Method for apparent editing of target spot and application

Abstract

The invention discloses an apparent editing target point method and application. Specifically, a method for regulating the expression level of a target gene and a nucleic acid binding molecule are provided, and the combination of the nucleic acid binding molecule and a gene expression regulating molecule has the ability to regulate the expression level of the target gene.

Description

A method and use of epigenetic editing targets Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an epigenetic editing target and its use. Background Art

[0002] Fusion of a catalytically inactive "deactivated Cas9" (dCas9) to a Kruppel-associated box (KRAB) domain can generate a gene expression repressor that can highly specifically and effectively regulate or silence target genes in cell culture experiments. However, in vivo, these repressors present certain challenges in achieving therapeutic effects. For example, safety, toxicity, immunogenicity, and off-target effects are other challenges that limit the use of synthetic repressors in vivo.

[0003] Therefore, there is a need in the art for an effective epigenetic editing target, which can be used to improve the efficacy and safety of epigenetic editing and reduce toxicity, immunogenicity and / or off-target effects.

[0004] Summary of the Invention

[0005] The present application provides an epigenetic editing target, which can be used to improve the efficacy and safety of epigenetic editing and reduce toxicity, immunogenicity and / or off-target effects. For example, targeting near the target gene and / or within the target gene regulatory element can effectively modify at least one nucleotide, thereby regulating (e.g., reducing or eliminating) the expression of the target gene product in the cell.

[0006] On the one hand, the present application provides a method for regulating the expression and / or activity of the ANGPTL3 (Angiopoietin Like 3) gene, the method comprising providing a gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule, wherein the gene expression regulatory molecule has the function of regulating the expression of the ANGPTL3 gene without changing its gene sequence.

[0007] On the other hand, the present application provides a method for treating and / or alleviating a disease associated with abnormal ANGPTL3 gene expression and / or ANGPTL3 gene activity, the method comprising providing a gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule, wherein the gene expression regulatory molecule has the function of regulating the expression of the ANGPTL3 gene without changing its gene sequence.

[0008] In some embodiments, the gene expression modulator molecule comprises a first functional domain that provides for modification of at least one nucleotide proximal to the ANGPTL3 gene and / or within a regulatory element of the ANGPTL3 gene.

[0009] In some embodiments, the modification of the at least one nucleotide comprises a methylation modification.

[0010] 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.

[0011] In some embodiments, the first functional domain comprises one or more of a DNA methyltransferase, a DNA demethylase, and functionally active fragments thereof.

[0012] In some embodiments, the DNA methyltransferase comprises one or more of DNMT 3A, DNMT 3B, DNMT 3L, DNMT 1, and DNMT 2.

[0013] In some embodiments, the DNMT 3A is derived from mouse.

[0014] In some embodiments, the DNMT 3L is derived from human and / or mouse.

[0015] In some embodiments, the DNMT 3A and the DNMT 3L are directly and / or indirectly linked.

[0016] In some embodiments, the gene expression regulatory molecule comprises a second functional domain, which comprises a zinc finger protein-based transcription factor or a functionally active fragment thereof, or comprises a substance capable of modifying histones.

[0017] In some embodiments, the second functional domain comprises Krab.

[0018] In some embodiments, the second functional domain comprises ZIM3 Krab or KOX1 Krab.

[0019] 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.

[0020] In some embodiments, the gene expression regulatory molecule comprises the first functional domain and the second functional domain, and the first functional domain is directly or indirectly connected to one end of the second functional domain, or the first functional domain is directly and / or indirectly connected to both ends of the second functional domain.

[0021] In some embodiments, the gene expression regulating molecule comprises a DNA binding domain.

[0022] In some embodiments, the gene expression regulating molecule comprises one or more DNA binding domains selected from the group consisting of a TALEN domain, a zinc finger domain, and a protein domain of a CRISPR / Cas system.

[0023] In some embodiments, the gene expression regulating molecule comprises a Cas enzyme.

[0024] In some embodiments, the gene expression regulating molecule comprises a Cas enzyme that has substantially no nuclease activity.

[0025] In some embodiments, the gene expression regulating molecule comprises a dCas9 enzyme.

[0026] In some embodiments, the first functional domain and the second functional domain are directly or indirectly connected to one end of the DNA binding domain, or the first functional domain and the second functional domain are directly and / or indirectly connected to both ends of the DNA binding domain.

[0027] In some embodiments, the gene expression regulating molecule is capable of binding 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.

[0028] In some embodiments, the gene expression regulatory molecule is capable of binding to the DNA region or fragment thereof where any one of SEQ ID NOs: 71-72 is located.

[0029] In some embodiments, the gene expression regulating molecule is capable of binding to one or more DNA regions near the following transcription start site (TSS) of the ANGPTL3 gene: between 180 bp upstream and 10 bp upstream of the TSS, between the TSS and 90 bp downstream of the TSS, and between 180 bp downstream and 240 bp downstream of the TSS.

[0030] In some embodiments, the gene expression regulating molecule is capable of binding to one or more of the following DNA regions near the transcription start site (TSS) of the ANGPTL3 gene: between 180 bp and 150 bp upstream of the TSS, between 120 bp and 90 bp upstream of the TSS, between 70 bp and 10 bp upstream of the TSS, between the TSS and 30 bp downstream of the TSS, between 60 bp and 90 bp downstream of the TSS, and between 180 bp and 240 bp downstream of the TSS.

[0031] In some embodiments, the method includes providing a nucleic acid binding molecule comprising the sequence of any one of SEQ ID NOs: 1-70.

[0032] In some embodiments, the gene expression regulating molecule and / or the nucleic acid binding molecule are formulated in the same or different delivery vehicles.

[0033] In some embodiments, the delivery vehicle comprises a liposome and / or a lipid nanoparticle.

[0034] In some embodiments, the expression regulating molecule and / or the nucleic acid binding molecule are formulated in the same or different recombinant vectors.

[0035] In some embodiments, the recombinant vector comprises a viral vector.

[0036] In some embodiments, the recombinant vector comprises an adeno-associated virus (AAV) vector.

[0037] In some embodiments, the gene expression regulating molecule comprises a nuclear localization sequence.

[0038] In some embodiments, the nuclear localization sequence comprises an amino acid having a positively charged group.

[0039] 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.

[0040] On the other hand, the present application provides a nucleic acid binding molecule comprising a sequence of any one of SEQ ID NOs: 1-70.

[0041] On the other hand, the present application provides a gene expression regulatory molecule, which has the function of regulating the expression of the ANGPTL3 gene without changing its gene sequence.

[0042] In some embodiments, the gene expression regulatory molecule is a gene expression regulatory molecule provided in the method of the present application.

[0043] In some embodiments, the gene expression regulating molecule and / or nucleic acid binding molecule are formulated in the same or different delivery vehicles, and the nucleic acid binding molecule comprises the sequence of any one of SEQ ID NOs: 1-70.

[0044] In some embodiments, the delivery vehicle comprises a liposome and / or a lipid nanoparticle.

[0045] In some embodiments, the expression regulating molecule and / or the nucleic acid binding molecule are formulated in the same or different recombinant vectors, and the nucleic acid binding molecule comprises the sequence of any one of SEQ ID NOs: 1-70.

[0046] In some embodiments, the recombinant vector comprises a viral vector.

[0047] In some embodiments, the recombinant vector comprises an adeno-associated virus (AAV) vector.

[0048] On the other hand, the present application provides a nucleic acid encoding the nucleic acid binding molecule of the present application and / or encoding the gene expression regulating molecule of the present application.

[0049] On the other hand, the present application provides a recombinant vector comprising the nucleic acid of the present application.

[0050] On the other hand, the present application provides a delivery vector, which comprises the nucleic acid binding molecule of the present application, the gene expression regulating molecule of the present application, the nucleic acid of the present application, and / or the recombinant vector of the present application, and optionally comprises liposomes and / or lipid nanoparticles.

[0051] On the other hand, the present application provides a composition comprising the nucleic acid binding molecule of the present application, the gene expression regulating molecule of the present application, the nucleic acid of the present application, the recombinant vector of the present application, and / or the delivery vector of the present application.

[0052] On the other hand, the present application provides a cell comprising the nucleic acid binding molecule of the present application, the gene expression regulating molecule of the present application, the nucleic acid of the present application, the recombinant vector of the present application, the delivery vector of the present application, and / or the composition of the present application.

[0053] On the other hand, the present application provides a kit comprising the nucleic acid binding molecule of the present application, the gene expression regulating molecule of the present application, the nucleic acid of the present application, the recombinant vector of the present application, the delivery vector of the present application, the composition of the present application, and / or the cell of the present application.

[0054] On the other hand, the present application provides a method for regulating the expression and / or activity of a target gene, which method comprises providing the nucleic acid binding molecule of the present application, the gene expression regulating molecule of the present application, the nucleic acid of the present application, the recombinant vector of the present application, the delivery vector of the present application, the composition of the present application, the cell of the present application, and / or the kit of the present application.

