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WO2024032678A1 - 一种表观编辑靶点的方法及用途 - Google Patents

一种表观编辑靶点的方法及用途 Download PDF

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Publication number
WO2024032678A1
WO2024032678A1 PCT/CN2023/112094 CN2023112094W WO2024032678A1 WO 2024032678 A1 WO2024032678 A1 WO 2024032678A1 CN 2023112094 W CN2023112094 W CN 2023112094W WO 2024032678 A1 WO2024032678 A1 WO 2024032678A1
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Prior art keywords
molecule
gene expression
nucleic acid
gene
sequence
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PCT/CN2023/112094
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English (en)
French (fr)
Inventor
张宝弘
罗浩
韦翔
王鹏程
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益杰立科(上海)生物科技有限公司
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Publication of WO2024032678A1 publication Critical patent/WO2024032678A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • This application relates to the field of biomedicine, specifically to an epigenetic editing target and its use.
  • Targeting this target can be used to improve the effect and safety of epigenetic editing and reduce toxicity, immunogenicity and/or off-target effects.
  • the present application provides an epigenetic editing target.
  • Targeting the target can be used to improve the epigenetic editing effect and safety, and reduce toxicity, immunogenicity and/or off-target effects.
  • targeting near the target gene and/or within the target gene regulatory element can effectively modify at least one nucleotide, thereby regulating (eg, reducing or eliminating) the expression of the target gene product in the cell.
  • the present application provides a method for regulating F11 (Coagulation Factor XI) gene expression and/or activity.
  • the method includes providing a gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule.
  • the gene The expression regulatory molecule has the function of regulating the expression of the F11 gene without changing its gene sequence.
  • the present application provides a method for treating and/or alleviating conditions associated with abnormal F11 gene expression and/or F11 gene activity, the method comprising providing a gene expression regulatory molecule or encoding the gene expression regulator
  • the nucleic acid of the molecule, the gene expression regulating molecule has the function of regulating the expression of the F11 gene without changing its gene sequence.
  • the gene expression modulating molecule comprises a DNA binding domain.
  • the gene expression regulatory molecule comprises one or more DNA binding domains selected from the group consisting of TALEN domains, zinc finger domains, and protein domains of the CRISPR/Cas system.
  • the gene expression regulatory molecule comprises a Cas enzyme.
  • the gene expression modulating molecule comprises a Cas enzyme that has substantially no nuclease activity.
  • the gene expression modulating molecule comprises a dCas9 enzyme.
  • the gene expression regulatory molecule is capable of binding to a DNA region or a fragment thereof within 500 bp upstream and/or downstream of the transcription start site (TSS) of the F11 gene.
  • TSS transcription start site
  • the gene expression regulatory molecule is capable of binding to the DNA region located in any one of SEQ ID NOs: 71-72 or a fragment thereof.
  • the gene expression regulatory molecule can bind to one or more DNA regions near the transcription start site (TSS) of the F11 gene: between 250 bp upstream of the TSS and 130 bp upstream of the TSS. Between 50bp upstream and 120bp downstream, and between 230bp downstream and 390bp downstream of TSS.
  • TSS transcription start site
  • the gene expression regulatory molecule can bind to one or more DNA regions near the transcription start site (TSS) of the F11 gene: between 250 bp upstream of the TSS and 230 bp upstream of the TSS. Between 210bp upstream and 180bp upstream, between 160bp upstream and 130bp upstream of TSS, between 50bp upstream and 10bp downstream of TSS, between 30bp downstream and 60bp downstream of TSS, between 90bp and 120bp downstream of TSS, and Between 230bp downstream and 260bp downstream, between 300bp downstream and 320bp downstream of TSS, and between 360bp downstream and 390bp downstream of TSS.
  • TSS transcription start site
  • the methods include providing a nucleic acid binding molecule comprising the sequence of any one of SEQ ID NOs: 1-70.
  • the gene expression modulating molecule and/or the nucleic acid binding molecule are formulated in the same or different delivery vehicles.
  • the delivery vehicle comprises liposomes and/or lipid nanoparticles.
  • the gene expression modulating molecule and/or the nucleic acid binding molecule are formulated in the same or different recombinant vectors.
  • the recombinant vector comprises a viral vector.
  • the recombinant vector comprises an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • the gene expression modulating molecule comprises a first functional domain that provides modification of at least one nucleotide near the F11 gene and/or within the F11 gene regulatory element.
  • the modification of at least one nucleotide comprises a methylation modification.
  • the regulatory elements comprise a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a border element, and/or a locus control region.
  • the first functional domain includes a DNA methyltransferase, a DNA demethylase, and a functional One or more active fragments.
  • the DNA methyltransferase comprises one or more of DNMT 3A, DNMT 3B, DNMT 3L, DNMT1 and DNMT 2.
  • the DNMT 3A is derived from mice.
  • the DNMT 3L is derived from human and/or mouse.
  • the DNMT 3A and the DNMT 3L are directly and/or indirectly connected.
  • the gene expression modulating molecule comprises a second functional domain comprising a zinc finger protein-based transcription factor or a functionally active fragment thereof, or a substance capable of modifying histones.
  • the second functional domain includes Krab.
  • the second functional domain comprises ZIM3 Krab or KOX1 Krab.
  • the second functional domain comprises one of a histone methyltransferase, a histone demethylase, a histone acetyltransferase, a histone deacetylase, and functionally active fragments thereof, or Various.
  • the first functional domain and the second functional domain are directly or indirectly linked to one end of the DNA binding domain.
  • the gene expression modulating molecule comprises a nuclear localization sequence.
  • the nuclear localization sequence comprises an amino acid having an electropositive group.
  • 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 DNA binding domain N-terminus and/or C-terminus.
  • the present application provides a nucleic acid binding molecule comprising the sequence of any one of SEQ ID NOs: 1-70.
  • the present application provides a gene expression regulatory molecule that has the function of regulating the expression of the F11 gene without changing its gene sequence.
  • the gene expression modulating molecule is as provided in the methods of the present application.
  • the gene expression modulating molecules and/or nucleic acid binding molecules comprising the sequence of any one of SEQ ID NOs: 1-70 are formulated in the same or different delivery vectors.
  • the delivery vehicle comprises liposomes and/or lipid nanoparticles.
  • the expression modulating molecule and/or the nucleic acid binding molecule comprising the sequence of any one of SEQ ID NOs: 1-70 is formulated in the same or different recombinant vectors.
  • the recombinant vector comprises a viral vector.
  • the recombinant vector comprises an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • 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.
  • the present application provides a recombinant vector comprising the nucleic acid of the present application.
  • the present application provides a delivery vector, the delivery vector comprising the nucleic acid binding molecule of the present application, the gene expression regulatory molecule of the present application, the nucleic acid of the present application, and/or the recombinant vector of the present application, and optionally containing liposomes and/or lipid nanoparticles.
  • 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 present application. Delivery vehicle.
  • 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 compositions of the present application.
  • the present application provides a kit, which includes 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.
  • the present application provides a method for regulating the expression and/or activity of a target gene, which method includes 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, and the recombinant of the present application.
  • 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, and a nucleic acid binding molecule of the present application.
  • the application of the cells and/or the kit of the present application in the preparation of medicaments for treating and/or alleviating disorders, including disorders associated with abnormal expression and/or activity of target genes.
  • Figure 1A shows a schematic diagram of the reporter plasmid constructed from the F11 gene fragment and fluorescent protein.
  • Figure 1B shows the flow cytometry results of the proportion of cells with low green fluorescence intensity in the transfected cell population.
  • Figure 1C shows the expression regulatory effect of the gene expression regulatory molecule of the present application in targeting different F11 gene regulatory regions.
  • nucleic acid is used interchangeably with “polynucleotide”, “nucleotide”, “nucleotide sequence” and “oligonucleotide”, which generally refers to nucleotides (e.g. , deoxyribonucleotides or ribonucleotides) and their polymers in single-stranded, double-stranded or multi-stranded form or their complements.
  • nucleotides may be ribonucleotides, deoxyribonucleotides, or modified versions thereof.
  • nucleotides can be single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules with mixtures of single- and double-stranded DNA and RNA.
  • nucleotides may include, but are not limited to, any type of RNA, such as mRNA, siRNA, miRNA, sgRNA, and guide RNA, as well as any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof.
  • the term also encompasses nucleic acids, whether synthetic, naturally occurring and non-naturally occurring, containing known nucleotide analogs or modified backbone residues or linkages.
  • 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 initiation and termination signals operably linked to regulatory elements comprising a promoter and a polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. .
  • the coding sequence can be codon optimized.
  • treatment for example, when applied to a disease, means when administering, for example, a gene expression modulating molecule as described herein or a nucleic acid encoding such a gene expression modulating molecule and/or a nucleic acid binding molecule as described herein (for example, gRNA) or a nucleic acid encoding the nucleic acid binding molecule, compared with when the gene expression regulating molecule or the nucleic acid encoding the gene expression regulating molecule and/or the nucleic acid binding molecule or the nucleic acid encoding the nucleic acid binding molecule has never been administered,
  • a subject eg, a human who has the disease, is at risk for the disease, and/or experiences symptoms of the disease will, in one embodiment, experience milder symptoms and/or will recover more quickly.
  • DNA-binding domain generally refers to an independently folded protein domain containing at least one motif that recognizes double-stranded or single-stranded DNA.
  • the DNA binding domain may recognize a specific DNA sequence (recognize or modulate sequence) or have general affinity for DNA.
  • other domains of the DNA-binding domain often modulate the activity of the DNA-binding domain; the DNA-binding function can be structural or include transcriptional regulation, and sometimes the two roles overlap.
  • the DNA binding domain may comprise a (DNA) nuclease, such as one capable of targeting DNA in a sequence-specific manner or capable of being directed or instructed to act in a sequence-specific manner.
  • Nucleases that target DNA in a sexual manner such as the CRISPR-Cas system, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or meganucleases.
  • the DNA binding domain is a DNA nuclease derived from the CRISPR-Cas system.
  • the DNA nuclease derived from the CRISPR-Cas system is a Cas protein.
  • Cas enzyme may be used with “Cas protein”, “CRISPR protein”, “CRISPR enzyme”, “CRISPR-Cas protein”, “CRISPR-Cas enzyme”, “Cas”, “CRISPR effector” or “Cas effector proteins” are used interchangeably and generally refer to a class of enzymes that are complementary to CRISPR sequences and are able to use the CRISPR sequences as guides to recognize and cut specific DNA strands.
  • Cas proteins include: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Cs f1 , Csf2, Csf3, Csf4, and/or their homologues, or modified forms thereof.
  • These proteins are known, for example, the amino acid sequence of the Streptococcus pyogen
  • dCas9 enzyme is also referred to as “inactivated Cas9 protein” or “inactivated Cas9 enzyme”.
  • inactivated Cas9 protein or “inactivated Cas9 enzyme”.
  • known methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are described, for example, Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-GuidedPlatform” for Sequence-Specific Control of Gene Expression," Cell. 28, 152(5):1173-83 (2013), the entire contents of which are incorporated herein by reference).
  • the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain.
  • the HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations in these subdomains silence the nuclease activity of Cas9.
  • 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 inactivating or nicking DNA binding domains include, but are not limited to, nuclease inactive variant Cas9 domains including D10A, D10A/D839A/H840A and D10A/D839A/H840A/N863A mutant domains, such as WO2015089406A1 , which is incorporated herein by reference.
  • endonuclease-inactive dCas9 from Streptococcus pyogenes has been targeted by gRNA to genes in bacteria, yeast, and human cells to silence gene expression through steric hindrance.
  • dCas may refer to a dCas protein or fragment thereof.
  • dCas9 may refer to a dCas9 protein or fragment thereof.
  • the term “iCas” Used interchangeably with “dCas” to refer to CRISPR-related proteins that have no catalytic activity.
  • the dCas protein contains one or more mutations in the DNA cleavage domain.
  • the dCas protein contains one or more mutations in the RuvC or domain.
  • the dCas molecule contains one or more mutations in both the RuvC and HNH domains.
  • the dCas protein is a fragment of wild-type Cas protein.
  • the dCas protein comprises a functional domain from a wild-type Cas protein, wherein the functional domain is selected from the group consisting of a Reel domain, a bridged helix domain, or a PAM interaction domain.
  • the nuclease activity of dCas is reduced by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% compared to the nuclease activity of the corresponding wild-type Cas protein. , at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%.
  • Suitable dCas can be derived from wild-type Cas protein.
  • Cas proteins can be derived from type I, type II, or type III CRISPR-Cas systems.
  • a suitable dCas may be derived from Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 or Cas10.
  • dCas is derived from Cas9 protein.
  • dCas9 can be obtained by introducing point mutations (eg, substitutions, deletions or additions) in the DNA cleavage domain (eg, nuclease domain, eg, RuvC and/or HNH domain) of the Cas9 protein.
  • introducing two point mutations in the RuvC and HNH domains reduced Cas9 nuclease activity while retaining Cas9 sgRNA and DNA binding activities.
  • the two point mutations within the RuvC and HNH active sites are the D10A and H840A mutations of S. pyogenes Cas9.
  • D10 and H840 of S. pyogenes Cas9 can be deleted to eliminate Cas9 nuclease activity while retaining its sgRNA and DNA binding activities.
  • the two point mutations within the RuvC and HNH active sites are the D10A and N580A mutations of S. pyogenes Cas9.
  • the present application relates to a dCas protein, or any variant or mutant thereof.
  • All variants and mutants of dCas9 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 isolated Cas9), 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.
  • the dCas9 sequences shown in SEQ ID NOs: 1162-1179 of this application only provide a few exemplary options and are not exclusive.
  • the dCas protein is Streptococcus pyogenes dCas9 comprising mutations at D10 and/or H840 (as set forth in SEQ ID NO: 1162) protein.
  • the dCas protein is a Streptococcus pyogenes dCas9 protein comprising the D10A and/or H840A mutations (as set forth in SEQ ID NO: 1162).
  • the dCas9 protein is a Staphylococcus aureus dCas9 protein comprising the amino acid sequence set forth in SEQ ID NO: 1163 or 1164, 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 higher sequence identity) sequence, or a sequence having 1, 2, 3, 4, 5 or more changes (eg, amino acid substitutions, insertions, or deletions) relative to SEQ ID NO: 11631164, or any fragment thereof.
  • dCas9 includes Streptococcus pyogenes dCas9, Staphylococcus aureus dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheria dCas9, E.
  • the application also provides vectors comprising nucleotides encoding the following protein molecules: Streptococcus pyogenes dCas9, Staphylococcus aureus dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheriae dCas9, Eubacterium aureus dCas9 dCas9, Streptococcus pasteurianus dCas9, Lactobacillus sausage dCas9, Coccidioides dCas9, Azospirillum (strain B510) dCas9, Gluconacetobacter diazophila dCas9, Neisseria griseus dCas9, R.
  • intestinalis bacterium dCas9 food detergent Corynebacterium parvum dCas9, brine nitrate lysing bacterium (strain DSM 16511) dCas9, Campylobacter gullum (strain CF89-12) dCas9, Streptococcus thermophilus (strain LMD-9) dCas9 or the above fragment.
  • 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., guide RNA), However, the enzyme may not significantly cleave the target phosphodiester bond (eg, no measurable phosphodiester bond cleavage under physiological conditions).
  • a nuclease-deficient RNA-guided DNA endonuclease retains DNA-binding ability (e.g., specific binding to a target sequence) but lacks significant intranuclease binding.
  • nuclease-deficient RNA-guided DNA endonucleases are dCas9, ddCpf1, nuclease-deficient Cas9 variants, or nuclease-deficient Cas9 endonucleases.
  • Class II CRISPR endonuclease For example, an RNA-guided DNA endonuclease lacking nuclease is dCas9.
  • dCas9 or "dCas9 protein” as referred to herein is a Cas9 protein in which both catalytic sites of endonuclease activity are defective or lack activity.
  • dCas9 has essentially no detectable endonuclease (eg, endodeoxyribonuclease) activity.
  • dCas9 comprises 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% of the dCas9 enzyme sequence of the present application.
  • a variant of the amino acid sequence that has %, 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, or homologues.
  • the term “capable of binding” is used interchangeably with “binds to”, “specifically recognizes”, “targets”, etc., and generally refers to a binding molecule (e.g., a gene expression modulating molecule of the present application) Able to interact with 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 non-covalent force, improving binding stability, etc.
  • transcription start site generally refers to the nucleic acid in the construct that corresponds to the first nucleic acid integrated into the primary transcript (i.e., pre-mRNA); the transcription start site may Overlaps with promoter sequence.
  • fragment thereof generally refers to a portion or fragment of the specified whole.
  • fragment thereof refers to a contiguous length of a specified nucleotide sequence that is shorter than the full-length sequence of a specified polynucleotide.
  • a portion of a specified nucleotide may 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 corresponding to the first position The position is N-terminal to the sequence position corresponding to the last position, and thus the sequence of that portion is a contiguous 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 The sequence position of the position ends.
  • a portion may also be defined by reference to a position in a specified polynucleotide sequence and the length of residues relative to the reference position, whereby the sequence of the portion is a contiguous sequence of nucleotides in the specified polynucleotide that has a defined length and located in a specified polynucleotide according to a defined position.
  • modification of nucleotides may mean that the nucleic acid described in the present invention is modified by methods mature in the art, such as “Current protocols innucleic acid chemistry” Beaucage, SL et al., (Edrs.), John Synthesis or modification is performed by methods described in Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated by reference.
  • the modification may include, but is not limited to: terminal modification, such as 5'-end modification (e.g., phosphorylation, conjugation, inverted linkage) or 3'-end modification (e.g., conjugation, DNA nucleotide , trans bond, etc.); base modification, such as replacement with stabilized bases, destabilized bases or bases paired with expanded pairing library bases, removal of bases (abasic nucleosides acid), or conjugated bases; sugar modifications (e.g., sugar modifications at the 2'-position or 4'-position) or substituted sugars; or backbone modifications, including phosphorus Acid diester bonds are modified or replaced.
  • terminal modification such as 5'-end modification (e.g., phosphorylation, conjugation, inverted linkage) or 3'-end modification (e.g., conjugation, DNA nucleotide , trans bond, etc.
  • base modification such as replacement with stabilized bases, destabilized bases or bases paired with expanded pairing library bases, removal of bases (abasic nucleoside
  • the term "substances that modify histones” usually refers to related enzymes that can modify histones and regulate gene transcription. Common modifications to histones can be methylation, acetylation, phosphorylation, etc. ionization, adenylation, ubiquitination, ADP ribosylation, etc.
  • methylation modification is used interchangeably with "DNA methylation” and "nucleic acid methylation”, which generally refers to making the gene fragments, nucleotides or bases thereof in this application have Methylation state, a process that often occurs inside cells that have been transfected with a nucleic acid containing a structural gene encoding a polypeptide operably linked to a promoter in which the promoter nucleic acid Cytosine is converted into 5-methylcytosine.
  • a promoter nucleic acid in which at least one cytosine is converted to 5-methylcytosine is called a "methylated" nucleic acid or DNA.
  • the DNA fragment in which the gene in this application is located may have methylation on one strand or multiple strands, or may have methylation on one site or multiple sites.
  • regulatory element refers to a genetic element capable of controlling the expression of a nucleic acid sequence.
  • splicing signals, promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites ("IRES"), enhancers, etc. which together provide the coding sequence with the Replication, transcription, and translation in recipient cells. Not all of these control sequences need to be present.
  • Transcription control signals in eukaryotes often contain "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 genetics by increasing the activity of the closest promoter located on the same DNA molecule. Regulatory elements for the rate of transcription, sequences that specifically interact with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 (1987), incorporated herein by reference in its entirety). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect and mammalian cells, and viruses (similar control sequences, known as promoters, are also found in prokaryotes). The choice of specific promoters and enhancers depends on the recipient cell type.
