WO2021037232A1 - Procédé d'édition de gène bcl11a dans des cellules souches/progénitrices hématopoïétiques - Google Patents

Procédé d'édition de gène bcl11a dans des cellules souches/progénitrices hématopoïétiques Download PDF

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WO2021037232A1
WO2021037232A1 PCT/CN2020/112224 CN2020112224W WO2021037232A1 WO 2021037232 A1 WO2021037232 A1 WO 2021037232A1 CN 2020112224 W CN2020112224 W CN 2020112224W WO 2021037232 A1 WO2021037232 A1 WO 2021037232A1
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seq
grna
cells
edited
hematopoietic stem
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焦娇
崔正之
栗飞红
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甘李药业股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • 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
    • C12N15/90—Stable introduction of foreign DNA into chromosome

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 HbF expression of artificial hematopoietic stem/progenitor cells after erythroid differentiation, and also relates to BCL11A edited hematopoietic stem/progenitor cells and the cells
  • a gRNA for editing hematopoietic stem/progenitor cells BCL11A and a kit containing the gRNA for the preparation of drugs or medical products for preventing or treating anemia diseases.
  • Beta 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), 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 the decrease in the quantity of hemoglobin or the lack of function can cause anemia.
  • Normal hemoglobin is composed of 4 subunits, the most common adult hemoglobin HbA is composed of 2 ⁇ subunits and 2 ⁇ subunits ( ⁇ 2 ⁇ 2), which is the main form of adult hemoglobin, accounting for more than 98%; the other It is fetal hemoglobin HbF, composed 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 total hemoglobin at birth, but with age, the expression of ⁇ subunits begins to decrease, while ⁇ subunits begin to increase, and HbF content generally does not exceed 1% of total hemoglobin in adulthood.
  • mutations in the gene encoding ⁇ -subunits result in insufficient ⁇ -subunit synthesis and decreased HbA, which in turn leads to severe anemia.
  • BCL11A B-cell lymphoma 11A
  • the BCL11A gene suppresses the expression of HbF.
  • the researchers used the CRISPR/Cas system to knock out the enhancer region of the BCL11A gene of the patient’s autologous hematopoietic stem cells to inhibit the expression of the BCL11A gene, thereby re-expressing HbF, compensating for the mutated HbA, in order to achieve the purpose of treating ⁇ -thalassemia .
  • CRISPR/Cas clustered regularly spaced short palindrome repeats (CRISPR)/CRISPR-associated (Cas) systems (clustered regularly interspaced palindromic repeats/CRISPR-associated proteins), which are currently found in most bacteria and all An acquired immune system in the ancient bacteria to destroy foreign plastids or bacteriophages.
  • DNAi DNA interference
  • TALEN transcription activator-like nuclease
  • NHEJ non-homologous end joining
  • CRISPR/Cas9 is an RNA-mediated DNA endonuclease, which is guided by a gRNA complementary to the target sequence to the target sequence of the genome.
  • This target sequence must be combined with a PAM (Protospacer Adjacent Motif) in the form of NGG or NAG. The sequence is adjacent.
  • PAM Protospacer Adjacent Motif
  • SSB single-strand break
  • DSB double-strand break
  • 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 enhancer +55 has a good effect on increasing HbF expression.
  • gene-edited hematopoietic stem/ The proportion of progenitor cells differentiated into HbF-positive red blood cells is greater than 70%.
  • the method includes: introducing one or more gRNAs targeting BCL11A gene enhancer +55 and one or more endonucleases into the cell, causing BCL11A enhancer +55 to be produced at or near Single-strand break (SSB) or double-strand break (DSB), for example, results in the deletion or inactivation of 50%-90% of the BCL11A enhancer + 55 in the cell, correspondingly causing the differentiation of the hematopoietic stem/progenitor cell
  • SSB Single-strand break
  • DSB double-strand break
  • the proportion of HbF-positive red blood cells is greater than 70%; wherein, the gRNA includes crRNA and tracrRNA, and the BCL11A enhancer +55 sequence is shown in SEQ ID NO: 94.
