Poster W15 - Intracellular Delivery of Triphosphorylated Stem-loop RNAs for Potent RIG-I Activation and Improved Anticancer Immune Responses

Introduction: Although immune checkpoint blockade (ICB) has demonstrated disease control in certain cancers, it has also indicated limited therapeutic efficacy in poorly immunogenic cancers. Through the activation of specific cellular pattern recognition receptors (PRRs), the immunosuppressive tumor microenvironment (TME) of these cancers can be reprogrammed to a more immunogenic, ‘hot’ phenotype with larger populations of infiltrating T cells, directly correlating with improved responses to ICB. Indeed, activation of the retinoic acid-inducible gene I (RIG-I) pathway has been found to elicit a downstream signaling cascade resulting in the production of type I interferons which can induce this shift in the TME. Unfortunately, most RIG-I-activating therapeutics are not able to freely cross the cell membrane where they are required to activate this cytosolic PRR. The goal of this work is to optimize the loading of 5’-triphosphorylated RNA (3pRNA), a potent RIG-I agonist, within pH-responsive polymeric nanocarriers via a flash nanoprecipitation (FNP) process and assess intracellular delivery and therapeutic efficacy of this platform in murine cancer models.

Materials and

Methods: Reversible addition-fragmentation chain transfer (RAFT) polymerization was utilized to synthesize a library of [PEG]-bl-[DMAEMA50%-co-AnMA50%] surface-forming copolymers and [DMAEMA50%-co-AnMA50%] core-stabilizing polymers. By inducing turbulent mixing within a confined impingement jet (CIJ) mixer of an organic copolymer solution and an aqueous solution containing solubilized 3pRNA, uniform 3pRNA-loaded nanocarriers were produced. Carriers were analyzed for size, polydispersity (PDI), and surface charge using a Malvern Nano ZetaSizer, morphology using cryogenic electron microscopy (cryoEM), and encapsulation efficiency using a Quanti-it RiboGreen assay (Thermo Fisher Scientific). Dose-dependent cytotoxicity was assessed in vitro using a CellTiter-Glo luminescent cell viability assay (Promega), and endosomolytic activity was measured using a galectin (Gal) reporter assay. To examine activation of RIG-I in vitro, reporter cells were treated with formulations and a QUANTI-Luc assay was performed to measure the amount of Lucia luciferase secreted from cells, a readout which is directly proportional to interferon production. Finally, therapeutic efficacy was assessed in a murine MC38 colon cancer model and a B16.F10 melanoma model by monitoring tumor volume, weight loss, and survival after intratumoral administration of 3pRNA-loaded nanocarriers.

Results, Conclusions, and Discussions: Nanocarriers were first assessed for size, polydispersity (PDI), and encapsulation efficiency (EE) of SLR14, a potent RIG-I-activating stem-loop RNA. All nanocarriers were found to be of desired size ( < 100 nm), uniformity (PDI < 0.3), and possess encapsulation efficiencies of ~50%. Transmission electron microscopy (TEM) and cryogenic electron microscopy (cryoEM) images indicated that all nanocarriers possessed vesicular morphologies. Galectin reporter assays were conducted to assess endosomolytic activity of the platform, and it was determined that all formulations were able to induce endosomal disruption and intracellularly deliver drug payloads. In vitro immune activation was assessed in A549-Dual reporter cells, bone-marrow derived macrophages, and splenocytes, and it was found that all formulations elicited greater immune responses compared to free drug. Finally, a therapy study was conducted in an MC38 murine colon cancer model in which mice were given three treatments of nanocarriers intratumorally three days apart. Mice were monitored for tumor volumes, weight loss, and survival, and it was found that nanocarriers were able to significantly inhibit tumor growth compared to PBS control mice. Future work will seek to gain a more thorough understanding of the behavior of the system in vivo. We plan to conduct uptake studies in which we will fluorescently label both the 3pRNA and the nanocarrier and monitor retention at the tumor site over time. Additionally, we will perform qPCR analysis in which we will harvest the tumors of treated mice and assess the expression of pro-inflammatory markers including Cxcl9, Cxcl10, Tnf, and Ifnb1. Finally, we will conduct a therapy study using a B16.F10 melanoma model in which we will monitor tumor burden, survival, and weight loss of mice over time. This will allow for us to assess therapeutic efficacy of our platform in both an immunogenic, 'hot' tumor microenvironment (MC38) and an immunosuppressive, 'cold' tumor microenvironment (B16.F10).

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