WO2022052909A1 - 编辑造血干/祖细胞中bcl11a基因的方法 - Google Patents

编辑造血干/祖细胞中bcl11a基因的方法 Download PDF

Info

Publication number
WO2022052909A1
WO2022052909A1 PCT/CN2021/116914 CN2021116914W WO2022052909A1 WO 2022052909 A1 WO2022052909 A1 WO 2022052909A1 CN 2021116914 W CN2021116914 W CN 2021116914W WO 2022052909 A1 WO2022052909 A1 WO 2022052909A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
grna
cells
hematopoietic stem
bases
Prior art date
Application number
PCT/CN2021/116914
Other languages
English (en)
French (fr)
Inventor
焦娇
崔正之
栗飞红
Original Assignee
甘李药业股份有限公司
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 甘李药业股份有限公司 filed Critical 甘李药业股份有限公司
Publication of WO2022052909A1 publication Critical patent/WO2022052909A1/zh

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
    • 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
    • C12N15/1135—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 against oncogenes or tumor suppressor genes
    • 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
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • 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
    • 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
    • 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]

Definitions

  • the invention belongs to the field of biomedicine. Specifically, it relates to a method for editing genes in hematopoietic stem/progenitor cells through a gene editing system and a method for increasing the expression of HbF after erythroid differentiation of human hematopoietic stem/progenitor cells.
  • ⁇ -thalassemia (abbreviated as ⁇ -thalassemia) is a common clinical hereditary hemolytic anemia. According to statistics, about 1.5% of the world's population carries the ⁇ -thalassemia gene (80 million to 90 million people), and at least tens of thousands of children with severe ⁇ -thalassemia are born every year, which has become a global public health problem.
  • the hemoglobin present in the blood is mainly responsible for the transport of oxygen, and a decrease or lack of function of the hemoglobin can cause anemia.
  • Normal hemoglobin is composed of 4 subunits, most commonly adult hemoglobin HbA, composed of 2 ⁇ subunits and 2 ⁇ subunits ( ⁇ 2 ⁇ 2), which is the main form of adult hemoglobin, accounting for more than 98%; another It is fetal hemoglobin HbF, consisting of 2 ⁇ subunits and 2 ⁇ subunits ( ⁇ 2 ⁇ 2), which is the main form of hemoglobin during fetal development and after birth.
  • HbF can account for 70% of the total hemoglobin at birth, but with age, the expression of ⁇ subunit begins to decrease, while the ⁇ subunit begins to increase, and the content of HbF in adulthood generally does not exceed 1% of total hemoglobin.
  • mutations in the gene encoding the ⁇ -subunit lead to insufficient synthesis of the ⁇ -subunit and lower HbA in the patient, leading to severe anemia.
  • BCL11A B-cell lymphoma 11A gene
  • the BCL11A gene suppresses the expression of HbF.
  • the researchers knocked out the enhancer region of the BCL11A gene of the patient's autologous hematopoietic stem cells through the CRISPR/Cas system to inhibit the expression of the BCL11A gene, thereby re-expressing HbF and compensating for the mutated HbA to achieve the purpose of treating ⁇ -thalassemia .
  • CRISPR/Cas system clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system (clustered regularly interspaced palindromic repeats/CRISPR-associated proteins), which is currently found in most bacteria and all An acquired immune system in archaea to destroy foreign plastids or phages. Due to its deoxyribonucleic acid interference properties, it is currently actively used in genetic engineering as a gene editing tool. Nuclease; TALEN) also utilizes the mechanism of non-homologous end joining (NHEJ) to generate double-strand breaks of deoxyribonucleic acid in the genome to facilitate editing.
  • NHEJ non-homologous end joining
  • Cas9 is an RNA-mediated DNA endonuclease, which is guided to the target sequence in the genome by a gRNA complementary to the target sequence.
  • the target sequence must be in the form of NGG or NAG. adjacent.
  • SSBs single-strand breaks
  • DSBs double-strand breaks
  • repair mechanisms such as non-homologous end joining (NHEJ) or homology repair pathway (HDR) in the body .
  • NHEJ non-homologous end joining
  • HDR homology repair pathway
  • the first aspect of the present invention discloses a method for editing the BCL11A gene in hematopoietic stem/progenitor cells.
  • the inventor unexpectedly found that editing the BCL11A gene enhancer +62 has a good effect on improving HbF expression.
  • the gene-edited hematopoietic stem The proportion of progenitor cells that differentiated into HbF positive erythrocytes was greater than 65%.
  • the method comprises: introducing one or more gRNAs targeting position +62 of the BCL11A gene enhancer and one or more endonucleases into the cells, resulting in the production of single strands within or near the BCL11A gene enhancer Breaks (SSBs) or double-strand breaks (DSBs), eg, resulting in deletion or inactivation of 45%-80% of the BCL11A gene enhancer in the cells, correspondingly causing the hematopoietic stem/progenitor cells to differentiate into HbF-positive erythrocytes
  • the ratio is greater than 65%; wherein, the gRNA comprises crRNA and tracrRNA, and the BCL11A enhancer+62 sequence is shown in SEQ ID NO: 14.
  • the guide sequence of the crRNA is selected from
  • 1-4 bases are optionally added or deleted at the 5' or 3' end, preferably, the added or deleted bases are complementary to the bases at the corresponding positions of the target DNA.
  • the guide sequence of the crRNA is selected from SEQ ID NO:1 or SEQ ID NO:2,
  • SEQ ID NO: 1 or SEQ ID NO: 2 wherein 1-4 bases are optionally added or deleted at the 5' or 3' end of SEQ ID NO: 1 or SEQ ID NO: 2, preferably, The added or deleted base is complementary to the base at the corresponding position of the target DNA,
  • the proportion of the hematopoietic stem/progenitor cells to differentiate into HbF positive erythrocytes is correspondingly greater than 70%.
  • the endonuclease is selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2 , Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15 , Csf1, Csf2, Csf3, Csf4 or Cpf1, homologs thereof, naturally occurring molecules thereof, codon-optimized forms thereof, recombinant forms thereof, or mutant forms thereof, and combinations thereof; preferably, the endo
  • sequence of the tracrRNA is as shown in SEQ ID NO: 7, or is formed with SEQ ID NO: 7 Nucleotide sequences with equivalent functions.
  • the gRNA is not modified with 2'-O-methylation and/or internucleotide 3' phosphorothioate.
  • most of the gRNAs in the existing technology are modified by 2'-O-methyl analogs and/or 3' thiols between nucleotides, such as chemical modifications to the 5' end of the gRNA by one, two and/or 2'-O-methylation of three bases and/or the last base at the 3' end.
  • the present invention unexpectedly found that the editing efficiency is higher without the above-mentioned chemical modification on the gRNA.
  • the method comprises premixing the endonuclease with the gRNA to form one or more RNP complexes.
  • the one or more RNP complexes are delivered into the hematopoietic stem/progenitor cells by electroporation transfection, and the method can ensure high editing efficiency of the gRNA, reduce costs, and meet the quality of drug application.
  • endonucleases and gRNAs are mostly introduced by means of lentivirus, and the method of lentivirus may lead to easy induction of leukemia in clinical practice.
  • the molar ratio of endonuclease to gRNA in the RNP complex is 1:(0.4-5.5), for example, 1:0.5; 1:1; 1:1.5; 1:2; 1:2.5; 1: 3;1:3.5;1:4;1;4.5;1:5, further preferably, the molar ratio of endonuclease to gRNA in the RNP complex is 1:(1-2.5).
  • the hematopoietic stem/progenitor cells are mammalian cells, primate cells, preferably primate cells, and more preferably human cells.
  • the second invention of this aspect provides a method for improving the expression of HbF in hematopoietic stem/progenitor cells after erythroid differentiation, comprising the following steps:
  • a third aspect of the present invention provides a hematopoietic stem/progenitor cell, the hematopoietic stem/progenitor cell being obtained by the method of any one of the first aspects, and the red blood cells differentiated from the hematopoietic stem/progenitor cell exhibiting
  • the HbF/(HbF+HbA) ratio is above 15% and, preferably, 15%-45%; more preferably 18%-25%.
  • the fourth aspect of the present invention provides the use of the hematopoietic stem/progenitor cells described in the third aspect in the preparation of a medicament or a medical product for increasing the expression of mammalian (preferably human) HbF.
  • the present invention provides the use of the hematopoietic stem/progenitor cells described in the third aspect in the preparation of medicines or medical products for preventing or treating anemia diseases, blood loss diseases, tumors or other Diseases requiring massive blood transfusion for prevention or treatment, preferably, the anemic disease is beta thalassemia or sickle cell anemia.
  • the fifth aspect of the present invention provides one or more guide ribonucleic acid (gRNA) for editing the BCL11A gene of hematopoietic stem/progenitor cells
  • the gRNA comprises crRNA and tracrRNA, and the form of the gRNA can be composed of crRNA and tracrRNA
  • the dimer can also be an artificially modified sgRNA fused by crRNA and tracrRNA; the guide sequence of the crRNA is selected from
  • 1-4 bases are optionally added or deleted at the 5' or 3' end, preferably, the bases added or deleted are complementary to the bases at the corresponding positions of the BCL11A gene;
  • the sequence of the tracrRNA is shown in SEQ ID NO: 7, or is equivalent to SEQ ID NO: 7 formed by substitution, deletion and/or optional addition of 1-4 bases at its 5' to 3' end functional nucleotide sequence.
  • the gRNA is not modified by 2'-O-methylation and/or internucleotide 3' phosphorothioate.
  • the present invention also provides a kit comprising the gRNA according to any one of the fifth aspects and one or more endonucleases selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 or Cpf1, their homologues, their natural
  • the present molecule its codon-optimized form,
  • kit is used to treat or prevent anemia diseases, blood loss diseases, tumors or other diseases that require massive blood transfusion for prevention or treatment in subjects.
  • the present invention also provides the application of a BCL11A gene fragment as a target for designing a drug for improving hemoglobin evaluation in a subject, wherein the BCL11A gene fragment is selected from the group consisting of sequences that are completely complementary to the following items:
  • 1-4 bases are optionally added or deleted at the 5' or 3' end, and the added or deleted bases are the bases at the corresponding positions of the BCL11A gene.
  • CD34+ cells CD34 molecules are highly glycosylated transmembrane glycoproteins selectively expressed on the surface of human and mammalian hematopoietic stem/progenitor cells.
  • the CD34 positive cells described herein therefore represent hematopoietic stem/progenitor cells.
  • B-cell lymphoma 11A (BCL11A) gene is a proto-oncogene, which is abundantly expressed in human lymph nodes, thymus and bone marrow tissues, and has low-level expression in most other tissues.
  • the enhancer of the BCL11A gene can negatively regulate the expression of fetal hemoglobin (HbF), wherein the enhancer + 55 (distance from the transcription start site) is defined according to the distance (kilobases) of the BCL11A enhancer from the transcription start site.
  • HbF fetal hemoglobin
  • kb indicates 1000 bases
  • +58 position indicates 1000 bases
  • +62 position indicates 1000 bases
  • the above position is a key regulatory region that negatively regulates fetal hemoglobin (HbF).
  • the base sequence of BCL11A+62 is located in the region from 60,717,492 to 60,718,860 of human chromosome 2 (relative to the human hg19 genome) (see Erythroid Enhancer of BCL11A Subject to Genetic Variation Determines Fetal Hemoglobin Level, Daniel E. Bauer et al., Science, vol. 342, Supplementary Material). These regions were edited to disrupt the expression of the BCL11A protein and were ultimately used to enrich for HbF.
  • CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated endonumclease 9) gene editing system described in this paper is an artificial endonuclease gene editing system, and a gene knockout method commonly used in the prior art .
  • CRISPR/Cas9 consists of two parts: 1) guide RNA, hereinafter also referred to as gRNA, and 2) endonuclease Cas9; the gRNA described herein includes crRNA (CRISPR-derived RNA) and tracrRNA (trans-activating RNA).
