Associate Professor University of Pennsylvania, United States
Introduction:: Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in recent years, attaining FDA approval for the treatment of B cell lymphoma, acute lymphoblastic leukemia (ALL), and mantle cell lymphoma. Our group has previously reported the design of ionizable lipid nanoparticles (LNPs) encapsulating mRNA for ex vivo engineering of T cells with transient CAR expression. Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed on T cells, with a well-established role in cancer immune evasion and reduced CAR T therapeutic efficacy. Herein, LNPs co-encapsulating mRNA and siRNA are developed for simultaneous potent gene expression and knockdown in T cells and used to deliver CAR mRNA and PD-1 siRNA to primary human T cells ex vivo to generate transient CAR T cells with temporary PD-L1 resistance for applications in cancer immunotherapy (Figure 1a).
Materials and Methods:: A library of LNPs was designed with formulations varying along a continuum from “siRNA-like” to “mRNA-like” to identify excipient compositions for enhanced mRNA and siRNA encapsulation and delivery to Jurkat (immortalized human T) cells. Following optimization of nucleic acid cargo composition, PD-1 knockdown kinetics of a top-performing LNP formulation were investigated in a time-course study in primary human T cells. This lead formulation was then adapted for the delivery of CAR mRNA and PD-1 siRNA to primary human T cells, with CAR expression and PD-1 knockdown quantified via flow cytometry along with simultaneous measurement of T cell activation markers CD25, CD62L (L-selectin), and CD69. Work is underway to assess CAR T functionality in coculture killing assays.
Results, Conclusions, and Discussions:: In the initial screen of lipid excipients in Jurkat cells, the LNP most closely resembling typical mRNA formulations demonstrated the greatest encapsulation and delivery of both mRNA and siRNA (Figure 1b-c), with subsequent adjustments to nucleic acid ratios resulting in further improvement of both mRNA and siRNA delivery when co-encapsulated compared to encapsulating either alone. A time-course study revealed durable mRNA expression (at least 8 d) and high transfection efficiency (up to 98%) in primary human T cells (Figure 1d), along with persistent (approximately 7 d) and potent (up to roughly 60%) PD-1 knockdown (Figure 1e). Subsequent transfection of primary human T cells with LNPs encapsulating CAR mRNA and PD-1 siRNA resulted in up to 70% CAR positivity (Figure 1f) and a roughly 30% reduction in PD-1 expression (Figure 1g), with no apparent changes in overall T cell activation state (Figure 1h–j). Moreover, cells transfected with dual-encapsulating LNPs demonstrated CAR positivity rates of roughly 70%, whereas cells transfected with an equal amount of CAR mRNA alone reached only approximately 50% positivity, suggesting that inclusion of small nucleic acids may be generally beneficial for mRNA LNP development, including in applications not strictly requiring RNA interference. In summary, this work reports the development of an LNP platform for the potent simultaneous delivery of mRNA and siRNA to primary human T cells ex vivo. These LNPs show promise to simultaneously transiently express and inhibit proteins and factors in T cells for a number of immunoengineering applications, including in the development of improved cancer immunotherapies, and reveal interesting interactions resulting from the co-encapsulation of RNA cargoes.
