Biallelic knockout of ANGPTL3

Abstract

An RNA molecule comprising a guide sequence portion consisting of 17 to 50 nucleotides and containing 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a composition, method and use thereof.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 492,808, filed Mar. 29, 2023; U.S. Provisional Application No. 63 / 385,944, filed Dec. 2, 2022; and U.S. Provisional Application No. 63 / 368,926, filed Jul. 20, 2022, the contents of each of which are incorporated herein by reference.

[0002] Throughout this application, various publications are referenced, including those within parentheses. The entire disclosure of all publications mentioned in this application is incorporated herein by reference to supplement the technology that can be used in the present invention and the technology related to the present invention.

[0003] Sequence Listing References This application was created on Jul. 20, 2023, in the form of an IBM-PC machine using an operating system compatible with MS-Windows® and was filed on Jul. 20, 2023, as part of this application. It is an XML file with a size of 18,030,806 bytes, and the nucleotide sequences in the file named "230720_91943-A-PCT_Sequence_Listing_AWG.xml" are incorporated herein by reference.

Background Art

[0004] The treatment of several lipid metabolism disorders, including familial hypercholesterolemia (FH), hypertriglyceridemia, and hyperlipidemia, may benefit from gene-targeted therapies. More specifically, familial hypercholesterolemia (FH) is a hereditary disease mainly caused by mutations in the low-density lipoprotein receptor (LDLR), which leads to elevated cholesterol and early onset of cardiovascular diseases. Hypertriglyceridemia is a lipid metabolism disorder characterized by high blood triglyceride levels. This can cause atherosclerosis and other life-threatening abnormalities even when cholesterol levels are normal. Hyperlipidemia is another example of a lipid metabolism disorder, characterized by elevated levels of any or all of the lipids and / or lipoproteins in the blood. A simple and specific gene therapy approach can improve treatment options for each of these abnormalities. Furthermore, since high triglyceride levels suppress insulin release, such an approach may be available for the treatment of type II diabetes symptoms.

[0005] Angiopoietin-like protein 3 (ANGPTL3) is a secreted protein that plays a role in regulating plasma lipid levels. After secretion, ANGPTL3 binds to lipoprotein lipase (LPL) and endothelial lipase (LIPG) and hydrolyzes triglycerides and phospholipids. Thus, in healthy individuals, ANGPTL3 functions as an inhibitor of lipoprotein lipase.

[0006] Loss-of-function mutations in the ANGPTL3 gene resulting in ANGPTL3 deficiency lead to a condition called "familial combined hypobetalipoproteinemia" (FHBL2, OMIM #605019), which is characterized by low concentrations of all classes of major lipoproteins in the blood. In fact, in families with loss-of-function mutations in ANGPTL3, it has been found that the levels of LDL, VLDL, and triglycerides are low. Thus, knockout of wild-type ANGPTL3 may reduce plasma lipoprotein concentrations after decreasing ANGPTL3 expression and may be a target for the treatment of combined hyperlipidemia. Summary of the Invention

[0007] Disclosed is a method for treating disorders including hypertriglyceridemia, hyperlipidemia, and hypercholesterolemia by knocking out the ANGPTL3 gene to increase lipid metabolism. Accordingly, knockout of both alleles of the ANGPTL3 gene in liver cells (e.g., hepatocytes) as described in this specification can be used for the treatment, inhibition, prevention, and / or amelioration of any of hypertriglyceridemia, hyperlipidemia, and hypercholesterolemia (e.g., familial hypercholesterolemia (FH)).

[0008] This disclosure provides a method for inactivating an allele of the angiopoietin-like 3 (ANGPTL3) gene in a cell, the method comprising: a CRISPR nuclease, or a nucleotide molecule encoding a CRISPR nuclease; and, an RNA molecule comprising a guide sequence portion having 17 to 50 nucleotides, or a DNA molecule encoding an RNA molecule, introducing into the cell a composition comprising the same, wherein a complex of the CRISPR nuclease and the RNA molecule double-strand breaks the allele of the ANGPTL3 gene.

[0009] In an aspect of this invention, there is provided an RNA molecule consisting of 17 to 50 consecutive nucleotides and comprising a guide sequence portion having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347.

[0010] In some aspects of this invention, there is provided a composition comprising an RNA molecule consisting of 17 to 50 consecutive nucleotides and comprising a guide sequence portion having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347 and a CRISPR nuclease.

[0011] In some aspects of this invention, a method for inactivating the ANGPTL3 allele within a cell is provided, the method comprising delivering to the cell a composition comprising an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease.

[0012] In some aspects, the cell is a liver cell. In some aspects, the cell is a hepatocyte. In some aspects, the cell is a stem cell. In some aspects, delivery to the cell is performed in vivo, ex vivo or in vitro. In some aspects, delivery to the cell is performed by in vivo delivery of a lentivirus, adeno-associated virus (AAV) or nanoparticle to the liver. In some aspects, the method is performed ex vivo and the cells are provided / explanted from an individual patient. In some aspects, the method further comprises introducing into an individual patient cells having a modified / knocked-out ANGPTL3 allele.

[0013] In some aspects of this invention, a method for treating and / or preventing hypertriglyceridemia, hyperlipidemia or hypercholesterolemia is provided, the method comprising delivering to the cells of a subject having or at risk of having hypertriglyceridemia, hyperlipidemia or hypercholesterolemia a composition comprising an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease.

[0014] In some aspects of the present invention, provided is the use of a composition comprising an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease, for inactivating the ANGPTL3 allele intracellularly, comprising delivering to the cell a composition comprising an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease.

[0015] In an aspect of the present invention, provided is a medicament comprising an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease, for use in inactivating the ANGPTL3 allele intracellularly, wherein the medicament is administered by delivering to the cell a composition comprising an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease.

[0016] In some aspects of the present invention, provided is the use of a composition comprising an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease, in the treatment, amelioration or prevention of hypertriglyceridemia, hyperlipidemia or hypercholesterolemia, wherein the use comprises delivering to the cells of a subject suffering from or at risk of suffering from hypertriglyceridemia, hyperlipidemia or hypercholesterolemia, a composition comprising an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease.

[0017] In some aspects, the method is performed in vivo and the cells are liver cells (e.g., hepatocytes).

[0018] In some embodiments, the composition is delivered to cells in vivo by lipid nanoparticle delivery, lentiviral delivery, or AAV delivery.

[0019] In some embodiments of the present invention, provided is a medicament for use in the treatment, amelioration, or prevention of hypertriglyceridemia, hyperlipidemia, or hypercholesterolemia, the medicament comprising an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides containing nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease, and the medicament is administered by delivering to cells of a subject suffering from or at risk of suffering from hypertriglyceridemia, hyperlipidemia, or hypercholesterolemia, a composition comprising an RNA molecule consisting of 17 to 50 consecutive nucleotides and comprising a guide sequence portion having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease.

[0020] In some embodiments of the present invention, provided is a kit for inactivating the ANGPTL3 allele in cells, the kit comprising an RNA molecule consisting of 17 to 50 consecutive nucleotides and comprising a guide sequence portion having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, a CRISPR nuclease and / or a tracrRNA molecule; and instructions for delivering the RNA molecule; the CRISPR nuclease and / or the tracrRNA to cells.

[0021] In some aspects of the present invention, there is provided a kit for treating hypertriglyceridemia, hyperlipidemia or hypercholesterolemia in a subject, the kit comprising an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, a CRISPR nuclease and / or a tracrRNA molecule; and instructions for delivering the RNA molecule; the CRISPR nuclease and / or the tracrRNA to cells of a subject suffering from or at risk of suffering from hypertriglyceridemia, hyperlipidemia or hypercholesterolemia.

Brief Description of the Drawings

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Figure 1A-1B

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Table A

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Table B

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Figure 2A-2D

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Table C

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Figure 3A-3C

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Table D

Mode for Carrying Out the Invention

[0029] Detailed Description Unless otherwise defined, all technical and / or scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of aspects of this invention, representative methods and / or materials are described below. In case of conflict, this specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0030] The term "one" is understood to refer to "one or more" of the listed components. The use of the singular form is clearly understood by those skilled in the art to include the plural form unless specifically stated otherwise. Thus, the terms "one" and "at least one" have the same meaning in this application.

[0031] For the purposes of better understanding this disclosure and without in any way limiting the scope of the disclosure, unless otherwise indicated, all numbers and other numerical values used in this specification and the claims to represent amounts, percentages, or ratios are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0032] Unless otherwise explained, adjectives such as "substantially" and "about" modifying a condition or relationship characteristic of an aspect of this invention are understood to mean that the condition or property is defined within an acceptable range for the operation of the aspect for its intended application. Unless otherwise indicated, the term "or" as used in this specification and the claims is to be construed as inclusive rather than exclusive or, and indicates at least one, or any combination, of the items it conjoins.

[0033] In this specification and the claims, the verbs "comprise", "comprising", "include" and "including" and their conjugations are used to indicate that the object of the verb does not necessarily enumerate every component, element or part of the subject of the verb. Other terms in this specification are intended to be defined by their well-known meanings in the relevant art.

[0034] In some embodiments of the invention, DNA nucleases are utilized to cleave DNA at a target site and induce a cellular repair mechanism, such as, but not limited to, non-homologous end joining (NHEJ). In classical NHEJ, the two ends of a double-strand break (DSB) site are ligated in a rapid but inaccurate manner (i.e., frequently resulting in mutations in the DNA at the break site in the form of small insertions or deletions).

