749. Effective Plasmid DNA Dose Range and Promoters for Gene Expression in Muscle Following Hydrodynamic Limb Vein Delivery of pDNA into Rodents and Primates
Authors: Christine I. Wooddell, Julia O. Hegge, Guofeng Zhang, Patricia Kleinpeter, Magdolna G. Sebestyén, Mark A. Noble, Loretta V. Pfannes, Hans Herweijer, Serge Braun, Thierry Huss, Jon A. Wolff
Journal: Molecular Therapy (2006)

Plasmid DNA (pDNA) is efficiently delivered to limb muscles of rodents and large animals by hydrodynamic limb vein (HLV) injection, a facile procedure that uses a tourniquet or blood pressure cuff placed distal to the injected vein to delimit the target tissue. In rhesus monkeys of various weights and ages we detected much higher reporter gene expression when the pDNA dose was above an apparent threshold level measured in pDNA dose/gram muscle of the target tissue. Studies in mice and rats were carried out to investigate these dose response effects and confirmed a threshold dose above which expression correlated directly with the dose and below which it did not. The percentages of transfected myofibers were also shown to increase as the pDNA dose increased. Co-localization of transgene expression and fiber type by immunohistochemistry showed that all types of myofibers were transfected at similar efficiencies, both in a primate and in the mdx dystrophic mouse model. Equal molar amounts of the human cytomegalovirus (CMV) promoter, the human desmin promoter and locus control region (DCR), and the mouse and human muscle creatine kinase (MCK) promoters were then compared for tissue specificity, for short-term efficiency of expression, and for stability of long-term expression in muscle. Tissue specificity was examined by use of hydrodynamic tail vein injection to deliver the plasmids to mouse liver and HLV injection to deliver the same plasmids to muscle. At lower pDNA doses, the DCR and MCK promoters had limited expression in the liver. Surprisingly, the DCR promoter was highly expressed in liver when a large dose of pDNA was injected. Short-term expression in muscle was evaluated 7 days after HLV injection of luciferase expression vectors into both mice and rats. The CMV promoter consistently gave the highest expression, two-fold higher than the mouse MCK promoter. The DCR, human MCK and mouse MCK promoters were similarly effective in rats, while the DCR and human MCK promoters were less effective in mice. A non-immunogenic mouse secreted alkaline phosphatase (mSEAP) reporter gene was then utilized to evaluate the time course and the long-term stability of expression from each promoter in mice. While high expression levels were attained by the CMV promoter within the first week, expression from both MCK promoters increased only gradually over the course of two weeks. The CMV promoter gave 2–3 fold higher long-term expression levels than the muscle promoters; however, all of these promoters gave sustained high levels of expression for at least 37 weeks. Furthermore, the amount of transgene expression could be additively increased by two to four injections into the same target area. These studies will help investigators determine effective pDNA doses and promoters for transgene expression in limb muscles.