Compositions for PCSK9 gene editing and methods of using same for treatment of disease

Abstract

Disclosed herein are methods of disease treatments by administering to a subject in need thereof a pharmaceutical composition comprising gene editing compositions. The methods disclosed herein can provide durable in vivo editing of PCSK9. More particularly, the compositions disclosed herein are capable of in vivo editing PCSK9 gene in humans and reducing PCSK9 protein and LDL-C for a potentially transformative treatment of cardiovascular disease, the leading cause of death in the United States and world-wide. Various aspects of the compositions, use and preparation are disclosed, including clinically supported therapeutically effective and safe human dosing of the compositions.

Description

WSGR Attorney Docket No. 53989-743.601COMPOSITIONS FOR PCSK9 GENE EDITING AND METHODS OF USING SAME FOR TREATMENT OF DISEASECROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of Provisional Application Serial No. 63 / 718,518, filed November 8, 2024; and Provisional Application Serial No. 63 / 788,053, filed April 13, 2025, each of which are incorporated herein by reference in its entirety.SUMMARY OF THE INVENTION

[0002] Disclosed are compositions capable of in vivo editing PCSK9 gene in humans and reducing PCSK9 protein and LDL-C for a potentially transformative treatment of cardiovascular disease, the leading cause of death in the United States and world-wide. Various aspects of the compositions, use and preparation are disclosed. Human clinical data on safety and efficacy of the compositions, including human subject dosing thereof is disclosed. Various aspects of the inventive subject matter are set forth herein including the claims and examples set forth herein. Such aspects include formulation of the pharmaceutical composition, composition of the active drug substances including the mRNA / base editor (including constituent components thereof) and gRNA (including constituent components thereof), compositions and / or physical attributes of the LNP, therapeutically effective and safety criteria, administration regimen, and dosing amounts (including based on mg / kg and / or total mg of RNA (active drug substances), with each aspect considered alone and / or in combination with one or more other aspects.BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0004] FIGs. 1A-1E shows the percentage reduction from baseline in blood PCSK9 protein level observed in human subjects following administration of the pharmaceutical composition described herein. The PCSK9 baseline level, which is illustrated by the horizontal dashed-lines in FIGs. 1A-1E, is calculated as the average of the PCSK9 level on pre-dose screening 1, pre-dose screening 2, and the day before treatment. FIG. 1A represents the % PCSK9 change in four patients (n=4) treated with 0.3 mg / kg dose and shows an average reduction of PCSK9 protein of 55% at Day 28. FIG. IB represents the % PCSK9 change in six patients (n=6) treated with 0.45 mg / kg dose and shows an average reduction of PCSK9 protein of 50% at Day 28. FIG. 1C represents the % PCSK9 change in four patients (n=4) treated with 0.6 mg / kg dose and shows an average reduction of PCSK9 protein of 60% at Day 28. FIG. IDWSGR Attorney Docket No. 53989-743.601 represents the % PCSK9 change in three patients (n=3) treated with 0.7 mg / kg dose and shows an average reduction of PCSK9 protein of 50% at Day 28. FIG. IE represents the % PCSK9 change in two patients (n=2) treated with 0.8 mg / kg dose and shows an average reduction of PCSK9 protein of 74% at Day 28. As noted on the Y axis of each graph, the error bars indicate standard error of the mean (SEM) of the percent reduction of the PCSK9 concentration level. Post 28-day time on study data, as applicable, is provided to illustrate durability trends.

[0005] FIGs. 2A-2E show the percentage reduction from baseline in blood LDL-C observed in human subjects following administration of the pharmaceutical composition described herein. The LDL-C baseline level, which is illustrated by the horizontal dashed-lines in FIGs. 2A-2E, is calculated as the average of the LDL-C level on pre-dose screening 1, pre-dose screening 2, and the day before treatment. FIG. 2A represents the percentage reduction in blood LDL-C observed in four patients (n=4) treated with 0.3 mg / kg dose of the pharmaceutical composition described herein and shows an average reduction of LDL-C level of 26% at Day 28. FIG. 2B represents the percentage reduction in blood LDL-C observed in six patients (n=6) treated with 0.45 mg / kg dose of the pharmaceutical composition described herein and shows an average reduction of LDL-C level of 42% at Day 28. FIG. 2C represents the percentage reduction in blood LDL-C observed in four patients (n=4) treated with 0.6 mg / kg dose of the pharmaceutical composition described herein and shows an average reduction of LDL-C level of 47% at Day 28. FIG. 2D represents the percentage reduction in blood LDL-C observed in four patients (n=4) treated with 0.7 mg / kg dose of the pharmaceutical composition described herein and shows an average reduction of LDL-C level of 53% at Day 28. FIG. 2E represents the percentage reduction in blood LDL- C observed in two patients (n=2) treated with 0.8 mg / kg dose of the pharmaceutical composition described herein and shows an average reduction of LDL-C level of 39% at Day 28. As noted on the Y axis of each graph, the error bars indicate standard error of the mean (SEM) of the percent reduction of the LDL-C concentration level. Post 28-day time on study data, as applicable, is provided to illustrate durability trends.

[0006] FIGs. 3A-3E show the levels of alanine aminotransferase (ALT) in blood in human subjects predosing and following administration of the pharmaceutical composition described herein. The dashed- lines in each graph represents an upper limit of normal for male (ULN (M)), an upper limit of normal for female (ULN (F)), and a lower limit of normal (LLN) as noted therein. FIG. 3A shows the blood level of ALT observed in four human subjects treated with the 0.3 mg / kg dose of the pharmaceutical composition described herein (participant 1, participant 2, participant 3, and participant 4). FIG. 3B shows the blood level of ALT observed in six human subjects treated with the 0.45 mg / kg dose of the pharmaceutical composition described herein (participant 5, participant 6, participant 7, participant 8, participant 9 and participant 10). FIG. 3C shows the blood level of ALT observed in four human subjects treated with the 0.6 mg / kg dose of the pharmaceutical composition described herein (participant 11, participant 12, participant 13, and participant 14). FIG. 3D shows the blood level of ALT observed in five human subjects treated with the 0.7 mg / kg dose of the pharmaceutical composition described herein (participant 15, participant 16, participant 17, participant 18 and participant 19). FIG. 3E shows the blood level ofWSGR Attorney Docket No. 53989-743.601ALT observed in three human subjects treated with the 0.8 mg / kg dose of the pharmaceutical composition described herein (participant 20, participant 21, participant and participant 22).

[0007] FIGs. 4A-4E show the levels of aspartate aminotransferase (AST) in blood in human subjects pre-dosing and following administration of the pharmaceutical composition described herein. FIG. 4A shows the blood level of AST observed in four human subjects treated with the 0.3 mg / kg dose of the pharmaceutical composition described herein (participant 1, participant 2, participant 3, and participant 4). FIG. 4B shows the blood level of AST observed in six human subjects treated with the 0.45 mg / kg dose of the pharmaceutical composition described herein (participant 5, participant 6, participant 7, participant 8, participant 9 and participant 10). FIG. 4C shows the blood level of AST observed in four human subjects treated with the 0.6 mg / kg dose of the pharmaceutical composition described herein (participant 11, participant 12, participant 13, and participant 14). FIG. 4D shows the blood level of AST observed in five human subjects treated with the 0.7 mg / kg dose of the pharmaceutical composition described herein (participant 15, participant 16, participant 17, participant 18 and participant 19). FIG. 4E shows the blood level of AST observed in three human subjects treated with the 0.8 mg / kg dose of the pharmaceutical composition described herein (participant 20, participant 21, participant and participant 22).

[0008] FIGs. 5A-5E show the platelet counts in human subjects pre dosing and following administration of the pharmaceutical composition described herein. The dashed-lines in each graph represents an upper limit of normal (ULN) and a lower limit of normal (LLN) as noted therein. FIG. 5A shows the blood platelet counts observed in four human subjects treated with the 0.3 mg / kg dose of the pharmaceutical composition described herein (participant 1, participant 2, participant 3, and participant 4). FIG. 5B shows the blood platelet counts observed in three human subjects treated with the 0.45 mg / kg dose of the pharmaceutical composition described herein (participant 5, participant 6, participant 7, participant 8, participant 9 and participant 10). FIG. 5C shows the platelet count observed in four human subjects treated with the 0.6 mg / kg dose of the pharmaceutical composition described herein (participant 11, participant 12, participant 13, and participant 14). FIG. 5D shows platelet count observed in five human subjects treated with the 0.7 mg / kg dose of the pharmaceutical composition described herein (participant 15, participant 16, participant 17, participant 18 and participant 19). FIG. 5E shows the platelet count observed in three human subjects treated with the 0.8 mg / kg dose of the pharmaceutical composition described herein (participant 20, participant 21, participant and participant 22).

[0009] FIGs. 6A-6E show the levels of alanine aminotransferase (ALT) in blood in human subjects predosing and following administration of the pharmaceutical composition described herein. The dashed-line in each graph represents an upper limit of normal (ULN). The dots represent the mean values at one time point with error bars indicating standard error of the mean (SEM). FIG. 6A shows the blood level of ALT observed in four human subjects treated with the 0.3 mg / kg dose of the pharmaceutical composition described herein. FIG. 6B shows the blood level of ALT observed in six human subjects treated with the 0.45 mg / kg dose of the pharmaceutical composition described herein. FIG. 6C shows the blood level of ALT observed in four human subjects treated with the 0.6 mg / kg dose of the pharmaceutical composition described herein. FIG. 6D shows the blood level of ALT observed in five subjects treated with the 0.7WSGR Attorney Docket No. 53989-743.601 mg / kg dose of the pharmaceutical composition described herein. FIG. 6E shows the blood level of ALT observed in three human subjects treated with the 0.8 mg / kg dose of the pharmaceutical composition described herein.

[0010] FIGs. 7A-7E show the levels of aspartate aminotransferase (AST) in blood in human subjects pre-dosing and following administration of the pharmaceutical composition described herein. The dashed-line in each graph represents an upper limit of normal (ULN). The dots represent the mean values at one time point with error bars indicating standard error of the mean (SEM). FIG. 7A shows the blood level of AST observed in four human subjects treated with the 0.3 mg / kg dose of the pharmaceutical composition described. FIG. 7B shows the blood level of AST observed in six human subjects treated with the 0.45 mg / kg dose of the pharmaceutical composition described herein. FIG. 7C shows the blood level of AST observed in four human subjects treated with the 0.6 mg / kg dose of the pharmaceutical composition described herein. FIG. 7D shows the blood level of AST observed in five subjects treated with the 0.7 mg / kg dose of the pharmaceutical composition described herein. FIG. 7E shows the blood level of AST observed in three human subjects treated with the 0.8 mg / kg dose of the pharmaceutical composition described herein.

[0011] FIGs. 8A-8E show the levels of total bilirubin in blood in human subjects pre-dosing and following administration of the pharmaceutical composition described herein. The dashed-line in each graph represents an upper limit of normal (ULN). The dots represent the mean values at one time point with error bars indicating standard error of the mean (SEM). FIG. 8A shows the blood level of bilirubin observed in four human subjects treated with the 0.3 mg / kg dose of the pharmaceutical composition described herein. FIG. 8B shows the blood level of bilirubin observed in six human subjects treated with the 0.45 mg / kg dose of the pharmaceutical composition described herein. FIG. 8C shows the blood level of bilirubin observed in four human subjects treated with the 0.6 mg / kg dose of the pharmaceutical composition described herein. FIG. 8D shows the blood level of bilirubin observed in five subjects treated with the 0.7 mg / kg dose of the pharmaceutical composition described herein. FIG. 8E shows the blood level of bilirubin observed in three human subjects treated with the 0.8 mg / kg dose of the pharmaceutical composition described herein.

[0012] FIGs. 9A-9E show the platelet counts in blood in human subjects pre-dosing and following administration of the pharmaceutical composition described herein. The dashed-lines in each graph represents an upper limit of normal (ULN) and a lower limit of normal (LLN), as noted therein. The dots represent the mean values at one time point with error bars indicating standard error of the mean (SEM). FIG. 9A shows the platelet counts in four human subjects treated with the 0.3 mg / kg dose of the pharmaceutical composition described herein. FIG. 9B shows the platelet counts in six human subjects treated with the 0.45 mg / kg dose of the pharmaceutical composition described herein. FIG. 9C shows the platelet counts in four human subjects treated with the 0.6 mg / kg dose of the pharmaceutical composition described herein. FIG. 9D shows the platelet counts observed in five subjects treated with the 0.7 mg / kg dose of the pharmaceutical composition described herein. FIG. 9E shows the platelet counts observed in three human subjects treated with the 0.8 mg / kg dose of the pharmaceutical composition described herein.WSGR Attorney Docket No. 53989-743.601

[0013] FIG. 10 shows the percentage reduction from baseline in blood PCSK9 protein level observed in human subjects following administration of the pharmaceutical composition described herein. Four human subjects were treated with the 0.3 mg / kg dose of the pharmaceutical composition described herein. Six human subjects were treated with the 0.45 mg / kg dose of the pharmaceutical composition described herein. Four human subjects were treated with the 0.6 mg / kg dose of the pharmaceutical composition described herein. Three human subjects were treated with the 0.7 mg / kg dose of the pharmaceutical composition described herein. Two human subjects were treated with the 0.8 mg / kg dose of the pharmaceutical composition described herein. The percentage of PCSK9 protein reduction were calculated using a time-weighted average (TWA) method as described in the Examples. Each bar represents a dose group as noted therein. The y-axis represents the percent change from baseline of the time-weighted average of PCSK9 from Day 28 onward. Day 28 value was used in participants where the Day 28 was the last timepoint. Single observations in 3 participants censored due to changes in background lipid-lowering therapy. The error bars indicate standard error of the mean (SEM). A dosedependent reductions in PCSK9 with a mean reduction of 76% observed in the highest dose cohort.

[0014] FIG. 11 shows the percentage reduction from baseline in blood LDL-C level observed in human subjects following administration of the pharmaceutical composition described herein. Four human subjects were treated with the 0.3 mg / kg dose of the pharmaceutical composition described herein. Six human subjects were treated with the 0.45 mg / kg dose of the pharmaceutical composition described herein. Four human subjects were treated with the 0.6 mg / kg dose of the pharmaceutical composition described herein. Four human subjects were treated with the 0.7 mg / kg dose of the pharmaceutical composition described herein. Two human subjects were treated with the 0.8 mg / kg dose of the pharmaceutical composition described herein. The percentage of LDL-C reduction were calculated using a time-weighted average (TWA) method as described in the Examples. Each bar represents a dose group as noted therein. The y-axis represents the percent change from baseline of the time-weighted average of PCSK9 from Day 28 onward. Day 28 value was used in participants where the Day 28 was the last timepoint. Single observations in 3 participants censored due to changes in background lipid-lowering therapy. The error bars indicate standard error of the mean (SEM). A dose-dependent reductions in LDL with a mean reduction of 43% observed in the highest dose cohort.

[0015] FIG. 12 shows the percentage reduction from baseline in blood LDL-C level observed in human subjects correlating to the total RNA dose received by the subjects. Each dot represents a single participant. The y-axis represents the percent change from baseline of the time-weighted average of PCSK9 from Day 28 onward. Day 28 value was used in participants where the Day 28 was the last timepoint. Single observations in 3 participants censored due to changes in background lipid-lowering therapy.

[0016] FIG. 13 shows the percentage reduction from baseline in blood PCSK9 protein level observed in human subjects following administration of the pharmaceutical composition described herein. The human subjects were grouped as follows: a) four human subjects were treated with less than 25mg total RNA dose of the pharmaceutical composition described herein; b) seven human subjects were treated withWSGR Attorney Docket No. 53989-743.601 between 25mg and 50mg total RNA dose of the pharmaceutical composition described herein; and c) eight human subjects were treated with between 50mg and 60mg total RNA dose of the pharmaceutical composition described herein. The y-axis represents the percent change from baseline of the time- weighted average of PCSK9 from Day 28 onward. Day 28 value was used in participants where the Day 28 was the last timepoint. Single observation in 2 participants censored due to changes in background lipid-lowering therapy. The error bars indicate standard error of the mean (SEM). In the highest dose group, who received 50 to 60 mg, an average 65% change from baseline in the change from baseline of PCSK9 was observed.

[0017] FIG. 14 shows the percentage reduction from baseline in blood LDL-C level observed in human subjects following administration of the pharmaceutical composition described herein. The human subjects were grouped as follows: a) four human subjects were treated with less than 25mg total RNA dose of the pharmaceutical composition described herein; b) seven human subjects were treated with between 25mg and 50mg total RNA dose of the pharmaceutical composition described herein; c) eight human subjects were treated with between 50mg and 60mg total RNA dose of the pharmaceutical composition described herein; and d) one human subject was treated with between 60mg and 70mg total RNA dose of the pharmaceutical composition described herein. The y-axis represents the percent change from baseline of the time-weighted average of LDL-C from Day 28 onward. Day 28 value was used in participants where the Day 28 was the last timepoint. Single observations in 3 participants censored due to changes in background lipid-lowering therapy. The error bars indicate standard error of the mean (SEM). In the participant in the highest dose group, who received 70 mg, an 81% change from baseline was observed.

[0018] Each of the foregoing figures represent various aspects of the corresponding patient clinical data set forth in Table 7 below. ULN and LLN for the safety related data set forth in the foregoing figures reflect accepted reference ranges for ALT, AST and bilirubin. For ALT: >1-3 x ULN, >3-5 x LN, > 5 x ULN, > 10 x ULN may be interpreted or otherwise considered as mild, moderate, significant and severe elevation of ALT, respectively, with significant elevation indicating potential hepatocellular injury and severe elevation indicating potential liver toxicity. For AST: >1-3 x ULN, >3-5 x ULN, > 5 x ULN, > 10 x ULN may be interpreted or otherwise considered as mild, moderate, marked, and severe elevation of AST, respectively, with severe elevation indicating possible acute hepatocellular or muscle injury. For bilirubin: >1-1.5 x ULN, >1.5-2 x ULN, > 2 x ULN, >3 x ULN may be respectively interpreted or otherwise considered as borderline elevation, mild hyperbilirubinemia, potentially clinically significant elevation, and potential liver injury to the extent that Hy’s law is met (e.g., if ALT is simultaneously >3 ULN). In drug development and gene-therapy trials, Hy’s Law defines potential drug-induced liver injury (DILI) as: ALT or AST > 3x ULN combined with total bilirubin > 2x ULN (without major alkaline phosphatase (ALP) elevation or hemolysis, such as no other cause (e.g., viral hepatitis, gallstones, hemolysis) can explain the finding). When Hy’s law criteria is met, such a findingWSGR Attorney Docket No. 53989-743.601 indicates possible serious hepatic injury. Notably, as set forth in the foregoing safety data, the levels of ALT, AST and bilirubin do not appear to be dose dependent and are relatively flat (with only mild elevations in ALT or AST observed post dosing in a small number of patients and no above normal bilirubin elevations observed) and hence do not meet Hy’s law. One of ordinary skill in the art, as contemplated by Hy’s law, would recognize that transient levels of ALT, AST, and bilirubin may vary independent of the clinical trial drug being administered, factors such as exercise, muscle injury, fasting, diet, consumption of alcohol, viral or bacterial infections, acute or general inflammation, anaesthesia or use of other drugs like acetaminophen, statins, antibiotics and NSAIDs, can have an impact on these levels apart from or independent from the clinical trial drug being administered. With respect to platelet counts, platelets (thrombocytes) are the cell fragments that help blood clot. Thrombocytosis refers to higher than normal platelet counts, whereas thrombocytopenia refers to lower than normal platelet counts. For thrombocytosis, elevated platelet counts between: 400-600 x 109 / L are considered mild, usually reactive, with patients typically having no symptoms; 600-1,000 x 109 / L are generally considered moderate with a general recommendation that patients be monitored for cause; and >1,000 x 109 / L are considered high, with patients generally evaluated for bone-marrow disease. For thrombocytopenia, severity of platelet count levels [x 109 / L] under the Common Terminology Criteria for Adverse Events (CTCAE) grade subject to clinical assessment is generally as follows: Grade 1 (mild) 75-150 Usually no symptoms, Grade 2 (moderate) 50-75 Mild risk of bruising or bleeding, Grade 3 (severe) 25-50 Increased bleeding risk; may require monitoring or transfusion; and Grade 4 (lifethreatening) < 25 High risk of spontaneous bleeding. Notably, the platelet data set forth in the foregoing figures, do not appear to be dose dependent and are relatively flat, with a few patients having mild variations outside normal range being observed post dosing. Thus, gene editing compositions described herein, as supported by the clinical data set forth in the foregoing figures and Table 7, exhibit a clinically significant dose dependent therapeutic effect on lowering LDL-C and PCSK9 and are remarkable well tolerated as evidenced by the ALT, AST, bilirubin and platelet safety data. It should be understood that where a patient starting AST, ALT, or total bilirubin value > ULN at baseline, the baseline value may be used in place of ULN for determining the elevation thresholds set forth above.DETAILED DESCRIPTION

[0019] Certain specific details of this description are set forth to provide a more thorough understanding of various aspects and embodiments. However, one skilled in the art will understand that the present disclosure may be practiced without certain of the details herein disclosed. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments.

[0020] Unless otherwise specified , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice orWSGR Attorney Docket No. 53989-743.601 testing of the present disclosure, suitable methods, and materials are described below. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.Definitions

[0021] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0022] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.

[0023] It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0024] The term “about” or “approximately” can mean within an acceptable error range for the 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 methodology employed, as is typically accepted by the governing regulatory agency. Thus “about” can be characterized by standard deviation (1 or more than 1 standard deviation of a given value, per the practice in the art), range, or percentage or magnitude of a given value, mean within 1 or more than 1 standard deviation, per the practice in the art. The values of RNA dosages administered to human participants that generated the clinical trial data described herein were measured using regulatory agency accepted conventional methods, such as RiboGreen fluorescence RNA quantification, with a qualified precision of 3%.

[0025] As used in this specification and claim(s), 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.

[0026] As used herein, “some embodiments,” “an embodiment,” “one embodiment,” “embodiments” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0027] The term “nucleic acid” as used herein refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form and includes DNA and RNA, hybrids of DNA and RNA, and combinations thereof. The term “nucleic acid” as usedWSGR Attorney Docket No. 53989-743.601 herein also refers to a polymer containing at least two chemically modified nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form and includes DNA and RNA, hybrids of DNA and RNA, and combinations thereof.

[0028] As used herein, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably and refer to a polymer of amino acid residues linked via peptide bonds and which may be composed of two or more polypeptide chains. The terms “polypeptide,” “protein,” and “peptide” refer to a polymer of at least two amino acid monomers joined together through amide bonds. An amino acid may be the L-optical isomer or the D-optical isomer. More specifically, the terms “polypeptide,” “protein,” and “peptide” refer to a molecule composed of two or more amino acids in a specific order; for example, the order as determined by the base sequence of nucleotides in the gene or RNA coding for the protein. Proteins are essential for the structure, function, and regulation of the body’s cells, tissues, and organs, and each protein has unique functions. Examples are hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein can be a portion of the protein, for example, a domain, a subdomain, or a motif of the protein. In some cases, a protein can be a variant (or mutation) of the protein, wherein one or more amino acid residues are inserted into, deleted from, and / or substituted into the naturally occurring (or at least a known) amino acid sequence of the protein. A protein or a variant thereof can be naturally occurring or recombinant. Methods for detection and / or measurement of polypeptides in biological material are well known in the art and include, but are not limited to, Western-blotting, flow cytometry, ELISAs, RIAs, and various proteomics techniques. An exemplary method to measure or detect a polypeptide is an immunoassay, such as an ELISA. This type of protein quantitation can be based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen. Exemplary assays for detection and / or measurement of polypeptides are described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

[0029] The term “sequence identity,” as used herein, refers to the amount of nucleotide or amino acid which match exactly between two different sequences. When comparing RNA and DNA sequences Uracil and Thymine bases are considered to be the same base. Gaps are not counted and the measurement is typically in relation to the shorter of the two sequences.For example, for nucleotide sequences:A: AAGGCTT B: AAGGC C: AAGGCATHere 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). So 100% identity does not mean two sequences are the same.For amino acid sequences:A: Ala Ala Gly Gly Gin His His B: Ala Ala Gly Gly Gin C: Ala Ala Gly Gly Gin Ala HisWSGR Attorney Docket No. 53989-743.601Here identity (A,B)=100% (5 identical amino acids / min(length(A), length(B))). Identity (B,C)=100%, but identity(A,C)=85% ((6 identical amino acids / 7).

[0030] The term “sequence similarity,” as used herein, can be described as an optimal matching problem that finds the minimal number of edit operations (inserts, deletes, and substitutions) in order to transform the one sequence into an exact copy of the other sequence being aligned (edit distance). Using this, the percentage sequence similarity of the examples above are sim(A,B)=60%, sim(B,C)=60%, sim(A,C)=86% (semi-global, sim=l-(edit distance / unaligned length of the shorter sequence)).

[0031] A “subject” in need thereof, refers to an individual who can benefit from the treatment, such as a person who has a disease, a symptom of the disease, or a predisposition toward the disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition toward the disease. In some embodiments, the subject has hypercholesterolemia. In some embodiments, the subject may have elevated LDL or LDL that is not at recommended goal. In some embodiments, the subject may have atherosclerotic vascular disease. In some embodiments, the subject has hypertriglyceridemia. In some embodiments, the subject has diabetes. In some embodiments, the subject may have intolerance to certain lipid lowering therapies, such as statins or other therapies. In some embodiments, the subject may have one or more of the foregoing. The term “subject” or “participant” encompasses mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. The data disclosed herein, specifically, discloses human clinical data and hence the participants are human subjects.

[0032] The term “condition,” as used herein, includes diseases, disorders, and susceptibilities. In some embodiments, the condition is an atherosclerotic vascular disease. In some embodiments, the condition is a hypertriglyceridemia. In some embodiments, the condition is a diabetes.

[0033] The term “atherosclerosis” or “atherosclerotic vascular disease,” as used herein, refers to a disease in which the inside of an artery narrows due to the buildup of plaque. In some instances, it may result in coronary artery disease, stroke, peripheral artery disease, or kidney problems.

[0034] The term “hypertriglyceridemia,” as used herein, refers to high (hyper-) blood levels (-emia) of triglycerides, the most abundant fatty molecule in most organisms. Elevated levels of triglycerides can be associated with atherosclerosis, even in the absence of hypercholesterolemia (high cholesterol levels), and can predispose to cardiovascular disease. Very high triglyceride levels can increase the risk of acute pancreatitis. Hypertriglyceridemia can be associated with overeating, obesity, diabetes mellitus and insulin resistance, excess alcohol consumption, kidney failure, nephrotic syndrome, genetic predisposition (e.g., familial combined hyperlipidemia, i.e., Type II hyperlipidemia), lipoprotein lipase deficiency, lysosomal acid lipase deficiency, cholesteryl ester storage disease, certain medications (e.g., isotretinoin, hydrochlorothiazide diuretics, beta blockers, protease inhibitors), hypothyroidism (underactive thyroid),WSGR Attorney Docket No. 53989-743.601 compositionic lupus erythematosus and associated autoimmune responses, glycogen storage disease type 1, propofol, or HIV medications.

[0035] The term “diabetes,” as used herein, refers to a group of metabolic disorders characterized by a high blood sugar level over a prolonged period of time. Diabetes can be type 1 diabetes that results from the pancreas’s failure to produce enough insulin due to loss of beta cells. Diabetes can be type 2 diabetes characterized by insulin resistance, a condition in which cells fail to respond to insulin properly. Diabetes can be gestational diabetes that occurs when pregnant women without a previous history of diabetes develop high blood sugar levels.

[0036] The term “low-density lipoprotein (LDL),” as used herein, refers to a microscopic blob made up of an outer rim of lipoprotein and a cholesterol center. LDL can have a highly hydrophobic core composed of a polyunsaturated fatty acid known as linoleate and hundreds to thousands esterified and unesterified cholesterol molecules. The core of LDL can also carry triglycerides and other fats and can be surrounded by a shell of phospholipids and unesterified cholesterol.

[0037] The term “high-density lipoprotein (HDL),” as used herein, refers to the smallest lipoprotein particles. Plasma enzyme lecithin-cholesterol acyltransferase (LCAT) can convert the free cholesterol into cholesteryl, which is then sequestered into the core of the lipoprotein particle, eventually causing the newly synthesized HDL to assume a spherical shape. HDL particles can increase in size as they circulate through the bloodstream and incorporate more cholesterol and phospholipid molecules from cells and other lipoproteins.

[0038] The term “cholesterol,” as used herein, refers to a lipid with a unique structure composed of four linked hydrocarbon rings forming the bulky steroid structure. The term “triglyceride,” as used herein, refers to a tri-ester composed of a glycerol bound to three fatty acid molecules. In some embodiments, the fatty acids are saturated or unsaturated fatty acids.

[0039] The terms “treat,” “treating,” or “treatment,” and its grammatical equivalents as used herein, can include alleviating, abating, or ameliorating at least one symptom of a disease or a condition, preventing additional symptoms, inhibiting the disease or the condition, e.g., delaying, decreasing, suppressing, attenuating, diminishing, arresting, or stabilizing the development or progression of a disease or the condition, relieving the disease or the condition, causing regression of the disease or the condition, relieving a condition caused by the disease or the condition, reducing disease severity, or stopping the symptoms of the disease or the condition either prophylactically and / or therapeutically. “Treating” also includes lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to a disease or condition and / or the side effects associated with the disease or condition. “Treating” does not necessarily require curative results. It is appreciated that, although not precluded, treating a disorder or condition also does not require that the disorder, condition, or symptoms associated therewith be completely eliminated. The term “treating” encompasses the concept of “managing” which refers to reducing the severity of a particular disease or disorder in a participant or delaying its recurrence, e.g., lengthening the period of remission in a participant who had suffered from the disease. “Treating”WSGR Attorney Docket No. 53989-743.601 may refer to the application or administration or a composition to a subject after the onset, or suspected onset, of a disease or condition.

[0040] The term “treating” encompasses the concept of “prevent,” “preventing,” and “prevention.” The terms “prevent,” “preventing,” and “prevention,” as used herein, refer to a decrease in the occurrence of pathology of a condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition. The prevention may be complete, e.g., the total absence of pathology of a condition in a subject. The prevention may also be partial, such that the occurrence of pathology of a condition in a subject is less than that which would have occurred without the present disclosure.

[0041] By “treating or preventing a condition,” for example, as compared with an equivalent untreated control, alleviating a symptom of a disorder may involve reduction or degree of prevention at least 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% as measured by any standard technique. In some embodiments, alleviating a symptom of a disorder may involve reduction or degree of prevention by 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 as compared with an equivalent untreated control.

[0042] As used therein, “delaying” the development of a disease means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0043] “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset.

[0044] As used herein “onset” or “occurrence” of a disease includes initial onset and / or recurrence.

[0045] “Administering” and its grammatical equivalents as used herein can refer to providing pharmaceutical compositions described herein to a subject or a participant. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the composition to the subject, depending upon the type of disease to be treated or the site of the disease. For example, the composition can be administered, e.g., orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or via intravenous infusion. One or more such routes can be employed.

[0046] By “co-administering” is meant administering one or more additional therapeutic regimens or agents or treatments and the composition of the disclosure sufficiently close in time to enhance the effectWSGR Attorney Docket No. 53989-743.601 of one or more additional therapeutic agents, or vice versa. In this regard, the composition of the disclosure described herein can be administered simultaneously with one or more additional therapeutic regimens or agents or treatments, at a different time, or on an entirely different therapeutic schedule (e.g., the first treatment can be daily, while the additional treatment is weekly). For example, in embodiments, the secondary therapeutic regimens or agents or treatments are administered simultaneously, prior to, or subsequent to the composition of the disclosure.

[0047] The terms “pharmaceutical composition” and its grammatical equivalents as used herein can refer to a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients, carriers, and / or a therapeutic agent to be administered to a subject, e.g., a human in need thereof. In some embodiments, the dosage in “mg / kg” of the pharmaceutical composition described herein is calculated by dividing the total amount of the RNA by the weight of the human subject who received the dose. In the context of the examples herein mg / kg represents the total amount of drug substances (i.e., the mRNA and guide RNA) in milligrams divided by the weight of the human participant.

[0048] The term “pharmaceutically acceptable” and its grammatical equivalents as used herein can refer to an attribute of a material which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for veterinary as well as human pharmaceutical use. “Pharmaceutically acceptable” can refer a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the pharmaceutical composition in which it is contained.

[0049] A “pharmaceutically acceptable excipient, carrier, or diluent” refers to an excipient, carrier, or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0050] A “pharmaceutically acceptable salt” may be an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication. Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2-hydroxyethyl sulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC- (C hjn-COOH where n is 0-4, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize from this disclosure and the knowledge in the art that further pharmaceuticallyWSGR Attorney Docket No. 53989-743.601 acceptable salts include those listed by Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985). In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent.

[0051] The term “clinically supported” (used independently or to modify the terms “safe” and / or “effective”) shall mean that initial data generated from a human clinical trial authorized by U.S. Food and Drug Administration, MHRA, Medsafe New Zealand, EMEA or other national regulatory agency, wherein such human clinical trial data supports that the subject drug product is effective and safe as it relates to composition, dose, dosage regimen, treatment or method of use of the pharmaceutical composition disclosed herein. As used herein, “clinically supported safe” can be shown by measuring levels of one or more liver biomarkers (ALT, AST, ALP, and bilirubin) and platelet counts alone and in combination and comparing them to acceptable norms or levels. Thus, demonstrating that the levels of a biomarker are within the ULN and LLN or within regulatory acceptable levels (e.g., mild and / or moderate variations relative thereto), or do not trigger Hy’s law, at a desired or selected therapeutically effective dose) are ways to characterize or define clinically supported safety. As used herein, clinically supported safe can also be shown as a change in biosafety metric level, wherein the resulting change does not constitute a safety event (e.g., an adverse event (NCI-CTCAE Grade 3 or higher), a serious adverse event, an adverse event of special interest, a treatment-emergent adverse event (CTCAE Grade 3 or higher), and / or an event that otherwise requires discontinuation of the study drug as determined by a clinician. As used herein, “clinically supported effective” can be shown by reductions (e.g., amount of reduction and / or percentage of reduction relative to baseline) of human subjects’ blood PSCK9 protein level and / or blood LDL-C level.

[0052] The term “therapeutic agent” can refer to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. Therapeutic agents can also be referred to as “actives” or “active agents.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids. The mRNA and gRNA of the drug product dosed in this application are considered therapeutic agents or drug substances by the regulatory authorities.

[0053] The term “protospacer,” or “target sequence” and their grammatical equivalents as used herein can refer to a DNA sequence of a target gene. In the native state, a protospacer is adjacent to a PAM (protospacer adjacent motif). The editing site (for the base editors and guide RNAs described in the examples herein) is located within a protospacer sequence.

[0054] The term “base editing,” “gene editing,” “genome editing,” or “gene modification” and its grammatical equivalents as used herein can refer to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome. Gene editing can be performed using a nuclease (e.g. , a natural-existing nuclease or an artificially engineered nuclease). Gene modification can includeWSGR Attorney Docket No. 53989-743.601 introducing a double stranded break, a non-sense mutation, a frameshift mutation, a splice site alteration, or an inversion in a polynucleotide sequence, e.g., a target polynucleotide sequence.

