Hepatocytes and skeletal myocytes shape immunity to mRNA-LNP encoded protein - PROJECT SUMMARY Applications for messenger RNA (mRNA) in a wide range of clinical indications—such as pathogen vaccination, cancer immunotherapy, protein replacement, and gene editing—are rapidly advancing. Since Moderna and Pfizer’s mRNA vaccines against SARS-CoV-2 highlighted the efficacy and potency of mRNA technology, there has been ongoing effort to optimize the formulation of the mRNA and the delivery vehicle, which is often a lipid nanoparticle (LNP), for novel applications. One of the major challenges faced by mRNA therapeutics, even those that have targeting moieties, is mRNA uptake and expression by off-target cells. Current mRNA vaccines depend on antigen presentation by antigen-presenting cells (APCs) to induce robust immunity, yet hepatocytes and skeletal myocytes are also transfected, leading to unintended immune modulation. To better understand how these two cell types influence adaptive immunity to the mRNA-encoded protein, I will regulate the cellular expression patterns of delivered mRNA by incorporating target sites complementary to cell-type specific, endogenous miRNA. My preliminary data indicate that hepatocytes dampen the magnitude of CD8+ T cell responses to the mRNA-encoded antigen, whereas skeletal myocytes enhance CD8+ T cell responses. Understanding the mechanisms underlying the immunomodulatory effects of these two cell types is crucial for optimizing mRNA-based therapeutics. My central hypothesis is that hepatocytes drive an immunoregulatory response to mRNA-encoded protein through abortive CD8+ T cell stimulation and regulatory T cell (Treg) induction, while skeletal myocytes can directly present antigen to and activate CD8+ T cells. I will engineer clinically-relevant mRNA formulations with regulated mRNA expression in these two cell types to enhance antigen-specific adaptive immunity while reducing deleterious off-target effects. In Aim 1, I will delve into the mechanisms by which hepatocytes and skeletal myocytes modulate antigen-specific T cell immunity against the mRNA-encoded protein. I will specifically evaluate the effect of Treg depletion and hepatocyte-restricted antigen presentation on CD8+ T cell responses. To assess the role skeletal myocytes may play, I will evaluate the duration of antigen presentation and whether skeletal myocytes are sufficient to induce T cell recall. I will also investigate whether exogenous antigen uptake by APCs can induce antigen-specific T cell immunity. In Aim 2, I will leverage the impact of mRNA expression by hepatocytes and skeletal myocytes to enhance antigen-specific adaptive immune responses to tumor-associated antigen (TAA) and SARS-CoV-2 spike mRNA vaccines, while reducing off-target cytotoxicity from CD8+ T cells, especially in the liver. I will design mRNA vaccines to boost anti-TAA CAR-T cells, induce exogenous anti-tumor CD8+ T cell activity, and enhance anti-spike antibody and T cell responses. Ultimately, this project aims to provide insight into design strategies for mRNA vaccines to tailor immunogenicity while minimizing adverse effects.
