Compositions and methods for treating hypercholesterolemia and / or cardiovascular diseases

Abstract

Described herein are compositions and methods for treating a subject suffering from hypercholesterolemia and / or cardiovascular disease.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to PCT / CN2022 / 120376 filed on September 22, 2022. The invention name of PCT / CN2022 / 120376 is “COMPOSITIONS AND METHODS FOR TREATMENT OF HYPERCHOLESTEROLEMIA AND / OR CARDIOVASCULAR DISEASE”, the contents of which are fully incorporated into this application by reference. Technical Field

[0003] The present application relates to compositions and methods for treating hypercholesterolemia and / or cardiovascular diseases associated with proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0004] Sequence Listing

[0005] This application contains a submitted sequence listing, which is incorporated in its entirety by reference in this application. The sequence listing is an xml version, generated on October 8, 2023, named "53333-0005WO1", and is 1,172,853 bytes in size. Background Art

[0006] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a serine protease that plays a regulatory role in cholesterol homeostasis, primarily by reducing the levels of hepatic and extrahepatic low-density lipoprotein (LDL) receptors (LDLRs) on the plasma membrane, thereby increasing plasma low-density lipoprotein (LDL) cholesterol. PCSK9 is ubiquitously expressed in many tissues and cell types, but is most abundantly expressed in the liver, small intestine, and kidney. PCSK9 is also highly expressed in arterial walls (e.g., endothelial cells), smooth muscle cells, and macrophages, with local effects that can regulate vascular homeostasis and atherosclerosis. PCSK9 binds to receptors on LDL particles, which typically carry 3,000 to 6,000 fat molecules (including cholesterol) per particle in the extracellular fluid. LDLRs on the membranes of liver and other cells bind to LDL particles in the extracellular fluid and initiate their uptake from the extracellular fluid into the cell, thereby reducing the concentration of LDL particles. If the activity of PCSK9 is inhibited (e.g., by mutation or drug intervention), more LDLRs are recycled and present on the cell surface, thereby removing LDL particles from the extracellular fluid. Therefore, inhibiting PCSK9 or reducing the abundance of PCSK9 can reduce the concentration of LDL particles in the blood.

[0007] In human patients, PCSK9 variants can reduce or increase circulating cholesterol. For example, PCSK9 gain-of-function mutations associated with hypercholesterolemia (such as R218S, F216L and D374Y) result in full or partial loss of processing of mature PCSK9 at the furin cleavage motif RFHR (SEQ ID NO: 974). In contrast, PCSK9 loss-of-function mutations associated with hypocholesterolemia (such as A443T and C679X) result in abnormal subcellular localization and increased sensitivity to furin cleavage (A443T) or PCSK9 cannot leave the endoplasmic reticulum (C679X).

[0008] Therefore, people have been exploring the possible use of PCSK9 inhibitors for the treatment of hypercholesterolemia. Antibody-based therapeutics alirocumab and evolocumab have been studied in Phase III clinical trials. In addition, RNAi-based therapeutics for inhibiting PCSK9 have also been studied. Although these therapeutics that inhibit PCSK9 have achieved encouraging results, there is still a need for therapies that can produce a lasting inhibitory effect on PCSK9 to treat hypercholesterolemia and cardiovascular disease. Summary of the invention

[0009] The present application relates to compositions and methods for reducing PCSK9 gene expression using a CRISPR / Cas system, thereby significantly reducing or eliminating the production of mutant PCSK9 protein or wild-type PCSK9 protein in, for example, liver, small intestine, kidney or vascular tissue. The present application is based at least in part on the following discovery: a novel guide RNA (gRNA) with high editing efficiency can knock out or knock down mutant or wild-type PCSK9 gene expression, thereby providing a durable treatment for hypercholesterolemia and / or cardiovascular disease.

[0010] In a first aspect, the present application describes a guide RNA comprising:

[0011] a) a sequence selected from the group consisting of SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932, and 935-940;

[0012] b) at least 15, 16, 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from SEQ ID NO: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940; or

[0013] c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940.

[0014] In a second aspect, the present application describes a vector comprising one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise:

[0015] a) one or more sequences selected from SEQ ID NO: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940;

[0016] b) at least 15, 16, 17, 18, 19 or 20 consecutive nucleotides of one or more sequences selected from SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940; or

[0017] c) one or more sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932, and 935-940.

[0018] In a third aspect, the present application describes a composition comprising:

[0019] (i) a nucleic acid encoding a guide RNA, or a vector comprising a nucleic acid encoding a guide RNA, wherein the guide RNA comprises:

[0020] a) a sequence selected from the group consisting of SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932, and 935-940;

[0021] b) at least 15, 16, 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from SEQ ID NO: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940; or

[0022] c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932, and 935-940; and

[0023] (ii) an RNA-guided DNA-binding agent, a nucleic acid encoding an RNA-guided DNA-binding agent, or a vector comprising a nucleic acid encoding an RNA-guided DNA-binding agent.

[0024] In a fourth aspect, the present application describes a method for modifying the human proprotein convertase subtilisin / kexin type 9 (PCSK9) gene and / or inducing double-strand breaks (DSBs) within the PCSK9 gene, the method comprising administering a composition of the present application to a cell, wherein the composition recognizes and cleaves the PCSK9 target sequence.

[0025] In the fifth aspect, the present application describes a method for treating hypercholesterolemia and / or cardiovascular disease in a subject, a method for reducing LDL levels in the subject's circulation, a method for reducing the subject's risk of atherosclerosis and / or a method for treating or preventing coronary artery disease in a subject, comprising administering the composition of the present application to cells of a subject in need thereof, wherein the composition recognizes and cleaves the PCSK9 target sequence, thereby reducing the expression and / or abundance of PCSK9 in cells of one or more tissues of the subject, reducing LDL levels in the subject's circulation, reducing the subject's risk of atherosclerosis, treating or preventing the subject's coronary artery disease and / or treating the subject's hypercholesterolemia and / or cardiovascular disease.

[0026] In some embodiments, the RNA-guided DNA binder includes a Cas nuclease or a Cas nickase. In some embodiments, the nucleic acid encoding the RNA-guided DNA binder is a Cas9 nucleic acid comprising a nucleic acid sequence shown in SEQ ID NO: 902 or 903. In some embodiments, the nucleic acid encoding the RNA-guided DNA binder is a nucleic acid encoding Cas9 comprising a polynucleotide sequence shown in one or more of SEQ ID NO: 941-953, 954-960 and 963-972. In some embodiments, the RNA-guided DNA binder is a Cas9 comprising an amino acid sequence shown in SEQ ID NO: 901. In some embodiments, the Cas nuclease is a Class 2 Cas nuclease. In some embodiments, the Cas nuclease is Cas9, Cpfl, C2cl, C2c2, and C2c3, or a modified protein thereof. In some embodiments, the Cas nuclease is a Streptococcus pyogenes (S.pyogenes) Cas9 nuclease or a Staphylococcus aureus (S.aureus) Cas9 nuclease or a modified protein thereof. In some embodiments, the Cas nuclease is from a type II CRISPR / Cas system.

[0027] In some embodiments, the compositions of the present application are used to edit the proprotein convertase subtilisin / kexin 9 (PCSK9) gene. In some embodiments, editing is calculated as the percentage (editing percentage) of the edited cell population. In some embodiments, about 30% to 99% of the cell population is edited. In some embodiments, the editing percentage is 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95% or 95% to 99% of the cell population.

[0028] In some embodiments, the composition of the present application increases the abundance of low-density lipoprotein receptor (LDLR) on the plasma membrane of at least one tissue or organ. In some embodiments, the tissue or organ is liver, small intestine, kidney or vascular tissue. In some embodiments, the composition of the present application reduces the amount of LDL cholesterol in the circulation of the subject. In some embodiments, the LDL cholesterol in the circulation is measured 8 weeks after the composition is applied. In some embodiments, the LDL cholesterol in the circulation is compared with the negative control or the level measured in the subject before the composition is applied. In some embodiments, the LDL cholesterol in the circulation is reduced by at least 20% relative to the corresponding negative control or the level measured in the subject before the composition is applied.

[0029] In some embodiments, the composition is administered or delivered at least once. In some embodiments, administration or delivery is performed at intervals of: (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days; or (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks; or (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months; or (d) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0030] In some embodiments, the guide RNA is at least partially complementary to a target sequence present in a human PCSK9 gene. In some embodiments, the target sequence is located in exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of a human PCSK9 gene. In some embodiments, the guide RNA sequence is complementary to a target sequence in the positive strand of the PCSK9 gene. In some embodiments, the guide RNA sequence is complementary to a target sequence in the negative strand of the PCSK9 gene. In some embodiments, the first guide sequence is complementary to a first target sequence in the positive strand of the PCSK9 gene, and wherein the composition further comprises a second guide sequence complementary to a second target sequence in the negative strand of the PCSK9 gene.

[0031] In some embodiments, the guide RNA comprises crRNA, and further comprises tracrRNA (trRNA) or a portion thereof, wherein the tracrRNA (trRNA) comprises the nucleotide sequence shown in SEQ ID NO: 904, wherein the trRNA is operably linked to the crRNA.

[0032] In some embodiments, the guide RNA is a dual guide RNA (dgRNA). In some embodiments, the guide RNA is a single guide RNA (sgRNA). In some embodiments, the guide RNA comprises at least one modification. In some embodiments, the at least one modification includes 2'-O-methyl (2'-O-Me) modified nucleotides, phosphorothioate (PS) bonds between nucleotides, 2'-fluoro (2'-F) modified nucleotides, or DNA-RNA hybrids (DNA-RNA hybrid). In some embodiments, the at least one modification includes one or more of the first five nucleotides at the 5' end of the guide RNA and / or one or more of the last five nucleotides at the 3' end of the guide RNA. In some embodiments, the at least one modification includes modification of at least 50% of the nucleotides of the guide RNA.

[0033] In some embodiments, the sgRNA comprises a guide sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932, and 935-940. In some embodiments, the sgRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 593-888. In some embodiments, the sgRNA comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence set forth in any one of SEQ ID NOs: 593-888.

[0034] In some embodiments, the guide RNA is associated with a lipid nanoparticle (LNP).In some embodiments, the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier.

[0035] In some embodiments, the composition reduces the risk of cardiovascular disease in a subject or prevents cardiovascular disease in a subject. In some embodiments, the composition reduces the risk of atherosclerosis in a subject or prevents atherosclerosis in a subject. In some embodiments, the composition reduces the risk of atherosclerotic plaque formation in a subject's vascular tissue or prevents atherosclerotic plaque formation in a subject's vascular tissue.

[0036] In some embodiments, the administration of the composition results in a deletion or insertion of one or more nucleotides in the PCSK9 gene. In some embodiments, the deletion or insertion of one or more nucleotides induces a frameshift or nonsense mutation in the PCSK9 gene. In some embodiments, a frameshift or nonsense mutation is induced in the PCSK9 gene of about 20% to about 30% of cells. In some embodiments, the cell is a hepatocyte, a kidney cell, an intestinal epithelial cell, or a vascular epithelial cell. In some embodiments, the deletion or insertion of one or more nucleotides occurring in the PCSK9 gene is at least 50 times or more than that in the off-target site.

[0037] In some embodiments, the composition reduces the level of PCSK9 protein in the subject's cells. In some embodiments, the level of PCSK9 protein is reduced by at least 30%. In some embodiments, the level of PCSK9 protein is measured in serum, plasma, blood, or cerebrospinal fluid. In some embodiments, the level of PCSK9 protein is measured in hepatocytes, kidney cells, intestinal epithelial cells, or vascular epithelial cells.

[0038] In some embodiments, the composition increases the level of LDL receptor protein on the plasma membrane of the subject's cells. In some embodiments, the level of the LDL receptor protein increases by at least 10%. In some embodiments, the level of LDL receptor protein in hepatocytes, kidney cells, intestinal epithelial cells, or vascular epithelial cells is measured.

[0039] In some embodiments, the composition reduces the level of circulating LDL cholesterol in a subject.In some embodiments, in some embodiments, the level of LDL cholesterol is measured in serum, plasma, or blood.

[0040] In some embodiments, the subject has hypercholesterolemia, familial hypercholesterolemia, or a family history of hypercholesterolemia. In some embodiments, the subject has cardiovascular disease, familial cardiovascular disease, or a family history of cardiovascular disease. In some embodiments, the subject has atherosclerosis, familial atherosclerosis, or a family history of atherosclerosis. In some embodiments, the subject exhibits cardiovascular symptoms of atherosclerotic plaques. In some embodiments, the subject exhibits cardiovascular symptoms of coronary artery disease.

[0041] In some embodiments, the subject expresses wild-type PCSK9 or PCSK9 with one or more mutations selected from the group consisting of R46L, S127R, Y142X, R218S, F216L, D374Y, A443T or C679X. In some embodiments, the subject is homozygous for wild-type PCSK9.

[0042] In some embodiments, after applying the composition of the present application, the subject shows improvement, stabilization or slowing of changes in symptoms of hypercholesterolemia. In some embodiments, lipid panel is used to measure improvement, stabilization or slowing of changes in hypercholesterolemia. In some embodiments, the subject shows improvement, stabilization or slowing of symptoms of hypercholesterolemia, cardiovascular disease, coronary artery disease or atherosclerosis.

[0043] In some embodiments, the composition or pharmaceutical formulation is administered via a viral vector. In some embodiments, the composition or pharmaceutical formulation is administered via a lipid nanoparticle.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs. Methods and materials for use in this application are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0045] Other features and advantages of the methods and materials described herein will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A graph showing the editing efficiency of various sgRNAs targeting the human PCSK9 gene in HepG2 cells.

[0047] Figure 2 Graph showing EC50 and maximal editing of human PCSK9 sgRNA delivered together with Cas9 mRNA in Cos-7 cells.

[0048] Figure 3 Graph showing EC50 and maximal editing of human PCSK9 sgRNA delivered with Cas9 mRNA in primary cynomolgus monkey hepatocyte (PCH) cells.

[0049] Figure 4 Graph showing EC50 and maximal editing of human PCSK9 sgRNA delivered with Cas9 mRNA containing various engineered untranslated regions (UTRs) in Huh7 cells.

[0050] Figure 5 Graph showing EC50 and maximal editing of human PCSK9 sgRNA delivered with Cas9 mRNA containing various engineered coding sequences in Huh7 cells. DETAILED DESCRIPTION

[0051] The present application describes compositions and methods for editing the human proprotein convertase subtilisin / kexin type 9 (PCSK9) gene. The compositions and methods described in the present application are used to treat subjects with hypercholesterolemia and / or cardiovascular disease associated with PCSK9.

[0052] Before describing the present teachings in detail, it should be understood that the application is not limited to specific compositions or process steps, as these may vary. It should be noted that as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a conjugate" includes a plurality of conjugates, reference to "a cell" includes a plurality of cells, and so forth.

[0053] definition

[0054] Unless otherwise stated, the following terms and phrases used herein shall have the following meanings:

[0055] As used herein, the term "nucleic acid" refers to a polymer compound having nucleosides or nucleoside analogs having nitrogenous heterocyclic bases or base analogs linked together along the backbone, including conventional RNA, DNA, hybrid RNA-DNA, and polymers as analogs thereof. The terms "nucleic acid", "polynucleotide", "nucleotide", "nucleotide sequence", and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. The following are non-limiting examples of nucleic acids: coding or non-coding regions of genes or gene fragments, one or more loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. The term also encompasses nucleic acid-like structures with synthetic backbones, see, e.g., Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97 / 03211; WO 96 / 39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure may be modified before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0056] The nucleic acid backbone can be composed of a variety of bonds, including one or more of a sugar-phosphodiester bond, a peptide-nucleic acid bond ("peptide nucleic acid" or PNA, such as those described in International Patent Publication No. WO1995032305), a phosphorothioate bond, a methylphosphonate bond, or a combination thereof. The sugar portion of the nucleic acid can be ribose, deoxyribose, or a similar compound having a substituent (e.g., 2' methoxy or 2' halide substitution). The nitrogenous base can be a conventional base (A, G, C, T, U), an analog thereof (e.g., a modified uridine such as 5-methoxyuridine, pseudouridine or N1-methylpseudouridine, etc.); inosine; a derivative of a purine or pyrimidine (e.g., N4-methyldeoxyguanosine, a deaza or azapurine, a deaza or azapyrimidine, a pyrimidine base having a substituent at the 5 or 6 position (e.g., 5-methylcytosine), a purine base having a substituent at the 2, 6 or 8 position, 2-amino-6-methylaminopurine, 06-methylguanine, 4-thiopyrimidine, 4-aminopyrimidine, 4-dimethylhydrazinepyrimidine and 04-alkylpyrimidine; (see, e.g., U.S. Pat. No. 5,378,825 and International Patent Publication No. WO1993013121). For a general discussion, see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992). Nucleic acids may include one or more "absic" residues, in which the backbone does not include a nitrogenous base at one or more positions of the polymer (see, e.g., U.S. Pat. No. 5,585,481). Nucleic acids may contain only conventional RNA or DNA sugars, bases, and bonds, or may include conventional components and substitutes (e.g., conventional bases with 2' methoxy bonds, or polymers containing both conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNA), which are analogs containing one or more LNA nucleotide monomers in which the bicyclic furanose unit is locked in an RNA-mimicking sugar conformation, thereby enhancing hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43 (42): 13233-41). RNA and DNA have different sugar moieties and may differ by the presence of uracil or its analogs in RNA and thymine or its analogs in DNA.

[0057] As used herein, the term "guide RNA" refers to a combination of CRISPR RNA (crRNA) and tracr RNA (trRNA). "Guide RNA" can be used interchangeably with "gRNA" or "guide". CrRNA and trRNA can be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" can refer to each type, i.e., sgRNA or dgRNA. TrRNA can be a naturally occurring sequence, or the trRNA sequence can have a modification or variation compared to a naturally occurring sequence. Guide RNA can include modified RNA as described herein.

[0058] As used herein, "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and is used to guide the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binder. "Guide sequence" may also be referred to as a "targeting sequence" or a "spacer sequence". The guide sequence may be about 20 base pairs in length, for example, in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences may also be used as guide sequences, for example, with a length of 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the guide sequence and the target sequence may be 100% complementary or identical to each other in sequence. In other embodiments, the guide sequence and the target sequence may include at least one mismatch. For example, the guide sequence and the target sequence may include 1, 2, 3, or 4 mismatches, wherein the total length of the targeting sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and the target sequence may comprise 1-4 mismatches, wherein the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target sequence may comprise 1, 2, 3 or 4 mismatches, wherein the guide sequence comprises at least 20 nucleotides.

[0059] In some embodiments, the guide RNA comprises a crRNA having a guide sequence (e.g., a guide sequence from Table 4), and further comprises a nucleotide sequence GUU UUA GAG CUA UGC UGU UUU G (SEQ ID NO: 889), wherein SEQ ID NO: 889 is immediately adjacent to the 3' end of the guide sequence. In some embodiments, the crRNA is any crRNA selected from the nucleotide sequences shown in SEQ ID NOs: 297-592. In some embodiments, the guide RNA comprises any crRNA nucleotide sequence shown in SEQ ID NOs: 297-592.

