Abstract LT08: Advancing Targeted Gene Therapy: Leveraging Dumbbell-Shaped DNA for Cell-Specific Precision in Cancer, Virus Infection and Mitochondrial Disease

Cell type-specific gene delivery, which restricts gene transfer to therapy-relevant cells and reduces unwanted side effects from ectopic transgene expression, remains a major challenge. To mitigate off-target effects, we developed a programmable cell type-specific gene therapy using an RNA trans-splicing-based approach for gene replacement or suicide gene therapy. This method selectively expresses therapeutic proteins or suicide signals based on the cell’s endogenous pre-mRNA expression profile. Targeting the liver, we developed an HCC-specific trans-splicing based HSVtk/GCV suicide gene therapy approach. We designed suicide RNAs targeting multiple HCC-related pre-mRNA biomarkers, increasing the concentration of targetable pre-mRNA. Non-viral, redosable dumbbell-shaped DNA vectors were designed, manufactured, and tested in tissue culture cells, 3D patient-derived xenograft spheroids, mouse and porcine models. Liver and lung-specific lipid nanoparticles (LNPs) enable precise delivery of therapeutic agents directly to these organs, enhancing treatment efficacy while minimizing systemic side effects. Meanwhile, conjugation of dumbbell vectors with GalNAc3 residues enabled targeted delivery into hepatoblastoma-derived human tissue culture cells. To deliver nucleic acids into mitochondria, we studied the long non-coding β2.7 RNA of the human cytomegalovirus, known to colocalize with mitochondrial complex I. Thermodynamic profiling identified four conserved structural subdomains within β2.7 RNA responsible for mitochondrial targeting. A vector with eight mitochondrial targeting domains showed three times higher activity than the parental β2.7 RNA. Combining mitochondrial targeting RNA and trans-splicing allows cell type-specific transcription of mRNA for mitochondrial gene therapy. Trans-splicing based gene therapy offers promising perspectives for suicide gene therapy of various cancers, incurable viral infections, and cell type-specific mitochondrial gene therapy. Citation Format: Pei She Loh (Rachel), Sushmita Poddar, Kaustav Chatterjee, Chiu Jing Wen, Derrick Wee Zan Liang, Fritz Lai, Chen Qingfeng and Volker Patzel. Advancing Targeted Gene Therapy: Leveraging Dumbbell-Shaped DNA for Cell-Specific Precision in Cancer, Virus Infection and Mitochondrial Disease [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr LT08.