WO2016086768A1 - Nucléase chimérique pour reconnaître spécifiquement et réparer le gène bêta-globine de la β-thalassémie - Google Patents

Nucléase chimérique pour reconnaître spécifiquement et réparer le gène bêta-globine de la β-thalassémie Download PDF

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WO2016086768A1
WO2016086768A1 PCT/CN2015/094901 CN2015094901W WO2016086768A1 WO 2016086768 A1 WO2016086768 A1 WO 2016086768A1 CN 2015094901 W CN2015094901 W CN 2015094901W WO 2016086768 A1 WO2016086768 A1 WO 2016086768A1
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protein
gene
nucleic acid
acid molecule
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纪家葵
林建飞
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清华大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the invention belongs to the field of genetic engineering technology, and particularly relates to a chimeric nuclease which specifically recognizes and repairs the beta-globin gene of beta thalassemia.
  • Thalassemia is a high-risk, widespread genetic disease in China and around the world.
  • Normal hematopoietic stem cells have the ability to differentiate into blood cells.
  • Thalassemia patients have abnormal red blood cells, are fragile and easy to die, and their oxygen carrying capacity is insufficient. Therefore, hematopoietic stem cell transplantation (HSCT) is currently curing severe ⁇ -mediterranean.
  • the most effective method of anemia includes bone marrow hematopoietic stem cell transplantation (BMT), peripheral blood stem cell transplantation (PB SCT), and cord blood stem cell transplantation (CBSCT).
  • BMT bone marrow hematopoietic stem cell transplantation
  • PB SCT peripheral blood stem cell transplantation
  • CBSCT cord blood stem cell transplantation
  • Beta-thalassemia is a type of anemia caused by mutations in the beta globin gene.
  • Normal hemoglobin consists of two parts of beta globin and two parts of alpha globin. If the expression level of ⁇ -globin is too small, it will result in agglomeration of ⁇ -globin and cause symptoms such as hemolysis.
  • Beta globin is expressed by the beta gene located on chromosome 11 of the chromosome. The beta gene contains multiple mutation hotspots, and the mutation hotspots are different in different regions.
  • I-SceI is a very specific homing endonuclease. It is a very specific and very safe endonuclease. It can combine DNA fragments of specific sequences on one hand and cleavage in recognized DNA sequences on the other hand. DNA double-strand breaks, the 18 sites of recognition and cleavage have very strong specificity. In many previous reports, I-SceI has been shown to be less toxic to the human genome. In published studies, the modification of homing endonucleases has been screened for specific recognition in bacteria or yeast. Of the 18 bases recognized by I-SceI, -7 bases to +11 bases are very important. The crystal structure shows that the specific recognition of these 18 bases and I-SceI proteins is formed by direct contact of many phosphates and bases and the regulation of water molecules between the two.
  • TALEN is currently the easiest to construct nucleases, and TALE, which specifically binds to any DNA, confers higher homologous recombination repair rates to TALEN.
  • TALE has the characteristic of specifically recognizing longer DNA sequences.
  • the current protein analysis and crystallization results show that the DNA binding domain of TALEs protein is highly conserved and consists of repeat fragments containing approximately 33-35 amino acids. In-depth research found that this section of ammonia The base acid is only two amino acids that are different, and these two amino acids are exactly the bases that specifically recognize the DNA. These two variable amino acids are referred to as repeatvariable di-residues (RVD).
  • RVD repeatvariable di-residues
  • HD specifically recognizes C bases
  • NI recognizes A bases
  • NN recognizes G or A bases
  • NG recognizes T bases.
  • nucleases include zinc finger protein (ZFN), TALEN and CRIPAR/Cas9 systems, and the like.
  • ZFN zinc finger protein
  • TALEN zinc finger protein
  • CRIPAR/Cas9 CRIPAR/Cas9 systems
  • ZFN zinc finger protein
  • TALEN technology has the characteristics of easy assembly, fast speed and strong identification.
  • FokI FokI
  • the CRISPR/Cas9 system has the characteristics of simple operation, but its binding to nucleic acids is very non-specific, which will hinder its safe application in clinical gene therapy.
  • CRISPR/CAS9 is not suitable for human genome targeting and modification of mutant genes (Wu et al., nature Biotechnology, 2014). , doi: 10.1038/nbt. 2889; Liang et al, Protein Cell, 2015, 6(5): 363-372).