[0060] In some embodiments, the guide RNA comprises a crRNA and further comprises a tracrRNA (trRNA) sequence comprising the nucleotide sequence shown in SEQ ID NO: 904 or a portion thereof. AAC AGC AUA GCAAGU UAA AAU AAG GCU AGU CCG UUA UCA ACU UGA AAA AGU GGC ACC GAG UCG GUG CUUUUU UU (SEQ ID NO: 904).

[0061] In some embodiments, the guide RNA comprises additional nucleotides to form an sgRNA, for example, having the following exemplary nucleotide sequence after the 3' end of the guide sequence: GUU UUA GAG CUA GAA AUA GCA AGU UAA AAU AAGGCU AGU CCG UUA UCA ACU UGA AAA AGU GGC ACC GAG UCG GUG CUU UU (SEQ ID NO: 890), in a 5' to 3' direction. In some embodiments, the sgRNA is any sgRNA selected from the nucleotide sequences shown in SEQ ID NOs: 593-888. In some embodiments, the gRNA comprises any one of the nucleotide sequences shown in SEQ ID NOs: 593-888. In some embodiments, the gRNA consists of any one of the nucleotide sequences shown in SEQ ID NOs: 593-888.

[0062] In some embodiments, the guide RNA comprises a portion of SEQ ID NO: 889 covalently linked to a trRNA. For example, a guide RNA comprises a guide sequence (e.g., a guide sequence from Table 4) linked to GUUUUAGAGCUA (SEQ ID NO: 905), which is further linked to a trRNA (SEQ ID NO: 904 or a portion thereof). For example, a guide RNA comprises a guide sequence (e.g., a guide sequence from Table 4) linked to GUU UUA GAG CUA (SEQ ID NO: 905), which is further linked to the nucleotide sequence AUA GCA AGU UAA AAU AAG GCU AGU CCG UUA UCA ACU UGA AAA AGU GGC ACCGAG UCG GUG CUU UU (SEQ ID NO: 906).

[0063] The targeting sequence of the Cas protein includes the positive and negative strands of the genomic DNA (i.e., a given sequence and the reverse complementary sequence of the sequence) because the nucleic acid substrate of the Cas protein is double-stranded. Therefore, when the guide sequence is referred to as "complementary to the target sequence", it should be understood that the guide sequence can guide the guide RNA to bind to the reverse complementary sequence of the target sequence. Therefore, in some embodiments in which the guide sequence binds to the reverse complementary sequence of the target sequence, in addition to replacing T with U in the guide sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., a target sequence excluding the protospacer adjacent motif (PAM)).

[0064] As used herein, "RNA-guided DNA binder" refers to a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binders (such as those described in International Patent Application No. WO2020198697, the entire contents of which are incorporated herein) include Cas nickases and inactivated forms thereof, such as dCas DNA binders".

[0065] As used herein, the term "Cas" refers to any Cas protein that can be operably used to perform gene editing using a guide molecule. "Cas nuclease" also covers Cas nickases, and endonuclease-deficient or inactivated Cas (dCas) DNA binders. Cas nickases and dCas DNA binders can include Csm or Cmr complexes of type III CRISPR systems, its Cas10, Csml or Cmr2 subunits, Cascade complexes of type I CRISPR systems, its Cas3 subunits, and 2 types of Cas nucleases. As used herein, "2 types of Cas nucleases" are single-chain polypeptides with RNA-guided DNA binding activity, such as Cas9 nucleases or Cpf1 nucleases. 2 types of Cas nucleases include 2 types of Cas nickases (e.g., H840A, D10A or N863A variants), which also have RNA-guided DNA nickase activity, and 2 types of dCas DNA binders, wherein the nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2cl, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9 (1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9 (1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9 and contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence is incorporated by reference in its entirety. See, e.g., Zetsche, Tables SI and S3. "Cas9" encompasses Spy Cas9, the Cas9 variants listed herein, and their equivalents. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0066] dCas DNA binders can be used for CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). In CRISPRi, dCas9 binds to its DNA target but does not cleave it. Without being bound by theory, it is believed that Cas9 alone will prevent the cell's transcription machinery from approaching the promoter, thereby inhibiting gene expression. On the other hand, the ability of dCas9 to bind to target DNA can be used for activation, i.e. CRISPRa. Transcription activators are fused to dCas9, which can activate gene expression without changing the DNA sequence. In some embodiments, dCas DNA binders are fused to repressors, such as Krüppel-associated boxes (KRAB).

[0067] "Modified uridine" as used herein refers to a nucleoside having the same hydrogen bond acceptor as uridine and having one or more structural differences with uridine, including but not limited to deoxythymidine. In some embodiments, the modified uridine is a substituted uridine, that is, a uridine in which one or more non-proton substituents (e.g., alkoxy, such as methoxy) replace protons. In some embodiments, the modified uridine is a pseudouridine. In some embodiments, the modified uridine is a substituted pseudouridine, that is, a pseudouridine in which one or more non-proton substituents (e.g., alkyl, such as methyl) replace protons, for example, N1-methyl pseudouridine. In some embodiments, the modified uridine is any one of a substituted uridine, a pseudouridine, or a substituted pseudouridine.

[0068] As used herein, a first sequence is said to "comprise a sequence at least X% identical to a second sequence" if an alignment of the first sequence with a second sequence shows that X% or more of the positions in the second sequence as a whole match the first sequence. For example, the sequence AAGA comprises a sequence that has 100% identity to the sequence AAG, since the alignment would yield 100% identity due to matches to all three positions in the second sequence. Differences between RNA and DNA (typically the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs (such as modified uridine) do not result in differences in identity or complementarity between polynucleotides, as long as related nucleotides (such as thymidine, uridine or modified uridine) are bound to the same complementary nucleotide (e.g., adenosine is the complement of all thymidine, uridine or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5'-AXG (wherein X is any modified uridine, such as pseudouridine, N1-methylpseudouridine or 5-methoxyuridine) is considered to be 100% identical to AUG, since both are fully complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. One skilled in the art will understand which algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences that are generally similar in length and have an expected identity of >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm using the default settings of the Needleman-Wunsch algorithm interface provided by EBI on the www.ebi.ac.uk web server is generally appropriate.

[0069] As used herein, the term "mRNA" refers to a polynucleotide that is an RNA or modified RNA that includes an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and aminoacylated tRNAs). The mRNA may include a phosphate-sugar backbone having a ribose residue or an analog thereof (e.g., a 2'-methoxyribose residue). In some embodiments, the sugar of the nucleic acid phosphate-sugar backbone consists essentially of a ribose residue, a 2'-methoxyribose residue, or a combination thereof.

[0070] As used herein, the term "PCSK9" refers to proprotein convertase subtilisin / kexin type 9, which is the expression product of the PCSK9 gene. The human wild-type PCSK9 sequence can be obtained at NCBI Gene ID: 255738; Ensembl: ENSG00000169174. PCSK9 contains four major components in the pre-processed protein: signal peptide (amino acid residues 1-30); N-terminal prodomain (residues 31-152); catalytic domain (residues 153-425); C-terminal domain (residues 426-692), which is further divided into three modules (Du F et al. Novel domain interaction regulates secretion of proprotein convertase subtilisin / kexin type 9 (PCSK9) protein. J Biol Chem. 2011 Dec 16; 286 (50): 43054-61.). The PCSK9 gene is located at the cytogenetic position 1p32.3 and contains a total of 14 exons that can be selectively spliced. The PCSK9 protein is a member of the subtilisin-like proprotein convertase family, which includes enzymes that process protein and peptide precursors that are transported through the regulated or constitutive branches of the secretory pathway. The encoded protein undergoes autocatalytic processing events in its pro-fragment in the ER and is constitutively secreted into the extracellular matrix and the trans-Golgi network in the form of an inactive protease. It is expressed in liver, intestine, vascular epithelium, and kidney tissues, and escorts specific receptors into lysosomes for degradation. It plays a role in cholesterol and fatty acid metabolism. Mutations in this gene are associated with autosomal dominant familial hypercholesterolemia. Alternative splicing produces a variety of transcript variants. The gene product of PCSK9 (i.e., PCSK9 protein) referred to in this article as "mutant PCSK9" has changed in the amino acid sequence of PCSK9 compared to the wild-type amino acid sequence of PCSK9. PCSK9 mutant forms that correlate with patient LDLR levels include, for example, R46L, S127R, Y142X, R218S, F216L, D374Y, A443T, and C679X.

[0071] As used herein, "low-density lipoprotein (LDL)" refers to particles containing a variety of proteins (e.g., about 80-100 proteins) that transfer lipids through aqueous fluids so that cells can use the lipids for receptor-mediated endocytosis. A single LDL particle may be about 220-275 angstroms in diameter, and typically transports about 3,000 to about 6,000 lipid molecules per particle, with the size varying depending on the number and composition of the lipid molecules contained within the particle. For example, LDL particles may carry a mixture of cholesterol, phospholipids, and triglycerides. It is well known in the art that elevated levels of LDL detected in the blood are associated with an increased risk of cardiovascular disease.

[0072] As used herein, "low-density lipoprotein receptor (LDLR)" refers to a cell surface receptor that mediates the endocytosis of LDL particles. For example, LDLR recognizes apolipoprotein B100 embedded in the outer phospholipid layer of LDL particles. The LDLR protein is encoded by the LDLR gene on chromosome 19 of the human genome. It is well known that the function of LDLR is related to cholesterol metabolism, and the disruption of LDLR increases the risk of diseases related to cholesterol metabolism.

[0073] As used herein, "hypercholesterolemia" refers to a higher than normal cholesterol level in a subject's blood. A normal blood cholesterol level is a number derived by laboratory analysis. A normal or ideal cholesterol level is defined as less than 200 mg of cholesterol per deciliter of blood (mg / dL). If the cholesterol level in the blood is within the range of 200 to 239 mg / dL, it is considered to be at a borderline level. An elevated cholesterol level is 240 mg / dL or more, but there is no absolute cutoff between normal and abnormal cholesterol levels, and cholesterol values ​​must be considered in conjunction with other health conditions and risk factors. Elevated blood cholesterol is considered hypercholesterolemia.

[0074] As used herein, "familial hypercholesterolemia" refers to an inherited form of hypercholesterolemia, which may be caused, for example, by an elevated polygenic risk of hypercholesterolemia or an inherited single gene mutation that increases the risk of hypercholesterolemia. It is known in the art that familial hypercholesterolemia may be inherited, for example, in an autosomal dominant or autosomal recessive manner.

[0075] As used herein, "atherosclerosis" refers to the buildup of fat, cholesterol, and other substances in and on the walls of your arteries. This buildup is called plaque. Plaque can cause your arteries to narrow, blocking blood flow. Plaque can also rupture, causing a blood clot.

[0076] As used herein, the term "pathological mutation" refers to a mutation that makes a gene product (eg, PCSK9 protein) more likely to cause, promote, contribute to, or fail to inhibit the development of a disease (such as hypercholesterolemia or cardiovascular disease).

[0077] As used herein, "indel" refers to an insertion / deletion mutation consisting of multiple nucleotides inserted into or deleted from a polynucleotide sequence. For example, an indel may occur at a double-strand break (DSB) site in a target nucleic acid.

[0078] As used herein, "knock down" refers to the reduction of expression of a specific gene product (e.g., protein, mRNA or both). The knock down of a protein can be measured by detecting a protein secreted by a tissue or cell group (e.g., in serum or cell culture medium) or by detecting the total cell amount of a protein from a tissue or cell group of interest before and after knocking down. The method of measuring mRNA knocking down is known in the art, and includes sequencing the mRNA separated from a tissue or cell group of interest. In some embodiments, "knock down" can refer to some losses of expression of a specific gene product, for example, the amount of mRNA transcribed by a cell group (including a cell group in vivo, such as a cell group present in a tissue) is reduced or the amount of protein expressed or secreted is reduced.

[0079] As used herein, "target sequence" refers to a nucleic acid sequence in a target gene that is complementary to the guide sequence of a gRNA. The interaction of the target sequence and the guide sequence allows the RNA-guided DNA binder to bind to the target sequence and may produce a nick or cleavage within the target sequence (depending on the activity of the binder).

[0080] As used herein, "treatment" or "treatment" refers to the improvement, alleviation or reduction of at least one symptom of a disclosed condition after administration or application of a therapeutic agent for the condition. The term includes inhibiting the condition or disease, preventing its development, alleviating one or more symptoms of the condition or disease, curing the condition or disease, or preventing the recurrence of one or more symptoms of the condition or disease. In the context of the present application, treatment of hypercholesterolemia and / or cardiovascular disease may include alleviating the symptoms of hypercholesterolemia and / or cardiovascular disease. If the treatment results in a reduction in the pathology of the condition, the treatment using the composition of the present application is said to have "treated" the condition.

[0081] As used herein, the term "lipid nanoparticle" (LNP) refers to a particle comprising a plurality (i.e., more than one) of lipid molecules that are physically associated with each other by intermolecular forces. LNPs can be, for example, microspheres (including unilamellar and multilamellar vesicles, such as "liposomes" - lamellar phase lipid bilayers, in some embodiments, substantially spherical - and in more specific embodiments, can include an aqueous core, for example, including a majority of RNA molecules), a dispersed phase in an emulsion, an internal phase in a micelle or a suspension. See also, for example, WO2015006747, WO2016118724, WO2021026358, WO2017173054, and WO2019067992, the contents of which are incorporated herein by reference in their entirety. Any LNP known to those skilled in the art capable of delivering nucleotides to a subject can be used with guide RNA and nucleic acids encoding RNA-guided DNA binders described herein.

[0082] As used herein, the term "pharmaceutically acceptable" refers to a biologically acceptable gaseous, liquid or solid formulation, or a mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. A "pharmaceutically acceptable" composition is a material that does not cause biological or other adverse effects, for example, the material can be administered to a subject without causing significant adverse biological effects.

[0083] As used herein, "infusion" refers to the active administration of one or more agents, and the infusion time is, for example, about 30 minutes to 12 hours. In some embodiments, the one or more agents include LNPs, such as mRNA encoding an RNA-guided DNA binder (such as Cas9) described herein and a gRNA described herein.

[0084] The term "about" or "approximately" means an acceptable error for a particular value determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In some embodiments, "about" refers to, for example, a difference of less than plus or minus 5% (e.g., less than plus or minus 1%, less than 0.5%, or less than 0.1%).

[0085] Numerical ranges include the numbers defining the range. Taking into account significant figures and errors associated with measurement, measured values ​​and measurable values ​​should be understood as approximate values. In addition, the use of "comprise, comprises, comprising", "contain, contains, containing" and "include, includes, including" is not intended to be limiting. It should be understood that the foregoing general description and detailed description are only exemplary and explanatory and do not limit the teaching.

[0086] Unless otherwise specified in the above description, the embodiments described in the description "including" various components should also be understood as "consisting of the components" or "consisting essentially of the components"; the embodiments described in the description "consisting of various components" should also be understood as "including" or "consisting essentially of the components"; the embodiments described in the description "consisting essentially of various components" should also be understood as "consisting of the components" or "including" the components (this interchangeability does not apply to the use of these terms in the claims). Unless the context clearly indicates otherwise, the term "or" is used in an inclusive sense, that is, equivalent to "and / or".

[0087] Compositions and methods targeting the PCSK9 gene

[0088] Disclosed herein are compositions for methods for targeting PCSK9 genes. The methods disclosed herein induce double-strand breaks (DSBs) in the PCSK9 gene of a subject, modify the PCSK9 gene of a cell or subject, treat hypercholesterolemia and / or cardiovascular disease associated with PCSK9 in a subject, reduce PCSK9 abundance in subject cells, increase the abundance of LDLR on the surface of subject cells, and / or reduce LDL levels in the circulation of a subject. In some embodiments, the disclosed compositions and methods inhibit the transcription of the PCSK9 gene and the translation of the PCSK9 protein, thereby preventing the accumulation of PCSK9 in tissues. In general, the disclosed compositions include: guide RNAs targeting PCSK9 (either by themselves or in a vector), and RNA-guided DNA binders, or nucleic acids encoding RNA-guided DNA binders (e.g., CRISPR / Cas systems). Subjects treated with such methods and compositions may have wild-type or non-wild-type PCSK9 gene sequences, such as, for example, subjects with hypercholesterolemia or familial hypercholesterolemia, wherein the patient may carry a genetic PCSK9 mutation. In some embodiments, the composition is administered by infusion for 0.5-6 hours. In some embodiments, the composition is administered by subcutaneous injection. In some embodiments, the composition is administered by intrathecal injection.

[0089] A. Guide RNA (gRNA)

[0090] The guide RNA used in the disclosed methods and compositions comprises a guide sequence that targets the PCSK9 gene. Exemplary guide sequences that target the PCSK9 gene are shown in SEQ ID NOs: 1-296 in Table 4. Guide sequences useful in the guide RNA compositions and methods described herein are shown in Table 4 and throughout the application.

[0091] Each guide sequence in Table 4 may further comprise additional nucleotides to form a crRNA, for example, immediately following the 3' end of the guide sequence with the following exemplary nucleotide sequence: GUU UUA GAG CUA UGC UGU UUU G (SEQ ID NO: 889). In the case of sgRNA, the guide sequence of Table 4 may further comprise additional nucleotides to form an sgRNA, for example, immediately following the 3' end of the guide sequence with the following exemplary nucleotide sequence, wherein the sgRNA has a custom-designed short crRNA component followed by a trRNA component: GUU UUA GAG CUA GAA AUA GCA AGU UAA AAU AAG GCU AGU CCG UUA UCA ACUUGA AAA AGU GGC ACC GAG UCG GUG CUU UU (SEQ ID NO: 890), in a 5' to 3' direction.

[0092] SEQ ID NO: 890 is attached to the 3' end of the guide sequence in a 5' to 3' direction. sgRNA sequences useful in the compositions and methods of the present application are described in Table 5.

[0093] In some embodiments, the sgRNA is modified. In some embodiments, the sgRNA comprises the modification pattern shown in SEQ ID NO: 907 below, wherein N is any natural or non-natural nucleotide, and wherein all N comprises a guide sequence as described herein, and the modified sgRNA comprises the following sequence: mN*mN*mN*NNN NNN NNN NNN NNNNNG UUU UAG AmGmCm UmAmGm AmAmAm UmAmGm CAA GUU AAA AUA AGG CUA GUC CGU UAUCAmAm CmUmUm GmAmAm AmAmAm GmUmGm GmCmAm CmCmGm AmGmUm CmGmGm UmGmCm U*mU*mU*mU (SEQ ID NO: 907), wherein "N" can be any natural or non-natural nucleotide; * = PS bond; 'm' = 2'-O-Me nucleotide. Although N is replaced by the guide nucleotide, the modifications are retained in SEQ ID NO: 907. That is, although the guide nucleotide replaces "N", the first three nucleotides are 2'OMe modified, and there are phosphorothioate bonds between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides.

