WO2019080920A1 - 一种提高胎儿血红蛋白表达水平的方法 - Google Patents

一种提高胎儿血红蛋白表达水平的方法

Info

Publication number
WO2019080920A1
WO2019080920A1 PCT/CN2018/112068 CN2018112068W WO2019080920A1 WO 2019080920 A1 WO2019080920 A1 WO 2019080920A1 CN 2018112068 W CN2018112068 W CN 2018112068W WO 2019080920 A1 WO2019080920 A1 WO 2019080920A1
Authority
WO
WIPO (PCT)
Prior art keywords
hematopoietic stem
sgrna
cell
cells
stem cells
Prior art date
Application number
PCT/CN2018/112068
Other languages
English (en)
French (fr)
Inventor
袁鹏飞
方日国
于玲玲
夏鹏辉
王佳
梁福才
Original Assignee
博雅辑因（北京）生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 博雅辑因（北京）生物科技有限公司 filed Critical 博雅辑因（北京）生物科技有限公司
Priority to CN202210443541.6A priority Critical patent/CN114875026A/zh
Priority to JP2020523710A priority patent/JP2021500058A/ja
Priority to EP18870256.7A priority patent/EP3702458A4/en
Priority to US16/758,824 priority patent/US20200384032A1/en
Priority to CN201880069637.2A priority patent/CN111278977B/zh
Publication of WO2019080920A1 publication Critical patent/WO2019080920A1/zh
Priority to PH12020550488A priority patent/PH12020550488A1/en
Priority to ZA2020/02204A priority patent/ZA202002204B/en
Priority to JP2022149587A priority patent/JP2022180511A/ja

Links

Images

Classifications

    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • 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
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • 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 RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/14—Erythropoietin [EPO]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C12N2501/392—Sexual steroids
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

