Triplet RNA Lipid Nanoparticles for Locoregional Cancer Immunotherapy

Ionizable lipid nanoparticles (LNPs) are a proven means of delivering nucleic acid-based therapeutics. This project aims to expand the LNP platform for the delivery of immunostimulatory polyinosinic-polycytidylic acid (pIpC). It is demonstrated that pIpC could be successfully incorporated into LNPs with minimal modification to existing protocols. LNPs encapsulating pIpC (pIpC-LNPs) exhibit a spherical shape with a diameter under 200 nm. When administered intratumorally, pIpC-LNPs are significantly more potent than the soluble adjuvant, resulting in complete remission in 25% of tumors. To identify potential synergistic targets, T cell activation markers are screened following pIpC-LNP treatment. OX40 and CD27 are strongly upregulated and associated with intratumoral pIpC-LNP administration. Furthermore, direct treatment of a cancer cell line with pIpC-LNPs results in upregulation of the immunosuppressive PDL1. To develop a comprehensive RNA-based immunotherapeutic strategy, LNPs are formulated with mRNAs encoding CD70 (the CD27 ligand) and OX40L, or with siRNA targeting PDL1, and are evaluated in combination. Tumor growth reduction is observed when pIpC-LNPs are combined with siPDL1. This study demonstrates the potential of a triplet RNA platform-comprising immunostimulatory RNA, mRNA, and siRNA, delivered via a single versatile LNP. The data support development of pIpC-LNPs as potent intratumoral therapeutics and highlight several potential synergistic targets.