Paul Szymanski

Development and validation of an improved inducer-regulator protein complex in the pBRES-regulated expression system.

Widespread adaptation of small molecule-regulated expression systems requires the development of selective inducer molecules that do not have any significant side effects on the endogenous receptors from which the regulated expression system is derived. Here we report the identification and in vitro validation of a novel inducer-receptor pair for the single-plasmid regulated expression system termed pBRES, which contains the ligand-binding domain from the human progesterone receptor (hPR). A small molecule inducer, BLX-913, has been identified as having a 30-fold lower IC(50) for the human progesterone receptor than mifepristone (MFP), the previously best characterized inducer for pBRES. Using modeling-guided protein engineering, compensatory mutations were installed at positions W755 and V729 (hPR numbering) in the ligand-binding pocket of the pBRES regulator protein (pBRES RP) to accommodate the new inducer and allow induction of transgene expression to levels previously seen with MFP. The improved inducer-pBRES RP complex was validated in vitro by monitoring the induction of luciferase, murine secreted alkaline phosphatase, and human interferon beta transgenes in mouse skeletal muscle cells. The engineered pBRES demonstrated low levels of transgene expression in the absence, and high expression levels in the presence, of the new BLX-913 inducer. Findings presented here allow induction of the pBRES-regulated gene expression system by a compound with markedly lower anti-hPR activity than MFP, the previously best characterized inducer.

768. Two Distinct Mechanisms, Silencing of RNA Expression and Loss of Vector DNA, Are Responsible for the Loss of Transgene Expression Following Delivery of Foreign Genes to Skeletal Muscle

Plasmid based delivery of foreign transgenes often results in transient expression in vivo. To investigate the cause of this transient expression we used quantitative PCR to measure the copy number of transgene RNA and vector DNA in the skeletal muscle of C57BL6 mice after electroporation of various plasmids. Gene transfer of a plasmid encoding the mouse interferon beta (mIFN-|[beta]|) gene driven by the CMV promoter (pCMV-mIFN) resulted in mIFN RNA levels between 1 and 5 |[times]| 106 copies per microgram (|[mu]|g) of total RNA that were stable for the first 10 days then dropped by only 10-fold by day 44. Plasmid vector DNA levels were also stable for the first 10 days then dropped 7.5-fold between day 10 and day 44. The ratio of mIFN RNA copies per vector DNA copy reached a peak of 1.8 at day 10 and declined slightly to 0.9 at day 44. These results show that following delivery of a non-foreign transgene, a slow decline in RNA levels occurred that was due to a gradual loss of plasmid DNA. Next, we compared plasmids encoding either the mIFN-|[beta]| or human interferon beta (hIFN-|[beta]|) genes driven by identical CMV promoters (pCMV-hIFN and pCMV-mIFN). At 50 days after electroporation of mouse skeletal muscle, the mean copy number of transgene RNA in the muscles that received the mouse or human IFN-|[beta]| plasmids was 1.1 |[times]| 105 and 7.4 |[times]| 103 copies per |[mu]|g of total RNA, respectively. In contrast, the mean plasmid DNA level was slightly higher in the muscles that received pCMV-hIFN. The mean IFN-|[beta]| RNA to vector DNA ratio was 1.3 in the muscles that received pCMV-mIFN, but only 0.04 (31-fold lower, p<0.001) in the muscles that received pCMV-hIFN. This demonstrates that the lower level of transgene RNA expression achieved with the foreign human IFN-|[beta]| gene as compared to the native mouse IFN-|[beta]| gene was due to reduced gene expression, not to reduced persistence of the vector DNA. When a mixture of pCMV-mIFN and a plasmid encoding human secreted alkaline phosphotase driven by the CMV promoter (pCMV-hSEAP) in a 14:1 ratio was delivered to mouse muscle, mIFN RNA levels fell 350-fold between day 1 and day 10 (p<0.01). The level of pCMV-mIFN plasmid DNA in the same muscles fell 100-fold by day 10 (p<0.01) and the ratio of mIFN RNA copies per copy of pCMV-mIFN DNA dropped slightly from 2.8 at day 1 to 1.1 at day 10, suggesting that there was only a small decrease in transcription. This data demonstrates that following co-delivery of pCMV-hSEAP and pCMV-mIFN, rapid clearance of the CMV-mIFN plasmid DNA from the muscle was primarily responsible for the large drop in mIFN RNA expression.

959. Pharmacokinetic Studies of Human IFN-|[szlig]| in Mice Following Gene-Based Delivery

Interferon-|[szlig]| (IFN-|[szlig]|) is an immunoregulatory cytokine that has been approved as a protein therapeutic for multiple sclerosis (MS). Pharmacokinetic studies have shown that IFN-|[szlig]| has an extremely short half-life in the circulation. Within a few hours following bolus delivery of the recombinant protein, hIFN-|[szlig]| levels in serum are undetectable. Gene-based delivery of IFN-|[szlig]| would offer advantages over bolus delivery of protein in providing sustained long-term expression of the protein resulting in therapeutic efficacy while minimizing side effects.