Compositions for the modification of the human APOC3 gene

Abstract

Provided herein are compositions and methods for modifying the human gene, APOC3. Such compositions and methods may result in the reduction of the protein, apolipoprotein C3 (apoC-III) when administered to a human subject. Compositions and methods provided herein may comprise a CRISPR-associated (Cas) protein or uses thereof. Compositions and methods of the present disclosure may be useful for treatment of APOC3 associated conditions, including persistent chylomicronemia, familial chylomicronemia syndrome (FCS) and severe hypertriglyceridemia (SHTG).

Description

Attorney Docket No.: MABI-051 / 03WO 344183-2344COMPOSITIONS FOR THE MODIFICATION OF THE HUMAN APOC3 GENECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application 63 / 688,271, filed August 28, 2024; U.S. Provisional Application 63 / 696,175, filed September 18, 2024; U.S. Provisional Application 63 / 745,289, filed January 14, 2025, the contents of each of which are incorporated herein by reference in their entireties.SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (MABI_051_03WO_SeqList_ST26.xml; Size 170,516 bytes; and Date of Creation: August 26, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0003] Apolipoprotein C3 (APOC3) protein, expressed in liver and intestine, is a key regulator of plasma triglyceride levels. APOC3 regulates liver uptake of triglyceride-rich lipoproteins through lipoprotein lipase (LPL)-dependent and LPL-independent mechanisms. It has been suggested that APOC3 exerts pro-atherogenic effects directly by enhancing vessel wall inflammation and indirectly by promoting hypertriglyceridemia. Individuals with loss of function mutations in APOC3 show ~40% reduction in both triglyceride levels and risk for atherosclerotic cardiovascular disease (ASCVD) compared with non-carriers. Furthermore, epidemiological studies have concluded that APOC3 levels predict risk of ASCVD and cardiovascular mortality.

[0004] Chylomicronemia is a condition characterized by chronically elevated levels of circulating fat particles called chylomicrons. Typically, patients with chylomicronemia have fasting triglyceride levels above 880 mg / dl. Chylomicronemia may occur sporadically, intermittently, or persistently. Chylomicronemia is generally considered persistent chylomicronemia when it is refractory to conventional lipid lowering therapies (e.g., statins, fibrates, niacin, omega-3 fatty acids, etc.). Chylomicronemia is associated with the risk of recurrent and potentially fatal pancreatitis and other complications. Familial chylomicronemia syndrome (FCS) is a rare autosomal recessive disease characterized by a buildup in the blood of chylomicrons (chylomicronemia). FCS is caused by mutations in the gene encoding LPL or, less frequently, by mutations in genes encoding other proteins necessary for LPL function. People with FCS are at high risk of unpredictable and potentially fatal acute pancreatitis. In addition to pancreatitis, FCS patients are at risk of chronic complications due to permanent organ damage, including chronic pancreatitis and pancreatogenic (Type 3c) diabetes. Patients with hypertriglyceridemia or chylomicronemia may experience symptoms including abdominal pain, yellowing of the skin and eyes, generalized fatigue, impaired vision, and impaired cognition thatAttorney Docket No.: MABI-051 / 03WO 344183-2344 affect their ability to work. People with FCS report major emotional and psychosocial effects including anxiety, social withdrawal, depression, and brain fog.

[0005] Severe hypertriglyceridemia (SHTG) is a common condition characterized by high levels of triglycerides in the bloodstream (> 1000 mg / dL) . SHTG can be caused by diet-derived chylomicronemia and excessive liver triglyceride production, often superimposed on genetic predisposition. It is often associated with acute pancreatitis (a life-threatening condition) and a higher risk of cardiovascular disease. Lowering of plasma triglycerides is desirable in SHTG patients. Hypertriglyceridemia can lead to conditions including atherosclerosis (hardening of the arteries), obesity, and insulin resistance, which all can contribute to increased risk of cardiovascular disease.SUMMARY

[0006] Disclosed herein, in some aspects, are compositions comprising a messenger RNA (mRNA) encoding an effector protein (e.g., a CRISPR associated (Cas) protein), and a guide nucleic acid for the modification of the human gene, APOC3. In some embodiments, the messenger RNA (mRNA) comprises or consists of a nucleotide sequence of SEQ ID NO: 25; and the guide nucleic acid comprises or consists of a nucleotide sequence of SEQ ID NO: 55. In some embodiments, the mRNA comprises a nucleotide sequence of SEQ ID NO: 23; and the guide nucleic acid comprises or consists of a nucleotide sequence selected from SEQ ID NOS: 52-55. In some embodiments, the mRNA encodes an amino acid sequence of SEQ ID NO: 21; and the guide nucleic acid comprises or consists of a nucleotide sequence selected from SEQ ID NOS: 52-55. In some embodiments, the mRNA comprises or consists of a nucleotide sequence selected from SEQ ID NO: 23 and SEQ ID NO: 25; and a guide nucleic acid comprises or consists of a nucleotide sequence selected from: i) mG*mU*mC*rUrUrGrArGrArG*mG*mA*mA*mArCrUrCrUrCrArArGrArC*mU*rArGrArUrUrGr CrUrCrCrUrUrArCrGrArGrGrArGrArCrCrCrUrGrArArArGrArCrUrArCrUrGrGmA*mG*mC (SEQ ID NO: 53), and ii) mG*mU*mC*rUrUrGrArGrArG*mG*mA*mA*mArCrUrCrUrCrArArGrArC*mU*rArGrArUrUrGr CrUrCrCrUrUrArCrGrArGrGrArGrArCrCrCrCrUrCrCrCrCrArGrArGrGrGrCmA*mU*mU (SEQ ID NO: 55), wherein r represents an unmodified nucleotide, m represents a 2’-0Me modified nucleotide, and * represents a phosphorothioate bond. In some embodiments, compositions comprise a lipid nanoparticle (LNP), wherein the mRNA and the guide nucleic acid are encapsulated in the LNP. In some embodiments, the mRNA comprises or is linked to the following chemical structure:Attorney Docket No.: MABI-051 / 03WO 344183-2344In some embodiments, the chemical structure is located at or linked to the 5’ end of the mRNA. Disclosed herein in some aspects, are pharmaceutical composition comprising a composition disclosed herein and a pharmaceutical acceptable excipient or carrier.

[0007] Also disclosed herein, in some aspects, are methods of modifying APOC3, comprising contacting APOC3, or a cell comprising APOC3, with a composition disclosed herein, wherein (i) an effector protein encoded by the mRNA and (ii) the guide RNA form a ribonucleoprotein (RNP) complex that binds to a target nucleotide sequence of APOC3. In some embodiments, modifying APOC3 reduces the expression of APO('3. optionally wherein modifying APOC3 permanently reduces the expression of the APOC3 gene. In some embodiments, modifying APOC3 comprises cleaving both strands of APOC3. In some embodiments, methods comprise modifying APOC3 in vivo. In some embodiments, methods comprise modifying APOC3 in a liver of a subject. Also disclosed herein, in some aspects, are methods of lowering triglycerides in a human subject with hypertriglyceridemia wherein the method comprises administering a composition disclosed herein to the human subject. Also disclosed herein, in some aspects, are methods of treating or preventing a disease comprising chylomicronemia in a human subject in need thereof, comprising administering a composition disclosed herein to the human subject. In some embodiments, the disease comprises persistent chylomicronemia. In some embodiments, the disease comprises SHTG. In some embodiments, the disease comprises FCS. Also disclosed herein, in some aspects, are cells, or populations of cells, comprising, or modified by, a composition or method disclosed herein.

[0008] Also disclosed herein, in some aspects, are engineered polypeptides or a nucleic acid encoding the same, wherein the engineered polypeptide comprises or consists of an amino acid sequence that is identical to SEQ ID NO: 19, with the exception of the following amino acid substitutions: L26R, T87G, Y220S, S223P, 147 IT, and D703G, optionally wherein the nucleic acid is DNA or RNA. Also disclosed herein, in some aspects, are mRNAs encoding an engineered effector protein, wherein the engineered protein comprises or consists of an amino acid sequence that is identical to SEQ ID NO: 19, with the exception of the following amino acid substitutions: L26R, T87G, Y220S, S223P, I471T, and D703G.Attorney Docket No.: MABI-051 / 03WO 344183-2344Also disclosed herein, in some aspects, are engineered polypeptides or a nucleic acid encoding the same, wherein the engineered polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 21, optionally wherein the nucleic acid comprises DNA, RNA or a combination thereof. Also disclosed herein, in some aspects, are engineered guide nucleic acids comprising or consisting of a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from SEQ ID NOS: 52-55. Also disclosed herein, in some aspects, are engineered guide nucleic acids comprising from 5' to 3' a) a stabilizer sequence, b) a repeat sequence, and c) a spacer sequence, wherein the stabilizer sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 65, 77-78, and wherein the repeat sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 5-11. In some embodiments, the spacer sequence comprises or consists of SEQ ID NO: 51. Also disclosed herein, in some aspects, are compositions comprising the engineered guide nucleic acid selected from SEQ ID NOS: 12-18, 49, 52-60, and an effector protein or nucleic acid encoding the same, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 19.

[0009] Also disclosed herein, in some aspects, are engineered guide nucleic acids according to the following formula: mN*mN*mN*rNrNrNrNrNrNrN*mN*mN*mN*mNrNrNrNrNrNrNrNrNrNrN*mN*rNrNrNrNrUrG rCrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNmN*mN*mN, wherein N is a nucleotide selected from A, G, C, and U or T, wherein * represents a phosphorothioate linkage, wherein m represents a 2’-OMe sugar modification, wherein r represents an unmodified ribonucleotide, and wherein italicized letters indicate a spacer sequence. Also disclosed herein, in some aspects, are engineered guide nucleic acids according to the following formula: mN*mN*mN*rNrNrNrNrNrNrN*mN*mN*mN*mNrNrNrNrNrNrNrNrNrNrN*mN*rNrNrNrNrUrG rCrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNmmN*mN*mN, wherein N is a nucleotide selected from A, G, C, and U or T, wherein * represents a phosphorothioate linkage, wherein m represents a 2’-OMe sugar modification, wherein r represents an unmodified ribonucleotide, wherein italicized letters indicate a spacer sequence, and wherein n = 13 to 19.

[0010] Also disclosed herein, in some aspects, are compositions for use in treating or preventing a disease in a human subject in need thereof, comprising administering the composition to the human subject, wherein the composition is disclosed herein. In some embodiments, the disease is selected from cardiovascular disease, persistent chylomicronemia, familial chylomicronemia syndrome, and hypertriglyceridemia. Also disclosed herein, in some aspects, are methods of producing (a) a messenger RNA (mRNA) comprising or consisting of a nucleotide sequence of SEQ ID NO: 25; and (b) a guide nucleic acid comprising or consisting of a nucleotide sequence of SEQ ID NO: 55, or a combinationAttorney Docket No.: MABI-051 / 03WO 344183-2344 thereof. In some embodiments, methods comprise encapsulating the mRNA and the guide nucleic acid in an LNP.INCORPORATION BY REFERENCE

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 shows nuclease activity (% indel) of multiple CasPhi.12 variants in HEK293T cells.

[0013] FIG. 2 shows off-target modifications (% indel) generated with multiple CasPhi. 12 variants.

[0014] FIG. 3 illustrates exemplary nuclear localization signals (NLS) that can be fused to effector proteins, including but not limited to CasPhi.12 and engineered variants thereof.

[0015] FIG. 4 shows the nuclease activity (% indel) of an APOC3 targeting guide nucleic acid, and CasPhi.12 variant L26R, 147 IT fused to various NLSs in primary human hepatocytes.

[0016] FIG. 5 shows % indel APOC3 generated in livers of hAPOC3 mice with mRNA253 and guide RNAs comprising a stabilizer sequence.

[0017] FIG. 6A shows % indel A POC3 generated in livers of hAPOC3 mice mRNA253 and guide RNAs comprising a stabilizer sequence.

[0018] FIG. 6B shows levels of serum APOC3 protein in livers of hAPOC3 mice after administration of mRNA253 and guide RNAs comprising a stabilizer sequence.

[0019] FIG. 7 shows the mean specificity ratio (specificity score) of CasPhi.12 L26R-T87G-Y220S- S223P-I471T-D703G (mRNA253 encoded protein).

[0020] FIG. 8 shows mean specificity ratios (specificity scores) of CasPhi.12 variants that include an S526N substitution.

[0021] FIGS. 9A and 9B show that dosing a humanized APOC3 mouse model of SHTG with a CasPhi.12 engineered variant and APOC3 guide RNA lowers serum levels of APOC3 protein (FIG. 9A) and triglycerides (FIG. 9B).

[0022] FIG. 10 shows the results (% indel in liver samples) of treating non-human primates with a CasPhi.12 variant and APOC3 targeting guide nucleic acid.

[0023] FIG. 11A shows % indel in livers of non-human primates one week after administration of mRNA253, and APOC3 targeting guide nucleic acid R22528 or R22529. FIG. 11B shows relative levels of APOC3 mRNA in livers of non-human primates one week after administration of a mRNA253, and APOC3 targeting guide nucleic acid R22528 or R22529.

[0024] FIG. 12 shows levels of APOC3 protein in livers of non-human primates 8, 15, and 29 days after administration of mRNA253 and an APOC3 targeting guide nucleic acid R22528 or R22529.Attorney Docket No.: MABI-051 / 03WO 344183-2344

[0025] FIG. 13 shows that reduction of serum APOC3 protein in non-human primates by mRNA253 and guide nucleic acid R22529 persists 89 days after administration.

[0026] FIGS. 14A-14C show % indel APOC3 (FIG. 14A), relative quantities of APOC3 mRNA (FIG. 14B) and APOC3 protein (FIG. 14C) in livers of non-human primates with mRNA253 and guide nucleic acid R22529 over time after administration.

[0027] FIGS. 15A-15D show the results of liver function tests in non-human primates at various time points after administration of mRNA253 and guide nucleic acid R22529 or R22528. Specifically, FIG. 15A shows the ALT group means, and FIG. 15B shows the total bilirubin group mean. FIG. 15C shows peak ALT levels and FIG. 15D shows peak total bilirubin levels 24-72 hours post-dosing.

[0028] FIG. 16 shows % indel APOC3 generated in livers of male and female non-human primates with mRNA253 and guide nucleic acid R22529 one week after administration.

[0029] FIG. 17 shows relative amounts of APOC3 mRNA in livers of female non-human primates at multiple time points after administration of mRNA253 and guide nucleic acid R22529.DETAILED DESCRIPTION OF THE INVENTION

[0030] It is to be understood that both the foregoing general description and the following detailed description are exemplary, and explanatory only, and are not restrictive of the disclosure.

[0031] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0032] All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.Definitions

[0033] Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated or obvious from context, the following terms have the following meanings:

[0034] The terms, “a,” “an,” and “the,” as used herein, include plural references unless the context clearly dictates otherwise.

[0035] The terms, “or” and “and / or,” as used herein, include any, and all, combinations of one or more of the associated listed items.

[0036] The terms, “including,” “includes,” “included,” and other forms, are not limiting.

[0037] The terms, “comprise” and its grammatical equivalents, as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.Attorney Docket No.: MABI-051 / 03WO 344183-2344

[0038] The term, “about,” as used herein in reference to a number or range of numbers, is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0039] The terms, “% identical,” “% identity,” and “percent identity,” or grammatical equivalents thereof, refer to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” can refer to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(l): 11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep l;25(17):3389-40), BLASTP, BLASTN, or GCG.

[0040] The term “cis cleavage,” as used herein, refers to cleavage (hydrolysis of a phosphodiester bond) of a target nucleic acid by an effector protein complexed with a guide nucleic acid (e.g. , an RNP complex), wherein at least a portion of the guide nucleic acid is hybridized to at least a portion of the target nucleic acid. Cleavage may occur within or directly adjacent to the region of the target nucleic acid that is hybridized to the guide nucleic acid.

[0041] The terms “complementary” and “complementarity,” as used herein, with reference to a nucleic acid molecule or nucleotide sequence, refer to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T or U) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is in general, understood as going in the direction from its 5'- to 3 '-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand. Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5'-end, while the ‘reverse complement’ sequence or the ‘reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3 '-end. Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.

[0042] The terms “cleave,” “cleaving,” and “cleavage,” as used herein, with reference to a nucleic acid molecule or nuclease activity of an effector protein, refer to the hydrolysis of a phosphodiester bond of a nucleic acid molecule that results in breakage of that bond. The result of this breakage can be a nick (hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule), single strand break (hydrolysis of a single phosphodiester bond on a single-stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double -stranded molecule) depending uponAttorney Docket No.: MABI-051 / 03WO 344183-2344 whether the nucleic acid molecule is single-stranded (e.g., ssDNA or ssRNA) or double-stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein.

[0043] The term “clustered regularly interspaced short palindromic repeats (CRISPR),” as used herein, refers to a segment of DNA found in the genomes of certain prokaryotic organisms, including some bacteria and archaea, that includes repeated short sequences of nucleotides interspersed at regular intervals between unique sequences of nucleotides derived from the DNA of a pathogen (e.g., virus) that had previously infected the organism and that functions to protect the organism against future infections by the same pathogen.

[0044] The terms “CRISPR RNA” or “crRNA,” as used herein, refer to a type of guide nucleic acid, wherein the nucleic acid is RNA comprising a first sequence that is capable of interacting with an effector protein either directly (by being bound by an effector protein) or indirectly (e.g., by hybridization with a second nucleic acid molecule that can be bound by an effector, such as a tracrRNA); and a second sequence that hybridizes to a target sequence of a target nucleic acid. In some embodiments, the first sequence is referred to as a repeat sequence and the second sequence is referred to as a spacer sequence. The first sequence and the second sequence are directly connected to each other or by a linker.

[0045] The term “effector protein,” as used herein, refers to a protein that is capable of interacting with a guide nucleic acid to form a complex (e.g., a RNP complex), wherein the complex interacts with a target nucleic acid. A complex between an effector protein and a guide nucleic acid may include multiple effector proteins or a single effector protein. An effector protein may modify a target nucleic acid by cleaving of a phosphodiester bond of the target nucleic acid. Additional examples of modifications an effector protein can make to target nucleic acids are described herein and throughout. Herein, reference to an effector protein includes reference to a nucleic acid encoding the effector protein, unless indicated otherwise.

[0046] The terms, “engineered modification,” and “chemical modification,” as used herein, refer to a structural change of an amino acid of an amino acid sequence or a nucleotide of a polynucleotide relative to a naturally occurring amino acid or nucleotide, respectively. Non-limiting examples of chemical modifications of a nucleic acid are replacement of a phosphodiester bond with a phosphorothioate bond; a 2’-0 Methyl (2’-0Me) modification of the sugar component of a nucleotide; and a 5’ cap on an mRNA. Such modifications can be made to an effector protein amino acid sequence or guide nucleic acid nucleotide sequence, or any sequence disclosed herein (e.g., a nucleic acid encoding an effector protein or a nucleic acid that encodes a guide nucleic acid). Methods of modifying a nucleic acid or amino acid sequence are known. One of ordinary skill in the art will appreciate that the engineered modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid, protein, or composition is not substantially decreased. Nucleic acids provided herein can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vfi -transcription, cloning, enzymatic, or chemicalAttorney Docket No.: MABI-051 / 03WO 344183-2344 cleavage, etc. In some embodiments, the nucleic acids provided herein are not uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures can exist at various positions within the nucleic acid.