[0035] As used herein, the term "modified cell" refers to a cell in which an RNA molecule and a CRISPR nuclease complex cause a double-strand break as a result of hybridization with a target sequence, i.e., on-target hybridization.

[0036] In this specification, the term "targeting array" or "targeting molecule" refers to a nucleotide sequence or molecule that includes a nucleotide sequence capable of hybridizing to a specific target sequence. For example, a targeting array has a nucleotide sequence that is at least partially complementary to the target sequence along its length. A targeting array or targeting molecule may be part of an RNA molecule that can form a complex with a CRISPR nuclease, either alone or in combination with other RNA molecules, and the targeting array serves as the targeting portion of the CRISPR complex. When a molecule having a targeting array is present simultaneously with a CRISPR molecule, the RNA molecule can direct the CRISPR nuclease to a specific target sequence, either alone or in combination with one or more other RNA molecules (e.g., a tracrRNA molecule). By way of non-limiting example, the guide sequence portion of a CRISPR RNA molecule or a single-guide RNA molecule may serve as a targeting molecule. Each possibility is a separate aspect. The targeting array can be custom-designed to target a desired sequence.

[0037] In this specification, the term "target, direct (to a target)" refers to preferentially hybridizing the targeting sequence of a targeting molecule to a nucleic acid having a target nucleotide sequence. It is understood that the term "target, direct (to a target)" includes various hybridization capabilities, such that a nucleic acid having a target nucleotide sequence is preferentially targeted, but off-target hybridization may also occur in addition to on-target hybridization. When an RNA molecule targets a sequence, it is understood that the complex of the RNA molecule and the CRISPR nuclease molecule targets that sequence for nuclease activity.

[0038] The "guide sequence portion" of an RNA molecule refers to a nucleotide sequence that can hybridize with a specific target DNA sequence. For example, the guide sequence portion has a nucleotide sequence that is partially or completely complementary to the target DNA sequence along its length. In some embodiments, the length of the nucleotides in the guide sequence portion is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, or about 17-50, 17-49, 17-48, 17-47, 17-46, 17-45, 17-44, 17-43, 17-42, 17-41, 17-40, 17-39, 17-38, 17-37, 17-36, 17-35, 17-34, 17-33, 17-31, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24, 17-22, 17-21, 18-25, 18-24, 18-23, 18-22, 18-21, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-22, 18-20, 20-21, 21-22 or 17-20. In some embodiments, the entire length of the guide sequence portion is completely complementary to the target DNA sequence along its length. The guide sequence portion may be a part of an RNA molecule that can form a complex with a CRISPR nuclease, and the guide sequence portion serves as the DNA targeting portion of the CRISPR complex. When an RNA molecule having a guide sequence portion is present simultaneously with a CRISPR molecule, either alone or in combination with one or more other RNA molecules (e.g., a tracrRNA molecule), the RNA molecule can direct the CRISPR nuclease to a specific target DNA sequence. Thus, the CRISPR complex can be formed by directly binding an RNA molecule having a guide sequence portion to a CRISPR nuclease, or by binding an RNA molecule having a guide sequence portion and one or more other RNA molecules to a CRISPR nuclease. Each possibility is a separate embodiment. The guide sequence portion can be custom-designed to target a desired sequence. Thus, a molecule containing the "guide sequence portion" is a type of targeting molecule.In some embodiments, the guide sequence portion comprises the same sequence as the guide sequence portions described in this specification (e.g., the guide sequences shown in any of SEQ ID NOs: 1-20347), or a different sequence that is 1, 2, 3, 4, or 5 nucleotides or less. Each possibility is a separate embodiment. In some of these embodiments, the guide sequence portion comprises the same sequence as the sequences shown in any of SEQ ID NOs: 1-20347. Throughout this application, the terms “guide molecule,” “RNA guide molecule,” “guide RNA molecule,” and “gRNA molecule” are synonymous with molecules that contain a guide sequence portion.

[0039] As used in this specification, the term “undifferentiated” refers to the guide sequence portion of an RNA molecule that targets a specific DNA sequence common to all alleles of a gene.

[0040] In embodiments of the invention, the RNA molecule comprises a guide sequence portion consisting of 17-50 contiguous nucleotides, including 17-22 nucleotides within the sequence shown in any of SEQ ID NOs: 1-20347.

[0041] The RNA molecule and / or the guide sequence portion of the RNA molecule may have modified nucleotides. Representative modifications to nucleotides / polynucleotides may be synthetic and may include polynucleotides having nucleotides with bases other than the naturally occurring adenine, cytosine, thymine, uracil, or guanine bases. Modifications to polynucleotides may include synthetic, non-naturally occurring nucleosides, such as polynucleotides having locked nucleic acids. Modifications to polynucleotides may be utilized to increase or decrease the stability of the RNA. An example of a modified polynucleotide is mRNA having 1-methylpseudouridine. Examples of modified polynucleotides and their uses are described in U.S. Patent No. 8,278,036, International Publication No. 2015 / 006747, and Weissman and Kariko (2015), which are incorporated herein by reference.

[0042] In this specification, "consecutive nucleotides" indicated by SEQ ID NO. refer to the nucleotides of the sequence in the order indicated by the SEQ ID NO. without any intervening nucleotides.

[0043] In an embodiment of the present invention, the guide sequence portion may be 50 nucleotides in length and may contain 20 to 22 consecutive nucleotides within the sequence shown in any of SEQ ID NOs: 1 to 20347. In an embodiment of the present invention, the guide sequence portion may be less than 22 nucleotides in length. For example, in an embodiment of the present invention, the guide sequence portion may be 17, 18, 19, 20, or 21 nucleotides in length. In such an embodiment, the guide sequence portion may consist of 17, 18, 19, 20, or 21 nucleotides, respectively, in the sequence of 17 to 22 consecutive nucleotides shown in any of SEQ ID NOs: 1 to 20347. For example, the guide sequence portion of the sequence of 17 consecutive nucleotides shown in SEQ ID NO: 20348 may be any of the following nucleotide sequences (the nucleotides removed from the consecutive sequence are strikethrough):

[0044]

Chemical Formula

[0045] In an embodiment of the present invention, the nucleotide length of the guide sequence portion may exceed 20. For example, in an embodiment of the present invention, the nucleotide length of the guide sequence portion may be 21, 22, 23, 24, or 25. In such an embodiment, the guide sequence portion may include 17 to 50 nucleotides having a sequence of 20, 21, or 22 consecutive nucleotides shown in any of SEQ ID NOs: 1 to 20347, and nucleotides that are completely complementary to the nucleotides or their sequences adjacent to the 3'-end, 5'-end, or both ends of the target sequence.

[0046] In aspects of this invention, a CRISPR nuclease and an RNA molecule comprising a guide sequence portion bind to a target DNA sequence to form a CRISPR complex that cleaves the target DNA sequence. The CRISPR nuclease (e.g., Cpf1) may form a CRISPR complex comprising the CRISPR nuclease and the RNA molecule without an additional tracrRNA molecule. Alternatively, the CRISPR nuclease (e.g., Cas9) may form a CRISPR complex between the CRISPR nuclease, the RNA molecule, and the tracrRNA molecule. The guide sequence portion having a nucleotide sequence capable of hybridizing to a specific target DNA sequence and the sequence portion involved in CRISPR nuclease binding (e.g., the tracrRNA sequence portion) may be present in the same RNA molecule. Alternatively, the guide sequence portion may be present in one RNA molecule, and the sequence portion involved in CRISPR nuclease binding (e.g., the tracrRNA portion) may be present in another RNA molecule. A single RNA molecule comprising a guide sequence portion (e.g., a DNA-targeting RNA sequence) and at least one CRISPR protein-binding RNA sequence portion (e.g., a tracrRNA sequence portion) can form a complex with the CRISPR nuclease and serve as a DNA-targeting molecule. In some aspects, a first RNA molecule comprising a DNA-targeting RNA portion comprising a guide sequence portion and a second RNA molecule comprising a CRISPR protein-binding RNA sequence interact by base pairing to form an RNA complex that directs the CRISPR nuclease to a DNA target site, or fuse with each other to form an RNA molecule that forms a complex with the CRISPR nuclease and directs the CRISPR nuclease to a DNA target site.

[0047] In aspects of the invention, an RNA molecule comprising a guide sequence portion may further comprise the sequence of a tracrRNA molecule. Such aspects may be designed as synthetic fusions of the guide portion of the RNA molecule and trans-activating crRNA (tracrRNA) (see Jinek et al., 2012). In such aspects, the RNA molecule is a single guide RNA (sgRNA) molecule. Some aspects of the invention may also form CRISPR complexes that utilize an individual tracrRNA molecule and an individual RNA molecule comprising a guide sequence portion. In such aspects, the tracrRNA may hybridize to the RNA molecule via base pairing, which may be advantageous in certain applications of the invention described herein.

[0048] The term "tracr mate sequence" refers to a sequence that is sufficiently complementary to a tracrRNA molecule to hybridize to the tracrRNA via base pairing and promote the formation of a CRISPR complex (see U.S. Patent No. 8,906,616). In aspects of the invention, the RNA molecule may further comprise a tracr mate sequence portion.