[0055] The term “base editing” as used herein can refer to an agent that binds a polynucleotide and has nucleobase modifying activity. The base editing can comprise a nucleobase modifying polypeptide (e.g., a deaminase) and a nucleic acid programmable nucleotide binding domain or protein in conjunction with a guide polynucleotide (e.g., guide RNA), or nucleic acids encoding the programmable nucleotide binding domain or protein and the deaminase. The agent can be a biomolecular complex comprising a protein domain having base editing activity, i.e., a domain capable of modifying a base (e.g., A, T, C, G, or U) within a nucleic acid molecule (e.g., DNA), or a nucleic acid encoding the same. The polynucleotide programmable DNA binding domain or protein can be fused or linked to a deaminase domain, resulting in a base editing protein. The base editing can comprise a nucleic acid encoding the base editing protein, e.g., an RNA encoding the base editing protein. The base editing protein can comprise one or more linkers, for example, peptide linkers. The agent can be a protein comprising a domain having base editing activity. The protein domain having base editing activity can be linked to the guide RNA (e.g., via an RNA binding motif on the guide RNA and an RNA binding domain fused to the deaminase). In some instances, the domain having base editing activity is capable of deaminating a base within a nucleic acid molecule. In some instances, the base editing is capable of deaminating one or more bases within a DNA molecule. In some instances, the base editing is capable of deaminating an adenosine (A) within DNA. The base editing can be an adenosine base editing (ABE). In some instances, the base editing is capable of deaminating a cytosine (C) within DNA. The base editing can be a cytosine base editing (CBE).

[0056] The term “base editing composition” refers to a gene editing composition for editing a single nucleobase of a target nucleotide sequence. In various embodiments, the base editing composition comprises (1) a polynucleotide programmable nucleotide binding domain or protein (e.g., Cas9); (2) a deaminase domain (e.g., an adenosine deaminase or a cytidine deaminase) for deaminating said nucleobase; and (3) one or more guide polynucleotide (e.g., guide RNA). In some embodiments, the base editing composition comprises a base editing protein comprising (1) and (2). In some embodiments, like those used to dose human participants from which the clinical trial data disclosed herein was observed, the base editing composition comprises an mRNA encoding the based editing protein comprising (1) and (2). In some embodiments, the polynucleotide programmable nucleotide binding domain or protein is a polynucleotide programmable DNA binding domain or protein. In some embodiments, the base editing is an adenine or adenosine base editing (ABE). In some embodiments, the base editing is a cytosine base editing (CBE).

[0057] The term “CRISPR RNA” (crRNA) herein refers to an RNA sequence that can form a complex with one or more Cas proteins (e.g., Cas9) and provides DNA binding specificity to the complex. A crRNA provides DNA binding specificity since it contains a “spacer sequence” that is complementary to a strand of a DNA target sequence. A crRNA further comprises a “repeat sequence” (“tracr RNA mate sequence”) encoded by a repeat region of the CRISPR locus from which the crRNA was derived. A repeat sequence of a crRNA can anneal to sequence at the 5 '-end of atracrRNA. crRNA in native CRISPRWSGR Attorney Docket No. 53989-743.601 compositions is derived from a “pre-crRNA” transcribed from a CRISPR locus. A pre-crRNA comprises spacer regions and repeat regions; spacer regions contain unique sequence complementary to a DNA target site sequence. Pre-crRNA in native compositions is processed to multiple different crRNAs, each with a guide sequence along with a portion of repeat sequence. CRISPR compositions utilize crRNA, for example, for DNA targeting specificity.

[0058] The term “trans-activating CRISPR RNA” (tracrRNA) herein refers to a non-coding RNA used in type II CRISPR compositions, and contains, in the 5'-to-3' direction, (i) a sequence that anneals with the repeat region of CRISPR type II crRNA and (ii) a stem loop-containing portion (Deltcheva et al., Nature 471:602-607). A modified tracrRNA refers to a tracrRNA with modified ribonucleotide (e.g., 2-OMe modified RNA).

[0059] As used herein, the term “guide nucleic acid”, relates to a polynucleotide sequence that can form a complex with a Cas endonuclease and enables the Cas endonuclease to recognize and optionally cleave a DNA target site. The guide nucleic acid can be comprised of a single molecule (like the ones set forth in the specific embodiments disclosed herein) or be comprised of plurality of molecules as described above. When comprised of a single molecule the guide nucleic acid or guide RNA extends from a 5 ’ end region comprising a spacer region to a 3’ end comprising atracr region that serves as a scaffold for the Cas protein. . The guide nucleic acid sequence can be RNA only (gRNA) or in some cases may be a hybrid of RNA and DNA molecules, such as in specific locations in the spacer region of the guide RNA. The guide nucleic acid may be modified or unmodified. Optionally, the guide nucleic acid can comprise at least one nucleotide, phosphodiester bond or linkage modification such as, but not limited, to Locked Nucleic Acid (LNA), 5-methyl dC, 2,6-Diaminopurine, 2'-Fluoro A, 2'-Fluoro U, 2'-O-Methyl RNA, Phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 (hexaethylene glycol chain) molecule, or 5' to 3' covalent linkage resulting in circularization.

[0060] As used herein, a spacer sequence that corresponds to a protospacer is capable of facilitating a base editor protein to make a modification to a base within the complimentary strand of the target protospacer nucleic acid sequence. A spacer sequence that corresponds to a protospacer sequence may be identical or substantially identical to the protospacer sequence.

[0061] As used herein, “corresponding” is intended to encompass the concept that two sequences may not be identical (i.e., may have one or more mis-matches), yet may effectuate the operable binding or hybridization desired to effectuate the desired edit. When determining the identicality of the two sequences, uracil is considered the same as thymine. For example, a spacer sequence corresponds to a protospacer sequence when the spacer sequence is sufficiently identical to the protospacer so as to operably bind to the strand complimentary to the protospacer sequence. In some embodiments, the spacer sequence is identical to the protospacer sequence. In some embodiments, the spacer sequence may be the same length, longer, or shorter than the protospacer sequence and may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mis-matches with the protospacer sequence.Pharmaceutical Compositions and DosageWSGR Attorney Docket No. 53989-743.601

[0062] In some aspects, provided herein is a pharmaceutical composition comprising a RNA (e.g., mRNA) encoding a base editor protein, a guide polynucleotide (e.g., gRNA) including a spacer that bind to proprotein convertase subtilisin-kexin type 9 (PCSK9) and LNP excipients. The subject that can be treated with pharmaceutical composition described herein based on methods described herein is a human subject.

[0063] The human subject can be prenatal (e.g., a fetus), a child (e.g., a neonate, an infant, a toddler, a preadolescent), an adolescent, a pubescent, or an adult (e.g., an early adult, a middle-aged adult, a senior citizen). The human subject can be between about 0 month and about 120 years old, or older. The human subject can be between about 0 and about 12 months old; for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months old. The human subject can be between about 0 and 12 years old; for example, between about 0 and 30 days old; between about 1 month and 12 months old; between about 1 year and 3 years old; between about 4 years and 5 years old; between about 4 years and 12 years old; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years old. The human subject can be between about 13 years and 19 years old; for example, about 13, 14, 15, 16, 17, 18, or 19 years old. The human subject can be between about 20 and about 39 years old; for example, about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 years old. The human subject can be between about 40 to about 59 years old; for example, about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 years old. The human subject can be greater than 59 years old; for example, about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 years old. The human subjects can include male subjects and / or female subjects.

[0064] In some embodiments, the dosage of the guide RNA and the mRNA administered is a fixed dose. In some embodiments, the fixed dose is at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 55 mg, at least about 60 mg, at least about 65 mg, at least about 70 mg, at least about 75 mg, at least about 80 mg, at least about 85 mg, at least about 90 mg, at least about 95 mg, at least about 100 mg. In some embodiments, the fixed dose is about 15 mg to about 45 mg. In some embodiments, the fixed dose is about 17.8mg, about 18.7mg, about 21. Img, about 23.1mg, about 26.6mg, about 28.4mg, about 41.5mg, about 42mg, about 45. Img, about 38.6mg, about 55.4mg, about 39.8mg, about 57.6mg or about 51.1 mg. In some embodiments, the fixed dose is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, lOOmg or more, or a fixed dosed amount therebetween (e.g. ,26,27, 28, 29, 31, 32, 33, 34, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 51, 52, 53, 54. 56, 57, 58, 59, 61,62, 63, 64, 66, 67, 68, 69, 71, 72, 73, 74, 77, 78, 79, 81, 82, 83, 84, 86, 87, 88, 89, 91, 92, 93, 94, 96, 97,98. 99 mg of total gRNA and mRNA). In some embodiments, the fixed dose may be between about 40-80 mg, about 50-60 mg, about 50-70 mg, about 50-80 mg, about 60-80 mg, about 60-70 mg, about 70-80 mg, about 70-90 mg, about 70-100 mg, about 80-90 mg, about 80-100 mg, about 90-100 mg. In some embodiments, the fixed dose may be about 40 mg + / - 5 mg, about 50 mg + / - 5 mg, about 60 mg + / - 5 mg, about 70 mg + / - 5 mg, about 80 mg + / - 5 mg, about 90 mg + / - 5 mg, about 100 mg + / - 5 mg,WSGR Attorney Docket No. 53989-743.601

[0065] In some embodiments, the dosage of the guide RNA and the mRNA administered is about 0.3 mg / kg. In some embodiments, the dosage of the guide RNA and the mRNA administered at about 0.45 mg / kg. In some embodiments, the dosage of the guide RNA and the mRNA administered is at about 0.6 mg / kg. In some embodiments, the dosage of the guide RNA and the mRNA administered is at about 0.01 mg / kg to about 5 mg / kg, about 0.02 mg / kg to about 5 mg / kg, about 0.03 mg / kg to about 5 mg / kg, about 0.04 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.06 mg / kg to about 5 mg / kg, about 0.07 mg / kg to about 5 mg / kg, about 0.08 mg / kg to about 5 mg / kg, about 0.09 mg / kg to about 5 mg / kg, about 0. 1 mg / kg to about 5 mg / kg, about 0.2 mg / kg to about 5 mg / kg, about 0.3 mg / kg to about 5 mg / kg, about 0.4 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.6 mg / kg to about 5 mg / kg, about 0.7 mg / kg to about 5 mg / kg, about 0.8 mg / kg to about 5 mg / kg, about 0.9 mg / kg to about 5 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1.5 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 2.5 mg / kg to about 5 mg / kg, about 3 mg / kg to about 5 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 5 mg / kg, or about 4.5 mg / kg to about 5 mg / kg.

[0066] In some embodiments, the dosage of the guide RNA and the mRNA administered is about 0.45- 1.00 mg / kg. In some embodiments, the dosage of the guide RNA and the RNA administered is about 0.50-0.60, 0.50-0.70, 0.50-0.80, 0.50-0.90, 0.55-0.60, 0.55-0.70, 0.55-0.80, 0.55-0.90, 0.60-0.70, 0.60- 0.80, 0.60-0.90, 0.65-0.70, 0.65-0.80, 0.65-0.90, 0.70-0.80, 0.70-0.90, 0.75-0.80, 0.75-0.90, 0.80-0.90, 0.85-0.90, 0.50-0.65, 0.50-0.75, 0.50-0.85, 0.50-0.95, 0.55-0.65, 0.55-0.75, 0.55-0.85, 0.55-0.95, 0.60- 0.75, 0.60-0.85, 0.60-0.95, 0.65-0.75, 0.65-0.85, 0.65-0.95, 0.70-0.85, 0.70-0.95, 0.75-0.85, 0.75-0.95, 0.80-0.95, or 0.85-0.95 mg / kg. In some embodiments, the dosage of the guide RNA and the mRNA administered is about 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00 mg / kg. In some embodiments, the dosage of the guide RNA and the mRNA administered is about 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00 mg / kg.Method of Treatment

[0067] The present disclosure further provides a method for treating or preventing an atherosclerotic cardiovascular disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the guide nucleic acid, gene editing composition, or the pharmaceutical composition disclosed herein. The subject in need thereof can be a subject that needs gene editing in PCSK9 gene. The gene editing composition disclosed herein can achieve the editing in PCSK9 gene in a subject with high on-target efficiency but low off-target effect. The guide nucleic acid can direct the gene editing protein to affect a nucleobase alternation in a PCSK9 gene in the subject.

[0068] Described herein are methods for reducing proprotein convertase subtilisin-kexin type 9 (PCSK9) level in a human subject to effectuate reduction in low-density lipoprotein cholesterol (LDL-C) level in the human subject, comprising: administering a pharmaceutical composition to the human subject comprising: (a) an RNA encoding a base editing protein; (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodesWSGR Attorney Docket No. 53989-743.601PCSK9, and wherein uracil is considered the same as thymine; and (c) a lipid nanoparticle (LNP), wherein the LNP comprises between about 43-52 mol% of an amino lipid, between about 2.9-3.2 mol% of a PEG- lipid, between about 0.03-0.07 mol% of a N-acetylgalactosamine (GalNAc) lipid, between about 35.9- 45.0 mol% of a sterol, and between about 8.7-9. 1 mol% of a phospholipid; wherein a total amount of the RNA and the guide polynucleotide is at least about 0.3 mg / kg.

[0069] Described herein are methods for reducing PCSK9 level in a human subject to effectuate reduction in low-density lipoprotein cholesterol (LDL-C) level in the human subject, comprising: administering a pharmaceutical composition to the human subject comprising: (a) an RNA encoding a base editing protein; (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine; and (c) a lipid nanoparticle (LNP), wherein the LNP comprises between about 43-52 mol% of an amino lipid, between about 2.9-3.2 mol% of a PEG-lipid, between about 0.03-0.07 mol% of a N-acetylgalactosamine (GalNAc) lipid, between about 35.9-45.0 mol% of a sterol, and between about 8.7-9.1 mol% of a phospholipid; wherein a total amount of the RNA and the guide polynucleotide is at least about 10 mg.

[0070] Described herein are methods for lowering low-density lipoprotein cholesterol (LDL-C) level in a human subject, comprising: administering a pharmaceutical composition in the human subject comprising: (a) an RNA encoding a base editing protein; (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on a gene that encodes PCSK9, and wherein uracil is considered the same as thymine; and (c) a lipid nanoparticle (LNP), wherein the LNP comprises between about 43-52 mol% of an amino lipid, between about 2.9-3.2 mol% of a PEG-lipid, between about 0.03-0.07 mol% of a N-acetylgalactosamine (GalNAc) lipid, between about 35.9-45.0 mol% of a sterol, and between about 8.7-9.1 mol% of a phospholipid; wherein a total amount of the RNA and the guide polynucleotide is at least about 0.3 mg / kg.

[0071] Described herein are methods for lowering low-density lipoprotein cholesterol (LDL-C) level in a human subject, comprising: administering a pharmaceutical composition in the human subject comprising: (a) an RNA encoding a base editing protein; (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on a gene that encodes PCSK9, and wherein uracil is considered the same as thymine; and (c) a lipid nanoparticle (LNP), wherein the LNP comprises between about 43-52 mol% of an amino lipid, between about 2.9-3.2 mol% of a PEG-lipid, between about 0.03-0.07 mol% of a N-acetylgalactosamine (GalNAc) lipid, between about 35.9-45.0 mol% of a sterol, and between about 8.7-9.1 mol% of a phospholipid; wherein a total amount of the RNA and the guide polynucleotide is at least about 10 mg.

[0072] Described herein are methods for treating or preventing a cardiovascular disease in a human subject, comprising: administering a pharmaceutical composition in the human subject comprising: (a) anWSGR Attorney Docket No. 53989-743.601RNA encoding a base editing protein; (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on a gene that encodes PCSK9, and wherein uracil is considered the same as thymine; and (c) a lipid nanoparticle (LNP), wherein the LNP comprises between about 43-52 mol% of an amino lipid, between about 2.9-3.2 mol% of a PEG-lipid, between about 0.03-0.07 mol% of a N-acetylgalactosamine (GalNAc) lipid, between about 35.9-45.0 mol% of a sterol, and between about 8.7-9.1 mol% of a phospholipid; wherein a total amount of the RNA and the guide polynucleotide is at least about 0.3 mg / kg.

[0073] Described herein are methods for treating or preventing a cardiovascular disease in a human subject, comprising: administering a pharmaceutical composition in the human subject comprising: an RNA encoding a base editing protein; a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on a gene that encodes PCSK9, and wherein uracil is considered the same as thymine; and a lipid nanoparticle (LNP), wherein the LNP comprises between about 43-52 mol% of an amino lipid, between about 2.9-3.2 mol% of a PEG-lipid, between about 0.03-0.07 mol% of a N-acetylgalactosamine (GalNAc) lipid, between about 35.9-45.0 mol% of a sterol, and between about 8.7-9.1 mol% of a phospholipid; wherein a total amount of the RNA and the guide polynucleotide is at least about 10 mg.

[0074] Described herein are methods comprising: administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising: (a) an RNA encoding a base editor protein, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3 - [4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)- 9,12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and wherein the pharmaceutical composition comprises a first dose of at least about 0.3 mg / kg of the RNA and the guide polynucleotide.

[0075] In some embodiments, the pharmaceutical composition comprises a first dose of at least about 0.45 mg / kg of the RNA and the guide polynucleotide. In some embodiments, the pharmaceutical composition comprises a first dose of at least about 0.6 mg / kg of the RNA and the guide polynucleotide. In some embodiments, the pharmaceutical composition comprises a first dose of about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2 mg / kg of the RNA and the guide polynucleotide. In some embodiments, the human subject in need thereof is administered a second dose of the pharmaceutical composition, and wherein the second dose is higher, the same or lower than the first dose. In some embodiments, the second dose is administered one to six days after the first dose.WSGR Attorney Docket No. 53989-743.601

[0076] Described herein are methods comprising: administering a pharmaceutical composition to a group of human subjects in need thereof having different body weights, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising: (a) an RNA encoding a base editor protein, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)-9,12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000, or c. a N- acetylgalactosamine (GalNAc) lipid; and wherein the pharmaceutical composition comprises a first fixed dose of the RNA and the guide polynucleotide.

[0077] In some embodiments, the first fixed dose is lower than about 25 mg. In some embodiments, the first fixed dose is about 25 mg to about 50 mg. In some embodiments, the first fixed dose is about 50 mg to about 60mg. In some embodiments, the first fixed dose is about 10 mg to about lOOmg, about 20 mg to about 90mg, about 30 mg to about 80mg, about 40 mg to about 70mg, or about 50 mg to about 60mg. In some embodiments, the first fixed dose is about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, or about lOOmg. In some embodiments, the first fixed dose is about 20 mg, about 25 mg, about 30 mg, about 36 mg, about 37 mg, about 42 mg, about 48 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg or about 80 mg.

[0078] In some embodiments, the first fixed dose is at least about 60 mg. In some embodiments, the first fixed dose is about 75, about 80, about 85, about 90, about 95 or about 100 mg. In some embodiments, the group of human subjects are administered a second fixed dose of the pharmaceutical composition, and wherein the second fixed dose is higher, the same or lower than the first fixed dose. In some embodiments, the group of human subjects that were administered a first dose are not administered a second fixed dose of the pharmaceutical composition. In some embodiments, the second fixed dose is administered after the first fixed dose by a period of time, and wherein the period of time may range fromWSGR Attorney Docket No. 53989-743.6011 day, 1 week, 1 month, 2 months, 3 months, 6 months, 9 months to over a year after the administration of the first fixed dose. Administration of a second fixed dose may be dependent on the efficacy and safety of the pharmaceutical composition on the human subject.

[0079] Described herein are methods comprising: administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising: (a) an RNA encoding a base editor protein, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3- [4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)- 9,12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. aN-acetylgalactosamine (GalNAc) lipid; and wherein the human subject in need thereof, before administration of the pharmaceutical composition, has received a prior medication comprising a steroid or an antihistamine.

[0080] In some embodiments, the guide RNA comprises a spacer having a nucleotide base sequence that is identical to protospacer on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine. In some embodiments, the guide RNA comprises a spacer having a nucleotide base sequence that is identical to protospacer on the gene that encodes PCSK9 except that it has 1, 2 3 or 4 nucleotide base mismatches, and wherein uracil is considered the same as thymine.

[0081] In some embodiments, the prior medication comprises a steroid and an antihistamine. In some embodiments, the steroid comprises dexamethasone. In some embodiments, the antihistamine comprises a Hl blocker or a H2 blocker. In some embodiments, the Hl blocker comprises diphenhydramine. In some embodiments, the H2 blocker comprises famotidine.

[0082] Described herein are methods comprising: administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising: (a) an RNA encoding a base editor protein, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3 - [4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)- 9,12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. aN-acetylgalactosamine (GalNAc) lipid; and wherein the human subject in need thereof, after administration of the pharmaceutical composition, receives a steroid. In some embodiments, the steroid comprises dexamethasone.

[0083] Described herein are methods comprising: administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticleWSGR Attorney Docket No. 53989-743.601(LNP) comprising: (a) an RNA encoding a base editor protein, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3- [4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)-9,12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. aN-acetylgalactosamine (GalNAc) lipid; and wherein the human subject in need thereof 1) had atherosclerotic cardiovascular disease (ASCVD), 2) had heterozygous familial hypercholesterolemia (HeFH) with a low-density lipoprotein-cholesterol (LDL-C) level of at least about 190 mg / dL, 3) had an LDL-C level above a standard LDL-C level, or 4) cannot tolerate a lipid lowering medication.

[0084] In some embodiments, the standard LDL-C level is about 160 mg / dL, about 140 mg / dL, about 130 mg / dL or about 100 mg / dL. In some embodiments, the lipid lowering medication comprises a statin, ezetimibe, a bile acid sequestrant, a fibrate, or niacin.

[0085] Described herein are methods comprising: administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising: (a) an RNA encoding a base editor protein, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3- [4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)-9,12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. aN-acetylgalactosamine (GalNAc) lipid; and wherein the human subject in need thereof has received a coronary revascularization, a coronary artery bypass grafting, a percutaneous coronary intervention or a lipid-lowering therapy that is not a PCSK9 inhibitor. In some embodiments, the lipid-lowering medication comprises a statin, ezetimibe, a bile acid sequestrant, a fibrate, or niacin.

[0086] Described herein are methods comprising: administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising: (a) an RNA encoding a base editor protein, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3 - [4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)-9,12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3-WSGR Attorney Docket No. 53989-743.601 methoxypolyethylene glycol-2000, or c. aN-acetylgalactosamine (GalNAc) lipid; and wherein the human subject in need thereof has at least one risk factor selected from the group consisting of: 1) atherosclerotic cardiovascular disease (ASCVD), 2) heterozygous familial hypercholesterolemia (HeFH) with a low- density lipoprotein-cholesterol (LDL-C) level of at least about 190 mg / dL, or 3) an LDL-C level above a standard LDL-C level. In some embodiments, the standard LDL-C level is about 160 mg / dL, about 140 mg / dL, about 130 mg / dL or about 100 mg / dL. In some embodiments, the ASCVD is represented by 1) a prior treatment comprising revascularization, coronary artery bypass grafting, or percutaneous coronary intervention, or 2) a prior condition comprising myocardial infarction, cardiac arrest, or stroke.

[0087] Described herein are methods comprising: administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising: (a) an RNA encoding a base editor protein, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3 - [4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)- 9,12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and wherein after administration of the pharmaceutical composition, i. a level of PCSK9 protein in the human subject in need thereof is reduced by at least 40% as compared to the level of PCSK9 protein prior to the administration, or ii. a level of LDL-C in the human subject in need thereof is reduced by at least 20% as compared to the level of LDL-C prior to the administration.

[0088] In some embodiments, the after administration of the pharmaceutical composition, the level of PCSK9 protein in the human subject in need thereof is reduced by at least 50% as compared to the level of PCSK9 protein prior to the administration. In some embodiments, the after administration of the pharmaceutical composition, the level of PCSK9 protein in the human subject in need thereof is reduced by at least 60% as compared to the level of PCSK9 protein prior to the administration. In some embodiments, the level of PCSK9 protein is reduced for at least 2 weeks after administration. In some embodiments, the level of PCSK9 protein is reduced for at least 4 weeks after administration. In some embodiments, the level of PCSK9 protein is measured in blood. In some embodiments, the after administration of the pharmaceutical composition, the level of LDL-C in the human subject in need thereof is reduced by at least 40% as compared to the level of LDL-C prior to the administration. In some embodiments, the after administration of the pharmaceutical composition, the level of LDL-C in the human subject in need thereof is reduced by at least 50% as compared to the level of LDL-C prior to the administration. In some embodiments, the level of LDL-C is reduced for at least 2 weeks after administration. In some embodiments, the level of LDL-C is reduced for at least 4 weeks after administration. In some embodiments, the level of LDL-C is measured in blood.WSGR Attorney Docket No. 53989-743.601

[0089] Described herein are methods comprising: administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising: (a) an RNA encoding a base editor protein, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3- [4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)- 9,12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. aN-acetylgalactosamine (GalNAc) lipid; and wherein after administration of the pharmaceutical composition, the human subject in need thereof i. has an alanine aminotransferase (ALT) level between 10-50 IU / L, ii. has an aspartate aminotransferase (AST) level between 10-40 IU / L, iii. has a bilirubin level between 0-1 mg / dL, or iv. has a platelet count between 150- 400xl0A9 / L.

[0090] Described herein are methods comprising: administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising: (a) an RNA encoding a base editor protein, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3 - [4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)- 9,12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. aN-acetylgalactosamine (GalNAc) lipid; and wherein after administration of the pharmaceutical composition, a level of a liver biomarker indicating the human subject’s liver function is lower than an upper limit of normal (ULN) or higher than a lower limit of normal (LLN). In some embodiments, the level of the liver biomarker indicating the human subject’s liver function is measured at least 28 days after administration of the pharmaceutical composition. In some embodiments, the liver biomarker comprises platelet count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), or bilirubin. In some embodiments, liver biomarkers include platelet count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, alkaline phosphatase (ALP), Gamma-Glutamyl Transferase (GGT), International Normalized Ratio (INR), prolonged prothrombin time (PT), albumin or lactate dehydrogenase (LDH).

[0091] In some embodiments, a ULN for platelet count is about 400x10A9 / L, and a LLN for platelet count is about 150xl0A9 / L. In some embodiments, a ULN for ALT is about 50 IU / L. In some embodiments, a ULN for AST is about 40 IU / L. In some embodiments, a ULN for bilirubin is about 1 mg / dL.WSGR Attorney Docket No. 53989-743.601

[0092] Described herein are methods comprising: 1) administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising: (a) an RNA encoding a base editor protein, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3- [4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z,12Z)- 9,12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. aN-acetylgalactosamine (GalNAc) lipid; and 2) measuring a level of a liver biomarker indicating the human subject’s liver function.

[0093] In some embodiments, the liver biomarker comprises platelet count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), or bilirubin.

[0094] Described herein are methods comprising: administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a clinically supported safe and clinically supported effective amount of a lipid nanoparticle (LNP), wherein the LNP comprises: (a) an RNA encoding a base editor protein, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2, (b) a guide polynucleotide, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1, and (c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a PEG lipid, aN-acetylgalactosamine (GalNAc) lipid, a sterol and a phospholipid; and wherein after administration, the human subject in need thereof demonstrates: (i) reduction of a level of PCSK9 protein by at least 40% as compared to a level of PCSK9 protein prior to the administration, or (ii) reduction of a level of LDL-C by at least 20% as compared to the level of LDL-C prior to the administration.

[0095] In some embodiments, after administration, the human subject in need thereof: (i) has an alanine aminotransferase (ALT) level between 10-50 IU / L, (ii) has an aspartate aminotransferase (AST) level between 10-40 IU / L, (iii) has a bilirubin level between 0-1 mg / dL, or (iv) has a platelet count between 150-400xl0A9 / L.

[0096] In some embodiments, the human subject has atherosclerotic cardiovascular disease, accelerated atherosclerotic cardiovascular disease, premature coronary artery disease, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, coronary heart disease, carotid artery disease, peripheral artery disease, microvascular disease, stroke, heart attack, sudden cardiac arrest, or an LDL-C level above a standard LDL-C level. In some embodiments, the standard LDL-C level is about 160 mg / dL, about 140 mg / dL, about 130 mg / dL or about 100 mg / dL. In some embodiments, the human subject has familial hypercholesterolemia (FH). In some embodiments, the human subject has heterozygous familial hypercholesterolemia (HeFH). In some embodiments, the human subject has severe HeFH. In some embodiments, the human subject has an LDL-C level of at least about 190 mg / dL. In some embodiments, the human subject has premature coronary artery disease. In some embodiments, the human subjectWSGR Attorney Docket No. 53989-743.601 cannot tolerate a lipid-lowering medication. In some embodiments, the lipid-lowering medication comprises a statin, ezetimibe, a bile acid sequestrant, a fibrate, or niacin. In some embodiments, (a) the amino lipid is present at about 39 to about 59 mol% of the total LNP excipients, the PEG-lipid is present at about 1.9 to about 4.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.02 to about 0.1 mol% of the total LNP excipients, the sterol is present at about 27.3 to about 50.2 mol% of the total LNP excipients, and the phospholipid is present at about 5.9 to about 13.4 mol% of the total LNP excipients, (b) the amino lipid is present at about 50 ± 12 mol% of the total LNP excipients, the PEG-lipid is present at about 3 ± 1.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.05 ± 0.04 mol% of the total LNP excipients, the sterol is present at about 37.95 ± 12.3 mol% of the total LNP excipients, and the phospholipid is present at about 9 ± 4.4 mol% of the total LNP excipients, or (c) the amino lipid is present at about 9.4 to about 26.3 mg / mL of the pharmaceutical composition, the PEG-lipid is present at about 2.1 to about 4. mg / mL of the pharmaceutical composition, the GalNAc lipid is present at about 0.03 to about 0. 14 mg / mL of the pharmaceutical composition, the sterol is present at about 4.5 to about 8.2 mg / mL of the pharmaceutical composition, and the phospholipid is present at about 1.8 to about 4.3 mg / mL of the pharmaceutical composition.

[0097] In some embodiments, the human subject has a body weight of at least 40kg, at least 0kg, at least 60kg, at least 70kg, at least 80kg, at least 90kg, at least 100kg, at least 120 kg, at least 140kg, or at least 160kg. In some embodiments, the human subject has a body weight of at most 160kg, at most 140 kg, at most 120 kg, at most 100kg, at most 90kg, at most 80 kg, at most 70kg, or at most 60kg. In some embodiments, the human subject has a body weight of about 40kg to about 200 kg. In some embodiments, the human subject has a body weight of about 50kg to about 150 kg. In some embodiments, the human subject has a body weight of about 50kg to about 120 kg. In some embodiments, the human subject has a body weight of about 60kg to about 100 kg.

[0098] In some embodiments, the ratio of the guide nucleotide and the RNA is about 1 : 1 by weight.

[0099] In some embodiments, the guide RNA comprises at least one modification. In some embodiments, the guide RNA comprises a sequence of 5'- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcG UUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu-3'. In some embodiments, the guide RNA comprises a sequence of 5'- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAA cuuGaaaaagugGcaccgagucggugcusususu -3'. In some embodiments, the guide RNA comprises a sequence of 5’-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGU UAucAAcuugaaaaagugGcaccgagucggugcusususu-3 ’ .

[0100] In some embodiments, the spacer sequence comprises a nucleotide base sequence having at least 80% sequence identity to CCCGCACCUUGGCGCAGCGG (SEQ ID No: 101), GGUGCUAGCCUUGCGUUCCG (SEQ ID NO: 102), or UUGGAAAGACGGAGGCAGCC (SEQ ID NO: 103), wherein the uppercase A, G, and C represent adenine, guanine, and cytosine, respectively, and wherein the uppercase U represents uracil or thymine.WSGR Attorney Docket No. 53989-743.601

[0101] In some embodiments, the pharmaceutical composition disclosed herein is administered only once to the human subject during treatment. In some embodiments, the pharmaceutical composition is administered to the human subject in need thereof via intravenous infusion. In some embodiments, the human subject has a mutation on an LDLR gene. In some embodiments, the LDLR gene has a splice site mutation or a mutation in an exon. In some embodiments, the human subject has atherosclerotic cardiovascular disease, accelerated atherosclerotic cardiovascular disease, premature coronary artery disease, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, coronary heart disease, carotid artery disease, peripheral artery disease, microvascular disease, stroke, heart attack, sudden cardiac arrest, or an LDL-C level above a standard LDL-C level. In some embodiments, the standard LDL-C level is about 160 mg / dL, about 140 mg / dL, about 130 mg / dL or about 100 mg / dL. In some embodiments, the human subject has familial hypercholesterolemia (FH). In some embodiments, the human subject has heterozygous familial hypercholesterolemia (HeFH). In some embodiments, the human subject has severe HeFH. In some embodiments, the human subject has an LDL-C level of at least about 190 mg / dL. In some embodiments, the human subject has premature coronary artery disease. In some embodiments, the human subject cannot tolerate a lipid-lowering medication. In some embodiments, the lipid-lowering medication comprises a statin, ezetimibe, a bile acid sequestrant, a fibrate, or niacin. In some embodiments, a PCSK9 gene is inactivated by the pharmaceutical composition. In some embodiments, the pharmaceutical composition is designed to be taken up by liver cells of the human subject in need thereof.

[0102] In some embodiments, the human subject or participant, before the administration of the pharmaceutical composition disclosed herein, has a low-density lipoprotein-cholesterol (LDL-C) level at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mg / dL. In some embodiments, the LDL-C level is between 61 mg / dL and 240 mg / dL. In some embodiments, the LDL-C level is at least 130 mg / dL. In some embodiments, the LDL-C level is at least 130 mg / dL. In some embodiments, the LDL-C level is at least 140 mg / dL. In some embodiments, the LDL-C level is at least 150 mg / dL. In some embodiments, the LDL-C level is at least 160 mg / dL. In some embodiments, the LDL-C level is at least 170 mg / dL. In some embodiments, the LDL-C level is at least 180 mg / dL. In some embodiments, the LDL-C level is at least 190 mg / dL. In some embodiments, the LDL-C level is at least 200 mg / dL. In some embodiments, the LDL-C level is at least 210 mg / dL. In some embodiments, the LDL-C level is at least 220 mg / dL. In some embodiments, the LDL-C level is at least 230 mg / dL. In some embodiments, the LDL-C level is at least 240 mg / dL. In some embodiments, the LDL-C level is at least 250 mg / dL. In some embodiments, the LDL-C level is at least 260 mg / dL. In some embodiments, the LDL-C level is at least 270 mg / dL. In some embodiments, the LDL-C level is at least 280 mg / dL. In some embodiments, the LDL-C level is at least 290 mg / dL. In some embodiments, the LDL-C level is at least 300 mg / dL. In some embodiments, the LDL-C level is at about 150-350 mg / dL. In some embodiments, the LDL-C level is at about 160-350 mg / dL. In some embodiments, the LDL-C level is at about 170-350 mg / dL. In some embodiments, the LDL-C level is at about 180-350 mg / dL. In some embodiments, the LDL-C level is at about 190-350 mg / dL. In someWSGR Attorney Docket No. 53989-743.601 embodiments, the LDL-C level is at about 200-350 mg / dL. In some embodiments, the LDL-C level is at about 210-350 mg / dL. In some embodiments, the LDL-C level is at about 220-350 mg / dL. In some embodiments, the LDL-C level is at about 230-350 mg / dL. In some embodiments, the LDL-C level is at about 240-350 mg / dL. In some embodiments, the LDL-C level is at about 250-350 mg / dL.

[0103] In some embodiments, the human subject has a LDLR gene variant. In some embodiments, the LDLR gene variant comprises a mutation in the LDLR gene. In some embodiments, only one allele of the LDLR gene in the participant is a variant, e.g., comprises a mutation. In some embodiments, both alleles of the LDLR gene in the participants are variant, e.g., comprises a mutation. In some embodiments, the LDLR gene has a splice site mutation or a mutation in an exon.

[0104] In some embodiments, the pharmaceutical composition is administered to the human subject via intravenous infusion. In some embodiments, the pharmaceutical composition is administered only once to the human subject during treatment. In some embodiments, the pharmaceutical composition is administered repeatedly. In some embodiments, the pharmaceutical composition administered repeatedly at an interval of one to sixty days. In some embodiments, the pharmaceutical composition administered at least twice, at least three times, at least five times, or at least ten times.

[0105] In some embodiments, the human subject, before administration of the pharmaceutical composition, receives a prior treatment comprising a steroid, an antihistamine, or a combination thereof. In some embodiments, the steroid comprises dexamethasone. In some embodiments, the antihistamine comprises a Hl blocker or a H2 blocker.

