Methods for epigenetic editing target sites and uses thereof

Abstract

A method for epigenetically editing a target site and its use are provided. In particular, a method for regulating the expression level of a target gene and a nucleic acid binding molecule are provided. The combination of the nucleic acid binding molecule and the gene expression regulatory molecule has the function of regulating the expression level of the target gene.

Description

[Technical Field]

[0001] Technical Field This application relates to the biomedical field, and in particular to epigenetic editing targets and uses thereof. [Background technology]

[0002] background Fusing catalytically inactive "dead Cas9" (dCas9) to a Krueppel-associated box (KRAB) domain generates gene repressors that can regulate or silence target genes with high specificity and efficiency in cell culture assays. However, in an in vivo environment, repressors face certain challenges in exerting therapeutic effects. For example, safety, toxicity, immunogenicity, and off-target effects are other challenges that limit the use of synthetic repressors in vivo.

[0003] Thus, there is a need in the field for effective epigenetic editing targets that can improve epigenetic editing efficacy and safety, and reduce toxicity, immunogenicity, and / or off-target effects. Summary of the Invention

[0004] overview The present application provides epigenetic editing targets, wherein targeting the targets can be used to improve epigenetic editing efficacy and safety, and reduce toxicity, immunogenicity, and / or off-target effects. For example, by targeting the vicinity of the target gene and / or within the regulatory element of the target gene, at least one nucleotide modification can be effectively performed to regulate (e.g., reduce or eliminate) the expression of the target gene product in cells.

[0005] In one aspect, the present application provides a method for regulating the expression and / or activity of the ANGPTL3 (angiopoietin-like 3) gene, comprising providing a gene expression regulatory molecule or a nucleic acid encoding a gene expression regulatory molecule, wherein the gene expression regulatory molecule has the function of regulating the expression of the ANGPTL3 gene without altering the genetic sequence of the ANGPTL3 gene.

[0006] In another aspect, the present application provides a method for treating and / or alleviating a pathology associated with abnormal expression and / or activity of the ANGPTL3 gene, the method comprising providing a gene expression regulatory molecule or a nucleic acid encoding a gene expression regulatory molecule, wherein the gene expression regulatory molecule has the function of regulating the expression of the ANGPTL3 gene without altering the genetic sequence of the ANGPTL3 gene.

[0007] In some embodiments, the gene expression regulatory molecule comprises a first functional domain that provides for modification of at least one nucleotide near the ANGPTL3 gene and / or within an ANGPTL3 gene regulatory element.

[0008] In some embodiments, the modification of at least one nucleotide comprises a methylation modification.

[0009] In some embodiments, the regulatory elements comprise a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region.

[0010] In some embodiments, the first functional domain comprises one or more of a DNA methyltransferase, a DNA demethylase, and functionally active fragments thereof.

[0011] In some embodiments, the DNA methyltransferase comprises one or more of DNMT 3A, DNMT 3B, DNMT 3L, DNMT 1, and DNMT 2.

[0012] In some embodiments, the DNMT 3A is from a mouse.

[0013] In some embodiments, the DNMT 3L is of human and / or mouse origin.

[0014] In some embodiments, DNMT 3A and DNMT 3L are directly and / or indirectly linked.

[0015] In some embodiments, the gene expression regulatory molecule comprises a zinc finger protein-based transcription factor or a functionally active fragment thereof, or a second functional domain comprising an agent capable of modifying histones.

[0016] In some embodiments, the second functional domain comprises Krab.

[0017] In some embodiments, the second functional domain comprises a ZIM3 Krab or a KOX1 Krab.

[0018] In some embodiments, the second functional domain comprises one or more of a histone methyltransferase, a histone demethylase, a histone acetyltransferase, a histone deacetylase, and functionally active fragments thereof.

[0019] In some embodiments, the gene expression regulatory molecule comprises a first functional domain and a second functional domain, wherein the first functional domain is directly or indirectly linked to one end of the second functional domain, or the first functional domain is directly and / or indirectly linked to both ends of the second functional domain.

[0020] In some embodiments, the gene expression regulatory molecule comprises a DNA binding domain.

[0021] In some embodiments, the gene expression regulatory molecule comprises one or more DNA binding domains selected from a TALEN domain, a zinc finger domain, and a protein domain of a CRISPR / Cas system.

[0022] In some embodiments, the gene expression regulatory molecule comprises a Cas enzyme.

[0023] In some embodiments, the gene expression regulatory molecule comprises a Cas enzyme that is substantially free of nuclease activity.

[0024] In some embodiments, the gene expression regulatory molecule comprises a dCas9 enzyme.

[0025] In some embodiments, the first functional domain and the second functional domain are directly or indirectly linked to one end of the DNA-binding domain, or the first functional domain and the second functional domain are directly and / or indirectly linked to both ends of the DNA-binding domain.

[0026] In some embodiments, the gene expression regulatory molecule can bind to a DNA region within 500 bp upstream and / or downstream of the transcription start site (TSS) of the ANGPTL3 gene or a fragment thereof.

[0027] In some embodiments, the gene expression regulatory molecule can bind to a DNA region in which any one of SEQ ID NOs: 71-72 is located, or a fragment thereof.

[0028] In some embodiments, the gene expression regulatory molecule can bind to one or more DNA regions near the transcription start site (TSS) of the ANGPTL3 gene, as described below: between 180 bp upstream of the TSS and 10 bp upstream, between the TSS and 90 bp downstream of the TSS, and between 180 bp downstream of the TSS and 240 bp downstream of the TSS.

[0029] In some embodiments, the gene expression regulatory molecule can bind to one or more DNA regions near the transcription start site (TSS) of the ANGPTL3 gene, as described below: between 180 bp upstream and 150 bp upstream of the TSS, between 120 bp upstream and 90 bp upstream of the TSS, between 70 bp upstream and 10 bp upstream of the TSS, between the TSS and 30 bp downstream of the TSS, between 60 bp downstream and 90 bp downstream of the TSS, and between 180 bp downstream and 240 bp downstream of the TSS.

[0030] In some embodiments, the method comprises providing a nucleic acid binding molecule comprising the sequence of any one of SEQ ID NOs: 1-70.

[0031] In some embodiments, the gene expression modulating molecule and / or the nucleic acid binding molecule are formulated in the same delivery vehicle or in different delivery vehicles.

[0032] In some embodiments, the delivery vehicle consists of a liposome and / or a lipid nanoparticle.

[0033] In some embodiments, the gene expression modulating molecule and / or the nucleic acid binding molecule are formulated in the same recombinant vector or in different recombinant vectors.

[0034] In some embodiments, the recombinant vector comprises a viral vector.

[0035] In some embodiments, the recombinant vector comprises an adeno-associated virus (AAV) vector.

[0036] In some embodiments, the gene expression regulatory molecule comprises a nuclear localization sequence.

[0037] In some embodiments, the nuclear localization sequence comprises an amino acid having a positively charged group.

[0038] In some embodiments, the nuclear localization sequence is located at the N-terminus and / or C-terminus of the first functional domain, the N-terminus and / or C-terminus of the second functional domain, and / or the N-terminus and / or C-terminus of the DNA binding domain.

[0039] In another aspect, the present application provides a nucleic acid-binding molecule comprising any one of the sequences of SEQ ID NOs: 1 to 70.

[0040] In another aspect, the present application provides gene expression regulatory molecules that have the function of regulating the ANGPTL3 gene without altering the gene sequence of the ANGPTL3 gene.

[0041] In some embodiments, the gene expression regulatory molecule is a gene expression regulatory molecule provided in the methods of the present application.

[0042] In some embodiments, the gene expression regulatory molecule and / or the nucleic acid binding molecule are formulated in the same delivery vehicle or in different delivery vehicles, and the nucleic acid binding molecule comprises the sequence of any one of SEQ ID NOs: 1-70.

[0043] In some embodiments, the delivery vehicle comprises a liposome and / or a lipid nanoparticle.

[0044] In some embodiments, the expression-regulating molecule and / or the nucleic acid-binding molecule are formulated in the same recombinant vector or in different recombinant vectors, and the nucleic acid-binding molecule comprises the sequence of any one of SEQ ID NOs: 1-70.

[0045] In some embodiments, the recombinant vector comprises a viral vector.

[0046] In some embodiments, the recombinant vector comprises an adeno-associated virus (AAV) vector.

[0047] In another aspect, the present application provides a nucleic acid encoding the nucleic acid-binding molecule of the present invention and / or a nucleic acid encoding the gene expression-regulating molecule of the present invention.

[0048] In another aspect, the present application provides a recombinant vector comprising a nucleic acid of the present invention.

[0049] In another aspect, the present application provides a delivery carrier comprising the nucleic acid binding molecule of the present invention, the gene expression regulatory molecule of the present invention, the nucleic acid of the present invention, and / or the recombinant vector of the present invention, and optionally comprising a liposome and / or a lipid nanoparticle.

[0050] In another aspect, the present application provides a composition comprising the nucleic acid binding molecule of the present invention, the gene expression regulatory molecule of the present invention, the nucleic acid of the present invention, the recombinant vector of the present invention, and / or the delivery vehicle of the present invention.

[0051] In another aspect, the present application provides a cell comprising the nucleic acid binding molecule of the present invention, the gene expression regulatory molecule of the present invention, the nucleic acid of the present invention, the recombinant vector of the present invention, the delivery vehicle of the present invention, and / or the composition of the present invention.

[0052] In another aspect, the present application provides a kit comprising the nucleic acid binding molecule of the present invention, the gene expression regulatory molecule of the present invention, the nucleic acid of the present invention, the recombinant vector of the present invention, the delivery carrier of the present invention, the composition of the present invention, and / or the cell of the present invention.

[0053] In another aspect, the present application provides a method for regulating the expression and / or activity of a target gene, comprising providing a nucleic acid binding molecule of the present invention, a gene expression regulatory molecule of the present invention, a nucleic acid of the present invention, a recombinant vector of the present invention, a delivery vehicle of the present invention, a composition of the present invention, a cell of the present invention, and / or a kit of the present invention.