[0049] In the present invention, "gene" includes a DNA region encoding a gene product and all DNA regions controlling the production of the gene product, and the sequence of the control region may or may not be adjacent to the coding sequence and / or the transcription sequence. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translational control sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0050] "Eukaryotic" cells include, but are not limited to, fungal cells (e.g., yeast), plant cells, animal cells, mammalian cells, and human cells.

[0051] In this specification, the term "nuclease" refers to an enzyme capable of cleaving the phosphodiester bond between nucleotide subunits of nucleic acids. The nuclease may be isolated from a natural product or may be derived from a natural product. The natural product may be a living organism. Alternatively, the nuclease may be a modified or synthetic protein that retains phosphodiester bond cleavage activity. Modification of genes can be achieved using nucleases (e.g., CRISPR nucleases).

[0052] In an aspect of this invention, there is provided an RNA molecule comprising a guide sequence portion (e.g., a targeting sequence) having a nucleotide sequence that is fully or partially complementary to a target sequence of the ANGPTL3 gene. In some aspects, the guide sequence portion is fully or partially complementary to a target sequence located within an exon of the ANGPTL3 gene or within 30 nucleotides upstream or downstream thereof. In some aspects, the guide sequence portion of the RNA molecule consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 nucleotides, or more than 26 nucleotides. In some aspects, the guide sequence portion is configured to direct a CRISPR nuclease to a target sequence and form a complex therewith, such that the CRISPR nuclease produces a double-strand break or a single-strand break within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides from the ANGPTL3 target site. In some aspects, the cleavage enables nonsense-mediated decay of the ANGPTL3 gene. In some aspects, the RNA molecule is a guide RNA molecule such as a crRNA molecule or a single-guide RNA molecule.

[0053] In some aspects, the target sequence of an allele of the ANGPTL3 gene is modified (e.g., by introduction of NHEJ-mediated indels (e.g., insertions or deletions)) to reduce or eliminate the expression of the gene product encoded by the allele of the ANGPTL3 gene. In some aspects, the reduction or elimination of expression is by nonsense-mediated mRNA decay, e.g., by a premature stop codon. In some aspects, the reduction or elimination of expression is by expression of a truncated form of the ANGPTL3 gene product.

[0054] In aspects of this invention, there is provided an RNA molecule comprising a guide sequence portion (e.g., a targeting sequence) having a nucleotide sequence that is fully or partially complementary to a target sequence located within or near the ANGPTL3 gene. In some aspects, the guide sequence portion is complementary to a target sequence located from 30 base pairs upstream to 30 base pairs downstream of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the ANGPTL3 gene. In some aspects, the guide sequence portion is complementary to a target sequence located from 50 base pairs upstream to 50 base pairs downstream of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the ANGPTL3 gene. Each possibility is a separate aspect. In some aspects, the target sequence of the ANGPTL3 gene is modified (e.g., by introduction of NHEJ-mediated insertion or deletion) to reduce or abolish the expression of the gene product encoded by the ANGPTL3 gene. In some aspects, the reduction or abolition of expression is by nonsense mutation-mediated mRNA degradation. In some aspects, the guide sequence portion of the RNA molecule consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 nucleotides, or more than 26 nucleotides. In some aspects, the guide sequence portion is configured to direct a CRISPR nuclease to the target sequence and, by the CRISPR nuclease complexed therewith, to cause double-strand breaks and single-strand breaks within 500, 400, 300, 200, 100, 50, 25, or 10 nucleotides of the ANGPTL3 target site. In some aspects, the cleavage enables nonsense mutation-mediated degradation of the ANGPTL3 gene. In some aspects, the RNA molecule is a guide RNA molecule such as a crRNA molecule or a single guide RNA molecule.

[0055] In some embodiments, the guide sequence portion is complementary to a target sequence located 30 base pairs upstream to 30 base pairs downstream of the exon of the ANGPTL3 gene. In some embodiments, the guide sequence portion is complementary to a target sequence located 50 base pairs upstream to 50 base pairs downstream of the exon of the ANGPTL3 gene. In some embodiments, the guide sequence portion is complementary to a target sequence located 30 base pairs upstream to 30 base pairs downstream of the exon of the ANGPTL3 gene.

[0056] In some embodiments, the exon is exon 1, and the guide sequence portion includes 17 to 22 nucleotides of a sequence shown in any of SEQ ID NOs: 6333 to 9188 or a sequence that is the same as or differs by 3 nucleotides or less in the same sequence.

[0057] In some embodiments, the exon is exon 2, and the guide sequence portion includes 17 to 22 nucleotides of a sequence shown in any of SEQ ID NOs: 9189 to 9978 or a sequence that is the same as or differs by 3 nucleotides or less in the same sequence.

[0058] In some embodiments, the exon is exon 3, and the guide sequence portion includes 17 to 22 nucleotides of a sequence shown in any of SEQ ID NOs: 9979 to 10786 or a sequence that is the same as or differs by 3 nucleotides or less in the same sequence.

[0059] In some embodiments, the exon is exon 4, and the guide sequence portion includes 17 to 22 nucleotides of a sequence shown in any of SEQ ID NOs: 10787 to 11633 or a sequence that is the same as or differs by 3 nucleotides or less in the same sequence.

[0060] In some embodiments, the exon is exon 5, and the guide sequence portion includes 17 to 22 nucleotides of a sequence shown in any of SEQ ID NOs: 11634 to 12384 or a sequence that is the same as or differs by 3 nucleotides or less in the same sequence.

[0061] In some embodiments, the exon is exon 6, and the guide sequence portion comprises 17 to 22 nucleotides that are the same as the sequence shown in any of SEQ ID NOs: 12385 to 14057 or sequences that differ by 3 nucleotides or less within the same sequence.

[0062] In some embodiments, the exon is exon 7, and the guide sequence portion comprises a sequence that is the same as or differs by 3 nucleotides or less from the sequence shown in any of SEQ ID NOs: 1 to 6332 and 14058 to 15412.

[0063] In aspects of the invention, there is provided a method for inactivating at least one allele of the angiopoietin-like 3 (ANGPTL3) gene in a cell, the method comprising: at least one CRISPR nuclease, or a nucleotide molecule encoding a CRISPR nuclease; and, introducing into the cell a composition comprising an RNA molecule comprising a guide sequence portion, or a DNA molecule encoding an RNA molecule, wherein the complex of the CRISPR nuclease and the RNA molecule double-strand breaks at least one allele of the ANGPTL3 gene, wherein the guide sequence portion of the RNA molecule consists of 17 to 50 contiguous nucleotides.

[0064] In some embodiments, the RNA molecule comprising a guide sequence portion is a crRNA molecule or an sgRNA molecule. In some embodiments, the RNA molecule is a crRNA molecule, and the composition further comprises a tracrRNA molecule that forms a complex with the crRNA molecule.

[0065] In some embodiments, the composition is introduced into a cell of a subject or a cell in culture.

[0066] In some embodiments, the cell is a cell of the liver, preferably a hepatocyte. In some embodiments, the cell is a stem cell.

[0067] In some embodiments, the CRISPR nuclease and the RNA molecule are introduced into the cell substantially simultaneously or at different times.

[0068] In some embodiments, alleles of the ANGPTL3 gene in cells undergo insertion or deletion mutations.

[0069] In some embodiments, the insertion or deletion mutation creates a premature stop codon.

[0070] In some embodiments, inactivation results in a truncated protein encoded by the inactivated allele.

[0071] In some embodiments, the guide sequence portion is complementary to a target sequence located from 50 base pairs upstream to 50 base pairs downstream of the exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the ANGPTL3 gene.

[0072] In some embodiments, the guide sequence portion is complementary to a target sequence located from 30 base pairs upstream to 30 base pairs downstream of an exon of the ANGPTL3 gene, and a) the exon is exon 1, and the guide sequence portion comprises 17-22 nucleotides in the same sequence as shown in any of SEQ ID NOs: 6333-9188 or in a sequence that differs by 3 nucleotides or less; b) the exon is exon 2, and the guide sequence portion comprises 17-22 nucleotides in the same sequence as shown in any of SEQ ID NOs: 9189-9978 or in a sequence that differs by 3 nucleotides or less; c) the exon is exon 3, and the guide sequence portion comprises 17-22 nucleotides in the same sequence as shown in any of SEQ ID NOs: 9979-10786 or in a sequence that differs by 3 nucleotides or less; d) the exon is exon 4, and the guide sequence portion comprises 17-22 nucleotides in the same sequence as shown in any of SEQ ID NOs: 10787-11633 or in a sequence that differs by 3 nucleotides or less; e) the exon is exon 5, and the guide sequence portion comprises 17-22 nucleotides in the same sequence as shown in any of SEQ ID NOs: 11634-12384 or in a sequence that differs by 3 nucleotides or less; f) The exon is exon 6, and the guide sequence portion contains 17 to 22 nucleotides in the same sequence as the sequence shown in any of SEQ ID NOs: 12385 to 14057 or in a sequence that differs by 3 nucleotides or less; or, g) The exon is exon 7, and the guide sequence portion contains 17 to 22 nucleotides in the same sequence as the sequence shown in any of SEQ ID NOs: 1 to 6332 and 14058 to 15412 or in a sequence that differs by 3 nucleotides or less.

[0073] In some embodiments, the guide sequence portion consists of 17 to 50 consecutive nucleotides and contains 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, or differs from the sequence shown in any one of SEQ ID NOs: 1 to 20347 by 3 nucleotides or less.

