Engineering strategies to safely drive CAR T-cells into the future

Abstract

Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.

Keywords: CAR T-cells; cell engineering; gene editing; gene modification; transgene delivery.

Figure 1

Schematic illustration of different gene delivery methods. Methodologies are divided into in vivo, ex vivo viral and ex vivo non-viral methodologies. Lentiviruses, retroviruses and transposons all are incorporated into the genome, and lead to stable CAR expression (upper half). In contrast, AAV LNP/Nanocarriers and mRNA all lead to transient CAR expression (lower half).

Figure 2

Schematic illustration depicting methods for gene editing and non-gene editing technologies. Gene editing strategies used in CAR T-cells depicted are divided into nuclease-dependent gene-editing technologies (CRISPR/Cas9, Base editing, prime editing, and PASTE) and nuclease-independent gene editing technologies (CAST). Non-gene editing technologies (CRISPRa, CRISPRi, CRISPR/Cas13, and RNAi) are also shown.