  • eukaryotic promoters and enhancers have a broad host range, whereas others are functional within a restricted subset of cell types (for review, see, e.g., Voss et al., Trends Biochem. Sci., 11:287 (1986); and Maniatis et al. (supra), incorporated herein by reference in their entirety).
  • the SV40 early gene enhancer is 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.
  • Promoters and enhancers may occur naturally alone or together.
  • reverse The viral long terminal repeat contains promoter and enhancer elements.
  • promoters and enhancers act independently of the gene being transcribed or translated.
  • the enhancers and promoters used may be "endogenous,”"exogenous,” or “heterologous” relative to the gene to which they are operably linked.
  • An “endogenous” enhancer/promoter is one that is naturally associated with a given gene in the genome.
  • a “foreign” or “heterologous” enhancer or promoter is one that is juxtaposed with a gene through genetic manipulation (i.e., molecular biology techniques) such that transcription of the gene is enhanced by the connection Sub/Promoter Guidance.
  • the presence of a "splicing signal" on an expression vector usually results in high-level expression of the recombinant transcript.
  • a “splicing signal” mediates removal of introns from the primary RNA transcript and consists of splice donor and acceptor sites (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989), pp. 16.7-16.8, incorporated herein by reference in its entirety).
  • splice donor and acceptor sites are the splice junctions of the 16S RNA from SV40.
  • a "transcription termination signal” typically exists downstream of the polyadenylation signal and is hundreds of nucleotides in length.
  • the term "poly A signal” or “poly A sequence” refers to a DNA sequence that directs the termination and polyadenylation of nascent RNA transcripts. Efficient polyadenylation of recombinant transcripts is often necessary because transcripts lacking poly A signals are unstable and rapidly degraded.
  • the poly A signal used in the expression vector can be "heterologous” or "endogenous.”
  • the 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 one that is isolated from one gene and operably linked to the 3' end of another gene.
  • a commonly used heterologous poly A signal is the SV40 poly A signal.
  • the SV40 poly A signal is contained on a 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).
  • DNA methyltransferase generally refers to an enzyme that catalyzes the transfer of methyl groups to DNA.
  • Non-limiting examples of DNA methyltransferases include DNMT1, DNMT 3A, DNMT 3B, and DNMT 3L.
  • DNA methyltransferases can modify the activity of DNA fragments (such as regulating gene expression) without changing the DNA sequence.
  • gene expression regulatory molecules may include one or more (eg, two) DNA methyltransferases.
  • the DNA methyltransferase domain comprises 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 A variant of the amino acid sequence that has %, 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, or homologues.
  • the DNA methyltransferase domain comprises 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 Amino acids with 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 Sequence variants or homologs.
  • 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.
  • a functionally active fragment may retain or partially retain the ability of the full-length protein to bind another molecule.
  • a functionally active fragment of a DNA methyltransferase may retain or partially retain the biologically active function of a full-length DNA methyltransferase to catalyze the transfer of methyl groups to DNA.
  • direct and/or indirect connection generally refer to the opposite “direct connection” or “indirect connection”.
  • the term “directly connected” generally means directly connected or directly coupled.
  • the direct connection can be a situation where the connected substances (such as amino acid sequence segments) are directly connected without spacing components (such as amino acid residues or derivatives thereof); for example, the amino acid sequence segment X and another amino acid sequence Segment Y is directly connected through an amide bond formed by the C-terminal amino acid of amino acid sequence segment X and the N-terminal amino acid of amino acid sequence segment Y.
  • “Indirect connection” usually refers to a situation where the connected substances (such as amino acid sequence segments) are indirectly connected by a spacer component (such as an amino acid residue or its derivative).
  • the term “Krab” is also referred to as "Krüppel-related box domain” or “Krüppel-related box domain”, which generally refers to the transcriptional repression domain present in transcription factors of human zinc finger proteins. About 45 to about 75 amino acid residues.
  • the Krab domain may comprise 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% amino acid sequence identity Sequence variants or homologues.
  • delivery vector generally refers to a transfer vehicle capable of delivering an agent (eg, a nucleic acid molecule) to a target cell.
  • Delivery vehicles can deliver agents to specific cell subtypes. For example, by means of inherent characteristics of the delivery vehicle or by means of a moiety coupled to, contained within (or a moiety bound to the carrier) such that the moiety and the delivery vehicle are maintained together, thereby rendering the moiety sufficient to target the target.
  • the delivery vehicle may also increase the in vivo half-life of the agent to be delivered and/or the bioavailability of the agent to be delivered.
  • Delivery vectors may include viral vectors, virus-like particles, polycationic vectors, peptide vectors, liposomes, and/or hybrid vectors.
  • the properties of the delivery vehicle e.g., size, charge, and/or pH
  • the properties of the delivery vehicle can effectively deliver the delivery vehicle and/or molecules entrapped therein to the target cells, reduce immune clearance and/or promote retention in the target cell.
  • the term "liposome” generally refers to a vesicle having an internal space separated from an external medium by a membrane of one or more bilayers.
  • the membrane of the bilayer may be formed by amphipathic molecules, such as synthetic or naturally derived lipids containing spatially separated hydrophilic and hydrophobic domains; in other embodiments, the bilayer
  • the film of layers can be formed by amphiphilic polymers and surfactants.
  • the liposomes are spherical vesicular structures composed of a single or multilamellar lipid bilayer surrounding an internal aqueous compartment, and a relatively impermeable outer lipophilic phospholipid bilayer. consists of sub-layers.
  • liposomes are biocompatible, nontoxic, can deliver hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their cargo across biological membranes and the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • Liposomes can be made from several different types of lipids such as phospholipids. Liposomes may contain natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, lecithin, monosialoganglioside lipids or any combination thereof.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphatidylcholine
  • sphingomyelin sphingomyelin
  • lecithin monosialoganglioside lipids or any combination thereof.
  • monosialoganglioside lipids monosialogangli
  • the liposomes can also contain cholesterol, sphingomyelin and/or 1,2-dioleoyl - sn-glycero-3-phosphoethanolamine (DOPE), for example, to increase stability and/or prevent leakage of cargo within liposomes.
  • DOPE 1,2-dioleoyl - sn-glycero-3-phosphoethanolamine
  • lipid nanoparticle generally refers to particles containing multiple (ie, more than one) lipid molecules physically bound to each other (eg, covalently or non-covalently) by intermolecular forces.
  • LNPs can be, for example, microspheres (including unilamellar and multilamellar vesicles, such as liposomes), dispersed phase in emulsions, micelles or internal phase in suspensions.
  • LNPs can encapsulate nucleic acids within cationic lipid particles (eg, liposomes) and can be delivered to cells relatively easily. In some instances, lipid nanoparticles do not contain any viral components, which helps 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 with cell populations of various sizes.
  • the LNPs of the present application can be readily prepared by various methods known in the art, such as by mixing an organic phase with an aqueous phase. Mixing of the two phases can be achieved using microfluidic devices and impinging flow reactors. The more thoroughly the organic phase and the aqueous phase are mixed, the better the embedding rate and particle size distribution of the LNP obtained.
  • the particle size of 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 will be.
  • LNPs can be used to deliver DNA molecules (eg, molecules containing coding sequences for DNA binding proteins and/or sgRNA) and/or RNA molecules (eg, Cas, sgRNA's mRNA). In some cases, LNPs can be used to deliver Cas/gRNA RNP complexes.
  • DNA molecules eg, molecules containing coding sequences for DNA binding proteins and/or sgRNA
  • RNA molecules eg, Cas, sgRNA's mRNA.
  • LNPs can be used to deliver Cas/gRNA RNP complexes.
  • LNPs are used to deliver mRNA and gRNA (eg, an mRNA fusion molecule comprising DNMT3A-DNMT3L(3A-3L)-dCas9-KRAB and at least one sgRNA targeting a target gene).
  • mRNA and gRNA eg, an mRNA fusion molecule comprising DNMT3A-DNMT3L(3A-3L)-dCas9-KRAB and at least one sgRNA targeting a target gene.
  • the term "recombinant vector” generally refers to a nucleic acid molecule capable of transporting it and another nucleic acid to which it is linked.
  • a vector refers to a circular double-stranded DNA ring within which additional DNA segments can be ligated.
  • the vector may be linear.
  • a viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication within the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the host cell's genome upon introduction into the host cell, and thereby replicate together with the host genome.
  • adeno-associated virus (AAV) vector generally refers to a vector having functional or partially functional ITR sequences and a transgene.
  • ITR refers to inverted terminal repeats. ITR sequences can be derived from adeno-associated virus serotypes, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13, and any AAV variant or mixture.
  • the ITR need not be a wild-type nucleotide sequence, and may be altered (eg, by insertion, deletion, or substitution of nucleotides) as long as the sequence retains functions that provide functional rescue, replication, and packaging.
  • the AAV vector may have one or more AAV wild-type genes, preferably the rep and/or cap genes, deleted in whole or in part, but retaining functional flanking ITR sequences. Functional ITR sequences serve, for example, to rescue, replicate and package AAV virions or particles. Therefore, an "AAV vector" is defined in this application to include at least those sequences required for insertion of a transgene into cells of a subject. Optionally included are those cis-sequences necessary for viral replication and packaging (eg, functional ITR).
  • nuclear localization sequence or “nuclear localization signal” or “NLS” generally refers to a peptide that directs a protein to the nucleus.
  • NLS includes five basic positively charged amino acids.
  • NLS can be located anywhere on the peptide chain.
  • complementary generally refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence via traditional Watson-Crick or other non-traditional types.
  • sequence A-G-T is complementary to the sequence T-C-A.
  • Percent complementarity 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., 50 out of 10, 6, 7, 8, 9, 10, respectively) %, 60%, 70%, 80%, 90% and 100% complementary).
  • perfect complementary means that all contiguous residues of a nucleic acid sequence will hydrogen bond to the same number of contiguous residues in a second nucleic acid sequence.
  • substantially complementary means that at 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, Within a region of 40, 45, 50 or more nucleotides, or refers to at least 60%, 65%, 70%, 75%, 80%, A degree of complementarity of 85%, 90%, 95%, 97%, 98%, 99% or 100%.
  • the term "gene” generally refers to a DNA segment designed to produce a protein.
  • the gene may also include regions before and after the coding region (leader and tail) as well as intervening sequences (introns) between individual coding segments (exons). Leaders, tails, and introns include regulatory elements necessary for gene transcription and translation.
  • a "protein gene product” may be a protein expressed by a specific gene.
  • polypeptide or amino acid residues.
  • a fusion protein may refer to a chimeric protein encoding two or more separate protein sequences recombinantly expressed as a single part.
  • identity refers to the use of the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below or by manual alignment and visual inspection. Measured by inspection.
  • identical or having a specified percentage of identical amino acid residues or nucleotides when comparing and aligning for maximum correspondence over a comparison window or specified region, approximately 60% identity within a specified region, preferably 65 %, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher consistency) of two or more Multiple sequences or subsequences, such sequences may be said to be "substantially identical".
  • guide RNA or "gRNA” generally refers to any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and specifically bind the CRISPR complex to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, about 60%, about 75%, about 80% when optimally aligned using a suitable alignment algorithm , about 85%, about 90%, about 95%, about 97.5%, about 99% or higher.
  • the specific protein may include any natural form of the protein or maintain the activity of the protein (for example, with 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).
  • 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.
  • linker is generally intended to include a linker that joins two or more moieties.
  • a linker is linked at the N-terminus and C-terminus to the amino acid sequence of the remainder of the compound (eg, a fusion protein provided herein).
  • XTEN XTEN linker
  • XTEN polypeptide refers to a recombinant polypeptide lacking hydrophobic amino acid residues.
  • detectable agent refers to a composition that can be detected by suitable means.
  • suitable means are, for example, spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging or other physical means.
  • useful detectable reagents include radioactive elements, fluorophores (eg, fluorescent dyes), electron-dense reagents, enzymes (eg, commonly used in ELISA), biotin, paramagnetic molecules, and the like.
  • the terms “inhibit”, “repress”, “silence” and the like generally refer to the reduction of gene expression and/or activity.
  • administration of a substance of the present application may negatively affect (e.g., decrease) the activity of a nucleic acid sequence relative to the activity of a nucleic acid sequence in the absence of the substance (e.g., fusion protein, complex, nucleic acid, vector) (control) .
  • suppression may refer to a reduction of a disease or symptoms of a disease.
  • inhibition includes at least partially, partially or completely blocking activation (eg, transcription) of a nucleic acid sequence, or reducing, preventing, or delaying activation of a nucleic acid sequence.
  • the inhibitory activity may be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less of the control.
  • selected from is generally intended to include selected objects and all combinations thereof.
  • selected from (:)A, “B and C” is meant to include all combinations of A, B and C, for example, A, B, C, A+B, A+C, B+C or A+B+C.
  • the term "about” generally refers to a variation within the range of 0.5% to 10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a method for regulating F11 gene expression and/or activity.
  • the method includes providing a gene expression regulating molecule or a nucleic acid encoding the gene expression regulating molecule.
  • the gene expression regulating molecule may have Modulate the expression of the F11 gene without changing the function of its gene sequence.
  • the methods of the present application may be non-therapeutic methods.
  • the method of the present application may not directly target the human body.
  • the methods of the present application may be in vitro or ex vivo methods.
  • the methods of the present application may be methods for therapeutic purposes.
  • the methods of the present application may be in vivo methods.
  • the present application provides a method for treating and/or alleviating conditions associated with abnormal F11 gene expression and/or F11 gene activity, the method comprising providing a gene expression regulatory molecule or encoding the gene expression regulator
  • the gene expression regulating molecule may have the function of regulating the expression of the F11 gene without changing its gene sequence.
  • the present application provides a gene expression regulatory molecule or a nucleic acid encoding the gene expression regulatory molecule.
  • the gene expression regulatory molecule may have the function of regulating the expression of the F11 gene without changing the gene sequence.
  • the nucleic acid includes DNA and/or mRNA.
  • the gene expression regulatory molecule or the nucleic acid encoding the gene expression regulatory molecule can be used to treat and/or alleviate conditions related to abnormal F11 gene expression and/or F11 gene activity.
  • the present application provides 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 F11 gene expression and/or F11 gene activity.
  • the gene expression regulating molecule may have the function of regulating the expression of the F11 gene without changing its gene sequence.
  • the nucleic acid encoding the gene expression regulatory molecule of the present application includes DNA and/or mRNA.
  • the present application provides a nucleic acid binding molecule or a nucleic acid encoding the nucleic acid binding molecule, the nucleic acid binding molecule comprising the sequence shown in any one of SEQ ID NOs: 1-70.
  • the nucleic acid encoding the nucleic acid binding molecule of the present application includes DNA and/or mRNA.
  • a recombinant vector comprising the nucleic acid of the present application.
  • a recombinant vector may refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • Recombinant vectors may include single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and Other types of polynucleotides known in the art. For example, you can use the disease poison carrier.
  • Viral vectors may contain virus-derived DNA or RNA sequences for packaging into viruses (eg, retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses (AAV)).
  • viruses eg, retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses (AAV)).
  • viruses eg, retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses (AAV)
  • viruses eg, retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses (AAV)
  • viruses eg, retroviruses, replication-deficient retroviruses, aden
  • the present application provides a delivery vector, the delivery vector comprising the nucleic acid binding molecule of the present application, the gene expression regulatory molecule of the present application, the nucleic acid of the present application, and/or the recombinant vector of the present application, and optionally containing liposomes and/or lipid nanoparticles (LNP).
  • a delivery vector may comprise one or more Cas proteins and one or more guide RNAs, for example, in the form of a ribonucleoprotein complex (RNP).
  • RNP ribonucleoprotein complex
  • ribonucleoproteins can be delivered via polypeptide-based shuttle agents.
  • ribonucleoproteins can be delivered using synthetic peptides.
  • the delivery vector can be introduced into the cell by physical delivery methods.
  • physical methods include microinjection, electroporation, and hydrodynamic delivery.
  • LNPs can encapsulate nucleic acids in cationic lipid particles (such as liposomes) and can be delivered to cells relatively easily.
  • lipid nanoparticles do not contain any viral components, which helps minimize safety and immunogenicity concerns.
  • Lipid particles can be used for in vitro, ex vivo and in vivo delivery.
  • the components of the LNP may include cationic lipids, ionizable lipids, pegylated lipids and/or support lipids, and optionally a cholesterol component.
  • the LNP can comprise ionizable lipid (20%-70%, molar ratio), pegylated lipid (0%-30%, molar ratio), support lipid (30%-50% , molar ratio) and cholesterol (10%-50%, molar ratio).
  • 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 present application. Delivery vehicle.
  • the nucleic acid binding molecule, the gene expression modulating molecule, the nucleic acid encoding the nucleic acid binding molecule, the nucleic acid encoding the gene expression modulating molecule, the recombinant vector and the delivery vector in the composition can be included in one composition at the same time, or separately. Contained in different compositions.
  • nucleic acid binding molecule when using a nucleic acid binding molecule, a gene expression regulating molecule, a nucleic acid encoding the nucleic acid binding molecule, a nucleic acid encoding the gene expression regulating molecule, a recombinant vector and/or a delivery vector in the composition, they can be used simultaneously, or Use separately.
  • 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 compositions of the present application.
  • the present application provides a kit, which includes 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.
  • the present application provides a method for regulating the expression and/or activity of a target gene, which method includes 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, and the recombinant of the present application. carrier, book The application's delivery vector, the application's composition, the application's cell, and/or the application's kit.
  • the methods can reduce the expression and/or activity of a gene of interest.
  • administration of a substance of the present application may negatively affect (e.g., reduce) the activity of a nucleic acid sequence compared to the expression and/or activity of a target gene in the absence of the substance of the present application, for example, may include at least partially, partially Block activation (e.g., transcription) of a nucleic acid sequence completely or completely, or reduce, prevent, or delay activation of a nucleic acid sequence.
  • 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 of the control.
  • 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, and a nucleic acid binding molecule of the present application.
  • the application of the cells and/or the kit of the present application in the preparation of medicaments for treating and/or alleviating disorders, including disorders associated with abnormal expression and/or activity of target genes.
  • 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, and a nucleic acid binding molecule of the present application.
  • the cells, and/or the kits of the present application are used to treat and/or alleviate disorders, including disorders associated with abnormal expression and/or activity of target genes.
  • the present application provides a method for treating and/or alleviating diseases, which method includes 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, In the delivery vector of the present application, the composition of the present application, the cells of the present application, and/or the kit of the present application, the disease includes a disease related to abnormal expression and/or activity of the target gene.
  • the treatment is a disease/disorder of the 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.
  • the gene expression molecules provided in the present application or the method of the present application have the function of regulating the expression of the F11 gene without changing its gene sequence.