[0074] In an embodiment of this invention, a composition is provided that includes an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, or a guide sequence portion that differs from the sequence shown in any one of SEQ ID NOs: 1 to 20347 by 3 nucleotides or less.

[0075] In some embodiments, the guide sequence portion is complementary to a target sequence located 50 base pairs upstream to 50 base pairs downstream of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the ANGPTL3 gene.

[0076] In some embodiments, the guide sequence portion is complementary to a target sequence located 30 base pairs upstream to 30 base pairs downstream of an exon of the ANGPTL3 gene, and a) The exon is exon 1, and the guide sequence portion contains 17 to 22 nucleotides in the same sequence as the sequence shown in any of SEQ ID NOs: 6333 to 9188 or in a sequence that differs by 3 nucleotides or less; b) The exon is Exon 2, and the guide sequence portion contains 17 to 22 nucleotides that are the same as the sequence shown in any of SEQ ID NOs: 9189 to 9978 or in a sequence that differs by 3 nucleotides or less; c) The exon is Exon 3, and the guide sequence portion contains 17 to 22 nucleotides that are the same as the sequence shown in any of SEQ ID NOs: 9979 to 10786 or in a sequence that differs by 3 nucleotides or less; d) The exon is Exon 4, and the guide sequence portion contains 17 to 22 nucleotides that are the same as the sequence shown in any of SEQ ID NOs: 10787 to 11633 or in a sequence that differs by 3 nucleotides or less; e) The exon is Exon 5, and the guide sequence portion contains 17 to 22 nucleotides that are the same as the sequence shown in any of SEQ ID NOs: 11634 to 12384 or in a sequence that differs by 3 nucleotides or less; f) The exon is Exon 6, and the guide sequence portion contains 17 to 22 nucleotides that are the same as the sequence shown in any of SEQ ID NOs: 12385 to 14057 or in a sequence that differs by 3 nucleotides or less; or, g) The exon is Exon 7, and the guide sequence portion contains 17 to 22 nucleotides that are the same as the sequence shown in any of SEQ ID NOs: 1 to 6332 and 14058 to 15412 or in a sequence that differs by 3 nucleotides or less.

[0077] In some embodiments, the composition further comprises a CRISPR nuclease.

[0078] In some embodiments, the composition further comprises a trans-activating CRISPR RNA (tracrRNA) molecule.

[0079] In some embodiments, the CRISPR nuclease and the RNA molecule form a complex, or the CRISPR nuclease, the RNA molecule, and the tracrRNA molecule form a complex.

[0080] In an aspect of this invention, there is provided a medicament for use in inactivating the ANGPTL3 allele intracellularly, which comprises any of the compositions described in this specification, and the medicament is administered by delivering the composition to the cell.

[0081] In an aspect of this invention, there is provided the use of any of the compositions described in this specification in the treatment, amelioration or prevention of hypertriglyceridemia, hyperlipidemia or hypercholesterolemia, and the use comprises delivering the composition to a subject suffering from or at risk of suffering from hypertriglyceridemia, hyperlipidemia or hypercholesterolemia.

[0082] In an aspect of this invention, there is provided a medicament for the treatment, amelioration or prevention of hypertriglyceridemia, hyperlipidemia or hypercholesterolemia, which comprises any of the compositions described in this specification, and the medicament is administered by delivering the composition to a subject suffering from or at risk of suffering from hypertriglyceridemia, hyperlipidemia or hypercholesterolemia.

[0083] In an aspect of this invention, there is provided a kit for inactivating the ANGPTL3 allele intracellularly, which comprises any of the compositions described in this specification and instructions for delivering the composition to the cell.

[0084] In an aspect of this invention, there is provided a kit for treating or preventing hypertriglyceridemia, hyperlipidemia or hypercholesterolemia in a subject, which comprises any of the compositions described in this specification and instructions for delivering the composition to a subject suffering from or at risk of suffering from hypertriglyceridemia, hyperlipidemia or hypercholesterolemia.

[0085] In an aspect of this invention, there is provided any of the compositions described in this specification for use in ameliorating or preventing hypertriglyceridemia, hyperlipidemia or hypercholesterolemia.

[0086] In an aspect of this invention, there is provided a method for treating hypertriglyceridemia, hyperlipidemia or hypercholesterolemia in a subject, the method comprising administering to a subject suffering from or at risk of suffering from hypertriglyceridemia, hyperlipidemia or hypercholesterolemia any of the compositions described in this specification.

[0087] In an aspect of this invention, there is provided a gene editing composition comprising an RNA molecule consisting of 17 to 50 consecutive nucleotides and comprising a guide sequence portion having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347. In some aspects, the RNA molecule further comprises a portion having a sequence that binds to a CRISPR nuclease. In some aspects, the sequence that binds to the CRISPR nuclease is a tracrRNA sequence.

[0088] In some aspects, the RNA molecule further comprises a portion having a tracr mate sequence.

[0089] In some aspects, the RNA molecule may further comprise one or more linker portions.

[0090] In an aspect of this invention, the length of the RNA molecule may be at most 1000, 900, 800, 700, 600, 500, 450, 400, 350, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110 or 100 nucleotides. Each possibility is a separate aspect. In an aspect of this invention, the length of the RNA molecule may be 17 to at most 300 nucleotides, 100 to at most 300 nucleotides, 150 to at most 300 nucleotides, 100 to at most 500 nucleotides, 100 to at most 400 nucleotides, 200 to at most 300 nucleotides, 100 to 200 nucleotides or 150 to at most 250 nucleotides. Each possibility is a separate aspect.

[0091] In some aspects of the present invention, a method for inactivating ANGPTL3 expression in cells is provided, the method comprising delivering to the cells a composition comprising an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease.

[0092] In some aspects of the present invention, a method for preventing hypertriglyceridemia, hyperlipidemia or hypercholesterolemia is provided, the method comprising delivering to the target cells a composition comprising an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, and a CRISPR nuclease.

[0093] In aspects of the present invention, at least one RNA molecule comprising at least one CRISPR nuclease and a guide sequence portion is delivered to a subject and / or cells, either substantially simultaneously or at different times.

[0094] In some aspects, a tracrRNA molecule is delivered to a subject and / or cells, either substantially simultaneously or at different times, with at least one RNA molecule comprising at least one CRISPR nuclease and a guide sequence portion.

[0095] The compositions and methods of the present invention can be used for the treatment, prevention, amelioration or deceleration of the progression of hypertriglyceridemia, hyperlipidemia or hypercholesterolemia.

[0096] Any one or combination of the strategies described above for inactivating the expression of ANGPTL3 can be used in the context of the present invention.

[0097] In aspects of this invention, to cause a double-strand break (DSB), an RNA molecule is used to direct a CRISPR nuclease to an exon or splice site of the ANGPTL3 allele, inducing the error-prone non-homologous end joining (NHEJ) mechanism to cause the insertion or deletion of one or more nucleotides, forming a frameshift mutation in the ANGPTL3 allele. The frameshift mutation can inactivate or knockout the ANGPTL3 allele, for example, by generating a premature stop codon and producing a truncated protein in the ANGPTL3 allele, or by nonsense-mediated mRNA decay of the allele's transcript. In another aspect, one RNA molecule is used to direct the CRISPR nuclease to the promoter of the ANGPTL3 allele.

[0098] In some aspects, this method is utilized to treat subjects at risk of hypertriglyceridemia, hyperlipidemia, or hypercholesterolemia. In such aspects, this method improves, alleviates, or prevents the disease phenotype by reducing or eliminating the expression of ANGPTL3.

[0099] Aspects of the compositions described herein include at least one CRISPR nuclease, at least one RNA molecule comprising a guide sequence portion, and a tracrRNA molecule that are simultaneously effective in a subject or cell. The at least one CRISPR nuclease, at least one RNA molecule comprising a guide sequence portion, and the tracrRNA molecule may be delivered substantially simultaneously or at different times but have an effect simultaneously. For example, this includes delivering the CRISPR nuclease to the subject or cell before the RNA guide molecule and / or tracrRNA is substantially present in the subject or cell.

[0100] In some aspects, human cells are modified by any of the methods described herein. In some aspects, the cells are cells of the liver. In some aspects, the cells are hepatocytes. In some aspects, the cells are stem cells.

[0101] ANGPTL3 Knockout Strategy The present invention provides a method for preventing inhibition of lipid metabolism without causing harm to a subject by knocking out the ANGPTL3 allele in cells of the subject, preferably liver cells (e.g., hepatocytes). The methods provided for knocking out the ANGPTL3 allele in cells may be used to treat, prevent, or ameliorate any of hypertriglyceridemia, hyperlipidemia, or hypercholesterolemia.

[0102] The ANGPTL3 knockout strategy includes, but is not limited to, targeting both alleles targeting any one or combination of exons 1-7 of ANGPTL3, including the regions of 30 nucleotides upstream and downstream of the exon, to flank the splice donor and acceptor sites. Frame-shift mutations in these exons lead to non-functional truncated ANGPTL3 proteins or nonsense-mediated decay (NMD) of mutant ANGPTL3 transcripts. For example, an RNA molecule containing a guide sequence portion containing nucleotides 17-24 in the sequence present in any one of SEQ ID NOs: 1-20347 may be used to direct a CRISPR nuclease to the ANGPTL3 target site to induce double-stranded DNA cleavage leading to nonsense-mediated decay (NMD) of non-functional truncated ANGPTL3 proteins or mutant ANGPTL3 transcripts.

