Abstract A036: Multiplex human T-cell engineering by Cas9 base editor technology
Authors: Lonetree Cara , Webber Beau R. , Diers Miechaleen D. , Lahr Walker S. , Kluesner Mitchell , Osborn Mark J. , Johnson Matthew J. , Moriarity Branden S. 
Published: February 2019

Chimeric antigen receptor (CAR)-engineered T-cells have mediated impressive outcomes in a subset of hematologic malignancies, yet this therapy remains highly personalized and largely ineffective against solid tumors. Genome editing strategies using targeted nucleases could overcome these limitations and have begun to enter clinical application. Multiplex editing strategies to develop off-the- shelf therapies are of high interest but remain limited by concerns of off-target effects and chromosomal translocations formed by simultaneous double-strand break (DSB) induction at multiple loci. The risk of genotoxic side effects is amplified when combining multiplex DSB induction with randomly integrating platforms for antigen-specific receptor delivery. An ideal strategy would allow for multigene disruption and targeted integration of antigen-specific receptors without introduction of multiple genomic DSBs. To this end, we evaluated the application of third- and fourth-generation Cas9 base editor technologies for gene disruption and integration in primary human T-cells. Chemically modified gRNAs and Cas9 base editor mRNA were delivered to stimulated T-cells by electroporation, followed by viral transduction for delivery of a DNA repair template as recombinant adeno-associated virus (rAAV). Base editing efficiencies were determined on the genomic level by PCR amplification, Sanger sequencing, and analysis of resultant traces using the EditR web app. Gene knock-out and knock-in efficiencies were analyzed on the protein level by flow cytometry. Through systematic reagent and dose optimization efforts, we achieved highly efficient C&gt;T base conversion and consequent protein knockout at multiple therapeutically relevant loci including TRAC (KO = 83.6 ± 3.3%), PD-1 (KO = 78.6 ± 2.3%), and B2M (KO = 80 ± 1.8%). We observed that fourth-generation base editor (BE4) achieved consistently higher C&gt;T conversion rates with reduced non-canonical editing (i.e., C&gt;A/G) compared to third-generation base editor (BE3). Targeted disruption of splice acceptor (SA) and splice donor (SD) sites resulted in higher frequency of protein knockout vs. induction of premature stop codons at all loci examined. Importantly, while multiplex editing using Cas9 nuclease resulted in detectable translocations between the targeted sites, we were unable to detect these translocations using BE3 and BE4 as measured by PCR. Finally, we exploited the single-strand nickase function of the base editors in conjunction with rAAV delivery to achieve simultaneous targeted integration of a gene expression cassette at the AAVS1 safe harbor locus. Staphylococcus aureus Cas9 (SaCas9) was also employed as an orthogonal approach to AAVS1 targeting, and yielded higher rates of donor integration. Collectively, we demonstrate that Cas9 base editor technology can be utilized to mediate efficient, multiplex gene disruption and targeted gene integration in primary human T-cells without associated translocations. This streamlined approach to genome engineering may be broadly applied for the development of safe and effective cell therapies.

Citation Format: Cara Lonetree, Beau R. Webber, Miechaleen D. Diers, Walker S. Lahr, Mitchell Kluesner, Mark J. Osborn, Matthew J. Johnson, Branden S. Moriarity. Multiplex human T-cell engineering by Cas9 base editor technology [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A036.