WO2024032678A1

WO2024032678A1 - Method for epigenome editing of targets and use thereof

Info

Publication number: WO2024032678A1
Application number: PCT/CN2023/112094
Authority: WO
Inventors: 张宝弘; 罗浩; 韦翔; 王鹏程
Original assignee: 益杰立科(上海)生物科技有限公司
Priority date: 2022-08-11
Filing date: 2023-08-10
Publication date: 2024-02-15

Abstract

A method for epigenome editing of targets and use thereof. Specifically, provided are a method for regulating the expression level of a target gene and a nucleic acid-binding molecule. The combination of the nucleic acid-binding molecule and a gene expression-regulating molecule has the capability of regulating the expression level of the target gene.

Description

A method and use for epigenetically editing targets

Technical field

This application relates to the field of biomedicine, specifically to an epigenetic editing target and its use.

Background technique

Fusion of catalytically inactive "deactivated Cas9 (dCas9)" to the Kruppel-associated box (KRAB) domain creates a gene expression repressor that is highly specific and selective for target genes in cell culture experiments. Effective conditioning or silencing. However, there are certain challenges for this repressor to exert therapeutic effects in the in vivo environment. For example, safety, toxicity, immunogenicity, and off-target effects are other challenges that limit the use of synthetic repressors in vivo.

Therefore, there is a need in this field for an effective epigenetic editing target. Targeting this target can be used to improve the effect and safety of epigenetic editing and reduce toxicity, immunogenicity and/or off-target effects.

Contents of the invention

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. For example, 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.

On the one hand, 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.

On the other hand, 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.

In some embodiments, the gene expression modulating molecule comprises a DNA binding domain.

In some embodiments, 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.

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

In some embodiments, the gene expression modulating molecule comprises a Cas enzyme that has substantially no nuclease activity.

In some embodiments, the gene expression modulating molecule comprises a dCas9 enzyme.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the methods include providing a nucleic acid binding molecule comprising the sequence of any one of SEQ ID NOs: 1-70.

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

In some embodiments, the delivery vehicle comprises liposomes and/or lipid nanoparticles.

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

In some embodiments, the recombinant vector comprises a viral vector.

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

In some embodiments, 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.

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

In some embodiments, 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.

In some embodiments, the first functional domain includes a DNA methyltransferase, a DNA demethylase, and a functional One or more active fragments.

In some embodiments, the DNA methyltransferase comprises one or more of DNMT 3A, DNMT 3B, DNMT 3L, DNMT1 and DNMT 2.

In some embodiments, the DNMT 3A is derived from mice.

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

In some embodiments, the DNMT 3A and the DNMT 3L are directly and/or indirectly connected.

In some embodiments, 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.

In some embodiments, the second functional domain includes Krab.

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

In some embodiments, 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.

In some embodiments, the first functional domain and the second functional domain are directly or indirectly linked to one end of the DNA binding domain.

In some embodiments, the gene expression modulating molecule comprises a nuclear localization sequence.

In some embodiments, the nuclear localization sequence comprises an amino acid having an electropositive group.

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

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

On the other hand, 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.

In some embodiments, the gene expression modulating molecule is as provided in the methods of the present application.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the recombinant vector comprises a viral vector.

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

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

On the other hand, 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.

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

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

On the other hand, 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.

On the other hand, 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. Vector, delivery vehicle of the present application, composition of the present application, cell of the present application, and/or kit of the present application.

On the other hand, the present application provides a nucleic acid binding molecule of the present application, a gene expression regulating molecule of the present application, a nucleic acid of the present application, a recombinant vector of the present application, a delivery vector of the present application, a composition of the present application, 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.

Those skilled in the art will readily appreciate other aspects and advantages of the present application from the detailed description below. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will realize, the contents of this application enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention covered by this application. Accordingly, the drawings and descriptions of the present application are illustrative only and not restrictive.

Description of drawings

The specific features of the invention to which this application relates are set forth in the appended claims. Described in detail below with reference to The exemplary embodiments and drawings provide a better understanding of the features and advantages of the invention to which this application relates. A brief description of the drawings is as follows:

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.

