通过肌肉注射修饰的前胰岛素原基因对链脲佐菌素诱导的糖尿病进行基因治疗。

Croze France, Prud'homme Gérald J

Department of Pathology, McGill University, 3775 University St., Room B13, Montreal, Quebec, Canada H3A 2B4.

J Gene Med. 2003 May;5(5):425-37. doi: 10.1002/jgm.359.

BACKGROUND

Despite improvements in insulin preparation and delivery, physiological normoglycemia is not easily achieved in diabetics. Therefore, there has been considerable interest in developing gene therapy approaches to supply insulin. We studied a nonviral muscle-based method of gene therapy and demonstrated that it could prevent hyperglycemia in murine streptozotocin (STZ)-induced diabetes.

METHODS

A plasmid encoding mouse furin-cleavable preproinsulin II cDNA (FI), or its B10-analogue (B10FI), and a plasmid encoding furin were coinjected into muscle of CD-1 mice, who were treated a day later with STZ to induce diabetes. Electroporation was applied to increase gene transfer. Blood glucose was measured in fed and fasting mice, and fasting plasma insulin was measured by radioimmunoassay. The form of insulin produced and the presence of C-peptide were analyzed by gel filtration chromatography.

RESULTS

A B10FI plasmid codelivered with a furin plasmid reduced fed and fasting blood glucose levels in STZ-treated diabetic mice. The (pro)insulin levels in plasma were increased by up to 70-fold versus blank plasmid-treated diabetic mice. The administration of FI with furin was less effective. (Pro)insulin levels were greatly increased by using two plasmids carrying different promoter elements (CMV and SV40). Insulin was identified in muscle cells by immunohistochemistry. In plasma, 40-70% of the (pro)insulin was processed to the mature form and free C-peptide was identified. Insulin gene-treated mice had improved growth rates and appeared healthier. A single injection of B10FI with SV40Furin DNA increased plasma (pro)insulin for at least 8 weeks and reduced fed blood glucose levels for 5 weeks and fasting levels for 8 weeks.

CONCLUSIONS

This is the first report that electroporation-enhanced intramuscular gene therapy with B10FI can prevent hyperglycemia in murine STZ-induced diabetes. Gene therapy using various routes and methods of furin-cleavable insulin gene delivery has been previously explored but, in muscle, results comparable to ours have not been reported.

背景

尽管胰岛素制剂和给药方式有所改进，但糖尿病患者仍难以实现生理性血糖正常。因此，开发基因治疗方法来供应胰岛素引起了广泛关注。我们研究了一种基于肌肉的非病毒基因治疗方法，并证明它可以预防小鼠链脲佐菌素（STZ）诱导的糖尿病中的高血糖。

方法

将编码小鼠弗林蛋白酶可裂解的前胰岛素原II cDNA（FI）或其B10类似物（B10FI）的质粒与编码弗林蛋白酶的质粒共注射到CD-1小鼠的肌肉中，一天后用STZ治疗以诱导糖尿病。应用电穿孔来增加基因转移。在喂食和禁食的小鼠中测量血糖，并通过放射免疫测定法测量空腹血浆胰岛素。通过凝胶过滤色谱分析产生的胰岛素形式和C肽的存在。

结果

与弗林蛋白酶质粒共递送的B10FI质粒降低了STZ治疗的糖尿病小鼠的喂食和空腹血糖水平。与空白质粒处理的糖尿病小鼠相比，血浆中（前）胰岛素水平增加了多达70倍。将FI与弗林蛋白酶一起给药效果较差。使用携带不同启动子元件（CMV和SV40）的两个质粒可大大提高（前）胰岛素水平。通过免疫组织化学在肌肉细胞中鉴定出胰岛素。在血浆中，40-70%的（前）胰岛素被加工成成熟形式，并鉴定出游离C肽。接受胰岛素基因治疗的小鼠生长速率提高，看起来更健康。单次注射B10FI与SV40弗林蛋白酶DNA可使血浆（前）胰岛素增加至少8周，并使喂食血糖水平降低5周，空腹水平降低8周。