  • the protein provided by the present invention is as follows 1) or 2):
  • the fusion protein shown is a protein obtained by replacing the TALE protein FokI with the modified I-SceI protein;
  • the amino acid sequence of the TALE protein is the sequence 4 in the sequence listing;
  • amino acid sequence of the engineered I-SceI protein is amino acid sequence 706-940 of the sequence 2 in the sequence listing;
  • amino acid sequence of the protein shown is the amino acid sequence 706-940 from the N' end of the sequence 2 in the sequence listing.
  • Nucleic acid molecules encoding the above proteins are also within the scope of the invention.
  • the above nucleic acid molecule is a DNA molecule according to any one of the following 1) to 4):
  • the coding region is a fusion protein represented by the DNA molecule (1) shown in SEQ ID NO: 1 in the sequence listing. Code area);
  • the coding region is the coding region of the protein represented by the DNA molecule (2) represented by nucleotides 2116-2823 of SEQ ID NO: 1 in the sequence listing);
  • the above stringent conditions may be to hybridize and wash the membrane at 65 ° C in a DNA or RNA hybridization experiment using a solution of 0.1 x SSPE (or 0.1 x SSC), 0.1% SDS.
  • An expression cassette, recombinant vector, recombinant strain, transgenic cell line or recombinant strain containing the above DNA molecule is also within the scope of protection of the present invention.
  • the above recombinant vector is a recombinant vector obtained by inserting the above nucleic acid molecule into an expression vector.
  • the expression vector used in the embodiment of the present invention is a PCS2 vector
  • the recombinant vector PCS2-TALE-I-SceI is a vector obtained by inserting the nucleotide represented by the sequence 1 in the sequence table into the NcoI and XbaI cleavage sites of the PCS2 vector.
  • This vector expresses the fusion protein TALE-I-SceI.
  • the recombinant strain is a recombinant strain obtained by introducing the recombinant vector into a target bacteria.
  • the above nucleic acid molecule an expression cassette containing the above nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain is also useful for improving gene efficiency of cleavage, improving gene efficiency of repairing target or reducing cytotoxicity
  • the scope of the invention is protected.
  • the target gene is cleaved so that the target gene after cleavage is self-repaired in the cell, and finally the normal gene sequence is produced.
  • the gene of interest is the beta thalassemia beta-globin gene.
  • the above protein, the above nucleic acid molecule, an expression cassette containing the above nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain are prepared for repairing a patient with ⁇ -thalassemia in vitro
  • the use of mutant products of genes carried by blood stem cells is also within the scope of the invention.
  • the gene is a ⁇ -thalassemia beta-globin gene.
  • Another object of the present invention is to provide a kit for cleavage of a gene of interest and/or repair of a gene of interest.
  • the kit provided by the present invention comprises the above protein, the above nucleic acid molecule, an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain.
  • a third object of the present invention is to provide a kit for improving the efficiency of gene gene for cleavage, improving the efficiency of repairing a gene of interest, or reducing cytotoxicity.
  • the kit provided by the present invention comprises the above protein, the above nucleic acid molecule, an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain.
  • a fourth object of the present invention is to provide a therapeutic or adjunctive treatment of a beta-thalassemia product.
  • the active ingredient of the product provided by the present invention is the above-mentioned protein, the above nucleic acid molecule, an expression cassette containing the above nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain.
  • a fifth object of the present invention is to provide a mutant product for repairing a gene carried by a hematopoietic stem cell of a patient with ⁇ -thalassemia in vitro, wherein the active ingredient is the above protein, the above nucleic acid molecule, an expression cassette containing the nucleic acid molecule, a recombinant vector, Recombinant bacteria, transgenic cell lines or recombinant bacteria.
  • the gene is a ⁇ -thalassemia beta-globin gene
  • the above products are drugs or kits.
  • Figure 1 shows a green fluorescent protein repair reporter system expressed in human cells.
  • Figure 2 is a scheme for engineering I-SceI to specifically recognize the beta-globin DNA sequence.
  • Figure 3 shows the position and screening results of the I-SceI protein modification.
  • Figure 4 is a schematic representation of the chimeric protein.
  • Figure 5 shows the targeting efficiency of the modified I-SceI protein and TALE-ISceI in the beta-globin DNA sequence, respectively.
  • Figure 6 shows the gene repair efficiency of the modified I-SceI protein and TALE-ISceI.
  • Figure 7 is a comparison of the cytotoxicity of the engineered I-SceI protein and TALE-ISceI.
  • Figure 8 shows the expression of the modified I-SceI protein and Tale-ISVBII by Western blot.
