Optimizing homology-directed repair of DNA breaks in hematopoietic stem cells

Abstract

Targeted nucleases such as CRISPR/Cas9 or zinc finger nucleases generate site-specific DNA breaks, whose repair by different cellular pathways can lead to different genetic outcomes. If the fast-acting but mutagenic NHEJ repair pathway is used, the outcome can be insertions or deletions (indels) at the break site that lead to gene disruption. In contrast, homology directed repair (HDR) pathways involve copying of genetic information from homologous DNA templates, and thereby allow for the possibility of correcting genetic mutations from an introduced wildtype sequence template. To achieve such outcomes, the exogenous DNA template must be introduced into the cell at the same time as the nuclease, typically by using oligonucleotides, plasmids or DNA virus vectors.

Promoting high levels of HDR in primary cells such as hematopoietic stem cells (HSC) is challenging. Exogenous DNA templates can be sensed by the cells and trigger cytotoxicity, while the NHEJ repair pathway is preferentially used over HDR during most stages of the cell cycle. To overcome these limitations, we have identified methods that create less toxic DNA delivery systems, including the removal of bacterial coding sequences, and the finding that AAV serotype 6 vectors are an especially effective template delivery system.

At the same time, we are attempting to reduce NHEJ-mediated gene disruption. One approach is based on ‘editing-at-a-distance’, where the nuclease is targeted to an adjacent intron, in order to reduce the chance that any indels will lead to ORF disruption. Other approaches are based on trying to skew DNA repair pathway choice towards HDR, by overexpressing HDR promoting factors that are not normally present outside of G2/S, and by blocking the activity of NHEJ promoting factors. A particularly effective strategy was identified based on targeting the master regulator, 53BP1. This protein promotes NHEJ over HDR by suppressing the formation of 3’ single-stranded DNA tails, which is the rate-limiting step in the initiation of HDR. 53BP1 activity was inhibited with a ubiquitin mimetic that prevents it from binding to ubiquitinylated histones at the site of a DNA break. Other strategies also enhanced HDR, and some synergism was possible. Carefully combining these approaches allows the titration of high levels of HDR while maintaining HSC function, and will thereby facilitate therapeutic applications of genome editing in HSC.

 

Biosketch
Paula Cannon, Ph.D. is a professor in the Keck School of Medicine of the University of Southern California. She earned a Ph.D. from the University of Liverpool and did post-doctoral training at Harvard and Oxford Universities. Her lab studies genome engineering in hematopoietic stem cells (HSC), with an emphasis on developing therapies for HIV/AIDS. In 2010, she first reported on the use of zinc finger nucleases to knock out the CCR5 gene in human HSC, which has now led to a clinical trial in HIV-infected individuals. More recently, she has shown that combining ZFNs with homologous sequences delivered using AAV vectors can achieve high levels of site-specific genome editing in HSCs, opening up the possibility of treating other diseases of the blood and immune system.