Ionizable Lipid Nanoparticles with Integrated Immune Checkpoint Inhibition for mRNA CAR T Cell Engineering

Abstract

The programmed cell death protein 1 (PD‐1) signaling pathway is a major source of dampened T cell activity in the tumor microenvironment (TME). While clinical approaches to inhibiting the PD‐1 pathway using antibody blockade have been broadly successful, these approaches lead to widespread PD‐1 suppression, increasing the risk of autoimmune reactions. This work reports the development of an ionizable lipid nanoparticle (LNP) platform for simultaneous therapeutic mRNA expression and RNA interference (RNAi)‐mediated transient gene knockdown in T cells. In developing this platform, interesting interactions were observed between the two RNA cargoes when co‐encapsulated, leading to improved expression and knockdown characteristics compared to delivering either cargo alone. This messenger RNA (mRNA)/small interfering RNA (siRNA) co‐delivery platform was adopted to deliver chimeric antigen receptor (CAR) mRNA and siRNA targeting PD‐1 to primary human T cells ex vivo and strong CAR expression and PD‐1 knockdown were observed without apparent changes to overall T cell activation state. This delivery platform shows great promise for transient immune gene modulation for a number of immunoengineering applications, including in the development of improved cancer immunotherapies.This article is protected by copyright. All rights reserved

High-Throughput In Vivo Screening Identifies Differential Influences on mRNA Lipid Nanoparticle Immune Cell Delivery by Administration Route

“…mRNA was synthesized via in vitro transcription (IVT) essentially as described previously. 46 FLuc or mCherry coding sequences (CDS) were cloned into an mRNA production plasmid with a 101 nt poly(A) tail. Sequences were transcribed with cotranscriptional capping using the CleanCap AG trinucleotide cap 1 analog, precipitated using lithium chloride, and purified using cellulose chromatography as described previously.…”

Prospects and challenges of CAR-T cell therapy combined with ICIs

“…In this way, they generated CAR-T cells with transient CAR expression and PD-1 interference, without altering the overall activation state of the T cells. This delivery method, which can restore the expression of immune checkpoints, can significantly reduce autoimmune risk ( 126 ).…”

Advances in research on factors affecting chimeric antigen receptor T‐cell efficacy

“…Researchers have also developed an engineered LNP platform capable of simultaneously delivering mRNA encoding CAR and siRNA targeting PD‐1 mRNA to T cells. By targeting both tumor cells and immune inhibitory signals, this platform transiently disrupts the PD‐1 signal, allowing the restoration of normal T cell function in patients 89 . Through continuous optimization and improvement, the engineered LNP platform holds promise as an effective and safe gene delivery system, bringing forth additional possibilities for clinical therapy.…”