[0106] In some embodiments, the human subject, before administration of the pharmaceutical composition, received prior treatment for the disease or condition related to PCSK9. In some embodiments, the prior treatment comprises coronary revascularization, coronary artery bypass grafting, or percutaneous coronary intervention.

[0107] In some embodiments, the human subject, before administration of the pharmaceutical composition, had myocardial infarction or cardiac arrest.

[0108] In some embodiments, the human subject, before administration of the pharmaceutical composition, had lipid-lowering medication. In some embodiments, the lipid-lowering medication comprises a statin, ezetimibe, a bile acid sequestrant, a fibrate, or niacin. In some embodiments, the lipid- lowering medication is oral lipid-lowering medication. In some embodiments, the human subject is tolerant or resistant to the lipid-lowering medication, for example, there is no significant change of the LDL-C level after treatment as compared to the LDL-C level before treatment.

[0109] In some embodiments, the human subject has familial hypercholesterolemia (FH). In some embodiments, the human subject has heterozygous familial hypercholesterolemia (HeFH). In some embodiments, the human subject has severe HeFH. The term “severe HeFH,” as used herein, refers to HeFH that significantly increase the risk of cardiovascular disease (CVD) for a participant, for example, a HeFH with untreated LDL-C levels above 8 mmol / L (309 mg / dl). In some embodiments, the human subject has premature coronary artery disease. In some embodiments, the human subject has atherosclerotic cardiovascular disease, accelerated atherosclerotic cardiovascular disease (ASCVD),WSGR Attorney Docket No. 53989-743.601 premature coronary artery disease, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, coronary heart disease, carotid artery disease, peripheral artery disease, microvascular disease, stroke, heart attack, sudden cardiac arrest.

[0110] The methods disclosed herein can achieve at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of on-target editing in a PCSK9 gene in the subject. The methods disclosed herein can achieve at least 40% of on-target editing in a PCSK9 gene in the subject. The methods disclosed herein can achieve at least about 45% of on-target editing in a PCSK9 gene in the subject. The methods disclosed herein can achieve at least about 50% of on-target editing in a PCSK9 gene in the subject. The methods disclosed herein can achieve at least about 55% of on-target editing in a PCSK9 gene in the subject. The methods disclosed herein can achieve at least about 60% of on-target editing in a PCSK9 gene in the subject. The methods disclosed herein can achieve at least about 65% of on-target editing in a PCSK9 gene in the subject. The methods disclosed herein can achieve about 50% of on-target editing in a PCSK9 gene in the subject. The methods disclosed herein can achieve about 55% of on-target editing in a PCSK9 gene in the subject. The methods disclosed herein can achieve about 60% of on-target editing in a PCSK9 gene in the subject.[oni] The methods disclosed herein can achieve at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of reductions in plasma PCSK9 protein in a subject. The methods disclosed herein can achieve at least about 70% of reductions in plasma PCSK9 protein in a subject. The methods disclosed herein can achieve at least about 80% of reductions in plasma PCSK9 protein in a subject. The methods disclosed herein can achieve at least about 90% of reductions in plasma PCSK9 protein in a subject. The methods disclosed herein can achieve at least about 95% of reductions in plasma PCSK9 protein in a subject. The methods disclosed herein can achieve at least about 96% of reductions in plasma PCSK9 protein in a subject. The methods disclosed herein can achieve at least about 97% of reductions in plasma PCSK9 protein in a subject. The methods disclosed herein can achieve at least about 98% of reductions in plasma PCSK9 protein in a subject. The methods disclosed herein can achieve at least about 99% of reductions in plasma PCSK9 protein in a subject.

[0112] In some embodiments, a level of PCSK9 protein in the human subject is reduced by at least 20%, 30%, 40%, 50%, 60%, or 70% after administration of the pharmaceutical composition as compared to the level of PCSK9 protein prior to the administration. In some embodiments, the level of PCSK9 protein is measured in blood. In some embodiments, the level of PCSK9 protein is reduced within 2, 3, or 4 weeks after administration. In some embodiments, the level of PCSK9 protein is reduced within 2, 3, or 4 weeks after administration. In some embodiments, the level of PCSK9 protein is reduced within 4 weeks after administration. In some embodiments, the level of PCSK9 protein is reduced by at least 40% after administration. In some embodiments, the level of PCSK9 protein is reduced by least 50% after administration. In some embodiments, the level of PCSK9 protein in the human subject is reduced by about 40%, 50%, 60%, or 70% after administration.WSGR Attorney Docket No. 53989-743.601

[0113] The methods disclosed herein can achieve at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of reductions in LDL-C in a subject. The methods disclosed herein can achieve at least about 20% of reductions in LDL-C in a subject. The methods disclosed herein can achieve at least about 30% of reductions in LDL-C in a subject. The methods disclosed herein can achieve at least about 40% of reductions in LDL-C in a subject. The methods disclosed herein can achieve at least about 50% of reductions in LDL-C in a subject. The methods disclosed herein can achieve at least about 60% of reductions in LDL-C in a subject.

[0114] In some embodiments, a level of LDL-C in the human subject is reduced by at least 30%, 40%, 50%, or 60% after administration of the pharmaceutical composition as compared to the level of LDL-C prior to the administration. In some embodiments, the level of LDL-C in the human subject is reduced by about 30%, 40%, 50%, or 60% after administration of the pharmaceutical composition as compared to the level of LDL-C prior to the administration. In some embodiments, the level of LDL-C is measured in blood. In some embodiments, the level of LDL-C is reduced within 2, 3, or 4 weeks after administration. In some embodiments, the level of LDL-C is reduced within 4 weeks after administration. In some embodiments, the level of LDL-C is reduced by at least 40% after administration. In some embodiments, the level of LDL-C is reduced by at least 20%, 30%, or 40% within 1 week after administration. In some embodiments, the level of LDL-C is reduced by at least 30% within 1 week after administration. In some embodiments, the level of LDL-C is reduced by at least 40% within 1 week after administration. In some embodiments, the level of LDL-C is reduced by about 20%, 30%, or 40% within 1 week after administration. In some embodiments, the level of LDL-C is reduced by about 30% within 1 week after administration. In some embodiments, the level of LDL-C is reduced by about 40% within 1 week after administration. In some embodiments, the level of LDL-C is reduced by at least 30%, 40%, or 50% within 2 weeks after administration. In some embodiments, the level of LDL-C is reduced by at least 40% within 2 weeks after administration. In some embodiments, the level of LDL-C is reduced by at least 50% within 2 weeks after administration. In some embodiments, the level of LDL-C is reduced by about 30%, 40%, or 50% within 2 weeks after administration. In some embodiments, the level of LDL-C is reduced by about 40% within 2 weeks after administration. In some embodiments, the level of LDL-C is reduced by at least 30%, 40%, or 50% within 3 weeks after administration. In some embodiments, the level of LDL-C is reduced by at least 40% within 3 weeks after administration. In some embodiments, the level of LDL-C is reduced by at least 50% within 3 weeks after administration. In some embodiments, the level of LDL-C is reduced by about 30%, 40%, or 50% within 3 weeks after administration. In some embodiments, the level of LDL-C is reduced by about 40% within 3 weeks after administration. In some embodiments, the level of LDL-C is reduced by about 50% within 3 weeks after administration. In some embodiments, the level of LDL-C is reduced by at least 30%, 40%, 50%, or 60% within 4 weeks after administration. In some embodiments, the level of LDL-C is reduced by at least 40% within 4 weeks after administration. In some embodiments, the level of LDL-C is reduced by at least 50% within 4 weeks after administration. In some embodiments, the level of LDL-C is reduced by at least 60% within 4 weeks after administration. In some embodiments, the level of LDL-C is reduced by about 30%, 40%, 50%, or 60% within 4 weeks afterWSGR Attorney Docket No. 53989-743.601 administration. In some embodiments, the level of LDL-C is reduced by about 40% within 4 weeks after administration. In some embodiments, the level of LDL-C is reduced by about 50% within 4 weeks after administration. In some embodiments, the level of LDL-C is reduced by about 60% within 4 weeks after administration. In some embodiments, the level of LDL-C is maintained at most 40%, 50%, or 60% of the level of LDL-C prior to administration from Week 4 to at least Week 10. In some embodiments, the level of LDL-C is maintained from Week 4 to at least Week 20. In some embodiments, the level of LDL-C is maintained from Week 4 to at least Week 25. In some embodiments, the level of LDL-C is maintained at most 50% of the level of LDL-C prior to administration. In some embodiments, the level of LDL-C is maintained at about 40%, 50%, or 60% of the level of LDL-C prior to administration from Week 4 to at least Week 10. In some embodiments, the level of LDL-C is maintained at about 40% of the level of LDL-C prior to administration.

[0115] In some embodiments, the human subject has accelerated atherosclerotic cardiovascular disease (ASCVD). In some embodiments, the PCSK9 gene is inactivated by the pharmaceutical composition disclosed herein. In some embodiments, one allele of the PCSK9 gene is inactivated by the pharmaceutical composition disclosed herein. In some embodiments, both alleles of the PCSK9 gene is inactivated by the pharmaceutical composition disclosed herein. In some embodiments, the PCSK9 gene inactivated by the pharmaceutical composition disclosed herein is from liver cells of the human subject. In some embodiments, the PCSK9 gene of at least 70%, 80%, or 90% of the liver cells of the human subject are inactivated.

[0116] The methods disclosed herein can effectively achieve on-target editing in a PCSK9 gene, reducing plasma or blood PCSK9 protein and / or reducing LDL-C levels in a subject without serious safety and / or toxicity concerns.Base Editing / Base Editing Composition

[0117] The pharmaceutical composition described herein comprises an RNA encoding a base editor protein. In some embodiments, base editing protein comprising a programmable DNA binding domain or protein (e.g., Cas9 or dCas9) and a deaminase. In some embodiments, the deaminase is an adenosine deaminase. The pharmaceutical composition described herein further comprises a guide polynucleotide. In some embodiments, the guide polynucleotide is a guide RNA.

[0118] In some embodiments, the guide polynucleotide directs the base editing protein to affect a nucleobase alteration in a PCSK9 gene in vivo when administered to a subject. In some embodiments, the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine. In some embodiments, the spacer sequence comprises a nucleotide base sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to CCCGCACCUUGGCGCAGCGG (SEQ ID No. 101)WSGR Attorney Docket No. 53989-743.601GGUGCUAGCCUUGCGUUCCG (SEQ ID NO: 102), or UUGGAAAGACGGAGGCAGCC (SEQ ID NO: 103), wherein the uppercase A, G, and C represent adenine, guanine, and cytosine, respectively, and wherein the uppercase U represents uracil or thymine. In some embodiments, the spacer sequence comprises a nucleotide sequence having at least 80% identity to cscscsGCACCUUGGCGCAGCGG (SEQ ID No: 108), gsgsusGCUAGCCUUGCGUUCCG (SEQ ID NO: 109), or ususgsGAAAGACGGAGGCAGCC (SEQ ID NO: 110), wherein 1) the uppercase A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) the lowercase a, u, g, and c represent 2’-O-Methyl- modified adenosine, uridine, guanosine, and cytidine, respectively, and 3) the lowercase s represents phosphorothioate (PS) linkage.

[0119] In some embodiments, the guide polynucleotide further comprises atracr sequence, wherein the tracr sequence comprises a nucleotide base sequence, wherein the nucleotide base sequence has at least 80% sequence identity to a nucleotide base sequence ofGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 107). In some embodiments, the guide polynucleotide further comprises a tracr sequence, wherein the tracr sequence comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to (i) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgcaccgAgUCggug cusususu (SEQ ID No. 104), (ii) gUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusu susu (SEQ ID No. 105), or (iii) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcus ususu (SEQ ID No. 106), and wherein 1) the uppercase A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) the lowercase a, u, g, and c represent 2’-O-Methyl- modified adenosine, uridine, guanosine, and cytidine, respectively, and 3) the lowercase s represents phosphorothioate (PS) linkage. In some embodiments, the tracr sequence comprises a nucleotide base sequence, wherein the nucleotide base sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to a functional portion of a nucleotide base sequence ofGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (nucleotide base sequence of SEQ ID NO: 107).

[0120] In some embodiments, the spacer sequence comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to cscscsGCACCUUGGCGCAGCGG (SEQ ID No. 108), gsgsusGCUAGCCUUGCGUUCCG (SEQ ID NO: 109), orWSGR Attorney Docket No. 53989-743.601 ususgsGAAAGACGGAGGCAGCC (SEQ ID NO: 110), wherein 1) the uppercase A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) the lowercase a, u, g, and c represent 2’-O-Methyl- modified adenosine, uridine, guanosine, and cytidine, respectively, and 3) the lowercase s represents phosphorothioate (PS) linkage.

[0121] In some embodiments, the protospacer sequence comprises a protospacer sequence. In some embodiments, the protospacer comprises the sequence of 5'-CCCGCACCTTGGCGCAGCGG-3'. In some embodiments, the protospacer comprises the sequence of 5'-CCGCACCTTGGCGCAGCGG-3'. In some embodiments, the protospacer comprises the sequence of 5'-AAGATACCTGAATAACTCTC-3'.

[0122] In some embodiments, the guide RNA comprises a sequence with at least 80%, 85%, 90%, or 95% sequence identity or sequence similarity to the sequence of 5'- CCCGCACCUUGGCGCAGCGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUC CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU -3'. In some embodiments, the guide RNA comprises the sequence 5'- CCCGCACCUUGGCGCAGCGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUC CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU -3'. In some embodiments, the guide RNA consists of the sequence 5'- CCCGCACCUUGGCGCAGCGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUC CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU -3 ' .

[0123] In some embodiments, the guide RNA comprises at least one modification. In some embodiments, the guide RNA comprises a sequence with at least 80%, 85%, 90%, or 95% sequence identity or sequence similarity to the sequence of 5'- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcG UUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu-3'. In some embodiments, the guide RNA comprises the sequence of 5'- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcG UUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu-3'. In some embodiments, the guide RNA consists of the sequence of 5'- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcG UUAucAAcuuGaaaaaguGgcaccg AgU Cggugcusususu-3 ' .

[0124] In some embodiments, the guide RNA comprises a sequence with at least 80%, 85%, 90%, or 95% sequence identity or sequence similarity to the sequence of 5'- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAA cuuGaaaaagugGcaccgagucggugcusususu -3'. In some embodiments, the guide RNA comprises the sequence of 5'- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAA cuuGaaaaagugGcaccgagucggugcusususu -3'. In some embodiments, the guide RNA consists of the sequence of 5'-WSGR Attorney Docket No. 53989-743.601 cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAA cuuGaaaaagugGcaccgagucggugcusususu -3 ' .

[0125] In some embodiments, the guide RNA comprises a sequence with at least 80%, 85%, 90%, or 95% sequence identity or sequence similarity to the sequence of 5'- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGU UAucAAcuugaaaaagugGcaccgagucggugcusususu-3'. In some embodiments, the guide RNA comprises the sequence of 5'- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGU UAucAAcuugaaaaagugGcaccgagucggugcusususu-3'. In some embodiments, the guide RNA consists of the sequence of 5'- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGU UAucAAcuugaaaaagugGcaccgagucggugcusususu-3 ' .

[0126] In some embodiments, the guide RNA comprises a sequence with at least 80%, 85%, 90%, or 95% sequence identity or sequence similarity to the sequence of 5’- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAacAA cuugaaaaagugGcaccgagucggugcusususu-3 ’ . In some embodiments, the guide RNA comprises the sequence of 5’- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAacAA cuugaaaaagugGcaccgagucggugcusususu-3 ’ . In some embodiments, the guide RNA consist of the sequence of 5’- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAacAA cuugaaaaagugGcaccgagucggugcusususu-3 ’ .

[0127] In some embodiments, the guide RNA comprises a sequence with at least 80%, 85%, 90%, or 95% sequence identity or sequence similarity to the sequence of 5’- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAA cuugaaaaagugGcaccgagucggugcusususuuuu-3 ’ . In some embodiments, the guide RNA comprises the sequence of 5’- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAA cuugaaaaagugGcaccgagucggugcusususuuuu-3 ’ . In some embodiments, the guide RNA consists of the sequence of 5’- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAA cuugaaaaagugGcaccgagucggugcusususuuuu-3 ’ .

[0128] In some embodiments, the guide RNA comprises a sequence with at least 80%, 85%, 90%, or 95% sequence identity or sequence similarity to the sequence of 5’- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAA cuugaaaaagugGcaccgagucggugcusususuuUu-3 ’ . In some embodiments, the guide RNA comprises the sequence of 5’- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAWSGR Attorney Docket No. 53989-743.601 cuugaaaaagugGcaccgagucggugcusususuuUu-3 ’ . In some embodiments, the guide RNA consists of the sequence of 5’- cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAA cuugaaaaagugGcaccgagucggugcusususuuUu-3 ’ .

[0129] In some embodiments, the guide polynucleotide comprises a nucleotide sequence of cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAA cuuGaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 1), wherein 1) the uppercase A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) the lowercase a, u, g, and c represent 2’-O-Methyl- modified adenosine, uridine, guanosine, and cytidine, respectively, and 3) the lowercase s represents phosphorothioate (PS) linkage.

[0130] In some embodiments, the RNA comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the RNA comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the RNA comprises a nucleic acid sequence of SEQ ID NO: 2.

[0131] In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD (TadA) or to any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to the amino acid sequence of TadA or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences of TadA or any of the adenosine deaminases provided herein.

[0132] In some embodiments, the adenosine deaminase is encoded by a nucleic acid with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity or sequence similarity of comprising a sequence of Au’GAGCGAGGu’GGAGu’u’CAGCCACGAGu’ACu’GGAu’GCGGCACGCCCu’GACCCu’GGCCA AGCGGGCCCGGGACGAGCGGGAGGu’GCCCGu’GGGCGCCGu’GCu’GGu’GCu’GAACAACCGGWSGR Attorney Docket No. 53989-743.601 Gu’GAu’CGGCGAGGGCu’GGAACCGGGCCAu’CGGCCu’GCACGACCCCACCGCCCACGCCGAG Au’CAu’GGCCCu’GCGGCAGGGCGGCCu’GGu’GAu’GCAGAACu’ACCGGCu’GAu’CGACGCCAC CCu’Gu’ACGu’GACCu’u’CGAGCCCu’GCGu’GAu’Gu’GCGCCGGCGCCAu’GAu’CCACAGCCGG Au’CGGCCGGGu’GGu’Gu’u’CGGCGu’GCGGAACGCCAAGACCGGCGCCGCCGGCAGCCu’GAu’ GGACGu’GCu’GCACCACCCCGGCAu’GAACCACCGGGu’GGAGAu’CACCGAGGGCAu’CCu’GG CCGACGAGu’GCGCCGCCCu’GCu’Gu’GCCGGu’u’Cu’u’CCGGAu’GCCCCGGCGGGu’Gu’u’CAA CGCCCAGAAGAAGGCCCAGAGCAGCACCGAC (TadA Coding Sequence).

[0133] In some embodiments, the molar ratio of the guide polynucleotide and the RNA encoding the base editing protein is about 500: 1 to about 1:500. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editing protein is about 1: 1.

[0134] In some embodiments, the ratio of the guide polynucleotide and the RNA encoding the base editing protein is about 1000: 1 to about 1 : 1000 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editing protein is about 1000: 1, 950: 1, 900: 1, 850: 1, 800: 1, 750: 1, 700: 1, 650: 1, 600: 1, 550: 1, 500: 1, 450: 1, 400: 1, 350: 1, 300: 1, 250: 1, 200: 1, 100: 1, 95: 1, 90: 1, 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1, 50: 1, 45: 1, 40: 1, 35: 1, 30: 1, 25: 1, 20: 1, 19: 1, 18: 1, 17: 1, 17: 1, 15: 1, 14: 1, 13: 1, 12: 1, 11: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2;1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1: 1, 1.0: 1, 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, 0.5: 1, 0.4: 1, 0.3: 1, 0.2: 1, or 0.1 by weight.

[0135] In some embodiments, the ratio of the guide polynucleotide and the RNA encoding the base editing protein is about 10: 1 to about 1 : 10 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editing protein is about 4: 1, 3; I, 2: 1, 1.5: 1, 1: 1, 1: 1.5, 1:2, 1:3, or 1:4 by weight.

[0136] In some embodiments, the base editing system comprises a protein comprising a programmable DNA binding protein fused to an adenosine deaminase. In some embodiments, the base editing comprises a protein comprising a Cas9 protein and an adenosine deaminase. In some embodiments, the base editing is a Cas9 nickase (nCas9) fused to an adenosine deaminase. In some embodiments, the base editing is a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, the base editing comprises a protein comprising a programmable DNA binding protein fused to a cytidine deaminase. In some embodiments, the base editing comprises a protein comprising a Cas9 protein and a cytidine deaminase. In some embodiments, the base editing is a Cas9 nickase (nCas9) fused to a cytidine deaminase. In some embodiments, the base editing is a nuclease-inactive Cas9 (dCas9) fused to a cytidine deaminase. In some embodiments, the base editing further comprises, an inhibitor of base excision repair, for example, a UGI domain. In some embodiments, the protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair, such as a UGI or dISN domain. In some embodiments, the dCas9 domain of the protein comprises a D10A and a H840A mutation as numbered in the wild type SpCas9 amino acid sequence. In some embodiments, the UGI comprises the following amino acid sequence:

[0137] >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitorWSGR Attorney Docket No. 53989-743.601MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT S D APE YKPW ALVIQDS NGENKIKML.

[0138] In some embodiments, the nucleobase editing protein ABE8.8 comprises the sequence as provided below: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPES SGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLV QTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTE ELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKAT AKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKEL LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGD.Proprotein convertase subtilisin-kexin type 9 (PCSK9)

[0139] The target gene for modification using the compositions and methods disclosed herein can be a gene encoding PCSK9. Proprotein convertase subtilisin-kexin type 9 (PCSK9), also known as neural apoptosis- regulated convertase 1 (NARC-I), is a proteinase K-like subtilase identified as the 9th member of the secretory subtilase family. “Proprotein convertase subtilisin / kexin type 9 (PCSK9)" refers to an enzyme encoded by the PCSK9 gene. PCSK9 binds to the receptor for low-density lipoprotein (LDL) particles. In the liver, the LDL receptor removes LDL particles from the blood through the endocytosis pathway. When PCSK9 binds to the LDL receptor, the receptor is channeled towards the lysosomalWSGR Attorney Docket No. 53989-743.601 pathway and broken down by proteolytic enzymes, limiting the number of times that a given LDL receptor is able to uptake LDL particles from the blood. Thus, blocking PCSK9 activity may lead to more LDL receptors being recycled and present on the surface of the liver cells to remove LDL cholesterol from the blood. Therefore, blocking PCSK9 can lower blood cholesterol levels.

[0140] PCSK9 orthologs are found across many species. The human gene for PCSK9 localizes to human chromosome Ip33-p34.3. PCSK9 is expressed in cells capable of proliferation and differentiation including, for example, hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic brain telencephalon neurons.

[0141] Original synthesis of PCSK9 is in the form of an inactive enzyme precursor, or zymogen, of 72- kDa, which undergoes autocatalytic, intramolecular processing in the endoplasmic reticulum (ER) to activate its functionality. This internal processing event has been reported to occur at the SSVFAQ jSIP motif, and has been reported as a requirement of exit from the ER. "j" indicates cleavage site. The cleaved protein is then secreted. The cleaved peptide remains associated with the activated and secreted enzyme.

[0142] The gene sequence for human PCSK9 is ~22-kb long with 12 exons encoding a 692 amino acid protein. Human, mouse and rat PCSK9 nucleic acid sequences have been deposited; see, e.g., GenBank Accession Nos.: AX127530 (also AX207686), AX207688, and AX207690, respectively, each of which sequence is incorporated herein in its entirety. The gene sequence of Macaca fascicularis can be found publicly, for example, NCBI Gene ID: 102142788, which sequence is incorporated herein in their entirety. Macaca fascicularis proprotein convertase subtilisin / kexin type 9 isoform X2 sequence can be found publicly, for example, at NCBI Reference Sequence: XP 005543317.1, which sequence is incorporated herein in its entirety.

[0143] The translated protein contains a signal peptide in the NH2 -terminus, and in cells and tissues an about 74 kDa zymogen (precursor) form of the full-length protein is found in the endoplasmic reticulum. During initial processing in the cell, the about 14 kDa prodomain peptide is autocatalytically cleaved to yield a mature about 60 kDa protein containing the catalytic domain and a C-terminal domain often referred to as the cysteine-histidine rich domain (CHRD). This about 60 kDa form of PCSK9 is secreted from liver cells. The secreted form of PCSK9 appears to be the physiologically active species, although an intracellular functional role of the about 60 kDa form has not been ruled out.

[0144] Several mutant forms of PCSK9 are well characterized, including S 127R, N157K, F216L, R218S, and D374Y, with S 127R, F216L, and D374Y being linked to autosomal dominant hypercholesterolemia (ADH). Benjannet et al. (J. Biol. Chem., 279(47):48865-48875 (2004)) demonstrated that the S 127R and D374Y mutations result in a significant decrease in the level of pro- PCSK9 processed in the ER to form the active secreted zymogen. As a consequence, it is believed that wild-type PCSK9 increases the turnover rate of the LDL receptor causing inhibition of LDL clearance (Maxwell et al, PNAS, 102(6):2069-2074 (2005); Benjannet et al, and Lalanne et al), while PCSK9 autosomal dominant mutations result in increased levels of LDLR, increased clearance of circulating LDL, and a corresponding decrease in plasma cholesterol levels. See, Rashid et al, PNAS, 102( 15) :5374-WSGR Attorney Docket No. 53989-743.6015379 (2005); Abifadel et al, 2003 Nature Genetics 34: 154-156; Timms et al, 2004 Hum. Genet. 114:349- 353; and Leren, 2004 Clin. Genet. 65:419-422, each of which are incorporated herein by reference.

[0145] A later-published study on the S127R mutation of Abifadel et al, reported that participants carrying such a mutation exhibited higher total cholesterol and apoB 100 in the plasma attributed to (1) an overproduction of apoB 100-containing lipoproteins, such as low density lipoprotein (LDL), very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL), and (2) an associated reduction in clearance or conversion of said lipoproteins. Together, the studies referenced above evidence the fact that PCSK9 plays a role in the regulation of LDL production. Expression or upregulation of PCSK9 is associated with increased plasma levels of LDL cholesterol, and inhibition or the lack of expression of PCSK9 is associated with low LDL cholesterol plasma levels. Significantly, lower levels of LDL cholesterol associated with sequence variations in PCSK9 have conferred protection against coronary heart disease; Cohen et al, 2006 N. Engl. J. Med. 354: 1264-1272.

[0146] Lalanne et al. demonstrated that LDL catabolism was impaired and apolipoprotein B-containing lipoprotein synthesis was enhanced in two participants harboring S 127R mutations in PCSK9 (J. Lipid Research, 46: 1312-1319 (2005)). Sun et al. also provided evidence that mutant forms of PCSK9 are also the cause of unusually severe dominant hypercholesterolaemia as a consequence of its effect of increasing apolipoprotein B secretion (Sun et al, Hum. Mol. Genet, 14(9): 1161-1169 (2005)). These results were consistent with earlier results which demonstrated adenovirus -mediated overexpression of PCSK9 in mice results in severe hypercholesteromia due to drastic decreases in the amount of LDL receptor Dubuc et al., Thromb. Vase. Biol., 24: 1454-1459 (2004), in addition to results demonstrating mutant forms of PCSK9 also reduce the level of LDL receptor (Park et al., J. Biol. Chem., 279:50630-50638 (2004). The overexpression of PCSK9 in cell lines, including liver-derived cells, and in livers of mice in vivo, results in a pronounced reduction in LDLR protein levels and LDLR functional activity without changes in LDLR mRNA level (Maxwell et al., Proc. Nat. Amer. Set, 101:7100-7105 (2004); Benjannet S. et al, J. Bio. Chem. 279: 48865-48875 (2004)).

[0147] In some embodiments, the loss of function mutation induced in PCSK9 e.g., G106R, L253F, A443T, R93C, etc. In some embodiments, the loss-of-function mutation is engineered (i.e., not naturally occurring), e.g., G24D, S47F, R46H, S 153N, H193Y, etc.

[0148] PCSK9 variants that can be useful in the present disclosure are loss-of-function variants that may boost LDL receptor-mediated clearance of LDL cholesterol, alone or in combination with other genes involved in the pathway, e.g., APOC3, LDL-R, or Idol. In some embodiments, the PCSK9 loss-of- function variants produced using the methods of the present disclosure express efficiently in a cell. In some embodiments, the PCKS9 loss-of-function variants produced using the methods of the present disclosure is activated and exported to engage the clathrin-coated pits from unmodified cells in a paracrine mechanism, thus competing with the wild-type PCSK9 protein. In some embodiments, the PCSK9 loss- of-function variant comprises mutations in residues in the LDL-R bonding region that make direct contact with the LDL-R protein. In some embodiments, the residues in the LDL-R bonding region that makeWSGR Attorney Docket No. 53989-743.601 direct contact with the LDL-R protein are selected from the group consisting of R194, R237, F379, S372, D374, D375, D378, R46, R237, and A443.

