CRISPR-Cas9-AAV versus lentivector transduction for genome modification of X-linked severe combined immunodeficiency hematopoietic stem cells

Abstract

<i>In vitro</i> differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI-<i>IL2RG</i> HSPCs with efficient T cell development following TI-<i>IL2RG</i> in all four patients' HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before (<i>in vitro</i>) or following transplant, in stark contrast to LV's non-targeted vector integration sites. Together, the improved efficiency and safety of <i>IL2RG</i> correction <i>via</i> CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.