  • the guide sequence of the crRNA is selected from
  • any one of a to f, optionally adding or deleting 1-4 bases at the 5'or 3'end, preferably, the added or deleted base is complementary to the base at the corresponding position of the target DNA.
  • crRNA is selected from
  • the proportion of red blood cells that differentiate into HbF-positive hematopoietic stem/progenitor cells is greater than 75%.
  • the guide sequence of the crRNA is selected from
  • the ratio of the hematopoietic stem/progenitor cells differentiated into HbF-positive red blood cells is greater than 80%.
  • the endonuclease is selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2 , Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx1, Csx10, Csx16, Csx16 , Csf1, Csf2, Csf3, Csf4 or Cpf1, its homologs, its naturally occurring molecules, its codon-optimized form, its modified form of recombinant form or its mutant form, and combinations thereof; preferably, the endonuclease It
  • sequence of the tracrRNA is shown in SEQ ID NO: 7, or is formed by substitution, deletion, and/or optionally adding 1-4 bases to the 5'to 3'end of the tracrRNA and SEQ ID NO: 7 Nucleotide sequences with equivalent functions.
  • the gRNA is not modified by 2'-O-methylation and/or internucleotide 3'phosphorothioate modification.
  • most of the existing technologies have undergone 2'-O-methylation modification and/or internucleotide 3'phosphorothioate modification on gRNA, such as chemical modification such as one, two and one before the 5'end of the gRNA. / Or 2'-O-methylation modification of three bases and/or the last base at the 3'end.
  • the present invention has unexpectedly discovered that without the above-mentioned chemical modification on gRNA, the editing efficiency is higher.
  • the method includes pre-mixing the endonuclease and gRNA to form one or more RNPs.
  • the one or more RNPs are delivered to the hematopoietic stem/progenitor cells by electroporation transfection, which can ensure high editing efficiency of gRNA, reduce costs, and meet the quality of drug declaration.
  • endonuclease and gRNA are introduced by means of lentivirus, and the lentivirus method can cause leukemia to be induced clinically.
  • the molar ratio of endonuclease to gRNA in the RNP is 1:0.4-1:5.5, such as 1:0.5; 1:1; 1:1.5; 1:2; 1:2.5; 1:3; 1:3.5; 1:4; 1:4.5, more preferably, the molar ratio is 1:1-1:2.5.
  • 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 increasing HbF expression of hematopoietic stem/progenitor cells after erythroid differentiation, which includes the following steps:
  • the third aspect of the present invention provides a hematopoietic stem/progenitor cell, the hematopoietic stem/progenitor cell is obtained by the method of any one of the first aspect, and the red blood cell differentiated into the hematopoietic stem/progenitor cell exhibits
  • the ratio of HbF/(HbF+HbA) is more than 20%, for example, 20%-50%.
  • the inventors unexpectedly found that the HbF/(HbF+HbA) ratio of red blood cells differentiated into hematopoietic stem/progenitor cells edited by gRNA-1 was 47.6%, which was significantly higher than the reported level.
  • the fourth aspect of the present invention provides the use of the hematopoietic stem/progenitor cells of the third aspect in the preparation of drugs or medical products that increase the expression of HbF in mammals (preferably humans).
  • the present invention provides the use of the hematopoietic stem/progenitor cells of the third aspect in the preparation of medicines or medical products for the prevention or treatment of anemic diseases, hemorrhagic diseases, tumors or other diseases A disease that requires a large amount of blood transfusion for prevention or treatment.
  • the anemic disease is beta thalassemia or sickle cell anemia.
  • the fifth aspect of the present invention provides one or more guide ribonucleic acids (gRNA) for editing the BCL11A gene of hematopoietic stem/progenitor cells.