[0149] Wild Type PCSK9 Gene (NG_009061.1), Homo sapiens proprotein convertase subtilisin / kexin type 9 (PCSK9), RefSeqGene (LRG 275) on chromosome 1GTCCGATGGGGCTCTGGTGGCGTGATCTGCGCGCCCCAGGCGTCAAGCACCCACACCCTAGAAGGTTTCCGCAGCGACGTCGAGGCGCTCATGGTTGCAGGCGGGCGCCGCCGTTCAGTTCAGGGTCTGAGCCTGGAGGAGTGAGCCAGGCAGTGAGACTGGCTCGGGCGGGCCGGGACGCGTCGTTGCAGCAGCGGCTCCCAGCTCCCAGCCAGGATTCCGCGCGCCCCTTCACGCGCCCTGCTCCTGAACTTCAGCTCCTGCACAGTCCTCCCCACCGCAAGGCTCAAGGCGCCGCCGGCGTGGACCGCGCACGGCCTCTAGGTCTCCTCGCCAGGACAGCAACCTCTCCCCTGGCCCTCATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGTGCGGGTGTAGGGATGGGAGGCCGGGGCGAACCCGCAGCCGGGACGGTGCGGTGCTGTTTCCTCTCGGGCCTCAGTTTCCCCCCATGTAAGAGAGGAAGTGGAGTGCAGGTCGCCGAGGGCTCTTCGCTTGGCACGATCTTGGGGACTGCAGGCAAGGCGGCGGGGGAGGACGGGTAGTGGGGAGCACGGTGGAGAGCGGGGACGGCCGGCTCTTTGGGGACTTGCTGGGGCGTGCGGCTGCGCTATTCAGTGGGAAGGTTCGCGGGGTTGGGAGACCCGGAGGCCGAGGAAGGGCGAGCAGAGCACTGCCAGGATATCCTGCCCAGATTTCCCAGTTTCTGCCTCGCCGCGGCACAGGTGGGTGAAGGAGTGAATGCCTGGAACGTACTGGGAACTGCACCAGGCACAGAGAAAGCGGGCTTGCCATTATAGTGGGTTCCGATTTGGTTTGGAAAACATGGGCAGCGGAGGGTGGAGGGCCTGGAGAGAAGGCCCTACCCGAGACAGGGGCGGGGTGGGAAGGACGGCAGATGCTGGGAGCACGAGGCAATTTCTTTATGACACAGAACTCATGCTCTAGTATTCCATCTGTTTCAGCCGAAGAAAAGAACCAGCTGAAGGGGCAGGGGAGAAGGGGCGGAGGTATTCTCGAGGCCCATTGGCGTCCTTTAGGACTCAGGCAGGGAAGGGCCCTTGGTGCTCTGGAGCCGGAGGTGGTGCGCCTGGTACTGGGACCCCGGAGCTGAGCCCGGCGCCTCAGCCCACCTGGCTGTCTGCCGACCGTGTGCGGGGCGAGTTTGCTCAACAACTCTGCCAGCTTCTGGCCCTCAGGCTGTGGGAAGCTTCTTCCCGGGGCGAGACCACTAGCTTTTTCTAAGTATTACCAGCCCAGGACTTGGCTGAGGTTCTGTGTCCCCCAGCTTGGAGTCAGATGTGGGGTTGAATCTTGGCTTCCTCTCACTAGCTGTGGTGCTTGACAAGTCACTTATCCTTGAGCCTCCATTGCCTAATCTTTAAAAGGGAGGTGACAATCGTCCCTACGGCTCAGTGGCAGCAGATGGGGAGATGAAGGGAAAGTTCTGTTGACCATGAGTGAACTTACAATGCAAGCCCCGGGGGGATCACTTGCAGTTTTGTCCCTGTCTGCAGTGTGACCTGTTGGTGACATTGTCTTTGCTCCAAACCACAGCTCCTGGGGCAGAGGGGAAAATTCTGCCACTCACAGCTGCCTGCCCACGCTTCTGTCTGAGTGTGCTGGGTGGCAGGATGGCAAGTCCTTACTCAGCTCAGTATAGCCCTCTTCCTTGTTCCCTGAGCCTTTGACTTTCTCGAGGGATGTTGTGGGGTTGTGGCCAGGATAAGAAAGGGCATTTCAAGTTACCACTGCTCCAAAACAACTGTTCTGGAAATAGTGAGTACCCCATCCTGAGAGGTGAGTAAGCAGAGGCTGTATGACCACCTGAACCAAGCCCTTGAGGATGTTTCTTCTCTGGTGGAAGTTTGGAACAGGAGCCTCCTCAAGTTCATTTATTWSGR Attorney Docket No. 53989-743.601CATTCATTCAATGGTTATTTTGTGGGAATCGAATTTAGAATGAAAATATTTTTTGGCAAGCAGAAAATAATTTTTAGACCAATCCTTTTCTTTTAGTCATGAGAAACTGAGGCCCAGAGAGAGGAGGTCACCCCAGGTGCATTAGAACTGGGTTTCCAGAACTGACACTCCACTGCACAGAGTACTCTCCCAATTCATTCAATTTTTATTTAGCGGAAGGCATTTTCAGATGGGTCTTTGAAGCATTAGTAGGAGTTCAGCGATGATGGTGTCATGAGAATTTTATTCTAGGATTAGGAGGTACCATGAACAAAGATACAGAGCTGGGAAAACCAGAGGTGGAAGATAAGGAGCACATGTCCACAGTTCTTTTTCTTTTTTTTTTGAGATGGAGTTTCGCTCTTGTTGCCCAGGCTGGAGTGCAATGGTGCAGTCTCAGCTCACTGCAACATCTGTCTCCCGGGTTCAAGTGGTTCTCCTGCCTCAGCCTCCCAAGAAGCTGGGATTACAGGTACCTGCCACCACGCCCGGCTAATTTTTGTATTTTTAGTAGAGAAGGGGTTTCACCACGTTGGCCAGGCTAGTCGCAAACTCCTGACCTCCTCAGTGGATCCGAGGAGGTGATCCTCCCGCCTCAGCCTCCCAAAGTGCTCGAATTACAGGTGTGAGCCACCACGCCTGGCCTCCACAGTTCTTTATCCACCGTCTGAAATGTAAAATGTTACGAAAACCAAAAGTTTTTTTTGTGATTTATTTGATGGTAGCACCTGACGTGAACTGACATGAGATTATTTTTAATTTAGTTGTGTGAATATGCATATTCATATATTTTGCTGCATAGATTACAGTATGCAGCTCCAGATTCTTCCAAGCAGACTCTGATTGCCCATTACTGCCTTTCTAAAATCCAAACAAGTTCTGAGGTTCAAAACCGTTTTGGCCCTAAGGCTTTGGGTAAAGGGGGTGGACTCTGTTCTACTCTGACTGGAGTCCAAGATGCATATATACAGAGATATGGGTGATGGGGCTGCAAGGTAGGTTGAGGTAGGGGCCAAGGAGGAGCATGGAGTTTGGACTTGATTCATGAGGCTGTGGGGAGCCAGTGAAGGTTCTTAAGCAGGTATGTCTGCCTGAGAGCAGTTGGAGCAGACAAGAGCTAAAAACCAAACAAATCACCATAGATAGTGGCTGCTATAATTTGTTTGTCCCCTCCAAATCTCATGTGGAAATTTGGTCCTCAGTGTTGGAAGTGGGGCCTAATGGGAGGTGTTTGGGTCATGGGGGAGGAACCCCTGTGAAAGGCTTGGTGCCGTCCTTGTGATAATGAGTAAGTTCTCCCGCTATGATTTCCCTTGAAGGCTGATTATTAAAAAGAGCTTGGCACCTCCCTCTCTTCTCTCTTGCTTCTTCTCTTGCCATGTGATTGATCTCTGCACATGTAGGCTCCCCTTCACCTTCTGCCATCAGTGAAAGCAGCTTAAGGCCCTCACCAGAAGCAGATGCTGGTGCCATGCTTCCTGGAGAGCTTGCAGAATCATGAGCTGAATAAATCCCTTTTCCTTGTAAATTACTCACCTTCAGGTATTCCTTTATATAGCAACACAAAAGGACTAAGACAGTGGCCTTGACTTTTCTCTCTCTTTAAGAAGTGTTGCCTTTGCTCACTTAGTCATCCCTTCTGCCTGCATTTGTAGAGCATCTGGATGGGAGATTTATATAACCGTCACTCTTGACTTTCCCAGCAGGCCTATGTCATAGGTACTGTGGTCTCTACAATACAGCAGAGGTATCTGAGGCTCCGAGAGGTTGAGTGACTTGCTCATGGCTGCACAACCAGTAAATATTGGAGCTGGAATTCAGGTCCACGGTTTCCTGGCTCCAAAGCCCATGATTTTTTCCCTCAATTTATTCTGACTGGGGCATGGGGGAGGGGGTGGCCTTTGGGCAGGGCCACCAGGAGCGACCAGGCCCGTAGAGAGCTGGGTGCAGGTACAGAGGAAAACCTGTTGTCGAGTGTGGCCCGTAGTTCCCATTTTTGCCTGAATGGCACATTTGAAAGTGTTATATAACCATGTGAATAATAATAGTTGGCCTATATGAGTTCTTTAATTTGCTTTTTGGTCCGCATTTGGTAACTTCTTTATCATCTACTATACTCTGTTGTGTCTCTTTTGTTGTAATTTGTAAGTAGGGGTGAGATAAAGTACACCTAGGGTTTGCTGGGTTTCTTCCATGTCATCATGTTCCTCCTTGCATGGGGCCAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGWSGR 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53989-743.601ATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGTGAGCCACCCTTTTTGGGAATGGCACTTCCTGATAGGGCTGGGCCACTGCATATACACTGGGGACTGTGCTTAGTAGGCCCATTGCTGAAAATCAGAAGGGGACAGCAAGTATGTATTGAGCACTTATCGGGTACCAAGCACAGTAACTACTGGCTTTCTGTATAGAATTCCCTTTAAGCCTGGCCATGCCCCAGTGGTACGTCTATCTTCATTTGAAAGACGAGGAGACTGAAGTTCAGAGGGGACCACACAGACAGCTAGGGGTAGAGCCTGGATCAAACCCATTGGTCTGCCTGCCAGCCATTCTTGTGCCAATGCATCTGCTGCCTACGGAAACCTGTAGGGACAAGGCCCTGGGATGTTCAGTGGAGCCTGAGTCATTTTATAAAAAAGCATGACTCTAGGGTCCAAAATTCCTTTGAAGCTGTTGCTATCCAGAGTGAAGTCCCTTCTTTAGGACAGGGTGGCCCTCCTCCCTCCTGGATGTCACATCTTCGGTGGAGGGGCAGAAAGGGGACTGGGTATTCTCCTCACCCTGGCCCTAGTGCTTCAAATCTTAAAAAAACGTTTTTATTTGTGCTTCTGCACCACCTTCTAGCCCACCTCGTTTCCTGGCCTCTAACTTGATGAGAGCGTGTGTCATTTTCACACTGATTCTCCACATGGCAGGCGGTGCTTCTTAGCCTCCTGCAGACAGTGAGGCCCCACGGTCTTGTCCAAGGTCACACAGCGTGTAATGGGCAGGGTCAGAGTCTGGAGTCTGGACCTGGGTCTCCTAGCTGCACTGCACTGCTGCCCCATGGGTTAATCAGCTCAGCATACCGTGGCTGAACAGCTACCTCATACCAAGGCCTGTGGCGCCATGACAGGGATTGACAGGGTCCCTGCCTTGGAAACCCGTAGTCTAAGTAGAGGAGACTGACAAGTCAATGCCTTCCATCAGTCTGCTCAACACACGTTTACCAAGTGCCTACTGTGTGCTGCAGAGGCGAAGATGACACAGCTCAGGCCTTTCCCTTGAGCTTACAGTTCAGGAGGAGAGACTGACCAGTGACTGCCAGTACAGTTGACTATGGGACAATGTGCTCAGCCTTGGGGAGAGACGAAGAAGGTACCCGTATAGCACCAGATGACAGGCACGAGCCCCACAGGCCAGGGCAGCTGCTCAGAGGAGAGTAGGCCAAGCAGAAGGCAAACAGAAGGCTGCAGGCATTTGCCATCGAGAGCTGGACTTCAAACTGGGCATCATACCAGCCTGGGTTCGAGTCCTGCCCAGCCCCTTATTGGCTGTCTAACCCTGAGCAAATCCCTTCACCTCTCTGAGCCTCATTCCTCTATCTGTAAACCAGTTATAATAATTGGAACATTCATTTAAGGACTAAATGAGGTCGTGAAGCATTCAGCAGATGCTAGGTACGGAAACTCGCTGAAGTGGGGGCAGGTTAAGAAGCCTCTGGGGATACGAAGGCATCCAGGGACTAGTTGTGGCAGGAGGCTGTTACCACTTAGGTCTGAAGGGTAAGGAGAGGGAATAGCTTTCCCTCTGCCCAGTTGGAGCCGGTGGCATGGAGGAGAGGCTGCCTGTGGGGAATCACCCGAGGGTTCACCGCTGCCATGCGCAGGGAGTCAGGAGGTAGGGAGGGAGTGGGGCAGATGCACACCATTTTTTTTTTTTTTTGAGACTCTGTTGCCCAGACTGGAGTGCAGTGGTGCCATATCTGCACCTCTGCCTCCCGGGTTCAAGCTCACTGCAACCTCTGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGTGTGCCACCATGCCTGGCTAATTTTTGTATTTTTAATAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCTCGACCTCAGGTGATCCCCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGTCACCGCTCCCAGCTGCTGATGCACTCTTGTCCTTCTAACTCCTGCTAGTGCCTCCCATTGGCTGAGCCCAACTGGAAGCTTTGCAAGGGAGCTGGTGCTGCAGTTTGCACTGAGCAGGCTGGAGAAGGCTGGAGAATAGACTAGGGGACAAACCGAATTGCCAGTGCTGTTATGTCATGATTTAGGCATGGAGTCCAGGGCCTGAGCTTCACTCCATGTCCATCCTGCCCAGAGCCTTGGCACAGCCTGGCTCCCAGACAAGATGTCAAGTTCAGAATCCTTCCTAAAAGGAATCCTCTATGCCAGACCGTGTTGCAWSGR Attorney Docket No. 53989-743.601GGGATATGGGAGTGCTGGGCTCCCAGCCTGATCAAGGAGCGAGAAAACTCAGGCTCCTAGTCTGTCCTCCGGGGCACTAGCAGGGACAAGGTGGGAGGCTGCTGGGCTGGGATGTGGGGACAGGTTTGATCAGGTAAGGCCAGGCTGTGGCTGTGTTTGCTGCTGTCCAAATGGCTTAAGCAGAGTCCCCCGGCCTCTCTGGCTTCTGCAGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGAGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTACCGGGCGGATGAATACCAGCCCCCCGGTAAGACCCCCATCTGTGCCCTGCCCCACCCCATCTGAGCTGAATCCATTTGCTCTGCCCTGGCCTGGCCTCCCTGCTGGTGGTTTCCACTTCTCGGGGGGCTTTGGGACTCAGCACCTCCACTGACCCCTTTTTTTCTGTCCCATCCCCATCCCCTGCAGCCCCCACTGCCTGCCTTCCTGTTGCCCCACAAATGCAAAAGTCTTGCCTTAAATGATCCTCTTTTCCTTCTTTTCTCTTGTTTTCCTTTTCTCACCATTTGGAATGGCCCAGCAGGCTGCACTTACCTTGGAAGGAGGGTTCATCTGATGGTGACTCTACCTAGGGCCCCCAGGCCTCTATAACTCCCAGTGCCCTGCAGACTGGACCAGATCCTTTAATGGGATAGACACAACCCTGTCTGGGATGCCTCTGCCTACCTTCCTGTTTTGCTGCTCCACCTGCCTCCAGCTCCGTTTGGCTTCCTGGGGCTCCCTGCCTGGGCCACTTTGTGTCTTCCCTCTAGGCCTTTCTTTCCACTGTTCCCTCTGCCTGGTGTGGCCTGGCTATGGAAGGGAGGGAGGAGGAGCGGCCATGGAAAACGGTCTGCATTCTAGCAGGGACTTGCAGGTGGCAATTCAGTCGGGGAAGACTCTAGATGCACCTGGCCTGAGGAGAGAATGAAGGGTTCTAGTTGGACTGTGTTAAGTTTGAGGTGCCCATGGTGTGAGGTCTGGAGCTCAGCGCAGAGATGATGCAATGTGGTGGGTCCATGCAACATGGTGCCAGGACGCAGAGCTTGGGGTGAACTCAGCTTTCACCCCTTACCGGTTCTCGTGGGATCTTGGGAAGCCACTTTCTTCTATGAGCTTTGTCGTTCTTGTCTGTAAAATGGGCACATAACCCTGTCCCTGTCCTTCTCACAGGTTGCTGTGAGACTCCAATGAGTTGAAGGATGTGCAGATGCTTTTGGAAGTGAAAAGTTGGGGGGCTACTGTGTGACTTTGCATACACCCAAACTGTGTGACCTTGCATATGTCTGAGTTGCTGCCATTGCAACAGATCAGAGCTGGTGGGCTGGGTGTGGAGAAAGGGTTTGTGTGGGGGACATCCTCTGGCAAGGGTGGCAGCAGCAGAAGTGAGGGGCCTGGTCGGTCATGTGTGCTGACCCGGCCTGGGCAGCCTGTGGCCAGGGAGAGGACAGCTCCTCTGTAGGAAGAGCCTGTTCCTTTCCAACCAGGTGAGACCTCTTCAGTGGAGCCCTGGAGCCCCCTGTACTCCACATCAGTGCCTCAGGGACCTCCCGGAGCAGGCTAATATCAGAGACCAAGAGGGACACTGGCAGAGGATCACAGAGACCCCAGTCCAGGCAGGGACTGAGAAGATCTTGCCCCCTAAGTTAGTTTCCTAGCACTGCTGTGACAAATTACCACCCCCTCGGTTGGAACAAGTTGATTCTCTGCAGTCCTGGAGGCCAGAAGCCTGAATCAGTGTCGGCAGGACCACTTTCTCCCGGGGGGCTCCAGGGAGAAGCTTCTCTTGCCTCTTCCGTGTCCCAACAGCGGCAGCACACCAATCCCAGCCTCTGTCTTCACACAGCCTTCTCTGTGTCTCTCTCCTCTTCATTGTCTCATAAGGACACTTGTCATTGGATTTAGGGCCCACTGGATCCTCCAGGATGATCTCATGTGGGGAACCTTAACCACATCTGCAAGGACCCTTTTTCCAAATAAGGTCACAGCCACAGGTTGTGGGGGTTAGGATGTGAGTGTATCTCTTTGGCAGCCACTGTTCCCTCCTCTCCCTTGGGCCAGAAGCAGACGTGGGGCCCTTTCTTCCCCATAGGATGCCCATGGATTGCCCCCCTTCCCGCTTCCCCCGAGTGTCTGTGGGAGGTGGCAGGAATGGCAGGCAGGGGTGTGGAACCCCTTCTGGAGTCATATCAAGGGCTTGGCTGGAGGAAGTCCTCCTGGAGCTGTTGGGCTGGCATGGGGCAGGCTGGCTGGGCCCAGCAGCAGCTTCTTCATTCATGGGGAGGCCACAAGCATGGGWSGR Attorney Docket No. 53989-743.601CCCTAGAGCTGGCTGCCGCCCTCAAACCCAGACCCTGCACTCTTAACTGTGTGACCTTGCATACGTCACTCACCCTCTCTGATCTTCAGGTTCCTCTGCAAAAGGGAGGTAATGATAACCCTCACTCTGGGGGGCTGTTTGGAGGGTTAAATCAGTTATTGCTGTAGCATGCATTTCTCTGTCAGGTATTGAGTGAGGTGCTGTGATTTTAGCCCTGCATTTTTCTTTTCTTACCATTCAATAATAACGTTTTGAGCACCCACTGTGCGCCAGGCACCATATTAGGTGCTGGGGATACAAATGTGAATGAAATGAATGTGGTCTCTTCCCCCAACAGTGTATCCAGAAGATTAATCCATTCCTTAAACAAATGCTACTTGACACAGATTAGTTCTGGATAGGCTGAGAGCTCTGAAGGAGTGCAGGCAGCTGCGAGCCTGTGTATCCAGCAGAAGGATCAGGAAAGGATTCCTGGAGGAAGCGCTGTTCTAGCCAAGACCTACGGGGGCATTATTAACCAGGCAAAGGGGACGGTGTCCAAGCAGTGGAATGAACGTGGATTGAAGCTGTGAGGCAGGAGGGAGTGTGGCCTGTGCAGAAGGGACCGAGGCTGGTGAGACCAGGAGGGCCTGGGTGGCCTCCAGGTCAGATGTGAAAGGAAGAACTTGGCCACAGTCTGAGCTTCTCAGGCGTATGGCAGGGCTGCCTGGTGAGAGGGAATGAGCTCCCTGCTCTGGAGGTATGCAAGCAGGACTGGGCTCTCACCTGCCAGAGGCCACAGAGCTTTCCAGAGGCTGGAAGAGGCCACTCCAAGGCCTCTTTGCCCCTGAGAGTGGTGGCTCTTCTTGAGGCCACCTTGCCACGCTGTCACAGGGAACTAGCAGCCCCTGCCTCACCCGGGGGTTTGGAAGATAGAGGGAGGCCTAGGAAGGGCCCTGTGTCTCATCCGAGCTGGGCCCCTTTCCAGCCTCTCACTGGAAGGAAGCCCAAGGATGTTCCTGTGGGGGCTTTTACCAGGCCCACCTGCCCTCTGCTGGCCATGCTTGCAGCCTCCTGACCCTGTCCCAGCAGGACAGTGGGCTGGTGTGAGCGGGCAGGAACCGCCTGCACTTAGAAGGTGTGGGGCTGCCTCCCCGAGCTTCCATCTGCCGCTGGGGCCACACCCCAGGCCCAGGGATGGGACCCCACAGTGGTCACATCATCTTGCAGCAGAACCCAGGTACAGCTCCTGGAGCAGATGGTGGTCCCAAGCACGGGTGGGACCAGAAAGGACTCTCACCTGGGCTAACTCAGCTGCAGCCTCAGTTCCCTCCTCACACACGACGAGGAACATGGACTGGAAGCCTGCCCAGCAGGCCTTCTGCTCGATGTGCGTTGTGTGGCTTACGTCCAGGGAGGGAAGCAGCCTCTGTGCTGTCTTCTAGATAAGCCTGTATTCCCCGGGCTGTCTGCCAATGTATCCAGTTGTCCCGTCAGCCTGGAAGCTCTGAGGGAAAACCTTGGGCTGCTTCCTGAGCACCTGTATCCCCTGCAGCCAGCCCGGGGCCTCTGCTAGGAGCAGACTGAGCATGGCTTATGGGCCTGGCACCATCTGGCCTCTGCCCACCTTGCTGGCCTTGTCTTGTGTCTGCCCCTTCGACATTCCATAGCCCAGCTCAATATCTAGTGGTTCCTCTAGGGTGGCGAGCACTGTTTGGTCTCCAGATGTCTTCAGGTCGGAGCTCACAGCGCTCTCAGCCACCCCTTCCCAGTGTAGCACCGGGCACATGGTAGATGCCTATTGATGAGTGAAAGCTCCTAACACACTCAGAGAGCAAGGACTCCGCCTCATCCCACAGCCTGGGAGGAGAGGCAGACTGCCAAGGACCTGCTCAGCATGCTACAGAAGAAACCAAAGTGCCCACGGGACTGATCAGTGGAGCTTCCTGCCGAGACTGGAGGCCTTAGGGCAGGGTAGACAGTGTGTGTGCAGGCTGGGGACTCACAGTTCGGACTGTGCCCAGACCTACTAGCATAGTGGGTGGGTGGGAGGATGCGGGACTGGGGGCCGACCTTGCCTGAAATTCATGTGGGATCTCAGAGCAGCCACTGAATTGCTCTGTAGGGGGCTAAATAGTGGCCCCCACAGATACACACACCCAGACAGAGCCTGTGAGCCAGACCTTATTTGGAGAAAAGGTCTTTGTAGATGTAATTAAGCATCTCAAGATGGCATCATCTGGATTATGCGGTGGGCTGTAAGTCCTGTGATGTGTCTTTATGAGAGAAAGGCAGAGGGAGATTTGACACACACAGGAGGGGCCACGTGGAGACAGAGGTGGAGATTGGAGAAATGTGGCCACAAGCCAGGGAAWSGR Attorney Docket No. 53989-743.601CACCAGCAGCCACCAGAAGCCGGAAGACGTGAGGCAGGGTTCTTCCCAGAGCCTTCGCTGCTGAGTCTGGGAATTTGTGACCGAAGCCATAAGAAGTGGGTACACGCCCTGAGCCTCCCACACTTGCTCACCTGTCCTGAGATGAGAATCTCTACTCTGCAGCATATTTGGAGGATCACTGCGGGGGCCACAGAGGTGCTGTTCAGATGGCACTTCAGAAGACTCAGGAGACCCTGGGGCAGGAGCAGTTTGACTGACAGCCCAGAGGGCTGCCCTCTGATTCCACCTGAGGCCCTGCTTTTCCTGGCTGCAGGGGTTCCAGGGCCAGGCCATTTCCGCTGGCGCAGGACTCTGCTAGCAGCAACCTGCCTGAAGTCTTCCTTTGGCCTGGCTGAGAGTTTCTGAGACCTGCGCTGGAGCGGAGGTGCTTCCTTCCTTGCTTCCTTTCTTCCTCTCTCCCTTCTCCATCCAGCAGGCTGGACCTGCCTGGCATCTGTGAGCTCTCCCTACTTTCTCCTATACCCTAACCTTTGTCCTGCATGGGCGACTCCCCCAGTGAGTCTCTTGCAGCTTTTACCCCAGTGCCTGCTTCTTGGAGAATCCAAACTGATCCAGTTAGGGATGATAAAGTGTAGGGTAGGCGCTCGGTGACTGTTTTCTCTGAGGTTGTGACTCGTGTGAGGCAGAAGCAGTCCCCGTGAGCCCTCCTGGTATCTTGTGGAGTGGAGAACGCTTGGACCTGGAGCCAGGAGGCCCAGACATACATCCTGTCCGAGCTGCAGCTTCCTGTCTCTAAAATGAGCCGGCCAGCGCAGGTGGCCAGACATCACTGTTATTCTCCTTTGAGTCTTTAAATCTTGTTGTCTTTCTTGCAGACTCGGTGAGCTGTGAAAGGCTATAATAGGGGCTTTATTTTACACTTTGATACTATTTTTTGAACATTCATATTATTGTTAGATATTGATATTCATATGAAGGAGCAGGATGACTTGGGTCCTTCTTGGCAGTAGCATTGCCAGCTGATGGCCTTGGACAGTTACCTGCCCTCTCTAGGCCTCCCTTTCCTTGTCTATGAAATACATTATAGAATAGGATGTAGTGTGTGAGGATTTTTTGGAGGTTAAACGAGTGAATATATTTAAGGCGCTTTCACCAGTGCCTGGGATGTGCTCTGTAGTTTCTGTGTGTTAACTATAAGGTTGACTTTATGCTCATTCCCTCCTCTCCCACAAATGTCGCCTTGGAAAGACGGAGGCAGCCTGGTGGAGGTGTATCTCCTAGACACCAGCATACAGAGTGACCACCGGGAAATCGAGGGCAGGGTCATGGTCACCGACTTCGAGAATGTGCCCGAGGAGGACGGGACCCGCTTCCACAGACAGGTAAGCACGGCCGTCTGATGGGAGGGCTGCCTCTGCCCATATCCCCATCCTGGAGGTGGGTGGGGACTGCCACCCCAGAGCGTTGCAGCTGTACTCCTGGGTTGCACCCCCCCCAGCTGTCACTGTCCCCTCCCTGCCATCAGTTGTGGGAAGGGCGTTCATCCATCCAGCCACCTGCTGATTTGTTATAGGGTGGAGGGGGGGTCTTTCTCATGTGGTCCTTGTGTTCGTCGAGCAGGCCAGCAAGTGTGACAGTCATGGCACCCACCTGGCAGGGGTGGTCAGCGGCCGGGATGCCGGCGTGGCCAAGGGTGCCAGCATGCGCAGCCTGCGCGTGCTCAACTGCCAAGGGAAGGGCACGGTTAGCGGCACCCTCATAGGTAAGTGATGGCCCCAGACGCTGGTCTCTCTCCATCTGGACCTGGCCTGGGAGGTGGCTTGGGCTGGGCCCAGGGAGAGCTAATGTCTCCTAACCAAGAATGCTGTGGCAGCCTCTGCCGCAGAGCCAGAGAACCAGAGTGCCAAGGCTGGCAGGGTTCCCAGTGGCCACGAGTGCAGATGAAGAAACCCAGGCCCCAAGAGGGTCATGCAGGTAGCCCAGGGAGTTCAGCCTTGACCCTGGGTCAATGACCTTTCCACAGTTCCACACTGCTCCCCTTTTAAAATCCGGTGATGTCTTTATGTCTTTTGTTATGTTATCTTCAATGTGGAGGGACTCGAGGTGATCTAAGCAAACTTTTTCTATCTTCTGCTTGCATACCTCTGAGACCAGGGGACTCACTCACTTGCATGACTGGGCCCTGCAGGTCACACTGGCCAGGCAGATGTGGTGGAGGAACTGGCAGAGGACTTTTTCTAGACTGTGACTACATTTAGTCCACCCAGCGGCCCCCCTATGAAGTCCAGTTGAGAACTAGGACTCTGGGGGCCGGTGGACAGAGAAGAGGGAGGGTTCTCTCCCTTACTGACTTCCTTCTGTGGCCAGAWSGR Attorney Docket No. 53989-743.601CATTGAGCAAGGCCTCTGTACAGCATGTCCTGGGGCTGGCCTTGCCGTAGCTGCTAAATAGTTGACGAAACCAGTCCAGAGAGGGGAGGTGACTGCCAGGGTCGCACAGCTCAAGCTGGGGAACTCGCTGGGAAAACTGTCAGCTCTGGGCAGCAGCTTGACTTCCACTGTAAGCCCCAGCCCCCAGGGTCAAACACTGGCTCTGGTGCTGGCAGAGGCAGCCCACTAGCCTGTTTCAAAGGCTGAGAAGGCCCAGGAGTCTGCCCTGTGCTCCACCAGTTCTGCCCTGAGACTTTCCTACAGAGTACAGGTTTTGATGTTCAGTTTTAAAGGCAAGAATCAATAACCTTCTGCCCCATCAGGTGACCCCTTGTGCCTGTCCCACCCCTTTATTGACTGACCTCGGCTCAGTCAGGTCAGTTCCTGAAGGTCAGTGTGTGGAGGGGAGGCTGTTCTTTCCCAGAAAGGCCTTCCCCAGGCCTGGTGCTCTGGCCTCTGGAGGACTTCCTGGAGAAGTCCCTTCTTTGGGGTCCCAGTCAGTGTATGGGAAGCCCTTATTGCATGACCTGGCACGGGGCAGGGGCTCAACAGTCACTATTGCCTTCCTTGCCACTGCCATTTCCTCCTCTGTAAGCAGGTGATTGTGTGTCCAGTCTGAGCACAGAGATAAGCACACAGCAGGTGCTTAATAACTAGCAGCTGTAGGCTGGGCGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGTTCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCAGGCATGGTGGTGGGTGTCTGTATCCCAGCTACTTGGGAGGCTAAGGCAGGAGAATCGCTTGAACCCAGGAGGTGGAGGTTGCAGTGAGCTGAGATCGTGCCACTGCAATCCAGCCTGAGTGATAGAGCGAGATTCCATCTCAAAAATAAATAAGTAAATAACTAGCAGCTGTAAATGTGGCTGTTGTTCTTCACCTCCACACTCAGTGCCACTCCACTCCCTCCCTCCGTGGTGTGAGGGGCCTCACTAGCTGTCTCCTAGGAGGAGCATGGCTGTGAGATTCCAGCTCCATCCTTGGCCACGGCTCCTGGAGACATCTTAGAGGCCAGGATCCAGAAGGCTCCCACACCTCATTTGACAGGGGAGAAGCTGTCAGTTCCAGGTCCCCTTGCACATCAGGGCCAGAGCTGCGTTAGGCCTCCAGTCTCCAGGCCACTGGGCCAGAGCTCACAGGCTGGCAGAGGGTTAGAACTGTTACTGGTGGCTGGGTGCAGTGGCTCACGCCTGTAATCTTAGCACTTTGGGAGGGCAAGGCGGGAGGATCATGAGGTCAGGACATCGAGACCATCCTTGCTAACACGGTGAAGCCCCGTCTCTACTAAAACTACAAAAAATTAGCCGGGCGTGGTGGCAGGCGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATTGCGCCACTGCACTCCAGCCTGGGCAATAGAGCGAGACTCCGTCTGGAAAGAAAAAAAAAAAAAAGAGCTGTTACTGTTGACAGTAGCATGAGGTAGACCATGGCCTGCACCAAAATGGGGGAGTGGAGTGCCACTGAGGCCAGAAGGAACCACACCCTCAAGGGTGGGGAGTTATGGTATGGGGGGTCCTAGGCATGGAGTCTTTTAATTCTTTAGACAATCCTGGGAGCAACTGTCCCTGTTTCACAGAGGGCGGGGCCACACAGCTGGTGAGTGGGCAGCCAAGACTCTGTTCAAGTTTGTGTGGGTCCAACACTTGCGGCCACGGTGGAGGGGCATCTGAGCCAGGCCTCAGAGAGTGGCGGGGGAAGTTGGGTGGGGAAGTGTGCCCTTCTCATTCCTCTGAGGCTCATCCTCTTGGTGCCTCTCTTTCATGGAAAGGGATAATAAGGTTATTGTGAGGATCCCCTGAGTTCGTATATTCAGACGCTTAGACAGAGCCAGGCACAGAGAAGGGCCCGGGGTTGGCTAGTTTGATTGCTGGTGTAATTGCTAATATCTTCCAGTTTGTATTGGTCAAGGTTCTGCAGAGAAGCAGAACCAGTAGGATGTATATATTAAGAGTTTCAAGCTCATGTGACCGTGCGGGCTGGCAAGTCTGAAATCCGCAGGGCAGGCCAGGCAGGCTGGCAATTCCTGCAGAATTTGATGTTGCAATACTGAGTCCTAAGGCAGTCCTGGGGCAGAATTCCTTCTTCCCTGGGAGGCCTCAGTCTGTTCTCTTAAGGCCTWSGR Attorney Docket No. 53989-743.601TCAACTGATTAAATGAGGCCTGCCCAAGTTATAGAGAGTAACCTGCCTTACTCCGTCTTCTGATTTAAATGTTAGTCACATCTAAAAAATATTTTCGCAGCAGCATTTCCACTGGCTTTTGACCAAACATCAGGCCACAAAGTTGATCCCCAAAATTAACCATCACTCTGTGCCTGTAAGGGAGGGGCTGGGAAAGGGGAGCAGGTCTCCCCAAGGGGTGACCTTGGCTTTGTTCCTCCCAGGCCTGGAGTTTATTCGGAAAAGCCAGCTGGTCCAGCCTGTGGGGCCACTGGTGGTGCTGCTGCCCCTGGCGGGTGGGTACAGCCGCGTCCTCAACGCCGCCTGCCAGCGCCTGGCGAGGGCTGGGGTCGTGCTGGTCACCGCTGCCGGCAACTTCCGGGACGATGCCTGCCTCTACTCCCCAGCCTCAGCTCCCGAGGTAGGTGCTGGGGCTGCTGCCCCAAGGCGCGGGTAGGGGGCGGAGGGCGGAGGGCGGAGGGAGGGCGGGCGGGCAGGCGGGCTTCTTGTGGCACGTGGGCTTCTTGTGGCACGTTCCTGGAGGCCGAACCCTTCTGGCTTTGGAAGGAGTCGTCAGAGACCCCCGCCATGCGGGAGGCTGGGGAGGAAGGGGCTCGAAACCTCCATCATCGCAGAGTCTGAATAGCAGTGGCCCCGCCATGCGCCCACGTAGCGGCGCCTACGTAGCCACGCCCCCACACCCCGTCCTGGCCACTCTCCCTCCTGAAGGTCTTCTGGTACCCGCCCCCTCCCCATCTCCATCCCCAGGCCCTGCGTCCTCTGCCCAATACTCTTTGGGCCTCCCTGTTGTCCAGCTCTCTCCGCGGCTCCATGACTGACAACTTGAGCAAGGCTAATGTGAATGGGAGCGGTTGAGGGCTCAGACCTCTCACCCGAGGAACATCCACAGAGTGTGCCGCATGCCCGGTGCAGTGTGGCTGCGGGGACACAGACACGGAGCCTCGGCCCTGAGGAGCTGGGGGGCAGTGACCGTCCCTCCTCTGACCCACCACTCCTCCAGTGTCAGGACACTGCGGGTATCTAGGGGAAGGAATCTTGTTCCACTTCAAGTCTGGAACTTCAAGTCTGTGTGTGTGCGTGCGCGCGCGCGCGTTGGGGGTGGGGGTTGCAGAGCAGATGCGTACCTGACAGCGGTAACCTAGGTCCCCCCTGGCCTATCAAGGCTTCCCTGGCGGCCGAATTTAAAGGCATCAAGCAAACAAAGCCCAACACATCTCTGCCTTGTCCTCTCAGTTTCCCCCCGTGGCACTTAGAACCACTTGATACACCGAATAGTTTCCTATCTCCCCCACTAGGATGTAAACTCCACAGGGGCATTGGGAATGCTGCCTGGCTATGGTAGGGACAGAGGGGAGCACCAGGGCGGGGCAGGGGTGCCAGAGTTCTGCCTGGGCAGTCAGATTTTCCTTAGGAGGGGACATTTGAGTGGGACCCAAACAGGTGTATAGCAGTTGTCCAGCCCAGCTGGCAAGGCCTGAGTCTGCCTCTGCAACCCCTCTCTTGGGCTCCTTTCTCTGCCACCCACCTCCTCACCTTTCCAGGTCATCACAGTTGGGGCCACCAATGCCCAAGACCAGCCGGTGACCCTGGGGACTTTGGGGACCAACTTTGGCCGCTGTGTGGACCTCTTTGCCCCAGGGGAGGACATCATTGGTGCCTCCAGCGACTGCAGCACCTGCTTTGTGTCACAGAGTGGGACATCACAGGCTGCTGCCCACGTGGCTGGTAAGTCACCACCCCACTGCCTCGGCCACCGTGATGCTAACAGCCCCTTTGGCAGTCAGGGTCTGTGCCGGGACCTCCAGTGCCAGGCTCTGTGCAGGGGGACCAGAGATGAAGTAGGCCTGATGGTGCCTTCAAGGACACTCAGTCTGATGAGGGAGGCGAGTGCACAGAGGAAACACGAGGTCAGGGCTGTATTAGAGGGAGCCCAGAGGAGGCACCTGCCCAGCCCGAGGGTCAGAGAAGGCATCTTGGAGGAGGGACATTTGATCGGGAGCTTGATGGATGAATAGGAGTTCACCTGGCCGATAAGACAGCAACTACCAAGGCTTAGAGGTGTGAGAGGAGGCTGTCTTACCTCACTGAGTAAGGACTGCAGGCGGCTTACCTTCGAGAAGAGAGCTTAGTGTCTGTGTGCACGTGTGTTTGTGTGTATGTGTGTGCGTGTGTGCACTGGCAGGAGTCCCCTGCTGGGGCAGGAGGGCCGGGCCATCACCATCTTTCACCATTCACCCCTGCACCAGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCACCCTGGCCGAGTTGAGGCAGAGACTGATCWSGR Attorney Docket No. 53989-743.601CACTTCTCTGCCAAAGATGTCATCAATGAGGCCTGGTTCCCTGAGGACCAGCGGGTACTGACCCCCAACCTGGTGGCCGCCCTGCCCCCCAGCACCCATGGGGCAGGTAAGCAGGATGGCAGGGTGGGCAAGTCCAGGCTGGGGCTTGGGAGGTCTGTGTGACCTTGACAGTCTCTCCCTTCTCCCTTGTCTGTGTAAGGAGGATGACGCCACCTTAAATAGGATTAAATGAGAATGGGGCTCTGAAAGGGCTGTGCAATATTTTCATAACGTGTTTTTATAGAGACAGTTGAGTATGTTCTTTAAGCCCTCCTCTCTCCTACCATGAACTAAAGATTTCTGTGGAGGTCCCCTCACTCCCAGCACCCCCTCCTCATCCCAGGCCCTTTTTGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCCACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGTGACTGTACCCCTCCTTCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCAGTGCTGGGCCCTCAGGGACCCCCAGCAAGCCCCTCCATCCTCCAGACTCCAGCTCTTCTGTAAGCTTACAGGGCTGGCCAGACCAGGAGTGGGGCACTCCTCACTTCACGCGGCTGGGGGCTGCTGGAGAGAGCCACAGCGGGAAGGGTTTCCTAGAGGCTGCAGGACAGTGCTGGATGGATTTTCAATGCTCACCTGGGTGTGAGCGTGCGGCAGGGCCGCGTGAGGGTCAGCGATCTGCTACTCTGGACTCAGCCATCTCTAGGCCCCTCTCACTCAGGTGCTCCATGGTTCTGGGAGCTGAGAAATCTCAAACCAGCAAAAAAGTGGAATTGATGTTGATGCTACAGGATAGTGCACAGATGCCATCTGGTTGCAGCATTTTGGTGGAAGGGCAGTGCCCAGCTAGGAGAGTGAGGAGGGGCAGGCATTTCTGGCTTGAGGAGATGGGGTCTTAATGCTCGTGTGAGAGGCAGAGTGGGTGGAGTGGAGCTGGCTGGATCCTTGCTTTGGCCTCCTGGATTTCTCTCTATCTCCATTTTGAAACCACTCTGTGTTTGGAAGAACTTTTGAGTATTCAGAGCTGCCCACTGGCAGAACAGTCTTCCTTGGGCAGGAGTGAGCTCCTTGTCCCCAGAAGGCTGGGTCTGGCTGGCCCCTGGCAGGGACACTGATGAGGGTGCTTGAGTTGATCCTGTCTAGTCCCTTTCTGTGTTTTCAAAGCCCATTCTAAAGCAGATTCCCATTTCCGTCTTTGACTCTAAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGTAGGAGGCTGGGCTTGCCCTGGGGTGAGGAGGGGTCTCTTTCTCCTTATGCACCCACTGCCCGCGAGGCTTGGTCCTCACAAGTGTGATCCATGAGACTCAAGCCTGACTTGCAGTTCCATACTCTGGTTCTGCCACTTCCATGCCCTTTGAGCCTGGGCAGGTGACCTTACTTCTCCTCATCTCAGCTTCCTCCTCCATAAGAGGGAAAAAGGTATTACCTGCCTCATTGTGTTGCAAGGAGATGGGCAGCATCTAGGGCACTGGCCTGGAGTATCGCAGGTGCTTTGCCTAAGGTGGTGCAGTCCAGGAGAGGCAGCTCCAGAGAGAGGCCCCCGGCTGGGGCTGAAAGGAGGGCAGACCTCGGTTTGAATTTCACCCTGCCGCTCTATAGCTGTGTGACTTGGGCAAATTACTTAACATCTCTGTATGAGGAAATGATGAGTGCTAAGCACTTAGCTTAGTGCCGGGACAATATAAATTCTAGCTATCGTTACTATTGTTTTCATCACCCGTTGCTTTAAAATCCAGCCTCTGGTATAGGCAACTATTGACGGGCTACCCTGTGTCGAAAACATGCCCAGGCAGGTAGCAGGAAGTCACAGATGGGGACCTCTTGGGGCATCAAGGGATGGTGCCCTGAGGCTGAGCTGTTCTGGTTGGGTGGAGCATGAGAGGTCTGGGAAGACAGTGGGACTCCAGCCTGGAATAAGAGGCTCAGAGTTGATTCTCGTCTGAGCACGTCCAGGGGAACCACTGAGGGTTTGGGAACAGGAGAGTGAGGGTGAGAACCTGGTTCTGGWSGR Attorney Docket No. 53989-743.601GCACAGCAGGCTGGCATGTAGGATGGATGTTCAGGAAAGATGAGCATAGTCAGGTGGCTGGTGCCCTTGTCCAGGGGAGAGGCTCCGTCAGGTTCAGGGGTCCTGGCTTGGAGGGAAGTCCGCCATGCTCTAATCACGCTCCCCTTTGGAAGTGCTCAGCCGATGAGCTCACAGGCACATGTCAGTTTGAAGTCATGGAATCTGACTCCATGAAGCGCACCTCAAAGAGCACCATTTTGCAGCTAAGGGAACTGCAGGCTGGACATGCTGAGTGGCTGCCCCGAGCCCTTGCAGCTAGGACATAGAGAATGCTAGTAACCACAACCCTACCATGTTCAGAGCACATGCCAGGCTCCATGCTGGGGCTTCGCACGTGTCATCTTCACAGTGTCCCTGTGAGTAGGTGTGGTTTCTCTTTCCATCTTACAAATGAGTAAACAGAGCCTCAGTGTAGCTAAGTAACCACTATTTTAGGTTTCTTAGCCAATGGGTGTGTCTGACTCCTAAGCCCATGGAGGGCATTCTGAGGTGGTTCAGACAGACCCCGGCTTACCCTTGAACTTCTGCCTGCTGGCTGCATAGGGAGGGGCTGGGGGGAGTTTGAGCATCTCAGGCCATAGAGCCCCTGCCTCACTGTCTCCATCTCTGGGTGGAAAGATGGTGTTTTCCCTGAGAAACTAAGGCTCAGAGAGGTTGAATGGCTCTCCCAAGGTCACACAGCTGGTCAGCTGCAGAGTTGAGAACACAGGAGTCCTGGTGCTCAGGCCAGCATCTCTTTTTTTCTTTGAGTTGTTTCTAGGTTTCCTAGCTCTTGCCTCAGACCTTAAAGAGAGAGGGTCTGATGGGGATGGGCACTGGAGACGGAGCATCCCAGCATTTCACATCTGAGCTGGCTTTCCTCTGCCCCAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGAAGAGGCCCGTGAGGCCGGGTGGGTGGGGTGCTGCGTGTCTCTCCTGCACAGCTTTTCTGTGTCAGTTTGTGCCACCACCATACCGCCATGCATCAGGGTGGCGGTTTGCCAGGTAGATGCTGTGGGCAGCTTCCGCCATTGTGTGGACAGCATGTATATGTGTCTCTGTGTGGCTGGGTCTGTTTTTGCTTTTGTCCAGATCAGTAAGGTTTGCTACCTGGGTACCCCACTCCACTTGGAGTAGAATGTGCATAAATATGGCATAAAGAAATGCAATATGCATGCATTTATTGATTGATCTATTTTTTTCTGAGATGGGGTCTTGCTGTGTTGCCCAGGCTGGTCTCAAATTCCTGGGCTCAAGCAATCCTCTGGTCTCAGCCTCCCCAAGTGTTGGGATTATAGGCATGAGCCGCTGCACCTGGCCTCTCTGATCTATTTAACAAACCTGCTGGGAGGGTCTCAGGGTCAGGAGCAGCACTGGGCTCTGAGGACACAGAGCTCACTCAGCCGTGACCCAGAGGGGGTGCCTGAGCTGCATGCTGAAGGTTGTTAGCATGACCAGCAAGGCAAGAAAAGGCCCTGCCGAGATTAGCAAGGCATGTGCCAAGCCCTGGAATGTGACAGCCGGGCCTTCTAGAAACCTGAGTGTATAACTCTCCTTAAAAGCCAGTAGGAGCTCCTCAAAAGGCAGCCCTAAGGAGTCCACTCTTAAATGAACTCAGAGTCAGTTTTAAAATGCAAGTCTGTGTTGATTCTGGTCTGGATGGTGCATTCCTCGAGAGCAAAAGACAGTCTTGGTCTTGGATCCACTTGCCCTGGGTACACTGAGGGCTGCTAGGTTCCAGGTGCTCTTCCTGGCACTGGGGAGGGATACAGGCCCAAGAGACATGCTGTTCTCCCTCCTGGAGCATCTATTTTAGTGGAGGAAGACAGAAAACAAACCATTAATATAGAGTACTGAAAAGATGCGATGGAGAAAACTATAGCAAGGAAGGGAATGGGGTGGGAGAGAGGTCAGGAGAGGTCTCGCTGACAAGGTGGACGAAACAGGCCATGAGGCAGAGAACATGTTCCAGGCAAAGCAAAGGCCCCCAGGTGGGGATGTGCAGGGAGTACCAGGAAACCAGAGAGGTGGGAATAGTTATGAGATGGGGGGTGCCTCAGAGGGGACAGGGCCAAGTCAGGTGAGACCTGAGGGTCACAGTCAGCAGTGAGCTGGGGCCATGCAGGGGTCTGGCCTCAGAGGAGTGTGGTCWSGR Attorney Docket No. 53989-743.601TGGCCTGGATCTGAACCTCTCACTGTGGCCTAGCTGCTGAGCTGAGAAGAGATGACAAGGACCTTGGGCAGAAGCAGGGAGACTGGAGGGAGGCGGTGGAGGGTCCAGGCGTTGGGGCGGGGCTCAGGCTGGAGTCTGAAGGGAGCCTGCAGGCCTGGTGGGTGGATGTGGGTGGGAGAGGGGGAGGATGGCACCAAGGCTCGGGCCCCTGGACAGATGGAGTTGCCATTAAGTGGGATGGGGCAGGCTATGGGGCCATCAGTTTCAGAGGGATGAGTTTGGCACTGGCATGGTAGGCATCTGTCTATCTCCACGGCCCTCAAACCAGGCATGAAGCAGGAGCTCACGTGTTTGGTCAGCCATGGTGCAGAACCGCCTGGGTGGGAGGTGCGGGGTGGGAGATACACGGTTGTGTCCCAAATGGGCTCTGAGCCAGCGAGGGCCGTCTGCACTTTGGCCTCACAGAAGGATGTCGGAGGGAGAAATGAAGTGTGGGTGGGGGTCCCGGGCCACGCTAGACATGTGCTTTCTTTTCCTCGGGCTCTGGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGGGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGACAGCCCCATCCCAGGATGGGTGTCTGGGGAGGGTCAAGGGCTGGGGCTGAGCTTTAAAATGGTTCCGACTTGTCCCTCTCTCAGCCCTCCATGGCCTGGCACGAGGGGATGGGGATGCTTCCGCCTTTCCGGGGCTGCTGGCCTGGCCCTTGAGTGGGGCAGCCTCCTTGCCTGGAACTCACTCACTCTGGGTGCCTCCTCCCCAGGTGGAGGTGCCAGGAAGCTCCCTCCCTCACTGTGGGGCATTTCACCATTCAAACAGGTCGAGCTGTGCTCGGGTGCTGCCAGCTGCTCCCAATGTGCCGATGTCCGTGGGCAGAATGACTTTTATTGAGCTCTTGTTCCGTGCCAGGCATTCAATCCTCAGGTCTCCACCAAGGAGGCAGGATTCTTCCCATGGATAGGGGAGGGGGCGGTAGGGGCTGCAGGGACAAACATCGTTGGGGGGTGAGTGTGAAAGGTGCTGATGGCCCTCATCTCCAGCTAACTGTGGAGAAGCCCCTGGGGGCTCCCTGATTAATGGAGGCTTAGCTTTCTGGATGGCATCTAGCCAGAGGCTGGAGACAGGTGCGCCCCTGGTGGTCACAGGCTGTGCCTTGGTTTCCTGAGCCACCTTTACTCTGCTCTATGCCAGGCTGTGCTAGCAACACCCAAAGGTGGCCTGCGGGGAGCCATCACCTAGGACTGACTCGGCAGTGTGCAGTGGTGCATGCACTGTCTCAGCCAACCCGCTCCACTACCCGGCAGGGTACACATTCGCACCCCTACTTCACAGAGGAAGAAACCTGGAACCAGAGGGGGCGTGCCTGCCAAGCTCACACAGCAGGAACTGAGCCAGAAACGCAGATTGGGCTGGCTCTGAAGCCAAGCCTCTTCTTACTTCACCCGGCTGGGCTCCTCATTTTTACGGGTAACAGTGAGGCTGGGAAGGGGAACACAGACCAGGAAGCTCGGTGAGTGATGGCAGAACGATGCCTGCAGGCATGGAACTTTTTCCGTTATCACCCAGGCCTGATTCACTGGCCTGGCGGAGATGCTTCTAAGGCATGGTCGGGGGAGAGGGCCAACAACTGTCCCTCCTTGAGCACCAGCCCCACCCAAGCAAGCAGACATTTATCTTTTGGGTCTGTCCTCTCTGTTGCCTTTTTACAGCCAACTTTTCTAGACCTGTTTTGCTTTTGTAACTTGAAGATATTTATTCTGGGTTTTGTAGCATTTTTATTAATATGGTGACTTTTTAAAATAAAAACAAACAAACGTTGTCCTAAC

