Abstract 2827: Non-viral gene editing enables multiplex single-step precision genome engineering for adoptive cell therapies
Authors: Lu William , Byers James , Tran Charles W. , Mass Michal , Shenoy Tanu , Sun Shirley , Jacoby Kyle , Mandl Stefanie 
Published: June 2022

Abstract
PACT Pharma has developed a robust single-step, targeted, non-viral method for the manufacturing of personalized adoptive cells therapies for the treatment of solid cancers (NCT03970382). For this, we insert and express a neoepitope-specific T cell receptor (neoTCR) from the endogenous locus while simultaneously abolishing the expression of endogenous TCR. This results in neoTCR-specific T cells in which neoTCR expression is naturally regulated and not impeded by competition for CD3 by the endogenous TCR.
We furthermore designed a system to additionally express CD8 coreceptor along with the neoTCR on the same RNA transcript from the native TCR promoter. Both methods, currently in clinical testing, allow for the timely generation of several billion T cells expressing neoTCRs using our GMP cell manufacturing procedure. Nevertheless, the suppressive nature of some tumor microenvironments may require cells engineered with further modifications for long lasting therapeutic outcomes in certain patients. Here we describe how the flexibility of a non-viral system allows for additional complex modifications in a single step.
First, we demonstrate an additional gene knock-out in the TGFBR2 gene with simultaneous TCR replacement, making the engineered cells resistant to the immunosuppressive effects of TGF-β while still maintaining neoepitope-specific recognition of the tumor.
Second, we demonstrate knock-down in addition to TCR replacement. For some targets, reduction of gene expression may be favored over complete knock-out. Accordingly, we have designed an shRNA expression construct that can efficiently knock-down transcript levels of a given gene in cells expressing the neoTCR without the generation of additional double-stranded breaks, allowing for enhanced T cell function in the absence of additional genomic breaks.
Third, we demonstrate a non-viral knock-in strategy to express transgenes with transcriptional regulation independent from the neoTCR. This strategy generates a product from which two genes, the neoTCR plus an additional transgene regulated by its own promoter, can be precision genome engineered into the same cell using homology directed templates that far exceed the size limitations of AAV. This technology can be successfully applied for the expression of intracellular, secreted, and membrane-bound proteins as well as proteins expressed only during the activated T cell state.
In conclusion, our single-step non-viral precision genome engineering technology is highly versatile with the ability to knock-out, knock-down, knock-in, and precisely regulate additional genes in a single step. These modifications have the potential to expand the applicability of T cell drug products and are broadly applicable to a variety of other cellular therapies and research models.
Citation Format: William Lu, James Byers, Charles W. Tran, Michal Mass, Tanu Shenoy, Shirley Sun, Kyle Jacoby, Stefanie Mandl. Non-viral gene editing enables multiplex single-step precision genome engineering for adoptive cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2827.