Gene therapy of urea cycle deficirncy

鸟氨酸转氨甲酰酶（OTC）缺乏症是人类最常见和最严重的先天性尿素循环缺陷，目前仍缺乏适当的治疗，死亡率很高。腺病毒载体提供了一种有效的基因传递系统，但也存在一些问题，包括毒性。需要开发减少治疗所需载体量的有效启动子。我们构建了两个重组腺病毒载体AdexCAGhOTC和AdexSRalphahOTC，它们分别携带在CAG（一种带有CMV IE增强子的修饰的鸡β-肌动蛋白启动子）和SR α（带有HTLV-1 LTR的R片段和部分U 5片段的SV 40早期启动子）转录控制下的人OTC基因。在成年spf+<ash>小鼠（人OTC缺乏症的动物模型）和OTC缺乏症的原代人肝细胞中对每种方法进行了测试。SPF级<ash>小鼠具有明显的乳清酸尿症，如在人类中所见。AdexCAGhOTC单独静脉给药后，在这些动物中观察到肝脏OTC活性完全恢复，组织损伤极小。蛋白质印迹分析证实了肝脏OTC表达，乳清酸尿症的正常化在60天内是明显的。AdexCAGhOTC感染的原代肝细胞酶活性是AdexSR α hOTC感染的10-40倍，因此，携带CAG等高效启动子的腺病毒载体为OTC缺陷症的基因治疗提供了进一步的实验依据。

Ornithine transcarbamylase (OTC) deficiency, the most commom and severe inborn error of the urea cycle in humans, remains without adequate treatment, and ortality rates are high. Adenoviral vectors provide an efficient system for gene delivery, but there are problems, including toxicity. Efficient promoters that reduce the amount of vector required for treatment need to be developed. We constructed two recombinant adenoviral vectors, AdexCAGhOTC and AdexSRalphahOTC,which harbor the human OTC gene under transcriptional control of CAG (a modified chicken beta-actin promoter with CMV-IE enhancer) and SRalpha (the SV 40 early prommoter with the R segment and part of the U5 segment of the HTLV-1 LTR) , respectively. Each was tested in adult spf^<ash> mice, an animal model of human OTC deficiency, and in primary human hepatocytes with OTC deficiency. Spf^<ash> mice have a pronounced orotic aciduria as seen in humans. A complete recovery of hepatic OTC activity with minimal tissue damage was observed in these animals following the intravenous administration of AdexCAGhOTC alone. Western blot analysis confirmed hepatic OTC expression and normalization of orotic aciduria was evident for 60 days. Enzyme activities of primary human hepatocytes infected with AdexCAGhOTC were 10-40 times higher than those with AdexSRalphahOTC.Thus, the adenoviral vector with an efficient promoter such as CAG,can be given further considerartion for possible gene therapy in humans with OTC deficiency.