[0047] The term “guide nucleic acid,” as used herein, refers to a nucleic acid comprising: a first nucleotide sequence that is capable of being non-covalently bound by an effector protein; and a second nucleotide sequence that hybridizes to a target nucleic acid. When in a complex with one or more proteins described herein (e.g. , an RNP complex), a guide nucleic acid may impart sequence selectivity to the complex when the complex interacts with a target nucleic acid. The first sequence may be referred to herein as a repeat sequence. The second sequence may be referred to herein as a spacer sequence. The term, “guide nucleic acid,” may be used interchangeably herein with the term “guide RNA” (gRNA). However, it is understood that guide nucleic acids may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof.

[0048] The term “heterologous,” as used herein, is used to refer to two or more polynucleotide or polypeptide sequences that are not found in a native nucleic acid or protein, respectively.

[0049] The term “linked” when used in reference to biopolymers (e.g., polynucleotides, polypeptides) refers to being covalently connected. In some embodiments, two biopolymers are linked by at least a covalent bond. In some embodiments, two polynucleotides are linked by at least one nucleotide. In some embodiments, two polypeptides are linked by at least one amino acid. The terms “fused” and “linked” are used interchangeably herein.

[0050] The term “linker,” as used herein, refers to a covalent bond or molecule that links a first polypeptide to a second polypeptide (e.g., by an amide bond, or one or more amino acids) or a first nucleic acid to a second nucleic acid (e.g., by a phosphodiester bond, or one or more nucleotides).

[0051] The terms “non-naturally occurring” and “engineered,” as used herein, are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid that is at least substantially free from at least one other feature with which it is naturally associated in nature and as found in nature, and / or contains a modification (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally occurring nucleic acid, nucleotide, protein, polypeptide, peptide, or amino acid. The terms, when referring to a composition described herein, refer to a composition having at least one component that is not naturally associated with the other components of the composition. By way of a non-limiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effectorAttorney Docket No.: MABI-051 / 03WO 344183-2344 protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man.

[0052] The term “nuclear localization signal (NLS),” as used herein, refers to an entity (e.g., peptide) that facilitates localization of a polynucleotide or polypeptide to the nucleus, when present in a cell that contains a nuclear compartment.

[0053] The term “nuclease activity,” as used herein, refers to the catalytic activity that results in nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), or deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).

[0054] The term “pharmaceutically acceptable excipient, carrier or diluent,” as used herein, refers to any substance formulated alongside the active ingredient of a pharmaceutical composition that allows the active ingredient to retain biological activity and is non-reactive with the subject’s immune system. Such a substance can be included for the purpose of long-term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, or enhancing solubility. The selection of appropriate substance can depend upon the route of administration and the dosage form, as well as the active ingredient and other factors. Compositions having such substances can be formulated by well-known conventional methods (see, e.g., Remington, The Science and Practice of Pharmacy 23rdedition, A. Adejare, ed., Elsevier Publishing Co., 2020).

[0055] The term “protospacer adjacent motif’ and “PAM,” as used herein, refers to a nucleotide sequence found in a target nucleic acid that directs an effector protein to modify the target nucleic acid at a specific location. In some embodiments, a PAM sequence is required for a complex of an effector protein and a guide nucleic acid (e.g. , an RNP complex) to hybridize to and edit the target nucleic acid. In some embodiments, the complex does not require a PAM to edit the target nucleic acid.

[0056] In some embodiments, the term “region” as used herein may be used to describe a portion of, or all of, a corresponding sequence. By way of non-limiting example, a second region may comprise or consist of a spacer sequence.

[0057] The term, “repeat sequence,” as used herein, refers to a sequence of nucleotides in a guide nucleic acid that is capable of, at least partially, interacting with an effector protein.

[0058] The terms, “ribonucleotide protein complex” and “RNP” as used herein, refer to a complex of one or more nucleic acids and one or more polypeptides described herein. While the term utilizes “ribonucleotides” it is understood that the one or more nucleic acid may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof.

[0059] The terms, “RuvC” and “RuvC domain,” as used herein, refer to a region of an effector protein that is capable of cleaving a target nucleic acid, and in certain embodiments, of processing a pre-crRNA. In some embodiments, the RuvC domain is located near the C-terminus of the effector protein. A singleAttorney Docket No.: MABI-051 / 03WO 344183-2344RuvC domain may comprise RuvC subdomains, for example a RuvCI subdomain, a RuvCII subdomain and a RuvCIII subdomain. The term “RuvC” domain can also refer to a “RuvC-like” domain. Various RuvC-like domains are known in the art and are easily identified using online tools such as InterPro (ebi.ac.uk / interpro / ). For example, a RuvC-like domain may be a domain which shares homology with a region of TnpB proteins of the IS605 and other related families of transposons

[0060] The term, “spacer sequence,” as used herein, refers to a nucleotide sequence in a guide nucleic acid that is capable of, at least partially, hybridizing to an equal length portion of a sequence (e.g., a target sequence) of a target nucleic acid. In general, a spacer sequence is complementary to the target sequence.

[0061] The term “target nucleic acid,” as used herein, refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. By way of non-limiting example, APOC3 is a target nucleic acid.

[0062] The terms “target nucleic acid sequence” and “target sequence,” as used herein, when used in reference to a target nucleic acid, refers to a sequence of nucleotides found within a target nucleic acid. Such a sequence of nucleotides can, for example, hybridize to an equal length portion of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid. By way of non-limiting example, a target sequence is a sequence within APOC3.

[0063] The terms “treatment” and “treating,” as used herein, are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.Introduction

[0064] Disclosed herein are compositions and methods for the modification of the APOC3 gene. APOC3 resides within the APOA5 / APOA4 / APOC3 / APOA1 multigene cluster on the long arm of the human chromosome 1 lq23. It comprises 4 exons and 3 introns and encodes a 99 amino acidAttorney Docket No.: MABI-051 / 03WO 344183-2344 glycoprotein called apoC-III (or APOC3). This apolipoprotein is mostly expressed in hepatocytes and enterocytes, where it undergoes an intracellular cleavage, yielding the mature 79 amino acid protein. Furthermore, it undergoes a post-translational modification leading to the formation of three distinct isoforms containing zero (apoC-IIIO), one (apoC-IIIl) or two (apoC-III2) sialic acid residues, and importantly, all these isoforms exhibit the same plasma half-life and catabolic mechanisms, suggesting similar physiological implications.

[0065] At the transcriptional level, the APOC3 gene expression is tightly regulated by several proposed pathways. A series of in vivo and in vitro studies have demonstrated that its expression is downregulated by insulin, peroxisome proliferator-activated receptor a, Rev-erb, and famesoid X receptor. Conversely, the positive responsiveness of the APOC3 promoter to glucose was reported. This factor stimulates the gene expression by the activation of the carbohydrate-responsive element binding protein, as well as the hepatocyte nuclear factor-4a. Hence, the opposite interplay between insulin and glucose on modulating APOC3 transcriptional activity may induce an enhanced apoC-III secretion under an insulin-resistant condition associated with hyperglycemia (as in type 2 diabetes). Also, the total apoC- III levels can be significantly modulated in hyperlipidemic individuals by the dietary intake of low saturated fat and high amounts of monosaturated and omega-3 polyunsaturated fatty acids. Dysregulated expression of APOC3 has been associated with dyslipidemia, hypertriglyceridemia, atherosclerosis, altered HDL functionality, and other cardiovascular disorders. Also, polymorphs of APOC3 (SstI, T-455C and C-482T) are known to associate with hypertriglyceridemia in mice, and the SstI and T-455C polymorphs significantly increased the susceptibility to CHD in humans.

[0066] In some embodiments, the present disclosure provides guide nucleic acids that are capable of binding to a target sequence in APOC3. In some embodiments, the present disclosure provides guide nucleic acids that are capable of binding to a target sequence in APOC3 and an effector protein. In some embodiments, the effector protein is a CRISPR-associated (Cas) protein. In general, Cas proteins bind and / or modify nucleic acids in a sequence-specific manner. Cas proteins with guide nucleic acids may modify DNA at a precise target location in the genome of a wide variety of cells and organisms, allowing for precise and efficient editing of DNA sequences of interest. In some embodiments, the present disclosure provides methods for treating a disease (e.g., coronary artery disease and other cardiovascular related disorders or a liver disease) by modifying a target gene (e.g., APOC3).