[0094] In some embodiments, the gRNA sequence has a modification pattern described in WO2016164356 and WO2016089433, each of which is incorporated herein in its entirety.

[0095] In some embodiments, the gRNA comprises a guide sequence that guides the RNA-guided DNA binder (which can be a nuclease (e.g., a Cas nuclease, such as Cas9)) to a target DNA sequence in PCSK9. The gRNA includes a crRNA having a guide sequence as shown in Table 4. The gRNA includes a guide sequence having at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides of any one of the guide sequences of SEQ ID NOs: 1-296 as shown in Table 4. In some embodiments, the gRNA comprises a guide sequence having a sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to at least 16, 17, 18, 19, or 20 consecutive nucleotides of any one of the guide sequences of SEQ ID NOs: 1-296 as shown in Table 4. The gRNA may also include a tracr RNA (trRNA). In each of the compositions and method embodiments described herein, crRNA and trRNA can be associated into a single RNA (sgRNA), or can be located on separate RNAs (dgRNA). In the case of sgRNA, the crRNA and trRNA components can be covalently linked, for example, via a phosphodiester bond or other covalent bond.

[0096] In each of the compositions, uses, and method embodiments described herein, the guide RNA may comprise two RNA molecules, as a "dual guide RNA" or "dgRNA". The dgRNA comprises a first RNA molecule comprising a crRNA having a guide sequence, such as shown in Table 4, and a second RNA molecule having a trRNA. The first and second RNA molecules may not be covalently linked, but may form an RNA duplex by base pairing between a portion of the crRNA and the trRNA.

[0097] In each composition, use and method embodiment described herein, the guide RNA may comprise a single RNA molecule as a "single guide RNA" or "sgRNA". The sgRNA may comprise a crRNA (or a portion thereof) having a guide sequence as shown in Table 3, which is covalently linked to the trRNA. The sgRNA may comprise at least 15, 16, 17, 18, 19 or 20 consecutive nucleotides of any guide sequence of SEQ ID NO: 1-296 shown in Table 4. In some embodiments, the crRNA and trRNA are covalently linked via a linker. In some embodiments, the sgRNA forms a stem-loop structure via base pairing between a portion of the crRNA and the trRNA. In some embodiments, the crRNA and the trRNA are covalently linked by one or more bonds that are non-phosphodiester bonds.

[0098] In some embodiments, trRNA may include all or part of a trRNA sequence derived from a naturally occurring CRISPR / Cas system. In some embodiments, trRNA includes truncated or modified wild-type trRNA. The length of trRNA depends on the CRISPR / Cas system used. In some embodiments, trRNA includes or is composed of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 nucleotides. In some embodiments, trRNA may include some secondary structures, such as, for example, one or more hairpins or stem-loop structures, or one or more protruding structures. In some embodiments, the composition comprises a gRNA comprising a guide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least 16, 17, 18, 19, or 20 consecutive nucleotides of any one of the guide sequences of SEQ ID NOs: 1-296 shown in Table 4.

[0099] In some embodiments, the composition comprises a guide RNA having a guide sequence selected from SEQ ID NO: 1-296. The guide RNA having a guide sequence selected from SEQ ID NO: 1-296 can be a chemically modified sgRNA, such as a terminally modified RNA. The guide RNA having a guide sequence selected from SEQ ID NO: 1-296 can be a dgRNA, such as a chemically modified dgRNA.

[0100] In other embodiments, the composition comprises at least one (e.g., at least two) gRNAs having a guide sequence selected from any two or more of the guide sequences of SEQ ID NOs: 1-296. In some embodiments, the composition comprises at least two gRNAs, each of which comprises a guide sequence that is at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any nucleic acid of SEQ ID NOs: 1-296.

[0101] In some embodiments, the gRNA is an sgRNA having any one of SEQ ID NOs: 593-888. In some embodiments, the gRNA is an sgRNA having any one of SEQ ID NOs: 593-888, but without the modifications described in the present application (ie, unmodified SEQ ID NOs: 593-888). In some embodiments, the gRNA is an sgRNA having any one of SEQ ID NOs: 593-888, but with at least one chemical modification. In some embodiments, the chemically modified SEQ ID NOs: 593-888 has a 5' and / or 3' end modification. In some embodiments, the gRNA is an sgRNA having any one of SEQ ID NOs: 593-888, but with a modification pattern shown in SEQ ID NO: 907.

[0102] The guide RNA provided herein can be used to identify (e.g., hybridize) a target sequence in a PCSK9 gene. For example, a PCSK9 target sequence can be recognized and cleaved by a Cas nuclease with a guide RNA provided. Therefore, an RNA-guided DNA binder (such as a Cas nuclease) can be guided to a target sequence of a PCSK9 gene by a guide RNA, wherein the guide sequence of the guide RNA hybridizes with the target sequence, and the RNA-guided DNA binder (such as a Cas nuclease) cleaves the target sequence.

[0103] In some embodiments, the selection of one or more guide RNAs is determined based on a target sequence within the PCSK9 gene. For example, one or more guide RNAs are based on a target sequence within any one of exons 1-14 or 5'UTR or 3'UTR of the PCSK9 gene.

[0104] Without being bound by any particular theory, mutations in certain regions of the gene (e.g., frameshift mutations due to insertions / deletions caused by nuclease-mediated DSBs) may be less tolerated than mutations in other regions of the gene, and therefore, the location of the DSB is an important factor in the amount or type of protein knockdown that may result. In some embodiments, a gRNA that is complementary or has complementarity to a target sequence within PCSK9 is used to guide an RNA-guided DNA binder to a specific location in the PCSK9 gene. In some embodiments, the gRNA is designed to have a guide sequence that is complementary or has complementarity to a target sequence in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, or exon 14 of PCSK9. In some embodiments, a frameshift or nonsense mutation is induced in the PCSK9 gene in about 10%, about 15%, about 20%, about 25%, about 30% to about 35% of the cells.

[0105] B. gRNA modification

[0106] In some embodiments, the gRNA is chemically modified. A gRNA having one or more modified nucleosides or nucleotides is referred to as a "modified" gRNA or a "chemically modified" gRNA to describe the presence of one or more non-natural and / or naturally occurring components or configurations that are used to replace or supplement typical A, G, C, and U residues. In some embodiments, a modified gRNA is synthesized with atypical nucleosides or nucleotides, referred to herein as "modified". Modified nucleosides and nucleotides may include one or more of the following: (i) alteration (e.g., replacement) of one or both non-linked phosphate oxygens and / or one or more linked phosphate oxygens in the phosphodiester backbone linkage (exemplary backbone modifications); (ii) alteration (e.g., replacement) of a ribose component (e.g., the 2' hydroxyl group on the ribose) (exemplary sugar modifications); (iii) global replacement of the phosphate moiety with a "dephosphorylated" linker (exemplary backbone modification); (iv) modification or replacement of naturally occurring nucleobases, including modification or replacement with atypical nucleobases (exemplary base modifications); (v) replacement or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) modification of the 3' or 5' end of the oligonucleotide, such as removal, modification or replacement of a terminal phosphate group or a conjugated moiety, cap or linker (such 3' or 5' cap modifications may include sugar and / or backbone modifications); and (vii) modification or replacement of the sugar (exemplary sugar modifications).

[0107] Chemical modifications such as those described above can be combined to provide modified gRNAs having nucleosides and nucleotides (collectively referred to as "residues") that may have two, three, four or more modifications. For example, the modified residues may have modified sugars and modified nucleobases. In some embodiments, each base of the gRNA is modified, for example, all bases have modified phosphate groups, such as thiophosphate groups. In certain embodiments, all or substantially all phosphate groups of the gRNA molecule are replaced by thiophosphate groups. In some embodiments, the modified gRNA comprises at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified gRNA comprises at least one modified residue at or near the 3' end of the RNA.

[0108] In some embodiments, the gRNA comprises one, two, three or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%) of the position in the modified gRNA is a modified nucleoside or nucleotide.

[0109] Unmodified nucleic acids may be susceptible to degradation by, for example, nucleases in cells or serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Therefore, in one aspect, the gRNA described herein may include one or more modified nucleosides or nucleotides, for example, to introduce stability to intracellular or serum nucleases. In some embodiments, the modified gRNA molecules described herein may show reduced innate immune responses when introduced into cell populations in vivo and in vitro. The term "innate immune response" includes cellular responses to exogenous nucleic acids (including single-stranded nucleic acids), which involve inducing cytokine expression and release (particularly interferon) and cell death.

[0110] In some embodiments of backbone modification, the phosphate group of the modified residue can be modified by replacing one or more oxygens with different substituents. In addition, the modified residue (e.g., the modified residue present in the modified nucleic acid) can include the comprehensive replacement of the unmodified phosphate moiety with the modified phosphate group described herein. In some embodiments, the backbone modification of the phosphate backbone can include the change that causes an uncharged joint or a charged joint with an asymmetric charge distribution.

[0111] The example of the phosphate group of modification comprises phosphorothioate, phosphoroselenate, boranophosphoric acid, boranophosphate, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester.The phosphorus atom in the unmodified phosphate group is achiral.But, replacing one of the non-bridging oxygen with one of the above-mentioned atoms or atomic groups can make the phosphorus atom have chirality.The stereoisomerism phosphorus atom can have " R " configuration (here Rp) or " S " configuration (here Sp).The main chain can also be modified by replacing the bridging oxygen (i.e. the oxygen connecting phosphate and nucleosides) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate) and carbon (bridging methylene phosphonate).Replacement can occur at any connection oxygen place or two connection oxygen places.

[0112] In some main chain modifications, the phosphate group can be replaced by a phosphorus-free linker. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of substitutable phosphate groups include, but are not limited to, for example, methylphosphonates, hydroxyamino groups, siloxanes, carbonates, carboxymethyl groups, carbamates, amides, thioethers, ethylene oxide joints, sulfonates, sulfonamides, thioformal, methylal, oximes, methyleneimino groups, methylenemethylimino groups, methylenehydrazine groups, methylenedimethylhydrazine groups, and methyleneoxymethylimino groups.

[0113] Can also construct the framework that can simulate nucleic acid, wherein phosphate joint and ribose are replaced by nuclease resistant nucleoside or nucleotide substitute.Such modification can comprise main chain and sugar modification.In some embodiments, core base can be connected by alternative main chain.Example can include but not limited to, morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside substitute.

[0114] Modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e., sugar modifications. For example, the 2' hydroxyl (OH) can be modified, for example, replaced with a number of different "oxygen" or "deoxy" substituents. In some embodiments, the modification of the 2' hydroxyl can enhance the stability of the nucleic acid because the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion.

[0115] Examples of 2'hydroxyl modifications can include alkoxy or aryloxy (OR, where "R" can be, for example, an alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), 0(CH2CH20)nCH2CH2OR, where R can be, for example, H or an optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20). In some embodiments, the 2'hydroxyl modification can be 2'-O-Me. In some embodiments, the 2'hydroxyl modification can be a 2'-fluoro modification, which replaces the 2'hydroxyl with fluoride. In some embodiments, the 2' hydroxyl modification may include a "locked" nucleic acid (LNA), wherein the 2' hydroxyl may be connected to the 4' carbon of the same ribose by, for example, a C1-6 alkylene or C1-6 heteroalkylene bridge, wherein exemplary bridges may include methylene, propylene, ether or amino bridges; O-amino (wherein the amino group may be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino) and aminoalkoxy, O(CH2)n-amino (wherein the amino group may be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino). In some embodiments, the 2' hydroxyl modification may include an "unlocked" nucleic acid (UNA), wherein the ribose ring lacks a C2'-C3' bond. In some embodiments, the 2' hydroxyl modification may include a methoxyethyl (MOE) (OCH2CH2OCH3, for example, a PEG derivative).

[0116] "Deoxy" 2' modifications can include hydrogen (i.e., deoxyribose, such as in the overhang of a portion of a dsRNA); halogen (e.g., bromine, chloride, fluorine, or iodine); amino (wherein the amino group can be, for example, NEE; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or an amino acid); NH(CH2CH2NH)nCH2CH2-amino (wherein the amino group can be, for example, as described herein), -NHC(0)R (wherein R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), cyano; thiol; alkylthioalkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl groups, which can be optionally substituted with, for example, amino groups as described herein.

[0117] Sugar modification can include sugar groups, and this sugar group can also include one or more carbons with stereochemical configuration opposite to the corresponding carbon in ribose.Therefore, the nucleic acid of modification can include nucleotides containing, for example, arabinose as sugar.The nucleic acid of modification can also include abasic sugars.These abasic sugars can also be further modified at one or more composition sugar atoms.The nucleic acid of modification can also include one or more L forms of sugar, for example L-nucleosides.

[0118] The modified nucleosides and modified nucleotides that can be incorporated into the modified nucleic acids described herein may include modified bases, also referred to as core bases. Examples of core bases include, but are not limited to, adenine (A), guanine (G), cytosine (C) and uracil (U). These core bases may be modified or completely replaced to provide modified residues that can be incorporated into the modified nucleic acids. The core bases of nucleotides may be independently selected from purine, pyrimidine, purine analogs or pyrimidine analogs. In some embodiments, core bases may include, for example, naturally occurring and synthetic derivatives of bases.

[0119] In embodiments employing dual guide RNAs, each of the crRNA and tracr RNA may include modifications. Such modifications may be located at one or both ends of the crRNA and / or tracr RNA. In embodiments with sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, or the entire sgRNA may be chemically modified. Certain embodiments include 5' end modifications. Certain embodiments include 3' end modifications. In certain embodiments, one or more or all nucleotides in the single-stranded overhang of the guide RNA molecule are deoxynucleotides.

[0120] In some embodiments, the gRNA may have one or more modifications. In some embodiments, the modification includes 2'-O-methyl (2'-O-Me) modified nucleotides. In some embodiments, the modification includes phosphorothioate (PS) bonds between nucleotides.

[0121] In some embodiments, the gRNA is a DNA-RNA hybrid. In some embodiments, the guide RNA is a hybrid DNA-RNA guide. In some embodiments, the hybrid DNA-RNA guide comprises a sequence selected from SEQ ID NOs: 908-940. In some embodiments, at least a portion of the sgRNA is a hybrid DNA-RNA guide. Exemplary DNA-RNA hybrid guide sequences are provided in Table 1 below. For the sequences provided in Table 1 below, "d" indicates that the base after the letter "d" is a deoxyribonucleotide, and the letter without a "d" in front is a ribonucleotide.

[0122] Table 1: Exemplary DNA-RNA hybrid guide sequences

[0123]

[0124]

[0125] The terms "mA," "mC," "mU," or "mG" may be used to denote a nucleotide that has been modified with 2'-O-Me.

[0126] In some embodiments, the guide RNA includes an sgRNA having a guide sequence selected from SEQ ID NOs: 1-296 and nucleotides of SEQ ID NO: 890, wherein the nucleotides of SEQ ID NO: 890 are located at the 3' end of the guide sequence, and wherein the guide sequence can be modified as shown in SEQ ID NO: 907.

[0127] Further examples of gRNA modifications are shown, for example, in WO2020198697, WO2016164356 and WO2016089433, which are incorporated herein by reference in their entirety.

[0128] C. PAM sequence

[0129] PAM, also known as the protospacer adjacent motif, is a short specific sequence complementary to a portion of the gRNA, located after the target DNA sequence necessary for Cas nuclease cleavage. The PAM is located about 2-8 nucleotides downstream of the DNA sequence targeted by the guide RNA, and Cas cuts 3-4 nucleotides upstream of it. The PAM sequence is illustrated in Table 2-3 below. The PAM in this application can be any of the sequences in Table 2-3 or any other sequence known in the art.

[0130] Table 2: PAMs of synthetic spCas9 variants

[0131]

[0132] N is A, G, C or T.

[0133] Table 3: PAMs of different Cas9 species

[0134] Cas9 species PAM Sequence Streptococcus pyogenes (Sp) NGG (SEQ ID NO: 891) Staphylococcus aureus (Sa) NGRRN (SEQ ID NO:896) Neisseria meningitidis (Nm or Nme) NNNNGATT (SEQ ID NO:897) Campylobacter jejuni (Cj) NNNNRYAC (SEQ ID NO:898) Streptococcus thermophilus (St) NNAGAAW (SEQ ID NO:899) Treponema denticola (Td) NAAAAC (SEQ ID NO:900)

[0135] N is A, G, C or T.

[0136] D. RNA-guided DNA binders

[0137] Any nucleic acid with an open reading frame encoding an RNA-guided DNA binder (e.g., Cas9 nuclease, such as Streptococcus pyogenes Cas9) can be combined with any gRNA disclosed herein in a composition or method. In some embodiments, the nucleic acid with an open reading frame encoding an RNA-guided DNA binder is mRNA. In some embodiments, the RNA-guided DNA binder is administered in its amino acid form (i.e., as a protein). In some embodiments, the nucleic acid encoding the RNA-guided DNA binder is part of a vector described herein. The nucleic acid encoding the RNA-guided DNA binder may have any of the features described in WO2020198697, which is incorporated herein by reference in its entirety.

[0138] In some embodiments, the RNA-guided DNA binder for the compositions and methods described herein is a Class 2 Cas nuclease. In some embodiments, the RNA-guided DNA binder has double-stranded endonuclease activity. In some embodiments, the RNA-guided DNA binder includes a Cas nuclease, such as a Class 2 Cas nuclease (which may be, for example, a II, V, or VI type Cas nuclease). Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, and C2c3 proteins and modifications thereof.

[0139] Examples of Cas9 nucleases include those of the type II CRISPR systems of Streptococcus pyogenes, Staphylococcus aureus, and other prokaryotes (see, e.g., the list in the next paragraph) and modified (e.g., engineered or mutated) versions thereof. See, e.g., US2016 / 0312198A1; US2016 / 0312199A1. Other examples of Cas nucleases include the Csm or Cmr complexes of the type III CRISPR system, or its Cas10, Csml, or Cmr2 subunits; and the Cascade complexes of the type I CRISPR system, or its Cas3 subunits. In some embodiments, the Cas nuclease may be from a type IIA, type IIB, or type IIC system. For discussion of various CRISPR systems and Cas nucleases, see, e.g., Makarova et al., Nat. Rev. Microbiol. 9:467-477 (2011); Makarova et al., Nat. Rev. Microbiol, 13:722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). In some embodiments, the RNA-guided DNA binder is a Cas nickase, such as a Cas9 nickase. In some embodiments, the RNA-guided DNA binder is a Streptococcus pyogenes Cas9 nuclease.

[0140] Non-limiting exemplary species from which RNA-guided DNA binders (e.g., Cas nucleases) can be derived include, but are not limited to, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp.), Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter Jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis), Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalene-degrading naphthalenivorans), Polaromonas sp., Crocosphaera watsonii, Cyanothece sp.), Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, 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., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp.), Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter Zari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae bacteria ND2006 and Acaryochloris marina.

