WO2020187268A1 - 一种增强基因编辑的融合蛋白及其应用 - Google Patents

一种增强基因编辑的融合蛋白及其应用 Download PDF

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WO2020187268A1
WO2020187268A1 PCT/CN2020/080032 CN2020080032W WO2020187268A1 WO 2020187268 A1 WO2020187268 A1 WO 2020187268A1 CN 2020080032 W CN2020080032 W CN 2020080032W WO 2020187268 A1 WO2020187268 A1 WO 2020187268A1
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fusion protein
gene editing
gene
present
cells
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French (fr)
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李大力
尹树明
张梅
陈曦
张晓辉
王立人
刘明耀
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上海邦耀生物科技有限公司
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Priority to EP20773078.9A priority Critical patent/EP3943512A4/en
Priority to US17/440,777 priority patent/US20220177529A1/en
Publication of WO2020187268A1 publication Critical patent/WO2020187268A1/zh

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    • C12Y305/04004—Adenosine deaminase (3.5.4.4)

Definitions

  • the invention relates to the field of biotechnology, in particular to a fusion protein for enhancing gene editing and its application.
  • Gene editing technology is a technology that artificially breaks double-stranded DNA and uses the repair mechanism of double-stranded DNA breaks to achieve gene manipulation.
  • the existing gene editing technologies include ZFN, TALEN and CRISPR/Cas9 technologies, of which CRISPR/Cas9 technologies are the most widely used.
  • CRISPR/Csa9 technology is an adaptive immune mechanism derived from bacteria or archaea. It uses a single-stranded guide RNA (sgRNA) and Csa9 protein to generate DNA double-strand breaks at specific locations in the genome, and then through endogenous non-identical Source end joining (NHEJ) or homologous recombination (HDR) repair mechanism to achieve target gene knockout or specific gene or fragment insertion.
  • sgRNA single-stranded guide RNA
  • NHEJ non-identical Source end joining
  • HDR homologous recombination
  • CRISPR/Cas9 technology has extremely powerful functions, it also has its shortcomings, such as: 1. Off-target problem; 2. The limitation of PAM leads to the limitation of target selection; 3. The editing efficiency of some new tools is generally low ( XCas9 and SpCas9-NG), and the current optimization and modification of gene editing tools are mainly based on enhancing the accuracy of the tools and enhancing the modification of the target range. There is no extensive improvement of various gene editing tools for the gene editing tools themselves. Methods.
  • the purpose of the present invention is to provide a new method for improving gene editing efficiency.
  • the first aspect of the present invention provides a fusion protein, the structure of the fusion protein is shown in the following formula I or I':
  • A is the gene editing protein
  • C is an optional base editor element
  • L is no or connecting peptide
  • Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond.
  • the non-peptide bond includes PEG.
  • the gene editing protein is selected from the following group: Cas9, Cas12, Cas12a, Cas12b, Cas13, Cas14, or a combination thereof.
  • the gene editing protein includes wild-type or mutant gene editing protein.
  • the gene editing protein is selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium, Or a combination.
  • amino acid sequence of the wild-type gene editing protein is shown in SEQ ID NO.: 1, 14 or 15.
  • amino acid sequence of the base editor element is shown in SEQ ID NO.: 2 or 12.
  • the DNA double-strand binding domain is a non-sequence-specific DNA double-strand binding domain.
  • the DNA double-strand binding domain is selected from the group consisting of HMG-D, Sac7d, or a combination thereof.
  • the DNA double-stranded binding domain includes a wild-type DNA double-stranded binding domain and a mutant type of DNA double-stranded binding domain.
  • the DNA double-strand binding domain is derived from Drosophila or Archaea.
  • amino acid sequence of the DNA double-strand binding domain is shown in SEQ ID NO.: 10 or 11.
  • the length of the connecting peptide is 1-100 aa, preferably, 15-85 aa, more preferably, 25-70 aa.
  • the connecting peptide is a sequence shown as Gly-Gly-Ser with n repeats, where n is 2-8, preferably n is 3-6.
  • amino acid sequence of the connecting peptide is selected from the following group:
  • the base editor element includes cytosine deaminase and adenine deaminase.
  • the cytosine deaminase includes Apobec1 and Apobec3A.
  • the adenine deaminase includes TadA.
  • the fusion protein has an amino acid sequence shown in any one of SEQ ID NO.: 8, 9, and 13.
  • the second aspect of the present invention provides a polynucleotide which encodes the fusion protein according to the first aspect of the present invention.
  • the polynucleotide additionally contains an accessory element selected from the group consisting of signal peptide, secretory peptide, tag sequence (such as 6His), or flank of the ORF of the mutein or fusion protein. Its combination.
  • the polynucleotide is selected from the following group: DNA sequence, RNA sequence, or a combination thereof.
  • the third aspect of the present invention provides a vector which contains the polynucleotide according to the second aspect of the present invention.
  • the vector includes one or more promoters, which are operably linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, and selectable marker , Nucleic acid restriction sites, and/or homologous recombination sites.
  • the vectors include plasmids and viral vectors.
  • the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.
  • AAV adeno-associated virus
  • adenovirus adenovirus
  • lentivirus lentivirus
  • retrovirus lentivirus
  • herpes virus SV40
  • poxvirus poxvirus
  • the vectors include expression vectors, shuttle vectors, and integration vectors.
  • the third aspect of the present invention provides a host cell, which contains the vector according to the third aspect of the present invention, or the polynucleotide according to the second aspect of the present invention is integrated into its genome.
  • the host cell is a eukaryotic cell, such as a yeast cell, a plant cell or a mammalian cell (including human and non-human mammals).
  • the host cell is a prokaryotic cell, such as Escherichia coli.
  • the yeast cell is selected from one or more yeasts from the following group: Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell includes:
  • the yeast Luveyi is more preferably Kluyveromyces marxianus, and/or Kluyveromyces lactis.
  • the host cell is selected from the group consisting of E. coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, yeast cells, or a combination thereof.
  • the fifth aspect of the present invention provides a method for producing the fusion protein according to the first aspect of the present invention, including the steps:
  • the fusion protein is isolated.
  • the sixth aspect of the present invention provides a gene editing reagent, which comprises the fusion protein according to the first aspect of the present invention.
  • the reagent further includes one or more reagents selected from the following group:
  • Template for homologous targeted repair single-stranded nucleotide sequence or plasmid vector.
  • the seventh aspect of the present invention provides a kit including the gene editing reagent according to the sixth aspect of the present invention.
  • the kit further includes one or more reagents selected from the following group:
  • Template for homologous targeted repair single-stranded nucleotide sequence or plasmid vector.
  • the kit further includes a label or instructions.
  • the eighth aspect of the present invention provides a use of the fusion protein according to the first aspect of the present invention to prepare reagents or kits for improving gene editing efficiency.
  • the ninth aspect of the present invention provides a pharmaceutical composition comprising:
  • the expression vector includes a viral vector.
  • the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.
  • AAV adeno-associated virus
  • adenovirus adenovirus
  • lentivirus lentivirus
  • retrovirus lentivirus
  • herpes virus SV40
  • poxvirus poxvirus
  • the vector is selected from the following group: lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof, preferably, the vector is adeno-associated virus (AAV).
  • the dosage form of the pharmaceutical composition is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.
  • the dosage form of the pharmaceutical composition is an injection dosage form.
  • the pharmaceutical composition also includes other drugs for gene therapy.
  • the other drugs for gene therapy are selected from the group consisting of antisense nucleotide drugs, EDIT-101 drugs, CTX001, or a combination thereof.
  • the pharmaceutical composition is a cell preparation.
  • the tenth aspect of the present invention provides a medicine kit including:
  • the kit further includes:
  • first container and the second container are the same or different containers.
  • the drug in the first container is a unilateral preparation containing the fusion protein of claim 1.
  • the medicine in the second container is a single preparation containing other medicines for gene therapy.
  • the dosage form of the drug is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.
