Biomedical subjects

Beat P Kramer

Publications and source records attributed to Beat P Kramer.

5 recordsLinked to original sources

PubMed2004

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.

Animals↗

PubMed2003

Artificial regulatory networks and cascades for discrete multilevel transgene control in mammalian cells.

Prototype drug-adjustable heterologous transcription control systems designed for gene therapy applications typically show sigmoid dose-response characteristics and enable fine-tuning of therapeutic transgenes only within a narrow inducer concentration range of a few nanograms. However, the design of clinical dosing regimes which achieve tissue-specific concentrations with nanogram precision is yet a "mission impossible." Therefore, most of today's transcription control systems operate as ON/OFF switches and not in a true adjustable mode. The availability of robust transcription control configurations which lock expression of a single therapeutic transgene at desired levels in response to fixed clinical doses of different inducers rather than minute concentration changes of a single inducer would be highly desirable. Based on in silico predictions, we have constructed a variety of mammalian artificial regulatory networks by interconnecting the tetracycline- (TET(OFF)), streptogramin- (PIP(OFF)), and macrolide- (E(OFF)) repressible gene regulation systems as linear (auto)regulatory cascades. These networks enable multilevel expression control of several transgenes in response to different antibiotics or allow titration of a single transgene to four discrete expression levels by clinical dosing of a single antibiotic: 1) high expression in the absence of any antibiotic (+++), 2) medium level expression following addition of tetracycline (++), 3) low level expression in response to the macrolide erythromycin (+), and 4) complete repression by streptogramins such as pristinamycin (-). The first-generation artificial regulatory networks exemplify modular interconnections of different heterologous gene regulations systems to achieve multigene expression, fine-tuning, or to design novel control networks with unprecedented transgene regulation properties. Such higher-level transcription control modalities will lead the way towards composite artificial regulatory networks able to effect complex therapeutic interventions in future gene therapy and tissue engineering scenarios.

Alkaline Phosphatase↗

PubMed2002

Versatile macrolide-responsive mammalian expression vectors for multiregulated multigene metabolic engineering.

The novel macrolide-inducible and -repressible mammalian gene regulation systems (E.REX) have been cloned into a variety of sophisticated expression configurations including (1) multi-purpose expression vectors, (2) pTRIDENT-based artificial operons, (3) dual-regulated expression strategies for independent control of two different transgenes, (4) autoregulated vectors for one-step installation of adjustable multigene expression, and (5) oncoretroviral and lentiviral plasmids for transduction of macrolide-, streptogramin- and tetracycline-dependent transactivators and production of cell lines supporting independent control of three different transgenes. This vector portfolio represents a construction kit-like toolbox for efficient installation of adjustable gene expression responsive to clinically licensed antibiotics and enables the design of multiregulated multigene metabolic engineering strategies required for biopharmaceutical manufacturing, gene therapy, and tissue engineering.

Animals↗

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↗

PubMedDate not supplied

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

BACKGROUND: 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. METHODS: In order to provide optimal regulation modalities for a wide variety of mammalian cell lines, we have performed a detailed analysis of E.REX and PIP systems modified in (i) the transactivation domains of the antibiotic-dependent transactivators, (ii) the type of minimal promoter used, and (iii) the spacing between the operator module and the minimal promoter. RESULTS: These novel E.REX and PIP regulation components showed not only dramatically improved regulation performance in some cell types, but also enabled their use in cell lines which had previously been inaccessible to regulated transgene expression. CONCLUSIONS: Due to their modular set-up the novel E.REX and PIP regulation systems presented here are most versatile and ready for future upgrades using different cell-specific key regulation components.

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