[0141] In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus thermophilus. In some embodiments, the Cas nuclease is a Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is a Cas9 nuclease from Staphylococcus aureus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella novicida. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Acidococcus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from the genus Acidococcus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from the bacterium ND2006 of the family Lachnospiraceae. In some embodiments, the Cas nuclease is from Francisella tularensis, Lachnospiraceae, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macaques. macacae). In some embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus or Lachnospiraceae.

[0142] Wild-type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, while the HNH domain cleaves the DNA target strand. In some embodiments, the Cas9 nuclease comprises more than one RuvC domain and / or more than one HNH domain. In some embodiments, the Cas9 nuclease is a wild-type Cas9. In some embodiments, Cas9 is capable of inducing double-strand breaks in the target DNA. In certain embodiments, the Cas nuclease can cleave one or both strands of dsDNA. In some embodiments, the Cas nuclease can cleave single-stranded DNA. In some embodiments, the Cas nuclease may not have DNA nickase activity. An exemplary Cas9 amino acid sequence is provided as SEQ ID NO: 901.

[0143]

[0144] An exemplary Cas9 mRNA ORF sequence (including start and stop codons) is provided as SEQ ID NO:902.

[0145]

[0146] An exemplary Cas9 mRNA coding sequence suitable for inclusion in a fusion protein is provided as SEQ ID NO:903.

[0147]

[0148] In some embodiments, a chimeric Cas nuclease is used in which a domain or region of a protein is replaced by a portion of a different protein. In some embodiments, the Cas nuclease domain can be replaced by a domain from a different nuclease (such as Fok1). In some embodiments, the Cas nuclease can be a modified nuclease.

[0149] In other embodiments, the Cas nuclease may be from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

[0150] In some embodiments, the Cas nuclease is a modified Cas nuclease. In some embodiments, the nucleic acid encoding the Cas nuclease includes one or more of the sequences of the modified 5' untranslated region, 3' untranslated region, coding region or polyA tail. In some embodiments, the nucleic acid encoding the Cas nuclease includes a 5' untranslated region (UTR) containing any one of SEQ ID NO:941-947. In some embodiments, the nucleic acid encoding the Cas nuclease includes a 3' untranslated region (UTR) containing any one of SEQ ID NO:948-953. In some embodiments, the nucleic acid encoding the Cas nuclease includes a coding region (CDS) containing any one of SEQ ID NO:954-960. In some embodiments, the nucleic acid encoding the Cas nuclease includes a polyA tail containing any one of SEQ ID NO:963-972. In some embodiments, the engineered Cas nuclease is provided to a cell together with one or more guide RNAs selected from SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932, and 935-940.

[0151] E. Determination of gRNA efficacy

[0152] In some embodiments, the efficacy of the gRNA is determined when delivered with other components (e.g., nucleic acids encoding RNA-guided DNA binders, such as any described herein). In some embodiments, the efficacy of a combination of a gRNA and a nucleic acid encoding an RNA-guided DNA binder is determined.

[0153] As described herein, the use of RNA-guided DNA nucleases and guide RNAs disclosed herein can cause DNA double-strand breaks, which can result in errors in the form of insertion / deletion (indel) mutations when the cell machinery repairs them. Many mutations caused by insertion / deletion will change the reading frame or introduce premature stop codons, thus producing non-functional proteins.

[0154] In some embodiments, the efficacy of a specific gRNA or combination is determined based on an in vitro model. In some embodiments, the in vitro model is a HEK293 cell. In some embodiments, the in vitro model is a HUH7 human liver cancer cell. In some embodiments, the in vitro model is a HepG2 cell. In some embodiments, the in vitro model is a primary human hepatocyte. In some embodiments, the in vitro model is a primary rodent hepatocyte. In some embodiments, the in vitro model is a primary cynomolgus monkey hepatocyte. With regard to the use of primary human hepatocytes, commercially available primary human hepatocytes can be used to provide greater consistency between experiments. In some embodiments, the number of off-target sites deleted or inserted in an in vitro model (e.g., in primary human hepatocytes) is determined, for example, by analyzing the genomic DNA of primary human hepatocytes transfected with Cas9mRNA and guide RNA in vitro. In some embodiments, this determination includes analyzing the genomic DNA of primary human hepatocytes transfected with Cas9 mRNA and guide RNA in vitro. Such exemplary procedures for determination are provided in the following working examples.

[0155] In some embodiments, the efficacy of a specific gRNA or combination is determined in multiple in vitro cell models to perform a gRNA selection process. In some embodiments, cell line comparisons are performed on the data for the selected gRNA. In some embodiments, cross-screening is performed in multiple cell models.

[0156] In some embodiments, the efficacy of a particular gRNA or combination is determined based on an in vivo model. In some embodiments, the in vivo model is a rodent model. In some embodiments, the rodent model is a mouse that expresses a human PCSK9 gene, which may be a mutated human PCSK9 gene. In some embodiments, the in vivo model is a non-human primate, such as a cynomolgus monkey.

[0157] In some embodiments, the efficacy of the guide RNA or combination is measured by the percentage of editing of PCSK9. In some embodiments, the percentage of editing of PCSK9 is compared to the percentage of editing required to achieve PCSK9 protein knockdown, for example, in cells or cell culture media in the case of an in vitro model or in serum, cells or tissues in the case of an in vivo model. In some embodiments, the percentage of editing is 30% to 99% of the cell population. In some embodiments, the percentage of editing is 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95% or 95% to 99% of the cell population. In some embodiments, the editing percentage is 30%-95%, 40%-90% or 50%-85%, 30%-60%, 40%-80%, 50%-75%, 60%-90%.

[0158] In some embodiments, the efficacy of a guide RNA or combination is measured by the number and / or frequency of insertions / deletions at off-target sequences within the genome of a target cell type. In some embodiments, effective guide RNAs and combinations are provided, which produce insertions / deletions at off-target sites at very low frequencies (e.g., <5%) in cell populations and / or relative to the frequency of insertions / deletions at the target site. Therefore, the application provides guide RNAs that do not show off-target insertions / deletions in target cell types (e.g., hepatocytes), or produce off-target insertions / deletions in cell populations and / or relative to the frequency of insertions / deletions at the target site. In some embodiments, the application provides guide RNAs and combinations that do not show any off-target insertions / deletions in target cell types (e.g., hepatocytes).

[0159] In some embodiments, guide RNAs and combinations are provided that produce insertions / deletions at less than 20 off-target sites, for example, as assessed by one or more methods described herein. In some embodiments, guide RNAs and combinations that produce insertions / deletions at less than or equal to 4, 3, 2, or 1 off-target sites are provided, for example, as assessed by one or more methods described herein. In some embodiments, the off-target sites do not occur in protein coding regions in the genome of target cells (e.g., hepatocytes).

[0160] In some embodiments, gene editing events, such as the formation of insertion / deletion ("indel") mutations and homology-directed repair (HDR) events in the target DNA, are detected by linear amplification of labeled primers and separation of labeled amplification products (hereinafter referred to as "LAM-PCR" or "linear amplification (LA)" method), as described in WO2018 / 067447 or Schmidt et al., Nature Methods 4: 1051-1057 (2007).

[0161] In some embodiments, gene editing events are detected, such as forming insertion / deletion ("indel") mutations and homology-directed repair (HDR) events in the target DNA, and also include sequencing the linear amplification product or further amplification product. Sequencing may include any method known to those skilled in the art, including next-generation sequencing, and cloning the linear amplification product or further amplification product into a plasmid and sequencing a portion of the plasmid or plasmid. Exemplary next-generation sequencing methods are discussed in, for example, Shendure et al., Nature 26: 1135-1145 (2008). In other aspects, gene editing events are detected, such as forming insertion / deletion ("indel") mutations and homology-directed repair (HDR) events in the target DNA, and also include performing digital PCR (dPCR) or droplet digital PCR (ddPCR) on the linear amplification product or further amplification product, or contacting the linear amplification product or further amplification product with a nucleic acid probe designed to identify DNA with homology-directed repair (HDR) template sequences and detecting probes that have been combined with the linear amplification product or further amplification product. In some embodiments, the method further comprises determining the position of the HDR template in the target DNA.

[0162] In some embodiments, the efficacy of gRNA or combination is measured by the amount of PCSK9 in cells (including cells from tissues). In some embodiments, the amount of PCSK9 in cells is measured using Western blotting. In some embodiments, the cells used are HUH7 cells. In some embodiments, the cells used are primary human hepatocytes. In some embodiments, the cells used are primary cells obtained from animals. In some embodiments, the amount of PCSK9 is compared with the amount of glyceraldehyde 3-phosphate dehydrogenase GAPDH (a housekeeping gene) to control changes in cell number.

[0163] In some embodiments, the amount of PCSK9 is reduced by 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% compared to PCSK9 detected in cells in the subject prior to administration of the composition. In some embodiments, the amount of PCSK9 is reduced by 30%-95%, 40%-90%, or 50%-85%, 30%-60%, 40%-80%, 50%-75%, or 60%-90% compared to PCSK9 detected in cells in the subject prior to administration of the composition.

[0164] In some embodiments, the efficacy of gRNA or combination is measured by the level or amount of LDL in the subject's circulation. In some embodiments, the level or amount of LDL in the subject's circulation is measured by methods known in the art. For example, the subject's LDL can be measured using a lipid profile test, which can include total cholesterol, LDL cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride measurements (Cooper GR et al. Blood lipid measurements. Variations and practical utility. JAMA. 1992 Mar 25; 267 (12): 1652-60.).

[0165] In some embodiments, the LDL in the subject's circulation is reduced by 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% compared to the LDL in the subject's circulation before administration of the composition. In some embodiments, the LDL in the subject's circulation is reduced by 30%-95%, 40%-90%, or 50%-85%, 30%-60%, 40%-80%, 50%-75%, or 60%-90% compared to the LDL in the subject's circulation before administration of the composition.

[0166] F. Treatment Methods

[0167] In some embodiments, the present application provides a method for treating hypercholesterolemia and / or cardiovascular disease, the method comprising administering a composition comprising a guide RNA having any one or more guide sequences of SEQ ID NO: 1-296, or any one or more sgRNAs of SEQ ID NO: 593-888, or any one or more crRNAs of SEQ ID NO: 297-592. In some embodiments, a gRNA having any one or more guide sequences of SEQ ID NO: 1-296, or any one or more sgRNAs of SEQ ID NO: 593-888 is administered to treat hypercholesterolemia and / or cardiovascular disease. The guide RNA is administered together with a nucleic acid or vector encoding an RNA-guided DNA nuclease described herein, such as a Cas nuclease (e.g., Cas9). The RNA-guided DNA nuclease can be Streptococcus pyogenes Cas9. In a specific embodiment, the guide RNA is chemically modified. In some embodiments, the guide RNA and the nucleic acid encoding the RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP with a CCD lipid (e.g., an amine lipid such as lipid A), a helper lipid (e.g., cholesterol), a stealth lipid (e.g., a PEG lipid such as PEG2k-DMG), and optionally a neutral lipid (e.g., DSPC).

[0168] In some embodiments, the present application provides a method for inducing a double-strand break (DSB) in a PCSK9 gene, the method comprising administering a composition having a guide RNA as described herein (e.g., having any one or more guide sequences of SEQ ID NO: 1-296) or any one or more sgRNAs of SEQ ID NO: 593-888. In some embodiments, a gRNA, such as any one or more guide sequences of SEQ ID NO: 1-296, is administered to recognize and bind to the PCSK9 gene. The guide RNA is administered together with a nucleic acid (e.g., mRNA) or a vector encoding an RNA-guided DNA nuclease as described herein, such as a Cas nuclease (e.g., Cas9). The RNA-guided DNA nuclease can be Streptococcus pyogenes Cas9. In specific embodiments, the guide RNA is chemically modified. In some embodiments, the guide RNA and a nucleic acid encoding an RNA-guided DNA nuclease. In some embodiments, a method of inducing a double-strand break (DSB) in a PCSK9 gene is provided, the method comprising administering a composition comprising a guide RNA (such as a chemically modified guide RNA), the guide RNA comprising any one or more guide sequences of SEQ ID NO: 1-296, or the composition comprising any one or more sgRNAs of SEQ ID NO: 593-888. In some embodiments, any one or more sgRNAs of SEQ ID NO: 593-888 or a gRNA comprising any one or more guide sequences of SEQ ID NO: 1-296 is administered to induce DSBs in the PCSK9 gene. The guide RNA is administered together with a nucleic acid or vector encoding an RNA-guided DNA nuclease as described herein, such as a Cas nuclease (e.g., Cas9). The RNA-guided DNA nuclease can be Streptococcus pyogenes Cas9. In a specific embodiment, the guide RNA is chemically modified. In some embodiments, the guide RNA and the nucleic acid encoding the RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid such as lipid A), a helper lipid (e.g., cholesterol), a stealth lipid (e.g., a PEG lipid such as PEG2k-DMG), and optionally a neutral lipid (e.g., DSPC).

[0169] In some embodiments, a method for modifying a PCSK9 gene is provided, the method comprising administering a composition comprising a guide RNA as described herein (e.g., having any one or more guide sequences of SEQ ID NO: 1-296) or any one or more sgRNAs of SEQ ID NO: 593-888. In some embodiments, a gRNA comprising any one or more guide sequences of SEQ ID NO: 1-296, or any one or more sgRNAs of SEQ ID NO: 593-888 is administered to modify the PCSK9 gene. The guide RNA is administered together with a nucleic acid or vector encoding an RNA-guided DNA nuclease as described herein, such as a Cas nuclease (e.g., Cas9). The RNA-guided DNA nuclease can be Streptococcus pyogenes Cas9. In a specific embodiment, the guide RNA is chemically modified. In some embodiments, the guide RNA and the nucleic acid encoding the RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid such as lipid A), a helper lipid (e.g., cholesterol), a stealth lipid (e.g., a PEG lipid such as PEG2k-DMG), and optionally a neutral lipid (e.g., DSPC).

[0170] In some embodiments, a method for modifying a PCSK9 gene is provided, the method comprising administering a composition comprising a guide RNA (the guide RNA comprises any one or more guide sequences of SEQ ID NO: 1-296) or any one or more sgRNAs of SEQ ID NO: 593-888. In some embodiments, a gRNA comprising any one or more guide sequences of SEQ ID NO: 1-296, or any one or more sgRNAs of SEQ ID NO: 593-888 is administered to modify the PCSK9 gene. The guide RNA is administered together with a nucleic acid or vector encoding an RNA-guided DNA nuclease described herein, such as a Cas nuclease (e.g., Cas9). The RNA-guided DNA nuclease can be Streptococcus pyogenes Cas9. In a specific embodiment, the guide RNA is chemically modified. In some embodiments, the guide RNA and the nucleic acid encoding the RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid such as lipid A), a helper lipid (e.g., cholesterol), a stealth lipid (e.g., a PEG lipid such as PEG2k-DMG), and optionally a neutral lipid (e.g., DSPC).

[0171] In some embodiments, a method for treating hypercholesterolemia and / or cardiovascular disease is provided, the method comprising administering a composition comprising a guide RNA as described herein (e.g., having any one or more guide sequences of SEQ ID NO: 1-296) or any one or more sgRNAs of SEQ ID NO: 593-888. In some embodiments, a gRNA comprising any one or more guide sequences of SEQ ID NO: 1-296, or any one or more sgRNAs of SEQ ID NO: 593-888 is administered to treat hypercholesterolemia and / or cardiovascular disease. The guide RNA is administered together with a nucleic acid or vector encoding an RNA-guided DNA nuclease described herein, such as a Cas nuclease (e.g., Cas9). The RNA-guided DNA nuclease can be Streptococcus pyogenes Cas9. In a specific embodiment, the guide RNA is chemically modified. In some embodiments, the guide RNA and the nucleic acid encoding the RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid such as lipid A), a helper lipid (e.g., cholesterol), a stealth lipid (e.g., a PEG lipid such as PEG2k-DMG), and optionally a neutral lipid (e.g., DSPC).

[0172] In some embodiments, the present application describes a method for reducing LDL levels in the circulation of a subject, the method comprising administering a guide RNA as described herein (e.g., a guide RNA having any one or more guide sequences of SEQ ID NO: 1-296) or any one or more sgRNAs of SEQ ID NO: 593-888. In some embodiments, a gRNA comprising any one or more guide sequences of SEQ ID NO: 1-296 or any one or more sgRNAs of SEQ ID NO: 593-888 is administered to reduce LDL levels in the circulation of the subject and / or prevent atherosclerosis in the vascular tissue of the subject. The gRNA is administered together with a nucleic acid or vector encoding an RNA-guided DNA nuclease described herein, such as a Cas nuclease (e.g., Cas9). The RNA-guided DNA nuclease can be Streptococcus pyogenes Cas9. In a specific embodiment, the guide RNA is chemically modified. In some embodiments, the guide RNA and the nucleic acid encoding the RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid such as lipid A), a helper lipid (e.g., cholesterol), a stealth lipid (e.g., a PEG lipid such as PEG2k-DMG), and optionally a neutral lipid (e.g., DSPC).

[0173] In some embodiments, the present application describes a method for reducing the risk of atherosclerosis in a subject, the method comprising administering a guide RNA as described herein (e.g., a guide RNA comprising any one or more guide sequences of SEQ ID NO: 1-296) or any one or more sgRNAs of SEQ ID NO: 593-888. In some embodiments, a gRNA comprising any one or more guide sequences of SEQ ID NO: 1-296, or any one or more sgRNAs of SEQ ID NO: 593-888 is administered to reduce or prevent the occurrence of atherosclerosis in the subject's vascular tissue. The gRNA is administered together with a nucleic acid or vector encoding an RNA-guided DNA nuclease described herein, such as a Cas nuclease (e.g., Cas9). The RNA-guided DNA nuclease can be Streptococcus pyogenes Cas9. In a specific embodiment, the guide RNA is chemically modified. In some embodiments, the guide RNA and the nucleic acid encoding the RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid such as lipid A), a helper lipid (e.g., cholesterol), a stealth lipid (e.g., a PEG lipid such as PEG2k-DMG), and optionally a neutral lipid (e.g., DSPC).

[0174] In some embodiments, the present application describes a method for treating or preventing coronary artery disease in a subject, the method comprising administering a composition comprising a guide RNA described herein (e.g., a guide RNA having any one or more guide sequences of SEQ ID NO: 1-296) or any one or more sgRNAs of SEQ ID NO: 593-888. In some embodiments, a method for treating or preventing coronary artery disease in a subject is provided, the method comprising administering a composition comprising any one or more sgRNAs in SEQ ID NO: 593-888. In some embodiments, a gRNA comprising any one or more guide sequences of SEQ ID NO: 1-296 or any one or more sgRNAs of SEQ ID NO: 593-888 is administered to treat or prevent coronary artery disease in a subject. The gRNA is administered together with a nucleic acid or vector encoding an RNA-guided DNA nuclease described herein, such as a Cas nuclease (e.g., Cas9). The RNA-guided DNA nuclease can be Streptococcus pyogenes Cas9. In a specific embodiment, the guide RNA is chemically modified. In some embodiments, the guide RNA and the nucleic acid encoding the RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid such as lipid A), a helper lipid (e.g., cholesterol), a stealth lipid (e.g., a PEG lipid such as PEG2k-DMG), and optionally a neutral lipid (e.g., DSPC).