  • Described crRNA and tracrRNA act in one of the following ways: 1) tracrRNA/crRNA dimer, the partial sequence of crRNA is complementary to the partial sequence of tracrRNA, and forms dimer, those skilled in the art can understand Yes, a part of the base sequence of the crRNA is a guide sequence complementary to the target DNA sequence, and the other part of the base sequence is combined with a part of the tracrRNA sequence through base pairing to form a chimeric RNA (that is, a tracrRNA/crRNA dimer); 2) The crRNA and tracrRNA are fused to form a chimeric single-stranded guide RNA (single guide RNA, sgRNA), and the crRNA is located at the 5' end of the tracrRNA, wherein the crRNA includes a guide sequence.
  • sgRNA single guide RNA
  • the tracrRNA sequence of gRNA can be the conventional tracrRNA sequence known to those skilled in the art, that is, as those skilled in the art know which tracrRNA sequence can be used to realize the present invention, as those skilled in the art know that SEQ ID NO: 7 can be used as tracrRNA; Further Typically, those skilled in the art know and routinely use tracrRNA sequences, such as SEQ ID NO: 7, with 50% to 99%, preferably 60% to 99%, more preferably 70% to 99%, still preferably 80% to 99% , even mutants of preferably 90% to 99% identity can be used to carry out the present invention.
  • the gRNA includes a guide sequence and a backbone sequence
  • the gRNA can be in the form of a dimer composed of crRNA and tracrRNA, or an artificially engineered chimeric single fused to crRNA and tracrRNA.
  • chain sgRNA so the backbone sequence can be a dimer composed of crRNA and tracrRNA, or an artificially modified chimeric single-stranded sgRNA fused by crRNA and tracrRNA, and the backbone sequence is known to those skilled in the art;
  • the Cas9 endonuclease is used to cleave the active domain of DNA, resulting in DNA fragmentation.
  • the ribonucleoprotein (RNP complex) described herein refers to a protein complex containing RNA, that is, a form in which nucleic acid and protein are combined together.
  • gRNA binds to Cas9 to form a Cas9-gRNA complex, which binds to the matched target DNA adjacent to the PAM, and Cas9 undergoes a conformational change to stimulate endonuclease activity and cause DNA single-strand breaks (SSBs). ) or double-strand break (DSB), cells will be repaired by means such as non-homologous end joining (NHEJ) or homology-directed repair (HDR).
  • NHEJ non-homologous end joining
  • HDR homology-directed repair
  • Ratio of hematopoietic stem/progenitor cells differentiated into HbF-positive erythrocytes The ratio was measured using flow cytometry to characterize the ratio of HbF-expressing erythrocytes to total erythrocytes (where the erythrocytes are hematopoietic stem/progenitor cells) obtained by differentiation), which directly reflects the ratio of HbF-expressing erythrocytes to the total number of erythrocytes.
  • HbF/(HbF+HbA) the ratio is obtained by high performance liquid chromatography (HPLC) detection, specifically, in the HPLC atlas, the normalized ratio of the peak area of HbF accounts for the normalized ratio of the peak area of HbF and HbA The ratio of the sum; used to characterize the ratio of the amount of HbF to the sum of the total amount of HbF and HbA.
  • HPLC high performance liquid chromatography
  • Figure 1 FACS scatter plot of the percentage of HbF-expressing cells in red blood cells differentiated from BCL11A gene-edited CD34+ cells using gRNA-1 in experimental group a.
  • the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents forward scattered light.
  • Figure 2 FACS scatter plot of the percentage of HbF-expressing cells in red blood cells differentiated from BCL11A gene-edited CD34+ cells using gRNA-2 in experimental group b.
  • the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents the forward scattered light.
  • Figure 3 FACS scatter plot of the percentage of HbF-expressing cells in red blood cells differentiated from BCL11A gene-edited CD34+ cells using gRNA-3 in experimental group c, the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents forward scattered light.
  • Figure 4 FACS scatter plot of the percentage of HbF-expressing cells in red blood cells differentiated from BCL11A gene-edited CD34+ cells using gRNA-4 in experimental group d, the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents the forward scattered light.
  • Figure 5 FACS scatter plot of the percentage of HbF-expressing cells in red blood cells differentiated from BCL11A gene-edited CD34+ cells using gRNA-5 in experimental group e, the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents the forward scattered light.
  • Figure 6 FACS scatter plot of the percentage of HbF-expressing cells in red blood cells differentiated from BCL11A gene-edited CD34+ cells using gRNA-6 in experimental group f, the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents the forward scattered light.
  • Figure 7 FACS scatter plot of the percentage of HbF-expressing cells in erythrocytes differentiated from CD34+ cells without editing the gBCL11A gene in the control group, the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents the forward scattered light.
  • Figure 8 HPLC detection results of HbF expression in erythrocytes differentiated from CD34+ cells edited with gRNA-1 gene in experimental group a.
  • the X-axis represents time (unit: min), and the Y-axis represents peak height.
  • Figure 9 HPLC detection results of HbF expression in erythrocytes differentiated from CD34+ cells edited with gRNA-2 gene in experimental group b.
  • the X-axis represents time (unit: min), and the Y-axis represents peak height.
  • SFEM II medium (Stemcell) was prepared for culturing CD34+ cells.
  • the prepared SFEM II medium was supplemented with 100ng/mL SCF (purchased from Nearshore Protein Technology Co., Ltd.), 100ng/mL Flt3-L ( purchased from Inshore Protein Technology Co., Ltd.), 100ng/mL TPO (purchased from Inshore Protein Technology Co., Ltd.), 100ng/mL IL-6 (purchased from Inshore Protein Technology Co., Ltd.), and 1% Penicillin-Streptomycin Bis Anti-(Gibco).
  • CD34+ cells were placed in the prepared SFEM II medium (Stemcell) for pre-stimulation at a concentration of 0.25 ⁇ 106 cells/mL. The cells were placed in a CO2 incubator at 37°C for pre-stimulation for 48h.
  • Example 2 Electroporation of CD34+ cells derived from peripheral blood
  • the gRNA used in the embodiment of the present invention is a tracrRNA/crRNA dimer formed by the annealing of two parts of crRNA and tracrRNA.
  • the tracrRNA of each gRNA used in the experimental group is shown in SEQ ID NO: 7, and the crRNA backbone sequence paired with tracrRNA For GUUUUAGAGCUAUGCU, the guide sequence of crRNA is shown in Table 2:
  • step (3) the cells in step (2) were centrifuged for 10 min under a centrifugal force of 100 g, and the supernatant was sucked off;
  • the electroporation transfection solution is to mix 16.4 ⁇ L of Nucleofector Solution in P3 Primary Cell 4D-NucleofectorTM X Kit S Kit (Lonza) with 3.6 ⁇ L of Supplement 1, and electroporate transfection. Place the solution at room temperature for use; use 20 ⁇ L of electroporation transfection solution to resuspend the cells in each group;
  • the amplified fragment is about 500-600 bp, and ensure that the editing site is not located in the middle of the amplified fragment, so that it can be cut out.
  • Two bands of different sizes, and those skilled in the art know to design and synthesize primer sequences for amplification based on the fragments to be amplified.
  • SEQ ID NO:8 and 9 are used to amplify the DNA fragment of about 565bp near the editing site of gRNA-1, gRNA-2 or gRNA-4;
  • SEQ ID NOs: 10 and 11 are used to amplify a DNA fragment of about 501 bp near the editing site of gRNA-3 or gRNA-6;
  • SEQ ID NOs: 12 and 13 are used to amplify a DNA fragment of about 564 bp near the editing site of gRNA-5.
  • T7E1 digestion to identify shearing efficiency use T7 endonuclease I kit (GenScript), wherein the digestion system is shown in Table 4:
  • the gradient annealing procedure was adopted: a. Pre-denaturation: 5min at 95°C; b. Gradient annealing: 95-85°C, -2°C/s; 85-25°C, -0.1°C/s; 4°C maintained until the annealing was complete. Add 1 ⁇ L of T7E1 enzyme to the annealed PCR product, incubate at 37°C for 15 minutes, and detect the results of enzyme cleavage by 1.5% agarose gel electrophoresis. For example, the electrophoresis result will show 3 bands, the sizes are about 565bp, 360bp and 200bp respectively.
  • Example 5 Erythroid differentiation of hematopoietic stem/progenitor cells
  • the erythroid differentiation basal medium includes IMDM (Gibco), 15% FBS, 1% L-Glutamine (Gibco), 1% BSA (Sigma), 1% ITS (Gibco), 1% penicillin-streptomycin Double antibody (Gibco).
  • DIF I Differentiation (days 0-4), cells were cultured in DIF I medium, DIF I medium including erythrocyte differentiation basal medium (EDM), 1 ⁇ M DEX (Sigma), 5ng/mL IL-3 (purchased from Inshore Protein Technology Co., Ltd.), 100ng/mL SCF (purchased from Inshore Protein Technology Co., Ltd.), 6U/mL EPO (purchased from Inshore Protein Technology Co., Ltd.), 100nM gw7647 (Sigma); Differentiation (DIF) II (Day 5-8), cells were cultured in DIF II medium, DIF II medium including erythroid differentiation basal medium (EDM), 50ng/mL SCF (purchased from Nearshore Protein Technology Co., Ltd.), 6U/mL EPO (purchased from Nearshore Protein Technology Co., Ltd.), 10 nM gw7647 (Sigma); differentiation (DIF) III (days 9-16), cells were cultured in DIF III medium including erythroid differentiation basal culture base
  • Differentiated erythrocytes were used as experimental materials.
  • the differentiated erythrocytes were labeled with PE-CD71 antibody (Invitrogen) and PB-CD235a antibody (Invitrogen) to label the erythrocyte population. After staining and fixation, the cells were ruptured, and APC-HbF monoclonal antibody ( Invitrogen), the HbF expression was detected by flow cytometry analysis.
  • the erythroid differentiated cells were used as the experimental material, 1 ⁇ 10 7 cells were collected, washed once with PBS, lysed with 100 ⁇ L 0.01% SDS on ice for 10 min; centrifuged at 12,000 rpm for 5 min and added 20 ⁇ L of supernatant to 80 ⁇ L of mobile phase A, Incubate on ice for 30min;
  • Mobile phase A 20mM bis-tris+2mM KCN, pH 6.9
  • Mobile phase B 20mM bis-tris+2mM KCN+200mM NaCl, pH 6.57
  • UV detection wavelength 415nm
  • HbF/(HbF+HbA) ratio of CD34+ cells differentiated into erythrocytes after editing the BCL11A gene with the gRNA of the present invention is above 15%, specifically in the range of 15%-45%.
  • Example 7 Effects of unmodified gRNA and modified gRNA on BCL11A gene editing efficiency
  • the gRNA used in the control group was a customized modified gRNA.
  • the modification method of the gRNA was as follows: 3 nucleotides at the 5' end of the gRNA used in the experimental group were modified by 2 nucleotides. '-O-methylation and internucleotide 3'-phosphorothioate modification. As shown in the chemical modifications below, the chemically modified gRNA is on the left and the unmodified gRNA is on the right. After the modified gRNA and Cas9 were used to prepare RNP complexes in vitro, hematopoietic stem/progenitor cells were electroporated to detect the editing efficiency. Experiments have shown that unmodified gRNAs have higher editing efficiency, as shown in Table 9:
  • Example 8 Influence of gRNA into hematopoietic stem/progenitor cells by electroporation transfecting RNP complex and packaging lentivirus on gene editing efficiency
  • control groups 3 and 4 package lentiviruses Introduction of gRNAs (gRNA-1 and gRNA-2) into hematopoietic stem/progenitor cells.
  • the packaging plasmid and the lentiviral vector carrying the CRSPR/Cas9 gene and gRNA sequence were co-transfected into 293T cells by the liposome method, the lentiviral vector was obtained from the supernatant, and the lentiviral vector was co-transfected with hematopoietic stem/progenitor cells. cultured to obtain infected hematopoietic stem/progenitor cells. The specific steps are:
  • step (3) Dilute the annealed oligonucleotide in step (3) into sterile water or EB at a dilution ratio of 1:200;
  • HEK293T cells were cultured with DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco) and passaged in 15 cm tissue culture dishes;
  • Table 10 The effect of gRNA into hematopoietic stem/progenitor cells by electroporation transfecting RNP complex and packaging lentivirus on gene editing efficiency
  • Example 9 Effects of different molar ratios of endonuclease Cas9 and gRNA on the gene editing efficiency of CD34+ cells and the positive rate of HbF in differentiated erythrocytes
  • Example 1-6 use gRNA-1 to edit the BCL11A gene, and the difference from experimental group a is that example group 5-10 (using the gRNA of experimental group a) changes the molar ratio of endonuclease Cas9 to gRNA-1 , the experiment proves that the gene editing efficiency is high under the ratio described in the present invention, and finally the red blood cells differentiated from the edited hematopoietic stem/progenitor cells have a high HbF positive rate. See Table 11 for details:

Landscapes

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

Abstract

提供了编辑造血干/祖细胞中BCL11A基因的方法及提高人造血干/祖细胞经红系分化后HbF表达的方法，还提供了BCL11A基因被编辑的造血干/祖细胞及所述细胞用于制备预防或治疗贫血性疾病的药物或医用制品中的用途，还提供了用于编辑造血干/祖细胞中BCL11A基因的gRNA及包含所述gRNA的试剂盒。

Description

编辑造血干/祖细胞中BCL11A基因的方法 技术领域
本发明属于生物医药领域。具体涉及通过基因编辑系统编辑造血干/祖细胞中基因的方法及提高人造血干/祖细胞经红系分化后HbF表达的方法。
背景技术
β地中海贫血(简称β地贫)是临床常见的遗传性溶血性贫血。据统计，世界范围内约1.5％的人口携带β地贫基因(8 000万～9 000万人)，每年至少有上万例重型β地贫患儿出生，已成为全球公共卫生问题。
存在于血液中的血红蛋白主要负责氧的运输，血红蛋白数量下降或功能缺失会引起贫血。正常血红蛋白由4个亚基构成，最常见为成人血红蛋白HbA，由2个α亚基和2个β亚基构成(α2β2)，这是成人血红蛋白的主要形式，约占98％以上；另一种为胎儿血红蛋白HbF，由2个α亚基和2个γ亚基组成(α2γ2)，这是胎儿发育期间及出生后血红蛋白的主要形式。刚出生时HbF可占全部血红蛋白70％，但随着年龄增长，γ亚基表达开始减少，而β亚基开始增多，成年后HbF含量一般不超过总血红蛋白的1％。β地中海贫血患者由于编码β亚基的基因出现突变，从而导致患者体内β亚基合成不足，HbA降低，进而导致严重贫血。
目前，大部分重症β地贫患者依靠长期输血与排铁剂连用进行治疗，然而这样的长期治疗会造成病人脏器损伤，肝脾肿大，预期寿命缩短。可能治愈中重度β地中海贫血的方法是异体造血干细胞移植，但其配型成功率低且费用十分昂贵，给家庭、社会都会带来很大的负担。自体造血干细胞基因疗法首先采集病人自体的携带变异基因的造血干细胞，在体外短暂培养并且导入正常基因和相应表达调控元件后，再把细胞移植入病人体内，治疗可以带来长期的改善。
近年来研究发现，B细胞淋巴瘤11A(BCL11A)基因是调节HbF表达的关键基因。在胎儿出生后，BCL11A基因抑制HbF的表达。对此，研究人员通过CRISPR/Cas系统敲除患者自体造血干细胞BCLl1A基因的增强子区域，抑制BCL11A基因的表达，从而使HbF重新表达，代偿发生突变的HbA，以达到治疗β地中海贫血的目的。
CRISPR/Cas系统全名为成簇的规律间隔的短回文重复(CRISPR)/ CRISPR相关(Cas)系统(clustered regularly interspaced palindromic repeats/CRISPR-associated proteins)，为目前发现存在于大多数细菌与所有的古菌中的一种后天免疫系统，以消灭外来的质体或者噬菌体。由于其对去氧核糖核酸干扰的特性，目前被积极地应用于遗传工程中，作为基因剪辑工具，与锌指核酸酶(Zinc Finger nuclease)及类转录活化因子核酸酶(Transcription Activator-Like Effect or Nuclease；TALEN)同样利用非同源性末端接合(Non-homologous end joining；NHEJ)的机制，于基因体中产生去氧核糖核酸的双股断裂以利剪辑。目前已发现三种不同类型的CRISPR/Cas系统存在，其中第二型的组成较为简单，以Cas9以及向导RNA(guide RNA，gRNA)为核心组成，并经由遗传工程的改造应用于哺乳类细胞及斑马鱼的基因体剪辑。Cas9是一种RNA介导的DNA内切酶，通过一个与目标序列互补的gRNA引导定位到基因组的目标序列上，此目标序列必须与一个以NGG或NAG形式的PAM(Protospacer Adjacent Motif)序列相邻。在与目标序列结合后，在特定基因组区域产生单链断裂(SSB)或双链断裂(DSB)，从而激活机体的非同源末端连接(NHEJ)或同源性修复途径(HDR)等修复机制。当在NHEJ修复机制下，由于没有模板，DNA只是随机修复以恢复其双链结构，这会使修复结果与原基因组序列不同，形成突变，导致目标序列的基因在所述细胞中减少或消除。近几年来，已被改造的CRISPR/Cas9系统已经用于真核细胞中的基因组编辑。
发明内容
本发明第一方面公开了一种编辑造血干/祖细胞中BCL11A基因的方法，发明人意外地发现编辑BCL11A基因增强子+62位对提高HbF表达量效果好，例如，基因编辑过的造血干/祖细胞分化成HbF阳性红细胞的比例大于65％。所述方法包括：将一种或多种靶向BCL11A基因增强子+62位的gRNA和一种或多种核酸内切酶引入所述细胞中，造成BCL11A基因增强子内或其附近产生单链断裂(SSB)或双链断裂(DSB)，例如，导致所述细胞中45％-80％的BCL11A基因增强子的删除或失活，相应地造成所述造血干/祖细胞分化成HbF阳性红细胞的比例大于65％；其中，所述gRNA包含crRNA和tracrRNA，所述BCL11A增强子+62位序列参见SEQ ID NO:14。在造血干/祖细胞中高效删除BCL11A基因，解除BCL11A基因对HbF的抑制作用，获得高水平表达的HbF，代偿发生突变的HbA，以达到治疗贫血性疾病的目的。
其中，所述crRNA的引导序列选自
a.SEQ ID NO:1，
b.SEQ ID NO:2，
c.SEQ ID NO:3，
d.SEQ ID NO:4，
e.SEQ ID NO:5，
f.SEQ ID NO:6，
g.a-f中任一项，其5’或3’端任选地添加或缺失1-4个碱基，优选地，添加或缺失的碱基与所述目标DNA对应位置的碱基互补。
优选地，所述crRNA的引导序列选自SEQ ID NO:1或SEQ ID NO:2，
或是SEQ ID NO:1或SEQ ID NO:2，其中在SEQ ID NO:1或SEQ ID NO:2的5’或3’端任选地添加或缺失1-4个碱基，优选地，添加或缺失的碱基与所述目标DNA对应位置的碱基互补，
其中相应地造成所述造血干/祖细胞分化成HbF阳性红细胞的比例大于70％。
进一步地，所述核酸内切酶选自Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csn1和Csx12)、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4或Cpf1，其同系物，其天然存在的分子、其密码子优化形式、其修饰形式的重组形式或其突变形式以及其组合；优选地，所述核酸内切酶为Cas9。
进一步地，所述tracrRNA的序列如SEQ ID NO:7所示，或经替换、缺失和/或在其5’至3’端任选地添加1-4个碱基形成的与SEQ ID NO:7具有同等功能的核苷酸序列。
进一步地，所述gRNA不经2’-O-甲基化和/或核苷酸间3’硫代磷酸酯修饰。而目前现有技术大多对gRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰，比如化学修饰为在gRNA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-甲基化。本发明意外地发现，对gRNA不进行上述化学修饰，编辑效率更高。
进一步地，所述方法包括将所述核酸内切酶与gRNA预先混合以形成一 种或多种RNP复合物。
进一步地，所述一种或多种RNP复合物通过电穿孔转染方式递送到所述造血干/祖细胞中，所述方法能够保证gRNA的编辑效率高，降低成本，符合药品申报的质量。而现有技术中多数是采用通过慢病毒的方式导入核酸内切酶和gRNA，所述慢病毒的方法在临床上会导致易诱发白血病。
优选地，所述RNP复合物中核酸内切酶与gRNA的摩尔比为1:(0.4-5.5)，例如1:0.5；1:1；1:1.5；1:2；1:2.5；1:3；1:3.5；1:4；1；4.5；1:5，进一步优选地，所述RNP复合物中核酸内切酶与gRNA的摩尔比为1:(1-2.5)。
进一步地，所述造血干/祖细胞是哺乳动物细胞、灵长类动物细胞，优选是灵长类动物细胞，进一步优选是人细胞。
本方面第二发明提供了一种提高造血干/祖细胞经红系分化后HbF表达的方法，包括以下步骤：
1)使用第一方面任一项所述的方法编辑造血干/祖细胞的BCL11A基因；
2)将步骤1)中所述的造血干/祖细胞进行分化。
本发明第三方面提供了一种造血干/祖细胞，所述造血干/祖细胞通过第一方面中任一项所述的方法得到，并且所述造血干/祖细胞分化成的红细胞表现出的HbF/(HbF+HbA)比值为15％以上与，优选地，为15％-45％；更优选为18％-25％。
本发明第四方面提供了第三方面所述的造血干/祖细胞在制备提高哺乳动物(优选人)HbF表达的药物或医用制品中的用途。
进一步地，本发明提供了第三方面所述的造血干/祖细胞在制备药物或医用制品中的用途，所述药物或医用制品用于预防或治疗贫血性疾病、失血性疾病、肿瘤或其他需要大量输血进行预防或治疗的疾病，优选地，所述贫血性疾病为β地中海贫血或镰刀形红细胞贫血。
本发明第五方面提供了一种或多种向导核糖核酸(gRNA)，其用于编辑造血干/祖细胞的BCL11A基因，所述gRNA包含crRNA和tracrRNA，gRNA的形式可以是由crRNA和tracrRNA组成的二聚体，也可以是人工改造的由crRNA和tracrRNA融合而成的sgRNA；所述crRNA的引导序列选自
a.SEQ ID NO:1，
b.SEQ ID NO:2，
c.SEQ ID NO:3，
d.SEQ ID NO:4，
e.SEQ ID NO:5，
f.SEQ ID NO:6，
g.a-f中任一项，其5’或3’端任选地添加或缺失1-4个碱基，优选地，添加或缺失的碱基与BCL11A基因对应位置的碱基互补；
所述tracrRNA的序列如SEQ ID NO:7所示，或经替换、缺失和/或在其5’至3’端任选地添加1-4个碱基形成的与SEQ ID NO:7具有同等功能的核苷酸序列。
进一步地，所述gRNA不经2’-O-甲基化修饰和/或核苷酸间3’硫代磷酯修饰。
本发明还提供了一种试剂盒，所述试剂盒包含第五方面任一项所述的gRNA和一种或多种核酸内切酶，所述核酸内切酶选自Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csn1和Csx12)、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4或Cpf1，其同系物，其天然存在的分子、其密码子优化形式、其修饰形式的重组形式或其突变形式以及其组合，更优选地，所述核酸内切酶为为Cas9。
进一步地，所述试剂盒用于治疗或预防受试者贫血性疾病、失血性疾病、肿瘤或其他需要大量输血进行预防或治疗的疾病。
本发明还提供了BCL11A基因片段作为靶点用于设计提高受试者体内血红蛋白朔评的药物的应用，所述BCL11A基因片段选自与以下各项完全互补的序列组成的组：
a.SEQ ID NO:1，
b.SEQ ID NO:2，
c.SEQ ID NO:3，
d.SEQ ID NO:4，
e.SEQ ID NO:5，
f.SEQ ID NO:6，
g.a-f中任一项，其5’或3’端任选地添加或缺失1-4个碱基，所述添加或 缺失的碱基是BCL11A基因对应位置的碱基。
定义：
CD34+细胞：CD34分子是高度糖基化的跨膜糖蛋白，选择性地表达于人类及其哺乳动物造血干/祖细胞表面。所以本文所述的CD34阳性细胞代表造血干/祖细胞。
B细胞淋巴瘤11A(BCL11A)基因：是一个原癌基因，它在人体淋巴结、胸腺和骨髓组织中表达较丰富，大部分其他组织中存在低水平的表达。BCL11A基因的增强子能够负向调控胎儿血红蛋白(HbF)的表达，其中，根据BCL11A增强子距转录起始位点的距离(千碱基数计)定义增强子+55位(距离转录起始位点+55kb的区域，kb表示1000个碱基)、+58位(距离转录起始位点+58kb的区域，kb表示1000个碱基)、+62位(距离转录起始位点+62kb的区域，kb表示1000个碱基)，上述位置是负调控胎儿血红蛋白(HbF)的关键调控区域。BCL11A+62位的碱基序列(参见序列SEQ ID NO:14)位于人类第二号染色体的第60,717,492至60,718,860的区域(相对于人hg19基因组)(参见参见Erythroid Enhancer of BCL11A Subject to Genetic Variation Determines Fetal Hemoglobin Level,Daniel E.Bauer et al.,Science,vol.342,Supplementary Material)。这些区域被编辑破坏后从而影响BCL11A蛋白质的表达，并最终用于富集HbF。
本文中所述的CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated endonumclease 9)基因编辑系统，是一种人工核酸内切酶基因编辑系统，是现有技术常用的一种基因敲除手段。CRISPR/Cas9由两部分构成：1)向导RNA，以下也称为gRNA，和2)核酸内切酶Cas9；本文所述gRNA，包括crRNA(CRISPR-derived RNA)和tracrRNA(trans-activating RNA)。所述crRNA和tracrRNA以以下所述方式中的一种起作用：1)tracrRNA/crRNA二聚体，crRNA的部分序列与tracrRNA的部分序列互补、并形成二聚体，本领域技术人员可以理解的是，crRNA的一部分碱基序列是与目标DNA序列互补的引导序列，另一部分碱基序列与tracrRNA的一部分序列通过碱基配对结合在一起，形成嵌合RNA(即tracrRNA/crRNA二聚体)；2)crRNA与tracrRNA融合成为一条嵌合型单链向导RNA(single guide RNA，sgRNA)，crRNA位于tracrRNA的5’端，其中所述crRNA包括 引导序列。gRNA的tracrRNA序列可以是本领域技术人员知晓的常规tracrRNA序列，即如本领域技术人员知晓何种tracrRNA序列能够用于实现本发明，如本领域技术人员知晓SEQ ID NO:7可以作为tracrRNA；进一步地，本领域技术人员知晓与常规使用的tracrRNA序列，如SEQ ID NO:7，具有50％～99％，优选60％～99％，更优选70％～99％，还优选80％～99％，甚至优选90％～99％的同一性的突变体也可以用于实现本发明。本领域技术人员可以理解的是，gRNA包含引导序列和骨架序列，gRNA的形式可以是由crRNA和tracrRNA组成的二聚体，也可以是人工改造的由crRNA和tracrRNA融合而成的嵌合型单链sgRNA，所以骨架序列可以是由crRNA和tracrRNA组成的二聚体，也可以人工改造的由crRNA和tracrRNA融合而成的嵌合型单链sgRNA，且骨架序列为本领域技术人员所知晓的；Cas9核酸内切酶用于切割DNA的活性结构域，使得DNA断裂。
本文中所述的核糖核酸蛋白(ribonucleoprotein，RNP复合物)，是指包含有RNA的蛋白复合物，即将核酸和蛋白质结合在一起的一种形式。在CRISPR/Cas9系统中，gRNA与Cas9结合形成Cas9-gRNA复合物，所述复合物与PAM毗邻的匹配靶DNA结合后，Cas9发生构象改变，激发内切酶活性，引起DNA单链断裂(SSB)或双链断裂(DSB)，细胞将通过非同源末端连接(NHEJ)或同源定向修复(HDR)等方式修复。
造血干/祖细胞分化成HbF阳性红细胞的比值：所述比值是使用流式细胞术检测得到，用于表征表达了HbF的红细胞占红细胞总数的比值(其中所述红细胞是经造血干/祖细胞分化获得的)，其直接地反应表达了HbF的红细胞占红细胞总数的比值。
HbF/(HbF+HbA)：所述比值通过高效液相色谱(HPLC)检测获得，具体地，其为在HPLC图谱中，HbF峰面积归一化比值占HbF与HbA峰面积归一化比值之和的比例；用于表征HbF的量占HbF与HbA总量之和的比值。
附图说明
图1：实验组a使用gRNA-1对BCL11A基因编辑后的CD34+细胞分化成的红细胞中表达HbF细胞的百分比的FACS散点图，横轴为HbF的荧光强度，FSC通道代表前向散射光。
图2：实验组b使用gRNA-2对BCL11A基因编辑后的CD34+细胞分化 成的红细胞中表达HbF细胞的百分比的FACS散点图，横轴为HbF的荧光强度，FSC通道代表前向散射光。
图3：实验组c使用gRNA-3对BCL11A基因编辑后的CD34+细胞分化成的红细胞中表达HbF细胞的百分比的FACS散点图，横轴为HbF的荧光强度，FSC通道代表前向散射光。
