Background To address the challenges of advanced cancers using cellular immunotherapy, it will likely require cutting-edge, multiplex genome engineered immune effector cells. Fortuitously, the development of ‘digital editors’, such as base editors (BEs), have enabled multiplex editing without induction of double strand breaks (DSBs) or need for a DNA donor to install gain- or loss-of-function edits with high efficiency. We previously deployed BE in lentiviral generated CAR-T cells, which demonstrated outstanding safety and efficacy of multiplex base editing that led to enhanced function. ¹ To enable others in this approach, we developed EditR software ² for analysis of base editing from Sanger sequencing and SpliceR³ for design of BE-gRNAs to knockout genes by editing splice sites. Since our pioneering work, multiplex base edited CAR-Ts have entered clinical trials in the academic and industry setting. ^{4 5}

Methods Recently, we combined base editing with two novel transgene cargo delivery approaches to generate advanced, multiplex genome engineered immune effector cells. First, we performed directed evolution of the TC Buster transposase that identified a variant (TcB-M) with enhanced transposition capacity, enabling stable integration of large, multicistronic transposons for the generation of CAR-T/CAR-NKs with >50% efficiency. ⁶ Additionally, we developed an all-in-one approach that leverages BE nickase activity for iterative nicking for synchronous engineered reprogramming of therapeutic cells (INSERT). INSERT utilizes juxtaposed gRNAs with BE to induce a DSB for site-specific homologous recombination (HR), achieving integration rates >30%. Both approaches reduce the cost and complexity of GMP manufacturing of advanced, multiplex genome engineered immune effector cells. Lastly, we developed novel BE approaches to install mutations for dominant negative receptors (TGFβRII and FAS) and non-cleavable Fc receptors (CD16A). Finally, in an effort to assess the upper limit of base editing, we contemporaneously installed 6 of these base edits and achieved 99–100% editing of all targets, indicating we have yet to reach the maximal number of base edits achievable in immune cells.

Results Using synergistic base edits, cytokine armoring, and CAR expression, we significantly enhanced immune effector functions in vitro using serial killing and spheroid assays. In vivo studies using xenografted NSG models also demonstrated enhanced persistence, resilience, and function. Beyond αβ CAR-T cells, we also demonstrate that these approaches are just as efficient in natural killer (NK) and γδT cells, two cellular chassis amenable to off-the-shelf use.

Conclusions Advanced multiplex engineering using BE and transposons is highly efficient in αβT, γδT, and NK cells for enhanced cancer immunotherapy.

References

1. Webber BR, et al. Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors. Nat. Commun 2019;10:5222.

2. Kluesner MG, et al. EditR: a method to quantify base editing from sanger sequencing. CRISPR J 2018;1:239–250.

3. Kluesner MG, et al. CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells. Nat. Commun 2021;12:2437.

4. Chiesa R, et al. Base-Edited CAR7 T Cells for relapsed T-Cell acute lymphoblastic leukemia. N Engl. J. Med. 2023;389:899–910.

5. Diorio C, et al. Cytosine base editing enables quadruple-edited allogeneic CART cells for T-ALL. Blood 2022;140:619–629.

6. Skeate JG, et al. Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies. Mol. Ther. 2024. doi:10.1016/j.ymthe.2024.04.024.