CRISPR/Cas9-mediated gene deletion efficiently retards the progression of Philadelphia-positive acute lymphoblastic leukemia in a p210 BCR-ABL1T315I mutation mouse model

Philadelphia-positive acute lymphoblastic leukemia (Ph ALL) refers to the subgroup of ALL with the Philadelphia chromosome, consisting of the t(9;22) translocation, which results in an oncogenic BCR-ABL1 fusion gene. For decades, Ph ALL has been regarded as an ALL subgroup with a poor outcome. Although the introduction of tyrosine kinase inhibitors (TKI) has contributed to improved outcomes, one of the major challenges that lies ahead is the emergence of resistant mutations of the BCR-ABL1 gene. One point mutation in particular, the T315I mutation, which influences the gatekeeper residue Thr315, is resistant to all currently approved firstand second-generation TKI. Ponatinib, a third-generation TKI, is the only TKI effective against the T315I BCR-ABL1 mutation. However, the recent 5-year results of the phase II PACE trial showed that more than half of Ph ALL patients bearing the T315I mutant responded poorly to ponatinib. We report here that in vivo delivery of CRISPR/Cas9 can effectively retard rapid progression of Ph ALL with the T315I mutation. This CRISPR/Cas9 method only disrupts the BCR-ABL1 fusion gene, and has the potential to destroy any point-mutated BCR-ABL1 fusion gene that may be drug-resistant. BCR-ABL1 is required for both induction and maintenance of leukemia, and switching-off this gene can result in rapid apoptosis of leukemic cells, a phenomenon referred to as “oncogene addiction”. As CRISPR/Cas9 technology has shown its undeniable power of genome editing by overcoming the limitations of earlier methods, we reasoned that disrupting the T315I-mutated BCRABL1 gene via CRISPR/Cas9 might revert the leukemia phenotype. Since normal BCR and ABL genes are also expressed in non-leukemc cells in Ph ALL patients, it is essential to destroy only the BCR-ABL1 fusion genes while leaving the expression of normal BCR and ABL genes unimpaired. Consequently, one strategy considered was to target introns rather than exons. Conceivably, paired single guide RNA (sgRNA) that target the introns of BCR and ABL could enable ablation of the BCR-ABL1 fusion gene while leaving the non-leukemic cells unaffected (Online Supplementary Figure S1A, B). Considering the fact that the CRISPR-mediated deletion frequency is inversely related to the deletion size, the targeted sequences we chose for paired sgRNA were adjacent to the BCR-ABL1 junction sequences (Figure 1A). Although BCR-ABL1 has diverse breakpoints, the fusion hybrids from the patient-derived pre-B ALL samples in our hands express p210 BCR-ABL1 isoforms, so we designed the sgRNA specifically against p210 BCR-ABL1. P210 fusion proteins usually comprise products of either the b2a2 or the b3a2 exon junction, corresponding to the fusion of BCR exon 13 and ABL1 exon 2 (e13a2) or e14a2. To save the effort of distinguishing the p210 subtype before CRISPR/Cas9 editing, we selected the commonly owned intron 12 by b3a2 and b2a2 p210 fusion gene as the target site for the BCR gene. For the ABL gene, the target site we chose was its intron 4, where the SH2 domain spans and is before the tyrosine kinase domain (TKD) of ABL kinase so that the size of the ablated BCR-ABL1 fragment would be around 10 kb (Figure 1A). Another thought was that the absence of the SH2-TKD interface, caused by ablation of the SH2 domain, would disable the oncogenic potential of BCRABL1. To avoid affecting RNA splicing, the targeted sites for sgRNA were at least 100 bp away from the 5’ or 3’ end of the introns. Publicly available tools (crispr.mit.edu and Benchling) were used to find sgRNA with minimal off-target DNA cuts. We also chose SaCas9 rather than the SpCas9 to further minimize the possibility of off-target effects. Eight sgRNA for either BCR intron 12 or ABL intron 4 were chosen and engineered into the GFPor mCherry-expressing pX601 plasmids that we had previously constructed. The plasmids were transfected into 293T cells followed by a Surveyor assay, and the sgRNA7 for BCR and sgRNA4 for ABL1were selected for the subsequent experiments because of their comparatively higher targeting efficiency, which was between 30% and 45% (Figure 1B). The frequency of indels was determined for the top five genomic off-target locations as predicted by the design tool, and no indels were detected by the Surveyor assay (Online Supplementary Table S1). Next, we examined the BCR-ABL1 ablation by transfecting the paired plasmids, pX601-BCR-intron12-GFP and pX601-ABL-intron4-mCherry, into K562 cells, which express b3a2 p210. GFP and mCherry double-positive cells were sorted and seeded into the individual wells, each of which contained 2000 3000 cells. The cells in each well were defined as “a clone” for the sake of descriptive convenience, although the clone was not necessarily a homogeneous population. The percentage of the ablated clone was defined as “ablation efficiency” and the percentage of non-homologous end joining (NHEJ) events in each ablated clone was defined as “on-target efficiency”. Polymerase chain reaction (PCR) and the subsequent Sanger sequencing with PCR products were performed 48 to 72 h after transfection, and the ablated BCR-ABL1 could be detected in about 50% of the clones (Online Supplementary Figure S1C). Since the K562 cell line contains multiple copies of BCR-ABL1, this could presumably account for the observation that only 50% of the clones were ablated. To further evaluate the NHEJ event in each ablated clone, we adopted the droplet digital PCR (ddPCR) method, which is a wellproven approach for detecting on-site editing of CRISPR/Cas9. The results showed that the efficiency of NHEJ-mediated targeting was 45%~50% on either the BCR or ABL1 site (Figure 1C). Of note, the majority of the cells in the seeded clones that were positive for BCRABL1 ablation underwent rapid cell death within 4 to 5 days after transfection, indicating the dominant role of BCR-ABL1 for the survival of leukemic cells addicted to this fusion gene. Considering the limited survival time of these targeted cells, it was hard to define the ablation efficiency at the single-cell level because the time required for the proliferation of a single cell to the cell population that could have enabled the PCR detection was far longer than the expected lifespan of this targeted single cell. Therefore, instead of resorting to the laborious single-cell sequencing method, we evaluated the ablation efficiency at the clonal level. Despite the acceptable efficiency, expression of two sgRNA from separate constructs would be impractical in vivo. We therefore incorporated two sgRNA into one construct that also simultaneously expressed SaCas9, and an improved ablation efficiency of around 55 65% was subsequently observed (Online Supplementary Figure S1D). For the purpose of in vivo genome editing, we engineered the lentiviral CRISPR vector encoding SaCas9 to express either one or two sgRNA. The observed ablation efficiency was around 40 50% for the dual vector system