Abstract 3300: Heat-induced expression of cytosine deaminase for enzyme-prodrug therapy.

Abstract

Abstract Introduction: Bacterial cytosine deaminase (CD) mediates the de novo conversion of the non-toxic prodrug, 5-fluorocytosine (5-FC) to its cytotoxic form, 5-fluorouracil (5-FU). Enzyme localization at the tumor site reduces systemic toxicity while increasing local drug availability. The in situ cellular expression of enzymes provides greater stability and control of enzyme activity as compared to isolated enzymes, and their on-demand expression can be achieved through transcriptional factors activated by stimuli such as heat. Bacterial λ PR promoter in conjunction with the cI857 repressor gene imparts thermoselectivity to enzyme expression - cloned vectors containing CD gene under the transcriptional control of this expression system preferentially produce CD at elevated temperatures, providing a thermal switch to trigger enzyme synthesis. Such enzyme-prodrug therapy, when timed to perform as an adjuvant to other treatment modalities such as magnetic nanoparticle hyperthermia, has the potential to enhance therapeutic index as a combination therapy. Methods: CD gene was PCR amplified from pbCD540FT vector and cloned into pLDR20 vector. The CD gene was placed under the transcriptional control of λ PR promoter and cI857 thermosensitive repressor cassette. The cloned vector was transformed into NM522 competent cells. Positive colonies selected from LB-Agar-Ampicillin plates were cultured in LB-Ampicillin medium. CD gene was characterized by PCR amplification and cycle sequencing of the isolated plasmid DNA. Protein expression in cells grown at 30 °C and 42 °C was analyzed by SDS-PAGE followed by Coomassie blue staining. Enzyme activity in cells grown at 30 °C and 42 °C was compared by incubating engineered cells with 5-FC and spectrophotometrically measuring its conversion to 5-FU. Results summary: The presence of CD gene in competent cells was verified by PCR amplification and cycle sequencing. We observed an enhanced expression of CD gene in the transformed cells at 42 °C as compared to 30 °C, from SDS-PAGE. Spectrophotometric analysis of enzyme activity showed an enhancement in cells grown at 42 °C. We are extending our work to combine enzyme-prodrug therapy with magnetic nanoparticle hyperthermia. We will coencapsulate our engineered cells with magnetic iron oxide nanoparticles (IONP) in immunoisolative sodium alginate microspheres. Localized heating during IONP hyperthermia can be a source of thermal stress for triggering enzyme expression resulting in the in situ synthesis of 5-FU. This combined therapy should enhance therapeutic index as compared to each of the therapies alone. Acknowledgement: pbCD540FT was a kind gift from Prof. J. M. Brown, Stanford University School of Medicine. This work is supported by the Dartmouth Center of Cancer Nanotechnology Excellence pilot project grant, NIH U54 CA151662 (BG, NVK). Citation Format: Venkata K. Nemani, Riley E. Ennis, Karl E. Griswold, Barjor Gimi. Heat-induced expression of cytosine deaminase for enzyme-prodrug therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3300. doi:10.1158/1538-7445.AM2013-3300