UCLA

Autologous hematopoietic stem cell (HSC) transplantation, combined with gene editing, could provide an ideal therapeutic option for the treatment of congenital blood diseases, such as hemoglobinopathies, primary immune deficiencies, and storage disorders. Gene editing relies on site-specific induction of a double stranded break (DSB) by targeted nucleases (such as Zinc Finger Nucleases (ZFNs) or CRISPR/Cas9 system), and subsequent gene correction using endogenous cellular repair mechanisms. The two main competing pathways to repair the break are non-homologous end joining (NHEJ), an often-imprecise pathway which can result in insertions and deletions (indels), or accurate homology-directed repair (HDR) pathway which uses a homologous donor template to seamlessly repair the break and incorporate the desired changes. For certain diseases, where a knockout of a gene can result in therapeutic benefit, repair by NHEJ pathway may be favorable. However, for conditions where disruption of a gene can result in an even more severe phenotype than the original disease (such as sickle cell anemia), repair via HDR pathway is critical.

Despite advances in nuclease technologies and the ability to efficiently achieve high frequency of site-specific gene disruption, the current progress to reach clinically relevant levels of precise HDR-mediated repair still remains elusive. Therefore, our translational goal is to improve the gene editing outcomes in HSCs, specifically, increase HDR and decrease NHEJ levels, which will be beneficial for treating many diseases of the blood. This dissertation aims to identify the hindrances that limit efficient HDR-mediated editing in HSCs, and investigates several approaches to address these impediments.

Our results indicate that one major reason for low gene correction in HSCs is their heightened susceptibility to cell toxicity resulting from the electroporation of the nuclease and homologous donor template. We demonstrate that co-electroporation of mRNA encoding the anti-apoptotic protein BCL2 with gene editing reagents significantly ameliorates the cytotoxicity and increases the yield of gene-corrected HSCs.

Next, we show that cell cycle-dependent control of nuclease activity and DNA repair pathways can influence gene editing outcomes to favor the precise DNA modification (HDR) over faulty repair events (NHEJ) in human HSCs. By using a modified version of Cas9 protein with reduced nuclease activity in G1 phase of cell cycle, when HDR cannot occur, and transiently increasing the proportion of cells in HDR-preferred phases (S/G2), we achieve a 4-fold improvement in HDR/NHEJ ratio over the control condition in vitro, and a significant improvement in long-term gene-modified engrafted cells after xenotransplantation of edited human HSCs into immune-deficient mice.

Finally, we investigate what cellular elements govern the DNA repair pathway choice and how they can be exploited to shift the balance toward HDR from NHEJ. We test the effects of manipulating the expression levels of several DNA repair factors, that are presumed to be important for pathway choice and progression, on HDR and NHEJ levels in K562 cell line and primary human hematopoietic stem and progenitor cells (HSPCs). Interestingly, we observe differential effects of DNA repair factor manipulation on gene editing outcomes dependent upon the delivery method employed and the types of cells used.

These strategies for improving gene editing outcomes in human HSCs have important implications for the field of gene therapy as a whole, and can be applicable to diseases where increased HDR/NHEJ ratio is critical for therapeutic success.