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WO2021037234A1 - Procédé pour réparer efficacement une mutation génétique responsable d'une anémie avec sidéroblastes en couronne - Google Patents

Procédé pour réparer efficacement une mutation génétique responsable d'une anémie avec sidéroblastes en couronne Download PDF

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WO2021037234A1
WO2021037234A1 PCT/CN2020/112227 CN2020112227W WO2021037234A1 WO 2021037234 A1 WO2021037234 A1 WO 2021037234A1 CN 2020112227 W CN2020112227 W CN 2020112227W WO 2021037234 A1 WO2021037234 A1 WO 2021037234A1
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sequence
gene
alas
sgrna
cells
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方日国
袁鹏飞
张英驰
杨卉慧
于玲玲
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Institute of Hematology and Blood Diseases Hospital of CAMS and PUMC
Edigene Guangzhou Inc
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Institute of Hematology and Blood Diseases Hospital of CAMS and PUMC
Edigene Guangzhou Inc
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Publication of WO2021037234A1 publication Critical patent/WO2021037234A1/fr
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention relates to the field of gene editing therapy. Specifically, the present invention relates to a method for efficiently repairing gene mutations that cause ring sideroblastic anemia by using gene editing technology, which includes using gene editing technology to efficiently and safely genetically modify human hematopoiesis
  • the specific point mutation of the ALAS-2 gene of the stem cells restores the expression of the ALAS-2 gene and achieves the purpose of treating diseases.
  • Hereditary sideroblastic anemia (Congenital sideroblastic anemia, CSA) is a group of inherited diseases of iron utilization disorders. It is characterized by the appearance of a large number of ring sideroblasts in the bone marrow, ineffective production of red blood cells, excessive iron reserves in the tissues, and small cell hypochromic anemia in the peripheral blood. At present, molecular level testing has found 7 disease mutations, which mainly lead to iron synthesis, iron-sulfur complex, and mitochondrial protein synthesis disorders. Among them, X-chain sideroblastic anemia is the most common type of disease (Kaneko K, et al. Haematologica. 2014).
  • X-linked sideroblastic anemia is a rare genetic disease of the blood system, which is a kind of CSA with insufficient incidence
  • ALAS-2 is a key regulator that regulates the synthesis of catalyzed heme in red blood cells.
  • the treatment plan of gene editing therapy is to use gene editing tools, such as CRISPR/Cas9, zinc finger nuclease (Zinc Finer Nulease, ZFN), and transcription activator-like effector nuclease (transcription activator-like effector nucleases, TALEN), etc.
  • Gene editing tools such as CRISPR/Cas9, zinc finger nuclease (Zinc Finer Nulease, ZFN), and transcription activator-like effector nuclease (transcription activator-like effector nucleases, TALEN), etc. Edit the patient’s autologous hematopoietic stem cells, repair the ALAS-2 gene mutation to restore the expression of the ALAS-2 gene, and then return the genetically modified autologous hematopoietic stem cells to the patient to make the patient’s heme synthesis and red blood cell status Return to normal level to achieve the purpose of curing diseases.
  • Gene editing technology is a genetic recombination technology that artificially uses encoded nucleases to insert, knock out, and mutate at specific sites in the DNA sequence to change the gene sequence.
  • Gene editing tools will first identify specific sequences in the genome, generate DNA double-strand break gaps through nucleases, and rely on endogenous repair mechanisms.
  • Non-homologous end joining (NHEJ) and homologous recombination (Homology-directed) repair, HDR) two repair mechanisms to repair.
  • the former introduces insertions and deletions (INDELs) into the genome through cell replication and repair, resulting in mutations; the latter precisely repairs the genome sequence due to the addition of foreign donor nucleic acid as a template (Dever, et al. Nature). .2016).
  • CRISPR/Cas9 has the advantages of simple operation, low cost, and large development space, which greatly improves the operability and work efficiency of gene editing (Cong, et al. Science. 2013; Jinek, et al. .Science.2012).
  • the present invention uses gene editing technology for the first time, such as CRISPR/Cas9 gene editing technology, to develop a new generation of hematopoietic stem cells, and successfully and efficiently repair the point mutation of the ALAS-2 gene in the hematopoietic stem cells derived from the bone marrow of XLSA patients.
  • the gene repair efficiency is as high as approx. 30%-40%, the ALAS-2 gene expression of the gene repaired cells reaches about 50% of the ALAS-2 expression of healthy donors, thereby promoting the differentiation of hematopoietic stem cells into mature red blood cells.
  • the present application provides a method for correcting 5-aminolevulinic acid synthase 2 (ALAS-2) gene mutations in stem cell chromosomes through gene editing, wherein the gene editing includes:
  • the present application provides a method for correcting ALAS-2 gene mutations through gene editing, thereby increasing the expression of functional ALAS-2, wherein the gene editing includes:
  • the present application provides a method for correcting ALAS-2 gene mutations in stem cell chromosomes through gene editing, increasing functional ALAS-2 expression, thereby increasing heme production in cells derived from the hematopoietic stem cells, wherein
  • the gene editing includes:
  • the present application provides a method for correcting ALAS-2 gene mutations in stem cell chromosomes through gene editing, increasing functional ALAS-2 expression, thereby increasing heme production in cells derived from the hematopoietic stem cells, thereby promoting The method for maturation of hematopoietic stem cells, wherein the gene editing includes:
  • the present application provides a method for correcting ALAS-2 gene mutations in stem cell chromosomes through gene editing, increasing the expression of functional ALAS-2, thereby increasing the production of hemoglobin in the cells derived from the hematopoietic stem cells, and promoting the A method for the maturation of hematopoietic stem cells to treat anemia (such as XLSA) in an individual, wherein the gene editing includes:
  • the present application provides a method for correcting ALAS-2 gene mutations in stem cell chromosomes through gene editing and increasing functional ALAS-2 expression, wherein said gene editing does not cause off-target or off-target rate in the genome of said hematopoietic stem cells Less than 1%, such as less than 0.5% or less than 0.1%, wherein the gene editing includes:
  • the hematopoietic stem cells are CD34 + hematopoietic stem and progenitor cells ("HSPC"), or human induced pluripotent stem cells (hiPSC).
  • HSPC CD34 + hematopoietic stem and progenitor cells
  • hiPSC human induced pluripotent stem cells
  • the hematopoietic stem cells are obtained from anemia patients, such as sideroblastic anemia patients, specifically, hereditary sideroblastic anemia patients, more specifically, XLSA patients.
  • the mutation is located in exon 5-11 or intron-1 of the ALAS-2 gene.
  • the mutation is located in intron-1 of the ALAS-2 gene.
  • the mutation is Int-1-GATA.
  • sequence-specific nuclease is selected from the group consisting of RNA guide nuclease, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN).
  • the sequence-specific nuclease is an RNA guide nuclease.
  • the RNA guide nuclease is Cas.
  • the RNA guide nuclease is Cas9.
  • it further comprises introducing a guide RNA (sgRNA) that recognizes the ALAS-2 gene into the CD34 + HSPC.
  • sgRNA guide RNA
  • the nuclease cleavage site is not more than about 20 nucleotides away from the mutation site, for example, about 15, 13, 12, 11, or about 10 nucleotides. About 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1 nucleotide, or nuclease cleavage site and the The mutation sites overlap.
  • the sgRNA is complementary to the chromosomal sequence of the mutation site on the chromosome or complementary to the chromosomal sequence adjacent to the mutation site on the chromosome.
  • the guide sequences in the sgRNA are about 10 to about 25, about 12 to about 24, about 14 to about 23, about 16 to about 22, about 17 to about 21 nucleotides long. In some specific embodiments of the above method, the guide sequence in the sgRNA is 20 nucleotides long.
  • the sgRNA is chemically modified.
  • the sgRNA is modified by nucleotide ribose 2'-O-methylation and/or internucleotide 3'phosphorothioate modification (also referred to as phosphorothioate modification) .
  • nucleotide ribose 2'-O-methylation and/or internucleotide 3'phosphorothioate modification also referred to as phosphorothioate modification
  • one, two and/or three bases before the 5'end of the sgRNA and/or the last nucleotide ribose base at the 3'end are 2'-O-methylated modifications.
  • the sgRNA comprises the first 3 nucleotides at the 5'end and the 3 nucleotides after the 3'end.
  • the sgRNA comprises the 2'-O-methylation modification of the nucleotide ribose in the first 3 nucleotides of the 5'end and the 3 nucleotides after the 3'end and the first 3 nucleotides of the 5'end The connection between the 3 nucleotides after the 3'end contains phosphorothioate modification.
  • the sgRNA includes a 2'-O-methylation modification in the first 5 nucleotides of the 5'end and the last 5 nucleotides of the 3'end of the ribose, and the first 5 nucleotides of the 5'end
  • the last 5 internucleotide linkages at the 3'end and the 3'end include phosphorothioate modification.
  • the sgRNA comprises a nucleic acid sequence complementary to a sequence in intron-1 of the ALAS-2 gene.
  • the nucleic acid sequence that is complementary to the sequence in intron-1 of the ALAS-2 gene contained in the sgRNA is selected from the following group: SEQ ID NO: 1-SEQ ID NO: 3.
  • the sgRNA is introduced into the hematopoietic stem cells by electroporation and transduction.
  • the donor DNA is circular.
  • the donor DNA is linear.
  • the donor DNA is ssODN.
  • the donor DNA contains phosphorylation modification at the 5'end, and phosphorothioate modification is included between the first three nucleotides of the 5'end and the last 3 nucleotides of the 3'end. .
