technique. For example, a clinical analyzer device with enzymatic direct measurement of cholesterol (total C), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) can be used to measure a "lipid panel" in a serum sample. Reagent kits specific to each analyte include buffers, calibrators, blanks, and controls. As used in this disclosure, cholesterol, triglycerides, and HDL-C can be quantified using absorbance measurements of specific enzyme reaction products. LDL-C can be determined indirectly. In some examples, the majority of cholesterol in the circulating system can be found in three major lipoprotein fractions: very low-density lipoprotein (VLDL), LDL, and HDL. In some embodiments, total circulating cholesterol may be estimated by the formula [total C] = [VLDL-C] + [LDL-C] + [HDL-C]. That is, LDL-C may be calculated from measurements of total cholesterol, triglycerides, and HDL-C according to the relationship: [LDL-C] = [total C] - [HDL-C] - [TG] / 5, where [TG] / 5 is an estimate of VLDL-cholesterol. A triglyceride specific reagent kit may be used that includes buffers, calibrators, blanks, and controls. As used herein, serum samples obtained in a study may be analyzed and triglycerides may be measured using a series of coupled enzymatic reactions. In some embodiments, H is used to quantify the analyte as the end product of the last enzymatic reaction and its absorbance at 500 nm. 2 O 2 may be used, with the color intensity being proportional to the triglyceride concentration.

[0085]

[0141] In some embodiments, the guide polynucleotide is a guide RNA and the guide R The NA comprises a spacer sequence that binds to the complementary strand of the protospacer sequence of the PCSK9 gene with 0, 1, or 2 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of the protospacer sequence of the PCSK9 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 PCSK9 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 PCSK9 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 PCSK9 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 PCSK9 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 PCSK9 gene with five mismatches.

[0086]

[0142] In some embodiments, the guide polynucleotide is a guide RNA and the guide R The NA comprises a spacer sequence that binds to the complementary strand of the protospacer sequence of the ANGPTL3 gene with zero, one, or 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 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 three mismatches. The guide RNA comprises a spacer sequence that binds to the complementary strand of the pacer sequence 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.

[0087]

[0143] In some embodiments, nucleobase alterations are measured by net nucleobase editing. , outside the protospacer sequence in less than 1% of all hepatocytes in the subject. In some embodiments, the nucleobase alterations are outside the protospacer sequence in less than 1% of hepatocytes in the subject, as measured by net nucleobase editing. In some embodiments, the nucleobase alterations are only within the protospacer sequence, as measured by net nucleobase editing.

[0088]

[0144] In some embodiments, nucleobase alterations are measured by net nucleobase editing. , 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 all hepatocytes in a subject are outside the protospacer sequence. In some embodiments, nucleobase alterations are outside the protospacer sequences 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 hepatocytes in the subject as measured by net nucleobase editing. In some embodiments, nucleobase alterations 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 cells in a subject as measured by net nucleobase editing.

[0089]

[0145] In some embodiments, the deaminase is an adenine deaminase. In some embodiments, the nucleobase alteration is an A·T to G·C alteration. In some embodiments, the deaminase is an adenine deaminase and the nucleobase alteration is an A·T to G·C alteration. In some embodiments, the programmable DNA binding domain comprises a nuclease-inactive Cas9 or Cas9 nickase. In some embodiments, the programmable DNA binding domain comprises Cas9.

[0090]

[0146] In some embodiments, the nucleobase alteration is at a splice site of the PCSK9 gene. In some embodiments, the nucleobase modification 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, referenced in SEQ ID NO:5. In some embodiments, the nucleobase modification is at a splice acceptor site of the PCSK9 gene. In some embodiments, the nucleobase modification results in a frameshift, a premature stop codon, an insertion or deletion in the transcript encoded by the PCSK9 gene. In some embodiments, the nucleobase modification 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 described in Table 1 or Table 24.

[0091]

[0147] In some embodiments, the nucleobase alteration is at a splice site of the ANGPTL3 gene. In some embodiments, the nucleobase modification 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, referenced in SEQ ID NO: 7. In some embodiments, the nucleobase modification is at a splice acceptor site of the ANGPTL3 gene. In some embodiments, the nucleobase modification results in a frameshift, a premature stop codon, an insertion or deletion in the transcript encoded by the ANGPTL3 gene. In some embodiments, the nucleobase modification 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 described in Table 1 or Table 24.

[0092]

[0148] In some embodiments, the guide RNA is a guide RNA as described in Table 1 or Table 24. In some embodiments, the guide RNA comprises the sequence 5'-5'-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcG UUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu-3' (SEQ ID NO: 9), 5'-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGU UAucAAcuugaaaaagugGcaccgagucggugcusususu-3' (SEQ ID NO: 9), 5'-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu-3' (SEQ ID NO: 9) (GA346), 5'-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAacAAcuugaaaaagugGcaccgagucggugcusususu-3' (SEQ ID NO: 10) (GA374), 5'-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauag caaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususuuuu-3' (SEQ ID NO: 11) (GA385), 5'-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususuuUu-3' (SEQ ID NO: 11) (GA386) or 5'-cscscsGCACCUUGGCGCAGCGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcuusususuuuu-3' (SEQ ID NO: 12) (GA387).

[0093]

[0149] In some embodiments, the protospacer sequence is set forth in Table 1 or Table 24. Protospacer sequences include, in some embodiments, the protospacer includes the sequences 5'-CCCGCACCTTGGCGCAGCGG-3' (SEQ ID NO: 13), AAGATACCTGAATAACTCTC-3' (SEQ ID NO: 14), and 5'-AAGATACCTGAATAACCCTC-3' (SEQ ID NO: 15).

[0094]

[0150] In some embodiments, the base editor fusion protein comprises the sequence of SEQ ID NO:3. In some embodiments, the adenosine deaminase has an amino acid sequence identical to that of any of the adenosine deaminases provided herein, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least The adenosine deaminase domains include those that are 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 adenosine deaminase domains described herein. It is recognized that the adenosine deaminase domains provided herein may include one or more mutations (e.g., any of the mutations provided herein). The present disclosure provides any deaminase domain having any percent identity and 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:3 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, at least 170 identical contiguous amino acid residues compared to any one of the amino acid sequences set forth in SEQ ID NO:3 or any of the adenine deaminases provided herein.

[0095]

[0151] In some embodiments, the nucleic acid encoding the base editor fusion protein is an mRNA. A. The mRNA may include modifications, such as modifications to the 3' or 5' end of the mRNA. In some embodiments, the mRNA includes a cap analog.

[0096]

[0152] In some embodiments, the mRNA comprises a 2'-hydroxyl group, a 2'-O-methyl group, , or further 2' chemical modifications, or combinations thereof. In some embodiments, the mRNA comprises at least one, two, or three nucleotides at the 5' end that comprise a 2'-hydroxyl group, a 2'-O-methyl group, or further 2' chemical modifications, or combinations thereof. In some embodiments, the mRNA comprises at least one nucleotide at the 5' end that comprises a 2'-hydroxyl group, a 2'-O-methyl group, or further 2' chemical modifications, or combinations thereof. In some embodiments, the mRNA comprises at least two nucleotides at the 5' end that comprise a 2'-hydroxyl group, a 2'-O-methyl group, or further 2' chemical modifications, or combinations thereof. In some embodiments, the mRNA comprises at least three nucleotides at the 5' end that comprise a 2'-hydroxyl group, a 2'-O-methyl group, or further 2' chemical modifications, or combinations thereof. In some embodiments, the mRNA comprises at least four nucleotides at the 5' end that contain a 2'-hydroxyl group, a 2'-O-methyl group, or a further 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least five nucleotides at the 5' end that contain a 2'-hydroxyl group, a 2'-O-methyl group, or a further 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least six nucleotides at the 5' end that contain a 2'-hydroxyl group, a 2'-O-methyl group, or a further 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least seven nucleotides at the 5' end that contain a 2'-hydroxyl group, a 2'-O-methyl group, or a further 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least eight nucleotides at the 5' end that contain a 2'-hydroxyl group, a 2'-O-methyl group, or a further 2' chemical modification, or a combination thereof. In some embodiments, the mRNA comprises at least 9 nucleotides at the 5' end that contain 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 contain a 2'-hydroxyl group, a 2'-O-methyl group, or an additional 2' chemical modification, or a combination thereof. The 5' end of the nucleic acid sequence may comprise at least 10 nucleotides, or an additional 2' chemical modification, or a combination thereof.

[0097]

[0153] In some embodiments, the mRNA comprises a polyA tail. It may be at the 3' end of the

[0154] In some embodiments, the GC% content of the mRNA sequence is 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% or higher. 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%.

[0098]

[0155] In some embodiments, the mRNA sequence is adenine tTNA deaminase (TadA In some embodiments, the GC% of the TadA region is 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% or more. 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%.

[0099]

[0156] In some embodiments, the mRNA sequence comprises a Cas9 region. , the GC% of the Cas9 region is 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% or more. 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%.

