(H-299) Engineered RIG-I Activating Nanoparticles for Glioblastoma Immunotherapy

Introduction:: Glioblastoma (GBM) is a rare form of brain cancer with over 13,000 new cases each year and a dismal outlook for patients who face a mean survival time of 12-18 months post-diagnosis. Further, there is a high recurrence rate with a five-year survival rate below 10%. The current standard of care – comprising surgical resection followed by radiation and chemotherapy – has not advanced significantly over the past 20 years, creating a need for new therapies. To this end, leveraging pattern recognition receptor (PRR) activation to elicit strong innate immune responses shows promise for cancer immunotherapy. RIG-I is one such PRR, and higher levels of RIG-I are associated with improved survival outcomes for the first year following GBM diagnosis. RIG-I recognizes short double-stranded RNA with a triphosphate (3p) group on the 5’ end (3pRNA), and RIG-I activation stimulates pro-inflammatory responses, including type-I interferons which trigger antitumor immunity. At the preclinical level, RIG-I activating therapies have shown promise for treating various solid tumors, yet pharmacological activation of RIG-I has not yet been explored for GBM immunotherapy. Despite their promise for cancer immunotherapy, the clinical utility of 3pRNA RIG-I agonists is currently limited by significant drug delivery barriers, including poor intracellular uptake, nuclease degradation in the endosome/lysosome, and an inability to reach the cytosol to bind RIG-I. To realize the promise of RIG-I, we will use RIG-I activating nanoparticles (RANs) to treat a mouse model of glioblastoma.

Materials and Methods:: Diblock polymer consisted of a first block of 10K polyethylene glycol (PEG) and a second block of poly[(DMAEMA-c-butyl methacrylate (BMA)) (DB). The DB block serves to complex with RNA and promote endosomal escape. To form micelles, the diblock copolymer was dissolved in ethanol at 100 mg/mL then diluted with sterile-filtered HEPES-buffered glucose solution (HBG, 50 mM HEPES, 5 mM EDTA, 5% glucose (w/v), pH 7.4) to a concentration of 10 mg/mL. RNA was added for 45 minutes at room temperature then dialyzed overnight to concentrate the solution. Ribogreen (Thermofisher) was conducted according to the manufacturer’s protocol to quantify RNA loading. Nanoparticle sizing used dynamic light scatter (DLS) on a Zetasizer Nano ZS instrument (Malvern Panalytical). Mouse Glioma261 (GL261) cells were plated in a 96 well plate at 10,000 cells per well. 24 hours later, cells were treated with transfected with lipofectamine + 3pRNA or lipofectamine + control RNA. 24 hours later, supernatants were collected for ELISA, and cells were stained for flow cytometry. The ELISA was conducted according to the manufacturer’s protocol. In vivo Experiments: Mice were inoculated with 1,000,000 GL261 cells on the right flank. When tumors reached ~50 mm^3, mice were treated intratumorally with PBS or RANs, with additional injections on days 3 and 6 post treatment initiation. Each treatment injection contained 10 μg of 3pRNA.

Results, Conclusions, and Discussions:: DLS showed that RANs were ~90 nm in diameter, and Ribogreen showed successful complexation of RNA to nanoparticles. We show that GL261 cells treated in vitro with RANs produce significantly more interferon beta (IFN-β) than mice treated with control RNA (Fig 1A). Elevated IFN-β levels suggest that tumor resident cells can produce inflammatory cytokines, which may increase immune activation/infiltration into the tumor, leading to reduced tumor size, but do not present antigen themselves. To this end, mice treated with RANs significantly delayed tumor growth (Fig. 1B) and prolonged survival (Fig. 1C). Specifically, RAN treatment extended the time to first death by 6 days and 60% of mice are long term survivors with tumor volumes below 500 mm³. Although the subcutaneous flank model of GBM does not fully represent the tumor microenvironment, our results provide proof-of-concept that RIG-I is a promising target for GBM immunotherapy and that RANs can elicit an antitumor innate immune response that can curtail GBM tumor growth.


Acknowledgements (Optional): :

References (Optional): :