BME MS Thesis Defense: Kenji Goto-Hardy

Title: "RNA Interference-enabled Self-amplifying RNA Platform for Combined Targeted Gene Silencing and Transgene Expression"

Advisory Committee: Chair: Alexander Green, PhD (BME) Advisor: Mark Grinstaff, PhD (BME, Chemistry, MSE, MED) Members: Wilson Wong, PhD (BME)

Abstract: Self-amplifying RNA (saRNA) is a novel mRNA platform technology that utilizes an RNA-dependent RNA polymerase to enable the specific amplification of a desired cargo for sustained potent expression. While saRNA is known for extended transgene expression, the capability of combining the platform with RNA interference (RNAi) technology remains largely unexplored. RNAi describes a mechanism of specific post-transcriptional gene regulation mediated through small RNA oligonucleotides, such as microRNA (miRNA) or small interfering RNA (siRNA), which utilizes sequence complementarity to induce translational repression, destabilize mRNA, or directly cleave target mRNA molecules. Our RNAi-enabled saRNA platform provides two distinct advantages over traditional RNAi methods for gene knockdown: 1) saRNA is an mRNA platform technology which introduces novel functionality through the integration of gene silencing with simultaneous sustained transgene expression and 2) saRNA is known to exhibit tissue tropism, namely negligible liver expression, demonstrating the potential to overcome therapeutic siRNA’s poor extra-hepatic delivery. This work applies two orthogonal design methodologies to insert a miRNA element into our group’s modified saRNA creating a novel self-amplifying miRNA (sa-miRNA) platform. To evaluate each design, we first transfected candidate constructs as DNA plasmids into cells providing access to canonical nuclear processing pathways and identified the best performing sequences. We then optimized RNA dosing by investigating the relationship between saRNA transfection dose and its effect on cell viability and transgene expression in vitro. Leveraging the knowledge gained in these initial experiments, we then synthesized our best designs as mCherry-targeting sa-miRNAs and successfully demonstrated targeted knockdown in an mCherry reporter cell line. Finally, we evaluated the performance of these designs targeting an endogenous gene (p53) in primary human cells in vitro. Our findings demonstrate that our sa-miRNA system further expands the functionality of the modified saRNA platform to address limitations facing traditional RNAi methods.