ABSTRACT MicroRNAs have a powerful and well described role in the control of biological processes, both in healthy and pathologic tissue. Yet, their success in gene therapy applications, particularly for cancer, has been very limited to date. This shortcoming is mainly due to the fact that single microRNA strategies have been implemented. On the contrary, the promise of microRNAs as a valid therapeutic tool relies on their unique property to function in clusters, whereby groups of closely associated microRNAs regulate intertwined, and often redundant, cellular pathways. This oncogenic redundancy is at the base for the failure of many targeted therapies, as it mediates rescue phenomena responsible for resistance. Ternalys Therapeutics, Inc., is a startup company based on intellectual property which allows the design and production of chimeric artificial RNA genes that can simultaneously modulate multiple microRNAs of choice, thus re-establishing expression of desired microRNA clusters, and achieve precision multitargeting. We have demonstrated the validity of this approach in glioblastoma, the most lethal and common of brain cancers in the adult population. This cancer relies on a complex of undruggable chromatin-modifying enzymes to enact resistance against genotoxic stress and sustain stemness. This complex, and the crucial biologic responses that it mediates, can be successfully targeted with a combination of multiple microRNAs encoded by recombinant RNA transgenes of our design. In this proposal we seek to fine tune the potency and applicability of our product, by pursuing two independent and correlated specific aims. In Aim 1, three transgenic RNAs with progressively higher microRNA-modulating capability will be compared for their ability to interfere with the glioblastoma epigenetic landscape, and to synergize with genotoxic stress provided by standard of care chemoradiation. In Aim 2 we will select a suitable delivery strategy for the candidate transgene, comparing delivery efficiency between Lentiviral and Adeno- Associated Virus vectors in a preclinical mouse model of intracranial glioblastoma. EXPECTED OUTCOME: By the end of Phase I, we will have sufficient data to select the best performing product, which will then undergo IND-enabling studies in a follow-up Phase II application