[0100]

[0157] In some embodiments, the mRNA sequence comprises an NLS region. The GC% of the NLS regions is 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% or greater. 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%.

[0101]

[0158] In some embodiments, the mRNA sequence links the TadA region and the Cas9 region. In some embodiments, the GC% of the first linker region is 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%, 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%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 1 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% or more. In some embodiments, the GC % content of the first linker region is 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% or more.

[0102]

[0159] In some embodiments, the mRNA sequence comprises a second sequence linking the Cas9 region and the NLS region. In some embodiments, the GC% of the second linker region is 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% or more. 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%.

[0103]

[0160] In some embodiments, the base editor systems provided herein comprise a guide The base editor system further comprises a lipid nanoparticle (LNP) encapsulating a polynucleotide or a nucleic acid encoding a guide polynucleotide (i). In some embodiments, the LNP further encapsulates a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding the same (ii). In some embodiments, the base editor system further comprises a second LNP encapsulating a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding the same (ii).

[0104]

[0161] The base editor systems provided herein include one or more LNPs. For example, the base editor system may include an LNP that encapsulates both a guide polynucleotide and a nucleic acid encoding a base editor fusion protein, such as an mRNA encoding a base editor fusion protein. In another example, the base editor system may include an LNP that encapsulates a guide polynucleotide, such as a guide RNA, and an LNP that encapsulates a nucleic acid encoding a base editor fusion protein, such as an mRNA. LNPs that encapsulate a guide polynucleotide and a base editor fusion protein or an mRNA encoding a base editor fusion protein separately allow for flexible dosing and administration of the base editor system. For example, an LNP that encapsulates a guide RNA can be administered first, followed by an LNP that encapsulates an mRNA encoding a base editor fusion protein. In some embodiments, an LNP that encapsulates a guide RNA and a second LNP that encapsulates an mRNA encoding a base editor fusion protein are administered to a subject simultaneously. In some embodiments, an LNP that encapsulates a guide RNA and an LNP that encapsulates an mRNA encoding a base editor fusion protein are administered to a subject sequentially. In some embodiments, a subject is administered an LNP encapsulating an mRNA encoding a base editor fusion protein, followed by multiple administrations or doses of a second LNP encapsulating 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, 12 weeks or more later. 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, 20 days or more apart.

[0105]

[0162] In some embodiments, the guide polynucleotide and the base editor fusion protein In some embodiments, the ratio of guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 1:10 to about 10:1 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 and 1:4 by weight. The ratio of polynucleotide to nucleic acid encoding a base editor fusion protein can be determined by titration of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein.

[0106]

[0163] In some embodiments, the guide polynucleotide and the base editor fusion protein The molar ratio of the nucleic acid encoding the target protein to the target nucleic acid is about 500:1 to about 1:500.

[0164] In some embodiments, the guide polynucleotide and the base editor fusion protein In some embodiments, the ratio of guide polynucleotides to nucleic acids encoding base editor fusion proteins is about 1000:1 to about 1:1000 by weight. In some embodiments, the ratio of guide polynucleotides to nucleic acids encoding base editor fusion proteins 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 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, 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 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, 1:500, 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.

[0107]

[0165] In some embodiments, the guide polynucleotide and the base editor fusion protein In some embodiments, the molar ratio of the guide polynucleotide to the nucleic acid encoding the base editor fusion protein is about 10,000:1 to about 1:10,000. In some embodiments, the molar ratio of the guide polynucleotide to the nucleic acid encoding the 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, 1:10 ... :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 :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 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, 9 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, 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, 1:500, 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, 1:4. 0, 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, 95 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.

[0108]

[0166] In some embodiments, a nucleic acid encoding a guide polynucleotide and a base edit In some embodiments, the ratio of nucleic acid encoding a guide polynucleotide to a nucleic acid encoding a base editor fusion protein is about 10:1 to about 1:10 by weight. In some embodiments, the ratio of nucleic acid encoding a guide polynucleotide to a nucleic acid encoding a 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, 7 The molar ratio of nucleic acid encoding a guide polynucleotide to nucleic acid encoding a base editor fusion protein is about 500:1 to about 1:500.

[0109]

[0167] In some embodiments, a nucleic acid encoding a guide polynucleotide and a base edit In some embodiments, the ratio of nucleic acid encoding a guide polynucleotide to a 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 a 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, 45:1, 55:1, 50:1, 45:1, 55:1, 50:1, 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, 1:2, 1:3, 1:4, 1:5, 1:19, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 ...15, 1:15, 1:16, 1:17, 1:18, 1:19, 1:19, 1:10, 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 nucleic acid encoding a 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, 55:1, 50:1, 45:1, 55:1, 50:1, 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 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.

[0110]

[0168] In some embodiments, a nucleic acid encoding a guide polynucleotide and a base edit In some embodiments, the molar ratio of the nucleic acid encoding the guide polynucleotide to the nucleic acid encoding the base editor fusion protein is about 10,000:1 to about 1:10,000. In some embodiments, the molar ratio of the nucleic acid encoding the guide polynucleotide to the nucleic acid encoding the 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, 1:8 000, 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 acid encoding a 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, 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 acid encoding a guide polynucleotide to a 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, 1:500, 1:600, 1:750, 1:800, 1:500, 1:450, 1:500, 1:500, 1:600, 1:500, 1:750, 1:800, 1:850, 1:600, 1:500, 1:500, 1:450, 1:500, 1:500, 1:500, 1:600, 1:750, 1:800, 1:850, 1:600, 1:500, 1:500, 1:500, 1:450, 1:500, 1:500, 1:500, 1:600, 1:750, 1:800, 1:85 :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, 1:4. 0, 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 acid encoding a 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, 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.

[0111]

[0169] Precision genome editing is a growing field with industrial, agricultural, and biomedical applications. One of the major genome editing systems available today is the integrated regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated 9 (Cas9). Cas9 can be used to create double-stranded breaks (DSBs) in target sequences by using guide RNAs (gRNAs) with sequences homologous to those of DNA (known as protospacers) in the target genome adjacent to specific protospacer adjacent motifs (PAMs) that contain the sequence NGG (where N is any standard base) in the NA. Non-homologous end joining (NHEJ) at DSBs can be used to create indels and knock-out genes at gene loci. Similarly, homology-directed repair (HDR) can be used with introduced template DNA to insert genes or modify target sequences. A variety of Cas9-based tools have been developed in recent years, including tools that methylate DNA, recognize larger sequence spaces, or create single-stranded 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 DSBs (Komor AC, Kim YB, Packer MS, et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature, 2016, 533:420-4, incorporated herein by reference in its entirety). After the cytidine deaminase domain of rat APOBEC1 was fused to the N-terminus of catalytically inactive 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, alternatively, 13-17 nucleotides upstream of the PAM). Remarkably, any cytosine base within this "window" was amenable to editing, with various outcomes depending on how many and which cytosines were edited. After DNA replication or repair, each uracil was replaced with a thymine, completing the C to T base editing.3 The next version of the base editor (BE2) incorporated a uracil glycosylase inhibitor fused to the C-terminus of dCas9 that helps inhibit base excision repair of uracil bases due to 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 Cas9 nickase rather than dCas9. The nickase cleaved the unedited strand opposite the edited C to T base and stimulated removal of the opposing guanidine through eukaryotic mismatch repair. BE2 and BE3 base editing were observed in both human and mouse cell lines. The specificity of the base editing has been further improved by the addition of mutations to the Cas9 nickase. Similarly, Cas9 has been mutated to narrow the width of its editing window from approximately 5 nucleotides to only 1-2 nucleotides (Rees HA, Komor AC, Yeh WH, et al., "Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery," Nat 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).

[0112]

[0170] An alternative cytosine base editing platform is activity-induced cytosine deaminase By linking main PmCDA1 to dCas9 (target-AID), they were able to demonstrate targeted C to T base editing in yeast. Furthermore, an alternative C to T editing strategy was also demonstrated without fusing a deaminase domain to Cas9. Instead, an SH3 (Src 3 homology) domain was added to the C-terminus of dCas9. , whereas SHL (SH3 interacting ligand) was added to PmCDA1. 6 Optimal efficiency was achieved by using Cas9 nickase rather than dCas9 Additionally, a uracil DNA glycosylase inhibitor was added to enhance base editing in mammalian CHO cell lines. The resulting platform was able to consistently edit bases within 3-5 bases of the 18th nucleotide upstream of the PAM sequence (Nishida K, Arazoe T, Yachie N, et al., "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive transcription," in Proc. Natl. Sci., 2014, 143:1111-1122, which is incorporated herein by reference in its entirety). “immune systems” Science, 2016, p. 353: aaf8729).

