5110 Development Of Exosome-Mediated Suicide Gene Therapy For Enhanced Cancer Treatment

Abstract Disclosure: J. Ku: None. R. Flojo: None. P. Zhang: None. B. Lu: None. Development of Exosome-mediated Suicide Gene Therapy for Enhanced Cancer Treatment Joy Ku#, Renceh Flojo#, Pengyan Zhang, and Biao Lu* Department of Bioengineering, Santa Clara University, 500 El Camino Real, Santa Clara, California 95053, USA#Authors contributed equally to this work. *Correspondence: [email protected] Existing suicide gene therapy strategies are designed to express prodrug-converting enzymes in the cytosol of cancer cells, limiting their therapeutic reach to gene-transfected cells and their immediate neighbors through bystander effects ([1]). This study proposes a novel approach to enhance cancer treatment by redirecting prodrug-converting enzymes into extracellular vesicles, also known as exosomes, potentially enabling metastatic cancer cells to receive therapeutic drugs through cancer-homing mechanisms (2, 3). To achieve this, we aimed to develop an exosome-based suicide gene therapy strategy employing human herpes simplex virus-thymidine kinase (HSV-TK) and cytosine deaminase-uracil phosphoribosyl transferase (CD-UPRT) enzymes. We designed and constructed two lentiviral vectors expressing chimeric fusion proteins, incorporating exosome-targeting motifs, green fluorescent protein, Gaussia luciferase, and HSV-TK/CD-UPRT. We demonstrated that both chimeric fusion proteins were properly shunted into the exosome pathways by transfection of cultured 293T and HepG2 cells, followed by real-time monitoring and confocal imaging. This was further corroborated by colocalizing these fusion proteins with well-established exosome markers, such as CD9, CD63, CD8[1], and XPACK. Furthermore, the isolation and characterization of secreted exosomes confirmed the successful loading of engineered enzymes onto exosomes, which were subsequently released into the conditioned media of both cell models. Importantly, initial experiments involving transfection and prodrug treatment showed that HepG2 cells were significantly inhibited (up to 84%) in a dose- and time-dependent manner when treated with the prodrug Ganciclovir in the HSV-TK model. Our study demonstrates the potential adaptation of prodrug therapy to an exosome-based platform, opening the door to a novel nano-treatment approach for cancer. Reference: ([1]) Thoidingjam et al., Translational Medicine communications. 2023. 8:[1][1]. (2) Lu et al., Advanced Drug Delivery Reviews. 2022. [1][1]4465. (3) Zhang et al., Signal Trnsductio and Targeted Therapy. 2023. 8:[1]24. Presentation: 6/2/2024