Biomedical subjects

Christoph Heinzen

Publications and source records attributed to Christoph Heinzen.

4 recordsLinked to original sources

PubMed2003

Streptomyces-derived quorum-sensing systems engineered for adjustable transgene expression in mammalian cells and mice.

Prokaryotic transcriptional regulatory elements have been adopted for controlled expression of cloned genes in mammalian cells and animals, the cornerstone for gene-function correlations, drug discovery, biopharmaceutical manufacturing as well as advanced gene therapy and tissue engineering. Many prokaryotes have evolved specific molecular communication systems known as quorum-sensing to coordinate population-wide responses to physiological and/or physicochemical signals. A generic bacterial quorum-sensing system is based on a diffusible signal molecule that prevents binding of a repressor to corresponding operator sites thus resulting in derepression of a target regulon. In Streptomyces, a family of butyrolactones and their corresponding receptor proteins, serve as quorum-sensing systems that control morphological development and antibiotic biosynthesis. Fusion of the Streptomyces coelicolor quorum-sensing receptor (ScbR) to a eukaryotic transactivation domain (VP16) created a mammalian transactivator (SCA) which binds and adjusts transcription from chimeric promoters containing an SCA-specific operator module (P(SPA)). Expression of erythropoietin or the human secreted alkaline phosphatase (SEAP) by this quorum-sensor-regulated gene expression system (QuoRex) could be fine-tuned by non-toxic butyrolactones in a variety of mammalian cells including human primary and mouse embryonic stem cells. Following intraperitoneal implantation of microencapsulated Chinese hamster ovary cells transgenic for QuoRex-controlled SEAP expression into mice, the serum levels of this model glycoprotein could be adjusted to desired concentrations using different butyrolactone dosing regimes.

4-Butyrolactone↗

PubMed2003

Conditional human VEGF-mediated vascularization in chicken embryos using a novel temperature-inducible gene regulation (TIGR) system.

Advanced heterologous transcription control systems for adjusting desired transgene expression are essential for gene function assignments, drug discovery, manufacturing of difficult to produce protein pharmaceuticals and precise dosing of gene-based therapeutic interventions. Conversion of the Streptomyces albus heat shock response regulator (RheA) into an artificial eukaryotic transcription factor resulted in a vertebrate thermosensor (CTA; cold-inducible transactivator), which is able to adjust transcription initiation from chimeric target promoters (P(CTA)) in a low-temperature- inducible manner. Evaluation of the temperature-dependent CTA-P(CTA) interaction using a tailored ELISA-like cell-free assay correlated increased affinity of CTA for P(CTA) with temperature downshift. The temperature-inducible gene regulation (TIGR) system enabled tight repression in the chicken bursal B-cell line DT40 at 41 degrees C as well as precise titration of model product proteins up to maximum expression at or below 37 degrees C. Implantation of microencapsulated DT40 cells engineered for TIGR-controlled expression of the human vascular endothelial growth factor A (hVEGF121) provided low-temperature-induced VEGF-mediated vascularization in chicken embryos.

Animals↗

PubMed2003

Novel macrolide-adjustable bidirectional expression modules for coordinated expression of two different transgenes in mice.

BACKGROUND: Precise control of transgene expression is essential for a variety of applications ranging from gene-function analysis, biopharmaceutical manufacturing to next-generation molecular interventions in gene therapy and tissue engineering. The regulation of gene expression is currently a key issue for clinical implementation of gene-therapy-based treatments since desired transgene expression may need to be maintained within a narrow therapeutic window for successful treatment of a particular human disease. METHODS: We have designed a novel bidirectional expression module that enables adjustable coregulation of two different transgenes in response to clinical doses of macrolide antibiotics. A bidirectional macrolide-responsive promoter consisting of a central operator module (ETR) specific for the macrolide-dependent transactivator (ET1) is flanked by two minimal promoters (P(hCMVmin); P(hsp70min)) which drive expression of two divergently oriented transgenes. Macrolide antibiotics modulate the binding affinity of ET1 to ETR and adjust expression of both transgenes to desired levels. RESULTS: Bidirectional expression configurations enabled excellent macrolide-adjustable coregulation profiles of two secreted reporter genes or one-vector-based autoregulated fine-tuning of a single transgene in various transgenic rodent and human cell lines. Following implantation of microencapsulated CHO-K1 cell derivatives transgenic for macrolide-controlled bidirectional expression of erythropoietin (EPO) and the human secreted alkaline phosphatase (SEAP) intraperitoneally into mice, serum EPO and SEAP levels could be coadjusted to desired levels by administration of different erythromycin doses. CONCLUSIONS: Based on their in vivo compatibility, the versatile bidirectional and macrolide-responsive expression modules represent an important advancement on the way to implementing targeted and conditional molecular interventions into a clinical reality.

Alkaline Phosphatase↗

PubMed2002

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 (E(OFF)) and inducible (E(ON)) mammalian gene regulation systems (E.REX) responsive to clinically licensed macrolide antibiotics (erythromycin, clarithromycin, and roxithromycin). The E(OFF) 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 (P(ETR)). Addition of macrolide antibiotic results in repression of transgene expression. The E(ON) 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 (E(OFF) and E(ON)) 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.

Alkaline Phosphatase↗