[0055] On the other hand, the present application provides a method for preparing a drug for treating and / or alleviating a disease, wherein the disease includes a disease associated with abnormal expression and / or activity of a target gene.

[0056] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0058] FIG1 shows the mRNA knockdown efficiency of the gene expression regulatory molecules of the present application in targeting different ANGPTL3 gene regulatory regions. DETAILED DESCRIPTION

[0059] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0060] Definition of terms

[0061] In this application, the term "nucleic acid" is used interchangeably with "polynucleotide", "nucleotide", "nucleotide sequence" and "oligonucleotide" and generally refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in single-stranded, double-stranded or multi-stranded form or their complements. For example, a nucleotide can be a ribonucleotide, a deoxyribonucleotide or a modified version thereof. For example, a nucleotide can be a single-stranded and double-stranded DNA, a single-stranded and double-stranded RNA, and a hybrid molecule having a mixture of single-stranded and double-stranded DNA and RNA. For example, a nucleotide can include, but is not limited to, any type of RNA, such as mRNA, siRNA, miRNA, sgRNA and guide RNA, and any type of DNA, genomic DNA, plasmid DNA and minicircle DNA, and any fragments thereof. The term also encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring.

[0062] As used herein, the term "sequence encoding" or "nucleic acid encoding" generally refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may also include start and stop signals operably linked to regulatory elements, including 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.

[0063] In this application, the term "treatment", for example, when applied to a disease, means that when a gene expression regulatory molecule as described herein or a nucleic acid encoding the gene expression regulatory molecule and / or a nucleic acid binding molecule (e.g., gRNA) as described herein or a nucleic acid encoding the nucleic acid binding molecule is administered, a subject (e.g., a human) who has the disease, is at risk of having the disease and / or experiences symptoms of the disease will, in one embodiment, experience less severe symptoms and / or will recover faster than when the gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule and / or the nucleic acid binding molecule or a nucleic acid encoding the nucleic acid binding molecule has never been administered.

[0064] In the present application, the term "DNA binding domain" generally refers to an independently folded protein domain that contains at least one motif that recognizes double-stranded or single-stranded DNA. For example, the DNA binding domain can recognize a specific DNA sequence (recognition or regulatory sequence) or has a general affinity for DNA. In some cases, other domains of the DNA binding domain typically regulate the activity of the DNA binding domain; the DNA binding function can be structural or include transcriptional regulation, and sometimes these two effects are overlapping. In certain embodiments of the methods and gene expression regulatory molecules provided herein, the DNA binding domain may include a (DNA) nuclease, such as a nuclease that can target DNA in a sequence-specific manner or can be guided or instructed to target DNA in a sequence-specific manner, such as a CRISPR-Cas system, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) or a large range of nucleases. In some embodiments, the DNA binding domain is a DNA nuclease derived from the CRISPR-Cas system. For example, the DNA nuclease derived from the CRISPR-Cas system is a Cas protein.

[0065] In this application, "Cas enzyme" can be used interchangeably with "Cas protein", "CRISPR protein", "CRISPR enzyme", "CRISPR-Cas protein", "CRISPR-Cas enzyme", "Cas", "CRISPR effector" or "Cas effector protein", which generally refers to a class of enzymes that are complementary to the CRISPR sequence and can use the CRISPR sequence as a guide to recognize and cut specific DNA strands. Non-limiting examples of Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and / or homologs thereof, or modified forms thereof. These proteins are known, for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0066] In this application, the term "dCas9 enzyme" is also referred to as "inactivated Cas9 protein" or "inactivated Cas9 enzyme". Methods for generating Cas9 proteins (or fragments thereof) with inactivated DNA cleavage domains are known, for example, 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 each of which are incorporated herein by reference). For example, it is known that the DNA cleavage domain of Cas9 includes two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cuts the strand complementary to the gRNA, while the RuvC1 subdomain cuts the non-complementary strand. Mutations in these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A 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 inactivation or nicking DNA binding domains include, but are not limited to, nuclease-inactivated variant 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 from Streptococcus pyogenes without endonuclease activity has been targeted by gRNA to genes in bacteria, yeast, and human cells to silence gene expression by 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, the terms "iCas" and "dCas" are used interchangeably to refer to a CRISPR-associated protein without catalytic activity. 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 HNH domain. In one embodiment, the dCas molecule comprises one or more mutations in both the RuvC and HNH domains. 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 from a wild-type Cas protein, wherein 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 the 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.

[0067] Suitable dCas can be derived from wild-type Cas protein. Cas protein can be from type I, type II or type III CRISPR-Cas system. In one embodiment, suitable dCas can be derived from Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 or Cas10. In one embodiment, dCas is derived from Cas9 protein. For example, dCas9 can be obtained by introducing point mutations (e.g., substitutions, deletions or additions) in the DNA cleavage domain (e.g., nuclease domain, e.g., RuvC and / or HNH domain) of Cas9 protein. See, e.g., Jinek et al., Science (2012) 337: 816-21, incorporated herein by reference in its entirety. For example, introducing two point mutations in RuvC and HNH domains reduces Cas9 nuclease activity while retaining Cas9sgRNA and DNA binding activity. In one embodiment, the two point mutations in RuvC and HNH active sites are 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 its sgRNA and DNA binding activity. In one embodiment, the two point mutations within the RuvC and HNH active sites are the D10A and N580A mutations of Streptococcus pyogenes Cas9.

[0068] In various embodiments, the present application relates to a dCas protein or any variant or mutant thereof. All variants and mutants of dCas9 can be used in the methods, compositions, fusion molecules, or kits disclosed herein, including but not limited to those derived from 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), and any variants and mutant forms of Cas9 listed above, such as high-fidelity Cas9 (Kleinstiver et al., Nature. January 28, 2016) and enhanced SpCas9 (Slaymaker et al., Sciences. January 1, 2016). For example, the dCas9 sequences shown in SEQ ID NOs: 1162-1179 of the present application only provide a few exemplary options and are not exclusive. In one embodiment, the dCas protein is a Streptococcus pyogenes dCas9 protein comprising 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 comprising a D10A and / or H840A mutation (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 1, 2, 3, 4, 5 or more alterations (e.g., amino acid substitutions, insertions or deletions) relative to SEQ ID NO: 1163 or 1164, or any fragment thereof.

[0069] Similar mutations can also be applied to any other naturally occurring Cas9 (e.g., Cas9 from other species) or engineered Cas9. In certain embodiments, the dCas9 comprises Streptococcus pyogenes dCas9, Staphylococcus aureus dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheria dCas9, Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, Azospirillum (e.g., strain B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia enterica dCas9, and Streptococcus pasteurianus dCas9. intestinalis) dCas9, Parvibaculum lavamentivorans) dCas9, Nitratifractor salsuginis (e.g., strain DSM 16511) dCas9, Campylobacter lari (e.g., strain CF89-12) dCas9, Streptococcus thermophilus (e.g., strain LMD-9) dCas9, or fragments thereof. In certain embodiments, the present application also provides a vector comprising a nucleotide encoding the following protein molecule: Streptococcus pyogenes dCas9, Staphylococcus aureus dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheriae dCas9, Eubacterium convexogastricum dCas9, Streptococcus pasteurianum dCas9, Lactobacillus farinae dCas9, Trichoderma sphaeroides dCas9, Azospirillum (strain B510) dCas9, Gluconacetobacter diazophila dCas9, Neisseria griseus dCas9, Roseburia intestinalis dCas9, Microbacterium edodes dCas9, Brine Nitrate Lysing Bacteria (strain DSM 16511) dCas9, Campylobacter seagulls (strain CF89-12) dCas9, Streptococcus thermophilus (strain LMD-9) dCas9 or a fragment thereof.

[0070] In this application, the term "Cas enzyme having substantially no nuclease activity" generally refers to an RNA-guided enzyme in which recognition of phosphodiester bonds is facilitated by a separate polynucleotide sequence (e.g., a guide RNA), but the enzyme may not significantly cleave the target phosphodiester bond (e.g., no measurable phosphodiester bond cleavage under physiological conditions). For example, when complexed with a polynucleotide (e.g., sgRNA), a DNA endonuclease guided by an RNA lacking a nuclease retains DNA binding ability (e.g., specific binding to a target sequence), but lacks significant endonuclease activity. For example, a DNA endonuclease guided by an RNA lacking a nuclease is dCas9, ddCpf1, a Cas9 variant lacking a nuclease, or a Class II CRISPR endonuclease lacking a nuclease. For example, a DNA endonuclease guided by an RNA lacking a nuclease is dCas9. As referred to herein, the term "dCas9" or "dCas9 protein" is a Cas9 protein in which both catalytic sites of endonuclease activity are defective or lack activity. For example, dCas9 has substantially no detectable endonuclease (e.g., deoxyribonuclease) activity. In various aspects, for example, dCas9 comprises a variant or homolog of an amino acid 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 dCas9 enzyme sequence of the present application.

