Figure 4. Workflow for CRISPR/Cas9 gene repair in expanded CD8+ T memory stem cells (TSCM) from patients with familial hemophagocytic lymphohistiocytosis (FHL).
Starting from aliquots of expanded, patient-derived frozen TSCM-like cells, T cells are reactivated and expanded for 2 days with IL-2, then electroporated with RNPs, and immediately infected with recombinant adenovirus-associated virus (AAV) serotype 6 (carrying the DNA donor repair templates) in the presence or absence of a small molecule NHEJ inhibitor [DNA-PK inhibitor M3814 (Nedisertib)]. Transfected, infected T cells are first expanded for 4 days with IL-2 and then for two more days in the presence of IL-7 and IL-15.