  • the gRNA includes crRNA and tracrRNA, and the form of gRNA can be composed of crRNA and tracrRNA
  • the dimer can also be an artificially modified sgRNA formed by the fusion of crRNA and tracrRNA; the guide sequence of the crRNA is selected from
  • any one of a-f optionally adding or deleting 1-4 bases at its 5'or 3'end, preferably, the added or deleted base is complementary to the base at the corresponding position of the BCL11A gene;
  • the sequence of the tracrRNA is shown in SEQ ID NO: 7, or is formed by substitution, deletion, and/or optionally adding 1-4 bases at its 5'to 3'end, which is equivalent to SEQ ID NO: 7 Functional nucleotide sequence.
  • the gRNA is not modified by 2'-O-methylation and/or internucleotide 3'phosphorothioate modification.
  • the guide sequence of the crRNA is SEQ ID NO: 1
  • the tracrRNA is SEQ ID NO: 7.
  • the present invention also provides a kit, for example, for the treatment or prevention of anemic diseases, hemorrhagic diseases, tumors or other diseases that require a large amount of blood transfusion for prevention or treatment in subjects, the kit comprising any one of the fifth aspect
  • the gRNA and one or more endonucleases described in item preferably the endonuclease is selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cbr6, Csb1, Csb2, Cs17 , Csx14, Csx10, Csx16, CsaX
  • the present invention also provides a method for increasing the level of hemoglobin in a subject, which comprises administering to the subject an effective amount of the hematopoietic stem/progenitor cells according to the third aspect.
  • the subject is a mammal, More preferred is human.
  • the present invention also provides the use of BCL11A gene fragments as targets for designing drugs that increase hemoglobin levels in subjects, the BCL11A gene fragments being selected from the group consisting of sequences that are completely complementary to the following:
  • Any one of a to f, optionally adding or deleting 1-4 bases 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 that are selectively expressed on the surface of human and mammalian hematopoietic stem/progenitor cells. Therefore, the CD34-positive cells described herein 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).
  • HbF fetal hemoglobin
  • the enhancer +55 position is defined according to the distance between the BCL11A enhancer and the transcription start site (in kilobases).
  • Point +55kb region, kb means 1000 bases), +58 (region +58kb from the transcription start site), +62 (region +62kb from the transcription start site), the above position is negative
  • the base sequence of BCL11A enhancer +55 (see SEQ ID NO: 94) is located in the region from 60,724,802 to 60,726,084 of human chromosome 2 (relative to the human hg19 genome) (see Erythroid Enhancer of BCL 11A Subject to Genetic Variation Determines Fetal Hemoglobin Level, Daniel E. Bauer et al., Science, vol. 342, Supplementary Material). These regions were edited and damaged to affect the expression of BCL11A, and ultimately used to enrich HbF.
  • the CRISPR/Cas9 gene editing system described herein is composed of two parts: 1) guide RNA, also referred to as gRNA hereinafter, and 2) endonuclease Cas9;
  • the gRNA described herein includes crRNA (CRISPR-derived RNA) and tracrRNA (trans-activating RNA), specifically one of the following 1) or 2): 1) tracrRNA/crRNA dimer, the partial sequence of crRNA is complementary to the partial sequence of tracrRNA, and forms a dimer, the technology in the art
  • a part of the base sequence of crRNA is a guide sequence complementary to the target DNA sequence, and the other part of the base sequence (3' end) and a part of the tracrRNA sequence (5' end) are joined together by base pairing ( About 13bp), forming a chimeric RNA (ie tracrRNA/crRNA dimer); 2) Single-stranded guide RNA (single guide RNA, sgRNA), fusion of crRNA and tracrRNA
  • the form of gRNA can be a dimer composed of crRNA and tracrRNA, or it can be an artificially modified fusion of crRNA and tracrRNA. Therefore, the skeleton sequence can be a dimer composed of crRNA and tracrRNA, or a chimeric single-stranded sgRNA formed by the fusion of crRNA and tracrRNA, and the skeleton sequence is a technology in the art Known to the person; Cas9 endonuclease is used to cut the active domain of double-stranded DNA, causing the double-stranded DNA to break.
  • the ribonucleoprotein (RNP complex) mentioned herein refers to a protein complex containing RNA, that is, a form of combining nucleic acid and protein.
  • RNA that is, a form of combining nucleic acid and protein.
  • Cas9 In the CRISPR/Cas9 system, gRNA and Cas9 combine to form a Cas9-gRNA complex. After the complex binds to the matched target DNA adjacent to PAM, Cas9 undergoes a conformational change, which stimulates endonuclease activity and causes DNA single-strand break (SSB). ) Or double-strand break (DSB), cells will be repaired through non-homologous end joining (NHEJ) or homologous directed repair (HDR).
  • NHEJ non-homologous end joining
  • HDR homologous directed repair
  • the ratio of hematopoietic stem/progenitor cells differentiated into HbF-positive red blood cells The ratio is detected by flow cytometry and used to characterize the ratio of HbF-expressing red blood cells to the total number of red blood cells (wherein the red blood cells are hematopoietic stem/progenitor cells). Differentiation), which reflects the ratio of red blood cells expressing HbF to the total number of red blood cells.
  • HbF/(HbF+HbA) The ratio is obtained by high-performance liquid chromatography (HPLC) detection. Specifically, it is the ratio of the normalized ratio of the peak area of HbF to the normalized ratio of the peak area of HbF and HbA in the HPLC spectrum The ratio of sum; used to characterize the ratio of the amount of HbF to 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 CD34+ cells edited by BCL11A gene using gRNA-1, the horizontal axis is the fluorescence intensity of HbF, and the SSC channel represents side-scattered light.
  • Figure 2 FACS scatter plot of the percentage of HbF-expressing cells in red blood cells differentiated from CD34+ cells after BCL11A gene editing using gRNA-2, the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents forward scattered light.
  • Figure 3 FACS scatter plot of the percentage of HbF-expressing cells in red blood cells differentiated from CD34+ cells after BCL11A gene editing using gRNA-3, 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 CD34+ cells after BCL11A gene editing using gRNA-4, the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents forward scattered light.
  • Figure 5 FACS scatter plot of the percentage of HbF-expressing cells in red blood cells differentiated from CD34+ cells after BCL11A gene editing using gRNA-5, the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents forward scattered light.
  • Figure 6 FACS scatter plot of the percentage of HbF-expressing cells in red blood cells differentiated into CD34+ cells after BCL11A gene editing using gRNA-6, the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents forward scattered light.
  • Figure 7 FACS scatter plot of the percentage of HbF-expressing cells in red blood cells differentiated from unedited CD34+ cells of the BCL11A gene.
  • the horizontal axis is the fluorescence intensity of HbF, and the FSC channel represents forward scattered light.
  • Figure 8 HPLC detection results of HbF expression of red blood cells differentiated from CD34+ cells edited with gRNA-1 gene, X-axis represents time (unit: min), Y-axis represents peak height.
  • Figure 9 HPLC detection results of HbF expression of red blood cells differentiated from CD34+ cells edited with gRNA-2 gene, X-axis represents time (unit: min), Y-axis represents peak height.
  • Figure 10 Genomic extraction of unedited CD34+ cells (Ctrl) and CD34+ cells (Edited) with gRNA-1 gene editing, and electrophoresis detection results of the obtained genome.
  • Figure 11a Electrophoresis detection results of PCR amplification products of 39 possible off-target sequences selected from CD34+ cells (Ctrl) and CD34+ cells (Edited) that have not undergone gene editing.
  • Figure 11b Electrophoresis detection results of PCR amplification products of some possible off-target sequences and target sites in CD34+ cells (Ctrl) without gene editing and CD34+ cells (Edited) after gene editing.