[0175] In some embodiments, the gRNA includes a guide sequence of Table 4, is used with an RNA-guided DNA nuclease (such as a Cas nuclease translated from a nucleic acid) to induce DSB, and non-homologous end joining (NHEJ) during repair results in a mutation in the PCSK9 gene. In some embodiments, NHEJ results in a deletion or insertion of nucleotides, thereby inducing a frameshift or nonsense mutation in the PCSK9 gene.

[0176] In some embodiments, administration of a guide RNA and a nucleic acid encoding an RNA-guided DNA binder (e.g., in a composition provided herein) reduces the abundance of PCSK9 in the subject's cells (e.g., in the subject's liver, intestine, kidney, or vascular epithelial tissue) and thereby reduces the level of LDL in the subject's circulation.

[0177] In some embodiments, reducing PCSK9 abundance in subject cells includes reducing PCSK9 abundance in cells of one or more tissues (such as liver, intestine, kidney or vascular epithelial tissue) of the subject. In some embodiments, vascular epithelial tissue includes blood vessels, for example, arteries. In some embodiments, reducing PCSK9 abundance in subject cells is inferred based on measuring LDL levels in the subject's circulation (e.g., measured by blood lipids). In some embodiments, PCSK9 abundance in cells of one or more tissues of the subject can result in reduced LDL levels in the subject's circulation, for example, when measured 8 weeks after administering the composition.

[0178] In some embodiments, the abundance of PCSK9 in the subject's cells is reduced by 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% compared to the abundance of PCSK9 in the subject's cells before administration of the composition. In some embodiments, the abundance of PCSK9 in the subject's cells is reduced by 30%-95%, 40%-90%, or 50%-85%, 30%-60%, 40%-80%, 50%-75%, or 60%-90% compared to the abundance of PCSK9 in the subject's cells before administration of the composition.

[0179] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a cow, pig, monkey, sheep, dog, cat, fish, or poultry. In some embodiments, the subject is a companion animal or a livestock animal.

[0180] In some embodiments, one or more guide RNAs described herein (e.g., including one or more guide sequences in Table 4 (e.g., in a composition provided herein)) and a nucleic acid (e.g., mRNA) encoding an RNA-guided DNA binder described herein are provided for the preparation of a medicament for treating a human subject with hypercholesterolemia and / or cardiovascular disease. The RNA-guided DNA binder can be Cas9, such as Streptococcus pyogenes Cas9. In specific embodiments, the guide RNA is chemically modified.

[0181] In some embodiments, the composition comprising the guide RNA and the nucleic acid is administered intravenously. In some embodiments, the composition comprising the guide RNA and the nucleic acid is administered into the hepatic circulation.

[0182] In some embodiments, a single administration of a composition comprising a guide RNA and a nucleic acid provided herein is sufficient to knock down the expression of a mutant protein (e.g., a mutated PCSK9). In some embodiments, a single administration of a composition comprising a guide RNA and a nucleic acid provided herein is sufficient to knock out the expression of a mutant protein in a cell population. In other embodiments, more than one administration of a composition comprising a guide RNA and a nucleic acid provided herein can be beneficial for maximizing editing by a cumulative effect. For example, a composition provided herein can be administered 2, 3, 4, 5 or more times, such as 2 times. Administration can be spaced for a period of time, for example, 1 day to 2 years, such as 1 to 7 days, 7 to 14 days, 14 days to 30 days, 30 days to 60 days, 60 days to 120 days, 120 days to 183 days, 183 days to 274 days, 274 days to 366 days or 366 days, 2 years, 5 years or 10 years.

[0183] In some embodiments, the effective amount of the composition administered is in the range of 0.01 to 20 mg / kg (mpk), for example 0.01 to 0.1 mpk, 0.1 to 0.3 mpk, 0.3 to 0.5 mpk, 0.5 to 1 mpk, 1 to 2 mpk, 2 to 3 mpk, 3 to 5 mpk, 5 to 10 mpk or 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5, 6, 8, 10, 15 or 20 mpk. In some embodiments, the amount of the composition administered is 2-4 mg / kg, such as 2.5-3.5 mg / kg. In some embodiments, the amount of the composition administered is about 3 mg / kg.

[0184] In some embodiments, the efficacy of treatment with the composition described herein is evaluated 1 year, 2 years, 3 years, 4 years, 5 years or 10 years after delivery. In some embodiments, the efficacy of treatment with the composition described herein is evaluated by measuring the LDL level in the subject's circulation before and after treatment. In some embodiments, the efficacy of treatment with the composition described herein is observed by reducing the LDL level in the subject's circulation at 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months or 11 months. In some embodiments, the LDL level in the subject's circulation is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

[0185] In some embodiments, treatment slows, stops, or reverses disease progression.

[0186] In some embodiments, treatment slows or stops the progression of cardiovascular disease. In some embodiments, treatment slows or stops the progression of coronary artery disease. In some embodiments, treatment slows or stops the progression of atherosclerosis. In some embodiments, treatment results in improvement, stabilization, or slowing of changes in the symptoms of cardiovascular disease.

[0187] In some embodiments, efficacy of treatment is measured by increased survival time of the subject.

[0188] Additional treatment

[0189] In some embodiments, combination therapies are described that include administering any one of the gRNAs described herein (e.g., including any one or more guide sequences disclosed in Table 4) and a nucleic acid encoding an RNA-guided DNA binder as described herein (such as a nucleic acid (e.g., mRNA) or vector encoding Streptococcus pyogenes Cas9 described herein) (e.g., in a composition provided herein), and an additional therapy suitable for alleviating symptoms of hypercholesterolemia and / or cardiovascular disease.

[0190] In some embodiments, the additional therapy is for the treatment of hypercholesterolemia and / or cardiovascular disease. In some embodiments, the treatment of hypercholesterolemia and / or cardiovascular disease is a statin, for example, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin or simvastatin. In some embodiments, the treatment of hypercholesterolemia and / or cardiovascular disease is a cholesterol absorption inhibitor, for example, ezetimibe. In some embodiments, the treatment of hypercholesterolemia and / or cardiovascular disease is bempedoic acid. In some embodiments, the treatment of hypercholesterolemia and / or cardiovascular disease is a bile acid binding resin, for example, cholestyramine, colesevelam or colestipol.

[0191] In some embodiments, the combination therapy includes administering any one of the gRNAs including any one or more guide sequences disclosed in Table 4 and a nucleic acid encoding an RNA-guided DNA binder (e.g., in a composition provided herein) and an antibody targeting PCSK9 and / or inhibiting PCSK9. In some embodiments, the antibody is an antibody composition that can further reduce PCSK9 abundance, thereby promoting the removal of LDL cholesterol from the circulation. In some embodiments, the antibody is eloumab, bococizumab, or alirocumab. In some embodiments, the antibody composition is administered after any one of the gRNAs including any one or more guide sequences disclosed in Table 4 (e.g., in a composition provided herein). In some embodiments, the antibody composition is administered regularly after treatment with any gRNA composition provided herein.

[0192] In some embodiments, the combination therapy includes administering any one of the gRNAs including any one or more guide sequences disclosed in Table 4 and nucleic acids encoding RNA-guided DNA binders described herein (e.g., in compositions provided herein) and siRNAs targeting PCSK9 or mutant PCSK9. In some embodiments, the siRNA is any siRNA that can further reduce or eliminate wild-type or mutant PCSK9 expression. In some embodiments, the siRNA is the drug inclisiran. In some embodiments, the siRNA is administered after any one of the gRNAs including any one or more guide sequences disclosed in Table 4 and nucleic acids encoding RNA-guided DNA binders (e.g., in compositions provided herein). In some embodiments, the siRNA is administered regularly after treatment with any gRNA composition provided herein.

[0193] In some embodiments, the combination therapy includes administering any one of the gRNAs including any one or more guide sequences described herein (e.g., disclosed in Table 4) and nucleic acids encoding RNA-guided DNA binders described herein (e.g., in compositions provided herein) and antisense nucleotides targeting PCSK9 or mutant PCSK9. In some embodiments, the antisense nucleotides are any antisense nucleotides that can further reduce or eliminate wild-type or mutant PCSK9 expression. In some embodiments, the antisense nucleotides are administered after any one of the gRNAs including any one or more guide sequences disclosed in Table 4 and nucleic acids encoding RNA-guided DNA binders (e.g., in compositions provided herein). In some embodiments, antisense nucleotides are administered periodically after treatment with any of the gRNA compositions provided herein.

[0194] In any of the foregoing embodiments, the guide sequence disclosed in Table 4, and / or the guide RNA may be a chemically modified guide RNA.

[0195] In some embodiments, the methods described herein include an infusion prophylactic agent. In some embodiments, the infusion prophylactic agent is administered to the subject before the gene editing composition. In some embodiments, the infusion prophylactic agent is administered to the subject 8-24 hours or 1-2 hours before the administration of the nucleic acid composition.

[0196] In some embodiments, the infusion preventive comprises a corticosteroid. In some embodiments, the infusion preventive comprises one or more or all of a corticosteroid, an antipyretic (e.g., oral acetaminophen (also known as paracetamol), which can relieve pain and fever and / or inhibit COX enzymes and / or prostaglandins), a HI blocker or an H2 blocker. In some embodiments, the infusion preventive comprises an intravenous corticosteroid (e.g., dexamethasone 8-12 mg, such as 10 mg, or equivalent) and an antipyretic (e.g., oral acetaminophen or paracetamol 500 mg). In some embodiments, HI blockers (e.g., diphenhydramine 50 mg or equivalent) and / or H2 blockers (e.g., ranitidine 50 mg or equivalent) are administered orally. In some embodiments, HI blockers (e.g., diphenhydramine 50 mg or equivalent) and / or H2 blockers (e.g., ranitidine 50 mg or equivalent) are administered intravenously. In some embodiments, the infusion prophylactic agent is administered intravenously 1-2 hours prior to infusion of the nucleic acid composition.

[0197] In some embodiments, an intravenous HI blocker and / or an intravenous H2 blocker is replaced by an oral equivalent. Infusion prophylactics can be used to reduce adverse reactions associated with the administration of nucleic acid compositions. In some embodiments, infusion prophylactics are administered as a premedication required before administering a nucleic acid composition. The dosage, frequency, and mode of administration of corticosteroids, infusion prophylactics, and compositions containing guide RNAs described herein can be independently controlled.

[0198] The corticosteroid used in the disclosed method can be administered according to protocols known in the art (e.g., protocols approved by the U.S. FDA). In some embodiments, for example, administered to a human subject or for a human subject, the amount of the corticosteroid administered can range from about 0.75 mg to about 25 mg. In some embodiments, for example, administered to a human subject or for a human subject, the amount of the corticosteroid administered can range from about 0.01-0.5 mg / kg, such as 0.1-0.40 mg / kg or 0.25-0.40 mg / kg.

[0199] In some embodiments, the corticosteroid is administered before the composition containing the guide RNA described herein. In some embodiments, the corticosteroid is administered after the composition containing the guide RNA described herein. In some embodiments, the corticosteroid is administered simultaneously with the composition containing the guide RNA described herein. In some embodiments, multiple doses of the corticosteroid are administered before or after the administration of the composition containing the guide RNA. In some embodiments, multiple doses of the composition containing the guide RNA are administered before or after the administration of the corticosteroid. In some embodiments, multiple doses of the corticosteroid and multiple doses of the composition containing the guide RNA are administered.

[0200] If appropriate, a dose of corticosteroid can be administered as at least two subdoses separated by appropriate intervals. In some embodiments, corticosteroids are administered at least twice before administering a composition containing a guide RNA as described herein. In some embodiments, a dose of corticosteroids is administered at least twice after administering a composition containing a guide RNA as described herein. In some embodiments, corticosteroids are administered at intervals of 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 weeks; or an interval of time within the range defined by any two of the foregoing values ​​(e.g., before, simultaneously and / or after administering a composition containing a guide RNA as described herein). In some embodiments, the corticosteroid is administered prior to administration of a composition containing a guide RNA described herein at an interval of 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks; or an amount of time within a range defined by any two of the foregoing values. In some embodiments, the corticosteroid is administered after administration of a composition containing a guide RNA described herein at an interval of 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks; or an amount of time within a range defined by any two of the foregoing values.

[0201] In some embodiments, the corticosteroid is administered at least twice. In some embodiments, the corticosteroid is administered at least three times. In some embodiments, the corticosteroid is administered at least four times. In some embodiments, the corticosteroid is administered up to five, six, seven, eight, nine, or ten times. The first dose can be oral, and the second or subsequent doses can be parenteral, such as infusion. Alternatively, the first dose can be parenteral, and the second or subsequent doses can be oral.

[0202] In some embodiments, the corticosteroid is administered orally prior to the intravenous administration of a composition containing a guide RNA described herein. In some embodiments, the corticosteroid is administered orally during or after the intravenous administration of a composition containing a guide RNA described herein.

[0203] In some embodiments, the corticosteroid is dexamethasone. In some embodiments, dexamethasone is administered intravenously 1-2 hours before the infusion of the nucleic acid composition. In some embodiments, 8-12 mg, such as 10 mg of dexamethasone, is administered intravenously 1-2 hours before the infusion of the nucleic acid composition. In some embodiments, dexamethasone is orally administered 8 to 24 hours before the infusion of the nucleic acid composition. In some embodiments, 8-12 mg, such as 8 mg of dexamethasone, is orally administered 8 to 24 hours before the infusion of the nucleic acid composition. In some embodiments, 8-12 mg, such as 8 mg of dexamethasone, is orally administered 8 to 24 hours before the infusion of the nucleic acid composition, and 8-12 mg, such as 10 mg of dexamethasone, is administered intravenously 1-2 hours before the infusion of the nucleic acid composition.

[0204] Delivery of Nucleic Acid Compositions

[0205] In some embodiments, the nucleic acid compositions described herein include gRNA and nucleic acids encoding RNA-guided DNA binders (encoded as RNA or on one or more vectors), which are formulated in or administered by lipid nanoparticles (LNPs); see, e.g., WO2017173054A1 and WO2019067992A1, the contents of which are incorporated herein by reference in their entirety. Any LNP known to those skilled in the art capable of delivering nucleotides to a subject can be used with guide RNAs described herein and nucleic acids encoding RNA-guided DNA nucleases.

[0206] In some embodiments, the guide RNA and the nucleic acid encoding the RNA-guided DNA nuclease are administered in an LNP described herein, such as an LNP comprising a CCD lipid (e.g., an amine lipid such as lipid A), a helper lipid (e.g., cholesterol), a stealth lipid (e.g., a PEG lipid such as PEG2k-DMG), and optionally a neutral lipid (e.g., DSPC).

[0207] Various embodiments of LNP preparations for RNA (including CRISPR / Cas payloads) are disclosed herein. Such LNP preparations may include (i) CCD lipids, such as amine lipids, (ii) neutral lipids, (iii) auxiliary lipids, and (iv) stealth lipids, such as PEG lipids. Some embodiments of LNP preparations include amine lipids, as well as auxiliary lipids, neutral lipids and stealth lipids (such as PEG lipids). In some embodiments, the LNP preparation includes less than 1% of neutral phospholipids. In some embodiments, the LNP preparation includes less than 0.5% of neutral phospholipids. "Lipid nanoparticles" can be particles comprising multiple (i.e., more than one) lipid molecules, which are physically associated with each other by intermolecular forces. CCD lipids, amine lipids, neutral lipids and other lipids that can be used in LNP preparations disclosed herein are described in WO2020198697, WO2015006747, WO2016118724 and WO2021026358, which are all incorporated herein in their entirety.

[0208] More technologies that can be used to deliver the composition of the present application include technologies that utilize biodegradable polymers, liposomes, virus-like particles or nanoparticles to encapsulate. In some embodiments, the composition of the present application is applied with any suitable delivery vehicle, including but not limited to polymers, engineered virus particles (e.g., adeno-associated virus), exosomes, liposomes, supercharged proteins, implantable devices or red blood cells. Suitable delivery methods are described in US10851357, US10709797 and US20170349914, each of which is incorporated herein in its entirety.

[0209] Example

[0210] Unless otherwise indicated, the practice of the methods and compositions of the present application employs conventional techniques of molecular biology (including recombinant techniques), cell culture, immunology, cell biology, and biochemistry, which are well within the capabilities of those skilled in the art. Such techniques are explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the methods and compositions of the present application. The following sections will discuss particularly useful techniques for specific embodiments. The materials, reagents and methods further described below are used in the following examples. The embodiments described in the following examples do not limit the scope of the claims.

[0211] Example 1. Design of guide RNA sequence

[0212] Initial guide design is completed using common computational tools and workflows, a human reference genome (e.g., GRCh38) and a user-defined target genomic region (e.g., PCSK9). The first step in determining a guide sequence (i.e., gRNA) is to scan the PAM in the region of interest. Then, candidate guides are sorted according to a number of criteria (cutting efficiency and binding specificity scores, GC content, poly-T, and free energy), which are expected to ensure high targeted cutting efficiency and low off-target probability. A total of 296 sgRNAs targeting the coding region of PCSK9 (ENST00000302118.5) exons 1-12 were generated. About 10% of these guides are 100% homologous in the reference genome of cynomolgus monkeys (Macaca fascularis). Guide sequences and genomic coordinates are provided in Table 4.

[0213] The selected guide sequences are shown in Table 4. Table 4 below shows the 296 guide sequences designed to target the PCSK9 gene. The corresponding sgRNAs are shown in Table 5.

[0214] Table 4: Boot sequence

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] Table 5: crRNA and sgRNA sequences corresponding to the guide sequences in Table 4

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258] The 296 sgRNA sequences shown in Table 5 (SEQ ID NOs: 593-888) were further tested in in vitro and in vivo assays.

[0259] Example 2. Target analysis

[0260] Targeting efficiency analysis: Targeted cleavage efficiency was assessed using deep amplicon sequencing. Targeted mutations introduced by the gene editing system disclosed herein were enumerated and visualized using in-house computational tools and workflows. The editing effects of coding and non-coding elements associated with the selected target region were assessed.

[0261] In addition, off-target cleavage was assessed. For example, cell-based oligonucleotide insertion assays were also performed in PHH, Huh7, and HepG2 cell lines (Tasi et al., 2015). Sites with high dsODN insertion efficiency were selected for further analysis using amplicon-based next-generation sequencing to more precisely assess off-target editing.