Detailed ways

The implementation of the invention of the present application will be described below with specific examples. Those familiar with this technology can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "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. For example, the nucleotides may be ribonucleotides, deoxyribonucleotides, or modified versions thereof. For example, the 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. For example, 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.

In this application, the term "sequence encoding" or "nucleic acid encoding" generally refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may also include 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.

In this application, the term "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.

In this application, the term "DNA-binding domain" generally refers to an independently folded protein domain containing at least one motif that recognizes double-stranded or single-stranded DNA. For example, the DNA binding domain may recognize a specific DNA sequence (recognize or modulate sequence) or have general affinity for DNA. In some cases, 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. In certain embodiments of the methods and gene expression modulating molecules provided in accordance with the present application, 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. In some embodiments, the DNA binding domain is a DNA nuclease derived from the CRISPR-Cas system. For example, the DNA nuclease derived from the CRISPR-Cas system is a Cas protein.

In this application, "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. Non-limiting examples of Cas proteins include: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, 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 pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

In this application, the term "dCas9 enzyme" is also referred to as "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). For example, 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. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28; 152(5): 1173-83 ( 2013)). Suitable CRISPR 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. In some cases, 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. As used herein, "dCas" may refer to a dCas protein or fragment thereof. As used herein, "dCas9" may refer to a dCas9 protein or fragment thereof. As used herein, the term "iCas" Used interchangeably with "dCas" to refer to CRISPR-related proteins that have no catalytic activity. In one embodiment, the dCas protein contains one or more mutations in the DNA cleavage domain. In one embodiment, the dCas protein contains one or more mutations in the RuvC or domain. In one embodiment, the dCas molecule contains one or more mutations in both the RuvC and HNH domains. In one embodiment, the dCas protein is a fragment of wild-type Cas protein. In one embodiment, the dCas protein comprises a functional domain from a wild-type Cas protein, wherein the functional domain is selected from the group consisting of a Reel domain, a bridged helix domain, or a PAM interaction domain. In one embodiment, the nuclease activity of dCas is reduced by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% 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. In one embodiment, a suitable dCas may be derived from Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 or Cas10. In one embodiment, dCas is derived from Cas9 protein. For example, dCas9 can be obtained by introducing point mutations (eg, substitutions, deletions or additions) in the DNA cleavage domain (eg, nuclease domain, eg, RuvC and/or HNH domain) of the Cas9 protein. See, eg, Jinek et al., Science (2012) 337:816-21, incorporated herein by reference in its entirety. For example, introducing two point mutations in the RuvC and HNH domains reduced Cas9 nuclease activity while retaining Cas9 sgRNA and DNA binding activities. In one embodiment, the two point mutations within the RuvC and HNH active sites are the D10A and H840A mutations of S. pyogenes Cas9. Alternatively, D10 and H840 of S. pyogenes Cas9 can be deleted to eliminate Cas9 nuclease activity while retaining its sgRNA and DNA binding activities. In one embodiment, the two point mutations within the RuvC and HNH active sites are the D10A and N580A mutations of S. pyogenes Cas9.

In various embodiments, 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. 28 January 2016) and those variants and mutants of enhanced SpCas9 (Slaymaker et al., Sciences. 1 January 2016). For example, the dCas9 sequences shown in SEQ ID NOs: 1162-1179 of this application only provide a few exemplary options and are not exclusive. In one embodiment, the dCas protein is Streptococcus pyogenes dCas9 comprising mutations at D10 and/or H840 (as set forth in SEQ ID NO: 1162) protein. In one embodiment, the dCas protein is a Streptococcus pyogenes dCas9 protein comprising the D10A and/or H840A mutations (as set forth in SEQ ID NO: 1162). In one embodiment, the dCas9 protein is a Staphylococcus aureus dCas9 protein comprising the amino acid sequence set forth in SEQ ID NO: 1163 or 1164, 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.

Similar mutations can also be applied to any other naturally occurring Cas9 (eg, Cas9 from other species) or engineered Cas9. In certain embodiments, dCas9 includes Streptococcus pyogenes dCas9, Staphylococcus aureus dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheria dCas9, E. Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, Azospirillum (e.g., strain B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia intestinalis dCas9, food detergent Parvibaculum lavamentivorans dCas9, brine nitric acid Nitratifractor salsuginis (e.g., strain DSM 16511) dCas9, Campylobacter lari (e.g., strain CF89-12) dCas9, Streptococcus thermophilus (e.g., strain LMD-9) dCas9 or all of the above fragments of the narrative. In certain embodiments, 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.

