Gene editing replaces the CD40LG gene at the endogenous locus with a GFP transgene. (A) Arrow shows position of TALEN target site within the 5′UTR of the human CD40LG gene; rAAV gene-editing templates are shown below; shaded dotted lines indicate regions of homology used to target the surrounding break site. Editing templates contain identical 1000 base pair 5′ and 3′ homology arms. The no-homology MND-BFP AAV template is diagrammed at right. (B) Basic experimental timeline for gene editing in human T cells: 48-hour CD3/CD28 activation followed by mRNA transfection and rAAV transduction. Gene-editing analysis took place 4 to 5 days following transfection/transduction. (C) GFP transgene expression following gene editing in human T cells. (Top row) AAV template only, no TALEN. (Bottom row) AAV template plus TALEN. No GFP signal is observed (day 5) in cells receiving gene-editing template only. (D) Genomic analysis of CD40LG following editing. Primers that bind outside the rAAV homology arms amplify the edited region (−165-Fwd:165 bp upstream, +26-Rvs: 26 base pairs downstream). Predicted amplicon size (base pairs): WT: 2422, CD40LG[GFP.3′UTR] HDR: 4122, and CD40LG[GFP.WPRE] HDR: 3634. HDR-editing bands are only observable in TALEN + AAV conditions; TALEN + AAV-edited cells were GFP enriched prior to PCR analysis. (E) CD40L surface expression on CD4⁺ T cells following gene editing. Resting (top) and P/I-activated (bottom). GFP⁺ cells do not express CD40L in response to P/I, due to replacement of CD40LG by GFP transgene. P/I activation increases MFI of GFP in gene-edited cells. (F) Editing by “homology-less” template (MND-BFP). GFP (x-axis) and BFP (y-axis) after transduction with MND-BFP AAV alone (middle), or cotransduction with an equal MOI of the CD40LG[GFP.WPRE] template. (Top row) No TALEN; (bottom row) +TALEN. Sustained GFP expression is ∼10-fold higher than BFP signal from “homology-less” MND-BFP template.