[0150] Human PCSK9 Amino Acid Sequence (NP 777596.2)MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQWSGR Attorney Docket No. 53989-743.601 GKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAAGNFRD DACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGTS QAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHG AGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHN AFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKP PVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPG TSHVLGAYAVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ.Lipid Nanoparticle (LNP) excipients

[0151] The pharmaceutical composition disclosed herein can further comprises LNP excipients.

[0152] The LNP excipients described herein can be designed for one or more specific applications or targets. The elements of a nanoparticle composition or a composition can be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, and availability. Similarly, the particular formulation of a nanoparticle composition is a composition comprising one or more described lipids, may be selected for the particular application or target. Suitable phosphate charge neutralizers to be used in formulations include, but are not limited to, Spermidine and 1,3- propanediamine.

[0153] In some embodiments, the LNP excipients comprise the amino lipid, the PEG lipid and the GalNac lipid. In some embodiments, the LNP excipients further comprise a sterol and a phospholipid. In some embodiments, (a) the amino lipid is present at about 39 to about 59 mol% of the total LNP excipients, the PEG-lipid is present at about 1.9 to about 4.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.02 to about 0.1 mol% of the total LNP excipients, the sterol is present at about 27.3 to about 50.2 mol% of the total LNP excipients, and the phospholipid is present at about 5.9 to about 13.4 mol% of the total LNP excipients, (b) the amino lipid is present at about 50 ± 12 mol% of the total LNP excipients, the PEG-lipid is present at about 3 ± 1.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.05 ± 0.04 mol% of the total LNP excipients, the sterol is present at about 37.95 ± 12.3 mol% of the total LNP excipients, and the phospholipid is present at about 9 ± 4.4 mol% of the total LNP excipients, or (c) the amino lipid is present at about 9.4 to about 26.3 mg / mL of the pharmaceutical composition, the PEG-lipid is present at about 2. 1 to about 4. mg / mL of the pharmaceutical composition, the GalNAc lipid is present at about 0.03 to about 0.14 mg / mL of the pharmaceutical composition, the sterol is present at about 4.5 to about 8.2 mg / mL of the pharmaceutical composition, and the phospholipid is present at about 1.8 to about 4.3 mg / mL of the pharmaceutical composition.

[0154] In some embodiments, the amino lipid is present at about 50 mol% of the total LNP excipients, the PEG-lipid is present at about 3 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.05 mol% of the total LNP excipients, the sterol is present at about 37.95 mol% of the total LNP excipients, and the phospholipid is present at about 9 mol% of the total LNP excipients. In some embodiments, the pharmaceutical composition has a particle size between 65 ± 15 nM Z-averageWSGR Attorney Docket No. 53989-743.601 hydrodynamic diameter. In some embodiments, the pharmaceutical composition has a particle size between 65 ± 5 nM Z-average hydrodynamic diameter. In some embodiments, the pharmaceutical composition has a particle size between 65 ± 10 nM Z-average hydrodynamic diameter. In some embodiments, the pharmaceutical composition has polydispersity index of at most 0.2 as determined by dynamic light scattering. In some embodiments, at least 85% of the total amount of the RNA and the guide polynucleotide is present in the pharmaceutical composition at pH 7.0-8.0. In some embodiments, at least 60% of the guide polynucleotide in the pharmaceutical composition is the full-length guide polynucleotide at pH 7.0-8.0. In some embodiments, at least 70% of the RNA in the pharmaceutical composition is the full-length RNA at pH 7.0-8.0. In some embodiments, In some embodiments, In some embodiments, In some embodiments, In some embodiments, In some embodiments,

[0155] In some embodiments, LNPs are composed of an amino lipid, a PEG-Lipid, aN- acetylgalactosamine (GalNAc) lipid, a sterol, and a phospholipid. In some embodiments, LNPs are composed of proprietary ionizable cationic lipid, l,2-distearoyl-sn-glycero-3 -phosphocholine, cholesterol, an ionizable amino lipid, and a PEG-lipid. In some embodiments, the LNP comprises about 50 mol% of the amino lipid, about 3 mol% of the PEG-lipid, about 0.05 mol% of the N-acetylgalactosamine (GalNAc) lipid, about 37.95 mol% of the sterol, and about 9.0 mol% of the phospholipid. In some embodiments, the LNP has a particle size between 50-80 nM Z-average hydrodynamic diameter. In some embodiments, the LNP has polydispersity index of <0.2 as determined by dynamic light scattering. In some embodiments, the LNP comprises at least 85% total RNA by weight at pH 7.0-8.0. In some embodiments, at least 60% of the guide polynucleotide in the LNP is the full-length guide polynucleotide at pH 7.0-8.0. In some embodiments, at least 70% of the RNA in the LNP is the full-length RNA at pH 7.0-8.0. In some embodiments, the ratio of the total lipid and total RNA is about (27 ± 5.4) : I by weight. In some embodiments, the ratio of (1) the total LNP excipients and (2) the total amount of the RNA and the guide polynucleotide is about (27 ± 2.7) : I by weight. In some embodiments, the ratio of (1) the total LNP excipients and (2) the total amount of the RNA and the guide polynucleotide is about (27 ± 1.35) : 1 by weight. In some embodiments, the ratio of the total lipid and total RNA is about 27: 1 by weight.

[0156] In some embodiments, LNPs have an average hydrodynamic diameter of about 30 - about 160, about 35 - about 160, about 40 - about 160, about 45 - about 160, about 50 - about 160, about 55 - about 160, about 60 - about 160, about 65 - about 160, about 70 - about 160, about 75 - about 160, about 80 - about 160, about 85 - about 160, about 90 - about 160, about 95 - about 160, about 100 - about 160, about 105 - about 160, about 110 - about 160, about 115 - about 160, about 120 - about 160, about 125 - about 160, about 130 - about 160, about 135 - about 160, about 140 - about 160, about 145 - about 160, about 150 - about 160, about 30 - about 155, about 30 - about 150, about 30 - about 145, about 30 - about 140, about 30 - about 135, about 30 - about 130, about 30 - about 125, about 30 - about 120, about 30 - about 115, about 30 - about 110, about 30 - about 105, about 30 - about 100, about 30 - about 95, about 30 - about 90, about 30 - about 85, about 30 - about 80, about 30 - about 75, about 30 - about 70, about 30 - about 65, about 30 - about 60, about 30 - about 55, about 30 - about 50, about 30 - about 45, about 30 - about 40, about 30 - about 45, about 35 - about 45, about 35 - about 50, about 40 - about 50, about 40 -WSGR Attorney Docket No. 53989-743.601 about 55, about 45 - about 55, about 45 - about 60, about 50 - about 60, about 50 - about 65, about 50 - about 80, about 55 - about 60, about 55 - about 65, about 55 - about 70, about 60 - about 70, about 60 - about 75, about 65 - about 75, about 65 - about 80, about 70 - about 80, about 70 - about 85, about 75 - about 85, about 75 - about 90, about 80 - about 90, about 80 - about 95, about 85 - about 95, about 85 - about 100, about 90 - about 100, about 90 - about 105, about 95 - about 105, about 95 - about 110, about 100 - about 110, about 100 - about 115, about 105 - about 115, about 105 - about 120, about 110 - about 120, about 110 - about 125, about 115 - about 125, about 115 - about 130, about 120 - about 130, about 120 - about 135, about 125 - about 135, about 125 - about 140, about 130 - about 140, about 130 - about 145, about 35 - about 140, about 45 - about 130, about 55 - about 120, about 65 - about 110, about 75 - about 100, or about 85 - about 90 nm.

[0157] In some embodiments, LNPs comprise about 1 - about 97, about 5 - about 97, about 10 - about 97, about 15 - about 97, about 20 - about 97, about 25 - about 97, about 30 - about 97, about 35 - about 97, about 40 - about 97, about 45 - about 97, about 50 - about 97, about 55 - about 97, about 60 - about 97, about 65 - about 97, about 70 - about 97, about 75 - about 97, about 80 - about 97, about 1 - about 95, about 1 - about 90, about 1 - about 85, about 1 - about 80, about 1 - about 75, about 1 - about 70, about 1 - about 65, about 1 - about 60, about 1 - about 55, about 1 - about 50, about 1 - about 45, about 1 - about 40, about 1 - about 35, about 1 - about 30, about 1 - about 25, about 1 - about 20, about 1 - about 15, about 1 - about 10, about 10 - about 30, about 10 - about 35, about 15 - about 35, about 15 - about 40, about 20 - about 40, about 20 - about 45, about 25 - about 45, about 25 - about 50, about 30 - about 50, about 30 - about 55, about 35 - about 55, about 35 - about 60, about 40 - about 60, about 40 - about 65, about 45 - about 65, about 45 - about 70, about 50 - about 70, about 50 - about 75, about 55 - about 75, about 55 - about 80, or about 60 - about 80% of amino lipids (in mol%).

[0158] In some embodiments, LNPs comprise about 1 - about 40, about 1 - about 38, about 1 - about 36, about 1 - about 34, about 1 - about 32, about 1 - about 30, about 1 - about 28, about 1 - about 26, about 1 - about 24, about 1 - about 22, about 1 - about 20, about 1 - about 18, about 1 - about 16, about 1 - about 14, about 1 - about 12, about 1 - about 10, about 1 - about 8, about 1 - about 6, about 1 - about 4, about 1 - about 2, about 2 - about 40, about 4 - about 40, about 6 - about 40, about 8 - about 40, about 10 - about 40, about 12 - about 40, about 14 - about 40, about 16 - about 40, about 18 - about 40, about 20 - about 40, about 22 - about 40, about 24 - about 40, about 26 - about 40, about 28 - about 40, about 30 - about 40, about 32 - about 40, about 34 - about 40, about 36 - about 40, about 38 - about 40, about 2 - about 35, about 2 - about 30, about 2 - about 25, about 2 - about 20, about 2 - about 15, about 2 - about 10, about 2 - about 8, about 2 - about 6, or about 2 - about 4% phospholipid (in mol%).

[0159] In some embodiments, LNPs comprise about 1 - about 20, about 1 - about 19, about 1 - about 18, about 1 - about 17, about 1 - about 16, about 1 - about 15, about 1 - about 14, about 1 - about 13, about 1 - about 12, about 1 - about 11, about 1 - about 10, about 1 - about 9, about 1 - about 8, about 1 - about 7, about 1 - about 6, about 1 - about 5, about 1 - about 4, about 1 - about 3, about 2 - about 6, about 3 - about 7, about 4 - about 8, about 5 - about 9, about 6 - about 10, about 7 - about 11, about 8 - about 12, about 9 -WSGR Attorney Docket No. 53989-743.601 about 13, about 10 - about 14, about 11 - about 15, about 12 - about 16, about 13 - about 17, about 14 - about 18, about 15 - about 19, or about 16 - about 20% PEG-Lipid (in mol%).

[0160] In some embodiments, LNPs comprise about 1 - about 97, about 5 - about 97, about 10 - about 97, about 15 - about 97, about 20 - about 97, about 25 - about 97, about 30 - about 97, about 35 - about 97, about 40 - about 97, about 45 - about 97, about 50 - about 97, about 55 - about 97, about 60 - about 97, about 65 - about 97, about 70 - about 97, about 75 - about 97, about 80 - about 97, about 1 - about 95, about 1 - about 90, about 1 - about 85, about 1 - about 80, about 1 - about 75, about 1 - about 70, about 1 - about 65, about 1 - about 60, about 1 - about 55, about 1 - about 50, about 1 - about 45, about 1 - about 40, about 1 - about 35, about 1 - about 30, about 1 - about 25, about 1 - about 20, about 1 - about 15, about 1 - about 10, about 10 - about 30, about 10 - about 35, about 15 - about 35, about 15 - about 40, about 20 - about 40, about 20 - about 45, about 25 - about 45, about 25 - about 50, about 30 - about 50, about 30 - about 55, about 35 - about 55, about 35 - about 60, about 40 - about 60, about 40 - about 65, about 45 - about 65, about 45 - about 70, about 50 - about 70, about 50 - about 75, about 55 - about 75, about 55 - about 80, or about 60 - about 80% of cholesterol (in mol%).

[0161] In some embodiments, LNPs comprise about 1 - about 97, 5 - about 97, 10 - about 97, 15 - about 97, 20 - about 97, 25 - about 97, 30 - about 97, 35 - about 97, 40 - about 97, 45 - about 97, 50 - about 97, 55 - about 97, 60 - about 97, 65 - about 97, 70 - about 97, 75 - about 97, 80 - about 97, 1 - about 95, 1 - about 90, 1 - about 85, 1 - about 80, 1 - about 75, 1 - about 70, 1 - about 65, 1 - about 60, 1 - about 55, 1 - about 50, 1 - about 45, 1 - about 40, 1 - about 35, 1 - about 30, 1 - about 25, 1 - about 20, 1 - about 15, 1 - about 10, 10 - about 30, 10 - about 35, 15 - about 35, 15 - about 40, 20 - about 40, 20 - about 45, 25 - about 45, 25 - about 50, 30 - about 50, 30 - about 55, 35 - about 55, 35 - about 60, 40 - about 60, 40 - about 65, 45 - about 65, 45 - about 70, 50 - about 70, 50 - about 75, 55 - about 75, 55 - about 80, or 60 - about 80% of amino lipids; 1 - about 40, 1 - about 38, 1 - about 36, 1 - about 34, 1 - about 32, 1 - about 30, 1 - about 28, 1 - about 26, 1 - about 24, 1 - about 22, 1 - about 20, 1 - about 18, 1 - about 16, 1 - about 14, 1 - about 12, 1 - about 10, 1 - about 8, 1 - about 6, 1 - about 4, 1 - about 2, 2 - about 40, 4 - about 40, 6- about 40, 8 - about 40, 10 - about 40, 12 - about 40, 14 - about 40, 16 - about 40, 18 - about 40, 20 - about 40, 22 - about 40, 24 - about 40, 26 - about 40, 28 - about 40, 30 - about 40, 32 - about 40, 34 - about 40, 36 - about 40, 38 - about 40, 2 - about 35, 2 - about 30, 2 - about 25, 2 - about 20, 2 - about 15, 2- about 10, 2 - about 8, 2 - about 6, or 2 - about 4% DSPC; 1 - about 20, 1 - about 19, 1 - about 18, 1 - about 17, 1 - about 16, 1 - about 15, 1 - about 14, 1 - about 13, 1 - about 12, 1 - about 11, 1 - about 10, 1 - about 9, 1 - about 8, 1 - about 7, 1 - about 6, 1 - about 5, 1 - about 4, 1 - about 3, 2 - about 6, 3 - about 7, 4- about 8, 5 - about 9, 6 - about 10, 7 - about 11, 8 - about 12, 9 - about 13, 10 - about 14, 11 - about 15, 12 - about 16, 13 - about 17, 14 - about 18, 15 - about 19, and about 16 - about 20% PEG-Lipid, with the balance being cholesterol (all in mol%).GalNAc lipid

[0162] In some embodiments, the LNPs comprise a GalNAc lipid of formula (V):WSGR Attorney Docket No. 53989-743.601Formula (V) wherein, a plurality of the A groups collectively comprising a receptor targeting ligand; each of L1, L2, L3, L4, L5, L6, L7, L8, L9, L10and L12is independently substituted or unsubstitutedC1-C12 alkylene, substituted or unsubstituted C1-C12 heteroalkylene, substituted or unsubstituted C2-C12 alkenylene, substituted or unsubstituted C2-C12 alkynylene, - (CH2CH2O)m-, -(OCH2CH2)m-, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR1)-, -C(=O)-, - C(=N-OR’)-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)N(R’)-, -N(R’)C(=O)-, - OC(=O)N(R1)-, -N(R1)C(=O)O-, -N(R1)C(=O)N(R1)-, -S(=O)2N(R1)-, -N(R1)S(=O)2-, - N(R’)-, or -N(OR’)-;L11is substituted or unsubstituted -(CH2CH2O)n- or substituted or unsubstituted -(OCH2CH2)n-; each R1is independently H or substituted or unsubstituted Ci-Ce alkyl;R is a lipid; m is an integer selected from 1 to 10; and n is an integer selected from 1 to 200, wherein each of the A groups is a N-acetylgalactosamine moiety

[0163] In some embodiments, each of L1, L4, and L7is independently C2-C6 alkylene; each of L2, L5, and L8is independently -C(=O)NR1- or -NR1C(=O)-; each of L3, L6, and L9is independently C2-C6 alkylene;L10is Ci-Ce alkylene, -CH2CH2O-, or -CH2O-;L11is -(CH2CH2O)n- or -(OCH2CH2)n-, where n is an integer selected from 1 to 50;L12is -C(=O)O-, -OC(=O)-, -C(=O)NR1-, -NR’C(=O)-, -OC(=O)NR1-, or -NR'C(=O)O-: andR is selected from the group consisting of dialkylglycerolyl, diacylglycerolyl, sterol, n-alkyl comprising C10-C30 carbon atoms, branched alkyl comprising C10-C30 carbon atoms or tocopherol.

[0164] In some embodiments, R is dialkylglycerolyl. In some embodiments, R iswherein n = 10-30. In some embodiments, RWSGR Attorney Docket No. 53989-743.601

[0165] In some embodiments,someWSGR Attorney Docket No. 53989-743.601 embodiments,, wherein each of the p and q is independently an integer from 1 to 5, and n is an integer from 1 to 50. In some embodiments, p = 1. In some embodiments, p = 2. In some embodiments, p = 3. In some embodiments, p = 4. In some embodiments, p = 5. In some embodiments, q = 1. In some embodiments, q = 2. In some embodiments, q = 3. In some embodiments, q = 4. In some embodiments, q = 5. In some embodiments, n is 1 to 10. In some embodiments, n is 10 to 20. In some embodiments, n is 20 to 30. In some embodiments, n is 30 to 40. In some embodiments, n is 40 to 50. In some embodiments, n is 33-37. In some embodiments, n is 33. In some embodiments, n is 34. In some embodiments, n is 35. In some embodiments, n is 36. In some embodiments, n is 37.

[0167] In some embodiments, the GalNAc lipid isWSGR Attorney Docket No. 53989-743.601wherein n= 33, 34, 35, 36, or 37. In some embodiments, the GalNAc lipid is, wherein n= 35.

[0169] In any of the above embodiments, the GalNAc-LNPs may comprise one or more the GalNAc lipids described herein.Amino lipid

[0170] Described herein are LNP compositions comprising an amino lipid, a phospholipid, a PEGlipid, a cholesterol or a derivative thereof, a payload, or any combination thereof. In some embodiments, the LNP composition comprises an amino lipid. In one aspect, disclosed herein is an amino lipid having the structure of. Formula (I), or a pharmaceutically acceptable salt or solvate thereof,Fonnula (I) wherein each of R1and R2is independently C7-C22 alkyl, C7-C22 alkenyl, Cs-Cs cycloalkyl,- C2-C10 allkylene-L-R6, or, wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted; each of X, Y, and Z is independently -C(=O)NR4-, -NR4C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, - NR4C(=O)O-, -OC(=O)NR4-, -NR4C(=O)NR4-, -NR4C(=NR4)NR4-, -C(=S)NR4-, -NR4C(=S)- , -C(=O)O-,WSGR Attorney Docket No. 53989-743.601-OC(=S)-, OC(=S)O-, -NR4C(=S)O-, -OC(=S)NR4-, -NR4C(=S)NR4-, -C(=O)S-, -SC(=O)-, -OC(=O)S-, - NR4C(=O)S-. -SC(=O)NR4- , -C(=S)S-, -SC(=S)-, -SC(=S)O-, -NR4C(=S)S-, -SC(=S)NR4-, -C(=S)S-, - SC(=S)-, -SC(=O)S-, -SC(=S)S-, -NR4C(=S)S-, - SC(=S)NR4- O, S, or a bond; each of L is independently -C(=O)NR4-, -NR4C(=O)-, -C(=O)O-. -OC(=O)O-, -NR4C(=O)O-, - OC(=O)NR4-, -NR4C(=O)NR4-, -NR4C(=NR4)NR4-, -C(=S)NR4-, -NR4C(=S)- , -C(=O)O-, -OC(=S)-, OC(=S)O-, -NR4C(=S)O-, -OC(=S)NR4-, -NR4C(=S)NR4-, -C(=O)S-, SC(=O)-, -OC(=O)S-, - NR4C(=O)S-, -SC(=O)NR4- , -C(=S)S-, -SC(=S)-, -SC(=S)O-, - NR4C(=S)S-, -SC(=S)NR4-, -C(=S)S-, - SC(=S)-, -SC(=O)S-, -SC(=S)S-, -NR4C(=S)S-, - SC(=S)NR4-, O, S, -Ci-Cw alkylene-O-, -C1-C10 alkylene-C(=O)O-, -C1-C10 alkylene-OC(=O)-, or a bond, wherein the alkylene is substituted or unsubstituted;R3is -Co-Cio alkylene-NR7R8, -Co-Cw alkylene-heterocycloalkyl, or -Co-Cio alkylene-heterocycloaryl, wherein the alkylene, heterocycloalkyl and heterocycloaiyl is independently substituted or unsubstituted; each of R4is independently hydrogen or substituted or unsubstituted Ci-Ce alkyl;R5is hydrogen or substituted or unsubstituted Ci-Ce alkyl; each of R6is independently substituted or unsubstituted C3-C22 alkyl or substituted or unsubstituted C3-C22 alkenyl; each of R7and R8is independently hydrogen or substituted or unsubstituted Ci-Ce alkyl, or R7and R8taken together with the nitrogen to which they are attached form a substituted or unsubstituted C2-C6 heterocyclyl; p is an integer selected from 1 to 10; and each of n, m, and q is independently 0, 1, 2, 3, 4, or 5.

[0171] In some embodiments of Formula (I), if the structure carries more than one asymmetric C- atom, each asymmetric C-atom independently represents racemic, chirally pure R and / or chirally pure S isomer, or a combination thereof.

[0172] In some embodiments, each of n, in, and q in Formula (I) is independently 0, 1, 2, or 3. In some embodiments, each of n, m, and q in Formula (I) is 1.

[0173] In some embodiments, the compound of Formula (1) has a structure of Formula (la), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof:Formula ( ) wherein each of R1and R2is independently C7-C22 alkyl, C7-C22 alkenyl, C3-C8 cycloalkyl, -C2-C10 alkylene-L-R6,or, wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted;WSGR Attorney Docket No. 53989-743.601 each of X, Y, and Z is independently C(=O)NR4-, -NR4C(D)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, - NR4C(=O)O-, -OC(=O)NR4-, -NR4C=O)NR4-, -NR4C(=NR4)NR4-. -C(=S)NR4-, -NR4C(=S)- , -C(E)O-, - OC(=S)-, OC(=S)O-, -NR4C(=S)O-, -OC(=S)NR4-, -NR4C(=S)NR4-, -C(=O)S-, -SC(=O)-, -OC(=O)S-, - NR4C(=O)S-, -SC(=O)NR4- -C(=S)S-, -SC(=S)-, -SC(=S)O-, - NR4C(=S)S-, -SC(=S)NR4-, -C(=S)S-. - SC(=S)-, -SC(=O)S-, -SC(=S)S-. -NR4C(=S)S-, - SC(=S)NR4-, O, S, -Ci-Cw alkylene-O-, or a bond, wherein the alkylene is substituted or unsubstituted; each of L is independently -C(=O)NR4-, -NR4C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, - NR4C(=O)O-, -OC(=O)NR4-, -NR4C(=O)NR4-, -NR4C(=NR4)NR4-. -C(=S)NR4-, -NR4C(=S)- , -C(=O)O-, -OC(=S)-, OC(=S)O-, -NR4C(=S)O-, -OC(=S)NR4-, -NR4C(=S)NR4-, -C(=O)S-, -SC(=O)-, -OC(=O)S-, - NR4C(=O)S-, -SC(=O)NR4- -C(=S)S-, -SC(=S)-, -SC(=S)O-, -NR4C(=S)S-, -SC(=S)NR4-, -C(=S)S-, - SC(=S)-, -SC(=O)S-, -SC(=S)S-, -NR4C(=S)S-, - SC(=S)NR4-, O, S. -C1-C10 alkylene-O-, -C1-C10 alkylene-C(=O)O-, -Ci-C 10 alkylene- OC(=O)-, or a bond, wherein the alkylene is substituted or unsubstituted;R3is - Co-Cio alkylene-NR7R8, - Co-Cio alkylene-heterocycloalkyl, or - Co-Cio alkylene-heterocyclowyl, wherein the alkylene, heterocycloalkyl and heterocycloaryl is independently substituted or unsubstituted; each of R4is independently hydrogen or substituted or unsubstituted CI-C6 alkyl;R5is hydrogen or substituted or unsubstituted Ci-Ce alkyl; each of R6is independently substituted or unsubstituted C3-C22 alkyl or substituted or unsubstituted C3-C22 alkenyl; each of R7and R8is independently hydrogen or substituted or unsubstituted Ci-Ce alkyl, or R7and R8taken together with the nitrogen to which they are attached form a substituted or unsubstituted C2-C6 heterocyclyl; and p is an integer selected from 1 to 10.

[0174] In some embodiments of Formula (la), if the structure carries more than one asymmetric Catom, each asymmetric C-atom independently represents racemic, chirally pure R and / or chirally pure S isomer, or a combination thereof.

