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Gene Therapy for Hemophilia B With Liver-specific Element Mediated by Rep-RBE Site-specific Integration System

Xu, Zhengxin MD, PhD*,†; Ye, Juan MS*,†; Zhang, Amin MS*,†; Xie, Linjun MS*,†; Shen, Qi MS*; Xue, Jinglun PhD*; Chen, Jinzhong MD, PhD*

Journal of Cardiovascular Pharmacology: February 2015 - Volume 65 - Issue 2 - p 153–159
doi: 10.1097/FJC.0000000000000172
Original Article

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.

*State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China; and

Department of Pharmacology, Medical School, Yangzhou University, Yangzhou, China.

Reprints: Jinzhong Chen, MD, PhD, State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China (e-mail:

The authors report no conflicts of interest.

Received July 12, 2014

Accepted September 18, 2014

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