  • the gene expression regulatory molecule may have the function of inhibiting gene expression.
  • the gene expression modulating molecule comprises a DNA binding domain.
  • the gene expression regulatory molecule may have the function of binding to a gene sequence.
  • the DNA binding domain includes a (DNA) nuclease, and the nuclease can be a nuclease that targets DNA in a sequence-specific manner; exemplarily, it can be a CRISPR-Cas system-related enzyme, a zinc finger nuclease ( ZFN), transcription activator-like effector nucleases (TALENs) or meganucleases.
  • the gene expression regulatory molecule may comprise the DNA binding domain of a TALEN, a zinc finger domain, and/or the DNA binding domain of a CRISPR/Cas system.
  • the DNA binding domain is a DNA nuclease derived from the CRISPR-Cas system.
  • the DNA-binding domain is a (modified) transcription activator-like effector nuclease (TALEN) system; while transcription activator-like effector (TALE) can be designed to bind almost any desired DNA sequence.
  • the DNA binding domain is or consists of a (modified) zinc finger nuclease (ZFN) system; the ZFN system uses an artificial protein generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. Restriction enzymes, DNA-cutting domains that can be engineered to target a desired DNA sequence.
  • the DNA binding domain is a (modified) meganuclease, which is an endodeoxyribonuclease characterized by a large recognition site (double-stranded DNA sequence of 12 to 40 base pairs) .
  • the gene expression regulatory molecule may comprise a Cas enzyme.
  • the gene expression modulating molecule may comprise a Cas enzyme that has substantially no nuclease activity.
  • a guide sequence can be any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence.
  • a nucleic acid-targeting Cas protein can be mutated relative to the corresponding wild-type enzyme such that the mutated nucleic acid-targeting Cas protein lacks the ability to cleave one or both strands of a target polynucleotide.
  • the DNA cleavage activity of a mutant enzyme is about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the nucleic acid cleavage activity of a non-mutated form of the enzyme.
  • a mutated Cas can have one or more mutations that result in reduced off-target effects.
  • the gene expression regulatory molecule may comprise a Cas9 enzyme.
  • Cas proteins include Class I (eg, Types I, III, and IV) and Class 2 (eg, Type II, V, and VI) Cas proteins, such as Cas9, Cas12 (eg, Cas12a, Cas12b, Cas12c, Cas12d), Cas13 (e.g., Cas13a, Cas13b, Cas13c, Cas13d), CasX, CasY, Cas14, variants thereof (e.g., mutated forms, truncated forms), their homologs, and their orthologs.
  • Cas proteins include Class I (eg, Types I, III, and IV) and Class 2 (eg, Type II, V, and VI) Cas proteins, such as Cas9, Cas12 (eg, Cas12a, Cas12b, Cas12c, Cas12d), Cas13 (e.g., Cas13a, Cas13b, Ca
  • the Cas protein is Cas9, Cas12a, Cas12b, Cas12c, or Cas12d.
  • Cas9 can be SpCas9, SaCas9, StCas9, and other Cas9 orthologs.
  • Cas12 can be Cas12a, Cas12b and Cas12c, including FnCas12a, or homologs or orthologs thereof.
  • the Cas9 enzyme can 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 ( For example, B510)dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia intestinalis dCas9, Parvibaculum lavamentivorans dCas9, Nitratifractor salsuginis (eg, DSM 16511) dCas9, Campylo
  • the gene expression regulatory molecule may comprise the amino acid sequence of dCas9.
  • the dCas9 sequence may have one, two, three, four, five or more changes, such as amino acid substitutions, insertions or deletions of the sequence, or any fragment thereof.
  • the gene expression regulatory molecule is capable of binding to a DNA region or a fragment thereof within 500 bp upstream and/or downstream of the transcription start site (TSS) of the F11 gene.
  • TSS transcription start site
  • the gene expression regulatory molecule can bind to about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp upstream of the TSS , about 1300bp, about 1400bp or about 1500bp.
  • the gene expression regulatory molecule can bind to about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp downstream of the TSS , about 1300bp, about 1400bp or about 1500bp.
  • the gene expression regulatory molecule can bind to the DNA region or fragments thereof located in any one of SEQ ID NOs: 71-72.
  • the gene expression regulatory molecule can bind to one or more DNA regions near the transcription start site (TSS) of the F11 gene described below: between 250 bp upstream of the TSS and 130 bp upstream. between 50bp upstream and 120bp downstream of TSS, and between 230bp downstream and 390bp downstream of TSS.
  • TSS transcription start site
  • the gene expression regulatory molecule is capable of binding to one or more DNA regions near the transcription start site (TSS) of the F11 gene: between 250 bp upstream and 230 bp upstream of the TSS (e.g., Between 248-229 bp upstream of TSS), between 210 bp upstream of TSS and 180 bp upstream (for example, between 202-185 bp upstream of TSS), between 160 bp upstream of TSS and 130 bp upstream (for example, 156-137 bp upstream of TSS between), between 50 bp upstream of TSS and 10 bp downstream (for example, between 44-25 bp upstream of TSS, and/or between 12 bp upstream and 8 bp downstream), between 30 bp downstream of TSS and 60 bp downstream (for example, between TSS Between 37-56 bp downstream of TSS), between 90 bp downstream of TSS and 120 bp downstream (for example, between 96-115 bp
  • TSS
  • the gene expression modulating molecule comprises a first functional domain that provides modification of at least one nucleotide near the F11 gene and/or within the F11 gene regulatory element.
  • the first functional domain can regulate target gene expression through epigenetic modification at a regulatory element of the target gene, such as a promoter, enhancer, or transcription start site, such as through histone acetylation or Methylation, or DNA methylation.
  • regulatory elements may include a transcription start site, a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, or a locus control region.
  • the epigenetic modification can be through any known epigenetic modification modulator that can be used for DNA methylation.
  • exemplary epigenetic modification modulators 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.
  • the first functional domain may provide modification of at least one nucleotide near the F11 gene and/or within the regulatory element of the F11 gene, and the modification of the at least one nucleotide includes a methylation modification.
  • the first functional domain comprises an epigenetic modification modulator, which may have DNA methylase activity.
  • the epigenetic modification modulator may have methylase activity, which involves the transfer of methyl groups to DNA, RNA, proteins, small molecules, cytosine, or adenine.
  • the modification can be located 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 upstream of the transcription start site of the target gene. , about 1300bp, about 1400bp or about 1500bp.
  • the modification can be located 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 downstream of the transcription start site of the target gene. , about 1300bp, about 1400bp or about 1500bp.
  • the first functional domain may comprise a DNA methyltransferase (DNMT) domain.
  • the first functional domain may comprise a DNMT 3A domain and/or a DNMT 3L domain.
  • the DNMT 3A domain and/or DNMT 3L domain is derived from mammals.
  • the DNMT 3A domain is derived from mice.
  • the gene expression regulatory molecule may comprise the amino acid sequence described in DNMT 3A.
  • the DNMT 3L domain is derived from human and/or mouse.
  • the gene expression regulatory molecule may comprise the amino acid sequence of DNMT 3L.
  • 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.
  • the DNMT 3A domain and the DNMT 3L domain are directly and/or indirectly connected.
  • the DNMT 3A domain and the DNMT 3L domain are connected through a linker.
  • the C-terminal of the DNMT 3A structural domain is connected to the N-terminal of the DNMT 3L, or the C-terminal of the DNMT 3L structural domain is connected to the N-terminal of the DNMT 3A.
  • the gene expression modulating molecule comprises a second functional domain comprising a zinc finger protein-based transcription factor or a functionally active fragment thereof, or a substance capable of modifying histones.
  • the second functional domain may include a gene expression repressor, which may be any known gene expression repressor.
  • Exemplary gene expression repressors may be selected from the group consisting of Krüppel-related box (KRAB) domains, mSin3 interaction structures 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 factors 1 and 2, Drosophila Groucho (Drosophila Gro protein), tripartite motif-containing 28 (TRTM28), nuclear receptor corepressor 1, nuclear receptor corepressor 2, or functionally active fragments or fusions thereof.
  • KRAB Krüppel-related box
  • SID mSin3 interaction structures domain
  • MXI1 MAX interacting protein 1
  • chromosome shadow domain chromosome shadow domain
  • SRDX EAR-repressor domain
  • EEF1
  • the second functional domain may comprise a substance capable of modifying histones. qualitative function.
  • the second functional domain can comprise a Krab domain.
  • the second functional domain may comprise a ZIM3 Krab domain or a KOX1 Krab domain.
  • the KRAB domain or fragment thereof can be fused to the C-terminus of the dCas9 molecule. In certain embodiments, the KRAB domain or fragment thereof can be fused to the N-terminus and C-terminus of the dCas9 molecule.
  • the second functional domain may comprise a KRAB domain that may comprise substantially the same (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 greater identity), or identical to ZIM3 Krab or KOX1 Krab , a sequence with 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.
  • a KRAB domain may comprise substantially the same (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 greater identity), or identical to ZIM3 Krab or KOX1 Krab ,
  • the second functional domain may include a gene expression activator, which may be any known gene expression activator.
  • exemplary gene expression activators may include VP16 activation domain, VP64 activation domain, p65 activation domain. Domain, Epstein-Barr virus R transactivator Rta molecule or fragment thereof.
  • the second functional domain may comprise a substance capable of modifying histones, which may comprise a histone acetyltransferase (e.g., p300 catalytic domain), a histone deacetylase, Histone methyltransferases (eg, SUV39H1 or G9a (EHMT2)), histone demethylases (eg, LSD1), and/or functionally active fragments thereof.
  • histone acetyltransferase e.g., p300 catalytic domain
  • a histone deacetylase eg, Histone methyltransferases (eg, SUV39H1 or G9a (EHMT2)
  • histone demethylases eg, LSD1
  • the gene expression regulatory molecule includes 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.
  • the C-terminal of the first functional domain is directly connected to the N-terminal of the second functional domain, or the C-terminal of the first functional domain is indirectly connected (for example, through a linker) to the N-terminal of the second functional domain.
  • the N-terminus, or the first functional domain is located on the N-terminal side of the second functional domain (for example, from the N-terminus to the C-terminus, the first functional domain, other parts such as the DNA binding domain, and the order of two functional domains).
  • the C-terminal of the second functional domain is directly connected to the N-terminal of the first functional domain, or the C-terminal of the second functional domain is indirectly connected (for example, through a linker) to the N-terminal of the first functional domain.
  • the N-terminus, or the second functional domain is located on the N-terminal side of the first functional domain (for example, from the N-terminus to the C-terminus, there may be the second functional domain, other parts such as the DNA-binding domain, and the a sequence of functional domains).
  • the first functional domain includes two or more functional domains, and they can be directly connected, indirectly connected (for example, through a linker), or located on the N-terminal side of the second functional domain.
  • first functional domain a other parts such as the DNA-binding domain, the second functional domain, and the first functional domain b, or the first functional domain
  • the first functional domain and the second functional domain are directly or indirectly linked to one end of the DNA binding domain.
  • the first functional domain and the second functional domain are directly or indirectly connected to the C-terminus of the DNA binding domain.
  • the gene regulatory molecule may include dCas9-DNMT 3A-DNMT 3L-Krab , dCas9-DNMT 3L-DNMT 3A-Krab, dCas9-Krab-DNMT 3A-DNMT 3L, dCas9-Krab-DNMT 3L-DNMT 3A.
  • the first functional domain and the second functional domain are directly or indirectly connected to the N-terminus of the DNA binding domain.
  • the gene regulatory molecule may include DNMT 3A-DNMT 3L-Krab-dCas9 , DNMT 3L-DNMT 3A-Krab-dCas9, Krab-DNMT 3A-DNMT 3L-dCas9, Krab-DNMT 3L-DNMT 3A-dCas9.
  • the first functional domain and the second functional domain are directly or indirectly connected at both ends of the DNA binding domain.
  • the first functional domain is directly or indirectly connected to the C-terminal of the DNA-binding domain
  • the second functional domain is directly or indirectly connected to the N-terminal of the DNA-binding domain.
  • the The gene regulatory molecule may include any one of Krab-dCas9-DNMT 3A-DNMT 3L and Krab-dCas9-DNMT 3L DNMT 3A.
  • the first functional domain is directly or indirectly connected to the N-terminal of the DNA-binding domain
  • the second functional domain is directly or indirectly connected to the C-terminal of the DNA-binding domain.
  • the The gene regulatory molecule may include any one of DNMT 3A-DNMT 3L-dCas9-Krab and DNMT 3L-DNMT 3A-dCas9-Krab.
  • the gene expression modulating molecule may further comprise a tag for detection, isolation and/or purification.
  • the gene expression regulatory molecule can include an HA tag.
  • the gene expression regulatory molecule may comprise the amino acid sequence of an HA tag. Alternatively, 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.
  • the gene expression modulating molecule may further comprise a nuclear localization sequence.
  • the nuclear localization sequence may comprise amino acids with electropositive groups.
  • the nuclear localization sequence may comprise the amino acid sequence of a nuclear localization sequence known in the art.
  • 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.
  • 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 of the DNA binding domain. and/or C-terminal.
  • the gene expression modulating molecule may further comprise a detectable moiety.
  • the detectable moiety may comprise blue fluorescent protein and/or green fluorescent protein.
  • the detectable moiety and the first functional domain, the second functional domain, the DNA binding domain, and/or the nuclear localization sequence can be linked by a self-cleaving peptide.
  • the self-cleaving peptide may comprise 2A peptide.
  • the first functional domain, the second functional domain, the DNA binding domain and other elements included in the gene regulatory molecule can be connected in an indirect manner, for example, through a certain length of Adapter sequence ligation.
  • the linker may include approximately 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.
  • the first functional domain and the second functional domain are connected by a linker of about 80 or more amino acids in length.
  • the first functional domain and the second functional domain are connected by a linker of about 92 or more amino acids in length.
  • the first functional domain and the second functional domain are connected through an XTEN linker.
  • the linker comprises the amino acid sequence of an XTEN linker.
  • the linker comprises the amino acid sequence of an XTEN linker.
  • the linker includes an XTEN linker that is 16 amino acids in length, an XTEN linker that is 80 amino acids in length, or an amino acid sequence of a longer XTEN linker.
  • the present application also provides a nucleic acid binding molecule, which may comprise the sequence of any one of SEQ ID NOs: 1-70.
  • the nucleic acid binding molecules and/or the gene expression modulating molecules of the present application can be delivered to the subject by local injection, systemic infusion, or a combination thereof.
  • the nucleic acid binding molecule can comprise at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% the sequence of any one of SEQ ID NOs: 1-70 , 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 of the sequence.
  • the combination of the nucleic acid binding molecule and the gene expression modulating molecule of the present application may have the ability to modulate the expression level of the F11 gene.
  • 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 of the F11 gene district.
  • the nucleic acid binding molecule and/or the gene expression regulating molecule of the present application can bind to the DNA region within 500 bp upstream and/or downstream of the transcription start site of the F11 gene or a fragment thereof.
  • 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.
  • the DNA region that can be combined can comprise at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% of the DNA region in any one of SEQ ID NOs: 71-72. %, 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 Sequences with 100% sequence identity.
  • the nucleic acid binding molecule and/or the gene expression regulating molecule of the present application can bind to the DNA region located in any one of SEQ ID NOs: 73-142 or a fragment thereof.
  • the DNA region that can be combined can comprise at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% of the DNA region in any one of SEQ ID NOs: 73-142. %, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, to Sequences that are less than 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical.
  • targeting any 20 bp region in the DNA region where any one of SEQ ID NOs: 73-142 is located can have the ability to regulate the expression level of the F11 gene.
  • administration of a substance of the present application may negatively affect (e.g., reduce) the activity of a nucleic acid sequence compared to target gene expression and/or activity in the absence of the substance of the present application, for example, may include at least partially, partially Either completely blocking the activation (eg, transcription) of the nucleic acid sequence, or reducing, preventing or delaying the activation of the nucleic acid sequence.
  • 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 of the control.
  • the guide sequence or spacer of the nucleic acid binding molecule can be 15 to 50 nucleotides in length.
  • the nucleic acid binding molecule is a guide RNA (gRNA), and the guide RNA can have a spacer length of at least 15 nucleotides.
  • 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. 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.
  • 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.
  • the target binding region can be between about 19 and about 21 nucleotides in length. In one embodiment, the target binding region may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In one embodiment, the target binding region may be complementary, eg, completely complementary, to the target region in the target gene. In one embodiment, the target binding region can be substantially complementary to the target region in the target gene.
  • the target binding region may comprise no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 regions that are different from the target region in the target gene.
  • complementary nucleotides in some embodiments, the nucleic acid binding molecules and/or gene expression modulating molecules of the present application can be formulated in liposomes or lipid nanoparticles. In some embodiments, the nucleic acid binding molecules and/or gene expression modulating molecules of the present application can be formulated in viral vectors.
  • a guide RNA can be included with one or more chemical modifications (e.g., by chemically linking two ribonucleotides or by replacing one or more ribonucleotides with one or more deoxyribonucleotides) of RNA-based molecules.
  • a target binding region of human origin can be targeted.
  • the DNA sequence of the reporter system in this example (standard font: promoter region, bold: F11 gene regulatory region, italics: green fluorescent protein (EGFP) sequence) is as follows:
  • DNA sequence of the editing tool EpiRegA (Dnmt3a-Dnmt3l-dCas9-KOX1 KRAB) (italic/underline italic: Dnmt3a/Dnmt3l, bold: dCas9, bold italic: KRAB, underline bold italic: blue fluorescent protein, standard font: junction sequence )as follows:
  • DNA sequence of the editing tool EpiRegB (Dnmt3a-Dnmt3l-ZIM3 KRAB-dCas9) (italic/underline italic: Dnmt3a/Dnmt3l, bold: dCas9, bold italic: KRAB, underline bold italic: blue fluorescent protein, standard font: junction sequence )as follows:
  • Amino acid sequence of editing tool EpiRegB (Dnmt3a-Dnmt3l-ZIM3 KRAB-dCas9) (italic/underline italic: Dnmt3a/Dnmt3l, bold: dCas9, bold italic: KRAB, underline bold italic: blue fluorescent protein, standard font: connection sequence , *: any amino acid) as follows:
  • DNA sequence of editing tool EpiRegC (dCas9-ZIM3 KRAB-DNMT3L-Dnmt3a) (italic/underline italic: Dnmt3a/Dnmt3l, bold: dCas9, bold italic: KRAB, underline bold italic: blue fluorescent protein, standard font: connection sequence )as follows:
  • Amino acid sequence of editing tool EpiRegC (dCas9-ZIM3 KRAB-DNMT3L-Dnmt3a) (italic/underline italic: Dnmt3a/Dnmt3l, bold: dCas9, bold italic: KRAB, underline bold italic: blue fluorescent protein, standard font: connection sequence , *: any amino acid) as follows:
  • Regional NT gRNA SEQ ID NO: 154) sample set to obtain the relative regulatory efficiency of different editing tools when using different gRNA.
  • SEQ ID NOs: 22, 23, 24, 26, 34, 35, 38, 45, 50 and 62 (their targeting regions 5' ends respectively start upstream of TSS
  • the gRNA shown in (44bp, upstream 248bp, downstream 37bp, downstream 96bp, upstream 12bp, upstream 156bp, downstream 233bp, upstream 204bp, downstream 301bp and downstream 366bp) combined with the epigenetic editing tool of this embodiment can effectively inhibit the expression of the F11 gene level.
  • the guide RNA in the epigenetic editing tool to target the target gene; at the same time, according to the gene expression regulatory molecules provided by this application, construct the table Epigenetic editing tools.
  • the region within 500 bp upstream and downstream of the F11 transcription start site (TSS) can be targeted.
  • the guide RNA plasmid and the gene expression regulatory molecule plasmid were co-transfected into mouse cell lines. After 72 hours, the top 10% of GFP+ and mCherry+ cells were sorted by FACS. RT-QPCR experiments were performed to evaluate the mRNA expression levels of target genes.
  • transfected cells showed reduced expression of the F11 expression product.
  • transfected cells may show reduced mRNA expression of F11 transcripts.
  • transfected cells may show reduced expression of the protein product of F11 expression.
  • diseases or conditions related to the F11 gene can be alleviated.
  • the epigenetic editing tool of the present application is used to regulate the expression of target points.
  • the domain in the gene expression regulating molecule that has the function of binding gene sequences can be replaced by the DNA-binding domain of TALEN, the zinc finger domain, the DNA-binding domain of tetR, a meganuclease and/or any CRISPR known in the art.
  • the nuclease domain of the Cas system such as dCas12 enzyme, etc.
  • the results show that the epigenetic target of the present application is versatile, and has a high effect of regulating gene expression using various gene sequence binding domains.