  • the donor DNA includes a phosphorylation modification at the 5'end, and phosphorothioate modification is included between the first three nucleotides of the 5'end and the last 3 nucleotides of the 3'end, And the first three nucleotides at the 5'end and the last three nucleotides at the 3'end also contain 2'-O-methylation modification of ribose.
  • the correction sequence has the same length as the mutation sequence. In some embodiments of the above methods, the donor DNA and the correction sequence are equal in length. In some embodiments of the above methods, the donor DNA is longer than the correction sequence.
  • the correction sequence is about 50 to about 300, about 60 to about 250, about 60 to about 240, about 60 to about 230, about 60 to about About 220, about 60 to about 210, about 60 to about 200 nucleotides long.
  • the correction sequence comprises a 5'arm that is substantially complementary to a target region located at the 3'end of the mutation site, and a 5'arm that is substantially complementary to a target region located at the 5'end of the mutation site. Complementary 3'arms on top.
  • the 5'arm or 3'arm of the correction sequence has at least about 85% homology with the target region at the 3'end or the target region at the 5'end of the mutation site, respectively, at least about 85%. 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% homology.
  • the 5'arm or 3'arm of the correction sequence has 100% homology with the target region at the 3'end or the target region at the 5'end of the mutation site, respectively.
  • the 5'arms of the correction sequence are about 30 to about 100 nucleotides long, for example, about 35 to about 80, about 40 to about 70, about 40. From about 60 nucleotides in length.
  • the 3'arms of the correction sequence are about 20 to about 100 nucleotides long, for example, about 20 to about 80, about 20 to about 70, about 20. From about 20 to about 50 nucleotides in length.
  • the 5'arm of the correction sequence is longer than the 3'arm of the correction sequence.
  • the 3'arm of the correction sequence is longer than the 5'arm of the correction sequence.
  • the 5'arm of the correction sequence and the 3'arm of the correction sequence have the same length.
  • the correction sequence is complementary to the target sequence at ChrX:55028172-55028268 except for the mutation site.
  • the correction sequence when the correction sequence includes a coding sequence, it encodes an amino acid sequence that is the same as the amino acid sequence encoded by the mutation sequence except for the mutation site.
  • the correction sequence corresponding to the mutation is SEQ ID NO: 4.
  • the donor DNA is introduced into the hematopoietic stem cells by electroporation.
  • introducing the sequence-specific nuclease includes introducing mRNA encoding the sequence-specific nuclease into stem cells.
  • the mRNA encoding the sequence-specific nuclease is introduced into the hematopoietic stem cell by electroporation.
  • the mRNA encoding the sequence-specific nuclease and the donor DNA are simultaneously introduced into the stem cell.
  • sgRNA is introduced into stem cells, and wherein the mRNA encoding the sequence-specific nuclease and the sgRNA are simultaneously introduced into the stem cells.
  • the sgRNA and the donor DNA are simultaneously introduced into the stem cell.
  • the sgRNA, the mRNA encoding the sequence-specific nuclease, and the donor DNA are separately or simultaneously introduced into the stem cells by means of electroporation or transduction.
  • the weight ratio of the sgRNA to the donor DNA is about 1:12 to about 12:1, for example, about 1:11 to about 11:1, about 1:10 to about 10:1, about 1:9 to about 9:1, about 1:8 to about 8:1, about 1:7 to about 7:1, about 1:6 to about 6:1.
  • the weight ratio of the mRNA encoding the sequence-specific nuclease to the single-stranded DNA is about 1:12 to about 12:1, for example, about 1:11 to about 11:1 , About 1:10 to about 10:1, about 1:9 to about 9:1, about 1:8 to about 8:1, about 1:7 to about 7:1, about 1:6 to about 6:1 .
  • the hematopoietic stem cells are obtained from a male individual.
  • the CD34 + HSPC is obtained from a male individual.
  • the human induced pluripotent stem cells are obtained from male individuals.
  • the hematopoietic stem cells are obtained from a female individual.
  • the CD34 + HSPC is obtained from a female individual.
  • the human induced pluripotent stem cells are obtained from a female individual.
  • This application also relates to a gene-edited CD34 + HSPC or human induced pluripotent stem cell (hiPSC) obtained by the above method, wherein the CD34 + HSPC or human induced pluripotent stem cell (hiPSC) is derived from an anemia patient and has been gene-edited,
  • the mutation of the ALAS-2 gene has been corrected.
  • the anemia is sideroblast anemia, such as hereditary sideroblast anemia, specifically, XLSA.
  • the ALAS-2 gene mutation is located in exon 5-11 or intron-1 of the ALAS-2 gene.
  • the mutation is Int-1-GATA.
  • a method for correcting 5-aminolevulinic acid synthase 2 (ALAS-2) gene mutations of hematopoietic stem cells by CRISPS/Cas9 gene editing comprising: adding a single-stranded correction sequence corresponding to the ALAS-2 mutation sequence
  • the donor DNA, the sgRNA that recognizes the ALAS-2 mutation sequence, and the nucleic acid sequence encoding the Cas9 protein are introduced into the hematopoietic stem cell, whereby the correction sequence in the donor DNA replaces the ALAS-2 mutation sequence in the hematopoietic stem cell.
  • ALAS-2 mutant sequence is a mutant sequence in exons 5-11 of the ALAS-2 gene and/or a mutant sequence in intron-1 of the ALAS-2 gene.
  • sequence of the sgRNA is selected from the following group: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
  • correction sequence comprises a 5'arm complementary to the target region 3'of the mutation site, and a 3'arm complementary to the target region 5'of the mutation site.
  • 'Arm wherein the 5'arm of the calibration sequence is about 40 to about 60 nucleotides long, and the 3'arm of the calibration sequence is about 20 to about 50 nucleotides long.
  • Figure 1 Point mutations in intron-1 of ALAS-2 gene on human X chromosome (X:55054635[ChrX(GRCh37/hg19):g.55054635A>G,NM 000032.4:c.-15-2187T> C) Schematic diagram of multiple sgRNAs and donor template single-stranded DNA designed at nearby locations.
  • Figure 2 Point mutations in intron-1 of ALAS-2 gene on human X chromosome (X:55054635 [Chr X(GRCh37/hg19):g.55054635 A>G,NM 000032.4:c.-15-2187 T> C) Sequence information of multiple sgRNAs and donor template single-stranded DNA designed at nearby locations.
  • Electrotransform Cas9 mRNA, sgRNA-1 near the point mutation of ALAS-2 intron-1 and the donor template ssODN enter the hiPSCs derived from XLSA patients according to the addition amount of Cas9, sgRNA and ssODN, and expand after 4 days Increase the target fragment and NGS, and analyze the ratio of NHEJ and HDR through bioinformatics methods.
  • NHEJ non-homologous end joining, representing the ratio of indels
  • HDR Homology-directed repair, representing the ratio of gene repair
  • n 3 experimental replicates.
  • FIG. 8 Electrotransformation of Cas9 mRNA, sgRNA-1 and donor template ssODN near the point mutation of ALAS-2 intron-1 into CD34+HSPC derived from the bone marrow of XLSA patients for 2 days, and in vitro clone formation experiment (CFU detection), 14 days later Count the number of clones in different blood systems, BFU-E, CFU-GM, CFU-E, CFU-GEMM represent the formation of clones of different blood system lineages such as erythroid, myeloid, and lymphatic systems.
  • healthy donors represent healthy donors that have not undergone gene editing
  • blank control represent cells that have not undergone gene editing
  • gene repair represent cells that have undergone gene repair
  • n 3 experimental replicates.
  • Figure 9 Electrotransform Cas9 mRNA and sgRNA-1 near the point mutation of ALAS-2 intron-1 and the donor template ssODN into CD34+HSPC derived from the bone marrow of XLSA patients for red blood cell differentiation. The 7th day after differentiation was detected. The expression ratio of human CD71 and human CD235a membrane proteins on the 13th and 18th days represents the efficiency of erythroid differentiation.
  • healthy donors represent healthy donors that have not undergone gene editing
  • blank control represent cells that have not undergone gene editing
  • gene repair represent cells that have undergone gene repair.
  • Figure 10 Electrotransformation of Cas9 mRNA and sgRNA-1 near the point mutation of ALAS-2 intron-1 and the donor template ssODN into CD34+HSPC derived from bone marrow of XLSA patients for red blood cell differentiation. 18 days later, Figure A is the post-differentiation Photograph of the posterior cell.
  • Figure B is a schematic diagram of Benzidine staining bright field.
  • Panel C is a schematic diagram of Wright-Giemsa staining bright field. Ruler: 20um.
  • healthy donors represent healthy donors that have not undergone gene editing
  • blank control represent cells that have not undergone gene editing
  • gene repair represent cells that have undergone gene repair.
  • FIG. 11 Electrotransform Cas9 mRNA and sgRNA-1 near the point mutation of ALAS-2 intron-1 and the donor template ssODN into CD34+HSPC derived from the bone marrow of XLSA patients for red blood cell differentiation. 18 days later, perform Benzidine staining and statistics Analyze the proportion of Benzidine positive.
  • healthy donors represent healthy donors that have not undergone gene editing
  • blank control represent cells that have not undergone gene editing
  • gene repair represent cells that have undergone gene repair
  • n 3 experimental replicates.