  • the dosage form of the drug is an injection dosage form.
  • the eleventh aspect of the present invention provides a use of the fusion protein according to the first aspect of the present invention to prepare a medicine for gene therapy.
  • the twelfth aspect of the present invention provides a method for improving gene editing efficiency, including the steps:
  • gene editing is performed on cells, thereby improving gene editing efficiency.
  • the cells include human or non-human mammalian cells (such as primates or livestock).
  • the cells include cancer cells or normal cells.
  • the cells are selected from the group consisting of kidney cells, liver cells, nerve cells, heart cells, epithelial cells, muscle cells, somatic cells, bone marrow cells, endothelial cells, or combinations thereof.
  • the cell is selected from the group consisting of 293 cells, A549 cells, SW626 cells, HT-3 cells, PA-1 cells, or a combination thereof.
  • the cell includes HEK293T.
  • the gene editing is performed in an in vitro reaction system.
  • the content of the fusion protein or gene editing reagent is 100ng-700ng, preferably, 200ng-600ng, more preferably, 300ng-500ng.
  • the method is non-diagnostic and non-therapeutic.
  • the cell is an in vitro cell.
  • FIG. 1 shows the editing efficiency for two different endogenous targets, indicating that the different connection methods of the DNA double-strand binding domain and Cas9, as well as linkers of different lengths, have differences in the improvement of efficiency.
  • Comprehensive selection of HMG-D The structural domain is best connected to the N-terminal of Cas9 through an L4 length linker.
  • HMG-D-L4-Cas9 where H stands for HMG-D; S stands for Sac7d; L1-L5 stands for linkers of different lengths; mutH stands for mutant HMG-D (V32A and T33A mutations reduce the binding activity); C stands for Cas9.
  • Figure 2 shows that the editing efficiency of HMG-D-L4-Cas9 on other endogenous targets can also be improved.
  • the efficiency is improved by> 20%, preferably,> 40%, and more preferably,> 60% (such as 80 %), up to 2 times, where H stands for HMG-D.
  • FIG 3 shows that the double-stranded binding domain HMG-D can increase the efficiency of Cas9 proteins from other sources (such as SaCas9), and the efficiency is increased by more than 20%.
  • Figure 4 shows that the double-stranded binding domain HMG-D can increase the efficiency of non-Cas9 proteins (such as AsCas12a) by 10-20%.
  • non-Cas9 proteins such as AsCas12a
  • FIG. 5 shows that the double-stranded binding domain HMG-D can increase the efficiency of apparent regulatory tools (such as CRISPR-VPR), and the efficiency can be increased by 2 times.
  • endCas9 is HMG-D-L4-dCas9.
  • FIG 6 shows that the double-stranded binding domain HMG-D can improve the efficiency of the single-base editing tool ABE, where H stands for HMG-D.
  • the inventors unexpectedly obtained an enhanced fusion protein.
  • the enhanced fusion protein of the present invention can significantly improve gene editing efficiency in vivo or in vitro, and the present invention also unexpectedly found that the gene editing protein and DNA double-strand binding domain, optional bases
  • the fusion protein formed by the editor element and the optional connecting peptide can significantly improve gene editing efficiency (increased by ⁇ 20%, such as 80%, or even up to 2 times).
  • the present invention also unexpectedly discovered that The fusion protein can be used in gene therapy. On this basis, the inventor completed the present invention.
  • the term “about” may refer to a value or composition within an acceptable error range of a particular value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
  • Sequence identity is passed along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) ) Compare two aligned sequences and determine the number of positions where the same residue appears. Normally, this is expressed as a percentage.
  • a predetermined comparison window which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein
  • EDIT-101 drug belongs to gene therapy drugs, which is a type of cell. Specifically, EDIT-101 is a drug that uses CRISPR gene editing technology to treat hereditary retinal degeneration disease (LCA10 disease). 101 is administered by subretinal injection, and the gene editing system is directly delivered to the photoreceptor cells to achieve the therapeutic effect.
  • CRISPR gene editing technology to treat hereditary retinal degeneration disease (LCA10 disease). 101 is administered by subretinal injection, and the gene editing system is directly delivered to the photoreceptor cells to achieve the therapeutic effect.
  • CTX001 belongs to gene therapy drugs, which is a type of cell. Specifically, CTX001 is based on CRISPR gene editing technology to achieve therapeutic purposes by cutting the BCL11A gene of patients with ⁇ -thalassemia.
  • wild-type gene editing protein refers to a naturally occurring gene editing protein that has not been artificially modified. Its nucleotides can be obtained through genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR ) Etc. The amino acid sequence can be derived from the nucleotide sequence.
  • the source of the wild-type gene editing protein includes (but is not limited to): Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium .
  • amino acid sequence of the wild-type gene editing protein is shown in SEQ ID NO.: 1 or 14 or 15.
  • the gene editing protein includes, but is not limited to, Cas9, Cas9a, Cas12, Cas12a, Cas12b, Cas13, and Cas14.
  • DNA double-strand binding domain is a DNA double-strand binding domain without sequence specificity.
  • the non-sequence-specific DNA double-stranded binding domain of the present invention is not limited by the DNA sequence, and theoretically can bind to any DNA sequence, so it can be applied to any position The binding of DNA.
  • the sequence of a preferred DNA double-strand binding domain is shown in SEQ ID NO.: 10 or 11.
  • any base editor provided herein can modify specific nucleotide bases without producing a significant proportion of indels.
  • insertion/deletion refers to the insertion or deletion of nucleotide bases within a nucleic acid. Such insertions or deletions can lead to frameshift mutations in the coding region of the gene.
  • any of the base editors provided herein are capable of producing a larger proportion of the intended modification (eg, point mutation or deamination) relative to indels.
  • Any base editor of the present invention can effectively generate intended mutations, such as point mutations, in nucleic acids (for example, nucleic acids in the genome) without generating a large number of unintended mutations, such as unintended point mutations.
  • the base editor includes cytosine deaminase and adenine deaminase, and other types of base editors as long as they have the function of the base editor of the present invention are also within the protection scope of the present invention.
  • ABE the structure after the gene editing protein is fused with the base editor
  • CBE the structure after the gene editing protein is fused with adenine deaminase
  • ABE is the structure after the gene editing protein is fused with adenine deaminase
  • CBE is the gene editing protein and the cell
  • sequence of a preferred base editor is shown in SEQ ID NO.: 2 or 12.
  • fusion protein of the present invention or “polypeptide” refers to the fusion protein described in the second aspect of the present invention.
  • the structure of the fusion protein of the present invention is shown in the following formula I or I':
  • A is the gene editing protein
  • C is an optional base editor element
  • L is no or connecting peptide
  • Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond.
  • the length of the connecting peptide has an effect on the activity of the fusion protein.
  • the preferred length of the connecting peptide is 1-100 aa, preferably, 15-85 aa, and more preferably, 25-70 aa.
  • a preferred connecting peptide is shown in SEQ ID NO.: 3-7.
  • the term "fusion protein” also includes variant forms shown in SEQ ID NO.: 8, 9, or 13 having the above-mentioned activity. These variants include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletion, insertion and/or substitution, and addition or addition at the C-terminal and/or N-terminal One or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids are deleted. For example, in this field, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. In addition, the term also includes the polypeptides of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).
  • the present invention also includes active fragments, derivatives and analogs of the above fusion protein.
  • fragment refers to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention.
  • polypeptide fragments, derivatives or analogues of the present invention can be (i) one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) in one or more A polypeptide with substitution groups in each amino acid residue, or (iii) a polypeptide formed by fusing an antigenic peptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (a fusion protein fused with a leader sequence, secretory sequence, or 6His tag sequence). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.
  • a preferred type of active derivative means that compared with the amino acid sequence of Formula I, there are at most 3, preferably at most 2, and more preferably at most 1 amino acid replaced by an amino acid with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by amino acid substitutions.
  • the invention also provides analogs of the fusion protein of the invention.