[0054] In another aspect, the present application provides use of a nucleic acid binding molecule of the present invention, a gene expression regulatory molecule of the present invention, a nucleic acid of the present invention, a recombinant vector of the present invention, a delivery vehicle of the present invention, a composition of the present invention, a cell of the present invention, and / or a kit of the present invention in the preparation of a medicament for treating and / or alleviating a pathology, including a pathology associated with aberrant expression and / or activity of a target gene.

[0055] Those skilled in the art may readily ascertain other aspects and advantages of the present application from the following detailed description. The following detailed description shows and describes only exemplary embodiments of the present invention. As those skilled in the art will recognize, the contents of this application will enable those skilled in the art to make modifications to the particular embodiments disclosed without departing from the spirit and scope of the invention disclosed herein. Accordingly, the descriptions in the drawings and specification are illustrative only and are not intended to be limiting.

[0056] BRIEF DESCRIPTION OF THE DRAWINGS Specific features of the invention involved in this application are set forth in the appended claims. The features and advantages of the invention involved in this application can be better understood by reference to the detailed description of exemplary embodiments and drawings provided below. A brief description of the drawings follows. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 shows the mRNA knockdown efficiency of the gene expression regulatory molecules of the present invention when targeting different regulatory regions of the ANGPTL3 gene. DETAILED DESCRIPTION OF THE INVENTION

[0058] Detailed Description The following aspects of the present invention are exemplified by specific examples, and other benefits and advantages of the present invention will be readily apparent to those skilled in the art from the disclosure herein.

[0059] Definition of Terms As used herein, the terms "nucleic acid," "polynucleotide," "nucleotide," "nucleotide sequence," and "oligonucleotide" are used interchangeably and generally refer to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and their polymers or complements in single-stranded, double-stranded, or multi-stranded form. For example, nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. For example, nucleotides can be single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, or hybrid molecules of a mixture of single-stranded and double-stranded DNA and RNA. For example, nucleotides can include, but are not limited to, any type of RNA, such as mRNA, siRNA, miRNA, sgRNA, guide RNA, and any type of DNA, genomic DNA, plasmid DNA, minicircle DNA, and fragments thereof. The term also covers nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, whether synthetic, naturally occurring, or non-naturally occurring.

[0060] As used herein, the terms "sequence encoding ..." or "nucleic acid encoding ..." generally refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein. The coding sequence may also comprise start and stop signals operably linked to regulatory elements comprising a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon optimized.

[0061] As used herein, the term "treatment," for example, when used in the context of a disease, refers to a condition in which a subject (e.g., a human) suffering from, at risk of, and / or experiencing symptoms of a disease recovers more quickly, in some embodiments, when a gene expression regulator molecule described herein or a nucleic acid encoding the same and / or a nucleic acid binding molecule (e.g., gRNA) or a nucleic acid encoding a nucleic acid binding molecule described herein is administered, compared to when such gene expression regulator molecule or nucleic acid and / or nucleic acid binding molecule or a nucleic acid encoding such nucleic acid binding molecule is not administered.

[0062] As used herein, the term "DNA-binding domain" generally refers to an independently folded protein domain containing at least one motif that recognizes double-stranded or single-stranded DNA. For example, a DNA-binding domain may recognize a specific DNA sequence (recognition sequence or regulatory sequence), or may have a general affinity for DNA. In some cases, other domains of the DNA-binding domain may control the activity of the DNA-binding domain. The DNA-binding function may be structural or may involve transcriptional regulation, and these two functions may overlap. In some embodiments of the methods and gene expression regulatory molecules provided herein, the DNA-binding domain may comprise a (DNA) nuclease, such as a CRISPR-Cas system, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or meganuclease, which can target DNA in a sequence-specific manner or can be induced or directed to target DNA in a sequence-specific manner. In some embodiments, the DNA-binding domain is a DNA nuclease derived from a CRISPR-Cas system. For example, the DNA nuclease derived from the CRISPR-Cas system is a Cas protein.

[0063] As used herein, "Cas enzyme" is used interchangeably with "Cas protein," "CRISPR protein," "CRISPR enzyme," "CRISPR-Cas protein," "CRISPR-Cas enzyme," "Cas," "CRISPR effector," or "Cas effector protein," and generally refers to a class of enzymes that are complementary to CRISPR sequences and can use the CRISPR sequence as a guide to recognize and cleave specific DNA strands. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csf1, Csf2, Csf3, Csf4, and / or homologs or modified forms thereof. For example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein is available in the SwissProt database under accession number Q99ZW2.

[0064] Herein, the term "dCas9 enzyme" is also known as "inactive Cas9 protein" or "inactive Cas9 enzyme." Methods for generating a Cas9 protein (or a fragment thereof) with an inactive DNA cleavage domain are known, for example, from Jinek et al., Science. 337: 816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression," Cell. 28, 152(5): 1173-83 (2013) (the entire contents of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, and the RuvC1 subdomain cleaves the strand that is not complementary to the gRNA. Mutation of these subdomains can silence the nuclease activity of Cas9. For example, the D10A and H840A mutations completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337: 816-821(2012); Qi et al., Cell. 28; 152(5): 1173-83(2013)). Suitable CRISPR-inactivating or nicked DNA-binding domains include, but are not limited to, nuclease-inactivating mutations of Cas9 domains, including D10A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains, as described in WO2015089406A1, which is incorporated herein by reference. In some cases, dCas9, which lacks endonuclease activity and is derived from Streptococcus pyogenes, has been targeted to genes in bacteria, yeast, and human cells using gRNA to silence gene expression through steric hindrance. As used herein, "dCas" may refer to a dCas protein or a fragment thereof. As used herein, "dCas9" may refer to a dCas9 protein or a fragment thereof.As used herein, "iCas" and "dCas" can be used interchangeably to refer to catalytically inactive CRISPR-associated proteins. In one embodiment, the dCas protein comprises one or more mutations in the DNA cleavage domain. In one embodiment, the dCas protein comprises one or more mutations in the RuvC or domain. In one embodiment, the dCas molecule comprises one or more mutations in both the RuvC domain and the HNH domain. In one embodiment, the dCas protein is a fragment of a wild-type Cas protein. In one embodiment, the dCas protein comprises a functional domain derived from a wild-type Cas protein, and the functional domain is selected from a Reel domain, a bridge helix domain, or a PAM interaction domain. In one embodiment, the nuclease activity of dCas is reduced by 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 at least 99% compared to the nuclease activity of the corresponding wild-type Cas protein.

[0065] A suitable dCas can be derived from a wild-type Cas protein. The Cas protein can be derived from a type I, type II, or type III CRISPR-Cas system. In one embodiment, a suitable dCas can be derived from Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, or Cas10. In one embodiment, the dCas is derived from a Cas9 protein. For example, dCas9 can be obtained by introducing point mutations (e.g., substitutions, deletions, or additions) into the DNA cleavage domain (e.g., the nuclease domain, e.g., the RuvC and / or HNH domain) of the Cas9 protein. See, e.g., Jinek et al., Science (2012) 337: 816-21. For example, introducing two point mutations into the RuvC and HNH domains reduces Cas9 nuclease activity while retaining Cas9 sgRNA and DNA binding activity. In one embodiment, the two point mutations in the RuvC and HNH active sites are the D10A and H840A mutations of Streptococcus pyogenes Cas9. Alternatively, D10 and H840 of Streptococcus pyogenes Cas9 can be deleted to eliminate Cas9 nuclease activity while retaining sgRNA and DNA binding activity. In one embodiment, the two point mutations in the RuvC and HNH active sites are the D10A and N580A mutations of Streptococcus pyogenes Cas9.

[0066] In various embodiments, the present application includes a dCas protein, or a variant or mutant thereof. All variants and mutants of dCas9 may be used in the methods, compositions, fusion molecules, or kits disclosed herein, including SpCas9 (Cas9 isolated from Streptococcus pyogenes), SaCas9 (Cas9 isolated from Staphylococcus aureus), StCas9 (Cas9 isolated from Streptococcus thermophilus), NmCas9 (Cas9 isolated from Neisseria meningitidis), FnCas9 (Cas9 isolated from Francisella novicida), CjCas9 (Cas9 isolated from Campylobacter jejuni), ScCas9 (Cas9 isolated from Streptococcus canis), as well as high-fidelity Cas9 (Kleinstiver et al., Nature, January 28, 2016) and enhanced SpCas9 (Slaymaker et al., Sciences, January 1, The dCas protein may be derived from any of the above-mentioned variants and mutants of Cas9, such as those described in SEQ ID NOs: 1162-1179 of the present application, but is not limited to these. For example, the dCas9 sequences shown in SEQ ID NOs: 1162-1179 of the present application are merely exemplary options and are not intended to be exhaustive. In one embodiment, the dCas protein is a Streptococcus pyogenes dCas9 protein having a mutation at D10 and / or H840 (as shown in SEQ ID NO: 1162). In one embodiment, the dCas protein is a Streptococcus pyogenes dCas9 protein having a mutation at D10A and / or H840A (as shown in SEQ ID NO: 1162).In one embodiment, the dCas9 protein is a Staphylococcus aureus dCas9 protein comprising the amino acid sequence set forth in SEQ ID NO: 1163 or 1164, a sequence substantially identical to SEQ ID NO: 1163 or 1164 (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity), or a sequence having one, two, three, four, five or more modifications (e.g., amino acid substitutions, insertions, or deletions) relative to SEQ ID NO: 1163 or 1164, or a fragment thereof.