[0113]

[0171] A notable cytosine base editing platform is RNA-guided end-targeting. Cpf1 (also known as Cas12a) was used instead of Cas9 as a nuclease. Catalytically inactive Cpf1 was fused to APOBEC1 (dLbCpf1-BE0) to generate C to T conversion in human cell lines. Cas9 base editor variants BE3 and target-AID recognize the PAM sequence NGG, while dLbCpf1-BE0 recognizes the T-rich PAM sequence TTTV. Base editing was observed between positions 8 and 13 of the protospacer sequence in dLbCpf1-BE0, but the window could be reduced from positions 10 to 12 by introducing additional mutations into Cpf1. However, narrowing the base editing window correlated with reduced editing efficiency (Li X, Wang Y, Liu Y, et al., "Base editing at positions 8 and 13 of the protospacer sequence," incorporated herein by reference in its entirety). with a Cpf1-cytidine deaminase fusion” Nat Biotechnol, 2018, 36: 324-7).

[0114]

[0172] Cytosine base editing is not completely predictable and has been observed in C-to-T editing editors. Indels can occur at the target site, albeit at a lower frequency than expected. Furthermore, cytosine base editors can sometimes induce C to A or C to G edits rather than the expected C to T edit. The cytosine base editor, designated "BE4," was improved by adding a linker length between the Cas9 nickase and the rat APOBEC1 cytosine deaminase domain from 16 to 32 amino acids, adding a linker between the Cas9 nickase and a uracil glycosylase inhibitor from 4 to 9 amino acids, and using a second uracil glycosylase inhibitor attached to the C-terminus of the new cytosine base editor using another 9 amino acid linker (Komor AC, Zhao KT, Packer MS, et al., "Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity." Sci Adv, 2017, 3:eaao4774. Incorporated by reference herein in its entirety).

[0115]

[0173] By fusing the Escherichia coli adenine tTNA deaminase TadA (ecTadA) to dCas9 and mutagenesis of the ecTadA domain combined with selection for editing activity, it was found that the A106V and D108N mutations result in a base editor, designated ABE7.10, capable of editing adenines to guanines in DNA (Gaudelli NM, Komor AC, Rees HA, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage," Nature, 2017, 551:464-71, incorporated herein by reference in its entirety). Koblan et al. improved the efficiency of ABE7.10 by modification of the nuclear localization signal and codon optimization, resulting in a version they called ABEmax. A similar approach improved the efficiency of the cytosine base editor BE4. 10 Huang et al. used an alternative PAM to extend the editing window to include adenine and cytosine. Further development of both cytidine and adenine base editors was carried out, thereby increasing their target coverage ("Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction," Nat Biotechnol, 2018, 36:843-6, incorporated herein by reference in its entirety).

[0116]

[0174] The same proved to be true for base editors 12-14, , Comparison of Cas9, cytosine base editors, and adenine base editors using the same gRNA shows clear off-target profiles.

[0117]

[0175] Various studies have demonstrated that the deaminase domain acts during isolation There has been growing interest in gRNA-free off-target base editing (which does not require DNA engagement by the Cas9-gRNA complex) that is used in gene editing. Further studies have shown that the gRNA-free off-target effects of base editors are not limited to DNA. RNA sequencing of cells treated with cytosine or adenine base editors revealed RNA off-target editing that was widespread in the transcriptome, and the introduction of amino acid substitutions (e.g., R106W) in the deaminase domain of adenine base editors reduced RNA off-target editing 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).

[0118]

[0176] Correction of disease-causing mutations through precision editing with standard Cas9 genome editing Positive editing has mainly required HDR. Precision editing of non-mitotic cells is difficult because HDR is limited to cells in the S or G2 phase of mitosis. However, base editors do not rely on HDR. Editing of postmitotic cochlear cells in mice can be performed using the cytosine base editor BE3. By injecting BE3 in the form of a preassembled ribonucleoprotein and gRNA via cationic liposomes, serine-33 of beta-catenin was edited to phenylalanine (TCT codon edited to TTT), allowing transdifferentiation of supporting cells into hair cells. Base editing in cochlear tissue was confirmed via sequencing, showing an editing rate of 0.7% to 3.0% depending on the region of the cochlea. In contrast, However, standard Cas9 editing via HDR showed signs of negligible efficacy in cochlear cells. A variant of SaBE3 delivered to the liver via an adeno-associated virus (AAV) vector was reported to directly correct a pathogenic T-to-C mutation in the Pah gene with an editing rate as high as 29%, thereby treating the disease phenylketonuria in adult mice. Adenine base editing has also been demonstrated 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 tyrosinaemia” 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.

[0119]

[0177] Modifying a nucleobase (e.g., A, T, C, G, or U) in a target nucleotide sequence Provided herein are compositions of nucleobase editor systems that include nucleobase editor proteins, complexes, or compounds that can enhance or convert a nucleic acid sequence.

[0120]

[0178] Nucleobase editors, or base editors (BEs), are used to modify nucleic acid sequences (e.g., DNA or RNA). In some embodiments, a base editor is capable of deaminating a base in a nucleic acid. In some embodiments, a base editor is capable of deaminating a base in a DNA molecule. In some embodiments, a base editor is capable of deaminating adenine (A) in DNA.

[0121]

[0179] In some embodiments, the base editor is a promoter fused to an adenosine deaminase. In some embodiments, the base editor comprises a fusion protein comprising a programmable DNA binding protein. 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 a cytidine 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 an inhibitor of deaminase and base excision repair, For example, it comprises a Cas9 nickase fused to a UGI or dISN domain. In some embodiments, the dCas9 domain of the fusion protein comprises the following mutations, numbered in the wild-type SpCas9 amino acid sequence: D10A and H840A. In some embodiments, the UGI comprises the following amino acid sequence:

[0180] >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase Enzyme inhibitors MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SD APE YKPW ALVIQDS NGENKIKML (SEQ ID NO: 16)

[0122]

[0181] In some embodiments, the base editor systems provided herein include base editors. The base editor fusion protein includes 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, a C2c3 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., 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 fusion protein comprising a deaminase, e.g., adenosine deaminase, and a programmable DNA binding protein, e.g., a Cas9 domain, joined 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, joined via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is 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 1-200 amino acids.In some embodiments, the adenosine deaminase and the programmable DNA binding protein are 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-80, 1-100, 1-150, 1-200, 5-10, 5-20, 5-30, 5-40, 5-60, 5-80, 5-100, 5-150, 5-200, 10-20, 10-30, 10-40, 10-50, 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, 50-80, 50-100, 50-150, 50-200, 60-80, 60-100, 60-150, 60-200, 80-100, 80-150, 80-200, 100-150, 100-200, or 150-200 amino acids. 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 SGSETPGTSESATPES (SEQ ID NO: 17), SGGS (SEQ ID NO: 18), SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 19), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 20), or GGSGGSPGSPAG. The linker is fused through a linker comprising the amino acid sequence of SPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 21). In some embodiments, the adenosine deaminase and the programmable DNA binding protein are fused through a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 17), 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: 23). 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: 24). 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: 25). 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: 26).

[0123]

[0182] In some embodiments, the base editor systems provided herein include base editing systems. In some embodiments, the base editor comprises a fusion protein comprising a cytidine deaminase and a programmable DNA binding domain, such as a Cas9 domain. In some embodiments, the base editor comprises a fusion protein comprising an adenosine deaminase and a programmable DNA binding domain, such as 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 an inosine excision enzyme from excising an inosine residue from DNA (e.g., by steric hindrance). For example, catalytically inactive inosine glycosylase (e.g., alkyladenine glycosylase [AAG]) binds to inosine but does not create an abasic site or remove the inosine, thereby sterically blocking the newly formed inosine moiety from potential DNA damage and / or repair mechanisms. That is, the present disclosure contemplates a fusion protein comprising a programmable DNA binding protein and an adenosine deaminase further fused to a dISN. The present disclosure contemplates a fusion protein 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 an adenosine deaminase that can catalyze the A to I change. For example, a fusion protein comprising a dISN domain can be efficient in deaminating A residues.

[0124]

[0183] In some embodiments, the base editors provided herein comprise one or more In some embodiments, the fusion protein further comprises a nuclear targeting sequence, such as 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 import of the protein comprising the NLS into the 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 the programmable DNA binding protein, such as Cas9. In some embodiments, the fusion protein further comprises a nuclear targeting sequence, such as a nuclear localization sequence (NLS), such as a nuclear localization sequence (NLS). In some embodiments, the fusion protein further 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 import of the protein comprising the NLS into the 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, such as Cas9. In some embodiments, the NLS is fused to the C-terminus of the programmable DNA binding protein. In some embodiments, the NLS is fused to the N-terminus of the adenosine deaminase. In some embodiments, the NLS is fused to the C-terminus of the 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 comprises 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: 27) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 28). 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.

[0125]

[0184] In some embodiments, the fusion proteins provided herein do not include 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 construction above indicates the presence of an optional linker.

