CRISPR/Cas9-Directed Gene Trap Constitutes a Selection System for Corrected BCR/ABL Leukemic Cells in CML

Abstract

Chronic myeloid leukaemia (CML) is a haematological neoplasm driven by the BCR/ABL fusion oncogene. The monogenic aspect of the disease and the feasibility of ex vivo therapies in haematological disorders make CML an excellent candidate for gene therapy strategies. The ability to abolish any coding sequence by CRISPR-Cas9 nucleases offers a powerful therapeutic opportunity to CML patients. However, a definitive cure can only be achieved when only CRISPR-edited cells are selected. A gene-trapping approach combined with CRISPR technology would be an ideal approach to ensure this. Here, we developed a CRISPR-Trap strategy that efficiently inserts a donor gene trap (SA-CMV-Venus) cassette into the BCR/ABL-specific fusion point in the CML K562 human cell line. The trapping cassette interrupts the oncogene coding sequence and expresses a reporter gene that enables the selection of edited cells. Quantitative mRNA expression analyses showed significantly higher level of expression of the BCR/Venus allele coupled with a drastically lower level of BCR/ABL expression in Venus+ cell fractions. Functional in vitro experiments showed cell proliferation arrest and apoptosis in selected Venus+ cells. Finally, xenograft experiments with the selected Venus+ cells showed a large reduction in tumour growth, thereby demonstrating a therapeutic benefit in vivo. This study represents proof of concept for the therapeutic potential of a CRISPR-Trap system as a novel strategy for gene elimination in haematological neoplasms.

Clinical Insights into Structure, Regulation, and Targeting of ABL Kinases in Human Leukemia

“…However, these risks may be addressed with autologous transplantation of gene-edited HSCs. The CRISPR/Cas9 system has been demonstrated to effectively disrupt BCR::ABL1 at the genomic level leading to leukemia cell death in vitro and in vivo with no off-target effects [ 126 , 127 , 128 , 129 ]. Despite high gene-editing efficiency using the CRISPR/Cas9 system, a major barrier to curative gene therapy in CML is the presence of unedited cells that have the potential to initiate relapse after transplantation [ 127 ].…”

“…The CRISPR/Cas9 system has been demonstrated to effectively disrupt BCR::ABL1 at the genomic level leading to leukemia cell death in vitro and in vivo with no off-target effects [ 126 , 127 , 128 , 129 ]. Despite high gene-editing efficiency using the CRISPR/Cas9 system, a major barrier to curative gene therapy in CML is the presence of unedited cells that have the potential to initiate relapse after transplantation [ 127 ]. To overcome this, a proof-of-concept study described the efficacy of a CRISPR-gene trapping strategy that would allow for CRISPR-edited cells to be selected before transplantation [ 127 ].…”

“…Despite high gene-editing efficiency using the CRISPR/Cas9 system, a major barrier to curative gene therapy in CML is the presence of unedited cells that have the potential to initiate relapse after transplantation [ 127 ]. To overcome this, a proof-of-concept study described the efficacy of a CRISPR-gene trapping strategy that would allow for CRISPR-edited cells to be selected before transplantation [ 127 ]. In this study, a donor gene trap cassette carrying a fluorescent reporter gene was inserted into the BCR::ABL1 fusion site in K562 cells [ 127 ].…”

Islands of genomic stability in the face of genetically unstable metastatic cancer

“…Recently, CRISPR-Cas systems have been used for cancer gene therapy. Strategies have included insertion of suicide genes(11), targeting mutations in oncogenic driver genes that contain PAM sites(12, 13), unique sequences in oncoviruses such as E6 and E7(14), and fusion oncogenes(15, 16). We recently reported that somatic single base substitutions (SBS) forming novel PAM sites specific to cancer cells are targetable by CRISPR/Cas9.…”

Advances in Molecular Research of Oncogenes

“…Vuelta and co-workers [ 4 ] presented a gene-trapping approach combined with CRISPR technology as an ideal methodology to increase the selection of CRISPR-edited cells as a potential approach for the treatment of BCR/ABL-driven chronic myelogenous leukemia (CML). Strikingly, both in vitro and in vivo experiments demonstrated a significant reduction in CML tumor cell proliferation and tumor growth, thus providing a proof-of-concept for the therapeutic potential of a CRISPR-trap system as a novel strategy for gene elimination in hematological neoplasms.…”