[0071] In the present application, the term "capable of binding" can be used interchangeably with "binding to", "specifically identifying", "targeting", etc., and generally refers to that the binding molecule (for example, the gene expression regulatory molecule of the present application) is able to interact with the nucleotides on the target gene or target site, or the binding molecule (for example, the gene expression regulatory molecule of the present application) has sufficient affinity for the target gene or target site. This interaction can be through conjugation, coupling, attachment, providing complementarity, providing covalent force or providing non-covalent force, improving binding stability, etc.

[0072] As used herein, the term "transcription start site" generally refers to the nucleic acid in a construct that corresponds to the first nucleic acid integrated into the primary transcript (ie, pre-mRNA); the transcription start site may overlap with a promoter sequence.

[0073] In this application, the term "fragment thereof" generally refers to a portion or fragment of a specified whole. For example, when used in this application relative to a specified nucleotide sequence, the term "fragment thereof" refers to a continuous length of a specified nucleotide sequence that is shorter than the full-length sequence of a specified polynucleotide. A portion of a specified nucleotide can be defined by its first position and its last position, wherein the first and last positions each correspond to a position in the sequence of the specified polynucleotide, wherein the sequence position corresponding to the first position is located at the N-terminus of the sequence position corresponding to the last position, and whereby the sequence of the portion is a continuous sequence of nucleotides in the specified polynucleotide that begins at the sequence position corresponding to the first position and ends at the sequence position corresponding to the last position. A portion can also be defined by reference to a position in the sequence of a specified polynucleotide and a residue length relative to the reference position, whereby the sequence of the portion is a continuous sequence of nucleotides in the specified polynucleotide that has a defined length and is positioned in the specified polynucleotide according to the defined position.

[0074] In this application, the term "nucleotide modification" may mean that the nucleic acids described in the present invention are synthesized or modified by methods well-established in the art, such as the methods described in "Current protocols in nucleic acid chemistry" Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA (which is incorporated herein by reference). The modification may include, but is not limited to, terminal modifications, such as 5'-terminal modifications (e.g., phosphorylation, conjugation, inverted linkage) or 3'-terminal modifications (e.g., conjugation, DNA nucleotides, inverted linkage, etc.); base modifications, such as substitution with stabilized bases, destabilized bases, or bases that pair with an extended pairing repertoire, removal of bases (absorptive nucleotides), or conjugated bases; sugar modifications (e.g., sugar modification at the 2'-position or 4'-position) or replacement of sugars; or backbone modifications, including modification or substitution of phosphodiester bonds.

[0075] In this application, the term "substance that modifies histones" generally refers to enzymes that can modify histones and regulate gene transcription. Common modifications to histones include methylation, acetylation, phosphorylation, adenylation, ubiquitination, ADP-ribosylation, etc.

[0076] In this application, the term "methylation modification" is used interchangeably with "DNA methylation" and "nucleic acid methylation", and generally refers to a process in which a gene fragment, nucleotide or base thereof in this application is methylated, which often occurs in a cell that has been transfected with a nucleic acid containing a structural gene encoding a polypeptide operatively linked to a promoter, during which the cytosine of the promoter nucleic acid is converted to 5-methylcytosine. A promoter nucleic acid in which at least one cytosine is converted to 5-methylcytosine is referred to as a "methylated" nucleic acid or DNA. In this application, the DNA fragment in which the gene is located may be methylated on one or more strands, and may also be methylated at one or more sites.

[0077] In the present application, 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, etc., together provide for the replication, transcription, and translation of coding sequences in recipient cells. Not all of these control sequences need to exist. Transcriptional control signals in eukaryotes typically include "promoter" and "enhancer" elements. Promoters and enhancers are composed of short arrays of DNA sequences. Promoters are regulatory elements that promote the initiation of transcription of operably linked coding regions. Enhancers increase the activity of the closest promoter on the same DNA molecule, thereby increasing the regulatory elements of genetic transcription speed. These sequences specifically interact with the cellular proteins involved in transcription (Maniatis et al., Science 236:1237 (1987), incorporated herein by reference in their entirety). Promoter and enhancer elements have been isolated from various eukaryotic sources, including genes in yeast, insect and mammalian cells, and viruses (similar control sequences, i.e., promoters, are also found in prokaryotes). The selection of a specific promoter and enhancer depends on the recipient cell type. Some eukaryotic promoters and enhancers have a broad host range, while others are functional in a limited subset of cell types (for review, see, e.g., Voss et al., Trends Biochem. Sci., 11: 287 (1986); and Maniatis et al. (ibid.), incorporated herein by reference in their entirety). For example, the SV40 early gene enhancer is very active in a variety of cell types from many mammalian species and has been used to express proteins in a variety of mammalian cells (Dijkema et al., EMBO J. 4: 761 (1985), incorporated herein by reference in its entirety). Promoter and enhancer elements derived from the human elongation factor 1-alpha 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 Rous 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 are incorporated herein by reference in their entirety. Promoters and enhancers can occur alone or together in nature. For example, retroviral long terminal repeats contain promoter and enhancer elements.In general, the role of promoters and enhancers is independent of the gene being transcribed or translated. Therefore, the enhancers and promoters used can be "endogenous", "exogenous" or "heterologous" relative to the genes to which they are operably connected. An "endogenous" enhancer / promoter is an enhancer / promoter naturally connected to a given gene in a genome. An "exogenous" or "heterologous" enhancer or promoter is an enhancer or promoter juxtaposed with a gene by genetic manipulation (i.e., molecular biology techniques), wherein the juxtaposition directs the transcription of the gene by the connected enhancer / promoter. The presence of a "splicing signal" on an expression vector generally results in high-level expression of recombinant transcripts. In certain embodiments, a "splicing signal" mediates the removal of introns from the primary RNA transcript and is composed of splicing donor and acceptor sites (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). Commonly used splice donor and acceptor sites are the splice junctions of 16S RNA from SV40. In certain embodiments, a "transcription termination signal" is typically present downstream of the polyadenylation signal and is hundreds of nucleotides in length. For example, the term "poly A signal" or "poly A sequence" refers to a DNA sequence that directs the termination and polyadenylation of a nascent RNA transcript. Efficient polyadenylation of recombinant transcripts is often necessary because transcripts lacking a poly A signal are unstable and rapidly degraded. The poly A signal used in an expression vector can be "heterologous" or "endogenous." An endogenous poly A signal is a signal naturally present at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is a signal isolated from a gene and operably linked to the 3' end of another gene. A commonly used heterologous poly A signal is the SV40 poly A signal. The SV40 poly A signal is contained on the 237 bp BamHI / BclI restriction fragment and directs termination and polyadenylation (Sambrook et al., supra, 16.6-16.7, incorporated by reference in its entirety).

[0078] In this application, the term "DNA methyltransferase" generally refers to an enzyme that catalyzes the transfer of a methyl group to DNA. Non-limiting examples of DNA methyltransferases include DNMT1, DNMT 3A, DNMT 3B, and DNMT 3L. For example, through DNA methylation, a DNA methyltransferase can modify the activity of a DNA fragment (e.g., regulate gene expression) without changing the DNA sequence. As described herein, a gene expression regulatory molecule can include one or more (e.g., two) DNA methyltransferases. When a DNA methyltransferase is included as part of a gene expression regulatory molecule, the DNA methyltransferase can be referred to as a "DNA methyltransferase domain." In various aspects, the DNA methyltransferase domain comprises a variant or homolog of an amino acid 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%, 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 various aspects, the DNA methyltransferase domain comprises a variant or homolog of an amino acid 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 DNMT 3L.

[0079] As used herein, the term "functionally active fragment" generally refers to a fragment that has a partial region of a full-length protein or nucleic acid but retains or partially retains the biological activity or function of the full-length protein or nucleic acid. For example, a functionally active fragment may retain or partially retain the ability of the full-length protein to bind to another molecule. For example, a functionally active fragment of a DNA methyltransferase may retain or partially retain the biological activity of the full-length DNA methyltransferase in catalyzing the transfer of methyl groups to DNA.

[0080] In the present application, the term "directly and / or indirectly connected" generally refers to the relative "direct connection" or "indirect connection". The term "direct connection" generally refers to direct connection or direct binding. For example, the direct connection can be a situation where there is no spacer component (such as an amino acid residue or its derivative) between the connected substances (such as amino acid sequence segments) and they are directly connected; for example, an amino acid sequence segment X is directly connected 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. "Indirect connection" generally refers to a situation where there is a spacer component (such as an amino acid residue or its derivative) between the connected substances (such as amino acid sequence segments) and they are indirectly connected.

[0081] In this application, the term "Krab", also referred to as "Krupp-associated box domain" or "Krüppel-associated box domain", generally refers to about 45 to about 75 amino acid residues of the transcriptional repression domain present in the transcription factors of human zinc finger proteins. In various aspects, the Krab domain can include a variant or homolog of an amino acid 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 ZIM3 Krab domain or the KOX1 Krab domain.