[0126]

[0185] Some embodiments of the present disclosure include a programmable DNA binding protein and at least Also provided are base editors or fusion proteins that comprise 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) can improve the ability (e.g., efficiency) of the fusion protein to modify a nucleobase, e.g., 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 comprises 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 comprises any one of the mutations provided herein numbered in SEQ ID NO: 1. In some embodiments, the second adenosine deaminase comprises any one of the mutations provided herein numbered in SEQ ID NO: 1. In some embodiments, the first adenosine deaminase comprises any one of the mutations provided herein numbered in SEQ ID NO: 1, 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 numbered in SEQ ID NO: 1, and the first adenosine deaminase comprises a wild-type adenosine deaminase sequence. As one example, a fusion protein can include a first adenosine deaminase and a second adenosine deaminase, both of which include the A106V, D108N, D147Y, and E155V mutations from ecTadA (SEQ ID NO:1).As another example, the fusion protein may comprise a first adenosine deaminase domain comprising the A106V, D108N, D147Y, and E155V mutations from ecTadA (SEQ ID NO: 1), and a second adenosine deaminase comprising the L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F mutations from ecTadA (SEQ ID NO: 1).

[0127]

[0186] In some embodiments, the adenosine deaminase has the amino acid sequence: Contains MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 1).

[0128]

[0187] In some embodiments, the fusion protein comprises two adenosine deaminases (e.g., 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 adenosine deaminase are fused directly or via a linker. In some embodiments, the linker is any of the linkers provided herein, such as 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 deaminases provided herein. In some embodiments, the second adenosine deaminase is any of the adenosine deaminases 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: 1 or 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:1 or any of the adenosine deaminases provided herein. In some embodiments, the second adenosine deaminase comprises the amino acid sequence of SEQ ID NO:1.

[0129]

[0188] In some embodiments, the fusion proteins provided herein do not include a linker. In some embodiments, 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).

[0130]

[0189] It will be appreciated 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, but are not limited to, biotin carboxylase carrier protein (BCCP) tag, myc tag, calmodulin tag, FLAG tag, hemagglutinin (HA) tag, polyhistidine tag, also referred to 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, soft tag (e.g., These include Softag1, Softag3), Strep tag, Biotin ligase tag, FlAsH tag, V5 tag, and SBP tag. Further suitable sequences will be apparent to those skilled in the art. In some embodiments, the fusion protein comprises one or more His tags.

[0131]

[0190] In some embodiments, the fusion protein comprises any of the amino acid sequences listed in Table 23. 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 one of the amino acid sequences listed in Table 23. In some embodiments, the fusion protein comprises any one of the amino acid sequences listed in Table 23. In some embodiments, the sequence of the fusion protein is any one of the amino acid sequences listed in Table 23. 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: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146, 2152, 2156, and 2160. In some embodiments, the fusion protein comprises any one of the amino acid sequences in SEQ ID NOs: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146, 2152, 2156, and 2160. In some embodiments, the sequence of the fusion protein is any one of the amino acid sequences in SEQ ID NOs: 2137, 2149, 2154, 2158, and 2188.

[0132]

[0191] In some embodiments, the fusion protein comprises a polynucleotide sequence listed in Table 23. 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 sequences. 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 23. In some embodiments, the fusion protein is expressed by any one of the polynucleotide sequences listed in Table 23. In some embodiments, the fusion protein is expressed by any one of the polynucleotide sequences listed in SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. The polypeptide 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 octide sequences.In some embodiments, the fusion protein comprises a polypeptide of any of the following SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In some embodiments, the fusion protein is encoded by a polynucleotide sequence comprising any one of the polynucleotide sequences set forth in SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189. In some embodiments, the fusion protein comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158 The polypeptide 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 octide sequences, 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: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158, or a combination thereof. In some embodiments, the fusion protein is expressed by any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189.In some embodiments, the polynucleotide sequence further comprises 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 in SEQ ID NOs: 2138, 2147, 2158, or a combination thereof. In some embodiments, the polynucleotide sequence further comprises any one of the polynucleotide sequences in SEQ ID NOs: 2138, 2147, 2158, or a combination thereof.

[0133]

[0192] In some embodiments, the nucleobase editor ABE8.8 comprises the sequence provided below. : MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV (SEQ ID NO:3).

[0134]

[0193] In some embodiments, the nucleobase editor comprises a polynucleotide sequence as provided below. (Also referred to as MA002 in this specification) Sequence: ATGAGCGAGGTCGAGTTCTCTCACGAATATTGGATGAGACACGCTCTCACCCTGGCTAAGAGAGCCAGGGACGAAAGAGAGGTGCCAGTTGGCGCTGTCCTGGTGTTGAACAATCGCGTCATCGGAGAAGGATGGAATCGCGCCATTGGCCTGCACGATCCAACCGCACATGCCGAAATTATGGCTCTGCGGCAAGGCGGCCTCGTGATGCAAAATTACAGACTGATCGATGCTACCCTCTACGTCACCTTCGAGCCCTGTGTCATGTGTGCTGGGGCAATGATTCACTCCCGGATTGGCCGCGTGGTGTTTGGAGTGCGGAATGCCAAGACTGGCGCCGCTGGATCTCTGATGGACGTCCTGCACcatCCTGGGATGAACCACCGGGTCGAGATCACAGAGGGAATTCTGGCTGACGAGTGCGCTGCCCTGCTGTGCaggTTCTTTAGAATGCCtAGAaggGTGTTCAACGCCCAGAAAAAAGCTCAGAGCAGCACCGATTCCGGCGGAAGCAGCGGAGGATCTTCTGGAAGCGAAACCCCAGGCACCAGCGAGTCTGCCACACCAGAATCATCTGGCGGTAGCTCCGGCGGCAGCGACAAGAAGTATTCTATCGGACTGGCCATCGGCACCAACTCTGTTGGATGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACAGGCACAGCATCAAGAAGAACCTGATCGGCGCACTGCTGTTCGACTCTGGCGAAACAGCCGAGGCCACCAGACTGAAGAGAACAGCCCGCAGACGGTACACCAGAAGAAAGAACCGGATCTGCTACCTCCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGACAAGAAGCACGAGAGACACCCCATCTTCGGCAACATC

[0135]

[0194] In one aspect, the nucleobase editor systems provided herein comprise a guide polypeptide. In some embodiments, the guide polynucleotide comprises a base editor fusion. The guide polynucleotide binds to the protein to form a complex. In some embodiments, the guide polynucleotide directs the base editor fusion protein to effect a modification in the target sequence. The guide polynucleotide may comprise a single nucleic acid sequence or two separated nucleic acid sequences. In some embodiments, the guide polynucleotide is a single guide, e.g., a single guide RNA. In some embodiments, the single guide RNA comprises a spacer sequence that can hybridize to the target sequence. In some embodiments, the single guide RNA comprises a tracrRNA sequence that binds to a programmable DNA binding protein, e.g., the Cas9 protein of the base editor fusion protein. In some embodiments, the single guide RNA comprises a stem-loop structure, a tracrRNA sequence, a crRNA sequence, a direct repeat, and / or an anti-repeat. In some embodiments, the single guide RNA comprises a chemical modification. In some embodiments, the guide polynucleotide is any one of the guide polynucleotides provided herein, as described in the "Guide Polynucleotides" section.

[0136] Deaminase domain

[0195] Salts for editing, modifying or altering a target nucleotide sequence of a polynucleotide Disclosed herein is a radical editor system.

[0137]

[0196] The base editor systems provided herein are capable of programmable DNA binding. It may include proteins and deaminases. As used herein, deaminase may refer to an enzyme that catalyzes the removal of amine groups from a molecule or deamination, for example, by hydrolysis. In some embodiments, the deaminase is a cytidine deaminase that catalyzes the deamination of cytidine (C) to uridine (U), deoxycytidine (dC) to deoxyuridine (dU), or 5-methyl-cytidine 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), which is incorporated herein by reference in its entirety. In some embodiments, the deaminase is a cytosine deaminase that 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 that 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, a chimpanzee, a gorilla, a monkey, a cow, a dog, a rat or a mouse. In some embodiments, the deaminase is a variant of a naturally occurring deaminase from an organism, and the variant does not occur naturally. 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.

[0138]

[0197] Cytidine deaminase (or cytosine deaminase) is a enzyme that catalyzes the chemical reaction "cytosine In the context of a gene, such nucleotide changes or mutations may in turn lead to amino acid changes in the protein, which may affect the function of the protein, e.g., loss of function or gain of function. Komor et al. (Nature, Programmable editing of a target base in genomic DNA, 1999, 144:131-132, which is incorporated herein by reference in its entirety) Subsequent DNA repair mechanisms ensure that uracil bases in the DNA are replaced by T, as described in A without double-stranded DNA cleavage, 533, 420-424 (2016)).

[0139]

[0198] One exemplary suitable class of cytosine deaminases is The Apolipoprotein B mRNA Editing Complex (APOBEC) family of cytosine deaminases encompasses 11 proteins that help initiate mutagenesis in a targeted manner. The Apolipoprotein B Editing Complex 3 (APOBEC3) enzymes provide protection to human cells from certain HIV-1 strains via 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. The 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 hAID or TTC in hAPOBEC3F. The recent crystal structure of the catalytic domain of APOBEC3G shows a secondary structure that includes a five-stranded β-sheet core flanked by six α-helices, which is believed to be conserved across the entire family. The active center 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, thus highlighting the importance of sequence-specific targeting. Another suitable cytosine deaminase is activity-induced cytidine deaminase (AID), which is involved in antibody maturation by converting cytosines in ssDNA to uracils in a transcription-dependent, strand-biased manner.

