title: Gene therapy for hemophilia B with liver-specific element mediated by Rep-RBE site-specific integration system.
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.
authors.affiliation: *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.
authors.forename: Zhengxin
authors.initials: Z
authors.lastname: Xu
authors.forename: Juan
authors.initials: J
authors.lastname: Ye
authors.forename: Amin
authors.initials: A
authors.lastname: Zhang
authors.forename: Linjun
authors.initials: L
authors.lastname: Xie
authors.forename: Qi
authors.initials: Q
authors.lastname: Shen
authors.forename: Jinglun
authors.initials: J
authors.lastname: Xue
authors.forename: Jinzhong
authors.initials: J
authors.lastname: Chen
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002888:Chromosomes, Human, Pair 19; D004268:DNA-Binding Proteins / Q000235:genetics*; D000229:Dependovirus / Q000502:physiology; D005164:Factor IX / Q000235:genetics; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D002836:Hemophilia B / Q000628:therapy*; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D010957:Plasmids / Q000502:physiology; D011401:Promoter Regions, Genetic; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D005164:Factor IX