Gene therapy for Fabry mice

我们最近开发了一种新的肾脏靶向基因转移技术，使用逆行肾静脉注射裸质粒DNA，并表明肾脏可以作为生产治疗性蛋白质的储存器官。法布里病是一种x连锁的隐性先天性代谢疾病，其特征是由溶酶体酶α-半乳糖苷酶a （α-Gal a）缺乏引起的球状三烷基神经酰胺（Gb3）的全身和血管积聚。基因疗法有望彻底改变法布里病的治疗方法。我们检测α-Gal A敲除小鼠（Fabry小鼠）α-Gal A转移。表达人α-Gal A的裸质粒DNA （pKSCX-α-Gal A）溶液经肾逆行静脉快速注入左肾。同样将pKSCX溶液注射到对照小鼠（pKSCX小鼠）。我们通过逆转录酶聚合酶链反应证实了左肾中存在载体衍生的α-Gal A mRNA，并观察了pKSCX-α-Gal A注射小鼠血浆α-Gal A水平。与pKSCX小鼠相比，pKSCX-α-Gal A小鼠表现出显著的治疗效果：经薄层色谱分析证实，注射后的肾、肝、心α-Gal A升高，心、双侧肾、肝、脾Gb3降低。这些结果表明，通过逆行肾静脉注射肾靶向pKSCX-α-Gal A基因转移可减少Fabry小鼠全身Gb3的积累。

We recently developed a novel kidney-targeted gene transfer technique, using the retrograde renal vein injection of naked plasmid DNA and indicated that the kidney serves as a depot organ for the production of therapeutic proteins. Fabry disease is an X-linked recessive inborn metabolic disorder characterized by systemic and vascular accumulation of globotriaosylceramide (Gb3) caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A). Gene therapy is expected to revolutionize the treatment of Fabry disease. We examined the α-Gal A transfer to the treatment of mice with α-Gal A knock out (Fabry mice). Naked plasmid DNA expressing human α-Gal A (pKSCX-α-Gal A) solution was rapidly injected into the left kidneys via the retrograde renal vein. pKSCX solution was similarly injected into control mice (pKSCX mice).We confirmed the presence of vector-derived α-Gal A mRNA in the left kidneys by reverse transcriptase polymerase chain reaction and observed plasma α-Gal A levels in pKSCX-α-Gal A-injected mice. Compared with the pKSCX mice, the pKSCX-α-Gal A mice showed significant therapeutic effects: increase of α-Gal A in injected kidney, liver, heart, and decrease of Gb3 in heart, bilateral kidneys, liver, spleen confirmed by thin-layer chromatography analysis.These results demonstrate that kidney-targeted pKSCX-α-Gal A gene transfer by retrograde renal vein injection reduces systemic accumulation of Gb3 in Fabry mice.