Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model.

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Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause severe X-linked retinitis pigmentosa (XLRP). More than 80% of the mutations are located in the terminal exon ORF15 of the RPGR gene. Genome editing, which represents a novel approach to treat monogenic disorders, is based on highly specific nucleases that cleave or nick at a chosen position within the complex genome followed by the repair of the double or single strand break (DSB or SSB) by endogenous repair mechanisms. The major pathways include error prone non-homologous end joining (NHEJ), microhomology-mediated end-joining (MMEJ), and homologous recombination (HR), the latter two with the help of a donor template. Currently, endonucleases for inducing the DSB are based on the CRISPR-Cas9 system or TALE proteins fused to the non-specific FokI nuclease (TALEN). However, specificity and toxicity of both endonuclease types raise concerns about their use for therapeutic in vivo applications. In order to study in vivo genome editing for the treatment of XLRP, our lab has generated a mouse model containing a point mutation in the ORF15 exon. In the present study, we characterize advanced variants of both endonuclease types (Cas9-FokI and TALE-MutH) for their activity and toxicity at the murine Rpgr-ORF15 locus for later usage in the mouse model. In total, ten sequences within or near the ORF15 exon have been targeted for the induction of DSB or SSB. Nine target sites for CRISPR/Cas9-FokI were chosen: three before, within, and behind the exon, respectively, and one target site for TALE-MutH within the exon. These sequences have been cloned into the traffic light reporter (TLR) gene expression system at the homing endonuclease I-SceI site. The TLR system has been modified to express either GFP in case of successful HR or BFP in case of NHEJ. Plasmids containing substrate, nucleases and template DNA were transfected into HEK293T cells. Efficiency of DNA modification was measured by FACS analysis and T7 surveyor assay, and toxicity was assessed by cell survival assay. In addition to the episomal TLR system within a human cell line (HEK293T), the genome of murine C2C12 cells was targeted by all endonuclease variants and toxicity was analysed via the T7 surveyor assay. Toxicity of Cas9-FokI and TALE-MutH are comparable to the golden standard ISceI while standard Cas9 nucleases showed slightly increased toxicity in HEK293T cells. Two different concentrations of the nucleases were used in a toxicity assay and were equally tolerated. Cas9-FokI showed preferences in its activity at the nine target sites with activities well above ISce-I level, while the one target site of TALE-MutH was as efficient as ISceI. Activity results were confirmed in the murine cell line C2C12. Off target toxicity in C2C12 cells was non-detectable. The characterization of the activity and toxicity of the tested endonucleases helped us to identify the most promising tailored nuclease and its target sequence in our gene targeting approach to treat XLRP. With the help of mouse reti...

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