[0082] In the present application, term " delivery vector " generally refers to the transfer vehicle that reagent (for example, nucleic acid molecule) can be delivered to target cell.Delivery vector can deliver reagent to specific cell subclass.For example, by the intrinsic characteristics of delivery vector or by the part coupled with carrier, the part contained therein (or the part combined with carrier, so that this part and this delivery vector are maintained together, and then make this part enough to target delivery vector) make delivery vector target certain type of cell.Delivery vector also can improve the half-life in vivo of the reagent to be sent and / or the bioavailability of the reagent to be sent.Delivery vector can comprise viral vector, virus-like particle, polycationic carrier, peptide carrier, liposome and / or hybrid carrier.For example, if target cell is hepatocyte, the character (for example, size, charge and / or pH) of described delivery vector can effectively be delivered to target cell, reduce immune clearance and / or promote to stay in this target cell by the molecule of described delivery vector and / or wherein encapsulated.

[0083] In this application, the term "liposome" generally refers to a vesicle with an internal space that is isolated from an external medium by one or more bilayer membranes. In some embodiments, the bilayer membrane can be formed by amphiphilic molecules, such as synthetic or naturally derived lipids comprising spatially isolated hydrophilic and hydrophobic domains; in other embodiments, the bilayer membrane can be formed by amphiphilic polymers and surfactants. In some embodiments, the liposome is a spherical vesicle structure consisting of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer. In some embodiments, liposomes are biocompatible, non-toxic, can deliver hydrophilic and lipophilic drug molecules, protect their cargo from being degraded by plasma enzymes, and transport their loads across biological membranes and the blood-brain barrier (BBB). Liposomes can be made of several different types of lipids, such as phospholipids. Liposomes can comprise natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, monosialoganglioside, or any combination thereof. In order to modify the structure and properties of the liposomes, several other additives can be added to the liposomes. For example, the liposomes can also comprise cholesterol, sphingomyelin, and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), for example, to increase stability and / or prevent leakage of cargo inside the liposomes.

[0084] The term "lipid nanoparticle (LNP)" generally refers to a particle comprising a plurality (i.e., more than one) lipid molecules that are physically bound to each other (e.g., covalently or non-covalently) by intermolecular forces. LNP can be, for example, a microsphere (including unilamellar and multilamellar vesicles, such as liposomes), a dispersed phase in an emulsion, a micelle, or an internal phase in a suspension. LNP can encapsulate nucleic acids in cationic lipid particles (e.g., liposomes) and can be delivered to cells relatively easily. In some instances, lipid nanoparticles do not contain any viral components, which helps to minimize safety and immunogenicity issues. The lipid particles can be used for in vitro, ex vivo, and in vivo delivery. The lipid particles can also be used for cell populations of various sizes. The LNP of the present application can be easily prepared by various methods known in the art, such as by mixing an organic phase with an aqueous phase. The mixing of the two phases can be achieved by a microfluidic device and an impinging stream reactor. The more fully the organic phase and the aqueous phase are mixed, the better the embedding efficiency and particle size distribution of the LNP obtained. 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 embedding efficiency can be optimized by adjusting the N / P (ionizable lipid / nucleic acid) ratio of the LNP system. In some instances, LNP can be used to deliver DNA molecules (e.g., molecules comprising the coding sequence of a DNA binding protein and / or sgRNA) and / or RNA molecules (e.g., mRNA of Cas, sgRNA). In some cases, LNP can be used to deliver the RNP complex of Cas / gRNA. In some embodiments, LNP is used to deliver mRNA and gRNA (e.g., mRNA fusion molecules comprising DNMT3A-DNMT3L (3A-3L)-dCas9-KRAB and at least one sgRNA targeting a target gene).

[0085] In the present application, term " recombinant vector " generally refers to the nucleic acid molecule that can transport it and another kind of nucleic acid connected thereto.One type of carrier is " plasmid ", and this refers to that other DNA segment can be connected to the circular double-stranded DNA loop in it.Alternately, carrier can be linear.Another type of carrier is viral vector, and wherein other DNA segment can be connected in viral genome.Specific carrier can be introduced into the host cell autonomous replication (for example, bacterial vector and additional mammalian vector with bacterial replication origin) in them.Other carriers (for example, non-additional mammalian vector) can be integrated into the genome of host cell after introducing in host cell, and thereby replicate together with host genome.

[0086] In this application, the term "adeno-associated virus (AAV) vector" generally refers to a vector with a functional or partially functional ITR sequence and a transgene. As used herein, the term "ITR" refers to an inverted terminal repeat sequence. The ITR sequence can be derived from an adeno-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13, and any AAV variant or mixture. However, ITR does not have to be a wild-type nucleotide sequence and can be changed (for example, by insertion, deletion or substitution of nucleotides) as long as the sequence retains the function of providing functional rescue, replication and packaging. The AAV vector may have one or more fully or partially deleted AAV wild-type genes, preferably rep and / or cap genes, but retains functional flanking ITR sequences. Functional ITR sequences play a role in, for example, rescuing, replicating and packaging AAV virus particles or particles. Thus, an "AAV vector" is defined herein as comprising at least those sequences required for insertion of a transgene into a subject's cells, and optionally comprising those cis sequences necessary for viral replication and packaging (e.g., functional ITRs).

[0087] In this application, 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 comprises five basic, positively charged amino acids. For example, an NLS can be located at any position along the peptide chain.

[0088] In this application, the terms "complementary" or "complementarity" generally refer to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional types. For example, the sequence AGT is complementary to the sequence TCA. The complementarity percentage indicates 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, 6, 7, 8, 9, 10 out of 10 are 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" refers to a degree of complementarity between two nucleic acids that hybridize under stringent conditions (i.e., stringent hybridization conditions) of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% 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 at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.

[0089] In this application, the term "gene" generally refers to a DNA fragment designed to produce a protein. For example, the gene can also include regions before and after the coding region (leader and tail) and insertion sequences (introns) between each coding segment (exon). Leaders, tails, and introns comprise essential regulatory elements in gene transcription and translation processes. In addition, a "protein gene product" can be a protein expressed by a specific gene.

[0090] In this application, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. For example, wherein the polymer can be bound to a part that is not composed of amino acids in various aspects. For example, a "fusion protein" can refer to a chimeric protein encoding two or more separate protein sequences that are expressed as a single part in a recombinant manner. In the case of two or more nucleic acids or peptide sequences, the term "identical" or percentage "consistency" refers to using BLAST or BLAST 2.0 sequence comparison algorithms or measured by manual comparison and visual inspection with the default parameters as described below. For example, two or more sequences or subsequences of identical or specified amino acid residues or nucleotides (when comparing and aligning for maximum correspondence on a comparison window or a specified region, there is about 60% consistency in a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher consistency) are referred to as "substantially identical".

[0091] In this application, the term "guide RNA" or "gRNA" generally refers to any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize with the target sequence and specifically bind the CRISPR complex to the target sequence. In various aspects, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between the guide sequence and its corresponding target sequence is about or greater than about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 99% or more.

[0092] In the present application, the specific protein (e.g., KRAB, dCas9, Dnmt3A, Dnmt3L) described herein may include any native form of the protein or a variant or homolog that maintains the activity of the protein (e.g., 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 native protein). In various aspects, the variant or homolog 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., 50, 100, 150 or 200 consecutive amino acid portions) compared to the naturally occurring form.

[0093] In this application, the term "linker" generally refers to a connexon that includes two or more parts. In various embodiments, the linker is connected to the amino acid sequence of the remainder of the compound at the N-terminus and the C-terminus (e.g., the fusion protein provided herein). For example, the term "XTEN," "XTEN linker," or "XTEN polypeptide" as used herein refers to a recombinant polypeptide lacking a hydrophobic amino acid residue.

[0094] As used herein, the term "detectable agent" or "detectable moiety" refers to a composition that can be detected by an appropriate means. For example, the means are, for example, 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., commonly used in ELISA), biotin, paramagnetic molecules, and the like.

[0095] In this application, the terms "inhibit", "repress", "silence" and the like generally refer to a reduction in gene expression and / or activity. For example, administration of the substance of the present application may negatively affect (e.g., reduce) the activity of a nucleic acid sequence relative to the activity of the nucleic acid sequence in the absence of a substance (e.g., a fusion protein, a complex, a nucleic acid, a vector) (control). For example, inhibition may refer to a reduction in a disease or a symptom of a disease. For example, inhibition includes at least partially, partially or completely blocking the activation (e.g., transcription) of a nucleic acid sequence, or reducing, preventing or delaying the activation of a nucleic acid sequence. For example, the inhibitory activity may be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of that in the control.

[0096] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0097] In this application, the term "selected from" generally refers to the selected objects and all combinations thereof. For example, "selected from (:) A, B and C" means all combinations of A, B and C, for example, A, B, C, A+B, A+C, B+C or A+B+C.

[0098] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 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% above or below the specified value.