[0262] In vitro delivery of Cas9 protein and sgRNA

[0263] HepG2 cell line. The human hepatocellular carcinoma cell line HepG2 was cultured in DMEM medium supplemented with 10% fetal bovine serum. 24 hours before electroporation, cells were seeded at a density of 1,000,000-1,500,000 cells / well in 6-well plates or 8,000-22,000 cells / well in 96-well plates. Cells were electroporated using a Celetrix electroporator (Celetrix, CTX-1500A) according to the supplier's protocol. Cells were electroporated using RNP complexes containing Cas9 nuclease (5-50 pmol), sgRNA (10-500 pmol), and Celetrix buffer.

[0264] In vitro delivery of Cas9 mRNA and sgRNA

[0265] HepG2 cell line. Human hepatocellular carcinoma cell line HepG2 was cultured in DMEM medium supplemented with 10% fetal bovine serum. 24 hours before electroporation, cells were seeded in 6-well plates at a density of 1,000,000-1,500,000 cells / well, or in 96-well plates at a density of 8,000-22,000 cells / well. Cells were transfected using Lipofectamine MessengerMAX (ThermoFisher, catalog number LMRNA003) according to the supplier's protocol. Cells were transfected with lipid complexes containing 1-500ng Cas9mRNA, 2-1,000ng sgRNA, and Lipofectamine MessengerMAX.

[0266] Huh7 cell line. Human hepatocellular carcinoma cell line Huh7 was cultured in DMEM medium supplemented with 10% fetal bovine serum. 24 hours before electroporation, cells were seeded in 6-well plates at a density of 500,000-1,500,000 cells / well, or in 96-well plates at a density of 5,000-15,000 cells / well. Cells were transfected using Lipofectamine MessengerMAX (ThermoFisher, catalog number LMRNA003) according to the supplier's protocol. Cells were transfected with lipid complexes containing 1-500ng Cas9mRNA, 2-1,000ng sgRNA, and Lipofectamine MessengerMAX.

[0267] Cos-7 cell line. Green monkey kidney cell line Cos-7 was cultured in DMEM medium supplemented with 10% fetal bovine serum. 24 hours before electroporation, cells were seeded in 96-well plates at a density of 5,000-15,000 cells / well. Cells were transfected using Lipofectamine MessengerMAX (ThermoFisher, catalog number LMRNA003) according to the supplier's protocol. Cells were transfected with lipid complexes containing 1-500ng Cas9 mRNA, 2-1,000ng sgRNA, and Lipofectamine MessengerMAX.

[0268] Primary hepatocytes. Primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH) were cultured according to the supplier's protocol (BioIVT). Briefly, cells were thawed and resuspended in hepatocyte thawing medium supplemented with supplements, then centrifuged at 100g for 10 minutes for human hepatocytes and 80g for 4 minutes for cynomolgus monkey hepatocytes. The supernatant was discarded and the precipitated cells were resuspended in hepatocyte seeding medium supplemented with a supplement pack. Cells were counted and seeded on Bio-coat collagen I-coated multiwell plates (ThermoFisher, catalog number 877272): seeded at a density of 60,000 cells / well in 96-well plates, or at a density of 125,000 cells / well in 24-well plates, or at a density of 270,000 cells / well in 6-well plates. The seeded cells were allowed to settle and grow adherently in a tissue culture incubator at 37°C and 5% CO2 atmosphere for 6 or 24 hours.

[0269] After culture, check whether the cells have formed a monolayer and replace the medium with hepatocyte medium containing a serum-free supplement pack.

[0270] Genomic DNA isolation. For in vitro studies, transfected cells were harvested 72 hours after transfection. Genomic DNA was extracted from each well of a 6-well plate / 24-well plate / 96-well plate using QuickExtract DNA Extraction Solution (LGC Lucigen, catalog number QE09050) according to the supplier's protocol. All DNA samples were subjected to subsequent Sanger sequencing analysis as described herein.

[0271] For in vivo studies, genomic DNA was extracted from mouse liver homogenates using the FastPure Blood / Cell / Tissue / Bacteria DNA Isolation Mini Kit (Vazyme, Cat. No. DC112) following the supplier's protocol.

[0272] Sanger sequencing analysis

[0273] To quantitatively determine the editing efficiency at target locations in the genome and rapidly screen potential gRNAs, Sanger sequencing was used to identify the efficiency of edits introduced by gene editing.

[0274] Primers are designed around a target site within a gene of interest (eg, PCSK9) and the genomic region of interest is amplified.

[0275] Sanger sequencing was performed on a 3730xl DNA Analyzer (ThermoFisher, catalog number 3730XL) according to the supplier's protocol. Raw sequencing files (.ab1) were analyzed using an online analysis tool to determine editing efficiency.

[0276] Next Generation Sequencing (NGS) Analysis

[0277] To quantitatively determine the efficiency and pattern of editing at target locations in the genome, sequencing was used to identify the presence of insertions and deletions introduced by gene editing.

[0278] Primers are designed around a target site within a gene of interest (eg, PCSK9) and the genomic region of interest is amplified.

[0279] According to the supplier's (Illumina) protocol, additional PCR was performed to add chemicals for sequencing. The amplicons were sequenced using an Illumina NovaSeq 6000 instrument. The resulting reads were aligned to a reference genome (e.g., a human reference genome (hg38), a cynomolgus monkey reference genome (mf5), a rat reference genome (rn6), or a mouse reference genome (mm10)), with reads of low quality scores eliminated before alignment. The resulting file containing the reads was mapped to a reference genome (BAM file), where those reads that overlapped with the target region of interest were selected, and the number of wild-type reads and the number of reads containing insertions, substitutions, or deletions were counted.

[0280] The editing percentage (e.g., "editing efficiency" or "editing percentage" or "indel frequency") is defined as the total number of sequence reads with insertions / deletions ("indels") or substitutions / the total number of sequence reads including wild-type reads.

[0281] PCSK9 ELISA assay for cell-based studies.

[0282] Cell (HepG2 or Huh7) lysates were collected and separated, and the expression level of PCSK9 was determined using a human PCSK9 ELISA kit (Abcam, catalog number ab209884) according to the manufacturer's protocol. In brief, when measuring human PCSK9, the samples were serially diluted with the kit sample diluent, and the final dilution factor was 5,000 times. 100uL of the prepared standard curve sample or diluted serum sample was added to the ELISA plate, incubated at room temperature for 30 minutes, and then washed 3 times with the provided wash buffer. Then, 100uL of detection antibody was added to each well, incubated at room temperature for 20 minutes, and then washed 3 times. 100uL of substrate was added, incubated at room temperature for 10 minutes, and then 100ul of stop solution was added. The absorbance of the contents was measured on a Spectramax M5 plate reader and analyzed using SoftmaxPro version 7.0 software. PCSK9 levels were calculated using a 4-parameter logistic fit to the standard curve and expressed as ng / mL in serum or as percent knockdown relative to control (vehicle-treated) cells.

[0283] sgRNA synthesis

[0284] sgRNA was synthesized on a 192-YiBo solid phase synthesizer. Controlled pore glass (CPG) was used as a solid support and each monomer was added in each cycle using TBDMS-modified phosphoramidite. After the synthesis process, the sgRNA was cleaved from the CPG and deprotected. Purification was performed in an AKTA purifier.

[0285] In other experiments, sgRNAs were ordered from vendors such as Genscript, General Biosystems, or Synthego. If sgRNAs are to be compared for potency or off-target effects, sgRNAs from the same vendor and of similar purity were used in each experiment.

[0286] mRNA codon optimization

[0287] The 004R sequence was optimized by Genscript using its in-house algorithm for optimal human protein production and low GC content to facilitate gene synthesis. Seq311 and Seq204 were from US11697806B2 for comparison. K1-1, K4-8, K8-1, and K10-2 were optimized for high codon adaptation index (CAI) and low minimum free energy (MFE). Since the calculation of MFE requires full-length mRNA, all tested sequences contained the same 5'UTR (5'UTR HSD, TCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT GCAGGCCTTATTC, SEQ ID NO: 961), 3'UTR (3'UTR ALB, CATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAA, SEQ ID NO: 962) and nuclear localization sequence (G3S-NLS). All codon-optimized CDSs were compared with the same UTR as described below.

[0288] mRNA plasmid construction and in vitro transcription (IVT)

[0289] Different sequence elements (such as UTR, CDS, polyA, see sequence list) are PCR amplified or de novo synthesized and cloned into the original plasmid used to produce Cas9 mRNA (Genscript, General Biosystem or GENEWIZ). The length of PolyA in the plasmid is verified by the gene synthesis provider through Sanger verification and is less than 3 different from the designed number.

[0290] In vitro transcription using linearized plasmid template and T7 RNA polymerase produced capped and polyadenylated Cas9 mRNA containing N1-methyl pseudouracil. Transcript concentration was determined by absorbance at 260 nm (Nanodrop), and transcripts were analyzed by capillary electrophoresis using a Bioanalyzer (Agilent).

[0291] Determination of PolyA length by mass spectrometry

[0292] To detect the length of polyA mRNA, the full-length mRNA is cleaved by RNase T1t to cut the phosphodiester bond between the 3'-phosphate group of the guanine ribonucleotide and the 5'-hydroxyl group of the adjacent ribonucleotide. This process releases a short polyA fragment from the parent mRNA molecule. The released polyA fragment is then purified using biotin-avidin magnetic beads. The molecular weight distribution of this polyA fragment is then analyzed by mass spectrometry.

[0293] LNP delivery in vivo

[0294] Compositions for delivering the protein and nucleic acid components of CRISPR / Cas to cells (such as cells of a patient) are needed. In particular, compositions that have useful properties for in vitro and in vivo delivery, that can stabilize and deliver RNA components are of interest.

[0295] Herein, we provide lipid nanoparticle (LNP)-based compositions having useful properties, particularly compositions for delivering CRISPR / Cas gene editing components. The LNP composition comprises: an RNA component; and a lipid component, wherein the lipid component comprises: (1) about 45-55 mol% amine lipids; (2) about 9-11 mol% neutral lipids; and (3) about 1-5 mol% PEG lipids, wherein the remainder of the lipid component is a helper lipid, and wherein the LNP composition has an N / P ratio of 3 to 8.

[0296] Unless otherwise stated, each study used PCSK9 humanized mice aged 6-15 weeks. Animals were weighed and grouped according to body weight to prepare dosing solutions according to group average body weight. LNP was administered via the tail vein, with a volume of 0.2 ml per animal (approximately 10 ml / kg body weight). Animals were observed every day to monitor status. Blood was collected by saphenous vein or cardiac puncture at the indicated time points. Liver tissue was collected after blood collection and immediately placed at -80 ° C for further analysis.

[0297] PCSK9 ELISA Assays Used in Animal Studies

[0298] Blood was collected and serum was separated. Total human PCSK9 serum levels were measured using a human KCSK9 ELISA kit (Abcam, catalog number ab209884) following the supplier's protocol.

[0299] Example 3. sgRNA sequence screening

[0300] PCSK9 guide RNA screening in HepG2 cells

[0301] As described in Example 2, sgRNA targeting the human PCSK9 gene was delivered to HepG2. The editing percentage of sgRNA containing each guide sequence was determined, and the guide sequences were then ranked according to the highest editing percentage. The editing data are listed in Table 6 below. The data are graphically displayed in Figure 1 middle.

[0302] Table 6: PCSK9 editing data using Cas9 protein and sgRNA in HepG2 cells

[0303]

[0304]

[0305]

[0306] Example 4. Dose response of sgRNA in Cos-7 and PCH cells

[0307] As described in Example 2, sgRNA and Cas9 mRNA targeting human and monkey PCSK9 were delivered to Cos-7 and PCH cells in a two-fold dose response curve of 8 points. Cells were lysed 72 hours after treatment for editing analysis as described in Example 2. The editing percentage was determined for the sgRNA containing each guide sequence, and the guide sequences were then ranked based on the EC50 value and the maximum editing percentage. The dose response curves for the guide sequences in Cos-7 and PCH cells are shown in Figure 2 and Figure 3 The EC50 values ​​and maximum editing percentages are listed in Tables 7 and 8 below.

[0308] Table 7 shows the EC50 and maximum editing (as a dose-response curve) of Cos-7 by the tested human PCSK9 sgRNA and Cas9 mRNA. The data are presented graphically in Figure 2 middle.

[0309] Table 7: PCSK9 editing data in Cos-7 cells treated with Cas9 mRNA and sgRNA

[0310] name EC50(nM) Maximum Edit (%) P9-hc-026 0.221 87 P9-hc-028 0.439 97 P9-hc-162 0.217 96 P9-hc-023 1.158 89

[0311] Table 8 below shows the EC50 and maximum editing (as a dose response curve) of the tested human PCSK9 sgRNA and Cas9 mRNA for PCH. The data are graphically displayed in Figure 3 middle.

[0312] Table 8: PCSK9 editing data in PCH cells treated with Cas9 mRNA and sgRNA

[0313]

[0314]

[0315] Example 5. Phenotypic analysis

[0316] ELISA analysis of intracellular PCSK9

[0317] HepG2 and Huh7 cells were transfected with Cas9 mRNA and sgRNA as described in Example 2. Transfected cell pools were maintained in tissue culture and passaged for further analysis. On day 5 after transfection, cell lysates were collected and analyzed by ELISA as described previously.

[0318] The percentage reduction of PCSK9 protein was calculated. The percentage reduction of PCSK9 protein was determined after the PCSK9 level was normalized to the scrambled control. The results are shown in Tables 9 and 10 below.

[0319] Table 9: Percent reduction of PCSK9 protein in HepG2 cells.

[0320]

[0321]

[0322] Table 10: Percent reduction of PCSK9 protein in Huh7 cells.

[0323]

[0324]

[0325] Example 6: Screening of sgRNA sequences in PHH

[0326] Primary human hepatocytes (PHH) were thawed in thawing medium (Gibco, catalog number CM7500). After centrifugation, the supernatant was discarded, and the precipitated cells were resuspended in hepatocyte inoculation medium (William's E Medium plus Plating Supplements CM3000, Thermofisher) with a supplement pack, and inoculated at a density of 35,000 cells / well in a collagen-coated flat 24-well plate (Stem cell technologies, catalog number 100-0365). The inoculated cells were allowed to settle and adhere to the wall for 4-6 hours in a tissue culture incubator at 37°C and 5% CO2 atmosphere. The culture medium was replaced with William's E Medium plus Maintenance Supplements CM4000 (Thermofisher) and used until the end of the experiment. Transfection was performed using RNAiMax reagent (Thermofisher) with 500ng of cas9 mRNA (Trilink) and 100ng of sgRNA (Synthego). Three days later, the cells were extracted with QuickExtract TM DNA extraction solution (Lucigen, catalog number QE0905T) was used for lysis, and the targeted genomic region was amplified for NGS sequencing. The results of PCSK9 protein reduction and gene editing efficiency are shown in Table 11 below.

[0327] Table 11: PCSK9 protein reduction and gene editing efficiency in primary human hepatocytes

[0328]

[0329]

[0330] Example 7. sgRNA Editing Efficiency in PHH

[0331] Primary human hepatocytes (PHH) from another donor were thawed in InvitroGRO CP medium containing 10% FBS and 1% Pen / Strep and seeded in collagen-coated flat 24-well plates at a density of 27,000 cells / well. After 4-6 hours, the medium was replaced with InvitroGRO CP medium. Transfection was performed using RNAiMax reagent (Thermofisher) with 500ng of cas9mRNA and 250ng of sgRNA (Genscript). On the day after transfection, the medium was replaced with 1% Pen / Strep InvitroGRO CP medium until cells were collected 3 days after transfection. The gene editing results are shown in Table 12 below.

[0332] Table 12: PCSK9 gene editing efficiency in primary human hepatocytes

[0333] name Gene editing efficiency P9-hc-212 65 P9-hc-095 63 P9-hc-162 58 P9-hc-023 58 P9-hc-028 53 P9-hc-026 52 P9-hc-223 51 P9-hc-010 37 P9-hc-026 32

[0334] Example 8. Off-target analysis by dsDNA insertion assay in HepG2

[0335] The potential genomic off-target sites cleaved by Cas9 and corresponding gRNA were screened using a double-stranded (dsDNA) insertion assay. HepG2 cells were maintained in MEM (Gibco) supplemented with 10% FBS (OPCEL) at 37 ° C and 5% CO2. 1 million HepG2 cells were electrotransfected with 200 pmol of dsDNA, 35 pmol of Cas9 (NEB, EnGen SpyCas9 NLS) protein and 200 pmol of gRNA (Genscript) in 4D-Nuclefector (LONZA, X-unit). Genomic DNA was extracted and processed for NGS determination in NextSeq 6000 sequencers (for example, see Tsai et al., Nature Biotechnology 33, 187-197; 2015). The dsDNA incorporation efficiency of each potential off-target site was calculated as the reading at the site divided by the reading at the target site (PCSK9). The efficiency sum of the first 30 off-target sites was divided by the efficiency sum of the targeted sites (top 30 off / on) and used as a semi-quantitative readout for comparing the off-target potential between different gRNAs. The total number of off-target sites and the top five sites with the highest dsODN incorporation efficiency are listed in Tables 13 and 14 below, which represent two independent replicate experiments.

[0336] Table 13: dsDNS incorporation efficiency and top 5 off-target sites in HepG2 cells

[0337]

[0338]

[0339] Table 14: dsDNS incorporation efficiency and top 5 off-target sites in HepG2

[0340]

[0341]

[0342] Example 9. Off-target analysis by dsDNA insertion assay in PHH

[0343] Primary human hepatocytes (PHH) were thawed in thawing medium (Gibco, catalog number CM7500). After centrifugation, the supernatant was discarded, and the precipitated cells were resuspended in hepatocyte inoculation medium (William's E Medium plus Plating Supplements CM3000, Thermofisher) with a supplement pack, and inoculated in a collagen-coated flat 24-well plate (Stem cell technologies, catalog number 100-0365) at a density of 35,000 cells / well. The inoculated cells were allowed to settle and adhere to the wall for 4-6 hours in a tissue culture incubator at 37°C and 5% CO2 atmosphere. Thereafter, the culture medium was replaced with William's E Medium plus Maintenance Supplements CM4000 (Thermofisher) and used until the end of the experiment. RNAiMax reagent (Thermofisher) was used to transfect with 500ng of cas9 mRNA (Trilink), 5pmol dsDNA and 100ng of sgRNA (Synthego). Three days later, genomic DNA was extracted with OceanNano Tech PureBind genomic DNA isolation kit and processed for NGS determination in NextSeq 2000 sequencer (for example, see Tsai et al., Nature Biotechnology 33, 187-197; 2015). The efficiency sum of the first 30 off-target sites was divided by the efficiency sum of the targeted sites (top 30off / on) and used as a semi-quantitative reading for comparing the off-target potential between different gRNAs. The total number of off-target sites and the first five sites with the highest dsODN incorporation efficiency are listed in Table 15 below.

[0344] Table 15: dsDNS incorporation efficiency and top 5 off-target sites in PHH

[0345]

[0346]

[0347] Example 10. Off-target analysis of sgRNA by amplicon-based NGS in PHH

[0348] Primary human hepatocytes (PHH) were thawed in InvitroGRO CP medium containing 10% FBS and 1% Pen / Strat and seeded in collagen-coated flat 24-well plates at a density of 270,000 cells / well. After 4-6 hours, the medium was replaced with InvitroGRO CP medium. 1.5 μL RNAiMax reagent (Thermofisher) was used for transfection with 400ng of cas9 mRNA and 200ng of sgRNA (Genscript). On the day after transfection, the medium was replaced with 1% Pen / Strep InvitroGRO CP medium until cells were collected 3 days after transfection. Genomic DNA was extracted with QuickExtractDNA extraction solution (Lucigen). Editing at the target site and the off-target site in front was amplified by PCR with Taq Pro Multiplex DNA polymerase (Vazyme). PCR products were purified with VAHTS DNA purification beads (Vazyme) and sequenced on the Illumina Novaseq6000 platform. The off-target site editing efficiency was divided by the targeted efficiency in the same experiment to standardize for different transfection efficiencies. The editing efficiency of the top off-target sites was divided by the targeted editing efficiency, which is shown in Table 16 below.

[0349] Table 16: Editing efficiency of the top off-target sites divided by the on-target editing efficiency

[0350] gRNA OT1 OT2 OT3 OT4 OT5 OT6 OT7 P9-hc-063 26.20％ 0.10％ 0.20％ 0.10％ 2.30％ 0.00％ 1.20％ P9-hc-175 13.40％ 3.90％ 0.10％ 0.00％ 0.00％ 0.10％ 0.00％ P9-hc-023 2.60％ 0.60％ 0.30％ 0.00％ N / A N / A N / A P9-hc-082 1.60％ 0.10％ 0.10％ 0.20％ 0.10％ N / A N / A P9-hc-010 1.10％ 0.50％ 0.20％ N / A N / A N / A N / A P9-h-057 0.50％ 0.30％ 0.60％ 0.30％ 3.20％ 0.50％ N / A P9-hc-212 0.30％ 0.30％ N / A N / A N / A N / A N / A P9-hc-026 0.20％ 1.60％ 1.80％ 0.10％ N / A N / A N / A P9-hc-223 0.20％ 0.00％ 0.00％ 0.00％ 0.00％ N / A N / A P9-hc-028 0.10％ 0.10％ 0.20％ 0.00％ N / A N / A N / A P9-hc-043 0.10％ 0.00％ 0.00％ N / A N / A N / A N / A P9-hc-162 0.10％ 0.10％ 0.10％ N / A N / A N / A N / A P9-hc-095 0.00％ 0.00％ 0.00％ N / A N / A N / A N / A

[0351] Example 11. On-target / off-target editing efficiency of DNA-RNA hybrid gRNA in Huh7 cells

[0352] Huh7 cells were seeded in 96-well plates at 8,000 cells / well. 0.4 μL RNAiMax reagent (Thermofisher) was used to transfect with 100 ng of cas9 mRNA and 100 ng of sgRNA (with or without deoxyribonucleotide substitution) (General Biosystems). Cells were collected three days after transfection. Genomic DNA was extracted with QuickExtract DNA extraction solution (Lucigen). Editing at the target site and the off-target site in front was performed by PCR amplification with Taq Pro Multiplex DNA polymerase (Vazyme). The PCR product was purified with VAHTSDNA purification beads (Vazyme) and sequenced on the illumina Novaseq6000 platform. The editing efficiency of the target site and the off-target site in front is listed in Table 17 below.

[0353] Table 17: Editing efficiency of on-target sites and top off-target sites

[0354]

[0355]

[0356] Example 12. On-target / off-target editing efficiency of DNA-RNA hybrid gRNA in PHH cells

[0357] Primary human hepatocytes (PHH) were thawed in InvitroGRO CP medium containing 10% FBS and 1% Pen / Strat and seeded in collagen-coated flat 48-well plates at 130,000 cells / well. After 4-6 hours, the medium was replaced with InvitroGRO CP medium. 0.75 μL RNAiMax reagent (Thermofisher) was used for transfection with 250 ng of cas9 mRNA and 250 ng of sgRNA (General BioL). On the day after transfection, the medium was replaced with 1% Pen / Strep InvitroGRO CP medium until cells were collected 3 days after transfection. Genomic DNA was extracted with QuickExtract DNA extraction solution (Lucigen). Editing at the target site and the off-target site in front was amplified by PCR with Taq Pro Multiplex DNA polymerase (Vazyme). PCR products were purified using VAHTS DNA purification beads (Vazyme) and sequenced on the Illumina Novaseq 6000 platform. The editing efficiencies of the targeted sites and the top off-target sites are listed in Table 18 below.

[0358] Table 18: Editing efficiency of targeted sites and top off-target sites

[0359] gRNA ON OT1 OT2 OT3 OT4 OT5 P9-hc-023 65.7％ 2.2％ 0.6％ 0.3％ 0.0％ N / A P9-hc-023-seq5 60.0％ 0.3％ 0.3％ 0.3％ 0.0％ N / A P9-hc-023-seq9 58.9％ 0.2％ 0.3％ 0.3％ 0.0％ N / A P9-hc-023-seq10 36.4％ 0.1％ 0.3％ 0.3％ 0.0％ N / A P9-hc-023-seq11 39.3％ 0.1％ 0.3％ 0.3％ 0.0％ N / A P9-hc-028-seq1 53.4％ 0.3％ 0.1％ 0.2％ 0.0％ N / A P9-hc-028-seq4 33.8％ 0.1％ 0.0％ 0.1％ 0.0％ N / A P9-hc-028-seq5 50.2％ 0.3％ 0.0％ 0.1％ 0.0％ N / A P9-hc-028-seq6 50.8％ 0.2％ 0.0％ 0.1％ 0.0％ N / A P9-hc-082 44.1％ 1.0％ 0.1％ 0.0％ 0.2％ 0.1％ P9-hc-082-seq4 39.0％ 0.1％ 0.0％ 0.0％ 0.1％ 0.0％ P9-hc-082-seq6 22.1％ 0.0％ 0.0％ 0.0％ 0.1％ 0.0％ P9-hc-082-seq7 22.5％ 0.0％ 0.0％ 0.0％ 0.1％ 0.0％ P9-hc-162 45.6％ 0.2％ 0.1％ 0.2％ N / A N / A P9-hc-162-seq2 45.6％ 0.2％ 0.1％ 0.2％ N / A N / A P9-hc-162-seq3 54.4％ 0.1％ 0.1％ 0.1％ N / A N / A P9-hc-162-seq5 55.3％ 0.1％ 0.1％ 0.1％ N / A N / A P9-hc-162-seq6 54.4％ 0.0％ 0.0％ 0.1％ N / A N / A P9-hc-212 60.8％ 1.2％ 0.2％ N / A N / A N / A P9-hc-212-seq2 56.6％ 0.4％ 0.1％ N / A N / A N / A P9-hc-212-seq3 53.8％ 0.3％ 0.2％ N / A N / A N / A P9-hc-212-seq6 49.3％ 0.3％ 0.3％ N / A N / A N / A

[0360] Example 13. Effects of different Cas9 mRNA elements in Huh7 and PHH cells

[0361] UTR screening in Huh7 cells. As described in Example 2, sgRNA P9-hc-162 targeting human PCSK9 and Cas9 mRNA containing different UTRs were delivered to Huh7 cells in a two-fold dose response curve of 8-12 points. Cells were lysed 72 hours after treatment and used for editing analysis as described in Example 2. Then, UTR elements were listed based on EC50 values ​​and maximum editing percentages. The dose response curve for guide sequences in Huh7 cells is shown in Figure 4The EC50 values ​​and maximum editing percentages are listed in Table 19 below.

[0362] Table 19: Efficiency of Cas9 mRNA containing different UTRs

[0363] Name EC50(nM) Maximum Edit (%) ART-UTR-16 0.797 96.83 ART-UTR-37 0.994 96.66 ART-UTR-21 0.721 95.51 ART-UTR-23 0.628 95.42 ART-UTR-26 0.576 95.68 ART-UTR-28 0.725 96.17 ART-UTR-33 1.094 95.32

[0364] CDS design using MFE and CAI. Considering three factors: high CAI, low MFE and medium GC content, CDS was designed. The same UTR and nuclear localization signal sequence were used to calculate MFE and perform cell experiments. The characteristics of the designed CDS are listed in Table 20 below. The percentage of similarity between the designed CDS is listed in Table 21 below. The sequences of the CDS are provided as SEQ ID NOs: 954-960 in Table 22 below.

[0365] Table 20: Characteristics of the designed CDS

[0366] MFE(kcal / mol) CAI Scoring GC content (%) ART-CDS-K1-1 -1824 0.923 58.6 ART-CDS-K4-8 -1776 0.968 59.5 ART-CDS-S311 -1745 0.992 61.8 ART-CDS-K10-2 -1742 0.991 60.3 ART-CDS-K8-1 -1729 0.990 60.2 ART-CDS-004R -1412 0.910 52.6 ART-CDS-S204 -1169 0.865 50.4

[0367] Table 21: Similarity percentage of designed CDS

[0368]

[0369] Table 22: Sequences of CDS design

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385] Efficacy of CDS variants in Huh7 cells. As described in Example 2, sgRNA with seed sequence NTLA-2001 (AAAGGCUGCUGAUGACACCU, SEQ ID NO: 973) and Cas9 mRNA containing different CDS were delivered to Huh7 cells, with 4-fold replication of 4 points. Cells were lysed 72 hours after treatment and used for editing analysis as described in Example 2. The editing efficiency is listed in Table 23 below.

[0386] Table 23: Editing efficiency of cas9 mRNA variants containing different CDS

[0387]

[0388] Efficacy of selected CDS variants in Huh7 cells. As described in Example 2, gRNA P9-hc-162 targeting human PCSK9 and Cas9 mRNA containing different CDS were delivered to Huh7 cells in a four-fold dose-response curve of 4 points or a two-fold dose-response curve of 8-10 points. Cells were lysed 72 hours after treatment and used for editing analysis as described in Example 1. CDS elements were then listed based on EC50 values ​​and maximum editing percentages. Dose-response curves for guide sequences in Huh7 cells are shown in Figure 5 The EC50 values ​​and maximum editing efficiencies are listed in Table 24 below.

[0389] Table 24: Editing efficiency of cas9 mRNA variants containing selected CDS in Huh7

[0390] name EC50 Maximum Edit % ART-CDS-004R 0.865 86.2 ART-CDS-K1-1 0.266 93.8 ART-CDS-K4-8 0.348 92.9 ART-CDS-K10-2 0.226 94.1

[0391] The efficacy of selected CDS variants in PHH cells. Primary human hepatocytes (PHH) were thawed in InvitroGRO CP medium containing 10% FBS and 1% Pen / Strat and seeded in collagen-coated flat 48-well plates at a density of 13,000 cells / well. After 4-6 hours, the culture medium was replaced with InvitroGRO CP medium. RNAiMax reagent (Thermofisher) was used to transfect with two-fold dilutions starting from 200ng of cas9 mRNA (Levostar) and 100ng of ART-001-g091 sgRNA per well. On the day after transfection, the culture medium was replaced with InvitroGRO CP medium containing 10% FBS and 1% Pen / Strep. After another two days, cells were collected for gene editing efficiency analysis, as expressed readouts, shown in Table 25 below.

[0392] Table 25: Editing efficiency of cas9 mRNA variants containing selected CDS in PHH

[0393] Total RNA ng ART-CDS-K1-1 ART-CDS-K10-2 ART-CDS-K4-8 ART-CDS-004R 37.5 31.3 39.8 37.7 34.4 75 58.1 60.0 59.1 53.6 150 71.7 71.7 74.4 70.7 300 74.7 71.3 76.8 67.0

[0394] Rationally designing the polyA tail to make its size distribution more precise

[0395] Rational design of PolyA sequences was evaluated in Huh7 cells. Huh7 cells were seeded in 96-well plates at 8,000 cells / well. 0.4 μL RNAiMax reagent (Thermofisher) was used to transfect with 100ng of cas9 mRNA and 100ng of sgRNA (NTLA-2001) (Genscript). Cells were collected three days after transfection. Genomic DNA was extracted with QuickExtract DNA extraction solution (Lucigen). Editing at the targeted site was performed by PCR amplification with Taq Pro Multiplex DNA polymerase (Vazyme). The PCR product was purified with VAHTSDNA purification beads (Vazyme) and sequenced on the Illumina Novaseq6000 platform. The editing efficiency of cas9mRNA variants containing different polyA tail designs in Huh7 cells is shown in Table 26 below. The sequences of different polyA tail designs are provided as SEQ ID NO:963-972 in Table 27 below.

[0396] Table 26: Editing efficiency of cas9 mRNA variants containing different polyA tail designs in Huh7 cells

[0397]

[0398] Table 27: Sequences of polyA tail design

[0399]

[0400] Determination of the length distribution of polyA tails in mRNA by mass spectrometry

[0401] As described in the Methods section, polyA tails of mRNAs of different designs were analyzed. The distribution of polyA lengths deviated from the predicted sizes in the plasmids and are shown in Table 28 as a percentage of the total detected events. Adding G or GG at the end reduced the width of the size distribution and the deviation of the peak from the predicted peak.

[0402] Table 28: Distribution of polyA length in mRNA

[0403] Deviation from predicted size pA-3Seg pA-3SegG pA-3SegGG -2 1.7 -1 3.5 8.6 5.8 0 8.5 17.3 15.6 +1 14.5 23.1 24.0 +2 18.4 21.4 19.6 +3 16.9 14.8 14.7 +4 13.8 10.0 14.1 +5 10.7 7.7 4.6 +6 8.8 +7 2.5 +8 2.4

[0404] Rational design of PolyA sequences was evaluated in PHH cells. Primary human hepatocytes (PHH) were thawed in InvitroGRO CP medium containing 10% FBS and 1% Pen / Strat and seeded in collagen-coated 48-well plates at a density of 13,000 cells / well. After 4-6 hours, the medium was replaced with InvitroGRO CP medium. Transfection was performed using RNAiMax reagent (Thermofisher) with 250ng of cas9 mRNA and 250ng of sgRNA (General Biosystem). On the day after transfection, the medium was replaced with 1% Pen / Strep InvitroGRO CP medium supplemented with 10% FBS until cells were collected 3 days after transfection. The editing efficiency of Cas9 mRNA variants containing different polyA tail designs in PHH cells is shown in Table 29 below.

[0405] Table 29: Editing efficiency of Cas9 mRNA variants containing different polyA tail designs in PHH cells

[0406] gRNA (ng) pA51C8A pA60C8A pA60C20A pA100 pA120 pA-3SegG pA-3SegGG 7.8 16.3 15.6 17.7 13.7 12.7 Not tested 19.1 15.6 27.1 26.2 30.4 27.3 26.3 Not tested 29.3 31.3 38.7 40.6 39.8 32.7 37.6 Not tested 36.9 62.5 44.7 42.3 45.3 46.2 38.9 38.1 41.5 125.0 53.5 49.2 51 53.8 39.7 45.1 44 250.0 53.2 52.5 22.3 56.6 34 44.1 51.2

[0407] Example 14. In vivo evaluation of sgRNA in humanized PCSK9 mice

[0408] Humanized PCSK9 mice were transformed so that the region of the endogenous mouse Pcsk9 locus was deleted and replaced with an orthologous human PCSK9 sequence, so that the locus encodes human PCSK9 protein. These PCSK9 gene humanized mice were administered with LNP formulations containing Cas9 mRNA (SEQ ID NO: 902) and sgRNA as shown in Table 30 below at a weight ratio of 2: 1. The LNPs contain ALC-0315, DSPC, cholesterol, and PEG2k-DMG. The dosing level was 1 mg / kg or 0.3 mg / kg (based on total RNA content) and was injected intravenously. Mice administered with a single vehicle (20 mM Tris buffer containing 7.5% sucrose) were used as negative controls.

[0409] Table 30: Experimental design for in vivo evaluation of sgRNAs in humanized PCSK9 mice

[0410]

[0411]

[0412] The results of liver editing were determined using primers designed to amplify the region of interest for NGS analysis, and the knockdown of serum human PCSK9 protein was detected using a specific human PCSK9 ELISA kit as described above. The results of liver gene editing and serum PCSK9 protein knockdown for each group are shown in Table 31 below. The editing of the PCSK9 gene and subsequent protein knockdown were demonstrated with a series of sgRNAs (including P9-hc-162, P9-hc-212, P9-h-057, P9-hc-082, and P9-hc-023). Using each sgRNA, a clear dose response was observed for both liver gene editing and serum PCSK9 protein reduction. Due to the low number of sequencing reads, some data were not obtained.

[0413] Table 31: Liver PCSK9 gene editing and serum PCSK9 (% KD) results for sgRNA screening

[0414]

[0415]

[0416] Example 15. In vivo evaluation of DNA / RNA hybrid sgRNA design in humanized PCSK9 mice

[0417] Humanized PCSK9 mice were transformed so that the region of the endogenous mouse Pcsk9 locus was deleted and replaced with an orthologous human PCSK9 sequence, so that the locus encodes human PCSK9 protein. These PCSK9 gene humanized mice were administered with Cas9 mRNA (SEQ ID NO: 902) at a weight ratio of 2: 1 and sgRNA as shown in Table 32 below. LNPs contain ALC-0315, DSPC, cholesterol, and PEG2k-DMG. The dosing level was 0.6 mg / kg or 0.2 mg / kg (based on total RNA content) and was administered by intravenous injection. Mice of the corresponding genotype as a negative control were administered with a single vehicle (20 mM Tris buffer containing 7.5% sucrose).

[0418] Table 32: Experimental design for in vivo evaluation of DNA / RNA hybrid sgRNA in humanized PCSK9 mice

[0419]

[0420]

[0421] The knockdown of serum human PCSK9 protein was detected using a specific human PCSK9 ELISA kit as described above. The reduction of serum PCSK9 protein in each group is shown in Table 33 below. The efficacy of protein knockdown was demonstrated using some sgRNAs (including P9-hc-162, P9-hc-162-seq5, P9-hc-162-seq6, P9-hc-023, and P9-hc-023-seq9).

[0422] Table 33: Serum PCSK9 (% KD) results for sgRNA screening

[0423]

[0424] Example 16. In vivo evaluation of UTR design in humanized PCSK9 mice

[0425] Humanized PCSK9 mice were transformed so that the region of the endogenous mouse Pcsk9 locus was deleted and replaced with an orthologous human PCSK9 sequence, so that the locus encodes human PCSK9 protein. These PCSK9 gene humanized mice were administered with Cas9 mRNA containing different UTR sequences (SEQ ID NO: 941-953 shown in Table 34 below) and sgRNA P9-hc-162 (SEQ ID NO: 805) as shown in Table 35 below at a weight ratio of 2: 1. LNP contains ALC-0315, DSPC, cholesterol and PEG2k-DMG. The administration level was 1 mg / kg or 0.3 mg / kg (based on total RNA content) by intravenous injection (N=1 / group). Mice of the corresponding genotype as a negative control were administered with a single vehicle (20mMTris buffer containing 7.5% sucrose).