[0067] Compositions disclosed herein are not naturally occurring. In general, guide nucleic acids disclosed herein are not found in nature. In some embodiments, compositions herein comprise at least one non-naturally occurring component. For example, compositions may comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid. In some embodiments, compositions comprise at least two components that do not naturally occur together. For example, compositions may comprise a guide nucleic acid comprising a repeat sequence and a spacer sequence which do not naturally occur together. Also, by way of example,Attorney Docket No.: MABI-051 / 03WO 344183-2344 composition may comprise a guide nucleic acid and an effector protein that do not naturally occur together.Certain EmbodimentsThe present disclosure provides the following non-limiting embodiments: Embodiment 1. A composition comprising: a messenger RNA (mRNA) comprising or consisting of a nucleotide sequence of SEQ ID NO: 25; a guide nucleic acid comprising or consisting of a nucleotide sequence selected from SEQ ID NO: 53 and SEQ ID NO: 55; and a lipid nanoparticle (LNP), wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 2. A composition comprising: a messenger RNA (mRNA) comprising a nucleotide sequence of SEQ ID NO: 23; a guide nucleic acid comprising or consisting of a nucleotide sequence selected from SEQ ID NOS: 52-55; and a lipid nanoparticle (LNP), wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 3. A composition comprising: a messenger RNA (mRNA) encoding an amino acid sequence of SEQ ID NO: 21; a guide nucleic acid comprising or consisting of a nucleotide sequence selected from SEQ ID NOS: 52-55; and a lipid nanoparticle (LNP), wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 4. A composition comprising: a messenger RNA (mRNA) comprising or consisting of a nucleotide sequence selected from SEQ ID NO: 23 and SEQ ID NO: 25; a guide nucleic acid comprising or consisting of a nucleotide sequence selected from: i) mG*mU*mC*rUrUrGrArGrArG*mG*mA*mA*mArCrUrCrUrCrArArGrArC*mU*rArGrArUrUrGr CrUrCrCrUrUrArCrGrArGrGrArGrArCrCrCrUrGrArArArGrArCrUrArCrUrGrGmA*mG*mC (SEQ ID NO: 53), and ii) mG*mU*mC*rUrUrGrArGrArG*mG*mA*mA*mArCrUrCrUrCrArArGrArC*mU*rArGrArUrUrGr CrUrCrCrUrUrArCrGrArGrGrArGrArCrCrCrCrUrCrCrCrCrArGrArGrGrGrCmA*mU*mU (SEQ ID NO: 55), wherein r represents an unmodified nucleotide, m represents a 2’-OMe modified nucleotide, and * represents a phosphorothioate bond; and a lipid nanoparticle (LNP), wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 5. A composition comprising: a messenger RNA (mRNA) comprising or consisting of a nucleotide sequence of SEQ ID NO: 25; a guide nucleic acid comprising or consisting of a nucleotide sequence of SEQ ID NO: 15; and a lipid nanoparticle (LNP), wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 6. A composition comprising: an mRNA comprising or consisting of a nucleotide sequence of SEQ ID NO: 24; a guide nucleic acid comprising or consisting of a nucleotide sequence of SEQ ID NO: 16; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Attorney Docket No.: MABI-051 / 03WO 344183-2344Embodiment 7. A composition comprising: an mRNA comprising or consisting of a nucleotide sequence of SEQ ID NO: 25; a guide nucleic acid comprising or consisting of a nucleotide sequence of SEQ ID NO: 17; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 8. A composition comprising: an mRNA comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 25; a guide nucleic acid comprising a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 15; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 9. A composition comprising: an mRNA comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 24; a guide nucleic acid comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 16; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 10. A composition comprising: an mRNA comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 25; a guide nucleic acid comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 17; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment IL composition comprising: an mRNA comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 25; a guide nucleic acid comprising a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 12; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 12. A composition comprising: an mRNA comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 24; a guide nucleic acid comprising a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 13; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 13. A composition comprising: an mRNA comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 25; a guide nucleic acid comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 14; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNPEmbodiment 14. The composition of any one of embodiments 11-13, wherein the guide RNA comprises at least one 2’-OMe modified nucleotide and at least one phosphorothioate linkage.Attorney Docket No.: MABI-051 / 03WO 344183-2344Embodiment 15. A composition comprising: an mRNA comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 25; a guide nucleic acid comprising a spacer sequence that is at least 90%, at least 95%, or 100% identical SEQ ID NO: 1 ; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 16. A composition comprising: an mRNA comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 24; a guide nucleic acid comprising a spacer sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical SEQ ID NO: 2; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 17. A composition comprising: an mRNA comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 25; a guide nucleic acid comprising a spacer sequence that is at least 90%, at least 95%, or 100% identical SEQ ID NO: 3 ; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 18. The composition of any one of embodiments 15-17, wherein the guide nucleic acid comprises a repeat sequence that is at least 90%, at least 95%, or 100% identical to a nucleotide sequence selected from SEQ ID NOS: 5-11.Embodiment 19. The composition of any one of embodiments 15-18, wherein the guide nucleic acid comprises at least one 2’-OMe modified nucleotide and at least one phosphorothioate linkage.Embodiment 20. A composition comprising: an mRNA comprising or consisting of a nucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from SEQ ID NO: 24 and SEQ ID NO: 25; a guide nucleic acid comprising a nucleotide that is at least 90%, at least 95%, or 100% identical a nucleotide sequence selected from any one of SEQ ID NOS: 1-18, 49-60, 65, 77-81, or a combination thereof; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 21. A composition comprising: an mRNA encoding an effector protein that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 19-21; a guide nucleic acid comprising a nucleotide that is at least 90%, at least 95%, or 100% identical a nucleotide sequence selected from any one of SEQ ID NOS: 1-18, 49-60, 65, 77-81, or a combination thereof; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.Embodiment 22. A composition comprising: an mRNA encoding an effector protein, wherein the effector protein comprises or consists of an amino acid sequence that is 100% identical to SEQ ID NO: 19, with the exception of the following amino acid substitution combinations:Attorney Docket No.: MABI-051 / 03WO 344183-2344L26R, T87G, Y220S, S223P, I471T, D703G; L26R, T87G, S186G, Y220S, S223P, I471T, M503K, D703G; S21L, L26R, T87G, N147K, Y220S, S223P, I471T, D703G; S21L, L26R, E68P, T87G, Q 1381, N147K, K184P, S186G, P187I, K189P, S196Q, Y220S, S223P, I240K, E258K, C405L, K435Q, I471T, D703G; S21L, L26R, E68P, T87G, N147K, K189P, Y220S, S223P, K435Q, I471T, D703G; S21L, L26R, T87G, N147K, K184P, S186G, Pl 871, K189P, Y220S, S223P, I471T, K634G, D703G; S21L, L26R, T87G, N147K, K189P, Y220S, S223P, C405L, K435Q, I471T, N568D, D703G; S21L, L26R, E68P, T87G, Q 1381, N147K, K184P, S186G, Pl 871, Y220S, S223P, E258K, K347A, A366V, I471T, I653A, D703G; S21L, L26R, T87G, N147K, K184P, S186G, Pl 871, Y220S, S223P, C405L, I471T, K516R, N568D, D703G; S21L, L26R, T87G, E119S, N147K, K189P, Y220S, S223P, A366V, I471T, P622N, A623P, D703G; S21L, L26R, E68P, T87G, N147K, Y220S, S223P, I240K, I471T, P622N, A623P, D703G; S21L, L26R, T87G, E119S, N147K, Y220S, S223P, I240K, K347A, C405L, I471T, D703G; S21L, L26R, T87G, N147K, K189P, Y220S, S223P, I471T, V521T, K634G, I653A, D703G; S21L, L26R, T87G, N147K, K184P, S186G, Pl 871, Y220S, S223P, E258K, I471T, I653A, D703G; S21L, L26R, E68P, T87G, E100K, Q 1381, N147K, K189P, Y220S, S223P, I471T, K516R, K634G, I653A, D703G; S21L, L26R, T87G, Q138I, N147K, K184P, S186G, P187I, Y220S, S223P, E258K, I471T, M503K, V521T, I653A, D703G; S21L, L26R, T87G, E100K, N147K, K189H, Y220S, S223P, I240K, E258K, L383I, I471T, K516R, P622T, A623P, D703G; S21L, L26R, E68P, T87G, E100K, N147K, K184P, S186G, P187I, Y220S, S223P, I471T, K516R, P622N, A623P, D703G; S21L, L26R, T87G, E100K, N147K, K189P, Y220S, S223P, A366V, K435Q, I471T, P622N, A623P, D703G; S21L, L26R, E68P, T87G, E100K, E119S, Q138I, N147K, Y220S, S223P, I240K, K435Q, I471T, P622N, A623P, K634G, D703G; S21L, L26R, T87G, N147K, K184P, S186G, P187I, Y220S, S223P, I240K, A366V, C405L, I471T, K516R, D703G; S21L, L26R, T87G, N147K, K184P, S186G, P187I, K189P, Y220S, S223P, E258K, I471T, D703G; or S21L, L26R, T87G, N147K, K184P, S186G, P187I, K189P, Y220S, S223P, R397L, I471T, K634G, D703G; a guide nucleic acid comprising a nucleotide that is at least 90%, at least 95%, or 100% identical a nucleotide sequence selected from any one of SEQ ID NOS: 1-18, 49-60, 65, 77-81, or a combination thereof; and an LNP, wherein the mRNA and the guide nucleic acid are encapsulated in the LNP. Embodiment 23. The composition of any one of embodiments 1-22, wherein the LNP comprises: an ionizable lipid; a neutral lipid; a structure lipid; and a polyethylene glycol (PEG) lipid.Embodiment 24. The composition of embodiment 23, wherein the ionizable lipid comprises the following chemical structure:Attorney Docket No.: MABI-051 / 03WO 344183-2344Embodiment 25. The composition of embodiment 23 or 24, wherein the neutral lipid comprises a phospholipid, optionally wherein the phospholipid comprises l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC). Embodiment 26. The composition of any one of embodiments 23-25, wherein the structure lipid comprises cholesterol.Embodiment 27. The composition of any one of embodiments 23-26, wherein the PEG lipid comprises the following chemical structure:Embodiment 28. The composition of any one of embodiments 1-27, wherein the mRNA comprises a 5’ cap selected from the following chemical structures:andAttorney Docket No.: MABI-051 / 03WO 344183-2344Embodiment 29. A pharmaceutical composition comprising the composition of any one of embodiments 1-28, and a pharmaceutical acceptable excipient or carrier.Embodiment 30. A method of modifying an APOC3 gene, comprising contacting the APOC3 gene, with the composition of any one of embodiments 1-29.Embodiment 31. The method of embodiment 30, wherein modifying the APOC3 gene reduces the expression of the APOC3 gene.Embodiment 32. The method of embodiment 30, wherein modifying the APOC3 gene permanently reduces the expression of the APOC3 gene.Embodiment 33. The method of any one of embodiments 30-32, wherein modifying the APOC3 gene comprises cleaving at least one strand of the APOC3 gene.Embodiment 34. The method of any one of embodiments 30-33, comprising modifying the APOC3 gene in vivo.Embodiment 35. The method of embodiment 34, comprising modifying the APOC3 gene in the liver. Embodiment 36. A method of lowering triglycerides in a human subject with hypertriglyceridemia, the method comprising delivering the composition of any one of embodiments 1-29 to the human subject. Embodiment 37. A method of treating or preventing a disease in a human subject in need thereof, comprising administering the composition of any one of embodiments 1-29.Embodiment 38. The method of embodiment 37, wherein the disease is selected from cardiovascular disease, familial chylomicronemia syndrome, and hypertriglyceridemia.Embodiment 39. A cell, or population of cells, comprising, or modified by, the composition or method of any one of embodiments 1-38.Embodiment 40. A composition comprising a guide nucleic acid, wherein the guide nucleic acid comprises: a first region that is capable of being non-covalently bound by an effector protein; and a second region that hybridizes to a target nucleic acid, wherein the guide nucleic acid comprises at least 20 nucleotides,Attorney Docket No.: MABI-051 / 03WO 344183-2344 and wherein 15 nucleotides at the 5’ end of the guide nucleic acid each comprise a 2’-0Me modification.Embodiment 41. The composition of embodiment 40, wherein the length of the guide nucleic acid is at least 30, at least 40, or at least 50 linked nucleotides, but not greater than 100, 150 or 200 nucleotides. Embodiment 42. The composition of embodiment 40 or 41, wherein the 3 nucleotides at the 3’ end of the guide nucleic acid each comprise a 2’-OMe modification.Embodiment 43. The composition of any one of embodiments 40-42, wherein the three 5’ most intemucleotide linkages and the two 3 ’ most intemucleotide linkages comprise a phosphorothioate bond and the remaining intemucleotide linkages are phosphodiester bonds.Embodiment 44. The composition of any one of embodiments 40-43, wherein the guide nucleic acid comprises a chemical modification pattern according to the following formula: 5 ’-mX*mX*mX*mXmXmXmXmXmXmXmXmXmXmXmXrXnmX*mX*mX-3 ’ ; wherein: X = a nucleotide selected from A, C, G, T, and U; r indicates an unmodified ribonucleotide, m indicates a 2’-OMe modified nucleotide, * indicates a phosphorothioate bond, and n = 1-40.Embodiment 45. The composition of any one of embodiments 40-43, wherein the guide nucleic acid comprises a chemical modification pattern according to the following formula: 5’- mX*mX*mX*mXmXmXmXmXmXmXmXmXmXmXmXrXrXrXrXrXrXrXrXrXrXrXrXrXrXrXrX rXrXrXrXrXrXrXrXrXrXrXrXrXrXrXrXrXrXrXrXmX*mX*mX-3 ’ .Embodiment 46. The composition of any one of embodiments 40-45, wherein the first region comprises or consists of a nucleotide sequence selected from TABLE 2.Embodiment 47. The composition of any one of embodiments 40-46, wherein the composition comprises an effector protein or a composition comprising an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 19.Embodiment 48. An engineered protein or a nucleic acid encoding the same, wherein the engineered protein comprises or consists of an amino acid sequence that is identical to SEQ ID NO: 19, with the exception of the following amino acid substitutions: L26R, T87G, Y220S, S223P, I471T, and D703G. Embodiment 49. A messenger RNA (mRNA) encoding an engineered effector protein, wherein the engineered protein comprises or consists of an amino acid sequence that is identical to SEQ ID NO: 19, with the exception of the following amino acid substitutions: L26R, T87G, Y220S, S223P, I471T, and D703G.Embodiment 50. An engineered protein or a nucleic acid encoding the same, wherein the engineered protein comprises or consists of an amino acid sequence that is 100% identical to SEQ ID NO: 19, with the exception of the following amino acid substitution combinations: L26R, T87G, Y220S, S223P, 147 IT, D703G; L26R, T87G, S186G, Y220S, S223P, 147 IT, M503K, D703G; S21L, L26R, T87G, N147K, Y220S, S223P, I471T, D703G; S21L, L26R, E68P, T87G, Q138I, N147K, K184P, S186G,Attorney Docket No.: MABI-051 / 03WO 344183-2344P187I, K189P, S196Q, Y220S, S223P, I240K, E258K, C405L, K435Q, I471T, D703G; S21L, L26R, E68P, T87G, N147K, K189P, Y220S, S223P, K435Q, I471T, D703G; S21L, L26R, T87G, N147K, K184P, S186G, P187I, K189P, Y220S, S223P, I471T, K634G, D703G; S21L, L26R, T87G, N147K, K189P, Y220S, S223P, C405L, K435Q, I471T, N568D, D703G; S21L, L26R, E68P, T87G, Q138I, N147K, K184P, S186G, Pl 871, Y220S, S223P, E258K, K347A, A366V, I471T, I653A, D703G; S21L, L26R, T87G, N147K, K184P, S186G, Pl 871, Y220S, S223P, C405L, I471T, K516R, N568D, D703G; S21L, L26R, T87G, E119S, N147K, K189P, Y220S, S223P, A366V, I471T, P622N, A623P, D703G; S21L, L26R, E68P, T87G, N147K, Y220S, S223P, I240K, I471T, P622N, A623P, D703G; S21L, L26R, T87G, E119S, N147K, Y220S, S223P, I240K, K347A, C405L, I471T, D703G; S21L, L26R, T87G, N147K, K189P, Y220S, S223P, I471T, V521T, K634G, I653A, D703G; S21L, L26R, T87G, N147K, K184P, S186G, P187I, Y220S, S223P, E258K, I471T, I653A, D703G; S21L, L26R, E68P, T87G, E100K, Q138I, N147K, K189P, Y220S, S223P, I471T, K516R, K634G, I653A, D703G; S21L, L26R, T87G, Q 1381, N147K, K184P, S186G, P 1871, Y220S, S223P, E258K, I471T, M503K, V521T, I653A, D703G; S21L, L26R, T87G, E100K, N147K, K189H, Y220S, S223P, I240K, E258K, L383I, I471T, K516R, P622T, A623P, D703G; S21L, L26R, E68P, T87G, E100K, N147K, K184P, S186G, P187I, Y220S, S223P, I471T, K516R, P622N, A623P, D703G; S21L, L26R, T87G, E100K, N147K, K189P, Y220S, S223P, A366V, K435Q, 147 IT, P622N, A623P, D703G; S21L, L26R, E68P, T87G, E100K, E119S, Q138I, N147K, Y220S, S223P, I240K, K435Q, I471T, P622N, A623P, K634G, D703G; S21L, L26R, T87G, N147K, K184P, S186G, P187I, Y220S, S223P, I240K, A366V, C405L, I471T, K516R, D703G; S21L, L26R, T87G, N147K, K184P, S186G, P187I, K189P, Y220S, S223P, E258K, I471T, D703G; or S21L, L26R, T87G, N147K, K184P, S186G, Pl 871, K189P, Y220S, S223P, R397L, 147 IT, K634G, D703G.Embodiment 51. An engineered polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO: 21Embodiment 52. An mRNA encoding the engineered protein of embodiment 50, wherein the mRNA comprises or consists of a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from SEQ ID NO: 23 and SEQ ID NO: 25Embodiment 53. A guide nucleic acid comprising or consisting of a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from SEQ ID NOS: 52-55.Embodiment 54. An engineered guide nucleic acid comprising from 5' to 3' a) a stabilizer sequence, b) a repeat sequence, and c) a spacer sequence, wherein the stabilizer sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NO: 65, 77-78, and wherein the repeat sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 5-11Attorney Docket No.: MABI-051 / 03WO 344183-2344Embodiment 55. The engineered guide nucleic acid of embodiment 54, wherein the spacer sequence comprises any one of SEQ ID NOs: 1-4, 50 or 51.Embodiment 56. An effector protein or a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19, wherein the mRNA does not encode an effector protein with an S526N amino acid substitution relative to SEQ ID NO: 19.Embodiment 57. A composition comprising an mRNA, wherein the mRNA comprises or is linked to a 5’ cap according to the following chemical structure:, wherein the mRNA comprises a nucleotide sequence that comprises or consists of a sequence selected from SEQ ID NO: 23 and SEQ ID NO: 25, or wherein the nucleotide sequence encodes an effector protein represented by SEQ ID NO: 21, optionally wherein the composition further comprises a guide nucleic acid, wherein the guide nucleic acid comprises or consists of a nucleotide sequence selected from SEQ ID NO: 54 and SEQ ID NO: 55 Embodiment 58. A composition comprising an mRNA, wherein the mRNA is represented by the following chemical structure:Attorney Docket No.: MABI-051 / 03WO 344183-2344 wherein “mRNA” represents an mRNA without a 5 ’ cap, and the remainder of the structure represents a 5 ’ cap, and wherein the 5 ’ cap is linked to the 5 ’ end of the mRNA, wherein the mRNA comprises a nucleotide sequence that comprises or consists of a sequence selected from SEQ ID NO: 23 and SEQ ID NO: 25, or wherein the nucleotide sequence encodes an effector protein represented by SEQ ID NO: 21, optionally wherein the composition further comprises a guide nucleic acid, wherein the guide nucleic acid comprises or consists of a nucleotide sequence selected from SEQ ID NO: 54 and SEQ ID NO: 55Embodiment 59. The composition of Embodiment 57 or 58, wherein the composition comprises a cation that associates with the mRNA, the 5’ cap, or a combination thereof, optionally wherein the cation is selected from sodium and ammonium.Embodiment 60. A lipid nanoparticle comprising the mRNA of embodiment of 49 or 52 and the guide nucleic acid of embodiment of 53.Embodiment 61. A lipid nanoparticle comprising the mRNA of embodiment of 49 or 52 and the engineered guide nucleic acid of embodiment of 54 or 55.Embodiment 62. A lipid nanoparticle comprising the mRNA encoding an effector protein that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOS: 19-21, and 61-67, and the guide nucleic acid comprising a sequence selected from SEQ ID NOS: 1-18, 49-60, 65, and 77-81.Embodiment 63. The lipid nanoparticle of any one of embodiments 60-62, further comprising an ionizable lipid; a neutral lipid; a structure lipid; and a polyethylene glycol (PEG) lipid.Embodiment 64. The lipid nanoparticle of embodiment 63, wherein the ionizable lipid comprise a hydrophobic tail and an ionizable head group.Embodiment 65. The lipid nanoparticle of embodiment 64, wherein the ionizable lipid comprises the following chemical structure:Embodiment 66. The lipid nanoparticle of embodiment 62, wherein the neutral lipid comprises a phospholipid.Embodiment 67. The lipid nanoparticle of embodiment 62, wherein the phospholipid is selected from a phospholipid selected from l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1,2-dipalmitoyl sn- glycero-3 -phosphocholine (DPPC), 1 ,2-dioleyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2-Attorney Docket No.: MABI-051 / 03WO 344183-2344 dioleyl-sn-glycero-3-phosphatidylcholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 phosphoethanolamine (DPPE), l,2-dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), 1,2- dioleoyl-sn-glycero-3-phospho-(l'-rac -glycerol (DOPG), or any combinations thereof.Embodiment 68. The lipid nanoparticle of any one of embodiments 60-67, wherein the structure lipid comprises cholesterol, cholesterol hemisuccinate, or any combination thereof.Embodiment 69. The lipid nanoparticle of any one of embodiments 60-68, wherein the PEG lipid is selected from dimyristoylglycerol-PEG (DMG-PEG), dilauroylglycerol-PEG, dipalmitoylglycerol- PEG, distearoylglycerol-PEG, dilaurylglycamide-PEG, dimyristylglycamide-PEG, dipalmitoylglycamide-PEG, distearoylglycamide-PEG, cholesterol-PEG, and methoxypolyethyleneglycoloxy(2000)-N,N-ditetradecylacetamide (ALC-0159).Embodiment 70. The lipid nanoparticle of any one of embodiments 60-69, wherein the PEG lipid comprises the following chemical structure:Embodiment 71. The lipid nanoparticle of any one of embodiments 60-70, wherein the mRNA comprises a 5’ cap selected from the following chemical structures:andAttorney Docket No.: MABI-051 / 03WO 344183-2344Embodiment 72. The lipid nanoparticle of any one of embodiments 60-71, wherein the mRNA further comprises a UTR, a nucleotide sequence encoding an NLS, and a polyA.Embodiment 73. The lipid nanoparticle of embodiment 72, wherein the NLS comprises an amino acid sequence selected from SEQ ID NOS: 26-31.Embodiment 74. An effector protein or a nucleic acid encoding an effector protein, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19, and an NLS comprising an amino acid sequence selected from SEQ ID NOS: 26-31 at the C terminus of the effector protein.Embodiment 75. An effector protein or a nucleic acid encoding an effector protein, comprising an amino acid sequence that is identical to SEQ ID NO: 19, with the exception of the following amino acid substitutions: L26R, T87G, Y220S, S223P, I471T, and D703G and an NLS comprising an amino acid sequence selected from SEQ ID NOS: 26-31 at the C terminus of the effector protein.Guide Nucleic Acids

[0068] The compositions and methods of the present disclosure may comprise a guide nucleic acid or a nucleic acid encoding a guide nucleic acid (such as DNA) and uses thereof. In general, guide nucleic acids comprise a nucleotide sequence and / or may be represented by a nucleotide sequence. Such a nucleotide sequence may also be referred to as a guide RNA. However, the term, guide RNA, is not limited to linked ribonucleotides. A guide RNA may comprise ribonucleotides, deoxyribonucleotides, and chemically modified versions thereof. Disclosure of the nucleotide sequences described herein also discloses a complementary nucleotide sequence, a reverse nucleotide sequence, and the reverse complement nucleotide sequence, any one of which may be a nucleotide sequence for use in a guide nucleic acid.

[0069] In general, a guide nucleic acid comprises a first region that is capable of being non-covalently bound by an effector protein and a second region that hybridizes to a target nucleic acid. The first region may be represented by a first nucleotide sequence and the second region may be represented by a secondAttorney Docket No.: MABI-051 / 03WO 344183-2344 nucleotide sequence. In some embodiments, the first nucleotide sequence is located 5’ to a second nucleotide sequence. In some embodiments, the first nucleotide sequence comprises a repeat sequence. In some embodiments, the first region comprises or forms an intramolecular secondary structure (e.g., hairpins, stem-loops, etc.) that is bound by the effector protein. In some embodiments, the second nucleotide sequence comprises a spacer sequence, wherein the spacer sequence can interact in a sequence-specific manner (e.g., has complementarity with, or can hybridize to, a target sequence) with a target nucleic acid (e.g., APOC3).

[0070] In general, guide nucleic acids disclosed herein are non-naturally occurring molecules. In some embodiments, guide nucleic acids comprise a non-natural nucleotide sequence. The non-natural sequence may comprise a portion of a naturally-occurring sequence, wherein the portion of the naturally-occurring sequence is not present in nature absent the remainder of the naturally-occurring sequence. In some embodiments, the nucleotide sequence of the guide nucleic acid is not found in nature. In some embodiments, guide nucleic acids comprise two naturally-occurring sequences arranged in an order or proximity that is not observed in nature. In some embodiments, guide nucleic acids comprise two heterologous sequences arranged in an order or proximity that is not observed in nature. The guide nucleic acid may comprise a sequence of a repeat sequence that occurs naturally in an organism and a spacer sequence that does not occur naturally in that organism. For example, a guide nucleic acid may comprise a sequence of a naturally-occurring repeat sequence and a spacer sequence that is complementary to a naturally-occurring eukaryotic sequence. In some embodiments, a guide nucleic acid is a crRNA, wherein the crRNA comprises a repeat sequence and a spacer sequence that is complementary to a eukaryotic target sequence. In some embodiments, compositions comprise an mRNA and a guide nucleic acid that do not occur together in nature. Therefore, guide nucleic acids and compositions described herein are not naturally occurring.

[0071] A guide nucleic acid may be chemically synthesized or recombinantly produced by any suitable methods. Guide nucleic acids may comprise a chemical modification to provide it with a new or enhanced feature, such as improved stability. Such modifications of a nucleic acid include anucleobase base modification, a backbone modification, a sugar modification, and combinations thereof. In some embodiments, uridines can be exchanged for pseudouridines (e.g., IN-Methyl-Pseudouridine). In some embodiments, the modification comprises a phosphorothioate (PS) linkage. In some embodiments, the modification comprises a 2’-O-Methyl (2’-0Me) nucleotide modification. In some embodiments, the modification comprises a locked nucleic acid (LNA). In some embodiments, the modification comprises a phosphorodiamidate morpholino oligonucleotide (PMO). In some embodiments, the modification comprises a peptide nucleic acid (PNA). In some embodiments, the first 15 and the last 3 nucleotides are 2’-0Me modified, and the first 3 and last 2 intranucleotide linkages are phosphorothioate linkages. In some embodiments, the sequence is modified according to the following formula: mN*mN*mN*mNmNmNmNmNmNmNmNmNmNmNmNNnmN*mN*mN, wherein N is a nucleotide, n is an integer between 1 and 100, m is a 2’-OMe modified sugar moiety, and the * denotesAttorney Docket No.: MABI-051 / 03WO 344183-2344 a PS linkage. In some embodiments, any or all uridines are exchanged for IN-Methyl-Pseudouridine. In some embodiments, U represents uracil. In some embodiments U represents IN-Methyl- Pseudouridine.

[0072] In some embodiments, an engineered guide nucleic acid is modified according to the following formula: mN*mN*mN*rNrNrNrNrNrNrN*mN*mN*mN*mNrNrNrNrNrNrNrNrNrNrN*mN*rNrNrNrNrNrN rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrArArArArArArArArArArJVrArJ\WVrAmJV*mJV*mJV, wherein N is a nucleotide selected from A, G, C, and U or T, wherein * represents a phosphorothioate linkage, wherein m represents a 2’-OMe sugar modification, wherein r represents an unmodified ribonucleotide, and wherein italicized letters indicate a spacer sequence.

[0073] In some embodiments, an engineered guide nucleic acid is modified according to the following formula: mN*mN*mN*rNrNrNrNrNrNrN*mN*mN*mN*mNrNrNrNrNrNrNrNrNrNrN*mN*rNrNrNrNrNrN rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrA„mA*mA*mA, wherein N is a nucleotide selected from A, G, C, and U or T, wherein * represents a phosphorothioate linkage, wherein m represents a 2’-0Me sugar modification, wherein r represents an unmodified ribonucleotide, wherein italicized letters indicate a spacer sequence, and wherein n = 13 to 19.

