| Hemophilia B is an X-linked bleeding disorder caused by the deficiency of clotting factor IX (FIX). Compared with the conventional blood transfusion treatment, gene therapy offers a more attractive approach to achieve the goal of prophylactic hemostasis without the extreme costs, infectious and thrombotyic risks. In this study, long-term, high-level expression of FIX mediated by recombinant AAV vectors in hemophilia B mice was reported. The bleeding symptom of the treated mice was significantly ameliorated. Firstly, high titer of recombinant AAV particles was prepared according to the rHSV/AAV helper virus method. After intramuscularly injection (2×1012 v.g./mouse) of viral vectors in the hind limb, a sustained elevated level (>370ng/ml) of murine FIX expression in the plasma of hemophilia B mice were detected and persisted for more than 500 days. The biological activity reached 30% of normal levels, and bleeding symptoms in the treated mice were significantly alleviated. No anti-FIX antibody (inhibitor) was detected, though anti-AAV antibodies were found at a very low level after single injection. Repeated injection with rAAV/mFIX lead to a variation in anti-AAV antibody levels between the two groups received different doses. Results from tissue analysis confirmed the skeletal muscle as the origin for circulating functional mFIX. Secondly, we constructed the rAAV vectors of human clotting factor IX minigene, regulated by CMV promoter, liver specific promoter and EF promoter respectively. Large quantity of rAAV/hFIX by using the rHSV/AAV hybrid helper virus method was prepared. The viral particles were purified by HPLC and titrated by QC-PCR. After intramuscularly injection of viral vectors (2.5-3×1011v.g./mouse), extreme elevations (350 ng/ml intramuscularly and 500 ng/ml via tail vein injection) of hFIX expression in the plasma of hemophilia B mice were detected andpersisted for more than 12 weeks. The level of inhibitory antibody in plasma aligned with the hFIX curve. Low level of anti-HSV and anti-AAV antibodies was detected in mouse serum. Up to 4320 ng/ml of hFIX expression was achieved via tail vein injection of 2×1012 v.g./mouse, however, no elongation of expression time was found due to the multiple increases of inhibitory antibodies. Meanwhile, the bleeding symptoms of the treated mice were greatly alleviated. Part of the data were even exciting than those from normal mice.We also introduced Cre-loxP system in the production of rAAV. Defective adenovirus AdLC was used as the helper virus. While approaching mass production of rAAV carrying EGFP or hFIX gene, the generation of helper virus was significantly limited, it increased the simplicity of rAAV purification. The results from in vivo study demonstrated the superiority of this method. Recombinant AAV carrying EGFP or hFIX gene via HSV amplicon method was also performed. The titer of AAV reached 109 v.g./ml. The rAAV particles were infection-competent. High level of hFIX expression (754±32 ng/106 cells/24 h) in supernatant was detected, and the rate of fluorescent cells was 70% (MOI=500).Finally, BHK cells were transfected with two EGFP plasmids with or without AAV ITRs. The rates of fluorescent cells were 20.27% and 11.08% after screening and 10 passages. These results suggested that the existence of AAV ITRs played an active role for long-term transgene expression even without tans elements.
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