116. MegaTAL Nucleases Outperform TALENs in Promoting Homology-Directed-Gene Modification in Primary Human T Cells

Abstract

Advances in rare cleaving nuclease technologies have dramatically increased the potential for seamless manipulation of virtually any gene (i.e. gene editing). Among these platforms, TALENs and Cas9-based nucleases have become broadly applied owing to ease in customizing their DNA recognition properties. In contrast, LAGLIDADG homing endonucleases (LHE) are more challenging to engineer, yet exhibit superior physical attributes, DNA recognition and hydrolysis properties. Recent advances have overcome hurdles in LHE engineering leading to the ability to more rapidly customize LHEs as therapeutic nucleases. In addition, LHEs can be fused to TAL effector arrays to create chimeric proteins [referred to here as megaTALs (MTAL)] that exhibit further enhancements in activity. We previously developed a CCR5-specific MTAL as a potential HIV therapeutic. In parallel, we generated high-activity TALEN pairs targeting the identical region in CCR5. In the current study, we directly compared the ability of these nuclease platforms to induce homology directed repair (HDR) in primary human T-cells. We first verified that both reagents facilitated efficient disruption of the CCR5 locus. We directly compared activity in primary CD4 T cells using mRNA nuclease delivery. Indel frequency was estimated using both the T7 endonuclease assay and a re-cleavage assay (RCA; where genomic DNA surrounding the cleavage site is amplified and digested in vitro with the original LHE used to create the MTAL). NHEJ rates were 65-82% for the TALEN vs. 44-61% (6 experiments) for the MTAL using these respective assays. Next, we coupled mRNA-mediated nuclease expression with AAV delivery of a donor template. We co-delivered mRNA with a range of doses of an AAV-CCR5-GFP donor (containing 1.3kb CCR5 homology arms and an internal MND-GFP expression cassette). Cell viability was not significantly different; and control experiments using AAV BFP (lacking homology arms) demonstrated that infection and expression rates were equivalent. Strikingly, the proportion of cells with sustained GFP expression (at day 16) was up to 5-fold higher in MTAL treated cells (across a range of AAV doses; n=6, independent donors); with the greatest differences observed when access to donor template was limiting. Molecular analysis verified HDR in both test groups. While the mechanism for these differences remains incompletely defined, preliminary comparisons of HR:NHEJ ratios using single cell analysis suggest that alternative DNA repair pathways may be preferentially utilized by the 3’ vs 5’ overhangs generated by LHE vs Fok1 nucleases, respectively. Taken together, our findings suggest that the MTAL platform and the co-delivery method described here may provide significant clinical utility in future editing applications in human hematopoietic cells.