[0103] CRISPR Nucleases and PAM Recognition In some embodiments, the sequence-specific nuclease is selected from a CRISPR nuclease or a functional variant thereof. In some embodiments, the sequence-specific nuclease is an RNA-guided DNA nuclease. In such embodiments, the RNA sequence that guides the RNA-guided DNA nuclease (e.g., Cpf1) binds to the RNA-guided DNA nuclease and / or directs it to all ANGPTL3 alleles in the cell. In some embodiments, the CRISPR complex further does not include a tracrRNA. In non-limiting examples where the RNA-guided DNA nuclease is a CRISPR protein, at least one nucleotide that differs between the dominant ANGPTL3 allele and the functional allele may be within and / or near the PAM site in the region designed such that the RNA molecule hybridizes. Those skilled in the art will understand that RNA molecules can be engineered in methods generally known in the art to bind to selected targets in the genome. In some embodiments, the RNA-guided DNA nuclease is part of a fusion protein. In non-limiting examples, the RNA-guided DNA nuclease is bound to a second enzyme (e.g., reverse transcriptase). In some embodiments, the RNA-guided DNA nuclease is a nickase that cleaves single-stranded DNA.

[0104] In this specification, the term "PAM" refers to the nucleotide sequence of the target DNA that is located near the target DNA sequence and is recognized by the CRISPR nuclease complex. The PAM sequence may vary depending on the type of nuclease. In addition, there are CRISPR nucleases that can target almost all PAMs. In some aspects of this invention, the CRISPR system forms Watson-Crick base pairs between the guide sequence portion and the protospacer of the target DNA site adjacent to the protospacer adjacent motif (PAM), which is an additional requirement for target recognition, to utilize one or more RNA molecules having a guide sequence portion that directs the CRISPR nuclease to the target DNA site. Subsequently, the CRISPR nuclease cleaves the target DNA site, resulting in a double-strand break within the protospacer. In a non-limiting example, the type II CRISPR system utilizes a mature crRNA:tracrRNA complex that forms Watson-Crick base pairs between the guide sequence portion of the crRNA and the protospacer on the target DNA adjacent to the protospacer adjacent motif (PAM) to direct the CRISPR nuclease (e.g., Cas9) to the target DNA. One skilled in the art will understand that the engineered RNA molecules of the present invention are further designed to bind to the target genomic DNA sequence of interest adjacent to a protospacer adjacent motif (PAM), e.g., a PAM corresponding to the sequence associated with the CRISPR nuclease utilized.The PAM is, for example, but not limited to, NGG or NAG (where N is any nucleobase) for Streptococcus pyogenes Cas9 WT (SpCAS9); NNGRRT for Staphylococcus aureus (SaCas9); NNNVRYM for Jejun Cas9 WT; NGAN or NGNG for SpCas9-VQR variant; NGCG for SpCas9-VRER variant; NGAG for SpCas9-EQR variant; NRRH (where N is any nucleobase, R is A or G, and H is A, C, or T) for SpCas9-NRRH variant; NRTH (where N is any nucleobase, R is A or G, and H is A, C, or T) for SpCas9-NRTH variant; NRCH (where N is any nucleobase, R is A or G, and H is A, C, or T) for SpCas9-NRCH variant; NG (where N is any nucleobase) for SpG variant of SpCas9; NG or NA (where N is any nucleobase) for SpCas9-NG variant of SpCas9; NR, NRN, or NYN (where N is any nucleobase, R is A or G, and Y is C or T) for SpRY variant of SpCas9; NNG (where N is any nucleobase) for Streptococcus canis Cas9 variant (ScCas9); NNNRRT (where N is any nucleobase and R is A or G) for SaKKH-Cas9 variant (SaCas9) of Staphylococcus aureus; NNNNGATT (where N is any nucleobase) for Neisseria meningitidis (NmCas9); TTN (where N is any nucleobase) for Alicyclobacillus acidiphilus Cas12b (AacCas12b); or TTTV (where V is A, C, or G) for Cpfl. The RNA molecules of the present invention are each designed to form a complex with one or more different CRISPR nucleases and are designed to target a polynucleotide sequence of interest using one or more different PAM sequences corresponding to the CRISPR nucleases.

[0105] In some embodiments, an RNA-guided DNA nuclease (e.g., a CRISPR nuclease) may be used to cause a double-stranded or single-stranded DNA break at a desired location in the genome of a cell. The most commonly used RNA-guided DNA nucleases are derived from the CRISPR system, although other RNA-guided DNA nucleases are also contemplated for use in the genome editing compositions and methods described herein. See, for example, US Patent Application Publication No. 2015 / 0211023, which is incorporated herein by reference.

[0106] The CRISPR systems that can be used in the practice of the present invention are diverse. The CRISPR system may be a type I, type II, or type III system. Non-limiting examples of suitable CRISPR proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, Casl0, Casl Od, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl0, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966.

[0107] In some embodiments, the RNA-guided DNA nuclease is a CRISPR nuclease (e.g., Cas9) derived from a type II CRISPR system. The CRISPR nuclease is from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Neisseria meningitidis, Treponema denticola, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp.) Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.) may also be derived from Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, or any species encoding a CRISPR nuclease having a known PAM sequence. CRISPR nucleases encoded by unculturable bacteria may also be used in the present invention (see Burstein et al. Nature, 2017). Variants of CRISPR proteins having a known PAM sequence, for example, the SpCas9 D1135E variant, the SpCas9 VQR variant, the SpCas9 EQR variant, or the SpCas9 VRER variant may also be used in the present invention.

[0108] Accordingly, an RNA-guided DNA nuclease of the CRISPR system such as Cas9 protein or modified Cas9, or a homolog or ortholog of Cas9, or another RNA-guided DNA nuclease belonging to another CRISPR system such as Cpf1 and its homologs and orthologs may be used in the compositions of the present invention. Other CRISPR nucleases, for example, the nucleases described in International Publication Nos. 2020 / 223514 and 2020 / 223553 (each incorporated herein by reference) may also be used.

[0109] In certain embodiments, the CRISPR nuclease may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound having biological properties substantially common to the native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of the native sequence and derivatives of the native sequence polypeptide and fragments thereof that share biological activity with the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses both amino acid sequence variants of the polypeptide and its covalent modifications and fusions. Suitable derivatives of the Cas polypeptide or fragment thereof include, but are not limited to, variants, fusions, and covalent modifications of the Cas protein or fragment thereof. The Cas protein, including the Cas protein or fragment thereof, in addition to derivatives of the Cas protein or fragment thereof, may be obtainable from cells, or may be chemically synthesized, or may be obtained by a combination of these methods. The cells may be cells that naturally produce the Cas protein, or may be genetically engineered cells that naturally produce the Cas protein and produce the endogenous Cas protein at a higher expression level, or produce the Cas protein from an introduced exogenous nucleic acid encoding the same or a different Cas as the endogenous Cas. In some cases, the cells do not naturally produce the Cas protein and are genetically engineered to produce the Cas protein.

[0110] In some embodiments, the CRISPR nuclease is Cpf1. Cpf1 is a single RNA-guided endonuclease that utilizes a T-rich protospacer adjacent motif. Cpf1 cleaves the twisted DNA double strand. Two Cpf1 enzymes derived from Acidaminococcus and Lachnospiraceae have been shown to efficiently perform genome editing in human cells. (See Zetsche et al., 2015).

[0111] Therefore, an RNA-guided DNA nuclease of a type II CRISPR system such as Cas9 protein or modified Cas9, or a homologue, orthologue or variant of Cas9, or another RNA-guided DNA nuclease belonging to another CRISPR system such as Cpf1 and its homologue, orthologue or variant may be used in the present invention.

[0112] In some embodiments, the guide molecule comprises one or more chemical modifications that confer new or improved properties (e.g., stability to degradation, hybridization energy, or binding to an RNA-guided DNA nuclease). Suitable chemical modifications include, but are not limited to, modified bases, modified sugars, or modified internucleoside linkages. Non-limiting examples of suitable chemical modifications include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methylpseudouridine, β,D-galactosylqueuosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, β,D-mannosylqueuosine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-β-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, N-((9-β-D-ribofuranosylpurin-6-yl)N-methylcarbamoyl)threonine, methyl ester of uridine-5-oxyacetic acid, uridine-5-oxyacetic acid, wybutoxosine, queuosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, N-((9-β-D-ribofuranosylpurin-6-yl)-carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine, 3-(3-amino-3-carboxypropyl)uridine, (acp3)u, 2'-O-methyl (M), 3'-phosphorothioate (MS), 3'-thioPACE (MSP), pseudouridine or 1-methylpseudouridine.The respective realizabilities of this invention are separate aspects.

[0113] In addition to targeting the ANGPTL3 allele with an RNA-guided CRISPR nuclease, other means of blocking the expression of ANGPTL3 in target cells, preferably cells of the liver (e.g., hepatocytes), include, but are not limited to, gapmers, shRNAs, siRNAs, customized TALENs, meganucleases, zinc finger nucleases, small molecule inhibitors, and the use of other methods known in the art for reducing or eliminating the expression of a gene in target cells. See, for example, U.S. Patent Nos. 6,506,559; 7,560,438; 8,420,391; 8,552,171; 7,056,704; 7,078,196; 8,362,231; 8,372,968; 9,045,754; International Publication Nos. 2004 / 067736; 2006 / 097853; 2003 / 087341; 2000 / 041566; 2003 / 080809; 2010 / 079430; 2010 / 079430; 2011 / 072246; 2018 / 057989 and 2017 / 164230 (the entire contents of which are incorporated herein by reference).

