Abstract PR12: Robust modulation of gene expression in aggressive glioblastoma mouse models: A new approach for in vivo target validation

Introduction: Glioblastoma (GBM) is an aggressive and highly malignant form of brain cancer with a devastatingly poor clinical prognosis. Despite years of research, there is a surprising lack of effective therapies and novel approaches for treatment. There exists a great, unmet need for strategies enabling efficient and targeted modulation of gene expression in established tumors. RNAi-mediated gene silencing is a promising strategy for straightforward modulation of gene expression, and siRNA-based therapies could result in more effective and less toxic treatment for GBM. The Khvorova laboratory has developed a fully chemically stabilized, self-delivering siRNA platform supporting efficient intratumoral uptake and target gene silencing in a GBM orthotopic mouse model. Methods: For all in vitro and in vivo studies, a patient-derived GBM8 cell line was used. Uptake in GBM8 cells was done by treating cells with 0.75 µM of fully chemically stabilized, Cy3-conjugated, hydrophobically-modified siRNA (hsiRNA) targeting the huntingtin mRNA (Cy3-FM-hsiRNAHTT). At different time points (0 to 5.5 hours) GBM8 cells were harvested by cytospin, fixed in 4% formaldehyde, and then imaged by confocal microscopy (40X oil). Next, levels of mRNA knockdown in GBM8 cells were determined by plating 50,000 cells/well in a 96-well plate and treating with increasing doses of FM-hsiRNAHTT (0.01 to 3 µM). After 72 hours, cells were lysed and mRNA was quantified using QuantiGene (Affymetrix) assay. In vivo distribution was done by unilaterally injecting Cy3-FM-hsiRNAHTT into the striatum (2 nmol) or the ventricle of nude mice (5 nmol), 4 weeks after GBM8 tumor implantation. 24 hours after injection, mice were sacrificed, perfused with 4% formaldehyde, and brains were sectioned into 4 µm sections. Knockout in nude mice following GBM8 tumor implantation was also conducted. Mice were treated with FM-hsiRNAHTT (5 nmoles, n=5), a non-targeting control hsiRNA (NTC, 5 nmoles, n=5), or vehicle (5 µL, n=5) and sacrificed after 5 days. 3 biopsies per animal were collected from the striatum of the tumor-bearing side. The levels of huntingtin mRNA expression were determined by QuantiGene® and normalized to a housekeeping gene and reported as percent of CSF. Results: We observed significant uptake in GBM8 cells treated with 0.75 µM Cy3-FM-hsiRNAHTT starting at 2 hours. In addition, huntingtin mRNA was reduced by 50% at 0.3 µM and was reduced by 80% when cells were treated with 1 µM. Following a direct intratumoral injection of Cy3-FM-hsiRNAHTT, we saw widespread distribution of Cy3-fluorescent co-localizing with the tumor. Intratumoral administration of 5 nmoles of FM-hsiRNAHTT resulted in 50% target silencing and was statistically significant when compared to the CSF or NTC treated groups. However, a single injection into the ventricle only resulted in 25% silencing and was only statistically significant relative to a non-targeting hsiRNA control. Summary/Conclusion: Here, we demonstrate that a single intratumoral injection of hydrophobically modified, metabolically stable siRNA (hsiRNA) results in robust and long-lasting silencing of targets in established tumors. Due to the high potency, long duration of effect, and predictable pharmacokinetics of hsiRNA, it is possible that medicine based on this technology can be tailored to a highly diverse patient population. As is, it is believed to be an ideal drug platform for treatment of cancer, as long as efficient delivery to the tumors and metastasis is achieved. Thus, this technology allows immediate modulation of expression in orthotropic tumor models and may contribute to our understating of tumor biology. In addition, this approach can, in the future, serve as a foundation for new therapeutic interventions for this devastating disease. This abstract is also presented as Poster B44. Citation Format: Andrew H. Coles, Maire F. Osborn, Diane Golebowski, Matthew Hassler, Miguel S. Esteves, Anastasia Khvorova. Robust modulation of gene expression in aggressive glioblastoma mouse models: A new approach for in vivo target validation. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR12.