  • the present invention relates to a cell treatment protocol for treating anemia diseases such as thalassemia and sickle cell anemia, which comprises gene-editing technology for efficiently and safely genetically modifying BH11A enhancer sites of human hematopoietic stem cells, up-regulating gamma globin and The expression of fetal hemoglobin is achieved for the purpose of treating diseases.
  • anemia diseases such as thalassemia and sickle cell anemia
  • anemia diseases such as thalassemia and sickle cell anemia
  • gene-editing technology for efficiently and safely genetically modifying BH11A enhancer sites of human hematopoietic stem cells, up-regulating gamma globin and The expression of fetal hemoglobin is achieved for the purpose of treating diseases.
  • Thalassemia also known as globin-forming anemia or maritime anemia, is a group of hemolytic anemia diseases caused by genetic factors such as genetic defects. Genetic genetic defects result in the loss or deficiency of synthesis of one or several globin peptide chains in hemoglobin, resulting in an anemia or hemolysis pathology. The reduction or deletion of the globin chain of synthetic hemoglobin leads to abnormal structure of hemoglobin.
  • the red blood cell containing abnormal hemoglobin has reduced deformability, shortened life span, and may cause in situ hemolysis in the bone marrow, and enter the peripheral blood circulation and be spleen and other organs. Destruction in advance, resulting in anemia, iron deposition in the body and even abnormal development. Due to the diversity and complexity of different globin chain gene mutations, the type, quantity and clinical symptom variability of the deleted globin are very large.
  • Thalassemia is named and classified according to the type and lack of globin chains that are missing. According to different types of globin peptide chain formation disorders can be divided into ⁇ type, ⁇ type, ⁇ type, ⁇ type.
  • thalassemia is one of the most common genetic defects in the world. According to statistics, 4.83% of the global population carries a globin mutant gene, including 1.67% of alpha and beta thalassemia heterozygotes, containing 1.92% hemoglobin with a sickle cell mutation, 0.95% carrying hemoglobin E, and 0.29% carrying hemoglobin C. Wait. The resulting global birth rate of abnormal hemoglobin with disease symptoms is not less than 0.024%, which is a common gene-deficient disease.
  • Beta thalassemia (hereinafter referred to as beta thalassemia) is a type of thalassemia.
  • the pathogenesis is due to mutations in the ⁇ -globin peptide chain.
  • Most patients have point mutations, and a few are large fragment gene deletions.
  • Gene deletion and some point mutations can lead to the complete inhibition of the synthesis of a part of the ⁇ -globin peptide chain, which is called ⁇ 0 thalassemia; and a few point mutations inhibit the synthesis of the ⁇ chain, still retain a part of the peptide chain synthesis.
  • This type is called ⁇ + thalassemia, and different combinations may have different clinical symptoms.
  • ⁇ thalassemia There are two types of mutated genes for heavy ⁇ thalassemia, one of which is ⁇ homozygote, the double heterozygote of Kiehl's ⁇ 0 and ⁇ + thalassemia, because ⁇ globin peptide chain formation is nearly completely inhibited, and ⁇ globin cannot be produced in vivo.
  • normal hemoglobin A synthesis consisting of an alpha chain and a beta chain is reduced or eliminated.
  • the excess ⁇ -chain protein can bind to the ⁇ chain in the red blood cell to form hemoglobin F, since the ⁇ synthesis is gradually inhibited after birth (also known as hemoglobin chain synthesis switching), the excess ⁇ chain will be deposited in the red blood cells to form inclusion.
  • the body attaches to the surface of the erythrocyte membrane, causing the erythrocyte membrane properties to change, the cells becoming stiff and causing a decrease in deformability, and destruction in the bone marrow results in "ineffective hematopoiesis".
  • some red blood cells of thalassemia can mature in the bone marrow and eventually be released into the peripheral blood circulation, when they pass through the peripheral microcirculation (such as the spleen and other organs), they will easily become mechanical due to the deformability. damage.
  • the child is clinically asymptomatic at birth, due to HbF (fetal hemoglobin) expression and red blood cell life span of up to 120 days after birth, often after 6 months due to physiological inhibition of ⁇ -chain synthesis, hemoglobin chain synthesis Switching to the ⁇ -strand, and unable to synthesize the ⁇ -chain due to gene defects, the pathological changes of the cells cause an increase in damage, showing chronic hemolytic anemia, which further leads to changes in bone marrow composition. During the treatment, repeated blood transfusions are required, resulting in hemosiderin, which affects important organ function.
  • HbF fetal hemoglobin
  • Sickle-shaped red blood cell anemia similar to ⁇ -thalassemia, belongs to an autosomal recessive genetic disease, except that the anemia mutation site is single, due to the single base mutation of ⁇ -globin, the normal beta gene
  • the 6-position codon is caused by the mutation of GAG (encoding glutamic acid) to GTG (valine).
  • GAG encoding glutamic acid
  • GTG valine
  • HbS tetrameric complex
  • the body is arranged in a direction parallel to the membrane and closely contacts the cell membrane.
  • the cell membrane changes from a normal concave shape to a sickle shape.
  • Sickle-shaped red blood cells poor deformability, easy to break and hemolysis, resulting in vascular blockage, damage, necrosis and so on.
  • Treatments for beta-thalassemia and sickle cell anemia include general supportive care, high-dose transfusions and regular de-iron therapy, hematopoietic stem cell transplantation, induction of fetal hemoglobin therapy, and exploratory gene therapy.
  • the only cure for all treatment options is allogeneic hematopoietic stem cell transplantation. Since Thomas first carried out the first hematopoietic stem cell transplantation in thalassemia patients in 1981, hematopoietic stem cell transplantation technology has been used in several thalassaemia research centers around the world. Developed and successfully replaced the classic blood transfusion and de-iron treatment options.
  • hematopoietic stem cell transplantation is still limited in the treatment of thalassemia.
  • researchers have been continuing to explore drugs for the treatment of thalassemia.
  • the only FDA approved oral drug for clinical treatment is hydroxyurea, which mainly relies on the induction of fetal hemoglobin expression to alleviate the clinical symptoms of the disease, but because of the clinical Inconsistent efficacy and large side effects, as well as dose-related myelosuppression, are urgently needed to develop new treatments for the treatment of anemia associated with beta-thalassemia and sickle cell anemia.
  • the invention utilizes gene editing technology, such as CRISPR/Cas9 gene editing technology, to develop a new generation of hematopoietic stem cells, which greatly increase the efficiency of knockout of the BCL11A enhancer gene compared with the prior art hematopoietic stem cells, thereby The expression of hemoglobin in red blood cells after differentiation and maturation is greatly improved, which solves the problem that the editing efficiency of BCL11A enhancer in the prior art is low and the expression of fetal hemoglobin cannot satisfy the clinical application.
  • gene editing technology such as CRISPR/Cas9 gene editing technology
  • the present invention further improves the strategy for culturing and differentiating hematopoietic stem cells after gene editing, which not only greatly shortens the differentiation process from hematopoietic stem cells to mature red blood cells, but also greatly increases the number of harvested mature red blood cells, thereby partially satisfying The requirements for clinical applications.
  • the invention relates to the following:
  • a method for increasing the expression of fetal hemoglobin (HbF) in human hematopoietic stem cells comprising:
  • the BCL11A genomic region from position 60,952,219 to 60,495,336 of chromosome 2 in the hematopoietic stem cells was disrupted by gene editing techniques.
  • the gene editing technique is a zinc finger nuclease-based gene editing technique, a TALEN gene editing technique, or a CRISPR/Cas gene editing technique.
  • a method for efficiently editing hematopoietic stem cells in vitro by a CRISPR/Cas9 system comprising introducing an sgRNA comprising a sequence selected from any one of SEQ ID NO: 3 to SEQ ID NO: 25 into the hematopoietic stem cell, wherein the sgRNA is 2 '-O-methyl analog and/or internucleotide 3' thio-modified.
  • Hematopoietic stem cells obtained by the method according to any one of items 1 to 14.
  • a human hematopoietic stem cell which is genetically engineered to increase fetal hemoglobin (HbF) expression, wherein one or more sites in the BCL11A genomic target sequence at positions 60,952,519 to 60,495,336 on chromosome 2 in the hematopoietic stem cell pass Gene editing techniques have been destroyed.
  • HbF fetal hemoglobin
  • a method of making mature red blood cells or precursor cells thereof that are genetically engineered to increase fetal hemoglobin (HbF) expression comprising:
  • the hematopoietic stem cell erythroid expansion and differentiation medium comprises a basal medium, and a composition of growth factors, wherein the composition of the growth factor comprises stem cell growth factor (SCF); interleukin 3 (IL-3) and Erythropoietin (EPO).
  • SCF stem cell growth factor
  • IL-3 interleukin 3
  • EPO Erythropoietin
  • the erythroid differentiation denucleation medium comprises a basal medium, a growth factor, and an antagonist and/or inhibitor of a progesterone receptor and a glucocorticoid receptor.
  • the growth factor in the erythroid differentiation denucleation medium comprises erythropoietin (EPO), an antagonist of the progesterone receptor and the glucocorticoid receptor and/ Or the inhibitor is any one or two or more selected from the following compounds (I) to (IV):
  • EPO erythropoietin
  • a composition comprising the hematopoietic stem cell of item 15 or 16, or the precursor cell of item 17 or 22, or the mature red blood cell of item 18 or 22.
  • a medical article comprising the hematopoietic stem cell of item 15 or 16, or the precursor cell of item 17 or 22, or the mature red blood cell of item 18 or 22.
  • hematopoietic stem cell of item 15 or 16, or the precursor cell of item 17 or 22, or the mature red blood cell of item 18 or 22 for preventing or treating a disease in a subject in need thereof.
  • Item 25 The use of Item 25, the disease being an anemia disease, a blood loss disease, a tumor or other disease requiring a large amount of blood transfusion for prevention or treatment.
  • Item 29 wherein the disease is an anemia disease, a blood loss disease, a tumor or other disease requiring a large amount of blood transfusion for prevention or treatment.
  • An sgRNA construct comprising a nucleotide sequence selected from one of SEQ ID NO: 3 to SEQ ID NO: 25.
  • a vector, host cell or preparation comprising the construct of any of items 33-35.
  • a method of treating or preventing an anemic disease, a hemorrhagic disease, a tumor or other disease requiring a large amount of blood transfusion for prevention or treatment in a subject comprising administering a hematopoietic stem cell of the subject 15 or 16, item 17 Or a precursor cell of 22, or a mature red blood cell of item 18 or 22.
  • the method of claim 38, wherein the disease is beta-thalassemia or sickle cell anemia.
  • kits for treating or preventing an anaemia, a hemorrhagic disease, a tumor or other disease requiring a large amount of blood transfusion for prevention or treatment in a subject the construct comprising the sgRNA of any one of items 33-35 Or the carrier in item 36.
  • kit of item 41 further comprising Cas9 mRNA.
  • the invention satisfies the requirements of clinical treatment by optimizing the electrotransformation system, genetically editing the enhancer site of BCL11A of hematopoietic stem cells of normal donors and anemia patients; and editing the hematopoietic stem cells to differentiate into erythroid cells can significantly increase gamma globin and fetal hemoglobin
  • the expression of (HbF) and the hematopoietic system of the reconstructed animal model; off-target analysis showed that the method was highly safe and almost no side effects caused by gene editing were detected.
  • Figure 1 Gene editing autologous hematopoietic stem cells for the treatment of ⁇ -thalassemia and sickle cell anemia.
  • Peripheral blood was mobilized from the patient, CD34-positive hematopoietic stem cells were obtained by in vitro isolation, and genetically edited in vitro to improve fetal hemoglobin expression, and the genetically modified autologous hematopoietic stem cells were returned to the patient.
  • Fig. 2 shows the best electroporation conditions in a multi-batch experiment on the cancer cell line K562, and a fluorescence microscope photograph of the cells after electrophoresis for 4 days.
  • V refers to the pulse voltage
  • ms refers to the pulse time.
  • FIG. 3 shows the optimal electroporation conditions in a multi-batch assay on the cancer cell line K562.
  • 7-AAD indicates cell viability
  • 7-AAD (7 -Amino-actinomycin D) is a nucleic acid dye that does not pass through the normal plasma membrane.
  • the permeability of the plasma membrane to 7-AAD is gradually increased, and excitation at a suitable wavelength. Under the excitation of light, bright red fluorescence can be emitted, 7-AAD is negative for normal viable cells; GFP is used for electrical conversion efficiency.
  • “250-1” means 250V voltage, 1ms pulse time
  • “250-1-1", “250-1-2” means 250V 1ms, two repetitions
  • “300-1-1", “300-1- 2" indicates 300v 1ms, respectively, two repetitions.
  • Fig. 4 Fluorescence micrographs of electroporated GFP mRNA into hematopoietic stem cells for 4 days under 300V, 1ms electroporation conditions, including bright field, green channel, red channel and bright field green channel superimposed four fields.
  • Figure 5 shows the expression of GPF and CD34 protein by flow-through analysis of GFP mRNA into hematopoietic stem cells for 4 days under 300V, 1ms electroporation.
  • Figure 6 is a schematic representation of multiple sgRNAs designed for the enhancer 58K position of human BCL11A.
  • Figure 7 is a 150 bp sequence information for the enhancer 58K site of human BCL11A.
  • Figure 8 shows specific DNA sequence information for 23 sgRNAs designed for the position of the enhancer 58K locus 150 bp of human BCL11A.
  • FIG. 10 Electroporation of Cas9 mRNA and BCL11A enhancer-2, 3, 4, 5, and 6 sgRNA (ie, Enhancer-2, 3, 4, 5, and 6 in the figure) into three different cord blood-derived CD34-positive hematopoietic stem cells After 4 days, TIDE software analysis produced a statistical analysis of Indels efficiency.
  • FIG. 11 Electroporation of Cas9 mRNA and BCL11A enhancer-2 sgRNA into cord blood-derived CD34-positive hematopoietic stem cells, 2 days later in vitro clone formation assay (CFU assay), 14 days later, the number of clones of different blood systems, BFU-E, CFU -M, CFU-GM, CFU-E, CFU-G, CFU-MM represent the clonal formation of different lineages of the erythroid, myeloid, lymphatic and other blood systems.
  • Mock represents cells that have not been genetically edited.
  • FIG. 12 Electroporation of Cas9 mRNA and BCL11A enhancer-2 sgRNA into hematopoietic stem cells, while transplanting genetically modified and unmodified cells into a NGF immunodeficient mouse model irradiated by an irradiator, 6 weeks, 8 weeks, 10 After weeks, 12 weeks, and 16 weeks, the proportion of human CD45-positive cells was detected in peripheral blood of mice, and the proportion of human CD45-positive cells was detected in mouse bone marrow and spleen after 16 weeks of transplantation, and the proportion of CD45-positive cells was calculated.
  • the method was human CD45 positive cell % / (human CD45 positive cell % + mouse CD45 positive cell %), human CD45 positive cell % and mouse CD45 positive cell % were respectively measured by flow cytometry experiments.
  • Mock Represents cells that have not been genetically edited.
  • Enhancer-2 represents genetically engineered cells.
  • FIG. 13 Electroporation of Cas9 mRNA and BCL11A enhancer-2 sgRNA into cord blood-derived hematopoietic stem cells, while transplanting genetically modified and unmodified cells into a NGF immunodeficient mouse model irradiated by an irradiator, 16 weeks later
  • the ratio of human cell membrane proteins such as CD3, CD4, CD8, CD33, CD56, and CD19 to human CD45 protein was detected in mouse bone marrow, spleen, and peripheral blood, respectively.
  • Mock Represents cells that have not been genetically edited.
  • Enhancer-2 represents genetically engineered cells.
  • FIG. 14 Electroporation of Cas9 mRNA and BCL11A enhancer-2 sgRNA into cord blood-derived hematopoietic stem cells, transplanted through genetically modified cells into an NGF immunodeficient mouse model irradiated by an irradiator, and 16 weeks later in mouse bone marrow and spleen In peripheral blood, flow cytometry was used to detect the expression of CD45, human CD45, CD3, CD4 and CD8 in mice.
  • the SSC-H channel represents lateral angular scattering, which represents the granularity of the cells. The larger the value, the larger the particle size of the cells, the granularity refers to the wrinkle on the cell surface, the number of intracellular subcellular organelles, particles, and the like.
  • FIG. 15 Electroporation of Cas9 mRNA and BCL11A enhancer-2 sgRNA into cord blood-derived hematopoietic stem cells, transplanted through genetically modified cells into an NGF immunodeficient mouse model irradiated by an irradiator, and 16 weeks later in mouse bone marrow and spleen
  • flow analysis analyzed human CD33, CD56, CD49 in one mouse.
  • the SSC-H channel represents lateral angular scattering, which represents the granularity of the cells. The larger the value, the larger the particle size of the cells, the granularity refers to the wrinkle on the cell surface, the number of intracellular subcellular organelles, particles, and the like.
  • FIG. 16 Electroporation of Cas9 mRNA and BCL11A enhancer-2 sgRNA into cord blood-derived hematopoietic stem cells, extracting pre-transplant cells and peripheral blood, bone marrow, and spleen genomes 16 weeks after transplantation, amplification of the target fragment and Sanger sequencing, by TIDE Software analysis of gene editing Indels efficiency.
  • FIG. 17 Electroporation of Cas9 mRNA and BCL11A enhancer-2 sgRNA into cord blood-derived hematopoietic stem cells for red blood cell differentiation, detected 12 days later.
  • Panel A represents a photograph of the red blood cells after differentiation;
  • Panel B represents the detection of two membrane protein expressions of human CD71 and human 235a, indicating the efficiency of erythroid differentiation.
  • Mock Represents cells that have not been genetically edited.
  • Enhancer-2 represents genetically engineered cells.
  • FIG. 18 Electroporation of Cas9 mRNA and BCL11A enhancer-2 sgRNA into cord blood-derived hematopoietic stem cells for erythrocyte differentiation. After 12 days, mRNA expression of BCL11A, HBB, HBG and other genes was detected by real-time PCR. Mock: Represents cells that have not been genetically edited. Enhancer-2 represents genetically engineered cells.
  • Figure 19 shows the expression of fetal hemoglobin HbF in cells after electrophoresis of 6 ⁇ g of Cas9 mRNA and 4 ug of BCL11A enhancer-2 sgRNA into umbilical cord blood-derived hematopoietic stem cells for 12 days.
  • Mock Represents cells that have not been genetically edited.
  • Enhancer-2 represents genetically engineered cells.
  • Figure 20 shows the expression of CD45 and CD34 in peripheral blood of freshly isolated patients with beta thalassemia.
  • the left side is the control group and the right side is the experimental group.
  • the experimental samples were from three patients with beta-thalassemia.
  • Figure 21 Peripheral blood separation of Cas9 mRNA and BCL11A enhancer-2, 3, 4, 5, and 6 sgRNAs (Enhancer-2, 3, 4, 5, and 6 in the figure) into three different patients with beta thalassemia After 4 days of CD34-positive hematopoietic stem cells, TIDE software analysis yielded a statistical analysis of Indels efficiency.
  • FIG. 22 Electroporation of Cas9 mRNA and BCL11A enhancer-2 sgRNA into CD34-positive hematopoietic stem cells isolated from peripheral blood of patients with beta thalassemia, genome extraction, and NGS deep sequencing analysis of 14 potential off-target sites.
  • FIG. 23 Electroporation of Cas9 mRNA and BCL11A enhancer-2, 3, 4, 5, and 6 sgRNA (ie, Enhancer-2, 3, 4, 5, and 6 in the figure) into hematopoietic stem cells derived from beta thalassemia patients, and erythrocytes Differentiation, tested after 12 days.
  • Panel A represents a photograph of the red blood cells after differentiation;
  • Panel B represents the detection of two membrane protein expressions of human CD71 and human 235a, indicating the efficiency of erythroid differentiation.
  • Mock Represents cells that have not been genetically edited.
  • Enhancer-2, 3, 4, 5 and 6 represent genetically edited cells.
  • Figure 24 Electroacupuncture of Cas9 mRNA and BCL11A enhancer-2, 3, 4, 5, and 6 sgRNA (ie, Enhancer-2, 3, 4, 5, and 6 in the figure) into peripheral blood isolated CD34 from patients with beta thalassemia Positive hematopoietic stem cells, 12 days after erythrocyte differentiation, detected gene expression of BCL11A and ⁇ -globin. Mock: Represents cells that have not been genetically edited. Enhancer-2, 3, 4, 5 and 6 represent genetically edited cells.
  • FIG. 25 Electroacupuncture of Cas9 mRNA and BCL11A enhancer-2 sgRNA into CD34-positive hematopoietic stem cells derived from ⁇ -thalassemia patients.
  • CFU assay In vitro clone formation assay (CFU assay) was performed 2 days later, and the number of clones of different blood systems was counted after 14 days, BFU- E, CFU-M, CFU-GM, CFU-E, CFU-G, CFU-MM represent the clonal formation of different lineages of the erythroid, myeloid, lymphatic and other blood systems.
  • FIG. 26 Electroporation of Cas9 mRNA and BCL11A enhancer-3 (Enhancer-3), Enhancer-4 or Enhancer-5 sgRNA into CD34-positive hematopoietic stem cells derived from ⁇ -thalassemia patients, evaluation of gene editing efficiency, BCL11A gene expression and ⁇ - Gene expression of globin.
  • Enhancer-3, Enhancer-4, and Enhancer-5 represent cells that have been genetically edited after introduction of Enhancer-3, Enhancer-4, and Enhancer-5 sgRNA.
  • Figure 27 shows the results of chromatography after representing cells that have not been genetically edited (Mock) and genetically engineered cells (edited with enhancer 2sg RNA), showing the expression levels of HbF and HbA.
  • Figure 28 shows the results of chromatography after representing cells that have not been genetically edited (Mock) and genetically engineered cells (edited with enhancers 3, 4, 5, and 6sg RNA), showing the expression levels of HbF and HbA.
  • Figure 29 shows the ratio of the expression levels of HbF and HbA after calculation based on the chromatographic results in Figures 27 and 28.
  • the present application relates to gene editing of BCL11A enhancer sites of CD34-positive hematopoietic stem cells, and by optimizing various conditions of the CRISPR/Cas system, such as sgRNA and transformation conditions, to obtain hematopoietic stem cells in which BCL11A enhancers are efficiently edited.
  • the experimental results prove that the hematopoietic stem cells obtained by the invention have normal functions in the erythroid differentiation and the reconstitution of the blood system in the animal model, and the off-target analysis proves that it has safety at the same time and meets the clinical treatment standard.
  • the present invention provides gene-edited hematopoiesis for treating or preventing anemia, hemorrhagic diseases, tumors or other diseases requiring a large amount of blood transfusion for prevention or treatment (such as ⁇ -thalassemia and sickle cell anemia) Stem cells, red blood cell precursors, or mature red blood cells.
  • the invention also provides sgRNAs (such as chemically modified sgRNAs) required for gene editing hematopoietic stem cells comprising a nucleotide sequence selected from any one of SEQ ID NOs: 3-25.
  • sgRNAs such as chemically modified sgRNAs
  • the present invention also provides an editing method for hematopoietic stem cells of patients with ⁇ -thalassemia and sickle cell anemia.
  • CD34 + hematopoietic stem cells based on CRISPR/Cas gene editing technology.
  • CD34-positive hematopoietic stem cells can be obtained from cord blood or bone marrow, and Cas9 and a given sgRNA can be introduced into hematopoietic stem cells through optimized electroporation conditions, gene editing of hematopoietic stem cells, and transplantation of genetically edited hematopoietic stem cells.
  • Experimental animals were used to evaluate the ability of the hematopoietic stem cells to rebuild the blood system.
  • the sgRNA can be designed for BCL11A enhancers, such as for the BCL11A (+58) site, by a variety of design software, such as the "CRISPR RGEN TOOLS" software.
  • the designed sgRNA is chemically modified, such as a 2'-O-methyl analog modification at its 5' and 3' ends 3 bases and a 3' thio modification between nucleotides .
  • Efficient sgRNA can be obtained by testing the combination of Cas9 mRNA and different sgRNAs.
  • the hematopoietic stem cells are obtained by using clinical grade magnetic column sorting, the Cas9 protein encoding nucleotides for gene editing, and the chemically modified sgRNA are obtained by in vitro transcription experiments.
  • the genetically edited hematopoietic stem cells are differentiated into erythroid cells, and the proportion of erythropoiesis is detected.
  • genetically edited hematopoietic stem cells can be transplanted into NPG mice irradiated by irradiators to evaluate the ability to rebuild the blood system, and the patients can be separated.
  • CD34-positive hematopoietic stem cells were evaluated for gene editing efficiency, erythroid differentiation ability, gamma globin, and fetal hemoglobin expression.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression, comprising: editing an enhanced sputum site of hematopoietic stem cell BCL11A by a CRISPR/Cas9 editing technology gene.
  • the editing process designed sgRNA for the enhancer site targeting sequence of hematopoietic stem cell BCL11A.
  • the enhancer of BCL11A has sites named +62, +58, and +55 according to the distance from the transcription start site (kilo-base number). It is reported that the erythroid enhancer of BCL11A gene can negatively regulate fetal hemoglobin.
  • HbF HebF expression
  • kb indicates 1000 bases
  • 58 kb, 62 kb position is a key regulatory region.
  • researchers have studied the +55, +58, and +62 loci, the gene sequences before and after the above sites are all above 1000bp, a total of about 6000bp, so which areas can be edited to achieve the desired editing effect. It is not known to those skilled in the art that even for a +58 locus, gene editing efficiency varies greatly (see: Bauer, DE et al. An erythroid enhancer of BCL11A subject to genetic variation decided fetal hemoglobin level. Science.
  • the inventors of the present application have found through intensive research that a 150 bp base sequence in the +58 position (for example, as shown in SEQ ID NO: 2) has a great influence on the efficiency of gene editing, and the region targeted by the sgRNA of the present invention is Achieve efficient, editable areas for clinical applications.
  • the 150 bp genomic sequence is located in the region from position 60495197 to position 60495346 of human chromosome 2 (herein, abbreviated as chr2: 60495197-60495346).
  • the sgRNA of the invention can efficiently realize the gene-editing of normal donors and hematopoietic stem cells derived from anemia patients, wherein the relatively more efficient candidate sgRNA can significantly increase the expression of fetal hemoglobin. It has been reported in the literature that by designing sgRNAs libraries for +55, +58, and +62 loci, and using CRISPR/Cas9 for screening on cell models, it was found that targeting the "GATAA" locus at +58 is a key regulatory sequence, including The sgRNA of the "GATAA" sequence is most effective in promoting fetal hemoglobin.
  • the region near +58k anchored by the sgRNA found in the present invention is different from the key "GATAA" base site disclosed in the prior art.
  • the gene editing introduced by the sgRNA of the present invention plays a significant role in promoting the expression of fetal hemoglobin, and is sufficient for clinical treatment.
  • the invention encompasses a technical scheme for the BCL11A gene target sequence selected from the sequences 3-25 identified by the present invention by any available gene editing method.
  • the invention relates to a technical solution for achieving efficient gene editing by the CRISPR/Cas9 gene editing technique.
  • CRISPR/Cas is a genetic editing technique including, but not limited to, a variety of naturally occurring or manually 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 by bacteria and archaea during long-term evolution and can be used against invading viruses and foreign DNA.
  • CRISPR/Cas9 works by crRNA (CRISPR-derived RNA) by base pairing with tracrRNA (trans-activating RNA) to form a tracrRNA/crRNA complex that directs the sequence of the nuclease Cas9 protein in pair with the crRNA.
  • the target site cleaves double-stranded DNA.
  • a sgRNA single guide RNA
  • the Cas9 effector nuclease is capable of co-localizing RNA, DNA and proteins, thus providing tremendous potential for transformation.
  • the CRISPR/Cas system can use one, two or three Cas proteins. In some embodiments of the invention, the method uses Cas9.
  • Other suitable CRISPR/Cas systems include, but are not limited to, the systems and methods described in WO2013176772, WO2014065596, WO2014018423, US8,697,359.
  • Another aspect of the invention relates to a series of sgRNA molecules of the invention that enable efficient gene editing.
  • sgRNA single guide RNA
  • gRNA guide RNA
  • the sgRNA of the invention comprises a guide sequence that targets a target sequence.
  • the sgRNA of the invention further comprises a tracr sequence and a tracr partner sequence.
  • a “guide sequence” in the present invention may be a sequence of about 17-20 bp specifying a targeting site, and may be used interchangeably with “guide sequence” or “spacer”.
  • a “target sequence” is, for example, a sequence in which a guide sequence is designed to be complementary thereto, wherein hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex that requires “target sequence”
  • the "coding sequence” or “guide sequence” is sufficiently complementary to cause hybridization and promote the formation of a CRISPR complex, and complete complementation is not necessary.
  • Complementary refers to a "director sequence” or “guide sequence” and a target nucleotide sequence (for the purposes of the present invention, a BCL11A genomic target nucleotide sequence in hematopoietic stem cells) that can be found by Watson and Crick's nucleus The glycosidic pairing principle hybridizes.
  • the "director sequence” can hybridize to the target nucleotide sequence without requiring 100% complete complementarity between them.
  • the degree of complementarity between the guide sequence and its corresponding target sequence can be about or greater than about 75%, 80%, 85%, 90%, 91% when optimal alignment is performed using an appropriate alignment algorithm.
  • the optimal alignment can be determined using any suitable algorithm for aligning sequences, including the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, the Burrows-Wheeler Transform based algorithm, and the like.
  • a CRISPR complex including a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins, results in or near the target sequence (eg, from target sequence 1)
  • target sequence eg, from target sequence 1
  • the cleavage of one or both strands within the range of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs.
  • the tracr sequence may comprise all or a portion of a wild-type tracr sequence, such as a wild-type tracr sequence of about or greater than about 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 70, 75, 80, 85 or more nucleotides) or a tracr sequence consisting of the above may also form part of a CRISPR complex, for example by at least along a tracr sequence A portion hybridizes to all or a portion of the tracr partner sequence operably linked to the leader sequence.
  • a wild-type tracr sequence such as a wild-type tracr sequence of about or greater than about 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 70, 75, 80, 85 or more nucleotides
  • a tracr sequence consisting of the above may also form part of a CRISPR complex, for example by at least along a trac
  • the tracr sequence is sufficiently complementary to the tracr partner sequence to hybridize and participate in the formation of a CRISPR complex. Similar to the case of "target sequence” and “guide sequence” or “guide sequence” hybridization, complete complementation is not necessary as long as it is sufficient to perform its function. In some embodiments, in the case of optimal alignment, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% complementarity along the length of the tracr partner sequence.
  • the BCL11A genomic region in the hematopoietic stem cells referred to in the present application which is located at position 60,495,536 of chromosome 2, is defined by position c in the GRCh38 standard human gene sequence.
  • position c in the GRCh38 standard human gene sequence.
  • the disrupting the BCL11A genomic region from position 60,952,519 to 60,495,336 of chromosome 2 in hematopoietic stem cells including "adding and/or deleting (indel)" of the nucleotide sequence introduced in the region, eg, introduction Any type (eg selected from A, T, C, G) and/or quantity (eg 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1- 5, 1-4, 1-3, 1-2) nucleotide indel.
  • the disrupting comprises replacing the original nucleotide sequence with a new nucleotide sequence.
  • the disrupting comprises knock-in or knock-out a nucleotide sequence in the region.
  • the hematopoietic stem cell BCL11A site targeting sequence is set forth in SEQ ID NO: 2.
  • the present application designs an sgRNA for the 150 bp base region set forth in SEQ ID NO: 2, requiring the sgRNA sequence to be complementary to at least 17, preferably 18, preferably 19, or preferably 20 contiguous nucleotides of the sequence of SEQ ID NO: 2. the sequence of.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495203 to 6049522 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular The BCL11A genomic region of position 60495219.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:9 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 9 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495203 to 60495222 on chromosome 2, particularly at position 60,952,519 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495208 to 60495227 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular, BCL11A genomic region at position 60495224.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:10 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 10 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495208 to 60495227 on chromosome 2, particularly at position 60092224 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the number 60,495,517 to 60,495,236 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular The BCL11A genomic region of position 60495233.
  • HbF fetal hemoglobin
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the sgRNA of NO:11 is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 11 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence on chromosome 2 from position 60,952,517 to 60,495,236, particularly at position 60495233 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting, by gene editing technology, the number 60,952,518 to 60,495,237 of chromosome 2 of the hematopoietic stem cell, particularly BCL11A genomic region at position 60495234.
  • HbF fetal hemoglobin
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:12 sgRNA is complementary.
  • the method of the present invention involves introducing an sgRNA comprising the sequence of SEQ ID NO: 12 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA into a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • a Cas9 encoding nucleotide such as mRNA
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at position 60,952,518 to 60,495,237 on chromosome 2, particularly at position 60495234 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the number 60,495,519 to 60,495,238 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular, B49511A genomic region at position 60495235.
  • HbF fetal hemoglobin
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the sgRNA of NO:13 is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 13 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence on chromosome 2 from position 60,952,219 to 60,495,238, particularly at position 60495235 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting, by gene editing technology, the number 60,952,521 to 60,495,240 of the chromosome 2 in the hematopoietic stem cell, in particular, The BCL11A genomic region at position 60495223.
  • HbF fetal hemoglobin
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:16 sgRNA is complementary.
  • the method of the present invention relates to introducing an sgRNA comprising the sequence of SEQ ID NO: 16 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at position 60,952,521 to 60,495,240 on chromosome 2, particularly at position 60495223 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 6049552 to 60495241 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular, The BCL11A genomic region of position 60495238.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:14 sgRNA is complementary.
  • the method of the present invention relates to introducing an sgRNA comprising the sequence of SEQ ID NO: 14 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495222 to 60495241 on chromosome 2, particularly at position 60495238 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495223 to 60495242 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular, BCL11A genomic region at position 60495239.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:15 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 15 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495223 to 60495242 on chromosome 2, particularly at position 60495239 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495228 to 60495247 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular BCL11A genomic region at position 60495244.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the sgRNA of NO:17 is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 17 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495228 to 60495247 on chromosome 2, particularly at position 60495244 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495229 to 60495248 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular BCL11A genomic region at position 60495245.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:18 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 18 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495229 to 60495248 on chromosome 2, particularly at position 60495245 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting, by gene editing technology, the number 60,952,530 to 60,495,249 of the chromosome 2 in the hematopoietic stem cell, in particular, BCL11A genomic region at position 60495246.
  • HbF fetal hemoglobin
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:19 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 19 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at position 60,952,028 to 60,495,249 on chromosome 2, particularly at position 60495246 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the number 60,495,521 to 60,495,250 of the chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular, The BCL11A genomic region of position 60495247.
  • HbF fetal hemoglobin
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:20 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 20 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at position 60,952,531 to 60,495,250 on chromosome 2, particularly at position 60495247 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495234 to 60495253 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular B49511A genomic region of 60495250.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:21 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 21 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495234 to 60495253 on chromosome 2, particularly at position 60495250 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495235 to 60495254 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular B49511A genomic region of position 60495251.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the sgRNA of NO:22 is complementary.
  • the method of the present invention involves introducing an sgRNA comprising the sequence of SEQ ID NO: 22 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA into a MSC9 encoding nucleotide (eg, mRNA) into the hematopoietic stem cell,
  • a MSC9 encoding nucleotide eg, mRNA
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495235 to 60495254 on chromosome 2, particularly at position 60495251 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495 236 to 60495255 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular The BCL11A genomic region of position 60495238.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:4 sgRNA is complementary.
  • the method of the present invention involves introducing an sgRNA comprising the sequence of SEQ ID NO: 4 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA into a Cas9 encoding nucleotide (eg, mRNA) into the hematopoietic stem cell,
  • a Cas9 encoding nucleotide eg, mRNA
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495236 to 60495255 on chromosome 2, particularly at position 60495238 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495495 to 60495266 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular BCL11A genomic region at position 60495263.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:3 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 3 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA into a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • a Cas9 encoding nucleotide such as mRNA
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495247 to 60495266 on chromosome 2, particularly at position 60495263 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495252 to 60495271 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular BCL11A genomic region at position 60495268.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:8 sgRNA is complementary.
  • the method of the present invention involves introducing an sgRNA comprising the sequence of SEQ ID NO: 8 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA into a Cas9 encoding nucleotide (eg, mRNA) into the hematopoietic stem cell,
  • a Cas9 encoding nucleotide eg, mRNA
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495252 to 60495271 on chromosome 2, particularly at position 60495268 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495253 to 60495272 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular The BCL11A genomic region of position 60495269.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:7 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 7 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence on chromosome 2 from position 60495253 to position 60495272, particularly at position 60495269 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495495 to 60495276 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular The BCL11A genomic region of position 60495273.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:6 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 6 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495257 to 60495276 on chromosome 2, particularly at position 60495273 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 60495th to 60495283 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular, BCL11A genomic region at position 60495280.
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:5 sgRNA is complementary.
  • the method of the present invention involves introducing an sgRNA comprising the sequence of SEQ ID NO: 5 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA into a MSC9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • a MSC9 encoding nucleotide such as mRNA
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at positions 60495264 to 60495283 on chromosome 2, particularly at position 60495280 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the 6049599 to 60,519,518 of the chromosome 2 in the hematopoietic stem cell by a gene editing technique, in particular, B49511A genomic region of position 60495301.
  • HbF fetal hemoglobin
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The guide sequence of NO:24 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 24 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence on chromosome 2 from position 60495299 to 60,495,318, especially at position 60495301 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting, by gene editing technology, the number 60,495,319 to 60,495,338 of chromosome 2 in the hematopoietic stem cell, particularly BCL11A genomic region at position 60495335.
  • HbF fetal hemoglobin
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the NO:25 sgRNA is complementary.
  • the method of the present invention relates to introducing a sgRNA comprising the sequence of SEQ ID NO: 25 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA and a Cas9 encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at position 60,495,519 to 60,495,338 on chromosome 2, particularly at position 60495335 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the present invention provides a method for increasing fetal hemoglobin (HbF) expression in a human hematopoietic stem cell, comprising: disrupting the number of chromosomes 60,495,520 to 60,495,339 of chromosome 2 in the hematopoietic stem cell by gene editing technology, in particular The BCL11A genomic region at position 60495336.
  • HbF fetal hemoglobin
  • the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology or a CRISPR/Cas gene editing technology, preferably a CRISPR/Cas9 gene editing technology, preferably the BCL11A genomic target nucleotide sequence and the SEQ ID comprising The leader sequence of the sgRNA of NO:23 is complementary.
  • the method of the present invention relates to introducing an sgRNA comprising the sequence of SEQ ID NO: 23 into a hematopoietic stem cell to effect editing of the BCL11A genome, preferably introducing the sgRNA into a MSC9-encoding nucleotide (such as mRNA) into the hematopoietic stem cell,
  • a MSC9-encoding nucleotide such as mRNA
  • the sgRNA and the Cas9 coding nucleotide are co-introduced into the hematopoietic stem cells by an electroporation method of electroporation conditions of 200-600 V, 0.5 ms-2 ms.
  • the sgRNA is 3' thio-modified via a 2'-O-methyl analog and/or an internucleotide, eg, chemically modified to the first and second of the 5' end of the sgRNA And/or a 2'-O-methyl analog modification of the last base of the three bases and/or the 3' end.
  • the present invention also relates to a hematopoietic stem cell having one or more sites in the BCL11A genomic target sequence at position 60,495,520 to 60,495,339 on chromosome 2, particularly at position 60495336 Editing techniques are being destroyed. Further, it relates to red blood cells obtained by culturing the hematopoietic stem cells by in vitro differentiation, and medical articles containing the same.
  • the 23 sgRNAs designed by the inventors can efficiently produce Indels, that is, the gene editing can be performed efficiently, and the preferred sgRNA of the present invention is selected from any one of SEQ ID NO: 3 to SEQ ID NO: 25.
  • the sequence of the cleavage site of the target sequence corresponding to the sgRNA is located at chr2: 60495219 to chr2: 60495336 (i.e., between positions 60,952,519 to 60,495,336 on chromosome 2).
  • the following table lists the genomic sequence positions on human chromosome 2 targeted by the 23 sg RNAs of the present invention, and the Cas9 cleavage site initiated by each sgRNA.
  • a specific embodiment of the present invention further comprises a composition of sgRNA comprising the above 23 sgRNAs or vectors thereof according to the present invention.
  • a guide sequence in an sgRNA is any polynucleotide sequence that is sufficiently complementary to a target polynucleotide sequence to hybridize to a target sequence and direct the sequence specific binding of the CRISPR complex to the target sequence.
  • the degree of complementarity between the leader sequence and its corresponding target sequence is about or greater than about 80%, 85%, 90%, 95%, 97.5%, 99% or more.
  • Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, the Burrows-Wheeler Transform-based algorithm (eg, 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).
  • any suitable algorithm for aligning sequences non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, the Burrows-Wheeler Transform-based algorithm (eg, Burrows Wheeler Aligner), ClustalW, Clustai X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn) and Maq (
  • the length of the guide sequence may 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. In some embodiments, the guide sequence is less than about 75, 70, 65, 60, 55 in length , 50, 45, 40, 35, 30, 25, 20, 15, 12 or fewer nucleotides.
  • the ability of the guide sequence to direct sequence-specific binding of the CR1 SPR complex to the target sequence can be assessed by any suitable assay For example, a host cell having a corresponding target sequence can be provided with a CRI sufficient to form a CRISPR complex.
  • the components of the SPR system can be performed, for example, by transfection with a vector encoding a CRISPR sequence component, followed by evaluation of preferential cleavage within the target sequence (as by the Surveyor assay as described herein).
  • Cleavage of the target polynucleotide sequence can be performed in a test tube by providing a target sequence, a CRISPR complex (containing a guide sequence to be tested, and a control guide sequence different from the guide sequence), and comparing the test and control guide sequences to the target The binding or cleavage rate of the sequences is evaluated by this.
  • the above assays and evaluations can also be carried out using other assay methods known to those skilled in the art.
  • the sgRNA used in the gene editing process is preferably chemically modified.
  • the "chemically modified sgRNA” pointer specifically modifies sgRNA, such as 2'-O-methyl analog modifications at the 5' and 3' ends 3' and / or 3' between nucleotides Thio modification.
  • the chemically modified sgRNA employed by the present inventors is considered to have the following two advantages.
  • Second, unmodified sgRNAs have poor ability to penetrate cell membranes and cannot effectively enter cells or tissues to function accordingly. The ability of chemically modified sgRNAs to penetrate cell membranes is generally enhanced.
  • Chemical modification methods commonly used in the art can be employed in the present invention, as long as the sgRNA stability (prolonged half-life) and the ability to enter the cell membrane can be improved.
  • other modification methods are also included, 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 enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol. 2015 Sep; 33(9): 985-989 The chemical modification method reported in the literature.
  • the sgRNA and/or Cas9 encoding nucleotide (eg, mRNA) is introduced into hematopoietic stem cells by electroporation, eg, by 250-360V, 0.5-1 ms; 250-300 V, 0.5-1 ms; 250 V 1 ms; 250 V 2ms; 300V 0.5ms; 300V 1ms; 360V 0.5ms; or 360V1ms electroporation conditions are introduced into hematopoietic stem cells.
  • the sgRNA and Cas9 encoding nucleotides are co-introduced into hematopoietic stem cells by electroporation.
  • the sgRNA is introduced into a hematopoietic stem cell expressing Cas9 by electroporation.
  • the Cas9 encoding nucleotide is an mRNA, such as an mRNA comprising an ARCA cap. In some embodiments, the Cas9 encoding nucleotide is in a viral vector, such as a lentiviral vector. In some embodiments, the Cas9 encoding nucleotide comprises the sequence set forth in SEQ ID NO:26. In some embodiments, the sgRNA is in the same vector as the Cas9 encoding nucleotide.
  • the present invention relates to a hematopoietic stem cell obtained by the genetic engineering method according to the present invention, and a precursor cell obtained by differentiating and culturing the hematopoietic stem cell obtained by the genetic modification, at different differentiation stages before the mature red blood cell, and a differentiation culture medium.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the method obtained by the method of the present invention, which comprises hematopoietic stem cells obtained by the genetic engineering method according to the present invention, or obtained by differentially culturing the genetically engineered hematopoietic stem cells, before mature red blood cells The precursor cells of different differentiation stages, or the mature red blood cells obtained by the genetically engineered hematopoietic stem cells.
  • the pharmaceutical composition according to the present invention can be administered to a subject in need thereof by a route conventionally used for administering a pharmaceutical preparation containing a cellular component, for example, an intravenous infusion route.
  • the dosage administered can be specifically determined based on the condition of the subject and general health conditions.
  • the invention provides methods of delivering a sgRNA and/or Cas9 encoding nucleotide of the invention to a hematopoietic stem cell.
  • the delivery can be introduced into a hematopoietic stem cell or other host cell using conventional viral and non-viral-based gene transfer methods.
  • Non-viral delivery systems include DNA plasmids, RNA (such as the vector transcripts described herein), naked nucleic acids, and liposomes.
  • Viral vector delivery systems include DNA and RNA viruses having a free or integrated genome for delivery to cells.
  • the inventors use the electroporation method to introduce the genes encoding Cas9 and sgRNA into hematopoietic stem cells for gene editing of the specific sequence of the hematopoietic stem cell BCL11A gene. After repeated experiments, the inventors found that the gene editing efficiency of co-introduction of Cas9 coding nucleotides and sgRNA into hematopoietic stem cells under electroporation conditions of 200-600v, 0.5ms-2ms was significantly higher than that of other electroporation conditions.
  • the chemically modified sgRNA and the Cas9 encoding gene are co-electrically transferred into CD34+ hematopoietic stem cells, resulting in efficient gene editing efficiency (expressed in Indels%).
  • Indels% the gene editing efficiency
  • the data in the examples show that if the sgRNA is electro-transformed with Cas9 mRNA, the Indels efficiency is only 2.7%, which is much lower than the Indels efficiency obtained when electro-transformed chemically-modified sgRNA (efficiency is at least 10%). the above).
  • index is referred to collectively as an insertion/deletion, ie, an insertion and deletion mutation.
  • hematopoietic stem and progenitor cells are the most primitive hematopoietic cells in which various blood cells occur. Its main characteristics are its proliferative potential, multi-directional differentiation ability and self-renewal ability. Therefore, it can not only differentiate and supplement various blood cells, but also maintain the characteristics and quantity of stem cells through self-renewal. Hematopoietic stem cells have different degrees of differentiation and proliferation, and are heterogeneous.
  • Pluripotent hematopoietic stem cells are the most primitive, first differentiated into pluripotent hematopoietic stem cells, such as myeloid hematopoietic stem cells capable of producing granulocyte, erythroid, mononuclear and megakaryocyte-platelet types, and lymphocytes capable of developing B lymphocytes and T lymphocytes. stem cell. These two types of stem cells not only retain the basic characteristics of hematopoietic stem cells, but also slightly differentiate them. They are responsible for the occurrence of "marrow bone components" and lymphocytes, respectively, so they are called pluripotent hematopoietic stem cells.
  • hematopoietic progenitor cells which are primitive blood cells, but have lost many of the basic features of hematopoietic stem cells, such as the ability to lose multi-directional differentiation, and can only differentiate toward a lineage or closely related secondary cells; Losing the ability of repeated self-renewal, but relying on the proliferation and differentiation of hematopoietic stem cells to supplement the number; proliferation potential is limited, can only be split several times.
  • hematopoietic stem cells encompasses pluripotent hematopoietic stem cells, directed pluripotent hematopoietic stem cells, and hematopoietic progenitor cells, and is a general term for hematopoietic stem cells having different heterogeneities.
  • the hematopoietic stem cells (HSPCs) to be subjected to gene editing used in the present invention may be derived from bone marrow, cord blood or Peripheral blood mononuclear cells (PBMC).
  • PBMC Peripheral blood mononuclear cells
  • CRISPR RGEN is the name of a website developed by the research team of Korean scientist Jin-Soo Kim for the design of sgRNA at www.rgenome.net/about/.
  • TIDE refers to the name of an instrumental website dedicated to analyzing the efficiency of Indels at tide-calculator.nki.nl.
  • CD34, CD45RA, CD3, CD4, CD8, CD33, CD19, CD56, CD71, CD235a are membrane protein markers of blood system cells.
  • BCL11A is a transcription factor first discovered in mice as a binding site for retroviruses, named Evi9, which was later found in the human genome and localized to chromosome 2.
  • the short arm 2p13 site is mainly expressed in the germinal center of B lymphocytes.
  • Hemoglobin is a protein that is responsible for carrying oxygen in higher organisms. Hemoglobin consists of four chains, two alpha chains and two beta chains, each of which has a cyclic heme containing an iron atom. Oxygen is bound to the iron atom and transported by red blood cells for use by the body.
  • the present invention relates to a hematopoietic stem cell which is a hematopoietic stem cell obtained by editing a specific sequence of hematopoietic stem cell BCL11A by a CRISPR/Cas9 editing technology gene using the method of the present invention described above.
  • articles comprising the hematopoietic stem cells are also within the scope of the invention.
  • the hematopoietic stem cells or preparations of the present invention can be used to treat a disease selected from the group consisting of an anemia disease, a hemorrhagic disease, a tumor, or other treatment requiring a large amount of blood transfusion.
  • a disease selected from the group consisting of an anemia disease, a hemorrhagic disease, a tumor, or other treatment requiring a large amount of blood transfusion.
  • it can be used to treat beta-thalassemia or sickle cell anemia.
  • the present invention relates to a red blood cell obtained by culturing the genetically edited hematopoietic stem cells obtained by the present invention by in vitro differentiation and culture.
  • the present invention relates to precursor cells from various stages of differentiation between hematopoietic stem cells and mature red blood cells, which are obtained by treating the genetically edited hematopoietic stem cells obtained by the present invention by the following hematopoietic stem cell erythroid expansion and differentiation steps.
  • the above in vitro differentiation culture comprises: a hematopoietic stem cell erythroid expansion and differentiation step; and a hematopoietic stem cell erythroid differentiation denucleation step.
  • the erythroid expansion and differentiation step cultures hematopoietic stem cells using a hematopoietic stem cell erythroid expansion and differentiation medium.
  • the erythroid differentiation denucleation step uses a erythroid differentiation denucleation medium.
  • the hematopoietic stem cell erythroid expansion and differentiation medium comprises: a basal medium, and a composition of a growth factor, wherein the composition of the growth factor comprises: a stem cell growth factor, ie, SCF; an interleukin 3, namely IL-3; and erythropoietin, EPO.
  • a stem cell growth factor ie, SCF
  • an interleukin 3 namely IL-3
  • EPO erythropoietin
  • the erythroid differentiation denucleation medium comprises a basal medium, a growth factor, and an antagonist and/or inhibitor of a progesterone receptor and a glucocorticoid receptor.
  • the hematopoietic stem cells after gene editing are cultured by the above method, and the hematopoietic stem cells can be differentiated into mature red blood cells by two steps, and compared with the prior art, the in vitro differentiation culture of the present invention is performed in only 14 days, and the time is shorter. It takes more than 21 days to compare with the prior art, and the cycle is greatly shortened.
  • the growth factor comprises erythropoietin, ie, EPO, the antagonist and/or inhibitor of the progesterone receptor and the glucocorticoid receptor is selected from the group consisting of the following compounds (I)-( Any one or two or more of IV):
  • the hematopoietic stem cell expansion and differentiation of erythroid medium comprises basal medium, the basal medium e.g. STEMSPAN TM SFEM II (STEM CELLS TECHNOLOGY Inc.), IMDM (Iscove's Modified Dulbecco's Medium), X- VIVO 15, alpha-MEM, RPMI 1640 and DF12, etc.
  • basal medium e.g. STEMSPAN TM SFEM II (STEM CELLS TECHNOLOGY Inc.), IMDM (Iscove's Modified Dulbecco's Medium), X- VIVO 15, alpha-MEM, RPMI 1640 and DF12, etc.
  • EPO erythropoietin
  • the basal medium is basal medium other than STEMSPAN TM SFEM II
  • 100X ITS insuin-transferrin-selenium
  • insulin concentration is 0.1mg/ml
  • human transferrin is 0.0055mg / ml, selenium 6.7 * 10 -6 mg / ml) (ie mainly including insulin, human transferrin and selenium)
  • 10-50 ⁇ g / ml vitamin C 0.5-5%
  • BSA Bovine serum albuin, bovine serum albumin
  • growth factors such as 50-200 ng/ml SCF, 10-100 ng/ml IL-3, 1-10 U/ml EPO.
  • basal media any of the commonly used basal media can be used.
  • basal media examples include conventional base medium, e.g. STEMSPAN TM SFEM II (available from STEM CELL TECHONOLOGIES); e.g. available from Thermo Fisher's IMDM, DF12, Knockout DMEM, RPMI 1640, Alpha MEM, DMEM like.
  • ITS i.e., mainly including insulin, human transferrin, and selenium
  • L-glutamine L-glutamine
  • vitamin C bovine serum albumin
  • ITS may be added to the IMDM medium, plus 2 mM L-glutamine, plus 10-50 ⁇ g/ml of vitamin C, and 0.5-5 mass% of BSA (bovine serum albumin).
  • BSA bovine serum albumin
  • the above DF12 may be added with the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin.
  • Knockout DMEM can be added with the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin.
  • RPMI 1640 can be added with the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin.
  • Alpha MEM can be added with the same concentration.
  • ITS, L-glutamine, vitamin C and bovine serum albumin, DMEM can also be added to the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin.
  • the concentration of the ITS added in various basal mediums may be: an insulin concentration of 0.1 mg/ml, a human transferrin of 0.0055 mg/ml, and a selenium element of 6.7 ⁇ 10 -6 mg/ml.
  • the concentration of each component of the added ITS can also be adjusted according to actual needs. ITS can be purchased from Thermofisher and adjusted to the appropriate end use concentration as needed.
  • the erythroid differentiation denucleation medium comprises a basal medium, a growth factor, and an antagonist of a progesterone receptor and a glucocorticoid receptor.