[0140]

[0199] Adenosine deaminase (or adenine deaminase) is adenine The adenosine deaminase includes an enzyme that catalyzes the hydrolytic deamination of adenine or deoxyadenosine to inosine or deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be derived from any organism, for example, bacteria. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase derived 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 derived from bacteria, 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 TadA deaminase is E. coli TadA deaminase. In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, the truncated ecTadA may be missing one or more N-terminal amino acids compared to full-length ecTadA. In some embodiments, the truncated ecTadA may be missing 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 compared to full-length ecTadA. In some embodiments, the truncated ecTadA is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 compared to full-length ecTadA. In some embodiments, the ecTadA deaminase is missing an N-terminal methionine.

[0141]

[0200] In some embodiments, the adenosine deaminase is an adenosine deaminase as set forth in SEQ ID NO:1. The adenosine deaminase domains include those that are 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 any of the adenosine deaminases provided herein. It should be appreciated that the adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The present disclosure provides any deaminase domain having any percent identity and 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:1 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, at least 170 identical contiguous amino acid residues compared to any one of the amino acid sequences set forth in SEQ ID NO:1 or any of the adenosine deaminases provided herein.

[0142]

[0201] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. D108X mutation or a corresponding mutation in another adenosine deaminase, X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, adenosine deaminase comprises D108G, D108N, D108V, D108A or D108Y mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. However, it should be recognized that additional deaminase can be aligned in the same manner to identify the homologous amino acid residues that can be mutated as provided herein.

[0143]

[0202] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. A106X mutation, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

[0144]

[0203] In some embodiments, the adenosine deaminase comprises an E155X mutation in SEQ ID NO:1. or a corresponding mutation in another adenosine deaminase, and the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E155D, E155G or E155V mutation in SEQ ID NO:1, or a corresponding mutation in another adenosine deaminase.

[0145]

[0204] In some embodiments, the adenosine deaminase comprises a D147X mutation in SEQ ID NO:1. or a corresponding mutation in another adenosine deaminase, and the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In the form, the adenosine deaminase comprises a D147Y mutation in SEQ ID NO:1, or a corresponding mutation in another adenosine deaminase.

[0146]

[0205] Any of the mutations provided herein (e.g., the ecTadA amine of SEQ ID NO:1) can be used in It should be recognized that the mutations identified in ecTadA (based on amino acid sequence) can be introduced into other adenosine deaminases, such as Staphylococcus aureus TadA (saTadA) or other adenosine deaminases (e.g., bacterial adenosine deaminases). Methods that are homologous to the mutated residues in ecTadA will be apparent to those skilled in the art. Thus, any of the mutations identified in ecTadA can be made in other adenosine deaminases that have homologous amino acid residues. It should also be recognized that any of the mutations provided herein can be made individually or in any combination in ecTadA or another adenosine deaminase. For example, an adenosine deaminase can contain D108N, A106V, E155V and / or D147Y mutations in ecTadA SEQ ID NO:1, or the corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the following groups of mutations (groups of mutations are separated by ";") in ecTadA SEQ ID NO:1, or corresponding mutations in another adenosine deaminase: D108N and A106V; D108N and E155V; D108N and D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D108N, A106V and E55V; D108N, A106V and D147Y; D108N, E55V and D147Y; A106V, E55V and D147Y; and D108N, A106V, E55V and D147Y. However, it should be recognized that any combination of the corresponding mutations provided herein can be made in an adenosine deaminase (eg, ecTadA).

[0147]

[0206] In some embodiments, the adenosine deaminase is H8X, T1 in SEQ ID NO:1. adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the H8Y, T17S, L18E, W23L, L34S, W45L, R51H, A56E or A56S, E59G, E85K or E85G, M94L, 1951, V102A, F104L, A106V, R107C or R107H or R107P, D108G or D108N or D108V or D108A or D108Y, K1101, M118K, N127S, A138V, F149Y, M151V, R153C, Q154L, I156D and / or K157R mutations in SEQ ID NO:1 or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the mutations corresponding to SEQ ID NO: 1 or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the mutation(s) in any construct shown in Table 23 corresponding to SEQ ID NO: 1 or the corresponding mutation(s) in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the H8X, D108X and / or N127X mutations in SEQ ID NO: 1 or one or more corresponding mutations in another adenosine deaminase, where X represents the presence of any amino acid. In some embodiments, the adenosine deaminase comprises one or more of the H8Y, D108N and / or N127S mutations in SEQ ID NO: 1 or one or more corresponding mutations in another adenosine deaminase.

[0148]

[0207] In some embodiments, the adenosine deaminase is H8X, R2 in SEQ ID NO:1. adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the H8Y, R26W, M61I, L68Q, M70V, A106T, D108N, A109T, N127S, D147Y, R152C, Q154H or Q154R, E155G or E155V or E155D, K161Q, Q163H and / or T166P mutations in SEQ ID NO:1 or one or more corresponding mutations in another adenosine deaminase.

[0149]

[0208] In some embodiments, the adenosine deaminase is H8X, D1 in SEQ ID NO:1. 1, 2, 3, 4, 5, or 6 mutations selected from the group consisting of H8X, M61X, M70X, D108X, N127X, Q154X, E155X, and Q163X, or the corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, M61X, M70X, D108X, N127X, Q154X, E155X, and Q163X, or the corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four or five mutations selected from the group consisting of H8X, D108X, N127X, E155X and T166X in SEQ ID NO: 1 or the corresponding mutation(s) in another adenosine deaminase, where X represents the presence of any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five or six mutations selected from the group consisting of H8X, A106X, D108X or the mutation(s) in another adenosine deaminase, where X represents the presence of any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven or eight mutations selected from the group consisting of H8X, R126X, L68X, D108X, N127X, D147X and E155X in SEQ ID NO:1 or the corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.In some embodiments, the adenosine deaminase comprises one, two, three, four or five mutations selected from the group consisting of H8X, D108X, A109X, N127X and E155X in SEQ ID NO:1 or the corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0150]

[0209] In some embodiments, the adenosine deaminase is H8Y, D1 in SEQ ID NO:1. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, M61I, M70V, D108N, N127S, Q154R, E155G, and Q163H in SEQ ID NO: 1, or the corresponding mutation(s) in another adenosine deaminase. In some embodiments, the adenosine deaminase is one, two or more selected from the group consisting of H8Y, D108N, N127S, E155V and T166P in SEQ ID NO:1 or the corresponding mutation(s) in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, A106T, D108N, N127S, E155D, and K161Q in SEQ ID NO: 1, or the corresponding mutation(s) in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, R126W, L68Q, D108N, N127S, D147Y, and E155V in SEQ ID NO: 1, or the corresponding mutation(s) in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four or five mutations selected from the group consisting of H8Y, D108N, A109T, N127S and E155G in SEQ ID NO:1 or a corresponding mutation(s) in another adenosine deaminase.

[0151]

[0210] In some embodiments, the adenosine deaminase comprises the sequence shown in SEQ ID NO: 1, D108N, D108G or D108V mutation or corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises A106V and D108N mutation in SEQ ID NO: 1 or corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises R107C and D108N in SEQ ID NO: 1 or corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises H8Y, D108N, N127S, D147Y and Q154H mutation in SEQ ID NO: 1 or corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises H8Y, R24W, D108N, N127S, D147Y and E155V mutation in SEQ ID NO: 1 or corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises D108N, D147Y and E155V mutations in SEQ ID NO: 1 or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises H8Y, D108N and S127S mutations in SEQ ID NO: 1 or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises A106V, D108N, D147Y and E155V mutations in SEQ ID NO: 1 or corresponding mutations in another adenosine deaminase.

[0152]

[0211] In some embodiments, the adenosine deaminase is S2X, H8 in SEQ ID NO:1. The adenosine deaminase comprises one or more of the following mutations: X, I49X, L84X, H123X, N127X, I156X and / or K160X, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the following mutations: S2A, H8Y, I49F, L84F, H123Y, N127S, I156F and / or K160S in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the following mutations: SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises any one of the mutation(s) of the clone corresponding to SEQ ID NO:1 or the corresponding mutation(s) in another adenosine deaminase.

