In humans, deficiency of the GLA enzyme is an inheritable, X-linked recessive disorder (Fabry disease) that results in accumulation of glycosphingolipid globotriaosylceramide (Gb3) within blood vessels, various tissues, and organs leading to severe pain, renal failure, cardiovascular disease and resultant premature mortality. Enzyme replacement therapy (ERT) is the only approved pharmacological treatment and is prohibitively expensive. Gene therapy may be a viable alternative to ERT. This study investigates the use of hydrodynamic gene delivery (HGD) of the GLA gene to GLA knockout mice, the Fabry disease animal model. The objective of this study is to evaluate the safety, efficacy and therapeutic outcome of administering GLA-containing plasmids via HGD to GLA knockout mice. The human GLA gene cDNA sequence was successfully inserted into the pAAV-MCS vector at the EcoR1 and Xho1 cloning sites resulting in pAAV-GLA plasmids and into the pLIVE vector at BamH1 and Xho1 sites resulting in pLIVE-GLA. It was determined that an 8% (of body weight) injection volume significantly increased GLA activity when compared to 6% (p<0.05) and injection volumes greater than 8% resulted in mortality. DNA doses of 5 to 15 µg/ml produced higher serum levels of GLA in the GLA knockout mice when compared to wild-type (WT) mice. The DNA dose used in this study was 15 µg/ml with an injection volume of 8%. Results show that pAAV-GLA generated greater activity (7600 %) in serum when compared to pLIVE-GLA (2500 %). With the pAAV-GLA plasmid, serum activity was maintained for two days. However, for pLIVE-GLA, at 18 days post-injection, GLA activity in liver and heart tissues in GLA knockout mice was significantly (p<0.05) increased when compared to WT. These levels were sustained for up to 66 days after the treatment when 3 treatments were administered, each 2 weeks apart. These data demonstrate that HGD of pLIVE-GLA safely and effectively corrected GLA deficiency in GLA knockout mice. Future studies should address Gb3 accumulation relative to GLA expression using the same model
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