[0074] In some embodiments, an engineered guide nucleic acid is modified according to the following formula: mN*mN*mN*(rN),, *mN*mN*mN*mN(rN)x*mN*(rN)v< / A',> / mA'*mA'*mA'. wherein N is a nucleotide selected from A, G, C, and U or T, wherein * represents a phosphorothioate linkage, wherein m represents a 2’-0Me sugar modification, wherein r represents an unmodified ribonucleotide, wherein italicized letters indicate a spacer sequence, and wherein (i) w= 5 to 9, preferably 7, (ii) x= 8 to 12, preferably 10, (iii) y= 21 to 25, preferably 23, and (iv) z= 13 to 19, preferably 19. crRNA

[0075] In some embodiments, the guide nucleic acid comprises a crRNA. In general, a crRNA comprises a first region (FR) and a second region (SR), wherein the FR of the crRNA comprises a repeat sequence, and the SR of the crRNA comprises a spacer sequence. In some embodiments, the spacer sequence follows the repeat sequence in a 5’ to 3’ direction. In some embodiments, the repeat sequence and the spacer sequences are directly connected to each other (e.g., covalent bond (phosphodiester bond)). In some embodiments, the repeat sequence and the spacer sequence are connected by a linker. In some embodiments, the linker comprises one or more nucleotides.

[0076] In such embodiments, the repeat sequence of the crRNA interacts with an effector protein. In some embodiments, the repeat sequence is non-covalently bound by an effector protein. In some embodiments, a crRNA is sufficient to form a complex with an effector protein (e.g., to form an RNP)Attorney Docket No.: MABI-051 / 03WO 344183-2344 through the repeat sequence and direct the effector protein to a target nucleic acid sequence through the spacer sequence.

[0077] A crRNA may include deoxyribonucleotides, ribonucleotides, chemically modified nucleotides, or any combination thereof. In some embodiments, a crRNA comprises at least: 30, 35, 40, 45, 50, 55, or 60 linked nucleotides. In some embodiments, the length of the crRNA is about 30 to about 80 linked nucleotides. In some embodiments, guide nucleic acids comprise a nucleotide linker, also simply referred to as a linker that links a first region of a guide nucleic acid to a second region of a guide nucleic acid. In some embodiments, the linker links a repeat sequence to a spacer sequence. A non-limiting example of a nucleotide linker is 5’-GAAA-3’.Spacer Sequences

[0078] Guide nucleic acids described herein generally comprise a spacer sequence. In some embodiments, a spacer sequence is capable of hybridizing to a target sequence of APOC3. In some embodiments, a spacer sequence comprises a nucleotide sequence that is hybridizable to an equal length of a target sequence of APOC3. In some embodiments, the spacer sequence may function to direct an RNP complex comprising the guide nucleic acid to the target nucleic acid. A spacer sequence may be complementary to a target sequence that is adjacent to a protospacer adjacent motif (PAM) that is recognizable by an effector protein described herein. Non-limiting examples of spacer sequences are provided in TABLE 1.

[0079] In some embodiments, the spacer sequence of a guide nucleic acid is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a target sequence. In general, the spacer sequence is capable of hybridizing to a target sequence of a target nucleic acid. It is understood that the spacer sequence need not be 100% complementary to that of a target sequence of a target nucleic acid to hybridize or hybridize specifically to the target sequence.

[0080] In some embodiments, the spacer sequence comprises or consists of 15-28 nucleotides. In some embodiments, the spacer sequence comprises or consists of 15-26, 15-24, 15-22, 15-20, 15-18, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20, 17-18, 18-26, 18-24, or 18-22 nucleotides. In some embodiments, the spacer sequence comprises or consists of 17 linked nucleotides. In some embodiments, the spacer sequence comprises or consists of 18 linked nucleotides. In some embodiments, the spacer sequence comprises or consists of 19 linked nucleotides. In some embodiments, the spacer sequence comprises or consists of 20 linked nucleotides.Exemplary APOC3 spacer sequences

[0081] TABLE 1 provides illustrative spacer sequences for use in the compositions and methods of the disclosure. Spacer sequences in TABLE 1 may be suitable for use in combination with an effector protein, wherein the effector protein comprises an amino acid sequence selected from SEQ ID NOs:Attorney Docket No.: MABI-051 / 03WO 344183-234419-21. In some embodiments, the spacer sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a sequence as set forth in TABLE 1.TABLE 1: Exemplary APOC3 Spacer Sequences

[0082] In some embodiments, spacer sequences comprise one or more nucleotide alterations at one or more positions in any one of the sequences of TABLE 1. Alternative nucleotides can be any one or more of A, C, G, T or U. By way of non-limiting example, a guanine nucleobase could be replaced with the nucleobase of any one of a cytosine, adenosine, thymine, and uracil. In some embodiments, spacer sequences comprise only one nucleobase alteration relative to a sequence of TABLE 1. In some embodiments, the spacer sequence comprises not more than 1, not more than 2, not more than 3, or not more than 4 nucleobase alterations relative to a sequence of TABLE 1. In some embodiments, the spacer sequence hybridizes to APOC3 within 200, within 100, within 50, or within 20 nucleotides of the sequence of APOC3 that a spacer sequence of TABLE 1 hybridizes to. In preferred embodiments, the spacer sequence is represented by SEQ ID NO: 51.Repeat Sequences

[0083] Guide nucleic acids described herein may comprise a repeat sequence. In some embodiments, a repeat sequence comprises a nucleotide sequence that interacts with an effector protein. In some embodiments, a repeat sequence includes a nucleotide sequence that can form a guide nucleic acideffector protein complex (e.g., a RNP complex). In some embodiments, the repeat sequence may also be referred to as a “protein-binding segment.” In some embodiments, a repeat sequence comprises a nucleotide sequence that is not complementary to a target sequence of a target nucleic acid. In some embodiments, the repeat sequence is between 10 and 40, 12 and 38, 14 and 36, 16 and 36, and 18 and 36 nucleotides in length. In some embodiments, repeat sequences consist of 36 nucleotides. In some embodiments, a repeat sequence is adjacent to a spacer sequence. In some embodiments, a repeat sequence is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a guide nucleic acid comprises a repeat sequence linked to a spacer sequence, which may be a direct link (e.g., a phosphodiester or phosphorothioate bond) or by any suitable linker (e.g., one or more nucleotides).

[0084] In some embodiments, guide nucleic acids comprises a repeat sequence that is at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99%, or 100% identical to a sequence that is provided in TABLE 2. In some embodiments, guide nucleic acids comprise a repeat sequence,Attorney Docket No.: MABI-051 / 03WO 344183-2344 wherein the repeat sequence comprises at least 14, at least 16, at least 18, at least 20, at least 24, at least 28, at least 32, at least 34, or 36 contiguous nucleotides of a sequence provided in TABLE 2.TABLE 2: Exemplary Repeat Sequences

[0085] In some embodiments, guide nucleic acids comprise a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the sequences as set forth in TABLE 1; and a repeat sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the sequences as set forth in TABLE 2.Stabilizer Sequences

[0086] Guide nucleic acid described herein may comprise a stabilizer sequence. In some embodiments, a stabilizer sequence comprises a nucleic acid sequence that stabilizes the sequence. In some embodiments, a guide nucleic acid that comprises a stabilizer sequence degrades slower than a guide nucleic acid that does not comprise a stabilizer sequence. In some embodiments, a guide nucleic acid that comprises a stabilizer sequence degrades slower than a guide nucleic acid that does not comprise the stabilizer sequence. In some embodiments, a stabilizer sequence increases the nuclease activity (as measured, e.g., by % indel) of the effector protein that it is bound by in a ribonucleoprotein (RNP) complex. In some embodiments, a stabilizer sequence increases the half-life of a guide RNA, thus the RNP complex may cleave more target nucleic acids over time. In some embodiments, a stabilizer sequence increases % indel generation induced by the effector protein by 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%, or at least 100%. In some embodiments, a stabilizer sequence increases % indel of the effector protein by as much as 100%, 200%, or 300%.

[0087] In some embodiments, the stabilizer sequence is adjacent to the repeat sequence, which is adjacent to a spacer sequence. In some embodiments, the stabilizer sequence is followed by the repeat and spacer sequences in the 5’ to 3’ direction. In some embodiments, a guide nucleic acid comprises a stabilizer sequence, a repeat sequence, and a spacer sequence, which may be a direct link (e.g., a phosphodiester or phosphorothioate bond) or by any suitable linker (e.g., one or more nucleotides).

[0088] Non-limiting examples of stabilizer sequences are provided in TABLE 2.1. In some embodiments, guide nucleic acids comprises a stabilizer sequence that is at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99%, or 100% identical to a sequence that is providedAttorney Docket No.: MABI-051 / 03WO 344183-2344 in TABLE 2.1. In some embodiments, guide nucleic acids comprise a stabilizer sequence, wherein the stabilizer sequence comprises at least 14, at least 16, at least 18, at least 20, at least 24, at least 28, at least 32, at least 34, or 36 contiguous nucleotides of a sequence provided in TABLE 2.1. Non-limiting, exemplary chemical modifications are provided in TABLE 2.1 as well (bold text indicates stabilizer sequence, italics indicates repeat sequence, and spacer is in regular font; “m” indicates a 2’-OMe sugar modification of the following nucleotide, “r” indicates that the following nucleotide is unmodified, and * indicates a phosphorothioate linkage between the two neighboring nucleotides, all other intemucleotide linkages are phosphodiester linkages).TABLE 2.1: Exemplary Stabilizer Sequences

[0089] In some embodiments, guide nucleic acids comprises a stabilizer sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, or 100% identical to a sequence selected from TABLE 2.1. In some embodiments, the stabilizer sequence comprises at least 15, at least 18, or at least 21, contiguous nucleotides of a sequence selected from TABLE 2.1. In some embodiments, guide nucleic acids comprises a stabilizer sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, or 100% identical to SEQ ID NO: 65. In some embodiments, the stabilizer sequence comprises at least 15, at least 18, or at least 21, contiguous nucleotides of SEQ ID NO: 65.

[0090] In some embodiments, disclosed herein is an engineered guide nucleic acid comprising from 5' to 3' a) a stabilizer sequence, b) a repeat sequence, and c) a spacer sequence. In some embodiments, the stabilizer sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from TABLE 2.1, and the repeat sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 5-11. In some embodiments, the stabilizer sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 65, and the repeat sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 5-11. In some embodiments, the spacer sequence comprises a sequence that is 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to any one of SEQ ID NOs: 1-4, 50 or 51.Attorney Docket No.: MABL051 / 03WO 344183-2344Exemplary Guide Nucleic Acids

[0091] Exemplary guide nucleic acids useful for compositions and methods described herein are provided in TABLE 3. TABLE 3 provides exemplary unmodified nucleotide sequences as well as exemplary chemical modifications for these guide nucleic acids. In TABLE 3, “m” represents a 2’- OMe modified ribonucleotide, “r” represents an unmodified ribonucleotide, and * represents a phosphorothioate bond between the two surrounding ribonucleotides. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the sequences in TABLE 3. In some embodiments, the guide nucleic acid comprises or consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the sequences of TABLE 3. In some embodiments, the guide nucleic acids provided in TABLE 3 comprise an additional “G” at the 5’ end of the nucleotide sequence.

[0092] The exemplary guide nucleic acids in TABLE 3 are shown without any chemical modifications, as well as with exemplary chemical modifications. A person of skill in the art understands that alternative chemical modification patterns could be employed.TABLE 3: Exemplary Guide Nucleic Acids Targeting APOC3Attorney Docket No.: MABI-051 / 03WO 344183-2344Effector Proteins

[0093] In some embodiments, compositions provided herein comprise an effector protein or a nucleic acid encoding the same. In some embodiments, the nucleic acid encoding the effector protein comprises RNA, DNA or a combination thereof. In some embodiments, compositions described herein comprise an effector protein that is similar to a naturally occurring effector protein. The effector protein may comprise an amino acid substitution relative to the naturally-occurring effector protein, wherein the amino acid substitution is not found in nature. In some embodiments, effector proteins disclosed herein are engineered proteins. Engineered proteins are not identical to a naturally-occurring protein. Engineered proteins may provide increased nuclease activity as compared to a corresponding naturally occurring nuclease. An effector protein may be small (e.g., less than 800 amino acids). The smaller nature of these effector proteins may allow for them to be more easily packaged and delivered with higher efficiency in the context of genome editing.

[0094] An effector protein may be brought into proximity of a target nucleic acid in the presence of a guide nucleic acid. The ability of an effector protein to modify a target nucleic acid may be dependent upon the effector protein being bound to a guide nucleic acid and the guide nucleic acid being hybridized to a target nucleic acid. An effector protein may also recognize a protospacer adjacent motif (PAM) sequence present in the target nucleic acid, which may direct genetic modification activity of the effector protein.

[0095] In some embodiments, the effector protein is a CRISPR-associated (Cas) protein or an engineered variant thereof. In some embodiments, the effector protein is a Type V Cas protein or an engineered variant thereof. In some embodiments, the effector protein is Cas 12 protein or an engineered variant thereof. In some embodiments, the effector protein is Cas 12J (CasPhi) protein or an engineeredAttorney Docket No.: MABI-051 / 03WO 344183-2344 variant thereof. In some embodiments, the effector protein is an engineered variant of CasPhi.12 (sometimes referred to simply in the figures as “CasPhi.”

[0096] In some embodiments, effector proteins comprise nuclease activity. In some embodiments, the nuclease activity comprises cis cleavage activity. In some embodiments, cis cleavage activity results in the cleavage of an intemucleotide linkage of a target nucleic acid when the effector protein is the proximity of the target nucleic acid. For example, an effector protein disclosed herein, when complexed in a human cell with a guide nucleic acid disclosed herein, may cleave one or both strands of APOC3. In some embodiments, effector proteins generate a double stranded break. In some embodiments, effector proteins comprise a RuvC domain. In some embodiments, the RuvC domain comprises nuclease activity. In some embodiments, effector proteins comprise multiple RuvC subdomains, which may form a functional RuvC domain when the effector protein folds in a physiological context (e.g., within a cell), wherein the functional RuvC domains comprises substrate binding and / or catalytic activity.

[0097] In some embodiments, effector proteins comprise or consist of an amino acid sequence that is at least 90%, at least 95%, at least, at least 97%, or at least 98%, at least 99%, or 100% identical to SEQ ID NO: 19. In some embodiments, the effector protein comprises at least one of the following amino acid substitutions L26R, T87G, Y220S, S223P, 147 IT, and D703G relative to SEQ ID NO: 19.

[0098] In some embodiments, the amino acid sequence comprises or consists of an amino acid sequence that is 100% identical to SEQ ID NO: 19, with the exception of the following amino acid substitution combinations:L26R / T87G / Y220S / S223P / I471T / D703G; L26R / T87G / S186G / Y220S / S223P / I471T / M503K / D703G; S21L / L26R / T87G / N147K / Y220S / S223P / I471T / D703G;S21L / L26R / E68P / T87G / Q138I / N147K / K184P / S186G / P187I / K189P / S196Q / Y220S / S223P / I240K / E2 58K / C405L / K435Q / I471T / D703G;S21L / L26R / E68P / T87G / N147K / K189P / Y220S / S223P / K435Q / I471T / D703G;S2 IL / L26R / T87G / NI47K / KI 84P / S 186G / PI 87I / KI 89P / Y220S / S223P / I47 IT / K634G / D703G; S21L / L26R / T87G / N147K / K189P / Y220S / S223P / C405L / K435Q / I471T / N568D / D703G;S21L / L26R / E68P / T87G / Q138I / N147K / K184P / S186G / P187I / Y220S / S223P / E258K / K347A / A366V / I 471T / I653A / D703G;S21L / L26R / T87G / N147K / K184P / S186G / P187I / Y220S / S223P / C405L / I471T / K516R / N568D / D703G; S2IL / L26R / T87G / EI I9S / NI47K / KI89P / Y220S / S223P / A366V / I47IT / P622N / A623P / D703G;S21L / L26R / E68P / T87G / N147K / Y220S / S223P / I240K / I471T / P622N / A623P / D703G; S2IL / L26R / T87G / EI I9S / NI47K / Y220S / S223P / I240K / K347A / C405L / I47IT / D703G; S21L / L26R / T87G / N147K / K189P / Y220S / S223P / I471T / V521T / K634G / I653A / D703G;S2 IL / L26R / T87G / NI47K / KI 84P / S 186G / P 187I / Y220S / S223P / E258K / I47 IT / I653A / D703G;S21L / L26R / E68P / T87G / E100K / Q138I / N147K / K189P / Y220S / S223P / I471T / K516R / K634G / I653A / D 703G;Attorney Docket No.: MABI-051 / 03WO 344183-2344S21LA26R / T87G / Q 138I / N147K / K184P / S 186G / P187I / Y220S / S223P / E258K / I471T / M503K / V52 IT / I653A / D703G;S21L / L26R / T87G / E100K / N147K / K189H / Y220S / S223P / I240K / E258K / L383I / I471T / K516R / P622T / A623P / D703G;S21L / L26R / E68P / T87G / E100K / N147K / K184P / S 186G / P187I / Y220S / S223P / I471T / K516R / P622N / A 623P / D703G;S21L / L26R / T87G / E100K / N147K / K189P / Y220S / S223P / A366V / K435Q / I471T / P622N / A623P / D703 G;S21L / L26R / E68P / T87G / E 100K / E 119S / Q 138I / N 147K / Y220S / S223P / I240K / K435Q / I471T / P622N / A 623P / K634G / D703G;S21L / L26R / T87G / N147K / K184P / S 186G / P187I / Y220S / S223P / I240K / A366V / C405L / I471T / K516R / D703G;S21L / L26R / T87G / N147K / K184P / S186G / P187I / K189P / Y220S / S223P / E258K / I471T / D703G; or S21L / L26R / T87G / N147K / K184P / S186G / P187I / K189P / Y220S / S223P / R397L / I471T / K634G / D703G.

[0099] TABLE 4 provides illustrative amino acid sequences of effector proteins. In some embodiments, the amino acid sequence of an effector protein is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to the sequence as set forth in TABLE 4. In some embodiments, an effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences as set forth in TABLE 4. In some embodiments, the effector protein consists of an amino acid sequence selected from the sequences as set forth in TABLE 4.TABLE 4: Exemplary Effector ProteinsAttorney Docket No.: MABI-051 / 03WO 344183-2344Attorney Docket No.: MABI-051 / 03WO 344183-2344

[0100] In some embodiments, compositions comprise an effector protein, wherein the amino acid sequence of the effector protein comprises at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400, at least about 420, at least about 440, at least about 460, at least about 480, at least about 500, at least about 520, at least about 540, at least about 560, at least about 580, at least about 600, at least about 620, at least about 640, at least about 660, at least about 680, at least about 700, at least about 710, at least about 711, at least about 712, at least about 713, at least about 714, at least about 715 or 717 contiguous amino acids or more of any one of the sequences as set forth in TABLE 4.

