Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells

Abstract

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.

Off the shelf T cell therapies for hematologic malignancies

“…Several different gene editing technologies have been used to prevent expression of endogenous TCRs. Zinc-finger nucleases (ZFN) [14], CRISPR/Cas9 [15,16], transcription activator-like effector nucleases (TALEN) [17,18], and engineered homing endonucleases [19] have been described that target an exon within the TCRα constant (TRAC) or TCRβ constant 1 (TRBC1) or 2 (TRBC2) loci for genetic knockout thereby preventing expression of α/β TCRs on the cell surface. ZFN, CRISPR/Cas9, or TALEN are two component systems in which sequence-specific DNA targeting and DNA cleavage functions are provided by separate molecular entities.…”

“…The DNA cleavage domain of FokI and Cas9 nucleases introduce a DSB at the target site specified by the Znfinger, TALE, or gRNA which, in the absence of a homology repair template, is repaired by nonhomologous end joining (NHEJ), an error prone cellular repair pathway that results in insertion or deletion of nucleotides at the cleavage site resulting in loss of functional gene expression [20,21]. In the case of the engineered homing endonuclease technology such as described by MacLeod et al [19], a guide sequence is not required in order to target nuclease activity to the intended genomic DNA sequence. T cells are electroporated with an mRNA that encodes the engineered nuclease which directly binds the DNA target sequence and makes a staggered cut resulting in 4 bp 3′ overhangs, a feature that may favor targeted insertion of a transgene via homology-directed repair (HDR) in the presence of a homology repair template (described below).…”

“…Hale et al [26] used a megaTAL (TALE covalently linked to a site-specific meganuclease) combined with a recombinant adeno-associated virus (rAAV) homology repair template to insert the CAR at the site of the DSB in TRAC by homologous recombination using cellular HDR machinery. Eyquem et al [27] used CRISPR/Cas9 and an rAAV homology repair template to insert an anti-CD19 CAR into TRAC or the β 2 -microglobulin (B2M) gene, and MacLeod et al [19] describe targeted insertion of an anti-CD19 CAR into TRAC using an engineered homing endonuclease and an rAAV homology repair template. Targeted insertion of a CAR transgene into a defined site within the cellular genome allows for controlled and homogeneous expression of the CAR.…”

Emerging applications of gene edited T cells for the treatment of leukemia

“…Other laboratory developments are delivering further refinements, including the possibility of targeted CAR insertion into the TRAC locus thereby introducing an element of regulatory control from relevant transcriptional machinery while simultaneously disrupting TCR expression. Adeno-associated virus was recently used for targeted integration of promoterless CAR19 cassettes directly into the TRAC locus following either CRISPR/Cas9 [17] or I-CreI homing endonuclease [10] mediated DNA breakage, and this may reduce theoretical risks of vector-mediated insertional mutagenesis. This approach harnesses homologous recombination pathways rather than default NHEJ repair, and although scalability may be challenging, the products are expected to be more homogeneous.…”

“…T-cells encode highly specific heterodimeric αβ T-cell receptors, and these are the key mediators of major histocompatibility complex (MHC) recognition leading to GVHD. Strategies to disrupt T-cell receptor (TCR) expression have used a variety of reagents including RNA interference (RNAi) [9] and targeted gene disruption using directed DNA nucleases such as Zinc Finger Nucleases [6,7], Meganucleases [10], MegaTALs [11,12], and Transcription activator like effector nucleases (TALENs) [8,13,14]. The latter operate at the genomic level, directing engineered endonucleases to highly specific loci to create double stranded DNA cleavage.…”