[0114] Advantageously, a guide RNA molecule comprising at least one guide sequence portion presented herein improves the ANGPTL3 knockout efficiency when forming a complex with a CRISPR nuclease intracellularly as compared to other guide RNA molecules. These specially designed sequences may also be useful for identifying target sites of ANGPTL3 for other nucleotide-targeting based gene editing or gene silencing methods (e.g., siRNA, TALEN, meganuclease or zinc finger nuclease).

[0115] Delivery into Cells Any of the compositions described in the specification may be delivered to target cells by appropriate means. The RNA molecule compositions of the present invention may be directed to cells that contain and / or express the ANGPTL3 allele, such as cells of the mammalian liver (e.g., hepatocytes). For example, in one aspect, the RNA molecule specifically targets the ANGPTL3 allele within the target cell, and the target cell is a liver cell (e.g., hepatocyte) or a stem cell. Delivery to the cell may be performed in vivo, ex vivo, or in vitro. The delivery may be an in vivo delivery of a composition packaged in a lentivirus, adeno-associated virus (AAV), or nanoparticle to the subject's liver or the cells of the subject's liver. The delivery may also be ex vivo to the subject's cells (e.g., liver cells, hepatocytes, or stem cells isolated from the subject). Furthermore, the nucleic acid compositions described in the specification may be delivered to the cell as one or more of a DNA molecule, an RNA molecule, a ribonucleoprotein (RNP), a nucleic acid vector, or a combination thereof.

[0116] In some aspects, any of the compositions described in the specification are delivered to cells in vivo. In some aspects, the cells are liver cells, such as hepatocytes. In some aspects, the composition is delivered to the subject's liver. The composition may be delivered to the cells by known in vivo delivery methods including, but not limited to, viral transduction (e.g., using lentivirus or adeno-associated virus (AAV)), nanoparticle delivery, etc. Details of the delivery methods are described throughout this section.

[0117] In some aspects, any of the compositions described in the specification are delivered to cells ex vivo. In some aspects, the cells are liver cells, such as hepatocytes. The composition may be delivered to the cells by known ex vivo delivery methods including, but not limited to, nucleofection, electroporation, viral transduction (e.g., using lentivirus or adeno-associated virus (AAV)), nanoparticle delivery, liposomes, etc. Details of the delivery methods are described throughout this section.

[0118] In some embodiments, the RNA molecule includes chemical modifications. Non-limiting examples of suitable chemical modifications include 2'-O-methyl (M), 2'-O-methyl 3'-phosphorothioate (MS) or 2'-O-methyl, 3'-thioPACE (MSP), pseudouridine, and 1-methylpseudouridine. Each of these realizations of the invention is a separate embodiment.

[0119] A suitable viral vector system may be used to deliver the nucleic acid composition (e.g., a composition of the RNA molecules of the invention). Conventional viral and non-viral based gene transfer methods can be used to introduce the nucleic acid into the target tissue. In certain embodiments, the nucleic acid is administered for in vivo or ex vivo gene therapy. Non-viral vector delivery systems include naked nucleic acids, nucleic acids complexed with delivery vehicles (e.g., liposomes or poloxamers). Reviews of gene therapy are provided in Anderson (1992); Nabel & Felgner (1993); Mitani & Caskey (1993); Dillon (1993); Miller (1992); Van Brunt (1988); Vigne (1995); Kremer & Perricaudet (1995); Haddada et al. (1995) and Yu et al. (1994).

[0120] Methods of non-viral delivery of nucleic acids and / or proteins include electroporation, lipofection, microinjection, particle bombardment, particle gun acceleration, virosomes, liposomes, immunoliposomes, lipid nanoparticles (LNP), polycations or lipid:nucleic acid conjugates, artificial virions, and uptake of nucleic acids by facilitators, or nucleic acids and / or proteins can be delivered to plant cells by bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizobium meliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus, and cassava vein mosaic virus). (See, e.g., Chung et al., 2006). For example, sonoporation using the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Delivery of proteins and / or nucleic acids by cationic lipids is also contemplated for in vivo, ex vivo, or in vitro delivery. (See Zuris et al. (2015); Coelho et al. (2013); Judge et al. (2006) and Basha et al. (2011)).

[0121] Non-viral vectors such as transposon-based systems (e.g., recombinant Sleeping Beauty transposon system or recombinant PiggyBac transposon system) may be delivered to target cells and used for translocation of the polynucleotide sequence of the molecule of the composition or the polynucleotide sequence encoding the molecule of the composition in the target cells.

[0122] Other representative nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, e.g., U.S. Patent No. 6,008,336). Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam®, Lipofectin®, and Lipofectamine® RNAiMAX). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those disclosed in International Publication Nos. 91 / 17424 and 91 / 16024. Delivery to cells (ex vivo administration) or target tissues (in vivo administration) is possible.

[0123] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of skill in the art. (See, for example, Crystal, Science (1995); Blaese et al., (1995); Behr et al., (1994); Remy et al. (1994); Gao and Huang (1995); Ahmad and Allen (1992); U.S. Patent Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787.)

[0124] Another method of delivery involves packaging the nucleic acid to be delivered into an EnGeneIC delivery vehicle (EDV). The EDV is specifically delivered to the target tissue using a bispecific antibody where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody transports the EDV to the surface of the target cell and the EDV is carried intracellularly by endocytosis. The contents are released after entering the cell. (See MacDiarmid et al., 2009).

[0125] Delivery vehicles include, but are not limited to, bacteria, preferably non-pathogenic vehicles, nanoparticles, exosomes, microvesicles, for example gene gun delivery by attachment of the composition to gold particles that are shot intracellularly by a “gene gun”, viral vehicles including lentiviruses, AAVs and retroviruses, virus-like particles (VLPs), large VLPs (LVLPs), lentivirus-like particles, transposons, viral vectors, naked vectors, DNA or RNA, and other delivery vehicles known in the art, but not limited thereto.

[0126] Delivery of the CRISPR nuclease and / or polynucleotide encoding the CRIPSR nuclease, and optionally additional nucleotide molecules and / or additional proteins or peptides, may be performed using a single delivery vehicle or method, or a combination of different delivery vehicles or methods. For example, the CRISPR nuclease may be delivered to cells using an LNP, and the crRNA molecule and tracrRNA molecule may be delivered to cells using an AAV. Alternatively, the CRISPR nuclease may be delivered to cells using AAV particles, and the crRNA molecule and tracrRNA molecule may be delivered to cells using a different AAV particle, which may be advantageous due to size limitations.

[0127] The use of RNA or DNA virus systems for the delivery of nucleic acids by viruses exploits highly evolved methods by which the virus targets specific cells in the body and transports the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo), or used for the treatment of cells in vitro, and the modified cells are administered to patients (ex vivo). Conventional virus systems for delivering nucleic acids include, but are not limited to, vectors of retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, and herpes simplex viruses for gene transfer.

[0128] RNA viruses may be utilized for the delivery of the RNA compositions described in the specification. Also, high transduction efficiencies have been observed in various cells and target tissues. The nucleic acids of the present invention may be delivered by non-integrating lentiviruses. If necessary, RNA delivery by lentiviruses is utilized. In some cases, the lentivirus contains nuclease mRNA, guide RNA. In some cases, the lentivirus contains nuclease mRNA, guide RNA, and donor template. In some cases, the lentivirus contains nuclease protein, guide RNA. In some cases, the lentivirus contains nuclease protein, guide RNA, and / or a donor template for gene editing, for example, by homology-directed repair. In some cases, the lentivirus contains nuclease mRNA, DNA-targeted RNA, and tracrRNA. In some cases, the lentivirus contains nuclease mRNA, DNA-targeted RNA, tracrRNA, and donor template. In some cases, the lentivirus contains nuclease protein, DNA-targeted RNA, and tracrRNA. In some cases, the lentivirus contains nuclease protein, DNA-targeted RNA, tracrRNA, and a donor template for gene editing, for example, by homology-directed repair.

[0129] As described previously, the compositions described in the specification may be delivered to target cells using a non-integrating lentiviral particle method (e.g., LentiFlash (registered trademark) system). mRNA or other RNA may be delivered to target cells using such methods such that the compositions described in the specification are assembled inside the target cells upon delivery of the RNA to the target cells. See also International Publication Nos. WO 2013 / 014537, WO 2014 / 016690, WO 2016 / 185125, WO 2017 / 194902, and WO 2017 / 194903.

[0130] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding the population of potential target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically have high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Lentiviral vectors are composed of cis-acting terminal repeat sequences and have the ability to package up to 6 - 10 kb of foreign sequence. The minimal cis-acting LTR is sufficient for vector replication and packaging and is used to integrate the therapeutic gene into target cells and to permanently express the transgene. Widely used lentiviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof. (See, for example, Buchschacher et al. (1992); Johann et al. (1992); Sommerfelt et al. (1990); Wilson et al. (1989); Miller et al. (1991); International Publication No. WO 94 / 26877).

[0131] For gene transfer in clinical trials, at least six viral vectors are currently available, and they are used to prepare transducing agents using an approach that involves complementation of defective vectors by genes inserted into helper cell lines.

