Search

Background: A model for in vivo gene therapy based on electroporation of human growth hormone (hGH)-coding naked DNA in the muscle of dwarf (lit/lit) and immunodeficient dwarf (lit/scid) mice is described., Methods: A plasmid containing the ubiquitin C promoter and the genomic hGH sequence was administered to the exposed quadriceps muscle, followed by electrotransfer using eight 50-V pulses of 20 ms at a 0.5-s interval. Serum hGH levels were determined after various days of DNA administration and a long-term body weight gain experiment was carried out., Results: Serum hGH, determined 3 days after DNA administration, revealed a significant dose-response curve (p < 0.01) in the 0-50 microg range. Because 50 microg of plasmid DNA produced circulating hGH levels of 2-3 ng/ml for at least 12 days, a long-term body weight gain assay was carried out. After 60 days, the weight of treated lit/scid mice increased 33.1% compared to a 4.2% weight decrease for the control group. hGH circulating levels were of the order of 1.5-3 ng/ml throughout the experiment and the average weight increase during the first 10 days was comparable to that obtained upon regular daily injection of 10 microg of recombinant hGH per mouse, producing comparable circulating levels of the hormone. A lower, but still significant increase in body weight was obtained upon repeating the experiment in immunocompetent dwarf mice (lit/lit)., Conclusions: We report for the first time sustained levels of circulating hGH after intramuscular naked DNA administration and, consequently, a highly significant weight increase of dwarf 'little' mice., (Copyright (c) 2010 John Wiley & Sons, Ltd.)

Background: Keratinocytes are a very attractive vehicle for ex vivo gene transfer and systemic delivery because proteins secreted by these cells may reach the circulation via a mechanism that mimics the natural process., Methods: An efficient retroviral vector (LXSN) encoding the mouse growth hormone gene (mGH) was used to transduce primary human keratinocytes. Organotypic raft cultures were prepared with these genetically modified keratinocytes and were grafted onto immunodeficient dwarf mice (lit/scid)., Results: Transduced keratinocytes presented a high and stable in vitro secretion level of up to 11 microg mGH/10(6)cells/day. Conventional epidermal sheets made with these genetically modified keratinocytes, however, showed a drop in secretion rates of > 80% due to detachment of the epithelium from its substratum. Substitution of conventional grafting methodologies with organotypic raft cultures completely overcame this problem. The stable long-term grafting of such cultures onto lit/scid mice could be followed for more than 4 months, and a significant weight increase over the control group was observed in the first 40 days. Circulating mGH levels revealed a peak of 21 ng/ml just 1 h after grafting but, unfortunately, these levels rapidly fell to baseline values., Conclusions: mGH-secreting primary human keratinocytes presented the highest in vitro expression and peak circulatory levels reported to date for a form of GH with this type of cells. Together with previous data showing that excised implants can recover a remarkable fraction of their original in vitro mGH secretion efficiency in culture, the factors that might still hamper the success of this promising model of cutaneous gene therapy are discussed.