Engineered AAV vectors with dual tumor-specific promoters for safer and more effective cancer therapy.

e15112 Background: Solid tumors remain a significant challenge in oncology due to recurrence, side effects, and resistance to existing therapies. Pyroptosis, a programmed cell death mechanism, has shown promise for inducing tumor cell death while stimulating anti-tumor immunity. However, achieving safe and tumor-specific delivery of pyroptosis-inducing genes remains a major obstacle. Methods: We engineered adeno-associated virus vectors by incorporating an RGD peptide into the capsid protein (AAV-RGD) to enhance transduction efficiency in cancer cells. Tumor-specific expression was further achieved by employing the PEG3 and CEA promoters to drive the expression of pyroptosis-related genes. A dual-promoter system, in which Cre recombinase and a reverse-oriented GSDMD-N cassette (GSDMD-N FLEX) were placed under separate tumor-specific promoters, was used to confine pyroptosis induction to tumor cells. Therapeutic efficacy and safety were evaluated with tunor cell lines and a liver cancer mouse model. Following safety evaluation studies, an Investigator-Initiated Trial (IIT) was conducted on a patient with lung cancer and bone metastasis. Results: Enhanced Tumor Targeting: AAV-RGD achieved 8- to 15-fold higher transduction efficiency in cancer cells compared to normal cells in vitro and in vivo. Tumor-Specific Promoters: The PEG3 and CEA promoters demonstrated robust activity in liver cancer cells with minimal off-target expression in normal hepatocytes. Selective Pyroptosis Induction: Co-delivery of Cre and GSDMD-N FLEX vectors led to tumor-specific inversion and expression of GSDMD-N, effectively inducing pyroptosis in cancer cells without affecting normal tissues. Therapeutic Efficacy: In a murine liver cancer model, systemic AAV-RGD administration significantly suppressed tumor growth, matching the efficacy of conventional chemotherapy while minimizing systemic toxicities. Immunogenicity and IIT Outcomes: AAV-RGD showed reduced immunogenicity compared to wild-type AAV. In the IIT study, PET-CT imaging of a patient with lung cancer and bone metastases revealed loss of activity in metastatic lesions after treatment with AAV-RGD. Conclusions: The combination of engineered AAV-RGD vectors and dual tumor-specific promoters represents a safe and effective approach for pyroptosis-based gene therapy. By ensuring precise tumor targeting and minimizing off-target effects, this approach holds the potential for improved clinical outcomes in solid tumors. Future directions include further vector optimization and assessment of translational feasibility for clinical applications.