In this application, the term "Cas enzyme having substantially no nuclease activity" generally refers to an RNA-guided enzyme in which recognition of phosphodiester bonds is facilitated by a separate polynucleotide sequence (e.g., guide RNA), However, the enzyme may not significantly cleave the target phosphodiester bond (eg, no measurable phosphodiester bond cleavage under physiological conditions). For example, when complexed with a polynucleotide (e.g., sgRNA), a nuclease-deficient RNA-guided DNA endonuclease retains DNA-binding ability (e.g., specific binding to a target sequence) but lacks significant intranuclease binding. Dicer activity. For example, 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. The term "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. For example, dCas9 has essentially no detectable endonuclease (eg, endodeoxyribonuclease) activity. In various aspects, for example, 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.

In this application, 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.

In this application, the term "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.

In this application, the term "fragment thereof" generally refers to a portion or fragment of the specified whole. For example, when used in this application with respect to a specified nucleotide sequence, the term "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.

In the present application, the term "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.

In this application, 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.

In this application, the term "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.

In this application, the term "regulatory element" refers to a genetic element capable of controlling the expression of a nucleic acid sequence. For example, splicing signals, promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites ("IRES"), enhancers, etc., 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. Some 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). For example, 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. USA 79:6777 (1982)), and human giant cells Viruses (Boshart et al., Cell 41:521 (1985)), which reference is incorporated by reference in its entirety, can also be used to express proteins in different mammalian cell types. Promoters and enhancers may occur naturally alone or together. For example, reverse The viral long terminal repeat contains promoter and enhancer elements. In general, promoters and enhancers act independently of the gene being transcribed or translated. Thus, 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. In certain embodiments, 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). Commonly used splice donor and acceptor sites are the splice junctions of the 16S RNA from SV40. In certain embodiments, a "transcription termination signal" typically exists downstream of the polyadenylation signal and is hundreds of nucleotides in length. For example, the term "poly A signal" or "poly A sequence" refers to a DNA sequence that directs the termination and polyadenylation of 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).

In this application, the term "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. For example, through DNA methylation, DNA methyltransferases can modify the activity of DNA fragments (such as regulating gene expression) without changing the DNA sequence. As described herein, gene expression regulatory molecules may include one or more (eg, two) DNA methyltransferases. When a DNA methyltransferase is included as part of a gene expression regulatory molecule, the DNA methyltransferase may be referred to as a "DNA methyltransferase domain." In various aspects, 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. In various aspects, 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.

In this application, the term "functionally active fragment" generally refers to a fragment that has a partial region of a full-length protein or nucleic acid, but retains or partially retains the biological activity or function of the full-length protein or nucleic acid. For example, a functionally active fragment may retain or partially retain the ability of the full-length protein to bind another molecule. For example, 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.

In this application, the terms "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. For example, 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).

In this application, 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. In various aspects, 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.

As used herein, the term "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. Targeting the delivery vector to certain types of cells. 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. For example, if the target cells are hepatocytes, the properties of the delivery vehicle (e.g., size, charge, and/or pH) 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.

In this application, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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). 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. In order to change the structure and properties of the liposomes, several other additives can be added to the liposomes. For example, 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.

The term "lipid nanoparticle (LNP)" 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. Preferably, 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. The entrapment efficiency can be optimized by adjusting the N/P (ionizable lipid/nucleic acid) ratio of the LNP system. In some examples, LNPs can be used to deliver DNA molecules (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. In some embodiments, 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).

In this application, the term "recombinant vector" generally refers to a nucleic acid molecule capable of transporting it and another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA ring within which additional DNA segments can be ligated. Alternatively, the vector may be linear. Another type of vector is 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.

In this application, the term "adeno-associated virus (AAV) vector" generally refers to a vector having functional or partially functional ITR sequences and a transgene. As used herein, the term "ITR" refers to inverted terminal 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. However, 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).

