Induction and Prevention of Severe Hyperammonemia in the spfash Mouse Model of Ornithine Transcarbamylase Deficiency Using shRNA and rAAV-mediated Gene Delivery
Induction and Prevention of Severe Hyperammonemia in the spfash Mouse Model of Ornithine Transcarbamylase Deficiency Using shRNA and rAAV-mediated Gene Delivery
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DOI：
10.1038/mt.2011.32
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发表时间：
2011-05-01
影响因子：
12.4
通讯作者：
Alexander, Ian E.
中科院分区：
文献类型：
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作者：
Cunningham, Sharon C.;Kok, Cindy Y.;Alexander, Ian E.
生命早期出现的尿素循环缺陷伴高氨血症仍然难以治疗，通常需要肝移植。基因治疗有可能在等待肝移植期间预防高氨血症发作，也可能完全避免移植的需要。鸟氨酸转氨甲酰酶（OTC）缺乏症是最常见的尿素循环障碍，为肝脏靶向基因治疗的发展提供了理想的模型。虽然我们和其他人已经使用腺相关病毒 (AAV) 载体成功治愈了 OTC 缺陷的 spf(ash) 小鼠模型，但该模型的主要限制是残留 OTC 酶活性的存在，该活性赋予温和的表型，而没有临床上显着的高氨血症。为了更好地模拟严重疾病，我们设计了一种策略，涉及 AAV2/8 介导的短发夹 RNA (shRNA) 递送，以特异性敲低残留的内源性 OTC 信使 RNA (mRNA)。事实证明，这种策略非常成功，经过媒介治疗的小鼠出现了严重的高氨血症和相关的神经损伤。使用该系统，我们表明，预防高氨血症所需的编码小鼠 OTC (mOTC) cDNA 的 AAV 救援构建体的剂量比控制乳清酸尿所需的剂量低五倍。这一结果有利于临床转化，因为它表明治疗益处的阈值可能低于早期研究表明的水平。
Urea cycle defects presenting early in life with hyperammonemia remain difficult to treat and commonly necessitate liver transplantation. Gene therapy has the potential to prevent hyperammonemic episodes while awaiting liver transplantation, and possibly also to avert the need for transplantation altogether. Ornithine transcarbamylase (OTC) deficiency, the most prevalent urea cycle disorder, provides an ideal model for the development of liver-targeted gene therapy. While we and others have successfully cured the spf(ash) mouse model of OTC deficiency using adeno-associated virus (AAV) vectors, a major limitation of this model is the presence of residual OTC enzymatic activity which confers a mild phenotype without clinically significant hyperammonemia. To better model severe disease we devised a strategy involving AAV2/8-mediated delivery of a short hairpin RNA (shRNA) to specifically knockdown residual endogenous OTC messenger RNA (mRNA). This strategy proved highly successful with vector-treated mice developing severe hyperammonemia and associated neurological impairment. Using this system, we showed that the dose of an AAV rescue construct encoding the murine OTC (mOTC) cDNA required to prevent hyperammonemia is fivefold lower than that required to control orotic aciduria. This result is favorable for clinical translation as it indicates that the threshold for therapeutic benefit is likely to be lower than indicated by earlier studies.