  • the hematopoietic stem cells release nuclear erythroid differentiation medium comprises a basal medium, e.g. STEMSPAN TM SFEM II (STEM CELLS TECHNOLOGY Inc.), IMDM (Iscove's Modified Dulbecco's Medium), X-VIVO 15, alpha- MEM, RPMI 1640 and DF12, etc.
  • a basal medium e.g. STEMSPAN TM SFEM II (STEM CELLS TECHNOLOGY Inc.), IMDM (Iscove's Modified Dulbecco's Medium), X-VIVO 15, alpha- MEM, RPMI 1640 and DF12, etc.
  • a growth factor for example if the choice of basal media STEMSPAN TM SFEM II, the need to add additional growth factors include 1-10U / ml EPO, 100-1000 ⁇ g / ml human transferrin ( human transferrin), small chemical molecule is 0.5 -10 ⁇ mol/
  • ITS insuin-transferrin-selenium
  • ITS medium wherein the final concentration of each substance is: insulin concentration is 0.1mg / ml, Human transferrin is 0.0055mg/ml, selenium 6.7 ⁇ 10 -6 mg/ml) (ie mainly including insulin, human transferrin and selenium), 10-50ug/ml vitamin C, 0.5-5% BSA ( Bovine serum albuin, bovine serum albumin), growth factors such as 1-10 U/ml EPO, 100-1000 ug/ml human transferrin, chemical small molecules such as 0.5-10 ⁇ mol//ml mifepristone.
  • basal media any of the commonly used basal media can be used.
  • basal media examples include conventional base medium, e.g. STEMSPAN TM SFEM II (available from STEM CELL TECHONOLOGIES); e.g. available from Thermo Fisher's IMDM, DF12, Knockout DMEM, RPMI 1640, Alpha MEM, DMEM like.
  • ITS i.e., mainly including insulin, human transferrin, and selenium
  • L-glutamine L-glutamine
  • vitamin C bovine serum albumin
  • ITS may be added to the IMDM medium, plus 2 mM L-glutamine, plus 10-50 ⁇ g/ml of vitamin C, and 0.5-5 mass% of BSA (bovine serum albumin).
  • BSA bovine serum albumin
  • the above DF12 may be added with the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin.
  • Knockout DMEM can be added with the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin.
  • RPMI 1640 can be added with the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin.
  • Alpha MEM can be added with the same concentration.
  • ITS, L-glutamine, vitamin C and bovine serum albumin, DMEM can also be added to the same concentration of ITS, L-glutamine, vitamin C and bovine serum albumin.
  • the concentration of the ITS added in various basal mediums may be: an insulin concentration of 0.1 mg/ml, a human transferrin of 0.0055 mg/ml, and a selenium element of 6.7 ⁇ 10 -6 mg/ml.
  • the concentration of each component of the added ITS can also be adjusted according to actual needs. ITS can be purchased from Thermofisher and adjusted to the appropriate end use concentration as needed.
  • the mifepristone used herein is a chemically synthesized small molecule that is an antagonist of the progesterone receptor and the glucocorticoid receptor, and has the following structural formula:
  • the mature red blood cells of the invention can be produced by a method comprising the following steps a) to c): a) genetically engineering CD34-positive HSPCs isolated from human umbilical cord blood using any of the methods described herein. b) The genetically engineered HSPCs are subjected to 5-10 days, for example 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, after amplification and differentiation to obtain red blood cell precursor cells, such as erythroblasts (erythroblast) ), nucleated red blood cells, young red blood cells, and reticulocytes, c) red blood cell precursor cells are further subjected to differentiation treatment for 7 days to obtain mature red blood cells.
  • red blood cell precursor cells such as erythroblasts (erythroblast)
  • the mature red blood cell matured red blood cells of the invention can be produced by a method comprising the following steps a) to d):
  • HEEDM HSPCs erythroid expansion and differentiatin medium
  • HSPCs erythroid differentiation enucleation medium HEDEM
  • the mature red blood cells can be obtained in two steps by the method of the present invention described above, and the whole process can be 10-18 days, 11-17 days, 12-16 days, 13 times. -15 days, 10-17 days, 10-16 days, 10-15 days, or 10-14 days, this time greatly shortens the period of at least 21 days in the prior art using three or four steps.
  • the present invention relates to a method of producing a mature red blood cell or a precursor cell thereof which is genetically engineered to increase fetal hemoglobin (HbF) expression, the method comprising: (a) using the genetic modification method of the present invention Hematopoietic stem cells are obtained; (b) genetically engineered hematopoietic stem cells are subjected to hematopoietic stem cell erythroid expansion and differentiation using the above-described hematopoietic stem cell erythroid expansion and differentiation medium.
  • HbF fetal hemoglobin
  • the present invention relates to a method for producing mature red blood cells or precursor cells thereof which are genetically engineered to increase fetal hemoglobin (HbF) expression, the method comprising: (a) obtaining hematopoiesis using the genetic modification method of the present invention Stem cells; (b) hematopoietic stem cell erythroid expansion and differentiation of genetically modified hematopoietic stem cells using the hematopoietic stem cell erythroid expansion and differentiation medium; and (c) hematopoietic stem cell red using erythroid differentiation denucleation medium Department of differentiation and denucleation.
  • HbF fetal hemoglobin
  • the present invention also relates to the use of the hematopoietic stem cell erythroid expansion and differentiation medium and/or the erythroid differentiation denucleation medium used in the above method for amplifying and/or differentiating hematopoietic stem cells or precursor cells of mature red blood cells.
  • differentiation refers to a phenomenon in which the structure or function of a cell is specialized in division, proliferation, and growth thereof, that is, changes in characteristics and functions of an organism's cells or tissues to perform functions imparted to the cells or tissues. . In general, it refers to the phenomenon in which a relatively simple system is divided into two or more partial systems having different properties.
  • Ficoll liquid density gradient centrifugation has the basic principle that there is a difference in the specific gravity of different components in the blood, and different cell layer powders can be removed during low-speed density gradient centrifugation.
  • the density of red blood cells and granulocytes is greater than that of stratified liquids, and because red blood cells encounter ficoll liquid, they will quickly aggregate into a string of money and accumulate at the bottom of the tube.
  • Only a single nuclear cell with a stratified liquid density is enriched between the plasma layer and the stratified liquid, that is, the white film layer, and the hematopoietic stem cells are present in this layer, which can be obtained by subsequent magnetic bead sorting.
  • mononuclear cells are obtained using a commercially available lymphocyte separation tube.
  • mature red blood cells refers to a type of cells that are most abundant in blood and have a function of carrying nutrients such as oxygen, amino acids, and carbon dioxide. Mature red blood cells have no mitochondria and nuclei.
  • interleukin 3 refers to a cytokine produced by activated CD4 and CD8-positive T lymphocytes whose primary biological function is to participate in the regulation of proliferation and differentiation of hematopoietic stem cells in the bone marrow. .
  • EPO Erythropoietin, EPO refers to a growth factor produced by erythroblasts, or erythroid precursor cells. In the adult, there are glycoproteins secreted by the interstitial cells surrounding the renal cortex and the liver. EPO can stimulate the differentiation of hematopoietic stem cells into red blood cells.
  • the stem cell factor comprises mast cell growth factor (MGF), Kit ligand (KL), and Steel factor (SLF).
  • hematopoietic stem cell erythroid expansion and differentiation medium refers to a system of interactions that aid in the massive expansion of cells of HSPCs and differentiation into erythroid progenitor cells.
  • the medium contains two main components, a basic medium and a growth factor additive.
  • the basal medium is a serum-free system, may be STEMSPAN TM SFEM II (STEM CELLS TECHNOLOGY Inc.), it may be IMDM (Iscove's Modified Dulbecco's Medium ), plus ITS (Thermofisher) and L-gulutamin (Thermofisher) and vitamin C, and bovine serum albumin.
  • the growth factor additive is a combination of different concentrations of IL-3, SCF, EPO.
  • hematopoietic stem cell erythroid differentiation denucleation medium refers to an erythrocyte culture medium that assists in further expansion and differentiation of erythroid progenitor cells into denucleation.
  • the medium comprises two main components, namely a basic medium and a growth factor and a chemical small molecule additive.
  • the basal medium is a serum-free system, may be STEMSPAN TM SFEM II (STEM CELLS TECHNOLOGY Inc.), it may be IMDM (Iscove's Modified Dulbecco's Medium ), plus ITS (Thermofisher) and L-gulutamin (Thermofisher) and vitamin C, and bovine serum albumin.
  • Growth factors and chemical small molecule additives include EPO and human transferrin, as well as the chemical small molecule mifepristone.
  • the hematopoietic stem cells are sorted by magnetic beads, for example, the cells are specifically labeled with superparamagnetic MACS MicroBeads (MACS microbeads), and after magnetic labeling, the cells are placed in one strong and stable state.
  • MACS microbeads superparamagnetic MACS MicroBeads
  • a sorting column in a magnetic field The matrix in the sorting column creates a high gradient magnetic field.
  • the magnetically labeled cells are retained in the column while the unlabeled cells are effluxed.
  • the sorting column is moved out of the magnetic field, the magnetically labeled cells in the stagnant column can be eluted, so that the labeled and unlabeled two cell components can be completely obtained.
  • three different cord blood-derived hematopoietic stem cells can be transformed by the genetic modification method described in the present invention, thereby achieving efficient gene editing and efficiency. It is at least 40% or more, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. For example, even for the preferred sgRNA, the average gene editing efficiency is 80%.
  • the hematopoietic stem cells obtained by the genetic editing method of the present invention are transplanted into a mouse, and after transplantation of the genetically edited hematopoietic stem cells, compared with the transplantation of the unedited hematopoietic stem cells.
  • the proportion of hCD45 expression in humans continued to increase in mice. In peripheral blood samples, it increased from 20% at week 6 to 60% at 16 weeks. The proportion of bone marrow in 16 weeks even reached 90%, and the spleen reached 70%.
  • the gene-edited hematopoietic stem cells can be rapidly and efficiently implanted into the hematopoietic system of the mouse model, and the differentiation function in the cells is normal.
  • the cord blood-derived hematopoietic stem cells are modified by the gene editing method of the present invention, and then differentiated by the "two-step method" differentiation method of the present invention, that is, the hematopoietic stem cell erythroid expansion is utilized.
  • the differentiation medium is expanded and differentiated, and then further differentiated by using a hematopoietic stem cell erythroid differentiation denucleation medium.
  • hemoglobin (HBG) in differentiated red blood cells increased by 20%-90% compared with the original red blood cells, and even increased by 100%, that is, increased by about 1 time (for the original 2) Double
  • fetal hemoglobin expression is also increased by 20%-90% compared to the original red blood cell, or even up to 100%, that is, about 1 times higher (2 times the original), even up to 200% , that is, about 2 times higher (3 times as much as the original), even up to 300%, that is, about 3 times higher (4 times the original), even up to 400%, that is, about 4 times higher (5 times the original) ), even up to 500% or more, that is, about 5 times or more (more than 6 times the original) and the like.
  • Example 1 Efficient gene editing of cord blood-derived CD34-positive hematopoietic stem cells
  • the cancer cell line K562 (purchased from ATCC Corporation, website: https://www.atcc.org) was selected as a model cell line for testing electroporation conditions.
  • the first batch of experiments electroporation 5 ⁇ 10 5 K562 cells, GFP mRNA (sequence shown in SEQ ID No. 1) amount of 5 ⁇ g, selected BTX830 electro-transformation instrument, respectively tested at 250V 1ms, 360V 1ms, 400V 1ms, 500V Under the condition of 1 ms, the expression of GFP and 7-AAD were detected by flow cytometry after 4 days, GFP was the electrotransfer efficiency, and 7-AAD was the viability of the cells after electroporation.
  • SEQ ID No. 1 Sequence information of GFP mRNA:
  • the second batch of experiments electroporation 5 ⁇ 10 5 K562 cells, the amount of GFP mRNA above was 5 ⁇ g, using BTX830 electro-transformation instrument, tested at 250V 1ms, 250V 2ms, 300V 0.5ms, 300V 1ms, 360V 0.5ms, 360V1ms, Four days later, flow cytometry analysis detected GFP expression and 7-AAD expression, GFP indicates electrotransfer efficiency, and 7-AAD indicates cell growth state or viability after electroporation.
  • the third batch of experiments electroporation 5 ⁇ 10 5 K562 cells, the amount of GFP mRNA above was 5 ⁇ g, using BTX830 electro-transformation instrument, testing at 250V 1ms, 250V 1ms, 300V 1ms, 300V 1ms, 4 days after flow analysis GFP expression and 7-AAD expression, GFP indicates electrotransfer efficiency, and 7-AAD indicates cell growth state or viability after electroporation.
  • the results are shown in Figures 2 and 3.
  • FIG. 2 shows a fluorescence microscope photograph taken after 4 days of electroporation under optimal electrotransfer conditions in a multi-batch experimental assay on a cancer cell line K562.
  • V refers to the pulse voltage
  • ms refers to the pulse time.
  • FIG. 3 shows the statistical analysis of 7-AAD and GFP expression in flow cytometry after 4 days of electroporation in the optimal electroporation conditions of multi-batch experiments on the cancer cell line K562.
  • the 7-AAD negative representative in the flow analysis Cell viability, 7-AAD (7-amino-actinomycin D) is a nucleic acid dye that does not pass through the normal plasma membrane. With the process of apoptosis and cell death, the plasma membrane is transparent to 7-AAD. The gradual increase in sex, can emit bright red fluorescence under the excitation of suitable wavelength excitation light, 7-AAD negative is normal viable cells; GFP efficiency indicates electrotransfer efficiency. Among them, taking "250-1" as an example, it represents 250V voltage, 1ms pulse time.
  • FIG. 4 is a fluorescence micrograph of the GFP mRNA after entering the hematopoietic stem cells for 4 days under 300V, 1ms electroporation conditions, including bright field, green channel, red channel and bright field green channel superimposed four fields of view.
  • Figure 5 shows the flow-through analysis of GPF and CD34 protein expression after electroporation of GFP mRNA into hematopoietic stem cells for 4 days under 300V, 1ms electroporation.
  • the control group in Fig. 5 was a hematopoietic stem cell, but no GFP mRNA was transferred, and the CD34 antibody was not stained by flow cytometry.
  • SEQ ID NO: 2 CL11A enhancer 58K site 150 bp sequence:
  • SEQ ID NO: 3 sgRNA (also sometimes referred to as enhancer-1) designated as enhancer-1 of BCL11A:
  • SEQ ID NO: 4 sgRNA (also sometimes referred to as enhancer-2) designated as enhancer-2 of BCL11A:
  • SEQ ID NO: 5 sgRNA (also sometimes referred to as enhancer-3) designated as enhancer-3 of BCL11A:
  • SEQ ID NO: 6 sgRNA (also sometimes referred to simply as enhancer-4) designated as enhancer-4 of BCL11A:
  • SEQ ID NO: 7 sgRNA (also sometimes referred to as enhancer-5) designated as enhancer-5 of BCL11A:
  • SEQ ID NO: 8 sgRNA (also sometimes referred to as enhancer-6) designated as enhancer-6 of BCL11A:
  • SEQ ID NO: 9 sgRNA (also sometimes referred to as enhancer-7) designated as enhancer-7 of BCL11A:
  • SEQ ID NO: 10 sgRNA (also sometimes referred to as enhancer-8) designated as enhancer-8 of BCL11A:
  • SEQ ID NO: 11 sgRNA (also sometimes referred to as enhancer-9) designated as enhancer-9 of BCL11A:
  • SEQ ID NO: 12 sgRNA (also sometimes referred to as enhancer-10) designated as enhancer-10 of BCL11A:
  • SEQ ID NO: 13 sgRNA (also sometimes referred to as enhancer-11) designated as enhancer-11 of BCL11A:
  • SEQ ID NO: 14 sgRNA (also sometimes referred to as enhancer-12) designated as enhancer-12 of BCL11A:
  • SEQ ID NO: 15 sgRNA (also sometimes referred to as enhancer-13) designated as enhancer-13 of BCL11A:
  • SEQ ID NO: 16 sgRNA (also sometimes referred to as enhancer-14) designated as enhancer-14 of BCL11A:
  • SEQ ID NO: 17 sgRNA (also sometimes referred to as enhancer-15) designated as enhancer-15 of BCL11A:
  • SEQ ID NO: 18 sgRNA (also sometimes referred to as enhancer-16) designated as enhancer-16 of BCL11A:
  • SEQ ID NO: 19 sgRNA (also sometimes referred to as enhancer-17) designated as enhancer-17 of BCL11A:
  • SEQ ID NO: 20 sgRNA (also sometimes referred to as enhancer-18) designated as enhancer-18 of BCL11A:
  • SEQ ID NO: 21 sgRNA (also sometimes referred to as enhancer-19) designated as enhancer-19 of BCL11A:
  • SEQ ID NO:22 sgRNA (also sometimes referred to as enhancer-20) designated as enhancer-20 of BCL11A:
  • SEQ ID NO: 23 sgRNA (also sometimes referred to simply as enhance-21) designated as enhancer-21 of BCL11A:
  • SEQ ID NO:24 sgRNA (also sometimes referred to as enhancer-22) designated as enhancer-22 of BCL11A:
  • SEQ ID NO:25 sgRNA (also sometimes referred to as enhancer-23) designated as enhancer-23 of BCL11A:
  • the above 23 sgRNAs were designed for the 150 bp sequence of the enhancer 58K site of BCL11A.
  • the electroporation conditions of 300 V for 1 ms were selected.
  • the inventors synthesized Cas9 mRNA (the sequence is shown in SEQ ID No. 26) and 23 sgRNAs of the above design chemically modified.
  • the chemical modification of 23 sgRNAs refers to the modification of the 2'-O-methyl analog and the internucleotide 3 for the first three bases of the 5' end of the sgRNA and the last three bases of the 3' end. 'Sulfur modification.
  • the left side is a chemically modified sgRNA
  • the right side is an unmodified sgRNA.
  • CD23-positive hematopoietic stem cells derived from cord blood (purchased from Aussell Biotech (Shanghai) Co., Ltd., www.allcells.com) were electroporated under the above-mentioned determined electroporation conditions, and the above 23 chemically modified sgRNAs were electroporated, 4 days later.
  • the Indels efficiency was analyzed by TIDE. The results are shown in Fig. 9.
  • the unschemically modified sgRNA was also electroporated by the same method, but the indels efficiency of the chemically modified sgRNA was only 2.7%, used in the following examples are chemically modified sgRNAs.
  • Figure 9 shows the electrophoresis of Cas9 mRNA and 23 sgRNA on CD34-positive hematopoietic stem cells.
  • the genome of the CD34-positive hematopoietic stem cells was extracted 4 days later, and a fragment of about 450 bp and a total length of 903 bp was amplified from the sgRNA cleavage site.
  • the sequence of the primer sequence used for amplification is shown below.
  • Reverse primer gggaagctccaaactctcaa (SEQ ID NO: 30)
  • Cutting site selection Take Enhancer-2 as an example, 5 '-ctaacagttg cttttatcac-3', and the cleavage site is to the right of the 3' end (Cong L, et al. Science. 2013).
  • SEQ ID NO:26 Cas9 mRNA sequence
  • Enhancer-2, Enhancer-3, Enhancer-4, Enhancer-5 and Enhancer-6 with relatively high gene editing efficiency were selected, and Cas9 mRNA and Enhancer-2 and Enhancer were electroporated at 300V 1ms.
  • Enhancer-4, Enhancer-5, and Enhancer-6 were tested for Indels efficiency after entering cord blood-derived hematopoietic stem cells for 4 days, and the results are shown in FIG.
  • Figure 10 shows electroporation of Cas9 mRNA and BCL11A enhancer-2 sgRNA, Enhancer-3 sgRNA, Enhancer-4 sgRNA, Enhancer-5 sgRNA, and Enhancer-6 sgRNA into three different cord blood-derived CD34-positive hematopoietic stem cells, 4 days later Statistical analysis of the efficiency of Indels was analyzed using TIDE software in the same manner as described above.
  • Example 2 Gene editing in vitro clonal formation of hematopoietic stem cells derived from cord blood
  • This experiment involves the detection of cloned formation units (CFU, colony-formation units) of gene-edited cord blood stem cells.
  • the electroporation conditions of 300V 1ms were selected, and Cas9 mRNA and Enhancer-2 were electroporated into cord blood-derived hematopoietic stem cells, and 800-1000 cells were resuspended in 1 ml of H4434 (purchased from STEM CELLS TECHNOLOGIES, Canada) and IMDM (purchased from Thermo Fisher).
  • H4434 purchased from STEM CELLS TECHNOLOGIES, Canada
  • IMDM purchased from Thermo Fisher
  • FBS purchased from Thermo Fisher
  • the number of clones of different forms such as CFU-M, BFU-E, CFU-E, CFU-G, CFU-GM, and GEMM was observed under microscope after 14 days. The results are shown in Fig. 11. Shown.
  • Figure 11 shows that the electrospread Cas9 mRNA and BCL11A enhancer-2 sgRNA enters cord blood-derived CD34-positive hematopoietic stem cells, and in vitro clone formation experiments (CFU test) after 2 days, and the number of clones of different blood systems are counted after 14 days, BFU- E, CFU-M, CFU-GM, CFU-E, CFU-G, CFU-MM represent the clonal formation of different lineages of the erythroid, myeloid, lymphatic and other blood systems.
  • Mock represents cells that have not been genetically edited.
  • the gene-edited cell function has a normal in vitro differentiation function compared to hematopoietic stem cells that have not been genetically edited, and is capable of differentiating clones of different blood system lineages.
  • Example 3 Gene editing cord blood stem cell-derived hematopoietic stem cell reconstruction mouse model hematopoietic system
  • the electroporation conditions of 300V 1ms were selected, and the Cas9 mRNA and Enhancer-2 were transferred into cord blood-derived hematopoietic stem cells and transplanted into the NGF immunodeficient mouse model irradiated by the irradiator (purchased from Beijing Vitalstar Biotechnology Co., Ltd.). , Inc.).
  • the expression of human CD45 and mouse CD45 was detected in peripheral blood at 6 weeks, 8 weeks, 10 weeks, 12 weeks, and 16 weeks after transplantation, and human CD45 and bone marrow and spleen were detected at 16 weeks after transplantation.
  • the expression of mouse CD45 was shown in Fig. 12 and Fig. 14.
  • the method of transplantation into mice was as follows: 1.0 Gy irradiation was performed 24 hours before cell transplantation to remove the bone marrow of the mouse model. 1.0 ⁇ 10 6 cells resuspended in 20 ⁇ L of 0.9% physiological saline were injected into the tail vein of the mouse, and then placed in a clean-grade animal house.
  • Figures 12 and 14 show that after genetically engineered hematopoietic stem cells, after transplantation into a mouse model, the genetically modified cells prolonged over time, and the proportion of human hCD45 expression persisted compared with unedited hematopoietic stem cells. Increased, in peripheral blood samples, increased from 20% in week 6 to 60% in 16 weeks, the proportion of bone marrow in 16 weeks even reached 90%, and the spleen reached 70%, indicating that genetically edited hematopoietic stem cells can be fast and efficient.
  • the hematopoietic system of the mouse model is implanted, and the differentiation function of the cells in vivo is normal.
  • cell membrane proteins such as human CD3, CD4, CD8, CD33, CD19, and CD56 was detected in mice transplanted with the gene-edited cells as shown in Figs. 13, 14, and 15 after 16 weeks.
  • the results showed that the genetically modified cells can express these proteins normally, indicating that they can differentiate into cells of the blood system such as T cells, B cells, macrophages, etc., and can efficiently reconstruct the hematopoietic system of the mouse model.
  • we first tested six cell membrane surface proteins such as CD3, CD4, CD8, CD33, CD19 and hCD56 to determine the expression of T cells, myeloid cells, B cells and NK cells in the blood of mice.
  • Fig. 16 The results of the gene editing of the cells reconstructed in the mouse model are shown in Fig. 16.
  • the results showed that the human cells of peripheral blood, bone marrow and spleen were genetically edited after 16 weeks of transplantation, and the efficiency was similar to that of pre-transplant cells, between 50 and 70%.
  • Example 4 Gene editing umbilical cord blood-derived hematopoietic stem cell erythroid differentiation assay gamma globin and Fetal hemoglobin expression
  • the electroporation conditions of 300v 1ms were selected, and Cas9 mRNA and Enhancer-2 were electroporated into cord blood-derived hematopoietic stem cells, and differentiated by the following two-step differentiation protocol.
  • the two-step method differentiates into differentiation using a hematopoietic stem cell erythroid expansion and differentiation medium, and then differentiates using a hematopoietic stem cell erythroid differentiation denucleation medium.
  • Erythroid hematopoietic stem cell proliferation and differentiation medium is a basal medium StemSpan TM SFEM II, growth factor 50-200ng / ml SCF, 10-100ng / ml IL-3,1-10U EPO / ml, culture conditions: using Hematopoietic stem cell erythroid expansion and differentiation medium The hematopoietic stem cells were cultured at 1.0 ⁇ 10 5 cells/ml and expanded for 7 days.
  • Erythroid differentiation of hematopoietic stem cells denucleation medium is basal medium STEMSPAN TM SFEM II, growth factor 1-10U EPO, 100-1000 ⁇ g / ml human transferrin, small chemical molecule is 0.5-10 ⁇ m mifepristone, to the use of a
  • STEMSPAN TM SFEM II basal medium
  • growth factor 1-10U EPO growth factor 1-10U EPO
  • 100-1000 ⁇ g / ml human transferrin 100-1000 ⁇ g / ml human transferrin
  • small chemical molecule is 0.5-10 ⁇ m mifepristone
  • CD71 and CD235a expression was detected as shown in FIG. Both the experimental group and the control group could efficiently differentiate into red blood cells, and the expression ratios of CD71 and CD235a were all above 90%.
  • the mRNA of the cells differentiated from the above red blood cells was extracted, reverse transcribed into cDNA, and the expression of genes such as BCL11A, HBB, HBG and the like were detected by real-time PCR, as shown in FIG.
  • the results showed that the expression of BCL11A gene in the BCL11A enhancher locus was down-regulated by 1 fold and the expression of HBG was doubled.
  • Example 5 Gene editing BCL11A of hematopoietic stem cells derived from ⁇ -thalassemia patients Enhancer site
  • CD34-positive hematopoietic stem cells were obtained by conventional magnetic bead sorting. The result is shown in FIG.
  • the electroporation conditions of 300V 1ms were used to convert the Cas9 mRNA and Enhancer-2, Enhancer-3, Enhancer-4, Enhancer-5 and Enhancer-6 into the peripheral blood-derived hematopoietic stem cells of patients with ⁇ -thalassemia.
  • the Indels efficiency was detected after 4 days.
  • Figure 21 It is indicated that 5 sgRNAs can achieve efficient gene editing on hematopoietic stem cells derived from peripheral blood of 3 different patients.
  • Enhancer-2 is the most efficient, reaching at least 70%.
  • Example 6 Gene editing anemia patients peripheral blood-derived hematopoietic stem cells erythroid differentiation detection ⁇ Embryo and fetal hemoglobin expression
  • Example 5 The gene-edited hematopoietic stem cells of Example 5 were subjected to in vitro erythroid differentiation, and the method is shown in Example 4-1. The results of differentiation are shown in Figure 22. The results showed that the control group, Enhancer-2, Enhancer-3, Enhancer-4, Enhancer-5 and Enhancer-6 erythroids had similar differentiation efficiency, and both expressed CD71 and CD235a cell membrane proteins.
  • Enhancer-2, Enhancer-3, Enhancer-4 and Enhancer-5 could down-regulate the expression of BCL11A gene in cells except Enhancer-6, and the Enhancer-2 effect was most significantly down-regulated by about 1 time.
  • Enhancer- 2, Enhancer-3, Enhancer-4, Enhancer-5 and Enhancer-6 can increase the expression of gamma globin, the most significant of which is Enhancer-2, gamma globin expression increased by 9 times, in line with clinical treatment standards.
  • HbF fetal hemoglobin
  • HbA normal hemoglobin
  • HPLC test High Performance Liquid Chromatography, high performance liquid chromatography
  • HbF The expression of HbF in the cells edited by Enhancer-2, Enhancer-3, Enhancer-4, Enhancer-5 and Enhancer-6 genes is higher (higher peak and larger peak area) than HbA
  • the lower expression level indicates that the gene editing BCL11A enhancer site increases HbF expression and down-regulates HbA expression.
  • the ratio of HbF/HbA of Enhancer-2 is about 5.28
  • the ratio of HbF/HbA of mock is about 0.79, which is 6.68 times higher, which is significantly higher than Enhancer-3, Enhancer-4, Enhancer-5 and Ratio ratio of Enhancer-6 to HbF/HbA.
  • hemoglobin in the blood is composed of a small amount of HbA and a large proportion of HbF due to the deletion of ⁇ -globin, and the hemoglobin of a patient with severe ⁇ -thalassaemia is about 20g / L, wherein the proportion of HbF is more than 90%.
  • the range of normal human hemoglobin expression is described in patients with severe beta thalassemia.
  • the present invention is the only invention on the hematopoietic stem cells of patients with beta thalassemia, which accurately evaluates the effectiveness of the gene editing BCL11A enhancer in improving fetal hemoglobin by HPLC experiments, and the results clearly improve the fetal hemoglobin.
  • Expression meeting the clinical requirements for treating patients with severe beta thalassemia (Chang et al. Methods & Clinical Development. 2017; Mattew C et al. Nature. 2015; Lin Ye, et al. PNAS. 2016; Matthew H. Porteus, Advances in Experimental Medicine and Biology. 2013).
  • Example 7 In vitro cloning of hematopoietic stem cells derived from peripheral blood of patients with gene editing anemia
  • the experimental data shows that compared with hematopoietic stem cells that have not been genetically edited, the gene-edited cell function has normal differentiation function in vitro and can differentiate into clones of different blood system lineages.
  • Example 8 Off-target effect of peripheral blood-derived hematopoietic stem cells from genetically edited anemia patients
  • This experiment involves gene sequencing methods, specifically analyzing the off-target effect of peripheral blood-derived hematopoietic stem cells from genetically edited anemia patients by second generation sequencing (NGS).
  • NGS second generation sequencing
  • the experimental data showed that the on-target efficiency of gene editing was 76%, and the ratio of 14 potential off-target sites was less than 0.3%, which was equivalent to the error of the second-generation sequencing itself. Within the scope, we did not detect off-targeting due to genetic editing, so the gene editing protocol is safe.
  • Enhancer-2 showed the best effect against cells of different origins.
  • the method of the present invention has the following advantages. Firstly, the method can genetically edit hematopoietic stem cells derived from thalassemia patients, fully satisfying the requirements for clinical treatment of thalassemia and sickle cell anemia; secondly, by using chemically modified sgRNA, gene editing efficiency, significantly improve fetal hemoglobin expression, cells can reconstitute the hematopoietic system of model mice; finally, off-target analysis shows high safety. Based on this, the method developed by the present invention will likely replace the traditional hematopoietic stem cell transplantation therapy to cure patients with severe thalassemia and sickle cell anemia.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Immunology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Diabetes (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

