Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 106; no. 26; pp. 10638 - 10643
• Main Authors: Gitzinger, Marc, Kemmer, Christian, El-Baba, Marie Daoud, Weber, Wilfried, Fussenegger, Martin
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 30.06.2009; National Acad Sciences
• Subjects: Alkaline Phosphatase - genetics; Alkaline Phosphatase - metabolism; Animals; Bacteria; Biological Sciences; Cell culture techniques; Cell Line; Cell Line, Tumor; Cell lines; Cell Transplantation - methods; Cells; Cercopithecus aethiops; CHO Cells; COS Cells; Cricetinae; Cricetulus; Dermatologic Agents - pharmacology; Dose-Response Relationship, Drug; Flavonoids; Flavonoids - pharmacology; Gene expression; Gene expression regulation; Gene Expression Regulation - drug effects; Genes; Genetic Vectors - genetics; Humans; Kidney cells; Malus; Malus - metabolism; Metabolism; Metabolites; Mice; NIH 3T3 Cells; Operon - genetics; Phloretin - pharmacology; Proteins; Pseudomonas putida; Pseudomonas putida - genetics; Response Elements - genetics; Skin; Transdermal application; Transfection; Transgenes; Transgenes - genetics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0901501106

Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida-mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies.