Ronald V. Abruzzese

Ligand-Dependent Regulation of Plasmid-Based Transgene Expression in Vivo

As gene therapy advances, the ability to regulate transgene expression will become paramount for safety and efficacy. In this study, we investigate the ability of the mifepristone-dependent GeneSwitch system to regulate the expression of trangenes delivered to mice by nonviral methods. Two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene for secreted human placental alkaline phosphatase (SEAP), were delivered to the hind-limb muscles of adult mice. Modulation of the level of secretion of the transgene product into serum was achieved by intraperitoneal administration of low doses of the drug mifepristone (MFP). The EC50 for induction of transgene expression by MFP was 0.03 +/- 0.005 mg/kg. The maximal level of transgene expression after induction was equal to or higher than that displayed by a plasmid driven by the CMV enhancer/promoter. The average magnitude of induction was 14- to 19-fold. Multiple rounds of drug-dependent regulation of transgene expression in vivo were demonstrated. In BALB/c mice, the ability to regulate transgene expression persisted for approximately 3 weeks, until the appearance of neutralizing antibodies to the secreted transgene product. In immune-deficient mice, the ability to repetitively regulate transgene expression persisted for at least 5 weeks. Although the dynamic range of regulation needs improvement, the plasmid-based GeneSwitch system has features that are attractive for gene therapy applications.

Optimization of Electroporation Parameters for the Intramuscular Delivery of Plasmids in Pigs

Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimum conditions are not well defined. Using a plasmid with a muscle-specific secreted embryonic alkaline phosphatase (SEAP) gene, we have optimized the electroporation conditions in a large mammal (pig). Parameters tested included electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. Electric pulses, between 0.4 and 0.6 Amp constant current, applied 80 sec after the injection of 0.5 mg SEAP-expressing plasmid in a total volume of 2 mL produced the highest levels of expression. Further testing demonstrated that electroporation of a nondelineated injection site reduces the levels of SEAP expression. These results demonstrate that electroporation parameters such as amperage, lag time, and the number of pulses are able to regulate the levels of reporter gene expression in pigs.

“Blue heart”: characterization of a mifepristone-dependent system for conditional gene expression in genetically modified animals

A detailed characterization of a cardiac muscle-specific, ligand-regulated gene expression system was performed in transgenic mice using the inducing ligand mifepristone (MFP). Several lines of double transgenic mice were created that expressed a bacterial lacZ reporter gene in the heart, under the control of a MFP-activated transcription factor constitutively expressed in cardiac muscle. The transgenic mice, which were administered MFP at a dose of 1 micromol/l in the drinking water, responded to the ligand within 24 h. Induction of beta-galactosidase enzyme activity in the heart continued for up to 21 days and resulted in an average 17-fold increase in enzyme activity. The highest individual animal response measured was a 94-fold increase in enzyme activity. The EC(50) for MFP induction of beta-galactosidase activity in the heart was 0.7 micromol/l when MFP was administered in the drinking water. Pharmacokinetic analysis of MFP dosing in wild-type FVB/N mice showed that absorption was very rapid (T(max) 1-10 min), bioavailability was modest ( approximately 10%) and the t(1/2) of MFP in mouse plasma was determined to be approximately 5 h. Thus, the system functions effectively in transgenic mouse heart where induction of gene expression is sensitive and can be accomplished by a simple and broadly applicable drinking water protocol.