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

Abstract

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.synthetic biology ͉ synthetic gene networks ͉ transdermal gene regulation ͉ gene therapy ͉ biopharmaceutical manufacturing

Recent advances in mammalian synthetic biology—design of synthetic transgene control networks

“…A panoply of different transgene control circuits have been designed simply by using different DNA-binding protein-operator sets, preferably of heterologous (mostly prokaryotic) origin to reduce pleiotropic effects in mammalian cells. Non-limiting examples include transgene control modalities responsive to antibiotics [13,[15][16][17], hormones, and hormone analogs [18], immunosuppressive and anti-diabetic drugs [7], vitamins [19], amino acids, and different (secondary) metabolites [20,21] including gaseous acetaldehyde [22] and the apple-derived phloretin mediating transdermal gene induction after topical application [23]. Since all of these control circuits share an identical genetic set-up they are compatible and could be functionally liked to construct higher order gene networks showing more complex control dynamics than the basic ON/OFF-type transcription switch.…”

Application of Synthetic Biology to Regenerative Medicine

“…The idea of controlling a synthetic circuit embedded into the body by externally-applied small molecules has been demonstrated in a proof-of-principle experiment by Gitzinger et al [54], who constructed a transdermally-controllable transcription system in mammalian cells using the apple-derived metabolite phloretin as a non-toxic trigger. This synthetic, phloretin-adjustable control element was able to adjust target gene expression reversibly in transgenic cells that had been implanted into mice, with the phloretin applied as a skin lotion.…”

Engineering Gene Circuits for Mammalian Cell–Based Applications

“…Encapsulated cells that contain ligand-responsive gene switches have shown the ability to regulate the production of therapeutic proteins in a ligand-dependent manner in mice. Although small molecules that are not found in an organism (e.g., antibiotics, Weber et al 2002;and drugs, Ye et al 2013) they must be invasively administered, gene-control systems based on radio waves (Stanley et al 2012), light (Ye et al 2011;Folcher et al 2014;Kim et al 2015), food additives (Gitzinger et al 2012;Rössger et al 2013b), and skin cream (Gitzinger et al 2009), providing more convenient, noninvasive options to manipulate gene expression. Accordingly, recent work has shown the use of a brain -computer interface to control the illumination of red-light-emitting diodes (LEDs) to control gene expression in living cells (Folcher et al 2014).…”

“…Because the ligandbinding domain is part of the protein, the binding of the ligand to the regulator dimer leads to the dissociation of the operator-dimer complex, thus influencing gene-expression rates. Based on this design strategy, mammalian cells have been engineered with various synthetic systems to sense different ligand inputs (e.g., antibiotics, Gossen and Bujard 1992;Fussenegger et al 2000;Urlinger et al 2000;Weber et al 2002Weber et al , 2003and Weber et al 2006Weber et al , 2009Hartenbach et al 2007;Gitzinger et al 2009Gitzinger et al , 2012Kemmer et al 2010;Bacchus et al 2013) and proven to be useful tools for diverse biological applications. Moreover, a synthetic system that enables dual-input gene-expression control was developed using a single engineered prokaryotic regulator protein fused to the transrepressor domain KRAB, termed KRAB-CbaR (Xie et al 2014).…”