图4：实验组d使用gRNA-4对BCL11A基因编辑后的CD34+细胞分化成的红细胞中表达HbF细胞的百分比的FACS散点图，横轴为HbF的荧光强度，FSC通道代表前向散射光。
图5：实验组e使用gRNA-5对BCL11A基因编辑后的CD34+细胞分化成的红细胞中表达HbF细胞的百分比的FACS散点图，横轴为HbF的荧光强度，FSC通道代表前向散射光。
图6：实验组f使用gRNA-6对BCL11A基因编辑后的CD34+细胞分化成的红细胞中表达HbF细胞的百分比的FACS散点图，横轴为HbF的荧光强度，FSC通道代表前向散射光。
图7：对照组gBCL11A基因未经编辑后的CD34+细胞分化成的红细胞中表达HbF细胞的百分比的FACS散点图，横轴为HbF的荧光强度，FSC通道代表前向散射光。
图8：实验组a利用gRNA-1基因编辑的CD34+细胞分化成的红细胞的HbF表达量HPLC检测结果，X轴表示时间(单位：min)，Y轴表示峰高。
图9：实验组b利用gRNA-2基因编辑的CD34+细胞分化成的红细胞的HbF表达量HPLC检测结果，X轴表示时间(单位：min)，Y轴表示峰高。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施方式中使用的缩写名称解释详见表1：
表1缩写名称解释
Figure PCTCN2021116914-appb-000001
实施例1：CD34+细胞培养
配制SFEM II培养基(Stemcell)用以培养CD34+细胞，配制的SFEM II培养基为在SFEM II培养基中添加100ng/mL SCF(购自近岸蛋白质科技有限公司)，100ng/mL Flt3-L(购自近岸蛋白质科技有限公司)，100ng/mL TPO(购自近岸蛋白质科技有限公司)，100ng/mL IL-6(购自近岸蛋白质科技有限公司)和1％青霉素-链霉素双抗(Gibco)。
CD34+细胞复苏后置于配制的SFEM II培养基(Stemcell)中预刺激，浓度为0.25×106细胞/mL，细胞在37℃下放置于CO2培养箱中，预刺激48h。
实施例2：电穿孔转染外周血来源的CD34+细胞
(1)取实施例1中获得的状态良好的CD34+细胞，吸吹混匀后进行细胞计数；
(2)分别吸取5×104个细胞至7个15mL离心管中，编号为a-g。后续将编号a-g的离心管分别采用以下条件进行电穿孔转染：
实验分组：a.gRNA-1
b.gRNA-2
c.gRNA-3
d.gRNA-4
e.gRNA-5
f.gRNA-6
g.对照(不进行电穿孔转染)
本发明实施例中使用的gRNA是由crRNA与tracrRNA两部分退火形成的tracrRNA/crRNA二聚体，实验组中使用的各个gRNA的tracrRNA如SEQ ID NO:7所示，与tracrRNA配对的crRNA骨架序列为GUUUUAGAGCUAUGCU，crRNA的引导序列见表2：
表2实验组中gRNA的crRNA的引导序列
Figure PCTCN2021116914-appb-000002
(3)分别将步骤(2)中的细胞在离心力为100g下离心10min，吸去上清；
(4)分别取1mL PBS重悬细胞沉淀，离心10min，吸去上清；
(5)制备RNP复合物：按照步骤2)中设计的电穿孔转染条件，分别将2.1μL PBS、1.2μL gRNA(120pmol)和1.7μL Cas9(IDT,105pmol)轻轻混匀，在室温下放置15min，制备好的RNP复合物放置于4度冰箱备用；
(6)配置20μL的电穿孔转染液，电穿孔转染液为将P3 Primary Cell 4D-NucleofectorTM X Kit S试剂盒(Lonza)中的16.4μL Nucleofector Solution与3.6μL Supplement 1混合，电穿孔转染液放置至常温使用；使用20μL电穿孔转染液重悬各组细胞；
(7)电穿孔转染
分别将各组20μL细胞悬液加入到5μL对应RNP复合物和3.85μM电穿孔增强剂(IDT)中，混匀后，将25μL细胞悬液加入P3 Primary Cell 4D-NucleofectorTM X Kit S试剂盒(Lonza)中提供的专用电击杯中；
将电击杯放入电穿孔转染仪(Lonza)中，执行电穿孔转染程序；
(8)电穿孔转染后，各组立刻加入75μL新鲜SFEM II培养基(Stemcell)，混匀，吸取电击杯中的细胞悬液，在培养箱恢复培养30min后，加入96孔板中继续培养。
实施例3：电穿孔转染后CD34+细胞基因组提取
(1)各实验组CD34+细胞电穿孔转染后继续培养72h后回收细胞；
(2)手动提取各实验组基因组：
在离心力为500g下离心5min，500μL DNA抽提缓冲液(50mM/L Tris-HCl(pH＝8.0),100mM/L EDTA(pH＝8.0),100mM/L NaCl,1％SDS)重悬细胞沉淀；向其中加入200μL饱和NaCl溶液，剧烈震荡混匀；在温度4℃，转速13000rpm条件下离心10min；将所得上清转移至含有700μL异丙醇的EP管中，上下颠倒混匀出现类似小羽毛的物质(DNA)；在温度4℃，转速13000rpm下离心5min，弃上清；向其中加入600μL 75％的乙醇，上下颠倒，13000rpm离心5min，弃上清；开盖3-5min，使乙醇挥发；最后加20μL灭菌双蒸水溶解DNA。
实施例4：剪切效率验证
(1)根据每个实验组gRNA不同序列，选取包含gRNA编辑位点的片段进行扩增，扩增片段为500～600bp左右，且保证编辑的位点不要位于扩增片段中间，这样能切出两个不同大小的条带，且本领域技术人员知晓根据拟扩增片段设计合成用于扩增的引物序列。
(2)利用所设计的引物进行PCR扩增(引物序列如SEQ ID NO:8-13)，具体使用
Figure PCTCN2021116914-appb-000003
HS DNA Polymerase(Takara)进行PCR扩增，对PCR产物进行回收，其中PCR体系见表3：
Figure PCTCN2021116914-appb-000004
Figure PCTCN2021116914-appb-000005
其中SEQ ID NO:8和9用于扩增gRNA-1，gRNA-2或gRNA-4编辑位点附近约565bp的DNA片段；
其中SEQ ID NO:10和11用于扩增gRNA-3或gRNA-6编辑位点附近约501bp的DNA片段；
其中SEQ ID NO:12和13用于扩增gRNA-5编辑位点附近约564bp的DNA片段。
表3 PCR体系
5ⅹ缓冲液 10μL
dNTP 4μL
正向引物 1μL
反向引物 1μL
模板 1μL
聚合酶 0.5μL
双蒸水 32.5μL
(3)T7E1酶切鉴定剪切效率：使用T7核酸内切酶I试剂盒(金斯瑞)，其中酶切体系见表4：
表4酶切体系
PCR产物 200ng
10x反应缓冲液 2μl
无核酸酶水 17μl
T7核酸内切酶I 1μL
采用梯度退火程序：a预变性：在95℃下5min；b.梯度退火：95-85℃，-2℃/s；85-25℃，-0.1℃/s；4℃保持至退火完全。向退火后的PCR产物中加入1μL T7E1酶，37℃孵育15min，1.5％琼脂糖凝胶电泳检测酶切结果，各实验组的基因编辑效率(以gRNA-1编辑位点的片段进行扩增结果为例，电泳结果会呈现3条带，大小分别约为565bp、360bp和200bp。用Image J软件对条带进行灰度分析，计算切割效率。编辑效率＝(360bp条带灰度值+200bp条带灰度值)/3条条带灰度值总和×100％)见表5：
表5实验组a-f的BCL11A基因编辑效率
编号 基因编辑效率
a 60.5％
b 62.2％
c 51.0％
d 48.6％
e 55.7％
f 46.7％
实施例5：造血干细胞/祖细胞红系分化
电穿孔转染3天后的细胞依次转移到不同阶段的红系分化培养基中，此方法由3个阶段组成：分化(DIF)I(第0-4天)，分化(DIF)II(第5-8天)和分化(DIF)III(第9-16天)。其中红系分化基础培养基(EDM)包括IMDM(Gibco)，15％FBS，1％L-Glutamine(Gibco)，1％BSA(Sigma)，1％ITS(Gibco)，1％青霉素-链霉素双抗(Gibco)。