  • Example 1 Preparation of a chimeric nuclease that specifically recognizes and repairs the beta-globin gene of beta thalassemia
  • the homing endonuclease I-SceI uses a specific fluorescent reporter system in human cells to screen for DNA sequences that specifically recognize the proximity of the beta thalassemia mutation site (Fig. 1). From the structure of the current I-SceI, it can be found that certain amino acids and DNA sequences in I-SceI are in direct contact, and the sequence of bases can be gradually changed, and then these directly contacted amino acids are screened, and the most specific new binding is found. The binding site for the amino acid (see Figure 2).
  • ATAAC in DNA sequences is an important part of protein cleavage. When these bases are changed, the cleavage efficiency will be significantly reduced. It can be screened by changing the amino acid of I-SceI. To the I-SceI engineered enzyme that recognizes the new sequence.
  • T at position -7 is directly in contact with N152 amino acid.
  • the strategy is to sequentially change the amino acid of I-SceI that is in direct contact with the four bases of DNA, and it is hoped that a new I-SceI amino acid capable of specifically recognizing the ⁇ -globin gene can be found.
  • I-SceI first recognition site TAG changed it to AAA of ⁇ -globin gene, then point mutation of N152 amino acid of I-SceI, changed to 19 other amino acids, and then used eGFP reporting system to screen the most Highly efficient HDR amino acids.
  • I-SceI Fig.
  • a I-SceI-recognized original sequence and post-engineered sequence recognized
  • b the three-dimensional structure map shows the need for altered nucleic acid sites and The position of interaction of the protein
  • cf which is the result of four rounds of screening
  • the altered nucleotide position of the engineered I-SceI coding gene is sequence 1 in the sequence listing from the 5' end of the 5' end, 45, 144, 456, 579, modified I -SceI amino acid sequence altered amino acid position as sequence Sequence 2 in the table is from position 15, 48, 152, 193 at the N' end.
  • the GFP Gene Targeting Systems (see Figure 4). This system consists of three parts: the first part, the cell line is stably integrated with one unit of GFP gene by viral transfection, and the coding region of this gene is inserted into a target sequence of 18 bp which is gradually transformed into ⁇ -globin, so that GFP cannot be expressed normally. In the second part, the engineered endonuclease I-SceI was expressed by viral transfection in cells. In the third part, the donor is transfected into a donor sequence containing a truncated GFP (tGFP) sequence.
  • tGFP truncated GFP
  • the homing endonuclease When the homing endonuclease cleaves to form a DNA double-strand break, it can induce homologous recombination of the donor fragment and the endonuclease recognition fragment, repair the gene, and repair the eGFP gene into the correct sequence to express the eGFP protein. .
  • the relative content of eGFP protein-positive cells was detected by flow cytometry, etc., so the relative repair efficiency of I-SceI protein can be judged to find the most suitable I-SceI engineering enzyme. In the absence of I-SceI, homologous repair is very inefficient and it is generally believed that one of the 10 million cells will be repaired. In the presence of I-SceI, this efficiency can be significantly improved, reaching more than 1000 times, which is why the high specificity of the target protein is very important.
  • TALE Three pairs of homologous enzymes are currently found on the repeats of TALE: BsaI (multiple sites are recognizable), SpeI and NheI, and BspEI and XmalI found.
  • BsaI multiple sites are recognizable
  • SpeI and NheI the fragment can be ligated by T4 ligase, and the fragment can be identified.
  • Two bases Two bases.
  • a TALE plasmid sequence capable of recognizing the 15 bp globin gene sequence corresponding to the TALE amino acid was constructed.
  • the 53 bp globin sequence to be recognized was inserted into the coding region of the eGFP gene using the "green fluorescent protein gene targeting system", and the biological activity of the constructed TALE plasmid was verified.
  • the forward primer "CACCATGGCTCCAAAGAAGAAGCGTAAGGTA” and the reverse primer “GGATCCGGCAACGCGA TGGGATGTGC” were used to obtain a 2119 bp TALE amplification product;
  • the above TALE amplification product was digested with NcoI and BamHI, and the obtained digested product was ligated with the same digested PCS2 vector (addgene, plasmid #17095) to obtain an intermediate vector PCS2-TALE (for sequence 1 from 5'
  • PCS2-TALE for sequence 1 from 5'
  • the 1-2115 nucleotide of the terminal is inserted between the NcoI and BamHI restriction sites of the PCS2 vector, and the PCS2 vector contains a CMV promoter, which is capable of initiating expression of the gene of interest in various human cell lines;
  • the above I-SceI amplification product was digested with BamHI and XbaI, and the digested product was ligated with the same digested intermediate vector PCS2-TALE to obtain a recombinant vector;
  • the recombinant vector is a vector obtained by inserting the nucleotide shown in SEQ ID NO: 1 in the sequence table into the NcoI and XbaI cleavage sites of the PCS2 vector, and named PCS2-TALE-I-SceI, which expresses the fusion protein TALE.
