TALEN^®-Based Gene Editing in Hematopoietic Stem Cells

Autologous gene therapy in human hematopoietic stem cells (HSCs) has shown the potential to transform the treatment of genetic diseases of the blood and immune system. However, viral vector delivery has shown issues with insertional mutagenesis, unregulated transgene expression and durability. Genome editing via homology directed repair (HDR) in human HSCs can mitigate these issues by correcting and repairing the endogenous gene permitting precise spatio-temporal gene expression. Transcription activator-like effector nuclease (TALEN^®) are a designer nuclease that enables the site-specific introduction of double-stranded breaks (DSBs) in the genome. Repair of these DSBs occurs largely through one of two pathways, non-homologous end joining (NHEJ) and HDR. NHEJ is an error-prone pathway that often results in insertions or deletions (indels) whereas HDR is a precise mechanism that uses a homologous piece of DNA (usually the homologous chromosome or sister chromatid) to accurately repair the lesion by recombination. However, HDR can be manipulated by supplying an exogenous DNA template with homology arms that start at the DSB as single-stranded oligonucleotides or as double-stranded DNA to introduce any genetic modifications encoded in the template DNA. Recent studies have shown efficient HDR in numerous cell types using adeno-associated virus (AAV) to deliver template DNA. Here we combined TALEN^® with AAV containing a repair template to identify the conditions required to efficiently modify disease-relevant alleles in HSCs. We first describe a strategy to remove the nuclease target site in the template DNA even when the target site was located in regulatory sequences. Optimization of the transfection, transduction and culturing conditions of HSCs shows highly efficient allele modification using low doses of AAV and nuclease. Importantly, alleles more often show evidence of HDR as opposed to NHEJ. Further, colony forming unit assays and a xenograft model confirm that modified HSCs are multipotent. Lastly, we show that differentiated progeny of modified HSCs produce similar amounts of mRNA from the modified allele as wild-type unmodified alleles. In total, our results show that combining TALEN^® with repair template delivery via AAV is a robust gene editing system for HSCs.