Gene Therapy for Hemophilia B With Liver-specific Element Mediated by Rep-RBE Site-specific Integration System
Gene Therapy for Hemophilia B With Liver-specific Element Mediated by Rep-RBE Site-specific Integration System
复制标题
Rep-RBE 位点特异性整合系统介导的肝脏特异性元件对 B 型血友病的基因治疗
DOI：
10.1097/fjc.0000000000000172
复制
发表时间：
2014-10
影响因子：
3
通讯作者：
薛京伦
中科院分区：
文献类型：
--
作者：
张阿敏;谢林俊;沈琦;薛京伦
摘要：腺相关病毒（Adeno-associated virus， AAV）是一种非致病性病毒，能够靶向人类19号染色体的AAVS1位点进行整合，在AAV调节蛋白（Rep）存在的情况下，AAV的16 bp Rep结合元件（RBE）序列足以介导这种特异性整合。在此之前，我们通过水动力注射将2个质粒pRBE-CMV-hFIX和pRC共转导到AAVS1转基因小鼠体内，观察了人凝血因子IX （human coagulation Factor IX, hFIX）的长期表达。通过巢式聚合酶链反应验证相应的AAVS1位点特异性整合。在这项研究中，我们建立了一个新的hFIX表达质粒，pRBE-HCR-hAAT-hFIX，由肝脏特异性启动子驱动，用HCR-hAAT组成的人源化启动子取代pRBE-CMV-hFIX的CMV启动子。瞬时转染pRBE-CMV-hFIX或pRBE-HCR-hAAT-hFIX后，hFIX在体外的表达几乎相同。在转染pRC/pRBE-CMV-hFIX鸡尾酒和pRC/pRBE-HCR-hAAT-hFIX鸡尾酒的小鼠中均发现了aavs1特异性整合。然而，pRBE-HCR-hAAT-hFIX小鼠的hFIX表达更高，持续时间更长。达到正常血浆hFIX浓度的1%以上，并维持240天。结果表明，RBE-HCR-hAAT元件可以提高hFIX的表达，并显示Rep-RBE位点特异性整合在血友病B基因治疗中的潜在应用。
Abstract: Adeno-associated virus (AAV) is a nonpathogenic virus capable of targeting human chromosome 19 for integration at AAVS1 site, and a 16 bp Rep binding element (RBE) sequence of AAV was sufficient for mediating this specific integration in the presence of AAV regulation proteins (Rep). Previously, we cotransduced 2 plasmids, pRBE-CMV-hFIX and pRC, into the AAVS1 transgenic mice by hydrodynamic injection, and a long-term expression of human coagulation Factor IX (hFIX) was observed. The corresponding AAVS1 locus site-specific integrations were verified by nested polymerase chain reaction. In this study, we established a novel hFIX expression plasmid, pRBE-HCR-hAAT-hFIX, driven by a liver-specific promoter by replacing the CMV promoter of pRBE-CMV-hFIX with a humanized promoter consisting of HCR-hAAT. The expression of hFIX in vitro was almost the same in transient transfection of pRBE-CMV-hFIX or pRBE-HCR-hAAT-hFIX. AAVS1-specific integrations were identified both in mice transfected with pRC/pRBE-CMV-hFIX cocktail and pRC/pRBE-HCR-hAAT-hFIX cocktail. However, the expression of hFIX of pRBE-HCR-hAAT-hFIX mice was higher and persisted longer. It achieved more than 1% of normal plasma hFIX concentration and maintained for 240 days. The result suggested that RBE-HCR-hAAT element could improve the expression of hFIX and present potential usage of Rep-RBE site-specific integration in gene therapy for hemophilia B.
登录
查看更多内容
DOI：
10.1038/mt.2008.161
发表时间：
2008-10
期刊：
Molecular therapy : the journal of the American Society of Gene Therapy
影响因子：
--
作者：
通讯作者：
--
影响因子：
56.9
作者：
KAY, MA;ROTHENBERG, S;WOO, SLC
通讯作者：
WOO, SLC
影响因子：
2.9
作者：
Douglas, Kimberly L.
通讯作者：
Douglas, Kimberly L.
影响因子：
5.1
作者：
Zhang, G;Song, YK;Liu, D
通讯作者：
Liu, D
DOI：
10.1002/jgm.1583
发表时间：
2011-07
期刊：
The Journal of Gene Medicine
影响因子：
--
作者：
H. Kim;Jong Chul Kim;Yeon Kyung Lee;Jung Seok Kim;Y. Park
通讯作者：
H. Kim;Jong Chul Kim;Yeon Kyung Lee;Jung Seok Kim;Y. Park