[0099] Detailed Description of the Invention

[0100] In one aspect, the present application provides a method for regulating ANGPTL3 gene expression and / or activity, comprising providing a gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule. The gene expression regulatory molecule may function to regulate the expression of the ANGPTL3 gene without altering its gene sequence. For example, the method of the present application may be a non-therapeutic method. For example, the method of the present application may be a method not directly used on the human body. For example, the method of the present application may be an in vitro or ex vivo method. For example, the method of the present application may be a therapeutic method. For example, the method of the present application may be an in vivo method.

[0101] On the other hand, the present application provides a method for treating and / or alleviating a condition associated with abnormal ANGPTL3 gene expression and / or ANGPTL3 gene activity, the method comprising providing a gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule, wherein the gene expression regulatory molecule can have the function of regulating the expression of the ANGPTL3 gene without changing its gene sequence.

[0102] In another aspect, the present application provides a gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule, which can regulate the expression of the ANGPTL3 gene without altering the gene sequence. For example, the nucleic acid comprises DNA and / or mRNA. For example, the gene expression regulatory molecule or the nucleic acid encoding the gene expression regulatory molecule can be used to treat and / or alleviate conditions associated with abnormal ANGPTL3 gene expression and / or ANGPTL3 gene activity.

[0103] In another aspect, the present application provides a use of a gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule in the preparation of a medicament for treating and / or alleviating conditions associated with abnormal ANGPTL3 gene expression and / or ANGPTL3 gene activity. The gene expression regulatory molecule can regulate the expression of the ANGPTL3 gene without altering its gene sequence. For example, the nucleic acid encoding the gene expression regulatory molecule of the present application comprises DNA and / or mRNA.

[0104] In another aspect, the present application 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 shown in any one of SEQ ID NOs: 1 to 70. For example, the nucleic acid encoding the nucleic acid binding molecule of the present application comprises DNA and / or mRNA.

[0105] On the other hand, the present application provides a recombinant vector comprising the nucleic acid of the present application. For example, a recombinant vector may refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. Recombinant vectors may include single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, without free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA or both; and other types of polynucleotides known in the art. For example, viral vectors may be used. Viral vectors may comprise virally derived DNA or RNA sequences for packaging into viruses (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses AAV). Viruses and viral vectors may be used for in vitro, ex vivo and / or in vivo delivery.

[0106] On the other hand, the application provides a delivery vector, the delivery vector comprises the nucleic acid binding molecules of the application, the gene expression regulating molecules of the application, the nucleic acid of the application and / or the recombinant vector of the application, and optionally comprises liposomes and / or lipid nanoparticles (LNP). For example, the delivery vector can comprise one or more Cas proteins and one or more guide RNAs, for example, in the form of ribonucleoprotein complexes (RNPs). For example, ribonucleoproteins can be delivered by shuttle agents based on polypeptides. For example, ribonucleoproteins can be delivered using synthetic peptides. For example, the delivery vector can be introduced into cells by physical delivery methods. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery. For example, LNPs can be wrapped in cationic lipid particles (such as liposomes) for nucleic acids and can be delivered to cells relatively easily. In some examples, lipid nanoparticles do not contain any viral components, which helps to minimize safety and immunogenicity issues. Lipid particles can be used for in vitro, ex vivo, and in vivo delivery. The components of LNPs may include cationic lipids, ionizable lipids, pegylated lipids, and / or support lipids, and optional cholesterol components. In some embodiments, LNPs may comprise ionizable lipids (20%-70% by molar ratio), PEGylated lipids (0%-30% by molar ratio), supporting lipids (30%-50% by molar ratio), and cholesterol (10%-50% by molar ratio).

[0107] On the other hand, the application provides a kind of composition, described composition comprises the nucleic acid binding molecule of the application, the gene expression regulating molecule of the application, the nucleic acid of the application, the recombinant vector of the application and / or the delivery vector of the application.For example, the nucleic acid binding molecule in the composition, 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 vector can be contained in a composition simultaneously, or are respectively contained in different compositions.For example, when using the nucleic acid binding molecule in the composition, 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 vector, it can be used simultaneously, or used separately.

[0108] On the other hand, the present application provides a cell comprising the nucleic acid binding molecule of the present application, the gene expression regulating molecule of the present application, the nucleic acid of the present application, the recombinant vector of the present application, the delivery vector of the present application, and / or the composition of the present application.

[0109] On the other hand, the present application provides a kit comprising the nucleic acid binding molecule of the present application, the gene expression regulating molecule of the present application, the nucleic acid of the present application, the recombinant vector of the present application, the delivery vector of the present application, the composition of the present application, and / or the cell of the present application.

[0110] On the other hand, the application provides a method for regulating target gene expression and / or activity, the method comprising providing the nucleic acid binding molecules of the application, the gene expression regulating molecules of the application, the nucleic acid of the application, the recombinant vector of the application, the delivery vector of the application, the composition of the application, the cell of the application, and / or the kit of the application. For example, the method can reduce the expression and / or activity of the target gene. For example, compared to the expression and / or activity of the target gene in the absence of the application substance, applying the material of the application can have a negative impact (such as reduction) on the activity of the nucleic acid sequence, for example, it can include at least partially, partially or completely blocking the activation (such as transcription) of the nucleic acid sequence, or reducing, preventing or delaying the activation of the nucleic acid sequence. For example, the inhibitory activity can be about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% or less in the control.

[0111] On the other hand, the present application provides a method for preparing a drug for treating and / or alleviating a disease, wherein the disease includes a disease associated with abnormal expression and / or activity of a target gene.

[0112] On the other hand, the present application provides a nucleic acid binding molecule of the present application, a gene expression regulating molecule of the present application, a nucleic acid of the present application, a recombinant vector of the present application, a delivery vector of the present application, a composition of the present application, a cell of the present application, and / or a kit of the present application, which are used to treat and / or alleviate diseases, including diseases related to abnormal expression and / or activity of target genes.

[0113] On the other hand, the present application provides a method for treating and / or alleviating a disease, the method comprising providing a nucleic acid binding molecule of the present application, a gene expression regulating molecule of the present application, a nucleic acid of the present application, a recombinant vector of the present application, a delivery vector of the present application, a composition of the present application, a cell of the present application, and / or a kit of the present application, wherein the disease comprises a disease associated with abnormal expression and / or activity of a target gene. In some embodiments, the treatment is for a disease / disorder of an organ, illustratively including liver disease, eye disease, muscle disease, heart disease, blood disease, encephalopathy, kidney disease, or may include treatment of autoimmune disease, central nervous system disease, cancer and other proliferative diseases, neurodegenerative diseases, inflammatory diseases, metabolic diseases, musculoskeletal diseases, etc.

[0114] Gene expression regulators

[0115] The gene expression molecules provided in the present application or the methods of the present application have the function of regulating the expression of the ANGPTL3 gene without changing its gene sequence. For example, the gene expression regulating molecule can have the function of inhibiting gene expression.

[0116] In some embodiments, the gene expression regulating 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. In certain embodiments, the first functional domain can regulate target gene expression through epigenetic modification, such as DNA methylation, at a regulatory element of the target gene, such as a promoter, enhancer, or transcription start site. For example, a regulatory element can comprise 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 by any known epigenetic modification regulator that can be used for DNA methylation. Exemplary epigenetic modification regulators can include DNA methyltransferases (e.g., DNMT3A or DNMT3A-DNMT3L), DNA demethylases (e.g., TET1 catalytic domain or TDG), and / or functionally active fragments thereof.

[0117] For example, the first functional domain can provide for modification of at least one nucleotide adjacent to the ANGPTL3 gene and / or within an ANGPTL3 gene regulatory element, wherein the modification of the at least one nucleotide comprises a methylation modification. In some embodiments, the first functional domain comprises an epigenetic modification regulator, which can have DNA methylase activity. For example, the epigenetic modification regulator can have methylase activity, which involves the transfer of a methyl group to DNA, RNA, a protein, a 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.

[0118] For example, the first functional domain may comprise a DNA methyltransferase (DNMT) domain. For example, the first functional domain may comprise a DNMT 3A domain and / or a DNMT 3L domain. For example, the DNMT 3A domain and / or the DNMT 3L domain are derived from mammals. For example, the DNMT 3A domain is derived from mice. For example, the gene expression regulatory molecule may comprise the amino acid sequence described in DNMT 3A. For example, the DNMT 3L domain is derived from humans and / or mice. For example, the gene expression regulatory molecule may comprise the amino acid sequence of DNMT 3L. Alternatively, there may be one, two, three, four, five or more changes relative to the above sequence, such as amino acid substitutions, insertions or deletions, or any fragments thereof.

[0119] For example, the DNMT 3A domain and the DNMT 3L domain are directly and / or indirectly connected. For example, the DNMT 3A domain and the DNMT 3L domain are connected via a linker. For example, the C-terminus of the DNMT 3A domain is connected to the N-terminus of the DNMT 3L domain, or the C-terminus of the DNMT 3L domain is connected to the N-terminus of the DNMT 3A domain.

