Overcoming recombination in semi-replicating retroviral vectors: A novel double suicide gene therapy for glioblastoma

One of the longstanding challenges in developing semi-replicating retroviral vector (sRRV) systems has been the loss of therapeutic genes due to non-homologous recombination during reverse transcription in dividing tumor cells. To address this, we developed a genetically stabilized sRRV platform capable of efficient combinatorial gene therapy, preserving the integrity of multiple therapeutic genes while replicating and spreading exclusively within tumors. Our system consists of two trans-complementing, replication-defective retroviral vectors: one encoding MuLV-Gag-Pol and cytosine deaminase (CD), and the other encoding GaLV-Env and HSV1-thymidine kinase (TK). These vectors co-infect tumor cells and propagate selectively within the tumor microenvironment. By eliminating homologous sequences between vectors, we achieved a recombination-resistant design, ensuring long-term genetic stability during serial replication. The antitumor efficacy of this platform was validated in two glioblastoma models. First, in a syngeneic rat orthotopic glioma model, C6 glioma cells were implanted intracranially into Wistar rats.The therapeutic vectors were directly injected into the tumor site, followed by systemic administration of prodrugs 5-fluorocytosine (5-FC) and ganciclovir (GCV). Histological analysis on day 98 revealed dose-dependent tumor regression, with complete tumor eradication in the high-dose, dual-prodrug cohort. Survival analysis confirmed significantly extended survival in treated groups. Second, in an intracranial xenograft model using athymic nude mice, human glioma cells were implanted in the brain parenchyma, followed by stereotactic delivery of the sRRV vectors and systemic prodrug treatment. Remarkably, complete tumor remission was again observed in the combination treatment group, despite the absence of adaptive immunity, highlighting the platform's intrinsic efficacy. Analysis of vector genomes recovered from tumor tissues revealed no evidence of recombination or gene loss, confirming the system's robust genetic stability. The vectors maintained persistent coexpression of both therapeutic genes, which are essential for synergistic prodrug activation and tumor cell killing. In conclusion, this study presents a next-generation sRRV platform that resolves a major technical barrier-recombination-mediated gene loss-while enabling stable and tumor-selective delivery of multiple therapeutic genes. Its potent efficacy in both immunocompetent and immunodeficient brain tumor models, including intracranial xenografts, positions it as a promising candidate for clinical translation in glioblastoma and other refractory solid tumors.