  • FIG. 12 Electrotransform Cas9 mRNA, sgRNA-1 near the point mutation of ALAS-2 intron-1 and donor template ssODN into CD34+HSPC derived from the bone marrow of XLSA patients for red blood cell differentiation, detected after 18 days, and quantified by fluorescence PCR was used to detect the mRNA expression of ALAS-2, GATA-1 and GAPDH genes.
  • healthy donors represent healthy donors that have not undergone gene editing
  • blank control represent cells that have not undergone gene editing
  • gene repair represent cells that have undergone gene repair
  • n 3 experimental replicates.
  • ALAS-2 gene and GATA-1 were normalized with GAPDH and healthy donors.
  • FIG. 13 Electrotransform Cas9 mRNA and sgRNA-1 near the point mutation of ALAS-2 intron-1 and the donor template ssODN into CD34+HSPC derived from the bone marrow of XLSA patients for red blood cell differentiation. After 18 days, the test was performed by Western Blot experiment. Detect the protein level expression of ALAS-2, GATA-1 and GAPDH genes. Among them, healthy donors: represent healthy donors that have not undergone gene editing, blank control: represent cells that have not undergone gene editing, and gene repair: represent cells that have undergone gene repair.
  • FIG. 14 Electrotransformation of Cas9 mRNA, sgRNA-1 and donor template ssODN near the point mutation of ALAS-2 intron-1 into CD34+HSPC derived from the bone marrow of XLSA patients.
  • the transplantation has undergone gene repair and has not undergone gene editing.
  • the cells from the irradiator enter the 6-week-old NPG immunodeficiency mouse model. After 10 weeks, 12 weeks, and 16 weeks, the proportion of human CD45-positive cells in the peripheral blood of the mouse is detected. At the same time, the proportion of human CD45 positive cells is detected in the mouse after 16 weeks of transplantation.
  • the proportion of CD45 positive cells is calculated as human CD45 positive cells%/(human CD45 positive cells%+mouse CD45 positive cells%), human CD45 positive cells% and small
  • the percentages of mouse CD45 positive cells were measured by flow cytometry experiments.
  • Blank control represents cells that have not undergone gene editing
  • gene repair represents cells that have undergone gene repair.
  • n 6 mice.
  • FIG. 15 Electrotransformation of Cas9 mRNA, sgRNA-1 and donor template ssODN near the point mutation of ALAS-2 intron-1 into CD34+ HSPC derived from the bone marrow of XLSA patients.
  • the transplantation has undergone gene repair and has not undergone gene editing.
  • the cells entered into a 6-week-old NPG immunodeficiency mouse model irradiated with an irradiator. After 16 weeks, the ratio of human cell membrane proteins such as CD3, CD33, CD56, and CD19 to human CD45 protein was detected in the mouse bone marrow and spleen.
  • Blank control represents cells that have not undergone gene editing
  • gene repair represents cells that have undergone gene repair.
  • n 6 mice.
  • FIG. 16 Electrotransformation of Cas9 mRNA, sgRNA-1 and donor template ssODN near the point mutation of ALAS-2 intron-1 into CD34+ HSPC derived from the bone marrow of XLSA patients. Two days after electrotransformation, the transplantation has undergone gene repair and has not undergone gene editing. The cells entered the 6-week-old NPG immunodeficiency mouse model irradiated by the irradiator. After 16 weeks, flow cytometry analysis of human CD45 in bone marrow, spleen and peripheral blood of 1 mouse in the blank control group and the gene repair group The proportion of positive cells.
  • FIG. 17 Electrotransformation of Cas9 mRNA, sgRNA-1 and donor template ssODN near the point mutation of ALAS-2 intron-1 into CD34+ HSPC derived from the bone marrow of XLSA patients.
  • the transplantation has undergone gene repair and has not undergone gene editing.
  • the cells enter the 6-week-old NPG immunodeficiency mouse model that has been irradiated by the irradiator. After 16 weeks, flow cytometry analysis of CD3, CD33, and CD56 in the bone marrow and spleen of 1 mouse in the blank control group and the gene repair group , CD19 and other human cell membrane proteins accounted for the proportion of human CD45 protein.
  • blank control represents cells that have not undergone gene editing
  • gene repair represents cells that have undergone gene repair.
  • FIG. 18 Electrotransformation of Cas9 mRNA, sgRNA-1 and donor template ssODN near the point mutation of ALAS-2 intron-1 into CD34+ HSPC derived from the bone marrow of XLSA patients.
  • the transplantation has undergone gene repair and has not undergone gene editing.
  • Cells from the irradiator enter the 6-week-old NPG immunodeficiency mouse model, extract the genome of the cells before transplantation and the bone marrow 16 weeks after transplantation, amplify the target fragment and NGS, and analyze the NHEJ and HDR by bioinformatics methods. proportion.
  • NHEJ non-homologous end joining
  • Indels represents the ratio of Indels
  • HDR Homology-directed repair
  • Figure 19 Isolation of bone marrow from NPG immunodeficient mice 16 weeks after one transplantation, and transplantation into new irradiated NPG immunodeficient mice for two transplantation experiments. Isolate bone marrow cells at 12 weeks after transplantation and detect the proportion of human CD45-positive cells. The proportion of CD45-positive cells is calculated as human CD45-positive cells%/(human CD45-positive cells%+mouse CD45-positive cells%), human CD45-positive cells % And mouse CD45 positive cells% are the results measured by flow cytometry experiments. Blank control: represents cells that have not undergone gene editing, and gene repair: represents cells that have undergone gene repair.
  • Figure 20 Isolation of bone marrow 16 weeks after one transplantation of NPG immunodeficiency mice, and then transplantation into new irradiated NPG immunodeficiency mice for two transplantation experiments.
  • the bone marrow cells were isolated 12 weeks after transplantation to test the gene repair efficiency and analyze the ratio of NHEJ and HDR.
  • NHEJ non-homologous end connection, representing the ratio of Indels
  • HDR homologous recombination repair, representing the ratio of gene repair.
  • FIG. 21 Electrotransform Cas9 mRNA and sgRNA-1 near the point mutation of ALAS-2 intron-1 into hiPSCs derived from XLSA patients, extract the genome 2 days after electrotransformation, and amplify through sequence similarity prediction analysis and unbiased whole-genome analysis Methods
  • the target fragments of 32 potential off-target sites predicted by the Digenome-Seq method were analyzed by NGS sequencing, and the mutation frequency of each off-target site was analyzed by bioinformatics methods.
  • gene repair represents cells that have undergone gene editing
  • POT potential off-target
  • On-target represents gene editing efficiency.
  • This application provides a method for correcting 5-aminolevulinic acid synthase 2 (ALAS-2) gene mutations through gene editing, which can efficiently repair the ALAS-2 gene mutations in hiPSC and CD34+HSPC derived from XLSA patients , Significantly increase the expression of ALAS-2 gene and protein, thereby significantly increasing the synthesis of heme in differentiated red blood cells, promoting the maturation of red blood cells and the ability to carry oxygen, improving the symptoms of anemia patients, so as to overcome the shortcomings of traditional treatment methods and meet the clinical needs Treatment requirements.
  • ALAS-2 5-aminolevulinic acid synthase 2
  • the "gene editing” mentioned in this application refers to the technique of site-specific modification of the genome to achieve site-specific deletion, insertion, and/or replacement of specific nucleotides and nucleotide fragments at the gene level.
  • well-known gene editing technologies include artificial nuclease-mediated zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, and RNA-guided CRISPR/Cas nuclease (CRISPR/Cas RGNs) technology. They can specifically recognize the target site, and after precise cutting of its single or double strand, the cell's endogenous repair mechanism completes the knockout and replacement of the target gene.
  • CRISPR/Cas technology is an emerging gene editing technology, which uses sgRNA complementary to the target sequence to guide Cas enzyme to cut DNA at a specific point.
  • a “mutated sequence” is a gene sequence whose nucleotide sequence has been changed compared to a normal natural sequence.
  • the nucleotide sequence that replaces the mutated sequence to achieve correction of the mutated sequence is called a "correction sequence”.
  • "Donor DNA” is DNA containing a "correction sequence”. After the donor DNA molecule containing the correction sequence is introduced into the cell by means of electroporation or transduction, homologous recombination can occur with the mutant sequence, so that the correction sequence can replace the mutant sequence to realize gene editing.
  • Stem cell refers to a cell population with vigorous proliferation potential, multi-differentiation ability and self-renewal ability.
  • Hematopoietic stem cells refer to cell populations with vigorous proliferation potential, multidirectional differentiation into blood cells, and self-renewal capabilities. Hematopoietic stem cells can not only differentiate and supplement various blood cells, but also maintain the characteristics and quantity of stem cells through self-renewal. The degree of differentiation and proliferation ability of hematopoietic stem cells are different and heterogeneous.
  • Pluripotent hematopoietic stem cells are the most primitive, and first differentiate into directed pluripotent hematopoietic stem cells, such as myeloid hematopoietic stem cells that can produce granulocytes, erythroid, mononuclear and megakaryocyte-platelet lines, and lymphoids that can produce B lymphocytes and T lymphocytes stem cell. These two types of stem cells not only maintain the basic characteristics of hematopoietic stem cells, but are also slightly differentiated. They are responsible for the occurrence of "bone marrow components" and lymphocytes, so they are called directed pluripotent hematopoietic stem cells. They further differentiate into hematopoietic progenitor cells.