In this application, the term "nuclear localization sequence" or "nuclear localization signal" or "NLS" generally refers to a peptide that directs a protein to the nucleus. For example, NLS includes five basic positively charged amino acids. For example, NLS can be located anywhere on the peptide chain.

In this application, the term "complementary" or "complementarity" 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. For example, the 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). For example, "perfectly 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. For example, "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%.

In this application, the term "gene" generally refers to a DNA segment designed to produce a protein. For example, 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. Additionally, a "protein gene product" may be a protein expressed by a specific gene.

In this application, the terms "polypeptide,""peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. For example, wherein the polymer may in various aspects be bound to a moiety that does not consist of an amino acid. For example, a "fusion protein" may refer to a chimeric protein encoding two or more separate protein sequences recombinantly expressed as a single part. In the case of two or more nucleic acid or polypeptide sequences, the term "identical" or percent "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. For example, 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".

As used herein, the term "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. . In various aspects, 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.

In this application, the specific protein (for example, KRAB, dCas9, Dnmt3A, Dnmt3L) 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). In various aspects, the variant or homolog has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% Amino acid sequence identity.

In this application, the term "linker" is generally intended to include a linker that joins two or more moieties. In various embodiments, 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). For example, the term "XTEN", "XTEN linker" or "XTEN polypeptide" as used herein refers to a recombinant polypeptide lacking hydrophobic amino acid residues.

In this application, the term "detectable agent" or "detectable moiety" refers to a composition that can be detected by suitable means. Such means are, for example, spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging or other physical means. For example, 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.

In this application, the terms "inhibit", "repress", "silence" and the like generally refer to the reduction of gene expression and/or activity. For example, 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) . For example, suppression may refer to a reduction of a disease or symptoms of a disease. For example, 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. For example, the inhibitory activity may be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less of the control.

In this application, the term "comprising" generally means the inclusion of explicitly specified features, but not the exclusion of other elements.

In this application, the term "selected from" is generally intended to include selected objects and all combinations thereof. For example "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.

In this application, 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%.

Detailed description of the invention

On the one hand, 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. For example, the methods of the present application may be non-therapeutic methods. For example, the method of the present application may not directly target the human body. For example, the methods of the present application may be in vitro or ex vivo methods. For example, the methods of the present application may be methods for therapeutic purposes. For example, the methods of the present application may be in vivo methods.

On the other hand, 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.

On the other hand, 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. For example, the nucleic acid includes DNA and/or mRNA. For example, the gene expression regulatory molecule or the nucleic acid encoding the gene expression regulatory molecule can be used to treat and/or alleviate conditions related to abnormal F11 gene expression and/or F11 gene activity.

On the other hand, 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. For purposes, the gene expression regulating molecule may have the function of regulating the expression of the F11 gene without changing its gene sequence. For example, the nucleic acid encoding the gene expression regulatory molecule of the present application includes DNA and/or mRNA.

On the other hand, 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. For example, the nucleic acid encoding the nucleic acid binding molecule of the present application includes DNA and/or mRNA.

On the other hand, the present application provides a recombinant vector comprising the nucleic acid of the present application. For example, a recombinant vector may refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is 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 and viral vectors can be used for in vitro, ex vivo and/or in vivo delivery.

On the other hand, 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). For example, 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). For example, ribonucleoproteins can be delivered via polypeptide-based shuttle agents. For example, ribonucleoproteins can be delivered using synthetic peptides. For example, the delivery vector can be introduced into the cell by physical delivery methods. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery. For example, LNPs can encapsulate nucleic acids in cationic lipid particles (such as liposomes) and can be delivered to cells relatively easily. In some cases, 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. In some embodiments, 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).

On the other hand, the present application provides a composition comprising the nucleic acid binding molecule of the present application, the gene expression regulating molecule of the present application, the nucleic acid of the present application, the recombinant vector of the present application, and/or the present application. Delivery vehicle. For example, 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. For example, 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.

On the other hand, 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. For example, the methods can reduce the expression and/or activity of a gene of interest. For example, 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. For example, the inhibitory activity can be about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or less of the control.

On the other hand, the present application provides a nucleic acid binding molecule of the present application, a gene expression regulating molecule of the present application, a nucleic acid of the present application, a recombinant vector of the present application, a delivery vector of the present application, a composition of the present application, 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.

