Date of Award

1-1-2023

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Richard Edelson

Abstract

Transimmunization (TI) is a dendritic cell (DC)-based immunotherapy derived from extracorporeal photochemotherapy (ECP), a widely used immunotherapy for cutaneous T cell lymphoma. TI builds upon the mechanistic understanding that ECP inducts large-scale monocyte-to-dendritic cell differentiation through physiologic platelet-monocyte interactions that occur during the passage of cells over a plastic plate. While TI and ECP have used apoptotic tumor cells treated with 8-methoxypsoralen (8-MOP) and ultraviolet-A (UVA) irradiation as the source of antigens by which to target its immunotherapeutic effects, there is investigational interest in alternative antigen sources, including mRNA-containing lipid nanoparticles (LNP). In this work, we developed a modified version of TI incorporating mRNA LNPs as its antigen source, dubbed mRNA-TI. We demonstrated that mRNA-TI-derived DCs are able to efficiently internalize mRNA LNPs, express encoded proteins, and process and present these proteins as antigens in a manner that effectively stimulates antigen-specific CD8+ T cells, using ovalbumin (OVA) as a model antigen. We also found that mRNA-TI vaccination of mice prior to tumor challenge with E.G7-OVA, an OVA-expressing lymphoma model, significantly inhibited tumor establishment and growth, demonstrating the potential of mRNA-TI as an immunotherapy. We also developed an mRNA-TI-based LNP screening and discovery platform that used nanoluciferase-encoding mRNA LNP treatment to rapidly and simultaneously quantify the relative transfection efficiency of multiple LNP formulations in murine and human TI-derived DCs. Using this method, we observed species-dependent differences in the transfection efficiency of LNP formulations based on cKK-e12 and SM102 lipid backbones. Finally, single-cell RNA sequencing analysis of TI-treated PBMCs revealed the emergence of a new population of monocyte-derived dendritic cells within hours of TI plate passage, with notable upregulations in genes not only associated with dendritic cell differentiation, but also with the uptake and processing of lipids by dendritic cells. Collectively, these findings suggest a synergy between TI-derived DCs and mRNA LNPs and demonstrate a proof-of-principle of mRNA-TI as an antitumor cellular immunotherapy and as a platform for LNP discovery.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 06/30/2024

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