1. Gene Therapy for Hemophilia B With Liver-specific Element Mediated by Rep-RBE Site-specific Integration System
- Author
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Linjun Xie, Juan Ye, Xu Zhengxin, Qi Shen, Jing-Lun Xue, Amin Zhang, and Chen Jinzhong
- Subjects
viruses ,Transgene ,Genetic enhancement ,Genetic Vectors ,Mice, Transgenic ,Biology ,Hemophilia B ,DNA-binding protein ,Virus ,Factor IX ,Mice ,Viral Proteins ,Plasmid ,medicine ,Animals ,Humans ,Promoter Regions, Genetic ,Cells, Cultured ,Pharmacology ,Expression vector ,Gene Transfer Techniques ,Genetic Therapy ,Transfection ,Dependovirus ,Molecular biology ,DNA-Binding Proteins ,Liver ,Cardiology and Cardiovascular Medicine ,Chromosomes, Human, Pair 19 ,Plasmids ,medicine.drug - 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.
- Published
- 2015