Cornelia Fux

Streptogramin-based gene regulation systems for mammalian cells

Here we describe repressible (PipOFF) as well as inducible (PipON) systems for regulated gene expression in mammalian cells, based on the repressor Pip (pristinamycin-induced protein), which is encoded by the streptogramin resistance operon of Streptomyces coelicolor. Expression of genes placed under control of these systems was responsive to clinically approved antibiotics belonging to the streptogramin group (pristinamycin, virginiamycin, and Synercid). The versatility of these systems was demonstrated by streptogramin-regulated expression of mouse erythropoietin (EPO), human placental secreted alkaline phosphatase (SEAP), or green fluorescent protein (GFP) in diverse cell lines (BHK, CHO, HeLa, and mouse myoblasts). Analysis of isogenic constructs in CHO cells demonstrated the PipOFF system gave lower background and higher induction ratios than the widely used tetracycline-repressible (TetOFF) expression systems. The streptogramin-based expression technology was functionally compatible with the TetOFF system, thus enabling the selective use of different antibiotics to independently control two different gene activities in the same cell.

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.

An Update of pTRIDENT Multicistronic Expression Vectors: pTRIDENTs Containing Novel Streptogramin-Responsive Promoters

We present an update on the pTRIDENT multicistronic mammalian expression vectors and their implications in various metabolic engineering and therapeutic applications. The pTRIDENT vector family has been expanded by construction of a new set of pTRIDENT‐based vectors containing constitutive promoters of human origin (ubiquitin C and EF‐1α promoters) and selectable markers (zeocin resistance) and expressing different reporter genes (secreted alkaline phosphatase (SEAP) and the secreted single‐chain urokinase‐type plasminogen activator (low‐Mr u‐PA)). In addition, we have constructed pTRIDENT derivatives with novel streptogramin‐repressible and streptogramin‐inducible promoters for simultaneous and adjustable expression of three different transgenes. Streptogramin‐inducible and tetracycline‐repressible pTRIDENT derivatives were used to simultaneously control expression of three fluorescent proteins in mammalian cells: the enhanced cyan fluorescent protein (CFP), the recently isolated red fluorescent protein (RFP, also designated dsRed), and the enhanced yellow fluorescent protein (YFP). Owing to their modular structure, the pTRIDENT vector family represents a construction kit for the design of novel multicistronic expression constructs.

Dual-regulated expression technology: a new era in the adjustment of heterologous gene expression in mammalian cells

On the basis of the compatible streptogramin‐ and tetracycline‐responsive expression systems, a series of dual‐regulated expression systems have been established for use in sophisticated biopharmaceutical manufacturing, advanced gene therapy, and tissue engineering.

Toward higher order control modalities in mammalian cells-independent adjustment of two different gene activities.

Heterologous higher order control modalities will be important tools for targeted multigene interventions in next‐generation gene therapy, tissue engineering, and sophisticated gene‐function studies. In this study, we present the design and rigorous quantitative analysis of a variety of different dual‐regulated gene transcription control configurations combining streptogramin‐ and tetracycline‐responsive expression systems in a one‐vector format. Quantitative assessment of dual‐regulated expression performance in various mammalian and human cell lines is based on two compatible secreted reporter genes, SEAP, the human placental secreted alkaline phosphatase, and the recently developed SAMY, the secreted α‐amylase. Assembly of streptogramin‐and tetracycline‐responsive transgene control units in consecutive (→ →), divergent (← →), and convergent (→ ←) orientation showed excellent regulation characteristics in most genetic arrangements exemplified by neglectable interference and high transgene induction ratios in all four control settings (ON/ON, OFF/ON, ON/OFF, OFF/OFF). The overall regulation performance of divergent dual‐regulated expression configurations could be substantially increased when placing noncoding stuffer fragments or insulator modules between the divergently oriented antibiotic‐responsive promoters. Dual‐regulated expression technology pioneers artificial higher order gene control networks that will likely enable new opportunities in multigene metabolic engineering and generate significant therapeutic impact.

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

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.

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

Advanced gene therapy, tissue engineering and biopharmaceutical manufacturing require sophisticated and well‐balanced multiregulated multigene interventions to reprogram desired mammalian cell phenotypes.

Improved Dual-Regulated Expression Systems for Independent Control of Two Different Transgenes

Unlike current gene therapy scenarios which focus on single gene replacement or overexpression to substitute for somatic genetic deficiencies, 3rd generation gene therapy strategies will require more complex multiregulated multigene interventions to reprogram or substitute entire regulatory networks and metabolic pathways to cure complex multifactorial human disease. Dual-regulated expression technology enables independent control of two different set(s) of transgenes and represents a first step towards multiregulated molecular interventions in mammalian and human cells. We have designed the dual-regulated expression vector pDuoRex? which contains divergently oriented streptogramin- (PPIR) and tetracycline- (PhCMV*-1) responsive promoters. A stuff er fragment of about 600 bp placed between PPIR and PhCMV*-1 prevents any interference between both expression units previously observed with other divergent promoter configurations. pDuoRex7 was engineered to express two marker genes encoding the yellow and cyan fluorescent proteins (pDuoRex8) in response to clinically licensed streptogramin- (PI) and tetracycline (Tet) andbiotics. pDuoRex8 enabled completely independent control of both fluorescent proteins in a variety of mammalian and human cell lines (CHO-K1, NIH/3T3 and K-562 cells) stably expressing a retroviral pTWIN vector (pRetroTwin3) driving constitutive expression of the streptogramin- (PIT) and tetracycline- (tTA) dependent transactivators. The combination of the pRetroTWIN vector series and pDuoRex vector families results in a dual-regulated expression concept with powerful impact on rationale reprogramming of mammalian cells. Here we present the latest generation of the dual-regulated expression technology.

Process Development for the GMP Production of N-Acetylgalactosamine-6-Sulfate Sulfatase (GALNS) Expressed by CHO Cells

The enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) is intended to be used as an Enzyme Replacement Therapy to treat the disorder Mucopolysaccharidosis IV A (MPS IVA or Morbus Morquio) caused by a deficiency of GALNS. GALNS is a lysosomal enzyme required to degrade glycosaminoglycans (GAGs), keratin sulphate (KS) and chondroitin-6-sulfate (C6S). The human GALNS cDNA encodes a polypeptide of 522 amino acid residues, whereas the GALNS protein is a 120 kDa homodimer with a molecular mass of 60 kDa for the monomer that is processed to polypeptides of 40 and 15 kDa.