342. Gene Edition for Wiskott-Aldrich Syndrome Gene Therapy

Abstract

Site Specific nucleases (SSN) are powerful tools for genome editing that are revolutionizing basic and applied science. However, for most therapeutic applications their efficiency and/or specificity must be still improved before translation into clinic. Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency (PID) caused by mutations in the WAS gene. An interesting characteristic of this disease is that, due the growth selective advantage of the WAS-expressing cells, a small number of corrected hematopoietic stem cells (HSCs) could be enough to achieve a complete cure of the patients. In this context, our final aim is to develop gene edition tools for WAS gene therapy with the idea to achieve safe genetic correction in a small numbers of patients’ HSCs. To study efficiency and safety of our tools, we first developed a WAS-specific GFP-reporter cellular model harboring GFP coding sequences disrupted by the intron 1 of the WAS gene. This reporter cell allowed us to have a direct measurement of the efficiency and specificity of WAS-specific nucleases to achieve genetic restoration. We constructed different CRISPR-based and ZFNs-based WAS-specific nucleases expressed by Lentiviral vectors (LV). With these set of plasmids we performed a side by side comparison of two different nucleases (ZFNs and CRISPR) and two different delivery systems (nucleofection versus Integrative deficient lentiviral vectors (IDLV)) to edit the WAS locus. We showed that CRISPR and ZFNs were equally efficient for WAS gene edition (over 60% gene disruption and 4% gene restoration) when delivered by plasmid nucleofection. However, IDLV-ZFNs were more efficient than IDLV-CRISPR, probably due to the low expression levels of Cas9 in the IDLV backbone. Our data indicates that CRISPR based systems can be as efficient as other SSN using standard delivery systems, however, their efficiency using IDLV is limited.