[0101] In some embodiments, compositions and methods described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more amino acid alterations relative to a sequence recited in TABLE 4. In some embodiments, an amino acid alteration comprises a deletion of an amino acid. In some embodiments, an amino acid alteration comprises an insertion of an amino acid. In some embodiments, an amino acid alteration comprises a conservative amino acid substitution. In some embodiments, an amino acid alteration comprises a nonconservative amino acid substitution. In some embodiments, one or more amino acid alterationsAttorney Docket No.: MABI-051 / 03WO 344183-2344 comprises a combination of one or more conservative amino acid substitutions and one or more nonconservative amino acid substitutions. When describing a conservative amino acid substitution herein, reference is made to the replacement of one amino acid for another such that the replacement takes place within a family of amino acids that are related in their side chains. Conversely, when describing anon-conservative alteration (e.g., non-conservative substitution), reference is made to the replacement of one amino acid residue for another that does not have a related side chain. It is understood that genetically encoded amino acids can be divided into four families having related side chains: (1) acidic (negatively charged): Asp (D), Glu (E); (2) basic (positively charged): Lys (K)Arg (R), His (H); (3) non-polar (hydrophobic): Cys (C), Ala (A), Vai (V), Leu (L), lie (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic: Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic: Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar: Asn (N), Gin (Q), Ser (S), Thr (T). Amino acids may be related by aliphatic side chains: Gly (G), Ala (A), Vai (V), Leu (L), lie (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic-hydroxyl. Amino acids may be related by aromatic side chains: Phe (F), Tyr (Y), Trp (W). Amino acids may be related by amide side chains: Asn (N), Gin (Q). Amino acids may be related by sulfur-containing side chains: Cys (C) and Met (M).

[0102] In some embodiments, an effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence selected from TABLE 4, wherein the effector protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions relative to the sequence selected from TABLE 4. In some embodiments, an effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence selected from TABLE4, wherein the effector protein comprises 1 to 10, 10 to 20, 20 to 30, or 30 to 40 conservative amino acid substitutions relative to the sequence selected from TABLE 4. In some embodiments, an effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence selected from TABLE 4, wherein the effector protein comprises not more than 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 non-conservative amino acid substitutions relative to the sequence selected from TABLE 4

[0103] In certain embodiments, compositions and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% similar to any one of the sequences selected from TABLE 4. The similarity of two amino acid sequences can be calculated by using a BLOSUM62 similarity matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA., 89: 10915-10919 (1992)) that is transformed so that any value > 1 is replaced with +1 and any value < 0 is replaced with 0. For example,Attorney Docket No.: MABI-051 / 03WO 344183-2344 an He (I) to Leu (L) substitution is scored at +2.0 by the BLOSUM62 similarity matrix, which in the transformed matrix is scored at +1. This transformation allows the calculation of percent similarity, rather than a similarity score. Alternately, when comparing two full protein sequences, the proteins can be aligned using pairwise MUSCLE alignment. Then, the % similarity can be scored at each residue and divided by the length of the alignment. For determining % similarity over a protein domain or motif, a multilevel consensus sequence (or PROSITE motif sequence) can be used to identify how strongly each domain or motif is conserved. In calculating the similarity of a domain or motif, the second and third levels of the multilevel sequence are treated as equivalent to the top level. Additionally, if a substitution could be treated as conservative with any of the amino acids in that position of the multilevel consensus sequence, +1 point is assigned. For example, given the multilevel consensus sequence: RLG and YCK, the test sequence Qlq would receive three points. This is because in the transformed BLOSUM62 matrix, each combination is scored as: Q-R: +1; Q-Y: +0; I-L: +1; I-C: +0; Q-G: +0; Q-K: +1. For each position, the highest score is used when calculating similarity. The % similarity can also be calculated using commercially available programs, such as the Geneious Prime software given the parameters matrix = BLOSUM62 and threshold > 1.Messenger RNA (mRNA)

[0104] In some embodiments, compositions comprise a messenger RNA (mRNA) encoding an effector protein described herein. In some embodiments, the mRNA encodes an effector protein described in TABLE 4. In some embodiments, the mRNA comprises a protein coding sequence, one or more untranslatable regions (UTR), one or more nuclear localization signals (NLS), one or more stop codons, and or more adenine bases that are encompassed in a polyA tail. Non-limiting examples of protein coding sequences are provided in TABLE 5. A person of skill in the art would understand that various UTRs, NLSs, stop codons and polyA tails can be used in combination with these protein coding sequences. A person of skill in the art also understands that the letter “U” represents uridine and modified forms thereof, such as pseudouridine.TABLE 5: Exemplary CasPhi.12 Variant mRNA Protein Coding SequencesAttorney Docket No.: MABI-051 / 03WO 344183-2344Attorney Docket No.: MABI-051 / 03WO 344183-2344Attorney Docket No.: MABI-051 / 03WO 344183-2344

[0105] In some embodiments, the mRNA comprises a UTR, a protein coding sequence comprising a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence as set forth in TABLE 5, a nucleotide sequence encoding an NLS, and a polyA. In some embodiments, the mRNA comprises a UTR, a protein coding sequence, a nucleic acid sequence encoding an NLS comprising an amino acidAttorney Docket No.: MABI-051 / 03WO 344183-2344 sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence as set forth in TABLE 7, and a polyA. In some embodiments, the mRNA comprises a UTR, a protein coding sequence comprising the nucleic acid sequence of SEQ ID NO: 22, a nucleic acid sequence encoding an NLS comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence as set forth in TABLE 7, and a polyA. In some embodiments, the mRNA comprises a UTR, a protein coding sequence comprising the nucleic acid sequence of SEQ ID NO: 23, a nucleic acid sequence encoding an NLS comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence as set forth in TABLE 7, and a polyA. In some embodiments, the mRNA comprises a UTR, a protein coding sequence comprising the nucleic acid sequence of SEQ ID NO: 62, a nucleic acid sequence encoding an NLS comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to a sequence as set forth in TABLE 7, and a polyA. In some embodiments, the mRNA comprises a UTR, a protein coding sequence comprising a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 23, a nucleic acid sequence encoding an NLS comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26, and a polyA. In some embodiments, the mRNA comprises a UTR, a protein coding sequence comprising the nucleic acid sequence of SEQ ID NO: 23, a nucleic acid sequence encoding an NLS comprising the amino acid sequence of SEQ ID NO: 26, and a polyA.

[0106] Exemplary mRNA nucleotide sequences are shown in TABLE 6 below (regular font = effector protein coding sequence; bold = UTRs; underlined = NLS; italics = stop codons; and series of As = polyA tail). In some embodiments, the mRNA comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from TABLE 6.TABLE 6: Exemplary CasPhi.12 Variant mRNAsAttorney Docket No.: MABI-051 / 03WO 344183-2344Attorney Docket No.: MABI-051 / 03WO 344183-2344Attorney Docket No.: MABI-051 / 03WO 344183-2344Attorney Docket No.: MABI-051 / 03WO 344183-2344Nuclear Localization Signals (NLS)

[0107] In some embodiments, the effector protein comprises or is linked to a nuclear localization signal (NLS). In some embodiments, compositions described herein may comprise an NLS sequence that is adjacent to the N terminal of the effector protein or that is adjacent to the C terminal of the effector protein, or both. In some embodiments, an NLS is linked to the C terminus of the effector protein, but not the N terminus of the effector protein.

[0108] TABLE 7 provides exemplary nuclear localization sequences. In TABLE 7, X is any naturally occurring amino acid, andAD / E is any naturally occurring amino acid except Asp or Glu. In some embodiments, the NLS comprises an amino acid sequence set forth in any of SEQ ID NOs: 26-46. In preferred embodiments, the NLS is linked to the C terminus of the effector protein, optionally via a linker, wherein the NLS comprises or consists of an amino acid sequence of SEQ ID NO: 26.TABLE 7: Exemplary Nuclear Localization SequencesAttorney Docket No.: MABI-051 / 03WO 344183-2344mRNA 5’ Cap

[0109] In general, mRNAs for therapeutic use, such as those disclosed herein, comprise or are linked to a 5’ cap. A 5’ cap may be described as a modified nucleotide at the 5’ end of the mRNA. In general, and without being bound by theory, the 5’ cap provides for regulation and stability of an mRNA. In some embodiments, compositions described herein comprise an mRNA, wherein the mRNA comprises or is linked to a 5’ cap. In some embodiments, compositions described herein comprise an mRNA, wherein the mRNA comprises or is linked to a 5’ cap according to the following chemical structure:Attorney Docket No.: MABL051 / 03WO 344183-2344

[0110] In some embodiments, an mRNA of the instant disclosure may be represented by the following chemical structure:wherein “mRNA” represents an mRNA without a 5 ’ cap, and the remainder of the structure represents a 5 ’ cap, and wherein the 5 ’ cap is linked to the 5 ’ end of the mRNA. In some embodiments the mRNA comprises a nucleotide sequence selected from TABLE 5 and TABLE 6. In some embodiments the mRNA comprises a nucleotide sequence selected from SEQ ID NO: 23 and SEQ ID NO: 25.[oni] In some embodiments, compositions comprise a cation that associates with the mRNA and / or the 5’ cap. In some embodiments, the cation is sodium. In some embodiments, the cation is ammonium. In some embodiments, the 5’ cap is represented by a chemical structure selected from:Attorney Docket No.: MABI-051 / 03WO 344183-2344

[0112] In some embodiments, compositions described herein comprise an mRNA, wherein the mRNA is linked to a 5’ cap according to any one of the following structures:, andAttorney Docket No.: MABI-051 / 03WO 344183-2344wherein “mRNA” represents an mRNA minus a 5’ cap, and the remainder of the structure represents a 5’ cap, and wherein the 5’ cap is linked to the 5’ end of the mRNA. In some embodiments the mRNA comprises a nucleotide sequence selected from TABLE 5 and TABLE 6. In some embodiments the mRNA comprises a nucleotide sequence selected from SEQ ID NO: 23 and SEQ ID NO: 25.Protospacer Adjacent Motif (PAM) Sequences

[0113] Effector proteins of the present disclosure may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, cleavage occurs within 10, 20, 30, 40 or 50 nucleotides of a 5’ or 3’ terminus of a PAM sequence. In some embodiments, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a 5’ or 3’ terminus of a PAM sequence. A target nucleic acid may comprise a PAM sequence adjacent to atarget sequence. In some embodiments, compositions and methods comprise a guide nucleic acid or use thereof, wherein the guide nucleic acid comprises a spacer sequence that is complementary to a target sequence that is adjacent to a PAM sequence. In some embodiments, the PAM is 5 ’-NTTN-3 ’, wherein N = any nucleic acid. In some embodiments, the PAM is located 5’ of the target sequence on a non-target strand of the target nucleic acid. In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 19, 20, or 21.Fusion Proteins

[0114] In some embodiments, compositions and methods comprise a fusion protein, a fusion partner, or uses thereof. A fusion protein generally comprises an effector protein and a fusion partner. In some embodiments, the effector protein of a fusion protein has no cleavage activity. In some embodiments, the effector protein is engineered from a naturally occurring protein to have reduced cleavage activity relative to the naturally occurring protein. In general, the fusion partner provides an enzymatic activityAttorney Docket No.: MABI-051 / 03WO 344183-2344 that is not provided by the effector protein. By way of non-limiting example, introducing any combination of amino acid substitutions D369A, D369N, D658A, D658N, E567A, and E567Q into CasPhi.12 (SEQ ID NO: 19) may reduce its nuclease activity.

[0115] In some embodiments, the fusion partner comprises a polypeptide or peptide that is linked to the effector protein. In some embodiments, the fusion partner is not linked to the effector protein but is brought into proximity of the effector protein by other means. By way of non-limiting example, a fusion partner protein may comprise a peptide that binds an aptamer of a guide nucleic acid, wherein the effector protein is also capable of binding the guide nucleic acid, the guide nucleic acid thereby bringing the fusion partner into proximity of the effector protein. In some embodiments, the fusion partner is capable of binding or being bound by an effector protein. In some embodiments, the fusion partner and the effector protein are both capable of binding or being bound by an additional protein or moiety, the additional protein or moiety thereby bringing the fusion partner into proximity of the effector protein. In some embodiments, the fusion protein is a heterologous peptide or polypeptide as described herein. In some embodiments, the amino terminus of the fusion partner is linked to the carboxy terminus of the effector protein. In some embodiments, the carboxy terminus of the fusion partner protein is linked to the amino terminus of the effector protein by the linker. Unless otherwise indicated, reference to effector proteins throughout the present disclosure include fusion proteins comprising the effector protein described herein and a fusion partner.

[0116] In some embodiments, a fusion partner imparts some function or activity to a fusion protein that is not provided by an effector protein. Such activities may include but are not limited to nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, dimer forming activity (e.g. , pyrimidine dimer forming activity), integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity, modification of a polypeptide associated with target nucleic acid (e.g., a histone), and / or signaling activity.

[0117] In some embodiments, a fusion partner provides signaling activity. In some embodiments, a fusion partner directly or indirectly edits a target nucleic acid. Edits can be of a nucleobase, nucleotide, or nucleotide sequence of a target nucleic acid. In some embodiments, the fusion partner interacts with additional proteins, or functional fragments thereof, to make modifications to a target nucleic acid. In some embodiments, the fusion partner modifies proteins associated with a target nucleic acid. In some embodiments, a fusion partner modulates transcription (e.g., inhibits transcription, increases transcription) of a target nucleic acid. In some embodiments, a fusion partner directly or indirectly inhibits, reduces, activates or increases expression of a target nucleic acid.Attorney Docket No.: MABI-051 / 03WO 344183-2344Certain Compositions

[0118] Disclosed herein, in some aspects, are compositions comprising (1) a guide RNA disclosed herein and (2) an mRNA encoding an effector protein disclosed herein. In some embodiments, the present disclosure provides compositions that comprise (1) a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that is capable of hybridizing to a target sequence in APOC3,- and (2) an mRNA encoding an effector protein, wherein the effector protein is an engineered variant of CasPhi.12 (SEQ ID NO: 19). In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is selected from SEQ ID NOS: 12-18, and 52-55. In some embodiments, the mRNA comprises a nucleotide sequence selected from SEQ ID NOS: 22-25. In some embodiments, the composition comprises a lipid nanoparticle (LNP), wherein the mRNA and guide nucleic acid are encapsulated in the LNP. In some embodiments, the mRNA further comprises a nucleotide sequence encoding a nuclear localization sequence (NLS).

[0119] In some embodiments, the composition comprises (1) a guide nucleic acid sequence that comprises a sequence that is 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% identical to any one of SEQ ID NOs: 12- 18, and 52-55; and (2) a mRNA that encodes (a) an effector protein comprising an amino acid sequence that is 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% identical to any one of SEQ ID NOs: 19-21, and (b) a NLS that comprises a sequence that is 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% identical to any one of SEQ ID NOs: 26- 46, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0120] In some embodiments, the composition comprises (1) a guide nucleic acid sequence that comprises any one of SEQ ID NOs: 12-18, and 52-55; and (2) a mRNA that encodes (a) a polypeptide sequence that comprises any one of SEQ ID NOs: 19-21, and (b) an NLS that comprises any one of SEQ ID NOs: 26-46, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0121] In some embodiments, the composition comprises (1) a guide nucleic acid sequence that consists of any one of SEQ ID NOs: 12-18, and 52-55; and (2) an mRNA that encodes (a) an effector protein comprising an amino acid sequence that consists of any one of SEQ ID NOs: 19-21, and (b) an NLS that consists of any one of SEQ ID NOs: 26-46, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0122] In some embodiments, the composition comprises (1) a guide nucleic acid sequence comprises a repeat sequence that that is 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% identical to any one of SEQ ID NOs: 5- 11 and a spacer sequence that is 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% identical to any one of SEQ ID NOs: 1-4, and 50-51; and (2) a mRNA that comprises a sequence that is 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%Attorney Docket No.: MABI-051 / 03WO 344183-2344 identical to any one ofSEQ ID NOs: 22-23, and a nucleotide sequence encoding an NLS that comprises a sequence that is 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% identical to any one of SEQ ID NOs: 26-46, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0123] In some embodiments, the composition comprises (1) a guide nucleic acid sequence comprises a repeat sequence that comprises any one of SEQ ID NOs: 5-11 and a spacer sequence that comprises any one of SEQ ID NOs: 1-4, and 50-51; and (2) a mRNA that comprises any one of SEQ ID NOs: 22-23, and a nucleotide sequence encoding an NLS that comprises any one of SEQ ID NOs: 26-46, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0124] In some embodiments, the composition comprises (1) a guide nucleic acid sequence comprises a repeat sequence that consists of any one of SEQ ID NOs: 5-11 and a spacer sequence that consists of any one of SEQ ID NOs: 1-4, and 50-51; and (2) a mRNA that consists of any one of SEQ ID NOs: 22-23, and a nucleotide sequence encoding an NLS that consists of any one of SEQ ID NOs: 26-46, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0125] In some embodiments, an mRNA further comprises a nuclear localization sequence (NLS). In some embodiments, the composition comprises (1) a guide nucleic acid sequence that comprises a sequence that is 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% identical to any one of SEQ ID NOs: 15-17, 53 and 55; and (2) a mRNA that encodes (a) an effector protein comprising an amino acid sequence that is 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% identical to any one of SEQ ID NOs: 20-21, and (b) an NLS that comprises a sequence that is 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% identical to SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0126] In some embodiments, the composition comprises (1) a guide nucleic acid sequence that comprises any one of SEQ ID NOs: 15-17, 53 and 55; and (2) an mRNA that encodes (a) an effector protein comprising an amino acid sequence that comprises any one of SEQ ID NOs: 20-21, and (b) an NLS that comprises SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0127] In some embodiments, the composition comprises (1) a guide nucleic acid sequence that consists of any one of SEQ ID NOs: 15-17, 53 and 55; and (2) an mRNA that encodes (a) an amino acid sequence that consists of any one of SEQ ID NOs: 20-21, and (b) an NLS that consists of SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0128] In some embodiments, the mRNA further comprises a nuclear localization sequence (NLS). In some embodiments, the composition comprises (1) a guide nucleic acid sequence that comprises a sequence that is 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% identical to SEQ ID NO: 15; and (2) a mRNAAttorney Docket No.: MABI-051 / 03WO 344183-2344 that encodes a sequence that is 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% identical to SEQ ID NO: 21, and a NLS that comprises a sequence that is 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% identical to SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0129] In some embodiments, the composition comprises (1) a guide nucleic acid sequence that comprises SEQ ID NO: 15; and (2) an mRNA that encodes (a) a polypeptide sequence that comprises SEQ ID NO: 21, and (b) an NLS that comprises SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0130] In some embodiments, a composition comprises (1) a guide nucleic acid sequence that consists of SEQ ID NO: 15; and (2) an mRNA that encodes (a) a polypeptide sequence that consists of SEQ ID NO: 21, and (b) an NLS that consists of SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0131] In some embodiments, an mRNA further comprises a nuclear localization sequence (NLS). In some embodiments, the composition comprises (1) a guide nucleic acid sequence that comprises a sequence that is 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% identical to SEQ ID NO: 16; and (2) a mRNA that encodes a polypeptide sequence that is 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% identical to SEQ ID NO: 20, and a NLS that comprises a sequence that is 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% identical to SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0132] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that comprises SEQ ID NO: 16; and (2) an mRNA that encodes a polypeptide sequence that comprises SEQ ID NO: 20, and the NLS that comprises SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0133] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that consists of SEQ ID NO: 16; and (2) an mRNA that encodes a polypeptide sequence that consists of SEQ ID NO: 20, and the NLS that consists of SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0134] In some embodiments, an mRNA further comprises a nucleotide sequence encoding a nuclear localization sequence (NLS). In some embodiments, compositions comprise (1) a guide nucleic acid sequence that comprises a sequence that is 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% identical to SEQ ID NO: 17; and (2) a mRNA that encodes (a) a polypeptide sequence that is 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% identical to SEQ ID NO: 21, and (b) a NLS that comprises a sequence that is at least 90%, at least,Attorney Docket No.: MABI-051 / 03WO 344183-234491%, 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% identical to SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0135] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that comprises SEQ ID NO: 17; and (2) an mRNA that encodes (a) a polypeptide sequence that comprises SEQ ID NO: 21, and (b) an NLS that comprises SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0136] In some embodiments, composition comprise (1) a guide nucleic acid sequence that consists of SEQ ID NO: 17; and (2) an mRNA that encodes (a) a polypeptide sequence that consists of SEQ ID NO: 21, and (b) an NLS that consists of SEQ ID NO: 26, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0137] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that comprises a sequence that is 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% identical to SEQ ID NO: 15; and (2) an mRNA that is 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% identical to SEQ ID NO: 25, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0138] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that comprises SEQ ID NO: 15; and (2) an mRNA comprises SEQ ID NO: 25, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0139] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that consists of SEQ ID NO: 15; and (2) an mRNA consists of SEQ ID NO: 25, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0140] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that comprises a sequence that is 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% identical to SEQ ID NO: 16; and (2) an mRNA that is 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% identical to SEQ ID NO: 24, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0141] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that comprises SEQ ID NO: 16; and (2) an mRNA comprises SEQ ID NO: 24, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0142] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that consists of SEQ ID NO: 16; and (2) an mRNA consists of SEQ ID NO: 24, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0143] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that comprises a sequence that is at least 90%, at least, 91%, at least 92%, at least 93%, at least 94%, at least 95%, atAttorney Docket No.: MABI-051 / 03WO 344183-2344 least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17; and (2) an mRNA that is 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% identical to SEQ ID NO: 25, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0144] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that comprises SEQ ID NO: 17; and (2) an mRNA comprises a nucleotide sequence of SEQ ID NO: 25, wherein the mRNA and the guide RNA are encapsulated in an LNP.

