Cell factories that manufacture microvesicles containing gene silencing RNA prodrugs

Among the vehicles investigated as delivery agents for antisense RNAs are extracellular vesicles (EVs) released from cultured cells. Arrestin domain-containing 1 (ARRDC1)-mediated microvesicles (ARMMs) are naturally occurring EVs that uniquely are formed by outward budding of cytoplasmic membranes. Previous work has shown that ARMMs production is quantitatively controlled by the ARRDC1 protein and that ARRDC1 and macromolecules attached to it are loaded into nascent ARMMs during ARMM formation. Here, we report a strategy for constructing cell factories that biologically manufacture both ARMMs and short hairpin RNA (shRNA)-like antisense RNA precursors, designated as shT-RNAs. Human HEK293T cells mutated in the endoribonuclease DICER1 were engineered to express a fusion protein containing components of ARRDC1 and the trans-activator of transcription (Tat) peptide encoded by the HIV-1 virus. Prodrug RNAs were constructed by replacing the canonical loop regions of shRNAs with a 24-nucleotide segment derived from the Tat-binding trans-activation response (TAR) element. We show that a truncated TAR sequence embedded within the structural framework of shT-RNAs enables their linkage to the ARRDC1-Tat fusion protein and consequent loading of the RNAs into nascent ARMMs, and that RNA is protected by ARMMs membranes from attack by external ribonucleases. We further show that prodrug modules consisting of shT-RNAs that target different genomic sequences of SARS-CoV-2 virus and are manufactured as components of a single transcript can, when delivered by ARMMs, be activated to suppress virus RNA production. Our results indicate that the ARMMs-based platform we have designed can deliver gene-silencing RNAs in the form of biologically produced shRNA-like prodrugs.