AAV-delivered miRNA-gated Cre circuits enable programmable gene activation in AFP-active liver tumor models.

Selective therapeutic gene activation in tumors remains challenging because tumor-enriched promoters often exhibit basal activity in non-target cells and may not fully capture intratumoral heterogeneity. In addition, direct promoter-driven expression of cytotoxic or tumor-suppressive payloads may be vulnerable to promoter leakiness, which can compromise specificity and safety. Here, we developed SLICER, a two-vector adeno-associated virus (AAV)-delivered Cre-loxP gene circuit that integrates an alpha-fetoprotein (AFP) promoter-driven sensor with post-transcriptional gating through microRNA (miRNA) recognition elements (MREs) for miR-21 and miR-122. This dual-layer design separates tumor-context sensing from downstream payload activation, converting context-dependent Cre accumulation into recombination-gated payload expression from the actuator vector. In AFP-active liver tumor cell models, including HepG2, Huh7, and PLC/PRF/5, SLICER induced robust Cre expression and loxP-dependent luciferase activation, whereas non-target/comparator cells showed minimal background activity and limited functional toxicity. Mutation of miR-21 and/or miR-122 recognition elements increased Cre accumulation and reporter output, supporting an MRE-dependent gating mechanism that contributes to circuit specificity. A pro-apoptotic BAX payload triggered caspase-dependent apoptosis in target cells, which was partially attenuated by Z-VAD-FMK, while non-target cells remained largely unaffected. Perturbation of miRNA inputs further tuned circuit output, consistent with miRNA-guided regulation. SLICER also accommodated modular tumor-suppressor payloads, including TP53 and PTEN, supporting actuator-layer interchangeability. In vivo, systemic AAV delivery of SLICER-TP53 suppressed tumor growth and reduced tumor burden in both HepG2 and Huh7 xenograft models, accompanied by increased intratumoral P53 expression. Together, these findings establish SLICER as a modular proof-of-concept platform for combinatorial transcriptional and post-transcriptional control of therapeutic gene activation in AFP-active liver tumor contexts, while supporting further development of logic-gated gene circuits for more precise tumor-selective payload delivery.