  • PCS2-TALE-I-SceI which expresses the fusion protein TALE.
  • -I-SceI which is a chimeric nuclease, the amino acid sequence of which is sequence 2 in the sequence listing.
  • the nucleotide sequence shown in SEQ ID NO: 1 includes the engineered I-SceI encoding gene and the TALE encoding gene ligated upstream thereof.
  • the nucleotide sequence of the I-SceI encoding gene after the transformation is sequence 1 from the 5' end.
  • the nucleotide sequence of the TALE-encoding gene is sequence 1 from nucleotides 1-2115 of the 5' end.
  • Sequence 2 in the sequence listing is from the N-terminal 1-705 amino acid residue to TALE, and from the N-terminal amino acid residues 706-940 is I-SceI.
  • a recombinant vector expressing I-SceI was constructed, and the transformed I-SceI coding gene (sequence 1 from the 5' end 2116-2823 nucleotide) was inserted into the NcoI and XbaI restriction sites of the PCS2 vector, and was named as PCS2-I-SceI;
  • TALE-encoding recombinant vector was constructed, and the TALE-encoding gene (SEQ ID NO: 1 to 1-5115 from the 5' end) was inserted into the vector of the NcoI and XbaI cleavage sites of the PCS2 vector, Named PCS2-TALE.
  • the engineered fusion protein has the effect of cutting genomic DNA. Without providing a donor, DSB will be repaired by NEHJ repair in vivo, producing indel or mutations at the cleavage site.
  • the expression of the protein was identified by anti-flag antibody by immunofluorescence technique, and the flag was tagged on the PCS2 vector, so it can be used to detect the expression of the target protein.
  • the target proteins of the three transgenic cells were expressed. (See Figure 8, western blot.)
  • PCR amplification was performed with the forward primer "GCCTAGTACATTACTATTTG” and the reverse primer "ATTAGGCAGAATCCAGATGC” to obtain a 768 bp target fragment, which is a beta-globin gene partial fragment of beta thalassemia (beta-globin gene)
  • the length is sequence 6, and the nucleotide sequence of this partial fragment is sequence 6 from the 5' end of nucleotides 892-1565).
  • the PCR product was ligated to the T3 (full-scale gold product) vector, and the monoclonal was selected for Sanger sequencing.
  • the fusion protein TALE-I-SceI is much more efficient at cutting the target fragment than the I-SceI alone.
  • the PCS2-eGFP vector (purchased by Addgene) was used as a template, and PCR amplification was carried out using the forward primer "CACCATGGTGAGCAAGGGCGAGGAGC” and the reverse primer "CTACTTGTACAGCTCGTCCATGC” to obtain a 724 bp green fluorescent protein eGFP gene (SEQ ID NO: 7).
  • a forward primer "ACGTCCAGGAGCGCACCATCTAAAGGATAACAGGGTAATTCTTCAAGGACGACGGCA” and a reverse primer “CTACTTGTACAGCTCGTCCATGC” were used to obtain a 461 bp PCR product 2;
  • CACCATGGTGAGCAAGGGCGAGGAGC and CTACTTGTACAGCTCGTCCATGC were used as primers to obtain a 742 bp DNA fragment eGFP-1.
  • nucleotide sequence of the DNA fragment is SEQ ID NO: 3 in the sequence listing, wherein the nucleotides 299-316 from the 5' end are I-SceI protein recognition sequences.
  • the DNA fragment eGFP-1 prepared in the above 2) was inserted into a D-TOPO vector (topological enzyme recombination, purchased by Life Technology) to obtain a recombinant vector D-TOPO-eGFP-1;
  • the above recombinant vector D-TOPO-eGFP-1 was subjected to LR recombination, and the eGFP-1 sequence was recombined into a p2k viral vector (purchased from Life Technology) to obtain a reporter vector;
  • 293 cells were transfected with the reporter vector to obtain transgenic cells containing eGFP-1 as a reporter system.
  • PCS2-TALE-I-SceI plasmid prepared in Example 1 and the plasmid containing the donor fragment were co-transfected (mass ratio of plasmid: 1:1) transgenic cells containing eGFP-1; after transfection for 36 hours, cells were collected. , using flow cytometry analysis.
