121. T Cell Receptor Modification by Highly Specific TALEN and CRISPR/Cas9

Abstract

Adoptive transfer of T cells with transgenic high avidity T cell receptors (TCR) is a promising therapeutic approach, however it comprises certain challenges. Endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially leading to autoimmunity. This can be prevented by designer nucleases such as transcription activator-like effector nucleases (TALEN) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system. Their ability to introduce specific DNA double strand breaks (DSB) at their target sites can be harnessed for targeted genome editing. DSB repair through non-homologous end joining (NHEJ) can result in permanent gene knockout due to frame shift mutations. Alternatively, if a homology-containing donor template is provided, transgenes can be integrated into the specific target locus by homology directed repair (HDR). In order to disrupt endogenous TCR expression, we assembled nine TALEN and two CRISPR/Cas9 guide RNA (gRNA) targeting the constant region of the TCR α-chain (TRAC) and four TALEN and three CRISPR/Cas9 gRNA targeting both constant regions of the TCR β-chain (TRBC1/TRBC2). Here we show specific DSB induction by TALEN and CRISPR/Cas9 in K562 and primary T cells using T7 endonuclease I targeting efficiency assay and deep sequencing. Electroporation of primary T cells with TRAC-TALEN or TRBC-TALEN mRNA led to successful elimination of surface TCR expression in about 76% of the cells. To analyze nuclease specificity, K562 cells were transduced with an integrase-defective lentiviral vector (IDLV) prior to nucleofection with TALEN- or CRISPR/Cas9-expressing plasmids. IDLV can be integrated into DSB during DNA repair, thereby serving as stable markers for transient DSB. IDLV-marked DSB were subsequently localized using LAM-PCR and deep sequencing. Clustered integration sites (CLIS) were detected around all nuclease target sites, confirming on-target activity. Although up to 3268 IDLV integration sites were analyzed for each TALEN and CRISPR/Cas9 gRNA, only one CLIS for one gRNA was mapped at an off-target position, indicating a very high level of specificity. To establish HDR-mediated targeted integration, we assembled donor templates containing a GFP expression cassette flanked by 800bp TRAC- or TRBC1-homologous sequences. We verified targeted gene addition in 10% of K562 cells treated with TALEN and the respective donor template. Delivery of the TRAC-donor packaged into IDLV resulted in targeted integration of the GFP expression cassette in 5% of primary T cells. To sum up, here we present highly efficient and specific TALEN and CRISPR/Cas9 and their utility for T cell engineering.