Non-Viral Genome Engineering Method Allows Highly Efficient, Single-Step Removal And Precise Insertion Of Multiple Large Genes

Recent advances in gene editing have enabled the targeted engineering of primary cells to insert entire transgenes without the use of viral vectors. Using these methods, novel genes can be inserted in a seamless manner into the specified genomic locus, realizing the goal of precise targeted genomic modifications. Non-viral modification of primary cells holds potential to significantly reduce costs and time needed for the generation of therapeutic cell products. It has also allowed the generation of personalized cell therapies involving patient-specific manufacturing of DNA constructs, such as for the developed of patient-derived, neoepitope-specific TCR-T cells. Proof-of-concept experiments have shown that non-viral genome engineering methods can integrate small genetic elements such as GFP, while the delivery of larger therapeutically relevant payloads at high efficiency has previously been challenging. Here we describe a proprietary single-step, DNA-mediated method developed for seamlessly engineering fully natural neoepitope-specific TCRs (neoTCR) into the endogenous TCR locus of primary human T cells at high efficiencies (i.e. \u003e50% gene editing efficiency). This method allows the delivery of the two genes comprising the neoTCR without the need for selection necessitated by less efficient approaches. To further evaluate this approach, fresh human donor T cells were engineered to express the patient-specific neoTCR plus two additional gene products, CD8α and CD8β (i.e. precise genome engineering of four ectopic genes). The data shows that these modifications were made at high efficiency, resulting in fully functional CD8 and CD4 T cells. Surface expression of CD8 coreceptor together with the neoE-targeted TCR increased T cell signaling sensitivity of the engineered neoTCR-T cells by 10-100 fold. The potential for off-target cleavage or unexpected genomic outcomes was assessed using multiple methods, including a newly developed primary T cell GUIDE-seq assay. Despite multi-locus editing, no evidence of off-target insertion or unexpected genomic rearrangements were observed. In summary, these results demonstrate the applicability of a single step, highly efficient method for manufacturing fresh human T cells into neoTCR-T cell therapies, engineered with multiple functionalities. This proprietary precision genome engineering technology supports the on-going Phase 1 clinical trial of personalized autologous, NeoTCR-P1 engineered T cell therapies for patients with solid tumors (NCT03970382). Citation Format: Kyle Jacoby, William Lu, Diana Nguyen, Barbara Sennino, Andrew Conroy, Bhamini Purandare, Alex Franzusoff, Stefanie Mandl. Non-viral genome engineering method allows highly efficient, single-step removal and precise insertion of multiple large genes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2192.