Project 3: Modified RNA and LNP formulation as immunomodulatory agents for cellular immunity

Brown University, Providence RI

Investigators

Abstract

PROJECT 3 ABSTRACT RNA has become a new and successful therapeutic modality that entails the introduction of in vitro transcribed (IVT) modified mRNA into cells via lipid nanoparticle (LNP) delivery vehicles. RNA modifications and LNP formulation both critically impact RNA translation, immunogenicity, biodistribution, and the efficacy of such therapeutics. While incorporation of N1-methylpseudouridine (m1ψ) modifications into IVT mRNA in COVID-19 vaccines has demonstrated safety and potent antibody generation, the fundamental concepts underlying RNA modification and LNP formulation for the generation of cellular immunity are unknown. Unlike vaccination, such cellular (macrophage and T cell) immunity will be required for efficacious application of mRNA-LNPs to cancer immunotherapy. Macrophages are ideal targets for mRNA-LNP cancer therapeutics due to their propensity to internalize nanoparticles, central role in modulating the tumor immune microenvironment, and capacity to present cancer antigen to T cells. We propose that optimization of mRNA modifications and LNP formulations will lead to the generation of improved effector anti-cancer chimeric antigen receptor-macrophages (CAR-M) (aim 1) or promote macrophage cross-presentation of cancer antigens leading to T cell immunity (aim 2). We propose simultaneously screening IVT mRNA modifications, beyond m1ψ, and LNP formulation parameters to optimize anti-cancer macrophage immunity. Anti-HER2 CAR sequences (aim 1) or model cancer antigen (aim 2) will be encoded into IVT mRNA, IVT reactions with T7 RNA polymerase will allow incorporation of modified nucleosides with expected variability in immune stimulatory to antagonistic behavior. These modified IVT mRNAs will be incorporated LNPs with varying immunostimulatory properties. MC3, a lipid used in the FDA approved RNA-LNP therapeutic Onpattro®, will be a ‘non-inflammatory’ control LNP, to compare experimental lipids with heterocyclic head groups, previously shown to promote immune activation. Libraries generated by varying both RNA modification and LNP formulation will be screened in vitro, to identify mRNA-LNPs that maximize CAR-M cancer phagocytosis (aim 1) or antigen presentation (aim 2). Finally, top mRNA-LNPs will be tested for therapeutic efficacy in murine models of peritoneal disseminated ovarian cancer (aim 1) or OVA-antigen expressing melanoma (aim 2). Ultimately, we expect to identify mRNA modification and LNP formulations capable of optimizing macrophage-driven anti-cancer cellular immunity. The insights generated by this proposal will be critical to understanding how IVT mRNA and LNPs modify immunity in the tumor microenvironment and will elucidate fundamental concepts underlying the design of RNA-LNP therapeutics for diverse clinical applications.