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E. González-Romero et al.
the genome.12 Multiplex CRISPR/Cas9 editing of genes mutated in human leukemias has been demonstrated in mouse and human cells using either lentiviral or ribonu- cleoprotein approaches. Edited cells were then transplant- ed into conditioned animals and the identity of the dis- rupted genes was revealed by next-generation sequencing from clones expanded in sick mice.19,35,47,48 Moreover, ex vivo CRISPR/Cas9 gene editing of HSPC is also useful for studying clonal hematopoiesis of indeterminate potential.19 Multiplex ribonucleoprotein-editing and tracking clonal dynamics by high-throughput sequencing revealed the expansion of mutant clones resembling human clonal hematopoiesis of indeterminate potential, some of which continued to expand and cause death, by hematopoietic failure or AML, in transplanted mice. Accordingly, multiplex CRISPR/Cas9 gene editing is an advantageous tool for functional genomics and for mod- eling the mutational complexity and co-occurrence pat- terns observed in hematologic patients at diagnosis, who in the case of AML, carry an average of 2.3 genomic mutations.49 A number of publications on the use of CRISPR/Cas9 gene editing in hematologic research are listed in Table 5.
Gene editing as a therapeutic application in hematologic disorders
Allogeneic HSC transplantation is the frontline treat- ment for many hematologic disorders; however, this
option is only available when a suitable donor exists. Nevertheless, transplanted patients can develop graft-ver- sus-host disease and die of transplant-associated causes. In this scenario, ex vivo gene therapy using viral vectors and ex vivo gene editing by TALEN or zinc-finger nucleas- es in hematopoietic cells followed by autologous HSC transplantation represent therapeutic alternatives that are currently being investigated in clinical trials.50 However, permanent viral integration into the host genome and/or insertional activation of proto-oncogenes that could lead to secondary leukemia are potential pitfalls related to integrative vector-based gene therapy.51 Site-specific endonucleases, especially CRISPR/Cas9, offer the possi- bility of delivering non-integrative editing components into target cells, such as mRNA and ribonucleoproteins, constituting a promising approach for HSC gene editing.
Inherited diseases
Clinically, CRISPR/Cas9 gene editing holds promise for monogenic hematologic disorders and, thus far, it has been mainly employed in hemoglobinopathies. β-tha- lassemia is caused by mutations in the human hemoglo- bin beta (HBB) gene and is characterized by reduced β- hemoglobin production, resulting in hemoglobin clump- ing, hemolytic anemia, and ineffective erythropoiesis. One strategy to remedy this defect using CRISPR/Cas9 is to repair the HBB mutation as has been achieved in iPSC from patients with β-thalassemia.52,53 Another strategy is to reactivate the fetal hemoglobin gene via disruption of the BCL11A gene, an erythroid enhancer regulator of the
Table 5. List of studies on CRISPR/Cas9 gene editing in hematologic diseases.
Disease Gene/s
Myeloid malignancies TET2, RUNX1, DNMT3A, NF1, EZH2
Myeloid malignancies
MDS
MDS, CMML, AML MLL
AML
AML
SCN
Pediatric AML
AML and MDS
AML
MDS XCGD CHIP CHIP
and SMC3 192 chromatin
regulatory domains
SRSF2
ASXL1 MLL and AF4 IDH2 IDH2 HAX1 MLL and ENL
Target cells
LSK
RN2 with constitutive
Format/Delivery
Two-vector system/Lentivirus
One-vector system/Lentivirus
CRISPR vector and ssODN/Electroporation
Reference
35
36
23
Aim/Repair pathway
Knock out/NHEJ
Knock out/NHEJ
Point mutation/HDR
Mutation correction/HDR
Chromosomal rearrangements/ HDR
Knock in /HDR
Mutation correction/HDR
Mutation correction/HDR
Chromosomal rearrangements/ NHEJ
Knock out/NHEJ
Chromosomal rearrangements/ NHEJ
Knock out/NHEJ Mutation correction/NHEJ Knock out/NHEJ Knock out/NHEJ
Cas9 expression K562
KBM5 HEK293
K562 Primary AML blasts iPSC Human HSPC
Human HSPC
Human HSPC
U937 PLB Human HSPC LSK
CRISPR vector and ssODN/Electroporation
CRISPR vector and template plastmid/Lipofection 31 CRISPR vector and template plasmid/Nucleofection 25 Two-vector system/Lentivirus 25 CRISPR vector and ssODN/Lipofectamine 27 One-vector system/Lentivirus 32
One-vector system/Lentivirus 48
One-vector system/ Electroporation 30
Two vector system/Electroporation 20 One vector system/Lentivirus 29 One vector system/Lentivirus 44
RNP/Electroporation 19
26
TET2, ASXL1, DNMT3A, RUNX1, TP53, NF1, EZH2, STAG2, SMC3, SRSF2 and U2AF1
RUNX1 and ETO ASXL1
CYBB
DNMT3A and TET2
FLT3, DNMT3A, SMC3,
EZH2, RUNX1 and NF1
MDS: myelodysplastic syndromes; CMML: chronic myelomonocytic leukemia; AML: acute myeloid leukemia; MLL: mixed lineage leukemia; SCN: severe congenital neutropenia; XCGD: X- linked chronic granulomatous disease; CHIP: clonal hematopoiesis of indeterminate potential; NHEJ: non-homologous end joining; HDR: homology-directed repair; LSK: Lin-Sca-1+c-Kit+; iPSC: induced pluripotent stem cells; HSPC: hematopoietic stem and progenitor cells; ssODN: single-stranded donor oligonucleotides; RPN: ribonucleoprotein.
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haematologica | 2019; 104(5)