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Abstract

一种表观编辑靶点的方法及用途。具体而言，提供了一种调节目标基因表达水平的方法以及一种核酸结合分子，所述核酸结合分子与基因表达调节分子的组合具有调节目标基因表达水平的能力。

Description

一种表观编辑靶点的方法及用途 技术领域
本申请涉及生物医学领域，具体地涉及一种表观遗传编辑靶点及用途。
背景技术
将无催化活性的“失活Cas9(dCas9)”融合到Kruppel相关框(KRAB)结构域可以产生一种基因表达阻遏物，该阻遏物能够可以在细胞培养实验中对靶基因进行高度特异性和有效的调节或沉默。然而在体内环境，该阻遏物发挥治疗效果具有一定的挑战。例如，安全性、毒性、免疫原性和脱靶效应是限制合成阻遏物在体内使用的其他挑战。
因此，本领域需要一种有效的表观遗传编辑靶点，靶向该靶点可以用于提高表观遗传编辑效果、安全性，降低毒性、免疫原性和/或脱靶效应。
发明内容
本申请提供一种表观遗传编辑靶点，靶向该靶点可以用于提高表观遗传编辑效果、安全性，降低毒性、免疫原性和/或脱靶效应。例如，靶向该目标基因附近和/或该目标基因调节元件内，可以有效地进行至少一种核苷酸的修饰，从而调节(例如，减少或消除)目标基因产物在细胞中的表达。
一方面，本申请提供了一种调控F11(Coagulation Factor XI)基因表达和/或活性的方法，所述方法包括提供一种基因表达调节分子或编码所述基因表达调节分子的核酸，所述基因表达调节分子具有调节所述F11基因的表达而不更改其基因序列的功能。
另一方面，本申请提供了一种治疗和/或缓解与F11基因表达和/或F11基因活性异常相关的病症的方法，所述方法包括提供一种基因表达调节分子或编码所述基因表达调节分子的核酸，所述基因表达调节分子具有调节所述F11基因的表达而不更改其基因序列的功能。
在一些实施方案中，所述基因表达调节分子包含DNA结合域。
在一些实施方案中，所述基因表达调节分子包含选自TALEN结构域、锌指结构域和CRISPR/Cas系统的蛋白结构域的一个或多个DNA结合域。
在一些实施方案中，所述基因表达调节分子包含Cas酶。
在一些实施方案中，所述基因表达调节分子包含基本上不具有核酸酶活性的Cas酶。
在一些实施方案中，所述基因表达调节分子包含dCas9酶。
在一些实施方案中，所述基因表达调节分子能够结合F11基因的转录起始位点(TSS)的上游和/或下游500bp范围内的DNA区域或其片段。
在一些实施方案中，所述基因表达调节分子能够结合SEQ ID NOs：71-72中任一项所在DNA区域或其片段。
在一些实施方案中，所述基因表达调节分子能够结合下列所述的F11基因的转录起始位点(TSS)附近的一个或多个DNA区域：TSS的上游250bp至上游130bp之间、TSS的上游50bp至下游120bp之间、和TSS的下游230bp至下游390bp之间。
在一些实施方案中，所述基因表达调节分子能够结合下列所述的F11基因的转录起始位点(TSS)附近的一个或多个DNA区域：TSS的上游250bp至上游230bp之间、TSS的上游210bp至上游180bp之间、TSS的上游160bp至上游130bp之间、TSS的上游50bp至下游10bp之间、TSS的下游30bp至下游60bp之间、TSS的下游90bp至下游120bp之间、TSS的下游230bp至下游260bp之间、TSS的下游300bp至下游320bp之间、和TSS的下游360bp至下游390bp之间。
在一些实施方案中，所述方法包括提供一种核酸结合分子，所述核酸结合分子包含SEQ ID NOs：1-70中任一项的序列。
在一些实施方案中，所述基因表达调节分子和/或所述核酸结合分子被配制在相同或不同的递送载体中。
在一些实施方案中，所述递送载体包含脂质体和/或脂质纳米颗粒。
在一些实施方案中，所述基因表达调节分子和/或所述核酸结合分子被配制在相同或不同的重组载体中。
在一些实施方案中，所述重组载体包含病毒载体。
在一些实施方案中，所述重组载体包含腺相关病毒(AAV)载体。
在一些实施方案中，所述基因表达调节分子包含第一功能域，所述第一功能域提供F11基因附近和/或F11基因调节元件内的至少一个核苷酸的修饰。
在一些实施方案中，所述至少一个核苷酸的修饰包含甲基化修饰。
在一些实施方案中，所述调节元件包含核心启动子、近端启动子、远端增强子、沉默子、绝缘子元件、边界元件和/或基因座控制区。
在一些实施方案中，所述第一功能域包含DNA甲基转移酶、DNA去甲基化酶、和其功 能活性片段中的一种或多种。
在一些实施方案中，所述DNA甲基转移酶包含DNMT 3A、DNMT 3B、DNMT 3L、DNMT1和DNMT 2的一种或多种。
在一些实施方案中，所述DNMT 3A来源于小鼠。
在一些实施方案中，所述DNMT 3L来源于人和/或小鼠。
在一些实施方案中，所述DNMT 3A和所述DNMT 3L直接和/或间接连接。
在一些实施方案中，所述基因表达调节分子包含第二功能域，所述第二功能域包含基于锌指蛋白的转录因子或其功能活性片段，或者包含能够修饰组蛋白的物质。
在一些实施方案中，所述第二功能域包含Krab。
在一些实施方案中，所述第二功能域包含ZIM3 Krab或KOX1 Krab。
在一些实施方案中，所述第二功能域包含组蛋白甲基转移酶、组蛋白去甲基化酶、组蛋白乙酰转移酶、组蛋白脱乙酰酶、和其功能活性片段中的一种或多种。
在一些实施方案中，所述第一功能域和所述第二功能域直接或间接地连接在所述DNA结合域的一端。
在一些实施方案中，所述基因表达调节分子包含核定位序列。
在一些实施方案中，所述核定位序列包含具有正电基团的氨基酸。
在一些实施方案中，所述核定位序列位于所述第一功能域的N端和/或C端、所述第二功能域的N端和/或C端、和/或所述DNA结合域的N端和/或C端。
另一方面，本申请提供了一种核酸结合分子，所述核酸结合分子包含SEQ ID NOs：1-70中任一项的序列。
另一方面，本申请提供了一种基因表达调节分子，所述基因表达调节分子具有调节所述F11基因的表达而不更改其基因序列的功能。
在一些实施方案中，所述基因表达调节分子如本申请的方法中所提供的基因表达调节分子。
在一些实施方案中，所述基因表达调节分子和/或核酸结合分子被配制在相同或不同的递送载体中，所述核酸结合分子包含SEQ ID NOs：1-70中任一项的序列。
在一些实施方案中，所述递送载体包含脂质体和/或脂质纳米颗粒。
在一些实施方案中，所述表达调节分子和/或所述核酸结合分子被配制在相同或不同的重组载体中，所述核酸结合分子包含SEQ ID NOs：1-70中任一项的序列。
在一些实施方案中，所述重组载体包含病毒载体。
在一些实施方案中，所述重组载体包含腺相关病毒(AAV)载体。
另一方面，本申请提供了一种核酸，所述核酸编码本申请的核酸结合分子和/或编码本申请的基因表达调节分子。
另一方面，本申请提供了一种重组载体，所述重组载体包含本申请的核酸。
另一方面，本申请提供了一种递送载体，所述递送载体包含本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、和/或本申请的重组载体，以及任选地包含脂质体和/或脂质纳米颗粒。
另一方面，本申请提供了一种组合物，所述组合物包含本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、和/或本申请的递送载体。
另一方面，本申请提供了一种细胞，所述细胞包含本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、本申请的递送载体、和/或本申请的组合物。
另一方面，本申请提供了一种试剂盒，所述试剂盒包含本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、本申请的递送载体、本申请的组合物、和/或本申请的细胞。
另一方面，本申请提供了一种调控目标基因表达和/或活性的方法，所述方法包括提供本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、本申请的递送载体、本申请的组合物、本申请的细胞、和/或本申请的试剂盒。
另一方面，本申请提供了一种本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、本申请的递送载体、本申请的组合物、本申请的细胞、和/或本申请的试剂盒在制备治疗和/或缓解病症的药物中的应用，所述病症包含与目标基因表达和/或活性异常相关的病症。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的 示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1A显示的是通过F11基因片段和荧光蛋白构建的报告质粒示意图。
图1B显示的是转染细胞群中绿色荧光强度较低的细胞比例的流式细胞仪检测结果。
图1C显示的是本申请的基因表达调节分子在靶向不同F11基因调控区域的表达调控效果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“核酸”与“多核苷酸”、“核苷酸”、“核苷酸序列”和“寡核苷酸”可互换地使用，其通常是指核苷酸(例如，脱氧核糖核苷酸或核糖核苷酸)和其呈单链、双链或多链形式的聚合物或其互补物。例如，核苷酸可以为核糖核苷酸、脱氧核糖核苷酸或其修饰版本。例如，核苷酸可以为单链和双链DNA、单链和双链RNA以及具有单链和双链DNA和RNA的混合物的杂交分子。例如，核苷酸可以包括但不限于任何类型的RNA，例如mRNA、siRNA、miRNA、sgRNA和引导RNA，以及任何类型的DNA、基因组DNA、质粒DNA和微环DNA以及其任何片段。所述术语还涵盖含有已知核苷酸类似物或经修饰的主链残基或键的核酸，所述核酸为合成的、天然存在的和非天然存在的。
在本申请中，术语“编码……的序列”或“编码……的核酸”通常是指包含编码蛋白质的核苷酸序列的核酸(RNA或DNA分子)。编码序列还可包括与调控元件可操作地连接的起始和终止信号，所述调控元件包含能够在对其施用了核酸的个体或哺乳动物的细胞中指导表达的启动子和多腺苷酸化信号。可对编码序列进行密码子优化。
在本申请中，术语“治疗”，例如，当用于疾病，意指当施用例如本文所述的基因表达调节分子或编码该基因表达调节分子的核酸和/或本文所述的核酸结合分子(例如gRNA)或编码该核酸结合分子的核酸时，与从未施用该基因表达调节分子或编码该基因表达调节分子的核酸和/或该核酸结合分子或编码该核酸结合分子的核酸时相比，患有疾病、有患疾病的风险和/或经历疾病症状的受试者(例如，人)在一个实施方案中将经历症状更轻和/或将更快恢复。
在本申请中，术语“DNA结合域”通常是指独立折叠的蛋白质结构域，其含有识别双链或单链DNA的至少一个基序。例如，所述DNA结合域可识别特异性DNA序列(识别或调 节序列)或具有对DNA的一般亲和性。在某些情形下，DNA结合域的其他结构域通常调节DNA结合域的活性；DNA结合功能可以是结构性的或者包括转录调节，有时这两种作用是重叠的。在根据本申请所提供的方法和基因表达调节分子的某些实施方案中，DNA结合域可包含(DNA)核酸酶，诸如能够以序列特异性方式靶向DNA或者能够被指导或指示以序列特异性方式靶向DNA的核酸酶，诸如CRISPR-Cas系统、锌指核酸酶(ZFN)、转录激活子样效应因子核酸酶(TALEN)或大范围核酸酶。在一些实施方案中，DNA结合域是源自CRISPR-Cas系统的DNA核酸酶。例如，该源自CRISPR-Cas系统的DNA核酸酶是Cas蛋白。
在本申请中，“Cas酶”可与“Cas蛋白”、“CRISPR蛋白”、“CRISPR酶”、“CRISPR-Cas蛋白”、“CRISPR-Cas酶”、“Cas”、“CRISPR效应子”或“Cas效应子蛋白”互换地使用，其通常是指与CRISPR序列互补的一类酶，能够使用CRISPR序列作为指导(guide)，从而识别和切割特定的DNA链。Cas蛋白的非限制性实例包括：Casl、CaslB、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csnl和Csxl2)、CaslO、Csyl、Csy2、Csy3、Csel、Cse2、Cscl、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmrl、Cmr3、Cmr4、Cmr5、Cmr6、Csbl、Csb2、Csb3、Csxl7、Csxl4、CsxlO、Csxl6、CsaX、Csx3、Csxl、Csxl5、Csf1、Csf2、Csf3、Csf4，和/或他们的同系物、或其修饰形式。这些蛋白是已知的，例如，化脓链球菌Cas9蛋白的氨基酸序列可见于SwissProt数据库登录号Q99ZW2下。
在本申请中，术语“dCas9酶”也被称为“失活Cas9蛋白”或“失活Cas9酶”。已知用于生成具有失活DNA切割结构域的Cas9蛋白(或其片段)的方法参见例如，Jinek等，Science.337：816-821(2012)；Qi等，“Repurposing CRISPR as an RNA-GuidedPlatform for Sequence-Specific Control of Gene Expression”，Cell.28，152(5)：1173-83(2013)，其各自的全部内容通过引用纳入本文)。例如，已知Cas9的DNA切割结构域包括两个亚结构域，HNH核酸酶亚结构域和RuvC1亚结构域。HNH亚结构域切割与gRNA互补的链，而RuvC1亚结构域切割非互补链。这些亚结构域中的突变可使Cas9的核酸酶活性沉默。例如，突变D10A和H840A使酿脓链球菌Cas9的核酸酶活性完全失活(Jinek等，Science.337：816-821(2012)；Qi等，Cell.28；152(5)：1173-83(2013))。合适的CRISPR失活或切口DNA结合结构域包括但不限于，核酸酶失活的变体Cas9结构域，其包括D10A，D10A/D839A/H840A和D10A/D839A/H840A/N863A突变结构域，如WO2015089406A1中所述，其通过引用纳入本文。在某些情形下，来自化脓性链球菌的无核酸内切酶活性的dCas9已被gRNA靶向细菌、酵母和人细胞中的基因，以通过空间位阻沉默基因表达。如本文中所用，“dCas”可指dCas蛋白或其片段。如本文中所用，“dCas9”可指dCas9蛋白或其片段。如本文中所用，术语“iCas” 和“dCas”可互换使用，指无催化活性的CRISPR相关蛋白。在一个实施方案中，dCas蛋白在DNA切割结构域中包含一个或多个突变。在一个实施方案中，dCas蛋白在RuvC或结构域中包含一个或多个突变。在一个实施方案中，dCas分子在RuvC和HNH结构域中都包含一个或多个突变。在一个实施方案中，dCas蛋白是野生型Cas蛋白的片段。在一个实施方案中，dCas蛋白包含来自野生型Cas蛋白的功能结构域，其中该功能结构域选自Reel结构域、桥螺旋结构域或PAM相互作用结构域。在一个实施方案中，与相应的野生型Cas蛋白的核酸酶活性相比，dCas的核酸酶活性降低了至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％或至少99％。
合适的dCas可衍生自野生型Cas蛋白。Cas蛋白可来自I型、II型或III型CRISPR-Cas系统。在一个实施方案中，合适的dCas可衍生自Cas1、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9或Cas10。在一个实施方案中，dCas衍生自Cas9蛋白。例如，可通过在Cas9蛋白的DNA切割结构域(例如核酸酶结构域，例如RuvC和/或HNH结构域)中引入点突变(例如，取代、缺失或添加)来获得dCas9。参见，例如，Jinek等人，Science(2012)337:816-21，通过引用以其整体并入本文。例如，在RuvC和HNH结构域中引入两个点突变降低了Cas9核酸酶活性，同时保留了Cas9sgRNA和DNA结合活性。在一个实施方案中，RuvC和HNH活性部位内的两个点突变是化脓性链球菌Cas9的D10A和H840A突变。或者，可以删除化脓性链球菌Cas9的D10和H840，以消除Cas9核酸酶活性，同时保留其sgRNA和DNA结合活性。在一个实施方案中，RuvC和HNH活性部位内的两个点突变是化脓性链球菌Cas9的D10A和N580A突变。
在各种实施方案中，本申请涉及dCas蛋白或其任何变体或突变体。dCas9的所有变体和突变体可用于本文公开的方法、组合物、融合分子或试剂盒，所述变体和突变体包括但不限于衍生自SpCas9(从化脓链球菌分离的Cas9)、SaCas9(从金黄色葡萄球菌分离的Cas9)、StCas9(从嗜热链球菌分离的Cas9)、NmCas9(从脑膜炎奈瑟菌(Neisseria meningitidis)分离的Cas9)、FnCas9(从诺维弗朗西斯菌(Francisella novicida)分离的Cas9)、CjCas9(从空肠弯曲杆菌分离的Cas9)、ScCas9(从犬链球菌(Streptococcus canis)分离的Cas9)以及上面列出的Cas9的任何变体和突变体形式，诸如高保真Cas9(Kleinstiver等人，Nature.2016年1月28日)和增强的SpCas9(Slaymaker等人，Sciences.2016年1月1日)的那些变体和突变体。例如，本申请SEQ ID NOs:1162-1179所示的dCas9序列仅提供几个示例性选项，并不是排他性的。在一个实施方案中，dCas蛋白是在D10和/或H840(如SEQ ID NO:1162所示的)处包含突变的化脓性链球菌dCas9 蛋白。在一个实施方案中，dCas蛋白是包含D10A和/或H840A突变(如SEQ ID NO:1162所示的)的化脓性链球菌dCas9蛋白。在一个实施方案中，dCas9蛋白是金黄色葡萄球菌dCas9蛋白，其包含SEQ ID NO:1163或1164所示的氨基酸序列、与SEQ ID NO:1163或1164基本相同的(例如，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％或更高的序列同一性)序列、或相对于SEQ ID NO:11631164具有1个、2个、3个、4个、5个或更多个改变(例如，氨基酸取代、插入或缺失)的序列或其任意片段。