[0153]

[0212] In some embodiments, the adenosine deaminase is an L84X mutant adenosine deaminase. adenosine deaminase, wherein X represents the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an L84F mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0154]

[0213] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. H123X mutation or a corresponding mutation in another adenosine deaminase, where X is a wild-type The term "adenosine deaminase" refers to any amino acid other than the corresponding amino acid in adenosine deaminase of the type 1. In some embodiments, the adenosine deaminase comprises an H123Y mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0155]

[0214] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. In some embodiments, the adenosine deaminase comprises an I157X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an I157F mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0156]

[0215] In some embodiments, the adenosine deaminase is selected from the group consisting of L84X, A The adenosine deaminase comprises one, two, three, four, five, six or seven mutations selected from the group consisting of 106X, D108X, H123X, D147X, E155X and I156X or the corresponding mutation(s) in another adenosine deaminase, where X represents the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five or six mutations selected from the group consisting of S2X, I49X, A106X, D108X, D147X and E155X or the corresponding mutation(s) in SEQ ID NO: 1, where X represents the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four or five mutations selected from the group consisting of H8X, A106X, D108X, N127X and K160X in SEQ ID NO:1 or the corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0157]

[0216] In some embodiments, the adenosine deaminase is selected from the group consisting of L84F, A The adenosine deaminase comprises one, two, three, four, five, six or seven mutations selected from the group consisting of 106V, D108N, H123Y, D147Y, E155V and I156F or the corresponding mutation(s) in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five or six mutations selected from the group consisting of S2A, I49F, A106V, D108N, D147Y and E155V in SEQ ID NO:1 or the corresponding amino acids in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four or five mutations selected from the group consisting of H8Y, A106T, D108N, N127S and K160S in SEQ ID NO: 1 or a corresponding mutation(s) in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of E25X, R26X, R107X, A142X and / or A143X mutations in SEQ ID NO: 1 or one or more corresponding mutations in another adenosine deaminase, where the presence of X denotes any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the E25M, E25D, E25A, E25R, E25V, E25S, E25Y, R26G, R26N, R26Q, R26C, R26L, R26K, R107P, R07K, R107A, R107N, R107W, R107H, R107S, A142N, A142D, A142G, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and / or A143R mutations in SEQ ID NO:1 or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the mutations provided in Table 23 corresponding to SEQ ID NO: 1 or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises any one of the mutation(s) shown in Table 23 corresponding to SEQ ID NO: 1 or one or more corresponding mutations in another adenosine deaminase. The corresponding mutation(s) in

[0158]

[0217] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. E25X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises E25M, E25D, E25A, E25R, E25V, E25S or E25Y mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0159]

[0218] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. The adenosine deaminase comprises an R26X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R26G, R26N, R26Q, R26C, R26L or R26K mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0160]

[0219] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. R107X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises R107P, R07K, R107A, R107N, R107W, R107H or R107S mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0161]

[0220] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. A142X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises A142N, A142D, A142G mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0162]

[0221] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. A143X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and / or A143R mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0163]

[0222] In some embodiments, the adenosine deaminase is selected from the group consisting of H36X, N 37X, P48X, I49X, R51X, M70X, N72X, D77X, E134X, S 146X, Q154X, K157X and / or K161X mutations or one or more corresponding mutations in another adenosine deaminase, where the presence of X denotes any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H36L, N37T, N37S, P48T, P48L, I49V, R51H, R51L, M70L, N72S, D77G, E134G, S 146R, S 146C, Q154H, K157N and / or K161T mutations in SEQ ID NO: 1 or one or more corresponding mutations in another adenosine deaminase.

[0164]

[0223] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. H36X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H36L mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises an N37X mutation in ecTadA SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an N37T or N37S mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0165]

[0224] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. P48X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48T or P48L mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0166]

[0225] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. R51X mutation, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R51H or R51L mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase.

[0167]

[0226] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. In some embodiments, the adenosine deaminase comprises an S146X mutation or a corresponding mutation in another adenosine deaminase, where X represents the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. 146C mutation or a corresponding mutation in another adenosine deaminase.

[0168]

[0227] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. In some embodiments, the adenosine deaminase comprises a K157X mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a K157N mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0169]

[0228] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. P48X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48S, P48T or P48A mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0170]

[0229] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. A142X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises the A142N mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0171]

[0230] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. It comprises a W23X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a W23R or W23L mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0172]

[0231] In some embodiments, the adenosine deaminase is ecTadA in SEQ ID NO:1. R152X mutation or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R152P or R52H mutation in SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0173]

[0232] Additional adenosine deaminase mutations and variants are herein incorporated by reference in their entirety. This is described in patent application WO2018119354, which is incorporated herein.

[0233] Additional adenosine deaminases useful in the present application will be apparent to those of skill in the art. , are within the scope of the present disclosure. For example, adenosine deaminase may be a homolog of AD AT. Exemplary AD AT homologs include, without limitation:

[0234] Staphylococcus aureus TadA: MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAH AEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCS GS LMNLLQQS NFNHRAIVDKG VLKE AC S TLLTTFFKNLRANKKS TN (SEQ ID NO: 30)

[0235] Bacillus subtilis TadA: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEML VIDE AC KALGT WRLEG ATLY VTLEPCPMC AG AV VLS R VEK V VFG AFDPKGGC S GTLMN LLQEERFNHQ AE V VS G VLEEEC GGMLS AFFRELRKKKK A ARKNLS E (SEQ ID NO: 31)

[0236] Salmonella typhimurium (S. typhimurium) TadA: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEG WNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIG RVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIK ALKKADRAEGAGPAV (SEQ ID NO: 32)

[0237] Shewanella putrefaciens ens) (S. putrefaciens) TadA: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE (SEQ ID NO: 33)

[0238] Haemophilus influenzae F3031 (H. influenzae) TadA: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK (SEQ ID NO: 34)

[0239] Caulobacter crescentus s) (C. crescentus) TadA: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI (SEQ ID NO: 35)

[0240] Geobacter sulfurreduces ucens) (G. sulfurreducens) TadA: MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALF IDERKVPPEP (SEQ ID NO: 36)

[0241] In some embodiments, the adenosine deaminase is an amino acid sequence listed in Table 23. 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%, at least 99.5%, at least 99.7% or at least 99.9% identical to any one of the sequences. In some embodiments, the adenosine deaminase comprises any one of the amino acid sequences listed in Table 23. In some embodiments, the sequence of the adenosine deaminase is any one of the amino acid sequences listed in Table 23. 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%, 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: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146, 2152, 2156 and 2160. In some embodiments, the adenosine deaminase comprises any one of the amino acid sequences in SEQ ID NOs: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146, 2152, 2156 and 2160. In some embodiments, the sequence of the adenosine deaminase is any one of the amino acid sequences in SEQ ID NOs: 2137, 2149, 2154, 2158, and 2188.

[0174]

[0242] In some embodiments, the adenosine deaminase is a polypeptide selected from the group consisting of those listed in Table 23. In some embodiments, the adenosine deaminase 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 adenosine deaminase sequences listed in Table 23. In some embodiments, the adenosine deaminase 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 adenosine deaminase sequences listed in Table 23. In some embodiments, the adenosine deaminase is expressed by any one of the polynucleotide sequences listed in Table 23. In some embodiments, the adenosine deaminase is expressed by any one of the polynucleotide sequences listed in SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In some embodiments, the adenosine deaminase 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 nucleotide sequences. 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In some embodiments, the adenosine deaminase is encoded by any one of the polynucleotide sequences in SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189. In some embodiments, the adenosine deaminase is a polynucleotide selected from the group consisting of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158 The polypeptides are 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 nucleotide sequences, or a combination thereof. In some embodiments, the adenosine deaminase is encoded by a polynucleotide sequence comprising any one of the polynucleotide sequences in SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158, or a combination thereof.In some embodiments, the adenosine deaminase is expressed by any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189. In some embodiments, the polynucleotide sequence further comprises 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 of SEQ ID NOs: 2138, 2147, 2158, or a combination thereof. In some embodiments, the polynucleotide sequence further comprises any one of the polynucleotide sequences of SEQ ID NOs: 2138, 2147, 2158, or a combination thereof.

[0175] Programmable DNA-binding proteins

[0243] Targets and binds to DNA sequences in any desired nucleotide sequence within a genome Provided herein are programmable DNA-binding proteins that can be programmed to bind to desired nucleotide sequences. To program the DNA-binding protein to bind to a desired nucleotide sequence, the DNA-binding protein can be modified to change its binding specificity, for example, zinc finger DNA-binding domain, zinc finger nuclease (ZFN), CRISPR-Cas9 protein, or transcription activator-like effector protein (TALE). ZFNs are artificial restriction enzymes created by fusing zinc finger DNA-binding domains to DNA cleavage domains. The zinc finger domains can be engineered to target specific desired DNA sequences, allowing the zinc fingers to bind to unique sequences within a complex genome. Transcription activator-like effector nucleases (TALENs) are engineered restriction enzymes that can be engineered to cleave specific sequences of DNA. They are created by fusing the TAL effector DNA-binding domain to a nuclease domain (e.g., Fokl). Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any desired DNA sequence. Methods for programming ZFNs and TALEs are well known to those skilled in the art. For example, such methods are described in Maeder et al., Mol. Cell 31 (2): 294-301, 2008; Carroll et al., Genetics Society of America, 188 (4): 773-782, 2011; Miller et al., Nature Biotechnology 25 (7): 778-785, 2007; Christian et al., Genetics 186 (2): 757-61, 2008; Li et al., Nucleic Acids Res. 39 (1): ...