[0145] In some embodiments, compositions comprise (1) a guide nucleic acid sequence that consists of SEQ ID NO: 17; and (2) an mRNA consists of SEQ ID NO: 25, wherein the mRNA and the guide RNA are encapsulated in an LNP.R22529 and mRNA253

[0146] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA encodes an effector protein, wherein the effector protein comprises an amino acid sequence of SEQ ID NO: 21; the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 54, and the mRNA and the guide RNA are encapsulated in the LNP.

[0147] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA comprises a nucleotide sequence of SEQ ID NO: 25; the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 54, and the mRNA and the guide RNA are encapsulated in the LNP. In some embodiments, the nucleotides at positions 1, 2, 3, 11, 12, 13, 14, 25, 63, 64, and 65 (from 5’ to 3’) comprise a 2’-0Me modification, the 1st, 2nd, 3rd, 10th- 13th, 24th, 25th, 63rd, and 64thintemucleotide linkages (from 5’ to 3’) are phosphorothioate bonds, and the remaining linkages are phosphodiester bonds.

[0148] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA comprises a nucleotide sequence of SEQ ID NO: 25; the guide nucleic acid is represented by: mG*mU*mC*rUrUrGrArGrArG*mG*mA*mA*mArCrUrCrUrCrArArGrArC*mU*rArGrArUrUrGr CrUrCrCrUrUrArCrGrArGrGrArGrArCrCrCrCrUrCrCrCrCrArGrArGrGrGrCmA*mU*mU (SEQ ID NO: 55), wherein r represents an unmodified nucleotide, m represents a 2’-0Me modified nucleotide, and * represents a phosphorothioate linkage; and the mRNA and the guide RNA are encapsulated in the LNP.R22528 and mRNA253

[0149] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA encodes an effector protein, wherein the effector protein comprises an amino acid sequence of SEQ ID NO: 21; the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 52, and the mRNA and the guide RNA are encapsulated in the LNP.Attorney Docket No.: MABI-051 / 03WO 344183-2344

[0150] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA comprises a nucleotide sequence of SEQ ID NO: 25; the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 52, and the mRNA and the guide RNA are encapsulated in the LNP. In some embodiments, the nucleotides at positions 1, 2, 3, 11, 12, 13, 14, 25, 64, 65 and 66 (from 5’ to 3’) comprise a 2’-OMe modification, the 1st, 2nd, 3rd, lO*-^*, 24th, 25th, 64th, and 65thintemucleotide linkages (from 5’ to 3’) are phosphorothioate bonds, and the remaining linkages are phosphodiester bonds.

[0151] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA comprises a nucleotide sequence of SEQ ID NO: 25; the guide nucleic acid is represented by: mG*mU*mC*rUrUrGrArGrArG*mG*mA*mA*mArCrUrCrUrCrArArGrArC*mU*rArGrArUrUrGr CrUrCrCrUrUrArCrGrArGrGrArGrArCrCrCrUrGrArArArGrArCrUrArCrUrGrGmA*mG*mC (SEQ ID NO: 53), wherein r represents an unmodified nucleotide, m represents a 2’-0Me modified nucleotide, and * represents a phosphorothioate linkage; and the mRNA and the guide RNA are encapsulated in the LNP.R21971 and mRNA253

[0152] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA encodes an effector protein, wherein the effector protein comprises an amino acid sequence of SEQ ID NO: 21; the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 12, and the mRNA and the guide RNA are encapsulated in the LNP.

[0153] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA comprises a nucleotide sequence of SEQ ID NO: 25; the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 12, and the mRNA and the guide RNA are encapsulated in the LNP. In some embodiments, the 15 nucleotides at the 5’ end and the 3 nucleotides at the 3’ end of the guide nucleic acid each comprise a 2’-0Me modification, and the first three intemucleotide linkages and the last two intemucleotide linkages are phosphorothioate bonds, and the remaining linkages are phosphodiester bonds.

[0154] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA comprises a nucleotide sequence of SEQ ID NO: 25; the guide nucleic acid is represented by: mC*mU*mU*mUmCmAmAmGmAmCmUmAmAmUmArGrArUrUrGrCrUrCrCrUrUrArCrGrArGr GrArGrArCrCrCrUrGrArArArGrArCrUrArCrUrGrGrAmG*mC*mA (SEQ ID NO: 15), wherein r represents an unmodified nucleotide, m represents a 2’-0Me modified nucleotide, and * represents a phosphorothioate linkage; and the mRNA and the guide RNA are encapsulated in the LNP.Attorney Docket No.: MABI-051 / 03WO 344183-2344R21969 and mRNA261

[0155] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA encodes an effector protein, wherein the effector protein comprises an amino acid sequence of SEQ ID NO: 20; the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO:13, and the mRNA and the guide RNA are encapsulated in the LNP.

[0156] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA comprises a nucleotide sequence of SEQ ID NO: 24; the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 13, and the mRNA and the guide RNA are encapsulated in the LNP. In some embodiments, the 15 nucleotides at the 5’ end and the 3 nucleotides at the 3’ end of the guide nucleic acid each comprise a 2’-0Me modification, and the first three intemucleotide linkages and the last two intemucleotide linkages are phosphorothioate bonds, and the remaining linkages are phosphodiester bonds.

[0157] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein the mRNA comprises a nucleotide sequence of SEQ ID NO: 24, wherein the guide nucleic acid is represented by: mC*mU*mU*mUmCmAmAmGmAmCmUmAmAmUmArGrArUrUrGrCrUrCrCrUrUrArCrGrArGr GrArGrArCrCrCrUrGrArArArGrArCrUrArCrUrGmG*mA*mG (SEQ ID NO: 16), wherein r represents an unmodified nucleotide, m represents a 2’-0Me modified nucleotide, and * represents a phosphorothioate linkage; and the mRNA and the guide RNA are encapsulated in the LNP.R21461 and mRNA253

[0158] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA encodes an effector protein, wherein the effector protein comprises an amino acid sequence of SEQ ID NO: 21; the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO:14, and the mRNA and the guide RNA are encapsulated in the LNP.

[0159] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein: the mRNA comprises a nucleotide sequence of SEQ ID NO: 25; the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 14, and the mRNA and the guide RNA are encapsulated in the LNP. In some embodiments, the 15 nucleotides at the 5’ end and the 3 nucleotides at the 3’ end of the guide nucleic acid each comprise a 2’-0Me modification, and the first three intemucleotide linkages and the last two intemucleotide linkages are phosphorothioate bonds, and the remaining linkages are phosphodiester bonds.

[0160] In some embodiments, compositions comprise an mRNA, a guide nucleic acid, and an LNP, wherein the mRNA comprises a nucleotide sequence of SEQ ID NO: 25, wherein the guide nucleic acid is represented by: mC*mU*mU*mUmCmAmAmGmAmCmUmAmAmUmArGrArUrUrGrCrUrCrCrUrUrArCrGrArGr GrArGrArCrCrCrUrGrArArArGrArCrUrArCrUmG*mG*mA (SEQ ID NO: 17), wherein r representsAttorney Docket No.: MABI-051 / 03WO 344183-2344 an unmodified nucleotide, m represents a 2’-0Me modified nucleotide, and * represents a phosphorothioate linkage; and the mRNA and the guide RNA are encapsulated in the LNP.Target Nucleic Acids

[0161] Disclosed herein are compositions and methods for modifying a target nucleic acid, wherein target nucleic acid comprises the human gene, APOC3. APOC3 encodes the apolipoprotein C3 protein (APOC3). APOC3 may also be referred to as apolipoprotein C-III (APOCIII), Apo-CIII, ApoC-III, and HALP2. APOC3 has a cytogenetic location of 1 lq23.3 and the genomic coordinates on Chromosome 11 on the forward strand are positions 116,829,706-116,833,072. In general, guide nucleic acids described herein comprise a sequence that is complementary to and / or hybridizes to a target sequence of APOC3. Exemplary reference sequences for APOC3 are provided in TABLE 8. Exemplary reference sequences for the APOC3 protein are listed in TABLE 9. The target sequence of the APOC3 gene may be a portion of the APOC3 gene that encodes the APOC3 protein (e.g., an exon).TABLE 8: Exemplary reference APOC3 genesTABLE 9: Exemplary reference APOC3 proteinsLipid Particles

[0162] Disclosed herein, in some aspects, are compositions comprising a lipid particle that encapsulates a nucleic acid encoding an effector protein (e.g., an mRNA), and a guide nucleic acid. In some embodiments, the lipid particle is a lipid nanoparticle (LNP). Also disclosed herein, in some aspects, are methods of administering such LNPs to a subject in order to introduce the effector proteins and the guide nucleic acid to a cell or tissue of a subject. Also disclosed herein, are methods of producing such LNPs. In some embodiments, the gRNAs described herein can be produced by in vitro transcription (IVT), chemical synthesis, or a combination thereof. In some embodiments, methods comprise at least one of enzymatic IVT, solid-phase synthesis, liquid-phase synthesis, small region synthesis, and ligation. In some embodiments, the guide nucleic acid is made using IVT enzymatic synthesis methods. Similarly, mRNAs and portions thereof may be produced with IVT, chemical synthesis, or a combination thereof. In some embodiments, methods comprise encapsulating a guide nucleic acid and an mRNA in an LNP.

[0163] LNPs are a non-viral delivery system and are particularly effective for delivery of nucleic acids to the liver. Beneficial properties of LNP include ease of manufacture, low cytotoxicity, low immunogenicity, high efficiency of nucleic acid encapsulation and cell transfection, and multi-dosing capabilities and flexibility of design (Kulkami et al., (2018) Nucleic Acid Therapeutics, 28(3): 146-157).Attorney Docket No.: MABI-051 / 03WO 344183-2344

[0164] Non-limiting examples of lipids and polymers are cationic polymers, cationic lipids, ionizable lipids, or bio-responsive polymers. In some embodiments, ionizable lipids exploit chemical-physical properties of the endosomal environment (e.g. , pH) offering improved delivery of nucleic acids. In some embodiments, the ionizable lipids are neutral at physiological pH. In some embodiments, the ionizable lipids are protonated under acidic pH. In some embodiments, the bio-responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space.

[0165] In some embodiments, a LNP comprises an outer shell and an inner core. In some embodiments, the outer shell comprises lipids. In some embodiments, the lipids comprise modified lipids. In some embodiments, the modified lipids comprise pegylated lipids. In some embodiments, the lipids comprise one or more of cationic lipids, anionic lipids, ionizable lipids, and non-ionic lipids. In some embodiments, the LNP comprises one or more of Nl,N3,N5-tris(3-(didodecylamino)propyl)benzene- 1,3,5-tricarboxamide (TT3), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l-palmitoyl-2- oleoylsn-glycero-3-phosphoethanolamine (POPE), l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), cholesterol (Choi), 1,2-dimyristoyl-sn-glycerol, and methoxypolyethylene glycol, derivatives, analogs, or variants thereof.

[0166] In some embodiments, the LNP has a negative net overall charge prior to complexation with one or more of a guide nucleic acid and a nucleic acid encoding the effector protein. In some embodiments, the inner core is a hydrophobic core. In some embodiments, the one or more of a guide nucleic acid and the nucleic acid encoding the effector protein forms a complex with one or more of the cationic lipids and the ionizable lipids. In some embodiments, the nucleic acid encoding the effector protein or the nucleic acid encoding the guide nucleic acid is self-replicating.

[0167] In some embodiments, an LNP comprises a lipid composition targeting to a specific organ. In some embodiments, the lipid composition comprises lipids having a specific alkyl chain length that controls accumulation of the LNP in the specific organ (e.g., liver or spleen). In some embodiments, the lipid composition comprises a biomimetic lipid that controls accumulation of the LNP in the specific organ (e.g., brain). In some embodiments, the lipid composition comprises lipid derivatives (e.g., cholesterol derivatives) that controls accumulation of the LNP in a specific cell (e.g., liver endothelial cells, Kupffer cells, hepatocytes).

[0168] In some embodiments, an LNP comprises an ionizable lipid, a structure lipid, a neutral lipid and a polyethylene glycol (PEG) molecule.Ionizable lipids

[0169] In general, ionizable lipids are a class of lipid molecules that can change their charge in response to pH. In some embodiments, ionizable lipids comprise a hydrophobic tail and an ionizable head group. At physiological pH, they are neutral, but when the pH is acidic, they become positively charged. ThisAttorney Docket No.: MABI-051 / 03WO 344183-2344 pH sensitivity makes ionizable lipids a key component of lipid nanoparticles (LNPs), which are used to deliver RNA therapeutics.

[0170] In some embodiments, compositions comprise an LNP, wherein the LNP comprises an ionizable lipid according to the following chemical structure:, which may also be referred to as the following chemical formula:(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also referred to as 3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. In some embodiments, this lipid is referred to as “LP01” or “Lipid A.”Neutral lipids

[0171] In general, neutral lipids function to stabilize an LNP. Often, neutral lipids comprise a phospholipid. In some embodiments, compositions comprise an LNP, wherein the LNP comprises a phospholipid selected from l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1,2-dipalmitoyl sn- glycero-3 -phosphocholine (DPPC), l,2-dioleyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2- dioleyl-sn-glycero-3-phosphatidylcholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 phosphoethanolamine (DPPE), l,2-dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), 1,2- dioleoyl-sn-glycero-3-phospho-(l'-rac -glycerol (DOPG), or any combinations thereof. In some embodiments, the phospholipid comprises DSPC.Structure lipids

[0172] In some embodiments, structure lipids improve the stability of LNPs. In some embodiments, compositions comprise an LNP, wherein the LNP comprises a structure lipid, wherein the structure lipid comprises a steroid or sterol. In some embodiments, the structure lipid comprises cholesterol, cholesterol hemisuccinate, or any combination thereof. In some embodiments, the structure lipid comprises cholesterol.PEG molecules

[0173] In some embodiments, LNPs comprise a polyethylene glycol (PEG) molecule, also referred to as a PEG lipid. In some embodiments, PEG lipids alter the length of time an LNP can exist in vivo. In some embodiments, PEG lipids assist in a formulation process of an LNP. In some embodiments, PEG lipids help reduce particle aggregation of LNPs. In some embodiments, PEG lipids help control particle size of LNPs. Non-limiting examples of PEG lipids are dimyristoylglycerol-PEG (DMG-PEG),Attorney Docket No.: MABI-051 / 03WO 344183-2344 dilauroylglycerol-PEG, dipalmitoylglycerol-PEG, distearoylglycerol-PEG, dilaurylglycamide-PEG, dimyristylglycamide-PEG, dipalmitoylglycamide-PEG, distearoylglycamide-PEG, cholesterol-PEG, and methoxypolyethyleneglycoloxy(2000)-N,N-ditetradecylacetamide (ALC-0159). In some embodiments, the PEG lipid comprises DMG-PEG. In some embodiments, the PEG lipid comprises DMG-PEG2k. DMG-PEG2k may be represented by the following chemical structure:

[0174] Additional LNPs are described in WO 2022 / 229903, WO 2023 / 144792, WO 2023 / 144798, WO 2021 / 081225, WO 2020 / 219941, WO 2022 / 011156, WO 2021 / 188389, WO 2021 / 163339, WO 2022 / 133344, WO 2019 / 067872, WO 2024 / 011206, WO 2023 / 114937, WO 2023 / 114943, WO 2024 / 089229, and WO 2023 / 144193, all of which are incorporated herein by reference.

[0175] Disclosed herein, in some aspects, are methods for delivering compositions disclosed herein to a target cell or tissue. In some embodiments, methods comprise delivering an LNP encapsulating an mRNA encoding an effector protein and a guide nucleic acid to a human subject, wherein the mRNA is translated into an effector protein in a cell of the liver of the human subject, and wherein the effector protein and the guide nucleic acid form an RNP complex that modifies APOC3 in the cell. In some embodiments, the LNPs of the present disclosure specifically bind to apolipoproteins. In some embodiments, the LNPs specifically bind to apolipoprotein (ApoE) in the blood. Apolipoproteins are proteins circulating in plasma that are important in regulating lipid transport. ApoE represents a class of apolipoproteins which interacts with cell surface heparin sulfate proteoglycans in the liver during the uptake of lipoprotein.Pharmaceutical Compositions and Modes of Administration

[0176] Disclosed herein are compositions comprising an effector protein described herein or a nucleic acids encoding the same, and a guide nucleic acid for the modification of APOC3. In some embodiments, compositions described herein comprise liposomes (e.g. , cationic lipids or neutral lipids), dendrimers, lipid nanoparticle (LNP), or cell-penetrating peptides. In some embodiments, compositions described herein comprise an LNP.