提供一种基因编辑造血干细胞BCL11A的增强子位点的方法。基因编辑改造后的造血干细胞具有正常细胞功能，能显著提高胎儿血红蛋白的表达，用于β-地中海贫血和镰刀红细胞贫血的治疗。

Description

一种提高胎儿血红蛋白表达水平的方法
交叉引用
本申请要求享有2017年10月27日提交的中国专利申请2017110277086的优先权，该中国专利申请的公开内容以其整体通过引用并入本申请。
技术领域
本发明涉及用于治疗地中海贫血和镰刀形红细胞贫血等贫血疾病的细胞治疗方案，其包含利用基因编辑技术，高效安全地基因修饰人的造血干细胞的BCL11A的增强子位点，上调γ珠蛋白和胎儿血红蛋白的表达，实现治疗疾病的目的。
背景技术
地中海贫血又称为珠蛋白生成障碍贫血或者海洋性贫血，是一组由于基因缺陷等遗传因素导致的溶血性贫血类疾病。遗传性基因缺陷致使血红蛋白中一种或几种珠蛋白肽链合成缺失或者不足，从而导致贫血或者溶血的病理状态。合成血红蛋白的珠蛋白链减少或缺失会导致血红蛋白结构异常，这种含有异常血红蛋白的红细胞变形性降低，寿命减短，在骨髓中可能出现原位溶血，进入到外周血液循环后被脾脏等脏器提前破坏，致使贫血、体内铁沉积甚至发育异常。由于不同珠蛋白链基因突变的多样性和复杂性，使缺失的珠蛋白类型、数量及临床症状变异性非常大。
地中海贫血根据所缺失的珠蛋白链种类及缺乏程度予以命名和分类。按照不同类型珠蛋白肽链生成障碍可分为α型、β型、δ型、δβ型。目前地中海贫血是全球最普遍的基因缺陷遗传性疾病之一。据统计，4.83％的全球人口携带珠蛋白突变基因，其中包括1.67％的α、β地中海贫血杂合子，同时包含1.92％携带镰刀形细胞突变的血红蛋白，0.95％携带血红蛋白E，0.29％携带血红蛋白C等。由此产生的有疾病症状的血红蛋白异常的全球人口出生率不少于0.024％，是一种常见的基因缺陷型疾病。
β地中海贫血(以下简称β地贫)是地中海贫血的一种，其发病机理是因为β珠蛋白肽链发生基因突变，多数病人是点突变，少数是大片段基因缺失。基因缺失和某些点突变可以导致一部分β珠蛋白肽链的合成完全被抑制，这类称为β 0地贫；而少数的点突变使β链的合成部分受到抑制，仍保留一部分肽链合成，这类称为β +地贫，不同的组合可能出现不同的临床症状。β地贫基因突变类型非常多，据统计全球约有300多种基因异常，已发现的突变点有100多种，目前国内临床报道的有28种。其中常见的突变有6种，β41-42(-TCTT缺失)，约占45％，IVS-II654(C到T点突变)，约占24％，β17(A到T点突变)，约占14％，TATA盒子-28(A到T点突变)，约占9％。Β71-72(+A插入突变)，约占2％，β26(G到A点突变)约占2％等。
重型β地贫的突变基因类型有两种，其一是β纯合子，契尔氏β 0与β +地贫双重杂合子，因为β珠蛋白肽链生成近于完全受到抑制，体内无法产生β珠蛋白链，由α链和β链组成的正常的血红蛋白A合成减少或消失。虽然多余的α链蛋白能与红细胞内γ链结合形成血红蛋白F，但是由于出生后，γ合成逐渐受到抑制(又称为血红蛋白链合成切换)，因此，多余的α链会沉积在红细胞内形成包涵体，附着在红细胞膜表面上，使红细胞膜特性发生改变，细胞变僵硬而导致变形能力下降，在骨髓中发生被破坏导致“无效造血”。部分地中海贫血的红细胞虽然能够在骨髓中生长发育成熟，并最终被释放到外周血循环中，但当它们通过外周局部微循环(如脾脏等脏器)时，就会因为变形能力下降，容易发生机械破坏。由于上述原因，患儿在临床上出现出生时无症状，出生后由于HbF(胎儿血红蛋白)表达而且红细胞寿命长达120天，往往当6个月后由于γ链合成被生理性抑制，血红蛋白链合成切换为β链，同时由于基因缺陷无法合成β链，细胞发生病理改变引起破坏增加，呈现慢性溶血性贫血表现，进一步导致骨髓构成改变。治疗过程中需反复输血，导致含铁血黄素沉着症，影响重要脏器功能。
镰刀形红细胞贫血，与β-地中海贫血类似，都属于一种常染色体隐性遗传病，不同的是该贫血症突变位点单一，由于β珠蛋白的单一碱基突变，正常的β基因的第6位密码子由GAG(编码谷氨酸)突变为GTG(缬氨酸)所致。在突变纯合子状态，正常的α珠蛋白和异常的β珠蛋白形成四聚体复合物，称为HbS,该四聚体携带氧气的能力是正常血红蛋白的一半，在脱氧状态下 聚集成多聚体，因形成的多聚体排列方向与膜平行，紧密接触细胞膜，当多聚体数量达到一定程度后，细胞膜由正常的凹形变成镰刀形。镰刀形红细胞，变形性差，容易破碎而发生溶血，造成血管堵塞，损伤，坏死等。
β-地中海贫血和镰刀形红细胞贫血的治疗方法包括：一般支持治疗、高剂量输血及规律去铁治疗、造血干细胞移植、诱导胎儿血红蛋白药物治疗和探索性的基因治疗。目前，所有治疗方案中唯一有治愈希望的方法是异基因造血干细胞移植，自1981年托马斯进行了首例地贫患者造血干细胞移植并取得成功后，造血干细胞移植技术在全球多个地贫研究中心开展并成功取代了经典的输血和去铁的治疗方案。然而，由于HLA全相合供者严重缺乏和移植后GVHD(graft-versus-host disease，移植物抗宿主反应)导致的死亡，依旧限制着造血干细胞移植治疗地中海贫血的广发应用。与此同时，一直以来研究者们在持续探索治疗地中海贫血的药物，目前FDA唯一批准用于临床治疗的口服药物是羟基脲，主要通过诱导胎儿血红蛋白表达缓解疾病临床症状，但由于该药物的临床疗效不一致且存在很大的副作用，以及剂量相关的骨髓抑制，亟待开发新的治疗方法治疗β-地中海贫血和镰刀形红细胞贫血等相关贫血疾病。
在本说明书的整篇文本中引用了数篇文件。此处的每篇文件(包括任何期刊文章或摘要、公开或未公开的专利申请、授权专利、制造商的说明书、使用说明等)通过提述并入本文。然而，并非认可此处引用的文件事实上是本发明的现有技术。
发明内容
本发明利用基因编辑技术，例如CRISPR/Cas9基因编辑技术，开发出了新一代的造血干细胞，该造血干细胞与现有技术的造血干细胞相比，对BCL11A增强子基因敲除的效率大大增加，从而使分化成熟后的红细胞胎儿血红蛋白的表达大大提升，一定程度上解决了现有技术中BCL11A增强子编辑效率低，胎儿血红蛋白表达量不能满足临床应用的问题。另外，本发明也进一步改善了对基因编辑后的造血干细胞的培养和分化策略，不仅大大缩短了从造血干细胞到成熟红细胞的分化进程，同时也使收获的成熟红细胞的数量大大增加，从而部分满足了临床应用的要求。
具体地本发明涉及如下各项：
1.一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：
通过基因编辑技术破坏所述造血干细胞中2号染色体第60495219位至第60495336位的BCL11A基因组区域。
2.项1所述的方法，其中所述基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术。
3.项2所述的方法，其中所述基因编辑技术为CRISPR/Cas9基因编辑技术。
4.项1-3中任一项所述的方法，其中所述BCL11A基因组靶核苷酸序列与选自SEQ ID No.3～SEQ ID No.25任一的序列互补。
5.项3或4的方法，将包含选自SEQ ID NO:3～SEQ ID NO:25任一序列的sgRNA导入所述造血干细胞以实现对所述BCL11A基因组的编辑。
6.项5的方法，其中所述sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的。
7.项6的方法，其中所述化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。
8.项3-7中任一项的方法，其中将所述sgRNA与Cas9编码核苷酸共同导入造血干细胞。
9.项8的方法，其中通过电转方法将sgRNA与Cas9编码核苷酸共同导入造血干细胞。
10.项9的方法，其中所述电转条件为200-600V，0.5ms-2ms。
11.一种通过CRISPR/Cas9系统体外高效编辑造血干细胞的方法，包括将含有选自SEQ ID NO：3～SEQ ID NO：25任一序列的sgRNA导入所述造血干细胞，其中该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的。
12.项11的方法，其中将所述sgRNA与Cas9编码核苷酸共同导入造血干细胞。
13.项12的方法，其中通过电转方法将sgRNA与Cas9编码核苷酸共同导入造血干细胞。
14.项13的方法，其中所述电转条件为200-600V，0.5ms-2ms。
15.通过项1～14中任一项所述的方法得到的造血干细胞。
16.一种通过基因改造使胎儿血红蛋白(HbF)表达升高的人造血干细胞，其中该造血干细胞中染色体2上第60495219位至第60495336位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。
17.通过分化培养项15或16所述的造血干细胞获得的、处于成熟红细胞之前的不同分化阶段的前体细胞。
18.通过分化培养项15或16所述的造血干细胞获得的成熟红细胞。
19.一种制造通过基因改造使胎儿血红蛋白(HbF)表达升高的成熟红细胞或其前体细胞的方法，该方法包括：
(a)使用项1-14中任一项所述的方法得到基因改造的造血干细胞；和
(b)使用造血干细胞红系扩增和分化培养基对所述基因改造的造血干细胞进行造血干细胞红系扩增和分化，
其中，所述造血干细胞红系扩增和分化培养基包括基础培养基，以及生长因子的组合物，其中所述生长因子的组合物包括干细胞生长因子(SCF)；白介素3(IL-3)和促红细胞生成素(EPO)。
20.根据项19所述的方法，该方法还包括：
使用红系分化脱核培养基进行造血干细胞红系分化脱核，
所述红系分化脱核培养基包含基础培养基、生长因子、以及孕酮受体和糖皮质激素受体的拮抗剂和/或抑制剂。
21.根据项20所述的方法，其中所述红系分化脱核培养基中的生长因子包括促红细胞生成素(EPO)，所述孕酮受体和糖皮质激素受体的拮抗剂和/或抑制剂为选自下述化合物(I)～(IV)中的任一种或两种及以上：
Figure PCTCN2018112068-appb-000001
Figure PCTCN2018112068-appb-000002
22.通过项19-21任意一项获得的成熟红细胞或其前体细胞。
23.一种组合物，包含项15或16的造血干细胞，或项17或22的前体细胞，或项18或22的成熟红细胞。
24.包含项15或16的造血干细胞，或项17或22的前体细胞，或项18或22的成熟红细胞的医用制品。
25.项15或16的造血干细胞，或项17或22的前体细胞，或项18或22的成熟红细胞在预防或治疗有需要的受试者的疾病中的用途。
26.项25的用途，所述疾病为贫血性疾病、失血性疾病、肿瘤或其它需要大量输血进行预防或治疗的疾病。
27.根据项26的用途，其中所述疾病为β-地中海贫血或镰刀形红细胞贫血。
28.项25-27任一项的用途，其中所述受试者为人。
29.项15或16的造血干细胞，或项17或22的前体细胞，或项18或22的成熟红细胞在制备预防或治疗受试者疾病中的药物或医用制品中的用途。
30.项29的用途，所述疾病为贫血性疾病、失血性疾病、肿瘤或其它需要大量输血进行预防或治疗的疾病。
31.根据项30的用途，其中所述疾病为β-地中海贫血或镰刀形红细胞贫血。
32.项29-31中任一项的用途，其中所述受试者为人。
33.一种sgRNA构建体，包含选自SEQ ID NO：3～SEQ ID NO：25之一的核苷酸序列。
34.项33的构建体，其包含2’-O-甲基类似物和/或核苷酸间3’硫代修饰。
35.项34的构建体，其中所述化学修饰为在选自SEQ ID NO：3～SEQ ID  NO：25之一的核苷酸序列的5’端的前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。
36.包含项33-35中任一项的构建体的载体、宿主细胞或制剂。
37.包含项33-35中任一项的构建体在基因编辑造血干细胞中的用途。
38.一种治疗或预防受试者贫血性疾病、失血性疾病、肿瘤或其它需要大量输血进行预防或治疗的疾病的方法，其包括给药受试者项15或16的造血干细胞，项17或22的前体细胞，或项18或22的成熟红细胞。
39.根据项38所述的方法，其中，所述疾病为β-地中海贫血或镰刀形红细胞贫血。
40.根据项39所述的方法，其中，所述受试者为人。
41.一种用于治疗或预防受试者贫血性疾病、失血性疾病、肿瘤或其它需要大量输血进行预防或治疗的疾病的试剂盒，包含项33-35中任一项的sgRNA的构建体，或项36中的载体。
42.项41所述的试剂盒，还包含Cas9 mRNA。
发明效果
本发明通过优化电转体系，基因编辑正常供体和贫血病人的造血干细胞的BCL11A的增强子位点，满足临床治疗的需求；且编辑的造血干细胞向红系分化能显著提升γ珠蛋白和胎儿血红蛋白(HbF)的表达，以及重建动物模型的造血系统；脱靶分析显示本方法安全性高，几乎检测不到基因编辑所引起的副作用。
附图简述
图1基因编辑自体造血干细胞治疗β-地中海贫血和镰刀红细胞贫血的治疗路线。从病人体内动员外周血，通过体外分离获得CD34阳性的造血干细胞，体外经过基因编辑，提高胎儿血红蛋白表达，将经过基因修饰的自体造血干细胞回输病人。
图2在癌症细胞系K562上多批次实验检测最佳电转条件，电转GFP 4天后的荧光显微镜拍照图。“V”指脉冲电压，“ms”指脉冲时间。
图3在癌症细胞系K562上多批次实验检测最佳电转条件，电转GFP 4 天后，流式分析7-AAD和GFP表达统计分析图，以7-AAD表示细胞活率，7-AAD(7-氨基-放线菌素D)是一种核酸染料，它不能通过正常质膜，随着细胞凋亡、细胞死亡过程，质膜对7-AAD的通透性逐渐增加，在合适波长的激发光的激发下可发出明亮的红色荧光，7-AAD阴性为正常活力细胞；用GFP表示电转效率。“250-1”表示250V电压，1ms脉冲时间，“250-1-1”，“250-1-2”分别表示250V 1ms,两个重复；“300-1-1”，“300-1-2”分别表示300v 1ms,两个重复。
图4在300V,1ms电转条件下，电转GFP mRNA进入造血干细胞4天后的荧光显微镜拍照图，分别包括明场、绿色通道、红色通道和明场绿色通道叠加四个视野。
图5在300V,1ms电转条件下，电转GFP mRNA进入造血干细胞4天后流式分析GPF和CD34蛋白表达情况。
图6针对人BCL11A的增强子58K位置设计的多条sgRNA的示意图。
图7针对人BCL11A的增强子58K位点的150bp序列信息。
图8针对人BCL11A的增强子58K位点150bp的位置设计的23条sgRNA的具体DNA序列信息。
图9在CD34阳性造血干细胞上，电转Cas9 mRNA和多条sgRNA 4天后提取基因组，扩增片段和Sanger测序，TIDE软件分析产生Indels效率的统计分析。
图10电转Cas9 mRNA和BCL11A的增强子-2、3、4、5和6 sgRNA(即图中的Enhancer-2、3、4、5和6)进入3个不同脐带血来源的CD34阳性造血干细胞，4天后，TIDE软件分析产生Indels效率的统计分析。
图11电转Cas9 mRNA和BCL11A的增强子-2 sgRNA进入脐带血来源的CD34阳性造血干细胞，2天后进行体外克隆形成实验(CFU检测)，14天后统计不同血液系统的克隆数目，BFU-E、CFU-M、CFU-GM、CFU-E、CFU-G、CFU-MM代表红系、髓系、淋巴系等血液系统不同谱系的克隆形成。其中，Mock:代表未经过基因编辑的细胞。
图12电转Cas9 mRNA和BCL11A的增强子-2 sgRNA进入造血干细胞，同时移植经过基因修饰和未经过基因修饰的细胞进入经过辐照仪照射的NPG免疫缺陷小鼠模型，6周、8周、10周、12周、16周后，在小鼠外周血检测 人CD45阳性细胞的比例，同时移植16周后在小鼠骨髓和脾脏中检测人CD45阳性细胞的比例，其中CD45阳性细胞的比例的计算方式为人CD45阳性细胞％/(人CD45阳性细胞％+小鼠CD45阳性细胞％)，人CD45阳性细胞％和小鼠CD45阳性细胞％分别是通过流式分析实验测得的结果。Mock:代表未经过基因编辑的细胞。Enhancer-2代表经过基因编辑的细胞。
图13电转Cas9 mRNA和BCL11A的增强子-2 sgRNA进入脐带血来源的造血干细胞，同时移植经过基因修饰和未经过基因修饰的细胞进入经过辐照仪照射的NPG免疫缺陷小鼠模型，16周后分别在小鼠骨髓、脾脏、外周血中检测CD3、CD4、CD8、CD33、CD56、CD19等人细胞膜蛋白占人CD45蛋白的比例。Mock:代表未经过基因编辑的细胞。Enhancer-2代表经过基因编辑的细胞。
图14电转Cas9 mRNA和BCL11A的增强子-2 sgRNA进入脐带血来源的造血干细胞，移植经过基因修饰细胞进入经过辐照仪照射的NPG免疫缺陷小鼠模型，16周后分别在小鼠骨髓、脾脏、外周血中，流式分析检测小鼠CD45、人CD45、CD3、CD4、CD8的表达结果。SSC-H通道代表侧向角散射，其代表细胞的颗粒度，值越大代表细胞的颗粒度越大，颗粒度指细胞表面的皱折度，细胞内亚细胞器、颗粒的数目等。
图15电转Cas9 mRNA和BCL11A的增强子-2 sgRNA进入脐带血来源的造血干细胞，移植经过基因修饰细胞进入经过辐照仪照射的NPG免疫缺陷小鼠模型，16周后分别在小鼠骨髓、脾脏、外周血中，流式分析检测一只小鼠的人CD33、CD56、CD49。SSC-H通道代表侧向角散射，其代表细胞的颗粒度，值越大代表细胞的颗粒度越大，颗粒度指细胞表面的皱折度，细胞内亚细胞器、颗粒的数目等。
图16电转Cas9 mRNA和BCL11A的增强子-2 sgRNA进入脐带血来源的造血干细胞，提取移植前细胞和移植后16周的外周血、骨髓、脾脏的基因组，扩增目的片段和Sanger测序，通过TIDE软件分析基因编辑Indels效率。
图17电转Cas9 mRNA和BCL11A的增强子-2 sgRNA进入脐带血来源的造血干细胞，进行红细胞分化，12天后检测。A图代表分化后的红细胞拍照图；B图代表检测人CD71和人235a两个膜蛋白表达，表示红系分化效率。Mock:代表未经过基因编辑的细胞。Enhancer-2代表经过基因编辑的细胞。
图18电转Cas9 mRNA和BCL11A的增强子-2 sgRNA进入脐带血来源的造血干细胞，进行红细胞分化，12天后通过荧光定量PCR检测BCL11A、HBB、HBG等基因mRNA表达。Mock:代表未经过基因编辑的细胞。Enhancer-2代表经过基因编辑的细胞。
图19电转6μg Cas9 mRNA和4ug BCL11A的增强子-2 sgRNA进入脐带血来源的造血干细胞向红细胞分化12天后的细胞内胎儿血红蛋白HbF的表达情况。左侧是流式分析图，右侧是胎儿血红蛋白表达统计分析图。Mock:代表未经过基因编辑的细胞。Enhancer-2代表经过基因编辑的细胞。
图20检测新鲜分离的β地中海贫血病人的外周血中CD45和CD34的表达，左侧是对照组，右侧是实验组。实验样品分别来自3名β-地中海贫血病人。
图21电转Cas9 mRNA和BCL11A的增强子-2、3、4、5和6 sgRNA(即图中的Enhancer-2、3、4、5和6)进入3个不同β地中海贫血病人的外周血分离的CD34阳性的造血干细胞，4天后，TIDE软件分析产生Indels效率的统计分析。
图22电转电转Cas9 mRNA和BCL11A的增强子-2 sgRNA进入β地中海贫血病人的外周血分离的CD34阳性的造血干细胞，提取基因组，NGS深度测序分析14个潜在的脱靶位点统计图。
图23电转Cas9 mRNA和BCL11A的增强子-2、3、4、5和6 sgRNA(即图中的Enhancer-2、3、4、5和6)进入β地中海贫血病人来源的造血干细胞，进行红细胞分化，12天后检测。A图代表分化后的红细胞拍照图；B图代表检测人CD71和人235a两个膜蛋白表达，表示红系分化效率。Mock:代表未经过基因编辑的细胞。Enhancer-2、3、4、5和6代表经过基因编辑的细胞。
图24电转电转Cas9 mRNA和BCL11A的增强子-2、3、4、5和6 sgRNA(即图中的Enhancer-2、3、4、5和6)进入β地中海贫血病人的外周血分离的CD34阳性的造血干细胞，红细胞分化12天后，检测BCL11A和γ-珠蛋白的基因表达。Mock:代表未经过基因编辑的细胞。Enhancer-2、3、4、5和6代表经过基因编辑的细胞。
图25电转Cas9 mRNA和BCL11A的增强子-2 sgRNA进入β地中海贫血病人来源的CD34阳性的造血干细胞，2天后进行体外克隆形成实验(CFU 检测)，14天后统计不同血液系统的克隆数目，BFU-E、CFU-M、CFU-GM、CFU-E、CFU-G、CFU-MM代表红系、髓系、淋巴系等血液系统不同谱系的克隆形成。
图26电转Cas9 mRNA和BCL11A的增强子-3(Enhancer-3)、Enhancer-4或Enhancer-5 sgRNA进入β地中海贫血病人来源的CD34阳性的造血干细胞，评估基因编辑效率，BCL11A基因表达和γ-珠蛋白的基因表达。A)电转4天后，TIDE软件分析不同sgRNAs产生Indels效率的统计分析；B)电转造血干细胞后，进行红细胞分化，12天后检测BCL11A基因表达。C)电转造血干细胞后，进行红细胞分化，12天后检测γ-珠蛋白基因表达。Mock:代表未经过基因编辑的细胞。Enhancer-3,Enhancer-4,Enhancer-5代表经过Enhancer-3,Enhancer-4,Enhancer-5 sgRNA导入后基因编辑的细胞。
图27显示代表未经过基因编辑的细胞(Mock)和经过基因编辑的细胞(利用增强子2sg RNA编辑的)后的色谱结果，显示HbF和HbA的表达量。
图28显示代表未经过基因编辑的细胞(Mock)和经过基因编辑的细胞(利用增强子3、4、5、以及6sg RNA编辑的)后的色谱结果，显示HbF和HbA的表达量。
图29显示根据图27和图28中的色谱结果进行计算后的HbF和HbA表达量的比值。
具体实施方式
本申请涉及对CD34阳性造血干细胞的BCL11A增强子位点进行基因编辑，通过优化CRISPR/Cas系统的各项条件，例如sgRNA和转化条件等，获得BCL11A增强子被高效编辑的造血干细胞。实验结果证明，本发明获得的造血干细胞体外红系分化和动物模型体内重建造血系统的功能正常，并通过脱靶分析证明其同时具备安全性，达到临床治疗标准。
本发明提供用于治疗或预防贫血性疾病、失血性疾病、肿瘤或其它需要大量输血进行预防或治疗的疾病(如β-地中海贫血和镰刀红细胞贫血)的满足临床治疗级别的基因编辑后的造血干细胞、红细胞前体、或成熟红细胞。
本发明还提供用于基因编辑造血干细胞所需的sgRNA(如经过化学修饰的sgRNA)，其包含选自SEQ ID NOs:3-25任一的核苷酸序列。
本发明还提供用于β-地中海贫血和镰刀红细胞贫血患者的造血干细胞的编辑方法。
根据本发明的目的之一是基于CRISPR/Cas基因编辑技术，高效基因修饰CD34 +造血干细胞。例如可以从脐带血或骨髓中获得CD34阳性的造血干细胞，并将Cas9和给定的sgRNA，通过优化的电转条件导入造血干细胞，对造血干细胞进行基因编辑，并将基因编辑后的造血干细胞移植进入实验动物以评价该造血干细胞重建造血系统的能力。所述sgRNA可以通过多种设计软件，例如“CRISPR RGEN TOOLS”软件，针对BCL11A的增强子，例如针对BCL11A(+58)位点进行设计。在一些实施方案中，对设计的sgRNA进行化学修饰，例如在其5’和3’末端3个碱基处的2’-O-甲基类似物修饰和核苷酸间的3’硫代修饰。可以通过测试Cas9 mRNA和不同sgRNA的组合，获取高效的sgRNA。在某些实施方案中，所述的造血干细胞是通过使用临床级别的磁柱分选获得，用于基因编辑的Cas9蛋白编码核苷酸以及经过化学修饰的sgRNA是经过体外转录实验获得。将基因编辑后的造血干细胞向红系分化，检测红细胞生成比例，例如，可以将基因编辑后的造血干细胞移植进入经过辐照仪照射的NPG小鼠，评价重建造血系统的能力，也可以分离病人的CD34阳性的造血干细胞，评价基因编辑效率、红系分化能力、γ珠蛋白和胎儿血红蛋白的表达。
首先，本发明提供一种提高胎儿血红蛋白(HbF)表达的方法，包括：通过CRISPR/Cas9编辑技术基因编辑造血干细胞BCL11A的增强子位点。编辑过程针对造血干细胞BCL11A的增强子位点靶向序列设计sgRNA。BCL11A的增强子根据其转录起始位点的距离(kilo-base数)存在命名为+62、+58、+55的位点，有报道称BCL11A基因的红系增强子能够负向调控胎儿血红蛋白(HbF)表达，其中距离转录起始位点55kb(kb表示1000个碱基)，58kb、62kb位置是关键的调控区域。虽然已经有研究人员针对+55、+58、+62位点进行研究，但是上述三个位点前后的基因序列均在1000bp以上，总共约6000bp，因此具体编辑哪些区域可以实现理想的编辑效果本领域技术人员并不知晓，即使针对一个+58位点而言，基因编辑效率也差别很大(参见：Bauer,D.E.et al.An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level.Science.342,253–257(2013)，以及Canver MC,et al. BCL11A的增强子dissection by Cas9-mediated in situ saturating mutagenesis.Nature.2015 Nov 12；527(7577):192-7.)。因此，寻找到对编辑效率至关重要的具体区域是本领域技术人员首先面临的问题。
本申请的发明人通过深入研究发现+58位置中150bp的碱基序列(例如，如SEQ ID NO:2所示的)对基因编辑的效率影响很大，而通过本发明sgRNA靶向的区域是实现高效的、能达到临床应用的可编辑区域。该150bp的基因组序列位于人类第2号染色体第60495197位至第60495346位的区域(在本文中，简写为chr2:60495197-60495346)。
本发明的sgRNA均能高效实现基因编辑正常供体和贫血病人来源的造血干细胞，其中相对效率更高的候选的sgRNA均能显著提高胎儿血红蛋白的表达。有文献报道通过针对+55、+58、+62位点设计sgRNAs文库，利用CRISPR/Cas9进行细胞模型上的筛选，发现靶向+58处中“GATAA”位点是关键的调控序列，即包含”GATAA”序列的sgRNA所导致的胎儿血红蛋白提升效果最明显。然而，值得注意的是，本发明中发现的sgRNA锚定的+58k附近的区域与现有技术中公开的关键的“GATAA”碱基位点是不同的。通过本发明sgRNA导入的基因编辑对胎儿血红蛋白的表达起到了显著的提升作用，足以应用于临床治疗。
本领域技术人员可以理解，在获知高效基因编辑区域后，本领域技术人员可以用任何基因编辑方法，例如基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术和CRISPR/Cas(如CRISPR/Cas9)基因编辑技术，以及今后发现的其它基因编辑方法，对所获知的高效基因编辑区域进行编辑，优化基因编辑条件，实现高效编辑的目的。因此，本发明涵盖通过任何可利用的基因编辑方法对本发明所鉴定的选自序列3-25的BCL11A基因靶序列的技术方案。在一些优选实施方案中，本发明涉及通过CRISPR/Cas9基因编辑技术实现高效基因编辑的技术方案。
如本申请使用的，“CRISPR/Cas”是一种基因编辑技术，包括但不限于各种自然存在或人工设计的CRISPR/Cas系统,如CRISPR/Cas9系统。自然存在的CRISPR/Cas系统(Naturally occurring CRISPR/Cas system)是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御，可用来对抗入侵的病毒及外源DNA。例如，CRISPR/Cas9的工作原理是crRNA(CRISPR-derived RNA) 通过碱基配对与tracrRNA(trans-activating RNA)结合形成tracrRNA/crRNA复合物，此复合物引导核酸酶Cas9蛋白在与crRNA配对的序列靶位点剪切双链DNA。而通过人工设计tracrRNA和crRNA，可以改造形成具有引导作用的sgRNA(single guide RNA)，足以引导Cas9对DNA的定点切割。作为一种RNA导向的dsDNA结合蛋白，Cas9效应物核酸酶能够共定位RNA、DNA和蛋白，从而拥有巨大的改造潜力。CRISPR/Cas系统可使用一类，二类或三类Cas蛋白。本发明的一些实施方式中，所述方法使用Cas9。其他适用的CRISPR/Cas系统包括但不限于WO2013176772，WO2014065596，WO2014018423，US8,697,359中所描述的系统和方法。
本发明的另一方面，涉及可实现高效基因编辑的本发明的一系列sgRNA分子。
本发明中，“sgRNA(single guide RNA)”和“gRNA(guide RNA)”或可以是“单指导RNA”、“合成的指导RNA”或“指导RNA”可互换使用。本发明的sgRNA包含靶向目标序列的指导序列。在优选实施方案中，本发明的sgRNA进一步包含tracr序列和tracr伴侣序列。
本发明中的“指导序列”(guide sequence)可以是指定靶向位点的约17-20bp的序列，且可与“引导序列”或“间隔子”互换使用。在形成CRISPR复合物的背景下，“靶序列”例如是指导序列经设计以与其具有互补性的序列，其中靶序列和指导序列间的杂交促进CRISPR复合物的形成，所述杂交要求“靶序列”和“指导序列”或称“引导序列”有足够的互补性，能引起杂交并促进CRISPR复合物形成即可，完全互补不是必须的。
“互补”是指“指导序列”或称“引导序列”与靶核苷酸序列(就本发明而言为造血干细胞中BCL11A基因组靶核苷酸序列)可以通过沃森和克里克发现的核苷酸配对原则杂交。本领域技术人员可以理解，只要具有足够的互补性，“指导序列”便可与靶核苷酸序列杂交，而不需要它们之间具有100％的完全互补。在一些实施方案中，当使用适当的比对算法最佳比对时，指导序列及其相应靶序列间的互补程度可为约或大于约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多。最佳比对可使用用于比对序列的任何适当的算法确定，包括Smith-Waterman算法、Needleman-Wimsch算法、基于Burrows-Wheeler Transform的算法等。
通常，在内源CRISPR系统的背景下，CRISPR复合物的形成(包括指导序列与靶序列杂交并与一种或多种Cas蛋白复合)导致靶序列中或靶序列附近(例如，距离靶序列1、2、3、4、5、6、7、8、9、10、20、50或更多碱基对的范围内)的一条链或两条链的切割。不希望受理论所限，tracr序列可包含野生型tracr序列的全部或其一部分，例如野生型tracr序列约或大于约20、23、26、29、32、35、38、41、44、47、50、53、56、59、62、65、70、75、80、85或更多个核苷酸)或由上述组成的tracr序列还可形成CRISPR复合物的一部分，例如通过沿tracr序列的至少一部分与指导序列可操作连接的tracr伴侣序列的全部或一部分杂交。
在一些实施方案中，tracr序列与tracr伴侣序列具有足够的互补性以杂交并参与CRISPR复合物的形成。与“靶序列”和“指导序列”或称“引导序列”杂交的情况类似，完全互补并不是必须的，只要足以发挥其功能即可。在一些实施方案中，在最佳对齐的情况下，tracr序列沿tracr伴侣序列的长度具有至少50％、60％、70％、80％、90％、95％或99％的互补性。
本申请中所指的造血干细胞中2号染色体第60495219位至第60495336位的BCL11A基因组区域是根据GRCh38标准人类基因序列中的位置定义的。本领域技术人员应明白该定位参照不同的标准人类基因组序列，可能有所不同，但本领域技术人员能够明白参照不同的标准人类基因序列时所述区域相应的位置。在一些实施例中，所述破坏造血干细胞中2号染色体第60495219位至第60495336位的BCL11A基因组区域，包括在此区域引入核苷酸序列的“添加和/或删除(indel)”，例如引入任何类型(例如选自A、T、C、G)和/或数量(例如1-20、1-15、1-10、1-9、1-8、1-7、1-6、1-5、1-4、1-3、1-2个)的核苷酸的indel。在一些实施例中，所述的破坏包括用新的核苷酸序列置换原有的核苷酸序列。在一些实施例中，所述的破坏包括在所述区域敲入(knock-in)或敲除(knock-out)一段核苷酸序列。