[0132] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (see Dunbar et al., 1995; Kohn et al., 1995; Malech et al., 1997). PA317 / pLASN was the first therapeutic vector used in gene therapy trials (Blaese et al., 1995). The transduction efficiency of the MFG-S packaging vector was greater than 50% (Ellem et al., (1997); Dranoff et al., 1997).

[0133] Packaging cells are used to form virus particles capable of infecting host cells. Such cells include 293 cells that package adenovirus, AAV, and Psi-2 cells or PA317 cells that package retrovirus. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into virus particles. The vector usually contains the minimal viral sequences necessary for packaging and (where applicable) subsequent integration into the host, and other viral sequences are replaced with an expression cassette encoding the protein to be expressed. The defective viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy usually have only the inverted terminal repeats (ITRs) derived from the AAV genome that are necessary for packaging and integration into the host genome. The viral DNA is packaged in a cell line that contains a helper plasmid lacking other AAV genes, i.e., the ITR sequences, but encoding rep and cap. The cell line is also infected with adenovirus as a helper. The helper virus promotes the replication of the AAV vector and the expression of the AAV genes from the helper plasmid. Due to the lack of ITR sequences, the helper plasmid is not packaged in large amounts. Contamination with adenovirus can be reduced, for example, by heat treatment in which adenovirus is more sensitive than AAV. In addition, AAV can be produced at the clinical scale using a baculovirus system (see U.S. Patent No. 7,479,554).

[0134] In many gene therapies, it is desirable for the gene therapy vector to be delivered with a high degree of specificity to a particular tissue. Thus, viral vectors can be modified to have specificity for a given cell by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is selected to have an affinity for a receptor known to be present on the target cell. For example, Han et al. (1995) reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and that the recombinant virus infects specific human breast cancer cells that express the human epidermal growth factor receptor. This principle can be extended to other virus-target cell combinations where the target cell expresses a receptor and the virus expresses a fusion protein containing a ligand for the cell surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) with specific binding affinity for virtually any selected cell receptor. This explanation applies mainly to viral vectors, but the same principle can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences that preferentially allow uptake by specific target cells.

[0135] As described below, gene therapy vectors can be delivered in vivo by administration to individual patients, for example, by systemic administration (e.g., intravitreal, intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial injection) or by local application. Preferably, to knock out ANGPTL3 expression in liver cells (e.g., hepatocytes), the compositions disclosed herein are delivered in vivo to cells within the liver of a subject. The compositions may be delivered to liver cells by several known means, including viral vehicles (e.g., lentivirus, adeno-associated virus (AAV), etc.), use of nanoparticles, or delivery of naked RNA compositions. For example, the compositions may be delivered in vivo to liver cells by lipid nanoparticle delivery, lentiviral delivery or AAV delivery.

[0136] Alternatively, the vector can be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells, and subsequently the cells can be re-transplanted into the patient after selection of the cells incorporating the vector. Representative ex vivo approaches, which are not limiting, may include removing tissue (e.g., peripheral blood, bone marrow, and spleen) from a patient for culture, introducing nucleic acid into cultured cells (e.g., hematopoietic stem cells), and then transplanting the cells into the patient's target tissue (e.g., bone marrow and spleen). In some embodiments, the stem cells or hematopoietic stem cells may be further treated with a survival enhancer.

[0137] Ex vivo cell transfection for diagnostic, research, or gene therapy (e.g., by re-injection of transfected cells into a host) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from a subject, transfected with a nucleic acid composition, and re-injected into the subject (e.g., a patient). A variety of cells suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney, “Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications (6th edition, 2010) and the references cited therein for a discussion of methods for isolating and culturing cells from patients).

[0138] Vectors (e.g., retroviruses, liposomes) having a therapeutic nucleic acid composition can also be administered directly to an organism for in vivo transduction of cells. Administration can be by any route commonly used to introduce molecules into ultimate contact with blood or tissue cells, including but not limited to injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well known to those of skill in the art, and multiple routes can be used to administer a particular composition, although a particular route may often provide a more rapid and effective response than another route. In some embodiments, the composition is delivered by IV injection.

[0139] Vectors suitable for introducing the transgene into cells include non-integrating lentiviral vectors. See, for example, U.S. Patent Application Publication No. 2009 / 0117617.

[0140] The pharmaceutically acceptable carrier is determined in part by the composition being administered and in addition by the method used to administer the composition. Thus, there are a wide variety of suitable formulations of pharmaceutical compositions available, as described, for example, in Remington's Pharmaceutical Sciences, 17th ed., 1989.

[0141] The compositions and methods of the present invention may also be used in the manufacture of a medicament for treating a dominant genetic disorder in a patient.

[0142] Examples of RNA Guide Sequence Portions Specifically Targeting Alleles of the ANGPTL3 Gene Numerous guide sequences can be designed to target the ANGPTL3 gene, but the nucleotide sequences shown in Table 1 and identified by SEQ ID NOs: 1 to 20347 were specifically selected to effectively carry out the methods described in this specification.

[0143] Table 1 shows guide arrays designed for use in the manner described heretofore to associate ANGPTL3 alleles. Each engineered guide molecule is further designed to bind to a target genomic DNA sequence adjacent to a protospacer adjacent motif (PAM) (e.g., a PAM corresponding to the sequence NGG or NAG (where N is any nucleobase)). The guide sequences are designed to work with one or more different CRISPR nucleases, including, but not limited to, SpCas9WT (PAM sequence: NGG), SpCas9.VQR.1 (PAM sequence: NGAN), SpCas9.VQR.2 (PAM sequence: NGNG), SpCas9.EQR (PAM sequence: NGAG), SpCas9.VRER (PAM sequence: NGCG), SaCas9WT (PAM sequence: NNGRRT), SpRY (PAM sequence: NRN or NYN), NmCas9WT (PAM sequence: NNNNGATT), Cpf1 (PAM sequence: TTTV), JeCas9WT (PAM sequence: NNNVRYM), OMNI-50 (PAM sequence: NGG), OMNI-79 (PAM sequence: NGG), OMNI-103 (PAM sequence: NNRACT), OMNI-159 (NNNNCMAN) or OMNI-124 (PAM sequence: NNGNRMNN).

[0144] OMNI-124 nuclease (SEQ ID NO: 20362) and OMNI-159 nuclease (SEQ ID NO: 20353) are further described in International Publication No. 2023 / 019269, the content of which is incorporated herein by reference. OMNI-103 nuclease (SEQ ID NO: 20354) is further described in International Publication Nos. 2022 / 170199 and 2023 / 107946, the content of each of which is incorporated herein by reference. OMNI-50 nuclease (SEQ ID NO: 20360) is further described in U.S. Patent No. 11,666,641, International Publication Nos. 2020 / 223514, 2022 / 098693 and 2023 / 019263, the content of each of which is incorporated herein by reference. OMNI-79 nuclease (SEQ ID NO: 20361) is further described in U.S. Patent Application Publication No. 2023 / 0122086, International Publication Nos. 2021 / 248016 and 2023 / 102407, the content of each of which is incorporated herein by reference.

[0145] The RNA molecules of the present invention are each designed to form a complex with one or more different CRISPR nucleases and utilize one or more different PAM sequences corresponding to the CRISPR nucleases to target a polynucleotide sequence of interest.

[0146] [Table 1]

[0147] Examples are shown below to facilitate a more complete understanding of the present invention. The following examples show representative ways of constructing and implementing the present invention. However, the scope of the present invention is not limited to the specific aspects disclosed in these examples, and these aspects are for illustrative purposes only. Examples

[0148] Details of Experiments Example 1 ANGPTL3 Target Analysis For the guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 20347, in a human cell line (e.g., HeLa cells), a compatible CRISPR nuclease (e.g., OMNI-103 or OMNI-159) is used to screen for high on-target activity. The on-target activity is determined by DNA capillary electrophoresis analysis.

[0149] Example 2 ANGPTL3-Targeting sgRNAs Tested in Liver Cells To reduce the expression of ANGPTL3 protein in liver cells, several single-guide RNAs (sgRNAs) were tested in Huh-7 human hepatoma cells using the OMNI-103 CRISPR nuclease expressed from mRNA. Six sgRNAs targeting exon 1 of the ANGPTL3 gene were screened for high on-target activity in Huh-7 cells (Table C and Figure 2A). Briefly, using the Lonza 4D-nucleofector system, 1 μg of OMNI-103 and 124 pmol of sgRNA were electroporated into the cells. Seventy-two hours after electroporation, the cells were harvested. Cell lysis and genomic DNA extraction were performed using Quick Extract (Lucigen), the endogenous genomic region was amplified using specific primers, and the on-target activity was measured by next-generation sequencing (NGS) (Figure 2B). To verify the changes in ANGPTL3 expression, the level of ANGPTL3 mRNA was evaluated by qRT-PCR and the level of ANGPTL3 secreted protein was evaluated by ELISA. At the mRNA level, total mRNA from edited and unedited cells was extracted using the Maxwell RSC automated RNA purification kit (Promega). Total cDNA was synthesized from mRNA using the High-Capacity RNA to cDNA kit (Applied Biosystems). The level of mRNA was measured by qRT-PCR using Sybr Green dye and the level of HPRT mRNA as a control (Figure 2C). To measure the level of secreted ANGPTL3 protein, 1 × 106 Individual Huh-7 cells were seeded and incubated for 24 hours. The collected medium was centrifuged at 800 g for 10 minutes to precipitate cells and residues, and then the expression of ANGPTL3 was analyzed using a human ANGPTL3 ELISA kit (Abcam) (Figure 2D). For all sgRNAs tested, the genome editing rate decreased by more than 90%, and the mRNA expression level decreased by more than 50%. For sgRNA29, 30, 32, and 36, the secretion of ANGPTL3 was below the detection limit.