[0067] Similar mutations can be applied to other naturally occurring Cas9s (e.g., Cas9s from other species) or recombinant Cas9s. In some embodiments, the dCas9 is selected from the group consisting of Streptococcus pyogenes dCas9, Staphylococcus aureus dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheriae dCas9, Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, Azospirillum sp. (e.g., strain B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia intestinalis dCas9, Parvibacrum lavamentivorans dCas9, Nitratifractor sarsuginis dCas9, and the like. Examples of suitable dCas9 include Bacillus salsugini (e.g., strain DSM16511) dCas9, Campylobacter lari (e.g., strain CF89-12) dCas9, Streptococcus thermophilus (e.g., strain LMD-9) dCas9, or fragments of the above.In some embodiments, the present application also provides vectors encoding the following protein molecules: Streptococcus pyogenes dCas9, Staphylococcus aureus dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheriae dCas9, Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, Azospirillum sp. (strain B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia intestinalis dCas9, Parvivacrum lavamentivorans dCas9, Nitratifractor sarsuginis dCas9, and salsuginis (strain DSM16511) dCas9, Campylobacter lari (strain CF89-12) dCas9, Streptococcus thermophilus (strain LMD-9) dCas9, or fragments of the above.

[0068] In the present application, the term "Cas enzyme substantially lacking nuclease activity" generally refers to an RNA-guided enzyme whose phosphodiester bond recognition is facilitated by another polynucleotide sequence (e.g., a guide RNA), but the enzyme does not significantly cleave the target phosphodiester bond (e.g., no measurable cleavage of the phosphodiester bond under physiological conditions). For example, when complexed with a polynucleotide (e.g., an sgRNA), an RNA-guided DNA endonuclease lacking nuclease activity retains DNA binding ability (e.g., specific binding to the target sequence), but significantly lacks endonuclease activity. For example, an RNA-guided DNA endonuclease lacking nuclease activity is dCas9, ddCpf1, a Cas9 variant lacking nuclease activity, or a class II CRISPR endonuclease lacking nuclease activity. For example, an RNA-guided DNA endonuclease lacking nuclease activity is dCas9. As used herein, the term "dCas9" or "dCas9 protein" refers to a Cas9 protein that is defective or lacks activity in both catalytic sites of endonuclease activity. For example, dCas9 substantially lacks detectable endonuclease (e.g., deoxyriboendonuclease) activity. In all aspects, for example, dCas9 includes variants or homologs having an amino acid sequence that shares 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the dCas9 enzyme sequence of the present application.

[0069] As used herein, the term "capable of binding" can be used interchangeably with "bind to," "specifically recognize," "target," etc., and generally means that a binding molecule (e.g., a gene expression regulatory molecule of the present invention) can interact with nucleotides on a target gene or target site, or that a binding molecule (e.g., a gene expression regulatory molecule of the present invention) has sufficient affinity for a target gene or target site, and this interaction can be achieved through conjugation, coupling, attachment, providing complementarity, providing covalent bonding, providing non-covalent bonding, enhancing binding stability, etc.

[0070] As used herein, the term "transcription start site" generally refers to a nucleic acid in a construct that corresponds to the first nucleic acid incorporated into the primary transcription product (i.e., pre-mRNA). The transcription start site may overlap with a promoter sequence.

[0071] As used herein, the term "fragment thereof" generally refers to a portion or segment of a specific whole. For example, when used in this application with respect to a specific nucleotide sequence, the term "fragment thereof" refers to a contiguous length of a specific nucleotide sequence that is shorter than the full-length sequence of the specific polynucleotide. A specific nucleotide portion can be defined by its initial position and its final position, where the initial and final positions correspond to positions in the sequence of the specific polynucleotide, and the sequence position corresponding to the initial position is N-terminal to the sequence position corresponding to the final position. Thus, the sequence of the portion is a contiguous nucleotide sequence in the specific polynucleotide that begins at the sequence position corresponding to the initial position and ends at the sequence position corresponding to the final position. A portion can also be defined by its position in a specified polynucleotide sequence and the length of residues relative to a reference position. Thus, the sequence of the portion is a contiguous nucleotide sequence in the specified polynucleotide, has a defined length, and is arranged in the specified polynucleotide according to the defined positions.

[0072] As used herein, the term "nucleotide modification" refers to a method well-established in the art, such as "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA (incorporated herein by reference). Modifications may include, but are not limited to, terminal modifications such as 5'-end modifications (e.g., phosphorylation, conjugation, reverse linkage) or 3'-end modifications (e.g., conjugation, DNA nucleotide, reverse linkage, etc.); base modifications such as stabilizing bases, destabilizing bases, or bases paired with extended pairing group library bases, base removal (base-free nucleotides), or substitution with conjugate bases; sugar modifications (e.g., sugar modifications at the 2'- or 4'-position, etc.) or sugar substitutions; or backbone modifications, including modification or substitution of phosphodiester bonds.

[0073] As used herein, the term "histone-modifying substance" generally refers to the relevant enzymes that can modify histones to regulate gene transcription. Common histone modifications can include methylation, acetylation, phosphorylation, adenylation, ubiquitination, ADP-ribosylation, etc.

[0074] As used herein, the term "methylation modification" is used interchangeably with "DNA methylation" and "nucleic acid methylation," and generally refers in this application to the methylation state of a gene fragment, nucleotide, or their base. This state often occurs in a cell transfected with a nucleic acid, where the cell is transfected with a nucleic acid containing a structural gene encoding a polypeptide operatively linked to a promoter. During this process, cytosines in the promoter nucleic acid are converted to 5-methylcytosines. A promoter nucleic acid in which at least one cytosine has been converted to 5-methylcytosine is referred to as a "methylated" nucleic acid or DNA. In this application, a DNA fragment in which a gene is located can be methylated on one or more strands and can have methylation at one or more sites.

[0075] As used herein, the term "regulatory element" refers to a genetic element that can control the expression of a nucleic acid sequence. For example, splicing signals, promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites ("IRES"), enhancers, and the like, work together to provide replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these regulatory sequences are necessary. Transcriptional control signals in eukaryotes typically include "promoter" and "enhancer" elements. Promoters and enhancers are composed of short arrays of DNA sequences. A promoter is a regulatory element that promotes the initiation of transcription of an operably linked coding region, while an enhancer is a regulatory element that increases the activity of the nearest promoter on the same DNA molecule, thereby increasing the transcription rate of the gene. These sequences specifically interact with cellular proteins involved in transcription (Maniatis et al., Science 236: 1237 (1987), the entire contents of which are incorporated herein by reference). Promoter and enhancer elements can be isolated from various eukaryotic organisms, including yeast, insect, and mammalian cell genes, as well as viruses (similar regulatory sequences, i.e., promoters, also exist in prokaryotes). The selection of a particular promoter and enhancer depends on the type of recipient cell. Some eukaryotic promoters and enhancers have a broad host range, while others function in a limited subgroup of cell types (for reviews, see, e.g., Voss et al., Trends Biochem. Sci., 11: 287 (1986); and Maniatis et al. (ibid., the entire contents of which are incorporated herein by reference). For example, the SV40 early gene enhancer is significantly active in various cell types of many mammalian species and has been used to express proteins in various mammalian cells (Dijkema et al., EMBO J. 4: 761 (1985), the entire contents of which are incorporated herein by reference).Promoter and enhancer elements from the human elongation factor 1-α gene (Uetsuki et al., J. Biol. Chem., 264: 5791 (1989); Kim et al., Gene 91: 217 (1990); and Mizushima and Nagata, Nucl. Acids Res., 18: 5322 (1990)), the long terminal repeat of Ras sarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79: 6777 (1982)), and human cytomegalovirus (Boshart et al., Cell 41: 521 (1985)) can also be used to express proteins in different mammalian cell types, and the references cited herein are incorporated by reference in their entireties. Promoters and enhancers can occur alone or together in nature. For example, retroviral long terminal repeats contain both promoter and enhancer elements. Generally, promoters and enhancers act independently of the gene being transcribed or translated. Therefore, the enhancer and promoter used may be "endogenous," "exogenous," or "heterologous" to the gene to which they are operably linked. An "endogenous" enhancer / promoter is one that is naturally linked to a gene in the genome. An "exogenous" or "heterologous" enhancer or promoter is one that is juxtaposed to a gene by genetic engineering (i.e., molecular biological techniques) so that transcription of the gene is directed by the linked enhancer / promoter. The presence of "splicing signals" on an expression vector usually results in high levels of expression of the recombinant transcript.In some embodiments, a "splicing signal" mediates the removal of introns from a primary RNA transcript and consists of a splice donor and acceptor site (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, New York (1989), pp. 16.7-16.8, incorporated herein by reference in its entirety). A commonly used splice donor and acceptor site is the SV40 16S RNA splice site. In some embodiments, a "transcription termination signal" is typically located downstream of a polyadenylation signal and is several hundred nucleotides in length. For example, the term "polyA signal" or "polyA sequence" refers to a DNA sequence that directs the termination and polyadenylation of newly synthesized RNA transcripts. Efficient polyadenylation of recombinant transcripts is often required because transcripts lacking a polyA signal are unstable and rapidly degraded. The polyA signal used in an expression vector is "heterologous" or "endogenous." An endogenous poly A signal is one that is naturally present at the 3' end of the coding region of a gene in the genome. A heterologous poly A signal is one that is isolated from one gene and operably linked to the 3' end of another gene. A commonly used heterologous poly A signal is the SV40 poly A signal, which is contained in a 237 bp BamHI / BclI restriction fragment and directs termination and polyadenylation (Sambrook et al., ibid., 16.6-16.7, incorporated herein by reference).

[0076] As used herein, the term "DNA methyltransferase" generally refers to an enzyme that catalyzes the transfer of methyl to DNA. Non-limiting examples of DNA methyltransferases include DNMT1, DNMT 3A, DNMT 3B, and DNMT 3L. For example, by DNA methylation, DNA methyltransferases can modify the activity of a DNA fragment (such as regulating gene expression) without altering the DNA sequence. As described herein, a gene expression regulatory molecule can contain one or more (e.g., two) DNA methyltransferases. When DNA methyltransferases are included as part of a gene expression regulatory molecule, they can be referred to as a "DNA methyltransferase domain." In all aspects, the DNA methyltransferase domain includes variants or homologs having an amino acid sequence with 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to DNMT 3A. In all aspects, the DNA methyltransferase domain includes variants or homologs having an amino acid sequence with 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to DNMT 3L.