在本申请中，造血干细胞BCL11A位点靶向序列如SEQ ID NO：2所示。本申请针对SEQ ID NO：2所示的150bp碱基区域设计sgRNA，要求sgRNA序列互补于SEQ ID NO：2序列的至少17个，优选18个，优选19个，或优选20个连续核苷酸的序列。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白 (HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495203位至第60495222位，尤其是第60495219位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:9的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：9的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495203位至第60495222位，尤其是第60495219位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495208位至第60495227位，尤其是第60495224位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:10的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：10的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体 2上第60495208位至第60495227位，尤其是第60495224位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495217位至第60495236位，尤其是第60495233位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:11的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：11的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495217位至第60495236位，尤其是第60495233位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495218位至第60495237位，尤其是第60495234位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:12的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：12的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同 导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495218位至第60495237位，尤其是第60495234位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495219位至第60495238位，尤其是第60495235位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:13的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：13的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495219位至第60495238位，尤其是第60495235位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495221位至第60495240位，尤其是第60495223位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:16的sgRNA的引导 序列互补。本发明方法涉及将包含SEQ ID NO：16的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495221位至第60495240位，尤其是第60495223位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495222位至第60495241位，尤其是第60495238位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:14的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：14的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495222位至第60495241位，尤其是第60495238位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色 体第60495223位至第60495242位，尤其是第60495239位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:15的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：15的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495223位至第60495242位，尤其是第60495239位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495228位至第60495247位，尤其是第60495244位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:17的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：17的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495228位至第60495247位，尤其是第60495244位的BCL11A基因 组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495229位至第60495248位，尤其是第60495245位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:18的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：18的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495229位至第60495248位，尤其是第60495245位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495230位至第60495249位，尤其是第60495246位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:19的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：19的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同 导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495230位至第60495249位，尤其是第60495246位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495231位至第60495250位，尤其是第60495247位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:20的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：20的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495231位至第60495250位，尤其是第60495247位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495234位至第60495253位，尤其是第60495250位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:21的sgRNA的引导 序列互补。本发明方法涉及将包含SEQ ID NO：21的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495234位至第60495253位，尤其是第60495250位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495235位至第60495254位，尤其是第60495251位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:22的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：22的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495235位至第60495254位，尤其是第60495251位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色 体第60495236位至第60495255位，尤其是第60495238位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:4的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：4的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495236位至第60495255位，尤其是第60495238位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495247位至第60495266位，尤其是第60495263位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:3的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：3的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495247位至第60495266位，尤其是第60495263位的BCL11A基因 组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495252位至第60495271位，尤其是第60495268位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:8的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：8的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495252位至第60495271位，尤其是第60495268位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495253位至第60495272位，尤其是第60495269位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:7的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：7的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似 物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495253位至第60495272位，尤其是第60495269位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495257位至第60495276位，尤其是第60495273位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:6的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：6的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495257位至第60495276位，尤其是第60495273位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495264位至第60495283位，尤其是第60495280位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:5的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：5的序列的sgRNA导入造血 干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495264位至第60495283位，尤其是第60495280位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495299位至第60495318位，尤其是第60495301位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:24的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：24的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495299位至第60495318位，尤其是第60495301位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495319位至第60495338位，尤其是第60495335位的BCL11A基因组 区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:25的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：25的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495319位至第60495338位，尤其是第60495335位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
在一些实施方式中，本发明提供一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：通过基因编辑技术破坏所述造血干细胞中2号染色体第60495320位至第60495339位，尤其是第60495336位的BCL11A基因组区域。该基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，优选为CRISPR/Cas9基因编辑技术，优选所述BCL11A基因组靶核苷酸序列与包含SEQ ID NO:23的sgRNA的引导序列互补。本发明方法涉及将包含SEQ ID NO：23的序列的sgRNA导入造血干细胞以实现对所述BCL11A基因组的编辑，优选将所述sgRNA与Cas9编码核苷酸(如mRNA)共同导入所述造血干细胞，优选通过电转条件为200-600V，0.5ms-2ms的电转方法来将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。在一些实施方案中，该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的，比如，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。在一些实施方案中，本发明还涉及一种造血干细胞，该造血干细胞中染色体2上第60495320位至第60495339位，尤其是第60495336位的BCL11A基因组靶序列中的一个或多个位点通过基因编辑技术被破坏。进一步，涉及通过 体外分化培养该造血干细胞后得到的红细胞，以及包含该红细胞的医用制品。
进一步，在本发明中，发明人设计的23条sgRNA均能有效产生Indels，即可以高效地进行基因编辑，本发明优选的sgRNA选自SEQ ID NO：3～SEQ ID NO：25中任一个，所述sgRNA对应的靶序列的切割位点的序列位于chr2:60495219～chr2:60495336(即染色体2上的第60495219位至第60495336之间)。
具体来说，下表列出了本发明涉及的23条sg RNA所靶向的人染色体2上的基因组序列位置，以及每条sgRNA引发的Cas9切割位点。
名称 人类基因组序列上的位置 切割位点
BCL11A的增强子-7 chr2:60495203-60495222 chr2:60495219
BCL11A的增强子-8 chr2:60495208-60495227 chr2:60495224
BCL11A的增强子-9 chr2:60495217-60495236 chr2:60495233
BCL11A的增强子-10 chr2:60495218-60495237 chr2:60495234
BCL11A的增强子-11 chr2:60495219-60495238 chr2:60495235
BCL11A的增强子-14 chr2:60495221-60495240 chr2:60495223
BCL11A的增强子-12 chr2:60495222-60495241 chr2:60495238
BCL11A的增强子-13 chr2:60495223-60495242 chr2:60495239
BCL11A的增强子-15 chr2:60495228-60495247 chr2:60495244
BCL11A的增强子-16 chr2:60495229-60495248 chr2:60495245
BCL11A的增强子-17 chr2:60495230-60495249 chr2:60495246
BCL11A的增强子-18 chr2:60495231-60495250 chr2:60495247
BCL11A的增强子-19 chr2:60495234-60495253 chr2:60495250
BCL11A的增强子-20 chr2:60495235-60495254 chr2:60495251
BCL11A的增强子-2 chr2:60495236-60495255 chr2:60495238
BCL11A的增强子-1 chr2:60495247-60495266 chr2:60495263
BCL11A的增强子-6 chr2:60495252-60495271 chr2:60495268
BCL11A的增强子-5 chr2:60495253-60495272 chr2:60495269
BCL11A的增强子-4 chr2:60495257-60495276 chr2:60495273
BCL11A的增强子-3 chr2:60495264-60495283 chr2:60495280
BCL11A的增强子-22 chr2:60495299-60495318 chr2:60495301
BCL11A的增强子-23 chr2:60495319-60495338 chr2:60495335
BCL11A的增强子-21 chr2:60495320-60495339 chr2:60495336
分析上述23条sg RNA的切割位点发现，这些sgRNA所引发的Cas9切割位置集中在BCL11A基因第60495219位至第60495336位基因组区域。
本发明的一个具体的实施方式还包括sgRNA的组合物，其包括本发明涉及的上述23条sgRNA或其载体。
一般而言，sgRNA中的指导序列是与靶多核苷酸序列具有足够的互补性以与靶序列杂交并指导CRISPR复合物与靶序列的序列特异性结合的任何多核苷酸序列。在一些实施方案中，当使用适当的比对算法最佳比对时，指导序列及其相应靶序列间的互补程度为约或大于约80％、85％、90％、95％、97.5％、99％或更多。最佳比对可使用用于比对序列的任何适当的算法确定，其非限制性实例包括Smith-Waterman算法、Needleman-Wimsch算法、基于Burrows-Wheeler Transform的算法(例如Burrows Wheeler Aligner)、ClustalW、Clustai X、BLAT、Novoalign(Novocraft Technologies,ELAND((Illumina,San Diego,CA)、SOAP(可在soap.genomics.org.cn获得)和Maq(可在maq.sourceforge.net获得)。在一些实施方案中，指导序列长度可以为约或大于约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或更多个核苷酸。在一些实施方案中，指导序列长度少于约75、70、65、60、55、50、45、40、35、30、25、20、15、12或更少的核苷酸。指导序列指导CR1SPR复合物与靶序列的序列特异性结合的能力可通过任何适当的测定方法评估。例如，可向具有相应靶序列的宿主细胞提供足以形成CRISPR复合物的CRISPR系统的组件(包括待测试的指导序列)，如可通过使用编码CRISPR序列组件的载体转染，随后评估靶序列内的优先切割(如通过如本文所述的Surveyor测定)来进行。同样地，靶多核苷酸序列的切割可在测试管中通过提供靶序列、CRISPR复合物(包含待测试的指导序列和不同于指导序列的对照指导序列)的组件，并比较测试和对照指导序列在靶序列的结合或切割率，以此进行评估。也可以使用本领域技术人员知道的其它测定方法进行上述测定和评估。
在本发明中，进行基因编辑过程中所使用的sgRNA优选是经过化学修饰的。“化学修饰的sgRNA”指针对sgRNA进行特殊的化学修饰，例如在其5’ 和3’末端3个碱基处的2’-O-甲基类似物修饰和/或核苷酸间的3’硫代修饰。
本发明人采用的经过化学修饰的sgRNA认为具有以下两个优点。第一、由于sgRNA是单链形式的RNA，其半衰期非常短，进入到细胞后，会迅速降解(最长不超过12小时)，而Cas9蛋白结合sgRNA发挥基因编辑作用则至少需要48hrs。因此，采用经过化学修饰的sgRNA，进入细胞后，稳定表达，与Cas9蛋白结合后，能高效基因编辑基因组，产生Indels。第二、未经修饰的sgRNA穿透细胞膜能力差，无法有效进入细胞或组织发挥相应功能。而经过了化学修饰的sgRNA穿透细胞膜的能力通常是增强的。在本发明中可以采用本领域中常用的化学修饰方法，只要能够提高sgRNA稳定性(延长半衰期)和提升进入细胞膜能力，均可以使用。除了实施例中使用的具体的化学修饰之外，还包括采用其它的修饰方法，例如，Deleavey GF1,Damha MJ.Designing chemically modified oligonucleotides for targeted gene silencing.Chem Biol.2012 Aug 24；19(8):937-54，以及Hendel et al.Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.Nat Biotechnol.2015 Sep；33(9):985-989文献中报道的化学修饰方法。
在一些实施方案中，所述sgRNA和/或Cas9编码核苷酸(如mRNA)通过电转导入造血干细胞，例如，通过250-360V，0.5-1ms；250-300V，0.5-1ms；250V 1ms；250V 2ms；300V 0.5ms；300V 1ms；360V 0.5ms；或360V1ms的电转条件导入造血干细胞。在一些实施方案中，所述sgRNA和Cas9编码核苷酸通过电转方式共同导入造血干细胞。在一些实施方案中，所述sgRNA通过电转方式导入表达Cas9的造血干细胞。
在一些实施方案中，所述Cas9编码核苷酸为mRNA，如含有ARCA帽的mRNA。在一些实施方案中，所述Cas9编码核苷酸在病毒载体中，如慢病毒载体。在一些实施方案中，所述Cas9编码核苷酸包含如SEQ ID NO：26所述的序列。在一些实施方案中，所述sgRNA与Cas9编码核苷酸在同一载体中。
本发明涉及利用本发明涉及的基因改造方法获得的造血干细胞，以及通过分化培养经所述基因改造得到的造血干细胞获得的、处于成熟红细胞之前的不同分化阶段的前体细胞，以及分化培养经所述基因改造得到的造血干细胞获得的成熟红细胞。
本发明涉及包含通过本发明的方法获得的药物组合物，其包括利用本发明涉及的基因改造方法获得的造血干细胞，或者通过分化培养经所述基因改造得到的造血干细胞获得的、处于成熟红细胞之前的不同分化阶段的前体细胞，或者分化培养经所述基因改造得到的造血干细胞获得的成熟红细胞。
本发明涉及的药物组合物可以通过给药包含细胞成分的医药制品常规使用的途径，例如静脉输注途径，给药有此需要的受试者。给药剂量可以基于受试者的病情和一般健康状况具体确定。
在一些方面，本发明提供向造血干细胞递送包含本发明所述sgRNA和/或Cas9编码核苷酸的方法。在一些实施方案中，所述递送可使用常规的病毒和不基于病毒的基因转移方法将所述构建体引入造血干细胞或其它宿主细胞。非病毒递送系统包括DNA质粒、RNA(例如本文所述的载体转录物)、裸核酸和脂质体。病毒载体递送系统包括DNA和RNA病毒，其具有用于递送至细胞的游离或整合的基因组。在一些实施方案中，发明人利用电转方法将Cas9和sgRNA的编码基因导入造血干细胞中对造血干细胞BCL11A基因的具体序列进行基因编辑。经过反复实验，发明人发现在200-600v，0.5ms-2ms的电转条件下将Cas9的编码核苷酸和sgRNA共同导入造血干细胞的基因编辑效率显著高于其它电转条件下的基因编辑效率。
在一些实施方案中，经过化学修饰的sgRNA与Cas9编码基因共同电转进入CD34+的造血干细胞，产生高效的基因编辑效率(以Indels％表示)。实施例中的数据显示，如果和Cas9 mRNA一起电转的是未经化学修饰的sgRNA，其Indels效率只有2.7％，远远低于电转经化学修饰的sgRNA时获得的Indels效率(效率至少为10％以上)。
如本申请使用的，“Indel”全称为插入/缺失,即插入和缺失突变。
如本文使用，“造血干细胞(hematopoietic stem and progenitor cells,HSPCs)”是发生各种血细胞的最原始的造血细胞。其主要特点是具有旺盛的增殖潜力、多向分化的能力和自我更新能力，因此，它不仅能分化、补充各种血细胞，还能通过自我更新保持干细胞的特性和数量。造血干细胞的分化程度和增殖能力不一，有异质性。多能造血干细胞最原始，先分化为定向多能造血干细胞，例如能生成粒系、红系、单核系和巨核-血小板系的髓系造血干细胞及能发生B淋巴细胞和T淋巴细胞的淋巴干细胞。这两类干细胞既保 持着造血干细胞基本特点，又略有分化，分别负责“骨髓成分”和淋巴细胞的发生，故称定向多能造血干细胞。它们进一步分化成为造血祖细胞，此细胞虽然也是原始的血细胞，但是它已丧失造血干细胞的许多基本特点，如已失去多向性分化能力，只能朝向一系或密切相关的二系细胞分化；失去了反复自我更新能力，而要依靠造血干细胞的增殖分化来补充数量；增殖潜力有限，只能分裂数次。根据造血祖细胞所能分化生成的血细胞系多少，又分为单能造血祖细胞(只分化为一个血细胞系)和寡能造血祖细胞(可分化为2～3个血细胞系)。本发明“造血干细胞”术语涵盖多能造血干细胞、定向多能造血干细胞和造血祖细胞，是具有不同异质性的造血干细胞的总称。
在本发明的具体的实施方式中，本发明使用的待进行基因编辑的造血干细胞(HSPCs)可来源于骨髓、脐带血或外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)。
如本申请使用的，“CRISPR RGEN”是由韩国科学家Jin-Soo Kim的科研团队开发的专门用于设计sgRNA的网站名称，其网址为www.rgenome.net/about/。
如本文使用，“TIDE”指专门用于分析Indels效率的工具性网站名称，其网址为tide-calculator.nki.nl。
如本文使用，“CD34、CD45RA、CD3、CD4、CD8、CD33、CD19、CD56、CD71、CD235a”是血液系统细胞的膜蛋白标志物。
如本文使用，“BCL11A”是一种转录因子，首先在小鼠中发现，作为逆转录病毒的结合位点，被命名为Evi9,后来在人类基因组中也发现这一基因，定位于2号染色体短臂2p13位点，主要在B淋巴细胞的生发中心表达。
如本文使用，“HBB/HBG”是血红蛋白的不同亚型。血红蛋白是高等生物体内负责运载氧气的一种蛋白质。血红蛋白由四条链组成，两条α链和两条β链，每一条链有一个包含一个铁原子的环状血红素。氧结合在铁原子上，由红细胞运输，供机体利用。
进一步，本发明还涉及一种造血干细胞，其是利用上述本发明的方法，通过CRISPR/Cas9编辑技术基因编辑造血干细胞BCL11A的具体序列之后得到的造血干细胞。此外，包含该造血干细胞的制品也在本发明的范围之内。
本发明涉及的造血干细胞或制品可以用于治疗选自贫血性疾病、失血性 疾病、肿瘤或其它需要大量输血进行治疗的疾病。尤其是可以用于治疗β-地中海贫血或镰刀形红细胞贫血。
进一步，本发明涉及一种红细胞，其是通过下述体外分化培养通过本发明获得的经过基因编辑的造血干细胞而获得的。
进一步，本发明涉及从造血干细胞到成熟红细胞之间各个分化阶段的前体细胞，其是通过下述造血干细胞红系扩增和分化步骤处理本发明获得的经过基因编辑的造血干细胞而获得的。
其中，上述体外分化培养包括：造血干细胞红系扩增和分化步骤；以及造血干细胞红系分化脱核步骤。
所述红系扩增和分化步骤使用造血干细胞红系扩增和分化培养基对造血干细胞进行培养。
所述红系分化脱核步骤使用红系分化脱核培养基。
在一些实施方案中，所述造血干细胞红系扩增和分化培养基包含：基础培养基，以及生长因子的组合物，其中，所述生长因子的组合物包括：干细胞生长因子，即SCF；白介素3，即IL-3；以及促红细胞生成素，即EPO。
在一些实施方案中，所述红系分化脱核培养基包含基础培养基、生长因子、以及孕酮受体和糖皮质激素受体的拮抗剂和/或抑制剂。
利用上述方法培养经基因编辑之后的造血干细胞，该造血干细胞可以通过两步向成熟的红细胞的分化，相比较现有技术，利用本发明的体外分化培养，只需14天，时间更短，相比于现有技术中必须要21天以上，该周期被大幅缩短。
在一些实施方案中，所述生长因子包括促红细胞生成素，即EPO，所述孕酮受体和糖皮质激素受体的拮抗剂和/或抑制剂为选自下述化合物(I)～(IV)中的任一种或两种及以上：
Figure PCTCN2018112068-appb-000003
Figure PCTCN2018112068-appb-000004
在一些实施方案中，所述造血干细胞红系扩增和分化培养基包含基础培养基，所述基础培养例如STEMSPAN TM SFEM II(STEM CELLS TECHNOLOGY Inc.),IMDM(Iscove's Modified Dulbecco's Medium),X-VIVO 15,alpha-MEM,RPMI 1640和DF12等。所述生长因子，例如基础培养基若选用STEMSPAN TM SFEM II，则需要额外添加的生长因子包括50-200ng/ml SCF,10-100ng/ml IL-3,1-10U/ml EPO，其中U的单位定义为：在特定的条件下，1min能转化1μmol底物的蛋白量，即1IU＝1μmol/min。目前国内外大多数临床实验室常省略国际二字，即将IU简写为U。EPO，促红细胞生成素，主要是由肾脏为了响应低氧或者贫血而分泌的糖蛋白，在本实施例中，能促进造血干细胞向分化。通常发挥作用时间是7天左右。
基础培养基若选用STEMSPAN TM SFEM II外其它的基础培养基，则需要添加有100X ITS(insuin-transferrin-selenium)(其中培养基中ITS中各物质的终浓度是：胰岛素浓度是0.1mg/ml、人转铁蛋白是0.0055mg/ml、硒元素6.7*10 -6mg/ml)(即主要包括胰岛素、人转铁蛋白以及硒元素),10-50μg/ml维生素C,0.5-5％BSA(Bovine serum albuin，牛血清白蛋白)，生长因子，例如50-200ng/ml SCF,10-100ng/ml IL-3,1-10U/ml EPO。
此外，本领域技术人员可以理解，任何常用的基础培养基均可以使用。例如可以列举本领域中常用的基础培养基，例如STEMSPAN TM SFEM II(购自STEM CELL TECHONOLOGIES)；例如购自Thermo Fisher的IMDM、DF12、Knockout DMEM、RPMI 1640、Alpha MEM、DMEM等。
此外，可以根据需要进一步在上述培养基中外加一些其他成分，例如可以外加ITS(即主要包括胰岛素、人转铁蛋白以及硒元素)、L-谷氨酰胺、维生素C以及牛血清白蛋白。例如可以在IMDM培养基中外加ITS、外加2mM L- 谷氨酰胺、外加10-50μg/ml维生素C以及0.5-5质量％的BSA(牛血清白蛋白)。此外，上述DF12可以外加同样浓度的ITS，L-谷氨酰胺，维生素C和牛血清白蛋白。Knockout DMEM可以外加同样浓度的ITS，L-谷氨酰胺，维生素C和牛血清白蛋白，RPMI 1640可以外加同样浓度的ITS，L-谷氨酰胺，维生素C和牛血清白蛋白，Alpha MEM可以外加同样浓度的ITS，L-谷氨酰胺，维生素C和牛血清白蛋白，DMEM也可以外加同样浓度的ITS，L-谷氨酰胺，维生素C和牛血清白蛋白。在此，各种基础培养基中外加的ITS的浓度可以是：胰岛素浓度是0.1mg/ml、人转铁蛋白是0.0055mg/ml、硒元素6.7×10 -6mg/ml。此外，外加的ITS各成分的浓度也可以根据实际需要来调整。ITS可以从Thermofisher购买，并根据需要调节成合适的最终使用浓度。
在一个实施方案中，所述红系分化脱核培养基包含基础培养基、生长因子和孕酮受体和糖皮质激素受体的拮抗剂。
在一个实施方案中，所述造血干细胞红系分化脱核培养基包含基础培养基，例如STEMSPAN TM SFEM II(STEM CELLS TECHNOLOGY Inc.),IMDM(Iscove's Modified Dulbecco's Medium),X-VIVO 15,alpha-MEM,RPMI 1640和DF12等。还包含生长因子，例如基础培养基若选用STEMSPAN TM SFEM II，则需要额外添加的生长因子包括1-10U/ml EPO,100-1000μg/ml human transferrin(人转铁蛋白)，化学小分子为0.5-10μmol//ml mifepristone。
基础培养基若选用STEMSPAN TM SFEM II外其它的基础培养基，则需要添加例如ITS(insuin-transferrin-selenium)(其中培养基中ITS中各物质的终浓度是：胰岛素浓度是0.1mg/ml、人转铁蛋白是0.0055mg/ml、硒元素6.7×10 -6mg/ml)(即主要包括胰岛素、人转铁蛋白以及硒元素),10-50ug/ml维生素C,0.5-5％BSA(Bovine serum albuin,牛血清白蛋白)，生长因子，例如1-10U/ml EPO，100-1000ug/ml人转铁蛋白，化学小分子例如0.5-10μmol//ml mifepristone。
此外，本领域技术人员可以理解，任何常用的基础培养基均可以使用。例如可以列举本领域中常用的基础培养基，例如STEMSPAN TM SFEM II(购自STEM CELL TECHONOLOGIES)；例如购自Thermo Fisher的IMDM、DF12、Knockout DMEM、RPMI 1640、Alpha MEM、DMEM等。
此外，可以根据需要进一步在上述培养基中外加一些其他成分，例如可 以外加ITS(即主要包括胰岛素、人转铁蛋白以及硒元素)、L-谷氨酰胺、维生素C以及牛血清白蛋白。例如可以在IMDM培养基中外加ITS、外加2mM L-谷氨酰胺、外加10-50μg/ml维生素C以及0.5-5质量％的BSA(牛血清白蛋白)。此外，上述DF12可以外加同样浓度的ITS，L-谷氨酰胺，维生素C和牛血清白蛋白。Knockout DMEM可以外加同样浓度的ITS，L-谷氨酰胺，维生素C和牛血清白蛋白，RPMI 1640可以外加同样浓度的ITS，L-谷氨酰胺，维生素C和牛血清白蛋白，Alpha MEM可以外加同样浓度的ITS，L-谷氨酰胺，维生素C和牛血清白蛋白，DMEM也可以外加同样浓度的ITS，L-谷氨酰胺，维生素C和牛血清白蛋白。在此，各种基础培养基中外加的ITS的浓度可以是：胰岛素浓度是0.1mg/ml、人转铁蛋白是0.0055mg/ml、硒元素6.7×10 -6mg/ml。此外，外加的ITS各成分的浓度也可以根据实际需要来调整。ITS可以从Thermofisher购买，并根据需要调节成合适的最终使用浓度。
本文中使用的mifepristone是一种化学合成的小分子，该小分子是孕酮受体和糖皮质激素受体的拮抗剂，结构式如下：
Figure PCTCN2018112068-appb-000005