[0150] Example 3 ANGPTL3-Targeting sgRNAs Tested in vivo To reduce the in vivo expression of ANGPTL3 protein in C57Bl6 mice, LNPs containing the guide molecule "sgRNA1m" or "sgRNA2m" and the mRNA molecule encoding OMNI-103 were injected into the tail vein of C57Bl6 mice at 2.5 mg / kg or 5 mg / kg (Table D). Serum was collected from the mice on day 0 (injection day) and 14 days later. To evaluate on-target editing, the entire liver was collected and a single cell suspension was isolated using a Liver Perfusion kit and a GentleMACS device (Miltenyi Biotec). Genomic DNA was extracted using a Maxwell RSC Cell kit (Promega), the endogenous genomic region was amplified using specific primers, and on-target activity was measured by next-generation sequencing (NGS) (Figure 3A). To verify the changes occurring in ANGPTL3 expression, the level of ANGPTL3 mRNA was evaluated by qRT-PCR and the level of ANGPTL3 secreted protein was evaluated by ELISA. For the evaluation of mRNA levels, total mRNA was extracted using a Maxwell RSC automated RNA purification kit (Promega). Total cDNA was synthesized from the mRNA using a High-Capacity RNA to cDNA kit (Applied Biosystems). The level of LDLR mRNA was measured by qRT-PCR using Sybr Green dye, and the level of HPRT mRNA was measured as a control (Figure 3B). To measure the level of secreted ANGPTL3 protein, serum collected from the mice before and 14 days after treatment was analyzed for ANGPTL3 expression using a mouse ANGPTL3 ELISA kit (Abcam) (Figure 3C). In all mice, the proportion of genomic editing decreased by more than 50% and the mRNA expression level decreased by more than 80% for any of the sgRNAs tested. The secretion of ANGPTL3 decreased by more than 80% in the sgRNA2m test group and by more than 95% in the sgRNA1m test group.

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Claims

1. A method for inactivating at least one allele of the angiopoietin-like protein 3 (ANGPTL3) gene in a cell, comprising: at least one CRISPR nuclease, or a nucleotide molecule encoding the CRISPR nuclease; and an RNA molecule comprising a guide sequence portion, or a DNA molecule encoding the RNA molecule introducing a composition comprising the same into the cell, a complex of the CRISPR nuclease and the RNA molecule double-strand breaks at least one allele of the ANGPTL3 gene, the guide sequence portion of the RNA molecule consists of 17 to 50 consecutive nucleotides.

2. The method according to claim 1, wherein the composition is introduced into cells of a subject or cells in culture.

3. The method according to claim 1 or 2, wherein the cells are liver cells, preferably hepatocytes, or stem cells.

4. The method according to any one of claims 1 to 3, wherein the CRISPR nuclease and the RNA molecule are introduced into the cell substantially simultaneously or at different times.

5. The method according to any one of claims 1 to 4, wherein the allele of the ANGPTL3 gene in the cell undergoes an insertion or deletion mutation.

6. The method according to claim 5, wherein the insertion or deletion mutation produces a premature stop codon.

7. The method according to any one of claims 1 to 6, wherein the inactivation results in a truncated protein encoded by the inactivated allele.

8. The guide sequence portion is complementary to a target sequence located 50 base pairs downstream to 50 base pairs upstream of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 or exon 7 of the ANGPTL3 gene. The method according to any one of claims 1 to 7.

9. The guide sequence portion is complementary to a target sequence located 30 base pairs downstream to 30 base pairs upstream of an exon of the ANGPTL3 gene, and a) when the exon is exon 1, the guide sequence portion comprises 17 to 22 nucleotides in the same sequence as shown in any of SEQ ID NOs: 6333 to 9188 or in a sequence with 3 or fewer nucleotides different; b) when the exon is exon 2, the guide sequence portion comprises 17 to 22 nucleotides in the same sequence as shown in any of SEQ ID NOs: 9189 to 9978 or in a sequence with 3 or fewer nucleotides different; c) The exon is exon 3, and the guide sequence portion contains 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 9979 to 10786 or in a sequence that differs by 3 nucleotides or less; d) The exon is exon 4, and the guide sequence portion contains 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 10787 to 11633 or in a sequence that differs by 3 nucleotides or less; e) The exon is exon 5, and the guide sequence portion contains 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 11634 to 12384 or in a sequence that differs by 3 nucleotides or less; f) The exon is exon 6, and the guide sequence portion contains 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 12385 to 14057 or in a sequence that differs by 3 nucleotides or less; or, g) The exon is exon 7, and the guide sequence portion contains 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 1 to 6332 and 14058 to 15412 or in a sequence that differs by 3 nucleotides or less; The method according to any one of claims 1 to 8.

10. The guide sequence portion consists of 17 to 50 consecutive nucleotides and contains 17 to 22 nucleotides in a sequence shown in any one of SEQ ID NOs: 1 to 20347, or differs from a sequence shown in any one of SEQ ID NOs: 1 to 20347 by 3 nucleotides or less. The method according to any one of claims 1 to 7.

11. A composition comprising an RNA molecule comprising a guide sequence portion consisting of 17 to 50 consecutive nucleotides and having 17 to 22 nucleotides in a sequence shown in any one of SEQ ID NOs: 1 to 20347, or a guide sequence portion that differs from a sequence shown in any one of SEQ ID NOs: 1 to 20347 by 3 nucleotides or less.

12. The composition according to claim 11, wherein the guide sequence portion is complementary to a target sequence located 50 base pairs upstream to 50 base pairs downstream of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or exon 7 of the ANGPTL3 gene.

13. The guide sequence portion is complementary to a target sequence located 30 base pairs upstream to 30 base pairs downstream of an exon of the ANGPTL3 gene, and a) The exon is exon 1, and the guide sequence portion comprises 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 6333 to 9188 or in a sequence that differs by 3 nucleotides or less; b) The exon is exon 2, and the guide sequence portion comprises 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 9189 to 9978 or in a sequence that differs by 3 nucleotides or less; c) The exon is exon 3, and the guide sequence portion comprises 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 9979 to 10786 or in a sequence that differs by 3 nucleotides or less; d) The exon is exon 4, and the guide sequence portion comprises 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 10787 to 11633 or in a sequence that differs by 3 nucleotides or less; e) The exon is exon 5, and the guide sequence portion comprises 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 11634 to 12384 or in a sequence that differs by 3 nucleotides or less; f) The exon is exon 6, and the guide sequence portion comprises 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 12385 to 14057 or in a sequence that differs by 3 nucleotides or less; or, g) The exon is exon 1, and the guide sequence portion comprises 17 to 22 nucleotides in a sequence identical to the sequence shown in any of SEQ ID NOs: 1 to 6332 and 14058 to 15412 or in a sequence that differs by 3 nucleotides or less; The composition according to claim 11 or 12.

14. The composition according to any one of claims 11 to 13, further comprising a CRISPR nuclease.

15. The composition according to any one of claims 11 to 14, further comprising a trans-activating CRISPR RNA (tracrRNA) molecule.

16. The composition according to any one of claims 11 to 15, wherein the CRISPR nuclease and the RNA molecule form a complex, or the CRISPR nuclease, the RNA molecule, and the tracrRNA molecule form a complex.

17. A medicament for use in inactivating the ANGPTL3 allele intracellularly, comprising the composition according to any one of claims 11 to 16, wherein the medicament is administered by delivering the composition according to any one of claims 11 to 16 to the cell.

18. Use of the composition according to any one of claims 11 to 16 in the treatment, improvement or prevention of hypertriglyceridemia, hyperlipidemia or hypercholesterolemia, comprising delivering the composition according to any one of claims 11 to 16 to a subject suffering from or at risk of suffering from hypertriglyceridemia, hyperlipidemia or hypercholesterolemia.

19. A medicament for use in the treatment, improvement or prevention of hypertriglyceridemia, hyperlipidemia or hypercholesterolemia, comprising the composition according to any one of claims 11 to 16, wherein the medicament is administered by delivering the composition according to any one of claims 11 to 16 to a subject suffering from or at risk of suffering from hypertriglyceridemia, hyperlipidemia or hypercholesterolemia.

20. A kit for inactivating the ANGPTL3 allele intracellularly, comprising the composition according to any one of claims 11 to 16 and instructions for delivering the composition to the cell.

21. A kit for treating or preventing hypertriglyceridemia, hyperlipidemia or hypercholesterolemia in a subject, comprising the composition according to any one of claims 11 to 16 and instructions for delivering the composition to a subject suffering from or at risk of suffering from hypertriglyceridemia, hyperlipidemia or hypercholesterolemia.

22. The composition according to any one of claims 11 to 16 for use in the treatment, improvement or prevention of hypertriglyceridemia, hyperlipidemia or hypercholesterolemia.

23. A method for treating hypertriglyceridemia, hyperlipidemia or hypercholesterolemia in a subject, comprising administering to a subject suffering from or at risk of suffering from hypertriglyceridemia, hyperlipidemia or hypercholesterolemia the composition according to any one of claims 11 to 16.

24. A composition, method, process, kit, modified cell or use characterized by one or more elements disclosed in the specification.

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