分化(DIF)I(第0-4天)，细胞培养在DIF I培养基中，DIF I培养基包括红细胞分化基础培养基(EDM)，1μM DEX(Sigma)，5ng/mL IL-3(购自近岸蛋白质科技有限公司)，100ng/mL SCF(购自近岸蛋白质科技有限公司)，6U/mL EPO(购自近岸蛋白质科技有限公司)，100nM gw7647(Sigma)；分化(DIF)II(第5-8天)，细胞培养在DIF II培养基中，DIF II培养基包括红系分化基础培养基(EDM)，50ng/mL SCF(购自近岸蛋白质科技有限公司)，6U/mL EPO(购自近岸蛋白质科技有限公司)，10nM gw7647(Sigma)；分化(DIF)III(第9-16天)，细胞培养在DIF III培养基中，DIF III培养基包括红系分化基础培养基(EDM)和2U/mL EPO(购自近岸蛋白质科技有限公司)。
实施例6：HbF表达量检测
6.1流式细胞术检测HbF阳性的红细胞比例
(1)采用分化后的红细胞为实验材料，分化后红细胞以PE-CD71抗体(Invitrogen)和PB-CD235a抗体(Invitrogen)标记红细胞群，染色固定后进行细胞破膜，加入APC-HbF单抗(Invitrogen)，上机进行流式分析检测HbF表达情况。
通过对分化后的红细胞进行流式检测，各个实验组的gRNA编辑 BCL11A基因后及未经编辑的CD34+细胞分化成HbF阳性红细胞的结果见附图1-7，具体地，实验组a-g的HbF阳性红细胞的比例见表6。
表6实验组a-g的HbF阳性红细胞的比例
编号 HbF阳性红细胞的比例
a 74.2％
b 71.9％
c 70.3％
d 70.2％
e 66.4％
f 65.8％
g 50.1％
上述结果说明经本发明所述的gRNA编辑的BCL11A基因后CD34+细胞分化成HbF阳性红细胞的比例均大于65％。
6.2 HPLC检测HbF表达量
(1)样品处理：
采用红系分化后的细胞为实验材料，收集1×10 7个细胞，用PBS洗一遍，100μL 0.01％SDS冰上裂解10min；12000rpm，离心5min后取上清20μL加入到80μL流动相A中，冰上孵育30min；
(2)HbF的HPLC检测条件：
色谱柱：PolyLC Inc.item#3.54CT0315
上样量：20μL
流速：1.2mL/min
流动相A：20mM bis-tris+2mM KCN,pH 6.9
流动相B：20mM bis-tris+2mM KCN+200mM NaCl,pH 6.57
紫外检测波长：415nm
通过整合HbF峰下的面积来进行血红蛋白的直接定量。实验组a-g中BCL11A基因被编辑后的或未编辑的CD34+细胞分化后的红细胞中表达的HbF的峰面积占总峰面积的比例见表7，经gRNA-1和gRNA-2编辑后的CD34+细胞分化的红细胞表达HbF的HPLC检测结果见附图8和9。
表7实验组a-g中CD34+细胞分化后的红细胞中表达的HbF的峰面积占总峰面积比例
编号 HbF峰面积占总峰面积的比例
a 19.8％
b 20.4％
c 17.0％
d 17.6％
e 16.9％
f 16.2％
g 14.9％
实验组a-g中CD34+细胞分化成的红细胞表现出的HbF/(HbF+HbA)比值见表8。
表8实验组a-g中CD34+细胞分化后的红细胞表现出的HbF/(HbF+HbA)比值
编号 HbF/(HbF+HbA)
a 21.9％
b 23.2％
c 20.8％
d 20.9％
e 18.9％
f 18.1％
g 17.4％
上述结果说明经本发明所述的gRNA编辑BCL11A基因后CD34+细胞分化成的红细胞表现出的HbF/(HbF+HbA)比值在15％以上，具体地在15％-45％范围内。
实施例7：未修饰的gRNA与经修饰过的gRNA对BCL11A基因编辑效率的影响
使用gRNA编辑BCL11A基因，与实验组不同的是，对照组使用的gRNA为定制的经修饰过的gRNA，gRNA的修饰方法为：在实验组使用的gRNA的5’末端3个核苷酸经2'-O-甲基化和核苷酸间3’-硫代磷酸酯修饰。如下述化学修饰所示，左侧是化学修饰后的gRNA，右侧是未经过修饰的gRNA。经修饰的gRNA与Cas9体外制备RNP复合物后，电穿孔转染造血干/祖细胞，检测编辑效率。实验证明，未经修饰的gRNA有更高的编辑效率，详见表9：
Figure PCTCN2021116914-appb-000006
表9未经修饰的gRNA与经修饰过的gRNA对BCL11A基因编辑效率的影响
Figure PCTCN2021116914-appb-000007
实施例8：gRNA以电穿孔转染RNP复合物和包装慢病毒两种方式导入造血干/祖细胞对基因编辑效率的影响
使用gRNA编辑BCL11A基因，与实验组a和b(采用电穿孔转染将gRNA导入造血干细胞)不同的是，对照组3和4(对应采用实验组a和b的gRNA)以包装慢病毒的方式将gRNA(gRNA-1和gRNA-2)导入造血干/祖细胞。具体地用脂质体方法将包装质粒以及携带CRSPR/Cas9基因和gRNA序列的慢病毒载体，共同转染293T细胞，从上清中得到慢病毒载体，将慢病毒载体与造血干/祖细胞共培养，得到被感染的造血干/祖细胞。具体步骤为：
(1)在37℃下用BsmBI(NEB)对5ug慢病毒CRISPR质粒(如LentiCRISPRv2，Addgene)进行消化和去磷酸化处理30分钟；
(2)使用凝胶提取试剂盒(Thermo)凝胶纯化酶切的质粒并在水中洗 脱；
(3)磷酸化并退火每对寡核苷酸：
使用以下参数将磷酸化/退火反应放入PCR仪中：
37℃30分钟；
95℃5分钟，然后以5℃/min的速度降至25℃；
(4)将步骤(3中)的退火寡核苷酸以1：200的稀释比例稀释到无菌水或EB中；
(5)设置连接反应并在16℃下孵育过夜：
(6)转化克隆感受态DH5α(全式金)；
(7)将长出的克隆送去测序，选择正确的克隆进行后续实验；
(8)将正确的克隆进行质粒大规模提取，用于慢病毒包装；
(9)为了产生慢病毒，将HEK293T细胞与补充了10％FBS(Gibco)和1％青霉素-链霉素(Gibco)的DMEM(Gibco)培养，并传代于15厘米组织培养培养皿中；
(10)使用脂质体(Lipofectamine 3000(Thermo))转染的方法包装慢病毒，接种细胞至70–90％汇合度时转染，使用Opti-MEM TM(Gibco)培养基稀释质粒，其中含有13.3mg psPAX2(addgene)、6.7mg VSV-G(addgene)和20mg目的慢病毒构建质粒，转染后48小时和72小时收集慢病毒上清液，然后通过超速离心，温度4℃，转速24,000rpm离心2小时，进行浓缩；
(11)用qPCR进行慢病毒滴度检测后，感染CD34+细胞后进行红系分化，测定编辑效率以及HbF的表达情况。
如实施例4所述检测编辑效率。实验表明将gRNA采用RNP复合物的方式比包装慢病毒的方式导入造血干/祖细胞基因编辑效率更高，详见表10：
表10 gRNA以电穿孔转染RNP复合物和包装慢病毒两种方式导入造血干/祖细胞对基因编辑效率的影响
编号 导入gRNA的方式 编辑效率
实验组a RNP 60.5％
实验组b RNP 62.2％
对照组3 慢病毒 49.3％
对照组4 慢病毒 51.8％
实施例9：核酸内切酶Cas9与gRNA不同摩尔比例对CD34+细胞的基 因编辑效率及分化成的红细胞中HbF阳性率的影响
重复实施例1-6的步骤，使用gRNA-1编辑BCL11A基因，与实验组a不同的是，示例组5-10(采用实验组a的gRNA)改变核酸内切酶Cas9与gRNA-1摩尔比例，实验证明在本发明所述比例下基因编辑效率高，且最终使编辑过的造血干/祖细胞分化成的红细胞的HbF阳性率高，详见表11：
表11 Cas9与gRNA-1不同摩尔比例对BCL11A基因的编辑效率及分化成的红细胞中HbF阳性率的影响
Figure PCTCN2021116914-appb-000008
本发明已经通过上述实施例进行了说明，但应当理解的是，上述实施例只是用于举例和说明的目的，而非意在将本发明限制于所描述的实施例范围内。此外本领域技术人员可以理解的是，本发明并不局限于上述实施例，根据本发明的教导还可以做出更多的变型和修改，这些变型和修改均落在本发明所要求保护的范围以内。本发明的保护范围由附属的权利要求书及其等效范围所界定。