  • the difference between these analogues and the polypeptide shown in SEQ ID NO.: 8 or 9 or 13 may be the difference in the amino acid sequence, the difference in the modification form that does not affect the sequence, or both.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modified (usually without changing the primary structure) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.
  • A is a gene editing protein
  • B is HMG-D or Sac7d
  • C is adenine deaminase or cytosine deaminase or none
  • L is L1 or L2 or L3 or L4 or L5 or none.
  • A is a gene editing protein
  • B is HMG-D
  • C is either adenine deaminase or cytosine deaminase or none
  • L is L4 or L5.
  • the fusion protein of the present invention may also include two or more of the A, B, C, and L elements in Formula I.
  • A is a gene editing protein
  • B is HMG-9
  • C is none
  • L is L4.
  • A is a gene editing protein
  • B is HMG-D
  • C is adenine deaminase
  • L is L5.
  • A is a gene editing protein
  • B is HMG-D
  • C is cytosine deaminase
  • L is L5.
  • amino acid sequence of the fusion protein of the present invention is shown in SEQ ID NO.: 8, 9 or 13.
  • Adeno-associated virus is smaller than other viral vectors, is non-pathogenic, and can transfect dividing and undivided cells, gene therapy methods for genetic diseases based on AAV vectors have been affected. Widespread concern.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the Parvoviridae dependent virus genus. It is the simplest type of single-stranded DNA-deficient virus found so far and requires a helper virus (usually adenovirus). Viruses) participate in replication. It encodes the cap and rep genes in the inverted repeat (ITR) at both ends. ITRs play a decisive role in virus replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.
  • Recombinant adeno-associated virus vector is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), and low immunogenicity, it can express foreign genes in vivo. Long and other characteristics, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially its application effects in various cells, tissues and in vivo experiments have accumulated a lot of data.
  • rAAV is used in the research of gene therapy for various diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, disease model construction, and gene preparation. Knockout mice and other aspects.
  • the vector is a recombinant AAV vector.
  • AAVs are relatively small DNA viruses that can integrate into the genome of the cells they infect in a stable and site-specific manner. They can infect a large range of cells without any effect on cell growth, morphology or differentiation, and they do not seem to be involved in human pathology.
  • the AAV genome has been cloned, sequenced and characterized.
  • AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of replication of the virus. The rest of the genome is divided into two important regions with encapsidation functions: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.
  • ITR inverted terminal repeat
  • AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described in, for example, PCT Application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for in vitro and in vivo gene transfer has been described (see, for example, International Patent Application Publication Nos. WO91/18088 and WO93/09239; U.S. Patent Nos.
  • Replication-deficient recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (such as adenovirus): the nucleic acid sequence of interest is flanked by two AAV inverted terminal repeats (ITR) Region plasmids, and plasmids carrying AAV encapsidation genes (rep and cap genes).
  • a human helper virus such as adenovirus
  • the recombinant vector is capsidized to viral particles (e.g., including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 And AAV virus particles of AAV16). Therefore, the present disclosure includes recombinant virus particles (recombinant because they contain recombinant polynucleotides) containing any of the vectors described herein. Methods of producing such particles are known in the art and are described in US Patent No. 6,596,535.
  • the present invention also relates to vectors containing the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors of the present invention or the fusion protein coding sequence of the present invention, and methods for producing the polypeptides of the present invention through recombinant technology.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant fusion protein. Generally speaking, there are the following steps:
  • polynucleotide (or variant) of the present invention encoding the fusion protein of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • the polynucleotide sequence encoding the fusion protein can be inserted into the recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus, or other vectors well known in the art. As long as it can replicate and stabilize in the host, any plasmid and vector can be used.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • the methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti Transcriptional virus LTRs and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
  • the host cell can be a prokaryotic cell (such as E. coli), or a lower eukaryotic cell, or a higher eukaryotic cell, such as yeast cells, plant cells or mammalian cells (including human and non-human mammals).
  • a prokaryotic cell such as E. coli
  • yeast cells such as Pichia pastoris, Kluyveromyces, or a combination thereof
  • yeast cells include: Kluyveromyces, more preferably Maxsk Luwei and/or Kluyveromyces lactis) are host cells.
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples that can be cited include the 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, the polyoma enhancer on the late side of the replication initiation point, and adenovirus enhancers.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be performed by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic cleavage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • Gene Therapy for genetic diseases refers to the application of genetic engineering technology to introduce normal genes into patient cells to correct defective genes and cure the disease.
  • the way of correction can be either to repair the defective gene in situ, or to transfer a functional normal gene into a certain part of the cell genome to replace the defective gene to play a role.
  • Gene is the basic functional unit that carries biological genetic information and is a specific sequence located on the chromosome. Certain technical methods or vectors must be used to introduce foreign genes into biological cells. The methods of gene transfer are divided into biological methods, physical methods and chemical methods. Adenovirus vectors are currently one of the most commonly used viral vectors for gene therapy.
  • Gene therapy is mainly used to treat diseases that pose a serious threat to human health, including, but not limited to: genetic diseases (such as hemophilia, cystic fibrosis, family hypercholesterolemia, etc.), malignant tumors, cardiovascular diseases, Infectious diseases (such as AIDS, rheumatoid, etc.).
  • Gene therapy is a high-tech biomedical technology that introduces human normal genes or therapeutic genes into human target cells in a certain way to correct gene defects or exert therapeutic effects, so as to achieve the purpose of curing diseases.
  • Gene therapy is different from conventional treatment methods: in general, the treatment of diseases is aimed at various symptoms caused by genetic abnormalities, while gene therapy is aimed at the root cause of the disease-the abnormal genes themselves.
  • Target cells for gene therapy include, but are not limited to, somatic cells, bone marrow cells, liver cells, nerve cells, endothelial cells, and muscle cells.
  • the target gene is subjected to efficient gene editing (including gene insertion, replacement, etc.) through gene therapy, so as to restore normal gene expression or enhance gene expression, thereby treating related diseases.
  • the present invention finds for the first time that the fusion protein of the present invention can significantly improve gene editing efficiency in vivo or in vitro.
  • the present invention finds for the first time that the fusion protein of the present invention can significantly improve gene editing efficiency in vivo or in vitro by ⁇ 20%, preferably,>40%, more preferably,>60% (such as 80%) , Up to 2 times.
  • the present invention is the first to discover the mRNA of an in vitro transcription-enhanced gene editing tool, which improves the success of animal model construction.
  • the present invention uses the AAV virus packaging the enhanced gene editing tool to express the protein of the enhanced gene editing tool to improve the effect of disease treatment.
  • the present invention utilizes fusion of double-stranded DNA binding domains to improve gene editing efficiency.
  • the present invention screens for the first time to find a double-stranded DNA binding domain (such as HMG-D) that efficiently improves gene editing efficiency, and its excellent fusion method.
  • a double-stranded DNA binding domain such as HMG-D
  • the present invention finds for the first time that the double-stranded DNA binding domain of the present invention can broadly improve the gene editing efficiency of various gene editing tools.
  • the enhanced gene editing tool of the present invention can improve the success rate of animal model construction and the efficiency of gene therapy.
  • the enhanced gene editing tool of the present invention can also be used for gene therapy.
  • Method 1 (Applicable to Example 1, Example 2 and Example 3)
  • the density of HEK293T cells in a 24-well plate grows to 60-70%, an equimolar amount of plasmid is transfected into the cells by the transfection reagent PEI, the medium is changed for 8-10 hours, and the cells are cultured for a period of time (Cas9 system culture for 72 hours, Culture the base editing system for 120 hours), harvest the cells and extract the genome.
  • Method 2 (Applicable to the construction of the animal model of Example 4)
  • Mouse embryos were injected with mRNA and sgRNA and transplanted to surrogate mother mice to obtain F0 generation mice, identify genes, count mouse genotype mutation rates, and calculate model construction success rates.