On the other hand, 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. In some embodiments, 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.

gene expression regulatory molecules

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. For example, the gene expression regulatory molecule may have the function of inhibiting gene expression.

In some embodiments, the gene expression modulating molecule comprises a DNA binding domain. For example, the gene expression regulatory molecule may have the function of binding to a gene sequence. For example, 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. For example, 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. In some cases, the DNA binding domain is a DNA nuclease derived from the CRISPR-Cas system. in some cases Below, 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. In some cases, 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. In some cases, 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) .

In some embodiments, the gene expression regulatory molecule may comprise a Cas enzyme. For example, the gene expression modulating molecule may comprise a Cas enzyme that has substantially no nuclease activity. Generally, a guide sequence (or spacer 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. In some cases, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 70%, 75%, 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, 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. For example, 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. In some embodiments, a mutated Cas can have one or more mutations that result in reduced off-target effects.

For example, the gene expression regulatory molecule may comprise a Cas9 enzyme. Examples of 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. In some embodiments, the Cas protein is Cas9, Cas12a, Cas12b, Cas12c, or Cas12d. In some embodiments, Cas9 can be SpCas9, SaCas9, StCas9, and other Cas9 orthologs. Cas12 can be Cas12a, Cas12b and Cas12c, including FnCas12a, or homologs or orthologs thereof. For example, 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, Campylobacter lari (eg, CF89-12) dCas9, and/or Streptococcus thermophilus (eg, strain LMD-9) dCas9. For example, the gene expression regulatory molecule may comprise the amino acid sequence of dCas9. Or, relative to the above 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.

In some embodiments, 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. For example, 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. For example, 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. For example, the gene expression regulatory molecule can bind to the DNA region or fragments thereof located in any one of SEQ ID NOs: 71-72.

Exemplarily, in some cases, 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.

In some embodiments, 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 downstream of TSS), between 230 bp downstream of TSS and 260 bp downstream (for example, between 233-252 bp downstream of TSS between), between 300 bp downstream of the TSS and 320 bp downstream of the TSS (e.g., between 301-320 bp downstream of the TSS), and between 360 bp downstream of the TSS and 390 bp downstream (e.g., between 366-385 bp downstream of the TSS).

In some embodiments, 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. In certain embodiments, 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. For example, 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. For example, 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.

For example, 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. In some embodiments, the first functional domain comprises an epigenetic modification modulator, which may have DNA methylase activity. For example, the epigenetic modification modulator may have methylase activity, which involves the transfer of methyl groups to DNA, RNA, proteins, small molecules, cytosine, or adenine. For example, 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. For example, 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.

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

For example, the DNMT 3A domain and the DNMT 3L domain are directly and/or indirectly connected. For example, the DNMT 3A domain and the DNMT 3L domain are connected through a linker. For example, 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.

In some embodiments, 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. For example, 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. For example, the second functional domain may comprise a substance capable of modifying histones. qualitative function. In certain embodiments, the second functional domain can comprise a Krab domain. Specifically, the second functional domain may comprise a ZIM3 Krab domain or a KOX1 Krab domain.

In certain embodiments, 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. In certain embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some of the above situations, 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. For example, 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 the order of two functional domains). For example, 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). For example, 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. and the C-terminal side (for example, from the N-terminus to the C-terminus, it can be the sequence of the 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 sequence of domain a, a second functional domain, other parts such as a DNA binding domain, and a first functional domain b, where a and b are different kinds of first functional domains).

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

In some embodiments, the gene expression modulating molecule may further comprise a tag for detection, isolation and/or purification. For example, the gene expression regulatory molecule can include an HA tag. For example, 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.

In some embodiments, the gene expression modulating molecule may further comprise a nuclear localization sequence. For example, the nuclear localization sequence may comprise amino acids with electropositive groups. For example, the nuclear localization sequence may comprise the amino acid sequence of a nuclear localization sequence known in the art. Alternatively, the sequence may have one, two, three, four, five or more changes relative to the above sequence, such as amino acid substitutions, insertions or deletions, or any fragment thereof. For example, the nuclear localization sequence may be located at the N-terminus and/or C-terminus of the first functional domain, the N-terminus and/or C-terminus of the second functional domain, and/or the N-terminus of the DNA binding domain. and/or C-terminal.

