862. Feasibility of Cutaneous Gene Therapy Using a Factor VIII Gene and a Marker Gene in Factor VIII_Deficient Mice by Non_Invasive Electroporation
Authors: Lei Zhang, Amy Goldbeck, Hongbo Lu, Edward Nolan, Dietmar Rabussay, Steve Fakharzadeh
Journal: Molecular Therapy (2006)

In vivo gene delivery to skin is potentially useful for a broad range of clinical purposes. We previously reported that in vivo electroporation (EP) is a simple and effective method for intracellular delivery of naked DNA to mouse skin. Furthermore, we have demonstrated that transgene_directed epidermal expression of FVIII achieved phenotypic correction in a mouse model for hemophilia A. Given these findings, our main objectives for the present study were: (i) to test the feasibility of producing circulating FVIII in hemophilia A mice by introducing a "B" domain_deleted murine FVIII gene (mFVIII) into skin cells via EP; and (ii) to compare levels and duration of transgene expression using two types of non_invasive electrodes (caliper vs. meander). To address these objectives, we co_ delivered independent plasmids carrying either a mFVIII cDNA or a secreted alkaline phosphatase (SEAP) marker gene to FVIII knockout mice. Both genes were under transcriptional control of CMV promoter/enhancer sequences. FVIII knockout mice were assigned to four treatment groups (N=4/group): i) skin DNA injection only, ii) skin DNA injection + EP (caliper), iii) skin DNA injection + EP (meander), and iv) muscle DNA injection + EP (caliper). A mixture containing 200 μg mFVIII plasmid DNA and 10 μg of SEAP plasmid DNA was injected at 10 sites on dorsal skin prior to EP. Muscle_ treated mice received injections of the DNA mixture into the tibialis muscle of each rear leg. Blood samples were obtained by tail bleeding at days 0, 2, 4, 7, 10, 15, and at 6 weeks. FVIII activity in plasma samples was measured using the COAMATIC assay and circulating SEAP was measured by ELISA. Skin biopsies were obtained from treated sites from additional mice at days 2, 4, 7, and 15 after treatment. Total RNA extracted from skin samples was used to assess expression of mFVIII and SEAP by RT_PCR. We detected transgene_specific mRNA for both mFVIII and SEAP in treated skin. Both genes showed similar expression patterns peaking at approximately day 4. The meander electrode appeared to yield more efficient EP than the caliper based on duration of transgene expression and band signal intensity. In addition, EP enhanced levels of SEAP in the circulation of skin_treated mice by one to two orders of magnitude compared to non_EP_treated mice. Furthermore, use of the meander electrode yielded an approximately twofold higher level of circulating SEAP than the caliper. Muscle_treated mice, however, displayed SEAP levels an order of magnitude higher than skin_treated mice at the time of peak expression on day 7. In contrast, mFVIII activity was not detectable in the circulation of skin_ or muscle_ treated mice despite expression of mFVIII mRNA and high expression of the co_ delivered SEAP plasmid. These results indicate that EP use of a meander electrode may be more efficient, in addition to its user_friendliness, than use of a caliper electrode. However, application of this technology to gene therapy for more complex gene products, such as FVIII, will require further optimization.