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Plasmid based gene delivery to skeletal muscle is an attractive method for the expression of therapeutic proteins. However, the level of transgene expression decreases over time and may be an obstacle to achieving sustained therapeutic levels of proteins. To investigate the mechanism responsible for loss of expression over time, a standard plasmid backbone containing the luciferase gene driven by the CMV promoter was injected into the skeletal muscle of the hind limb of C57Bl/6 mice at either a high dose (125|[mu]|g) or a low dose (25|[mu]|g) followed by electroporation. Transgene expression was monitored over time using in situ CCCD imaging. At the end of the study the quantity of transgene DNA and luciferase mRNA in the muscle was measured by quantitative PCR. At early time points (day 3 and day 7) luciferase activity in the high dose group was 2.5- fold higher than in the low dose group. However, luciferase activity in mice that received the high dose fell 230-fold between day 7 and day 42, while in mice that received the low dose of the same plasmid it fell only 4-fold. Vector DNA levels in the injected muscles at the end of the study (day 84) were not significantly different between the two doses indicating that the 27-fold lower luciferase activity in the high dose group compared to the low dose group at day 84 was not due to differences in vector DNA levels but rather to a difference in gene expression. Indeed, the luciferase mRNA to vector DNA ratio was 9-fold higher in the low dose group than in the high dose group at this time point. In the same study, a plasmid containing the identical CMV-luciferase expression cassette flanked by matrix attachment sites (MARs) and placed into a plasmid backbone from which all CpG dinucleotides had been removed, was tested at the same doses. At the low dose the CpG-depleted/MARs plasmid exhibited a similar luciferase expression profile to that of the standard plasmid at the same dose. However, in the high dose group the CpG- depleted/MARs plasmid partially prevented the decrease in luciferase expression over time that was observed with the standard plasmid at the high dose. At 28, 42 and 84 days after plasmid injection the CpG- depleted/MAR plasmid expressed 10-, 13- and 9-fold higher luciferase activity respectively than the standard plasmid. Vector DNA levels in the muscle at day 84 were only 2-fold higher in the CpG-depleted/ MAR group than in the mice that received the standard plasmid. These results demonstrate that reducing the plasmid dose can result in a significant improvement in the duration of transgene expression, and that this effect was not due only to improved persistence of the plasmid DNA in the muscle. In addition, use of a CpG-free plasmid backbone plus MARs partially prevented the decline of transgene expression that occurred at the high plasmid dose, suggesting that the presence of CpG's and/or absence of MARs may contribute to loss of expression over time.

The goal of the present study was to explore the tropism of Virus-Like Particles (VLP) originating from the human polyoma JC- virus (JCV) in vitro and in vivo. VLP have been known for several years to be a promising alternative to the existing viral and non-viral DNA delivery systems. VLP are self-assembled structures composed only of proteins necessary for capsid formation, DNA packaging and gene delivery. They are replication deficient, exhibit a similar morphology and presumably share the same cell tropism, cellular uptake and intracellular trafficking mechanisms compared to their viral origin. VLP from JCV are being evaluated as a delivery vector for the central nervous system (CNS) because JCV preferentially infects both oligodendrocytes and astrocytes.

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.