Claims

1. (i) an mRNA encoding a protein containing a programmable DNA-binding domain and a deaminase; and (ii) a guide polynucleotide comprising a spacer sequence wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, the protospacer being present on a gene encoding angiopoietin-like protein 3, and uracil is understood to be identical to thymine.

2. (i) (a) a protein comprising a programmable DNA-binding domain and an adenosine deaminase; or (b) a nucleic acid encoding the protein; and (ii) a guide polynucleotide comprising a spacer sequence wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, the protospacer being present on a gene encoding angiopoietin-like protein 3, and uracil is understood to be identical to thymine.

3. (i) (a) a protein comprising a programmable DNA-binding domain and a deaminase; or (b) a nucleic acid encoding the protein; and (ii) a guide polynucleotide comprising a spacer sequence wherein the spacer sequence comprises at least 13 nucleotide bases of the nucleotide base sequence of the protospacer, the protospacer is present on a gene encoding angiopoietin-like protein 3, and uracil is understood to be equivalent to thymine; The spacer sequence is (a) GCCAAUGGCCUCCUUCAGUU (nucleotides 1-20 of SEQ ID NO: 287, without any chemically modified nucleotides); (b) GGCCUCCUUCAGUUGGGACA (nucleotides 1-20 of SEQ ID NO: 285, without any chemically modified nucleotides); or (c) AAGAUACCUGAAUAACCCUC (nucleotides 1-20 of SEQ ID NO:59, without any chemically modified nucleotides); comprising a nucleotide base sequence having at least 80% sequence identity to The capital letters A, G, and C represent adenine, guanine, and cytosine, respectively, and the capital letter U represents uracil or thymine.

4. (i) (a) a protein comprising a programmable DNA-binding domain and a deaminase; or (b) a nucleic acid encoding the protein; and (ii) a guide polynucleotide comprising a spacer sequence wherein the spacer sequence comprises at least 13 nucleotide bases of the nucleotide base sequence of the protospacer, the protospacer is present on a gene encoding angiopoietin-like protein 3, and uracil is understood to be equivalent to thymine; The protospacer is (a) GCCAATGGCCTCCTTCAGTT (SEQ ID NO: 109), (b) GGCCTCCTTCAGTTGGGACA (SEQ ID NO: 107), or (c) AAGATACCTGAATAACCCTC (SEQ ID NO: 15) has at least 80% sequence identity to A composition in which the capital letters A, T, G, and C represent adenosine, thymine, guanosine, and cytidine, respectively.

5. (i) (a) a protein comprising a programmable DNA-binding domain and a deaminase; or (b) a nucleic acid encoding the protein; and (ii) a guide polynucleotide comprising a tracr sequence and a spacer sequence wherein the spacer sequence comprises at least 13 nucleotide bases of the nucleotide base sequence of the protospacer, the protospacer is present on a gene encoding angiopoietin-like protein 3, and uracil is understood to be equivalent to thymine; The tracr sequence is GUUUUAGAGCUAGAAAAUAGCAAGUUAAAAAAAAAGGCUAGUCCGUUAUCAACUUGAAAAAAGUGGCACCGAGUCGGUGCU UUU (nucleotide base sequence of SEQ ID NO:61), a nucleotide base sequence having at least 80% sequence identity to a functional portion of the nucleotide base sequence of The capital letters A, G, and C represent adenine, guanine, and cytosine, respectively, and the capital letter U represents uracil or thymine.

6. (i) a protein comprising a programmable DNA-binding domain, or a nucleic acid encoding said protein; and (ii) a guide polynucleotide comprising a spacer sequence wherein the spacer sequence comprises at least 13 nucleotide bases of the nucleotide base sequence of the protospacer, the protospacer is present on a gene encoding angiopoietin-like protein 3, and uracil is understood to be equivalent to thymine; The composition, wherein the protein affects a nucleobase pair modification in a gene encoding angiopoietin-like protein 3, the nucleobase pair modification resulting in an A.T to G.C base pair modification.

7. (i) (a) a protein that comprises a programmable DNA-binding domain, or (b) a nucleic acid encoding the protein; and (ii) a guide polynucleotide comprising a spacer sequence wherein the spacer sequence comprises at least 13 nucleotide bases of the nucleotide base sequence of the protospacer, the protospacer is present on a gene encoding angiopoietin-like protein 3, and uracil is understood to be equivalent to thymine; the protein affects nucleotide editing of a gene encoding angiopoietin-like protein 3; The composition, wherein the nucleotide edit is at nucleotide 12 of the nucleotide sequence set forth in SEQ ID NO:

76.

8. (i) (a) a protein comprising a programmable DNA-binding domain and a deaminase; or (b) a nucleic acid encoding the protein; and (ii) a guide polynucleotide comprising a spacer sequence wherein the spacer sequence comprises at least 13 nucleotide bases of the nucleotide base sequence of the protospacer, the protospacer is present on a gene encoding angiopoietin-like protein 3, and uracil is understood to be equivalent to thymine; A composition, wherein the protospacer is present at a splice site of a gene encoding angiopoietin-like protein 3.

9. The composition of claim 8 , wherein the splice site is a splice donor site.

10. The composition of claim 9, wherein the splice donor site is the 5' end of intron 6 of the gene encoding angiopoietin-like protein 3 as set forth in SEQ ID NO:

7.

11. (i) (a) a protein comprising a programmable DNA-binding domain, or a nucleic acid encoding said protein; and (ii) a guide polynucleotide comprising a tracr sequence and a spacer sequence wherein the spacer sequence comprises at least 13 nucleotide bases of the nucleotide base sequence of the protospacer, the protospacer is present on a gene encoding angiopoietin-like protein 3, and uracil is understood to be equivalent to thymine; The tracr sequence is the following chemically modified sequence: (a) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu (SEQ ID NO: 57); (b) gUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 2249) or (c) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 3164) has at least 80% identity with any of 1) capital letters A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) lower case letters a, u, g, and c represent 2'-O-methyl modified adenosine, uridine, guanosine, and cytidine, respectively; and 3) lower case s represents a phosphorothioate (PS) linkage.

12. 2. The composition of claim 1 for use in editing a gene encoding angiopoietin-like protein 3.

13. 3. The composition of claim 2 for use in editing a gene encoding angiopoietin-like protein 3.

14. The composition of claim 6 for use in editing a gene encoding angiopoietin-like protein 3.

15. 8. The composition of claim 7 for use in editing a gene encoding angiopoietin-like protein 3.

16. 9. The composition of claim 8 for use in editing a gene encoding angiopoietin-like protein 3.

17. The composition of claim 16 , wherein the splice site is a splice donor site.

18. The composition of claim 17, wherein the splice donor site is the 5' end of intron 6 of the gene encoding angiopoietin-like protein 3 as set forth in SEQ ID NO:

7.

19. The composition of any one of claims 1 to 18, wherein the programmable DNA binding domain is selected from the group consisting of Cas9, CasX, CasY, Cas12a (Cpf1), Cas12b, C2c1, C2c2, C2C3, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Argonautes.

20. 19. The composition of any one of claims 1 to 18, wherein the programmable DNA binding domain is selected from the group consisting of a nuclease-inactive spCas9 domain or a spCas9 nickase.

21. 16. The composition of any one of claims 6, 7, 11, 14, and 15, wherein the protein further comprises a deaminase.

22. 22. The composition of claim 21, wherein the deaminase comprises a protein selected from the group consisting of adenosine deaminase and cytidine deaminase.

23. The composition of any one of claims 1 to 18, wherein the guide polynucleotide is a guide RNA.

24. A composition according to any one of claims 2 to 11 and 13 to 18, comprising a nucleic acid encoding said protein.

25. The composition of claim 24 , wherein the nucleic acid encoding the protein is mRNA.

26. 26. The composition of claim 25, wherein the GC% content of the region of the mRNA encoding the deaminase is at least 60%.

27. 26. The composition of claim 25, wherein the region of the mRNA encoding the programmable DNA binding domain has a GC% content of at least 45%.

28. The composition of claim 25, wherein the GC% content of the mRNA is at least 47%.

29. 26. The composition of any one of claims 1, 12 and 25, wherein the ratio of guide nucleotides to mRNA is about 1:10 to about 10:1 by weight.

30. 26. The composition of any one of claims 1, 12 and 25, wherein the ratio of guide nucleotides to mRNA is about 1:3 to about 3:1 by weight.

31. 26. The composition of any one of claims 11, 12 and 25, wherein the ratio of guide nucleotides to mRNA is about 1:2 to about 2:1 by weight.

32. 26. The composition of any one of claims 1, 12 and 25, wherein the ratio of guide nucleotides to mRNA is about 1:1.5 to about 1.5:1 by weight.