In some embodiments, the gene expression modulating molecule may further comprise a detectable moiety. For example, the detectable moiety may comprise blue fluorescent protein and/or green fluorescent protein. For example, 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. For example, the self-cleaving peptide may comprise 2A peptide.

In this application, 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. For example, 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. For example, the first functional domain and the second functional domain are connected by a linker of about 80 or more amino acids in length. For example, the first functional domain and the second functional domain are connected by a linker of about 92 or more amino acids in length. For example, the first functional domain and the second functional domain are connected through an XTEN linker. For example, the linker comprises the amino acid sequence of an XTEN linker. For example, the linker comprises the amino acid sequence of an XTEN linker. For example, the linker 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. 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.

nucleic acid binding molecules

The present application also provides a nucleic acid binding molecule, which may comprise the sequence of any one of SEQ ID NOs: 1-70. For example, 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. In various aspects, 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.

In some embodiments, 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. For example, the nucleic acid binding molecule and/or the gene expression regulating molecule of the present application can bind to the core promoter, proximal promoter, distal enhancer, silencer, insulator element, boundary element and/or locus control of the F11 gene district. For example, 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. For example, the nucleic acid binding molecule and/or the gene expression regulating molecule of the present application can bind to the DNA region or fragment thereof located in any one of SEQ ID NOs: 71-72. In various aspects, the DNA region that can 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.

For example, 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. In various aspects, 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. For example, 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. For example, 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. For example, the inhibitory activity can be about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or less of the control.

In certain embodiments, the guide sequence or spacer of the nucleic acid binding molecule can be 15 to 50 nucleotides in length. In certain embodiments, the nucleic acid binding molecule is a guide RNA (gRNA), and the guide RNA can have a spacer length of at least 15 nucleotides. In certain embodiments, the spacer length can be 15 to 17 nucleotides in length, 17 to 20 nucleotides in length, 20 to 24 nucleotides in length, 23 to 25 nucleotides in length. length, 24 to 27 nucleotides in length, 27 to 30 nucleotides in length, 30 to 35 nucleotides in length, or greater than 35 nucleotides in length. In some embodiments, the number of gRNAs administered can be at least 1 gRNA, at least 2 different gRNAs, at least 3 different gRNAs, at least 4 different gRNAs, at least 5 different gRNAs. In certain embodiments, the 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. In one embodiment, 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. For example, 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. For example, a target binding region of human origin can be targeted.

Without intending to be limited by any theory, the following examples are only to illustrate the products, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1

The apparent regulatory efficiency of the gene regulatory molecules of the present application in reporter cell lines

(1) Experimental method: In this example, a sequence of 500 bp before and after the transcription start site of the human F11 gene (1000 bp in total) was inserted in front of the fluorescent protein expressing the green fluorescent vector to construct a reporter plasmid (Figure 1A), and then the reporter plasmid was The plasmid was stably integrated into HEK293T cells to obtain a reporter system cell line. At the same time, by constructing a guide RNA (gRNA) plasmid that inserts the red fluorescent protein mCherry+, multiple guide sequences are designed on the above 1000 bp sequence to combine different epigenetic editing tools (editing tool plasmids with blue fluorescent protein ) are guided to the target sites corresponding to these gRNAs. This guided process co-transfects a blue fluorescent editing tool plasmid and a red fluorescent gRNA plasmid into a reporter cell line via cell transfection.

(2)Experimental materials and design

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:

Amino acid sequence of editing tool EpiRegA (Dnmt3a-Dnmt3l-dCas9-KOX1 KRAB) (italics/underlined italics) Font: Dnmt3a/Dnmt3l, bold: dCas9, bold italic: KRAB, underlined bold italic: blue fluorescent protein, standard font: connecting sequence, *: any amino acid) 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:

(3) Test results: The gRNA of the positive control (PC) group used in this example directly targets EGFP (SEQ ID NO: 153). When the expression of the target F11 gene is inhibited, the intensity of EGFP will decrease accordingly. Use flow cytometry to detect the proportion of cells with lower green fluorescence in the cell population that was simultaneously transfected with blue light and red light (Figure 1B), and then subtract the transfection non-targeting regulation from the proportion of samples transfected with different gRNAs. Regional NT gRNA (SEQ ID NO: 154) sample set to obtain the relative regulatory efficiency of different editing tools when using different gRNA. From the regulatory efficiency results shown in Figure 1C, it can be seen that 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.

