569. Precision Editing of the WAS Locus via Homologous Recombination in Primary Human Hematopoietic Cells Mediated by Either TALEN or CRISPR/Cas Nucleases

Abstract

Wiskott-Aldrich Syndrome (WAS) is an inherited primary immunodeficiency caused by mutations in the WAS gene which encodes a protein (WASp) that regulates the actin cytoskeleton in multiple hematopoietic cell lineages. Currently, allogeneic stem cell transplantation constitutes the only available cure for WAS, although phase I/II clinical trials using lentiviral gene therapy of autologous stem cells are currently underway and have shown improvement in immune system defects. Although effective, the gene therapy approach carries a risk of insertional mutagenesis, and unpredictable expression due to promoter choice as well as influences of the random integration site, including epigenetic status and the presence of neighboring transcription regulatory elements. As a refinement, our goal is to develop gene editing methodologies that would allow the specific targeting of a WASp cDNA to a position within the WAS locus allowing transcriptional regulation by the endogenous WAS promoter. To this end, we first designed and tested the cleavage efficiency of several guide RNAs (delivered as self-complementary AAV along with Cas9, delivered as mRNA), as well as candidate TALEN pairs (delivered as mRNAs), in primary human T cells. Using the T7 endonuclease assay, we identified candidate nucleases from both platforms (CRISPR/Cas9 and TALEN) that achieved a high Indel frequency: 73 and 85%, respectively. We next created a synthetic AAV6 donor template for homology-directed repair (HDR) that contained an MND promoter driven GFP cDNA with 1kb of WAS homology arms flanking it. When delivered with the TALEN or CRISPR/Cas9 nucleases, we observed stable integration of the GFP reporter within >25% of primary human T cells. Subsequently, we utilzed the identical reagents to target integration of the reporter cDNA into the WAS locus in adult human mobilized CD34+ cells, albeit at lower efficiencies. The off-target cleavage sites for TALENs identified using the Prognos software were amplified and sequenced, with no evidence of off-target cleavage observed at any of the predicted loci. Thus, we have generated genome editing tools that possess a high degree of specificity, providing the foundation for site-specific modification of the WAS locus as a therapeutic option.