Preparation of a recombinant adeno-associated viral vector with a mutationof human factor IX in large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human f actor IX (hFIXR338A) with different regulation elements were constructed an d used to transduce cell lines. The plasmids and the stable transduction ce ll clones with high expression level of hFIXR338A were obtained by selectin g and optimizing, and then, the recombinant adeno-associated viral vector w ith hFIXR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for p roducing rAAV-hFIX R338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAVhF IXR338A was more than 1.25x10(12) particle/mL, and then, a mammalian cell l ine, C2C12 and the factor IX knock-out mice were transfected with the rAAV- hFIXR338A in vitro and in vivo. The results show that the high-level expres sion of rAAV-hFIXR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32 +/- 92.14) ng . (10(6) cells)-(1) . (24 h)(-1) in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFIX R338A, which was as high as the expression of rAAV-hFIX -wt (2565.76 +/- 64.36) ng . (10(6) cells)-(1) . (24 h)(-1) in C2CI2 and 453.9 2 ng/mL in the mice treated with rAAV-hFDC-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFIXR 338A is about 2.46 times higher than that of hFIX-wt. It was first reported that a mutation of human factor DC was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for p reparation of rAAV-hFIX R338A on a large scale, which laid the foundation o f industrial production for applying rAAV viral stocks to gene therapy clin ical trial for hemophilia B mediated with rAAV-hFIX.