Rep-RBE Vivo Studies Mediated Site-specific Integration System Hemophilia B Gene Therapy

Objects:We have developed liver-specific expression cassettes containing a hepatic locus control region (HCR) from the ApoE gene locus, an al-anti-trypsin promoter and target gene hFIX based on RBE-Rep site-specific integration system. The hFIX level was evaluated in transgenic mice. The distribution, transcription, site-specific integration and safety were revealed.Methods:Plasmid of RBE-HCR-hAAT-hFIX was constructed and transfected different cell lines to detected the expression of hFIX; Delivered plasmid of RBE-HCR-hAAT-hFIX and Rep-donored plasmid to transgenic mice by hydrodynamic injection, hFIX expression at different time point by ELISA and plasmid of RBE-CMV-hFIX used as control group.The distribution and transcription were detected by PCR and RT-PCR, site-specific integration was verified by nested-PCR and liver damage was tested by ALT reagent kit and pathological section.Result:The expression of hFIX protein in RBE-HCR-hAAT-hFIX group and in the RBE-CMV-hFIX group was at the same level initially, but the former group was almost4-fold higher than that of the RBE-CMV-hFIX group at day120.Liver toxicity was acute and transient. Moreover, the damage was caused by the rapid injection of a large volume rather than the plasmids themselves.Conclusion:Liver-specific element can be a substitute of CMV promoter in gene therapy for hemophilia B mediated by RBE-Rep site-specific integration system and would achieve a higher-expression level of hFIX with a long-term.