Megatal, Crispr/Cas9, and Talen T-Cell Receptor Gene Editing
Authors: Osborn Mark J. , Webber Beau R , Knipping Friederike , Lonetree Cara-lin , DeFeo Anthony , McElroy Amber , Starker Colby , Gabriel Richard , Merkel Sarah , Hippen Keli L. , Voytas Daniel , von Kalle Christof , Schmidt Manfred , Miller Jeffrey S. , Jarjour Jordan , Astrakhan Alexander , Scharenberg Andrew M , Tolar Jakub , Blazar Bruce R. 
Published: October 2019

Abstract
T-cell based immunotherapy holds great promise for targeted tumor therapies and includes the use of tumor infiltrating lymphocytes or cells engineered to express chimeric antigen receptors. Each have resulted in promising therapeutic responses; however, they are largely reliant on autologous, ex vivo cellular expansion, manipulation, and reinfusion. This individualized process may result in non-uniformity and may be limiting for urgent and widespread use. An attractive strategy would be to generate a universal donor pool of cells at sufficient scale to be broadly applicable. Allogeneic approaches to this hold the risk of off tumor reactivity toward host tissue as part of graft versus host disease (GVHD), a pathogenic process mediated by the T-cell receptor (TCR). The TCR is a heterodimeric complex comprised of alpha and beta chains encoded by the TRAC and TRBC genes, respectively. Because the alpha chain is encoded by a single gene, while the beta chain is encoded by two, it has become a prime candidate for genome editing strategies aimed at disrupting the formation of the TCR and diminishing the risk of GVHD.
Four major classes of gene editing proteins exist that share the commonality of binding a target sequence of DNA and generating a double stranded DNA break (DSB). The DSB is subsequently repaired by non- homologous endjoining an error prone pathway resulting in insertions/deletions that can disrupt open reading frames. Zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are heterodimeric arrays that are tethered to the Fok I nuclease domain that co-localize at a target DNA site. ZFNs are comprised of 3-6 finger subunits that bind DNA and TALENs are comprised of repeating DNA binding units containing a hypervariable two amino acid sequence (repeat variable diresidue; RVD) that governs DNA base recognition. Meganucleases are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases that have been fused with TAL RVDs to generate megaTAL (MT) proteins. The clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas9 nuclease platform is comprised of a small guide RNA (gRNA) transcript that contacts a target DNA sequence via Watson-Crick base pairing and the Cas9 nuclease that cleaves the DNA.
No evaluation has been performed between the different platforms for TCR disruption in the context of a translatable manufacturing process. Critical components of such an evaluation are reagent optimization, interrogation of off-target (OT) effects, and scalable expansion of gene edited cell populations. Utilizing Jurkat cells we observed that polyadenylated TALEN and MT mRNA and Cas9 with a gRNA encoded on a plasmid were the most effective in TRAC gene disruption (Fig 1A). A comparison of the optimized expression platforms in primary human T-cells revealed maximal editing rates were achieved with the MT reagent (Fig 1B). We then sorted the TCR negative cells to purity and re-introduced the TRAC gene mRNA that resulted in transient reconstitution of the CD3 complex that allowed for CD3/CD28 bead stimulation and a 5-fold expansion (Fig 1C). This represents a pool of TCR null cells for archival. Finally, we performed a genome level assessment of the potential off target (OT) activity of the nuclease candidates. Surprisingly, the CRISPR/Cas9 and TALEN reagents did not display OT effects (Fig 2A). At saturation levels of MT treatment we observed one bona fide OT site in both Jurkats and primary T-cells (Fig 2B); importantly, this occurred within an intron and did not impact proper gene splicing or cellular viability.
In summary, we evaluated three different TRAC -targeting nucleases, and developed an optimized process for generating and expanding TCR/CD3-deficient cells. Our study demonstrates the utility of performing unbiased genome wide assessment of editing for identification of off target cleavage, and the importance of achieving a high efficiency of gene editing and cellular expansion to facilitate clinical translation.
Figure 1 and 2. Figure 1 and 2.

Disclosures
No relevant conflicts of interest to declare.