  • Method 3 (Applicable to the gene therapy of Example 4)
  • AAV adeno-associated virus
  • AAA H-L4-C-L4-S, H-L4-C-L5-S
  • AAAA H-L3-C, C-L4-S, C-L5-S, C-L4-H, H-L4-C-L4-H, H-L4-C-L5-H, H-L4- H-L4-C, C-L5-H-L5-H
  • AAAAA H-L4-C, C-L5-H
  • this enhanced gene editing tool that enhances gene editing efficiency through the fusion of double-stranded DNA binding domains can also be compared to other types of double-stranded DNA binding domains, such as the widely used zinc finger protein (ZFP) and other DNA binding domains of transcription factors and HMG-D or Sac7d from other species, etc.
  • ZFP zinc finger protein
  • the present invention can also improve gene editing efficiency through these double-stranded DNA binding domains. Therefore, the most important thing of the present invention is the discovery that a double-stranded DNA binding domain fusion gene editing tool can improve gene editing efficiency.
  • DNA binding domain HMG-D DNA binding domain HMG-D.
  • the obtained enhanced gene editing tool ie the fusion protein of the present invention, such as HMG-D-L4-SpCas9 is further compared in terms of effects on more endogenous targets, and 293T cells are transfected at an equimolar ratio It was found that compared with SpCas9, the editing efficiency of the fusion protein of the present invention was improved by more than 20% (or 60%, or 80%) in the compared targets, up to 2 times ( Figure 2).
  • Example 3 Improve the gene editing efficiency of Cas9 from other species (such as SaCas9)
  • Example 4 Improve the gene editing efficiency of other non-Cas9 proteins (such as AsCas12a)
  • Example 5 Improve the transcriptional activation efficiency of transcriptional regulatory tools (CRISPR/Cas9)
  • the editing efficiency of the fusion protein fused with other types of base editors is similar or equivalent to that of the fusion protein fused with ABE.
  • Example 7 Improve the success rate of animal model construction and the efficiency of gene therapy
  • the success rate of animal model construction is increased.
  • the efficiency of disease treatment can also be improved by packaging the AAV virus with enhanced gene editing tools.
  • Comparative example 1 does not work well after fusion with non-DNA binding domain

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Abstract

本发明涉及一种增强基因编辑的融合蛋白及其应用。具体地，本发明提供一种增强型融合蛋白。相比野生型的基因编辑蛋白，本发明的增强型融合蛋白可显著提高体内或体外的基因编辑效率。

Description

一种增强基因编辑的融合蛋白及其应用 技术领域
本发明涉及生物技术领域，具体地涉及一种增强基因编辑的融合蛋白及其应用。
背景技术
基因编辑技术是通过人为地实现双链DNA的断裂，利用双链DNA断裂的修复机制来实现基因操纵的一门技术。目前已有的基因编辑技术包括ZFN、TALEN和CRISPR/Cas9技术，其中以CRISPR/Cas9技术应用最为广泛。CRISPR/Csa9技术是来源于细菌或古细菌的一种获得性免疫机制，利用一条单链向导RNA(sgRNA)和Csa9蛋白定向的在基因组特定位置产生DNA双链断裂，进而通过内源的非同源末端连接(NHEJ)或同源重组(HDR)修复机制，来实现目的基因的敲除或特定基因或片段的插入。
CRISPR/Cas9技术虽然有着无比强大的功能，但也有其缺点，如：1.脱靶问题；2.PAM的限制导致靶点选择的有限性；3.一些新出来的工具的编辑效率总体偏低(XCas9和SpCas9-NG)，并且目前对于基因编辑工具的优化和改造主要是基于增强工具的精确性和增强靶向范围的改造，还没有针对于基因编辑工具本身来广泛的提高各类基因编辑工具的方法。
因此，本领域迫切需与开发一种新的提高基因编辑效率的方法。
发明内容
本发明的目的是提供一种新的提高基因编辑效率的方法。
本发明第一方面提供了一种融合蛋白，所述融合蛋白的结构如下式I或I’所示：
C-A-L-B    (I)
B-L-A-C    (I’)
式中，
A为基因编辑蛋白，
B为DNA双链结合结构域，
C为任选的碱基编辑器元件；
L为无或连接肽，
各“-”独立地为连接肽或肽键或非肽键。
在另一优选例中，当融合蛋白的结构如式I’所示时，C为无。
在另一优选例中，所述非肽键包括PEG。
在另一优选例中，所述基因编辑蛋白选自下组：Cas9、Cas12、Cas12a、Cas12b、Cas13、Cas14、或其组合。
在另一优选例中，所述基因编辑蛋白包括野生型或突变型的基因编辑蛋白。
在另一优选例中，所述基因编辑蛋白选自下组：酿脓链球菌(Streptococcus pyogenes)、葡萄球菌(Staphylococcus aureus)、氨基酸球菌属(Acidaminococcus sp)、毛螺科菌(Lachnospiraceae bacterium)、或其组合。
在另一优选例中，所述野生型的基因编辑蛋白的氨基酸序列如SEQ ID NO.:1、14或15所示。
在另一优选例中，所述碱基编辑器元件的氨基酸序列如SEQ ID NO.:2或12所示。
在另一优选例中，所述DNA双链结合结构域为非序列特异性的DNA双链结合结构域。
在另一优选例中，所述DNA双链结合结构域选自下组：HMG-D、Sac7d、或其组合。
在另一优选例中，所述DNA双链结合结构域包括野生型的DNA双链结合结构域和突变型的DNA双链结合结构域。
在另一优选例中，所述DNA双链结合结构域来源于果蝇或古细菌。
在另一优选例中，所述DNA双链结合结构域的氨基酸序列如SEQ ID NO.:10或11所示。
在另一优选例中，所述连接肽的长度为1-100aa，较佳地，15-85aa，更佳地，25-70aa。
在另一优选例中，所述连接肽为具有n个重复的如Gly-Gly-Ser所示的序列，其中n为2-8，优选地n为3-6。
在另一优选例中，所述连接肽的氨基酸序列选自下组：
(1)氨基酸序列如SEQ ID NO.:3-7中任一所示的多肽；
(2)将SEQ ID NO.:3-7中任一所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-8个、更优选1-3个、最优选1个氨基 酸残基的取代、缺失或添加而形成的，具有(1)所述多肽功能的由SEQ ID NO.:3-7中任一所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，所述碱基编辑器元件包括胞嘧啶脱氨酶、腺嘌呤脱氨酶。
