Co-expression of MGMT(P140K) and alpha-L-iduronidase in primary hepatocytes from mucopolysaccharidosis type I mice enables efficient selection with metabolic correction.
Co-expression of MGMT(P140K) and alpha-L-iduronidase in primary hepatocytes from mucopolysaccharidosis type I mice enables efficient selection with metabolic correction.
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MGMT(P140K) 和 α-L-艾杜糖醛酸酶在粘多糖贮积症 I 型小鼠的原代肝细胞中共表达，能够通过代谢校正进行有效选择。
DOI：
10.1002/jgm.1141
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Pan,Dao
中科院分区：
文献类型：
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作者：
Wang,Daren;Worsham,DNicole;Pan,Dao
背景：在动物模型中，当在出生时接受系统霉素体内基因治疗时，已经导致了广泛的纠正。然而，在出生后治疗的动物中观察到有限的改善，主要针对肝脏和骨髓。研究表明，O6 -甲基鸟嘌呤- DNA -甲基转移酶变异（MGMTP140K）介导了动物转导造血干细胞（HSC）的体内选择。方法研究MGMTP140K介导的慢病毒载体在粘多糖病（MPS I）小鼠原代肝细胞中筛选的可行性。结果我们发现，O6‐苄基鸟嘌呤(BG)/1,3‐双（2‐氯乙基）‐1‐亚硝基脲（BCNU）多次循环处理，在体外hsc选择的有效剂量下，在最小细胞扩增的培养条件下，MGMT‐表达的原代肝细胞增加了两倍。这种富集水平与选择肝脏最大耐受剂量的BCNU后获得的富集水平相当。无论初始转导频率如何，也无论MGMT（内部核糖体进入位点的上游或下游）在载体构建中的位置如何，均观察到类似水平的增加。此外，我们发现延伸因子1α启动子在原代肝细胞中的转基因表达优于来自脾灶形成病毒的长末端重复启动子。此外，转导的酶缺陷肝细胞中的治疗性转基因表达水平与BCNU的剂量直接相关，导致转导肝细胞的代谢纠正和邻近非转导的MPS I细胞的代谢交叉纠正。这些结果表明，MGMTP140K的表达在原代肝细胞中具有成功的保护/选择作用，并为MGMTP140K介导的BG/BCNU治疗肝细胞和HSC的共选择前景提供了“概念证明”。版权所有©2007 John Wiley & Sons， Ltd
BackgroundSystemicin vivogene therapy has resulted in widespread correction in animal models when treated at birth. However, limited improvement was observed in postnatally treated animals with mainly targeting to the liver and bone marrow. It has been shown that an O6‐methylguanine‐DNA‐methyltransferase variant (MGMTP140K) mediatedin vivoselection of transduced hematopoietic stem cells (HSC) in animals.MethodsWe investigated the feasibility of MGMTP140K‐mediated selection in primary hepatocytes from a mouse model of mucopolysaccharidosis type I (MPS I)in vitrousing lentiviral vectors.ResultsWe found that multiple cycles of O6‐benzylguanine (BG)/1,3‐bis(2‐chloroethyl)‐1‐nitrosourea (BCNU) treatment at a dosage effective forex vivoHSC selection led to a two‐fold increase of MGMT‐expressing primary hepatocytes under culture conditions with minimum cell expansion. This enrichment level was comparable to that obtained after selection at a hepatic maximal tolerated dose of BCNU. Similar levels of increase were observed regardless of initial transduction frequency, or the position of MGMT (upstream or downstream of internal ribosome entry site) in the vector constructs. In addition, we found that elongation factor 1α promoter was superior to the long‐terminal repeat promoter from spleen focus‐forming virus with regard to transgene expression in primary hepatocytes. Moreover, the levels of therapeutic transgene expression in transduced, enzyme‐deficient hepatocytes directly correlated with the doses of BCNU, leading to metabolic correction in transduced hepatocytes and metabolic cross‐correction in neighbouring non‐transduced MPS I cells.ConclusionsThese results demonstrate that MGMTP140Kexpression confers successful protection/selection in primary hepatocytes, and provide ‘proof of concept’ to the prospect of MGMTP140K‐mediated co‐selection for hepatocytes and HSC using BG/BCNU treatment. Copyright © 2007 John Wiley & Sons, Ltd.