Table 1 sgRNA targeting human F11 gene

Example 2

Refer to the nucleotide sequence shown in any one of SEQ ID NOs: 1-70 to construct 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. For example, 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.

The results showed that the transfected cells showed reduced expression of the F11 expression product. For example, transfected cells may show reduced mRNA expression of F11 transcripts. For example, transfected cells may show reduced expression of the protein product of F11 expression. For example, using the editing method of the present application, diseases or conditions related to the F11 gene can be alleviated.

Example 3

The versatility of epigenetic targets

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.

The foregoing detailed description is provided by way of explanation and example, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments described herein will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims

A method for regulating the expression and/or activity of the F11 gene, the method comprising providing a gene expression regulating molecule or a nucleic acid encoding the gene expression regulating molecule, the gene expression regulating molecule having the function of regulating the expression of the F11 gene Does not change the function of its genetic sequence.
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 a nucleic acid encoding the gene expression regulatory molecule, the gene expression The regulatory molecule has the function of regulating the expression of the F11 gene without changing its gene sequence.
According to the method of any one of claims 1-2, the gene expression regulatory molecule comprises a DNA binding domain.
According to the method of any one of claims 1-3, 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.
According to the method of any one of claims 1-4, the gene expression regulatory molecule comprises a Cas enzyme.
According to the method of any one of claims 1-5, the gene expression regulatory molecule comprises a Cas enzyme that substantially has no nuclease activity.
According to the method of any one of claims 1-6, the gene expression regulating molecule comprises a dCas9 enzyme.
According to the method of any one of claims 1 to 7, the gene expression regulatory molecule is capable of binding to the DNA region within 500 bp upstream and/or downstream of the transcription start site (TSS) of the F11 gene or a fragment thereof.
According to the method of any one of claims 1-8, the gene expression regulatory molecule can bind to the DNA region or fragment thereof where any one of SEQ ID NOs: 71-72 is located.
According to the method of any one of claims 1-9, 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: upstream of the TSS Between 250bp and 130bp upstream, between 50bp upstream and 120bp downstream of TSS, and between 230bp downstream and 390bp downstream of TSS.
According to the method of any one of claims 1-10, 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: upstream of the TSS Between 250bp and 230bp upstream, between 210bp upstream and 180bp upstream of TSS, between 160bp upstream and 130bp upstream of TSS, between 50bp upstream and 10bp downstream of TSS, between 30bp downstream and 60bp downstream of TSS, and downstream of TSS Between 90bp and 120bp downstream, between 230bp and 260bp downstream of TSS, between 300bp and 320bp downstream of TSS, and between 360bp and 390bp downstream of TSS.
The method according to any one of claims 1-11, comprising providing a nucleic acid binding molecule comprising the sequence of any one of SEQ ID NOs: 1-70.
According to the method of claim 12, the gene expression modulating molecule and/or the nucleic acid binding molecule is formulated in the same or different delivery vectors.
The method of claim 13, wherein the delivery vehicle comprises liposomes and/or lipid nanoparticles.
According to the method of any one of claims 12-14, the expression regulatory molecule and/or the nucleic acid binding molecule are formulated in the same or different recombinant vectors.
According to the method of claim 15, the recombinant vector comprises a viral vector.
According to the method of any one of claims 15-16, the recombinant vector comprises an adeno-associated virus vector.
The method according to any one of claims 1 to 17, the gene expression regulatory molecule comprising a first functional domain providing at least one nucleoside near the F11 gene and/or within the F11 gene regulatory element Acid modification.
According to the method of claim 18, the modification of at least one nucleotide comprises a methylation modification.
According to the method of any one of claims 18-19, the regulatory element comprises a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a border element and/or a locus control region.