[0177] In some embodiments, compositions described herein are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions comprise compositions described herein and a pharmaceutically acceptable carrier or diluent. Non-limiting examples of pharmaceutically acceptable carriers and diluents suitable for the pharmaceutical compositions disclosed herein include buffers (e.g. , neutral buffered saline, phosphate buffered saline); carbohydrates (e.g., glucose, mannose, sucrose, dextran, mannitol); polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA, glutathione); adjuvants (e.g., aluminum hydroxide); surfactants (Polysorbate 80, PolysorbateAttorney Docket No.: MABI-051 / 03WO 344183-234420, or Pluronic F68); glycerol; sorbitol; mannitol; polyethylene glycol; and preservatives. In some embodiments, the vector is formulated for delivery through injection by a needle carrying syringe. In some embodiments, the composition is formulated for delivery by electroporation. In some embodiments, the composition is formulated for delivery by chemical method.

[0178] Pharmaceutical compositions described herein may comprise a salt. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is a potassium salt. In some embodiments, the salt is a magnesium salt. In some embodiments, the salt is NaCl. In some embodiments, the salt is KNOs. In some embodiments, the salt is Mg2+SO42

[0179] Pharmaceutical compositions described herein may be in the form of a solution (e.g., a liquid). In some embodiments, the solution is formulated for injection, e.g., intravenous or subcutaneous injection. In some embodiments, the pH of the solution is about 7, about 7.1, about 7.2, about 7.3, about7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9. In some embodiments, the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some cases, the pH of the solution is less than 7. In some cases, the pH is greater than 7.

[0180] Pharmaceutical compositions may be used to modify a target nucleic acid or the expression thereof in a cell, or a population of cells, in vitro, in vivo, or ex vivo. Pharmaceutical compositions may be used to modify a target nucleic acid or the expression thereof in a cell or progeny of the cell, in vitro, in vivo, or ex vivo. In some embodiments, pharmaceutical compositions comprise one or more nucleic acids encoding an effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent. The effector protein, fusion effector protein, fusion partner protein, or combination thereof may be any one of those described herein.Methods of Nucleic Acid Modification

[0181] Disclosed herein, in some aspects, are methods of modifying APOC3. In some embodiments, methods comprise contacting APOC3 with an effector protein disclosed herein or a nucleic acid encoding the same and a guide nucleic acid disclosed herein. In some embodiments, the nucleic acid encoding the effector protein is RNA, DNA or a combination thereof. In some embodiments, compositions comprise an mRNA encoding an effector protein and a guide nucleic acid disclosed herein, wherein the mRNA and the guide nucleic acid are encapsulated in an LNP. Methods may comprise introducing such a composition to a cell or subject, wherein (i) an effector protein encoded by the mRNA and (ii) the guide RNA form a ribonucleoprotein (RNP) complex that binds to a target nucleotide sequence of the APOC3 gene. In some embodiments, methods comprise contacting a cell comprising APOC3 with an mRNA disclosed herein and a guide nucleic acid disclosed herein. In some embodiments, methods comprise contacting a cell comprising APOC3 with an mRNA disclosed herein and a guide nucleic acid disclosed herein, wherein the mRNA and guide nucleic acid are encapsulatedAttorney Docket No.: MABI-051 / 03WO 344183-2344 in an LNP. In some embodiments, methods comprise (a) introducing a composition disclosed herein comprising (i) an mRNA encoding an effector protein and (ii) a guide nucleic acid to a cell, and (b) contacting a target nucleotide sequence within the APOC3 gene with an RNP complex comprising the effector protein encoded by the mRNA and the guide nucleic acid. In some embodiments, the mRNA and the guide nucleic acid are encapsulated in an LNP for introduction to the cell. In some embodiments, the effector protein cleaves the target nucleotide sequence in the APOC3 gene. In some embodiments, the mRNA comprises a nucleotide sequence selected from SEQ ID NOS: 22-25, and the guide nucleic acid comprises a nucleotide sequence selected from SEQ ID NOS: 12-18 and 52-55. In some embodiments, the mRNA comprises a nucleotide sequence of SEQ ID NO: 25 and the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 17. In some embodiments, the mRNA comprises a nucleotide sequence of SEQ ID NO: 24 and the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 16. In some embodiments, the mRNA comprises a nucleotide sequence of SEQ ID NO: 25 and the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 15. In some embodiments, the mRNA comprises a nucleotide sequence of SEQ ID NO: 25 and the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 53. In some embodiments, the mRNA comprises a nucleotide sequence of SEQ ID NO: 25 and the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 55

[0182] Methods of modifying APOC3 may comprise cleaving an intemucleotide linkage (e.g., a phosphodiester bond) of APOC3. Modifying APOC3 may comprise cleaving an intemucleotide linkage on each strand of the APOC3 gene, otherwise referred to as a “double stranded break” (DSB). Modifying may comprise deleting one or more nucleotides of APOC3. Modifying APOC3 may result in disrupting the translation of a functional APOC3 protein. Modifying may result in reducing or abolishing expression of APOC3 in a cell. The cell may be a cell in a liver. The cell may be a hepatocyte.

[0183] Methods of modifying APOC3 may comprise deleting one or more nucleotides of APOC3 and / or inserting one or more nucleotides into APOC3. An indel, sometimes referred to as an insertiondeletion or indel mutation, is a type of genetic mutation that results from the insertion and / or deletion of nucleotides in a target nucleic acid. An indel can vary in length (e.g. , 1 to 1,000 nucleotides in length) and be detected using methods well known in the art, including nucleic acid sequencing. The term, % indel, refers to the percentage of sequencing reads that comprise at least one nucleotide insertion or deletion. By way of non-limiting example, 50% indels means that 50% of the sequencing reads corresponding to a sequence of interest had at least one nucleotide insertion or deletion, and the remaining 50% of the sequencing reads looked normal (unmodified). In certain embodiments, methods may result in at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% indels.

[0184] Methods of modifying a target nucleic acid or modulating the expression of a target nucleic acid may be performed in vivo. Methods may be performed in a cell. Methods may be performed in a human subject. Methods may comprise administering to a human subject a composition disclosedAttorney Docket No.: MABI-051 / 03WO 344183-2344 herein. Methods may comprise assessing indel formation in a cell or an organ of the human subject. The cell may be a hepatocyte. The organ may be the liver.Methods of Treating a Disease

[0185] Disclosed herein, in some aspects, are methods for treating and / or preventing a disease in a subject in need thereof comprising administering to the subject a composition disclosed herein. In some aspects, the compositions disclosed herein are for use in treating or preventing a disease in a human subject in need thereof, comprising administering the composition to the human subject. In some embodiments, the disease is selected from cardiovascular disease, familial chylomicronemia syndrome, and hypertriglyceridemia. The term, “disease,” may be used interchangeably with the terms, “disorder” and “syndrome”, all terms indicating a subject’s condition or symptom thereof that is undesirable or presents a health risk to the subject. In some embodiments, treating and / or preventing a disease comprises modifying APOC3. In some embodiments, the disease is a cardiovascular disease (CVD). In some embodiments, the cardiovascular disease is coronary artery disease (CAD). In some embodiments, the disease is chronic kidney disease (CKD). In some embodiments, the disease is chylomicronemia. In some embodiments, chylomicronemia is persistent chylomicronemia. In some embodiments, the disease is Familial chylomicronemia syndrome (FCS). In some embodiments, the disease is lipodystrophy. In some embodiments, the disease is hypertriglyceridemia. In some embodiments, the hypertriglyceridemia is severe hypertriglyceridemia (SHTG).

[0186] Hypertriglyceridemia (HTG) is a clinical diagnosis defined when plasma triglyceride (TG) concentrations rise above a threshold value, such as the 90th or 95th percentile for age and sex. In some embodiments, the methods provided herein comprise lowering triglyceride levels in a subject with hypertriglyceridemia comprising administering to the subject a composition disclosed herein. In some embodiments, methods lower plasma TG concentrations at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or at least 70% relative to the subject’s plasma TG concentrations before administration of the composition. Triglycerides may be measured by known methods including that described in Example 10 of the instant disclosure.

[0187] In some embodiments, methods of treating or preventing a disease in a subject comprises administering the subject a composition comprising: an mRNA disclosed herein and a guide nucleic acid disclosed herein, wherein the mRNA and guide nucleic acid are encapsulated in an LNP. In some embodiments, the mRNA comprises a nucleotide sequence selected from SEQ ID NOS: 22-25, and the guide nucleic acid comprises a nucleotide sequence selected from SEQ ID NOS: 12-18 and 52-55. In some embodiments, the mRNA comprises a nucleotide sequence of SEQ ID NO: 25 and the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 17. In some embodiments, the mRNA comprises a nucleotide sequence of SEQ ID NO: 24 and the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 16. In some embodiments, the mRNA comprises a nucleotide sequence of SEQ ID NO: 25 and the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 15. InAttorney Docket No.: MABI-051 / 03WO 344183-2344 some embodiments, the mRNA comprises a nucleotide sequence of SEQ ID NO: 25 and the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 53. In some embodiments, the mRNA comprises a nucleotide sequence of SEQ ID NO: 25 and the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 55EXAMPLES

[0188] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1: Engineered CasPhi.12 variants and human APOC3 guides demonstrate APOC3 editing across multiple mammalian cell types

[0189] HepG2 cells, CYN0M-K1 cells, primary monkey hepatocytes or primary human hepatocytes are transfected with CasPhi.12 variant encoding mRNA and APOC3 targeting gRNA. As a control, cells are similarly transfected with a Cas9 encoding mRNA and a guide nucleic acid (R15779) according to the following sequence (spacer italicized): 5-GG4GGGCCCGGAL4GriCGriCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3’ (SEQ ID NO: 47). The Cas9 guide nucleic acid may be represented by the following formula: 5’- mU*mC*mA*rGrUrUrCrCrCrUrGrArArArGrArCrUrArCrGrUrUrUrUrArGrArGrCrUrArGrArArAr UrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArAr ArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3’ (SEQ ID NO: 48), wherein r represents an unmodified nucleotide, m represents a 2’-OMe modified nucleotide, and * represents a phosphorothioate linkage wherein as a control. Cells are harvested after 48 hours and analyzed by NGS.Example 2. Modification of APOC3 in non-human primates (NHP) with Engineered CasPhi.12 Variants

[0190] Cynomolgus macaques receive an LNP formulation comprising mRNA encoding an engineered variant of CasPhi. 12 (SEQ ID NO: 19), e.g., SEQ ID NO: 20 or SEQ ID NO: 21, with an NLS at the C terminus, and a guide RNA targeting APOC3 (e.g., a guide RNA according to any one of SEQ ID NOs: 12-18 and 52-55) via IV infusion. mRNA / gRNA ratio is 1 :3, and the dose is 2.0 mg / kg. Additional NHPs are administered control effector proteins (e.g., SpyCas9) and comparator guide RNAs (e.g., SEQ ID NO: 48)

[0191] The efficacy and tolerability of the mRNA and guide RNA are assessed. A liver biopsy is collected about 1 week after injection, and animals are sacrificed about 4 weeks after IV infusion and tissue samples (including liver) are collected. Indels in the NHP APOC3 gene from the collected tissue samples are determined by NGS. Liver and / or serum levels of APOC3 protein may be quantified. Serum triglycerides may also be quantified. Other factors quantified or analyzed may include cytokines, liver enzymes, and LDL-C.Attorney Docket No.: MABI-051 / 03WO 344183-2344Example 3. mRNA253 CasPhi.12 variant is more active than CasPhi.12 variants with a smaller subset of its amino acid substitutions

[0192] HEK293T at 10k seeding density (200ul / well) were transfected with plasmids encoding CasPhi.12 variants and various guides RNAs. The DNA dose was lOOng total DNA (1: 1 nuclease / guide). Cells were harvested 72 hours post transfection. Nuclease activity of the variants were compared based on % indel generated. The CasPhi.12 variants tested were CasPhi.12 L26R, 147 IT; CasPhi.12 L26R, S223P, 147 IT, D703G; and CasPhi.12 L26R, T87G, Y220S, S223P, 147 IT, D703G (corresponding to the variant of mRNA253). The nucleotide sequences of the gRNAs are provided in TABLE 10 (spacer shown in bold font). Results are shown in FIG. 1. FIG. 1 shows that CasPhi.12 L26R, T87G, Y220S, S223P, I471T, D703G (corresponding to the variant of mRNA253) was the CasPhi.12 variant that was most effective at inducing indel formation in this experiment.TABLE 10. Nucleotide sequences of the gRNAs tested in Example 3Example 4. CasPhi.12 L26R-T87G-Y220S-S223P-I471T-D703G is highly specific, generating fewer off target modifications relative to CasPhi.12 L26R-T87G-S186G-Y220S-S223P-I471T-M503K- D703G

[0193] The off-target profdes of CasPhi.12 variants, CasPhi.12 L26R-T87G-Y220S-S223P-I471T- D703G (corresponding to mRNA253) and CasPhi.12 L26R-T87G-S186G-Y220S-S223P-I471T- M503K-D703G (corresponding to mRNA261), were compared in this experiment. Both variants were expressed with a C-terminal NLS, CasPhi.12 L26R-T87G-S186G-Y220S-S223P-I471T-M503K- D703G is represented by SEQ ID NO: 66 and CasPhi.12 L26R-T87G-Y220S-S223P-I471T-D703G is represented by SEQ ID NO: 67. Briefly, HEK293T cells were transfected with plasmids encoding the CasPhi.12 variants and w APOC3 targeting guide nucleic acid R21969 (SEQ ID NO: 13). Cells were harvested 72 hours later and NGS was used to analyze % indel on multiple sites including APOC3. Results are provided in FIG. 2, which shows that while % indel of APOC 3 (on-target) were comparable for these variants, there were detectable off target modifications generated with CasPhi. 12 L26R-T87G- S186G-Y220S-S223P-I471T-M503K-D703G (corresponding to mRNA261), and no detectable off- target indel generated with CasPhi.12 L26R-T87G-Y220S-S223P-I471T-D703G (corresponding to mRNA253). The CasPhi.12 L26R-T87G-Y220S-S223P-I471T-D703G is a robust effector protein for gene editing providing effective target cleavage with minimal off-target activity thereby demonstrating that the CasPhi.12 L26R-T87G-Y220S-S223P-I471T-D703G variant is an effector protein with favorable properties for gene editing.Attorney Docket No.: MABI-051 / 03WO 344183-2344Example 5. Evaluating the effects of NLS designs on the activity of CasPhi.12 variants in primary human hepatocytes

[0194] Primary human hepatocytes were transfected with plasmids encoding R17566; and an engineered CasPhi.12 variant comprising amino acid substitutions L26R and 147 IT, and having various NLS configurations shown in FIG. 3. The sequence of R17566 is AUAGAUUGCUCCUUACGAGGAGACCUCAGGAGCUUCAGAGG (SEQ ID NO: 18). Cells were harvested after 48 hours and % indel were quantified with NGS. Results are shown in the FIG. 4. Similar experiments are performed with mRNA and guide nucleic acids described herein.Example 6. R22529 guide modifications and stabilizer sequence provide improved activity relative to APOC3 guides with standard modifications without stabilizer sequence in hAPOC3 mice

[0195] Humanized APOC3 (hAPOC3) mice with severe plasma hypertriglyceridemia and significantly increased plasma cholesterol (B6;CBA-Tg(APOC3)3707Bres / J), were intravenously injected with an LNP containing (a) a CasPhi. 12 variant mRNA253 (SEQ ID NO: 25), encoding SEQ ID NO: 21; and (b) a guide RNA targeting APOC3, at doses of 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, and 2.0 mg / kg. The guide RNAs are described in TABLE 11 (bold text indicates stabilizer sequence, italics indicates repeat sequence, and spacer is in regular font; “m” indicates a 2’-OMe sugar modification of the following nucleotide, “r” indicates that the following nucleotide is unmodified, and * indicates a phosphorothioate linkage between the two neighboring nucleotides, all other intemucleotide linkages are phosphodiester linkages). Levels of human APOC3 mRNA and protein (electro-chemi-luminescence assay run on the Meso Scale Discovery (MSD) system) in serum were quantified after 5 days. Results are shown in FIG. 5. R21461 and R22149 show comparable potency. R22148 with the same stabilizer sequence (SEQ ID NO: 65) and chemical modifications as R22529 has higher potency at all doses than R21461 and R22149.TABLE 11. Guide RNAs tested in Example 5Attorney Docket No.: MABI-051 / 03WO 344183-2344Example 7. R22529 guide modifications and stabilizer sequence provide improved activity relative to APOC3 guides with standard modifications without stabilizer sequence in hAPOC3 mice

[0196] Humanized APOC3 (hAPOC3) mice with severe plasma hypertriglyceridemia and significantly increased plasma cholesterol (Biocytogen, C\^7BL / 6N-4 / Jocj'"' / '|l / Y -J' / >’t's'e7Bcgcn). were intravenously injected with an LNP containing (a) CasPhi.12 variant mRNA253 (SEQ ID NO: 25), encoding SEQ ID NO: 21; and (b) a guide RNA targeting APOC3, at a ratio of 1: 1 or 1:3, at a dose of 0.5 mg / kg. The guide RNAs are described in TABLE 12 (bold text indicates stabilizer sequence, italics indicates repeat sequence, and spacer is in regular font; “m” indicates a 2’-0Me sugar modification of the following nucleotide, “r” indicates that the following nucleotide is unmodified, and * indicates a phosphorothioate linkage between the two neighboring nucleotides, all other intemucleotide linkages are phosphodiester linkages). Animals were euthanized at day 5. % Indels in liver APOC3 were analyzed with NGS, and levels of human APOC3 protein in serum were quantified by an electro-chemiluminescence assay run on a Meso Scale Discovery (MSD) system. Results are shown in FIG. 6A and FIG. 6B, respectively. Guide RNAs with stabilizer sequences (SEQ ID NOs: 65, 77-78) and corresponding chemical modifications (SEQ ID NOs: 79-81) were more potent than the guide RNA that did not have a stabilizer sequence (R21969).TABLE 12. Guide RNAs tested in Example 6Example 8. CasPhi.12 L26R-T87G-Y220S-S223P-I471T-D703G is highly specific, generating fewer off target modifications relative to similar variants

[0197] The off-target profiles of several CasPhi. 12 variants were characterized in primary hepatocytes from human donors.