[0426] Table 34: UTR sequence of Cas9 mRNA

[0427]

[0428] Table 35: Experimental design for in vivo screening of UTRs in humanized PCSK9 mice

[0429]

[0430] The results of liver editing were determined using primers designed to amplify the region of interest for NGS analysis, and the knockdown of serum human PCSK9 protein was detected using a specific human PCSK9 ELISA kit as described above. The results of liver gene editing and serum PCSK9 protein knockdown 7 days after treatment of each group are shown in Table 36 below. Efficient editing and protein knockdown of PCSK9 sequences were demonstrated using the tested LNPs containing Cas9 mRNA with different UTR sequences (including ART-UTR-21, ART-UTR-26, ART-UTR-28, ART-UTR-28, and ART-UTR-37).

[0431] Table 36: Gene editing efficiency and serum PCSK9 protein reduction with different UTR designs

[0432]

[0433]

[0434] Example 17. In vivo evaluation of different CDS designs in humanized PCSK9 mice

[0435] Humanized PCSK9 mice were transformed so that the region of the endogenous mouse Pcsk9 locus was deleted and replaced with an orthologous human PCSK9 sequence, so that the locus encodes human PCSK9 protein. These mice humanized with respect to the PCSK9 gene were administered with a 2:1 weight ratio of Cas9 mRNA containing a series of CDS designs (provided as SEQ ID NO: 954-960 in Table 22) and sgRNA P9-hc-162 (SEQ ID NO: 805) as shown in Table 37 below. LNP contains ALC-0315, DSPC, cholesterol and PEG2k-DMG in a molar ratio of 49.5: 9.5: 38.5: 2.5. The administration level was 0.3 mg / kg or 0.1 mg / kg (based on total RNA content) by intravenous injection (N = 1 / group). Mice of the corresponding genotype as a negative control were administered with a single vehicle (20 mM Tris buffer containing 7.5% sucrose).

[0436] Table 37: Experimental design for in vivo evaluation of different CDS designs in humanized PCSK9 mice

[0437]

[0438] The results of liver editing were evaluated using primers designed to amplify the region of interest followed by NGS analysis. In addition, the reduction of serum human PCSK9 protein was detected using a specific human PCSK9 ELISA kit as described above. The results of liver gene editing and serum PCSK9 protein knockdown 7 days after treatment for each group are shown in Table 38 below. Efficient editing and protein knockdown of PCSK9 sequences were demonstrated with all CDS sequences tested.

[0439] Table 38: Efficacy of gene editing and serum PCSK9 reduction with different CDS designs

[0440]

[0441] Other Implementations

[0442] It should be understood that the foregoing description is intended to illustrate and not to limit the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims

1. A guide RNA comprising: a. A sequence selected from SEQ ID NO: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940; b. at least 15, 16, 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from SEQ ID NO: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940; or c. a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940.

2. A vector comprising one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise: a. one or more sequences selected from SEQ ID NO:915, 933, 934, 1-296, 908-914, 916-932 and 935-940; b. at least 15, 16, 17, 18, 19 or 20 consecutive nucleotides of one or more sequences selected from SEQ ID NO: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940; or c. one or more sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940.

3. A composition comprising: (i) a nucleic acid encoding a guide RNA, or a vector comprising a nucleic acid encoding a guide RNA, wherein the guide RNA comprises: a. A sequence selected from SEQ ID NO: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940; b. at least 15, 16, 17, 18, 19 or 20 consecutive nucleotides of a sequence selected from SEQ ID NO: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940; or c. a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from SEQ ID NO: 915, 933, 934, 1-296, 908-914, 916-932 and 935-940; and (ii) an RNA-guided DNA-binding agent, a nucleic acid encoding an RNA-guided DNA-binding agent, or a vector comprising a nucleic acid encoding an RNA-guided DNA-binding agent.

4. The composition of claim 3, wherein the RNA-guided DNA binder comprises a Cas nuclease or a Cas nickase.

5. The composition of any one of claims 3-4, wherein the nucleic acid encoding the RNA-guided DNA binder is a Cas9 encoding nucleic acid comprising a polynucleotide sequence shown in one or more of SEQ ID NOs: 902, 903, 941-953, 954-960, and 963-972.

6. The composition of any one of claims 3-5, wherein the RNA-guided DNA binder is Cas9 comprising the amino acid sequence shown in SEQ ID NO:

901.

7. The composition of claim 4, wherein the Cas nuclease is a Class 2 Cas nuclease.

8. A composition according to claim 4 or 7, wherein the Cas nuclease is Cas9, Cpf1, C2cl, C2c2 and C2c3 or a modified protein thereof.

9. A composition according to any one of claims 4, 7 or 8, wherein the Cas nuclease is Streptococcus pyogenes (S. pyogenes) Cas9 nuclease or Staphylococcus aureus (S. aureus) Cas9 nuclease or a modified protein thereof.

10. The composition of any one of claims 4, 7, 8 or 9, wherein the Cas nuclease is from a type II CRISPR / Cas system.

11. The composition according to any one of claims 3 to 10, which is used for editing the proprotein convertase subtilisin / kexin type 9 (PCSK9) gene.

12. The composition of claim 11, wherein the editing is calculated as the percentage of the cell population that is edited (percent editing).

13. The composition of claim 12, wherein about 30% to 99% of the cell population is edited.

14. The composition of claim 13, wherein the percentage of editing is 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% of a cell population.

15. The composition of any one of claims 3-14, wherein the composition reduces circulating LDL levels in a subject.

16. The composition of claim 15, wherein the level of LDL in the subject's circulation is measured 8 weeks after administration of the composition.

17. The composition of claim 16, wherein the level of LDL in the subject's circulation is compared to a negative control or to a level determined in the subject prior to administration of the composition.

18. The composition of claim 17, wherein the level of LDL in the subject's circulation is reduced by at least 20% relative to a corresponding negative control or a level measured in the subject prior to administration of the composition.

19. The composition of claim 16, wherein the composition is administered or delivered at least once.

20. The composition of claim 19, wherein the administration or delivery is performed at the following intervals: (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days; or (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 weeks; or (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 months; or (d) 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.

21. The composition of any one of claims 3-20, wherein the guide RNA is at least partially complementary to a target sequence present in the human PCSK9 gene.

22. The composition of claim 21, wherein the target sequence is located in exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the human PCSK9 gene.

23. The composition of claim 21, wherein the guide RNA sequence is complementary to a target sequence in the positive strand of the PCSK9 gene.

24. The composition of claim 21, wherein the guide RNA sequence is complementary to a target sequence in the negative strand of PCSK9.

25. The composition of claim 21, wherein the first guide sequence is complementary to a first target sequence in the positive strand of the PCSK9 gene, and wherein the composition further comprises a second guide sequence complementary to a second target sequence in the negative strand of the PCSK9 gene.

26. The composition of any one of claims 3-25, wherein the guide RNA comprises crRNA, and further comprises tracrRNA (trRNA) or a portion thereof, wherein the tracrRNA (trRNA) comprises the nucleotide sequence shown in SEQ ID NO: 904, wherein the trRNA is operably linked to the crRNA.

27. The composition of any one of claims 3-26, wherein the guide RNA is a dual guide RNA (dgRNA).

28. The composition of any one of claims 3-27, wherein the guide RNA is a single guide RNA (sgRNA).

29. The composition of any one of claims 3-28, wherein the guide RNA comprises at least one modification.

30. The composition of claim 29, wherein the at least one modification comprises 2'-O-methyl (2'-O-Me) modified nucleotides, phosphorothioate (PS) bonds between nucleotides, 2'-fluoro (2'-F) modified nucleotides, or a DNA-RNA hybrid.

31. The composition of claim 30, wherein the at least one modification comprises a modification at one or more of the first five nucleotides at the 5' end of the guide RNA and / or at one or more of the last five nucleotides at the 3' end of the guide RNA.

32. The composition of claim 29, wherein the at least one modification comprises modification of at least 50% of the nucleotides in the guide RNA.

33. The composition of claim 28, wherein the sgRNA comprises a guide sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932, and 935-940.

34. The composition of claim 28, wherein the sgRNA comprises the nucleotide sequence shown in any one of SEQ ID NOs: 593-888.

35. The composition of claim 28, wherein the sgRNA comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 593-888.

36. The composition of any one of claims 3-35, wherein the sgRNA is associated with a lipid nanoparticle (LNP).

37. The composition according to any one of claims 3-36, wherein the composition is a pharmaceutical preparation and further comprises a pharmaceutically acceptable carrier.

38. The composition of any one of claims 3-37, wherein the composition reduces or prevents atherosclerosis in vascular tissue of a subject.

39. The composition of any one of claims 3-38, wherein administration of the composition results in a deletion or insertion of one or more nucleotides in the PCSK9 gene.

40. The composition of claim 39, wherein the deletion or insertion of one or more nucleotides induces a frameshift or nonsense mutation in the PCSK9 gene.

41. The composition of claim 40, wherein a frameshift or nonsense mutation is induced in the PCSK9 gene of about 20% to about 30% or more of the cells.

42. The composition of claim 41, wherein the cell is a hepatocyte, a kidney cell, an intestinal epithelial cell, or a vascular epithelial cell.

43. The composition of claim 39, wherein the deletion or insertion of one or more nucleotides in the PCSK9 gene is at least 50 times greater than that in the off-target site or more.

44. The composition of any one of claims 3-43, wherein administration of the composition increases LDLR levels in cells of the subject.

45. The composition of any one of claims 3-44, wherein the LDL level in the blood of the subject is measured.

46. ​​A composition according to any one of claims 3-45, wherein the subject suffers from hypercholesterolemia and / or cardiovascular disease.

47. The composition of claim 46, wherein the subject has familial hypercholesterolemia.

48. The composition of claim 46 or 47, wherein the subject exhibits symptoms of atherosclerosis.

49. The composition of any one of claims 3-48, wherein the subject expresses PCSK9 with one or more mutations selected from the group consisting of: R46L, Y142X, R218S, F216L, D374Y, A443T, or C679X.

50. The composition of any one of claims 3-49, wherein after administration, the subject exhibits improvement, stabilization, or slowing of changes in symptoms of hypercholesterolemia and / or cardiovascular disease.

51. The composition of claim 50, wherein improvement, stabilization or reduction in changes in symptoms of hypercholesterolemia and / or cardiovascular disease is measured using blood lipid testing or patient reported outcomes.

52. A composition according to any one of claims 3-51, wherein the composition or pharmaceutical preparation is administered via a viral vector.

53. A composition according to any one of claims 3-52, wherein the composition or pharmaceutical formulation is administered via lipid nanoparticles.

54. A method for modifying a human proprotein convertase subtilisin / kexin type 9 (PCSK9) gene and / or inducing double-strand breaks (DSBs) within a PCSK9 gene, comprising administering a composition as described in any one of claims 3 to 53 into a cell, wherein the composition recognizes and cleaves a PCSK9 target sequence.

55. A method for reducing a subject's blood LDL level and / or treating a subject's hypercholesterolemia and / or cardiovascular disease, comprising administering to a subject in need thereof a composition according to any one of claims 3-53, wherein the composition recognizes and cleaves a PCSK9 target sequence, thereby reducing the subject's blood LDL level and / or treating the subject's hypercholesterolemia and / or cardiovascular disease.

56. The method according to claim 54 or 55, which is used for editing the PCSK9 gene.

57. The method of any one of claims 54-56, wherein the RNA-guided DNA binder comprises a Cas nuclease or a Cas nickase.

58. The method of any one of claims 54-57, wherein the nucleic acid encoding the RNA-guided DNA binder is a Cas9 encoding nucleic acid comprising a nucleic acid sequence shown in one or more of SEQ ID NOs: 902, 903, 941-953, 954-960, and 963-972.

59. The method of any one of claims 54-58, wherein the RNA-guided DNA binder is Cas9 comprising the amino acid sequence shown in SEQ ID NO:

901.

60. The method of claim 57, wherein the Cas nuclease is a Class 2 Cas nuclease.

61. The method of claim 57 or 60, wherein the Cas nuclease is Cas9, Cpfl, C2cl, C2c2 and C2c3 or a modified protein thereof.

62. The method of claim 57, 60 or 61, wherein the Cas nuclease is Streptococcus pyogenes Cas9 nuclease or Staphylococcus aureus Cas9 nuclease or a modified protein thereof.

63. The method of claim 57, 60, 61 or 62, wherein the Cas nuclease is from a type II CRISPR / Cas system.

64. The method according to any one of claims 54-63, which is used for editing the proprotein convertase subtilisin / kexin type 9 (PCSK9) gene.

65. The method of claim 64, wherein the editing is calculated as the percentage of the cell population that is edited (percent editing).

66. The method of claim 65, wherein between about 30% and 99% of the cell population is edited.

67. The method of claim 66, wherein the percentage of editing is between 30% and 35%, between 35% and 40%, between 40% and 45%, between 45% and 50%, between 50% and 55%, between 55% and 60%, between 60% and 65%, between 65% and 70%, between 70% and 75%, between 75% and 80%, between 80% and 85%, between 85% and 90%, between 90% and 95%, or between 95% and 99% of a cell population.

68. The method of any one of claims 54-67, wherein the composition reduces PCSK9 abundance in cells of at least one tissue or organ and increases LDLR abundance in cells of at least one tissue or organ.

69. The method of claim 68, wherein the at least one tissue or organ is selected from the group consisting of liver, kidney, intestinal epithelium, or vascular epithelium.

70. The method of any one of claims 54-69, wherein the composition reduces LDLR abundance in cells of at least one tissue or organ and increases LDLR abundance in cells of at least one tissue or organ, and reduces LDL levels in blood.

71. The method of claim 70, wherein the LDL level in the blood is determined 8 weeks after administration of the composition.

72. The method of claim 71, wherein the LDL level in the blood is compared to a negative control or a level determined in the subject prior to administration of the composition.

73. The method of claim 72, wherein the LDL level in the blood is reduced by at least 20% relative to a corresponding negative control or the level determined in the subject prior to administration of the composition.

74. The method of claim 71, wherein the composition is administered or delivered at least once.

75. The method of claim 74, wherein the administering or delivering is performed at the following intervals: (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days; or (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 weeks; or (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 months; or (d) 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.

76. The method of any one of claims 54-75, wherein the guide RNA is at least partially complementary to a target sequence present in a human PCSK9 gene.

77. The method of claim 76, wherein the target sequence is located in exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the human PCSK9 gene.

78. The method of claim 76, wherein the guide RNA sequence is complementary to a target sequence in the positive strand of the PCSK9 gene.

79. The method of claim 76, wherein the guide RNA sequence is complementary to a target sequence in the negative strand of PCSK9.

80. The method of claim 76, wherein the first guide sequence is complementary to a first target sequence in the positive strand of the PCSK9 gene, and wherein the composition further comprises a second guide sequence complementary to a second target sequence in the negative strand of the PCSK9 gene, or vice versa.

81. The method of any one of claims 54-80, wherein the guide RNA comprises crRNA, and further comprises tracrRNA (trRNA) or a portion thereof, wherein the tracrRNA (trRNA) comprises the nucleotide sequence shown in SEQ ID NO: 904, wherein the trRNA is operably linked to the crRNA.

82. The method of any one of claims 54-80, wherein the guide RNA is a dual guide RNA (dgRNA).

83. The method of any one of claims 54-81, wherein the guide RNA is a single guide (sgRNA).

84. The method of any one of claims 54-83, wherein the guide RNA comprises at least one modification.

85. The method of claim 84, wherein the at least one modification comprises 2'-O-methyl (2'-O-Me) modified nucleotides, phosphorothioate (PS) bonds between nucleotides, or 2'-fluoro (2'-F) modified nucleotides.

86. The method of claim 84, wherein the at least one modification comprises a modification at one or more of the first five nucleotides at the 5' end of the guide RNA and / or at one or more of the last five nucleotides at the 3' end of the guide RNA.

87. The method of claim 84, wherein the at least one modification comprises modification of at least 50% of the nucleotides in the guide RNA.

88. The method of claim 83, wherein the sgRNA comprises a guide sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 915, 933, 934, 1-296, 908-914, 916-932, and 935-940.

89. The method of claim 83, wherein the sgRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 593-888.

90. The method of claim 83, wherein the sgRNA comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 593-888.

91. The method of any one of claims 54-90, wherein the guide RNA is associated with a lipid nanoparticle (LNP).

92. The method of any one of claims 54-91, wherein the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier.

93. The method of any one of claims 54-92, wherein administration of the composition results in the deletion or insertion of one or more nucleotides in the PCSK9 gene.

94. The method of claim 93, wherein the deletion or insertion of one or more nucleotides induces a frameshift or nonsense mutation in the PCSK9 gene.

95. The method of claim 94, wherein a frameshift or nonsense mutation is induced in the PCSK9 gene in about 20% to about 30% or more of the cells.

96. The method of claim 95, wherein the cell is a hepatocyte, a kidney cell, an intestinal epithelial cell, or a vascular epithelial cell.

97. The method of claim 93, wherein the deletion or insertion of one or more nucleotides in the PCSK9 gene is at least 50 times greater than that in the off-target site or sites.

98. The method of any one of claims 54-97, wherein administering the composition increases LDLR levels in cells of the subject.

99. The method of claim 98, wherein the LDLR levels in the subject's cells are increased by at least 10%.

100. The method of claim 99, wherein the LDL level in the subject's blood is measured.

101. The method of any one of claims 54-100, wherein the subject suffers from hypercholesterolemia and / or cardiovascular disease.

102. The method of claim 101, wherein the subject has familial hypercholesterolemia.

103. The method of claim 101 or 102, wherein the subject exhibits symptoms of atherosclerosis.

104. The method of any one of claims 54-103, wherein the subject expresses PCSK9 with one or more mutations selected from the group consisting of: R46L, Y142X, R218S, F216L, D374Y, A443T, and C679X.

105. The method of claim 104, wherein following administration, the subject exhibits improvement, stabilization, or a reduction in changes in symptoms of hypercholesterolemia and / or cardiovascular disease.

106. The method of claim 105, wherein improvement, stabilization, or reduction in changes in symptoms of hypercholesterolemia and / or cardiovascular disease is measured using blood lipid testing or patient reported outcomes.

107. The method of any one of claims 54-106, wherein the composition or pharmaceutical formulation is administered via a viral vector or via lipid nanoparticles.

108. Use of a composition according to any one of claims 54-107 in the preparation of a medicament for treating a human subject suffering from hypercholesterolemia and / or cardiovascular disease.

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