  • directed pluripotent hematopoietic stem cells such as myeloid hematopoietic stem cells that can produce granulocytes, erythroid, mononuclear and megakaryocyte-platelet lines, and lymphoids that can produce B lymphocytes and
  • hematopoietic progenitor cells Although this cell is also a primitive blood cell, it has lost many of the basic characteristics of hematopoietic stem cells. Lost the ability of repeated self-renewal, and rely on the proliferation and differentiation of hematopoietic stem cells to supplement the number; the proliferation potential is limited and can only divide several times. According to the number of blood cell lines that hematopoietic progenitor cells can differentiate, they are divided into unipotent hematopoietic progenitor cells (differentiated into only one blood cell line) and oligopotent hematopoietic progenitor cells (differentiated into 2 to 3 blood cell lines).
  • hematopoietic stem cells refer to cell populations that can form granulocytes, erythroid, monocytes, megakaryocytes-platelet cells and/or lymphoid cells through differentiation or directed differentiation. They are pluripotent hematopoietic stem cells and are multipotent. Generic term for hematopoietic stem cells and hematopoietic progenitor cells.
  • Hematopoietic stem cells can be derived from bone marrow (bone marrow hematopoietic stem cells), peripheral blood (peripheral hematopoietic stem cells), umbilical cord blood (umbilical cord blood hematopoietic stem cells), and can also be derived from placental stem cells or hiPSC.
  • flow cytometry and fluorescently labeled anti-CD34 antibodies can be used to detect and count CD34-positive hematopoietic stem/progenitor cells (HSPC).
  • CRISPR/Cas is a gene editing technology, including but not limited to various naturally occurring or artificially designed CRISPR/Cas systems, such as the CRISPR/Cas9 system.
  • the naturally occurring CRISPR/Cas system (Naturally occurring CRISPR/Cas system) is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be used to fight invading viruses and foreign DNA.
  • a simple CRISPR/Cas9 system includes three components: Cas9 enzyme, crRNA (CRISPR-derived RNA) and tracrRNA (trans-activating crRNA).
  • crRNA contains a guide sequence and a sequence partially complementary to tracrRNA.
  • tracrRNA is trans-activating RNA (trans-activating RNA), which contains a long constant base sequence and provides a "stem-loop" structure bound by CRISPR nuclease (such as Cas9 enzyme).
  • crRNA combines with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, through which crRNA and the target sequence are complementary, and through the "stem-loop" structure in tracrRNA, Cas9 nuclease can be guided Cut the double-stranded DNA to the target site of the target sequence.
  • tracrRNA and crRNA can be combined to transform into a guiding sgRNA (single guide RNA), so that the sgRNA is sufficient to guide Cas9's targeted cleavage of DNA.
  • a guiding sgRNA single guide RNA
  • Cas9 nuclease can co-localize RNA, DNA and protein, and has great potential for transformation.
  • the CRISPR/Cas system can use type 1, type 2 or type 3 Cas proteins. In some embodiments of the invention, the method uses Cas9.
  • CRISPR/Cas systems include but are not limited to the systems and methods described in WO2013176772, WO2014065596, WO2014018423, US8,697,359, PCT/CN2018/112068, PCT/CN2018/112027.
  • Cell “differentiation” refers to the process in which cells from the same source gradually produce cell groups with different morphological structures and functional characteristics.
  • the "differentiation" from hematopoietic stem cells to erythrocytes includes hematopoietic stem cell stage, erythroid progenitor cell stage, erythroid precursor cell (primary red blood cell to late red blood cell) proliferation and differentiation stage, reticulocyte proliferation and maturation process, and reticulum Red blood cells are released from peripheral blood to mature into red blood cells.
  • Hematopoietic stem cell stage It is currently known that hematopoietic stem cells mainly exist in bone marrow, spleen, liver and other hematopoietic tissues. There is also a small amount of circulation in the peripheral blood.
  • Erythroid progenitor cell stage In the progenitor cell stage, cells are a cell group between hematopoietic stem cells and erythroid precursor cells. Hematopoietic stem cells differentiate into erythroid progenitor cells under the influence of bone marrow hematopoietic microenvironment. The hematopoietic microenvironment includes the microvascular system, nervous system and hematopoietic interstitium. Humoral factors and cytokines have a special effect and influence on the differentiation of hematopoietic stem cells. Erythroid precursor cell stage: including primitive red blood cells, early young red blood cells, middle young red blood cells, late young red blood cells and reticulocytes to reach mature red blood cells.
  • Non-homologous end joining is also abbreviated as NHEJ (Non-homologous end joining), which refers to eukaryotic cells that do not rely on DNA homology to avoid the retention of DNA or chromosome breaks and the resulting DNA The effect of degradation is a DNA double-strand break repair mechanism that forcibly connects two DNA ends to each other.
  • NHEJ may produce insertions and deletions (Indels, Insertions and deletions), leading to gene mutations.
  • HDR Homology-directed repair
  • HDR homology-mediated double-stranded DNA repair
  • “Anemia” refers to a clinical symptom in which the volume of human peripheral blood red blood cells decreases below the lower limit of the normal range.
  • “Sideroblastic anemia” is a disorder of iron utilization. It is characterized by the appearance of a large number of ring sideroblasts in the bone marrow, ineffective production of red blood cells, excessive tissue iron reserves, and small cell hypochromic anemia in the peripheral blood. Sideroblastic anemia is mainly divided into acquired and hereditary sideroblastic anemia. Among them, hereditary sideroblastic anemia is mostly adolescents, males and have family history. Poor iron utilization, impaired heme synthesis and ineffective production of red blood cells are the main links in the pathogenesis of this disease.
  • the result of poor iron utilization and heme synthesis disorder is the formation of hypochromic anemia.
  • a large amount of iron accumulates in red blood cells and various tissues, which damages the morphology and function of red blood cells and causes premature destruction of red blood cells.
  • a large amount of iron deposits in various tissues, forming hemochromatosis, affecting the functions of various tissues and organs.
  • This application relates to genetic repair of specific mutations in the ALAS-2 gene, such as mutations in exons 5-11 or intron-1 of the ALAS-2 gene, such as Int-1-GATA point mutations, to improve the source of iron Myeloblastic anemia patients, such as hereditary sideroblast anemia patients, specifically, CD34 + HSPC or human induced pluripotent stem cells (hiPSC) in XLSA patients with ALAS-2 gene and protein expression, so that the treatment includes hereditary sideroblasts Immature red blood cell anemia, such as sideroblast anemia of XLSA.
  • specific mutations in the ALAS-2 gene such as mutations in exons 5-11 or intron-1 of the ALAS-2 gene, such as Int-1-GATA point mutations
  • iron Myeloblastic anemia patients such as hereditary sideroblast anemia patients, specifically, CD34 + HSPC or human induced pluripotent stem cells (hiPSC) in XLSA patients with ALAS-2 gene and
  • the present invention relates to a method for correcting 5-aminolevulinic acid synthase 2 (ALAS-2) gene mutations in the chromosomes of hematopoietic stem cells through gene editing, wherein the gene editing includes: (a) A donor DNA containing a single-stranded correction sequence corresponding to the mutant sequence is introduced into the hematopoietic stem cell; (b) a sequence-specific nuclease that cleaves the ALAS-2 gene is introduced into the hematopoietic stem cell, wherein the donor The correction sequence on the somatic DNA replaces the mutation sequence on the chromosome of the hematopoietic stem cell, thereby correcting the mutation.
  • the gene editing includes: (a) A donor DNA containing a single-stranded correction sequence corresponding to the mutant sequence is introduced into the hematopoietic stem cell; (b) a sequence-specific nuclease that cleaves the ALAS-2 gene is introduced into the hematopoietic
  • the invention also relates to a method for correcting ALAS-2 gene mutations through gene editing, thereby increasing the expression of functional ALAS-2.
  • the present invention also relates to a method for correcting ALAS-2 gene mutations through gene editing and increasing the expression of functional ALAS-2, thereby increasing the production of heme in cells derived from the hematopoietic stem cells.
  • the present invention also relates to a method for correcting ALAS-2 gene mutations through gene editing, increasing the expression of functional ALAS-2, thereby increasing the production of heme in the cells derived from the hematopoietic stem cells, thereby promoting the maturation of the hematopoietic stem cells .
  • the present invention also relates to a method for correcting ALAS-2 gene mutations through gene editing, increasing the expression of functional ALAS-2, thereby increasing the production of heme in the cells derived from the hematopoietic stem cells, and promoting the maturation of the hematopoietic stem cells, thereby Methods of treating individuals include hereditary sideroblast anemia, such as XLSA sideroblast anemia.
  • the hematopoietic stem cells are obtained from patients with sideroblast anemia, including hereditary sideroblast anemia, such as XLSA.
  • the patient is a male individual or a female individual, and the obtained stem cells are CD34 + hematopoietic stem and progenitor cells ("HSPC"), or human induced pluripotent stem cells (hiPSC).
  • HSPC hematopoietic stem and progenitor cells
  • hiPSC human induced pluripotent stem cells
  • CD34-positive hematopoietic stem/progenitor cells are isolated from an organism (individual) containing cells of hematopoietic origin. "Separate” means to remove from its original environment. For example, a cell is isolated if it is separated from some or all of the components that normally accompany it in its natural state. Hematopoietic stem cells/progenitor cells can be obtained or isolated from unfractionated or fractionated bone marrow of adults, including femurs, hip bones, ribs, sternum and other bones.
  • Hematopoietic stem cells and progenitor cells can be directly obtained or separated from the hip bone using a needle and syringe, or obtained from the blood, usually obtained from the blood after pretreatment with a hematopoietic stem cell mobilizer such as G-CSF (granulocyte colony stimulating factor) .
