Thulé P M, Liu J M
Department of Medicine, Emory University School of Medicine, and Atlanta VA Medical Center, GA 30033, USA.
Gene Ther. 2000 Oct;7(20):1744-52. doi: 10.1038/sj.gt.3301297.
Effective and safe insulin gene therapy will require regulation of transgenic insulin secretion. We have created a liver-targeted insulin transgene by engineering glucose responsive elements into a hepatic promoter containing an inhibitory insulin response sequence. In this work, we demonstrate application of this transgene for the treatment of diabetes mellitus in vivo, by administering a recombinant adenovirus vector, Ad/(GIRE)3BP-1 2xfur, to rats made diabetic with streptozotocin. We verified hepatic expression of transgenic insulin by RT-PCR, and confirmed glucose responsive stimulation of transgenic insulin secretion in vivo by serum RIA. Following a portal system injection of either Ad/(GIRE)3BP-1 2xfur, or an empty adenoviral vector, animals made diabetic with either low (120 mg/kg), or high (290 mg/kg) dose streptozotocin (STZ) were monitored for changes in body weight, and blood glucose. Without subcutaneous insulin injections, blood glucose values of sham-treated animals (n = 8) remained elevated, and animals failed to gain weight (n = 4), or died (n = 4). In contrast, body weight of Ad/(GIRE)3BP-1 2xfur-treated animals (n = 13) increased, and blood glucose remained at near normal levels from one to 12 weeks. Glucose values <50 mg/dl were infrequently observed, and no Ad/(GIRE)3BP-1 2xfur-treated animal succumbed to hypoglycemia. Treatment with the insulin transgene enabled diabetic animals to reduce blood sugars following a glucose load, and to maintain blood sugar levels during a 10-h fast. Hepatic production of human insulin produced near normal glycemia, and weight gain, without exogenous insulin, and without lethal hypoglycemia. In conclusion, we have demonstrated the feasibility of utilizing transcription to control transgenic insulin production in a rodent model of diabetes mellitus.
有效且安全的胰岛素基因疗法需要对转基因胰岛素的分泌进行调控。我们通过将葡萄糖反应元件导入含有抑制性胰岛素反应序列的肝脏启动子中，构建了一个肝脏靶向性胰岛素转基因。在这项研究中，我们通过向经链脲佐菌素诱导糖尿病的大鼠体内注射重组腺病毒载体Ad/(GIRE)3BP-1 2xfur，证明了该转基因在体内治疗糖尿病的应用。我们通过逆转录聚合酶链反应（RT-PCR）验证了转基因胰岛素在肝脏中的表达，并通过血清放射免疫分析（RIA）证实了体内转基因胰岛素分泌的葡萄糖反应性刺激。在门静脉系统注射Ad/(GIRE)3BP-1 2xfur或空腺病毒载体后，对用低剂量（120 mg/kg）或高剂量（290 mg/kg）链脲佐菌素（STZ）诱导糖尿病的动物监测体重和血糖变化。未经皮下注射胰岛素，假处理动物（n = 8）的血糖值持续升高，动物体重未增加（n = 4）或死亡（n = 4）。相比之下，Ad/(GIRE)3BP-1 2xfur处理的动物（n = 13）体重增加，血糖在1至12周内维持在接近正常水平。很少观察到血糖值<50 mg/dl，且没有Ad/(GIRE)3BP-1 2xfur处理的动物死于低血糖。胰岛素转基因治疗使糖尿病动物在葡萄糖负荷后能够降低血糖，并在10小时禁食期间维持血糖水平。肝脏产生的人胰岛素在无外源性胰岛素且无致命低血糖的情况下产生了接近正常的血糖水平和体重增加。总之，我们已经证明了在糖尿病啮齿动物模型中利用转录控制转基因胰岛素产生的可行性。
