Beat P. Kramer

An engineered epigenetic transgene switch in mammalian cells

In multicellular systems cell identity is imprinted by epigenetic regulation circuits, which determine the global transcriptome of adult cells in a cell phenotype–specific manner. By combining two repressors, which control each others expression, we have developed a mammalian epigenetic circuitry able to switch between two stable transgene expression states after transient administration of two alternate drugs. Engineered Chinese hamster ovary cells (CHO-K1) showed toggle switch–specific expression profiles of a human glycoprotein in culture, as well as after microencapsulation and implantation into mice. Switch dynamics and expression stability could be predicted with mathematical models. Epigenetic transgene control through toggle switches is an important tool for engineering artificial gene networks in mammalian cells.

Macrolide-based transgene control in mammalian cells and mice

Heterologous mammalian gene regulation systems for adjustable expression of multiple transgenes are necessary for advanced human gene therapy and tissue engineering, and for sophisticated in vivo gene-function analyses, drug discovery, and biopharmaceutical manufacturing. The antibiotic-dependent interaction between the repressor (E) and operator (ETR) derived from an Escherichia coli erythromycin-resistance regulon was used to design repressible (EOFF) and inducible (EON) mammalian gene regulation systems (E.REX) responsive to clinically licensed macrolide antibiotics (erythromycin, clarithromycin, and roxithromycin). The EOFF system consists of a chimeric erythromycin-dependent transactivator (ET), constructed by fusing the prokaryotic repressor E to a eukaryotic transactivation domain that binds and activates transcription from ETR-containing synthetic eukaryotic promoters (PETR). Addition of macrolide antibiotic results in repression of transgene expression. The EON system is based on E binding to artificial ETR-derived operators cloned adjacent to constitutive promoters, resulting in repression of transgene expression. In the presence of macrolides, gene expression is induced. Control of transgene expression in primary cells, cell lines, and microencapsulated human cells transplanted into mice was demonstrated using the E.REX (EOFF and EON) systems. The macrolide-responsive E.REX technology was functionally compatible with the streptogramin (PIP)–regulated and tetracycline (TET)–regulated expression systems, and therefore may be combined for multiregulated multigene therapeutic interventions in mammalian cells and tissues.

Novel promoter/transactivator configurations for macrolide‐ and streptogramin‐responsive transgene expression in mammalian cells

The recently developed heterologous macrolide‐ (E.REX system) and streptogramin‐ (PIP system) responsive gene regulation systems show significant differences in their regulation performance in diverse cell lines.

Synergies of microtissue design, viral transduction and adjustable transgene expression for regenerative medicine.

In the past decade, regenerative medicine has evolved as an interdisciplinary field, integrating expertise from the medical, life‐ and material‐science communities. Recent advances in tissue engineering, gene therapy, gene‐function analysis, animal‐free drug testing, drug discovery, biopharmaceutical manufacturing and cell‐phenotype engineering have capitalized on a core technology portfolio including artificial microtissue design, viral transduction and precise transcription dosing of therapeutic or phenotype‐modulating transgenes. We provide a detailed overview on recent progress in these core technologies and comment on their synergistic impact on current and future human therapies.

715. Artificial Mammalian Gene Networks

Next-generation gene therapy approaches for the treatment of complex disease phenotypes will have to deal with the precise titration of key (regulatory) proteins and complementation of entire gene regulatory networks. A growing set of adjustable gene regulation systems, operating as ON/OFF switches and responsive to small-molecule drugs, pave the way for next-generation clinical interventions. Currently available binary transcription control systems enable fine-tuning of therapeutic transgene levels only within a narrow inducer concentration range of a few nanograms/ml. We have developed a gene regulatory network tailored to lock transgene expression at desired levels in response to clinical doses of different inducers rather than different concentrations of a single inducer. The regulatory cascade was designed by interconnecting streptogramin-, macrolide- and tetracycline- repressible gene regulation systems. Four different expression levels could be achieved by clinical dosing of a single antibiotic: high expression level in the absence of any antibiotic (+++), medium level expression following addition of tetracycline (++), low level expression in response to the macrolide erythromycin (+), and complete repression by streptogramins (-). We have furthermore developed a mammalian epigenetic transcription control system, which can be switched between two stable expression states following transient integration of pharmacological signals, thus eliminating the need for sustained inducer administration. We will present the latest progress in the development of gene regulation systems, gene regulatory networks and their building blocks, as well as advances in in vivo applications and tissue engineering.