[0077] As used herein, the term "functionally active fragment" generally refers to a fragment having a partial region of a full-length protein or nucleic acid, which retains or partially retains the biological activity or function of the full-length protein or nucleic acid. For example, a functionally active fragment can retain or partially retain the ability of the full-length protein to bind to other molecules. For example, a functionally active fragment of a DNA methyltransferase can retain or partially retain the biological activity function of the full-length DNA methyltransferase, which catalyzes the transfer of methyl to DNA.

[0078] As used herein, the term "directly and / or indirectly linked" generally refers to the relative terms "directly linked" or "indirectly linked." The term "directly linked" generally refers to a direct link or bond. For example, a direct link can be a situation in which no spacer component (such as an amino acid residue or a derivative thereof) exists between directly linked linking materials (e.g., amino acid sequence segments). For example, an amino acid sequence segment X is directly linked to another amino acid sequence segment Y through an amide bond formed by the C-terminal amino acid of the amino acid sequence segment X and the N-terminal amino acid of the amino acid sequence segment Y. An "indirect link" generally refers to a situation in which a spacer component (such as an amino acid residue or a derivative thereof) exists between indirectly linked linking materials (e.g., amino acid sequence segments).

[0079] As used herein, the term "Krab" is also known as a "Kruppel-associated box domain" and generally refers to approximately 45 to 75 amino acid residues present in the transcriptional repression domain of human zinc finger transcription factors. In all aspects, the Krab domain may include variants or homologs having an amino acid sequence that shares 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the ZIM3 Krab domain or the KOX1 Krab domain.

[0080] As used herein, the term "delivery carrier" generally refers to a delivery vehicle capable of delivering a reagent (e.g., a nucleic acid molecule) to a target cell. A delivery carrier can deliver a reagent to a specific subclass of cells. For example, a delivery carrier can target a certain type of cell through its inherent properties or through a moiety conjugated to or contained within the carrier (or bound to the carrier, thereby holding the moiety and the delivery carrier together and thus making the moiety sufficient to target the delivery carrier). A delivery carrier can also increase the in vivo half-life and / or bioavailability of the delivered reagent. Delivery carriers can include viral vectors, virus-like particles, polycationic carriers, peptide carriers, liposomes, and / or hybrid carriers. For example, when the target cell is a hepatocyte, the characteristics of the delivery carrier (e.g., size, charge, and / or pH) can effectively deliver the delivery carrier and / or the molecule it carries to the target cell, reduce immune clearance, and / or promote retention in the target cell.

[0081] As used herein, the term "liposome" generally refers to a vesicle having an internal space separated from the external medium by one or more bilayers. In some embodiments, the bilayer may be formed by amphiphilic molecules, such as synthetic or naturally occurring lipids, containing spatially separated hydrophilic and hydrophobic structural domains. In other embodiments, the bilayer may be formed by amphiphilic polymers and surfactants. In some embodiments, liposomes are spherical vesicular structures consisting of a unilamellar or multilamellar lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer. In some embodiments, liposomes are biocompatible and nontoxic, can deliver hydrophilic and lipophilic drug molecules, protect cargo from degradation by plasma enzymes, and transport their contents across biological membranes and the blood-brain barrier (BBB). Liposomes may be composed of several types of lipids, including phospholipids. Liposomes may contain natural phospholipids and lipids, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), sphingomyelin, phosphatidylcholine, monosialoganglioside, or any combination thereof. To alter the structure and properties of liposomes, several additives can be added to the liposomes. For example, liposomes may contain, for example, cholesterol, sphingomyelin, and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to enhance stability and / or prevent leakage of cargo within the liposomes.

[0082] The term "lipid nanoparticle (LNP)" generally refers to a particle containing multiple (i.e., two or more) lipid molecules physically bound to each other by intermolecular forces (e.g., covalent or noncovalent bonds). LNPs can be, for example, microspheres (including unilamellar and multilamellar vesicles such as liposomes), the dispersed phase in an emulsion, micelles, or the internal phase in a suspension. LNPs can encapsulate nucleic acids within cationic lipid particles (e.g., liposomes) and deliver them relatively easily to cells. In some instances, lipid nanoparticles do not contain viral components, minimizing safety and immunogenicity concerns. Lipid particles can be used for in vitro, ex vivo, and in vivo delivery. Lipid particles can also be used to deliver cells at various scales. The LNPs of the present application can be easily prepared by various methods known in the art, such as mixing an organic phase with an aqueous phase. Mixing of the two phases can be achieved using microfluidic devices and impinging flow reactors. The more complete the mixing of the organic and aqueous phases, the better the encapsulation efficiency and particle size distribution of the resulting LNPs. Preferably, the particle size of the LNP can be adjusted by changing the mixing speed of the organic phase and the aqueous phase. The faster the mixing speed, the smaller the particle size of the prepared LNP. The encapsulation efficiency can be optimized by adjusting the N / P (ionizable lipid / nucleic acid) ratio of the LNP system. In some examples, LNPs can be used to deliver DNA molecules (e.g., molecules containing sequences encoding DNA-binding proteins and / or sgRNAs) and / or RNA molecules (e.g., Cas mRNA, sgRNA mRNA). In some cases, LNPs can also be used to deliver Cas / sgRNA RNP complexes. In some embodiments, LNPs are used to deliver mRNA and gRNA (e.g., mRNA fusion molecules containing DNMT3A-DNMT3L (3A-3L)-dCas9-KRAB and at least one sgRNA targeting a target gene).

[0083] As used herein, the term "recombinant vector" generally refers to a nucleic acid molecule capable of transporting itself and another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Alternatively, the vector can be linear. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell genome after introduction into a host cell, and thereby are replicated along with the host genome.

[0084] As used herein, the term "adeno-associated virus (AAV) vector" generally refers to a vector having functional or partially functional ITR sequences and a transgene. As used herein, the term "ITR" refers to inverted terminal repeat sequences. The ITR sequences can be derived from adeno-associated virus serotypes, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13, as well as any AAV variant or mixture. However, the ITRs need not be wild-type nucleotide sequences and can be modified (e.g., by nucleotide insertion, deletion, or substitution) so long as they retain the ability to provide functional rescue, replication, and packaging. AAV vectors may be completely or partially deleted from one or more AAV wild-type genes, preferably the rep gene and / or cap gene, but retain functional flanking ITR sequences. Functional ITR sequences play a role in, for example, rescuing, replicating, and packaging AAV viral particles or particles. Therefore, the term "AAV vector" is defined herein as including at least the sequences necessary for inserting a transgene into a subject's cells. If necessary, it also includes the cis sequences (e.g., functional ITRs) necessary for viral replication and packaging.

[0085] As used herein, the term "nuclear localization sequence" or "nuclear localization signal" or "NLS" generally refers to a peptide that directs a protein to the cell nucleus. For example, an NLS contains five basic, positively charged amino acids. For example, an NLS can be located anywhere along the peptide chain.

[0086] As used herein, the term "complementary" generally refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either by conventional Watson-Crick or other non-conventional methods. For example, the sequence AGT is complementary to the sequence TCA. The percentage of complementarity refers to the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5 out of 10, 6 out of 10, 7 out of 10, 8 out of 10, 9 out of 10, and 10 out of 10 represent 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). For example, "fully complementary" means that all consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. For example, "substantially complementary" means that two nucleic acids have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complementarity over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or that two nucleic acids that hybridize under stringent conditions (i.e., stringent hybridization conditions) with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complementarity.

[0087] As used herein, the term "gene" generally refers to a DNA fragment designed to produce a protein. For example, a gene may also include regions before and after the coding region (leader and trailer), as well as intervening sequences (introns) between each coding fragment (exon). The leader, trailer, and introns contain regulatory elements necessary for the transcription and translation processes of a gene. Additionally, the term "protein gene product" may refer to the protein expressed by a particular gene.

[0088] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. For example, a polymer can be linked in all respects to moieties not composed of amino acids. For example, a "fusion protein" refers to a chimeric protein of two or more separate protein sequences, each encoded as an individual moiety and expressed in a recombinant manner. In the case of two or more nucleic acid or polypeptide sequences, the term "identical" or percent "identity" refers to the measurement obtained using the BLAST or BLAST 2.0 sequence alignment algorithm, with default parameters as described below, or by manual alignment and visual inspection. For example, two or more sequences or subsequences (having about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity when compared and aligned for maximum correspondence over a comparison window or designated region) in which identical amino acid residues or nucleotides are present in the same or a specified percentage can be said to be "substantially identical."

[0089] As used herein, the term "guide RNA" or "gRNA" generally refers to any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target polynucleotide sequence and allow a CRISPR complex to specifically bind to the target sequence. In all aspects, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 99%, or more.

[0090] In this application, a particular protein (e.g., KRAB, dCas9, Dnmt3A, Dnmt3L) can include any naturally occurring form of the protein or a variant or homolog that maintains the activity of the protein (e.g., having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% activity compared to the naturally occurring protein). In all aspects, a variant or homologue has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the native form.

[0091] As used herein, the term "linker" generally refers to a conjugate that connects two or more moieties. In various embodiments, the linker is attached to the N-terminus and C-terminus of the amino acid sequence of the remainder of the compound (e.g., the fusion proteins provided herein). For example, the term "XTEN," "XTEN linker," or "XTEN polypeptide" refers to a recombinant polypeptide lacking hydrophobic amino acid residues.

[0092] As used herein, the term "detectable agent" or "detectable moiety" refers to a composition that can be detected by appropriate means, such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. For example, useful detectable agents include radioactive elements, fluorophores (e.g., fluorescent dyes), electron-dense reagents, enzymes (e.g., those commonly used in ELISAs), biotin, paramagnetic molecules, and the like.