  • Figure 12 Clonal growth status and counting results of unedited and gRNA-1 edited CD34+ cells after in vitro differentiation observed under the microscope.
  • the two pictures on the left show unedited CD34+ cells (Ctrl) after in vitro differentiation
  • the two pictures on the right show the growth status of clones observed at different positions under the microscope after in vitro differentiation of gRNA-1 edited CD34+ cells (Edited).
  • Figure 13 Comparison of the number of clones of unedited CD34+ cells (Ctrl) and gRNA-1 edited CD34+ cells (Edited) into CFU-E, BFU-E, CFU-GM and CFU-GEMM .
  • Figure 14 In vitro tumorigenicity microscopic examination results of Hela cells, MRC-5 cells and CD34+ cells edited with gRNA-1.
  • the prepared SFEM II medium is to add 100ng/mL SCF (purchased from Nearshore Protein Technology Co., Ltd.) to SFEM II medium, 100ng/mL Flt3-L( (Purchased from Nearshore Protein Technology Co., Ltd.), 100ng/mL TPO (purchased from Nearshore Protein Technology Co., Ltd.), 100ng/mL IL-6 (purchased from Nearshore Protein Technology Co., Ltd.) and 1% penicillin-streptomycin double Anti (Gibco).
  • CD34+ cells were placed in the prepared SFEM II medium (Stemcell) for pre-stimulation at a concentration of 0.25 ⁇ 10 6 cells/mL. The cells were placed in a CO 2 incubator at 37° C. for pre-stimulation for 48 hours.
  • the gRNA used in the embodiment of the present invention is a tracrRNA/crRNA dimer formed by 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, the crRNA backbone sequence paired with tracrRNA For GUUUUAGAGCUAUGCU, the guide sequence of crRNA is shown in Table 1:
  • step (3) Centrifuge the cells in step (2) at a centrifugal force of 100g for 10 minutes, and aspirate the supernatant;
  • the electroporation transfection solution is to mix 16.4 ⁇ L Nucleofector Solution in P3 Primary Cell 4D-Nucleofector TM X Kit S (Lonza) with 3.6 ⁇ L Supplement 1 and electroporate the transfection solution. Place the staining solution at room temperature for use; use 20 ⁇ L of electroporation transfection solution to resuspend each group of cells;
  • CD34+ cells in each experimental group were transfected and cultured for 72 hours and then recovered;
  • the amplified fragments are 500 to 600 bp, and ensure that the edited sites are not in the middle of the amplified fragments, so that two can be cut out.
  • Bands of different sizes, and those skilled in the art know to design and synthesize primer sequences for amplification according to the fragments to be amplified.
  • T7E1-F1 CACTGGTTTCTCCCCTTGTCA (SEQ ID NO: 8)
  • T7E1-R1 CAAGCAGGAAGGGCCTCTAT (SEQ ID NO: 9)
  • SEQ ID NO: 8 and 9 are used to amplify DNA fragments of about 570 bp near the editing site of gRNA-1, gRNA-2 or gRNA-4;
  • SEQ ID NOs: 10 and 11 are used to amplify DNA fragments of about 530 bp near the editing site of gRNA-3 or gRNA-6;
  • SEQ ID NOs: 12 and 13 were used to amplify DNA fragments of approximately 520 bp near the gRNA-5 editing site.