在另一优选例中，所述胞嘧啶脱氨酶包括Apobec1、Apobec3A。
在另一优选例中，所述腺嘌呤脱氨酶包括TadA。
在另一优选例中，所述融合蛋白具有SEQ ID NO.:8、9、13中任一所示的氨基酸序列。
本发明第二方面提供了一种多核苷酸，所述的多核苷酸编码本发明第一方面所述的融合蛋白。
在另一优选例中，所述的多核苷酸在所述突变蛋白或融合蛋白的ORF的侧翼还额外含有选自下组的辅助元件：信号肽、分泌肽、标签序列(如6His)、或其组合。
在另一优选例中，所述的多核苷酸选自下组：DNA序列、RNA序列、或其组合。
本发明第三方面提供了一种载体，所述的载体含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体包含一个或多个启动子，所述启动子可操作地与所述核酸序列、增强子、转录终止信号、多腺苷酸化序列、复制起点、选择性标记、核酸限制性位点、和/或同源重组位点连接。
在另一优选例中，所述载体包括质粒、病毒载体。
在另一优选例中，所述的病毒载体选自下组：腺相关病毒(AAV)、腺病毒、慢病毒、逆转录病毒、疱疹病毒、SV40、痘病毒、或其组合。
在另一优选例中，所述载体包括表达载体、穿梭载体、整合载体。
本发明第三方面提供了一种宿主细胞，所述宿主细胞含有本发明第三方面所述的载体，或其基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞为真核细胞，如酵母细胞、植物细胞或哺乳动物细胞(包括人和非人哺乳动物)。
在另一优选例中，所述的宿主细胞为原核细胞，如大肠杆菌。
在另一优选例中，所述酵母细胞选自下组的一种或多种来源的酵母：毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母。
在另一优选例中，所述宿主细胞选自下组：大肠杆菌、麦胚细胞，昆虫细胞， SF9、Hela、HEK293、CHO、酵母细胞、或其组合。
本发明第五方面提供了一种产生本发明第一方面所述的融合蛋白的方法，包括步骤：
在适合表达的条件下，培养本发明第四方面所述的宿主细胞，从而表达融合蛋白；和/或
分离所述融合蛋白。
本发明第六方面提供了一种基因编辑试剂，所述基因编辑试剂包含本发明第一方面所述的融合蛋白。
在另一优选例中，所述的试剂还包括选自下组的一种或多种试剂：
(a1)gRNA、crRNA、或用于产生所述gRNA或crRNA的载体；
(a2)用于同源定向修复的模板：单链核苷酸序列或质粒载体。
本发明第七方面提供了一种试剂盒，包括本发明第六方面所述的基因编辑试剂。
在另一优选例中，所述试剂盒还包括选自下组的一种或多种试剂：
(a1)gRNA、crRNA、或用于产生所述gRNA或crRNA的载体；
(a2)用于同源定向修复的模板：单链核苷酸序列或质粒载体。
在另一优选例中，所述试剂盒还包括标签或说明书。
本发明第八方面提供了一种本发明第一方面所述的融合蛋白的用途，用于制备试剂或试剂盒，所述试剂或试剂盒用于提高基因编辑效率。
本发明第九方面提供了一种药物组合物，包括：
(a)本发明第一方面所述的融合蛋白、或其编码基因、或其表达载体(vector)；和
(b)药学上可接受的载体(carrier)。
在另一优选例中，所述表达载体包括病毒载体。
在另一优选例中，所述的病毒载体选自下组：腺相关病毒(AAV)、腺病毒、慢病毒、逆转录病毒、疱疹病毒、SV40、痘病毒、或其组合。
在另一优选例中，所述的载体选自下组：慢病毒、腺病毒、腺相关病毒(AAV)、或其组合，较佳地，所述载体为腺相关病毒(AAV)。
在另一优选例中，所述药物组合物的剂型选自下组：冻干制剂、液体制剂、或其组合。
在另一优选例中，所述药物组合物的剂型为注射剂型。
在另一优选例中，所述药物组合物中还包括其他用于基因治疗的药物。
在另一优选例中，所述其他用于基因治疗的药物选自下组：反义核苷酸药物、EDIT-101药物、CTX001、或其组合。
在另一优选例中，所述药物组合物为细胞制剂。
本发明第十方面提供了一种药盒，包括：
(a1)第一容器，以及位于所述第一容器中的本发明第一方面所述的融合蛋白、其编码基因、或其表达载体，或含有本发明第一方面所述的融合蛋白的药物。
在另一优选例中，所述药盒还包括：
(a2)第二容器，以及位于所述第二容器中的其他用于基因治疗的药物，或含有其他用于基因治疗的药物的药物。
在另一优选例中，所述的第一容器和第二容器是相同或不同的容器。
在另一优选例中，所述的第一容器的药物是含权利要求1所述的融合蛋白的单方制剂。
在另一优选例中，所述的第二容器的药物是含其他用于基因治疗的药物的单方制剂。
在另一优选例中，所述药物的剂型选自下组：冻干制剂、液体制剂、或其组合。
在另一优选例中，所述药物的剂型为注射剂型。
本发明第十一方面提供了一种本发明第一方面所述的融合蛋白的用途，用于制备用于基因治疗的药物。
本发明第十二方面提供了一种提高基因编辑效率的方法，包括步骤：
在本发明第一方面所述的融合蛋白或本发明第六方面所述的基因编辑试剂存在下，对细胞进行基因编辑，从而提高基因编辑效率。
在另一优选例中，所述细胞包括人或非人哺乳动物细胞(如灵长类动物或家畜)。
在另一优选例中，所述细胞包括癌细胞或正常细胞。
在另一优选例中，所述细胞选自下组：肾脏细胞、肝脏细胞、神经细胞、心脏细胞、上皮细胞、肌细胞、体细胞、骨髓细胞、内皮细胞、或其组合。
在另一优选例中，所述细胞选自下组：293细胞、A549细胞、SW626细胞、HT-3细胞、PA-1细胞、或其组合。
在另一优选例中，所述细胞包括HEK293T。
在另一优选例中，所述的基因编辑在一体外反应体系中进行。
在另一优选例中，所述的体外反应体系中，所述融合蛋白或基因编辑试剂 的含量为100ng－700ng，较佳地，200ng－600ng，更佳地，300ng—500ng。
在另一优选例中，所述方法为非诊断性和非治疗性的。
在另一优选例中，所述细胞为体外的细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了对于两个不同的内源靶点的编辑效率，说明DNA双链结合结构域和Cas9的不同连接方式，以及不同长度的linker对效率的提高都有区别，综合选出HMG-D结构域通过L4长度的linker连接在Cas9的N端最好。即：HMG-D-L4-Cas9，其中，H代表HMG-D；S代表Sac7d；L1-L5代表不同长度的linker；mutH代表突变的HMG-D(V32A和T33A突变，降低了结合活性)；C代表Cas9。
图2显示了HMG-D-L4-Cas9在其他内源性靶点的编辑效率也是可以提高的，效率提高＞20％，较佳地，＞40％，更佳地，＞60％(如80％)，最高可达2倍，其中，H代表HMG-D。
图3显示了双链结合结构域HMG-D可以提高其他来源Cas9蛋白(如：SaCas9)的效率，效率提高了20％以上。
图4显示了显示了双链结合结构域HMG-D可以提高非Cas9蛋白(如：AsCas12a)的效率，效率提高了10-20％。
图5显示了双链结合结构域HMG-D可以提高表观调控工具(如：CRISPR-VPR)的效率，效率可以提高2倍。其中，endCas9即为HMG-D-L4-dCas9。
图6显示了双链结合结构域HMG-D可以提高单碱基编辑工具ABE的效率，其中，H代表HMG-D。
具体实施方式
本发明人经过广泛而深入的研究，意外地获得一种增强型融合蛋白。相比野生型基因编辑蛋白，本发明的增强型融合蛋白可显著提高体内或体外的基因编辑效 率，并且本发明还意外的发现，基因编辑蛋白和DNA双链结合结构域、任选的碱基编辑器元件以及任选的连接肽所形成的融合蛋白，可显著提高基因编辑效率(提高≥20％，如80％，甚至可达2倍)，此外，本发明还意外的发现，本发明的融合蛋白可用于基因治疗。在此基础上，本发明人完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
序列同一性(或同源性)通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
如本文所用，术语“EDIT-101药物”属于基因治疗药物，是一类细胞，具体地，EDIT-101是一种利用CRISPR基因编辑技术治疗遗传性视网膜衰退疾病(LCA10疾病)的药物，EDIT-101通过视网膜下注射给药，将基因编辑系统直接输送到感光细胞中而达到治疗效果。
如本文所用，术语“CTX001”属于基因治疗药物，是一类细胞，具体地，CTX001是基于CRISPR基因编辑技术通过剪切β-地中海贫血患者的BCL11A基因实现治疗目的。
野生型的基因编辑蛋白
如本文所用，“野生型的基因编辑蛋白”是指天然存在的、未经过人工改造的基因编辑蛋白，其核苷酸可以通过基因工程技术来获得，如基因组测序、聚合酶链式 反应(PCR)等，其氨基酸序列可由核苷酸序列推导而得到。所述野生型的基因编辑蛋白的来源包括(但并不限于)：酿脓链球菌(Streptococcus pyogenes)、葡萄球菌(Staphylococcus aureus)、氨基酸球菌属(Acidaminococcus sp)、毛螺科菌(Lachnospiraceae bacterium)。
在本发明的一个优选例中，所述野生型基因编辑蛋白的氨基酸序列如SEQ ID NO.:1或14或15所示。
在本发明的一个优选例中，所述基因编辑蛋白包括，但并不限于，Cas9、Cas9a、Cas12、Cas12a、Cas12b、Cas13、Cas14。