Claims (18)

  1. 一种编辑造血干/祖细胞中BCL11A基因的方法，所述方法包括：
    将一种或多种靶向BCL11A基因增强子+62位的gRNA和一种或多种核酸内切酶引入所述细胞中；
    其中，所述BCL11A增强子+62位序列参见SEQ ID NO:14，所述gRNA包含crRNA和tracrRNA；
    其中，所述crRNA包括与目标DNA配对的引导序列，该引导序列选自
    a.SEQ ID NO:1，
    b.SEQ ID NO:2，
    c.SEQ ID NO:3，
    d.SEQ ID NO:4，
    e.SEQ ID NO:5，
    f.SEQ ID NO:6，
    g.a-f中任一项，其5’或3’端任选地添加或缺失1-4个碱基，优选地，添加或缺失的碱基与所述目标DNA对应位置的碱基互补。
  2. 如权利要求1所述的方法，所述crRNA的引导序列选自
    SEQ ID NO:1或SEQ ID NO:2，
    或是SEQ ID NO:1或SEQ ID NO:2，其中在SEQ ID NO:1或SEQ ID NO:2的5’或3’端任选地添加或缺失1-4个碱基，优选地，添加或缺失的碱基与所述目标DNA对应位置的碱基互补。
  3. 如权利要求1-2任一项所述的方法，所述核酸内切酶选自Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4或Cpf1，其同系物，其天然存在的分子、其密码子优化形式、其修饰形式的重组形式或其突变形式以及其组合，优选地，所述核酸内切酶为Cas9。
  4. 如权利要求1-3任一项所述的方法，所述tracrRNA的序列如SEQ ID NO:7所示，或经替换、缺失和/或在其5’至3’端任选地添加1-4个碱基形成的与SEQ ID NO:7具有同等功能的核苷酸序列。
  5. 如权利要求1-4任一项所述的方法，所述gRNA不经2’-O-甲基化和/或核苷酸间3’硫代磷酸酯修饰。
  6. 如权利要求1-5任一项所述的方法，所述方法包括将所述核酸内切酶与所述gRNA预先混合以形成一种或多种RNP复合物。
  7. 如权利要求1-6任一项所述的方法，所述一种或多种RNP复合物通过电穿孔转染的方式递送到所述造血干/祖细胞中。
  8. 如权利要求6所述的方法，所述RNP复合物中核酸内切酶与gRNA的摩尔比为1:(0.4-5.5)，优选地，所述RNP中核酸内切酶与gRNA的摩尔比为1:(1-2.5)。
  9. 如权利要求1-8任一项所述的方法，所述造血干/祖细胞是哺乳动物细胞、灵长类动物细胞，优选是灵长类动物细胞，进一步优选是人细胞。
  10. 一种提高造血干/祖细胞经红系分化后HbF表达的方法，包括以下步骤：
    1)使用权利要求1-9中任一项所述的方法编辑造血干/祖细胞的BCL11A基因；
    2)将步骤1)中所述造血干/祖细胞进行分化。
  11. 一种造血干/祖细胞，所述造血干/祖细胞通过权利要求1-9中任一项所述的方法得到，并且所述造血干/祖细胞分化成的红细胞表现出的HbF/(HbF+HbA)比值为15％以上，优选地，为15％-45％；更优选为18％-25％。
  12. 权利要求11所述的细胞在制备提高哺乳动物(优选人)HbF表达的药物或医用制品中的用途。
  13. 权利要求11所述的细胞在制备药物或医用制品中的用途，所述药物或医用制品用于预防或治疗贫血性疾病、失血性疾病、肿瘤或其他需要大量输血进行预防或治疗的疾病，优选地，所述贫血性疾病为β地中海贫血或镰刀形红细胞贫血。
  14. 一种或多种向导核糖核酸(gRNA)，所述gRNA用于编辑造血干/祖细胞的BCL11A基因，所述gRNA包含crRNA和tracrRNA，所述crRNA的引导序列选自
    a.SEQ ID NO:1，
    b.SEQ ID NO:2，
    c.SEQ ID NO:3，
    d.SEQ ID NO:4，
    e.SEQ ID NO:5，
    f.SEQ ID NO:6，
    a-f中任一项，其5’或3’端任选地添加或缺失1-4个碱基，优选地，添加或缺失的碱基与所述目标DNA对应位置的碱基互补；
    所述tracrRNA的序列如SEQ ID NO:7所示，或经替换、缺失和/或在其5’或3’端任选地添加1-4个碱基形成的与SEQ ID NO:7具有同等功能的核苷酸序列。
  15. 如权利要求14所述的gRNA，所述gRNA不经2’-O-甲基化和/或核苷酸间3’硫代磷酸酯修饰。。
  16. 一种试剂盒，所述试剂盒包含权利要求14-15任一项所述的gRNA和一种或多种核酸内切酶，所述核酸内切酶选自Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csn1和Csx12)、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4或Cpf1，其同系物，其天然存在的分子、其密码子优化形式、其修饰形式的重组形式或其突变形式以及其组合；更优选地，所述核酸内切酶为Cas9。
  17. 如权利要求16所述的试剂盒，所述试剂盒用于治疗或预防受试者贫血性疾病、失血性疾病、肿瘤或其他需要大量输血进行预防或治疗的疾病。
  18. BCL11A基因片段作为靶点用于设计提高受试者体内血红蛋白水平的药物的应用，所述BCL11A基因片段选自与以下各项完全互补的序列组成的组：
    a.SEQ ID NO:1，
    b.SEQ ID NO:2，
    c.SEQ ID NO:3，
    d.SEQ ID NO:4，
    e.SEQ ID NO:5，
    f.SEQ ID NO:6，
    g.a-f中任一项，其5’或3’端任选地添加或缺失1-4个碱基，所述添加或缺失的碱基是BCL11A基因对应位置的碱基。
PCT/CN2021/116914 2020-09-08 2021-09-07 编辑造血干/祖细胞中bcl11a基因的方法 WO2022052909A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010933224.3 2020-09-08
CN202010933224.3A CN114149990A (zh) 2020-09-08 2020-09-08 编辑造血干/祖细胞中bcl11a基因的方法