  • a hematopoietic stem cell mobilizer such as G-CSF (granulocyte colony stimulating factor)
  • G-CSF granulocyte colony stimulating factor
  • Other sources of hematopoietic stem and progenitor cells include cord blood, placental blood, and peripheral blood of mobilized individuals.
  • the cell population After the cell population is isolated from an individual (such as bone marrow or peripheral blood), it can be further purified to obtain CD34-positive hematopoietic stem cells/progenitor cells.
  • mature lineage-directed cells in an isolated cell population can be removed by immunization, for example, by using antibodies that bind to a set of "lineage" antigens (eg CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a)
  • the solid matrix is labeled, and then the original hematopoietic stem cells and progenitor cells are separated with antibodies that bind to CD34 positive antigen.
  • Kits for purifying hematopoietic stem and progenitor cells from a variety of cell sources are commercially available, and in specific embodiments, these kits can be used with the methods of the present invention.
  • CD34 positive hematopoietic stem cells/progenitor cells can represent at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the cell population rich in CD34 positive cells Or 100% CD34-positive hematopoietic stem/progenitor cells (HSPC).
  • the gene editing includes introducing a donor DNA containing a single-stranded correction sequence corresponding to the mutant sequence into the hematopoietic stem cell.
  • the single-stranded correction sequence replaces the mutant sequence in the ALAS-2 gene.
  • the donor DNA has the same nucleotide composition and length as the single-stranded correction sequence. In some embodiments, the donor DNA is longer than the single-stranded correction sequence, for example, one or more nucleotides are added to one or both ends of the single-stranded correction sequence. In some embodiments, the sequence composed of the added nucleotides is a nuclease specific recognition site. In some embodiments, both ends of the calibration sequence in the donor DNA may further include protective bases for the specific recognition site of the nuclease. In some embodiments, the donor DNA further comprises one or more LNA nucleosides. In some embodiments, the donor DNA is single-stranded. In some embodiments, the donor DNA is circular.
  • the donor DNA is provided in the form of a plasmid or viral vector.
  • the donor DNA is ssODN (single-stranded donor oligonucleotides).
  • the donor DNA is shown in SEQ ID NO: 4.
  • the donor DNA is chemically modified, such as 2'-O-methylation modification on nucleotide ribose, 3'phosphorothioate modification between nucleotides, and 5' End phosphorylation modification.
  • the chemical modification is a 2'-O-methylation modification and/or a ribose 3 nucleotides before the 5'end and 3 nucleotides after the 3'end of the donor DNA. 3'phosphorothioate modification between glycidyl acids.
  • the chemical modification is one, two and/or three bases before the 5'end of the donor DNA and/or the 2'-O of the last nucleotide ribose at the 3'end.
  • the donor DNA contains the 2'-O-methylation modification of the nucleotide ribose in the 3 nucleotides before the 5'end and the 3 nucleotides after the 3'end, and the donor DNA contains the 2'-O-methylation modification of the nucleotide ribose before the 5'end.
  • the three internucleotide linkages after the 3 and 3'ends contain phosphorothioate modification.
  • the donor DNA contains a 2'-O-methylation modification in the ribose of the first 5 nucleotides at the 5'end and the last 5 nucleotides at the 3'end, and the ribose at the 5'end
  • the first 5 and the last 5 internucleotide linkages at the 3'end contain phosphorothioate modification.
  • the donor DNA contains phosphorylation modification at the 5'end, and phosphorothioate modification is included between the first three nucleotides at the 5'end and the last 3 nucleotides at the 3'end.
  • the donor DNA contains a phosphorylation modification at the 5'end, and phosphorothioate modification is included between the first three nucleotides of the 5'end and the last 3 nucleotides of the 3'end, and The first three nucleotides at the 5'end and the last 3 nucleotides at the 3'end also contain the 2'-O-methylation modification of ribose.
  • sequence-specific nucleases include RNA guide nuclease, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN).
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • the sequence-specific nuclease may be, for example, an RNA guide nuclease, that is, a Cas nuclease, and specifically may be Cas9.
  • the nuclease cleavage site is no more than about 11 nucleotides away from the mutation site.
  • mRNA encoding Cas9 such as mRNA containing an ARCA cap, is introduced into stem cells (e.g., by electroporation or other means of gene transduction).
  • the nucleotide encoding the Cas nuclease e.g., Cas9
  • a viral vector e.g., a lentiviral vector.
  • the sgRNA and the Cas9-encoding nucleic acid are present in the same vector.
  • the sgRNA and the Cas9-encoding nucleic acid are in different vectors.
  • it further includes introducing sgRNA that recognizes the ALAS-2 gene into the hematopoietic stem cells, such as CD34 + HSPC.
  • the "guide sequence" in sgRNA is any polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence and direct the sequence-specific binding of the CRISPR complex to the target sequence.
  • the degree of complementarity between the guide sequence and its corresponding target sequence is about or greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.
  • the optimal alignment can be determined using any appropriate algorithm for aligning sequences.
  • Non-limiting examples include Smith-Waterman algorithm, Needleman-Wimsch algorithm, Burrows-Wheeler Transform-based algorithms (such as Burrows Wheeler Aligner), ClustalW, Clustai X, BLAT, Novoalign (Novocraft Technologies, ELAND ((Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn) and Maq (available at maq.sourceforge.net).
  • the guide sequence length can be about or greater than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides.
  • the guide sequence length is less than about 75, 70, 65, 60, 55 , 50, 45, 40, 35, 30, 25, 20, 15, 12 or fewer nucleotides.
  • the ability of the guide sequence to direct the sequence-specific binding of the CRISPR complex to the target sequence can be assessed by any appropriate assay method
  • a host cell with the corresponding target sequence can be provided with the components of the CRISPR system (including the guide sequence to be tested) sufficient to form a CRISPR complex, for example, by transfection with a vector encoding the CRISPR sequence component, and then evaluate the target sequence.
  • the preferential cleavage (such as by the Surveyor assay as described herein) is carried out.
  • the cleavage of the target polynucleotide sequence can be performed in the test tube by providing the target sequence, the CRISPR complex (containing the guide sequence to be tested and the other The evaluation is carried out by comparing the binding or cleavage rate of the test and the control guide sequence in the target sequence.
  • Other methods known to those skilled in the art can also be used for the above determination and evaluation.
  • the sgRNA may be modified, for example, it may be chemically modified, specifically, the sgRNA is modified by nucleotide ribose 2'-O-methylation and/ Or internucleotide 3'phosphorothioate modified.
  • “Chemically modified sgRNA” refers to the special chemical modification of sgRNA, such as the 2'-O-methylation modification of ribose with 3 nucleotides at the 5'and 3'ends and/or the 3'between nucleotides. 'Phosphorothioate modification.
  • the chemical modification is a 2'-O-methylation modification of one, two and/or three bases before the 5'end of the sgRNA and/or the last nucleotide ribose at the 3'end.
  • the sgRNA comprises 2'-O-methyl modification of ribose at the first 3 nucleotides at the 5'end and 3 nucleotides at the rear of the 3'end and/or an internucleotide 3'sulfur Phosphorylation modification.
  • the sgRNA comprises the 2'-O-methylation modification of the nucleotide ribose in the first 3 nucleotides of the 5'end and the 3 nucleotides after the 3'end and the first 3 nucleotides of the 5'end
  • the connection between the 3 nucleotides after the 3'end contains phosphorothioate modification.
  • the sgRNA includes a 2'-O-methylation modification in the first 5 nucleotides of the 5'end and the last 5 nucleotides of the 3'end of the ribose, and the first 5 nucleotides of the 5'end
  • the last 5 internucleotide linkages at the 3'end and the 3'end include phosphorothioate modification.
  • the chemically modified sgRNA has at least the following two advantages.
  • chemical modification methods commonly used in the art can be used, as long as they can improve the stability of sgRNA (extend the half-life) and enhance the ability to enter the cell membrane.
  • it also includes the use of other modification methods, for example, Deleavey GF1, Damha MJ. Designing chemically modified oligonucleotides for targeted gene silencing. Chem Biol. 2012 Aug 24; 19(8): 937-54, and Hendel et al. Chemically modified guide RNAs enhancement CRISPR-Cas genome editing in human primary cells. Nat Biotechnol. 2015 Sep; 33(9):985-989 The chemical modification methods reported in the literature.
  • the sgRNA may be complementary to the chromosomal sequence of the mutation site on the chromosome or complementary to the chromosomal sequence adjacent to the mutation site on the chromosome.
  • the sgRNA may include a nucleic acid sequence complementary to the sequence in intron-1 of the ALAS-2 gene.
  • the nucleic acid sequence that is complementary to the sequence in intron-1 of the ALAS-2 gene contained in the sgRNA may be about 17 to about 20 nucleotides long.
  • the sgRNA is selected from the following group: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.
  • This application uses CRISPR/Cas9 gene editing technology to develop a method for efficiently repairing ALAS-2 gene mutations.
  • the gene repair efficiency is as high as about 30%-40%.
  • the ALAS-2 gene expression of the cells after the gene repair reaches the healthy donor ALAS -2 expression is about 50%, which can significantly alleviate the clinical manifestations of patients with sideroblast anemia (such as XLSA).
  • using the method can produce extremely high mutation frequency at the target site (On-target), far exceeding the mutation frequency of the blank control group, and close to 100%, while at potential off-target sites Then the significant difference between the gene editing group and the blank control group cannot be measured.
