A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ-ated viral vector containing hFIXR338A...A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ-ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27%±3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeho-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.展开更多
Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein ...Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with GTiIX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTiIX were obtained. At the same time, previous normal adenoviral vector pAdSPiIX containing viral genome and hFIX mini-gene was constructed, and then previous ade-novirus (pre-Ad) AdSPiIX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTiIX was less than 0.8%. 3T3 cells were transfected with AdGTiIX and AdSPiIX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 mg /106·24 h and 1.6±0.3 mg/106·24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 11010pfu AdGTiIX or AdSPiIX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 mL to 60 mL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTiIX was obviously weaker than that triggered by AdSPiIX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector sys-tem prolonged the expression time of hFIX and reduced immune response, thus offering a prom-ising result for further pre-clinical study.展开更多
基金the State High Technology Development Program (Grant No.Z20-02-01), Shanghai Post-doctoral Fellowship Foundation the National "863" High-Tech Program+1 种基金 the National Natural Science Foundation of China (Grant No. 39880019) Fok Ying Tung Foundation (Gra
文摘A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ-ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27%±3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeho-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.
基金supported by the State High Technology Development Program(863).
文摘Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with GTiIX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTiIX were obtained. At the same time, previous normal adenoviral vector pAdSPiIX containing viral genome and hFIX mini-gene was constructed, and then previous ade-novirus (pre-Ad) AdSPiIX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTiIX was less than 0.8%. 3T3 cells were transfected with AdGTiIX and AdSPiIX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 mg /106·24 h and 1.6±0.3 mg/106·24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 11010pfu AdGTiIX or AdSPiIX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 mL to 60 mL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTiIX was obviously weaker than that triggered by AdSPiIX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector sys-tem prolonged the expression time of hFIX and reduced immune response, thus offering a prom-ising result for further pre-clinical study.