[0120] In some embodiments, the gene expression regulatory molecule comprises a second functional domain, which comprises a zinc finger protein-based transcription factor or a functionally active fragment thereof, or comprises a substance capable of modifying histones. For example, the second functional domain can comprise a gene expression repressor, which can be any known gene expression repressor, and exemplary gene expression repressors can be selected from Krüppel-associated box (KRAB) domain, mSin3 interaction domain (SID), MAX interacting protein 1 (MXI1), chromosome shadow domain, EAR-repressor domain (SRDX), eukaryotic release factor 1 (ERF1), eukaryotic release factor 3 (ERF3), tetracycline repressor, lad repressor, Catharanthus roseus G-box binding factor 1 and 2, Drosophila Groucho (fruit fly Gro protein), tripartite motif-containing 28 (TRTM28), nuclear receptor co-repressor 1, nuclear receptor co-repressor 2, or their functionally active fragments or fusions. For example, the second functional domain can comprise the function of a substance capable of modifying histones. In certain 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.

[0121] In certain embodiments, the KRAB domain or a fragment thereof can be fused to the C-terminus of the dCas9 molecule. In certain embodiments, the KRAB domain or a fragment thereof can be fused to the N-terminus and C-terminus of the dCas9 molecule. In certain embodiments, the second functional domain may comprise a KRAB domain, which may 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 higher identity), or has one, two, three, four, five or more changes (e.g., amino acid substitutions, insertions or deletions relative to ZIM3 Krab or KOX1 Krab), or any fragment thereof.

[0122] In some embodiments, the second functional domain may comprise a gene expression activator, which may be any known gene expression activator, and exemplary gene expression activators may comprise a VP16 activation domain, a VP64 activation domain, a p65 activation domain, an Epstein-Barr virus R transactivator Rta molecule or a fragment thereof.

[0123] In some embodiments, the second functional domain may comprise a substance capable of modifying histones, and the substance that modifies histones may comprise a histone acetyltransferase (e.g., 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.

[0124] In some of the above situations, the gene expression regulatory molecule comprises the first functional domain and the second functional domain, and the first functional domain is directly or indirectly connected to one end of the second functional domain, or the first functional domain is directly and / or indirectly connected to both ends of the second functional domain. For example, the C-terminus of the first functional domain is directly connected to the N-terminus of the second functional domain, or the C-terminus of the first functional domain is indirectly connected (for example, through a joint) to the N-terminus of the second functional domain, or the first functional domain is located on the N-terminal side of the second functional domain (for example, the order of the first functional domain, other parts such as DNA binding domains, and the second functional domain can be sequentially from the N-terminus to the C-terminus). For example, the C-terminus of the second functional domain is directly connected to the N-terminus of the first functional domain, or the C-terminus of the second functional domain is indirectly connected (for example, through a joint) to the N-terminus of the first functional domain, or the second functional domain is located on the N-terminal side of the first functional domain (for example, the order of the second functional domain, other parts such as DNA binding domains, and the first functional domain can be sequentially from the N-terminus to the C-terminus). For example, the first functional domain comprises two or more functional domains, and they can be directly connected to, indirectly connected to (for example, through a linker), or located on the N-terminal side and the C-terminal side of the second functional domain (for example, the order from N-terminus to C-terminus can be the first functional domain a, other parts such as DNA binding domains, the second functional domain, and the first functional domain b, or the order can be the first functional domain a, the second functional domain, other parts such as DNA binding domains, and the first functional domain b, where a and b are different types of first functional domains).

[0125] In some embodiments, the gene expression regulatory molecule comprises a DNA binding domain. For example, the gene expression regulatory molecule may have a function of binding to a gene sequence. For example, the DNA binding domain comprises a (DNA) nuclease, which may be a nuclease that targets DNA in a sequence-specific manner; illustratively, it may be a CRISPR-Cas system-related enzyme, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) or a large-range nuclease. For example, the gene expression regulatory molecule may comprise a DNA binding domain, a zinc finger domain, and / or a DNA binding domain of a CRISPR / Cas system of TALEN. In some cases, the DNA binding domain is a DNA nuclease derived from a CRISPR-Cas system. In some cases, the DNA binding domain is a (modified) transcription activator-like effector nuclease (TALEN) system; and 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; the ZFN system uses an artificial restriction enzyme generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain that can be engineered 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).

[0126] In some embodiments, the gene expression regulatory molecule may include a Cas enzyme. For example, the gene expression regulatory molecule may include a Cas enzyme that has substantially no nuclease activity. Typically, the guide sequence (or spacer sequence) can be any polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence and guide the sequence-specific binding of the CRISPR complex to the target sequence. In some cases, when an appropriate alignment algorithm is used for optimal alignment, the degree of complementarity between the guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the Cas protein targeting nucleic acid can be mutated relative to the corresponding wild-type enzyme so that the mutated Cas protein targeting nucleic acid lacks the ability to cut one or both chains of the target polynucleotide. For example, the DNA cleavage activity of the mutant enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01% or less of the nucleic acid cleavage activity of the non-mutated form of the enzyme. In some embodiments, the mutant Cas may have one or more mutations that result in reduced off-target effects.

[0127] For example, the gene expression regulating molecule can include a Cas9 enzyme. Examples of Cas proteins include Class I (e.g., Type I, Type III, and Type IV) and Class 2 (e.g., Type II, Type V, and Type VI) Cas proteins, such as Cas9, Cas12 (e.g., Cas12a, Cas12b, Cas12c, Cas12d), Cas13 (e.g., Cas13a, Cas13b, Cas13c, Cas13d), CasX, CasY, Cas14, their variants (e.g., mutant form, truncated form), their homologs, and their orthologs. In some embodiments, the Cas protein is Cas9, Cas12a, Cas12b, Cas12c, or Cas12d. In certain embodiments, Cas9 can be SpCas9, SaCas9, StCas9, and other Cas9 orthologs. Cas12 can be Cas12a, Cas12b, and Cas12c, including FnCas12a, or its homologs or orthologs. For example, the Cas9 enzyme may include Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheria 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, Parvibaculum lavamentivorans dCas9, Nitratifractor salsuginis (e.g., DSM 16511) dCas9, Campylobacter lari (e.g., CF89-12) dCas9, and / or Streptococcus thermophilus (e.g., strain LMD-9) dCas9. For example, the gene expression regulating molecule can comprise the amino acid sequence of dCas9. Alternatively, the dCas9 sequence can have one, two, three, four, five or more changes, such as amino acid substitutions, insertions or deletions, or any fragment thereof, relative to the above dCas9 sequence.

[0128] In some embodiments, the first functional domain and the second functional domain are directly or indirectly connected to one end of the DNA binding domain. For example, the first functional domain and the second functional domain are directly or indirectly connected to the C-terminus of the DNA binding domain. Exemplarily, the gene regulatory molecule may include any one of dCas9-DNMT 3A-DNMT 3L-Krab, dCas9-DNMT 3L-DNMT 3A-Krab, dCas9-Krab-DNMT 3A-DNMT 3L, and dCas9-Krab-DNMT 3L-DNMT 3A. For example, the first functional domain and the second functional domain are directly or indirectly connected to the N-terminus of the DNA binding domain. For example, the gene regulatory molecule may include any one of DNMT 3A-DNMT 3L-Krab-dCas9, DNMT 3L-DNMT 3A-Krab-dCas9, Krab-DNMT 3A-DNMT 3L-dCas9, and Krab-DNMT 3L-DNMT 3A-dCas9. In other embodiments, the first functional domain and the second functional domain are directly and / or indirectly connected to both ends of the DNA binding domain. For example, the first functional domain is directly or indirectly connected to the C-terminus of the DNA binding domain, and the second functional domain is directly or indirectly connected to the N-terminus of the DNA binding domain. For example, the gene regulatory molecule may include any one of Krab-dCas9-DNMT 3A-DNMT 3L and Krab-dCas9-DNMT 3L DNMT 3A. For example, the first functional domain is directly or indirectly connected to the N-terminus of the DNA binding domain, and the second functional domain is directly or indirectly connected to the C-terminus of the DNA binding domain. Exemplarily, the gene regulatory molecule may include any one of DNMT 3A-DNMT 3L-dCas9-Krab and DNMT 3L-DNMT 3A-dCas9-Krab.

[0129] In some embodiments, the gene expression regulatory molecule is capable of binding to a DNA region or fragment thereof within 500 bp upstream and / or downstream of the transcription start site (TSS) of the ANGPTL3 gene. For example, the gene expression regulatory molecule is capable of binding to a DNA region or fragment thereof 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 to 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 the DNA region or fragment thereof located in any one of SEQ ID NOs: 71-72.

[0130] Exemplarily, in some cases, the gene expression regulatory molecule is capable of binding to one or more DNA regions near the following transcription start site (TSS) of the ANGPTL3 gene: between 180 bp upstream and 10 bp upstream of the TSS, between the TSS and 90 bp downstream of the TSS, and between 180 bp downstream and 240 bp downstream of the TSS.