Skeletal muscle has several features that make it attractive as a site for therapeutic gene expression - it is accessible to most of the current gene delivery systems, abundant, and displays a relatively slow turn over rate. A variety of gene delivery systems have been tested for high levels of sustainable gene expression from the skeletal muscle. Unfortunately, these studies have generally focused on optimization of one of the systems and have not made direct comparisons to other currently available gene delivery systems. To better understand the potential of these different systems to achieve high, sustained levels of gene expression from the skeletal muscle, we compared the viral-based gene delivery systems derived from human adenovirus (Ad) and adeno-associated virus (AAV) to optimized (electroporation, synthetic polymers) plasmid-based delivery. Using the HCMV promoter driven luciferase gene expression cassette as a reporter system for these studies, gene expression levels in the murine skeletal muscle were quantitated by both direct measurement of luciferase gene expression from excised skeletal muscle and by in vivo CCD camera intensity images. Initial expression levels (day 2 post infection/transduction) were highest from plasmid-based delivery by electroporation, slightly (approx. 2 fold ) higher than Ad and AAV-1 and significantly (100-1000X) higher than plasmid + poloxamer or plasmid alone. By day 6, the rank order of expression had changed, with AAV-1-based expression generating levels superior to the other systems tested (AAV-1>plasmid+electroporation>Ad>plasmid+poloxamer>plasmid). Gene expression has been detected out to 8 months for all the vectors examined. AAV-1 mediated expression levels at the 8 month time point are approximately 2-logs over both plasmid + electroporation and Ad as measured by CCD camera imaging. Consequently, AAV-1 based gene delivery results in superior expression levels and duration in the skeletal muscle relative to many of the other current gene delivery systems available.

Interferon beta (IFN-) has been shown to be an effective drug for patients with multiple sclerosis (MS). However dose-dependent side effects from administration of IFN- protein have limited its therapeutic efficacy and clinical utility. IFN- gene therapy offers an alternative approach to overcome these obstacles, and it would be advantageous over bolus delivery of protein. A critical issue in developing such a new approach is in vivo detection of biologically active IFN- in pre-clinical animal models. Several biomarkers have been established in humans undergoing IFN- therapy, including MxA (myxovirus resistant protein) an antiviral protein induced by IFN-. The goal of the present study was to establish and validate a quantitative PCR-based assay to measure RNA expression of Mx1, the murine homologue of human MxA, in peripheral blood mononuclear cells (PBMC) of mice following IFN- protein administration or gene-based delivery. In parallel it was investigated whether IP10, which was recently reported to be up-regulated following IFN- treatment in MS patients, could also be used as biomarker in mice. IP-10 has the advantage that it can be assayed directly by ELISA in plasma samples.

Top of pageAbstract At present, the most methods to evaluate in vivo gene expression are limited to the use of tissue obtained from sacrificed animals. To develop a simple, non-invasive and quantitative method for the detection of gene expression in the mouse central nervous system (CNS) while the animal is living, we evaluated a method of repetitively tracking in vivo gene expression of firefly luciferase (Luc) using a Cooled Charged Couple Detector (CCCD) camera. To evaluate the sensitivity of the bioluminescence imaging system, the initial experiment was performed by stereotactic injection of different doses of recombinant E1-deleted type 5 adenovirus expressing the Luc, Ad5.Luc, into the striatum of anesthetized adult C57BL/6 mice. The real-time in vivo gene expression of luciferase was monitored by CCCD camera at day 2, 7, 14, 28, and day 60 post gene transfer. The results indicated that mice treated with middle dose of Ad5.Luc (4|[times]|107 VP), a bioluminescence signal was detected at the site of injection as early as 2 days post injection. In contrast, the mice treated with higher dose of adenovirus (4|[times]|108 VP), the expression level reached its peak at day 7 and declined after day14. There was no detectable signal with the lowest dose of adenovirus (4|[times]|106 VP) at any of the time point post injection. To evaluated two recombinant adeno-associated virus (AAV) serotypes (AAV2/2, and AAV 2/5) with the bioluminescence imaging system in the mouse CNS, a subsequent experiment was performed using the similar protocol, stereotactic-guide injected 5|[times]|109 VP of AAV.Luc in 4 ul of PBS into the striatum of C57BL/6 mice. The results showed comparable expression of Luc in the mice treated with both AAV 2/2 and AAV 2/5 on day 2 post gene transfer. Between day 7 to day 28, the luciferase imaging in the mice treated with AAV 2/5 was two to five fold higher than in the mice treated with AAV 2/2. Gene expression of both AAV 2/2 and AAV2/5 have been persistent over two months in the live mice. Our results demonstrate that real-time in vivo bioluminescence imaging is a promising method to measure gene expression in the CNS of mice.

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