Abstract 2068: Engineering of hairpin loop enhances the loading of endogenously expressed pre-miRNA into extracellular vesicles

Extracellular vesicles (EVs) hold tremendous potential as drug delivery carriers for therapeutic nucleic acids such as microRNA (miRNA) due to their natural composition and ability to be engineered to contain targeting peptides on their surface. We have engineered HEK293T cells to secrete EVs by overexpressing Lamp-2A protein containing liver cancer targeting peptide PC94 and therapeutic pre-miR-199. The novel feature of this system is that both the cargo and the microvesicles are synthesized by the same cells, thus abrogating the need for loading synthetic oligonucleotides into EVs. The pre-miR-199a loop region was modified such that it resembled the HIV-1 transactivation response (TAR) RNA which was engineered into the Lamp2A intron. Correct splicing of the intron portion and processing of the mature miRNA was evaluated after its transfection into HEK293T cells. Computational modeling was used to study the interaction between the modified pre-miR-199a loop (TAR RNA) and the TAT peptide. This study exhibited a stable interaction between the two and also showed that the peptide is not bound to the stem portion of the RNA loop, permitting the insertion of any pre-miRNA sequence. EMSA gel shift, rDICER processing and luciferase assays were performed to study the correct binding, processing and functionality of the modified sequence. These results show that the modified loop is actively involved in binding with the TAT peptide and it enables the modified miRNA to be loaded in the microvesicles. In an effort to assess the loading of the therapeutic miR-199a-3p relative to other endogenous miRNAs contained within the EVs, we performed small RNA sequencing on RNA isolated from both the producing cells and purified EVs. Triplicate samples of RNA isolated from 3 different HEK293T producing cells were sequenced: wild type HEK293T and those stably transfected with the empty vector (empty) or the TAT/TAR pre-miR-199a (full). The expression of the miRNAs were ranked from the RNA sequenced in both the cells and EVs. Producing cells engineered to express miR-199a-3p containing the TAT/TAR loading motif ranked third when comparing the ratio of miRNAs loaded in the EVs from the full to empty cells. Interestingly, certain mature miRNAs were preferentially loaded into the EVs, including miR-451a, miR-122 and miR-1246. As reported by Villarroya-Beltri, et al., (Nature Comm., 2013) in human peripheral blood mononuclear cells, all three mature miRNAs contained 4 nucleotide “ex