According to the method of any one of claims 18-20, the first functional domain includes one or more of DNA methyltransferase, DNA demethylase, and functionally active fragments thereof.
The method according to claim 21, the DNA methyltransferase comprises one or more of DNMT 3A, DNMT 3B, DNMT3L, DNMT 1 and DNMT 2.
According to the method of claim 22, the DNMT 3A is derived from mice.
According to the method of any one of claims 22-23, the DNMT 3L is derived from human and/or mouse.
According to the method according to any one of claims 22-24, the DNMT 3A and the DNMT 3L are directly and/or indirectly connected.
The method according to any one of claims 1 to 25, wherein the gene expression regulatory molecule comprises a second functional domain comprising a zinc finger protein-based transcription factor or a functionally active fragment thereof, or comprising a zinc finger protein-based transcription factor capable of Substances that modify histone proteins.
According to the method of claim 26, the second functional domain includes Krab.
According to the method of any one of claims 26-27, the second functional domain comprises ZIM3 Krab or KOX1 Krab.
The method of claim 26, wherein the second functional domain includes histone methyltransferase, histone demethylase, histone acetyltransferase, histone deacetylase, and functionally active fragments thereof. one or more.
The method according to any one of claims 26-29, the first functional domain and the second functional domain are directly or indirectly connected at one end of the DNA binding domain, or the first functional domain and The second functional domain is directly and/or indirectly connected to both ends of the DNA binding domain.
The method of any one of claims 1-30, wherein the gene expression regulatory molecule comprises a nuclear localization sequence.
The method of claim 31, wherein the nuclear localization sequence comprises an amino acid having an electropositive group.
The method according to any one of claims 31-32, the nuclear localization sequence is located at the N-terminal and/or C-terminal of the first functional domain and the N-terminal and/or C-terminal of the second functional domain. , and/or the N-terminus and/or C-terminus of the DNA binding domain.
A nucleic acid binding molecule comprising the sequence of any one of SEQ ID NOs: 1-70.
A gene expression regulatory molecule has the function of regulating the expression of the F11 gene without changing its gene sequence.
The gene expression regulatory molecule according to claim 35, which is as provided in the method of any one of claims 1-33.
The gene expression regulatory molecule according to any one of claims 35-36, the gene expression regulatory molecule and/or the nucleic acid binding molecule is formulated in the same or different delivery vectors, the nucleic acid binding molecule comprises SEQ ID NOs : A sequence of any item from 1-70.
The gene expression regulating molecule according to claim 37, said delivery vehicle comprising liposomes and/or lipid nanoparticles.
The gene expression regulating molecule according to any one of claims 35-38, the expression regulating molecule and/or the nucleic acid binding molecule is formulated in the same or different recombinant vectors, the nucleic acid binding molecule comprises SEQ ID NOs: Sequence of any one from 1-70.
The gene expression regulatory molecule according to claim 39, wherein the recombinant vector comprises a viral vector.
The gene expression regulatory molecule according to any one of claims 39-40, wherein the recombinant vector comprises an adeno-associated virus vector.
A nucleic acid encoding the nucleic acid binding molecule of claim 34 and/or encoding the gene expression regulating molecule of any one of claims 35-41.
A recombinant vector comprising the nucleic acid of claim 42.
A delivery vector comprising the nucleic acid binding molecule of claim 34, the gene expression regulating molecule of any one of claims 35-41, the nucleic acid of claim 42, and/or the The recombinant vector of 43, and optionally comprising liposomes and/or lipid nanoparticles.
A composition comprising the nucleic acid binding molecule of claim 34, the gene expression regulating molecule of any one of claims 35-41, the nucleic acid of claim 42, the nucleic acid of claim 43 The recombinant vector, and/or the delivery vector of claim 44.
A cell comprising the nucleic acid binding molecule of claim 34, the gene expression regulating molecule of any one of claims 35-41, the nucleic acid of claim 42, and the recombinant of claim 43 The carrier, the delivery vehicle of claim 44, and/or the composition of claim 45.
A kit, said kit comprising the nucleic acid binding molecule of claim 34, the gene expression regulating molecule of any one of claims 35-41, the nucleic acid of claim 42, the nucleic acid of claim 43 The recombinant vector, the delivery vector of claim 44, the composition of claim 45, and/or the cell of claim 46.