其它孕酮受体和糖皮质激素受体的拮抗剂和抑制剂也可应用到本发明中，包括Cyproterone Acetate，Geldanamycin，CORT 108297等。化学结构式如下：
Figure PCTCN2018112068-appb-000006
Figure PCTCN2018112068-appb-000007
在一些实施方案中，本发明的成熟红细胞可通过包括下列a)-c)步骤的方法产生：a)使用本发明所述的任一方法对从人脐带血中分离CD34阳性的HSPCs进行基因改造；b)将基因改造后的HSPCs经过5-10天，例如5天、6天、7天、8天、9天、10天，扩增和分化后获得红细胞前体细胞，例如成红细胞(erythroblast)、有核红细胞、幼红细胞和网织红细胞，c)将红细胞前体细胞再经过7天的分化处理获得成熟的红细胞。
在一些实施方案中，本发明的成熟红细胞成熟的红细胞可通过包括下列步骤a)-d)的方法产生：
a)从人脐带血中通过磁珠分选的方法分离CD34阳性的HSPCs；
b)使用本发明所述的任一方法对所述的CD34阳性的HSPCs进行基因改造；
c)在无血清培养基(Serum-free medium(SFME))中，添加额外的生长因子，经过5-10天，例如5天、6天、7天、8天、9天、10天扩增和分化后，将HSPCs分化成为红细胞前体细胞，该阶段的培养基命名为造血干细胞红系扩增和分化培养基(HSPCs erythroid expansion and differentiatin medium；缩写为HEEDM)；
d)在无血清培养基(Serum-free medium(SFME))中，添加额外的生长因子，经过7天分化后，即可获得成熟的红细胞，该阶段的培养基命名为：造血干细胞红系分化脱核培养基(HSPCs erythroid differentiation enucleation medium,缩写为HEDEM)。
即针对经过编辑的CD34阳性的造血干细胞，利用上述本发明的方法可以通过两步就可以获得成熟的红细胞，整个过程的时间可以为10-18天、11-17 天、12-16天、13-15天、10-17天、10-16天、10-15天或10-14天，这个时间将现有技术中利用三步或者四步的方法中的至少21天的周期大大缩短。
如上所述，本发明涉及一种制造通过基因改造使胎儿血红蛋白(HbF)表达升高的成熟红细胞或其前体细胞的方法，该方法包括：(a)使用本发明的所涉及的基因改造方法得到造血干细胞；(b)使用上述造血干细胞红系扩增和分化培养基对基因改造过的造血干细胞进行造血干细胞红系扩增和分化。
进一步，本发明涉及一种制造通过基因改造使胎儿血红蛋白(HbF)表达升高的成熟红细胞或其前体细胞的方法，该方法包括：(a)使用本发明的所涉及的基因改造方法得到造血干细胞；(b)使用上述造血干细胞红系扩增和分化培养基对基因改造过的造血干细胞进行造血干细胞红系扩增和分化；以及(c)使用红系分化脱核培养基进行造血干细胞红系分化脱核。本发明还涉及上述方法中使用的造血干细胞红系扩增和分化培养基和/或红系分化脱核培养基在扩增和/或分化造血干细胞或成熟红细胞的前体细胞中的用途。
如本申请使用的，“分化”指其中细胞的结构或功能在分裂、增殖和其生长过程中特化的现象，即，生物体细胞或组织的特征和功能改变以实施给予细胞或组织的功能。一般而言，其指其中相对简单的系统分为两种或多种性质不同的部分系统的现象。
如本申请使用的，“Ficoll液密度梯度离心法”，其基本原理是血液中的不同成分的比重存在差异，在低速密度梯度离心时，可将不同的细胞层粉离开。红细胞和粒细胞密度大于分层液体，并且因红细胞遇到ficoll液之后会迅速凝集成串钱状而积于管底。唯有于分层液体密度相当的单个核细胞富集在血浆层和分层液之间，即白膜层，造血干细胞即存在于此层，通过后续磁珠分选即可获得。在本发明中，单个核细胞是利用商售的淋巴细胞分离管获得。
如本申请使用的，“成熟红细胞”是指血液中含量最多的一类细胞，具有携带氧气、氨基酸、二氧化碳等营养物质的功能。成熟的红细胞没有线粒体和细胞核。
如本申请使用的，“白介素3,IL-3”是指由激活的CD4和CD8阳性的T淋巴细胞产生的一种细胞因子，其主要生物学功能是参与调控骨髓中造血干细胞的增殖和分化。
如本申请使用的，“促红细胞生成素，Erythropoietin，EPO”是指 erythroblasts即红细胞前体细胞分泌产生的一种生长因子。在成体中，是有肾皮质肾小管周围间质细胞和肝脏分泌产生的糖蛋白。EPO能刺激造血干细胞分化形成成红细胞。
在一些实施方案中，干细胞因子，stem cell factor(SCF)包括肥大细胞生长因子(MGF)，Kit配体(KL)及Steel因子(SLF)。
如本申请使用的，“造血干细胞红系扩增和分化培养基”是指帮助HSPCs的细胞大量扩增和向红系祖细胞分化的配演体系。本发明中该培养基包含两种主要成分，分别是基础培养基和生长因子添加剂。基础培养基是无血清体系，可以是STEMSPAN TM SFEM II(STEM CELLS TECHNOLOGY Inc.),也可以是IMDM(Iscove's Modified Dulbecco's Medium)，外加ITS(Thermofisher)和L-gulutamin(Thermofisher)和维生素C和牛血清白蛋白。生长因子添加剂为IL-3,SCF,EPO的不同浓度的组合。
如本申请使用的，“造血干细胞红系分化脱核培养基”是指帮助红系祖细胞进一步扩增分化成为脱核的红细胞培养基。本发明中该培养基包含两种主要成分，分别是基础培养基和生长因子及化学小分子添加剂。基础培养基是无血清体系，可以是STEMSPAN TM SFEM II(STEM CELLS TECHNOLOGY Inc.),也可以是IMDM(Iscove's Modified Dulbecco's Medium)，外加ITS(Thermofisher)和L-gulutamin(Thermofisher)和维生素C和牛血清白蛋白。生长因子及化学小分子添加剂包括EPO和人转铁蛋白，以及化学小分子mifepristone。
在一些实施方案中，造血干细胞通过磁珠分选，例如把细胞用超级顺磁性的MACS MicroBeads(MACS微型磁珠)特异性地标记，磁性标记完后，把这些细胞通过一个放在强而稳定磁场中的分选柱。分选柱里的基质造成一个高梯度磁场。
被磁性标记的细胞滞留在柱里而未被标记的细胞则流出。当分选柱移出磁场后，滞留柱内的磁性标记细胞就可以被洗脱出来，这样就完全可以获得标记和未标记的两个细胞组份。
在本发明的一些具体的实施方式中，利用本发明的所述的sgRNA，通过本发明描述的基因改造方法，对3个不同的脐带血来源的造血干细胞进行改造，可以实现高效基因编辑，效率达到至少40％以上，例如50％以上、60％ 以上、70％以上、80％以上、或90％以上。例如，甚至于针对于优选的sgRNA，平均基因编辑效率达到80％。
在本发明的一些具体实施方式中，向小鼠移植了经本发明基因编辑方法得到的造血干细胞，与移植了未经过基因编辑的造血干细胞相比，在移植了经基因编辑的造血干细胞之后，小鼠人源的hCD45表达比例持续增高，在外周血样品中，由第6周的20％提高到16周的60％，16周的骨髓中比例甚至达到90％，脾脏达到70％,表明经过基因编辑的造血干细胞能够快速、高效地植入小鼠模型的造血系统，细胞体内分化功能正常。
在本发明的一些具体实施方式中，利用本发明的基因编辑方法对脐带血来源的造血干细胞进行改造，然后利用本发明的“两步法”分化方法进行分化，即利用造血干细胞红系扩增和分化培养基进行扩增和分化，然后利用造血干细胞红系分化脱核培养基进行进一步分化。通过检测显示，分化后的红细胞其血红蛋白(hemoglobin，HBG)表达相比于原始的红细胞的HBG表达提高了20％-90％、甚至于提高了100％，即提高约1倍(为原始的2倍)，胎儿血红蛋白表达相比于原始的红细胞的胎儿血红蛋白的表达也提高了20％-90％、甚至于高达100％，即提高约1倍(为原始的2倍)，甚至于高达200％，即提高约2倍(为原始的3倍)，甚至于高达300％，即提高约3倍(为原始的4倍)，甚至于高达400％，即提高约4倍(为原始的5倍)，甚至于高达500％以上，即提高约5倍以上(为原始的6倍以上)等。
实施例
此后，本发明将参照实施例进一步说明。对本领域的技术人员显而易见的是这些实施例仅出于说明目的而不应理解为对本发明保护范围的限制。因此，本发明的实质性保护范围将通过所附权利要求和其等同物限定。
实施例1：高效基因编辑脐带血来源的CD34阳性的造血干细胞
1-1:利用K562细胞测试电转条件
由于造血干细胞来源少且单次分离成本高，因此在本实施例中选择癌症细胞系K562(购买自ATCC公司，网址：https://www.atcc.org)作为测试电转条件的模型细胞系。
具体地，以下为具体实施步骤。
第一批实验：电转5×10 5个K562细胞，GFP mRNA(序列如SEQ ID No.1所示)的量为5μg,选用BTX830电转仪器，分别测试在250V 1ms,360V 1ms,400V 1ms,500V 1ms的条件下，4天后流式分析实验检测GFP表达和7-AAD表达，GFP表示电转效率，7-AAD表示电转后细胞的生长状态即活力(viability)。
SEQ ID No.1：GFP mRNA的序列信息：
Figure PCTCN2018112068-appb-000008
第二批实验：电转5×10 5K562细胞，上述GFP mRNA的量为5μg,选用BTX830电转仪器，测试分别在250V 1ms,250V 2ms,300V 0.5ms,300V 1ms，360V 0.5ms,360V1ms条件下，4天后流式分析实验检测GFP表达和7-AAD表达，GFP表示电转效率，7-AAD表示电转后细胞的生长状态即活力(viability)。
第三批实验：电转5×10 5K562细胞，上述GFP mRNA的量为5μg,选用BTX830电转仪器，测试分别在250V 1ms,250V 1ms,300V 1ms,300V 1ms条件下，4天后流式分析实验检测GFP表达和7-AAD表达，GFP表示电转效率，7-AAD表示电转后细胞的生长状态即活力(viability)。结果如图2和3所示。
其中，图2显示在癌症细胞系K562上进行多批次实验检测的最佳电转条件下，在电转GFP 4天后的荧光显微镜拍照图。“V”指脉冲电压，“ms”指脉冲时间。
图3在癌症细胞系K562上进行多批次实验检测的最佳电转条件下，在电转GFP 4天后，流式分析7-AAD和GFP表达统计分析图，流式分析中的 7-AAD阴性代表细胞活率，7-AAD(7-氨基-放线菌素D)是一种核酸染料，它不能通过正常质膜，随着细胞凋亡、细胞死亡过程，质膜对7-AAD的通透性逐渐增加，在合适波长激发光的激发下可发出明亮的红色荧光，7-AAD阴性为正常活力细胞；GFP效率表示电转效率。其中，以“250-1”为例，其表示250V电压，1ms脉冲时间。
本实验的数据表明针对癌症细胞系K562，在“300V 1ms”的电转条件下，电转效率和细胞活率的综合指标最高，在以后的实验中将作为后续实验的电转条件。
1-2.利用300v 1ms电转GFP mRNA进入造血干细胞
选取上一步骤中效率和活率最高的条件300V 1ms，向5×10 5造血干细胞(购买自澳赛尔斯生物技术(上海)有限公司,www.allcells.c)电转上述GFP mRNA，4天后检测GFP和CD34的表达情况。结果如图4、5所示。
其中，图4为在300V,1ms电转条件下，电转上述GFP mRNA进入造血干细胞4天后的荧光显微镜拍照图，分别包括明场、绿色通道、红色通道和明场绿色通道叠加四个视野。
图5为在300V,1ms电转条件下，电转GFP mRNA进入造血干细胞4天后流式分析GPF和CD34蛋白表达情况。其中图5中的对照组是造血干细胞，但是没有转入GFP mRNA,且流式分析不染CD34抗体的情况。
根据图4和5的结果可以看出，本实验的数据显示利用“300V 1ms”的电转条件，在脐带血来源的造血干细胞上GFP的表达比例高。
1-3:利用CRISPR/Cas9电转脐带血来源的造血干细胞来编辑BCL11A 位点
A、针对BCL11A的增强子58K位点(该目标58K位点的序列如SEQ ID NO:2所示)，利用“CRISPR RGEN TOOLS”软件设计针对BCL11A(+58)位点的多条sgRNA并合成经过化学修饰的sgRNA，信息如图6、7、8所示。
SEQ ID NO:2：CL11A enhancer 58K位点150bp序列：
Figure PCTCN2018112068-appb-000009
SEQ ID NO:3：名称为BCL11A的增强子-1的sgRNA(有时也简称为enhancer-1)：
Figure PCTCN2018112068-appb-000010
SEQ ID NO:4：名称为BCL11A的增强子-2的sgRNA(有时也简称为enhancer-2)：
Figure PCTCN2018112068-appb-000011
SEQ ID NO:5：名称为BCL11A的增强子-3的sgRNA(有时也简称为enhancer-3)：
Figure PCTCN2018112068-appb-000012
SEQ ID NO:6：名称为BCL11A的增强子-4的sgRNA(有时也简称为enhancer-4)：
Figure PCTCN2018112068-appb-000013
SEQ ID NO:7：名称为BCL11A的增强子-5的sgRNA(有时也简称为enhancer-5)：
Figure PCTCN2018112068-appb-000014
SEQ ID NO:8：名称为BCL11A的增强子-6的sgRNA(有时也简称为enhancer-6)：
Figure PCTCN2018112068-appb-000015
SEQ ID NO:9：名称为BCL11A的增强子-7的sgRNA(有时也简称为enhancer-7)：
Figure PCTCN2018112068-appb-000016
SEQ ID NO:10：名称为BCL11A的增强子-8的sgRNA(有时也简称为enhancer-8)：
Figure PCTCN2018112068-appb-000017
SEQ ID NO:11：名称为BCL11A的增强子-9的sgRNA(有时也简称为enhancer-9)：
Figure PCTCN2018112068-appb-000018
SEQ ID NO:12：名称为BCL11A的增强子-10的sgRNA(有时也简称为enhancer-10)：
Figure PCTCN2018112068-appb-000019
SEQ ID NO:13：名称为BCL11A的增强子-11的sgRNA(有时也简称为enhancer-11)：
Figure PCTCN2018112068-appb-000020
SEQ ID NO:14：名称为BCL11A的增强子-12的sgRNA(有时也简称为enhancer-12)：
Figure PCTCN2018112068-appb-000021
SEQ ID NO:15：名称为BCL11A的增强子-13的sgRNA(有时也简称为enhancer-13)：
Figure PCTCN2018112068-appb-000022
SEQ ID NO:16：名称为BCL11A的增强子-14的sgRNA(有时也简称为enhancer-14)：
Figure PCTCN2018112068-appb-000023
SEQ ID NO:17：名称为BCL11A的增强子-15的sgRNA(有时也简称为enhancer-15)：
Figure PCTCN2018112068-appb-000024
SEQ ID NO:18：名称为BCL11A的增强子-16的sgRNA(有时也简称为enhancer-16)：
Figure PCTCN2018112068-appb-000025
SEQ ID NO:19：名称为BCL11A的增强子-17的sgRNA(有时也简称为enhancer-17)：
Figure PCTCN2018112068-appb-000026
SEQ ID NO:20：名称为BCL11A的增强子-18的sgRNA(有时也简称为enhancer-18)：
Figure PCTCN2018112068-appb-000027
SEQ ID NO:21：名称为BCL11A的增强子-19的sgRNA(有时也简称为enhancer-19)：
Figure PCTCN2018112068-appb-000028
SEQ ID NO:22：名称为BCL11A的增强子-20的sgRNA(有时也简称为enhancer-20)：
Figure PCTCN2018112068-appb-000029
SEQ ID NO:23：名称为BCL11A的增强子-21的sgRNA(有时也简称为enhancer-21)：
Figure PCTCN2018112068-appb-000030
SEQ ID NO:24：名称为BCL11A的增强子-22的sgRNA(有时也简称为enhancer-22)：
Figure PCTCN2018112068-appb-000031
SEQ ID NO:25：名称为BCL11A的增强子-23的sgRNA(有时也简称为enhancer-23)：
Figure PCTCN2018112068-appb-000032
如上所述，针对BCL11A的增强子58K位点的150bp序列设计了上述23条sgRNA。
B、选取300V 1ms的电转条件，发明人合成了Cas9 mRNA(序列如SEQ ID No.26所示)和经过化学修饰的上述设计的23条sgRNA。其中，对于23条sgRNA的化学修饰是指对于sgRNA的5’端的前三个碱基以及3’端的最后三个碱基进行了2’-O-甲基类似物修饰和核苷酸间的3’硫代修饰。如下述化学式所示，左侧是化学修饰后的sgRNA,右侧是未经过修饰的sgRNA。
Figure PCTCN2018112068-appb-000033
在上述确定的电转条件下向脐带血来源的CD34阳性的造血干细胞(购买自澳赛尔斯生物技术(上海)有限公司,www.allcells.com，电转了上述23条经过化学修饰sgRNA，4天后通过TIDE分析Indels效率。结果如图9所示。同样，在本实施例中，作为对比，将未经化学修饰的sgRNA也通过同样的方 法电转，但是未经化学修饰的sgRNA，其Indels效率只有2.7％，在以后的实施例中使用的均是经化学修饰的sgRNA。
图9显示在CD34阳性造血干细胞上，电转Cas9 mRNA和23条sgRNA，4天后提取该CD34阳性的造血干细胞的基因组，选取sgRNA切割位点左右各约450bp，总长度为903bp的片段进行扩增，用于扩增的引物序列序列如下所示。
正向引物:cacctcagcagaaacaaagttatc(SEQ ID NO:29)
反向引物：gggaagctccaaactctcaa(SEQ ID NO:30)
切割位点选择：以Enhancer-2为例,5‘-ctaacagttg cttttatcac-3’,切割位点在3’端右侧(Cong L,et al.Science.2013)。
针对上述扩增片段进行Sanger测序，测序时使用的上下游引物序列如SEQ ID NO:27和SEQ ID NO:28所示。针对测序结果利用TIDE软件分析产生Indels效率的统计分析。其中，TIDE软件是在线分析Indels效率的软件，以一代测序结果为基础，分析Indels引起的双峰突变的效率，可以参考tide.deskgen.com。
结果表明，在该电转条件下，本实施例中合成的23条sgRNA均能够成功基因编辑造血干细胞，都可以有效地产生Indels，效率至少10％。其中效率最高的是Enhancer-2。
SEQ ID NO:26：Cas9 mRNA序列
Figure PCTCN2018112068-appb-000034
Figure PCTCN2018112068-appb-000035
Figure PCTCN2018112068-appb-000036
SEQ ID NO:27测序引物
Figure PCTCN2018112068-appb-000037
SEQ ID NO:28测序引物
Figure PCTCN2018112068-appb-000038
C、根据上述实验结果，选取基因编辑效率相对高效的Enhancer-2、Enhancer-3、Enhancer-4、Enhancer-5以及Enhancer-6，在300V 1ms的电转条件，电转Cas9 mRNA和Enhancer-2、Enhancer-3、Enhancer-4、Enhancer-5以及Enhancer-6进入脐带血来源的造血干细胞4天后检测Indels效率，结果如图10所示。图10显示电转Cas9 mRNA和BCL11A的增强子-2 sgRNA、Enhancer-3 sgRNA、Enhancer-4 sgRNA、Enhancer-5 sgRNA以及Enhancer-6 sgRNA进入3个不同脐带血来源的CD34阳性的造血干细胞，4天后，采用与上述相同的方法利用TIDE软件分析产生Indels效率的统计分析。
图10的结果表明在3个不同的脐带血来源的造血干细胞上，5条sgRNA均实现高效基因编辑，效率达到至少40％以上，其中效率最高的是Enhancer-2 sgRNA,平均基因编辑效率达到80％，显著高于Enhancer-3、Enhancer-4、Enhancer-5以及Enhancer-6，同时比现有的文献报道的在造血干细胞上基因编辑效率高(Xu,et al.Molecular Therapy.2017；DeWitt,et al.Sci Transl Med. 2016)。
基于此，后续实验以Enhancer-2 sgRNA为目的靶点开展。
实施例2：基因编辑脐带血来源的造血干细胞的体外克隆形成
本实验涉及基因编辑脐带血来源的造血干细胞的克隆形成单位(CFU,colony-formation units)检测。
选取300V 1ms的电转条件，电转Cas9 mRNA和Enhancer-2进入脐带血来源的造血干细胞,将800-1000个细胞重悬入1ml H4434(购自加拿大STEM CELLS TECHNOLOGIES)和IMDM(购自Thermo Fisher)以及FBS(购自Thermo Fisher)的混合液中，14天后显微镜下观察CFU-M、BFU-E、CFU-E、CFU-G、CFU-GM、GEMM等不同形态的克隆形成数目，结果如图11所示。其中，图11显示电转Cas9 mRNA和BCL11A的增强子-2 sgRNA进入脐带血来源的CD34阳性造血干细胞，2天后进行体外克隆形成实验(CFU检测)，14天后统计不同血液系统的克隆数目，BFU-E、CFU-M、CFU-GM、CFU-E、CFU-G、CFU-MM代表红系、髓系、淋巴系等血液系统不同谱系的克隆形成。其中，Mock:代表未经过基因编辑的细胞。
根据实施例2的数据表明，与未经过基因编辑的造血干细胞相比，基因编辑后的细胞功能体外分化功能正常，能分化不同血液系统谱系的克隆。
实施例3：基因编辑脐带血来源的造血干细胞重建小鼠模型的造血系统
选取300V 1ms的电转条件，电转Cas9 mRNA和Enhancer-2进入脐带血来源的造血干细胞，移植进入经过辐照仪照射的NPG免疫缺陷小鼠模型(购自北京维通达生物技术有限公司(Beijing Vitalstar Biotechnology,Inc.)。在移植6周、8周、10周、12周、16周后的外周血中检测人CD45和小鼠CD45的表达情况，同时检测移植16周后骨髓、脾脏的人CD45和小鼠CD45的表达情况，其结果如图12和图14所示。其中移植到小鼠中的方法为：在细胞移植24小时前，进行1.0Gy射线照射，清除小鼠模型的骨髓。随后将用20μL0.9％的生理盐水重悬的1.0×10 6的细胞注射到小鼠的尾静脉中，随后放入洁净级别的动物房中饲养。
图12和图14的结果表明经过基因编辑的造血干细胞，移植进入小鼠模 型后，与未经过基因编辑的造血干细胞相比，经过基因修饰的细胞随着时间延长，人源的hCD45表达比例持续增高，在外周血样品中，由第6周的20％提高到16周的60％，16周的骨髓中比例甚至达到90％，脾脏达到70％,表明经过基因编辑的造血干细胞能够快速、高效地植入小鼠模型的造血系统，细胞体内分化功能正常。而现有的文献报道表明，6周后外周血中CD45表达比例在1-10％之间，16周后CD45表达比例在20-40％之间，骨髓中CD45表达比例在50％左右，因此，本发明中的动物实验结果明显好于现有领域中报道的移植实验结果(Xu,et al.Molecular Therapy.2017；DeWitt,et al.Sci Transl Med.2016；Mettananda,et al.Nature Communications.2017)。
同时，在移植基因编辑的细胞的小鼠中，16周后检测了人源的CD3、CD4、CD8、CD33、CD19、CD56等细胞膜蛋白的表达如图13、14、15所示。结果表明，经过基因修饰的细胞能正常表达这些蛋白，表明可以分化为T细胞、B细胞、巨噬细胞等血液系统的细胞，能高效重建小鼠模型的造血系统。与现有文献报道结果相比，首先我们检测了CD3、CD4、CD8、CD33、CD19、hCD56等6个细胞膜表面蛋白判定T细胞、髓系细胞、B细胞和NK细胞等在小鼠血液中表达情况，更准确地评估移植的人造血干细胞重建小鼠造血系统的能力，而现有文献中仅测试了CD3、CD19、CD56、CD33等蛋白表达。其次我们检测了外周血、骨髓和脾脏等三个重要的血液系统相关组织中以上6个蛋白的表达谱，更全面地评估了重建小鼠造血系统的能力,而现有文献中只以骨髓作为检测的组织(Chang,et al.Methods & Clinical Development.2017；DeWitt,et al.Sci Transl Med.2016；Mettananda,et al.Nature Communications.2017)。
此外，虽然基因编辑的细胞能够快速、高效重建小鼠模型的造血系统。针对重建小鼠模型的细胞是否发生了基因编辑的判断结果如图16所示，提取移植前细胞、移植16周后外周血、骨髓和脾脏的基因组，扩增目的片段和Sanger测序，通过TIDE分析，检测Indels效率。结果表明移植16周后的外周血、骨髓和脾脏的人源细胞均发生了基因编辑，效率与移植前细胞相似，在50～70％之间。
实施例4：基因编辑脐带血来源的造血干细胞红系分化检测γ珠蛋白和 胎儿血红蛋白表达
4-1.红细胞分化
选取300v 1ms的电转条件，电转Cas9 mRNA和Enhancer-2进入脐带血来源的造血干细胞，利用下述“两步法”分化方案进行分化。
其中两步法分化为利用造血干细胞红系扩增和分化培养基进行分化，然后利用造血干细胞红系分化脱核培养基进行分化。
造血干细胞红系扩增和分化培养基为基础培养基为StemSpan TM SFEM II,生长因子为50-200ng/ml SCF,10-100ng/ml IL-3,1-10U EPO/ml，培养条件：利用造血干细胞红系扩增和分化培养基培养造血干细胞1.0×10 5cells/ml，扩增7天。
造血干细胞红系分化脱核培养基为基础培养基为STEMSPAN TM SFEM II,生长因子为1-10U EPO,100-1000μg/ml人转铁蛋白，化学小分子为0.5-10μm mifepristone，将利用上一步骤培养的1.0×10 6cells/ml细胞在造血干细胞红系分化脱核培养基分化5天。
然后，检测CD71和CD235a表达，如图17所示。实验组和对照组均能高效分化成为红细胞，CD71和CD235a的表达比例均在90％以上。
4-2.检测γ珠蛋白和胎儿血红蛋白表达
提取上述红细胞分化后的细胞的mRNA,反转录成cDNA，通过荧光定量PCR检测BCL11A、HBB、HBG等基因的表达，如图18所示。结果表明，基因敲除BCL11A enhancher位点的细胞BCL11A基因表达下调1倍，HBG表达提高一倍。
检测红系分化后的细胞的胎儿血红蛋白表达，如图19所示，流式分析结果表明基因敲除BCL11A enhancher位点的细胞，胎儿血红蛋白表达提高约1倍。
实施例5：基因编辑β-地中海贫血病人来源的造血干细胞的BCL11A的 增强子位点
5-1.β-地中海贫血病人外周血来源的造血干细胞分离
通过常规的磁珠分选获得CD34阳性的造血干细胞。结果如图20所示。
5-1.电转β-地中海贫血病人外周血来源的造血干细胞
选取300V 1ms的电转条件，电转Cas9 mRNA和Enhancer-2、Enhancer-3、Enhancer-4、Enhancer-5以及Enhancer-6进入β-地中海贫血病人外周血来源的造血干细胞，4天后检测Indels效率，结果如图21所示。表明在3个不同的病人外周血来源的造血干细胞上，5条sgRNA均可实现高效基因编辑，其中，效率最高的是Enhancer-2，达到至少70％以上。
实施例6：基因编辑贫血病人外周血来源的造血干细胞红系分化检测γ 珠蛋白和胎儿血红蛋白表达
将实施例5中经过基因编辑的造血干细胞进行体外红系分化，方法见实施例4-1。分化结果如图22所示，实验结果表明对照组、Enhancer-2、Enhancer-3、Enhancer-4、Enhancer-5以及Enhancer-6红系分化效率相似，均高表达CD71和CD235a细胞膜蛋白。
12天后，提取红细胞分化后的细胞的mRNA,反转录成cDNA，通过荧光定量PCR检测BCL11A、HBB、HBG、HBA等基因的表达，如图23所示。结果表明，除Enhancer-6以外，Enhancer-2、Enhancer-3、Enhancer-4和Enhancer-5均能下调细胞中BCL11A基因表达，其中Enhancer-2效果最为显著下调约1倍左右；此外，Enhancer-2、Enhancer-3、Enhancer-4、Enhancer-5以及Enhancer-6均能够提高γ珠蛋白表达，其中效果最显著的是Enhancer-2，γ珠蛋白表达提高9倍，符合临床治疗的标准。与现有文献中的结果相比，首先我们以重症地贫病人来源的造血干细胞作为起始的评估细胞来源，精确验证了本发明的方法符合临床治疗的要求，而现有文献只使用了正常人的骨髓或者外周血的样本；其次，现有文献中报道的提高胎儿血红蛋白的倍数只在4-7倍之间，低于本发明中提高胎儿血红蛋白的表达比例。由此表明，本发明的实验结果显著优于现有的文献报道(Chang et al.Methods& Clinical Development.2017；Mattew C et al.Nature.2015)。
此外，为了更精确地评估Enhancer-2、Enhancer-3、Enhancer-4、Enhancer-5以及Enhancer-6基因编辑BCL11A enhancer位点对于胎儿血红蛋白(HbF)和正常血红蛋白(HbA)的蛋白表达量的影响，在本发明中，我们提取了造血干细胞分化12天后获得的红细胞的蛋白，进行了HPLC试验(High Performance Liquid  Chromatography,高效液相色谱法)，如图27、28和29所示。结果表明，1)保留时间约为5.4min和10min处的色谱峰分别代表HbF和HbA。2)与未经过基因编辑的细胞相比，Enhancer-2、Enhancer-3、Enhancer-4、Enhancer-5以及Enhancer-6基因编辑的细胞HbF表达量更高(峰更高和峰面积更大),HbA的表达量更低(峰更低和峰面积更小)，表明基因编辑BCL11A enhancer位点提高了HbF的表达，且下调了HbA的表达。3)其中，Enhancer-2的HbF/HbA的比例为约为5.28，mock的HbF/HbA的比例为约为0.79，提高了6.68倍，显著高于Enhancer-3、Enhancer-4、Enhancer-5以及Enhancer-6对于HbF/HbA的比例倍数。在临床上，如本发明的背景中所述重型β地贫患者由于β珠蛋白缺失，血液中的血红蛋白由少量的HbA和占绝大部分的HbF组成，通常重型β地贫患者的血红蛋白约为20g/L，其中HbF占比达到90％以上。我们按照占比90％计算，经过基因编辑后的细胞HbF表达量提高了6.68倍，最终的血红蛋白量为20*0.9*6.68+20*0.1＝122.24g/L,正常人的血红蛋白表达量的范围是115-150g/L之间，该结果完全符合临床治疗的标准(Antonio Cao,et al.Genes in Medicine.2010；2010年《重型β地中海贫血的诊断和治疗指南》)。与现有文献相比，本发明是唯一在β地贫患者的造血干细胞上，通过HPLC实验精确评估了基因编辑BCL11A enhancer提高胎儿血红蛋白的有效性的发明研究，且结果表明显著提高了胎儿血红蛋白的表达，达到治疗重型β地贫患者的临床要求(Chang et al.Methods& Clinical Development.2017；Mattew C et al.Nature.2015；Lin Ye，et al.PNAS.2016；Matthew H.Porteus,Advances in Experimental Medicine and Biology.2013)。
实施例7：基因编辑贫血病人外周血来源的造血干细胞体外克隆形成
选取300V 1ms的电转条件，电转Cas9 mRNA和Enhancer-2进入贫血病人外周血来源的造血干细胞,将500-800个细胞重悬入1ml H4434(购自加拿大STEM CELLS TECHNOLOGIES)和IMDM(购自Thermo Fisher)以及FBS(购自Thermo Fisher)的混合液中，14天后显微镜下观察CFU-M、BFU-E、CFU-E、CFU-G、CFU-GM、GEMM等不同形态的克隆形成数目，结果如图25所示。
本实验数据表明，与未经过基因编辑的造血干细胞相比，基因编辑后的细胞功能体外分化功能正常，能分化不同血液系统谱系的克隆。
实施例8：基因编辑贫血病人外周血来源的造血干细胞的脱靶效应
本实验涉及基因测序方法，具体是通过2代测序技术(Next generation sequencing,NGS)分析基因编辑贫血病人外周血来源的造血干细胞的脱靶效应(off-target effect)。
选取300V 1ms的电转条件，电转Cas9 mRNA和Enhancer-2进入贫血病人外周血来源的造血干细胞,扩增4天后，提取基因组进行2代测序分析。我们通过软件预测了14个潜在的脱靶位点，结果如图22所示。
本实验数据表明，基因编辑中靶(on-target)效率为76％，而14个潜在的脱靶位点的比率均低于0.3％，与二代测序本身误差相当，由此可见，在测序误差范围内，我们并没有检测到由于基因编辑而导致的脱靶现象，所以该基因编辑方案是安全的。
根据上述实施例的数据可以看出，针对不同来源的细胞，Enhancer-2均显示出最好的效果。
工业实用性
根据本发明，本发明的方法有以下几个优点，首先本方法能够基因编辑地中海贫血病人来源的造血干细胞，完全满足临床治疗地中海贫血和镰刀型红细胞贫血的要求；其次，通过使用经过化学修饰的sgRNA,基因编辑效率高，显著提高胎儿血红蛋白的表达，细胞能够重建模型小鼠的造血系统；最后，脱靶分析显示安全性高。基于此，本发明开发的方法将有可能代替传统的造血干细胞移植治疗技术来治愈重型地中海贫血和镰刀型红细胞贫血的患者。
尽管已提及具体特征对本发明进行详细描述，但对本领域的技术人员显而易见的是此描述仅用于优选的实施方案而非限制本发明的范围。因此，本发明的实质范围会通过所附权利要求和其等同物来进行限定。