  • the plasmid containing the donor fragment is a vector obtained by inserting a donor fragment (nucleotide sequence is sequence 8) into a T3 (full-scale product) carrier.
  • the plasmid containing the donor fragment was separately transfected into control 2;
  • the PCS2-TALEN vector is a vector obtained by inserting the TALEN encoding gene shown in SEQ ID NO: 5 into the NcoI and XbaI cleavage sites of the PCS2 vector.
  • the transfection rate of transfected cells (TALE-ISVBII) transfected with PCS2-TALE-I-SceI vector was 0.21%.
  • the GFP positive cell rate in transgenic cells (ISVBII) infected with PCS2-I-SceI vector was 0.10%; the GFP positive cell rate in transgenic cells (TALEN) transfected with PCS2-TALEN vector was 0.22%; 0, it can be seen that TALE-ISVBII expression of TALE-ISceI protein can significantly improve the repair efficiency of 0.21%, ISVBII expression of I-SceI protein repair efficiency of 0.1%.
  • the fusion protein TALE-I-SceI has low cytotoxicity
  • Both ⁇ -H2AX and 53BP1 proteins are used to detect the biomarkers that produce DSB in cells, and are characterization proteins for cytotoxicity by detecting these two biomarker experiments. Immunofluorescence staining of ⁇ -H2AX and 53BP1 showed that the gene toxicity of TALEN was high.
  • the experimental method is as follows:
  • the cells were cultured for 36 hours.
  • the cells of the 6-well plate were subjected to immunofluorescence staining.
  • the experimental procedure can be briefly as follows: 4% formaldehyde for 15 min, 0.1% Triton X-100 for 10 min, 10% FBS for 1 h, anti- ⁇ -H2AX and 53BP1 protein antibodies were incubated for 1 h, washed with PBS for 3 min for 5 min, secondary antibody for 1 h, washed with PBS for 5 min, DAPI stained for 15 min, washed with PBS for 3 min for 5 min, and the resulting cells were used for microscopic observation or flow analysis.
  • A is the result of ⁇ -H2AX and b is the result of 53BP1. It can be seen that the transfected cells transfected with PCS2-TALE-I-SceI can be compared with the transfected cells transfected with PCS2-I-SceI. Significantly reduced cytotoxicity, reflected in a significant decrease in the expression of ⁇ -H2AX and 53BP1 protein levels.
  • the experiments of the present invention prove that the present invention utilizes the low genotoxicity of homing endonuclease I-SceI and the high-efficiency targeting advantage of TALEN nuclease to construct a fusion endonuclease TALE-ISceI, which is a hematopoietic stem cell of a patient with ⁇ -thalassemia disease. Model to repair its mutant genes.
  • the enzyme plays a role in hematopoietic stem cells of patients with ⁇ -thalassemia in vitro and is expected to be widely used in clinical practice. At the same time, the enzyme can be screened so that the enzyme recognizes and repairs more nucleic acid fragments and promotes them to other genetic diseases.
  • the novel gene targeting technology is used to repair mutations in genes carried by hematopoietic stem cells of patients with ⁇ -thalassemia in vitro, and to differentiate the repaired hematopoietic stem cells into red blood cells capable of normally expressing ⁇ -globin.
  • Direct gene repair on hematopoietic stem cells has the following advantages: avoidance of iPS transgene instability and possible carcinogenicity of iPS cells, while avoiding immunological rejection of transplanted cells. Therefore, the acquisition of this engineered enzyme provides a practical basis for the radical cure of patients with beta thalassemia.

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Abstract

Cette invention concerne une nucléase chimérique TALE-ISceI, dans laquelle FokI dans TALEN est remplacé par une protéine de fusion obtenue par une I-SceI transformée. La nucléase chimérique TALE-ISceI peut spécifiquement reconnaître et réparer un gène β-globine de la β-thalassémie et peut être utilisée pour traiter la β-thalassémie.
PCT/CN2015/094901 2014-12-04 2015-11-18 Nucléase chimérique pour reconnaître spécifiquement et réparer le gène bêta-globine de la β-thalassémie WO2016086768A1 (fr)

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CN201410730391.2A CN105713885B (zh) 2014-12-04 2014-12-04 特异识别并修复β地中海贫血症beta-globin基因的嵌合核酸酶

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CN111088253A (zh) * 2018-10-24 2020-05-01 广州鼓润医疗科技有限公司 针对hbb-28地中海贫血基因的crispr单碱基供体修复体系

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CN108165581B (zh) * 2017-12-14 2021-06-08 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） 采用单链核苷酸片段体外修复hba2基因突变的方法

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