DNA damage responses to genome editing in hematopoietic stem cells - Project Summary Several genome editing strategies have been developed for the treatment of genetic diseases that affect the hematopoietic system, such as sickle cell disease and beta-thalassemia. Although there has been extensive research on detecting off-target effects of genome editors, there is limited information on the genotoxicities of on-target effects. The understanding of how hematopoietic stem and progenitor cells (HSPCs) resolve errors of mitosis following DNA damage to prevent persistent chromosomal abnormalities is incomplete. For safer clinical genome editing applications, understanding the frequency of such events is vital. We have shown in HSPCs that Cas9-induced double-stranded DNA breaks (DSBs) resulting from genome editing of therapeutic loci can cause chromosome segregation errors during cell division, leading to micronucleus formation and copy number abnormalities of the telomeric chromosomal segment. Micronuclei (MN) can contribute to chromothripsis, a genomic rearrangement phenomenon observed in cancer and other diseases. This proposal explores the potential for persistent aneuploidies, including chromothripsis, in HSPCs resulting from genome editing and the mechanisms of DNA repair in response to genotoxicities. Preliminary data generated from K01-supported research demonstrates that genome editing of HSPCs at specific targets allows for the enrichment of edited cells using pharmacologic and flow cytometric methods. Aim 1 will extend this observation both in vitro and in vivo by performing genome editing of therapeutically relevant genes centromeric of the selectable gene. Isolated cells will be subject to whole genome sequencing to reveal the nature of chromosomal rearrangements. This analysis will reveal the capacity of aneuploid HSPCs to survive and the heritability of rearrangements during the process of hematopoiesis. Secondly, our preliminary data show the inhibition of p53-mediated responses to DNA damage or caspase inhibition reduces the frequency of MN. Aim 2 will study how p53 and caspases may contribute to genome instability as a means of preventing persistent errors of DNA repair, allowing for the elimination of HSPCs with somatic mutations. Together, the proposed studies will provide a deeper understanding of on-target adverse events related to genome editing to better inform future genetic therapies. Data generated through this R03 award will provide preliminary data for future R01 applications on mechanisms of DNA repair in human HSPCs while supporting my transition from K01 recipient to independent investigator.