  • the method will not cause off-target in the hematopoietic stem cell genome.
  • the off-target rate is less than 1%, for example, less than 0.5% or less than 0.1%. This extremely low off-target rate can improve the safety of the method for gene repair of hematopoietic stem cells.
  • the present application provides a method for correcting ALAS-2 gene mutations through gene editing, thereby increasing the expression of functional ALAS-2, wherein the gene editing includes: (a) comprising: The donor DNA of the single-stranded correction sequence of the mutant sequence is introduced into the hematopoietic stem cell; (b) the sequence-specific nuclease that cuts the ALAS-2 gene is introduced into the hematopoietic stem cell, wherein the correction on the donor DNA The sequence replaces the mutant sequence on the chromosome of the hematopoietic stem cell, thereby correcting the mutation.
  • the hematopoietic stem cells are CD34 + hematopoietic stem and progenitor cells ("HSPC"), or human induced pluripotent stem cells (hiPSC).
  • the sequence-specific nuclease is an RNA-guided nuclease, specifically Cas9.
  • the inventors found that the closer the nuclease cleavage site is to the mutation site, the more helpful it is to improve the repair efficiency.
  • the nuclease cleavage site is no more than about 11 nucleotides away from the mutation site, for example, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1 nucleotide, or nuclease cleavage site overlaps the mutation site.
  • the sgRNA is complementary to the chromosomal sequence of the mutation site on the chromosome or complementary to the chromosomal sequence adjacent to the mutation site on the chromosome.
  • the sgRNA comprises a nucleic acid sequence complementary to the sequence in intron-1 of the ALAS-2 gene.
  • the nucleic acid sequence complementary to the sequence in intron-1 of the ALAS-2 gene contained in the sgRNA is 17-20 nucleotides long.
  • the sgRNA is selected from the following group: SEQ ID NO: 1-SEQ ID NO: 3, preferably SEQ ID NO: 1.
  • the sgRNA is chemically modified. Specifically, the sgRNA is modified by 2'-O-methylation of nucleotide ribose and/or internucleotide 3'phosphorothioate modification, for example, the chemical modification is that of the sgRNA 2'-O-methylation modification of one, two and/or three bases before the 5'end and/or the last nucleotide ribobase at the 3'end. In some embodiments, the sgRNA comprises the first 3 nucleotides at the 5'end and the 3 nucleotides after the 3'end.
  • the sgRNA comprises the 2'-O-methylation modification of the nucleotide ribose in the first 3 nucleotides of the 5'end and the 3 nucleotides after the 3'end and the first 3 nucleotides of the 5'end The connection between the 3 nucleotides after the 3'end contains phosphorothioate modification.
  • the sgRNA includes a 2'-O-methylation modification in the first 5 nucleotides of the 5'end and the last 5 nucleotides of the 3'end of the ribose, and the first 5 nucleotides of the 5'end
  • the last 5 internucleotide linkages at the 3'end and the 3'end include phosphorothioate modification.
  • the donor sequence is longer than the correction sequence.
  • the donor sequence is the same length as the correction sequence, and is about 60 to about 200 nucleotides in length, for example, 60 to about 180, 60 to about 160, 60 to about 140. One, 60 to about 120, 60 to about 100, 60 to about 80 nucleotides long.
  • the correction sequence comprises a 5'arm that is substantially complementary to a target region located at the 3'end of the mutation site, and a 5'arm that is substantially complementary to a target region located at the 5'end of the mutation site. 3'arm.
  • the substantially complementary means that the 5'arm or the 3'arm of the correction sequence has high homology with the target region at the 3'end or the target region at the 5'end of the mutation site, for example, at least about 90%. , At least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% homology. Most preferably, the 5'arm or 3'arm of the correction sequence has 100% homology with the target region at the 3'end or the target region at the 5'end of the mutation site, respectively.
  • the 5'arm of the correction sequence is longer than the 3'arm of the correction sequence, the 3'arm of the correction sequence is longer than the 5'arm of the correction sequence, or the 5'arm of the correction sequence and the The 3'arms of the calibration sequence have the same length.
  • the sequence of the donor DNA is shown in SEQ ID NO: 4.
  • the donor DNA is chemically modified, such as 2'-O-methylation modification on nucleotide ribose, 3'phosphorothioate modification between nucleotides, and 5' End phosphorylation modification.
  • the chemical modification is a 2'-O-methylation modification and/or a ribose 3 nucleotides before the 5'end and 3 nucleotides after the 3'end of the donor DNA. 3'phosphorothioate modification between glycidyl acids.
  • the chemical modification is one, two and/or three bases before the 5'end of the donor DNA and/or the 2'-O of the last nucleotide ribose at the 3'end.
  • the donor DNA contains the 2'-O-methylation modification of the nucleotide ribose in the 3 nucleotides before the 5'end and the 3 nucleotides after the 3'end, and the donor DNA contains the 2'-O-methylation modification of the nucleotide ribose before the 5'end.
  • the three internucleotide linkages after the 3 and 3'ends contain phosphorothioate modification.
  • the donor DNA contains a 2'-O-methylation modification in the ribose of the first 5 nucleotides at the 5'end and the last 5 nucleotides at the 3'end, and the ribose at the 5'end
  • the first 5 and the last 5 internucleotide linkages at the 3'end contain phosphorothioate modification.
  • the donor DNA contains phosphorylation modification at the 5'end, and phosphorothioate modification is included between the first three nucleotides at the 5'end and the last 3 nucleotides at the 3'end.
  • the donor DNA contains a phosphorylation modification at the 5'end, and phosphorothioate modification is included between the first three nucleotides of the 5'end and the last 3 nucleotides of the 3'end, and The first three nucleotides at the 5'end and the last 3 nucleotides at the 3'end also contain the 2'-O-methylation modification of ribose.
  • the donor DNA, sgRNA and/or mRNA encoding Cas9 are introduced sequentially or simultaneously into the hematopoietic stem cells by electroporation (or electrotransduction).
  • the weight ratio of the sgRNA to the donor DNA is about 1:12 to about 12:1, for example, about 1:11 to about 11:1, about 1. :10 to about 10:1, about 1:9 to about 9:1, about 1:8 to about 8:1, about 1:7 to about 7:1, about 1:6 to about 6:1.
  • the weight ratio of the mRNA encoding the sequence-specific nuclease to the single-stranded DNA is about 1:12 to about 12:1, for example, about 1:11 to about 11: 1.
  • the Cas9 mRNA: sgRNA-1: ssODN is 6 ⁇ g: 4 ⁇ g: 6 ⁇ g, 6 ⁇ g: 4 ⁇ g: 8 ⁇ g, 6 ⁇ g: 4 ⁇ g: 10 ⁇ g, 6 ⁇ g: 4 ⁇ g: 12 ⁇ g.
  • the donor DNA, sgRNA and/or mRNA encoding Cas9 are introduced sequentially or simultaneously into the hematopoietic stem cells by electroporation (or electrotransduction).
  • the electrical transduction conditions are, for example, 250-360V, 0.5-1ms; 250-300V, 0.5-1ms; 250V, 1ms; 250V, 2ms; 300V, 0.5ms; 300V, 1ms; 360V, 0.5ms; or 360V, 1ms.
  • the hematopoietic stem cells gene-repaired by the above-mentioned method of this application that is, the ALAS-2 gene mutation (for example, the gene mutation in exons 5-11 or intron-1 of the gene, specifically the Int-1-GATA mutation) is corrected or
  • the repaired hematopoietic stem cells can be returned to patients with sideroblast anemia (for example, hereditary sideroblast anemia, specifically XLSA).
  • sideroblast anemia for example, hereditary sideroblast anemia, specifically XLSA
  • the hematopoietic stem cells genetically repaired by the above method of the present application are returned to the patient, the hematopoietic stem cells can be colonized in the bone marrow of the patient for a long time, and the hematopoietic system of the patient can be successfully reconstructed.
  • the ALAS-2 gene mutation repaired CD34 + HSPC is derived from the peripheral blood of the individual to be treated (with or without bone marrow hematopoietic stem cell mobilization) or obtained from the bone marrow of the individual.
  • the CD34+HSPC population is subjected to erythroidization using hematopoietic stem cell erythroid expansion and differentiation medium.
  • the hematopoietic stem cell erythroid expansion and differentiation medium includes a basal medium and a composition of growth factors, wherein the composition of growth factors includes stem cell growth factor (SCF); interleukin 3 (IL-3) and erythropoietin (EPO).
  • SCF stem cell growth factor
  • IL-3 interleukin 3
  • EPO erythropoietin
  • erythroid differentiation and denucleation medium for erythroid differentiation and denucleation of hematopoietic stem cells, the erythroid differentiation and denucleation medium comprising a basal medium, growth factors, and progesterone receptors and glucocorticoids Antagonists and/or inhibitors of hormone receptors.
  • the growth factor in the erythroid differentiation and denucleation medium includes erythropoietin (EPO), and the antagonist and/or inhibitor of the progesterone receptor and glucocorticoid receptor is selected From any one or two or more of the following compounds (I) to (IV):
  • the hematopoietic stem cell erythroid expansion and differentiation medium comprises a basal medium and growth factor additives, wherein the basal medium can be selected from any serum-free basal medium, such as STEMSPAN TM SFEM II (STEM CELLS TECHNOLOGY Inc.), IMDM (Iscove's Modified Dulbecco's Medium), optionally supplemented with ITS (Thermofisher), L-gulutamin (Thermofisher), vitamin C and/or bovine serum albumin; wherein the growth factor additive is selected from IL-3 A combination of one or more of, SCF and EPO.