[0198] mRNA253 encoding the CasPhi.12 L26R-T87G-Y220S-S223P-I471T-D703G variant and guide nucleic acid R22529 targeting APOC3 were delivered to cells via LNP. Pooled Amp-Seq (interrogating 355 rhAmpSeq sites) was used to screen for % indel on multiple genes including APOC3.Attorney Docket No.: MABI-051 / 03WO 344183-2344

[0199] Separately, nucleases shown in FIG. 8 and guide nucleic acid R15595 targeting APOC3 (repeat lowercase, spacer uppercase: auagauugcuccuuacgaggagacCAGGGAACUGAAGCCAU (SEQ ID NO: 70)) were delivered to cells via MessengerMAX™.

[0200] In both experiments, the mean specificity ratio was calculated as follows: specificity ratio = on- target editing / (sum of significant off-target indels + on-target indels). The specificity ratio is on scale from 0-1, 0 being worst and 1 being best (high on target and no off targets).

[0201] FIG. 7 shows off target data for CasPhi.12 L26R-T87G-Y220S-S223P-I471T-D703G (mRNA253 encoded protein) and FIG. 8 shows off target data for other CasPhi. 12 variants that include an S526N substitution. FIG. 8 shows that, in general, S526N reduces specificity. FIG. 8 shows that variants with S526N have a specificity score of about 0.3 to about 0.45, whereas variants without S526N have a higher specificity score (0.7 to 0.85). For instance, Nucl5 which encodes L26R-T87G-Y220S- S223P-I471T-S526N-D703G has a specificity score of less than 0.4. FIG. 7 shows that CasPhi.12 L26R-T87G-Y220S-S223P-I471T-D703G (mRNA253 encoded variant, the same as Nucl5 minus S526N) has a particularly high specificity score (0.93 to 0.99).Example 9. CasPhi.12 variants reduce expression of human APOC3 and triglycerides in humanized APOC3 mice with severe plasma hypertriglyceridemia and hypercholesterolemia

[0202] Humanized APOC3 (hAPOC3) mice with severe plasma hypertriglyceridemia and significantly increased plasma cholesterol (B6;CBA-Tg(APOC3)3707Bres / J), were dosed with CasPhi.12 variant mRNA253 (SEQ ID NO: 25), encoding SEQ ID NO: 21; and (2) axi APOC3 guide RNA (R22148, SEQ ID NO: 57). Doses included 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg and 2 mg / kg. An LNP was used to deliver the mRNA and guide RNA. Liver tissue samples were collected from mice at various time points. Levels of human APOC3 protein in serum were quantified by ELISA. Results are shown in FIGS. 9A and 9B, showing a 95% reduction in serum triglycerides (FIG. 9B) and 90% reduction in serum APOC3 protein (FIG. 9A).Example 10. mRNA253 and R22529 reduce serum APOC3 protein by 95% and reduce serum triglycerides by 93%> in hAPOC3 mice.

[0203] Humanized APOC3 (hAPOC3) mice with severe plasma hypertriglyceridemia and significantly increased plasma cholesterol (B6;CBA-Tg(APOC3)3707Bres / J), were intravenously injected with an LNP encapsulating (a) CasPhi.12 variant mRNA253 (SEQ ID NO: 25), encoding SEQ ID NO: 21; and (b) an APOC3 guide RNAs R22528 (SEQ ID NO: 53) and R22529 (SEQ ID NO: 55), at doses 0. 1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, and 1.0 mg / kg. Two different ratios of mRNA to gRNA were tested, 1: 1 and 1:3. Levels of human APOC3 mRNA and protein in serum were quantified by ELISA. Triglyceride levels were also measured. Briefly, triglycerides were hydrolyzed by a combination of microbial lipases to give glycerol and fatty acids. The glycerol is phosphorylated by adenosine triphosphate (ATP) in the presence of glycerol kinase (GK) to produce glycerol-3 -phosphate. The glycerol-3 -phosphate was oxidized by molecular oxygen in the presence of GPO (glycerol phosphateAttorney Docket No.: MABL051 / 03WO 344183-2344 oxidase) to produce hydrogen peroxide (H202) and dihydroxyacetone phosphate. The formed H2O2 reacts with 4-aminophenazone and N,N-bis(4-sulfobutyl)-3, 5 -dimethylaniline, disodium salt (MADB) in the presence of peroxidase (POD) to produce a chromophore, which is read at 660 / 800 nm. The increase in absorbance at 660 / 800 nm is proportional to the triglyceride content of the sample.

[0204] Results are shown in TABLE 13. Five days after injection, mRNA253 and R22529, at a dose of 1 mg / kg, achieved a 95% reduction in serum triglycerides and 93% reduction in serum APOC3 protein.TABLE 13. Percent Reduction of Serum APOC3 mRNA, APOC3 protein and triglycerides in humanized APOC3 mice treated with mRNA253 and R22528 or R22529Example 11. Modification of APOC3 in non-human primates (NHP) with Engineered CasPhi.12 Variant (L26R-I471 T-S223P-D 703G- Y220S)

[0205] Cynomolgus macaques (n=3) received an intravenous infusion of an LNP formulation (5mL / kg) comprising mRNA encoding an engineered variant of CasPhi.12 (mRNA180, SEQ ID NO: 63), and guide RNA R15596 (SEQ ID NO: 58) targeting exon 4 of APOC3. or R17161 (SEQ ID NO:60), targeting exon 3 of APOC3. Animals are dosed at 0.5 mg / kg, 1.0 mg / kg or 2.0 mg / kg at a 1:3 ratio of mRNA to gRNA. Additional animals were similarly treated, but with a guide RNA targeting PCSK9Attorney Docket No.: MABI-051 / 03WO 344183-2344(R14051) having a sequence of: mA*mU*mA*rGrArUrUrGrCrUrCrCrUrUrArCrGrArGrGrArGrArCrGrCrGrCrArGrCrGrGrUrGrGr ArAmG*mG*mU (SEQ ID NO: 64). As a positive control, additional animals were dosed with mRNA encoding SpCas9 and corresponding guide R15779 (SEQ ID NO: 47) at a ratio of 1 : 1 and dose of 2.0 mg / kg.

[0206] The efficacy and tolerability of the mRNA and guide RNA were assessed by measuring % indel in liver samples. Liver biopsies were performed 15-17 days after infusion, and animals were sacrificed 29-30 days after infusion, and tissue samples were collected at both time points. Indels in the NHP APOC3 gene from the collected tissue samples were determined by NGS. Results from animals treated with the 2mg / kg dose are shown in FIG. 10.Example 12. Modification of APOC3 in non-human primates (NHP) with Engineered CasPhi.12 Variant (L26R-T87G-Y220S-S223P-I471T-D703G)

[0207] Male cynomolgus macaques (macaca fascicularis (mf), n=3) received an intravenous infusion of an LNP formulation (5mL / kg) comprising mRNA encoding an engineered variant of CasPhi.12 (mRNA253, SEQ ID NO: 25), and guide RNA R22528 (SEQ ID NO: 53, targets exon 4 of APOC3) or R22529 (SEQ ID NO: 55, targets intron 1 of APOC3). Animals were dosed at 0.5 mg / kg (mpk), 1.0 mg / kg (mpk) or 3.0 mg / kg (mpk) at a 1:3 ratio of mRNA to gRNA.

[0208] A liver biopsy was collected 1 week after injection. Indels in the NHP APOC3 gene of the liver biopsy samples were determined by NGS and relative quantities of APOC3 mRNA (mf= macaca fascicularis) analyzed by dPCR. Initial results are provided in FIG. HA (indel) and FIG. 11B (mRNA). Serum APOC3 protein measured at days 8, 15, and 29 are provided in FIG. 12. FIG. 13 shows that reduction of serum APOC3 protein by mRNA253 and R22529 persists after 89 days. Protein levels were assessed with Western blot.

[0209] The efficacy and tolerability of the mRNA and guide RNA are further assessed in additional samples and time points. Animals are sacrificed about 12 weeks or about 24 weeks after IV infusion and tissue samples (including liver) are collected. Indels in the NHP APOC3 gene from the collected tissue samples are determined by NGS. Liver and / or serum levels of APOC3 protein may be quantified. Serum cholesterol and triglycerides may also be quantified. Other factors quantified or analyzed may include cytokines, liver enzymes, and LDL-C.

[0210] FIG. 14A and FIG.14B show % indel APOC3 (FIG. 14A) and relative levels of APOC3 mRNA (FIG. 14B) in liver (biopsy) samples 1, 8, 20, and 24 weeks post administration of mRNA253 and R22529 to assess genome editing efficiency in liver. FIG. 14C shows relative levels of APOC3 protein in liver samples 1, 2, 4, 8, and 12 weeks post administration. 1 week post-dose, both dose levels achieved robust and comparable mean liver editing efficiencies- 65% for the 1.0 mg / kg group and 67% for the 3.0 mg / kg group. These editing levels remained stable or showed a slight increase at 8, 20, and 24 weeks post-dose, indicating durable and sustained editing over time. In contrast, PBS- treatedAttorney Docket No.: MABI-051 / 03WO 344183-2344 animals showed no detectable editing, confirming the specificity and effectiveness of mRNA253 and R22529.

[0211] Liver function tests were performed at multiple time points during this study, measuring ALT and total bilirubin. Results are shown in FIGS. 15A-15D. Transient increases were observed in ALT (FIG. 15A) at the 3 mpk dose with both guides, although less pronounced with R22529 as compared to R22528 (see arrows), with levels returning to baseline by Day 8. FIG. 15C and FIG. 15D shows peak ALT levels (FIG. 15C) and peak total bilirubin levels (Fig. 15D), which were closer to levels seen with vehicle for R22529 relative to R22528.Example 13. Modification of APOC3 in female non-human primates (NHP) with Engineered CasPhi.12 Variant (L26R-T87G-Y220S-S223P-I471T-D703G)

[0212] Female cynomolgus macaques (macaco fascicularis (mf), n=3) and male cynomogus macaques (n=3) received an intravenous infusion of an LNP formulation comprising mRNA encoding an engineered variant of CasPhi. 12 (mRNA253, SEQ ID NO: 25), and guide RNA R22529 (SEQ ID NO: 55). Animals were dosed at 1.0 mg / kg or 3.0 mg / kg at a 1:3 ratio of mRNA to gRNA.

[0213] A liver biopsy was collected 1 week after injection. Indels in the NHP APOC3 gene of the liver biopsy samples were determined by NGS. Results are provided in FIG. 16.

[0214] FIG. 16 shows % indel APOC3 in liver (biopsy) samples 1 week post administration of mRNA253 and R22529 to assess genome editing efficiency in livers of male and female cynomolgus macaques. 1 week post-dose, both dose levels achieved robust and comparable mean liver editing efficiencies- -60-65% for the 1.0 mg / kg group and -70% for the 3.0 mg / kg group. FIG. 17 shows that there was a corresponding decrease in liver APOC3 mRNA (females shown), remaining stable up to 4 weeks post-dose, indicating durable and sustained editing over time. In contrast, PBS- treated animals showed no detectable editing, confirming the specificity and effectiveness of mRNA253 and R22529.Example 14. Additional engineered CasPhi.12 variants demonstrate gene editing in mammalian cells

[0215] HEK293T cells were transfected with plasmids encoding various CasPhi.12 engineered variants and APOC3 targeting guide nucleic acid R15596 (AUAGAUUGCUCCUUACGAGGAGACCCUGAAAGACUACUGGA (SEQ ID NO: 49) Briefly, lOOng total DNA (50ng nuclease / 50ng guide) was transfected into HEK293T cells with TransIT and harvested 72. hours post transfection. Indel levels were analyzed via NGS. The CasPhi engineered variants are described in TABLE 14.

[0216] HepG2 cells were transfected with mRNA encoding various CasPhi .12 engineered variants and APOC3 targeting guide R15596. Briefly, lOOng (1:3 nuclease / guide) or 400ng ( 1:3 nuclease / guide) total DNA was transfected into HepG2 cells with MessengerMax and harvested 48 hours post transfection. Indel levels were analyzed via NGS.Attorney Docket No.: MABI-051 / 03WO 344183-2344

[0217] Results from both experiments are provided in TABLE 14.TABLE 14. CasPhi.12 Engineered Variant Indel Formation

Claims

Attorney Docket No.: MABI-051 / 03WO 344183-2344CLAIMSWhat is claimed is:

1. A composition comprising: a) a messenger RNA (mRNA) comprising or consisting of a nucleotide sequence of SEQ ID NO: 25; and b) a guide nucleic acid comprising or consisting of a nucleotide sequence of SEQ ID NO: 55;2. A composition comprising: a) an mRNA comprising a nucleotide sequence of SEQ ID NO: 23; and b) a guide nucleic acid comprising or consisting of a nucleotide sequence selected from SEQ ID NOS: 52-553. A composition comprising: a) an mRNA encoding an amino acid sequence of SEQ ID NO: 21; and b) a guide nucleic acid comprising or consisting of a nucleotide sequence selected from SEQ ID NOS: 52-554. A composition comprising: a) an mRNA comprising or consisting of a nucleotide sequence selected from SEQ ID NO: 23 and SEQ ID NO: 25; and b) a guide nucleic acid comprising or consisting of a nucleotide sequence selected from: i) mG*mU*mC*rUrUrGrArGrArG*mG*mA*mA*mArCrUrCrUrCrArArGrArC*mU* rArGrArUrUrGrCrUrCrCrUrUrArCrGrArGrGrArGrArCrCrCrUrGrArArArGrArCrU rArCrUrGrGmA*mG*mC (SEQ ID NO: 53), and ii) mG*mU*mC*rUrUrGrArGrArG*mG*mA*mA*mArCrUrCrUrCrArArGrArC*mU* rArGrArUrUrGrCrUrCrCrUrUrArCrGrArGrGrArGrArCrCrCrCrUrCrCrCrCrArGrA rGrGrGrCmA*mU*mU (SEQ ID NO: 55) wherein r represents an unmodified nucleotide, m represents a 2’-0Me modified nucleotide, and * represents a phosphorothioate bond.

5. The composition of any one of claims 1-4, comprising a lipid nanoparticle (LNP), wherein the mRNA and the guide nucleic acid are encapsulated in the LNP.

6. The composition of any one of claims 1-5, wherein the mRNA comprises or is linked to the following chemical structure:Attorney Docket No.: MABI-051 / 03WO 344183-23447. The composition of claim 6. wherein the chemical structure is located at or linked to the 5’ end of the mRNA.

8. A pharmaceutical composition comprising the composition of any one of claims 1-7, and a pharmaceutical acceptable excipient or carrier.

9. A method of modifying APOC3, comprising contacting APOC3, or a cell comprising APOC3, with the composition of any one of claims 1-8, wherein (i) an effector protein encoded by the mRNA and (ii) the guide RNA form a ribonucleoprotein (RNP) complex that binds to a target nucleotide sequence of APOC3.

10. The method of claim 9, wherein modifying APOC3 reduces the expression of APOC3, optionally wherein modifying APOC3 permanently reduces the expression of APOC3.

11. The method of claim 9 or 10, wherein modifying APOC3 comprises cleaving both strands of APOC3.

12. The method of any one of claims 9-11, comprising modifying APOC3 in vivo.

13. The method of claim 12, comprising modifying APOC3 in a liver of a subject.

14. A method of lowering triglycerides in a human subject with hypertriglyceridemia, comprising administering the composition of any one of claims 1-8 to the human subject.

15. A method of treating or preventing a disease comprising chylomicronemia in a human subject in need thereof, comprising administering the composition of any one of claims 1-8 to the human subject.

16. The method of claim 15, wherein the disease comprises persistent chylomicronemia.

17. The method of claim 15, wherein the disease comprises severe hypertriglyceridemia.

18. The method of claim 15, wherein the disease comprises familial chylomicronemia syndrome (FCS).

19. A cell, or population of cells, comprising the composition of any one of claims 1-8, or modified by the method of any one of claims 9-18.Attorney Docket No.: MABI-051 / 03WO 344183-234420. An engineered polypeptide or a nucleic acid encoding the same, wherein the engineered polypeptide comprises or consists of an amino acid sequence that is identical to SEQ ID NO: 19, with the exception of the following amino acid substitutions: L26R, T87G, Y220S, S223P, 147 IT, and D703G, optionally wherein the nucleic acid is DNA or RNA.

21. A messenger RNA (mRNA) encoding an engineered effector protein, wherein the engineered protein comprises or consists of an amino acid sequence that is identical to SEQ ID NO: 19, with the exception of the following amino acid substitutions: L26R, T87G, Y220S, S223P, 147 IT, and D703G.

22. An engineered polypeptide or a nucleic acid encoding the same, wherein the engineered polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 21, optionally wherein the nucleic acid comprises DNA, RNA or a combination thereof23. An engineered guide nucleic acid comprising or consisting of a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from SEQ ID NOS: 52-55.

24. An engineered guide nucleic acid comprising from 5' to 3' a) a stabilizer sequence, b) a repeat sequence, and c) a spacer sequence, wherein the stabilizer sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOS: 65 and 77-78, and wherein the repeat sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOS: 5-11.

25. The engineered guide nucleic acid of claim 24, wherein the spacer sequence comprises or consists of SEQ ID NO: 5126. A composition comprising the engineered guide nucleic acid of claim 24 or 25, and an effector protein or nucleic acid encoding the same, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 19.

27. An engineered guide nucleic acid according to the following formula: mN*mN*mN*rNrNrNrNrNrNrN*mN*mN*mN*mNrNrNrNrNrNrNrNrNrNrN*mN*rNrNrN rNrUrGrCrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrArArArArArArArArArArArArArArAmA* mN*mN, wherein N is a nucleotide selected from A, G, C, and U or T, wherein * represents a phosphorothioate linkage, wherein m represents a 2’-OMe sugar modification, wherein r represents an unmodified ribonucleotide, and wherein italicized letters indicate a spacer sequence.

28. An engineered guide nucleic acid according to the following formula: mN*mN*mN*rNrNrNrNrNrNrN*mN*mN*mN*mNrNrNrNrNrNrNrNrNrNrN*mN*rNrNrN rNrUrGrCrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrAr„mA*mA*mA, wherein N is a nucleotide selected from A, G, C, and U or T, wherein * represents a phosphorothioate linkage, wherein m represents a 2’-OMe sugar modification, wherein r represents an unmodified ribonucleotide, wherein italicized letters indicate a spacer sequence, and wherein n = 13 to 19.Attorney Docket No.: MABI-051 / 03WO 344183-234429. A composition for use in treating or preventing a disease in a human subject in need thereof, comprising administering the composition to the human subject, wherein the composition is selected from any one of claims 1-7.

30. The composition of claim 29, wherein the disease is selected from cardiovascular disease, persistent chylomicronemia, familial chylomicronemia syndrome, and hypertriglyceridemia.

31. A method of producing (a) a messenger RNA (mRNA) comprising or consisting of a nucleotide sequence of SEQ ID NO: 25; and (b) a guide nucleic acid comprising or consisting of a nucleotide sequence of SEQ ID NO: 55, or a combination thereof.

32. The method of claim 31, comprising encapsulating the mRNA and the guide nucleic acid in an LNP.

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