History of the GeneSwitch System

GeneSwitch Regulator Protein, version 3.0
(GS 3.0, GLp65)

The VP16 activation domain has been linked to toxicity due to squelching, and, since it is of viral origin, it is potentially immunogenic in humans. To make the GeneSwitch® system more suitable for human gene therapy applications, the viral VP16 activation domain of GS 2.0 was replaced by the activation domain (amino acids 285-551) of the p65 subunit of human NF-kB. (Burcin et al., 1999). 83% of the sequence of GS 3.0 is human in origin. The structure of GS 3.0, including the amino acid sequence of the linker regions, is shown below.

MDSQQPDL / yGAL4 (2-93) / EFPGVDQ / hPR-LBD D19 (640-914) / GSTRYQA / hNF-kB p65 (285-551)

In transient transfection studies, basal transgene expression with GS 3.0 was significantly lower than with GS 2.0. The level of induced expression in response to mifepristone was about half of that observed with GS 2.0. The combination of a lower level of basal expression and maintenance of a relatively high level of induced expression make GS 3.0 an attractive choice.

When a gene for GS 3.0, controlled by a transthyretin promoter, and a regulated hGH gene, controlled by a TATA box promoter linked to four GAL4 sites, was incorporated into a "gutless" adenovirus vector, extremely effective drug-dependent regulation of transgene expression was observed. In cultured HepG2 cells, hGH expression was induced 19,000-fold. When the recombinant virus was administered to mice by i.v. injection, serum levels of hGH were induced over 50,000-fold.

GeneSwitch Regulator Protein, version 3.1
(GS 3.1, GLp65.1)

GS 3.1 and GS 3.0 are identical except for the linker between the hPR-LBD and the NF-kB p65 domains. In GS 3.0, this linker is 7 amino acids (GSTRYQA). In GS 3.1 the linker is 3 amino acids (GST). The structure of GS 3.1, including the amino acid sequence of the linker regions, is shown below.

MDSQQPDL / yGAL4 (2-93) / EFPGVDQ / hPR-LBD D19 (640-914) / GST / hNF-kB p65 (285-551)

In transient transfection studies, the performance of GS 3.1 is indistinguishable from that of GS 3.0.

The first non viral gene therapy studies with the GeneSwitch® system were performed with two plasmids (one plasmid expressed GS 3.1 from a CMV promoter, the other expressed SEAP (secreted alkaline phosphatase) from an inducible promoter) that were delivered to the skeletal muscle of mice with electroporation. Induction of secretion of SEAP into serum was achieved by intraperitoneal administration of low doses of MFP. The EC50 for induction of SEAP expression by MFP was 0.03 mg/kg. The maximal level of SEAP induction was equal to or higher than that displayed by the CMV promoter. The average magnitude of induction was 14- to 19-fold. Multiple rounds of drug-dependent regulation of SEAP expression were demonstrated. Although the studies showed that the dynamic range of regulation needs improvement, the data demonstrate that the plasmid-based GeneSwitch® system has features that are highly attractive for gene therapy applications.