  • serum-free basal medium such as STEMSPAN TM SFEM II (STEM CELLS TECHNOLOGY Inc.), IMDM (Iscove's Modified Dulbecco's Medium), optionally supplemented with ITS (Thermofisher), L-gulutamin (Thermofisher), vitamin C and/or bovine serum albumin; wherein
  • any commonly used basic medium can be used in the above hematopoietic stem cell erythroid expansion and differentiation medium, such as STEMSPAN TM SFEM II (purchased from STEM CELL TECHONOLOGIES); for example, IMDM, DF12, Knockout DMEM, RPMI 1640 from Thermo Fisher , Alpha MEM, DMEM, etc.
  • other components can be further added to these basic media as needed, for example, ITS (that is, mainly including insulin, human transferrin, and selenium), L-glutamine, vitamin C, and bovine serum albumin can be added.
  • ITS 2mM L-glutamine, 10-50 ⁇ g/ml vitamin C and 0.5-5 mass% BSA (bovine serum albumin)
  • BSA bovine serum albumin
  • DF12 can be added with the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin.
  • Knockout DMEM can be supplemented with the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin
  • RPMI 1640 can be supplemented with the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin
  • Alpha MEM can be supplemented with the same concentration ITS, L-glutamine, vitamin C and bovine serum albumin
  • DMEM can also be added with the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin.
  • the concentration of additional ITS in various basal media can be: insulin concentration is 0.1 mg/ml, human transferrin is 0.0055 mg/ml, selenium element is 6.7 ⁇ 10 -6 mg/ml.
  • the concentration of each component of ITS added can also be adjusted according to actual needs. ITS can be purchased from Thermofisher and adjusted to the appropriate final use concentration as required.
  • the above-mentioned hematopoietic stem cells repaired by gene editing can be directly or cultured for one or more days and then returned to the sideroblast anemia (for example, XLSA) patient for treatment.
  • the hematopoietic stem cells are cultured for one or more days (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days) before being administered to the individual.
  • the hematopoietic stem cells are stored in a frozen condition for at least 24 hours before the hematopoietic stem cells are returned to the individual patient.
  • the cells are cultured for one or more days (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days) before being stored under freezing conditions.
  • the treatment comprises administering to the individual (such as intravenous injection, comprising a single intravenous injection) ⁇ 2x10 6, ⁇ 5x10 6, ⁇ 1x10 7, ⁇ 2x10 7 cells / kg body weight above ALAS-2 Hematopoietic stem cells repaired by genetic mutations.
  • hematopoietic stem cells With the proliferation and differentiation of hematopoietic stem cells, the production of heme can be detected. It can be stained with Benzidine: benzidine staining. In the presence of hydrogen peroxide, benzidine can bind and react with the heme in hemoglobin to produce a brown or blue precipitate to detect the synthesis of heme. Assess the effect of gene therapy. The effect of gene therapy can also be evaluated by detecting the expression of ALAS-2 gene and/or protein by conventional methods in the art.
  • the differentiated cells can also be evaluated by, for example, Benzidine staining and Wright-Giemsa staining.
  • the red blood cells differentiated from healthy donors and hematopoietic stem cells that have undergone gene repair are mainly mature red blood cells and reticulocytes, while the red blood cells differentiated from hematopoietic stem cells that have not undergone gene repair are mainly promyelocytic red blood cells, indicating that differentiation is stagnant. In the early stage, mature red blood cells cannot be differentiated.
  • the detection methods known in the art can determine the differentiation of hematopoietic stem cells, so as to determine whether the ALAS-2 gene mutation of hematopoietic stem cells has been corrected.
  • Example 1 Efficient gene repair of ALAS-2 intron-1 point in hiPSC derived from XLSA patients mutation
  • This embodiment relates to the use of CRISPR/Cas9 system to edit XLSA patient-derived human induced pluripotent stem cells (Human induced pluripotent stem cells, hiPSC) to efficiently repair the ALAS-2 intron-1 point mutation, the location is (X: 55054635 [Chr X(GRCh37/hg19):g.55054635 A>G,NM 000032.4:c.-15–2187 T>C), because this site is the junction of GATA-1 and ALAS-2 genes, so The point mutation is named Int-1-GATA.
  • sgRNA-1 aactctggcaactttacctg (SEQ ID NO: 1)
  • sgRNA-2 caactttacctgtggtctgc
  • sgRNA-3 gggctgagcctgcagaccac (SEQ ID NO: 3)
  • tcccacgccctggtctcagcttggggagtggtcagaccccaatggcgataaactctggcaactttacctgtggtctgcaggctcagccccaagtgct (SEQ ID NO: 4), the
  • Cas9 mRNA encoding information is as follows: gacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttctccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatctggtggaggaggagg
  • sgRNA refers to the modification of the ribose of the first three nucleotides of the 5'end and the last three nucleotides of the 3'end of the sgRNA with 2'-O-methylation and the 3'sulfur between the nucleotides. Phosphorylation modification. As shown in the following chemical formula, the left side is the chemically modified sgRNA, and the right side is the unmodified sgRNA.
  • the donor DNA contains phosphorylation modification at the 5'end, and phosphorothioate modification is included between the first three nucleotides at the 5'end and the last 3 nucleotides at the 3'end.
  • Cas9 mRNA and sgRNA were purchased from Trilink Biotechnologies, USA.
  • Reverse primer catatggcaacctccttcatc (SEQ ID NO: 7)
  • the Indels efficiency of sgRNA-1, sgRNA-2, and sgRNA-3 are about 35%, 10%, and 40%, respectively, as shown in FIG. 3.
  • efficient Indels efficiency is the prerequisite for gene repair, so we choose sgRNA-1 and sgRNA-3 as the preferred sgRNA;
  • second the closer the sgRNA cutting site is to the gene repair site, the closer the gene repair is. The higher the efficiency (Xiquan Liang, et al. Journal of Biotechnology. 2016; Mark A. et al. Science Translational Medicine. 2017).
  • sgRNA-1 we optimized the amount of Cas9 and sgRNA to electrotransform the same amount of hiPSC (1.0*10 ⁇ 6 cells), that is, electrotransform Cas9mRNA: sgRNA, 1 ⁇ g:1 ⁇ g, 2 ⁇ g:2 ⁇ g, 3 ⁇ g, respectively: 3 ⁇ g, 4 ⁇ g: 4 ⁇ g, 6 ⁇ g: 6 ⁇ g enter the hiPSC, 4 days after electroporation, extract the genome of the hiPSC, select about 450bp around the sgRNA cleavage site, and a total length of 905bp fragments for amplification and Sanger sequencing. Analyze the efficiency of Indels through the "Synthego ICE Analysis" online software. The results showed that with the increase of Cas9 mRNA and sgRNA, the efficiency of Indels increased, and the efficiency of 4ug:4ug was the highest, reaching about 50% gene editing efficiency, as shown in Figure 4.
  • the test volume of Cas9 mRNA:sgRNA:ssODN is 1 ⁇ g:1 ⁇ g:1 ⁇ g, 2 ⁇ g:2 ⁇ g:2 ⁇ g, 3 ⁇ g:3 ⁇ g:3 ⁇ g, 4 ⁇ g:4 ⁇ g:4 ⁇ g, 6 ⁇ g:6 ⁇ g:6 ⁇ g.
  • the results show that as the amount of addition increases, The gene repair efficiency HDR is improved.
  • the highest gene repair efficiency HDR is 6 ⁇ g:6 ⁇ g:6 ⁇ g, which is about 25%. This proves that we have successfully repaired the point mutations in hiPSC derived from XLSA patients at the Int-1-GATA position. Point successfully realized the repair from C to T.
  • Cas9 mRNA:sgRNA:ssODN is 6 ⁇ g:6 ⁇ g:6 ⁇ g, which induces a higher Indels efficiency (%NHEJ, as shown in Figure 5).
  • Example 2 Efficient gene repair of ALAS-2 in CD34+HSPC derived from bone marrow of XLSA patients Intron-1 point mutation
  • Example 1 we achieved an efficient repair of the Int-1-GATA point mutation of hiPSC derived from XLSA. Refer to the amount of Cas9 mRNA, sgRNA-1 and ssODN added in Example 1. In this experiment, we tried gene repair CD34+HSPC from bone marrow of XLSA patients.
  • Cas9 mRNA sgRNA-1: ssODN is 6 ⁇ g:4 ⁇ g: 12 ⁇ g, 4 days later, the HSPC genome was extracted, and the gene repair efficiency (HDR) and Indels efficiency (NHEJ) were analyzed by the next-generation sequencing bioinformatics method. As shown in Figure 7, the gene repair efficiency reached about 40%.
  • Example 2 Refer to the addition amount of Cas9 mRNA, sgRNA-1 and ssODN found in Example 2 (6 ⁇ g:4 ⁇ g:12 ⁇ g), select 300v 1ms electroporation conditions, and electrotransform Cas9 mRNA, sgRNA-1 and ssODN into HSPC derived from the bone marrow of XLSA patients, respectively , Use the following "two-step method" differentiation protocol for red blood cell differentiation experiments. In addition, our erythroid differentiation of healthy donors mobilized CD34+HSPC derived from peripheral blood as a positive control.
  • the two-step method of differentiation is to use HSPC erythroid amplification and differentiation medium for differentiation, and then use HSPC erythroid differentiation and denucleation medium for differentiation.