[0175] In some embodiments, R1and R2in Formula (I) and Formula (la) is independently C7-C22 alkyl,C7-C22 alkenyl, -C2-C10 alkylene-L- R6, orwherein each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R1and R2in Formula (I) and Formula (la) is independently C10-C20 alkyl, C10-C20 alkenyl. - G-C? alkylene-L- R6, orwherein each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R1in Formula (I) and Formula (la) isWSGR Attorney Docket No. 53989-743.601

[0176] In some embodiments, each of L in Formula (I) and Formula (la) is independently O, S, -C1-C10 alkylene-O-, - C1-C10 alkylene-C(=O)O-, - C1-C10 alkylene-OC(=O)-, or a bond, wherein the alkylene is substituted or unsubstituted. In some embodiments, each of L in Formula (I) and Formula (la) is independently O, S, - C1-C3 alkylene-O-, - C1-C3 alkylene-C(=O)O-, - C1-C3 alkylene-OC(=O)-, or a bond, wherein the alkylene is substituted or unsubstituted. In some embodiments, each of L in Formula (I) and Formula (la) is independently O, S, - C1-C3 alkylene-O-, - C1-C3 alkylene-C(=O)O-, -C1-C3 alkylene- OC(=O)-, or a bond, wherein the alkylene is linear or branched unsubstituted alkylene.

[0177] In some embodiments, each of R6in Formula (I) and Formula (la) is independently substituted or unsubstituted linear C3-C22 alkyl or substituted or unsubstituted linear C3-C22 alkenyl. In some embodiments, each of R6in Formula (I) and Formula (la) is independently substituted or unsubstituted C3- C20 alkyl or substituted or unsubstituted C3-C20 alkenyl. In some embodiments, each of R6in Formula (I) and Formula (la) is independently substituted or unsubstituted C3-C10 alkyl or substituted or unsubstituted C3-C10 alkenyl. In some embodiments, each of R6in Formula (I) and Formula (la) is independently substituted or unsubstituted C3-C10 alkyl. In some embodiments, each of R6in Formula (I) and Formula (la) is independently substituted or unsubstituted linear C3-C10 alkyl. In some embodiments, each of R6in Formula (I) and Formula (la) is independently substituted or unsubstituted n-pentyl, n-hexyl, n-heptyl, n- octyl, n-nonyl, n-decyl, n-undecyl, or n-dodecyl. In some embodiments, each of R6in Formula (I) and Formula (la) is independently substituted or unsubstituted n-octyl. In some embodiments, each of R6in Formula (I) and Formula (la) is n-octyl.

[0178] In some embodiments, each of L in Formula (I) and Formula (la) is independently -C(=O)O-, - OC(=O)-, -C1-C10 alkylene-O-, or O. In some embodiments, each of. L in Formula (I) and Formula (la) is O. In some embodiments, each of L in Formula (I) and Formula (la) is -C1-C3 alkylene-O-. In some embodiments, p in Formula (I) and Formula (la) is 1, 2, 3, 4, or 5. In some embodiments, p in Formula (I) and Formula (la) is 2.

[0179] In some embodiments, R1in Formula (I) and Formula (la) isWSGR Attorney Docket No. 53989-743.601

[0180] In some embodiments, each of R4in Formula (I) and Formula (la) is independently H or substituted or unsubstituted C1-C4 alkyl. In some embodiments, each of. R4in Formula (I) and Formula (la) is independently substituted or unsubstituted linear C1-C4 alkyl. In some embodiments, each of R4in Formula (1) and Formula (la) is H. In some embodiments, each of R4in Formula (I) and Formula (la) is independently H, -CH3, -CH2CFL, -CH2CH2CFL, or -CH(CH3)2. In some embodiments, each of R4in Formula (I) and Formula (la) is independently H or -CH3. In some embodiments, each of R4in Formula (1) and Formula (la) is -CH3.

[0181] In some embodiments, X in Formula (I) and Formula (la) is -C(=O)O- or -OC(=O))-. In some embodiments, X in Formula (I) and Formula (la) is -C(=O)NR4- or -NR4C(=O)-. In some embodiments, X in Formula (I) and Formula (la) is -C(=O)N(CH3)-, -N(CH3)C(=O)-, -C(=O)NH-, or -NHC(=O)-. In some embodiments, X in Formula (I) and Formula (la) is -C(=O))NH-, -C(=O)N(CH3)-. -OC(=O))-, -NHC(=O)-, -N(CH3)C(=O))-, -C(=O)O-, -OC(=O)O-, -NHC(=O)O-, -N(CH3)C(=O)O-, - OC(=O))NH-, -OC(=O)N(CH3)-, -NHC(=O)NH-, -N(CH3)C(=O))NH-, -NHC(=O)N(CH3)-, - N(CH3)C(=O)N(CH3)-,NHC(=NH)NH-, -N(CH3)C(=NH)NH-, -NHC(=NH)N(CH3)-, - N(CH3)C(=NH)N(CH3)-,NHC(=NMe)NH-, -N(CH3)C(=NMe)NH-, -NHC(=NMe)N(CH3)-, or - N(CH3)C(=NMe)N(CH3)-.

[0182] In some embodiments. R2in Formula (I) and Formula (la) is C7-C22 alkyl, C7-C22 alkenyl, - C2-C10 alkylene-L- R6, or;, wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R2in Formula (I) and Formula (la) is substituted or unsubstituted C7-C22 alkyl or substituted or unsubstituted C7-C22 alkenyl. In some embodiments, R2in Formula (I) and Formula (la) is substituted or unsubstituted linear C7-C22 alkyl or substituted or unsubstituted linear C7-C22 alkenyl. In some embodiments, R2in Formula (I) and Formula (la) is substituted or unsubstituted C10-C20 alkyl or substituted or unsubstituted C10-C20 alkenyl. In some embodiments, R2in Formula (I) and Formula (la) is unsubstituted C10-C20 alkyl. In some embodiments, R2in Formula (I) and Formula (la) is unsubstituted C10-C20 alkenyl. In some embodiments, R2in Formula (I) and Formula (la) is -C2-C10 alkylene-L- R6. In some embodiments, R2in Formula (I) and Formula (la) is - C2-C10 alkylene- C(=O)O- R6or -C2-C10 alkylene-OC(=O)- R6.WSGR Attorney Docket No. 53989-743.601

[0183] In some embodiments, R2in Formula (I) and Formula (la) is

[0184] In some embodiments, Y in Formula (I) and Formula (la) is -C(=O)O- or -OC(=O)-. In some embodiments, Y in Formula (I) and Formula (la) is -C(=O)NR4- or -NR4C(=O)-. In some embodiments, Y in Formula (I) and Formula (la) is -C(=O)N(CH3)-, -N(CH3)C(=O)-, -C(=O)NH-, or -NHC(=O)-. In some embodiments, Y in Formula (I) and Formula (la) is -OC(=O)O-, -NR4C(=O)O-, -OC(=O)NR4-, or - NR4C(=O)NR4-. In some embodiments. Y in Formula (I) and Formula (la) is - OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -N(CH3)C(=O)O-. -OC(=O)N(CH3)-, - N(CH3)C(=O)N(CH3)- or - N(CH3)C(=O)NH-. In some embodiments, Y in Formula (I) and Formula (la) is -OC(=O)O-, -NHC(=O)0- , -OC(=O)NH-, or -NHC(=O)NH-.

[0185] In some embodiments, R3in Formula (I) and Formula (la) is -Co-Cio alkylene -NR7R8or -Co-Cio alkylene-heterocycloalkyl, wherein the alkylene and heterocycloalkyl is independently substituted or unsubstituted. In some embodiments, R3in Formula (I) and Formula (Ta) is -Co-Cio alkylene-NR7R8. Tn some embodiments, R3in Formula (I) and Formula (la) is -Ci-Ce alkylene-NR7R8. In some embodiments, R3in Formula (I) and Formula (la) is - C1-C4 alkylene-NR7R8. In some embodiments, R3in Formula (I) and Formula (la) is -Ci- alkylene-NR7R8. In some embodiments, R3in Formula (I) and Formula (la) is - C2— alkylene-NR7R8. Tn some embodiments, R3in Formula (I) and Formula (la) is -C3- alkylene-NR7R8. In some embodiments, R3in Formula (I) and Formula (la) is -C4- alkylene- NR7R8. In some embodiments, R3in Formula (I) and Formula (la) is -C5- alkylene-NR7R8. In some embodiments, R3in Formula (I) and Formula (la) is - Co-Cio alkylene-heterocycloalkyl. In some embodiments, R3in Formula (I) and Formula (la) is - Ci-Ce alkylene-heterocycloalkyl, wherein the heterocycloalkyl comprises 1 to 3 nitrogen and 0-2 oxygen. In some embodiments, R3in Formula (I) and Formula (la) is -Ci-Ce alkylene-heterocycloaryl.

[0186] In some embodiments, each of R7and R8in Formula (I) and Formula (la) is independently hydrogen or substituted or unsubstituted Ci-Ce alkyl. Tn some embodiments, each of R7and R8is independently hydrogen or substituted or unsubstituted C1-C3 alkyl. In some embodiments, each of R7andWSGR Attorney Docket No. 53989-743.601R8is independently substituted or unsubstituted C1-C3 alkyl. In some embodiments, each of R7and R8is independently -CH3, -CH2CH3, -CH2CH2CH3, or -CH( 013)2. In some embodiments, each of R and R8is CH3. In some embodiments, each of R7and R8is -CH2CH3.

[0187] In some embodiments, R7and R8in Formula (I) and Formula (la) taken together with the nitrogen to which they are attached form a substituted or unsubstituted C2-C6 heterocyclyl. In some embodiments, R7and R8taken together with the nitrogen to which they are attached form a substituted or unsubstituted C2-C6 heterocycloalkyl. In some embodiments, R7and R8taken together with the nitrogen to which they are attached form a substituted or unsubstituted 3-7 membered heterocycloalkyl.

[0188] In some embodiments, R3in Formula (I) and Formula (la) is

[0189] In some embodiments. R3in Formula (I) and Formula (la) is

[0190] In some embodiments. R3in Formula (1) and Formula (la) is

[0191] In some embodiments, Z in Formula (I) and Formula (la) is -C(=O)O- or -OC(=O)-. In some embodiments, Z in Formula (1) and Formula (la) is -C(=O)NR4- or -NR4C(=O)-. In some embodiments, Z in Formula (I) and Formula (la) is -C(=O)N(CH3)-, -N(CH3)C( =O)-, -C(=O)NH-, or -NHC(=O)-. In some embodiments, Z in Formula (I) and Formula (la) is -OC(=O)O-, -NR4C(=O)O-, -OC(O)NR4-, or - NR4C(=O)NR4-. In some embodiments. Z in Formula (I) and Formula (la) is -OC(=O)O-, - NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -N(CH3)C(=O)O-, -OC(=O)N(CH3)-, - N(CH3)C(=O)N(CH3)-, - NHC(=O)N(CH3)- or -N(CH3)C(=O)NH-. In some embodiments, Y in Formula (I) and Formula (la) is - OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, or -NHC(=O)NH-.

[0192] In some embodiments, R5in Formula (I) and Formula (la) is hydrogen or substituted or unsubstituted C1-C3 alkyl. In some embodiments, R5in Formula (I) and Formula (la) is H, -CH3, - CH- )CH3, -CH2CH2CH3, or -CH(CH3)2. In some embodiments, R5in Formula (I) and Formula (la) is H.

[0193] In some embodiments, the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate.WSGR Attorney Docket No. 53989-743.601

[0194] In some embodiments, the amino lipid is

[0195] In some embodiments, the LNP comprises a plurality of amino lipids. For example, the LNP composition can comprise 2, 3, 4, 5, 6.7, 8, 9. 10, or more amino lipids. For another example, the LNP composition can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, or at least 20 amino lipids. For yet another example, the LNP composition can comprise at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 9, at most 10, at most 20, or at most 30 amino lipids.

[0196] In some embodiments, the amino lipid is an ionizable lipid. An ionizable lipid can comprise one or more ionizable nitrogen atoms. In some embodiments, at least one of the one or more ionizable nitrogen atoms is positively charged. Tn some embodiments, at least 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%. 90 mol%, 95 mol%, or 99 mol% of the ionizable nitrogen atoms in the LNP composition are positively charged. In some embodiments, the amino lipid comprises a primary amine, a secondary amine, a tertiary amine, an imine, an amide, a guanidine moiety, a histidine residue, a lysine residue, an arginine residue, or any combination thereof. In some embodiments, the amino lipid comprises a primary amine, a secondary amine, a tertiary amine, a guanidine moiety, or any combination thereof. In some embodiments, the amino lipid comprises a tertiary amine.

[0197] In some embodiments, the amino lipid is a cationic lipid. Tn some embodiments, the amino lipid is an ionizable lipid. In some embodiments, the amino lipid comprises one or more nitrogen atoms. In some embodiments, the amino lipid comprises one or more ionizable nitrogen atoms. Exemplary cationic and / or ionizable lipids include, but are not limited to, 3 -(didodecylamino)- Nl,Nl,4-tri dodecyl-1- piperazineethan amine (KL10), N142-(didodecylamino)ethyl]-Nl,N4,N4- tridodecyl- 1,4- piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21 ,24-tetraaza-octatriacontane (KL25), 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]- dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen- 19-yl 4- (dimethylamino)butanoate (DLin- MC 3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]- dioxolane (DLin-KC2-DMA), 1,2- dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3[3)- cholest-5-en-3-yloxy]octyl}oxy)-N,N- dimethy l-3-[(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA), (2R)-2-( { 8-[(3 (3)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l- amine (Octyl-CLinDMA (2R)), and (2S)-2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA (2S)).

[0198] In some embodiments, an amino lipid described herein can take the form of a salt, such as a pharmaceutically acceptable salt. All pharmaceutically acceptable salts of the amino lipid are encompassed by this disclosure. As used herein, amino lipid also includes its pharmaceutically acceptable salts, and its diastereomeric, enantiomeric, and epimeric forms.WSGR Attorney Docket No. 53989-743.601

[0199] In some embodiments, an amino lipid described herein, possesses one or more stereocenters and each stereocenter exists independently in either the R or S configuration. The lipids presented herein include all diastereomeric, enantiomeric, and epimeric forms as well as the appropriate mixtures thereof. The lipids provided herein include all cis. trans, syn, anti, entgegen (E), and zusammen (Z) isomers as well as the appropriate mixtures thereof. Tn certain embodiments, lipids described herein are prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds / salts, separating the diastereomers and recovering the optically pure enantiomers. In some embodiments, resolution of enantiomers is carried out using covalent diastereomeric derivatives of the compounds described herein. In another embodiment, diastereomers are separated by separation / resolution techniques based upon differences in solubility. In other embodiments, separation of stereoisomers is performed by chromatography or by the forming diastereomeric salts and separation by recrystallization, or chromatography, or any combination thereof.

[0200] In some embodiments, the lipids such as the amino lipids are substituted based on the structures disclosed herein. In some embodiments, the lipids such as the amino lipids are unsubstituted. In another embodiment, the lipids described herein are labeled isotopically (e.g. with a radioisotope) or by another other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.PEG-Lipid

[0201] As used herein, a “PEG lipid” or “PEG-lipid” refers to a lipid comprising a polyethylene glycol component.

[0202] In some embodiments, the described LNP composition comprises a PEG-lipid. In some embodiments, the described LNP composition comprises two or more PEG-lipids. Exemplary PEG- lipids include, but are not limited to, the lipids. Exemplary PEG-lipids also include, but are not limited to, PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG- modified ceramides, PEG- modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, the one or more PEG-lipids can comprise PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, a PEG-DSPE lipid, or a combination thereof. In some embodiments, PEG moiety is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, the PEG moiety is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, the PEG moiety includes PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, e.g., j. Milton Harris, Polyethylene glycol) chemistry: biotechnical and biomedical applications (1992)). In some embodiments, the PEG moiety does not include PEG copolymers, e.g., it may be a PEG monopolymer. Exemplary PEG-lipids include, but are not limited to, PEG-dilauroylglycerol, PEG- dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG- distearoylgiycerol (PEG-DSPE), PEG-dipalmitoylglycerol, PEG-disteiylglycerol, PEG-dilawylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol, and PEG-DMB (3,4- Ditetradecoxylbenzyl- [omega] -methyl -poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-WSGR Attorney Docket No. 53989-743.601 glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol) -2000]). In some embodiments, the PEG-lipid is l,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene gly col-2000.

[0203] In some embodiments, a PEG-lipid is a PEG-lipid conjugate, for example, PEG coupled to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)- lipid conjugates (e.g. POZ-DAA conjugates; see, e.g., WO 2010 / 006282), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof.

[0204] A PEG-lipid can comprise one or more ethylene glycol units, for example, at least 1, at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, or at least 150 ethylene glycol units. In some embodiments, a number average molecular weight of the PEG-lipids is from about 200 Da to about 5000 Da. In some embodiments, a number average molecular weight of the PEG-lipids is from about 500Da to about 3000 Da. In some embodiments, a number average molecular weight of the PEG-lipids is from about 750 Da to about 2500 Da. In some embodiments, a number average molecular weight of the PEG- lipids is from about 750 Da to about 2500 Da. In some embodiments, a number average molecular weight of the PEG- lipids is about 500 Da, about 750 Da, about 1000 Da, about 1250 Da, about 1500 Da, about 1750 Da, or about 2000 Da. In some embodiments, a polydispersity index (PD1) of the one or more PEG-lipids is smaller than 2. In some embodiments, a PDI of the one or more PEG-lipids is at most 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3. 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In some embodiments, a PDI of the one or more PEG-lipids is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, or 3.0.

[0205] In some embodiments, the PEG-lipid is 1, 2-dimyristoyl-rac-glycero-3 -methoxypolyethylene gly col-2000.

[0206] In some embodiments, the PEG-lipid is

[0207] In some embodiments, the PEG-lipid comprises from about 0.1 mol% to about 10 mol% of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises from about 0. 1 mol% to about 6 mol% of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises from about 0.5 mol% to about 5 mol% of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises from about 1 mol% to about 3 mol% of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises about 2.0 mol% to about 2.5 mol% of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises about 1 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol%, about 2.9 mol%, or about 3.0 mol% of the total lipid present in the particle.WSGR Attorney Docket No. 53989-743.601Phospholipid

[0208] As used herein, a “phospholipid” refers to a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds. In some embodiments, a phospholipid may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of an LNP to pass through the membrane, i.e., delivery of the one or more elements to a cell.

[0209] In some embodiments, the described LNP composition comprises a phospholipid. In some embodiments, the phospholipid comprises a lipid selected from the group consisting of: phosphatidylcholine (PC), phosphatidylethanolamine amine, glycerophospholipid, sphingophospholipids, Guriserohosuhono, sphingolipids phosphono lipids, natural lecithins, and hydrogenated phospholipid. In some embodiments, the phospholipid comprises a phosphatidylcholine. Exemplary phosphatidylcholines include, but are not limited to, soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoyl phosphatidylcholine, dipalmitoyl -sn-glycero-3 -phosphocholine (DPPC), 2-Oleoyl-l-palmitoyl-sn- glycero-3 -phosphocholine (POPC), dimyristoyl phosphatidylcholine (DMPC), and dioleoyl phosphatidylcholine (DOPC). In certain specific embodiments, the phospholipid is DSPC.

[0210] In some embodiments, the phospholipid comprises a phosphatidylethanolamine amine. In some embodiments, the phosphatidylethanolamine amine is distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), dimyristoyl phosphoethanolamine (DMPE), 16-0-Monome Le PE, 16-0-dimethyl PE, 18-1 -trans PE, palmitoyl oleoyl -phosphatidylethanolamine (POPE), or 1 -stearoyl -2- oleoyl-phosphatidyl ethanolamine (SOPE). In some embodiments, the phospholipid comprises a glycerophospholipid. In some embodiments, the glycerophospholipid is plasmalogen, phosphatidate, or phosphatidylcholine. In some embodiments, the glycerophospholipid is phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyl oleoyl phosphatidylglycerol (POPG), or lysophosphatidylcholine. In some embodiments, the phospholipid comprises a sphingophospholipid. In some embodiments, the sphingophospholipid is sphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol, or ceramide phosphoglycerophosphoric acid. In some embodiments, the phospholipid comprises a natural lecithin. In some embodiments, the natural lecithin is egg yolk lecithin or soybean lecithin. Tn some embodiments, the phospholipid comprises a hydrogenated phospholipid. In some embodiments, the hydrogenated phospholipid is hydrogenated soybean phosphatidylcholine. In some embodiments, the phospholipid is selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.WSGR Attorney Docket No. 53989-743.601

[0211] In some embodiments, the phospholipid comprises a lipid selected from: 1,2-distearoyl-sn- glycero-3 -phosphocholine (DSPC). l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 2-Oleoyl-l- pahnitoyl-sn-gly cero-3 - phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), 1 -oleoyl - 2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-gly cero-3 - phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-di arachidonoyl-sn- glycero-3 -phosphocholine, 1 ,2-di docosahexaenoyl-sn- glycero-3-phosphocholine, 1,2-diphytanoyl-sn- glycero-3 -phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1,2- diaracliidonoyl-sn-gly cero-3- phosphoethanol amine, 1,2-di docosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-diol eoy I -sn- glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin.

[0212] A phospholipid can comprise a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety can comprise phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, or a sphingomyelin. A fatty acid moiety can comprise lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, or docosahexaenoic acid. In some specific embodiments, a phospholipid can be functionalized with or cross-linked to one or more alkynes, which may undergo a copper-catalyzed cycloaddition upon exposure to an azide.

[0213] In some embodiments, the LNP composition comprises a plurality of phospholipids, for example, at least 2, 3, 4, 5, or more distinct phospholipids. In some embodiments, the phospholipid comprises from 1 mol% to 20 mol% of the total lipid present in the particle. In some embodiments, the phospholipid comprises from about 5 mol% to about 15 mol% of the total lipid present in the particle. In some embodiments, the phospholipid comprises from about 8 mol% to about 12 mol% of the total lipid present in the particle. In some embodiments, the phospholipid comprises from about 9 mol%, 10 mol%, or 11 mol% of the total lipid present in the particle.

[0214] In some embodiments, the phospholipid is 1,2-distearoyl-sn-gly cero-3 -phosphocholine.

[0215] In some embodiments, the phospholipid isCholesterol

[0216] In some embodiments, the LNP composition comprises a cholesterol or a derivative thereof. In some embodiments, the LNP composition comprises a structural lipid. The structural lipid can be selected from steroid, sterol, alkyl resorcinol, cholesterol or derivative thereof, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alphatocopherol, and aWSGR Attorney Docket No. 53989-743.601 combination thereof. In some embodiments, the structural lipid is a corticosteroid such as prednisolone, dexamethasone, prednisone, and hydrocortisone. In some embodiments, the cholesterol or derivative thereof is cholesterol, 5- heptadecylresorcinol, or cholesterol hemisuccinate. Tn some embodiments, the cholesterol or derivative thereof is cholesterol.

[0217] In some embodiments, the cholesterol is a plant cholesterol. In some embodiments, the plant cholesterol is

[0218] In some embodiments, the cholesterol or derivative thereof is a cholesterol derivative. In some embodiments, the cholesterol derivative is a polar cholesterol analogue. In some embodiments, the polar cholesterol analogue is 5a-cholestanol, 513-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholestelyl- (4'-hydroxy)-butyl ether, or 6-ketocholestanol. In some embodiments, the polar cholesterol analogue is cholesteryl -(4'-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is a non-polar cholesterol analogue. In some embodiments, the non-polar cholesterol analogue is 5acholestane, cholestenone, 5a-cholestanone, 5[3-cholestanone, or cholestetyl decanoate.

[0219] In some embodiments, the cholesterol or the derivative thereof comprises from 20 mol% to 50 mol% of the total lipid present in the particle. In some embodiments, the cholesterol or the derivative thereof comprises about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%. about 45 mol%. about 46 mol%, about 47 mol%, about 48 mol%, or about 50 mol% of the total lipid present in the particle.EXAMPLES

[0220] The following examples are not intended to limit the scope of what the inventors regard as various aspects of the present invention. LNPs were made using RNA MA079 and gRNA GA346 and formulated in the manner detailed herein and dosed to human participants as described herein to effectuate in vivo editing of the pcsk9 gene. Reductions of PCSK9 protein and LDL-C were observed and the LNPs were well tolerated by the participants as set forth in the data below and in the drawings.

[0221] Generally, as to the data presented herein, it should be understood that while various gRNA:RNA ratios may be employed, unless otherwise expressly specified to the contrary, the gene editing compositions set forth in the studies and data described and presented herein, employ 1 : 1 gRNA to RNA ratio by weight. Similarly, while various dosing methods may be employed, unless otherwise expressly specified to the contrary, dosing specified by mg / kg in the studies and data described and presented hereinWSGR Attorney Docket No. 53989-743.601 refers to mg of the combined total gRNA and RNA per kg weight of the dosed subject that is contained in the LNPs and intravenously dosed to the subject.Example 1. PCSK9 Base Editing to Reduce LDL-C Level in Human Subject

[0222] LNPs with RNA MA079 and gRNA GA346 were formulated according to the method described in U.S. Patent No. 11,207,416 and U.S. Patent Application No. 18 / 420,112. Formulated LNP excipient compositions are summarized in Tables 1-2. Formulated LNP excipient properties are summarized in Table 3. Sequences of the drug substances (e.g. MA079 and GA346) are listed in Table 4. Exemplary iLipid excipients used for LNP preparations are listed in Table 5. Exemplary PEG-Lipid excipients used for LNP preparations are listed in Table 6. Chemical names and structures of the LNP excipients used for LNP preparations are listed in Table 8. The ratio of the total LNP excipients and the total amount of the RNA and the guide polynucleotide is about 27 ± 5.4 : 1 (e.g., target ratio equals 27: 1 + / - 20%) by weight.Table 1. Formulated LNP excipients (in Mol%)Table 2. Formulated LNP excipients (mg / ml)Table 3. Formulated LNP PropertiesWSGR Attorney Docket No. 53989-743.601* Test descriptions noted in parentheticals. Particle size and encapsulation assessed at specified pH.Table 4. Sequences of drug substances (e.g., gene editing proteins and nucleic acids encoding thereof)WSGR Attorney Docket No. 53989-743.601WSGR Attorney Docket No. 53989-743.601WSGR Attorney Docket No. 53989-743.601WSGR Attorney Docket No. 53989-743.601WSGR Attorney Docket No. 53989-743.601WSGR Attorney Docket No. 53989-743.601WSGR Attorney Docket No. 53989-743.601Table 5. Exemplary iLipid excipients used for LNP preparationsTable 6. Exemplary PEG-Lipid excipients used for LNP preparationsWSGR Attorney Docket No. 53989-743.601able 7: Participant characterization, dosage received, and clinical efficacy and safety data= missing (result pending), C = censored (result is censored in time-weighted reduction calculation due to changes in background lipid lowering therapy, initiatio f a procedure that may have affected PD endpoints, receiving prohibited medication, or with any important protocol deviation). ote: Calculated LDL-C results by Friedewald formula are imputed with directly measured LDL-C by beta quantification when calculated LDL-C results are missin ie, samples are out of stability, or triglycerides are greater than 400 mg / dL) or below 40 mg / dL. If LDL-C by beta quantification result is also missing, the omogenous LDL-C result is used. articipant 1 stopped taking ezetimibe 10 mg daily on Day 263 and restarted on Day 269. Therefore, their day 270 LDL-C / PCSK9 results are impacted and these da oints were precluded from the time-weighted reduction calculation. articipant 5 stopped taking rosuvastatin 20 mg daily on Day 34 and restarted on Day 53. Therefore, their day 60 LDL-C / PCSK9 results are impacted and these da oints were precluded from the time-weighted reduction calculation. articipant 8 started taking fenofibrates on Day 28. Therefore, results after Day 28 are impacted and these datapoints were excluded from the time-weighted reducti alculation.WSGR Attorney Docket No. 53989-743.601Table 8. LNP excipients used for LNP preparations

[0223] Eighteen participants diagnosed with heterozygous familial hypercholesterolemia (HeFH) and four patients with premature coronary artery disease (CAD) were treated with the pharmaceutical composition disclosed herein. The participants had a mean age of 50. Sixteen participants were males and six were females. The participants had a mean weight of 79kg and a mean baseline LDL-C level of 210 mg / dL. Table 9 shows the summary of the participant baseline characteristics.Table 9: Participant baseline characteristicsWSGR Attorney Docket No. 53989-743.601

[0224] Dexamethasone 8mg was administered orally in the evening before treatment. At least 60 minutes before the treatment, dexamethasone lOmg, a Hl blocker ((e.g. diphenhydramine 50 mg or equivalent), and a H2 blocker (e.g. famotidine 20 mg or equivalent) were administered via intravenous infusion. Subsequently, single doses of 0.3 mg / kg (n=4), 0.45 mg / kg (n=6), 0.6 mg / kg (n=4), 0.7 mg / kg (n=5), or 0.8 mg / kg (n=3) of the pharmaceutical formulation comprising the LNP as described above were administered via peripheral intravenous infusion for two to four hours. After the single dose treatment of the pharmaceutical formulation, PCSK9 protein level, LDL-C protein level, ALT level, AST level, total bilirubin level, and platelet counts in blood were measured at different time points in each participant.

[0225] The baseline levels of PCSK9 and LDL-C were calculated by averaging the levels of pre-dose screening 1, pre-dose screening 2, and protein level on the day before treatment. The percentage of PCSK9 protein reduction and LDL-C reduction were calculated using a time-weighted average (TWA) method. First, the trapezoidal rule was used to calculate the area under the curve (AUC). For participants who had data beyond day 28, the average of PCSK9 protein or LDL-C value of two visits was taken and multiplied by the number of days between these two visits. The sum of these intervals was the AUC. Second, the TWA is calculated by dividing the AUC with the total number of days between the first and last timepoint. As these calculations were performed on the actual values, the % change from baseline was computed using the following formula: 100*((TWA value -baseline value ) / baseline value). For participants that only had data until day 28, the % change from baseline was calculated at that timepoint. It is worth mentioning that the days 8, 14, 28, 60, 90, 180, 270 in Table 7 are target dates and a participant might come in for a blood sample a day or two or three off these target days, the TWA calculations described above took these variances into consideration.

[0226] Following a single infusion, PCSK9 level in the blood was measured at various timepoints post treatment by ELISA assay. FIGs. 1A-1E depicts selected datapoints that were shown in Table 7. FIG. 1A shows, at day 28, PCSK9 level was reduced by 55% on average in four participants who received 0.3 mg / kg dosage. FIG. 1A shows, at day 28, PCSK9 level was reduced by 55% on average in four participants who received 0.3 mg / kg dosage. FIG. IB shows, at day 28, PCSK9 level was reduced byWSGR Attorney Docket No. 53989-743.60150% on average in six participants who received 0.45 mg / kg dosage. FIG. 1C shows, at day 28, PCSK9 level was reduced by 60% on average in four participants who received 0.6 mg / kg dosage. FIG. ID shows, at day 28, PCSK9 level was reduced by 50% on average in three participants who received 0.7 mg / kg dosage. FIG. IE shows, at day 28, PCSK9 level was reduced by 74% on average in two participants who received 0.8 mg / kg dosage. All the data suggest successful editing at the intended genomic target.

[0227] LDL-C level was measured at various timepoints post treatment. FIGs. 2A-2E depict selected datapoints that were shown in Table 7. In participants treated with 0.3 mg / kg dose, LDL-C level was reduced by 26% on average on day 28 (FIG. 2A). In participants treated with 0.45 mg / kg dose, LDL-C level was reduced by 42% on average on day 28 (FIG. 2B). In participants treated with 0.6 mg / kg dose, LDL-C level was reduced by 47% on average on day 28 (FIG. 2C). In participants treated with 0.7 mg / kg dose, LDL-C level was reduced by 53% on average on day 28 (FIG. 2D). In participants treated with 0.8 mg / kg dose, LDL-C level was reduced by 39% on average on day 28 (FIG. 2E).

[0228] The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in blood were monitored post treatment as a clinical safety measurement. FIGs. 3A-3E and 4A-4E depict selected datapoints that were shown in Table 7. FIGs. 6A-6E and 7A-7E show the mean value of selected datapoints that were shown in Table 7. As shown in FIGs. 3A-3E, FIGs. 4A-4E, FIGs. 6A-6E, and FIGs. 7A-7E, no clinically significant changes in ALT and AST level were observed at any of the dose levels. The phrase clinically significant in the context of this disclosure for ALT, AST, and bilirubin may be characterized as:Type of Elevation Typical CriteriaMild / transient ALT or AST < 3 * ULN, bilirubin normalClinically significant ALT or AST > 3 x ULN ± symptoms, or > 5 x ULN if asymptomaticSerious / Hy’s Law ALT or AST > 3 x ULN + bilirubin > 2x ULN

[0229] Total bilirubin levels were monitored post treatment as a clinical safety measurement. FIGs. BASE show the mean value of selected datapoints that were shown in Table 7. As shown in FIGs. 8A-8E, no clinically significant changes in bilirubin level were observed at any of the dose levels.

[0230] Blood platelets were counted at various time points post treatment. FIGs. 5A-5E depict selected datapoints that were shown in Table 7. FIGs. 9A-9E show the mean value of selected datapoints that were shown in Table 7. As shown in FIGs. 5A-5E and 9A-9E, no clinically significant changes in platelet numbers were observed at any of the dose level. The phrase clinically significant in the context of this disclosure for platelet drops (thrombocytopenia) may be characterized by using standard criteria, such as Criteria for Adverse Events (CTCAE v5.0) as previously described, which characterizes platelet count levels less than 50 x 109 / L as potentially clinically significant. The phrase clinically significant in the context of this disclosure for platelet elevations above normal (thrombocytosis) may be characterized as a platelet count approaching or exceeding 1,000 x 109 / L.

[0231] PCSK9 level in the blood was measured on different days post treatment. FIG. 9 shows the percentage reduction from baseline in blood PCSK9 protein level observed in human subjects followingWSGR Attorney Docket No. 53989-743.601 administration of the pharmaceutical composition described herein. The percentage of PCSK9 protein reduction were calculated using a time-weighted average (TWA) method as described in the Examples. Each bar represents a dose group. The y-axis represents the percent change from baseline of the time- weighted average of PCSK9 from Day 28 onward. Day 28 value was used in participants where the Day 28 was the last timepoint. Single observation in 3 participants censored due to changes in background lipid-lowering therapy. The error bars indicate standard error of the mean (SEM). As shown in FIG. 10, PCSK9 level was reduced by 49% on average in four participants who received 0.3 mg / kg dosage, reduced by 57% on average in the six participants who received 0.45 mg / kg dosage, reduced by 63% on average in the four participants who received 0.6 mg / kg dosage, reduced by 56% on average in the three participants who received 0.7 mg / kg dosage, and reduced by 76% in the two participants who received 0.8 mg / kg dosage. This suggests that PCSK9 editing at the intended genomic target was successful across all dose levels.

[0232] LDL-C level was measured on different days post treatment. FIG. 11 shows the percentage reduction from baseline in blood LDL-C level observed in human subjects following administration of the pharmaceutical composition described herein. The percentage of LDL-C reduction were calculated using a time-weighted average (TWA) method as described in the Examples. Each bar represents a dose group. The y-axis represents the percent change from baseline of the time-weighted average of PCSK9 from Day 28 onward. Day 28 value was used in participants where the Day 28 was the last timepoint. Single observation in 3 participants censored due to changes in background lipid-lowering therapy. The error bars indicate standard error of the mean (SEM). As shown in FIG. 11, LDL-C level was reduced by 13% on average in the four participants treated with 0.3 mg / kg dose, by 43% on average in the six participants treated with 0.45 mg / kg dose, by 47% on average in the four participants treated with 0.6 mg / kg dose, by 52% on average in the four participants treated with 0.7 mg / kg dose, and by 43% on average in the two participants treated with 0.8 mg / kg dose.

[0233] The percentage of LDL-C reduction was plotted against the total RNA amount received by the participant. FIG. 12 shows that the level of LDL-C reduction is correlated with the amount of total RNA received by the participant. Each dot represents a participant. The y-axis represents the percent change from baseline of the time-weighted average of PCSK9 from Day 28 onward. Day 28 value was used in participants where the Day 28 was the last timepoint. Single observation in 3 participants censored due to changes in background lipid-lowering therapy.

[0234] Participants were re-grouped to the first group receiving less than 25mg total RNA (four participants), the second group receiving between 25mg and 50mg total RNA (seven participants), and the third group receiving between 50mg and 60mg total RNA (eight participants). FIG.13 depicts percentages of PCSK9 reduction and percentages of LDL-C level reduction in each group. The y-axis represents the percent change from baseline of the time-weighted average of PCSK9 from Day 28 onward. Day 28 value was used in participants where the Day 28 was the last timepoint. Single observation in 2 participants censored due to changes in background lipid-lowering therapy. The error bars indicate standard error of the mean (SEM). As shown in FIG. 13, PCSK9 level was reduced by 49% in the fourWSGR Attorney Docket No. 53989-743.601 participants who received less than 25mg total RNA, reduced by 56% on average in the seven participants who received between 25mg and 50mg total RNA, and reduced by 65% on average in the three participants who received between 50mg and 60mg total RNA.

[0235] FIG. 14 depicts percentages of LDL-C level reduction in each group. The y-axis represents the percent change from baseline of the time-weighted average of PCSK9 from Day 28 onward. Day 28 value was used in participants where the Day 28 was the last timepoint. Single observation in 3 participants censored due to changes in background lipid-lowering therapy. The error bars indicate standard error of the mean (SEM). As shown in FIG. 14, LDL-C level was reduced by 13% in the four participants who received less than 25mg total RNA, reduced by 41% on average in the seven participants who received between 25mg and 50mg total RNA, reduced by 46% on average in the eight participants who received between 50mg and 60mg total RNA, and reduced by 81% in the participants who received between 60mg and 70 mg total RNA.Example 2. PCSK9 Base Editing with Fixed Doses in Human Subject

[0236] LNPs with RNA MA079 and gRNA GA346 are formulated according to the method described in U.S. Patent No. 11,207,416 and U.S. Patent Application No. 18 / 420,112. Formulated LNP excipient compositions are summarized in Tables 1-2. Formulated LNP excipient properties are summarized in Table 3. Sequences of the drug substances (e.g. MA079 and GA346) are listed in Table 4. Exemplary iLipid excipients used for LNP preparations are listed in Table 5. Exemplary PEG-Lipid excipients used for LNP preparations are listed in Table 6. Chemical names and structures of the LNP excipients used for LNP preparations are listed in Table 8. The ratio of the total LNP excipients and the total amount of the RNA and the guide polynucleotide is about 27 ± 5.4 : 1 (e.g., target ratio equals 27: 1 + / - 20%) by weight.

[0237] Dexamethasone is administered before treatment. At least 60 minutes before the treatment, dexamethasone, a Hl blocker ((e.g. diphenhydramine 50 mg or equivalent), and a H2 blocker (e.g. famotidine 20 mg or equivalent) are administered via intravenous infusion.Subsequently, a fixed dose of or about 20 mg + / - 5 mg, 30 mg + / - 5 mg, 40 mg + / - 5 mg, 50 mg + / - 5 mg, 60 mg + / - 5 mg, 70 mg + / - 5 mg, 80 mg + / - 5 mg, 90 mg + / - 5 mg, or 100 mg + / - 5 mg of the pharmaceutical formulation comprising the formulated LNPs as described above are administered via peripheral intravenous infusion. After the single dose treatment of the pharmaceutical formulation, PCSK9 protein level, LDL-C protein level, ALT level, AST level, total bilirubin level, and platelet counts in blood are measured at different time points in each participant. The dose selected may be based on the clinically supported therapeutically effective and safe amount of the pharmaceutical composition that corresponds to a desired clinically supported reduction of LDL-C and / or PCSK9, (e.g., TWA mean percent reduction as compared to baseline at a specified time, e.g., Day 7, 14, 21, 28, 90, 180, 270, 365 or later relative day of dosing).

[0238] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art withoutWSGR Attorney Docket No. 53989-743.601 departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Attorney Docket No. 53989-743.601CLAIMSWHAT IS CLAIMED IS:

1. A method comprising: administering a clinically supported and therapeutically effective pharmaceutical composition to lower LDL-C to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and wherein the pharmaceutical composition comprises a first dose of at least about 0.3 mg / kg of the RNA and the guide polynucleotide.

2. The method of claim 1, wherein the pharmaceutical composition comprises a first dose of at least about 0.45 mg / kg of the RNA and the guide polynucleotide.

3. The method of claim 1, wherein the pharmaceutical composition comprises a first dose of at least about 0.6 mg / kg of the RNA and the guide polynucleotide.

4. The method of claim 3, wherein the pharmaceutical composition comprises a first dose of about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2 mg / kg of the RNA and the guide polynucleotide.

5. The method of any one of claims 1-4, wherein the human subject in need thereof is administered a second dose of the pharmaceutical composition, and wherein the second dose is higher, the same or lower than the first dose.

6. The method of claim 5, wherein the second dose is administered one to six days after the first dose.

7. A method comprising: administering a clinically supported and therapeutically effective pharmaceutical composition to lower LDL-C to a group of human subjects in need thereof having different body weights, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequenceWSGR Attorney Docket No. 53989-743.601 of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and wherein the pharmaceutical composition comprises a first fixed dose of the RNA and the guide polynucleotide.

8. The method of claim 7, wherein the first fixed dose is lower than about 25 mg.

9. The method of claim 7, wherein the first fixed dose is about 25 mg to about 50 mg.

10. The method of claim 7, wherein the first fixed dose is about 50 mg to about 60mg.

11. The method of claim 7, wherein the first fixed dose is about 20 mg, about 25 mg, about 30 mg, about 36 mg, about 37 mg, about 42 mg, about 48 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg or about 80 mg.

12. The method of claim 7, wherein the first fixed dose is at least about 60 mg.

13. The method of claim 10, wherein the first fixed dose is about 75, about 80, about 85, about 90, about 95 or about 100 mg.

14. The method of any one of claims 7-13, wherein the group of human subjects are administered a second fixed dose of the pharmaceutical composition, and wherein the second fixed dose is higher, the same or lower than the first fixed dose.

15. The method of claim 14, wherein the second fixed dose is administered one to six days after the first fixed dose.

16. A method comprising: administering a clinically supported and therapeutically effective pharmaceutical composition to lower LDL-C to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, orWSGR Attorney Docket No. 53989-743.601 c. a N-acetylgalactosamine (GalNAc) lipid; and wherein the human subject in need thereof, before administration of the pharmaceutical composition, has received a prior medication comprising a steroid or an antihistamine.

17. The method of claim 16, wherein the prior medication comprises a steroid and an antihistamine.

18. The method of claim 16 or 17, wherein the steroid comprises dexamethasone.

19. The method of claim 16 or 17, wherein the antihistamine comprises a Hl blocker or a H2 blocker.

20. The method of claim 19, wherein the Hl blocker comprises diphenhydramine.

21. The method of claim 19, wherein the H2 blocker comprises famotidine.

22. A method comprising: administering a clinically supported and therapeutically effective pharmaceutical composition to lower LDL-C to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and wherein the human subject in need thereof, after administration of the pharmaceutical composition, receives a steroid.

23. The method of claim 22, wherein the steroid comprises dexamethasone.

24. A method comprising: administering a clinically supported and therapeutically effective pharmaceutical composition to lower LDL-C to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate,WSGR Attorney Docket No. 53989-743.601 b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and wherein the human subject in need thereof 1) had atherosclerotic cardiovascular disease (ASCVD), 2) had heterozygous familial hypercholesterolemia (HeFH) with a low-density lipoproteincholesterol (LDL-C) level of at least about 190 mg / dL, 3) had an LDL-C level above a standard LDL- C level, or 4) cannot tolerate a lipid lowering medication.

25. The method of claim 24, wherein the standard LDL-C level is about 160 mg / dL, about 140 mg / dL, about 130 mg / dL or about 100 mg / dL.

26. The method of claim 24, wherein the lipid lowering medication comprises a statin, ezetimibe, a bile acid sequestrant, a fibrate, or niacin.

27. A method comprising: administering a clinically supported and therapeutically effective pharmaceutical composition to lower LDL-C to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and wherein the human subject in need thereof has received a coronary revascularization, a coronary artery bypass grafting, a percutaneous coronary intervention or a lipid-lowering medication that is not a PSCK9 inhibitor.

28. The method of claim 27, wherein the lipid-lowering medication comprises a statin, ezetimibe, a bile acid sequestrant, a fibrate, or niacin.

29. A method comprising: administering a clinically supported and therapeutically effective pharmaceutical composition to lowering LDL-C to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequenceWSGR Attorney Docket No. 53989-743.601 of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and wherein the human subject in need thereof has at least one risk factor selected from the group consisting of: 1) atherosclerotic cardiovascular disease (ASCVD), 2) heterozygous familial hypercholesterolemia (HeFH) with a low-density lipoprotein-cholesterol (LDL-C) level of at least about 190 mg / dL, or 3) an LDL-C level above a standard LDL-C level.

30. The method of claim 29, wherein the standard LDL-C level is about 160 mg / dL, about 140 mg / dL, about 130 mg / dL or about 100 mg / dL.

31. The method of claim 29 or 30, wherein the ASCVD is represented by 1) a prior treatment comprising revascularization, coronary artery bypass grafting, or percutaneous coronary intervention, or 2) a prior condition comprising myocardial infarction, cardiac arrest, or stroke.

32. A method comprising: administering a clinically supported and therapeutically effective pharmaceutical composition to lowering LDL-C to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and wherein after administration of the pharmaceutical composition, i. a level of PCSK9 protein in the human subject in need thereof is reduced by at least 40% as compared to the level of PCSK9 protein prior to the administration, or ii. a level of LDL-C in the human subject in need thereof is reduced by at least 20% as compared to the level of LDL-C prior to the administration.WSGR Attorney Docket No. 53989-743.60133. The method of claim 32, wherein the after administration of the pharmaceutical composition, the level of PCSK9 protein in the human subject in need thereof is reduced by at least 50% as compared to the level of PCSK9 protein prior to the administration.

34. The method of claim 32, wherein the after administration of the pharmaceutical composition, the level of PCSK9 protein in the human subject in need thereof is reduced by at least 60% as compared to the level of PCSK9 protein prior to the administration.

35. The method of any one of claims 32-34, wherein the level of PCSK9 protein is reduced for at least 2 weeks after administration.

36. The method of any one of claims 32-34, wherein the level of PCSK9 protein is reduced for at least 4 weeks after administration.

37. The method of any one of claims 32-34, wherein the level of PCSK9 protein is measured in blood.

38. The method of claim 32, wherein the after administration of the pharmaceutical composition, the level of LDL-C in the human subject in need thereof is reduced by at least 40% as compared to the level of LDL-C prior to the administration.

39. The method of claim 32, wherein the after administration of the pharmaceutical composition, the level of LDL-C in the human subject in need thereof is reduced by at least 50% as compared to the level of LDL-C prior to the administration.

40. The method of any one of claims 32-39, wherein the level of LDL-C is reduced for at least 2 weeks after administration.

41. The method of any one of claims 32-39, wherein the level of LDL-C is reduced for at least 4 weeks after administration.

42. The method of claim 41, wherein the level of LDL-C is measured in blood.

43. A method comprising: administering a clinically supported and therapeutically effective pharmaceutical composition to lowering LDL-C to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and wherein after administration of the pharmaceutical composition, the human subject in need thereofWSGR Attorney Docket No. 53989-743.601 i. has an alanine aminotransferase (ALT) level between 10-50 IU / L, ii. has an aspartate aminotransferase (AST) level between 10-40 IU / L, iii. has a bilirubin level between 0-1 mg / dL, or iv. has a platelet count between 150-400xl0A9 / L.

44. A method comprising: administering a clinically supported and therapeutically effective pharmaceutical composition to lowing LDL-C to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and wherein after administration of the pharmaceutical composition, a level of a liver biomarker indicating the human subject’s liver function is lower than an upper limit of normal (ULN) or higher than a lower limit of normal (LLN).

45. The method of claim 44, wherein the level of the liver biomarker indicating the human subject’s liver function is measured at least 28 days after administration of the pharmaceutical composition.

46. The method of claim 44 or 45, wherein the liver biomarker comprises platelet count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), or bilirubin.

47. The method of claim 46, wherein a ULN for platelet count is about 400x10A9 / L, and a LLN for platelet count is about 150xl0 9 / L.

48. The method of claim 46, wherein a ULN for ALT is about 50 IU / L.

49. The method of claim 46, wherein a ULN for AST is about 40 IU / L.

50. The method of claim 46, wherein a ULN for bilirubin is about 1 mg / dL.

51. A method comprising:1) administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequenceWSGR Attorney Docket No. 53989-743.601 of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a. an amino lipid, wherein the amino lipid is 3-[4,4-Bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl (9Z, 12Z)-9, 12-octadecadienoate, b. a PEG lipid, wherein the PEG-lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000, or c. a N-acetylgalactosamine (GalNAc) lipid; and2) measuring a level of a liver biomarker indicating the human subject’s liver function.

52. The method of claim 51, wherein the liver biomarker comprises platelet count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), or bilirubin.

53. The method of any one of claims 1 to 52, wherein the LNP excipients comprise the amino lipid, the PEG lipid and the GalNac lipid.

54. The method of claim 53, wherein the LNP excipients further comprise a sterol and a phospholipid.

55. The method of claim 54, wherein(a) the amino lipid is present at about 39 to about 59 mol% of the total LNP excipients, the PEG-lipid is present at about 1.9 to about 4.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.02 to about 0.1 mol% of the total LNP excipients, the sterol is present at about 27.3 to about 50.2 mol% of the total LNP excipients, and the phospholipid is present at about 5.9 to about 13.4 mol% of the total LNP excipients,(b) the amino lipid is present at about 50 ± 12 mol% of the total LNP excipients, the PEG- lipid is present at about 3 ± 1.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.05 ± 0.04 mol% of the total LNP excipients, the sterol is present at about 37.95 ± 12.3 mol% of the total LNP excipients, and the phospholipid is present at about 9 ± 4.4 mol% of the total LNP excipients, or(c) the amino lipid is present at about 9.4 to about 26.3 mg / mL of the pharmaceutical composition, the PEG-lipid is present at about 2.1 to about 4. mg / mL of the pharmaceutical composition, the GalNAc lipid is present at about 0.03 to about 0. 14 mg / mL of the pharmaceutical composition, the sterol is present at about 4.5 to about 8.2 mg / mL of the pharmaceutical composition, and the phospholipid is present at about 1 .8 to about 4.3 mg / mL of the pharmaceutical composition.

56. The method of claim 55, wherein the amino lipid is present at about 50 mol% of the total LNP excipients, the PEG-lipid is present at about 3 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.05 mol% of the total LNP excipients, the sterol is present at about 37.95 mol% of the total LNP excipients, and the phospholipid is present at about 9 mol% of the total LNP excipients.

57. The method of any one of claims 1-56, wherein the pharmaceutical composition has a particle size between 65 ± 15 nM Z-average hydrodynamic diameter.WSGR Attorney Docket No. 53989-743.60158. The method of claim 57, wherein the pharmaceutical composition has a particle size between 65 ± 10 nM Z-average hydrodynamic diameter.

59. The method of claim 57, wherein the pharmaceutical composition has a particle size between 65 ± 5 nM Z-average hydrodynamic diameter.

60. The method of any one of claims 1-59, wherein the pharmaceutical composition has polydispersity index of at most 0.2 as determined by dynamic light scattering.

61. The method of any one of claims 1-60, wherein at least 85% of the total amount of the RNA and the guide polynucleotide is present in the pharmaceutical composition at pH 7.0-8.0.

62. The method of any one of claims 1-61, wherein at least 60% of the guide polynucleotide in the pharmaceutical composition is the full-length guide polynucleotide at pH 7.0-8.0.

63. The method of any one of claims 1-62, wherein at least 70% of the RNA in the pharmaceutical composition is the full-length RNA at pH 7.0-8.0.

64. The method of any one of claims 1-63, wherein the ratio of (1) the total LNP excipients and (2) the total amount of the RNA and the guide polynucleotide is about (27 ± 5.4) : 1 by weight.

65. The method of claim 64, wherein the ratio of (1) the total LNP excipients and (2) the total amount of the RNA and the guide polynucleotide is about (27 ± 2.7) : 1 by weight.

66. The method of claim 65, wherein the ratio of (1) the total LNP excipients and (2) the total amount of the RNA and the guide polynucleotide is about (27 ± 1.35) : 1 by weight.

67. The method of any one of claims 1-66, wherein the ratio of the guide polynucleotide and the RNA is about 1 : 1 by weight.

68. The method of any one of claims 1-67, wherein the spacer sequence comprises a nucleotide base sequence having at least 80% sequence identity to CCCGCACCUUGGCGCAGCGG (SEQ ID No: 101), GGUGCUAGCCUUGCGUUCCG (SEQ ID NO: 102), or UUGGAAAGACGGAGGCAGCC (SEQ ID NO: 103), wherein the uppercase A, G, and C represent adenine, guanine, and cytosine, respectively, and wherein the uppercase U represents uracil or thymine.

69. The method of any one of claims 1-67, wherein the spacer sequence comprises a nucleotide sequence having at least 80% identity to cscscsGCACCUUGGCGCAGCGG (SEQ ID No: 108), gsgsusGCUAGCCUUGCGUUCCG (SEQ ID NO: 109), or ususgsGAAAGACGGAGGCAGCC (SEQ ID NO: 110), wherein 1) the uppercase A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) the lowercase a, u, g, and c represent 2’-O-Methyl- modified adenosine, uridine, guanosine, and cytidine, respectively, and 3) the lowercase s represents phosphorothioate (PS) linkage.

70. The method of any one of claims 1-69, wherein the guide polynucleotide further comprises a tracr sequence, wherein the tracr sequence comprises a nucleotide base sequence, wherein the nucleotide base sequence has at least 80% sequence identity to a nucleotide base sequence of GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA AGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 107).WSGR Attorney Docket No. 53989-743.60171. The method of any one of claims 1 -69, wherein the guide polynucleotide further comprises a tracr sequence, wherein the tracr sequence comprises a nucleotide sequence having at least 80% sequence identity to(i) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcGUUAucAAcuuGaaaaagu GgcaccgAgUCggugcusususu (SEQ ID No. 104),(ii) gUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugG caccgagucggugcusususu (SEQ ID No. 105), or(iii) gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagug Gcaccgagucggugcusususu (SEQ ID No. 106), wherein 1) the uppercase A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) the lowercase a, u, g, and c represent 2’-O-Methyl- modified adenosine, uridine, guanosine, and cytidine, respectively, and 3) the lowercase s represents phosphorothioate (PS) linkage.

72. The method of any one of claims 1-71, wherein the guide polynucleotide comprises a nucleotide sequence of cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAu cAAcuuGaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 1), wherein 1) the uppercase A, U, G, and C represent adenosine, uridine, guanosine, and cytidine, respectively; 2) the lowercase a, u, g, and c represent 2’-O-Methyl- modified adenosine, uridine, guanosine, and cytidine, respectively, and 3) the lowercase s represents phosphorothioate (PS) linkage.

73. The method of any one of claims 1-72, wherein the RNA comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 2.

74. The method of claim 73, wherein the RNA comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2.

75. The method of claim 74, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2.

76. The method of any one of claims 1-75, wherein the GalNAc lipid comprises a compound of Formula (V):Formula (V) wherein, a plurality of the A groups collectively comprising a receptor targeting ligand; each of L1, L2, L3, L4, L5, L6, L7, L8, L9, L10and L12is independently substituted or unsubstituted C1-C12 alkylene, substituted or unsubstituted C1-C12 heteroalkylene, substituted or unsubstituted C2-C12 alkenylene, substituted or unsubstituted C2-C12 alkynylene, -WSGR Attorney Docket No. 53989-743.601(CH2CH2O)m-, -(OCH2CH2)m-, -0-, -S-, -S(=0)-, -S(=0)2-, -S(=O)(=NR1)-, -C(=0)-, - C(=N-OR’)-, -C(=0)0-, -0C(=0)-, -C(=O)C(=O)-, -C(=O)N(R’)-, -N(R’)C(=O)-, - OC(=O)N(R1)-, -N(R1)C(=O)O-, -N(R1)C(=O)N(R1)-, -S(=O)2N(R1)-, -N(R1)S(=O)2-,N(R’)-, or -N(OR’)-;L11is substituted or unsubstituted -(CH2CH2O)n- or substituted or unsubstituted -(OCH2CH2)n-; each R1is independently H or substituted or unsubstituted Ci-Cg alkyl;R is a lipid; m is an integer selected from 1 to 10; and n is an integer selected from 1 to 200, wherein each of the A groups is a N-acetylgalactosamine moiety77. The method of claim 76, wherein, each of L1, L4, and L7is independently C2-Ce alkylene; each of L2, L5, and L8is independently -C(=O)NR1- or -NR1C(=O)-; each of L3, L6, and L9is independently C2-Ce alkylene;L10is Ci-Ce alkylene, -CH2CH2O-, or -CH2O-;L11is -(CH2CH2O)n- or -(OCH2CH2)n-, where n is an integer selected from 1 to 50;L12is -C(=O)O-, -OC(=O)-, -C(=O)NR1-, -NR'C(=O)-. -OC(=O)NR1-, or -NR'C(=O)O-: and R is selected from the group consisting of dialkylglycerolyl, diacylglycerolyl, sterol, n-alkyl comprising C10-C30 carbon atoms, branched alkyl comprising C10-C30 carbon atoms or tocopherol.

78. The method of claim 76 or 77, wherein79. The method of any one of claims 76 to 78, whereinWSGR Attorney Docket No. 53989-743.60180. The method of any one of claims 1 to 79, wherein the GalNAc lipid iswherein each of the p and q is independently an integer from 1 to 5, and n is an integer from 1 to 50, from 20 to 45, or from 30 to 40.

81. The method of claim 80, wherein the GalNAc lipid iswherein n is an integer selected from 33 to 37.

82. The method of claim 81, wherein n = 34, 35 or 36.

83. The method of claim 82, wherein n = 35.

84. The method of any one of claims 1 to 83, wherein the phospholipid is l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC).WSGR Attorney Docket No. 53989-743.60185. The method of any one of claims 1 to 83, wherein the phospholipid is86. The method of any one of claims 1 to 85, wherein the PEG lipid is88. The method of any one of claims 1 to 87, wherein the cholesterol is a plant cholesterol.

89. The method of claim 88, wherein the plant cholesterol is90. The method of any one of claims 1 to 89, wherein the pharmaceutical composition is administered to the human subject in need thereof via intravenous infusion.

91. The method of any one of claims 1 to 90, wherein the human subject has a mutation on an LDLR gene.

92. The method of claim 91, wherein the LDLR gene has a splice site mutation or a mutation in an exon.

93. The method of any one of claims 1-26 and 32-92, wherein the human subject has atherosclerotic cardiovascular disease, accelerated atherosclerotic cardiovascular disease, premature coronary artery disease, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, coronary heart disease, carotid artery disease, peripheral artery disease, microvascular disease, stroke, heart attack, sudden cardiac arrest, or an LDL-C level above a standard LDL-C level.

94. The method of claim 93, wherein the standard LDL-C level is about 160 mg / dL, about 140 mg / dL, about 130 mg / dL, about 100 mg / dL, about 70 mg / dL or about 50 mg / dL.

95. The method of claim 93, wherein the human subject has familial hypercholesterolemia (PH).WSGR Attorney Docket No. 53989-743.60196. The method of claim 95, wherein the human subject has heterozygous familial hypercholesterolemia (HeFH).

97. The method of claim 96, wherein the human subject has severe HeFH.

98. The method of claim 96, wherein the human subject has an LDL-C level of at least about 190 mg / dL.

99. The method of claim 93, wherein the human subject has premature coronary artery disease.

100. The method of any one of claims 1-23 and 29-99, wherein the human subject cannot tolerate a lipid-lowering medication.

101. The method of claim 98, wherein the lipid-lowering medication comprises a statin, ezetimibe, a bile acid sequestrant, a fibrate, or niacin.

102. The method of any one of claims 1-101, wherein a PCSK9 gene is inactivated by the pharmaceutical composition.

103. The method of any one of claims 1-102, wherein the pharmaceutical composition is designed to be taken up by liver cells of the human subject in need thereof.

104. A method comprising: administering a pharmaceutical composition to a human subject in need thereof, wherein the pharmaceutical composition comprises a clinically supported safe and clinically supported effective amount of a lipid nanoparticle (LNP), wherein the LNP comprises:(a) an RNA encoding a base editor protein, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a PEG lipid, aN-acetylgalactosamine (GalNAc) lipid, a sterol and a phospholipid; wherein after administration of the clinically supported effective amount of the LNP, the human subject in need thereof demonstrates:(i) reduction of a level of PCSK9 protein by at least 40% as compared to a level of PCSK9 protein prior to the administration, or(ii) reduction of a level of LDL-C by at least 20% as compared to the level of LDL-C prior to the administration, and wherein after administration of the clinically supported safe amount of the LNP, the human subject in need thereof demonstrates:(i) has an alanine aminotransferase (ALT) level between 10-50 IU / L,(ii) has an aspartate aminotransferase (AST) level between 10-40 IU / L,(iii) has a bilirubin level between 0- 1 mg / dL, or(iv) has a platelet count between 150-400xl0A9 / L.

105. The method of claim 104, wherein the human subject has atherosclerotic cardiovascular disease, accelerated atherosclerotic cardiovascular disease, premature coronary artery disease, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, coronary heart disease, carotidWSGR Attorney Docket No. 53989-743.601 artery disease, peripheral artery disease, microvascular disease, stroke, heart attack, sudden cardiac arrest, or an LDL-C level above a standard LDL-C level.

106. The method of claim 105, wherein the standard LDL-C level is about 160 mg / dL, about 140 mg / dL, about 130 mg / dL or about 100 mg / dL.

107. The method of claim 106, wherein the standard LDL-C level is about 130 mg / dL.

108. The method of claim 106, wherein the human subject has familial hypercholesterolemia (FH).

109. The method of claim 108, wherein the human subject has heterozygous familial hypercholesterolemia (HeFH).

110. The method of claim 109, wherein the human subject has severe HeFH.

111. The method of claim 109, wherein the human subject has an LDL-C level of at least about 190 mg / dL.

112. The method of claim 106, wherein the human subject has premature coronary artery disease.

113. The method of any one of claims 104-112, wherein the human subject cannot tolerate a lipid- lowering medication.

114. The method of claim 111, wherein the lipid-lowering medication comprises a statin, ezetimibe, a bile acid sequestrant, a fibrate, or niacin.

115. The method of any one of claims 104-114, wherein(a) the amino lipid is present at about 39 to about 59 mol% of the total LNP excipients, the PEG-lipid is present at about 1.9 to about 4.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.02 to about 0.1 mol% of the total LNP excipients, the sterol is present at about 27.3 to about 50.2 mol% of the total LNP excipients, and the phospholipid is present at about 5.9 to about 13.4 mol% of the total LNP excipients,(b) the amino lipid is present at about 50 ± 12 mol% of the total LNP excipients, the PEG- lipid is present at about 3 ± 1.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.05 ± 0.04 mol% of the total LNP excipients, the sterol is present at about 37.95 ± 12.3 mol% of the total LNP excipients, and the phospholipid is present at about 9 ± 4.4 mol% of the total LNP excipients, or(c) the amino lipid is present at about 9.4 to about 26.3 mg / mL of the pharmaceutical composition, the PEG-lipid is present at about 2.1 to about 4. mg / mL of the pharmaceutical composition, the GalNAc lipid is present at about 0.03 to about 0.14 mg / mL of the pharmaceutical composition, the sterol is present at about 4.5 to about 8.2 mg / mL of the pharmaceutical composition, and the phospholipid is present at about 1.8 to about 4.3 mg / mL of the pharmaceutical composition.

116. A pharmaceutical composition comprising :(a) an RNA encoding a base editor protein, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide baseWSGR Attorney Docket No. 53989-743.601 sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a N- acetylgalactosamine (GalNAc) lipid, wherein the GalNAc lipid iswherein n is an integer selected from 33 to 37.

117. The pharmaceutical composition of claim 116, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1.

118. The pharmaceutical composition of any one of claims 116-117, wherein the LNP excipients further comprises an amino lipid, a phospholipid, a PEG lipid and a sterol.

119. The pharmaceutical composition of claim 118, wherein the amino lipid is present at a concentration of about 9.4 to about 26.3 mg / mL, the PEG-lipid is present at a concentration of about 2.1 to about 4.4 mg / mL, the GalNAc lipid is present at a concentration of about 0.03 to about 0.14 mg / mL, the sterol is present at a concentration of about 4.5 to about 8.2 mg / mL, and the phospholipid is present at a concentration of about 1.8 to about 4.3 mg / mL.

120. The pharmaceutical composition of any one of claims 116-119, wherein the ratio of (1) total LNP excipients and (2) a total amount of the RNA and the guide polynucleotide is about (27 ± 5.4) : 1 by weight.

121. A pharmaceutical composition comprising :(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise a N- acetylgalactosamine (GalNAc) lipid, wherein the GalNAc lipid isWSGR Attorney Docket No. 53989-743.601wherein n is an integer selected from 33 to 37.

122. The pharmaceutical composition of claim 121, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2.

123. The pharmaceutical composition of claim 121 or 122, wherein the LNP excipients further comprises an amino lipid, a phospholipid, a PEG lipid and a sterol.

124. The pharmaceutical composition of claim 123, wherein the amino lipid is present at a concentration of about 9.4 to about 26.3 mg / mL, the PEG-lipid is present at a concentration of about 2. 1 to about 4.4 mg / mL, the GalNAc lipid is present at a concentration of about 0.03 to about 0. 14 mg / mL, the sterol is present at a concentration of about 4.5 to about 8.2 mg / mL, and the phospholipid is present at a concentration of about 1.8 to about 4.3 mg / mL.

125. The pharmaceutical composition of any one of claims 121-124, wherein the ratio of (1) total LNP excipients and (2) a total amount of the RNA and the guide polynucleotide is about (27 ± 5.4) : 1 by weight.

126. A pharmaceutical composition comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprises an amino lipid, a phospholipid, a PEG lipid, a sterol and a N-acetylgalactosamine (GalNAc) lipid, and wherein the amino lipid is present at a concentration of about 9.4 to about 26.3 mg / mL, the PEG-lipid is present at a concentration of about 2. 1 to about 4.4 mg / mL, the GalNAc lipid is present at a concentration of about 0.03 to about 0. 14 mg / mL, the sterol is present at a concentration of about 4.5 to about 8.2 mg / mL, and the phospholipid is present at a concentration of about 1.8 to about 4.3 mg / mL.

127. The pharmaceutical composition of claim 126, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2.

128. The pharmaceutical composition of claim 126 or 127, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1.WSGR Attorney Docket No. 53989-743.601129. The pharmaceutical composition of any one of claims 126-128, wherein the ratio of (1) total LNP excipients and (2) a total amount of the RNA and the guide polynucleotide is about (27 ± 5.4) : 1 by weight.

130. A pharmaceutical composition comprising :(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprises a N- acetylgalactosamine (GalNAc) lipid, and wherein the ratio of (1) total LNP excipients and (2) a total amount of the RNA and the guide polynucleotide is about (27 ± 5.4) : 1 by weight.

131. The pharmaceutical composition of claim 130, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2.

132. The pharmaceutical composition of claim 130 or 131, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1.

133. The pharmaceutical composition of any one of claims 130-132, wherein the LNP excipients further comprises an amino lipid, a phospholipid, a PEG lipid and a sterol.

134. The pharmaceutical composition of claim 133, wherein the amino lipid is present at a concentration of about 9.4 to about 26.3 mg / mL, the PEG-lipid is present at a concentration of about 2. 1 to about 4.4 mg / mL, the GalNAc lipid is present at a concentration of about 0.03 to about 0. 14 mg / mL, the sterol is present at a concentration of about 4.5 to about 8.2 mg / mL, and the phospholipid is present at a concentration of about 1.8 to about 4.3 mg / mL.

135. A method of treating a human subj ect in need thereof comprising :(1) administering a pharmaceutical composition to the human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients aN-acetylgalactosamine (GalNAc) lipid; and(2) administering a steroid to the human subject in need thereof after administration of the pharmaceutical composition.

136. The method of claim 135, wherein the steroid comprises dexamethasone.

137. The method of claim 135 or 136, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1.WSGR Attorney Docket No. 53989-743.601138. The method of any one of claims 135-137, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2.

139. The method of any one of claims 135-138, wherein the LNP excipients further comprises an amino lipid, a phospholipid, a PEG lipid and a sterol.

140. The method of claim 139, wherein the amino lipid is present at a concentration of about 9.4 to about 26.3 mg / mL, the PEG-lipid is present at a concentration of about 2.1 to about 4.4 mg / mL, the GalNAc lipid is present at a concentration of about 0.03 to about 0.14 mg / mL, the sterol is present at a concentration of about 4.5 to about 8.2 mg / mL, and the phospholipid is present at a concentration of about 1.8 to about 4.3 mg / mL.

141. The method of any one of claims 135-140, wherein the ratio of (1) total LNP excipients and (2) a total amount of the RNA and the guide polynucleotide is about (27 ± 5.4) : 1 by weight.

142. A method of treating a human subject in need thereof comprising: administering a pharmaceutical composition to the human subject in need thereof, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) comprising:(a) an RNA encoding a base editor protein,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a spacer sequence, wherein the spacer sequence comprises at least 13 nucleotide bases of a nucleotide base sequence of a protospacer, wherein the protospacer is on the gene that encodes PCSK9, and wherein uracil is considered the same as thymine, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients a N-acetylgalactosamine (GalNAc) lipid; and wherein the human subject in need thereof 1) had heterozygous familial hypercholesterolemia (HeLH) with a low-density lipoprotein-cholesterol (LDL-C) level of at least about 190 mg / dL, 2) had an LDL-C level above a standard LDL-C level, 3) cannot tolerate a lipid lowering medication, or 4) has received a coronary revascularization, a coronary artery bypass grafting, a percutaneous coronary intervention or a lipid-lowering medication that is not a PSCK9 inhibitor.

143. The method of claim 142, wherein the standard LDL-C level is about 160 mg / dL, about 140 mg / dL, about 130 mg / dL or about 100 mg / dL.

144. The method of claim 142 or 143, wherein the lipid lowering medication comprises a statin, ezetimibe, a bile acid sequestrant, a fibrate, or niacin.

145. The method of any one of claims 142-144, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1.

146. The method of any one of claims 142-145, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2.

147. The method of any one of claims 142-146, wherein the LNP excipients further comprises an amino lipid, a phospholipid, a PEG lipid and a sterol.

148. The method of claim 147, wherein the amino lipid is present at a concentration of about 9.4 to about 26.3 mg / mL, the PEG-lipid is present at a concentration of about 2. 1 to about 4.4 mg / mL, theWSGR Attorney Docket No. 53989-743.601GalNAc lipid is present at a concentration of about 0.03 to about 0.14 mg / mL, the sterol is present at a concentration of about 4.5 to about 8.2 mg / mL, and the phospholipid is present at a concentration of about 1.8 to about 4.3 mg / mL.

149. The method of any one of claims 142-148, wherein the ratio of (1) total LNP excipients and (2) a total amount of the RNA and the guide polynucleotide is about (27 ± 5.4) : 1 by weight.

150. A method of treating a human subject in need thereof comprising administering a pharmaceutical composition of any one of claims 116-134 to the human subject in need thereof.

