Oncolytic adenovirus incorporating TetO sites in the E1A promoter for controlled replication in TetR-expressing mesenchymal stem cells.

Oncolytic viruses are therapeutic agents that combine self-amplification, lytic activity, and immunostimulatory properties. However, their systemic administration is limited by inefficient tumor delivery. Although mesenchymal stem cell (MSC)-based carrier systems have been proposed to overcome these barriers, their efficacy is restricted by rapid MSC lysis following viral replication, which limits the time available for tumor homing. Here, we describe a tetracycline operator-regulated oncolytic adenovirus designed to temporally control viral replication within MSC carriers and enhance intratumoral viral delivery. In this system, menstrual blood-derived MSCs (MenSCs) are engineered to stably express the tetracycline repressor, while the adenoviral genome incorporates TetO sites upstream of the E1A promoter, enabling reversible pharmacological control of viral replication through tetracycline or doxycycline administration. TetO incorporation did not compromise viral fitness, and viral replication was effectively repressed in TetR-expressing MenSCs, with restoration upon inducer addition. Importantly, once released within the tumor microenvironment, viral progeny replicated unrestrictedly in TetR-negative cancer cells. In lung adenocarcinoma model, TetO-regulated oncolytic adenoviruses delivered by TetR-MenSCs achieved enhanced intratumoral viral accumulation and improved antitumor efficacy. Overall, these findings establish a controllable MSC-based oncolytic adenovirus delivery strategy that addresses a key limitation of systemic virotherapy and supports its clinical translation across solid tumors.