Poster 136 - RNA Co-Delivery Nanoparticle-Mediated Concurrent Restoration of Tumor Suppressor and Inhibition of Tumorigenic Driver for Synergistic Cancer Treatment

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Ryan rafarokhzad@gmail.com (he/him/his)

Research Trainee
Brigham and Women's Hospital
Boston, Massachusetts, United States

Co-Author(s)

YZ

Yang Zhang

Postdoctoral fellow
Brigham and Women's Hospital
Boston, Massachusetts, United States

Last Author(s)

JS

Jinjun Shi, PhD

Associate Professor of Anaesthesia
Brigham and Women's Hospital, Harvard Medical School
Boston, Massachusetts, United States

Introduction: Cancer is commonly caused by gain of function in proto-oncogenes and simultaneous loss of function in tumor suppressor genes. For example, mutations in both phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a well-known tumor suppressor, and androgen receptor (AR), a pro-tumorigenic transcription factor, are often linked with advanced prostate cancer (PCa), the second leading cause of cancer death among men in the United States. However, few therapeutic approaches can directly address the loss of tumor suppressor functions, and there are no therapies for adequately addressing the simultaneous correction of oncogenic promotion and suppressor depletion. Thus, we hypothesized that concurrent PTEN restoration with mRNA and AR silencing with siRNA may be highly beneficial for the treatment of advanced PCa, as the AR and PI3K-AKT (mediated by PTEN loss) pathways regulate each other by reciprocal feedback. Here, we explored first-in-field concurrent modulation of PTEN mRNA (mPTEN) and AR siRNA (siAR) to PCa tumor cells using an RNA lipid nanoparticle (LNP) platform.

Materials and

Methods: We began our experiments by optimizing our LNP delivery platform for both siRNA and mRNA delivery. We formulated a library of 12 LNPs by varying the ratio of two distinct ionizable lipids, polyethylene glycol (PEG)-lipid conjugates, phospholipids, and cholesterols. The examined ionizable lipids were MC3 and SM102, both of which are used in FDA-approved LNP products. The LNPs were evaluated for their biocompatibility in cytotoxicity assays and efficacy for RNA delivery and bioactivity. We further optimized the LNPs for RNA delivery and bioactivity by changing the ratio of Nitrogen (N; from the MC3) to Phosphorus (P; from the RNA). We explored three distinct N to P ratios (N:P) and selected a final formation for our delivery platform. We performed cell viability experiments with four concentrations of siAR and mPTEN in two cell lines: LNCaP and C4-2, both of which are PTEN null and AR-positive. We performed experiments with either siRNA or mPTEN and subsequently explored concurrent delivery of siAR and mPTEN LNPs. We observed and quantified synergistic effects with several concentrations of mPTEN and siAR in the concurrently delivered groups. We explored the mechanism of siAR and mPTEN activity by performing deep, unbiased cellular proteomics analysis using the Proteograph XT assay and Orbitrap Astral mass spectrometer. We measured the perturbation of nearly 9,000 proteins in C4-2 cells in response to the administration of siAR, mPTEN, and the combination of siAR and mPTEN.

Results, Conclusions, and Discussions: In LNCaP and C4-2 cells, we saw a much better anti-tumor effect in vitro by LNP mPTEN + LNP siAR vs. LNP(mPTEN) or LNP(siAR). More impressively, the co-delivery strategy showed a synergistic effect under certain combinations of mPTEN and siAR concentrations, and among the three LNPs, LNP mPTEN + LNP siAR also showed the highest apoptosis in both cell lines. We further did western blot analysis to demonstrate successful concurrent PTEN reconstitution and AR silencing by the LNPs, as well as the inhibition of their downstream targets. We are now analyzing the proteome of PCa cells after treatment with these RNA LNPs for an in-depth biological understanding of the co-targeting strategy that could lead to the development of a more effective treatment for advanced PCa and potentially other malignancies. Bioinformatic analysis of nearly 9,000 proteins was performed, and we identified up and downregulation of distinct biological pathways after administration of siAR, mPTEN, and the combination of siAR and mPTEN.

Acknowledgements (Optional): I want to thank Professor Jinjun Shi and Dr. Yang Zhang for their guidance and support.

References (Optional):