EXTH-31. Systematic identification of lipid nanoparticles for mRNA-based epigenetic and immune glioblastoma therapies

Messenger RNA-based therapeutics, including cancer vaccines and gene editors, have potential to revolutionize treatment of malignant gliomas. We recently adapted a CRISPR-based epigenetic editing therapeutic, CRISPRoff, to induce long-term silencing of MGMT in glioblastoma preclinical models, enhancing sensitivity to temozolomide. However, in vivo delivery of epigenetic editors is a barrier to clinical translation. Here, we developed an iterative screening strategy to optimize delivery of mRNA-based therapeutics for gliomas using lipid nanoparticles (LNPs), which have demonstrated promise for mRNA vaccines and epigenetic editing in other diseases. Using high-throughput flow cytometry in glioblastoma patient-derived tumor cultures, we first performed in vitro screening of 54 LNPs, comprising ionizable and novel lipids combined with DOPE, Cholesterol, and DMG-PEG. Each formulation was complexed with GFP mRNA and delivered to GBM6 and SF14259. Efficiency of mRNA delivery was assessed at 24h, identifying 13 LNPs with >50% efficiency in both GBM cultures. To then identify LNPs capable of high efficiency delivery to glioma tumors in vivo, we performed a pooled screen of different LNP compositions whose delivery efficiencies could be measured by next-generation sequencing. 25 top LNPs were coupled with unique mRNA barcodes, and this pool of barcoded LNPs was injected intracranially into patient-derived xenografts, followed by targeted RNA-sequencing of LNP barcodes in tumor and normal adjacent brain 24h post-treatment. Three LNPs (cKK-E12, Lipid A9, Lipid CL1) were validated for high efficiency delivery of mRNA encoding CRISPRoff and sgRNAs targeting MGMT, achieving >99% MGMT silencing in glioblastoma cells, reducing temozolomide IC₅₀ by 48.8-fold. LNP cKK-E12 was also capable of delivering IL-2 mRNA to SF14259 cells, resulting in >60-fold IL-2 secretion. LNP cKK-E12 effectively delivered mRNA to glioblastoma xenografts in vivo and slice cultures derived from patient tumors ex vivo. Overall, this study presents a systematic strategy to optimize LNPs for mRNA-based therapies for glioma.