类似的突变也可以应用于任何其它天然存在的Cas9(例如，来自其它物种的Cas9)或工程化的Cas9。在某些实施方案中，dCas9包括化脓性链球菌(Streptococcus pyogenes)dCas9、金黄色葡萄球菌(Staphylococcus aureus)dCas9、空肠弯曲菌(Campylobacter jejuni)dCas9、白喉棒状杆菌(Corynebacterium diphtheria)dCas9、凸腹真杆菌(Eubacterium ventriosum)dCas9、巴氏链球菌(Streptococcus pasteurianus)dCas9、香肠乳杆菌(Lactobacillus farciminis)dCas9、球形球毛菌(Sphaerochaeta globus)dCas9、固氮螺菌属(Azospirillum，例如，菌株B510)dCas9、嗜重氮葡糖醋杆菌(Gluconacetobacter diazotrophicus)dCas9、灰色奈瑟球菌(Neisseria cinerea)dCas9、肠道罗斯拜瑞氏菌(Roseburia intestinalis)dCas9、食清洁剂细小棒菌(Parvibaculum lavamentivorans)dCas9、卤水硝酸盐裂解菌(Nitratifractor salsuginis，例如，菌株DSM 16511)dCas9、海鸥弯曲菌(Campylobacter lari，例如，菌株CF89-12)dCas9、嗜热链球菌(Streptococcus thermophilus，例如，菌株LMD-9)dCas9或以上所述的片段。在某些实施方案中，本申请还提供了包含编码以下蛋白分子的核苷酸的载体：化脓性链球菌dCas9、金黄色葡萄球菌dCas9、空肠弯曲菌dCas9、白喉棒状杆菌dCas9、凸腹真杆菌dCas9、巴氏链球菌dCas9、香肠乳杆菌dCas9、球形球毛菌dCas9、固氮螺菌属(菌株B510)dCas9、嗜重氮葡糖醋杆菌dCas9、灰色奈瑟球菌dCas9、肠道罗斯拜瑞氏菌dCas9、食清洁剂细小棒菌dCas9、卤水硝酸盐裂解菌(菌株DSM 16511)dCas9、海鸥弯曲菌(菌株CF89-12)dCas9、嗜热链球菌(菌株LMD-9)dCas9或以上所述的片段。
在本申请中，术语“基本上不具有核酸酶活性的Cas酶”通常是指一种RNA引导的酶，其中磷酸二酯键的识别通过单独的多核苷酸序列(例如，引导RNA)促进，但所述酶可以不将目标磷酸二酯键显著地裂解(例如在生理条件下没有可测量的磷酸二酯键裂解)。例如，当与多核苷酸(例如，sgRNA)复合时，缺乏核酸酶的RNA引导的DNA核酸内切酶保留了DNA结合能力(例如，与靶序列的特异性结合)，但是缺乏显著的核酸内切酶活性。例如，缺乏核酸酶的RNA引导的DNA核酸内切酶为dCas9、ddCpf1、缺乏核酸酶的Cas9变异体或缺乏核 酸酶的II类CRISPR核酸内切酶。例如，缺乏核酸酶的RNA引导的DNA核酸内切酶为dCas9。如本文提及的术语“dCas9”或“dCas9蛋白”是其中核酸内切酶活性的两个催化位点都有缺陷或缺乏活性的Cas9蛋白。例如，dCas9基本上不具有可检测核酸内切酶(例如，脱氧核糖核酸内切酶)活性。在各方面中，例如，dCas9包含与本申请dCas9酶序列具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列一致性的氨基酸序列的变异体或同源物。
在本申请中，术语“能够结合”可与“结合于”、“特异性地识别”、“靶向”等互换地使用，通常是指结合分子(例如，本申请的基因表达调节分子)能够与靶基因或靶位点上的核苷酸相互作用，或者该结合分子(例如，本申请的基因表达调节分子)对靶基因或靶位点具有足够的亲和力，这种相互作用可以是通过缀合、偶联、附着、提供互补性、提供共价作用力或提供非共价作用力、提高结合稳定性等方式。
在本申请中，术语“转录起始位点”通常是指构建体中的核酸，其对应于整合到初级转录本(即，mRNA前体)中的第一个核酸；转录起始位点可以与启动子序列重叠。
在本申请中，术语“其片段”通常是指指定整体的部分或片段。例如，当在本申请中、相对于指定的核苷酸序列使用时，术语“其片段”是指短于指定的多核苷酸的全长序列的一段连续长度的指定核苷酸序列。指定核苷酸的一部分可以通过它的第一位置和它的最后位置得到限定，其中所述第一和最后位置各自对应于指定多核苷酸的序列中的位置，其中对应于第一位置的序列位置位于对应于最后位置的序列位置的N端，并且由此该部分的序列是指定多核苷酸中的连续的核苷酸序列，其在对应于第一位置的序列位置开始并且在对应于最后位置的序列位置结束。也可以通过参照指定多核苷酸序列中的位置和相对于参照位置的残基长度来限定一部分，由此该部分的序列是指定多核苷酸中的连续的核苷酸序列，其具有限定的长度并且根据限定的位置定位在指定多核苷酸中。
在本申请中，术语“核苷酸的修饰”可意指本发明中所述的核酸通过本领域成熟的方法，例如“Current protocols innucleic acid chemistry”Beaucage，S.L.等人，(Edrs.)，John Wiley&Sons，Inc.，NewYork，NY，USA中所描述的方法(其在此通过引用并入本文)进行合成或修饰。该修饰可包括但不限于：末端修饰，如5’-末端修饰(例如，磷酸化，缀合，反式键(inverted linkage))或3’-末端修饰(例如，缀合，DNA核苷酸，反式键等)；碱基修饰，如用稳定化的碱基、去稳定化的碱基或者与扩展的配对组库碱基配对的碱基进行置换，除去碱基(无碱基核苷酸)，或者缀合的碱基；糖修饰(例如，在2’-位或4’-位进行糖修饰)或置换糖；或者骨架修饰，包括对磷 酸二酯键进行修饰或置换。
在本申请中，术语“修饰组蛋白的物质”通常是指可对组蛋白产生修饰作用的、调节基因转录的相关酶，常见的对组蛋白产生的修饰可以是甲基化、乙酰化、磷酸化、腺苷酸化、泛素化、ADP核糖基化等。
在本申请中，术语“甲基化修饰”与“DNA甲基化”和“核酸甲基化”可互换地使用，其通常是指使本申请中基因片段、核苷酸或其碱基具有甲基化状态，该状态常出现在已经用核酸转染的细胞内部的过程，所述细胞已转染了包含与启动子有效连接的编码多肽的结构基因的核酸，在该过程中启动子核酸的胞嘧啶转化成5-甲基胞嘧啶。其中至少一个胞嘧啶转化成5-甲基胞嘧啶的启动子核酸称作“甲基化”核酸或DNA。本申请中基因所在的DNA片段可以在一条链或多条链上具有甲基化，还可以在一个位点或多个位点上具有甲基化。
在本申请中，术语“调节元件”是指能够控制核酸序列表达的遗传元件。例如，剪接信号、启动子序列、多腺苷酸化信号、转录终止序列、上游调节结构域、复制起点、内部核糖体进入位点(“IRES”)、增强子等，它们共同提供了编码序列在受体细胞中的复制、转录和翻译。并非所有这些控制序列都需要存在。真核生物中的转录控制信号通常包含“启动子”和“增强子”元件。启动子和增强子由DNA序列的短阵列组成，启动子是促进可操作连接的编码区转录起始的调控元件，增强子则通过增加位于同一DNA分子上最接近的启动子的活性从而增加遗传转录速度的调控元件，所述的这些序列特异性地与参与转录的细胞蛋白质相互作用(Maniatis等人，Science 236:1237(1987)，通过引用以其整体并入本文)。启动子和增强子元件已可以从各种真核生物来源中分离出来，包括酵母、昆虫和哺乳动物细胞以及病毒中的基因(类似的控制序列，即启动子，也在原核生物中发现)。特定启动子和增强子的选择取决于受体细胞类型。一些真核启动子和增强子具有广泛的宿主范围，而其它启动子和增强子在有限的细胞类型的亚组内具有功能(关于综述，参见，例如，Voss等人，Trends Biochem.Sci.,11:287(1986)；和Maniatis等人(同上)，通过引用以其整体并入本文)。例如，SV40早期基因增强子在来自许多哺乳动物物种的多种细胞类型中非常活跃，并已被用于在多种哺乳动物细胞中表达蛋白质(Dijkema等人，EMBO J.4:761(1985)，通过引用以其整体并入本文)。源自人延伸因子1-α基因的启动子和增强子元件(Uetsuki等人，J.Biol.Chem.,264:5791(1989)；Kim等人，Gene 91:217(1990)；和Mizushima and Nagata,Nucl.Acids.Res.,18:5322(1990))、劳斯肉瘤病毒的长末端重复序列(Gorman等人，Proc.Natl.Acad.Sci.U.S.A.79:6777(1982))和人巨细胞病毒(Boshart等人，Cell 41:521(1985))也可用于在不同的哺乳动物细胞类型中表达蛋白质，所述参考文献通过引用以其整体并入本文。启动子和增强子可以单独或一起天然存在。例如，逆转 录病毒长末端重复序列包含启动子和增强子元件。一般来说，启动子和增强子的作用独立于被转录或翻译的基因。因此，所用的增强子和启动子相对于它们可操作连接的基因可以是“内源的”、“外源的”或“异源的”。“内源性”增强子/启动子是与基因组中给定的基因天然连接的增强子/启动子。“外源”或“异源”增强子或启动子是通过遗传操作(即，分子生物学技术)与基因并置的增强子或启动子，所述并置使得该基因的转录由连接的增强子/启动子指导。表达载体上“剪接信号”的存在通常导致重组转录物的高水平表达。在某些实施方式中，“剪接信号”介导从初级RNA转录物上去除内含子，由剪接供体和受体位点组成(Sambrook等人，Molecular Cloning:A Laboratory Manual，第2版，Cold Spring Harbor Laboratory Press,New York(1989),第16.7-16.8页，通过引用以其整体并入本文)。常用的剪接供体和受体位点是来自SV40的16S RNA的剪接点。在某些实施方式中，“转录终止信号”通常存在于多腺苷酸化信号的下游，长度为数百个核苷酸。例如，术语“poly A信号”或“poly A序列”表示指导新生RNA转录物的终止和多聚腺苷酸化的DNA序列。重组转录物的高效多腺苷酸化往往是必要的，因为缺少poly A信号的转录物是不稳定的，并且会被快速降解。表达载体中使用的poly A信号可以是“异源的”或“内源的”。内源性poly A信号是天然存在于基因组中给定基因的编码区的3’末端的信号。异源poly A信号是从一个基因中分离出来并与另一个基因的3’末端可操作地连接的信号。常用的异源poly A信号是SV40poly A信号。SV40poly A信号包含在237bp的BamHI/BclI限制性片段上，并指导终止和多聚腺苷酸化(Sambrook等人，同上，16.6-16.7，通过引用以其整体并入)。
在本申请中，术语“DNA甲基转移酶”通常是指催化甲基转移至DNA的酶。DNA甲基转移酶的非限制性实例包括DNMT1、DNMT 3A、DNMT 3B和DNMT 3L。例如，通过DNA甲基化，DNA甲基转移酶可以在不更改DNA序列的情况下修饰DNA片段的活性(例如调控基因表达)。如本文所述，基因表达调节分子可以包括一个或多个(例如两个)DNA甲基转移酶。当DNA甲基转移酶作为基因表达调节分子的一部分包括在内时，DNA甲基转移酶可以被称为“DNA甲基转移酶结构域”。在各方面中，DNA甲基转移酶结构域包含与DNMT 3A具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列一致性的氨基酸序列的变异体或同源物。在各方面中，DNA甲基转移酶结构域包含与DNMT 3L具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列一致性的氨基酸 序列的变异体或同源物。
在本申请中，术语“功能活性片段”通常是指具有全长蛋白质或核酸的部分区域，但保留或部分保留全长蛋白质或核酸的生物活性或功能的片段。例如，功能活性片段可以保留或部分保留全长蛋白质结合另一种分子的能力。例如，DNA甲基转移酶的功能活性片段，可以保留或部分保留全长DNA甲基转移酶的催化甲基基团转移到DNA的生物活性功能。
在本申请中，术语“直接和/或间接连接”通常是指相对的“直接连接”或“间接连接”。术语“直接连接”通常是指直接相连或直接结合。例如，所述直接相连可以为相连的物质(例如氨基酸序列区段)之间没有间隔成分(例如氨基酸残基或其衍生物)而直接相连的情况；例如氨基酸序列区段X与另一氨基酸序列区段Y通过氨基酸序列区段X的C端氨基酸与氨基酸序列区段Y的N端氨基酸形成的酰胺键直接相连。“间接连接”通常是指相连的物质(例如氨基酸序列区段)之间有间隔成分(例如氨基酸残基或其衍生物)而间接相连的情况。
在本申请中，术语“Krab”也称为“克鲁珀相关盒结构域”或“Krüppel相关盒结构域”，其通常是指存在于人锌指蛋白的转录因子中的转录抑制结构域的约45至约75个氨基酸残基。在各方面中，Krab结构域可以包括与ZIM3 Krab结构域或KOX1 Krab结构域具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列一致性的氨基酸序列的变异体或同源物。
在本申请中，术语“递送载体”通常是指能够将试剂(例如，核酸分子)递送至靶细胞的转移媒介物。递送载体可以将试剂递送到特定的细胞亚类。例如，借助递送载体的固有特征或者通过与载体相偶联的部分、包含在其内的部分(或者与载体结合的部分，从而使得该部分和该递送载体维持在一起，进而使得该部分足以靶向递送载体)使递送载体靶向某些类型的细胞。递送载体还可提高要递送的试剂的体内半衰期和/或要递送的试剂的生物利用度。递送载体可包括病毒载体、病毒样颗粒、聚阳离子载体、肽载体、脂质体和/或杂交载体。例如，如果靶细胞是肝细胞，所述递送载体的性质(例如，尺寸、电荷和/或pH)可以有效地将所述递送载体和/或其中包载的分子递送至靶细胞、降低免疫清除和/或促进在该靶细胞中停留。
在本申请中，术语“脂质体”通常是指通过一个或多个双层的膜与外部介质隔离的具有内部空间的囊泡。在一些实施方案中，所述双层的膜可以通过两性分子形成，如包含空间隔离的亲水性和疏水性结构域的合成或天然来源的脂质；在另一些实施方案中，所述双层的膜可以通过两亲性聚合物和表面活性剂形成。在一些实施方案中，所述脂质体是球形囊泡结构，其由围绕内部水性区室的单层或多层脂质双分子层、和相对不可渗透的外部亲脂性磷脂双分 子层组成。在一些实施方案中，脂质体是生物相容的、无毒的，可以递送亲水性和亲脂性药物分子，保护它们的运载物不被血浆酶降解，并且将它们的负载运输穿过生物膜和血脑屏障(BBB)。脂质体可由几种不同类型的脂质例如磷脂制成。脂质体可包含天然磷脂和脂质(诸如1,2-二硬脂酰基-sn-甘油-3-磷脂酰胆碱(DSPC)、鞘磷脂、卵磷脂酰胆碱、单唾液酸神经节苷脂或其任意组合。为了改变脂质体的结构和性质，可向脂质体中加入几种其它添加剂。例如，脂质体还可包含胆固醇、鞘磷脂和/或1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)，例如，以增加稳定性和/或防止脂质体内部运载物的泄漏。
术语“脂质纳米颗粒(LNP)”通常是指包含通过分子间力彼此物理结合(例如，共价或非共价)的多个(即多于一个)脂质分子的颗粒。LNP可以是例如微球(包括单层和多层囊泡，例如脂质体)、乳液中的分散相、胶团或悬浮液中的内相。LNP可将核酸包封在阳离子脂质颗粒(例如，脂质体)内，并且可被相对容易地递送至细胞。在一些实例中，脂质纳米颗粒不含任何病毒组分，这有助于最小化安全性和免疫原性问题。所述脂质颗粒可用于体外、离体和体内递送。所述脂质颗粒还可用于各种规模的细胞群。本申请的LNP可通过本领域已知的各种方法，例如通过混合有机相与水相来容易地制备。两相的混合可通过微流体装置和撞击流反应器来实现。有机相和水相混合越充分，获得的LNP的包埋率和粒径分布就越好。优选地，LNP的粒径可通过改变有机相与水相的混合速度来调节。混合速度越快，制备的LNP的粒径将越小。包埋效率可通过调节LNP系统的N/P(可电离脂质/核酸)比值来优化。在一些实例中，LNP可用于递送DNA分子(例如，包含DNA结合蛋白和/或sgRNA的编码序列的分子)和/或RNA分子(例如，Cas、sgRNA的mRNA)。在某些情况下，LNP可用于递送Cas/gRNA的RNP复合物。在一些实施方案中，LNP用于递送mRNA和gRNA(例如，包含DNMT3A-DNMT3L(3A-3L)-dCas9-KRAB和至少一种靶向靶基因的sgRNA的mRNA融合分子)。