  • T7E1 restriction enzyme digestion to identify the cutting efficiency use T7 Endonuclease I kit (GenScript), and the restriction enzyme digestion system is shown in Table 3:
  • Example 5 Erythroid differentiation of hematopoietic stem cells/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 (day 0-4), cells are cultured in DIF I medium, DIF I medium includes red blood cell differentiation basal medium (EDM), 1 ⁇ M DEX (Sigma), 5ng/mL IL-3 (purchase From Nearshore Protein Technology Co., Ltd.), 100ng/mL SCF (purchased from Nearshore Protein Technology Co., Ltd.), 6U/mL EPO (purchased from Nearshore Protein Technology Co., Ltd.), 100nM gw7647 (Sigma); Differentiation (DIF) II (Day 5-8), cell culture in DIF II medium, DIF II medium includes 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.), 10nM gw7647 (Sigma); differentiation (DIF) III (day 9-16), cell culture in DIF III medium, DIF III medium includes erythroid differentiation basic culture Base (EDM) and 2U
  • red blood cells were used as experimental materials. After differentiation, the red blood cells were labeled with PE-CD71 antibody (Invitrogen) and PB-CD235a antibody (Invitrogen). After staining and fixing, the cells were ruptured, and APC-HbF monoclonal antibody (Invitrogen) was added. Flow cytometry detection of HbF expression, the specific steps are as follows:
  • the differentiated red blood cells were centrifuged at a centrifugal force of 500g for 5 minutes, the supernatant was removed, and 50 ⁇ L FACS Buffer (PBS containing 2% FBS) was added to the PE-CD71 antibody (Invitrogen) and PB-CD235a antibody (Invitrogen), and the cells were resuspended ,Dyeing for 15min in the dark at 4°C;
  • FACS Buffer PBS containing 2% FBS
  • erythroid differentiated cells as experimental materials, collect 1 ⁇ 10 7 cells, wash them with PBS, and lyse 100 ⁇ L 0.01% SDS on ice for 10 min; centrifuge at 12000 rpm for 5 min and take 20 ⁇ L of the supernatant and add it to 80 ⁇ L mobile phase A. Incubate on ice for 30 minutes;
  • 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
  • Direct quantification of hemoglobin is performed by integrating the area under the HbF peak.
  • the ratio of the peak area of HbF expressed in red blood cells after differentiation of BCL11A or unedited CD34+ cells to the total peak area in the experimental group ag is shown in Table 6.
  • the differentiation of CD34+ cells edited by gRNA-1 and gRNA-2 The ratio of the peak area of HbF expressed in red blood cells to the total peak area is about 32.0% and 29.5%, respectively.
  • the HPLC detection results of HbF expressed by red blood cells differentiated by CD34+ cells edited by gRNA-1 and gRNA-2 are shown in the attached drawings. 8 and Figure 9.
  • Table 6 The ratio of HbF peak area expressed in red blood cells after differentiation of CD34+ cells in experimental groups a-g to the total peak area
  • the ratio of HbF/(HbF+HbA) shown by the red blood cells differentiated from CD34+ cells in the experimental groups a-g is shown in Table 7. According to the experiment of the present invention, the red blood cells differentiated into CD34+ cells edited by gRNA-1 show a HbF/(HbF+HbA) value of 47.6%.
  • Table 7 The ratio of HbF/(HbF+HbA) displayed by red blood cells after differentiation of CD34+ cells in experimental groups a-g
  • the red blood cells differentiated into CD34+ cells after the BCL11A gene edited by the gRNA of the present invention exhibit a HbF/(HbF+HbA) ratio of more than 20%, specifically in the range of 20%-50%.
  • Example 7 Influence of unmodified gRNA and modified gRNA on BCL11A gene editing efficiency
  • the gRNA used in the control group is a customized modified gRNA.
  • the gRNA modification method is: 3 nucleotides at the 5'end of the gRNA used in the experimental group '-O-methylation and internucleotide 3'-phosphorothioate modification.
  • the left side is the chemically modified gRNA
  • the right side is the unmodified gRNA.
  • Example 8 Effect of gRNA into hematopoietic stem/progenitor cells by electroporation transfecting RNA and packaging lentivirus on gene editing efficiency
  • the control group 3-4 (corresponding to the use of experimental groups a and b gRNA) is packaged
  • the lentivirus method introduces gRNA (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 liposome method, the lentiviral vector was obtained from the supernatant, and the lentiviral vector was combined with hematopoietic stem/progenitor cells. Co-culture to obtain infected hematopoietic stem/progenitor cells. The specific steps are:
  • step (3) Dilute the annealed oligonucleotides 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 9 The effect of gRNA into hematopoietic stem/progenitor cells by electroporation transfection RNP and packaging lentivirus on gene editing efficiency
  • Experimental group a 83.1% Experimental group b RNP 72.7% Control group 3 Lentivirus 63.0% Control group 4 Lentivirus 53.1%
  • Example 9 The effect 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 red blood cells
  • Example 1-6 use gRNA-1 to edit the BCL11A gene.