Claims (39)

  1. 一种提高人造血干细胞胎儿血红蛋白(HbF)表达的方法，包括：
    通过基因编辑技术破坏所述造血干细胞中2号染色体第60495219位至第60495336位的BCL11A基因组区域。
  2. 权利要求1所述的方法，其中所述基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术。
  3. 权利要求2所述的方法，其中所述基因编辑技术为CRISPR/Cas9基因编辑技术。
  4. 权利要求1-3任一项所述的方法，其中，所述BCL11A基因组靶核苷酸序列与选自SEQ ID NO:3～SEQ ID NO:25任一序列互补。
  5. 权利要求3或4的方法，将包含选自SEQ ID NO：3～SEQ ID NO：25任一序列的sgRNA导入所述造血干细胞以实现对所述BCL11A基因组的编辑。
  6. 权利要求5的方法，其中所述sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的。
  7. 权利要求6的方法，其中所述化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。
  8. 权利要求3-7任一项的方法，其中将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。
  9. 权利要求8的方法，其中通过电转方法将sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。
  10. 权利要求9的方法，其中所述电转条件为200-600V，0.5ms-2ms。
  11. 一种通过CRISPR/Cas9系统体外高效编辑造血干细胞的方法，包括将含有选自SEQ ID NO：3～SEQ ID NO：25任一序列的sgRNA导入造血干细胞，其中该sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的。
  12. 权利要求11的方法，其中将所述sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。
  13. 权利要求12的方法，其中通过电转方法将sgRNA与Cas9编码核苷酸共同导入所述造血干细胞。
  14. 权利要求13的方法，其中所述电转条件为200-600V，0.5ms-2ms。
  15. 通过权利要求1～14中任一项所述的方法得到的造血干细胞。
  16. 一种通过基因改造使胎儿血红蛋白(HbF)表达升高的人造血干细胞，其中该造血干细胞中第2号染色体第60495219位至第60495336位的BCL11A基因组区域通过基因编辑技术被破坏。
  17. 通过分化培养权利要求15或16所述的造血干细胞获得的、处于成熟红细胞之前的不同分化阶段的前体细胞。
  18. 通过分化培养权利要求15或16所述的造血干细胞获得的成熟红细胞。
  19. 一种制造通过基因改造使胎儿血红蛋白(HbF)表达升高的成熟红细胞或其前体细胞的方法，该方法包括：
    (a)使用权利要求1～14中任一项所述的方法得到基因改造的造血干细胞；
    (b)使用造血干细胞红系扩增和分化培养基对所述基因改造的造血干细胞进行造血干细胞红系扩增和分化，
    其中，所述造血干细胞红系扩增和分化培养基包括基础培养基，以及生长因子的组合物，其中所述生长因子的组合物包括干细胞生长因子(SCF)；白介素3，(IL-3)和促红细胞生成素(EPO)。
  20. 根据权利要求19所述的方法，该方法还包括：
    使用红系分化脱核培养基进行造血干细胞红系分化脱核，
    所述红系分化脱核培养基包含基础培养基、生长因子、以及孕酮受体和糖皮质激素受体的拮抗剂和/或抑制剂。
  21. 根据权利要求20所述的方法，其中所述红系分化脱核培养基中的生长因子包括促红细胞生成素(EPO)，所述孕酮受体和糖皮质激素受体的拮抗剂和/或抑制剂为选自下述化合物(I)～(IV)中的任一种或两种及以上：
    Figure PCTCN2018112068-appb-100001
    Figure PCTCN2018112068-appb-100002
  22. 一种组合物，包含权利要求15或16的造血干细胞或权利要求17的前体细胞或权利要求18的成熟红细胞。
  23. 包含权利要求15或16的造血干细胞或权利要求17的前体细胞或权利要求18的成熟红细胞的医用制品。
  24. 权利要求15或16的造血干细胞或权利要求17的前体细胞或权利要求18的成熟红细胞在预防或治疗有需要的受试者的疾病中的用途。
  25. 权利要求24的用途，所述疾病为贫血性疾病、失血性疾病、肿瘤或其它需要大量输血进行预防或治疗的疾病。
  26. 根据权利要求25的用途，其中所述疾病为β-地中海贫血或镰刀形红细胞贫血。
  27. 权利要求24-26中任一项的用途，其中所述受试者为人。
  28. 权利要求15或16的造血干细胞或权利要求17的前体细胞或权利要求18的成熟红细胞在制备预防或治疗受试者疾病中的药物或医用制品中的用途。
  29. 权利要求28的用途，所述疾病为贫血性疾病、失血性疾病、肿瘤或其它需要大量输血进行预防或治疗的疾病。
  30. 根据权利要求29的用途，其中所述疾病为β-地中海贫血或镰刀形红细胞贫血。
  31. 权利要求28-30中任一项的用途，其中所述受试者为人。
  32. 一种sgRNA构建体，包含选自SEQ ID NO：3～SEQ ID NO：25之一的核苷酸序列。
  33. 权利要求32的构建体，其包含2’-O-甲基类似物和/或核苷酸间3’硫代修饰。
  34. 权利要求33的构建体，其中所述化学修饰为在选自SEQ ID NO：3～SEQ ID NO：25之一的核苷酸序列的5’端的前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。
  35. 包含权利要求32-34中任一项的构建体的载体、宿主细胞或制剂。
  36. 包含权利要求32-34中任一项的构建体在基因编辑造血干细胞中的用途。
  37. 一种治疗或预防受试者贫血性疾病、失血性疾病、肿瘤或其它需要大量输血进行预防或治疗的疾病的方法，其包括给药受试者权利要求15或16的造血干细胞，权利要求17的前体细胞或权利要求18的成熟红细胞。
  38. 根据权利要求37所述的方法，其中，所述疾病为β-地中海贫血或镰刀形红细胞贫血。
  39. 根据权利要求38所述的方法，其中，所述受试者为人。
PCT/CN2018/112068 2017-10-27 2018-10-26 一种提高胎儿血红蛋白表达水平的方法 WO2019080920A1 (zh)

Priority Applications (8)

Application Number Priority Date Filing Date Title
CN202210443541.6A CN114875026A (zh) 2017-10-27 2018-10-26 一种提高胎儿血红蛋白表达水平的方法
JP2020523710A JP2021500058A (ja) 2017-10-27 2018-10-26 胎児ヘモグロビンの発現レベルを増加させる方法
EP18870256.7A EP3702458A4 (en) 2017-10-27 2018-10-26 METHOD FOR INCREASING THE LEVEL OF FETAL HEMOGLOBIN EXPRESSION
US16/758,824 US20200384032A1 (en) 2017-10-27 2018-10-26 Method for increasing fetal hemoglobin expression level
CN201880069637.2A CN111278977B (zh) 2017-10-27 2018-10-26 一种提高胎儿血红蛋白表达水平的方法
PH12020550488A PH12020550488A1 (en) 2017-10-27 2020-04-24 Method for increasing fetal hemoglobin expression level
ZA2020/02204A ZA202002204B (en) 2017-10-27 2020-05-04 Method for increasing fetal hemoglobin expression level
JP2022149587A JP2022180511A (ja) 2017-10-27 2022-09-20 胎児ヘモグロビンの発現レベルを増加させる方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711027708.6 2017-10-27
CN201711027708 2017-10-27

Publications (1)

Publication Number Publication Date
WO2019080920A1 true WO2019080920A1 (zh) 2019-05-02

Family

ID=66246216

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2018/112068 WO2019080920A1 (zh) 2017-10-27 2018-10-26 一种提高胎儿血红蛋白表达水平的方法
PCT/CN2018/112027 WO2019080917A1 (zh) 2017-10-27 2018-10-26 一种提高胎儿血红蛋白表达的方法

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/112027 WO2019080917A1 (zh) 2017-10-27 2018-10-26 一种提高胎儿血红蛋白表达的方法

Country Status (9)

Country Link
US (2) US20200384032A1 (zh)
EP (2) EP3702459B1 (zh)
JP (4) JP2021502062A (zh)
CN (10) CN109722415B (zh)
MA (2) MA52665A (zh)
PH (2) PH12020550488A1 (zh)
TW (2) TW201930592A (zh)
WO (2) WO2019080920A1 (zh)
ZA (2) ZA202002203B (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023107675A3 (en) * 2021-12-10 2023-10-26 The Children's Medical Center Corporation Combination bcl11a enhancer editing

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2018007987A (es) * 2015-12-28 2019-01-10 Novartis Ag Composiciones y metodos para el tratamiento de hemoglobinopatias.
CN109486814A (zh) * 2017-10-31 2019-03-19 广东赤萌医疗科技有限公司 一种用于修复HBB1基因点突变的gRNA、基因编辑系统、表达载体和基因编辑试剂盒
CN112011576A (zh) * 2019-05-31 2020-12-01 华东师范大学 Crispr基因编辑技术在治疗地中海贫血中的应用
CN112391410B (zh) * 2020-02-17 2024-04-02 华东师范大学 一种sgRNA及其在修复内含子异常剪接中的应用
CN110511909B (zh) * 2019-07-29 2022-01-04 吉林大学 体外扩增造血干细胞的生长因子组合物及其应用
WO2021037232A1 (zh) * 2019-08-28 2021-03-04 甘李药业股份有限公司 编辑造血干/祖细胞中bcl11a基因的方法
WO2021037234A1 (zh) * 2019-08-29 2021-03-04 广州辑因医疗科技有限公司 一种高效修复环状铁粒幼细胞性贫血基因突变的方法
CN112442516A (zh) * 2019-08-29 2021-03-05 广州辑因医疗科技有限公司 一种高效修复环状铁粒幼细胞性贫血基因突变的方法
JP2022547105A (ja) * 2019-09-04 2022-11-10 博雅▲輯▼因（北京）生物科技有限公司 オフターゲット評価に基づく遺伝子編集治療の評価方法
WO2021049617A1 (ja) * 2019-09-11 2021-03-18 国立大学法人 東京大学 ヒト造血幹細胞を培養するために適したアルブミンフリーの無血清培地およびアルブミンフリーの培養方法
CN112746072A (zh) * 2019-10-31 2021-05-04 广州瑞风生物科技有限公司 用于β-血红蛋白病基因编辑的sgRNA及应用
CN111876416B (zh) * 2020-07-01 2021-09-03 广州瑞风生物科技有限公司 激活γ-珠蛋白基因表达的方法和组合物
CN114149990A (zh) * 2020-09-08 2022-03-08 甘李药业股份有限公司 编辑造血干/祖细胞中bcl11a基因的方法
US11970713B2 (en) * 2020-12-04 2024-04-30 Ocgene Therapeutics Corporation Method for long-term ex vivo maintenance or expansion of human erythroblast, human megakaryocyte-erythroid progenitor, or human common myeloid progenitor cell and application thereof
CN113046330B (zh) * 2021-03-23 2023-03-21 中吉智药(南京)生物技术有限公司 携带红系基因编辑系统的慢病毒及药物
IL306119A (en) * 2021-04-02 2023-11-01 Univ Shanghai Tech Gene therapy for the treatment of beta-hemoglobinopathies
CN114032240A (zh) * 2021-11-02 2022-02-11 珠海横琴爱姆斯坦生物科技有限公司 一种用于提高基因敲除效率的方法
CN114317436A (zh) * 2021-12-30 2022-04-12 中国人民解放军军事科学院军事医学研究院 一种重新激活人γ-珠蛋白基因表达的方法
CN116426472A (zh) * 2023-06-08 2023-07-14 呈诺再生医学科技(北京)有限公司 一种促进造血干细胞或造血祖细胞分化为红细胞的诱导分化体系及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013176772A1 (en) 2012-05-25 2013-11-28 The Regents Of The University Of California Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
WO2014018423A2 (en) 2012-07-25 2014-01-30 The Broad Institute, Inc. Inducible dna binding proteins and genome perturbation tools and applications thereof
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
WO2014065596A1 (en) 2012-10-23 2014-05-01 Toolgen Incorporated Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US20160369262A1 (en) * 2015-06-18 2016-12-22 Sangamo Biosciences, Inc. Nuclease-mediated regulation of gene expression
WO2017182881A2 (en) * 2016-04-18 2017-10-26 Crispr Therapeutics Ag Materials and methods for treatment of hemoglobinopathies

Family Cites Families (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100506978C (zh) * 2001-09-21 2009-07-01 中国人民解放军军事医学科学院野战输血研究所 一种新的造血干/祖细胞的富集方法及其体外定向诱导分化
CN101045914A (zh) * 2006-03-29 2007-10-03 中国人民解放军军事医学科学院野战输血研究所 体外诱导造血干/祖细胞分化为成熟红细胞的方法与应用
CN102802412A (zh) * 2009-12-08 2012-11-28 海玛奎斯特医药公司 用于治疗红细胞病症的方法及低剂量方案
JP6170080B2 (ja) * 2012-02-24 2017-07-26 フレッド ハッチンソン キャンサー リサーチ センター 異常ヘモグロビン症の治療のための組成物および方法
CN102643784B (zh) * 2012-03-29 2016-05-25 中国人民解放军第四军医大学 一种造血干/祖细胞的体外扩增体系
CN103667188B (zh) * 2012-09-21 2016-02-24 北京市红十字血液中心 一种制备成熟红细胞的方法
US9822355B2 (en) * 2012-11-27 2017-11-21 Children's Medical Center Corporation Targeting BCL11A distal regulatory elements for fetal hemoglobin reinduction
PT3068881T (pt) * 2013-11-13 2019-05-31 Childrens Medical Center Regulação da expressão de genes mediada por nucleases
CA2938419A1 (en) * 2014-01-30 2015-08-06 Children's Hospital Medical Center An improved fetal hemoglobin for genetic correction of sickle cell disease
AU2015249381B2 (en) * 2014-04-25 2020-04-30 Children's Medical Center Corporation Compositions and methods to treating hemoglobinopathies
CN113699113A (zh) * 2014-09-16 2021-11-26 桑格摩治疗股份有限公司 用于造血干细胞中核酸酶介导的基因组工程化和校正的方法和组合物
US11827904B2 (en) * 2015-04-29 2023-11-28 Fred Hutchinson Cancer Center Modified stem cells and uses thereof
US11572543B2 (en) * 2015-05-08 2023-02-07 The Children's Medical Center. Corporation Targeting BCL11A enhancer functional regions for fetal hemoglobin reinduction
CN104877965B (zh) * 2015-05-27 2018-04-20 上海厚东生物科技有限公司 一种制备成熟红细胞的方法
CN105238758A (zh) * 2015-09-17 2016-01-13 中国科学院广州生物医药与健康研究院 一种体外获得造血干/祖细胞的方法
MX2018007987A (es) * 2015-12-28 2019-01-10 Novartis Ag Composiciones y metodos para el tratamiento de hemoglobinopatias.
CN105854017A (zh) * 2016-03-01 2016-08-17 扬州大学 一种治疗β-地中海贫血的试剂及其应用
CN105754939A (zh) * 2016-04-15 2016-07-13 广州市天河诺亚生物工程有限公司 一种提高脐带血红系祖细胞定向分化效果的方法
EP3596217A1 (en) * 2017-03-14 2020-01-22 Editas Medicine, Inc. Systems and methods for the treatment of hemoglobinopathies
CN107151655A (zh) * 2017-04-10 2017-09-12 中国人民解放军第三O七医院 一种细胞扩增的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013176772A1 (en) 2012-05-25 2013-11-28 The Regents Of The University Of California Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
WO2014018423A2 (en) 2012-07-25 2014-01-30 The Broad Institute, Inc. Inducible dna binding proteins and genome perturbation tools and applications thereof
WO2014065596A1 (en) 2012-10-23 2014-05-01 Toolgen Incorporated Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US20160369262A1 (en) * 2015-06-18 2016-12-22 Sangamo Biosciences, Inc. Nuclease-mediated regulation of gene expression
WO2017182881A2 (en) * 2016-04-18 2017-10-26 Crispr Therapeutics Ag Materials and methods for treatment of hemoglobinopathies

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
ANTONIO CAO ET AL., GENES IN MEDICINE, 2010
BAUER, D. E. ET AL.: "An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level", SCIENCE, vol. 342, 2013, pages 253 - 257, XP055568654, DOI: 10.1126/science.1242088
CANVER MC ET AL.: "Enhancer section by Cas9-mediated in situ saturating mutagenesis", NATURE, vol. 527, no. 7577, 12 November 2015 (2015-11-12), pages 192 - 7, XP055274680, DOI: 10.1038/nature15521
CHANG ET AL., METHODS & CLINICAL DEVELOPMENT, 2017
CONG L ET AL., SCIENCE, 2013
DELEEVEY GFLDAMHA MJ.: "Designing chemically modified oligonucleotides for targeted gene silencing", CHEM BIOL., vol. 19, no. 8, 24 August 2012 (2012-08-24), pages 937 - 54, XP055107150, DOI: 10.1016/j.chembiol.2012.07.011
DEWITT ET AL., SCI TRANSL MED., 2016
GUIDELINES FOR DIAGNOSIS AND TREATMENT OF SEVERE B THALASSEMIA, 2010
HENDEL ET AL.: "Chemically modified guide RNAs enhance CRISPR-Cas gene editing in human primary cells", NATBIOTECHNOL., vol. 33, no. 9, September 2015 (2015-09-01), pages 985 - 989
LIN YE ET AL., PNAS, 2016
MATTEW C ET AL., NATURE, 2015
MATTHEW H. PORTEUS, ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY, 2013
METTANANDA ET AL., NATURE COMMUNICATIONS, 2017
WANG, YEBO: "The Optimization of CRISPR/Cas9 and Its Application in Human Stem Cells", DOCTORAL DISSERTATION, no. 6, 15 June 2017 (2017-06-15), pages 1 - 134, XP009520648, ISSN: 1674-022X *
XU ET AL., MOLECULAR THERAPY, 2017

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023107675A3 (en) * 2021-12-10 2023-10-26 The Children's Medical Center Corporation Combination bcl11a enhancer editing

Also Published As

Publication number Publication date
US20200339979A1 (en) 2020-10-29
CN111278977A (zh) 2020-06-12
CN111278976B (zh) 2022-03-01
PH12020550488A1 (en) 2021-03-22
CN109722415A (zh) 2019-05-07
PH12020550487A1 (en) 2021-03-22
CN111278977B (zh) 2022-03-22
MA50844A (fr) 2020-09-02
WO2019080917A1 (zh) 2019-05-02
ZA202002204B (en) 2022-10-26
US20200384032A1 (en) 2020-12-10
CN114657140A (zh) 2022-06-24
JP2022180510A (ja) 2022-12-06
EP3702459A4 (en) 2021-07-21
JP2021502062A (ja) 2021-01-28
CN109735497B (zh) 2021-11-02
CN109735497A (zh) 2019-05-10
ZA202002203B (en) 2023-05-31
CN115747165A (zh) 2023-03-07
JP2021500058A (ja) 2021-01-07
EP3702458A4 (en) 2021-08-25
CN114921415A (zh) 2022-08-19
EP3702459B1 (en) 2023-10-04
EP3702458A1 (en) 2020-09-02
TW201922777A (zh) 2019-06-16
CN114875026A (zh) 2022-08-09
EP3702459A1 (en) 2020-09-02
CN111278976A (zh) 2020-06-12
TW201930592A (zh) 2019-08-01
CN109735574A (zh) 2019-05-10
CN109722414A (zh) 2019-05-07
CN109722415B (zh) 2021-01-26
JP2022180511A (ja) 2022-12-06
MA52665A (fr) 2020-09-02

Similar Documents

Publication Publication Date Title
WO2019080920A1 (zh) 一种提高胎儿血红蛋白表达水平的方法
US20200121721A1 (en) Methods for genetic modification of hematopoietic cells
US20220193142A1 (en) Method for predicting effectiveness of treatment of hemoglobinopathy
WO2021043278A1 (zh) 基于脱靶评估评价基因编辑治疗的方法
WO2020147272A1 (zh) 一种利用非动员外周血制备异质性造血干祖细胞的方法
TW202100752A (zh) 一種異常血紅素症治療有效性預測方法
CN112442516A (zh) 一种高效修复环状铁粒幼细胞性贫血基因突变的方法
Zhang et al. Nanotechnology: A New Strategy for Lung Cancer Treatment Targeting Pro-Tumor Neutrophils
WO2021037234A1 (zh) 一种高效修复环状铁粒幼细胞性贫血基因突变的方法
CA3237748A1 (en) Biomarkers of megakaryocyte-derived extracellular vesicles
CN112447264A (zh) 基于脱靶评估评价基因编辑治疗的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18870256

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020523710

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2018870256

Country of ref document: EP

Effective date: 20200527

WWE Wipo information: entry into national phase

Ref document number: 520411830

Country of ref document: SA