DNA双链结合结构域
如本文所用，术语“DNA双链结合结构域”为无序列特异性的DNA双链结合结构域。与序列特异性的DNA双链结合结构域相比，本发明的无序列特异性的DNA双链结合结构域不受DNA序列的限制，理论上可以结合任何DNA序列，因此，可以适用于任何位置的DNA的结合。一种优选的DNA双链结合结构域的序列如SEQ ID NO.:10或11所示。
碱基编辑器
本文提供的任何碱基编辑器能够修饰特定核苷酸碱基而不产生显著比例的插入/缺失。如本文所用，“插入/缺失”指核酸内的核苷酸碱基的插入或缺失。此类插入或缺失可以导致基因编码区内的移码突变。在一些实施方案中，期望产生有效修饰(例如突变或脱氨基化)核酸内的特定核苷酸，而不在核酸中产生大量插入或缺失(即插入/缺失)的碱基编辑器。在某些实施方案中，本文提供的任何碱基编辑器能够产生相对于插入/缺失更大比例的意图修饰(例如，点突变或脱氨基化)。
本发明的任何碱基编辑器能够有效地在核酸(例如基因组内的核酸)中产生意图的突变，如点突变，而不产生大量的非意图突变，诸如非意图点突变。
在本发明中，碱基编辑器包括胞嘧啶脱氨酶和腺嘌呤脱氨酶，其他类型的碱基编辑器只要具备本发明的碱基编辑器的功能也在本发明的保护范围内。
在本发明中，将基因编辑蛋白与碱基编辑器融合后的结构称之为ABE或CBE，其中，ABE为基因编辑蛋白与腺嘌呤脱氨酶融合后的结构，CBE为基因编辑蛋白与胞嘧啶脱氨酶融合后的结构。
一种优选的碱基编辑器的序列如SEQ ID NO.:2或12所示。
融合蛋白
如本文所用，“本发明融合蛋白”、或“多肽”均指本发明第二方面所述的融合蛋白。本发明融合蛋白的结构如下式I或I’所示：
C-A-L-B    (I)
B-L-A-C    (I’)
式中，
A为基因编辑蛋白，
B为DNA双链结合结构域，
C为任选的碱基编辑器元件；
L为无或连接肽，
各“-”独立地为连接肽或肽键或非肽键。
在本发明中，连接肽的长度对融合蛋白的活性有影响，优选的连接肽的长度为1-100aa，较佳地，15-85aa，更佳地，25-70aa。
一种优选的连接肽如SEQ ID NO.:3-7所示。
如本文所用，术语“融合蛋白”还包括具有上述活性的、SEQ ID NO.:8、9、或13所示的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明还包括上述融合蛋白的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明融合蛋白的功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)抗原肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式I的氨基酸序列相比，有至多3个，较佳地至多2个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供本发明融合蛋白的类似物。这些类似物与SEQ ID NO.:8或9或13所示的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
在本发明中，在式I中，A为基因编辑蛋白，B为HMG-D或Sac7d，C为腺嘌呤脱氨酶或胞嘧啶脱氨酶或无，L为L1或L2或L3或L4或L5或无。
在一优选实施方式中，在式I中，A为基因编辑蛋白，B为HMG-D，C为或腺嘌呤脱氨酶或胞嘧啶脱氨酶或无，L为L4或L5。
在一优选实施方式中，本发明的融合蛋白还可以包括式I中的A、B、C、L元件中的两种或多种。
在一优选实施方式中，在式I中，A为基因编辑蛋白，B为HMG-9，C为无，L为L4。
在一优选实施方式中，在式I中，A为基因编辑蛋白，B为HMG-D，C为腺嘌呤脱氨酶，L为L5。
在一优选实施方式中，在式I中，A为基因编辑蛋白，B为HMG-D，C为胞嘧啶脱氨酶，L为L5。
在一优选实施方式中，本发明的融合蛋白的氨基酸序列如SEQ ID NO.:8、9或13所示。
腺相关病毒
因腺相关病毒(Adeno-associated virus,AAV)较其他病毒载体小，无致病性，可转染正在分裂和未分裂的细胞等特性，基于AAV载体的针对遗传性疾病的基因治疗方法受到了广泛的关注。
腺相关病毒(adeno-associated virus，AAV),也称腺伴随病毒,属于微小病毒科依赖病毒属,是目前发现的一类结构最简单的单链DNA缺陷型病毒，需要辅助病毒(通常为腺病毒)参与复制。它编码两个末端的反向重复序列(ITR)中的cap和rep基因。ITRs对于病毒的复制和包装具有决定性作用。cap基因编码病毒衣壳蛋白，rep基因参与病毒的复制和整合。AAV能感染多种细胞。
重组腺相关病毒载体(rAAV)源于非致病的野生型腺相关病毒，由于其安全性好、宿主细胞范围广(分裂和非分裂细胞)、免疫源性低，在体内表达外源基因时间长等特点，被视为最有前途的基因转移载体之一，在世界范围内的基因治疗和疫苗研究中得到广泛应用。经过10余年的研究，重组腺相关病毒的生物学特性己被深入了解，尤其是其在各种细胞、组织和体内实验中的应用效果方面已经积累了许多资料。在医学研究中，rAAV被用于多种疾病的基因治疗的研究(包括体内、体外实验)；同时作为一种有特点的基因转移载体，还广泛用于基因功能研究、构建疾病模型、制备基因敲除鼠等方面。
在本发明一个优选的实施例中，载体为重组AAV载体。AAV是相对较小的DNA病毒，其可以稳定和位点特异性方式整合到它们所感染的细胞的基因组中。它们能够感染一大系列的细胞而不对细胞生长、形态或分化产生任何影响，并且它们似乎并不涉及人体病理学。AAV基因组己被克隆、测序及表征。AAV在每个末 端包含约145个碱基的反向末端重复序列(ITR)区域，其作为病毒的复制起点。该基因组的其余被分成两个带有衣壳化功能的重要区域：包含涉及病毒复制和病毒基因表达的rep基因的基因组左边部分；以及包含编码病毒衣壳蛋白的cap基因的基因组右边部分。
AAV载体可采用本领域的标准方法制备。任何血清型的腺相关病毒均是合适的。用于纯化载体的方法可见于例如美国专利No.6566118、6989264和6995006，它们的公开内容整体以引用方式并入本文。杂合载体的制备在例如PCT申请No.PCT/US2005/027091中有所描述，该申请的公开内容整体以引用方式并入本文。用于体外和体内转运基因的衍生自AAV的载体的使用己有描述(参见例如国际专利申请公布No.WO91/18088和WO93/09239；美国专利No.4,797,368、6,596,535和5,139,941，以及欧洲专利No.0488528，它们均整体以引用方式并入本文)。这些专利公布描述了其中rep和/或cap基因缺失并被所关注的基因替换的各种来源于AAV的构建体，以及这些构建体在体外(进入培养的细胞中)或体内(直接进入生物体)转运所关注的基因的用途。复制缺陷重组AAV可通过将以下质粒共转染进被人类辅助病毒(例如腺病毒)感染的细胞系而制备：所含的所关注核酸序列的侧翼为两个AAV反向末端重复序列(ITR)区域的质粒，和携带AAV衣壳化基因(rep和cap基因)的质粒。然后通过标准技术纯化所产生的AAV重组体。
在一些实施方案中，重组载体被衣壳化到病毒粒子(例如包括但不限于AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15和AAV16的AAV病毒粒子)中。因此，本公开包括含有本文所述的任何载体的重组病毒粒子(因其包含重组多核苷酸而为重组的)。产生这样的粒子的方法是本领域己知的，并在美国专利No.6,596,535中有所描述。
表达载体和宿主细胞
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明融合蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的融合蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明融合蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码融合蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明融合蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞(如大肠杆菌)，或是低等真核细胞，或是高等真核细胞，如酵母细胞、植物细胞或哺乳动物细胞(包括人和非人哺乳动物)。代表性例子有：大肠杆菌、麦胚细胞，昆虫细胞，SF9、Hela、HEK293、CHO、酵母细胞等。在本发明的一个优选实施方式中，选择酵母细胞(如毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母)为宿主细胞。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
基因治疗