  • the example group 5-10 (using the gRNA of experimental group a) changes the molar ratio of endonuclease Cas9 to gRNA-1
  • the gene editing efficiency is high at the ratio of the present invention, and the HbF positive rate of the red blood cells differentiated into the edited hematopoietic stem/progenitor cells is high, as shown in Table 10
  • Table 10 The effect of different molar ratios of Cas9 and gRNA-1 on the gene editing efficiency of BCL11A and the positive rate of HbF in differentiated red blood cells
  • Example 10 CD34+ cell off-target detection after gRNA-1 editing
  • CD34+ cells After gene-edited CD34+ cells, extract genomic DNA, CD34+ cells that have not been gene-edited as a control, perform whole-genome sequencing, where the control measures 90G data volume, and the edited sample measures 150G data volume;
  • the target position number of gRNA-1 is 24, and the target DNA sequence is shown in SEQ ID NO: 118.
  • the two sites BCRP2 and POM121L8P are two ncRNAs, and there are variant frequencies in both Ctrl and Edited samples.
  • the mutations at BCRP2 and POM121L8P sites disappeared, indicating that these two sites have background mutations in the Ctrl sample, not off-target mutations caused by Cas9 gene editing.
  • Design primers for the selected first 39 off-target sites for PCR amplification of the possible off-target regions and design primers for the targeted sites to PCR amplify the corresponding targeted regions.
  • the aforementioned primer sequence is shown in Table 13 (the primer sequence is shown in SEQ ID NO: 14-93), where OT-24 is the on-target site.
  • Example 11 In vitro differentiation experiment of CD34+ cells after gRNA-1 editing
  • CD34+ cells Take an appropriate amount of CD34+ cells after gene editing (Edited group, using gRNA-1) and resuspend the cells with IMDM (Gibco) + 2% FBS (Gibco) to a concentration of 500 cells/0.1 mL, unedited CD34+ Cells are used as controls (Ctrl group);
  • CD34+ cells edited by gRNA-1 differentiated into CFU-E and BFU- in vitro.
  • the experimental results show that the differentiation ability of the edited cells in vitro is not affected.
  • Example 12 In vitro tumorigenicity detection of CD34+ cells after gRNA-1 editing
  • Example 10 showed no serious off-target phenomenon; the in vitro differentiation experiment results of Example 11 showed that the differentiation ability of the edited CD34+ cells in vitro was not affected; the softness of Example 12 The results of the agar clone formation experiment indicated that CD34+ cells edited by gRNA-1 were not tumorigenic in the in vitro experiment.

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Abstract

L'invention concerne un procédé d'édition du gène BCL11A dans des cellules souches/progénitrices hématopoïétiques et un procédé pour augmenter l'expression de HbF de cellules souches/progénitrices hématopoïétiques humaines après différenciation érythroïde; l'invention concerne également des cellules souches/progénitrices hématopoïétiques BCL11A modifiées et l'utilisation desdites cellules dans la préparation de médicaments ou de produits médicaux pour la prévention ou le traitement de maladies anémiques; et l'invention concerne en outre un ARNg pour l'édition de BCL11A dans les cellules souches/progénitrices hématopoïétiques et un kit de réactifs contenant l'ARNg.
PCT/CN2020/112224 2019-08-28 2020-08-28 Procédé d'édition de gène bcl11a dans des cellules souches/progénitrices hématopoïétiques WO2021037232A1 (fr)

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