[0093] As used herein, the terms "inhibition," "suppression," "silencing," and the like generally refer to a reduction in gene expression and / or activity. For example, administration of a substance (e.g., a fusion protein, complex, nucleic acid, vector) described herein can negatively affect (e.g., decrease) the activity of a nucleic acid sequence compared to the activity in the absence of the substance (control). For example, inhibition can refer to the alleviation of a disease or disease symptom. For example, inhibition includes at least partially, partially, or completely blocking activation of a nucleic acid sequence (e.g., transcription), or reducing, preventing, or delaying activation of a nucleic acid sequence. For example, inhibitory activity can be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less than the control.

[0094] As used herein, the term "comprising" generally means including the specifically named features but does not exclude other elements.

[0095] As used herein, the term "selected" generally means to include selected objects and all combinations thereof. For example, "selected from A, B, and C" means to include all combinations of A, B, and C, such as A, B, C, A+B, A+C, B+C, or A+B+C.

[0096] As used herein, the term "about" generally refers to a variation within 0.5% to 10% upper or lower limits of a stated value, for example, a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% upper or lower limits of a stated value.

[0097] Detailed Description of the Invention In one aspect, the present application provides a method for regulating the expression and / or activity of ANGPTL3 gene, comprising providing a gene expression regulatory molecule or a nucleic acid encoding a gene expression regulatory molecule, wherein the gene expression regulatory molecule can have the function of regulating the expression of ANGPTL3 gene without changing the gene sequence of ANGPTL3 gene.For example, the method of the present invention can also be a method that is not for therapeutic purposes.For example, the method of the present invention can be a method that does not directly target the human body.For example, the method of the present invention can be an in vitro or ex vivo method.For example, the method of the present invention can also be a method for therapeutic purposes.For example, the method of the present invention can be an in vivo method.

[0098] In another aspect, the present invention provides a method for treating and / or alleviating a pathology associated with abnormal expression and / or activity of the ANGPTL3 gene, comprising providing a gene expression regulatory molecule or a nucleic acid encoding a gene expression regulatory molecule, wherein the gene expression regulatory molecule can function to regulate the expression of the ANGPTL3 gene without altering the genetic sequence of the ANGPTL3 gene.

[0099] In another aspect, the present invention provides gene expression regulating molecule or the nucleic acid that encodes gene expression regulating molecule, wherein gene expression regulating molecule can regulate the expression of ANGPTL3 gene without changing the gene sequence of ANGPTL3 gene.For example, nucleic acid comprises DNA and / or mRNA.For example, gene expression regulating molecule or the nucleic acid that encodes gene expression regulating molecule can be used to treat and / or alleviate the pathology that is related to the abnormal expression and / or activity of ANGPTL3 gene.

[0100] In another aspect, the present invention provides the use of a gene expression regulatory molecule or a nucleic acid encoding a gene expression regulatory molecule in the preparation of a medicament for treating and / or alleviating a pathology associated with the abnormal expression and / or activity of the ANGPTL3 gene, wherein the gene expression regulatory molecule can have the function of regulating the expression of the ANGPTL3 gene without changing the gene sequence of the ANGPTL3 gene.For example, the nucleic acid encoding the gene expression regulatory molecule of the present invention comprises DNA and / or mRNA.

[0101] In another aspect, the present invention provides a nucleic acid-binding molecule or a nucleic acid encoding the nucleic acid-binding molecule, wherein the nucleic acid-binding molecule comprises a sequence set forth in any one of SEQ ID NOs: 1 to 70. For example, the nucleic acid encoding the nucleic acid-binding molecule of the present invention comprises DNA and / or mRNA.

[0102] In another aspect, the present invention provides a recombinant vector comprising a nucleic acid of the present invention. For example, a recombinant vector can refer to a nucleic acid molecule capable of transporting another nucleic acid molecule linked to it. A recombinant vector can comprise a single-stranded, double-stranded, or partially double-stranded nucleic acid molecule; a nucleic acid molecule with one or more free ends, a nucleic acid molecule with no free ends (e.g., a loop); a nucleic acid molecule comprising DNA, RNA, or both; and other types of polynucleotides known in the art. For example, a viral vector can be used. A viral vector can comprise a viral-derived DNA or RNA sequence for packaging into a virus (e.g., retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, adeno-associated virus AAV). Viruses and viral vectors can be used for in vitro, ex vivo, and / or in vivo delivery.

[0103] In another aspect, the present application provides a delivery vehicle comprising a nucleic acid-binding molecule of the present invention, a gene expression-regulating molecule of the present invention, a nucleic acid of the present invention, and / or a recombinant vector of the present invention, and optionally comprising a liposome and / or a lipid nanoparticle (LNP). For example, the delivery vehicle may comprise one or more Cas proteins and one or more guide RNAs, e.g., in the form of a ribonucleoprotein complex (RNP). For example, ribonucleoproteins can be delivered via polypeptide-based shuttle agents. For example, ribonucleoproteins can be delivered using synthetic peptides. For example, the delivery vehicle can be introduced into cells via physical delivery methods. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery. For example, LNPs can encapsulate nucleic acids within cationic lipid particles (e.g., liposomes), allowing for relatively easy delivery into cells. In some instances, lipid nanoparticles do not contain viral components, thereby minimizing safety and immunogenicity issues. Lipid particles can be used for in vitro, ex vivo, and in vivo delivery. Components of LNPs can include cationic lipids, ionizable lipids, PEGylated lipids and / or helper lipids, and optionally, cholesterol components. In some embodiments, LNPs can include ionizable lipids (20%-70%, molar ratio), PEGylated lipids (0%-30%, molar ratio), helper lipids (30%-50%, molar ratio), and cholesterol (10%-50%, molar ratio).

[0104] In another aspect, the present application provides a composition comprising a nucleic acid binding molecule of the present invention, a gene expression regulating molecule of the present invention, a nucleic acid of the present invention, a recombinant vector of the present invention, and / or a delivery carrier of the present invention. For example, the nucleic acid binding molecule, the gene expression regulating molecule, the nucleic acid encoding the nucleic acid binding molecule, the nucleic acid encoding the gene expression regulating molecule, the recombinant vector, and the delivery carrier in the composition may be contained in one composition or in separate compositions. For example, when the nucleic acid binding molecule, the gene expression regulating molecule, the nucleic acid encoding the nucleic acid binding molecule, the nucleic acid encoding the gene expression regulating molecule, the recombinant vector, and / or the delivery carrier are used in the composition, they may be used simultaneously or separately.

[0105] In another aspect, the present application provides a cell comprising the nucleic acid binding molecule of the present invention, the gene expression regulatory molecule of the present invention, the nucleic acid of the present invention, the recombinant vector of the present invention, the delivery vehicle of the present invention, and / or the composition of the present invention.

[0106] In another aspect, the present application provides a kit comprising the nucleic acid binding molecule of the present invention, the gene expression regulatory molecule of the present invention, the nucleic acid of the present invention, the recombinant vector of the present invention, the delivery carrier of the present invention, the composition of the present invention, and / or the cell of the present invention.

[0107] In another aspect, the present application provides a method for regulating the expression and / or activity of a target gene, comprising providing a nucleic acid binding molecule of the present invention, a gene expression regulating molecule of the present invention, a nucleic acid of the present invention, a recombinant vector of the present invention, a delivery vehicle of the present invention, a composition of the present invention, a cell of the present invention, and / or a kit of the present invention. For example, the method of the present invention can reduce the expression and / or activity of a target gene. For example, administration of a substance of the present invention can negatively affect (e.g., reduce) the activity of a nucleic acid sequence, compared to the expression and / or activity of a target gene in the absence of the substance of the present invention, including at least partially, partially, or completely inhibiting activation (e.g., transcription) of the nucleic acid sequence, or reducing, preventing, or delaying activation of the nucleic acid sequence. For example, activity inhibition can be about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or less of the control.

[0108] In another aspect, the present application provides use of a nucleic acid binding molecule of the present invention, a gene expression regulatory molecule of the present invention, a nucleic acid of the present invention, a recombinant vector of the present invention, a delivery vehicle of the present invention, a composition of the present invention, a cell of the present invention, and / or a kit of the present invention in the preparation of a medicament for treating and / or alleviating a pathology, including a pathology associated with aberrant expression and / or activity of a target gene.

[0109] In another aspect, the present application provides a nucleic acid binding molecule of the present invention, a gene expression regulatory molecule of the present invention, a nucleic acid of the present invention, a recombinant vector of the present invention, a delivery vehicle of the present invention, a composition of the present invention, a cell of the present invention, and / or a kit of the present invention for use in the treatment and / or alleviation of a pathology, including a pathology associated with aberrant expression and / or activity of a target gene.

[0110] In another aspect, the present application provides a method for treating and / or alleviating a pathology, comprising providing a nucleic acid-binding molecule of the present invention, a gene expression-regulating molecule of the present invention, a nucleic acid of the present invention, a recombinant vector of the present invention, a delivery carrier of the present invention, a composition of the present invention, a cell of the present invention, and / or a kit of the present invention, wherein the pathology includes a pathology associated with abnormal expression and / or activity of a target gene. In some embodiments, the treatment targets an organ disease / disorder, exemplary of which may include treatment of liver disease, eye disease, muscle disease, heart disease, blood disease, brain disease, kidney disease, or autoimmune disease, central nervous system disease, cancer and other proliferative diseases, neurodegenerative diseases, inflammatory diseases, metabolic diseases, musculoskeletal diseases, etc.

[0111] gene expression regulatory molecules The gene expression molecules provided in the present invention or the methods of the present invention have the function of regulating the expression of the ANGPTL3 gene without changing the gene sequence of the ANGPTL3 gene. For example, the gene expression regulatory molecule has the function of inhibiting gene expression.