[0131] In some embodiments, the gene expression regulating molecule can bind to one or more DNA regions near the following transcription start site (TSS) of the ANGPTL3 gene: between 180 bp and 150 bp upstream of the TSS (e.g., between 177-158 bp upstream of the TSS), between 120 bp and 90 bp upstream of the TSS (e.g., between 112-93 bp upstream of the TSS), between 70 bp and 10 bp upstream of the TSS (e.g., between 67-48 bp, 65-46 bp, 61-47 bp, etc. upstream of the TSS), or between 120 bp and 90 bp upstream of the TSS (e.g., between 112-93 bp upstream of the TSS). 2bp, between 57-38bp, and / or between 32-13bp), between TSS to 30bp downstream of TSS (e.g., between 2-21bp downstream of TSS), between 60bp downstream of TSS to 90bp downstream of TSS (e.g., between 68-87bp downstream of TSS), and between 180bp downstream of TSS to 240bp downstream of TSS (e.g., between 185-204bp, between 186-205bp, between 197-216bp, between 200-219bp, and / or between 221-240bp downstream of TSS).

[0132] In some embodiments, the gene expression regulatory molecule may further comprise a tag for detection, isolation, and / or purification. For example, the gene expression regulatory molecule may comprise an HA tag. For example, the gene expression regulatory molecule may comprise the amino acid sequence of an HA tag. Alternatively, the gene expression regulatory molecule may comprise a sequence having one, two, three, four, five, or more changes relative to the above sequence, such as amino acid substitutions, insertions, or deletions, or any fragment thereof.

[0133] In some embodiments, the gene expression regulating molecule may further comprise a nuclear localization sequence. For example, the nuclear localization sequence may comprise an amino acid having a positively charged group. For example, the nuclear localization sequence may comprise an amino acid sequence of a nuclear localization sequence known in the art. Alternatively, the sequence may have one, two, three, four, five or more changes relative to the above sequence, such as amino acid substitutions, insertions or deletions, or any fragment thereof. 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.

[0134] In some embodiments, the gene expression regulating molecule may further comprise a detectable portion. For example, the detectable portion may comprise blue fluorescent protein and / or green fluorescent protein. For example, the detectable portion may be 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.

[0135] In this application, the first functional domain, the second functional domain, the DNA binding domain, and other elements contained in the gene regulatory molecule can be connected indirectly, for example, by a linker sequence of a certain length. For example, the linker can comprise 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 by a linker of about 80 or more amino acids in length. For example, the first functional domain and the second functional domain are connected by a linker of about 92 or more amino acids in length. For example, the first functional domain and the second functional domain are connected by 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. In some embodiments, the present invention provides the amino acid sequence of the XTEN linker of claim 1, or ...2, or the amino acid sequence of the XTEN linker of claim 3, or the amino acid sequence of the XTEN linker of claim 4, or the amino acid sequence of the XTEN linker of claim 5. For example, the joint comprises an XTEN linker of 16 amino acid lengths, an XTEN linker of 80 amino acid lengths, or a longer XTEN linker. Perhaps, relative to the above sequence, there is one, two, three, four, five or more variations, for example amino acid replacements, insertions or deletions, or any fragment thereof.

[0136] Nucleic acid binding molecules

[0137] The present application also provides a nucleic acid binding molecule, which may comprise a sequence of any one of SEQ ID NOs: 1-70. For example, the nucleic acid binding molecule and / or the gene expression regulating molecule of the present application may be delivered to a subject by local injection, systemic infusion, or a combination thereof. In various aspects, the nucleic acid binding molecule 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 a sequence of any one of SEQ ID NOs: 1-70.

[0138] In some embodiments, the combination of the nucleic acid binding molecule and the gene expression regulating molecule of the present application can have the ability to regulate the expression level of the ANGPTL3 gene. For example, the nucleic acid binding molecule and / or the gene expression regulating molecule of the present application can 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 the gene expression regulating molecule of the present application can bind to a DNA region or fragment thereof within 500 bp upstream and / or downstream of the transcription start site of the ANGPTL3 gene. For example, the nucleic acid binding molecule and / or the gene expression regulating molecule of the present application can bind to the DNA region or fragment thereof located in any one of SEQ ID NOs: 71-72. In various aspects, the DNA region that can bind can comprise a sequence that 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 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 where any one of SEQ ID NOs: 71-72 is located.

[0139] For example, the nucleic acid binding molecules and / or gene expression regulating molecules of the present application can bind to the DNA region or fragment thereof located in any of SEQ ID NOs: 73-142. In various aspects, the DNA region that can bind can comprise a sequence that 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 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 located in any of SEQ ID NOs: 73-142. For example, targeting any region of approximately 20 bp in the DNA region located in any of SEQ ID NOs: 73-142 can modulate the expression level of the ANGPTL3 gene. For example, compared to the target gene expression and / or activity in the absence of the present application substance, the application of the substance can have a negative impact on the activity of the nucleic acid sequence (e.g., reduce), for example, including at least partially, partially or completely blocking the activation (e.g., transcription) of the nucleic acid sequence, or reducing, preventing or delaying the activation of the nucleic acid sequence. For example, the inhibitory activity can be about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% or less in the control.

[0140] In certain embodiments, the guide sequence or spacer length of the nucleic acid binding molecule can be 15 to 50 nucleotides. In certain embodiments, the nucleic acid binding molecule is a guide RNA (gRNA), and the spacer length of the guide RNA can be at least 15 nucleotides. In certain embodiments, the spacer length can be 15 to 17 nucleotides in length, 17 to 20 nucleotides in length, 20 to 24 nucleotides in length, 23 to 25 nucleotides in length, 24 to 27 nucleotides in length, 27 to 30 nucleotides in length, 30 to 35 nucleotides in length, or greater than 35 nucleotides in length. In some embodiments, the number of gRNAs administered can be at least 1 gRNA, at least 2 different gRNAs, at least 3 different gRNAs, at least 4 different gRNAs, at least 5 different gRNAs. In certain 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 can be complementary to the target region in the target gene, for example, completely complementary. In one embodiment, the target binding region can be substantially complementary to the target region in the target gene. In one embodiment, the target binding region can include no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides that are not complementary to the target region in the target gene. In some embodiments, the nucleic acid binding molecules and / or the gene expression regulating molecules of the present application can be formulated in liposomes or lipid nanoparticles. In some embodiments, the nucleic acid binding molecules and / or the gene expression regulating molecules of the present application can be formulated in viral vectors. For example, guide RNA can include an RNA-based molecule with one or more chemical modifications (for example, by chemically connecting two ribonucleotides or by replacing one or more ribonucleotides with one or more deoxyribonucleotides). For example, it is possible to target the target binding region derived from people.

[0141] Without intending to be bound by any theory, the following examples are merely intended to illustrate the products, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

[0142] Example

[0143] Example 1

[0144] The gene regulatory molecules of the present application can inhibit the mRNA expression of the human ANGPTL3 gene

[0145] (1) Experimental method: Corresponding sgRNAs were designed for the 500 bp sequence before and after the transcription start site of the human ANGPTL3 gene (a total of 1000 bp), and different epi-editing tools were directed to the corresponding locations in the genome. The mRNAs of different epi-editing tools (EpiRegA: Dnmt3a-Dnmt3l-dCas9-KOX1 KRAB, SEQ ID NO: 143, and EpiRegB: Dnmt3a-Dnmt3l-ZIM3 KRAB-dCas9, SEQ ID NO: 145) and the corresponding sgRNAs were prepared by LNP embedding at a mass ratio of 1:1 to obtain LNP test samples containing the corresponding editing tools combined with different sgRNAs (LNP preparation reference: https: / / doi.org / 10.1038 / s41586-021-03534-y). The test cells used were the human liver cancer cell line Huh7. 50,000 cells were seeded into a 24-well plate at a rate of 50,000 cells / well. 12 hours later, the LNP sample was added to the plate at a dose of 5 ug / mL. 4-6 hours later, the medium was replaced with fresh DMEM+10% FBS, and the cells were cultured. 3 days later, the cells were harvested for mRNA extraction and reverse transcribed into cDNA. qPCR was used to detect the mRNA expression level of the target gene ANGPTL3. The knockdown efficiency corresponding to each sgRNA was obtained by comparison with the control group with an empty package (no epigenetic editing tool mRNA and sgRNA). From the mRNA knockdown results shown in Figure 1, it can be seen that the sgRNAs represented by SEQ ID NOs: 28, 29, 39, 41, 42, 50, 54, 55, 57, 59, 60, 63, 67 and 69 (their targeting regions 5' end 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) (Table 1) combined with the epigenetic editing tool of this example can effectively inhibit the mRNA expression level of the ANGPTL3 gene.

[0146] Table 1 sgRNA targeting human ANGPTL3 gene

[0147] Example 2

[0148] Guide RNAs for epigenetic editing tools are constructed by referring to the nucleotide sequences set forth in any one of SEQ ID NOs: 1-70 to target the target gene. Simultaneously, epigenetic editing tools are constructed based on the gene expression regulatory molecules provided herein. For example, the epigenetic editing tool can be targeted within 500 bp upstream and downstream of the ANGPTL3 transcription start site (TSS).