151. The method of claim 150, wherein the human subject in need thereof is administered a first does of the pharmaceutical composition, and wherein the first dose is at least about 0.3 mg / kg of a total amount of the RNA and the guide polynucleotide.

152. The method of claim 150, wherein the first dose is about 0.45 mg / kg of the total amount of the RNA and the guide polynucleotide.

153. The method of claim 150, wherein the first dose is about 0.6 mg / kg of the total amount of the RNA and the guide polynucleotide.

154. The method of claim 150, wherein the first dose is about 0.8 mg / kg of the total amount of the RNA and the guide polynucleotide.

155. The method of claim 150, wherein the first dose is about 1 mg / kg of the total amount of the RNA and the guide polynucleotide.

156. The method of any one of claims 150-155, wherein the human subject in need thereof is administered a second dose of the pharmaceutical composition, and wherein the second dose is higher, the same or lower than the first dose.

157. The method of claim 156, wherein the second dose is administered one to six days after the first dose.

158. A method comprising administering a pharmaceutical composition of any one of claims 116-134 to a human subject in need thereof, wherein the pharmaceutical composition comprises a clinically supported effective and clinically supported safe amount of the LNP, wherein after administration of the clinically supported effective amount of the LNP, the human subject in need thereof demonstrates:(i) reduction of a level of PCSK9 protein by at least 40% as compared to a level of PCSK9 protein prior to the administration, or(ii) reduction of a level of LDL-C by at least 20% as compared to the level of LDL-C prior to the administration, and wherein after administration of the clinically supported safe amount of the LNP, the human subject in need thereof demonstrates:(i) has an alanine aminotransferase (ALT) level between 10-50 IU / L,(ii) has an aspartate aminotransferase (AST) level between 10-40 IU / L,(iii) has a bilirubin level between 0-1 mg / dL, orWSGR Attorney Docket No. 53989-743.601(iv) has a platelet count between 150-400xl0A9 / L.

159. The method of claim 158, wherein the first fixed dose is lower than about 25 mg of a total amount of the RNA and the guide polynucleotide.

160. The method of claim 158, wherein the first fixed dose is about 25 mg to about 100 mg of a total amount of the RNA and the guide polynucleotide.

161. The method of claim 158, wherein the first fixed dose is about 20 mg, about 25 mg, about 30 mg, about 36 mg, about 37 mg, about 42 mg, about 48 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 95 mg or about 100 mg of a total amount of the RNA and the guide polynucleotide.

162. The method of claim 158, wherein the first fixed dose is at least about 60 mg of a total amount of the RNA and the guide polynucleotide.

163. The method of claim 158, wherein the first fixed dose is about 75, about 80, about 85, about 90, about 95 or about 100 mg of a total amount of the RNA and the guide polynucleotide.

164. The method of any one of claims 158-163, wherein the group of human subjects are administered a second fixed dose of the pharmaceutical composition, and wherein the second fixed dose is higher, the same or lower than the first fixed dose.

165. The method of claim 164, wherein the second fixed dose is administered one to six days after the first fixed dose.

166. A pharmaceutical composition comprising:(a) an RNA encoding a base editor protein, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a phospholipid, a PEG lipid, a sterol and a N-acetylgalactosamine (GalNAc) lipid, and wherein(i) the amino lipid is(ii) the phospholipid is(iii) the PEG lipid isWSGR Attorney Docket No. 53989-743.601, wherein p is an integer selected from 42 to 48,(iv) the sterol is(v) the GalNAc lipid iswherein n is an integer selected from 33 to 37.

167. A pharmaceutical composition comprising:(a) an RNA encoding a base editor protein, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a phospholipid, a PEG lipid, a sterol and aN-acetylgalactosamine (GalNAc) lipid, wherein(i) the amino lipid is(ii) the phospholipid is(iii) the PEG lipid isWSGR Attorney Docket No. 53989-743.601, wherein p is an integer selected from 42 to 48, (iv) the sterol iswherein n is an integer selected from 33 to 37; and wherein the amino lipid is present at about 50 ± 12 mol% of the total LNP excipients, the PEG-lipid is present at about 3 ± 1.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.05 ± 0.04 mol% of the total LNP excipients, the sterol is present at about 37.95 ± 12.3 mol% of the total LNP excipients, and the phospholipid is present at about 9 ± 4.4 mol% of the total LNP excipients.

168. A pharmaceutical composition comprising:(a) an RNA encoding a base editor protein, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a phospholipid, a PEG lipid, a sterol and aN-acetylgalactosamine (GalNAc) lipid, wherein (i) the amino lipid is(ii) the phospholipid isWSGR Attorney Docket No. 53989-743.601(iii)the PEG lipid is, wherein p is an integer selected from 42 to 48, (iv) the sterol is(v) the GalNAc lipid iswherein n is an integer selected from 33 to 37; and wherein the ratio of (1) total LNP excipients and (2) a total amount of the RNA and the guide polynucleotide is about (27 ± 5.4) : 1 by weight.

169. A pharmaceutical composition comprising :(a) an RNA encoding a base editor protein, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO: 2,(b) a guide polynucleotide, wherein the guide polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1, and(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a phospholipid, a PEG lipid, a sterol and aN-acetylgalactosamine (GalNAc) lipid, wherein (i) the amino lipid isWSGR Attorney Docket No. 53989-743.601(iii)the PEG lipid is, wherein p is an integer selected from 42 to 48,(iv) the sterol is(v) the GalNAc lipid iswherein n is an integer selected from 33 to 37, wherein the amino lipid is present at about 50 ± 12 mol% of the total LNP excipients, the PEG-lipid is present at about 3 ± 1.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.05 ± 0.04 mol% of the total LNP excipients, the sterol is present at about 37.95 ± 12.3 mol% of the total LNP excipients, and the phospholipid is present at about 9 ± 4.4 mol% of the total LNP excipients, and wherein the ratio of (1) total LNP excipients and (2) a total amount of the RNA and the guide polynucleotide is about (27 ± 5.4) : 1 by weight.

170. The pharmaceutical composition or the method of any one of claims 118-120, 123-129, 133-134, 139-141, and 147-169, wherein the amino lipid is present at about 39 to about 59 mol% of the totalWSGR Attorney Docket No. 53989-743.601LNP excipients, the PEG-lipid is present at about 1.9 to about 4.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.02 to about 0.1 mol% of the total LNP excipients, the sterol is present at about 27.3 to about 50.2 mol% of the total LNP excipients, and the phospholipid is present at about 5.9 to about 13.4 mol% of the total LNP excipients.

171. The pharmaceutical composition or the method of any one of claims 118-120, 123-129, 133-134, 139-141, and 147-169, wherein the amino lipid is present at about 50 ± 12 mol% of the total LNP excipients, the PEG-lipid is present at about 3 ± 1.4 mol% of the total LNP excipients, the GalNAc lipid is present at about 0.05 ± 0.04 mol% of the total LNP excipients, the sterol is present at about 37.95 ± 12.3 mol% of the total LNP excipients, and the phospholipid is present at about 9 ± 4.4 mol% of the total LNP excipients.

172. The pharmaceutical composition or the method of any one of claims 116-171, wherein the pharmaceutical composition has a particle size between 65 ± 15 nM Z-average hydrodynamic diameter.

173. The pharmaceutical composition or the method of any one of claims 116-171, wherein the pharmaceutical composition has a particle size between 65 ± 10 nM Z-average hydrodynamic diameter.

174. The pharmaceutical composition or the method of any one of claims 116-171, wherein the pharmaceutical composition has a particle size between 65 ± 5 nM Z-average hydrodynamic diameter.

175. The pharmaceutical composition or the method of any one of claims 116-174, wherein the pharmaceutical composition has polydispersity index of at most 0.2 as determined by dynamic light scattering.

176. The pharmaceutical composition or the method of any one of claims 116-175, wherein at least 85% of the total amount of the RNA and the guide polynucleotide is present in the pharmaceutical composition at pH 7.0-8.0.

177. The pharmaceutical composition or the method of any one of claims 116-175, wherein at least 60% of the guide polynucleotide in the pharmaceutical composition is the full-length guide polynucleotide at pH 7.0-8.0.

178. The pharmaceutical composition or the method of any one of claims 116-175, wherein at least 70% of the RNA in the pharmaceutical composition is the full-length RNA at pH 7.0-8.0.

179. The pharmaceutical composition or the method of any one of claims 116-178, wherein the ratio of (1) the total LNP excipients and (2) the total amount of the RNA and the guide polynucleotide is about (27 ± 2.7) : 1 by weight.

180. The pharmaceutical composition or the method of any one of claims 116-178, wherein the ratio of (1) the total LNP excipients and (2) the total amount of the RNA and the guide polynucleotide is about (27 ± 1.35) : 1 by weight.

181. The pharmaceutical composition or the method of any one of claims 116-178, wherein the ratio of the guide polynucleotide and the RNA is about 1 : 1 by weight.WSGR Attorney Docket No. 53989-743.601182. The pharmaceutical composition or the method of any one of claims 118-120, 123-129, 133-134, 139-141, and 147-181, wherein the phospholipid is183. The pharmaceutical composition or the method of any one of claims 118-120, 123-129, 133-134, 139-141, and 147-181, wherein the PEG lipid is, wherein p is an integer selected from 42 to 48184. The pharmaceutical composition or the method of any one of claims 118-120, 123-129, 133-134, 139-141, and 147-181, wherein the amino lipid is185. The pharmaceutical composition or the method of any one of claims 118-120, 123-129, 133-134, 139-141, and 147-181, wherein the cholesterol is a plant cholesterol.

186. The pharmaceutical composition or the method of claim 185, wherein the plant cholesterol is187. The method of any one of claims 135-165, wherein the human subject in need thereof, before administration of the pharmaceutical composition, has received a prior medication comprising a steroid or an antihistamine.

188. The method of claim 187, wherein the prior medication comprises a steroid and an antihistamine.

189. The method of claim 188, wherein the steroid comprises dexamethasone.

190. The method of claim 188, wherein the antihistamine comprises a Hl blocker or a H2 blocker.

191. The method of claim 190, wherein the Hl blocker comprises diphenhydramine.

192. The method of claim 190, wherein the H2 blocker comprises famotidine.

193. The method of any one of claims 135-165 and 187-192, wherein the human subject in need thereof, after administration of the pharmaceutical composition, receives a steroid.WSGR Attorney Docket No. 53989-743.601194. The method of claim 193, wherein the steroid comprises dexamethasone.

195. The method of any one of claims 135-165 and 187-194, wherein the human subject in need thereof had atherosclerotic cardiovascular disease (ASCVD).

196. The method of claim 195, wherein the ASCVD is represented by 1) a prior treatment comprising revascularization, coronary artery bypass grafting, or percutaneous coronary intervention, or 2) a prior condition comprising myocardial infarction, cardiac arrest, or stroke.

197. The method of any one of claims 135-165 and 187-194, wherein the human subject has premature coronary artery disease.

198. The method of any one of claims 135-165 and 187-197, wherein after administration of the pharmaceutical composition, i. a level of PCSK9 protein in the human subject in need thereof is reduced by at least 40% as compared to the level of PCSK9 protein prior to the administration, or ii. a level of LDL-C in the human subject in need thereof is reduced by at least 20% as compared to the level of LDL-C prior to the administration.

199. The method of claim 198, wherein the after administration of the pharmaceutical composition, the level of PCSK9 protein in the human subject in need thereof is reduced by at least 50% as compared to the level of PCSK9 protein prior to the administration.

200. The method of claim 198, wherein the after administration of the pharmaceutical composition, the level of PCSK9 protein in the human subject in need thereof is reduced by at least 60% as compared to the level of PCSK9 protein prior to the administration.

201. The method of any one of claims 198-200, wherein the level of PCSK9 protein is reduced for at least 2 weeks after administration.

202. The method of any one of claims 198-200, wherein the level of PCSK9 protein is reduced for at least 4 weeks after administration.

203. The method of any one of claims 198-202, wherein the level of PCSK9 protein is measured in blood.

204. The method of any one of claims 198-203, wherein the after administration of the pharmaceutical composition, the level of LDL-C in the human subject in need thereof is reduced by at least 40% as compared to the level of LDL-C prior to the administration.

205. The method of any one of claims 198-203, wherein the after administration of the pharmaceutical composition, the level of LDL-C in the human subject in need thereof is reduced by at least 50% as compared to the level of LDL-C prior to the administration.

206. The method of any one of claims 198-205, wherein the level of LDL-C is reduced for at least 2 weeks after administration.

207. The method of any one of claims 198-205, wherein the level of LDL-C is reduced for at least 4 weeks after administration.

208. The method of any one of claims 198-207, wherein the level of LDL-C is measured in blood.WSGR Attorney Docket No. 53989-743.601209. The method of any one of claims 135-165 and 187-208, wherein after administration of the pharmaceutical composition, the human subject in need thereof i. has an alanine aminotransferase (ALT) level between 10-50 IU / L, ii. has an aspartate aminotransferase (AST) level between 10-40 IU / L, iii. has a bilirubin level between 0-1 mg / dL, or iv. has a platelet count between 150-400xl0A9 / L.

210. The method of any one of claims 135-165 and 187-209, wherein after administration of the pharmaceutical composition, a level of a liver biomarker indicating the human subject’s liver function is lower than an upper limit of normal (ULN) or higher than a lower limit of normal (LLN).

211. The method of claim 210, wherein the level of the liver biomarker indicating the human subject’s liver function is measured at least 28 days after administration of the pharmaceutical composition.

212. The method of any one of claims 210-211, wherein the liver biomarker comprises platelet count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), or bilirubin.

213. The method of any one of claims 210-212, wherein a ULN for platelet count is about 400x10A9 / L, and a LLN for platelet count is about 150xl0A9 / L.

214. The method of any one of claims 210-212, wherein a ULN for ALT is about 50 IU / L.

215. The method of any one of claims 210-212, wherein a ULN for AST is about 40 IU / L.

216. The method of any one of claims 210-212, wherein a ULN for bilirubin is about 1 mg / dL.

217. The method of any one of claims 135-165 and 187-216, wherein the method further comprising measuring a level of a liver biomarker indicating the human subject’s liver function.

218. The method of claim 217, wherein the liver biomarker comprises platelet count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), or bilirubin.

219. The method of any one of claims 135-165 and 187-218, wherein the pharmaceutical composition is administered to the human subject in need thereof via intravenous infusion.

220. The method of any one of claims 135-165 and 187-219, wherein the human subject has a mutation on an LDLR gene.

221. The method of claim 220, wherein the LDLR gene has a splice site mutation or a mutation in an exon.

222. The method of any one of claims 135-165 and 187-221, wherein the human subject has atherosclerotic cardiovascular disease, accelerated atherosclerotic cardiovascular disease, premature coronary artery disease, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, coronary heart disease, carotid artery disease, peripheral artery disease, microvascular disease, stroke, heart attack, sudden cardiac arrest, or an LDL-C level above a standard LDL-C level.

223. The method of claim 222, wherein the standard LDL-C level is about 160 mg / dL, about 140 mg / dL, about 130 mg / dL or about 100 mg / dL.

224. The method of any one of claims 135-165 and 187-223, wherein the human subject has familial hypercholesterolemia (FH).WSGR Attorney Docket No. 53989-743.601225. The method of any one of claims 135-165 and 187-224, wherein the human subject has heterozygous familial hypercholesterolemia (HeFH).

226. The method of any one of claims 135-165 and 187-225, wherein the human subject has severe HeFH.

227. The method of any one of claims 135-165 and 187-226, wherein the human subject has an LDL-C level of at least about 190 mg / dL.

228. The method of any one of claims 135-165 and 187-223, wherein the human subject has premature coronary artery disease.

229. The method of any one of claims 135-165 and 187-223, wherein the human subject cannot tolerate a lipid-lowering medication.

230. The method of claim 229, wherein the lipid-lowering medication comprises a statin, ezetimibe, a bile acid sequestrant, a fibrate, or niacin.

231. The method of any one of claims 135-165 and 187-231, wherein a PCSK9 gene is inactivated by the pharmaceutical composition.

232. The method of any one of claims 135-165 and 187-232, wherein the pharmaceutical composition is designed to be taken up by liver cells of the human subject in need thereof.

233. A method comprising: administering a therapeutically effective amount of a pharmaceutical composition to a human subject in need of lowering LDL-C levels, wherein the pharmaceutical composition comprises lipid nanoparticles (LNPs) comprising:(a) an mRNA encoding a base editor protein,(b) a guide polynucleotide comprising a nucleotide sequence specified by SEQ ID No: 1(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a phospholipid, a plurality of PEG lipids, a sterol and a plurality of N-acetylgalactosamine (GalNAc) lipids, and wherein(i) the amino lipid is(iii)the plurality of PEG lipids isWSGR Attorney Docket No. 53989-743.601, wherein p is an integer selected from 42 to 48, (iv) the sterol is(v) the plurality of GalNAc lipids iswherein n is an integer selected from 33 to 37. wherein the LNPs are formulated in accordance with Tables 1, 2 and 3; and wherein the therapeutically effective amount as measured by total mass of the mRNA and guide polynucleotide of the pharmaceutical composition administered corresponds to a clinically supported time-weighted average (TWA) mean percent reduction of LDL-C of 40% or greater as compared to baseline.

234. The method of claim 233, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 45% or greater as compared baseline235. The method of claim 233, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 50% or greater as compared to baseline.

236. The method of claim 233, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 55% or greater as compared to baseline.

237. The method of claim 233, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 60% or greater as compared to baseline.WSGR Attorney Docket No. 53989-743.601238. The method of claim 233, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 65% or greater as compared to baseline.

239. The method of claim 233, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 70% or greater as compared to baseline.

240. The method of claim 233, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 75% or greater as compared to baseline.

241. The method of claim 233, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 80% or greater as compared to baseline.

242. A method comprising: administering a therapeutically effective amount of a pharmaceutical composition to a human subject in need of lowering LDL-C levels, wherein the pharmaceutical composition comprises lipid nanoparticles (LNPs) comprising:(a) an mRNA encoding a base editor protein,(b) a guide polynucleotide comprising a nucleotide sequence specified by SEQ ID No: 1(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a phospholipid, a plurality of PEG lipids, a sterol and a plurality of N-acetylgalactosamine (GalNAc) lipids, and wherein(i) the amino lipid is(iii) the plurality of PEG lipids is, wherein p is an integer selected from 42 to 48,WSGR Attorney Docket No. 53989-743.601 (iv) the sterol is(v) the plurality of GalNAc lipids iswherein n is an integer selected from 33 to 37. wherein the LNPs are formulated in accordance with Tables 1, 2 and 3; and wherein the therapeutically effective amount as measured by total mass of the mRNA and guide polynucleotide of the pharmaceutical composition administered corresponds to a clinically supported time-weighted average TWA mean percent reduction of PCSK9 of 40% or greater as compared baseline.

243. The method of claim 242, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 45% or greater as compared to baseline.

244. The method of claim 242, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 50% or greater as compared to baseline.

245. The method of claim 242, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 55% or greater as compared to baseline.

246. The method of claim 242, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 60% or greater as compared to baseline.

247. The method of claim 242, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 65% or greater as compared to baseline.

248. The method of claim 242, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 70% or greater as compared to baseline.WSGR Attorney Docket No. 53989-743.601249. The method of claim 242, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 75% or greater as compared to baseline.

250. A method comprising: administering a therapeutically safe amount of a pharmaceutical composition to a human subject in need of lowering LDL-C levels, wherein the pharmaceutical composition comprises lipid nanoparticles (LNPs) comprising:(a) an mRNA encoding a base editor protein,(b) a guide polynucleotide comprising a nucleotide sequence specified by SEQ ID No: 1(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a phospholipid, a plurality of PEG lipids, a sterol and a plurality of N-acetylgalactosamine (GalNAc) lipids, and wherein(i) the amino lipid is(iii) the plurality of PEG lipids is, wherein p is an integer selected from 42 to 48, (iv) the sterol is(v) the plurality of GalNAc lipids isWSGR Attorney Docket No. 53989-743.601wherein n is an integer selected from 33 to 37. wherein the LNPs are formulated in accordance with Tables 1, 2 and 3; and wherein the therapeutically safe amount is measured by total mass of the mRNA and guide polynucleotide of the pharmaceutical composition, and wherein after administration of the therapeutically safe amount of the LNP, the human subject in need thereof demonstrates no clinically significant changes in the level(s) of liver biomarkers.

251. The method of claim 250, wherein the human subject in need thereof demonstrates no clinically significant changes in the level(s) of liver biomarkers as specified by an alanine aminotransferase (ALT) level less than or equal to three-fold of an upper limit of normal (ULN).

252. The method of claim 250, wherein the human subject in need thereof demonstrates no clinically significant changes in the level(s) of liver biomarkers as specified by an aspartate aminotransferase (AST) level less than equal to three-fold of an upper limit of normal (ULN).

253. The method of claim 250, wherein the human subject in need thereof demonstrates no clinically significant changes in the level(s) of liver biomarkers as specified by a platelet count between 50 x lO’ / L and 1,000 x l09 / L.

254. The method of claim 250, wherein the human subject in need thereof demonstrates no clinically significant changes in the level(s) of liver biomarkers as specified by a bilirubin level between 0-1 mg / dL.

255. The method of any one of claims 233 to 254, wherein the base editor protein comprises an adenosine base editor.

256. The method of claim 255, wherein the adenosine base editor comprises a TadA.

257. The method of claim 255, wherein the TadA comprises an amino acid sequence of SEQ ID NO: 6.

258. The method of any one of claims 233 to 257, wherein the base editor protein comprises an amino acid sequence of SEQ ID NO: 3.

259. The method of any one of claims 233 to 258, wherein the mRNA comprises a nucleic acid sequence of SEQ ID NO: 2.

260. The method of any one of claims 233 to 259, wherein the amino lipid is present at a concentration of about 9.4 to about 26.3 mg / mL, the PEG-lipid is present at a concentration of about 2. 1 to about 4.4 mg / mL, the GalNAc lipid is present at a concentration of about 0.03 to about 0.14 mg / mL, the sterolWSGR Attorney Docket No. 53989-743.601 is present at a concentration of about 4.5 to about 8.2 mg / mL, and the phospholipid is present at a concentration of about 1.8 to about 4.3 mg / mL.

261. The method of any one of claims 233 to 260, wherein the ratio of (1) total LNP excipients and (2) a total amount of the RNA and the guide polynucleotide is about (27 ± 5.4) : 1 by weight.

262. The method of any one of claims 233 to 261, wherein the human subject in need thereof is administered a first does of the pharmaceutical composition.

263. The method of claim 262, wherein the first dose is at least about 0.3 mg / kg of a total amount of the RNA and the guide polynucleotide.

264. The method of claim 262, wherein the first dose is at least about 0.45 mg / kg of a total amount of the RNA and the guide polynucleotide.

265. The method of claim 262, wherein the first dose is at least about 0.6 mg / kg of a total amount of the RNA and the guide polynucleotide.

266. The method of claim 262, wherein the first dose is at least about 0.8 mg / kg of a total amount of the RNA and the guide polynucleotide.

267. The method of claim 262, wherein the first dose is at least about 1 mg / kg of a total amount of the RNA and the guide polynucleotide.

268. The method of any one of claims 262-267, wherein the human subject in need thereof is administered a second dose of the pharmaceutical composition, and wherein the second dose is higher, the same or lower than the first dose.

269. The method of claim 268, wherein the second dose is administered one to six days after the first dose.

270. The method of any one of claims 233 to 261, wherein the human subject in need thereof is administered a first fixed dose of the pharmaceutical composition.

271. The method of claim 270, wherein the first fixed dose is about 20 mg, about 25 mg, about 30 mg, about 36 mg, about 37 mg, about 42 mg, about 48 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 95 mg or about 100 mg of a total amount of the RNA and the guide polynucleotide.

272. The method of claim 270, wherein the first fixed dose is at least about 60 mg of a total amount of the RNA and the guide polynucleotide.

273. The method of claim 270, wherein the first fixed dose is about 75, about 80, about 85, about 90, about 95 or about 100 mg of a total amount of the RNA and the guide polynucleotide.

274. The method of any one of claims 270-273, wherein the group of human subjects are administered a second fixed dose of the pharmaceutical composition, and wherein the second fixed dose is higher, the same or lower than the first fixed dose.

275. The method of claim 274, wherein the second fixed dose is administered one to six days after the first fixed dose.WSGR Attorney Docket No. 53989-743.601276. The method of any one of claims 233-275, wherein the pharmaceutical composition has a particle size between 65 ± 15 nM Z-average hydrodynamic diameter.

277. The method of any one of claims 233-275, wherein the pharmaceutical composition has a particle size between 65 ± 10 nM Z-average hydrodynamic diameter.

278. The method of any one of claims 233-275, wherein the pharmaceutical composition has a particle size between 65 ± 5 nM Z-average hydrodynamic diameter.

279. The method of any one of claims 233-278, wherein the pharmaceutical composition has polydispersity index of at most 0.2 as determined by dynamic light scattering.

280. The method of any one of claims 233-279, wherein the pharmaceutical composition has a pH of 7.5 + / - 0.5.

281. The method of any one of claims 233-280, wherein at least 85% of the total amount of the RNA and the guide polynucleotide is present in the pharmaceutical composition at pH 7.0-8.0.

282. The method of any one of claims 233-281, wherein at least 60% of the guide polynucleotide in the pharmaceutical composition is the full-length guide polynucleotide at pH 7.0-8.0.

283. The method of any one of claims 233-282, wherein at least 70% of the RNA in the pharmaceutical composition is the full-length RNA at pH 7.0-8.0.

284. A therapeutically effective amount of a pharmaceutical composition for administration to a human subject in need of lowering LDL-C levels, wherein the pharmaceutical composition comprises lipid nanoparticles (LNPs) comprising:(a) an mRNA encoding a base editor protein,(b) a guide polynucleotide comprising a nucleotide sequence specified by SEQ ID No: 1(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a phospholipid, a plurality of PEG lipids, a sterol and a plurality of N-acetylgalactosamine (GalNAc) lipids, and wherein(i) the amino lipid is(iii) the plurality of PEG lipids isWSGR Attorney Docket No. 53989-743.601, wherein p is an integer selected from 42 to 48, (iv) the sterol is(v) the plurality of GalNAc lipids iswherein n is an integer selected from 33 to 37. wherein the LNPs are formulated in accordance with Tables 1, 2 and 3; and wherein the therapeutically effective amount as measured by total mass of the mRNA and guide polynucleotide of the pharmaceutical composition administered corresponds to a clinically supported time-weighted average (TWA) mean percent reduction of LDL-C of 40% or greater as compared to baseline.

285. The pharmaceutical composition of claim 284, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 45% or greater as compared baseline286. The pharmaceutical composition of claim 284, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 50% or greater as compared to baseline.

287. The pharmaceutical composition of claim 284, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 55% or greater as compared to baseline.

288. The pharmaceutical composition of claim 284, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 60% or greater as compared to baseline.WSGR Attorney Docket No. 53989-743.601289. The pharmaceutical composition of claim 284, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 65% or greater as compared to baseline.

290. The pharmaceutical composition of claim 284, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 70% or greater as compared to baseline.

291. The pharmaceutical composition of claim 284, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 75% or greater as compared to baseline.

292. The pharmaceutical composition of claim 284, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of LDL-C of 80% or greater as compared to baseline.

293. A therapeutically effective amount of a pharmaceutical composition for administration to a human subject in need of lowering LDL-C levels, wherein the pharmaceutical composition comprises lipid nanoparticles (LNPs) comprising:(a) an mRNA encoding a base editor protein,(b) a guide polynucleotide comprising a nucleotide sequence specified by SEQ ID No: 1(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a phospholipid, a plurality of PEG lipids, a sterol and a plurality of N-acetylgalactosamine (GalNAc) lipids, and wherein(i) the amino lipid is(iii) the plurality of PEG lipids is, wherein p is an integer selected from 42 to 48,(iv) the sterol isWSGR Attorney Docket No. 53989-743.601(v) the plurality of GalNAc lipids iswherein n is an integer selected from 33 to 37. wherein the LNPs are formulated in accordance with Tables 1, 2 and 3; and wherein the therapeutically effective amount as measured by total mass of the mRNA and guide polynucleotide of the pharmaceutical composition administered corresponds to a clinically supported time-weighted average TWA mean percent reduction of PCSK9 of 40% or greater as compared baseline.

294. The pharmaceutical composition of claim 293, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 45% or greater as compared to baseline.

295. The pharmaceutical composition of claim 293, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 50% or greater as compared to baseline.

296. The pharmaceutical composition of claim 293, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 55% or greater as compared to baseline.

297. The pharmaceutical composition of claim 293, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 60% or greater as compared to baseline.

298. The pharmaceutical composition of claim 293, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 65% or greater as compared to baseline.

299. The pharmaceutical composition of claim 293, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 70% or greater as compared to baseline.WSGR Attorney Docket No. 53989-743.601300. The pharmaceutical composition of claim 293, wherein the therapeutically effective amount of the pharmaceutical composition corresponds to a clinically supported TWA mean percent reduction of PCSK9 of 75% or greater as compared to baseline.

301. A therapeutically safe amount of a pharmaceutical composition for administration to a human subject in need of lowering LDL-C levels, wherein the pharmaceutical composition comprises lipid nanoparticles (LNPs) comprising:(a) an mRNA encoding a base editor protein,(b) a guide polynucleotide comprising a nucleotide sequence specified by SEQ ID No: 1(c) lipid nanoparticle (LNP) excipients, wherein the LNP excipients comprise an amino lipid, a phospholipid, a plurality of PEG lipids, a sterol and a plurality of N-acetylgalactosamine (GalNAc) lipids, and wherein(i) the amino lipid is(iii) the plurality of PEG lipids is, wherein p is an integer selected from 42 to 48, (iv) the sterol is(v) the plurality of GalNAc lipids isWSGR Attorney Docket No. 53989-743.601wherein n is an integer selected from 33 to 37. wherein the LNPs are formulated in accordance with Tables 1, 2 and 3; and wherein the therapeutically safe amount is measured by total mass of the mRNA and guide polynucleotide of the pharmaceutical composition, and wherein after administration of the therapeutically safe amount of the LNP, the human subject in need thereof demonstrates no clinically significant changes in the level(s) of liver biomarkers.

302. The pharmaceutical composition of claim 301, wherein the human subject in need thereof demonstrates no clinically significant changes in the level(s) of liver biomarkers as specified by an alanine aminotransferase (ALT) level less than or equal to three-fold of an upper limit of normal (ULN).

303. The pharmaceutical composition of claim 301, wherein the human subject in need thereof demonstrates no clinically significant changes in the level(s) of liver biomarkers as specified by an aspartate aminotransferase (AST) level less than equal to three-fold of an upper limit of normal (ULN).

304. The pharmaceutical composition of claim 301, wherein the human subject in need thereof demonstrates no clinically significant changes in the level(s) of liver biomarkers as specified by a platelet count between 50 x 109 / L and 1,000 x 109 / L.

305. The pharmaceutical composition of claim 301, wherein the human subject in need thereof demonstrates no clinically significant changes in the level(s) of liver biomarkers as specified by a bilirubin level between 0-1 mg / dL.

306. The pharmaceutical composition of any one of claims 284-305, wherein the base editor protein comprises an adenosine base editor.

307. The pharmaceutical composition of claim 306, wherein the adenosine base editor comprises a TadA.

308. The pharmaceutical composition of claim 307, wherein the TadA comprises an amino acid sequence of SEQ ID NO: 6.

309. The pharmaceutical composition of any one of claims 284-308, wherein the base editor protein comprises an amino acid sequence of SEQ ID NO: 3.

310. The pharmaceutical composition of any one of claims 284-309, wherein the mRNA comprises a nucleic acid sequence of SEQ ID NO: 2.WSGR Attorney Docket No. 53989-743.601311. The pharmaceutical composition of any one of claims 284-310, wherein the amino lipid is present at a concentration of about 9.4 to about 26.3 mg / mL, the PEG-lipid is present at a concentration of about 2.1 to about 4.4 mg / mL, the GalNAc lipid is present at a concentration of about 0.03 to about0. 14 mg / mL, the sterol is present at a concentration of about 4.5 to about 8.2 mg / mL, and the phospholipid is present at a concentration of about 1.8 to about 4.3 mg / mL.

312. The pharmaceutical composition of any one of claims 284-311, wherein the ratio of (1) total LNP excipients and (2) a total amount of the RNA and the guide polynucleotide is about (27 ± 5.4) : 1 by weight.

313. The pharmaceutical composition of any one of claims 284-312, wherein the human subject in need thereof is administered a first does of the pharmaceutical composition.

314. The pharmaceutical composition of claim 313, wherein the first dose is at least about 0.3 mg / kg of a total amount of the RNA and the guide polynucleotide.

315. The pharmaceutical composition of claim 313, wherein the first dose is at least about 0.45 mg / kg of a total amount of the RNA and the guide polynucleotide.

316. The pharmaceutical composition of claim 313, wherein the first dose is at least about 0.6 mg / kg of a total amount of the RNA and the guide polynucleotide.

317. The pharmaceutical composition of claim 313, wherein the first dose is at least about 0.8 mg / kg of a total amount of the RNA and the guide polynucleotide.

318. The pharmaceutical composition of claim 313, wherein the first dose is at least about 1 mg / kg of a total amount of the RNA and the guide polynucleotide.

319. The pharmaceutical composition of any one of claims 313-318, wherein the human subject in need thereof is administered a second dose of the pharmaceutical composition, and wherein the second dose is higher, the same or lower than the first dose.

320. The pharmaceutical composition of claim 319, wherein the second dose is administered one to six days after the first dose.

321. The pharmaceutical composition of any one of claims 284-312, wherein the human subject in need thereof is administered a first fixed dose of the pharmaceutical composition.

322. The pharmaceutical composition of claim 321, wherein the first fixed dose is about 20 mg, about 25 mg, about 30 mg, about 36 mg, about 37 mg, about 42 mg, about 48 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 95 mg or about 100 mg of a total amount of the RNA and the guide polynucleotide.

323. The pharmaceutical composition of claim 321, wherein the first fixed dose is at least about 60 mg of a total amount of the RNA and the guide polynucleotide.

324. The pharmaceutical composition of claim 321, wherein the first fixed dose is about 75, about 80, about 85, about 90, about 95 or about 100 mg of a total amount of the RNA and the guide polynucleotide.WSGR Attorney Docket No. 53989-743.601325. The pharmaceutical composition of any one of claims 321-324, wherein the group of human subjects are administered a second fixed dose of the pharmaceutical composition, and wherein the second fixed dose is higher, the same or lower than the first fixed dose.

326. The pharmaceutical composition of claim 325, wherein the second fixed dose is administered one to six days after the first fixed dose.

327. The pharmaceutical composition of any one of claims 284-326, wherein the pharmaceutical composition has a particle size between 65 ± 15 nM Z-average hydrodynamic diameter.

328. The pharmaceutical composition of any one of claims 284-326, wherein the pharmaceutical composition has a particle size between 65 ± 10 nM Z-average hydrodynamic diameter.

329. The pharmaceutical composition of any one of claims 284-326, wherein the pharmaceutical composition has a particle size between 65 ± 5 nM Z-average hydrodynamic diameter.

330. The pharmaceutical composition of any one of claims 284-329, wherein the pharmaceutical composition has polydispersity index of at most 0.2 as determined by dynamic light scattering.

331. The pharmaceutical composition of any one of claims 284-330, wherein the pharmaceutical composition has a pH of 7.5 + / - 0.5.

332. The pharmaceutical composition of any one of claims 284-331, wherein at least 85% of the total amount of the RNA and the guide polynucleotide is present in the pharmaceutical composition at pH 7.0-8.0.

333. The pharmaceutical composition of any one of claims 284-332, wherein at least 60% of the guide polynucleotide in the pharmaceutical composition is the full-length guide polynucleotide at pH 7.0- 8.0.

334. The pharmaceutical composition of any one of claims 284-333, wherein at least 70% of the RNA in the pharmaceutical composition is the full-length RNA at pH 7.0-8.0.

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