[0112] In some embodiments, the gene expression regulatory molecule comprises a first functional domain, which provides modification of at least one nucleotide in the vicinity of the ANGPTL3 gene and / or within the ANGPTL3 gene regulatory element. In some embodiments, the first functional domain can regulate the expression of a target gene in a regulatory element of the target gene, such as a promoter, enhancer, or transcription start site, through epigenetic modification, such as DNA methylation. For example, regulatory elements include a transcription start site, a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, or a locus control region. For example, the epigenetic modification can be performed by any known epigenetic modifier that can be used for DNA methylation, and exemplary epigenetic modifiers can include DNA methyltransferases (e.g., DNMT3A or DNMT3A-DNMT3L), DNA demethylases (e.g., TET1 catalytic domain, TDG), and / or functionally active fragments thereof.

[0113] For example, the first functional domain can provide a modification of at least one nucleotide in the vicinity of the ANGPTL3 gene and / or in the ANGPTL3 gene regulatory element, and the modification of the at least one nucleotide includes a methylation modification. In some embodiments, the first functional domain includes an epigenetic modifier that can have DNA methylase activity. For example, the epigenetic modifier has methylase activity that transfers a methyl group to DNA, RNA, protein, small molecule, cytosine, or adenine. For example, the modification can be located within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp upstream of the transcription start site of the target gene. For example, the modification can be located within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp downstream of the transcription start site of the target gene.

[0114] For example, the first functional domain may include a DNA methyltransferase (DNMT) domain. For example, the first functional domain may include a DNMT3A domain and / or a DNMT3L domain. For example, the DNMT3A domain and / or the DNMT3L domain are derived from mammals. For example, the DNMT3A domain is derived from a mouse. For example, the gene expression regulatory molecule may include the amino acid sequence of DNMT3A. For example, the DNMT3L domain is derived from a human and / or a mouse. For example, the gene expression regulatory molecule may include the amino acid sequence of DNMT3L. Alternatively, the gene expression regulatory molecule may have one, two, three, four, five, or more mutations compared to the above sequence, such as a sequence having amino acid substitutions, insertions, or deletions, or a fragment thereof.

[0115] For example, the DNMT3A domain and the DNMT3L domain may be directly and / or indirectly linked. For example, the DNMT3A domain and the DNMT3L domain may be linked via a linker. For example, the C-terminus of the DNMT3A domain may be linked to the N-terminus of DNMT3L, or the C-terminus of the DNMT3L domain may be linked to the N-terminus of DNMT3A.

[0116] In some embodiments, the gene expression regulatory molecule comprises a second functional domain, wherein the second functional domain comprises a transcription factor based on a zinc finger protein or a functionally active fragment thereof, or a substance capable of modifying histones. For example, the second functional domain may comprise a gene repressor, which may be any known gene repressor; exemplary gene repressors may be selected from Krueppel-associated box (KRAB) domain, mSin3-interacting domain (SID), MAX-interacting protein 1 (MXI1), chromoshadow domain, EAR-repression domain (SRDX), eukaryotic release factor 1 (ERF1), eukaryotic release factor 3 (ERF3), tetracycline repressor, Rad repressor, Catharanthus roseus G-box binding factors 1 and 2, Drosophila Groucho (Drosophila Gro protein), tripartite motif-containing 28 (TRTM28), nuclear receptor corepressor 1, nuclear receptor corepressor 2, or functionally active fragments or fusions thereof. For example, the second functional domain may comprise a substance capable of modifying histones. In some embodiments, the second functional domain may comprise a Krab domain. Specifically, the second functional domain may comprise a ZIM3 Krab domain or a KOX1 Krab domain.

[0117] In some embodiments, the KRAB domain or a fragment thereof can be fused to the C-terminus of the dCas9 molecule. In some embodiments, the KRAB domain or a fragment thereof can be fused to both the N-terminus and C-terminus of the dCas9 molecule. In some embodiments, the second functional domain can comprise a KRAB domain, which can comprise a sequence that is substantially identical to ZIM3 Krab or KOX1 Krab (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical), or a sequence with one, two, three, four, five or more mutations (e.g., amino acid substitutions, insertions, deletions, etc. relative to ZIM3 Krab or KOX1 Krab), or a fragment of either thereof.

[0118] In some embodiments, the second functional domain can comprise a gene expression activator, which can be any known gene expression activator, and exemplary gene expression activators can include a VP16 activation domain, a VP64 activation domain, a p65 activation domain, an Epstein-Barr virus R transactivator Rta molecule, or a fragment thereof.

[0119] In some embodiments, the second functional domain may comprise a substance capable of modifying a histone, which may include a histone acetyltransferase (e.g., a p300 catalytic domain), a histone deacetylase, a histone methyltransferase (e.g., SUV39H1 or G9a (EHMT2)), a histone demethylase (e.g., LSD1), and / or functionally active fragments thereof.

[0120] In some aspects, the gene expression regulatory molecule comprises a first functional domain and a second functional domain, and the first functional domain is directly or indirectly linked to one end of the second functional domain, or the first functional domain is directly and / or indirectly linked to both ends of the second functional domain. For example, the C-terminus of the first functional domain is directly linked to the N-terminus of the second functional domain, or the C-terminus of the first functional domain is indirectly linked to the N-terminus of the second functional domain (e.g., via a linker), or the first functional domain is located on the N-terminal side of the second functional domain (e.g., the order from N-terminus to C-terminus may be the first functional domain, another portion such as a DNA binding domain, and the second functional domain). For example, the C-terminus of the second functional domain can be directly linked to the N-terminus of the first functional domain, or the C-terminus of the second functional domain can be indirectly linked to the N-terminus of the first functional domain (e.g., via a linker), or the second functional domain can be located N-terminally of the first functional domain (e.g., the order from N-terminus to C-terminus can be the second functional domain, another moiety such as a DNA-binding domain, and the first functional domain). For example, a first functional domain may include two or more functional domains, each of which may be directly linked to the N-terminus and C-terminus of a second functional domain, or may be indirectly linked (e.g., via a linker), or may be arranged N-terminus and C-terminus of the second functional domain (e.g., the order from N-terminus to C-terminus may be first functional domain a, another portion such as a DNA-binding domain, a second functional domain, and first functional domain b, or first functional domain a, a second functional domain, another portion such as a DNA-binding domain, and first functional domain b, where a and b are different types of first functional domains).

[0121] In some embodiments, the gene expression regulatory molecule comprises a DNA binding domain. For example, the gene expression regulatory molecule can have the function of binding to a gene sequence. For example, the DNA binding domain comprises a (DNA) nuclease, which can be a nuclease that specifically targets DNA. Exemplary nucleases can be CRISPR-Cas system-related enzymes, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or meganucleases. For example, the gene expression regulatory molecule can comprise a DNA binding domain of a TALEN, a zinc finger domain, and / or a DNA binding domain of a CRISPR / Cas system. In some cases, the DNA binding domain is a DNA nuclease from a CRISPR-Cas system. In some cases, the DNA binding domain is a (modified) transcription activator-like effector nuclease (TALEN) system. Transcription activator-like effectors (TALEs) can be designed to bind to almost any desired DNA sequence. In some cases, the DNA-binding domain is or consists of a (modified) zinc finger nuclease (ZFN) system. ZFN systems use artificial restriction enzymes produced by fusing a zinc finger DNA-binding domain with a DNA cleavage domain, and the DNA cleavage domain can be designed to target a desired DNA sequence. In some cases, the DNA-binding domain is a (modified) meganuclease, which is an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of 12 to 40 base pairs).

[0122] In some embodiments, the gene expression regulatory molecule may comprise a Cas enzyme. For example, the gene expression regulatory molecule may comprise a Cas enzyme that substantially lacks nuclease activity. Generally, the guide sequence (or spacer sequence) may be any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target polynucleotide sequence and guide a CRISPR complex to bind to the target sequence in a sequence-specific manner. In some cases, the degree of complementarity between the guide sequence and its corresponding target sequence may be about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more when a best match is determined using an appropriate alignment algorithm. In some embodiments, the nucleic acid-targeting Cas protein may be mutated relative to the corresponding wild-type enzyme, and the mutated nucleic acid-targeting Cas protein lacks the ability to cleave one or both strands of the target polynucleotide. For example, the DNA cleavage activity of the mutated enzyme can be 25% or less, 10% or less, 5% or less, 1% or less, 0.1% or less, or 0.01% or less of the DNA cleavage activity of the unmutated enzyme. In some embodiments, the mutant Cas may have one or more mutations that result in reduced off-target effects.

[0123] For example, the gene expression regulatory molecule may include a Cas9 enzyme. Examples of Cas proteins include class I (e.g., types I, III, and IV) and class 2 (e.g., types II, V, and VI) Cas proteins, such as Cas9, Cas12 (e.g., Cas12a, Cas12b, Cas12c, Cas12d), Cas13 (e.g., Cas13a, Cas13b, Cas13c, Cas13d), CasX, CasY, Cas14, mutants thereof (e.g., mutants, truncated forms), homologs thereof, and orthologs thereof. In some embodiments, the Cas protein is Cas9, Cas12a, Cas12b, Cas12c, or Cas12d. In some examples, Cas9 can be SpCas9, SaCas9, StCas9, and other Cas9 orthologs. The Cas12 is Cas12a, Cas12b, Cas12c, or a homolog or ortholog thereof, including FnCas12a. For example, Cas9 enzymes include Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheriae dCas9, Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, Azospirillum (e.g., B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia intestinalis dCas9, Parvibacrum lavamentivorans dCas9, and Nitratifractor salus ginseng dCas9. For example, the gene expression regulatory molecule can include the amino acid sequence of dCas9.Alternatively, the dCas9 sequence may have one, two, three, four, five or more mutations, such as amino acid substitutions, insertions, or deletions, compared to the above dCas9 sequences, or a fragment of any of these.

[0124] In some embodiments, the first functional domain and the second functional domain are directly or indirectly linked to one end of the DNA binding domain.For example, the first functional domain and the second functional domain are directly or indirectly linked to the C-terminus of the DNA binding domain, and exemplary gene regulatory molecules can include any one of dCas9-DNMT3A-DNMT3L-Krab, dCas9-DNMT3L-DNMT3A-Krab, dCas9-Krab-DNMT3A-DNMT3L, and dCas9-Krab-DNMT3L-DNMT3A. For example, the first and second functional domains are directly or indirectly linked to the N-terminus of the DNA-binding domain, and exemplary gene regulatory molecules may include any one of DNMT3A-DNMT3L-Krab-dCas9, DNMT3L-DNMT3A-Krab-dCas9, Krab-DNMT3A-DNMT3L-dCas9, and Krab-DNMT3L-DNMT3A-dCas9. In other embodiments, the first and second functional domains are directly and / or indirectly linked to both ends of the DNA-binding domain. For example, the first functional domain is directly or indirectly linked to the C-terminus of the DNA-binding domain, and the second functional domain is directly or indirectly linked to the N-terminus of the DNA-binding domain, and exemplary gene regulatory molecules may include any one of Krab-dCas9-DNMT3A-DNMT3L and Krab-dCas9-DNMT3L-DNMT3A. For example, the first functional domain is directly or indirectly linked to the N-terminus of the DNA-binding domain, and the second functional domain is directly or indirectly linked to the C-terminus of the DNA-binding domain, and exemplary gene regulatory molecules may include any one of DNMT 3A-DNMT 3L-dCas9-Krab and DNMT 3L-DNMT 3A-dCas9-Krab.

[0125] In some embodiments, the gene expression regulatory molecule can bind to a DNA region within about 500 bp upstream and / or downstream of the transcription start site (TSS) of the ANGPTL3 gene or a fragment thereof. For example, the gene expression regulatory molecule can bind within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp upstream of the TSS. For example, the gene expression regulatory molecule can bind within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp downstream of the TSS. For example, the gene expression regulatory molecule can bind to a DNA region in which any one of SEQ ID NOs: 71-72 is located, or a fragment thereof.

[0126] Illustratively, in some aspects, the gene expression regulatory molecule can bind to one or more DNA regions near the transcription start site (TSS) of the ANGPTL3 gene, as described below: between 180 bp upstream of the TSS and 10 bp upstream, between the TSS and 90 bp downstream of the TSS, and between 180 bp downstream of the TSS and 240 bp downstream of the TSS.

[0127] In some embodiments, the gene expression regulatory molecule can bind to one or more DNA regions near the transcription start site (TSS) of the ANGPTL3 gene, as described below: between 180 bp upstream and 150 bp upstream of the TSS (e.g., between 177-158 bp upstream of the TSS), between 120 bp upstream and 90 bp upstream of the TSS (e.g., between 112-93 bp upstream of the TSS), between 70 bp upstream and 10 bp upstream of the TSS (e.g., between 67-48 bp, between 65-46 bp, between 61-42 bp), between the TSS, between 57-38 bp upstream, and / or between 32-13 bp upstream of the TSS), between the TSS and 30 bp downstream of the TSS (e.g., between 2-21 bp downstream of the TSS), and between 60 bp downstream and 90 bp downstream of the TSS (e.g., between 68-87 bp downstream), and between 180 bp downstream and 240 bp downstream of the TSS (e.g., between 185-204 bp, 186-205 bp, 197-216 bp, 200-219 bp, and / or 221-240 bp downstream of the TSS).

[0128] In some embodiments, the gene expression regulatory molecule may also include a tag for detection, isolation, and / or purification. For example, the gene expression regulatory molecule may include an HA tag. For example, the gene expression regulatory molecule may include the amino acid sequence of an HA tag. Alternatively, the gene expression regulatory molecule may have one, two, three, four, five, or more mutations compared to the above sequence, such as a sequence with amino acid substitutions, insertions, or deletions, or a fragment thereof.

[0129] In some embodiments, the gene expression regulatory molecule may also include a nuclear localization sequence. For example, the nuclear localization sequence may include an amino acid having a positively charged group. For example, the nuclear localization sequence may include the amino acid sequence of a nuclear localization sequence known in the art. Alternatively, the nuclear localization sequence may have one, two, three, four, five, or more mutations compared to the above sequence, such as a sequence or fragment thereof having amino acid substitutions, insertions, or deletions. For example, the nuclear localization sequence may be located at the N-terminus and / or C-terminus of the first functional domain, the N-terminus and / or C-terminus of the second functional domain, and / or the N-terminus and / or C-terminus of the DNA binding domain.

[0130] In some embodiments, the gene expression regulatory molecule may also comprise a detectable moiety. For example, the detectable moiety may comprise blue fluorescent protein and / or green fluorescent protein. For example, the detectable moiety is linked to the first functional domain, the second functional domain, the DNA binding domain, and / or the nuclear localization sequence via a self-cleaving peptide. For example, the self-cleaving peptide may comprise a 2A peptide.

[0131] In the present invention, the first functional domain, the second functional domain, the DNA binding domain, and other elements contained in the gene regulatory molecule described herein may be indirectly connected, for example, via a linker sequence of a certain length.For example, the linker may contain about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100 or more amino acids.For example, the first functional domain and the second functional domain are connected via a linker of about 80 or more amino acids in length.For example, the first functional domain and the second functional domain are connected via a linker of about 92 or more amino acids in length. For example, the first functional domain and the second functional domain are connected via an XTEN linker. For example, the linker comprises the amino acid sequence of an XTEN linker. For example, the linker comprises the amino acid sequence of an XTEN linker. For example, the linker comprises the amino acid sequence of a 16-amino acid XTEN linker, an 80-amino acid XTEN linker, or a longer XTEN linker. Alternatively, the linker may have one, two, three, four, five, or more mutations compared to the above sequence, such as a sequence with amino acid substitution, insertion, deletion, or fragment thereof.

[0132] nucleic acid binding molecules The present application also provides nucleic acid binding molecules, wherein the nucleic acid binding molecules may comprise any one of the sequences of SEQ ID NOs: 1-70. For example, the nucleic acid binding molecules and / or gene expression regulating molecules of the present invention may be delivered to a subject by local injection, systemic infusion, or a combination thereof. In all aspects, the nucleic acid binding molecules may comprise a sequence having 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOs: 1-70.

[0133] In some embodiments, the combination of a nucleic acid binding molecule and a gene expression regulatory molecule of the present invention may be capable of regulating the expression level of the ANGPTL3 gene. For example, the nucleic acid binding molecule and / or gene expression regulatory molecule of the present invention may bind to the core promoter, proximal promoter, distal enhancer, silencer, insulator element, boundary element, and / or locus control region of the ANGPTL3 gene. For example, the nucleic acid binding molecule and / or gene expression regulatory molecule of the present invention may bind to a DNA region within approximately 500 bp upstream and / or downstream of the transcription start site of the ANGPTL3 gene, or a fragment thereof. For example, the nucleic acid binding molecule and / or gene expression regulatory molecule of the present invention may bind to a DNA region or a fragment thereof in which any one of SEQ ID NOs: 71 to 72 is located. In all aspects, the DNA region to be linked may comprise a sequence having 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the DNA region in which any one of SEQ ID NOs: 71-72 is located.

[0134] For example, the nucleic acid binding molecules and / or gene expression regulating molecules of the present invention can bind to the DNA region in which any one of SEQ ID NOs: 73-142 is located, or a fragment thereof. In all aspects, the DNA region to which it can bind can include a sequence having 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the DNA region in which any one of SEQ ID NOs: 73-142 is located. For example, targeting any region of approximately 20 bp in the DNA region in which any one of SEQ ID NOs: 73-142 is located can have the ability to regulate the expression level of the ANGPTL3 gene. For example, administration of a substance of the invention can negatively affect (e.g., decrease) the activity of a nucleic acid sequence, including at least partially, partially, or completely inhibiting activation (e.g., transcription) of the nucleic acid sequence, or reducing, preventing, or delaying activation of the nucleic acid sequence, compared to the expression and / or activity of the target gene in the absence of the substance of the invention. For example, activity inhibition is about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or less of control.

[0135] In some embodiments, the length of the guide sequence or spacer of the nucleic acid binding molecule can be 15 to 50 nucleotides. In some embodiments, the nucleic acid binding molecule is a guide RNA (gRNA), and the length of the spacer of the guide RNA can be at least 15 nucleotides. In some embodiments, the length of the spacer can be 15 to 17 nucleotides, 17 to 20 nucleotides, 20 to 24 nucleotides, 23 to 25 nucleotides, 24 to 27 nucleotides, 27 to 30 nucleotides, 30 to 35 nucleotides, or 35 nucleotides or more. In some embodiments, the number of gRNAs administered can be at least one gRNA, at least two different gRNAs, at least three different gRNAs, at least four different gRNAs, or at least five different gRNAs. In some embodiments, the length of the target binding region can be between about 19 and about 21 nucleotides. In one embodiment, the length of the target binding region can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In one embodiment, the target binding region may be complementary to the target region of the target gene, for example, completely complementary. In one embodiment, the target binding region may be substantially complementary to the target region of the target gene. In one embodiment, the target binding region may contain 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, or 10 or less nucleotides that are not complementary to the target region of the target gene. In some embodiments, the nucleic acid binding molecules and / or gene expression regulating molecules of the present invention may be formulated in liposomes or lipid nanoparticles. In some embodiments, the nucleic acid binding molecules and / or gene expression regulating molecules of the present invention may be formulated in viral vectors. For example, the guide RNA may comprise an RNA-based molecule having one or more chemical modifications (e.g., two ribonucleotides linked via a chemical bond, or one or more ribonucleotides replaced with one or more deoxyribonucleotides).For example, a target binding region of human origin can be targeted.