Publications (1)

Publication Number Publication Date
WO2022052909A1 true WO2022052909A1 (zh) 2022-03-17

Family

ID=80461752

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/116914 WO2022052909A1 (zh) 2020-09-08 2021-09-07 编辑造血干/祖细胞中bcl11a基因的方法

Country Status (2)

Country Link
CN (1) CN114149990A (zh)
WO (1) WO2022052909A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160369262A1 (en) * 2015-06-18 2016-12-22 Sangamo Biosciences, Inc. Nuclease-mediated regulation of gene expression
CN108779462A (zh) * 2015-12-28 2018-11-09 诺华股份有限公司 用于治疗血红蛋白病的组合物和方法
CN109706148A (zh) * 2017-09-30 2019-05-03 广东赤萌医疗科技有限公司 一种用于敲除BCL11A基因或者BCL11A基因增强子的gRNA、gRNA组合物以及电转方法
US20190169597A1 (en) * 2016-08-19 2019-06-06 Bluebird Bio, Inc. Genome editing enhancers
US20190177709A1 (en) * 2016-08-24 2019-06-13 Sangamo Therapeutics, Inc. Regulation of gene expression using engineered nucleases
CN111278976A (zh) * 2017-10-27 2020-06-12 博雅辑因（北京）生物科技有限公司 一种提高胎儿血红蛋白表达的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160369262A1 (en) * 2015-06-18 2016-12-22 Sangamo Biosciences, Inc. Nuclease-mediated regulation of gene expression
CN108779462A (zh) * 2015-12-28 2018-11-09 诺华股份有限公司 用于治疗血红蛋白病的组合物和方法
US20190169597A1 (en) * 2016-08-19 2019-06-06 Bluebird Bio, Inc. Genome editing enhancers
US20190177709A1 (en) * 2016-08-24 2019-06-13 Sangamo Therapeutics, Inc. Regulation of gene expression using engineered nucleases
CN109706148A (zh) * 2017-09-30 2019-05-03 广东赤萌医疗科技有限公司 一种用于敲除BCL11A基因或者BCL11A基因增强子的gRNA、gRNA组合物以及电转方法
CN111278976A (zh) * 2017-10-27 2020-06-12 博雅辑因（北京）生物科技有限公司 一种提高胎儿血红蛋白表达的方法

Also Published As

Publication number Publication date
CN114149990A (zh) 2022-03-08

Similar Documents

Publication Publication Date Title
CN113939591A (zh) 编辑rna的方法和组合物
KR102338993B1 (ko) 인위적으로 조작된 조작면역세포
CN114007655A (zh) 用于细胞疗法的环状rna
CN112662674B (zh) 靶向编辑VEGFA基因外显子区域的gRNA及其应用
US20240042025A1 (en) Biallelic knockout of b2m
US20240175013A1 (en) Biallelic knockout of trac
WO2022150974A1 (en) Targeted rna editing by leveraging endogenous adar using engineered rnas
WO2022228471A1 (zh) 一种基因编辑的造血干细胞及其与car-t细胞的联合应用
US20230383275A1 (en) Sgrna targeting aqp1 rna, and vector and use thereof
WO2021243058A1 (en) Biallelic knockout of sarm1
EP3912629A1 (en) Method for delivering gene in cells
WO2021037232A1 (zh) 编辑造血干/祖细胞中bcl11a基因的方法
WO2022052909A1 (zh) 编辑造血干/祖细胞中bcl11a基因的方法
WO2024064623A2 (en) Biallelic knockout of cish
WO2024064683A2 (en) Biallelic knockout of ciita
WO2024064613A2 (en) Biallelic knockout of hla-e
WO2024064607A2 (en) Biallelic knockout of tet2
WO2024064633A2 (en) Biallelic knockout of pdcd1
WO2024064606A2 (en) Biallelic knockout of ctla4
WO2024064637A2 (en) Biallelic knockout of faslg
AU2022403003A1 (en) Reduced expression of sarm1 for use in cell therapy
WO2023102478A2 (en) Reduced expression of sarm1 for use in cell therapy
WO2022192508A1 (en) Strategies for knock-ins at c3 safe harbor sites
WO2022170007A1 (en) Fusion proteins for crispr-based transcriptional repression
WO2024020484A2 (en) Biallelic knockout of angptl3

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: 21865963

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21865963

Country of ref document: EP

Kind code of ref document: A1