β-Hydroxy Thioether-Derived Ionizable Lipids for Spleen-Tropic mRNA Delivery and <i>In Vivo</i> Chimeric Antigen Receptor T Cell Engineering.

Abstract

The delivery of nucleic acids via lipid nanoparticles (LNPs) to generate chimeric antigen receptor (CAR) T cells <i>in vivo</i> represents a promising therapeutic strategy for a broad spectrum of diseases. Nevertheless, the low efficiency of <i>in vivo</i> gene delivery to T cells remains a major obstacle. In this study, we established a structurally diverse combinatorial library of ionizable lipids based on a β-hydroxy thioether scaffold. From this library, 300 distinct ionizable lipids were synthesized and systematically evaluated for their mRNA delivery efficacy using a luciferase reporter assay in both <i>in vitro</i> and <i>in vivo</i> settings. We identified 113-AA-C8C14 as a leading candidate, which demonstrated a tropism for spleen-specific mRNA delivery. The 113-AA-C8C14 LNPs achieved a 57-fold enhancement in mRNA expression in the spleen compared to a benchmark selective organ-targeting (SORT) LNP formulation. Furthermore, when loaded with mRNA encoding a CD19-specific CAR, the 113-AA-C8C14 LNPs generated 2.7 times more CAR T cells <i>in vivo</i>. This was accompanied by an enhanced T cell response, evidenced by a 2.9-fold and 3.7-fold increase in the secretion of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), respectively. Consequently, this approach led to a 2.3-fold greater inhibition of tumor growth in a mouse model of pancreatic cancer. Our study demonstrates the potential of the 113-AA-C8C14 LNP for <i>in vivo</i> CAR T cell therapy in the future.