Macrolide- and tetracycline-adjustable siRNA-mediated gene silencing in mammalian cells using polymerase II-dependent promoter derivatives

RNA interference has emerged as a powerful technology for downregulation of specific genes in cells and animals. We have pioneered macrolide- and tetracycline-adjustable short interfering RNA (siRNA) expression for conditional target gene translation fine-tuning in mammalian/human cell lines based on modified RNA polymerase II promoters. Established macrolide- and tetracycline-dependent transactivators/trans-silencers bound and activated modified target promoters tailored for optimal siRNA expression in response to clinical antibiotics' dosing regimes and modulated desired target genes in Chinese hamster ovary (CHO-K1) and human fibrosarcoma (HT-1080) cells with high precision. Further optimization of adjustable RNA polymerase II-based siRNA-specific promoters as well as their combination with various transmodulators enabled near-perfect regulation configurations in specific cell types. Devoid of major genetic constraints compared to basic RNA polymerase III-based siRNA-specific promoters, we expect RNA polymerase II counterparts to significantly advance siRNA-based molecular interventions in biopharmaceutical manufacturing and gene-function analysis as well as gene therapy and tissue engineering.     

Dual-regulated myoD- and msx1-based interventions in C2C12-derived cells enable precise myogenic/osteogenic/adipogenic lineage control

BACKGROUND: Advanced gene therapy, tissue engineering and biopharmaceutical manufacturing require sophisticated and well-balanced multiregulated multigene interventions to reprogram desired mammalian cell phenotypes. METHODS: We have combined the streptogramin (PIP)- and tetracycline (TET)-responsive gene regulation systems for independent expression control of the differentiation determinants myoD and msx1 in C2C12-derived cells. RESULTS: Different dual-regulated expression scenarios which induce either both, only one or none of the lineage control genes triggered differential differentiation and precise control of myogenic, osteogenic or adipogenic cell phenotypes. CONCLUSIONS: Our findings substantiate the use of multiregulated multigene interventions in reprogramming cellular differentiation pathways in a desired manner.     

Approaches for trigger-inducible viral transgene regulation in gene-based tissue engineering

Recent advances in mammalian transgene expression dosing have resulted in a portfolio of mutually compatible systems that can adjust therapeutic transgene levels in response to antibiotics, hormone analogues, quorum-sensing messengers and secondary metabolites. The molecular merger of trigger-inducible expression technology with the latest generation of virus-derived transduction systems has enabled unmatched clinical interventions to shape desired therapeutic cell and tissue phenotypes for the treatment of complex human diseases.