在本申请中，术语“重组载体”通常是指能够转运它以及与之连接的另一种核酸的核酸分子。一种类型的载体是“质粒”，这指另外的DNA区段可以连接到其内的环状双链DNA环。可替代地，载体可以是线性的。另一种类型的载体是病毒载体，其中另外的DNA区段可以连接到病毒基因组内。特定载体能够在它们引入其内的宿主细胞内自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如，非附加型哺乳动物载体)可以在引入宿主细胞内后整合到宿主细胞的基因组内，并且从而连同宿主基因组一起复制。
在本申请中，术语“腺相关病毒(AAV)载体”通常是指具有功能性或部分功能性ITR序列和转基因的载体。如本文中所用，术语“ITR”是指反向末端重复序列。ITR序列可源自腺相关病毒血清型，包括但不限于AAV-1、AAV-2、AAV-3、AAV-4、AAV-5、AAV-6 6、AAV7、 AAV8、AAV9、AAV10、AAV11、AAV12和AAV13，以及任何AAV变体或混合物。然而，ITR不必是野生型核苷酸序列，并且可被改变(例如，通过核苷酸的插入、缺失或取代)，只要序列保留提供功能性拯救、复制和包装的功能。AAV载体可具有一个或多个全部或部分删除的AAV野生型基因，优选为rep和/或cap基因，但保留功能性侧翼ITR序列。功能性ITR序列起到例如拯救、复制和包装AAV病毒粒子或颗粒的作用。因此，“AAV载体”在本申请中被定义为至少包括将转基因插入受试者细胞所需的那些序列。任选包括病毒复制和包装(例如，功能性ITR)所必需的那些顺式序列。
在本申请中，术语“核定位序列”或“核定位信号”或“NLS”通常是指将蛋白导向至细胞核的肽。例如，NLS包括五个碱性带正电的氨基酸。例如，NLS可以位于肽链上的任何位置。
在本申请中，术语“互补的”或“互补性”通常是指核酸通过传统的Watson-Crick或其它非传统类型与另一核酸序列形成氢键的能力。例如，序列A-G-T与序列T-C-A互补。互补性百分比指示可以与第二核酸序列形成氢键(例如，Watson-Crick碱基配对)的核酸分子中的残基百分比(例如，十分之5、6、7、8、9、10分别为50％、60％、70％、80％、90％以及100％互补)。例如，“完全互补”是指核酸序列的所有连续残基将与第二核酸序列中相同数目的连续残基氢键键合。例如，“基本上互补”是指在8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50或更多个核苷酸的区域内，或指在严格条件(即严格杂交条件)下杂交的两个核酸至少有60％、65％、70％、75％、80％、85％、90％、95％、97％、98％、99％或100％的互补性程度。
在本申请中，术语“基因”通常是指设计产生蛋白的DNA片段。例如，所述基因还可以包括编码区之前和之后的区域(前导和尾部)以及各个编码片段(外显子)之间的插入序列(内含子)。前导、尾部和内含子包括基因转录和转译过程中必需的调节元件。此外，“蛋白基因产物”可以为由特定基因表达的蛋白。
在本申请中，术语“多肽”、“肽”和“蛋白”在本文中可互换地使用以指代氨基酸残基的聚合物。例如，其中所述聚合物可以在各方面中结合至不由氨基酸组成的部分。例如，“融合蛋白”可以是指编码以重组方式表达为单个部分的两个或更多个单独蛋白序列的嵌合蛋白。在两个或更多个核酸或多肽序列的情况下，术语“相同”或百分比“一致性”是指以如下所述的默认参数来使用BLAST或BLAST 2.0序列比较算法或通过人工比对和目测检查而测量的。例如，相同的或具有指定百分比的相同氨基酸残基或核苷酸(在比较窗口或指定区域上针对最大对应性进行比较和比对时，在指定区域内具有约60％的一致性，优选65％、70％、 75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的一致性)的两个或更多个序列或子序列，此类序列可以被称为“基本上相同”。
在本申请中，术语“引导RNA”或“gRNA”通常是指与靶多核苷酸序列具有足够互补性以与靶序列杂交，并且将CRISPR复合物特异性结合至靶序列的任何多核苷酸序列。在各方面中，当使用合适的比对算法最佳比对时，引导序列与其对应的靶序列之间的互补性程度为约或大于约50％、约60％、约75％、约80％、约85％、约90％、约95％、约97.5％、约99％或更高。
本申请中，所述的特定蛋白(例如KRAB、dCas9、Dnmt3A、Dnmt3L)，可以包括所述蛋白的任何天然形式或维持所述蛋白活性(例如，与天然蛋白相比，具有至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％的活性)的变异体或同源物。在各方面中，与天然存在的形式相比，变异体或同源物在整个序列或一部分序列(例如50、100、150或200个连续氨基酸部分)上具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％的氨基酸序列一致性。
在本申请中，术语“接头”通常是指包括连接2个或更多部分的连接子。在各实施例中，接头在N端和C端连接至化合物的其余部分的氨基酸序列(例如本文所提供的融合蛋白)。例如，如本文所使用的术语“XTEN”、“XTEN接头”或“XTEN多肽”是指缺乏疏水性氨基酸残基的重组多肽。
在本申请中，术语“可检测剂”或“可检测部分”是可以通过适当方式检测的组合物。例如，所述方式为，例如光谱、光化学、生物化学、免疫化学、化学、磁共振成像或其它物理方式。例如，有用的可检测试剂包括放射性元素、荧光团(例如荧光染料)、电子致密试剂、酶(例如在ELISA中常用的)、生物素、顺磁性分子等等。
在本申请中，术语“抑制”、“阻遏”、“沉默”等通常是指基因表达和/或活性的降低。例如，相对于在无物质(例如融合蛋白、复合物、核酸、载体)存在的情况下(对照)的核酸序列的活性，施用本申请的物质可以对核酸序列的活性产生负面影响(例如降低)。例如，抑制可以是指疾病或疾病症状的减少。例如，抑制包括至少部分地、部分地或完全地阻断核酸序列的活化(例如转录)，或降低、预防或延迟核酸序列的活化。例如，抑制活性可以为对照中的90％、80％、70％、60％、50％、40％、30％、20％、10％或更低。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“选自”通常是指包括选择的对象以及其所有组合。例如“选自(：)A、 B和C”意指包括A、B和C的所有组合，例如，A、B、C、A+B、A+C、B+C或A+B+C。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下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％、或10％的范围内变动。
发明详述
一方面，本申请提供了一种调控F11基因表达和/或活性的方法，所述方法包括提供一种基因表达调节分子或编码所述基因表达调节分子的核酸，所述基因表达调节分子可以具有调节所述F11基因的表达而不更改其基因序列的功能。例如，本申请的方法可以是非治疗目的的方法。例如，本申请的方法可以是不直接以人体为使用对象的方法。例如，本申请的方法可以是体外的或离体的方法。例如，本申请的方法可以是治疗目的的方法。例如，本申请的方法可以是体内的方法。
另一方面，本申请提供了一种治疗和/或缓解与F11基因表达和/或F11基因活性异常相关的病症的方法，所述方法包括提供一种基因表达调节分子或编码所述基因表达调节分子的核酸，所述基因表达调节分子可以具有调节所述F11基因的表达而不更改其基因序列的功能。
另一方面，本申请提供了一种基因表达调节分子或编码所述基因表达调节分子的核酸，所述基因表达调节分子可以具有调节所述F11基因的表达而不更改起基因序列的功能。例如，所述核酸包含DNA和/或mRNA。例如，所述基因表达调节分子或编码所述基因表达调节分子的核酸可用于治疗和/或缓解F11基因表达和/或F11基因活性异常相关的病症。
另一方面，本申请提供了一种基因表达调节分子或编码所述基因表达调节分子的核酸在制备用于治疗和/或缓解与F11基因表达和/或F11基因活性异常相关的病症的药物中的用途，所述基因表达调节分子可以具有调节所述F11基因的表达而不更改其基因序列的功能。例如，所述编码本申请的基因表达调节分子的核酸包含DNA和/或mRNA。
另一方面，本申请提供一种核酸结合分子或编码所述核酸结合分子的核酸，所述核酸结合分子包含SEQ ID NOs：1-70中任一项所示的序列。例如，所述编码本申请的核酸结合分子的核酸包含DNA和/或mRNA。
另一方面，本申请提供了一种重组载体，所述重组载体包含本申请的核酸。例如，重组载体可以是指能够转运与其连接的另一种核酸的核酸分子。重组载体可以包括单链、双链或部分双链的核酸分子；包含一个或多个游离端，没有游离端(例如，环状)的核酸分子；包含DNA、RNA或两者的核酸分子；和本领域已知的其他种类的多核苷酸。例如，可以使用病 毒载体。病毒载体可包含病毒衍生的DNA或RNA序列，用于包装成病毒(例如逆转录病毒、复制缺陷型逆转录病毒、腺病毒、复制缺陷型腺病毒、和腺相关病毒AAV)。病毒和病毒载体可用于体外、离体和/或体内递送。
另一方面，本申请提供了一种递送载体，所述递送载体包含本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、和/或本申请的重组载体，以及任选地包含脂质体和/或脂质纳米颗粒(LNP)。例如，递送载体可以包含一种或多种Cas蛋白和一种或多种指导RNA，例如，以核糖核蛋白复合物(RNP)的形式。例如，核糖核蛋白可以通过基于多肽的穿梭剂递送。例如，核糖核蛋白可以使用合成肽递送。例如，可以通过物理递送方法将递送载体引入细胞。物理方法的例子包括显微注射、电穿孔和流体动力学递送。例如，LNPs可以将核酸包裹在阳离子脂质颗粒(例如脂质体)中，并且可以相对容易地递送至细胞。在一些例子中，脂质纳米颗粒不含任何病毒成分，这有助于最大限度地减少安全性和免疫原性问题。脂质颗粒可用于体外、离体和体内递送。LNP的成分可包括阳离子脂质，可电离的脂质，聚乙二醇化脂质和/或支持脂质，以及任选的胆固醇组分。在一些实施方案中，LNP可包含可电离脂质(20％-70％，摩尔比)、聚乙二醇化脂质(0％-30％，摩尔比)、支持脂质(30％-50％，摩尔比)和胆固醇(10％-50％，摩尔比)。
另一方面，本申请提供了一种组合物，所述组合物包含本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、和/或本申请的递送载体。例如，组合物中的核酸结合分子、基因表达调节分子、编码所述核酸结合分子的核酸、编码所述基因表达调节分子的核酸、重组载体和递送载体可以同时包含在一个组合物中，或者分别包含在不同组合物中。例如，当使用组合物中的核酸结合分子、基因表达调节分子、编码所述核酸结合分子的核酸、编码所述基因表达调节分子的核酸、重组载体和/或递送载体时，可以同时使用，或者分开使用。
另一方面，本申请提供了一种细胞，所述细胞包含本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、本申请的递送载体、和/或本申请的组合物。
另一方面，本申请提供了一种试剂盒，所述试剂盒包含本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、本申请的递送载体、本申请的组合物、和/或本申请的细胞。
另一方面，本申请提供了一种调控目标基因表达和/或活性的方法，所述方法包括提供本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、本 申请的递送载体、本申请的组合物、本申请的细胞、和/或本申请的试剂盒。例如，所述方法可以降低目标基因的表达和/或活性。例如，相比于本申请物质不存在的情况下的目标基因的表达和/或活性，施用本申请的物质可以对核酸序列的活性产生负面影响(例如降低)，例如可以包括至少部分地、部分地或完全地阻断核酸序列的活化(例如转录)，或降低、预防或延迟核酸序列的活化。例如，抑制活性可以为对照中的约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约10％或更低。
另一方面，本申请提供了一种本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、本申请的递送载体、本申请的组合物、本申请的细胞、和/或本申请的试剂盒在制备治疗和/或缓解病症的药物中的应用，所述病症包含与目标基因表达和/或活性异常相关的病症。
另一方面，本申请提供了一种本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、本申请的递送载体、本申请的组合物、本申请的细胞、和/或本申请的试剂盒，其用于治疗和/或缓解病症，所述病症包含与目标基因表达和/或活性异常相关的病症。
另一方面，本申请提供了一种治疗和/或缓解病症的方法，所述方法包括提供本申请的核酸结合分子、本申请的基因表达调节分子、本申请的核酸、本申请的重组载体、本申请的递送载体、本申请的组合物、本申请的细胞、和/或本申请的试剂盒，所述病症包含与目标基因表达和/或活性异常相关的病症。在一些实施方案中，治疗是针对器官的疾病/病症，示例性地包括肝病、眼病、肌肉病、心脏病、血液病、脑病、肾病，或可以包括对自身免疫病、中枢神经系统疾病的治疗、癌症和其他增殖性疾病、神经退行性疾病、炎症性疾病、代谢疾病、肌肉骨骼疾病等。
基因表达调节分子
本申请或本申请方法中提供的基因表达分子具有调节F11基因的表达而不更改其基因序列的功能。例如，所述基因表达调节分子可以具有抑制基因表达的功能。
在一些实施方案中，所述基因表达调节分子包含DNA结合域。例如，所述基因表达调节分子可以具有结合基因序列的功能。例如，所述DNA结合域包含(DNA)核酸酶，所述核酸酶可以是以序列特异性方式靶向DNA的核酸酶；示例性地，可以是CRISPR-Cas系统相关酶、锌指核酸酶(ZFN)、转录激活因子样效应核酸酶(TALEN)或大范围核酸酶。例如，所述基因表达调节分子可以包含TALEN的DNA结合域、锌指域、和/或CRISPR/Cas系统的DNA结合域。在某些情形下，DNA结合域是源自CRISPR-Cas系统的DNA核酸酶。在某些情形 下，DNA结合域是(修饰的)转录激活因子样效应核酸酶(TALEN)系统；而转录激活因子样效应子(TALE)可以被设计成几乎可以结合任何所需的DNA序列。在某些情形下，DNA结合域是(修饰的)锌指核酸酶(ZFN)系统或由其组成；所述ZFN系统使用通过将锌指DNA结合结构域与DNA切割结构域融合而产生的人工限制酶，该DNA切割结构域可通过工程改造以靶向所需的DNA序列。在某些情形下，DNA结合域是(修饰的)大范围核酸酶，其是以大的识别位点(12至40个碱基对的双链DNA序列)为特征的内切脱氧核糖核酸酶。
在一些实施方案中，所述基因表达调节分子可包含Cas酶。例如，所述基因表达调节分子可包含基本上不具有核酸酶活性的Cas酶。通常，指导序列(或间隔序列)可以是与靶多核苷酸序列具有足够互补性以与靶序列杂交并指导CRISPR复合物与靶序列的序列特异性结合的任何多核苷酸序列。在某些情形下，当使用合适的比对算法进行最佳比对时，指导序列与其对应的靶序列之间的互补程度为约或大于约50％、60％、70％、75％、80％、85％,90％,91％,92％,93％,94％,95％,96％,97％,98％,99％或更多。在一些实施方案中，靶向核酸的Cas蛋白可以相对于相应的野生型酶发生突变，使得突变的靶向核酸的Cas蛋白缺乏切割靶多核苷酸的一条或两条链的能力。例如，突变酶的DNA切割活性约为酶的非突变形式的核酸切割活性的不超过25％、10％、5％、1％、0.1％、0.01％或更少。在一些实施方案中，突变的Cas可具有一个或多个突变，导致脱靶效应降低。
例如，所述基因表达调节分子可以包含Cas9酶。Cas蛋白的实例包括I类(例如I型、III型和IV型)和2类(例如II型、V型和VI型)Cas蛋白，例如Cas9、Cas12(例如Cas12a、Cas12b、Cas12c、Cas12d)、Cas13(例如，Cas13a、Cas13b、Cas13c、Cas13d)、CasX、CasY、Cas14、它们的变体(例如，突变形式、截短形式)、它们的同源物和它们的直系同源物。在一些实施方案中，Cas蛋白是Cas9、Cas12a、Cas12b、Cas12c或Cas12d。在一些实施例中，Cas9可以是SpCas9、SaCas9、StCas9和其他Cas9直系同源物。Cas12可以是Cas12a、Cas12b和Cas12c，包括FnCas12a，或其同源物或直系同源物。例如，Cas9酶可以包含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(例如，B510)dCas9、Gluconacetobacter diazotrophicus dCas9、Neisseria cinerea dCas9、Roseburia intestinalis dCas9、Parvibaculum lavamentivorans dCas9、Nitratifractor salsuginis(例如，DSM 16511)dCas9、Campylobacter lari(例如，CF89-12)dCas9、和/或Streptococcus thermophilus(e.g.,strain LMD-9)dCas9。例如，所述基因表达调节分子可以包含dCas9的氨基酸序列。或者，相对于以上 dCas9序列可以具有一个、两个、三个、四个、五个或更多个变化，例如氨基酸取代、插入或缺失的序列，或其任何片段。