  • the erythroid expansion and differentiation medium of hematopoietic stem cells is StemSpan TM SFEM II, the growth factor is 50-200ng/ml SCF, 10-100ng/ml IL-3, 1-10U EPO/ml, culture conditions: use Hematopoietic stem cell erythroid expansion and differentiation medium culture hematopoietic stem cells 1.0 ⁇ 10 ⁇ 5 cells/ml for 7 days.
  • the erythroid differentiation and denucleation medium for hematopoietic stem cells is STEMSPAN TM SFEM II, the growth factor is 1-10 U EPO, 100-1000 ⁇ g/ml human transferrin, and the chemical small molecule is 0.5-10 ⁇ m mifepristone.
  • the 1.0 ⁇ 10 ⁇ 6 cells/ml cells cultured in one step were differentiated in the hematopoietic stem cell erythroid differentiation denucleation medium for 11 days.
  • the differentiation efficiency of the gene repaired cells in the second stage of differentiation is significantly higher than that of the unrepaired cells, indicating that the former differentiated cells are more mature. This is because the ALAS-2 gene is involved in ferrous iron. Heme synthesis and red blood cell maturation (Zhang, et al. Nucleic Acids Research. 2017; Liu, et al. Nature Communications. 2018), so when the ALAS-2 gene mutation is repaired, the degree of red blood cell differentiation increases. 2) The differentiation efficiency of gene repaired cells in the second stage of differentiation is lower than that of healthy donor-derived cells. This is because the ALAS-2 Int-1-GATA mutation is partially repaired by the gene, and the efficiency is about 40%.
  • Benzidine staining hematopoietic stem cells derived from healthy donors and red blood cells differentiated from hematopoietic stem cells that have undergone gene repair. After Benzidine staining, the proportion of positive cells (shown by the red arrow) is significantly higher than that of hematopoietic stem cells that have not undergone gene repair. Red blood cells. The results of statistical analysis further showed that the percentage of Benzidine-positive cells derived from healthy donors and red blood cells differentiated from hematopoietic stem cells that have undergone gene repair is about 60%, while the red blood cells differentiated from hematopoietic stem cells that have not undergone gene repair are only 20%. .
  • red blood cells differentiated from healthy donors and hematopoietic stem cells that have undergone gene repair are mainly mature red blood cells and reticulocytes, while red blood cells differentiated from hematopoietic stem cells that have not undergone gene repair are promyelocytic erythrocytes. Mainly, indicating that differentiation is stagnant in the early stage, and mature red blood cells cannot be differentiated.
  • Example 2 Extract cell proteins from the red blood cells differentiated from CD34+HSPC in Example 2.2, and perform Western Blot experiment to detect the protein expression of ALAS-2, GATA-1, and GAPDH genes, as shown in FIG. 12.
  • Example 3 In vitro clone formation of CD34+HSPC derived from bone marrow of XLSA patients with gene repair
  • This experiment involves the detection of colony-formation units (CFU) of CD34+HSPC derived from the bone marrow of gene-edited XLSA patients.
  • CFU colony-formation units
  • Select the 300V 1ms electroporation conditions refer to the Cas9 mRNA, sgRNA-1 and ssODN additions found in Example 2, electroporate Cas9 mRNA, sgRNA-1 and ssODN into CD34+HSPC derived from the bone marrow of the XLSA patient, and 500 cells Resuspend in 1ml H4434 (purchased from Canada STEM CELLS Technologies), IMDM (purchased from Thermo Fisher) and FBS (purchased from Thermo Fisher) in a mixed solution, 14 days later, observe CFU-M, BFU-E, CFU-E under the microscope The number of clones with different morphologies such as CFU-G, CFU-GM, GEMM, etc., and the results are shown in Figure 13.
  • BFU-E, CFU-GM, CFU-E, CFU-MM represent the clonal formation of different blood system lineages such as erythroid, myeloid, and lymphatic system.
  • healthy donors represent healthy donors mobilizing CD34+HSPC derived from peripheral blood, blank control: represents cells that have not undergone gene repair, and gene repair represents cells that have undergone gene repair.
  • the experimental results show that: compared with cells that have not undergone gene repair, the CFU-GM, BFU-E, and CFU-E of the cells that have undergone gene repair are significantly increased.
  • BFU-E and CFU-E represent the erythroid pre-clone and terminal, respectively
  • the differentiated erythroid clones further proved that gene repairing the mutation site of ALAS-2 Int-1-GATA restored the ability of the CD34+HSPC erythroid to become mature red blood cells.
  • the gene repair efficiency is about 40%, the total number of clones formed by the gene repaired cells and the number of different subclones are lower than the number of clones formed by cells from healthy donors, which is in line with experimental expectations.
  • 300V 1ms electroporation conditions were selected, referring to the addition of Cas9 mRNA, sgRNA-1 and ssODN found in Example 2, electroporation of Cas9 mRNA, sgRNA-1 and ssODN into the bone marrow-derived CD34+HSPC of XLSA patients, the transplantation process
  • the NPG immunodeficiency mouse model irradiated by the irradiator purchased from Beijing Vitalstar Biotechnology, Inc.
  • Human CD45 and small blood cells were detected in the peripheral blood 10, 12, and 16 weeks after transplantation.
  • mice CD45 The expression of mouse CD45, and the expression of human CD45 in bone marrow and spleen and mouse CD45 in the bone marrow and spleen 16 weeks after transplantation were detected at the same time.
  • the results are shown in Figure 14.
  • the method of transplantation into mice is: 24 hours before cell transplantation , Irradiated with 1.0Gy rays to clear the bone marrow of the mouse model. Then, 1.0 ⁇ 10 ⁇ 6 cells resuspended with 20 ⁇ L of 0.9% normal saline were injected into the tail vein of the mouse, and then put into a clean animal room Medium feeding.
  • blank control represents cells that have not undergone gene repair, and gene repair represents cells that have undergone gene repair.
  • FIG. 14 and Figure 15 show that after the gene repaired CD34+HSPC, after transplantation into the mouse model, compared with the non-genetically repaired CD34+HSPC, the peripheral blood, bone marrow and spleen of the animal after the genetically modified cell transplantation
  • the increased expression ratio of human hCD45 in humans indicates that the gene repaired CD34+HSPC can be quickly and efficiently implanted into the hematopoietic system of the mouse model, and the differentiation function of the cells in vivo is normal, while the CD34+HSPC that has not undergone gene repair is implanted in the mouse model.
  • the mouse model has abnormal functions in the hematopoietic system, and the implantation efficiency is low.
  • the cells that have undergone gene repair have high expression of CD19 protein, reaching a ratio of about 90%, while the expression of CD19 protein in cells without gene repair is significantly reduced, and the expression ratio is less than 5%, which indicates that cells that have undergone gene repair It can express CD19 protein normally and differentiate into B cells normally, but the B cells of cells that have not undergone gene repair have significantly abnormal differentiation.
  • the above results further prove that the gene repaired cells can efficiently rebuild the hematopoietic system of the mouse model.
  • gene-edited cells can quickly and efficiently rebuild the hematopoietic system of the mouse model.
  • the results of determining whether gene editing occurred in the reconstructed mouse model cells are shown in Figure 18.
  • the genome of the cells before transplantation and the bone marrow 16 weeks after transplantation was extracted, the target fragment was amplified, and the gene repair was analyzed by the next-generation sequencing bioinformatics method. Efficiency (HDR) and Indels efficiency (NHEJ).
  • HDR High-efficiency
  • NHEJ Indels efficiency
  • the genome of the bone marrow of the mice after the second transplantation was extracted 12 weeks, the target fragment was amplified, and the gene repair efficiency (HDR) and the Indels efficiency (NHEJ) were analyzed by second-generation sequencing bioinformatics methods.
  • the results showed that the human-derived cells in the bone marrow 12 weeks after transplantation all had high-efficiency gene editing, and the gene repair efficiency was similar to that of the cells before transplantation, about 40%, as shown in Figure 20.
  • the method of the present invention has the following advantages.
  • the method can gene-edit and efficiently repair hiPSC derived from XLSA patients and CD34+HSPC derived from bone marrow, which meets the needs of clinical treatment of X-chain cyclic iron particles.
  • Immature red blood cell anemia treatment requirements second, high gene repair efficiency, significantly increase the expression of ALAS-2 gene and protein, and significantly increase the synthesis of heme in differentiated red blood cells;
  • gene repaired hematopoietic stem cells can efficiently reconstruct models The hematopoietic system of mice; fourth, the cells after gene editing have no potential off-target phenomenon.
  • the method developed by the present invention may replace traditional hematopoietic stem cell transplantation treatment techniques to cure patients with X-chain ring sideroblast anemia.

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Abstract

L'invention concerne un procédé pour réparer efficacement la mutation du gène ALAS-2 qui provoque une anémie avec sidéroblastes en couronne au moyen d'une technologie d'édition génomique, consistant à utiliser une technologie d'édition génomique pour modifier génétiquement de manière efficace et sûre la mutation ponctuelle spécifique du gène ALAS-2 dans les cellules souches hématopoïétiques de patients ; restaurer l'expression du gène ALAS-2 ; et restaurer la synthèse de ferrohèmes et la maturation des globules rouges à des niveaux normaux, de manière à atteindre l'objectif de traiter la maladie.
PCT/CN2020/112227 2019-08-29 2020-08-28 Procédé pour réparer efficacement une mutation génétique responsable d'une anémie avec sidéroblastes en couronne Ceased WO2021037234A1 (fr)

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