遗传病的基因治疗(Gene Therapy)是指应用基因工程技术将正常基因引入患者细胞内，以纠正缺陷基因而根治疾病。纠正的途径既可以是原位修复有缺陷的基因，也可以是用有功能的正常基因转入细胞基因组的某一部位，以替代缺陷基因来发挥作用。基因是携带生物遗传信息的基本功能单位，是位于染色体上的一段特定序列。将外源的基因导入生物细胞内必须借助一定的技术方法或载体，基因转移的方法分为生物学方法、物理方法和化学方法。腺病毒载体是目前基因治疗最为常用的病毒载体之一。基因治疗主要是治疗那些对人类健康威胁严重的疾病，包括，但并不限于：遗传病(如血友病、囊性纤维病、家庭性高胆固醇血症等)、恶性肿瘤、心血管疾病、感染性疾病(如艾滋病、类风湿等)。基因治疗是将人的正常基因或有治疗作用的基因通过一定方式导入人体靶细胞以纠正基因的缺陷或者发挥治疗作用，从而达到治疗疾病目的的生物医学高技术。基因治疗与常规治疗方法不同：一般意义上疾病的治疗针对的是因基因异常而导致的各种症状，而基因治疗针对的是疾病的根源--异常的基 因本身。基因治疗的靶细胞包括，但并不限于，体细胞、骨髓细胞、肝细胞、神经细胞、内皮细胞、肌细胞。
在本发明中，通过基因治疗将靶基因进行高效的基因编辑(包括基因插入、替换等)，从而恢复基因的正常表达或增强基因的表达，从而治疗相关疾病。
本发明的主要优点包括：
(1)本发明首次发现，本发明的融合蛋白可显著提高体内或体外的基因编辑效率。
(2)本发明首次发现，本发明的融合蛋白可显著提高体内或体外的基因编辑效率，提高幅度≥20％，较佳地，＞40％，更佳地，＞60％(如80％)，最高可达2倍。
(3)本发明首次发现体外转录增强型基因编辑工具的mRNA，提高动物模型构建的成功性。
(4)本发明使用包装增强型基因编辑工具的AAV病毒，表达增强型基因编辑工具的蛋白，提高疾病治疗效果。
(5)本发明首次利用通过融合双链DNA结合结构域来提高基因编辑效率。
(6)本发明首次筛选找到了一种高效提高基因编辑效率的双链DNA结合结构域(如HMG-D),以及其非常优异的融合方式。
(7)本发明首次发现，本发明的双链DNA结合结构域可以广泛的提高各种基因编辑工具的基因编辑效率。
(8)本发明的增强型基因编辑工具可以提高动物模型构建的成功率，以及基因治疗效率。
(9)本发明的增强型基因编辑工具还可用于基因治疗。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非有特别说明，否则本发明实施例中的试剂和材料均为市售产品。
通用方法
方法1：(适用于实施例1、实施例2和实施例3)
1、24孔板的HEK293T细胞密度长至60-70％，等摩尔量的质粒通过转染试剂PEI转染进细胞，8-10小时换液，细胞培养一段时间后(Cas9系统培养72小时，碱基编辑系统培养120小时)，收细胞，提基因组。
2、设计合适引物扩增靶点左右共150-180bp的序列，进行Hitom建库，深度测序，分析计算编辑效率。
方法2：(适用于实施例4的动物模型构建)
1、体外转录增强型基因编辑工具的mRNA和对应靶点的sgRNA。
2、小鼠胚胎注射mRNA和sgRNA，移植到代孕母鼠，获得F0代小鼠，鉴定基因，统计小鼠基因型突变率，计算模型构建成功率。
方法3：(适用于实施例4的基因治疗)
1、包装增强型基因编辑工具的腺相关病毒(AAV)和治疗基因的sgRNA，或者同源修复模板。
2、通过静脉注射(尾静脉)或局部注射(肌肉等)包装的AAV至疾病动物模型(小鼠或大鼠)，同时注射对照病毒。
3、定期观察，检测治疗组的动物模型和对照组动物模型的表型，评价治疗效率。
实施例1筛选增强型基因编辑工具
分别合成不同种类的双链DNA结合结构域(HMG-D和Sac7d)，通过设计5种不同长度的linker(L1、L2、L3、L4和L5)(表1)，以及融合在Cas9的N端和C端，在两个内源性靶点(VEGF和HBG1/2)上进行比较，根据结果统计，对这一系列优化所得到的增强型的基因编辑工具进行打分(在效果均很好的基础上所做的相对评分)(不好：A；欠好：AA；较好：AAA；好：AAAA；非常好：AAAAA),结果如下：(注：C表示Cas9；L1-L5表示不同linker；H表示HMG-D结构域；S表示sac7d。)
A：S-L1-C,H-L1-C
AA:S-L2-C,S-L3-C,S-L4-C,H-L2-C
AAA:H-L4-C-L4-S,H-L4-C-L5-S
AAAA:H-L3-C,C-L4-S,C-L5-S,C-L4-H,H-L4-C-L4-H,H-L4-C-L5-H,H-L4-H-L4-C,C-L5-H-L5-H
AAAAA:H-L4-C,C-L5-H
综合统计，发现HMG-D的效果非常好，并且L4和L5长度(32个和64个氨基酸)的linker是最优的linker，通过比较N端和C端连接，发现在最优linker条件下，N端和C端连接的效果相都很好，因此本发明人得到增强型的基因编辑工具，即HMG-D通过L4或L5的linker融合在Cas9的N端或C端是非常好的融合方式(图1)。
同理，这种通过双链DNA结合结构域融合来增强基因编辑效率的增强型基因编辑工具，也可以类比到其他类型的双链DNA结合结构域，如应用广泛锌指蛋白(ZFP)、其他转录因子的DNA结合结构域和来源其他物种的HMG-D或Sac7d，等等。本发明同样可以通过这些双链DNA结合结构域来提高基因编辑效率，因此，本发明最重要的是在于发现了双链DNA结合结构域融合基因编辑工具可以提高基因编辑效率，而其中优选双链DNA结合结构域HMG-D。
实施例2提高SpCas9的基因编辑效率
将获得的增强型基因编辑工具(即本发明的融合蛋白，如HMG-D-L4-SpCas9)进一步在更多的内源性靶点上进行效果方面的比较，通过等摩尔比转染293T细胞，发现在所比较的靶点中和SpCas9相比较，本发明的融合蛋白的编辑效率都有大于20％(或60％、或80％)的提高，最高可达2倍(图2)。
实施例3提高其他种属来源的Cas9(如：SaCas9)的基因编辑效率
通过构建HMG-D-L4-SaCas9表达载体，针对内源性靶点，等摩尔比转染293T细胞，发现融合HMG-D后SaCas9编辑效率也有类似的效果的提升。(图3)
实施例4提高其他非Cas9蛋白(如：AsCas12a)的基因编辑效率
通过构建HMG-D-L4-AsCas12a表达载体，针对内源性靶点，等摩尔比转染293T细胞，发现融合HMG-D后AsCas12a编辑效率也有类似的效果的提升。(图4)
实施例5提高转录调控工具(CRISPR/Cas9)的转录激活效率
通过构建HMG-D-L4-dCas9-VPR表达载体，针对内源性靶点，等摩尔比转染293T细胞，发现融合HMG-D后dCas9-VPR对于内源性基因的转录激活效率也有类似的效果的提升。(图5)
实施例6提高碱基编辑器(base editor)的编辑效率
由于碱基编辑器(base editor)是通过在Cas9的N端融合了胞嘧啶脱氨酶(CBE)和腺嘌呤脱氨酶(ABE)，所以，在碱基编辑器的Cas9的c端通过L5linker来融合HMG-D，开发出增强型单碱基编辑工具。通过内源性靶点比较，融合HMG-D后碱基编辑器(如ABE)的编辑效率有了极大的提升，如提高了＞1.5－2倍(图6)。
融合了其他类型碱基编辑器的融合蛋白的编辑效率的提升幅度与融合了ABE的融合蛋白的编辑效率类似或相当。
实施例7提高提高动物模型构建的成功率，以及基因治疗效率
通过体外转录增强型基因编辑工具的mRNA，增加了动物模型构建的成功率。同时，通过包装增强型的基因编辑工具的AAV病毒，也可以提高疾病治疗效率。
对比例1与不是DNA结合结构域融合后效果不好
为了进一步验证DNA结合结构域融合后可以提高基因编辑效率，我们把DNA结合结构域改为GFP蛋白(一种非DNA结合结构域)这种无关的蛋白，发现融合了GFP蛋白后不能提高基因编辑效率(图1)。同时，还对HMG-D结构域进行突变，破坏其DNA结合的能力，构建了3个氨基酸突变的HMG-D结构域(mutHMG-D，简称mutH),通过实验比较，mutHMG-D结构域也不能提高基因编辑效率(图1)。
对比例2HMG-D、Sac7d之外的DNA结合结构域效果不好
为了扩宽DNA结合结构域的范围，又测试了一些单链DNA结合结构域(如Rad51)，融合Rad51后同样不能提高基因编辑效率(图1)。因此，结果表明，本发明上述筛选的HMG-D和Sac7d的双链DNA结合结构域是非常有效的。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被 单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种融合蛋白，其特征在于，所述融合蛋白的结构如下式I或I’所示：
    C-A-L-B  (I)
    B-L-A-C  (I’)
    式中，
    A为基因编辑蛋白，
    B为DNA双链结合结构域，
    C为任选的碱基编辑器元件；
    L为无或连接肽，
    各“-”独立地为连接肽或肽键或非肽键。
  2. 一种多核苷酸，其特征在于，所述的多核苷酸编码权利要求1所述的融合蛋白。
  3. 一种载体，其特征在于，所述的载体含有权利要求2所述的多核苷酸。
  4. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求3所述的载体，或其基因组中整合有权利要求2所述的多核苷酸。
  5. 一种产生权利要求1所述的融合蛋白的方法，其特征在于，包括步骤：
    在适合表达的条件下，培养权利要求4所述的宿主细胞，从而表达融合蛋白；和/或
    分离所述融合蛋白。
  6. 一种基因编辑试剂，其特征在于，所述基因编辑试剂包含权利要求1所述的融合蛋白。
  7. 一种试剂盒，其特征在于，包括权利要求6所述的基因编辑试剂。
  8. 一种权利要求1所述的融合蛋白的用途，其特征在于，用于制备试剂或试剂盒，所述试剂或试剂盒用于提高基因编辑效率。