在一些实施方案中，所述基因表达调节分子能够结合F11基因的转录起始位点(TSS)的上游和/或下游500bp范围内的DNA区域或其片段。例如，所述基因表达调节分子能够结合所述TSS上游的约100bp、约200bp、约300bp、约400bp、约500bp、约600bp、约700bp、约800bp、约900bp、约1000bp、约1100bp、约1200bp、约1300bp、约1400bp或约1500bp内。例如，所述基因表达调节分子能够结合所述TSS下游的约100bp、约200bp、约300bp、约400bp、约500bp、约600bp、约700bp、约800bp、约900bp、约1000bp、约1100bp、约1200bp、约1300bp、约1400bp或约1500bp内。例如，所述基因表达调节分子能够结合SEQ ID NOs：71-72中任一项所在DNA区域或其片段。
示例性地，在某些情形下，所述基因表达调节分子能够结合下列所述的F11基因的转录起始位点(TSS)附近的一个或多个DNA区域：TSS的上游250bp至上游130bp之间、TSS的上游50bp至下游120bp之间、和TSS的下游230bp至下游390bp之间。
在一些实施方案中，所述基因表达调节分子能够结合下列所述的F11基因的转录起始位点(TSS)附近的一个或多个DNA区域：TSS的上游250bp至上游230bp之间(例如，TSS的上游248-229bp之间)、TSS的上游210bp至上游180bp之间(例如，TSS的上游202-185bp之间)、TSS的上游160bp至上游130bp之间(例如，TSS的上游156-137bp之间)、TSS的上游50bp至下游10bp之间(例如，TSS的上游44-25bp之间、和/或上游12bp至下游8bp之间)、TSS的下游30bp至下游60bp之间(例如，TSS的下游37-56bp之间)、TSS的下游90bp至下游120bp之间(例如，TSS的下游96-115bp之间)、TSS的下游230bp至下游260bp之间(例如，TSS的下游233-252bp之间)、TSS的下游300bp至下游320bp之间(例如，TSS的下游301-320bp之间)、和TSS的下游360bp至下游390bp之间(例如，TSS的下游366-385bp之间)。
在一些实施方案中，所述基因表达调节分子包含第一功能域，所述第一功能域提供F11基因附近和/或F11基因调节元件内的至少一个核苷酸的修饰。在某些实施方案中，所述第一功能域在靶基因的调节元件例如启动子、增强子或转录起始位点处可以通过表观遗传修饰调节靶基因表达，例如通过组蛋白乙酰化或甲基化，或DNA甲基化。例如，调控元件可以包含转录起始位点、核心启动子、近端启动子、远端增强子、沉默子、绝缘子元件、边界元件或基因座控制区。例如，所述表观遗传修饰可以是通过任何已知的可用于DNA甲基化的表观遗传修饰调节剂，示例性的表观遗传修饰调节剂可包含DNA甲基转移酶(例如，DNMT3A或 DNMT3A-DNMT3L)、DNA去甲基化酶(例如，TET1催化结构域或TDG)和/或它们的功能活性片段。
例如，所述第一功能域可以提供F11基因附近和/或F11基因调节元件内的至少一个核苷酸的修饰，所述至少一个核苷酸的修饰包含甲基化修饰。在一些实施方案中，所述第一功能域包含表观遗传修饰调节剂，其可以具有DNA甲基化酶活性。例如，所述表观遗传修饰调节剂可能具有甲基化酶活性，其涉及将甲基基团转移到DNA、RNA、蛋白质、小分子、胞嘧啶或腺嘌呤。例如，修饰可以位于目标基因的转录起始位点上游的约100bp、约200bp、约300bp、约400bp、约500bp、约600bp、约700bp、约800bp、约900bp、约1000bp、约1100bp、约1200bp、约1300bp、约1400bp或约1500bp内。例如，修饰可以位于目标基因的转录起始位点下游的约100bp、约200bp、约300bp、约400bp、约500bp、约600bp、约700bp、约800bp、约900bp、约1000bp、约1100bp、约1200bp、约1300bp、约1400bp或约1500bp内。
例如，所述第一功能域可以包含DNA甲基转移酶(DNMT)结构域。例如，所述第一功能域可以包含DNMT 3A结构域和/或DNMT 3L结构域。例如，所述DNMT 3A结构域和/或DNMT 3L结构域来源于哺乳动物。例如，所述DNMT 3A结构域来源于小鼠。例如，所述基因表达调节分子可以包含DNMT 3A所述的氨基酸序列。例如，所述DNMT 3L结构域来源于人和/或小鼠。例如，所述基因表达调节分子可以包含DNMT 3L的氨基酸序列。或者，相对于以上序列可以具有一个、两个、三个、四个、五个或更多个变化，例如氨基酸取代、插入或缺失的序列，或其任何片段。
例如，所述DNMT 3A结构域和所述DNMT 3L结构域直接和/或间接连接。例如，所述DNMT 3A结构域和所述DNMT 3L结构域通过接头连接。例如，所述DNMT 3A结构域的C端与所述DNMT 3L的N端连接，或者所述DNMT 3L结构域的C端与所述DNMT 3A的N端连接。
在一些实施方案中，所述基因表达调节分子包含第二功能域，所述第二功能域包含基于锌指蛋白的转录因子或其功能活性片段，或者包含能够修饰组蛋白的物质。例如，所述第二功能域可以包含基因表达阻遏物，其可以是任何已知的基因表达阻遏物，示例性的基因表达阻遏物可选自Krüppel相关框(KRAB)结构域、mSin3相互作用结构域(SID)、MAX相互作用蛋白1(MXI1)、染色体阴影结构域、EAR-阻遏结构域(SRDX)、真核释放因子1(ERF1)、真核释放因子3(ERF3)、四环素阻遏物、lad阻遏物、长春花G-box结合因子1和2、Drosophila Groucho(果蝇Gro蛋白)、含有三方基序的28(TRTM28)、核受体共阻遏物1、核受体共阻遏物2、或它们的功能活性片段或融合物。例如，所述第二功能域可以包含能够修饰组蛋白的物 质的功能。在某些实施方案中，所述第二功能域可以包含Krab结构域。具体地，所述第二功能域可以包含ZIM3 Krab结构域或KOX1 Krab结构域。
在某些实施方案中，KRAB结构域或其片段可以融合到dCas9分子的C端。在某些实施方案中，KRAB结构域或其片段可以融合到dCas9分子的N端和C端。在某些实施方案中，所述第二功能域可以包含一个KRAB结构域，该结构域可以包含与ZIM3 Krab或KOX1 Krab基本上相同(例如，至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或更高同一性)、或者与ZIM3 Krab或KOX1 Krab具有一、二、三、四、五或更多个变化(例如，相对于ZIM3 Krab或KOX1 Krab的氨基酸取代、插入或缺失)的序列，或其任何片段。
在一些实施方案中，所述第二功能域可包含基因表达激活剂，其可以是任何已知的基因表达激活剂，示例性的基因表达激活剂可包含VP16激活域、VP64激活域、p65激活域、爱泼斯坦-巴尔病毒R反式激活因子Rta分子或其片段。
在一些实施方案中，所述第二功能域可包含能够修饰组蛋白的物质，所述修饰组蛋白的物质可包含组蛋白乙酰转移酶(例如，p300催化结构域)、组蛋白脱乙酰酶、组蛋白甲基转移酶(例如，SUV39H1或G9a(EHMT2))、组蛋白去甲基化酶(例如LSD1)、和/或它们的功能活性片段。
在上述的某些情形下，所述基因表达调节分子包含所述第一功能域和所述第二功能域，且所述第一功能域直接或间接地连接在所述第二功能域的一端，或者所述第一功能域直接和/或间接地连接在所述第二功能域的两端。例如，所述第一功能域的C端直接连接在所述第二功能域的N端，或者所述第一功能域的C端间接地连接(例如，通过接头)在所述第二功能域的N端，或者所述第一功能域位于所述第二功能域的N端一侧(例如，可以从N端到C端依次是第一功能域、其他例如DNA结合域的部分、以及第二功能域的顺序)。例如，所述第二功能域的C端直接连接在所述第一功能域的N端，或者所述第二功能域的C端间接地连接(例如，通过接头)在所述第一功能域的N端，或者所述第二功能域位于所述第一功能域的N端一侧(例如，可以从N端到C端依次是第二功能域、其他例如DNA结合域的部分、以及第一功能域的顺序)。例如，所述第一功能域包含两种或两种以上的功能域，且它们可分别直接连接在、间接连接在(例如，通过接头)、或者位于所述第二功能域的N端一侧和C端一侧(例如，可以从N端到C端依次是第一功能域a、其他例如DNA结合域的部分、第二功能域、以及第一功能域b的顺序，或者是第一功能域a、第二功能域、其他例如DNA结合域的部分、以及第一功能域b的顺序，其中a和b为不同种类的第一功能域)。
在一些实施方案中，所述第一功能域和所述第二功能域直接或间接地连接在所述DNA结合域的一端。例如，所述第一功能域和所述第二功能域直接或间接地连接在所述DNA结合域的C端，示例性地，所述基因调节分子可包含dCas9-DNMT 3A-DNMT 3L-Krab、dCas9-DNMT 3L-DNMT 3A-Krab、dCas9-Krab-DNMT 3A-DNMT 3L、dCas9-Krab-DNMT 3L-DNMT 3A中的任一项。例如，所述第一功能域和所述第二功能域直接或间接地连接在所述DNA结合域的N端，示例性地，所述基因调节分子可包含DNMT 3A-DNMT 3L-Krab-dCas9、DNMT 3L-DNMT 3A-Krab-dCas9、Krab-DNMT 3A-DNMT 3L-dCas9、Krab-DNMT 3L-DNMT 3A-dCas9中的任一项。在另一些实施方案中，所述第一功能域和所述第二功能域直接或间接地连接在所述DNA结合域的两端。例如，所述第一功能域直接或间接地连接在所述DNA结合域的C端，且所述第二功能域直接或间接地连接在所述DNA结合域的N端，示例性地，所述基因调节分子可包含Krab-dCas9-DNMT 3A-DNMT 3L和Krab-dCas9-DNMT 3L DNMT 3A中的任一项。例如，所述第一功能域直接或间接地连接在所述DNA结合域的N端，且所述第二功能域直接或间接地连接在所述DNA结合域的C端，示例性地，所述基因调节分子可包含DNMT 3A-DNMT 3L-dCas9-Krab和DNMT 3L-DNMT 3A-dCas9-Krab中的任一项。
在一些实施方案中，所述基因表达调节分子还可以包含用于检测、分离和/或纯化的标签。例如，所述基因表达调节分子可以包含HA tag。例如，所述基因表达调节分子可以包含HA tag的氨基酸序列。或者，相对于以上序列具有一个、两个、三个、四个、五个或更多个变化，例如氨基酸取代、插入或缺失的序列，或其任何片段。
在一些实施方案中，所述基因表达调节分子还可以包含核定位序列。例如，所述核定位序列可以包含具有正电基团的氨基酸。例如，所述核定位序列可以包含本领域已知的核定位序列的氨基酸序列。或者，相对于以上序列可以具有一个、两个、三个、四个、五个或更多个变化，例如氨基酸取代、插入或缺失的序列，或其任何片段。例如，所述核定位序列可以位于所述第一功能域的N端和/或C端、所述第二功能域的N端和/或C端、和/或所述DNA结合域的N端和/或C端。
在一些实施方案中，所述基因表达调节分子还可以包含可检测部分。例如，所述可检测部分可以包含蓝色荧光蛋白和或绿色荧光蛋白。例如，所述可检测部分与所述第一功能域、所述第二功能域、所述DNA结合域、和/或所述核定位序列可以通过自剪切肽连接。例如，所述自剪切肽可以包含2A肽。
在本申请中，涉及的所述第一功能域、所述第二功能域、所述DNA结合域以及其他所述基因调节分子所包含的元件可通过间接的方式连接，例如可通过一定长度的接头序列连接。 例如，所述接头可包含约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个或更多个氨基酸。例如，所述第一功能域和第二功能域通过长度为约80个或更多个氨基酸的接头连接。例如，所述第一功能域和第二功能域通过长度为约92个或更多个氨基酸的接头连接。例如，所述第一功能域和第二功能域通过XTEN接头连接。例如，所述接头包含XTEN接头的氨基酸序列。例如，所述接头包含XTEN接头的氨基酸序列。例如，所述接头包含16个氨基酸长度的XTEN接头、80个氨基酸长度的XTEN接头或更长的XTEN接头的氨基酸序列。或者，相对于以上序列具有一个、两个、三个、四个、五个或更多个变化，例如氨基酸取代、插入或缺失的序列，或其任何片段。
核酸结合分子
本申请还提供一种核酸结合分子，所述核酸结合分子可以包含SEQ ID NOs：1-70中任一项的序列。例如，所述核酸结合分子和/或本申请的基因表达调节分子可以通过局部注射、全身输注或其组合递送至受试者。在各方面中，所述核酸结合分子可以包含与SEQ ID NOs：1-70中任一项的序列具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列一致性的序列。
在一些实施方案中，所述核酸结合分子与本申请的基因表达调节分子的组合可具有调节F11基因表达水平的能力。例如，所述核酸结合分子和/或本申请的基因表达调节分子可以结合F11基因的核心启动子、近端启动子、远端增强子、沉默子、绝缘子元件、边界元件和/或基因座控制区。例如，所述核酸结合分子和/或本申请的基因表达调节分子可以结合F11基因的转录起始位点的上游和/或下游500bp范围内的DNA区域或其片段。例如，所述核酸结合分子和/或本申请的基因表达调节分子可以结合SEQ ID NOs：71-72中任一项所在DNA区域或其片段。在各方面中，可以结合的DNA区域可以包含与SEQ ID NOs：71-72中任一项所在DNA区域具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列一致性的序列。
例如，所述核酸结合分子和/或本申请的基因表达调节分子可以结合SEQ ID NOs：73-142中任一项所在DNA区域或其片段。在各方面中，可以结合的DNA区域可以包含与SEQ ID NOs：73-142中任一项所在DNA区域具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至 少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列一致性的序列。例如，靶向SEQ ID NOs：73-142中任一项所在DNA区域中任意20bp左右的区域，可以具有调节F11基因表达水平的能力。例如，相比于本申请物质不存在的情况下的目标基因表达和/或活性，施用本申请的物质可以对核酸序列的活性产生负面影响(例如降低)，例如可以包括至少部分地、部分地或完全地阻断核酸序列的活化(例如转录)，或降低、预防或延迟核酸序列的活化。例如，抑制活性可以为对照中的约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约10％或更低。
在某些实施方案中，所述核酸结合分子的指导序列或间隔区长度可以为15至50个核苷酸。在某些实施方案中，所述核酸结合分子为指导RNA(gRNA)，所述指导RNA的间隔区长度可以为至少15个核苷酸。在某些实施方案中，间隔区长度可以为15至17个核苷酸的长度、17至20个核苷酸的长度、20至24个核苷酸的长度、23至25个核苷酸的长度、24至27个核苷酸的长度、27至30个核苷酸的长度、30至35个核苷酸的长度，或大于35个核苷酸的长度。在一些实施方案中，施用的gRNA的数量可以是至少1种gRNA、至少2种不同gRNA、至少3种不同gRNA、至少4种不同gRNA、至少5种不同gRNA。在某些实施方案中，靶结合区的长度可以在约19至约21个核苷酸之间。在一个实施方案中，靶结合区的长度可以为15、16、17、18、19、20、21、22、23、24或25个核苷酸。在一个实施方案中，靶结合区可以与靶基因中的靶区互补，例如完全互补。在一个实施方案中，靶结合区可以与靶基因中的靶区基本上互补。在一个实施方案中，靶结合区可以包含不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个与靶基因中的靶区不互补的核苷酸。在一些实施方案中，所述核酸结合分子和/或本申请的基因表达调节分子可以被配制在脂质体或脂质纳米颗粒中。在一些实施方案中，所述核酸结合分子和/或本申请的基因表达调节分子可以被配制在病毒载体中。例如，指导RNA可以包括具有一种或多种化学修饰(例如，通过化学连接两个核糖核苷酸或通过用一种或多种脱氧核糖核苷酸替换一种或多种核糖核苷酸)的基于RNA的分子。例如，可以靶向来源于人的靶结合区。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的产品、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1
本申请的基因调节分子在报告细胞系中的表观调控效率
(1)实验方法：本实施例将human F11基因转录起始位点前后500bp(共1000bp)的序列，插入到表达绿色荧光载体的荧光蛋白前，构建了报告质粒(图1A)，然后将报告质粒稳定整合到HEK293T细胞中，得到报告系统细胞系。同时，通过构建插入红色荧光蛋白mCherry+的引导RNA(gRNA)质粒，在上述这段1000bp的序列上设计多条引导序列，用以将不同表观编辑工具(带有蓝色荧光蛋白的编辑工具质粒)引导到这些gRNA对应的靶位点上。该引导过程通过细胞转染将带有蓝色荧光的编辑工具质粒和带有红色荧光的gRNA质粒共同转染到报告细胞系中。
(2)实验材料及设计
本实施例的报告系统DNA序列(标准字体：启动子区域，粗体：F11基因调控区，斜体：绿色荧光蛋白(EGFP)序列)如下：