[0136] Without being bound by any particular theory, the examples set forth below are merely illustrative of the products, methods of preparation and use of the present invention and are not intended to limit the scope of the invention. [Example]

[0137] Example 1 The gene regulatory molecule of the present invention can inhibit the expression of human ANGPTL3 gene mRNA. (1) Experimental Method: We designed sgRNAs targeting the 500 bp (total of 1,000 bp) sequence before and after the transcription start site of the human ANGPTL3 gene, and then targeted different epigenetic editing tools to the corresponding locations in the genome. The mRNAs and corresponding sgRNAs for the different epigenetic editing tools (EpiRegA: Dnmt3a-Dnmt3l-dCas9-KOX1KRAB, SEQ ID NO: 143, and EpiRegB: Dnmt3a-Dnmt3l-ZIM3KRAB-dCas9, SEQ ID NO: 145) were encapsulated in LNPs at a 1:1 mass ratio to prepare LNP test samples combining the corresponding editing tools with different sgRNAs (see reference for LNP preparation: https: / / doi.org / 10.1038 / s41586-021-03534-y). The human hepatoma cell line Huh7 was used as the test cell line, seeded at 50,000 cells per well in a 24-well plate. After 12 hours, LNP samples were added to the wells at a dose of 5 μg / mL. After 4-6 hours, the medium was replaced with fresh DMEM + 10% FBS, and the cells were continued to be cultured. After 3 days, the cells were harvested, mRNA was extracted, reverse-transcribed into cDNA, and the mRNA expression level of the target gene ANGPTL3 was detected by qPCR. The knockdown efficiency corresponding to each sgRNA was obtained by comparing with the control group containing empty packaging (no epigenetic editing tool mRNA or sgRNA). From the mRNA knockdown results shown in Figure 1, by combining SEQ ID NOs: 28, 29, 39, 41, 42, 50, 54, 55, 57, 59, 60, 63, 67 and 69 (their target regions start at 65 bp upstream, 112 bp upstream, 177 bp upstream, 221 bp downstream, 61 bp upstream, 200 bp downstream, 57 bp upstream, 197 bp downstream, 185 bp downstream, 68 bp downstream, 32 bp upstream, 186 bp downstream, 2 bp downstream, and 67 bp upstream of the TSS, respectively), with the epigenetic editing tool of this example, the mRNA expression level of the ANGPTL3 gene can be effectively inhibited.

[0138] [Table 1]

[0139] Example 2 The guide RNA of the epigenetic editing tool is constructed to target a target gene by referring to any of the nucleotide sequences set forth in SEQ ID NOs: 1-70; on the other hand, the epigenetic editing tool is constructed based on the gene expression regulatory molecule provided in the present application. For example, it can target the region within 500 bp upstream and downstream of the transcription start site (TSS) of ANGPTL3.

[0140] Guide RNA plasmids and gene expression regulatory molecule plasmids were co-transfected into mouse cell lines. After 72 hours, the top 10% of GFP + Cells and mCherry + Cells were sorted by FACS. RT-QPCR experiments were performed to evaluate the mRNA expression levels of the target genes.

[0141] As a result, the transfected cells exhibit reduced expression of the ANGPTL3 expression product. For example, the transfected cells may exhibit reduced mRNA expression of ANGPTL3 transcription. For example, the transfected cells may exhibit reduced expression of the ANGPTL3 protein product. For example, the editing method of the present invention can be used to alleviate diseases or conditions associated with the ANGPTL3 gene.

[0142] Example 3 Universality of epigenetic targets The epigenetic editing tool of the present application was used to regulate the expression of a target. The domain in the gene expression regulatory molecule that functions to bind to a gene sequence can be replaced with any CRISPR / Cas-based nuclease domain known in the art, such as a known TALEN DNA binding domain, a zinc finger domain, a tetR DNA binding domain, a meganuclease and / or a dCas12 enzyme.

[0143] As a result, it was shown that the epigenetic target of the present invention is universal, and that the use of various gene sequence binding domains is highly effective in controlling gene expression.

[0144] The above detailed description is for purposes of explanation and example, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments described herein may be apparent to those skilled in the art and are encompassed within the scope of the appended claims and their equivalents.

Claims

1. A composition comprising a gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule for (a) regulating the expression and / or activity of the ANGPTL3 gene, or (b) treating and / or alleviating a disease condition associated with the abnormal expression and / or activity of the ANGPTL3 gene, wherein the gene expression regulatory molecule has the function of regulating the expression of the ANGPTL3 gene without altering the gene sequence of the ANGPTL3 gene, and the gene expression regulatory molecule is located near the ANGPTL3 gene and / or within the ANGPTL3 gene regulatory element. A composition comprising: a first functional domain that provides modification of another nucleotide, wherein the first functional domain comprises one or more of DNA methyltransferases, DNA demethylases, and functionally active fragments thereof; a second functional domain comprising a zinc finger protein-based transcription factor or a functionally active fragment thereof, or a substance capable of modifying histones; and a DNA-binding domain selected from the protein domain of the CRISPR / Cas system, the TALEN domain, and the zinc finger domain.

2. The composition according to claim 1, wherein the modification of at least one nucleotide includes a methylation modification, optionally the regulatory element includes a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, and / or a locus regulatory region, optionally the DNA methyltransferase includes one or more of DNMT 3A, DNMT 3L, DNMT 3B, DNMT 1, and DNMT 2, optionally DNMT 3A is derived from mouse and / or DNMT 3L is derived from human and / or mouse, and optionally DNMT 3A and DNMT 3L are directly and / or indirectly linked.

3. The composition according to claim 1, wherein the second functional domain comprises Krab, optionally comprising ZIM3 Krab or KOX1 Krab, and / or comprising one or more histone methyltransferases, histone demethylases, histone acetyltransferases, histone deacetylases, and their functionally active fragments.

4. The composition according to claim 1, wherein the first functional domain is directly or indirectly linked to one end of the second functional domain, or the first functional domain is directly and / or indirectly linked to both ends of the second functional domain, and / or the first functional domain and the second functional domain are directly or indirectly linked to one end of the DNA-binding domain, or the first functional domain and the second functional domain are directly and / or indirectly linked to both ends of the DNA-binding domain.

5. The composition according to claim 1, wherein the gene expression regulatory molecule comprises a Cas enzyme, optionally comprising a Cas enzyme that substantially lacks nuclease activity, and optionally comprising a dCas9 enzyme.

6. Gene expression regulatory molecules can bind to DNA regions or fragments within 500 bp upstream and / or downstream of the transcription start site (TSS) of the ANGPTL3 gene, and / or A gene expression regulatory molecule can bind to the DNA region or fragment thereof where any one of sequence numbers 71-72 is located, and / or A gene expression regulatory molecule can bind to one or more DNA regions near the transcription start site (TSS) of the ANGPTL3 gene, as described below: (i) between 180bp upstream of the TSS and 10bp upstream, between the TSS and 90bp downstream of the TSS, and between 180bp downstream of the TSS and 240bp downstream; or (ii) Between 180bp upstream of TSS and 150bp upstream, between 120bp upstream of TSS and 90bp upstream, between 70bp upstream of TSS and 10bp upstream, between TSS and 30bp downstream of TSS, between 60bp downstream of TSS and 90bp downstream, and between 180bp downstream of TSS and 240bp downstream, The composition according to claim 1.

7. The composition according to any one of claims 1 to 6, wherein the adjustment, treatment and / or relaxation comprises providing a nucleic acid binding molecule comprising any one sequence of SEQ ID NOs: 1 to 70.

8. (i) gene expression regulatory molecules and / or nucleic acid binding molecules are formulated in the same delivery carrier or in different delivery carriers, and optionally the delivery carriers include liposomes and / or lipid nanoparticles, or (ii) Gene expression regulatory molecules and / or nucleic acid binding molecules are formulated in the same recombinant vector or in different recombinant vectors, and optionally the recombinant vector contains a viral vector, and optionally the recombinant vector contains an adeno-associated virus vector. The composition according to claim 7.

9. The composition according to any one of claims 1 to 6, wherein the gene expression regulatory molecule comprises a nuclear localization sequence, optionally comprising an amino acid having a positively charged group, and optionally the nuclear localization sequence being located at the N-terminus and / or C-terminus of a first functional domain, the N-terminus and / or C-terminus of a second functional domain, and / or the N-terminus and / or C-terminus of a DNA-binding domain.

10. A nucleic acid-binding molecule containing one of the sequences from sequence numbers 1 to 70.

11. A gene expression regulatory molecule that has the function of regulating the expression of the ANGPTL3 gene without altering the gene sequence of the ANGPTL3 gene.

12. The gene expression regulatory molecule is the gene expression regulatory molecule described in claim 1, and optionally, (i) The gene expression regulatory molecule and / or nucleic acid binding molecule are formulated in the same delivery carrier or in different delivery carriers, the nucleic acid binding molecule contains one of the sequences of SEQ ID NOs: 1 to 70, and optionally the delivery carrier contains liposomes and / or lipid nanoparticles, or (ii) The gene expression regulatory molecule and / or nucleic acid binding molecule are formulated in the same recombinant vector or in different recombinant vectors, the nucleic acid binding molecule contains one of the sequences of SEQ ID NOs: 1 to 70, optionally the recombinant vector contains a viral vector, optionally the recombinant vector contains an adeno-associated virus vector. The gene expression regulatory molecule according to claim 11.

13. A nucleic acid encoding a gene expression regulatory molecule as described in claim 11.

14. A recombinant vector comprising the nucleic acid described in claim 13.

15. A delivery carrier comprising a nucleic acid binding molecule according to claim 10, a gene expression regulatory molecule according to claim 11, a nucleic acid according to claim 13, and / or a recombinant vector according to claim 14, and optionally comprising liposomes and / or lipid nanoparticles.

16. A composition comprising a nucleic acid binding molecule according to claim 10, a gene expression regulatory molecule according to claim 11, a nucleic acid according to claim 13, and / or a recombinant vector according to claim 14.

17. A cell comprising the nucleic acid binding molecule according to claim 10, the gene expression regulatory molecule according to claim 11, the nucleic acid according to claim 13, and / or the recombinant vector according to claim 14.

18. A kit comprising the nucleic acid binding molecule according to claim 10, the gene expression regulatory molecule according to claim 11, the nucleic acid according to claim 13, and / or the recombinant vector according to claim 14.