Zinc Finger Nuclease Induced DNA Double Stranded Breaks and Repair in the Chromosome 11q23 Mixed Lineage Leukemia Gene

Cancers such as therapy related acute leukemia and de novo infant acute leukemia have been shown to correlate with the introduction of double stranded breaks (DSB) within the mixed lineage leukemia (MLL ) gene. This dissertation presents the use of zinc finger nucleases (ZFNs) to introduce cuts within MLL to examine how a single DNA DSB might lead to leukemogenic chromosomal rearrangements.;A ZFN, targeting exon 13 at the telomeric portion of the break cluster region of MLL, was transiently expressed in a human lymphoblast cell line originating from a chronic myeloid leukemia patient. Fluorescence in situ hybridization results suggest that a single ZFN induced DSB can increase the occurrence of gross chromosomal rearrangements. However, inverse PCR was unable to detect any leukemogenic translocations. A majority of the rearrangements were determined to be interstitial deletions, insertions or base pair substitutions.;The role various repair pathways played in the mis-repair of ZFN DSB targeting MLL exon 13 was evaluated using the DNA-dependent protein kinase (DNA-PK) inhibitor (Nu7026) and by measuring RAD51 expression levels. Inhibition of the classical non-homologous end joining (NHEJ) repair pathway with the addition of Nu7026 appeared to have not only increased both the single strand annealing and homologous repair pathway, but also further increased the presence of gross chromosomal rearrangements, small interstitial deletions, insertions and base substitutions. This suggests that ZFN induced DSB within exon 13 of MLL is sensitive to NHEJ repair and the alternative end joining pathway may also play an integral role in faithful processing of open DNA end strands.;The full potential of ZFN to induce a single targeted DSB in the evaluation of pre-cancerous chromosomal rearrangements and translocations is realized, as is demonstrated here for the MLL gene.