33. 26. The composition of any one of claims 1, 12 and 25, wherein the ratio of guide nucleotides and mRNA is about 1:1 by weight.

34. 26. The composition of any one of claims 1, 12 and 25, wherein the ratio of guide nucleotides to mRNA is from about (1±0.2):1 to about 1:(1±0.2) by weight.

35. 12. A composition according to any one of claims 1 to 11 for use in a method of treating or preventing a condition in a subject in need thereof.

36. 36. The composition of claim 35, wherein the condition is homozygous familial hypercholesterolemia.

37. The composition of claim 5, wherein the nucleotide base sequence has at least 90% sequence identity to a functional portion of the nucleotide base sequence of SEQ ID NO:

61.

38. The composition of claim 5, wherein the nucleotide base sequence has at least 80% sequence identity to the nucleotide base sequence of SEQ ID NO:

61.

39. 39. The composition of claim 38, wherein the nucleotide base sequence has at least 90% sequence identity to the nucleotide base sequence of SEQ ID NO:

61.

40. The composition of claim 5, wherein the tracr sequence comprises at least one chemically modified nucleotide.

41. The tracr sequence is the following chemically modified sequence: (a) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu (SEQ ID NO: 57); (b) gUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 2249) or (c) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 3164) has at least 80% sequence identity with any of 6. The composition of claim 5, wherein: 1) capital letters A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) lower case letters a, u, g, and c represent 2'-O-methyl modified adenosine, uridine, guanosine, and cytidine, respectively; and 3) lower case letter s represents a phosphorothioate (PS) linkage.

42. The composition of claim 6, wherein the nucleobase pair modification is a splice donor site of the gene encoding angiopoietin-like protein 3 as set forth in SEQ ID NO:

7.

43. The composition of claim 42, wherein the splice donor site is the 5' end of intron 6 of the gene encoding angiopoietin-like protein 3 as set forth in SEQ ID NO:

7.

44. The composition of claim 6, wherein the nucleobase pair modification is at nucleotide 11722 of the gene encoding angiopoietin-like protein 3 as set forth in SEQ ID NO:

7.

45. The composition of claim 6, wherein the nucleobase pair modification is at nucleotide 12 of the nucleotide sequence set forth in SEQ ID NO:

76.

46. The spacer sequence is (a) GCCAAUGGCCUCCUUCAGUU (nucleotides 1-20 of SEQ ID NO: 287, without any chemically modified nucleotides); (b) GGCCUCCUUCAGUUGGGACA (nucleotides 1-20 of SEQ ID NO: 285, without any chemically modified nucleotides); or (c) AAGAUACCUGAAUAACCCUC (nucleotides 1-20 of SEQ ID NO:59, without any chemically modified nucleotides); comprising a nucleotide base sequence having at least 80% sequence identity to The composition of any one of claims 1, 2, and 4-18, wherein: 1) capital A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) lower case a, u, g, and c represent 2'-O-methyl modified adenosine, uridine, guanosine, and cytidine, respectively; and 3) lower case s represents a phosphorothioate (PS) linkage.

47. The protospacer is (a) GCCAATGGCCTCCTTCAGTT (SEQ ID NO: 109), (b) GGCCTCCTTCAGTTGGGACA (SEQ ID NO: 107), or (c) AAGATACCTGAATAACCCTC (SEQ ID NO: 15) has at least 80% sequence identity to The composition of any one of claims 1 to 3 and 5 to 18, wherein the capital letters A, T, G, and C represent adenosine, thymine, guanosine, and cytidine, respectively.

48. The composition of any one of claims 1 to 11, formulated for intravenous infusion.

49. The composition of any one of claims 12 to 18, wherein administration is by intravenous infusion.

50. The composition of any one of claims 1, 12, and 25-34, wherein the mRNA has at least 95% sequence identity to SEQ ID NO: 2148, 2153 or 2192.

51. The composition of any one of claims 1, 12, and 25-34, wherein the mRNA has at least 95% sequence identity to the coding sequence of SEQ ID NO: 2148, 2153, or 2192.

52. 19. The composition of any one of claims 1 to 18, wherein the spacer sequence comprises at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 nucleotide bases of the protospacer nucleotide base sequence.

53. 53. The composition of any one of claims 1 to 52, wherein the protospacer is flanked by a NGG protospacer adjacent motif (PAM), where N is adenine, thymine, guanine, or cytosine, and G is guanine.

54. The composition of any one of claims 12 to 18, wherein the subject has homozygous familial hypercholesterolemia.

55. (i) (a) an mRNA encoding a base editor protein comprising a programmable DNA-binding domain and a deaminase, the mRNA comprising a sequence having at least 90% sequence identity to SEQ ID NO: 2148, 2153, or 2192; and (ii) a guide polynucleotide comprising a spacer sequence and a tracr sequence Including, a) the spacer sequence is (a) GCCAAUGGCCUCCUUCAGUU (nucleotides 1-20 of SEQ ID NO: 287, without any chemically modified nucleotides); (b) GGCCUCCUUCAGUUGGGACA (nucleotides 1-20 of SEQ ID NO: 285, without any chemically modified nucleotides); or (c) AAGAUACCUGAAUAACCCUC (nucleotides 1-20 of SEQ ID NO:59, without any chemically modified nucleotides); comprising a nucleotide base sequence selected from the group consisting of: capital letters A, G, and C represent adenine, guanine, and cytosine, respectively; capital letter U represents uracil or thymine; and b) the tracr sequence is (a) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu (SEQ ID NO: 57); (b) gUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 2249) or (c) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 3164) comprising a chemically modified sequence selected from the group consisting of: 1) capital letters A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) lower case letters a, u, g, and c represent 2'-O-methyl modified adenosine, uridine, guanosine, and cytidine, respectively; and 3) lower case s represents a phosphorothioate (PS) linkage.

56. (i) mRNA, a) the 5' untranslated region, b) a first region encoding a deaminase comprising a protein selected from the group consisting of adenosine deaminase and cytidine deaminase; c) a second region encoding a programmable DNA-binding domain selected from the group consisting of Cas9, CasX, CasY, Cas12a (Cpf1), Cas12b, C2c1, C2c2, C2C3, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and Argonaute; d) a third region encoding a nuclear localization sequence; and e) 3' untranslated region The mRNA comprising: (ii) a guide polynucleotide comprising a spacer sequence and a tracr sequence Including, a) the spacer sequence is (a) GCCAAUGGCCUCCUUCAGUU (nucleotides 1-20 of SEQ ID NO: 287, without any chemically modified nucleotides); (b) GGCCUCCUUCAGUUGGGACA (nucleotides 1-20 of SEQ ID NO: 285, without any chemically modified nucleotides); or (c) AAGAUACCUGAAUAACCCUC (nucleotides 1-20 of SEQ ID NO:59, without any chemically modified nucleotides); comprising a nucleotide base sequence selected from the group consisting of: The capital letters A, G, and C represent adenine, guanine, and cytosine, respectively, and the capital letter U represents uracil or thymine; b) the tracr sequence is (d) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu (SEQ ID NO: 57); (e) gUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 2249) or (f) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 3164) comprising a chemically modified sequence selected from the group consisting of: 1) capital letters A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) lower case letters a, u, g, and c represent 2'-O-methyl modified adenosine, uridine, guanosine, and cytidine, respectively; and 3) lower case s represents a phosphorothioate (PS) linkage.

57. 57. The composition of claim 56, wherein (1) the deaminase comprises an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 2140, and (2) the first region encoding the deaminase comprises a nucleotide sequence having at least 90% or at least 95% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2139, 2162, 2169, and 2175.

58. 58. The composition of claim 56 or 57, wherein (1) the programmable DNA-binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 40, 2152, 2156, and 2160, or (2) the second region encoding the programmable DNA-binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 2142, 2151, 2155, 2159, 2164, 2171, and 2177.

59. the nuclear localization sequence comprises an amino acid sequence having at least 90% or at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 2146; or 59. The composition of any one of claims 56-58, wherein the third region comprises a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2145, 2167, 2173, and 2179.

60. The composition of any one of claims 56 to 59, wherein the 5' untranslated region comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% sequence identity to SEQ ID NO:2138.

61. The composition of any one of claims 56 to 59, wherein the 5' untranslated region comprises a nucleotide sequence of AGGA at the 5' end, where A and G represent adenosine and guanosine, respectively.

62. The composition of any one of claims 56 to 61, wherein the 3' untranslated region comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% sequence identity to SEQ ID NO:2147.

63. The 3' untranslated region comprises a nucleotide sequence of u'Cu'AGA at the 3' end, where u' is N. １ -methylpseudouridine, where the capital letters A, G and C represent adenosine, guanosine and cytidine, respectively.

64. The first region of the mRNA encoding the deaminase has a GC% content of at least 60%; the second region encoding the programmable DNA binding domain has a GC% content of at least 45%, and The composition of any one of claims 56 to 63, wherein the GC% content of the mRNA is at least 47%.

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