编辑工具EpiRegA(Dnmt3a-Dnmt3l-dCas9-KOX1 KRAB)的DNA序列(斜体/下划线斜体：Dnmt3a/Dnmt3l，粗体：dCas9，粗斜体：KRAB，下划线粗斜体：蓝色荧光蛋白，标准字体：连接序列)如下：




编辑工具EpiRegA(Dnmt3a-Dnmt3l-dCas9-KOX1 KRAB)的氨基酸序列(斜体/下划线斜 体：Dnmt3a/Dnmt3l，粗体：dCas9，粗斜体：KRAB，下划线粗斜体：蓝色荧光蛋白，标准字体：连接序列，*：任意氨基酸)如下：

编辑工具EpiRegB(Dnmt3a-Dnmt3l-ZIM3 KRAB-dCas9)的DNA序列(斜体/下划线斜体：Dnmt3a/Dnmt3l，粗体：dCas9，粗斜体：KRAB，下划线粗斜体：蓝色荧光蛋白，标准字体：连接序列)如下：




编辑工具EpiRegB(Dnmt3a-Dnmt3l-ZIM3 KRAB-dCas9)的氨基酸序列(斜体/下划线斜体：Dnmt3a/Dnmt3l，粗体：dCas9，粗斜体：KRAB，下划线粗斜体：蓝色荧光蛋白，标准字体：连接序列，*：任意氨基酸)如下：


编辑工具EpiRegC(dCas9-ZIM3 KRAB-DNMT3L-Dnmt3a)的DNA序列(斜体/下划线斜体：Dnmt3a/Dnmt3l，粗体：dCas9，粗斜体：KRAB，下划线粗斜体：蓝色荧光蛋白，标准字体：连接序列)如下：




编辑工具EpiRegC(dCas9-ZIM3 KRAB-DNMT3L-Dnmt3a)的氨基酸序列(斜体/下划线斜体：Dnmt3a/Dnmt3l，粗体：dCas9，粗斜体：KRAB，下划线粗斜体：蓝色荧光蛋白，标准字体：连接序列，*：任意氨基酸)如下：


(3)检测结果：本实施例采用的阳性对照(PC)组的gRNA直接靶向EGFP(SEQ ID NO：153)，当目标F11基因的表达受到抑制时，EGFP的强度将相应地下降。通过流式细胞仪检测同时转染了蓝光和红光的细胞群中的绿色荧光较低的细胞比例(图1B)，再将转染了不同gRNA的样品的比例减去转染非靶向调控区域的NT gRNA(SEQ ID NO：154)样品组，得到不同编辑工具在使用不同gRNA时的相对调控效率。从图1C所示的调控效率结果可知，SEQ ID NOs：22、23、24、26、34、35、38、45、50和62(它们的靶向区域5’端分别起始于TSS的上游44bp、上游248bp、下游37bp、下游96bp、上游12bp、上游156bp、下游233bp、上游204bp、下游301bp和下游366bp)所示的gRNA结合本实施例的表观编辑工具，可以有效抑制F11基因的表达水平。
表1靶向人F11基因的sgRNA
实施例2
参照序列SEQ ID NOs：1-70中任一项所示的核苷酸序列，构建表观遗传编辑工具中的引导RNA，靶向目标基因；同时根据本申请提供的基因表达调节分子，构建表观遗传编辑工具。例如，可以靶向F11转录起始位点(TSS)上游和下游500bp内的区域。
将引导RNA质粒与基因表达调节分子质粒共转染到小鼠细胞系中。72小时后，前10％的GFP+和mCherry+细胞通过FACS进行分类。进行RT-QPCR实验以评估目标基因的mRNA表达水平。
结果显示，转染的细胞可以显示出F11的表达产物的表达量降低。例如，转染的细胞可以显示出F11转录的mRNA表达量降低。例如，转染的细胞可以显示出F11表达的蛋白产物的表达量降低。例如，使用本申请的编辑方法，可以缓解F11基因相关的疾病或病症。
实施例3
表观遗传靶点的通用性
将本申请的表观遗传编辑工具，用于靶点的表达调控。其中基因表达调节分子中具有结合基因序列的功能的结构域，可以替换为本领域已知的TALEN的DNA结合域、锌指域、tetR的DNA结合域、大范围核酸酶和/或任意的CRISPR/Cas系统的核酸酶域，如dCas12酶等等。
结果显示，本申请的表观遗传靶点具有通用性，使用各种基因序列结合结构域都具有较高的调控基因表达的效果。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (47)

  1. 一种调控F11基因表达和/或活性的方法，所述方法包括提供一种基因表达调节分子或编码所述基因表达调节分子的核酸，所述基因表达调节分子具有调节所述F11基因的表达而不更改其基因序列的功能。
  2. 一种治疗和/或缓解与F11基因表达和/或F11基因活性异常相关的病症的方法，所述方法包括提供一种基因表达调节分子或编码所述基因表达调节分子的核酸，所述基因表达调节分子具有调节所述F11基因的表达而不更改其基因序列的功能。
  3. 根据权利要求1-2中任一项所述的方法，所述基因表达调节分子包含DNA结合域。
  4. 根据权利要求1-3中任一项所述的方法，所述基因表达调节分子包含选自TALEN结构域、锌指结构域和CRISPR/Cas系统的蛋白结构域的一个或多个DNA结合域。
  5. 根据权利要求1-4中任一项所述的方法，所述基因表达调节分子包含Cas酶。
  6. 根据权利要求1-5中任一项所述的方法，所述基因表达调节分子包含基本上不具有核酸酶活性的Cas酶。
  7. 根据权利要求1-6中任一项所述的方法，所述基因表达调节分子包含dCas9酶。
  8. 根据权利要求1-7中任一项所述的方法，所述基因表达调节分子能够结合F11基因的转录起始位点(TSS)的上游和/或下游500bp范围内的DNA区域或其片段。
  9. 根据权利要求1-8中任一项所述的方法，所述基因表达调节分子能够结合SEQ ID NOs：71-72中任一项所在DNA区域或其片段。
  10. 根据权利要求1-9中任一项所述的方法，所述基因表达调节分子能够结合下列所述的F11基因的转录起始位点(TSS)附近的一个或多个DNA区域：TSS的上游250bp至上游130bp之间、TSS的上游50bp至下游120bp之间、和TSS的下游230bp至下游390bp之间。
  11. 根据权利要求1-10中任一项所述的方法，所述基因表达调节分子能够结合下列所述的F11基因的转录起始位点(TSS)附近的一个或多个DNA区域：TSS的上游250bp至上游230bp之间、TSS的上游210bp至上游180bp之间、TSS的上游160bp至上游130bp之间、TSS的上游50bp至下游10bp之间、TSS的下游30bp至下游60bp之间、TSS的下游90bp至下游120bp之间、TSS的下游230bp至下游260bp之间、TSS的下游300bp至下游320bp之间、和TSS的下游360bp至下游390bp之间。
  12. 根据权利要求1-11中任一项所述的方法，所述方法包括提供一种核酸结合分子，所述核酸结合分子包含SEQ ID NOs：1-70中任一项的序列。
  13. 根据利要求12所述的方法，所述基因表达调节分子和/或所述核酸结合分子被配制在相同或不同的递送载体中。
  14. 根据权利要求13所述的方法，所述递送载体包含脂质体和/或脂质纳米颗粒。
  15. 根据权利要求12-14中任一项所述的方法，所述表达调节分子和/或所述核酸结合分子被配制在相同或不同的重组载体中。
  16. 根据权利要求15所述的方法，所述重组载体包含病毒载体。
  17. 根据权利要求15-16中任一项所述的方法，所述重组载体包含腺相关病毒载体。
  18. 根据权利要求1-17中任一项所述的方法，所述基因表达调节分子包含第一功能域，所述第一功能域提供F11基因附近和/或F11基因调节元件内的至少一个核苷酸的修饰。
  19. 根据权利要求18所述的方法，所述至少一个核苷酸的修饰包含甲基化修饰。
  20. 根据权利要求18-19中任一项所述的方法，所述调节元件包含核心启动子、近端启动子、远端增强子、沉默子、绝缘子元件、边界元件和/或基因座控制区。
  21. 根据权利要求18-20中任一项所述的方法，所述第一功能域包含DNA甲基转移酶、DNA去甲基化酶、和其功能活性片段中的一种或多种。
  22. 根据权利要求21所述的方法，所述DNA甲基转移酶包含DNMT 3A、DNMT 3B、DNMT3L、DNMT 1和DNMT 2的一种或多种。
  23. 根据权利要求22所述的方法，所述DNMT 3A来源于小鼠。
  24. 根据权利要求22-23中任一项所述的方法，所述DNMT 3L来源于人和/或小鼠。
  25. 根据权利要求22-24中任一项所述的方法，所述DNMT 3A和所述DNMT 3L直接和/或间接连接。
  26. 根据权利要求1-25中任一项所述的方法，所述基因表达调节分子包含第二功能域，所述第二功能域包含基于锌指蛋白的转录因子或其功能活性片段，或者包含能够修饰组蛋白的物质。
  27. 根据权利要求26所述的方法，所述第二功能域包含Krab。
  28. 根据权利要求26-27中任一项所述的方法，所述第二功能域包含ZIM3 Krab或KOX1 Krab。
  29. 根据权利要求26所述的方法，所述第二功能域包含组蛋白甲基转移酶、组蛋白去甲基化酶、组蛋白乙酰转移酶、组蛋白脱乙酰酶、和其功能活性片段中的一种或多种。
  30. 根据权利要求26-29中任一项所述的方法，所述第一功能域和所述第二功能域直接或间接地连接在所述DNA结合域的一端，或者所述第一功能域和所述第二功能域直接和/或间接地连接在所述DNA结合域的两端。
  31. 根据权利要求1-30中任一项所述的方法，所述基因表达调节分子包含核定位序列。
  32. 根据权利要求31所述的方法，所述核定位序列包含具有正电基团的氨基酸。
  33. 根据权利要求31-32中任一项所述的方法，所述核定位序列位于所述第一功能域的N端和/或C端、所述第二功能域的N端和/或C端、和/或所述DNA结合域的N端和/或C端。
  34. 一种核酸结合分子，所述核酸结合分子包含SEQ ID NOs：1-70中任一项的序列。
  35. 一种基因表达调节分子，所述基因表达调节分子具有调节所述F11基因的表达而不更改其基因序列的功能。
  36. 根据权利要求35所述的基因表达调节分子，所述基因表达调节分子如权利要求1-33中任一项所述的方法中所提供的基因表达调节分子。
  37. 根据权利要求35-36中任一项所述的基因表达调节分子，所述基因表达调节分子和/或核酸结合分子被配制在相同或不同的递送载体中，所述核酸结合分子包含SEQ ID NOs：1-70中任一项的序列。
  38. 根据权利要求37所述的基因表达调节分子，所述递送载体包含脂质体和/或脂质纳米颗粒。
  39. 根据权利要求35-38中任一项所述的基因表达调节分子，所述表达调节分子和/或所述核酸结合分子被配制在相同或不同的重组载体中，所述核酸结合分子包含SEQ ID NOs：1-70中任一项的序列。
  40. 根据权利要求39所述的基因表达调节分子，所述重组载体包含病毒载体。
  41. 根据权利要求39-40中任一项所述的基因表达调节分子，所述重组载体包含腺相关病毒载体。
  42. 一种核酸，所述核酸编码权利要求34所述的核酸结合分子和/或编码权利要求35-41中任一项所述的基因表达调节分子。
  43. 一种重组载体，所述重组载体包含权利要求42所述的核酸。
  44. 一种递送载体，所述递送载体包含权利要求34所述的核酸结合分子、权利要求35-41中任一项所述的基因表达调节分子、权利要求42所述的核酸、和/或权利要求43所述的重组载体，以及任选地包含脂质体和/或脂质纳米颗粒。
  45. 一种组合物，所述组合物包含权利要求34所述的核酸结合分子、权利要求35-41中任一项所述的基因表达调节分子、权利要求42所述的核酸、权利要求43所述的重组载体、和/或权利要求44所述的递送载体。
  46. 一种细胞，所述细胞包含权利要求34所述的核酸结合分子、权利要求35-41中任一项所述的基因表达调节分子、权利要求42所述的核酸、权利要求43所述的重组载体、权利要求44所述的递送载体、和/或权利要求45所述的组合物。
  47. 一种试剂盒，所述试剂盒包含权利要求34所述的核酸结合分子、权利要求35-41中任一项所述的基因表达调节分子、权利要求42所述的核酸、权利要求43所述的重组载体、权利要求44所述的递送载体、权利要求45所述的组合物、和/或权利要求46所述的细胞。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104769112A (zh) * 2012-11-01 2015-07-08 菲克特生物科学股份有限公司 用于在细胞中表达蛋白质的方法和产品
CN108220274A (zh) * 2017-12-11 2018-06-29 上海交通大学医学院附属瑞金医院 一种高活性凝血因子xi突变体及其基因治疗/编辑载体、重组/融合蛋白的制备与应用
CN113195002A (zh) * 2018-10-02 2021-07-30 桑格摩生物治疗股份有限公司 工程化的遗传调节物
WO2022028457A1 (zh) * 2020-08-04 2022-02-10 上海拓界生物医药科技有限公司 抑制凝血因子XI表达的siRNA、组合物及其医药用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104769112A (zh) * 2012-11-01 2015-07-08 菲克特生物科学股份有限公司 用于在细胞中表达蛋白质的方法和产品
CN108220274A (zh) * 2017-12-11 2018-06-29 上海交通大学医学院附属瑞金医院 一种高活性凝血因子xi突变体及其基因治疗/编辑载体、重组/融合蛋白的制备与应用
CN113195002A (zh) * 2018-10-02 2021-07-30 桑格摩生物治疗股份有限公司 工程化的遗传调节物
WO2022028457A1 (zh) * 2020-08-04 2022-02-10 上海拓界生物医药科技有限公司 抑制凝血因子XI表达的siRNA、组合物及其医药用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NUÑEZ, J.K. ET AL.: "Genome-wide Programmable Transcriptional Memory by CRISPR-based Epigenome Editing", CELL, vol. 184, 29 April 2021 (2021-04-29), XP086555165, DOI: 10.1016/j.cell.2021.03.025 *
ZHANG, WENLU: "EDITAS In-Vitro Gene Editing Medicine Enters Clinical Practice", CAITONG SECURITIES - NUCLEIC ACID DRUG INDUSTRY BIWEEKLY NEWSPAPER, 2 August 2022 (2022-08-02), pages 1 - 9, XP009553984 *

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