[0149] Guide RNA plasmids were co-transfected with gene expression regulator plasmids into mouse cell lines. After 72 hours, the top 10% of GFP+ and mCherry+ cells were sorted by FACS. RT-qPCR was performed to assess target gene mRNA expression levels.

[0150] The results show that the transfected cells can show reduced expression of ANGPTL3 expression products. For example, the transfected cells can show reduced expression of ANGPTL3 mRNA. For example, the transfected cells can show reduced expression of ANGPTL3 protein products. For example, using the editing methods of the present application, diseases or conditions associated with the ANGPTL3 gene can be alleviated.

[0151] Example 3

[0152] The versatility of epigenetic targets

[0153] The epigenetic editing tool of the present application is used for target expression regulation. The domain in the gene expression regulatory molecule that has the function of binding to the gene sequence can be replaced by the DNA binding domain of TALEN, zinc finger domain, DNA binding domain of tetR, large-range nuclease and / or any nuclease domain of the CRISPR / Cas system known in the art, such as dCas12 enzyme, etc.

[0154] The results show that the epigenetic targets of the present application are universal, and the use of various gene sequence binding domains has a high effect of regulating gene expression.

[0155] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to be within the scope of the appended claims and their equivalents.

Claims

1. A method for regulating the expression and / or activity of an ANGPTL3 gene, the method comprising providing a gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule, wherein the gene expression regulatory molecule has the function of regulating the expression of the ANGPTL3 gene without changing its gene sequence.

2. A method for treating and / or alleviating a condition associated with abnormal ANGPTL3 gene expression and / or ANGPTL3 gene activity, the method comprising providing a gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule, wherein the gene expression regulatory molecule has the function of regulating the expression of the ANGPTL3 gene without changing its gene sequence.

3. The method according to any one of claims 1-2, wherein 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 the regulatory element of the ANGPTL3 gene. The method according to claim 3 , wherein the modification of at least one nucleotide comprises a methylation modification.

5. The method according to any one of claims 3 to 4, wherein the regulatory element comprises a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element and / or a locus control region.

6. The method according to any one of claims 3 to 5, wherein the first functional domain comprises one or more of DNA methyltransferase, DNA demethylase, and functionally active fragments thereof.

7. The method according to claim 6, wherein the DNA methyltransferase comprises one or more of DNMT 3A, DNMT 3B, DNMT3L, DNMT 1 and DNMT 2. The method according to claim 7 , wherein the DNMT 3A is derived from mouse.

9. The method according to any one of claims 7-8, wherein the DNMT 3L is derived from human and / or mouse.

10. The method according to any one of claims 7 to 9, wherein the DNMT 3A and the DNMT 3L are directly and / or indirectly linked.

11. The method according to any one of claims 1 to 10, wherein the gene expression regulatory molecule comprises a second functional domain, and the second functional domain comprises a zinc finger protein-based transcription factor or a functionally active fragment thereof, or comprises a substance capable of modifying histones.

12. The method of claim 11, wherein the second functional domain comprises Krab.

13. The method according to any one of claims 11-12, wherein the second functional domain comprises ZIM3 Krab or KOX1 Krab. 14 . The method according to claim 11 , wherein 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.

15. The method according to any one of claims 11 to 14, wherein the gene expression regulatory molecule comprises the first functional domain and the second functional domain, and the first functional domain is directly or indirectly connected to one end of the second functional domain, or the first functional domain is directly and / or indirectly connected to both ends of the second functional domain.

16. The method according to any one of claims 1 to 15, wherein the gene expression regulatory molecule comprises a DNA binding domain.

17. The method according to any one of claims 1 to 16, wherein the gene expression regulatory molecule comprises one or more DNA binding domains selected from the group consisting of a TALEN domain, a zinc finger domain, and a protein domain of a CRISPR / Cas system.

18. The method according to any one of claims 1-17, wherein the gene expression regulatory molecule comprises a Cas enzyme.

19. The method according to any one of claims 1-18, wherein the gene expression regulatory molecule comprises a Cas enzyme that has substantially no nuclease activity.

20. The method according to any one of claims 1-19, wherein the gene expression regulating molecule comprises a dCas9 enzyme.

21. The method according to any one of claims 16 to 20, wherein the first functional domain and the second functional domain are directly or indirectly connected to one end of the DNA-binding domain, or the first functional domain and the second functional domain are directly and / or indirectly connected to both ends of the DNA-binding domain.

22. The method according to any one of claims 1 to 21, wherein the gene expression regulatory molecule is capable of binding 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.

23. The method according to any one of claims 1 to 22, wherein the gene expression regulatory molecule is capable of binding to the DNA region or fragment thereof where any one of SEQ ID NOs: 71 to 72 is located.

24. The method according to any one of claims 1 to 23, wherein the gene expression regulating molecule is capable of binding to one or more DNA regions near the following transcription start site (TSS) of the ANGPTL3 gene: between 180 bp upstream and 10 bp upstream of the TSS, between the TSS and 90 bp downstream of the TSS, and between 180 bp downstream and 240 bp downstream of the TSS.

25. The method according to any one of claims 1 to 24, wherein the gene expression regulating molecule is capable of binding to one or more of the following DNA regions near the transcription start site (TSS) of the ANGPTL3 gene: between 180 bp and 150 bp upstream of the TSS, between 120 bp and 90 bp upstream of the TSS, between 70 bp and 10 bp upstream of the TSS, between the TSS and 30 bp downstream of the TSS, between 60 bp and 90 bp downstream of the TSS, and between 180 bp and 240 bp downstream of the TSS.

26. The method of any one of claims 1-25, comprising providing a nucleic acid binding molecule comprising the sequence of any one of SEQ ID NOs: 1-70.

27. The method according to claim 26, wherein the gene expression regulatory molecule and / or the nucleic acid binding molecule are formulated in the same or different delivery vehicles.

28. The method of claim 27, wherein the delivery vehicle comprises a liposome and / or a lipid nanoparticle.

29. The method according to any one of claims 26 to 28, wherein the expression regulating molecule and / or the nucleic acid binding molecule are formulated in the same or different recombinant vectors.

30. The method of claim 29, wherein the recombinant vector comprises a viral vector.

31. The method according to any one of claims 29-30, wherein the recombinant vector comprises an adeno-associated virus vector.

32. The method of any one of claims 1-31, wherein the gene expression regulatory molecule comprises a nuclear localization sequence.

33. The method of claim 32, wherein the nuclear localization sequence comprises an amino acid having a positively charged group.

34. The method according to any one of claims 32-33, wherein 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.

35. A nucleic acid binding molecule comprising the sequence of any one of SEQ ID NOs: 1-70.

36. A gene expression regulatory molecule, which has the function of regulating the expression of the ANGPTL3 gene without changing its gene sequence.

37. The gene expression regulatory molecule according to claim 36, which is the gene expression regulatory molecule provided in the method according to any one of claims 1 to 34.

38. The gene expression regulatory molecule according to any one of claims 36-37, wherein the gene expression regulatory molecule and / or nucleic acid binding molecule are formulated in the same or different delivery vectors, and the nucleic acid binding molecule comprises the sequence of any one of SEQ ID NOs: 1-70. The gene expression regulating molecule according to claim 38 , wherein the delivery vehicle comprises a liposome and / or a lipid nanoparticle.

40. The gene expression regulatory molecule according to any one of claims 36-39, wherein the expression regulatory molecule and / or the nucleic acid binding molecule are formulated in the same or different recombinant vectors, and the nucleic acid binding molecule comprises the sequence of any one of SEQ ID NOs: 1-70. The gene expression regulatory molecule according to claim 40 , wherein the recombinant vector comprises a viral vector.

42. The gene expression regulatory molecule according to any one of claims 40-41, wherein the recombinant vector comprises an adeno-associated virus vector.

43. A nucleic acid encoding the nucleic acid binding molecule of claim 35 and / or encoding the gene expression regulating molecule of any one of claims 36-42. A recombinant vector comprising the nucleic acid of claim 43 .

45. A delivery vector comprising the nucleic acid binding molecule of claim 35, the gene expression regulating molecule of any one of claims 36-42, the nucleic acid of claim 43, and / or the recombinant vector of claim 44, and optionally comprising liposomes and / or lipid nanoparticles.

46. ​​A composition comprising the nucleic acid binding molecule of claim 35, the gene expression regulating molecule of any one of claims 36-42, the nucleic acid of claim 43, the recombinant vector of claim 44, and / or the delivery vector of claim 45.

47. A cell comprising the nucleic acid binding molecule of claim 35, the gene expression regulating molecule of any one of claims 36-42, the nucleic acid of claim 43, the recombinant vector of claim 44, the delivery vector of claim 45, and / or the composition of claim 46.

48. A kit comprising the nucleic acid binding molecule of claim 35, the gene expression regulating molecule of any one of claims 36-42, the nucleic acid of claim 43, the recombinant vector of claim 44, the delivery vector of claim 45, the composition of claim 46, and / or the cell of claim 47.

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