Tallon, A.; Laspidea, V.; Ausejo, I.; de la Nava, D.; Labiano, S.; Gonzalez-Huarriz, M.; Zalacain, M.; Patino-Garcia, A.; Villanueva, H.; Fueyo, J.; Gomez-Manzano, C.; Melero, I.; Pastor, F.; Alonso, M. M.; Garcia-Moure, M.

Viroimmunotherapy leverages oncolytic viruses to induce antitumor immunity and is increasingly explored for solid tumors. Their activity can be enhanced by arming them with immunostimulatory payloads, but most approaches rely on protein-based transgenes that are constrained by viral genome packaging limits. Here, we establish a replication-competent Delta 24 RGD-based platform for localized production of immunotherapeutic RNA aptamers at the tumor site. RNA aptamers provide compact, highly specific ligands that can, in principle, target diverse immune receptors. As a model, we engineered a Delta-24-RGD derivative encoding circular 4-1BB targeting aptamers and show that infected tumor cells sustain aptamer transcription and release, which is associated with a pro-inflammatory remodeling of the tumor microenvironment and measurable antitumor activity in different mouse models with a comparable effect to that achieved with a 4-1BBL expressing adenovirus used as a benchmark. Overall, this work delivers a proof of concept that replication competent adenoviruses can serve as in situ factories for extracellularly active RNA aptamers, supporting their development as flexible platforms for localized non coding cancer immunotherapy.