Nanoparticle Delivery of Immunostimulatory Alu RNA for Cancer Immunotherapy

Introduction:: Melanoma is a skin cancer with almost 100,000 new cases and 8,000 deaths each year. Although the standard of care consisting of surgical resection and checkpoint inhibition has shown success, 50% of melanoma patients do not respond to ICB in part due to insufficient T cell infiltrate. One strategy to overcome this challenge is to exploit the innate immune system's RNA sensing pathways through pattern recognition receptors (PRRs), which activate pro-inflammatory signaling cascades. To avoid aberrant immune activation by self-RNAs, cells employ adenosine deaminases acting on RNA (ADARs) to base edit adenosine to inosine, a process known as A-to-I editing. When A-to-I editing is defective, detection of RNA results in type I interferon and NF-κB pro-inflammatory responses. Lack of A-to-I editing is implicated autoimmune diseases including multiple sclerosis (MS) where PRRs recognize RNA derived from endogenous Alu elements dispersed within the human genome. We thereby hypothesized that localized pharmacological activation of PRRs via Alu RNA may be harnessed for cancer immunotherapy by stimulating an antitumor innate immune response. However, RNA requires a carrier as it is poorly internalized or degraded in the endosome before it can reach PRRs in the cytosol. Nanoparticles (NPs) are a strategy to overcome these challenges. Here we use endosomal lytic poly[(DMAEMA)-block-(PAA-co-DMAEMA-co-BMA)] (D-PDB) NPs to intratumorally deliver Alu RNA (Alu-NP) for treatment of mouse B16.F10 melanomas.

Materials and Methods:: Lyophilized stocks of D-PDB were dissolved in ethanol at 50 mg/mL and then diluted to 10mg/mL in phosphate buffer (pH=7.0), resulting in the formulation of micelles. Micelles were concentrated, diluted to 1 mg/mL, added to Alu RNA, rapidly mixed by pipetting, and incubated for 20 minutes at room temperature to allow for electrostatic complexation. Nanoparticle sizing used a Zetasizer Nano ZS instrument (Malvern Panalytical), morphology was evaluated via transmission electron microscopy and gel electrophoresis to determine RNA complexation. THP-1 Dual cells and RAW Dual cells were treated with RNA-polymer conjugates then supernatants were collected for Quanti-Luc^TM (InvivoGen) assay, which provides a readout of type I interferon production. In vivo experiments: 6-8 week-old C57BL/6 mice were inoculated with 500,000 B16.F10 or 1,000,000 B16.F10 IFN-LUC mouse melanoma cells on their rear right flank. Treatment utilized 2 μg of RNA in D-PDB NPs at an N/P ratio of 4 and began when tumor volume was between 50-100 mm³. Additional treatment injections took place three and six days post-initiation. IVIS imaging quantified interferon production in the luciferase expressing cancer cells, while PCR and nanostring quantified gene expression

Results, Conclusions, and Discussions:: Alu-NPs were ~100 nm in diameter, and the morphology was similar between loaded and unloaded NPs. Alu-NPs activate THP-1-Dual reporter human monocytes and RAW-Dual mouse macrophages in vitro, while empty NPs, free RNA, and an edited, inactive Alu RNA did not produce a type I interferon response. These data show that only delivery of active RNA in a NP induces a PRR response as neither polymer alone or RNA alone induce a type I interferon response. In vivo, Alu-NPs elevated Ifnb1, Cxcl10, and Tnf RNA levels in the tumor microenvironment compared to PBS treatment when evaluated by qPCR. Furthermore, Alu-NPs administration resulted in elevated interferon levels in the tumor as measured by IVIS imaging in mice with B16.F10 tumors expressing interferon-inducible luciferase (Fig. 1A). Shifting the tumor microenvironment from an immunosuppressive to a pro-inflammatory state may make the tumor more susceptible to immune-mediated killing. To this end, Alu-NPs inhibited tumor growth and extended survival compared to PBS, D-PDB alone, and inactive Alu RNA (Fig. 1B-C). Overall, our study demonstrates that NP-mediated delivery of an Alu RNA discovered in MS patients can prolong survival by increasing the pro-inflammatory cytokine levels in the tumor microenvironment.

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