  9. 一种药物组合物，其特征在于，包括：
    (a)权利要求1所述的融合蛋白、或其编码基因、或其表达载体；和
    (b)药学上可接受的载体。
  10. 一种药盒，其特征在于，包括：
    (a1)第一容器，以及位于所述第一容器中的权利要求1所述的融合蛋白、或其编码基因、或其表达载体，或含有权利要求1所述的融合蛋白的药物。
    在另一优选例中，所述药盒还包括：
    (a2)第二容器，以及位于所述第二容器中的其他用于基因治疗的药物，或含有其他用于基因治疗的药物的药物。
  11. 一种权利要求1所述的融合蛋白的用途，其特征在于，用于制备用于基因治疗的药物。
  12. 一种提高基因编辑效率的方法，其特征在于，包括步骤：
    在权利要求1所述的融合蛋白或权利要求6所述的基因编辑试剂存在下，对细胞进行基因编辑，从而提高基因编辑效率。
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Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4797368A (en) 1985-03-15 1989-01-10 The United States Of America As Represented By The Department Of Health And Human Services Adeno-associated virus as eukaryotic expression vector
WO1991018088A1 (en) 1990-05-23 1991-11-28 The United States Of America, Represented By The Secretary, United States Department Of Commerce Adeno-associated virus (aav)-based eucaryotic vectors
EP0488528A1 (en) 1990-10-30 1992-06-03 Applied Immune Sciences, Inc. Recombinant adeno-associated virus vectors
US5139941A (en) 1985-10-31 1992-08-18 University Of Florida Research Foundation, Inc. AAV transduction vectors
WO1993009239A1 (en) 1991-11-08 1993-05-13 Research Corporation Technologies, Inc. Adeno-associated virus-2 basal vectors
US6566118B1 (en) 1997-09-05 2003-05-20 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US6596535B1 (en) 1999-08-09 2003-07-22 Targeted Genetics Corporation Metabolically activated recombinant viral vectors and methods for the preparation and use
US6989264B2 (en) 1997-09-05 2006-01-24 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
WO2016057961A1 (en) * 2014-10-10 2016-04-14 Editas Medicine, Inc. Compositions and methods for promoting homology directed repair
CN109152808A (zh) * 2016-04-29 2019-01-04 生物辐射实验室股份有限公司 用于核酸序列的特异性靶向的二聚蛋白质
WO2019014230A1 (en) * 2017-07-11 2019-01-17 Sigma-Aldrich Co. Llc USE OF DOMAINS OF PROTEINS INTERACTING WITH NUCLEOSOMES TO IMPROVE THE TARGETED MODIFICATION OF THE GENOME

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5241493B2 (ja) * 2005-07-15 2013-07-17 アジレント・テクノロジーズ・インク Ｄｎａ結合タンパク質−ポリメラーゼのキメラ
WO2017031483A1 (en) * 2015-08-20 2017-02-23 Applied Stemcell, Inc. Nuclease with enhanced efficiency of genome editing
US20200291370A1 (en) * 2016-03-18 2020-09-17 President And Fellows Of Harvard College Mutant Cas Proteins

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4797368A (en) 1985-03-15 1989-01-10 The United States Of America As Represented By The Department Of Health And Human Services Adeno-associated virus as eukaryotic expression vector
US5139941A (en) 1985-10-31 1992-08-18 University Of Florida Research Foundation, Inc. AAV transduction vectors
WO1991018088A1 (en) 1990-05-23 1991-11-28 The United States Of America, Represented By The Secretary, United States Department Of Commerce Adeno-associated virus (aav)-based eucaryotic vectors
EP0488528A1 (en) 1990-10-30 1992-06-03 Applied Immune Sciences, Inc. Recombinant adeno-associated virus vectors
WO1993009239A1 (en) 1991-11-08 1993-05-13 Research Corporation Technologies, Inc. Adeno-associated virus-2 basal vectors
US6566118B1 (en) 1997-09-05 2003-05-20 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US6989264B2 (en) 1997-09-05 2006-01-24 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US6995006B2 (en) 1997-09-05 2006-02-07 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US6596535B1 (en) 1999-08-09 2003-07-22 Targeted Genetics Corporation Metabolically activated recombinant viral vectors and methods for the preparation and use
WO2016057961A1 (en) * 2014-10-10 2016-04-14 Editas Medicine, Inc. Compositions and methods for promoting homology directed repair
CN109152808A (zh) * 2016-04-29 2019-01-04 生物辐射实验室股份有限公司 用于核酸序列的特异性靶向的二聚蛋白质
WO2019014230A1 (en) * 2017-07-11 2019-01-17 Sigma-Aldrich Co. Llc USE OF DOMAINS OF PROTEINS INTERACTING WITH NUCLEOSOMES TO IMPROVE THE TARGETED MODIFICATION OF THE GENOME

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SAMBROOK ET AL.: "Molecular Cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP3943512A4

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